Milk fat globule membrane isolated from buttermilk or whey cream and their lipid components inhibit infectivity of rotavirus in vitro.

Milk fat is encapsulated in a milk fat globule membrane (MFGM) that contains bioactive glycoproteins and glycolipids. The MFGM inhibits infectivity of rotavirus (RV), activity that has been attributed to its glycoprotein and carbohydrate components. However, previous studies of proteins and oligosaccharides in the MGFM have not accounted for all the bioactivity associated with the complete MFGM. The lipid fraction of the MFGM accounts for half of its composition by weight, and we postulate that this fraction should be tested by itself to determine if it plays a role in antiviral activity. Herein, the anti-RV activity of an organic extract of MFGM was tested. Natural and whey buttermilk powders containing bovine MFGM enriched in polar lipids were prepared by microfiltration and supercritical fluid extraction treatment to reduce the triglyceride content of the powders. Lipid fractions were then extracted from the MFGM using both single- and dual-phase extraction methods. Whole MFGM and organic extracts were screened in MA-104 cells for anti-infective activity against a neuraminidase-sensitive rotavirus using a focus-forming unit assay. Dose-dependent inhibition was observed for whole buttermilk and cheese whey MFGM against the rotavirus. In general, buttermilk MFGM exhibited greater RV percentage inhibition than cheese whey MFGM. Organic-soluble anti-RV compounds were identified in bovine MFGM. The most active fraction, isolated by dual-phase extraction and iatrobead chromatography, was free of proteins and highly nonpolar. Further separation of this fraction in a less polar solvent (30:1 chloroform:methanol) resolved at least 5 lipid-containing compounds, which likely contribute to the anti-RV activity associated with bovine MFGM. In summary, lipid components associated with MFGM appear to contribute in large part to the anti-RV activity associated with the bovine MFGM.

Effect of corn distillers dried grains with solubles and Eimeria acervulina infection on growth performance and the intestinal microbiota of young chicks.

Chicks were used to determine whether dietary corn distillers dried grains with solubles (DDGS) may prevent or ameliorate Eimeria acervulina (EA) infection. The experiment had a completely randomized design with a factorial arrangement of 3 diets (inclusion of 0, 10, or 20% DDGS) × 2 challenge treatments: inoculation with distilled water or with 10(6) sporulated EA oocysts. Each treatment was replicated with 8 pens of 5 chicks each. Experimental diets were fed from 7 to 21 d of age. Inoculation occurred on d 10 of age, considered postinoculation (PI) d 0. Feed intake and BW were measured on PI d 0, 7, and 14. Excreta samples were collected on PI d 0, 5 to 10, 12, and 14 to detect oocysts. On PI d 14, mucosal samples were collected for the analysis of bacterial populations by denaturing gradient gel electrophoresis, using the V3 region of bacterial 16S ribosome. The EA challenge reduced (P < 0.001) ADG by 17%, ADFI by 12%, and G:F by 6% from PI d 0 to 7, and by smaller percentages from PI d 7 to 14. Diet and challenge treatments did not interact in the chick performance, so dietary DDGS did not alleviate EA infection. Oocysts in excreta were detected PI only in EA chicks and no dietary effects were found. Cecal bacterial population was changed (P < 0.05) by effect of dietary DDGS and EA infection. The cecal bacterial diversity among chicks within treatments and homogeneity among chicks within treatments were reduced by EA infection (P = 0.02 to 0.001) and increased by feeding 10% DDGS (diet quadratic, P < 0.001). In summary, feeding up to 20% DDGS to young chicks did not prevent or ameliorate EA infection. Changes in cecal microbiota of chicks fed 10% DDGS can be interpreted as beneficial for intestinal health.

Cytidine-5'-monophosphate-N-acetylneuraminic acid. Asialoglycoprotein sialic acid transferase activity in liver and serum of patients with juvenile hepatic cirrhosis and alpha-1-antitrypsin deficiency.

