Your browser doesn't support javascript.
loading
Show: 20 | 50 | 100
Results 1 - 17 de 17
Filter
Add more filters










Publication year range
1.
Physiol Rev ; 81(4): 1689-723, 2001 Oct.
Article in English | MEDLINE | ID: mdl-11581500

ABSTRACT

Eukaryotes are characterized by endomembranes that are connected by vesicular transport along secretory and endocytic pathways. The compositional differences between the various cellular membranes are maintained by sorting events, and it has long been believed that sorting is based solely on protein-protein interactions. However, the central sorting station along the secretory pathway is the Golgi apparatus, and this is the site of synthesis of the sphingolipids. Sphingolipids are essential for eukaryotic life, and this review ascribes the sorting power of the Golgi to its capability to act as a distillation apparatus for sphingolipids and cholesterol. As Golgi cisternae mature, ongoing sphingolipid synthesis attracts endoplasmic reticulum-derived cholesterol and drives a fluid-fluid lipid phase separation that segregates sphingolipids and sterols from unsaturated glycerolipids into lateral domains. While sphingolipid domains move forward, unsaturated glycerolipids are retrieved by recycling vesicles budding from the sphingolipid-poor environment. We hypothesize that by this mechanism, the composition of the sphingolipid domains, and the surrounding membrane changes along the cis-trans axis. At the same time the membrane thickens. These features are recognized by a number of membrane proteins that as a consequence of partitioning between domain and environment follow the domains but can enter recycling vesicles at any stage of the pathway. The interplay between protein- and lipid-mediated sorting is discussed.


Subject(s)
Golgi Apparatus/metabolism , Intracellular Membranes/metabolism , Protein Transport/physiology , Sphingolipids/metabolism , Animals , Cell Polarity , Humans , Intracellular Membranes/chemistry , Membrane Microdomains/chemistry , Membrane Microdomains/metabolism , Molecular Structure , Signal Transduction/physiology , Sphingolipids/chemistry , Sphingolipids/genetics
2.
Eur J Biochem ; 267(17): 5646-54, 2000 Sep.
Article in English | MEDLINE | ID: mdl-10951226

ABSTRACT

Vacuolar H+-ATPases (V-ATPases) are multisubunit enzymes that acidify various intracellular organelles, including secretory pathway compartments. We have examined the effects of the specific V-ATPase inhibitor bafilomycin A1 (Baf) on the intracellular transport, sorting, processing and release of a number of neuroendocrine secretory proteins in primary Xenopus intermediate pituitary cells. Ultrastructural examination of Baf-treated intermediate pituitary cells revealed a reduction in the amount of small dense-core secretory granules and the appearance of vacuolar structures in the trans-Golgi area. Pulse-chase incubations in combination with immunoprecipitation analysis showed that in treated cells, the proteolytic processing of the newly synthesized prohormone proopiomelanocortin, prohormone convertase PC2 and secretogranin III (SgIII) was inhibited, and an intracellular accumulation of intact precursor forms and intermediate cleavage products became apparent. Moreover, we found that treated cells secreted considerable amounts of a PC2 processing intermediate and unprocessed SgIII in a constitutive fashion. Collectively, these data indicate that in the secretory pathway, V-ATPases play an important role in creating the microenvironment that is essential for proper transport, sorting, processing and release of regulated secretory proteins.


Subject(s)
Endocytosis , Macrolides , Proteins/metabolism , Proton-Translocating ATPases/antagonists & inhibitors , Vacuolar Proton-Translocating ATPases , Animals , Anti-Bacterial Agents/metabolism , Cell Compartmentation , Golgi Apparatus/metabolism , Hydrolysis , Pituitary Gland/cytology , Pituitary Gland/enzymology , Pituitary Gland/metabolism , Protein Biosynthesis , Protein Processing, Post-Translational , Xenopus laevis
3.
Biochim Biophys Acta ; 1486(1): 145-70, 2000 Jun 26.
Article in English | MEDLINE | ID: mdl-10856719

ABSTRACT

Sphingolipids constitute a sizeable fraction of the membrane lipids in all eukaryotes and are indispensable for eukaryotic life. First of all, the involvement of sphingolipids in organizing the lateral domain structure of membranes appears essential for processes like protein sorting and membrane signaling. In addition, recognition events between complex glycosphingolipids and glycoproteins are thought to be required for tissue differentiation in higher eukaryotes and for other specific cell interactions. Finally, upon certain stimuli like stress or receptor activation, sphingolipids give rise to a variety of second messengers with effects on cellular homeostasis. All sphingolipid actions are governed by their local concentration. The intricate control of their intracellular topology by the proteins responsible for their synthesis, hydrolysis and intracellular transport is the topic of this review.


