Purification and characterization of cathepsin L in arrowtooth flounder (Atheresthes stomias) muscle

A predominant, heat-activated proteinase in muscle extract of arrowtooth flounder (Atheresthes stomias) was purified to 55-fold by heat treatment, followed by a series of chromatographic separations. The apparent molecular mass of the purified enzyme was 27 kDa by size exclusion chromatography and sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The proteinase had high affinity and activity toward Z-Phe-Arg-NMec with K(m) and k(cat) values of 8.2 microM and 12.2/s, respectively. Activity was inhibited by sulfhydryl reagents and activated by reducing agents. The purified proteinase displayed optimal activity at pH 5.0-5.5 and 60 degrees C, respectively. Consistent with the properties of proteases from other species, the heat-activated proteinase in arrowtooth flounder can be identified as cathepsin L.     

Female maturity,reproductive potential,relative distribution,and growth compared between arrowtooth flounder (Atheresthes stomias) and Kamchatka flounder (A. evermanni) indicating concerns for management

Arrowtooth flounder (Atheresthes stomias) and Kamchatka flounder (A. evermanni), major piscivorous predators in the eastern Bering Sea and Aleutian Islands, are morphologically similar. Consequently the two species have been managed together as a species complex using the length‐ and age‐at‐maturity derived from Gulf of Alaska arrowtooth flounder, which had been the only available maturity estimates. However, there could be serious management consequences if the two species matured at significantly different ages and fork lengths. Therefore, this study was conducted during 2007 and 2008 to determine if there were significant differences in maturation between the two species. Significant differences in size and age of female maturation and growth were found. The age and length of 50% maturity (A_50,L_50, respectively) for arrowtooth flounder females is 7.6 years of age and 480 mm in body length. In comparison, A_50,L₅₀ of Kamchatka flounder females is 10.1 years of age and 550 mm, meaning that Kamchatka flounder has a significantly lower reproductive potential than arrowtooth flounder. The large difference in reproductive potential indicates that managing the two species together as a species complex using the reproductive characteristics of arrowtooth flounder, was not conservative for Kamchatka flounder. This study also determined that arrowtooth flounder maturation was consistent between the Gulf of Alaska and eastern Bering Sea populations.     

Distribution of neuropeptides in the porcine stellate ganglion

The localization and distribution of neuropeptides including neuropeptide Y (NPY), [Met⁵]enkephalin-Arg⁶-Gly⁷-Leu⁸ (MEAGL), vasoactive intestinal polypeptide (VIP), calcitonin gene-related peptide (CGRP), substance P and somatostatin (SOM) were analyzed in the stellate ganglion of the pig by use of the indirect immunofluorescence technique. NPY, MEAGL, SOM, VIP and CGRP immunoreactivities were found to exist in subpopulations of neuronal cell bodies of the stellate ganglion. A population of the small intensely fluorescent (SIF) cells showed MEAGL immunoreactivity. In addition, the presence of NPY-, MEAGL-, CGRP-, SP-, SOM- and VIP-immunoreactive nerve fibers and axonal varicosities were observed in the stellate ganglion. The localization and pattern of distribution of these peptides in the porcine stellate ganglion were compared with studies carried out on stellate ganglia of other mammalian species.     

Quantitative Estimation and Comparison of the Trophic Niches of the Kamchatka Flounder Atheresthes evermanni,the Pacific Halibut Hippoglossus stenolepis,and the Greenland Halibut Reinhardtius matsuurae,by the Isotope Composition of Carbon and Nitrogen of the Food Components

Journal of Ichthyology - The width of trophic niches of the Kamchatka flounder Atheresthes evermanni, the Pacific halibut Hippoglossus stenolepis, and the Greenland halibut Reinhardtius matsuurae...     

