RESUMEN
15-Lipoxygenase (15-LO) is a nonheme iron-containing dioxygenase that has both pro- and anti-inflammatory roles in many tissues and disease states. 15-LO is thought to influence macrophage phenotype, and silencing 15-LO reduces fibrosis after acute inflammatory triggers. The goal of the present study was to determine whether altering 15-LO expression influences inflammation and fibrogenesis in a murine model of unilateral ureteral obstruction (UUO). C57BL/6J mice, 15-LO knockout (Alox15-/-) mice, and 15-LO transgenic overexpressing (15LOTG) mice were subjected UUO, and kidneys were analyzed at 3, 10, and 14 days postinjury. Histology for fibrosis, inflammation, cytokine quantification, flow cytometry, and metabolomics were performed on injured tissues and controls. PD146176, a specific 15-LO inhibitor, was used to complement experiments involving knockout animals. Compared with wild-type animals undergoing UUO, Alox15-/- mouse kidneys had less proinflammatory, profibrotic message along with less fibrosis and macrophage infiltration. PD146176 inhibited 15-LO and resulted in reduced fibrosis and macrophage infiltration similar to Alox15-/- mice. Flow cytometry revealed that Alox15-/- UUO-injured kidneys had a dynamic change in macrophage phenotype, with an early blunting of CD11bHiLy6CHi "M1" macrophages and an increase in anti-inflammatory CD11bHiLy6CInt "M2c" macrophages and reduced expression of the fractalkine receptor chemokine (C-X3-C motif) receptor 1. Many of these findings were reversed when UUO was performed on 15LOTG mice. Metabolomics analysis revealed that wild-type kidneys developed a glycolytic shift postinjury, while Alox15-/- kidneys exhibited increased oxidative phosphorylation. In conclusion, 15-LO manipulation by genetic or pharmacological means induces dynamic changes in the inflammatory microenvironment in the UUO model and appears to be critical in the progression of UUO-induced fibrosis.NEW & NOTEWORTHY 15-Lipoxygenase (15-LO) has both pro- and anti-inflammatory functions in leukocytes, and its role in kidney injury and repair is unexplored. Our study showed that 15-LO worsens inflammation and fibrosis in a rodent model of chronic kidney disease using genetic and pharmacological manipulation. Silencing 15-LO promotes an increase in M2c-like wound-healing macrophages in the kidney and alters kidney metabolism globally, protecting against anaerobic glycolysis after injury.
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Araquidonato 12-Lipooxigenasa/metabolismo , Araquidonato 15-Lipooxigenasa/metabolismo , Citocinas/metabolismo , Metabolismo Energético , Mediadores de Inflamación/metabolismo , Riñón/enzimología , Metaboloma , Nefritis/etiología , Obstrucción Ureteral/complicaciones , Animales , Araquidonato 12-Lipooxigenasa/genética , Araquidonato 15-Lipooxigenasa/genética , Microambiente Celular , Citocinas/genética , Modelos Animales de Enfermedad , Metabolismo Energético/efectos de los fármacos , Fibrosis , Riñón/efectos de los fármacos , Riñón/patología , Leucocitos/enzimología , Inhibidores de la Lipooxigenasa/farmacología , Macrófagos/enzimología , Masculino , Ratones Endogámicos C57BL , Ratones Noqueados , Nefritis/enzimología , Nefritis/patología , Nefritis/prevención & control , Fenotipo , Obstrucción Ureteral/tratamiento farmacológico , Obstrucción Ureteral/enzimología , Obstrucción Ureteral/patologíaRESUMEN
For a long time, proteins with enzymatic activity have not been usually considered to carry out other functions different from catalyzing chemical reactions within or outside the cell. Nevertheless, in the last few years several reports have uncovered the participation of numerous enzymes in other processes, placing them in the category of moonlighting proteins. Some moonlighting enzymes have been shown to participate in complex processes such as cell adhesion. Cell adhesion plays a physiological role in multiple processes: it enables cells to establish close contact with one another, allowing communication; it is a key step during cell migration; it is also involved in tightly binding neighboring cells in tissues, etc. Importantly, cell adhesion is also of great importance in pathophysiological scenarios like migration and metastasis establishment of cancer cells. Cell adhesion is strictly regulated through numerous switches: proteins, glycoproteins and other components of the cell membrane. Recently, several cell membrane enzymes have been reported to participate in distinct steps of the cell adhesion process. Here, we review a variety of examples of membrane bound enzymes participating in adhesion of immune cells.
