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Biomedical subjects

M Pech

Publications and source records attributed to M Pech.

At least 37 records · Page 2Linked to original sources
PubMed1995

Mechanism of inhibition of neointimal formation by the angiotensin-converting enzyme inhibitor cilazapril. A study in balloon catheter-injured rat carotid arteries.

We investigated the mechanism of inhibition of neointima formation by the angiotensin-covering enzyme the carotid artery. We looked for the effects of cilazapril on all phases of the response to injury, ie, on proliferation of smooth muscle cells (SMCs) in the media, their migration, their proliferation in the neointima, and their disposition of extracellular matrix in the neointima. Although treatment was discontinued after 2 weeks, the inhibitory effect of cilazapril on neointimal formation was evident even 52 weeks after injury. The amount of extracellular matrix deposited in the intima during cilazapril treatment was decreased by 20% 2 weeks after injury, but no effect was seen if tissues were analyzed at 4 or 52 weeks. [3H]Thymidine-labeled cells (pulse labeling as well as 14-day continuous labeling) showed a decrease in SMC labeling in the tunica medica by 50%, but no inhibition in the labeling indices was seen in the neointima. The fraction of unlabeled neointimal cells in the cilazapril-treated rats as judged from continuous labeling experiments was inhibited by 86%. Taken together, these data suggest an antiproliferative effect on medial SMCs and an inhibition of SMC migration into the intima by cilazapril. Since intimal extracellular matrix deposition was only delayed, the decrease in medial SMC proliferation and subsequent migration seems to be the main reason for the reduction of neointima formation.

Angiotensin-Converting Enzyme Inhibitors↗
PubMed1994

The role of platelet-derived growth factor production by tumor-associated macrophages in tumor stroma formation in lung cancer.

Lung cancer is the most common cause of death by cancer in developed countries. Since a tumor cannot develop without the parallel expansion of a tumor stroma, a better understanding of its formation could lead to new therapeutical approaches. In this respect, since platelet-derived growth-factor (PDGF) is a chemotactic and growth factor for mesenchymal and endothelial cells, lung tumors of patients undergoing surgery for non-small cell lung cancer were evaluated for their replication rate using iododeoxyuridine incorporation, and for the expression of PDGF genes and the presence of PDGF A and B chains and of PDGF receptor alpha and beta subunits. This observation demonstrates that: (a) tumor cells and stroma mesenchymal cells, but not tumor-associated macrophages, display a high replication rate; (b) 1 of 3 tumors are characterized by cancer cells expressing the genes for PDGF A and/or B chains, while 1 of 2 tumors are composed of tumor cells presenting PDGF receptors alpha and beta subunits on their surface, and in only 1 of 6 tumors, tumor cells coexpress PDGF and its receptor; (c) in almost all tumors, tumor-associated macrophages express PDGF A and/or B chain genes; (d) mesenchymal cells, as well as endothelial cells, do not express PDGF A and B chain genes but do express PDGF receptor alpha and beta subunits; and (e) an ongoing active process was suggested in the periphery of the tumor by the simultaneous strong expression of PDGF A and B chain genes by tumor-associated macrophages and the high replication rate of mesenchymal and endothelial cells in the same area. Thus, PDGF is likely to have a limited autocrine role in tumor cell replication but is a potential player, in a paracrine fashion, in tumor stroma development.

Carcinoma, Non-Small-Cell Lung↗
PubMed1994

PDGF-BB modulates endothelial proliferation and angiogenesis in vitro via PDGF beta-receptors.

To delineate potential angiogenic roles of platelet-derived growth factor (PDGF), we have investigated PDGF and its receptors on bovine aortic endothelial cells that exhibit spontaneous angiogenesis in vitro (angiogenic endothelial cells). Initiation of cord/tube formation by angiogenic endothelial cells required bovine or human serum. Neutralization of PDGF-BB in human serum with a monoclonal anti-PDGF-BB antibody reduced cord/tube formation by 37 +/- 10%, whereas neutralizing anti-PDGF-AA and an IgG isotype-matched control antibody had no effect. DNA synthesis in response to PDGF-BB increased as the cords and tubes developed; furthermore, PDGF-BB induced the incorporation of BrdU in the nuclei of cells associated with these structures. PDGF beta-receptor (PDGF-beta) mRNA increased concomitantly with cord/tube formation, and PDGFR-beta were specifically localized by immunocytochemistry to developing and mature cords and tubes. However, PDGFR-beta transcripts and protein were undetectable in nonangiogenic endothelial cells, and PDGF alpha-receptor mRNA was not expressed in either endothelial cell strain. In contrast to nonangiogenic endothelial cells, angiogenic endothelial cells did not express the PDGF B-chain, the required ligand for the PDGFR-beta. We conclude that (a) PDGF-BB can contribute to angiogenesis in vitro, (b) PDGFR-beta are specific for cord/tube-forming endothelial cells and mediate endothelial proliferation and cord/tube formation, and (c) in angiogenic and nonangiogenic endothelial cells, the expression of PDGFR-beta and PDGF B-chain is inversely correlated. We therefore suggest that paracrine PDGF might amplify angiogenesis via direct action on endothelially expressed PDGFR-beta.

