RESUMO
Activatable probes with a higher signal-to-background ratio and accuracy are essential for monitoring liver cancer as well as intraoperative fluorescence navigation. However, the presence of only one biomarker is usually not sufficient to meet the high requirement of a signal-to-background ratio in cancer surveillance, leading to the risk of misdiagnosis. In this work, a dual-locked activation response probe, Si-NTR-LAP, for nitroreductase and leucine aminopeptidase was reported. This dual-locked probe provides better tumor recognition and a higher signal-to-noise ratio than that of single-locked probes (Si-LAP and Si-NTR). In both the subcutaneous tumor model and the more complex orthotopic hepatocellular carcinoma model, the probe was able to identify tumor tissue with high specificity and accurately differentiate the boundaries between tumor tissue and normal tissue. Therefore, the dual-locked probe may provide a new and practical strategy for applying to real patient tumor tissue samples.
Assuntos
Leucil Aminopeptidase , Neoplasias Hepáticas , Nitrorredutases , Neoplasias Hepáticas/diagnóstico , Neoplasias Hepáticas/metabolismo , Humanos , Animais , Leucil Aminopeptidase/metabolismo , Leucil Aminopeptidase/análise , Nitrorredutases/metabolismo , Nitrorredutases/análise , Carcinoma Hepatocelular/diagnóstico , Carcinoma Hepatocelular/metabolismo , Camundongos , Corantes Fluorescentes/química , Imagem ÓpticaRESUMO
This article discusses the importance of early tumor detection, particularly in liver cancer, and the role of leucine aminopeptidase (LAP) as a potential marker for liver cancer diagnosis and prognosis assessment. The article highlights the limitations of current tumor markers and the need for new markers and multi-marker approaches to improve accuracy. The authors introduce a novel near-infrared fluorescent probe, NTAP, designed for LAP detection. They describe the synthesis of the probe and evaluate its spectral properties, including the LOD was 0.0038 U/mL, and QY was 0.32 %. The kinetic properties of NTAP, such as the relationship between LAP concentration (0-0.08 U/mL), reaction time (3 min), and fluorescence excitation spectra (475 nm) and emission spectra (715 nm) are investigated. The article also discusses the stability and selectivity of the probe and its ability to detect LAP in complex samples. Cellular imaging experiments demonstrate the NATP specificity and selectivity in detecting LAP activity and its inhibition. Animal models of liver and lung metastasis are used to evaluate the probe's imaging capabilities, showing its ability to accurately locate and detect metastatic lesions. The article concludes by emphasizing the potential applications of the NTAP probe in early tumor diagnosis, treatment monitoring, and the study of tumor metastasis mechanisms.
Assuntos
Corantes Fluorescentes , Leucil Aminopeptidase , Metástase Neoplásica , Animais , Humanos , Camundongos , Progressão da Doença , Corantes Fluorescentes/química , Corantes Fluorescentes/síntese química , Raios Infravermelhos , Leucil Aminopeptidase/metabolismo , Leucil Aminopeptidase/análise , Neoplasias Hepáticas/diagnóstico por imagem , Neoplasias Pulmonares/diagnóstico por imagem , Neoplasias Pulmonares/patologia , Neoplasias Pulmonares/metabolismo , Imagem ÓpticaRESUMO
Fluorescence analysis is an increasingly important contributor to the early diagnosis of kidney diseases. To achieve precise visualization of the kidneys and early diagnosis of related diseases, an asymmetric pyrrolopyrrolidone (DPP) dye platform with C-aromatic substituents and N-lipophilic/hydrophilic modification was constructed. Based on these, we developed the renal-clearable, water-soluble, and kidney injury biomarker leucine aminopeptidase (LAP) activated ratiometric fluorescent probe DPP-S-L. In the mouse model of cisplatin-induced acute kidney injury and during the development of type 2 diabetes to diabetic kidney disease, we visualized for the first time the upregulation of LAP in the kidney and urine by dual-channel ratiometric fluorescence signal and diagnosed the kidney injury earlier and more sensitively than blood/urine enzyme detection and tissue analysis. This study showcases an excellent asymmetric DPP dye platform and renal-clearable ratiometric fluorescent probe design strategy that is extended to determination and visualization of other biomarkers for early disease diagnosis.
