RESUMEN
Our understanding of the pathogenesis of Alzheimer's disease (AD) comes primarily from the study of rare inherited forms of the disease. Mutations that cause familial AD appear to act by a common mechanism: that of increasing production of A beta 42/43, one of the family of A beta peptides deposited in senile plaques. However, increased A beta 42/43 production has not been demonstrated to occur in most cases of sporadic AD, suggesting that genetic and environmental factors acting at other stages of the disease process can modify the risk for disease. Such factors most likely include those affecting A beta aggregation or clearance, the inflammatory response, cerebrovascular disease, or susceptibility of neurons to injury. Identifying these factors will lead to a better understanding of the etiology of the disease and provide additional targets for therapeutic intervention.
Asunto(s)
Enfermedad de Alzheimer/etiología , Edad de Inicio , Anciano , Alelos , Enfermedad de Alzheimer/epidemiología , Enfermedad de Alzheimer/genética , Enfermedad de Alzheimer/metabolismo , Enfermedad de Alzheimer/patología , Precursor de Proteína beta-Amiloide/biosíntesis , Precursor de Proteína beta-Amiloide/genética , Apolipoproteína E4 , Apolipoproteínas E/genética , Biomarcadores , Encéfalo/patología , Química Encefálica , Butirilcolinesterasa/genética , Calcio/metabolismo , Enfermedades Cardiovasculares/complicaciones , Susceptibilidad a Enfermedades , Genes Dominantes , Humanos , Inflamación , Proteínas de la Membrana/genética , Persona de Mediana Edad , Mitocondrias/metabolismo , Ovillos Neurofibrilares/ultraestructura , Neuroglía/patología , Neurotoxinas/efectos adversos , Estrés Oxidativo , Placa Amiloide/química , Presenilina-1 , Presenilina-2 , Factores de Riesgo , alfa 1-Antiquimotripsina/genéticaRESUMEN
Alzheimer disease (AD), the most common cause of dementia in the elderly, exists in both familial and sporadic forms. Genetic studies have led to the identification of 3 genes, beta-amyloid precursor protein (APP), presenilin-1 (PS1), and presenilin-2 (PS2), which, when mutated, can cause familial forms of AD. Mutations in each of these genes result in elevated levels of A beta42/43, a proteolytic processing fragment of APP that is deposited in brains of AD patients. Transgenic mice carrying AD-causing mutations in APP develop spontaneous age-related beta-amyloid (A beta) deposition and memory impairment. Genetic linkage and association studies have also shown that the epsilon4 allele of the apolipoprotein E (APOE) gene increases risk for AD in a dose-dependent manner in both familial and sporadic forms of AD and may account for as much as 50% of the attributable risk. APOE genotyping may be useful both as an adjunct to diagnosis and in identifying patient groups for therapeutic intervention. While the biological basis for the association of APOE epsilon4 with AD is not known, age of onset and A beta deposition are positively correlated with epsilon4 allele dosage in some cases, suggesting that this risk may also be mediated directly or indirectly through A beta. Because 50% of AD cases have no APOE epsilon4 alleles and families showing mendelian inheritance of AD exist in whom there are no mutations in any of the APP, PS1, or PS2 genes, it is likely that there are additional AD risk factors, both genetic and environmental, still to be identified.
Asunto(s)
Enfermedad de Alzheimer/genética , Precursor de Proteína beta-Amiloide/genética , Apolipoproteínas E/genética , Proteínas de la Membrana/genética , Mutación , Anciano , Enfermedad de Alzheimer/epidemiología , Enfermedad de Alzheimer/etiología , Animales , Genotipo , Humanos , Ratones , Ratones Transgénicos , Presenilina-1 , Presenilina-2 , Factores de RiesgoRESUMEN
Excretory/secretory (E/S) material of Trichuris muris was found to contain 2 major peptidases, M(r) 85 and 105 kDa, which degrade gelatin optimally at pH 6.0 in sodium dodecyl sulphate-polyacrylamide gels. The peptidases were inactivated by diisopropylfluorophosphate, leupeptin and soybean trypsin inhibitor, but were unaffected by inhibitors of aspartic-, cysteine- and metallo-peptidases, indicating that they are serine peptidases. Both enzymes were detectable within 5 h after incubation of worms in culture medium and showed a time-dependent increase in levels. Neither peptidase was detected in worm extracts, suggesting that they are activated during or following secretion from worms. Live worms degraded a radio-isotope labelled extracellular matrix protein substratum derived from mammalian cells. Aminopeptidase activities capable of catalysing hydrolysis of amino acyl aminomethylcoumarin (MCA) substrates and a Z-Phe-Arg-MCA-hydrolysing cysteine peptidase activity, were detected in extracts of adult worms but not in E/S material.
