Decrease In Sialic Acid Content Research Articles

Characteristics and qualities of edible bird's nest beverage as affected by thermal pasteurization and sterilization.

Edible bird's nest beverage (B-nest-Bev) was produced from edible bird's nest (B-nest) flakes using different thermal processes. Pasteurization of B-nest-Bev at a low temperature for a longer time (LTLT) or at a high temperature for a shorter time (HTST) resulted in lower CIE L * , CIE a * , CIE b * -values, and drained weight (p < 0.05) than sterilization (118 or 121°C). Sterilized and pasteurized B-nest-Bev had similar soluble solid contents and pH (p < 0.05). Nevertheless, acidified beverages pasteurized via either LTLT or HTST process had a marked decrease in sialic acid content. In addition, drastic protein degradation occurred in pasteurized acidified beverages, regardless of the conditions used, ascertained by the disappearance of major protein bands. However, polymerization of proteins took place in sterilized samples, irrespective of the temperature used. After digestion in a gastrointestinal tract model system (GIMs), all samples had increased (p < 0.05) antioxidant activities including DPPH and ABTS radical scavenging activities, ferric reducing antioxidant power (FRAP) and oxygen radical absorbance capacity (ORAC). B-nest-Bev subjected to HTST pasteurization or sterilization at 121°C had the best appearance and acceptability among all the treatments used. Therefore, thermal processes directly affected the properties and acceptability of B-nest-Bev.

Characterization and minimization of sialic acid degradation in an Fc-fusion protein-producing CHO cell bioprocess

Abstract Sialic acid content generally affects many biochemical properties of therapeutic glycoproteins. More specific properties, such as charge distribution, circulating half-life and bioactivity, are directly impacted by sialic acid content. Undesirable loss of protein sialylation is commonly observed in CHO bioprocesses, and sialic acid degradation is one of the root causes. In this work, sialic acid degradation by extracellular sialidase was investigated in an Fc-fusion protein production bioprocess. First, cell-free incubation with the cell culture supernatant (CCS) collected at the end of culture revealed a 12.46% drop in sialic acid content by sialidase after 6 days of incubation, while other monosaccharides on the glycans remained stable. Then, potential factors that could affect the action of sialidase were studied, and a pH of over 7.05 was found to have a strong inhibitory effect on the degradation of sialic acid in the CCS. The results were further validated in bioreactors with different pH controls to demonstrate that a minimization of sialic acid loss could be achieved by controlling pH at higher values. Our work provides a simple and effective way to alleviate the decrease in sialic acid content and improves the understanding of the relationship between process control and product sialylation in CHO bioprocesses.

Influence of myocardial infarction on membrane structure and hemorheological characteristics of erythrocytes

The study shows that cholesterol, phospholipids and c/p ratio in erythrocytes are increased in MI patients. The individual phospholipid composition is also altered with increase of phosphatidylcholine and sphingomyelin in plasma, and phosphatidylcholine in erythrocytes. There is no significant change in protein composition except a significant decrease in sialic acid content of cell. The membrane enzyme such as Na + , K + ATPase and Ca 2+ , Mg 2+ ATPase are decreased and there is no change in cytosolic calmodulin stimulatory activity observed. Whole blood viscosity and osmotic fragility are increased. The deformability of erythrocytes is significantly decreased

Galactose supplementation enhance sialylation of recombinant Fc-fusion protein in CHO cell: an insight into the role of galactosylation in sialylation.

Sialic acid levels of therapeutic glycoprotein play an important role in plasma half-life. An undesirable decrease of sialic acid content was observed when we increased Fc-fusion protein productivity fourfold in a GS-CHO cell line by bioprocess optimization. We investigated the potential mechanism for the sialic acid content reduction. We found that limited nucleotide sugar precursor and the extracellular sialidase were not responsible for the reduction of the sialic acid content after titer improvement. Oligosaccharide analysis revealed that the lack of protein galactosylation was the potential cause for the reduction of sialic acid content. Thus we validated this notion by evaluated galactose supplementation in 2 L bioreactors. Cell culture performance was not impacted by addition of up to 40 mM galactose except for the glucose consumption rate. Addition of 20 mM galactose to the bioreactor resulted in the increase of 44 % for total sialic acid content and 20.3 % for sialylated glycans. These data were further validated when the process was run on 200 L scaled bioreactor. These data together show that the galactosylation plays an apparent role in sialylation in our current system.