Assuntos
Diabetes Mellitus Tipo 2 , Sondas Moleculares , Animais , Camundongos , Corantes Fluorescentes , Leucil Aminopeptidase/análise , Biomarcadores , Rim/química , Diagnóstico Precoce , Imagem ÓpticaRESUMO
Leucine aminopeptidase (LAP) is a vital proteolytic enzyme, and its overexpression is often associated with many physiological diseases, such as liver dysfunction and breast cancer. Therefore, the accurate measurement of LAP concentrations in cells is critical for the diagnosis and prevention of related diseases. Herein, a new ratiometric fluorescent probe, DPP-Leu, based on diketopyrrolopyrrole (DPP) was designed and synthesized for LAP detection based on the specific enzymatic cleavage of the N-terminal leucine residue. The fluorescence intensity ratio of DPP-Leu (I548/I651) showed a remarkable change in the presence of LAP, with a limit of detection of 0.011 U L-1, and DPP-Leu was successfully applied to detect LAP in fetal bovine serum (FBS) and artificial urine. Cell imaging experiments revealed that DPP-Leu could target mitochondria and distinguish tumor cells with high LAP content from normal cells. Importantly, benefiting from the structural transformation of DPP-Leu to the photosensitizer 4 under LAP catalysis, the probe could kill tumor cells under light irradiation without damaging normal cells.
Assuntos
Antineoplásicos/farmacologia , Corantes Fluorescentes/farmacologia , Cetonas/farmacologia , Leucil Aminopeptidase/análise , Imagem Óptica , Fotoquimioterapia , Fármacos Fotossensibilizantes/farmacologia , Pirróis/farmacologia , Antineoplásicos/síntese química , Antineoplásicos/química , Proliferação de Células/efeitos dos fármacos , Células Cultivadas , Ensaios de Seleção de Medicamentos Antitumorais , Corantes Fluorescentes/síntese química , Corantes Fluorescentes/química , Humanos , Concentração de Íons de Hidrogênio , Cetonas/síntese química , Cetonas/química , Leucil Aminopeptidase/metabolismo , Fármacos Fotossensibilizantes/síntese química , Fármacos Fotossensibilizantes/química , Pirróis/síntese química , Pirróis/química , Espécies Reativas de Oxigênio/metabolismoRESUMO
Both protease overexpression and local pH changes are key signatures of cancer. However, the sensitive detection of protease activities and the accurate measurement of pH in a tumor environment remain challenging. Here, we develop a dual-response DNA probe that can simultaneously monitor protease activities and measure the local pH by translocation through α-hemolysin (αHL). The DNA probe bears a short peptide containing phenylalanine at a pre-designed position. Enzymatic cleavage of the peptide either exposes or removes the N-terminal phenylalanine that can form a complex with cucurbit[7]uril. Translocation of the DNA hybrid through αHL generates current signatures that can be used to quantify protease activities. Furthermore, the current signatures possess a pH-dependent pattern that reflects the local pH. Our results demonstrate that the versatile DNA probe may be further explored for simultaneously measuring multiple parameters of a complex system such as single cells in the future.
Assuntos
Técnicas Biossensoriais/métodos , Catepsina B/análise , Sondas de DNA/química , Leucil Aminopeptidase/análise , Nanoporos , Proteínas Hemolisinas/química , Concentração de Íons de Hidrogênio , Limite de DetecçãoRESUMO
Drug-induced liver injury (DILI) is a frequent cause of hepatic dysfunction as well as the single most frequent reason for removing approved medications from the market, and multispectral optoacoustic tomography (MSOT) is an emerging and noninvasive imaging modality for diagnosing and monitoring diseases. Herein, we report an activatable optoacoustic probe for imaging DILI through detecting the activity of leucine aminopeptidase (LAP). In this probe, an N-terminal leucyl moiety serving as the LAP recognition element is linked with a chromene-benzoindolium chromophore via 4-aminobenzylalcohol group. The elevated expression of hepatic LAP as a result of DILI cleaves the leucyl moiety and causes the red-shift of the probe's absorption band, thereby generating prominent optoacoustic signals for MSOT imaging. During this process, the probe also exhibits prominent NIR fluorescence, which can be utilized for fluorescent imaging. More importantly, by rendering stacks of cross-sectional images as maximal intensity projection (MIP) images, we could precisely locate the focus of drug-induced liver injury in mice. This probe is expected to serve a powerful tool for studying physiological and pathological processes related to LAP.
