Differential expression of interferon responsive genes in rodent models of transmissible spongiform encephalopathy disease

Background

Alzheimer's disease (AD) is characterized by a decline in cognitive function and accumulation of amyloid-β peptide (Aβ) in extracellular plaques. Mutations in amyloid precursor protein (APP) and presenilins alter APP metabolism resulting in accumulation of Aβ42, a peptide essential for the formation of amyloid deposits and proposed to initiate the cascade leading to AD. However, the role of Aβ40, the more prevalent Aβ peptide secreted by cells and a major component of cerebral Aβ deposits, is less clear. In this study, virally-mediated gene transfer was used to selectively increase hippocampal levels of human Aβ42 and Aβ40 in adult Wistar rats, allowing examination of the contribution of each to the cognitive deficits and pathology seen in AD.

Results

Adeno-associated viral (AAV) vectors encoding BRI-Aβ cDNAs were generated resulting in high-level hippocampal expression and secretion of the specific encoded Aβ peptide. As a comparison the effect of AAV-mediated overexpression of APPsw was also examined. Animals were tested for development of learning and memory deficits (open field, Morris water maze, passive avoidance, novel object recognition) three months after infusion of AAV. A range of impairments was found, with the most pronounced deficits observed in animals co-injected with both AAV-BRI-Aβ40 and AAV-BRI-Aβ42. Brain tissue was analyzed by ELISA and immunohistochemistry to quantify levels of detergent soluble and insoluble Aβ peptides. BRI-Aβ42 and the combination of BRI-Aβ40+42 overexpression resulted in elevated levels of detergent-insoluble Aβ. No significant increase in detergent-insoluble Aβ was seen in the rats expressing APPsw or BRI-Aβ40. No pathological features were noted in any rats, except the AAV-BRI-Aβ42 rats which showed focal, amorphous, Thioflavin-negative Aβ42 deposits.

Conclusion

The results show that AAV-mediated gene transfer is a valuable tool to model aspects of AD pathology in vivo, and demonstrate that whilst expression of Aβ42 alone is sufficient to initiate Aβ deposition, both Aβ40 and Aβ42 may contribute to cognitive deficits.     

Routes to improving the reliability of low level DNA analysis using real-time PCR

Background  

Accurate quantification of DNA using quantitative real-time PCR at low levels is increasingly important for clinical, environmental and forensic applications. At low concentration levels (here referring to under 100 target copies) DNA quantification is sensitive to losses during preparation, and suffers from appreciable valid non-detection rates for sampling reasons. This paper reports studies on a real-time quantitative PCR assay targeting a region of the human SRY gene over a concentration range of 0.5 to 1000 target copies. The effects of different sample preparation and calibration methods on quantitative accuracy were investigated.     

Impact of a glossy collard trap crop on diamondback moth adult movement, oviposition, and larval survival

One component of developing a systematic approach for deployment of trap crops is to understand how the trap crop modifies pest behavior. Glossy‐leafed collards, Brassica oleracea L. var. acephala (Brassicaceae), were evaluated as a potential trap crop for diamondback moth, Plutella xylostella (L.) (Lepidoptera: Plutellidae), because they are attractive to P. xylostella adults and are a poor host for P. xylostella larvae compared to cabbage, Brassica oleracea L. var. capitata. We used large field plots to measure the changes in adult, egg, and larval P. xylostella densities in cabbage when the trap crop was planted in the field. Furthermore, we planted the trap crop in dispersed and concentrated spatial arrangements to determine the impact of trap crop arrangement on the behavior of P. xylostella. In 2002, results showed that the presence of collards within a cabbage field reduced larval density on cabbage. In 2003, neither trap crop arrangement had a significant impact on P. xylostella larval density on cabbage. Adult moths aggregated in proximity to collards in 2002, but not in 2003. Egg and larval data in both years in all treatments showed that total oviposition was highest near a central release point, indicating that females lay many eggs before dispersing very far when suitable host plants are available. The mean direction of P. xylostella movement and oviposition from a central release point was not consistent or correlated to wind direction. Plant size of the trap crop in relation to the main crop and environmental factors may have been responsible for the inconsistent effectiveness of the trap crop.     