Subject(s)
Cell Membrane/metabolism , Sphingolipids/metabolism , Animals , Biological Transport , Diffusion , Erythrocyte Membrane/metabolism , Eukaryotic Cells , Evolution, Molecular , Glycosphingolipids/metabolism , Humans , Intracellular Membranes/metabolism , Liver/metabolism , Phosphatidylcholines/metabolism , Second Messenger Systems , Sphingolipids/chemistry , Sphingomyelins/metabolism , Subcellular Fractions/metabolism
4.
Traffic ; 1(3): 226-34, 2000 Mar.
Article in English | MEDLINE | ID: mdl-11208106

ABSTRACT

Membrane lipids do not spontaneously exchange between the two leaflets of lipid bilayers because the polar headgroups cannot cross the hydrophobic membrane interior. Cellular membranes, notably eukaryotic plasma membranes, are equipped with special proteins that actively translocate lipids from one leaflet to the other. In addition, cellular membranes contain proteins that facilitate a passive equilibration of lipids between the two membrane halves. In recent years, a growing number of proteins have been put forward as lipid translocators or facilitators. Unexpectedly, some of these appear to be required for efficient translocation of lipids lacking bulky headgroups, like cholesterol and fatty acids. The candidate lipid translocators identified so far belong to large protein families whose other members include pumps for amphiphilic molecules like bile salts and drugs.


Subject(s)
Carrier Proteins/metabolism , Lipid Bilayers/metabolism , Membrane Lipids/metabolism , Membrane Proteins/metabolism , Phospholipid Transfer Proteins , ATP Binding Cassette Transporter, Subfamily B, Member 1/metabolism , ATP-Binding Cassette Transporters/metabolism , Adenosine Triphosphatases/metabolism , Adenosine Triphosphate/metabolism , Animals , Biological Transport, Active , Carrier Proteins/genetics , Chemical Phenomena , Chemistry, Physical , Cholesterol/metabolism , Endoplasmic Reticulum/metabolism , Fatty Acids/metabolism , Golgi Apparatus/metabolism , Humans , Intracellular Membranes/metabolism , Membrane Proteins/genetics , Models, Biological , Models, Molecular , Molecular Structure , Multigene Family
5.
Eur J Biochem ; 262(2): 484-91, 1999 Jun.
Article in English | MEDLINE | ID: mdl-10336633

ABSTRACT

Vacuolar H+-ATPases (V-ATPases) mediate the acidification of multiple intracellular compartments, including secretory granules in which an acidic milieu is necessary for prohormone processing. A search for genes coordinately expressed with the prohormone proopiomelanocortin (POMC) in the melanotrope cells of Xenopus intermediate pituitary led to the isolation of a cDNA encoding the complete amino-acid sequence of the type I transmembrane V-ATPase accessory subunit Ac45 (predicted size 48 kDa). Comparison of Xenopus and mammalian Ac45 sequences revealed conserved regions in the protein that may be of functional importance. Western blot analysis showed that immunoreactive Ac45 represents a approximately 40-kDa product that is expressed predominantly in neuroendocrine tissues; deglycosylation resulted in a approximately 27-kDa immunoreactive Ac45 product which is smaller than predicted for the intact protein. Biosynthetic studies revealed that newly synthesized Xenopus Ac45 is an N-glycosylated protein of approximately 60 kDa; the nonglycosylated, newly synthesized form is approximately 46 kDa which is similar to the predicted size. Immunocytochemical analysis showed that in Xenopus pituitary, Ac45 is highly expressed in the biosynthetically active melanotrope cells. We conclude that the regionally conserved Xenopus Ac45 protein is synthesized as an N-glycosylated approximately 60-kDa precursor that is intracellularly cleaved to an approximately 40-kDa product and speculate that it may assist in the V-ATPase-mediated acidification of neuroendocrine secretory granules.