Assuntos
Doença Hepática Induzida por Substâncias e Drogas/diagnóstico por imagem , Leucil Aminopeptidase/análise , Animais , Benzopiranos/química , Desenho de Equipamento , Corantes Fluorescentes/química , Células Hep G2 , Humanos , Masculino , Camundongos , Sondas Moleculares/química , Imagem Óptica , Técnicas Fotoacústicas , TomografiaRESUMO
Near-infrared (NIR) fluorescence imaging technique is garnering increasing research attention due to various advantages. However, most NIR fluorescent probes still suffer from a false signals problem owing to their instability in real application. Especially in a pathological environment, many NIR probes can be easily destroyed due to the excessive generation of highly reactive species and causing a distorted false signal. Herein, we proposed an approach for developing a new stable NIR dye platform with an optically tunable group to eliminate false signals using the combination of dyes screening and rational design strategy. The conception is validated by the construction of two high-fidelity NIR fluorescent probes (NIR-LAP and NIR-ONOO-) sensing leucine aminopeptidase (LAP) and peroxynitrite (ONOO-), the markers of hepatotoxicity. These probes (NIR-LAP and NIR-ONOO-) were demonstrated to sensitively and accurately monitor LAP and ONOO- (detection limit: 80 mU/L for LAP and 90 nM for ONOO-), thereby allowing one to precisely evaluate drug-induced hepatotoxicity. In addition, based on the fluctuation of LAP, the therapeutic efficacy of six hepatoprotective medicines for acetaminophen-induced hepatotoxicity was analyzed in vivo. We anticipate the high-fidelity NIR dye platform with an optically tunable group could provide a convenient and efficient tool for the development of future probes applied in the pathological environment.
Assuntos
Doença Hepática Induzida por Substâncias e Drogas/tratamento farmacológico , Corantes Fluorescentes/química , Substâncias Protetoras/farmacologia , Acetaminofen , Biomarcadores/análise , Sobrevivência Celular/efeitos dos fármacos , Doença Hepática Induzida por Substâncias e Drogas/patologia , Corantes Fluorescentes/síntese química , Corantes Fluorescentes/farmacologia , Células Hep G2 , Humanos , Raios Infravermelhos , Leucil Aminopeptidase/análise , Leucil Aminopeptidase/metabolismo , Estrutura Molecular , Imagem Óptica , Ácido Peroxinitroso/análise , Ácido Peroxinitroso/farmacologiaRESUMO
The effects of varying concentrations (ranging from 0 to 10 µM) of two different metals that is, iron (Fe) and copper (Cu) on indigenous bacterial populations and their hydrolytic enzyme activities within the bacterioplankton assemblages from the surface waters of the Kalamazoo River were examined under controlled microcosm conditions. The two metals were added to water samples collected from the Kalamazoo River and examined for bacterial abundance and leucine aminopeptidase activities at various time intervals over a 48 h incubation period in the dark. Results revealed no concentration effects on the bacterial populations in the presence of both Fe and Cu, although the bacterial numbers varied significantly over time in both microcosms. Conversely, leucine aminopeptidase activities based on post-hoc tests using Bonferroni correction revealed significant differences to increasing concentrations of both metals over the study period. These results further validate previous knowledge regarding the importance of various metal ions in regulating bacterial community structures and also suggest that aminopeptidase have the potential of effectively functioning using diverse trace and heavy metals as extracellular peptidase cofactors in aquatic systems.
