RESUMO
Purpose: To detect the differential expression of long non-coding RNAs (lncRNAs) in the ocular posterior poles of two guinea pig myopia models and explore the pathogenic role of lncRNAs in myopia. Methods: Form-deprived myopia (FDM) and lens-induced myopia (LIM) models were induced in guinea pig right eyes by wearing a translucent latex balloon head mask and a -10.00 diopter (D) lens, respectively. Ocular biometric parameters were measured biweekly. At 6 weeks after the induction of myopia, the guinea pig eyeballs were processed for hematoxylin and eosin staining to examine the ocular morphology. The ocular posterior poles from the normal control, FDM, and LIM groups were collected to analyze the differential expression of lncRNAs between the groups with high-throughput RNA sequencing (RNA-seq). Further, the lncRNA-mRNA colocation network was delineated to predict the functions of the differentially expressed lncRNAs. Last, Gene Ontology (GO) functional enrichment analysis and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis were performed on the colocated mRNAs of the differentially expressed lncRNAs. Additionally, the expression of the most differentially expressed lncRNAs in the myopia-induced eyes and the contralateral eyes was validated with quantitative real-time PCR (qPCR). Results: Compared with the normal controls and the contralateral eyes, the myopia-induced eyes in the FDM and LIM groups exhibited decreased scleral and choroidal thicknesses, reduced refraction, and increased ocular axial length but without changes in the corneal curvature radius at 6 weeks after myopia was induced. RNA-seq showed that 372 and 247 lncRNAs were differentially expressed in the FDM and LIM groups, respectively, in comparison to the normal counterparts. There were 380 differentially expressed lncRNAs in the LIM group compared to the FDM group. The GO and KEGG analyses showed that the colocated mRNAs of the differentially expressed lncRNAs were enriched in cellular components such as the extracellular matrix (ECM) structural constituent; in molecular functions such as kinase activity, metabolism, and growth; as well as in pathways including ECM-receptor interaction, glycosaminoglycan degradation, and mucin type O-Glycan biosynthesis. The expression patterns of the selected lncRNAs were verified with qPCR. Conclusions: High-throughput RNA-seq revealed previously undescribed lncRNA expression profiling in guinea pig FDM and LIM models. These results may shed light on the molecular pathogenesis of myopia and provide clues for interventional targets for this highly prevalent visual disorder.
Assuntos
Regulação da Expressão Gênica/genética , Miopia/metabolismo , RNA Longo não Codificante/metabolismo , Retina/metabolismo , Esclera/metabolismo , Animais , Biometria , Modelos Animais de Doenças , Matriz Extracelular/metabolismo , Ontologia Genética , Glicosaminoglicanos/metabolismo , Cobaias , Masculino , Miopia/genética , Miopia/patologia , Polimorfismo de Nucleotídeo Único , Polissacarídeos/biossíntese , RNA Longo não Codificante/genética , RNA-Seq , Reação em Cadeia da Polimerase em Tempo Real , Refração Ocular/genética , Refração Ocular/fisiologia , Retina/patologia , Esclera/patologia , Transdução de Sinais/genéticaRESUMO
Pulmonary arterial hypertension (PAH) is a devastating disease characterized by high pulmonary artery pressure. It is reported that microRNA-204 (miR-204) plays an important role in the development of PAH. Calcitriol [1,25-dihydroxyvitamin D3, 1,25(OH)2D3] mediates multiple pathophysiological processes. The aim of the current study was to explore the role of 1,25(OH)2D3 in PAH. PAH was induced in rats and rat pulmonary arterial endothelial cells (PAECs) were isolated as in vitro PAH model. The mean pulmonary artery pressure, morphologic changes, and expressions of transforming growth factor-beta1 (Tgfbr2), Smad2/7, alpha smooth muscle actin (α-SMA), and p21 were then measured. Furthermore, the effect of 1,25(OH)2D3 on rat PAECs with or without hypoxia treatment was also assessed by measuring the proliferation, migration, and cell cycle distribution of PAECs. The potential targets of miR-204 were also predicted and validated with a dual-luciferase reporter system. Then the role of miR-204 and Tgfbr2 in the anti-PAH effect of 1,25(OH)2D3 was further explored by modulating the expression of the two genes. The overall pulmonary hypertension and hypoxia-induced proliferation and migration of PAECs were attenuated by administration of 1,25(OH)2D3, which was associated with the suppressed expressions of Tgfbr2, α-SMA, and Smad7 and induced expressions of miR-204, p21 and Smad2 both in vitro and in vivo. Moreover, the luciferase reporter assay identified Tgfbr2 as a novel direct target of miR-204. Both overexpression of miR-204 and inhibition of Tgfbr2 would strengthen the effect of 1,25(OH)2D3 administration. Findings outlined in the current study demonstrated that 1,25(OH)2D3 was a promising therapeutic modality for treatment of PAH, function of which was exerted through miR-204 mediated Tgfbr2 signaling.
