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Biomedical subjects

B Durand

Publications and source records attributed to B Durand.

At least 55 records · Page 3Linked to original sources
PubMed1997

Oligodendrocyte precursor cells count time but not cell divisions before differentiation.

During vertebrate development, many types of precursor cell divide a limited number of times before they stop and terminally differentiate. It is unclear what limits cell proliferation and causes the cells to stop dividing when they do. The stopping mechanisms are important as they influence both the number of differentiated cells generated and the timing of differentiation. We have been studying the 'stopping' problem in the oligodendrocyte cell lineage [1] [2], which is responsible for myelination in the vertebrate central nervous system. Previous studies demonstrated that the proliferation of oligodendrocyte precursor cells isolated from the developing rat optic nerve is limited by an intrinsic 'clock' mechanism [3], which consists of two components: a counting mechanism that counts time or cell divisions, and an effector mechanism that arrests the cell cycle and initiates cell differentiation when the appropriate time is reached [4] [5]. In the present study, we address the question of whether the counting mechanism operates by counting cell divisions. We show that precursor cells cultured at 33 degrees C divide more slowly but stop dividing and differentiate sooner, after fewer cell divisions, than when they are cultured at 37 degrees C, indicating that the counting mechanism does not count cell divisions but measures time in some other way. In addition, we show that the levels of the cyclin-dependent kinase inhibitor p27(Kip1) (p27) rise faster at 33 degrees C than at 37 degrees C, consistent with previous evidence [6] that the accumulation of p27 may be part of the counting mechanism.

Animals↗
PubMed1997

Accumulation of the cyclin-dependent kinase inhibitor p27/Kip1 and the timing of oligodendrocyte differentiation.

Many types of vertebrate precursor cells divide a limited number of times before they stop and terminally differentiate. In no case is it known what causes them to stop dividing. We have been studying this problem in the proliferating precursor cells that give rise to postmitotic oligodendrocytes, the cells that make myelin in the central nervous system. We show here that two components of the cell cycle control system, cyclin D1 and the Cdc2 kinase, are present in the proliferating precursor cells but not in differentiated oligodendrocytes, suggesting that the control system is dismantled in the oligodendrocytes. More importantly, we show that the cyclin-dependent kinase (Cdk) inhibitor p27 progressively accumulates in the precursor cells as they proliferate and is present at high levels in oligodendrocytes. Our findings are consistent with the possibility that the accumulation of p27 is part of both the intrinsic counting mechanism that determines when precursor cell proliferation stops and differentiation begins and the effector mechanism that arrests the cell cycle when the counting mechanism indicates it is time. The recent findings of others that p27-deficient mice have an increased number of cells in all of the organs examined suggest that this function of p27 is not restricted to the oligodendrocyte cell lineage.

Animals↗
PubMed1997

The impact of knee joint flexion on infrainguinal vascular grafts: an angiographic study.

OBJECTIVES: To characterise the morphologic behaviour of infrainguinal vascular grafts during flexion of the knee. DESIGN: A prospective angiographic study. MATERIALS AND METHODS: In 64 infrainguinal bypass grafts, intravenous digital subtraction angiography was performed within the first postoperative week. Frontal and lateral projection angiograms with the knee joint extended and with a 80-90 degrees flexion were taken. The distal anastomosis of the bypass was performed below-knee in 49 cases (18 in situ veins, 8 reversed veins with an anatomic course, 2 reversed veins with an extra-anatomic course, 4 composite grafts, 15 synthetic grafts with an anatomic course (14 polytetrafluoroethylene (ePTFE), 1 polyurethane), and two synthetic grafts with an extra-anatomic course. Fifteen ePTFE prostheses were implanted in the above-knee position. RESULTS: Out of 64 cases a total of 16 grafts showed stenotic kinking during flexion: two of the 18 in situ vein grafts, four of the 12 reversed vein grafts implanted with an anatomic course, one of the two reversed vein grafts implanted in an extra-anatomic site, eight of the 15 synthetic grafts crossing the knee, 0 of the two extra-anatomic ePTFE grafts, and one of the 15 cases of above-knee femoropopliteal ePTFE grafts. CONCLUSIONS: Stenotic kinking due to knee flexion can affect all kinds of bypass grafts including vein grafts placed anatomically and above-knee prostheses.

Anastomosis, Surgical↗
PubMed1997

[Contribution of dual CD13/CD14 markers in combination with CD34 for the collection of peripheral hematopoietic stem cells].

