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1.  Human Leukocyte Antigens and Systemic Lupus Erythematosus: A Protective Role for the HLA-DR6 Alleles DRB1*13:02 and *14:03 
PLoS ONE  2014;9(2):e87792.
Many studies on associations between human leukocyte antigen (HLA) allele frequencies and susceptibility to systemic lupus erythematosus (SLE) have been performed. However, few protective associations with HLA-DRB1 alleles have been reported. Here, we sought protective, as well as predispositional, alleles of HLA-DRB1 in Japanese SLE patients. An association study was conducted for HLA-DRB1 in Japanese SLE patients. Relative predispositional effects were analyzed by sequential elimination of carriers of each allele with the strongest association. We also explored the association of DRB1 alleles with SLE phenotypes including the presence of autoantibody and clinical manifestations. Significantly different carrier frequencies of certain DRB1 alleles were found to be associated with SLE as follows: increased DRB1*15:01 (P = 5.48×10−10, corrected P (Pc) = 1.59×10−8, odds ratio [OR] 2.17, 95% confidence interval [CI] 1.69–2.79), decreased DRB1*13:02 (P = 7.17×10−5, Pc = 0.0020, OR 0.46, 95% CI 0.34–0.63) and decreased DRB1*14:03 (P = 0.0010, Pc = 0.0272, OR 0.34, 95% CI 0.18–0.63). Additionally, the “*15:01/*13:02 or *14:03” genotype tended to be negatively associated with SLE (P = 0.4209, OR 0.66), despite there being significant positive associations with *15:01 when present together with alleles other than *13:02 or *14:03 (P = 1.79×10−11, OR 2.39, 95% CI 1.84–3.10). This protective effect of *13:02 and *14:03 was also confirmed in SLE patients with different clinical phenotypes. To the best of our knowledge, this is the first report of a protective association between the carrier frequencies of HLA-DRB1*13:02 and *14:03 and SLE in the Japanese population.
doi:10.1371/journal.pone.0087792
PMCID: PMC3912000  PMID: 24498373
2.  Purification, crystallization and X-ray diffraction study of extracellular dermal glycoprotein from carrot and the inhibition complex that it forms with an endo-β-glucanase from Aspergillus aculeatus  
Crystallization of extracellular dermal glycoprotein and the inhibition complex that it forms with endo-β-glucanase.
Extracellular dermal glycoprotein (EDGP) may play an important role in the plant defence system of the carrot (Daucus carota) as it has inhibitory activity against endo-β-glucanase produced by invading pathogens. Here, EDGP and the inhibition complex that it forms with FI-CMCase, a carboxyl methyl cellulase from Aspergillus aculeatus, were successfully crystallized. The hex­agonal crystal of EDGP belonged to space group P62, with unit-cell parameters a = b = 130.4, c = 44.5 Å, γ = 120°. The monoclinic crystal of the complex of EDGP with FI-CMCase belonged to space group C2, with unit-cell parameters a = 169.5, b = 143.0, c = 63.0 Å, β = 110.9°.
doi:10.1107/S1744309111020045
PMCID: PMC3144808  PMID: 21795806
extracellular dermal glycoproteins; Daucus carota; carboxyl methyl cellulases; Aspergillus aculeatus
3.  Purification, crystallization and X-ray diffraction study of basic 7S globulin from soybean 
Crystallization of basic 7S globulin from soybean is presented.
Basic 7S globulin (Bg7S) is expressed by soybeans in response to biotic or abiotic stress. Bg7S is capable of binding to a 4 kDa protein which is supposedly involved in cell proliferation. Bg7S is widely found not only in legumes, but also in other plants; however, its function is still unclear. Here, Bg7S was successfully crystallized. Orthorhombic and monoclinic crystals of Bg7S were obtained under different conditions and belonged to space groups P21212, with unit-cell parameters a = 111.9, b = 130.1, c = 287.8 Å, and P21, with unit-cell parameters a = 85.3, b = 137.6, c = 162.1 Å, β = 91.2°, respectively.
doi:10.1107/S1744309110046178
PMCID: PMC3079980  PMID: 21206032
basic 7S globulin; soybean
4.  Crystallization and X-ray diffraction studies of DNA-free and DNA-bound forms of EcoO109I DNA methyltransferase 
The crystallization and diffraction studies of EcoO109I DNA methyltransferase are described.