Effect of membrane-bound IgG and desialysation in the interaction of monocytes with senescent erythrocytes

Determination of the erythrocyte lifespan is a complex process affected by many cellular parameters. In the present study we measured and characterised the red blood cell (RBC) membrane proteins, mainly band 3, and quantified membrane-bound IgG in senescent RBC (SeRBC) and young RBC (YRBC). We also investigated, through a functional assay, the interaction between SeRBC and peripheral blood monocytes. We applied this erythrophagocytosis assay to study the phagocytosis of desialysed RBC. The results obtained showed no changes in the protein content between SeRBC and YRBC and no differences when examining membrane proteins by SDS-PAGE. Then, considering that the accumulation of autologous IgG on RBC membrane provides a direct mechanism for the removal of SeRBC, we measured the IgG content of intact RBC using an enzyme-linked anti-immunoglobulin test finding that the number of IgG molecules bound to SeRBC was significantly higher than that observed for YRBC. The increase observed in the percentage of erythrophagocytosis with SeRBC and sensitised RBC (SRBC) confirmed the involvement of autologous IgG in the selective removal of erythrocytes. We also observed a higher percentage of monocytes with phagocytosed and adherent RBC (AM) obtained with neuraminidase-treated RBC than those obtained with YRBC. This finding suggests that a decrease in sialic acid content of SeRBC may be involved in physiological erythrophagocytosis.

Ethanol-Induced Changes in Glycolipids of Saccharomyces cerevisiae

Total glycolipid content of Saccharomyces cerevisiae cells increased in ethanol-treated yeast cells. Sialic acid and hexosamine contents of glycolipids from ethanol-treated cells decreased, whereas those of hexoses increased. Increased sialidase activity in the presence of ethanol may be responsible for the decrease in sialic acid content of glycolipids. The saccharide moieties of glycolipids of S. cerevisiae consisted of fucose, mannose, galactose, and glucose. Ethanol treatment of yeast cells caused an increase in glucose and a decrease in galactose content of glycolipids. The changes in glucose content can be related to changes in beta-glucosidase activity under alcohol stress. The content of cerebrosides, sulfatides, and monoglucosyldiglycerides was enhanced following ethanol treatment. An increase in cerebroside as well as in sulfatide content during alcohol stress might play an important role in stabilizing the membrane both physically and structurally. Such variations in glycolipid content and composition of S. cerevisiae cells may represent an adaptive response to ethanol stress.

Higher reactivity of apolipoprotein B-100 and α-tocopherol compared to sialic acid moiety of low-density lipoprotein (LDL) in radical reaction

Radical reaction of low-density lipoprotein (LDL) is a key step in atherogenesis and causes both a decrease in the sialic acid moiety and modification of apolipoprotein B-100 (apoB). Although apoB modification (cross-link and fragmentation) increases in atherosclerosis, the change in apoB-bound sialic acid in atherosclerosis is controversial. To elucidate the physiological implications of desialylation of LDL by radical reaction, the reactivity of sialic acid of LDL was compared with that of apoB, which underwent facile fragmentation in radical reactions. ApoB was determined by immunoblot analysis with anti-apoB antiserum, and the sialic acid moiety was measured by blot analysis with a biotin-bound lectin [biotin-SSA from Japanese elderberry (Sambucus sieboldiana)] specific to sialic acid. When human LDL was oxidized with Cu 2+ at 37 °C, apoB and apoB-attached sialic acid decreased simultaneously. Comparison of the staining bands with anti-apoB and with biotin-SSA shows that sialic acid moieties still remain on fragmented apoB proteins, indicating that the decrease in sialic acid is much slower than that of apoB fragmentation. In addition, human plasma was oxidized with 400 μM of Cu 2+ at 37 °C. Similar analysis indicates that the decrease in sialic acid attached to apoB also results from the fragmentation of apoB. This study indicates that the fragmentation of apoB proceeds at a much faster rate than the decrease in sialic acid content when a free radical reaction is induced in isolated LDL as well as in plasma LDL exposed to Cu 2+-induced oxidative stress. On the basis of these results, the modification of apoB is much more sensitive than the decrease in sialic acid as an indicator of oxidative stress.