On the presence of hepatic stellate cells in portal spaces

Previous studies in mice with hypervitaminosis A have demonstrated that fat-storing cells (hepatic stellate cells-HSCs) participate in schistosomal granuloma fibrogenesis. The origin of such cells in portal areas, away from the Disse spaces, was herein investigated. HSCs were identified in frozen sections of the liver by means of Sudan III staining. They appeared as red-stained cells disposed along the sinusoids of normal mice, but were never found within portal spaces. However, in the chronically inflamed portal spaces of Capillaria hepatica-infected mice, Sudan III-positive cells were frequently present among leukocytes and fibroblast-like cells. Thus, there are no resident HSCs in portal spaces, but their presence there in chronic inflammatory processes indicates that they are able to migrate from peri-sinusoidal areas in order to reach the portal areas.     

Apoptosis in activated rat pancreatic stellate cells

Proliferation and matrix synthesis by activated pancreatic stellate cells (PSC) participate in the development of chronic pancreatitis. Apoptosis of PSC may terminate this process but has not yet been studied in this particular cell type and was the aim of the present study. PSC were isolated from rat pancreas and characterized for expression of glial fibrillary acidic protein, alpha-smooth muscle actin, CD95, and tumor necrosis factor-alpha-related apoptosis-inducing ligand (TRAIL) receptors. Apoptosis was determined by TdT-UTP nick end-labeling reaction, annexin V binding, and caspase-8 activation. Both CD95L and TRAIL induced apoptosis in PSC. The apoptotic response was minor in PSC cultured for 7 days but increased markedly thereafter. Sensitization of PSC with culture duration was accompanied by increased expression of CD95 and TRAIL receptor 2 and no alterations of Flip expression or protein kinase B phosphorylation but was paralleled by the appearance of a COOH-terminal cleavage product of receptor-interacting protein. PSC apoptosis was also induced by PK-11195, a ligand of the peripheral benzodiazepine receptor. PSC apoptosis may be important in terminating the wound-healing response after pancreas injury and exhibits features distinct from apoptosis induction in hepatic stellate cells.     

胰腺星状细胞活化相关的信号转导通路

胰腺纤维化是慢性胰腺炎（chronic pancreatitis,CP）和胰腺癌主要的病理学特征,活化的胰腺星状细胞（pancreatic stellate cells,PSCs）是公认的致胰腺纤维化的主要效应细胞。PSCs的活化涉及到几个重要的信号转导通路：有丝分裂原活化蛋白激酶（mitogen-activated protein kinases,MAPK）、磷酯酰肌醇3激酶（phosphatidylinositol 3-kinase,PI3K）=、Smad信号转导蛋白、过氧化物酶体增生物激活受体-γ（PPAR-γ）、Rho-ROCK等细胞内信号途径。探讨这些信号通路在胰腺纤维化中所起的作用对慢性胰腺炎、胰腺癌及糖尿病的治疗有重要意义。现就与PSCs激活有关的信号通路的研究结合最新进展作一综述。     

Phagocytic activity of the stellate cells in the anuran pars intermedia

Summary In an attempt to study further the stellate cell and its functions, the ultrastructure of this cell type in the neurointermediate lobe of the bullfrog, Rana catesbeiana, was examined in both organ and dissociated-cell culture. The cytoplasmic activity of stellate cells from neurointermediate lobes incubated 3 1/2 or 5 1/2 h was greater than that of those in vivo. Mitochondria and bundles of cytoplasmic filaments were numerous, in addition to prominent, well-developed Golgi complexes with associated vesicles. The most striking ultrastructural feature was the presence of phagocytic vacuoles that contain cellular debris. The stellate cells were seen to form cytoplasmic processes that phagocytosed this extracellular debris identifiable as belonging to the secretory cells of the pars intermedia. The stellate cells from the dissociated-cell preparations were also seen to contain debris within phagocytic vacuoles. In those neurointermediate lobes transplanted for 3 1/2 to 4 days into the anterior chamber of the eye, the stellate cells demonstrated similar phagocytic ability, but the phagocytic vacuoles contained material that seemed to be at a later stage of degradation. In all three of these conditions, the stellate cells were not seen to release this cellular debris nor were they seen to undergo cell division. These glial-like stellate cells of the pars intermedia acted as macrophages in all three of these experiments. There is now, therefore, a need to determine under what conditions, if any, these stellate cells function in vivo as macrophages.Supported by NSF Program for Small College Faculty Engaged in Research at Larger Institutions and Department of Energy — Associated Western Universities Faculty Participation Program. The authors thank Dr. W. Ferris and Dr. J. Berliner for the use of the electron microscopy facilities at the University of Arizona and Nuclear Medicine Laboratory, UCLA, respectively. Warm thanks are due to Ms. Ruth Cole for technical assistance     

