Cell-by-cell flow cytometric analysis of chromosomes

The authors discuss various aspects of a recently developed method permitting a detailed flow cytometric analysis of the individual cell karyotypes such as instrumentation, histochemistry, data proceeding algorithms. Possible drawbacks of the method and the ways of their overcoming are considered. Results of analysis of the Chinese hamster cells are presented that illustrate the possibilities of the method, including the metaphase chromosome distribution according to their fluorescence intensity, the analysed cell distribution according to their chromosomes number, the table in which the individual cell karyotypes are distributed according to their fluorescence. The results obtained show that the developed method may be successfully used for investigating chromosomal iNstability and heterogeneity of the mammalian cells.     

Common antigens of mouse oval and biliary epithelial cells. Expression on newly formed hepatocytes

Two antigens - A6 and G7 - shared by mouse biliary epithelial and oval cells were revealed by monoclonal antibodies raised in rat immunized with oval-cell-enriched liver fraction. Oval cells were induced in CBA or F1 (CBA x C57BL6) mice by a combination of a single injection of the alkylating drug Dipin with partial hepatectomy. In normal liver A6 antigen was localized, using light and electron microscopy, in biliary epithelial cells of all ducts including Hering canals. Some bile ductal and Hering cells were A6-negative. Occasionally, A6 antigen was present in single hepatocytes forming the periportal ends of hepatic cords. In preneoplastic and tumorous liver A6 antigen was present in bile ductal and oval cells and in a fraction of newly formed hepatocytes and tumor cells. G7 antigen was revealed in normal, precancerous and tumorous liver in biliary epithelial and oval cells but not in hepatocytes. A6 and G7 antigens were not liver-specific: they were expressed in various normal organs and tissues, especially in epithelia. In studies of mouse liver lineages A6 antigen can be used as a common marker of biliary epithelial and oval cells and hepatocytes at certain stages of differentiation. G7 antigen is a marker of oval and biliary epithelial cells. There was a striking similarity in A6 antigen localization to that of human blood group antigens in normal liver and liver tumors. A6 antigen may thus provide a useful tool for the study of neoexpression of human blood group antigens in liver tumors.     

Recovery of acetylcholinesterase activity after irreversible inhibition by organophosphorous compounds in embryonic development

1. Recovery of acetylcholinesterase (AChE) activity was studied using the embryos of sea urchins Strongylocentrotus intermedius and S. nudus, embryos of axolotl Ambystoma mexicanum and in the chick embryo muscle culture treated by "irreversible" organophosphorous inhibitors (OPI). 2. AChE activity was assayed by a modified Ellman's procedure. 3. It follows from the data obtained that, unlike the plutei of sea urchins and the monolayer culture of chick embryo muscle cells, the embryos of axolotl show a compensatory increase in AChE biosynthesis after inhibition by OPI. 4. This mechanism is assumed to be related to the presence of a well developed neuromuscular system in the A. mexicanum embryos. 5. It is possible that acetylcholine accumulated as a result of partial AChE inhibition is responsible for the compensatory increase in AChE biosynthesis.     

Cloning and inactivation of a chromosomal copy of the imidazole glycerophosphate dehydratase (HIS) gene from Hansenula polymorpha]

A gene for imidazole glycerophosphate dehydratase (HIS) has been selected from the library of Hansenula polymorpha genes by complementation of Escherichia coli hisB mutations or his3 mutation of Saccharomyces cerevisiae. Inactivation of the gene in Hansenula polymorpha strain 48V, as well as Leu(-)-Leu+ conversion of phenotype and arising of antibiotic G418 resistance resulted from transformation of the strain by the linear DNA molecules of the cloned HIS-gene with the in vitro inserted LEU2 gene from Saccharomices cerevisiae and KmR gene. The Southern hybridization analysis of the Leuf+His- G418R clones representing 1-2% of transformants population together with the DNA analysis of the monospore clones from the hybrid Leu+His-G418R transformant tetrad and tester strain have revealed the locus specific nature of the clones.     

