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A Yayon

Publications and source records attributed to A Yayon.

At least 73 records · Page 4Linked to original sources
PubMed1992

Promoter region of the murine fibroblast growth factor receptor 2 (bek/KGFR) gene.

Fibroblast growth factors (FGFs) and their receptors play an important role in cell growth, angiogenesis and embryonal development. Four distinct genes encoding fibroblast growth factor receptors (FGFRs) were identified: flg, encoding FGFR1, bek encoding FGFR2, and the genes for FGFR3 and FGFR4. Both FGFR2 and keratinocyte growth factor receptor (KGFR) are encoded by the same gene, bek. To study the regulation of expression of the FGF receptors we analysed the DNA sequence flanking the 5' region of the cDNA of murine FGFR2 to seek elements that control its transcription. A 5-kbp fragment containing the 5' end of the cDNA was isolated from mouse genomic library and used to map the promoter region. We found that the sequence encoding the 5' non-translated region of the FGFR2/KGFR cDNA contains an intron located 210 bp upstream from the translation start site. Using RNAase protection and primer extension, we identified the mRNA start 37 bp upstream from the beginning of the bek cDNA. The promoter activity was found to reside in a 1.3-kbp fragment upstream from the cDNA, and deletion mapping further localized the promoter to a 0.7-kbp fragment. The sequence of this region shows high G+C content (62%), which is particularly emphasized in the 200 bp upstream from the mRNA start (80% G+C). This region contains the CCGCCC, GGGCGG AND GGAGG motifs also found in promoters of other growth factor receptors. Neither TATA nor CAAT boxes were found near the RNA start site. The characterization of this promoter will allow studies of the regulation of expression of the FGFR2 during development and in pathophysiological states. The differences between the promoter sequence of the gene for FGFR2 (bek) and FGFR1 (flg) may explain their differential expression during development.

Animals↗
PubMed1992

Complexity of FGF receptors: genetic basis for structural diversity and functional specificity.

Since 1989, the receptors for fibroblast growth factors (FGFs) were cloned and characterized as a subgroup of the family of receptor tyrosine kinases. Four FGF receptor genes were identified, all of which encode membrane-bound glycoproteins containing three immunoglobulin (Ig) -like domains at the extracellular region, where only two of these domains are involved in ligand binding. Three unique features characterize the FGF receptors: 1) overlapping recognition and redundant specificity, where one receptor may bind with a similar affinity several of the seven known FGFs and one FGF may bind similarly to several distinct receptors. 2) The binding of FGFs to their receptors is dependent on the interaction of FGF with cell surface heparan sulfate proteoglycans. 3) A multitude of isoforms of cell-bound or secreted receptors are produced by the same gene. The gene structure of these receptors revealed two major mechanisms that are responsible for the formation of the diverse forms: alternative mRNA splicing, resulting in deletions or alternate exons usage, and internal polyadenylation, resulting in truncated products. These are reminiscent of mechanisms that also operate in the immunoglobulin family to generate diversity and to produce either secreted or cell-bound molecules. Tissue-specific alternative splicing in FGF receptors allows for the generation of two distinct receptors from a single gene because alternative exons determine the sequence of the COOH-terminal half of the third Ig-like domain involved in ligand binding. This represents a novel genetic mechanism to generate receptor diversity and specificity and to increase receptor repertoire.

Animals↗
PubMed1991

Cell surface, heparin-like molecules are required for binding of basic fibroblast growth factor to its high affinity receptor.

The role of low affinity, heparin-like binding sites for basic fibroblast growth factor (bFGF) was investigated in CHO cells mutant in their metabolism of glycosaminoglycans. Heparan sulfate-deficient mutants transfected to express a cloned mouse FGF receptor cDNA are not able to bind bFGF. It is demonstrated that free heparin and heparan sulfate can reconstitute a low affinity receptor that is, in turn, required for the high affinity binding of bFGF. These studies suggest that the low affinity receptor is an accessory molecule required for binding of bFGF to the high affinity site. Such an obligatory interaction of low and high affinity FGF receptors suggests a physiological role for heparin-like, low affinity receptors and constitutes a novel mechanism for the regulation of growth factor-receptor interactions.

Animals↗
PubMed1991

Developmental expression of two murine fibroblast growth factor receptors, flg and bek.

