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Les ribosomes sont des macrocomplexes ribonucléoprotéiques sophistiqués, essentiels pour décoder l’information génétique et la traduire en protéines fonctionnelles. Chez les organismes eucaryotes, le ribosome est constitué de deux sous-unités, la petite (40S) et la grande (60S). Leur biogenèse est un processus fondamental, très complexe, qui mène à la synthèse et l’assemblage de 4 ARNr et 80 protéines ribosomiques (79 chez la levure). La biogenèse du ribosome a longtemps été étudiée chez Saccharomyces cerevisiae. Près de 20 ans de recherches ont été nécessaires à la communauté scientifique pour identifier les quelques 200 facteurs de synthèse du ribosome levurien. Alors que le schéma global de cette voie de biosynthèse semble conservé chez les organismes eucaryotes, de nombreux éléments suggèrent qu’elle serait plus élaborée chez l’homme et nécessiterait un plus grand nombre de facteurs que chez la levure. De plus, la caractérisation de nombreuses ribosomopathies, ou maladies du ribosome prédisposant aux cancers, a suscité un intérêt accru pour l’étude de la voie de biosynthèse du ribosome dans le paradigme expérimental le plus approprié, la cellule humaine.Au cours de ma thèse de doctorat, j’ai contribué à un projet systématique d’identification de facteurs d’assemblage (FA) du ribosome chez l’homme. Pratiquement, nous avons identifié 286 FA humains, dont beaucoup sont homologues aux facteurs levuriens connus, et 74 sont sans équivalent chez la levure. Par ailleurs, j’ai caractérisé en détail certains facteurs. En particulier, Trm112 pour lequel j’ai montré qu’il agit comme un stabilisateur de la méthyltransférase (MTase) Bud23, spécifique à l’ARNr 18S de la sous-unité levurienne 40S. J’ai également participé à la caractérisation de mutations à l’interface du complexe Bud23-Trm112. Enfin, j’ai contribué à l’étude de trois FA que nous avons identifiés chez l’homme, DIMT1L et WBSCR22-TRMT112. J’ai montré que ces protéines sont les orthologues des MTases levurie, Doctorat en sciences, Spécialisation biologie moléculaire, info:eu-repo/semantics/nonPublished

La biogenèse des ribosomes est un processus hautement complexe impliquant plusieurs centaines de facteurs de synthèse dont la finalité est la production de toutes les protéines de la cellule. Chaque étapes de la ribogenèse est une source potentielle d’erreur et est vérifiée par des mécanismes de contrôle de qualité redondants et rigoureux. Dans la première partie de ma thèse, j’ai collaboré à une meilleure compréhension d’une des voies de la surveillance nucléolaire, celle qui dégrade les pré-ribosomes défectueux recrutée à partir de l’extrémité 3’ des ARN ribosomiques (ARNr). Suite à une erreur d’assemblage, les pré-ARNr sont polyadénylés par le complexe nucléaire TRAMP, ce dernier est recruté cotranscriptionnellement. Les ARNr polyadénylés deviennent alors des substrats pour l’exosome et sont dégradés. On ignore comment la synthèse des ARNr, leur maturation et la surveillance nucléolaire sont intégrées mais on suspecte l’existence d’une interface physico-fonctionnelle à l’ADNr. Dans une deuxième partie de ce travail, nous avons testé si des cofacteurs de l’exosome, les protéines Nrd1/Nab3 étaient impliquées dans la surveillance des pré-ARNr. Nous rapportons que, chez la levure S. cerevisiae, le facteur d’élongation Spt5 interagit avec l’ARN polymérase (Pol) I et avec Nrd1. L’interaction entre Spt5 et ces deux protéines requiert la présence d’un domaine particulier situé à l’extrémité C-terminal ressemblant au « Carboxy-terminal domain » (CTD) de la Pol II appelé « Carboxy terminal repeat » (CTR). Spt4/Spt5 et Nrd1/Nab3 interagissent fonctionnellement avec Rrp6, sous-unité catalytique de l’exosome. Ces complexes colocalisent à l’ADNr, comme déterminé par ChIP. Des mutations dans le domaine de liaison à l’ARN (RRM – « RNA recognition motif ») de Nrd1 mais pas dans son domaine de liaison au CTD (CID – « carboxy-terminal interacting domain ») de la Pol II et dans le RRM de Nab3 mènent à l’accumulation de transcrits ribosomiques aberrants polyadénylés. Ceci, Doctorat en Sciences, info:eu-repo/semantics/nonPublished

La biogenèse des ribosomes est un processus hautement complexe impliquant plusieurs centaines de facteurs de synthèse dont la finalité est la production de toutes les protéines de la cellule. Chaque étapes de la ribogenèse est une source potentielle d’erreur et est vérifiée par des mécanismes de contrôle de qualité redondants et rigoureux. Dans la première partie de ma thèse, j’ai collaboré à une meilleure compréhension d’une des voies de la surveillance nucléolaire, celle qui dégrade les pré-ribosomes défectueux recrutée à partir de l’extrémité 3’ des ARN ribosomiques (ARNr). Suite à une erreur d’assemblage, les pré-ARNr sont polyadénylés par le complexe nucléaire TRAMP, ce dernier est recruté cotranscriptionnellement. Les ARNr polyadénylés deviennent alors des substrats pour l’exosome et sont dégradés. On ignore comment la synthèse des ARNr, leur maturation et la surveillance nucléolaire sont intégrées mais on suspecte l’existence d’une interface physico-fonctionnelle à l’ADNr. Dans une deuxième partie de ce travail, nous avons testé si des cofacteurs de l’exosome, les protéines Nrd1/Nab3 étaient impliquées dans la surveillance des pré-ARNr. Nous rapportons que, chez la levure S. cerevisiae, le facteur d’élongation Spt5 interagit avec l’ARN polymérase (Pol) I et avec Nrd1. L’interaction entre Spt5 et ces deux protéines requiert la présence d’un domaine particulier situé à l’extrémité C-terminal ressemblant au « Carboxy-terminal domain » (CTD) de la Pol II appelé « Carboxy terminal repeat » (CTR). Spt4/Spt5 et Nrd1/Nab3 interagissent fonctionnellement avec Rrp6, sous-unité catalytique de l’exosome. Ces complexes colocalisent à l’ADNr, comme déterminé par ChIP. Des mutations dans le domaine de liaison à l’ARN (RRM – « RNA recognition motif ») de Nrd1 mais pas dans son domaine de liaison au CTD (CID – « carboxy-terminal interacting domain ») de la Pol II et dans le RRM de Nab3 mènent à l’accumulation de transcrits ribosomiques aberrants polyadénylés. Ceci, Doctorat en Sciences, info:eu-repo/semantics/nonPublished

