Biomedical subjects

J Thyberg

Publications and source records attributed to J Thyberg.

At least 73 records · Page 4Linked to original sources

PubMed1990

Phenotype modulation in primary cultures of rat aortic smooth muscle cells. Effects of drugs that interfere with the functions of the vacuolar system and the cytoskeleton.

The transition of adult rat aortic smooth muscle cells from a contractile to a synthetic phenotype during the first week of primary culture on a substrate of fibronectin in serum-free medium was studied by light and electron microscopy. The weak base chloroquine and the carboxylic ionophore monensin were both found to inhibit the spreading of the cells and the accompanying changes in cellular fine structure. The exchange of myofilament bundles for a prominent rough endoplasmic reticulum and Golgi complex was delayed and vacuoles filled with incompetely degraded material accumulated in the cytoplasm. The microtubule-disruptive drugs colchicine and nocodazole likewise opposed the spreading and fine structural reorganization of the cells. Most typically, the Golgi stacks were small and widely dispersed. In addition, vacuoles of the type mentioned above increased in number. On the other hand, there was surprisingly little effect of cytochalasin B, a drug that is supposed to interfere with the assembly of actin filaments. The observations suggest that the phenotypic modulation of arterial smooth muscle cells is dependent on: (a) lysosomal degradation of discarded cellular constituents, (b) active vesicular transport along the exocytic pathway to provide the expanding cell surface with new membrane, and (c) a normal microtubular cytoskeleton to ensure the establishment of a new and functionally efficient intracellular organization.

Animals↗

PubMed1990

Changes in expression and organization of smooth-muscle-specific alpha-actin during fibronectin-mediated modulation of arterial smooth muscle cell phenotype.

The spreading of freshly isolated arterial smooth muscle cells on a substrate of fibronectin is mediated by an integrin receptor on the cell surface. It is associated with organization of actin filaments in stress fibers and marked changes in cell morphology and function, collectively referred to as a transition from a contractile to a synthetic phenotype. To study further how extracellular matrix components affect smooth muscle phenotype, we have analyzed the expression and organization of smooth-muscle-specific alpha-actin in freshly isolated rat aortic smooth muscle cells cultured on a substrate of fibronectin under serum-free conditions. Northern-blot analysis showed that the expression of mRNA for smooth muscle alpha-actin, but not for nonmuscle actin, was strongly repressed during primary culture. On the other hand, the cellular content of alpha-actin was only moderately changed during the same period. Indirect immunofluorescence staining revealed that nonmuscle actin was rapidly organized in stress fibers, which did not stain with a monoclonal antibody against smooth muscle alpha-actin. Filament bundles containing alpha-actin were most prominent in the central parts of the cytoplasm and gradually disappeared as the spreading of the cells progressed. In contrast to the situation with nonmuscle actin, there was no apparent overlap in the staining for alpha-actin and the fibronectin receptor (alpha 5 beta 1), indicating that this receptor interacted with nonmuscle actin during the initial spreading process. Taken together, the results show that the expression and organization of smooth muscle alpha-actin are changed during interaction of the cells with fibronectin early in primary culture. They support the notion that integrin-mediated interactions between extracellular matrix components and arterial smooth muscle cells take part in the control of smooth muscle phenotype.

Actins↗

PubMed1990

Localization of platelet-derived growth factor (PDGF) in CHO cells transfected with PDGF A- or B-chain cDNA: retention of PDGF-BB in the endoplasmic reticulum and Golgi complex.

