Genomewide high-density SNP linkage analysis of 236 Japanese families supports the existence of schizophrenia susceptibility loci on chromosomes 1p, 14q, and 20p

The Japanese Schizophrenia Sib-Pair Linkage Group (JSSLG) is a multisite collaborative study group that was organized to create a national resource for affected sib pair (ASP) studies of schizophrenia in Japan. We used a high-density single-nucleotide–polymorphism (SNP) genotyping assay, the Illumina BeadArray linkage mapping panel (version 4) comprising 5,861 SNPs, to perform a genomewide linkage analysis of JSSLG samples comprising 236 Japanese families with 268 nonindependent ASPs with schizophrenia. All subjects were Japanese. Among these families, 122 families comprised the same subjects analyzed with short tandem repeat markers. All the probands and their siblings, with the exception of seven siblings with schizoaffective disorder, had schizophrenia. After excluding SNPs with high linkage disequilibrium, we found significant evidence of linkage of schizophrenia to chromosome 1p21.2-1p13.2 (LOD=3.39) and suggestive evidence of linkage to 14q11.2 (LOD=2.87), 14q11.2-q13.2 (LOD=2.33), and 20p12.1-p11.2 (LOD=2.33). Although linkage to these regions has received little attention, these regions are included in or partially overlap the 10 regions reported by Lewis et al. that passed the two aggregate criteria of a meta-analysis. Results of the present study—which, to our knowledge, is the first genomewide analysis of schizophrenia in ASPs of a single Asian ethnicity that is comparable to the analyses done of ASPs of European descent—indicate the existence of schizophrenia susceptibility loci that are common to different ethnic groups but that likely have different ethnicity-specific effects.     

Application of fluorophore-assisted carbohydrate electrophoresis to analysis of disaccharides and oligosaccharides derived from glycosaminoglycans

Various combinations of fluorescent dyes, polyacrylamide gels, and electrophoresis buffers were tested by fluorophore-assisted carbohydrate electrophoresis (FACE) for the purpose of analyzing sulfated and nonsulfated glycosaminoglycan (GAG) oligosaccharides in which disaccharides and low-molecular weight oligosaccharides were included. A nonionic fluorescent dye was found to be suitable for analyzing sulfated disaccharides derived from sulfated GAGs (e.g., chondroitin sulfate, dermatan sulfate) because sulfated disaccharides themselves had enough anionic potential for electrophoresis. The migration rates of chondroitin sulfate (CS) disaccharides in polyacrylamide gels were affected by the number of sulfate residues and the conformation of each disaccharide. When an anionic fluorescent dye, 8-aminonaphthalene-1,3,6-trisulfonic acid disodium salt (ANTS), was coupled with sulfated GAG oligosaccharides, nearly all of the conjugates migrated at the electrophoretic front due to the added anionic potential. Nonsulfated hyaluronan (HA) oligosaccharides (2-16 saccharides) were subjected to electrophoresis by coupling with a nonionic fluorescent dye, 2-aminoacridone (AMAC), but did not migrate in the order of their molecular size. Especially di-, tetra-, hexa-, and octasaccharides of HA migrated in the reverse order of their molecular size. HA/CS oligosaccharides were able to migrate in the order of their chain lengths by coupling with an anionic fluorescent dye in a nonborate condition.     

Indispensable role of Stat5a in Stat6-independent Th2 cell differentiation and allergic airway inflammation

It is well-recognized that Stat6 plays a critical role in Th2 cell differentiation and the induction of allergic inflammation. We have previously shown that Stat5a is also required for Th2 cell differentiation and allergic airway inflammation. However, it is the relative importance and redundancy of Stat6 and Stat5a in Th2 cell differentiation and allergic airway inflammation are unknown. In this study we addressed these issues by comparing Stat5a-deficient (Stat5a(-/-)) mice, Stat6(-/-) mice, and Stat5a- and Stat6 double-deficient (Stat5a(-/-) Stat6(-/-)) mice on the same genetic background. Th2 cell differentiation was severely decreased in Stat6(-/-)CD4+ T cells, but Stat6-independent Th2 cell differentiation was still significantly observed in Stat6(-/-)CD4+ T cells. However, even in the Th2-polarizing condition (IL-4 plus anti-IFN-gamma mAb), no Th2 cells developed in Stat5a(-/-)Stat6(-/-) CD4+ T cells. Moreover, Ag-induced eosinophil and lymphocyte recruitment in the airways was severely decreased in Stat5a(-/-)Stat6(-/-) mice compared with that in Stat6(-/-) mice. These results indicate that Stat5a plays an indispensable role in Stat6-independent Th2 cell differentiation and subsequent Th2 cell-mediated allergic airway inflammation.     

