RESUMO
Vitamin A (retinyl palmitate) was injected subcutaneously or administered to rats by tube feeding. After subcutaneous injection, vitamin A was taken up and stored in cells of the lamina propria mucosae of the rat intestine. After oral administration, vitamin A was absorbed by the intestinal absorptive epithelial cells and transferred to cells of the lamina propria mucosae, where cells took up and stored the transferred vitamin A. The morphology of these cells was similar to that of hepatic stellate cells (also called vitamin A-storing cells, lipocytes, interstitial cells, fat-storing cells or Ito cells). Thus, these cells in the intestine could take up vitamin A from the systemic circulation and as well as by intestinal absorption, and store the vitamin in the lipid droplets in their cytoplasm. The data suggest that these cells are extrahepatic stellate cells of the digestive tract that may play roles in both the absorption and homeostasis of vitamin A.
Assuntos
Citoplasma/metabolismo , Mucosa Intestinal/citologia , Mucosa Intestinal/metabolismo , Lipídeos/química , Vitamina A/metabolismo , Administração Oral , Animais , Forma Celular , Células Epiteliais/citologia , Células Epiteliais/metabolismo , Células Epiteliais/ultraestrutura , Fluorescência , Injeções Subcutâneas , Mucosa Intestinal/ultraestrutura , Jejuno/metabolismo , Fígado/metabolismo , Masculino , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Perilipina-2 , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Ratos , Ratos Wistar , Coloração e Rotulagem , Vitamina A/administração & dosagemRESUMO
GGA (geranylgeranoic acid) is a natural polyprenoic acid, derivatives of which has been shown to prevent second primary hepatoma. GGA induces mitochondria-mediated PCD (programmed cell death), which may be relevant to cancer prevention. To gain further insights into GGA-induced PCD, autophagy processes were examined in human hepatoma-derived HuH-7 cells. Treatment of HuH-7/GFP (green fluorescent protein)-LC3 cells with GGA induced green fluorescent puncta in the cytoplasm within 30 min and their massive accumulation at 24 h. After 15 min of GGA treatment, a burst of mitochondrial superoxide production occurred and LC3ß-I was appreciably converted into LC3ß-II. GGA-induced early stages of autophagy were unequivocally confirmed by electron-microscopic observation of early/initial autophagic vacuoles. On the other hand, LC3ß-II as well as p62/SQSTM1 (sequestosome 1) continuously accumulated and co-localized in the cytoplasmic puncta after GGA treatment. Furthermore, GGA treatment of HuH-7/mRFP (monomeric red fluorescent protein)-GFP-LC3 cells showed yellow fluorescent puncta, whereas glucose deprivation of the cells gave red fluorescent puncta. These results strongly suggest that GGA induces the initial phase of autophagy, but blocks the maturation process of autolysosomes or late stages of autophagy, insomuch that GGA provides substantial accumulation of autophagosomes under serum-starvation conditions in human hepatoma cells.
Assuntos
Autofagia/efeitos dos fármacos , Carcinoma Hepatocelular/fisiopatologia , Diterpenos/farmacologia , Neoplasias Hepáticas/fisiopatologia , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Linhagem Celular Tumoral , Humanos , Proteínas Associadas aos Microtúbulos/metabolismo , Mitocôndrias/efeitos dos fármacos , Proteína Sequestossoma-1RESUMO
We investigated the characteristics of extracellular matrix (ECM) in the soft tissue of two frozen baby woolly mammoths (Mammuthus primigenius) that died and were buried in Siberian permafrost approximately 40,000 years ago. Morphological and biochemical analyses of mammoth lung and liver demonstrated that those soft tissues were preserved at the gross anatomical and histological levels. The ultrastructure of ECM components, namely a fibrillar structure with a collagen-characteristic pattern of cross-striation, was clearly visible with transmission and scanning electron microscopy. Type I and type IV collagens were detected by immunohistochemical observation. Quantitative amino acid analysis of liver and lung tissues of the baby mammoths indicated that collagenous protein is selectively preserved in these tissues as a main protein. Type I and type III collagens were detected as major components by means of liquid chromatography-mass spectrometry analysis after digestion with trypsin. These results indicate that the triple helical collagen molecule, which is resistant to proteinase digestion, has been preserved in the soft tissues of these frozen mammoths for 40,000 years.