We evaluated the reliability of a flow cytometry technique for counting mononuclear cells (MNCs) in cytapheresis products. Eighty freshly-prepared samples of peripheral stem cells were studied using a dual immunolabeling technique with antibodies to CD13/CD14, and were also labeled with anti-CD34. Results of this immunophenotype determination were compared to those of the conventional method for counting MNCs under the microscope. Dual CD13/CD14 labeling was found to be a simple and reliable method for counting MNCs in the presence of immature and stimulated cells. When used in combination with CD34 labeling, the dual immunolabeling method helped improve the evaluation of the quality of peripheral stem cell grafts.

Antigens, CD34↗
PubMed1996

RFX proteins, a novel family of DNA binding proteins conserved in the eukaryotic kingdom.

Until recently, the RFX family of DNA binding proteins consisted exclusively of four mammalian members (RFX1-RFX4) characterized by a novel highly conserved DNA binding domain. Strong conservation of this DNA binding domain precluded a precise definition of the motif required for DNA binding. In addition, the biological systems in which these RFX proteins are implicated remained obscure. The recent identification of four new RFX genes has now shed light on the evolutionary conservation of the RFX family, contributed greatly to a detailed characterization of the RFX DNA binding motif, and provided clear evidence for the function of some of the RFX proteins. RFX proteins have been conserved throughout evolution in a wide variety of species, including Saccharomyces cerevisiae, Schizosaccharomyces pombe, Caenorhabditis elegans, mouse and man. The characteristic RFX DNA binding motif has been recruited into otherwise very divergent regulatory factors functioning in a diverse spectrum of unrelated systems, including regulation of the mitotic cell cycle in fission yeast, the control of the immune response in mammals, and infection by human hepatitis B virus.

Amino Acid Sequence↗
PubMed1995

Purified X2 binding protein (X2BP) cooperatively binds the class II MHC X box region in the presence of purified RFX, the X box factor deficient in the bare lymphocyte syndrome.

The conserved X2 box sequence of MHC class II promoters is homologous to TRE/CRE elements, and is required for B cell expression and IFN-gamma induction of MHC class II genes. The X2 binding protein (X2BP) was initially identified as a DNA-binding activity that specifically interacts with the conserved X2 box sequence in both the MHC HLA-DRA and HLA-DRB promoters. To begin to demonstrate that X2BP is the X2 box factor responsible for class II expression in B cells, we have purified X2BP to homogeneity from B cell nuclear extracts using DNA-affinity chromatography. X-box DNA-affinity purification indicates that X2BP is most likely composed of two polypeptides of 120 kDa and 46 kDa. The 120-kDa protein was specifically cross-linked to an X-box probe by exposure to UV irradiation. The 46-kDa subunit of X2BP cross-reacted with anti-rat CREB polyclonal Abs but not to anti-human CREB Abs in Western analysis and supershift assays, indicating that it may be a novel member of the ATF/CREB family. Purified X2BP interacted with purified RFX, a factor that binds to the adjacent X1 box and is absent in some cell lines that are mutant for MHC class II transcription. This interaction increases the DNA-binding half-life of RFX from 5 to at least 60 min, suggesting that X2BP functions in class II MHC gene expression by forming a stable complex with RFX.

Base Sequence↗
PubMed1995

A novel DNA-binding regulatory factor is mutated in primary MHC class II deficiency (bare lymphocyte syndrome).

Regulation of MHC class II gene expression is an essential aspect of the control of the immune response. Primary MHC class II deficiency is a genetically heterogeneous disease of gene regulation that offers the unique opportunity of a genetic approach for the identification of the functionally relevant regulatory genes and factors. Most patients exhibit a characteristic defect in the binding of a nuclear complex, RFX, to the X box motif of MHC class II promoters. Genetic complementation of a B-lymphocyte cell line from such a patient with a cDNA expression library has allowed us to isolate RFX5, the regulatory gene responsible for the MHC class II deficiency. This gene encodes a novel DNA-binding protein that is indeed a subunit of the RFX complex. Mutations in the RFX5 gene have been characterized in two patients. Transfection of the patient's cells with the RFX5 cDNA repairs the binding defect and fully restores expression of all the endogenous MHC class II genes in vivo.

Amino Acid Sequence↗
PubMed1995

Endogenous retinoic acid receptor (RAR)-retinoid X receptor (RXR) heterodimers are the major functional forms regulating retinoid-responsive elements in adult human keratinocytes. Binding of ligands to RAR only is sufficient for RAR-RXR heterodimers to confer ligand-dependent activation of hRAR beta 2/RARE (DR5).

