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Biomedical subjects

R Holme

Publications and source records attributed to R Holme.

18 recordsLinked to original sources
PubMed1996

Intercellular adhesion molecule-1 (ICAM-1; CD54) expression in human hepatocytic cells depends on protein kinase C.

BACKGROUND/AIMS: Intracellular regulation of intercellular adhesion molecule-1 has mainly been studied in lymphoid, endothelial, and epithelial cells. Intercellular adhesion molecule-1 plays a central role in many immune responses, and we have previously studied its regulation in hepatocytes. Here we report how manipulation of intracellular signal systems influenced its expression. METHODS: The constitutive and cytokine-induced expression of intercellular adhesion molecule-1 mRNA and protein was studied in the human hepatocytic cell lines Hep G2 and SK-Hep-1. RESULTS: When agonists and antagonists of protein kinase C, calmodulin, and protein kinase A were introduced in addition to prostaglandin E2 and a cyclooxygenase inhibitor, only the protein kinase C activator phorbol 12-myristate 13-acetate resulted in a rapid and dose-dependent increase in intercellular adhesion molecule-1 protein and mRNA. Phorbol 12-myristate 13-acetate stimulated sustained high levels of intercellular adhesion molecule-1 protein, whereas the corresponding mRNA response was biphasic, peaking at 3 h. Actinomycin D blocked the stimulatory mRNA phase, suggesting that de novo transcription was induced. Coincubation with phorbol 12-myristate 13-acetate and the protein synthesis inhibitor cycloheximide gave considerably higher mRNA levels than with phorbol 12-myristate 13-acetate alone. Protein kinase C may therefore even stimulate synthesis of proteins that speed up the turnover of intercellular adhesion molecule-1 mRNA. The protein kinase C inhibitor staurosporine abrogated the induction of intercellular adhesion molecule-1 by phorbol 12-myristate 13-acetate, indicating that this effect was indeed exerted by protein kinase C. More original was our observation that staurosporine also completely blocked the stimulatory effects of interferon-gamma, tumour necrosis factor-alpha, and interleukin-1. Recent reports have noted that these cytokines apparently use receptors which activate different intracellular pathways. We also noted that the glucocorticoid dexamethasone partially inhibited the stimulation of intercellular adhesion molecule-1 by these cytokines. This phenomenon could be important for the immunosuppressive effects of corticosteroids in patients with liver disease. CONCLUSIONS: Our data suggest that a certain level of protein kinase C activity is mandatory for liver cells in cytokine-mediated upregulation of intercellular adhesion molecule-1.

Cell Line↗
PubMed1985

Thromboplastin (tissue factor) in plasma membranes of human monocytes.

The synthesis of thromboplastin, a potent trigger of blood coagulation, can be induced in human peripheral blood monocytes. Indirect evidence suggests that newly synthesized thromboplastin becomes in part available on the cell surface. We have attempted to study the localization and availability of thromboplastin more directly by isolating plasma membranes from isolated human peripheral blood monocytes. The specific activities of the plasma membrane markers increased 16-22-fold in these preparations with a recovery of about 15%. The contamination by mitochondria, lysosomes, nuclei and endoplasmic reticulum was low as estimated by marker enzymes and electron microscopy. In both unstimulated and stimulated monocytes thromboplastin was largely recovered in this plasma membrane fraction, providing direct evidence for its membrane localization. Phospholipase C (E.C. 3.1.4.3) is a potent inactivator of thromboplastin through its hydrolysis of the phospholipids necessary for thromboplastin activity [Otnaess, Prydz, Bjørklid & Berre (1972) Eur. J. Biochem. 27, 238-243]. About 70% of the total membrane thromboplastin activity was inactivated when whole cells were treated with phospholipase C and the membranes subsequently isolated. Following stimulation to induce thromboplastin synthesis, the plasma membranes showed a shift in their relative content of phosphatidylcholine and phosphatidylethanolamine consistent with a transmethylation process.

