BACE is degraded via the lysosomal pathway

Amyloid plaques are formed by aggregates of amyloid-beta-peptide, a 37-43-amino acid fragment (primarily Abeta(40) and Abeta(42)) generated by proteolytic processing of the amyloid precursor protein (APP) by beta- and gamma-secretases. A type I transmembrane aspartyl protease, BACE (beta-site APP cleaving enzyme), has been identified to be the beta-secretase. BACE is targeted through the secretory pathway to the plasma membrane where it can be internalized to endosomes. The carboxyl terminus of BACE contains a di-leucine-based signal for sorting of transmembrane proteins to endosomes and lysosomes. In this study, we set out to determine whether BACE is degraded by the lysosomal pathway and whether the di-leucine motif is necessary for targeting BACE to the lysosomes. Here we show that lysosomal inhibitors, chloroquine and NH(4)Cl, lead to accumulation of endogenous and ectopically expressed BACE in a variety of cell types, including primary neurons. Furthermore, the inhibition of lysosomal hydrolases results in the redistribution and accumulation of BACE in the late endosomal/lysosomal compartments (lysosome-associated membrane protein 2 (LAMP2)-positive). In contrast, the BACE-LL/AA mutant, in which Leu(499) and Leu(500) in the COOH-terminal sequence (DDISLLK) were replaced by alanines, only partially co-localized with LAMP2-positive compartments following inhibition of lysosomal hydrolases. Collectively, our data indicate that BACE is transported to the late endosomal/lysosomal compartments where it is degraded via the lysosomal pathway and that the di-leucine motif plays a role in sorting BACE to lysosomes.     

RNA interference-mediated silencing of X11alpha and X11beta attenuates amyloid beta-protein levels via differential effects on beta-amyloid precursor protein processing

Processing of the beta-amyloid precursor protein (APP) plays a key role in Alzheimer disease neuropathogenesis. APP is cleaved by beta- and alpha-secretase to produce APP-C99 and APP-C83, which are further cleaved by gamma-secretase to produce amyloid beta-protein (Abeta) and p3, respectively. APP adaptor proteins with phosphotyrosine-binding domains, including X11alpha (MINT1, encoded by gene APBA1) and X11beta (MINT2, encoded by gene APBA2), can bind to the conserved YENPTY motif in the APP C terminus. Overexpression of X11alpha and X11beta alters APP processing and Abeta production. Here, for the first time, we have described the effects of RNA interference (RNAi) silencing of X11alpha and X11beta expression on APP processing and Abeta production. RNAi silencing of APBA1 in H4 human neuroglioma cells stably transfected to express either full-length APP or APP-C99 increased APP C-terminal fragment levels and lowered Abeta levels in both cell lines by inhibiting gamma-secretase cleavage of APP. RNAi silencing of APBA2 also lowered Abeta levels, but apparently not via attenuation of gamma-secretase cleavage of APP. The notion of attenuating gamma-secretase cleavage of APP via the APP adaptor protein X11alpha is particularly attractive with regard to therapeutic potential given that side effects of gamma-secretase inhibition due to impaired proteolysis of other gamma-secretase substrates, e.g. Notch, might be avoided.     

Chloroplast DNA phylogeography of the argan tree of Morocco

Polymorphisms in the chloroplast genome of the argan tree (Sapotaceae), an endemic species of south-western Morocco, have been detected by restriction site studies of PCR-amplified fragments. A total of 12 chloroplast DNA (cpDNA) and two mitochondrial DNA (mtDNA) fragments were amplified and digested with a single restriction enzyme ( Hin fI). Polymorphisms were identified in six of the cpDNA fragments, whereas no mtDNA polymorphisms were detected in a survey of 95 individuals from 19 populations encompassing most of the natural range of the species. The cpDNA polymorphisms allowed the identification of 11 haplotypes. Two lineages, one in the south-east and the other in the north-west, divide the range of the argan tree into two distinct areas. The level of genetic differentiation measured at the haplotype level ( G _STc= 0.60) (i.e. with unordered haplotypes) was smaller than when phylogenetic relationships were taken into account ( N _STc= 0.71–0.74) (ordered haplotypes), indicating that population history must be considered in the study of the geographical distribution of cpDNA lineages in this species. If contrasted with the level of nuclear genetic differentiation measured in a previous study with isozymes ( G _STn= 0.25), the results indicate a relatively high level of gene flow by seeds, or conversely a relatively low level of gene flow by pollen, as compared with other tree species. Goats and camels could have played an important role in disseminating the fruits of this tree.     

Physiological Responses of Spring Durum Wheat Cultivars to Early-season Drought in a Mediterranean Environment

The Mediterranean climate of North Africa is characterized byuncertain rainfall immediately after seedling emergence, leadingto drought early in the growing season which depresses durumwheat production. However, there is limited understanding ofthe physiological basis of resistance of spring durum wheatto drought in rainfed Mediterranean regions. The objectivesof this study were to examine differences in some physiologicalcharacters among spring durum wheat cultivars in response toduration of early-season drought, and to determine the relationshipof these characters to drought resistance. In two field experiments(1995 and 1996 growing seasons) and a glasshouse experiment(1996), six spring sown durum wheat cultivars were evaluatedunder four water regimes: well irrigated and three differentwater deficits from emergence until the onset of tillering,mid-tillering or at the end of tillering. Cultivars differedin their response. Decreases in photosynthesis soon after droughtstress was imposed resulted mainly from reduced stomatal conductance.Continued water deficits also reduced mesophyll photosyntheticactivity. Possible factors determining the drought-resistanceof a cultivar are lower sensitivity of CO₂exchange rate, netCO₂uptake to water loss ratio, stomatal resistance, relativewater content and greater osmotic adjustment under stress. Furthermore,there is sufficient intraspecific variation in these physiologicalattributes to suggest their use as selection tools.Copyright1998 Annals of Botany Company Wheat;Triticum durumDesf.; early-season drought; physiological responses.     

