Three-dimensional ultrastructure of normal rat hepatocytes by quick-freezing and deep-etching method

The three-dimensional ultrastructure of nuclei and cell organelles including rough-surfaced endoplasmic reticulum (RER), mitochondria, and cytoskeleton were studied in normal rat hepatocytes by the quick-freezing and deep-etching method. Peroxisomes and mitochondria were observed as spherical structures with granular matrices. Peroxisomes were identified by their size and matrices, which were more condensed than those of mitochondria. Ribosomes were identified as granular structures and were attached to the surface of endoplasmic reticulum. Cytoskeletal filaments were identified by their differences in diameter on the replica membranes, as well as in conventional ultrathin sections. Microfilaments were mainly localized around the bile canaliculi and adjacent to sinusoids. Intermediate filaments were observed around the bile canalicular microfilaments. Only a few filaments were observed near the lateral plasma membranes. Cross-bridges measuring 5-7 nm in diameter were localized between the lamellae of RER and the surface of mitochondria. The quick-freezing and deep-etching method could be used to clarify the three-dimensional association between the cytoskeleton and membrane-bound organelles in hepatocytes.     

Ultrastructural studies of hepatocyte cytoskeletons of phalloidin-treated rats by quick-freezing and deep-etching method

We observed hepatocyte cytoskeletons in phalloidin-treated rats by the quick-freezing and deep-etching method in three dimensions and compared them with the ultrastructural findings on conventional ultrathin sections. The numbers of microvilli in dilated bile canaliculi were decreased in the rats treated with phalloidin for 1 wk. In hepatocytes the cytoplasm around bile canaliculi could be divided into three layers, increased microfilament layer, cell organelle layer of secretory system and increased smooth surface endoplasmic reticulum layer. In the rats treated with phalloidin for 4 wk, microfilaments were extended into the cytoplasm near the nucleus in addition to the increased number of large lysosomes and microtubules. In both groups, three-dimensional structures of microfilaments could be visualized around bile canaliculi and along cell borders by the quick-freezing and deep-etching method. The branching microfilaments with the diameters of 7 to 10 nm were directly attached to other filaments, cell organelles or cytoplasmic sides of cell membranes. Moreover, bundled intermediate filaments were increased around peribiliary microfilaments associated with long-term cholestasis. It is suggested that excessive accumulation of peribiliary microfilaments disturb the secretion of bile components into bile canaliculi. The cytoskeletal reorganizations of intermediate filaments seem to alter the arrangements of various cell organelles.     

Hepatic macrophage malfunction in rats with obstructive jaundice and its biological significance.

The present study was designed to investigate the pathophysiology of obstructive jaundice by analyzing the function of hepatic macrophages and their role in immune responses and homeostasis in rats. The phagocytic index, determined by the rate of disappearance of 51Cr-endotoxin from the peripheral blood after intravenous injection, was increased in obstructive jaundice 2 weeks after bile duct ligation. The superoxide production of isolated hepatic macrophages and peripheral blood monocytes, measured by the superoxide dismutase inhibitable ferricytochrome c reduction method, was increased. Prostaglandin E2 release, measured by RIA, was markedly increased in rats with obstructive jaundice, but there was no significant difference in interleukin-1 release between jaundiced and control rats. The flow-cytometric analysis of surface molecules of hepatic macrophages showed decreased expression of interleukin-2 receptor in rats with obstructive jaundice. Thus, the functions of hepatic macrophages in rats with obstructive jaundice were impaired. This malfunction may disturb the immunoregulatory network and metabolism, although the exact implications of the altered function of hepatic macrophages have not yet been clarified.     

Intermyofibrillar and nuclear-myofibrillar connections in human and canine myocardium. An ultrastructural study

Cytoplasmic filaments, which measure 100 Å in diameter, and which differ morphologically from actin and myosin filaments, are described in normal canine myocardium and in normal and hypertrophied human myocardium. These filaments were found between Z bands, near the sarcolemma, in regions of intercalated discs, and in the vicinity of nuclear membranes. The 100 Å filaments appeared to form transverse connections between adjacent Z bands and between Z bands and outer nuclear membranes. These filaments probably function as a cytoskeleton.     

