RESUMO
The functionality of all metabolic processes in chloroplasts depends on a balanced integration of nuclear- and chloroplast-encoded polypeptides into the plastid's proteome. The chloroplast chaperonin machinery is an essential player in chloroplast protein folding under ambient and stressful conditions, with a more intricate structure and subunit composition compared to the orthologous GroEL/ES chaperonin of Escherichia coli. However, its exact role in chloroplasts remains obscure, mainly because of very limited knowledge about the interactors. We employed the competition immunoprecipitation method for the identification of the chaperonin's interactors in Chlamydomonas reinhardtii. Co-immunoprecipitation of the target complex in the presence of increasing amounts of isotope-labelled competitor epitope and subsequent mass spectrometry analysis specifically allowed to distinguish true interactors from unspecifically co-precipitated proteins. Besides known substrates such as RbcL and the expected complex partners, we revealed numerous new interactors with high confidence. Proteins that qualify as putative substrate proteins differ from bulk chloroplast proteins by a higher content of beta-sheets, lower alpha-helical conformation and increased aggregation propensity. Immunoprecipitations targeted against a subunit of the co-chaperonin lid revealed the ClpP protease as a specific partner complex, pointing to a close collaboration of these machineries to maintain protein homeostasis in the chloroplast.
Assuntos
Chaperonina 60 , Cloroplastos , Cloroplastos/metabolismo , Chaperonina 60/análise , Chaperonina 60/química , Chaperonina 60/metabolismo , Dobramento de Proteína , Proteínas de Cloroplastos/metabolismoRESUMO
Anaplasma platys is a tick-transmitted rickettsial pathogen, which is known to be the etiologic agent for cyclic thrombocytopenia in its primary canine host. Infections with this pathogen are also reported in cats, cattle and people. Similarly, Ehrlichia canis is another tick-borne rickettsial pathogen responsible for canine monocytic ehrlichiosis and is also reported to cause infections in people. We describe infections in dogs with these two pathogens on the Caribbean island of Grenada, West Indies by detection using molecular methods. We utilized a 16S rRNA gene-based PCR assay to detect both Ehrlichia and Anaplasma species by screening 155 canine blood samples from asymptomatic dogs. We found 18.7 % of the dogs to be positive for A. platys and 16.8 % for E. canis. Samples that tested positive for A. platys were further assessed by sequence analysis targeting 16S rRNA, 23S rRNA, citrate synthase (gltA) and heat shock protein (groEL) genes. Phylogenetic analysis revealed high correlation of A. platys 16S rRNA and gltA gene sequences with the geographic origins, while 23S rRNA and groEL gene sequences clustered independent of the geographic origins. This study represents an important step in defining the widespread distribution of active rickettsial infections in Caribbean dogs with no apparent clinical signs, thus posing a high risk for canine health and to a lesser extent to humans, as most dogs in the Caribbean are free-roaming.
Assuntos
Anaplasma/isolamento & purificação , Anaplasmose/epidemiologia , Doenças do Cão/epidemiologia , Ehrlichia canis/isolamento & purificação , Ehrlichiose/veterinária , Anaplasma/enzimologia , Anaplasma/genética , Anaplasmose/microbiologia , Animais , Proteínas de Bactérias/análise , Chaperonina 60/análise , Citrato (si)-Sintase/análise , Doenças do Cão/microbiologia , Cães , Ehrlichia canis/enzimologia , Ehrlichia canis/genética , Ehrlichiose/epidemiologia , Ehrlichiose/microbiologia , Granada/epidemiologia , Prevalência , RNA Bacteriano/análise , RNA Ribossômico 16S/análise , RNA Ribossômico 23S/análiseRESUMO
In anti-neutrophilic cytoplasmic antibody (ANCA)-associated vasculitis (AAV) genetic predisposition, ANCA autoantibodies, neutrophil extracellular traps (NETs), complement activation, and toll-like receptor signaling are implicated in AAV pathogenesis. Heat shock proteins (HSPs), a highly conserved group of small-sized molecular chaperones, take part in protein folding during cellular stress. Although HSPs were initially observed intracellularly, it has been shown that they can be secreted in the extracellular space and modulate the immune response in various autoimmune diseases including AAV. The scope of the present study is to investigate the role of heat shock protein 60 (HSP60) and 70 (HSP70) in the long renal effects in an ANCA vasculitis cohort. In this cohort of ANCA-associated vasculitis, 29 patients were followed up over 20 years. At diagnosis, immunohistochemistry was performed for HSP60 and HSP70 within the various nephron compartments. Higher renal HSP60 expression was associated with increased interstitial inflammatory infiltrates at diagnosis, while HSP70 expression was associated with a greater extent of interstitial fibrosis at diagnosis. Notably, intense tissue expression of HSP70 at the time of biopsy was associated with a worsened kidney survival. Renal HSP70 expression was associated with poor renal outcomes during long-term follow-up. This finding may indicate a role of HSPs in renal disease progression in ANCA vasculitis. Further validating studies are needed to verify a causative association between HSP70 expression and renal outcomes in ANCA-associated vasculitis.