EcoO109I DNA methyltransferase (M.EcoO109I) is a type II modification enzyme from the EcoO109I restriction-modification system identified in Escherichia coli strain H709c. M.EcoO109I recognizes double-stranded RGGNCCY (where R = A or G, Y = T or C and N is any base) and transfers a methyl group to the C5 of the inner cytosines from S-adenosylmethionine. To reveal the mechanism of substrate recognition by M.EcoO109I, DNA-free and DNA-bound forms of M.EcoO109I were successfully crystallized. Crystals of the DNA-free and DNA-bound forms belonged to space groups P42212, with unit-cell parameters a = b = 120.5, c = 79.8 Å, and P21, with unit-cell parameters a = 55.8, b = 77.4, c = 117.4 Å, β = 93.5°, respectively.
doi:10.1107/S174430911003753X
PMCID: PMC3001666  PMID: 21045313
DNA methytransferases; restriction-modification systems; DNA complexes
5.  The development of N-α-(2-carboxyl)benzoyl-N5-(2-fluoro-1-iminoethyl)-L-ornithine amide (o-F-amidine) and N α-(2-carboxyl)benzoyl-N5-(2-Chloro-1-iminoethyl)-L-ornithine amide (o-Cl-amidine) as second generation Protein Arginine Deiminase (PAD) inhibitors.† 
Journal of medicinal chemistry  2011;54(19):6919-6935.
Protein Arginine Deiminase (PAD) activity is upregulated in a number of human diseases, including rheumatoid arthritis, ulcerative colitis, and cancer. These enzymes, there are five in humans (PADs 1-4 and 6) regulate gene transcription, cellular differentiation, and the innate immune response. Building on our successful generation F- and Cl-amidine, which irreversibly inhibit all of the PADs, a structure activity relationship was performed to develop second generation compounds with improved potency and selectivity. Incorporation of a carboxylate ortho to the backbone amide resulted in the identification of N-α-(2-carboxyl)benzoyl-N5-(2-fluoro-1-iminoethyl)-L-ornithine amide (o-F-amidine) and Nα-(2-carboxyl)benzoyl-N5-(2-chloro-1-iminoethyl)-L-ornithine amide (o-Cl-amidine), as PAD inactivators with improved potency (up to 65-fold) and selectivity (up to 25-fold). Relative to F- and Cl-amidine, the compounds also show enhanced potency in cellulo. As such, these compounds will be versatile chemical probes of PAD function.
doi:10.1021/jm2008985
PMCID: PMC3196593  PMID: 21882827
6.  Expression, purification and crystallization of Swi5 and the Swi5–Sfr1 complex from fission yeast 
The crystallization of the homologous recombination mediators Swi5 and Sfr1 from fission yeast is reported.
The assembly of the presynaptic filament of recombinases represents the most important step in homologous recombination. The formation of the filament requires assistance from mediator proteins. Swi5 and Sfr1 have been identified as mediators in fission yeast and these proteins form a complex that stimulates strand exchange. Here, the expression, purification and crystallization of Swi5 and its complex with an N-terminally truncated form of Sfr1 (ΔN180Sfr1) are presented. Analytical ultracentrifugation of the purified samples showed that Swi5 and the protein complex exist as tetramers and heterodimers in solution, respectively. Swi5 was crystallized in two forms belonging to space groups C2 and R3 and the crystals diffracted to 2.7 Å resolution. Swi5–ΔN180Sfr1 was crystallized in space group P21212 and the crystals diffracted to 2.3 Å resolution. The crystals of Swi5 and Swi5–ΔN180Sfr1 are likely to contain one tetramer and two heterodimers in the asymmetric unit, respectively.
doi:10.1107/S1744309110032239
PMCID: PMC2935244  PMID: 20823543
homologous recombination mediators; Swi5; Sfr1; fission yeast
7.  En bloc transfer of polyubiquitin chains to PCNA in vitro is mediated by two different human E2–E3 pairs  
Nucleic Acids Research  2012;40(20):10394-10407.
Post-replication DNA repair in eukaryotes is regulated by ubiquitination of proliferating cell nuclear antigen (PCNA). Monoubiquitination catalyzed by RAD6–RAD18 (an E2–E3 complex) stimulates translesion DNA synthesis, whereas polyubiquitination, promoted by additional factors such as MMS2–UBC13 (a UEV–E2 complex) and HLTF (an E3 ligase), leads to template switching in humans. Here, using an in vitro ubiquitination reaction system reconstituted with purified human proteins, we demonstrated that PCNA is polyubiquitinated predominantly via en bloc transfer of a pre-formed ubiquitin (Ub) chain rather than by extension of the Ub chain on monoubiquitinated PCNA. Our results support a model in which HLTF forms a thiol-linked Ub chain on UBC13 (UBC13∼Ubn) and then transfers the chain to RAD6∼Ub, forming RAD6∼Ubn+1. The resultant Ub chain is subsequently transferred to PCNA by RAD18. Thus, template switching may be promoted under certain circumstances in which both RAD18 and HLTF are coordinately recruited to sites of stalled replication.
doi:10.1093/nar/gks763
PMCID: PMC3488225  PMID: 22904075
8.  Major risk factors for the appearance of white-matter lesions on MRI in hypertensive patients with controlled blood pressure 
Purpose
Blood pressure (BP), age, and reduced renal function are major risk factors for white-matter lesions (WMLs) in the general population. However, it remains unclear whether or not the BP itself or other parameters related to the BP are associated with WMLs in hypertensive patients with well-controlled BP. We investigated the relationships of the presence of WMLs with the central systolic BP (cSBP) and estimated glomerular filtration rate (eGFR) in treated hypertensive patients.
