RESUMO
Environmental pollutants are recognized as one of the major concerns for public health. The free-living nematode Caenorhabditis elegans are widely used to evaluate the toxicity of environmental contaminants in biomonitoring researches. In the present study, a new transgenic strain, rps-30-/- ;RFP-RPS-30UbL was generated, with constitutively active rps-30 promoter used to control the expression of RFP-RPS-30UbL fusion protein. We found RFP-RPS-30UbL would accumulate to form 'rod-like' structures, when worms were exposed to environmental contaminants, including Cd, Hg, Pb, As, Paraquat and Dichlorvos. The number of the 'rod-like' structures was induced by environmental contaminants in a concentration- and time-dependent manner. The 'rod-like' structure formation could be detectable in response to the concentration of each contaminant as low as 24-h LC50 × 10-7 , and the detectable time could be within 2 h. Detecting the transcription and expression levels of RFP-RPS-30UbL in worms exposed to different kinds of environmental contaminants showed that the expression level of RFP-RPS-30UbL was not regulated by environmental contaminants, and the number differences of 'rod-like' structures were just due to the morphological change of RFP-RPS-30UbL from dispersion to accumulation induced by environmental contaminants. In addition, this transgenic strain was developed in rps-30-/- homozygous worm, which was a longevity strain. Detection of lifespan and brood size showed that rps-30-/- ;RFP-RPS-30UbL transgenic worm was more suitable to be cultured and used further than N2;GFP-RPS-30UbL , for expressing RPS-30UbL in wild type N2 worms shortened the lifespan and deceased the brood size. Therefore, rps-30-/- ;RFP-RPS-30UbL transgenic worm might play a potential role in versatile environmental biomonitoring, with the advantage of not only the convenient and quick fluorescence-based reporter assay, but also the quantificational evaluation of the toxicities of environmental contaminants using 'rod-like' structures with high sensitivity, off-limited the expression level of the reporter protein.
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Proteínas de Caenorhabditis elegans , Poluentes Ambientais , Nematoides , Animais , Caenorhabditis elegans/genética , Poluentes Ambientais/toxicidade , Nematoides/metabolismo , Regiões Promotoras Genéticas , Proteínas de Caenorhabditis elegans/genética , Proteínas de Caenorhabditis elegans/metabolismoRESUMO
BACKGROUND: In addition to Mycoplasma haemocanis and Candidatus Mycoplasma haematoparvum, a few hemoplasma species that mainly infect other livestock have been detected in dogs. 'Candidatus Mycoplasma haemobos' (Ca. M. haemobos) has been found in a variety of animals in China. The present study was aimed to investigate the occurrence of 'Ca. M. haemobos' infections in dogs and ticks collected from the Henan province, China. RESULTS: Overall, 55 dog blood samples and 378 ticks on skins were collected from anemic and healthy dogs, and these samples were subjected to PCR, sequence analysis, and identification. The results showed that Haemaphysalis longicornis (266) and Rhipicephalus (Boophilus) microplus (112) were the only two parasitic ticks on dogs. Molecular detection revealed that 163 M. haemocanis, 88 'Ca. M. haemobos' and 32 Anaplasma platys positive amplicons could be amplified from dogs, H. longicornis and R. (B.) microplus. In addition, co-infections (M. haemocanis + A. platys and 'Ca. M. haemobos'+ A. platys) could be also detected. CONCLUSIONS: To the best of our knowledge, this is the first molecular evidence of 'Ca. M. haemobos' natural infection in dogs and tick species identified as H. longicornis and R. (B.) microplus from China.
