Development of a novel tocopheryl ester for suppression of lipid accumulation without cytotoxicity by optimization of dicarboxylic ester moiety

Tocopheryl succinate (Tsuc) is a succinic acid ester of the well-known antioxidant α-tocopherol (T). Tsuc exhibits various biological activities, including tumor growth suppression via activation of cell signaling and prevention of lipid accumulation in mouse adipocyte 3T3-L1 cells. The latter findings suggest that Tsuc may be a drug candidate for the treatment of obesity. However, Tsuc was found to induce apoptosis of normal cells (in addition to cancer cells), demonstrating the need to reduce the cytotoxicity of Tsuc without losing the suppression effect on lipid accumulation. Based on our previous findings, we focused on the ester structure of Tsuc for controlling cytotoxicity. Herein, we examined the cytotoxicity and lipid accumulation suppression effect of various T ester derivatives. We found that the terminal carboxylic group is necessary for suppression of lipid accumulation. We synthesized tocopheryl glutarate (Tglu) and tocopheryl adipate (Tadi) by elongation of carbon atoms 1 and 2 of the dicarboxylic moiety, respectively. Tglu and Tadi did not show any cytotoxicity, and both esters suppressed lipid accumulation, although their suppression activities were weaker than that of Tsuc. Tadi showed a more potent lipid accumulation inhibitory effect than Tglu. Although Tadi inhibited lipogenesis and promoted lipolysis, lipolysis was induced at lower concentrations than inhibition of lipogenesis, suggesting that Tadi mainly affects lipolysis. Taken together, we succeeded in the reduction of cytotoxicity, without loss of the suppression effect on lipid accumulation, by elongation of the dicarboxylic moiety of Tsuc. Tadi may be a promising candidate as an anti-obesity drug.     

cGMP-dependent protein kinase phosphorylates and inactivates RhoA

Small GTPase Rho and cGMP/cGMP-dependent protein kinase (cGK) pathways exert opposing effects in specific systems such as vascular contraction and growth. However, the direct interaction between these pathways has remained elusive. We demonstrate that cGK phosphorylates RhoA in vitro at Ser188, the same residue phosphorylated by cAMP-dependent protein kinase. In HeLa cells transfected with constitutively active cGK (C-cGK), stress fiber formation induced by lysophosphatidic acid or V14RhoA was blocked. By contrast, C-cGK failed to inhibit stress fiber formation in cells transfected with mutant RhoA with substitution of Ser188 to Ala. C-cGK did not affect actin reorganization induced by Rac1 or Rho-associated kinase, one of the effectors for RhoA. Furthermore, C-cGK expression inhibited the membrane translocation of RhoA. Collectively, our findings suggest that cGK phosphorylates RhoA at Ser188 and inactivates RhoA signaling. The physiological relevance of the direct interaction between RhoA and cGK awaits further investigation.     

Brain natriuretic peptide appears to act locally as an antifibrotic factor in the heart

In addition to cardiac myocyte hypertrophy, proliferation and increased extracellular matrix production of cardiac fibroblasts occur in response to cardiac overload. This remodeling of the cardiac interstitium is a major determinant of pathologic hypertrophy leading to ventricular dysfunction and heart failure. Atrial and brain natriuretic peptides (ANP and BNP) are cardiac hormones produced primarily by the atrium and ventricle, respectively. Plasma ANP and BNP concentrations are elevated in patients with hypertension, cardiac hypertrophy, and acute myocardial infarction, suggesting their pathophysiologic roles in these disorders. ANP and BNP exhibit diuretic, natriuretic, and vasodilatory activities via a guanylyl cyclase-coupled natriuretic peptide receptor subtype (guanylyl cyclase-A or GC-A). Here we report the generation of mice with targeted disruption of BNP (BNP-/- mice). We observed focal fibrotic lesions in ventricles from BNP-/- mice with a remarkable increase in ventricular mRNA expression of ANP, angiotensin converting enzyme (ACE), transforming growth factor (TGF)-beta3, and pro-alpha1(I) collagen [Col alpha1(I)], which are implicated in the generation and progression of ventricular fibrosis. Electron microscopic examination revealed supercontraction of sarcomeres and disorganized myofibrils in some ventricular myocytes from BNP-/- mice. No signs of cardiac hypertrophy and systemic hypertension were noted in BNP-/- mice. In response to acute cardiac pressure overload induced by aortic constriction, massive fibrotic lesions were found in all the BNP-/- mice examined, accompanied by further increase of mRNA expression of TGF-beta3 and Col alpha1(I). We postulate that BNP acts as a cardiocyte-derived antifibrotic factor in the ventricle.     