We have examined how retinoic acid receptors (RARs) and retinoid X receptors (RXRs) at physiological concentrations regulate distinct retinoid-responsive elements, hRAR beta 2/beta RARE (DR5) and rCRBPII/RXRE (DR1), in keratinocytes from human skin, a major retinoid target. In vitro, endogenous RAR gamma and RXRs bound to these elements as heterodimers (RAR.RXR) but not homodimers (RAR.RAR or RXR.RXR). In cultured keratinocytes, all-trans retinoic acid, 9-cis retinoic acid, and CD367 activated beta RARE but not RXRE via endogenous RAR.RXR (ED50 = 2.3, 3.8, and 0.3 nM, respectively) whereas SR11237 showed no significant effect. All-trans retinoic acid, 9-cis retinoic acid, and SR11237 activated RXRE via overexpressed RXR.RXR (ED50 = 110, 120, and 11 nM, respectively), indicating interconversion between retinoic acid isomers, whereas co-overexpression of RAR alpha or RAR gamma suppressed this activation. Unlike 9cRA, CD367 neither induced formation of nor activated RXR.RXR. Overexpression of RAR or RXR mutated in transactivation domain AF-2 suppressed endogenous receptor activity over beta RARE. Our data suggest that 1) in keratinocytes, RAR.RXR-mediated pathway dominates over that mediated by RXR.RXR; 2) RAR-selective CD367 and RXR-selective SR11237 can be used to identify these two distinct pathways, respectively; 3) beta RARE is mainly regulated by RAR.RXR, in which RAR alone confers ligand inducibility whereas AF-2 of unliganded RXR is required for transactivation by liganded RAR AF-2; 4) lack of RXRE activity in keratinocytes is due to low endogenous levels of RXR.RXR and inhibition by RAR.RXR; and 5) interaction among RXRs is much lower than that between RAR and RXR.

Adult↗
PubMed1995

[Psychologic management of extreme prematurity].

The ongoing progress in neonatal intensive care is modifying the psychic context of prematurity for all the partners, infants as well as parents and physicians. Comfort and prognosis of preterm infants have much improved. Since newborns under 24 weeks of gestational age are now surviving, they spend approximately half the duration of pregnancy out of the maternal uterus. All the psychological issues of such an early separation have to be considered, including the developmental outcome of a sensorial environment which is quite different from the intra-uterine one. Research has been developing in this field. The cooperation between neonatalogists and psychologists has been profitable to parents. Problems linked to the separation, such as difficulty in representing the infant, are no more frequent owing to the attention paid to the mother-child bond and subsequent early contacts. What is forward now is the impact of an hyper technical world of intensive care on the parents, and of the strange aspect of the tiny baby surrounded by engines and tubes. Such an overpresence of reality often results in a reaction of traumatic daziness among parents. The cooperation of the whole staff is necessary for the resumption of an imaginary process of psychic functioning. Finally, the survival of very-low-birth-weight infants confronts the neonatalogists with some delicate ethical questions. Psychiatrists and psychologists might have an important part to play in aiding the profession in its sorting out of these ethical issues.

Ethics, Medical↗
PubMed1994

Long-term generation of human mast cells in serum-free cultures of CD34+ cord blood cells stimulated with stem cell factor and interleukin-3.

The generation of murine mast cells is supported by several cytokines, and mast cell lines are frequently established in long-term cultures of normal murine marrow cells. In contrast, growth of human mast cells was initially dependent on coculture with murine fibroblasts. The growth factor produced by murine fibroblasts and required to observe differentiation of human mast cells is attributable in part to stem cell factor (SCF). However, other factors are likely involved. We have previously shown that the combination of SCF and interleukin-3 (IL-3) efficiently sustains proliferation and differentiation of colony-forming cells (CFCs) from pre-CFC enriched from human umbilical cord blood by CD34+ selection. With periodic medium changes and the addition of fresh growth factors, five consecutive cultures of different cord blood samples gave rise to differentiated cells and CFCs for more than 2 months. Although differentiated cells continued to be generated for more than 5 months, CFCs were no longer detectable by day 50 of culture. The cells have the morphology of immature mast cells, are Toluidine blue positive, are karyotypically normal, are CD33+, CD34-, CD45+, c-kit-, and c-fms-, and die in the absence of either SCF or IL-3. These cells do not form colonies in semisolid culture and are propagated in liquid culture stimulated with SCF and IL-3 at a seeding concentration of no less than 10(4) cells/mL. At refeedings, the cultures contain a high number (> 50%) of dead cells and have a doubling time ranging from 5 to 12 days. This suggests that subsets of the cell population die because of a requirement for a growth factor other than SCF or IL-3. These results indicate that the combination of cord blood progenitor and stem cells, plus a cocktail of growth factors including SCF and IL-3, is capable with high efficiency of giving rise in serum-deprived culture to human mast cells that behave like factor-dependent cell lines. These cells may represent a useful tool for studies of human mast cell differentiation and leukemia.

