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1.
Bull Exp Biol Med ; 158(1): 118-22, 2014 Nov.
Article in English | MEDLINE | ID: mdl-25403412

ABSTRACT

Replacement cell therapy with transplantation of stem cells is a promising approach for the therapy of various neurodegenerative diseases, e.g. Alzheimer's disease. However, the behavior of transplanted cells in the damaged tissue should be thoroughly studied before introduction of this method into clinical practice. We studied the pathways of migration of human multipotent mesenchymal stromal cells after their systemic transplantation into the brain of bulbectomized mice characterized by the development of Alzheimer-type neurodegenerative process. Immunohistochemical analysis with antibodies to human nuclear antigen (HNA) and immunofluorescent analysis of the results of transplantation of multipotent mesenchymal stromal cells carrying green fluorescent protein (GFP) gene showed that these cells can cross the blood-brain barrier and penetrate into some structures of recipient brain. Analysis of differentiation of transplanted human cells using antibodies to neurospecific enolase (NSE) or astroglial marker (GFAP) with parallel staining for human nuclear antigen revealed no neural differentiation of transplanted cells in the brains of bulbectomized animals. However, some of these cells differentiated into astrocytes, which brought us to an assumption on important role of astroglial abnormalities in the pathogenesis of Alzheimer's disease.


Subject(s)
Cell Differentiation , Mesenchymal Stem Cell Transplantation , Mesenchymal Stem Cells/physiology , Alzheimer Disease/therapy , Animals , Cell Movement , Cells, Cultured , Hippocampus/pathology , Humans , Mice , Olfactory Bulb/pathology
2.
Planet Space Sci ; 77(100): 3-11, 2013 Mar.
Article in English | MEDLINE | ID: mdl-23585696

ABSTRACT

During the years 2000-2011 the radio instruments onboard Cassini, Wind and STEREO spacecraft have recorded a large amount of the Jovian decametric radio emission (DAM). In this paper we report on the analysis of the new type of Jovian periodic radio bursts recently revealed in the decametric frequency range. These bursts, which are non-Io component of DAM, are characterized by a strong periodic reoccurrence over several Jovian days with a period [Formula: see text] longer than the rotation rate of the planet's magnetosphere (System III). The bursts are typically observed between 4 and 12 MHz and their occurrence probability has been found to be significantly higher in the sector of Jovian Central Meridian Longitude between 300° and 60° (via 360°). The stereoscopic multispacecraft observations have shown that the radio sources of the periodic bursts radiate in a non-axisymmetric hollow cone-like pattern and sub-corotate with Jupiter remaining active during several planet's rotations. The occurrence of the periodic non-Io DAM bursts is strongly correlated with pulses of the solar wind ram pressure at Jupiter. Moreover the periodic bursts exhibit a tendency to occur in groups every [Formula: see text] days. The polarization measurements have shown that the periodic bursts are right hand polarized radio emission associated with the Northern magnetic hemisphere of Jupiter. We suggest that periodic non-Io DAM bursts may be connected with the interchange instability in Io plasma torus triggered by the solar wind.

7.
Matrix Biol ; 20(8): 527-41, 2001 Dec.
Article in English | MEDLINE | ID: mdl-11731270

ABSTRACT

Transforming growth factor beta (TGF-beta) stimulates protein complex formation on a TGF-beta response element (TAE) found in the distal portion (-1624) of the collagen alpha 1(I) promoter. To identify the fibroblast proteins in this complex, an expression library constructed from human embryonic lung fibroblasts mRNA was screened using a tetramer of TAE. Y-box binding protein (YB-1), was identified as a protein in the TAE-protein complex. The protein expressed by phage clones formed a specific complex with labeled TAE but not mutated TAE (mTAE) similar to the complex formed with nuclear protein. Nuclear protein-TAE complexes isolated from native gels contained YB-1 by Western analysis. TGF-beta treatment increased the amount of YB-1 protein in nuclear extracts, decreased its amount in cytoplasm, but did not alter the steady state levels of YB-1 mRNA. A full-length YB-1 protein expressed in human lung fibroblasts was primarily located in the nucleus with punctate staining in cytoplasmic regions. The expression of YB-1 decreased in the cytoplasm after 2 h of TGF-beta treatment. Therefore, the increased binding activity seen in TGF-beta-stimulated nuclear extracts was due primarily to relocalization of YB-1 from the cytoplasm to the nuclear compartment. Co-transfection of YB-1 cDNA with a collagen promoter-reporter construct caused a dose-dependent activation of collagen promoter activity in rat fibroblasts whereas the promoter with a mutation in the TAE element was not sensitive to YB-1 co-expression. In conclusion, we have identified YB-1 as a protein that interacts with a TGF-beta response element in the distal region of the collagen alpha 1(I) gene. YB-1 protein activates the collagen promoter and translocates into the nucleus during TGF-beta addition to fibroblasts, suggesting a role for this protein in TGF-beta signaling.


