RESUMEN
Enhancing the accumulation and retention of small-molecule probes in tumors is an important way to achieve accurate cancer diagnosis and therapy. Enzyme-stimulated macrocyclization of small molecules possesses great potential for enhanced positron emission tomography (PET) imaging of tumors. Herein, we reported an 18F-labeled radiotracer [18F]AlF-RSM for legumain detection in vivo. The tracer was prepared by a one-step aluminum-fluoride-restrained complexing agent ([18F]AlF-RESCA) method with high radiochemical yield (RCY) (88.35 ± 3.93%) and radiochemical purity (RCP) (>95%). More notably, the tracer can be transformed into a hydrophobic macrocyclic molecule under the joint action of legumain and reductant. Simultaneously, the tracer could target legumain-positive tumors and enhance accumulation and retention in tumors, resulting in the amplification of PET imaging signals. The enhancement of radioactivity enables PET imaging of legumain activity with high specificity. We envision that, by combining this highly efficient 18F-labeled strategy with our intramolecular macrocyclization reaction, a range of radiofluorinated tracers can be designed for tumor PET imaging and early cancer diagnosis in the future.
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Cisteína Endopeptidasas , Radioisótopos de Flúor , Tomografía de Emisión de Positrones , Tomografía de Emisión de Positrones/métodos , Radioisótopos de Flúor/química , Cisteína Endopeptidasas/metabolismo , Cisteína Endopeptidasas/análisis , Animales , Ciclización , Ratones , Humanos , Radiofármacos/química , Línea Celular Tumoral , Ratones Endogámicos BALB C , Fluoruros/química , Ratones DesnudosRESUMEN
BACKGROUND: Bleomycin hydrolase (BH), which is expressed in the stratum granulosum and lower stratum corneum (SC), is involved in final filaggrin degradation. Furthermore, BH plays an essential role in producing free amino acids, which constitute the majority of natural moisturizing factors (NMF). However, the effects of BH expression and protease activity on human skin aging remain unclear. OBJECTIVE: This study was designed to evaluate the activity and expression patterns of BH in SC extracts from healthy young and elderly individuals. METHODS: SC samples were collected by tape stripping. BH activity was assessed by measuring the citrulline aminopeptidase activity. BH expression was determined by Western blotting, and NMF was quantified by liquid chromatography/mass spectrometry. Skin barrier function was determined by measuring SC hydration, transepidermal water loss (TEWL), and skin pH. RESULTS: The activity and expression of BH were higher in the elderly skin than in young skin, and BH activity was correlated with BH expression levels. Evaluation of the NMF showed that the levels of total amino acids, such as glycine, serine, aspartic acid, citrulline, pyrrolidone carboxylic acid (a metabolite of glutamic acid), and trans-urocanic acid (a metabolite of histidine), were significantly higher in elderly skin than in young skin. Moreover, SC hydration and TEWL were significantly lower in elderly, indicating dry skin, and pH was significantly higher in elderly, indicating greater skin alkalinization. CONCLUSION: These results suggest that BH activity and expression, as well as NMF amino acids, increase in elderly people as compensatory mechanisms against dry skin.
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Citrulina , Piel , Humanos , Anciano , Citrulina/análisis , Citrulina/metabolismo , Citrulina/farmacología , Piel/metabolismo , Cisteína Endopeptidasas/análisis , Epidermis/metabolismo , Agua/análisisRESUMEN
Legumains, also known as asparaginyl endopeptidases (AEPs), cleave peptide bonds after Asn/Asp (Asx) residues. In plants, certain legumains also have ligase activity that catalyzes biosynthesis of Asx-containing cyclic peptides. An example is the biosynthesis of MCoTI-I/II, a squash family-derived cyclic trypsin inhibitor, which involves splicing to remove the N-terminal prodomain and then N-to-C-terminal cyclization of the mature domain. To identify plant legumains responsible for the maturation of these cyclic peptides, we have isolated and characterized a legumain involved in splicing, McPAL1, from Momordica cochinchinensis (Cucurbitaceae) seeds. Functional studies show that recombinantly expressed McPAL1 displays a pH-dependent, trimodal enzymatic profile. At pH 4 to 6, McPAL1 selectively catalyzed Asp-ligation and Asn-cleavage, but at pH 6.5 to 8, Asn-ligation predominated. With peptide substrates containing N-terminal Asn and C-terminal Asp, such as is found in precursors of MCoTI-I/II, McPAL1 mediates proteolysis at the Asn site and then ligation at the Asp site at pH 5 to 6. Also, McPAL1 is an unusually stable legumain that is tolerant of heat and high pH. Together, our results support that McPAL1 is a splicing legumain at acidic pH that can mediate biosynthesis of MCoTI-I/II. We purport that the high thermal and pH stability of McPAL1 could have applications for protein engineering.