Subject(s)
Pituitary Gland/enzymology , Proton-Translocating ATPases/biosynthesis , Vacuolar Proton-Translocating ATPases , Amino Acid Sequence , Animals , Base Sequence , Biological Evolution , Blotting, Western , Cloning, Molecular , DNA, Complementary , Humans , Hydrolysis , Immunohistochemistry , Molecular Sequence Data , Proton-Translocating ATPases/chemistry , Proton-Translocating ATPases/metabolism , Sequence Homology, Amino Acid , Transfection , Xenopus laevis
6.
Mol Biol Cell ; 9(12): 3383-97, 1998 Dec.
Article in English | MEDLINE | ID: mdl-9843576

ABSTRACT

Tlg1p and Tlg2p, members of the syntaxin family of SNAREs in yeast, have been implicated in both endocytosis and the retention of late Golgi markers. We have investigated the functions of these and the other endocytic syntaxins Pep12p and Vam3p. Remarkably, growth is possible in the absence of all four proteins. In the absence of the others, Pep12p and Tlg1p can each create endosomes accessible to the endocytic tracer dye FM4-64. However, although Pep12p is required for the ligand-induced internalization of the alpha factor receptor and its passage via Pep12p-containing membranes to the vacuole, Tlg1p is not. In contrast, Tlg1p is required for the efficient localization of the catalytic subunit of chitin synthase III (Chs3p) to the bud neck, a process that involves endocytosis and polarized delivery of Chs3p. In wild-type cells, internalized Chs3p cofractionates with Tlg1p and Tlg2p, and in a strain lacking the other endocytic syntaxins, either Tlg1p or Tlg2p is sufficient for correct localization of the enzyme. Pep12p is neither necessary nor sufficient for this process. We conclude that there are two endocytic routes in yeast that can operate independently and that Tlg1p is located at the junction of one of these with the polarized exocytic pathway.


Subject(s)
Chitin Synthase/metabolism , Fungal Proteins/metabolism , Membrane Proteins/metabolism , Saccharomyces cerevisiae/growth & development , Saccharomyces cerevisiae/metabolism , Vesicular Transport Proteins , Biological Transport, Active , Cell Polarity , Endocytosis , Endosomes/metabolism , Exocytosis , Golgi Apparatus/metabolism , Membrane Fusion , Microscopy, Electron , Qa-SNARE Proteins , SNARE Proteins , Saccharomyces cerevisiae/cytology
7.
J Cell Sci ; 111 ( Pt 20): 2999-3006, 1998 Oct.
Article in English | MEDLINE | ID: mdl-9739073

ABSTRACT

Ac45 is a type I transmembrane protein associated with vacuolar H+-ATPase, a proton pump mediating the acidification of multiple intracellular organelles. In this study, we examined the intracellular routing of Ac45 in transfected CV-1 fibroblasts. Steady state immunolabeling showed that Ac45 is located on the plasma membrane and in a vacuolar compartment in the juxtanuclear region. Antibody internalization experiments revealed that Ac45 is rapidly retrieved from the cell surface and is targeted to the vacuolar structures. The 26-residue cytoplasmic tail of Ac45 was intrinsically capable of mediating endocytosis of the cell surface protein Tac, indicating that the tail contains an autonomous internalization signal. Immunolocalization studies on cells expressing carboxy-terminally truncated Ac45 mutants showed the presence of essential routing information in the membrane-distal region of the cytoplasmic tail. Further mutational analysis of this region, which lacks the recognized tyrosine- or di-leucine-based sorting motifs, suggested that multiple sites rather than a short linear sequence are responsible for the internalization. Collectively, our results indicate that the cytoplasmic tail of Ac45 contains autonomous targeting information distinct from previously described routing determinants.


