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Detergent-based workflows incorporating sodium dodecyl sulfate (SDS) necessitate additional steps for detergent removal ahead of mass spectrometry (MS). These steps may lead to variable protein recovery, inconsistent enzyme digestion efficiency, and unreliable MS signals. To validate a detergent-based workflow for quantitative proteomics, we herein evaluate the precision of a bottom-up sample preparation strategy incorporating cartridge-based protein precipitation with organic solvent to deplete SDS. The variance of data-independent acquisition (SWATH-MS) data was isolated from sample preparation error by modelling the variance as a function of peptide signal intensity. Our SDS-assisted cartridge workflow yield a coefficient of variance (CV) of 13%-14%. By comparison, conventional (detergent-free) in-solution digestion increased the CV to 50%; in-gel digestion provided lower CVs between 14% and 20%. By filtering peptides predicting to display lower precision, we further enhance the validity of data in global comparative proteomics. These results demonstrate the detergent-based precipitation workflow is a reliable approach for in depth, label-free quantitative proteome analysis.
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Precipitación Química , Detergentes , Proteómica , Dodecil Sulfato de Sodio , Flujo de Trabajo , Proteómica/métodos , Dodecil Sulfato de Sodio/química , Detergentes/química , Proteoma/análisis , Proteoma/química , Humanos , Péptidos/química , Péptidos/análisisRESUMEN
INTRODUCTION: Given the lack of data to help caregivers in the follow-up of Hirschsprung disease (HD), this study aimed to compare the functional outcomes of isolated Hirschsprung disease (I-HD) to syndrome-associated Hirschsprung disease (SA-HD) at 1, 3, 5, and 10 years. METHODS: A retrospective chart review of patients diagnosed with HD between January 1990 and May 2021 at our pediatric center was performed to collect data on patient characteristics, investigations, and treatments. Ninety-five patients were identified, of whom 76 were included in the study. SA-HD is defined as a syndrome known to be associated with HD or cognitive impairment. RESULTS: Patient characteristics were comparable between groups ( P > 0.05). There were 52 patients with I-HD and 24 with SA-HD. The patients median age was 9 days at diagnosis and 1.5 month at surgery. SA-HD patients became bowel continent at a significantly older age (mean age 8.43 vs 4.94 years, P = 0.0471) and received more bowel continence medications. At 5 years, SA-HD patients requiring ≥2 medications for bowel continence represented 54.5% versus 11.1% of I-HD patients ( P = 0.009). Lastly, SA-HD patients had urinary incontinence at a significantly older age ( P = 0.0136, 5 years). CONCLUSION: Clinicians should be aware that SA-HD patients are more prone to bladder dysfunction and became bowel continent at an older age than I-HD patients. They need more and prolonged bowel management medications, and other important complications need to be addressed in patient care. These results should prompt a longer follow-up period for these patients, especially in SA-HD.
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Enfermedad de Hirschsprung , Niño , Humanos , Enfermedad de Hirschsprung/complicaciones , Enfermedad de Hirschsprung/cirugía , Resultado del Tratamiento , Estudios Retrospectivos , Estreñimiento/terapia , Intestinos , SíndromeRESUMEN
Necrotizing enterocolitis (NEC) is a life-threatening condition for premature infants in neonatal intensive care units. Finding indicators that can predict NEC development before symptoms appear would provide more time to apply targeted interventions. In this study, stools from 132 very-low-birth-weight (VLBW) infants were collected daily in the context of a multi-center prospective study aimed at investigating the potential of fecal biomarkers for NEC prediction using proteomics technology. Eight of the VLBW infants received a stage-3 NEC diagnosis. Stools collected from the NEC infants up to 10 days before their diagnosis were available for seven of them. Their samples were matched with those from seven pairs of non-NEC controls. The samples were processed for liquid chromatography-tandem mass spectrometry analysis using SWATH/DIA acquisition and cross-compatible proteomic software to perform label-free quantification. ROC curve and principal component analyses were used to explore discriminating information and to evaluate candidate protein markers. A series of 36 proteins showed the most efficient capacity with a signature that predicted all seven NEC infants at least a week in advance. Overall, our study demonstrates that multiplexed proteomic signature detection constitutes a promising approach for the early detection of NEC development in premature infants.