Assuntos
Colágeno/análise , Matriz Extracelular/ultraestrutura , Fígado/metabolismo , Pulmão/metabolismo , Mamutes/metabolismo , Animais , Cromatografia Líquida , Colágeno/genética , Colágeno Tipo I/análise , Colágeno Tipo I/genética , Colágeno Tipo IV/análise , Colágeno Tipo IV/genética , Matriz Extracelular/metabolismo , Feminino , Fósseis/ultraestrutura , Fígado/ultraestrutura , Pulmão/ultraestrutura , Espectrometria de Massas , Pergelissolo , Preservação Biológica , Análise de Sequência de Proteína , SibériaRESUMO
HSCs (hepatic stellate cells) (also called vitamin A-storing cells, lipocytes, interstitial cells, fat-storing cells or Ito cells) exist in the space between parenchymal cells and liver sinusoidal endothelial cells of the hepatic lobule and store 50-80% of vitamin A in the whole body as retinyl palmitate in lipid droplets in the cytoplasm. In physiological conditions, these cells play pivotal roles in the regulation of vitamin A homoeostasis. In pathological conditions, such as hepatic fibrosis or liver cirrhosis, HSCs lose vitamin A and synthesize a large amount of extracellular matrix components including collagen, proteoglycan, glycosaminoglycan and adhesive glycoproteins. Morphology of these cells also changes from the star-shaped SCs (stellate cells) to that of fibroblasts or myofibroblasts. The hepatic SCs are now considered to be targets of therapy of hepatic fibrosis or liver cirrhosis. HSCs are activated by adhering to the parenchymal cells and lose stored vitamin A during hepatic regeneration. Vitamin A-storing cells exist in extrahepatic organs such as the pancreas, lungs, kidneys and intestines. Vitamin A-storing cells in the liver and extrahepatic organs form a cellular system. The research of the vitamin A-storing cells has developed and expanded vigorously. The past, present and future of the research of the vitamin A-storing cells (SCs) will be summarized and discussed in this review.
Assuntos
Biologia Celular/tendências , Células Estreladas do Fígado/fisiologia , Fígado/citologia , Fígado/fisiologia , Animais , Células Estreladas do Fígado/classificação , Células Estreladas do Fígado/metabolismo , Células Estreladas do Fígado/ultraestrutura , Hepatócitos/citologia , Hepatócitos/fisiologia , Humanos , Fígado/metabolismo , Fígado/ultraestrutura , Modelos Biológicos , Terminologia como Assunto , Vitamina A/metabolismoRESUMO
Hepatic stellate cells (HSCs) are the major site of retinoid storage, and their activation is a key process in liver fibrogenesis. We have previously shown that expression of the retinoic acid receptor alpha (RARalpha) is upregulated in activated rat HSCs at a posttranscriptional level and that these RARalpha proteins showed a speckled distribution in the cytosol, despite their possession of a nuclear localization signal (NLS). In this report, we further characterize these cytosolic RARalpha proteins by using exogenously expressed RARalpha protein fragments or mutants tagged with a green fluorescent protein. Substitution of four amino acids, 161-164 from lysine to alanine, abolished the NLS. Exogenously expressed RARalpha protein fragments containing an NLS were localized exclusively in the nuclei of activated rat HSCs and never colocalized with the endogenous RARalpha proteins in the cytosol, suggesting that the NLS of endogenous RARalpha proteins is masked. Biochemical analysis showed that 65% of RARalpha proteins in activated HSCs were insoluble in a mixture of detergents. The insolubility of RARalpha proteins makes it difficult to identify RARalpha proteins in activated HSCs. Therefore, we propose that insoluble, speckled cytosolic distribution of RARalpha proteins represents a new marker of HSC activation.