Immunohistochemical characterization of pituitary stellate cells in rats

Pituitary stellate cells from the normal adult male rats were immunohistochemically investigated at the light microscopical level by the use of rat TSH-beta, porcine ACTH1-39, porcine ACTH17-39, rat FSH and ovine FSH antisera. They were characterized by the stellate shape and a mimic engulfment of acidophils. In the present study, they were identified to be the ACTH cells but some were TSH cells. Although most of the corticotrophs showed a peripheral fringe immunostained with the porcine ACTH17-39 antiserum, some others were stained diffusively throughout the cytoplasm. The latter cells coincided, in shape and in homogenous stainability of the cytoplasm, with the stellate TSH cells. Both cells did not correspond but were independent in distribution at the same site of the gland on the adjacent two sections. The stellate type of FSH cells could react with the ovine FSH antiserum, but not with the rat FSH antiserum. Absorption tests of the ovine FSH, procine ACTH1-39 and procine ACTH17-39 antisera were carried out by an application of procine ACTH. In consequence, the porcine ACTH)-39 and porcine ACTH17-39 antisera were absorbed efficaciously by the ACTH antigen at the dose of 10 micrograms/ml, but the ovine FSH antiserum was not enough absorbed by ACTH in the doses of less than 1 mg/ml. It was not finally concluded whether or not the single stellate cells produced ACTH and FSH.     

Nestin expression in pancreatic stellate cells and angiogenic endothelial cells

Nestin is an intermediate filament protein expressed by neuroepithelial stem cells and which has been proposed to represent also a marker for putative islet stem cells. The aim of this study was to characterize the cell type(s) expressing nestin in the rat pancreas. By immunohistochemistry, nestin positivity was localized exclusively in mesenchymal cells of normal and regenerating adult pancreas. In the latter condition, the number of nestin-positive cells and the intensity of nestin immunoreactivity were greatly increased. Most nestin-positive cells had the morphology of stellate cells, a type of pericyte associated with blood vessels which has been previously reported to occur in liver and pancreas. In addition, nestin positivity was present in endothelial cells from neocapillaries during pancreas regeneration, and in all blood vessels during morphogenesis in fetal pancreas. Nestin expression was not found in the ductal epithelial cells from which islet cells originate in fetal and regenerating pancreas. In primary pancreatic tissue explants, nestin-positive mesenchymal cells rapidly attached to plastic and proliferated. These cells also expressed desmin, vimentin, and glial fibrillary acidic protein which are known to represent stellate cell markers. In summary, nestin in the pancreas is primarily a marker for reactive stellate cells, or pericytes, and endothelial cells during active angiogenesis.     

Pancreatic stellate cells promote epithelial-mesenchymal transition in pancreatic cancer cells

