Quantitative and qualitative variations in the glycoproteins in the chick embryo brain

Sugar content was examined in soluble and insoluble glycoproteins extracted from the chick embryo brain at different developmental stages. The content of hexosamines and uronic acids in the soluble fraction is higher during the whole period examined. The difference between the two fractions reaches a maximum at the 15th day. The insoluble fraction shows the highest content of sialic acid and fucose in comparison with the soluble one, especially toward hatching. The sialic acid/fucose ratio shows a different pattern in the two fractions examined, particularly in the soluble glycoproteins. The patterns of sialic acid and fucose indicate that quantitative and qualitative developmental changes occur in the soluble and insoluble glycoproteins. All sugars examined show significant changes on the 15th day, suggesting that this stage may represent a critical period in the development of the chick embryo brain.     

Developmental changes in the activity of membrane-bound gamma-glutamyl transpeptidase and in the sialylation of synaptosomal membranes from the chick embryonic brain.

gamma-Glutamyl transpeptidase (GGT) is a membrane-bound sialoglycoprotein. The developmental changes in GGT activity and in sialic acid content were determined in a crude synaptosomal membrane fraction from the cerebral hemispheres of the chick embryo between days 11 and 19 of incubation. The GGT activity increased almost eightfold during the examined developmental period, while sialic acid content rose significantly only between days 11 and 15. Cortical administered on day 13 significantly increased GGT activity. On the other hand, the content of membrane bound sialic acid was not substantially affected. The value of the GGT apparent Michaelis constant (Kmapp) for gamma-glutamyl-p-nitroanilide in the presence of 20 mmol.l-1 glycylglycine was 1.5 mmol.l-1 and cortisol did not influence it. However, Vmax was increased by this hormone. The affinity of GGT to concanavalin A (ConA) did not change during development. Neither the administration of cortisol nor neuroaminidase treatment had any effect on the interaction of GGT with ConA. Desialylation of crude synaptosomal fraction did not change GGT activity. The results presented here suggest no developmental nor functional relationship between the activity of GGT and the level of sialylation in synaptosomal membranes from the cerebral hemispheres of the chick embryo.     

Lectin histochemistry of the embryonic heart: expression of terminal and penultimate galactose residues in developing rats and chicks

Rat embryos at days 10-18 of gestation and chicken embryos at days 3-6 of incubation were fixed and processed for lectin histochemistry. The distribution of binding sites for a lectin from the peanut Arachis hypogaea (PNA) conjugated to horseradish peroxidase (HRP) was determined on tissue sections both before and after enzymatic cleavage of sialic acid with neuraminidase (sialidase). Endocardial cushion tissue in the rat, but not in the chick, reacted with PNA-HRP prior to digestion with sialidase. Endocardium of both species (12 and 13 days in rat, 5 and 6 days in chick), particularly at the level of endocardial cushions, reacted strongly with the sialidase-PNA sequence; this staining decreased markedly after day 14 of gestation in the rat. PNA binding sites capped by sialic acid were most abundant in the developing rat heart during the critical period of endocardial cushion formation and decreased as development proceeded. The marked changes in the appearance and distribution of cardiac cell and tissue glycoconjugates during cardiogenesis support the concept that rapid changes occur in the structure of complex carbohydrates during embryonic and fetal development. The findings also suggest that such glycosylation-related events may be species specific.     

Density-Dependent Changes in Gangliosides and Sialidase Activity of Murine Neuroblastoma Cells

Abstract: Density-dependent changes in ganglioside composition, Vibrio cholerae neuraminidase (VCN)-susceptible sialyl residues, and membrane- associated sialidase activity were determined for the cholinergic murine neuroblastoma cell line S20Y. A decrease in total ganglioside sialic acid and VCN-releasable sialic acid was observed with increasing cell density. G^M3 was the major ganglioside component of preconfluent S20Y cells, whereas G_DIA was predominant in postconfluent cells. Sialidase activity increased in confluent and postconfluent cells and may account for the reduction in total ganglioside sialic acid observed with increasing cell density. In contrast, while adrenergic N115 cells showed a decrease in VCN-susceptible sialic acid residues with increasing cell density, there was no significant change in ganglioside composition or ganglioside sialic acid levels.     

