scholarly journals Isolation of autophagic vacuoles from rat liver: morphological and biochemical characterization.

1982 ◽  
Vol 93 (1) ◽  
pp. 144-154 ◽  
Author(s):  
L Marzella ◽  
J Ahlberg ◽  
H Glaumann
Keyword(s):  
Proteolytic Activity ◽  
Rat Liver ◽  
Biochemical Characterization ◽  
Secretory Granules ◽  
Density Lipoprotein ◽  
Marker Enzymes ◽  
Cell Organelles ◽  
Protein Ratio ◽  
Autophagic Vacuoles

The induction of autophagy caused by vinblastine (VBL) has been found to be concomitant with a stimulation of proteolysis in a mitochondrial-lysosomal (ML) fraction from the rat liver (Marzella and Glaumann, 1980, Lab. Invest., 42: 8-17. Marzella and Glaumann, 1980, Lab. Invest., 42:18-27). In this fraction the enhanced proteolysis is associated with a threefold increase in the relative fractional volume of autophagic vacuoles (AVs). In an attempt to isolate the AVs, we subfractionated the ML suspension at different intervals after the induction of autophagy by VBL by centrifugation on a discontinuous Metrizamide gradient ranging from 50% to 15%. The material banding at the 24 to 20% and the 20 to 15% interphases was collected. Morphological analysis reveals that 3 h after induction of autophagy these fractions consist predominantly (approximately 90%) of intact autophagic vacuoles. These autophagic vacuoles contain cytosol, mitochondria, portions of endoplasmic reticulum, and occasional very low density lipoprotein, particles either free or in Golgi apparatus derivatives, in particular secretory granules. The sequestered materials show ultrastructural signs of ongoing degradation. In addition to containing typical autophagic vacuoles, the isolated fractions consist of lysosomes lacking morphologically recognizable cellular components. Contamination from nonlysosomal material is only a few percent as judged from morphometric analysis. Typical lysosomal "marker" enzymes are enriched 15-fold, whereas the proteolytic activity is enriched 10- to 20-fold in the isolated AV fraction as compared to the homogenate. Initially, the yield of nonlysosomal mitochondrial and microsomal enzyme activities increases in parallel with the induction of autophagy but, later on, decreases with advanced degradation of the sequestered cell organelles. Therefore, in the case of AVs the presence of nonlysosomal marker enzymes cannot be used for calculation of fraction purity, since newly sequestered organelles are enzymatically active. Isolated autophagic vacuoles show proteolytic activity when incubated in vitro. The comparatively high phospholipid/protein ratio (0.5) of the AV fraction suggests that phospholipids are degraded more slow than proteins. Is it concluded that AVs can be isolated into a pure fraction and are the subcellular site of enhanced protein degradation in the rat liver after induction of autophagy.

Uptake of cholesterol from the very low density lipoprotein or its remnants by the perfused rat liver

1981 ◽  
Vol 59 (6) ◽  
pp. 447-453 ◽  
Author(s):  
Simon-Pierre Noël ◽  
David Rubinstein
Keyword(s):  
Rat Liver ◽  
Lipid Composition ◽  
Low Density Lipoprotein ◽  
Liver Perfusion ◽  
Density Lipoprotein ◽  
Hepatic Uptake ◽  
Low Density ◽  
Perfused Liver ◽  
Very Low Density Lipoproteins

