RESUMO
Antimicrobial Peptides (AMPs) are immune effectors that are key components of the invertebrate innate immune system providing protection against pathogenic microbes. Parasitic helminths (phylum Nematoda and phylum Platyhelminthes) share complex interactions with their hosts and closely associated microbiota that are likely regulated by a diverse portfolio of antimicrobial immune effectors including AMPs. Knowledge of helminth AMPs has largely been derived from nematodes, whereas the flatworm AMP repertoire has not been described. This study highlights limitations in the homology-based approaches, used to identify putative nematode AMPs, for the characterisation of flatworm AMPs, and reveals that innovative algorithmic AMP prediction approaches provide an alternative strategy for novel helminth AMP discovery. The data presented here: (i) reveal that flatworms do not encode traditional lophotrochozoan AMP groups (Big Defensin, CSαß peptides and Myticalin); (ii) describe a unique integrated computational pipeline for the discovery of novel helminth AMPs; (iii) reveal >16,000 putative AMP-like peptides across 127 helminth species; (iv) highlight that cysteine-rich peptides dominate helminth AMP-like peptide profiles; (v) uncover eight novel helminth AMP-like peptides with diverse antibacterial activities, and (vi) demonstrate the detection of AMP-like peptides from Ascaris suum biofluid. These data represent a significant advance in our understanding of the putative helminth AMP repertoire and underscore a potential untapped source of antimicrobial diversity which may provide opportunities for the discovery of novel antimicrobials. Further, unravelling the role of endogenous worm-derived antimicrobials and their potential to influence host-worm-microbiome interactions may be exploited for the development of unique helminth control approaches.
Assuntos
Anti-Infecciosos , Nematoides , Animais , Peptídeos Catiônicos Antimicrobianos , AntibacterianosRESUMO
BACKGROUND: An early detection tool for EOC was constructed from analysis of biomarker expression data from serum collected during the UKCTOCS. METHODS: This study included 49 EOC cases (19 Type I and 30 Type II) and 31 controls, representing 482 serial samples spanning seven years pre-diagnosis. A logit model was trained by analysis of dysregulation of expression data of four putative biomarkers, (CA125, phosphatidylcholine-sterol acyltransferase, vitamin K-dependent protein Z and C-reactive protein); by scoring the specificity associated with dysregulation from the baseline expression for each individual. RESULTS: The model is discriminatory, passes k-fold and leave-one-out cross-validations and was further validated in a Type I EOC set. Samples were analysed as a simulated annual screening programme, the algorithm diagnosed cases with >30% PPV 1-2 years pre-diagnosis. For Type II cases (~80% were HGS) the algorithm classified 64% at 1 year and 28% at 2 years tDx as severe. CONCLUSIONS: The panel has the potential to diagnose EOC one-two years earlier than current diagnosis. This analysis provides a tangible worked example demonstrating the potential for development as a screening tool and scrutiny of its properties. Limits on interpretation imposed by the number of samples available are discussed.
Assuntos
Biomarcadores Tumorais/sangue , Proteínas Sanguíneas/análise , Proteína C-Reativa/análise , Antígeno Ca-125/sangue , Carcinoma Epitelial do Ovário/diagnóstico , Detecção Precoce de Câncer/métodos , Fosfatidilcolina-Esterol O-Aciltransferase/sangue , Algoritmos , Carcinoma Epitelial do Ovário/sangue , Feminino , Seguimentos , Humanos , Prognóstico , Estudos Prospectivos , Estudos RetrospectivosRESUMO
BACKGROUND: There is an urgent need for biomarkers for the early detection of ovarian cancer (OC). The purpose of this study was to assess whether changes in serum levels of lecithin-cholesterol acyltransferase (LCAT), sex hormone-binding globulin (SHBG), glucose-regulated protein, 78 kDa (GRP78), calprotectin and insulin-like growth factor-binding protein 2 (IGFBP2) are observed before clinical presentation and to assess the performance of these markers alone and in combination with CA125 for early detection. METHODS: This nested case-control study used samples from the United Kingdom Collaborative Trial of Ovarian Cancer Screening trial. The sample set consisted of 482 serum samples from 49 OC subjects and 31 controls, with serial samples spanning up to 7 years pre-diagnosis. The set was divided into the following: (I) a discovery set, which included all women with only two samples from each woman, the first at<14 months and the second at >32 months to diagnosis; and (ii) a corroboration set, which included all the serial samples from the same women spanning the 7-year period. Lecithin-cholesterol acyltransferase, SHBG, GRP78, calprotectin and IGFBP2 were measured using ELISA. The performance of the markers to detect cancers pre-diagnosis was assessed. RESULTS: A combined threshold model IGFBP2 >78.5 ng ml-1 : LCAT <8.831 µg ml-1 : CA125 >35 U ml-1 outperformed CA125 alone for the earlier detection of OC. The threshold model was able to identify the most aggressive Type II cancers. In addition, it increased the lead time by 5-6 months and identified 26% of Type I subjects and 13% of Type II subjects that were not identified by CA125 alone. CONCLUSIONS: Combined biomarker panels (IGFBP2, LCAT and CA125) outperformed CA125 up to 3 years pre-diagnosis, identifying cancers missed by CA125, providing increased diagnostic lead times for Type I and Type II OC. The model identified more aggressive Type II cancers, with women crossing the threshold dying earlier, indicating that these markers can improve on the sensitivity of CA125 alone for the early detection of OC.
