Vascular O-GlcNAcylation augments reactivity to constrictor stimuli by prolonging phosphorylated levels of the myosin light chain

O-GlcNAcylation is a modification that alters the function of numerous proteins. We hypothesized that augmented O-GlcNAcylation levels enhance myosin light chain kinase (MLCK) and reduce myosin light chain phosphatase (MLCP) activity, leading to increased vascular contractile responsiveness. The vascular responses were measured by isometric force displacement. Thoracic aorta and vascular smooth muscle cells (VSMCs) from rats were incubated with vehicle or with PugNAc, which increases O-GlcNAcylation. In addition, we determined whether proteins that play an important role in the regulation of MLCK and MLCP activity are directly affected by O-GlcNAcylation. PugNAc enhanced phenylephrine (PE) responses in rat aortas (maximal effect, 14.2±2 vs 7.9±1 mN for vehicle, n=7). Treatment with an MLCP inhibitor (calyculin A) augmented vascular responses to PE (13.4±2 mN) and abolished the differences in PE-response between the groups. The effect of PugNAc was not observed when vessels were preincubated with ML-9, an MLCK inhibitor (7.3±2 vs 7.5±2 mN for vehicle, n=5). Furthermore, our data showed that differences in the PE-induced contractile response between the groups were abolished by the activator of AMP-activated protein kinase (AICAR; 6.1±2 vs 7.4±2 mN for vehicle, n=5). PugNAc increased phosphorylation of myosin phosphatase target subunit 1 (MYPT-1) and protein kinase C-potentiated inhibitor protein of 17 kDa (CPI-17), which are involved in RhoA/Rho-kinase-mediated inhibition of myosin phosphatase activity. PugNAc incubation produced a time-dependent increase in vascular phosphorylation of myosin light chain and decreased phosphorylation levels of AMP-activated protein kinase, which decreased the affinity of MLCK for Ca2+/calmodulin. Our data suggest that proteins that play an important role in the regulation of MLCK and MLCP activity are directly affected by O-GlcNAcylation, favoring vascular contraction.

Biochemical characterization of the GM2 gangliosidosis B1 variant

The deficiency of the A isoenzyme of á-hexosaminidase (Hex) produced by different mutations of the gene that codes for the alpha subunit (Tay-Sachs disease) has two variants with enzymological differences: the B variant consists of the absence of Hex A isoenzyme and the B1 variant produces an inactive Hex A isoenzyme for the hydrolysis of the GM2 ganglioside and synthetic substrates with negative charge. In contrast to the early childhood form of the B variant, the B1 variant appears at a later clinical stage (3 to 7 years of age) with neurodegenerative symptoms leading to the death of the patient in the second decade of life. The most frequent mutation responsible for the GM2 gangliosidosis B1 variant is R178H, which has a widespread geographic and ethnic distribution. The highest incidence has been described in Portugal, which has been suggested as the point of origin of this mutation. Biochemical characterization of this lysosomal disease is carried out using negatively charged synthetic alpha subunit-specific sulfated substrates, since Hex A isoenzyme heat-inactivation assays are not applicable. However, the determination of the apparent activation energy of Hex using the neutral substrate 3,3'-dichlorophenolsulfonphthaleinyl N-acetyl-á-D-glucosaminide, may offer a valid alternative. The presence of an alpha subunit in the alphaá heterodimer Hex A means that its activation energy (41.8 kJ/mol) is significantly lower than that of the áá homodimer Hex B (75.1 kJ/mol); however, as mutation inactivates the alpha subunit, the Hex A of the B1 variant presents an activation energy that is similar to that of the Hex B isoenzyme.

Serum hexosaminidase and á-glucuronidase activities in infants: effects of age and sex

We investigated the effect of age and sex on the serum activity of hexosaminidase (HEX) and á-glucuronidase (BGLU) in 275 normal term infants aged 12 h to 12 months. Up to six weeks of life, HEX was significantly higher in boys (P<=0.023). During the age period of 1-26 weeks, BGLU was also higher in boys, but differences were significant only at 2-6 and 7-15 weeks (P<=0.016). The developmental pattern of HEX and BGLU was sex dependent. HEX activity increased in both sexes from 4-7 days of life, reaching a maximum of 1.4-fold the birth value at 2-6 weeks of age in boys (P<0.001) and a maximum of 1.6-fold at 7-15 weeks in girls (P<0.001). HEX activity gradually decreased thereafter, reaching significantly lower levels at 27-53 weeks than during the first three days of life in boys (P = 0.002) and the same level of this age interval in girls. BGLU increased in both sexes from 4-7 days of age, showing a maximum increase at 7-15 weeks (3.3-fold in boys and 2.9-fold in girls, both P<0.001). Then BGLU decreased in boys to a value similar to that observed at 4-7 days of age. In girls, BGLU remained elevated until the end of the first year of life. These results indicate a variation of HEX and BGLU activities during the first year of life and a sex influence on their developmental pattern. This observation should be considered in the diagnosis of GM2 gangliosidosis and mucopolysaccharidosis type VII

