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 phosphatase activity of the plasma membrane Ca2+ pump. Activation by acidic lipids in the absence of Ca2+ increases the apparent affinity for Mg2+

The purified PMCA supplemented with phosphatidylcholine was able to hydrolyze pNPP in a reaction media containing only Mg(2+) and K(+). Micromolar concentrations of Ca(2+) inhibited about 75% of the pNPPase activity while the inhibition of the remainder 25% required higher Ca(2+) concentrations. Acidic lipids increased 5-10 fold the pNPPase activity either in the presence or in the absence of Ca(2+). The activation by acidic lipids took place without a significant change in the apparent affinities for pNPP or K(+) but the apparent affinity of the enzyme for Mg(2+) increased about 10 fold. Thus, the stimulation of the pNPPase activity of the PMCA by acidic lipids was maximal at low concentrations of Mg(2+). Although with differing apparent affinities vanadate, phosphate, ATP and ADP were all inhibitors of the pNPPase activity and their effects were not significantly affected by acidic lipids. These results indicate that (a) the phosphatase function of the PMCA is optimal when the enzyme is in its activated Ca(2+) free conformation (E2) and (b) the PMCA can be activated by acidic lipids in the absence of Ca(2+) and the activation improves the interaction of the enzyme with Mg(2+).     

A Statistical Approach to Optimize the Spray Drying of Starch Particles: Application to Dry Powder Coating

This article describes the preparation of starch particles, by spray drying, for possible application to a dry powder coating process. Dry powder coating consists of spraying a fine powder and a plasticizer on particles. The efficiency of the coating is linked to the powder morphological and dimensional characteristics. Different experimental parameters of the spray-drying process were analyzed, including type of solvent, starch concentration, rate of polymer feeding, pressure of the atomizing air, drying air flow, and temperature of drying air. An optimization and screening of the experimental parameters by a design of the experiment (DOE) approach have been done. Finally, the produced spray-dried starch particles were conveniently tested in a dry coating process, in comparison to the commercial initial starch. The obtained results, in terms of coating efficiency, demonstrated that the spray-dried particles led to a sharp increase of coating efficiency value.     

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.      

The plasma membrane Ca pump catalyzes the hydrolysis of ATP at low rate in the absence of Ca

The plasma membrane Ca²⁺ ATPase catalyzed the hydrolysis of ATP in the presence of millimolar concentrations of EGTA and no added Ca²⁺ at a rate near 1.5% of that attained at saturating concentrations of Ca²⁺. Like the Ca-dependent ATPase, the Ca-independent activity was lower when the enzyme was autoinhibited, and increased when the enzyme was activated by acidic lipids or partial proteolysis. The ATP concentration dependence of the Ca²⁺-independent ATPase was consistent with ATP binding to the low affinity modulatory site. In this condition a small amount of hydroxylamine-sensitive phosphoenzyme was formed and rapidly decayed when chased with cold ATP. We propose that the Ca²⁺-independent ATP hydrolysis reflects the well known phosphatase activity which is maximal in the absence of Ca²⁺ and is catalyzed by E₂-like forms of the enzyme. In agreement with this idea pNPP, a classic phosphatase substrate was a very effective inhibitor of the ATP hydrolysis.     

The cytokine and chemokine expression profile of nucleus pulposus cells: implications for degeneration and regeneration of the intervertebral disc

Introduction

The aims of these studies were to identify the cytokine and chemokine expression profile of nucleus pulposus (NP) cells and to determine the relationships between NP cell cytokine and chemokine production and the characteristic tissue changes seen during intervertebral disc (IVD) degeneration.

Methods

Real-time q-PCR cDNA Low Density Array (LDA) was used to investigate the expression of 91 cytokine and chemokine associated genes in NP cells from degenerate human IVDs. Further real-time q-PCR was used to investigate 30 selected cytokine and chemokine associated genes in NP cells from non-degenerate and degenerate IVDs and those from IVDs with immune cell infiltrates (‘infiltrated’). Immunohistochemistry (IHC) was performed for four selected cytokines and chemokines to confirm and localize protein expression in human NP tissue samples.

Results

LDA identified the expression of numerous cytokine and chemokine associated genes including 15 novel cytokines and chemokines. Further q-PCR gene expression studies identified differential expression patterns in NP cells derived from non-degenerate, degenerate and infiltrated IVDs. IHC confirmed NP cells as a source of IL-16, CCL2, CCL7 and CXCL8 and that protein expression of CCL2, CCL7 and CXCL8 increases concordant with histological degenerative tissue changes.

Conclusions

Our data indicates that NP cells are a source of cytokines and chemokines within the IVD and that these expression patterns are altered in IVD pathology. These findings may be important for the correct assessment of the ‘degenerate niche’ prior to autologous or allogeneic cell transplantation for biological therapy of the degenerate IVD.