Design and synthesis of phthalimide-type histone deacetylase inhibitors

Several hydroxamic acid derivatives with a substituted phthalimide group as a linker and/or cap structure, prepared during structural development studies based on thalidomide, were found to have histone deacetylase (HDAC)-inhibitory activity. Structure-activity relationship studies indicated that nature of the substituent introduced at the phthalimide nitrogen atom, introduction of a hydroxamic acid structure, and distance between the N-hydroxyl group and the cap structure are important for HDAC-inhibitory activity.     

Efficient cleavage of RNA, enhanced cellular uptake, and controlled intracellular localization of conjugate DNAzymes

Conjugate DNAzymes with polyamines and peptides were successfully prepared by solid phase fragment condensation (SPFC) and showed up to 4.2 times higher catalytic efficiency (k(cat)/K(m)) and enhanced tolerance against DNase 1digestion. To be pointed out, intracellular localization of DNAzymes could be controlled by conjugated with naturally occurring signal peptides responsible for nuclear cytoplasmic transport of proteins.     

Solution structure of the rhodanese homology domain At4g01050(175-295) from Arabidopsis thaliana

The three-dimensional structure of the rhodanese homology domain At4g01050(175-195) from Arabidopsis thaliana has been determined by solution nuclear magnetic resonance methods based on 3043 upper distance limits derived from NOE intensities measured in three-dimensional NOESY spectra. The structure shows a backbone root mean square deviation to the mean coordinates of 0.43 A for the structured residues 7-125. The fold consists of a central parallel beta-sheet with five strands in the order 1-5-4-2-3 and arranged in the conventional counterclockwise twist, and helices packing against each side of the beta-sheet. Comparison with the sequences of other proteins with a rhodanese homology domain in Arabidopsis thaliana indicated residues that could play an important role in the scaffold of the rhodanese homology domain. Finally, a three-dimensional structure comparison of the present noncatalytic rhodanese homology domain with the noncatalytic rhodanese domains of sulfurtransferases from other organisms discloses differences in the length and conformation of loops that could throw light on the role of the noncatalytic rhodanese domain in sulfurtransferases.     

Radical scavenging activity of dicaffeoyloxycyclohexanes: contribution of an intramolecular interaction of two caffeoyl residues

Six regio- and stereoisomers of dicaffeoyloxycyclohexanes and 2,4-di-O-caffeoyl-1,6-anhydro-beta-D-glucose were synthesized as model compounds of dicaffeoylquinic acids, and their radical scavenging activity was evaluated by DPPH (2,2-diphenyl-1-picrylhydrazyl) and ABTS (2,2'-azinobis(3-ethylbenzthiazoline-6-sulfonic acid) diammonium salt) radical scavenging tests. Both DPPH and ABTS radical scavenging reactions of these compounds consisted of two different steps. In the first step, catechol moieties of the caffeoyl residues were rapidly converted to o-quinone structures and no significant difference in the reactivity was observed among the tested compounds. In the second step, however, the rate of the reaction increased as the intramolecular distance of the two caffeoyl residues decreased. A novel intramolecular coupling product, which could scavenge additional radicals, was isolated from the reaction mixture of trans-1,2-dicaffeoyloxycyclohexane and DPPH radical. The result suggests that the second step of the radical scavenging reaction is arising from an intramolecular interaction between the two caffeoquinone residues to regenerate catechol structures, and that the closer their distance is, the more rapidly they react. The radical scavenging activity of natural dicaffeoylquinic acids in a biological aqueous system might also depend on the positions of caffeoyl ester groups.     

New RNAi strategy for selective suppression of a mutant allele in polyglutamine disease

In gene therapy of dominantly inherited diseases with small interfering RNA (siRNA), mutant allele specific suppression may be necessary for diseases in which the defective gene normally has an important role. It is difficult, however, to design a mutant allele-specific siRNA for trinucleotide repeat diseases in which the difference of sequences is only repeat length. To overcome this problem, we use a new RNA interference (RNAi) strategy for selective suppression of mutant alleles. Both mutant and wild-type alleles are inhibited by the most effective siRNA, and wild-type protein is restored using the wild-type mRNA modified to be resistant to the siRNA. Here, we applied this method to spinocerebellar ataxia type 6 (SCA6). We discuss its feasibility and problems for future gene therapy.     

