Sequence analysis of 193.4 and 83.9 kbp of mouse and chicken genomic DNAs containing the entire Prkdc (DNA-PKcs) gene

The catalytic subunit of DNA-dependent protein kinase plays critical roles in nonhomologous end joining in repair of DNA double-strand breaks and V(D)J recombination. In addition to the SCID phenotype, it has been suggested that the molecule contributes to the polymorphic variations in radiosensitivity and susceptibility to cancer in mouse strains. Here we show the nucleotide sequence of approximately 193-kbp and 84-kbp genomic regions encoding the entire Prkdc gene (also known as DNA-PKcs) in the mouse and chicken, respectively. A large retroposon was found in intron 51 in the mouse but not in the human or chicken. Comparative analyses of the genome strongly suggested that the region contains only two genes for Prkdc and Mcm4; however, several conserved sequences and cis elements were also predicted.     

Asymmetric coiled-coil structure with Guanine nucleotide exchange activity

Vesicular traffic during exocytosis is regulated by Rab GTPase, Sec4p in yeast, which is activated by a guanine nucleotide exchange factor (GEF) called Sec2p. The GEF activity is localized in the N-terminal 160 residues of Sec2p, which lacks sequence similarity with any other GEFs with known structures, and thereby the guanine nucleotide exchange mechanism by Sec2p remains unknown. Here, we report the crystal structure of the Sec2p GEF domain at 3.0 A resolution. The structure unexpectedly consists of a homodimeric, parallel coiled coil that extends over 180 A. Pull-down and guanine nucleotide exchange analyses on a series of deletion and point mutants of Sec2p unveiled the catalytic residues for its GEF activity as well as the Sec4p binding site, thus presenting a nucleotide exchange mechanism by a simple coiled coil. The present functional analyses allow us to build the Sec2p:Sec4p complex model, which explains the specificity for Rab GTPases by their respective GEF proteins.     

Vasculitis induced by immunization with Bacillus Calmette-Guérin followed by atypical mycobacterium antigen: a new mouse model for Kawasaki disease

Kawasaki disease causes systemic vasculitis. The development of skin lesions at the vaccination site with Bacillus Calmette-Guérin (BCG) is an important diagnostic symptom. We hypothesized that infection with ubiquitous microorganisms immunogenically related to BCG might induce an immunopathologic reaction leading to the development of Kawasaki disease. Mice were first inoculated with BCG, and then secondarily inoculated 4 weeks later with crude extract from Mycobacterium intracellulare (cMI), an abundant atypical mycobacterium. Animals inoculated with BCG followed by cMI developed coronary arteritis with infiltration of inflammatory cells, whereas control animals inoculated with only cMI or BCG did not, suggesting that the immune response to the mycobacteria induced autoimmunity to the vascular wall. Intravenous injection with antibodies to peroxiredoxin II, a modulator of vascular remodeling and a suggested target for autoimmune vasculitis, also resulted in coronary arteritis, but only after prior inoculation with BCG. Tumor necrosis factor-alpha, MCP1 and interferon-gamma production were significantly higher in the animals inoculated with BCG than in the control groups (P<0.05). BCG immunization was required for the development of coronary arteritis, suggesting that these cytokines might play important roles. The results indicate that BCG induces primary autoimmunity and stimulates cytokine induction, and that atypical mycobacterial infection boosts the autoimmunity resulting in coronary arteritis.     

Microscopic study on resorption of β-tricalcium phosphate materials by osteoclasts

Sintered compounds prepared with β-tricalcium phosphate (β-TCP) are commonly used as biocompatible materials for bone regenerative medicine. Although implanted β-TCP is gradually replaced with new bone after resorption by osteoclasts, exactly how osteoclasts resorb β-TCP is not well understood. To elucidate this mechanism, we analyzed the structure of β-TCP discs on which mouse mature osteoclasts were cultured using scanning electron microscopy. We found that β-TCP was resorbed by mature osteoclasts on one side of each disc, as evidenced by the formation of multiple spine-like crystals at the exposed areas. Because osteoclasts secrete acid to resorb bone minerals, we mimicked this acidification by dipping β-TCP slices into HCl solution (pH 2.0). However, no spine-like crystals appeared even though the size of each β-TCP particle was reduced. On dentin slices, osteoclasts formed clear actin rings, which are cytoskeletal structures characteristic of bone-resorbing osteoclasts. No clear actin rings were observed in osteoclasts cultured on β-TCP slices, although small actin dots were observed. Analysis by transmission electron microscopy showed that osteoclasts attached to β-TCP particles. These results suggest that osteoclasts resorb β-TCP particles independently of clear actin ring formation.     