Assuntos
Citosol/metabolismo , Células Estreladas do Fígado/metabolismo , Receptores do Ácido Retinoico/metabolismo , Citoesqueleto de Actina/metabolismo , Animais , Biomarcadores/metabolismo , Linhagem Celular , Proteínas de Fluorescência Verde/genética , Humanos , Masculino , Mutação , Sinais de Localização Nuclear , Ratos , Ratos Wistar , Receptores do Ácido Retinoico/genética , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Receptor alfa de Ácido Retinoico , SolubilidadeRESUMO
To investigate the mechanisms involved in the atrophy of the intestines in lampreys (Lampetra japonica) during the spawning migration stage, we examined by morphological methods their intestines with special reference to degradation of extracellular matrix (ECM) components. Stellate cells are known to be distributed not only in the liver (hepatic stellate cells) but also in other organs, such as the pancreas, intestine, lung, and kidney (extrahepatic stellate cells). Hepatic stellate cells are well known to be able to biosynthesize, secrete, and degrade ECM. Therefore, we investigated the cellular and molecular mechanisms involved in the atrophy of the intestines by focusing on these intestinal extrahepatic stellate cells. The cells were found to contain phagocytosed and degraded collagen fibrils, which are one of the ECM components. A positive reaction for trimetaphosphatase (TMPase, a cytochemical marker of lysosomes) was preferentially detected in round or elongated vesicles in the intestinal extrahepatic stellate cells and the deposits of the reaction products coexisted with the degraded collagen fibrils. However, the basement membrane of the intestine, which membrane is also an ECM component, was preserved throughout the spawning migration stage of the lamprey and accumulated as a mass of thick membrane, suggesting the existence of a special mechanism for selective digestion of ECM components. These results indicate that the intestinal extrahepatic stellate cells in Lampetra japonica during its spawning migration stage might play an important mechanistic role in the atrophy of lamprey intestines by phagocytizing collagen fibrils and digesting the phagocytized collagen fibrils in their lysosomes.
Assuntos
Adipócitos/citologia , Matriz Extracelular/metabolismo , Intestinos/citologia , Lampreias/fisiologia , Fígado/citologia , Adipócitos/metabolismo , Adipócitos/ultraestrutura , Migração Animal/fisiologia , Animais , Colágeno/metabolismo , Matriz Extracelular/ultraestrutura , Mucosa Intestinal/metabolismo , Intestinos/ultraestrutura , Lampreias/anatomia & histologia , Fígado/metabolismo , Microscopia Eletrônica de Transmissão , Fagocitose/fisiologia , Comportamento Sexual Animal/fisiologiaRESUMO
Membrane proteins were obtained from the mitochondrial fraction of HL-60 cells by solubilization with octyl glucoside and bound to heparin-gels. Bound proteins were successively eluted with solutions containing increasing concentrations of Mg(2+) in the first and increasing concentrations of Ca(2+) in the second chromatography. After SDS-PAGE and subsequent N-terminal amino acid analysis of proteins on each band, 13 proteins were identified. Fifteen out of the 37 proteins analysed were modified at their N-termini. These results show that this two-step affinity chromatography method using divalent cations as eluents can be applied to a variety of membranes for the isolation of specific proteins.
Assuntos
Proteínas de Transporte/análise , Cromatografia de Afinidade/métodos , Heparina/metabolismo , Proteínas de Membrana/análise , Proteínas Mitocondriais/análise , Sequência de Aminoácidos , Proteínas de Transporte/metabolismo , Cátions Bivalentes , Fracionamento Celular , Eletroforese em Gel de Poliacrilamida , Células HL-60 , Humanos , Proteínas de Membrana/metabolismo , Proteínas Mitocondriais/química , Proteínas Mitocondriais/metabolismo , Reprodutibilidade dos Testes , Análise de Sequência de ProteínaRESUMO
Short bowel (SB) syndrome causes the malabsorption of various nutrients. Among these, vitamin A is important for a number of physiological activities. Vitamin A is absorbed by epithelial cells of the small intestine and is discharged into the lymphatic vessels as a component of chylomicrons and is delivered to the liver. In the present study, we used a rat model of SB syndrome in order to assess its effects on the expression of genes associated with the absorption, transport and metabolism of vitamin A. In the rats with SB, the intestinal mRNA expression levels of cellular retinol-binding protein II (CRBP II, gene symbol Rbp2) and apolipoprotein A-IV (gene symbol Apoa4) were higher than those in the sham-operated rats, as shown by RT-qPCR. Immunohistochemical analysis revealed that absorptive epithelial cells stained positive for both CRBP II and lecithin retinol acyltransferase, which are both required for the effective esterification of vitamin A. In the rats with SB, the retinol content in the ileum and the retinyl ester content in the jejunum were lower than those in the sham-operated rats, as shown by quantitative analysis of retinol and retinyl esters by high performance liquid chromatography. These results suggest that the elevated mRNA expression levels of Rbp2 and Apoa4 in the rats with SB contribute to the effective esterification and transport of vitamin A.
