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Biomedical subjects

A Baird

Publications and source records attributed to A Baird.

At least 145 records · Page 8Linked to original sources
PubMed1991

Internalization and processing of basic fibroblast growth factor by neurons and astrocytes.

The fate of iodinated basic fibroblast growth factor (FGF) after its binding to cultured astrocytes and hippocampal neurons was studied. Autoradiography after light and electron microscopy establishes that, if cells are returned to 37 degrees C, the 125I-basic FGF bound internalizes into vesicles in the cytoplasm, localizes to the perinuclear cytoplasm, and is translocated to chromatin structures of the nucleus. The radiolabeled protein is long-lived, a finding confirmed by biochemical analyses. Polyacrylamide gel electrophoresis and autoradiography of both hippocampal neurons and astrocyte extracts reveal that these cells internalize 125I-basic FGF and then metabolize it to three major heparin-binding peptides with molecular weights of 15.5, 9, and 4 kDa. These peptides are initially detected 16 hr after binding to neurons and 4 hr after binding to astrocytes but are still detectable 48 and 16 hr, respectively, after initial binding (though present at lower levels). Immunoprecipitation with sequence-specific antisera to basic FGF reveals that the 15.5-kDa fragment is generated by cleavage at the carboxyl terminus, that the 9-kDa peptide contains the sequences between residues 30 and 87, and the 4-kDa peptide is a C-terminus fragment containing the sequence of basic FGF(106-120) but without basic FGF(139-146) immunoreactivity. The internalization of basic FGF is required for this processing; the treatment of cells with trypsin and 2 M NaCl at different times after binding can only prevent the metabolism of basic FGF if it is performed immediately after binding. Similarly, WGA, which inhibits basic FGF binding to its high-affinity receptor, prevents the metabolism of basic FGF. The possible significance of a metabolic pathway that is responsible for the processing of basic FGF after its internalization by cells in the CNS is discussed in light of its potential function as a neurotrophic factor.

Animals↗
PubMed1990

Endothelin releasing activity in calf serum and porcine follicular fluid.

Bovine calf serum (CS) and porcine follicular fluid (PFF) added to bovine aortic endothelial cells (BAEC) in culture stimulate endothelin (ET) release. Sorbent extraction of acidified calf serum and PFF using Bond Elut C18 yields a product with an activity 30% that of the starting material. Fractionation of Bond Elut C18 extracted CS or PFF using HPLC yields peaks of activity with retention times similar to those of TGF beta and activin A, respectively. TGF beta is 10-fold more potent than activin A to stimulate ET release from BAECs. The observation that TGF beta and activin A are dose-additive but not effect-additive to stimulate ET release is compatible with the conclusion that these two substances act through a similar mechanism. Thus, TGF beta and/or activin A-related peptides may contribute to the ET releasing activity observed in acidified Bond Elut C18 extracted CS and PFF. The identity of ET-releasing activity in native CS and PFF remains to be established.

Activins↗
PubMed1990

Mediation of virion penetration into vascular cells by association of basic fibroblast growth factor with herpes simplex virus type 1.

Herpes simplex virus type-1 (HSV-1) is a ubiquitous pathogen that is associated with considerable morbidity in the general population. Although it is known that the virion uses a basic fibroblast growth factor (FGF) receptor to penetrate vascular cells, it is not known how the viral particle recognizes and binds to this cell surface protein. Here we report that an immunoreactive basic FGF-like protein is associated with the viral particle and that this association appears responsible for viral uptake. Accordingly, HSV-1 infection of Swiss 3T3 cells stimulates the tyrosine phosphorylation of the specific substrate that characterizes the initial cellular response to basic FGF. Antibodies to basic FGF prevent this phosphorylation and inhibit HSV-1 uptake. Because no basic FGF sequence is found in the HSV-1 genome, a model for the infection for some target cells is presented whereby the viral particle uses host cell-derived basic FGF to ensure subsequent infectivity of newly replicated virus.

Animals↗
PubMed1990

Identification of a ligand for the c-kit proto-oncogene.