Assuntos
Biomarcadores Tumorais/sangue , Antígeno Ca-125/sangue , Detecção Precoce de Câncer/métodos , Proteína 2 de Ligação a Fator de Crescimento Semelhante à Insulina/sangue , Neoplasias Ovarianas/sangue , Neoplasias Ovarianas/diagnóstico , Fosfatidilcolina-Esterol O-Aciltransferase/sangue , Estudos de Casos e Controles , Chaperona BiP do Retículo Endoplasmático , Feminino , Proteínas de Choque Térmico/sangue , Humanos , Complexo Antígeno L1 Leucocitário/sangue , Neoplasias Ovarianas/patologia , Globulina de Ligação a Hormônio Sexual/metabolismo , Fatores de TempoRESUMO
Kaposi's sarcoma-associated herpesvirus (KSHV) has been linked to the development of Kaposi's sarcoma, primary effusion lymphoma, and multicentric Castleman's disease (MCD). We have characterized the role of KSHV-encoded viral FLICE inhibitory protein (vFLIP) K13 in the modulation of anti-IgM-induced growth arrest and apoptosis in B cells. We demonstrate that K13 protects WEHI 231, an immature B-cell line, against anti-IgM-induced growth arrest and apoptosis. The protective effect of K13 was associated with the activation of the NF-κB pathway and was deficient in a mutant K13 with three alanine substitutions at positions 58 to 60 (K13-58AAA) and a structural homolog, vFLIP E8, both of which lack NF-κB activity. K13 upregulated the expression of NF-κB subunit RelB and blocked the anti-IgM-induced decline in c-Myc and rise in p27(Kip1) that have been associated with growth arrest and apoptosis. K13 also upregulated the expression of Mcl-1, an antiapoptotic member of the Bcl2 family. Finally, K13 protected the mature B-cell line Ramos against anti-IgM-induced apoptosis through NF-κB activation. Inhibition of anti-IgM-induced apoptosis by K13 may contribute to the development of KSHV-associated lymphoproliferative disorders.
Assuntos
Apoptose , Linfócitos B/imunologia , Linfócitos B/virologia , Herpesvirus Humano 8/patogenicidade , Interações Hospedeiro-Patógeno , Fator de Transcrição RelB/metabolismo , Proteínas Virais/metabolismo , Animais , Linhagem Celular , Camundongos , Proteína de Sequência 1 de Leucemia de Células Mieloides , NF-kappa B/metabolismo , Proteínas Proto-Oncogênicas c-bcl-2/metabolismoAssuntos
Regulação Neoplásica da Expressão Gênica , Síndromes Mielodisplásicas/genética , Proteínas Proto-Oncogênicas/biossíntese , Proteínas Repressoras/biossíntese , Transativadores/biossíntese , Antimetabólitos Antineoplásicos/farmacologia , Antimetabólitos Antineoplásicos/uso terapêutico , Azacitidina/farmacologia , Azacitidina/uso terapêutico , Biomarcadores , Biomarcadores Tumorais , Medula Óssea/metabolismo , Medula Óssea/patologia , Transformação Celular Neoplásica/genética , Metilação de DNA/efeitos dos fármacos , Progressão da Doença , Regulação para Baixo , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Humanos , Leucemia Mieloide Aguda/genética , Leucemia Mieloide Aguda/metabolismo , Síndromes Mielodisplásicas/tratamento farmacológico , Síndromes Mielodisplásicas/metabolismo , Síndromes Mielodisplásicas/fisiopatologia , Prognóstico , Proteínas Proto-Oncogênicas/genética , Proteínas Repressoras/genética , Transativadores/genética , Regulação para Cima/efeitos dos fármacosRESUMO
Clostridium difficile is a serious nosocomial pathogen whose prevalence worldwide is increasing. Postgenomic technologies can now be deployed to develop understanding of the evolution and diversity of this important human pathogen, yet little is known about the adaptive ability of C. difficile. We used iTRAQ labeling and 2D-LC-MS/MS driven proteomics to investigate the response of C. difficile 630 to a mild, but clinically relevant, heat stress. A statistically validated list of 447 proteins to which functional roles were assigned was generated, allowing reconstruction of central metabolic pathways including glycolysis, γ-aminobutyrate metabolism, and peptidoglycan biosynthesis. Some 49 proteins were significantly modulated under heat stress: classical heat shock proteins including GroEL, GroES, DnaK, Clp proteases, and HtpG were up-regulated in addition to several stress inducible rubrerythrins and proteins associated with protein modification, such