The influence of changing composition and content of RNA on the results of expression profiling was studied in the group-synchronous ovaries of roach (Rutilus rutilus) over the course of their maturation. The highest yield of total RNA was detected in the primary growth and early cortical alveolus stages. The total RNA yield gradually decreased through the late cortical alveolus and late vitellogenic stages. In the primary growth and early cortical alveolus stages, total RNA was characterized by a low percentage of 18S and 28S rRNA and a high percentage of smaller-sized RNAs (tRNA, 5S and 5.8S rRNA), whereas 18S and 28S rRNA had increased by the late cortical alveolus stage and dominated by the late vitellogenic stage. The ratio of mRNA to total RNA was highest at the primary growth stage but decreased significantly in later ovarian stages. When total RNA was used for reverse transcription (RT), the shift in the mRNA/total RNA ratio influenced the results of qPCR expression profiling of several commonly used reference genes (ribosomal protein L8, elongation factor-1α, RNA polymerase-subunit B5, and β2-microglobulin) and of two target genes, gonad-type aromatase (cyp19a1a) and follistatin (fst). We conclude that the expression of target genes should be related to the mRNA pool using the same input of either mRNA to RT or cDNA to qPCR. Furthermore, gene expression was related to tissue-specific RNA yield per body mass (RNA yield x ovary mass x body mass⁻¹) thereby reflecting the massive increase in the size and cellular composition of the ovary during the reproductive cycle., info:eu-repo/semantics/published

The final outcome of tube elongation and branching is to maximize the epithelial exchange surfaces in tubular organs. The molecular and cellular basis of these processes is actively studied in model organs such as mammary glands, liver and kidney, but they remain almost unexplored in the male reproductive tract. Here, we report that the orphan G protein-coupled receptor LGR4/GPR48 plays a role in the postnatal tissue remodeling needed for elongation and convolution of the efferent ducts and epididymis. In LGR4 knockout male mice, tube elongation fails, resulting in a hypoplastic and poorly convoluted tract. Cell proliferation is dramatically reduced in KO affected tissues, providing an explanation to the observed phenotype. Detailed analysis showed that LGR4 inactivation manifests differently in the affected organs. In efferent ducts, immune cells infiltrate the epithelium and reach the lumen, blocking the transit of sperm and testicular fluid. In addition, the hypoplasia and low convolution result in a reduction of the epithelial area involved in liquid reabsorption. Both phenomena contribute in tissue swelling upstream the blockade due to liquid and sperm accumulation, with secondary damaging effects on the germinal epithelium. In the epididymis, the thin and highly convoluted duct is replaced by a large cystic tube which is surrounded by a thick condensation of mesenchymal cells. The abnormal organization of the cellular compartments in and around the ducts suggests that LGR4 might play a role in epithelial-mesenchymal interactions. Altogether, our data identify LGR4 as an important signaling molecule implicated in the tube morphogenesis of the male reproductive tract., (Note: * F. Mendive and P. Laurent contributed equally to this study.), info:eu-repo/semantics/published

TIA-1 and TIAR are closely related RNA-binding proteins involved in several mechanisms of RNA metabolism, including alternative hnRNA splicing and mRNA translation regulation. In particular, TIA-1 represses tumor necrosis factor (TNF) mRNA translation by binding to the AU-rich element (ARE) present in the mRNA 3' untranslated region. Here, we demonstrate that TIA proteins interact with FUSE-binding proteins (FBPs) and that fbp genes are co-expressed with tia genes during Xenopus embryogenesis. FBPs participate in various steps of RNA processing and degradation. In Cos cells, FBPs co-localize with TIA proteins in the nucleus and migrate into TIA-enriched cytoplasmic granules upon oxidative stress. Overexpression of FBP2-KH3 RNA-binding domain fused to EGFP induces the specific sequestration of TIA proteins in cytoplasmic foci, thereby precluding their nuclear accumulation. In cytosolic RAW 264.7 macrophage extracts, FBPs are found associated in EMSA to the TIA-1/TNF-ARE complex. Together, our results indicate that TIA and FBP proteins may thus be relevant biological involved in common events of RNA metabolism occurring both in the nucleus and the cytoplasm., Journal Article, Research Support, Non-U.S. Gov't, SCOPUS: ar.j, info:eu-repo/semantics/published

TIA-1 and TIAR are closely related RNA-binding proteins involved in several mechanisms of RNA metabolism, including alternative hnRNA splicing and mRNA translation regulation. In particular, TIA-1 represses tumor necrosis factor (TNF) mRNA translation by binding to the AU-rich element (ARE) present in the mRNA 3' untranslated region. Here, we demonstrate that TIA proteins interact with FUSE-binding proteins (FBPs) and that fbp genes are co-expressed with tia genes during Xenopus embryogenesis. FBPs participate in various steps of RNA processing and degradation. In Cos cells, FBPs co-localize with TIA proteins in the nucleus and migrate into TIA-enriched cytoplasmic granules upon oxidative stress. Overexpression of FBP2-KH3 RNA-binding domain fused to EGFP induces the specific sequestration of TIA proteins in cytoplasmic foci, thereby precluding their nuclear accumulation. In cytosolic RAW 264.7 macrophage extracts, FBPs are found associated in EMSA to the TIA-1/TNF-ARE complex. Together, our results indicate that TIA and FBP proteins may thus be relevant biological involved in common events of RNA metabolism occurring both in the nucleus and the cytoplasm., Journal Article, Research Support, Non-U.S. Gov't, SCOPUS: ar.j, info:eu-repo/semantics/published