Cytochalasin B and the sialic acids of Ehrlich ascites cells

The effect of cytochalasin B (CB) on the electrophoretic mobility and density of ionized sialic acid groups at the surface of Ehrlich ascites cells was examined together with a biochemical assay of the total sialic acid content of treated and control cells. Sialic acid assays indicated that CB-treated cells had a greater amount of total sialic acid and sialic acid sensitive to neuraminidase than control cells/cell. Equal amounts of sialic acid were removable by neuraminidase treatment from control cells and cells pretreated with neuraminidase and subsequently cultured with CB. The electrophoresis results showed a decrease in electrophoretic mobility in the presence of CB which could be reversed by growth in CB-free medium. Neuraminidase treatment did not make a significant additional reduction in the mobility of CB-treated cells. CB also prevented the recovery of electrophoretic mobility of neuraminidase treated cells. The results suggest that while CB does not inhibit sialic acid synthesis, it does alter the expression of ionized sialic acid groups at the electrokinetic surface. CB-containing culture media could be re-utilized several times suggesting that CB is not significantly bound or metabolized by Ehrlich ascites cells.     

Quantitative and qualitative changes of plasma membrane glycoproteins in the early period of liver regeneration

Alterations of cell surface glycoconjugates have been observed in many developing systems and may be important in the physiological control of growth and differentiation. Liver regeneration after partial hepatectomy is a suitable model in which to study the regulatory mechanisms of cell proliferation in vivo. We have isolated the sinusoidal plasma membrane of hepatocytes at different times after partial hepatectomy. The sialic acid content and the SDS-polyacrylamide gel electrophoresis pattern of glycoproteins were determined. A decrease of periodic acid-Schiff-profiles, a change in the binding capacities of 125I-concanavalin A, a reduction of the sialic acid content and the appearance and disappearance of specific components have been observed during the pre-replicative phase of liver regeneration. These findings during this early period are consistent with the active involvement of the plasma membrane glycoproteins in the transition of cells to the proliferative state.     

Neuraminidase activity and cell surface sialic acid turnover in Rous sarcoma virus transformed chick embryo fibroblasts

Neuraminidase activity of Rous sarcoma virus transformed chick embryo fibroblasts (RSV-CEF) was assayed using an exogenous substrate, neuraminlactitol-[3H], and endogenous, cell surface [14C]-N]-acetyl-neuraminic acid. RSV-CEF had higher neuraminidase activity toward both substrates than did chick embryo fibroblasts (CEF) or nontransformed, Rous associated virus infected CEF (RAV-CEF). The total sialic acid content of RSV-CEF was lower than CEF or RAV-CEF, and more of the total sialic acid was accessible to extracellular Clostridium perfringens neuraminidase. Activity of the enzymes synthesizing and degrading the substrate for sialyltransferase, cytidine-5'-monophosphate-N-acetyl-neuraminic acid (CMP-AcNeu) was measured in order to determine whether control of substrate levels for sialyltransferase might contribute to the decreased levels of glycoprotein bound sialic acid. No change in activity of these enzymes was found in RSV-CEF as compared to CEF or RAV-CEF.     

Cell surface charge alterations occurring during dimethylsulfoxide-induced erythrodifferentiation of Friend leukemia cells

Dimethylsulfoxide-induced erythrodifferentiation of Friend leukemia cells caused a decrease in net negative cell surface charge which began two days after exposure to the polar solvent and continued throughout the maturation process. Neuraminidase treatment caused a marked reduction in mobility of both untreated and dimethylsulfoxide-treated cells suggesting that sialic acid residues are the major anionogenic moieties of the surface membrane of Friend cells. A decrease in the content of total glycosidically bound sialic acid in dimethylsulfoxide-treated cells also occurred. The findings provide evidence to support an association between erythrodifferentiation of Friend cells and net negative surface charge dependent upon sialic acid residues.     

Development of sucrase in the chick small intestine

Development of sucrase in the chick small intestine was studied biochemically and immunologically using antiserum prepared against purified chick intestinal sucrase. Sucrase activity was first detectable at 10 days of incubation and increased with age. After a transient drop at 20 days, the activity rapidly increased to the adult level. Immunodiffusion and polyacrylamide gel electrophoretic studies suggested that the sucrase of the embryonic and hatched chick intestines was identical except for a difference in the content of sialic acids. In immunofluorescence and immunoelectron microscopy, sucrase was found to appear on the luminal surface of epithelial cells at 8-10 days of incubation, soon after the start of morphological differentiation from an undifferentiated thick epithelium to a thin simple epithelium.     