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Mycoplasma , Rhipicephalus , Animais , China/epidemiologia , Cães , Gado , Mycoplasma/genética , Reação em Cadeia da Polimerase/veterináriaRESUMO
Hepatic glucose production (HGP) is an important component of glucose homeostasis, and deregulated HGP, particularly through gluconeogenesis, contributes to hyperglycemia and pathology of type-2 diabetes (T2D). It has been shown that the gluconeogenic gene expression is governed primarily by the transcription factor cAMP-response element (CRE)-binding protein (CREB) and its coactivator, CREB-regulated transcriptional coactivator 2 (CRTC2). Recently, we have discovered that Sam68, an adaptor protein and Src kinase substrate, potently promotes hepatic gluconeogenesis by promoting CRTC2 stability; however, the detailed mechanisms remain unclear. Here we show that in response to glucagon, Sam68 increases CREB/CRTC2 transactivity by interacting with CRTC2 in the CREB/CRTC2 complex and occupying the CRE motif of promoters, leading to gluconeogenic gene expression and glucose production. In hepatocytes, glucagon promotes Sam68 nuclear import, whereas insulin elicits its nuclear export. Furthermore, ablation of Sam68 in hepatocytes protects mice from high-fat diet (HFD)-induced hyperglycemia and significantly increased hepatic and peripheral insulin sensitivities. Thus, hepatic Sam68 potentiates CREB/CRTC2-mediated glucose production, contributes to the pathogenesis of insulin resistance, and may serve as a therapeutic target for T2D.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal , Diabetes Mellitus Tipo 2 , Gluconeogênese , Glucose , Hepatócitos , Resistência à Insulina , Proteínas de Ligação a RNA , Proteínas Adaptadoras de Transdução de Sinal/genética , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Animais , Proteínas de Ciclo Celular/metabolismo , Proteína de Ligação ao Elemento de Resposta ao AMP Cíclico/genética , Proteína de Ligação ao Elemento de Resposta ao AMP Cíclico/metabolismo , Diabetes Mellitus Tipo 2/genética , Diabetes Mellitus Tipo 2/metabolismo , Expressão Gênica , Glucagon/metabolismo , Gluconeogênese/genética , Gluconeogênese/fisiologia , Glucose/metabolismo , Hepatócitos/metabolismo , Hepatócitos/fisiologia , Homeostase , Hiperglicemia/metabolismo , Insulina/metabolismo , Resistência à Insulina/genética , Resistência à Insulina/fisiologia , Fígado/metabolismo , Camundongos , Proteínas de Ligação a RNA/genética , Proteínas de Ligação a RNA/metabolismo , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Quinases da Família src/genética , Quinases da Família src/metabolismoRESUMO
Objective: To clone and express the galectin-1 gene of Angiostrongylus cantonensis, and test the agglutination property of its protein. Methods: The three-dimensional structure of galectin-1 was analyzed with Swiss Model. Total RNA was extracted from male worms of A. cantonensis. Primers were designed for galectin-1 based on its coding region (GenBank Accession No. JN133961.1). RT-PCR was performed, and the PCR products were subcloned to pCold â ¢ plasmid and transduced into Escherichia coli BL21 strain. The recombinant plasmid was extracted from positive clones on LB plate containing 100 µg/ml Kanamycin, and validated with double digestion, PCR identification and sequencing. The confirmed positive clones of E. coli BL21 with the recombinant plasmid were grown in LB medium containing ampicillin (100 µg/ml, 100 µl). IPTG was added to induce expression of the plasmid. The galectin-1 recombinant protein was purified with Ni-NTA beads, and analyzed with SDS-PAGE and Western blotting using anti-serum of mouse immunized with whole worms of A. cantonensis. The agglutination reaction with red blood cells in fresh blood of ICR mouse was observed for the 10-fold serial dilutions of recombinant proteins (5.55 × 10(-1)-5.55 × 10(-5) ng/µl). Results: The Swiss Model analysis showed that the functional galectin-1 had a non-dimeric form. As was expected, the RT-PCR products had a size of 850 bp. Results of double digestion, PCR and sequencing showed successful construction of the pCold â ¢-galectin-1 plasmid. SDS-PAGE revealed expression of soluble recombinant fusion protein with molecular weight of ~36 000. Western blotting showed that the galectin-1 protein was recognized by mouse anti-serum. In addition, the minimun concentration of galectin-1 that showed significant agglutination reactions with mouse red blood cells was 5.55 × 10(-4) ng/µl. Conclusion: The galectin-1 clone can be expressed in the pCold â ¢ plasmid, and its protein product has agglutination property.
Assuntos
Angiostrongylus cantonensis , Clonagem Molecular , Aglutinação , Animais , Western Blotting , Eletroforese em Gel de Poliacrilamida , Escherichia coli , Galectina 1 , Expressão Gênica , Camundongos , Camundongos Endogâmicos ICR , Plasmídeos , Reação em Cadeia da Polimerase , Proteínas RecombinantesRESUMO
Aminopeptidase H11 present in the surface of intestine microvilli in Haemonchus contortus was identified as the most effective antigen candidate. However, its recombinant forms produced in Escherichiacoli, insect cells and yeast could not provide promising protection against H. contortus challenge, probably due to the inappropriate glycosylation and/or conformational folding. Herein, partial H11 containing the potential zinc-binding domain and two predicted glycosylation sites (nt 1 bp-1710 bp, Trans-HPS) was subcloned downstream of 5' flanking region of Caenorhabditis elegans cpr-1 gene in pPD95.77 vector, with the deletion of GFP gene. The recombinant was expressed in C. elegans and verified by blotting with anti-H11 and anti-Trans-HPS rabbit polyclonal antibodies and anti-His monoclonal antibody. Stably inherited Trans-HPS in worm descendants was achieved by integration using UV irradiation. Immunization with the crude Trans-HPS extracted from transgenic worms resulted in 37.71% reduction in faecal egg counts (FEC) (P<0.05) and 24.91% reduction in worm burden, but an upward curve with moderate rate of daily FEC in goats. These results suggested an apparent delay against H. contortus egg-laying in goats, which differed from that with bacteria-origin form of partial H11 (nt 670 bp-1710 bp, HPS) (26.04% reduction in FEC and 18.46% reduction in worm burden). These findings indicate the feasibility of sufficient C. elegans-expressed H11 for the immunological research and vaccine development.
