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1.
Eur J Neurol ; 28(2): 540-547, 2021 02.
Artigo em Inglês | MEDLINE | ID: mdl-33043545

RESUMO

BACKGROUND AND PURPOSE: B-type natriuretic peptide (BNP) and N-terminal pro-brain natriuretic peptide (NT-proBNP) are well-known surrogates of atrial fibrillation (AF) detection but studies usually present data on either BNP or NT-proBNP. The aim was to determine and directly compare the validity of the two biomarkers as a tool to predict AF and guide prolonged cardiac monitoring in cryptogenic stroke patients. METHODS: Non-lacunar acute ischaemic stroke (<72 h) patients over 55 years of age with cryptogenic stroke after standard evaluation were included in the Crypto-AF study and blood was collected. BNP and NT-proBNP levels were determined by automated immunoassays. AF was assessed by 28 days' monitoring. Highest (optimizing specificity) and lowest (optimizing sensitivity) quartiles were used as biomarker cut-offs to build predictive models adjusted by sex and age. The integrated discrimination improvement index (IDI) and DeLong test were used to compare the performance of the two biomarkers. RESULTS: From 320 patients evaluated, 218 were included in the analysis. AF was detected in 50 patients (22.9%). NT-proBNP (P < 0.001) and BNP (P < 0.001) levels were higher in subjects with AF and their levels correlated (r = 0.495, P < 0.001). BNP showed an increased area under the curve (0.720 vs. 0.669; P = 0.0218) and a better predictive capacity (IDI = 3.63%, 95% confidence interval 1.36%-5.91%) compared to NT-proBNP. BNP performed better than NT-proBNP in a specific model (IDI = 3.7%, 95% confidence interval 0.87%-6.5%), whilst both biomarkers performed similarly in the case of a sensitive model. CONCLUSIONS: Both BNP and NT-proBNP were increased in cryptogenic stroke patients with AF detection. Interestingly, BNP outperforms NT-proBNP, especially in terms of specificity.


Assuntos
Fibrilação Atrial , Isquemia Encefálica , AVC Isquêmico , Acidente Vascular Cerebral , Fibrilação Atrial/complicações , Fibrilação Atrial/diagnóstico , Fibrilação Atrial/epidemiologia , Biomarcadores , Humanos , Peptídeo Natriurético Encefálico , Fragmentos de Peptídeos , Acidente Vascular Cerebral/complicações
2.
Eur J Neurol ; 27(8): 1618-1624, 2020 08.
Artigo em Inglês | MEDLINE | ID: mdl-32347993

RESUMO

BACKGROUND AND PURPOSE: Covert paroxysmal atrial fibrillation (pAF) is the most frequent cause of cardiac embolism. Our goal was to discover parameters associated with early pAF detection with intensive cardiac monitoring. METHOD: Crypto-AF was a multicentre prospective study (four Comprehensive Stroke Centres) to detect pAF in non-lacunar cryptogenic stroke continuously monitored within the first 28 days. Stroke severity, infarct pattern, large vessel occlusion (LVO) at baseline, electrocardiography analysis, supraventricular extrasystolia in the Holter examination, left atrial volume index and brain natriuretic peptide level were assessed. The percentage of pAF detection and pAF episodes lasting more than 5 h were registered. RESULTS: Out of 296 patients, 264 patients completed the monitoring period with 23.1% (61/264) of pAF detection. Patients with pAF were older [odds ratio (OR) 1.04, 95% confidence interval (CI) 1.01-1.08], they had more haemorrhagic infarction (OR 4.03, 95% CI 1.44-11.22), they were more likely to have LVO (OR 4.29, 95% CI 2.31-7.97) (P < 0.0001), they had a larger left atrial volume index (OR 1.03, 95% CI 1.01-1.1) (P = 0.0002) and they had a higher level of brain natriuretic peptide (OR 1.01, 95% CI 1.0-1.1). Age and LVO were independently associated with pAF detection (OR 1.06, 95% CI 1.00-1.16, and OR 4.58, 95% CI 2.27- 21.38, respectively). Patients with LVO had higher cumulative incidence of pAF (log rank P < 0.001) and more percentage of pAF > 5 h [29.6% (21/71) vs. 8.3% (12/144); OR 4.62, 95% CI 2.11-10.08; P < 0.001]. In a mean follow-up of 26.82 months (SD 10.15) the stroke recurrence rate was 4.6% (12/260). CONCLUSIONS: Large vessel occlusion in cryptogenic stroke emerged as an independent marker of pAF.