Tracking melanosomes inside a cell to study molecular motors and their interaction

Cells known as melanophores contain melanosomes, which are membrane organelles filled with melanin, a dark, nonfluorescent pigment. Melanophores aggregate or disperse their melanosomes when the host needs to change its color in response to the environment (e.g., camouflage or social interactions). Melanosome transport in cultured Xenopus melanophores is mediated by myosin V, heterotrimeric kinesin-2, and cytoplasmic dynein. Here, we describe a technique for tracking individual motors of each type, both individually and in their interaction, with high spatial (approximately 2 nm) and temporal (approximately 1 msec) localization accuracy. This method enabled us to observe (i) stepwise movement of kinesin-2 with an average step size of 8 nm; (ii) smoother melanosome transport (with fewer pauses), in the absence of intermediate filaments (IFs); and (iii) motors of actin filaments and microtubules working on the same cargo nearly simultaneously, indicating that a diffusive step is not needed between the two systems of transport. In concert with our previous report, our results also show that dynein-driven retrograde movement occurs in 8-nm steps. Furthermore, previous studies have shown that melanosomes carried by myosin V move 35 nm in a stepwise fashion in which the step rise-times can be as long as 80 msec. We observed 35-nm myosin V steps in melanophores containing no IFs. We find that myosin V steps occur faster in the absence of IFs, indicating that the IF network physically hinders organelle transport.     

Expression of rat renal cortical OAT1 and OAT3 in response to acute biliary obstruction

Renal function in the course of obstructive jaundice has been the subject of great interest; however, little is known about the expression of renal organic anion transporters. The objective of this work was to study, in rats with acute extrahepatic cholestasis, the cortical renal expression of the organic anion transporter 1 (OAT1) and the organic anion transporter 3 (OAT3), in association with the pharmacokinetics and renal excretion of furosemide (FS). Male Wistar rats underwent bile duct ligation (BDL rats). Pair-fed sham-operated rats served as controls. All studies were carried out 21 hours after surgery. Rats were anesthetized and the pharmacokinetic parameters of FS and the renal elimination of FS were determined. Afterwards, the kidneys were excised and processed for immunoblot (basolateral membrane and renal homogenates) or immunocytochemical (light microscopic and confocal immunofluorescence microscopic analysis) techniques. The systemic and renal clearance of FS as well as the excreted and secreted load of FS increased in BDL rats. In kidneys from BDL rats, immunoblotting showed a significant increase in the abundance of both OAT1 and OAT3 in homogenates from renal cortex. In basolateral membranes from kidney cortex of BDL rats, OATI abundance was also increased and OAT3 abundance was not modified. Immunocytochemical techniques confirmed these results. In conclusion, acute obstructive jaundice is associated with an upregulation of OAT1 and OAT3, which might explain, at least in part, the increased systemic and renal elimination of FS.     

Fate of senescent megakaryocytes in the bone marrow

S ummary . Degenerating senescent megakaryocytes have been identified in mouse bone marrow by light and electron microscopy. The ultrastructural changes which occur as degeneration proceeds are characteristic of death by apoptosis, although most cells appear to round up rather than undergo fragmentation. A hitherto unreported finding in degenerating cells was the presence of bundles of approximately 7 nm diameter parallel filaments in nuclei and membrane-bound nuclear fragments. Structurally, they resembled bundles of filaments induced in nuclei with dimethyl sulphoxide and identified as actin. Often a bundle appeared to terminate at the inner membrane. In the cytoplasm the presence of microtubules and centrioles indicates that not all the latter organelles are lost by the megakaryocyte during platelet release. Degenerating senescent megakaryocytes are rare in the marrow of normal mice but increase in frequency during 5-fluorouracil stimulated thrombocytosis. The dying cells are eventually phagocytosed by macrophages, a process that can occur extravascularly, showing that entry of senescent megakaryocytes into the circulation is not necessary for their disposal.     

