Development of a novel neodymium compound for in vivo fluorescence imaging.

We developed a novel fluorescent probe that contains the neodymium(III) complex moiety and fluorescein moiety. This probe can emit long-lived near-infrared luminescence derived from a Nd ion through excitation of the fluorescein moiety with visible light (lambda(ex) = 488 nm, lambda(em) = 880 nm, lifetime = 2.3 micros). These results indicate the possibility of the probe as a candidate for in vivo fluorescence molecular imaging.     

First determination of the inhibitor complex structure of human hematopoietic prostaglandin D synthase

Hematopoietic prostaglandin (PG) D synthase (H-PGDS) is responsible for the production of PGD(2) as an allergy or inflammation mediator in mast and Th2 cells. We determined the X-ray structure of human H-PGDS complexed with an inhibitor, 2-(2'-benzothiazolyl)-5-styryl-3-(4'-phthalhydrazidyl) tetrazolium chloride (BSPT) at 1.9 A resolution in the presence of Mg(2+). The styryl group of the inhibitor penetrated to the bottom of the active site cleft, and the tetrazole ring was stabilized by the stacking interaction with Trp104, inducing large movement around the alpha5-helix, which caused the space group of the complex crystal to change from P2(1) to P1 upon binding of BSPT. The phthalhydrazidyl group of BSPT exhibited steric hindrance due to the cofactor, glutathione (GSH), increasing the IC(50) value of BSPT for human H-PGDS from 36.2 micro M to 98.1 micro M upon binding of Mg(2+), because the K(m) value of GSH for human H-PGDS was decreased from 0.60 micro M in the presence of EDTA to 0.14 micro M in the presence of Mg(2+). We have to avoid steric hindrance of the GSH molecule that was stabilized by intracellular Mg(2+) in the mM range in the cytosol for further development of structure-based anti-allergic drugs.     

Identification and characterization of a novel component of the cornified envelope, cornifelin

Psoriasis is recognized as a chronic inflammatory disease characterized by epidermal hyperproliferation. To identify psoriasis-related genes, we compared the mRNA populations of normal and psoriatic skin. We identified one gene, designated as cornifelin, which showed increased expression in psoriatic skin. Human cornifelin contains 112 amino acids and is expressed in the uterus, cervix, and skin. In situ hybridization analysis demonstrated the presence of human cornifelin in the granular cell layer of the epidermis. To investigate the function of cornifelin, we established a transgenic mouse line overexpressing human cornifelin. Using these mice, we have shown that cornifelin is directly or indirectly cross-linked to at least two other cornified envelope proteins, loricrin and involucrin, in vivo. Overexpression of human cornifelin correlated with decreased loricrin expression and increased involucrin expression in the transgenic mouse. However, abnormality of epidermal differentiation was not observed in the transgenic mouse.     

Interaction of D-lactate dehydrogenase protein 2 (Dld2p) with F-actin: implication for an alternative function of Dld2p

D-Lactate dehydrogenase protein 2 [Yeast 15 (1999) 1377; Biochem. Biophys. Res. Commun. 295 (2002) 910] was initially identified as the actin interacting protein 2 (Aip2p) using a two-hybrid screen to search for proteins that interact with actin [Nat. Struct. Biol. 2 (1995) 28], but no other evidence indicating an interaction between Aip2p and actin cytoskeleton has been reported so far. During our search for the protein conformation modifying activity, we serendipitously identified Aip2p isolated from Saccharomyces cerevisiae as exhibiting an interaction with F-actin both in vitro and in vivo. Incubation with Aip2p facilitated the formation of the circular form of F-actin in vitro, which exhibited an aberrant trypsin susceptibility. Overexpression of Aip2p induced multi-buds in yeast cells, whereas reduced expression interfered with the formation of the cleavage furrow for the cell division, which was rescued by the introduction of wild-type Aip2p. While Aip2p-treated F-actin in the circular form was negligibly stained by rhodamine-labeled phalloidin (rhodamine-phalloidin) in vitro, rhodamine-phalloidin staining profiles in actin interacting protein 2 gene (AIP2)-modified cells suggested a correlation between the conformation of F-actin and the expression of Aip2p in vivo. AIP2-deleted cells became sensitive to osmotic conditions, a hallmark of actin dysfunction. Finally, immunoprecipitation of yeast cells using anti-Aip2p antibody demonstrated that Aip2p associates with actin. These properties suggest that Aip2p may interact with F-actin in vivo and play an important role in the yeast cell morphology.     