The final outcome of tube elongation and branching is to maximize the epithelial exchange surfaces in tubular organs. The molecular and cellular basis of these processes is actively studied in model organs such as mammary glands, liver and kidney, but they remain almost unexplored in the male reproductive tract. Here, we report that the orphan G protein-coupled receptor LGR4/GPR48 plays a role in the postnatal tissue remodeling needed for elongation and convolution of the efferent ducts and epididymis. In LGR4 knockout male mice, tube elongation fails, resulting in a hypoplastic and poorly convoluted tract. Cell proliferation is dramatically reduced in KO affected tissues, providing an explanation to the observed phenotype. Detailed analysis showed that LGR4 inactivation manifests differently in the affected organs. In efferent ducts, immune cells infiltrate the epithelium and reach the lumen, blocking the transit of sperm and testicular fluid. In addition, the hypoplasia and low convolution result in a reduction of the epithelial area involved in liquid reabsorption. Both phenomena contribute in tissue swelling upstream the blockade due to liquid and sperm accumulation, with secondary damaging effects on the germinal epithelium. In the epididymis, the thin and highly convoluted duct is replaced by a large cystic tube which is surrounded by a thick condensation of mesenchymal cells. The abnormal organization of the cellular compartments in and around the ducts suggests that LGR4 might play a role in epithelial-mesenchymal interactions. Altogether, our data identify LGR4 as an important signaling molecule implicated in the tube morphogenesis of the male reproductive tract., (Note: * F. Mendive and P. Laurent contributed equally to this study.), info:eu-repo/semantics/published

We designed a low-density microarray carrying 132 DNA capture sequences highly specific for genes known to be differentially expressed among breast tumors and BCC lines or associated with specific tumor properties (cell-cycle alteration, proteolysis, adhesion, hormone sensitivity, etc). We analyzed gene expression in 11 BCC lines among which 6 had already been extensively studied (BT-474, Hs578T, MCF-7, MDA-MB-231, MDA-MB-453, T-47D) and 5 were still poorly characterized (Evsa-T, IBEP-1, IBEP-2, IBEP-3, KPL-1). Some data obtained were verified or extended by real-time polymerase chain reaction (real-time PCR), Northern blotting, Western blotting, immunohistochemistry and cell growth studies. Clustering analysis of the low-density microarray data allowed the sorting of BCC lines into two classes and supported a major discriminatory role for ER alpha, confirming data from previous studies. A few genes that are highly and specifically expressed in one cell line were identified, such as MGB1 (mammaglobin 1) in Evsa-T cells, and PIP (prolactin-inducible protein) in MDA-MB-453 BCC, suggesting an apocrine origin for these latter cells. Two BCC lines (IBEP-1 and IBEP-3) that had been previously characterized as ER alpha-negative, were classified by the low-density microarray among ER alpha-positive lines (MCF-7, T-47D, IBEP-2, BT-474, KPL-1) and were indeed confirmed as receptor-positive (at both mRNA and protein levels) and hormone-responsive cells. In conclusion, our results support the utility of a low-density microarray approach in cases where the cost and exhaustiveness of high-density microarrays may constitute a drawback; for instance, in obtaining a rapid phenotype evaluation in cell populations freshly isolated from breast tumors., Journal Article, Research Support, Non-U.S. Gov't, SCOPUS: ar.j, info:eu-repo/semantics/published

p66shc-/- mice exhibit prolonged lifespan and increased resistance to oxidative and hypoxic stress. To investigate p66shc involvement in human longevity, p66shc mRNA and protein were evaluated in fibroblasts from young people, elderly and centenarians, exposed to oxidative or hypoxic stress. Unexpectedly, centenarians showed the highest basal levels of p66shc. Oxidative stress induced p66shc in all samples. At variance, hypoxic stress caused p66shc reduction only in cells from centenarians. These changes occurred in absence of any modification of p66shc promoter methylation pattern. Intriguingly, in cells from centenarians, p66shc induction was affected by p53 codon 72 polymorphism. Thus, cells from centenarians present a peculiar regulation of p66shc, suggesting that its role in mammalian longevity is more complex than previously thought. © 2005 Elsevier Ireland Ltd. All rights reserved., SCOPUS: ar.j, info:eu-repo/semantics/published

We designed a low-density microarray carrying 132 DNA capture sequences highly specific for genes known to be differentially expressed among breast tumors and BCC lines or associated with specific tumor properties (cell-cycle alteration, proteolysis, adhesion, hormone sensitivity, etc). We analyzed gene expression in 11 BCC lines among which 6 had already been extensively studied (BT-474, Hs578T, MCF-7, MDA-MB-231, MDA-MB-453, T-47D) and 5 were still poorly characterized (Evsa-T, IBEP-1, IBEP-2, IBEP-3, KPL-1). Some data obtained were verified or extended by real-time polymerase chain reaction (real-time PCR), Northern blotting, Western blotting, immunohistochemistry and cell growth studies. Clustering analysis of the low-density microarray data allowed the sorting of BCC lines into two classes and supported a major discriminatory role for ER alpha, confirming data from previous studies. A few genes that are highly and specifically expressed in one cell line were identified, such as MGB1 (mammaglobin 1) in Evsa-T cells, and PIP (prolactin-inducible protein) in MDA-MB-453 BCC, suggesting an apocrine origin for these latter cells. Two BCC lines (IBEP-1 and IBEP-3) that had been previously characterized as ER alpha-negative, were classified by the low-density microarray among ER alpha-positive lines (MCF-7, T-47D, IBEP-2, BT-474, KPL-1) and were indeed confirmed as receptor-positive (at both mRNA and protein levels) and hormone-responsive cells. In conclusion, our results support the utility of a low-density microarray approach in cases where the cost and exhaustiveness of high-density microarrays may constitute a drawback; for instance, in obtaining a rapid phenotype evaluation in cell populations freshly isolated from breast tumors., Journal Article, Research Support, Non-U.S. Gov't, SCOPUS: ar.j, info:eu-repo/semantics/published