The molecular basis for the accumulation of a substance which displays the immunological reactivity of alpha-1-antitrypsin within vesicles of liver parenchymal cells of individuals with hepatic cirrhosis and serum alpha-1-antitrypsin deficiency remains unclear. We recently reported that serum from a patient with alpha-1-antitrypsin deficiency and hepatic cirrhosis was substantially deficient in sialyltransferease (EC 2.4.99.1) an enzyme which transfers sialic acid from cytidine 5'-monophosphate-N-acetylneuraminic acid to a variety of asialoglycoprotein acceptors. In the present report we have extended these studies to include serum from five additional patients with alpha-1-antitrypsin deficiency and juvenile hepatic cirrhosis as well as a liver specimen obtained at autopsy of one of these patients. We find the sialytransferase activity in serum from six patients with alpha-1-antitrypsin deficiency and hepatic cirrhosis to be 50% of healthy pediatric control values and 30% of pediatric patients with liver disease. However, serum from family members homozygous for alpha-1-antitrypsin deficiency but without hepatic cirrhosis, and serum from patients with a variety of other kinds of liver disease, failed to exhibit the marked sialytransferase deficiency. Similar assays carried out on a homogenate of a liver sample from one patient with alpha-1-antitrypsin deficiency and hepatic cirrhosis indicated that the deficiency of sialyltransferase activity was not demonstrable in liver. Furthermore, a comparative kinetic analysis of serum and liver sialytransferase in normal and afflicted individuals failed to detect differences in substrate affinities which might account for a decrease in functional sialyltransferase capacity in individuals with alpha-1-antitrypsin deficiency and hepatic cirrhosis. These observations suggest that the serum sialyltransferase deficiency in such patients probably arises after chronic and extensive liver disease involving hepatic accumulation of alpha-1-antitrypsin rather than the enzyme deficiency being the primary cause of the hepatic cirrhosis and alpha-1-antitrypsin deficiency.

Uptake and subcellular localization of tritiated dihydro-microcystin-LR in rat liver.

Microcystin-LR (MC-LR), a cyclic heptapeptide hepatotoxin (mol. wt = 994) produced by the blue-green alga (cyanobacterium), Microcystis aeruginosa, was reduced with tritium labeled sodium borohydride, converted to [3H]-dihydro-microcystin-LR ( [3H]-2HMC-LR), and purified to greater than 99% purity by C-18 reverse-phase high-performance liquid chromatography. The uptake and subcellular distribution of [3H]-2HMC-LR were determined in suspensions of hepatocytes at 0 degrees C and 37 degrees C, or following rifampicin pretreatment, and in perfused rat liver. The remaining cells were homogenized and subfractionated using sucrose gradient centrifugation. Suspensions of 7.5 x 10(6) hepatocytes also were incubated with 10 micrograms/ml of toxin, solubilized in Triton X-100, and ultracentrifuged to pellet the detergent insoluble fraction (containing actin). Isolated rat livers were perfused with media containing [3H]-2HMC-LR and the uptake of radiolabel was determined. Sequential biopsy samples were collected for histologic examination. The remaining liver was homogenized and subcellular fractions prepared. Uptake of radiolabel was rapid in both cell suspension at 37 degrees C and perfused liver; however, uptake in cell suspensions was reduced by about 50% at 0 degrees C and by rifampicin (50 micrograms/ml) pretreatment. Hepatocyte necrosis was observed in isolated perfused livers 45 min after initiation of perfusion with [3H]-2HMC-LR. In both hepatocyte suspensions and perfused livers 65 to 77% of the radiolabel was in the cytosolic fraction. In the hepatocyte suspensions, 13 to 18% of the radiolabel was present in the plasma membrane/nuclear fraction with lesser amounts in the other fractions. Trichloroacetic acid treatment of cytosolic fractions indicated that in hepatocyte suspensions, 50-60% of the radiolabel was bound to cytosolic protein. Studies using the perfused liver confirmed that the majority of the radiolabeled MCLR (78-88%) was bound to cytosolic protein. These data suggest that the uptake of [3H]-2HMC-LR occurs primarily by an energy-dependent transport process involving the rifampicin-sensitive hepatic bile acid carrier and that once inside the hepatocyte, the toxin binds to a cytosolic protein(s).

Application of intestinal xenografts to the study of enteropathogenic infectious disease.

