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1.
Science ; 277(5333): 1802-5, 1997 Sep 19.
Artículo en Inglés | MEDLINE | ID: mdl-9295267

RESUMEN

Classical late-infantile neuronal ceroid lipofuscinosis (LINCL) is a fatal neurodegenerative disease whose defective gene has remained elusive. A molecular basis for LINCL was determined with an approach applicable to other lysosomal storage diseases. When the mannose 6-phosphate modification of newly synthesized lysosomal enzymes was used as an affinity marker, a single protein was identified that is absent in LINCL. Sequence comparisons suggest that this protein is a pepstatin-insensitive lysosomal peptidase, and a corresponding enzymatic activity was deficient in LINCL autopsy specimens. Mutations in the gene encoding this protein were identified in LINCL patients but not in normal controls.


Asunto(s)
Lisosomas/enzimología , Mutación , Lipofuscinosis Ceroideas Neuronales/genética , Péptido Hidrolasas/química , Péptido Hidrolasas/genética , Secuencia de Aminoácidos , Aminopeptidasas , Mapeo Cromosómico , Cromosomas Humanos Par 11 , Codón , Dipeptidil-Peptidasas y Tripeptidil-Peptidasas , Endopeptidasas , Femenino , Glicosilación , Humanos , Punto Isoeléctrico , Masculino , Manosafosfatos/análisis , Datos de Secuencia Molecular , Peso Molecular , Lipofuscinosis Ceroideas Neuronales/enzimología , Pepstatinas/farmacología , Péptido Hidrolasas/deficiencia , Reacción en Cadena de la Polimerasa , Serina Proteasas , Tripeptidil Peptidasa 1
2.
Int J Biochem Cell Biol ; 31(7): 751-7, 1999 Jul.
Artículo en Inglés | MEDLINE | ID: mdl-10467731

RESUMEN

Active oxygen species are thought to be involved in many physiological and pathological processes and are known to oxidatively modify DNA, lipids and proteins. One such modification is the addition of carbonyl groups to amino acid residues in proteins. The number of carbonyl groups on proteins can be quantitated spectrophotometrically using 2,4-dinitrophenylhydrazine (DNPH). The DNPH assay described in the literature was found to be unreliable in samples containing high amounts of chromophore (e.g. hemoglobin, myoglobin, retinoids). By using an HCl-acetone wash, hemes from the chromophores could be extracted, enabling the determination of carbonyl content to be made even in highly colored tissue extracts. Residual DNPH, which was also found to interfere with the assay, was removed by additional washes with trichloroacetic acid and ethanol-ethylacetate. These improvements are known to remove lipids, do not lengthen the time required to do the assay, permit quantification of carbonyl content in 1-4 mg protein from a variety of tissue types and provide a sensitive and reliable method for assessing oxidative damage to tissue proteins.


Asunto(s)
Proteínas/química , Proteínas/metabolismo , Animales , Técnicas In Vitro , Indicadores y Reactivos , Ratones , Oxidación-Reducción , Fenilhidrazinas , Ratas , Especies Reactivas de Oxígeno/metabolismo , Solubilidad , Espectrofotometría , Distribución Tisular
3.
Eur J Paediatr Neurol ; 5 Suppl A: 57-62, 2001.
Artículo en Inglés | MEDLINE | ID: mdl-11589009

RESUMEN

The ability of aminoglycoside antibiotics to promote readthrough of eukaryotic stop codons has attracted interest in these drugs as potential therapeutic agents in human disorders caused by nonsense mutations. One disease for which such a therapeutic strategy may be viable is classical late infantile neuronal ceroid lipofuscinosis (LINCL), a fatal childhood neurodegenerative disorder with currently no effective treatment. Premature stop codon mutations in the gene CLN2 encoding the lysosomal tripeptidyl-peptidase 1 (TPP-I) are associated with disease in approximately half of children diagnosed with LINCL. The aim of this study was to examine the ability of the aminoglycoside gentamicin to restore TPP-I activity in LINCL cell lines. In one patient-derived cell line that was compound heterozygous for a commonly seen nonsense mutation, Arg208Stop and a different rare nonsense mutation, approximately 7% of normal levels of TPP-I were maximally restored with gentamicin treatment. In other cell lines from patients that were compound heterozygous for Arg208Stop and a splice junction mutation, approximately 0.5% of maximal activity was restored. These results suggest that pharmacological suppression of nonsense mutations by aminoglycosides or functionally similar pharmaceuticals may have therapeutic potential in LINCL.