We report the purification and N-terminal amino acid sequence of a novel mast cell growth factor, termed MGF, from the supernatants of a murine stromal cell line. A panel of interleukin 3-dependent cell lines were screened for responsiveness to partially purified MGF in [3H]thymidine incorporation assays; proliferative stimulation of these cells in response to MGF correlated with expression of mRNA for the c-kit protooncogene. MGF was shown to be a ligand for c-kit by cross-linking 125I-labeled MGF to c-kit-expressing cells with subsequent immunoprecipitation of the complex with antiserum specific for the C-terminus of c-kit. This establishes MGF as a ligand for the c-kit protein.

Amino Acid Sequence↗
PubMed1990

Molecular cloning of mast cell growth factor, a hematopoietin that is active in both membrane bound and soluble forms.

We have previously reported the identification of a novel mast cell growth factor (MGF) that was shown to be a ligand for c-kit and is encoded by a gene that maps near the steel locus on mouse chromosome 10. We now report the cloning of cDNAs encoding the MGF protein. The MGF protein encoded by this cDNA can be expressed in a biologically active form as either a membrane bound protein or as a soluble factor. The soluble protein promotes the proliferation of MGF-responsive cell lines and, in the presence of erythropoietin, stimulates the formation of macroscopic [corrected] erythroid and multilineage hematopoietic colonies.

Amino Acid Sequence↗
PubMed1990

Basic fibroblast growth factor in Alzheimer's disease.

We have examined the presence of basic fibroblast growth factor (FGF) in normal and in Alzheimer brains, studied the distribution of the mitogen by immunohistochemical techniques, measured the quantities of growth factor in selected areas of the brain (Brodmann areas 10/11 and 20/21), characterized the molecular forms by Western blotting and determined its sites of synthesis by in situ hybridization. Although the same molecular forms of basic FGF are found in control and Alzheimer brains, basic FGF is increased in the brains of Alzheimer's patients. Furthermore, basic FGF is not distributed in an identical fashion to normal and Alzheimer brains, but is found in association with the lesions that characterize this disease. In normal controls (n = 5), basic FGF was found to be widely distributed throughout the three brain regions examined (prefrontal cortex, hippocampus, and hypothalamus). Immunoreactivity was observed within astrocytes in both the grey and white matter, as well as within neuronal perikarya. Brain tissues that were obtained from Alzheimer patients (N = 4) showed a substantial increase in the overall specific staining of astrocytes and neurons, particularly in areas of reactive gliosis. Focal concentration of immunoreactive basic FGF was evident within the neuritic plaques, and could be clearly seen in association with the neurofibrillary tangles present within neuronal perikarya. The possibility that basic FGF expression in the CNS is linked to the pathogenesis of the disease is discussed.

Alzheimer Disease↗
PubMed1990

Fibroblast growth factor receptor is a portal of cellular entry for herpes simplex virus type 1.

Herpes simplex virus type 1 (HSV-1) is a ubiquitous pathogen responsible for considerable morbidity in the general population. The results presented herein establish the basic fibroblast growth factor (FGF) receptor as a means of entry of HSV-1 into vertebrate cells. Inhibitors of basic FGF binding to its receptor and competitive polypeptide antagonists of basic FGF prevented HSV-1 uptake. Chinese hamster ovary (CHO) cells that do not express FGF receptors are resistant to HSV-1 entry; however, HSV-1 uptake is dramatically increased in CHO cells transfected with a complementary DNA encoding a basic FGF receptor. The distribution of this integral membrane protein in vivo may explain the tissue and cell tropism of HSV-1.

Adsorption↗
PubMed1990

Characterization of endothelin secretion by vascular endothelial cells.

