Biomedical subjects

F Altruda

Publications and source records attributed to F Altruda.

At least 37 records · Page 2Linked to original sources

PubMed1998

Differential onset of expression of alpha 7 and beta 1D integrins during mouse heart and skeletal muscle development.

beta 1D is a recently identified isoform of the beta 1 integrin subunit selectively expressed in skeletal and cardiac muscles. In the present study we determined the temporal expression of beta 1D and its association with alpha subunits during mouse development. By immunohistochemistry and western blot analysis we demonstrated that beta 1D begins to be expressed in skeletal muscles of 17 days embryo (stage E17). Its level progressively increases reaching maximal values few days after birth and remaining high in adult mice. At earlier stages of development (E11-E17) the beta 1A isoform is expressed in skeletal muscle cells. After E17 beta 1A is downregulated and disappears from muscle fibers few days after birth. In cardiac muscle the regulation of the beta 1D expression is different: beta 1D and beta 1A are coexpressed in the heart of E11 embryo. Subsequently expression of beta 1A declines, while beta 1D increases until it becomes the unique beta 1 isoform in cardiomyocytes few days after birth. Previous studies (Belkin et al J. Cell Biol. 132: 211-226, 1996) demonstrated that beta 1D in adult mouse cardiomyocytes is exclusively associated with alpha 7B. Western blot analysis shows that alpha 7B starts to be expressed in the heart only at stage E17, while beta 1D is expressed already at E11 embryo, indicating that alpha subunits other than alpha 7 should associate with beta 1D in early developmental stages. To investigate this aspect, beta 1 associated alpha subunits were identified by western blotting from cardiomyocytes integrin complexes immunoprecipitated with alpha subunit specific antibodies. We found that, during cardiomyocyte development, beta 1D associates with several alpha subunits namely with alpha 5, alpha 6A and alpha 7B. In conclusion these data show that the expression of the beta 1D muscle specific integrin during development occurs much earlier in heart than in skeletal muscle and it can dimerize with different alpha subunits.

Alternative Splicing↗

PubMed1997

Soluble integrin ligands and growth factors independently rescue neuroblastoma cells from apoptosis under nonadherent conditions.

We have investigated the role of extracellular matrix (ECM) and growth factors in the survival of nonadherent human neuroblastoma cells (line SK-N-BE). Cells cultured in serum-free medium under nonadherent conditions died with apoptotic-like features (chromatin condensation and nuclear fragmentation). SK-N-BE cells underwent neuronal differentiation in response to retinoic acid (RA). While RA itself did not induce apoptosis, differentiation increased the susceptibility of SK-N-BE cells to detachment-induced apoptosis. The appearance of the apoptotic-like phenotype required the maintenance in suspension of SK-N-BE cells for at least 16 h (12.43 +/- 1.40% of cells undergoing apoptosis) and the percentage increased up to 46.84 +/- 3.15% after 24 h. Suspension-induced apoptosis did not depend on increased intracellular Ca2+ levels nor on de novo protein synthesis and was not associated with extensive DNA degradation. Stimulation by soluble collagen I rescued suspended cells from apoptosis, even in the absence of cell adhesion and spreading. The survival promoting effect of ECM was mediated by the integrin receptors, since (1) the protective effect of soluble collagen I was blocked by anti-integrin antibodies to beta 1 and alpha 1 subunits and (2) the antibody-induced clustering of alpha 1, alpha 3, alpha v, beta 1, and beta 3 integrins rescued SK-N-BE cells cultured in suspension from apoptosis. As expected, adhesion on immobilized ECM proteins, collagen I, or laminin (0.1 to 10 micrograms/ml) also rescued SK-N-BE cells from apoptosis in a dose-dependent manner. The de novo protein synthesis was required to promote the survival effect of ECM, since cycloheximide completely abolished the protective effect of collagen I and protection from apoptosis by ECM or by anti-beta 1 antibody was associated with the increased expression of bcl-2. In addition to integrin stimulation, serum, insulin, and nerve growth factor inhibited suspension-induced apoptosis of SK-N-BE cells. The survival effect of serum and growth factors did not require the synthesis of new proteins, unlike the ECM effect. These data show that matrix proteins can promote cell survival in neuronal cells via integrin receptors. This effect does not require cell adhesion and the subsequent changes in cell shape as it can be mediated by soluble integrin ligands in suspended cells and involves a signaling pathway different from that triggered by growth factors.

