Insulin is an hepatic mitogen that promotes liver regeneration. Actions of insulin are mediated by the insulin receptor, which is a receptor tyrosine kinase. It is currently thought that signaling via the insulin receptor occurs at the plasma membrane, where it binds to insulin. Here we report that insulin induces calcium oscillations in isolated rat hepatocytes, and that these calcium signals depend upon activation of phospholipase C and the inositol 1,4,5-trisphosphate receptor, but not upon extracellular calcium. Furthermore, insulin-induced calcium signals occur in the nucleus, and are temporally associated with selective depletion of nuclear phosphatidylinositol bisphosphate and translocation of the insulin receptor to the nucleus. These findings suggest that the insulin receptor translocates to the nucleus to initiate nuclear, inositol 1,4,5-trisphosphate-mediated calcium signals in rat hepatocytes. This novel signaling mechanism may be responsible for insulin's effects on liver growth and regeneration. ( I nsulin regulates a wide variety of biological functions in the liver, including glucose uptake, 1 regulation of gene expression, 2 and promotion of cell growth. [3][4][5] The biological actions of insulin are initiated by binding to the insulin receptor, a heterotetrameric receptor tyrosine kinase (RTK) composed of two extracellular ␣-subunits and two transmembrane ␤-subunits. 6 The ␣-subunit possesses insulin-binding activity whereas the ␤-subunit has intrinsic protein tyrosine kinase activity. Binding of insulin to the ␣-subunit of its receptor activates the protein tyrosine kinase and results in phosphorylation of tyrosine residues of the ␤-subunit and of several endogenous substrates. These substrates include proteins containing a src-homology 2 domain such as phosphatidylinositol 3-kinase and phospholipase C (PLC). 7 PLC hydrolyzes phosphatidylinositol 4,5-bisphosphate (PIP 2 ), generating two intracellular products: inositol 1,4,5-trisphosphate (InsP3), a universal calcium-mobilizing second messenger, and diacylglycerol, an activator of protein kinase C. Like insulin, Ca 2ϩ also regulates glucose metabolism, 8 gene expression, 9,10 and cell growth. 11,12 Although it has not been established how a single second messenger coordinates such diverse effects within a cell, there is increasing evidence that the spatial and temporal patterns of Ca 2ϩ signals may determine their specificity. Ca 2ϩ signaling patterns can vary in different regions of the cell, and increases in Ca 2ϩ in the nucleus have specific biological effects that differ from the effects of increases in cytosolic Ca 2ϩ . 9,10,[13][14][15] The mechanisms and pathways that promote localized increases in free Ca 2ϩ levels in the nucleus have not been entirely defined. It is currently thought that signaling via the insulin receptor occurs only at the plasma membrane, where it binds to insulin. 16 Here we investigate whether and how insulin signaling occurs in the nucleus of hepatocytes, where its downstream messenger Ca 2ϩ may act. Materials...

Development of RNA interference (RNAi) technology utilizing short interfering RNA sequences (siRNA) has focused on creating methods for delivering siRNAs to cells and for enhancing siRNA stability in vitro and in vivo. Here, we describe a novel approach for siRNA cellular delivery using siRNA coiling into carboxyl-functionalized single-wall carbon nanotubes (SWCNTs). The CNT-siRNA delivery system successfully demonstrates nonspecific toxicity and transfection efficiency greater than 95%. This approach offers the potential for siRNA delivery into different types of cells, including hard-to-transfect cells, such as neuronal cells and cardiomyocytes. We also tested the CNT-siRNA system in a non-metastatic human hepatocellular carcinoma cell line (SKHep1). In all types of cells used in this work the CNT-siRNA delivery system showed high efficiency and apparent no side effects for various in vitro applications.

Subcellular Ca2+ signals control a variety of responses in the liver. For example, mitochondrial Ca2+ regulates apoptosis while Ca2+ in the nucleus regulates cell proliferation. Since apoptosis and cell growth can be related, we investigated whether mitochondrial Ca2+ also affects liver regeneration. The Ca2+ buffering protein, Parvalbumin (PV), targeted to the mitochondrial matrix and fused to green fluorescent protein (PV-MITO-GFP) was expressed in the SKHep1 liver cell line. This construct properly localized to and effectively buffered Ca2+ signals in the mitochondrial matrix. Additionally, expression of PV-MITO-GFP reduced apoptosis induced by both the intrinsic and extrinsic pathways. The reduction in cell death correlated with increased expression of anti-apoptotic genes bcl-2, mcl-1, and bcl-xL, and decreased expression of the pro-apoptotic genes p53, bax, apaf-1 and caspase-6. PV-MITO-GFP was also expressed in hepatocytes in vivo using an adenoviral delivery system. Buffering mitochondrial Ca2+ in hepatocytes accelerated liver regeneration after partial hepatectomy, an effect that was associated with increased expression of Bcl-2 and decreased expression of Bax. Conclusion Together, these results reveal an essential role for mitochondrial Ca2+ in hepatocyte proliferation and liver regeneration, which may be mediated by regulating apoptosis.

