RESUMEN
The induction of experimental obstructive cholestasis is a reliable model for cholestatic liver diseases in rodents. Bile duct ligation (BDL) in mice provokes typical time-dependent morphological and structural changes in the liver, ranging from liver cell injury and elevated serum enzyme levels after several days, to a severe inflammatory response in the liver after 5-7 days, up to an advanced hepatic fibrosis as soon as three to four weeks after surgical ligation of the common biliary duct. Upon BDL induction, hepatic stellate cells become activated and transdifferentiate into myofibroblasts that produce extracellular matrix proteins such as collagen. In principle, the periportal fibrosis induced by BDL in rat livers is reversible. After the relief of a biliary obstruction, the liver has the capacity to revert to a nearly normal histological architecture and a fully normal biochemical function. When BDL surgery is performed by an experienced scientist, this model has very high reproducibility among all fibrotic models. All these factors corroborate the outstanding value of this model for basic and translational research in biomedicine and hepatology. Nevertheless, this model can result in significant variations when surgery is carried out by untrained personnel or when unconscious modifications are implemented that affect the quality of the intervention. A detailed protocol is provided here for the provision of reliable and reproducible BDL in mice.
Asunto(s)
Conductos Biliares/cirugía , Colestasis/etiología , Modelos Animales de Enfermedad , Ciencia de los Animales de Laboratorio , Animales , Colestasis/patología , Colestasis/fisiopatología , Colestasis/cirugía , Guías como Asunto , Humanos , Ciencia de los Animales de Laboratorio/normas , Ligadura , RatonesRESUMEN
Previously the expression of Protein Tyrosine Phosphatase Interacting Protein 51 (PTPIP51) in mouse brain was reported. Here, we investigated PTPIP51 mRNA and protein in two of the brain regions namely the hippocampus and the cerebellum of mouse brains. On a cellular level both the protein and the mRNA were related to the pyramidal cells of the hippocampal formation, the granular cells of the dentate gyrus and the cells of the adjacent strata. In the cerebellum PTPIP51 was traced in Purkinje cells, the cells of the molecular layer and the granular layer. On a subcellular level only partial co-localization was seen for the endoplasmic reticulum, but not with mitochondria. In addition the interactome of PTPIP51 was analysed. In hippocampal cells a strong interaction with PTP1B and vesicle-associated membrane protein-associated protein B (VAPB) was detected. A somewhat differing interaction profile was found in the cerebellum, where high interaction levels were found for 14-3-3, diacylglycerol kinase α (DGKα), NFκB and PTP1B. These interaction partners represent specific signalling pathways linked to building memory. PTPIP51 can be associated with nerve growth factor signalling, dendritic and axonal growth, synaptogenesis, and all processes needed for memory formation. Moreover, in HT-22 mouse hippocampal cells PTPIP51 expression was induced by administrating the fibroblast growth factor 1 (FGF-1), which is known to take part in learning/memory processes. Knocking down p38-MAPK also led to an up-regulation of PTPIP51 probably resembling a compensative mechanism. Thus, a possible connection to the processing of memories can be anticipated. Differences in the interaction profile in both regions may be attributed to the actual/local differences in memory formation.