Assuntos
Apolipoproteínas A/genética , Regulação da Expressão Gênica , Intestino Delgado/metabolismo , Proteínas Celulares de Ligação ao Retinol/genética , Síndrome do Intestino Curto/genética , Síndrome do Intestino Curto/metabolismo , Vitamina A/metabolismo , Animais , Apolipoproteínas A/metabolismo , Modelos Animais de Doenças , Intestino Delgado/cirurgia , Fígado/metabolismo , Transporte Proteico , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Ratos , Proteínas Celulares de Ligação ao Retinol/metabolismo , Regulação para CimaRESUMO
HSCs showed myofibroblast-like shapes when cultured on polystyrene surface or on type I collagen-coated surface, whereas HSCs cultured on type I collagen gel were induced to elongate cellular processes, suggesting that HSCs recognize 3-D structure of extracellular type I collagen fibrils and change their morphology and function. In this study we examined the differentially regulated gene expression by extracellular matrix (ECM) components by PCR-differential display (PCR-DD) analysis followed by cloning and FASTA homology search, and identified the mRNA species as a transcription factor SP1, breast cancer resistant protein (BCRP), dystonin, and KAP3B. Regulation of dystonin and KAP3B expression was confirmed by RT-PCR analysis. Thus, cell surface-binding to extracellular interstitial collagen may trigger intracellular signaling and alteration in gene expression, and HSCs not only produce various ECM components but also change their morphology and gene expression in response to ECM components adhering to the cells.
RESUMO
To investigate whether or not hepatic stellate cells can form intercellular junctions with each other, we cultured human stellate cells (LI90) on different kinds of substrata. Intercellular junctions were detected between these cultured stellate cells by transmission electron microscopy (TEM). The molecular components of the intercellular adhesive structures were identified by immunofluorescence microscopy. Immunofluorescence for cadherin and catenins was detected at the adhesion sites between the cultured stellate cells. Thus, the intercellular junctions were indicated to be adherens junctions at the molecular level. The junctions developed in the cultured stellate cells irrespective of the type of substratum. These data suggest that the junctional formation between the stellate cells occurs in vivo as well as in vitro.
RESUMO
We examined the liver of adult polar bears, arctic foxes, and rats by gold chloride staining, fluorescence microscopy for the detection of autofluorescence of vitamin A, hematoxylin-eosin staining, staining with Masson's trichrome, Ishii and Ishii's silver impregnation, and transmission electron microscopical morphometry. The liver lobules of the arctic animals showed a zonal gradient in the storage of vitamin A. The density (i.e., cell number per area) of hepatic stellate cells was essentially the same among the zones. These results indicate that the hepatic stellate cells of the polar bears and arctic foxes possess heterogeneity of vitamin A-storing capacity in their liver lobules.
RESUMO
Vitamin A (retinol and retinyl ester) distribution and content in tissues of a lamprey (Lampetra japonica) were analyzed by morphological methods, namely, gold chloride staining, fluorescence microscopy to detect specific vitamin A autofluorescence, and electron microscopy, as well as high-performance liquid chromatography (HPLC). Hepatic stellate cells showed an abundance of vitamin A stored in lipid droplets in their cytoplasm. Similar cells storing vitamin A were present in the intestine, kidney, gill, and heart in both female and male lampreys. Morphological data obtained by gold chloride staining method, fluorescence microscopy, transmission electron microscopy, and HPLC quantification of retinol were consistent. The highest level of total retinol measured by HPLC was found in the intestine. The second and third highest concentrations of vitamin A were found in the liver and the kidney, respectively. These vitamin A-storing cells were not epithelial cells, but mesoderm-derived cells. We propose as a hypothesis that these cells belong to the stellate cell system (family) that stores vitamin A and regulates homeostasis of the vitamin in the whole body in the lamprey. Fibroblastic cells in the skin and somatic muscle stored little vitamin A. These results indicate that there is difference in the vitamin A-storing capacity between the splanchnic and intermediate mesoderm-derived cells (stellate cells) and somatic and dorsal mesoderm-derived cells (fibroblasts) in the lamprey. Stellate cells derived from the splanchnic and intermediate mesoderm have high capacity and fibroblasts derived from the somatic and dorsal mesoderm have low capacity for the storage of vitamin A in the lamprey.