We have developed, characterized and utilized paired segments of fetal intestine subcutaneously transplanted into heterogenic nude or SCID mice as a model system for the study of viral, bacterial and protozoal pathogens. The xenografted intestine matures in the recipient mouse and is biochemically and anatomically comparable to intestine from age-matched, whole-animal controls. The grafted tissue is free of ingesta, intestinal flora, extra-intestinal secretions and host immune functions. The transplanted intestine is long-lived and easily accessible to manipulation and harvest. Tissue from a single fetal donor can be used to create numerous xenografts allowing for tightly controlled experiments. Xenografts enable the study of species-specific intestinal pathogens in the homologous intestinal tissue thus preserving biological applicability of results. Xenografts can be used to study pathogenesis, pathophysiology and therapeutics of enteric disease in situations where such study might otherwise be prohibitively expensive or confounded by intercurrent variables inherent to whole animals. Xenografts have important advantages over in vitro models that may not approximate the in vivo biology of the intestine in the disease process.

Characterization of a porcine enterocyte receptor for group A rotavirus.

We have identified, purified to apparent homogeneity and chemically characterized a biologically-relevant porcine enterocyte receptor for group A porcine rotavirus. Ceramide glycanase digestion followed by acid hydrolysis and monosaccharide compositional analyses indicated the receptor is a family of two GM, gangliosides, one containing N-glycolyl-neuraminic acid and the other N-acetylneuraminic acid. Both gangliosides displayed dose-dependent inhibition of rotavirus binding to, and infectivity of, host cells. Inhibition of infectivity in a focus-forming-unit-reduction assay was achieved with as little as 2 nmols of NeuGcGM3 (50% inhibition with 3.97 nmol) or NeuAcGM3 (50% inhibition with 9.84 nmol) per 10(4) FFU of virus. Preliminary data suggest specific porcine GM3 carbohydrate fine structure or spatial orientation of the sialyloligosaccharide epitopes of the holoGM3 gangliosides may be crucial to enterocyte receptor recognition by rotavirus. We have quantified both NeuGcGM3 and NeuAcGM3 in enterocytes of various-aged pigs from newborn through 16 weeks and have found with increasing age the amount of both GM3 derivatives, especially NeuGcGM3 per gram (dry weight) intestinal brush border decreases rapidly from newborn through 4 weeks of age. These results may help explain the age-sensitivity of piglets to severe rotavirus diarrhea.

Sialic acid dependence and independence of group A rotaviruses.

We have found (1), in contrast to previous reports, the human rotavirus Wa strain is sialic acid-dependent for binding to and infectivity of MA-104 cells and (2), a dual carbohydrate binding specificity is associated with both human Wa and Porcine OSU rotaviruses. One carbohydrate binding activity is associated with triple-layered virus particles (TLP) and the other with double-layered virus particles (DLP). In binding and infectivity studies, we found that gangliosides were the most potent inhibitors of both the human and procine rotavirus TLP. Furthermore, glycosylation mutant cells deficient in sialylation or neuraminidase-treated MA104 cells, did not bind rotavirus TLP from either strain. Our results show that human Wa binding and infectivity cannot be distinguished from the porcine OSU strain and appears to be sialic acid-dependent. Direct binding of human or porcine TLP to a variety of intact gangliosides was demonstrated in an thin-layer chromatographic (TLC) overlay assay. Human or porcine rotavirus DLP did not bind to any of the intact gangliosides but surprisingly bound asialogangliosides. This binding was abolished by prior treatment of the glycolipids with ceramide glycanase suggesting the intact asialoglycolipid was required for DLP binding. After treatment of either human or porcine TLP with EDTA to remove the outer shell, virus particles bound only to the immobilized asialogangliosides. These results suggest that rotavirus sugar binding specificity can be interpreted either as sialic acid-dependent or independent based on whether the virus preparation consists primarily of triple-layered or double-layered particles. Of perhaps greater interest is the possibility that sialic acid-independent carbohydrate binding activity plays a role in virus maturation or assembly.

Identification of antigens of two isolates of Anaplasma marginale, using a western blot technique.

