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Biomedical subjects

L Silengo

Publications and source records attributed to L Silengo.

At least 55 records · Page 3Linked to original sources
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

Alpha 6.beta 1 integrin (laminin receptor) is down-regulated by tumor necrosis factor alpha and interleukin-1 beta in human endothelial cells.

Endothelial cells from human umbilical vein (HEC) express several distinct integrin complexes that mediate the interaction with the basal membrane components. In this paper we show that treatment with tumor necrosis factor alpha (TNF alpha) down-regulates the expression of the laminin receptor alpha 6.beta 1 integrin in cultured HEC. After 48 h of treatment with TNF alpha, the level of expression of the alpha 6.beta 1 complex reached 20% of the control value. The down-regulation of the alpha 6.beta 1 integrin is caused by a decreased expression of the alpha 6 subunit, whereas the synthesis of the beta 1 subunit remains constant. Northern blot analysis shows that the decreased level of alpha 6 subunit synthesis is caused by down-regulation of alpha 6 mRNA in TNF alpha-treated HEC. TNF alpha treatment does not alter the expression of alpha 2, alpha 3, and alpha 5 integrins, also present on endothelial cell surface, thus showing that this cytokine has a selective action on distinct integrin complexes. Down-regulation of alpha 6.beta 1 correlates with pronounced reduction in adhesion of TNF alpha-treated HEC to laminin, but not to fibronectin-coated culture dishes. In addition to TNF alpha, interleukin-1 beta also decreases the expression of the alpha 6.beta 1 integrin and reduces adhesion to laminin, thus suggesting that this regulation plays an important role in inflammation.

Amino Acid Sequence↗
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

Electrophoretic analysis of neuronal genomic DNA from hypertrophic spinal ganglia during lizard tail regeneration.

Cytoplasmic and nuclear hypertrophy in neurons from the last 3 pairs of sensory ganglia left in situ cranially to the plane of amputation occurs during lizard tail regeneration. Cytophotometry after Feulgen staining demonstrated the presence of some neurons, from hypertrophic ganglia, whose quantity of DNA exceeded the diploid level (hyperdiploid neurons). In the present work agarose gel electrophoresis of total genomic DNA extracted from hypertrophic ganglia showed one or two bands migrating below the high molecular weight DNA, pointing to a selective amplification of discrete DNA segments.

Amputation, Surgical↗
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↗
PubMed1991

Cloning and expression of human ciliary neurotrophic factor.

Ciliary neurotrophic factor (CNTF) is a survival factor for avian ciliary ganglion neurons and a variety of other neuronal cell types in vitro. We report here the cloning of the entire genomic sequence encoding human CNTF and its primary structure. Biologically active CNTF has been expressed in Chinese hamster ovary cells from a human genomic DNA clone. Human CNTF has no significant sequence similarity to any previously reported protein, although approximately 84% similarity exists compared with rat and rabbit CNTF. The lack of both an N-terminal signal sequence and consensus sequences for glycosylation or hydrophobic regions, and the fact that active CNTF is expressed but not released into the culture medium of transfected cells, argue in favour of human CNTF as a cytosolic protein. These data provide a basis for understanding the role of CNTF in nervous system physiology and pathology.

Amino Acid Sequence↗
PubMed1991

Tumor necrosis factor alpha and interferon gamma modulate the expression of the vitronectin receptor (integrin beta 3) in human endothelial cells.

In this paper we show that tumor necrosis factor alpha (TNF alpha) and interferon gamma (IFN gamma) alter the expression of extracellular matrix receptors (integrins) in cultured human endothelial cells. Endothelial cells express at their surface integrins of the beta 1 and beta 3 groups that include receptors for fibronectin, collagen, laminin, and vitronectin. After treatment for 72 h with a combination of TNF alpha and IFN gamma, the level of the vitronectin receptor (alpha v beta 3) at the cell surface decreases by 70%, whereas the amounts of the beta 1 integrins remain unchanged. The decreased expression of the alpha v beta 3 complex at the cell surface is due to a selective effect of TNF alpha and IFN gamma on the regulation of the beta 3 subunit synthesis at the translational level. In fact, although the steady state levels of the mRNA for the beta 3 subunit are comparable in control and treated cells, the overall synthesis of the beta 3 subunit is decreased by a factor of 70%. No significant alteration of the synthesis of the companion alpha v subunit is detectable in cytokine-treated cells. As a consequence of the decreased expression of the receptor, cytokine-treated cells show decreased ability to adhere to vitronectin but adhere normally to fibronectin. These data show that two important inflammatory mediators, TNF alpha and IFN gamma, can modify the interaction of endothelial cells with the extracellular matrix by selectively altering the expression of specific cell surface integrin complexes.

Blotting, Northern↗
PubMed1991

Differential distribution and modulation of expression of alpha 1/beta 1 integrin on human endothelial cells.