as prolyl isomerases and proline racemase. The flagellar filament protein, FliC, was down-regulated, possibly as an energy conservation measure, as was the SecA1 preprotein translocase. The up-regulation of hydrogenases and various oxidoreductases suggests that electron flux across these pools of enzymes changes under heat stress. This work represents the first comparative proteomic analysis of the heat stress response in C. difficile strain 630, complementing the existing proteomics data sets and the single microarray comparative analysis of stress response. Thus we have a benchmark proteome for this pathogen, leading to a deeper understanding of its physiology and metabolism informed by the unique functional and adaptive processes used during a temperature upshift mimicking host pyrexia.
Assuntos
Proteínas de Bactérias/análise , Clostridioides difficile/metabolismo , Proteínas de Choque Térmico/análise , Resposta ao Choque Térmico/fisiologia , Proteômica/métodos , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Clostridioides difficile/química , Proteínas de Choque Térmico/genética , Proteínas de Choque Térmico/metabolismo , Reprodutibilidade dos Testes , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Espectrometria de Massas em TandemRESUMO
Murine models are amongst the most widely used systems to study biology and pathology. Targeted quantitative proteomic analysis is a relatively new tool to interrogate such systems. Recently the need for relative quantification on hundreds to thousands of samples has driven the development of Data Independent Acquisition methods. One such technique is SWATH-MS, which in the main requires prior acquisition of mass spectra to generate an assay reference library. In stem cell research, it has been shown pluripotency can be induced starting with a fibroblast population. In so doing major changes in expressed proteins is inevitable. Here we have created a reference library to underpin such studies. This is inclusive of an extensively documented script to enable replication of library generation from the raw data. The documented script facilitates reuse of data and adaptation of the library to novel applications. The resulting library provides deep coverage of the mouse proteome. The library covers 29519 proteins (53% of the proteome) of which 7435 (13%) are supported by a proteotypic peptide.
Assuntos
Reprogramação Celular , Bases de Dados de Proteínas , Camundongos , Proteoma , Animais , Espectrometria de Massas/métodos , Camundongos/genética , Camundongos/metabolismo , Camundongos/fisiologia , Análise Serial de Proteínas/métodos , Proteômica/métodosRESUMO
There are few good sources of natural food folates apart from green leafy vegetables and these may have a limited potential to increase folate status because of substantial losses that can occur during cooking. Fortified foods can overcome this but are controversial because of safety concerns regarding chronic exposure to high-dose folic acid (FA; the synthetic form). The aim of the present study was to develop eggs with an enriched natural folate content and minimal unmetabolised FA. Forty-eight, 30-week-old laying hens were randomised to receive the basal feed (formulated to provide 1 mg folate/kg feed) to which had been added one of the following FA levels (0, 2, 4, 8, 16, 32 mg/kg feed). Total folate was measured in eggs collected throughout the 12-week study period and the FA content estimated at 12 weeks. Results showed that the maximal egg folate content was achieved by adding 16 mg FA/kg feed. At this optimal dose, the total folate content per egg was 75 microg (compared with 32 microg in a regular egg) of which FA represented at most 10%, a level which would probably be converted into natural folates by humans after ingestion. The results demonstrate that it is possible to use synthetic FA at high doses to produce novel animal foods enriched with natural folates in a cost-efficient process. Such foods may be particularly relevant to European populations without access to FA fortification and therefore dependent on natural food folate sources for the primary prevention of folate-related disease.
