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1.
Food Chem ; 304: 125415, 2020 Jan 30.
Artigo em Inglês | MEDLINE | ID: mdl-31479995

RESUMO

The aim of our study was to characterize the proteolytic activity of 170 Lactobacillus strains isolated from traditional Mongolian dairy products (yogurt and fermented milk), and to investigate their capacity to generate bioactive peptides during milk fermentation. All isolates were screened for proteolytic activity using skim milk agar-well diffusion test. Fifteen strains (9 Lactobacillus helveticus and 6 Lactobacillus delbrueckii subsp. bulgaricus) were then selected and further evaluated using an original strategy based on multiparametric analysis, taking into account growth rate, acidification capacity, proteolytic activity, cell envelope associated peptidase (CEP) profile and LC-MS/MS analysis of peptides. All parameters were analyzed using principal component analysis (PCA). Results showed that strain growth and acidification correlate with peptide production and that Mongolian L. helveticus strains differ from Western strains in terms of CEP distribution. The PCA revealed that CEP profiles are major determinants of ß-casein hydrolysis patterns. Strains with distinctive proteolytic activities were identified.

2.
Front Microbiol ; 9: 2354, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-30386307

RESUMO

To compensate for their amino acid auxotrophy, lactobacilli have developed the ability to hydrolyze proteins present in their environment. This proteolytic activity not only generates the free amino acids needed by the bacteria, but also a large variety of peptides, some of which are endowed with biological activities. These so-called "bioactive peptides" (BAPs) are interesting from a nutrition and healthcare perspective. The use of lactic acid bacteria (LAB) such as lactobacilli is an effective strategy for production and valorization of new BAPs. The proteolytic activity of lactobacilli is exerted in a strain- and species-dependent manner: each species exhibits different proteinase content, leading to a large variety of proteolytic activities. This underlines the high potential of Lactobacillus strains to produce novel hydrolysates and BAPs of major interest. This review aims at discussing the potential of different Lactobacillus species to release BAPs from fermentation media and processes. Strategies used for peptide production are presented. Additionally, we propose a methodology to select the most promising Lactobacillus strains as sources of BAPs. This methodology combines conventional approaches and in silico analyses.

3.
Anaerobe ; 22: 50-6, 2013 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-23791918

RESUMO

Human intestinal microbiota plays an important role in the maintenance of host health by providing energy, nutrients, and immunological protection. Intestinal dysfunction is a frequent complaint in myalgic encephalomyelitis/chronic fatigue syndrome (ME/CFS) patients, and previous reports suggest that dysbiosis, i.e. the overgrowth of abnormal populations of bacteria in the gut, is linked to the pathogenesis of the disease. We used high-throughput 16S rRNA gene sequencing to investigate the presence of specific alterations in the gut microbiota of ME/CFS patients from Belgium and Norway. 43 ME/CFS patients and 36 healthy controls were included in the study. Bacterial DNA was extracted from stool samples, PCR amplification was performed on 16S rRNA gene regions, and PCR amplicons were sequenced using Roche FLX 454 sequencer. The composition of the gut microbiota was found to differ between Belgian controls and Norwegian controls: Norwegians showed higher percentages of specific Firmicutes populations (Roseburia, Holdemania) and lower proportions of most Bacteroidetes genera. A highly significant separation could be achieved between Norwegian controls and Norwegian patients: patients presented increased proportions of Lactonifactor and Alistipes, as well as a decrease in several Firmicutes populations. In Belgian subjects the patient/control separation was less pronounced, however some abnormalities observed in Norwegian patients were also found in Belgian patients. These results show that intestinal microbiota is altered in ME/CFS. High-throughput sequencing is a useful tool to diagnose dysbiosis in patients and could help designing treatments based on gut microbiota modulation (antibiotics, pre and probiotics supplementation).


