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Biomedical subjects

J Thyberg

Publications and source records attributed to J Thyberg.

At least 145 records · Page 8Linked to original sources
PubMed1979

Comparison of the in vitro effects of colchicine and its derivative colchiceine on chondrocyte morphology and function.

Colchiceine is a colchicine-metabolite which has been reported to inhibit axonal transport although not binding to brain tubulin. In the present study, colchiceine was shown not to depolymerize cytoplasmic microtubules, nor to mimic other effects of colchicine on the ultrastructure of cultured chondrocytes. In addition, the synthesis of proteoglycans was inhibited by colchicine but slightly stimulated by colchiceine. These results support the idea that the disturbances in cultured chondrocytes caused by colchicine are specifically related to a loss of cytoplasmic microtubules.

Animals↗
PubMed1979

Bone formation in cartilage produced by transplanted epiphyseal chondrocytes.

Chondrocytes were isolated from rat epiphyseal cartilage, cultured in vitro, and exposed to exogenous tracers which accumulated in their lysosomes. The cells were then injected into the posterior tibial muscle of animals from the same outbred strain, where they reconstructed calcifying hyaline cartilage. The mineralization of the tissue was followed by ingrowth of blood capillaries from the host bed. Macrophage-like cells surrounding the vessels phagocytized degenerated chondrocytes and unmineralized matrix, whereas multinucleated chondroclasts removed some of the mineralized cartilage matrix. Mesenchyme-like cells accompanying the invading vessels attached to the remaining septa of calcified cartilage matrix and developed into osteoblasts depositing bone matrix on the surface of these septa. The apparent lack of inherent tracer labeling of the lysosomes in the different bone cells indicate that they were derived from the host. No signs of transformation of chondrocytes into bone cells were observed. When isolated rat epiphyseal chondrocytes were injected into the wall of the hamster cheek pouch, calcifying cartilage was reconstructed without signs of subsequent ossification. Transplantation of cartilage reconstructed in the hamster into the dorsal muscles of rats was, however, followed by formation of bone by a sequence analogous to that described above. Such an osteogenetic response was also obtained when the cartilage had been devitalized before transplantation. These experiments show that calcified cartilage, developing in or grafted into an intramuscular site, is able to induce and serve as a substrate for endochondral bone formation, similar to that occurring during normal development. They further indicate that bone induction by calcified cartilage does not require the presence of living chondrocytes.

Animals↗
PubMed1979

Electron microscopic and cytochemical studies of rat aorta. Intracellular vesicles containing elastin- and collagen-like material.

Small, rounded vesicles with a dense core of amorphous material were observed in all cell types in the young rat aorta, that is, endothelial cells, smooth muscle cells and fibroblasts. They were particularly numerous in the Golgi complex but were also found in the cell periphery. The content of the vesicles had staining characteristics identical to those of elastin. Material of the same type was also found in cisternae on the maturing side of the dictyosomes and in vesicles budding from them. Reaction product for thiamine pyrophosphatase was present in both these structures, indicating that the Golgi complex is responsible for the formation of the dense-cored vesicles. This was further supported by the absence of reaction product for acid phosphatase in the cisternae and in the vesicles. Moreover, no uptake of exogenous markers was noted in the latter. On the basis of these findings it is suggested that the dense-cored vesicles have a secretory function and contain precursors of elastin. Elongated vesicles or profiles containing collagen fibrils were observed in smooth muscle cells and fibroblasts. In the cell periphery, these vesicles were often found to communicate with the extracellular space. Further inside the cells, they showed a close spatial relationship to the Golgi complex. Neither thiamine pyrophosphatase nor acid phosphatase activity was demonstrated in the elongated vesicles. Like the plasma membrane, their limiting membrane was positively stained for alkaline phosphatase. On the basis of these findings and the absence of uptake of exogenous markers in them, it is suggested that the elongated vesicles represent a means for collagen secretion in the growing aortic wall. The Golgi complex is believed to be involved in the transfer of collagen to these vesicles.

