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Kunihiko Tajima

Publications and source records attributed to Kunihiko Tajima.

17 recordsLinked to original sources
PubMed2006

Identification and characterization of L-arabonate dehydratase, L-2-keto-3-deoxyarabonate dehydratase, and L-arabinolactonase involved in an alternative pathway of L-arabinose metabolism. Novel evolutionary insight into sugar metabolism.

Azospirillum brasiliense possesses an alternative pathway of L-arabinose metabolism, different from the known bacterial and fungal pathways. In the preceding articles, we identified and characterized L-arabinose-1-dehydrogenase and alpha-ketoglutaric semialdehyde dehydrogenase, which catalyzes the first and final reaction steps in this pathway, respectively (Watanabe, S., Kodaki, T., and Makino, K. (2006) J. Biol. Chem. 281, 2612-2623 and Watanabe, S., Kodaki, T., and Makino, K. (2006) J. Biol. Chem. 281, 28876-28888). We here report the remaining three enzymes, L-arabonate dehydratase, L-2-keto-3-deoxyarabonate (L-KDA) dehydratase, and L-arabinolactonase. N-terminal amino acid sequences of L-arabonate dehydratase and L-KDA dehydratase purified from A. brasiliense cells corresponded to those of AraC and AraD genes, which form a single transcriptional unit together with the L-arabinose-1-dehydrogenase gene. Furthermore, the L-arabinolactonase gene (AraB) was also identified as a component of the gene cluster. Genetic characterization of the alternative L-arabinose pathway suggested a significant evolutional relationship with the known sugar metabolic pathways, including the Entner-Doudoroff (ED) pathway and the several modified versions. L-arabonate dehydratase belongs to the ILVD/EDD family and spectrophotometric and electron paramagnetic resonance analysis revealed it to contain a [4Fe-4S](2+) cluster. Site-directed mutagenesis identified three cysteine ligands essential for cluster coordination. L-KDA dehydratase was sequentially similar to DHDPS/NAL family proteins. D-2-Keto-3-deoxygluconate aldolase, a member of the DHDPS/NAL family, catalyzes the equivalent reaction to L-KDA aldolase involved in another alternative L-arabinose pathway, probably associating a unique evolutional event between the two alternative L-arabinose pathways by mutation(s) of a common ancestral enzyme. Site-directed mutagenesis revealed a unique catalytic amino acid residue in L-KDA dehydratase, which may be a candidate for such a natural mutation.

Amino Acid Sequence↗
PubMed2005

Solution structure of marinostatin, a natural ester-linked protein protease inhibitor.

Marinostatin is a unique protein protease inhibitor containing two ester linkages. We have purified a 12-residue marinostatin [MST(1-12), (1)FATMRYPSDSDE(12)] and determined the residues involved in the formation of the ester linkages and the solution structure by (1)H NMR spectroscopy and restrained molecular dynamics calculation. The two ester linkages of MST(1-12) are formed between hydroxyl and carboxyl groups, Thr(3)-Asp(9) and Ser(8)-Asp(11), indicating that MST(1-12) has two cyclic regions which are fused at the residues of Ser(8) and Asp(9). A strong NOE cross-peak between Tyr(6) H(alpha) and Pro(7) H(alpha) was observed, indicating that the Pro(7) residue takes a cis-conformation. Well-converged structures and hydrogen-deuterium experiments of MST(1-12) showed that the backbone NH proton of the P1'residue, Arg(5), is hydrogen-bonded to the carbonyl oxygen of the ester linkage between Thr(3) and Asp(9). To reveal the significance of the ester linkages, a marinostatin analogue, MST-2SS ((1)FACMRYPCCSCE(12)) with two disulfide bridges of Cys(3)-Cys(9) and Cys(8)-Cys(11), was also synthesized. The inhibitory activity of MST-2SS was as strong as that of MST(1-12), and the Pro(7) residue of MST-2SS also takes a cis-conformation. However, the exchange rate of the Arg(5) NH proton of MST-2SS was about 100 times faster than that of MST(1-12), and the structure calculation of MST-2SS was not converged on account of the small number of NOEs, indicating that MST-2SS takes a more flexible structure. The hydrogen acceptability of the ester linkage formed by the P2 position residue, Thr(3), is crucial for suppressing the fluctuation of the reactive site and sustaining the inhibitory activity, which enables marinostatin to be one of the smallest protease inhibitors in nature.