The interaction between pancreatic cancer cells and pancreatic stellate cells (PSCs), a major profibrogenic cell type in the pancreas, is receiving increasing attention. There is accumulating evidence that PSCs promote the progression of pancreatic cancer by increasing cancer cell proliferation and invasion as well as by protecting them from radiation- and gemcitabine-induced apoptosis. Because epithelial-mesenchymal transition (EMT) plays a critical role in the progression of pancreatic cancer, we hypothesized that PSCs promote EMT in pancreatic cancer cells. Panc-1 and SUIT-2 pancreatic cancer cells were indirectly co-cultured with human PSCs isolated from patients undergoing operation for pancreatic cancer. The expression of epithelial and mesenchymal markers was examined by real-time PCR and immunofluorescent staining. The migration of pancreatic cancer cells was examined by scratch and two-chamber assays. Pancreatic cancer cells co-cultured with PSCs showed loose cell contacts and a scattered, fibroblast-like appearance. The expression of E-cadherin, cytokeratin 19, and membrane-associated β-catenin was decreased, whereas vimentin and Snail (Snai-1) expression was increased more in cancer cells co-cultured with PSCs than in mono-cultured cells. The migration of pancreatic cancer cells was increased by co-culture with PSCs. The PSC-induced decrease of E-cadherin expression was not altered by treatment with anti-TGF-β-neutralizing antibody, excluding a central role of TGF-β in this process. In conclusion, PSCs promoted EMT in pancreatic cancer cells suggesting a novel mechanism by which PSCs contribute to the aggressive behavior of pancreatic cancer cells.     

Ontogenetic development and composition of the mucous cells and the occurrence of saccular cells in the epidermis of Atlantic halibut

An increase in the number of mucous cells of the epidermis, as well as qualitative changes of the mucus composition from predominantly neutral to a mixture of neutral and sulphated glycoproteins occurred during the development from a pelagic larva to a bottom-dwelling flatfish. Numerous saccular cells were observed in the epidermis of the yolk-sac larvae that disappeared simultaneously as the mucous cells increased in number in the epidermis of the metamorphosed halibut. These findings may help to understand the protective role of the mucus layer of Atlantic halibut during development as compared to other fish species in aquaculture.     

Three-dimensional co-culture of hepatocytes and stellate cells

Hepatocytes self-assemble in culture to form compacted spherical aggregates, or spheroids, that mimic the structure of the liver by forming tight junctions and bile canalicular channels. Hepatocyte spheroids thus resemble the liver to a great extent. However, liver tissue contains other cell types and has bile ducts and sinusoids formed by endothelial cells. Reproducing 3-D co-culture in vitro could provide a means to develop a more complex tissue-like structure. Stellate cells participate in revascularization after liver injury by excreting between hepatocytes a laminin trail that endothelial cells follow to form sinusoids. In this study we investigated co-culture of rat hepatocytes and a rat hepatic stellate cell line, HSC-T6. HSC-T6, which does not grow in serum-free spheroid medium, was able to grow under co-culture conditions. Using a three-dimensional cell tracking technique, the interactions of HSC-T6 and hepatocyte spheroids were visualized. The two cell types formed heterospheroids in culture, and HSC-T6 cell invasion into hepatocyte spheroids and subsequent retraction was observed. RT-PCR revealed that albumin and cytochrome P450 2B1/2 expression were better maintained in co-culture conditions. These three-dimensional heterospheroids provide an attractive system for in vitro studies of hepatocyte-stellate cell interactions.     

Beta-carotene storage, conversion to retinoic acid, and induction of the lipocyte phenotype in hepatic stellate cells

Hepatic stellate cells (HSCs) are the major site of retinol (ROH) metabolism and storage. GRX is a permanent murine myofibroblastic cell line, derived from HSCs, which can be induced to display the fat-storing phenotype by treatment with retinoids. Little is known about hepatic or serum homeostasis of beta-carotene and retinoic acid (RA), although the direct biogenesis of RA from beta-carotene has been described in enterocytes. The aim of this study was to identify the uptake, metabolism, storage, and release of beta-carotene in HSCs. GRX cells were plated in 25 cm(2) tissue culture flasks, treated during 10 days with 3 micromol/L beta-carotene and subsequently transferred into the standard culture medium. beta-Carotene induced a full cell conversion into the fat-storing phenotype after 10 days. The total cell extracts, cell fractions, and culture medium were analyzed by reverse phase high-performance liquid chromatography for beta-carotene and retinoids. Cells accumulated 27.48 +/- 6.5 pmol/L beta-carotene/10(6) cells, but could not convert it to ROH nor produced retinyl esters (RE). beta-Carotene was directly converted to RA, which was found in total cell extracts and in the nuclear fraction (10.15 +/- 1.23 pmol/L/10(6) cells), promoting the phenotype conversion. After 24-h chase, cells contained 20.15 +/- 1.12 pmol/L beta-carotene/10(6) cells and steadily released beta-carotene into the medium (6.69 +/- 1.75 pmol/ml). We conclude that HSC are the site of the liver beta-carotene storage and release, which can be used for RA production as well as for maintenance of the homeostasis of circulating carotenoids in periods of low dietary uptake.     

