Fluctuations in levels of various gangliosides in the different tracts of the oviduct of the frog (Rana esculenta) during the reproductive cycle.

The ganglioside content and pattern have been followed in the different tracts (rectus, convoluted and uterine) of the frog oviduct during the reproductive cycle. The main variations we observed are: a) average higher levels of ganglioside sialic acid in the preovulatory phase, with two peaks in March and April for the convoluted and rectus tract, respectively, and a more homogenous behaviour for the uterine tract; in all three tracts of the oviduct a minimum coincident with the ovulation has been found; b) a balanced presence of sulfolipids and gangliosides in the uterine tract: in fact sulfolipids, whose variations have been determined in a previous work, are higher when gangliosides are lower and vice versa, maintaining nearly constant the total negative charge due to these glycolipids; c) an alternate fluctuation of monosialo- and disialo-gangliosides in the preovulatory phase and a net trend toward the increase of monosialo- and the decrease of disialogangliosides in the postovulatory phase; trisialo-gangliosides are in general less represented and show less marked variations; d) the presence of particular gangliosides in particular moments of the reproductive cycle: Fuc-GM1, a fucosylated ganglioside, is higher than the more represented GM1 during the ovulation, while GD1 alpha, a ganglioside with a sialic acid residue linked to GalNAc, is steadily present in all three tracts after ovulation.(ABSTRACT TRUNCATED AT 250 WORDS)

A rapid procedure for elimination of non-polar lipids hampering the usual polar lipid extraction and TLC separation.

A new and rapid method is proposed for extraction of non-polar lipids from tissues where they are present as abundant components which can interfere with the usual procedures of lipid extraction and TLC separation, and hamper, in particular, sulphatide visualization. A solvent more hydrophobic than chloroform, i.e. n-hexane, was utilized to remove the neutral lipids from samples of female rabbit parotid gland, and the n-hexane phase was used for TLC which showed considerable amounts of cholesterol esters, in addition to triglycerides, diglycerides and monoglycerides. The methanol phase, now devoid of non-polar lipids, was utilized to prepare TLC plates in order to separate and visualize the polar lipid fractions, in particular the sulphatides.

Effects of low-power 632 nm radiation (HeNe laser) on a human cell line: influence on adenylnucleotides and cytoskeletal structures.

HeNe (632 nm) irradiation (5, 15 and 30 min) of an embryonal human cell line (EUE) was used to study the short-term effects on energy charge and the rapid, energy-dependent, remodelling processes of cytoskeletal and adhesion structures. The adenosine triphosphate (ATP) concentration, tested by luminometric and high performance liquid chromatography (HPLC) procedures, is constant after 15 and 30 min of HeNe treatment; the lower phosphorylated nucleotides, i.e. adenosine diphosphate (ADP) and adenosine monophosphate (AMP), change after 30 min in opposite directions: the ADP concentration decreases by 39% whilst that of AMP increases about sixfold. The adenylate energy charge (AEC) decreases by 21.7% in treated EUE cells (AEC = 0.65) in comparison with untreated EUE cells (AEC = 0.83). In HeNe-treated cells, the remodelling of cytoskeletal and adhesion molecules becomes evident after 15 min of treatment. The following events are important: (1) modification of stress fibre assembly and increase in vinculin-containing adhesion plaques; (2) assembly and bundling of intermediate filaments; (3) increase in laminin and L-cell adhesion molecules (L-CAM) expression. The lowered energy charge in irradiated cells is related to the increase in AMP production at the expense of ADP. ATP is dynamically constant despite its requirement in short-time remodelling processes of the cytoskeletal network which are enhanced in irradiated cells.

Effects of glycosaminoglycans on U-937 leukemia cell proliferation and differentiation: structure-function relationship.