Subject(s)
Cell Membrane/enzymology , Membrane Proteins/metabolism , Proton-Translocating ATPases/metabolism , Vacuolar Proton-Translocating ATPases , Vacuoles/enzymology , Xenopus Proteins , Amino Acid Sequence , Amino Acid Substitution , Animals , Biological Transport , Cell Line , Cytoplasm/enzymology , DNA Mutational Analysis , Endocytosis , Intracellular Membranes/enzymology , Membrane Proteins/chemistry , Membrane Proteins/genetics , Microscopy, Fluorescence , Molecular Sequence Data , Mutation , Protein Sorting Signals , Proton-Translocating ATPases/chemistry , Proton-Translocating ATPases/genetics , Receptors, Interleukin-2/metabolism , Recombinant Fusion Proteins/metabolism , Transfection
8.
EMBO J ; 17(1): 113-26, 1998 Jan 02.
Article in English | MEDLINE | ID: mdl-9427746

ABSTRACT

Intracellular membrane traffic is thought to be regulated in part by SNAREs, integral membrane proteins on transport vesicles (v-SNAREs) and target organelles (t-SNAREs) that bind to each other and mediate bilayer fusion. All known SNARE-mediated fusion events involve a member of the syntaxin family of t-SNAREs. Sequence comparisons identify eight such proteins encoded in the yeast genome, of which six have been characterized. We describe here the remaining two, Tlg1p and Tlg2p. These have the expected biochemical properties of t-SNAREs, and are located in separable compartments which correspond to a putative early endosome and the yeast equivalent of the TGN, respectively. They co-precipitate with the v-SNARE Vti1p, which is implicated in Golgi-endosome traffic and, remarkably, binds to five different syntaxins. Tlg1p also binds the plasma membrane v-SNARE Snc1p. Both Tlg1p and Tlg2p are required for efficient endocytosis and to maintain normal levels of TGN proteins. However, neither is required for intra-Golgi traffic. Since no further syntaxins have been identified in yeast, this implies that the Golgi apparatus can function with a single syntaxin, Sed5p.


Subject(s)
Endosomes/metabolism , Golgi Apparatus/metabolism , Membrane Proteins/metabolism , Saccharomyces cerevisiae/metabolism , Amino Acid Sequence , Endocytosis , Endosomes/ultrastructure , Fungal Proteins/metabolism , Golgi Apparatus/ultrastructure , Membrane Proteins/chemistry , Microscopy, Electron , Molecular Sequence Data , Phenotype , Qa-SNARE Proteins , Saccharomyces cerevisiae/genetics , Sequence Homology, Amino Acid
9.
Eur J Cell Biol ; 77(4): 263-8, 1998 Dec.
Article in English | MEDLINE | ID: mdl-9930650

ABSTRACT

SNAREs are compartmentally specific membrane proteins required for intracellular membrane fusion. Homologues of the Saccharomyces cerevisiae protein Sec1p interact with, and are likely to be involved in regulation of, the syntaxin family of SNAREs. In yeast there are 7 functionally distinct syntaxins but only four clearly identifiable homologues of Sec1p. One of these, Vps45p, is required for transport from Golgi to late endosomes, and has been implicated in the function of the late endosomal syntaxin Pep12p. However, there is evidence that not all the functions of Pep12p are equally dependent on Vps45p, and conversely that the phenotypes of vps45 mutants cannot be explained entirely by loss of Pep12p activity. We have recently characterised two yeast syntaxins which function in trans-Golgi or endosomal compartments, Tlg1p and Tlg2p. We show here that the principal binding site for Vps45p on intracellular membranes is provided by Tlg2p rather than Pep12p, and that Vps45p is required for stable expression of Tlg2p. Vps45p is also associated with Tlg1p as part of a triple complex containing both Tlg1p and Tlg2p. Since a deltavps45 deltatlg2 double mutant has a more severe vacuolar protein sorting defect than a deltatlg2 mutant, Vps45p cannot only interact with Tlg2p. It appears that the role of Vps45p in protein traffic is more complex than has previously been assumed.