Asunto(s)
Hipocampo/metabolismo , Memoria , Proteínas Tirosina Fosfatasas/metabolismo , Células de Purkinje/metabolismo , Proteínas 14-3-3/genética , Proteínas 14-3-3/metabolismo , Animales , Línea Celular , Diacilglicerol Quinasa/genética , Diacilglicerol Quinasa/metabolismo , Retículo Endoplásmico/metabolismo , Femenino , Factor 1 de Crecimiento de Fibroblastos/farmacología , Hipocampo/citología , Masculino , Proteínas de la Membrana/genética , Proteínas de la Membrana/metabolismo , Ratones , Ratones Endogámicos C57BL , Mitocondrias/metabolismo , FN-kappa B/genética , FN-kappa B/metabolismo , Transporte de Proteínas , Proteína Tirosina Fosfatasa no Receptora Tipo 1/genética , Proteína Tirosina Fosfatasa no Receptora Tipo 1/metabolismo , Proteínas Tirosina Fosfatasas/genética , Células Piramidales/efectos de los fármacos , Células Piramidales/metabolismo , ARN Mensajero/genética , ARN Mensajero/metabolismo , Proteínas de Transporte Vesicular , Proteínas Quinasas p38 Activadas por Mitógenos/genética , Proteínas Quinasas p38 Activadas por Mitógenos/metabolismoRESUMEN
AIMS: Our previous experiments revealed an association of PTPIP51 (protein tyrosine phosphatase interacting protein 51) with the insulin signalling pathway through PTP1B and 14-3-3beta. We aimed to clarify the role of PTPIP51 in adipocyte metabolism. METHODS: Four groups of ten C57Bl/6 mice each were used. Two groups were fed a standard diet; two groups were fed a high-fat diet. Two groups (one high-fat diet and one standard diet) were submitted to endurance training, while the remaining two groups served as untrained control groups. After ten weeks, we measured glucose tolerance of the mice. Adipose tissue samples were analyzed by immunofluorescence and Duolink proximity ligation assay to quantify interactions of PTPIP51 with either insulin receptor (IR) or PKA. RESULTS: PTPIP51 and the IR and PTPIP51 and PKA, respectively, were colocalized in all groups. Standard diet animals that were submitted to endurance training showed low PTPIP51-IR and PTPIP51-PKA interactions. The interaction levels of both the IR and PKA differed between the feeding and training groups. CONCLUSION: PTPIP51 might serve as a linking protein in adipocyte metabolism by connecting the IR-triggered lipogenesis with the PKA-dependent lipolysis. PTPIP51 interacts with both proteins, therefore being a potential gateway for the cooperation of both pathways.
Asunto(s)
Tejido Adiposo/metabolismo , Proteínas Quinasas Dependientes de AMP Cíclico/metabolismo , Proteínas Tirosina Fosfatasas/fisiología , Receptor de Insulina/metabolismo , Tejido Adiposo/química , Animales , Peso Corporal , Proteínas Quinasas Dependientes de AMP Cíclico/análisis , Dieta Alta en Grasa , Prueba de Tolerancia a la Glucosa , Lípidos/biosíntesis , Lipólisis , Masculino , Ratones , Ratones Endogámicos C57BL , Resistencia Física , Proteínas Tirosina Fosfatasas/análisis , Receptor de Insulina/análisis , Transducción de SeñalRESUMEN
OBJECTIVE: We investigated the expression of protein tyrosine phosphatase-interacting protein 51 (PTPIP51) and its interaction with protein tyrosine phosphatase 1B (PTP1B) and 14-3-3ß in mice exhibiting insulin resistance and obesity. DESIGN: A total of 20 mice were included in the study. Eight control animals were fed a normal standard diet, six animals were fed a high-fat diet and six animals were submitted to a treadmill training parallel to the feeding of a high-fat diet. After 10 weeks, a glucose tolerance test was performed and abdominal adipose tissue samples of the animals were collected. RESULTS: PTPIP51 protein was identified in the adipocytes of all samples. PTPIP51 interacted with PTP1B and with 14-3-3ß protein. Compared with untrained mice fed a standard diet, the interaction of PTPIP51 with PTP1B was reduced in high-fat diet-fed animals. The highest interaction of PTPIP51 with 14-3-3ß was seen in trained animals on high-fat diet, whereas untrained animals on high-fat diet displayed lowest values. CONCLUSION: PTPIP51 is expressed in adipose tissue of humans, rats and mice. Obesity with enhanced insulin resistance resulted in a reduction of PTPIP51 levels in adipocytes and influenced the interactions with PTP1B and 14-3-3ß. The interaction of PTPIP51 with PTP1B suggests a regulatory function of PTPIP51 in insulin receptor signal transduction. The interaction of PTPIP51 with 14-3-3ß, especially in trained individuals, hints to an involvement of PTPIP51 in the downstream regulation of insulin action.