Assuntos
Aminopeptidases/metabolismo , Caenorhabditis elegans/enzimologia , Endopeptidases/metabolismo , Haemonchus/enzimologia , Abomaso/parasitologia , Aminopeptidases/genética , Aminopeptidases/imunologia , Animais , Anticorpos Anti-Helmínticos/sangue , Antígenos de Helmintos/genética , Antígenos de Helmintos/imunologia , Antígenos de Helmintos/metabolismo , Caenorhabditis elegans/genética , Caenorhabditis elegans/imunologia , Endopeptidases/genética , Endopeptidases/imunologia , Fezes/parasitologia , Feminino , Regulação Enzimológica da Expressão Gênica , Cabras , Imunoglobulina G/sangue , Isoenzimas/genética , Isoenzimas/metabolismo , Masculino , Contagem de Ovos de Parasitas , Coelhos , Proteínas Recombinantes/genética , Proteínas Recombinantes/imunologia , Proteínas Recombinantes/metabolismo , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por MatrizRESUMO
Enterocytozoon bieneusi is a widespread intracellular fungus that can infect both humans and animals, making it a significant zoonotic threat. In the current study, a total of 208 fecal samples were assayed to investigate the prevalence of E. bieneusi in pigs reared in Zhejiang Province, China. Employing polymerase chain reaction (PCR) amplification techniques specifically designed to target the internal transcribed spacer (ITS) region of the small subunit ribosomal RNA (rRNA) gene, the results revealed that 78 samples (37.5â¯%) tested positive for the presence of E. bieneusi. A total of 19 different genotypes of E. bieneusi were detected. Nine of these genotypes were already known: EbpC (n = 36), KIN-1 (n = 10), PigEbITS7 (n = 8), EbpA (n = 6), Henan III (n = 3), PigEbITS5 (n = 2), Henan-IV (n = 1), EbpD (n = 1), and TypeIV (n = 1), and 10 were novel: ZJP-I to ZJP-X (one each). The present investigation revealed that all the nine known genotypes identified in pigs here, have also been previously discovered in humans. Additionally, the novel genotypes of E. bieneusi discovered here were all classified as belonging to Group 1. These findings suggest the potential for cross-species transmission between humans and pigs.
Assuntos
Enterocytozoon , Genótipo , Doenças dos Suínos , Zoonoses , Animais , Enterocytozoon/genética , Enterocytozoon/isolamento & purificação , China/epidemiologia , Suínos , Doenças dos Suínos/microbiologia , Doenças dos Suínos/epidemiologia , Zoonoses/microbiologia , Filogenia , Medição de Risco , Fezes/microbiologia , Humanos , Prevalência , Microsporidiose/veterinária , Microsporidiose/epidemiologia , Microsporidiose/microbiologia , Reação em Cadeia da Polimerase/veterinária , DNA Espaçador Ribossômico/genética , DNA Fúngico/genéticaRESUMO
Haemonchus contortus infections in small ruminants are of major economic importance worldwide. Heat shock proteins (HSPs) are a family of molecular chaperones that play important roles in the process of invasion and survival of nematodes. Although HSP70 has been identified in several parasitic nematodes, little is known of its distribution and function in Haemonchus contortus. The aims of this study were to characterize HSP70 from Haemonchus contortus (designed as Hc-hsp70), express Hc-hsp70 and analyse the promoter activity in Caenorhabditis elegans. Bioinformatic analysis revealed that the open reading frame of the Hc-hsp70 cDNA encodes a 646-amino acid peptide, which is highly conserved in comparison to HSP70 in other nematodes. Phylogenetic analysis indicated that H. contortus is closely related to Caenorhabditis. The 5'-flanking region promoted green fluorescence protein (GFP) expression in the intestine in all larval stages and adult with 2 expression patterns in C. elegans. Expression of Hc-hsp70 mRNA transcripts in C. elegans increased following 2, 4, 6 h of heat shock and peaked at 4 h. However, its expression induced down-regulation of hsp-1 of C. elegans. These results suggest that the H. contortus hsp70 might have a similar function to that of C. elegans hsp-1.