Assuntos
Calcitriol/administração & dosagem , Hipertensão Pulmonar/tratamento farmacológico , MicroRNAs/genética , Proteínas Serina-Treonina Quinases/genética , Receptores de Fatores de Crescimento Transformadores beta/genética , Animais , Apoptose , Proliferação de Células , Humanos , Hipertensão Pulmonar/genética , Hipertensão Pulmonar/patologia , Hipóxia , Músculo Liso Vascular , Artéria Pulmonar/efeitos dos fármacos , Artéria Pulmonar/patologia , Ratos , Receptor do Fator de Crescimento Transformador beta Tipo II , Transdução de Sinais , Proteína Smad2/genética , Proteína Smad7/genéticaRESUMO
Short chain fatty acids acetate and propionate have been demonstrated protective function in the intestinal mucosa. However, their impact on gastric mucosa has not yet been elucidated. The current study aimed to investigate the potential protective effects of acetate and propionate against ethanol-induced gastric mucosal lesion and the underlying mechanism in mice. ICR mice were orally treated with acetate and propionate, respectively, 30 min prior to the establishment of gastric mucosal injury model by challenge with absolute ethanol. The gastric samples were collected for the detection of oxidative, inflammatory and apoptotic related parameters. Acetate, but not propionate, attenuated the severity of gastric mucosal damage as evidenced by the gross changes of gastric mucosa, pathological aberrations. Acetate alleviated oxidative stress as shown by the increase in glutathione (GSH) content and superoxide dismutase (SOD) activities, and the decrease of malondialdehyde (MDA) level. The elevated concentrations of interleukin (IL)-1ß, tumor necrosis factor (TNF)-α and IL-6, and the activation of nuclear factor-kappaB (NF-κB) p65 by ethanol stimulation was also reduced by acetate. Moreover, the anti-inflammatory factors, IL-4, LXA4 and IL-10, were up-regulated in acetate treated group. With respect to gastric mucosal apoptosis, acetate suppressed caspase-3 activity and BAX expression in favor of cell survival. These favorable actions were maybe associated with up-regulation of the gastric MUC5AC, the key defense factor of gastric mucosal system. These findings accentuate the gastroprotective actions of acetate in ethanol-induced gastric injury which were mediated via concerted multi-prolonged actions, including suppression of gastric oxidation, inflammation and apoptosis and promotion of MUC5AC expression.
Assuntos
Depressores do Sistema Nervoso Central/antagonistas & inibidores , Depressores do Sistema Nervoso Central/toxicidade , Etanol/antagonistas & inibidores , Etanol/toxicidade , Ácidos Graxos Voláteis/farmacologia , Mucosa Gástrica/efeitos dos fármacos , Mucosa Gástrica/patologia , Úlcera Gástrica/induzido quimicamente , Úlcera Gástrica/prevenção & controle , Acetatos/farmacologia , Animais , Apoptose/efeitos dos fármacos , Caspase 3/metabolismo , Sobrevivência Celular/efeitos dos fármacos , Citocinas/metabolismo , Inflamação/induzido quimicamente , Inflamação/patologia , Inflamação/prevenção & controle , Masculino , Camundongos , Camundongos Endogâmicos ICR , Mucina-5AC/biossíntese , Estresse Oxidativo/efeitos dos fármacos , Propionatos , Úlcera Gástrica/patologia , Relação Estrutura-AtividadeRESUMO
Myopia is a major public health issue. However, interventional modalities for nonpathologic myopia are limited due to its complicated pathogenesis and the lack of precise targets. Here, we show that in guinea pig form-deprived myopia (FDM) and lens-induced myopia (LIM) models, the early initiation, phenotypic correlation, and stable maintenance of cochlin protein upregulation at the interface between retinal photoreceptors and retinal pigment epithelium (RPE) is identified by a proteomic analysis of ocular posterior pole tissues. Then, a microarray analysis reveals that cochlin upregulates the expression of the secreted frizzled-related protein 1 (SFRP1) gene in human RPE cells. Moreover, SFRP-1 elevates the intracellular Ca2+ concentration and activates Ca2+/calmodulin-dependent protein kinase II (CaMKII) signaling in a simian choroidal vascular endothelial cell line, and elicits vascular endothelial cell dysfunction. Furthermore, genetic knockdown of the cochlin gene and pharmacological blockade of SFRP1 abrogates the reduced choroidal blood perfusion and prevents myopia progression in the FDM model. Collectively, this study identifies a novel signaling axis that may involve cochlin in the retina, SFRP1 in the RPE, and CaMKII in choroidal vascular endothelial cells and contribute to the pathogenesis of nonpathologic myopia, implicating the potential of cochlin and SFRP1 as myopia interventional targets.
