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.

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.

Structure and function of a ganglioside receptor for porcine rotavirus.

A ganglioside fraction isolated from pooled intestines from newborn to 4-week-old piglets, which we previously partially characterized and showed to specifically inhibit the binding of porcine rotavirus (OSU strain) to host cells (M. D. Rolsma, H. B. Gelberg, and M. S. Kuhlenschmidt, J. Virol. 68:258-268, 1994), was further purified and found to contain two major monosialogangliosides. Each ganglioside was purified to apparent homogeneity, and their carbohydrate structure was examined by high-pH anion-exchange chromatography coupled with pulsed amperometric detection and fast atom bombardment mass spectroscopy. Both gangliosides possessed a sialyllactose oligosaccharide moiety characteristic of GM3 gangliosides. Compositional analyses indicated that each ganglioside was composed of sialic acid, galactose, glucose, and sphingosine in approximately a 1:1:1:1 molar ratio. Each ganglioside differed, however, in the type of sialic acid residue it contained. An N-glycolylneuraminic acid (NeuGc) moiety was found in the more polar porcine GM3, whereas the less polar GM3 species contained N-acetylneuraminic acid (NeuAc). Both NeuGcGM3 and NeuAcGM3 displayed dose-dependent inhibition of virus binding to host cells. NeuGcGM3 was approximately two to three times more effective than NeuAcGM3 in blocking virus binding. Inhibition of binding occurred with as little as 400 pmol of NeuGcGM3/50 ng of virus (approximately 2 x 10(7) virions) and 2 x 10(6) cells/ml. Fifty percent inhibition of binding was achieved with 0.64 and 1.5 microM NeuGcGM3 and NeuAcGM3, respectively. The free oligosaccharides 3'- and 6'-sialyllactose inhibited binding 50% at millimolar concentrations, which were nearly 1,000 times the concentration of intact gangliosides required for the same degree of inhibition. Direct binding of infectious, triple-layer rotavirus particles, but not noninfectious, double-layered rotavirus particles, to NeuGcGM3 and NeuAcGM3 was demonstrated by using a thin-layer chromatographic overlay assay. NeuGcGM3 and NeuAcGM3 inhibited virus infectivity of MA-104 cells by 50% at concentrations of 3.97 and 9. 84 microM, respectively. NeuGcGM3 (700 nmol/g [dry weight] of intestine) was found to be the predominant enterocyte ganglioside (comprising 75% of the total lipid-bound sialic acid) in neonatal piglets, followed by NeuAcGM3 (200 nmol/g [dry weight] of intestine). NeuGcGM3 and NeuAcGM3 together comprised nearly 100% of the lipid-bound sialic acid in the neonatal intestine, but their quantities rapidly diminished during the first 5 weeks of life. These data support the hypothesis that porcine NeuGcGM3 and NeuAcGM3 are physiologically relevant receptors for porcine rotavirus (OSU strain). Further support for this hypothesis was obtained from virus binding studies using mutant or neuraminidase-treated cell lines. Lec-2 cells, a mutant clone of CHO cells characterized by a 90% reduction in sialyllation of its glycoconjugates, bound less than 5% of the virus compared to control cell binding. In contrast, Lec-1 cells, a mutant CHO clone characterized by a deficiency in glycosylation of N-linked oligosaccharides, still bound rotavirus. Furthermore, exogenous addition of NeuGcGM3 to the Lec-2 mutant cells restored their ability to bind rotavirus in amounts equivalent to that of their parent (CHO) cell line. In the virus-permissive MA-104 cell line, NeuGcGM3 was also able to partially restore rotavirus infectivity in neuraminidase-treated cells. These data suggest that gangliosides play a major role in recognition of host cells by porcine rotavirus (OSU strain).

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.

Characterization and sequencing of a respiratory burst-inhibiting acid phosphatase from Francisella tularensis.

Acid phosphatases (Acp) of intracellular pathogens have recently been implicated as virulence factors that enhance intracellular survival through suppression of the respiratory burst. We describe here the identification, purification, characterization, and sequencing of a novel burst-inhibiting acid phosphatase from the facultative intracellular bacterium, Francisella tularensis. Similar to other the burst-inhibiting Acps, F. tularensis Acp (AcpA) is tartrate-resistant and has broad substrate specificity. The AcpA enzyme is unique, however, in that it is easily released from the bacterial cell in soluble form, is a basic enzyme, suppresses the respiratory burst of not only fMet-Leu-Phe but also phorbol 12-myristate 13-acetate-stimulated neutrophils and does not fit into any of the three currently recognized classes of acid phosphatase. We also report the complete nucleotide sequence of the gene acpA, encoding AcpA, and the deduced primary structure of its encoded polypeptide. Comparative sequence analyses of AcpA is discussed. To our knowledge, this is the first report describing the cloning and sequencing of a burst-inhibiting acid phosphatase.

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)

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.

An intestinal xenograft model for Cryptosporidium parvum infection.