Cell Membrane↗
PubMed1985

Effect of high-density lipoproteins on cholesterol efflux and esterification in lipid-enriched human skin fibroblasts.

The ability of high-density lipoprotein (HDL) to reduce the cholesterol content was studied in cultured fibroblasts enriched with cholesterol esters. Incubation of cholesterol-enriched cells with HDL in a final concentration of 1 g protein/l for 24 h reduced the total and esterified cholesterol content by 23% as compared with control fibroblasts incubated with albumin. Similar cholesterol efflux was obtained with HDL isolated from lecithin:cholesterol acyltransferase (LCAT)-deficient plasma. The HDL3 subfraction isolated by rate-zonal ultracentrifugation contained the major part of the cholesterol-depleting effect. HDL or HDL3 decreased CoA:cholesterol acyltransferase (ACAT) activity to 5% of the level found in control fibroblasts within 8 h of incubation. These findings suggest that ACAT activity is sensitive to a pool of intracellular cholesterol, which can be mobilized by the addition of HDL to the culture medium, and that ACAT activity is a useful measure of cholesterol efflux from cultured fibroblasts.

Cells, Cultured↗
PubMed1981

Familial lecithin:cholesterol acyltransferase deficiency. Report of a fourth family from northwestern Norway.

The tenth Norwegian patient with familial LCAT deficiency is reported. Her family lives in the same area as the three previously reported Norwegian families. The patient is a 26-year-old female with typical findings of the disease--proteinuria and corneal opacities. Total cholesterol was normal, but the main part was present in the free form. Triglycerides were slightly elevated. Kidney function was normal. A large molecular weight fraction of LDL was present in plasma.

Adult↗
PubMed1978

The effect of lipoprotein lipase and hepatic lipase on the electrophoretic mobility of lipoprotein-X.

Lipoprotein-X containing plasma from a patient with familial lecithin:cholesterol acyltransferase (LCAT) deficiency, was used as substrate and incubated with postheparin plasma or partly purified lipases. LP-X could not be demonstrated by agar gel electrophoresis after incubation with postheparin plasma from a healthy subject, from a patient with chronic active hepatitis deficient in hepatic lipase, or with partly purified lipoprotein lipase. After incubation a marked increase in free fatty acids (FFA) was observed. In contrast LP-X was still present after incubation when postheparin plasma deficient in lipoprotein lipase or partly purified hepatic lipase was added to the substrate. Only minor changes in the concentration of FFA occurred. After addition of oleic acid to the substrate LP-X could not be demonstrated by agar gel electrophoresis. However, in the isolated low density lipoproteins, LP-X like particles were still present as viewed by electron microscopy. Our results strongly suggest that the change in electrophoretic mobility of LP-X was induced by the release of FFA. This was achieved by lipoprotein lipase, but not by hepatic lipase.

Adult↗
PubMed1978

In vitro interaction between cultured cells and human blood platelets.

The effects of washed human cultured cells (tumour cells and Chang liver cells) on human blood platelets in heparinized plasma were studied. Platelet aggregation was induced by suspensions of the tumour cells. Ultrastructural examination showed that the platelets, especially in the central regions of the aggregates, were tightly packed and the alpha-granules were mostly present. In the periphery of the aggregates the platelets appeared swollen and devoid of organelles, and fibrin strands were seen. The platelet aggregation was not completely abolished by incubation with apyrase. The washing fluids from the tumour cells also induced platelet aggregation, but the aggregation could be abolished by incubation with apyrase. When three different lines of the Chang cells were used, suspensions of two lines of these cells induced platelet aggregation, but the third line did not. Presence of ADP could be demonstrated in the washing fluids from the cultured cells, except for the one line of Chang cells which did not induce platelet aggregation. The experiments indicated that the platelet aggregation induced by the various types of cells was mediated via ADP, but with a possible additional effect of coagulation activity.