Assuntos
Ração Animal , Galinhas/metabolismo , Ovos/análise , Ácido Fólico/metabolismo , Animais , Relação Dose-Resposta a Droga , Feminino , Ácido Fólico/administração & dosagem , Ácido Fólico/análise , Distribuição Aleatória , Fatores de TempoRESUMO
The etiology of thrombocytopenia is important in treatment and management of the condition. Most platelet parameters that are routinely analyzed in the diagnostic laboratory have not proven useful in identifying the etiology, while specialized assays suffer from poor standardization and lack of agreement between laboratories. The immature platelet fraction (IPF), which indirectly provides a measure of bone marrow function, is showing promise as a valuable marker of thrombopoietic responses. This study set out determine whether the IPF could effectively identify specific underlying etiologies of thrombocytopenia in a large thrombocytopenic cohort, to allow for quicker, more effective management of the condition. The IPF was analyzed in a large cohort of 637 thrombocytopenic patients and 171 healthy control patients on the Sysmex XN 10 hematology analyzer using the specialized fluorescence optical analysis. The thrombocytopenic patients were divided into six cohorts based on etiology. The IPF% was significantly higher in cases of increased platelet consumption (medianâ¯=â¯9.55, minâ¯=â¯1.1, maxâ¯=â¯77.9) or pseudothrombocytopenia (medianâ¯=â¯13.1, minâ¯=â¯0.4, maxâ¯=â¯28.8) compared with control (medianâ¯=â¯4.2, minâ¯=â¯1.3, maxâ¯=â¯12.8). Furthermore, the IPF% was also able to identify idiopathic thrombocytopenic purpura (ITP) (p < 0.05) (medianâ¯=â¯13.4, minâ¯=â¯2.8, maxâ¯=â¯77.9) from other causes of increased platelet consumptive disorders (infection: medianâ¯=â¯6.4, minâ¯=â¯1.1, maxâ¯=â¯21.6; hemorrhage: medianâ¯=â¯8.9, minâ¯=â¯1.2, maxâ¯=â¯20.2). By use of this large thrombocytopenic cohort, the IPF% has been found to be of significant diagnostic value, providing a useful rapid test in the etiological investigation of platelet disorders.
Assuntos
Plaquetas , Trombocitopenia , Adulto , Idoso , Idoso de 80 Anos ou mais , Biomarcadores/sangue , Plaquetas/metabolismo , Plaquetas/patologia , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Trombocitopenia/sangue , Trombocitopenia/diagnóstico , Trombocitopenia/patologiaRESUMO
Essentials The BCR-ABL negative myeloproliferative neoplasms are subjected to unknown phenotypic modifiers. GATA-1 is upregulated in ET patients, regardless of treatment regimen or mutational status. Myelofibrosis (MF) megakaryocytes displayed decreased GATA-1 staining. GATA-1 may have utility as a diagnostic marker in ET and in its differential diagnosis from MF. ABSTRACT: Background The BCR-ABL-negative myeloproliferative neoplasms, i.e., polycythemia vera, essential thrombocythemia (ET), and myelofibrosis (MF), are characterized by mutations in JAK2, CALR, or MPL. However, an as yet unknown factor drives the precise disease phenotype. The hematopoietic transcription factor GATA-1 and its downstream targets NFE2 and FLI1 are responsible for determining erythroid and megakaryocyte lineages during hematopoietic stem cell differentiation. Previous studies have demonstrated a low level of GATA-1 expression in megakaryocytes from patients with MF. Objectives and methods The expression of GATA-1, NFE2 and FLI1 was studied for changes in the peripheral blood (PB) of ET patients. Peripheral blood samples were obtained from 36 ET patients, 14 MF patients, and seven healthy control donors. Total RNA from PB mononuclear cells (PBMCs) was extracted, and quantitative polymerase chain reaction was used to determine relative changes in gene expression. Protein levels of GATA-1 were also determined in bone marrow sections from ET and MF patients. Results GATA-1 mRNA was upregulated in ET patients, regardless of treatment regimen or mutational status. FLI1 expression was significantly downregulated, whereas NFE2 expression was unaffected by changes in GATA-1 mRNA levels. Megakaryocytes from ET patients showed increased protein levels of GATA-1 as compared with those from MF patients. Conclusions Our results confirmed, in PB, our previous data demonstrating elevated levels of GATA-1 mRNA in total bone marrow of ET patients. GATA-1 mRNA levels are independent of cytoreductive therapies, and may have utility as a diagnostic marker in ET and in its differential diagnosis from MF.