Alterations of tumor necrosis factor-alpha and interleukin 6 production and activity of the reticuloendothelial system in experimental obstructive jaundice in rats

BackgroundImmunological changes are well recognised in obstructive jaundice. The aim of this study was to monitor plasma tumor necrosis factor α (TNF-α) and interleukin-6 (IL-6) levels in rats with obstructive jaundice.MethodsThe ability of splenocytes and peritoneal exudate cells (PEC) to produce these cytokines both spontaneously and on induction with lipopolysaccharide (LPS), was compared in rats with and without obstructive jaundice (OJ). The activity of the reticuloendothelial system (RES) was also measured.ResultsSerum cytokine levels in OJ rats were higher than in control rats. PEC cultures produced significantly more IL-6, compared with control rats, declining thereafter. TNF-α activity in the splenocyte cultures of OJ rats was also higher than in the control group. Pronounced differences were found in the ability to produce TNF-α by PEC, i.e., TNF-α production was much stronger on day 7 in OJ rats than in controls. On day 14 TNF-α production was much lower and the spontaneous response was equal to the LPS-induced one. On day 21 the cells of OJ rats partially regained the ability to produce TNF-α RES activity of OJ rats was significantly suppressed in the liver and spleen, whereas the phagocytic activity in the lungs was elevated.ConclusionWe have demonstrated that the immune reactivity of OJ rats, intially elevated, underwent subsequent depression. The study also revealed a major effect of the operation alone on the studied parameters.     

From the Cover: Dynamin at actin tails

Dynamin, the product of the shibire gene of Drosophila, is a GTPase critically required for endocytosis. Some studies have suggested a functional link between dynamin and the actin cytoskeleton. This link is of special interest, because there is evidence implicating actin dynamics in endocytosis. Here we show that endogenous dynamin 2, as well as green fluorescence protein fusion proteins of both dynamin 1 and 2, is present in actin comets generated by Listeria or by type I PIP kinase (PIPK) overexpression. In PIPK-induced tails, dynamin is further enriched at the interface between the tails and the moving organelles. Dynamin mutants harboring mutations in the GTPase domain inhibited nucleation of actin tails induced by PIPK and moderately reduced their speed. Although dynamin localization to the tails required its proline-rich domain, expression of a dynamin mutant lacking this domain also diminished tail formation. In addition, this mutant disrupted a membrane-associated actin scaffold (podosome rosette) previously shown to include dynamin. These findings suggest that dynamin is part of a protein network that controls nucleation of actin from membranes. At endocytic sites, dynamin may couple the fission reaction to the polymerization of an actin pool that functions in the separation of the endocytic vesicles from the plasma membrane.     

Experimental study on cell damage of ischemic liver in obstructive jaundiced rats

The hepatic cellular damage induced by liver ischemia was investigated in rats with obstructive jaundice. Hepatic tissue blood flow in obstructive jaundice was decreased in the relation to the duration of jaundice. The value of lipid peroxide and cathepsin D activity of the hepatic tissue increased in the obstructive jaundice. Therefore, it was suggested that cell membrane and lysosomal membrane injury were induced in obstructive jaundice. The value of lipid peroxide of hepatic tissue in obstructive jaundice was more increased after partial liver ischemia. The survival rate following hepatic ischemia in jaundiced rats was remarkably lower than that of normal rats, and also it related to the duration of jaundice. In addition, histological changes of the liver after partial ischemia are severe in obstructive jaundiced liver. These data suggest that more remarkable hepatic cellular damage than in normal liver may be induced by liver ischemia in obstructive jaundice.     