Method
We studied 185 hypertensive patients with median duration of hypertension, 10.0 years, whose BP is controlled to SBP and diastolic BP (DBP) of 139 ± 17 and 79 ± 10 mmHg, respectively. We measured cSBP and brain magnetic resonance imaging (MRI) was examined within 2 weeks after last BP and biological measurements.
Results
Patients with higher-grade WMLs, as assessed by the presence of Scheltens deep white-matter hyperintensity (SDWMH) in the frontal (grade 0–2 vs 3–6) and parietal areas (grade 0–2 vs 3–6) where small arteries are affected at earlier stage of hypertension, as well as that of Fazekas deep white-matter hyperintensity (FDWMH) (grade 2–3 vs 0–1) and Fazekas periventricular hyperintensity (FPVH) (grade 1–3 vs 0) were older, had higher serum creatinine levels, a longer duration of hypertension, and lower eGFR values. The grade of the WMLs was not associated with either the cSBP or the brachial SBP. In logistic regression analyses after adjustment for age, sex, cSBP, and hypertension duration, showed significant association between eGFR and WMLs. The patients with lower eGFR (<60 mL/minute/1.73 m2) tended to have higher grade WMLs. The odds ratio was 2.87 for FDWMH (P = 0.017), 1.99 for FPVH (P = 0.131), and 2.33 for SDWMH in the parietal area (P = 0.045).
Conclusion
Presence of WMLs was associated with eGFR, but not with either the brachial SBP or cSBP in hypertensive patients with well-controlled BP.
doi:10.2147/VHRM.S30507
PMCID: PMC3310357  PMID: 22454561
white-matter lesions; central systolic blood pressure; estimated glomerular filtration rate
9.  Purification, crystallization and initial X-ray diffraction study of human REV7 in complex with a REV3 fragment 
The crystallization and initial X-ray diffraction of REV7 in complex with REV3 is reported.
REV7 is involved in various cellular functions including DNA replication, signal transduction and cell-cycle regulation. In DNA replication, REV7 interacts with REV3 and forms DNA polymerase ζ, which plays a central role in error-prone DNA synthesis. REV3 is a catalytic subunit and its activity is stimulated by REV7. To clarify the structural basis of the interaction between REV7 and REV3, human REV7 was crystallized in complex with a REV3 fragment. Two crystal forms were obtained. Crystal forms I and II belonged to space groups P21, with unit-cell parameters a = 43.8, b = 50.0, c = 107.3 Å, β = 96.9°, and P41212 or P43212, with unit-cell parameters a = b = 76.6, c = 118.4 Å, respectively.
doi:10.1107/S1744309109046181
PMCID: PMC2802887  PMID: 20054135
REV7; REV3; DNA polymerases
10.  Fission Yeast Swi5-Sfr1 Protein Complex, an Activator of Rad51 Recombinase, Forms an Extremely Elongated Dogleg-shaped Structure* 
The Journal of Biological Chemistry  2011;286(50):43569-43576.
Background: The Swi5-Sfr1 protein complex is an activator of Rad51 recombinase, which mediates DNA strand exchange in homologous recombination.
Results: Swi5 and Sfr1 form a 1:1 complex, which exhibits an extremely elongated dogleg-shaped structure in solution.
Conclusion: The Swi5-Sfr1 structure is suitable for binding within the helical groove of the Rad51 filament.
Significance: A structural model will advance our understanding of the molecular mechanism of homologous recombination.
In eukaryotes, DNA strand exchange is the central reaction of homologous recombination, which is promoted by Rad51 recombinases forming a right-handed nucleoprotein filament on single-stranded DNA, also known as a presynaptic filament. Accessory proteins known as recombination mediators are required for the formation of the active presynaptic filament. One such mediator in the fission yeast Schizosaccharomyces pombe is the Swi5-Sfr1 complex, which has been identified as an activator of Rad51 that assists in presynaptic filament formation and stimulates its strand exchange reaction. Here, we determined the 1:1 binding stoichiometry between the two subunits of the Swi5-Sfr1 complex using analytical ultracentrifugation and electrospray ionization mass spectrometry. Small-angle x-ray scattering experiments revealed that the Swi5-Sfr1 complex displays an extremely elongated dogleg-shaped structure in solution, which is consistent with its exceptionally high frictional ratio (f/f0) of 2.0 ± 0.2 obtained by analytical ultracentrifugation. Furthermore, we determined a rough topology of the complex by comparing the small-angle x-ray scattering-based structures of the Swi5-Sfr1 complex and four Swi5-Sfr1-Fab complexes, in which the Fab fragments of monoclonal antibodies were specifically bound to experimentally determined sites of Sfr1. We propose a model for how the Swi5-Sfr1 complex binds to the Rad51 filament, in which the Swi5-Sfr1 complex fits into the groove of the Rad51 filament, leading to an active and stable presynaptic filament.