Assuntos
Vasculite Associada a Anticorpo Anticitoplasma de Neutrófilos/patologia , Proteínas de Choque Térmico HSP70/análise , Rim/patologia , Adulto , Idoso , Chaperonina 60/análise , Feminino , Fibrose , Humanos , Masculino , Pessoa de Meia-Idade , Proteínas Mitocondriais/análise , Estudos ProspectivosRESUMO
Borrelia burgdorferi sensu lato (s.l.) is the etiological agent of Lyme disease, transmitted by ticks of the genus Ixodes Latreille. Diagnosis of Lyme disease in humans is often difficult and a detailed knowledge of the circulation of B. burgdorferi s.l. in tick hosts is therefore fundamental to support clinical procedures. Here we developed a molecular approach for the detection of B. burgdorferi s.l. in North Italian Ixodes ricinus (Linnaeus). The method is based on the amplification of a fragment of the groEL gene, which encodes a heat-shock protein highly conserved among B. burgdorferi s.l. species. The tool was applied in both qualitative and Real-time PCR approaches testing ticks collected in a North Italian area. The obtained results suggest that this new molecular tool could represent a sensitive and specific method for epidemiological studies aimed at defining the distribution of B. burgdorferi s.l. in I. ricinus and, consequently, the exposure risk for humans.
Assuntos
Proteínas de Bactérias/análise , Grupo Borrelia Burgdorferi/isolamento & purificação , Chaperonina 60/análise , Ixodes/microbiologia , Reação em Cadeia da Polimerase em Tempo Real/métodos , Animais , Grupo Borrelia Burgdorferi/genética , Feminino , Itália , Ixodes/crescimento & desenvolvimento , Masculino , Ninfa/crescimento & desenvolvimento , Ninfa/microbiologia , Sensibilidade e Especificidade , Alinhamento de Sequência , Análise de Sequência de ProteínaRESUMO
BACKGROUND: Heat shock protein 60 has been reported to have a high diagnostic value for digestive system cancers. We sought to systematically evaluate the diagnostic value of HSP60 in patients with gastric cancer (GC), colorectal cancer (CRC), and hepatocellular carcinoma (HCC). METHODS: Relevant literature was adopted from the online databases. The pooled sensitivity, specificity, and diagnostic odds ratio (DOR) were pooled using random effects models. Summary receiver operating characteristic curve and the area under the curve (AUC) were used to express the overall test performance. Statistical analysis was performed by STATA 14.0 and Meta-DiSc 1.4 software. RESULTS: We merged 12 studies in a meta-analysis, including 1 GC, 5 CRC, and 6 HCC. Overall, the pooled sensitivity, specificity, and DOR to predict GC/CRC/HCC patients were 70%, 71%, and 8.49, respectively, corresponding to an AUC of 0.81. In subgroup analysis, the 82% specificity prompted a more advanced diagnostic accuracy for diagnosing CRC than HCC. CONCLUSIONS: HSP60 was an advanced biomarker for digestive system cancers and its abnormal expression might have implications for early diagnosis in screening of GC/CRC/HCC.
Assuntos
Biomarcadores Tumorais/metabolismo , Chaperonina 60/análise , Neoplasias do Sistema Digestório/metabolismo , Trato Gastrointestinal/metabolismo , Proteínas Mitocondriais/análise , Neoplasias do Sistema Digestório/diagnóstico , Trato Gastrointestinal/patologia , Humanos , Razão de Chances , Curva ROC , Sensibilidade e EspecificidadeRESUMO
In camels and their infesting ectoparasites, specific detection of pathogenic Anaplasma platys and genetically related strains (A. platys-like strains) remains problematic. This requires sequencing of the hemi-nested PCR products specific to A. platys and related strains. In this study, a PCR/RFLP method, earlier developed for specific detection of A. platys-like strains in animal species other than camels, was adapted in order to subtype A. platys-like strains isolated from camels and their ticks and to differentiate them from pathogenic A. platys without going through a sequencing step. This approach was used for investigating the infections with A. platys and related strains in 412 Camelus dromedarius camels and 334 feeding ticks from five Tunisian governorates. Microscopic examination using Giemsa-stained blood smears was performed in order to specify which types of cells were infected. Ticks were identified as Hyalomma dromedarii (nâ¯=â¯164, 49%), H. impeltatum (nâ¯=â¯161, 48.3%) and H. excavatum (nâ¯=â¯9, 2.7%). A. platys was not detected in any of the tested camels or ticks. The overall prevalence of A. platys-like strains was 5.6% (23/412) in camels and microscopic examination of infected cells showed a tropism for neutrophil granulocytes. One tick identified as H. dromedarii out of 327 analyzed ticks was found to be infected with A. platys-like strains (0.3%). Alignment, identity comparison and phylogenetic analysis of the 16S rRNA partial sequences obtained in this study suggest that Tunisian dromedaries and feeding ticks are infected with different Anaplasma strains genetically related to A. platys. Sequence analysis and phylogenetic study based on the groEL gene confirm the RFLP results and show that camel strains formed a separate sub-cluster relatively close to A. platys-like strains infecting Tunisian cattle. This adapted RFLP assay allows fast and specific detection of pathogenic A. platys and A. platys-like strains in camels and infesting ticks and has the intrinsic potential of revealing co-infections with these two types of bacteria in the same sample, reducing the time and costs associated with cloning and sequencing during molecular diagnosis.
