Biomedical subjects

Dominique Arion

Publications and source records attributed to Dominique Arion.

8 recordsLinked to original sources

PubMed2026

Alternative splicing in layer 3 pyramidal neurons differs across regions of the human cortical hierarchy.

The primate neocortex is organized as hierarchical networks of functionally distinct regions. In the dorsal visual stream network, information is conveyed from primary visual (V1) to posterior parietal (PPC) and dorsolateral prefrontal (DLPFC) cortices. This information transfer is mediated primarily by layer 3 pyramidal neurons (L3PNs), which differ across these regions in morphology, excitability, and intracellular Ca2+ regulation. These region-specific L3PN properties may be influenced by alternative splicing (AS) of pre-mRNA, which occurs extensively in the human brain. To explore the potential impact of AS of region-specific L3PN properties, we analyzed RNA-seq data from pools of L3PNs dissected from human V1, PPC, and DLPFC. We found that <6% of genes with regional differences in expression also differed in AS. This finding indicates that the absence of transcriptional differences is insufficient to conclude that a gene does not contribute functional differences between regions. Additionally, there were numerous regional differences in AS, particularly between V1 and DLPFC or PPC L3PNs, which involved genes associated with neuron morphology and Ca2+ regulation; > 90% of these AS differences involved functionally relevant sequences (eg phosphorylation sites, etc.). These findings suggest AS contributes to region-specific L3PN properties relevant to the function of the dorsal visual stream.

Humans↗

PubMed2006

Correlation of transcriptome profile with electrical activity in temporal lobe epilepsy.

The biology underlying epileptic brain activity in humans is not well understood and likely depends on changes in gene expression. We performed a microarray transcriptome profiling of 12 anterolateral temporal cortical samples originating from five individuals who suffered with temporal lobe epilepsy for at least 10 years. Prior to partial lobectomy, intraoperative electrocorticography was performed on the cortical surface of each patient. These recordings showed characteristic differences in frequency and amplitude that were defined as "spiking" (abnormal) or "non-spiking" (normal). Between the transcriptome of the two sample groups, transferrin (TF) was the most differentially expressed gene. Furthermore, gene expression profiling also revealed a downregulation of multiple GABA system-related genes (GABRA5, GABRB3, ABAT) in the spiking samples and an upregulation of oligodendrocyte and lipid metabolism transcripts (MOG, CA2, CNP, SCD, PLP1, FA2H, ABCA2). In addition, several transcripts related to the classical MAPK cascade showed expression level alterations between the spiking and non-spiking samples (G3BP2, MAPK1, PRKAR1A, and MAP4K4). Out of 12 genes chosen for verification by RT qPCR, 9 showed significant expression changes in the microarray-predicted direction. Furthermore, the microarray and qPCR data were highly correlated (r = 0.98; P < 0.001). We conclude that abnormal electrical brain activity in the spiking samples is strongly correlated with gene expression changes and we speculate that some of the observed transcriptome changes may be directly involved in the induction or prevention of the ictal events seen in epilepsy.

Action Potentials↗

PubMed2005

The 3'-azido group is not the primary determinant of 3'-azido-3'-deoxythymidine (AZT) responsible for the excision phenotype of AZT-resistant HIV-1.

The mechanism of human immunodeficiency virus (HIV) 1 resistance to 3'-azido-3'-deoxythymidine (AZT) involves reverse transcriptase (RT)-catalyzed phosphorolytic excision of the chain-terminating AZT-5'-monophosphate (AZTMP). Primers terminated with AZTMP are generally better substrates for this reaction than those terminated with 2',3'-dideoxynucleoside-5'-monophosphate (2',3'-ddNMP) analogs that lack a 3'-azido moiety. This led to the hypothesis that the 3'-azido group is a major structural determinant for maintaining the primer terminus in the appropriate site for phosphorolytic excision of AZTMP by AZT-resistant (AZT(R)) RT. To test this hypothesis, we evaluated the incorporation, phosphorolytic excision, and antiviral activity of a panel of 3'-azido-2',3'-ddN including 3'-azido-2',3'-ddA (AZddA), 3'-azido-2',3'-ddC (AZddC), 3'-azido-2',3'-ddG (AZddG), AZT, and 3'-azido-2',3'-ddU (AZddU). The results indicate that mutations correlated with resistance to AZT (D67N/K70R/T215F/K219Q) confer resistance to the 3'-azidopyrimidine nucleosides (AZddC, AZT, and AZddU) but not to the 3'-azidopurine nucleosides (AZddA and AZddG). The data suggest that the presence of a 3'-azido group on the 3'-terminal nucleotide of the primer does not confer increased phosphorolytic excision by AZT(R) RT for all 3'-azido-ddNMP analogs. Thus, the 3'-azido group cannot be the only structural determinant important for the enhanced phosphorolytic excision of AZTMP associated with HIV resistance to AZT. Other structural components, such as the base, must play a role in defining the specificity of the excision phenotype arising from AZT resistance mutations.