The characterization of mechanisms that regulate ET-LP secretion from bovine adrenal cortical capillary endothelial cells (ACE) in culture was performed by developing radioimmunoassays that distinguish between ET1-21 (AbET1-21) and ET1-39 (AbET1-39). The conditioned media (DMEM) content of ET-like immunoreactivity (ET1-21LI) increased from 50 to 350 pg/ml over a 24 h period. Addition of 10% calf serum or 0.1% BSA enhanced ET1-21LI release 2-3 fold. Authenticity of ET1-21LI was examined using reversed phase liquid chromatography. All ET1-21LI co-eluted with authentic ET-1. Examination of ET1-39IR by liquid chromatography revealed two peaks of immunoreactivity, one co-eluting with authentic ET22-39 and a later running peak co-eluting with authentic ET1-39. Neither ET1-21LI nor ET1-39LI was detected in the extracts of sonicated ACE cells. Treatment of cells with various forms of TGF beta significantly augmented ET1-21LI release. These data suggest that ACE secretion of ET-LP in vitro spontaneously and can be enhanced by TGFss. Since neither ET1-21 LI nor ET1-39 LI was detectable detectable in ACE cells it is unlikely that ET-LP are stored prior to their secretion.

Animals↗
PubMed1990

Mitotoxins: growth factor-targeted cytotoxic molecules.

When the selective specificity and exquisite affinity of growth factors for their receptors is conferred to protein toxins, the chimeric molecules so generated become potent cytotoxins. Chimera are produced by the chemical conjugation of the two proteins or by expression of fusion proteins in bacterial expression systems. The toxic moiety, usually a ribosome-inactivating protein or a fragment of a bacterial toxin, is internalized into target cells by receptor-mediated endocytosis. Release of the receptor's ligand in the endosome allows the toxic moiety to exert its action on protein synthesis. Accordingly, the potent catalytic activity of the toxin results in a sensitivity of some cells to picomolar quantities of the mitotoxin. This review discusses the numerous growth factor-toxins that have been created and describes some of their applications.

Animals↗
PubMed1990

Distribution of basic fibroblast growth factor in the 18-day rat fetus: localization in the basement membranes of diverse tissues.

Immunohistochemical methods were used to study the distribution of basic FGF in the 18-d rat fetus. The results reveal a pattern of widespread yet specific staining that is consistent with the wide distribution of basic FGF. Immunoreactive basic FGF is associated with mesenchymal structures, mesoderm- and neuroectoderm-derived cells, and their extracellular matrices. As an example, skeletal and smooth muscle cells are strongly positive. The basement membrane underlying the epithelia always contain basic FGF. In some tissues (i.e., cartilage and bone) the intensity of immunostaining is dependent on the stage of cell differentiation. Although the staining of tissues is primarily associated with the extracellular matrix, there is significant intracellular staining in various cell types. This is particularly evident in the endocrine cells of the adrenal cortex, testis, and ovary. The histochemical findings reported here support the notion that basic FGF has the characteristics required to mediate many of the effects of the mesenchyme on cell growth and differentiation. The significance of these findings in understanding the role of basic FGF in regulating cell proliferation and differentiation is discussed.

Animals↗
PubMed1990

Selective elimination of fibroblasts from pancreatic islet monolayers by basic fibroblast growth factor-saporin mitotoxin.

Fibroblast proliferation regularly impedes the initiation and maintenance of pancreatic islet monolayers in culture. We recently characterized a specific cytotoxin to cells expressing the basic fibroblast growth factor receptor by conjugating the growth factor to saporin-6, a ribosome-inactivating protein. In contrast to untreated islets, isolated adult rat islets grown on a substrate prepared from bovine corneal endothelial cells and incubated with the mitotoxin at 10-nM concentration for 96 h were free of contaminating fibroblasts. Histological and functional studies revealed there was no damage to the islets. The results suggest that treatment of this cell type with basic fibroblast growth factor mitotoxins may be an important tool for culture of pure islets for physiological and clinical studies.

Animals↗
PubMed1989

Biological and chemical characterization of basic FGF-saporin mitotoxin.

Basic fibroblast growth factor (FGF) and saporin-6, a ribosome-inactivating protein, were chemically conjugated and characterized as a cytotoxin to cells expressing the basic FGF receptor. Structural and Western blot analysis of the conjugate showed that it contained saporin and basic FGF in equimolar amounts. The conjugate inhibited protein synthesis in a cell-free system and had potent cytotoxic activity (ID50 = 25pM) for cells expressing the basic FGF receptor. It is equipotent with basic FGF in radioreceptor assays and elutes from heparin Sepharose columns with 2M NaCl. The activity of the mitotoxin can be inhibited by competition with an excess of basic FGF but not nerve growth factor. The possibility that this mitotoxin can be used as an anti-angiogenic factor in paradigms that involve basic FGF is discussed.

