Search PubMed⌕ Search

Biomedical subjects

Jacques Mallet

Publications and source records attributed to Jacques Mallet.

54 records · Page 3Linked to original sources
PubMed2003

Optimization of transgene expression at the posttranscriptional level in neural cells: implications for gene therapy.

Gene delivery vectors need to fulfill several efficacy and safety criteria before they can be used in humans. Successful clinical application requires effective transgene expression with a minimum of vector-associated toxicity. We describe the use of posttranscriptional regulatory elements in plasmid and lentiviral vectors coding for luciferase. These constructs allow high-level gene expression in both neuronal and glial cells. Of the several elements that we tested, WPRE gave the highest level of expression. Further enhancements were obtained when WPRE was combined with sequences corresponding to the 3' or 5' untranslated regions (UTR) of eukaryotic mRNAs (tau 3'UTR, TH 3'UTR, and APP 5'UTR). In neuronal cells, WPRE and both tau 3'UTR and APP 5'UTR had an additive effect on expression. The combination of the three elements increased the basal level of expression by up to 26-fold. In glial cells, WPRE and APP 5'UTR had additive effects on expression, and their combination increased expression up to 10-fold. These results provide important information regarding the development of optimal CNS gene transfer vectors not only for gene therapy but also for the study of gene function.

Adenoviridae↗
PubMed2003

Expression of PRiMA in the mouse brain: membrane anchoring and accumulation of 'tailed' acetylcholinesterase.

We analysed the expression of PRiMA (proline-rich membrane anchor), the membrane anchor of acetylcholinesterase (AChE), by in situ hybridization in the mouse brain. We compared the pattern of PRiMA transcripts with that of AChE transcripts, as well as those of choline acetyltransferase and M1 muscarinic receptors which are considered pre- and postsynaptic cholinergic markers. We also analysed cholinesterase activity and its molecular forms in several brain structures. The results suggest that PRiMA expression is predominantly or exclusively related to the cholinergic system and that anchoring of cholinesterases to cell membranes by PRiMA represents a limiting factor for production of the AChE tailed splice variant (AChET)-PRiMA complex, which represents the major AChE component in the brain. This enzyme species is mostly associated with cholinergic neurons because the pattern of PRiMA mRNA expression largely coincides with that of ChAT. We also show that, in both mouse and human, PRiMA proteins exist as two alternative splice variants which differ in their cytoplasmic regions.

Acetylcholinesterase↗
PubMed2003

Gene networks inference using dynamic Bayesian networks.

This article deals with the identification of gene regulatory networks from experimental data using a statistical machine learning approach. A stochastic model of gene interactions capable of handling missing variables is proposed. It can be described as a dynamic Bayesian network particularly well suited to tackle the stochastic nature of gene regulation and gene expression measurement. Parameters of the model are learned through a penalized likelihood maximization implemented through an extended version of EM algorithm. Our approach is tested against experimental data relative to the S.O.S. DNA Repair network of the Escherichia coli bacterium. It appears to be able to extract the main regulations between the genes involved in this network. An added missing variable is found to model the main protein of the network. Good prediction abilities on unlearned data are observed. These first results are very promising: they show the power of the learning algorithm and the ability of the model to capture gene interactions.

Algorithms↗
PubMed2002

Long-term fate of human telencephalic progenitor cells grafted into the adult mouse brain: effects of previous amplification in vitro.

We assessed the developmental potential of human telencephalic progenitor cells, with and without previous amplification in vitro, following grafting into the nonlesioned adult mouse CNS. Cell suspensions, shown to contain neuroepithelium-like and neuroblast-like cells, were injected into the subventricular zone (SVZ) and the striatum. These two regions were selected for comparative studies because one, the SVZ, is mitotically active, whereas the other, the striatum, is mitotically inactive. In situ hybridization with a human-specific Alu probe showed that the cells survived for up to 30 weeks in both targets and migrated away from the injection site. Fresh cells continued to proliferate and gave rise to very extended grafts before differentiating into neurons and glia. We further show that, when grown in vitro prior to grafting, human cells acquired new properties: Their proliferation was very limited, and they differentiated more rapidly. This study therefore provides new information about the use of these cells, which are a potential tool for both cellular and gene therapy.