Apoptosis↗

PubMed1997

Green fluorescent protein as a reporter of gene expression in transgenic mice.

We used the green fluorescent protein (GFP) from the jellyfish Aequorea victoria as a reporter of gene expression in transgenic mice. The GFP coding sequence was placed under the control of the human hemopexin and the mouse beta1 integrin promoter that were previously studied in transgenic mice using the lacZ reporter gene. We showed that GFP has a higher degree of sensitivity compared to the lacZ reporter gene allowing to identify cells with low and otherwise undetectable beta-galactosidase activity. Thus we showed the potentiality of GFP in replacing lacZ as a reporter gene to investigate promoter mapping and gene regulation in transgenic mice.

Animals↗

PubMed1996

Specific expression in brain and liver driven by the hemopexin promoter in transgenic mice.

Transgenic mice harboring the human hemopexin promoter sequences linked to the lacZ reporter gene were generated and analyzed for temporal and spatial distribution of beta-galactosidase. Upstream sequences spanning from -1800, -700 and -500 bp to the transcription start point direct regulated beta-galactosidase expression specifically to the liver and to the brain of transgenic mice. These results suggest that the 500 bp DNA fragment flanking the 5'end of the human hemopexin gene contains the cis-acting elements required for tissue and developmental stage-specific expression in vivo and provide evidence for a new extrahepatic site of expression of the hemopexin gene.

Acute-Phase Reaction↗

PubMed1996

Integrin subunit expression associated with epithelial-mesenchymal interactions during murine tooth development.

The initial information for patterning of early tooth development resides in the epithelium. Later, this is shifted to the mesenchyme. The process is governed by multiple epithelial-mesenchymal interactions. Integrins are cell surface receptors for extracellular matrix components. Expression of the beta 5 integrin subunit alternates between epithelium and mesenchyme during early tooth development (Yamada et al. [1994] Int. J. Dev. Biol. 38: 553-556). By immunofluorescence and in situ hybridization we show here a remarkably similar oscillating expression pattern of the alpha v integrin subunit. This subunit is known to associate with beta 5, and we therefore suggest that integrin alpha v beta 5 is involved in epithelial-mesenchymal interactions during tooth development. We also demonstrate that the developing tooth epithelium expresses the alpha 6, beta 1 and beta 4 subunits. The laminin receptors alpha 6 beta 1 and alpha 6 beta 4 may thus in part mediate the effect of basement membranes on tooth epithelial development. Interestingly, the enamel knot region expressed very little alpha 6 integrin subunit, whereas some expression was seen transiently in the condensing mesenchyme. During early tooth development, integrins possessing the alpha 6 subunit might also be involved in cell-cell interactions independently of laminins.

Animals↗

PubMed1996

Beta 1D integrin displaces the beta 1A isoform in striated muscles: localization at junctional structures and signaling potential in nonmuscle cells.

The cytoplasmic domains of integrins provide attachment of these extracellular matrix receptors to the cytoskeleton and play a critical role in integrin-mediated signal transduction. In this report we describe the identification, expression, localization, and initial functional characterization of a novel form of beta 1 integrin, termed beta 1D. This isoform contains a unique alternatively spliced cytoplasmic domain of 50 amino acids, with the last 24 amino acids encoded by an additional exon. Of these 24 amino acids, 11 are conserved when compared to the beta 1A isoform, but 13 are unique (Zhidkova, N. I., A. M. Belkin, and R. Mayne. 1995. Biochem. Biophys. Res. Commun. 214:279-285; van der Flier, A., I. Kuikman, C. Baudoin, R, van der Neuf, and A. Sonnenberg. 1995. FEBS Lett. 369:340-344). Using an anti-peptide antibody against the beta 1D integrin subunit, we demonstrated that the beta 1D isoform is synthesized only in skeletal and cardiac muscles, while very low amounts of beta 1A were detected by immunoblot in striated muscles. Whereas beta 1A could not be detected in adult skeletal muscle fibers and cardiomyocytes by immunofluorescence, beta 1D was localized to the sarcolemma of both cell types. In skeletal muscle, beta 1D was concentrated in costameres, myotendinous, and neuromuscular junctions. In cardiac muscle this beta 1 isoform was found in costamers and intercalated discs. beta 1D was associated with alpha 7A and alpha 7B in adult skeletal muscle. In cardiomyocytes of adult heart, alpha 7B was the major partner for the beta 1D isoform. beta 1D could not be detected in proliferating C2C12 myoblasts, but it appeared immediately after myoblast fusion and its amount continued to rise during myotube growth and maturation. In contrast, expression of the beta 1A isoform was downregulated during myodifferentiation in culture and it was completely displaced by beta 1D in mature differentiated myotubes. We also analyzed some functional properties of the beta 1D integrin subunit. Expression of human beta 1D in CHO cells led to its localization at focal adhesions. Clustering of this integrin isoform on the cell surface stimulated tyrosine phosphorylation of pp125FAK (focal adhesion kinase) and caused transient activation of mitogen-activated protein (MAP) kinases. These data indicate that beta 1D and beta 1A integrin isoforms are functionally similar with regard to integrin-mediated signaling.