Assuntos
Bacteroidetes/genética , DNA Bacteriano/genética , DNA Bacteriano/isolamento & purificação , Disbiose/microbiologia , Síndrome de Fadiga Crônica/microbiologia , Enteropatias/microbiologia , RNA Ribossômico 16S/genética , Adulto , Bélgica , Contagem de Colônia Microbiana , Disbiose/diagnóstico , Síndrome de Fadiga Crônica/complicações , Fezes/microbiologia , Feminino , Genes de RNAr , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Enteropatias/diagnóstico , Intestinos/microbiologia , Masculino , Pessoa de Meia-Idade , Noruega , Análise de Sequência de RNA
4.
In Vivo ; 27(2): 177-87, 2013 Mar-Apr.
Artigo em Inglês | MEDLINE | ID: mdl-23422476

RESUMO

Myalgic encephalomyelitis (ME) is a debilitating illness of unknown etiology characterized by neurocognitive dysfunction, inflammation, immune abnormalities and gastrointestinal distress. An increasing body of evidence suggests that disruptions in the gut may contribute to the induction of neuroinflammation. Therefore, reports of human endogenous retroviral (HERV) expression in association with neuroinflammatory diseases prompted us to investigate the gut of individuals with ME for the presence of HERV proteins. In eight out of 12 individuals with ME, immunoreactivity to HERV proteins was observed in duodenal biopsies. In contrast, no immunoreactivity was detected in any of the eight controls. Immunoreactivity to HERV Gag and Env proteins was uniquely co-localized in hematopoietic cells expressing the C-type lectin receptor CLEC4C (CD303/BDCA2), the co-stimulatory marker CD86 and the class II major histocompatibility complex HLA-DR, consistent with plasmacytoid dendritic cells (pDCs). Although the significance of HERVs present in the pDCs of individuals with ME has yet to be determined, these data raise the possibility of an involvment of pDCs and HERVs in ME pathology. To our knowledge, this report describes the first direct association between pDCs and HERVs in human disease.


Assuntos
Anticorpos Antivirais/sangue , Células Dendríticas/imunologia , Duodeno/imunologia , Síndrome de Fadiga Crônica/imunologia , Plasmócitos/imunologia , Proteínas dos Retroviridae/imunologia , Anticorpos Antivirais/imunologia , Células Dendríticas/patologia , Duodeno/patologia , Síndrome de Fadiga Crônica/patologia , Técnica Indireta de Fluorescência para Anticorpo , Humanos , Plasmócitos/patologia
5.
In Vivo ; 23(2): 209-13, 2009 Mar-Apr.
Artigo em Inglês | MEDLINE | ID: mdl-19414405

RESUMO

BACKGROUND: Human herpesvirus-6 (HHV-6), Epstein-Barr virus and parvovirus B19 have been suggested as etiological agents of chronic fatigue syndrome but none of these viruses is consistently detected in all patients. However, active viral infections may be localized in specific tissues, and, therefore, are not easily detectable. The aim of this study was to investigate the presence of HHV-6, HHV-7, EBV and parvovirus B19 in the gastro-intestinal tract of CFS patients. PATIENTS AND METHODS: Using real-time PCR, viral DNA loads were quantified in gastro-intestinal biopsies of 48 CFS patients and 35 controls. RESULTS: High loads of HHV-7 DNA were detected in most CFS and control biopsies. EBV and HHV-6 were detected in 15-30% of all biopsies. Parvovirus B19 DNA was detected in 40% of the patients versus less than 15% of the controls. CONCLUSION: Parvovirus B19 may be involved in the pathogenesis of CFS, at least for a subset of patients. The gastro-intestinal tract appears as an important reservoir of infection for several potentially pathogenic viruses.


Assuntos
Síndrome de Fadiga Crônica/virologia , Mucosa Gástrica/virologia , Regulação Viral da Expressão Gênica , Herpesviridae/metabolismo , Mucosa Intestinal/virologia , Parvovirus B19 Humano/metabolismo , Adulto , Biópsia , Primers do DNA/química , DNA Viral/metabolismo , Síndrome de Fadiga Crônica/metabolismo , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Reação em Cadeia da Polimerase Via Transcriptase Reversa
6.
Biochem Biophys Res Commun ; 376(1): 231-3, 2008 Nov 07.
Artigo em Inglês | MEDLINE | ID: mdl-18774769

RESUMO

Chronic fatigue syndrome (CFS) is characterized by immune dysfunctions including chronic immune activation, inflammation, and alteration of cytokine profiles. T helper 17 (Th17) cells belong to a recently identified subset of T helper cells, with crucial regulatory function in inflammatory and autoimmune processes. Th17 cells are implicated in allergic inflammation, intestinal diseases, central nervous system inflammation, disorders that may all contribute to the pathophysiology of CFS. IL-17F is one of the pro-inflammatory cytokines secreted by Th17 cells. We investigated the association between CFS and the frequency of rs763780, a C/T genetic polymorphism leading to His161Arg substitution in the IL-17F protein. The His161Arg variant (C allele) antagonizes the pro-inflammatory effects of the wild-type IL-17F. A significantly lower frequency of the C allele was observed in the CFS population, suggesting that the His161Arg variant may confer protection against the disease. These results suggest a role of Th17 cells in the pathogenesis of CFS.