Aspectos genéticos e clínico-terapêuticos da Doença de Tay-Sachs / Genetic and clinical therapeutics aspects of Tay-Sachs disease

A doença de Tay-Sachs apresenta uma freqüência elevada em determinados grupos étnicos, sobretudo nos judeus ashkenazi. É uma desordem neurodegenerativa, presente principalmente em crianças, decorrente de uma atividade deficiente da enzima lisossomal hexosaminidase A, acarretando um acúmulo intracelular de substratos e um progressivo déficit neurológico. O tratamento é discutível, entretanto, resultados promissores têm sido obtidos com a utilização da NB-DNJ e, principalmente, com a terapia genética

Valor diagnóstico dos marcadores bioquímicos da detecçäo da dependência e do abuso de álcool / Diagnostic value of biochemical markers in dependency and of alcohol abuse detection

A prevalência de transtornos relacionados à ingesta de álcool está aumentando e constitui sério problema de Saúde Pública. No entanto, a maioria dos pacientes nega ou não fornece informações suficientes para a efetivação de uma anamnese completa, dificultando a realização do diagnóstico precoce. Propõe-se, então, a utilização de dosagens bioquímicas que permitam auxiliar a detecção da ocorrência do abuso de álcool. Os testes laboratoriais disponíveis são: aspartato aminotransferase (AST), alanina aminotransferase (ALT), beta-hexosaminidade (B-HEX), gamaglutamiltranspeptidade (y-GT) e transferina deficiente de carboidrato (CDT). Contudo, a CDT destaca-se como marcador do alcoolismo, apresentando sensibilidade e especificidade superiores às outras dosagens e aplicabilidade clínica comprovadamente reconhecida

Effect of collection, transport, processing and storage of blood specimens on the activity of lysosomal enzymes in plasma and leukocytes

This study was designed to evaluate the effect of different conditions of collection, transport and storage on the quality of blood samples from normal individuals in terms of the activity of the enzymes Beta-glucuronidase, total hexosaminidase, hexosaminidase A, arylsulfatase A and Beta-galactosidase. The enzyme activities were not affected by the different materials used for collection (plastic syringes or vacuum glass tubes). In the evaluation of different heparin concentrations (10 percent heparin, 5 percent heparin, and heparinized syringe) in the syringes, it was observed that higher doses resulted in an increase of at least 1-fold in the activities of Beta-galactosidase, total hexosaminidase and hexosaminidase A in leukocytes, and Beta-glucuronidase in plasma. When the effects of time and means of transportation were studied, samples that had been kept at room temperature showed higher deterioration with time (72 and 96 h) before processing, and in this case it was impossible to isolate leukocytes from most samples. Comparison of heparin and acid citrate-dextrose (ACD) as anticoagulants revealed that Beta-glucuronidase and hexosaminidase activities in plasma reached levels near the lower normal limits when ACD was used. In conclusion, we observed that heparin should be used as the preferable anticoagulant when measuring these lysosomal enzyme activities, and we recommend that, when transport time is more than 24 h, samples should be shipped by air in a styrofoam box containing wet ice

Determinación de la ß-N-acetilhexosaminidasa en orina utilizando un método espectrofotométrico continuo automatizado / Determination of ß-N-acetylhexaminidase in urine using an automated continuous spectrophotometric method

Se adaptó a un analizador centrífugo Cobas Bio un método continuo para la determinación de la ß-N-acetilhexosaminidasa (Hex, EC 3.2.1.52) en orina utilizando 3,3'-diclorofenolsulfonftaleinil-N-acetil-ß-D-glucosaminido como sustrato. En el estudio de la precisión intraserie se obtuvieron unos coeficientes de variación de 0,77-2,10 por ciento y en el de precisión interserie de 0,89-2,83 por ciento. En todos los casos los coeficientes de variación fueron inferiores a los médicamente aceptables según criterios del Colegio de Patólogos Americanos. Se encontró una excelente correlación entre los resultados obtenidos con esta técnica y con un método discontinuo utilizando 2-metoxi-4-(2'-nitrovinil)-fenil-N-acetil-ß-D-glucosaminido como sustrato (n = 100, r = 0,997)> No se ha podido confirmar el hecho señalado por algunos autores, de que el efecto inhibidor de la urea sobre la actividad enzimática de la Hex sea mayor al aumentar el pH en que se lleva a cabo la hidrólisis de estos sustratos cromogénicos

Determinación de la actividad enzimática de la hexosaminidasa en fluidos biológicos / Determination of the enzimatic activity of hexosaminidase in biological fluids

Este estudio describe un método electroforético que emplea el sustrato fluorogénico 4-metilumbelliferil 2-acetamida 2-deoxi-ß-D-glucopiranosido, para determinación enzimática de la hexosaminidasa (HEX) en linfocitos de sangre períferica, líquido amniótico, suero y orina. Se propone que la electroforésis sirva como método confirmatorio del diagnóstico de la gangliosidosis GM2, ya que permite determinar la actividad enzimática de la HEX y hacer la diferenciación de las isoenzimas (HEX A y HEX B). La deficiencia de estas isoenzimas se detecta por disminución o ausencia de fluorescencia