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Enterocolitis Necrotizante , Enfermedades del Recién Nacido , Enfermedades del Prematuro , Biomarcadores/análisis , Enterocolitis Necrotizante/diagnóstico , Humanos , Lactante , Recién Nacido , Recién Nacido de muy Bajo Peso , Espectrometría de Masas , Estudios Prospectivos , ProteómicaRESUMEN
Mouse mast cell protease 4 (mMCP-4), the murine functional analog to the human chymase, is a serine protease synthesized and stored in mast cell secretory granules. Our previous studies reported physiologic and pathologic roles for mMCP-4 in the maturation and synthesis of the vasoactive peptide endothelin-1 (ET-1) from its precursor, big ET-1. The aim of this study was to investigate the impact of mast cell degranulation or stabilization on mMCP-4-dependent pressor responses after the administration of big ET-1 or angiotensin I (Ang I). In anesthetized mice, mast cell degranulation induced by compound 48/80 (C48/80) or stabilization by cromolyn enhanced or repressed, respectively, the dose-dependent vasopressor responses to big ET-1 in wild-type (WT) mice but not in mMCP-4 knockout mice in a chymase inhibitor (TY-51469)-sensitive fashion. In addition, mMCP-4-dependent hydrolysis of the fluorogenic substrate Suc-Leu-Leu-Val-Tyr-7-amino-4-methylcoumarin was depleted or enhanced in peritoneal mast cells isolated from mice pretreated with C48/80 or cromolyn, respectively. Furthermore, C48/80 or cromolyn markedly increased or abolished, respectively, ET-1 (1-31) conversion from exogenous big ET-1 in WT mice peritoneal fluid-isolated mast cells, in vitro. Finally, the vasopressor responses to Ang I were unaffected by mast cell activation or stabilization, whereas those induced by the angiotensin-converting enzyme-resistant Ang I analog, [Pro11, D-Ala12] Ang I, were potentiated by C48/80. Altogether, the present study shows that mast cell activation enhances the mMCP-4-dependent vasoactive properties of big ET-1 but not Ang I in the mouse model. SIGNIFICANCE STATEMENT: The current work demonstrates a significant role for mast cell stability in the cardiovascular pharmacology of big endothelin-1 but not angiotensin I in the murine systemic circulation.
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Angiotensina I/farmacología , Presión Sanguínea , Degranulación de la Célula , Endotelina-1/farmacología , Mastocitos/fisiología , Serina Endopeptidasas/metabolismo , Animales , Células Cultivadas , Quimasas/antagonistas & inhibidores , Cromolin Sódico/farmacología , Inhibidores Enzimáticos/farmacología , Masculino , Estabilizadores de Mastocitos/farmacología , Mastocitos/efectos de los fármacos , Mastocitos/metabolismo , Ratones , Ratones Endogámicos C57BL , Peritoneo/citología , Serina Endopeptidasas/genética , Sulfonamidas/farmacología , Tiofenos/farmacologíaRESUMEN
RATIONALE: Oxidative stress is an imbalance between reactive free radical oxygen species and antioxidant defenses. Its consequences can lead to numerous pathologies. Regulating oxidative stress is the complex interplay between antioxidant recycling and thiol-containing regulatory proteins. Understanding these regulatory mechanisms is important for preventing onset of oxidative stress. The aim of this study was to investigae S-thiol protein chemistry associated with oxidized vitamin C (dehydroascorbate, DHA), homocysteine (HcySH) and glutathione (GSH) using mass spectrometry. METHODS: Glutaredoxin-1 (Grx-1) was incubated with DHA, with and without GSH and HcySH. Disulfide formation was followed by electrospray ionization mass spectrometry (ESI-MS) of intact proteins and by LC/ESI-MS/MS of peptides from protein tryptic digestions. The mechanism of DHA-mediated S-thiolation was investigated using two synthetic peptides: AcFHACAAK and AcFHACE. Three proteins, i.e. human hemoglobin (HHb), recombinant peroxiredoxin 2 (Prdx2) and Grx-1, were S-homocysteinylated followed by S-transthiolyation with GSH and investigated by ESI-MS and ESI-MS/MS. RESULTS: ESI-MS analysis reveals that DHA mediates disulfide formation and S-thiolation by HcySH as well as GSH of Grx-1. LC/ESI-MS/MS analysis allows identification of Grx-1 S-thiolated cysteine adducts. The mechanism by which DHA mediates S-thiolation of heptapeptide AcFHACAAK is shown to be via initial formation of a thiohemiketal adduct. In addition, ESI-MS of intact proteins shows that GSH can S-transthiolate S-homocysteinylated Grx-1_ HHb and Prdx2. The GS-S-protein adducts over time dominate the ESI-MS spectrum profile. CONCLUSIONS: Mass spectrometry is a unique analytical technique for probing complex reaction mechanisms associated with oxidative stress. Using model proteins, ESI-MS reveals the mechanism of DHA-facilitated S-thiolation, which consists of thiohemiketal formation, disulfide formation or S-thiolation. Furthermore, protein S-thiolation by HcySH can be reversed by reversible GSH thiol exchange. The use of mass spectrometry with in vitro models of protein S-thiolation in oxidative stress may provide significant insight into possible mechanisms of action occurring in vivo.