Animals↗
PubMed1979

Effects of colchicine on endocytosis and cellular inactivation of horseradish peroxidase in cultured chondrocytes.

Horseradish peroxidase (HRP) was used as a marker to study the effects of microtubule-disruptive drugs on uptake and cellular inactivation of exogenous material in cultures of embryonic chick chondrocytes. HRP was ingested by fluid endocytosis, and intracellular enzyme activity subsequently diminished exponentially with time. Cytochemically, reaction product for HRP was found in vesicles often located close to the dictyosomes of the Golgi complex. Colchicine and vinblastine caused disappearance of cytoplasmic microtubules and disorganization of the Golgi complex with concomitant reduction in the cellular uptake of HRP to about half of that in the controls. Lumicolchicine, on the other hand, left cell fine structure and HRP uptake unaffected. These results indicate that microtubules are of considerable importance in the process of fluid endocytosis in cultured chondrocytes although the exact mechanism remains to be elucidated. The rate of intracellular inactivation of ingested HRP was not affected by colchicine or vinblastine. Double-labeling experiments with colloidal thorium dioxide and HRP likewise indicated that fusion of endocytic vesicles and lysosomes is not dependent on intact microtubules. The total specific activities of the three lysosomal enzymes examined were weakly or not at all changed by treatment of the cultures with colchicine or vinblastine. It therefore seems unlikely that microtubular organization plays an important role in the production or degradation of lysosomal enzymes in cultured chondrocytes.

Acetylglucosaminidase↗
PubMed1979

Further studies on splenic material inhibiting the growth of lymphocytes in vitro.

A material inhibiting growth of lymphocytes from different lymphoid organs and from different species was purified from calf spleen. A factor with a molecular weight of about 45,000 dal (estimated by gel chromatography) inhibited DNA synthesis and mitosis irreversibly in thymocytes and caused degenerative changes in the nucleus and the cytoplasm of the thymocytes as judged by electron microscopy. However, no decrease in cell number and no increase in dye uptake in a dye exclusion test were found. Growth inhibition was also demonstrated for human melanoma cells, but not for rat liver cells. The cytotoxicity and the lack of absolute lymphocyte specificity of the factor speak against its character as a lymphocyte chalone. A probably identical factor was purified from calf liver, but was not found in extracts of calf thymus.

Animals↗
PubMed1978

Cartilage proteoglycan aggregates. Electron-microscopic studies of native and fragmented molecules.

1. Proteoglycan aggregates from bovine nasal cartilage were studied by using electron microscopy of proteoglycan/cytochrome c monolayers. 2. The aggregates contained a variably long central filament of hyaluronic acid with an average length of 1037nm. The proteoglycan monomers attached to the hyaluronic acid appeared as side chain filaments varying in length (averaging 249nm). They were distributed along the central filament at an average distance of about 36nm. 3. Chondroitin sulphate side chains were removed from the proteoglycan monomers of the aggregates by partial chondroitinase digestion. The molecules obtained had the same general appearance as intact aggregates. 4. Proteoglycan aggregates were treated with trypsin and the largest fragment, which contains the hyaluronic acid, link protein and hyaluronic acid-binding region, was recovered and studied with electron microscopy. Filaments that lacked the side chain extensions and had the same length as the central filament in the intact aggregate were observed. 5. Hyaluronic acid isolated after papain digestion of cartilage extracts gave filaments with similar length and size distribution as observed for the central filament both in the intact aggregate and in the trypsin digests. 6. Umbilical-cord hyaluronic acid was also studied and gave electron micrographs similar to those described for hyaluronic acid from cartilage. However, the length of the filament was somewhat shorter. 7. The electron micrographs of both intact and selectively degraded proteoglycans corroborate the current model of cartilage proteoglycan structure.

