Blocking of acidosis-mediated apoptosis by a reduction of lactate dehydrogenase activity through antisense mRNA expression.

Lactic acid produced from the cells is a potential cause of extra- and intracellular acidification. Due to scarce technical tools, lactic acid that leads to acidification could not be reduced and direct evidence of the relationship between metabolic lactate and apoptosis has not yet been elucidated. In this study, we designed a cellular pH regulation system in CHO cells by a reduction of lactate dehydrogenase (LDH) activity through LDH antisense mRNA expression. This inhibited lactate production and, therefore, acidification of the cytosol. Under HCO3(-)-buffered growth conditions, both the parent CHO cells and the engineered CHO cells maintained their extracellular pH and intracellular pH fairly well. However, upon acidification of the cytosol, only the parent CHO cells underwent apoptosis under HCO3(-)-free conditions. In fact, we observed a number of apoptosis-related events only in control cells, including mitochondrial dysfunction, cytochrome c release, and an increase in caspase-3 enzymatic activity.     

Effectiveness of Losartan-Loaded Hyaluronic Acid (HA) Micelles for the Reduction of Advanced Hepatic Fibrosis in C3H/HeN Mice Model

Advanced hepatic fibrosis therapy using drug-delivering nanoparticles is a relatively unexplored area. Angiotensin type 1 (AT1) receptor blockers such as losartan can be delivered to hepatic stellate cells (HSC), blocking their activation and thereby reducing fibrosis progression in the liver. In our study, we analyzed the possibility of utilizing drug-loaded vehicles such as hyaluronic acid (HA) micelles carrying losartan to attenuate HSC activation. Losartan, which exhibits inherent lipophilicity, was loaded into the hydrophobic core of HA micelles with a 19.5% drug loading efficiency. An advanced liver fibrosis model was developed using C3H/HeN mice subjected to 20 weeks of prolonged TAA/ethanol weight-adapted treatment. The cytocompatibility and cell uptake profile of losartan-HA micelles were studied in murine fibroblast cells (NIH3T3), human hepatic stellate cells (hHSC) and FL83B cells (hepatocyte cell line). The ability of these nanoparticles to attenuate HSC activation was studied in activated HSC cells based on alpha smooth muscle actin (α-sma) expression. Mice treated with oral losartan or losartan-HA micelles were analyzed for serum enzyme levels (ALT/AST, CK and LDH) and collagen deposition (hydroxyproline levels) in the liver. The accumulation of HA micelles was observed in fibrotic livers, which suggests increased delivery of losartan compared to normal livers and specific uptake by HSC. Active reduction of α-sma was observed in hHSC and the liver sections of losartan-HA micelle-treated mice. The serum enzyme levels and collagen deposition of losartan-HA micelle-treated mice was reduced significantly compared to the oral losartan group. Losartan-HA micelles demonstrated significant attenuation of hepatic fibrosis via an HSC-targeting mechanism in our in vitro and in vivo studies. These nanoparticles can be considered as an alternative therapy for liver fibrosis.     

Modulation of Intracellular Calcium Levels by Calcium Lactate Affects Colon Cancer Cell Motility through Calcium-Dependent Calpain

Cancer cell motility is a key phenomenon regulating invasion and metastasis. Focal adhesion kinase (FAK) plays a major role in cellular adhesion and metastasis of various cancers. The relationship between dietary supplementation of calcium and colon cancer has been extensively investigated. However, the effect of calcium (Ca²⁺) supplementation on calpain-FAK-motility is not clearly understood. We sought to identify the mechanism of FAK cleavage through Ca²⁺ bound lactate (CaLa), its downstream signaling and role in the motility of human colon cancer cells. We found that treating HCT116 and HT-29 cells with CaLa immediately increased the intracellular Ca²⁺ (iCa²⁺) levels for a prolonged period of time. Ca²⁺ influx induced cleavage of FAK into an N-terminal FAK (FERM domain) in a dose-dependent manner. Phosphorylated FAK (p-FAK) was also cleaved in to its p-N-terminal FAK. CaLa increased colon cancer cells motility. Calpeptin, a calpain inhibitor, reversed the effects of CaLa on FAK and pFAK cleavage in both cancer cell lines. The cleaved FAK translocates into the nucleus and modulates p53 stability through MDM2-associated ubiquitination. CaLa-induced Ca²⁺ influx increased the motility of colon cancer cells was mediated by calpain activity through FAK and pFAK protein destabilization. In conclusion, these results suggest that careful consideration may be given in deciding dietary Ca²⁺ supplementation to patient undergoing treatment for metastatic cancer.     

Lipase-catalyzed synthesis of lysophosphatidic acid in a solvent free system

Summary The direct, lipase-catalyzed esterifications of glycerol-3-phosphate in an organic solvent system and in a solvent free system were carried out. In a solvent free system only, LPA synthesis could be achieved within the acceptable reaction time. Open reaction system was preferable to closed reaction system for LPA synthesis. Yield of LPA isolated by silica gel column chromatography was 32.3%.     

Leucocytosis in Biomphalaria glabrata sensitized and resensitized to Echinostoma lindoense

Leucocytosis was shown to occur in the pulmonate gastropod Biomphalaria glabrata exposed to the trematode Echinostoma lindoense. In these sensitized snails, the leukocyte count in the hemolymph was elevated 3 to 5 days postexposure to miracidia, and prior to complete encapsulation of sporocysts. This increase continued 1 to 5 days after destruction of sensitizing, irradiated E. lindoense sporocysts. Counts returned to normal levels after this period. A significant and more rapid increase in numbers of circulating leukocytes occurred 1 to 6 hr after reexposure of snails to a sensitizing dose of nonirradiated E. lindoense sporocysts. The leukocyte counts usually returned to normal levels after this period, except in snails in which some resensitizing sporocysts remained alive.