Heart slice NMR

Nuclear magnetic resonance (NMR) spectroscopy of the heart is normally carried out using whole heart preparations under coronary perfusion. In such preparations, either radical changes in ionic composition of the perfusate or applications of numerous drugs would affect coronary microcirculation. This report communicates the first (31)P NMR spectroscopy study using a heart slice preparation (left ventricular slices) superfused with extracellular medium. The ratio of phosphocreatine concentration to ATP concentration was approximately 2.1. Also, intracellular pH and Mg(2+) concentration ([Mg(2+)](i)), estimated from the chemical shifts of inorganic phosphate and ATP, were comparable with those under retrograde perfusion. [Mg(2+)](i) was significantly increased by the removal of extracellular Na(+), supporting the essential role of Na(+)-coupled Mg(2+) transport in Mg(2+) homeostasis of the heart. Heart slice preparation could also be used to evaluate the potency of cardiac drugs, regardless of their possible effects on coronary microcirculation.     

Edaravone mimics sphingosine-1-phosphate-induced endothelial barrier enhancement in human microvascular endothelial cells

Edaravone is a potent scavenger of hydroxyl radicals and is quite successful in patients with acute cerebral ischemia, and several organ-protective effects have been reported. Treatment of human microvascular endothelial cells with edaravone (1.5 microM) resulted in the enhancement of transmonolayer electrical resistance coincident with cortical actin enhancement and redistribution of focal adhesion proteins and adherens junction proteins to the cell periphery. Edaravone also induced small GTPase Rac activation and focal adhesion kinase (FAK; Tyr(576)) phosphorylation associated with sphingosine-1-phosphate receptor type 1 (S1P(1)) transactivation. S1P(1) protein depletion by the short interfering RNA technique completely abolished edaravone-induced FAK (Tyr(576)) phosphorylation and Rac activation. This is the first report of edaravone-induced endothelial barrier enhancement coincident with focal adhesion remodeling and cytoskeletal rearrangement associated with Rac activation via S1P(1) transactivation. Considering the well-established endothelial barrier-protective effect of S1P, endothelial barrier enhancement as a consequence of S1P(1) transactivation may at least partly be the potent mechanisms for the organ-protective effect of edaravone and is suggestive of edaravone as a therapeutic agent against systemic vascular barrier disorder.     

Functional domain analysis of human HP1 isoforms in Drosophila

Three subtypes of HP1, a conserved non-histone chromosomal protein enriched in heterochromatin, have been identified in humans, HP1alpha, beta and gamma. In the present study, we utilized a Drosophila system to characterize human HP1 functions. Over-expression of HP1beta in eye imaginal discs caused abnormally patterned eyes, with reduced numbers of ommatidia, and over-expression of HP1gamma in wing imaginal discs caused abnormal wings, in which L4 veins were gapped. These phenotypes were specific to the HP1 subtypes and appear to reflect suppressed gene expression. To determine the molecular domains of HP1 required for each specific phenotype, we constructed a series of chimeric molecules with HP1beta and HP1gamma. Our data show that the C-terminal chromo shadow domain (CSD) of HP1gamma is necessary for HP1gamma-type phenotype, whereas for the HP1beta-type phenotype both the chromo domain and the CSD are required. These results suggest human HP1 subtypes use different domains to suppress gene expression in Drosophila cells.     

Gene expression and distribution of Swi6 in partial aneuploids of the fission yeast Schizosaccharomyces pombe

Imbalances of gene expression in aneuploids, which contain an abnormal number of chromosomes, cause a variety of growth and developmental defects. Aneuploid cells of the fission yeast Schizosaccharomyces pombe are inviable, or very unstable, during mitotic growth. However, S. pombe haploid cells bearing minichromosomes derived from the chromosome 3 can grow stably as a partial aneuploid. To address biological consequences of aneuploidy, we examined the gene expression profiles of partial aneuploid strains using DNA microarray analysis. The expression of genes in disomic or trisomic cells was found to increase approximately in proportion to their copy number. We also found that some genes in the monosomic regions of partial aneuploid strains increased their expression level despite there being no change in copy number. This change in gene expression can be attributed to increased expression of the genes in the disomic or trisomic regions. However, even in an aneuploid strain that bears a minichromosome containing no protein coding genes, genes located within about 50 kb of the telomere showed similar increases in expression, indicating that these changes are not a secondary effect of the increased gene dosage. Examining the distribution of the heterochromoatin protein Swi6 using DNA microarray analysis, we found that binding of Swi6 within ~50 kb from the telomere occurred less in partial aneuploid strains compared to euploid strains. These results suggest that additional chromosomes in aneuploids could lead to imbalances in gene expression through changes in distribution of heterochromatin as well as in gene dosage.     