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Cisteína Endopeptidasas/metabolismo , Momordica/metabolismo , Proteínas de Plantas/metabolismo , Secuencia de Aminoácidos , Ciclización , Ciclotidas/genética , Ciclotidas/metabolismo , Cisteína Endopeptidasas/análisis , Cisteína Endopeptidasas/genética , Modelos Moleculares , Momordica/química , Momordica/genética , Péptidos Cíclicos/genética , Péptidos Cíclicos/metabolismo , Proteínas de Plantas/análisis , Proteínas de Plantas/genética , Ingeniería de Proteínas , TranscriptomaAsunto(s)
Cisteína Endopeptidasas/metabolismo , Dermatitis Atópica/patología , Epidermis/enzimología , Estaciones del Año , Adolescente , Estudios de Casos y Controles , Niño , Preescolar , Cisteína Endopeptidasas/análisis , Epidermis/patología , Femenino , Voluntarios Sanos , Humanos , Japón , Masculino , Índice de Severidad de la EnfermedadRESUMEN
Two pathophysiological different experimental models for multiple sclerosis were analyzed in parallel using quantitative proteomics in attempts to discover protein alterations applicable as diagnostic-, prognostic-, or treatment targets in human disease. The cuprizone model reflects de- and remyelination in multiple sclerosis, and the experimental autoimmune encephalomyelitis (EAE, MOG1-125) immune-mediated events. The frontal cortex, peripheral to severely inflicted areas in the CNS, was dissected and analyzed. The frontal cortex had previously not been characterized by proteomics at different disease stages, and novel protein alterations involved in protecting healthy tissue and assisting repair of inflicted areas might be discovered. Using TMT-labelling and mass spectrometry, 1871 of the proteins quantified overlapped between the two experimental models, and the fold change compared to controls was verified using label-free proteomics. Few similarities in frontal cortex between the two disease models were observed when regulated proteins and signaling pathways were compared. Legumain and C1Q complement proteins were among the most upregulated proteins in cuprizone and hemopexin in the EAE model. Immunohistochemistry showed that legumain expression in post-mortem multiple sclerosis brain tissue (n = 19) was significantly higher in the center and at the edge of white matter active and chronic active lesions. Legumain was associated with increased lesion activity and might be valuable as a drug target using specific inhibitors as already suggested for Parkinson's and Alzheimer's disease. Cerebrospinal fluid levels of legumain, C1q and hemopexin were not significantly different between multiple sclerosis patients, other neurological diseases, or healthy controls.
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Encefalomielitis Autoinmune Experimental/diagnóstico , Lóbulo Frontal/patología , Esclerosis Múltiple/diagnóstico , Adulto , Anciano , Anciano de 80 o más Años , Animales , Biomarcadores/análisis , Biomarcadores/metabolismo , Complemento C1q/análisis , Complemento C1q/metabolismo , Cuprizona/administración & dosificación , Cuprizona/toxicidad , Cisteína Endopeptidasas/análisis , Cisteína Endopeptidasas/metabolismo , Encefalomielitis Autoinmune Experimental/inducido químicamente , Encefalomielitis Autoinmune Experimental/inmunología , Encefalomielitis Autoinmune Experimental/patología , Femenino , Lóbulo Frontal/efectos de los fármacos , Lóbulo Frontal/inmunología , Regulación de la Expresión Génica/inmunología , Hemopexina/análisis , Hemopexina/metabolismo , Humanos , Inmunohistoquímica , Masculino , Ratones , Persona de Mediana Edad , Esclerosis Múltiple/inducido químicamente , Esclerosis Múltiple/inmunología , Esclerosis Múltiple/patología , Proteómica , Adulto JovenRESUMEN
BACKGROUND: Skin ageing is associated with structure-functional changes in the extracellular matrix, which is in part caused by proteolytic degradation. Since cysteine cathepsins are major matrix protein-degrading proteases, we investigated the age-dependent expression of elastolytic cathepsins K, S, and V in human skin, their in vitro impact on the integrity of the elastic fibre network, their cleavage specificities, and the release of bioactive peptides. METHODS: Cathepsin-mediated degradation of human skin elastin samples was assessed from young to very old human donors using immunohistochemical and biochemical assays, scanning electron microscopy, and mass spectrometry. RESULTS: Elastin samples derived from patients between 10 and 86â¯years of age were analysed and showed an age-dependent deterioration of the fibre structure from a dense network of thinner fibrils into a beaded and porous mesh. Reduced levels of cathepsins K, S, and V were observed in aged skin with a