Subject(s)
Fungal Proteins/metabolism , Membrane Proteins/metabolism , Membrane Transport Proteins , Saccharomyces cerevisiae Proteins , Vesicular Transport Proteins , Animals , Carrier Proteins/metabolism , Fungal Proteins/genetics , Membrane Proteins/chemistry , Membrane Proteins/genetics , Munc18 Proteins , Nerve Tissue Proteins , Qa-SNARE Proteins , Rabbits
10.
J Biol Chem ; 271(30): 17755-60, 1996 Jul 26.
Article in English | MEDLINE | ID: mdl-8663421

ABSTRACT

Secretogranin III (SgIII) is an acidic protein of unknown function that is present in the storage vesicles of many neuroendocrine cells. It is coexpressed with the prohormone proopiomelanocortin in the intermediate pituitary of Xenopus laevis. We developed an antiserum to investigate the biosynthesis of SgIII in pulse-chase incubated Xenopus neurointermediate lobes. SgIII was synthesized as a 61- or 63-kDa (N-glycosylated) protein and processed to a 48-kDa form which, in turn, was partially cleaved to fragments of 28 and 20 kDa. The 48-, 28-, and 20-kDa cleavage products, but not their precursors, were secreted. This secretion is regulated and can be blocked in parallel with that of proopiomelanocortin-derived peptides by the hypothalamic factors dopamine, gamma-aminobutyric acid, and neuropeptide Y. Coexpression of Xenopus SgIII with prohormone convertase (PC)1 or PC2 in transfected fibroblasts was sufficient to reconstitute the processing events observed in the neurointermediate lobes. Site-directed mutagenesis revealed that Xenopus SgIII is cleaved at two dibasic sites, namely Lys68-Arg69 and Arg237-Arg238. Pulse-chase incubations of lobes with Na2[35S]SO4 showed that SgIII is sulfated in the trans-Golgi network before it is processed. Finally, SgIII processing was found in several neuroendocrine cell types from various species. We conclude that SgIII is a precursor protein and that the intact molecule can only have an intracellular function, whereas an extracellular role can only be attributed to its cleavage products.


Subject(s)
Pituitary Gland/metabolism , Protein Precursors/metabolism , Protein Processing, Post-Translational , Proteins/metabolism , Sulfuric Acid Esters/metabolism , Adaptation, Physiological , Animals , Aspartic Acid Endopeptidases/metabolism , Base Sequence , Cell Compartmentation , Cell Line , Chromogranins , Color , Endocrine Glands/metabolism , Melanocyte-Stimulating Hormones/metabolism , Mice , Molecular Sequence Data , Neurons/metabolism , Pituitary Gland/cytology , Proprotein Convertase 2 , Proprotein Convertases , Subtilisins/metabolism , Xenopus laevis
11.
J Neurochem ; 66(6): 2248-56, 1996 Jun.
Article in English | MEDLINE | ID: mdl-8632145

ABSTRACT

Chromogranins and secretogranins are acidic secretory proteins of unknown function that represent major constituents of neuroendocrine secretary granules. Using a differential screening strategy designed to identify genes involved in peptide hormone biosynthesis and secretion, we have isolated cDNA clones encoding the first nonmammalian homologues of secretogranin II (SgII) and secretogranin III (SgIII) from a Xenopus intermediate pituitary cDNA library. A comparative analysis of the Xenopus and mammalian proteins revealed a striking regional conservation with an overall sequence identity of 48% for SgII and 61% for SgIII. One of the highly conserved and thus potentially functional domains in SgII corresponds to the bioactive peptide secretoneurin. However, in SgII and especially in SgIII, a substantial portion of the potential dibasic cleavage sites is not conserved, arguing against the idea that these granins serve solely as peptide precursors. Moreover, SgIII contains a conserved and repeated motif (DSTK) that is reminiscent of a repeat present in the trans-Golgi network integral membrane proteins TGN38 and TGN41, a finding more consistent with an intracellular function for this protein. When Xenopus intermediate pituitary cells were stimulated in vivo, the mRNA levels of SgII and SgIII increased dramatically (15- and 35-fold, respectively) and in parallel with that of the prohormone proopiomelanocortin (30-fold increase). Our results indicate that the process of peptide hormone production and release in a neuroendocrine cell involves multiple members of the granin family.


