Biomedical subjects

J Clos

Publications and source records attributed to J Clos.

At least 37 records · Page 2Linked to original sources

PubMed1995

A member of the ClpB family of stress proteins is expressed during heat shock in Leishmania spp.

We have identified and isolated the Leishmania major homologue to the bacterial ClpB gene and to the yeast Hsp104 gene. ClpB in Leishmania major is a single-copy gene and encodes a low-abundance mRNA which is induced several-fold during a heat stress. We raised antibodies against the product of the recombinant gene and show that the leishmanial ClpB encodes a predominantly cytoplasmic protein of approx. 100 kDa which is detectable in Leishmania promastigotes of various species after exposure to elevated temperatures. We, therefore, term this protein Hsp100.

Amino Acid Sequence↗

PubMed1995

Thyroid hormone promotes BCL-2 expression and prevents apoptosis of early differentiating cerebellar granule neurons.

Programmed cell death is a basic cellular process that has aroused much interest in recent years. Like immune cells, cultures of cerebellar granule neurons are very homogeneous and provide a unique opportunity for quantifying by flow cytometry one form of programmed cell death in the CNS, the apoptosis, and for studying its regulation by neurotrophic factors. We found that thyroid hormone promoted postmitotic survival by preventing the apoptosis of newly formed and early differentiated granule neurons in a dose-dependent manner. This regulation could be through the protein bcl-2, which is known to prevent cell death. This protein was present at all stages of granule neuron differentiation and appeared to be developmentally regulated. It was underexpressed in apoptotic granule neurons. The protein content of the cerebellum in hypothyroid rats was drastically reduced. In contrast, thyroid hormone caused a marked dose-dependent increase in the amounts of this protein in granule neuron cultures. The possibility that thyroid hormone may be directly or indirectly required to promote cell survival is discussed, in terms of the hormone control of the local delivery of neurotrophins, such as NGF and NT-3, as well as the expression of their low affinity receptors, gp75. We suggest that thyroid hormone has a permissive action on the developing CNS.

Animals↗

PubMed1994

Autocrine regulation of proliferation of cerebellar granule neurons by nerve growth factor.

Premigratory cerebellar granule neurons, which highly express nerve growth factor (NGF), low (gp75NGFR) and high (gp140trkA) affinity NGF receptors, were used as a physiological model to investigate the effects of NGF on neuronal replication. Studies in vivo and on cultures showed that NGF stimulates DNA synthesis, mitotic activity and related cell acquisition by initiating the entry of cells into the S phase and regulating their time in the G1 and S phases. The NGF-induced effects were blocked in vivo and in vitro by both monoclonal anti-blocked in vivo and in vitro by both monoclonal anti-NGF and anti-gp75NGFR antibodies. These results clearly demonstrate that NGF is essential for the crucial first step of cerebellar ontogenesis and support the idea that low affinity receptors are involved in the biological response, possibly by interacting with gp140trkA. By comparison with a number of well known mitogens, the high affinity form could be the main transducer of the mitogenic signal pathway. The early developing cerebellum appears therefore to be the first autocrine (and/or paracrine) model of NGF action on neurogenesis in the CNS.

Animals↗

PubMed1994

pJC20 and pJC40--two high-copy-number vectors for T7 RNA polymerase-dependent expression of recombinant genes in Escherichia coli.

We report the construction of two plasmid vectors, pJC20 and pJC40, for the expression of recombinant genes in Escherichia coli under the control of T7 RNA polymerase. Their small sizes of ca. 2.4 kb ease the subcloning of large inserts and the high copy numbers obtained result in satisfactory yields in all plasmid preparations. A multiple-cloning site offers sites for directional cloning and nested deletions. In addition, pJC40 encodes a cleavable amino-terminal histidine tail of 10 residues which is added to the gene product, thus allowing purification by metal chelate chromatography. Observed expression yields are in the range of 10% of total bacterial protein for all genes tested in our laboratory.

Animals↗

PubMed1993

Induction temperature of human heat shock factor is reprogrammed in a Drosophila cell environment.