Assuntos
Fator de Transcrição GATA1/metabolismo , Mielofibrose Primária/diagnóstico , Trombocitemia Essencial/diagnóstico , Adulto , Idoso , Idoso de 80 Anos ou mais , Biomarcadores/metabolismo , Medula Óssea/metabolismo , Estudos de Casos e Controles , Diagnóstico Diferencial , Feminino , Fator de Transcrição GATA1/genética , Expressão Gênica , Humanos , Masculino , Pessoa de Meia-Idade , Subunidade p45 do Fator de Transcrição NF-E2/genética , Mielofibrose Primária/genética , Mielofibrose Primária/metabolismo , Proteína Proto-Oncogênica c-fli-1/genética , RNA Mensageiro/sangue , RNA Mensageiro/genética , Trombocitemia Essencial/genética , Trombocitemia Essencial/metabolismoRESUMO
Research into snake venom components has intensified over the last number of decades, particularly that work directed towards the discovery of novel agents with potential applications in clinical therapy. In the present study we report, for the first time, defined patterns observed in the G-50 chromatographic elution profiles from 30 snake venoms taken from Elapidae and Viperidae families, as well as previously unreported patterns within subfamilies of these snake species. Development of this chromatographic technique thus offers a rapid method for the general classification of snakes within these families as well as providing insights into hitherto uncharacterised trends within the venoms of snake subfamilies that have opened new avenues for further investigation.
Assuntos
Cromatografia em Gel , Elapidae/classificação , Venenos de Serpentes/química , Viperidae/classificação , Animais , FilogeniaRESUMO
It is now more than 10 years since the publication of the first microbial genome sequence and science is now moving towards a post genomic era with transcriptomics and proteomics offering insights into cellular processes and function. The ability to assess the entire protein network of a cell at a given spatial or temporal point will have a profound effect upon microbial science as the function of proteins is inextricably linked to phenotype. Whilst such a situation is still beyond current technologies rapid advances in mass spectrometry, bioinformatics and protein separation technologies have produced a step change in our current proteomic capabilities. Subsequently a small, but steadily growing, number of groups are taking advantage of this cutting edge technology to discover more about the physiology and metabolism of microorganisms. From this research it will be possible to move towards a systems biology understanding of a microorganism. Where upon researchers can build a comprehensive cellular map for each microorganism that links an accurately annotated genome sequence to gene expression data, at a transcriptomic and proteomic level.In order for microbiologists to embrace the potential that proteomics offers, an understanding of a variety of analytical tools is required. The aim of this review is to provide a basic overview of mass spectrometry (MS) and its application to protein identification. In addition we will describe how the protein complexity of microbial samples can be reduced by gel-based and gel-free methodologies prior to analysis by MS. Finally in order to illustrate the power of microbial proteomics a case study of its current application within the Bacilliaceae is given together with a description of the emerging discipline of metaproteomics.
RESUMO
PURPOSE: Ovarian cancer (OC) is the most lethal gynaecological cancer. Early detection is required to improve patient survival. Risk estimation models were constructed for Type I (Model I) and Type II (Model II) OC from analysis of Protein Z, Fibronectin, C-reactive protein and CA125 levels in prospectively collected samples from the United Kingdom Collaborative Trial of Ovarian Cancer Screening (UKCTOCS). RESULTS: Model I identifies cancers earlier than CA125 alone, with a potential lead time of 3-4 years. Model II detects a number of high grade serous cancers at an earlier stage (Stage I/II) than CA125 alone, with a potential lead time of 2-3 years and assigns high risk to patients that the ROCA Algorithm classified as normal. MATERIALS AND METHODS: This nested case control study included 418 individual serum samples serially collected from 49 OC cases and 31 controls up to six years pre-diagnosis. Discriminatory logit models were built combining the ELISA results for candidate proteins with CA125 levels. CONCLUSIONS: These models have encouraging sensitivities for detecting pre-clinical ovarian cancer, demonstrating improved sensitivity compared to CA125 alone. In addition we demonstrate how the models improve on ROCA for some cases and outline their potential future use as clinical tools.