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Adhesión Celular/fisiología , Leucocitos/enzimología , 5'-Nucleotidasa/inmunología , 5'-Nucleotidasa/fisiología , Proteínas ADAM/inmunología , Proteínas ADAM/fisiología , ADP-Ribosil Ciclasa/inmunología , ADP-Ribosil Ciclasa/fisiología , ADP-Ribosil Ciclasa 1/inmunología , ADP-Ribosil Ciclasa 1/fisiología , Antígenos CD/inmunología , Antígenos CD/fisiología , Antígenos CD13/inmunología , Antígenos CD13/fisiología , Adhesión Celular/inmunología , Membrana Celular/enzimología , Membrana Celular/inmunología , Dipeptidil Peptidasa 4/inmunología , Dipeptidil Peptidasa 4/fisiología , Proteínas Ligadas a GPI/inmunología , Proteínas Ligadas a GPI/fisiología , Humanos , Leucocitos/inmunología , Leucocitos/fisiología , Glicoproteínas de Membrana/inmunología , Glicoproteínas de Membrana/fisiología , Proteínas de la Membrana/inmunología , Proteínas de la Membrana/fisiología , Modelos BiológicosRESUMEN
BACKGROUND: Polycyclic aromatic hydrocarbons (PAHs) exposure had been reported to be a risk factor of mtDNAcn in our early study. However, the effect of metabolic enzymes' genetic polymorphisms on mtDNAcn in PAHs-Exposure workers has not been fully evaluated. AIM: The aim of the study was to explore the effect of metabolic enzymes' genetic polymorphisms on mtDNAcn in PAHs-Exposure. METHODS AND RESULTS: We investigated the effects of metabolic enzymes' genetic polymorphisms on mtDNAcn among 544 coke oven workers and 238 office staffs. The mtDNAcn of peripheral blood leukocytes was measured using the Real-time quantitative polymerase chain reaction (PCR) method. PCR and restriction fragment length was used to detect five polymorphisms in GSTT1, GSTM1, GSTP1 rs1695, CYP2E1 rs6413432, and CYP2E1 rs3813867. The mtDNAcn in peripheral blood leukocytes was significantly lower in the exposure group than that in the control group (p < .001). The 1-OHPYR had an increasing trend with the genotypes AAâAG â GG of GSTP1 rs1695 in the control group. Generalized linear model indicated that the influencing factors of mtDNAcn were PAHs-exposure [ß (95% CI) = -0.420 (-0.469, -0.372), p < .001], male [ß (95% CI) = -0.058 (-0.103, -0.012), p = .013], and AA genotype for GSTP1 rs1695 [ß (95% CI) = -0.051 (-0.095, -0.008), p = .020]. CONCLUSION: The individuals carrying the AA genotype of GSTP1 rs1695 may have a lower mtDNAcn due to their weaker detoxification of PAHs.
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Coque/efectos adversos , Variaciones en el Número de Copia de ADN/efectos de los fármacos , Gutatión-S-Transferasa pi/genética , Glutatión Transferasa/genética , Exposición Profesional/efectos adversos , Hidrocarburos Policíclicos Aromáticos/efectos adversos , Adolescente , Adulto , Estudios de Casos y Controles , China , Citocromo P-450 CYP2E1/genética , Citocromo P-450 CYP2E1/metabolismo , ADN Mitocondrial/genética , Femenino , Gutatión-S-Transferasa pi/metabolismo , Glutatión Transferasa/metabolismo , Voluntarios Sanos , Humanos , Leucocitos/enzimología , Masculino , Persona de Mediana Edad , Hidrocarburos Policíclicos Aromáticos/metabolismo , Polimorfismo de Nucleótido Simple , Adulto JovenRESUMEN
Shortened telomeres have been linked to numerous chronic diseases, most importantly coronary artery disease, but the underlying mechanisms remain ill defined. Loss-of-function mutations and deletions in telomerase both accelerate telomere shortening but do not necessarily lead to a clinical phenotype associated with atherosclerosis, questioning the causal role of telomere length in cardiac pathology. The differential extranuclear functions of the 2 main components of telomerase, telomerase reverse transcriptase and telomerase RNA component, offer important clues about the complex relationship between telomere length and cardiovascular pathology. In this review, we critically discuss relevant preclinical models, genetic disorders, and clinical studies to elucidate the impact of telomerase in cardiovascular disease and its potential role as a therapeutic target. We suggest that the antioxidative function of mitochondrial telomerase reverse transcriptase might be atheroprotective, making it a potential target for clinical trials. Graphic Abstract: A graphic abstract is available for this article.
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Enfermedades Cardiovasculares/enzimología , Enfermedades Cardiovasculares/terapia , Telomerasa/metabolismo , Animales , Biomarcadores/sangre , Enfermedades Cardiovasculares/sangre , Ensayos Clínicos como Asunto , Medicamentos Herbarios Chinos/uso terapéutico , Ejercicio Físico , Estudio de Asociación del Genoma Completo , Humanos , Inhibidores de Hidroximetilglutaril-CoA Reductasas/uso terapéutico , Leucocitos/enzimología , Ratones , Modelos Cardiovasculares , Mutación , ARN/genética , Telomerasa/sangre , Telomerasa/genética , Homeostasis del Telómero/fisiología , Acortamiento del Telómero/fisiologíaRESUMEN
Troxerutin is a natural flavonoid present abundantly in tea, coffee, olives, wheat, and a variety of fruits and vegetables. Due to its diverse pharmacological properties, this flavonoid has aroused interest for treatment of various diseases, and consequently prompted investigation into its toxicological characteristics. The aim of this study was to evaluate the genotoxic and mutagenic effects and chemoprotective activity attributed to troxerutin using human peripheral blood leukocytes (PBLs) through several well-established experimental protocols based upon different parameters. Data demonstrated that troxerutin (100 to 1000 µM) induced no marked cytotoxic effect on PBLs after 24 hr, and did not produce strand breaks and mutagenicity. Regarding chemoprevention, this flavonoid attenuated cytotoxicity, genotoxicity, and mutagenicity initiated by hydrogen peroxide (H2O2) in human PBLs. Further, troxerutin demonstrated no marked cytotoxic effect on PBLs and exerted a protective effect against oxidative stress induced by H2O2 through modulation of GSH-dependent enzymes.