RED BLOOD CELL DEFORMABILITY AND ALCOHOL DEPENDENCE IN HUMANS

Studies of DPH fluorescence polarization and deformability have shown that alcohol induces rigidification of the red blood cell (RBC) membrane. We investigated a possible link between RBC membrane fluidity and deformability by studying both parameters simultaneously in samples from alcohol-dependent patients (group 1, N = 19), social drinkers (group 2, N = 12) and long-term abstaining alcoholics (group 3, N = 8). The active drinkers showed disturbances of several RBC membrane parameters, including abnormal microorganization of the membrane surface, a decrease in sialic acid content, and resistance to the fluidizing effect of ethanol, that were not completely corrected in the abstinent alcoholics. The RBC transit time was significantly longer in the active drinkers than in the abstainers but not the social drinkers. There were no significant differences between the groups with regard to membrane lipid core fluidity. The main abnormality (fluidization) in RBC from the active alcoholics involved the polar surface of the membrane (probed using TMA-DPH), and correlated with the decrease in sialic acid content but not with RBC deformability.

Measurements and analyses of electrophoretic mobilities of RAW 117 lymphosarcoma cells and their variant cells

The electrophoretic mobilities of the cells of malignant lymphosarcoma cell line RAW117-P and its variant H10 with a highly metastatic property to the liver have been measured at various ionic strengths. The cells of parental cell line (RAW117-P) show higher mobility values in magnitude than those of its variant line (RAW117-H10) in the whole range of electrolyte concentration measured. We have also measured the sialic acid amount carried by cells of both lines. The content of sialic acids in RAW117-H10 cells is observed to be about 27% less than that in RAW117-P cells. The mobility data obtained have been analyzed by a novel mobility formula for colloidal particles with ion-penetrable surface charge layers. The observed mobility difference between RAW117-P cells and RAW117-H10 cells is found to be due to the difference in friction exerted by the cell surface layers on the liquid flow around the cells between these two types of cells and to the difference in fixed-charge density in their surface layers, which is caused by the 27% decrease in sialic acid content. A possible explanation for this mobility difference between these two types of cells is given.

Decrease in erythrocyte glycophorin sialic acid content is associated with increased erythrocyte aggregation in human diabetes.

1. Sialic acid moieties of erythrocyte membrane glycoproteins are the principal determinants of the negative charge on the cell surface. The resultant electrostatic repulsion between the cells reduces erythrocyte aggregation and hence the low shear rate viscosity and yield stress of blood. 2. Using g.c.-m.s., a decrease in sialic acid content has been observed in the major erythrocyte membrane glycoprotein, glycophorin A, obtained from nine diabetic patients compared with that from seven normal control subjects [median (range): 3.30 (0.01-11.90) versus 18.60 (3.20-32.60) micrograms/100 micrograms of protein, P less than 0.02]. 3. Erythrocyte aggregation, measured by viscometry as the ratio of suspension viscosity to supernatant viscosity (LS/S) in fibrinogen solution, was increased in ten diabetic patients compared with ten normal control subjects (mean +/- SEM, 37.6 +/- 1.3 versus 33.8 +/- 0.6, P less than 0.02). 4. In the patients in whom both viscometry and carbohydrate analysis were performed, the decrease in erythrocyte glycophorin sialylation and the increase in erythrocyte aggregation in fibrinogen solution were related statistically (LS/S correlated negatively with glycophorin sialic acid content, r = 0.73, P less than 0.05). 5. Decreased glycophorin sialylation provides an explanation at the molecular level for increased erythrocyte aggregation and it may be important in the pathogenesis of vascular disease in diabetes.

