Secretory activity and oncogenicity of a cell line (MDCK) derived from canine kidney.

A cell line (MDCK) of dog kidney origin grows on a glass surface as a mosaic of epithelium with many multicellular hemispherical vesicles. The cells lining the blisters actively secrete into the cyst cavities. Suspensions of these cells injected intravenously in the chick embryo produce brain metastases resembling adenocarcinoma.

Activation of intracisternal A particles in mouse liver by diethylnitrosamine.

Livers of 6- to 7-week-old male C3H/He, CBA, A, and BALB/c mice were examined by electron microscopy for the presence of intracisternal A particles (ICAP) after administration of diethylnitrosamine (DEN) in drinking water. In control mice, ICAP were extremely rare; they were found in the livers of only 2 mice (strains C3H/He and A; none in the other strains). By contrast, the treatment of mice with DEN greatly enhanced the appearance of ICAP in the liver cells of all strains. Within 2 weeks of the treatment, ICAP were found in 8-26% of liver cells examined in all mice and the number of ICAP/cell ranged from 3 to 12. Aside from mild disorganization of the rough endoplasmic reticulum, such as segmentation and vesiculation, liver cells of carcinogen-treated mice showed none of the consistent abnormalities that characterize the appearance of ICAP. The reactivation of ICAP (which are usually suppressed in adult mice) by DEN may become a useful marker for analysis of the sequential alterations of the liver that lead to the development of hepatoma during carcinogenesis.

Isolation of oval cells and transitional cells from the livers of rats fed the carcinogen DL-ethionine.

For the characterization of the metabolic and biologic properties of oval cells (i.e., cells emerging in the livers of rats treated with chemical carcinogens due to proliferation of bile ductular and/or duct cells) and transitional cells (i.e., cells having properties intermediate between those of oval cells and hepatocytes), these cells were isolated from the livers of Sprague-Dawley rats fed DL-ethionine for 4-5 weeks. The livers were dissociated into single cells by perfusion in situ with collagenase, and total cell suspensions were allowed to stand at unit gravity for 10 minutes to separate parenchymal (hepatocytes) from nonparenchymal cells. Nonparenchymal cells were centrifuged in linear gradients of Metrizamide (8-24% wt/vol), and 2-ml fractions were collected from the gradients. The cells in the fractions were defined by light microscopy, electron microscopy, and histochemical and immunofluorescence methods. A cell isolate was thus obtained consisting of Kupffer's cells (approximately 20%), bile ductular and/or duct cells and oval cells (approximately 30%), and transitional cells (approximately 50%). A twofold enrichment of bile ductular and/or duct cells and their derivatives was achieved over that found in the nonparenchymal cell fraction before isopyknic gradient centrifugation.

Reduced response to phenobarbital by the liver of rats fed a choline-deficient diet.

1. The response of the liver to phenobarbital administration was compared in rats fed either laboratory chow, a semipurified choline-supplemented diet or a semipurified choline-deficient diet. 2. The liver contents of proteins, lipids and cytochrome P-450, as well as the activity of aminopyrine and ethylmorphine demethylases, were measured after 5 days of feeding and five daily injections of phenobarbital. Liver sections were examined electron microscopically. 3. In rats fed the choline-supplemented diet, phenobarbital administration caused increases in cellular constituents, enzyme activities and smooth endoplasmic reticulum membranes as great as those seen in rats fed laboratory chow. However, in rats fed the choline-deficient diet, the response of the liver to phenobarbital administration was severely reduced in comparison to that in rats fed the other diets. 4. It is concluded that dietary choline and an adequate synthesis of lecithins are necessary for the induction of microsomal mixed-function oxidases and the concomitant accumulation of smooth endoplasmic reticulum in hepatocytes.

Fetal rat hepatic ultrastructural changes associated with organ culture and glucocorticoid treatment.

Fetal hepatic ultrastructure was examined from 18 days gestation to term. Such structure was compared to that of tissue obtained at 18 days gestation which had been maintained in organ culture for 24, 48, and 72 h. In addition, the effects of maternal glucocorticoid administration in vivo and in vitro organ culture glucocorticoid exposure upon fetal hepatic morphogenesis were examined. Enhanced fetal hepatocyte ultrastructural maturation, characterized by a reduction in hematopoietic percursors, an increased frequency of hepatocyte-hepatocyte contact, a complete compliment of intracellular organelles, and the development of bile canaliculi by either maternal in vivo or in vitro fetal organ culture glucocorticoid exposure, was demonstrated. These studies extend previous observations concerning bile salt synthesis and secretion by fetal hepatic tissue both in vivo and in vitro by providing an ultrastructural basis for the functional changes observed and reported to occur in response to steroid therapy

Cardiac transplantation in the rat. I. The effect of histocompatibility differences on graft arteriosclerosis.