Assuntos
Bactérias/efeitos dos fármacos , Metais Pesados/farmacologia , Fitoplâncton/efeitos dos fármacos , Rios/microbiologia , Microbiologia da Água , Poluentes Químicos da Água/farmacologia , Bactérias/enzimologia , Bactérias/crescimento & desenvolvimento , Proteínas de Bactérias/análise , Cobre/metabolismo , Cobre/farmacologia , Ferro/metabolismo , Ferro/farmacologia , Leucil Aminopeptidase/análise , Metais Pesados/metabolismo , Michigan , Fitoplâncton/enzimologia , Fitoplâncton/crescimento & desenvolvimento , Rios/química , Poluentes Químicos da Água/metabolismoRESUMO
Extracellular enzymatic activity (EEA) is performed by cell-associated and cell-free (i.e., "dissolved") enzymes. This cell-free fraction is operationally defined as passing through a 0.22⯵m filter. The contribution of cell-free to total EEA is comparable to the cell-associated counterpart, so it is critical to understand what controls the relative importance of cell-free versus cell-associated EEA. However, attempts to tease apart the contribution of EEAs in the so-called dissolved fraction (<0.22⯵m) in general, and of the nanoparticle size fraction (0.020-0.20⯵m) in particular, to the total EEA pool are lacking. Here we performed experiments with Northern and Southern Hemisphere coastal waters to characterize the potential contribution of that nanoparticle fraction to the total EEA fraction of alkaline phosphatase, beta-glucosidase and leucine aminopeptidase. We found a significant contribution (in both hemispheres) of the nanoparticle fraction to the total EEA pool (up to 53%) that differed depending on the enzyme type and location. Collectively, our results indicate that a significant fraction of the so-called "dissolved EEA" is not really dissolved but associated to nanoparticles, colloidal nanogels and/or viruses. Thus, the total marine EEA pool can actually be divided into a cell-associated, undissolved-cell-free (associated to nano-particle of different origins such as viruses and nanogels) and a dissolved-cell-free pools. Our results also imply that the dissolved EEA pool is more complex than thus far anticipated. Future research will be now needed to further characterize the factors controlling the relative importance of these different pools of EEA, which are key in the recycling of organic matter in the ocean.
Assuntos
Monitoramento Ambiental , Água do Mar/microbiologia , Microbiologia da Água , Fosfatase Alcalina/análise , Bactérias , Leucil Aminopeptidase/análise , Oceanos e Mares , Vírus , beta-Glucosidase/análiseRESUMO
Abnormally-expressed leucine aminopeptidase (LAP) is associated with diverse physiological and pathological disorders; hence developing a highly selective and sensitive detection system for LAP is of great significance. Herein, a fluorescent light-up system with aggregation-induced emission (AIE) characteristic, (DPA-TPE-Leu) has been developed for detecting LAP, in which the recognition unit l-leucine amide group also acts as the hydrophilic moiety. Upon LAP-triggered enzymatic reaction, l-leucine amide moiety is cleaved from the probe molecule, resulting in the formation and aggregation of the hydrophobic reaction product (DPE-TPE-OH) with AIE effect and thus giving out the turn-on green fluorescence. The system features excellent photostability, large Stokes shift (194â¯nm), good water solubility, high sensitivity with the detection limit of 0.16 Uâ¯L-1, favorable specificity and low cytotoxicity. It has been effectively utilized in fluorescent imaging of endogenous LAP in living cells, and also successfully applied for fluorescent imaging of HepG2 xenograft tumor. Such a fluorescent assay could provide a convenient and sensitive method for detecting LAP activity and might aid in the auxiliary diagnosis of hepatocellular carcinoma and related pathological analysis in biopsy.
Assuntos
Corantes Fluorescentes/química , Leucil Aminopeptidase/análise , Neoplasias/diagnóstico , Imagem Óptica , Animais , Difenilamina/química , Células Hep G2 , Humanos , Interações Hidrofóbicas e Hidrofílicas , Leucil Aminopeptidase/química , Limite de Detecção , Camundongos , Camundongos Nus , Microscopia de Fluorescência , Nanoestruturas/química , Neoplasias/diagnóstico por imagem , Neoplasias/metabolismo , Espectrometria de Fluorescência , Estilbenos/química , Transplante HeterólogoRESUMO
ERAAP is an intracellular amino-peptidase that plays a central role in determining the repertoire of peptides displayed by cells by MHC class I molecules, and dysfunctions in ERAAP are linked to a variety of diseases. There is therefore great interest in developing probes that can image ERAAP in cells. In this report we present a fluorescent probe, termed Ep, that can image ERAAP activity in live cells. Ep is composed of a 10 amino acid ERAAP substrate that has a donor quencher pair conjugated to it, composed of BODIPY and dinitro-toluene. Ep undergoes a 20-fold increase in fluorescence after ERAAP cleavage, and was able to image ERAAP activity in cell culture via fluorescence microscopy. In addition, we used Ep to develop a high throughput screen for ERAAP inhibitors, and screened an electrophile library containing 1460 compounds. From this Ep based screen we identified aromatic alkyne-ketone as a lead fragment that can irreversibly inhibit ERAAP activity. We anticipate numerous applications of Ep given its unique ability to image ERAAP within cells.
