Electrophoretic pattern and distribution of cytoskeletal proteins in flat-epitheloid and stellate process-bearing astrocytes in primary culture

One- and two-dimensional electrophoresis patterns and distribution of major cytoskeletal proteins were studied in primary astrocytes with either flat-epitheloid or stellate appearance. No major differences in the electrophoretic patterns of actin, tubulin, glial fibrillary acidic protein (GFAP) and vimentin were detected between flat-epitheloid and stellate process-bearing astrocytes produced by the exposure of cultures to dibutyryl cyclic AMP (dBcAMP). However the morphological changes of astrocytes were accompanied by marked changes in the quantitative distribution of cytoskeletal proteins. The most prominent change was a large and specific decrease in the amount of actin, detected by [³⁵S]methionine incorporation, densitometric scanning of one-dimensional gels and DNase inhibition assay. In stellate astrocytes produced by a 4 day treatment with dibutyryl cyclic AMP, the amount of actin decreased by 50%. This decrease was not apparently related to the depolymerization of actin.     

Hepatic stellate cells and astrocytes

Scar formation inhibits tissue repair and regeneration in the liver and central nervous system. Activation of hepatic stellate cells (HSCs) after liver injury or of astrocytes after nervous system damage is considered to drive scar formation. HSCs are the fibrotic cells of the liver, as they undergo activation and acquire fibrogenic properties after liver injury. HSC activation has been compared to reactive gliosis of astrocytes, which acquire a reactive phenotype and contribute to scar formation after nervous system injury, much like HSCs after liver injury. It is intriguing that a wide range of neuroglia-related molecules are expressed by HSCs. We identified an unexpected role for the p75 neurotrophin receptor in regulating HSC activation and liver repair. Here we discuss the molecular mechanisms that regulate HSC activation and reactive gliosis and their contributions to scar formation and tissue repair. Juxtaposing key mechanistic and functional similarities in HSC and astrocyte activation might provide novel insight into liver regeneration and nervous system repair.     

Microtubular organization in protoplasts and cells of somatic embryo-regenerating and non-regenerating cultures of Larix

Protoplasts were isolated from both somatic embryo-regenerating and non-regenerating cultures of hybrid larch ( Larix x eurolepis Henry) and fractionated on a discontinuous Percoll density gradient, whereby a highly embryogenic protoplast fraction could be enriched. This fraction was cultured for 14 days, and the differentiating protoplasts, cells, proetmbryos and embryo-like cell clusters sampled at days 3, 5 and 14. Immunofluorescence studies showed that the microtubules became organized into parallel and helical arrays in protoplast-derived cells of the embryogenic tissue as early as day 3 in culture, at which time the protoplast-derived cells started to elongate. In most of the protoplasts from non-regenerating tissue the microtubules retained a random orientation for a longer period. Preprophase bands were observed in both lines. Mitotic spindles consisted mainly of kinetochore-associated microtubules and displayed broad polar regions at metaphase. The spindle poles contracted at anaphase, giving the spindles a pointed appearance. A difference between the two tissue lines was observed at telophase, when the phragmoplast in the non-regenerating tissue had a normal appearance, while a proportion of the phragmoplasts from the embryo-regenerating line were branched or Y-shaped. Y-shaped phragmoplasts resulted in two nucleated cells and one enucleated cell after fusion of the cell plate with the plasma membrane. The early rearrangement of cortical microtubules is an indication that organized growth is occurring but, as this phenomenon has been observed also in regenerating non-emhryogenic cells, it appears to be a doubtful indicator of the distinction between emhryogenic and non-embryogenic development.     

Microtubular organization during asymmetrical division of the generative cell inGagea lutea

Video microscopy and conventional or Confocal Laser Scanning Microscopy after DAPI staining and anti-α-tubulin labelling were used to study the asymmetrical division of the generative cell (GC) inGagea lutea. Pollen was cultured for up to 8 hr in a medium containing 10% poly (ethylene glycol), 3.0% to 3.8% sucrose, 0.03% casein acid hydrolysate, 15 mM 2-(N-morpholinoethane)-sulphonic acid-KOH buffer (pH 5.9) and salts. In the pollen grain, the GC had a spherical or ovoid shape and contained a fine network of intermingled microtubules. As the GC entered into the pollen tube, it assumed a cylindrical shape with a length often exceeding 250 μm. A cage of microtubules then developed around the nucleus. The presence of dense and thick microtubular bundles in front of the generative nucleus within the GC coincided with the translocation of the nucleus to the leading end of the GC. No pre-prophase band was ever detected, but a distinct prophase spindle of microtubules was formed. In some GCs a tubulin-rich dot became visible at each pole of the spindle. After nuclear envelope breakdown, the bundles of microtubules spread between the chromosomes and became oriented into parallel arrays. The spindle became shorter at metaphase, and there was no tubulin labelling at the site of the metaphase plate. At anaphase, the microtubular apparatus lost its spindle-shape and a bridge of prominent bundles of microtubules connected the two daughter nuclei. At telophase, the site of the cell plate remained unstained by the anti-α-tubulin antibody, but a distinct phragmoplast of microtubules was formed more closely to the leading nucleus, resulting in the formation of unequal sperm cells (SCs). The leading SC was up to 2.5 times smaller than the following SC and it contained a smaller or equal number of nucleoli.     