Subject(s)
Gene Expression Regulation , Pituitary Gland/physiology , Pro-Opiomelanocortin/genetics , Proteins/genetics , Xenopus laevis/genetics , Adaptation, Physiological/genetics , Animals , Base Sequence , Blotting, Northern , Chromogranins , Conserved Sequence , DNA, Complementary/isolation & purification , Gene Expression Regulation/physiology , Molecular Sequence Data , Neurosecretory Systems/physiology , RNA, Messenger/physiology , Sequence Homology, Amino Acid , Transcription, Genetic/physiology
12.
Biochem J ; 312 ( Pt 1): 205-13, 1995 Nov 15.
Article in English | MEDLINE | ID: mdl-7492314

ABSTRACT

In the intermediate pituitary gland of Xenopus laevis, the expression levels of the prohormone pro-opiomelanocortin (POMC) can be readily manipulated. When the animal is placed on a black background, the gene for POMC is actively transcribed, whereas on a white background the gene is virtually inactive. In this study, we characterized two genes whose transcript levels in the intermediate pituitary are regulated in coordination with that for POMC. One of these codes for a protein homologous to translocon-associated protein TRAP delta, a subunit of a transmembrane protein complex located at the site where nascent secretory proteins enter the endoplasmic reticulum (ER). Both Xenopus and mice were found to express an alternatively spliced transcript that gives rise to a previously unknown version of the TRAP delta protein. The product of the second gene is a novel and highly conserved protein with structural similarity to glycoprotein gp25L, a constituent of another translocon-associated protein complex. A database search revealed the existence of a novel family of gp25L-related proteins whose members occur throughout the animal kingdom. Together, our data imply that (i) the group of ER proteins surrounding translocating polypeptide chains may be far more complex than previously expected, and (ii) a number of the accessory components of the translocon participate in early steps of prohormone biosynthesis.


Subject(s)
Calcium-Binding Proteins/genetics , Membrane Glycoproteins/genetics , Pituitary Gland/metabolism , Pro-Opiomelanocortin/genetics , Receptors, Cytoplasmic and Nuclear/genetics , Receptors, Peptide/genetics , Adaptation, Physiological , Alternative Splicing , Amino Acid Sequence , Animals , Base Sequence , Calcium-Binding Proteins/chemistry , Cloning, Molecular , DNA Primers , DNA, Complementary/chemistry , DNA, Complementary/genetics , Endoplasmic Reticulum/metabolism , Fungal Proteins/chemistry , Fungal Proteins/genetics , Gene Expression Regulation/genetics , Membrane Glycoproteins/chemistry , Molecular Sequence Data , Multigene Family , Pro-Opiomelanocortin/metabolism , RNA, Messenger/analysis , RNA, Messenger/genetics , Receptors, Cytoplasmic and Nuclear/chemistry , Receptors, Peptide/chemistry , Ribonucleases/metabolism , Sequence Alignment , Xenopus laevis
13.
J Cell Sci ; 108 ( Pt 10): 3295-305, 1995 Oct.
Article in English | MEDLINE | ID: mdl-7593290

ABSTRACT

The biosynthetic machinery in the melanotrope cells of the Xenopus intermediate pituitary is primarily dedicated to the generation of proopiomelanocortin (POMC)-derived, melanophore-stimulating peptides. Transfer of the animal to a black background stimulates the production of these peptides and causes a dramatic increase in POMC mRNA levels. To identify genes involved in the biosynthesis and regulated release of peptide hormones, we differentially screened an intermediate pituitary cDNA library of toads adapted to a black background with cDNA probes derived from intermediate pituitary mRNA of black- and white-adapted animals. Here we report the identification of twelve distinct genes whose expression levels in the melanotropes are regulated in coordination with that of POMC. Four of these genes are novel while the others code for translocon-associated proteins, a lumenal cysteine protease of the endoplasmic reticulum, prohormone-processing enzymes, members of the granin family and a transmembrane protein presumably involved in the assembly and/or specific functioning of vacuolar H(+)-ATPase from secretory granules. Our results indicate that a wide variety of both soluble and membrane-associated components of the secretory pathway is recruited in physiologically activated, peptide hormone-producing cells.