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Glutatión/metabolismo , Peróxido de Hidrógeno/farmacología , Hidroxietilrutósido/análogos & derivados , Leucocitos/fisiología , Oxidantes/farmacología , Estrés Oxidativo/efectos de los fármacos , Sustancias Protectoras/farmacología , Anticoagulantes/farmacología , Humanos , Hidroxietilrutósido/farmacología , Leucocitos/efectos de los fármacos , Leucocitos/enzimologíaRESUMEN
Pompe disease is a lysosomal and neuromuscular disorder caused by deficiency of acid alpha-glucosidase (GAA), and causes classic infantile, childhood onset, or adulthood onset phenotypes. The biochemical diagnosis is based on GAA activity assays in dried blood spots, leukocytes, or fibroblasts. Diagnosis can be complicated by the existence of pseudodeficiencies, i.e., GAA variants that lower GAA activity but do not cause Pompe disease. A large-scale comparison between these assays for patient samples, including exceptions and borderline cases, along with clinical diagnoses has not been reported so far. Here we analyzed GAA activity in a total of 1709 diagnostic cases over the past 28 years using a total of 2591 analyses and we confirmed the clinical diagnosis in 174 patients. We compared the following assays: leukocytes using glycogen or 4MUG as substrate, fibroblasts using 4MUG as substrate, and dried blood spots using 4MUG as substrate. In 794 individuals, two or more assays were performed. We found that phenotypes could only be distinguished using fibroblasts with 4MUG as substrate. Pseudodeficiencies caused by the GAA2 allele could be ruled out using 4MUG rather than glycogen as substrate in leukocytes or fibroblasts. The Asian pseudodeficiency could only be ruled out in fibroblasts using 4MUG as substrate. We conclude that fibroblasts using 4MUG as substrate provides the most reliable assay for biochemical diagnosis and can serve to validate results from leukocytes or dried blood spots.
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Pruebas Enzimáticas Clínicas/métodos , Pruebas con Sangre Seca/métodos , Pruebas Genéticas/métodos , Enfermedad del Almacenamiento de Glucógeno Tipo II/genética , Células Cultivadas , Pruebas Enzimáticas Clínicas/estadística & datos numéricos , Pruebas con Sangre Seca/estadística & datos numéricos , Fibroblastos/enzimología , Fibroblastos/metabolismo , Pruebas Genéticas/estadística & datos numéricos , Enfermedad del Almacenamiento de Glucógeno Tipo II/diagnóstico , Enfermedad del Almacenamiento de Glucógeno Tipo II/metabolismo , Humanos , Himecromona/análogos & derivados , Himecromona/metabolismo , Leucocitos/enzimología , Leucocitos/metabolismo , Mutación , alfa-Glucosidasas/genética , alfa-Glucosidasas/metabolismoRESUMEN
Urinary tract infection (UTI) is one of the most common bacterial infections in children. The symptoms of UTI in young children are nonspecific, therefore urine should be examined whenever UTI cannot be ruled out. In clinical settings, however, collecting urine from children who are not toilet trained is sometimes difficult, presenting a challenge in UTI management. Here, we developed a "diaper UTI test", which enables the quick detection of pyuria in ordinary diapers, and investigated its sensitivity and specificity in a clinical study. The diaper UTI test is based on a leukocyte esterase reaction. Reagent was prepared in liquid form so that it can be absorbed by disposable diapers, where it will produce a violet color in the presence of pyuria. For the clinical study, we enrolled children younger than 3 years with potential UTI who underwent bladder catheterization for urine culture and urinalysis. Of the 65 children included, 21 were diagnosed with UTI. The sensitivity and specificity of the diaper UTI test were 90.5% (95% CI 69.6-98.8) and 93.2% (95% CI 81.3-98.6), respectively. Because of its convenience and good sensitivity, the diaper UTI test may be useful in the screening of pediatric UTI.
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Pañales Infantiles , Infecciones Urinarias/diagnóstico , Hidrolasas de Éster Carboxílico/química , Preescolar , Color , Femenino , Humanos , Hidrólisis , Lactante , Recién Nacido , Leucocitos/enzimología , Masculino , Polímeros/química , Estudios Prospectivos , Sensibilidad y Especificidad , Urinálisis/métodos , Cateterismo UrinarioRESUMEN
The nuclear receptor-binding SET domain (NSD) family of histone methyltransferases is associated with various malignancies, including aggressive acute leukemia with NUP98-NSD1 translocation. While NSD proteins represent attractive drug targets, their catalytic SET domains exist in autoinhibited conformation, presenting notable challenges for inhibitor development. Here, we employed a fragment-based screening strategy followed by chemical optimization, which resulted in the development of the first-in-class irreversible small-molecule inhibitors of the nuclear receptor-binding SET domain protein 1 (NSD1) SET domain. The crystal structure of NSD1 in complex with covalently bound ligand reveals a conformational change in the autoinhibitory loop of the SET domain and formation of a channel-like pocket suitable for targeting with small molecules. Our covalent lead-compound BT5-demonstrates on-target activity in NUP98-NSD1 leukemia cells, including inhibition of histone H3 lysine 36 dimethylation and downregulation of target genes, and impaired colony formation in an NUP98-NSD1 patient sample. This study will facilitate the development of the next generation of potent and selective inhibitors of the NSD histone methyltransferases.