Amino Acid Sequence↗

PubMed1996

Ciliary neurotrophic factor constitutively expressed in the nervous system of transgenic mice protects embryonic dorsal root ganglion neurons from apoptosis.

Ciliary neurotrophic factor (CNTF) is a potent survival factor for several neuronal populations. It is expressed postnatally by Schwann cells in the peripheral nervous system and by some glial and neuronal cells in the central nervous system. We used the promoter of the neurofilament light chain gene to produce transgenic mice that express CNTF in neurons from the beginning of neuronal differentiation. These transgenic animals may represent a suitable model to identify neuronal cell types responsive to CNTF in vivo and to study the mechanism of action of this neurotrophic factor. We show that dorsal root ganglion neurons of transgenic mice expressing CNTF in neurons are protected from apoptosis during embryonic development: 40% of these cells undergo apoptosis between embryonic day 12.5 and postnatal day 5 in transgenic mice whereas 60% do so in control animals. However, protection from apoptosis does not result in an increase in the total number of neurons at the end of development. We discuss our results with regard to CNTF potentialities in vivo and the significance of programmed cell death during development.

Animals↗

PubMed1995

Analysis of regulatory regions of the ciliary neutrophic factor gene in transgenic mice.

In order to study the regulatory regions of the human ciliary neurotrophic factor (CNTF) gene we made constructs containing sequences upstream and downstream of CNTF coding regions and the lacZ gene and analysed their expression in transgenic mice. We show that 240 bp upstream of the translation start codon are sufficient for the transcription of the lacZ gene. A further 4 kb upstream sequence is required for the expression of the transgene in Schwann cells. These two upstream regions together with a 2 kb downstream fragment drive high level of expression of the lacZ gene in the sciatic nerve. Our results indicate that these three fragments contain regulatory regions able to mimic the CNTF expression pattern in the mouse peripheral nervous system.

Animals↗

PubMed1995

A single cis-acting element in a short promoter segment of the gene encoding the interphotoreceptor retinoid-binding protein confers tissue-specific expression.

Interphotoreceptor retinoid-binding protein (IRBP) is the major protein component of the interphotoreceptor matrix. IRBP has a highly restricted tissue-specific expression in retinal photoreceptor cells and in a subgroup of pinealocytes. With the purpose of understanding how transcriptional regulation contributes to the expression of human IRBP, we have studied a short promoter fragment (from -123 to +18, relative to the transcription start site). We demonstrate, by analysis of the expression of the lacZ reporter gene fused to this short promoter fragment in transgenic mice, that it is sufficient to confer tissue-specific expression in retinal photoreceptors and in pinealocytes. DNA/protein binding assays, performed to identify binding sites for tissue-specific trans-acting factors, have shown that an element between -45 and -58 binds a factor present only in nuclear extracts of retinoblastoma-derived cell lines, which express IRBP. An element further upstream, between -86 and -106, binds apparently ubiquitous factors. Site-directed mutagenesis was performed to disrupt a GATTAA motif included in the -45 to -58 binding site and a second inverted GATTAA motif present shortly upstream. In transgenic mice bearing the mutated version of the promoter fragment, the expression of the reporter gene was completely abolished, thus suggesting that this element is essential for tissue-specific expression. A GATTAA motif appears in the 5'-flanking regions of several photoreceptor-specific genes, suggesting that this could be the recognition site for a photoreceptor-specific factor.