Platelet-derived growth factor (PDGF) is a powerful mitogen for connective tissue cells. It is made up of two polypeptide chains (A and B) and exists in three dimeric forms (AA, AB, and BB). Transfection experiments have indicated that PDGF-AA and -AB are secreted as 30 x 10(3) Mr products, whereas PDGF-BB is processed into a 24 x 10(3) Mr product and remains associated with the cells. Here, CHO cells were transfected with PDGF B- or A-chain cDNA and the intracellular distributions of the respective gene products were compared by indirect immunofluorescence and immunoelectron microscopy, using primary antibodies specific for PDGF B- and A-chain homodimers. PDGF-BB was most conspicuous in stacked Golgi cisternae. It was also found in the endoplasmic reticulum and in lysosomes. Upon treatment of the cells with the microtubule-disruptive drug nocodazole, the Golgi complex was broken up and its stacks of cisternae were dispersed throughout the cytoplasm together with clusters of lysosomes. After this structural disorganization, the concentration of PDGF-BB to the Golgi stacks was even more prominent than before. Weak reactivity for PDGF-AA was detected in the endoplasmic reticulum and groups of vacuoles, both in control and nocodazole-treated cells, whereas Golgi stacks and lysosomes only seldom were positive. The observations suggest that PDGF-BB is processed and retained within the endoplasmic reticulum and Golgi complex. Eventually, it may also be transferred to lysosomes for degradation. In contrast, PDGF-AA is likely to follow a pathway for bulk flow, including rapid passage through the endoplasmic reticulum and Golgi complex, package in secretory vacuoles, and extracellular release by exocytosis.

Animals↗

PubMed1990

Expression of PDGF alpha- and beta-receptors in rat arterial smooth muscle cells is phenotype and growth state dependent.

Adult rat arterial smooth muscle cells were shown to express mRNA for the platelet-derived growth factor (PDGF) alpha- and beta-receptors and to bind radioiodinated PDGF-AA and PDGF-BB in a phenotype-dependent and growth state-dependent manner. PDGF alpha-receptor mRNA was not detected in the intact aortic media, but appeared as the cells converted from a contractile to a synthetic phenotype during serum-free primary culture. PDGF beta-receptor mRNA was expressed already in vivo, and increased further as the cells were isolated and cultured in vitro. Exposure of the cells to human platelet PDGF resulted in increased PDGF alpha-receptor mRNA levels, decreased PDGF beta-receptor mRNA levels, and decreased binding of both PDGF-AA and PDGF-BB. Following removal of the exogenous mitogen, the content of PDGF alpha- and beta-receptor mRNA increased, as did the binding of PDGF-AA and PDGF-BB. Subsequently, the content of PDGF A-chain mRNA started to rise, and the cells retained a high rate of DNA synthesis in a serum-free medium. As a result of this autocrine stimulation, the PDGF receptors were down-regulated. Although smooth muscle cells in serum-free primary cultures bound the different PDGF isoforms to a varying extent (AA less than AB less than BB), the replicative response was of a similar magnitude. Subcultured cells bound the different PDGF isoforms in similar proportions as the primary cells. Contrary to the situation in primary cells, there was a direct correlation between the binding level and the DNA synthetic response. Moreover, the subcultured cells did not replicate in a serum-free medium. These observations support the idea that the phenotypic modulation of arterial smooth muscle cells in primary culture prepares the cells to activate autocrine growth mechanisms. When stimulated with an exogenous mitogen, they enter the cell cycle and are thereafter able to stimulate their own growth in an autocrine manner by production of PDGF-AA or a closely related molecule.

Animals↗

PubMed1990

Disorganization and reorganization of the Golgi complex and the lysosomal system in association with mitosis.

The fate of the Golgi complex and the lysosomal system during mitosis was studied in L929 mouse fibroblasts by staining with wheat germ agglutinin conjugated to horseradish peroxidase (WGA-HRP) and transmission electron microscopy. In interphase WGA-HRP stained a circumscribed juxtanuclear region encompassing the stacked Golgi cisternae, the trans Golgi network, secretory vacuoles, endosomes, and lysosomes. As the cells entered prophase, WGA-HRP reactive material was spread around the perimeter of the nucleus. Fine structurally, the Golgi complex was found to be disorganized and its stacks were scattered throughout the cytoplasm together with clusters of lysosomes. At the same time, the trans Golgi network atrophied and secretory vacuoles and endosomes decreased in number. The disperse and structurally modified state of the Golgi complex and the lysosomal system persisted until late telophase, when they were reorganized in a defined region, most frequently on the outside of the nucleus as related to the midbody. The disorganization and reorganization of the Golgi complex and the lysosomal system coincided with the breakdown and rebuilding of the microtubular cytoskeleton, and showed striking similarities to the previously described modifications of these organelles during treatment with and after withdrawal of microtubule-disruptive drugs. Based on the findings, a model is presented that links the division of the Golgi complex and the lysosomal system to the loss of the radiating array of interphase microtubules.

