NMR determination of the 2:1 binding complex of naphthyridine carbamate dimer (NCD) and CGG/CGG triad in double-stranded DNA

Trinucleotide repeat (TNR) diseases are caused by the aberrant expansion of CXG (X = C, A, G and T) sequences in genomes. We have reported two small molecules binding to TNR, NCD, and NA, which strongly bind to CGG repeat (responsible sequence of fragile X syndrome) and CAG repeat (Huntington''s disease). The NMR structure of NA binding to the CAG/CAG triad has been clarified, but the structure of NCD bound to the CGG/CGG triad remained to be addressed. We here report the structural determination of the NCD-CGG/CGG complex by NMR spectroscopy and the comparison with the NA-CAG/CAG complex. While the NCD-CGG/CGG structure shares the binding characteristics with that of the NA-CAG/CAG complex, a significant difference was found in the overall structure caused by the structural fluctuation at the ligand-bound site. The NCD-CGG/CGG complex was suggested in the equilibrium between stacked and kinked structures, although NA-CAG/CAG complex has only the stacked structures. The dynamic fluctuation of the NCD-CGG/CGG structure at the NCD-binding site suggested room for optimization in the linker structure of NCD to gain improved affinity to the CGG/CGG triad.     

Intrinsic kinase activity and SQ/TQ domain of Chk2 kinase as well as N-terminal domain of Wip1 phosphatase are required for regulation of Chk2 by Wip1

The anti-oncogenic Chk2 kinase plays a crucial role in DNA damage-induced cell cycle checkpoint regulation. Recently, we have shown that Chk2 associates with the oncogenic Wip1 (PPM1D) phosphatase and that Wip1 acts as a negative regulator of Chk2 during DNA damage response by dephosphorylating phosphorylated Thr-68 in activated Chk2 (Fujimoto, H., Onishi, N., Kato, N., Takekawa, M., Xu, X. Z., Kosugi, A., Kondo, T., Imamura, M., Oishi, I., Yoda, A., and Minami, Y. (2006) Cell Death Differ. 13, 1170-1180). Here, we performed structure-function analyses of Chk2 and Wip1 by using a series of deletion or amino acid-substituted mutant proteins of Chk2 and Wip1. We show that nuclear localization of both Chk2 and Wip1 is required for their association in cultured cells and that the serine-glutamine (SQ)/threonine-glutamine (TQ) domain of Chk2, containing Thr-68, and the N-terminal domain of Wip1, comprising about 100 amino acids, are necessary and sufficient for the association of both molecules. However, it was found that an intrinsic kinase activity of Chk2, but not phosphatase activity of Wip1, is required for the association of fulllength Chk2 and Wip1. Interestingly, we also show that the mutant Wip1 proteins, bearing the N-terminal domain of Wip1 alone or lacking an intrinsic phosphatase activity, exhibit dominant negative effects on the functions of the wild-type Wip1, i.e. ectopic expression of either of these Wip1 mutants inhibits dephosphorylation of Thr-68 in Chk2 by Wip1 and anti-apoptotic function of Wip1. These results provide a molecular basis for developing novel anti-cancer drugs, targeting oncogenic Wip1 phosphatase.     

Enhanced expression of human cDNA by phosphoglycerate kinase promoter-puromycin cassette in the mouse transthyretin locus

To produce a humanized mouse, it is critical to obtain a correct expression of a human gene/cDNA after insertion into a mouse locus. We previously generated a targeted allele in which the PGK-neo cassette, flanked by lox71 and loxP, was inserted into the first exon of the mouse endogenous transthyretin (Ttr) gene in ES cells. Using these ES cells, we showed that a human transthyretin (TTR) cDNA with the PGK-puro cassette can be efficiently inserted into this locus by Cre-mediated recombination, and that the human TTR cDNA was expressed in a tissue-specific manner under the control of the mouse endogenous Ttr promoter. To examine whether the PGK-puro cassette or IRES could affect the expression of human TTR cDNA, we generated four mouse lines using Cre and Flp-mediated recombination. The mouse line containing the PGK-puro cassette, but not IRES, exhibited quantitatively and temporally similar expression of human TTR cDNA. Removal of the PGK-puro cassette significantly downregulated the expression of the cDNA. The insertion of IRES sequence upstream of the human TTR cDNA resulted in decreased expression, even in the presence of the PGK-puro cassette. The mouse line containing IRES, but not PGK-puro, showed the lowest level of expression. These results suggest that the PGK-puro cassette is necessary to obtain the enhanced expression of a co-existing human cDNA in the mouse Ttr locus, even though the expression of co-existing cDNA was under the control of the mouse endogenous promoter.     

Accumulation of cyclitols functioning as compatible solutes in the haptophyte alga Pavlova sp.

Using nuclear magnetic resonance spectroscopy, we identified and characterized accumulated compatible solutes in cells of the haptophyte alga Pavlova sp. strain CCMP504. The predominant organic solutes were d ‐1,4/2,5‐cyclohexanetetrol (CHT), 1,3,5/2,4‐cyclohexanepentol (CHP) and scyllo‐inositol. We then profiled the intracellular organic solutes present in Pavlova sp. grown in medium with salinity ranging from 23 practical salinity units (PSU) (hyposaline) to 35 PSU (optimum salinity for growth), to 47 PSU (hypersaline). The results of these analyses reveal progressive accumulation of CHT and CHP in response to increasing growth medium salinity. We also observed altered accumulation of CHT and CHP in samples subjected to salinity shock. To further characterize the CHT and CHP biosynthesis in Pavlova sp., we carried out stable isotope feeding experiments. Specific labeling of CHT and CHP with d ‐¹³C‐glucose suggested that d ‐glucose is a biosynthetic precursor of these cyclitols. The salinity‐induced accumulation of CHT and CHP suggests that these cyclitols act as compatible solutes. Our results therefore provide new evidence supporting classification of CHP as a compatible solute.     