Asunto(s)
Endopeptidasas/metabolismo , Trichuris/enzimología , Secuencia de Aminoácidos , Aminopeptidasas/aislamiento & purificación , Aminopeptidasas/metabolismo , Animales , Cumarinas/química , Cisteína Endopeptidasas/aislamiento & purificación , Cisteína Endopeptidasas/metabolismo , Endopeptidasas/aislamiento & purificación , Concentración de Iones de Hidrógeno , Datos de Secuencia Molecular , Peso Molecular , Oligopéptidos/química , Inhibidores de Proteasas/farmacología , Serina Endopeptidasas/aislamiento & purificación , Serina Endopeptidasas/metabolismo , Especificidad por SustratoRESUMEN
The data presented here describe the first unequivocable characterization of a pore-forming protein in any helminth parasite. The excretory/secretory (E/S) material of the human whipworm T. trichiura contains a highly abundant protein of molecular mass 47 kDa (TT47); the murine model parasite, T. muris, contains a similarly abundant protein of molecular mass 43 kDa (TM43). When purified to homogeneity, these proteins induce ion-conducting pores in lipid bilayers. Antibodies raised against TM43 abolish channel activity. Pore formation in epithelial cell membranes may facilitate invasion of the host gut by Trichuris and enable the parasite to maintain its syncytial environment in the caecal epithelium. The observation that this activity may be inhibited by antibody opens a possible avenue for drug and vaccine development.
Asunto(s)
Proteínas del Helminto/aislamiento & purificación , Proteínas de la Membrana/aislamiento & purificación , Trichuris/fisiología , Animales , Anticuerpos , Membrana Celular/fisiología , Electroforesis en Gel de Poliacrilamida , Proteínas del Helminto/fisiología , Humanos , Membrana Dobles de Lípidos , Proteínas de la Membrana/fisiología , Ratones , Peso Molecular , Fosfatidilcolinas , Trichuris/químicaRESUMEN
Deposition of beta-amyloid peptide in the brain is an early event in Alzheimer's disease, the most common cause of dementia. Studies of the beta-amyloid precursor protein (APP), which gives rise to beta-amyloid, are rapidly leading to a better understanding of the biochemical basis of the disease--a prerequisite for rational drug development.
Asunto(s)
Enfermedad de Alzheimer/metabolismo , Precursor de Proteína beta-Amiloide/fisiología , Anciano , Anciano de 80 o más Años , Enfermedad de Alzheimer/etiología , Enfermedad de Alzheimer/genética , Secuencia de Aminoácidos , Precursor de Proteína beta-Amiloide/química , Precursor de Proteína beta-Amiloide/genética , Encéfalo/metabolismo , Humanos , Persona de Mediana Edad , Datos de Secuencia Molecular , Mutación Puntual , Procesamiento Proteico-PostraduccionalAsunto(s)
ADN Protozoario/aislamiento & purificación , Parasitología/métodos , ARN Protozoario/aislamiento & purificación , Trypanosoma cruzi/química , Animales , Enfermedad de Chagas/epidemiología , Humanos , Ratones , Fenol , Fenoles , Poli A/aislamiento & purificación , Prevalencia , ARN Mensajero/aislamiento & purificaciónRESUMEN
Protease of carp retina were examined by electrophoresis and fluorogenic assays. A 70 kD serine protease with an alkaline pH optimum was detected in gelatin-containing polyacrylamide gels. A similar enzyme was found in carp brain and muscle, but not in lens. Using aminomethylcoumarin (MCA) substrates, activities that hydrolysed Z-Phe-Arg-MCA, Boc-Ala-Gly-Pro-Arg-MCA and various aminoacyl-MCAs were detected. The Z-Phe-Arg-MCA hydrolase was an acidic cysteine protease, whereas the Boc-Ala-Gly-Pro-Arg-MCA hydrolase was an alkaline cysteine protease. All aminoacyl hydrolase activities tested were inhibited by bestatin and o-phenanthroline, but not by inhibitors of serine, cysteine and aspartic proteases, suggesting they are metalloaminopeptidases. Of the substrates tested, Tyr-MCA was the most readily hydrolysed aminoacyl substrate. Preliminary evidence was obtained suggesting that levels of these activities do not differ between light- and dark-adapted retinae. The proteases have a potential involvement in retinal functioning and show similarities to other proteases known to act in the central nervous system. In particular, the Tyr-MCA hydrolase may be related to an enzyme known to remove the N-terminal tyrosine residue from enkephalin.
