Search PubMed⌕ Search

Biomedical subjects

F Grosveld

Publications and source records attributed to F Grosveld.

At least 37 records · Page 2Linked to original sources
PubMed2000

A distal Schwann cell-specific enhancer mediates axonal regulation of the Oct-6 transcription factor during peripheral nerve development and regeneration.

The POU domain transcription factor Oct-6 is a major regulator of Schwann cell differentiation and myelination. During nerve development and regeneration, expression of Oct-6 is under the control of axonal signals. Identification of the cis-acting elements necessary for Oct-6 gene regulation is an important step in deciphering the complex signalling between Schwann cells and axons governing myelination. Here we show that a fragment distal to the Oct-6 gene, containing two DNase I-hypersensitive sites, acts as the Oct-6 Schwann cell-specific enhancer (SCE). The SCE is sufficient to drive spatially and temporally correct expression, during both normal peripheral nerve development and regeneration. We further demonstrate that a tagged version of Oct-6, driven by the SCE, rescues the peripheral nerve phenotype of Oct-6-deficient mice. Thus, our isolation and characterization of the Oct-6 SCE provides the first description of a cis-acting genetic element that responds to converging signalling pathways to drive myelination in the peripheral nervous system.

Animals↗
PubMed2000

Transcription factor Sp3 is essential for post-natal survival and late tooth development.

Sp3 is a ubiquitously expressed transcription factor closely related to Sp1 (specificity protein 1). We have disrupted the mouse Sp3 gene by homologous recombination. Sp3-deficient embryos are growth retarded and invariably die at birth of respiratory failure. The cause for the observed breathing defect remains obscure since only minor morphological alterations were observed in the lung, and surfactant protein expression is indistinguishable from that in wild-type mice. Histological examinations of individual organs in Sp3(-/-) mice show a pronounced defect in late tooth formation. In Sp3 null mice, the dentin/enamel layer of the developing teeth is impaired due to the lack of ameloblast-specific gene products. Comparison of the Sp1 and Sp3 knockout phenotype shows that Sp1 and Sp3 have distinct functions in vivo, but also suggests a degree of functional redundancy.

Ameloblasts↗
PubMed2000

Gata3 loss leads to embryonic lethality due to noradrenaline deficiency of the sympathetic nervous system.

Mouse embryos deficient in Gata3 die by 11 days post coitum (d.p.c.) from pathology of undetermined origin. We recently showed that Gata3-directed lacZ expression of a 625-kb Gata3 YAC transgene in mice mimics endogenous Gata3 expression, except in thymus and the sympathoadrenal system. As this transgene failed to overcome embryonic lethality (unpublished data and ref. 3) in Gata3-/- mice, we hypothesized that a neuroendocrine deficiency in the sympathetic nervous system (SNS) might cause embryonic lethality in these mutants. We find here that null mutation of Gata3 leads to reduced accumulation of Th (encoding tyrosine hydroxylase, Th) and Dbh (dopamine beta-hydroxylase, Dbh) mRNA, whereas several other SNS genes are unaffected. We show that Th and Dbh deficiencies lead to reduced noradrenaline in the SNS, and that noradrenaline deficiency is a proximal cause of death in mutants by feeding catechol intermediates to pregnant dams, thereby partially averting Gata3 mutation-induced lethality. These older, pharmacologically rescued mutants revealed abnormalities that previously could not be detected in untreated mutants. These late embryonic defects include renal hypoplasia and developmental defects in structures derived from cephalic neural crest cells. Thus we have shown that Gata3 has a role in the differentiation of multiple cell lineages during embryogenesis.

Animals↗
PubMed2000

A multi-domain protein for beta1 integrin-targeted DNA delivery.