Assuntos
Lampreias/anatomia & histologia , Lampreias/metabolismo , Vitamina A/metabolismo , Animais , Encéfalo/metabolismo , Cromatografia Líquida de Alta Pressão , Ésteres , Olho/metabolismo , Brânquias/metabolismo , Brânquias/ultraestrutura , Compostos de Ouro , Gônadas/metabolismo , Mucosa Intestinal/metabolismo , Intestinos/ultraestrutura , Rim/metabolismo , Rim/ultraestrutura , Fígado/metabolismo , Fígado/ultraestrutura , Microscopia Eletrônica , Microscopia de Fluorescência , Músculo Esquelético/metabolismo , Músculo Esquelético/ultraestrutura , Miocárdio/metabolismo , Miocárdio/ultraestrutura , Pele/ultraestrutura , Vitamina A/sangue , Vitamina A/farmacocinéticaRESUMO
Solubilized membrane proteins from HL-60 cells were separated by two-step affinity chromatography. Proteins eluted with MgCl2 in the first heparin-gel were applied to the second heparin-gel and eluted with CaCl2. The eluted proteins were analysed and purified by electrophoresis. N-terminal amino acid sequences of eight proteins on the characteristic bands were determined. Homology search for the sequences indicated that three microsomal proteins, two nuclear proteins and a glycolytic enzyme were eluted with divalent cations, whereas a nuclear ribonucleoprotein and a membrane-cytoskelton linker protein were not dissociated with divalent cations, but with 2 M NaCl. Heparin affinity chromatography combined with differential elution with divalent cations can be a useful method for separation of membrane proteins.
Assuntos
Proteínas Sanguíneas/isolamento & purificação , Proteínas de Transporte/isolamento & purificação , Cátions Bivalentes , Cromatografia de Afinidade/métodos , Proteínas de Membrana/isolamento & purificação , Sequência de Aminoácidos , Peptídeos Catiônicos Antimicrobianos , Proteínas Sanguíneas/química , Proteínas de Transporte/química , Eletroforese em Gel de Poliacrilamida , Células HL-60 , Humanos , Proteínas de Membrana/químicaRESUMO
Hepatic stellate cells (vitamin A-storing cells, lipocytes, interstitial cells, fat-storing cells, Ito cells) exist in the perisinusoidal space of the hepatic lobule and store 80% of the body's retinoids as retinyl palmitate in lipid droplets in the cytoplasm. Under physiological conditions, these cells play pivotal roles in the regulation of retinoid homeostasis; they express specific receptors for retinol-binding protein (RBP), a binding protein specific for retinol, on their cell surface, and take up the complex of retinol and RBP by receptor-mediated endocytosis. However, in pathological conditions such as liver fibrosis, these cells lose retinoids and synthesize a large amount of extracellular matrix (ECM) components including collagen, proteoglycan and adhesive glycoproteins. The morphology of these cells also changes from star-shaped stellate cells to that of fibroblasts or myofibroblasts. The three-dimensional structure of ECM components was found to regulate reversibly the morphology, proliferation and functions of hepatic stellate cells. Molecular mechanisms in the reversible regulation of stellate cells by ECM imply cell surface integrin binding to ECM components followed by signal transduction processes and then cytoskeleton assembly.