Hepatic protein synthesis and cytokines in obstructive jaundice]

To clarify the mechanism of the increase of hepatic protein synthesis observed in the obstructive jaundiced rats, hepatocellular protein synthesis (HPS) and secretory protein synthesis (SPS) were estimated in the rats with obstructive jaundice and the contents of the following in the peripheral blood were determined in 21 patients with obstructive jaundice before and two weeks after percutaneous transhepatic biliary drainage (PTBD): interleukin-1 beta (IL-1 beta), interleukin-6 (IL-6), tumor necrosis factor-alpha (TNF alpha), endotoxin (Et), acute-phase protein (APP) and negative acute-phase protein (NAPP). The results: (1) HPS and SPS were markedly increased by obstructive jaundice; (2) IL-1 beta and IL-6 were significantly high and were reduced after PTBD; (3) neither TNF alpha nor Et was detected; (4) APP were significantly high and failed to decline after PTBD; (5) NAPP were significantly low and the contents were restored to the normal levels after PTBD. These results suggest that increased hepatic protein synthesis observed in the rats with obstructive jaundice correspond to the increased hepatic production of APP in patients with obstructive jaundice.     

The structure and organization of the bile canalicular cytoskeleton with special reference to actin and actin-binding proteins

The distribution of actin filaments and actin-binding proteins in the bile canaliculus (BC) of normal human hepatocytes was determined as a means of establishing the structure and organization of the BC cytoskeleton. Immunoblots demonstrated that actin, and the actinbinding proteins, myosin II, tropomyosin, vinculin, aactinin, villin, were present, as were the non—actin-related proteins β-tubulin, and cytokeratins. Three actin filament regions were identified: microvillus core filaments, a membrane-associated microfilamentous network, and a circumferential pericanalicular actin filament band. Actin-binding proteins were nonrandomly associated with actin in these regions. In the case of the pericanalicular band, there was also association with the zonula adherens junction. Intermediate filaments inserted into desmosomes. The ultrastructural localization of the actin-binding proteins was fundamentally linked to the arrangement and organization of the major canaliculus-associated microfilament structures. Structural organization of the cytoskeleton was also linked to distinct components of the intercellular junctions. It is notable that tropomyosin and a-actinin, which in muscle cells are regulatory proteins of contractile activity, and myosin II are associated with the pericanalicular actin microfilament band; it is the BC counterpart of the contractile actin filament band found in the apical region of other secretory cells. The outer sheath of noncontractile intermediate filaments likely stabilizes the canalicular compartment.     

Obstructive Jaundice Results in Increased Liver Expression of Uncoupling Protein 2 and Intact Skeletal Muscle Glucose Metabolism in the Rat

Background: A majority of patients with pancreatic cancer have obstructive jaundice and diabetes with skeletal muscle insulin resistance. Surgery for these patients is associated with significant morbidity. Uncoupling protein 2 (UCP2) has been proposed to regulate energy expenditure and promote liver vulnerability. The effects of obstructive jaundice on muscle glucose metabolism and expression of UCP2 in liver and muscle are unknown. Methods: Rats were operated with bile duct ligation (BDL). After 7 days, UCP2 mRNA levels were determined in liver and muscle. Simultaneously, insulin-stimulated glucose transport and glycogen synthesis in skeletal muscle were analyzed in vitro. Results: The jaundiced rats lost more weight than pair-fed controls. UCP2 mRNA levels were increased 5-fold in liver but not in muscle in jaundiced rats compared to pair-fed controls. The jaundiced rats were hypoglycemic and hypoinsulinemic but demonstrated intact or enhanced insulin action on skeletal muscle glucose transport and glycogen synthesis in vitro. Muscle glycogen content was increased in the jaundiced rats. Conclusions: Experimental obstructive jaundice in the rat is associated with increased liver expression of UCP2, rapid weight loss, and intact insulin action on skeletal muscle glucose metabolism. Obstructive jaundice, by upregulated liver UCP2, may contribute to the cachexia and high surgical morbidity observed in these patients, but not to skeletal muscle insulin resistance in pancreatic cancer patients.     