Assuntos
Anaplasma/isolamento & purificação , Anaplasmose/epidemiologia , Camelus , Ixodidae/microbiologia , Infestações por Carrapato/veterinária , Anaplasma/genética , Anaplasmose/microbiologia , Animais , Proteínas de Bactérias/análise , Sequência de Bases , Chaperonina 60/análise , DNA Bacteriano/análise , Feminino , Ixodidae/fisiologia , Masculino , Filogenia , Prevalência , RNA Bacteriano/análise , RNA Ribossômico 16S/análise , Infestações por Carrapato/epidemiologia , Tunísia/epidemiologiaRESUMO
Ehrlichia spp. are obligatory intracellular microorganisms that infect hematopoietic, endothelial or blood cells of mammals. Ticks are the only vectors of these agents in nature. To date, the role of birds and their associated ticks as reservoirs of ehrlichiae remains almost unexplored. In this study, we performed a molecular screening for bacteria of Anaplasmataceae family in samples of spleen (nâ¯=â¯72) and lung (nâ¯=â¯17), recovered from 72 carcasses of Magellanic penguins (Spheniscus magellanicus) in Brazil and Chile. One apparently unengorged tick (Ixodes uriae) was also collected while wandering upon one of the carcasses and submitted to molecular analyses as well. Through conventional and nested PCR protocols three genes (16S rRNA, dsb and groEL) of a new Ehrlichia sp. were partially characterized upon organs of three penguins and in the tick coming from Magdalena Island (Chile). First matches after BLASTn comparisons showed that our sequences share 99.4% (16S rRNA), 94.6% (groEL) and 79.3% (dsb) of identity with "Candidatus Ehrlichia ornithorhynchi", Ehrlichia sp. NS101 and Ehrlichia canis CCZ, respectively. Matrixes of genetic distance including other representatives of the Ehrlichia genus point a 99.4%, 94.0%, and 80.0% of identity with 16S rRNA, groEL and dsb genes from Ehrlichia sp. It25, Ehrlichia sp. NS101, and Ehrlichia chaffeensis San Louis, respectively. A Bayesian phylogenetic analysis of Anaplasmataceae 16S rRNA gene places the detected Ehrlichia sp. into a group with Ehrlichia sp. BAT and Ehrlichia sp. Natal. Although depicting different topologies, Bayesian unrooted phylogenetic trees constructed for groEL and dsb genes position this Ehrlichia sp. into well-supported branches, which reinforces the finding of a new taxon. For the moment, any pathogenic effect of this new Ehrlichia sp. on penguins is still unknown. However, this fact becomes important to assess from a conservation point of view since populations of Magellanic penguins are currently threatened and in an ongoing decrease.
Assuntos
Ehrlichia/classificação , Ixodes/microbiologia , Spheniscidae/microbiologia , Animais , Proteínas de Bactérias/análise , Chaperonina 60/análise , Chile , Ehrlichia/fisiologia , Feminino , Filogenia , RNA Bacteriano/análise , RNA Ribossômico 16S/análiseRESUMO
Mycobacteria are a diverse bacterial group ubiquitous in many soil and aquatic environments. Members of this group have been associated with human and other animal diseases, including the nontuberculous mycobacteria (NTM), which are of growing relevance to public health worldwide. Although soils are often considered an important source of environmentally acquired NTM infections, the biodiversity and ecological preferences of soil mycobacteria remain largely unexplored across contrasting climates and ecosystem types. Using a culture-independent approach by combining 16S rRNA marker gene sequencing with mycobacterium-specific hsp65 gene sequencing, we analyzed the diversity, distributions, and environmental preferences of soil-dwelling mycobacteria in 143 soil samples collected from a broad range of ecosystem types. The surveyed soils harbored highly diverse mycobacterial communities that span the full extent of the known mycobacterial phylogeny, with most soil mycobacteria (97% of mycobacterial clades) belonging to previously undescribed lineages. While mycobacteria tended to have higher relative abundances in cool, wet, and acidic soil environments, several individual mycobacterial clades had contrasting environmental preferences. We identified the environmental preferences of many mycobacterial clades, including the clinically relevant Mycobacterium avium complex that was more commonly detected in wet and acidic soils. However, most of the soil mycobacteria detected were not closely related to known pathogens, calling into question previous assumptions about the general importance of soil as a source of NTM infections. Together, this work provides novel insights into the diversity, distributions, and ecological preferences of soil mycobacteria and lays the foundation for future efforts to link mycobacterial phenotypes to their distributions.IMPORTANCE Mycobacteria are common inhabitants of soil, and while most members of this bacterial group are innocuous, some mycobacteria can cause environmentally acquired infections of humans and other animals. Human infections from nontuberculous mycobacteria (NTM) are increasingly prevalent worldwide, and some areas appear to be "hotspots" for NTM disease. While exposure to soil is frequently implicated as an important mode of NTM transmission, the diversity, distributions, and ecological preferences of soil mycobacteria remain poorly understood. We analyzed 143 soils from a range of ecosystems and found that mycobacteria and lineages within the group often exhibited predictable preferences for specific environmental conditions. Soils harbor large amounts of previously undescribed mycobacterial diversity, and lineages that include known pathogens were rarely detected in soil. Together, these findings suggest that soil is an unlikely source of many mycobacterial infections. The biogeographical patterns we documented lend insight into the ecology of this important group of soil-dwelling bacteria.
