Biomedical subjects

M Jaspers

Publications and source records attributed to M Jaspers.

29 records · Page 2Linked to original sources

PubMed1995

Localization of alpha 4m integrin at sites of mesenchyme condensation during embryonic mouse development.

The expression and distribution of the murine alpha 4 (alpha 4m) integrin subunit and of one of its ligands, VCAM-1, were examined in the developing mouse embryo at different development stages. Transcription of the mRNA was investigated by in situ hybridization using single-stranded sense and anti-sense cDNA probes and by Northern blotting. In parallel sections integrin was identified by immunohistochemistry using the alpha 4m-specific antibody R1/2. In general both methods gave similar distributions. The results demonstrate that alpha 4m and vascular cell adhesion molecule-1 (VCAM-1) are expressed in mouse embryonic liver (E11-E15) and are developmentally down-regulated, consistent with the haematopoietic properties of embryonic liver. Developmental-stage-dependent expression of alpha 4m was also observed in lymphoid organs, such as spleen and thymus, and in some non-lymphoid organs or tissues, such as skeletal and tongue muscles, smooth muscles of the blood vessels, the outflow tract of the embryonic heart, the papilla of the tooth, the glomeruli of the kidney and the stroma of the gonads. In the latter tissues, the expression of alpha 4m correlated with the transient condensation of particular mesenchymal structures. We also confirm that VCAM-1 and alpha 4 beta 1 are co-distributed only in some tissues, suggesting that during mouse development, VLA-4 interacts mainly with another ligand, probably fibronectin.

Animals↗

PubMed1994

Stable expression of VLA-4 and increased maturation of the beta 1-integrin precursor after transfection of CHO cells with alpha 4m cDNA.

A full-length cDNA coding for the murine alpha 4 integrin subunit (alpha 4m) was transfected into CHO-K1 cells and cell lines that expressed VLA-4 at their surface as a result of the association of transfected alpha 4m with endogenous hamster beta 1 were selected. Functionality of the expressed alpha 4m beta 1 was shown by adhesion assays on VCAM-1 and antibody (anti-VCAM-1) inhibition. Pulse chase experiments indicated that transfection of the murine alpha 4 cDNA into CHO cells led to an increase in maturation and a decrease in degradation of the beta 1 precursor subunit compared to control CHO-K1 cells. This was supported by FACS analysis, using an anti-hamster beta 1 monoclonal antibody, which showed that more beta 1 subunit was expressed at the surface of these stably transfected alpha 4m expressing cells. These results support the hypothesis that degradation of precursor beta 1 is at least partly determined by the quantity of alpha subunits available intracellulary for heterodimer formation.

Animals↗

PubMed1994

Cloning and characterization of the promoter region of the murine alpha-4 integrin subunit.

To study the differential expression of the murine VLA-4 (alpha 4 beta 1) integrin, the 5'-flanking region of the gene for the alpha subunit (alpha 4m) was isolated and a cDNA for alpha 4m was obtained with reverse transcriptase polymerase chain reaction (RT-PCR). The cDNA sequence contained a difference in the signal peptide region compared to the previously described cDNA (Neuhaus et al., 1991). As a consequence, another start codon is predicted, resulting in a decrease in size of the signal peptide. This was confirmed by genomic sequencing. The promoter region was delimited by ribonuclease protection assay (RPA) and transfection experiments fusing 5'-upstream fragments to the luciferase gene. A fragment extending from -936 to +221 was capable of controlling the expected cell-type-specific expression. Sequence comparison of the mouse alpha 4m promoter region with the human alpha 4h promoter revealed little homology. Like most integrin subunits, alpha 4m lacks TATA anc CCAAT boxes. Putative recognition sites for DNA-binding nuclear factors (AP1, AP2, Sp1, and PU1) were identified. The characterization of the promoter region and further identification of the transcription regulatory elements should provide insight in the regulation of alpha 4m integrin gene expression.

Amino Acid Sequence↗

PubMed1993

Inhibition of the degradation of the precursor and of the mature beta 1 integrin subunit by different protein synthesis inhibitors and by ATP depletion.

A stabilization of both the precursor and the mature beta 1 integrin subunit was observed in metabolically labeled human skin fibroblasts and Molt-4 T lymphocytes upon addition of protein synthesis inhibitors or by ATP depletion. Differential effects of protein synthesis inhibitors are reported since the slow degradation of the mature beta 1 subunit was sensitive to cycloheximide but not to puromycin. We also show that the half-life of the mature subunit was not dependent on intracellular lysosomal degradation or on ubiquitination suggesting that VLA turn-over occurs at the cell surface and might involve proteins or proteases with short half-life.

Adenosine Triphosphate↗

PubMed1993

A monoclonal antibody to the alpha 2 integrin subunit cross-reacts with RGD-dependent epitopes in fibrinogen.

Monoclonal antibodies were raised against platelet integrins purified by affinity chromatography. Two monoclonal antibodies (5C5 and 3H8) reacted with the purified alpha 2 subunit of VLA-2 in Western blotting. The monoclonal antibody 3H8 also reacted with fibrinogen in Western blots and in ELISA tests. CNBr fragmentation of the alpha chain of fibrinogen generated two peptides which were still recognized by this monoclonal antibody in Western blotting. N-terminus sequencing of these two fragments showed that they were non-overlapping fragments of the fibrinogen alpha-chain with as only common epitope an RGD(F)/RGD(S) sequence. Dot blots and ELISA tests showed that the antibody 3H8 also recognized, however with lower affinity, fibronectin and collagen IV, which are RGDS containing extracellular matrix proteins. The assumption that Mab 3H8 recognizes an RGD sequence, was further supported by the findings that the binding of Mab 3H8 to fibrinogen was partially inhibited by RGDF containing peptides and that the antibody was able to inhibit platelet aggregation.

