Terje B. Christensen

Uptake and degradation of bovine testes β-galactosidase by parenchymal and nonparenchymal rat liver cells

The plasma half-life of beta-galactosidase in rat was about 1.5 min. Ten minutes after in vivo injection, 45% of the enzyme was recovered in liver, with hepatocytes and endothelial cells as the predominant cell types responsible for uptake. In vitro uptake of beta-galactosidase in hepatocytes and nonparenchymal liver cells was saturable, Ca2+-dependent and it could be partly inhibited by mannose or alpha-methyl-mannoside.

The distribution of protein-bound carbohydrates in submicrosomal fractions from rat liver

Abstract 1. 1. Subfractions of washed rough and smooth microsomes were prepared by sonication and deoxycholate treatment. The amount of protein-bound mannose, galactose, glucosamine and sialic acid (NANA) in the subfractions has been determined. 2. 2. The vesicle protein obtained from rough and smooth microsomes contained the same amounts of mannose, whereas the galactose and glucosamine content was significantly larger in the vesicle protein from smooth microsomes. The Galsmooth vesicles/Galrough vesicles ratio was 3.3, and GlcNsmooth vesicles/GlcNrough vesicles ratio was 1.9. Sialic acid was found only in the smooth vesicle protein. The results support the “multi-site” hypothesis for attachment of carbohydrate to the polypeptide chain. 3. 3. Mannose and glucosamine were found in similar amounts in total protein from rough and smooth membranes. The Galsmooth membranes/Galrough membranes ratio was 3.3, and the NANAsmooth membranes/NANArough membranes ratio was 2.7. 4. 4. The amino acid composition and the disc electrophoretic pattern of the vesicle proteins from rough and smooth microsomes were similar. A high degree of similarity in amino acid composition and the disc electrophoretic pattern was also found for the membrane proteins from rough and smooth microsomes, although different from that obtained for the vesicle protein.

Effect of dextran and dextran modifications on the thermal and proteolytic stability of conjugated bovine testis β-galactosidase and human serum albumin

In order to study carbohydrate-induced protein stabilization bovine testis beta-galactosidase and human serum albumin were conjugated with dextran, partially acetylated dextran and partially methylated dextran. The conjugates and the free proteins were compared with respect to thermal stability at 50 degrees C and resistance to proteolytic digestion by subtilopeptidase A. Both beta-galactosidase and serum albumin were stabilized by conjugation with polysaccharide. However, higher stability was achieved by conjugating the proteins with the hydrophilic polysaccharides, dextran and acetylated dextran, than by conjugation with the hydrophobic polysaccharide, methylated dextran. The results are discussed in relation to possible explanations of carbohydrate-induced protein stabilization.

Single-strand breaks in the DNA of human cells exposed to visible light from phototherapy lamps in the presence and absence of bilirubin

Clinical evidence indicates that phototherapy of hyperbilirubinaemia in newborn infants is a safe and efficient form of therapy. The short-term side effects are not serious and seem to be well controlled. There are few long-term follow-up studies of phototherapy-treated infants. Therefore one cannot completely exclude the possibility that side effects can be found in future studies. With this background we undertook the present study of possible genotoxic effects of phototherapy. Human cells of the established glioblastoma cell line TMG-1 were used. The cells were exposed to visible light in the presence of different concentrations of bilirubin or in the absence of bilirubin. DNA was unwound in alkaline solution and the induction of strand breaks was assayed by a method taking advantage of the fluorescence from the dye Hoechst 33258. Blue light induced single-strand breaks in the DNA of cells in culture in the absence of bilirubin. During irradiation of bilirubin solutions with blue and green phototherapy light, long-lived toxic photoproducts were formed under in vitro conditions. At high and clinically relevant bilirubin concentrations, the effects of blue and green light were relatively similar. At low concentrations, there was a smaller effect of green light as expected from the absorption spectrum of bilirubin. It remains to be seen whether the genotoxic effect observed in the present studies can occur in vivo.

Hematoporphyrin derivative: chemical composition, photochemical and photosensitizing properties.

