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Biomedical subjects

D L Knook

Publications and source records attributed to D L Knook.

At least 73 records · Page 4Linked to original sources
PubMed1985

Perisinusoidal fat-storing cells are the main vitamin A storage sites in rat liver.

Highly purified sinusoidal (fat-storing, Kupffer and endothelial cells) and parenchymal cells were isolated to assess the cellular distribution of vitamin A in liver of adult vitamin A-sufficient rats. A modified simple procedure was developed for the purification of fat-storing cells from rat liver. This was achieved by a single centrifugation step in a two-layer density Nycodenz gradient. Endothelial and Kupffer cells were obtained from the same gradient and further purified by centrifugal elutriation. Reverse-phase HPLC analysis showed that fat-storing cells contained about 300-fold the amount of retinyl esters present in parenchymal cells on a mg cell protein basis. In fat-storing cells, the same retinyl esters, viz. retinyl palmitate, retinyl stearate and retinyl oleate, were present as in whole liver. It was also observed that, within 12 h after intravenous injection of chylomicron [3H]retinyl ester, most of the radioactivity had accumulated in the fat-storing cells. It is concluded that fat-storing cells are the main storage sites for vitamin A in rat liver.

Aging↗
PubMed1985

UV-induced unscheduled DNA synthesis in fibroblasts of aging inbred rats.

Because of the suggested relationship between the lifespan of an organism and the amount of unscheduled DNA synthesis (UDS) occurring in its cells after treatment with genotoxic agents, we initiated a lifespan study of this step of the nucleotide excision repair pathway in female Wistar (WAG/Rij) rats. Skin fibroblasts were isolated at 2 time points, separated by a 9-month interval, from rats of various ages. The isolated cells were cultured for 1 passage, irradiated with ultraviolet light (UV) and analyzed by autoradiography for their capacity to perform UDS. The results of the two cross-sectional series of determinations were identical: small variations among individual animals and a slight, but statistically significant age-related decrease in the initial rate but not in the end level of UV-induced UDS. The small variation among individual inbred rats as compared with the large variation reported for UDS in human populations suggests that the latter is largely due to genetic differences. The lack of a more pronounced age-related decrease along with the small individual variation suggests that the activity of the DNA nucleotide excision repair pathway is not an important single determinant of individual longevity in inbred rats of the same strain and sex.

Aging↗
PubMed1985

Variation in restriction fragment length and methylation pattern of rat MHC class I genes.

Rat MHC (RT.1) class I genes were analyzed by performing Southern blot analysis of digests of genomic DNA isolated from three inbred rat strains using a human derived HLA cDNA probe specific for class I genes. A large number of hybridizing restriction fragments indicated that the rat MHC is a large multigene family. Polymorphic class I DNA restriction fragments specific for the different haplotypes were found for three restriction enzymes used, indicating a high degree of restriction fragment length polymorphism of RT.1 class I genes. Analysis of the methylation state of class I RT.1 genes showed that they are hypermethylated to an extent that is haplotype specific. Furthermore, variation in number and size of Hpa II fragments were observed when comparing liver and spleen DNA from two rat strains sharing the same haplotype. This indicates methylation variation between these organs for RT.1 class I genes.

Animals↗
PubMed1985

Retinoids, retinoid-binding proteins, and retinyl palmitate hydrolase distributions in different types of rat liver cells.