In vitro protein import of a putative amino acid transporter from Arabidopsis thaliana into chloroplasts and its suborganellar localization

We identified a gene product of At5g19500 (At5g19500p) from Arabidopsis thaliana that is homologous to EcTyrP, a tyrosine-specific transporter from Escherichia coli. Computational analyses of the amino acid sequence of At5g19500p predicted 11 transmembrane domains (TMDs) and a potential plastid targeting signal at its amino terminus. As a first step toward understanding the possible role of At5g19500p in plant cells, we attempted to determine the localization of At5g19500p by an in vitro chloroplastic import assay using At5g19500p translated in a cell-free wheat germ system (Madin et al., Proc. Natl. Acad. Sci. USA, 97, 559-564 (2000)), followed by subfractionation of the chloroplasts. At5g19500p was successfully imported into chloroplasts, and the newly transported mature form of At5g19500p was recovered from the inner envelope membrane.     

Fused p47phox and p67phox truncations efficiently reconstitute NADPH oxidase with higher activity and stability than the individual components

Activation of the neutrophil NADPH oxidase occurs via assembly of the cytosolic regulatory proteins p47(phox), p67(phox), and Rac with the membrane-associated flavocytochrome b(558). Following cell-free activation, enzymatic activity is highly labile (Tamura, M., Takeshita, M., Curnutte, J. T., Uhlinger, D. J., and Lambeth, J. D. (1992) J. Biol. Chem. 267, 7529-7538). To try to stabilize the activity and investigate the nature of the complex, fusion proteins between p47N-(1-286) and p67N-(1-210) were constructed. In a cell-free system, a fusion protein, p67N-p47N, had an 8-fold higher efficiency and produced a higher activity than the individual proteins, and also resulted in an 8-fold improved efficiency for Rac and a lowered K(m) for NADPH. O(2) generating activity was remarkably stabilized by using p67N-p47N. The cytosolic proteins fused in the opposite orientation, p47N-p67N, showed similar activity and stability as individual proteins, but with a 4-fold improved efficiency compared with the individual cytosolic factors. In the system efficiency for Rac and affinity for NADPH were also higher than those with the nonfused components. Interestingly, the p67N-p47N showed nearly full activation in the absence of an anionic amphifile in a cell-free system containing cytochrome b(558) relipidated with phosphatidylinositol- or phosphatidylserine-enriched phospholipid mixtures. From the results we consider multiple roles of anionic amphifiles in a cell-free activation, which could be substituted by our system. The fact that a fusion produces a more stable complex indicates that interactions among components determine the longevity of the complex. Based on the findings we propose a model for the topology among p47N, p67N, and cytochrome b(558) in the active complex.     

Frequent retention of heterozygosity for point mutations in p53 and Ikaros in N-ethyl-N-nitrosourea-induced mouse thymic lymphomas

In agreement with Knudson's two-hit theory, recent findings indicate that the inactivation of tumor suppressor genes is not only mediated by the loss of function but also by the dominant-negative or gain-of-function activity. The former generally accompanies loss of a wild-type allele whereas in the latter a wild-type allele is retained. N-Ethyl-N-nitrosourea (ENU), which efficiently induces point mutations, reportedly leads to the development of tumors by activating ras oncogenes. Little is known about how ENU affects tumor suppressor genes and, therefore, we examined ENU-induced mutations of p53 and Ikaros in thymic lymphomas and compared these with mutations of Kras. In addition, loss of heterozygosity was examined for chromosome 11 to which both p53 and Ikaros were mapped. The frequency of point mutations in p53 and Ikaros was 30% (8/27) and 19% (5/27), respectively, comparable to that observed in Kras (33%: 9/27). In total, 14 of the 27 thymic lymphomas examined (52%) harbored mutations in at least one of these genes. One Ikaros mutation was located at the splice donor site, generating a novel splice isoform lacking zinc finger 3, Ik (F3del). Interestingly, 90% (10/11) of the tumors with point mutations retained wild-type alleles of p53 and Ikaros. Sequence analysis revealed that the most common nucleic acid substitutions were T>A (4/8) in p53, T>C (4/5) in Ikaros and G>A/T (8/9) in Kras, suggesting that the spectrum of mutations was gene dependent. These results suggest that point mutations in tumor suppressor genes without loss of the wild-type allele play an important role in ENU-induced lymphomagenesis.     

