Subfractionation of rat liver plasma membrane: Uneven distribution of plasma membrane-bound enzymes on the liver cell surface

Plasma membranes were islotaed from rat liver mainly under isotonic conditions. As marker enzymes for the plasma membrane, 5′-nucleotidase and (Na⁺+K⁺)-ATPase were used. The yield of plasma membrane was 0.6–0.9 mg protein per g wet weight of liver. The recovery of 5′-nucleotidase and (Na⁺+K⁺)-ATPase activity was 18 and 48% of the total activity of the whole-liver homogenate, respectively. Judged from the acitvity of glucose-6 phosphatase and succinate dehydrogenase in the plasma membrane, and from the electron microscopic observation of it, the contamination by microsomes and mitochondria was very low. A further homogenization of the plasma membrane yielded two fractions, the light and heavy fractions, in a discontinuous sucrose gradient centrifugation. The light fraction showed higher specific activities of 5′-nucleotidase, alkaline phosphatase, (Na⁺+K⁺)-ATPase and Mg²⁺-ATPase, whereas the heavy one showed a higher specific activity of adenylate cyclase. Ligation of the bile duct for 48 h decreased the specific activities of (Na⁺+K⁺)-ATPase and Mg²⁺-ATPase in the light fraction, whereas it had no significant influence on the activities of these enzymes in the heavy fraction. The specific activity of alkaline phosphatase was elevated in both fractions by the obstruction of the bile flow. Electron microscopy on sections of the plasma membrane subfractions showed that the light fraction consisted of vesicles of various sizes and that the heavy fractions contained membrane sheets and paired membrane strips connected by junctional complexes, as well as vesicles. The origin of these two fractions is discussed and it is suggested that the light fraction was derived from the bile front of the liver cell surface and the heavy one contained the blood front and the lateral surface of it.     

Subfractionation of rat liver plasma membrane. Uneven distribution of plasma membrane-bound enzymes on the liver cell surface.

Plasma membranes were isolated from rat liver mainly under isotonic conditions. As marker enzymes for the plasma membrane, 5'-nucleotidase and (Na+ + K+)-ATPase were used. The yield of plasma membrane was 0.6-0.9 mg protein per g wet weight of liver. The recovery of 5'-nucleotidase and (Na+ +K+)-ATPase activity was 18 and 48% of the total activity of the whole-liver homogenate, respectively. Judged from the activity of glucose-6-phosphatase and succinate dehydrogenase in the plasma membrane, and from the electron microscopic observation of it, the contamination by microsomes and mitochondria was very low. A further homogenization of the plasma membrane yielded two fractions, the light and heavy fractions, in a discontinuous sucrose gradient centrifugation. The light fraction showed higher specific activities of 5'-nucleotidase, alkaline phosphatase, (Na+ +K+)-ATPase and Mg2+-ATPase, whereas the heavy one showed a higher specific activity of adenylate cyclase. Ligation of the bile duct for 48 h decreased the specific activities of (Na2+ +K+)-ATPase and Mg2+-ATPase in the light fraction, whereas it had no significant influence on the activities of these enzymes in the heavy fraction. The specific activity of alkaline phosphate was elevated in both fractions by the obstruction of the bile flow. Electron microscopy on sections of the plasma membrane subfractions showed that the light fraction consisted of vesicles of various sizes and that the heavy fractions contained membrane sheets and paired membrane strips connected by junctional complexes, as well as vesicles. The origin of these two fractions is discussed and it is suggested that the light fraction was derived from the bile front of the liver cell surface and the heavy one contained the blood front and the lateral surface of it.     

Purification and identification of intermediate catabolic products in the in vivo degradation of pig liver phosphofructokinase

Phosphofructokinase (ATP:D-fructose-6-phosphate 1-phosphotransferase, EC 2.7.1.11) was purified to homogeneity from pig livers. Polyclonal antibody against the enzyme was induced in a rabbit, and the IgG fraction was obtained by chromatography on a Protein A-Sepharose CL-4B column. The specific antibody was purified further by immunoaffinity chromatography on a phosphofructokinase-conjugated affinity column. Intermediate catabolic products of phosphofructokinase were extracted from fresh pig livers under conditions of inhibition of proteinases, concentrated by chromatography on an anti-phosphofructokinase IgG-conjugated affinity column, and purified by two-dimensional polyacrylamide gel electrophoresis. Their cross-reactivities to the purified phosphofructokinase were assessed by an immunoelectrotransfer blot method. The intact form of phosphofructokinase in pig liver was demonstrated as the major spot of 84 kDa on the blot. Polypeptides of 68, 64, 56, and 51 kDa showed apparent cross-reactivities to phosphofructokinase. The structural homology among them was confirmed by proteinase V8 digestion followed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The possibility of artifacts in preparation was ruled out by an internal tracer method. Thus, it is concluded that the predominant isozyme of phosphofructokinase in pig liver (84 kDa) is in vivo degraded via intermediate catabolic products of 68, 64, 56, and 51 kDa.     