Assuntos
Síndrome de Fadiga Crônica/genética , Interleucina-17/genética , Linfócitos T Auxiliares-Indutores/imunologia , Adulto , Substituição de Aminoácidos/genética , Arginina/genética , Arginina/metabolismo , Síndrome de Fadiga Crônica/imunologia , Feminino , Frequência do Gene , Histidina/genética , Histidina/metabolismo , Humanos , Interleucina-17/imunologia , Masculino , Pessoa de Meia-Idade
7.
In Vivo ; 22(1): 115-21, 2008 Jan-Feb.
Artigo em Inglês | MEDLINE | ID: mdl-18396793

RESUMO

This study examined possible interactions between immunological abnormalities and symptoms in CFS. Sixteen CFS patients filled in a battery of questionnaires, evaluating daily functioning, and underwent venous blood sampling, in order to analyse immunological abnormalities. Ribonuclease (RNase) L cleavage was associated with RNase L activity (rs=0.570; p=0.021), protein kinase R (PKR) (rs=0.716; p=0.002) and elastase activity (rs=0.500; p=0.049). RNase L activity was related to elastase (rs=0.547; p=0.028) and PKR activity (rs=0.625; p=0.010). RNase L activity (rs=0.535; p=0.033), elastase activity (rs=0.585; p=0.017) and RNase L cleavage (rs=0.521; p=0.038) correlated with daily functioning. This study suggests that in CFS patients an increase in elastase activity and subsequent RNase L cleavage is accompanied by increased activity of both the PKR and RNase L enzymes. RNase L and elastase activity are related to daily functioning, thus evidence supporting the clinical importance of these immune dysfunctions in CFS patients was provided.


Assuntos
Endorribonucleases/metabolismo , Síndrome de Fadiga Crônica/enzimologia , Sistema Imunitário/metabolismo , Elastase Pancreática/metabolismo , eIF-2 Quinase/metabolismo , Adolescente , Adulto , Idoso , Avaliação da Deficiência , Síndrome de Fadiga Crônica/sangue , Síndrome de Fadiga Crônica/fisiopatologia , Feminino , Nível de Saúde , Humanos , Sistema Imunitário/fisiopatologia , Linfócitos/enzimologia , Masculino , Pessoa de Meia-Idade , Monócitos/enzimologia , Qualidade de Vida , Inquéritos e Questionários
8.
Expert Opin Ther Targets ; 12(3): 281-9, 2008 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-18269338

RESUMO

BACKGROUND: Evidence in support of intracellular immune dysfunctions in people with myalgic encephalomyelitis (ME)/chronic fatigue syndrome (CFS) is accumulating, but few studies have addressed intracellular immunity as a potential therapeutic target. OBJECTIVE: To provide an overview of our present understanding of intracellular immunity in ME/CFS, to relate the intracellular immune dysfunctions to other aspects of the illness like decreased natural killer cell function, the presence of infections and poor exercise performance, and to point to potential therapeutic targets. METHODS: An in-depth review of the scientific literature of intracellular immunity in people with ME/CFS was performed. RESULTS/CONCLUSION: From the scientific literature it is concluded that proteolytic cleavage of the native RNase L enzyme is characteristic of the dysregulation of intracellular immunity in people with ME/CFS, but the origin of the dysregulation is speculative. There is increasing evidence for immune cell apoptosis and upregulation of various aspects of the 2'-5' oligoadenylate (2-5A) synthetase/RNase L pathway in ME/CFS. This review provides the theoretical rationale for conducting studies examining the effectiveness of direct or indirect drug targeting of the 2-5A synthetase/RNase L pathway in ME/CFS patients.