Animals↗
PubMed1993

Dimerization of extracellular domains of platelet-derived growth factor receptors. A revised model of receptor-ligand interaction.

The alpha- and beta-subunits of the receptor for platelet-derived growth factor (PDGFR) were found to be autophosphorylated in the absence of ligands at high expression levels which suggests a propensity of PDGFRs to dimerize spontaneously. When the extracellular domains (ED) of both receptors were expressed and purified to homogeneity, they could be dimerized specifically by the different PDGF isoforms. PDGF-BB-induced dimerization was dependent on uncleaved loop I sequences present on both chains. Whereas, in solution, the EDs were weak competitors for PDGF binding to cellular PDGFRs, they formed high and low affinity complexes upon immobilization on solid phase. Cross-competition experiments defined two distinct binding sites on PDGFR alpha-ED. PDGF-AB bound only to the low affinity form of immobilized PDGFR beta-ED and could not dimerize PDGFR beta-ED. Cross-linking studies, however, revealed that both chains of PDGF-AB can interact with a PDGFR beta-ED monomer. Cross-linking of PDGF homodimers with EDs also yielded complexes which contained more than two ligand chains. These results led to a revised model of receptor-ligand interaction and indicate that monomeric PDGF should be able to dimerize PDGF receptors.

Amino Acid Sequence↗
PubMed1993

Conservation in sequence and affinity of human and rodent PDGF ligands and receptors.

Platelet-derived growth factor (PDGF) consists of two chains, PDGF-A and -B, which activate as homo- or heterodimers two receptors, alpha and beta. To test PDGF function in vivo we have generated neutralizing monoclonal antibodies. When analyzed with rat PDGFs only antibodies raised against human PDGF-AA showed cross-species activity. This correlated with complete amino acid sequence conservation of PDGF-A whereas rat PDGF-B differed in six positions when cloned rat PDGF cDNAs were compared with their human homologs within the receptor binding region. Extracellular domains of cloned rat PDGF alpha- and beta-receptor cDNAs did not reflect this difference in cross-species ligand conservation. When rat extracellular domains were expressed as soluble proteins they bound human PDGF-BB with high affinity after immobilization of the purified proteins on solid phase. Dissociation constants were identical to those of their human homologs. Thus, high affinity binding of human PDGF-BB to extracellular domains does not depend on species origin but only on receptor type.

Amino Acid Sequence↗
PubMed1993

Expression of a rat PDGF receptor beta ectodomain generates a low affinity ligand antagonist.

Platelet-derived growth factor (PDGF) controls migration and proliferation of mesenchymal cells and is thought to be involved in the pathophysiology of different diseases such as arteriosclerosis and tumorigenesis. In order to investigate the role of PDGF in rat models for such diseases, sufficient amounts of PDGF antagonists are needed. For this purpose we expressed the extracellular domain (ectodomain) of the rat PDGF receptor beta (PDGFR beta) in insect cells using a baculovirus vector. A hydrophilic octapeptide was added onto the N-terminus of the receptor ectodomain to follow its expression with specific anti-FLAG antibodies. The FLAG tag was also utilized to design a rapid two-step purification protocol. Purified FLAG-tagged rat PDGFR beta ectodomain had an affinity to PDGF-BB identical to the untagged ectodomain as determined by Scatchard analysis. FLAG-tagged PDGFR beta ectodomain in solution, however, did not compete for PDGF-BB binding to full length cellular receptors at concentrations expected for an high affinity antagonist.

Animals↗
PubMed1992

Emergence of the keratinocyte growth factor multigene family during the great ape radiation.