predominant epidermal expression. Cathepsin V was the most potent elastase followed by cathepsin K and S. Biomechanical analysis of degraded elastin fibres corroborated the destructive activity of cathepsins. Mass spectrometric determination of the cleavage sites in elastin revealed that all three cathepsins predominantly cleaved in hydrophobic domains. The degradation of elastin was efficiently inhibited by an ectosteric inhibitor. Furthermore, the degradation of elastin fibres resulted in the release of bioactive peptides, which have previously been associated with various pathologies. CONCLUSION: Cathepsins are powerful elastin-degrading enzymes and capable of generating a multitude of elastokines. They may represent a viable target for intervention strategies to reduce skin ageing.
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Catepsina K/metabolismo , Catepsinas/metabolismo , Cisteína Endopeptidasas/metabolismo , Elastina/metabolismo , Envejecimiento de la Piel , Piel/metabolismo , Adolescente , Adulto , Anciano , Anciano de 80 o más Años , Catepsina K/análisis , Catepsinas/análisis , Niño , Cisteína Endopeptidasas/análisis , Elastina/análisis , Elastina/ultraestructura , Femenino , Humanos , Persona de Mediana Edad , Proteolisis , Adulto JovenRESUMEN
AIMS: Cathepsin V (CTSV/CTSL2) is a lysosomal cysteine proteinase and plays a role in extracellular matrix degradation. It is associated with poor prognosis in invasive breast cancer (IBC), but its role in breast ductal carcinoma in situ (DCIS) remains unclear. In this study, we aimed to evaluate the prognostic significance of CTSV in DCIS. METHODS: CTSV protein expression was immunohistochemically assessed in a well-characterised and annotated cohort of DCIS comprising pure DCIS (n=776) and DCIS coexisting with IBC (n=239). CTSV expression was analysed in tumour cells and surrounding stroma, including its association with clinicopathological parameters and outcome. RESULTS: In pure DCIS, high CTSV expression was observed in 29% of epithelial tumour cells and 20% of surrounding stroma. High expression in both components was associated with features of poor prognosis including higher nuclear grade, hormone receptor negativity and HER2 positivity. In addition, stromal CTSV expression was associated with larger DCIS size, comedo-type necrosis and high proliferation index. DCIS associated with IBC showed higher CTSV expression than pure DCIS either within the epithelial tumour cells or surrounding stroma (p<0.0001 and p=0.001, respectively). In DCIS/IBC, CTSV expression was higher in the invasive component than DCIS component either in tumour cells or surrounding stroma (both p<0.0001). CTSV stromal expression was associated with invasive recurrence independent of other prognostic factors in patients treated with breast conserving surgery (HR=3.0, p=0.005). CONCLUSION: High expression of CTSV is associated with poor outcome in DCIS and is a potential marker to predict DCIS progression to invasive disease.
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Biomarcadores de Tumor/análisis , Neoplasias de la Mama/enzimología , Carcinoma Intraductal no Infiltrante/enzimología , Catepsinas/análisis , Cisteína Endopeptidasas/análisis , Biomarcadores de Tumor/genética , Neoplasias de la Mama/genética , Neoplasias de la Mama/patología , Neoplasias de la Mama/terapia , Carcinoma Intraductal no Infiltrante/patología , Catepsinas/genética , Cisteína Endopeptidasas/genética , Progresión de la Enfermedad , Femenino , Humanos , Inmunohistoquímica , Persona de Mediana Edad , Pronóstico , Regulación hacia ArribaRESUMEN
trans-Sialidase and cruzipain are important virulence factors from Trypanosoma cruzi, the etiological agent of Chagas disease, that have highly antigenic domains in their structure and were reported as potential tools for diagnosis of the illness. The aim of the present study is to assess the possibility of using cruzipain and the catalytic domain of trans-sialidase in a Surface Plasmon Resonance-based immunosensor for the diagnosis of chronic Chagas disease. Immunoassays carried out with canine sera verified that cruzipain allows the detection of anti-Trypanosoma cruzi antibodies whereas recombinant trans-sialidase did not yield specific detections, due to the high dilutions of serum used in the immunoassays that hinder the possibility to sense the specific low titer antibodies. The developed cruzipain-based biosensor, whose price per assay is comparable to a commercial enzyme-linked immunosorbent assay (ELISA), was successfully applied for the rapid quantification of specific antibodies against Trypanosoma cruzi in fresh human sera showing an excellent agreement with ELISA.
