Effects of sesquiterpenes and amino acid-sesquiterpene conjugates from the roots of Saussurea lappa on inducible nitric oxide synthase and heat shock protein in lipopolysaccharide-activated macrophages

The methanolic extract of the roots of Saussurea lappa CLARKE, a Chinese medicinal herb Saussureae Radix, was found to inhibit nitric oxide (NO) production in lipopolysaccharide (LPS)-activated mouse peritoneal macrophages. Among the constituents from the methanolic extract, two sesquiterpene lactones (costunolide and dehydrocostus lactone) and two amino acid-sesquiterpene conjugates (saussureamines A and B) potently inhibited LPS-induced NO production (IC(50)=1.2-2.8 microM). Saussureamines A and B in addition to costunolide and dehydrocostus lactone did not inhibit iNOS enzyme activity, but they inhibited both induction of inducible NO synthase and activation of nuclear factor-kappaB in accordance with induction of heat shock protein 72.     

Reduction of methamphetamine-induced sensitization and reward in matrix metalloproteinase-2 and -9-deficient mice

Matrix metalloproteinases (MMPs) and their inhibitors (TIMPs) function to remodel the pericellular environment. Their activation and regulation are associated with synaptic physiology and pathology. Here, we investigated whether MMP-2 and MMP-9 are involved in the rewarding effects of and sensitization to methamphetamine (METH) in animals, in which the remodelling of neural circuits may play a crucial role. Repeated METH treatment induced behavioural sensitization, which was accompanied by an increase in MMP-2 and MMP-9 activity in the brain. In MMP-2- and MMP-9-deficient mice [MMP-2-(-/-) and MMP-9-(-/-)], METH-induced behavioural sensitization and conditioned place preference, a measure of the rewarding effect, as well as METH-increased dopamine release in the nucleus accumbens (NAc) were attenuated compared with those in wild-type mice. In contrast, infusion of purified human MMP-2 into the NAc significantly potentiated the METH-increased dopamine release. The [(3)H]dopamine uptake into striatal synaptosomes was reduced in wild-type mice after repeated METH treatment, but METH-induced changes in [(3)H]dopamine uptake were significantly attenuated in MMP-2-(-/-) and MMP-9-(-/-) mice. These results suggest that both MMP-2 and MMP-9 play a crucial role in METH-induced behavioural sensitization and reward by regulating METH-induced dopamine release and uptake in the NAc.     

Role of matrix metalloproteinase and tissue inhibitor of MMP in methamphetamine-induced behavioral sensitization and reward: implications for dopamine receptor down-regulation and dopamine release

Matrix metalloproteinases (MMPs) and its inhibitors (TIMPs) function to remodel the pericellular environment. We have demonstrated that methamphetamine (METH)-induced behavioral sensitization and reward were markedly attenuated in MMP-2- and MMP-9 deficient [MMP-2-(-/-) and MMP-9-(-/-)] mice compared with those in wild-type mice, suggesting that METH-induced expression of MMP-2 and MMP-9 in the brain plays a role in the development of METH-induced sensitization and reward. In the present study, we investigated the changes in TIMP-2 expression in the brain after repeated METH treatment. Furthermore, we studied a role of MMP/TIMP system in METH-induced behavioral changes and dopamine neurotransmission. Repeated METH treatment induced behavioral sensitization, which was accompanied by an increase in TIMP-2 expression. Antisense TIMP-2 oligonucleotide (TIMP-AS) treatment enhanced the sensitization, which was associated with the potentiation of METH-induced dopamine release in the nucleus accumbens (NAc). On the other hand, MMP-2/-9 inhibitors blocked the METH-induced behavioral sensitization and conditioned place preference, a measure of the rewarding effect, and reduced the METH-increased dopamine release in the NAc. Dopamine receptor agonist-stimulated [(35)S]GTPgammaS binding was reduced in the frontal cortex of sensitized rats. TIMP-AS treatment potentiated, while MMP-2/-9 inhibitor attenuated, the reduction of dopamine D2 receptor agonist-stimulated [(35)S]GTPgammaS binding. Repeated METH treatment also reduced dopamine D2 receptor agonist-stimulated [(35)S]GTPgammaS binding in wild-type mice, but such changes were significantly attenuated in MMP-2-(-/-) and MMP-9-(-/-) mice. These results suggest that the MMP/TIMP system is involved in METH-induced behavioral sensitization and reward, by regulating dopamine release and receptor signaling.     