Assuntos
Sistemas de Liberação de Medicamentos/tendências , Síndrome de Fadiga Crônica/enzimologia , Síndrome de Fadiga Crônica/imunologia , Líquido Intracelular/imunologia , Animais , Sistemas de Liberação de Medicamentos/métodos , Síndrome de Fadiga Crônica/tratamento farmacológico , Síndrome de Fadiga Crônica/patologia , Humanos , Imunidade Celular/efeitos dos fármacos , Líquido Intracelular/efeitos dos fármacos , Líquido Intracelular/enzimologia
9.
Life Sci ; 78(16): 1845-56, 2006 Mar 13.
Artigo em Inglês | MEDLINE | ID: mdl-16324719

RESUMO

The interferon-inducible, double-stranded (ds)RNA-dependent protein kinase (PKR) plays a major role in antiviral defense mechanisms where it down-regulates translation via phosphorylation of eukaryotic translation initiation factor 2alpha. PKR is also involved in the activation of nuclear factor kappaB (NFkappaB) through activation of the IkappaB kinase complex. Activation of PKR can occur in the absence of dsRNA and in such case is controlled by intracellular regulators like the PKR-activating protein (PACT), the PKR inhibitor p58(IPK), or heat-shock proteins (Hsp). These regulators are activated by stress stimuli, supporting a role for PKR in response to stress; however the final outcome of PKR activation in stress situations is unclear. We present here evidence that expression and activation of PKR contributes to an increased cellular resistance to mercury cytotoxicity. In two cell lines constitutively expressing PKR (THP-1 and Molt-3), treatment with the PKR inhibitor 2-aminopurine increases their sensitivity to mercury. In contrast, Ramos cells, which do not constitutively express PKR, present an increased resistance to mercury when PKR expression is induced by polyIC or interferon-beta treatment. This protective effect is inhibited by 2-aminopurine. We also show that exposure of Ramos cells to mercury leads to the induction of Hsp70. Treatment of cells with Hsp70 or NFkappaB inhibitors suppresses the PKR-dependent protection. We propose a model where PKR, modulated by Hsp70, activates a NFkappaB-mediated protective pathway. Because the cytotoxicity of mercury is primarily due to the generation of reactive oxygen species, our results suggest a more general function of PKR in the mechanisms of cellular response to oxidative stress.


Assuntos
Resistência a Medicamentos , Mercúrio/toxicidade , NF-kappa B/metabolismo , Estresse Oxidativo , eIF-2 Quinase/metabolismo , 2-Aminopurina/farmacologia , Linfócitos B/efeitos dos fármacos , Linfócitos B/enzimologia , Células Cultivadas , Citometria de Fluxo , Proteínas de Choque Térmico HSP70/biossíntese , Humanos , Interferon beta/farmacologia , Linfoma de Células B/tratamento farmacológico , Linfoma de Células B/enzimologia , Poli I-C/farmacologia , RNA Mensageiro/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Linfócitos T/efeitos dos fármacos , Linfócitos T/enzimologia
11.
Exp Mol Pathol ; 78(3): 239-46, 2005 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-15924878

RESUMO

A dysregulation in the 2',5'-oligoadenylate (2-5A)-dependent RNase L antiviral pathway has been detected in peripheral blood mononuclear cells (PBMC) of chronic fatigue syndrome (CFS) patients, which is characterized by upregulated 2-5A synthetase and RNase L activities, as well as by the presence of a low molecular weight (LMW) 2-5A-binding protein of 37-kDa related to RNase L. This truncated protein has been shown to originate from proteolytic cleavage of the native 83-kDa RNase L by m-calpain and human leukocyte elastase (HLE). We investigated the possible role of 2-5A oligomers in the proteolytic action toward the endonuclease and show that incubation of CFS PBMC extracts with 2-5A trimer and tetramer, but not with the dimer, results in a significant protection of the native 83-kDa RNase L against cleavage by endogenous and purified proteases. Similar results are obtained with a purified recombinant RNase L. An analysis of the size of 2-5A oligomers produced by the catalytic activity of the 2-5A synthetase present in PBMC extracts further shows that samples containing the 37-kDa RNase L preferentially produce 2-5A dimers instead of higher oligomers. Taken together, our results indicate that homodimerization of RNase L by 2-5A oligomers higher than the dimer prevents its cleavage by proteolytic enzymes. The presence of the truncated 37-kDa RNase L in PBMC extracts is therefore likely to result, not only from the abnormal activation of inflammatory proteases, but also from a dysregulation in 2-5A synthetase induction or activation towards the preferential production of 2-5A dimers.


Assuntos
Nucleotídeos de Adenina/metabolismo , Endorribonucleases/metabolismo , Síndrome de Fadiga Crônica/metabolismo , Leucócitos Mononucleares/enzimologia , Oligorribonucleotídeos/metabolismo , Dimerização , Eletroforese em Gel de Poliacrilamida , Ativação Enzimática/fisiologia , Humanos , Immunoblotting , Peptídeos/metabolismo , Proteínas Recombinantes/metabolismo , Especificidade por Substrato
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