The Interactomes of Influenza Virus NS1 and NS2 Proteins Identify New Host Factors and Provide Insights for ADAR1 Playing a Supportive Role in Virus Replication

Influenza A NS1 and NS2 proteins are encoded by the RNA segment 8 of the viral genome. NS1 is a multifunctional protein and a virulence factor while NS2 is involved in nuclear export of viral ribonucleoprotein complexes. A yeast two-hybrid screening strategy was used to identify host factors supporting NS1 and NS2 functions. More than 560 interactions between 79 cellular proteins and NS1 and NS2 proteins from 9 different influenza virus strains have been identified. These interacting proteins are potentially involved in each step of the infectious process and their contribution to viral replication was tested by RNA interference. Validation of the relevance of these host cell proteins for the viral replication cycle revealed that 7 of the 79 NS1 and/or NS2-interacting proteins positively or negatively controlled virus replication. One of the main factors targeted by NS1 of all virus strains was double-stranded RNA binding domain protein family. In particular, adenosine deaminase acting on RNA 1 (ADAR1) appeared as a pro-viral host factor whose expression is necessary for optimal viral protein synthesis and replication. Surprisingly, ADAR1 also appeared as a pro-viral host factor for dengue virus replication and directly interacted with the viral NS3 protein. ADAR1 editing activity was enhanced by both viruses through dengue virus NS3 and influenza virus NS1 proteins, suggesting a similar virus-host co-evolution.     

Critical review on quantitative autoradiography of D1 and D2 dopaminergic receptors in the striatum of the mammalian brain: differential localization and plastic changes after pharmacological manipulation and dopaminergic input disruption

Major technical progress in the development of computer-based image analysis systems has made possible the entry of autoradiographic and immunohistochemical techniques into a new era where quantification via densitometry and morphometry has become easily accessible. In this context, quantitative biochemical data can be adapted to anatomical and histological resolution. This adaptation is most efficient in the neuroscience fields because of the huge importance of cellular communication via neuronal networks in the nervous system. Therefore, any experimental approach to the brain which considers the brain as a 'black box' appears now as very crude. In fact, subtle heterogeneity in the distribution of biochemical markers can now be demonstrated, as illustrated here by the use of quantitative autoradiography of D1 and D2 dopaminergic receptors in the striatum of the mammalian brain. Also, local adaptive changes resulting from chronic blockade of the dopaminergic input can be detected after repeated treatments with dopaminergic antagonists selective for D1 or D2 receptors or with surgical lesioning of the dopaminergic nigrostriatal pathway. The resulting plastic changes are unevenly distributed throughout the striatal target organ and vary according to the mode of suppressing the dopaminergic flow: direct destruction of the dopaminergic pathway or selective pharmacological manipulation without physical elimination of the dopaminergic cells themselves. All these results are discussed and reviewed in light of the most recent reports in this field.     

Reduction in neuronal L-type calcium channel activity in a double knock-in mouse model of Alzheimer's disease

Increased function of neuronal L-type voltage-sensitive Ca^2 + channels (L-VSCCs) is strongly linked to impaired memory and altered hippocampal synaptic plasticity in aged rats. However, no studies have directly assessed L-VSCC function in any of the common mouse models of Alzheimer's disease where neurologic deficits are typically more robust. Here, we used cell-attached patch-clamp recording techniques to measure L-VSCC activity in CA1 pyramidal neurons of partially dissociated hippocampal “zipper” slices prepared from 14-month-old wild-type mice and memory-impaired APP/PS1 double knock-in mice. Surprisingly, the functional channel density of L-VSCCs was significantly reduced in the APP/PS1 group. No differences in voltage dependency and unitary conductance of L-VSCCs were observed. The results suggest that mechanisms for Ca^2 + dysregulation can differ substantially between animal models of normal aging and models of pathological aging.     

Chronic 1alpha,25-(OH)2 vitamin D3 treatment reduces Ca2+ -mediated hippocampal biomarkers of aging

Aging in the hippocampus of several species is characterized by alterations in multiple Ca(2+)-mediated processes, including an increase in L-type voltage-gated Ca(2+) channel (L-VGCC) current, an enhanced Ca(2+)-dependent slow afterhyperpolarization (AHP), impaired synaptic plasticity and elevated Ca(2+) transients. Previously, we found that 1alpha,25-dihydoxyvitamin D(3) (1,25VitD), a major Ca(2+) regulating hormone, down-regulates L-VGCC expression in cultured hippocampal neurons. Here, we tested whether in vivo treatment of aged F344 rats with 1,25VitD would reverse some of the Ca(2+) -mediated biomarkers of aging seen in hippocampal CA1 neurons. As previously reported, L-VGCC currents and the AHP were larger in aged than in young neurons. Treatment with 1,25VitD over 7 days decreased L-VGCC activity in aged rats, as well as the age-related increase in AHP amplitude and duration. In addition, reduced L-VGCC activity was correlated with reduced AHPs in the same animals. These data provide direct evidence that 1,25VitD can regulate multiple Ca(2+)-dependent processes in neurons, with particular impact on reducing age-related changes associated with Ca(2+) dysregulation. Thus, these results may have therapeutic implications and suggest that 1,25VitD, often taken to maintain bone health, may also retard some consequences of brain aging.     