Curcumin blocks activation of pancreatic stellate cells

Activated pancreatic stellate cells (PSCs) play a pivotal role in the pathogenesis of pancreatic fibrosis and inflammation. Inhibition of activation and cell functions of PSCs is a potential target for the treatment of pancreatic fibrosis and inflammation. The polyphenol compound curcumin is the yellow pigment in curry, and has anti-inflammatory and anti-fibrotic properties. We here evaluated the effects of curcumin on the activation and cell functions of PSCs. PSCs were isolated from rat pancreas tissue and used in their culture-activated, myofibroblast-like phenotype unless otherwise stated. The effects of curcumin on proliferation, alpha-smooth muscle actin gene expression, monocyte chemoattractant protein (MCP)-1 production, and collagen expression were examined. The effect of curcumin on the activation of freshly isolated cells in culture was also assessed. Curcumin inhibited platelet-derived growth factor (PDGF)-induced proliferation, alpha-smooth muscle actin gene expression, interleukin-1beta- and tumor necrosis factor (TNF)-alpha-induced MCP-1 production, type I collagen production, and expression of type I and type III collagen genes. Curcumin inhibited PDGF-BB-induced cyclin D1 expression and activation of extracellular signal-regulated kinase (ERK). Curcumin inhibited interleukin-1beta- and TNF-alpha-induced activation of activator protein-1 (AP-1) and mitogen-activated protein (MAP) kinases (ERK, c-Jun N-terminal kinase (JNK), and p38 MAP kinase), but not of nuclear factor-kappaB (NF-kappaB). In addition, curcumin inhibited transformation of freshly isolated cells to myofibroblast-like phenotype. In conclusion, curcumin inhibited key cell functions and activation of PSCs.     

Intralobular heterogeneity of perisinusoidal stellate cells in porcine liver

The aim of the present investigation was to elucidate the intralobular heterogeneity of the perisinusoidal stellate cells (fat-storing cells, lipocytes) in the porcine liver. Their three-dimensional structure, desmin immunoreactivity and vitamin-A storage were studied by use of the Golgi silver, immunocytochemical and gold chloride methods. In order to locate the stellate cells, the hepatic lobules were divided into 10 zones. The stellate cells were readily identified in Golgi preparations by their striking dendritic appearance with branching processes encompassing the sinusoids. The stellate cells in the centrolobular zones were conspicuously dendritic with longer processes in conspicuously dendritic with longer processes in comparison to those emitted by periportal elements. Such arborizations were studded with numerous thorn-like microprojections. Desmin immunoreaction in the periportal zones was stronger than that in the centrolobular zones. Vitamin-A storage in the stellate cells was well developed in zones 2–4, but reduced gradually toward the central region. The perisinusoidal etellate cells display marked heterogeneity in morphology and function based on their zonal location in the hepatic lobule.     

Ciliated epithelium in the gut of larval Atlantic halibut, Hippoglossus hippoglossus

The presence of cilia was documented in the hindgut of larval Atlantic halibut ( Hippoglossus hippoglossus ) with the use of electron microscopy. Cilia were shown to be present as late as 8 and 9 day post hatch. These observations were compared to similar observations in more primitive teleosts.