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1.  Purification, crystallization and preliminary X-ray analysis of uracil-DNA glycosylase from Sulfolobus tokodaii strain 7 
Uracil-DNA glycosylase from S. tokodaii strain 7 was overexpressed and purified. Crystals of the apo form and of a complex with uracil were obtained.
Uracil-DNA glycosylase (UDG) specifically removes uracil from DNA by catalyzing hydrolysis of the N-glycosidic bond, thereby initiating the base-excision repair pathway. Although a number of UDG structures have been determined, the structure of archaeal UDG remains unknown. In this study, a deletion mutant of UDG isolated from Sulfolobus tokodaii strain 7 (stoUDGΔ) and stoUDGΔ complexed with uracil were crystallized and analyzed by X-ray crystallography. The crystals were found to belong to the orthorhombic space group P212121, with unit-cell parameters a = 52.2, b = 52.3, c = 74.7 Å and a = 52.1, b = 52.2, c = 74.1 Å for apo stoUDGΔ and stoUDGΔ complexed with uracil, respectively.
doi:10.1107/S1744309112030278
PMCID: PMC3433208  PMID: 22949205
family 4 uracil-DNA glycosylases; Sulfolobus tokodaii strain 7
2.  Nutritional Effect of Oral Supplement Enriched in ω-3 Fatty Acids, Arginine, RNA on Immune Response and Leukocyte–platelet Aggregate Formation in Patients Undergoing Cardiac Surgery 
The aim of the present study was to investigate the influence of a supplement enriched in ω-3 fatty acids on immune responses and platelet–leukocyte complex formation in patients undergoing cardiac surgery. Patients in the supplement group (n = 7) took a supplement enriched in ω-3 fatty acids (Impact®) in addition to a hospital diet for five successive days before surgery; those in the control group (n = 7) took only hospital diet and did not take Impact®. Blood samples in both groups were collected at same time points. Before surgery, samples were collected five days before surgery, at the start of supplementation (baseline), and the end of supplementation (postoperative day (POD)-0). After surgery, samples were collected on POD-1 and POD-7. The expression of human leukocyte antigen (HLA)-DR, the ratio of CD4-/CD8-positive cells, the production of interferon (IFN)-γ by CD4-positive cells, plasma levels of cytokines, and leukocyte–platelet aggregates were measured. Before surgery (POD-0), the supplement caused significant increases in HLA-DR expression, CD4/CD8 ratio, and plasma levels of IFN-γ; these levels were significantly higher compared to those in the control group (P < 0.05, respectively). After surgery (POD-1), all values dramatically decreased in comparison with those of POD-0; however, the values in the supplement group were significantly higher compared to their respective markers in the control group (P < 0.05, respectively). Significant differences of HLA-DR expression and CD4/CD8 ratio persisted through POD-7. Before surgery (POD-0), plasma levels of interleukin (IL)-10 in the supplement group decreased significantly compared with those in the control group (P < 0.05). After surgery (POD-1), plasma levels of IL-10 in both the control and supplement groups increased; these levels in the supplement group were significantly lower than those in the control group (P < 0.05). Significant decreases in the percentage of leukocyte–platelet aggregates were found after supplementation; the difference between the supplement and the control groups was found on POD-0 and POD-1 (P < 0.05, respectively). In conclusion, the dietary supplement increased HLA-DR expression, the CD4/CD8 ratio, and the production of IFN-γ by CD4-positive cells; conversely, the levels of IL-10 and the formation of leukocyte–platelet aggregates before and after surgery were suppressed. These beneficial effects may decrease the incidence of complications after surgery.
doi:10.4137/NMI.S13810
PMCID: PMC4051814  PMID: 24932104
dietary supplement; HLA-DR; CD4/CD8 ratio; platelet–leukocyte aggregates; interferon-γ
3.  S46 Peptidases are the First Exopeptidases to be Members of Clan PA 
Scientific Reports  2014;4:4977.