Effect of aluminum on iron-induced lipid peroxidation and protein oxidative modification of mouse brain homogenate

In the present study the authors report on the enhancing effect of aluminum(III) (Al[III]) on iron(II)(Fe[II])-induced lipid peroxidation (LPO) of mice brain homogenate, which occurs in a concentration and time-dependent manner. No evidence of LPO caused by Al alone was found. Both Al(III) and Fe(II) ions induced protein oxidative modifications in mice brain homogenate, in a time and concentrationdependent manner. Aluminum enhances Fe(II)-induced protein oxidative modification at a concentration of 2:1 and 1:1 Al:Fe molar ratios. However, Al suppress Fe(II)-induced protein oxidative modification at a concentration of 0.5:1 Al:Fe molar ratio. Addition of ethylenediaminetetraacetic acid (EDTA) inhibits both LPO and protein oxidative modifications induced by Al(III) and Fe(II) ions. Addition of mannitol and of Superoxide dismutase (SOD) did not show such effects. It is concluded that in mice brain homogenate, Al accelerates Fe(II)-induced LPO. Protein oxidative modifications caused by Fe(II) and/or Al ions are enhanced at high, but suppressed at low concentrations of Al ions. The latter observation suggests a possible biological role of Al as an antioxidant.     

Intra-strain biological and epidemiological characterization of plum pox virus

Plum pox virus (PPV) is one of the most important plant viruses causing serious economic losses. Thus far, strain typing based on the definition of 10 monophyletic strains with partially differentiable biological properties has been the sole approach used for epidemiological characterization of PPV. However, elucidating the genetic determinants underlying intra-strain biological variation among populations or isolates remains a relevant but unexamined aspect of the epidemiology of the virus. In this study, based on complete nucleotide sequence information of 210 Japanese and 47 non-Japanese isolates of the PPV-Dideron (D) strain, we identified five positively selected sites in the PPV-D genome. Among them, molecular studies showed that amino acid substitutions at position 2,635 in viral replicase correlate with viral titre and competitiveness at the systemic level, suggesting that amino acid position 2,635 is involved in aphid transmission efficiency and symptom severity. Estimation of ancestral genome sequences indicated that substitutions at amino acid position 2,635 were reversible and peculiar to one of two genetically distinct PPV-D populations in Japan. The reversible amino acid evolution probably contributes to the dissemination of the virus population. This study provides the first genomic insight into the evolutionary epidemiology of PPV based on intra-strain biological variation ascribed to positive selection.     

Plant growth regulators and Axl and immune checkpoint inhibitors from the edible mushroom Leucopaxillus giganteus

ABSTRACT

A novel compound, (R)-4-ethoxy-2-hydroxy-4-oxobutanoic acid (1), and six known compounds (2–7) were isolated from the fruiting bodies of the wild edible mushroom Leucopaxillus giganteus. The planar structure of 1 was determined by the interpretation of spectroscopic data analysis. The absolute configuration of 1 was determined by comparing specific rotation of the synthetic compounds. In the plant regulatory assay, the isolated compounds (1–7) and the chemically prepared compounds (8–10) were evaluated their biological activity against the lettuce (Lactuca sativa) growth. Compounds 1 and 3–10 showed the significant regulatory activity of lettuce growth. 1 showed the strongest inhibition activity among the all the compounds tested. In the lung cancer assay, all the compounds were assessed the mRNA expression of Axl and immune checkpoints (PD-L1, PD-L2) in the human A549 alveolar epithelial cell line by RT-PCR. Compounds 1–10 showed significant inhibition activity against Axl and/or immune checkpoint.     

Dietary GABA induces endogenous synthesis of a novel imidazole peptide homocarnosine in mouse skeletal muscles

Amino Acids - Carnosine (β-alanyl-l-histidine) is an imidazole dipeptide present at high concentrations in skeletal muscles, where it plays a beneficial role. However, oral intake of carnosine...     

Automatic exposure control at MDCT based on the contrast-to-noise ratio: Theoretical background and phantom study

PurposeTo develop a new automatic exposure control (AEC) technique based on the contrast-to-noise ratio (CNR) and provide constant lesion detectability.MethodsLesion detectability is affected by factors such as image noise, lesion contrast, and lesion size. We performed ROC analysis to assess the relationship between the optimum CNR and the lesion diameter at various levels of lesion contrast. We then developed a CNR-based AEC algorithm based on lesion detectability. Using CNR- based AEC algorithm, we performed visual evaluation of low-contrast detectability by 5 radiologists on a low-contrast module of the Catphan phantom, a contrast-difference level of 1.0% (difference in the CT number = 10 HU), and objects 3.0–9.0 mm in diameter.ResultsOn step-and-shoot scans the mean detection fraction with CNR-based AEC remained almost constant from 88 to 99 % regardless of the lesion size. We observed the same trend on helical scans, the mean detection fraction with CNR-based AEC exhibited a high score from 91 to 100%. Although CNR-based AEC maintains higher CNR for smaller size or lower contrast lesion, radiation dose on 3 mm lesion resulted in about 13 times larger than that of 9 mm lesion size. CTDI_vol for the CNR-based AEC technique changed dramatically with the SD_Z from 7.5 to 100.0 mGy for step-and-shoot scans and from 9.1 to 121.5 mGy for helical scans.ConclusionsFrom the viewpoint of ROC analysis-based CNR for lesion detection, CNR-based AEC potentially provide image quality advantages for clinical implementation.