Assuntos
Caenorhabditis elegans/genética , Regulação da Expressão Gênica , Proteínas de Choque Térmico HSP70/genética , Haemonchus/genética , Regiões Promotoras Genéticas/genética , Sequência de Aminoácidos , Animais , Sequência de Bases , Caenorhabditis elegans/citologia , Caenorhabditis elegans/metabolismo , Mapeamento Cromossômico , Biologia Computacional , DNA de Helmintos/química , DNA de Helmintos/genética , Regulação para Baixo , Escherichia coli/genética , Escherichia coli/metabolismo , Fezes/parasitologia , Feminino , Proteínas de Choque Térmico HSP70/metabolismo , Haemonchus/metabolismo , Proteínas de Helminto/genética , Proteínas de Helminto/metabolismo , Temperatura Alta , Larva , Masculino , Dados de Sequência Molecular , Filogenia , Alinhamento de Sequência , Análise de Sequência de DNA , Homologia de Sequência de Aminoácidos , OvinosRESUMO
Exosomes are a subtype of extracellular vesicles (EVs) with a diameter of 30~150 nm (averaging ~100 nm) that are primarily produced through the endosomal pathway, and carry various components such as lipids, proteins, RNA, and other small molecular substances. Exosomes can mediate intercellular communication through the bioactive substances they carry, thus participating in different physiological activities. Metabolic syndrome (MS) is a disease caused by disturbances in the body's metabolism, mainly including insulin resistance (IR), diabetes, obesity, non-alcoholic fatty liver disease (NAFLD), hyperlipidemia, and atherosclerosis (AS). Recent studies have shown that exosomes are closely related to the occurrence and development of MS. Exosomes can act as messengers to mediate signaling transductions between metabolic cells in the organism and play a bidirectional regulatory role in the MS process. This paper mainly reviews the components, biogenesis, biological functions and potential applications of exosomes, and exosomes involved in the pathogenesis of MS as well as their clinical significance in MS diagnosis.
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BACKGROUND: Angiostrongylus cantonensis L5, parasitizing human cerebrospinal fluid, causes eosinophilic meningitis, which is attributed to tissue inflammatory responses caused primarily by the high percentage of eosinophils. Eosinophils are also involved in killing helminths, using the peroxidative oxidation and hydrogen peroxide (H2O2) generated by dismutation of superoxide produced during respiratory burst. In contrast, helminthic worms have evolved to attenuate eosinophil-mediated tissue inflammatory responses for their survival. In previous study, we demonstrated the extracellular function of Acan-Gal-1 in inducing the apoptosis of macrophages. Here, the intracellular functions of Acan-Gal-1 were investigated, aiming to further reveal the mechanism involved in A. cantonensis L5 worms surviving inflammatory responses in the human central nervous system. METHODS: In this study, a model organism, Caenorhabditis elegans, was used as a surrogate to investigate the intracellular functions of Acan-Gal-1 in protecting the worm from its host's immune attacks. First, structural characterization of Acan-Gal-1 was analyzed using bioinformatics; second, qRT-PCR was used to monitor the stage specificity of Acan-gal-1 expression in A. cantonensis. Microinjections were performed to detect the tissue specificity of lec-1 expression, the homolog of Acan-gal-1 in C. elegans. Third, microinjection was performed to develop Acan-gal-1::rfp transgenic worms. Then, oxidative stress assay and Oil Red O fat staining were used to determine the functions of Acan-Gal-1 in C. elegans. RESULTS: The results of detecting the stage specificity of Acan-gal-1 expression showed that Acan-Gal-1 was upregulated in both L5 and adult worms. Detection of the tissue specificity showed that the homolog of Acan-gal-1 in C. elegans, lec-1 was expressed ubiquitously and mainly localized in cuticle. Investigating the intracellular functions of Acan-Gal-1 in the surrogate C. elegans showed that N2 worms expressing pCe-lec-1::Acan-gal-1::rfp, with lipid deposition reduced, were significantly resistant to oxidative stress; lec-1 mutant worms, where lipid deposition increased, showed susceptible to oxidative stress, and this phenotype could be rescued by expressing pCe-lec-1::Acan-gal-1::rfp. Expressing pCe-lec-1::Acan-gal-1::rfp or lec-1 RNAi in fat-6;fat-7 double-mutant worms, where fat stores were reduced, had no significant effect on the oxidative stress tolerance. CONCLUSION: In C. elegans worms, upregulated Acan-Gal-1 plays a defensive role against damage due to oxidative stress for worm survival by reducing fat deposition. This might indicate the mechanism by which A. cantonensis L5 worms, with upregulated Acan-Gal-1, survive the immune attack of eosinophils in the human central nervous system.
Assuntos
Angiostrongylus cantonensis , Caenorhabditis elegans/parasitologia , Galectina 1 , Metabolismo dos Lipídeos , Estresse Oxidativo , Tecido Adiposo , Angiostrongylus cantonensis/genética , Animais , Caenorhabditis elegans/genética , Galectina 1/genética , Peróxido de HidrogênioRESUMO
Seventy-eight isolates of avian infectious bronchitis virus (IBV) were obtained from different field outbreaks in China in 2009 and genotyped with 34 reference strains. Four genotypes of IBV and three new isolates were identified by phylogenetic analysis and BLAST searches of the entire S1 gene. The results showed that most IBV strains that have circulated in China in recent years belong to the genotype of QX-like strains, and that they could be grouped further into two clusters, regardless of the level of genetic variation displayed. A study of pathogenicity that used three QX-like strains--ck/CH/LSD/091003, ck/CH/LDL/091022 and ck/CH/LJL/090330--showed that the isolates caused the most severe lesions in the kidneys and were therefore nephropathogenic strains with various levels of virulence in specific pathogen free chickens. A vaccination-challenge test that was performed using the three QX-like strains showed that the commercially available H120 vaccine did not provide sufficient protection against challenge with the QX-like isolates, as demonstrated by comparison of the clinical signs, pathological lesions and virus recovery from the trachea and kidney of unvaccinated-challenged and vaccinated-challenged birds.
