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.     

Efficacy of hyaluronic acid binding assay in selecting motile spermatozoa with normal morphology at high magnification

Background  

The present study aimed to evaluate the efficacy of the hyaluronic acid (HA) binding assay in the selection of motile spermatozoa with normal morphology at high magnification (8400x).     

The pathologies associated with functional titration of phosphatidylinositol transfer protein alpha activity in mice

Phosphatidylinositol transfer proteins (PITPs) bind phosphatidylinositol (PtdIns) and phosphatidylcholine and play diverse roles in coordinating lipid metabolism/signaling with intracellular functions. The underlying mechanisms remain unclear. Genetic ablation of PITPalpha in mice results in neonatal lethality characterized by intestinal and hepatic steatosis, spinocerebellar neurodegeneration, and glucose homeostatic defects. We report that mice expressing a PITPalpha selectively ablated for PtdIns binding activity (Pitpalpha(T59D)), as the sole source of PITPalpha, exhibit phenotypes that recapitulate those of authentic PITPalpha nullizygotes. Analyses of mice with graded reductions in PITPalpha activity reveal proportionately graded reductions in lifespan, demonstrate that intestinal steatosis and hypoglycemia are apparent only when PITPalpha protein levels are strongly reduced (>or=90%), and correlate steatotic and glucose homeostatic defects with cerebellar inflammatory disease. Finally, reconstitution of PITPalpha expression in the small intestine substantially corrects the chylomicron retention disease and cerebellar inflammation of Pitpalpha(0/0) neonates, but does not rescue neonatal lethality in these animals. These data demonstrate that PtdIns binding is an essential functional property of PITPalpha in vivo, and suggest a causal linkage between defects in lipid transport and glucose homeostasis and cerebellar inflammatory disease. Finally, the data also demonstrate intrinsic neuronal deficits in PITPalpha-deficient mice that are independent of intestinal lipid transport defects and hypoglycemia.     

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.     

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.      

Psychosocial and physical impairment in overweight adolescents at high risk for eating disorders

Objective: Many overweight adolescents display elevated risk for the development of eating disorders, as seen in higher rates of weight/shape concerns and disordered eating behaviors, but the extent of impairment in this subset of high‐risk adolescents has not been explored. Research Methods and Procedures: Eighty‐one overweight adolescents (63% girls) presenting for an Internet‐based weight loss program were assessed at baseline using the Eating Disorder Examination Questionnaire, the Depression, Anxiety, and Stress Scale, and the Pediatric Quality of Life questionnaire. Adolescents who earned elevated scores on both the Weight Concern and Shape Concern subscales of the Eating Disorder Examination Questionnaire were considered at high risk for the development of eating disorders (56.8%). Results: Comparisons of high‐ and normal‐risk groups revealed that high‐risk adolescents reported higher levels of depression [F(3,76) = 5.75, p = 0.019], anxiety [F(3,76) = 5.67, p = 0.020], and stress [F(3,75) = 8.50, p = 0.005], and greater impairments in physical health [F(3,77) = 10.7, p = 0.002], emotional functioning [F(3,77) = 5.3, p = 0.024], and social functioning [F(3,77) = 10.0, p = 0.002]. There were no differences in school functioning [F(3,77) = 1.5, p = 0.219]. Among the high‐risk adolescents, over half (52.2%) reported binge eating at least once in the past month. Discussion: Results suggest that overweight adolescents at high risk for the development of eating disorders also experience elevated levels of negative affect, impairment in health‐related quality of life, and eating disturbances, although prospective data are needed to determine the directionality between eating disorder pathology and general psychopathology. Further research is warranted to evaluate whether behavioral weight loss interventions should be enhanced for this high‐risk subset.     

Overweight Children's Barriers to and Support for Physical Activity

Objective: As the epidemic of overweight increases among youth, research needs to examine factors that may influence children's participation in weight‐related health behaviors. This study examined overweight children's perceived barriers to and support for physical activity compared with nonoverweight children. Research Methods and Procedures: Barriers to and support for physical activity were examined among 84 overweight children attending a summer fitness camp or a university‐based weight loss clinic. Barriers and support levels were then compared with those of 80 nonoverweight children of a similar age range. Results: Body‐related barriers were the most predominant barrier type among overweight youth, especially among overweight girls. Overweight children, particularly girls, reported significantly higher body‐related, resource, and social barriers to physical activity compared with nonoverweight children and lower levels of adult support for physical activity. Discussion: Overweight children may be particularly vulnerable to body‐related barriers to physical activity, and reducing such barriers may serve as physical activity intervention points most relevant for overweight youth. Future interventions may also benefit from enhancing support for physical activity from adults and peers.