The dipeptidyl aminopeptidase BII (DAP BII) belongs to a serine peptidase family, S46. The amino acid sequence of the catalytic unit of DAP BII exhibits significant similarity to those of clan PA endopeptidases, such as chymotrypsin. However, the molecular mechanism of the exopeptidase activity of family S46 peptidase is unknown. Here, we report crystal structures of DAP BII. DAP BII contains a peptidase domain including a typical double β-barrel fold and previously unreported α-helical domain. The structures of peptide complexes revealed that the α-helical domain covers the active-site cleft and the side chain of Asn330 in the domain forms hydrogen bonds with the N-terminus of the bound peptide. These observations indicate that the α-helical domain regulates the exopeptidase activity of DAP BII. Because S46 peptidases are not found in mammals, we expect that our study will be useful for the design of specific inhibitors of S46 peptidases from pathogens.
doi:10.1038/srep04977
PMCID: PMC4021333  PMID: 24827749
4.  Crystallization and preliminary X-ray crystallographic analysis of human autotaxin 
The α isoform of human autotaxin has been crystallized. Diffraction data were collected to 3.0 Å resolution using synchrotron radiation.
Autotaxin (ATX), which is also known as ectonucleotide pyrophosphatase/phosphodiesterase 2 (NPP2 or ENPP2) or phosphodiesterase Iα (PD-Iα), is an extracellular lysophospholipase D which generates lysophosphatidic acid (LPA) from lysophosphatidylcholine (LPC). ATX stimulates tumour-cell migration, angiogenesis and metastasis and is an attractive target for cancer therapy. For crystallographic studies, the α isoform of human ATX was overproduced in Escherichia coli, purified and crystallized using the hanging-drop vapour-diffusion method. X-ray diffraction data were collected to 3.0 Å resolution from a monoclinic crystal form belonging to space group C2, with unit-cell parameters a = 311.4, b = 147.9, c = 176.9 Å, β = 122.6°.
doi:10.1107/S174430911005311X
PMCID: PMC3080147  PMID: 21505238
autotaxin; lysophosphatidic acid; lysophospholipase D; ectonucleotide pyrophosphatase/phosphodiesterase 2
5.  Unique gangliosides synthesized in vitro by sialyltransferases from marine bacteria and their characterization: ganglioside synthesis by bacterial sialyltransferases 
Journal of Lipid Research  2013;54(3):571-580.
On the basis of the results outlined in our previous report, bacterial sialyltransferases (ST) from marine sources were further characterized using glycosphingolipids (GSL), especially ganglio-series GSLs, based on the enzymatic characteristics and kinetic parameters obtained by Line weaver-Burk plots. Among them, GA1 and GA2 were found to be good substrates for these unique STs. Thus, new gangliosides synthesized by α2-3 and α2-6STs were structurally characterized by several analytical procedures. The ganglioside generated by the catalytic activity of α2-3ST was identified as GM1b. On the other hand, when enzyme reactions by α2-6STs were performed using substrates GA2 and GA1, very unique gangliosides were generated. The structures were identified as NeuAcα2-6GalNAcβ1-4Galβ1-4Glcβ-Cer and NeuAcα2-6Galβ1-3GalNAcβ1-4Galβ1-4Glcβ-Cer, respectively. The synthesized ganglioside NeuAcα2-6GalNAcβ1-4Galβ1-4Glcβ-Cer showed binding activity to the influenza A virus {A/Panama/2007/99 (H3N2)} at a similar level to purified sialyl(α2-3)paragloboside (S2-3PG) and sialyl(α2-6)paragloboside (S2-6PG) from mammalian sources. The evidence suggests that these STs have unique features, including substrate specificities restricted not only to lacto-series but also to ganglio-series GSLs, as well as catalytic potentials for ganglioside synthesis. This evidence demonstrates that effective in vitro ganglioside synthesis could be a valuable tool for selectively synthesizing sialic acid (Sia) modifications, thereby preparing large-scale gangliosides and permitting the exploration of unknown functions.
doi:10.1194/jlr.M026955
PMCID: PMC3617933  PMID: 23220479
ceramides; influenza A virus; glycolipids; mass spectrometry; membranes
6.  Crystallization and preliminary X-ray crystallographic studies of an exo-β-d-glucosaminidase from Trichoderma reesei  
An exo-β-d-glucosaminidase from T. reesei (Gls93) has been crystallized by the hanging-drop vapour-diffusion method. Diffraction data have been collected using synchrotron radiation.
