Biomedical subjects

J Thyberg

Publications and source records attributed to J Thyberg.

At least 55 records · Page 3Linked to original sources

PubMed1995

Characterization of stable complexes involving apolipoprotein E and the amyloid beta peptide in Alzheimer's disease brain.

Genetic evidence suggests a role for apolipoprotein E (apoE) in Alzheimer's disease (AD) amyloidogenesis. Here, amyloid-associated apoE from 32 AD patients was purified and characterized. We found that brain amyloid-associated apoE apparently exists not as free molecules but as complexes with polymers of the amyloid beta peptide (A beta). Brain A beta-apoE complexes were detected irrespective of the apoE genotype, and similar complexes could be mimicked in vitro. The fine structure of purified A beta-apoE complexes was fibrillar, and immunogold labeling revealed apoE immunoreactivity along the fibrils. Thus, we conclude that A beta-apoE complexes are principal components of AD-associated brain amyloid and that the data presented here support a role for apoE in the pathogenesis of AD.

Aged↗

PubMed1995

Inhibitors of ADP-ribosylation suppress phenotypic modulation and proliferation of smooth muscle cells cultured from rat aorta.

The effects of hexamethylenebisacetamide (HMBA), an inhibitor of poly-ADP-ribosylation, and meta-iodobenzylguanidine (MIBG), an inhibitor of mono-ADP-ribosylation, on the phenotypic properties and proliferation of cultured rat aortic smooth muscle cells were studied using a combination of structural and chemical methods. The results show that HMBA and MIBG both slowed down the transition of the cells from a contractile to a synthetic phenotype in primary culture. While the control cells rapidly lost most of their myofilaments and built up an extensive endoplasmic reticulum and Golgi complex, a conspicuous fraction of the drug-treated cells retained a characteristic smooth muscle morphology for at least 6 days. Moreover, most of the treated cells remained positive for smooth muscle alpha-actin and desmin throughout this period. In contrast, the drugs lacked distinct effects on cell morphology and cytoskeletal organization in secondary cultures. Nevertheless, they strongly inhibited serum-stimulated cell growth both in primary and secondary cultures. The ability of serum-starved cells to synthesize DNA after exposure to platelet-derived growth factor or serum was also restrained. Notably, the drugs could be added several hours after the mitogens without loss of effect, suggesting that they did not prevent the entrance into but rather the progression through the cell cycle. Accordingly, the expression of early response genes like c-fos, c-jun and c-myc was not blocked by the drugs. On the other hand, HMBA reduced the expression of transcripts for smooth muscle alpha-actin, type IV collagenase, collagen type I, and osteopontin both in primary and secondary cultures. Weaker and more variable effects were obtained with MIBG. Taken together, the findings support the notion that poly- and mono-ADP-ribosylation of proteins are involved in the control of smooth muscle cell differentiation and growth.

3-Iodobenzylguanidine↗

PubMed1994

The Alzheimer A beta peptide develops protease resistance in association with its polymerization into fibrils.

An intriguing property of the polypeptide constituents of amyloid is that they apparently can escape the proteolytic mechanisms that normally catalyze turnover and prevent abnormal tissue accumulation of polypeptides. Here, we demonstrate that the A beta peptide, the principal component of cerebrovascular amyloid deposits in Alzheimer's disease, becomes resistant to an array of proteases as a result of structural changes associated with its polymerization into amyloid fibrils. It is further demonstrated that fibril formation per se does not lead to protease resistance but probably structural changes associated with polymerization. The results suggest that higher order structural changes, regulated by the primary structure, enable amyloidogenic polypeptides to escape proteolytic degradation and accumulate in tissues.

Alzheimer Disease↗

PubMed1994

The metabolic pathway generating p3, an A beta-peptide fragment, is probably non-amyloidogenic.

The Alzheimer a beta amyloid precursor protein is metabolized by at least two secretory pathways. One generates the A beta peptide and the other a N-terminally truncated A beta fragment termed p3 that is considered non-amyloidogenic. However, direct evidence is missing. We have undertaken to synthesize and purify p3. Pure p3 polymerizes in vitro, forming a lattice with an ultrastructure distinct from the linear fibrils of A beta. In contrast to amyloid, polymerized p3 does not bind thioflavine T. It is therefore concluded that amino acids in the N-terminal part of the A beta molecule are required for formation of typical amyloid fibrils and that the metabolic pathway generating p3 probably is non-amyloidogenic

Amino Acid Sequence↗

PubMed1994

Human neutrophil phagocytic granules contain a truncated soluble form of the Alzheimer beta/A4 amyloid precursor protein (APP).

