The C-terminal fragment of the Alzheimer's disease amyloid protein precursor is degraded by a proteasome-dependent mechanism distinct from gamma-secretase.

The beta-amyloid protein (Abeta) is derived by proteolytic processing of the amyloid protein precursor (APP). Cleavage of APP by beta-secretase generates a C-terminal fragment (APP-CTFbeta), which is subsequently cleaved by gamma-secretase to produce Abeta. The aim of this study was to examine the cleavage of APP-CTFbeta by gamma-secretase in primary cortical neurons from transgenic mice engineered to express the human APP-CTFbeta sequence. Neurons were prepared from transgenic mouse cortex and proteins labelled by incubation with [35S]methionine and [35S]cysteine. Labelled APP-CTFbeta and Abeta were then immunoprecipitated with a monoclonal antibody (WO2) specific for the transgene sequences. Approximately 30% of the human APP-CTFbeta (hAPP-CTFbeta) was converted to human Abeta (hAbeta), which was rapidly secreted. The remaining 70% of the hAPP-CTFbeta was degraded by an alternative pathway. The cleavage of hAPP-CTFbeta to produce hAbeta was inhibited by specific gamma-secretase inhibitors. However, treatment with proteasome inhibitors caused an increase in both hAPP-CTFbeta and hAbeta levels, suggesting that the alternative pathway was proteasome-dependent. A preparation of recombinant 20S proteasome was found to cleave a recombinant cytoplasmic domain fragment of APP (APPcyt) directly. The study suggests that in primary cortical neurons, APP-CTFbeta is degraded by two distinct pathways, one involving gamma-secretase, which produces Abeta, and a second major pathway involving direct cleavage of APP-CTFbeta within the cytoplasmic domain by the proteasome. These results raise the possibility that defective proteasome function could lead to an increase in Abeta production in the AD brain.     

A Bayesian approach to a logistic regression model with incomplete information

Summary .   We consider a set of independent Bernoulli trials with possibly different success probabilities that depend on covariate values. However, the available data consist only of aggregate numbers of successes among subsets of the trials along with all of the covariate values. We still wish to estimate the parameters of a modeled relationship between the covariates and the success probabilities, e.g., a logistic regression model. In this article, estimation of the parameters is made from a Bayesian perspective by using a Markov chain Monte Carlo algorithm based only on the available data. The proposed methodology is applied to both simulation studies and real data from a dose–response study of a toxic chemical, perchlorate.     

The tortoise and the hare II: relative utility of 21 noncoding chloroplast DNA sequences for phylogenetic analysis

Chloroplast DNA sequences are a primary source of data for plant molecular systematic studies. A few key papers have provided the molecular systematics community with universal primer pairs for noncoding regions that have dominated the field, namely trnL-trnF and trnK/matK. These two regions have provided adequate information to resolve species relationships in some taxa, but often provide little resolution at low taxonomic levels. To obtain better phylogenetic resolution, sequence data from these regions are often coupled with other sequence data. Choosing an appropriate cpDNA region for phylogenetic investigation is difficult because of the scarcity of information about the tempo of evolutionary rates among different noncoding cpDNA regions. The focus of this investigation was to determine whether there is any predictable rate heterogeneity among 21 noncoding cpDNA regions identified as phylogenetically useful at low levels. To test for rate heterogeneity among the different cpDNA regions, we used three species from each of 10 groups representing eight major phylogenetic lineages of phanerogams. The results of this study clearly show that a survey using as few as three representative taxa can be predictive of the amount of phylogenetic information offered by a cpDNA region and that rate heterogeneity exists among noncoding cpDNA regions.     

The genetics and genomics of Syngnathidae: pipefishes, seahorses and seadragons

The goal of this review was to provide a historical overview of how molecular techniques have increased the understanding of the ecology and evolution of the family Syngnathidae (pipefishes, seahorses and seadragons). Molecular studies based primarily on mitochondrial DNA markers have proved their worth by elucidating complex phylogenetic relationships within the family. Phylogeographic studies, which have revealed how life-history traits and past climatic events shape geographic distributions and patterns of genetic variation within syngnathid species, also provide interesting case studies for the conservation and management of threatened species. The application of microsatellite DNA markers has opened a floodgate of studies concerned with the breeding biology of these fishes, which are interesting due to their unique reproductive mode of male pregnancy. Research in this area has contributed significantly to the understanding of mating patterns and sexual selection. Molecular markers may also be employed in studies of demography, migration and local breeding population sizes. Genomic studies have identified genes that are probably involved in male pregnancy and promise additional insights into various aspects of syngnathid biology at the level of the gene. Despite these advances, much more remains to be explored. Goals for future research should include: (1) a more inclusive phylogeny to resolve outstanding issues concerning the relationships within the family and higher order taxa, (2) a broader use of molecular studies to aid management and conservation efforts, (3) the inclusion of more genera in comparative behavioural studies and (4) the continued development of genomic resources for syngnathids to facilitate comparative genomic work.     