Quantitative Trait Locus Analysis of Plasma Cholesterol and Triglyceride Levels in C57BL/6J × RR F2 Mice

A highly significant cholesterol quantitative trait locus (QTL) (Cq6) was identified on chromosome 1 in C57BL/6J x RR F2 mice. The Cq6 was located over the gene for apolipoprotein A-Il (Apoa2), and the RR allele was associated with increased plasma cholesterol. C57BL/6J has Apoa2a alleles and RR has Apoa2b alleles. Three different Apoa2 alleles are known on the basis of amino acid substitutions at four residues. Analysis with partial Apoa2 congenic strains possessing Apoa2a, Apoa2b, and Apoa2C alleles revealed that the Apoa2b allele is unique in the ability to increase cholesterol among the three Apoa2 alleles, and that the Ala-to-Val substitution at residue 61 may be crucial as far as cholesterol metabolism is concerned. We also investigated the question of whether the Apoa1 gene is responsible for the cholesterol QTLs (Cq4 and Cq5) that had been identified previously on chromosome 9 in C57BL/6J x KK-Ay/a F2 and in KK x RR F2, but not in C57BL/6J x RR F2 mice. Similar to Apoa2 alleles, three different Apoal alleles with two successive amino acid substitutions were revealed among the strains. However, we could not correlate Apoal polymorphisms with the occurrence of QTLs in these three sets of F2 mice.     

Crystal structures of collagen model peptides with Pro-Hyp-Gly repeating sequence at 1.26 A resolution: implications for proline ring puckering

Triple-helical structures of (Pro-Hyp-Gly)n (n = 10, 11) at 100 K and room temperature (RT) were analyzed at 1.26 A resolution by using synchrotron radiation data. Totals of 49 and 42 water molecules per seven triplets in an asymmetric unit were found for the structures at 100 K and RT, respectively. These water molecules were classified into two groups, those in the first and second hydration shells. Although there was no significant difference between water molecules in the first shell at 100 K and those at RT, a significant difference between those in the second shell was observed. That is, the number of water molecules at RT decreased to one half and the average distance from peptide chains at RT became longer by about 0.3 A. On the other hand, of seven triplets in an asymmetric unit, three proline residues at the X position at 100 K clearly showed an up-puckering conformation, as opposed to the recent propensity-based hypothesis for the stabilization and destabilization of triple-helical structures by proline hydroxylation. This puckering was attributed to the interaction between proline rings and the surrounding water molecules at 100 K, which is much weaker at RT, as shown by longer average distance from peptide chains.     

The GTP-binding release factor eRF3 as a key mediator coupling translation termination to mRNA decay

GTP is essential for eukaryotic translation termination, where the release factor 3 (eRF3) complexed with eRF1 is involved as the guanine nucleotide-binding protein. In addition, eRF3 regulates the termination-coupled events, eRF3 interacts with poly(A)-binding protein (Pab1) and the surveillance factor Upf1 to mediate normal and nonsense-mediated mRNA decay. However, the roles of GTP binding to eRF3 in these processes remain largely unknown. Here, we showed in yeast that GTP is essentially required for the association of eRF3 with eRF1, but not with Pab1 and Upf1. A mutation in the GTP-binding motifs of eRF3 impairs the eRF1-binding ability without altering the Pab1- or Upf1-binding activity. Interestingly, the mutation causes not only a defect in translation termination but also delay of normal and nonsense-mediated mRNA decay, suggesting that GTP/eRF3-dependent termination exerts its influence on the subsequent mRNA degradation. The termination reaction itself is not sufficient, but eRF3 is essential for triggering mRNA decay. Thus, eRF3 is a key mediator that transduces termination signal to mRNA decay.