Developmental expression of two closely related fibroblast growth factor receptors, bek and flg, is described from early postimplantation until advanced organogenesis. Transcripts of bek and flg were first seen in the primitive ectoderm of egg-cylinder-stage embryos. Later, starting with somitogenesis, and then throughout embryogenesis, they were actively transcribed both in the mesoderm and neuroectoderm. Bek was expressed also in the surface ectoderm and in various epithelia, whereas flg expression was restricted mainly to the mesenchyme. In the limb bud bek transcripts displayed a gradient-like distribution and appeared earlier than flg. The two receptors, in contrast to their almost identical ligand binding specificity, displayed distinct spatial specificities throughout development, suggesting that developmental localization may contribute to functional specificity. The role of bek and flg in gastrulation and in epithelial-mesenchymal interactions of organogenesis will be discussed.

Abdomen↗
PubMed1991

Flg-2, a new member of the family of fibroblast growth factor receptors.

Two genes, flg and bek, have been recently shown to encode receptors for fibroblast growth factors (FGF). Here we report the molecular cloning and sequence of a new member of the FGF receptor family, denoted flg-2, which was isolated from a human keratinocyte cDNA library. The cDNA sequence predicts an extracellular region possessing three immunoglobulin-like domains, a single transmembrane region and a cytoplasmic portion containing the tyrosine kinase domain split by a short inter-kinase segment. The amino acid sequence of flg-2 shows 68% and 64% identity with bek and flg, respectively. The most variable domain among the three genes is the amino-terminal immunoglobulin-like domain. Comparison with the chicken FGF receptor genes suggests that flg-2 is homologous to cek-2, whereas flg and bek are homologous to cek-1 and cek-3, respectively. Analysis of mRNA from various tissues shows that flg-2 is expressed predominantly in skin, brain and lung.

Amino Acid Sequence↗
PubMed1990

Autocrine regulation of cell growth and transformation by basic fibroblast growth factor.

Basic FGF (bFGF) and acidic FGF (aFGF) are multipotential factors that stimulate and support proliferation, migration and differentiation. Both bFGF and aFGF are non-secreted growth factors consistent with the lack of a signal peptide. However, bFGF and aFGF are deposited in extracellular matrix (ECM) suggesting that an alternative mechanism for FGF release exists. Four oncogenes, int-2, hst/K-fgf, FGF-5 and FGF-6 have been isolated that are highly homologous to aFGF and bFGF. Unlike bFGF and aFGF, they possess signal peptides and are secreted. These oncogenes transform cells and induce tumors, ostensibly via an autocrine mechanism. The involvement of bFGF and aFGF in autocrine transformation has been clarified by studies using FGF cDNA transfection. NIH-3T3 cells transfected with native bFGF cDNA and expressing 20 to 100 times as much bFGF as parental 3T3 cells acquire an enhanced proliferation rate and higher saturation density. NIH cells transfected with a construct in which bFGF cDNA is altered by addition of a signal peptide, undergo autocrine transformation and exhibit morphological and biochemical alterations characteristic of highly transformed cells. Injection of cells expressing native bFGF even at levels 100 times greater than parental 3T3 cells fails to induce tumors or lung metastasis in syngeneic mice. Signal peptide bFGF-transected cells on the other hand, acquire a high tumorigenic and metastatic potential with tumor incidence and numbers comparable to those induced by ras transformed cells. Acquisition of a signal peptide converts bFGF into a transforming protein analogous to FGF-related oncogenes which naturally have signal peptide sequences.

Animals↗
PubMed1990

Autocrine transformation by chimeric signal peptide-basic fibroblast growth factor: reversal by suramin.

NIH 3T3 cells transfected with basic fibroblast growth factor (bFGF) fused to an immunoglobulin signal peptide sequence are transformed in vitro and tumorigenic in vivo. The transformed phenotype of chimeric signal peptide-bFGF (spbFGF) cells is characterized by an enhanced proliferation rate compared to parental NIH 3T3 cells, density- and anchorage-independent growth, a transformed morphology, and lack of cell adhesion. The rate of spbFGF cell proliferation is not diminished by anti-bFGF neutralizing antibodies. 125I-labeled bFGF receptor cross-linking and binding studies suggest that surface FGF receptors in spbFGF cells are unavailable and down-regulated. The FGF receptors are also down-regulated in K-fgf-transformed cells but not in parental 3T3, native bFGF-transfected, and ras-transformed NIH 3T3 cells. The addition of suramin to spbFGF and K-fgf cells rapidly promotes the up-regulation of FGF receptors. Suramin also induces lowering of the proliferation rate to that of parental cells, anchorage-dependent growth, assembly of cytoskeletal filaments, cellular adhesion, and spreading. These results suggest that spbFGF cells undergo autocrine transformation, possibly by an internal autocrine loop, in which there is constitutive activation of the FGF receptor. Suramin inhibits autocrine transformation, leading to a normal untransformed phenotype.