Adenosine Diphosphate↗
PubMed1978

Triglyceride lipase activity in postheparin plasma and plasma lipoproteins in liver disease.

Hepatic lipase activity and lipoprotein lipase activity were studied in postheparin plasma from 14 patients with various liver disorders. Plasma lecithin: cholesterol acyltransferase (LCAT) activity and lipoprotein composition and structure were also estimated. Five patients had lower hepatic lipase activity than the lowest control value, and in three of these no hepatic lipase activity was detected. Lipoprotein lipase was low in 5 patients, but in only one of them was hepatic lipase activity also low. Hepatic lipase was not significantly correlated to the concentration of plasma triglycerides, either in controls or in patients, whereas lipoprotein lipase was negatively correlated with plasma triglycerides both in controls and patients. Lipoprotein lipase and LCAT activity, but not hepatic lipase, was negatively correlated to the triglyceride content of the low density lipoproteins (density 1.019-1.063 g/ml) from the patients. No specific lipid or lipoprotein pattern was found in plasma from the patients with a low or without any hepatic lipase activity. The results suggest an important role of lipoprotein lipase and LCAT, for the increased content of triglycerides in the low density lipoproteins in patients with liver disease. The role of hepatic lipase remains unclear.

Adult↗
PubMed1978

Plasma cholesterol esterification and plasma lipoproteins in bile-duct-ligated dogs.

To study the lipoprotein changes in cholestasis while the capacity for plasma cholesterol esterification was normal, the common bile duct was ligated in dogs and plasma investigated 8 h and 48 h later. The plasma concentration of cholesteryl esters was slightly increased, concomitant with a tendency toward an increase in the activity of lecithin:cholesterol acyltransferase (LCAT). The content of cholesteryl esters in the main lipoprotein classes was normal. Marked alteration in the low density lipoproteins (LDL) and the high density lipoproteins (HDL) took place and were essentially similar 8 h and 48 h after bile duct ligation. In LDL, (density 1.006--1.019 g/ml) and LDL2 (density 1.019--1.063 g/ml) an increase in the content of polar lipids was observed, and in LDL2 heterogeneity in particle size was demonstrated by gelfiltration on 2% agarose and by electron microsopcy. Large myelin structures, flattened disc-shaped particles, and particles with the appearance of normal LDL2 were present. HDL isolated after operation was characterized by a decreased protein/lipid ratio and an increased content of phospholipids. By gelfiltration on Sephadex G-200 and by electron microscopy changes in particle size were observed, with the presence of disc-shaped particles with a tendency in form rouleaux. These results demonstrate that marked lipoprotein changes occur as early as 8 h after bile duct-ligation in dogs and indicate that a deficient LCAT mechanism is present in cholestasis even with normal or high plasma LCAT activity.

Animals↗
PubMed1978

Familial lecithin:cholesterol acyltransferase deficiency. Further studies on plasma lipoproteins and plasma postheparin lipase activity of a patient with normal renal function.

Plasma lipoproteins and postheparin plasma were investigated in a patient with familial LCAT deficiency with normal renal function and without proteinuria. As revealed by gelfiltration the large molecular weight LDL2 was not present, and myelin structures were not found in LDL1 or LDL2 when she was on her ordinary diet. After 60--65% fat diet for one week the large molecular LDL2 was found, but only in low concentration. We have no explanation for the difference in the lipoprotein abnormalities of this patient and others with this disease. There is no major difference in the fat content in the ordinary diet of the Norwegian patients with familial LCAT deficiency, nor has our patient any clinical signs of malabsorption. Furthermore, there was no difference in lipoprotein lipase or hepatic lipase activity in postheparin plasma between our patient and others with the same disease. However, whereas hepatic lipase activity was within the reference values, lipoprotein lipase activity was rather low in all patients investigated. We suggest that impaired VLDL catabolism in plasma, because of LCAT deficiency and low lipoprotein lipase activity, may partly explain the low HDL concentration consistently found in patients with familial LCAT deficiency.