Assuntos
Modelos Estatísticos , Neoplasias Ovarianas/epidemiologia , Algoritmos , Biomarcadores Tumorais , Detecção Precoce de Câncer/métodos , Fatores Epidemiológicos , Feminino , Humanos , Programas de Rastreamento , Estadiamento de Neoplasias , Neoplasias Ovarianas/diagnóstico , Curva ROC , Reprodutibilidade dos Testes , RiscoRESUMO
Seven novel peptides were isolated from the skin secretions of the North American dusky gopher frog, Rana sevosa, on the basis of antimicrobial activity and histamine release from rat peritoneal mast cells. The peptides were purified to homogeneity using HPLC and characterized by electrospray ion-trap mass spectrometry, MALDI-TOF mass spectrometry and Edman sequencing. Bioinformatic analysis of primary structures revealed that the novel peptides could be assigned to four established families of ranid frog antimicrobial peptides, namely esculentin-1, esculentin-2, brevinin-1 and ranatuerin-2. The peptides were named in accordance with accepted terminology as ranatuerin 2SEa, etc., reflecting the peptide family name, the species of origin (SE for sevosa) and the isotype (a). Of major interest was the fact that brevinin 1SE displayed significant structural similarity to ponericin W5, an antibacterial venom peptide from the ant, Pachyconyla goeldii. This is a further example of amphibian skin defensive peptides showing striking structural similarities to peptides from insects. These data may shed some light on the functional biological relevance of defensive peptides that possess both antimicrobial and histamine-releasing activities.
Assuntos
Peptídeos Catiônicos Antimicrobianos/química , Histamina/química , Pele/metabolismo , Animais , Formigas , Cromatografia Líquida de Alta Pressão , Feminino , Masculino , Peptídeos/química , Ranidae , Ratos , Espectrometria de Massas por Ionização por Electrospray , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por MatrizRESUMO
An octadecapeptide was isolated from the skin secretions of the dusky gopher frog (Rana sevosa) on the basis of histamine release from rat peritoneal mast cells. This peptide was purified to homogeneity by HPLC and found to have the following primary structure, YLKGCWTKSYPPKPCFSR, using both Edman degradation chemistry and peptide sequencing using high-resolution mass spectrometry (Q-TOF MS). The peptide, named peptide Tyrosine Arginine (pYR) shares 77.8% homology with peptide Leucine Arginine (pLR). The effects of the natural amidated peptide, non-amidated peptide and C-loop region of pYR on granulopoiesis and neutrophil apoptosis were investigated. All three analogues inhibited the early development of granulocyte macrophage colonies from bone marrow stem cells but did not induce apoptosis of the end stage granulocytes, the mature neutrophil. Thus, pYR is a novel member of an important and emerging new class of amphibian peptides with hemopoietic actions.
Assuntos
Proteínas de Anfíbios/química , Proteínas de Anfíbios/farmacologia , Fatores Imunológicos/química , Fatores Imunológicos/farmacologia , Sequência de Aminoácidos , Proteínas de Anfíbios/isolamento & purificação , Animais , Ensaio de Unidades Formadoras de Colônias , Granulócitos/efeitos dos fármacos , Liberação de Histamina/efeitos dos fármacos , Fatores Imunológicos/isolamento & purificação , Macrófagos/efeitos dos fármacos , Masculino , Mastócitos/efeitos dos fármacos , Dados de Sequência Molecular , Peptídeos/química , Peptídeos/isolamento & purificação , Ranidae/metabolismo , Ratos , Pele/metabolismoRESUMO
Peptides in the skin secretion of frogs have been studied for some time now because they frequently possess important biological activity such as antibiotic, antimicrobial, or anticancer properties. In this paper, we present a computational approach for measuring the degree of similarity between the entire peptide complement of the skin secretion of specimens from the same or different species. The first step in the analysis is the generation of a mass spectral profile from an experimental high-performance liquid chromatography/electrosparay ionization analysis of the sample. An "overlap" between the mass spectral profiles of different specimens is then proposed as a measure of their similarity. Analysis of specimens from three species of the genus Litoria, viz., L. Aurea, L. Caerulea, and L. Infrafrenata, and Rana Capito of genus Rana shows that the degree of similarity is highest between specimens from the same species, lower for specimens from different species of the same genus, and lowest between specimens from different genera. This indicates that comparison of skin peptide profiles (i.e., mass spectral profiles of skin secretion) is potentially a useful aid in the taxonomic study of amphibian species.