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Antineoplásicos/farmacología , Inhibidores Enzimáticos/farmacología , Regulación Leucémica de la Expresión Génica , N-Metiltransferasa de Histona-Lisina/antagonistas & inhibidores , Leucocitos/efectos de los fármacos , Proteínas de Complejo Poro Nuclear/genética , Proteínas de Fusión Oncogénica/antagonistas & inhibidores , Antineoplásicos/síntesis química , Sitios de Unión , Inhibidores Enzimáticos/síntesis química , N-Metiltransferasa de Histona-Lisina/genética , N-Metiltransferasa de Histona-Lisina/metabolismo , Proteínas de Homeodominio/genética , Proteínas de Homeodominio/metabolismo , Humanos , Cinética , Leucemia/tratamiento farmacológico , Leucemia/enzimología , Leucemia/genética , Leucemia/patología , Leucocitos/enzimología , Leucocitos/patología , Modelos Moleculares , Proteína 1 del Sitio de Integración Viral Ecotrópica Mieloide/genética , Proteína 1 del Sitio de Integración Viral Ecotrópica Mieloide/metabolismo , Proteínas de Complejo Poro Nuclear/metabolismo , Proteínas de Fusión Oncogénica/genética , Proteínas de Fusión Oncogénica/metabolismo , Unión Proteica , Conformación Proteica en Hélice alfa , Conformación Proteica en Lámina beta , Dominios y Motivos de Interacción de Proteínas , Transducción de Señal , Especificidad por Sustrato , Células Tumorales CultivadasRESUMEN
OBJECTIVE: Experimental evidence suggests a close link between PARP (poly[ADP-ribose] polymerase) activation and diabetic endothelial dysfunction. Here, we tested whether PARP activity in circulating leukocytes was associated with coronary artery disease (CAD) among patients with type 2 diabetes mellitus (T2DM). Approach and Results: We performed observational and bidirectional Mendelian randomization studies of 3149 Chinese individuals with T2DM who underwent coronary angiography, with leukocyte PARP activity, 16 tag single-nucleotide polymorphisms in PARP1 and PARP2, and 17 CAD risk single-nucleotide polymorphisms analyzed. Of 3149 participants, 1180 who further received percutaneous coronary intervention were prospectively followed for 1 year to track major adverse cardiovascular and cerebrovascular events. Overall, greater PARP activity was cross-sectionally associated with an odds ratio of 1.23 for obstructive CAD, and prospectively with a hazard ratio of 1.34 for 1-year major adverse cardiovascular and cerebrovascular events after percutaneous coronary intervention (both P<0.001). Using a genetic score of 5 screened single-nucleotide polymorphisms in PARP1 and PARP2 as the instrumental variable, genetically predicted elevation in PARP activity showed a causal association with obstructive CAD (odds ratio=1.35, P<0.001). In contrast, the genetic risk of CAD had no significant effect on PARP activity. Ex vivo and in vitro cultures of human monocytes showed that rs747657, as the lead single-nucleotide polymorphism strongly associated with PARP activity, caused the differential binding of transcription factor GATA2 (GATA-binding protein 2) to an intronic regulatory region in PARP1, thus modulating PARP1 expression and PARP activity. CONCLUSIONS: Greater PARP activity may have causal roles in the development of obstructive CAD among patients with diabetes mellitus.
