Ultrastructural features of the columellar muscle and contractile protein analyses in different muscle groups of Megalobulimus abbreviatus (Gastropoda, Pulmonata)

In Megalobulimus abbreviatus, the ultrastructural features and the contractile proteins of columellar, pharyngeal and foot retractor muscles were studied. These muscles are formed from muscular fascicles distributed in different planes that are separated by connective tissue rich in collagen fibrils. These cells contain thick and thin filaments, the latter being attached to dense bodies, lysosomes, sarcoplasmic reticulum, caveolae, mitochondria and glycogen granules. Three types of muscle cells were distinguished: T1 cells displayed the largest amount of glycogen and an intermediate number of mitochondria, suggesting the highest anaerobic metabolism; T2 cells had the largest number of mitochondria and less glycogen, which suggests an aerobic metabolism; T3 cells showed intermediate glycogen volumes, suggesting an intermediate anaerobic metabolism. The myofilaments in the pedal muscle contained paramyosin measuring between 40 and 80 nm in diameter. Western Blot muscle analysis showed a 46-kDa band that corresponds to actin and a 220-kDa band that corresponds to myosin filaments. The thick filament used in the electrophoresis showed a protein band of 100 kDa in the muscles, which may correspond to paramyosin.     

Changes in E-NTPDase 3 expression and extracellular nucleotide hydrolysis during the myofibroblast/lipocyte differentiation

Hepatic stellate cells (HSC) play a critical role in the development and maintenance of liver fibrosis. HSC are lipocytes that displayed the capacity to develop into myofibroblast-like cells. Ecto-nucleoside triphosphate diphosphohydrolases (E-NTPDases) regulate the concentration of extracellular nucleotides, signaling molecules that play a role in the pathogenesis of hepatic fibrosis. In the present study, we identified and compared the expressions of E-NTPDase family members in two different phenotypes of the mouse hepatic stellate cell line (GRX) and evaluated the nucleotide hydrolysis by these cells. We show that both phenotypes of GRX cell line expressed NTPDase 3 and 5. However, only activated cells expressed NTPDase 6. In quiescent-like cells, the hydrolysis of triphosphonucleosides was significantly higher, and was related to an increase in Entpd3 mRNA expression. The diphosphonucleosides were hydrolyzed at a similar rate by two phenotypes of GRX cells. We suggest that up-regulation of Entpd3 mRNA expression modulates the extracellular concentration of nucleotides/nucleosides and affect P2-receptor signaling differently in quiescent-like cells and may play a role in the regulation of HSC functions.     

Hepatic stellate cell activation in vitro: cell cycle arrest at G2/M and modification of cell motility

Hepatic fibrosis is a common response to chronic liver injury and is characterized by increased production of extracellular matrix components, whose major part is produced by hepatic stellate cells activated by inflammatory mediators to proliferate and migrate into the injured regions. GRX cells are a model of hepatic stellate cells characterized as myofibroblasts by morphological and biochemical criteria. We have recently shown that they respond to inflammatory mediators and cytokines present in the concanavalin A-activated spleen cell supernatant (SCS) by quantitative changes in the expression of intermediate filaments. The present study investigated the effects of SCS and TNF-alpha on the GRX cell proliferation and on the organization of the actin cytoskeleton. SCS and TNF-alpha diminished the culture cell density, with an increase of cell [(3)H]thymidine incorporation and of cellular protein content, indicating an arrest in the G2/M phase of the cell cycle, which was reversible 48 h after removal of SCS. This effect was abrogated by dibutiryl-cAMP. Actin cytoskeleton reorganization was observed after 24 h treatment, indicating increased cell motility. Our results suggest that inflammation-dependent activation of stellate cells occurs in ordered interaction and coordination of proinflammatory agents. The increase of cAMP levels activates the conversion of lipocytes into myofibroblasts and increases the number of cells that can participate in repair. Since cAMP retains cells in the G1 phase, cytokines of the TNF-alpha group are required for cell proliferation inducing the entry into the S phase. The progression through the G2/M checkpoint is mediated again by increased cAMP levels.     