We have characterized the molecular species and subcellular distribution of Alzheimer beta/A4 amyloid precursor protein (APP) in neutrophilic granulocytes purified from human peripheral blood. APP was readily detectable in these cells. Immunochemical analysis with a panel of antibodies revealed that this APP species lacked the transmembrane and cytoplasmic domains previously demonstrated in cell-associated APP. However, it contained a protease inhibitor domain of the Kunitz type, indicating that neutrophil APP is a potent inhibitor of certain serine proteases. Upon subcellular fractionation, APP was primarily localized to azurophilic granules, which are neutrophil-specific phagocytic organelles assigned to enzymatic digestion of invading microbes and dead or injured tissue. Apparently, in the neutrophil, a nonsecretory organelle stores truncated, soluble APP, a species previously found only in blood plasma and cerebrospinal fluid or in conditioned medium of cultured cells. Soluble APP in neutrophils may therefore have intracellular functions in addition to its previously described extracellular functions. These findings also indicate that there are previously uncharacterized cell-specific differences in processing, trafficking, and storage of the APP molecule. Finally, the precise subcellular localization of APP to neutrophil-specific phagocytic organelles is suggestive of a role for APP in the nonimmunological host defense.

Amyloid beta-Protein Precursor↗

PubMed1994

Fibronectin and the basement membrane components laminin and collagen type IV influence the phenotypic properties of subcultured rat aortic smooth muscle cells differently.

A substrate of the extracellular matrix protein fibronectin has previously been found to promote the modulation of freshly isolated rat aortic smooth muscle cells from a contractile to a synthetic phenotype early in primary culture. In contrast, substrates of the basement membrane proteins laminin and collagen type IV were found to retain the cells in a contractile phenotype. Here, we have studied whether rat aortic smooth muscle cells tht have already adopted a synthetic phenotype are also affected differently by these proteins. For this sake, subcultured cells were detached with trypsin, seeded on substrates of either fibronectin or laminin plus collagen type IV, and incubated in a serum-free medium for one to three days. RNA blot and immunoblot analyses indicated that cells grown on laminin plus collagen type IV expressed smooth muscle alpha-actin transcripts and protein at higher levels than cells grown on fibronectin. Moreover, immunocytochemical and electron-microscopic analyses revealed that cells positively stained for smooth muscle alpha-actin and cells with a cytoplasm dominated by large microfilament bundles were more numerous on laminin plus collagen type IV than on fibronectin. Finally, thymidine autoradiography showed that the DNA synthetic response to stimulation with platelet-derived growth factor or serum was weaker in cells grown on laminin plus collagen type IV than in cells grown on fibronectin. These findings confirm the notion that a substrate of laminin and collagen type IV stimulates the in vitro expression of differentiated smooth muscle traits at a higher level than does a substrate of fibronectin.(ABSTRACT TRUNCATED AT 250 WORDS)

Actin Cytoskeleton↗

PubMed1994

Functions of the Golgi complex in cell division: formation of cell-matrix contacts and cell-cell communication channels in the terminal phase of cytokinesis.