In Catharanthus roseus cell suspensions, the expression of several terpenoid indole alkaloid biosynthetic genes, including two genes encoding strictosidine synthase (STR) and tryptophan decarboxylase (TDC), is coordinately induced by fungal elicitors such as yeast extract. To identify molecular mechanisms regulating the expression of these genes, a yeast one-hybrid screening was performed with an elicitor-responsive part of the TDC promoter. This screening identified three members of the Cys(2)/His(2)-type (transcription factor IIIA-type) zinc finger protein family from C. roseus, ZCT1, ZCT2, and ZCT3. These proteins bind in a sequence-specific manner to the TDC and STR promoters in vitro and repress the activity of these promoters in trans-activation assays. In addition, the ZCT proteins can repress the activating activity of APETALA2/ethylene response-factor domain transcription factors, the ORCAs, on the STR promoter. The expression of the ZCT genes is rapidly induced by yeast extract and methyljasmonate. These results suggest that the ZCT proteins act as repressors in the regulation of elicitor-induced secondary metabolism in C. roseus., Journal Article, Research Support, Non-U.S. Gov't, info:eu-repo/semantics/published

We produced and analyzed mice deficient for Na/Ca exchanger 3 (NCX3), a protein that mediates cellular Ca(2+) efflux (forward mode) or Ca(2+) influx (reverse mode) and thus controls intracellular Ca(2+) concentration. NCX3-deficient mice (Ncx3(-/-)) present a skeletal muscle fiber necrosis and a defective neuromuscular transmission, reflecting the absence of NCX3 in the sarcolemma of the muscle fibers and at the neuromuscular junction. The defective neuromuscular transmission is characterized by the presence of electromyographic abnormalities, including low compound muscle action potential amplitude, a decremental response at low-frequency nerve stimulation, an incremental response, and a prominent postexercise facilitation at high-frequency nerve stimulation, as well as neuromuscular blocks. The analysis of quantal transmitter release in Ncx3(-/-) neuromuscular junctions revealed an important facilitation superimposed on the depression of synaptic responses and an elevated delayed release during high-frequency nerve stimulation. It is suggested that Ca(2+) entering nerve terminals is cleared relatively slowly in the absence of NCX3, thereby enhancing residual Ca(2+) and evoked and delayed quantal transmitter release during repetitive nerve stimulation. Our findings indicate that NCX3 plays an important role in vivo in the control of Ca(2+) concentrations in the skeletal muscle fibers and at the neuromuscular junction., Journal Article, Research Support, Non-U.S. Gov't, SCOPUS: ar.j, info:eu-repo/semantics/published

In Catharanthus roseus cell suspensions, the expression of several terpenoid indole alkaloid biosynthetic genes, including two genes encoding strictosidine synthase (STR) and tryptophan decarboxylase (TDC), is coordinately induced by fungal elicitors such as yeast extract. To identify molecular mechanisms regulating the expression of these genes, a yeast one-hybrid screening was performed with an elicitor-responsive part of the TDC promoter. This screening identified three members of the Cys(2)/His(2)-type (transcription factor IIIA-type) zinc finger protein family from C. roseus, ZCT1, ZCT2, and ZCT3. These proteins bind in a sequence-specific manner to the TDC and STR promoters in vitro and repress the activity of these promoters in trans-activation assays. In addition, the ZCT proteins can repress the activating activity of APETALA2/ethylene response-factor domain transcription factors, the ORCAs, on the STR promoter. The expression of the ZCT genes is rapidly induced by yeast extract and methyljasmonate. These results suggest that the ZCT proteins act as repressors in the regulation of elicitor-induced secondary metabolism in C. roseus., Journal Article, Research Support, Non-U.S. Gov't, info:eu-repo/semantics/published

We produced and analyzed mice deficient for Na/Ca exchanger 3 (NCX3), a protein that mediates cellular Ca(2+) efflux (forward mode) or Ca(2+) influx (reverse mode) and thus controls intracellular Ca(2+) concentration. NCX3-deficient mice (Ncx3(-/-)) present a skeletal muscle fiber necrosis and a defective neuromuscular transmission, reflecting the absence of NCX3 in the sarcolemma of the muscle fibers and at the neuromuscular junction. The defective neuromuscular transmission is characterized by the presence of electromyographic abnormalities, including low compound muscle action potential amplitude, a decremental response at low-frequency nerve stimulation, an incremental response, and a prominent postexercise facilitation at high-frequency nerve stimulation, as well as neuromuscular blocks. The analysis of quantal transmitter release in Ncx3(-/-) neuromuscular junctions revealed an important facilitation superimposed on the depression of synaptic responses and an elevated delayed release during high-frequency nerve stimulation. It is suggested that Ca(2+) entering nerve terminals is cleared relatively slowly in the absence of NCX3, thereby enhancing residual Ca(2+) and evoked and delayed quantal transmitter release during repetitive nerve stimulation. Our findings indicate that NCX3 plays an important role in vivo in the control of Ca(2+) concentrations in the skeletal muscle fibers and at the neuromuscular junction., Journal Article, Research Support, Non-U.S. Gov't, SCOPUS: ar.j, info:eu-repo/semantics/published

Hairy-related transcription factor (HRT/Hey) genes encode a novel subfamily of basic helix-loop-helix (bHLH) transcription factors related to the Drosophila hairy and Enhancer-of-split (E(spl)) and the mammalian HES proteins that function as downstream mediators of Notch signaling. Using the yeast two-hybrid approach, a previously uncharacterized protein was identified in Xenopus that interacts with XHRT1 (originally referred to as bc8), one member of the HRT/Hey subclass. This protein is evolutionarily conserved in chordates. It binds to sequences adjacent to the bHLH domain of XHRT1 known as the Orange domain and has been named bc8 Orange interacting protein (BOIP). BOIP shows a rather uniform subcellular localization and is recruited to the nucleus upon binding to XHRT1. In Xenopus, XBOIP mRNA is detected by RNase protection analysis throughout embryogenesis. In the adult, the strongest expression is detected in testis. In the mouse, high levels of BOIP mRNA are also found in adult testis. No expression is detected in the embryo and in any of the other adult organs tested. In situ hybridization revealed that BOIP transcripts were detected almost exclusively in round spermatids and that this expression overlaps with that of Hey1 (HRT1), which is expressed throughout spermatogenesis. In view of the importance of the Orange domain for HRT/Hey function, the newly identified BOIP proteins may serve as regulators specifically of HRT1/Hey1 activity., Journal Article, Research Support, Non-U.S. Gov't, SCOPUS: ar.j, FLWIN, info:eu-repo/semantics/published