Assuntos
Corantes Fluorescentes/química , Ensaios de Triagem em Larga Escala , Leucil Aminopeptidase/análise , Imagem Óptica , Peptídeos/química , Animais , Leucil Aminopeptidase/deficiência , Leucil Aminopeptidase/metabolismo , Camundongos , Camundongos Knockout , Microscopia de FluorescênciaRESUMO
Brown tides of Aureococcus anophagefferens have occurred annually in the coastal waters of Qinhuangdao since 2009. High levels of dissolved organic matter (DOM) are always measured during bloom periods. Study focusing on the effect of DOM on the occurrences of brown tides in this area is scare by far. To analyze the efficiency of DOM hydrolysis by different groups of microorganisms and the possible influence of DOM on the formation of brown tides, extracellular enzymes such as α, ß-glucosidases (α, ß-GLUs), leucine aminopeptidase (LAP) and alkaline phosphatase (AP) as well as other environmental parameters were analyzed during a pre-bloom period of A. anophagefferens in June 2014. Dissolved organic nitrogen (DON) and phosphorus (DOP) contributed more than half of the total dissolved nutrient pools. Approximately 60-70% of the enzyme activities were associated with phytoplankton of size >5⯵m. The hydrolysis rates of LAP were approximately 5 to 20 fold higher than those of AP and α, ß-GLUs. The ratios of ß-GLU activities: LAP activities indicated the hydrolysis potential related to proteins rather than polysaccharides. The differences in turnover time among the enzymes suggested that DOP was firstly hydrolyzed and recycled in the water in the early minutes, followed by the hydrolysis of DON and dissolved organic carbon (DOC)(in hours). Results suggest that the hydrolysis of DOM, in particular DOP, might significantly contribute to the occurrences of brown tides in the coastal waters of Qinhuangdao, China.
Assuntos
Proteínas de Algas/análise , Fosfatase Alcalina/análise , Glucosidases/análise , Proliferação Nociva de Algas , Leucil Aminopeptidase/análise , Estramenópilas/enzimologia , China , Substâncias Húmicas , Hidrólise , Água do Mar/químicaRESUMO
Leucine aminopeptidase (LAP) is a kind of proteolytic enzymes and associated closely with pathogenesis of cancer and liver injury. Accurate detection of LAP activity with high sensitivity and selectivity is imperative to detect its distribution and dynamic changes for understanding LAP's function and early diagnosing the disease states. However, fluorescent detection of LAP in living systems is challenging. To date, rarely fluorescent probes have been reported for imaging LAP in vivo. In this study, a novel probe (TMN-Leu) was developed by conjugating a near-infrared dicyanoisophorone derivative fluorophore with LAP activatable l-leucine amide moiety for the first time. TMN-Leu featured large Stokes shift (198 nm), favorable water solubility, ultrasensitive sensitivity (detection limit of â¼0.38 ng/mL), good specificity, excellent cell membrane permeability, low toxicity, and a prominent near-infrared emission (658 nm) in response to LAP. TMN-Leu has been successfully applied to track LAP of cancer cells and normal cells, monitor LAP changes in different disease models, and rapidly evaluate LAP inhibitor in cell-based assay. Notably, this probe firstly revealed that HCT116 cells with higher LAP activity were more invasive than LAP siRNA transfected HCT116 cells, suggesting that LAP might serve as an indicator reflecting the intrinsic invasion ability of cancer cells. Finally, TMN-Leu was also employed for in vivo real-time imaging LAP in living tumor-bearing nude mice with low background interference. All together, our probe possesses potential value as a promising tool for diagnostic application, cell-based screening inhibitors and in vivo real-time tracking enzymatic activity in preclinical applications.