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Enfermedad de la Arteria Coronaria/enzimología , Estenosis Coronaria/enzimología , Diabetes Mellitus Tipo 2/enzimología , Leucocitos/enzimología , Poli(ADP-Ribosa) Polimerasa-1/sangre , Poli(ADP-Ribosa) Polimerasas/sangre , Anciano , China , Enfermedad de la Arteria Coronaria/sangre , Enfermedad de la Arteria Coronaria/genética , Enfermedad de la Arteria Coronaria/terapia , Estenosis Coronaria/sangre , Estenosis Coronaria/genética , Estenosis Coronaria/terapia , Estudios Transversales , Diabetes Mellitus Tipo 2/sangre , Diabetes Mellitus Tipo 2/genética , Femenino , Predisposición Genética a la Enfermedad , Humanos , Masculino , Análisis de la Aleatorización Mendeliana , Persona de Mediana Edad , Intervención Coronaria Percutánea/efectos adversos , Fenotipo , Poli(ADP-Ribosa) Polimerasa-1/genética , Poli(ADP-Ribosa) Polimerasas/genética , Polimorfismo de Nucleótido Simple , Estudios Prospectivos , Medición de Riesgo , Factores de Riesgo , Células THP-1 , Resultado del TratamientoRESUMEN
BACKGROUND: End-stage renal disease is an irreversible status of kidney dysfunction that reduces both renal and non-renal drug clearance. Accumulation of uremic toxins seems to modify the activities of drug-metabolizing cytochrome P450 (CYP) enzymes. The aim of the present work was to refine gene expression analysis for efficient and accurate quantification of CYP mRNAs in patients' leukocytes. METHODS: We compared six liquid-liquid extraction reagents for RNA isolation and five reverse transcriptase kits for RNA-to-cDNA conversion, and developed quantitative polymerase chain reaction methods for duplex measurements of CYP target genes and the reference gene. The expression of CYP1A2, CYP2C9, CYP2C19 and CYP3A4 in patients with end-stage kidney disease (N = 105) and in organ donors with healthy kidney function (N = 110) was compared. RESULTS: Regarding the RNA yield and purity, TRIzol, Trizolate and TRI reagents were equal; however, TRI reagent was the most advantageous in terms of financial cost. Reverse transcription using Maxima First Strand cDNA Synthesis kit appeared to be the most efficient with the widest range for quantification of the target transcript. The refined method with the detection of various CYPs and the reference gene in duplex PCR efficiently quantified even the low-level CYP expression. In leukocytes of patients with end-stage renal disease, all four CYPs were expressed at significantly lower level than in organ donors with normal kidney function (p < 0.0001). CONCLUSIONS: Reduced CYP expression was a direct evidence of transcriptional down-regulation of CYP genes in patients with impaired kidney function.
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Sistema Enzimático del Citocromo P-450/genética , Regulación Enzimológica de la Expresión Génica , Fallo Renal Crónico/fisiopatología , Adolescente , Adulto , Anciano , Estudios de Casos y Controles , Regulación hacia Abajo , Femenino , Humanos , Fallo Renal Crónico/enzimología , Pruebas de Función Renal , Leucocitos/enzimología , Masculino , Persona de Mediana Edad , ARN Mensajero/metabolismo , Adulto JovenRESUMEN
OBJECTIVE: This study was designed to investigate the effects of leukocyte Rho kinase activity and serum Cystatin C (CysâC) on cardiovascular events in patients with acute coronary syndrome (ACS). METHODS: A total of 48 patients with ST-segment elevation myocardial infarction (STEMI), 23 patients with non-ST-segment elevation myocardial infarction (NSTEMI), 25 patients with unstable angina (UA) and 20 patients with no-acute coronary syndrome as control from January 2017 to June 2018 in Tianyou Hospital affiliated to Wuhan University of Science and Technology were selected in this study. Western blot was used to detect the leukocyte Rho kinase activity and Elisa kit was used to measure serum Cys C. Univariate and multivariate analysis were used to analyze the influencing factors of cardiovascular events in ACS patients. RESULTS: The activity of leukocyte Rho kinase and serum Cys C were gradually reduced in the STEMI, NSTEMI and UA patients, but all significantly higher than that in No-ASC patients, and there was a positive correlation between leukocyte Rho kinase activity and serum Cys C in ACS patients (râ=â0.516, Pâ<â.001). The activity of leukocyte Rho kinase was positively correlated with the levels of serum TNF-α (râ=â0.634, Pâ<â.001), IL-6 (râ=â0.578, Pâ<â.001), IL-8 (râ=â0.582, Pâ<â.001) in ACS patients, and the level of Cys C was positively correlated with the levels of serum TNF-α (râ=â0.634, Pâ<â.001), IL-6 (râ=â0.578, Pâ<â.001), IL-8 (râ=â0.582, Pâ<â.001) in ACS patients. Univariate and multivariate analysis showed that the leukocyte Rho kinase activity (HRâ=â2.994, 95%CIâ=â1.328-6.054, Pâ<â.0001) and the levels of serum Cys C (HRâ=â1.692, 95%CIâ=â1.028-2.124, Pâ<â.0001) were independent influencing factors of cardiovascular events in ACS patients. CONCLUSION: The leukocyte Rho kinase activity and serum Cystatin C are high in acute coronary syndrome patients, and are the independent influencing factors of cardiovascular events in ACS patients.