The Influence of Plant-Growth Factors on the Initiation and Growth of Crown-Gall Tumours on Primary Pinto Bean Leaves

The number of tumours developing on primary pinto bean leaves(Phaseolus vulgaris L. variety Pinto) inoculated with Agrobacteriumtumefaciens (Smith and Town.) Conn, strain B6, was increasedby as much as 100 per cent through the addition of several plant-growthfactors and antagonists of some of these factors to the freshlyinoculated leaves. Naphthylacetic acid, gibberellic acid, (2-chloroethyl)trimethylammonium chloride, and adenine gave a biphasic responsewith optimal promotions of tumour initiation at 10^-5 to 10^-4mg/ml. Tri-iodobenzoic acid and 4-chlorophenoxyisobutyric acidwere most active at 10^-1 mg/ml. Mean tumour diameter showeda direct correlation with tumour number in these experiments.The results show tumour initiation to be sensitive to growthfactor changes, possibly through a heightened traumatic responseof the wounded leaves. The growth of the tumours was unaffectedby these additions except as they altered tumour number andthis secondarily affected tumour growth.     

Localization of the Huntington's disease gene to a small segment of chromosome 4 flanked by D4S10 and the telomere

Huntington's disease (HD) is an autosomal dominant neurodegenerative disorder of late onset, characterized by progressive motor disturbance, psychological manifestations, and intellectual deterioration. The HD gene has been genetically mapped by linkage to the DNA marker D4S10, but the exact physical location of the HD defect has remained uncertain. To delineate critical recombination events revealing the physical position of the HD gene, we have identified restriction fragment length polymorphisms for two recently mapped chromosome 4 loci, RAF2 and D4S62, and determined the pattern of segregation of these markers in both reference and HD pedigrees. Multipoint linkage analysis of the new markers with D4S10 and HD establishes that the HD gene is located in a very small physical region at the tip of the chromosome, bordered by D4S10 and the telomere. A crossover within the D4S10 locus orients this segment on the chromosome, providing the necessary information for efficient application of directional cloning strategies for progressing toward, and eventually isolating, the HD gene.     

Assessment of amyloid β-protein precursor gene mutations in a large set of familial and sporadic Alzheimer disease cases

A genetic locus associated with familial Alzheimer disease (FAD) and a candidate gene, APP, encoding the amyloid protein precursor have both been assigned previously to chromosome 21, and, in a few FAD families, mutations of APP have been detected. However, obligate crossovers between APP and FAD have also been reported in several FAD pedigrees, including FAD4, a large kindred showing highly suggestive evidence for linkage of the disorder to chromosome 21. In case the apparent APP crossover in FAD4 actually represented an intragenic recombination event or segregation of different mutations in different family branches, we have performed a more detailed assessment of APP as a candidate gene in this family. The entire coding region of the APP gene was sequenced for FAD4 and for FAD1, a second large kindred. No mutations were found, indicating that, in at least one chromosome 21-linked FAD pedigree, the gene defect is not accounted for by a mutation in the known coding region of the APP gene. A total of 25 well-characterized early- and late-onset FAD pedigrees were typed for genetic linkage to APP, to assess the percentage of FAD families predicted to carry mutations in the APP gene. None of the FAD families yielded positive lod scores at a recombination fraction of 0.0. To estimate the overall prevalence of FAD-associated mutations in the beta A4 domain of APP, we sequenced exons 16 and 17 in 30 (20 early- and 10 late-onset) FAD kindreds and in 11 sporadic AD cases, and we screened 56 FAD kindreds and 81 cases of sporadic AD for the presence of the originally reported FAD-associated mutation, APP717 Val----Ile (by BclI digestion). No APP gene mutations were found in any of the FAD families or sporadic-AD samples examined in this study, suggesting that the mutations in exons 16 and 17 are a rare cause of FAD. Overall, these data suggest that APP gene mutations account for a very small portion of FAD.     

The ETS genes on chromosome 21 are distal to the breakpoint of the acute myelogenous leukemia translocation (8;21)

The definition of the genetic linkage map of human chromosomes may be helpful in the analysis of cancer-specific chromosome abnormalities. In the translocation (8;21)(q22;q22), a nonrandom cytogenetic abnormality of acute myelogenous leukemia (AML), we previously observed the transposition of the ETS2 gene located at the 21q22 region from chromosome 21 to chromosome 8. However, no ETS2 rearrangements were detected in the DNA of t(8;21)-positive AML cells. Genetic linkage analysis has allowed us to locate the ETS2 gene relative to other loci and to establish that the breakpoint is at an approximate genetic distance of 17 cM from ETS2. When the information from the linkage map is combined with that from molecular studies, it is apparent that (a) the t(8;21) breakpoint does not affect the ETS2 gene structure or the structure of the other four loci proximal to ETS2: D21S55, D21S57, D21S17, and ERG, and ETS-related gene; and (b) the actual DNA sequence involved in the t(8;21) must reside in a 3-cM genetic region between the D21S58 and the D21S55/D21S57 loci, and remains to be identified.