Isolation and subfractionation on ficoll gradients of adult rat hepatocytes. Size, morphology, and biochemical characteristics of cell fractions.

The recirculating perfusion of adult rat liver with a Ca++ free Hanks' solution produces a release of the adhesiveness of cells and a cleaving of the desmosomes. The addition of collagenase and hyaluronidase to the perfusion medium leads to complete dissociation of the liver tissue into a mixture of isolated cells and cell cords in which the hepatocytes remain connected with specific junctional differentiations, namely the gap and tight junctions. Individual cells are released by submitting the suspension of cell trabeculae to a gentle rolling. The gap junctions are ruptured at least in 1 of the 2 adjacent cells and remain generally attached to the other cell taking with them a small portion of cytoplasm. This technique of isolation of hepatocytes yields about 60 to 65% of the parenchymal cells contained in a liver; endothelial cells and other cells of the connective tissue are recovered. The ultrastructural preservation of the isolated hepatocytes is excellent and the glucose 6 phosphatase activity, confined to the endoplasmic reticulum, appears unaltered in most cells. Protein, DNA and RNA recovery in the preparations of isolated hepatocytes is satisfactory, amounting to 70% of that found in liver homogenate; glycogen, the most labile component examined, is partly lost or degraded during the manipulations. Cell diameters measured by different methods confirm the preservation of the original volume of the in situ hepatocytes and the presence of more than 1 type of parenchymal cell. By submitting this heterogenous cell population to an isopycnic density gradient centrifugation, 2 types of hepatocyes can be distinguished: the light hepatocytes, with a mean diameter of 20.5 μm and a mean density of 1.10, are characterized by an extended smooth walled endoplasmic reticulum entrapping dispersed α glycogen particles; the heavy hepatocytes, with a mean diameter of 19.0 μm and a mean density of 1.14, present a relatively reduced compartment of smooth endoplasmic reticulum, but large accumulations of glycogen. It is suggested that the cell fraction of low density is enriched in centrolobular cells and the high density fraction in perilobular hepatocytes.SCOPUS: ar.jinfo:eu-repo/semantics/publishe

Adult rat hepatocytes in primary monolayer culture. Ultrastructural characteristics of intercellular contacts and cell membrane differentiations.

Primary monolayer cultures were obtained in 60 mm petri dishes by incubating 3 x 106 isolated hepatocytes at 37°C in Dulbecco's medium supplemented with 17% fetal calf serum. The ultrastructure of monolayer cells was examined after various incubation periods. Within 4 h of plating, the isolated spherical cells adhere to the plastic surface, establish their first contacts by numerous intertwined microvilli, and form a new hemidesmosomes. After 12 h of culture, wide branched trabeculae of flattened polyhedral cells extend in all directions. Finally, after 24 h of culture, bile canaliculi are reconstituted, and a biliary polarity is recovered: the Golgi elements, which are scattered throughout the cytoplasm in the isolated cells, are reassembled in front of the newly formed bile canaliculi, symmetrically in the adjacent cells; lysosomes are concentrated in that region, and microtubules reappear. Concomitantly, plasma membrane differentiations, namely desmosomes and tight junctions, develop. Tight junctions sealing the bile ducts constitute a barrier to the passage of ruthenium red and horseradish peroxidase. De novo formation of these junctions was studied by the freeze etching technique: 10 nm particles compose a network of anastomosed linear arrays in the vicinity of the bile canalculi; in the next step of differentiation, the particles fuse, form short ridge segments and finally continuous branched smooth strands, characteristic of the mature tight junction.SCOPUS: ar.jinfo:eu-repo/semantics/publishe

Structure and biochemical composition of desmosomes and tonofilaments isolated from calf muzzle epidermis.

Complexes of plasma membrane segments with desmosomes and attached tonofilaments were separated from the stratum spinosum cells of calf muzzle by means of moderately alkaline buffers of low ionic strength and mechanical homogenization. These structures were further fractionated by the use of various treatments including sonication, sucrose gradient centrifugaton, and extraction with buffers containing high concentrations of salt, urea, citric acid, or detergents. Subfractions enriched in desmosome-tonofilament-complexes and tonofilament fragments were studied in detail. The desmosome structures such as the midline, the trilaminar membrane profile, and the desmosomal plaque appeared well preserved and were notably resistant to the various treatments employed. Fractions containing desmosome-tonofilament complexes were invariably dominated by the nonmembranous proteins of the tonofilaments which appeared as five major polypeptide bands (apparent molecular weights: 48,000; 51,000; 58,000; 60,000; 68,000) present in molar ratios of approx. 2:1:1:2:2. Four of these polypeptide bands showed electrophoretic mobilities similar to those of prekeratin polypeptides from bovine hoof. However, the largest polypeptide (68,000 mol wt) migrated significantly less in polyacrylamide gels than the largest component of the hoof prekeratin (~ 63,000 mol wt). In addition, a series of minor bands, including carbohydrate-containing proteins, were identified and concluded to represent constituents of the desmosomal membrane. The analysis of protein-bound carbohydrates (total 270 μg/mg phospholipid in desmosomeenriched desmosome-enriched showed the presence of relatively high amounts of glucosamine, mannose, galactose, and sialic acids. These data as well as the lipid composition (e.g. high ratio of cholesterol to phospholipids, relatively high contents of sphingomyelin and gangliosides, and fatty acid pattern) indicate that the desmosomal membrane is complex in protein and lipid composition and has a typical plasma membrane character. The similarity of the desmosome-associated tonofilaments to prekeratin filaments and other forms of intermediate-sized filaments is discussed.SCOPUS: ar.jinfo:eu-repo/semantics/publishe