In this paper we report that the integrin complex alpha 1/beta 1, a laminin/collagen receptor, is expressed on cultured foreskin microvascular endothelium, but is absent on endothelial cells from large vessels such as the aorta and umbilical and femoral veins. The restricted expression of integrin alpha 1/beta 1 to microvascular endothelium was also demonstrated in vivo, by immunohistochemical staining of human tissue sections. Alpha 1 specific antibodies reacted strongly with endothelial cells of small blood vessels and capillaries in several tissues, but not with endothelium of vein and arteries of umbilical cord. Expression of integrin alpha 1 can be induced in cultured umbilical vein endothelial cells by treatment with 5 ng/ml tumor necrosis factor alpha (TNF alpha). Induction of alpha 1 subunit expression also occurred after treatment of umbilical vein endothelium with 10(-5) M retinoic acid or with 10 nM PMA; Maximal induction of alpha 1 integrin was reached after 48 h of treatment and costimulation with TNF alpha and PMA resulted in a synergistic effect. The induction of alpha 1 integrin changed the adhesive properties of umbilical vein endothelial cells, by increasing the adhesiveness to collagen, laminin, and laminin fragment P1, while adhesion to fibronectin and laminin fragment E8 remained constant. The alpha 1 integrin is thus a marker of a specific population of endothelial cells and its expression confers distinctive properties of interaction with the underlying basal membrane.

Amino Acid Sequence↗
PubMed1991

Up-regulation of the integrin alpha 1/beta 1 in human neuroblastoma cells differentiated by retinoic acid: correlation with increased neurite outgrowth response to laminin.

Retinoic acid (RA) is known to induce differentiation of neuroblastoma cells in vitro. Here we show that treatment of two human neuroblastoma cell lines, SY5Y and IMR32, with RA resulted in a fivefold increase of the integrin alpha 1/beta 1 expression. The effect was selective because expression of the alpha 3/beta 1 integrin, also present in these cells, was not increased. The up-regulation of the alpha 1/beta 1 differentiated SY5Y cells correlated with increased neurite response to laminin. In fact, RA-treated SY5Y cells elongated neurites on laminin-coated substratum more efficiently compared with untreated cells or cells treated with nerve growth factor, insulin, or phorbol 12-myristate 13-acetate. These three agents induced partial morphological differentiation but did not increase alpha 1 integrin expression. Neurite extension in RA-treated cells was more efficient on laminin than on fibronectin or collagen type I and was inhibited with beta 1 integrin antibodies on all three substrates. Affinity chromatography experiments showed that alpha 1/beta 1 is the major laminin receptor in both untreated and RA-treated SY5Y cells. These data show that RA, a naturally occurring morphogen implicated in embryonic development, can selectively regulate the expression of integrin complexes in neuronal cells and suggest an important role of the alpha 1/beta 1 laminin receptor in the morphological differentiation of nerve cells.

Cell Differentiation↗
PubMed1990

A human integrin beta 1 subunit with a unique cytoplasmic domain generated by alternative mRNA processing.

The integrin subunit (beta 1) is common to a group of plasma membrane glycoprotein heterodimers that include the fibronectin, laminin and collagen receptors. These receptors span the plasma membrane, providing a transmembrane linkage between the extracellular matrix and the cytoskeleton. Here, we describe a variant of the human beta 1 differing from the previously described beta 1 in the cytoplasmic domain. The variant beta 1 transcript (beta 13'v) is present in different cell types and is synthesized at lower levels compared to the beta 1 mRNA. The cytoplasmic domain of the beta 13'v is characterized by a unique 12-amino acid C-terminal sequence. A Tyr residue present in this region, and known to be phosphorylated in the beta 1, is no longer part of a consensus sequence for phosphorylation by Tyr kinases. The integrin cytoplasmic domain anchors actin fibrils to the plasma membrane by interacting with cytoskeletal proteins such as talin and fibulin. The integrin beta 13'v with the variant cytoplasmic domain is likely to mediate a new type of membrane-cytoskeleton interaction during cell-cell and cell-matrix adhesion. Analysis of genomic clones showed that the new sequences of the variant mRNA are identical to an intron located between the last two exons of the beta 1 gene, indicating that the alternative message is generated either by premature transcription termination or by lack of splicing at this site.

Amino Acid Sequence↗
PubMed1990

Nerve growth factor induces increased expression of a laminin-binding integrin in rat pheochromocytoma PC12 cells.