Heat shock factor (HSF), the transcriptional activator of eukaryotic heat shock genes, is induced to bind DNA by a monomer to trimer transition involving leucine zipper interactions. Although this mode of regulation is shared among many eukaryotic species, there is variation in the temperature at which HSF binding activity is induced. We investigated the basis of this variation by analysing the response of a human HSF expressed in Drosophila cells and Drosophila HSF expressed in human cells. We report here that the temperature that induces DNA binding and trimerization of human HSF in Drosophila was decreased by approximately 10 degrees C to the induction temperature for the host cell, whereas Drosophila HSF expressed in human cells was constitutively active. The results indicate that the activity of HSF in vivo is not a simple function of the absolute environmental temperature.

Animals↗

PubMed1993

Regulation of heat shock factor trimer formation: role of a conserved leucine zipper.

The human and Drosophila heat shock transcription factors (HSFs) are multi-zipper proteins with high-affinity binding to DNA that is regulated by heat shock-induced trimerization. Formation of HSF trimers is dependent on hydrophobic heptad repeats located in the amino-terminal region of the protein. Two subregions at the carboxyl-terminal end of human HSF1 were identified that maintain the monomeric form of the protein under normal conditions. One of these contains a leucine zipper motif that is conserved between vertebrate and insect HSFs. These results suggest that the carboxyl-terminal zipper may suppress formation of trimers by the amino-terminal HSF zipper elements by means of intramolecular coiled-coil interactions that are sensitive to heat shock.

Amino Acid Sequence↗

PubMed1992

Thyroid hormone promotes transient nerve growth factor synthesis in rat cerebellar neuroblasts.

Primary cultures of cerebellum from 5-day-old rats indicated that proliferating neuroblasts synthesize and release nerve growth factor (NGF). Since NGF promotes DNA synthesis in these cells, our findings demonstrate that the early developing cerebellum is a suitable physiological model for studying the autocrine mitogenic action of NGF. Thyroid deficiency led to a greater reduction in the NGF content of the cerebellum than of the olfactory bulbs or hippocampus. Cerebellar NGF mRNA was also very sensitive to hormone deprivation. Physiological amounts of thyroid hormone stimulated both the mitotic activity and NGF production of cultured cerebellar neuroblasts. A lack of thyroid hormone is known to markedly alter cell formation in the cerebellum where postnatal neurogenesis is highly significant, in contrast to the olfactory bulbs and hippocampus. Taken together, these results suggest that the hormonal control of cell formation in the cerebellum is, at least partly, mediated by the autocrine mitogenic action of NGF. The thyroid hormone could temporally regulate the transient NGF synthesis by cerebellar neuroblasts directly and/or through its ontogenetic action, and hence all the NGF-dependent trophic effects.

Animals↗

PubMed1991

Stress-induced oligomerization and chromosomal relocalization of heat-shock factor.

The induction of heat-shock transcription factor (HSF) binding to DNA is accomplished by a heat-induced oligomerization. The transition to the induced state is accompanied by a chromosomal redistribution of HSF to the heat-shock puff sites. Over 150 additional chromosomal sites also accumulate HSF, including developmental loci that are repressed during heat shock. These findings suggest an unforeseen role for HSF as a repressor of normal gene activity during heat stress.

Animals↗

PubMed1991

Molecular cloning and expression of a human heat shock factor, HSF1.

Human cells respond to heat stress by inducing the binding of a preexisting transcriptional activator (heat shock factor, HSF) to DNA. We have isolated recombinant DNA clones for a human HSF (HSF1) by screening cDNA libraries with a human cDNA fragment. The human HSF1 probe was produced by the PCR with primers deduced from conserved amino acids in the Drosophila and yeast HSF sequences. The human HSF1 mRNA is constitutively expressed in HeLa cells under nonshock conditions and encodes a protein with four conserved leucine zipper motifs. Like its counterpart in Drosophila, human HSF1 produced in Escherichia coli in the absence of heat shock is active as a DNA binding transcription factor, suggesting that the intrinsic activity of HSF is under negative control in human cells. Surprisingly, an independently isolated human HSF clone, HSF2, is related to but significantly different from HSF1 [Schuetz, T. J., Gallo, G. J., Sheldon, L., Tempst, P. & Kingston, R. E. (1991) Proc. Natl. Acad. Sci. USA 88, 6911-6915].