Antigens of the Illinois (IAM) and Florida (FAM) isolates of Anaplasma marginale were analyzed, using the western blot technique and antiserum from A marginale-infected calves. Crude antigens were prepared from the parasitemic blood of each. Antiserum was collected after the primary and recrudescent parasitemias. Antigens were separated, using sodium dodecyl sulfate polyacrylamide gel electrophoresis. Antigens were then transferred onto nitrocellulose membranes and exposed to test sera. Antibodies attached to the membrane-bound antigens were detected, using an avidin/biotin peroxidase assay and biotinylated rabbit anti-goat immunoglobulin G. Antigens detected were of a high molecular weight group (108 to 91 kilodaltons [kd]) or of a low molecular weight group (47 to 27 kd). The IAM antigens were 100 kd, 96 kd, 47 kd, 38 to 43 kd, and 27 kd; these antigens were detected, using anti-IAM and anti-FAM antibodies, but the anti-FAM antibodies had a strong reaction to only the 100-kd and 38- to 43-kd antigens of IAM. The FAM antigens were 108 kd, 91 kd, 47 kd, 38 to 43 kd, and 27 kd; these antigens were detected, using anti-FAM antibodies and, except the 91 kd antigen, anti-IAM antibodies. Because the 91-kd antigen was detected only in the FAM antigen and detected only by sera from FAM-infected calves, this isolate-specific antigen may be associated with the ability of FAM to induce disease in an IAM-immune animal. Sheep anti-A ovis antibodies reacted only to the 38- to 43-kd antigens of each isolate, indicating that these antigens may be genus-specific.(ABSTRACT TRUNCATED AT 250 WORDS)

Hepatic total 3 alpha-hydroxy bile acids concentration and enzyme activities in prednisone-treated dogs.

High serum alkaline phosphatase (ALP) activity is considered a sensitive marker of cholestasis in most mammalian species, including dogs. Induction of high serum ALP activity in association with cholestasis is dependent on high hepatic bile acids concentrations. Treatment of dogs with glucocorticoids also results in high serum ALP activity. The possible causal relation between serum ALP activity and bile acids concentration was investigated in dogs treated with glucocorticoids. The relation of glucocorticoid treatment to changes in the activity of individual ALP isoenzymes, alanine transaminase (ALT) and gamma-glutamyltransferase (GGT) also was investigated. Eight conditioned dogs were given 4 mg of prednisone/kg of body weight, i.m., daily for 10 days. Blood samples were taken prior to treatment and on treatment days 3, 5, 7, and 10. Liver tissue was then taken from each dog. Serum total ALP activity was significantly (P < 0.05) high at day 3 in prednisone-treated dogs. Isoenzyme analysis indicated that this increase was attributable to an increase in the liver ALP isoenzyme (LALP). Significant increases in serum corticosteroid-induced ALP (CALP) and bone ALP were first observed on days 7 and 10, respectively. Serum ALT and GGT activities were significantly increased by day 5. Increased serum or hepatic tissue bile acids concentrations were not observed in prednisone-treated dogs, compared with values in 8 clinically normal (control) dogs, but were high in 3 dogs with complete bile duct ligation. Hepatic activities of LALP, CALP, and GGT were higher in prednisone-treated dogs than values in controls, indicating probable increased hepatic synthesis of these enzymes. Hepatic ALT activity was not increased.(ABSTRACT TRUNCATED AT 250 WORDS)

Purification and comparison of corticosteroid-induced and intestinal isoenzymes of alkaline phosphatase in dogs.

Corticosteroid-induced alkaline phosphatase (CALP) and intestinal alkaline phosphatase (IALP) from dogs were purified to homogeneity, as determined by polyacrylamide gel electrophoresis. Purification involved an un-interrupted system using DEAE-cellulose, concanavalin A-agarose, and monoclonal antibody affinity columns. The monoclonal antibody was prepared by use of IALP as the antigen. The 2 isoenzymes were compared, using molecular weight determinations, amino acid analyses, peptide mapping, N-terminal sequencing of the first 10 amino acids, carbohydrate analyses, and recognition by anti-IALP monoclonal antibody. The data indicated that canine IALP and CALP are identical with regard to recognition by monoclonal antibody and N-terminal amino acid sequence, nearly identical in amino acid content and peptide maps, but different in carbohydrate content. It was concluded that CALP is a product of the same gene as IALP and that differences in glycosyl transferase activities between liver and intestines or the presence of glycosidase activities in or around the intestinal mucosae result in the marked difference in carbohydrate content.

Isolate of Anaplasma marginale not transmitted by ticks.