Tetrapod phylogeny inferred from 18S and 28S ribosomal RNA sequences and a review of the evidence for amniote relationships [published erratum appears in Mol Biol Evol 1991 May;8(3):398]

The 18S ribosomal RNAs of 21 tetrapods were sequenced and aligned with five published tetrapod sequences. When the coelacanth was used as an outgroup, Lissamphibia (living amphibians) and Amniota (amniotes) were found to be statistically significant monophyletic groups. Although little resolution was obtained among the lissamphibian taxa, the amniote sequences support a sister-group relationship between birds and mammals. Portions of the 28S ribosomal RNA (rRNA) molecule in 11 tetrapods also were sequenced, although the phylogenetic results were inconclusive. In contrast to previous studies, deletion or down- weighting of base-paired sites were found to have little effect on phylogenetic relationships. Molecular evidence for amniote relationships is reviewed, showing that three genes (beta-hemoglobin, myoglobin, and 18S rRNA) unambiguously support a bird-mammal relationship, compared with one gene (histone H2B) that favors a bird- crocodilian clade. Separate analyses of four other genes (alpha- crystallin A, alpha-hemoglobin, insulin, and 28S rRNA) and a combined analysis of all sequence data are inconclusive, in that different groups are defined in different analyses and none are strongly supported. It is suggested that until sequences become available from a broader array of taxa, the molecular evidence is best evaluated at the level of individual genes, with emphasis placed on those studies with the greatest number of taxa and sites. When this is done, a bird-mammal relationship is most strongly supported. When regarded in combination with the morphological evidence for this association, it must be considered at least as plausible as a bird-crocodilian relationship.      

Underreporting of AIDS cases in Canada: a record linkage study.

To estimate the rate of underreporting of AIDS (acquired immune deficiency syndrome) to the Federal Centre for AIDS (FCA), in 1988 the initials, date of birth and place of residence of 66 patients with AIDS known to the Toronto Sexual Contact Study (TSCS), 65 patients with AIDS known to the Vancouver Lymphadenopathy-AIDS Study (VLAS) and other participants in both studies who did not have AIDS were sent to the Bureau of Epidemiology and Surveillance, FCA. The FCA conducted a manual record linkage to link these data to the national registry of reported cases. The rate of underreporting was 12% (8/65) for the VLAS and 18% (12/66) for the TSCS. The specific diagnosis was not related to the rate of underreporting. For the TSCS the rate of underreporting had increased from 0% in 1983-84 to 44% in 1987-88 (p = 0.001). Differences in the observed rates of underreporting between the two studies are likely the result of differences in the reporting responsibilities of physicians involved in the studies.     

Expression of neutral endopeptidase (enkephalinase) in heterologous COS-1 cells. Characterization of the recombinant enzyme and evidence for a glutamic acid residue at the active site

Neutral endopeptidase (EC 3.4.24.11) is an integral membrane protein found in the plasma membrane of many cell types. The cDNA coding for the complete primary structure of neutral endopeptidase has recently been cloned and sequenced (Devault, A. Lazure, C., Nault, C., Le Moual, H., Seidah, N. G., Chretien, M., Kahn, P., Powell, J., Mallet, J., Beaumont, A., Roques, B. P., Crine, P., and Boileau, G. (1987) EMBO J. 6, 1317-1322). Comparison of the sequence of neutral endopeptidase with that of thermolysin, a bacterial Zn-metalloendopeptidase, suggests that Glu-584 in neutral endopeptidase probably corresponds to Glu-143 in thermolysin, which is an essential amino acid involved in catalysis. To test directly the importance of Glu-584 in the catalytic activity of neutral endopeptidase by site-directed metagenesis, we have constructed an expression vector in which the rabbit kidney cDNA encoding the entire neutral endopeptidase sequence is introduced downstream from the SV40 virus early promotor. After transfection in COS-1 monkey kidney cells, this vector was found to promote the expression of a protein with biochemical and catalytic properties identical to kidney neutral endopeptidase. Oligonucleotide-directed mutagenesis of Glu-584 to either valine or aspartic acid completely abolished the enzymatic activity of the recombinant protein without changing its affinity for the substrate-related tritiated inhibitor [3H]N-[(2R,2S)-3-hydroxyamino-carbonyl-2-benzyl-1-oxopropyl]-glycine. This observation clearly identifies Glu-584 as one of the important residues responsible for the catalytic activity of the enzyme.     