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Cistatina C/sangre , Isquemia Miocárdica/sangre , Quinasas Asociadas a rho/metabolismo , Anciano , Anciano de 80 o más Años , Estudios de Casos y Controles , Femenino , Humanos , Leucocitos/enzimología , Masculino , Persona de Mediana EdadRESUMEN
BACKGROUND: Metachromatic Leukodystrophy (MLD, OMIM 250100) is a neurodegenerative disease caused by mutations in the ARSA gene (OMIM 607574) that lead to deficiency in Arylsulfatase A (ASA). ASA pseudodeficiency (PD-ASA) is a biochemical condition that substantially diminishes ASA activity but is not associated with clinical manifestations. PD-ASA is associated with the c.1055A>G (p.Asn352Ser) (rs2071421) and c.*96A>G (rs6151429) variants, which have an estimated frequency of 2% in the population. OBJECTIVE: To determine the activity of Arylsulfatase A and to identify variants and haplotypes in the ARSA gene in Mexican individuals with pseudodeficiency. METHODS: Two-hundred apparently healthy individuals were included to determine the enzymatic activity of ASA in leukocytes by spectrophotometric analysis, and identification of the PD-ASA alleles was performed by PCR-RFLP assays. Genotypes were confirmed by semi-automated Sanger sequencing. Haplotypes were constructed using Arlequin v.10.04, and linkage disequilibrium analysis was performed with Cube X. RESULTS: The enzymatic activity of ASA was determined to be 1.74-2.09 nmol/mg protein/min and later correlated with genotypes and haplotypes. For the (p.Asn352Ser) variant, we found 126 (0.63) individuals with the AA genotype, 62 with AG (0.31) and 12 with GG (0.06); the frequency of the polymorphic allele was 0.215 (86 alleles, 21.5%), and the variant was in HWE (p = .2484). The variant c.*96A>G was also in HWE (p = .2105): 185 individuals (0.925) with the AA genotype, 14 (0.07) with AG, and 1 (0.005) with (GG), with a frequency of 0.04 (4%) for the polymorphic allele. The inference of haplotypes resulted in 312 (0.78) AA, 72 (0.18) GA, and 16 (0.04) GG haplotypes. The AG haplotype was not found. The variants were found to be in linkage disequilibrium (D' = 1). Of the nine possible diplotypes, AA/AG, AA/GG, and AG/GG were not found, in concordance with the hypothesis that the G allele of c.*96A>G does not occur in the absence of the G allele of c.1055A>G. We found a slight correlation between ASA biochemical activity and variants, mainly due to the G allele of c.*96A>G in either genotypes or haplotypes. CONCLUSIONS: In Northwestern Mexico, the presence of PD-ASA alleles was biochemically and molecularly determined, and the frequencies were found to be in HWE. The frequency of PD-ASA for the North Western Mexican mestizo is 8%.
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Cerebrósido Sulfatasa/genética , Haplotipos , Leucodistrofia Metacromática/genética , Adolescente , Adulto , Cerebrósido Sulfatasa/metabolismo , Femenino , Humanos , Leucocitos/enzimología , Leucodistrofia Metacromática/sangre , Desequilibrio de Ligamiento , Masculino , México , Polimorfismo de Nucleótido SimpleRESUMEN
BACKGROUND: Rho-associated protein kinase (ROCK) plays a vital role in the pathogenesis of many cardiovascular diseases. Previous studies have demonstrated that ROCK is overactivated and involved in myocardial ischemia/reperfusion in vivo. But the role of ROCK in circulating leukocytes during myocardial ischemia/reperfusion is not well studied. MATERIAL AND METHODS: This study was performed to evaluate ROCK activity in circulating leukocytes in rats with myocardial ischemia/reperfusion injury. Myocardial ischemia/reperfusion Wistar rats were subjected to 30-min ischemia followed by 180-min reperfusion. ROCK activity in circulating leukocytes was examined by the phosphorylation state of myosin phosphatase targeting subunit 1, a substrate of ROCK. RESULTS: ROCK activity significantly increased in leukocytes in rat ischemia/reperfusion models compared to the sham group. ROCK1 not ROCK2 level in circulating leukocytes was significantly elevated in ischemia/reperfusion. Administration of the selective inhibitor of ROCK, fasudil, significantly reduced myocardial infarct size, myocyte apoptosis, and inflammatory cytokine, including interleukin 6 and tumor necrosis factor α. Furthermore, fasudil upregulated ischemia/reperfusion-induced reduction of nitric oxide production. CONCLUSION: Increased ROCK1 not ROCK2 in circulating leukocytes plays a role in the pathogenesis of myocardial ischemia/reperfusion injury. Inhibition of ROCK1 in circulating leukocytes has an important role in fasudil-induced cardioprotective effects. ROCK1 in circulating leukocytes might be a new biomarker in myocardial ischemia/reperfusion injury.
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Leucocitos/metabolismo , Daño por Reperfusión Miocárdica/patología , Quinasas Asociadas a rho/metabolismo , Animales , Femenino , Leucocitos/enzimología , Daño por Reperfusión Miocárdica/sangre , Ratas , Ratas Wistar , Quinasas Asociadas a rho/sangreRESUMEN
Leukocyte migration is essential for exerting self-defense mechanisms. During the extravasation process, leukocytes transmigrate through the endothelial lining and the subendothelial basement membrane. Accumulating evidence supports the involvement of heparanase in this process. Altered cellular distribution resulting in relocalization of heparanase to the leading edge of migration is a key event to rapidly turn on the function of the enzyme during migration. This review presents current research investigating the cellular machinery that builds up a functional subcellular structure for leukocyte attachment to and degradation of the extracellular matrix. Recent advances in the understanding of the roles of heparanase in inflammatory diseases and pharmacological approaches to control heparanase-mediated actions during inflammation are also discussed.