SIALIC ACIDS ON THE PLASMA MEMBRANE OF CULTURED HUMAN LYMPHOID CELLS : Chemical Aspects and Biosynthesis

From 61 to 92% of the total sialic acid of a variety of human lymphoid cell lines maintained in tissue culture is present on the cell surface as measured by its susceptibility to cleavage by Clostridium perfringens neuraminidase. These cells contain from 1.22 x 10⁸ to 6.99 x 10⁸ molecules of surface sialic acid per cell. In synchronized cultures synthesis of surface sialic acid occurs only during a limited time in the late G₂ phase of the cell cycle. The amount and density of surface sialic acid vary considerably throughout the cell cycle.     

Abnormal diastolic currents in ventricular myocytes from spontaneous hypertensive heart failure rats

Hypertension is a common cause of heart failure, and ventricular arrhythmias are a major cause of death in heart failure. The spontaneous hypertension heart failure (SHHF) rat model was used to study altered ventricular electrophysiology in hypertension and heart failure. We hypothesized that a reduction in the inward rectifier K(+) current (I(K1)) and expression of pacemaker current (I(f)) would favor abnormal automaticity in the SHHF ventricle. SHHF ventricular myocytes were isolated at 2 and 8 mo of age and during end-stage heart failure (>/=17 mo); myocytes from age-matched rats served as controls. Inward I(K1) was significantly reduced at both 8 and >/=17 mo in SHHF rats compared with controls. There was a reduction in inward I(K1) due to aging in the controls only at >/=17 mo. We found a significant increase in I(f) at all ages in the SHHF rats, compared with young controls. In controls, there was an age-dependent increase in I(f). Action potential recordings in the SHHF rats demonstrated abnormal automaticity, which was abolished by the addition of an I(f) blocker (10 muM zatebradine). Increased I(f) during hypertension alone or combined increases in I(f) with reduced I(K1) during the progression to hypertensive heart failure contribute to a substrate for arrhythmogenesis.     

Cellular sialic acid level and phenotypic expression in B16 melanoma cells: comparison of spontaneous variations and bromodeoxyuridine- and theophylline-induced changes

The relation of the total cellular content of sialic acid to phenotypic expression of B16 mouse melanoma cells was examined by using phenotype-modifying reagents and more than 10 cloned cell lines with spontaneous phenotypic variations. The sialic acid content changed in a growth phase-dependent manner with a peak in the early log phase of growth. This peak completely disappeared when cells were treated with 5-bromodeoxyuridine (BrdU), suggesting its relation to quasi-normal phenotypes of the treated cells. BrdU treatment also reduced the cellular sialic acid content itself and resulted in the suppression of the activity of tyrosinase, the key enzyme for melanogenesis, and a considerable increase in cell-to-substratum adhesiveness. Treatment with theophylline, in contrast, markedly elevated the sialic acid content, which was accompanied by dramatic increments in tyrosinase activity and pigmentation as well as a slight increase in adhesiveness. The results show a correlation of sialic acid level with tyrosinase expression but not with cell adhesion. From comparison of spontaneous phenotypic variations, the correlation of sialic acid level with tyrosinase activity was confirmed, while there was only a slight correlation with adhesiveness. It is thus suggested that sialylation/desialylation, being reflected as variations in cellular sialic acid content, is implicated in melanoma cell differentiation in terms of tyrosinase expression.     

Age- and hormone-dependent ganglioside patterns of rat hepatocytes in primary culture

Using an improved procedure for the quantitative extraction of all glycolipids from small tissue samples the hepatic ganglioside pattern of rats was analysed during development. While this parameter remained fairly constant in adult animals, hepatocytes in primary culture showed drastic changes both in content and relative distribution among the various ganglioside species. The content of lipid-bound sialic acid increased several-fold during 6 days in monolayer and the pattern changed in favour of the higher sialylated forms. Dexamethasone delayed this transition and enhanced the content of GD1a and GM1 relative to GM3. The ganglioside content was also dependent on the density of hepatocytes in the primary culture. If the cell density was insufficient for formation of a confluent monolayer, higher ganglioside-sialic acid contents were found and the relative amount of GD3 increased after 3-4 days. These results support the notion that gangliosides are involved in cellular differentiation and cell-cell contact.     