Assuntos
Proteína Quinase Tipo 2 Dependente de Cálcio-Calmodulina , Miopia , Humanos , Animais , Cobaias , Proteína Quinase Tipo 2 Dependente de Cálcio-Calmodulina/genética , Células Endoteliais , Proteômica , Miopia/genética , Miopia/prevenção & controle , Epitélio Pigmentado da Retina , Proteínas de Membrana/genética , Peptídeos e Proteínas de Sinalização IntercelularRESUMO
The low reproductive efficiency (RE) of geese limits their production in the poultry industry. To select ganders with high breeding potential, the effect of 3 sperm mobility ranks (SMRs; high-, medium-, and low-SMR) on the RE of naturally mating geese was determined. To exclude the confounding effect of social rank (SR) on RE in naturally mating flocks, a 2-factor nested experimental design was used to differentiate the effects of SMR and SR on RE. Twenty-seven ganders and 135 geese (Zi geese, Anser cygnoides L.) at approximately 1 yr of age were divided into 3 flocks, each of which included the 3 SMR groups. Each SMR group included 3 ganders and 15 female geese. Relative genetic contribution (RGC) is defined as the number of offspring sired by 1 male as a percentage of the entire goslings in each flock, and it was used to compare the differences in RE among ganders. The frequency of agonistic behavioral interactions (ABIs) among the ganders was video recorded in each SMR group, and the SR of each gander was determined. In total, 1,026 eggs were incubated, and 609 goslings hatched. Parent-offspring relationships among 771 individuals from the 2 generations were identified using 20 microsatellite markers, and the RGC was calculated. Results showed that the SMR and SR had significant effects on RGC in naturally mating geese (P = 0.001 and P = 0.000, respectively). Significant differences in RGC were observed among the high- and medium- and low-SMR groups, with average RGCs of 14.3, 10.6, and 8.4%, respectively. The high-SMR group had the highest RGCs in each flock, and the ganders with high SR had the highest RGCs among the 3 SMRs. The study showed that in a naturally mating geese population, selecting for the sperm mobility traits of a gander can effectively improve the RE.
Assuntos
Gansos , Sêmen , Masculino , Animais , Feminino , Gansos/genética , Galinhas , Óvulo , Espermatozoides , GenótipoRESUMO
In the original publication [...].
RESUMO
TGF-ß signaling plays a pivotal role in promoting tumor cell migration and cancer metastasis. ΔNp63α and TAp63α are two major isoforms of p53-related p63 protein. Our recent study has shown that TGF-ß1 promotes ΔNp63α protein degradation to facilitate cancer metastasis. However, whether TAp63α is involved in TGF-ß1-induced cancer metastasis remains unclear. In this study, we show that, in human pancreatic cancer MIA PaCa-2 cells harboring p53-R248W allele, TGF-ß1 can significantly inhibit TAp63α protein stability in a Smad pathway-independent manner. Lysosome inhibitor, chloroquine, but not proteasome inhibitor MG132, can rescue TGF-ß1-induced downregulation of TAp63α protein. In addition, we show that either TGF-ß1 treatment or silencing of TAp63α can dramatically increase migration of MIA PaCa-2 cells. Importantly, the restored expression of TAp63α can effectively block TGF-ß1-induced migration of MIA PaCa-2 cells. Mechanistically, we show that TGF-ß1 promotes TAp63α protein degradation, leading to upregulation of p53-R248W protein expression, and consequently resulting in elevated MIA PaCa-2 cell migration. Together, this study indicates that lysosomal degradation is an important way for regulating TAp63α protein fate and highlights that TGF-ß1-TAp63α-mutant p53 axis is critically important in pancreatic cancer metastasis.
RESUMO
Proliferative diabetic retinopathy (PDR) is a severe complication of diabetes and can cause blindness. However, the available therapeutic modalities to PDR have unsatisfactory efficacies and incur adverse effects, which is due to the paucity in the understanding of pathogenic mechanisms responsible for the disease. In this study, tandem mass tag labeling technology combined with liquid chromatography and tandem mass spectrometry were utilized to identify differentially expressed proteins in vitreous humor of patients with rhegmatogenous retinal detachment and PDR. The data are available via ProteomeXchange with identifier PXD021788. Afterwards, the downregulated protein expression of Cathepsin B, D, and L was verified in vitreous and serum of another cohort. The gene expression profiling of the 3 cathepsins was confirmed in blood cells of an extra cohort. Furthermore, in high glucose (HG)-treated retinal vascular endothelial cell cultures recapitulating the cathepsin expression patterns, Cathepsin B or D downregulation mediated the HG-induced anti-autophagic and pro-apoptotic effects, thereby may contribute to vascular lesions under hyperglycemia. This study demonstrates previously undescribed expression patterns of cathepsins, reveals a novel cathepsin-involved pathogenic mechanism under PDR, and sheds light on potential therapeutic targets to this debilitating retinal disease.