Modificaciones bioquímicas en el suero de pacientes con criptorquidea / Biochemical alterations in serum of patients with cryptorchidism

Teniendo en cuenta que el criptorquismo es un factor de riesgo de malignidad testicular, fue estudiado el perfil isoenzimático de la fosfatasa alcalina sérica (FAL), en 42 pacientes con criptocardia unilateral o bilateral; los mismos fueron divididos en dos grupos, según que dicho perfil correspondiera al encontrado en dadores sanos (grupo A) o al de pacientes con alto riesgo de desarrollar cáncer (grupo B). En ambos grupos se efectuó el estudio de la actividad sérica de fosfatasa alcalina total (FAT), fosfatasa ácida total (FAcT), fosfatasa ácida prostática (FAcP), hexosaminidasa (Hex) y fracciones proteicas, comparándose los resultados con los obtenidos para el grupo C o control. Fue observado un incremento de la actividad de FAT en los grupos A y B, notándose niveles elevados de FAcT y FAcP en el 28,0% y 19,0% respectivamente, del grupo A y en el 43,0% y 25,0% del grupo B. La actividad de Hex presentó niveles elevados en el 50,0% y 63,6% de los grupos A y B respectivamente. La relación albúmina/globulinas estuvo disminuida en el 33,3% del grupo A y en el 85,0% del B, a expensas del incremento de las fracciones globulínicas y al descenso de albúmina. Los pacientes del grupo B generalmente no responden a la terapia hormonal ni quirúrgica, mientras los del grupo A sí. Los resultados sugieren la existencia de marcadas modificaciones en el metabolismo proteico, como asimismo en la actividad de algunas enzimas séricas en los pacientes criptorquídicos

Alteraciones bioquímicas y citoquímicas de hidrolasas ácidas lisosomales en leucemias / Biochemical and citochemical alterations of lisosomal acid hydrolases in leukemias

La actividad enzimática total de ß-galactosidasa (ß-gal), hexosaminidasa (hex) y fosfatasa ácida (Fac) fue determinada bioquímicamente, tanto en suero como en sobrenadantes de homogenatos celulares de sujetos normales y pacientes portadores de leucemias, empleándose sustratos paranitrofenilados específicos. La actividad de ß-gal en leucemias mieloides, tanto agudas (LMA-M1) como crónicas (LMC), sólo mostró un incremento significativo en sobrenadantes de homogenato celulares, respecto a los valores de neutrófilos normales. En leucemias linfoidales agudas (LLA), como crónicas (LLC), su comportamiento no ofreció variaciones. Tanto los sueros como los sobrenadantes de homogenatos celulares de LMA-M1 y LMC mostraron un franco incremento en la actividad de hex, mientras en LLA y LLC esta actividad no mostró variaciones. La actividad sérica de fosfatasa ácida estuvo incrementada en el 86% de las LMC. En los sobrenadantes de homogenatos celulares de LLA y LLC, esta actividad enzimática se mostró significativamente disminuida respecto de los valores de linfocitos normales. En los tres casos de LMA-M4 analizados, fue observado en el contenido celular niveles elevados de ß-gal, hex y Fac (lo que estaría correlacionado con la presencia de monocitos y/o monoblastos normales, con alto contenido de hidrolasas ácidas). Citoquímicamente fue demostrado en médula ósea y sangre periférica de pacientes con LMA-M1 una ligera o nula actividad de hex, en tanto que en LLA la reacción fue localizada asimétricamente en un polo celular o en gránulos citoplasmáticos. Los resultados encontrados demuestran una gran heterogeneidad en el contenido lisosomal de los diferentes tipos de leucemias

Deficiencia cronica de hexosaminidasa A asociada a neuropatia periferica sensitiva pura / Chronic hexosaminidase A deficiency associated to pure sensory peripheral neuropathy

Se presenta a un paciente con deficiencia de hexosaminidasa A (Hx A), que conduce a la gangliosidosis GM2, que desarrolla un cuadro neurológico progresivo cuyo comienzo pudo fijarse a los 10 años y que se caracterizó por deterioro intelectual, compromiso cerebeloso, alteración de neuromas motoras superior e inferior y neuropatía sensitiva sin aparente compromiso de las fibras motoras que integran el nervio mixto. La biopsia del nervio safeno externo mostró pérdida de fibras mielínicas, en especial de aquellas de mayor y menor diámetro, agrupamiento axonal, axones con cubiertas mielínicas anormalmente finas en relación con su diámetro, degeneración axonal, desmielinización segmentaria y paranodal y remielinización. La microscopia electrónica reveló cuerpos de inclusión electrodensos no específicos e incusiones laminares concéntricas dentro del citoplasma de las células de Schwann. Los hallazgos hechos señalan que la neuropatía sensitiva pura puede formar parte del espectroclínico de la deficiencia de HxA