Common variants in CDKN2B-AS1 associated with optic-nerve vulnerability of glaucoma identified by genome-wide association studies in Japanese

Background

To date, only a small portion of the genetic variation for primary open-angle glaucoma (POAG), the major type of glaucoma, has been elucidated.

Methods and Principal Findings

We examined our two data sets of the genome-wide association studies (GWAS) derived from a total of 2,219 Japanese subjects. First, we performed a GWAS by analyzing 653,519 autosomal common single-nucleotide polymorphisms (SNPs) in 833 POAG patients and 686 controls. As a result, five variants that passed the Bonferroni correction were identified in CDKN2B-AS1 on chromosome 9p21.3, which was already reported to be a significant locus in the Caucasian population. Moreover, we combined the data set with our previous GWAS data set derived from 411 POAG patients and 289 controls by the Mantel-Haenszel test, and all of the combined variants showed stronger association with POAG (P<5.8×10⁻¹⁰). We then subdivided the case groups into two subtypes based on the value of intraocular pressure (IOP)—POAG with high IOP (high pressure glaucoma, HPG) and that with normal IOP (normal pressure glaucoma, NPG)—and performed the GWAS using the two data sets, as the prevalence of NPG in Japanese is much higher than in Caucasians. The results suggested that the variants from the same CDKN2B-AS1 locus were likely to be significant for NPG patients.

Conclusions and Significance

In this study, we successfully identified POAG-associated variants in the CDKN2B-AS1 locus using a Japanese population, i.e., variants originally reported as being associated with the Caucasian population. Although we cannot rule out that the significance could be due to the differences in sample size between HPG and NPG, the variants could be associated specifically with the vulnerability of the optic nerve to IOP, which is useful for investigating the etiology of glaucoma.     

Th2 polarization enhanced by oral administration of higher doses of antigen

Although oral administration of a soluble proteinantigen can induce various immune responses, theeffect of the dosage of oral antigen on thepredominance of Th2-type cytokine and antibodyresponses has not been well clarified yet. In thepresent study, we fed T cell receptor (TCR) transgenic(tg) mice various amounts of chicken ovalbumin (0.1,5, and 250 mg) and examined the resulting immuneresponses to this antigen. In these TCR tg mice, theresponses of antigen-specific T cells were greatlyamplified concomitantly with significantantigen-specific cytokine secretion. We found that ahigh dose (250 mg) of antigen significantlyupregulated the serum antigen-specific IgG1 and IgAantibody responses in mice later intraperitoneallyinjected with antigen plus adjuvant. The miceadministered the same oral dose but not immunizedshowed upregulation of Th2-type IL-4 and IL-5secretion and downregulation of Th1-type IL-2 andIFN-. This enhancement of Th2-type cytokineand antibody responses was more marked when largerdoses of antigen orally administered. These resultsdemonstrated that antigen feeding induces thedevelopment of T cells secreting Th2-type cytokines ina dose-dependent manner and that these T cells have ahelper function for the production of antibodies ofthe Th2-type isotypes. This experimental system shouldbe useful to screen foods and other substances thatcan modulate Th2-type responses relating to allergy.     

Mutational and structural-based analyses of the osmolyte effect on protein stability

It is known that several naturally occurring substances known as osmolytes increase the conformational stability of proteins. Bolen and co-worker proposed the osmophobic theory, which asserts the osmolyte effect occurs because of an unfavorable interaction of osmolytes mainly with the protein backbone, based on the results on the transfer Gibbs energy of amino acids (Deltag) [Bolen and Baskakov (2001) J. Mol. Biol. 310, 955-963]. In this paper, we report the effect of sarcosine on the conformational stability (DeltaG) of RNase Sa (96 residues and one disulfide bond) and four mutant proteins. The thermal denaturation curves for RNase Sa in sarcosine fitted a two-state model on nonlinear least-squares analysis. All the RNase Sa proteins were stabilized by sarcosine. For example, the increase in stability of the wild-type protein in 4 M sarcosine due to the osmolyte effect (Delta(o)DeltaG) is 3.2 kcal/mol. Mutational analysis of the osmolyte effect indicated that the changed Delta(o)DeltaG values upon mutation (Delta(m)Delta(o)DeltaG), as estimated from the Deltag values, are similar to the experimental values. Structural-based analysis of the osmolyte effect was also performed using model denatured structures: (a) a fully extended model (single chain) with no disulfide bond, (b) two-part, unfolded models (two chains) with a disulfide bond constructed through molecular dynamic (MD) simulation, and (c) a two-part, folded model (two chains). The two-part, unfolded models were expected to be more suitable as denatured structures. The Delta(o)DeltaG values calculated using the two-part, unfolded models were more consistent with experimental values than those calculated using the fully extended and two-part, folded models. This suggests that MD simulation is useful for testing denatured structures. These results indicate that the osmophobic theory can explain the osmolyte effect on protein stability.     