Asunto(s)
Anticuerpos Antiprotozoarios/sangre , Enfermedad de Chagas/diagnóstico , Enfermedad de Chagas/veterinaria , Ensayo de Inmunoadsorción Enzimática/métodos , Trypanosoma cruzi/aislamiento & purificación , Animales , Enfermedad de Chagas/sangre , Enfermedad de Chagas/parasitología , Cisteína Endopeptidasas/análisis , Cisteína Endopeptidasas/genética , Cisteína Endopeptidasas/inmunología , Enfermedades de los Perros/sangre , Enfermedades de los Perros/diagnóstico , Enfermedades de los Perros/parasitología , Perros , Glicoproteínas/análisis , Glicoproteínas/genética , Glicoproteínas/inmunología , Humanos , Neuraminidasa/análisis , Neuraminidasa/genética , Neuraminidasa/inmunología , Proteínas Protozoarias/análisis , Proteínas Protozoarias/genética , Proteínas Protozoarias/inmunología , Trypanosoma cruzi/genética , Trypanosoma cruzi/inmunología , Factores de Virulencia/sangre , Factores de Virulencia/genética , Factores de Virulencia/inmunologíaRESUMEN
Legumain, which is also known as vacuolar processing enzyme (VPE) or asparaginyl endopeptidase (AEP), is a cysteine protease that was first discovered and characterized in the leguminous seeds of the moth bean in the early 1990s. Later, this enzyme was also detected in higher organisms, including eukaryotes. This pH-dependent protease displays the highest activity in acidic endolysosomal compartments; however, legumain also displays nuclear, cytosolic and extracellular activity when stabilized by other proteins or intramolecular complexes. Based on the results from over 25 years of research, this protease is involved in multiple cellular events, including protein degradation and antigen presentation. Moreover, when dysregulated, this protease contributes to the progression of several diseases, with cancer being the well-studied example. Research on legumain biology was undoubtedly facilitated by the use of small molecule chemical tools. Therefore, in this review, I present the historical perspectives and most current strategies for the development of small molecule substrates, inhibitors and activity-based probes for legumain. These tools are of paramount importance in elucidating the roles of legumain in multiple biological processes. Finally, as this enzyme appears to be a promising molecular target for anticancer therapies, the development of legumain-activated prodrugs is also described.
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Cisteína Endopeptidasas/análisis , Inhibidores Enzimáticos/química , Sondas Moleculares/química , Péptidos/química , Profármacos/química , Animales , Cisteína Endopeptidasas/metabolismo , Inhibidores Enzimáticos/farmacología , Fabaceae/enzimología , Humanos , Péptidos/farmacología , Profármacos/farmacología , Semillas/enzimologíaAsunto(s)
Contaminantes Atmosféricos/análisis , Alérgenos/análisis , Antígenos Dermatofagoides/análisis , Antígenos Fúngicos/análisis , Antígenos de Plantas/análisis , Proteínas de Artrópodos/análisis , Líquido del Lavado Bronquioalveolar/química , Cisteína Endopeptidasas/análisis , Proteínas Fúngicas/análisis , Proteínas de Plantas/análisis , Animales , Broncoscopía , HumanosRESUMEN
Subcutaneous allergen immunotherapy (SCIT) with non-standardized house dust (HD) extracts has been used in Japan since 1963 for house dust mite (HDM)-allergic patients. Since the potencies of HD extracts are unknown, the allergenic potency of HD extracts was examined by comparing with a standardized HDM allergen extracts. The major allergen content of HDM in the extracts was measured using a sandwich enzyme-linked immunosorbent assay (ELISA). The immunoglobulin E (IgE) inhibitory activities of the extracts were measured by a competitive ELISA. The extract concentrations giving 50% inhibition of IgE binding (log10 IC50) were determined from dose-response curves and defined as inhibitory activities. A linear regression line was constructed from the log10 IC50 values of the standardized HDM extract to interpolate the relative potency of the HD extract with strength of 1 : 10 w/v (HD 1 : 10). The amounts of major allergens (Der f 1, Der p 1 and Der 2) were 116.3 µg/mL in the HDM allergen extract (100000 Japanese Allergy Units [JAU]/mL) and 0.77 µg/mL in the HD 1 : 10. The inhibitory activity (log10 IC50 values) of HD 1 : 10 was 2.389 ± 0.078, indicating the allergenic potency was between 200 and 2000 JAU/mL. Based on regression analysis (R2 >0.99), the allergenic potency of HD 1 : 10 was estimated to be 842 ± 128 JAU/mL. The present study determined the major allergen content of HD extract, which contributes to its allergenic potency. The allergenic potency of HD 1 : 10 was ca. 100-fold less than that of HDM allergen extract.