Animals↗
PubMed2002

No major schizophrenia locus detected on chromosome 1q in a large multicenter sample.

Reports of substantial evidence for genetic linkage of schizophrenia to chromosome 1q were evaluated by genotyping 16 DNA markers across 107 centimorgans of this chromosome in a multicenter sample of 779 informative schizophrenia pedigrees. No significant evidence was observed for such linkage, nor for heterogeneity in allele sharing among the eight individual samples. Separate analyses of European-origin families, recessive models of inheritance, and families with larger numbers of affected cases also failed to produce significant evidence for linkage. If schizophrenia susceptibility genes are present on chromosome 1q, their population-wide genetic effects are likely to be small.

Africa↗
PubMed2002

Adenoviral retrograde gene transfer in motoneurons is greatly enhanced by prior intramuscular inoculation with botulinum toxin.

Retrograde axonal transport of recombinant adenoviral vectors has been used successfully to deliver genes to motoneurons in rodents after injection of the vectors into muscles. However, only a small proportion of motoneurons take up and retrogradely transport adenoviral particles, limiting the value of this gene delivery method for the treatment of motor neuron diseases (MNDs). Here we validate a new pharmacological approach for enhancing motoneuronal gene transfer after intramuscular injection of recombinant adenoviruses. We injected botulinum neurotoxin A (BoNT) into muscles of normal C57BL/6 mice and transgenic mice expressing the G93A mutation in the superoxide dismutase 1 gene (SOD1-G93A mutation, a model of amyotrophic lateral sclerosis) several days before inoculation with adenoviruses. Treatment with BoNT significantly enhanced gene transfer to motoneurons innervating the injected muscles. Modifications in motoneuron transduction appear to be a consequence of toxin-induced nerve sprouting at the end plates. These findings have major implications for devising protocols for preclinical and clinical studies using intramuscular injection of retrogradely transported gene vectors.

Adenoviridae↗
PubMed2002

Paradoxical methylation of the tyrosine hydroxylase gene in mouse preimplantation embryos.

The mouse tyrosine hydroxylase (Th) gene is located in an evolutionarily conserved imprinted gene cluster on distal chromosome 7. It is associated with a CpG island that spans the promoter of the gene. Using a bisulfite sequencing method we show that the Th promoter is fully methylated in both male and female mouse germ cells and in human spermatozoa, suggesting that it belongs to the newly identified category of CpG islands, the similarly methylated regions (SMRs). Contrary to other tissue-specific gene sequences, the mouse Th promoter escapes the initial wave of genome demethylation during the first few cell cycles, but becomes demethylated between the morula and the blastocyst stages. This unusual methylation ontogeny may be a characteristic of the SMRs and/or related to the localization of the Th gene in an imprinted gene cluster.

Animals↗
PubMed2002

Long-term expression of beta-glucuronidase by genetically modified human neural progenitor cells grafted into the mouse central nervous system.

Mucopolysaccharidosis type VII (MPS VII) is an inherited disease caused by beta-glucuronidase (beta-glu) deficiency. This deficiency results in the lysosomal accumulation of glycosaminoglycans in all tissues and affects a wide range of organs, including the central nervous system (CNS). Gene transfer is a promising approach to therapy for MPS VII because it allows extensive delivery of the enzyme to the affected tissues. We studied neurotransplantation of primary human cells to supply beta-glucuronidase to the CNS. Human neural progenitor cells (HNPC) were amplified and cotransduced with two lentiviral vectors, one encoding the green fluorescent protein and the other the human beta-glu. We show that these cells strongly expressed both transgenes in culture. When grafted into the mouse striatum, HNPC differentiated into neurons and astrocytes and expressed the two transgenes for at least 6 months. This study therefore paves the way for the treatment of MPS VII by long-term delivery of the appropriate enzyme.