Assuntos
Fibrilação Atrial , Acidente Vascular Cerebral , Fibrilação Atrial/complicações , Fibrilação Atrial/diagnóstico , Fibrilação Atrial/epidemiologia , Eletrocardiografia , Eletrocardiografia Ambulatorial , Humanos , Incidência , Estudos Prospectivos
3.
Neurologia (Engl Ed) ; 2023 Apr 26.
Artigo em Inglês | MEDLINE | ID: mdl-37116689

RESUMO

BACKGROUND: In addition to stent retrievers, direct aspiration has become a reasonable thrombectomy strategy. OBJECTIVES: We carried out the thrombectomy by guiding the aspiration catheter fully over the clot and performing immediate manual aspiration; we call this procedure "embed aspiration". METHODS: In this prospective, non-randomised, single-centre study, we included all patients treated at a high volume-of-care stroke centre between 2017 and 2018 for the TRIANA (Thrombectomy in Andalusia using Aspiration) registry. Thrombectomy was carried out by embed aspiration. Patients were classified according to the success (eTICI 2b67-2c-3) or failure (eTICI 0-1-2a-2b50) of the procedure. Baseline clinical data and outcomes were compared, and multivariate analysis was performed. RESULTS: The embed aspiration technique was used in 370 patients. Treatment was successful in 90.3% of patients. Mean puncture-to-recanalisation time was 25 minutes. The overall rate of good outcomes (mRS 0-2) at 3 months was 64%. CONCLUSIONS: This study supports real-life evidence that standardised embed aspiration may be an alternative to stent retrievers for thrombectomy.

4.
Sci Rep ; 11(1): 11582, 2021 06 02.
Artigo em Inglês | MEDLINE | ID: mdl-34078936

RESUMO

Pines are the dominant conifers in Mediterranean forests. As long-lived sessile organisms that seasonally have to cope with drought periods, they have developed a variety of adaptive responses. However, during last decades, highly intense and long-lasting drought events could have contributed to decay and mortality of the most susceptible trees. Among conifer species, Pinus pinaster Ait. shows remarkable ability to adapt to different environments. Previous molecular analysis of a full-sib family designed to study drought response led us to find active transcriptional activity of stress-responding genes even without water deprivation in tolerant genotypes. To improve our knowledge about communication between above- and below-ground organs of maritime pine, we have analyzed four graft-type constructions using two siblings as rootstocks and their progenitors, Gal 1056 and Oria 6, as scions. Transcriptomic profiles of needles from both scions were modified by the rootstock they were grafted on. However, the most significant differential gene expression was observed in drought-sensitive Gal 1056, while in drought-tolerant Oria 6, differential gene expression was very much lower. Furthermore, both scions grafted onto drought-tolerant rootstocks showed activation of genes involved in tolerance to abiotic stress, and is most remarkable in Oria 6 grafts where higher accumulation of transcripts involved in phytohormone action, transcriptional regulation, photosynthesis and signaling has been found. Additionally, processes, such as those related to secondary metabolism, were mainly associated with the scion genotype. This study provides pioneering information about rootstock effects on scion gene expression in conifers.


Assuntos
Regulação da Expressão Gênica de Plantas , Genes de Plantas , Pinus/genética , Raízes de Plantas/metabolismo , Adaptação Fisiológica , Secas
5.
Biochim Biophys Acta ; 649(2): 229-38, 1981 Dec 07.
Artigo em Inglês | MEDLINE | ID: mdl-7317394

RESUMO

A procedure of the isolation of platelets from the blood of adult sheep (Ovis aries L. var domestica) is reported. This procedure is based on differential centrifugation and a specific lysis for elimination of erythrocytes. We have obtained platelets with a purity at least 99% and a relative high yield (2.3 +/- 0.4 g wet/l whole blood identical to 235 +/- 40 mg platelet proteins/l whole blood). After disruption, homogenisation and ultracentrifugation onto a discontinuous sucrose gradient (1.6 M, 1.1 M, 1.0 M and 0.6 M sucrose), four fractions were obtained. We have separated, for the first time, a particulate preparation enriched in the whole sheep plasma membrane. This fraction was characterized by: (i) the typical membrane morphology as shown by electron micrographs; (ii) the highest activities in membrane marker enzymes such as bis(p-nitrophenyl)phosphate phosphodiesterase (EC 3.1.16.1) and 5'-dTMP-p-nitrophenyl ester phosphodiesterase (EC 3.1.3.35), and the relatively low activity for marker enzymes associated to other subcellular fractions; (iii) the highest sialic acid, cholesterol and phospholipid concentrations. The chemical composition of the platelet membrane isolated is: total proteins, 49%; lipids, 47%; carbohydrates, congruent to 3.4% (the content of hexoses is twice as high as that of hexosamines and sialic acid). The similarities and differences of this preparation with others from several sources are discussed.