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Ácido Deshidroascórbico , Glutatión , Homocisteína , Espectrometría de Masa por Ionización de Electrospray/métodos , Compuestos de Sulfhidrilo/análisis , Ácido Deshidroascórbico/análisis , Ácido Deshidroascórbico/química , Ácido Deshidroascórbico/metabolismo , Glutatión/análisis , Glutatión/química , Glutatión/metabolismo , Homocisteína/análisis , Homocisteína/química , Homocisteína/metabolismo , Humanos , Estrés Oxidativo/fisiología , Proteínas/análisis , Proteínas/química , Proteínas/metabolismo , Compuestos de Sulfhidrilo/química , Compuestos de Sulfhidrilo/metabolismo , Espectrometría de Masas en Tándem/métodosAsunto(s)
Anticuerpos Monoclonales Humanizados , Esofagitis Eosinofílica , Humanos , Esofagitis Eosinofílica/tratamiento farmacológico , Esofagitis Eosinofílica/diagnóstico , Anticuerpos Monoclonales Humanizados/efectos adversos , Anticuerpos Monoclonales Humanizados/uso terapéutico , Niño , Masculino , Femenino , Enfermedades del Sistema Nervioso Periférico/inducido químicamente , Enfermedades del Sistema Nervioso Periférico/diagnósticoRESUMEN
Macrophages provide the first line of host immune defense. Their activation triggers the secretion of pro-inflammatory cytokines and chemokines recruiting other immune cells. In cancer, macrophages present an M2 anti-inflammatory phenotype promoting tumor growth. In this way, strategies need to be develop to reactivate macrophages. Previously thought to be expressed only in cells with a neural/neuroendocrine phenotype, the proprotein convertase 1/3 has been shown to also be expressed in macrophages and regulated as a function of the Toll-like receptor immune response. Here, we investigated the intracellular impact of the down-regulation of the proprotein convertase 1/3 in NR8383 macrophages and confirmed the results on macrophages from PC1/3 deficient mice. A complete proteomic study of secretomes and intracellular proteins was undertaken and revealed that inhibition of proprotein convertase 1/3 orient macrophages toward an M1 activated phenotype. This phenotype is characterized by filopodial extensions, Toll-like receptor 4 MyD88-dependent signaling, calcium entry augmentation and the secretion of pro-inflammatory factors. In response to endotoxin/lipopolysaccharide, these intracellular modifications increased, and the secreted factors attracted naïve T helper lymphocytes to promote the cytotoxic response. Importantly, the application of these factors onto breast and ovarian cancer cells resulted in a decrease viability or resistance. Under inhibitory conditions using interleukin 10, PC1/3-knockdown macrophages continued to secrete inflammatory factors. These data indicate that targeted inhibition of proprotein convertase 1/3 could represent a novel type of immune therapy to reactivate intra-tumoral macrophages.
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Inmunoterapia/métodos , Macrófagos Alveolares/inmunología , Macrófagos Peritoneales/inmunología , Proproteína Convertasa 1/antagonistas & inhibidores , Animales , Linfocitos T CD4-Positivos/efectos de los fármacos , Linfocitos T CD4-Positivos/inmunología , Linfocitos T CD4-Positivos/patología , Linfocitos T CD8-positivos/efectos de los fármacos , Linfocitos T CD8-positivos/inmunología , Linfocitos T CD8-positivos/patología , Calcio/metabolismo , Línea Celular , Línea Celular Tumoral , Citocinas/biosíntesis , Citocinas/inmunología , Regulación de la Expresión Génica , Humanos , Células Asesinas Naturales/efectos de los fármacos , Células Asesinas Naturales/inmunología , Células Asesinas Naturales/patología , Lipopolisacáridos/farmacología , Macrófagos Alveolares/citología , Macrófagos Alveolares/efectos de los fármacos , Macrófagos Peritoneales/citología , Macrófagos Peritoneales/efectos de los fármacos , Ratones , Ratones Noqueados , Factor 88 de Diferenciación Mieloide/genética , Factor 88 de Diferenciación Mieloide/inmunología , Cultivo Primario de Células , Proproteína Convertasa 1/genética , Proproteína Convertasa 1/inmunología , Análisis por Matrices de Proteínas , ARN Interferente Pequeño/genética , ARN Interferente Pequeño/metabolismo , Ratas , Transducción de Señal , Linfocitos T Colaboradores-Inductores/efectos de los fármacos , Linfocitos T Colaboradores-Inductores/inmunología , Linfocitos T Colaboradores-Inductores/patología , Receptor Toll-Like 4/genética , Receptor Toll-Like 4/inmunologíaRESUMEN
Inflammatory bowel disease (IBD) flare-ups exhibit symptoms that are similar to other diseases and conditions, making diagnosis and treatment complicated. Currently, the gold standard for diagnosing and monitoring IBD is colonoscopy and biopsy, which are invasive and uncomfortable procedures, and the fecal calprotectin test, which is not sufficiently accurate. Therefore, it is necessary to develop an alternative method. In this study, our aim was to provide proof of concept for the application of Sequential Window Acquisition of All Theoretical Mass Spectra-Mass spectrometry (SWATH-MS) and machine learning to develop a non-invasive and accurate predictive model using the stool proteome to distinguish between active IBD patients and symptomatic non-IBD patients. Proteome profiles of 123 samples were obtained and data processing procedures were optimized to select an appropriate pipeline. The differentially abundant analysis identified 48 proteins. Utilizing correlation-based feature selection (Cfs), 7 proteins were selected for proceeding steps. To identify the most appropriate predictive machine learning model, five of the most popular methods, including support vector machines (SVMs), random forests, logistic regression, naive Bayes, and k-nearest neighbors (KNN), were assessed. The generated model was validated by implementing the algorithm on 45 prospective unseen datasets; the results showed a sensitivity of 96% and a specificity of 76%, indicating its performance. In conclusion, this study illustrates the effectiveness of utilizing the stool proteome obtained through SWATH-MS in accurately diagnosing active IBD via a machine learning model.