Amino Acid Sequence↗

PubMed1992

Assignment of three rat integrin genes to chromosome 19 (ITGB1), chromosome 3 (ITGA4), and chromosome 7 (ITGA5).

By means of somatic cell hybrids segregating rat chromosomes, we determined the chromosome localization of three rat beta 1 family integrin genes. ITGB1 was assigned to Chromosome (Chr) 19, ITGA4 to Chr 3, and ITGA5 to Chr 7. These chromosome assignments reveal or confirm homology between two pairs of rat and human chromosomes (rat Chr 3-human Chr 2; rat Chr 7-human Chr 12).

Animals↗

PubMed1991

The gene for the alpha 4 subunit of the VLA-4 integrin maps to chromosome 2Q31-32.

The VLA-4 integrin (CD49d/CD29), initially discovered on lymphoid cells, is actually known to be highly expressed on T cells, B cells, monocytes, and derived cell lines. Unlike other VLA integrins, mainly involved in cell-matrix adhesive interactions, VLA-4 has also been implicated in several cellular interactions. Based on the published alpha 4 cDNA sequence, a 1,142-bp alpha 4 cDNA fragment was amplified using the polymerase chain reaction. This fragment was used to isolate three overlapping genomic clones from a phage library. By Southern analysis with the cDNA probe, and using the polymerase chain reaction on DNA isolated from a panel of human/mouse somatic cell hybrids, the alpha 4 gene was mapped to chromosome 2. Fluorescence in situ hybridization confirmed this assignment and allowed a more precise mapping to chromosome 2q31-32.

Animals↗

PubMed1991

Localization of the gene encoding the alpha 2 subunit of the human VLA-2 receptor to chromosome 5q23-31.

The alpha 2 subunit of the VLA-2 receptor (CD49B) was mapped to human chromosome 5 by several independent approaches. First, the expression of the alpha 2 subunit at the protein level was investigated in a panel of human-mouse hybrid cell lines. Cell surface expression was detected by indirect immunofluorescence with monoclonal anti-alpha 2 antibody 12F1. Intracellular alpha 2 antigen was detected by immunostaining of whole cell extracts or of immunoprecipitated 12F1 antigen with the monoclonal antibodies 3H8 and 5C5. Second, the presence of human genomic alpha 2 sequences in the panel of human-mouse hybrids was detected by PCR, using primers derived from the published alpha 2 cDNA sequence. The specificity of the amplification product was shown by direct sequencing. The results of the PCR study were confirmed by amplifying a CD14 gene fragment, known to map to chromosome 5. Finally, in situ hybridization with a 3H-labeled 1040-bp cDNA probe, also obtained by PCR, confirmed and refined the localization of CD49B on chromosome 5 at q23-31.

Animals↗

PubMed1989

Distribution of the beta 1 subgroup of the integrins in human cells and tissues.

We studied the distribution of the beta 1 integrin subfamily in human tissues and cells by light microscopy, electron microscopy, and immunoblotting, using monoclonal antibody DH12, previously shown to react with the beta 1 subunit of the human fibronectin receptor. Crossreaction with the other beta subunits of the integrin family, which have 45% and 47% primary amino acid sequence identity with the beta 1 subunit, was excluded, as MAb DH12 did not react with the beta 2 subunit in granulocytes and the beta 3 subunit in thrombocytes. Reactivity with the anti-beta 1 antibody was found in skin, lung, heart, striated and smooth muscle, blood cells, liver, kidney, intestine, spleen and placenta. Thus, cells of mesodermal, ectodermal, and entodermal origin express the beta 1 subunit. In skin fibroblasts cultured in vitro, beta 1 subunit was also detected intracellularly. The wide distribution of the beta 1 family, originally detected in activated T-lymphocytes after prolonged culture in vitro, contrast with the restricted distribution of the beta 2 integrins on leucocytes.

Antibodies, Monoclonal↗

PubMed1988

Post-translational modification of the beta-subunit of the human fibronectin receptor.

Monoclonal antibody DH12, directed against the beta-subunit of the fibronectin receptor recognizes a doublet of proteins (100 and 110 kDa) in Western blots of solubilized whole fibroblasts. Pulse-chase experiments with [35S]methionine in human skin fibroblasts suggested that the two proteins might be metabolically related as precursor (100 kDa) and product (110 kDa). Endo H digestion and [3H]fucose labeling suggested that maturation converted the high-mannose oligosaccharides (100 kDa) to the endoglycosidase H resistant complex type (110 kDa). This was supported by N-glycanase digestion and by chemical deglycosylation which showed a single polypeptide. Surface iodination of intact cells labeled only the presumed mature beta-subunit.

Antibodies, Monoclonal↗

PubMed1988

Monoclonal antibody DH12 reacts with a cell surface and a precursor form of the beta subunit of the human fibronectin receptor.

Monoclonal and polyclonal antibodies were raised against a placenta plasma membrane protein preparation, which was obtained by fractionation on Blue B dye matrix and by HPLC-anionexchange, and which was shown to contain fibronectin receptors. Immunochemical and functional evidence showed that monoclonal antibody DH12 recognized the beta subunit of the human fibronectin receptor on fibroblasts. This monoclonal antibody reacted with two proteins in Western blots and in double immune precipitations of whole cell preparations. Only the higher Mr protein became labeled by surface iodination of intact fibroblasts. The lower Mr protein is thought to be an intracellular precursor of the beta subunit of the fibronectin receptor.

Animals↗