Aged↗
PubMed1978

Plasma lipoprotein alterations and morphologic changes with lipid deposition in the kidney of patients with hepatorenal syndrome.

Four patients with advanced liver disease and progressive renal failure compatible with the diagnosis of hepatorenal syndrome have been studied. All four patients had low lecithin:cholesterol acyltransferase activity in plasma, and the concentration of cholesteryl esters was markedly reduced. The main lipoprotein classes were abnormal with an increased content of polar lipids. Electron microscopy of negatively stained lipoproteins from two of the patients (H.K. and I.A.) revealed large particles with layered membranes (700 to 2000 A in diameter) corresponding to the large molecular weight fraction of the low density lipoproteins. These structures were not present in the low density lipoproteins from the other two patients. In the renal biopsy from H.K. and in the necropsy specimen from I.A. deposition of osmophilic material was found in the glomeruli (especially located subendothelially), in the basement membrane, and in the mesangial regions. The deposits were similar to those we have previously described in patients with familial lecithin:cholesterol acyltransferase deficiency and most probably represent cholesterol and phospholipids. It is suggested that the renal deposition of lipid may be related to the large molecular weight low density lipoprotein fraction and that the mechanisms involved in this lipid deposition are similar to those occurring in familial lecithin:cholesterol acyltransferase deficiency.

Female↗
PubMed1977

The electrophoretic mobility of lipoprotein X in postheparin plasma.

After incubation whole plasma and low density lipoproteins (LDL) taken before and 10 min after intravenous administration of heparin, from a patient with primary biliary cirrhosis and a patient with familial lecithin:cholesterol acyltransferase (LCAT) deficiency, have been tested for the presence of lipoprotein X (LP-X) by agar gel electrophoresis. LP-X was present in preheparin whole plasma and LDL. No precipitation lines on the cathodal side of the wells, indicating the absence of LP-X, were seen after electrophoresis of postheparin plasma and LDL. Immunodiffusion revealed the presence of apo-beta-lipoproteins and LP-X in preheparin as well as postheparin LDL. After gel filtration three subfractions and similar patterns were observed in the preheparin and postheparin LDL. Electronmicroscopical examination of the intermediate subfractions showed LP-X-like particles in preheparin and postheparin samples. These observations indicate a changed electrophoretic mobility in agar gel of postheparin LP-X, giving a false negative LP-X test by the conventional agar gel electrophoresis.

Electrophoresis, Agar Gel↗
PubMed1975

Isolation and studies of the granules of the amebocytes of Limulus polyphemus, the horseshoe crab.

Granules were isolated from the cytoplasm of the amebocytes of Limulus polyphemus, the horseshoe crab, by disruption of cells obtained from blood which had been drawn into 2 mM propranolol. The granules subsequently were purified by centrifugation through a sucrose gradient that contained heparin. Extracts of the granules were prepared by freezing and thawing the granule preparations in distilled water. Transmission and scanning electron microscopy of the granules revealed round or ovoid particles. However, only one type of granule appeared to be present. The ultraviolet spectrum of the extract of amebocyte granules demonstrated a peak at 277 nm at pH 7.4, and a shift into two peaks of 281 nm and 290 nm at alkaline pH. Analytical ultracentrifugation revealed a pattern similar to that observed with lysates prepared from intact amebocytes. Polyacrylamide gel electrophoresis, in the presence of urea at pH 4.5, demonstrated patterns similar to those observed with amebocyte lysate. Extracts of the granules were gelled by bacterial endotoxin. The blood of the horseshoe crab contains only one type of cell, the amebocyte. Previous studies have shown that the blood coagulation mechanism of Limulus is contained entirely within amebocytes. The current studies suggest that the granules, which pack the cytoplasm of these cells, contain all of the factors required for the coagulation of blood, including the clottable protein. The intracellularly localized coagulation system is released from amebocytes when their granules rupture during cell aggregation.

Animals↗