Assuntos
Apoptose/genética , Autofagia/genética , Catepsina B/metabolismo , Catepsina D/metabolismo , Catepsina L/metabolismo , Retinopatia Diabética/metabolismo , Corpo Vítreo/metabolismo , Adulto , Idoso , Animais , Catepsina B/genética , Catepsina D/genética , Catepsina L/genética , Catepsinas/genética , Catepsinas/metabolismo , Linhagem Celular , Cromatografia Líquida , Análise por Conglomerados , Retinopatia Diabética/genética , Células Endoteliais/metabolismo , Oftalmopatias Hereditárias/metabolismo , Feminino , Humanos , Macaca mulatta , Masculino , Pessoa de Meia-Idade , Mapas de Interação de Proteínas , Proteômica , Retina/citologia , Descolamento Retiniano/metabolismo , Espectrometria de Massas em Tandem , TranscriptomaRESUMO
We report here on the clinical, genetic, and molecular characterization of three Han Chinese pedigrees with aminoglycoside-induced and nonsyndromic hearing loss. Clinical evaluation revealed the variable phenotype of hearing impairment including severity, age-at-onset, audiometric configuration in these subjects. The penetrance of hearing loss in WZD8, WZD9, and WZD10 pedigrees were 46%, 46%, and 50%, respectively, when aminoglycoside-induced deafness was included. When the effect of aminoglycosides was excluded, the penetrance of hearing loss in these pedigrees were 23%, 31%, and 37.5%, respectively. Mutational analysis of the complete mitochondrial genomes showed the homoplasmic A1555G mutation and distinct sets of mitochondrial DNA variants belonging to haplogroups D4b2b, B5b1, and F2, respectively. Of these, the tRNA(Cys) T5802C, tRNA(Thr) A15924C, and ND5 T12338C variants are of special interest as these variants occur at positions which are highly evolutionarily conserved nucleotides of tRNAs or amino acid of polypeptide. These homoplasmic mtDNA variants were absent among 156 unrelated Chinese controls. The T5802C and G15927A variants disrupted a highly conserved A-U or C-G base-pairing at the anticodon-stem of tRNA(Cys) or tRNA(Thr), while the ND5 T12338C mutation resulted in the replacement of the translation-initiating methionine with a threonine, and also located in two nucleotides adjacent to the 3' end of the tRNA(Leu(CUN)). Thus, mitochondrial dysfunctions, caused by the A1555G mutation, would be worsened by these mtDNA variants. Therefore, these mtDNA mutations may have a potential modifier role in increasing the penetrance and expressivity of the deafness-associated 12S rRNA A1555G mutation in those Chinese pedigrees.
Assuntos
DNA Mitocondrial/genética , Surdez/genética , Mutação , Linhagem , RNA Ribossômico/genética , Aminoglicosídeos/farmacologia , Sequência de Bases , China , Conexina 26 , Conexinas/genética , Surdez/induzido quimicamente , Surdez/fisiopatologia , Variação Genética , Haplótipos , Humanos , Proteínas Mitocondriais/genética , Dados de Sequência Molecular , Penetrância , Fenótipo , RNA de Transferência de Cisteína/química , RNA de Transferência de Cisteína/genética , RNA de Transferência de Treonina/química , RNA de Transferência de Treonina/genética , Índice de Gravidade de Doença , tRNA Metiltransferases/genéticaRESUMO
We report here the clinical and genetic evaluations as well as mutational analysis of mitochondrial DNA (mtDNA) in a four-generation Chinese Han family with aminoglycoside-induced and nonsyndromic hearing loss. Strikingly, this family exhibited a high penetrance and expressivity of hearing loss. The penetrances of hearing loss in this family were 75% and 41.7% respectively, when aminoglycoside-induced deafness was included or was excluded. The severity of hearing loss in matrilineal relatives varied from profound hearing loss to normal hearing. Mutational analysis of mtDNA identified the homoplasmic A1555G mutation and a distinct set of mtDNA variants belonging to the Asian haplogroup B5b. Of these, the G15927A mutation absent in 156 Chinese controls is localized at the anticodon-stem of tRNAThr at conventional position 42. The guanine at this position (G42) of tRNAThr is highly conserved from bacteria to human mitochondria. Thus, it is antici-pated that the G15927A disrupted the highly conserved C-G base-pairing at the anticodon-stem of tRNAThr. The alteration of structure of this tRNA likely leads to a failure in tRNA metabolism, thereby worsens the mitochondrial dysfunction asso-ciated with the A1555G mutation. Thus, the G15927A mutation has a potential modifying role in increasing the penetrance and expressivity of hearing loss associated with the deafness-associated 12S rRNA A1555G mutation in this Chinese pedigree.