Paired segments of near-term fetal rabbit small intestine were transplanted subcutaneously into athymic nude mice. At 5 weeks postsurgery, the xenografts were inoculated intraluminally with Cryptosporidium parvum sporozoites. Parasites rapidly and reliably infected the xenograft mucosal epithelium. Lesions typical of cryptosporidiosis were readily apparent by light microscopy and scanning and transmission electron microscopy. Xenografts are well suited to the study of the early events of C. parvum infection and are of potential value in the evaluation of anticryptosporidial chemotherapeutic agents.

Behavior of the idiotypic network in conventional immune responses. I. Kinetics of idiotypic and anti-idiotypic antibodies following immunization with T-independent and T-dependent antigens.

A minimal requirement in investigations of the behavior of the idiotypic network during immunization is the ability to quantitate both the idiotypic (Ab1) and anti-idiotypic (Ab2) responses. Quantitation of Ab2 in serum is complicated by the simultaneous presence of Ab1, so that Ab1-Ab2 immune complexes escape detection. In contrast, immune complexes should not complicate the enumeration of Ab2-producing lymphocytes in a hemolytic plaque assay. This study utilizes a procedure that allows detection of Ab2-producing cells in such an assay. The procedure relies upon the insertion of the appropriate antibody (Ab1) into the membrane of indicator SRBC through a covalently attached dipalmitoyl phosphatidylethanolamine (DPPE) tail. When the Ab2 response following murine immunization with DNP-Ficoll was analyzed using such an assay, peak plaque-forming cell (PFC) numbers were found to coincide with peak Ab1 PFC numbers in both the primary and secondary response. In addition, this Ab2 response was found to be T independent. The murine immune response to DNP-HGG demonstrated a peak Ab2 PFC response which followed the peak Ab1 PFC response after both primary and secondary immunization. This Ab2 response appeared to be T dependent. The secondary responses to both DNP-Ficoll and DNP-HGG showed increased levels of Ab2 PFC and decreased levels of Ab1 PFC in comparison to the primary responses to the same antigens, suggesting that immunoregulation may occur within these idiotypic networks.

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.

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.

Actin filament alterations in rat hepatocytes induced in vivo and in vitro by microcystin-LR, a hepatotoxin from the blue-green alga, Microcystis aeruginosa.

The morphologic effects of microcystin-LR (MCLR) were examined in vitro and in vivo to identify the specific cell type(s) affected and to characterize the actin filament changes occurring in hepatocytes. Male Sprague Dawley rats were used for all studies. For in vitro studies, hepatic cells were isolated by collagenase perfusion of liver, while parenchymal cells (hepatocytes) and nonparenchymal cells were prepared by pronase digestion and metrimazide gradient centrifugation. Cell suspensions and and primary hepatocyte monolayer cultures were treated with MCLR at doses up to 10 micrograms/ml; cultured hepatocytes were also treated with phalloidin or cytochalasin B at a dose of 10 micrograms/ml; and rats were treated intraperitoneally with MCLR at 180 mg/kg. Cultured hepatocyte preparations and frozen liver sections were stained with rhodamine-labeled phalloidin for filamentous actin. In cell suspensions, MCLR did not affect nonparenchymal cells but caused rapid, progressive, blebbing of the plasma membrane in hepatocytes. In cultured hepatocytes, MCLR caused plasma membrane blebbing as well as marked reorganization of actin microfilaments. These alterations were dose and time dependent. Cultured hepatocytes treated with phalloidin or cytochalasin B also showed extensive plasma membrane blebbing and actin filament alterations; however, actin filament changes were morphologically distinct from those induced by MCLR. In vivo, MCLR-induced hepatocyte actin alterations occurred at the same time as, or slightly preceded, histologic changes that began 30 minutes after dosing. These studies suggest that early MCLR-induced morphologic changes occurring both in vivo and in vitro are due to alterations in hepatocyte actin filaments.

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.

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).

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.

Effect of glucocorticoids on alkaline phosphatase, alanine aminotransferase, and gamma-glutamyltransferase in cultured dog hepatocytes.

Glucocorticoid(GC)-induced hepatopathy in the dog is characterized by abnormal liver morphology and increases in serum alanine aminotransferase (ALT), gamma-glutamyltransferase (GGT), and the liver alkaline phosphatase isoenzyme (LALP) and by the appearance of an unusual isoenzyme of alkaline phosphatase known as the corticosteroid-induced alkaline phosphatase isoenzyme (CALP). It has not been shown whether the increases in serum ALT, GGT, and LALP are as a result of an increase in production of these enzymes or as a result of the GC-induced hepatocellular swelling and possible membrane alterations. Also, it has been assumed that the mechanism of production of CALP is via GC-induced gene derepression and de novo protein synthesis; however, this hypothesis has not been directly tested. Using isolated dog hepatocytes maintained in a confluent monolayer culture in the presence and absence of GC or cyclic AMP, no statistical increase in serum ALT, GGT, or LALP was observed. A combination of GC and cyclic AMP also caused no statistical increase in ALT and GGT; however, we demonstrate that these conditions clearly stimulated the de novo synthesis of LALP. These conditions do not induce the synthesis of CALP as determined by a sensitive immunoassay. The data obtained using this in vitro model suggest that the primary mechanism(s) of the in vivo increase of serum ALT and GGT in GC treated dogs may be other than that of de novo protein synthesis. Likewise, in vitro production of CALP may be a mechanism more complex than the conditions tested in this study.

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.