Animals↗

PubMed1995

Ciliary neurotrophic factor-induced gene expression in human neuroblastoma cell lines.

We have analyzed the response of the human neuroblastoma cell lines SK-N-SH (clone SY5Y) and SK-N-BE to the ciliary neurotrophic factor CNTF. In both cell lines CNTF induced the expression of the mRNA for two transcription factors, c-fos and NGF1A. The induction was rapid and transient reaching a maximum between 30 and 60 min after exposure to CNTF and subsequently declining. The level of induction of both c-fos and NGF1A mRNAs was much higher in SK-N-BE neuroblastoma cells compared to the SY5Y. Both cells express comparable levels of the transcript for the CNTF receptor-alpha. This mRNA was down regulated after 5 days of CNTF stimulation in both cell lines. CNTF also induced increased levels of the transcript for the growth cone associated protein GAP43 in SK-N-BE, but not in SY5Y cells. Induction followed a slower kinetic compared to that observed for c-fos and NGF1A. In fact, the GAP43 mRNA levels increased during 2 days of exposure to CNTF. Morphological analysis of CNTF treated cells showed that SK-N-BE undergo significant differentiation in response to CNTF (increased number of cells with neurites and increased neurite length) while SY5Y did not show appreciable morphological differentiation. These data shows that CNTF may elicit different response in neuroblastoma cell lines.

Cell Division↗

PubMed1994

Alpha v integrin subunit is predominantly located in nervous tissue and skeletal muscle during mouse development.

Alpha v integrin subunit can dimerize with different beta subunits to form receptors for several matrix proteins. The function of these receptors in vivo is still largely unknown. We examined the localization of alpha v integrin during mouse development and showed that its distribution is dynamically regulated in the glia of the central nervous system and in skeletal muscle. Immunoreactivity in the neural tube was firstly localized at embryonic day 10.5 (E10.5) around cell bodies lining the lumen and along tiny fibres extending towards the outer margin. At E12.5 alpha v distribution follows the highly defined pattern of the radial glia: fascicles of immunoreactive fibres form parallel palisades, in particular along the hindbrain and the spinal cord. At E15.5, although with weaker intensity, alpha v was still detectable in radial glia fibres, and it codistributed with glial fibrillary acidic protein positive fascicles. After birth (P8) alpha v immunoreactivity in the brain and spinal cord decreased dramatically, but remained high in the radial glia of the cerebellum. In adult mice alpha v reactivity in the central nervous system disappeared. During myogenesis alpha v appears at E10.5 in myotomal cells and from E12.5 alpha v was evident in myoblasts and in myotubes. In the developing skeletal muscle of E15.5 embryos, immunoreactivity became more concentrated in the apical portion of the myotubes. In adult striated muscle the amount of alpha v subunit dramatically declined and immunostaining was no longer detectable. During development, alpha v was weakly evident in other sites including heart and endothelia of blood vessels, mesonephric tubula, smooth muscle of the digestive tract, and bronchia. Comparative analysis of the localization of alpha v, alpha 3, and alpha 5 integrin subunits indicated that alpha v has a unique and highly regulated distribution pattern. The distribution in the nervous system is consistent with a role of alpha v in neuron-glia interaction during the organization of the neuronal layers in the brain cortex and in the cerebellum. Moreover, alpha v is likely to be involved in the myotendinous junction during embryonic life, suggesting a dual functional role of this integrin in muscle and nervous tissue.

Amino Acid Sequence↗

PubMed1993

Human beta 1-integrin gene expression is regulated by two promoter regions.