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Infecções por Coronavirus/veterinária , Vírus da Bronquite Infecciosa/genética , Vírus da Bronquite Infecciosa/imunologia , Filogenia , Doenças das Aves Domésticas/virologia , Vacinas Virais , Animais , Galinhas , China/epidemiologia , Infecções por Coronavirus/prevenção & controle , Infecções por Coronavirus/virologia , Surtos de Doenças/prevenção & controle , Surtos de Doenças/veterinária , Feminino , Vírus da Bronquite Infecciosa/isolamento & purificação , Vírus da Bronquite Infecciosa/patogenicidade , Vigilância da População , Doenças das Aves Domésticas/prevenção & controle , Vacinação/veterináriaRESUMO
Hepatic gluconeogenesis is essential for glucose homeostasis and also a therapeutic target for type 2 diabetes, but its mechanism is incompletely understood. Here, we report that Sam68, an RNA-binding adaptor protein and Src kinase substrate, is a novel regulator of hepatic gluconeogenesis. Both global and hepatic deletions of Sam68 significantly reduce blood glucose levels and the glucagon-induced expression of gluconeogenic genes. Protein, but not mRNA, levels of CRTC2, a crucial transcriptional regulator of gluconeogenesis, are >50% lower in Sam68-deficient hepatocytes than in wild-type hepatocytes. Sam68 interacts with CRTC2 and reduces CRTC2 ubiquitination. However, truncated mutants of Sam68 that lack the C- (Sam68ΔC) or N-terminal (Sam68ΔN) domains fails to bind CRTC2 or to stabilize CRTC2 protein, respectively, and transgenic Sam68ΔN mice recapitulate the blood-glucose and gluconeogenesis profile of Sam68-deficient mice. Hepatic Sam68 expression is also upregulated in patients with diabetes and in two diabetic mouse models, while hepatocyte-specific Sam68 deficiencies alleviate diabetic hyperglycemia and improves insulin sensitivity in mice. Thus, our results identify a role for Sam68 in hepatic gluconeogenesis, and Sam68 may represent a therapeutic target for diabetes.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Gluconeogênese/fisiologia , Fígado/metabolismo , Proteínas de Ligação a RNA/metabolismo , Fatores de Transcrição/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/genética , Animais , Glicemia/metabolismo , Proteínas de Ligação a DNA , Diabetes Mellitus Tipo 2/metabolismo , Regulação da Expressão Gênica , Glucagon/metabolismo , Gluconeogênese/genética , Glucose/metabolismo , Hepatócitos/metabolismo , Homeostase , Humanos , Hiperglicemia , Resistência à Insulina , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Proteínas de Ligação a RNA/genética , Fatores de Transcrição/genética , Regulação para CimaRESUMO
Bovine pestiviruses include Pestivirus A (BVDV-1), Pestivirus B (BVDV-2), and Pestivirus H, which was originally called HoBi-like pestivirus. We conducted an epidemiological investigation for pestiviruses circulating in backyard cattle farms in central China. RT-PCR assays and sequences analysis were conducted on 54 nasal swabs, 26 serum samples, and three lung samples from cattle with respiratory infections and identified 29 pestivirus strains, including 24 Pestivirus A and five Pestivirus H strains. Phylogenetic analysis based on partial 5'-UTR and Npro sequences showed that the genotypes of 24 Pestivirus A strains included Pestivirus A 1b (six isolates), Pestivirus A 1m (six isolates), Pestivirus A 1q (two isolates), Pestivirus A 1u (one isolates), and Pestivirus A 1o (nine isolates, a putative new sub-genotype). In addition, a single Pestivirus H agenotype included all five Pestivirus H strains. This study revealed extensive genetic variations within bovine pestivirus isolates derived from cattle in backyard farms in Central China, and this epidemiological information improves our understanding of the epidemics of bovine Pestiviruses, as well as will be useful in designing and evaluating diagnostic methods and developing more effective vaccines.
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Stipa breviflora Grisb. (S. breviflora) is a dominant species in the desert steppe of northern China. Its function and role at the plant community level increases with increasing stocking rate. However, the response of spatial stability remains unclear. We selected treatment areas representing no grazing (CK), light grazing (LG), moderate grazing (MG) and heavy grazing (HG) in a long-term grazing experiment (2004-2017) in a S. breviflora desert steppe in Inner Mongolia, northern China. Using a mechanical sampling method, 40 m × 40 m representative sample plots were selected to obtain the height, coverage and density of the S. breviflora population and community, and we computed the standing crop of mechanical sampling quadrats based on a random sample of cutting quadrats. Analysis of standing crop, density of S. breviflora population and its ratio in the plant community showed that the dominant role of S. breviflora population in the plant community increased with increasing grazing intensity, while the spatial stability of S. breviflora population not only had many dimensions, but also many states. The dimension or combination of dimensions of its stability performance and its adaptive state varied under different disturbance intensities and frequencies.