The Golgi complex of mammalian cells is disorganized into dispersed vesicular and tubular elements during mitosis and is then reorganized into an interconnected system of cisternal stacks in each daughter cell during cytokinesis. Recent studies further indicate that the Golgi complex is typically relocated from the proximal to the distal side of the nucleus in the terminal phase of cytokinesis (as related to the intercellular bridge). Here, the functional role of this shift in position was approached using rat embryo fibroblasts synchronized with thymidine and nocodazole. Mitotic cells were collected by shaking and seeded in medium without or with brefeldin A (a fungal metabolite that inhibits protein secretion). They were fixed after one or two hours and stained for immunocytochemical demonstration of mannosidase II (a Golgi protein), fibronectin (an extracellular matrix protein), the fibronectin receptor (a member of the integrin family of proteins), and connexin 43 (a member of the connexin family of gap junction proteins). One hour after seeding, the cells had completed mitosis and progressed into cytokinesis. The Golgi complex was now usually located on the proximal side of the nucleus and overlapping fibrillar arrays of fibronectin and fibronectin receptors were observed in the contact zone between the daughter cells, while connexin 43 mainly occurred in fine dispersed spots. Two hours after seeding, the cells had spread out on the substrate and started to move apart. The Golgi complex was now usually located on the distal side of the nucleus. Moreover, fibronectin and fibronectin receptors were found to codistribute both in the contact zone between the daughter cells and in adhesive contacts beneath them, while connexin 43 was concentrated to plaques in the former zone. After treatment with brefeldin A, there was a diffuse cytoplasmic staining for mannosidase II and fibronectin and no distinct extracellular staining for fibronectin was noted. In addition, the connexin 43 positive plaques were reduced in size and number. Although the cells completed cytokinesis in the presence of the drug, they showed an increased tendency to detach from the substrate and locate on top of each other rather than to move apart normally. Taken together, the observations suggest that the change in position of the Golgi complex during cytokinesis serves the function to direct transport of secretory proteins as well as membrane constituents to different parts of the cell surface at different times.(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗

PubMed1993

Identification of the Alzheimer beta/A4 amyloid precursor protein in clathrin-coated vesicles purified from PC12 cells.

The Alzheimer beta/A4 amyloid precursor protein (APP) can be proteolytically processed by at least two separate pathways in PC12 cells: chloroquine-insensitive secretory cleavage and chloroquine-sensitive intracellular degradation, presumably in the endosomal/lysosomal system. To further investigate the possibility of APP processing in the endosomal/lysosomal system, we have examined whether APP is present in clathrin-coated vesicles (CCVs), which mediate the transport of many proteins to the endosomal compartment. Using a procedure derived from established protocols for the purification of CCVs from mammalian organs, we obtained from PC12 cells highly purified CCVs that displayed the same morphological features as described for CCVs purified from other sources. The CCVs were enriched in full-length mature (fully post-translationally modified) forms of APP, as well as in the carboxyl-terminal APP fragment produced by the secretory cleavage pathway. As CCVs are known to be involved in only two intracellular pathways (trafficking from the plasma membrane to early endosomes, and from the trans-Golgi network to late endosomes/prelysosomes), these findings provide direct evidence that APP is transported to the endosomal/lysosomal system. Furthermore, the presence in CCVs of the carboxyl-terminal fragment resulting from APP secretory cleavage suggests that APP secretory processing occurs in a pre-CCV compartment.

Amyloid beta-Protein Precursor↗

PubMed1993

Relationship between the Golgi complex and microtubules enriched in detyrosinated or acetylated alpha-tubulin: studies on cells recovering from nocodazole and cells in the terminal phase of cytokinesis.

Double immunofluorescence microscopy was used to study the relationship between the Golgi complex and microtubules enriched in posttranslationally modified tubulins in cultured mouse L929 fibroblasts. In interphase cells, the elements of the Golgi complex were grouped around the microtubule-organizing center. From here, tyrosinated microtubules extended to the periphery of the cells, whereas the distribution of detyrosinated and acetylated microtubules largely overlapped with that of the Golgi complex. Treatment of cells with 10 microM nocodazole led to the disruption of all microtubules and dispersion of the Golgi elements. Following withdrawal of the drug, tyrosinated microtubules reformed first, followed by acetylated and then detyrosinated microtubules. In parallel, the Golgi elements moved back toward the juxtanuclear region and reestablished a close spatial relationship first with the acetylated and later also with the detyrosinated microtubules. Long-term recovery in the presence of 0.15 or 0.3 microM nocodazole allowed partial reformation of tyrosinated and acetylated microtubules, whereas no or only a few detyrosinated microtubules were detected. At the same time, the Golgi elements were grouped closer together around or on one side of the nucleus in close relation to acetylated microtubules. In synchronized cells released from a mitotic block, a radiating array of tyrosinated microtubules was first formed, followed by acetylated and detyrosinated microtubules. The Golgi elements initially came together in a few groups and thereafter took an overall morphology similar to that in interphase cells. During this reunification, they showed a close spatial relationship to acetylated microtubules, whereas detyrosinated microtubules appeared only later. Microtubules enriched in acetylated and/or detyrosinated tubulin thus appear to take part in establishing and maintaining the organization of the Golgi elements within an interconnected supraorganellar system. Whether the acetylation and detyrosination of tubulin are directly involved in this process or merely represent two modifications within this subpopulation of microtubules remains unknown.