A study was conducted to determine the levels and distributions of retinoids, retinol-binding protein (RBP), retinyl palmitate hydrolase (RPH), cellular retinol-binding protein (CRBP), and cellular retinoic acid-binding protein (CRABP) in different types of isolated liver cells. Highly purified fractions of parenchymal, fat-storing (stellate), endothelial, and Kupffer cells were isolated in high yield from rat livers. The retinoid content of each fraction was measured by HPLC analysis. RBP, CRBP, and CRABP were measured by sensitive and specific radioimmunoassays, and RPH activity was measured by a sensitive microassay. The concentrations of each parameter expressed per 10(6) parenchymal or fat-storing cells were, respectively: retinoids, 1.5 and 83.9 micrograms of retinol equivalents; RBP, 138 and 7.4 ng; RPH, 826 and 1152 pmol FFA formed hr-1; CRBP, 470 and 236 ng; and CRABP, 5.6 and 8.7 ng. When these data were expressed on the basis of per unit mass of cellular protein, the concentrations of RPH, CRBP, and CRABP in the fat-storing cells, which contain 10-fold less protein than the large parenchymal cells, were seen to be greatly enriched over parenchymal cells. The parenchymal cells contained approximately 9% of the total retinoids, 98% of the total RBP, 90% of the total RPH activity, 91% of the total CRBP, and 71% of the total CRABP found in the liver. The fat-storing cells accounted for approximately 88% of the total retinoids, 0.7% of the total RBP, 10% of the RPH activity, 8% of the total CRBP, and 21% of the CRABP in the liver. The endothelial and Kupffer cell fractions contained very low levels of all of these parameters. Thus, the large and abundant parenchymal cells account for greater than 70% of the liver's RBP, RPH, CRBP, and CRABP; but the much smaller and less abundant fat-storing cells contain the majority of hepatic retinoids and greatly enriched concentrations of RPH, CRBP, and CRABP.

Animals↗
PubMed1984

Lysosomal activity in the aging rat liver: II. Morphometry of acid phosphatase positive dense bodies.

Hepatocytes from 3-, 12- and 36-month-old rats were studied in situ with cytochemical and morphometrical techniques to obtain information about the intralobular and age-dependent variation in lysosomal volume. An increase with age was found for the lysosomal volume density. This increase is evident throughout the lobule, but occurs at different rates in the centrolobular and peripheral areas. It is most pronounced in the centrolobular area, resulting in a lysosomal volume density of 7.1% in old rats, compared to 3.6% in the peripheral area of the liver lobule. Both the size and the number of individual lysosomes contribute to the overall effect. Most dense bodies, including lipofuscin-containing aggregates in senescent animals, exhibit acid phosphatase activity. Our results suggest that the use of enzyme activity as a marker may yield higher values for lysosomal volume density than morphologic characteristics alone. The alleged homogeneity of the liver as a source of hepatocytes for studies on isolated cells must be questioned when lysosomal functions are concerned.

Acid Phosphatase↗
PubMed1984

Distribution of thiol-protein disulfide oxidoreductase, insulin-glucagon proteinase and cathepsin D in different cell types of the rat liver.

Cathepsin D (EC 3.4.23.5), the insulin and glucagon degrading proteinase (IGP, EC 3.4.22.-) and the thiol-protein disulfide oxidoreductase (TPO, EC 1.8.4.2, 5.3.4.1) participate in the intracellular protein degradation, the last one also in post-protein-synthetic processing. The distribution of these enzymes was determined in isolated liver parenchymal cells, Kupffer cells and endothelial cells by means of immunochemical methods in order to further characterize these cell types. The cathepsin D content, expressed as microgram enzyme per mg protein, is about 3 fold higher in endothelial cells and about 5 to 24 fold higher in Kupffer cells than in parenchymal cells. This result confirms an earlier report which is based on the activity determination. The TPO concentration is highest in parenchymal cells with half of that concentration in Kupffer cells and one third in endothelial cells. About 0.5% of the total liver protein is represented by this enzyme. The IGP has been found to be totally absent in non-parenchymal cells. It represents, therefore, together with the glucose-6-phosphatase a valuable marker enzyme for parenchymal cells of rat liver.

Animals↗
PubMed1983

Lysosomal activity in aging rat liver: I. Variation in enzyme activity within the liver lobule.