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Poaceae , China , Ecossistema , Meio Ambiente , PradariaRESUMO
BACKGROUND: Angiostrongylus cantonensis can cause severe symptoms of central nervous system infections. In the host, this parasite localizes in the blood and cerebrospinal fluid, and its secreted components can impact immune responses. Our previous study demonstrated that immune responses were inhibited in A. cantonensis-infected mice immunized with Ac-Galectin-1 (AcGal-1). However, the mechanisms by which AcGal-1 regulates the immune responses remain unclear. Macrophages are innate immune cells that rapidly respond to infection. The direct impact of AcGal-1 on macrophages may affect the immune responses. METHODS: AcGal-1 protein was purified by nickel ion affinity chromatography. The effect of AcGal-1 on the apoptosis of macrophages was detected using CCK-8 assay, flow cytometry and western blot. Macrophage membrane proteins bound to AcGal-1 were obtained using the His-tag-based pull-down assay and identified via mass spectrometry. Co-localization of AcGal-1 and the macrophage membrane protein Annexin A2 was observed by immunofluorescence microscopy, and their interaction was validated by co-immunoprecipitation experiments. SiRNA-mediated knockdown of Annexin A2 was used to determine if AcGal-1-induced macrophage apoptosis required interaction with Annexin A2. The phosphorylation level of apoptotic signal pathway protein was detected by phospho-antibody microarray and western blot. RESULTS: Our study showed that AcGal-1 caused apoptosis of the macrophages. AcGal-1 increased the expression of apoptosis proteins caspase-3, caspase-9, Bax, but reduced the expression of anti-apoptosis protein Bcl-2. AcGal-1 interacted with the membrane protein Annexin A2, and knockdown of Annexin A2 expression increased Bcl-2 but decreased Bax levels in AcGal-1-treated cells. Moreover, AcGal-1 increased JNK phosphorylation and the inhibition of JNK phosphorylation in AcGal-1-treated cells decreased the expression of caspase-3, -9, Bax and almost restored Bcl-2 to the level observed in control cells. CONCLUSIONS: AcGal-1 can induce the apoptosis of macrophages by binding to Annexin A2 and activating JNK downstream the apoptotic signaling pathway.
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Anexina A2/imunologia , Apoptose , Galectina 1/imunologia , Sistema de Sinalização das MAP Quinases , Macrófagos/parasitologia , Angiostrongylus cantonensis , Animais , Proliferação de Células , Técnicas de Silenciamento de Genes , Humanos , Macrófagos/imunologia , Ligação Proteica , RNA Interferente Pequeno , Células THP-1RESUMO
BACKGROUND: Eosinophilic meningitis, caused by fifth-stage larvae of the nematode (roundworm) Angiostrongylus cantonensis, is mainly attributed to the contribution of eosinophils to tissue inflammatory responses in helminthic infections. Eosinophils are associated with the killing of helminths via peroxidative oxidation and hydrogen peroxide generated by the dismutation of superoxide produced during respiratory bursts. In contrast, when residing in the host with high level of eosinophils, helminthic worms have evolved to attenuate eosinophil-mediated tissue inflammatory responses for their survival in the hosts. In a previous study we demonstrated that the expression of the A. cantonensis RPS 30 gene (Acan-rps-30) was significantly downregulated in A. cantonensis L5 roundworms residing in cerebrospinal fluid with a high level of eosinophils. Acan-RPS-30 is a protein homologous to the human Fau protein that plays a pro-apoptotic regulatory role and may function in protecting worms from oxidative stress. METHODS: The isolation and structural characterization of Acan-RPS-30 were performed using rapid amplification of cDNA ends (RACE), genome walking and bioinformatics. Quantitative real-time-PCR and microinjection were used to detect the expression patterns of Acan-rps-30. Feeding RNA interference (RNAi) was used to knockdown the apoptosis gene ced-3. Microinjection was performed to construct transgenic worms. An oxidative stress assay was used to determine the functions of Acan-RPS-30. RESULTS: Our results showed that Acan-RPS-30 consisted of 130 amino acids. It was grouped into clade V with C. elegans in the phylogenetic analysis. It was expressed ubiquitously in worms and was downregulated in both L5 larvae and adult A. cantonensis. Worms expressing pCe-rps30::Acan-rps-30::rfp, with the refractile "button-like" apoptotic corpses, were susceptible to oxidative stress. Apoptosis genes ced-3 and ced-4 were both upregulated in the transgenic worms. The phenotype susceptible to oxidative stress could be converted with a ced-3 defective mutation and RNAi. rps-30-/- mutant worms were resistant to oxidative stress, with ced-3 and ced-4 both downregulated. The oxidative stress-resistant phenotype could be rescued and inhibited by through the expression of pCe-rps30::Acan-rps-30::rfp in rps-3-/- mutant worms. CONCLUSION: In C. elegans worms, downregulated RPS-30 plays a defensive role against damage due to oxidative stress, facilitating worm survival by regulating downregulated ced-3. This observation may indicate the mechanism by which A. cantonensis L5 worms, with downregulated Acan-RPS-30, survive in the central nervous system of humans from the immune response of eosinophils.