Characterization of the Escherichia coli protein-export gene secB

The Escherichia coli secB gene product is required for normal export of envelope proteins out of the cell cytoplasm. In this report, we present the identification and nucleotide sequence of the secB coding sequence. The secB structural gene overlaps almost completely with a predicted open reading frame (ORF) that is encoded on the opposite strand. To establish the identity of the secB ORF, we characterized a secB mutation that caused total loss of secB function, based upon its phenotype. This mutation resulted from a nucleotide change that caused an ochre mutation in one ORF (the secB gene) and a silent (no amino acid change) codon change in the opposite ORF.     

Importance of color stimuli in host-finding by Dalbulus leafhoppers

The importance of color stimuli in host-finding by Dalbulus maidis (DeLong & Wolcott), D. gelbus DeLong, and D. quinquenotatus DeLong & Nault (Homoptera: Cicadellidae) was determined by comparing pre- and post-contact responses of leafhoppers to maize seedlings (Zea mays L.) with those to vertical models varying in hue or value (total amount of reflected light). For each species, more contacts were made with maize by jumping/flying than by walking, and both sexes remained on maize for similar times after contact. When presented simultaneously with seven models (vertical wooden rods) painted in different colors, each species made more contacts with yellow. Yellow also received more contacts when it was present in a group of four colors. There were no differences in contacts made with the yellow model by jumping/flying or by walking for D. maidis and D. gelbus, but D. quinquenotatus made more contacts by jumping/flying than by walking. If yellow was not present in the group, the color reflecting maximally between 500–580 nm received more contacts compared to the other three colors. Responses to a series of neutrals indicated contacts were elicited primarily because of hue, and not changes in value. Yellow also elicited tenure as long or significantly longer than other colors after contact with a model. For each species, there were no differences in tenure between males and females on models. The importance of color stimuli in host-finding by Dalbulus leafhoppers is suggested to be influenced by differences in flight behavior and habitat stability of field hosts.

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Résumé Nous avons examiné l'importance de la couleur dans la découverte de l'hôte, en comparant au laboratoire les réactions avant et après contacts avec des plants de maïs,-à 4 feuilles-, et avec des leurres peints présentant des différences de nuance ou de quantité de lumière réfléchie, chez 3 espèces de Dalbulus: D. maidis,-spécialiste du maïs-, D. gelbus,-qui consomme le maïs et Tripsacum, et D. quinquenotatus,-spécialiste de Tripsacum. Les espèces entrent en contact avec le maïs plus en voltigeant que par la marche, mâles et femelles séjournant plusieurs heures après être entrés en contact. En utilisant des leurres végétaux,-des morceaux de bois verticaux peints de différentes couleurs-, une forte réponse au jaune, due à la teinte plus qu'à l'intensité, a été mise en évidence. Il n'y a pas de différence dans les contacts après voltige ou après marche avec des leurres jaunes pour D. maidis et D. gelbus, tandis que chez D. quinquenotatus, ils sont plus fréquents après voltige. Ces 3 espèces de Dalbulus continuent à être influencées par la couleur après contact, puisque chaque espèce reste plus longtemps sur le jaune que sur les autres couleurs. L'importance de la stimulation par la couleur dans la découverte des hôtes par Dalbulus est semble-t-il due au comportement de vol et à la stabilité de l'habitat dans la nature.