Assuntos
Plaquetas/ultraestrutura , Membrana Celular/ultraestrutura , Lipídeos de Membrana/sangue , Animais , Fracionamento Celular , Centrifugação com Gradiente de Concentração/métodos , Colesterol/sangue , Hexoses/sangue , Proteínas de Membrana/sangue , Microscopia Eletrônica , Fosfolipídeos/sangue , Ovinos , Ácidos Siálicos/sangue
6.
Biochim Biophys Acta ; 999(2): 171-5, 1989 Nov 30.
Artigo em Inglês | MEDLINE | ID: mdl-2597705

RESUMO

Using linear sucrose-density ultracentrifugation analysis of Triton-solubilized Newcastle Disease Virus envelopes, we have evidenced, for the first time, the existence of interactions between the outer hemagglutinin-neuraminidase transmembrane glycoprotein and the inner non-glycosylated peripheral matrix protein. Such interactions seem to be electrostatic. These conclusions are based on the behavior of both proteins at different ionic strengths. When in low ionic strength buffer, hemagglutinin-neuraminidase and matrix proteins band together in the sucrose gradient, whereas at high ionic strength both proteins band at different rates in the gradient. The behavior of the inner matrix protein in our conditions was the expected one for a peripheral protein. The results of these 'in vitro' studies are also discussed in terms of the possible 'in vivo' role of such interactions.


Assuntos
Proteína HN/isolamento & purificação , Vírus da Doença de Newcastle/metabolismo , Proteínas do Envelope Viral/isolamento & purificação , Proteínas da Matriz Viral/isolamento & purificação , Animais , Centrifugação com Gradiente de Concentração , Embrião de Galinha , Eletroforese em Gel de Poliacrilamida , Proteína HN/metabolismo , Peso Molecular , Neuraminidase/metabolismo , Proteínas do Envelope Viral/metabolismo , Proteínas da Matriz Viral/metabolismo
7.
Biochim Biophys Acta ; 616(2): 228-38, 1980 Dec 04.
Artigo em Inglês | MEDLINE | ID: mdl-7213637

RESUMO

Neuraminidase (acylneuraminyl hydrolase, EC 3.2.1.18) from the influenza virus A/Hong Kong/68 (H3N2) was purified after treatment of the purified virus with sarcosyl (sodium laurylsarcosinate), centrifugation at 110 000 x g, and chromatography on DEAE-Sephadex and Sephadex G-200. It migrated as a single component during electrophoresis on polyacrylamide gel, and its molecular weight was estimated about 270 000. The enzyme was thermolabile, the activity being reduced to 60% in 10 min at 50 degrees C. The purified neuraminidase had an apparent Km value of 4.1 . 10(-3) M for 5-N-acetyl-2-O-(3-methoxyphenyl)-alpha-D-neuraminic acid and was able to release sialic acid with linkages alpha 2-3, alpha 2-6 and alpha 2-8 (with very different efficiency) from fetuin, gangliosides, colominic acid, and bovine and porcine submaxillary mucins. The enzymic activity was measured by several procedures: (A) spectrophotometric determination at 340 nm of the NADH produced in the reaction catalysed by beta-galactose dehydrogenase on beta-galactose + NAD+, this beta-galactose was the product released from lactose by beta-galactosidase and lactose was the product of the neuraminidase activity on N-acetylneuraminyl-lactose; (B) determination of the colored quinone yielded by the liberated methoxyphenol with 4-aminoantipyrine (Santer, U.V., Yee-Foon, J. and Glick, M.C. (1978) Biochim. Biophys. Acta 523, 435-442); (C) periodate-thiobarbiturate procedures (Warren, L. (1959) J. Biol. Chem 234, 1971-1975 or Aminoff, D. (1961) Biochem. J. 81, 384-391). Some peculiarities of these methods are discussed.