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Alérgenos/inmunología , Antígenos Dermatofagoides/inmunología , Proteínas de Artrópodos/inmunología , Cisteína Endopeptidasas/inmunología , Desensibilización Inmunológica , Polvo , Pyroglyphidae/inmunología , Alérgenos/análisis , Animales , Antígenos Dermatofagoides/análisis , Proteínas de Artrópodos/análisis , Mezclas Complejas/análisis , Mezclas Complejas/farmacología , Cisteína Endopeptidasas/análisis , Vivienda , Inmunoglobulina E/inmunología , Inyecciones SubcutáneasRESUMEN
Periodontal disease is an oral inflammatory disease that destroys the tooth supporting periodontal tissues resulting in tooth loss. Porphyromonas gingivalis is a keystone pathogen that plays a significant role in periodontitis. In previous studies, resveratrol has shown significant results by targeting inflammatory and adhesive markers. Virulence factors of P. gingivalis play an important role in the bacterial adhesion and colonization. In this study, we aimed to demonstrate the anti-biofilm and anti-bacterial activity of resveratrol and also study the effect of resveratrol on the expression of virulence factor genes of P. gingivalis using reverse transcriptase polymerase chain reaction (RT-PCR). The anti-microbial and anti-biofilm activity of resveratrol on P. gingivalis was carried out by broth microdilution assay and biofilm adhesion reduction-crystal violet assay, respectively. We carried out the gene expression analysis by RT-PCR with the P. gingivalis treated compound to analyze the change in the expression of virulence factors: fimbriae and gingipain. Minimal inhibitory concentrations (MIC) of resveratrol against P. gingivalis and other clinical strains are in the range of 78.12-156.25 µg/mL. Resveratrol dose-dependently prevented the biofilm formation and also attenuated the virulence of P. gingivalis by reducing the expression of virulence factor genes such as fimbriae (type II and IV) and proteinases (kgp and rgpA). Resveratrol demonstrated superior anti-bacterial and anti-biofilm activity against P. gingivalis. There was significant reduction in the expression of fimbriae and gingipain with the resveratrol-treated compound. The results suggest that resveratrol, due to its multiple actions, may become a simple and inexpensive therapeutic strategy for treating periodontal disease.
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Antibacterianos/farmacología , Biopelículas/efectos de los fármacos , Porphyromonas gingivalis/efectos de los fármacos , Resveratrol/farmacología , Factores de Virulencia/antagonistas & inhibidores , Adhesinas Bacterianas/análisis , Infecciones por Bacteroidaceae/microbiología , Cisteína Endopeptidasas/análisis , Proteínas Fimbrias/análisis , Perfilación de la Expresión Génica , Violeta de Genciana/análisis , Cisteína-Endopeptidasas Gingipaínas , Humanos , Pruebas de Sensibilidad Microbiana , Enfermedades Periodontales/microbiología , Porphyromonas gingivalis/aislamiento & purificación , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Coloración y EtiquetadoRESUMEN
An electrostatic nanocomplex between naturally occurring ε-poly-l-lysine (εPL) and ß-cyclodextrin sulphate (sCD) was designed, and its capacity to entrap four model proteins with high or low molecular weight and isoelectric point, i.e., lactoferrin, albumin, actinidin, and lysozyme, was investigated. The optimal formulations gave nanocomplexes with an average diameter around 276⯱â¯16â¯nm, a ζ-potential of -39⯱â¯1.5â¯mV, and a spherical shape with a core-shell structure. Different strategies were pursued to increase the entrapment efficiency for selected proteins, which led to 40-100% entrapment depending on the protein type. Under simulated gastric conditions with pepsin, the complexes protected lactoferrin and albumin against proteolysis, whereas actinidin and lysozyme were intrinsically stable. In Caco-2 cells, these complexes transiently decreased the trans-epithelial electrical resistance, indicating the potential to enhance the paracellular permeability of bioactive macromolecules. Thus, these εPL-sCD complexes would be a promising system for loading diverse proteins for gastric protection and enhancing intestinal absorption.