Hairy-related transcription factor (HRT/Hey) genes encode a novel subfamily of basic helix-loop-helix (bHLH) transcription factors related to the Drosophila hairy and Enhancer-of-split (E(spl)) and the mammalian HES proteins that function as downstream mediators of Notch signaling. Using the yeast two-hybrid approach, a previously uncharacterized protein was identified in Xenopus that interacts with XHRT1 (originally referred to as bc8), one member of the HRT/Hey subclass. This protein is evolutionarily conserved in chordates. It binds to sequences adjacent to the bHLH domain of XHRT1 known as the Orange domain and has been named bc8 Orange interacting protein (BOIP). BOIP shows a rather uniform subcellular localization and is recruited to the nucleus upon binding to XHRT1. In Xenopus, XBOIP mRNA is detected by RNase protection analysis throughout embryogenesis. In the adult, the strongest expression is detected in testis. In the mouse, high levels of BOIP mRNA are also found in adult testis. No expression is detected in the embryo and in any of the other adult organs tested. In situ hybridization revealed that BOIP transcripts were detected almost exclusively in round spermatids and that this expression overlaps with that of Hey1 (HRT1), which is expressed throughout spermatogenesis. In view of the importance of the Orange domain for HRT/Hey function, the newly identified BOIP proteins may serve as regulators specifically of HRT1/Hey1 activity., Journal Article, Research Support, Non-U.S. Gov't, SCOPUS: ar.j, FLWIN, info:eu-repo/semantics/published

Iron-mediated oxidative stress has been implicated in the pathology of the neurodegenerative disease Friedreich ataxia (FRDA). Here, we show that normal upregulation of the stress defense protein manganese superoxide dismutase (MnSOD) fails to occur in FRDA fibroblasts exposed to iron. This impaired induction was observed at iron levels in which increased activation of the redox-sensitive factor NF-kappaB was absent. Furthermore, MnSOD induction could only be partially suppressed by antioxidants. We conclude that an NF-kappaB-independent pathway that may not require free radical signaling is responsible for the reduction of MnSOD induction. This impairment could constitute both a novel defense mechanism against iron-mediated oxidative stress in cells with mitochondrial iron overload and conversely, an alternative source of free radicals that could contribute to the disease pathology., Journal Article, Research Support, Non-U.S. Gov't, Research Support, U.S. Gov't, P.H.S., info:eu-repo/semantics/published

Iron-mediated oxidative stress has been implicated in the pathology of the neurodegenerative disease Friedreich ataxia (FRDA). Here, we show that normal upregulation of the stress defense protein manganese superoxide dismutase (MnSOD) fails to occur in FRDA fibroblasts exposed to iron. This impaired induction was observed at iron levels in which increased activation of the redox-sensitive factor NF-kappaB was absent. Furthermore, MnSOD induction could only be partially suppressed by antioxidants. We conclude that an NF-kappaB-independent pathway that may not require free radical signaling is responsible for the reduction of MnSOD induction. This impairment could constitute both a novel defense mechanism against iron-mediated oxidative stress in cells with mitochondrial iron overload and conversely, an alternative source of free radicals that could contribute to the disease pathology., Journal Article, Research Support, Non-U.S. Gov't, Research Support, U.S. Gov't, P.H.S., info:eu-repo/semantics/published

Although the TSH receptor and Galpha(s), which activate the cAMP cascade in the thyroid gland have been much studied, nothing is known about the adenylyl cyclase (AC) isoforms which are actually involved in this pathway. To characterize the cAMP generation in the dog and human thyroid gland, resulting from the presence of distinct adenylyl cyclase families, the responses to various agents (Ca2+, calmodulin (CaM), phorbol esters (TPA) and thapsigargin (Tg)) were studied. These experiments suggest a role of at least two families of cyclases: cyclases negatively modulated by Ca2+ (ACV or ACVI) and cyclases positively modulated by PKC (ACII, ACIII or ACVII). To further analyze by other experimental procedures the expression pattern of the cyclase isoforms in the thyroid gland, Northern blotting, Western blotting and RT-PCR experiments were performed. The results clearly suggest that in both species, three different adenylyl cyclases ACIII, ACVI and ACIX are mainly expressed in thyrocytes., Journal Article, Research Support, Non-U.S. Gov't, info:eu-repo/semantics/published

Although the TSH receptor and Galpha(s), which activate the cAMP cascade in the thyroid gland have been much studied, nothing is known about the adenylyl cyclase (AC) isoforms which are actually involved in this pathway. To characterize the cAMP generation in the dog and human thyroid gland, resulting from the presence of distinct adenylyl cyclase families, the responses to various agents (Ca2+, calmodulin (CaM), phorbol esters (TPA) and thapsigargin (Tg)) were studied. These experiments suggest a role of at least two families of cyclases: cyclases negatively modulated by Ca2+ (ACV or ACVI) and cyclases positively modulated by PKC (ACII, ACIII or ACVII). To further analyze by other experimental procedures the expression pattern of the cyclase isoforms in the thyroid gland, Northern blotting, Western blotting and RT-PCR experiments were performed. The results clearly suggest that in both species, three different adenylyl cyclases ACIII, ACVI and ACIX are mainly expressed in thyrocytes., Journal Article, Research Support, Non-U.S. Gov't, info:eu-repo/semantics/published