Asunto(s)
Proteínas 14-3-3/metabolismo , Tejido Adiposo/metabolismo , Resistencia a la Insulina , Obesidad/metabolismo , Proteína Tirosina Fosfatasa no Receptora Tipo 1/metabolismo , Proteínas Tirosina Fosfatasas/metabolismo , Tejido Adiposo/patología , Animales , Dieta Alta en Grasa , Regulación de la Expresión Génica , Prueba de Tolerancia a la Glucosa , Ratones , Ratas , Reacción en Cadena en Tiempo Real de la PolimerasaRESUMEN
This study demonstrates the expression of the novel protein protein tyrosine phophatase-interacting protein 51 (PTPIP51) in mammalian brain tissue. Serial sections of the whole adult mouse brain were analyzed for PTPIP51 protein and mRNA by immunohistochemistry, immunoblotting, RT-PCR, and in situ hybridization. Recent investigations by Yu et al. (2008) describe PTPIP51 as being capable of activating Raf-1, thereby modulating the MAPK pathway. The role of Raf-1, as well as of 14-3-3, in neurological disorders is well established. PTPIP51 expression was confined to neurons in the following structures: the piriform cortex and their connections to the anterior commissure, nucleus accumbens, paraventricular and supraoptical nuclei, neurohypophysis, superior colliculus, genu of facialis nerve, spinal trigeminal tract, inferior cerebellar peduncle, and cerebellum. In the cerebellum, a subpopulation of Purkinje cells and their dendrites was strongly PTPIP51 positive. Moreover, PTPIP51 was found to be colocalized with vasopressin and its transport protein neurophysin II in the neuroendocrine nuclei and their connections to the neurohypophysis. The data presented here suggest a role of PTPIP51 in neuronal homeostasis, axonal growth, and transport.
Asunto(s)
Encéfalo/metabolismo , Proteínas Tirosina Fosfatasas/metabolismo , Animales , Dermoscopía , Femenino , Immunoblotting , Inmunohistoquímica , Hibridación in Situ , Ratones , Neurofisinas/metabolismo , ARN Mensajero/metabolismo , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Vasopresinas/metabolismoRESUMEN
The surface chemical and physical character of offset paper was studied before and after application of model fountain solutions based on isopropyl alcohol and an alcohol-free surfactant solution. The paper surface features were characterised with atomic force microscopy and the surface energies were determined by contact angle measurements. Changes in the surface chemical properties induced by the fountain solutions were investigated with X-ray photoelectron spectroscopy and time-of-flight secondary ion mass spectroscopy. Coated papers wetted with the surfactant solution revealed a slight increase in the root mean square roughness, but the isopropyl alcohol solution led to no observable changes. The change in sub-micro roughness is ascribed not only to substrate swelling or migration of coating constituents but also to the presence of surfactant on the surface. A change in the surface energy and particularly the polar contribution was observed after application of the surfactant solution. The X-ray photoelectron spectroscopy showed an increase in the oxygen-to-carbon ratio, which confirms the presence of surfactant on the surface. Time-of-flight secondary ion mass spectroscopy showed that the isopropyl alcohol solution did not change the elemental composition of the surface whereas the surfactant solution clearly did so. The distribution of surfactant on the surface was confirmed by mapping the characteristic fragments of the molecule.
RESUMEN
Hepatic stellate cells (HSCs) are pericytes of liver sinusoidal endothelial cells (LSECs) and activation of HSC into a myofibroblast-like phenotype (called transdifferentiation) is involved in several hepatic disease processes including neovascularization during liver metastasis, chronic and acute liver injury. While early smooth muscle cell (SMC) differentiation markers including SM alpha-actin and SM22alpha are expressed in a variety of non-SMC, expression of late-stage markers is far more restricted. Here, we found that in addition to early SMC markers, activated rat HSC express a large panel of characteristic late vascular SMC markers including SM myosin heavy chain, h1-calponin and h-caldesmon. Furthermore, myocardin, which is present exclusively in SMCs and cardiomyocytes and controls the transcription of a subset of early and late SMC markers, is highly expressed in activated HSC. We further studied activated HSC in a functional three-dimensional spheroidal co-culture system together with endothelial cells (EC). Co-culture spheroids of EC and SMC differentiate spontaneously and organize into a core of SMC and a surface layer of EC representing an inside-outside model of the physiological assembly of blood vessels. Replacing SMC by in vitro activated HSC resulted in a similar organized spheroid with differentiated, von-Willebrand factor producing, surface lining quiescent human umbilical vein endothelial cell and a core of HSC. In an in vitro angiogenesis assay, activated HSC induced quiescence in vascular EC-the hallmark of vascular SMC function. Co-spheroids of LSEC and activated HSC formed capillary-like sprouts in gel angiogenesis assays expressing the vascular EC marker VE-cadherin. Our findings indicate that activated HSC are capable to adapt a functional SMC phenotype and to induce formation of tubular sprouts by LSEC and vascular endothelial cells. Since tumors and tumor metastasis induce HSC activation, HSC may take part in tumor-induced neoangiogenesis by adapting SMC-like functions.