Animals↗
PubMed1978

Effects of antimicrotubular agents on the fine structure of the Golgi complex in embryonic chick osteoblasts.

Embryonic chick frontal bones were cultured in the presence of colchicine or vinblastine and subsequently examined by tranmission electron microscopy. In control cultures the osteoblasts showed a large Golgi complex consisting of dictyosomes arranged in a well-defined juxtanuclear area. Microtubules were particularly numerous within this Golgi area although they could be observed throughout the cytoplasm. Colchicine and vinblastine caused the disappearance of cytoplasmic microtubules, while bundles of 10 nm diameter filaments appeared more frequently. In addition, cell polarity was lost and the Golgi complex became disorganized, with the dictyosomes randomly dispersed in the cytoplasm and showing a decreased number of cisternae and an increased number of vacuoles, the latter generally lacking stainable material. Increased number of autophagosomes were also noted. These findings indicate that microtubules function in the organization of the Golgi complex in osteoblasts. In view of the well documented role of this organelle system in collagen secretion it is suggested that previously observed secretory disturbances produced by antimicrotubular drugs may be due to a defective transfer of material to the dictyosomes and/or a defect in the packaging and transport of such material away from them.

Animals↗
PubMed1977

In vitro effects of chlorpromazine on microtubules and the Golgi complex in embryonic chick spinal ganglion cells: an electron microscopic study.

Spinal ganglia from 11-day-old chick embryos were incubated in media containing chlorpromazine (CPZ), lidocaine or D-amphetamine and subsequently examined by transmission electron microscopy. CPZ and lidocaine both caused a structural modification of the neuroblasts similar to that induced by the microtubular-disrupting drugs colchicine and vinblastine. That is, there was a partial disappearance of cytoplasmic microtubules with a concomitant increase in the number of microfilaments and the dictyosomes of the Golgi complex changed, with the number of narrow cisternae decreasing and the number of associated vacuoles increasing. Moreover, the dictyosomes were more distinctly separated from each other than in control ganglia. D-Amphetamine did not give rise to any clear changes in either the microtubular system or the Golgi complex and, furthermore, opposed the colchicine-like effects of CPZ. On the basis of these results it is suggested that the subcellular mechanism of action of CPZ involves the cytoplasmic microtubular system and thus secondarily leads to structural and functional alterations in the Golgi complex, viz., in the case of nerve cells, a disturbance in the production and packaging of material destined for the axon terminals.

Animals↗
PubMed1977

Fine structure of rabbit ear chondrocytes in vitro and after autotransplantation.

Chondrocytes were isolated enzymatically from rabbit ear cartilage, grown in vitro or as autotransplants for 1, 2 or 5 weeks and then examined by transmission electron microscopy. A confluent monolayer formed rapidly in vitro and the cells later grew in multiple overlapping layers, producing thick sheets of cartilaginous tissue. The cells retained a normal structure throughout the period of observation and, like the chondrocytes in intact cartilage, showed numerous microfilaments, and extensive granular endoplasmic reticulum and a prominent Golgi complex. Large amounts of intercellular matrix were laid down in vitro consisting of thin collagen fibrils, small rounded or polygonal granules believed to represent proteoglycans and patches and fibres of elastin. Chondrocytes in intramuscular autotransplants reconstituted an elastic cartilage. The exogenous origin of the cells in the transplants was verified by labeling of the lysosomes by exposure of the cells to colloidal thorium dioxide particles prior to injection. Structurally, the cells and the matrix of the transplants conformed to the above description. Accumulations of elastin-like material were sometimes observed in the Golgi vacuoles of the cells. Extracellularly, such conglomerates aggregated in connection with bundles of microfibrils, building up mature elastic fibres with a dense amorphous structure. The culture and transplant systems characterized here provide suitable experimental models for studies on development, growth and aging of elastic cartilage, including various aspects of the formation and turnover of elastic fibres and other macromolecular matrix components.