We recently synthesized a novel cationic amphiphile (N-t-butyl-N'-tetradecyl-3-tetradecylaminopropionamidine or Vectamidine (previously described as diC14-amidine)) that associates with DNA and RNA and facilitates their entry and expression into eukaryotic cells. Among several parameters that have been shown to influence the transfection process, the surface charge density plays a key role. Quantitative information about that charge density associated to the cationic amphiphiles organized in liposomal structure is not yet available. We provide here evidence by titration and microelectrophoresis measurements that an evaluation of the intrinsic acidity constants, the surface pH and the counterion binding constants allows to determine the charge density at physiological pH of Vectamidine liposomes. The knowledge of this superficial charge is a prerequisite to a molecular understanding of the DNA-cationic amphiphile complex formation. The method described could be extended to any kind of cationic amphiphile., Journal Article, info:eu-repo/semantics/published

We report the characterization of a Trypanosoma brucei 75-kDa protein of the RGG (Arg-Gly-Gly) type, termed TBRGG1. Dicistronic and monocistronic transcripts of the TBRGG1 gene were produced by both alternative splicing and polyadenylation. TBRGG1 was found in two or three forms that differ in their electrophoretic mobility on SDS-polyacrylamide gel electrophoresis gels, one of which was more abundant in the procyclic form of the parasite. TBRGG1 was localized to the mitochondrion and appeared to be more abundant in bloodstream intermediate and stumpy forms in which the mitochondrion reactivates and during the procyclic stage, which possesses a fully functional mitochondrion. This protein was characterized to display oligo(U) binding characteristics and was found to co-localize with an in vitro RNA editing activity in a sedimentation analysis. TBRGG1 most likely corresponds to the 83-kDa oligo(U)-binding protein previously identified by UV cross-linking of guide RNA to mitochondrial lysates (Leegwater, P. Speijer, D. and Benne, R. (1995) Eur. J. Biochem. 227, 780-786)., Journal Article, Research Support, Non-U.S. Gov't, info:eu-repo/semantics/published

We recently synthesized a novel cationic amphiphile (N-t-butyl-N'-tetradecyl-3-tetradecylaminopropionamidine or Vectamidine (previously described as diC14-amidine)) that associates with DNA and RNA and facilitates their entry and expression into eukaryotic cells. Among several parameters that have been shown to influence the transfection process, the surface charge density plays a key role. Quantitative information about that charge density associated to the cationic amphiphiles organized in liposomal structure is not yet available. We provide here evidence by titration and microelectrophoresis measurements that an evaluation of the intrinsic acidity constants, the surface pH and the counterion binding constants allows to determine the charge density at physiological pH of Vectamidine liposomes. The knowledge of this superficial charge is a prerequisite to a molecular understanding of the DNA-cationic amphiphile complex formation. The method described could be extended to any kind of cationic amphiphile., Journal Article, info:eu-repo/semantics/published

We report the characterization of a Trypanosoma brucei 75-kDa protein of the RGG (Arg-Gly-Gly) type, termed TBRGG1. Dicistronic and monocistronic transcripts of the TBRGG1 gene were produced by both alternative splicing and polyadenylation. TBRGG1 was found in two or three forms that differ in their electrophoretic mobility on SDS-polyacrylamide gel electrophoresis gels, one of which was more abundant in the procyclic form of the parasite. TBRGG1 was localized to the mitochondrion and appeared to be more abundant in bloodstream intermediate and stumpy forms in which the mitochondrion reactivates and during the procyclic stage, which possesses a fully functional mitochondrion. This protein was characterized to display oligo(U) binding characteristics and was found to co-localize with an in vitro RNA editing activity in a sedimentation analysis. TBRGG1 most likely corresponds to the 83-kDa oligo(U)-binding protein previously identified by UV cross-linking of guide RNA to mitochondrial lysates (Leegwater, P. Speijer, D. and Benne, R. (1995) Eur. J. Biochem. 227, 780-786)., Journal Article, Research Support, Non-U.S. Gov't, info:eu-repo/semantics/published

Complexity is one of the hallmarks that applies to C2H2 type zinc finger proteins (ZFPs). Structurally distinct clusters of zinc finger modules define an extremely large superfamily of nucleic acid binding proteins with several hundred, perhaps thousands of different members in vertebrates. Recent discoveries have provided new insights into the biochemistry of RNA and DNA recognition, into ZFP evolution and genomic organization, and also into basic aspects of their biological function. However, as much as we have learned, other fundamental questions about ZFP function remain highly enigmatic. This essay is meant to define what we personally feel are important questions, rather than trying to provide a comprehensive, encyclopaedic review., Journal Article, Research Support, Non-U.S. Gov't, Review, info:eu-repo/semantics/published

Complexity is one of the hallmarks that applies to C2H2 type zinc finger proteins (ZFPs). Structurally distinct clusters of zinc finger modules define an extremely large superfamily of nucleic acid binding proteins with several hundred, perhaps thousands of different members in vertebrates. Recent discoveries have provided new insights into the biochemistry of RNA and DNA recognition, into ZFP evolution and genomic organization, and also into basic aspects of their biological function. However, as much as we have learned, other fundamental questions about ZFP function remain highly enigmatic. This essay is meant to define what we personally feel are important questions, rather than trying to provide a comprehensive, encyclopaedic review., Journal Article, Research Support, Non-U.S. Gov't, Review, info:eu-repo/semantics/published

Male rats underwent either portacaval shunt or portacaval transposition; in both cases, sham-operated pair-fed rats served as controls. Three weeks after a portacaval shunt, fasting serum values of glucose (-35%) and cholesterol (-24%) were lower, and fasting plasma glucagon was higher (+65%). The wet weight of the liver and its total content in DNA, RNA, and protein decreased by 43, 40, 43, and 48%, respectively. The supernatant of liver obtained after centrifugation at 700 g incorporated less [1-14C]acetate (-56%) into fatty acids and less [1-14C]acetate (-94%) and [2-14C]mevalonate (-37%) into cholesterol. The activity of acetyl CoA carboxylase was reduced by 56%. The in vivo incorporation of [3H]H2O into liver fatty acids was 83% lower and that into liver cholesterol was 39% lower than in pair-fed controls. Several of the preceding parameters, including in vitro and in vivo labeling of hepatic fatty acids and cholesterol, were found to be mostly normal in rats with portacaval transposition. These data suggest that the reduction of fatty acid and cholesterol biosynthesis in the liver of rats with portacaval shunt was due to the reduction of total hepatic blood flow rather than to the diversion of portal blood constituents., Journal Article, SCOPUS: NotDefined.j, SCOPUS: ar.j, info:eu-repo/semantics/published