An integrated map of p53-binding sites and histone modification in the human ENCODE regions

TP53 (tumor protein p53; p53) regulates its target genes under various cellular stresses. By combining chromatin immunoprecipitation with oligonucleotide microarrays, we have mapped binding sites of p53 (p53-BS) in the genome of HCT116 human colon carcinoma cells, along with those of acetylated H3, acetylated H4, and methylated H3-K4. We analyzed a 30-Mb portion of the human genome selected as a representative model by the ENCODE Consortium. In the region, we found 37 p53-BS, of which the p53-binding motif was present in 32 (86%). Acetylated histone H3 and H4 were detected at 14 (38%) and 33 (89%) of the p53-BS, respectively. A significant portion (58%) of H4 acetylation in the p53-BS was not accompanied by H3 acetylation. Acetyl H3 were preferentially located at the 5' and 3' ends of genes, whereas acetyl H4 were distributed widely across the genome. These results provide novel insights into how p53 binding coordinates with histone modification in human.     

Role of cysteine residues in 4-oxalomesaconate hydratase from Pseudomonas ochraceae NGJ1

4-Oxalomesaconate hydratase from Pseudomonas ochraceae NGJ1 is unstable in the absence of reducing reagents such as dithiothreitol, and strongly inhibited by 5,5'-dithiobis(2-nitrobenzoic acid) (DTNB). To study the role of cysteine residues in enzyme catalysis, the eight individual cysteine residues of the enzyme were replaced with serine residues by site-directed mutagenesis. The catalytic properties and chemical modification of wild- and mutant type-enzymes by DTNB showed that (i) none of eight cysteine residues was essential for enzyme catalysis; (ii) the inhibition by DTNB was mostly due to modification of Cys-186; (iii) Cys-96 might be another residue reacting with DTNB, and its modification caused an increase in the K(m)-value for 4-oxalomesaconate; (iv) the other six cysteine residues were inaccessible to DTNB, but susceptible to HgCl(2); and (v) only replacement of Cys-186 remarkably improved the stability of the enzyme in the absence of reducing reagent.     

Local conformational transition of Hydrogenobacter thermophilus cytochrome c552 relevant to its redox potential

In order to elucidate the molecular mechanisms responsible for the apparent nonlinear behavior of the temperature dependence of the redox potential of Hydrogenobacter thermophilus cytochrome c552 [Takahashi, Y., Sasaki, H., Takayama, S. J., Mikami, S., Kawano, S., Mita, H., Sambongi, Y., and Yamamoto, Y. (2006) Biochemistry 45, 11005-11011], its heme active site structure has been characterized using variable-temperature and -pressure NMR techniques. The study revealed a temperature-dependent conformational transition between protein structures, which slightly differ in the conformation of the loop bearing the Fe-bound axial Met residue. The heme environment in the protein structure which arises at lower temperature was found to be more polar, as a result of the altered orientation of the loop with respect to the heme due to its conformational change, than that arising at higher temperature. The present study demonstrated the importance of the structural and dynamic properties of the polypeptide chain in close proximity to the heme for redox regulation of the protein.     

Abnormal nodal flow precedes situs inversus in iv and inv mice.

We examined the nodal flow of well-characterized mouse mutants, inversus viscerum (iv) and inversion of embryonic turning (inv), and found that their laterality defects are always accompanied by an abnormality in nodal flow. In a randomized laterality mutant, iv, the nodal cilia were immotile and the nodal flow was absent. In a situs inversus mutant, inv, the nodal cilia was motile but could only produce very weak leftward nodal flow. These results consistently support our hypothesis that the nodal flow produces the gradient of putative morphogen and triggers the first L-R determination event.     

Astrocytic contributions to blood-brain barrier (BBB) formation by endothelial cells: A possible use of aortic endothelial cell for In vitro BBB model

Astrocytic contribution of endothelial cell monolayer permeability was examined in two blood-brain barrier (BBB) models, using the coculture in a double chamber system: rat astrocytes and bovine aortic endothelial cells (BAECs) or bovine brain endothelial cells (BBECs). In system 1, where astrocytes were separated from endothelial cells, a 40% reduction in -glucose permeability of the BBEC monolayer, but not the BAEC monolayer, was observed by cocultivation with astrocytes. Although several passages of BBEC in culture elicited morphological transformation from spindle-shapes to cobblestone-like features, the passaged BBECs remained responsive to astrocytes in coculture in system 1 (37% reduction of the -glucose permeability). By contrast, in system 2, where respective endothelial cells and astrocytes layered on the upper and lower surfaces of a membrane, the permeability of both BAEC and BBEC monolayers was reduced by cocultivation with astrocytes (75% reduction for BAEC and 40% reduction for BBEC). BAECs in this contiguous coculture (system 2) with astrocytes showed numerous tight junction-like structures characteristic of the BBB in vivo. These results suggest that primary cultured BBECs, which had been primed by astrocytes in vivo, retain a higher sensitivity to astrocytes possibly through an astrocytic soluble factor (s) to exhibit BBB-specific phenotypes, and that even BAEC from extra-neural tissues, when cultured with astrocytes in close proximity in vitro, may acquire the similar phenotypes and serve for an extensive use of BBB model in vitro.