Assuntos
Vírus da Influenza A Subtipo H3N2 , Vírus da Influenza A/enzimologia , Neuraminidase/metabolismo , Gangliosídeos , Glicoproteínas , Cinética , Ácidos Neuramínicos , Especificidade por Substrato
8.
Mech Ageing Dev ; 99(2): 95-107, 1997 Dec 15.
Artigo em Inglês | MEDLINE | ID: mdl-9483485

RESUMO

The activities of 21 enzymes (belonging to four classes of enzymes) involved in different metabolic pathways were assayed in blood sera of healthy young and adult/elderly groups of humans, rats and pigs, to determine whether activity changes coinciding with changes in age and aging could be detected. In all three species analysed, measurable activities (performed by highly specific and sensitive techniques, generally spectrofluorimetric procedures) were found, usually following a decreasing order of: among glycosidases, beta-N-acetylglucosaminidase (NAG) > alpha-L-fucosidase > alpha-mannosidase > beta-glucuronidase > beta-galactosidase > alpha-galactosidase. In addition, among esterases very high values were found for arylesterase and acid phosphatase. By contrast, no measurable activity was found for the remaining enzymes assayed (8 hydrolases, 1 oxidoreductase, 3 transferases and 1 lyase). In the elderly group of humans, significantly higher activities (P < or = 0.05) were found for NAG, alpha-mannosidase and beta-glucuronidase in comparison to the adult and young groups. However, several activities in rats and all activities in pigs decreased with age. In conclusion, differences in the activities of 6 lysosomal glycosidases and 2 esterases (but no significant differences for another 13 enzymes belonging to several enzyme classes) are found in the sera of healthy humans, rats and pigs. These differences coincide with changes observed in aging.


Assuntos
Envelhecimento/metabolismo , Hidrolases/metabolismo , Lisossomos/enzimologia , Fosfatase Ácida/metabolismo , Adulto , Idoso , Idoso de 80 Anos ou mais , Animais , Proteínas Sanguíneas/análise , Hidrolases de Éster Carboxílico/metabolismo , Criança , Feminino , Glicosídeo Hidrolases/metabolismo , Humanos , Masculino , Pessoa de Meia-Idade , Ratos , Ratos Wistar , Suínos
9.
Mech Ageing Dev ; 66(2): 121-9, 1992 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-1365839

RESUMO

The activity of beta-N-acetylglucosaminidase (NAG), beta-galactosidase, alpha-L-fucosidase, beta-glucuronidase, beta-glucosidase and alpha-mannosidase was determined in the urine of rats at progressive ages from newborn to old animals. The age-dependence of urinary creatinine, protein and pH values was also studied. Enzyme activity, related to urinary creatinine, was significantly higher in the newborn group than other ages. The excretion of NAG increased significantly in adult rats (3-6 months old) compared to young rats (1 month old). Most of the enzyme activities were diminished in old rats (25 months old). Increased proteinuria and creatinine excretion were observed in rats since 3 months of age. Age-related differences among enzyme activities therefore should be considered when these urinary glycosidases are to be studied in rats.


Assuntos
Envelhecimento/urina , Glicosídeo Hidrolases/urina , Acetilglucosaminidase/urina , Envelhecimento/fisiologia , Animais , Animais Recém-Nascidos , Creatinina/urina , Glucuronidase/urina , Concentração de Íons de Hidrogênio , Rim/fisiologia , Masculino , Manosidases/urina , Proteinúria/urina , Ratos , Ratos Wistar , alfa-L-Fucosidase/urina , alfa-Manosidase , beta-Galactosidase/urina , beta-Glucosidase/urina
10.
Mech Ageing Dev ; 71(3): 189-98, 1993 Oct 15.
Artigo em Inglês | MEDLINE | ID: mdl-8133676

RESUMO

This study examined the profile in sheep erythrocyte fatty acids from animals of different ages during storage at 4 degrees C in a nutritive medium for up to 6 days. The changes found in the fatty acyl profile were a decrease (P < 0.01) in the percentage of arachidonic acid and an increase (P < 0.01) in the percentage of minor fatty acids (representing < 2% in each case; 20:2, 22:0 and 22:1) with respect to fresh erythrocytes in all age groups. However, the saturated/unsaturated ratios and unsaturated index started almost constant in all cases. The changes observed occurred after 24-48 h of storage, with significant increases (P < 0.01) in the fluorescence detected in the lipid extracts from stored erythrocytes during this period. The above findings suggest peroxidative damage and changes in the erythrocyte lipid membrane during storage.