Asunto(s)
Albúminas/metabolismo , Cisteína Endopeptidasas/metabolismo , Sistemas de Liberación de Medicamentos , Tracto Gastrointestinal/efectos de los fármacos , Lactoferrina/metabolismo , Muramidasa/metabolismo , Polilisina/farmacología , Sustancias Protectoras/farmacología , beta-Ciclodextrinas/farmacología , Albúminas/análisis , Células CACO-2 , Supervivencia Celular/efectos de los fármacos , Células Cultivadas , Cisteína Endopeptidasas/análisis , Relación Dosis-Respuesta a Droga , Humanos , Lactoferrina/análisis , Estructura Molecular , Muramidasa/análisis , Tamaño de la Partícula , Polilisina/química , Sustancias Protectoras/química , Relación Estructura-Actividad , Propiedades de Superficie , beta-Ciclodextrinas/químicaRESUMEN
Fluorescent labeling of proteins is a critical requirement for single-molecule imaging studies. Many protein labeling strategies require harsh conditions or large epitopes that can inactivate the target protein, either by decreasing the protein's enzymatic activity or by blocking protein-protein interactions. Here, we provide a detailed protocol to efficiently label CRISPR-Cas complexes with a small fluorescent peptide via sortase-mediated transpeptidation. The sortase tag consists of just a few amino acids that are specifically recognized at either the N- or the C-terminus, making this strategy advantageous when the protein is part of a larger complex. Sortase is active at high ionic strength, 4°C, and with a broad range of organic fluorophores. We discuss the design, optimization, and single-molecule fluorescent imaging of CRISPR-Cas complexes on DNA curtains. Sortase-mediated transpeptidation is a versatile addition to the protein labeling toolkit.
Asunto(s)
Proteínas Asociadas a CRISPR/análisis , Sistemas CRISPR-Cas , Cisteína Endopeptidasas/análisis , Proteínas de Escherichia coli/análisis , Escherichia coli/química , Colorantes Fluorescentes/análisis , Repeticiones Palindrómicas Cortas Agrupadas y Regularmente Espaciadas , Escherichia coli/citología , Modelos Moleculares , Imagen Óptica/métodos , Coloración y Etiquetado/métodosRESUMEN
Legumain is a proteolytic enzyme that plays a role in the regulation of cell proliferation in invasive breast cancer. Studies evaluating its role in ductal carcinoma in situ (DCIS) are lacking. Here, we aimed to characterize legumain protein expression in DCIS and evaluate its prognostic significance. Legumain was assessed immunohistochemically in a tissue microarray of a well-characterized cohort of DCIS (n = 776 pure DCIS and n = 239 DCIS associated with invasive breast cancer (DCIS-mixed)). Legumain immunoreactivity was scored in tumor cells and surrounding stroma and related to clinicopathological parameters and patient outcome. High legumain expression was observed in 23% of pure DCIS and was associated with features of high-risk DCIS including higher nuclear grade, comedo necrosis, hormone receptor negativity, HER2 positivity, and higher proliferation index. Legumain expression was higher in DCIS associated with invasive breast cancer than in pure DCIS (p < 0.0001). In the DCIS-mixed cohort, the invasive component showed higher legumain expression than the DCIS component (p < 0.0001). Legumain was an independent predictor of shorter local recurrencefree interval for all recurrences (p = 0.0003) and for invasive recurrences (p = 0.002). When incorporated with other risk factors, legumain provided better patient risk stratification. High legumain expression is associated with poor prognosis in DCIS and could be a potential marker to predict DCIS progression to invasive disease.