Animals↗
PubMed1985

Susceptibility of human malaria parasites to chloroquine is pH dependent.

Chloroquine (CQ) accumulates in the acidic food vacuole of intraerythrocytic malaria parasites (Plasmodium falciparum) by virtue of its weak base properties. In the present work, the extent of CQ accumulation was determined by the transvacuolar pH gradient: modification of the latter--either by changing the external pH or by adding the acidotropic agent NH4Cl--led to a corresponding change in CQ distribution between cells and medium. Changes in pH gradient provoked a change in the susceptibility of parasites to CQ: at external pH values of 8.0, 7.4, and 6.8, the IC50 values for CQ were 0.48 X 10(-7) M, 1.8 X 10(-7) M, and 3.3 X 10(-7) M, respectively. Marked resistance to CQ (IC50 = 9.8 X 10(-7) M) was conferred upon cells by exposing them simultaneously to CQ and 10 mM NH4Cl, at pH 7.4. The final concentration of CQ attained within the acidic compartment of the parasite was correlated with inhibition of parasite growth. At therapeutic drug levels, CQ accumulation caused minor changes in the food vacuole pH, whereas at higher CQ concentrations substantial alkalinization was observed. The antimalarial activity of CQ is suggested to be exerted by the interference of the high concentrations of the accumulated drug with vital functions of the food vacuole.

Ammonium Chloride↗
PubMed1984

The malarial pigment in rat infected erythrocytes and its interaction with chloroquine. A Mössbauer effect study.

Mössbauer studies of rat erythrocytes infected by Plasmodium berghei malaria parasites, using 57Fe-enriched rat red blood cells, were carried out in order to determine the physical parameters which characterize the malarial pigment iron and to test the effect of the widely used antimalaria drug, chloroquine, on these parameters. The iron in the malarial pigment which is derived from hemoglobin digestion by the intracellular parasite was found to be trivalent, high spin, with Mössbauer parameters which are significantly different from those of any known iron porphyrin containing compound. No difference was found between the parameters obtained in erythrocytes infected by drug-sensitive and drug-resistant strains of P. berghei, both before and after the treatment with chloroquine. The iron compound consists of microaggregates, about 30 A in diameter. These are somewhat larger in chloroquine-resistant strains and tend to increase in size in chloroquine-sensitive strains upon treatment with the drug. Mössbauer spectra of erythrocytes infected by a chloroquine-resistant strain revealed pigment iron in relative amounts invariable of those found in chloroquine-sensitive strains, demonstrating that drug-resistant parasites indeed digest hemoglobin.

Animals↗
PubMed1984

Identification of the acidic compartment of Plasmodium falciparum-infected human erythrocytes as the target of the antimalarial drug chloroquine.

Chloroquine (CQ), the most widely used antimalarial drug, is an acidotropic agent (De Duve, 1983) which accumulates to high levels in malaria-infected erythrocytes. A possible site of accumulation of the drug, the parasite's food vacuole, has been implicated in the mode of action of CQ. We have defined the various compartments of Plasmodium falciparum-parasitized human erythrocytes in terms of their pH and capacity to accumulate bases. The host cell and the parasite cytosols were differentially labeled in situ with pH-sensitive fluorescein, and the parasite food vacuole was revealed by targeting fluoresceinated dextran via endocytosis. The pH of the various compartments obtained from fluorescence excitation spectra were 6.9 for the cytosol of normal and infected erythrocytes and 5.2 for the parasite food vacuole. Determination of CQ and methylamine accumulation in infected erythrocytes, in conjunction with morphometric determination of the relative sizes of the various cellular compartments, provided an independent assessment of the vacuolar pH, yielding a value of 5.0-5.2. Perturbation of the proton gradient, either by lowering extracellular pH or by alkalinization of the food vacuole with NH4Cl or monensin, resulted in a concomitant and reversible decrease in accumulation of the probe. We conclude that drug accumulation in malaria-infected erythrocytes can be fully accounted for by the steady-state proton gradients across the barriers delineating the various cellular compartments and the acidotropic properties of the drug.