Assuntos
Proteínas de Bactérias/análise , Chaperonina 60/análise , Microbiota , Mycobacterium/fisiologia , Microbiologia do Solo , Mycobacterium/genética , Infecções por Mycobacterium não Tuberculosas/epidemiologia , Infecções por Mycobacterium não Tuberculosas/microbiologia , Infecções por Mycobacterium não Tuberculosas/transmissão , Micobactérias não Tuberculosas/genética , Micobactérias não Tuberculosas/fisiologia , RNA Bacteriano/análise , RNA Ribossômico 16S/análiseRESUMO
The UL47 gene product, VP8, is a major tegument protein of BoHV-1. While VP8 is not essential for virus replication in cell culture, a UL47-deleted virus exhibits a smaller tegument structure and is avirulent in cattle. To obtain pure VP8 protein for structural analysis, we expressed a N-terminally truncated version of VP8 in Eschericia coli. However, the recombinant VP8 was consistently co-purified with a tightly associated bacterial protein; this protein was identified by mass spectrometry as GroEL, which has considerable homology with mammalian heat shock protein-60 (HSP60), thus suggesting a new role for VP8 in virus-host interaction. A physical interaction of HSP60 and VP8 in both VP8-transfected and BoHV-1-infected cells was demonstrated by immunoprecipitation. Analysis of different truncated VP8 constructs revealed that amino acids 259-482 and 632-741 are involved in binding to HSP60. Full-length VP8 and VP8 219-741 (containing both interacting domains, 259-482 and 632-741) co-localized with HSP60 and mitochondria. VP8 was localized in the mitochondria from 2 to 14 h post infection in BoHV-1-infected cells. The mitochondrial membrane potential was reduced in both VP8-transfected and BoHV-1-infected cells and was further diminished by overexpression of HSP60 in the presence of VP8. In addition, VP8 expression decreased the ATP concentration during transfection, as well as BoHV-1 infection. Thus, VP8 may play a role in the deregulation of mitochondrial function through interaction with HSP60. This is consistent with the fact that BoHV-1 infection is known to promote mitochondrial dysfunction.
Assuntos
Proteínas do Capsídeo/metabolismo , Chaperonina 60/metabolismo , Herpesvirus Bovino 1/fisiologia , Interações Hospedeiro-Patógeno , Mitocôndrias/patologia , Mapeamento de Interação de Proteínas , Trifosfato de Adenosina/análise , Animais , Proteínas do Capsídeo/análise , Bovinos , Linhagem Celular , Chaperonina 60/análise , Células Epiteliais/virologia , Humanos , Imunoprecipitação , Potenciais da Membrana , Mitocôndrias/química , Membranas Mitocondriais/fisiologia , Ligação ProteicaRESUMO
Heat shock protein 60 (HSP60) overexpresses in various types of cancer, but its expression levels and functions in hepatocellular carcinoma (HCC) are still in dispute. We aim to clarify this issue and examine whether HSP60 could be a therapeutic target for HCC. We found drastically enhanced cell apoptosis and suppressed cell proliferation in two HCC cell lines with HSP60-silencing, and also indicated survivin was involved in this regulatory process in vitro and in vivo. However, HSP60-silencing in normal human hepatocytes only resulted in a minimal reduction of cell proliferation but without effects on cell apoptosis. We also showed HSP60 interacted with cytosolic but not mitochondrial survivin by immunoprecipitation assay. A rigorous method was used to standardize quantification from immunoblot assay to obtain more precise expression levels of HSP60 and survivin. The expression of HSP60 and survivin positively correlated in both cancerous and non-cancerous liver tissues (P < 0.001) after analyzing 145 surgically removed HCC tissues. A total of 56.6% of HCC patients overexpressed HSP60 in cancerous tissues, and 40.0% under-expressed HSP60. Higher expression of HSP60 and survivin in non-cancerous tissues both correlated with shorter overall survival (P = 0.029 and P < 0.001, respectively). Finally, we evaluated the therapeutic potential of HSP60 using extraneous delivery of jetPEI/shHSP60 complexes. The treatment results showed significant reduction of tumor weight by 44.3% (P < 0.05), accompanied by under-expression of survivin. These studies suggested that HSP60 not only served as a prognostic marker but also served as a novel therapeutic target for HCC.