Storage and release of ATP from astrocytes in culture

ATP is released from astrocytes and is involved in the propagation of calcium waves among them. Neuronal ATP secretion is quantal and calcium-dependent, but it has been suggested that ATP release from astrocytes may not be vesicular. Here we report that, besides the described basal ATP release facilitated by exposure to calcium-free medium, astrocytes release purine under conditions of elevated calcium. The evoked release was not affected by the gap-junction blockers anandamide and flufenamic acid, thus excluding purine efflux through connexin hemichannels. Sucrose-gradient analysis revealed that a fraction of ATP is stored in secretory granules, where it is accumulated down an electrochemical proton gradient sensitive to the v-ATPase inhibitor bafilomycin A(1). ATP release was partially sensitive to tetanus neurotoxin, whereas glutamate release from the same intoxicated astrocytes was almost completely impaired. Finally, the activation of metabotropic glutamate receptors, which strongly evokes glutamate release, was only slightly effective in promoting purine secretion. These data indicate that astrocytes concentrate ATP in granules and may release it via a regulated secretion pathway. They also suggest that ATP-storing vesicles may be distinct from glutamate-containing vesicles, thus opening up the possibility that their exocytosis is regulated differently.     

Hepatic stellate cells and astrocytes: Stars of scar formation and tissue repair

Scar formation inhibits tissue repair and regeneration in the liver and central nervous system. Activation of hepatic stellate cells (HSCs) after liver injury or of astrocytes after nervous system damage is considered to drive scar formation. HSCs are the fibrotic cells of the liver, as they undergo activation and acquire fibrogenic properties after liver injury. HSC activation has been compared to reactive gliosis of astrocytes, which acquire a reactive phenotype and contribute to scar formation after nervous system injury, much like HSCs after liver injury. It is intriguing that a wide range of neuroglia-related molecules are expressed by HSCs. We identified an unexpected role for the p75 neurotrophin receptor in regulating HSC activation and liver repair. Here we discuss the molecular mechanisms that regulate HSC activation and reactive gliosis and their contributions to scar formation and tissue repair. Juxtaposing key mechanistic and functional similarities in HSC and astrocyte activation might provide novel insight into liver regeneration and nervous system repair.Key words: p75 neurotrophin receptor, transforming growth factor-β, neurotrophins, epidermal growth factor, extracellular matrix, collagen, chondroitin sulfate proteoglycans, matrix metalloproteinases, scar, neurons, hepatocytes     

Microtubular organization and its involvement in the biogenetic pathways of plasma membrane proteins in Caco-2 intestinal epithelial cells

We characterized the three-dimensional organization of microtubules in the human intestinal epithelial cell line Caco-2 by laser scanning confocal microscopy. Microtubules formed a dense network approximately 4-microns thick parallel to the cell surface in the apical pole and a loose network 1-micron thick in the basal pole. Between the apical and the basal bundles, microtubules run parallel to the major cell axis, concentrated in the vicinity of the lateral membrane. Colchicine treatment for 4 h depolymerized 99.4% of microtubular tubulin. Metabolic pulse chase, in combination with domain-selective biotinylation, immune and streptavidin precipitation was used to study the role of microtubules in the sorting and targeting of four apical and one basolateral markers. Apical proteins have been recently shown to use both direct and transcytotic (via the basolateral membrane) routes to the apical surface of Caco-2 cells. Colchicine treatment slowed down the transport to the cell surface of apical and basolateral proteins, but the effect on the apical proteins was much more drastic and affected both direct and indirect pathways. The final effect of microtubular disruption on the distribution of apical proteins depended on the degree of steady-state polarization of the individual markers in control cells. Aminopeptidase N (APN) and sucrase-isomaltase (SI), which normally reach a highly polarized distribution (110 and 75 times higher on the apical than on the basolateral side) were still relatively polarized (9 times) after colchicine treatment. The decrease in the polarity of APN and SI was mostly due to an increase in the residual basolateral expression (10% of control total surface expression) since 80% of the newly synthesized APN was still transported, although at a slower rate, to the apical surface in the absence of microtubules. Alkaline phosphatase and dipeptidylpeptidase IV, which normally reach only low levels of apical polarity (four times and six times after 20 h chase, nine times and eight times at steady state) did not polarize at all in the presence of colchicine due to slower delivery to the apical surface and increased residence time in the basolateral surface. Colchicine-treated cells displayed an ectopic localization of microvilli or other apical markers in the basolateral surface and large intracellular vacuoles. Polarized secretion into apical and basolateral media was also affected by microtubular disruption. Thus, an intact microtubular network facilitates apical protein transport to the cell surface of Caco-2 cells via direct and indirect routes; this role appears to be crucial for the final polarity of some apical plasma membrane proteins but only an enhancement factor for others.     