doi:10.1074/jbc.M111.303339
PMCID: PMC3234860  PMID: 22033972
DNA Repair; Homologous Recombination; Mass Spectrometry (MS); Ultracentrifugation; X-ray Scattering; Yeast; Swi5-Sfr1; Fission Yeast
11.  Purification, crystallization and initial X-ray diffraction study of the zinc-finger domain of zebrafish Nanos 
Crystallization of Nanos.
Nanos is a highly conserved RNA-binding protein in higher eukaryotes and acts as a key regulator protein involved in translational control utilizing the 3′ untranslated region of mRNA. The C-terminal domain of Nanos has two conserved and novel CCHC-type zinc-finger motifs that are responsible for the function of Nanos. To clarify the structural basis of the function of Nanos, the C-­terminal domain (residues 59–159) of zebrafish Nanos was overexpressed, purified and crystallized. The crystal belonged to space group P63, with unit-cell parameters a = b = 100.9, c = 71.5 Å, γ = 120°. Structure determination by the MAD/SAD method is now in progress.
doi:10.1107/S1744309109032163
PMCID: PMC2795612  PMID: 19724144
Nanos; zinc-finger domains; zebrafish
12.  A marked increase in gastric fluid volume during cardiopulmonary bypass 
Major physiological stress occurs during cardiac surgery with cardiopulmonary bypass. This is related to hypothermia and artificial organ perfusion. Thus, serious gastrointestinal complications, particularly upper gastrointestinal bleeding, sometimes follow cardiac surgery. We have compared the antisecretory effects of a preanesthetic H2 antagonist (roxatidine, cardiopulmonary bypass-H2 group, n = 15) and a proton pump inhibitor (rabeprazole, cardiopulmonary bypass-PPI group, n = 15) in patients undergoing cardiac surgery with cardiopulmonary bypass, and also compared in patients undergoing a off-pump coronary artery bypass graft surgery (off-pump cardiopulmonary bypass-H2 group, n = 15). Gastric pH (5.14 ± 0.61) and gastric fluid volume (13.2 ± 2.4 mL) at the end of surgery in off-pump cardiopulmonary bypass-H2 groups was significantly lower and higher than those in both cardiopulmonary bypass-H2 (6.25 ± 0.54, 51.3 ± 8.0 mL) and cardiopulmonary bypass-PPI (7.29 ± 0.13, 63.5 ± 14.8 mL) groups, respectively although those variables did not differ between groups after the induction of anesthesia. Plasma gastrin (142 ± 7 pg/mL) at the end of surgery and maximal blood lactate levels (1.50 ± 0.61 mM) in off-pump cardiopulmonary bypass-H2 group were also significantly lower than those in both cardiopulmonary bypass-H2 (455 ± 96 pg/mL, 3.97 ± 0.80 mM) and cardiopulmonary bypass-PPI (525 ± 27 pg/mL, 3.15 ± 0.44 mM) groups, respectively. In addition, there was a significant correlation between gastric fluid volume and maximal blood lactate (r = 0.596). In conclusion, cardiopulmonary bypass may cause an increase in gastric fluid volume which neither H2 antagonist nor PPI suppresses. A significant correlation between gastric fluid volume and maximal blood lactate suggests that gastric fluid volume may predict degree of gastrointestinal tract hypoperfusion.