RESUMEN
The proprotein convertase 1/3 is expressed in the regulated secretory pathway of neural and endocrine cells. Its major function is in the post-translational processing and activation of precursor proteins. The PC1/3 knock-out (KO) mouse model has allowed us to elucidate its physiological functions in studies focused primarily on neuroendocrine tissues. However, PC1/3 is also expressed in cells of the immune system, mainly in macrophages. The present study explores the effects of innate immune challenge in the PC1/3 KO mouse. PC1/3 KO mice have an enlarged spleen with marked disorganization of the marginal zone and red pulp. Immunohistochemical studies using various markers demonstrate a depletion of dendritic cells in PC1/3 KO spleens. When challenged with lipopolysaccharide, PC1/3 KO mice are more susceptible to septic shock than wild-type controls or other PC KO mice, such as PC2 and PC7 null mice. Plasma levels of proinflammatory cytokines (IL-6, IL-1ß, and TNF-α) were very significantly elevated in PC1/3 KO mice, consistent with a hypercytokinemia, i.e. indicative of a major systemic uncontrolled inflammatory response or cytokine storm. Peritoneal macrophages isolated from PC1/3 KO mice also demonstrate elevated cytokine secretion when treated with LPS. Electron micrographs show morphological features indicating a prolonged activation of these cells following LPS stimulation. We also present evidence that the proinflammatory T(h)1 pathway is dominant in the PC1/3 KO mouse model. We conclude that aside from its important role in neuroendocrine functions PC1/3 also has an important role in the regulation of the innate immune system, most likely through the regulation of cytokine secretion in macrophages.
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Citocinas/inmunología , Regulación Enzimológica de la Expresión Génica/inmunología , Inmunidad Innata , Macrófagos Peritoneales/inmunología , Proproteína Convertasa 1/inmunología , Animales , Citocinas/metabolismo , Regulación Enzimológica de la Expresión Génica/efectos de los fármacos , Regulación Enzimológica de la Expresión Génica/genética , Enfermedades del Sistema Inmune/genética , Enfermedades del Sistema Inmune/inmunología , Enfermedades del Sistema Inmune/metabolismo , Enfermedades del Sistema Inmune/patología , Inflamación/genética , Inflamación/inmunología , Inflamación/patología , Lipopolisacáridos/farmacología , Macrófagos Peritoneales/metabolismo , Macrófagos Peritoneales/ultraestructura , Ratones , Ratones Noqueados , Proproteína Convertasa 1/biosíntesis , Proproteína Convertasa 1/genética , Células TH1/enzimología , Células TH1/metabolismoRESUMEN
Ascorbate is an important water-soluble antioxidant, which when oxidized by reactive oxygen species is converted into dehydroascorbate (DHA). If not rapidly reduced back to ascorbate, DHA decomposes to a reactive 5-carbon compound (DHA*, +130 Da) that can modify reduced cysteinyl residues in peptides and proteins in vitro. The formation of cysteine adducts by DHA* was characterized by mass spectrometry using reduced insulin B-chain, α-lactalbumin, and hemoglobin. Mass spectrometry of DHA* modified insulin B-chain revealed the presence of one and two DHA* adducts. Enzymatic cleavage and tandem mass spectrometry of modified peptides allowed unambiguous localization of DHA* to the two cysteine residues in positions 7 and 19 of the insulin B-chain. Incubations of DHA with α-lactalbumin revealed that approximately 25% of the protein population was in a reduced state and could be modified by DHA*. The adduct was assigned to the N-terminally located cysteinyl residue in position 6. Incubation of hemoglobin with DHA followed by pepsin digestion and electrospray ionization tandem mass spectrometry (ESI-MSMS) of the peptide mixture allowed for the identification of three modified peptides. Tandem mass spectrometry of the modified peptides, two from the hemoglobin A-chain with identical mass and one from the hemoglobin B-chain, gave a complete series of y-type fragment ions, which were assigned to the cysteine containing peptides (100)LLSHCL(105) (A-chain), (101)LSHCLL(106) (A-chain), and (111)VCVLAHHFGKE(121) (B-chain). Although the DHA* adduct was lost from the peptides derived from α-lactalbumin and hemoglobin before fragmentation of the peptide bond, carbamidomethylation of the proteins prior to incubation with DHA abolished the formation of DHA*-protein adducts and confirmed that the target was indeed the cysteine thiol group. Future studies are focused on the modification of proteins by DHA* in cells and in vivo.