Priming effect of lipopolysaccharide on acetyl-coenzyme A:lyso-platelet-activating factor acetyltransferase is MyD88 and TRIF independent

LPS has a priming effect on various stimuli. For instance, LPS priming enhances the production of platelet-activating factor (PAF), a proinflammatory lipid mediator that is induced by PAF itself. Among various enzymes responsible for PAF biosynthesis, acetyl-coenzyme A:1-O-alkyl-2-lyso-sn-glycero-3-phosphocholine acetyltransferase is one of the enzymes activated by PAF receptor stimulation. In this study we investigated the priming effect of LPS on the acetyltransferase activation by PAF in TLR4-knockout (KO) mice, MyD88-KO mice, and Toll/IL-1R domain-containing adaptor inducing IFN-beta (TRIF)-KO mice. This enzyme was biphasically activated by LPS. Although the first peak occurred within 30 min in wild-type (WT), but not TLR4-KO or MyD88-KO, macrophages, the second phase reached a maximum within hours in WT, MyD88-KO, and TRIF-KO, but not in TLR4-KO, macrophages. Only in the second phase was the increase in acetyltransferase activity upon PAF receptor activation remarkably enhanced in WT, MyD88-KO, and TRIF-KO cells, but not in TLR4-KO cells. These data demonstrated that LPS exerted a priming effect on PAF receptor-mediated acetyltransferase activation through the TLR4-dependent, but MyD88- and TRIF-independent, pathway.     

Muramyl dipeptide enhances osteoclast formation induced by lipopolysaccharide, IL-1 alpha, and TNF-alpha through nucleotide-binding oligomerization domain 2-mediated signaling in osteoblasts

Muramyl dipeptide (MDP) is the minimal essential structural unit responsible for the immunoadjuvant activity of peptidoglycan. As well as bone-resorbing factors such as 1alpha,25-dihydroxyvitamin D3 (1alpha,25(OH)2D3) and PGE2, LPS and IL-1alpha stimulate osteoclast formation in mouse cocultures of primary osteoblasts and hemopoietic cells. MDP alone could not induce osteoclast formation in the coculture, but enhanced osteoclast formation induced by LPS, IL-1alpha, or TNF-alpha but not 1alpha,25(OH)2D3 or PGE2. MDP failed to enhance osteoclast formation from osteoclast progenitors induced by receptor activator of NF-kappaB ligand (RANKL) or TNF-alpha. MDP up-regulated RANKL expression in osteoblasts treated with LPS or TNF-alpha but not 1alpha,25(OH)2D3. Osteoblasts expressed mRNA of nucleotide-binding oligomerization domain 2 (Nod2), an intracellular sensor of MDP, in response to LPS, IL-1alpha, or TNF-alpha but not 1alpha,25(OH)2D3. Induction of Nod2 mRNA expression by LPS but not by TNF-alpha in osteoblasts was dependent on TLR4 and MyD88. MDP also enhanced TNF-alpha-induced osteoclast formation in cocultures prepared from Toll/IL-1R domain-containing adapter protein (TIRAP)-deficient mice through the up-regulation of RANKL mRNA expression in osteoblasts, suggesting that TLR2 is not involved in the MDP-induced osteoclast formation. The depletion of intracellular Nod2 by small interfering RNA blocked MDP-induced up-regulation of RANKL mRNA in osteoblasts. LPS and RANKL stimulated the survival of osteoclasts, and this effect was not enhanced by MDP. These results suggest that MDP synergistically enhances osteoclast formation induced by LPS, IL-1alpha, and TNF-alpha through RANKL expression in osteoblasts, and that Nod2-mediated signals are involved in the MDP-induced RANKL expression in osteoblasts.     

Murine plasmacytoid dendritic cells produce IFN-gamma upon IL-4 stimulation

IL-4 plays a key role in inducing IL-4 production in CD4+ T cells, functioning as an important determinant for Th2 cell differentiation. We show here that IL-4 induces IFN-gamma production in B220+ plasmacytoid dendritic cells (PDCs). By searching for cell populations that produce IFN-gamma upon IL-4 stimulation, we found that PDCs were a major IFN-gamma-producing cell upon IL-4 stimulation in wild-type and Rag-2-/- splenocytes. Isolated PDCs, but not CD11b+ DCs or CD8+ DCs, produced IFN-gamma upon IL-4 stimulation. In vivo, the depletion of PDCs by anti-Ly6G/C Ab prevented IFN-gamma production induced by IL-4 administration. We also found that IL-4 induced IFN-gamma production, but not IL-12 or IFN-alpha production, in PDCs and also strongly enhanced CpG oligodeoxynucleotide-induced IFN-gamma production, but not CpG oligodeoxynucleotide-induced IL-12 or IFN-alpha production. However, IL-4 did not induce IFN-gamma production in Stat6-/- PDCs. Moreover, IL-4 induced Stat4 expression in PDCs through a Stat6-dependent mechanism, and only the Stat4-expressing PDCs produced IFN-gamma. Furthermore, IL-4 did not induce IFN-gamma production in Stat4-/- PDCs. These results indicate that PDCs preferentially produce IFN-gamma upon IL-4 stimulation by Stat6- and Stat4-dependent mechanisms.     