Journal Article, Research Support, Non-U.S. Gov't, SCOPUS: ar.j, info:eu-repo/semantics/published

Activation of unfertilized Chaetopterus eggs by treatment with an excess of KCl may lead to the production of unicellular ciliated larvae (Lillie's differentiation without cleavage). The effects of a number of inhibitors of protein (puromycin, cycloheximide, emetin), RNA (actinomycin D), and DNA (hydroxyurea) synthesis of differentiation without cleavage and on normal development have been studied in Chaetopterus. Incorporation of radioactive leucine, uridine, and thymine has been followed by biochemical methods and by autoradiography. The DNA content of the large polyploid nucleus has been estimated by cytophotometry. The initial pseudocleavage period of differentiation without cleavage is characterized by a burst in DNA and protein synthesis; the inhibitors have little or no effect on this burst and on pseudocleavage itself. Protein and DNA synthesis levels off during the following phase (segregation), but the inhibitors become more effective. RNA synthesis is almost linear for 20 h. These results are compared with those obtained on eggs from other species., Journal Article, SCOPUS: ar.j, info:eu-repo/semantics/published

Historical Article, Journal Article, SCOPUS: NotDefined.j, info:eu-repo/semantics/published

The murine sarcoma virus 3611 contains the transforming v-raf gene that has partial nucleotide homology with the src family of tyrosine kinase-encoding oncogenes. Although this virus induces fibrosarcomas in mice, a recombinant murine retrovirus carrying both the raf and myc oncogenes induces immunoblastic lymphomas and immortalizes mouse macrophages in vitro. The present study has thus monitored the expression of c-raf in human hematopoietic cells. The results demonstrate the presence of a 3.6-kb c-raf transcript in HL-60 promyelocytic leukemic cells. The induction of HL-60 cell differentiation along the monocytic or granulocytic lineages had no detectable effect on the level of c-raf transcripts. Furthermore, in contrast to c-myc and c-fms expression, inhibition of protein synthesis with cycloheximide had no detectable effect on c-raf expression. Similar levels of c-rafRNA were also found in other human cell lines derived from myeloid, B cell, and T cell tumors, as well as in normal granulocytes, monocytes, and macrophages. These findings suggest that the c-raf protooncogene is widely expressed in multiple hematopoietic lineages., Journal Article, Research Support, Non-U.S. Gov't, Research Support, U.S. Gov't, P.H.S., info:eu-repo/semantics/published

Phorbol esters induce the human HL-60 promyelocytic cell line to differentiate along a monocytic pathway. This induction of differentiation may involve phorbol ester-induced activation of the phospholipid- and calcium-dependent protein kinase C. Bryostatin 1, a macrocyclic lactone, has been shown to compete with phorbol esters for binding to protein kinase C. We have confirmed that bryostatin 1 translocates activity of protein kinase C from the cytosolic to membrane fractions of HL-60 cells. The present results also demonstrate that bryostatin 1 (10 nmol/L) induces monocytic differentiation of HL-60 cells as determined by adherence, growth inhibition, appearance of monocyte cell surface antigens, and alpha-naphthyl acetate esterase staining. Furthermore, bryostatin 1 (10 nmol/L) downregulated c-myc expression and induced c-fos, c-fms, and tumor necrosis factor transcripts. These changes in gene expression induced by bryostatin 1 are similar to those associated with phorbol ester-induced monocytic differentiation of HL-60 cells. In contrast, exposure to a higher concentration of bryostatin 1 (100 nmol/L) had less of an effect on growth inhibition of HL-60 cells and changes in gene expression. Moreover, 100 nmol/L bryostatin 1 antagonized the cytostatic effects and adherence induced by phorbol esters. Our results thus suggest that bryostatin 1 activates HL-60 cell protein kinase C and that this effect is associated with induction of monocytic differentiation., Journal Article, Research Support, Non-U.S. Gov't, Research Support, U.S. Gov't, P.H.S., info:eu-repo/semantics/published

Activation of unfertilized Chaetopterus eggs by treatment with an excess of KCl may lead to the production of unicellular ciliated larvae (Lillie's differentiation without cleavage). The effects of a number of inhibitors of protein (puromycin, cycloheximide, emetin), RNA (actinomycin D), and DNA (hydroxyurea) synthesis of differentiation without cleavage and on normal development have been studied in Chaetopterus. Incorporation of radioactive leucine, uridine, and thymine has been followed by biochemical methods and by autoradiography. The DNA content of the large polyploid nucleus has been estimated by cytophotometry. The initial pseudocleavage period of differentiation without cleavage is characterized by a burst in DNA and protein synthesis; the inhibitors have little or no effect on this burst and on pseudocleavage itself. Protein and DNA synthesis levels off during the following phase (segregation), but the inhibitors become more effective. RNA synthesis is almost linear for 20 h. These results are compared with those obtained on eggs from other species., Journal Article, SCOPUS: ar.j, info:eu-repo/semantics/published

Historical Article, Journal Article, SCOPUS: NotDefined.j, info:eu-repo/semantics/published