The tick-borne transmissibility of 2 isolates of Anaplasma marginale was compared. Dermacentor variabilis were exposed to A marginale as nymphs by feeding on 1 of 4 splenectomized calves during periods of ascending parasitemia (maximum 49% to 81% parasitized erythrocytes) induced by injection of a stabilate. Tick-borne transmission was attempted, using 26 to 224 adult ticks within 30 to 220 days after molting. Adult D variabilis did not transmit an Illinois isolate of A marginale in 7 tick-borne transmission experiments (P = 0.0047), including 2 experiments in which calves were inoculated IV with homogenates of adult ticks. In contrast, a Virginia isolate of A marginale was readily transmitted by the same tick colony. Thus, previously reported morphologic and immunologic differences among A marginale isolates may extend to tick-borne transmissibility. The Virginia and Illinois A marginale isolates had an inclusion appendage that was not a marker for tick transmissibility.

Demonstration of acid phosphatase in Eimeria spp.: partial characterization of the enzyme in E. vermiformis.

Sporozoite extracts of E. vermiformis, E. stiedai, and E. tenella are rich in acid phosphatase activity. They contain specific enzyme activities equal to or greater than those reported for other highly virulent protozoan parasites. The absolute amount of enzyme activity per oocyst dramatically increases during sporulation of E. stiedai and E. vermiformis. Partial characterization of the acid phosphatase activity of E. vermiformis indicates that sporozoites account for greater than 92% of the total activity in sporulated oocysts, that the enzyme is resistant to inhibition by tartrate, and that it can be separated into two forms by anion exchange chromatography.

Improved method for high-yield excystation and purification of infective sporozoites of Eimeria spp.

This report describes a new, gentle procedure for rapid and efficient excystation of large numbers of infective sporozoites of Eimeria vermiformis and Eimeria stiedai. Excysted sporozoites are purified using modifications of a previously described ion-exchange chromatography method. The procedure avoids physical breakage of oocysts and results in greater than 70% recovery of the sporozoites present as sporulated oocysts (i.e. 5-6 sporozoites per sporulated oocyst). The recovered sporozoites are greater than 95% pure and are infective in vivo. We routinely isolate greater than 2 x 10(8) sporozoites without the use of specialized or expensive equipment.

Adhesive specificity of juvenile rat and chicken liver cells and membranes.

Liver cells, isolated from either juvenile rats or chickens by a collagenase perfusion technique, reaggregated when maintained in suspension. The cells exhibited marked adhesive specificity; when suspensions contained both cell types, the aggregates consisted primarily of either rat or chicken cells. Adhesive specificity was also observed with plasma membrane fractions isolated from rat liver homogenates, and with comparable fractions from chicken liver. These membranes stimulated aggregation of the homologous but not the heterologous cell type. Other membrane fractions had little or no effect on the aggregation of the homologous cell type. These and other properties of the liver cell and membrane preparations suggest that biochemical studies on cell-cell recognition and adhesion can most effectively be conducted with cells from juvenile and adult animals.

Assay for evaluation of rotavirus-cell interactions: identification of an enterocyte ganglioside fraction that mediates group A porcine rotavirus recognition.

A virus-host cell-binding assay was developed and used to investigate specific binding between group A porcine rotavirus and MA-104 cells or porcine enterocytes. A variety of glycoconjugates and cellular components were screened for their ability to block rotavirus binding to cells. During these experiments a crude ganglioside mixture was observed to specifically block rotavirus binding. On the basis of these results, enterocytes were harvested from susceptible piglets and a polar lipid fraction was isolated by solvent extraction and partitioning. Throughout subsequent purification of this fraction by Sephadex partition, ion-exchange, silicic acid, and thin-layer chromatography, blocking activity behaved as a monosialoganglioside (GMX) that displayed a thin-layer chromatographic mobility between those of GM2 and GM3. The blocking activity of GMX was inhibited by treatment with neuraminidase and ceramide glycanase but not by treatment with protease or heat (100 degrees C). Further purification of GMX by high-pressure liquid chromatography resulted in the resolution of two monosialogangliosides, GMX and a band which comigrated with GM1 on thin-layer chromatography. These data suggest that a cell surface monosialoganglioside or family of monosialogangliosides may function as an in vivo relevant receptor for group A porcine rotavirus and that sialic acid is a required epitope for virus-binding activity.

Glucocorticoid hepatopathy: effect on receptor-mediated endocytosis of asialoglycoproteins.

