Hepatic metabolism of oxidatively modified apo E-free high-density lipoproteins

ABSTRACT— Aims/Background: The metabolism of rat apo E-free high-density lipoproteins (HDL) was contrasted with oxidatively modified apo E-free high-density lipoproteins (OX-HDL) in the rat hepatoma cell, Fu5AH. Results: When 10–100 μg/ml [¹²⁵I]-HDL or [¹²⁵I]-OX-HDL were incubated with cells for 4 h at 37°C, cellular uptake of oxidized lipoproteins was twice control. In contrast, protein degradation was equal. [¹²⁵I]-HDL or [¹²⁵I]-OX-HDL were incubated with the cells for 4 h followed by a 4 h chase with unlabeled HDL and OX-HDL, respectively. In these experiments, 80% of [¹²⁵I]-HDL was resecreted from the cell within 30 min while 50% of [¹²⁵I]-OX-HDL was retained by the cell after 2 h. Electron microscopy was used to determine if the OX-HDL was retained in lysosomes. Cells were incubated with gold-labeled OX-HDL, and lysosomes were stained with acid phosphatase. Gold-labeled OX-HDL was abundant in intracellular vesicles that were not reactive to acid phosphatase. However, vesicles with a high content of OX-HDL frequently stained positively for 3,3′-diaminobenzidine, a stain that reacts with catalase and is used to detect peroxisomes. Conclusions: The present evidence indicates that the cellular metabolism of OX-HDL is different from that of unmodified HDL.     

巨噬细胞过氧化物酶体与鼠伤寒沙门菌相互作用的初步分析

目的观察巨噬细胞过氧化物酶体在鼠伤寒沙门菌感染中的作用。方法将感染及未感染鼠伤寒沙门菌的鼠巨噬细胞RAW264.7以氮气压迫法裂解,逐级离心,用制备的TassC多抗磁珠纯化得到粗提液,用Western blot法鉴定TassC多抗磁珠与细胞结合物的性质;用pDs Red2-Perxi质粒转染RAW264.7细胞以标记细胞中的过氧化物酶体,用三维荧光显微镜观察感染细胞中过氧化物酶体(红色)与表达绿色荧光蛋白的鼠伤寒沙门菌突变株的相互作用。结果Western blot分析显示TassC多抗磁珠可以结合细胞内的TassC,并与细胞内的过氧化物酶体结合,也可以与感染细胞中的iNOS结合,而未感染样本中未检测到iNOS。磁珠洗脱物中未检测到溶酶体标志物LAMP1,也未检测到受染菌标志物;免疫荧光显示感染的巨噬细胞中过氧化物酶体聚集在鼠伤寒沙门菌突变株SCV周围,甚至发生重叠,随感染时间延长(1h或24h),过氧化物酶体数量增加(1.2倍或1.3倍),而胞内菌数量下降。结论TassC可能定位于过氧化物酶体而不是溶酶体;感染鼠伤寒沙门菌的巨噬细胞中iNOS可能与过氧化物酶体结合而参与杀灭微生物的作用。     

Localization of mRNAs coding for peroxisomal proteins in the yeast,Saccharomyces cerevisiae

Targeted mRNA trafficking and local translation may play a significant role in controlling protein localization. Here we examined for the first time the localization of all (≈50) mRNAs encoding peroxisomal proteins (mPPs) involved in peroxisome biogenesis and function. By using the bacteriophage MS2-CP RNA-binding protein (RBP) fused to multiple copies of GFP, we demonstrated that >40 endogenously expressed mPPs tagged with the MS2 aptamer form fluorescent RNA granules in vivo. The use of different RFP-tagged organellar markers revealed 3 basic patterns of mPP granule localization: to peroxisomes, to the endoplasmic reticulum (ER), and nonperoxisomal. Twelve mPPs (i.e., PEX1, PEX5, PEX8, PEX11–15, DCI1, NPY1, PCS60, and POX1) had a high percentage (52%–80%) of mRNA colocalization with peroxisomes. Thirteen mPPs (i.e., AAT2, PEX6, MDH3, PEX28, etc.) showed a low percentage (30%–42%) of colocalization, and 1 mPP (PEX3) preferentially localized to the ER. The mPPs of the nonperoxisomal pattern (i.e., GPD1, PCD1, PEX7) showed ≪30% colocalization. mPP association with the peroxisome or ER was verified using cell fractionation and RT-PCR analysis. A model mPP, PEX14 mRNA, was found to be in close association with peroxisomes throughout the cell cycle, with its localization depending in part on the 3′-UTR, initiation of translation, and the Puf5 RBP. The different patterns of mPP localization observed suggest that multiple mechanisms involved in mRNA localization and translation may play roles in the importation of protein into peroxisomes.     