Age Factors↗
PubMed2002

Neuronal transfer of the human Cu/Zn superoxide dismutase gene increases the resistance of dopaminergic neurons to 6-hydroxydopamine.

Several mechanisms are thought to be involved in the progressive decline in neurons of the substantia nigra pars compacta (SNpc) that leads to Parkinson's disease (PD). Neurotoxin 6-hydroxydopamine (6-OHDA), which induces parkinsonian symptoms in experimental animals, is thought to be formed endogenously in patients with PD through dopamine (DA) oxidation and may cause dopaminergic cell death via a free radical mechanism. We therefore investigated protection against 6-OHDA by inhibiting oxidative stress using a gene transfer strategy. We overexpressed the antioxidative Cu/Zn-superoxide dismutase (SOD1) enzyme in primary culture dopaminergic cells by infection with an adenovirus carrying the human SOD1 gene (Ad-hSOD1). Survival of the dopaminergic cells exposed to 6-OHDA was 50% higher among the SOD1-producing cells than the cells infected with control adenoviruses. In contrast, no significant increased survival of (6-OHDA)-treated dopaminergic cells was observed when they were infected with an adenovirus expressing the H(2) O(2) -scavenging glutathione peroxidase (GPx) enzyme. These results underline the major contribution of superoxide in the dopaminergic cell death process induced by 6-OHDA in primary cultures. Overall, this study demonstrates that the survival of the dopaminergic neurons can be highly increased by the adenoviral gene transfer of SOD1. An antioxidant gene transfer strategy using viral vectors expressing SOD1 is therefore potentially beneficial for protecting dopaminergic neurons in PD.

Adenoviridae↗
PubMed2002

Manic-depressive illness: an association study with the inositol polyphosphate 1-phosphatase and serotonin transporter genes.

Association studies with candidate genes may contribute towards the understanding of the etiopathogenesis of bipolar disorder. Candidate genes in bipolar disorders are those related to aminergic neurotransmission, which is the target of the effects of antipsychotics and antidepressants, as well as genes related to signal transduction pathways, reporting the target for the mood-stabilizing effects of lithium. Association with such candidate genes may provide clues towards the understanding of the biological components of bipolar disorder. An association study was performed between the 5' regulatory region of the serotonin transporter gene (5-HTTLPR), the inositol polyphosphate 1-phosphatase gene (INPP1) and bipolar disorder using our sample of proband/parent trios. A total of 101 bipolar probands were considered eligible for the study. Since both parents had to be available, mean age at onset of bipolar disorder in probands was relatively young. However, the mean duration of illness and the number of episodes were consistent with a stable diagnosis. In our trios sample, the transmission disequilibrium test revealed no preferential transmission of alleles of the 5-HTTLPR and INPP1 from heterozygous parents to probands. Therefore, additional family-based data are warranted, possibly with a more complete subdivision of 5-HTTLPR alleles, since short and long alleles have recently been divided into four and six kinds of allelic variant, respectively, with significant ethnic differences in allele and genotype distributions.

Adult↗
PubMed2002

Neurotensin induces tyrosine hydroxylase gene activation through nitric oxide and protein kinase C signaling pathways.

The regulation of tyrosine hydroxylase (TH) represents an effective means to control the level of catecholamines, because TH is the major limiting enzyme of monoamine biosynthesis. The neuropeptide neurotensin (NT) is a neuromodulator of dopaminergic systems, and a direct interaction between NT and TH expression has been demonstrated in vivo and in vitro. In the present work, the molecular mechanisms and signaling pathways responsible for TH gene activation have been explored. In N1E-115 cells, NT agonist induced a TH protein level increase, correlating with a significant increase in TH mRNA abundance. This cellular response was the result of TH promoter activation, via c-fos and Jun D binding at the AP-1 responsive element. Using selective protein kinase C and nitric oxide synthase inhibitors, we demonstrate, by quantitative reverse transcription-polymerase chain reaction, gel shift, and protein assays, that TH gene activation by NT agonist requires both protein kinase C stimulation and nitric oxide production. The two pathways exert distinct roles; whereas nitric oxide synthase inhibitors blocked c-fos expression, protein kinase C inhibitors blocked that of Jun D. The requirement for two distinct and concomitant pathways by NT demonstrates a very fine level of control of specificity on TH gene activation.