Rheological characteristics of desialylated erythrocytes in relation to fibrinogen-induced aggregation

The effect of fibrinogen and sialic acid content of erythrocytes on the aggregation of erythrocytes was quantitatively examined by using a rheoscope combined with a television image analyzer and a computer. (1) The electrophoretic mobility of erythrocytes was proportional to the sialic acid content of erythrocytes (the surface potential of erythrocytes could be expressed by the sialic acid content). (2) The aggregation of erythrocytes was accelerated by increasing fibrinogen concentration in the medium (due to the increased bridging force among erythrocytes) or by decreasing the sialic acid content (due to the reduction of the electrostatic repulsive force among erythrocytes). (3) An empirical equation expressing the velocity of aggregate formation (ν, in μm²/min) by the concentration of fibrinogen ($\text{F}$, in g/dl) and the sialic acid content ($\text{S}$, in μmol/ml red blood cells), log $\text{ν = ?0.065 F}{}^{\mathrm{?1.2}}\text{S + 2.2 F}{}^{0.35}$, was deduced. (4) The contribution of the bridging force of fibrinogen to the erythrocyte aggregation was much greater than that of the electrostatic repulsive force produced by sialic acid on the surface of erythrocytes.     

The isolation and characterization of plasma membrane from cultured cells V. The chemical composition of plasma membranes isolated from chicken tumors initiated with virus-transformed cells

Sarcomas were initiated in chicken muscle and wing web by Bratislava 77 and morph^r Fujinami virus. Plasma membrane was isolated from the virus-induced tumor cells by differential centrifugation and flotation equilibrium centrifugation. The levels of neutral sugar and sialic acid in these isolated plasma membranes were very similar to the levels found in cultured chick embryo fibroblasts transformed in vitro with the same oncogenic viruses and differed markedly from the levels found in uninfected and leukosis virus-infected fibroblasts.The phospholipid content of the isolated cell membranes from tumors was less than the quantity of lipid found in the plasma membrane of cultured cells and differed with the site of the tumor. Breast muscle tumors contained less plasma membrane phospholipid than did wing tumors.The similarities in the neutral sugar and the sialic acid content of these two different sources of plasma membrane indicate that oncogenic transformation in cell culture reproduces in situ neoplastic change to a large extent, for at least this one parameter of cell surface change.     

Histochemical detection of sugar residues in the chick embryo mesonephros with lectin-horseradish peroxidase conjugates

Summary Fragments of mesonephros were taken from chick embryos and studied from the 4th to the 21st day of incubation. A battery of seven different horseradish peroxidase-labelled lectins was used to study the distribution of carbohydrate residues in glycoconjugates along the mesonephric nephron during the period of excretory activity and the period of involution. ConA and WGA reacted at every site of the nephron thus showing the ubiquitous presence of -D-mannose andN-acetyl-d-glucosamine. SBA was a good marker of the proximal tubule. Other lectins, such as PNA and LTA, reacted only for a short time at some sites during the considered period of incubation. The presence of sialic acid was detected in the podocytes, capillary wall and mesangial cells. From the 10th-11th day of incubation changes were noted in the proximal tubule as shown by PNA reactivity. This may be significant as regards the exact stage of incubation during which the involution of mesonephros begins.     

Glucose utilization, pH reduction and density dependent inhibition in cultures of chick embryo fibroblasts.