Erythrocyte aggregation: Bridging by macromolecules and electrostatic repulsion by sialic acid

Relation between aggregating force (of fibrinogen and IgG) and disaggregating force (due to electrostatic repulsion among erythrocytes) in erythrocyte aggregation was investigated with a rheoscope combining a video camera, an image analyzer and a computer. (i) Erythrocyte aggregation was augmented with the increase of molecular weight of bridging macromolecules as far as examined for fibrinogen and the degradation products and IgG and the related macromolecules, and the augmentation seemed to be dependent on the molecular length of macromolecules. In accelerating the erythrocyte aggregation, fibrinogen was more effective than IgG, and some interaction between fibrinogen and IgG in their coexistence was suggested. (ii) The decrease of sialic acid content on the erythrocyte surface accelerated IgG-induced erythrocyte aggregation much greater than fibrinogen-induced one. (iii) Counteraction between aggregating force and disaggregating force in leading to erythrocyte aggregation was discussed relating to molecular length of bridging macromolecule and electrostatic repulsive force by sialic acid.

Influenza virus changes cell-surface glycoproteins including major histocompatibility complex determinants on lymphocytes

The effect of influenza virus infection on the expression of major histocompatibility complex (MHC) antigens was investigated. Infection with influenza virus resulted in an increase of the binding of anti-MHC class I and class II antibodies to resting T cells. The binding of anti-MHC class II antibodies to activated T cells was increased approximately threefold. The binding of anti-MHC class I and class II antibodies to Epstein-Barr virus — transformed B cells appeared unaffected after influenza virus infection. Recombinant human interferon-α and/or-γ added to T cells did not enhance the binding of anti-MHC antibodies. Biochemical analysis revealed no increase in the amount of class I and class II antigens as a consequence of viral infection, but a marked decrease in sialic acid content was found, most probably caused by the viral neuraminidase. Pulse-chase experiments suggest that the viral neuraminidase can catalyze the removal of sialic acids both en route to and at the cell surface. The absence of sialic acid residues can explain the increased binding of anti-MHC antibodies, because neuraminidase (clostridium perfringens) treatment of T and Epstein-Barr virus-transformed B cells resulted in a shift in both isoelectric point and antibody binding similar to that observed after influenza virus infection.

Developmental aspects of the rat brain insulin receptor: loss of sialic acid and fluctuation in number characterize fetal development.

In this study, I have investigated the structure of the rat brain insulin receptor during fetal development. There is a progressive decrease in the apparent molecular size of the brain alpha-subunit during development: 130K on day 16 of gestation, 126K at birth, and 120K in the adult. Glycosylation was investigated as a possible reason for the observed differences in the alpha-subunit molecular size. The results show that the developmental decrease in the brain alpha-subunit apparent molecular size is due to a parallel decrease in sialic acid content. This was further confirmed by measuring the retention of autophosphorylated insulin receptors on wheat germ agglutinin (WGA)-Sepharose. An inverse correlation between developmental age and retention of 32P-labeled insulin receptors on the lectin column was observed. Insulin binding increases 6-fold between 16 and 20 days of gestation [61 +/- 25 (+/- SE) fmol/mg protein and 364 +/- 42 fmol/mg, respectively]. Thereafter, binding in brain membranes decreases to 150 +/- 20 fmol/mg by 2 days after birth, then reaches the adult level of 63 +/- 15 fmol/mg. In addition, the degree of insulin-stimulated autophosphorylation closely parallels the developmental changes in insulin binding. Between 16 and 20 days of fetal life, insulin-stimulated phosphorylation of the beta-subunit increases 6-fold. Thereafter, the extent of phosphorylation decreases rapidly, reaching adult values identical with those in 16-day-old fetal brain. These results suggest that the embryonic brain possesses competent insulin receptors whose expression changes markedly during fetal development. This information should be important in defining the role of insulin in the developing nervous system.

Alterations in the sialic acid content of the rat glomerular filter in aminonucleoside nephrosis.