Male rats underwent either portacaval shunt or portacaval transposition; in both cases, sham-operated pair-fed rats served as controls. Three weeks after a portacaval shunt, fasting serum values of glucose (-35%) and cholesterol (-24%) were lower, and fasting plasma glucagon was higher (+65%). The wet weight of the liver and its total content in DNA, RNA, and protein decreased by 43, 40, 43, and 48%, respectively. The supernatant of liver obtained after centrifugation at 700 g incorporated less [1-14C]acetate (-56%) into fatty acids and less [1-14C]acetate (-94%) and [2-14C]mevalonate (-37%) into cholesterol. The activity of acetyl CoA carboxylase was reduced by 56%. The in vivo incorporation of [3H]H2O into liver fatty acids was 83% lower and that into liver cholesterol was 39% lower than in pair-fed controls. Several of the preceding parameters, including in vitro and in vivo labeling of hepatic fatty acids and cholesterol, were found to be mostly normal in rats with portacaval transposition. These data suggest that the reduction of fatty acid and cholesterol biosynthesis in the liver of rats with portacaval shunt was due to the reduction of total hepatic blood flow rather than to the diversion of portal blood constituents., Journal Article, SCOPUS: NotDefined.j, SCOPUS: ar.j, info:eu-repo/semantics/published

The murine sarcoma virus 3611 contains the transforming v-raf gene that has partial nucleotide homology with the src family of tyrosine kinase-encoding oncogenes. Although this virus induces fibrosarcomas in mice, a recombinant murine retrovirus carrying both the raf and myc oncogenes induces immunoblastic lymphomas and immortalizes mouse macrophages in vitro. The present study has thus monitored the expression of c-raf in human hematopoietic cells. The results demonstrate the presence of a 3.6-kb c-raf transcript in HL-60 promyelocytic leukemic cells. The induction of HL-60 cell differentiation along the monocytic or granulocytic lineages had no detectable effect on the level of c-raf transcripts. Furthermore, in contrast to c-myc and c-fms expression, inhibition of protein synthesis with cycloheximide had no detectable effect on c-raf expression. Similar levels of c-rafRNA were also found in other human cell lines derived from myeloid, B cell, and T cell tumors, as well as in normal granulocytes, monocytes, and macrophages. These findings suggest that the c-raf protooncogene is widely expressed in multiple hematopoietic lineages., Journal Article, Research Support, Non-U.S. Gov't, Research Support, U.S. Gov't, P.H.S., info:eu-repo/semantics/published

Phorbol esters induce the human HL-60 promyelocytic cell line to differentiate along a monocytic pathway. This induction of differentiation may involve phorbol ester-induced activation of the phospholipid- and calcium-dependent protein kinase C. Bryostatin 1, a macrocyclic lactone, has been shown to compete with phorbol esters for binding to protein kinase C. We have confirmed that bryostatin 1 translocates activity of protein kinase C from the cytosolic to membrane fractions of HL-60 cells. The present results also demonstrate that bryostatin 1 (10 nmol/L) induces monocytic differentiation of HL-60 cells as determined by adherence, growth inhibition, appearance of monocyte cell surface antigens, and alpha-naphthyl acetate esterase staining. Furthermore, bryostatin 1 (10 nmol/L) downregulated c-myc expression and induced c-fos, c-fms, and tumor necrosis factor transcripts. These changes in gene expression induced by bryostatin 1 are similar to those associated with phorbol ester-induced monocytic differentiation of HL-60 cells. In contrast, exposure to a higher concentration of bryostatin 1 (100 nmol/L) had less of an effect on growth inhibition of HL-60 cells and changes in gene expression. Moreover, 100 nmol/L bryostatin 1 antagonized the cytostatic effects and adherence induced by phorbol esters. Our results thus suggest that bryostatin 1 activates HL-60 cell protein kinase C and that this effect is associated with induction of monocytic differentiation., Journal Article, Research Support, Non-U.S. Gov't, Research Support, U.S. Gov't, P.H.S., info:eu-repo/semantics/published

Journal Article, Research Support, Non-U.S. Gov't, SCOPUS: ar.j, info:eu-repo/semantics/published

Effects of x-rays on the uptake of adenine-14C and phenylalanine-14C have been studied. An inhibition of about 20 per cent of the uptake of phenylalanine is found after 300 rads, and there is no further inhibition if the dosage of x-rays is increased. A similar response is sometimes found in the case of the uptake of adenine, but there is often no inhibition at all and even sometimes an increase in adenine uptake. It had been postulated that this behaviour might be explained by the existence of various RNA or protein fractions having different functions and responding differently to x-radiation. Fractionation experiments in order to test this hypothesis have been done; but no clear-cut results have been obtained. It has also been shown that incubating irradiated nuclei with high concentration of denatured DNA restores the uptake of the amino acids. © 1962 Informa UK Ltd All rights reserved: reproduction in whole or part not permitted., SCOPUS: ar.j, info:eu-repo/semantics/published

Journal Article, info:eu-repo/semantics/published

Journal Article, info:eu-repo/semantics/published

Our present knowledge of the cell structure, which is largely based on electron microscopy, is compared with what was known a few decades ago, when only light microscopy was available to the cytologist. The importance of cytochemical methods for the detection and localization of macromolecules (nucleic acids, proteins) is stressed. But it is pointed out that further analysis, with biochemical techniques, was required in order to understand the actual mechanisms of macromolecule synthesis in the cell (in particular, the relationships existing between nucleic acids and protein synthesis). The importance of genetical analysis in simple systems such as viruses and bacteria for the development of ‘molecular’ biology is then emphasized: in particular, the work of Avery identifying the ‘transforming principle’ with DNA, of Beadle leading to the ‘one gene, one enzyme’ theory, of the virologists who demonstrated that it is the nucleic acid component of viruses which carries the genetical information, have been of fundamental importance for the development of modern biology. No less important has been the work of the X‐ray crystallographers (Crick and Watson, Perutz, Kendrew, etc.) who established the fine structure of nucleic acids and of proteins. A brief review and a schematic representation of present ideas regarding the control exerted by DNA on the synthesis of specific proteins are then given: the main characteristics of the different kinds of RNA's, their interactions for the formation of polysomes, the role of the latter in protein synthesis, the main principles of the genetic codes, are briefly summarized. But cells are, in many respects, more complicated than bacteria. The concepts of molecular biology cannot be applied to cell differentiation without a recognition of the greater complexity of animal and plant cells. They represent, however, a most useful and powerful guide for research in that area: for instance, many aspects of morphogenesis in the unicellular al, Journal Article, Review, info:eu-repo/semantics/published

Journal Article, SCOPUS: ar.j, info:eu-repo/semantics/published