The development of effective receptor-targeted nonviral vectors for use in vivo is complicated by a number of technical problems. One of these is the low efficiency of the conjugation procedures used to couple protein ligands to the DNA condensing carrier molecules. We have made and characterized a multi-domain protein (SPKR)4inv, that is designed to target plasmid DNA to beta1 integrins in remodeling tissue. It contains a nonspecific DNA-binding domain (SPKR)4, a rigid alpha-helical linker, and the C-terminal beta1 integrin binding domain (aa 793-987) of the Yersinia pseudotuberculosis invasin protein. (SPKR)4inv could be purified at high yields using a bacterial expression system. We show that (SPKR)4inv binds with high affinity to both plasmid DNA and beta1 integrins. In a cell attachment assay, the apparent affinity of (SPKR)4inv for beta1 integrins is three orders of magnitude higher than that of the synthetic peptide integrin ligand RGDS. (SPKR)4inv-plasmid complexes are not active in an in vitro transfection assay. However, transfection efficiencies of plasmid complexes with a cationic lipid micelle (DOTAP/Tween-20) or a cationic polymer (polyethylenimine), are significantly increased in combination with (SPKR)4inv. (SPKR)4inv-mediated transfection can be inhibited by a soluble form of beta1 integrin, which is evidence for its receptor specificity. In conclusion, (SPKR)4inv allows beta1 integrin-specific targeting of plasmid-carrier complexes, while avoiding inefficient and cumbersome coupling chemistry. The modular design of the expression vector allows production of similar multi-domain proteins with a different affinity. The further development of such complexes for use in vivo is discussed.

Antigen-Antibody Reactions↗
PubMed2000

Human plasma phospholipid transfer protein increases the antiatherogenic potential of high density lipoproteins in transgenic mice.

Plasma phospholipid transfer protein (PLTP) transfers phospholipids between lipoprotein particles and alters high density lipoprotein (HDL) subfraction patterns in vitro, but its physiological function is poorly understood. Transgenic mice that overexpress human PLTP were generated. Compared with wild-type mice, these mice show a 2.5- to 4.5-fold increase in PLTP activity in plasma. This results in a 30% to 40% decrease of plasma levels of HDL cholesterol. Incubation of plasma from transgenic animals at 37 degrees C reveals a 2- to 3-fold increase in the formation of pre-beta-HDL compared with plasma from wild-type mice. Although pre-beta-HDL is normally a minor subfraction of HDL, it is known to be a very efficient acceptor of peripheral cell cholesterol and a key mediator in reverse cholesterol transport. Further experiments show that plasma from transgenic animals is much more efficient in preventing the accumulation of intracellular cholesterol in macrophages than plasma from wild-type mice, despite lower total HDL concentrations. It is concluded that PLTP can act as an antiatherogenic factor preventing cellular cholesterol overload by generation of pre-beta-HDL.

Animals↗
PubMed2000

Functional analysis of CLIP-115 and its binding to microtubules.

Cytoplasmic linker proteins (CLIPs) bind to microtubules and are proposed to link this cytoskeletal network to other intracellular structures. We are interested in CLIP-115, since this protein is enriched in neuronal dendrites and may operate in the control of brain-specific organelle translocations. Each CLIP monomer is characterized by two microtubule-binding (MTB) motifs, surrounded by basic, serine-rich regions. This head domain is connected to the C-terminal tail through a long coiled-coil structure. The MTB domains are conserved as a single domain in other proteins involved in microtubule based transport and dynamics, such as p150(Glued). Here we provide evidence that efficient binding of CLIP-115 to microtubules is sensitive to phosphorylation and is not mediated by the conserved MTB domains alone, but requires the presence of the basic, serine rich regions in addition to the MTB motifs. In transfected COS-1 cells, CLIP-115 initially accumulates at the distal ends of microtubules and coincides with CLIP-170, indicating that both proteins mark growing microtubule ends. However, when expressed at higher levels, CLIP-115 and -170 affect the microtubule network differently. This might be partly due to the divergent C-termini of the two proteins. We demonstrate that, similar to CLIP-170, CLIP-115 forms homodimers, which, at least in vitro, are linked by disulfide bridges. Cysteine(391) of CLIP-115, however, is specific in that it controls the microtubule bundling capacity of certain mutant CLIP-115 molecules. Therefore, both similar and specific mechanisms appear to regulate the conformation of CLIPs as well as their binding to microtubules.

Amino Acid Sequence↗
PubMed2000

Characterisation of transcriptionally active and inactive chromatin domains in neurons.