Obstructive jaundice results in increased liver expression of uncoupling protein 2 and intact skeletal muscle glucose metabolism in the rat

BACKGROUND: A majority of patients with pancreatic cancer have obstructive jaundice and diabetes with skeletal muscle insulin resistance. Surgery for these patients is associated with significant morbidity. Uncoupling protein 2 (UCP2) has been proposed to regulate energy expenditure and promote liver vulnerability. The effects of obstructive jaundice on muscle glucose metabolism and expression of UCP2 in liver and muscle are unknown. METHODS: Rats were operated with bile duct ligation (BDL). After 7 days, UCP2 mRNA levels were determined in liver and muscle. Simultaneously, insulin-stimulated glucose transport and glycogen synthesis in skeletal muscle were analyzed in vitro. RESULTS: The jaundiced rats lost more weight than pair-fed controls. UCP2 mRNA levels were increased 5-fold in liver but not in muscle in jaundiced rats compared to pair-fed controls. The jaundiced rats were hypoglycemic and hypoinsulinemic but demonstrated intact or enhanced insulin action on skeletal muscle glucose transport and glycogen synthesis in vitro. Muscle glycogen content was increased in the jaundiced rats. CONCLUSIONS: Experimental obstructive jaundice in the rat is associated with increased liver expression of UCP2, rapid weight loss, and intact insulin action on skeletal muscle glucose metabolism. Obstructive jaundice, by upregulated liver UCP2, may contribute to the cachexia and high surgical morbidity observed in these patients, but not to skeletal muscle insulin resistance in pancreatic cancer patients.     

Evidence for myosin motors on organelles in squid axoplasm.

Squid axoplasm has proved a rich source for the identification of motors involved in organelle transport. Recently, squid axoplasmic organelles have been shown to move on invisible tracks that are sensitive to cytochalasin, suggesting that these tracks are actin filaments. Here, an assay is described that permits observation of organelles moving on unipolar actin bundles. This assay is used to demonstrate that axoplasmic organelles move on actin filaments in the barbed-end direction, suggesting the presence of a myosin motor on axoplasmic organelles. Indeed, axoplasm contains actin-dependent ATPase activity, and a pan-myosin antibody recognized at least four bands in Western blots of axoplasm. An approximately 235-kDa band copurified in sucrose gradients with KI-extracted axoplasmic organelles, and the myosin antibody stained the organelle surfaces by immunogold electron microscopy. The myosin is present on the surface of at least some axoplasmic organelles and thus may be involved in their transport through the axoplasm, their movement through the cortical actin in the synapse, or some other aspect of axonal function.     

Mini-thin filaments regulated by troponin-tropomyosin

Striated muscle thin filaments contain hundreds of actin monomers and scores of troponins and tropomyosins. To study the cooperative mechanism of thin filaments, "mini-thin filaments" were generated by isolating particles nearly matching the minimal structural repeat of thin filaments: a double helix of actin subunits with each strand approximately seven actins long and spanned by a troponin-tropomyosin complex. One end of the particles was capped by a gelsolin (segment 1-3)-TnT fusion protein (substituting for normal TnT), and the other end was capped by tropomodulin. EM showed that the particles were 46 +/- 9 nm long, with a knob-like mass attributable to gelsolin at one end. Average actin, tropomyosin, and gelsolin-troponin composition indicated one troponin-tropomyosin attached to each strand of the two-stranded actin filament. The minifilaments thus nearly represent single regulatory units of thin filaments. The myosin S1 MgATPase rate stimulated by the minifilaments was Ca2+-sensitive, indicating that single regulatory length particles are sufficient for regulation. Ca2+ bound cooperatively to cardiac TnC in conventional thin filaments but noncooperatively to cardiac TnC in minifilaments in the absence of myosin. This suggests that thin filament Ca2+-binding cooperativity reflects indirect troponin-troponin interactions along the long axis of conventional filaments, which do not occur in minifilaments. Despite noncooperative Ca2+ binding to minifilaments in the absence of myosin, Ca2+ cooperatively activated the myosin S1-particle ATPase rate. Two-stranded single regulatory units therefore may be sufficient for myosin-mediated Ca2+-binding cooperativity. Functional mini-thin filaments are well suited for biochemical and structural analysis of thin-filament regulation.     