Histologic and electron microscopic examination of liver tissue from glucocorticoid-treated dogs (GT dogs) showed a markedly abnormal hepatocellular morphology which consisted of severe hepatocellular swelling, vacuolation, and peripheral displacement of subcellular organelles. The abnormal cell morphology was typical of that seen in clinical cases of canine Cushing's Syndrome. The hepatocyte isolation procedure used here works equally well for the preparation of viable hepatocytes from both normal and GT dogs even though GT dogs displayed a pronounced hepatopathy. Cell yields (10(9) cells from a 30-cm3 section of liver) are similar to those reported for rat hepatocytes using whole liver in situ perfusion and cell viability is routinely greater than 85%. The isolation procedure preserved the "abnormal" state or swollen morphology of the hepatocytes from GT dogs and thus can be used in pathophysiological studies of glucocorticoid-induced hepatopathy. The isolated hepatocytes were 3.2 times greater in cell volume than normal hepatocytes. We also observed over a 12.3-fold increase in alkaline phosphatase activity and the appearance in both the liver and the serum of GT dogs of the unique, corticosteroid alkaline phosphatase isozyme (CALP). In spite of the obvious abnormal liver morphology and elevated serum and liver alkaline phosphatase activities, the function of the hepatic cell surface carbohydrate binding protein, the Gal/GalNAc or asialoglycoprotein receptor, was not impaired. We found a trend of about a 1.5-fold increase in the initial rate of ligand uptake as well as 1.6-fold more receptors on GT dog hepatocytes compared to normal hepatocytes. The ligand binding affinity of these receptors, as well as the rate of ligand degradation, was identical in hepatocytes isolated from normal and diseased dogs. When intestinal alkaline phosphatase (IALP) is used as the ligand, approximately 25% was exocytosed intact following endocytosis. These results demonstrate that dogs with glucocorticoid hepatopathy possess a normally functioning Gal/GalNAc receptor. Furthermore, these data are consistent with the hypothesis that structurally related IALP and CALP isozymes may also be metabolically related through the Gal/GalNAc receptor endocytosis pathway. That is, a portion of the IALP normally endocytosed through the Gal/GalNAc receptor pathway in glucocorticoid-treated dogs may be recycled and converted (hyperglycosylated) to the abnormal serum CALP isozyme rather than being degraded.

Development, characterization, and utilization of an intestinal xenograft model for infectious disease research.

Paired xenografts of near-term fetal rabbit jejunum were subcutaneously implanted in the backs of athymic nude (nu/nu) mice. At 3 to 4 weeks post-implantation, the grafts had histologic, ultrastructural, and biochemical (lactase, sucrase, alkaline phosphatase, leucine aminopeptidase) parameters comparable to age-matched control rabbits. Four weeks post-transplantation the xenografts were intraluminally inoculated with various strains of lapine attaching and effacing E. coli or group A rotavirus. Infection with 2 strains of E. coli resulted in typical light microscopic and ultrastructural lesions of attachment and effacement. Immunohistochemical analysis of rotavirus-infected xenografts demonstrated rotavirus antigen within enterocytes. These lesions are comparable to those in conventional rabbits. Intestinal xenografts are a novel, highly controlled, and reproducible model which may have unique applications in the study of enteric diseases. The model provides anatomically and biochemically correct intestinal mucosal epithelium uncomplicated by variables such as enteric flora, host immune response, gastric, hepatic, and pancreatic secretions and is susceptible to infection by specific enteropathogens. Xenografts, therefore, may be a viable alternative in certain investigations where whole animals, ligated intestinal loops, organ cultures, or cell cultures might otherwise be chosen.

Influences of structural factors of biological media on the acoustic nonlinearity parameter B/A.

The influence of structural factors of biological media on the acoustic nonlinearity parameter B/A have been studied at the tissue, cellular, and molecular levels, using the thermodynamic and finite amplitude methods. B/A was determined as the structural factors of the media were altered physically and biochemically, while chemical composition was maintained unchanged. Significant structural dependencies of B/A were observed at all three levels; 26% of the dry weight contribution to the total B/A (the B/A value with water contribution subtracted) is due to the cell-cell adhesive force in liver tissue, 20% is due to the hepatocyte cellular structure, and 15% is due to secondary and tertiary protein structure.

Binding and endocytosis of glycoproteins by isolated chicken hepatocytes.