Acetylation↗

PubMed1993

Cervical ripening with mifepristone (RU 486) in first trimester abortion. An electron microscope study.

Mifepristone (RU 486) is a steroid that binds to the progesterone receptor and acts as a progesterone antagonist. It has been used clinically to terminate an early pregnancy. In the present investigation, the effects of mifepristone on the fine structure of the cervix were evaluated in two groups of women undergoing a first trimester abortion, using a triple-blind randomized protocol. In group I (n = 18), a cervical biopsy was taken after two doses of 100 mg mifepristone or placebo orally 24 and 12 h before vacuum aspiration. In group II (n = 20), cervical biopsies were taken before and after two doses of 100 mg mifepristone or placebo orally 48 and 36 h before vacuum aspiration. Smooth muscle cells, fibroblasts and an extracellular matrix with large bundles of collagen fibrils were the main structural components in all cervical biopsies. In group I, an increased number of mast cells and signs of new blood capillary formation and collagenolysis were observed in several of the biopsies after mifepristone treatment (67% correctly classified). In group II, the effects were less evident and in most of the cases (70%) no definite differences were noted between the biopsies taken before and after mifepristone treatment. Nevertheless, mifepristone treatment had induced softening in the cervical tissue, as judged by the decrease in resistance to mechanical dilatation.

Abortion, Induced↗

PubMed1992

Retention of the transforming growth factor-beta 1 precursor in the Golgi complex in a latent endoglycosidase H-sensitive form.

Transforming growth factor-beta 1 (TGF-beta 1) is synthesized as a latent high molecular weight complex in a human erythroleukemia cell line, HEL, treated with phorbol 12-myristate 13-acetate. The complex is comprised of three components: mature TGF-beta 1, the TGF-beta 1 latency-associated peptide (beta 1-LAP), and the latent TGF-beta 1-binding protein (LTBP). LTBP plays an important role in the assembly and secretion of the latent TGF-beta 1 complex; if the TGF-beta 1 precursor fails to bind to LTBP, much of it remains inside the cells and may contain anomalous disulfide bond(s) between beta 1-LAP and the mature TGF-beta 1 molecule (Miyazono, K., Olofsson, A., Colosetti, P., and Heldin, C.-H. (1991) EMBO J. 10, 1091-1101). In the present work, we have investigated the subcellular localization and properties of the TGF-beta 1 precursor retained intracellularly. When the HEL cells were metabolically labeled and chased for up to 72 h, a considerable part of the TGF-beta 1 precursor was still observed intracellularly in an unprocessed form. The secreted form of the TGF-beta 1 precursor was resistant to endoglycosidase H, whereas the intracellular form of the TGF-beta 1 precursor was sensitive to endoglycosidase H, regardless of the presence or absence of swainsonine, an inhibitor of mannosidase II. Indirect immunofluorescence microscopy revealed that the TGF-beta 1 precursor co-localized with mannosidase II, a marker for the Golgi complex, but not with protein disulfide isomerase, a marker for the endoplasmic reticulum. The intracellular TGF-beta 1 precursor was prepared from phorbol 12-myristate 13-acetate-treated HEL cells and tested for TGF-beta 1 bioactivity. Half-maximal inhibition of the DNA synthesis in mink lung epithelial cells, Mv1Lu, was observed at 80 pM of the acid-treated TGF-beta 1 precursor, whereas nontreated material showed minimal growth inhibitory activity. Taken together, these results indicate that the TGF-beta 1 precursor is retained inside the cells in the Golgi complex, mainly in a latent, immature form.

Animals↗

PubMed1992

Nuclear colocalization of c-myc protein and hsp70 in cells transfected with human wild-type and mutant c-myc genes.