Rat pheochromocytoma PC12 cells exposed to nerve growth factor differentiate as sympathetic neurons and extend neurites on laminin and to a much lesser extent on fibronectin. Analysis of laminin fragments indicated that neurite outgrowth occurs mainly on fragment P1, corresponding to the center of the cross, and only poorly on fragment E8, a long arm structure that is active with other neuronal cells. Integrin antibodies prevented adhesion and neurite sprouting of these cells on laminin, fragment P1, and fibronectin. By affinity chromatography we isolated an integrin-type receptor for laminin consisting of two subunits with molecular massess of 180 and 135 kDa. The latter is recognized by an antiserum to integrin beta 1 subunit. The bound laminin receptor could be displaced by EDTA, but not by Arg-Gly-Asp or Tyr-Ile-Gly-Ser-Arg peptides. Affinity chromatography on laminin fragments showed that the 180/135 kDa receptor binds to P1. The expression of the 180-kDa alpha subunit of the laminin receptor at the cell surface was increased 10-fold after NGF treatment. The effect of NGF is specific since the amount of a 150-kDa fibronectin-binding integrin alpha subunit remained unchanged. Moreover, the increased expression of the 180/135 kDa receptor at the cell surface corresponded to a selective increase in cell adhesion to laminin and to fragment P1. The 180/135-kDa complex is thus an integrin-type receptor for laminin whose expression and binding specificity correlates with the capacity of NGF-induced PC12 cells to extend neurites on laminin.

Adrenal Gland Neoplasms↗
PubMed1990

Expression of receptors for extracellular matrix proteins in human endothelial cells.

The interaction with matrix components of the basal membrane is an important factor in the control of vascular endothelial cell function in both normal and pathological conditions. In the present work we define integrin receptors in vascular endothelial cells and analyze whether their expression is affected by endothelial cell activators. Using in vitro adhesion tests we show that human endothelial cells (HEC) can attach and spread on substrates coated with several matrix components including fibronectin, laminin, collagen type IV, fibrinogen and vitronectin. Using specific antibodies we detect integrin receptor complexes of the beta 1 and beta 3 families that can support adhesion of HEC to the above matrix proteins. These are the alpha 2/beta 1 collagen receptor, the alpha 3/beta 1 receptor for fibronectin, collagens and laminin, the alpha 5/beta 1 fibronectin receptor, the alpha 6/beta 1 laminin receptor and the alpha V/beta 3 receptor for vitronectin and fibrinogen. When HEC are exposed to a combination of tumor necrosis factor alpha (TNF alpha) and immune interferon (IFN-gamma) the amount of the alpha V/beta 3 vitronectin receptor at the cell surface was decreased by a factor of 50-70%, while the beta 1 integrin complexes were not affected. HEC cells thus express receptors for several matrix components and inflammatory mediators such as TNF alpha and IFN-gamma can selectively alter the expression of some of them.

Cell Adhesion↗
PubMed1989

Cloning of cDNA for a novel mouse membrane glycoprotein (gp42): shared identity to histocompatibility antigens, immunoglobulins and neural-cell adhesion molecules.

A full-length clone encoding a murine membrane glycoprotein, gp42, was selected from a mouse fibroblast cDNA expression library by screening with a polyclonal antiserum. The deduced amino acid (aa) sequence indicates that gp42 is a transmembrane protein of 273 aa with a large N-terminal portion exposed outside the cell and a short cytoplasmic domain. Computer assisted analysis shows that gp42 is distinct from previously characterized proteins, but shares a number of structural features with the class II histocompatibility antigens. The sizes of the extracellular domains of gp42 and of class II histocompatibility antigens are similar, the position of four cysteines and the location of several aa residues are conserved. Some of these conserved residues are also present in immunoglobulins (Ig) and in the neural-cell adhesion molecule, thus indicating that gp42 is a new member of the Ig superfamily.

Amino Acid Sequence↗
PubMed1989

The analysis of the human hemopexin promoter defines a new class of liver-specific genes.

Hemopexin (Hpx) is a plasma glycoprotein which is expressed only in the liver. It is synthesized at a lower rate in the fetal liver than in the adult, and its level increases during acute infections. As shown here, a fragment of the human hemopexin promoter spanning from positions -130 to +22 relative to the cap site is sufficient to direct cell-specific transcription of a reporter gene. Within this segment a short sequence, located between positions -120 and -104, is responsible for this effect. This positive cis-acting element, the Hpx A site, interacts with a family of nuclear proteins, some of which are present only in hepatoma cells. The potential meaning of these complex DNA-protein interactions and the homology with elements present on the promoter of other liver-specific and acute phase genes are discussed.

Base Sequence↗
PubMed1988

Structure of the human hemopexin gene and evidence for intron-mediated evolution.

The human hemopexin gene was isolated and its structure determined. The gene spans approximately 12 kb and is interrupted by nine introns. When the intron/exon pattern was examined with respect to the polypeptide segments they encode, a direct correspondence between exons and the 10 repeating units in the protein was observed. The introns are not randomly placed; they fall in the middle of the region of amino acid sequence homology in strikingly similar locations in 6 of the 10 units and in a symmetrical position in the two halves of the coding sequence. These features strongly support the hypothesis that the gene evolved through intron-mediated duplications of a primordial sequence to a five-exon cluster. A more recent gene duplication led to the present-day gene organization.

Amino Acid Sequence↗