Assuntos
Angiostrongylus cantonensis/genética , Angiostrongylus cantonensis/metabolismo , Regulação para Baixo , Proteínas de Helminto/química , Proteínas de Helminto/genética , Estresse Oxidativo , Animais , Animais Geneticamente Modificados , Apoptose , Caenorhabditis elegans/genética , Sistema Nervoso Central , Eosinófilos/imunologia , Feminino , Perfilação da Expressão Gênica , Humanos , Masculino , Filogenia , Reação em Cadeia da Polimerase em Tempo Real , Ativação TranscricionalRESUMO
Understanding how grazing activity drives plant community structure or the distribution of specific species in a community remains a major challenge in community ecology. The patchiness or spatial aggregation of specific species can be quantified by analyzing their relative coordinates in the community. Using variance and geostatistical analysis methods, we examined the quantitative characteristics and spatial distribution of Stipa breviflora in a desert steppe in northern China under four different grazing intensities (no grazing, NG, light grazing, LG, moderate grazing, MG, and heavy grazing, HG) at three small spatial scales (10 × 10 cm, 20 × 20 cm, 25 × 25 cm). We found that grazing significantly increased cover, density, and proportion in standing crop of S. breviflora, but decreased height. The spatial distribution of S. breviflora was strongly dependent upon the sampling unit and grazing intensity. The patchiness of S. breviflora reduced with sampling scale, and spatial distribution of S. breviflora was mainly determined by structural factors. The intact clusters of S. breviflora were more fragmented with increasing grazing intensity and offspring clusters spread out from the center of the parent plant. These findings suggest that spatial aggregation can enhance the ability of S. breviflora to tolerate grazing and that smaller isolated clusters are beneficial to the survival of this dominant species under heavy grazing.
RESUMO
'Candidatus Mycoplasma haemobos' is an emerging pathogen in the genus Mycoplasma. The Rhipicephalus (Boophilus) microplus tick has been suspected to be the vector of 'C. M. haemobos'. To determine the role of R. (B.) microplus in transmission of 'C. M. haemobos', we tested the competence of R. (B.) microplus larvae to acquire 'C. M. haemobos' from positive female ticks and to serve as a 'C. M. haemobos' vector in mice. Using PCR and sequencing, we also analyzed the epidemic strains of 'C. M. haemobos' among R. (B.) microplus ticks collected from goats and sheep in southern Henan Province, central China. Our results identified three epidemic strains of 'C. M. haemobos', and the positive female ticks naturally infected could pass 'C. M. haemobos' at egg and larval stages. Furthermore, 'C. M. haemobos' infected larvae could transmit the pathogens to mice during feeding, and the negative larvae could acquire 'C. M. haemobos' from infected mice. Our study shows that R. (B.) microplus ticks could serve as a vector and reservoir of 'C. M. haemobos'.
Assuntos
Vetores Aracnídeos/microbiologia , Mycoplasma/isolamento & purificação , Rhipicephalus/fisiologia , Animais , China , Reservatórios de Doenças , Feminino , Larva , Camundongos , Camundongos Endogâmicos BALB CRESUMO
BACKGROUND: Angiostrongylus cantonensis is a human zoonotic nematode parasite. Our previous studies found that PAS-5 and Galectin-1 (Gal-1) proteins of A. cantonensis could be strongly recognized by sera from mice infected with A. cantonensis. In this study, we further evaluated the potential roles of these two proteins in the induction of immune response in mice. METHODS: Mice were immunized with recombinant PAS-5 or Gal-1 and then challenged with 30 infective A. cantonensis larvae following the last immunization. We then examined the infected mice for changes in serum antibodies and cytokines by ELISA, CD4+ T cells and CD4+CD25+FoxP3+ regulatory T cells (Tregs) by flow cytometry, and tissue damage severity by hematoxylin-eosin (H&E) staining. RESULTS: Compared with control mice, the PAS-5-immunized mice exhibited increased levels of serum antibodies and cytokines (except for IL-10) at different time points post-infection. PAS-5 immunization promoted significant proliferation of CD4+ T cells, and caused more damage in the brain tissue. Vaccination with Gal-1 inhibited the production of antibodies (except for IgG1) and IFN-γ, but promoted the expression of IL-4 and IL-10. Gal-1 immunization results in significant increases in the levels of CD4+CD25+FoxP3+ Tregs, and mild inflammatory changes. CONCLUSIONS: Taken together, our findings show that PAS-5 enhances, but Gal-1 inhibits the immune response in the early stage of A. cantonensis infections.