Glycosaminoglycans (heparins, dermatan sulfate, chondroitin sulfate) with different structures and physicochemical properties were evaluated for their capacity to influence proliferation and differentiation of U-937 cell line. The contrasting and specific effects of glycosaminoglycans (depending on their structures and properties) on a leukemia cell line could help explain the regulation of proliferative and/or differentiative processes of hematopoietic cells in order to clarify the control of development and differentiation of hematopoietic progenitor cells by bone marrow extracellular matrix. Heparin from beef intestinal mucosa, heparan sulfate from beef spleen, dermatan sulfate from beef intestinal mucosa, and chondroitin sulfate from bovine trachea were extracted and purified, and their purity, structures, and physicochemical properties were evaluated. Fast-moving heparin was obtained by its selective precipitation as barium salt, and partially desulfated and re-N-sulfated heparin was produced by chemical modifications. Different glycosaminoglycans were tested to evaluate their effects on proliferation and differentiation processes of a monoblastic leukemia cell line (U-937). Heparin and derivatives (from 0.1 to 100 micrograms/ml) inhibit cell proliferation; heparan sulfate does not produce modifications, while chondroitin sulfate and dermatan sulfate (from 0.01 to 100 micrograms/ml) significantly stimulate cell growth. Cell differentiation was evaluated by cytoenzymatic determination of alpha-naphthyl butyrate esterase and by fluorescein-labeled anti-HLA-DR, anti-CD11b, and anti-CD14 antibodies. Nitro blue tetrazolium reduction and phagocytosis were also evaluated. Heparin and derivatives significantly increase U-937 differentiation. Heparin sulfate has no effect, while chondroitin sulfate and, to a lesser extent, dermatan sulfate, induce a strong decrease of differentiative markers. The regulation of U-937 cell properties appears to be related to charge density and to the amount of N-sulfate and N-acetyl groups. In particular, glycosaminoglycans with lower sulfate-to-carboxyl ratios and N-sulfate group percentages (chondroitin sulfate and dermatan sulfate) stimulate proliferation and produce a decrease of differentiative markers; on the contrary, polysaccharides with high charge density and N-sulfate group amounts (heparin and derivatives) inhibit U-937 proliferation and induce terminal differentiation. A previous paper (N. Volpi, L. Bolognani, A. Conte, and M. Petrini, (1993) Leukemia Res. 17, 789-798) demonstrates dissimilar effects on U-937 cells by chondroitin sulfates with different structures and physicochemical properties. In this study we confirm the importance of glycosaminoglycan structures and physicochemical properties in regulating cell functions. Possible mechanisms of action are discussed.

ATPase activity of melanosomes in liver pigment cells of Rana esculenta L.

Luminometric methods show that melanosomes in liver pigment cells of Rana esculenta L. have endogenous ATP and ATPase activity. The Km value of ATPase is 0.42 x 10(-8) mol/l at pH 7.0. Inhibition of ATPase by antimycin and by ouabain is not effective. In the presence of an excess of ADP and Pi, ATP synthesis was observed.

Effects of chondroitin sulfates with different structures on leukemia cells: U-937 cell proliferation and differentiation.

Chondroitin sulfates extracted and purified by different manufacturers were tested to evaluate their effects on proliferation and differentiation processes of U-937 cells. The different chondroitin sulfates were evaluated for purity, structure and physicochemical properties. The three chondroitin sulfates utilized did not present other contaminant glycosaminoglycans and proteins and had about the same relative molecular mass but different disaccharide patterns and charge density. Chondroitin sulfates with small amounts of disulfated disaccharides and low charge density, at 5 micrograms/ml concentration, doubled (about + 133%) cell proliferation in comparison to controls. In contrast, chondroitin sulfates with large amounts of disulfated disaccharides and high sulfate to carboxyl ratio were less effective (about + 15%) in stimulating cell proliferation at low concentration. A decrease of U-937 cell proliferation was observed in proportion to the increased amounts of chondroitin sulfate with low sulfate to carboxyl ratio. On the contrary, chondroitin sulfate with large amounts of disulfated disaccharides produced increased cell proliferation depending on concentration. Small amounts (5-10 micrograms/ml) of chondroitin sulfates with low charge density reduced the differentiative process of U-937 cells. Chondroitin sulfate with large amounts of disulfated disaccharides and high charge density seemed to be able to produce a significant decrease of differentiative processes only at very high concentrations (1000 micrograms/ml). These contrasting effects of chondroitin sulfates with different disaccharide patterns (and structure) and charge density on a leukemia cell line could help to explain the regulation of proliferative and/or differentiative processes of hemopoietic cells. This is underlined by the changes of types, physicochemical properties and structure of glycosaminoglycans induced by different extracellular factors and agents.

Glycosaminoglycans and proteins: different behaviours in high-performance size-exclusion chromatography.