Asunto(s)
Células Endoteliales/citología , Hígado/citología , Músculo Liso Vascular/citología , Animales , Biomarcadores/metabolismo , Diferenciación Celular , Técnicas de Cocultivo , Células Endoteliales/metabolismo , Humanos , Masculino , Músculo Liso Vascular/metabolismo , Fenotipo , Ratas , Ratas Sprague-Dawley , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Factor A de Crecimiento Endotelial Vascular/metabolismoRESUMEN
Endothelial cells line the blood vessel and precursor endothelial cells appear to have a pivotal effect on the organ formation of the heart, the embryonic development of the kidney, and the liver. Several growth factors including the fibroblast growth factors (FGF) seem to be involved in these processes. Ligands such as basic FGF produced and secreted by endothelial cells may also coordinate cellular migration, differentiation, and proliferation under pathological conditions including wound healing, tumorgenesis, and fibrogenesis in the adult. Recently we demonstrated the expression of two secreted FGFs, FGF16, and FGF18, in HUVEC and in rat aortic tissue. In the present report, we confirmed by RT-PCR analysis that FGF18 is wildly expressed in the cardiovascular tissue, while FGF16 showed a more restricted expression pattern. HUVEC clearly demonstrated chemotaxis towards FGF16 and FGF18. Both FGFs also enhanced cell migration in response to mechanical damage. However, recombinant FGF16 and FGF18 failed to induce endothelial cell proliferation or sprouting in a three-dimensional in vitro angiogenesis assay. Fgf18 expression was earlier reported in the liver, and we detected FGF18 expression in liver vascular and liver sinusoidal endothelial cells (LSECs), but not in hepatic parenchymal cells. Recombinant FGF18 stimulated DNA synthesis in primary hepatocytes, suggesting, that endothelial FGF18 might have a paracrine function in promoting growth of the parenchymal tissue. Interestingly, FGF2, which is mitogenic on endothelial cells and hepatocytes stimulates a sustained MAPK activation in both cell types, while FGF18 causes a short transient activation of the MAPK pathway in endothelial cells but a sustained activation in hepatocytes. Therefore, the difference in the time course of MAPK activation by the different FGFs appears to be the cause for the different cellular responses.
Asunto(s)
Endotelio Vascular/metabolismo , Factores de Crecimiento de Fibroblastos/biosíntesis , Factores de Crecimiento de Fibroblastos/fisiología , Animales , Movimiento Celular/efectos de los fármacos , Movimiento Celular/fisiología , Proliferación Celular/efectos de los fármacos , Endotelio Vascular/citología , Factor 2 de Crecimiento de Fibroblastos/farmacología , Hepatocitos/efectos de los fármacos , Humanos , Hígado/efectos de los fármacos , Proteínas Quinasas Activadas por Mitógenos/metabolismo , Músculo Liso Vascular/metabolismo , Neovascularización Fisiológica/fisiología , Ratas , Receptores de Factores de Crecimiento de Fibroblastos/biosíntesisRESUMEN
Different ways of calculating surface energy components for substrates used in offset printing are compared. The results of the very useful van Oss-Chaudhury-Good bi-bidentate model (vOCG) are simplified to mono-bidentate and mono-monodentate models. The unbalance in the acid-base values often obtained by the vOCG model is strongly reduced when applying the simple mono-monodentate model. Moreover, the frequently encountered problem of negative square roots of the acid and base components is removed. An attempt to describe the ink transfer during offset printing by calculating theoretical works of adhesion between ink/plate and ink/paper is also made. The effect of paper roughness on the wetting was studied with atomic force microscopy (AFM).