Assuntos
Algoritmos , Anuros/classificação , Anuros/metabolismo , Perfilação da Expressão Gênica/métodos , Mapeamento de Peptídeos/métodos , Proteoma/metabolismo , Pele/metabolismo , Proteínas de Anfíbios/metabolismo , Animais , Classificação/métodos , Cromatografia Gasosa-Espectrometria de Massas/métodos , Especificidade da EspécieRESUMO
BACKGROUND: Kaposi's sarcoma associated herpesvirus encoded viral FLICE inhibitory protein (vFLIP) K13 activates the NF-κB pathway by binding to the NEMO/IKKγ subunit of the IκB kinase (IKK) complex. However, it has remained enigmatic how K13-NEMO interaction results in the activation of the IKK complex. Recent studies have implicated TRAF6, TAK1 and linear ubiquitin chains assembled by a linear ubiquitin chain assembly complex (LUBAC) consisting of HOIL-1, HOIP and SHARPIN in IKK activation by proinflammatory cytokines. METHODOLOGY/PRINCIPAL FINDINGS: Here we demonstrate that K13-induced NF-κB DNA binding and transcriptional activities are not impaired in cells derived from mice with targeted disruption of TRAF6, TAK1 and HOIL-1 genes and in cells derived from mice with chronic proliferative dermatitis (cpdm), which have mutation in the Sharpin gene (Sharpin(cpdm/cpdm)). Furthermore, reconstitution of NEMO-deficient murine embryonic fibroblast cells with NEMO mutants that are incapable of binding to linear ubiquitin chains supported K13-induced NF-κB activity. K13-induced NF-κB activity was not blocked by CYLD, a deubiquitylating enzyme that can cleave linear and Lys63-linked ubiquitin chains. On the other hand, NEMO was required for interaction of K13 with IKK1/IKKα and IKK2/IKKß, which resulted in their activation by "T Loop" phosphorylation. CONCLUSIONS/SIGNIFICANCE: Our results demonstrate that K13 activates the NF-κB pathway by binding to NEMO which results in the recruitment of IKK1/IKKα and IKK2/IKKß and their subsequent activation by phosphorylation. Thus, K13 activates NF-κB via a mechanism distinct from that utilized by inflammatory cytokines. These results have important implications for the development of therapeutic agents targeting K13-induced NF-κB for the treatment of KSHV-associated malignancies.
Assuntos
Herpesvirus Humano 8/metabolismo , MAP Quinase Quinase Quinases/metabolismo , Complexos Multiproteicos/metabolismo , NF-kappa B/metabolismo , Sarcoma de Kaposi/metabolismo , Fator 6 Associado a Receptor de TNF/metabolismo , Proteínas Virais/metabolismo , Animais , Doença Crônica , Dermatite/genética , Dermatite/metabolismo , Dermatite/patologia , Embrião de Mamíferos/metabolismo , Embrião de Mamíferos/patologia , Fibroblastos/metabolismo , Fibroblastos/patologia , Herpesvirus Humano 8/genética , Humanos , Quinase I-kappa B/genética , Quinase I-kappa B/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular/genética , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Células Jurkat , MAP Quinase Quinase Quinases/genética , Camundongos , Camundongos Knockout , Complexos Multiproteicos/genética , NF-kappa B/genética , Sarcoma de Kaposi/genética , Sarcoma de Kaposi/patologia , Fator 6 Associado a Receptor de TNF/genética , Proteínas Virais/genéticaRESUMO
Mass spectrometry based proteomics is now widely used in the microbial sciences. In conjunction with transcriptomics it has greatly enhanced the field of microbial biology and has provide microbiologists with unparalleled insights into cellular processes and functions. Proteomics allows the dynamic nature of the entire protein network to be mapped providing a deeper understanding of microbial systems, their evolution and role in disease states. This review is intended to provide an overview of mass spectrometry and its application to the field of microbial proteomics. Background is provided on the core mass analyzers, including the Orbitrap mass spectrometer, and novel fragmentation processes such as Electron Transfer Dissociation which leave post-translational modifications intact on peptide backbones allowing for their identification and localization. The review will also provide information on current key quantitative technologies and the state of the art in microbial metaproteomics.