RESUMO
Four types of materials, type I collagen coat (Coat), acid-soluble type I collagen gel (Hardgel), pepsin-treated acid-soluble type I collagen gel (Softgel), and an extract of extracellular matrix of the murine Engelbreth-Holm-Swarm sarcoma (Matrigel), were used as matrices to culture rat hepatic parenchymal cells, and their morphological changes and adhesion were compared to the matrices by electron microscopic observations. Hepatic parenchymal cells cultured on Coat and Hardgel were extended and flattened, whereas cells cultured on Softgel and Matrigel assembled and formed aggregates. Such aggregates consisted of several hepatic parenchymal cells, with a recognizable bile duct-like alveolus on the inside. Morphologically, the aggregates were more spherical on Matrigel and oval shaped on Softgel. Microvilli of the cell surface were parallel to the matrix on Matrigel, but invaded into the gel on Softgel. Subsequently, investigation into how these morphological features affected the liver-specific functions, including secretion of albumin and induction of P450 by 3-methylcholanthrene, demonstrated that a high level of liver function was maintained in a long-term culture in hepatic parenchymal cells on Softgel. These results suggest that hepatic parenchymal cell interactions were stronger with Softgel than with Matrigel, and that Softgel appears to closely mimic the in vivo environment.
RESUMO
The primary function of hepatic stellate cells (HSCs) is the storage of vitamin A. However, they are also responsible for liver fibrosis and are therapeutic targets for treatment of liver cirrhosis. Among the many molecular markers that define quiescent or activated states of HSCs, the characteristics of type III intermediate filaments are of particular interest. Whereas vimentin and desmin are upregulated in activated HSCs, glial fibrillary acidic protein is downregulated in activated HSCs. The functional differences between vimentin and desmin are poorly understood. By time-course quantifications of several molecular markers for HSC activation, we observed that the expression of vimentin preceded that of desmin during the transdifferentiation of HSCs. The immunoreactivity of vimentin in transdifferentiated HSCs was more intense in perinuclear regions compared to that of desmin. We propose that the delayed expression of desmin following the expression of vimentin and the peripheral localization of desmin compared to vimentin are both related to the more extended phenotype of transdifferentiating HSCs observed in vitro.
RESUMO
We performed a systematic characterization of the hepatic vitamin A storage in mammals and birds of the Svalbard Archipelago and Greenland. The liver of top predators, including polar bear, Arctic fox, bearded seal, and glaucous gull, contained about 10-20 times more vitamin A than the liver of all other arctic animals studied, as well as their genetically related continental top predators. The values are also high compared to normal human and experimental animals like mouse and rat. This massive amount of hepatic vitamin A was located in large autofluorescent lipid droplets in hepatic stellate cells (HSCs; also called vitamin A-storing cells, lipocytes, interstitial cells, fat-storing cells, or Ito cells). The droplets made up most of the cells' cytoplasm. The development of such an efficient vitamin A-storing mechanism in HSCs may have contributed to the survival of top predators in the extreme environment of the arctic. These animals demonstrated no signs of hypervitaminosis A. We suggest that HSCs have capacity to take-up and store large amounts of vitamin A, which may play a pivotal role in maintenance of the food web, food chain, biodiversity, and eventually ecology of the arctic.
Assuntos
Células Estreladas do Fígado/química , Células Estreladas do Fígado/metabolismo , Comportamento Predatório/fisiologia , Vitamina A/química , Vitamina A/metabolismo , Animais , Regiões Árticas , Charadriiformes/metabolismo , Rena/metabolismo , Focas Verdadeiras/metabolismo , Especificidade da Espécie , Sobrevida/fisiologia , Ursidae/metabolismoRESUMO
Transforming growth factor (TGF) ß is a pro-fibrotic cytokine. While three isoforms (TGF-ß1, 2 and 3) are known, the functional differences between them are obscure. To investigate the roles of TGF-ß isoforms during liver fibrogenesis, male Wistar rats were administrated carbon tetrachloride (CCl4) subcutaneously twice a week for two months. Livers were excised and sectioned for histochemical examinations. These livers were also used to quantitate the expression of genes associated with fibrogenesis, including TGF-ß isoforms, as well as those associated with retinoid metabolism. Expression levels of Tgfb1 and Tgfb3 were up-regulated in CCl4-treated rat livers while that of Tgfb2 was not changed. The mRNAs for lecithin-retinol acyltransferase (Lrat) and retinoic acid hydroxylase, Cyp26a1, were also elevated. By immunohistochemical staining, TGF-ß3 protein was found to be localized mainly in liver parenchymal cells (hepatocytes). These results indicate that retinoid mobilization likely takes place within the rat's liver following CCl4 treatment, and suggest the possibility that the expression of Tgfb mRNA is regulated by retinoic acid receptors. Reporter analyses of a region of the Tgfb3 gene were performed using the rat liver parenchymal cell line, RLC-16, and a positively responsive region was identified within its intron.