Mechanism of caveolin filament assembly

Caveolin-1 was the first protein identified that colocalizes with the approximately 10-nm filaments found on the inside surface of caveolae membranes. We have used a combination of electron microscopy (EM), circular dichroism, and analytical ultracentrifugation to determine the structure of the oligomers that form when the first 101 aa of caveolin-1 (Cav(1-101)) are allowed to associate. We determined that amino acids 79-96 in this caveolin-1 fragment are arranged in an alpha-helix. Cav(1-101) oligomers are approximately 11 nm in diameter and contain seven molecules of Cav(1-101). These subunits, in turn, are able to assemble into 50 nm long x 11 nm diameter filaments that closely match the morphology of the filaments in the caveolae filamentous coat. We propose that the heptameric subunit forms in part through lateral interactions between the alpha-helices of the seven Cav(1-101) units. Caveolin-1, therefore, appears to be the structural molecule of the caveolae filamentous coat.     

急慢性大鼠梗阻性黄疸模型的建立

目的:探讨急慢性SD大鼠梗阻性黄疸模型的建立,评价其模拟临床梗阻性黄疸的价值,提供一种新型的慢性梗阻性黄疸模型.方法:SD大鼠随机分成3组,每组每时段6只:急性梗阻性黄疸模型组(A组),慢性梗阻性黄疸模型组(B组),对照组(C组).造模后于第1、2、3、4、5周分别抽取大鼠静脉血检查肝功能,显微镜下刻度目镜检测大鼠胆总管直径,光学显微镜观察肝组织的病理改变,其中第4周行胆总管十二指肠吻合再通.结果:A组造模术后4wk内黄疸进行性加重(TB:749.38μmol/L±38.99μmol/Lvs84.86μmol/L±49.09μmol/L,P<0.05),胆总管直径扩张明显(1.50cm±0.30cmvs0.35cm±0.15cm,P<0.05),肝细胞变性、坏死明显,伴有增生.第4周行胆总管与十二指肠端侧吻合后黄疸消退明显(TB:153.93μmol/L±57.36μmol/Lvs749.38μmol/L±38.99μmol/L).B组造模术后黄疸呈现波动性,胆总管直径扩张(0.20cm±0.15cmvs0.30cm±0.10cm,P<0.05),肝细胞以变性为主,并有纤维组织增生.第4周行胆总管与十二指肠端...     

Intestinal ischemia-reperfusion injury augments intestinal mucosal injury and bacterial translocation in jaundiced rats

BACKGROUND/AIMS: The aim of this study was to evaluate local effects and degree of bacterial translocation related with intestinal ischemia-reperfusion injury in a rat obstructive jaundice model. METHODOLOGY: Thirty adult Sprague-Dawley rats (200-250 g) were divided into three groups; including Group 1 (jaundice group), Group 2 (jaundice-ischemia group) and Group 3 (ischemia group). All rats had 2 laparotomies. After experimental interventions, tissue samples for translocation; liver and ileum samples for histopathological examination, 25 cm of small intestine for mucosal myeloperoxidase and malondialdehyde levels and blood samples for biochemical analysis were obtained. RESULTS: Jaundiced rats had increased liver enzyme levels and total and direct bilirubin levels (p<0.05). Intestinal mucosal myeloperoxidase and malondialdehyde levels were found to be high in intestinal ischemia-reperfusion groups (p<0.05). Intestinal mucosal damage was more severe in rats with intestinal ischemia-reperfusion after bile duct ligation (p<0.05). Degree of bacterial translocation was also found to be significantly high in these rats (p<0.05). CONCLUSIONS: Intestinal mucosa is disturbed more severely in obstructive jaundice with the development of ischemia and reperfusion. Development of intestinal ischemia-reperfusion in obstructive jaundice increases bacterial translocation.