Lack of evidence for a pyruvate kinase isozyme shift in hepatocytes of the regenerating rat liver

1. Pyruvate kinase isozyme shift in a regenerating rat liver was studied. Rats were subjected to a 70% hepatectomy and the liver homogenate or hepatocyte preparations were obtained from the regenerating liver. 2. Using thin layer polyacrylamide gel electrophoresis, liver homogenates from an intact normal rat appeared to contain the L-type isozyme in the greatest number and M2-type to a lesser extent. 3. The ratio of the M2- to L-type increased in the preparations obtained from the regenerating liver. 4. In the hepatocyte preparations prepared from a regenerating rat liver by the conventional method, a small amount of M2-type isozyme was detected. 5. However, the M2-type isozyme was hardly detected in the highly purified hepatocyte preparations prepared using Percoll. 6. Similar results were obtained by separation of the enzyme by DEAE cellulose column chromatography. 7. These results suggest that there is no pyruvate kinase isozyme shift from L- to M2-type in hepatocytes in the course of regeneration. 8. The increased M2-type isozyme in the regenerating rat liver is considered to originate from nonparenchymal cells.     

Oxygen concentration dependence of lipid peroxidation and lipid-derived radical generation: application of profluorescent nitroxide switch

Lipid-derived radicals and peroxides are involved in the pathogenesis of oxidative stress diseases and, although lipid peroxide production is a required reaction between a lipid radical and molecular oxygen, a useful lipid radical detection method has remained tentative. Also, the effect of oxygen concentration on lipid peroxide production must be considered because of the hypoxic conditions in cancer and ischemic regions. In this study, the focus was on nitroxide reactivity, which allows spin trapping with carbon-centred radicals via radical-radical reactions and fluorophore quenching through interactions with nitroxide's unpaired electron. Thus, the aim here was to demonstrate a useful detection method for lipid-derived radicals as well as to clarify the effects of oxygen concentration on lipid peroxide production using profluorescent nitroxide. This latter compound reacted with lipid-derived radicals in a manner inversely dependent on oxygen concentration, resulting in fluorescence due to alkoxyamine formation and, conversely, lipid peroxide concentrations decreased with lower oxygen in the reaction system. Furthermore, nitroxide inhibited lipid peroxide production and stopped oxygen consumption in the same solution. These results suggested that the novel application of profluorescent nitroxide could directly and sensitively detect lipid-derived radicals and that radical and peroxide production were dependent on oxygen concentration.     

Suppression of discoidin domain receptor 1 by RNA interference attenuates lung inflammation

Discoidin domain receptor 1 (DDR1) is a receptor tyrosine kinase whose ligand is collagen. Recently, we have reported the association of DDR1 in the cytokine production of human leukocytes in in vitro and in vivo expression in idiopathic pulmonary fibrosis. However, its role in in vivo inflammation has not been fully elucidated. Small interference RNA (siRNA) can induce specific suppression of in vitro and in vivo gene expression. In this study, using a bleomycin-induced pulmonary fibrosis mouse model, we administered siRNA against DDR1 transnasally and evaluated histological changes, cytokine expression, and signaling molecule activation in the lungs. Histologically, siRNA against DDR1 successfully reduced in vivo DDR1 expression and attenuated bleomycin-induced infiltration of inflammatory cells. Furthermore, it significantly reduced inflammatory cell counts and concentrations of cytokines such as MCP-1, MIP-1alpha, and MIP-2 in bronchoalveolar lavage fluid. Subsequently, bleomycin-induced up-regulation of TGF-beta in bronchoalveolar lavage fluid was significantly inhibited, and collagen deposition in the lungs was reduced. Furthermore, siRNA against DDR1 significantly inhibited bleomycin-induced P38 MAPK activation in the lungs. Considered together, we propose that DDR1 contributes to the development of bleomycin-induced pulmonary inflammation and fibrosis.     

Clithioneine,an amino acid betaine from clitocybe acromelalga

A new amino acid betaine, clithioneine, was isolated from a poisonous mushroom Clitocybe acromelalga, and its structure was established on the basis of spectral analyses and chemical degradation studies.     

The induction of accelerated murine amyloid with human splenic extract. Probable role of amyloid enhancing factor

Amyloid enhancing factor (AEF) is derived from the tissues of pre-amyloidotic and amyloidotic animals and, when transferred, greatly accelerates amyloid induction in the recipient murine models. It has also been reported that similarly accelerated amyloid induction can be achieved in mice by injection of human splenic homogenates from patients with amyloidosis. The present study has attempted to characterize further the mechanism of this "heterologous transfer of amyloid". Treatment of mice with the "tissue homogenate" or the "AEF extract" of AA-, AL- and A prealbumin-laden human spleens followed by daily subcutaneous casein injections induced amyloidosis in an accelerated fashion. The resultant amyloid deposits in mice had strongly positive immunohistochemical reactions with anti-mouse AA, and negative reaction with anti-human AA or anti-human prealbumin. The results lend support to the idea that accelerated amyloid induction in the recipient mice is unlikely to be due to transfer of human amyloid substance, but rather to formation of "native" murine amyloid under the influence of a human AEF factor similar to or identical with AEF described in mouse-to mouse transfer models.