The influence of the conformation of globular proteins and glycosaminoglycans in high-performance size-exclusion chromatography (HPSEC) was studied. Glycosaminoglycans (heparin, chondroitin sulphate and dermatan sulphate) with different primary structures, sulphate-to-carboxyl ratios and physico-chemical properties were extracted and purified. Their physico-chemical properties and purity were evaluated by several analytical techniques. Glycosaminoglycans with different relative molecular masses (M(r)) were prepared by a chemical depolymerization process. These heteropolysaccharides were evaluated by HPSEC and compared with globular proteins of known relative molecular mass. The two third-degree polynomial regression curves for proteins and glycosaminoglycans have different coefficients and the columns present different exclusion limits. In particular, under the experimental conditions, the M(r) exclusion limits for high M(r) are 44,000 for glycosaminoglycans and 240,000 for globular proteins. In contrast, the behaviours of these two classes of macromolecules are similar for lower M(r). In fact, the two third-degree polynomial curves show the same regression below about M(r) = 1000. The behaviour in HPSEC is discussed in relation to the different steric conformations for proteins and glycosaminoglycans with different relative molecular masses.

Effects of monocarboxylic and dicarboxylic acids on myosin ATPase activity tested by luminometric procedure.

L(+)Lactic acid enhances myosin ATPase in vitro. Different organic acids were tested for activation of myosin ATPase activity. L(+)Lactic is more effective in stimulating ATPase than D(-)Lactic. D(+) and L(-)Malic acids were also effective at the concentration of 2.5 x 10(-2)-5.0 x 10(-2) mmoles/l. At 3.0 x 10(-2) mmoles/l concentration the following acids are activators: acetic, oxalic, malonic, oxaloacetic, pyruvic, glyoxylic, glycolic; succinic is an inhibitor and acetoacetic is without effect. The activation is not in relation with the pKa of these acids. The inhibitory effects of organic acids are evident at the concentration of 5.0 x 10(-2) mmoles/l. This inhibitory effect is linearly increasing with their pKa. The results are discussed in connection with the possible role of these metabolites in controlling not only ATPase activity towards splitting of ATP, but also in controlling the removal of its hydrolytic products.

Competitive inhibition of myosin ATPase activity by different molecular weight heparins.

Myosin ATPase activity was measured, by continuous luminometric method, in presence of different molecular weight heparins. ATPase activity decreases in the presence of heparin, when simultaneous incubation with ATP is carried out; the percentage of inhibition is proportional to polysaccharide concentration. Heparins of different molecular weights (1.75 KD to 11.6 KD) are competitive inhibitors of enzymatic activity; the inhibitory effects is also appreciable with trisulphated disaccharide. The possible mechanisms of interaction between heparin and myosin ATPase are discussed.

Changes induced by progesterone treatment in the sulphatides of the frog oviduct.

Sulphatides have been studied by histochemical and biochemical procedures in the oviduct of the frog in different experimental conditions. In ovariectomized or hypophysectomized animals, compared to sham-operated, an increase in sulphatides was observed. The progesterone treatment did not significantly modify this lipid fraction in ovariectomized frogs, while in hypophysectomized frogs it induced a further increase. Densitographic profiles of the sulphatides, obtained by thin-layer chromatography (TLC) and also recorded by Tesak equipment, were similar in ovariectomized or hypophysectomized frogs following hormone treatment because they showed three distinct fractions in both experimental groups of animals. The appearance of a third fraction never previously observed was probably induced by the progesterone treatment. Moreover, under the effects of this hormone, the phospholipid fractions (phosphatidylcholine and phosphatidylethanolamine) also showed different densitographic profiles.

Sialic acid and neuraminidase activity in the frog oviduct: comparative biochemical investigation in the different tracts during the reproductive cycle.

1. A biochemical study was carried out on the protein-bound and lipid-bound sialic acid, and neuraminidase activity in the different tracts of the oviduct of the frog Rana esculenta during the reproductive cycle. 2. Plasma sexual steroids were also investigated by RIA. 3. Fluctuations in neuraminidase activity are related to that of glycoprotein sialic acid and plasma estradiol. Glycolipid sialic acid does not have a close relationship either with neuraminidase or plasma estradiol. 4. Very high plasma concentration of progesterone before ovulation and, on the contrary, its drop after ovulation were observed. 5. The results are discussed and hypotheses advanced to explain fluctuations of the studied parameters during the reproductive cycle.