The structural gene for human keratinocyte growth factor (KGF), a member of the fibroblast growth factor family, consists of three coding exons and two introns typical of other fibroblast growth factor loci. A portion of the KGF gene, located on chromosome 15, is amplified to approximately 16 copies in the human genome, and these highly related copies (which consist of exon 2, exon 3, the intron between them, and a 3' noncoding segment of the KGF transcript) are dispersed to multiple human chromosomes. The KGF-like sequences are transcriptionally active, differentially regulated in various tissues, and composed of three distinct classes of coding sequences that are 5% divergent from each other and from the authentic KGF sequence. Multiple copies of KGF-like genes were also discovered in the genomic DNAs of chimpanzee and gorilla but were not found in lesser apes (gibbon), Old World monkeys (African green monkey and macaques), mice, or chickens. The pattern of evolutionary occurrence suggests that a primordial KGF gene was amplified and chromosomally dispersed subsequent to the divergence of orangutan from African apes but before the trichotomous divergence of human, chimpanzee, and gorilla 5-8 million years ago. The appearance of a transcriptionally active and chromosomally dispersed multigene KGF family may have implications in the evolution of the great apes and humans.

Animals↗
PubMed1991

Presence of platelet-derived growth factor in normal and fibrotic lung is specifically associated with interstitial macrophages, while both interstitial macrophages and alveolar epithelial cells express the c-sis proto-oncogene.

Normal lung structure is maintained by the presence of mesenchymal cells and their extracellular matrix products. The slow normal turnover of these cells is disrupted in fibrotic disorders, resulting in the in situ accumulation of mesenchymal cells and their extracellular matrix leading to a progressive alveolar wall thickening. Idiopathic pulmonary fibrosis (IPF) is a chronic fibrotic disorder of the lung characterized by a diffuse interstitial and intra-alveolar inflammation dominated by macrophages and polymorphonuclear neutrophils. Evaluation of alveolar macrophages (AM) obtained by bronchoalveolar lavage has previously shown that AM from normal individuals spontaneously release small amounts of platelet-derived growth factor (PDGF), a chemotactic and growth factor for mesenchymal cells, whereas AM from IPF patients spontaneously release increased amounts of biologically active PDGF, suggesting its involvement in mesenchymal cell accumulation. However, other cells such as endothelial cells and vascular smooth muscle cells can also release PDGF in vitro. In order to specify PDGF location in lung parenchyma, open lung biopsies from normal individuals and IPF patients were examined by immunohistochemistry using an anti-PDGF antibody and by in situ hybridization using PDGF A-chain and B-chain gene probes. In normal as well as in fibrotic lung, PDGF was only present in relation with interstitial macrophages but not with any other inflammatory cells or mesenchymal cells. Furthermore, the percentage of PDGF-positive macrophages in IPF was 3-fold increased in comparison to normal lung. In addition, the percentage of PDGF-positive macrophages was the same in fibrotic and nonfibrotic areas of IPF lungs.(ABSTRACT TRUNCATED AT 250 WORDS)

Aged↗
PubMed1990

Expression of protein kinase CI in NIH 3T3 cells increases its growth response to specific activators.

In order to investigate the effects of protein kinase C (PKC) expression on cellular growth and morphology, we established mouse fibroblast cell populations which expressed the rat pkc-gamma gene under the control of a retroviral promoter. NIH 3T3 stable transfectants displayed a three-fold increase in total PKC levels. These cells appeared morphologically unaltered but exhibited a stronger mitogenic response to 12-O-tetradecanoylphorbol-13-acetate (TPA) and cardiolipin (CL) as well as enhanced growth in semisolid medium in the presence of TPA. Thus, at these enzyme levels, PKC conferred growth advantages to NIH 3T3 cells only in response to specific activators.

Animals↗
PubMed1990

SV40 DNA amplification and reintegration in surviving hamster cells after 60Co gamma-irradiation.

SV40-transformed Chinese hamster embryo cells were exposed to 60Co gamma-irradiation and the fate of the integrated SV40 sequences was pursued over a period of 20 days following radiation exposure. As shown by colony hybridization, integrated SV40 sequences were amplified in surviving and non-surviving cells. At later times, however, clonal sublines of surviving cells grown for 20-30 cell generations after irradiation had lost most of their amplified SV40 copies but showed altered restriction fragment patterns indicating reintegration of SV40 sequences at new sites of the hamster genome. This suggests that 60Co gamma-irradiation can generate mutations by inducing over-replication of chromosome segments that are then substrates of enzymatic rearrangements.

