Methylene blue protection against hypoxic injury in primary cultures of rat hepatocyte monolayers

Mitochondrial respiration is inhibited in cells exposed to hypoxia, and the oxidation of NADH to NAD(+) is blocked. As a result, oxidation reactions requiring NAD(+) are blocked, disrupting cellular metabolism. We studied the influence of methylene blue, which oxidizes NADH, on hypoxic damage to primary cultures of rat hepatocyte monolayers. During hypoxic treatment of hepatocytes, aspartate aminotransferase leaked out of the cells into the culture medium. However, addition of methylene blue to the medium repressed the hypoxic leakage of the enzyme. The exposure of hepatocytes to hypoxia decreased the acetoacetate/beta-hydroxybutyrate ratio which reflects the redox state of the cell. The level of the acetoacetate/beta-hydroxybutyrate ratio in hypoxic cells was increased by the addition of methylene blue. These results suggest that methylene blue protects against hypoxic injury due to its oxidation of NADH.     

Focal neurometabolic alterations in mice deficient for succinate semialdehyde dehydrogenase

Metabolite profiling in succinate semialdehyde dehydrogenase (SSADH; Aldh5a1-/-) deficient mice previously revealed elevated gamma-hydroxybutyrate (GHB) and total GABA in urine and total brain and liver extracts. In this study, we extend our metabolic characterization of these mutant mice by documenting elevated GHB and total GABA in homogenates of mutant kidney, pancreas and heart. We quantified beta-alanine (a GABA homolog and putative neurotransmitter) to address its potential role in pathophysiology. We found normal levels of beta-alanine in urine and total homogenates of mutant brain, heart and pancreas, but elevated concentrations in mutant kidney and liver extracts. Amino acid analysis in mutant total brain homogenates revealed no abnormalities except for significantly decreased glutamine, which was normal in mutant liver and kidney extracts. Regional amino acid analysis (frontal cortex, parietal cortex, hippocampus and cerebellum) in mutant mice confirmed glutamine results. Glutamine synthetase protein and mRNA levels in homogenates of mutant mouse brain were normal. We profiled organic acid patterns in mutant brain homogenates to assess brain oxidative metabolism and found normal concentrations of Kreb's cycle intermediates but increased 4,5-dihydroxyhexanoic acid (a postulated derivative of succinic semialdehyde) levels. We conclude that SSADH-deficient mice represent a valid metabolic model of human SSADH deficiency, manifesting focal neurometabolic abnormalities which could provide key insights into pathophysiologic mechanisms.     

Non-proteolytic house dust mite allergen, Der p 2, upregulated expression of tight junction molecule claudin-2 associated with Akt/GSK-3β/β-catenin signaling pathway

Non-proteolytic group 2 allergen, Der p 2 (DP2) is known as a major allergen derived from house dust mite Dermatophagoides pteronyssinus.Paracellular epithelial barrier, being composed of a number of tight junction (TJ) molecules, plays pivotal roles in resistance of pathogen invading. However, whether DP2 affects epithelial TJ molecules is unclear. Therefore, we aimed to investigate the effects of DP2 on epithelial TJ molecules, and the mechanism by which expression of junction molecules is regulated by DP2. Cell cycle and mRNA expression of TJ proteins of lung alveolar cell A549 were analyzed by RT-PCR and flow cytometry. Level of claudin-2, subcellular distribution of b-catenin and kinase activation was determined using immunoblot. Our findings revealed that DP2 had no significant influence on cell cycle distribution but affected mRNA expression of TJ molecules including claudin-2, occludin, and ZO-1 in A549 cells. Our results showed that DP2 significantly elevated level of claudin-2 and increased expression and nuclear translocation of b-catenin. Moreover, DP2 enhanced the phosphorylation of glycogen synthase kinase-3b (GSK-3b) and its potential upstream regulator Akt. The DP2-induced claudin-2 expression was also suppressed by GSK-3b inhibitor (lithium chloride) and phosphatidyl inositol 3-phosphate kinase (PI3K) inhibitor (wortamannin). Taken together, these findings showed that DP2 increased claudin-2 expression and its cell surface distribution in A549 cells, which may attribute to phosphorylation of GSK-3b and Akt and the consequent increase and nuclear translocation of b-catenin. It is suggested that presence of DP2 may alter epithelial junction by regulating expression of TJ molecules.     

Perspectives on the metabolic management of epilepsy through dietary reduction of glucose and elevation of ketone bodies

Brain cells are metabolically flexible because they can derive energy from both glucose and ketone bodies (acetoacetate and beta-hydroxybutyrate). Metabolic control theory applies principles of bioenergetics and genome flexibility to the management of complex phenotypic traits. Epilepsy is a complex brain disorder involving excessive, synchronous, abnormal electrical firing patterns of neurons. We propose that many epilepsies with varied etiologies may ultimately involve disruptions of brain energy homeostasis and are potentially manageable through principles of metabolic control theory. This control involves moderate shifts in the availability of brain energy metabolites (glucose and ketone bodies) that alter energy metabolism through glycolysis and the tricarboxylic acid cycle, respectively. These shifts produce adjustments in gene-linked metabolic networks that manage or control the seizure disorder despite the continued presence of the inherited or acquired factors responsible for the epilepsy. This hypothesis is supported by information on the management of seizures with diets including fasting, the ketogenic diet and caloric restriction. A better understanding of the compensatory genetic and neurochemical networks of brain energy metabolism may produce novel antiepileptic therapies that are more effective and biologically friendly than those currently available.     