RESUMEN
Pleiotrophin (PTN) is a secretory heparin binding protein with various biological activities including mitogenesis, angiogenesis, and tissue repair after injury. Recent studies have shown that PTN is a strong mitogen of hepatocytes and involved in liver regeneration. In adult liver cells Ptn gene is mainly expressed by quiescent hepatic stellate cells (HSCs). Although we have been able to demonstrate mRNA and protein expression of the anaplastic lymphoma kinase-the receptor tyrosine kinase for PTN-on HSCs, PTN did not act as a mitogen of HSCs in contrast to hepatocytes. PTN immunoreactivity was markedly increased in experimental fibrogenesis by common bile duct ligation and observed in sinusoidal HSCs. In primary HSC cultures, Ptn transcription was significantly increased by PDGF-BB, and under hypoxic atmosphere. Mechanistically, hypoxia and PDGF mediated induction of PTN expression in sinusoidal HSCs may provide a strong mitogenic signal for hepatocytes to limit the damage to the parenchymal cells in biliary-type liver fibrogenesis.
Asunto(s)
Proteínas Portadoras/metabolismo , Hipoxia de la Célula/fisiología , Citocinas/metabolismo , Hepatocitos/efectos de los fármacos , Hepatocitos/metabolismo , Cirrosis Hepática/metabolismo , Factor de Crecimiento Derivado de Plaquetas/farmacología , Regulación hacia Arriba/efectos de los fármacos , Animales , Becaplermina , Proteínas Portadoras/genética , Células Cultivadas , Chlorocebus aethiops , Citocinas/genética , ADN/biosíntesis , ADN/genética , Células Endoteliales/citología , Células Endoteliales/metabolismo , Hepatocitos/citología , Hígado/citología , Hígado/metabolismo , Cirrosis Hepática/patología , Mitosis , Proteínas Proto-Oncogénicas c-sis , Ratas , Transcripción Genética/genéticaRESUMEN
Fibroblast growth factors (FGFs) are important angiogenic growth factors. While basic FGF (FGF2) is well established as a potent inducer of angiogenesis much less is known about other FGFs possibly expressed by EC. We investigated the expression of all known FGFs, their main tyrosine kinase receptors and antagonists by RT-PCR analysis in human umbilical vascular endothelial cells (HUVECs) to obtain a complete expression profile of this important growth factor system in model endothelial cells (EC). In addition to FGFR1IIIc, which is considered as the major FGF receptor in EC, HUVECs express similar levels of FGFR3IIIc, detectable amounts of FGFR2IIIc and a new FGF receptor without an intracellular kinase domain (FGFR5). HUVECs express several secreted FGFs, including FGF5, 7, 8, 16 and 18 and two members of the fibroblast growth factor homologous factors (FHFs), not yet reported to be expressed in EC. The expression panel was compared with that obtained from human vascular smooth muscle cells (VSMCs) and human aortic tissue. Human umbilical artery smooth muscle cells (HUASMCs) and HUVECs express the identical FGF receptor and ligand panel implicating that both cell types act, according the FGF signals more as an entity than as individual cell types. Expression of Fgf1, 2, 7, 16 and 18 and the antagonists Sprouty 2,3 and 4 was demonstrated for all analysed cDNAs. The IIIc isoforms of FGFR1 and 2 and the novel FGFR5 were expressed in the aorta, but expression of the FGF receptor 3 was not detected in cDNAs derived from aortic tissue. In the VSMC of rat aortic tissue and in HUASM cultured cells we could demonstrate FGF18 immunoreactivity in the nucleus of the cells. The expression of several secreted FGFs by EC may focus the view more on their paracrine effects on neighbouring cells during tissue regeneration or tumor formation.