doi:10.3164/jcbn.10-101
PMCID: PMC3128360  PMID: 21765601
cardiopulmonary bypass; gastrointestinal ischemia; gastric acidity; H2 antagonists; proton pump inhibitors
13.  Identification of a novel REV1-interacting motif necessary for DNA polymerase κ function 
When a replicative DNA polymerase (Pol) is stalled by damaged DNA, a 'polymerase switch' recruits specialized translesion synthesis (TLS) DNA polymerase(s) to sites of damage. Mammalian cells have several TLS DNA polymerases, including the four Y-family enzymes (Polη, Polι, Polκ and REV1) that share multiple primary sequence motifs, but show preferential bypass of different DNA lesions. REV1 interacts with Polη, Polι, and Polκ and therefore appears to play a central role during TLS in vivo. Here we have investigated the molecular basis for interactions between REV1 and Polκ. We have identified novel REV1-interacting regions (RIRs) present in Polκ, Polι and Polη. Within the RIRs, the presence of two consecutive phenylalanines (FF) is essential for REV1-binding. PCNA-binding motifs found in many proteins also contain FF with some conserved residues N-terminal to FF, as frequently represented by Q-x-x-(I,L,M)-x-x-F-F (x, any residue). In contrast, our results show that the critical FF in RIR motifs are not flanked by specific conserved residues. Instead, the consensus sequence for REV1-binding is denoted by x-F-F-y-y-y-y (y, any residue but not proline). Our results identify structural requirements that are necessary for FF-flanking residues to confer interactions with REV1. A Polκ mutant lacking REV1-binding activity did not complement the genotoxin-sensitivity of Polk-null mouse embryonic fibroblast cells, thereby demonstrating that the REV1-interaction is essential for Polκ function in vivo.
doi:10.1111/j.1365-2443.2008.01255.x
PMCID: PMC3103050  PMID: 19170759
14.  TLR7 single-nucleotide polymorphisms in the 3' untranslated region and intron 2 independently contribute to systemic lupus erythematosus in Japanese women: a case-control association study 
Introduction
The Toll-like receptor 7 (TLR7) gene, encoded on human chromosome Xp22.3, is crucial for type I interferon production. A recent multicenter study in East Asian populations, comprising Chinese, Korean and Japanese participants, identified an association of a TLR7 single-nucleotide polymorphism (SNP) located in the 3' untranslated region (3' UTR), rs3853839, with systemic lupus erythematosus (SLE), especially in males, although some difference was observed among the tested populations. To test whether additional polymorphisms contribute to SLE in Japanese, we systematically analyzed the association of TLR7 with SLE in a Japanese female population.
Methods
A case-control association study was conducted on eight tag SNPs in the TLR7 region, including rs3853839, in 344 Japanese females with SLE and 274 healthy female controls.
Results
In addition to rs3853839, two SNPs in intron 2, rs179019 and rs179010, which were in moderate linkage disequilibrium with each other (r2 = 0.53), showed an association with SLE (rs179019: P = 0.016, odds ratio (OR) 2.02, 95% confidence interval (95% CI) 1.15 to 3.54; rs179010: P = 0.018, OR 1.75, 95% CI 1.10 to 2.80 (both under the recessive model)). Conditional logistic regression analysis revealed that the association of the intronic SNPs and the 3' UTR SNP remained significant after we adjusted them for each other. When only the patients and controls carrying the risk genotypes at the 3' UTR SNPpositionwere analyzed, the risk of SLE was significantly increased when the individuals also carried the risk genotypes at both of the intronic SNPs (P = 0.0043, OR 2.45, 95% CI 1.31 to 4.60). Furthermore, the haplotype containing the intronic risk alleles in addition to the 3' UTR risk allele was associated with SLE under the recessive model (P = 0.016, OR 2.37, 95% CI 1.17 to 4.80), but other haplotypes were not associated with SLE.
Conclusions
The TLR7 intronic SNPs rs179019 and rs179010 are associated with SLE independently of the 3' UTR SNP rs3853839 in Japanese women. Our findings support a role of TLR7 in predisposition for SLE in Asian populations.
doi:10.1186/ar3277
PMCID: PMC3132023  PMID: 21396113
15.  Initial crystallographic study of human PCNA in complex with a peptide containing the noncanonical PIP-box sequence of human DNA polymerase ι 
Crystallization and diffraction studies of a human PCNA–Polι peptide complex are reported.
Human DNA polymerase ι (Polι) is one of the Y-family DNA polymerases involved in translesion synthesis (TLS), which allows continued replication at damaged DNA templates. Polι has a noncanonical PCNA-interacting protein box (PIP-box) within an internal region of the protein. Polι activity is stimulated by PCNA binding through the noncanonical PIP-box. To clarify the interaction of PCNA with the noncanonical PIP-box of Polι, PCNA and a Polι peptide carrying the noncanonical PIP-box complex have been cocrystallized. The crystal belongs to space group C2, with unit-cell parameters a = 167.1, b = 68.7, c = 90.0 Å, β = 95.1°. Structural analysis by molecular replacement is in progress.
doi:10.1107/S1744309108028522
PMCID: PMC2564886  PMID: 18931444
PCNA; DNA replication; translesion DNA synthesis; DNA polymerase ι
16.  Association of TNFAIP3 interacting protein 1, TNIP1 with systemic lupus erythematosus in a Japanese population: a case-control association study 
Arthritis Research & Therapy  2010;12(5):R174.