Asymmetric reduction of β-ketoesters and chiral β-iminoesters: impact of a α-quaternary stereocenter

Diastereomeric reduction of nonactivated, hindered β-keto and chiral β-iminoesters are described. The influence of a α-stereocontrolled center on the efficiency and stereoselectivity of the reduction was studied. Reaction conditions were optimized to synthesize β-hydroxy- and β-aminoesters in good yields. In the case of chiral β-iminoesters, influence of matched/mismatched diastereomeric pairs has been assessed.     

TGF-β regulates β-catenin signaling and osteoblast differentiation in human mesenchymal stem cells

Human adult bone marrow-derived skeletal stem cells a.k.a mesenchymal stem cells (hMSCs) have been shown to be precursors of several different cellular lineages, including osteoblast, chondrocyte, myoblast, adipocyte, and fibroblast. Several studies have shown that cooperation between transforming growth factor β (TGF-β) and Wnt/β-catenin signaling pathways plays a role in controlling certain developmental events and diseases. Our previous data showed that agents like TGF-β, cooperation with Wnt signaling, promote chondrocyte differentiation at the expense of adipocyte differentiation in hMSCs. In this study, we tested mechanisms by which TGF-β activation of β-catenin signaling pathway and whether these pathways interact during osteoblast differentiation of hMSCs. With selective small chemical kinase inhibitors, we demonstrated that TGF-β1 requires TGF-β type I receptor ALK-5, Smad3, phosphoinositide 3-kinases (PI3K), and protein kinase A (PKA) to stabilize β-catenin, and needs ALK-5, PKA, and JNK to inhibit osteoblastogenesis in hMSCs. Knockdown of β-catenin with siRNA stimulated alkaline phosphatase activity and antagonized the inhibitory effects of TGF-β1 on bone sialoprotein (BSP) expression, suggested that TGF-β1 cooperated with β-catenin signaling in inhibitory of osteoblastogenesis in hMSCs. In summary, TGF-β1 activates β-catenin signaling pathway via ALK-5, Smad3, PKA, and PI3K pathways, and modulates osteoblastogenesis via ALK5, PKA, and JNK pathways in hMSCs; the interaction between TGF-β and β-catenin signaling supports the view that β-catenin signaling is a mediator of TGF-β's effects on osteoblast differentiation of hMSCs.     

Role of endogenous TGF-β family in myogenic differentiation of C2C12 cells

The present study evaluated endogenous activities and the role of BMP and transforming growth factor-β (TGF-β), representative members of the TGF-β family, during myotube differentiation in C2C12 cells. Smad phosphorylation at the C-terminal serines was monitored, since TGF-β family members signal via the phosphorylation of Smads in a ligand-dependent manner. Expression of phosphorylated Smad1/5/8, which is an indicator of BMP activity, was higher before differentiation, and rapidly decreased after differentiation stimulation. Differentiation-related changes were consistent with those in the expression of Ids, well-known BMP-responsive genes. Treatment with inhibitors of BMP type I receptors or noggin in C2C12 myoblasts down-regulated the expression of myogenic regulatory factors, such as Myf5 and MyoD, leading to impaired myotube formation. Addition of BMP-2 during the myoblast phase also inhibited myotube differentiation through the down-regulation of Myf5 and MyoD. In contrast to endogenous BMP activity, the phosphorylation of Smad2, a TGF-β-responsive Smad, was higher 8-16 days after differentiation stimulation. A-83-01, an inhibitor of TGF-β type I receptor, increased the expression of Myf5 and MyoD, and enhanced myotube formation. The present results reveal that endogenous activities of the TGF-β family are changed during myogenesis in a pathway-specific manner, and that the activities are required for myogenesis.     

Wnt3a upregulates transforming growth factor-β-stimulated VEGF synthesis in osteoblasts

It is recognized that Wnt3a affects bone metabolism via the canonical Wnt/β-catenin signalling pathway. We have previously shown that transforming growth factor-β (TGF-β) stimulates the synthesis of vascular endothelial growth factor (VEGF) via p44/p42 mitogen-activated protein (MAP) kinase, stress-activated protein kinase (SAPK)/c-Jun N-terminal kinase (JNK) and p38 MAP kinase in osteoblast-like MC3T3-E1 cells. In the present study, we investigated the effect of Wnt3a on TGF-β-stimulated VEGF synthesis in these cells. Wnt3a, which alone had little effect on the VEGF levels, significantly enhanced the TGF-β-stimulated VEGF release. Lithium chloride and SB216763, inhibitors of glycogen synthase kinase 3β, markedly amplified the TGF-β-stimulated VEGF release. Wnt3a failed to affect the TGF-β-induced phosphorylation of Smad2, p44/p42 MAP kinase, p38 MAP kinase or SAPK/JNK. Wnt3a and lithium chloride strengthened the VEGF mRNA expression induced by TGF-β. These results strongly suggest that Wnt3a upregulates VEGF synthesis stimulated by TGF-β via activation of the canonical pathway in osteoblasts.