Animals↗
PubMed1992

Role of Dopaminergic D2 Receptors in the Regulation of Glutamic Acid Decarboxylase Messenger RNA in the Striatum of the Rat.

Levels of messenger RNA (mRNA) encoding glutamic acid decarboxylase (GAD) and preproenkephalin (PPE) were measured by Northern blot and in situ hybridization analyses in the striatum of the rat, after chronic injections of two neuroleptics, sulpiride and haloperidol. The Northern blot analysis showed that the chronic injection of sulpiride at high doses (80 mg/kg, twice a day, 14 days) increased striatal GAD and PPE mRNA levels by 120% and 78% respectively, when compared to vehicle-injected rats. Haloperidol injections at relatively low doses (1 mg/kg, once a day, 14 days) produced parallel increases in GAD (40%) and PPE (52%) mRNA levels. After in situ hybridization densitometric measurements were performed on autoradiograms from rats treated with sulpiride, haloperidol or vehicle. The distribution of GAD and PPE mRNA signals in control rats was homogeneous along the rostrocaudal extension of the striatum. A similar increase was found along this axis after sulpiride (20%) and haloperidol (30%) treatments. The cellular observation of hybridization signals showed that grain density for GAD mRNA was increased in a majority of striatal cells after both treatments. By contrast, the PPE mRNA hybridization signal only increased in a subpopulation of neurons. The effects of such treatments were also analysed by measuring GAD activity in the striatum and in its output structures, the globus pallidus and the substantia nigra. After the administration of sulpiride, GAD activity was not modified in the striatum but increased in the globus pallidus (by 17%). After haloperidol treatment, GAD activity was increased in the globus pallidus (20%) and the substantia nigra (17%). It is concluded that the interruption of dopaminergic transmission, more precisely the D2 receptor blockade, promotes in striatopallidal neurons an increase in GAD mRNA accompanied by an increase in GAD activity and PPE mRNA. A possible regulation of GAD mRNA and GAD activity in striatonigral neurons is also discussed.

Journal Article↗
PubMed1990

Molecular Cloning of Forebrain mRNAs which are Modulated by Sleep Deprivation.

Sleep deprivation (SD) experiments have suggested that specific endogenous substances mediate the control of sleep and waking. However, such 'sleep substances' have not yet been unambiguously identified. The isolation of specific molecular markers would make it possible to obtain new insights into the regulatory mechanism underlying sleep and waking. For this purpose, we have used a molecular genetical approach based on subtractive cDNA cloning. Using these techniques, we were able to detect and isolate in rat forebrain four cDNA clones whose corresponding transcripts are expressed at a lower level after 24 h of SD, and six cDNA clones whose corresponding transcripts are expressed at a higher level. For two of the former transcripts, the level showed a significant reduction of approximately 50% after 24 h of SD and a non-significant reduction after 12 h of SD. A significant reduction was also observed after 12 h of cold exposure. A regional analysis of their level under baseline conditions revealed variation during the 24-h cycle. The highest levels tended to occur at the onset of darkness, the beginning of the rat's activity period. Our results are compatible with the hypothesis that the cloned transcripts are associated with the regulation of the sleep-waking cycle. Analysis of their primary structure indicated that these mRNAs have not yet been characterized. The in vivo distribution of these transcripts in the forebrain shows some correspondence to that of receptors of excitatory amino acids, suggesting an association between the functional role of the cloned sequences and this neurotransmission system.

Journal Article↗
PubMed1990

Behavioural Effect of Engineered Cells that Synthesize l-dopa or Dopamine after Grafting into the Rat Neostriatum.