Using immunofluorescence and electron microscopy we have studied the localization of wild-type and mutant c-myc proteins transiently expressed in CV-1 cells. In agreement with our previous observations, wild-type c-myc protein accumulated in large amorphous globules in the nucleus. All mutant proteins tested accumulated in the nucleus as well, but gave rise to morphologically different inclusion bodies. Many small globules appeared in cells transfected with D145-262 (deletion of amino acids 145-262), while cells transfected with D371-412 or D414-433 generated structures looking like a fine network or like beads on a string. In addition, a particulate cytoplasmic staining appeared in some cells transfected with the wild-type gene and in cells transfected with mutants D145-262 or D414-433. Since the c-myc protein has been reported to stimulate expression of exogenous hsp70 protein, we also examined the intracellular distribution of hsp70 in the transfected cells. Double immunofluorescence microscopy revealed that hsp70 codistributed with the c-myc protein in distinct globules in the nucleus of many but not all myc-positive cells. However, the levels of hsp70 transcripts were not significantly raised compared to nontransfected and vector-transfected cells. Likewise, the levels of hsp70 protein did not vary significantly. These findings indicate that overexpression of c-myc stimulates translocation of preexisting hsp70 from the cytoplasm into the nucleus, rather than influencing hsp70 expression. Conceivably, this may represent one of several mechanisms whereby the cell deals with excessive amounts of c-myc protein.

Animals↗

PubMed1992

PDGF-AA and PDGF-BB biosynthesis: proprotein processing in the Golgi complex and lysosomal degradation of PDGF-BB retained intracellularly.

Platelet-derived growth factor is a potent mitogen for cells of mesenchymal origin. It is made up of two polypeptide chains (A and B) combined in three disulfide-linked dimeric forms (AA, AB, and BB). Here, the biosynthesis and proteolytic processing of the two homodimeric forms of PDGF (AA and BB) were studied in CHO cells stably transfected with A-chain (short splice version) or B-chain cDNA. PDGF-AA was processed to a 30-kD molecule which was secreted from the cells. In contrast, PDGF-BB formed two structurally distinct end products; a minor secreted 30-kD form and a major cell-associated 24-kD form. Immunocytochemical studies at light- and electron-microscopical levels revealed presence of PDGF in the Golgi complex, in lysosomes, and to a smaller extent in the ER. From analysis of cells treated with brefeldin A, an inhibitor of ER to Golgi transport, it was concluded that dimerization occurs in the ER, whereas the proteolytic processing of PDGF-AA and PDGF-BB precursors normally occurs in a compartment distal to the ER. Exposure of the cultures to the lysosomal inhibitor chloroquine led to an increased cellular accumulation of PDGF-BB, as determined both by metabolic labeling experiments and immunocytochemical methods, indicating that the retained form of PDGF-BB is normally degraded in lysosomes. Structural analysis of the two end products of PDGF-BB revealed that the secreted 30-kD form is a dimer of peptides processed as the B-chain of PDGF purified from human platelets, and that the retained 24-kD form is made up of subunits additionally processed in the NH2-terminus. Also, the 24-kD form was shown to be composed of proteolytic fragments held together by disulfide bridges. Taken together these findings suggest that the newly synthesized PDGF A- and B-chains are dimerized in the ER and thereafter transferred to the Golgi complex for proteolytic processing. From there, PDGF-AA is carried in vesicles to the cell surface for release extracellularly by exocytosis. A smaller part of PDGF-BB (the 30-kD form) is handled in a similar way, whereas the major part (the 24-kD form) is generated by additional proteolysis in the Golgi complex, from which it is slowly carried over to lysosomes for degradation.

Animals↗

PubMed1992

Disorganization of the Golgi complex and the cytoplasmic microtubule system in CHO cells exposed to okadaic acid.

A combination of immunocytochemical and electron microscopic methods was used to study the effects of okadaic acid, a specific inhibitor of protein phosphatase types 1 and 2A, on the Golgi complex and the microtubule system of interphase CHO cells. At a concentration of 0.25 microM and within 2-3 h of exposure, okadaic acid caused a reversible disorganization of the Golgi complex, observed as a disintegration of the stacks of cisternae and formation of clusters of tubules and vesicles dispersed in the cytoplasm. At the same time, staining for mannosidase II was shifted from the Golgi stacks to the endoplasmic reticulum, whereas the clusters of tubules and vesicles for the main part were negative. This change in localization of the enzyme was not blocked by cycloheximide and thus not dependent on ongoing protein synthesis. The changes in the morphology of the Golgi complex were coordinated in time with a remodelling of the microtubule system, observed as a reduction in the number of microtubules, a tendency of the remaining microtubules to arrange in an aster-like pattern, and an increased sensitivity to low concentrations of the microtubule-disruptive drug nocodazole. After removal of the drug, the microtubule system was rapidly normalized (1-2 h) and subsequently also the Golgi complex (4-8 h). The results suggest that okadaic acid induces a redistribution of the Golgi stacks into the endoplasmic reticulum, leaving the trans-most elements behind as tubules and vesicles.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗

PubMed1992

Synchronized shift in localization of the Golgi complex and the microtubule organizing center in the terminal phase of cytokinesis.