Introduction
TNFAIP3 interacting protein 1, TNIP1 (ABIN-1) is involved in inhibition of nuclear factor-κB (NF-κB) activation by interacting with TNF alpha-induced protein 3, A20 (TNFAIP3), an established susceptibility gene to systemic lupus erythematosus (SLE) and rheumatoid arthritis (RA). Recent genome-wide association studies revealed association of TNIP1 with SLE in the Caucasian and Chinese populations. In this study, we investigated whether the association of TNIP1 with SLE was replicated in a Japanese population. In addition, association of TNIP1 with RA was also examined.
Methods
A case-control association study was conducted on the TNIP1 single nucleotide polymorphism (SNP) rs7708392 in 364 Japanese SLE patients, 553 RA patients and 513 healthy controls.
Results
Association of TNIP1 rs7708392C was replicated in Japanese SLE (allele frequency in SLE: 76.5%, control: 69.9%, P = 0.0022, odds ratio [OR] 1.40, 95% confidence interval [CI] 1.13-1.74). Notably, the risk allele frequency in the healthy controls was considerably greater in Japanese (69.9%) than in Caucasians (24.3%). A tendency of stronger association was observed in the SLE patients with renal disorder (P = 0.00065, OR 1.60 [95%CI 1.22-2.10]) than in all SLE patients (P = 0.0022, OR 1.40 [95%CI 1.13-1.74]). Significant association with RA was not observed, regardless of the carriage of human leukocyte antigen DR β1 (HLA-DRB1) shared epitope. Significant gene-gene interaction between TNIP1 and TNFAIP3 was detected neither in SLE nor RA.
Conclusions
Association of TNIP1 with SLE was confirmed in a Japanese population. TNIP1 is a shared SLE susceptibility gene in the Caucasian and Asian populations, but the genetic contribution appeared to be greater in the Japanese and Chinese populations because of the higher risk allele frequency. Taken together with the association of TNFAIP3, these observations underscore the crucial role of NF-κB regulation in the pathogenesis of SLE.
doi:10.1186/ar3134
PMCID: PMC2991001  PMID: 20849588
17.  High-resolution fluorescence microscopy based on a cyclic sequential multiphoton process 
Biomedical Optics Express  2010;1(3):791-797.
We demonstrate high-resolution fluorescence microscopy based on a cyclic sequential multiphoton (CSM) process, which gives rise to fluorescence emission following a sequence of cyclic transitions between the bright and dark states of a fluorophore induced by pump and reverse light. By temporally modulating the reverse intensity, we can extract the fluorescence signal generated through the CSM process. We show that the demodulated fluorescence signal is nonlinearly proportional to the excitation intensities and it gives a higher spatial resolution than that of a confocal microscope.
doi:10.1364/BOE.1.000791
PMCID: PMC3018047  PMID: 21258510
(180.2520) Fluorescence microscopy; (180.6900) Three-dimensional microscopy; (120.1880) Detection
18.  Crystallographic study of G178S mutant of human proliferating cell nuclear antigen 
Crystallization and diffraction studies of human PCNA G178S mutant are reported.
Proliferating cell nuclear antigen (PCNA) is an evolutionarily conserved protein that forms a ring-shaped homotrimer that functions as a sliding clamp for DNA replication. The rev6-1 mutation of Saccharomyces cerevisiae, which inactivates both translesion DNA synthesis and damage-avoidance pathways while having little effect on normal cell growth, has a G178S substitution in the PCNA protein. Human PCNA protein carrying the G178S substitution was crystallized. Two crystal forms were obtained under similar conditions. Crystal forms I and II belong to space groups P21, with unit-cell parameters a = 84.1, b = 130.2, c = 97.8 Å, β = 113.4°, and P212121, with unit-cell parameters a = 68.1, b = 100.2, c = 131.2 Å, respectively. Structural analyses by molecular replacement are now in progress.
doi:10.1107/S174430910802277X
PMCID: PMC2531271  PMID: 18765913
PCNA; DNA replication; DNA damage tolerance; translesion DNA synthesis
19.  Crystallographic characterization of the N-terminal domain of a plant NADPH oxidase 
A crystal of the N-terminal domain of a plant NADPH oxidase was obtained and X-ray diffraction data were collected on a synchrotron beamline to a maximum resolution of 2.4 Å.
Respiratory burst oxidase homologue (Rboh), which is found in the plasma membrane, is a generator of reactive oxygen species (ROS) in plants. Many studies have indicated that the ROS produced by Rboh play critical roles in various cellular activities, including plant defence against pathogens. Crystals of the N-­terminal domain of Oryza sativa RbohB (OsRbohB) have been obtained. The crystals belonged to space group P212121, with unit-cell parameters a = 60.4, b = 72.2, c = 118.9 Å. An intensity data set was collected to 2.4 Å resolution.
doi:10.1107/S1744309108026535
PMCID: PMC2531284  PMID: 18765925
NADPH oxidases; EF-hand motifs; Rac small GTPase
20.  Measurement of two-photon excitation spectrum used to photoconvert a fluorescent protein (Kaede) by nonlinear Fourier-transform spectroscopy 
Biomedical Optics Express  2010;1(2):687-693.