Cell lines in which tyrosine hydroxylase was introduced either by infection or transfection were used in grafting experiments in a rat model of Parkinson's disease obtained by unilateral lesion of the substantia nigra. A neuroblastoma NS20 Y cell line which synthesizes only l-dopa and a neuroendocrine AtT-20 cell line which produces dopamine were obtained. They were grafted into denervated striata and their ability to compensate for the dopaminergic deficit was studied. Both modified cell types displayed a rapid partial reversal of apomorphine-induced turning behaviour. No effect was observed with the control unmodified cell lines. We discuss the usefulness of engineered cell lines to address the fundamental issues in grafting experiments and more particularly in the therapy of Parkinson's disease.

Journal Article↗
PubMedDate not supplied

More than one way to toy with ChAT and VAChT.

Expression of choline acetyltransferase (ChAT) and of the vesicular acetylcholine transporter (VAChT) is required for the acquisition and the maintenance of the cholinergic phenotype. The ChAT and VAChT genes have been demonstrated to share a common gene locus and this suggests a coordinate regulation of their expression. In the present work, we examined the effects of several differentiating treatments on the modulation of ChAT and VAChT expression at the mRNA and protein levels in growing and differentiating NG108-15 cells. In cells grown in the presence of serum, all the agents tested-retinoic acid, dexamethasone and dibutyrylcyclicAMP (dbcAMP)-induced an increase of ChAT and VAChT mRNA levels but with different efficacy. Treatment with dbcAMP plus dexamethasone resulted in the largest increase of VAChT mRNA amount while retinoic acid mostly enhanced ChAT mRNA level. However, while ChAT activity was increased by all agents, no change in the VAChT protein level was detected. In cells differentiated by serum deprivation, only retinoic acid was effective in inducing an increase of VAChT and ChAT mRNA and ChAT activity, while we observed a downregulation by dbcAMP and dexamethasone. Treatment with the antimitotic agent cytosine arabinoside led to an increase of ChAT activity which was further largely enhanced by the addition of dbcAMP plus dexamethasone, but to only a slight change in VAChT activity. We further showed that complex glycosylation processes which might play a role in targeting and/or stability of the membrane protein VAChT are deficient in these cells. Indeed, in transient transfection assays with the reporter soluble enzyme luciferase placed under regulatory and promoter regions of the VAChT gene, we observed a modulation of luciferase expression by differentiating agents. These data illustrate the complexity of the processes which participate to the expression of the ChAT and VAChT genes, both at the transcriptional and posttranslational levels.

Antineoplastic Agents↗
PubMedDate not supplied

Post-genomic era and gene discovery for psychiatric diseases: there is a new art of the trade? The example of the HUMTH01 microsatellite in the Tyrosine Hydroxylase gene.

The microsatellite HUMTH01, located in the first intron of the Tyrosine Hydroxylase (TH) gene (encoding the rate-limiting enzyme in the synthesis of catecholamines), is characterized by a TCAT repeated motif and has been used in genetic studies of neuropsychiatric and other complex diseases, in which catecholaminergic neurotransmission is implicated. After reporting a positive association between HUMTH01 and bipolar disorder as well as schizophrenia, the authors established that HUMTH01 alleles display the features of regulatory elements. Thereafter, they cloned two proteins (ZNF191 and HBP1), specifically binding to HUMTH01, and demonstrated that allelic variations of HUMTH01 have a quantitative silencing effect on TH gene expression in vitro, and correlate with quantitative and qualitative changes in the binding by ZNF191. The authors aim to characterize the transduction pathway impinging on the HUMTH01 microsatellite and establish its relevance for TH gene regulation in vivo. Since the TCAT repeated sequence is widespread throughout the genome, their approach may lead to the dissection of the mechanisms underlying the quantitative expression of several genes implicated in complex genetic traits, both normal and pathological. Thus, these investigations on the possible contribution and potential role of the HUMTH01 microsatellite in neuro-pathological conditions may represent an example of the different approaches needed to validate genetic targets in the "post-genomic era."

Animals↗