“Hematoporphyrin derivative” (HPD) is a product prepared by treating acetylated hematoporphyrin (HPA) with alkali. Acetylated hematoporphyrin is prepared by treating hematoporphyrin dihydrochloride with a mixture of sulphuric acid and acetic acid (Lipson et al. 1961). HPD has been reported by a number of investigators to be preferentially retained in tumors (for a review, see Moan and Christensen, 1980). HPD is now being evaluated for use in cancer detection and therapy. The therapeutic aspect is based on the fact that many porphyrins are efficient photosensitizers. Tumor tissue, which after injection contains more HPD than surrounding tissue, is selectively destroyed when exposed to visible light (Dougherty et al., 1975).

The significance of the carbohydrate constituents of bovine thrombin for the clotting activity

Abstract Bovine thrombin, which is glycoprotein, has been purified and subjected to incubation with glycosidases. This treatment released approximately all the sialic acid, one third of the galactose content and one quarter of the glucosamine content without having any apparent effect on the clotting activity as compared to the controls. The clotting activity of bovine thrombin, as measured in vitro , does not therefore, seem to be dependent upon the carbohydrate constituents being intact.

Enhanced stability of β-galactosidase in parenchymal and nonparenchymal liver cells by conjugation with dextran

In order to enhance the stability of beta-galactosidase, we conjugated the enzyme with dextran T-10 (Mr approx. 10 000). The conjugate contained 9-10 mol dextran/mol protein (beta-galactosidase, Mr 68 000), and the specific activity retained after conjugation was 90 +/- 4% (n = 3) of the initial activity. Uptake and degradation of native and conjugated beta-galactosidase in isolated hepatocytes and nonparenchymal liver cells was studied. There was a marked increase in stability against degradation in both cell types when beta-galactosidase was conjugated with Dextran. The degradation of dextran-conjugated enzyme was reduced by 35% in hepatocytes and by 43% in nonparenchymal cells, after 80 and 40 min, respectively, as compared with the free enzyme. However, there was insignificant difference between the uptake of native and conjugated enzyme into the liver cells. Upon intravenous infusion into rats, native and conjugated enzyme were cleared from plasma with only a slight difference in the clearance rate. The observed stability of dextran-conjugated beta-galactosidase towards cellular degradation was in accordance with the in vitro experiments. The conjugate showed marked thermal stability at 50 degrees C and enhanced resistance towards proteolysis by the broad specific protease subtilopeptidase A. This demonstrates that dextran conjugation may be used as a means of stabilizing lysosomal enzymes for therapeutic purposes.

Heparin coupled to albumin, dextran and ficoll: Influence on blood coagulation and platelets, and in vivo duration

Abstract Heparin coupled to albumin, ficoll and dextran by the CNBr method exerts anticoagulant and blood platelet aggretating effects which deviate from that of free heparin. Heparin-albumin complex had the same anticoagulant and aggregating effect as heparin. Heparin-dextran complex had a weaker anticoagulant and aggregating effect than heparin. Heparin-ficoll complex had the same effect as heparin in the thrombin clotting time and Thrombotest systems, but a stronger anticoagulant effect in the APTT-system, and a weaker platelet aggregating effect than heparin. After intra venous injection in rabbits, the half life of the heparin-albumin, heparin-dextran and heparin-ficoll complexes were about 120, 110 and 160 per cent, respectively, of the half life of uncomplexed heparin.

Photodynamic Effects And Hyperthermia In Vitro

Cells from the established human line NHIK 3025 were labelled with hematoporphyrin derivative in vitro. Subsequently, the cells were treated with light and hyperthermia. The cells could be irradiated either before, during or after the incubation at a hyperthermic temperature. It was shown that hyperthermia given shortly after the light exposure gave a synergistic killing effect. In spite of some loss of porphyrins from the cells, the light sensitivity increased 20 min after a light irradiation. At later times, the cells apparently repaired some of the photodynamic damage at 37 degrees C. At higher temperatures, the repair was inhibited.

On the significance of the carbohydrate moieties of bovine prothrombin for clotting activity

Purified bovine prothrombin has been treated with different mixtures of glycosidases. Upon incubation of the prothrombin for 30 h with a combination of neuraminidase, alpha- and beta-galactosidase and beta-N-acetylglucosaminidase in 4 mM diisopropylfluorophosphate at pH 5.3 and 30 degrees C, about 70% of the carbohydrates were removed without affecting the coagulation activity. All the sialic acid and about half of the mannose, galactose and glucosamine residues were removed by this treatment.