Interactions between plexin-A2, plexin-A4, and semaphorin 6A control lamina-restricted projection of hippocampal mossy fibers

Hippocampal mossy fibers project preferentially to the stratum lucidum, the proximal-most lamina of the suprapyramidal region of CA3. The molecular mechanisms that govern this lamina-restricted projection are still unknown. We examined the projection pattern of mossy fibers in mutant mice for semaphorin receptors plexin-A2 and plexin-A4, and their ligand, the transmembrane semaphorin Sema6A. We found that plexin-A2 deficiency causes a shift of mossy fibers from the suprapyramidal region to the infra- and intrapyramidal regions, while plexin-A4 deficiency induces inappropriate spreading of mossy fibers within CA3. We also report that the plexin-A2 loss-of-function phenotype is genetically suppressed by Sema6A loss of function. Based on these results, we propose a model for the lamina-restricted projection of mossy fibers: the expression of plexin-A4 on mossy fibers prevents them from entering the Sema6A-expressing suprapyramidal region of CA3 and restricts them to the proximal-most part, where Sema6A repulsive activity is attenuated by plexin-A2.     

Effects of Japanese Torreya (Torreya nucifera) seed oil on the activities and mRNA expression of lipid metabolism-related enzymes in rats

The mechanism for the plasma and liver triacylglycerol-reducing effects of Japanese torreya (Torreya nucifera) seed oil containing sciadonic acid (all-cis-5, 11, 14-eicosatrienoic acid) is reported. Male SD rats were fed experimental diets containing 10% torreya, corn, or soybean oil for 4 weeks, and the activities and mRNA expression of the enzymes involved in lipid metabolism were measured in the liver. The activities of some hepatic enzymes involved in fatty acid synthesis were lower in the rats fed torreya oil.     

Dietary pulverized konjac glucomannan prevents the development of allergic rhinitis-like symptoms and IgE response in mice

Konjac is a traditional Japanese food with a peculiar texture, and it has been suggested that its main ingredient, konjac glucomannan (KGM), ameliorates metabolic disorders such as diabetes and hypercholesteremia. We have found that feeding with pulverized KGM (PKGM) prevents skin inflammation in a murine model of atopic dermatitis. Here, we show that dietary PKGM suppresses allergic rhinitis-like symptoms in mice upon immunization and nasal sensitization with ovalbumin (OVA). The PKGM-fed mice showed a much lower frequency of sneezing than in control animals. We also found that PKGM supplementation exclusively suppressed OVA-specific IgE response without affecting IgG1/IgG2a responses as well as systemic Th1/Th2 cytokine production. These results suggest that PKGM can be a beneficial foodstuff in preventing nasal allergy such as seasonal pollinosis.     

Orally active 4-amino-5-diarylurea-furo[2,3-d]pyrimidine derivatives as anti-angiogenic agent inhibiting VEGFR2 and Tie-2

During our effort to develop dual VEGFR2 and Tie-2 inhibitors as anti-angiogenic agents for cancer therapy, we discovered 4-amino-5-(4-((2-fluoro-5-(trifluoromethyl)phenyl)- aminocarbonylamino)phenyl)furo[2,3-d]pyrimidine (8a) possessing strong inhibitory activity at both the enzyme and cellular level against VEGFR2 and Tie-2. Compound 8a demonstrated high pharmacokinetic exposure through oral administration, and showed marked tumor growth inhibition and anti-angiogenic activity in mouse HT-29 xenograft model via once-daily oral administration.     

Liver X receptor ligands inhibit the lipopolysaccharide-induced expression of microsomal prostaglandin E synthase-1 and diminish prostaglandin E2 production in murine peritoneal macrophages

Microsomal prostaglandin E synthase (mPGES)-1, which is dramatically induced in macrophages by inflammatory stimuli such as lipopolysaccharide (LPS), catalyzes the conversion of cyclooxygenase-2 (COX-2) reaction product prostaglandin H(2) (PGH(2)) into prostaglandin E(2) (PGE(2)). The mPGES-1-derived PGE(2) is thought to help regulate inflammatory responses. On the other hand, excess PGE(2) derived from mPGES-1 contributes to the development of inflammatory diseases such as arthritis and inflammatory pain. Here, we examined the effects of liver X receptor (LXR) ligands on LPS-induced mPGES-1 expression in murine peritoneal macrophages. The LXR ligands 22(R)-hydroxycholesterol (22R-HC) and T0901317 reduced LPS-induced expression of mPGES-1 mRNA and mPGES-1 protein as well as that of COX-2 protein. However, LXR ligands did not influence the expression of microsomal PGES-2 (mPGES-2) or cytosolic PGES (cPGES) protein. Consequently, LXR ligands suppressed the production of PGE(2) in macrophages. These results suggest that LXR ligands diminish PGE(2) production by inhibiting the LPS-induced gene expression of the COX-2-mPGES-1 axis in LPS-activated macrophages.     

Regulation of mitochondrial fusion and division

In many organisms, ranging from yeast to humans, mitochondria fuse and divide to change their morphology in response to a multitude of signals. During the past decade, work using yeast and mammalian cells has identified much of the machinery required for fusion and division, including the dynamin-related GTPases--mitofusins (Fzo1p in yeast) and OPA1 (Mgm1p in yeast) for fusion and Drp1 (Dnm1p) for division. However, the mechanisms by which cells regulate these dynamic processes have remained largely unknown. Recent studies have uncovered regulatory mechanisms that control the activity, assembly, distribution and stability of the key components for mitochondrial fusion and division. In this review, we discuss how mitochondrial dynamics are controlled and how these events are coordinated with cell growth, mitosis, apoptosis and human diseases.