Subject(s)
CCAAT-Enhancer-Binding Proteins/metabolism , Collagen Type I/genetics , DNA-Binding Proteins , Transcription Factors , Animals , Base Sequence , Binding Sites/genetics , CCAAT-Enhancer-Binding Proteins/genetics , Cell Line , Chloramphenicol O-Acetyltransferase/genetics , DNA, Complementary/genetics , DNA, Complementary/metabolism , Gene Expression Regulation , Genes, Reporter , Humans , NFI Transcription Factors , Nuclear Proteins , Promoter Regions, Genetic , RNA, Messenger/genetics , RNA, Messenger/metabolism , Rats , Recombinant Fusion Proteins/genetics , Recombinant Fusion Proteins/metabolism , Signal Transduction , Transfection , Transforming Growth Factor beta/pharmacology , Y-Box-Binding Protein 1
8.
Am J Physiol Lung Cell Mol Physiol ; 281(4): L766-75, 2001 Oct.
Article in English | MEDLINE | ID: mdl-11557580

ABSTRACT

Previously, we have demonstrated that basic fibroblast growth factor (bFGF) decreases elastin gene transcription in confluent rat lung fibroblasts via the binding of a Fra-1-c-Jun heterodimer to an activator protein-1-cAMP response element in the distal region of the elastin promoter. In the present study, we show that bFGF activates the mitogen-activated protein kinase extracellular signal-regulated kinase 1/2, resulting in the translocation of phosphorylated extracellular signal-regulated kinase 1/2 into the nucleus followed by increased binding of Elk-1 to the serum response element of the c-Fos promoter, transient induction of c-Fos mRNA, and sustained induction of Fra-1 mRNA. The addition of PD-98059, an inhibitor of mitogen-activated protein kinase kinase, abrogates the bFGF-dependent repression of elastin mRNA expression. Comparative analyses of confluent and subconfluent fibroblast cultures reveal significant differences in elastin mRNA levels and activator protein-1 protein factors involved in the regulation of elastin transcription. These findings suggest that bFGF modulates specific cellular events that are dependent on the state of the cell and provide a rationale for the differential responses that can be expected in development and injury or repair situations.


Subject(s)
DNA-Binding Proteins , Elastin/genetics , Fibroblast Growth Factor 2/pharmacology , Fibroblasts/enzymology , Lung/cytology , MAP Kinase Signaling System/drug effects , Transcription Factors , Animals , Elastin/metabolism , Enzyme Inhibitors/pharmacology , Flavonoids/pharmacology , Gene Expression/drug effects , Mitogen-Activated Protein Kinases/metabolism , Phosphorylation , Proto-Oncogene Proteins/metabolism , Proto-Oncogene Proteins c-fos/genetics , Proto-Oncogene Proteins c-fos/metabolism , RNA, Messenger/analysis , Rats , Rats, Sprague-Dawley , Transcription Factor AP-1/metabolism , Transcription, Genetic/drug effects , ets-Domain Protein Elk-1
11.
Am J Physiol Cell Physiol ; 278(1): C92-C101, 2000 Jan.
Article in English | MEDLINE | ID: mdl-10644516