Animals↗

PubMed1989

Suramin inhibits binding and degradation of platelet-derived growth factor in arterial smooth muscle cells but does not interfere with autocrine stimulation of DNA synthesis.

During in vitro culture arterial smooth muscle cells of adult rats are able to produce a platelet-derived growth factor (PDGF)-like protein and to promote their own growth in an autocrine manner. Here, this process has been studied using suramin, a polyanionic drug that has been reported to interfere with the cellular binding of several growth factors. Our results indicate that suramin speeds up the transition of the cells from a contractile to a synthetic phenotype early in primary culture. It inhibits the binding of PDGF to the cells, displaces PDGF bound to the cell surface, and slows down the degradation of PDGF internalized by the cells. It reduces the specific activities of the lysosomal enzymes acid phosphatase, beta-N-acetylglucosaminidase and beta-glucuronidase, and gives rise to an accumulation of lysosomes with myelin-like inclusions. It blocks PDGF- and serum-induced DNA synthesis and cellular proliferation in secondary cultures, but lacks a distinct inhibitory effect on DNA synthesis in primary cultures under serum-free conditions. The results suggest that the PDGF-like protein produced by the smooth muscle cells under the latter conditions may bind to its receptor and exert its autocrine effect intracellularly, without prior release into the pericellular space.

Animals↗

PubMed1989

A substrate of the cell-attachment sequence of fibronectin (Arg-Gly-Asp-Ser) is sufficient to promote transition of arterial smooth muscle cells from a contractile to a synthetic phenotype.

Extracellular matrix components strongly influence the differentiated properties of isolated rat arterial smooth muscle cells during in vitro cultivation. The attachment and spreading of the cells on a substrate of fibronectin or a 105-kDa cell-binding fragment of fibronectin are accompanied by a structural and functional transformation, referred to as a transition or modulation from a contractile to a synthetic phenotype. Here, the ability of the cell-attachment sequence of fibronectin, Arg-Gly-Asp-Ser (RGDS), to promote this process was studied. The results demonstrate that freshly isolated smooth muscle cells attached to a substrate of the synthetic peptide Gly-Arg-Gly-Asp-Ser-Cys (GRGDSC) in a specific manner and as well as to substrates of fibronectin and the 105-kDa fragment. Subsequent spreading of the cells on the peptide substrate followed the same kinetics and was as extensive as on fibronectin, even if protein synthesis was blocked by treatment of the cultures with cycloheximide. Like fibronectin, the peptide substrate induced formation of actin filament bundles, again without ongoing protein synthesis. Moreover, it was as efficient as fibronectin in supporting the transition of the cells from a contractile to a synthetic phenotype as analyzed by electron microscopy. Antibodies against the beta subunit of the fibronectin receptor interfered with the attachment, spreading, and fine structural reorganization of the cells in a similar manner on substrates of fibronectin, the 105-kDa fragment, and GRGDSC. Taken together, the findings indicate that the cell-attachment sequence (RGDS) mimics intact fibronectin in promoting a change in the differentiated properties of arterial smooth muscle cells and does so by interacting with a cell surface receptor for fibronectin.

Actins↗

PubMed1989

Integrin-type fibronectin receptors of rat arterial smooth muscle cells: isolation, partial characterization and role in cytoskeletal organization and control of differentiated properties.

The spreading of freshly isolated rat arterial smooth muscle cells (SMCs) on a substrate of fibronectin (FN) is associated with marked changes in fine structure and function of the cells, collectively referred to as a modulation from a contractile to a synthetic phenotype. Recent studies have indicated that this process is mediated via an interaction between the minimal cell-attachment sequence of FN (RGDS) and cell surface receptors. Here, we report the isolation of such receptors by sequential chromatography on affinity columns of wheat germ agglutinin (WGA) and a 105-kDa cell-binding fragment of FN (105-kDa fragment). The receptor was composed of two proteins with electrophoretic mobilities in SDS-polyacrylamide gels of 160 and 115 kDa under nonreducing conditions and 150 and 130 kDa under reducing conditions. Immunoprecipitation of surface-labeled cells with a rabbit antiserum against the beta chain of the rat hepatocyte FN receptor similarly yielded two proteins of 160 and 115 kDa. In metabolically labeled cells an additional component of 105 kDa was precipitated, presumably representing a precursor of the 115-kDa protein. Immunocytochemical studies demonstrated that SMCs grown on laminin formed FN fibrils and actin filament bundles in close alignment with cell surface receptors after a few days of culture. In cells seeded on the 105-kDa fragment, the receptors were already arranged in parallel with actin filaments on the first day of culture. Later on, the cells secreted FN and laid down FN fibrils along the receptors on the cell surface and the actin filament bundles in the cytoplasm. Taken together, the findings indicate that arterial SMCs are equipped with FN receptors that belong to the integrin family of proteins and consists of alpha (160-kDa) and beta (115-kDa) subunits. The receptor complexes apparently play an important role in determining the differentiated characteristics of the cells, possibly by mediating a linkage between the extracellular matrix and the cytoskeleton.