The tandemly organised ribosomal DNA (rDNA) repeats are transcribed by a dedicated RNA polymerase in a specialised nuclear compartment, the nucleolus. There appears to be an intimate link between the maintenance of nucleolar structure and the presence of heterochromatic chromatin domains. This is particularly evident in many large neurons, where a single nucleolus is present, which is separated from the remainder of the nucleus by a characteristic shell of heterochromatin. Using a combined fluorescence in situ hybridisation and immunocytochemistry approach, we have analysed the molecular composition of this highly organised neuronal chromatin, to investigate its functional significance. We find that clusters of inactive, methylated rDNA repeats are present inside large neuronal nucleoli, which are often attached to the shell of heterochromatic DNA. Surprisingly, the methylated DNA-binding protein MeCP2, which is abundantly present in the centromeric and perinucleolar heterochromatin, does not associate significantly with the methylated rDNA repeats, whereas histone H1 does overlap partially with these clusters. Histone H1 also defines other, centromere-associated chromatin subdomains, together with the mammalian Polycomb group factor Eed. These data indicate that neuronal, perinucleolar heterochromatin consists of several classes of inactive DNA, that are linked to a fraction of the inactive rDNA repeats. These distinct chromatin domains may serve to regulate RNA transcription and processing efficiently and to protect rDNA repeats against unwanted silencing and/or homologous recombination events.

Animals↗
PubMed1999

GATA-3 is involved in the development of serotonergic neurons in the caudal raphe nuclei.

The GATA-3 transcription factor shows a specific and restricted expression pattern in the developing and adult mouse brain. In the present study we investigated the role of GATA-3 in the caudal raphe system, which is known to operate as a modulator of motor activity. We demonstrate that virtually all neurons in the caudal raphe nuclei that express GATA-3 also produce serotonin. Absence of GATA-3, as analyzed in chimeric -/- mice, affects the cytoarchitecture of serotonergic neurons in the caudal raphe nuclei. As a result the chimeras show a serious defect in their locomotor performance on a rotating rod. In sum, we conclude that GATA-3 plays a major role in the development of the serotonergic neurons of the caudal raphe nuclei, and that it is crucial for their role in locomotion.

Animals↗
PubMed1999

Early arrest in B cell development in transgenic mice that express the E41K Bruton's tyrosine kinase mutant under the control of the CD19 promoter region.

Bruton's tyrosine kinase (Btk) is a nonreceptor protein kinase that is defective in X-linked agammaglobulinemia in humans and in X-linked immunodeficiency in mice. To study the effect of Btk activation in early B cell development in vivo, we have created transgenic mouse strains expressing Btk under the control of the human CD19 promoter region. The transgenic expression of wild-type human Btk corrected all X-linked immunodeficiency features in mice carrying a targeted disruption of the Btk gene. In contrast, expression of an activated form of Btk, the E41K mutant, resulted in an almost complete arrest of B cell development in the immature IgM+IgD- B cell stage in the bone marrow, irrespective of the presence of the endogenous intact Btk gene. Immature B cells were arrested at the progression from IgMlow into IgMhigh cells, which reflects the first immune tolerance checkpoint at which autoreactive B cells become susceptible to apoptosis. As the constitutive activation of Btk is likely to mimic B cell receptor occupancy by autoantigens in the bone marrow, our findings are consistent with a role for Btk as a mediator of B cell receptor-induced apoptotic signals in the immature B cell stage. Whereas the peripheral mature B cell pool was reduced to <1% of the normal size, significant numbers of IgM-secreting plasma cells were present in the spleen. Serum IgM levels were substantial and increased with age, but specific Ab responses in vivo were lacking. We conclude that the residual peripheral B cells were efficiently driven into IgM+ plasma cell differentiation, apparently without functional selection.

Agammaglobulinaemia Tyrosine Kinase↗
PubMed1999

Inefficient processing impairs release of RNA from the site of transcription.