ABSTRACT

Heme oxygenase-1 (HO-1) catalyzes the rate-limiting step in heme catabolism and presumably is involved in cellular iron homeostasis. It is induced by a variety of cellular stresses, including oxygen deprivation and free radical-mediated stress. We examined induction of HO-1 mRNA in skin fibroblasts and investigated the mechanism by which it occurs. Hypoxia did not appear to act via induction of oxygen free radicals: induction of HO-1 was not sensitive to the free radical scavenger GSH or other antioxidants. Moreover, hypoxia did not increase steady-state levels of free radicals generated by fibroblasts. In contrast, HO-1 induction by the oxidants, H(2)O(2) and carbonyl cyanide m-chlorophenylhydrazone (CCCP) was significantly attenuated in the presence of free radical scavengers. This correlated with increased levels of free radical production in fibroblasts treated with these oxidants. Iron depletion by desferrioxamine mesylate, a specific iron complexon, completely inhibited hypoxic stimulation of HO-1 but did not attenuate the effect of H(2)O(2) and CCCP on HO-1 mRNA. Addition of Fe(2+), Fe(3+), or holo-transferrin to fibroblasts increased levels of HO-1 mRNA. Treatment of cells with hypoxia, but not H(2)O(2) or an exogenous source of iron, significantly increased the half-life of HO-1 mRNA. The data suggest hypoxia regulates HO-1 gene expression by a specific posttranscriptional mechanism: stabilization of mRNA. Hypoxia has previously been shown to increase fibroblast collagen synthesis and is thought to play a role in pathogenesis of systemic sclerosis (SSc). Skin fibroblasts isolated from patients with SSc demonstrated significantly stronger induction of HO-1 by hypoxia than did fibroblasts from normal controls. We hypothesize that exposure of SSc fibroblasts to hypoxic conditions leads to in vivo selective proliferation of cells that adapt to hypoxia.


Subject(s)
Dermis/cytology , Dermis/enzymology , Heme Oxygenase (Decyclizing)/genetics , Heme Oxygenase (Decyclizing)/metabolism , Carbonyl Cyanide m-Chlorophenyl Hydrazone/pharmacology , Cell Hypoxia/drug effects , Cell Hypoxia/physiology , Cells, Cultured , Chelating Agents/pharmacology , Deferoxamine/pharmacology , Fibroblasts/cytology , Fibrosis , Free Radicals/metabolism , Gene Expression Regulation, Enzymologic , Glutathione/pharmacology , Heme Oxygenase-1 , Humans , Hydrogen Peroxide/pharmacology , Iron/metabolism , Membrane Proteins , Oxidants/pharmacology , Oxidative Stress/drug effects , Oxidative Stress/physiology , RNA, Messenger/metabolism , Scleroderma, Systemic/metabolism , Scleroderma, Systemic/pathology , Uncoupling Agents/pharmacology
12.
Vopr Med Khim ; 44(4): 347-52, 1998.
Article in Russian | MEDLINE | ID: mdl-9845921

ABSTRACT

Using immunoblotting with specific antibodies, we have identified beta 1- and beta 2-subunits of Gi-proteins in membrane and cytosolic fractions of pig lung. It has been shown that beta 1-subunit is present both in membrane and cytosolic fractions, whereas beta 2-subunit is associated only with membranes. Activation of membrane-bound G proteins with non-hydrolysable GTP analogues have led to partial release from membrane of beta 1-, but not beta 2-subunits. When depolymerisation of F-actin during fractionation was prevented, both beta 1- and beta 2-subunits were found in fraction containing total membranes and F-actin. Dialysis of this fraction into low ionic strength buffer caused depolymerization of the bulk of actin and release of about 1/4 of beta 1-subunits into solution. The data presented here suggest that distribution of beta 1-subunits between membrane and cytosol could depend on the state of actin cytoskeleton.


Subject(s)
Actins/metabolism , Cytoskeleton/metabolism , GTP-Binding Proteins/metabolism , Animals , Cell Membrane/metabolism , Cytosol/metabolism , Lung/metabolism , Osmolar Concentration , Protein Binding , Swine
13.
J Biol Chem ; 273(43): 28298-304, 1998 Oct 23.
Article in English | MEDLINE | ID: mdl-9774453