Animals↗

PubMed1989

Effects of leukotrienes on phenotypic properties and growth of arterial smooth muscle cells in primary culture.

During the first few days in primary culture arterial smooth muscle cells (SMCs) go through a transition from a contractile to a synthetic phenotype. Morphologically, this process includes loss of myofilaments and formation of an extensive rough endoplasmic reticulum and a large Golgi complex. Functionally, it leads to the cells losing their contractility, beginning to secrete extracellular matrix components, and dividing in response to growth factor stimulation. Similar changes in the structure and function of the SMCs occur in the initial stages of atherogenesis. The object of the present investigation was to study the effects of leukotrienes on the differentiated properties and growth of rat aortic SMCs in primary culture. Enzymically isolated cells were seeded directly on a plastic surface in serum-containing medium or on a substratum of plasma fibronectin in serum-free medium. The change in cell morphology was followed by transmission electron microscopy, and the activation of cell growth by thymidine autoradiography and cell counting. The results demonstrate that 10 pM-LTB4, -LTC4, -LTD4 and -LTE4 all speeded up the shift of the SMCs into a synthetic phenotype, whereas 5S,12S-DHETE (an isomer of LTB4) lacked effect. Further, LTB4, LTC4 and LTD4 stimulated the SMCs to enter the cell cycle earlier than in the controls, enhanced the proliferative response to serum mitogens, and under serum-free conditions induced DNA synthesis by themselves. Indomethacin did not interfere with the effect of LTB4 on the structural transformation of the cells but blocked its effect on DNA replication, suggesting that only the latter involved endogenous production of a cyclo-oxygenase product.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗

PubMed1989

Subpopulations of microtubules with differential sensitivity to nocodazole: role in the structural organization of the Golgi complex and the lysosomal system.

The role of cytoplasmic microtubules (MTs) in the structural organization of the Golgi complex and the lysosomal system was studied in L929 mouse fibroblasts using a combination of cytochemical and electron microscopic methods. Immunocytochemical staining demonstrated a radiating pattern of MTs, originating in the juxtanuclear region containing the centrioles. The stacks of Golgi cisternae with associated vesicles and tubules (the trans Golgi network), as well as most of the endosomes and lysosomes, were also located in and immediately around this region. Wheat germ agglutinin (WGA) conjugated to horseradish peroxidase (HRP) bound to the plasma membrane at 4 degrees C. After warming to 37 degrees C, the conjugate was rapidly internalized by endocytosis and via small vesicles transferred to endosomes and lysosomes in the juxtanuclear region. Slightly later it was also found in the trans Golgi network. A weaker but otherwise similar staining was obtained if the conjugate was applied to fixed and permeabilized cells. Treatment with 3.0 microM nocodazole induced depolymerization of MTs and rounding up of the cells. At the same time, the Golgi complex was disorganized with dispersion of its stacks of cisternae throughout the cytoplasm and atrophy of the trans Golgi network. The lysosomes gathered around the dispersed cisternal stacks and the endosomes became fewer in number. Moreover, the uptake of WGA-HRP was markedly inhibited. Following withdrawal of the drug, MTs reformed and the structural organization of the cell was again normalized. If the recovery was allowed to take place in the presence of low concentrations of nocodazole (0.03 or 0.15 microM), MTs of short and medium length were formed, the latter showing a curly path. In parallel, a partial or nearly complete normalization of cell morphology occurred. Internalization and transport of WGA-HRP labeled membrane to the lysosomes and the trans Golgi network was also resumed. In contrast, the cells were not able to pass through mitosis in a normal manner and multimicronucleated cells accumulated. Taken together, the findings indicate that L929 cells contain a subset of nocodazole-resistant MTs which play an important role in the structural organization of the cell, especially with regard to the Golgi complex and the lysosomal system. They support recent notions that there exists subpopulations of MTs with different physicochemical properties and functions.