Assuntos
Corantes Fluorescentes/química , Leucil Aminopeptidase/análise , Água/química , Animais , Sobrevivência Celular , Inibidores Enzimáticos/química , Inibidores Enzimáticos/farmacologia , Humanos , Raios Infravermelhos , Leucina/análogos & derivados , Leucina/química , Leucina/farmacologia , Leucil Aminopeptidase/antagonistas & inibidores , Leucil Aminopeptidase/metabolismo , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Nus , Microscopia de Fluorescência , Neoplasias Experimentais/diagnóstico por imagem , Imagem Óptica , Solubilidade , Fatores de Tempo , Células Tumorais CultivadasRESUMO
Leucine aminopeptidase (LAP) and cathepsin L1 (CL1) are important enzymes for the pathogenesis and physiology of Fasciola hepatica. These enzymes were analysed in silico to design a chimeric protein containing the most antigenic sequences of LAP (GenBank; AAV59016.1; amino acids 192-281) and CL1 (GenBank CAC12806.1; amino acids 173-309). The cloned 681-bp chimeric fragment (rFhLAP-CL1) contains 270 bp from LAP and 411 bp from CL1, comprising three epitopes, DGRVVHLKY (amino acids 54-62) from LAP, VTGYYTVHSGSEVELKNLV (amino acids 119-137) and YQSQTCLPF (amino acids 161-169) from CL1. The ~25 kDa rFhLAP-CL1 chimeric protein was expressed from the pET15b plasmid in the Rosetta (DE3) Escherichia coli strain. The chimeric protein rFhLAP-CL1, which showed antigenic and immunogenic properties, was recognized in Western blot assays using F. hepatica-positive bovine sera, and induced strong, specific antibody responses following immunization in rabbits. The newly generated chimeric protein may be used as a diagnostic tool for detection of antibodies against F. hepatica in bovine sera and as an immunogen to induce protection against bovine fasciolosis.
Assuntos
Catepsina L/genética , Fasciola hepatica/genética , Fasciolíase/veterinária , Proteínas de Helminto/genética , Leucil Aminopeptidase/genética , Fígado/enzimologia , Animais , Catepsina L/análise , Catepsina L/imunologia , Bovinos , Doenças dos Bovinos/parasitologia , Epitopos/análise , Epitopos/genética , Epitopos/imunologia , Fasciola hepatica/imunologia , Fasciolíase/parasitologia , Expressão Gênica , Proteínas de Helminto/análise , Proteínas de Helminto/imunologia , Imunização , Leucil Aminopeptidase/análise , Leucil Aminopeptidase/imunologia , Coelhos , Proteínas Recombinantes de Fusão/análise , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/imunologiaRESUMO
A three-component probe harnesses the extraordinary properties of a solid-state fluorophore for the detection of living cells exhibiting a particular peptidase activity. The off-on mode by which the probe operates, the bright fluorescence of the resulting precipitate, and the rapid response allow an exceptional signal-to-background ratio during microscopic imaging. A tertiary carbamate link between the spacer and phenolic fluorophore is at the heart of the probe's long-term stability. The degree of chlorination of the probe determines its response time and thus its suitability for live-cell analysis. Our probe also allows highly resolved localization of peptidase activity during gel analysis or on agar. In comparison, probes releasing soluble fluorophores demonstrate complete diffusion of the fluorescent signal. These results demonstrate the probe's potential for diverse biomedical applications, including high-fidelity flow cytometry and sensitive colony assays.
Assuntos
Corantes Fluorescentes/análise , Leucil Aminopeptidase/análise , Leucil Aminopeptidase/metabolismo , Sobrevivência Celular , Fluorescência , Corantes Fluorescentes/metabolismo , Células HeLa , Humanos , Microscopia de Fluorescência , Espectrometria de FluorescênciaRESUMO
AIM: Application of quantitative stable isotope-labelling chemistries and mass spectrometry (MS) to determine alterations in gingival crevicular fluid (GCF) proteome in periodontal disease. MATERIAL AND METHODS: Quantitative proteome of GCF from 40 healthy individuals versus 40 patients with periodontal disease was established using 320 GCF samples and stable isotope-labelling reagents, ICAT and mTRAQ, with MS technology and validated by enzyme-linked immunosorbent methods. RESULTS: We have identified 238 distinct proteins of which 180 were quantified in GCF of both healthy and periodontal patients with additional 26 and 32 distinct proteins that were found only in GCF of healthy or periodontal patients. In addition, 42 pathogenic bacterial proteins and 11 yeast proteins were quantified. The data highlighted a series of proteins not quantified previously by large-scale MS approaches in GCF with relevance to periodontal disease, such as host-derived Ig alpha-2 chain C, Kallikrein-4, S100-A9, transmembrane proteinase 13, peptidase S1 domain, several collagen types and pathogenic bacterial proteins, e.g. formamidase, leucine aminopeptidase and virulence factor OMP85. CONCLUSIONS: The innovative analytical approaches provided detailed novel changes in both host and microbial derived GCF proteomes of periodontal patients. The study defined 50 host and 16 pathogenic bacterial proteins significantly elevated in periodontal disease most of which were novel with significant potential for application in the clinical arena of periodontal disease.
