Immunoelectrophoretic pattern of native mucosal intracellular glycoproteins of hog healthy and drug-intoxicated stomachs and of hog body fluids.

Naturally occurring glycoproteins have been extracted from fundic and antral mucosal tissue of the hog stomach by means of nondegrading techniques. Major and retarded glycoprotein fractions separated by gel filtration were further dissociated from appreciable amounts of noncovalently bound proteins by CsCl density gradient centrifugation. Antisera to glycoprotein fractions of fundic and antral regions of the stomach were prepared in rabbits. The major fractions from both gastric regions have similar molecular mass (approximately 2 x 10(6)), sedimentation coefficient (approximately 31.5 s), and specific viscosity (approximately 1.6). Purified fractions from each region were further separated into two subfractions by affinity chromatography on wheat germ lectin. Glycoprotein subfractions from antrum and fundus differ appreciably in their carbohydrate and amino acids content, share antigenic determinants, but do not cross-react with anti-hog serum protein antisera. Further diversity in native mucin glycoproteins was observed by the use of one-(D) and two-dimensional (2D) immunoelectrophoresis; subfractions that cross-react with specific anti-hog gastric glycoproteins were found to contain three or more components. D-Immunoelectrophoretic analyses demonstrated (1) in vivo degradation of glycoprotein components of the major fundic fraction isolated from mucosal tissue of alcohol/acetyl salicylate-intoxicated hog stomachs and (2) in vitro catabolism of major fundic glycoproteins by corresponding mitochondrial lysosomal (ML) acid hydrolases. Furthermore, 2D-immunoelectrophoretic analyses showed that (1) hog synovial fluid and plasma proteins have similar prosthetic moieties as either reacted with anti-hog serum proteins antisera. Nonetheless, locations, shapes, and staining intensities of the immunoprecipitate lines differed, which is indicative of different structures of the carbohydrate moieties of components of synovial fluid and plasma proteins, and (2) only a minor fraction of hog cerebrospinal fluid cross-reacted with anti-hog serum protein antisera. This is contrary to the generally accepted deduction based on high-resolution 2D-electrophoresis, indicative of different compositional patterns of plasma and cerebrospinal fluids.

Acute-phase plasma protein response to cholera intoxication in healthy and diabetic rats.

The aim of the present study is twofold: to establish the response of hepatic machinery of plasma protein biosynthesis to cholera intoxication, and to examine the same response of alloxan-diabetic hepatocytes with minimal capacity of synthesis of plasma proteins. Direct lesion of hepatic plasma membranes via ip administration of cholera toxin to male rats resulted in a typical acute-phase response (APR) of plasma proteins, which had regressed to levels similar to those of healthy controls approximately at 240 h postintoxication. The d 2 response to a single 0.16 mg/kg body weight dose was typified by a 23% reduction in the level of albumin, but a 6- and 24-fold increase in the levels of fibrinogen and alpha-1-acid glycoproteins, respectively. This response was similar (in direction but not in magnitude) to the acute-phase reaction to a simple subcutaneous administration of carrageenan. The intoxication was accompanied by a massive leakage, into the peritoneal cavity, of plasma fluid, which embraced the complete profile of acute-phase reactants. A three-step mechanism is proposed to account for the observations as follows: (1) There is a rapid formation of a stable complex between subunit B of the toxin and ganglioside GM1 of hepatic plasma membrane. An APR is induced in response to the alteration(s) of hepatic plasma membranes. (2) The release, from the choleragen-membrane complex, of polypeptide A1 and its subsequent penetration of the hepatic membrane result in both activation of adenylate cyclase and increased vascular permeability of hepatic membranes. This leads, in turn, to exudation of components of plasma fluid in the peritoneal cavity of intoxicated rats. An alternate rationale for this exudation is the slow leakage of plasma proteins out of the blood vascular system (possibly through microvesicles) into the peritoneal cavity of cholera intoxicated rats. The spectrum of acute-phase hepatic secretory components was mirrored in the corresponding peritoneal exudate. (3) The increased hepatic membrane flow provides the continued renewal of plasma membrane proteins required for its eventual repair by either endocytosis or sloughing off the toxin-bound membrane segments into the circulatory system, thus producing regression of APR. Livers of diabetic rats, an already established model in terms of APR, responded to ip administration of cholera toxin by increased biosynthesis of the identified plasma proteins and a marked reduction in total free-glucose in serum.(ABSTRACT TRUNCATED AT 400 WORDS)

Partially hepatectomized rats: a model for the study of the effect of toxins on the plasma protein profiles of nascent hepatocytes.