Asunto(s)
Antibacterianos/farmacología , Codón sin Sentido , Gentamicinas/farmacología , Lipofuscinosis Ceroideas Neuronales/tratamiento farmacológico , Lipofuscinosis Ceroideas Neuronales/genética , Línea Celular , Codón de Terminación/efectos de los fármacos , Fibroblastos/citología , Expresión Génica/efectos de los fármacos , Humanos , Lactante , Supresión Genética/efectos de los fármacos , Tripeptidil Peptidasa 1
4.
Eur J Paediatr Neurol ; 5 Suppl A: 43-5, 2001.
Artículo en Inglés | MEDLINE | ID: mdl-11589006

RESUMEN

We recently showed that a form of neuronal ceroid lipofuscinosis (NCL) in white Swedish landrace sheep is caused by a missense mutation in the cathepsin D gene resulting in complete inactivation of the enzyme. Despite the lack of cathepsin D activity, the brains of the cathepsin D deficient sheep showed strongly increased staining for cathepsin D in immunohistochemistry. By Western blotting, a 5-10 fold increase in the level of cathepsin D was confirmed. These results indicate that the missense mutation in congenital NCL sheep results in the synthesis of an inactive yet stable cathepsin D.


Asunto(s)
Catepsina D/deficiencia , Lipofuscinosis Ceroideas Neuronales/enzimología , Animales , Catepsina D/análisis , Catepsina D/genética , Lóbulo Frontal/enzimología , Lisosomas/enzimología , Mutación Missense , Degeneración Nerviosa/congénito , Degeneración Nerviosa/enzimología , Lipofuscinosis Ceroideas Neuronales/congénito , Ovinos
5.
In Vivo ; 7(5): 431-4, 1993.
Artículo en Inglés | MEDLINE | ID: mdl-8110987

RESUMEN

A tricyclic compound tetrahydroaminoacridine is known to improve the cognitive function in Alzheimer's disease. The possible mechanism of action of acridine and structurally related tricyclic compounds was studied on the bivalent cation content of bacterial membrane, rat brain acetylcholinesterase and some tissue proteases in model experiments. Acridine orange and disubstituted chlorpromazine (CPZ) derivatives lowered Ca2+ and Mg2+ binding and membrane polarization in the simplest biological membrane (E. coli), as revealed by reactor neutron activation analysis. Acetylcholinesterase (AChE) was inhibited by CPZ, 3,7,8-trihydroxy-CPZ, acridine orange partially saturated desipramine, imipramine, trans-clopenthixol and tetrahydrocannabidiolic at 10(-4) to 10(-5). A metalloproteinase, MMP-7-ase, was inhibited by tetrahydrocannabidiolic acid, 3,7,8-trihydroxy-CPZ, acridine orange but other tissue proteinases, ATN-ase and cathepsin B, were less sensitive to these compounds. (ATN-ase is an acetyltyrosine-p-nitroanilide splitting enzyme, a serine protease). The chelate complex forming ability and electron donor capacity of the compounds may play a role in the biological effects tested. It is assumed that compounds which do not displace bivalent cations in membranes may exert an inhibitory effect on AChE, and that metalloproteinase enzymes may be promising for the treatment of degenerative brain diseases.