Animals↗
PubMed1989

Characterization of the neuronal receptor for basic fibroblast growth factor and comparison to receptors on mesenchymal cells.

The receptor for basic fibroblast growth factor (bFGF) was characterized in highly enriched cultures of fetal hippocampal neurons. Two major components of binding could be distinguished. One component comprising about 70% of total binding was removed by 2 M NaCl, and by analogy to other cells could be presumptively attributed to glycosaminoglycans. The remaining 30% of binding which was resistant to 2 M NaCl reflects a high affinity receptor. Scatchard analysis indicated that the two components have Kd values of 500 and 120 pM, and densities of 165,000 and 35,000-60,000 sites/neuron, respectively. Cross-linking 125I-bFGF to neuronal cultures with disuccinimidyl suberate labeled a major membrane protein of 135 kDa and a minor protein of 85 kDa. Examination of neuronal cultures derived from multiple brain regions and membrane preparations from fetal brain suggested that the larger protein was widely distributed. The neuronal bFGF receptor was not blocked by heparin at concentrations up to 100 micrograms/ml. Twelve synthetic peptide fragments of bFGF were examined to determine the domain of bFGF interacting with the neuronal receptor. Inhibition was observed chiefly with a peptide including the sequence 103-146. The properties of the neuronal bFGF receptor were compared directly to those of the receptors characterized on BHK cells and other mesenchymal cells. Specific differences observed between neuronal and mesenchymal bFGF receptors are discussed.

Animals↗
PubMed1989

Interactions between growth hormone-releasing hormone and glucocorticoids in male rats.

While chronic glucocorticoid treatment increases pituitary growth hormone (GH) content in rats and primates and increases pituitary GH release in response to growth hormone-releasing hormone (GHRH) in rats, it also inhibits somatic growth. We investigated these opposite actions in rats using the synthetic glucocorticoid dexamethasone. Seven days of dexamethasone treatment (40 micrograms/animal per day) did not alter the frequency of spontaneous GH pulses in conscious, freely-moving animals. The amplitude of the GH pulses in saline and dexamethasone-treated rats was different (P less than 0.01), the latter group having a higher incidence of GH levels less than 95 ng/ml, a lower incidence of GH levels between 96 and 251 ng/ml, and a higher incidence of GH values greater than 480 ng/ml. A 20 microgram/kg per day dose of dexamethasone was sufficient to significantly inhibit growth but was inadequate in enhancing the GH response to an acute injection of GHRH in anesthetized animals. These results support the concept that glucocorticoids exert their catabolic effects on somatic growth in peripheral tissues and not at the pituitary level.

Animals↗
PubMed1989

Basic fibroblast growth factor is a substrate for protein phosphorylation and is phosphorylated by capillary endothelial cells in culture.

A phosphorylated basic fibroblast growth factor (FGF) can be detected in extracts of bovine capillary endothelial cells and human hepatoma cells. Accordingly, human basic FGF contains consensus sequences that account for its phosphorylation on Thr-112 by the catalytic subunit of the cAMP-dependent protein kinase A (PK-A) and on Ser-64 by the calcium- and phospholipid-dependent protein kinase C (PK-C). A kinetic analysis of both of these reactions revealed that basic FGF is among the better substrates for these enzymes. Although the kinase responsible for the phosphorylation in vivo has not yet been identified, we examined the effects of phosphorylation on the biological activity, heparin-binding capacity, and receptor-binding capacity of phosphorylated basic FGF. No effects of phosphorylation were observed when the mitogen was phosphorylated by PK-C. In contrast, when basic FGF was phosphorylated in the receptor-binding domain with PK-A, the growth factor was 3-8 times better at displacing radiolabeled basic FGF in the radioreceptor assay. No effects were seen on the binding of this FGF to immobilized heparin or cell-associated glycosaminoglycans, suggesting that this phosphorylation modifies the affinity of basic FGF for its receptor. Biological assays for basic FGF failed to identify differences between the phosphorylated and unphosphorylated forms of recombinant basic FGFs presumably because of the presence of ectophosphatases and the experimental conditions of proliferation and mitogenic assays (37 degrees C, 24-96 hr). Because the relative affinity of basic FGF for its receptor and cell-associated glycosaminoglycans may regulate its activity, the identification of a modified form of basic FGF may be of particular importance in understanding the mechanisms that regulate its biological activity, bioavailability, and processing to and from the extracellular matrix.