Chitosan is degraded to glucosamine (GlcN) by chitosanase and exo-β-d-glucosaminidase (GlcNase). GlcNase from Trichoderma reesei (Gls93) is a 93 kDa extracellular protein composed of 892 amino acids. The enzyme liberates GlcN from the nonreducing end of the chitosan chain in an exo-type manner and belongs to glycoside hydrolase family 2. For crystallographic investigations, Gls93 was overexpressed in Pichia pastoris cells. The recombinant Gls93 had two molecular forms of ∼105 kDa (Gls93-F1) and ∼100 kDa (Gls93-F2), with the difference between them being caused by N-glycosylation. Both forms were crystallized by the hanging-drop vapour-diffusion method. Crystals of Gls93-F1 belonged to the orthorhombic space group P212121, with unit-cell parameters a = 98.27, b = 98.42, c = 108.28 Å, and diffracted to 1.8 Å resolution. Crystals of Gls93-F2 belonged to the ortho­rhombic space group P212121, with unit-cell parameters a = 67.84, b = 81.62, c = 183.14 Å, and diffracted to 2.4 Å resolution. Both crystal forms were suitable for X-ray structure analysis at high resolution.
doi:10.1107/S1744309110000606
PMCID: PMC2833044  PMID: 20208168
exo-β-d-glucosaminidases; exochitosanases; Gls93; Trichoderma reesei
7.  Crystallization of mouse S-adenosyl-l-homocysteine hydrolase 
Mouse S-adenosyl-l-homocysteine hydrolase has been crystallized in the presence of the reaction product adenosine. Diffraction data to 1.55 Å resolution were collected using synchrotron radiation.
S-Adenosyl-l-homocysteine hydrolase (SAHH; EC 3.3.1.1) catalyzes the reversible hydrolysis of S-adenosyl-l-homocysteine to adenosine and l-homo­cysteine. For crystallographic investigations, mouse SAHH (MmSAHH) was overexpressed in bacterial cells and crystallized using the hanging-drop vapour-diffusion method in the presence of the reaction product adenosine. X-ray diffraction data to 1.55 Å resolution were collected from an orthorhombic crystal form belonging to space group I222 with unit-cell parameters a = 100.64, b = 104.44, c = 177.31 Å. Structural analysis by molecular replacement is in progress.
doi:10.1107/S1744309110000771
PMCID: PMC2833045  PMID: 20208169
S-adenosyl-l-homocysteine hydrolase; SAHH
8.  Crystallization and preliminary X-ray crystallographic study of 1-deoxy-d-xylulose 5-­phosphate reductoisomerase from Plasmodium falciparum  
1-Deoxy-d-xylulose 5-phosphate reductoisomerase from P. falciparum has been crystallized in the presence of NADPH. Diffraction data to 1.85 Å resolution have been collected using synchrotron radiation.
The nonmevalonate pathway of isoprenoid biosynthesis present in Plasmodium falciparum is known to be an effective target for antimalarial drugs. The second enzyme of the nonmevalonate pathway, 1-deoxy-d-xylulose 5-phosphate reductoisomerase (DXR), catalyzes the transformation of 1-deoxy-d-xylulose 5-phosphate (DXP) to 2-C-methyl-d-erythritol 4-phosphate (MEP). For crystallographic studies, DXR from the human malaria parasite P. falciparum (PfDXR) was overproduced in Escherichia coli, purified and crystallized using the hanging-drop vapour-diffusion method in the presence of NADPH. X-ray diffraction data to 1.85 Å resolution were collected from a monoclinic crystal form belonging to space group C2 with unit-cell parameters a = 168.89, b = 59.65, c = 86.58 Å, β = 117.8°. Structural analysis by molecular replacement is in progress.
doi:10.1107/S1744309110001739
PMCID: PMC2833050  PMID: 20208174
1-deoxy-d-xylulose 5-phosphate reductoisomerase; malaria; nonmevalonate pathway
9.  Crystallization and preliminary X-ray crystallographic study of phosphoglucose isomerase from Plasmodium falciparum  
Phosphoglucose isomerase from P. falciparum has been crystallized. Diffraction data to 1.8 Å resolution have been collected using synchrotron radiation.