Asunto(s)
Calcio/metabolismo , Membrana Celular/efectos de los fármacos , Inhibidores de la Colinesterasa/farmacología , Metaloendopeptidasas/antagonistas & inhibidores , Fenotiazinas/farmacología , Enfermedad de Alzheimer/tratamiento farmacológico , Enfermedad de Alzheimer/metabolismo , Secuencia de Aminoácidos , Animales , Calmodulina/antagonistas & inhibidores , Escherichia coli/efectos de los fármacos , Magnesio/metabolismo , Datos de Secuencia Molecular , Proteínas Musculares/antagonistas & inhibidores , Proteínas del Tejido Nervioso/antagonistas & inhibidores , Ratas , Relación Estructura-Actividad , betaendorfina/metabolismo
6.
Gen Physiol Biophys ; 10(5): 505-14, 1991 Oct.
Artículo en Inglés | MEDLINE | ID: mdl-1816030

RESUMEN

The activity of different cathepsins and neutral proteinases was measured in normal and vitamin E-deficient rabbit muscles using specific substrates. Among the changes of enzyme activities in dystrophy caused by vitamin E-deficiency the increase in the activity of cathepsin B is the most striking. The activity of cathepsin H, both in the fast and slow muscles and that of MMP-ase in the slow muscle remains practically unchanged. Activities of other proteases significantly increase. The change in the activity of proteolytic enzymes in striated muscle of vitamin E-deficient rabbits seems to be selective. As a rule the increase in the activity is higher in fast than in slow muscles.


Asunto(s)
Catepsina B/metabolismo , Cisteína Endopeptidasas , Endopeptidasas/metabolismo , Músculos/enzimología , Deficiencia de Vitamina E/enzimología , Animales , Catepsina H , Catepsinas/metabolismo , Electroforesis en Gel de Poliacrilamida , Distrofia Muscular Animal/enzimología , Distrofia Muscular Animal/etiología , Conejos , Estadística como Asunto
7.
Acta Biol Hung ; 42(1-3): 183-201, 1991.
Artículo en Inglés | MEDLINE | ID: mdl-1844310

RESUMEN

Two weeks of immobilization in shortened state caused 50% decrease in muscle mass and an increase in lysosomal proteinase activities. In this study causes of elevated protease activities in muscle are studied. Many factors could play a role in these elevated proteinase activities. We have found that redox state, ATP content, fuel supply and glucocorticoid receptor number were important in this period. Testosterone, insulin or proteinase inhibitors were not proved to play role in elevated proteinase activities. These practical results are explained by the results achieved in other types of cells. We conclude that changes in redox potential and/or oxygen free radical content of muscle elements can cause a post-translational covalent modification of cysteine proteinases and -SH dependent metalloproteinases, leading thereby to their activation. Lysosomal cysteine proteinases can activate procathepsin D that can damage lysosomal cysteine proteinase inhibitors and in another path it activates procathepsin B, L and H reversely. This feed-back regulation and the activation of cysteine proteinases by metalloproteinases might accelerate the proteinase activities in skeletal muscles.


Asunto(s)
Endopeptidasas/metabolismo , Músculos/enzimología , Animales , Activación Enzimática , Radicales Libres , Lisosomas/enzimología , Oxidación-Reducción
8.
Acta Biol Hung ; 42(1-3): 285-95, 1991.
Artículo en Inglés | MEDLINE | ID: mdl-1688230

RESUMEN

Proteinase activities in rat thioglycollate elicited peritoneal cells and the cell-free supernatant (lavage fluid) were measured by using the following substrates: Suc-Ala-Ala-Pro-Phe-Methyl-Coumarin-Amide (for cathepsin G or chymase), Suc-Ala-Ala-Ala-AMC (for elastase or elastase-like), Z-Arg-Arg-AMC (for cathepsin B), haemoglobin (for cathepsin D) and Ala-AMC (for alanine-aminopeptidase: AAP). The enzyme activities were correlated to the quantitative distribution of various cell types in the exudate from 0 to 192 nd h. In the supernatant all the examined activities showed a higher value at 72nd h. In the cells activity of chymase and AAP proved to be very high at 0 h but after four h the activities were dropped. From this time all enzyme activities started to elevate till the 24th h. At the 96th h only the activity of cathepsin B and AAP had a high value. We conclude that the intracellular activation and secretion of proteolytic enzymes characteristic for the various peritoneal cell types involved in the acute and chronic inflammatory reaction can be followed by activity measurements using enzyme-specific substrates and inhibitors.