Assuntos
Envelhecimento/fisiologia , Eritrócitos/química , Ácidos Graxos/química , Animais , Cromatografia Gasosa , Eritrócitos/fisiologia , Ácidos Graxos/fisiologia , Lipídeos/química , Lipídeos/fisiologia , Ovinos/fisiologia
11.
Mech Ageing Dev ; 87(1): 1-13, 1996 May 24.
Artigo em Inglês | MEDLINE | ID: mdl-8735902

RESUMO

The patterns of urinary proteins in rats of different ages were examined on SDS gradient polyacrylamide gel electrophoresis coupled with silver staining. Proteins were fractionated into at least 26 bands. Densitometric measurements were used to characterize protein excretion patterns. The results showed that proteinuria in newborn, young and adult rats is predominantly tubular, consisting of low molecular-weight species. Conversely, late adults and old rats had a mixed glomerular pattern, with a steadily increasing excretion of albumin, IgG and transferrin, as was the case of other high molecular-weight proteins. Fragments of both immunoglobulins and albumin were found in all urine samples assayed. In 1 month old rats the percentage of Tamm-Hörsfall (T-H) protein was higher (P < 0.01) than in the remaining groups studied. In newborns, relatively high albumin, IgG and transferrin percentages were detected, as well as an alpha 1-acid glycoprotein and carbonic anhydrase excretion (P < 0.05 and P < 0.01 respectively) higher than that observed in the other age groups studied.


Assuntos
Envelhecimento/urina , Proteinúria/urina , Albuminúria/urina , Animais , Animais Recém-Nascidos , Anidrases Carbônicas/urina , Eletroforese em Gel de Poliacrilamida , Fragmentos de Imunoglobulinas/urina , Masculino , Mucoproteínas/urina , Orosomucoide/urina , Ratos , Ratos Wistar , Prata , Dodecilsulfato de Sódio , Coloração e Rotulagem , Transferrina/urina , Uromodulina
12.
Biochimie ; 66(4): 291-304, 1984 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-6234944

RESUMO

Two beta-N-acetylhexosaminidases have been purified to homogeneity and characterized, from the digestive gland of the slug A. rufus L., showing very high specific activities. Hexosaminidase A (Hex A) was purified 1300-fold with a yield of 12%, and hexosaminidase B (Hex B) was purified 1400-fold with a yield of 20%. Purified Hex A or Hex B run as a single protein band in polyacrylamide gel disc electrophoresis, showing different mobilities. The purified preparations do not show any of the other glycosidase activities present in the crude extract. beta-N-acetylglucosaminidase (GlcNAc-ase) and beta-N-acetylgalactosaminidase (GalNAc-ase) activities are always associated in a single peak for each enzyme form, with constant activity ratio, in all the purification steps, since they are catalyzed by the same enzyme (Hex A or Hex B). The optimal pH for both forms are 4.5 for GlcNAc-ase and 4.0 for GalNAc-ase activity. Hex B shows thermal and pH-stability higher than Hex A. The isoelectric points are 4.5 and 5.5 for A and B forms, respectively. The molecular weight is 150 000 for Hex A and 320 000 for Hex B. The amino acid composition of purified Hex A and B presents some differences concerning particularly Cys, Thr, Ser, Glu and Ile. The ratios Vmax/Km show that GlcNAc-ase is the main activity of both enzyme forms. beta-N-acetylglucosides and beta-N-acetylgalactosides completely compete for a common active site in mixed-substrates experiments. The Ki values are always coincident for GlcNAc-ase and GalNAc-ase activities, using competitive inhibitors (the corresponding lactones). These results strongly suggest that both activities are catalyzed by the same active site in both Hex A and B. Inhibition of the enzyme activities was found with the corresponding lactones, N-acetyl hexosamines, mannose, mannosides, HgCl2 and lead acetate; activation, with ribose, and with some chlorides and sulphates of divalent cations.