Assuntos
Carcinoma Hepatocelular/terapia , Chaperonina 60/genética , Neoplasias Hepáticas/terapia , RNA Interferente Pequeno/uso terapêutico , Terapêutica com RNAi , Survivina/genética , Animais , Carcinoma Hepatocelular/genética , Carcinoma Hepatocelular/patologia , Linhagem Celular Tumoral , Proliferação de Células , Chaperonina 60/análise , Citoplasma/genética , Citoplasma/patologia , Regulação Neoplásica da Expressão Gênica , Injeções Subcutâneas , Fígado/metabolismo , Fígado/patologia , Neoplasias Hepáticas/genética , Neoplasias Hepáticas/patologia , Masculino , Camundongos Endogâmicos BALB C , Camundongos Nus , RNA Interferente Pequeno/administração & dosagem , RNA Interferente Pequeno/genética , Terapêutica com RNAi/métodos , Survivina/análiseRESUMO
Hsp60 (also called Cpn60) is a chaperonin with essential functions for cell physiology and survival. Additionally, its involvement in the pathogenesis of a variety of diseases (e.g., some autoimmune disorders and cancer) is becoming evident with new research. For example, the distribution and levels of Hsp60 in cells and tissues have been found altered in many pathologic conditions, and the significance of these alterations is being investigated in a number of laboratories. The aim of this ongoing research is to determine the meaning of these Hsp60 alterations with regard to pathogenetic mechanisms, diagnosis, classification of lesions, and assessing prognosis and response to treatment.Hsp60 occurs in the mitochondria, i.e., its typical residence according to classic knowledge, and also in other locales, such as the cytosol, the cell membrane, the intercellular space, and biological fluids (e.g., blood and cerebrospinal fluid). Detection and quantitative determinations in all these locations are becoming essential components of laboratory pathology in clinics and research. Consequently, immunohistochemistry targeting Hsp60 is also becoming essential for pathologists and researchers interested in disorders involving this chaperonin.In this chapter, we summarize some recent discoveries on the participation of Hsp60 in the pathogenesis of human diseases, and describe in detail how to perform immunohistochemical reactions for detecting the chaperonin, determining its location, and measuring its quantitative levels.
Assuntos
Chaperonina 60/análise , Imuno-Histoquímica/métodos , Autoimunidade , Humanos , Neoplasias/tratamento farmacológico , Neoplasias/metabolismo , PrognósticoRESUMO
Scrub typhus, caused by Orientia tsutsugamushi, is a common cause of acute undifferentiated febrile illness in the Asia-Pacific region. However, its nonspecific clinical manifestation often prevents early diagnosis. We propose the use of PCR and serologic tests as diagnostic tools. Here, we developed a multiplex real-time PCR assay using hydrolysis (TaqMan) probes targeting O. tsutsugamushi 47-kDa, groEL, and human interferon beta (IFN-ß gene) genes to improve early diagnosis of scrub typhus. The amplification efficiency was higher than 94%, and the lower detection limit was 10 copies per reaction. We used a human gene as an internal DNA quality and quantity control. To determine the sensitivity of this PCR assay, we selected patients with confirmed scrub typhus who exhibited a clear 4-fold increase in the level of IgG and/or IgM. The PCR assay result was positive in 45 of 52 patients, indicating a sensitivity of 86.5% (95% confidence interval [CI]: 74.2 to 94.4). The PCR assessment was negative for all 136 non-scrub typhus patients, indicating a specificity of 100% (95% CI: 97.3 to 100). In addition, this test helped diagnose patients with inconclusive immunofluorescence assay (IFA) results and using single blood samples. In conclusion, the real-time PCR assay proposed here is sensitive and specific in diagnosing scrub typhus. Combining PCR and serologic tests will improve the diagnosis of scrub typhus among patients presenting with acute febrile illness.