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Biomarcadores de Tumor/análisis , Neoplasias de la Mama/enzimología , Carcinoma Intraductal no Infiltrante/enzimología , Cisteína Endopeptidasas/análisis , Biomarcadores de Tumor/genética , Neoplasias de la Mama/genética , Neoplasias de la Mama/patología , Neoplasias de la Mama/terapia , Carcinoma Intraductal no Infiltrante/genética , Carcinoma Intraductal no Infiltrante/patología , Carcinoma Intraductal no Infiltrante/terapia , Cisteína Endopeptidasas/genética , Progresión de la Enfermedad , Femenino , Humanos , Persona de Mediana Edad , Invasividad Neoplásica , Recurrencia Local de Neoplasia , Supervivencia sin Progresión , Medición de Riesgo , Factores de Riesgo , Factores de Tiempo , Regulación hacia ArribaRESUMEN
The sortase-catalyzed coupling reaction is an efficient strategy to incorporate chemically defined modifications into proteins of interest. Despite its widespread applications in protein chemistry, the conventional bulk fluorescence measurement is not sufficient to characterize sortase due to the fluorescence inner filter effect-induced self-quenching. Herein, we develop a new method to visualize and quantify sortase A (SrtA) activity at the single-molecule level by using transpeptidation-directed intramolecular Förster resonance energy transfer (FRET). This assay utilizes two cyanine dye-peptide conjugates, in which one carries an LPXTG motif and a donor fluorophore while the other harbors an oligoglycine nucleophile and an acceptor fluorophore as the substrate of SrtA. The presence of SrtA catalyzes the fusion of two conjugates and allows for the occurrence of intramolecular FRET. The FRET signal is recorded at the single-molecule level via total internal reflection fluorescence (TIRF)-based imaging. The proposed assay not only can accurately determine the kinetic parameters of SrtA but also can characterize the inhibition of SrtA activity by berberine chloride both in vitro and in Staphylococcus aureus ( S. aureus) cells. Moreover, the assay is very specific, and it can sensitively measure SrtA down to 7.08 pM, which is much lower than most of the reported methods. This strategy may provide a valuable tool for an in-depth study of sortases and for the discovery of anti-infective agents.
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Aminoaciltransferasas/análisis , Proteínas Bacterianas/análisis , Cisteína Endopeptidasas/análisis , Transferencia Resonante de Energía de Fluorescencia/métodos , Imagen Individual de Molécula/métodos , Aminoaciltransferasas/química , Proteínas Bacterianas/química , Carbocianinas/química , Cisteína Endopeptidasas/química , Colorantes Fluorescentes/química , Cinética , Staphylococcus aureus/enzimologíaRESUMEN
Legumain is one of the cysteine proteases which can serve as an essential indicator for cancer diagnosis. Near-infrared (NIR) nanoprobes with fluorescence "Turn On" property are advantageous in cancer diagnosis. However, to the best of our knowledge, using a completely organic NIR nanoprobe to image legumain activity either in vitro or in vivo has not been reported. Herein, employing a CBT-Cys click condensation reaction, we used a rationally designed NIR probe Cys(StBu)-Ala-Ala-Asn-Lys(Cy5.5)-CBT (1) to synthesize its nanoprobes 1-NPs with self-quenched fluorescence. Cell and animal experiments indicated that our nanoprobes were able to specifically image legumain activity in living cells and tumors with a NIR fluorescence "Turn On" manner. We envision that the nanoprobes could be applied for the diagnosis of legumain-related diseases in the near future.
Asunto(s)
Carbocianinas/química , Neoplasias del Colon/diagnóstico por imagen , Cisteína Endopeptidasas/análisis , Colorantes Fluorescentes/química , Oligopéptidos/química , Imagen Óptica/métodos , Animales , Carbocianinas/síntesis química , Química Clic , Neoplasias del Colon/enzimología , Colorantes Fluorescentes/síntesis química , Células HCT116 , Humanos , Rayos Infrarrojos , Ratones , Microscopía Fluorescente/métodos , Oligopéptidos/síntesis químicaRESUMEN
Household dust contains an array of constituents, including house dust mites (HDM) and the HDM allergen, Der p 1, which can cause sensitivities such as asthma and eczema. Vacuuming can help alleviate symptoms, yet little is understood about cleaning behaviour in different households. This pilot study investigated the contents of dust from four household types (students; over 65 s; and families with and without pets). This was then related to cleaning behaviours and perceptions of cleanliness. Our investigation found that HDMs and Der p 1 were present in all households and sampling locations, including participants' cars. The median Der p 1 was greatest in the living room, though results varied. Demographic group was a determinant for the number of human and pet hairs present in dust. Surprisingly, vacuuming was the most disliked task overall. This information requires consideration when developing cleaning products and advising individuals with dust-related health issues.