Kinetic studies of site-directed mutational isomalto-dextranase-catalyzed hydrolytic reactions on a 27 MHz quartz-crystal microbalance

A quartz-crystal microbalance (QCM) technique was applied to analyze effects of site-directed mutagenesis of a glycosidase (isomalto-dextranase) on the hydrolysis mechanism of the substrate binding (k(on), k(off), and K(d)) and the catalytic process (k(cat)), separately, by using a dextran-immobilized QCM in buffer solution. D266N, D198N, and D313N mutants, which are predicted as critical residues of the isomalto-dextranase hydrolytic activity, dramatically decreased the apparent enzyme activity. The D266N mutant, however, did not change the substrate binding ability (K(d)), and the D198N and D313N mutants largely increased K(d) values due to the increase of k(off) and/or the decrease of k(on) values, as well as the negatively small k(cat) values. From these results, we estimate the reaction mechanism, in which Asp266 acts as only a general acid in the catalytic process, Asp198 acts as both nucleophile in the catalytic process and binding the substrate, and Asp313 acts as only the substrate binding.     

Cytologic features of chordoid meningioma. A case report

BACKGROUND: Chordoid meningioma is a rare subtype of meningioma characterized by myxoid matrices deposited among epithelioid or vacuolated tumor cells and infiltrates of inflammatory cells, and its cytologic features have rarely been reported. CASE: A 57-year-old man with a history of headache and visual disturbance presented with a tumor in the suprasellar region. Intraoperative touch smear cytology of the tumor disclosed a cord-like arrangement of polygonal tumor cells occasionally containing intranuclear inclusions. Furthermore, periodic acid-Schiff-positive, mucinous matrices were deposited among the tumor cells. Also, infiltrates of lymphocytes and plasma cells were noted. Histologic, immunohistochemical and ultrastructural examination confirmed the diagnosis of chordoid meningioma. CONCLUSION: Intraoperative smear cytology in a case of chordoid meningioma showed distinctive cytologic features suggestive of the histologic patterns. The cytologic features, together with a histologic examination, are useful for its diagnosis.     

siRNA-based inhibition specific for mutant SOD1 with single nucleotide alternation in familial ALS, compared with ribozyme and DNA enzyme

In many of autosomal dominant diseases such as familial amyotrophic lateral sclerosis (ALS) with SOD1 mutation, a missense point mutation may induce the disease by its gain of adverse property. Reduction of such a mutant protein expression is expected to improve the disease phenotype. Duplex of 21-nt RNA, known as siRNA, has recently emerged as a powerful tool to silence gene, but the sequence specificity and efficacies have not been fully studied in comparison with ribozyme and DNA enzyme. We could make the siRNA which recognized even a single nucleotide alternation and selectively suppress G93A SOD1 expression leaving wild-type SOD1 intact. In mammalian cells, the siRNA much more efficiently suppressed the expression of mutant SOD1 than ribozyme or DNA enzyme. Furthermore, these siRNAs could suppress cell death of Neuro2a induced by over-expression of mutant SOD1s with stress of proteasome inhibition. Our results support the feasibility of utilizing siRNA-based gene therapy of familial ALS with mutant SOD1.     

TNF receptor-associated factor 5 limits the induction of Th2 immune responses

The TNF receptor-associated factor (TRAF) family of molecules acts as adapter proteins for signaling pathways initiated by several members of the TNF receptor (TNFR) superfamily. TRAF5(-/-) animals are viable and have normal development of the immune system despite interacting with several TNFR family members. A clear role for TRAF5 has yet to emerge. OX40 (CD134) interacts with TRAF5, suggesting that this pathway could be involved in regulating T cell differentiation into Th1 or Th2 cells. In tissue culture, OX40 stimulation of TRAF5(-/-) T cells resulted in a pronounced Th2 phenotype with elevated levels of IL-4 and IL-5. Similarly, in vivo immunization with protein in adjuvant in the presence of an agonist anti-OX40 Ab resulted in enhanced Th2 development in TRAF5(-/-) mice. Additionally, lung inflammation induced by T cells, which is critically controlled by OX40, was more pronounced in TRAF5(-/-) mice, characterized by higher levels of Th2 cytokines. These results suggest that TRAF5 can limit the induction of Th2 responses, and that TRAF5 can play a role in modulating responses driven by OX40 costimulation.