Ammonium Chloride↗
PubMed1984

Effects of chloroquine on the feeding mechanism of the intraerythrocytic human malarial parasite Plasmodium falciparum.

Ultrastructural investigations of P. falciparum cultivated in vitro in human erythrocytes revealed new features of the feeding mechanism of the parasite. Mature trophozoites and schizonts take up a portion of the host cytosol by endocytosis which is restricted to cytostomes and which involves the invagination of both parasitophorous and parasite membranes. The resulting endocytic vesicles, surrounded by two concentric membranes, migrate towards the central food vacuole membrane. The external membrane of the endocytic vesicles apposes that of the food vacuole, leading to the internalization of vesicles bounded by a single membrane into the vacuole space where they are rapidly degraded. We conclude from this sequence of events that endocytic vesicles fuse with the food vacuole. Treatment of infected cells with therapeutic concentrations of chloroquine inhibited the last step of the feeding process, i.e. vacuolar degradation. This was manifested by the accumulation within the vacuolar space of intact vesicles bounded by single membranes. The implications of these findings for the antimalarial activity of chloroquine are discussed.

Animals↗
PubMed1984

Effects of mitochondrial protein synthesis inhibitors on the incorporation of isoleucine into Plasmodium falciparum in vitro.

Plasmodium falciparum was grown in human erythrocytes in vitro and the effect of chloramphenicol, erythromycin, and tetracycline on growth and maturation of the parasites and on their ability to incorporate [3H]isoleucine into protein was observed. Exposure of rings to high concentrations of chloramphenicol had little effect on subsequent maturation of the rings whereas brief (4 h) exposure of trophozoites caused a dose-dependent inhibition of subsequent ring formation. Incorporation of [3H]isoleucine into protein was not affected during at least 6 h of exposure to high concentration of the three drugs examined, but appreciable inhibition was observed after 21 h, with chloramphenicol being the least effective inhibitor. These results suggest that there is a stage-specific effect of inhibition of mitochondrial protein synthesis on subsequent development and that the mitochondria are essential for growth and development even though they lack a functional Krebs cycle.

Animals↗
PubMed1983

Stage-dependent effects of chloroquine on Plasmodium falciparum in vitro.

The erythrocytic developmental cycle of Plasmodium falciparum can be conveniently divided into the ring, trophozoite, and schizont stages based on morphology and metabolism. Using highly synchronous cultures of P. falciparum, considerable variation was demonstrated among these stages in sensitivity to chloroquine. The effects of timed, sequential exposure to several clinically relevant concentrations of chloroquine were monitored by three techniques: morphological analysis, changes in the rate of glucose consumption, and changes in the incorporation of 3H-hypoxanthine into parasite nucleic acids. All three techniques gave essentially identical results. The trophozoite and schizont stages were considerably more sensitive to the drug than ring-stage parasites. Chloroquine sensitivity decreased as nuclear division neared completion. The increase in chloroquine sensitivity was coincident with a marked rise in the rate of glucose consumption and nucleic acid synthesis. The rate of nucleic acid synthesis decreased as schizogony progressed while glucose consumption continued at high rates during this process. The degree of chloroquine sensitivity was not highly correlated with either metabolic activity.

Animals↗
PubMed1982

The transport of chloroquine across human erythrocyte membranes is mediated by a simple symmetric carrier.

The kinetic properties of the mediated transport of chloroquine in human erythrocytes are investigated. The high rates of translocation across the cell membrane and high adsorbance properties to glass surfaces have led to the development of new techniques for measuring initial rates of transport. Three different methodological procedures are used to accomplish a complete kinetic characterization of the system. All measurements were done at 25 degrees C. Under zero-trans conditions the system displays complete symmetry, the Michaelis constants being 39.2 +/- 2.4 microM for influx and 36.6 +/- 5.6 microM for efflux. The respective maximal velocities are 206.4 +/- 36.0 microM . min-1 and 190.0 +/- 7.8 microM . min-1. Under equilibrium-exchange conditions the Michaelis constant is 108.6 +/- 15.6 microM and the maximal velocity is 630.3 +/- 50.4 microM . min-1. This 3-fold increase in both K and V over the zero-trans values indicates that the rate-limiting step in the transport of chloroquine is the movement of the unloaded carrier. The kinetic data are consistent with the prediction of a simple carrier model.

Biological Transport↗