Phosphoglucose isomerase (PGI) is a key enzyme in glycolysis and glycogenesis that catalyses the interconversion of glucose 6-phosphate (G6P) and fructose 6-­phosphate (F6P). For crystallographic studies, PGI from the human malaria parasite Plasmodium falciparum (PfPGI) was overproduced in Escherichia coli, purified and crystallized using the hanging-drop vapour-diffusion method. X-ray diffraction data to 1.5 Å resolution were collected from an orthorhombic crystal form belonging to space group P212121 with unit-cell parameters a = 103.3, b = 104.1, c = 114.6 Å. Structural analysis by molecular replacement is in progress.
doi:10.1107/S1744309110001740
PMCID: PMC2833051  PMID: 20208175
glucose 6-phosphate isomerase; malaria; phosphoglucose isomerase; phosphohexose isomerase
10.  Purification, crystallization and preliminary X-ray analysis of the PCNA2–PCNA3 complex from Sulfolobus tokodaii strain 7 
A PCNA2−PCNA3 complex which has recently been identified from S. tokodaii strain 7 was overexpressed, purified and crystallized in two crystal forms.
Crenarchaeal PCNA is known to consist of three subunits (PCNA1, PCNA2 and PCNA3) that form a heterotrimer (PCNA123). Recently, another heterotrimeric PCNA composed of only PCNA2 and PCNA3 was identified in Sulfolobus tokodaii strain 7 (stoPCNAs). In this study, the purified stoPCNA2–stoPCNA3 complex was crystallized by hanging-drop vapour diffusion. The crystals obtained belonged to the orthorhombic space groups I222 and P21212, with unit-cell parameters a = 91.1, b = 111.8, c = 170.9 Å and a = 91.1, b = 160.6, c = 116.6 Å, respectively. X-ray diffraction data sets were collected to 2.90 Å resolution for the I222 crystals and to 2.80 Å resolution for the P21212 crystals.
doi:10.1107/S1744309109044479
PMCID: PMC2802881  PMID: 20054129
PCNA; Sulfolobus tokodaii
11.  Molecular basis of fosmidomycin's action on the human malaria parasite Plasmodium falciparum 
Scientific Reports  2011;1:9.
The human malaria parasite Plasmodium falciparum is responsible for the deaths of more than a million people each year. Fosmidomycin has been proven to be efficient in the treatment of P. falciparum malaria by inhibiting 1-deoxy-D-xylulose 5-phosphate reductoisomerase (DXR), an enzyme of the non-mevalonate pathway, which is absent in humans. However, the structural details of DXR inhibition by fosmidomycin in P. falciparum are unknown. Here, we report the crystal structures of fosmidomycin-bound complete quaternary complexes of PfDXR. Our study revealed that (i) an intrinsic flexibility of the PfDXR molecule accounts for an induced-fit movement to accommodate the bound inhibitor in the active site and (ii) a cis arrangement of the oxygen atoms of the hydroxamate group of the bound inhibitor is essential for tight binding of the inhibitor to the active site metal. We expect the present structures to be useful guides for the design of more effective antimalarial compounds.
doi:10.1038/srep00009
PMCID: PMC3216497  PMID: 22355528
12.  Positive correlation of pigment epithelium‐derived factor and total antioxidant capacity in aqueous humour of patients with uveitis and proliferative diabetic retinopathy 
The British Journal of Ophthalmology  2007;91(9):1133-1134.
Background/aims
There are several animal studies to suggest that pigment epithelium‐derived factor (PEDF) may exert beneficial effects on diabetic retinopathy and uveitis by acting as an endogenous antioxidant. However, the interrelationship between PEDF and total antioxidant capacity in the human eye remains to be elucidated. In this study, PEDF and total antioxidant levels were determined in the aqueous humour of patients with proliferative diabetic retinopathy (PDR) and uveitis, and the relationship between these two markers was investigated.
