To establish the process by which transplanted cells integrate into the liver parenchyma, we used dipeptidyl peptidase IV-deficient F344 rats as hosts. On intrasplenic injection, transplanted hepatocytes immediately entered liver sinusoids, along with attenuation of portal vein radicles on angiography. However, a large fraction of transplanted cells (Ͼ70%) was rapidly cleared from portal spaces by phagocyte/macrophage responses. On the other hand, transplanted hepatocytes entering the hepatic sinusoids showed superior survival. These cells translocated from sinusoids into liver plates between 16 and 20 hours after transplantation, during which electron microscopy showed disruption of the sinusoidal endothelium. Interestingly, production of vascular endothelial growth factor was observed in hepatocytes before endothelial disruptions. Portal hypertension and angiographic changes resulting from cell transplantation resolved promptly. Integration of transplanted hepatocytes in the liver parenchyma required cell membrane regenesis, with hybrid gap junctions and bile canaliculi forming over 3 to 7 days after cell transplantation. We propose that strategies to deposit cells into distal hepatic sinusoids, to disrupt sinusoidal endothelium for facilitating cell entry into liver plates, and to accelerate cell integrations into liver parenchyma will advance applications of hepatocyte transplantation. (HEPATOLOGY 1999;29:509-519.)

Phosphatase cytochemistry was used to distinguish between the Golgi apparatus and GERL (considered as a specialized region of endoplasmic reticulum [ER] at the inner [trans] aspect of the Golgi stack) in pancreatic exocrine cells of guinea pig, rat, rabbit, and hamster. The trans element of the Golgi stack exhibits thiamine pyrophosphatase (TPPase) but no acid phosphatase (AcPase) activity. In contrast, GERL shows AcPase but no TPPase activity. The nascent secretory granules, or condensing vacuoles, are expanded cisternal portions of GERL. Continuities of condensing vacuoles with rough ER are suggested, and it is proposed that some secretory components may have direct access to the condensing vacuoles from ER. Connections of Golgi apparatus with GERL were not seen.

Mannosidase H is involved in the trimming of a-1,6-mannosyl residues during the biosynthesis of glycoproteins containing N-linked oligosaccharides of the complex type. A highly specific polyclonal antibody (IgG) was isolated from rabbits immunized with a homogeneous preparation of mannosidase II prepared from rat liver. With this antibody, light and electron microscopic immunocytochemical studies on rat liver reveal that essentially all mannosidase H in hepatocytes is localized in the Golgi apparatus, the only other site with reaction product being the endoplasmic reticulum. The indirect immunocytochemical method used in this study involved three major steps: exposure of aldehyde-fixed tissue to immune and nonimmune IgG, treatment with staphylococcal protein A labeled with horseradish peroxidase, and incubation in diaminobenzidine to reveal sites of peroxidase activity. The procedures described overcome major problems in immunocytochemistry, allowing preservation of antigenic sites and maintaining adequate ultrastructural integrity. The in situ localization of other carbohydrate-processing enzymes, involved in either trimming or attachment of sugar residues, should be possible with this procedure. Because biosynthetic precursors of the processing enzymes may be revealed by an immunocytochemical approach, it is potentially significant that mannosidase II reaction product is present in areas of the endoplasmic reticulum as well as in the Golgi apparatus.The Golgi apparatus is involved in the post-translational modification of glycoproteins. This includes proteolytic cleavages as well as glycosylation reactions (1). Secretory and membrane proteins containing N-linked oligosaccharides are synthesized on the endoplasmic reticulum (ER) as precursors whose common oligosaccharide moiety is modified during subsequent processing events. a-1,2-Mannosyl residues are removed by mannosidases IA and IB, and a-1,3-and a-1,6-mannosyl residues are removed from GlcNAcMan5(GlcNAc)2 species by mannosidase II to produce GlcNAcMan3(GlcNAc)2 species, which are then converted to complex oligosaccharides by the addition of various sugars (2). Evidence for the Golgi localization of the mannosyl trimming reactions consists of: (i) the finding of mannosidases in purified Golgi membrane preparations (2-5); and (ii) pulse-chase experiments on glycoprotein biosynthesis (6).The in situ visualization of these enzymes would provide direct confirmation of their intracellular localization and perhaps do so in a more precise manner than possible by biochemical methods. The possibility of cross-contamination of isolated organelle fractions can rarely be excluded unequivocally, particularly contamination by as-yet-undefined cellular elements. Reliable ultrastructural enzyme cytochemical methods for demonstrating a-D-mannosidases and other glycoprotein processing enzymes have not been developed to date.Rat liver mannosidase II has been isolated in homogeneous form and shown to be different from other liver mannosidases (7). With the availabili...