     

Effect of temperature and host plant on the development of the blackfaced leafhopper

Population dynamics of the blackfaced leafhopper, Graminella nigrifrons (Forbes) (Homoptera: Cicadellidae), was studied at five temperatures (18, 21, 24, 27, & 30°C) in the laboratory on seedling maize (Zea mays L.), oats (Avena sativa L.), and the perennial johnsongrass (Sorghum halepense (L.) Pers.). Effects of temperature and host plant on egg to adult mean development time, adult size and weight, and fecundity were determined. Leafhoppers on all three hosts developed fastest at the highest temperature tested (21.3 days), and slowest at the lowest temperature tested (73.2 days). The duration from first to last adult eclosion was shortest at 30°C, (11.5 days) and longest at 18°C (43 days). The sex ratio of males to females did not differ from 1:1, but males developed an average of 1.2 days faster than females on all three hosts. Mean percent development/day ranged from 1.4% at 18°C to 4.7% at 30°C. The relationship of this development rate and temperature was determined using both a linear model and a variable parameter biophysical model. Based on these models, the developmental threshold is estimated at 12–15°C. The lowest temperature yielded larger and heavier adults (312 g, dry weight) than did the highest temperature (225 g). Fewer leafhoppers developed on the perennial than the annuals at 30°C and fewer on the annuals than the perennial at 18°C. Our results suggest that early in the season johnsongrass and perhaps other perennials are the superior developmental hosts for this leafhopper, whereas in midsummer when temperatures are highest, annuals are the better hosts.

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Résumé La dynamique des populations (durée de développement de l'uf à l'adulte, poids et taille des adultes, fécondité) de G. nigrifrons Forbes (Homop. Cicadellidae) a été étudiée au laboratoire à 5 températures sur plantules de maïs (Zea mays L.), avoine (Avena sativa L.) et sorgho vivace (Shorgum halepense (L.) Pers.). Sur les 3 plantes, les mâles se développent en moyenne 1,2 j plus vite que les femelles. Les relations entre vitesse de développement et température ont été déterminées en utilisant à la fois un modèle linéaire et le modèle biophysique à 2 paramètres de Sharpe & DeMichele (1977).Les températures plus basses donnent des adultes des 2 sexes plus gros et plus lourds. Moins de G. nigrifrons se sont développés sur la graminée vivace que sur les 2 graminées annuelles à la température la plus élevée (30°C), tandis qu'à la température la plus basse (18°C) moins de cicadelles se sont développées sur les graminées annuelles. La température semble jouer un rôle significatif en déterminant l'adéquation des plantes comme hôtes convenant au développement de G. nigrifrons. Le potentiel de ponte de cette cicadelle avait été sous-estimé par les étudies précédentes.

     

Chemical Changes During 6 Years of Decomposition of 11 Litters in Some Canadian Forest Sites. Part 1. Elemental Composition, Tannins, Phenolics, and Proximate Fractions

Slowing or even cessation of litter decomposition with time is well-known, but there is insufficient understanding of the chemical changes that contribute to increasing recalcitrance. Samples from the Canadian Intersite Decomposition Experiment (CIDET) were used to determine 6-year chemical changes for all 11 litters from a site with rapid initial decomposition (Morgan Arboretum, MAR) and for three litters at three colder sites. Six-year mass remaining was 17–37% at MAR, with higher values at the colder sites. Atomic C/N ratios declined and phenolics and condensed tannins generally decreased to minimal values. However, for the three species compared across four sites, phenolics and tannins showed small increases for species with the lowest initial values and also tended to increase with increasing mass loss. For the foliar litters at MAR, there was an average increase in proportion of acid-unhydrolyzable residue (AUR) and decreases in proportions of acid-hydrolyzable (ACID) and extractable fractions, with final AUR/(ACID + AUR) ratios within 0.55–0.66. Principal component analysis showed that foliar litters (and to a lesser extent wood) became more alike after 6 years, decomposition being associated with increase of Fe, Al, N, and AUR concentrations and decrease of K, Mg, tannins, phenolics, and non-polar and water-soluble fractions. However, litters were also affected by site soil chemistry, with some high 6-year accumulations of Ca, Mg, Fe, Al, Mn, and Mg at two sites. Increasing recalcitrance likely arises from increasing dominance of complex, less-soluble organic structures, collectively represented by AUR, together with increases in heavy elements such as Al and Fe, which also specifically bind and stabilize organic matter.