Asunto(s)
Carpas , Endopeptidasas/metabolismo , Retina/enzimología , Secuencia de Aminoácidos , Animales , Cumarinas , Concentración de Iones de Hidrógeno , Leucina/análogos & derivados , Leucina/farmacología , Leupeptinas/farmacología , Datos de Secuencia Molecular , Péptidos/metabolismo , Fenantrolinas/farmacología , Inhibidores de Proteasas/farmacología , Serina Endopeptidasas/metabolismo , Clorometilcetona Tosilisina/farmacologíaRESUMEN
Detergent extracts of Trypanosoma cruzi epimastigotes catalysed the hydrolysis of a range of amino-acyl and peptidyl p-nitro-anilides and aminomethylcoumarins. At least three enzymes were detected that cleave Z-Phe-Arg-MCA. Two of these were optimally active at alkaline pH, the other at pH 4.0. Of the two enzymes with alkaline pH optima, one was a cysteine peptidase and was unable to cleave Bz-Arg-MCA readily, whilst the other cleaved Bz-Arg-MCA and was inhibited by diisopropyl fluorophosphate. The acidic enzyme was similar to cathespin L of other eukaryotes with respect to its pH profile, substrate-specificity and inhibitor-sensitivity. Evidence was presented that epimastigotes contain a cysteine-type dipeptidyl aminopeptidase, one or more aminopeptidases, and a serine peptidase that cleaves Boc-Ala-Ala-pNA. Digitonin solubilization of the activities from cells supports the hypothesis that the cathespin L-like enzyme and the dipeptidyl aminopeptidase are lysosomal, whilst the Bz-Arg-MCA hydrolase, the aminopeptidases and the Boc-Ala-Ala-pNA serine peptidase are cytosolic.
Asunto(s)
Péptido Hidrolasas/análisis , Trypanosoma cruzi/enzimología , Secuencia de Aminoácidos , Aminopeptidasas/análisis , Aminopeptidasas/metabolismo , Animales , Compuestos Cromogénicos , Cumarinas/metabolismo , Dipéptidos/metabolismo , Electroforesis en Gel de Poliacrilamida , Colorantes Fluorescentes , Concentración de Iones de Hidrógeno , Hidrólisis , Datos de Secuencia Molecular , Péptido Hidrolasas/metabolismo , Inhibidores de Proteasas/farmacología , Especificidad por SustratoRESUMEN
Proteases from infective larval (L3) and adult stages of Nippostrongylus brasiliensis were investigated with a combination of techniques involving gelatin degradation and cleavage of fluorogenic substrates. Analysis of L3 excretory-secretory (ES) products revealed enzymes of Mr 51, 58, 79, approximately 150 and approximately 250 kDa. Inhibition profiles indicate that the major 51 kDa protease is a metallo-enzyme. Significantly, little activity was present in larval somatic extracts, suggesting the synthesis of zymogens or precursor forms prior to secretion. Adult ES contained a distinct enzyme, of 50 kDa, and a number of other proteases were detected in somatic extracts of this stage, ranging from 51 to greater than 300 kDa. The largest of these adult somatic enzymes is also a putative metallo-protease. While nearly all enzymes from both L3 and adult are heat labile, incubation at 100 degrees C generated a previously unobserved activity at 20 kDa. Furthermore, a protease of similar size may be found in uninfected rat intestinal tissue, suggesting specific uptake of a host-associated enzyme by the parasite in the form of an inactive, heat-labile complex.