Effect of acute and chronic deprivation of catecholaminergic system activity induced by 6-hydroxydopamine on the behavior of the rat

On 202 male rats of Wistar line, a study was carried out of the effect of chronic and acute deprivations of the brain CA-systems activity resulting from administration of 6-OHDA on investigating behaviour and learning. Chronic deprivation of CA-systems activity by neonatal administration of 6-OHDA (100 mg/kg subcutaneously) and their acute deprivation by intracerebral administration of 6-OHDA to adult rats (150 mkg in each lateral ventriculus) was accompanied by similar deep changes of behaviour. Both forms of deprivation reduced the investigating activity of the animals in the open field. In both cases, the above 6-OHDA dozes sharply impeded the learning of animals with emotionally negative reinforcement, with no significant influence on learning with emotionally positive reinforcement. Both forms of deprivation of CA-systems activity weakened the reaction of frustration elicited by a sharp reduction of food reinforcement.     

Effect of the segregation of neutral red, acridine orange and ammonium chloride by L cells (subline LSM) on lysosomal hydrolase activity

Kinetics of Neutral red (NR) and Acridine orange (AO) uptake by cultured L cells (subline LSM) has been studied. It was found that the uptake of both NR and AO, with their constant concentrations in the medium was characterized as a two-phase process. During 2 hours, these cells concentrated as much as 90% of the total amount of NR and AO taken up during the whole incubation period. The segregation and accumulation of NR, AO as well as NH4Cl took place in lysosomes. NR and AO concentrations within the cells exceed by 600 and 400 times, respectively, those in the medium. NR, AO and NH4+ accumulation in cells resulted in inhibition of the activity of the following lysosomal hydrolases: cathepsins B and D, acid lipase, N-acetyl-beta,D-glucosaminidase, beta-galactosidase, acid phosphatase and galactosyltransferase, the latter being a marker of Golgi apparatus. The effect of lysosomal enzyme activity inhibition on the cell economy, and a possible role of lysosomotropic agents as regulators of the lysosomal apparatus functional activity are discussed.     

Effect of neonatal 6-hydroxydopamine administration on the catecholaminergic structures of the hypothalamus and on the behavior of rats of various ages

A neurotoxin 6-hydroxydopamine (6-OHDA) was introduced to 1, 2 and 3 day old male Wistar rats. Falck-Hillarp histochemical fluorescent method was used to analyze hypothalamic and hypophysial structures containing catecholamines. Statistically reliable decrease in catecholamine fluorescence intensity in hypothalamus and hypophysis, accumulation of catecholamines in nerve cells of supraoptic nucleus and in tanycytes of median eminence differed in 25- and 90-days rats. Disturbance of catecholaminergic systems of hypothalamus and hypophysis induced by neonatal introduction of 6-OHDA leads to reliable relaxation of orienting reaction and deep violation of learning that becomes stronger with age.     

Embryonic stem cells derived from morulae,inner cell mass,and blastocysts of mink: Comparisons of their pluripotencies

A characterization of cell lines that we derived from morulae (three lines), blastocysts (two lines), and the inner cell mass (ICM) is given. The karyotype of all the lines was normal; the genotype of four lines was XX, and four lines were genotypically XY. The pluripotencies and commitment status of the derived lines were estimated. First, there were not less than two-thirds of cells in the populations of the lines derived from morulae and the lCM with both Xs active; 70–100% of cells of the blastocyst-derived lines had one of the Xs in an inactive state. The activity of glucose-6-phosphate dehydrogenase (G6PD) in the lines (genotype XX) derived from morulae and ICM was found to be twofold higher than in lines with genotype XY, and G6PD activity was the same in the blastocyst-derived XX lines and XY lines. Second, when injected intraperitoneally into athymic mice, morulae- and ICM-derived cells gave rise to simple and complex embryoid bodies (EB) resembling to typical “cystic” mouse EBs. Third, when injected subcutaneously to athymic mice, the ICM- or morula-derived cells gave rise to typical teratomas containing derivatives of the three germ layers and components of organogenesis. Comparisons of cell lines of different derivations demonstrated that the pluripotencies of the ES cells derived from morulae or the ICM are higher than those of blastocyst derivation. © 1993 Wiley-Liss, Inc.