We report the cloning of two full-length cDNAs coding for the human beta 1-integrin which diverge from each other for their 5'-untranslated sequences. Characterization of a genomic clone containing these two sequences showed that they are contiguous, spaced by 261 nucleotides, and both followed by donor splice sites. Analysis by primer extension and transient transfection in a human osteogenic sarcoma cell line (MG-63) demonstrated the existence of two independent promoters for transcription initiation. The two promoter regions are very G+C-rich, and lack both a TATA box and a CAAT box. Northern blot analysis showed that transcripts starting from the distal promoter (with respect to the first coding exon) are at least 20-fold more abundant than transcripts originating from the proximal one. The levels of both transcripts increase after transforming growth factor-beta 1 induction, however, mRNAs originating from the proximal promoter increase at an higher extent. Reverse transcriptase/polymerase chain reaction analysis performed on different human tissues and cell lines revealed that, while the distal promoter is ubiquitously active, the proximal promoter is not. These findings suggest a possible complex pattern for regulation of the human beta 1-integrin gene expression.

Base Sequence↗

PubMed1993

Expression and functional analysis of a cytoplasmic domain variant of the beta 1 integrin subunit.

We have previously described a variant form of the integrin beta 1 subunit (beta 1B)1 characterized by an altered sequence at the cytoplasmic domain. Using polyclonal antibodies to a synthetic peptide corresponding to the unique sequence of the beta 1B, we analyzed the expression of this molecule in human tissues and cultured cells. Western blot analysis showed that the beta 1B is expressed in skin and liver and, in lower amounts, in skeletal and cardiac muscles. The protein was not detectable in brain, kidney, and smooth muscle. In vitro cultured keratinocytes and hepatoma cells are positive, but fibroblasts, endothelial cells, and smooth muscle cells are negative. An astrocytoma cell line derived from immortalized fetal astrocytes was found to express beta 1B. In these cells beta 1B represent integral of 30% of the beta 1 and form heterodimers with alpha 1 and alpha 5 subunits. To investigate the functional properties of beta 1B, the full-length cDNA coding for this molecule was transfected into CHO cells. Stable transfectants were selected and the beta 1B was identified by a mAb that discriminate between the transfected human protein and the endogenous hamster beta 1A. Immunoprecipitation experiments indicated that the beta 1B was exported at the cell surface in association with the endogenous hamster alpha subunits. The alpha 5/beta 1B complex bound to a fibronectin-affinity matrix and was specifically released by RGD-containing peptides. Thus beta 1B and beta 1A are similar as far as the alpha/beta association and fibronectin binding are concerned. The two proteins differ, however, in their subcellular localization. Immunofluorescence studies indicated, in fact, that beta 1B, in contrast to beta 1A, does not localize in focal adhesions. The restricted tissue distribution and the distinct subcellular localization, suggest that beta 1B has unique functional properties.

Amino Acid Sequence↗

PubMed1993

Expression of beta 1 integrin complexes on the surface of unfertilized mouse oocyte.

Integrins are a family of cell surface receptors that mediate cell-cell and cell-matrix interactions in a variety of different cellular systems. Here we show that unfertilized mouse oocytes express beta 1 class integrins both at mRNA and protein levels. Using the reverse transcription polymerase chain reaction and oligonucleotide primers based on the DNA sequence of mouse integrins, the RNA transcripts for the beta 1, alpha 5 and alpha 6 subunits were detected in unfertilized oocytes. The expression of the mRNAs is paralleled by the expression of the corresponding proteins, in fact, the alpha 5/beta 1 and the alpha 6/beta 1 complexes can be immunoprecipitated with specific antibodies form 125I-surface-labeled oocytes. Using subunit-specific antibodies we also demonstrate the presence of the alpha 3/beta 1 at the oocyte surface but alpha 1, alpha 2, alpha 4 or alpha V subunits were not detectable. Since the mouse alpha 3 DNA sequence is not available, we have not tested for the corresponding transcript. Integrin subunits alpha 6 and beta 1 were differently distributed on the oocyte surface, as visualized by immunofluorescence staining and by immunoelectron microscopy. alpha 6 antigen was mainly confined to the microvillous area of the oocyte surface, while beta 1 was more homogeneously distributed over the whole oolemma. These data demonstrate for the first time the expression of three beta 1 integrin complexes in unfertilized mouse oocytes. Such proteins may have a role in sperm-egg interaction or during very early steps of embryogenesis.

Animals↗

PubMed1993

The beta 1 integrin distal promoter is developmentally regulated in transgenic mice.

