Arginase,an s-phase enzyme in a human cell line

Suspension cultures of ‘Chang liver’ cells were synchronized by preincubation in a glutamine-deficient medium or by thymidine blockade. Specific arginase activity varied in the synchronized cultures, being high when the number of S-phase cells was maximal. A relationship between high arginase activity and a high percentage of (S+G₂) cells was also found when unsynchronized cells were separated by velocity sedimentation. The increase in arginase activity near the G₁/S border was totally inhibited in the presence of cycloheximide. The rate of decrease in activity after addition of the drug indicated that the variations in the rate of synthesis of the enzyme, while the rate of degradation was more or less constant, corresponding to 4–6% per h. The role of arginase in cells lacking a urea cycle and the regulation of arginase activity in ‘Chang liver’ cells is discussed.     

Early changes in mitochondrial monoamine oxidase activity of rat liver during 2-acetylaminofluorene feeding

The activities of mitochondrial type A and B monoamine oxidase were determined in the liver of rats fed a diet containing 2-acetylaminofluorene (AAF). Three days after the initiation of AAF-feeding, there was a significant decrease of type B monoamine oxidase activity without affect on type A enzyme. The decreased activity of type B monoamine oxidase, which reached a minimum after three weeks, was sustained for as long as AAF-feeding was continued. Sex-related difference in response to AAF was seen in the rat with respect to the onset and the intensity of the decreased type B monoamine oxidase activity, male rats being more sensitive to the carcinogen than female rats. In contrast to the in vivo effect, AAF showed a potent inhibitory effect on type A monoamine oxidase, rather than on type B enzyme, when added in vitro. The pI₅₀ values were estimated to be 7.5 against type A monoamine oxidase and 4.1 against type B enzyme, respectively. The in vitro inhibition of both types of monoamine oxidase by AAF was competitive. The K_i values for AAF were calculated to be 9.51 · 10^?9 M for type A monoamine oxidase and 1.30 · 10^?5 M for type B enzyme, respectively. In accordance with the potent inhibitory effect of AAF on type A monoamine oxidase in vitro, a single administration of the carcinogen, at a dose of 50 mg/kg, resulted in a marked and temporal decrease of the enzyme activity in the mitochondria of male rat liver. Recovery of the decreased type B monoamine oxidase activity was slow, and the enzyme activity did not return to control levels, even if rats were fed the basal diet for 2 or 4 weeks after the cessation of AAF-feeding.     

Kinetic characteristics of lipids of mammalian tissues in autooxidation reactions

We generalize the results of multiyear studies of the level of antioxidative activity of lipids isolated from tissues of laboratory rodents of different species and lines. A classification of lipids according to their ability to inhibit thermal autooxidation of methyl oleate is proposed. The involvement of lipids in low-temperature autooxidation reactions at the radical initiation and chain propagation stages was proved using the model developed. In addition to antioxidative activity, the initial content of peroxides in lipids, determined by their degree of unsaturation, and the antiperoxide activity of lipids are proposed for quantitative estimation of the kinetic characteristics of lipids of mammalian tissues. The dependence of effects on the rate of radical initiation in the system is shown, which is determined by the influence of the physicochemical properties of lipids on the coordination of relationships and balance of biochemical functions in biological objects differing in the intensity of oxidation processes.     

Differential expression of microRNAs in mouse liver under aberrant energy metabolic status

Despite years of effort, exact pathogenesis of nonalcoholic fatty liver disease (NAFLD) remains obscure. To gain an insight into the regulatory roles of microRNAs (miRNAs) in aberrant energy metabolic status and pathogenesis of NAFLD, we analyzed the expression of miRNAs in livers of ob/ob mice, streptozotocin (STZ)-induced type 1 diabetic mice, and normal C57BL/6 mice by miRNA microarray. Compared with normal C57BL/6 mice, ob/ob mice showed upregulation of eight miRNAs and downregulation of four miRNAs in fatty livers. Upregulation of miR-34a and downregulation of miR-122 was found in livers of STZ-induced diabetic mice. These results demonstrate that distinct miRNAs are strongly dysregulated in NAFLD and hyperglycemia. Comparison between miRNA expressions in livers of ob/ob mice and STZ-administered mice further revealed upregulation of four miRNAs and downregulation of two miRNAs in livers of ob/ob mice, indicating that these miRNAs may represent a molecular signature of NAFLD. A distinctive miRNA expression pattern was identified in ob/ob mouse liver, and hierarchical clustering of this pattern could clearly discriminate ob/ob mice from either normal C57BL/6 mice or STZ-administered mice. These findings suggest an important role of miRNAs in hepatic energy metabolism and implicate the participation of miRNAs in the pathophysiological processes of NAFLD.     