[3H]Cholesterol labelled very low density lipoproteins ([3H]chol-VLDL) were prepared to study the hepatic uptake of cholesterol associated with VLDL and its remnants in the perfused liver system. [3H]Chol-VLDL was incubated with rat postheparin plasma to produce labelled remnants in vitro. The degree of lipolysis of [3H]chol-VLDL depended on the ratio of triacylglycerols to lipase in the incubation medium. Therefore, the produced remnant of d < 1.019 g∙mL−1 had a variable lipid composition depending on the degree of lipolysis. [3H]Chol-VLDL or its remnants were added to liver perfusate and the radioactivity remaining in the perfusate was measured. The kinetic disappearance of [3H]chol-VLDL and its remnants in the perfused liver system indicated that remnant of d < 1.019 g∙mL−1 was taken up by the liver faster than the original VLDL preparation (t1/2 of 8 min vs. 51 min). Appearance of the label in bile during the perfusion was significantly faster when livers were perfused with [3H]chol-VLDL remnants as opposed to uncatabolized [3H]chol-VLDL.The results indicate that first of all, VLDL remnants produced in vitro and reisolated at density less than 1.019 g∙mL−1 do not have a fixed lipid composition but a rather variable one depending on the degree of lipolysis. Secondly, the rat liver may preferentially recognize this VLDL remnant of d < 1.019 g∙mL−1 and take it up more readily than uncatabolized VLDL. Finally when equimolar amount of cholesterol from VLDL or VLDL remnants are circulated in the liver perfusion, the VLDL remnants convey a significantly greater mass of cholesterol to the bile.

scholarly journals Altered hepatic catabolism of low-density lipoprotein subjected to lipid peroxidation in vitro

1994 ◽  
Vol 297 (3) ◽  
pp. 573-579 ◽  
Author(s):  
W L Stone ◽  
M Heimberg ◽  
R L Scott ◽  
I LeClair ◽  
H G Wilcox
Keyword(s):  
Lipid Peroxidation ◽  
Rat Liver ◽  
Low Density Lipoprotein ◽  
Liver Perfusion ◽  
Density Lipoprotein ◽  
Low Density ◽  
Perfusion System ◽  
Oxidatively Modified ◽  
Generating System

Recent evidence suggests that oxidatively modified forms of low-density lipoprotein (LDL) may be particularly atherogenic. In this investigation, the catabolism of human LDL modified by lipid peroxidation in vitro was studied with a recirculating rat liver perfusion system. A dual-labelling technique was used that permitted native LDL and modified LDL to be studied simultaneously in the liver perfusion system. Native human LDL was found to have a fractional catabolic rate (FCR) of 1.00 +/- 0.21%/h, in agreement with other investigators. Subjecting LDL to oxidation for 12 h in the presence of 30 microM FeEDTA did not significantly affect its FCR. LDL treated with a superoxide-generating system (xanthine oxidase, hypoxanthine, O2) in the presence of 30 microM FeEDTA did, however, show a significant increase in FCR (3.23 +/- 0.19%/h). The hepatic uptakes of native LDL and LDL oxidized with FeEDTA+O2 were similar, but both were significantly lower than the hepatic uptake of LDL treated with the superoxide-radical-generating system. The proteolysis of LDL with pancreatin did not influence either its susceptibility to oxidation or its FCR. LDL oxidation resulted in the preferential loss of alpha-tocopherol rather than gamma-tocopherol. These data indicate that the rat liver effectively catabolizes LDL oxidatively modified by treatment with the superoxide-generating system. Furthermore, our results suggest that only very low plasma levels of highly oxidized LDL could be found under conditions in vivo. The liver may therefore play a major role in protecting the arterial vasculature from highly atherogenic forms of LDL.

scholarly journals Characterization of binding sites for acetylated low density lipoprotein in the rat liver in vivo and in vitro.

1985 ◽  
Vol 4 (5) ◽  
pp. 1157-1162 ◽  
Author(s):  
H.A. Dresel ◽  
E. Friedrich ◽  
D.P. Via ◽  
G. Schettler ◽  
H. Sinn
Keyword(s):  
Rat Liver ◽  
Binding Sites ◽  
Low Density Lipoprotein ◽  
Density Lipoprotein ◽  
Low Density

Abstract 390: VLDL Exits From The Endoplasmic Reticulum In A Specialized VLDL-transporting-vesicle (VTV) In Rat Primary Hepatocytes.