Assuntos
Líquido do Sulco Gengival/química , Doenças Periodontais/metabolismo , Proteoma/análise , Adulto , Albuminas/análise , Amidoidrolases/análise , Proteínas da Membrana Bacteriana Externa/análise , Proteínas de Bactérias/análise , Calgranulina B/análise , Cromatografia Líquida , Colágeno/análise , Eletroforese em Gel de Poliacrilamida , Feminino , Proteínas Fúngicas/análise , Humanos , Cadeias alfa de Imunoglobulina/análise , Isótopos , Calicreínas/análise , Leucil Aminopeptidase/análise , Masculino , Espectrometria de Massas , Proteínas de Membrana/análise , Pessoa de Meia-Idade , Doenças Periodontais/microbiologia , Serina Endopeptidases/análise , Albumina Sérica/análise , Espectrometria de Massas por Ionização por Electrospray , Espectrometria de Massas em Tandem , Adulto JovemRESUMO
BACKGROUND: Perinatal care of esophageal atresia (EA) may be improved by prenatal diagnosis. Ultrasound findings (polyhydramnios and/or nonvisualization of fetal stomach) lead to a detection rate of ~50%. An amniotic fluid (AF) biochemical pattern characterized by high total protein, γ-glutamyl transpeptidase (GGTP), and normal l-leucine-aminopeptidase (AMP) leads to a 100% detection rate. The aim of this study was to explain this specific pattern. METHODS: On the basis of enzyme activities assay, the following four objectives were sought: (i) comparing AF markers between EA and other digestive tract atresias, (ii) determining local GGTP synthesis in the esophagus (immunohistobiochemistry), (iii) determining the presence of a specific AF-AMP activity inhibitor, and (iv) comparing AF-AMP and AF-GGTP half-lives. RESULTS: The AF-EA pattern was similar to that observed in upper duodenal atresia (above the Oddi sphincter). No local synthesis of GGTP was observed in the esophagus. No AF-AMP activity inhibitor was found. AF-GGTP had a longer half-life than AF-AMP. CONCLUSION: Due to the swallowing anomaly observed in EA, GGTP and AMP values physiologically observed at 18 wk will decrease on the basis of the half-lives of markers, with a flat slope for GGTP and a sharp slope for AMP, therefore explaining the differences observed in the AF-EA pattern.
Assuntos
Líquido Amniótico/química , Biomarcadores/química , Atresia Esofágica/diagnóstico , Leucil Aminopeptidase , Diagnóstico Pré-Natal/métodos , Proteínas , gama-Glutamiltransferase , Atresia Esofágica/metabolismo , Atresia Esofágica/patologia , Meia-Vida , Humanos , Imuno-Histoquímica , Cariotipagem , Leucil Aminopeptidase/análise , Proteínas/análise , Estudos Retrospectivos , Estatísticas não Paramétricas , gama-Glutamiltransferase/análiseRESUMO
A new method to estimate the leucine aminopeptidase (LAP, EC 3.4.11.1) activity using capillary electrophoresis coupled with electrochemiluminescence (ECL) is described. The liberated proline produced by LAP catalyzing the hydrolysis reaction of leucin-proline was used as an ECL coreagent to enhance Ru(bpy)3 (2+) ECL signals efficiently. The detection limit for proline was 2.88 × 10(-6) m (signal-to-noise ratio 3), which was equal to 9.60 × 10(-8) units of LAP being used to catalyze leucin-proline for 1 min. The Michaelis constant Km (2.07 × 10(-2) mol/L) and the maximum reaction velocity Vmax (1.06 × 10(-5) mol/L/min) of LAP for leucin-proline are reported. The reaction conditions including the concentration of metal ions, incubation temperature and pH were optimized. This method was successfully applied to detect LAP activity in plasma and the results were in good agreement with that obtained by the clinical method.