Specific inhibition of hepatitis C virus replication by cyclosporin A

The difficulty in eradicating hepatitis C virus (HCV) infection is attributable to the limited treatment options against the virus. Recently, cyclosporin A (CsA), a widely used immunosuppressive drug, has been reported to be effective against HCV infection [J. Gastroenterol. 38 (2003) 567], although little is understood about the mechanism of its action against HCV. In this study, we investigated the anti-viral effects of CsA using an HCV replicon system. Human hepatoma Huh7 cells were transfected with an HCV replicon expressing a chimeric gene encoding a luciferase reporter and neomycin phosphotransferase (Huh7/Rep-Feo). Treatment of the Huh7/Rep-Feo cells with CsA resulted in suppression of the replication of the HCV replicon in a dose-dependent manner, with an IC50 of approximately 0.5 microg/ml. There were no changes in the rate of cell growth or viability, suggesting that the effect of CsA against HCV is specific and not due to cytotoxicity. In contrast, FK506, another immunosuppressive drug, did not suppress HCV replication. CsA did not activate interferon-stimulated gene responses, suggesting that its action is independent of that of interferon. In conclusion, CsA inhibits HCV replication in vitro specifically at clinical concentrations. Further defining its mode of action against HCV replication potentially may be important for identifying novel molecular targets to treat HCV infection.     

The endothelin receptor antagonist ameliorates the hypertensive phenotypes of transgenic hypertensive mice with renin-angiotensin genes and discloses roles of organ specific activation of endothelin system in transgenic mice

Endothelin (ET)-1 and ET-2 are potent vasoconstrictor peptides with mitogenic activity. In this study, we investigated roles of ET system in renin-angiotensin system (RAS)-mediated hypertension, using transgenic hypertensive mice (THM) with over-expression of both human renin and angiotensinogen genes. In the first step, it was revealed that expression of ET system was locally enhanced, i.e. increases in cardiac preproET-1 mRNA and renal preproET-2 mRNA in THM, compared with the control (wild type) mice. In the next step, we studied the chronic effects of an ET antagonist (SB209670) on THM. Blood pressure (BP) in THM was significantly higher than that in the normal mice during the investigation. However, in the later phase of the study, from 12 to 20 weeks of treatment, THM receiving SB 209670 showed significantly lower BP than that in THM receiving saline. SB 209670 treatment for 20 weeks significantly attenuated phenotypes of cardiac hypertrophy, vascular wall thickening and hypertensive nephropathy observed in THM, suggesting that the ETA/B receptor antagonist is also effective even in the extraordinarily activated RAS condition. These findings suggest that organ specifically activated ET system in THM develops the phenotypes, hypertension, cardiac hypertrophy, and hypertensive nephropathy.     

The Q223R polymorphism in LEPR is associated with obesity in Pacific Islanders

Various Pacific Island populations have experienced a marked increase in the prevalence of obesity in past decades. This study examined the association of a promoter polymorphism of the leptin gene (LEP), G-2548A (rs7799039), and two non-synonymous single nucleotide polymorphisms of the leptin receptor gene (LEPR), K109R (rs1137100) and Q223R (rs1137101), with body weight, body mass index (BMI) and obesity (BMI ≥ 30) in Pacific Islanders. A total of 745 Austronesian (AN)-speaking participants were analyzed after adjusting for age, gender, and population differences. The results revealed that carriers of the 223Q alleles of LEPR had significantly higher body weight (P = 0.0009) and BMI (P = 0.0022) than non-carriers (i.e., 223R homozygotes); furthermore, the 223Q carriers also had a significantly higher risk of obesity in comparison to non-carriers (P = 0.0222). The other two polymorphisms, G-2548A and K109R, were associated with neither body weight, BMI, nor obesity. The 223Q allele was widely found among the AN-speaking study subjects, thus suggesting that the LEPR Q223R polymorphism is one of the factors contributing to the high prevalence of obesity in the Pacific Island populations.