Animals↗
PubMed1989

Generation of fibrosarcomas in vivo by a retrovirus that expresses the normal B chain of platelet-derived growth factor and mimics the alternative splice pattern of the v-sis oncogene.

A retrovirus containing the entire human platelet-derived growth factor B-chain (PDGF-B) gene was constructed in order to investigate the in vivo biological activity of its encoded growth factor. When this virus was introduced into newborn mice, it reproducibly generated fibrosarcomas at the site of inoculation. Proviruses in each fibrosarcoma analyzed had lost 149 nucleotides downstream of the PDGF-B coding region. This deletion originated from an alternative or aberrant splice event that occurred within exon 7 of the PDGF-B gene and mimicked the v-sis oncogene. Thus, deletion of this region may be necessary for efficient retrovirus replication or for more potent transforming function. Evidence that the normal growth factor coding sequence was unaltered derived from RNase protection studies and immunoprecipitation analysis. Tumors were generally polyclonal but demonstrated clonal subpopulations. Moreover, tumor-derived cell lines became monoclonal within a few tissue culture passages and rapidly formed tumors in vivo. These findings argue that overexpression of the normal human PDGF-B gene product under retrovirus control can induce the fully malignant phenotype.

Abelson murine leukemia virus↗
PubMed1989

Functional identification of regulatory elements within the promoter region of platelet-derived growth factor 2.

Human platelet-derived growth factor (PDGF) is composed of two polypeptide chains, PDGF-1 and PDGF-2, the human homolog of the v-sis oncogene. Deregulation of PDGF-2 expression can confer a growth advantage to cells possessing the cognate receptor and, thus, may contribute to the malignant phenotype. We investigated the regulation of PDGF-2 mRNA expression during megakaryocytic differentiation of K562 cells. Induction by 12-O-tetradecanoylphorbol-13-acetate (TPA) led to a greater than 200-fold increase in PDGF-2 transcript levels in these cells. Induction was dependent on protein synthesis and was not enhanced by cycloheximide exposure. In our initial investigation of the PDGF-2 promoter, a minimal promoter region, which included sequences extending only 42 base pairs upstream of the TATA signal, was found to be as efficient as 4 kilobase pairs upstream of the TATA signal in driving expression of a reporter gene in uninduced K562 cells. We also functionally identified different regulatory sequence elements of the PDGF-2 promoter in TPA-induced K562 cells. One region acted as a transcriptional silencer, while another region was necessary for maximal activity of the promoter in megakaryoblasts. This region was shown to bind nuclear factors and was the target for trans-activation in normal and tumor cells. In one tumor cell line, which expressed high PDGF-2 mRNA levels, the presence of the positive regulatory region resulted in a 30-fold increase in promoter activity. However, the ability of the minimal PDGF-2 promoter to drive reporter gene expression in uninduced K562 cells and normal fibroblasts, which contained no detectable PDGF-2 transcripts, implies the existence of other negative control mechanisms beyond the regulation of promoter activity.

Base Sequence↗
PubMed1988

The Z family, a group of transposed human immunoglobulin V kappa genes.

A group of highly homologous transposed human V kappa I genes, which we call the Z family, was characterized. To date four members, ZI-ZIV, comprising about 230 kb, have been analyzed on cosmid clones. The largest region (ZI) has a length of 85 kb. The Z regions show extensive homology to each other according to restriction maps and hybridization data. In each Z region a solitary V kappa I gene was found. No V kappa genes of other subgroups were detected by hybridization. The nucleotide sequence of the ZI gene revealed a non-processed V kappa I pseudogene. Hybridization experiments with DNAs from rodent/human cell hybrids and other experimental data indicate that some and possibly all members of the Z family lie outside of the kappa locus which is located on chromosome 2; they have been transposed to other chromosomes. Because of their separation from the J kappa C kappa gene segment, the Z genes can be classified as pseudogenes independent of their sequences. We postulate that the Z family arose by amplification event(s). The Z regions can also be regarded as a small family of very long repetitive sequences.

Amino Acid Sequence↗
PubMed1988

The human VK locus. Characterization of a duplicated region encoding 28 different immunoglobulin genes.