The development of arteriosclerosis is the most serious and common complication in long-term survivors of cardiac transplantation. We have used a variety of inbred rat strains with selected histocompatibility differences to examine the influence of prolonged, mild rejection reactions on the development of pathological changes in long-term cardiac allografts. Heterotopic cardiac allografts were exchanged between rat strains that differed for MHC class I (RT1.A and/or RT1.E) antigens or groups of minor, non-MHC antigens in MHC-compatible congenic combinations. Our results demonstrate that in strain combinations in which the allograft reaction is mild and prolonged, the donor hearts exhibit pathological changes that include a diffuse, interstitial myocardial fibrosis, perivascular fibrosis, and intimal proliferation in arteries of the graft myocardium. The lesions were less prominent in animals with more active rejection and infrequent in strains that differ for class I histocompatibility antigens or syngeneic controls. These results suggest that the comparable pathological changes seen in long-term human cardiac survivors may reflect low-level, persistent allograft reactions rather than factors associated with graft anoxia or effects of immunotherapy to prevent graft rejection.

Studies on acute methionine toxicity. I. Nucleolar disaggregation in guinea pig hepatic cells with methionine or ethionine and its reversal with adenine.

The effects of methionine and ethionine on the fine structure of hepatic cell nucleoli of guinea pigs and rats were investigated. A single intraperitoneal injection of methionine into guinea pigs results in the disruption of nucleolonema as early as 2 hours after the injection. By 4 hours, nucleoli show complete fragmentation consisting of many small fragments and small remnants of nucleoli. Large aggregates of interchromatinic granules and condensation of chromatin appear in the nucleoplasm. These changes are remarkably similar to the lesions induced by ethionine in the liver of the rat or the guinea pig. The methionine-induced nuclear and nucleolar lesions persist up to 10 hours after the injection. The administration of adenine 4 hours after the methionine injection reverses the nucleolar lesions by 8 hours. The appearance of incompletely reconstructed nucleoli with twisted ropelike structures suggests a pattern of recovery very similar to the adenine-induced nucleolar reformation in ethionine-treated rats. Injecting methionine into rats induced no nucleolar abnormalities. It is suggested that the mechanism of nucleolar fragmentation induced by methionine or ethionine is related to the accumulation of S-adenosyl compounds with concomitant ATP deficiency in the liver.

Acute hemorrhagic pancreatitis (massive necrosis) with fat necrosis induced in mice by DL-ethionine fed with a choline-deficient diet.

Female, albino mice were fed a choline-deficient diet containing 0.5% DL-ethionine. All animals died within 5 days due to the development of an acute hemorrhagic pancreatis with fat necrosis throughout the peritoneal cavity. The apancreatitis was characterized by a massive necrosis of the exocrine parenchyma with intense hemorrhage and inflammatory reaction of the stroma. The sequence of histologic and ultrastructural alterations occurring in the acinar cells of the pancreas were studied in mice fed the diet for 1, 2, and 3 days. Major findings consited of accumulation of zymogen granules, vacuolation due to foci of cytoplasmic degradation, and alterations in the morphology of the zymogen granules. The pancreatitis appears to be due to intraparenchymal activation of zymogens, resulting from a synergistic action of choline deficiency with the basic toxicity of ethionine toward the acinar cells of the pancreas. The experimental model simulates closely the acute hemorrhagic pancreatitis with fat necrosis occurring in humans and may prove useful for exploring the pathogenesis of this condition.

Method for the isolation of surfactant from homogenates and lavages of lung of adult, newborn, and fetal rats.

A method is presented for the isolation of pulmonary surfactant from rat lung homogenates and lavages using differential discontinuous density gradient centrifugation. The isolated surfactant was characterized by electron microscopy, assay of enzyme markers for different subcellular organelles, and its chemical composition. Surfactant that was isolated from lung homogenates appeared to be free of other contaminating cellular organelles. Electron microscopic examination of surfactant isolated from whole homogenates showed the presence of lamellar bodies and tubular myelin figures and, hence, represented total lung surfactant. On the other hand, surfactant prepared from lung lavages had mostly tubular myelin figures and, thus, represented extracellular surfactant. Both preparations were rich in phospholipids, especially lecithins and phosphatidylglycerol, and showed high phospholipid to protein ratios. The method was used to study the quantity and composition of surfactant during lung development in rats. Surfactant was isolated from lung homogenates of fetuses at days 19 to 21 of gestation and from newborn and adult rats. During the period of 19 to 21 days of gestation, there is a 10- and 20-fold increase in the amount of protein- and phospholipids (milligrams per gram of wet lung), respectively. A further 2-fold increase occurs after birth. Phosphatidylcholines account for 65% of the total phospholipids at day 20, 71% on day 21, and 81% at term. A progressive increase in the phosphatidylcholine to sphingomyelin ratios and in the amount of phosphatidylglycerol occurs during this period. The amount of disaturated lecithins (expressed as percentage of total phosphatidylcholines) in the lung surfactant increases from 49% at day 20 to 53% at birth. There is no change in the amount of disaturated lecithins after the newborn stage. The present method for the isolation of lung surfactant is reproducible, is less time consuming, and can be used to isolate quantitatively surfactant from small lung aliquots.