Very low density lipoprotein (VLDL) particles are packaged by the Golgi apparatus into vacuoles which move to the plasma membrane and empty the particles into the space of Disse, via exocytosis. Traditionally, all lipoproteincontaining cisternae and vacuoles are thought to be parts of this pathway. Observations reported here demonstrate that there is a second population of lipoprotein-containing cisternae and vacuoles. This population is part of GERL, an organelle we consider to be a specialized hydrolase-rich region of the endoplasmic reticulum (ER). To our knowledge, this is the first systematic study of GERL in normal rat hepatocytes.KEY WORDS lipoproteins 9 GERL 9 apparatus hepatocytes 9 acid phosphatase 9 thiamine pyrophosphatase GolgiIn a variety of cell types it has been possible to discriminate between the innermost (trans [10]) element of the Golgi apparatus and adjacent GERL, considered by us to be a specialized area of endoplasmic reticulum (ER); that distinction is based on different cytochemically demonstrable phosphatase (Pase) activities. The trans Golgi element exhibits thiamine pyrophosphatase (TPPase) activity, but not acid phosphatase (AcPase) activity. The reverse is true of GERL; it shows AcPase and no TPPase activity.Recently, we reported the cytochemical identification of GERL in hepatocytes of rats in experiments in which the organelle is much enlarged. By feeding rats orotic acid (OA), markedly fatty livers develop, and subsequent addition of cl'dorophenoxyisobutyrate (clofibrate or CPIB) to the OA diet is followed by clearing of the lipid from the hepatocytes. The morphological signs of Golgi apparatus involvement in very low density lipoprotein (VLDL) secretion disappear in OA-fed rats (38) and return to normal when the rats are fed the reversal (CPIB + OA) diet (36); also, apoprotein B disappears from the serum during OA feeding and reappears with CPIB + OA feeding. With the CPIB + OA diet, GERL elements are much enlarged, distended with lipoprotein (LP) particles. Morphological identification of GERL was recently confirmed by cytochemistry (35,39).These studies led us to extend fragmentary observations made in various laboratories, including our own, on hepatocytes of normal rats. To our knowledge, the present report is the first systematic study of GERL in untreated rats. In normal hepatocytes, the organelle is less extensive than in rats on the CPIB + OA diet, but the pattern of cytochemically demonstrable phosphatase activities is the same. This study demonstrates that hepatocytes, as in other cell types (34, 29,

Plasma concentrations of immunoreactive melatonin, estradiol, progesterone, follicle stimulating hormone (FSH), and beta-human chorionic gonadotropin (beta hCG) were studied between 1000 and 1230 h in 105 Chinese females during six periods of normal pregnancy and 1-5 min after normal delivery. We have also examined the midday levels of immunoreactive melatonin in the cord blood of fetuses and plasma collected 1-5 min after and 24 h after delivery from their mothers. Concentrations of hormone immunoreactivities were determined by radioimmunoassay, and distinct fluctuations of all hormones were recorded during pregnancy. In the pregnant females, there were significant negative correlations between melatonin and estradiol, melatonin and progesterone, beta hCG and progesterone, and beta hCG and estradiol, and positive correlations between melatonin and FSH and progesterone and estradiol. Furthermore, plasma melatonin levels in the cord blood demonstrated no sex difference and were significantly lower than and correlated positively with the levels in their mothers. Our results suggest that sex steroids may inhibit and FSH may potentiate circulating melatonin levels in gravid women; changes in the levels of melatonin during pregnancy may affect the in utero development of the human embryo; and circulating melatonin in the mother may be the major source of blood melatonin in the fetus before parturition.