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).     

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.      

Molecular determinants for the distinct pH sensitivity of Kir1.1 and Kir4.1 channels

Kir1.1 (ROMK1) is inhibited by hypercapnia andintracellular acidosis with midpoint pH for channel inhibition(pK_a) of ~6.7. Another close relative,Kir4.1 (BIR10), is also pH sensitive with much lower pH sensitivity(pK_a ~6.0), although it shares a high sequencehomology with Kir1.1. To find the molecular determinants for thedistinct pH sensitivity, we studied the structure-functional relationship using site-directed mutagenesis. AnNH₂-terminal residue (Lys-53) was found to be responsiblefor the low pH sensitivity in Kir4.1. Mutation of this lysine to valine(K53V), a residue seen at the same position in Kir1.1, markedlyincreased channel sensitivity to CO₂/pH. Reverse mutationon Kir1.1 (V66K) decreased the CO₂/pH sensitivities.Interestingly, mutation of these residues to glutamate greatly enhancedthe pH sensitivity in both channels. Other contributors to the distinctpH sensitivity were histidine residues in the COOH terminus, whosenumbers are fewer in Kir4.1 than Kir1.1. Mutation of two of thesehistidine residues in Kir1.1 (H342Q/H354N) reduced CO₂/pHsensitivities, whereas the creation of two histidines (S328H/G340H) inKir4.1 increased the CO₂/pH sensitivities. Combinedmutations of the lysine and histidine residues in Kir4.1(K53V/S328H/G340H) gave rise to a channel that had CO₂/pHsensitivities almost identical to those of the wild-type Kir1.1. Thusthe residues demonstrated in our current studies are likely themolecular basis for the distinct pH sensitivity between Kir1.1 andKir4.1.

     

Involvement of histidine residues in proton sensing of ROMK1 channel

ROMK channels are inhibited by intracellular acidification. This pH sensitivity is related to several amino acid residues in the channel proteins such as Lys-61, Thr-51, and His-206 (in ROMK2). Unlike all other amino acids, histidine is titratable at pH 6-7 carrying a positive charge below pH 6. To test the hypothesis that certain histidine residues are engaged in CO(2) and pH sensing of ROMK1, we performed experiments by systematic mutations of all histidine residues in the channel using the site-directed mutagenesis. There are two histidine residues in the N terminus. Mutations of His-23, His-31, or both together did not affect channel sensitivity to CO(2). Six histidine residues are located in the C terminus. His-225, His-274, His-342, and His-354 were critical in CO(2) and pH sensing. Mutation of either of them reduced CO(2) and pH sensitivities by 20-50% and approximately 0.2 pH units, respectively. Simultaneous mutations of all of them eliminated the CO(2) sensitivity and caused this mutant channel to respond to only extremely acidic pH. Similar mutations of His-280 had no effect. The role of His-270 in CO(2) and pH sensing is unclear, because substitutions of this residue with either a neutral, negative, or positive amino acid did not produce any functional channel. These results therefore indicate that histidine residues contribute to the sensitivity of the ROMK1 channel to hypercapnia and intracellular acidosis.     

Distinct histidine residues control the acid-induced activation and inhibition of the cloned K(ATP) channel

The modulation of K(ATP) channels during acidosis has an impact on vascular tone, myocardial rhythmicity, insulin secretion, and neuronal excitability. Our previous studies have shown that the cloned Kir6.2 is activated with mild acidification but inhibited with high acidity. The activation relies on His-175, whereas the molecular basis for the inhibition remains unclear. To elucidate whether the His-175 is indeed the protonation site and what other structures are responsible for the pH-induced inhibition, we performed these studies. Our data showed that the His-175 is the only proton sensor whose protonation is required for the channel activation by acidic pH. In contrast, the channel inhibition at extremely low pH depended on several other histidine residues including His-186, His-193, and His-216. Thus, proton has both stimulatory and inhibitory effects on the Kir6.2 channels, which attribute to two sets of histidine residues in the C terminus.     

Direct activation of cloned K(atp) channels by intracellular acidosis

ATP-sensitive K(+) (K(ATP)) channels may be regulated by protons in addition to ATP, phospholipids, and other nucleotides. Such regulation allows a control of cellular excitability in conditions when pH is low but ATP concentration is normal. However, whether the K(ATP) changes its activity with pH alterations remains uncertain. In this study we showed that the reconstituted K(ATP) was strongly activated during hypercapnia and intracellular acidosis using whole-cell recordings. Further characterizations in excised patches indicated that channel activity increased with a moderate drop in intracellular pH and decreased with strong acidification. The channel activation was produced by a direct action of protons on the Kir6 subunit and relied on a histidine residue that is conserved in all K(ATP). The inhibition appeared to be a result of channel rundown and was not seen in whole-cell recordings. The biphasic response may explain the contradictory pH sensitivity observed in cell-endogenous K(ATP) in excised patches. Site-specific mutations of two residues showed that pH and ATP sensitivities were independent of each other. Thus, these results demonstrate that the proton is a potent activator of the K(ATP). The pH-dependent activation may enable the K(ATP) to control vascular tones, insulin secretion, and neuronal excitability in several pathophysiologic conditions.