A method for studying the sequence-specific binding of proteins to DBA is described. The technique is a simple conjoining of the Maxam-Gilbert DNA-sequencing method and the technique of DNAase-protected fragment isolation. Fragments of a 5' end-labelled, double-stranded DNA segment, partially degraded by DNAase in the presence and absence of the binding protein, are visualized by electrophoresis and autoradiography alongside the base-specific reaction products of the Maxam-Gilbert sequencing method. It is then possible to see the protective "footprint" of the binding protein on the DNA sequence. The binding of lac repressor to lac operator is visualized by "footprinting" as an example. Equillibrium estimates indicate that 10-fold sequence-specificity (differential binding constant) could be studied easily using this technique.
The impact of microalbuminuria on mortality as well as other risk factors was investigated in a 10-year follow-up study of 503 predominantly non-insulin-dependent diabetic patients of whom 265 had died. Using Cox's regression analysis the prognostic influence of age, sex, age at diagnosis, known diabetes duration, blood pressure, fasting plasma glucose, relative weight, serum creatinine, retinopathy, and treatment was evaluated as well as morning urine albumin concentration (UAC) in four categories, i.e. UAC less than or equal to 15 micrograms/ml (normal), 15 micrograms/ml less than UAC less than or equal to 40 micrograms/ml, 40 micrograms/ml less than UAC less than or equal to 200 micrograms/ml and UAC greater than 200 micrograms/ml. Age, UAC, known duration, and serum creatinine were the only significant risk factors. After correction for the other three independent risk factors, the hazard ratios in the elevated UAC categories relative to the group with UAC less than or equal to 15 micrograms/ml were 1.53 (p = 0.007), 2.28 (p = 0.000002), and 1.82 (p = 0.02). The statistically significant correlations with UAC were: age (r = 0.09, p less than 0.05), duration (r = 0.14, p less than 0.01), systolic blood pressure (r = 0.12, p less than 0.01), serum creatinine (r = 0.33, p less than 0.001), and fasting plasma glucose (r = 0.12, p less than 0.01). Increased UAC was associated also with retinopathy (p = 0.01). Fifty-eight per cent of the deaths were caused by cardiovascular disease or stroke; only 3% died from uraemia. A reinvestigation including blood pressure, fasting plasma glucose, and UAC was made on 208 survivors.
Presenilin is implicated in the pathogenesis of Alzheimer's disease. It is thought to constitute the catalytic subunit of the ␥-secretase complex that catalyzes intramembrane cleavage of -amyloid precursor protein, the last step in the generation of amyloidogenic A peptides. The latter are major constituents of amyloid plaques in the brain of Alzheimer's disease patients. Inhibitors of ␥-secretase are considered potential therapeutics for the treatment of this disease because they prevent production of A peptides. Recently, we discovered a family of presenilin-type aspartic proteases. The founding member, signal peptide peptidase, catalyzes intramembrane cleavage of distinct signal peptides in the endoplasmic reticulum membrane of animals. In humans, the protease plays a crucial role in the immune system. Moreover, it is exploited by the hepatitis C virus for the processing of the structural components of the virion and hence is an attractive target for anti-infective intervention. Signal peptide peptidase and presenilin share identical active site motifs and both catalyze intramembrane proteolysis. These common features let us speculate that ␥-secretase inhibitors directed against presenilin may also inhibit signal peptide peptidase. Here we demonstrate that some of the most potent known ␥-secretase inhibitors efficiently inhibit signal peptide peptidase. However, we found compounds that showed higher specificity for one or the other protease. Our findings highlight the possibility of developing selective inhibitors aimed at reducing A generation without affecting other intramembranecleaving aspartic proteases. Alzheimer's disease (AD)1 is characterized by the formation of senile plaques in the brain. Major constituents of these plaques are the amyloidogenic 40-and 42-residue-long A peptides A40 and A42, respectively (1). The amyloid cascade hypothesis casually links the generation of amyloid plaques with the neuropathological changes accompanying the symptoms typical of this disease (2). A peptides are generated from the type I transmembrane protein -APP (-amyloid precursor protein) by sequential proteolysis (3). The protein is first cleaved in the exoplasmic domain by the -site APP-cleaving enzyme (BACE) to release the ectodomain (4, 5). The residual membrane-anchored stub of 99 residues (C99) is subsequently cleaved in the center of the transmembrane region by ␥-secretase (6). The resulting cleavage products, an A peptide and the amyloid intracellular domain (AICD), are liberated from the lipid bilayer toward the exoplasm and cytosol, respectively (7-9).To date, the majority of characterized familial AD mutations are clustered along the presenilin-1 (PS1) gene (10, 11). They are thought to accelerate disease onset by increasing the A42/ A40 ratio (12). It is not well understood how these mutations, which are essentially scattered along the entire PS1 gene, can lead to a specific increase in the production of the 42-residuelong peptide that corresponds to the most amyloidogenic form of A (13). ...
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