Amino Acid Sequence↗

PubMed1991

FTZ-F1, a steroid hormone receptor-like protein implicated in the activation of fushi tarazu.

The Drosophila homeobox segmentation gene fushi tarazu (ftz) is expressed in a seven-stripe pattern during early embryogenesis. This characteristic pattern is largely specified by the zebra element located immediately upstream of the ftz transcriptional start site. The FTZ-F1 protein, one of multiple DNA binding factors that interacts with the zebra element, is implicated in the activation of ftz transcription, especially in stripes 1, 2, 3, and 6. An FTZ-F1 complementary DNA has been cloned by recognition site screening of a Drosophila expression library. The identity of the FTZ-F1 complementary DNA clone was confirmed by immunological cross-reaction with antibodies to FTZ-F1 and by sequence analysis of peptides from purified FTZ-F1 protein. The predicted amino acid sequence of FTZ-F1 revealed that the protein is a member of the nuclear hormone receptor superfamily. This finding raises the possibility that a hormonal ligand affects the expression of a homeobox segmentation gene early in embryonic development.

Amino Acid Sequence↗

PubMed1991

Nerve growth factor enhances DNA synthesis in cultured cerebellar neuroblasts.

The cerebellar neuroblasts in primary cultures from five-day-old rats bore NGF receptor immunoreactivity, suggesting a potential responsive to NGF. At low plating density, NGF was found to enhance DNA synthesis in these cells in a dose-dependent manner. As these cells synthesize NGF, one possibility to account for the lack of response of neuroblasts plated at high density is that the amount of endogenous trophic agent produced in this culture condition is sufficient to ensure an optimal effect. The results demonstrate that premitotic neuroblasts in the CNS, as well postmitotic neurons, are responsive to NGF. At the early stage of its development, the cerebellum therefore appears to be a very good autocrine model of NGF action.

Animals↗

PubMed1991

Biochemical, immunocytochemical and morphological evidence for an interaction between thyroid hormone and nerve growth factor in the developing cerebellum of normal and hypothyroid rats.

The effects of treatment with L-thyroxine (T4), 2.5 S nerve growth factor (NGF), monoclonal anti-NGF and monoclonal anti-NGF receptor antibodies, separately or together, on the two main processes of cerebellar histogenesis, the disappearance of the external granular layer (egl) and Purkinje cell (PC) morphogenesis, were studied in 10-day-old (1 day after the last injection) and 15-day-old normal and hypothyroid rats. The results provide the following information. (1) Anti-NGF antibodies given to normal rats alter more markedly the growth of PC soma and dendrite than the developmental pattern of egl. In contrast, anti-NGF receptor antibodies mainly delay the disappearance of egl, with minor changes in PC morphogenesis. This is the first evidence for a physiological role of NGF in neuronal maturation in both pre- and postmigratory phases. (2) The delays in the disappearance of egl and hypotrophy of PC due to hypothyroidism are greater than those induced in normal rats by anti-NGF antibodies, and T4 therapy in hypothyroid rats is more effective than that with NGF. The effects of combined T4/NGF treatment on PC size (including soma and dendrite) were approximately the sum of individual effects, with no apparent positive cooperation. Moreover, the effects of NGF treatment, but not those of T4, disappear over the long term. (3) Thyroid deficiency strongly reduces NGF receptor immunoreactivity. Anti-NGF antibodies given to thyroid-deficient rats partly counteract T4 therapy on the cerebellar growth and cortex layering, whereas they potentiate the action of the T4 on the growth of PC nuclei. The PC somas of thyroid-deficient rats assume a normal shape only after T4/NGF treatment. The perisomatic processes of immature PC in thyroid-deficient rats disappear after T4 therapy whereas they grow after NGF treatment. These results strongly suggest that NGF is complementary to thyroid hormone, and that the T4 action is partly mediated and regulated by NGF. Finally, thyroid hormone appears to have a long-term permissive role, while NGF may be a local and short-term limiting neurotrophic factor. Such a balance is essential for ensuring a normal time course of cerebellar histogenesis.