Assuntos
Espectrometria de Massas/métodos , Microbiologia , Proteômica/métodos , Modelos TeóricosRESUMO
OBJECTIVE: Fusion genes involving the platelet-derived growth factor receptor-beta (PDGFRbeta) are found in a subgroup of myeloproliferative neoplasms, with one such fusion, Tel/PDGFRbeta found in a subset of chronic myelomonocytic leukemia patients. Tel/PDGFRbeta results in constitutive activation of several signaling pathways and induces a myeloproliferative disease in mice, with signals via tyrosines 579/581 identified as being important for this phenotype. In this study, we have used a tetracycline-regulated system to express wild-type and the mutated F2 Tel/PDGFRbeta to identify the key signaling pathways, which drive Tel/PDGFRbeta-induced differentiation of embryonic stem (ES) cells. MATERIALS AND METHODS: The leukemic oncogene Tel/PDGFRbeta and Tel/PDGFRbeta-F2 were inducibly expressed in ES cells and their effects on self-renewal, signal transduction, and gene expression patterns analyzed. RESULTS: Tel/PDGFRbeta activated several major signal transduction pathways (signal transducers and activators of transcription [STAT] 3, STAT5, mitogen-activated protein kinases, phosphatidylinositol-3 kinase) in ES cells, but only specific inhibition of the mitogen-activated protein kinase kinase/extracellular regulated kinase (MEK/ERK) or STAT5 pathways was able to significantly prevent Tel/PDGFRbeta-induced differentiation and restore ES-cell self-renewal. Inhibiting the tyrosine kinase activity of the oncogene using Gleevec or PDGFRbeta inhibitor III also substantially prevented Tel/PDGFRbeta-induced differentiation and its ability to upregulate key genes involved in myelopoiesis. Tyrosines 579/581 played a critical role in mediating signals via the Ras/ERK and STAT5 pathways, with dual targeting of the tyrosine kinase activity of Tel/PDGFRbeta and the MEK/ERK pathway completely preventing Tel/PDGFRbeta-induced differentiation. CONCLUSION: These findings suggest that targeted disruption of key signaling pathways in combination with the tyrosine kinase activity of leukemic oncogenes, such as Tel/PDGFRbeta, may result in more efficacious therapies for suppressing leukemic progression in the clinical setting.
Assuntos
Diferenciação Celular , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Sistema de Sinalização das MAP Quinases , Células-Tronco Neoplásicas/metabolismo , Proteínas de Fusão Oncogênica/metabolismo , Proteínas Proto-Oncogênicas c-ets/metabolismo , Receptor beta de Fator de Crescimento Derivado de Plaquetas/metabolismo , Proteínas Repressoras/metabolismo , Fator de Transcrição STAT5/metabolismo , Proteínas ras/metabolismo , Animais , Diferenciação Celular/genética , Linhagem Celular , MAP Quinases Reguladas por Sinal Extracelular/genética , Humanos , Leucemia Mielogênica Crônica BCR-ABL Positiva/metabolismo , Leucemia Mielogênica Crônica BCR-ABL Positiva/patologia , Camundongos , Células-Tronco Neoplásicas/patologia , Proteínas de Fusão Oncogênica/genética , Fosfatidilinositol 3-Quinases/genética , Fosfatidilinositol 3-Quinases/metabolismo , Proteínas Proto-Oncogênicas c-ets/genética , Receptor beta de Fator de Crescimento Derivado de Plaquetas/genética , Proteínas Repressoras/genética , Fator de Transcrição STAT3/genética , Fator de Transcrição STAT3/metabolismo , Fator de Transcrição STAT5/genética , Proteínas ras/genética , Variante 6 da Proteína do Fator de Translocação ETSRESUMO
Given the key role of Zn in many physiological functions, optimal Zn status could be a predictive parameter of successful ageing. However, the benefit of Zn supplementation is still a matter of debate since Zn supplementation has been reported to be associated with the alteration of Cu status and lipid metabolism. As part of the Zenith Project, the present study aimed to investigate, in free-living healthy European middle-aged and older subjects, the effect of Zn supplementation on the biochemical status of Zn, Fe and Cu and on lipid profile. Volunteers aged 55-70 (n 188) and 70-85 (n 199) years old participated in a double-blinded, randomised study and received a daily placebo, or Zn as 15 or 30 mg for 6 months. Zn supplementation did not significantly modify erythrocyte Zn levels or erythrocyte Cu,Zn-superoxide dismutase activity. But Zn supplementation at 15 or 30 mg/d for 6 months increased significantly serum Zn levels and Zn urinary excretion with no major adverse effects on Fe and Cu status or on lipid metabolism. However, Zn supplementation at 30 mg/d showed some age- and sex-dependent alterations in Fe status or lipid profile. Therefore, with respect to the key role of an optimal Zn status in successful ageing, Zn supplementation at 15 mg/d, when necessary, could be safely proposed regarding lipids and the risk of interaction with Fe and Cu.