ABSTRACT

The betagamma subunits of the heterotrimeric GTP-binding proteins (G proteins) that couple heptahelical, plasma membrane-bound receptors to intracellular effector enzymes or ion channels directly regulate several types of effectors, including phospholipase Cbeta and adenylyl cyclase. The beta subunit is made up of two structurally different regions: an N-terminal alpha helix followed by a toroidal structure made up of 7 blades, each of which is a twisted beta sheet composed of four anti-parallel beta strands (Wall, M. A., Coleman, D. E., Lee, E., Iñiguez-Lluhi, J. A., Posner, B. A., Gilman, A. G., and Sprang, S. R. (1995) Cell 83, 1047-1058; Lambright, D. G., Sondek, J., Bohm, A., Skiba, N. P., Hamm, H. E., and Sigler, P. B. (1996) Nature 379, 311-319). We have previously shown that sites for activation of PLCbeta2, PLCbeta3, and adenylyl cyclase II overlap on the "top" surface of the propeller, where Galpha also binds (Li, Y., Sternweis, P. M., Charnecki, S., Smith, T. F., Gilman, A. G., Neer, E. J., and Kozasa, T. (1998) J. Biol. Chem. 273, 16265-16272). The present study was undertaken to identify the regions on the side of the torus that might be important for effector interactions. We made mutations in each of the outer beta strands of the G protein beta1 propeller, as well as mutations in the loops that connect the outer strands to the adjacent beta strands. Our results suggest that activation of PLCbeta2 involves residues in the outer strands of blades 2, 6, and 7 of the propeller. We tested three of the mutations that most severely affected PLCbeta2 activity against two forms of adenylyl cyclase (ACI and ACII). Both inhibition of ACI and activation of ACII were unaffected by these mutations, suggesting that if ACI and ACII contact the outer strands, the sites of contact are different from those for PLCbeta2. We propose that distinct sets of contacts along the sides of the propeller will define the specificity of the interaction of betagamma with effectors.


Subject(s)
GTP-Binding Proteins/metabolism , Isoenzymes/metabolism , Type C Phospholipases/metabolism , Adenylyl Cyclases/metabolism , Animals , Binding Sites , Enzyme Activation , GTP-Binding Proteins/genetics , Humans , Models, Molecular , Mutagenesis , Phospholipase C beta , Protein Binding , Protein Conformation , Rats , Recombinant Proteins/metabolism
14.
Biochim Biophys Acta ; 1403(1): 115-25, 1998 May 27.
Article in English | MEDLINE | ID: mdl-9622608

ABSTRACT

Alveolar epithelial type 2 (T2) cells isolated from the lungs of adult rats responded to exogenous atrial natriuretic peptide (ANP) by two signalling mechanisms. First, ANP induced a dose-dependent reduction of ligand-stimulated adenylyl cyclase activity and cAMP accumulation. This effect was inhibited by the addition of GDPbetaS or by pretreatment with pertussis toxin (PT), consistent with mediation by a Gi protein(s). PT-catalyzed [32P]ADP-ribosylation, immunoblots with specific antisera, and Northern blot analysis demonstrated that T2 cells contain the G-proteins Gi2 and Gi3 which could transduce this signal. ANP also promoted PT-insensitive, dose-dependent accumulation of cGMP, consistent with activation of a receptor guanylyl cyclase. Isoproterenol-stimulated phosphatidylcholine secretion was markedly attenuated by ANP, and this effect was inhibited by PT pretreatment, consistent with mediation by a Gi protein(s). These data indicate that in addition to the lung being a major clearance organ for circulating ANP, lung parenchymal cells are targets of ANP action.


Subject(s)
Adenylyl Cyclases/drug effects , Atrial Natriuretic Factor/pharmacology , Epithelial Cells/cytology , Epithelial Cells/drug effects , Guanylate Cyclase/drug effects , Pulmonary Alveoli/cytology , Surface-Active Agents/metabolism , Adenylate Cyclase Toxin , Adenylyl Cyclases/metabolism , Animals , Atrial Natriuretic Factor/administration & dosage , Bronchodilator Agents/pharmacology , Cell Membrane/drug effects , Cell Membrane/enzymology , Cyclic AMP/metabolism , Cyclic GMP/metabolism , Dose-Response Relationship, Drug , Epithelial Cells/enzymology , GTP-Binding Proteins/drug effects , GTP-Binding Proteins/metabolism , Guanylate Cyclase/metabolism , Isoproterenol/pharmacology , Ligands , Pertussis Toxin , Phosphatidylcholines/metabolism , Pulmonary Alveoli/drug effects , Pulmonary Alveoli/enzymology , Rats , Virulence Factors, Bordetella/pharmacology
15.
Biochemistry ; 35(44): 13985-94, 1996 Nov 05.
Article in English | MEDLINE | ID: mdl-8909296