A daily injection protocol with puromycin aminonucleoside (PAN) causes loss of sialic acid from the glomerular filter. These changes have been studied previously by colloidal iron staining, but we have recently shown that phosphotungstic acid (PTA) at low pH allows the demonstration of sialic acid groups in the glomerular basement membrane in ultrathin sections of glycolmethacrylate(GMA)-embedded rat kidney. With this technique the slit diaphragm is seen as a continuation of the luminal cell coat and the method also gives an idea of the sialic acid distribution at the podocyte plasma membrane. The availability of this method made it possible to reevaluate the results obtained earlier in aminonucleoside (PAN) nephrosis indicating a decrease in the sialic acid content of the glomerulus. Although there are changes in the epithelial architecture, the ultrastructural appearances of the basement membrane are only slightly altered in PAN nephrosis. Detachment of epithelial cells was variable in different animals. Seven days after the first injection of PAN, staining with PTA revealed local defects in the lamina rara externa which later became more extensive. In PAN-treated animals the luminal cell coat showed reduced staining and large areas of the plasma membrane were completely devoid of a cell coat. These changes coincided with the onset of heavy proteinuria. The results indicate that both the basement membrane and the epithelial plasma membrane are affected in PAN nephrosis, as revealed by decreased staining for sialic acid-containing molecules in the basement membrane and by changes in the epithelial cell coat. The defects in the cell coat material point to functional alterations at the level of the slit pores and it is suggested that the decrease in sialic acid content of the lamina rara externa may be partly responsible for defects in the size-selective filtration barrier in PAN nephrosis.

Studies on kidney sialidase in normal and diabetic rats

Rat kidney cortex sialidase was studied using α- sialyl-(2→3)-[ 3 H] lactitol and α- sialyl-(2→6)-[ 3 H] lactitol as substrates. The enzyme was found mainly in the lysosomal fraction. Only 23% of the sialidase activity of this fraction could be solubilized by a combination of freezing-thawing, sonication and Triton X-100 treatment. The optimal pH for the lysosomal enzyme activity was 4.2 and the enzyme's K m values for αsialyl-(2→3)-lactitol and α-sialyl-(2→6)-lactitol were 0.28 and 0.41 mM, respectively. The specific activity was twice as high with the former substrate than with the latter. Sialidase activities in dialyzed kidney cortex homogenates of streptozotocin-diabetic rats and of age-matched control rats were compared. The specific activity was found to be significantly increased in the diabetic animals when using both substrates 5950±720 (S.E.) dpm/h per mg protein ( n=7) vs. 3970±370 in the controls ( n = 8 with α-sialyl-(2-→3)-lactitol ( P <0.025) and 2870±300 vs. 1820±170 with α-sialyl-(2→6)-lactitol ( P<0.02). The activities were also found to be increased when expressed per whole kidney cortex ( P<0.005 and P<0.001, respectively. The elevated sialidase activity in diabetic kidney cortex may be related to the reported decrease in sialic acid content of the glomerular basement membrane, which lowers its negative charges and which may contribute to an increased permeability to proteins.

Topological distribution of different forms of neural cell adhesion molecule in the developing chick visual system.

The cell adhesion molecule isolated from neural tissue (N-CAM) is a membrane glycoprotein which is directly involved in calcium-independent adhesion among nerve cells and their processes (for review see refs 1,2). N-CAM has an unusual carbohydrate moiety containing a large and variable amount of sialic acid, the variation reflecting both the type of tissue and its developmental age. N-CAM is believed to be a ligand in the formation of cell-cell bonds and a decrease in sialic acid content from 30% to 10% is associated with a marked enhancement of the molecule's binding activity. Antibodies to N-CAM block its function and inhibit or alter bundling of nerve fibres, retinal cell development and nerve-muscle interaction. Here we use micro-gel electrophoresis to compare N-CAM from several parts of the developing chick visual system. The results indicate that N-CAM from the retina of 5-10-day-old embryos already exists in a relatively sialic acid-poor form, whereas the tectum and optic nerve beyond the eye contain sialic acid-rich N-CAM until much later in development. These studies suggest that the perikaryon and proximal axon shaft of retinoganglion cells have N-CAM with a lower sialic acid content than the distal portion of the axons, and that resulting differences in neurite adhesivity may be an important factor in the formation of the optic system.