Animals↗

PubMed1990

Molecular cloning and expression of a hexameric Drosophila heat shock factor subject to negative regulation.

We report the cloning of the transcriptional activator of heat shock genes, HSF, from Drosophila. The predicted sequence of Drosophila HSF protein is surprisingly divergent from that of yeast HSF, except in regions important for DNA binding and oligomerization. A segment of the DNA binding domain of HSF bears an intriguing similarity to the putative DNA recognition helix of bacterial sigma factors, while the oligomerization domain contains an unusual arrangement of conserved hydrophobic heptad repeats. Drosophila HSF produced in E. coli under nonshock conditions forms a hexamer that binds specifically to DNA with high affinity and activates transcription from a heat shock promoter in vitro. In contrast, when HSF is expressed in Xenopus oocytes, maximal DNA binding affinity is observed only after heat shock induction. These results suggest that Drosophila HSF has an intrinsic affinity for DNA, which is repressed under nonshock conditions in vivo.

Amino Acid Sequence↗

PubMed1990

Isolation and functional characterization of TIF-IB, a factor that confers promoter specificity to mouse RNA polymerase I.

The murine ribosomal gene promoter contains two cis-acting control elements which operate in concert to promote efficient and accurate transcription initiation by RNA polymerase I. The start site proximal core element which is indispensable for promoter recognition by RNA polymerase I (pol I) encompasses sequences from position -39 to -1. An upstream control element (UCE) which is located between nucleotides -142 and -112 stimulates the efficiency of transcription initiation both in vivo and in vitro. Here we report the isolation and functional characterization of a specific rDNA binding protein, the transcription initiation factor TIF-IB, which specifically interacts with the core region of the mouse ribosomal RNA gene promoter. Highly purified TIF-IB complements transcriptional activity in the presence of two other essential initiation factors TIF-IA and TIF-IC. We demonstrate that the binding efficiency of purified TIF-IB to the core promoter is strongly enhanced by the presence in cis of the UCE. This positive effect of upstream sequences on TIF-IB binding is observed throughout the purification procedure suggesting that the synergistic action of the two distant promoter elements is not mediated by a protein different from TIF-IB. Increasing the distance between both control elements still facilitates stable factor binding but eliminates transcriptional activation. The results demonstrate that TIF-IB binding to the rDNA promoter is an essential early step in the assembly of a functional transcription initiation complex. The subsequent interaction of TIF-IB with other auxiliary transcription initiation factors, however, requires the correct spacing between the UCE and the core promoter element.

Animals↗

PubMed1990

An interaction between thyroid hormone and nerve growth factor promotes the development of hippocampus, olfactory bulbs and cerebellum: a comparative biochemical study of normal and hypothyroid rats.