Animals↗
PubMed1977

Electron microscopy of cartilage proteoglycans.

The proteoglycans of cartilage are complex molecules in which chondroitin sulphate and keratan sulphate chains are covalently linked to a protein core, forming a polydisperse population of proteoglycan monomers. By interaction with hyaluronic acid and link proteins, the monomers form large macromolecular complexes. In vivo the proteoglycans mainly occur in such aggregates. In the electron microsope, the cartilaginous matrix can be seen to be made up of thin collagen fibrils and polygonal granules about 10-50 nm in diameter Addition of the polyvalent cationic dye Ruthenium Red to glutaraldehyde and osmium tetroxide fixatives yields a dense selective staining of the matrix granules. Following a short digestion of cartilage slices with either of the chondroitin sulphate-degrading enzymes hyaluronidase and chondroitinase or with the proteolytic enzyme papain, the matrix granules were few in number or completely absent and the proteoglycan content, measured as hexosamine, decreased by up to 90%. Similarly, extraction of the cartilage with 4 M guanidine-HCl removed all matrix granules and most of the proteoglycans. From these findings, it can be concluded that the matrix granules represent proteoglycans, most probably in aggregate form, and that Ruthenium Red staining may be used to study the distribution of these macromolecules in thin sections. As a complement to chemical studies on proteoglycan structure, it is also possible to observe and measure individual molecules in the electron microscope after spreading them into a monomolecular layer with cytochrome c. This technique has been applied in investigations on proteoglycans isolated from bovine nasal cartilage and other hyaline cartilages. The molecules in the monomer fractions appeared as an extended central core filament to which about 25--30 side-chain filaments were attached at various intervals. The core filament, averaging about 300 nm in length, was interpreted as representing the polysaccharide binding part of the protein core and the side-chain filaments, averaging about 45 nm in length, as representing the clusters of chondroitin sulphate chains. Statistical treatment of the collected data indicated that no distinct subpopulations existed within the monomer fractions. The electron microscopic results correlated well with chemical data for the corresponding fractions and together with recent observations on various aggregate fractions strongly support present concepts of proteoglycan structure.

Animals↗
PubMed1977

Electron microscopic studies on embryonic chick spinal ganglion cells: relationship between microtubules and the Golgi complex.

Spinal ganglia from 11 day chick embryos were fixed immediately after removal, or after a short incubation in media with or without nerve growth factor (NGF), and were subsequently examined by transmission electron microscopy. Qualitatively, incubation did not effect the fine structure of the neuroblasts. Morphometrically, however, NGF was found to cause a marked increase in the amounts of microtubules and microfilaments in the perikarya of the cells with a threefold increase in the volume density of these organelles after 4 h. The cells displayed a prominent Golgi complex, mostly with dictyosomes organized in one continuous band or area. Individual dictyosomes consisted of a stack of 3-6 parallel cisternae associated with small vesicles and occasional larger vacuoles. Microtubules were observed in all parts of the cyotplasm but were particularly numerous within the Golgi area. They occurred both on the forming and maturing sides of the dictyosomes and in the latter site were closely associated with small vesicles and peripheral dilatations of the cisternae. These observations indicate a possible role of microtubules in the organization and function of the Golgi complex.

Animals↗
PubMed1977

Electron microscopic studies on embryonic chick spinal ganglion cells: in vitro effects of antimicrotubular agents on the Golgi complex.

Spinal ganglia from 11 day chick embryos were incubated in media containing colchicine or vinblastine and subsequently examined by transmission electron microscopy. Both drugs caused a partial disappearance of cytoplasmic microtubules and a concomitant growth in the number of microfilaments in the neuroblasts. In ganglia treated with vinblastine for 4 h these effects were recorded morphometrically as a decrease of about 80% in the volume density of microtubules and a more than tenfold increase in the volume density of microfilaments. Furthermore, the cells displayed marked structural changes in the Golgi complex. The dictyosomes were mostly distinctly separated from each other and individual dictyosomes showed a decrease number of cisternae and an increased number of closely associated large vacuoles. The results are discussed with regard to the role of microtubules in the organization and function of the Golgi complex in nerve cells.

