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.     

Positive selection of novel peroxisome biogenesis-defective mutants of the yeast Pichia pastoris

We have developed two novel schemes for the direct selection of peroxisome-biogenesis-defective (pex) mutants of the methylotrophic yeast Pichia pastoris. Both schemes take advantage of our observation that methanol-induced pex mutants contain little or no alcohol oxidase (AOX) activity. AOX is a peroxisomal matrix enzyme that catalyzes the first step in the methanol-utilization pathway. One scheme utilizes allyl alcohol, a compound that is not toxic to cells but is oxidized by AOX to acrolein, a compound that is toxic. Exposure of mutagenized populations of AOX-induced cells to allyl alcohol selectively kills AOX-containing cells. However, pex mutants without AOX are able to grow. The second scheme utilizes a P. pastoris strain that is defective in formaldehyde dehydrogenase (FLD), a methanol pathway enzyme required to metabolize formaldehyde, the product of AOX. AOX-induced cells of fld1 strains are sensitive to methanol because of the accumulation of formaldehyde. However, fld1 pex mutants, with little active AOX, do not efficiently oxidize methanol to formaldehyde and therefore are not sensitive to methanol. Using these selections, new pex mutant alleles in previously identified PEX genes have been isolated along with mutants in three previously unidentified PEX groups.     

Development of a transformation system for the flavinogenic yeast Candida famata

Riboflavin-overproducing mutants of the flavinogenic yeast Candida famata are used for industrial riboflavin production. This paper describes the development of an efficient transformation system for this species. Leucine-deficient mutants have been isolated from C. famata VKM Y-9 wild-type strain. Among them leu2 mutants were identified by transformation to leucine prototrophy with plasmids YEp13 and PRpL2 carrying the Saccharomyces cerevisiae LEU2 gene. DNA fragments (called CfARSs) conferring increased transformation frequencies and extrachromosomal replication were isolated from a C. famata gene library constructed on the integrative vector containing the S. cerevisiae LEU2 gene as a selective marker. The smallest cloned fragment (CfARS16) has been sequenced. This one had high adenine plus thymine (A+T) base pair content and a sequence homologous to the S. cerevisiae ARS Consensus Sequence. Methods for spheroplast transformation and electrotransformation of the yeast C. famata were optimized. They conferred high transformation frequencies (up to 10(5) transformants per microg DNA) with a C. famata leu2 mutant using replicative plasmids containing the S. cerevisiae LEU2 gene as a selective marker. Riboflavin-deficient mutants were isolated from the C. famata leu2 strain and their biochemical identification was carried out. Using the developed transformation system, several C. famata genomic fragments complementing mutations of structural genes for riboflavin biosynthesis (coding for GTP cyclohydrolase, reductase, dihydroxybutanone phosphate synthase and riboflavin synthase, respectively) have been cloned.     

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 pleiotropic nature of <Emphasis Type="Italic">rib80, hit1</Emphasis>, and <Emphasis Type="Italic">red6</Emphasis> mutations affecting riboflavin biosynthesis in the yeast <Emphasis Type="Italic">Pichia guilliermondii</Emphasis>

The yeast Pichia guilliermondii is capable of riboflavin overproduction under iron deficiency. The rib80, hit1, and red6 mutants of this species, which exhibit impaired riboflavin regulation, are also distinguished by increased iron concentrations in the cells and mitochondria, morphological changes in the mitochondria, as well as decreased growth rates (except for red6) and respiratory activity. With sufficient iron supply, the rib80 and red6 mutations cause a 1.5–1.8-fold decrease in the activity of such Fe-S cluster proteins as aconitase and flavocytochrome b ₂, whereas the hit1 mutation causes a six-fold decrease. Under iron deficiency, the activity of these enzymes was equally low in all of the studied strains.     

Deficiency in frataxin homologue YFH1 in the yeast Pichia guilliermondii leads to missregulation of iron acquisition and riboflavin biosynthesis and affects sulfate assimilation

Pichia guilliermondii is a representative of yeast species that overproduce riboflavin (vitamin B₂) in response to iron deprivation. P. guilliermondii YFH1 gene coding for frataxin homologue, eukaryotic mitochondrial protein involved in iron trafficking and storage, was identified and deleted. Constructed P. guilliermondii Δyfh1 mutant grew very poorly in a sucrose-containing synthetic medium supplemented with sulfate or sulfite as a sole sulfur source. Addition of sodium sulfide, glutathione, cysteine, methionine, N-acetyl-l-cysteine partially restored growth rate of the mutant suggesting that it is impaired in sulfate assimilation. Cellular iron content in Δyfh1 mutant was ~3–3.5 times higher as compared to the parental strain. It produced 50–70 times more riboflavin in iron sufficient synthetic media relative to the parental wild-type strain. Biomass yield of the mutant in the synthetic glutathione containing medium supplemented with glycerol as a sole carbon source was 1.4- and 2.6-fold increased as compared to sucrose and succinate containing media, respectively. Oxygen uptake of the Δyfh1 mutant on sucrose, glycerol or succinate, when compared to the parental strain, was decreased 5.5-, 1.7- and 1.5-fold, respectively. Substitution of sucrose or glycerol in the synthetic iron sufficient medium with succinate completely abolished riboflavin overproduction by the mutants. Deletion of the YFH1 gene caused hypersensitivity to hydrogen peroxide and exogenously added riboflavin and led to alterations in superoxide dismutase activities. Thus, deletion of the gene coding for yeast frataxin homologue has pleiotropic effect on metabolism in P. guilliermondii.     

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.