Arylsulphatase activity and cerebroside sulphates in the frog oviduct during the reproductive cycle.

The presence of arylsulphatase A and cerebroside sulphates in different tracts of Rana esculenta oviduct during different phases of the reproductive cycle were investigated by histochemical and biochemical procedures. The results indicate that enzyme activity shows seasonal fluctuations connected with the phase of the sexual cycle. The concentrations of cerebroside sulphates (the natural substrates of arylsulphatase A) is related to the activity of this hydrolytic enzyme. The role of arylsulphatase A activity in regulating the substrate concentration, and particularly that of sulphatides, is discussed.

Histochemical research on metabolic pathways of glucose in some species of Mollusca Gastropoda.

The metabolic pathways of glucose were studied by histochemical reactions in some species of gastropods living in different habitats. The glycolytic pathway is histochemically indicated by positive results for glucose-6-phosphate isomerase, fructose-1,6-biphosphate aldolase, glyceraldehyde-3-phosphate dehydrogenase, and D-lactate dehydrogenase. The enzymes of the Krebs cycle gave different responses: isocitrate dehydrogenase and L-malate dehydrogenase were positive, whilst succinate dehydrogenase was constantly negative. Malate synthetase activity was also demonstrated. Despite L-glutamate dehydrogenase is undetectable, the presence of transaminase indicates the gluconeogenetic route. Phosphoglucomutase and glucose-6-phosphate phosphatase appear also positive. The metabolic meaning of our results were discussed.

A rapid method for purification of arylsulphatase A from rabbit uterus.

The arylsulphatase A from rabbit uterus has been purified with a rapid method using Fast Protein Liquid Chromatography (FPLC). The enzyme is an acid glycoprotein with an apparent molecular weight of 110,000. The enzymatic activity is competitively inhibited by various phosphoesters and various ions such as SO4(2-), PO4(3-) and P2O7(4-).

Fluctuations in arylsulphatase activity in the rabbit endometrium during the sexual cycle.

Histochemical and biochemical studies were performed to verify the presence of arylsulphatase A (ASA) and B (ASB) in the rabbit uterus. Fluctuations in the activity of these sulphatases during the sexual cycle were also studied. Some structural and functional properties of purified ASA were determined. The results indicate that arylsulphatases are active in the endometrium during both the estrogenic and progesteronic phases. The activity of ASA was much more intense than that of ASB; it increased during estrus and decreased during the post-ovulatory phase. ASB activity, however, decreased during estrus and increased during the post-ovulatory phase. The significance of these fluctuations is discussed in relation to the action of sexual hormones and physiological substrates of arylsulphatases.

Enzymatic characterization of E.U.E. (embryonal human explants) cells adapted to hypertonic media.

E.U.E. cells (general population) were submitted to biochemical and cytoenzymatic tests to compare the enzymatic profile of E.U.E. cells (controls) with that of E.U.E. adapted to hypertonic medium. The adapted cells are characterized by very high oxoreductase activity (LDH, HBDH, G-6-P DH) and very high alkaline-phosphatase activity. Clones derived from general population were also submitted to biochemical tests to characterize those more strictly related to the enzymatic profile of adapted cells. The profile of clone N. 13 resembles on this respect that of the adapted cells. The high redox activity is a prerequisite supporting energy supply for osmotic work. The increased activity of plasma membrane enzymes of the adapted cells is also demonstrable in cells exposed for short time to salinity.

Polar lipids extraction from rat primary lysosomes and their possible role in regulating lysosomal hydrolases.

The polar lipids in primary lysosomes prepared from rat liver were extracted and evaluated by qualitative and quantitative procedures. Phospholipid phosphorus ranges between 1.50 and 2.6 micrograms/mg protein and sulfolipids varied from 12 to 60 micrometer/mg protein. The TLD of the organic phase have nine lipid fractions. Glycolipids were also present: GM1, GM3 and GD1a gangliosides could be separated by TLC. Sialic acid was about 0.24 micrograms/mg protein. The cytochemical interest of these components lies not only in the chemical composition of lysosomal structures, but also in the regulatory mechanism due to some of them, such as sulfolipids, which are likely to be involved in inactivating lysosomal enzymes by cross-inhibition.