Assuntos
Intoxicação por Tetracloreto de Carbono/metabolismo , Cirrose Hepática Experimental/metabolismo , Retinoides/metabolismo , Fator de Crescimento Transformador beta3/biossíntese , Animais , Intoxicação por Tetracloreto de Carbono/genética , Intoxicação por Tetracloreto de Carbono/patologia , Linhagem Celular , Perfilação da Expressão Gênica , Regulação da Expressão Gênica , Imuno-Histoquímica , Fígado/metabolismo , Fígado/patologia , Cirrose Hepática Experimental/induzido quimicamente , Cirrose Hepática Experimental/genética , Cirrose Hepática Experimental/patologia , Masculino , Camundongos , Reação em Cadeia da Polimerase , Isoformas de Proteínas , Ratos , Ratos Wistar , Retinoides/genética , Transdução de Sinais , Fator de Crescimento Transformador beta3/genética , Fator de Crescimento Transformador beta3/metabolismoRESUMO
Lampreys are ancestral representatives of vertebrates known as jawless fish. The Japanese lamprey, Lethenteron japonicum, is a parasitic member of the lampreys known to store large amounts of vitamin A within its body. How this storage is achieved, however, is wholly unknown. Within the body, the absorption, transfer and metabolism of vitamin A are regulated by a family of proteins called retinoid-binding proteins. Here we have cloned a cDNA for cellular retinol-binding protein (CRBP) from the Japanese lamprey, and phylogenetic analysis suggests that lamprey CRBP is an ancestor of both CRBP I and II. The lamprey CRBP protein was expressed in bacteria and purified. Binding of the lamprey CRBP to retinol (Kd of 13.2 nM) was identified by fluorimetric titration. However, results obtained with the protein fluorescence quenching technique indicated that lamprey CRBP does not bind to retinal. Northern blot analysis showed that lamprey CRBP mRNA was ubiquitously expressed, although expression was most abundant in the intestine. Together, these results suggest that lamprey CRBP has an important role in absorbing vitamin A from the blood of host animals.
Assuntos
Lampreias/genética , Proteínas Celulares de Ligação ao Retinol/genética , Animais , Northern Blotting , Western Blotting , Escherichia coli/metabolismo , Regulação da Expressão Gênica , Fígado/metabolismo , Filogenia , Ligação Proteica , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Ratos , Proteínas Celulares de Ligação ao Retinol/isolamento & purificação , Proteínas Celulares de Ligação ao Retinol/metabolismo , Espectrometria de Fluorescência , Distribuição Tecidual/genética , Titulometria , Vitamina ARESUMO
Activated hepatic stellate cells (HSCs) play crucial roles in liver fibrosis. In the course of liver injury, HSCs, which reside in perisinusoidal spaces and lose lipid droplets, morphologically change into a myofibroblastic phenotype and acquire an increased proliferation activity in what is known as the activated state. We have investigated therapeutic strategies for liver fibrosis by promoting spontaneous reversion or inducing apoptosis in activated HSCs. Vitamin E consists of four tocopherols and four tocotrienols, all of which are well-known antioxidants. In this study, the antiproliferative and proapoptotic effects of a tocol, which lacks methyl groups attached to the chromanol ring, and four tocopherols were investigated using activated HSCs. δ-Tocopherol and tocol exhibited relatively high proliferation inhibitory and proapoptotic abilities. However, they did not show proliferation inhibition ability on primary hepatocytes or HepG2 cells. Significant cell detachment was also observed in δ-tocopherol- and tocol-treated HSCs. Decreased protein expressions of α-smooth muscle actin and ß1 integrin were observed in a dose-dependent manner. These results indicate that δ-tocopherol and tocol induce anoikis in activated HSCs.