We describe here for the first time the site of retention within the nucleus of pre-mRNA processing mutants unable to be exported to the cytoplasm. Fluorescence in situ hybridization was used to detect transcripts from human beta-globin genes that are either normal or defective in splicing or 3' end formation. Nuclear transcripts of both wild-type and mutant RNAs are detected only as intranuclear foci that colocalize with the template gene locus. The kinetics of transcript release from the site of transcription was assessed by treatment of cells with the transcriptional inhibitors actinomycin D, alpha-amanitin and DRB. These drugs induce the rapid disappearance of nuclear foci corresponding to wild-type human beta-globin RNA. In contrast, pre-mRNA mutants defective in either splicing or 3' end formation and which fail to be transported to the cytoplasm, are retained at the site of transcription. Therefore, 3' end processing and splicing appear to be rate limiting for release of mRNA from the site of transcription.

Amanitins↗
PubMed1999

Gp96/GRP94 is a putative high density lipoprotein-binding protein in liver.

We have previously shown that three high density lipoproteins (HDL)-binding proteins in liver, of 90, 110 and 180 kDa, are structurally related. In this study, these proteins are identified as gp96/GRP94. This protein is known to occur as a homodimer and has a dual subcellular localization: it is both an endoplasmic reticulum resident protein, where it is supposed to act as a chaperonin, and a plasma membrane protein, whose significance is unknown. In ultrastructural studies the plasma membrane localization of the homodimeric form was verified. The 90-kDa protein was abundantly present at the membranes of the endosomal/lysosomal vesicles as well as at the apical hepatocyte membranes, comprising the bile canaliculi. The monomeric protein is scarcely present at the basolateral membrane of the hepatocytes, but could be demonstrated in coated pits, suggesting involvement in receptor-mediated endocytosis. Labeling of the endoplasmic reticulum was virtually absent. Gp96/GRP94 was transiently expressed in COS-1 cells. However, the expressed protein was exclusively localized in the endoplasmic reticulum. Transfection with constructs in which the C-terminal KDEL sequence had been deleted, resulted in plasma membrane localized expression of protein, but only in an extremely low percentage of cells. In order to evaluate the HDL-binding capacities of this protein, stably transfected cells were generated, using several cell types. It appeared to be difficult to obtain a prolonged high level expression of gp96. In these cases, however, a marked increase of HDL-binding activity compared with the control cells could be observed.

Amino Acid Sequence↗
PubMed1999

Deletion of a region that is a candidate for the difference between the deletion forms of hereditary persistence of fetal hemoglobin and deltabeta-thalassemia affects beta- but not gamma-globin gene expression.

The analysis of a number of cases of beta-globin thalassemia and hereditary persistence of fetal hemoglobin (HPFH) due to large deletions in the beta-globin locus has led to the identification of several DNA elements that have been implicated in the switch from human fetal gamma- to adult beta-globin gene expression. We have tested this hypothesis for an element that covers the minimal distance between the thalassemia and HPFH deletions and is thought to be responsible for the difference between a deletion HPFH and deltabeta-thalassemia, located 5' of the delta-globin gene. This element has been deleted from a yeast artificial chromosome (YAC) containing the complete human beta-globin locus. Analysis of this modified YAC in transgenic mice shows that early embryonic expression is unaffected, but in the fetal liver it is subject to position effects. In addition, the efficiency of transcription of the beta-globin gene is decreased, but the developmental silencing of the gamma-globin genes is unaffected by the deletion. These results show that the deleted element is involved in the activation of the beta-globin gene perhaps through the loss of a structural function required for gene activation by long-range interactions.

Animals↗
PubMed1999

Mechanisms of developmental control of transcription in the murine alpha- and beta-globin loci.