The intralobular distribution of three lysosomal enzymes, acid phosphatase, N-acetyl-beta-D-glucosaminidase, and beta-D-glucuronidase, was investigated in hepatocytes of 3-, 12-, 24-, and 36-month-old rats. The distribution pattern changed with age. In young rats, the activity was higher in the peripheral area than in the central area, whereas this situation was reversed in old rats. At all ages, the intermediate area showed the lowest activity. The overall activity increased with age. The reaction product was always present in granules located mainly around the bile canaliculi.

Acetylglucosaminidase↗
PubMed1982

Endocytosis of lactate dehydrogenase isoenzyme M4 in rats in vivo. Experiments with enzyme labelled with O-(4-diazo-3,5-di[125I]iodobenzoyl)sucrose.

1. Pig lactate dehydrogenase isoenzyme M4 was labelled with O-(4-diazo-3,5-di[125I]iodobenzoyl)sucrose and injected intravenously into rats. Previous work has shown that this label does not influence the clearance of the enzyme (half-life about 26 min) and that it is retained within the lysosomes for several hours after endocytosis and breakdown of the protein [De Jong, Bouma & Gruber (1981) Biochem. J. 198, 45--51]. 2. The distribution of the radioactivity over a large number of tissues was determined 2 h after injection. A high percentage of the injected dose was found in liver (41%), spleen (10%) and bone including marrow (21%). 3. Autoradiography indicated uptake of the enzyme mainly by Kupffer cells of the liver, by spleen macrophages and by bone marrow macrophages. 4. Liver cells were isolated 1 h after injection of the enzyme. Kupffer cells, endothelial cells and parenchymal cells were found to endocytose the enzyme at rates corresponding to 4230, 35 and 25 ml of plasma/day per g of cell protein, respectively. 5. Previous injection of carbon particles greatly reduced the uptake of the enzyme by liver and spleen, but the uptake by bone marrow was not significantly changed.

Animals↗
PubMed1982

Primary cultures of endothelial cells of the rat liver: a model for ultrastructural and functional studies.

A new isolation and purification procedure for endothelial cells of the rat liver and the conditions for large scale survival of these cells in maintenance culture are reported. Cells isolated by this new method and cultured with homologous rat serum on a collagen matrix show the restoration of several ultrastructural characteristics typical of rat liver endothelial cells in situ, including the broad cytoplasmic extensions that contain the sieve plates. These fenestrated cytoplasmic projections, which cover the liver sinusoids in vivo, are well preserved and are reformed in a manner reminiscent of the situation in situ. Reformation of specific membrane receptors is indicated by the reappearance of the capacity to take up horseradish peroxidase by adsorptive endocytosis, a characteristic that is lost during the cell isolation procedure. From the results obtained in this study, maintenance culture of rat liver endothelial cells seems to be a promising system for studying the regulation of pore size of the fenestrated sieve plates by alcohol and certain hormones, for studying the interaction of endothelial cells with other liver cells and tumor cells, and for studying the mechanisms of adsorptive endocytosis.

Animals↗
PubMed1982

Effects of age on rat liver enzymes. A study using isolated hepatocytes, endothelial and Kupffer cells.

Hepatocytes, endothelial and Kupffer cells were isolated from young adult (3 month) and old (24 month) rat livers and the activities of some plasma membrane, endoplasmic reticulum, mitochondrial, lysosomal and soluble enzymes compared using biochemical and electron microscope cytochemical techniques. Age-associated changes included: a decrease in glucose-6-phosphatase activity both in hepatocytes and sinus lining cells; and increase in alkaline phosphatase in endothelial cells but a decrease in hepatocytes; reduced basal and glucagon-induced adenyl cyclase in hepatocytes and endothelial cells and an increase in the number of hepatocytes with gamma-glutamyl transferase reaction. Cytochemistry showed that heterogeneity may also be characteristic of senescence particularly with regard to hepatocyte glucose-6-phosphatase which was absent in some cells, low in many cells but high in some and gamma-glutamyl transferase which was normally lacking from hepatocytes but localised as large deposits of reaction product on the plasma membranes of occasional cells isolated from old donors.