ABSTRACT

The family of WD-repeat proteins comprises over 30 different proteins that share a highly conserved repeating motif [Neer, E. J., Schmidt, C. J., Nambudripad, R., & Smith, T. F. (1994) Nature 371, 297-300]. Members of this family include the signal-transducing G protein beta subunit, as well as other proteins that regulate signal transduction, transcription, pre-mRNA splicing, cytoskeletal organization, and vesicular fusion. The crystal structure of one WD-repeat protein (G beta) has now been solved (Wall et al., 1995; Sondek et al, 1996) and reveals that the seven repeating units form a circular, propeller-like structure with seven blades each made up of four beta strands. It is very likely that all WD-repeat proteins form a similar structure. If so, it will be possible to use information about important surface regions of one family member to predict properties of another. If WD proteins form structures similar to G beta, their hydrodynamic properties should be those of compact, globular proteins, and they should be resistant to cleavage by trypsin. However, the only studied example of a WD-repeat protein, G beta, synthesized in vitro in a rabbit reticulocyte lysate, is unable to fold into a native structure without its partner protein G gamma. The non-WD-repeat amino terminal alpha helix of G beta does not inhibit folding because G beta does not fold even when this region is removed. It is not known whether all WD-repeat proteins are unable to fold when synthesized in an in vitro system. We synthesized seven members of the family in a rabbit reticulocyte lysate, determined their Stokes radius, sedimentation coefficient, and frictional ratio, and assayed their stability to trypsin. Our working definition of folding was that the proteins from globular, trypsin-resistant structures because, except for G beta gamma, their functions are not known or cannot be assayed in reticulocyte lysates. We chose proteins that include amino and carboxyl extensions as well as proteins that are made up entirely of WD-repeats. We show that unlike G beta, several proteins with WD-repeats are able to fold into globular proteins in a rabbit reticulocyte lysate. One protein, beta Trcp, formed large aggregates like G beta, suggesting that it may also require a partner protein. Despite the presence of many potential tryptic cleavage sites, all of the proteins that did fold gave stable large products on tryptic proteolysis, as predicted on the basis of the structure of G beta. These studies suggest that other WD-repeat proteins are likely to form propeller structures similar to G beta.


Subject(s)
GTP-Binding Proteins/chemistry , Microtubule-Associated Proteins , Proteins/chemistry , 1-Alkyl-2-acetylglycerophosphocholine Esterase , Amino Acid Sequence , Animals , Fungal Proteins/chemistry , Fungal Proteins/genetics , GTP-Binding Proteins/genetics , Humans , Membrane Proteins/chemistry , Membrane Proteins/genetics , Microfilament Proteins/chemistry , Microfilament Proteins/genetics , Molecular Sequence Data , Nuclear Pore Complex Proteins , Peptides/chemistry , Peptides/genetics , Protein Conformation , Protein Folding , Proteins/genetics , Rabbits , Receptors for Activated C Kinase , Recombinant Proteins/chemistry , Recombinant Proteins/genetics , Repetitive Sequences, Nucleic Acid , Saccharomyces cerevisiae Proteins , beta-Transducin Repeat-Containing Proteins
16.
Am J Physiol ; 270(4 Pt 1): L547-58, 1996 Apr.
Article in English | MEDLINE | ID: mdl-8928814

ABSTRACT

Type II and type I receptor serine-threonine kinases (RSTK) are important components of the transmembrane signaling machinery that allow cells to respond to the transforming growth factor-beta (TGF-beta) superfamily of cytokines. We have cloned from rat lung and report here a 3,935-base pair (bp) cDNA encoding a type I RSTK previously identified as R-3 (rat) or ALK-1 (human). Northern blot analysis reveals that the R-3 mRNA is more abundant in lung than in other adult rat tissues. With the use of in situ hybridization, the R-3 transcripts are found exclusively in the pulmonary vessels of all sizes, as well as in aorta, vena cava, and certain blood vessels of kidney, spleen, heart and intestine. In most blood vessels, a higher level of gene expression is found in endothelium than in adjacent smooth muscle. The R-3 transcripts are also found in splenic macrophages, as well as within cells of marginal zone of the splenic lymphoid tissue. In fetal rat lung, the expression of R-3 transcripts differs from the expression patterns of two other type 1 RSTK. The R-3 is expressed in vessels; the activin type IB receptor (R-2) is preferentially expressed in putative developing airways, whereas the TGF-beta type I receptor (R-4) transcripts appear to be ubiquitous. Our data suggest that in vivo R-3 may propagate signaling of TGF-beta in selected cell types. The differential expression of multiple type I receptors within different cell lineages may therefore define cell specific responses to TGF-beta.