Assuntos
Fenótipo , RNA de Transferência de Treonina , Surdez/genética , Perda Auditiva/genética , Humanos , Mutação , LinhagemRESUMO
Mutations in mitochondrial DNA have been associated with a wide spectrum of clinical abnormalities. We reported here the clinical and genetic evaluations as well as mutational analysis of mitochondrial DNA(mtDNA) in a three-generation Chinese Han family with maternally transmitted hearing loss and epilepsy. Of 14 matrilineal relatives, three suffered from hearing loss, three had epilepsy, and other did not have significant clinical abnormalities. Sequence analysis of mitochondrial genome in this family identified the novel 7472delC in tRNASer(UCN) and 33 variants belonging to Asian haplogroup B4b1a2. The 7472delC locates at the highly conserved residue of T-arm of this tRNA. In fact, the 7472insC at the same position of this tRNA has been associated with hearing loss and epilepsy in several genetically unrelated families. The 7472insC has been shown to lead to a failure in tRNA metabolism and mitochondrial dysfunction. Thus, 7472delC mutation, similar to 7472insC mutation, may result in the mitochondrial dysfunctions responsible for the hearing loss and epilepsy. Furthermore, none of mutation in deafness-associated GJB2 gene was detected in this Chinese family. Therefore, the 7472delC is likely a new mitochondrial mutation with hearing loss and epilepsy.
Assuntos
DNA Mitocondrial/genética , Epilepsia/genética , Predisposição Genética para Doença/genética , Perda Auditiva/genética , RNA de Transferência de Serina/genética , Deleção de Sequência/genética , Adolescente , Adulto , Povo Asiático/genética , Conexina 26 , Conexinas , Análise Mutacional de DNA , DNA Mitocondrial/química , Epilepsia/etnologia , Feminino , Predisposição Genética para Doença/etnologia , Perda Auditiva/etnologia , Humanos , Masculino , Pessoa de Meia-Idade , Conformação de Ácido Nucleico , LinhagemRESUMO
We generated a bifunctional enzyme chimera containing the xylanase and lichenase coupled with SpyTag between them. Meanwhile, we generated another chimera containing SpyCatcher and elastin-like polypeptides (ELPs). As ELPs could bond to the xylanase-lichenase chimera through SpyTag/SpyCatcher spontaneous reaction in mild condition, which would lead to the formation of a 3-arm star multifunctional chimera. We purified the xylanase-lichenase by the non-chromatographic purification tag of ELPs. Interestingly, 57.5% of the xylanase and 47.2% of the lichenase in chimera self-assembled into insoluble active particles during the process of purification, which could serve as immobilized bifunctional enzymes. Notably, the immobilized chimera xylanase-lichenase showed a remarkable stability even after 10 reaction cycles, which retained around 56% (lichenase) and 44% (xylanase) of their initial activities, respectively. Moreover, the enhanced thermostability of the immobilized enzymes was also achieved. After incubating at 60⯰C for 60â¯min, the residual activity of the immobilized lichenase was 35%, while the free one was only 24%. Unexpectedly, the free xylanase almost lost its activity when incubated at 55⯰C for 60â¯min, whereas the immobilized xylanase retained 10% of its activity. However, the catalytic efficiency (kcat/Km) of the free xylanase was 1.7-fold higher than the immobilized one, while the free lichenase was 1.1-fold higher than the immobilized one. This is among the first known reports that two enzymes are purified and immobilized in one-step. This novel strategy is easy to scale up and may meet the demands of biofuel industry. It would have great potentials in other biotechnological fields, such as the multifunctional biomaterials systems.
Assuntos
Endo-1,4-beta-Xilanases/isolamento & purificação , Enzimas Imobilizadas/metabolismo , Glicosídeo Hidrolases/isolamento & purificação , Proteínas Recombinantes de Fusão/isolamento & purificação , Catálise , Elastina/química , Elastina/metabolismo , Endo-1,4-beta-Xilanases/genética , Endo-1,4-beta-Xilanases/metabolismo , Estabilidade Enzimática , Enzimas Imobilizadas/química , Glicosídeo Hidrolases/genética , Glicosídeo Hidrolases/metabolismo , Concentração de Íons de Hidrogênio , Peptídeos/química , Peptídeos/metabolismo , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismoRESUMO
Mutations in mitochondrial DNA (mtDNA) have been found to be associated with sensorineural hearing loss. We report here the clinical, genetic and molecular characterizations of seven Han Chinese pedigrees with aminoglycoside-induced and nonsyndromic bilateral hearing loss. Clinical evaluation revealed the variable phenotype of hearing impairment including severity, age-at-onset and audiometric configuration in these subjects. The penetrance of hearing loss in these pedigrees ranged from 3% to 29%, with an average of 13.6%, when aminoglycoside-induced deafness was included. When the effect of aminoglycosides was excluded, the penetrances of hearing loss in these seven pedigrees varied from 0% to 17%, with an average of 5.3%. Sequence analysis of the complete mitochondrial genomes in these pedigrees showed the presence of the deafness-associated 12S rRNA A1555G mutation, in addition to distinct sets of mtDNA polymorphism belonging to East Asian haplogroups B4, D4, D5 and F1, respectively. This suggested that the A1555G mutation occurred sporadically and multiplied through evolution of the mtDNA in China. Despite the presence of several evolutionary conservative variants in protein-encoding genes, there was the absence of functionally significant mutations in tRNA and rRNAs or secondary LHON mutations in these seven Chinese families. These suggest that these mtDNA haplogroup-specific variants may not play an important role in the phenotypic expression of the A1555G mutation in those Chinese families with very low penetrance of hearing loss. However, aminoglycosides appear to be a major modifier factor for the phenotypic manifestation of the A1555G mutation in these Chinese families.