Animals↗
PubMed1977

Fine structure of the golgi complex during mitosis of cartilaginous cells in vitro.

Chondrocytes were isolated enzymatically from guinea-pig epiphyses and grown in vitro. The fate of the Golgi complex during mitosis in relation to changes in the cytoplasmic microtubules was then studied by transmission electron microscopy. Interphase cells were observed to be polarized, with the Golgi complex occupying a well-defined juxtanuclear area of the cell's cytoplasmic pole. During prophase the cytoplasmic microtublues were largely lost, the nucleus moved to the center of the cell and the Golgi complex dissolved into single dictyosomes spread diffusely throughout the cytoplasm. The distribution of other organelles also changed to a more random pattern. In telophase, i.e. after the completion of nuclear division, the mitotic spindle decomposed and cytoplasmic microtubules reappeared. Furthermore, the organization of the Golgi complex and other organelles returned to that characteristic of interphase cells. Previous studies on cells treated with colchicine have indicated that the polarized distribution of cell organelles is dependent on the presence of intact cytoplasmic microtubules. It is suggested that the disappearance of such tubules observed here to be coupled with the disorganization of cell interphase structure fulfills the double function of providing free tubulin units from which to build the mitotic spindle and ensuring an approximately equal distribution of dictyosomes and other organelles to the daughter cells during cytokinesis.

Anaphase↗
PubMed1977

Effects of colchicine and vinblastine on the phytohaemagglutinin-induced transformation of lymphocytes.

The effects of 2 microtubular-disruptive drugs, colchicine and vinblastine, on the phytohaemagglutinin (PHA)-induced blast transformation and mitogenic stimulation of human lymphocytes were studied. Both drugs markedly inhibited cell growth and DNA synthesis and lowered the mitotic index. No microtubules were seen with the electron microscope in cells treated with PHA plus colchicine or vinblastine. Moreover, the PHA-induced development of all organelles was partially inhibited by these drugs, especially that of the Golgi complex. As compared to cells treated with PHA alone, the dictyosomes were fewer, not so clearly localized in one area of the cytoplasm, and contained a decreased number of cisternae and an increased number of vacuoles. These results indicate that cytoplasmic microtubules play an important role in the PHA-induced blast transformation and mitogenic stimulation of lymphocytes. It is suggested that the microtubules function in the structural organization of the cell and particularly the Golgi complex. In the drug-induced absence of microtubules this and other organelle systems do not respond as usual to PHA stimulation, which could largely explain the decreased cell growth. This in turn suggests that lowered mitotic activity is a result of inhibition of cell growth, as a critical amount of G1-associated cell growth is believed to be required for the initiation of DNA synthesis and thus mitosis.

Colchicine↗
PubMed1976

Heterotopic allotransplantation of isolated aortic cells. An electron microscopic study.

Smooth muscle cells were enzymatically isolated from the tunica media of the aorta of 5-day-old rats. Following in vitro exposure to colloidal thorium dioxide particles the cells displayed numerous lysosomes filled with these particles. Intramuscular injection of isolated cells into the tongues of young rats of the same strain, resulted in the formation by both freshly isolated and thorium dioxide-labeled cells of a characteristic tissue similar to that found in the intact aortic wall. Thus, the transplants consisted of typical smooth muscle cells surrounded by an extracellular matrix consisting of a microfibrillar network, patches and fibers of elastin, collagen fibrils and small polygonal granules believed to represent proteoglycans. This system can be used for experimental studies on the production of extracellular matrix components and on other functions of arterial smooth muscle cells growing outside the vascular wall.

Animals↗