Subject(s)
Pro-Opiomelanocortin/metabolism , Xenopus/metabolism , Amino Acid Sequence , Animals , DNA, Complementary/analysis , DNA, Complementary/genetics , Gene Expression Regulation , Molecular Probe Techniques , Molecular Sequence Data , Pro-Opiomelanocortin/genetics , Proton-Translocating ATPases/metabolism , RNA, Messenger/biosynthesis
14.
Biochim Biophys Acta ; 1219(1): 184-8, 1994 Sep 13.
Article in English | MEDLINE | ID: mdl-8086462

ABSTRACT

Sea urchin fascin and the Drosophila singed gene product form a unique class of actin cross-linking proteins involved in the bundling of filamentous actin by an as yet unknown mechanism. From a Xenopus laevis intermediate pituitary cDNA library we have isolated a cDNA encoding a 53-kDa protein that shares approximately 36% amino acid sequence identity with both fascin and the singed gene product, and thus likely represents a vertebrate homolog of these actin-bundling proteins. RNase-protection experiments revealed that in Xenopus the gene is expressed in a wide variety of tissues but with the highest levels of expression in oocytes and testis. This raises the possibility that fascin has a role in microfilament dynamics associated with the formation and/or fertilization of vertebrate germ cells.


Subject(s)
Carrier Proteins/genetics , Microfilament Proteins/genetics , Xenopus laevis/genetics , Amino Acid Sequence , Animals , Base Sequence , DNA, Complementary/genetics , Female , Gene Library , Male , Molecular Sequence Data , Oocytes/physiology , Pituitary Gland/physiology , Sequence Homology, Amino Acid , Testis/physiology , Tissue Distribution
15.
Article in English | MEDLINE | ID: mdl-7994377

ABSTRACT

Modification of hemoglobin (Hb) by crosslinking and polymerization results in an improved oxygen release capacity and a prolonged vascular retention time. Modification improves the efficacy and prevents certain side effects. It eliminates leakage of Hb through the kidneys and accumulation in the tubuli. Another important issue is the degree of purification of Hb solutions. Traces of membrane fragments may cause immunogenic and thrombogenic side effects. To determine the contamination with erythrocyte membrane fragments, we developed assays for glycophorin-alpha and phospholipids. Special models were evaluated for testing the maximum allowable level of membrane contamination. As an in vitro model for thrombogenicity we used confluent monolayers of human umbilical vein endothelial cells. These cells were incubated with Hb solutions and subsequently tested on tissue factor (TF) procoagulant activity. TF was tested by the factor VII-catalyzed activation of factor X. The lower detection limit of this assay for endotoxin was 0.5 ng/ml. Hb did not cause any tissue factor expression even after prolonged incubation. No cooperation was found within endotoxin. As an in vivo test on thrombogenicity we developed a guinea pig model in which we can follow the generation of fibrinopeptide A (FPA). This is one of the most sensitive markers for thrombin activation in vivo. When slightly contaminated Hb solutions (phospholipid content 2 nmol/ml) were infused in the presence of factor Xa at a dose (9 micrograms/kg) which in itself did not induce FPA generation, we observed an increase in FPA levels in the plasma from 1.2 +/- 0.4 ng/ml to 5.2 +/- 0.7 ng/ml. Factor Xa is used to mimic a stressed clinical condition with activated coagulation.(ABSTRACT TRUNCATED AT 250 WORDS)


Subject(s)
Blood Substitutes/toxicity , Hemoglobins/toxicity , Thrombosis/etiology , Animals , Cells, Cultured , Disease Models, Animal , Endothelium, Vascular/drug effects , Exchange Transfusion, Whole Blood , Fibrinopeptide A/metabolism , Guinea Pigs , Humans , Models, Biological , Solutions
16.
Endocrinology ; 124(4): 2029-41, 1989 Apr.
Article in English | MEDLINE | ID: mdl-2538320