Asunto(s)
Endotelio Vascular/citología , Factores de Crecimiento de Fibroblastos/biosíntesis , Regulación de la Expresión Génica , Músculo Liso Vascular/citología , Secuencia de Aminoácidos , Animales , Aorta/metabolismo , Northern Blotting , Células COS , Línea Celular , Línea Celular Tumoral , Células Cultivadas , Técnicas de Cocultivo , ADN/metabolismo , ADN Complementario/metabolismo , Factores de Crecimiento de Fibroblastos/metabolismo , Células HeLa , Humanos , Inmunohistoquímica , Ligandos , Microscopía Fluorescente , Datos de Secuencia Molecular , Músculo Liso/citología , Miocitos del Músculo Liso , Neovascularización Patológica , Reacción en Cadena de la Polimerasa , Isoformas de Proteínas , Estructura Terciaria de Proteína , Proteínas Tirosina Quinasas/metabolismo , ARN/metabolismo , Ratas , Receptor Tipo 5 de Factor de Crecimiento de Fibroblastos , Receptores de Factores de Crecimiento de Fibroblastos/metabolismo , Proteínas Recombinantes/química , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Homología de Secuencia de AminoácidoRESUMEN
High concentrations of protein tyrosine phosphatase (PTP) were found with secretory vesicles of glucagon-producing INR1G9 cells by electron microscopic immunocytochemistry, using a polyclonal antiserum specific for the PTP1B/T-cell (TC)PTP subfamily of PTP. Since TCPTP protein and mRNA were below the detection limit in the cells but significant amounts of PTP1B and mRNA were recognised by a specific monoclonal antibody and a mRNA probe we conclude, that the PTP associated with the vesicles is PTP1B. Only reverse transcriptase (RT)-PCR with primers specific for PTP1B yielded a product of the expected nucleotide sequence. Thus, we conclude that the PTP associated with the vesicles is PTP1B. The presence of vanadate for 48 h attenuated PTP1B expression and caused reduction of steady-state levels of the phosphatase. These conditions also led to a continuing increase in the steady-state rate of glucagon release by the cells. This rate and tyrosine phosphatase levels showed an inverse relationship, suggesting a suppressive role of PTP1B on the regulated secretion of glucagon by INR1G9 cells.
Asunto(s)
Glucagón/metabolismo , Páncreas/enzimología , Proteínas Tirosina Fosfatasas/análisis , Animales , Northern Blotting/métodos , Línea Celular Tumoral , Cricetinae , Immunoblotting/métodos , Inmunohistoquímica/métodos , Páncreas/metabolismo , Proteína Tirosina Fosfatasa no Receptora Tipo 1 , Proteínas Tirosina Fosfatasas/antagonistas & inhibidores , Proteínas Tirosina Fosfatasas/genética , ARN Mensajero/análisis , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Tasa de Secreción , Vanadatos/farmacologíaRESUMEN
OBJECTIVES: Real time polymerase chain reaction followed by melting curve analysis using hybridization probes has become an important tool in routine diagnosis of the HFE mutations, which are associated with hereditary hemochromatosis. DESIGN AND METHODS: We used the LightCycler technology for simultaneous detection of the H63D and C282Y mutations of the HFE gene in patients with a higher prevalence for hemochromatosis. RESULTS: In our cohort we identified two siblings with a variant pattern of the HFE-LightCycler melting profiles preventing allelic discrimination. CONCLUSIONS: As a consequence, in these patients DNA sequencing or RFLP analysis is necessary to unequivocally assign the correct HFE genotype.
Asunto(s)
Pruebas Genéticas/métodos , Hemocromatosis/diagnóstico , Hemocromatosis/genética , Mutación , Sustitución de Aminoácidos , Artefactos , Estudios de Cohortes , Análisis Mutacional de ADN , Frecuencia de los Genes , Genotipo , Alemania/epidemiología , Hemocromatosis/epidemiología , Heterocigoto , Homocigoto , Humanos , Desnaturalización de Ácido Nucleico , Reacción en Cadena de la Polimerasa/métodos , Polimorfismo de Longitud del Fragmento de Restricción , Prevalencia , Valores de ReferenciaRESUMEN
Latent transforming growth factor beta binding protein (LTBP), a high-molecular-weight glycoprotein of the large latent TGF-beta complex is suggested to serve as an anchor for latent TGF-beta in the extracellular matrix and as a component of microfibrillar structures. Proteolytic cleavage of LTBP is supposed to be a prerequisite for the release and generation of bioactive (mature) TGF-beta. We investigated the expression of LTBP isoforms in normal and fibrotic rat liver and in cultured rat hepatic stellate cells (HSC) transdifferentiating to myofibroblasts (MFB). We further determined their interaction with the matrix and some of their basic functions. Immunostainings of normal and fibrotic livers demonstrate intense signals for LTBP-1 and -2, preferably in parenchymal, but also nonparenchymal, cells and in fibrotic extracellular matrix. However, in situ hybridization points to a restriction of transcripts to nonparenchymal cells from fibrotic livers, whereas hepatocytes were always devoid of LTBP transcripts. The findings were confirmed by real-time quantitative reverse-transcription polymerase chain reaction (RT-PCR), which showed isoform-specific increases of LTBP transcripts in cultured stellate cells transdifferentiating to MFB and by Northern blot analyses showing the absence of LTBP-1 mRNA in freshly isolated hepatocytes. Using a cell enzyme-linked immunosorbent assay (ELISA), a differential increase of partly deoxycholate (DOC)-resistant, matrix-bound LTBP-1 and -2 was measured in cultured stellate cells. Treatment with plasmin generated soluble LTBP-1 and bioactive TGF-beta, which was able to induce Smad7 expression in an autocrine fashion. Our data propose (transdifferentiating) stellate cells, respectively MFB, as the major source of LTBP in normal and fibrotic liver, which here probably fulfills structural and TGF-beta-regulating functions as suggested for nonhepatic tissues.