A role for Hrs in endosomal sorting of ligand-stimulated and unstimulated epidermal growth factor receptor

Ligand-stimulated growth factor receptors are rapidly internalized and transported to early endosomes. Unstimulated receptors are also internalized constitutively, although at a slower rate, and delivered to the same organelle. At early endosomes, stimulated receptors are sorted for the lysosomal degradation pathway, whereas unstimulated receptors are mostly recycled back to the cell surface. To investigate the role of Hrs, an early endosomal protein, in this sorting process, we overexpressed Hrs in HeLa cells and examined the intracellular trafficking of epidermal growth factor receptor (EGFR) in EGF-stimulated and unstimulated cells. Overexpression of Hrs inhibited the trafficking of EGFR from early endosomes, resulting in an accumulation of EGFR on early endosomes in both ligand-stimulated and unstimulated cells. On the other hand, overexpression of Hrs mutants with a deletion or a point mutation within the FYVE domain did not inhibit the trafficking. These results suggest that Hrs regulates the sorting of ligand-stimulated and unstimulated growth factor receptors on early endosomes, and that the FYVE domain, which is required for Hrs to reside in a microdomain of early endosomes, plays an essential role in the function of Hrs.     

Effective reduction of antigenicity of hen egg lysozyme by site-specific glycosylation

Various mutant lysozymes were constructed by genetic modification and secreted in yeast expression system to evaluate the changes in the antigenicity of hen egg lysozyme (HEL). Although Arg68, the most critical residue to antigenicity of HEL, was substituted with Gln, the binding of monoclonal antibodies (mAbs) with the mutant lysozyme did not critically reduce, remaining 60% of the binding with mAb. In contrast, glycosylated mutant lysozyme G49N whose glycine was substituted with asparagine dramatically reduced the binding with mAb. The oligomannosyl type of G49N lysozyme reduced binding with mAb to one-fifth, while the polymannosyl type of G49N lysozyme completely diminished the binding with mAb. This suggests that the site-specific glycosylation of lysozyme in the interfacial region of lysozyme-antibody complex is more effective to reduce the antigenicity than the mutation of single amino acid substitution in the interfacial region.     

Systematic single base-pair substitution analysis of DNA binding by the cAMP receptor protein in cyanobacterium Synechocystis sp. PCC 6803

The cAMP receptor protein SYCRP1 in cyanobacterium Synechocystis sp. PCC 6803 is a regulatory protein that binds to the consensus DNA sequence (5'-AAATGTGATCTAGATCACATTT-3') for the cAMP receptor protein CRP in Escherichia coli. Here we examined the effects of systematic single base-pair substitutions at positions 4-8 (TGTGA) of the consensus sequence on the specific binding of SYCRP1. The consensus sequence exhibited the highest affinity, and the effects of base-pair substitutions at positions 5 and 7 were the most deleterious. The result is similar to that previously reported for CRP, whereas there were differences between SYCRP1 and CRP in the rank order of affinity for each substitution.     

Synthesis, structures, and magnetic properties of dicopper(II) and dinickel(II) complexes with a new bis(macrocycle), 7,7-(2-hydoxypropane-1,3-diyl)bis{3,7,11,17-tetraazabicyclo[11.3.1]- heptadeca-1(17),13,15-triene}

A new bis(macrocycle) ligand, 7,7^′-(2-hydoxypropane-1,3-diyl)-bis{3,7,11,17-tetraazabicyclo[11.3.1]heptadeca-1(17),13,15-triene} (HL), and its dicopper(II) ([Cu₂(HL)Cl₂](NO₃)₂ · 4H₂O (4a), [Cu₂(HL)I₂]I₂ · H₂O (4b)) and dinickel(II) ([Ni₂(L)(OH₂)](ClO₄)₃ (5a), [Ni₂(L)(OH₂)]I₃ · 2H₂O (5b), [Ni₂(L)N₃](N₃)₂ · 7H₂O (5c)) complexes have been synthesized. The alkoxide bridged face-to-face structure of the dinickel(II) complex 5c has been revealed by X-ray crystallography, as well as the “half-opened clamshell” form of the bis(macrocyclic) dicopper(II) complex 4b. Variable temperature magnetic susceptibility studies have indicated that there exists intramolecular antiferromagnetic coupling (J=−33.8 cm⁻¹ (5a), −32.5 cm⁻¹ (5b), and −29.7 cm⁻¹ (5c)) between the two nickel(II) ions in the nickel(II) complexes.