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Ácido Ascórbico/química , Cisteína/química , Hemoglobinas/química , Lactalbúmina/química , Receptor de Insulina/química , Compuestos de Sulfhidrilo/química , Ácido Ascórbico/metabolismo , Cisteína/metabolismo , Hemoglobinas/metabolismo , Lactalbúmina/metabolismo , Receptor de Insulina/metabolismo , Compuestos de Sulfhidrilo/metabolismoRESUMEN
BACKGROUND: In many parts of the world, it is common for secondary school students to be involved in part-time employment. Research shows that working can have a negative impact on school engagement. However, the majority of studies have focused on the amount of time that students spend working rather than on the quality of work experience and its influence on school engagement. AIMS: This study explored the relation of part-time work and school experiences to dropout intentions among secondary school and junior college students. The study was conceptualized from a self-determination theory perspective (Deci & Ryan, 2000). SAMPLE: Participants were 3,248 students from rural and suburban schools in the greater region of Montreal, Canada. METHOD: Questionnaires were used to assess the number of hours worked, the extent to which work interfered with or facilitated school functioning, autonomy, competence, and relatedness experienced in the work and school domains. School performance and school dropout intentions were also assessed. RESULTS: A curvilinear relation between work hours and dropout intentions was found, reflecting that part-time work began to be associated with higher dropout intentions only when students worked more than 7 hr per week. Analyses also showed that work-school interference was related to dropout intentions, and that this variable served to mediate the relation of employer autonomy support to dropout intentions. CONCLUSIONS: These results suggest that both the quantity and the quality of students' part-time work experiences need to be considered when examining the relation of work to school engagement.
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Logro , Empleo/psicología , Motivación , Autonomía Personal , Abandono Escolar/psicología , Adolescente , Adulto , Femenino , Humanos , Intención , Masculino , Quebec , Análisis de Regresión , Población Rural , Población Suburbana , Tolerancia al Trabajo Programado , Adulto JovenRESUMEN
Calnuc is an ubiquitous Ca(++)-binding protein found in the cytoplasm where it binds different Galpha subunits, in the Golgi lumen where it constitutes a major Ca(++) storage pool, and outside the cell. We identified LDLR-related protein 9 (LRP9) as the first transmembrane protein shown to interact directly with Calnuc. LRP9 is a member of a new subfamily of the LDLR superfamily that cycles between the trans-Golgi network (TGN) and endosomes through a mechanism dependent on clathrin adaptor GGA proteins. The aim of the present study was to characterize the interaction between Calnuc and LRP9. Various biochemical assays showed that the N-terminus of Calnuc interacts with an arginine-rich region in the cytosolic tail of LRP9. Confocal microscopy showed that Calnuc colocalizes with LRP9 at the surface of the TGN and early endosomes. Depletion of Calnuc by small interfering RNA (siRNA) missorted LRP9 in the late endosome/lysosome compartments and enhanced its lysosomal degradation. This phenotype was rescued by the expression of siRNA-resistant wild-type Calnuc as well as cytoplasmic Calnuc, indicating that the cytoplasmic pool of Calnuc is involved in LRP9 endosomal sorting to prevent the delivery of LRP9 to lysosomes. This is the first report showing that Calnuc plays a role in receptor trafficking.