Assuntos
Eletroforese Capilar/métodos , Leucil Aminopeptidase/análise , Leucil Aminopeptidase/metabolismo , Medições Luminescentes/métodos , Estudos de Casos e Controles , Colestase/sangue , Colestase/enzimologia , Estabilidade Enzimática , Feminino , Hepatite/sangue , Hepatite/enzimologia , Humanos , Concentração de Íons de Hidrogênio , Leucina/análise , Leucina/metabolismo , Leucil Aminopeptidase/sangue , Leucil Aminopeptidase/química , Limite de Detecção , Neoplasias Hepáticas/sangue , Neoplasias Hepáticas/enzimologia , Masculino , Prolina/análise , Prolina/metabolismo , Reprodutibilidade dos Testes , Razão Sinal-RuídoRESUMO
We have synthesized and evaluated a series of hydroxymethyl rhodamine derivatives and found an intriguing difference of intramolecular spirocyclization behavior: the acetylated derivative of hydroxymethyl rhodamine green (Ac-HMRG) exists as a closed spirocyclic structure in aqueous solution at physiological pH, whereas HMRG itself takes an open nonspirocyclic structure. Ac-HMRG is colorless and nonfluorescent, whereas HMRG is strongly fluorescent. On the basis of these findings, we have developed a general design strategy to obtain highly sensitive fluorescence probes for proteases and glycosidases, by replacing the acetyl group of Ac-HMRG with a substrate moiety of the target enzyme. Specific cleavage of the substrate moiety in the nonfluorescent probe by the target enzyme generates a strong fluorescence signal. To confirm the validity and flexibility of our strategy, we designed and synthesized fluorescence probes for leucine aminopeptidase (Leu-HMRG), fibroblast activation protein (Ac-GlyPro-HMRG), and ß-galactosidase (ßGal-HMRG). All of these probes were almost nonfluorescent due to the formation of spirocyclic structure, but were converted efficiently to highly fluorescent HMRG by the target enzymes. We confirmed that the probes can be used in living cells. These probes offer great practical advantages, including high sensitivity and rapid response (due to regulation of fluorescence at a single reactive site), as well as resistance to photobleaching, and are expected to be useful for a range of biological and pathological investigations.
Assuntos
Corantes Fluorescentes/química , Gelatinases/análise , Leucil Aminopeptidase/análise , Proteínas de Membrana/análise , Rodaminas/química , Serina Endopeptidases/análise , Compostos de Espiro/síntese química , beta-Galactosidase/análise , Animais , Bovinos , Ciclização , Endopeptidases , Corantes Fluorescentes/síntese química , Gelatinases/metabolismo , Células HEK293 , Células HeLa , Humanos , Concentração de Íons de Hidrogênio , Leucil Aminopeptidase/metabolismo , Proteínas de Membrana/metabolismo , Microscopia Confocal , Estrutura Molecular , Rodaminas/síntese química , Serina Endopeptidases/metabolismo , Espectrometria de Fluorescência , Compostos de Espiro/química , beta-Galactosidase/metabolismoRESUMO
We determined the total and dissolved extracellular enzymatic activity (EEA) of α-glucosidase and ß-glucosidase (AGase and BGase), alkaline phosphatase (APase) and leucine aminopeptidase (LAPase) activities in the epi-, meso- and bathypelagic waters of the subtropical Northeast Atlantic. EEA was also determined in treatments in which bacterial EEA was inhibited by erythromycin. Additionally, EEA decay experiments were performed with surface and deep waters to determine EEA lifetimes in both water masses. The proportion of dissolved to total EEA (66-89 %, 44-88 %, 57-82 % and 86-100 % for AGase, BGase, APase and LAPase, respectively) was generally higher than the cell-associated (i.e., particulate) EEA. The percentage of dissolved to total EEA was inversely proportional to the percentage of erythromycin-inhibited to total EEA. Since erythromycin-inhibited plus dissolved EEA equaled total EEA, this tentatively suggests that cell-associated EEA in the open oceanic water column is almost exclusively of bacterial origin. The decay constants of dissolved EEA were in the range of 0.002-0.048 h(-1) depending on the type of extracellular enzyme, temperature and depth in the water column. Although dissolved EEA can have different origins, the major contribution of Bacteria to cell-associated EEA and the long life-time of dissolved EEA suggest that Bacteria-and not mesophilic Archaea-are essentially the main producers of EEA in the open subtropical Northeast Atlantic down to bathypelagic layers.