DIETARY FAT, ACYL COA LEVELS AND PEROXISOMAL β-OXIDATION

Peroxisomal β-oxidation of long-chain fatty acids is influenced by both amounts and types of dietary fat. The fat oxidation activity of hepatic peroxisomes appears to be correlated with acyl CoA levels.     

Dengue and Zika Virus Capsid Proteins Contain a Common PEX19-Binding Motif

Flaviviruses such as dengue virus (DENV) and Zika virus (ZIKV) have evolved sophisticated mechanisms to suppress the host immune system. For instance, flavivirus infections were found to sabotage peroxisomes, organelles with an important role in innate immunity. The current model suggests that the capsid (C) proteins of DENV and ZIKV downregulate peroxisomes, ultimately resulting in reduced production of interferons by interacting with the host protein PEX19, a crucial chaperone in peroxisomal biogenesis. Here, we aimed to explore the importance of peroxisomes and the role of C interaction with PEX19 in the flavivirus life cycle. By infecting cells lacking peroxisomes we show that this organelle is required for optimal DENV replication. Moreover, we demonstrate that DENV and ZIKV C bind PEX19 through a conserved PEX19-binding motif, which is also commonly found in cellular peroxisomal membrane proteins (PMPs). However, in contrast to PMPs, this interaction does not result in the targeting of C to peroxisomes. Furthermore, we show that the presence of C results in peroxisome loss due to impaired peroxisomal biogenesis, which appears to occur by a PEX19-independent mechanism. Hence, these findings challenge the current model of how flavivirus C might downregulate peroxisomal abundance and suggest a yet unknown role of peroxisomes in flavivirus biology.     

Effects of di-isononyl phthalate, di-2-ethylhexyl phthalate, and clofibrate in cynomolgus monkeys.

The effects of the peroxisome proliferators di-isononyl phthalate (DINP) and di-2-ethylhexyl phthalate (DEHP) were evaluated in young adult male cynomolgus monkeys after 14 days of treatment, with emphasis on detecting hepatic and other effects seen in rats and mice after treatment with high doses of phthalates. Groups of 4 monkeys received DINP (500 mg/kg/day), DEHP (500 mg/kg/day), or vehicle (0.5% methyl cellulose, 10 ml/kg) by intragastric intubation for 14 consecutive days. Clofibrate (250 mg/kg/day), a hypolipidemic drug used for cholesterol reduction in human patients was used as a reference substance. None of the test substances had any effect on body weight or liver weights. Histopathological examination of tissues from these animals revealed no distinctive treatment-related effects in the liver, kidney, or testes. There were also no changes in any of the hepatic markers for peroxisomal proliferation, including peroxisomal beta-oxidation (PBOX) or replicative DNA synthesis. Additionally, in situ dye transfer studies using fresh liver slices revealed that DINP, DEHP, and clofibrate had no effect on gap junctional intercellular communication (GJIC). None of the test substances produced any toxicologically important changes in urinalysis, hematology, or clinical chemistry; however, clofibrate produced some emesis, small increases in serum triglyceride, decreased calcium, and decreased weights of testes/epididymides and thyroid/parathyroid. The toxicological significance of these small changes is questionable. The absence of observable hepatic effects in monkeys at doses that produce hepatic effects in rodents suggests that DINP, DEHP, and clofibrate would also not elicit in primates other effects such as liver cancer. These data, along with results from in vitro hepatocyte studies, indicate that rodents are not good animal models for predicting the hepatic effects of phthalates in primates, including humans.     