Effect of hydrocortisone on the desialylation of intestinal brush-border enzymes of the rat during postnatal development

Hydrocortisone acetate or hemisuccinate (75 mg kg body mass) applied to rats i.m. and/or s.c. on the 9th and 10th postnatal days causes a precocious decrease of sialic acid content of the small intestinal brush-border membrane. On the 15th postnatal day the bound sialic acid of the whole membrane fraction drops to almost half of the values of control animals and to one third of the control values for the papainsolubilized membrane proteins. The hydrocortisone effect is manifested on isoelectric focusing zymograms by a faster increase of p I of the solubilized brush-border enzymes on the 12th and 15th postnatal days.

Synthesis of sialoglycoconjugates during dimethylsufoxide-induced erythrodifferentiation of friend leukemia cells

We have previously shown that erythroid differentiation of Friend murine leukemia cells by dimethylsulfoxide results in a decrease in sialic acid content and net negative surface charge. The mechanism responsible for the decrease in sialic acid content was examined by measuring the synthesis of sialic acid from N-acetylmannosamine and its catabolic removal from sialoconjugates during the maturation process. A decrease in the incorporation of N-[ 3 H] acetylmannosamine into sialoglycoconjugates occured as early as 12 h after exposure to dimethyl-sulfoxide. Radioactivity incorporated into sialoglycoconjugates was relatively stable in untreated and dimethylsulfoxide-treated cells, implying that catabolic removal of sialic acid residues was not a factor in the decreased surface sialic acid content of differentiated erythroleukemia cells. In addition, no difference existed between control and treated cells in sialyltransferase activity. Significant decreases occurred, however, in the incorporation of radioactivity from N-[ 3 H] acetylmannosamine into N- acetylneuraminic acid, CMP-N- acetylneuraminic acid and a material tentatively identified as N-acetylmannosamine-6-phosphate, 48 h after the addition of dimethylsulfoxide. The decrease in sialic acid biosynthesis in differentiated erythroleukemia cells was reflected by an 83% decrease in the amount of radioactively-labeled sialic acid released by neuraminidase treatment of cells exposed to dimethylsulfoxide. These findings are consistent with a cellular aging phenomenon triggered by the polar solvent-induced differentiation of the leukemic cells into more mature forms.

Effect of modification of synaptosomal membrane glycoproteins on adenylate cyclase activity.

The effect of the modification of synaptosomal membrane glycoproteins on the activity of adenylate cyclase was studied. It was found that the binding of concanavalin A to unmodified guinea pig cerebral cortex synaptosomal membrane did not change adenylate cyclase activity. Concanavalin A binding to synaptosomal membrane of hypoxic brain cortex resulted in no decrease of enzyme activity. The level of protein-bound sialic acid in these synaptosomal fractions was 20% lower than in the control. Treatment of synaptosomal membranes with neuraminidase resulted in a decrease of sialic acid content by about 70%, but it had no significant effect on adenylate cyclase activity. The modification with concanavalin A of sugar end groups exposed by neuraminidase treatment resulted in significant decrease of both basal and fluoride-stimulated adenylate cyclase activity. These results seem to indicate that some component of the adenylate cyclase complex of brain synaptosomal membranes is closely interacting with a carbohydrate-containing macromolecule on the cell surface.

Age-dependent physiochemical and biochemical studies of human red cell membranes

Erythrocyte ghosts of male persons of two age groups, younger than thirty and older than seventy years, were analyzed to investigate the influence of age on lipid composition, the physical state of the lipid phase, Na +K +-ATPase activity and sialic acid content. The phospholipid content in red cell membranes of old donors is significantly lower than in young ones. Cholesterol and fatty acid compositions shows no difference between the two donor groups. The membrane fluidity in liposomes prepared from total lipids of old donors decreased. No significant difference in lipid composition and membrane fluidity reflected by the spin labels was observed between blood group A and O. The activity of Na +K +-ATPase of the young donors with blood group A is significantly higher than those of old donors. The results also demonstrate a decrease of sialic acid content of red cells of old donors.