In previous studies hepatocytes undergoing cell death by apoptosis but not normal hepatocytes in rat liver showed immunostaining for transforming growth factor ,1 (TGF-13). Staining was much stronger with antibodies recognizing the pro-region of TGF-I31 than the mature peptide itself. Therefore we investigated the ability of both forms of TGF-31 to induce apoptosis in primary cultures of rat hepatocytes.Mature TGF-fi1 induced rounding up of the cells and fragmentation into multiple vesicles. As revealed by the DNAspecific stain H33258, the chromatin of these cells condensed and segregated into masses at the nuclear membrane; this was obviously followed by fragmentation of the nucleus. Ultrastructurally the cytoplasm was well preserved, as demonstrated by the presence of intact cell organelles. These features strongly suggest the occurrence of apoptosis. Quantification of nuclei with condensed chromatin revealed that mature TGF-131 was 30-fold more effective than the TGF-P1 latency-associated protein complex. Finally, we administered TGF-131 in vivo using an experimental model in which regression of rat liver was initiated by a short preceding treatment with the hepatomitogen cyproterone acetate. Two doses of TGF-P1, each 1 nM/kg, augmented the incidence of apoptotic hepatocytes 5-fold. Equimolar doses of TGF-fi1 latency-associated protein complex were ineffective. These studies suggest that TGF-P1 is involved in the initiation of apoptosis in the liver and that the mature form of TGF-fi1 is the active principle.

Abstract. Chromatin condensation paralleled by DNA fragmentation is one of the most important criteria which are used to identify apoptotic cells. However, comparable changes are also observed in interphase nuclei which have been treated with cell extracts from mitotic cells. In this respect it is known that in mitosis, the lamina structure is broken down as a result of lamin solubilization and it is possible that a similar process is happening in apoptotic cells. The experiments described in this study have used confluent cultures of an embryonic fibroblast cell line which can be induced to undergo either apoptosis at low serum conditions or mitosis.Solubilization of lamin A+B was analyzed by immunoblotting and indirect immunofluorescence. These studies showed that in mitotic cells lamina breakdown is accompanied by lamin solubilization. In apoptotic cells, a small amount of lamin is solubilized before the onset of apoptosis, thereafter, chromatin condensation is accompanied by degradation of lamin A+B to a 46-kD fragment. Analysis of cellular lysates by probing blots with anti-PSTAIR followed by anti-phosphotyrosine showed that in contrast to mitosis, dephosphorylation on tyrosine residues did not occur in apoptotic cells. At all timepoints after the onset of apoptosis there was no significant increase in the activation of p34 ~2 as determined in the histone H1 kinase assay. Coinduction of apoptosis and mitosis after release of cells from aphidicolin block showed that apoptosis could be induced in parallel with S-phase.The sudden breakdown of chromatin structure may be the result of detachment of the chromatin loops from their anchorage at the nuclear matrix, as bands of 50 kbp and corresponding multimers were detectable by field inversion gel electrophoresis (FIGE). In apoptotic cells all of the DNA was fragmented, but only 14% of the DNA was smaller than 50 kbp. DNA strand breaks were detected at the periphery of the condensed chromatin by in situ tailing (ISTAIL). Chromatin condensation during apoptosis appears to be due to a rapid proteolysis of nuclear matrix proteins which does not involve the p34 ~c2 kinase.OPTOSIS waS originally defined as a program of morphological changes accompanying cell death during which the cells round up and the chromatin condenses, leading to the formation of crescent-shaped masses aggregating at the membrane. In parallel the nucleolus dissolves (Kerr, 1971). These initial changes are followed by cytoplasmic and nuclear compaction, and ultimately by fragmentation of the cell. The nuclear fragments formed in this process are still surrounded by the nuclear membranes (Kerr, 1971;Oberhammer et al., 1993a). As a common biochemical marker for apoptosis, an activation of a nonlysosomal, Ca2+/MgZ÷-dependent endonuclease has been suggested (Wyllie, 1980;Cohen and Duke, 1984). The response is so readily identifiable that endonuclease activation

The high density lipoprotein (HDL) receptor, scavenger receptor class B, type I (SR-BI), mediates selective cholesteryl ester uptake from lipoproteins into liver and steroidogenic tissues but also cholesterol efflux from macrophages to HDL. Recently, we demonstrated the uptake of HDL particles in SR-BI overexpressing Chinese hamster ovarian cells (ldlA7-SRBI) using ultrasensitive microscopy. In this study we show that this uptake of entire HDL particles is followed by resecretion. After uptake, HDL is localized in endocytic vesicles and organelles en route to the perinuclear area; many HDL-positive compartments were classified as multivesiculated and multilamellated organelles by electron microscopy. By using 125 I-labeled HDL, we found that ϳ0.8% of the HDL added to the media is taken up by the ldlA7-SRBI cells within 1 h, and almost all HDL is finally resecreted.125 I-Labeled low density lipoprotein showed a very similar association, uptake, and resecretion pattern in ldlA7-SRBI cells that do not express any low density lipoprotein receptor. Moreover, we demonstrate that the process of HDL cell association, uptake, and resecretion occurs in three physiologically relevant cell systems, the liver cell line HepG2, the adrenal cell line Y1BS1, and phorbol myristate acetate-differentiated THP-1 cells as a model for macrophages. Finally, we present evidence that HDL retroendocytosis represents one of the pathways for cholesterol efflux.Numerous studies have demonstrated the protective role of HDL 4 in the development of atherosclerosis and coronary artery disease (for review see Ref. 1). HDL exerts this atheroprotective effect mainly by transporting cholesterol from peripheral tissues back to the liver for biliary secretion, in a process referred to as "reverse cholesterol transport" (2). Moreover, HDL represents an important source of cholesterol for adrenal steroid hormone synthesis. The molecular details of the efflux of cellular cholesterol in the periphery and of cholesterol delivery to hepatocytes and adrenal cells are not completely understood. In particular, the fate and route of HDL particles taken up by cells and the physiological relevance of this process have not been delineated.The scavenger receptor class B, type I (SR-BI), a cell surface glycoprotein that binds HDL, LDL, very low density lipoprotein, modified LDL, and anionic phospholipids (3-6), can mediate the last step in reverse cholesterol transport, namely the delivery of cholesteryl esters from HDL to liver without HDL degradation, termed selective cholesteryl ester uptake (7-9). SR-BI is highly expressed in liver, adrenals, and ovaries with the highest mass of SR-BI protein localized in the liver (7). In addition to cholesterol uptake, SR-BI participates in the internalization of hepatitis C virus particles (10, 11) and lipopolysaccharide (12, 13). Serum amyloid A, a ligand of SR-BI, blocks the selective cholesteryl ester uptake (14). Furthermore, several small chemical inhibitors termed BLTs (block lipid transports) have been described to enh...

The mechanism of transport through the Golgi complex is not completely understood, insofar as no single transport mechanism appears to account for all of the observations. Here, we compare the transport of soluble secretory proteins (albumin and α1-antitrypsin) with that of supramolecular cargoes (e.g., procollagen) that are proposed to traverse the Golgi by compartment progression–maturation. We show that these soluble proteins traverse the Golgi much faster than procollagen while moving through the same stack. Moreover, we present kinetic and morphological observations that indicate that albumin transport occurs by diffusion via intercisternal continuities. These data provide evidence for a transport mechanism that applies to a major class of secretory proteins and indicate the co-existence of multiple intra-Golgi trafficking modes.DOI: http://dx.doi.org/10.7554/eLife.02009.001

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