Mapping ultra-weak protein-protein interactions between heme transporters of Staphylococcus aureus

Iron is an essential nutrient for the proliferation of Staphylococcus aureus during bacterial infections. The iron-regulated surface determinant (Isd) system of S. aureus transports and metabolizes iron porphyrin (heme) captured from the host organism. Transportation of heme across the thick cell wall of this bacterium requires multiple relay points. The mechanism by which heme is physically transferred between Isd transporters is largely unknown because of the transient nature of the interactions involved. Herein, we show that the IsdC transporter not only passes heme ligand to another class of Isd transporter, as previously known, but can also perform self-transfer reactions. IsdA shows a similar ability. A genetically encoded photoreactive probe was used to survey the regions of IsdC involved in self-dimerization. We propose an updated model that explicitly considers self-transfer reactions to explain heme delivery across the cell wall. An analogous photo-cross-linking strategy was employed to map transient interactions between IsdC and IsdE transporters. These experiments identified a key structural element involved in the rapid and specific transfer of heme from IsdC to IsdE. The resulting structural model was validated with a chimeric version of the homologous transporter IsdA. Overall, our results show that the ultra-weak interactions between Isd transporters are governed by bona fide protein structural motifs.     

Identification of Tau and MAP2 as novel substrates of Rho-kinase and myosin phosphatase

Rho-kinase and myosin phosphatase are implicated in the phosphorylation-state of myosin light chain downstream of Rho, which is thought to induce smooth muscle contraction and stress fibre formation in non-muscle cells. Here, we found that microtubule-associated proteins, Tau and MAP2, interacted with the myosin-binding subunit (MBS) of myosin phosphatase, and were the possible substrates of both Rho-kinase and myosin phosphatase. We determined the phosphorylation sites of Tau (Thr245, Thr377, Ser409) and MAP2 (Ser1796) by Rho-kinase. We also found that Rho-kinase phosphorylated Tau at Ser262 to some extent. Phosphorylation by Rho-kinase decreased the activity of Tau to promote microtubule assembly in vitro. Substitutions of Ala for Ser/Thr at the phosphorylation sites of Tau (Tau-AAA) did not affect the activity to promote microtubule assembly, while substitutions of Asp for Ser/Thr (Tau-DDD), which are expected to mimic the phosphorylation-state of Tau, slightly reduced the activity. When Tau, or mutated forms of Tau, were expressed in PC12 cells, followed by treatment with cytochalasin D, they promoted extension of the cell process in a cytochalasin-dependent manner. However, Tau-DDD showed the weaker activity in this capacity than wild-type Tau or Tau-AAA. These results suggest that the phosphorylation-state of these residues of Tau affects its activity both in vitro and in vivo. Thus, it is likely that the Rho-kinase/MBS pathway regulates not only the actin-myosin system but also microtubule dynamics.     