We demonstrate the measurement of two-photon excitation (TPE) spectra, used not only for fluorescence but also for photoconversion in green-to-red photoconvertible Kaede, using nonlinear Fourier-transform spectroscopy. It was found that in unphotoconverted Kaede, the TPE spectrum for photoconversion is much different to that for green-fluorescence. This is similar to the difference between the one-photon excitation of photoconversion in the neutral form and that of green-fluorescence in the ionized form.
doi:10.1364/BOE.1.000687
PMCID: PMC3017996  PMID: 21258500
(300.6420) Spectroscopy, nonlinear; (170.2520) Fluorescence microscopy
21.  Microscopic Polyangiitis in Systemic Sclerosis 
AAV in SSc is described from the point of view of MPA. Some of reported SSc cases with AAV are thought to exhibit the characteristic clinical manifestations of MPA, although ANCA positivity in SSc is uncommon. MPA is clinically characterized by a multisystemic disease such as RPGN, pulmonary hemorrhage, mononeuritis, and skin involvement, as well as other manifestations in conjunction with high levels of inflammatory activity such as high ESR or CRP. It is also characterized by a high frequency of MPO-ANCA, showing predominant pANCA by IIF. When rapid renal failure or RPGN with active urine sediments, pulmonary hemorrhage and/or systemic inflammatory manifestations are observed in patients with SSc having positive ANCA, the possibility of MPA should always be considered. If SSc patients with MPA have life-threatening visceral involvement such as the above clinical manifestations, the patients should be treated with induction therapy using cyclophosphamide, methotrexate, corticosteroids, or plasmapheresis, etc. according to the severity of the disease soon after the diagnosis of MPA. It is important not to overlook characteristic clinical manifestations of AAV during the course of the disease in SSc in order to diagnose MPA early.
doi:10.1155/2010/148528
PMCID: PMC2958497  PMID: 20981310
22.  Association of TNFAIP3 Polymorphism with Susceptibility to Systemic Lupus Erythematosus in a Japanese Population 
Recent genome-wide association studies demonstrated association of single nucleotide polymorphisms (SNPs) in the TNFAIP3 region at 6q23 with systemic lupus erythematosus (SLE) in European-American populations. In this study, we investigated whether SNPs in the TNFAIP3 region are associated with SLE also in a Japanese population. A case-control association study was performed on the SNPs rs13192841, rs2230926, and rs6922466 in 318 Japanese SLE patients and 444 healthy controls. Association of rs2230926 G allele with SLE was replicated in Japanese (allelic association P = .033, odds ratio [OR] 1.47, recessive model P = .023, OR 8.52). The association was preferentially observed in the SLE patients with nephritis. When the TNFAIP3 mRNA levels of the HapMap samples were examined using GENEVAR database, the presence of TNFAIP3 rs2230926 G allele was associated with lower mRNA expression of TNFAIP3 (P = .013). These results indicated that TNFAIP3 is a susceptibility gene to SLE both in the Caucasian and Asian populations.
doi:10.1155/2010/207578
PMCID: PMC2896654  PMID: 20617138
23.  Expression of mitochondrial transcription factor A in endometrial carcinomas: clinicopathologic correlations and prognostic significance 
Virchows Archiv  2010;456(4):387-393.
Mitochondrial transcription factor A (mtTFA) is necessary for both transcription and maintenance of mitochondrial DNA. This study was conducted to elucidate the clinicopathologic and prognostic significance of mtTFA in patients with endometrial carcinoma. This study investigated the relationship between the immunohistochemical expression of mtTFA and various clinicopathological variables in 276 endometrial carcinomas, including 245 endometrioid adenocarcinomas and 31 nonendometrioid carcinomas (21 serous carcinomas and 10 clear cell adenocarcinomas). Both uni- and multivariate regression analyses were performed. The mtTFA labeling index of endometrioid adenocarcinomas ranged from 0% to 98%, with a median value of 32%, which was selected as the cut-off point for mtTFA expression. The mtTFA expression in endometrioid adenocarcinomas was significantly associated with the surgical stage, myometrial invasion, lymphovascular space invasion, cervical invasion, and lymph node metastasis. In contrast, no correlation between clinicopathologic variables and mtTFA expression was found in nonendometrioid carcinomas. Correlation analysis between mtTFA and p53 expression by using the Pearson test showed significant correlation in endometrioid adenocarcinomas (P = 0.007), but no significant correlation in nonendometrioid carcinomas (P = 0.947). A univariate survival analysis showed that the 10-year overall survival rate of the patients with mtTFA-positive endometrioid adenocarcinoma was significantly worse than that of patients with mtTFA-negative endometrioid adenocarcinoma (80.8% vs. 93.8%, P = 0.012). However, the multivariate analysis revealed that mtTFA expression in endometrioid adenocarcinomas was no independent prognostic factor. The positive mtTFA expression is a useful maker for progression of the tumors and the poor prognosis of the patients in endometrioid adenocarcinomas.