Isolation and culture of hepatic stellate cells from mouse liver

Hepatic stellate cells （HSCs） are the primary extracellular matrix-producing cells within the river and have numerous vital functions. A robust protocol for the isolation and culture of HSCs is important for further investigations of cell functions and related mechanisms in river disease. The volume of the mouse river is much smaller than that of the rat river, which makes it much more difficult to isolate mouse HSCs （mHSCs） than rat HSCs. At present, isolating mHSCs is still a challenge because there is no efficient, robust method to isolate and culture these cells. In the present study, C57BL/6J mice were intravenously injected with riposomeencapsulated dichloromethylene diphosphate （CL2MDP） to selectively eliminate Kupffer cells from the river. The mouse livers were then perfused in situ, and the mHSCs were isolated with an optimized density gradient centrifugation technique. In the phosphate buffer solution （PBS）-liposome group, the yield of mHSCs was （1.37 ±0.23） × 10^6/g river, the cell purity was （90.18 ± 1.61）%, and the cell survival rate was （94.51 ±1.61）%. While in the CL2MDP-liposome group, the yield of mHSCs was （1.62 ±0.34）× 10^6/g liver, the cell purity was （94.44 ± 1.89）%, and the cell survival rate was （94.41 ±1.50）%. Based on the yield and purity of mHSCs, the CL2MDP-riposome treatment was superior to the PBS-liposome treatment （P 〈 0.05, P 〈 0.01）. This study established successfully a robust and efficient protocol for the separation and purification of mHSCs, and both a high purity and an adequate yield of mHSCs were obtained.     

Microtubular ATPase in Carcinus and Helix nerve

• 1.1. Fixed Carcinus leg nerves and Helix suboesophageal ganglia were incubated in a Wachstein-Meisel incubation medium to localize sites of ATPase activity.
• 2.2. Lead phosphate precipitation indicating the location of an ATPase, was specifically deposited on mitochondrial inner and outer membranes, axonal membranes and on microtubules of both Carcinus and Helix nerves.
• 3.3. The glutaraldehyde concentration affected the ATPase activity. 3% glutaraldehyde allowed location of the mitochondrial and axonal ATPase, whilst 1% glutaraldehyde allowed location of microtubular ATPase as well as the mitochondrial and axonal ATPase.

     

Binding constants of soluble NGF-receptors in rat oligodendrocytes and astrocytes in culture

The neuronotrophic factor NGF binds to peripheral neurons of the dorsal root ganglion and the sympathetic nervous system. NGF binds to a cell surface receptor, NGFR, on these cells and displays Kd's of 10(-9) and 10(-11)M. NGF receptors have also been reported for basal forebrain magnocellular neurons. In addition, NGF specifically binds to NGFR on Schwann cells although the biological significance of this binding is not known. Here we report that NGF binds in a saturable and specific fashion to receptors on cultured isolated populations of rat astrocytes but not to oligodendrocytes. The binding to astrocytes in culture displayed a Kd of 2.7 +/- 1.0 nM with 36,000 receptors per cell.     

Laminin is produced by early rat astrocytes in primary culture

The production of laminin by early rat astrocytes in primary culture was investigated by double immunofluorescence staining for laminin and the glial fibrillary acidic protein (GFAP), a defined astrocyte marker. In early cultures (3 d in vitro; 3 DIV) cytoplasmic laminin was detected in all the GFAP-positive cells which formed the major population (80%) of the nonneuronal cells present in cultures from 20- 21-d embryonic, newborn, or 5-d-old rat brains. Monensin treatment (10 microM, 4 h) resulted in accumulation of laminin in the Golgi region, located using labeled wheat germ agglutinin. Laminin started gradually to disappear from the cells with the time in culture, was absent in star-shaped, apparently mature astrocytes, but remained as pericellular matrix deposits. The disappearance of cellular laminin was dependent on the age of the animal and the time in culture so that it started earlier in cultures from 5-d-old rat brains (5 DIV) and approximately following the in vivo age difference in cultures from newborn (12 DIV) and embryonic (14 DIV) rat brains. Our results indicate that laminin is a protein of early astrocytes and also deposited by them in primary culture, thus suggesting a role for this glycoprotein in the development of the central nervous system.     

Muramyl dipeptide-elicited production of PGD2 from astrocytes in culture

We used primary cultures of rat brain astroglial cells in order to investigate the interrelationship between PGD2 and other sleep-promoting substances such as muramyl dipeptide (MDP), lipopolysaccharide (LPS), delta-sleep-inducing peptide (DSIP), uridine, and interleukin 1 (IL-1). A large amount of PGD2 was released into the culture medium by stimulation with MDP, LPS, and IL-1 but DSIP and uridine failed to stimulate such release. These results suggest that PGD2 may be part of the series of biochemical steps involved in induction of sleep by MDP, LPS, and IL-1.