A useful framework is proposed for unifying the synthesis of plasma proteins and their degradation by, or release from, liver cells of intact and partially hepatectomized rats, in which synthesis and release of acute-phase plasma proteins occur in synchrony with the internalization and catabolism of plasma and extracellular proteins. The catabolism of proteins and other hepato-intracellular glycoproteins during sepsis or trauma is essential to provide constituent amino acids and carbohydrates for the synthesis of acute-phase plasma proteins. Increases in the plasma levels of acute-phase response proteins in sham-operated rats reached a maximum between 1 and 2 d after mock surgery, and had returned virtually to control levels within 6 d. By contrast, acute-phase proteins in the plasma of partially hepatectomized rats were decreased by 10-20% of their initial values after 24 h. A maximum acute-phase response on d 7 after the operation was characterized by an increase of 181, 445, and 19% for alpha-1-acid glycoprotein, hepatoglobin, and hemopexin, whereas other acute-phase proteins remained below control levels, for example, by 11, 25, and 38% for albumin, transferrin, and prealbumin, respectively. This delayed response suggests that the nascent liver cells had inherited the capacity of the parent cells to respond to inflammatory signal and had synthesized acute-phase plasma proteins. Accordingly, a time frame for the application of toxin to nascent hepatocytes is suggested. An increased activity (300 +/- 50%) for both bound and free neuraminidase in remnant liver tissue 19 h post partial hepatectomy suggested that hepatic regenerating factor(s) were produced in liver tissue via the hepatic bound and/or free neuraminidase-mediated desialylation of humoral substrates. By contrast, circulating levels of lysosomal enzymes alpha-fucosidase and beta-N-acetyl-D-glucosaminidase were increased marginally after 24 h but had returned nearly to control levels after 7 d, suggesting that lysosomal acid hydrolases do not play a major role in regenerative DNA synthesis, mitosis, or in the synthesis of acute-phase plasma proteins.

On the genesis of gastric haemorrhage.

An hypothesis involving a three step mechanism to account for the initiation of gastric lesions is described. The mechanism necessitates: (a) A drop in the internal energy (ATP) of the mucosal cells of the stomach upon being subjected to stress; either pathological or psychological. (b) Membranes of mucosal "suicidal sacs" containing potent lysosomal acid-hydrolases are rendered fragile and burst, thus releasing hydrolytic acid-hydrolases into the cytoplasm of the mucosal cells as the latter develop an energy deficit under stress. (c) Gastric mucosal cell necrosis, via the degradation of cytoplasmic and mucinous gastric glycoproteins by these lysosomal acid- hydrolases and subjection of the submucosal tissue to the corrosive effects of the luminal fluid containing hydrochloric acid and pepsin, i.e., initiation of gastric haemorrhage. The above mechanism is a general one that describes events associated with the development of gastric lesions regardless of the factor(s) or the agent(s) initiating gastric mucosal haemorrhage.

Study of hepatotoxicity in isolated perfused liver versus cultures of rat hepatocytes.

Isolated perfused liver and cultures of rat hepatocytes were assessed for the quantitative evaluation of hepatotoxicity. Release of de novo biosynthesized plasma proteins and acid hydrolases into perfusion or culture media was taken as an indication of the integrity of hepatocytes in both systems. The activities of six acid hydrolases, alpha-L-fucosidase, alpha-D-galactosidase, beta-D-galactosidase, beta-D-N-acetylgalactosaminidase, beta-D-N-acetylglucosaminidase, and cathepsin D, were assayed in collagenase-segregated hepatocytes and in monolayer cultures of rat liver cells obtained via collagenase perfusion of rat liver. In situ, liver perfusion with collagenase led to a loss of 45 +/- 5% of the total acid hydrolase activity in the mitochondrial-lysosomal pellet of the liver cells with concomitant increase of these enzymes in the cytosol. In monolayer cultures over a period of 30 h, increased activity of cathepsin D, beta-D-galactosidase, and beta-D-N-acetylglucosaminidase in the mitochondrial-lysosomal pellet and the cytosol fraction was evident with concurrent biosynthesis of plasma proteins. The use of radioactive tracing techniques with the isolated perfused liver revealed that the rate of catabolism of intracellular protein was approximately 5 times that of plasma protein synthesis. Both methods described here are suitable for the study of the effects of toxins on the function of hepatocytes.

Inhibition by the mushroom toxins alpha-amanitin and phalloidin of hepatopoietin-induced 3H-thymidine incorporation into rat liver DNA and of plasma protein production in hepatocyte cultures.

In primary cultures of rat hepatocytes the induction of 3H-thymidine uptake into DNA of liver cells by the liver cell proliferation factor hepatopoietin demonstrates that this factor is active not only in vivo but also in vitro. Addition of the mushroom toxins alpha-amanitin or phalloidin to liver cell culture decreased the uptake of 3H-thymidine into hepatocytes (in the absence or presence of hepatopoietin) as well as the attachment of the hepatocyte cultures. Mushroom toxins also inhibited the production of plasma proteins in hepatocyte cultures. The inhibition, observed at toxin concentrations from 10(-5) to 10(-7) M, was dose-dependent. At low concentrations of phalloidin the inhibition appears to be selective for certain proteins.

Determination of plasma bupropion and its relationship to therapeutic effect.

An analytical method to determine the concentration of bupropion in human plasma has been developed using a deuterium-labeled analog as internal standard and selected ion monitoring applied to an extract of plasma samples taken as part of a clinical trial of this antidepressant. In all, 15 depressed outpatients were randomly assigned to bupropion in a double-blind study which included weekly evaluation of their clinical condition. A good correlation was found between the results obtained by this assay and by a radioimmunoassay technique currently in use. While no simple correlation between plasma concentration and therapeutic improvement was noted, the majority of subjects showed mild to marked amelioration of symptoms and those having mean concentrations above 35 ng ml-1 had significant improvement in their test scores. Interpretation of the mass spectra of both the labeled and unlabeled drug revealed an apparent violation of the 'even-electron rule'.