Epistasis effects of COMT and MTHFR on inter-individual differences in mental health: Under the inverted U-shaped prefrontal dopamine model

Higher cognitive performance, maintenance of mental health and psychological well-being require adequate prefrontal cortex (PFC) function. “Inverted U-shaped” dopamine model indicates optimal PFC dopamine level is important to attain its function while high or low levels have adverse effects. Catechol-O-methyltransferase (COMT) and methylenetetrahydrofolate reductase (MTHFR) may be involved in this complex non-linear PFC dopamine regulation. We addressed whether genetic variation reflecting COMT and MTHFR activities can explain the inter-individual mental health differences in healthy Japanese men (n = 188). The mental health was measured by Mental Health Inventory (MHI)-5 score. The rs4633–rs4818–rs4680 haplotypes were used to represent the multilevel COMT activities, while for MTHFR, the functional single polymorphism, rs1801133 (C677T), was used. We examined the effectiveness of haplotype-based association analysis of COMT on mental health together with studying its interaction with MTHFR-C677T. As a result, the relation between activity-ranked COMT genotype and MHI-5 score showed a tendency to fit into an “inverted U-shaped” quadratic curve (P = 0.054). This curvilinear correlation was significant in the subjects with MTHFR-CC (P < 0.001), but not with MTHFR T-allele carriers (P = 0.793). Our pilot study implies a potential influence of COMT and MTHFR genotypic combination on normal variation of mental health.     

DNA stable pentaploid H1 (ES) cells obtained from an octaploid cell induced from tetraploid cells polyploidized using demecolcine

Pentaploid H1 (ES) cells (5H1 cells) were accidentally obtained through one‐cell cloning of octaploid H1 (ES) cells (8H1 cells) that were established from tetraploid H1 (ES) cells (4H1 cells) polyploidized using demecolcine. The number of chromosomes of 5H1 cells was 100, unlike the 40 of diploid H1 (ES) cells (2H1 cells), 80 of 4H1, and 160 of 8H1 cells. The durations of G₁, S, and G₂/M phases of 5H1 cells were 3, 7, and 6 h, respectively, almost the same as those of 2H1, 4H1, and 8H1 cells. The cell volume of 5H1 cells was half of that of 8H1 cells, suggesting that 5H1 cells were created through abnormal cell divisions of 8H1 cells. The morphology of growing 5H1 cells was a spherical cluster similar to that of 2H1 cells and differing from the flagstone‐like shape of 4H1 and 8H1 cells. Pentaploid solid tumors were formed from 5H1 cells after interperitoneal injection into the mouse abdomen, and they contained endodermal, mesodermal, and ectodermal cells as well as undifferentiated cells, suggesting both that the DNA content of 5H1 cells was retained during tumor formation and that the 5H1 cells were pluripotent. The DNA content of 5H1 cells was stable in long‐term culturing as 2H1 cells, meaning that 5H1 and 2H1 cells shared similarities in DNA structure. The excellent stability of the DNA content of 5H1 cells was explained using a hypothesis for the DNA structure of polyploid cells because the pairing of homologous chromosomes in 5H1 cells is spatially forbidden. J. Cell. Physiol. 223: 369–375, 2010. © 2010 Wiley‐Liss, Inc.     

Characterization of the carotenoid-binding protein of the Y-gene dominant mutants of Bombyx mori

Carotenoids play important and diverse roles in insects. Recently, we purified and partially characterized a carotenoid-binding protein (CBP) from the wild type of Bombyx mori. In this report, we utilized immunoblotting, ELISA and immunocytochemistry to further characterize and localize the expression of CBP in the larval midgut and silk gland obtained from the wild type and four naturally occurring mutants linked to carotenoids transport. CBP was expressed throughout the 5th stadium, with highest expressions on days 4-5 in the silk gland and days 3-5 in the midgut. Immunoblotting analyses demonstrated the presence of CBP along the middle part of the midgut. Microscopic immunocytochemistry demonstrated that the 33 kDa CBP was uniformly expressed along the brush border of columnar cells in the epithelium of the midgut typifying its function in aiding absorption of dietary carotenoids. Similarly, CBP was highly expressed along the distal membrane of the middle part of the silk gland demonstrating its function in uptake of carotenoids from lipophorin. When the middle silk glands and midguts of the four mutants were incubated with rabbit anti-CBP antibody, only proteins of the Y-gene dominant mutants cross reacted with the antibody further accentuating the hypothesis that the CBP is a Y-gene dependent protein.