Anti-HIV Agents↗

PubMed2005

Presenilin-1-dependent transcriptome changes.

Familial forms of Alzheimer's disease (FADs) are caused by the expression of mutant presenilin 1 (PS1) or presenilin 2. Using DNA microarrays, we explored the brain transcription profiles of mice with conditional knock-out of PS1 (cKO PS1) in the forebrain. In parallel, we performed a transcription profiling of the hippocampus and frontal cortex of the FAD-linked DeltaE9 mutant transgenic (TG) mice and matched controls [TG mice expressing wild-type human PS1 (hPS1)]. When the TG and cKO datasets were cross-compared, the majority of the 30 common expression alterations were in opposite direction, suggesting that the FAD-linked PS1 variant produces transcriptome changes primarily by gain of aberrant function. Our microarray studies also revealed an unanticipated inverse correlation of transcript levels between the brains of mice that coexpress DeltaE9 hPS1+ amyloid precursor protein (APP)695 Swe and DeltaE9 hPS1 single transgenic mice. The opposite directionality of these changes in transcript levels must be a function of APP and/or APP derivatives.

Alzheimer Disease↗

PubMed2004

Proteolytic processing of an HIV-1 pol polyprotein precursor: insights into the mechanism of reverse transcriptase p66/p51 heterodimer formation.

HIV-1 reverse transcriptase (RT) is a heterodimer comprising a 66 kDa subunit (p66) and a p66-derived 51 kDa subunit (p51). RT is translated as part of a larger gag-pol polyprotein and subsequently processed to the p66/p51 heterodimer by HIV-1 protease (PR) during viral maturation. The processing events involved in the formation of the RT p66 and p51 subunits and the pathway(s) of RT dimer formation are poorly characterized. Attempts to study the kinetics of PR-catalyzed formation of p66/p51 HIV-1 RT in isolated HIV virions produced in the presence of HIV PR inhibitors were unsuccessful due to difficulties in removal of the tight-binding inhibitor to initiate proteolytic processing. Accordingly, an inducible bacterial expression vector encoding a 90 kDa pol polyprotein fragment was constructed. Following expression in Escherichia coli, the pol polyprotein underwent time-dependent proteolytic processing to the RT p66/p51 heterodimer. This processing was catalyzed entirely by HIV-1 PR since mutations that inactivate PR prevented RT heterodimer formation. The kinetics of RT processing follow an ordered sequential pathway in which RT p66 is first excised from the pol polyprotein, followed by formation of the p51 subunit. Processing of the p66 subunit to form p51 apparently proceeds through a p66/p66 RT homodimer intermediate since the L234A mutation in RT, a mutation that prevents RT dimerization, resulted in the formation of RT p66 only. These results provide the first experimental data defining the pathway for the HIV-1 PR catalyzed formation of the p66/p51 HIV-1 RT heterodimer.

Animals↗

PubMed2002

Destabilization of the HIV-1 reverse transcriptase dimer upon interaction with N-acyl hydrazone inhibitors.