Animals↗
PubMed1989

Differential effects of heparin, fibronectin, and laminin on the phosphorylation of basic fibroblast growth factor by protein kinase C and the catalytic subunit of protein kinase A.

Basic fibroblast growth factor (FGF) is synthesized as a phosphoprotein by both bovine capillary endothelial and human hepatoma cells in culture. Because basic FGF is characterized by its high affinity for heparin and its association in vivo with the extracellular matrix, we examined the possibility that the phosphorylation of this growth factor by purified protein kinase C (PK-C) and the catalytic subunit of cAMP-dependent protein kinase-A (PK-A) can be modulated by components of the extracellular matrix. Heparin and other glycosaminoglycans (GAGs) inhibit the ability of PK-C to phosphorylate basic FGF. In contrast, heparin can directly increase the phosphorylation of basic FGF by PK-A. While fibronectin, laminin, and collagen IV have no effect on the ability of PK-C to phosphorylate basic FGF, they all can inhibit the effects of PK-A. Thus, there is a differential effect of extracellular matrix-derived proteins and GAGs on the phosphorylation of basic FGF. The enhanced phosphorylation of basic FGF that is mediated by heparin is associated with a change in the kinetics of the reaction and the identity of the amino acid targeted by this enzyme. The amino acids that are targeted by PK-C and PK-A have been identified by phosphopeptide analyses as Ser64 and Thr112, respectively. In the presence of heparin, basic FGF is no longer phosphorylated by PK-A at the usual PK-A consensus site (Thr112), but instead is phosphorylated at the canonical PK-C site (Ser64). Accordingly, heparin inhibits the phosphorylation of basic FGF by PK-C presumably by masking the PK-C dependent consensus sequence surrounding Ser64. Thus, when basic FGF is no longer phosphorylated by PK-A in the receptor binding domain (Thr112), it loses the increased receptor binding ability that characterizes PK-A phosphorylated basic FGF. The results presented here demonstrate three novel features of basic FGF. First, they identify a functional effect of the binding of heparin to basic FGF. Second, they establish that the binding of heparin to basic FGF can induce structural changes that alter the substrate specificity of protein kinases. Third, and perhaps most important, the results demonstrate the existence of a novel interaction between basic FGF, fibronectin, and laminin. Although the physiological significance of this phosphorylation is not known, these results clearly suggest that the biological activities of basic FGF are regulated by a complex array of biochemical interactions with the proteins, proteoglycans, and glycosaminoglycans present in the extracellular milieu and the cytoplasm.

Amino Acid Sequence↗
PubMed1989

Fibroblast growth factors.

The existence of fibroblast growth factors (FGFs) was proposed over 40 years ago to account for the ability of tissue extracts to stimulate fibroblast proliferation. In the 1970s it became clear that preparations containing FGF activity were in fact pleiotropic, affecting the growth and function of a wide variety of mesenchymal, endocrine and neural cells. Their angiogenic effects have promoted research in cardiology and neurology because of their proposed role in stimulating collateral vascularisation and recovery from ischemia. Their identity with a component of tumour angiogenesis factor activity has stimulated research in oncology and their capacity to enhance wound healing, nerve regeneration and cartilage repair has affected research in neurology, orthopaedic medicine and pathology. The potential therapeutic value of FGFs is just beginning to be realized and will be dependent on a concerted effort to establish their function in the regulation of normal cell homeostasis and the pathophysiology of disease.

Animals↗