As mammalian cells enter mitosis, the Golgi complex is disorganized and the remnants are dispersed throughout the cytoplasm in the form of a few short cisternae and small clusters of vesicles. Once the separation of the chromosomes is completed and nuclei reform, stacks of flattened cisternae reappear and a united Golgi complex of interphase type starts to be reorganized. This process is believed to ensure an approximately equal partitioning of the Golgi complex on the daughter cells. Here, the configuration of the Golgi complex and its relation to the cytoplasmic microtubule system was studied at the end of cytokinesis using synchronized cultures of L929 mouse fibroblasts and rat dermal fibroblasts. One hour after the release of the mitotic block, the Golgi complex (visualized immunocytochemically with antibodies against mannosidase II) was most frequently located on the proximal side of the nucleus as related to the intercellular bridge (visualized immunocytochemically with antibodies against tyrosinated alpha-tubulin). One hour later, it was preferentially found on the distal side of the nucleus as related to the intercellular bridge. Immunocytochemical demonstration of the radiating pattern of microtubules, and direct demonstration of the centrioles using antibodies against detyrosinated or acetylated alpha-tubulin, showed that the microtubule organizing center (MTOC) shifted position in a similar manner as the Golgi complex. Moreover, double staining with antibodies against mannosidase II and tyrosinated alpha-tubulin revealed that the Golgi complex and the MTOC codistributed at both times after the release of the mitotic block. Electron microscopic analysis confirmed that the reforming Golgi stacks first gathered close to the centrosome (a pair of centrioles with associated structures, constituting the main MTOC in the cell) on the proximal side of the nucleus and that the Golgi stacks and the centrosome were subsequently both relocated to the distal side of the nucleus as related to the intercellular bridge. Taken together, the findings indicate that the Golgi complex goes through a characteristic translocation in the terminal phase of cytokinesis and confirm the idea that the cytoplasmic microtubule system plays an important role in the organization of this organelle system. A possible function of the shift in location of the Golgi complex at the end of cytokinesis could be to direct membrane traffic first to the elongating intercellular bridge and thereafter to the leading edge as the cells are about to separate and move away from each other.

Animals↗

PubMed1992

Reorganization of the Golgi complex in association with mitosis: redistribution of mannosidase II to the endoplasmic reticulum and effects of brefeldin A.

Previous studies have shown that the Golgi complex is broken down into dispersed clusters of vesiculotubular elements as mammalian cells enter mitosis and is reformed in each daughter cell in telophase/cytokinesis. In the present investigation, mannosidase II (a membrane-bound enzyme involved in oligosaccharide processing) was used as a marker to explore the fate of the Golgi complex in dividing L929 and CHO cells in some additional detail. Immunofluorescence microscopy demonstrated a juxta- or perinuclear staining for mannosidase II (man II) in interphase and immunoelectron microscopy revealed that it was restricted to the stacked Golgi cisternae at this stage. As the cells entered mitosis, the staining for man II assumed a pattern of dispersed elements in prophase and then turned into a diffuse pattern during metaphase and anaphase. At the electron microscopic level, this corresponded to a successive disorganization of the Golgi complex, first into structurally modified stacks scattered throughout the cytoplasm, and thereafter into small clusters of vesicles and tubules. In parallel, most of the immunoreactivity for man II was shifted into partially fragmented cisternae of endoplasmic reticulum, and only small amounts were found in the clusters just mentioned. During telophase/cytokinesis a circumscribed staining for man II reappeared in each daughter cell. At the electron microscopic level, cisternal stacks positive for man II were found to reform at the same time as immunoreactivity disappeared from the endoplasmic reticulum. Typically, the Golgi region was first located on the proximal side of the nucleus as related to the intercellular bridge, and then moved to the distal side of the nucleus before the cells were about to separate. Treatment of synchronized mitotic cells with brefeldin A, a fungal metabolite that inhibits endoplasmic reticulum to Golgi transport, prevented reformation of the Golgi complex in telophase/cytokinesis. Nevertheless, the separation of the daughter cells was completed at a similar rate as in the controls. On the basis of these findings, an extended model of the disorganization and reorganization of the Golgi complex in association with mitosis is presented. According to this model the disorganization of the Golgi complex at the onset of mitosis is a two-step process: the Golgi stacks are first separated from each other and spread out in the cytoplasm; thereafter the Golgi stacks disintegrate, at least in part by return of Golgi components to the endoplasmic reticulum. In interphase cells, similar changes in the organization of the Golgi complex are produced by microtubule-disruptive drugs (dispersion of the stacks) and brefeldin A (redistribution of Golgi proteins into the endoplasmic reticulum), respectively.