Transgenic mice harbouring 5' flanking sequences of the human beta 1 integrin gene linked to the Escherichia coli lacZ gene have been generated to examine spatial and temporal distribution of the promoter activity during development. Our previous data showed that this regulatory region is composed by two promoters, called distal and proximal, located closely on the human genome. To determine the role of each promoter region during development we generated transgenic mice using these two sequences linked to the lacZ reporter gene. Their analysis shows that these two sequences, as determined by in vitro studies, have different efficiencies in promoting transcription. Actually mice carrying the proximal promoter region exhibit a weak lacZ expression resulting in an undetectable beta-galactosidase activity in both embryonic and adult tissues. On the other hand, transgenic mice carrying the distal promoter express beta-galactosidase at high efficiency during embryonic development. The pattern of transgene expression is consistent with the localization of beta 1 protein on mouse embryos evidenced by immunohistochemistry. Moreover the distal promoter is subjected to a temporal modulation since in adult transgenic mice lacZ expression decreases to a level detected only by RT-PCR analysis. We have determined a similar down-regulation analysing by Northern blot beta 1 mRNA in adult and embryonic organs such as heart and gut.

Animals↗

PubMed1992

A recombinant human 'mini'-hexokinase is catalytically active and regulated by hexose 6-phosphates.

Mammalian hexokinase type I is a 100 kDa enzyme that has been considered to be evolved from an ancestral 50 kDa yeast-type hexokinase, insensitive to product inhibition, by gene duplication and fusion. According to this model, and based on many experimental data, the catalytic site is associated with the C-terminal half of the enzyme, although an allosteric site for the binding of glucose 6-phosphate could be present on the N-terminal half of the molecule. We have isolated a cDNA clone of hexokinase from a lambda gt11 human placenta library comprising 2658 bp, containing a single open reading frame of 1893 nucleotides, which encodes a truncate form of hexokinase starting from asparagine-287 to the terminal serine-917. This clone was further digested with restriction enzyme NcoI to obtain almost only the C-terminal half of human hexokinase starting from methionine-455 to the terminal amino acid and was overexpressed in active form in Escherichia coli and purified by ion-exchange h.p.l.c. The overexpressed 'mini'-hexokinase was found not only to catalyse glucose phosphorylation, but also to be inhibited by glucose 6-phosphate and other mono- and bis-phosphate sugars exactly like the complete mammalian enzyme. These results suggest that the C-terminal half of human hexokinase, in addition to the catalytic site, also contains the regulatory site and that the evolutionary relationship between the hexokinases should be reconsidered by including the appearance of a regulatory site before the gene duplication.

Amino Acid Sequence↗

PubMed1992

Mapping of human hexokinase 1 gene to 10q11----qter.

Two partial-length cDNAs encoding the type 1 human hexokinase (ATP:D-hexose 6-phosphotransferase) were isolated from a placenta cDNA library using a 50-bp oligonucleotide synthesized according to the known sequence of human HK1. Using the larger (1.8 kb) cDNA insert as a probe and a panel of human-hamster somatic cell hybrids, we were able to assign the HK1 gene to the long arm of chromosome 10.

Animals↗

PubMed1991

Cloning and expression of a new human polypeptide which regulates protein phosphorylation in Escherichia coli.

A 1,820 bp full-length clone encoding for a new human protein was isolated from a lambda gt11 placental cDNA library using anti-human hexokinase antibodies. The cDNA complete sequence includes a 12 bp 5' non-coding region, a single open reading frame encoding a protein of 55 KDa (HP-10) and a 177 bp non-coding with two putative polyadenylation signals upstream of 3' poly(A)tail. The deduced amino acid sequence reveals a sequence of 492 amino acids that contains a stretch of 7 glutamic acid from position 169 and one potential glycosylation site at position 274. Although antibodies against hexokinase recognize the fusion protein and antibodies against the fusion protein recognize hexokinase, HP-10 is not human hexokinase, by a number of criteria including the alignment of determined amino acid sequences. In searching for a possible functional role of HP-10 its cDNA was inserted into a procaryotic vector which allows the expression of the non-fused protein. Bacteria expressing the HP-10 encoded protein were isolated and found to have a dramatic increase in endogenous phosphorylated proteins. Since HP-10 does not have a protein kinase activity per se it should be considered a new regulatory phosphorylation protein which is active in E. coli.

Amino Acid Sequence↗