Background and Aims Nucleoplasmic Ca2+ regulates cell growth in the liver, but the proteins through which this occurs are unknown. Methods We used Rapid Subtraction Hybridization (RaSH) to subtract genes in SKHep1 liver cells expressing the Ca2+ buffer protein parvalbumin (PV) targeted to the nucleus, from genes in cells expressing a mutated form of nuclear-targeted PV which has one of two Ca2+-binding site inactivated. The subtraction permitted selection of genes whose expression was affected by a small alteration in nuclear Ca2+ concentration. Results The asparaginyl endopeptidase legumain (LGMN) was identified in this screening. When Ca2+ was buffered in the nucleus of SKHep1 cells, LGMN mRNA was decreased by 97%, in part by a transcriptional mechanism, and decreased expression at the protein level was observed by immunoblot and immunofluorescence. Treatment with Hepatocyte Growth Factor increased LGMN expression. Knockdown of LGMN by siRNA decreased proliferation of SKHep1 cells by ~50% as measured both by BrdU uptake and mitotic index, although an inhibitor of LGMN activity did not affect BrdU incorporation. A significant reduction in the fraction of cells in G2/M phase was seen as well. This was associated with increases in expression of cyclins A and E. Furthermore, LGMN expression was increased in hepatocellular carcinoma cells relative to normal hepatocytes in the same specimens. Conclusions These findings suggest a new role for LGMN and provide evidence that nuclear Ca2+ signals regulate cell proliferation in part through modulation of LGMN expression. Increased expression of LGMN may be involved in liver carcinogenesis.

In this study, our aims were to investigate transient receptor potential melastatin-8 channels (TRPM8) involvement in rotundifolone induced relaxation in the mesenteric artery and to increase the understanding of the role of these thermosensitive TRP channels in vascular tissue. Thus, message and protein levels of TRPM8 were measured by semi-quantitative PCR and western blotting in superior mesenteric arteries from 12 week-old Spague-Dawley (SD) rats. Isometric tension recordings evaluated the relaxant response in mesenteric rings were also performed. Additionally, the intracellular Ca2+ changes in mesenteric artery myocytes were measured using confocal microscopy. Using PCR and western blotting, both TRPM8 channel mRNA and protein expression was measured in SD rat mesenteric artery. Rotundifolone and menthol induced relaxation in the isolated superior mesenteric artery from SD rats and improved the relaxant response induced by cool temperatures. Also, this monoterpene induced an increase in transient intracellular Ca2+. These responses were significantly attenuated by pretreatment with capsazepine or BCTC, both TRPM8 channels blockers. The response induced by rotundifolone was not significantly attenuated by ruthenium red, a non-selective TRP channels blocker, or following capsaicin-mediated desensitization of TRPV1. Our findings suggest that rotundifolone induces relaxation by activating TRPM8 channels in rat superior mesenteric artery, more selectively than menthol, the classic TRPM8 agonist, and TRPM8 channels participates in vasodilatory pathways in isolated rat mesenteric arteries.

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RESUMO.-[Atividade antimicrobiana, toxicidade aguda e crônica do óleo essencial de Lippia origanoides.]Atualmente nota-se um aumento do interesse pelas plantas medicinais, fruto da grande procura por terapias alternativas. Neste trabalho foi avaliada a atividade antimicrobiana e a toxicidade do óleo essencial da Lippia origanoides (alecrim-pimenta). O óleo essencial de alecrim-pimenta foi obtido por arraste com vapor d'água e seus constituintes foram identificados por cromatografia acoplada a espectrofotômetro de massa (GC/MS). Entre os 15 compostos identificados os mais abundantes foram o carvacrol (29%), o-cimeno (25,57%) e metil timol éter (11,50%). Os óleos foram submetidos a ensaios antimicrobianos para determinação da CIM e da CBM. Os resultados mostraram que a dose de 120μl/mL de qualquer um dos óleos testados foi eficiente em inibir o crescimento dos micro-organismos Escherichia coli (ATCC 25922) Currently, there is a growing interest in medicinal plants, because of an increased demand for alternate therapies. In this study, the antimicrobial activity and toxicity of the essential oil of Lippia origanoides (L. origanoides) were investigated. The essential oil of L. origanoides was extracted by steam-dragging distillation and its constituents were identified by chromatography coupled with mass spectrometry. Among the 15 compounds identified, the most abundant were carvacrol (29.00%), o-cymene (25.57%), and thymol methyl ether (11.50%). The essential oil was studied in antimicrobial assays to determine the MIC and MBC. The results indicated that a concentration of 120μL/mL of oil was sufficient to inhibit the growth of the following microorganisms: Escherichia coli (ATCC 25922), Staphylococcus aureus (ATCC 25923) and Salmonella cholerasuis (ATCC 10708). Acute and chronic toxic effects of orally administered oil were investigated in Wistar rats by using standard methods. Doses of 30, 60 and 120mg/kg of the essential oil did not induce significant changes in weight, behavior or hematological and biochemical parameters in the animals. There were no signs of any histopathological changes to the liver, kidneys or heart of the treated rats, suggesting that Lippia origanoides oil is non--toxic after oral administration in acute or chronic toxicity studies. The results obtained in this study show that the essential oil of L. origanoides has a high safety margin, with no detectable toxic effects in rats treated with doses to 120mg/kg. In addition, L. origanoides oil demonstrated potent antimicrobial activity against S. aureus, E. coli and S. cholerasuis. Based on these findings, this essential oil may have practical application as a veterinary antimicrobial.INDEX TERMS: Lippia origanoides, essential oil, antimicrobial, acute toxicity, chronic toxicity.

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