doi:10.1007/s00428-010-0895-7
PMCID: PMC2852529  PMID: 20232213
Endometrial cancer; Mitochondria; Mitochondrial transcription factor A; p53; Immunohistochemistry
24.  Role of STAT4 polymorphisms in systemic lupus erythematosus in a Japanese population: a case-control association study of the STAT1-STAT4 region 
Arthritis Research & Therapy  2008;10(5):R113.
Introduction
Recent studies identified STAT4 (signal transducers and activators of transcription-4) as a susceptibility gene for systemic lupus erythematosus (SLE). STAT1 is encoded adjacently to STAT4 on 2q32.2-q32.3, upregulated in peripheral blood mononuclear cells from SLE patients, and functionally relevant to SLE. This study was conducted to test whether STAT4 is associated with SLE in a Japanese population also, to identify the risk haplotype, and to examine the potential genetic contribution of STAT1. To accomplish these aims, we carried out a comprehensive association analysis of 52 tag single nucleotide polymorphisms (SNPs) encompassing the STAT1-STAT4 region.
Methods
In the first screening, 52 tag SNPs were selected based on HapMap Phase II JPT (Japanese in Tokyo, Japan) data, and case-control association analysis was carried out on 105 Japanese female patients with SLE and 102 female controls. For associated SNPs, additional cases and controls were genotyped and association was analyzed using 308 SLE patients and 306 controls. Estimation of haplotype frequencies and an association study using the permutation test were performed with Haploview version 4.0 software. Population attributable risk percentage was estimated to compare the epidemiological significance of the risk genotype among populations.
Results
In the first screening, rs7574865, rs11889341, and rs10168266 in STAT4 were most significantly associated (P < 0.01). Significant association was not observed for STAT1. Subsequent association studies of the three SNPs using 308 SLE patients and 306 controls confirmed a strong association of the rs7574865T allele (SLE patients: 46.3%, controls: 33.5%, P = 4.9 × 10-6, odds ratio 1.71) as well as TTT haplotype (rs10168266/rs11889341/rs7574865) (P = 1.5 × 10-6). The association was stronger in subgroups of SLE with nephritis and anti-double-stranded DNA antibodies. Population attributable risk percentage was estimated to be higher in the Japanese population (40.2%) than in Americans of European descent (19.5%).
Conclusions
The same STAT4 risk allele is associated with SLE in Caucasian and Japanese populations. Evidence for a role of STAT1 in genetic susceptibility to SLE was not detected. The contribution of STAT4 for the genetic background of SLE may be greater in the Japanese population than in Americans of European descent.
doi:10.1186/ar2516
PMCID: PMC2592800  PMID: 18803832
25.  Expression Profiling of PBMC-based Diagnostic Gene Markers Isolated from Vasculitis Patients 
Vasculitis (angiitis) is a systemic autoimmune disease that often causes fatal symptoms. We aimed to isolate cDNA markers that would be useful for diagnosing not only vasculitis but also other autoimmune diseases. For this purpose, we used stepwise subtractive hybridization and cDNA microarray analyses to comprehensively isolate the genes whose expressions are augmented in peripheral blood mononuclear cells (PBMCs) pooled from vasculitis patients. Subsequently, we used quantitative real-time polymerase chain reaction (qRT–PCR) to examine the mRNA levels of each candidate gene in individual patients. These analyses indicated that seven genes exhibit remarkably augmented expression in many vasculitis patients. Of these genes, we analyzed G0/G1 switch gene 2 (G0S2) further because G0S2 expression is also enhanced in the PBMCs of patients with systemic lupus erythematodes (SLE). We generated G0S2 transgenic mice that ubiquitously overexpress human G0S2. Although we did not observe any obvious vasculitis-related histopathologic findings in these mice, these mice are unhealthy as they produce only few offspring and showed elevated serum levels of two autoimmunity-related antibodies, anti-nuclear antibody, and anti-double strand DNA antibody. Thus, our large-scale gene profiling study may help finding sensitive and specific DNA markers for diagnosing autoimmune diseases including vasculitis and SLE.
doi:10.1093/dnares/dsn014
PMCID: PMC2575881  PMID: 18562305
vasculitis; angiitis; G0S2; EGR1; amphiregulin; hemoglobin delta

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