Asunto(s)
Aminopeptidasas/metabolismo , Catepsinas/metabolismo , Elastasa Pancreática/metabolismo , Peritonitis/enzimología , Animales , Antígenos CD13 , Modelos Animales de Enfermedad , Activación Enzimática , Inhibidores Enzimáticos/farmacología , Estabilidad de Enzimas , Exudados y Transudados , Concentración de Iones de Hidrógeno , Cavidad Peritoneal/citología , Peritonitis/inducido químicamente , Ratas , Ratas Wistar , Especificidad por Sustrato , Tioglicolatos
9.
Acta Biol Hung ; 42(1-3): 265-74, 1991.
Artículo en Inglés | MEDLINE | ID: mdl-1844314

RESUMEN

Previously, MMP-7ases were isolated from rat skeletal muscle by gel filtration and anion exchange chromatography. The enzyme that hydrolyzed succinyl-Ala-Ala-Pro-Phe-AMC (AMC: 7-amino-4-methyl-coumarin) was inhibited by EDTA. In this study we attempted to isolate MMP-7ase from mouse kidney. The isolation procedure was the same as that previously used for skeletal muscle. Kidneys of ICR mice were homogenized and, after centrifugation, the supernatant fraction was subjected to gel filtration chromatography. The fraction with the highest activity (Mr 67-72 kDa) was subjected to anion exchange chromatography, which showed three peaks of activity. The second peak hydrolyzed succ-Ala-Ala-Pro-Phe-AMC, but had low activity against Arg- or Ala-AMC. This peak was a single protein (Mr 68-72 kDa) and its activity could be inhibited with EDTA. Several tri- and tetrapeptide derivatives were tested as substrates for this enzyme and the best was found to be succ-Ala-Ala-Pro-Phe-AMC. We can conclude that mouse kidney cytosol contains a metalloendopeptidase similar to muscle MMP-7ase.


Asunto(s)
Riñón/enzimología , Metaloendopeptidasas/aislamiento & purificación , Animales , Cromatografía en Gel , Cromatografía por Intercambio Iónico , Cumarinas/metabolismo , Citosol/enzimología , Ácido Edético/farmacología , Inhibidores Enzimáticos , Metaloproteinasa 7 de la Matriz , Metaloendopeptidasas/antagonistas & inhibidores , Ratones , Ratones Endogámicos C3H , Ratones Endogámicos ICR , Peso Molecular , Oligopéptidos/metabolismo , Solubilidad , Especificidad por Sustrato
13.
Biol Chem Hoppe Seyler ; 373(7): 567-72, 1992 Jul.
Artículo en Inglés | MEDLINE | ID: mdl-1515085

RESUMEN

A lot of new results have been published on activation of one or more proteinases in mammalian tissues. Explanation of proteinase activation without invasion of leukocyte during atrophy of mammalian tissues is the subject of the present study. The mechanism of activation process can be divided into two parts. The first one consists of modification of proteinases and their inhibitors by oxidation or reduction including oxygen free radical reactions. The second part of activation is a cascade or cycle including limited proteolysis of proenzymes and/or proteinase inhibitors. The hypothetic system of proteinase activation can give a hope for regulation of proteinase activity in mammalian tissues under pathological- or pathologic-like conditions as during exhausting exercise.


Asunto(s)
Endopeptidasas/metabolismo , Animales , Activación Enzimática/efectos de los fármacos , Humanos , Oxidación-Reducción
14.
Acta Paediatr Hung ; 28(3-4): 175-8, 1987.
Artículo en Inglés | MEDLINE | ID: mdl-3454203

RESUMEN

Lysosomal cysteine proteinase (cathepsin B, H, L) and metalloproteinase (MMP-7-ase) activities were measured from serum of 19 cystic fibrosis (CF) homozygotes and of 13 healthy children, as control group. The activity of cathepsin B and H significantly increased in the CF-group.


Asunto(s)
Catepsinas/sangre , Fibrosis Quística/enzimología , Metaloendopeptidasas/sangre , Niño , Fibrosis Quística/genética , Homocigoto , Humanos
15.
Acta Physiol Hung ; 70(4): 403-7, 1987.
Artículo en Inglés | MEDLINE | ID: mdl-3326400

RESUMEN

Goldblatt hypertension was induced in rats by constricting the renal artery on one side. In one group of animals the contralateral kidney remained untouched (two-kidney hypertension), while in the other it was removed (one-kidney hypertension). In the two-kidney hypertension group, renin activity was higher than in the control animals, the fibrinogen was normal both in arterial and venous blood while in one-kidney hypertension the PRA was normal, but the fibrinogen was increased. A close significant correlation could be demonstrated between blood pressure and fibrinogen.