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Proteínas de Unión al Calcio/metabolismo , Proteínas de Unión al ADN/metabolismo , Endosomas/metabolismo , Proteínas Relacionadas con Receptor de LDL/metabolismo , Proteínas de Transporte de Membrana/metabolismo , Transporte de Proteínas/fisiología , Secuencia de Aminoácidos , Animales , Proteínas de Unión al Calcio/genética , Línea Celular , Proteínas de Unión al ADN/genética , Aparato de Golgi/metabolismo , Aparato de Golgi/ultraestructura , Humanos , Proteínas Relacionadas con Receptor de LDL/genética , Lisosomas/metabolismo , Proteínas de Transporte de Membrana/genética , Ratones , Datos de Secuencia Molecular , Proteínas del Tejido Nervioso , Nucleobindinas , Unión Proteica , ARN Interferente Pequeño/genética , ARN Interferente Pequeño/metabolismo , Ratas , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/metabolismo , Alineación de Secuencia , Técnicas del Sistema de Dos Híbridos , Red trans-Golgi/metabolismoRESUMEN
TRPC proteins become involved in Ca2+ entry following the activation of Gq-protein coupled receptors. TRPC6 is inserted into the plasma membrane upon stimulation and remains in the plasma membrane as long as the stimulus is present. However, the mechanism that regulates the trafficking of TRPC6 is unclear. In the present study, we highlighted the involvement of two Rab GTPases in the trafficking of TRPC6. Rab9 co-localized in vesicular structures with TRPC6 in HeLa cells and co-immunoprecipitated with TRPC6. When co-expressed with TRPC6, Rab9(S21N), a dominant negative mutant, caused an increase in the level of TRPC6 at the plasma membrane and in TRPC6-mediated Ca2+ entry upon activation by a muscarinic receptor agonist. Similarly, the expression of Rab11 also caused an increase in TRPC6 expression at the cell surface and an increase in TRPC6-mediated Ca2+ entry. The co-expression of TRPC6 with the dominant negative mutant Rab11(S25N) abolished CCh-induced TRPC6 activation and reduced the level of TRPC6 at the plasma membrane. This study demonstrates that the trans-Golgi network and recycling endosomes are involved in the intracellular trafficking of TRPC6 by regulating channel density at the cell surface.
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Canales Catiónicos TRPC/metabolismo , Proteínas de Unión al GTP rab/metabolismo , Animales , Transporte Biológico/fisiología , Membrana Celular/metabolismo , Células HeLa , Humanos , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/metabolismo , Canales Catiónicos TRPC/genética , Canal Catiónico TRPC6 , Proteínas de Unión al GTP rab/genéticaRESUMEN
Furin plays an important role in various pathological states, especially in bacterial and viral infections. A detailed understanding of the structural requirements for inhibitors targeting this enzyme is crucial to develop new therapeutic strategies in infectious diseases, including an urgent unmet need for SARS-CoV-2 infection. Previously, we have identified a potent furin inhibitor, peptide Ac-RARRRKKRT-NH 2 (CF1), based on the highly pathogenic avian influenza hemagglutinin. The goal of this study was to determine how its N-terminal part (the P8-P5 positions) affects its activity profile. To do so, the positional-scanning libraries of individual peptides modified at the selected positions with natural amino acids were generated. Subsequently, the best substitutions were combined together and/or replaced by unnatural residues to expand our investigations. The results reveal that the affinity of CF1 can be improved (2-2.5-fold) by substituting its P5 position with the small hydrophobic residues (Ile or Val) or a basic Lys.
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Self-determination theory posits that satisfaction of three basic psychological needs-autonomy, competence, and relatedness-are required for psychological well-being, and a recent study showed that the balance in the satisfaction of these three needs independently affects well-being. The present investigation builds on these findings by examining the balance of adolescents' need satisfaction across distinct life contexts. The results of three studies show that adolescents who experience a balance of need satisfaction across important life contexts, including at school, at home, with friends, and in part-time jobs, reported higher well-being and better school adjustment. This finding emerged consistently across four countries and across multiple measures of school adjustment, including teacher reports. Together, these results support previous research that highlights the importance of consistency for psychological functioning.
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Autonomía Personal , Psicología del Adolescente , Calidad de Vida/psicología , Autoeficacia , Ajuste Social , Identificación Social , Logro , Adolescente , California , Niño , China , Comparación Transcultural , Depresión/psicología , Femenino , Francia , Amigos/psicología , Humanos , Individualidad , Intención , Control Interno-Externo , Satisfacción en el Trabajo , Masculino , Determinación de la Personalidad , Quebec , Medio Social , Abandono Escolar/psicologíaRESUMEN
Ascorbic acid (vitamin C) plays a significant role in the prevention of oxidative stress. In this process, ascorbate is oxidized to dehydroascorbate (DHA). We have investigated the impact of DHA on peptide/protein intramolecular disulfide formation as well as S-glutathionylation and S-homocysteinylation. S-glutathionylation of peptides/proteins is a reversible, potential regulatory mechanism in oxidative stress. Although the exact role of protein S-homocysteinylation is unknown, it has been proposed to be of importance in pathobiological processes such as onset of cardiovascular disease. Using an in vitro model system, we demonstrate that DHA causes disulfide bond formation within the active site of recombinant human glutaredoxin (Grx-1). DHA also facilities the formation of S-glutathionylation and S-homocysteinylation of a model peptide (AcFHACAAK) as well as Grx-1. We discuss the possible mechanisms of peptide/protein S-thiolation, which can occur either via thiol exchange or a thiohemiketal intermediate. A thiohemiketal DHA-peptide adduct was detected by mass spectrometry and its location on the peptide/protein cysteinyl thiol group was unambiguously confirmed by tandem mass spectrometry. This demonstrates that peptide/protein S-thiolation mediated by DHA is not limited to thiol exchange reactions but also takes place directly via the formation of a thiohemiketal peptide intermediate. Finally, we investigated a potential reducing role of glutathione (GSH) in the presence of S-homocysteinylated peptide/protein adducts. S-homocysteinylated AcFHACAAK, human hemoglobin α-chain and Grx-1 were incubated with GSH. Both peptide and proteins were reduced, and homocysteine replaced with GS-adducts by thiol exchange, as a function of time.