Subject(s)
Protein Serine-Threonine Kinases , Pulmonary Circulation , Receptors, Growth Factor/metabolism , Receptors, Transforming Growth Factor beta , Aging/metabolism , Animals , Animals, Newborn/growth & development , Animals, Newborn/metabolism , Base Sequence , Blood Vessels/metabolism , Cloning, Molecular , DNA, Complementary/genetics , Lung/metabolism , Molecular Probes/genetics , Molecular Sequence Data , RNA, Messenger/metabolism , Rats , Receptors, Growth Factor/genetics , Tissue Distribution
17.
J Biol Chem ; 271(12): 7113-9, 1996 Mar 22.
Article in English | MEDLINE | ID: mdl-8636146

ABSTRACT

Lysyl oxidase is secreted from fibrogenic cells as a 50-kDa proenzyme that is proteolytically processed to the mature enzyme in the extracellular space. To characterize the secreted proteinase activity, a truncated, recombinant form of lysyl oxidase was prepared as a proteinase substrate containing the sequence of the propeptide cleavage region. The processing proteinase activity secreted by cultured fibrogenic cells resists inhibitors of serine or aspartyl proteinases as well as tissue inhibitor of matrix metalloproteinases-2 (MMP-2) but is completely inhibited by metal ion chelators. Known metalloproteinases were tested for their activity toward this substrate. Carboxyl-terminal procollagen proteinase (C-proteinase), MMP-2, and conditioned fibrogenic cell culture medium cleave the lysyl oxidase substrate to the size of the mature enzyme. The NH2-terminal sequence generated by arterial smooth muscle conditioned medium and the C-proteinase but not by MMP-2, i.e. Asp-Asp-Pro-Tyr, was identical to that previously identified in mature lysyl oxidase isolated from connective tissue. The C-proteinase activity against the model substrate was inhibited by a synthetic oligopeptide mimic of the cleavage sequence (Ac-Met-Val-Gly-Asp-Asp-Pro-Tyr-Asn-amide), whereas this peptide also inhibited the generation of lysyl oxidase activity in the medium of fetal rat lung fibroblasts in culture. In toto, these results identify a secreted metalloproteinase activity participating in the activation of prolysyl oxidase, identify inhibitors of the processing activity, and implicate procollagen C-proteinase in this role.


Subject(s)
Bone Morphogenetic Proteins , Metalloendopeptidases/metabolism , Protein Precursors/metabolism , Protein Processing, Post-Translational , Protein-Lysine 6-Oxidase/metabolism , Amino Acid Sequence , Animals , Base Sequence , Bone Morphogenetic Protein 1 , Cells, Cultured , Connective Tissue/enzymology , Humans , Hydrolysis , Molecular Sequence Data , Muscle, Smooth, Vascular/enzymology , Oligodeoxyribonucleotides , Rats , Sequence Homology, Amino Acid
18.
J Cell Biochem ; 59(3): 329-38, 1995 Nov.
Article in English | MEDLINE | ID: mdl-8567751

ABSTRACT

Rat aortic lysyl oxidase cDNA was expressed under a metallothionein promoter in Chinese hamster ovary cells using a dihydrofolate reductase selection marker. One methotrexate-resistant cell line, LOD-06, generated by transfecting with full-length cDNA, yielded lysyl oxidase proteins consistent with the 50 kDa proenzyme and a 29 kDa mature catalyst. A second cell line, LOD32-2, was generated by transfection with a truncated cDNA lacking sequences which code for the bulk of the propeptide region. Both cell lines secreted apparently identical, 29 kDa forms of mature lysyl oxidase each of which catalyzed the deamination of human recombinant tropoelastin and alkylamines, consistent with the known specificity of lysyl oxidase. The secreted enzyme forms were inhibited by chemical inhibitors of lysyl oxidase activity, including beta-aminopropionitrile, phenylhydrazine, ethylenediamine, alpha, alpha'-dipyridyl, and diethyldithiocarbamate. Sensitivity to these agents is consistent with the presence of copper and carbonyl cofactors in the expressed enzymes, characteristic of lysyl oxidase from connective tissues. These results indicate the lack of essentiality of the deleted proprotein sequence for the proper folding, generation of catalytic function, and secretion of lysyl oxidase.