Animals↗

PubMed1989

Monensin and chloroquine inhibit transfer to lysosomes of endocytosed macromolecules in cultured mouse peritoneal macrophages.

The effects of the Na+/H+ ionophore monensin and the weak base chloroquine on lysosomal uptake of endocytosed macromolecules were studied in cultured mouse peritoneal macrophages using horseradish peroxidase (HRP) and ferritin as exogenous tracers. The lysosomes were first loaded with HRP using a pulse-chase protocol. The cells were then exposed to ferritin for 30 to 120 min, either in control medium or in medium containing 3 microM monensin or 50 microM chloroquine. Semiquantitative electron microscopic analyses indicated that the uptake of ferritin into HRP-labeled lysosomes was inhibited in the drug-treated cells, and that the tracer particles accumulated in endosomes. At the same time the volume density of the endosomes was increased, fourfold by monensin and threefold by chloroquine; with the latter drug there was also an increase in lysosome volume density. Further, both drugs decreased the rate of endocytosis as measured biochemically, but not in proportion to the reduction of lysosomal ferritin uptake. After withdrawal of the drugs, cell morphology returned to normal and transfer of ferritin from endosomes to HRP-labeled lysosomes was resumed. The recovery was more rapid and complete in monensin-treated than in chloroquine-treated cells. On the basis of these findings and earlier investigations demonstrating that monensin and chloroquine both raise the pH in acid cell compartments, it is suggested that the transfer of soluble and not only membrane-bound macromolecules from endosomes to lysosomes is modulated by the pH in these organelles.

Animals↗

PubMed1988

Chloroquine and monensin inhibit induction of DNA synthesis in rat arterial smooth muscle cells stimulated with platelet-derived growth factor.

The weak base chloroquine and the Na+/H+ ionophore monensin were used to study the role of lysosomes in the induction of DNA synthesis by platelet-derived growth factor (PDGF) in rat arterial smooth muscle cells cultivated in vitro. The results show that PDGF initiates DNA synthesis in a defined, serum-free medium. This indicates that a single factor may control, directly or indirectly, the transition from the G0 to the G1 phase, the progress through the G1 phase, and the entrance into the S phase of the cell cycle. It is further demonstrated that PDGF has to be present throughout most of the prereplicative period (12-16 h) to induce DNA synthesis in the maximum number of cells, suggesting that one or more processes need to be stimulated continually or successively to push the cell into the S phase. Chloroquine and monensin inhibit induction of DNA replication by PDGF, with maximum effect at 50 microM and 5 microM, respectively. To be fully active, the drugs have to be added within 4-8 h after the growth factor, but a partial inhibition persists if they are added at any time during the prereplicative period. Both drugs reduce PDGF-stimulated RNA and protein synthesis, and suppress degradation of [3H]leucine-labeled cellular protein and [125I]-labeled PDGF. Fine-structurally, they give rise to an accumulation of lysosomes or prelysosomal vacuoles with inclusions of incompletely degraded material. These findings suggest that the mitogenic effect of PDGF is dependent on a normal function of lysosomes during the prereplicative phase, especially its first half (0-8 h).

Animals↗

PubMed1988

Arterial smooth muscle cells express platelet-derived growth factor (PDGF) A chain mRNA, secrete a PDGF-like mitogen, and bind exogenous PDGF in a phenotype- and growth state-dependent manner.