The multiplication rate of sparse cultures of chick embryo cells is only slightly lower at pH 6.9 than at pH 7.4. There is, however, a marked reduction in the multiplication rate of the pH 6.9 cultures before they reach confluency. Cultures at pH 7.4 continue to multiply beyond confluency with only a slight decrease in the multiplication rate. Eighty to ninety percent of the glucose taken up by the cells growing at each pH is converted to lactic acid which is released into the medium. Metabolic reduction in pH of the medium is almost entirely accounted for by the amount of lactic acid produced by the cells. Neither the intracellular nor extracellular accumulation of lactic acid nor the accompanying reduction in pH is sufficient to explain density dependent inhibition of the rate of multiplication of chick cells. The rate of lactic acid production and the multiplication rate of chick cells are independent of glucose concentration in the range of 2--16 mM. In view of the kinetic parameters for the uptake of glucose, this shows that glycolysis is not limited by the rate of glucose uptake and that depletion of glucose from the medium cannot account for the onset of density dependent inhibition of multiplication. However, when cells reach very high population densities, conventional glucose concentrations of 5 mM can be depleted overnight by chick cells. Since the multiplication rate of cells is dependent on glucose concentration when it falls below 2 mM, depletion of glucose may cause some growth inhibition in crowded cultures supplied with conventional medium.     

Histochemical detection of sugar residues in the chick embryo mesonephros with lectin-horseradish peroxidase conjugates

Fragments of mesonephros were taken from chick embryos and studied from the 4th to the 21st day of incubation. A battery of seven different horseradish peroxidase-labelled lectins was used to study the distribution of carbohydrate residues in glycoconjugates along the mesonephric nephron during the period of excretory activity and the period of involution. ConA and WGA reacted at every site of the nephron thus showing the ubiquitous presence of alpha-D-mannose and N-acetyl-D-glucosamine. SBA was a good marker of the proximal tubule. Other lectins, such as PNA and LTA, reacted only for a short time at some sites during the considered period of incubation. The presence of sialic acid was detected in the podocytes, capillary wall and mesangial cells. From the 10th-11th day of incubation changes were noted in the proximal tubule as shown by PNA reactivity. This may be significant as regards the exact stage of incubation during which the involution of mesonephros begins.     

Erythrocyte membrane bound and plasma sialic acid during aging

Sialic acid, a nine-carbon sugar, is an acetylated derivative of neuraminic acid predominantly found in vertebrates, a few higher invertebrates, and certain types of bacteria. Red blood cells (RBCs) have a net negative surface charge and this bulk charge is due to ionized sialic acid. Decreased surface charge and sialic acid content have been reported in older erythrocytes, and it is postulated that the decreased electro-negativity may be related to cell senescence. In the present study we report the RBC and plasma sialic acid content during aging in rats. Our results show a significant decrease in RBC sialic acid content and increase in plasma sialic acid as a function of rat aging. The decreased sialic acid in erythrocyte membrane with increasing rat age presents a good biomarker of the aging process. The elevated plasma sialic acid may be a manifestation of several factors including increased expression of acute phase proteins and increased damage to various organs.     

Sialidase activities of cultured human fibroblasts and the metabolism of GM3 ganglioside

Free sialic acid has been found in the cell-conditioned medium of human foreskin fibroblasts. It is proposed that the accumulation of extracellular sialic acid may result from the hydrolysis of GM3 ganglioside on the cell surface of these fibroblasts. Sialidase activities with GM3 ganglioside and sialyllactitol as substrates were demonstrated in cell-conditioned medium, and the levels of their activities correlated positively with cell density. The GM3 sialidase activity at pH 4.5 was 4.1 and 38 pmol/h/ml of medium at sparse and confluent densities, respectively; the corresponding activities with sialyllactitol as the substrate were 12 and 75 pmol/h/ml of medium (pH 4.5). The pH versus activity profiles with GM3 as the substrate suggested the presence of a second sialidase with an optimal activity at pH 6.5 in the conditioned medium of preconfluent cells. This activity was virtually absent in the medium of contact-inhibited cells and could not be assayed with sialyllactitol as the substrate. The turnover of cell surface GM3 was assessed by pulse labeling human foreskin fibroblasts with a radioactive precursor of sialic acid ([1-14C]N-acetylmannosamine) and a radioactive precursor of ceramide ([3,3-3H2]serine). During a chase period of 24 h turnover of the doubly labeled cellular GM3 was observed; there was a loss of about 35% of the 14C-labeled sialic acid without any measureable loss of 3H-labeled ceramide from GM3. We have speculated that the enzyme-catalyzed removal of sialic acid from the GM3 ganglioside on the extracellular aspect of the plasma membrane may be a necessary event involved in the modulation of cell growth.