Asunto(s)
Proteínas Portadoras/metabolismo , Matriz Extracelular/metabolismo , Péptidos y Proteínas de Señalización Intracelular , Cirrosis Hepática/metabolismo , Hígado/citología , Hígado/metabolismo , Animales , Conductos Biliares , Proteínas Portadoras/genética , Proteínas Portadoras/fisiología , Diferenciación Celular/fisiología , Células Cultivadas , Proteínas de Unión al ADN/metabolismo , Fibrinolisina/farmacología , Proteínas de Unión a TGF-beta Latente , Ligadura , Masculino , ARN Mensajero/metabolismo , Ratas , Ratas Sprague-Dawley , Valores de Referencia , Proteína smad7 , Distribución Tisular , Transactivadores/metabolismo , Factor de Crecimiento Transformador beta/metabolismoRESUMEN
The secretory vesicles of some cells of the islets of Langerhans of the pancreas contain high amounts of immunoreactive tyrosine phosphatase of the PTP1B/TCPTP subfamily. The cells are located in the peripheral parts of the islets and were identified as glucagon- and pancreatic polypeptide-forming cells. The tyrosine phosphatase is also enriched in some of the somatostatin-producing cells but is not elevated either in insulin-producing B-cells or in the exocrine pancreas. Virtually the same patterns were found in pancretic tissues of rats, guinea pigs, pigs, and mice. High levels of detergent-soluble tyrosine phosphatase were measured in the particular fraction of pancreatic islets with a substrate preferred by PTP1B/TCPTP-type protein tyrosine phosphatases.
Asunto(s)
Glucagón/análisis , Islotes Pancreáticos/enzimología , Polipéptido Pancreático/análisis , Proteínas Tirosina Fosfatasas/análisis , Animales , Glucagón/metabolismo , Cobayas , Humanos , Inmunohistoquímica , Islotes Pancreáticos/citología , Islotes Pancreáticos/ultraestructura , Ratones , Polipéptido Pancreático/metabolismo , Ratas , PorcinosRESUMEN
Protein tyrosine phosphatases were analyzed in oocytes of Ascaris suum. Phosphatases dephosphorylating modified acidic lysozyme were present in high-molecular-weight form (M(r) > 600,000) and as a 50- to 55-kDa protein in the soluble fraction. The low-molecular-weight form of the phosphatase cross-reacted with an antiserum raised against human T-cell protein tyrosine phosphatase and was not distinguishable from the 50- to 55-kDa protein tyrosine phosphatase previously described in the muscular layer of the adult worms (B. Schmid et al. 1996, Molecular and Biochemical Parasitology 77, 183-192). The low-molecular-weight form was also present on immunoblots of high-molecular-weight protein tyrosine phosphatase preparations after denaturing electrophoresis. The same or a similar form of the tyrosine phosphatase was also found in detergent extracts from the pelletal fraction. In addition, another tyrosine phosphatase of 180 kDa molecular mass that dephosphorylated myelin basic protein was also found in extracts from the soluble compartment as well as in detergent extracts from the pelletal fraction. It showed no cross-reactivity with antisera raised against soluble mammalian phosphatases and was resistant to inhibition by vanadate. While the activities of the myelin basic protein-dephosphorylating protein phosphatase remained fairly constant during early development of the oocytes, the activity of the enzyme dephosphorylating modified lysozyme in the pelletal fraction decreased to less than 10% of the initial activity between days 3 and 28 of incubation. Immunocytochemical studies of unfertilized and developing Ascaris eggs revealed association of protein tyrosine kinase and protein tyrosine phosphatase with the egg shell, in addition to their presence in the neighborhood of mitochondria. The amount of enzyme changed with the stage of development. In the larval stage (21 days) protein tyrosine kinase had increased in the chitin layer of the shell and in the nuclei while the relative amount of tyrosine phosphatase decreased in accordance with the biochemical data.