Two large regions of the human multigene family coding for the variable parts of the immunoglobulin light chains of the K type (VK) have been characterized on cosmid clones. The two germline regions, called Aa and Ab, span together 250,000 base-pairs and comprise 28 different VK gene segments, nine of which have been sequenced. There is a preponderance of VKII genes but genes belonging to subgroups I and III, and genes that cannot be easily assigned to one of the known subgroups, are interspersed within the VKII gene clusters. A number of pseudogenes have been identified. Within the Aa and Ab regions, all gene segments are organized in the same transcriptional orientation. The regions Aa and Ab, whose restriction maps are highly homologous, were shown not to be allelic structures; they must have arisen by a duplication event. Taken together with previous results, one can conclude that the major part of the VK locus exists in duplicated form. One individual has been found who has only one copy of some of the duplicated regions. By chromosomal walking, the A regions could be linked to the O regions, an analysis of which has been reported. The A regions contribute about one-third of the VK genes so far identified.

Base Sequence↗
PubMed1988

The 5' untranslated sequence of the c-sis/platelet-derived growth factor 2 transcript is a potent translational inhibitor.

c-sis/platelet-derived growth factor 2 (PDGF-2) is a prototype growth factor with transforming potential. The c-sis/PDGF-2 transcript contains a long 5' untranslated sequence (UTS) that is highly G.C rich. To examine the influence of this sequence on sis/PDGF-2 expression, we localized the c-sis/PDGF-2 promoter and used this promoter or the simian virus 40 early promoter to drive expression of the bacterial chloramphenicol acetyltransferase or sis/PDGF-2 gene. The 5' UTS of c-sis/PDGF-2 mRNA had no effect on RNA expression but was shown to exert a potent inhibitory effect on translation. By deletion analysis, we demonstrated that the 5' UTS inhibited protein expression by as much as 40-fold. The inhibitory effect was independent of reporter gene, cell type, or promoter used. A highly G.C-rich 140-base-pair sequence immediately preceding the c-sis/PDGF-2 initiation codon was shown to be nearly as effective as the entire 5' UTS in translational inhibition. Transfection analysis demonstrated that the 5' UTS significantly reduced the transforming efficiency of the sis/PDGF-2 gene as well. Thus, our findings raise the possibility that changes in regulation at the level of sis/PDGF-2 translation may play a role in development of the neoplastic phenotype.

Amino Acid Sequence↗
PubMed1987

The human V kappa locus. Characterization of extended immunoglobulin gene regions by cosmid cloning.

As part of the ongoing work in our laboratory on the structural organization of the human V kappa locus we screened cosmid libraries with V kappa gene probes and obtained numerous V kappa gene-containing cosmid clones. Several genomic regions of the V kappa locus were reconstructed from overlapping cosmid inserts and were extended by one step of chromosomal walking. The regions that are called Wa, Wb, Oa, Ob and Ob' comprise about 370 kb (10(3) bases) of DNA and contain 24 V kappa genes and pseudogenes. The V kappa genes belong to the three dominant subgroups (V kappa I, V kappa II, V kappa III) and are arranged to form mixed clusters with members of the different subgroups being intermingled with each other. The distances between the genes range from 1 to 15 kb. Three genes of the Wa and Wb regions that were sequenced turned out to be pseudogenes. Terminal parts of the regions Wa and Ob that do not contain V kappa genes of one of the known subgroups may represent extended spacer regions within the V kappa locus. Wa and Wb are duplicated regions located at different positions of the locus. Region Wb was found to comprise inversely repeated sections of at least 14 kb each that contain V kappa genes oriented in opposite polarity. This finding is consistent with inversion-deletion models of V-J joining; it also shows that the V kappa locus contains not only unique and duplicated but also triplicated parts. The data on the W and O regions are discussed together with those on the L regions and on other regions established in our laboratory. Although the picture of the human V kappa locus with, to date, about 70 different non-allelic V kappa genes is still incomplete, some general features with respect to the organization of the genes and the limited duplication of genomic regions have emerged.

Base Sequence↗
PubMed1986

Characterization of a group of transposed human V kappa genes.

A genomic region with three V kappa pseudogenes which has been transposed to chromosome 22 is characterized by detailed restriction mapping. A number of subclones are described one of which proved useful to establish an allelic restriction fragment length polymorphism (RFLP) in the region. Allelic and duplication-derived restriction site differences in cosmid clones are discussed with respect to possible problems in genomic walking experiments.

Autoradiography↗