Asunto(s)
Fibrinógeno/metabolismo , Hipertensión Renovascular/fisiopatología , Renina/sangre , Animales , Arterias , Presión Sanguínea , Hipertensión Renovascular/sangre , Hipertensión Renovascular/etiología , Masculino , Ratas , Venas
16.
Am J Physiol ; 259(2 Pt 1): C232-40, 1990 Aug.
Artículo en Inglés | MEDLINE | ID: mdl-2382700

RESUMEN

Intermittent repetitive mechanical stimulation of differentiated avian skeletal muscle cells in vitro for 48 h stimulates skeletal muscle growth [Am. J. Physiol. 256 (Cell Physiol. 25): C674-C682, 1989]. During the first 2-3 h of stimulation, temporary muscle damage occurs based on increases in creatine kinase efflux, total protein degradation rates, and several proteinase activites. With continued mechanical stimulation for several days in serum-containing medium, the proteinase activities return to control levels, and total protein degradation rates decrease to levels less than static controls. Decreased protein degradation thus contributes to stretch-induced cell growth. The efflux of prostaglandins (PG) E2 and F2 alpha but not 6-keto-PGF1 alpha increase with mechanical stimulation. During the first 5 h of stimulation, PGE2 and PGF2 alpha efflux rates increase 101 and 41%, respectively. PGE2 efflux returns to control levels by 24 h of mechanical stimulation, whereas PGF2 alpha efflux is continuously elevated (41-116%) for at least 48 h. The long-term stretch-induced elevation of PGF2 alpha efflux correlates with a 52-98% long-term increase in total protein synthesis rates. The prostaglandin synthesis inhibitor indomethacin partially blocks early stretch-induced cell damage and long-term stretch-induced cell growth. The results indicate that both of these processes are partially dependent on stretch-induced increases in prostaglandin synthesis.


Asunto(s)
Catepsinas/metabolismo , Metaloendopeptidasas/metabolismo , Contracción Muscular , Músculos/fisiología , Prostaglandinas/metabolismo , Proteínas/metabolismo , Animales , Células Cultivadas , Embrión de Pollo , Dinoprost/farmacología , Dinoprostona/farmacología , Indometacina/farmacología , Cinética , Músculos/efectos de los fármacos , Estimulación Física , Biosíntesis de Proteínas
17.
Biol Chem Hoppe Seyler ; 369 Suppl: 277-9, 1988 May.
Artículo en Inglés | MEDLINE | ID: mdl-3202967

RESUMEN

Lysosomal cysteine proteinase (cathepsin B, H, and L) and MMP-7ase muscle metalloproteinase activities were measured in serum from Duchenne muscular dystrophic male patients and their mothers as gene-carriers. The activity of cathepsin H significantly increased in the Duchenne muscular dystrophic (DMD)-hemizygotes group and in the group of DMD heterozygotes. Significant positive correlation was found between the activity of serum creatine kinase (which previously has been proven to be a marker of muscular dystrophy) and of cathepsin L in the DMD-hemizygotes group. Furthermore, correlations were found between the activity of creatine kinase and MMP-7ase or between activity of creatine kinase and cathepsin H in the DMD heterozygotes. The changes in activity of proteolytic enzymes in serum of dystrophic patients can be explained by the elevated proteolytic enzyme activity in dystrophic muscle observed previously.