AM-3K, an anti-macrophage antibody, recognizes CD163, a molecule associated with an anti-inflammatory macrophage phenotype.

CD163 is a member of the scavenger receptor cysteine-rich superfamily restricted to the monocyte/macrophage lineage and is thought to be a useful marker for anti-inflammatory or alternatively activated macrophages. In this study we used mass spectrometric analysis to determine that the antigen recognized by the antibody AM-3K, which we previously generated as a tissue macrophage-specific monoclonal antibody, was CD163. An anti-inflammatory subtype of macrophages stimulated by dexamethasone or interleukin-10 showed strong reactivity for AM-3K and increased expression of CD163 mRNA. Immunohistochemical staining of routinely processed pathological specimens revealed that AM-3K recognized a specialized subpopulation of macrophages. In granulomatous diseases such as tuberculosis, sarcoidosis, or foreign body reactions, tissue macrophages around granulomas, but not component cells of the granulomas such as epithelioid cells and multinucleated giant cells, showed positive staining for AM-3K. In atherosclerotic lesions, scattered macrophages in diffuse intimal lesions were strongly positive for AM-3K, whereas foamy macrophages in atheromatous plaques demonstrated only weak staining. We therefore suggest that, in routine pathological specimens, AM-3K is a useful marker for anti-inflammatory macrophages because these cells can be distinguished from inflammatory or classically activated macrophages. Because AM-3K cross-reacts with macrophage subpopulations in different animal species including rats, guinea pigs, rabbits, cats, dogs, goats, pigs, bovine species, horses, monkeys, and cetaceans, it will have wide application for detection of CD163 in various animals.     

Dysregulation of Src family kinases in mast cells from epilepsy-resistant ASK versus epilepsy-prone EL mice

EL mice have been used as a model of epilepsy, whereas ASK mice are an epilepsy-resistant variant originating from a colony of EL mice. Mast cell-dependent anaphylaxis is easily inducible by stimulation with IgE and Ag in ASK mice, whereas EL mice are resistant to such stimuli. In this study we have characterized mast cells derived from these two strains. ASK mast cells proliferated more vigorously than EL cells in response to IL-3 and stem cell factor. Although ASK mast cells degranulated less vigorously than EL mast cells upon stimulation with IgE and Ag, ASK cells produced and secreted several-fold more TNF-alpha and IL-2 than EL cells. Consistent with the similarities of these ASK and EL mast cell responses with phenotypes of lyn(-/-) and wild-type mast cells, respectively, Lyn activity was reduced in ASK cells. In addition to the impaired Lyn activity, ASK cells just like lyn(-/-) cells exhibited reduced Syk activity, prolonged activation of ERK and JNK, and enhanced activation of Akt. Furthermore, the lipid raft-resident transmembrane adaptor protein Cbp/PAG that associates with Lyn was hypophosphorylated in ASK cells. Importantly, similar to lyn(-/-) cells, Fyn was hyperactivated in ASK cells. Therefore, these results are consistent with the notion that Lyn-dependent phosphorylation of Cbp/PAG negatively regulates Src family kinases. This study also suggests that reduced activity of Lyn, a negative regulator of mast cell activation, underlies the susceptibility of ASK mice to anaphylaxis and implies that dysregulation of Lyn and other Src family kinases contributes to epileptogenesis.     

Location of PsbY in oxygen-evolving photosystem II revealed by mutagenesis and X-ray crystallography

PsbY is one of the low molecular mass subunits of oxygen-evolving photosystem II (PSII). Its location, however, has not been identified in the current crystal structure of PSII. We constructed a PsbY-deletion mutant of Thermosynechococcus elongatus, crystallized, and analyzed the crystal structure of the mutant PSII dimer. The results obtained showed that PsbY is located in the periphery of PSII close to the alpha- and beta-subunits of cytochrome b559, which corresponded to an unassigned helix in the 3.7A structure of T. vulcanus or helix X2 in the 3.0A structure of T. elongatus. Our results also indicated that the C-terminal loop of PsbY is protruded toward the stromal side, instead of the lumenal side predicted previously.     

Unraveling tissue regeneration pathways using chemical genetics

Identifying the molecular pathways that are required for regeneration remains one of the great challenges of regenerative medicine. Although genetic mutations have been useful for identifying some molecular pathways, small molecule probes of regenerative pathways might offer some advantages, including the ability to disrupt pathway function with precise temporal control. However, a vertebrate regeneration model amenable to rapid throughput small molecule screening is not currently available. We report here the development of a zebrafish early life stage fin regeneration model and its use in screening for small molecules that modulate tissue regeneration. By screening 2000 biologically active small molecules, we identified 17 that specifically inhibited regeneration. These compounds include a cluster of glucocorticoids, and we demonstrate that transient activation of the glucocorticoid receptor is sufficient to block regeneration, but only if activation occurs during wound healing/blastema formation. In addition, knockdown of the glucocorticoid receptor restores regenerative capability to nonregenerative, glucocorticoid-exposed zebrafish. To test whether the classical anti-inflammatory action of glucocorticoids is responsible for blocking regeneration, we prevented acute inflammation following amputation by antisense repression of the Pu.1 gene. Although loss of Pu.1 prevents the inflammatory response, regeneration is not affected. Collectively, these results indicate that signaling from exogenous glucocorticoids impairs blastema formation and limits regenerative capacity through an acute inflammation-independent mechanism. These studies also demonstrate the feasibility of exploiting chemical genetics to define the pathways that govern vertebrate regeneration.