The effects of treatment with L-thyroxine (T4;20 ng/g body weight, given subcutaneously on days 1, 3, 5, 7 and 9), 2.5 S nerve growth factor (NGF; 2 ng/mg brain weight, given intracerebroventricularly on days 1, 3, 5, 7 and 9), monoclonal anti-NGF (2 ng/mg wet weight, given intracerebroventricularly on days 1, 3, 5, 7 and 9), and monoclonal anti-NGF receptor (192 IgG; 2 ng/mg wet weight, injected daily from day 1 to day 9) antibodies, separately or together, were studied on the biochemical development of hippocampal formation, olfactory bulbs and cerebellum in 10-day-old and 15-day-old normal and hypothyroid rats. The results provide the following information: (1) CNS structures other than the basal forebrain are sensitive to NGF during early development. (2) Both normal and hypothyroid rats are more sensitive to NGF deprivation than NGF supplementation. (3) The effects of anti-NGF antibodies in normal rats are similar to those induced by anti-NGFr antibodies. (4) NGF alone had little or no effect, but interacts with T4 in promoting cell maturation, especially in hypothyroid rats. (5) Hypothyroid rats are more sensitive to T4 and to T4 plus NGF than are normal ones. (6) The synergistic action of both trophic factors, but not that of T4, tend to disappear at long term in hypothyroid rats. (7) The differential sensitivity of the brain areas to T4, NGF, or both trophic factors correlates with their cell acquisition rate, especially in hypothyroid rats. (8) T4 and NGF together act more markedly (but not exclusively) on the cholinergic structures in both normal and hypothyroid rats. (9) RNA appears to be very sensitive to NGF, especially in hypothyroid rats. In close correlation with preliminary morphological observations, the results clearly demonstrate that an interaction between T4 and NGF regulates the ontogeny of a number of neuronal structures in CNS independently of their neurotransmitter phenotype, but with a regional specificity. The possibilities of accounting for this interaction, in particular the major role of thyroxine, are discussed.

Acetylcholinesterase↗

PubMed1989

Effects of hypothyroidism on high-affinity vasopressin binding sites in developing hippocampal synaptosomes.

Binding sites of vasopressin (VP) have been characterized in the hippocampal synaptic plasma membranes of developing normal and hypothyroid rats using a highly specific tritiated VP antagonist, d(CH2)5Tyr(Me)VP (V1 type). This antagonist bound to an apparently homogeneous population of specific sites with an affinity ten times higher than that of VP itself. The total amount of synaptosomal protein in 15-day-old normal rats represented about 50% of the adult value, but the density of binding sites was already maximal and remained constant thereafter, supporting the concept of an early development of an extrahypophyseal vasopressinergic hippocampal system. Thyroid deficiency specifically reduced the amount of synaptosomal protein. However, the binding site density in the synaptsomal fraction appeared to be relatively well preserved from hypothyroidism, although there was a transient decrease in the apparent affinity of the ligand. These data suggest that thyroid hormones are slightly involved in the early development of high-affinity VP receptors.

Animals↗

PubMed1989

A thyroid hormone-vasopressin interaction promotes survival and maturation of hippocampal neurons dissociated postnatally.

Hippocampal cells dissociated from 5-day-old rat pups were grown in a suitable chemically defined basal medium, supplemented or not with 3,3',5-triiodo-L-thyronine (T3), [Arg8]-vasopressin (AVP), or both, at different concentrations. Four days after plating, neuron-like cells began to degenerate drastically in the basal medium. Although AVP alone had no effect, T3, and to a greater extent T3 and AVP together, prevented their death. Moreover, T3 and AVP also acted synergically in promoting the maturation of surviving cells, especially AchE-positive neurons, either directly or through glial cells.

Animals↗

PubMed1989

The time course of hippocampal cholinergic innervation in the developing hypothyroid rat. A combined histochemical and biochemical study of acetylcholinesterase activity.

Development of cholinergic innervation in the hippocampal formation of normal and hypothyroid rats was studied by a combined biochemical and histochemical analysis of acetylcholinesterase (AChE) activity in normal and hypothyroid rats. The normal developmental pattern of cholinergic activity suggests an entrance of septal cholinergic fibers from the fimbria to different zones of the hippocampal formation mainly during the first postnatal week. By 10 days of age, the regional distribution of staining was similar to that in the adult. Thereafter, the intensity of staining increased without major changes in the laminar organization. As shown by closely related histochemical and biochemical findings, hypothyroidism led to a delay in the arrival of cholinergic afferences and a possible subsequent cholinergic hyperinnervation of the hippocampal formation in adulthood. These results are discussed taking into account the time course of structural development in the two synaptic compartments, namely the extrinsic septal neurons and intrinsic pyramidal and granule cells, in comparison with the development of thyroid function.

Acetylcholinesterase↗