Asunto(s)
Cisteína Endopeptidasas/sangre , Metaloendopeptidasas/sangre , Distrofias Musculares/enzimología , Adulto , Niño , Femenino , Genotipo , Heterocigoto , Humanos , Masculino
18.
Biochem Biophys Res Commun ; 134(3): 1269-75, 1986 Feb 13.
Artículo en Inglés | MEDLINE | ID: mdl-2936345

RESUMEN

In several myopathic disorders, the internal muscle cell calcium concentration increases significantly as compared to normal muscle cells. We report that in the presence of elevated calcium levels, the calcium-binding proteins troponin C and calmodulin are protected from digestion by the chymotrypsin-like serine proteinase that co-purifies with isolated myofibrils. Degradation of the 67k calcimedin in the presence of calcium shows altered major cleavage fragments while degradation of myosin is unaffected by the presence of calcium. A role for this serine proteinase in muscle-wasting diseases is suggested.


Asunto(s)
Proteínas de Unión al Calcio/metabolismo , Calcio/fisiología , Endopeptidasas/metabolismo , Animales , Anexina A6 , Anexinas , Calmodulina/metabolismo , Catálisis , Masculino , Músculos/enzimología , Miosinas/metabolismo , Ratas , Serina Endopeptidasas , Troponina/metabolismo , Troponina C
19.
Biomed Biochim Acta ; 48(5-6): S422-5, 1989.
Artículo en Inglés | MEDLINE | ID: mdl-2667517

RESUMEN

Physical activity can be studied by various kind of exercises. Many works have been published in field of proteolytic enzyme activities in skeletal muscle during endurance training. In this work we used high jumping as a dynamic force velocity training to study the changes in proteolytic enzyme activities during this type of exercise. The activity of cathepsin D, cathepsin L and ATN-ase (Acetyl-Tyrosine-paranitroanilide-splitting enzyme) in vastus lateralis muscle was measured after one, 3, 7 or 11 weeks of high jumping exercise. The results demonstrated that proteinase activity began to increase when the load, i.e. number of jumping and the weight put on the rat's back was too much for their muscles. They could carry out the task consuming the energy originating from muscle tissue in the first period of the experiment, but in the second period (after 7 weeks) the type of training with this load became equal with an endurance training.


Asunto(s)
Músculos/enzimología , Péptido Hidrolasas/metabolismo , Condicionamiento Físico Animal , Animales , Masculino , Ratas , Ratas Endogámicas
20.
J Biol Chem ; 276(3): 2249-55, 2001 Jan 19.
Artículo en Inglés | MEDLINE | ID: mdl-11054422

RESUMEN

The CLN2 gene mutated in the fatal hereditary neurodegenerative disease late infantile neuronal ceroid lipofuscinosis encodes a lysosomal protease with tripeptidyl-peptidase I activity. To understand the enzymological properties of the protein, we purified and characterized C-terminal hexahistidine-tagged human CLN2p/tripeptidyl-peptidase I produced from insect cells transfected with a baculovirus vector. The N terminus of the secreted 66-kDa protein corresponds to residue 20 of the primary CLN2 gene translation product, indicating removal of a 19-residue signal peptide. The purified protein is enzymatically inactive; however, upon acidification, it is proteolytically processed and concomitantly acquires enzymatic activity. The N terminus of the final 46-kDa processed form (Leu196) corresponds to that of mature CLN2p/tripeptidyl-peptidase I purified from human brain. The activity of the mature enzyme is irreversibly inhibited by the serine esterase inhibitor diisopropyl fluorophosphate, which specifically and stoichiometrically reacts with CLN2p/tripeptidyl-peptidase I at Ser475, demonstrating that this residue represents the active site nucleophile. Expression of wild type and mutant proteins in CHO cells indicate that Ser475, Asp360, Asp517, but not His236 are essential for activity. These data indicate that the CLN2 gene product is synthesized as an inactive proenzyme that is autocatalytically converted to an active serine protease.


Asunto(s)
Concentración de Iones de Hidrógeno , Péptido Hidrolasas/metabolismo , Serina Endopeptidasas/metabolismo , Ácidos , Secuencia de Aminoácidos , Aminopeptidasas , Animales , Sitios de Unión , Células CHO , Catálisis , Cricetinae , Dipeptidil-Peptidasas y Tripeptidil-Peptidasas , Endopeptidasas , Activación Enzimática , Humanos , Datos de Secuencia Molecular , Péptido Hidrolasas/química , Homología de Secuencia de Aminoácido , Serina Endopeptidasas/química , Serina Proteasas , Tripeptidil Peptidasa 1
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