Population genomics of the filarial nematode parasite Wuchereria bancrofti from mosquitoes

Wuchereria bancrofti is a parasitic nematode and the primary cause of lymphatic filariasis – a disease specific to humans. W. bancrofti currently infects over 90 million people throughout the tropics and has been acknowledged by the world health organization as a vulnerable parasite. Current research has focused primarily on the clinical manifestations of disease and little is known about the evolutionary history of W. bancrofti. To improve upon knowledge of the evolutionary history of W. bancrofti, we whole genome sequenced 13 W. bancrofti larvae. We circumvent many of the difficulties of multiple infections by sampling larvae directly from mosquitoes that were experimentally inoculated with infected blood. To begin, we used whole genome data to reconstruct the historical population size. Our results support a history of fluctuating population sizes that can be correlated with human migration and fluctuating mosquito abundances. Next, we reconstructed the putative pedigree of W. bancrofti worms within an infection using the kinship coefficient. We deduced that there are full‐sib and half‐sib relationships residing within the same larval cohort. Through combined analysis of the mitochondrial and nuclear genomes we concluded that this is likely a results of polyandrous mating, the first time reported for W. bancrofti. Lastly, we scanned the genomes for signatures of natural selection. Annotation of putative selected regions identified proteins that may have aided in a parasitic life style or may have evolved to protect against current drug treatments. We discuss our results in the greater context of understanding the biology of an animal with a unique life history and ecology.     

Surface Tensiometry of Apolipoprotein B Domains at Lipid Interfaces Suggests a New Model for the Initial Steps in Triglyceride-rich Lipoprotein Assembly

Apolipoprotein B (apoB) is the principal protein component of triacylglyceride (TAG)-rich lipoproteins, including chylomicrons and very low density lipoprotein, which is the precursor to LDL (the “bad cholesterol”). TAG-rich lipoprotein assembly is initiated by the N-terminal βα1 superdomain of apoB, which co-translationally binds and remodels the luminal leaflet of the rough endoplasmic reticulum. The βα1 superdomain contains four domains and is predicted to interact directly with lipids. Using drop tensiometry, we examined the interfacial properties of the α-helical and C-sheet domains and several subdomains to establish a detailed structure-function relationship at the lipid/water interface. The adsorption, stress response, exchangeability, and pressure (Π)-area relationship were studied at both triolein/water and triolein/1-palmitoyl, 2-oleoylphosphatidylcholine/water interfaces that mimic physiological environments. The α-helical domain spontaneously adsorbed to a triolein/water interface and formed a viscoelastic surface. It was anchored to the surface by helix 6, and the other helices were ejected and/or remodeled on the surface as a function of surface pressure. The C-sheet instead formed an elastic film on a triolein/water interface and was irreversibly anchored to the lipid surface, which is consistent with the behavior of amphipathic β-strands. When both domains were adsorbed together on the surface, the C-sheet shielded a portion of the α-helical domain from the surface, which retained its globular structure. Overall, the unique secondary and tertiary structures of the N-terminal domains of apoB support the intrinsic capability of co-translational lipid recruitment. The evidence presented here allows the construction of a detailed model of the initiation of TAG-rich lipoprotein assembly.     

Isolation and Characterization of Pyrophosphate:D-Fructose-6-phosphate 1-Phosphotransferase from Cucumber Seeds

Pyrophosphate:D-fructose-6-phosphate 1-phosphotransferase waspurified over 700-fold from germinating cucumber (Cucumis sativuscv. Fletcher) seeds. The purified enzyme has a specific activityof 5.2 µmol.min^–1.mg protein^–1 in the presenceof 1 µM fru-2,6-P₂. The pH optima is similar for boththe forward and reverse reactions (pH 7.5–7.8). Magnesium,manganese and cobalt activate the enzyme, with the highest affinitybeing for magnesium. The enzyme exhibits normal Michaelis-Mentenkinetics in both the presence and absence of fru-2,6-P₂. Half-maximumactivation of the enzyme was obtained with 35 nM fru-2,6-P2.Fru-2,6-P₂ stimulates activity by increasing V_max and increasingthe affinity for fru-6-P, fru-1,6-P₂ and PPi. Phosphate causesnoncompetitive inhibition with respect to both fru-6-P and PPi.On the basis of the steadystate substrate interaction and Piinhibition data a sequential ternary complex mechanism is proposed. (Received April 28, 1986; Accepted July 9, 1986)