Biochemical and enzymatic characterization of thymic and splenic lymphocyte plasma membranes from inbred rats.

Purified splenic and thymic lymphocytes from the ACI and F344 strains of inbred rats were disrupted by controlled hypotonic treatment, and their plasma membranes were prepared by sucrose density gradient centrifugation. The plasma membrane preparations were highly purified as judged by the structural appearance of the smooth membrane vesicles, by the 10- to 15-fold enrichment of 5'-nucleotidase, which cytochemically localized exclusively in the plasma membranes of intact lymphocytes, by the high cholesterol to phospholipid molar ratio (0.7-1.0), and by the very low specific activities of the enzymes associated predominantly with mitochondria, lysosomes, and endoplasmic reticulum. The protein and the lipid contents of the membranes were 48-55 and 37-48%, respectively. The total lipid content of plasma membranes was characteristically higher in thymic than splenic lymphocytes from both ACI and F344 strains. The specific activity of 5'-nucleotidase was similar in splenic lymphocyte membranes of the ACI strain, and in both the thymic and splenic lymphocyte membranes of the F344 strain. In contrast, the thymic lymphocyte membranes in the ACI strain showed half as much 5'-nucleotidase specific activity. Cytochemical results indicated that the 5'-nucleotidase is located on the outside surface of the lymphocyte plasma membranes.

Leishmania donovani: cell-surface heparin receptors of promastigotes are recruited from an internal pool after trypsinization.

With the use of [3H]heparin, we recently demonstrated that Leishmania donovani promastigotes express a cell-surface receptor that is specific for the glycosaminoglycan heparin (Mukhopadhyay et al. 1989, The Biochemical Journal, 264, 517-525.). Treatment of the parasite with trypsin abolishes 75-90% of this [3H]heparin-binding activity. When trypsinized promastigotes were resuspended in fresh culture medium in the absence and presence of cycloheximide (10 micrograms/ml), approximately 25-30% of the original heparin-binding capacity was restored within 1 hr, indicating that recruitment of receptors from an internal pool occurred without de novo protein synthesis. Scatchard analysis of the regenerated receptor revealed that the number of regenerated binding sites per cell was 2.3 x 10(5); these sites have a binding affinity of 6.7 x 10(-7) M. Like the native heparin receptors on the surface of freshly isolated cells, the receptors recruited after trypsinization are also highly specific for heparin, as a 25-fold excess of four other glycosaminoglycans displaced less than 10% of bound [3H]heparin from the trypsinized cells. The structural requirements of the ligand heparin, namely the number of monosaccharide units and degree of sulfation, were compared for both the native and regenerated receptor: for both receptors, oversulfated polysaccharide heparin fragments of at least six to eight sugar residues were most efficient at displacing [3H]heparin. The concentrations of oligosaccharide fragments required to displace 50% of [3H]heparin were 0.32 and 0.035 microM for the hexa- and octasaccharides, respectively. Colloidal gold-labeled heparin was bound to promastigotes and visualized by electron microscopy. This analysis revealed that the heparin bound almost exclusively to the flagella of control cells (not subjected to trypsin) and those which had regenerated receptor after trypsinization. The physiological significance of this heparin-binding activity on the surface of promastigotes is discussed.

Ethanol--its nephrotoxic effect in the rat.

The nephrotoxic effect of ethanol feeding on renal structure and function was evaluated in rats and compared to that in dextrose-fed isocaloric control animals. Alcohol-fed animals had larger kidneys than their controls. Despite this increase in renal mass, the alcohol-fed animals had a 50% reduction in creatinine clearance and a 67% reduction in osmolar clearance compared to their controls. When specific renal constituents were compared, the alcohol-fed animals were found to have twice the renal protein and a 50% increase in renal lipid. Despite these marked structural and functional differences, the light microscopic appearance of the kidneys of the two groups did not appear significantly different. In contrast, the electron microscopic differences were substantial. The renal epithelial cells, particularly of the distal tubules and Henle's loops, were found to show varying degrees of cellular injury and were observed to be sloughing into the lumens. These electron microscopic observations are similar to those obtained in tubular necrosis due to a variety of nephrotoxic agents. We propose, therefore, that chronic alcohol feeding of rats produces significant renal dysfunction and abnormalities of structure such that ethanol should be considered a true nephrotoxin.