Assuntos
Povo Asiático/genética , Surdez/genética , Perda Auditiva Bilateral/genética , Mutação/genética , Linhagem , Penetrância , RNA Ribossômico/genética , Adenina , Adolescente , Adulto , Aminoglicosídeos , Criança , China , Conexina 26 , Conexinas/genética , Análise Mutacional de DNA , DNA Mitocondrial/genética , Surdez/induzido quimicamente , Feminino , Genoma Humano/genética , Guanina , Perda Auditiva Bilateral/induzido quimicamente , Humanos , MasculinoRESUMO
We report here the clinical, genetic and molecular characterization of three Han Chinese pedigrees with maternally transmitted aminoglycoside-induced and nonsyndromic bilateral hearing loss. Clinical evaluation revealed the wide range of severity, age-at-onset and audiometric configuration of hearing impairment in matrilineal relatives in these families. The penetrances of hearing loss in these pedigrees were 28%, 20%, and 15%, with an average of 21%, when aminoglycoside-induced deafness was included. When the effect of aminoglycosides was excluded, the penetrances of hearing loss in these seven pedigrees were 21%, 13% and 8%, with an average of 14%. Sequence analysis of the complete mitochondrial genomes in these pedigrees showed the presence of the deafness-associated 12S rRNA C1494T mutation, in addition to distinct sets of mtDNA polymorphism belonging to Eastern Asian haplogroups F1a1, F1a1 and D5a2, respectively. This suggested that the C1494T mutation occurred sporadically and multiplied through evolution of the mtDNA. The absence of functionally significant mutations in tRNA and rRNAs or secondary LHON mutations in their mtDNA suggests that these mtDNA haplogroup-specific variants may not play an important role in the phenotypic expression of the C1494T mutation in those Chinese families. In addition, the lack of significant mutation in the GJB2 gene ruled out the possible involvement of GJB2 in the phenotypic expression of the C1494T mutation in those affected subjects. However, aminoglycosides and other nuclear modifier genes play a modifying role in the phenotypic manifestation of the C1494T mutation in these Chinese families.
Assuntos
Povo Asiático/genética , Perda Auditiva Bilateral/genética , Mutação , Linhagem , RNA Ribossômico/genética , Aminoglicosídeos/toxicidade , China , Conexina 26 , Conexinas , DNA Mitocondrial/genética , Perda Auditiva Bilateral/induzido quimicamente , Humanos , PenetrânciaRESUMO
Previous studies have revealed that the probiotic Clostridium butyricum (C. butyricum) can attenuate cirrhosis in chronic non-alcoholic liver disease. However, the effects of C. butyricum on acute liver injury (ALI) remain unclear. Therefore, the present study aims to examine the hepatoprotective effects and the underlying mechanisms employed by C. butyricum in a carbon tetrachloride (CCl4)-induced ALI murine model. Here, we evaluated the survival rate and the levels of alanine aminotransferase (ALT), aspartate aminotransferase (AST), anti-oxidants, cytokines and the gut microbiota to elucidate the potential mechanisms by which C. butyricum is hepatoprotective. Our results show that five days of prophylactic C. butyricum treatment significantly reduced mortality by 40% and decreased the CCl4-induced levels of ALT and AST in the serum of these mice. Additionally, prophylactic treatment with C. butyricum increased the activity of both superoxide dismutase (SOD) and catalase (CAT), and substantially reduced malondialdehyde (MDA) levels, which were deteriorated in the untreated ALI mice compared to normal control mice. Furthermore, C. butyricum up-regulated the nuclear factor (erythroid-derived 2)-like 2 (NRF2) content. CCl4-induced mice also exhibited considerable increases of phosphorylation of nuclear factor-kappa B (NF-κB) p65 and pro-inflammatory cytokines, including interleukin-1ß (IL-1ß), interleukin-6 (IL-6) and tumor necrosis factor-α (TNF-α). However, the inflammatory responses of the liver induced by CCl4 were significantly alleviated by C. butyricum pretreatment. Additionally, we found that interleukin-10 (IL-10), an anti-inflammatory mediator, was increased in the C. butyricum-pretreated group. Microbiota analysis in these mice revealed crosstalk between the gut microbial metabolites and ALI. The intestinal flora was changed by CCl4 administration and was shifted by the probiotic C. butyricum toward more beneficial bacteria, particularly the Clostridia orders, which are the known producers of the anti-inflammatory and anti-oxidative metabolite butyrate. In conclusion, we found that the intestinal flora changes after the intraperitoneal injection of CCl4. We also offer novel insights into the mechanism by which probiotic C. butyricum pretreatment alleviates the CCl4-induced inflammation and oxidative stress of the liver via the modulation of NRF2, NF-κB p65, IL-10 and the intestinal microbiota in mice.