Assuntos
Hexosaminidases/isolamento & purificação , Acetilglucosaminidase/metabolismo , Aminoácidos/análise , Animais , Eletroforese Descontínua , Hexosaminidase A , Hexosaminidase B , Hexosaminidases/metabolismo , Ponto Isoelétrico , Cinética , Peso Molecular , Moluscos , beta-N-Acetil-Galactosaminidase , beta-N-Acetil-Hexosaminidases
13.
Biochimie ; 72(10): 745-50, 1990 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-2078591

RESUMO

Development and aging processes in mammals are associated with changes in several physiological parameters. The aim of the present study was to investigate the changes in erythrocyte lipid composition during sheep development. In all the age groups studied, cholesterol/phospholipid ratios remained constant, at close to unity, while phospholipid patterns (sphingomyelin: 45-51%, phosphatidylethanolamine: 26-33%, phosphatidylserine: 13-19% and phosphatidylcholine: less than 2%) changed during development, with a statistically significant decrease (P less than 0.01) in phosphatidylserine and an increase in sphingomyelin content. These data suggest an increase in the rigidity of the erythrocyte lipid bilayer in adult sheep when compared with 1-month-old animals due to a decrease in the phosphatidylserine/sphingomyelin ratio. Fatty acid profiles consistently showed 5 main acids: oleic (52-54%), stearic (17-18%), linoleic (9-15%), palmitic (8.5-11%) and arachidonic acid (2-3%), mainly with significant variations (P less than 0.01) in palmitic and linoleic acid contents, respectively reaching the highest and lowest percentages in the youngest sheep. However, the developmental process seems to have no influence on the aminophospholipid topology of erythrocytes. This study suggests that the animals' developmental process has a marked effect on the lipid composition of erythrocyte membranes, which could affect cell functions.


Assuntos
Envelhecimento/fisiologia , Envelhecimento Eritrocítico/fisiologia , Membrana Eritrocítica/química , Ácidos Graxos/metabolismo , Bicamadas Lipídicas/química , Lipídeos de Membrana/química , Fosfolipídeos/metabolismo , Animais , Membrana Eritrocítica/metabolismo , Fluidez de Membrana , Lipídeos de Membrana/análise , Fosfatidilserinas/análise , Ovinos/crescimento & desenvolvimento , Esfingomielinas/análise , Ácido Trinitrobenzenossulfônico
14.
Biochimie ; 68(2): 251-60, 1986 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-3015254

RESUMO

A neutral beta-galactosidase has been purified by concanavalin A-Sepharose affinity chromatography, DEAE-cellulose chromatography, Sephadex G-200 gel filtration and hydroxylapatite chromatography. The enzyme was purified 126-fold with a yield of about 21%. This form has a neutral optimal pH (7.5) and it is located in the cytosolic fraction. It shows a wide pH stability from pH 4.5 to 8.0, but it is very unstable at low pH values. Its isoelectric point is 4.9 and this value does not change on neuraminidase treatment. The estimated molecular weight was 47 000. The neutral form shows beta-D-galactosidase, beta-D-fucosidase and beta-D-glucosidase activities, all of them associated in a single peak in all the purification steps. p-Nitrophenyl beta-D-galactosides, p-nitrophenyl beta-D-fucosides and p-nitrophenyl beta-D-glucosides competed fully for a common active site in mixed-substrate experiments. Using gamma-D-galactonolactone as competitive inhibitor the Ki values were always coincident for the three activities. The effect of NaCl, methyl mannoside and some sugars (fucose, galactose and glucose) was studied.


Assuntos
Galactosidases/metabolismo , Rim/enzimologia , beta-Galactosidase/metabolismo , Animais , Cromatografia , Cromatografia de Afinidade , Cromatografia DEAE-Celulose , Cromatografia em Gel , Durapatita , Feminino , Concentração de Íons de Hidrogênio , Hidroxiapatitas , Cinética , Masculino , Coelhos , beta-Galactosidase/isolamento & purificação
15.
Biochimie ; 70(2): 227-36, 1988 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-2969264

RESUMO

N-Acetyl-beta-D-hexosaminidase A was purified from rat urine by ion-exchange chromatography on DEAE-cellulose, followed by concanavalin A chromatography, and finally by chromatography on 2-acetamido-N-(epsilon-aminocaproyl)-2-deoxy-beta-glucosylamine-Se pharose 4B. The enzyme was purified 482-fold with a yield of about 7%. The optimal pH was 4.5 for N-acetyl-glucosaminidase activity and 4.0-4.5 for N-acetylgalactosaminidase activity. The enzyme was heat-labile and stable from pH 4.5 to pH 7.0 but it was very unstable at lower pH values. Km values were 0.55 mM and 0.059 mM, respectively. The glycoprotein nature of the enzyme was deduced from its behavior on concanavalin A. The effect of some carbohydrates and ionic compounds on the activities of the enzyme was studied. When N-acetyl-D-glucosaminolactone and N-acetyl-D-galactosaminolactone were used as inhibitors, Ki values were also calculated.