Assuntos
Proteínas de Bactérias/genética , Chaperonina 60/genética , Orientia tsutsugamushi/genética , Reação em Cadeia da Polimerase em Tempo Real/métodos , Tifo por Ácaros/diagnóstico , Ensaios de Anticorpos Bactericidas Séricos/métodos , Adulto , Anticorpos Antibacterianos/sangue , Carga Bacteriana/métodos , Proteínas de Bactérias/análise , Chaperonina 60/análise , Febre/diagnóstico , Febre/microbiologia , Amplificação de Genes , Humanos , Imunoglobulina G/sangue , Imunoglobulina M/sangue , Interferon beta/genética , Técnicas de Amplificação de Ácido Nucleico/métodos , Orientia tsutsugamushi/isolamento & purificação , Tifo por Ácaros/microbiologia , Sensibilidade e EspecificidadeRESUMO
CONTEXT: The effects of icariin, a chief constituent of ï¬avonoids from Epimedium brevicornum Maxim (Berberidaceae), on the levels of HIF-1α, HSP-60 and HSP-70 remain unknown. OBJECTIVE: To explore the effects of icariin on the levels of HSP-60, HIF-1α and HSP-70 neuron-specific enolase (NSE) and cell viability. MATERIALS AND METHODS: PC12 cells were treated with icariin (10-7, 10-6 or 10-5 mol/L) for 3 h (1 h before oxygen-glucose deprivation (OGD) plus 2 h OGD). HSP-60, HIF-1α, HSP-70 and NSE were measured using enzyme-linked immunosorbent assay (ELISA). Cell viability was determined by metabolic 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. RESULTS: After 2 h OGD, levels of HIF-1α, HSP-60, HSP-70 and NSE were increased significantly (HIF-1α: 33.3 ± 1.9 ng/L, HSP-60: 199 ± 16 ng/L, HSP-70: 195 ± 17 ng/L, NSE: 1487 ± 125 ng/L), and cell viability was significantly decreased (0.26 ± 0.03), while icariin (10-7, 10-6, or 10-5 mol/L) significantly reduced the contents of HIF-1α, HSP-60, HSP-70 and NSE (HIF-1α: 14.1 ± 1.4, 22.6 ± 1.8, 15.7 ± 2.1, HSP-60: 100 ± 12, 89 ± 6, 113 ± 11, HSP-70: 139 ± 9, 118 ± 7, 95 ± 9 and NSE: 1121 ± 80, 1019 ± 52, 731 ± 88), and improved cell viability (0.36 ± 0.03, 0.38 ± 0.04, 0.37 ± 0.03) in OGD-treated PC12 cells. DISCUSSION AND CONCLUSION: These results indicate that the protective mechanisms of icariin against OGD-induced injury may be related to down-regulating the expression of HIF-1α, HSP-60 and HSP-70.
Assuntos
Chaperonina 60/análise , Flavonoides/farmacologia , Proteínas de Choque Térmico HSP70/análise , Subunidade alfa do Fator 1 Induzível por Hipóxia/análise , Fármacos Neuroprotetores/farmacologia , Animais , Hipóxia Celular , Sobrevivência Celular/efeitos dos fármacos , Regulação para Baixo , Ensaio de Imunoadsorção Enzimática , Células PC12 , Fosfopiruvato Hidratase/análise , RatosAssuntos
Articulações/patologia , Osteoartrite/patologia , Proteína ADAMTS5/análise , Animais , Chaperonina 60/análise , Citocinas/análise , Humanos , Neovascularização Patológica/epidemiologia , Neovascularização Patológica/patologia , Neovascularização Patológica/terapia , Osteoartrite/epidemiologia , Osteoartrite/terapia , Receptores de Citocinas/análise , Fatores de Transcrição SOX9/análiseRESUMO
Heat shock proteins (Hsps) are a class of highly conserved proteins produced in virtually all living organisms from bacteria to humans. Hsp60 and Hsp10, the most important mitochondrial chaperones, participate in environmental stress responses. In this study, the full-length complementary DNAs (cDNAs) of Hsp60 (PmHsp60) and Hsp10 (PmHsp10) were cloned from Penaeus monodon. Sequence analysis showed that PmHsp60 and PmHsp10 encoded polypeptides of 578 and 102 amino acids, respectively. The expression profiles of PmHsp60 and PmHsp10 were detected in the gills and hepatopancreas of the shrimps under pH challenge, osmotic stress, and heavy metal exposure, and results suggested that PmHsp60 and PmHsp10 were involved in the responses to these stimuli. ATPase and chaperone activity assay indicated that PmHsp60 could slow down protein denaturation and that Hsp60/Hsp10 may be combined to produce a chaperone complex with effective chaperone and ATPase activities. Overall, this study provides useful information to help further understand the functional mechanisms of the environmental stress responses of Hsp60 and Hsp10 in shrimp.
Assuntos
Proteínas de Artrópodes/metabolismo , Chaperonina 10/metabolismo , Chaperonina 60/metabolismo , Penaeidae/fisiologia , Sequência de Aminoácidos , Animais , Proteínas de Artrópodes/análise , Proteínas de Artrópodes/genética , Sequência de Bases , Chaperonina 10/análise , Chaperonina 10/genética , Chaperonina 60/análise , Chaperonina 60/genética , Regulação da Expressão Gênica , Brânquias/química , Brânquias/fisiologia , Hepatopâncreas/química , Hepatopâncreas/fisiologia , Concentração de Íons de Hidrogênio , Metais Pesados/metabolismo , Pressão Osmótica , Penaeidae/química , Penaeidae/genética , Filogenia , Estresse FisiológicoRESUMO
BACKGROUND: Heat shock protein 60 (Hsp60) is a chaperonin involved in tumorigenesis, but its participation in tumor development and progression is not well understood and its value as a tumor biomarker has not been fully elucidated. In the current study, the authors presented evidence supporting the theory that Hsp60 has potential as a biomarker as well as a therapeutic target in patients with large bowel cancer. METHODS: The authors studied a population of 97 subjects, including patients and controls. Immunomorphology, Western blot analysis, and quantitative real-time polymerase chain reaction were performed on tissue specimens. Exosomes were isolated from blood and characterized by electron microscopy, biochemical tests, and Western blot analysis. RESULTS: Hsp60 was found to be increased in cancerous tissue, in which it was localized in the tumor cell plasma membrane, and in the interstitium associated with cells of the immune system, in which it was associated with exosomes liberated by tumor cells and, as such, circulated in the blood. An interesting finding was that these parameters returned to normal shortly after tumor removal. CONCLUSIONS: The data from the current study suggested that Hsp60 is a good candidate for theranostics applied to patients with large bowel carcinoma and encourage similar research among patients with other tumors in which Hsp60 has been implicated.