Effect of divalent cations on antiparallel G-quartet structure of d(G4T4G4)

The thermodynamic parameters of an antiparallel G-quartet formation of d(G4T4G4) with 1 mM divalent cation (Mg(2+), Ca(2+), Mn(2+), Co(2+), and Zn(2+)) were obtained. The thermodynamic parameters showed that the divalent cation destabilizes the antiparallel G-quartet of d(G4T4G4) in the following order: Zn(2+)>Co(2+)>Mn(2+)>Mg(2+)>Ca(2+). In addition, a higher concentration of a divalent cation induced a transition from an antiparallel to a parallel G-quartet structure. These results indicate that these divalent cations are a good tool for regulating the G-quartet structures.     

Immature multipotent hemopoietic progenitors lacking long-term bone marrow-reconstituting activity in the aorta-gonad-mesonephros region of murine day 10 fetuses

Previous studies indicated that multipotent progenitors exist in early fetuses that do not contain long-term reconstituting (LTR) activity. However, it remained unclear whether these multipotent progenitors are committed to the hemopoietic lineage or are immature mesodermal cells or hemangioblasts. In this study, we have succeeded in enriching the multipotent progenitors that are capable of generating myeloid, T, and B cells in the LFA-1(-) subpopulation of TER-119(-)c-kit(+)CD45(+) cells from the aorta-gonad-mesonephros (AGM) region of day 10 fetuses. We found that these day 10 AGM LFA-1(-) cells do not show the LTR activity, whereas day 11 AGM LFA-1(-) cells do have such an activity. These results strongly suggest that multipotent progenitors lacking LTR activity emerge as CD45(+) hemopoietic progenitor cells in the AGM region on the 10th day of gestation, and such p-Multi mature into hemopoietic stem cells by acquiring LTR activity.     

Structural and biochemical analyses of hemimethylated DNA binding by the SeqA protein

The Escherichia coli SeqA protein recognizes the 11 hemimethylated G-^mA-T-C sites in the oriC region of the chromosome, and prevents replication over-initiation within one cell cycle. The crystal structure of the SeqA C-terminal domain with hemimethylated DNA revealed the N⁶-methyladenine recognition mechanism; however, the mechanism of discrimination between the hemimethylated and fully methylated states has remained elusive. In the present study, we performed mutational analyses of hemimethylated G-^mA-T-C sequences with the minimal DNA-binding domain of SeqA (SeqA_71–181), and found that SeqA_71–181 specifically binds to hemimethylated DNA containing a sequence with a mismatched ^mA:G base pair [G-^mA(:G)-T-C] as efficiently as the normal hemimethylated G-^mA(:T)-T-C sequence. We determined the crystal structures of SeqA_71–181 complexed with the mismatched and normal hemimethylated DNAs at 2.5 and 3.0 Å resolutions, respectively, and found that the mismatched ^mA:G base pair and the normal ^mA:T base pair are recognized by SeqA in a similar manner. Furthermore, in both crystal structures, an electron density is present near the unmethylated adenine, which is only methylated in the fully methylated state. This electron density, which may be due to a water molecule or a metal ion, can exist in the hemimethylated state, but not in the fully methylated state, because of steric clash with the additional methyl group.     

Phosphorylation of Rab proteins from the brain of Bombyx mori

Rab proteins play fundamental roles in the regulation of membrane traffic. Previously, from the brain of Bombyx mori we isolated two cDNA clones (BRab1 and BRab14), each of which encoded a different member of Rab-protein family and was expressed in Escherichia coli and purified using an affinity chromatography. In this study, one cDNA clone (BRab8) was isolated from a cDNA library from the brain of B. mori. The recombinant protein was expressed in E. coli and purified. Next, the phosphorylations of these three purified BRab proteins were examined, using mammalian protein kinases in vitro. Protein kinase C (PKC) phosphorylated BRab8 and BRab14 proteins. Protein kinase A faintly phosphorylated BRab8 and BRab14 proteins. Calcium/calmodulin-dependent protein kinase faintly phosphorylated BRab8 protein. Next, brains of B. mori were dissected and homogenized. The homogenate showed a calcium-dependent protein kinase activity of BRab8 and BRab14 proteins. So PKC from the brain of B. mori was partially purified by a sequence of chromatographies on DEAE-Cellulofine and affinity chromatography. This PKC phosphorylated BRab8 and BRab14 proteins. These results suggest that the function of Rab proteins in the brain of B. mori is regulated by calcium-dependent protein kinases.