Adult rat arterial smooth muscle cells are shown to express platelet-derived growth factor (PDGF) A chain mRNA, to secrete a PDGF-like mitogen, and to bind exogenous PDGF in a phenotype- and growth state-dependent manner. In the intact aortic media, where the cells are in a contractile phenotype, only minute amounts of PDGF A chain and no B chain (c-sis) RNA were detected. After cultivation and modulation of the cells into a synthetic phenotype, the A chain gene was distinctly expressed, whereas the B chain gene remained unexpressed. Cells kept in serum-free medium on a substrate of plasma fibronectin showed high levels of A chain RNA and high PDGF receptor activity, but did not secrete detectable amounts of PDGF-like mitogen. After exposure to PDGF, which is itself sufficient to initiate DNA synthesis and mitosis in these cells, a PDGF-like mitogen was released into the extracellular medium. Concomitantly, the amount of A chain transcripts per cell and the ability of the cells to bind radioactive PDGF decreased. Similarly, smooth muscle cells initially grown in the presence of serum released more PDGF-like mitogen, contained fewer A chain transcripts, and bound more radioactive PDGF in proliferating than in stationary cultures. The findings confirm the notion that adult rat arterial smooth muscle cells are able to promote their own growth in an autocrine or paracrine manner. Furthermore, they reveal some basic principles in the control of this process.

Age Factors↗

PubMed1988

Diverse effects of fibronectin and laminin on phenotypic properties of cultured arterial smooth muscle cells.

Plasma fibronectin promotes modulation of rat arterial smooth muscle cells from a contractile to a synthetic phenotype during the first few days in primary culture. This process includes cell adhesion and spreading, loss of myofilaments, and formation of a widespread rough endoplasmic reticulum and a prominent Golgi complex. The structural reorganization is accompanied by activation of overall RNA and protein synthesis. Moreover, the cells gain the ability to replicate their DNA and divide in response to platelet-derived growth factor. Here, it is demonstrated that the power of fibronectin to bring about this change in the differentiated properties of the smooth muscle cells resides in a 105-kD cell-binding fragment, whereas a 70-kD collagen-binding fragment and a 31-kD heparin-binding fragment are inactive in this respect. Laminin, another adhesive glycoprotein and a component of the basement membrane that normally surrounds arterial smooth muscle, was contrarily found to maintain the cells in a contractile phenotype. However, with increasing time more and more cells went through the modulation into a synthetic phenotype. This "catch-up" was counteracted by a peptide that contained the cell-attachment sequence of fibronectin (Arg-Gly-Asp-Ser). Hence, it is possible that the delayed modulation on laminin was due to production of fibronectin by the cells themselves. In support of this notion, fibronectin isolated from smooth muscle cultures was found to be as effective as plasma fibronectin in stimulating the phenotypic modulation. Moreover, using a combination of chemical, immunochemical, and immunocytochemical methods, it was demonstrated that the cells secreted fibronectin as well as laminin at an increasing rate during the first 4 d in primary culture and, notably, cells cultured on laminin produced more fibronectin than cells cultured on fibronectin. Newly synthesized fibronectin was incorporated into a network of pericellular and intercellular fibrils, whereas laminin formed a more diffuse layer covering the cells in a basement membrane-like manner. Taken together, the findings suggest diverse roles for fibronectin and laminin in the control of the differentiated properties of arterial smooth muscle cells. They further indicate that the ability of arterial smooth muscle cells to produce fibronectin and laminin early in primary culture is not directly related to the phenotypic state as determined morphologically and by measurement of overall rates of RNA and protein synthesis. This may be due to the cells being able to sense the macromolecular composition of the pericellular matrix and to modify their secretory activity accordingly.(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗

PubMed1988

Elevated expression of c-myc and N-myc produces distinct changes in nuclear fine structure and chromatin organization.

The proto-oncogenes c-myc and N-myc encode nuclear phosphoproteins with unknown function. Here, c-myc or N-myc, or hybrid constructs of the two, were transfected into fibroblastic cells (CV-1) using SV40-based high expression vectors. The cells were studied by indirect immunofluorescence microscopy and transmission electron microscopy to determine the localization of the two myc proteins within the nucleus and their influence on nuclear fine structure and chromatin organization. In c-myc transfected cells the overproduced protein product accumulated in large amorphous globules that displaced the normal chromatin and did not stain for DNA. In N-myc transfected cells condensed chromatin loops were formed. They were attached to the nuclear envelope and by traction in the latter they may have contributed to give the nucleus its irregular shape in these cells. During mitosis the chromatin loops persisted as clearly identifiable entities within the chromosomes, suggesting a rigid conformation that did not allow normal chromosome packaging. These findings suggest that the c-myc and N-myc proteins bind to different structures and may have different functions. Observations on cells transfected with hybrid constructs indicated that both the second and third exon of c-myc were required to yield a product that behaved like the c-myc protein. In contrast, domains encoded by the second exon of N-myc were sufficient to give rise to a product that morphologically behaved like the N-myc protein.