Using budding yeast to screen for anti-prion drugs

Prions are misfolded proteins capable of propagating their altered conformation which are commonly considered as the causative agent of transmissible spongiform encephalopathies, a class of fatal neurodegenerative diseases. Currently, no treatment for prion-based diseases is available. Recently we have developed a rapid, yeast-based, two-step assay to screen for anti-prion drugs [1]. This new method allowed us to identify several compounds that are effective in vivo against budding yeast [PSI+] and [URE3] prions but also able to promote mammalian prion clearance in three different cell culture-based assays. Taken together, these results validate our method as an economic and efficient high-throughput screening approach to identify novel prion inhibitors or to carry on comprehensive structure-activity studies for already isolated anti-mammalian prion drugs. These results suggest furthermore that biochemical pathways controlling prion formation and/or maintenance are conserved from yeast to human and thus amenable to pharmacological and genetic analysis. Finally, it would be very interesting to test active drugs isolated using the yeast-based assay in models for other diseases (neurodegenerative or not) involving amyloid fibers like Huntington's, Parkinson's or Alzheimer's diseases.     

Mapping of the dopa decarboxylase gene to the 11A band of the murine genome.

A human DOPA decarboxylase (DDC) cDNA probe of 747 base pairs has been used to map the DDC gene by in situ hybridization on mouse metaphase chromosomes. This result indicates that the gene is located on band 11A, near the erythroblastosis oncogene B (erb b) locus. This provides evidence for a synteny group on mouse chromosome 11 and human chromosome 7.     

Effects of territorial intrusions, courtship feedings and mate fidelity on the copulation behaviour of the osprey

We studied copulation behaviour of the osprey, Pandion haliaetus, a semicolonial, fish-eating raptor, in Corsica (Mediterranean). Pairs copulated over a long period (45 days) and at a high rate, with, on average, 288 within-pair copulations (WPCs) for a clutch. Pairs breeding at higher density faced more frequent territorial intrusions than others and were potentially at an increased cuckoldry risk. However, and contrary to predictions of the ‘paternity assurance’ hypothesis for frequent copulations, we found that WPC rate decreased with increasing frequency of territorial intrusions. Male territory attendance increased with territorial intrusion frequency, to the detriment of the food provisioning of the female. Both attempted and successful WPC rates were positively related to the amount of food delivered by the male. Thus, the more frequent the territorial intrusions, the more time the male spent within his territory, the less he courtship fed and the smaller the fish he delivered, and the less the pair copulated successfully. WPC rate was also higher in newly formed pairs than in established pairs, and decreased with increasing pair bond length. The results suggest that males rely on mate guarding rather than frequent copulations to ensure paternity, and do not support the idea that sperm competition is the main cause of frequent WPCs. Nonfertilization functions of frequent copulations, such as pair bonding, mate assessment and mate retention, were likely early in the prelaying period. The findings that WPC rate decreased with mate fidelity and that females traded copulations for food suggest that mate retention was a possible function of frequent copulations in this species. Copyright 2002 The Association for the Study of Animal Behaviour. Published by Elsevier Science Ltd. All rights reserved.     

Partial characterization and solubilization of receptors for atrial natriuretic factor in rat glomeruli

Specific receptors for atrial natriuretic factor (ANF) have been identified and solubilized in glomeruli from rat kidney. Radioiodinated synthetic ANF (Arg 101-Tyr 126) bound to a single class of high affinity (Kd 27 +/- 24 pM) sites with a density of 390 +/- 230 fmole/mg protein. The binding was time- and temperature-dependent, saturable and reversible. The ANF-receptor complex was not affected by angiotensin II, ACTH or vasopressin. Solubilization with 10 mM 3-[(3-cholamidopropyl)-dimethylammonio]- 1-propane sulfonate (CHAPS) slightly increased the affinity for ANF (Kd 5.0 +/- 3.3 pM) without affecting the density (250 +/- 110 fmole/mg protein). Similar results were found with 1% Triton X-100. ANF-related peptides interact generally in the same way with non-solubilized and solubilized receptors, indicating a fully preserved specificity of the receptors.     

Identification of a biologically active circulating form of rat atrial natriuretic factor

An atrial natriuretic peptide has been isolated from plasma of morphine treated rats by means of glass beads extraction, immunoaffinity chromatography, and reverse phase HPLC. 1.3 micrograms of immunoreactive material was obtained. The biological activity of this material was found comparable to that of ANF (Arg 101 - Tyr 126) on the inhibition of basal aldosterone secretion by rat adrenal zona glomerulosa cells and the displacement curve of iodinated ANF from ANF receptors in a mesenteric artery preparation. Gas phase amino acid sequencing indicated that it is related to ANF (Ser 99 - Tyr 126). These results suggest that the maturation of ANF may require a tryptic-like cleavage after a single Arg residue.