We have characterized mRNA expression and transcription of the mouse alpha- and beta-globin loci during development. S1 nuclease and primary transcript in situ hybridization analyses demonstrate that all seven murine globin genes (zeta, alpha1, alpha2, epsilony, betaH1, betamaj, and betamin) are transcribed during primitive erythropoiesis, however transcription of the zeta, epsilony, and betaH1 genes is restricted to the primitive erythroid lineage. Transcription of the betamaj and betamin genes in primitive cells is EKLF-dependent demonstrating EKLF activity in embryonic red cells. Novel kinetic analyses suggest that multigene expression in the beta locus occurs via alternating single-gene transcription whereas coinitiation cannot be ruled out in the alpha locus. Transcriptional activation of the individual murine beta genes in primitive cells correlates inversely with their distance from the locus control region, in contrast with the human beta locus in which the adult genes are only activated in definitive erythroid cells. The results suggest that the multigene expression mechanism of alternating transcription is evolutionarily conserved between mouse and human beta globin loci but that the timing of activation of the adult genes is altered, indicating important fundamental differences in globin gene switching.

Animals↗
PubMed1999

Expression of the transcription factor GATA-3 is required for the development of the earliest T cell progenitors and correlates with stages of cellular proliferation in the thymus.

GATA-3 is a zinc-finger transcription factor that is essential for both early T cell development and Th2 cell differentiation. To quantify GATA-3 expression during T cell development in vivo in the mouse, the GATA-3 gene was targeted by insertion of a lacZ reporter by homologous recombination in embryonic stem (ES) cells. Although we could detect GATA-3+ cells throughout T cell development in the thymus, the proportions of GATA-3+ cells varied considerably between the distinct differentiation stages. The two periods of TCR alpha and beta gene recombination, which occur in quiescent or slowly dividing cells, were associated with low proportions of GATA-3+ cells. Conversely, the stage of rapidly proliferating cells, which insulates these two waves of TCR rearrangement, was characterized by a large proportion of GATA-3+ cells. In addition, we generated chimeric mice by injection of GATA-3-deficient, lacZ-expressing ES cells into wild-type blastocysts. In this in vivo competition analysis, no contribution of GATA-3-deficient cells to the T cell lineage was detected, not even in the earliest CD44+CD25- double-negative (CD4-CD8-) cell stage in the thymus. These results parallel data implicating other GATA family members as key regulators of proliferation and survival of early hematopoietic cells. We therefore propose that GATA-3 is required for the expansion of T cell progenitors, and for the control of subsequent proliferation steps, which alternate periods of TCR recombination in the thymus.

Animals↗
PubMed1999

Activation by locus control regions?

On the basis of homologous recombination experiments to delete the murine beta-globin locus control region (LCR) in embryonic stem cells, it was recently suggested that the LCR is not required for the activation of the murine beta-globin locus. This conclusion is in direct contradiction to the findings and conclusions that have been obtained with the human beta-globin LCR; thus the murine and human LCR may functionally be different or there may be a different interpretation of the results.

Animals↗
PubMed1999

Nucleotide changes in the gamma-globin promoter and the (AT)xNy(AT)z polymorphic sequence of beta LCRHS-2 region associated with altered levels of HbF.

We have studied 31 beta-thalassaemia intermedia, 30 beta-thalassaemia major patients and 50 normal individuals from Turkey, determining the relationship between the nucleotide variations in beta-globin gene cluster, the altered levels of foetal haemoglobin and the relative ratios of beta- and gamma mRNAs. We have found in beta-thalassaemia intermedia patients with high foetal haemoglobin expression that the three nucleotide variations in the 5' sequences of the gamma globin genes, A-->G at G gamma - 1396, the T-->C at A gamma - 228, and the GA-->AG at A gamma - 603/4, are linked to haplotype II in haplotypic homozygotes and the (AT)8N14(AT)7 motif in beta LCR. Conversely, the three single nucleotide substitutions in the 5' sequences of gamma globin genes, the G-->A at G gamma - 1225, the A-->G at A gamma + 25 and the C-->G at A gamma - 369, which have a strong linkage with haplotype I, V or VI in haplotypic homozygotes and the (AT)10N12(AT)12 and the (AT)9N12(AT)12 motifs in HS-2 of beta LCR are all associated with low foetal haemoglobin levels. The number of nucleotide changes in beta-globin gene cluster implied in our study are not the primary cause of the differences in haemoglobin F levels. They perhaps may contribute to the variations in the clinical severity observed among beta thalassaemia intermedia and major patients with other yet unknown gene conversions.

Adolescent↗