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Quimiotaxis de Leucocito , Glucuronidasa/metabolismo , Leucocitos/citología , Leucocitos/enzimología , Matriz Extracelular , HumanosRESUMEN
Liquid chromatography-tandem mass spectrometry (LC-MS/MS) assays were developed to measure arylsulfatase A (ARSA) activity in leukocytes and dried blood spots (DBS) using deuterated natural sulfatide substrate. These new assays were highly specific and sensitive. Patients with metachromatic leukodystrophy (MLD) and multiple sulfatase deficiency (MSD) displayed a clear deficit in the enzymatic activity and could be completely distinguished from normal controls. The leukocyte assay reported here will be important for diagnosing MLD and MSD patients and for monitoring the efficacy of therapeutic treatments. ARSA activity was measured in DBS for the first time without an antibody. This new ARSA DBS assay can serve as a second-tier test following the sulfatide measurement in DBS for newborn screening of MLD. This leads to an elimination of most of the false positives identified by the sulfatide assay.
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Cerebrósido Sulfatasa/análisis , Pruebas con Sangre Seca , Leucocitos/enzimología , Leucodistrofia Metacromática/sangre , Enfermedad por Deficiencia de Múltiples Sulfatasas/sangre , Cerebrósido Sulfatasa/metabolismo , Cromatografía Liquida , Humanos , Leucodistrofia Metacromática/diagnóstico , Leucodistrofia Metacromática/enzimología , Estructura Molecular , Enfermedad por Deficiencia de Múltiples Sulfatasas/diagnóstico , Enfermedad por Deficiencia de Múltiples Sulfatasas/enzimología , Sulfoglicoesfingolípidos/química , Espectrometría de Masas en TándemRESUMEN
BACKGROUND AND AIMS: Neutrophil elastase and myeloperoxidase enzymes protect us from infection by killing pathogens. However, exaggerated activities of these enzymes can induce tissue damage, inflammation and oxidative stress. The present study was aimed to explore the expressions of neutrophil elastase and myeloperoxidase mRNA in the peripheral blood leukocytes (PBL) in patients with newly diagnosed type 2 diabetes mellitus. METHODS: In this cross-sectional study, 104 participants including 65 normoglycemic control subjects and 39 newly diagnosed type 2 diabetes patients were recruited. Glycemic and metabolic markers were evaluated from fasting blood samples. The mRNA levels of neutrophil elastase and myeloperoxidase genes in the PBL were quantified by real-time quantitative PCR. RESULTS: Compared to control subjects, diabetes patients showed a significant down regulation of both neutrophil elastase (p = 0.039) and myeloperoxidase (p = 0.023) mRNA expressions in the PBL. The neutrophil elastase and myeloperoxidase mRNA levels showed a negative trend with fasting glucose levels but did not show any significant correlations with HbA1c, insulin level, insulin resistance or sensitivity status. CONCLUSIONS: It was concluded that type 2 diabetes mellitus is associated with a decrease in neutrophil elastase and myeloperoxidase gene expression in the PBL.
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Diabetes Mellitus Tipo 2/enzimología , Elastasa de Leucocito/metabolismo , Peroxidasa/metabolismo , Adulto , Estudios de Casos y Controles , Estudios Transversales , Femenino , Humanos , Leucocitos/enzimología , Masculino , Persona de Mediana EdadRESUMEN
Leukocyte inflammatory responses require integrin cell-adhesion molecule signaling through spleen tyrosine kinase (Syk), a non-receptor kinase that binds directly to integrin ß-chain cytoplasmic domains. Here, we developed a high-throughput screen to identify small molecule inhibitors of the Syk-integrin cytoplasmic domain interactions. Screening small molecule compound libraries identified the ß-lactam antibiotics cefsulodin and ceftazidime, which inhibited integrin ß-subunit cytoplasmic domain binding to the tandem SH2 domains of Syk (IC50 range, 1.02-4.9 µM). Modeling suggested antagonist binding to Syk outside the pITAM binding site. Ceftazidime inhibited integrin signaling via Syk, including inhibition of adhesion-dependent upregulation of interleukin-1ß and monocyte chemoattractant protein-1, but did not inhibit ITAM-dependent phosphorylation of Syk mediated by FcγRI signaling. Our results demonstrate a novel means to target Syk independent of its kinase and pITAM binding sites such that integrin signaling via this kinase is abrogated but ITAM-dependent signaling remains intact. As integrin signaling through Syk is essential for leukocyte activation, this may represent a novel approach to target inflammation.