A novel multi-coaxial hollow fiber bioreactor for adherent cell types. Part 1: hydrodynamic studies.

A novel multi-coaxial bioreactor for three-dimensional cultures of adherent cell types, such as liver, is described. It is composed of four tubes of increasing diameter placed one inside the other, creating four spatially isolated compartments. Liver acinar structure and physiological parameters are mimicked by sandwiching cells in the space between the two innermost semi-permeable tubes, or hollows fibers, and creating a radial flow of media from an outer compartment (ECC), through the cell mass compartment, and to an inner compartment (ICC). The outermost compartment is created by gas-permeable tubing, and the housing is used to oxygenate the perfusion media to periportal levels in the ECC. Experiments were performed using distilled water to correlate the radial flow rate (Q(r)) with (1) the pressure drop (DeltaP) between the media compartments that sandwich the cell compartment and (2) the pressure in the cell compartment (P(c)). These results were compared with the theoretical profile calculated based on the hydraulic permeability of the two innermost fibers. Phase-contrast velocity-encoded magnetic resonance imaging was used to visualize directly the axial velocities inside the bioreactor and confirm the assumptions of laminar flow and zero axial velocity at the boundaries of each compartment in the bioreactor. Axial flow rates were calculated from the magnetic resonance imaging results and were similar to the measured axial flow rates for the previously described experiments.     

Fasciola hepatica: effect of 4-amino-6-trichloroethenyl-1,3-benzenedisulfonamide on glycolysis in vitro

In vitro, 4-amino-6-trichloroethenyl-1,3-benzenedisulfonamide, a potent fasciolicide, causes a potent concentration-dependent inhibition of glucose uptake by mature Fasciola hepatica. In F. hepatica treated with the disulfonamide and then fed [U-¹⁴C]glucose, there was a 60% inhibition of glucose utilization and a corresponding inhibition of acetate and propionate formation. Treated fluke parasites possessed much lower levels of adenosine triphosphate, phosphoenolpyruvate, glucose 6-phosphate, and fructose 6-phosphate than untreated parasites and contained higher levels of glycerol and the free sugars fructose and mannose. Direct measurement of the effect of the disulfonamide on the glycolytic enzymes of F. hepatica demonstrated that 3-phosphoglycerate kinase (EC 2.7.2.3) and phosphoglyceromutase (EC 2.7.5.3) were inhibited. It is therefore suggested that the fasciolicidal activity of 4-amino-6-trichloroethenyl-1, 3-benzenedisulfonamide is due to inhibition of the enzymes 3-phosphoglycerate kinase and phosphoglyceromutase which effectively blocks the Embden-Myerhof glycolytic pathway.     

Fluorescence anisotropy from diphenylhexatriene in rat liver plasma membranes

Rat livers were fractionated to obtain intracellular membrane preparations and a highly purified preparation of bile canaliculi. The fraction containing bile canaliculi was homogenized and subfractionated to give fractions representing fragments of contiguous membrane and of canalicular microvilli. The relative purity and extent of contamination of each preparation was determined. When the fluorescent probe 1,6-diphenyl-1,3,5-hexatriene was incorporated into aliquots of each fraction at the same probe: lipid ratio and the steady-state anisotropy of its fluorescence measured, it was found that the plasma membrane preparations were much more ordered than the intracellular membrane preparations. Of the plasma membrane preparations, that containing the canalicular microvilli was the most ordered, even allowing for any contribution of contaminants. Thus the microvillus membrane of the bile canaliculus appears to be the most ordered domain of the plasma membrane of the hepatocyte. The high order in this domain may be a factor in reducing the susceptibility to bile salt damage during bile secretion, since it is this region which is exposed to high concentrations of bile salts in vivo.