Role of mitochondrial raft-like microdomains in the regulation of cell apoptosis

Lipid rafts are envisaged as lateral assemblies of specific lipids and proteins that dissociate and associate rapidly and form functional clusters in cell membranes. These structural platforms are not confined to the plasma membrane; indeed lipid microdomains are similarly formed at subcellular organelles, which include endoplasmic reticulum, Golgi and mitochondria, named raft-like microdomains. In addition, some components of raft-like microdomains are present within ER-mitochondria associated membranes. This review is focused on the role of mitochondrial raft-like microdomains in the regulation of cell apoptosis, since these microdomains may represent preferential sites where key reactions take place, regulating mitochondria hyperpolarization, fission-associated changes, megapore formation and release of apoptogenic factors. These structural platforms appear to modulate cytoplasmic pathways switching cell fate towards cell survival or death. Main insights on this issue derive from some pathological conditions in which alterations of microdomains structure or function can lead to severe alterations of cell activity and life span. In the light of the role played by raft-like microdomains to integrate apoptotic signals and in regulating mitochondrial dynamics, it is conceivable that these membrane structures may play a role in the mitochondrial alterations observed in some of the most common human neurodegenerative diseases, such as Amyotrophic lateral sclerosis, Huntington's chorea and prion-related diseases. These findings introduce an additional task for identifying new molecular target(s) of pharmacological agents in these pathologies

The effects of acute and elective cardiac surgery on the anxiety traits of patients with Marfan syndrome

BACKGROUND: Marfan syndrome is a genetic disease, presenting with dysfunction of connective tissues leading to lesions in the cardiovascular and skeletal muscle system. Within these symptoms, the most typical is weakness of the connective tissue in the aorta, manifesting as aortic dilatation (aneurysm). This could, in turn, become annuloaortic ectasia, or life-threatening dissection. As a result, life-saving and preventative cardiac surgical interventions are frequent among Marfan syndrome patients. Aortic aneurysm could turn into annuloaortic ectasia or life-threatening dissection, thus life-saving and preventive cardiac surgical interventions are frequent among patients with Marfan syndrome. We hypothesized that patients with Marfan syndrome have different level of anxiety, depression and satisfaction with life compared to that of the non-clinical patient population. METHODS: Patients diagnosed with Marfan syndrome were divided into 3 groups: those scheduled for prophylactic surgery, those needing acute surgery, and those without need for surgery (n = 9, 19, 17, respectively). To examine the psychological features of the patients, Spielberger's anxiety (STAI) test, Beck's Depression questionnaire (BDI), the Berne Questionnaire of Subjective Well-being, and the Satisfaction with Life scale were applied. RESULTS: A significant difference was found in trait anxiety between healthy individuals and patients with Marfan syndrome after acute life-saving surgery (p 0.1). Finally, a significant, medium size effect was found between patient groups on the Joy in Living scale (F (2.39) = 3.51, p = 0.040, eta2 = 0.15). CONCLUSIONS: Involving psychiatric and mental-health care, in addition to existing surgical treatment interventions, is essential for more successful recovery of patients with Marfan syndrome

Control of serum protein production in hepatocyte hybridomas: immortalization and expression of normal hepatocyte genes.

"Hepatocyte hybridomas" have been isolated after fusion of adult hepatocytes and alpha-fetoprotein (AFP)-producing mouse hepatoma cells. The yield of viable hybrid clones was low but could be increased by culturing the cells in the presence of insulin. On the basis of their chromosome constitution, the hybrids were classified into two groups characterized by either a single or a double set of mouse (hepatoma) chromosomes. The hybrids segregated rat chromosomes and thus constitute an excellent material for gene mapping studies in the rat. Most of the hepatocyte hybridomas retained the production of one or more rat serum proteins, indicating that the corresponding structural genes, contributed by the normal hepatocyte parent, have been immortalized and maintained in the active state after fusion. However, these hybrids do not produce rat AFP, although mouse AFP synthesis is maintained. This result strongly suggests that silent rat (hepatocyte) AFP genes coexist in hepatocyte hybridoma nuclei with active mouse (hepatoma) AFP genes. Finally, on the basis of certain properties of these hepatocyte-hepatoma hybrids, we suggest that the nondividing state of the hepatocytes is actively controlled by a regulatory mechanism which prevents DNA synthesis or entry into mitosis or both

Transmembrane potential of rat hepatocytes in primary monolayer culture

Transmembrane potentials of rat hepatocytes in primary monolayer culture on collagen gels were measured with glass microelectrodes. Potentials for cells in culture for 23-30 h comprised two populations. The mean ± SD for a population of stable low potentials was -9.7 ± 2.0 mV (n = 93). This was compared with -23.6 ± 9.4 mV (n = 42), the mean value for stable potentials that followed spontaneous increases in the low potentials, 0.5-2.0 min after the impalement. The estimated input resistance increased during these spontaneous hyperpolarizations. In some cells, after 48 h in culture, the transmembrane potential oscillated rhythmically, with an amplitude of 25 mV and a period of 7 min. Suffusing the cells with 120 mM potassium chloride decreased the potential and eliminated the oscillations. The stable high potentials were considered more accurate estimates of the hepatocyte transmembrane potential, based on comparison with values for intact liver. Low potentials may have resulted from current leaking through an electrode shunt resistance, followed by an increase in potential as the membrane \u27sealed\u27 the shunt pathway. However, these events may also reflect cells capable of two stable transmembrane potentials