Aging↗
PubMed1982

Relation between localization and function of rat liver Kupffer cells.

Kupffer cells were found to be distributed over zone 1 (periportal), zone 2 (midzonal), and zone 3 (perivenous) of the rat liver acinus in a ratio of 4:3:2. After pronase digestion of the liver, two purified tractions of Kupffer cells could be obtained by centrifugal elutriation. Liver zone marking with methylene blue prior to cell isolation revealed that one fraction contained Kupffer cells from the periportal area; the other consisted mainly of such cells from the midzonal and perivenous areas. Periportal Kupffer cells were larger and showed higher lysosomal enzyme activities on a per cell basis as compared with midzonal and perivenous Kupffer cells. The immediate uptake of 0.31-micrometer. latex particles during a 2-minute perfusion of the liver was mainly accomplished by periportal Kupffer cells. The higher phagocytic activity of these cells was independent of the direction which latex was flushed through the liver. Seven days after in vivo administration of latex particles, at least 80 per cent of all Kupffer cells contained latex, but again periportal Kupffer cells showed a higher phagocytic activity in that they accumulated a relatively larger number of particles per cell. Latex phagocytosis increased the activity of cathepsin D in all Kupffer cells. Titration with pepstatin revealed that periportal Kupffer cells contained many more cathepsin D molecules than pepstatin revealed that periportal Kupffer cells contained many more cathepsin D molecules than midzonal and perivenous Kupffer cells. Latex particle uptake stimulated an increase in the molecular activity of cathepsin D in periportal cells as well as in the number of cathepsin D molecules in perivenous and midzonal cells. Kupffer cells show a functional heterogeneity that is related to their position in the liver acinus. Kupffer cells with a high endocytic activity, large and heterogeneous lysosomes, and high lysosomal enzyme activities are found in the periportal zone. This zonal heterogeneity can be reduced after phagocytosis of a triggering dose of latex.

Animals↗
PubMed1982

Endocytosis of heat-denatured albumin by cultured rat Kupffer cells.

Purified Kupffer cells were obtained by centrifugal elutriation of sinusoidal cells isolated by pronase treatment of the rat liver. The endocytosis of radioactively labeled heat-aggregated colloidal albumin (CA 125I) was investigated in maintenance cultures of the purified Kupffer cells. The endocytic capacity of the cells was studied during 4 days of culture. Maximum uptake was observed after 24 hr of culture, with a gradual decline during the following days. When the uptake was measured after incubation with increasing concentrations of CA 125I, a saturation effect was observed. This finding and the observed high rate of uptake are strong indications that receptor sites on the cell membrane are involved in the mechanism of endocytosis. The uptake of CA 125I by Kupffer cells was inhibited by the metabolic inhibitors fluoride and antimycin A, indicating that endocytosis of CA 125I is dependent on energy derived from both glycolysis and mitochondrial respiration. The mechanism of internalization may also require the action of microfilaments as well as intact microtubules, since both cytochalasin B and colchicine inhibited the uptake of CA 125I. The intracellular degradation of CA 125I by Kupffer cells was strongly inhibited by chloroquine but not by colchicine. The degradation of ingested CA 125I occurred within the Kupffer cell lysosomes.

Animals↗
PubMed1981

Sex and strain dependency of age-related changes in protein synthesis of isolated rat hepatocytes.

In previous studies, a decrease in protein synthesis by hepatocytes isolated from female WAG/Rij rats was observed in the first year of life, while an increase was seen in advanced age. In the present study, no change in protein synthesis in early age was found for hepatocytes isolated from male WAG/Rij and female BN/Bi rats, suggesting that the decline in protein synthesis is sex and strain dependent. In contrast, the increase in protein synthesis in advanced age could be demonstrated with hepatocytes from male WAG/Rij and female BN/Bi rats and therefore seems to be independent of strain and sex. The increase in protein synthesis in advanced age could be attributed to increased excretion of protein in the urine.

Age Factors↗