The binding and endocytosis of glycoproteins containing different terminal sugars by isolated chicken hepatocytes were studied. At 2 degrees C, where there is no endocytosis, the hepatocyte surface bound 30 800 GlcNAc44-AI-BSA molecules [a bovine serum albumin (BSA) derivative which contains 44 residues of N-octylglucosamine (GlcNAc)] [Lee, Y.C., Stowell, C.P., & Krantz, M.J. (1976) Biochemistry 15, 3956-3963] and 32 900 asialoagalactoorosomucoid (AGOR) molecules per cell with estimated dissociation constants of 5 X 10(-10) and 4 X 10(-9) M, respectively. In the presence of digitonin or Triton X-100, each hepatocyte bound 7-18 times more ligand than in the absence of these detergents. Bound 125I-AGOR could be dissociated from the cell surface by 5.5 X 10(-5) M GlcNAc44-AI-BSA with a t 1/2 of 30 min, while GlcNAc (10 mM) or ethylene glycol bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid (4 mM) could dissociate over 98% of the surface-bound radioactivity within 10 min. Several neoglycoproteins inhibited the binding of 125I-AGOR, requiring for 50% inhibition 2.1 X 10(-9), 4.0 X 10(-7), 1.6 X 10(-6), and 2 X 10(-6) M for GlcNAc44-, Glc37-, Man43-, and L-Fuc28-AI-BSA, respectively. The bound AGOR and neoglycoproteins were internalized and degraded at 37 degrees C. [125I]Iodide was the only labeled degradation product found. When the hepatocytes were exposed to 250 nM AGOR at 37 degrees C, ca. 100 000 molecules of AGOR were associated with the cell surface at the steady state of endocytosis.(ABSTRACT TRUNCATED AT 250 WORDS)

Adhesion of chicken hepatocytes to polyacrylamide gels derivatized with N-acetylglucosamine.

Complex carbohydrates on the surfaces of eukaryotic cells are thought to participate in a wide variety of cell-cell interactions. A model system has therefore been developed to study these processes. In the present experiments, the ability of chicken hepatocytes to recognize and adhere to sugars covalently linked to polyacrylamide gels was investigated. The gels were snythesized by two methods. Type I gels were prepared from a co-polymer of an active ester of acrylic acid (N-succinimidyl acrylate), acrylamide, and bisacrylamide. The "activated" polyacrylamide gel was then treated with the desired ligand containing an amino group, such as 6-aminohexyl O- or S-glycoside. Type II gels were formed by treating similar ligands with acryloyl chloride, followed by co-polymerization of the resulting N-substituted acrylamide with acrylamide and N,N'-methylenebisacrylamide. These polyacrylamide derivatives offer many advantages for studies with intact cells. They are not toxic to any cell type studied, can be cast in any desired shape, are transparent and stable over a wide range of pH values, and contain no cationic and low to negligible levels of anionic charge (charged groups can be introduced if desired), and the polyacrylamide matrix is stable to common biological agents such as bacteria and enzymes. In addition, type I gels can be synthesized using a broad range of molecules containing amino groups, such as glycopeptides, proteins, etc. The hepatocytes were prepared by collagenase perfusion of intact chicken livers. The rate and extent of adhesion of the cells to the derivatized gels was determined by measuring lactate dehydrogenase in these cells. This enzyme was also used to assay viability and cell "leakiness." At 37 degrees C, 70 to 100% of the cells adhered within 60 min to gels derivatized with N-acetylglucosamine, i.e. gels derivatized with 6-aminohexyl 2-acetamido-2-deoxy-beta-D-glucopyranoside (or the corresponding thioglycoside). By contrast, less than 5% of the cells adhered to polyacrylamide or to gels derivatized with 6-aminohexanol or the 6-aminohexyl glycosides of beta-D-glucose, beta-D-galactose, alpha-D-mannose, beta-D-maltose, beta-D-melibiose, beta-D-cellobiose, and (alpha or beta)-D-lactose. Kinetic studies with the chicken hepatocytes and N-acetylglucosamine gels showed that cell-gel binding was dependent upon Ca2+ and was decreased at low temperatures. Binding was inhibited by N-acetylglucosamine or by glycosides of this sugar, the most effective inhibitor being orosomucoid (alpha1-acid glycoprotein) pretreated with sialidase and beta-galactosidase. The cell surface receptor(s) involved in this interaction is not known, but may be related or identical to the chicken liver binding protein described by Lunney and Ashwell (Lunney, J., and Ashwell, G. (1976) Proc. Natl. Acad. Sci. U. S. A. 73, 341--343). The present results suggest that this model system should prove useful in delineating cell surface interactions with carbohydrates.