Antigens, CD↗
PubMed1994

Activation function 2 (AF-2) of retinoic acid receptor and 9-cis retinoic acid receptor: presence of a conserved autonomous constitutive activating domain and influence of the nature of the response element on AF-2 activity.

A motif essential for the transcriptional activation function 2 (AF-2) present in the E region of retinoic acid receptor (RAR) alpha and 9-cis retinoic acid receptor (RXR) alpha has been characterized as an amphipathic alpha-helix whose main features are conserved between transcriptionally active members of the nuclear receptor superfamily. This conserved motif, which can activate autonomously in the absence of ligand in animal and yeast cells, can be swapped between nuclear receptors without affecting the ligand dependency for activation of transcription, thus indicating that a ligand-dependent conformational change is necessary to reveal the AF-2 activation potential within the E region of the nuclear receptor. Interestingly, we show that the precise nature of the direct repeat response element to which RAR/RXR heterodimers are bound can affect the activity of the AF-2s of the heterodimeric partners, as well as the relative efficiency with which all-trans and 9-cis retinoic acids activate the RAR partner.

Amino Acid Sequence↗
PubMed1994

Pure and functionally homogeneous recombinant retinoid X receptor.

Mouse retinoid X receptor alpha (RXR alpha) lacking the amino-terminal region A/B (RXR alpha delta AB) has been purified to more than 98% purity and functional homogeneity from bacterial and baculovirus-based recombinant expression systems with yields of 2-8 mg/liter of culture. The purified protein is soluble, and fluorescence quenching analysis demonstrated that it binds its cognate ligand 9-cis-retinoic acid (9-cis-RA) stoichiometrically, and with high affinity. Compared with RXR delta AB expressed in COS-1 cells, bacterially and baculovirus-expressed proteins bind approximately 10 and 5 times less efficiently to direct repeat 1 (DR1) DNA elements, respectively, suggesting that animal cell-specific modification of RXR or interaction with other animal cell-specific factors may modulate DNA binding. 9-cis-RA did not stimulate DR1 binding of functional RXR delta AB expressed in Escherichia coli, Sf9 or COS-1 cells. The previously reported ligand effect that can be observed with in vitro made receptor may therefore be a consequence of a conformational stabilization of improperly folded in vitro synthesized protein.

Amino Acid Sequence↗
PubMed1994

The cellular retinoic acid binding protein I is dispensable.

The cellular retinoic acid binding proteins I and II (CRABPI and CRABPII) bind retinoic acid with high affinity, exhibit distinct patterns of expression during embryonic development, and are thought to play important roles in the RA signaling pathway. We have generated a targeted mutation of the CRABPI gene using the "hit-and-run" strategy and shown that it prevents the production of a functional CRABPI protein. Homozygous mutant mice were normal, indicating that CRABPI does not play a crucial role in the RA signaling pathway.

Amino Acid Sequence↗
PubMed1994

Cooperative binding between factors RFX and X2bp to the X and X2 boxes of MHC class II promoters.

Transcription of major histocompatibility complex (MHC) class II genes is controlled primarily by the promoter, which contains several conserved cis-acting elements, including the X, X2, and Y boxes. We show here that RFX, the X box-binding protein that is deficient in certain MHC class II regulatory mutants, binds cooperatively with an X2 box-binding protein (X2bp) to form an RFX.X2bp.DNA complex in which the interaction of the two factors with their target sites is strongly stabilized. A functional role of this RFX.X2bp complex is consistent with mutational analysis of the X and X2 boxes of the DRA and DRB1 class II promoters. Together with previous results demonstrating cooperative binding between RFX and the Y box-binding protein NF-Y, our results indicate that RFX plays a central role in promoting cooperative binding interactions required for stable occupation of the MHC class II promoter. This may explain why the highly specific defect in binding of RFX observed in certain MHC class II regulatory mutants is associated in vivo with a bare promoter in which all of the cis-acting elements, including the X, X2, and Y boxes, are unoccupied.

Base Sequence↗