Asunto(s)
Endopeptidasas/química , Nippostrongylus/enzimología , Animales , Electroforesis en Gel de Poliacrilamida , Endopeptidasas/metabolismo , Concentración de Iones de Hidrógeno , Larva/enzimología , Peso Molecular , Especificidad por SustratoRESUMEN
Enhancement of trypanosomatid metacyclogenesis by insect urine components led us to test the effect of human urine as a culture additive. The addition of 1-5% urine to Schneider's Drosophila medium containing 10% foetal calf serum enhanced the growth of 11 Leishmania strains representing 8 different taxonomic groups. Cell division was stimulated in cultures with non-dividing organisms. Peak cell density was increased, as was the efficiency with which L. donovani could be isolated from infected hamsters. Preliminary work suggested that the modified medium would be useful for field isolation of L. donovani and L. braziliensis. The active nutrients or growth factors are not known.
Asunto(s)
Leishmania/crecimiento & desarrollo , Orina , Animales , Medios de Cultivo , Humanos , Leishmania donovani/crecimiento & desarrolloRESUMEN
An alkaline peptidase of Trypanosoma cruzi and Crithidia fasciculata, previously shown to cleave on the carboxyl side of arginine and lysine residues, was examined for its ability to cleave various fluorogenic substrates and for its sensitive to peptidase inhibitors. The enzyme of both T. cruzi and C. fasciculata has a preference for cleavage of substrates with basic amino acids at the P2 as well as the P1 position of the peptide chain. Arginine and lysine are equally acceptable at P2, whereas the enzyme prefers arginine to lysine at P1. An influence of the P3 amino acid residue on substrate cleavability was also apparent. The peptidase was highly susceptible to inactivation by peptidylfluoromethanes, peptidyldiazomethanes and peptidylsulphonium salts that contained arginine or lysine at P1. Additionally, diisopropylfluorophosphate inhibited the enzyme, whereas trans-epoxysuccinylleucylamido(4-guanidino)butane and iodoacetic acid were relatively weak inhibitors. Various reversible inhibitors of the enzyme were also examined. Inhibition by members of the primary aliphatic amine series, methylamine to n-heptylamine, showed a peak of inhibition at n-butylamine, which most closely resembles the lysine side chain. Agmatine, which resembles the arginine side chain, also strongly inhibited the peptidase. The kinetics of inhibition by these basic compounds were of the competitive type. Pentamidine and hirudonin, which resemble two arginine side chains joined together, were more effective inhibitors of the trypanosomatid peptidase than bases resembling only one arginine or lysine side chain.
Asunto(s)
Crithidia/enzimología , Cisteína Endopeptidasas/metabolismo , Trypanosoma cruzi/enzimología , Aminas/farmacología , Secuencia de Aminoácidos , Animales , Cisteína Endopeptidasas/aislamiento & purificación , Inhibidores de Cisteína Proteinasa , Dipéptidos/farmacología , Cinética , Datos de Secuencia Molecular , Oligopéptidos , Inhibidores de Proteasas/farmacología , Especificidad por SustratoRESUMEN
The genes which encode glycosomal glyceraldehyde-phosphate dehydrogenase (gGAPDH) of Trypanosoma cruzi are arranged as a tandemly repeated pair on a single chromosome and are identical at the level of nucleotide sequence. They are separated by an intergenic region which contains a 317 base pair sequence with the properties of a retroposon. The genes express a 1.5 kb mRNA and a 38 kd protein. The amino acid sequence contains features characteristic of glycosomal enzymes such as peptide insertions and a C-terminal extension. However, T. cruzi gGAPDH lacks one of the positively charged 'hotspot' motifs which have been proposed as topogenic signals for import into the glycosome, a unique microbody-like organelle. Molecular modelling of the T. cruzi and T. brucei enzymes suggests that neither structure would fulfil the requirements of the 'hotspot' glycosomal import model.