Asunto(s)
Ascaris suum/enzimología , Proteínas Tirosina Fosfatasas/análisis , Animales , Ascaris suum/crecimiento & desarrollo , Cromatografía en Gel , Femenino , Immunoblotting , Inmunohistoquímica , Peso Molecular , Oocitos/enzimología , Fosforilación , Proteínas Tirosina Fosfatasas/químicaRESUMEN
Two forms of protein tyrosine phosphatases were partially purified from the musculo-cutaneous layer of Ascaris suum. A 50-55-kDa soluble form of the phosphatase cross-reacted with antisera raised against human PTP-1B and TC-PTP. Like the enzyme of human origin the phosphatase from Ascaris exhibited a preference for anionic substrates (tyrosine-phosphorylated carboxymethylated and maleylated lysozyme) and was inhibited by micromolar concentrations of vanadate, molybdate, Zn2+, heparin, and poly(Glu4Tyr). As revealed by immuno-cytochemistry, the phosphatase was mainly localized and appeared equally distributed in the cytoplasm, apart from the myofibrils, possibly in loose association with cytoskeletal elements. A second tyrosine phosphatase of 180 kDa molecular mass was mainly found in detergent extracts from a microsomal fraction. It showed no cross-reactivity with antisera raised against soluble mammalian phosphatases and dephosphorylated a basic substrate (Tyr-phosphorylated myelin basic protein). It was resistant to common inhibitors of mammalian tyrosine phosphatases except Zn2+ and thiol reagents.
Asunto(s)
Ascaris suum/enzimología , Proteínas Tirosina Fosfatasas , Animales , Fraccionamiento Celular , Citosol/enzimología , Inhibidores Enzimáticos/farmacología , Femenino , Cinética , Microsomas/enzimología , Peso Molecular , Muramidasa/metabolismo , Músculos/enzimología , Proteína Básica de Mielina/metabolismo , Proteínas Tirosina Fosfatasas/análisis , Proteínas Tirosina Fosfatasas/antagonistas & inhibidores , Proteínas Tirosina Fosfatasas/química , Proteínas Tirosina Fosfatasas/aislamiento & purificación , Proteínas Tirosina Fosfatasas/metabolismo , Especificidad por SustratoRESUMEN
The inhibition potentials of products and substrate on the growth ofClostridium butyricum and Klebsiella pneumoniae in the glycerol fermentation are examined from experimental data and with a mathematicalmodel. Whereas the inhibition potential of externally added and self-produced 1,3-propanediol is essentially the same, butyric acid produced by the culture is more toxic than that externally added. The same seems to apply for acetic acid. The inhibitory effect of butyric acid is due tothe total concentration instead of its undissociated form. For acetic acid, it cannot be distinguished between the total concentration and the undissociated formThe inhibition effects of products and substrate in the glycerol fermentation are irrespective of the strains, and, therefore, the same growth model can be used. The maximum product concentrations tolerated (critical concentrations C(*) (pi)) are 0.35 g/Lfor undissociated acetic acid, 10.1 g/L for total butyric acid, 16.6 g/L for ethanol, 71.4 g/L for 1,3-propanediol, and 187.6 g/L for glycerol, which are applicable to C. butyricum and K. pneumoniae grown under a variety of conditions. For 55 steady-states, which were obtained from different types of continuous cultures over a pHrange of 5.3-8.5 and under both substrate limitation and substrate excess, the proposed growth model fits the experimental data with an average deviation of 17.0%. The deviation of model description from experimental values reduces of 11.4% if only the steady-states with excessive substrate are considered. (c) 1994 John Wiley & Sons, Inc.