ABSTRACT

P19 EPI-7, a differentiated murine embryonal carcinoma cell line with an epithelioid morphology, does not require external growth factors for proliferation under clonal and subconfluent conditions. At saturation density, however, cells become quiescent in the G1/G0 phase of the cell cycle from which they can be restimulated, particularly upon addition of epidermal growth factor. Medium conditioned by confluent P19 EPI-7 cultures is able to enhance clonal outgrowth of this cell line, suggesting that autocrine growth factor loops may be acting in these cells. Analysis of conditioned serum-free medium shows that this cell line produces a platelet-derived growth factor-like growth factor, next to a type beta transforming growth factor and large amounts of insulin-like growth factor II (IGF-II) and an IGF-binding protein with high specificity for IGF-II. This latter observation has been confirmed by the use of a specific bioassay for IGFs, based on their ability to specifically stimulate proliferation of MCF-7 human breast cancer cells. The amount of IGF-II produced (0.5 mg/liter conditioned medium) makes P19 EPI-7 one of the best producing cell lines for this factor described so far. Receptor cross-linking analysis shows that this cell line contains IGF-I receptors, but no specific receptors for IGF-II. Depending on the conditions tested, transforming growth factor-beta 1 either act as a growth-stimulating factor or as a strong growth inhibitory factor. These data demonstrate that upon cellular differentiation, embryonal carcinoma cells can be formed which produce polypeptide growth factors and are also able to respond to such factors. These observations are discussed in the light of the role of autocrine and paracrine growth stimulation processes during early murine development.


Subject(s)
Cell Transformation, Neoplastic/drug effects , Neoplastic Stem Cells/pathology , Platelet-Derived Growth Factor/metabolism , Somatomedins/metabolism , Transforming Growth Factors/metabolism , Animals , Cell Division/drug effects , Cell Line , Culture Media/analysis , Embryonal Carcinoma Stem Cells , Mice , Neoplastic Stem Cells/metabolism , Neoplastic Stem Cells/ultrastructure , Platelet-Derived Growth Factor/pharmacology , Platelet-Derived Growth Factor/physiology , Receptors, Cell Surface/analysis , Receptors, Cell Surface/metabolism , Receptors, Somatomedin , Somatomedins/pharmacology , Somatomedins/physiology , Teratoma/pathology , Transforming Growth Factors/pharmacology , Transforming Growth Factors/physiology , Tumor Cells, Cultured
17.
J Cell Biol ; 108(3): 1115-25, 1989 Mar.
Article in English | MEDLINE | ID: mdl-2537833

ABSTRACT

High levels of the neuron-specific protein kinase C substrate, B-50 (= GAP43), are present in neurites and growth cones during neuronal development and regeneration. This suggests a hitherto nonelucidated role of this protein in neurite outgrowth. Comparable high levels of B-50 arise in the pheochromocytoma PC12 cell line during neurite formation. To get insight in the putative growth-associated function of B-50, we compared its ultrastructural localization in naive PC12 cells with its distribution in nerve growth factor (NGF)- or dibutyryl cyclic AMP (dbcAMP)-treated PC12 cells. B-50 immunogold labeling of cryosections of untreated PC12 cells is mainly associated with lysosomal structures, including multivesicular bodies, secondary lysosomes, and Golgi apparatus. The plasma membrane is virtually devoid of label. However, after 48-h NGF treatment of the cells, B-50 immunoreactivity is most pronounced on the plasma membrane. Highest B-50 immunoreactivity is observed on plasma membranes surrounding sprouting microvilli, lamellipodia, and filopodia. Outgrowing neurites are scattered with B-50 labeling, which is partially associated with chromaffin granules. In NGF-differentiated PC12 cells, B-50 immunoreactivity is, as in untreated cells, also associated with organelles of the lysosomal family and Golgi stacks. B-50 distribution in dbcAMP-differentiated cells closely resembles that in NGF-treated cells. The altered distribution of B-50 immunoreactivity induced by differentiating agents indicates a shift of the B-50 protein towards the plasma membrane. This translocation accompanies the acquisition of neuronal features of PC12 cells and points to a neurite growth-associated role for B-50, performed at the plasma membrane at the site of protrusion.


Subject(s)
Membrane Proteins/analysis , Nerve Growth Factors/pharmacology , Nerve Tissue Proteins/analysis , Neurons/analysis , Pheochromocytoma/analysis , Animals , Axons/analysis , Bucladesine/pharmacology , Cell Differentiation , Cell Line , Cell Membrane/analysis , Chromaffin System/analysis , Chromaffin System/cytology , Chromaffin System/ultrastructure , GAP-43 Protein , Golgi Apparatus/analysis , Immunohistochemistry , Lysosomes/analysis , Microscopy, Electron , Microvilli/analysis , Neurons/ultrastructure , Pheochromocytoma/ultrastructure , Pseudopodia/analysis
SELECTION OF CITATIONS
SEARCH DETAIL
...