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Antiinflamatorios/farmacología , Cefsulodina/farmacología , Ceftazidima/farmacología , Cadenas beta de Integrinas/efectos de los fármacos , Leucocitos/efectos de los fármacos , Quinasa Syk/antagonistas & inhibidores , Antiinflamatorios/química , Cefsulodina/química , Ceftazidima/química , Ensayos Analíticos de Alto Rendimiento , Humanos , Cadenas beta de Integrinas/química , Cadenas beta de Integrinas/metabolismo , Leucocitos/enzimología , Masculino , Fosforilación , Unión Proteica , Dominios y Motivos de Interacción de Proteínas , Transducción de Señal , Bibliotecas de Moléculas Pequeñas , Quinasa Syk/química , Quinasa Syk/metabolismo , Células THP-1RESUMEN
BACKGROUND: Sporadic Parkinson's disease (PD) patients have lower α-galactosidase A (α-GAL A) enzymatic activity and Fabry disease (FD) patients potentially carry an increased risk of PD. OBJECTIVE: Determination of PD prevalence in FD and clinical, biochemical and vascular neuroimaging description of FD pedigrees with concomitant PD. METHODS: Clinical screening for PD in 229 FD patients belonging to 31 families, harbouring GLA gene mutation p.F113L, and subsequent pedigree analysis. Gender-stratified comparison of FD+/PD+ patients with their family members with FD but without PD (FD+/PD-) regarding Mainz scores, plasma & leukocytes α-GAL A enzymatic activity, urinary Gb3 and plasma Lyso-Gb3, vascular brain neuroimaging. RESULTS: Prevalence of PD in FD was 1.3% (3/229) (3% in patients aged ≥50 years). Three FD patients, one female (73 years old) (P1) and two males (60 and 65 years old) (P2 and P3), three different pedigrees, presented akinetic-rigid PD, with weak response to levodopa (16% - 36%), and dopaminergic deficiency on 18F-DOPA PET. No pathogenic mutations were found in a PD gene panel. FD+/PD+ patients had worse clinical severity of FD (above upper 75% IQR in Mainz scores), and cortico-subcortical white matter/small vessel lesions. P3 patient was under enzyme therapy, started 1 year before PD diagnosis. P2-P3 patients had higher leucocyte α-GAL A activity (2,2-3 vs.1,0 (median)(nmol/h/mg)). CONCLUSION: We have shown a high prevalence of PD in a late-onset phenotype of FD, presenting high cerebrovascular burden and weak response to levodopa. Further studies will untangle how much of this PD phenotype is due to Gb3 deposition versus cerebrovascular lesions in the nigro-striatal network.
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Encéfalo/diagnóstico por imagen , Enfermedad de Fabry , Glucolípidos/metabolismo , Leucocitos/enzimología , Enfermedad de Parkinson , Esfingolípidos/metabolismo , alfa-Galactosidasa/metabolismo , Adulto , Anciano , Estudios de Cohortes , Comorbilidad , Enfermedad de Fabry/diagnóstico por imagen , Enfermedad de Fabry/enzimología , Enfermedad de Fabry/epidemiología , Enfermedad de Fabry/fisiopatología , Femenino , Glucolípidos/sangre , Glucolípidos/orina , Humanos , Imagen por Resonancia Magnética , Masculino , Persona de Mediana Edad , Neuroimagen , Enfermedad de Parkinson/diagnóstico por imagen , Enfermedad de Parkinson/enzimología , Enfermedad de Parkinson/epidemiología , Enfermedad de Parkinson/fisiopatología , Linaje , Fenotipo , Prevalencia , Esfingolípidos/sangre , Esfingolípidos/orina , alfa-Galactosidasa/sangre , alfa-Galactosidasa/genéticaRESUMEN
The lower urinary tract is routinely exposed to microbes residing in the gastrointestinal tract, yet the urothelium resists invasive infections by gut microorganisms. This infection resistance is attributed to innate defenses in the bladder urothelium, kidney epithelium, and resident or circulating immune cells. In recent years, surmounting evidence suggests that these cell types produce and secrete soluble host defense peptides, including members of the Ribonuclease (RNase) A Superfamily, to combat invasive bacterial challenge. While some of these peptides, including RNase 4 and RNase 7, are abundantly produced by epithelial cells, the expression of others, like RNase 3 and RNase 6, increase at infection sites with immune cell recruitment. The objective of this mini-review is to highlight recent evidence showing the biological importance and responses of RNase A Superfamily members to infection in the kidney and bladder.
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Familia de Multigenes , Ribonucleasa Pancreática/genética , Ribonucleasa Pancreática/metabolismo , Infecciones Urinarias/enzimología , Infecciones Urinarias/etiología , Animales , Péptidos Catiónicos Antimicrobianos , Humanos , Inmunidad Innata , Leucocitos/enzimología , Membrana Mucosa/metabolismoRESUMEN
We report the case of a boy who was diagnosed with mucopolysaccharidosis (MPS) VII at two weeks of age. He harbored three missense ß-glucuronidase (GUSB) variations in exon 3: two novel, c.422A>C and c.424C>T, inherited from his mother, and the rather common c.526C>T, inherited from his father. Expression of these variations in transfected HEK293T cells demonstrated that the double mutation c.422A>C;424C>T reduces ß-glucuronidase enzyme activity. Enzyme replacement therapy (ERT), using UX003 (vestronidase alfa), was started at four months of age, followed by a hematopoietic stem cell allograft transplantation (HSCT) at 13 months of age. ERT was well tolerated and attenuated visceromegaly and skin infiltration. After a severe skin and gut graft-versus-host disease, ERT was stopped six months after HSCT. The last follow-up examination (at the age of four years) revealed a normal psychomotor development, stabilized growth curve, no hepatosplenomegaly, and no other organ involvement. Intriguingly, enzyme activity had normalized in leukocytes but remained low in plasma. This case report illustrates: (i) The need for an early diagnosis of MPS, and (ii) the possible benefit of a very early enzymatic and/or cellular therapy in this rare form of lysosomal storage disease.