Assuntos
Tetracloreto de Carbono/efeitos adversos , Doença Hepática Induzida por Substâncias e Drogas/tratamento farmacológico , Clostridium butyricum/fisiologia , Fígado/efeitos dos fármacos , Probióticos/administração & dosagem , Substâncias Protetoras/administração & dosagem , Alanina Transaminase/metabolismo , Animais , Aspartato Aminotransferases/metabolismo , Doença Hepática Induzida por Substâncias e Drogas/genética , Doença Hepática Induzida por Substâncias e Drogas/metabolismo , Doença Hepática Induzida por Substâncias e Drogas/microbiologia , Microbioma Gastrointestinal/efeitos dos fármacos , Humanos , Interleucina-10/genética , Interleucina-10/metabolismo , Interleucina-6/genética , Interleucina-6/metabolismo , Fígado/metabolismo , Masculino , Malondialdeído/metabolismo , Camundongos , NF-kappa B/metabolismo , Estresse Oxidativo/efeitos dos fármacosRESUMO
OBJECTIVE: To analyze the expression and its promoter methylation of chemokine CXC ligand 14 (CXCL14) in peripheral blood mononuclear cells (PBMCs) from patients with systemic lupus erythematosus (SLE). METHODS: The RNAs of PBMCs from 28 SLE patients and 20 healthy controls were isolated and reversely transcribed into cDNAs. Using GAPDH as the internal reference, the levels of CXCL14 ex-pression were detected by real-time polymerase chain reaction (PCR). The correlation between CXCL14 expression and the clinic pathological fe atures of SLE were further analyzed. DNA methylation was analyzed by bisulfite sequencing PCR (BSP). RESULTS: Our data indicated that the level of CXCL14 in the PBMC of SLE patients was statistically lower than that in healthy controls (P < 0.05). Further analysis showed that CXCL14 expression was negatively correlated with anti-Sj gren syndrome B antibody(anti-SSB antibody, P < 0.01) and albuminuria(P < 0.05). However, CXCL14 expression was not significantly correlated with the indexes of SLE activity, renal damage, the level of anti-ds-DNA antibodies, complement C3 and C-reactive protein. In addition, we further demonstrated that the CXCL14 promoter hypermethylation expres-sion was significant higher than healthy controls. CONCLUSIONS: Down-regulated of CXCL14 expression in PBMC maybe involved in the occur-rence or development of SLE disease. The loss of CXCL14 expression was regulated by promoter hypermethylation.
Assuntos
Quimiocinas CXC/genética , Metilação de DNA , Lúpus Eritematoso Sistêmico/genética , Regiões Promotoras Genéticas , Estudos de Casos e Controles , Humanos , Leucócitos MononuclearesRESUMO
BACKGROUND: SpyTag is a peptide that can form an irreversible covalent linkage to its 12 kDa partner SpyCatcher via a spontaneous isopeptide bond. Herein, we fused SpyTag at the N-terminal of lichenase and SpyCatcher at C-terminal so that the termini of lichenase were locked together by the covalent interaction between the partners. In addition, an elastin-like polypeptides tag was subsequently attached to the C-terminus of SpyCatcher, thereby facilitating the non-chromatographic purification of cyclized lichenase. RESULTS: The study showed that the optimum temperature of the cyclized lichenase was about 5 °C higher in comparison to its linear counterpart. Moreover, nearly 80 % of the cyclized lichenase activities were retained after 100 °C exposure, whereas the linear form lost almost all of its activities. Therefore, the cyclized variant displayed a significantly higher thermal stability as temperature elevated and was resistant to hyperthermal denaturation. Besides, the Km value of the cyclized lichenase (7.58 ± 0.92 mg/mL) was approximately 1.7-fold lower than that of the linear one (12.96 ± 1.93 mg/mL), indicating a higher affinity with substrates. CONCLUSIONS: This new SpyTag/SpyCatcher cyclization strategy is deemed as a generalized reference for enhancing enzyme stability and can be effectively customized to the cyclization of various enzymes, hence a tremendous potential for successful application in the biocatalytic conversion of biomass to produce fuels and chemicals.