A feature selection approach for identification of signature genes from SAGE data

Background  

One goal of gene expression profiling is to identify signature genes that robustly distinguish different types or grades of tumors. Several tumor classifiers based on expression profiling have been proposed using microarray technique. Due to important differences in the probabilistic models of microarray and SAGE technologies, it is important to develop suitable techniques to select specific genes from SAGE measurements.     

Variations of ganglioside biosynthetic pathways in the phenotype conversion from myofibroblasts to lipocytes in murine hepatic stellate cell line

GRX cell line represents hepatic stellate cell and can be transformed from an actively proliferation myofibroblast phenotype into a quiescent fat-storing lipocyte phenotype. Both express the same gangliosides (GM3, GM2, GM1 and GD1a), which are resolved as doublets on HPTLC. Upper/lower band ratio is increased in lipocyte-like cells and the upper band is composed by ceramides with long-chain fatty acids. This study evaluated the contribution of de novo synthesis, sphingosine and Golgi recycling pathways on ganglioside biosynthesis, in both phenotypes. Cells were preincubated with 5 mM β-chloroalanine (SPT: serine palmitoyltransferase inhibitor) or with 25 μM fumonisin B1 (ceramide synthase inhibitor) and then radiolabeled with [U-¹⁴C]galactose in the continued presence of inhibitors. Gangliosides were extracted, purified and analyzed by HPTLC. In myofibroblast-like cells, simple gangliosides use the de novo pathway while complex gangliosides are mainly synthesized by recycling pathways. In lipocyte-like cells, de novo pathway has a lesser contribution and this is in agreement with the lower activity of the committed enzyme of sphingolipid synthesis (SPT) detected in this phenotype. SPT mRNA has an identical expression in both phenotypes. It was also observed that gangliosides doublets from myofibroblast-like cells have the same distribution between triton soluble and insoluble fractions (upper band > lower band) while the gangliosides doublets from lipocyte-like cells show an inversion in the insoluble fraction (lower band > upper band) in comparison to soluble fraction. These results indicate that myofibroblast- and lipocyte-like cells have important differences between the glycosphingolipid biosynthetic pathways, which could contribute with the respective glycosphingolipid-enriched membrane microdomain’s composition.     

Alterations of membrane lipids and in gene expression of ganglioside metabolism in different brain structures in a mouse model of mucopolysaccharidosis type I (MPS I)

Mucopolysaccharidosis I (MPS I) is a congenital disorder caused by the deficiency of α-l-iduronidase (IDUA), with the accumulation of glycosaminoglycans (GAGs) in the CNS. Although GAG toxicity is not fully understood, previous works suggest a GAG-induced alteration in neuronal membrane composition. This study is aimed to evaluate the levels and distribution of gangliosides and cholesterol in different brain regions (cortex, cerebellum, hippocampus and hypothalamus) in a model using IDUA knockout (KO) mice (C57BL/6). Lipids were extracted with chloroform–methanol and then total gangliosides and cholesterol were determined, followed by ganglioside profile analyses. While no changes in cholesterol content were observed, the results showed a tissue dependent ganglioside alteration in KO mice: a total ganglioside increase in cortex and cerebellum, and a selective presence of GM3, GM2 and GD3 gangliosides in the hippocampus and hypothalamus. To elucidate this, we evaluated gene expression of ganglioside synthesis (GM3, GD3 and GM2/GD2 synthases) and degradation of (Neuraminidase1) enzymes in the cerebellum and hippocampus by RT-sq-PCR. The results obtained with KO mice showed a reduced expression of GD3 and GM2/GD2 synthases and Neuraminidase1 in cerebellum; and a decrease in GM2/GD2 synthase and Neuraminidase1 in the hippocampus. These data suggest that the observed ganglioside changes result from a combined effect of GAGs on ganglioside biosynthesis and degradation.