N-(4-tert-butylbenzoyl)-2-hydroxy-1-naphthaldehyde hydrazone (BBNH) inhibits both the DNA polymerase and ribonuclease H (RNase H) activities of the human immunodeficiency virus type 1 reverse transcriptase. In this study, we show that BBNH binding impacts on the stability of the human immunodeficiency virus type 1 (HIV-1) reverse transcriptase (RT) heterodimer. The Gibbs free energy of dimer dissociation of HIV-1 RT is decreased in the presence of increasing concentrations of BBNH, resulting in a loss in stability of 3.8 kcal mol(-1). To evaluate whether this observed phenomenon was mediated by BBNH binding to one or more sites in RT, we synthesized a variety of BBNH analogs and identified (4-t-butylbenzoyl)-2-hydroxy-1-salicylyl hydrazone (BBSH) and (4,N,N-dimethylaminobenzoyl)-2-hydroxy-1-naphthyl hydrazone as specific inhibitors of RT DNA polymerase or RT RNase H activity, respectively. Interestingly, only BBSH provided significant destabilization of the HIV-1 RT dimer. The identification of these specific inhibitors, in combination with other biochemical data, suggests a model in which two molecules of BBNH bind per RT heterodimer. In this regard, only the binding of hydrazone molecules in the DNA polymerase domain activity elicits the observed destabilization of the HIV-1 RT heterodimer.

DNA-Directed DNA Polymerase↗

PubMed2001

Mutational analysis of Tyr-501 of HIV-1 reverse transcriptase. Effects on ribonuclease H activity and inhibition of this activity by N-acylhydrazones.

N-(4-tert-Butylbenzoyl)-2-hydroxynaphthaldehyde hydrazone (BBNH) is a potent inhibitor of the ribonuclease H (RNase H) activity of human immunodeficiency virus (HIV)-1 reverse transcriptase (RT). Molecular modeling predicted that BBNH binds to the HIV-1 RT RNase H active site via two major interactions, coordination to the metal ion cofactor (Mg(2+) or Mn(2+)) in the enzyme active site and aromatic ring-stacking interaction between the naphthyl ring of BBNH and amino acid Tyr-501. The latter residue equivalent is conserved in virtually all RNases H, suggesting the need for an aromatic or pi-stacking interaction in this region. To assess the importance of Tyr-501 in the binding of BBNH for the inhibition of RT RNase H activity, we used site-specific mutagenesis to generate RT with a variety of substitutions at this position. Most substitutions resulted virtually in a complete loss of RNase H activity. However, three mutants, Y501F, Y501W, and Y501R, possessed RNase H activities comparable with wild-type enzyme. Whereas BBNH inhibited Y501F RT RNase H activity with potency equivalent to wild-type RT, the Y501W mutant showed a 6-fold resistance to inhibition by BBNH, and the Y501R mutant was completely resistant to inhibition by BBNH. The replication "fitness" of HIV molecular clones with the Y501W or Y510R mutation was significantly compromised compared with wild-type virus. Importantly, BBNH was an effective inhibitor of the DNA polymerase activity of all Y501X mutants tested. Our results highlight the importance of Tyr-501 in RT RNase H activity and in N-acylhydrazone inhibitor binding and suggest that drugs that target critical residues in HIV-1 proteins may be a useful approach in new antiviral development.

Binding Sites↗

PubMed1999

HIV resistance to zidovudine: the role of pyrophosphorolysis.

Zidovudine-resistant strains of HIV became apparent in many patients soon after advent of zidovudine (AZT) monotherapy. While this resistance could be unequivocally correlated with multiple mutations in HIV reverse transcriptase (D67N, K70R, T215F/Y, K219Q), the mechanism or phenotype for this resistance has remained obscure for more than a decade, despite active investigation. Recent studies indicate that AZT resistance may be related to removal of chain-terminating AZT from the 3'-terminus of the primer, by a process known as pyrophosphorolysis. This process is catalyzed by HIV-1 reverse transcriptase (RT), and is the reverse reaction of DNA polymerization. The D67N/K70R mutations result in a significantly increased rate of RT-catalyzed pyrophosphorolysis at physiological levels of pyrophosphate, which leads to a decrease in the extent of AZT chain termination of nascent viral DNA. The potential replication deficit of an increased reverse reaction during DNA synthesis is compensated by increased DNA synthesis processivity, a phenotype that results from the T215F/Y/K219Q mutations in RT. The net result of these multiple phenotypes imparted by the multiple mutations in RT is the facile synthesis of full-length viral DNA in the presence of AZT. Copyright 1999 Harcourt Publishers Ltd.

Journal Article↗