Assuntos
Adenocarcinoma/patologia , Chaperonina 60/metabolismo , Neoplasias do Colo/patologia , Exossomos/metabolismo , Proteínas Mitocondriais/metabolismo , Adenocarcinoma/metabolismo , Adenocarcinoma/cirurgia , Idoso , Idoso de 80 Anos ou mais , Biomarcadores Tumorais/análise , Western Blotting , Chaperonina 60/análise , Neoplasias do Colo/metabolismo , Neoplasias do Colo/cirurgia , Ensaio de Imunoadsorção Enzimática , Feminino , Humanos , Imuno-Histoquímica , Masculino , Pessoa de Meia-Idade , Proteínas Mitocondriais/análise , Reação em Cadeia da Polimerase em Tempo RealRESUMO
Liver fibrosis occurring as an outcome of non-alcoholic steatohepatitis (NASH) can precede the development of cirrhosis. We investigated the effects of sorafenib in preventing liver fibrosis in a rodent model of NASH. Adult Sprague-Dawley rats were fed a choline-deficient high-fat diet and exposed to diethylnitrosamine for 6 weeks. The NASH group (n=10) received vehicle and the sorafenib group (n=10) received 2.5 mg·kg(-1)·day(-1) by gavage. A control group (n=4) received only standard diet and vehicle. Following treatment, animals were sacrificed and liver tissue was collected for histologic examination, mRNA isolation, and analysis of mitochondrial function. Genes related to fibrosis (MMP9, TIMP1, TIMP2), oxidative stress (HSP60, HSP90, GST), and mitochondrial biogenesis (PGC1α) were evaluated by real-time quantitative polymerase chain reaction (RT-qPCR). Liver mitochondrial oxidation activity was measured by a polarographic method, and cytokines by enzyme-linked immunosorbent assay (ELISA). Sorafenib treatment restored mitochondrial function and reduced collagen deposition by nearly 63% compared to the NASH group. Sorafenib upregulated PGC1α and MMP9 and reduced TIMP1 and TIMP2 mRNA and IL-6 and IL-10 protein expression. There were no differences in HSP60, HSP90 and GST expression. Sorafenib modulated PGC1α expression, improved mitochondrial respiration and prevented collagen deposition. It may, therefore, be useful in the treatment of liver fibrosis in NASH.
Assuntos
Cirrose Hepática/tratamento farmacológico , Mitocôndrias Hepáticas/efeitos dos fármacos , Niacinamida/análogos & derivados , Hepatopatia Gordurosa não Alcoólica/complicações , Compostos de Fenilureia/uso terapêutico , Inibidores de Proteínas Quinases/uso terapêutico , Animais , Chaperonina 60/análise , Chaperonina 60/genética , Dieta Hiperlipídica/métodos , Dietilnitrosamina , Modelos Animais de Doenças , Colágenos Fibrilares/efeitos dos fármacos , Glutationa Transferase/análise , Glutationa Transferase/genética , Proteínas de Choque Térmico HSP90/análise , Proteínas de Choque Térmico HSP90/genética , Interleucina-10/análise , Interleucina-10/genética , Interleucina-6/análise , Interleucina-6/genética , Cirrose Hepática/etiologia , Cirrose Hepática/patologia , Metaloproteinase 9 da Matriz/análise , Metaloproteinase 9 da Matriz/genética , Mitocôndrias Hepáticas/metabolismo , Niacinamida/uso terapêutico , Hepatopatia Gordurosa não Alcoólica/induzido quimicamente , Coativador 1-alfa do Receptor gama Ativado por Proliferador de Peroxissomo , Polarografia , RNA Mensageiro/isolamento & purificação , Ratos Sprague-Dawley , Sorafenibe , Inibidor Tecidual de Metaloproteinase-1/análise , Inibidor Tecidual de Metaloproteinase-1/genética , Inibidor Tecidual de Metaloproteinase-2/análise , Inibidor Tecidual de Metaloproteinase-2/genética , Fatores de Transcrição/análise , Fatores de Transcrição/genéticaRESUMO
PURPOSE: Chlamydophila pneumoniae has been implicated in atherosclerosis/restenosis; however, clear evidence is missing. Therefore, the aim of our study was to examine the influence of intimal infection and systemic inflammation on cardiovascular complications after coronary intervention. METHODS: 45 atheroma specimens from patients with symptomatic coronary artery disease who underwent directional endatherectomy with stent implantation were analyzed by immunohistochemistry to detect chlamydial (c) and human (h) heat shock protein (HSP) 60. The antibodies used against cHSP60 and hHSP60 were characterized by specificity and lack of cross immunoreactivity. In addition, serum Ig antibodies against Chlamydophila pneumoniae and against mycobacterial (m) HSP65 as well as serum CRP levels were measured. At follow-up of 6 months, quantitative coronary angiography was performed and major adverse cardiac events (MACE) were assessed. RESULTS: Atheroma specimens of all 10 patients with MACE were positive for cHSP60 with overall higher cHSP60 tissue expressions (1.1 ± 0.4 %) and serum CRP levels (2.18 ± 0.85 mg/dl) compared to the remaining 35 patients without MACE (7 of 35 specimens positive for cHSP60, mean cHSP60 expression: 0.4 ± 0.1 %, CRP levels: 0.67 ± 0.16 mg/dl, p < 0.05). Colocalization of both HSP60 homologues was more frequent in the MACE group. Anti-mHSP65 serum titers were significantly higher in MACE (1:510) versus non-MACE patients (1:335) and correlated positively with plaque expressions of cHSP60 and hHSP60 (r = 0.54, p < 0.05; r = 0.46, p < 0.05; resp.). CONCLUSIONS: Intimal presence of cHSP60, systemic CRP and antibodies against mHSP65 are predictors for occurrence of MACE after coronary intervention.