Animals↗

PubMed1991

On the mechanism of induction of DNA synthesis in cultured arterial smooth muscle cells by leukotrienes. Possible role of prostaglandin endoperoxide synthase products and platelet-derived growth factor.

The induction of DNA replication in rat aortic smooth muscle cells (SMCs) by leukotrienes (LTs) was studied in order to elucidate the mechanisms of action in further detail. The effect of LTB4 was blocked by the prostaglandin (PG) synthesis inhibitor indomethacin and the effects of LTC4 and LTD4 were blocked by the cysteinyl-containing leukotriene receptor antagonists FPL 55712 and ICI 198615. These observations suggest that LTB4 and the cysteinyl-containing leukotrienes act via distinct receptors and point to a role for prostaglandin endoperoxide synthase products in bringing about the effect of LTB4. Radioimmunological determinations and analyses of [3H]arachidonic acid metabolism showed that the SMCs were able to synthesize PGI2 (measured as the stable metabolite 6-keto-PGF1 alpha), PGE2, PGF2 alpha and 15(S)hydroxy-eicosatetraenoic acid (15(S)HETE). Moreover, picomolar concentrations of arachidonic acid, PGI2, PGE2, PGF2 alpha and 15(S)HETE induced DNA replication in the SMCs under serum-free conditions, whereas linoleic acid, 6-keto-PGF1 alpha and 5(S)HETE were inactive in this respect. Analysis of conditioned media for mitogenic activity (with or without antibodies against platelet-derived growth factor, PDGF) and for the presence of material competing with radioiodinated PDGF for binding to specific cell surface receptors indicated that LTB4 stimulated release of PDGF or a PDGF-like molecule from the cells. These findings suggest that the growth-promoting effect of LTB4 is mediated via a prostaglandin endoperoxide synthase product and/or PDGF produced by the cells themselves.

Animals↗

PubMed1991

Cyclosporine A inhibits induction of DNA synthesis by PDGF and other peptide mitogens in cultured rat aortic smooth muscle cells and dermal fibroblasts.

The immunosuppressive drug cyclosporine A was recently shown to inhibit smooth muscle proliferation in the vascular response to injury. To examine if this may be due to a direct effect of the drug on the smooth muscle cells (SMCs), we have studied its influence on the phenotypic modulation of rat aortic SMCs in primary cultures and on the induction of DNA synthesis by peptide mitogens in serum-starved subcultures. The results demonstrate that cyclosporine A does not interfere with the transition of the SMCs from a contractile to a synthetic phenotype, an early step in the preparation for cell division. On the other hand, it inhibits induction of DNA synthesis by recombinant platelet-derived growth factor-BB (PDGF-BB), basic fibroblast growth factor (bFGF), epidermal growth factor (EGF), and insulin-like growth factor-I (IGF-I). Maximum effect was obtained at a concentration of 1-3 micrograms/ml and the drug could be added 4-6 h after the growth factors with full inhibitory effect. No distinct effect on the stimulation of overall RNA and protein synthesis by PDGF-BB was observed, indicating that the drug was not of general cytotoxicity at the concentrations used. Throughout this part of the investigation, similar results were obtained with rat dermal fibroblasts. The findings indicate that cyclosporine A inhibits induction of DNA synthesis by peptide mitogens, and suggest that the inhibitory effect of cyclosporine A on smooth muscle proliferation in vivo at least in part may be due to a direct action on these cells.

Animals↗