Assuntos
Angiostrongylus cantonensis/imunologia , Galectina 1/imunologia , Proteínas de Helminto/imunologia , Infecções por Strongylida/imunologia , Angiostrongylus cantonensis/química , Angiostrongylus cantonensis/patogenicidade , Animais , Encéfalo/parasitologia , Encéfalo/patologia , Citocinas/metabolismo , Modelos Animais de Doenças , Ensaio de Imunoadsorção Enzimática , Feminino , Citometria de Fluxo , Galectina 1/metabolismo , Proteínas de Helminto/metabolismo , Humanos , Imunidade Celular , Imunidade Humoral , Camundongos , Camundongos Endogâmicos C57BL , Organismos Livres de Patógenos Específicos , Baço/parasitologia , Baço/patologia , Infecções por Strongylida/parasitologia , Infecções por Strongylida/patologia , Linfócitos T Reguladores/imunologiaRESUMO
The insulin-like signaling (IIS) pathway is considered to be significant in regulating fat metabolism, dauer formation, stress response and longevity in Caenorhabditis elegans. "Dauer hypothesis" indicates that similar IIS transduction mechanism regulates dauer development in free-living nematode C. elegans and the development of infective third-stage larvae (iL3) in parasitic nematodes, and this is bolstered by a few researches on structures and functions of the homologous genes in the IIS pathway cloned from several parasitic nematodes. In this study, we identified the insulin-like receptor encoding gene, Acan-daf-2, from the parasitic nematode Angiostrongylus cantonensis, and determined the genomic structures, transcripts and functions far more thorough in longevity, stress resistance and dauer formation. The sequence of Acan-DAF-2, consisting of 1413 amino acids, contained all of the characteristic domains of insulin-like receptors from other taxa. The expression patterns of Acan-daf-2 in the C. elegans surrogate system showed that pAcan-daf-2:gfp was only expressed in intestine, compared with the orthologue in C. elegans, Ce-daf-2 in both intestine and neurons. In addition to the similar genomic organization to Ce-daf-2, Acan-DAF-2 could also negatively regulate Ce-DAF-16A through nuclear/cytosolic translocation and partially restore the C. elegans daf-2(e1370) mutation in longevity, dauer formation and stress resistance. These findings provided further evidence of the functional conservation of DAF-2 between parasitic nematodes and the free-living nematode C. elegans, and might be significant in understanding the developmental biology of nematode parasites, particularly in the infective process and the host-specificity.
Assuntos
Angiostrongylus cantonensis/metabolismo , Caenorhabditis elegans/metabolismo , Proteínas de Helminto/metabolismo , Longevidade/efeitos dos fármacos , Estresse Fisiológico/efeitos dos fármacos , Sequência de Aminoácidos , Animais , Caenorhabditis elegans/genética , DNA/genética , Regulação da Expressão Gênica/fisiologia , Proteínas de Helminto/genética , Temperatura Alta/efeitos adversos , Organismos Geneticamente Modificados , Filogenia , Especificidade da EspécieRESUMO
Fork head box transcription factors subfamily O (FoxO) is regarded to be significant in cell-cycle control, cell differentiation, ageing, stress response, apoptosis, tumour formation and DNA damage repair. In the free-living nematode Caenorhabditis elegans, the FoxO transcription factor is encoded by Ce-daf-16, which is negatively regulated by insulin-like signaling (IIS) and involved in promoting dauer formation through bringing about its hundreds of downstream genes expression. In nematode parasites, orthologues of daf-16 from several species have been identified, with functions in rescue of dauer phenotypes determined in a surrogate system C. elegans. In this study, we identified the FoxO encoding gene, Acan-daf-16, from the parasitic nematode Angiostrongylus cantonensis, and determined the genomic structures, transcripts and functions far more thorough in longevity, stress resistance and dauer formation. Acan-daf-16 encodes two proteins, Acan-DAF-16A and Acan-DAF-16B, consisting of 555 and 491 amino acids, respectively. Both isoforms possess the highly conserved fork head domains. Acan-daf-16A and Acan-daf-16B are expressed from distinct promoters. The expression patterns of Acan-daf-16 isoforms in the C. elegans surrogate system showed that p Acan-daf-16a:gfp was expressed in all cells of C. elegans, including the pharynx, and the expression of p Acan-daf-16b:gfp was restricted to the pharynx. In addition to the same genomic organization to the orthologue in C. elegans, Ce-daf-16, both Acan-DAF-16 isoforms could restore the C. elegans daf-16(mg54) mutation in longevity, dauer formation and stress resistance, in spite of the partial complementation of Acan-DAF-16B isoform in longevity. These findings provide further evidence of the functional conservation of DAF-16s between parasitic nematodes and the free-living nematode C. elegans.