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Ácido Deshidroascórbico/química , Glutarredoxinas/química , Glutatión/química , Homocisteína/química , Péptidos/química , Compuestos de Sulfhidrilo/química , Antioxidantes/química , Dominio Catalítico , Cisteína/química , Dimerización , Disulfuros/química , Hemoglobinas/química , Humanos , Oxidación-Reducción , Estrés OxidativoRESUMEN
Although the self-concordance of goals has been repeatedly shown to predict better goal progress, recent research suggests potential problems with aggregating autonomous and controlled motivations to form a summary index of self-concordance (Judge, Bono, Erez, & Locke, 2005). The purpose of the present investigation was to further examine the relations among autonomous motivation, controlled motivation, and goal progress to determine the relative importance of autonomous motivation and controlled motivation in the pursuit of personal goals. The results of three studies and a meta-analysis indicated that autonomous motivation was substantially related to goal progress whereas controlled motivation was not. Additionally, the relation of autonomous motivation to goal progress was shown to involve implementation planning. Together, the three studies highlight the importance for goal setters of having autonomous motivation and developing implementation plans, especially ones formulated in terms of approach strategies rather than avoidance strategies. The present research suggests that individuals pursuing goals should focus relatively greater attention on enhancing their autonomous motivation rather than reducing their controlled motivation.
Asunto(s)
Objetivos , Individualidad , Control Interno-Externo , Motivación , Solución de Problemas , Logro , Adolescente , Adulto , Femenino , Humanos , Intención , Masculino , Pérdida de Peso , Adulto JovenRESUMEN
The eosinophilia-myalgia syndrome (EMS) outbreak of 1989 that occurred in the USA and elsewhere was caused by the ingestion of l-Tryptophan (L-Trp) solely manufactured by the Japanese company Showa Denko K.K. (SD). Six compounds present in the SD L-Trp were reported to be case-associated contaminants. However, "one" of these compounds, Peak AAA has remained structurally uncharacterized, despite the fact that it was described as "the only statistically significant (p=0.0014) contaminant". Here, we employ on-line microcapillary-high performance liquid chromatography-electrospray ionization mass spectrometry (LC-MS), and tandem mass spectrometry (MS/MS) to determine that Peak AAA is in fact two structurally related isomers. Peak AAA1 and Peak AAA2 differed in LC retention times, and were determined by accurate mass-LC-MS to both have a protonated molecular ion (MH+) of mass 343.239Da (Da), corresponding to a molecular formula of C21H30N2O2, and possessing eight degrees of unsaturation (DoU) for the non-protonated molecule. By comparing the LC-MS and LC-MS-MS retention times and spectra with authentic synthetic standards, Peak AAA1 was identified as the intermolecular condensation product of L-Trp with anteiso 7-methylnonanoic acid, to afford (S)-2-amino-3-(2-((S,E)-7-methylnon-1-en-1-yl)-1H-indol-3-yl)propanoic acid. Peak AAA2 was determined to be a condensation product of L-Trp with decanoic acid, which produced (S)-2-amino-3-(2-((E)-dec-1-en-1-yl)-1H-indol-3-yl)propanoic acid.