Subject(s)
Gene Expression , Protein Folding , Protein Precursors/chemistry , Protein-Lysine 6-Oxidase/chemistry , Protein-Lysine 6-Oxidase/genetics , Amino Acid Sequence , Animals , Base Sequence , Blotting, Western , CHO Cells , Catalysis , Cricetinae , DNA, Complementary/genetics , Enzyme Inhibitors/pharmacology , Humans , Metallothionein/genetics , Mice , Molecular Sequence Data , Molecular Weight , Promoter Regions, Genetic , Protein Precursors/metabolism , Protein-Lysine 6-Oxidase/metabolism , Rats , Structure-Activity Relationship , Transfection
19.
Biochim Biophys Acta ; 1261(1): 19-24, 1995 Mar 14.
Article in English | MEDLINE | ID: mdl-7893757

ABSTRACT

The effect of PGE2 on TGF-beta receptor binding was assessed for both signaling (type I and type II) and non-signaling (type III) TGF-beta receptors. We found in cross-linking studies that PGE2 treatment (24 h) decreased binding of TGF-beta to the type I and type II receptors by approx. 50% and markedly increased binding of TGF-beta to type III receptors nearly 10-fold. Northern analyses indicated that PGE2 decreased type I receptor mRNA levels by approximately 30-50%, decreased type II receptor mRNA levels by approximately 60%, and markedly increased type III receptor mRNA levels. Coincubation with cycloheximide partially blocked the PGE2-induced inhibition of type II receptor mRNA. In contrast, cycloheximide minimally affected PGE2-induced type III receptor mRNA levels. Activation of protein kinase C by phorbol esters had no apparent effect on TGF-beta receptor mRNA levels. Our data suggest that alterations in TGF-beta receptor expression could modify the response of tissues to TGF-beta during injury.


Subject(s)
Dinoprostone/physiology , Receptors, Transforming Growth Factor beta/metabolism , Transforming Growth Factor beta/metabolism , Animals , Cells, Cultured , DNA, Complementary/genetics , Fibroblasts/metabolism , Humans , Lung , Plasmids , RNA, Messenger/biosynthesis , Rats , Receptors, Transforming Growth Factor beta/classification , Recombinant Fusion Proteins/biosynthesis
20.
J Biol Chem ; 269(39): 24446-53, 1994 Sep 30.
Article in English | MEDLINE | ID: mdl-7929107

ABSTRACT

Exposure of endothelial cells (EC) to hypoxia results in the increased expression of a distinct set of proteins with molecular masses of 56, 47, 39, 36, and 34 kDa. Their induction appears to be unique to EC and the stress of decreased oxygen tension. To understand the mechanism(s) and significance of the up-regulation of these proteins we have identified the 36-kDa protein by limited amino-terminal amino acid sequencing. The 21-amino acid sequence from the bovine protein exhibited 90.5% identity with the human sequence of the glycolytic enzyme glyceraldehyde-3-phosphate dehydrogenase (GAPDH). Northern blot analysis showed that the time course and extent of EC GAPDH mRNA up-regulation correlated with the increase in 36-kDa protein synthesis. Nuclear runoff analysis demonstrated that this increase in GAPDH expression is regulated, in part, at the transcriptional level; however, the increase in the rate of transcription did not account for the entire mRNA accumulation, suggesting that GAPDH, like other hypoxia-regulated proteins, is posttranscriptionally regulated. Subcellular fractionation of hypoxic EC showed up-regulation of the 36-kDa protein in the cytoplasmic fraction and, to a lesser extent, in the nuclear fraction. The up-regulation of GAPDH in EC may be related to their relative hypoxia tolerance. Alternatively, the up-regulation of GAPDH in EC during hypoxia may be related to the potential nonglycolytic functions of this enzyme.


Subject(s)
Endothelium, Vascular/enzymology , Glyceraldehyde-3-Phosphate Dehydrogenases/metabolism , Amino Acid Sequence , Animals , Cattle , Cell Hypoxia , Cells, Cultured , Electrophoresis, Polyacrylamide Gel , Gene Expression Regulation, Enzymologic , Glyceraldehyde-3-Phosphate Dehydrogenases/genetics , Glyceraldehyde-3-Phosphate Dehydrogenases/isolation & purification , Molecular Sequence Data , RNA, Messenger/metabolism , Subcellular Fractions/metabolism
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