Assuntos
beta-N-Acetil-Hexosaminidases/urina , Animais , Cromatografia de Afinidade , Cromatografia DEAE-Celulose , Eletroforese em Gel de Poliacrilamida , Estabilidade Enzimática , Glicosídeo Hidrolases/metabolismo , Concentração de Íons de Hidrogênio , Focalização Isoelétrica , Cinética , Masculino , Ratos , Ratos Endogâmicos , Temperatura , beta-N-Acetil-Hexosaminidases/isolamento & purificação
16.
Virus Res ; 25(1-2): 145-53, 1992 Sep 01.
Artigo em Inglês | MEDLINE | ID: mdl-1413991

RESUMO

Influenza virus type C (Johannesburg/1/66) was used as a source for the enzyme O-acetylesterase (EC 3.1.1.53) with several natural sialoglycoconjugates as substrates. The resulting products were immediately employed as substrates using influenza virus type A [(Singapore/6/86) (H1N1) or Shanghai/11/87 (H3N2)] as a source for sialidase (neuraminidase, EC 3.2.1.18). A significant increase in the percentage of sialic acid released was found when the O-acetyl group was cleaved by O-acetylesterase activity from certain substrates (bovine submandibular gland mucin, rat serum glycoproteins, human saliva glycoproteins, mouse erythrocyte stroma, chick embryonic brain gangliosides and bovine brain gangliosides). A common feature of all these substrates is that they contain N-acetyl-9-O-acetylneuraminic acid residues. By contrast, no significant increase in the release of sialic acid was detected when certain other substrates could not be de-O-acetylated by the action of influenza C esterase, either because they lacked O-acetylsialic acid (human glycophorin A, alpha 1-acid glycoprotein from human serum, fetuin and porcine submandibular gland mucin) or because the 4-O-acetyl group was scarcely cleaved by the viral O-acetylesterase (equine submandibular gland mucin). The biological significance of these facts is discussed, relative to the infective capacity of influenza C virus.


Assuntos
Acetilesterase/metabolismo , Gammainfluenzavirus/enzimologia , Vírus da Influenza A/enzimologia , Neuraminidase/metabolismo , Ácidos Siálicos , Especificidade por Substrato
17.
Cancer Lett ; 141(1-2): 73-7, 1999 Jul 01.
Artigo em Inglês | MEDLINE | ID: mdl-10454245

RESUMO

The activities of several glycosidases and cathepsin L were determined in the blood serum of a control group of ten healthy humans in comparison with a group (group I: 32 subjects) of preoperative colorectal cancer patients (1 week before surgical exeresis) and with another two groups: group II, comprising 18 operated subjects (1 week after surgery), and group III, of 15 operated subjects (4 months after surgery). All subjects were 48-88 years old. Both 'enzyme activity' and 'specific activity' determinations of serum beta-galactosidase, alpha-L-fucosidase and cathepsin L revealed peculiar profiles that differed from one another. Control values differed from those of some stages of the pathological groups, but not of others. These values were compared also with the levels of total, lipid- and glycoprotein-associated serum sialic acid. The usefulness of some assays (especially cathepsin L activity measurement) in the follow-up of the health status of humans operated for colorectal cancer is discussed.


Assuntos
Catepsinas/sangue , Neoplasias Colorretais/sangue , Endopeptidases , alfa-L-Fucosidase/sangue , beta-Galactosidase/sangue , Idoso , Idoso de 80 Anos ou mais , Antígeno Carcinoembrionário/sangue , Catepsina L , Neoplasias Colorretais/enzimologia , Cisteína Endopeptidases , Humanos , Modelos Lineares , Pessoa de Meia-Idade , Estadiamento de Neoplasias , Ácidos Siálicos/sangue
18.
J Biochem ; 104(1): 66-71, 1988 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-3146570