Circulation ◽  
2007 ◽  
Vol 116 (suppl_16) ◽  
Author(s):  
Shadab A Siddiqi
Keyword(s):  
Endoplasmic Reticulum ◽  
Rat Liver ◽  
Density Lipoprotein ◽  
Protein Composition ◽  
Secretory Protein ◽  
Proteinase K ◽  
Primary Hepatocytes ◽  
Density Region ◽  
Continuous Sucrose Gradient

Transport of very low-density lipoprotein (VLDL) from its site of synthesis, the endoplasmic reticulum (ER), to the Golgi is required for its eventual secretion from hepatocytes. This step represents a potential therapeutic target in controlling VLDL export and thus control of its metabolic derivative, LDL, the major carrier of cholesterol and determinant of atherosclerosis. The present study was designed to understand how VLDL exits from the ER at the molecular level. We developed an in vitro ER-budding assay in which rat liver ER (500 ug) was pre-loaded with 14 C-triacylglycerol (TAG) to mark VLDL and 3 H-proteins to mark newly synthesized proteins. The ER was incubated with rat liver cytosol (1 mg), GTP, and ATP at 37 o C for 30 min. The reaction mix was fractionated on a continuous sucrose gradient and the distribution of 14 C-TAG and 3 H-protein across the gradient was determined. 14 C-TAG was found in the light density region of the gradient, the expected place for TAG-rich VLDL carrying vesicles whereas 3 H-proteins appeared in the mid portion, the expected place in the gradient for protein vesicles. We examined the distribution of apolipoprotein B100 (apoB100), a marker for VLDL and albumin (a typical liver secretory protein) across the same gradient by Western blotting. As expected, apoB100 was distributed in light fractions whereas albumin was mainly in the mid portion of the gradient. These data show that VLDL and albumin are transported in vesicles of differing density. We hypothesize that a specialized vesicle is utilized for VLDL transport, which we name the VLDL-transporting-vesicle (VTV). Our results show that the release of VTV from rat liver ER requires cytosol, GTP, Sar1 (a GTPase), ATP, and incubation at 37 o C. VTV was sealed as judged by apoB100 signal post proteinase K treatment. VTVs concentrate ApoB100, Sar1, and exclude ER-resident protein calnexin. VTV fuses with liver cis -Golgi and delivers its cargo, VLDL, to the Golgi lumen. 2D-gels and electron microscopy data reveal that VTVs are different in their protein composition and are larger in size when compared to albumin carrying vesicles. In conclusion, we have identified and characterized a new ER-derived vesicle, VTV, which transports nascent VLDL from the ER to the Golgi in primary hepatocytes.

The in vivo distribution and dynamics of DNA topoisomerase II in Drosophila embryonic nuclei and chromosomes

1993 ◽  
Vol 51 ◽  
pp. 74-75
Author(s):  
Jason R. Swedlow ◽  
Neil Osheroff ◽  
Tim Karr ◽  
John W. Sedat ◽  
David A. Agard
Keyword(s):  
Topoisomerase Ii ◽  
Biochemical Characterization ◽  
Developmental Cycle ◽  
Dna Topoisomerase Ii ◽  
Chromosomal Distribution ◽  
Dna Topoisomerase ◽  
Ideal System ◽  
Dnase Treatment

DNA topoisomerase II is an ATP-dependent double-stranded DNA strand-passing enzyme that is necessary for full condensation of chromosomes and for complete segregation of sister chromatids at mitosis in vivo and in vitro. Biochemical characterization of chromosomes or nuclei after extraction with high-salt or detergents and DNAse treatment showed that topoisomerase II was a major component of this remnant, termed the chromosome scaffold. The scaffold has been hypothesized to be the structural backbone of the chromosome, so the localization of topoisomerase II to die scaffold suggested that the enzyme might play a structural role in the chromosome. However, topoisomerase II has not been studied in nuclei or chromosomes in vivo. We have monitored the chromosomal distribution of topoisomerase II in vivo during mitosis in the Drosophila embryo. This embryo forms a multi-nucleated syncytial blastoderm early in its developmental cycle. During this time, the embryonic nuclei synchronously progress through 13 mitotic cycles, so this is an ideal system to follow nuclear and chromosomal dynamics.

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