Methods
Aqueous humour levels of PEDF and total antioxidant capacity were determined by an ELISA system in 34 uveitis and 9 PDR samples.
Results
Aqueous humour levels of PEDF and total antioxidant capacity were significantly lower in patients with PDR than those with uveitis (1.8±0.2 μg/ml vs 6.4±0.8 μg/ml and 0.17±0.03 mmol/l vs 0.85±0.05 mmol/l, respectively, p<0.01). A positive correlation between PEDF and total antioxidant capacity was found in patients with PDR and uveitis (r = 0.33, p<0.05).
Conclusion
This study demonstrated that PEDF levels were associated with total antioxidant capacity in aqueous humour levels in humans. These observations suggest that substitution of PEDF may be a therapeutic target for oxidative stress‐involved eye diseases, especially PDR.
doi:10.1136/bjo.2007.115188
PMCID: PMC1954896  PMID: 17389742
13.  Positive correlation between pigment epithelium‐derived factor and monocyte chemoattractant protein‐1 levels in the aqueous humour of patients with uveitis 
Aim
To evaluate whether aqueous humour levels of pigment epithelium‐derived factor (PEDF) are associated with monocyte chemoattractant protein‐1 (MCP‐1) in patients with uveitis.
Methods
Aqueous humour levels of MCP‐1 and PEDF were determined by ELISA in 34 uveitis samples and 9 cataract control samples.
Results
Aqueous humour MCP‐1 and PEDF levels were significantly higher in patients with infectious or non‐infectious uveitis than in controls (mean (SD) 32.3 (10.7) ng/ml vs 4.48 (1.10) ng/ml vs 0.47 (0.10) ng/ml, and 8.40 (1.30) μg/ml vs 5.01 (0.92) μg/ml vs 1.32 (0.22) μg/ml, respectively, p<0.001). A positive correlation between PEDF and MCP‐1 was found in patients with uveitis (r = 0.39, p<0.01).
Conclusion
The results demonstrated that aqueous humour levels of PEDF were positively associated with MCP‐1 in patients with uveitis. The present observations suggest that aqueous humour levels of PEDF may be a marker of inflammation in uveitis.
doi:10.1136/bjo.2006.109843
PMCID: PMC1955592  PMID: 17166895
14.  Increased levels of pigment epithelium‐derived factor in aqueous humor of patients with uveitis 
Aim
To evaluate whether aqueous humor levels of pigment epithelium‐derived factor (PEDF) are increased in patients with uveitis
Methods
Aqueous humor levels of PEDF and tumour necrosis factor α (TNFα) were determined by ELISA in 34 uveitis samples and 9 cataract control samples.
Results
Aqueous humor PEDF and TNFα levels were significantly higher in patients with uveitis than in controls (mean (SD) 6.4 (0.8) v 1.3 (0.2) μg/ml and 14.7 (3.8) v 4.2 (0.4) pg/ml, respectively; p<0.01). A positive correlation between PEDF and TNFα was found in patients with uveitis (r = 0.40, p<0.01). Furthermore, PEDF levels in aqueous humor were increased in proportion to the disease activity of uveitis.
Conclusion
The results show that aqueous humor levels of PEDF are increased in patients with uveitis. Our observations suggest that aqueous humor levels of PEDF may be increased as a countersystem against inflammation in uveitis.
doi:10.1136/bjo.2006.103804
PMCID: PMC1857622  PMID: 16973658
15.  Crystallization and preliminary X-ray crystallographic studies of pig heart carbonyl reductase 
Pig heart carbonyl reductase has been crystallized in the presence of NADPH. Diffraction data have been collected using synchrotron radiation.