Asunto(s)
Contaminación de Medicamentos , Síndrome de Eosinofilia-Mialgia/inducido químicamente , Triptófano/análogos & derivados , Triptófano/química , Cromatografía Líquida de Alta Presión , Suplementos Dietéticos , Humanos , Espectrometría de Masas , Espectrometría de Masas en Tándem , Triptófano/efectos adversos , Triptófano/aislamiento & purificaciónRESUMEN
The eosinophilia-myalgia syndrome (EMS) outbreak that occurred in the USA and elsewhere in 1989 was caused by the ingestion of Showa Denko K.K. (SD) L-tryptophan (L-Trp). "Six compounds" detected in the L-Trp were reported as case-associated contaminants. Recently the final and most statistically significant contaminant, "Peak AAA" was structurally characterized. The "compound" was actually shown to be two structural isomers resulting from condensation reactions of L-Trp with fatty acids derived from the bacterial cell membrane. They were identified as the indole C-2 anteiso (AAA1-343) and linear (AAA2-343) aliphatic chain isomers. Based on those findings, we utilized a combination of on-line HPLC-electrospray ionization mass spectrometry (LC-MS), as well as both precursor and product ion tandem mass spectrometry (MS/MS) to facilitate identification of a homologous family of condensation products related to AAA1-343 and AAA2-343. We structurally characterized eight new AAA1-XXX/AAA2-XXX contaminants, where XXX represents the integer molecular ions of all the related homologs, differing by aliphatic chain length and isomer configuration. The contaminants were derived from the following fatty acids of the bacterial cell membrane, 5-methylheptanoic acid (anteiso-C8:0) for AAA1-315; n-octanoic acid (n-C8:0) for AAA2-315; 6-methyloctanoic acid (anteiso-C9:0) for AAA1-329; n-nonanoic acid (n-C9:0) for AAA2-329; 10-methyldodecanoic acid (anteiso-C13:0) for AAA1-385; n-tridecanoic acid (n-C13:0) for AAA2-385; 11-methyltridecanoic acid (anteiso-C14:0) for AAA1-399; and n-tetradecanoic acid (n-C14:0) for AAA2-399. The concentration levels for these contaminants were estimated to be 0.1-7.9⯵g / 500â¯mg of an individual SD L-Trp tablet or capsule The structural similarity of these homologs to case-related contaminants of Spanish Toxic Oil Syndrome (TOS) is discussed.
Asunto(s)
Suplementos Dietéticos/análisis , Síndrome de Eosinofilia-Mialgia/inducido químicamente , Ácidos Grasos/toxicidad , Contaminación de Alimentos , Indoles/toxicidad , Triptófano/análogos & derivados , Bacillus amyloliquefaciens/metabolismo , Caprilatos/análisis , Caprilatos/química , Caprilatos/aislamiento & purificación , Caprilatos/toxicidad , Centers for Disease Control and Prevention, U.S. , Cromatografía Líquida de Alta Presión , Suplementos Dietéticos/efectos adversos , Ácidos Grasos/análisis , Ácidos Grasos/química , Ácidos Grasos/aislamiento & purificación , Fermentación , Ácidos Heptanoicos/análisis , Ácidos Heptanoicos/química , Ácidos Heptanoicos/aislamiento & purificación , Ácidos Heptanoicos/toxicidad , Humanos , Indoles/análisis , Indoles/química , Indoles/aislamiento & purificación , Ácidos Láuricos/análisis , Ácidos Láuricos/química , Ácidos Láuricos/aislamiento & purificación , Ácidos Láuricos/toxicidad , Metilación , Estructura Molecular , Miristatos/análisis , Miristatos/química , Miristatos/aislamiento & purificación , Miristatos/toxicidad , Espectrometría de Masa por Ionización de Electrospray , Estereoisomerismo , Triptófano/análisis , Triptófano/química , Triptófano/aislamiento & purificación , Estados UnidosRESUMEN
Chymase, a mast cell serine protease involved in the generation of multiple cardiovascular factors, such as angiotensin II and endothelin-1 (ET-1), is elevated and participates in tissue degeneration after permanent myocardial infarction (PMI). Anesthetized 4-month old male wild-type (WT) C57BL/6J mice and mouse mast cell protease-4 knockout (mMCP-4 KO) congeners were subjected to ligation of the left anterior descending (LAD) coronary artery. A group of mice was then subjected to Kaplan-Meier 28-day survival analysis. In another group of mice, 18F-fluorodeoxyglucose positron emission tomography (PET) was performed to evaluate heart function and the infarcted zone 3 days post-PMI surgery. Cardiac morphology following PMI was evaluated on formalin-fixed heart slices and glycoproteomic analysis was performed using mass spectrometry. Finally, cardiac and lung tissue content of immunoreactive ET-1 was determined. PMI caused 60% mortality in WT mice, due to left ventricular wall rupture, and 7% in mMCP-4 KO mice. Cardiac PET analysis revealed a significant reduction in left ventricular volume (systolic and diastolic) and preserved the ejection fraction in mMCP-4 KO compared to WT animals. The infarcted area, apoptotic signaling and wall remodeling were significantly decreased in mMCP-4 KO mice compared to their WT congeners, while collagen deposition was increased. Glycoproteomic analysis showed an increase in apolipoprotein A1, an established chymase substrate in mMCP-4 KO mice compared to WT mice post-PMI. ET-1 levels were increased in the lungs of WT, but not mMCP-4 KO mice, 24 h post-PMI. Thus, the genetic deletion of mMCP-4 improved survival and heart function post-PMI.