RESUMO

beta-D-Galactosidase has been purified to apparent homogeneity from rabbit spleen. The purification steps involved ammonium sulphate precipitation, DEAE-cellulose, concanavalin A-Sepharose, Sephadex G-200, and Sepharose 4B-(epsilon-aminocaproyl)-2-deoxy-beta-D-glucosylamine affinity chromatographies. In the DEAE-cellulose step, the beta-D-galactosidase was separated into two molecular forms, designated I and II, with similar pH optimum, Km, substrate specificity, and sensitivity to substrate analogues and other substances. Form I was purified 1,800-fold with a yield of about 2% of the total activity. This form is heat-labile, it has an acid optimal pH (4.0), an isoelectric point of 6.7 and a molecular weight of 75,000 daltons. Form II has an optimal pH of 3.6 and three different pI values (5.3, 5.7, and 6.7) whose relative proportions can be modified by treatment with neuraminidase. Form II appeared to be a multimeric form (IIA) of about 600,000 daltons at pH 4.0, which was reversibly dissociated to an oligomeric form (IIB) with an apparent molecular weight of 120,000 at neutral pH values. Both IIA and IIB were purified separately and showed an acid pH optimum and an heterogeneous pI (from 4.6 to 7.2). The dissociation of IIA into IIB can be generated spontaneously, but is increased by the presence of urea in the elution buffer, suggesting that both are aggregates of a common subunit.


Assuntos
Galactosidases/isolamento & purificação , Baço/metabolismo , beta-Galactosidase/isolamento & purificação , Animais , Cromatografia de Afinidade , Cromatografia DEAE-Celulose , Cromatografia em Gel , Eletroforese em Gel de Poliacrilamida , Estabilidade Enzimática , Concentração de Íons de Hidrogênio , Focalização Isoelétrica , Cinética , Peso Molecular , Coelhos , beta-Galactosidase/metabolismo
19.
Clin Chim Acta ; 146(2-3): 129-34, 1985 Mar 15.
Artigo em Inglês | MEDLINE | ID: mdl-3987046

RESUMO

Some glycosidase activities have been determined in blood sera from 21 patients who ingested a toxic oil (rapeseed oil denatured with aniline(s) and treated by a thermal process). The samples were collected from the same patients on 3 or 4 occasions during a period of 11-12 mth. During this period, the clinical state of the patients improved and, generally, they were restored to health. beta-N-Acetylglucosaminidase, alpha-L-fucosidase, beta-D-glucuronidase, beta-D-glucosidase and alpha-D-mannosidase activities, which were higher in patients than in 17 controls during the first mth decreased to normal values in the period studied, 11-12 mth. In contrast, beta-D-galactosidase, alpha-D-galactosidase and alpha-D-glucosidase activities, which were initially lower in patients than in controls, were finally similar or higher than in controls. One explanation for these results could be the possible alteration of the cell membrane(s) by the toxic substance(s).


Assuntos
Compostos de Anilina/intoxicação , Glicosídeo Hidrolases/sangue , Óleos/efeitos adversos , Óleos de Plantas , Convalescença , Ácidos Graxos Monoinsaturados , Contaminação de Alimentos , Humanos , Óleo de Brassica napus , Espectrometria de Fluorescência , Fatores de Tempo
20.
Clin Chim Acta ; 119(1-2): 15-9, 1982 Feb 26.
Artigo em Inglês | MEDLINE | ID: mdl-6800674

RESUMO

The specific activities of several glycosidases (beta-N-acetylglucosaminidase, beta-D-glucosidase, alpha-D-glucosidase, beta-D-fucosidase, alpha-L-fucosidase and beta-D-galactosidase) were determined in human sera from a control group to 10 normal subjects and in four groups, each of 10 patients, with acute viral hepatitis, acute pancreatitis, acute myocardial infarction and breast cancer. The results show significantly higher activities in acute viral hepatitis for beta-N-acetylglucosaminidase, beta-D-glucosidase and alpha-D-glucosidase (p less than 0.001); in acute pancreatitis for the first two of these enzymes (p less than 0.001); and in breast cancer for beta-D-glucosidase (p less than 0.001). Further, lower differences were found in the patients with acute viral hepatitis for beta-D-fucosidase and alpha-L-fucosidase (p less than 0.01); in acute myocardial infarction for beta-N-acetylglucosaminidase, beta-D-glucosidase, alpha-D-glucosidase, beta-D-fucosidase and beta-D-galactosidase (p less than 0.01, p less than 0.05, p less than 0.05, p less than 0.01 and p less than 0.01, respectively); and in breast cancer for beta-N-acetylglucosaminidase (p less than 0.01). No significant differences were found for the other glycosidases.


Assuntos
Neoplasias da Mama/enzimologia , Glicosídeo Hidrolases/sangue , Hepatite Viral Humana/enzimologia , Infarto do Miocárdio/enzimologia , Pancreatite/enzimologia , Acetilglucosaminidase/sangue , Doença Aguda , Humanos , alfa-Glucosidases/sangue , alfa-L-Fucosidase/sangue , beta-Galactosidase/sangue , beta-Glucosidase/sangue
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