Pig heart carbonyl reductase (PHCR), which belongs to the short-chain dehydrogenase/reductase (SDR) family, has been crystallized by the hanging-drop vapour-diffusion method. Two crystal forms (I and II) have been obtained in the presence of NADPH. Form I crystals belong to the tetragonal space group P42, with unit-cell parameters a = b = 109.61, c = 94.31 Å, and diffract to 1.5 Å resolution. Form II crystals belong to the tetragonal space group P41212, with unit-cell parameters a = b = 120.10, c = 147.00 Å, and diffract to 2.2 Å resolution. Both crystal forms are suitable for X-ray structure analysis at high resolution.
doi:10.1107/S1744309106037535
PMCID: PMC2225176  PMID: 17012807
carbonyl reductase; retinal; retinol; SDR family
16.  Structural basis for recognition of cognate tRNA by tyrosyl-tRNA synthetase from three kingdoms 
Nucleic Acids Research  2007;35(13):4289-4300.
The specific aminoacylation of tRNA by tyrosyl-tRNA synthetases (TyrRSs) relies on the identity determinants in the cognate tRNATyrs. We have determined the crystal structure of Saccharomyces cerevisiae TyrRS (SceTyrRS) complexed with a Tyr-AMP analog and the native tRNATyr(GΨA). Structural information for TyrRS–tRNATyr complexes is now full-line for three kingdoms. Because the archaeal/eukaryotic TyrRSs–tRNATyrs pairs do not cross-react with their bacterial counterparts, the recognition modes of the identity determinants by the archaeal/eukaryotic TyrRSs were expected to be similar to each other but different from that by the bacterial TyrRSs. Interestingly, however, the tRNATyr recognition modes of SceTyrRS have both similarities and differences compared with those in the archaeal TyrRS: the recognition of the C1-G72 base pair by SceTyrRS is similar to that by the archaeal TyrRS, whereas the recognition of the A73 by SceTyrRS is different from that by the archaeal TyrRS but similar to that by the bacterial TyrRS. Thus, the lack of cross-reactivity between archaeal/eukaryotic and bacterial TyrRS-tRNATyr pairs most probably lies in the different sequence of the last base pair of the acceptor stem (C1-G72 vs G1-C72) of tRNATyr. On the other hand, the recognition mode of Tyr-AMP is conserved among the TyrRSs from the three kingdoms.
doi:10.1093/nar/gkm417
PMCID: PMC1934993  PMID: 17576676
17.  Serum Levels of sRAGE, the Soluble Form of Receptor for Advanced Glycation End Products, Are Associated with Inflammatory Markers in Patients with Type 2 Diabetes 
Molecular Medicine  2007;13(3-4):185-189.
Advanced glycation end products (AGEs) and their receptor (RAGE) play an important role in accelerated atherosclerosis in diabetes. We have recently found that the soluble form of RAGE (sRAGE) levels are significantly higher in type 2 diabetic patients than in nondiabetic subjects and positively associated with the presence of coronary artery disease in diabetes. In this study, we examined whether serum levels of sRAGE correlated with inflammatory biomarkers in patients with type 2 diabetes. Eighty-six Japanese type 2 diabetic patients (36 men and 50 women, mean age 68.4 ± 9.6 years) underwent a complete history and physical examination, determination of blood chemistries, sRAGE, monocyte chemotactic protein-1 (MCP-1), adiponectin, tumor necrosis factor-α (TNF-α), and interleukin-6 (IL-6). Univariate regression analysis showed that serum levels of sRAGE positively correlated with alanine aminotransferase (ALT) (r = 0.437, P = 0.0001), MCP-1 (r = 0.359, P = 0.001), TNF-α (r = 0.291, P = 0.006), and hyperlipidemia medication (r = 0.218, P = 0.044). After multiple regression analyses, ALT (P < 0.0001), MCP-1 (P = 0.007), and TNF-α (P = 0.023) remained significant. The present study demonstrates for the first time that serum levels of sRAGE are positively associated with MCP-1 and TNF-α levels in type 2 diabetic patients. These observations suggest the possibility that sRAGE level may become a novel biomarker of vascular inflammation in type 2 diabetic patients.
doi:10.2119/2006-00090.Nakamura
PMCID: PMC1892766  PMID: 17592553

Results 1-17 (17)