Analytical subcellular fractionation of normal human skeletal muscle by sucrose density gradient centrifugation

The principle organelle marker enzymes and various adenosine triphosphatase (ATPase) activities were studied in human skeletal muscle. The reproducibility of each assay was established under optimal and linear assay conditions. Whole homogenates of normal human quadriceps muscle were fractionated by centrifugation on a continuous sucrose density gradient. Gradient fractions were assayed for organelle marker enzymes and frequency-density histograms were constructed for each enzyme. Good resolution of the principal organelles was obtained. Adenosine triphosphatase (ATPase) was assayed under conditions of maximal stimulation by Ca2+, or Mg2+ or Na2+, K+ + Mg2+. The distribution of these activities was compared with those of the organelle marker enzymes. Both Ca2+-ATPase and Mg2+-ATPase were distributed to both the mitochondrial and myofibrillar fractions but could be distinguished by the inhibition of mitochondrial ATPase with sodium azide. The distribution of Na+, K+-activated, Mg2+-dependent ATPase (Na+, K+ ATPase) activity suggested a sarcolemmal localization. The results of electron microscopy of gradient fractions were consistent with the organelle content of the fractions as determined by enzymic analyses. These studies provide reference information for the subsequent investigation of organelle pathology of human muscle disorders.     

衰老对肌肉组织中过氧化体增殖物激活型受体-γ及葡萄糖转运子-4基础表达水平的影响

目的　观察衰老对肌肉组织中过氧化体增殖物激活型受体 γ(PPARγ)及葡萄糖转运子 4 (GLUT 4 )基础表达水平的影响,探讨其在胰岛素抵抗形成中的意义。方法　用Bergman创立的最小模型技术评价青年鼠(10～12周龄)和老年鼠(2 4月龄)的胰岛素敏感性,采用半定量逆转录聚合酶链反应(RT PCR)法检测肌肉组织中PPARγ1和PPARγ2 mRNA及GLUT 4mRNA的表达水平。结果　与青年鼠组比较,老年鼠组存在明显的胰岛素抵抗[胰岛素抵抗指数:(11.4 9±6 .92 )vs(5 .2 8±1.94 ) ,P <0 .0 5 ;胰岛素敏感指数:- (0 .0 5±0 .0 3)vs - (0 .0 2±0 .0 2 ) ,P <0 .0 5 ]。用RT PCR方法检测到骨骼肌中PPARγ1和PPARγ2 mRNA均有表达,而以PPARγ1mRNA为主要表达形式;与青年鼠组比较,老年鼠组PPARγ1mRNA [(0 .79±0 .0 4 ) vs(0 .5 2±0 .0 3) ,P <0 .0 1]及GLUT 4mRNA[(0 .73±0 .0 4 )vs(0 .6 1±0 .0 3) ,P <0 .0 5 ]的表达水平均明显降低。结论　衰老降低肌肉组织中PPARγmRNA及GLUT 4mRNA的基础表达水平,直接或间接使骨骼肌糖代谢作用受损,可能参与胰岛素抵抗的形成。     

Mitochondrial abnormalities in CLN2 and CLN3 forms of batten disease

The storage of subunit c of mitochondrial ATP synthase, other hydrophobic peptides, and autofluorescent pigment in both late infantile (CLN2) and juvenile (CLN3) neuronal ceroid lipofuscinosis, but not in infantile (CLN1), has raised the question of abnormal mitochondrial function. We now report a partial deficiency in three types of fatty acid oxidation in intact skin fibroblasts from CLN2 and CLN3 patients, but not CLN1. We observed a statistically significant 33% reduction in palmitate (β-oxidation; mainly mitochondrial) and lignocerate (β-oxidation; mainly peroxisomal), and a 50% reduction in phytanic acid (α-oxidation; mainly peroxisomal) in the absence of exogenous carnitine. In contrast, when we measured fatty acid β-oxidation (lignoceric acid and palmitic acid), in the same human skin fibroblasts, following lysis in the presence of carnitine, we found no difference in enzyme activity among normal, CLN1, CLN2, and CLN3. However, we did observe a 40% reduction in peroxisomal particulate (bound) catalase activity in CLN1 and CLN2 fibroblasts, which typically results from organellar lipid accumulation or a membrane abnormality. However, total catalase levels were normal, and western blot analysis of this and three other major oxidant protective enzymes (Mn-dependent superoxide dismutase [MnSOD], CuZn-dependent superoxide dismutase [CuZnSOD], and glutathione peroxidase) were normal in CLN1, CLN2, and CLN3, as well as in liver from an animal (English Setter dog) model for CLN, which shows similar pathology and subunit c storage. Our data showing differences between CLN1 and forms CLN2 and CLN3 suggest some type of mitochondrial membrane abnormality as the source of the pathology in CLN2 and CLN3.