RESUMO
Here, we explored the effects of suberoylanilide hydroxamic acid (SAHA) on the viability and apoptosis of rheumatoid arthritis of fibroblast-like synoviocytes (rheumatoid arthritis (RA) fibroblast-like synoviocytes (FLS)). FLS obtained from RA patients were treated with SAHA. SAHA significantly inhibited the viability of RA FLS in a concentration-dependent manner up to 5 µM. SAHA-treated FLS showed a significant increase in the percentage of apoptosis and the expression and activity of caspase-3 and higher intracellular ROS levels. N-acetyl-l-cysteine (NAC) pretreatment significantly attenuated SAHA-induced apoptosis, decreasing the percentage of apoptosis by about 60 %. A significant decline in phosphorylated IκBα and nuclear factor kappa B (NF-κB) p65 and concomitant increase in total IκBα were shown in SAHA-treated FLS. Additionally, the levels of anti-apoptotic Bcl-2 proteins (Bcl-xL and Mcl-1) were significantly reduced by SAHA. Collectively, SAHA induces apoptosis of RA FLS, at least partially, through generation of ROS and suppression of NF-κB activation and Bcl-xL and Mcl-1 expression.
Assuntos
Apoptose/efeitos dos fármacos , Artrite Reumatoide/tratamento farmacológico , Fibroblastos/metabolismo , Inibidores de Histona Desacetilases/farmacologia , Ácidos Hidroxâmicos/farmacologia , Membrana Sinovial/metabolismo , Acetilcisteína/farmacologia , Caspase 3/metabolismo , Sobrevivência Celular/efeitos dos fármacos , Células Cultivadas , Humanos , Proteína de Sequência 1 de Leucemia de Células Mieloides/metabolismo , Inibidor de NF-kappaB alfa/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Membrana Sinovial/citologia , Fator de Transcrição RelA/metabolismo , Vorinostat , Proteína bcl-X/metabolismoRESUMO
Enzyme immobilization is the core technology of biocatalysis. Over the past few decades, enzyme immobilization research mainly focused on single enzyme immobilization. In recent years, multi-enzyme immobilization attracts more and more attention as it could increase the local concentration of reaction and improve the reaction yield. In this review, a summary of the recent progress, together with our research, is presented. Special emphasis is placed on four methods in multi-enzymes co-immobilization, namely, the nonspecific covalent co-immobilization, the nonspecific non-covalent co-immobilization, the non-covalent encapsulation co-immobilized and the site specificity co-immobilized. Finally, some industrial uses of immobilized multi-enzymes were addressed and the application prospect of multi-enzyme immobilization was highlighted.
Assuntos
Enzimas Imobilizadas , BiocatáliseRESUMO
To verify the reliability of targeted detecting HER2 positive cancer cells and clinical pathological tissue specimens with a recombinant anti HER2 single chain antibody in single chain Fv fragment (scFv) format, we have constructed the fusion variable regions of the ScFv specific for HER2/neu. labeled a green-fluorescent protein(GFP). The humanized recombinant Anti HER2 ScFv-GFP gene was inserted into pFast Bac HT A, and expressed in insect cells sf9. Then the recombinant fusion protein Anti HER2 ScFv-GFP was properly purified with Ni2+-NTA affinity chromatography from the infected sf9 cells used to test the specificity of the fusion antibody for HER2 positive cancer cells. Firstly, the purified antibody incubated with HER2 positive breast cancer cells SKBR3, BT474 and HER2 negative breast cancer cells MCF7 for 12 h/24 h/48 h at 37 degrees C, in order to confirm targeted detecting HER2 positive breast cancer cells by Laser Confocal Microscopy. Furthermore, the same clinical pathological tissue samples were assessed by immunohistochemistry (IHC) and the fusion antibody Anti HER2 ScFv-GFP in the meanwhile. The data obtained indicated that the recombinant eukaryotic expression plasmid pFast Bac HT A/Anti HER2 ScFv-GFP was constructed successfully In addition, obvious green fluorescent was observed in insect cells sf9. When the purified fusion antibody was incubated with different cancer cells, much more green fluorescent was observed on the surface of the HER2 positive cancer cells SKBR3 and BT474. In contrast, no green fluorescent on the surface of the HER2 negative cancer cells MCF7 was detected. The concentration of the purified fusion antibody was 115.5 microg/mL, of which protein relative molecular weight was 60 kDa. The analysis showed the purity was about 97% and the titer was about 1:64. The detection results of IHC and fusion antibody testing indicated the conformity. In summary, the study showed that the new fusion antibody Anti HER2 ScFv-GFP can test HER2 positive cancer cells, indicating a potential candidate method for clinical HER2 positive specimens detection.