Animals↗

PubMed1988

On the role of the nucleus in the structural organization of the cell: dispersion and rearrangement of the Golgi complex in cytoplasts treated with antimicrotubular drugs.

Using cytochemical and electron microscopic techniques, it was shown that enucleated L929 fibroblasts retained a radiating pattern of microtubules as well as a large and circumscribed Golgi complex for at least one day. At the same time, the number of ribosomes and the overall size of the rough endoplasmic reticulum were reduced, probably as a result of the arrest in production of new mRNA. Treatment of the cytoplasts with either of the microtubule-disruptive drugs colchicine or nocodazole led to loss of microtubules, aggregation of intermediate filaments in large bundles, and a characteristic disorganization of the Golgi complex with spreading of its constituent stacks of cisternae throughout the cytoplasm. After withdrawal of the drugs, microtubules reappeared, intermediate filament bundles disaggregated, and the Golgi complex resumed a morphology comparable to that of cells kept in normal medium during the entire experiment. In all these respects, the cytoplasts behaved in a similar way as nucleated cells. The observations confirm earlier notions of a role of the microtubular cytoskeleton in the organization of the Golgi complex. They further indicate that the information needed to support the normal structure and interaction of these organelle systems is present in the cytoplasm and does not require the physical presence of the nucleus, nor continuous nuclear activity, i.e. transcription and processing of mRNA.

Animals↗

PubMed1987

Modulation of arterial smooth muscle cells from contractile to synthetic phenotype requires induction of ornithine decarboxylase activity and polyamine synthesis.

The role of polyamines in modulating cultivated arterial smooth muscle cells from contractile to synthetic phenotype was studied by biochemical and electron microscopic methods. Ornithine decarboxylase (ODC), the first and overall rate-limiting enzyme in polyamine biosynthesis, showed no detectable activity in freshly isolated contractile cells, rose to a peak of activity after 2 days of culture, simultaneously with the most rapid phase of phenotypic modulation, and thereafter returned to a lower level of activity. The increase in ODC activity was accompanied by a gradual rise in the cellular concentrations of the polyamines putrescine, spermidine and spermine. The polyamine synthesis inhibitors alpha-difluoromethylornithine and methylglyoxal-bis(guanylhydrazone) prevented transition into synthetic phenotype as well as initiation of cell growth. The results indicate that polyamines play an important role in the control of smooth muscle phenotype and growth.

Animals↗

PubMed1987

Induction of ornithine decarboxylase activity and putrescine synthesis in arterial smooth muscle cells stimulated with platelet-derived growth factor.

Cultivated arterial smooth muscle cells were used to study the effects of platelet-derived growth factor (PDGF) on polyamine synthesis and of polyamine synthesis inhibitors on initiation of DNA synthesis by PDGF. Quiescent (serum-starved) cells showed no detectable activity of ornithine decarboxylase (ODC), the first and overall rate-limiting enzyme in polyamine synthesis, and had low levels of the polyamines putrescine, spermidine and spermine. PDGF caused a marked increase in ODC activity, with a peak after 6 h followed by a rapid decline. The rise in enzyme activity was blocked by actinomycin D and cycloheximide, suggesting control at the transcriptional and translational levels, alpha-Difluoromethylornithine (DFMO), a catalytic and irreversible inhibitor of ODC, prevented the appearance of enzyme activity. The cellular content of putrescine increased distinctly in response to PDGF, with a peak after 8 h, abolished by simultaneous treatment with DFMO. In contrast, the concentrations of spermidine and spermine changed but little within the 20-h period examined here. DFMO inhibited the initiation of DNA synthesis without affecting the length of the prereplicative lag phase and was fully active if added within 4 h after PDGF. The effect of DFMO on DNA synthesis was counteracted by addition of polyamines to the culture medium and exogenous polyamines were also found to stimulate DNA synthesis in PDGF-free medium (spermine greater than spermidine greater than putrescine). It is concluded that induction of ODC and putrescine synthesis are integral parts in the mitogenic response of arterial smooth muscle cells to PDGF.

Animals↗