Assuntos
Infecções por Chlamydophila/complicações , Chlamydophila pneumoniae , Doença da Artéria Coronariana/complicações , Doença da Artéria Coronariana/microbiologia , Idoso , Anticorpos Antibacterianos/sangue , Proteínas de Bactérias/análise , Proteínas de Bactérias/imunologia , Proteína C-Reativa/análise , Chaperonina 60/análise , Chaperonina 60/imunologia , Infecções por Chlamydophila/epidemiologia , Infecções por Chlamydophila/imunologia , Infecções por Chlamydophila/microbiologia , Doença da Artéria Coronariana/epidemiologia , Vasos Coronários/química , Vasos Coronários/patologia , Feminino , Humanos , Imuno-Histoquímica , Masculino , Pessoa de Meia-Idade , Proteínas Mitocondriais/análise , Proteínas Mitocondriais/imunologia , Placa Aterosclerótica/química , Placa Aterosclerótica/microbiologiaRESUMO
Microglial activation has been recognized as being vital in the pathogenesis of several neurodegenerative disorders. Therefore, the identification of therapeutic drugs to prevent microglial activation and thus protect against inflammationmediated neuronal injury, is required. In the present study, dextromethorphan (DM), a compound widely used in antitussive remedies that has been demonstrated to possess neuroprotective effects, was shown to reduce proinflammatory mediator production in lipopolysaccharide (LPS)stimulated BV2 mouse microglial cells. Western blot analysis revealed that DM markedly suppressed the activation of nuclear factorκB (NFκB), caspase3 signaling and the expression of another inflammationinducing factor, heat shock protein 60 (HSP60) and heat shock factor1, induced by LPS in BV2 cells. Results from ELISA assay demonstrated that DM reduced the release of HSP60, nitric oxide (NO), inducible NO synthase, tumor necrosis factorα, interleukin (IL)1ß and IL6 induced by LPS in BV2 microglia. These results were confirmed by immunofluorescence, suggesting that DM may exert a neuroprotective and antiinflammatory effect by inhibiting microglial activation through the HSP60NFκB signaling pathway. Therefore, DM may offer substantial therapeutic benefits in the treatment of neurodegenerative diseases that are accompanied by microglial activation.
Assuntos
Dextrometorfano/farmacologia , Expressão Gênica/efeitos dos fármacos , Fármacos Neuroprotetores/farmacologia , Animais , Linhagem Celular , Sobrevivência Celular/efeitos dos fármacos , Chaperonina 60/análise , Ensaio de Imunoadsorção Enzimática , Interleucina-1beta/análise , Interleucina-6/análise , Lipopolissacarídeos/toxicidade , Camundongos , Microglia/citologia , Microglia/efeitos dos fármacos , Microglia/metabolismo , Proteínas Mitocondriais/análise , NF-kappa B/metabolismo , Óxido Nítrico/metabolismo , Óxido Nítrico Sintase Tipo II/metabolismo , Transdução de Sinais/efeitos dos fármacos , Fator de Necrose Tumoral alfa/análiseRESUMO
The production of IFN- I (IFN-α/ß) is one of the earliest and most important host-protective responses. Interferon regulatory factor 3 (IRF3) is a critical transcriptional factor in the IFN-ß signaling pathway. Although significant progress has been achieved in the regulation of IRF3, the process may be more complicated than previously considered. In the present study, heat shock protein 60 (HSP60, HSPD1) was identified as a novel IRF3-interacting protein. Overexpression of HSPD1 facilitated the phosphorylation and dimerization of IRF3 and enhanced IFN-ß induction induced by SeV infection. In contrast, knockdown of endogenous HSPD1 significantly inhibited the signaling pathway. Furthermore, HSPD1 enhanced activation of the IFN-ß promoter mediated by RIG-I, MDA-5, MAVS, TBK1 and IKKε but not IRF3/5D, a mock phosphorylated form of IRF3. The present study indicated that HSPD1 interacted with IRF3 and it contributed to the induction of IFN-ß.