Asunto(s)
Alérgenos/análisis , Antígenos Dermatofagoides/análisis , Proteínas de Artrópodos/análisis , Cisteína Endopeptidasas/análisis , Polvo/análisis , Contaminación del Aire Interior/análisis , Animales , Monitoreo del Ambiente , Cabello , Vivienda , Humanos , Higiene , Percepción , Mascotas , Pyroglyphidae , Encuestas y CuestionariosRESUMEN
BACKGROUND: Filaggrin is central to the pathogenesis of atopic dermatitis (AD). The cheeks are a common initiation site of infantile AD. Regional and temporal expression of levels of filaggrin degradation products [natural moisturizing factors (NMFs)], activities of filaggrin-processing enzymes [bleomycin hydrolase (BH) and calpain-1 (C-1)] and plasmin, and corneocyte envelope (CE) maturity in early life are largely unknown. OBJECTIVES: We conducted a cross-sectional, observational study investigating regional and age-dependent variations in NMF levels, activity of proteases and CE maturity in stratum corneum (SC) from infants to determine whether these factors could explain the observed predilection sites for AD in early life. METHODS: We measured NMF using a tape-stripping method at seven sites in the SC of 129 children (aged < 12 months to 72 months) and in three sites in 56 neonates and infants (< 48 h to 3 months). In 37 of these neonates and infants, corneocyte size, maturity, BH, C-1 and plasmin activities were determined. RESULTS: NMF levels are low at birth and increase with age. Cheek SC, compared with elbow flexure and nasal tip, has the lowest NMF in the first year of life and is the slowest to reach stable levels. Cheek corneocytes remain immature. Plasmin, BH and C-1 activities are all elevated by 1 month of age in exposed cheek skin, but not in elbow skin. CONCLUSIONS: Regional and temporal differences in NMF levels, CE maturity and protease activities may explain the predilection for AD to affect the cheeks initially and are supportive of this site as key for allergen priming in early childhood. These observations will help design early intervention and treatment strategies for AD.
Asunto(s)
Dermatitis Atópica/patología , Proteínas de Filamentos Intermediarios/metabolismo , Piel/metabolismo , Factores de Edad , Calpaína/análisis , Calpaína/metabolismo , Mejilla , Preescolar , Estudios Transversales , Cisteína Endopeptidasas/análisis , Cisteína Endopeptidasas/metabolismo , Dermatitis Atópica/diagnóstico , Dermatitis Atópica/genética , Codo , Femenino , Fibrinolisina/análisis , Fibrinolisina/metabolismo , Proteínas Filagrina , Humanos , Lactante , Recién Nacido , Proteínas de Filamentos Intermediarios/análisis , Proteínas de Filamentos Intermediarios/genética , Masculino , Mutación , Piel/química , Piel/citología , Piel/patologíaRESUMEN
Cancer procoagulant (CP), a direct activator of coagulation factor X, is among one of the tumour cell products or activities which may promote fibrin formation and has been suggested to be selectively associated with the malignant phenotype. At present, the most reliable assay for the quantification of CP activity is the three-stage chromogenic assay which utilises the ability of CP to activate factor X. In this assay, the activation of factor X leads to the formation of activated thrombin from prothrombin and the eventual hydrolyses of a thrombin chromogenic substrate which contains a p-nitroaniline leaving group. The complexity of the three-stage chromogenic assay suggests a need for a direct method of assaying CP activity. This study focuses on the design of a fluorogenic substrate that would enable the direct quantification of CP activity. The results of the study show two promising substrates for the determination of CP activity: Boc-PQVR-AMC and PQVR-AMC. Further analysis showed that Boc-PQVR-AMC could be excluded as a potential substrate for CP since it was also cleaved by collagenase.