ATP-Binding Cassette Transporters↗
PubMed2005

Prognostic factors in patients with advanced hepatocellular carcinoma receiving hepatic arterial infusion chemotherapy.

BACKGROUND: The prognosis of patients with advanced hepatocellular carcinoma (HCC) is poor. We aimed to clarify the prognostic factors in patients with advanced HCC receiving hepatic arterial infusion chemotherapy (HAIC). METHODS: Forty-four HCC patients were treated with HAIC, using low-dose cisplatin (CDDP) and 5-fluorouracil (5-FU) with/without leucovorin (or isovorin). Of these 44 patients, 15 received low-dose CDDP and 5-FU, and 29 received low-dose CDDP, 5-FU, and leucovorin or isovorin. Prognostic factors were evaluated by univariate and multivariate analyses of patient and disease characteristics. RESULTS: Of all patients, 5 and 12 patients respectively, exhibited a complete response (CR) and a partial response (PR) (response rate, 38%). The response rate (48.3%) in the low-dose CDDP and 5-FU with leucovorin/isovorin group was significantly better than that (20%) in the low-dose CDDP and 5-FU group (P = 0.002). The 1-, 2-, 3-, and 5-year cumulative survival rates of the 44 patients were 39%, 18%, 12%, and 9%, respectively. The regimen using low-dose CDDP and 5-FU with leucovorin/isovorin tended to improve survival rates (P = 0.097). Univariate and multivariate analyses showed the same variables--the Child-Pugh score (P = 0.013, P = 0.018), alpha-fetoprotein (AFP) level (P = 0.010, P = 0.009), and therapeutic effect after HAIC (P = 0.003, P = 0.01), respectively, to be significant prognostic factors. CONCLUSIONS: Patients who had advanced HCC with favorable hepatic reserve capacity and a lower AFP level were suitable candidates for HAIC. Moreover, the regimen using low-dose CDDP and 5-FU with leucovorin/isovorin may be suitable for advanced HCC patients, because of the improvement in the response rate and survival compared with the low-dose CDDP and 5-FU regimen without leucovorin/isovorin.

Adult↗
PubMed2005

Percutaneous radiofrequency ablation with cooled electrodes combined with hepatic arterial balloon occlusion in hepatocellular carcinoma.

BACKGROUND: We have reported that percutaneous radiofrequency ablation (RFA) with balloon occlusion of the hepatic artery (balloon-occluded RFA), using an expandable electrode, increases the coagulation area. In this study, we investigated the efficacy of balloon-occluded RFA and balloon-microcatheter-occluded RFA, using a cool RF single electrode. METHODS: We studies 41 patients with 47 hepatocellular carcinoma (HCC) lesions. We treated 28 patients (32 nodules) with balloon-occluded RFA, 5 patients (6 nodules) with balloon-microcatheter-occluded RFA, and 8 patients (9 nodules) with standard RFA. Initial therapeutic efficacy was evaluated with dynamic computed tomography performed 1 week after one session of treatment. RESULTS: One session of treatment was done for 20 nodules (62.5%) in the balloon-occluded RFA group and for 4 nodules (66.7%) in the balloon-microcatheter-occluded RFA group. We compared the coagulation diameter for balloon-occluded RFA (7 nodules), balloon-microcatheter-occluded RFA (6 nodules), and standard RFA (9 nodules) after one application cycle (12 min). The greatest dimension of the area coagulated by balloon-occluded RFA was significantly larger (greatest long-axis dimension, 47.6 +/- 7.8 mm; greatest short-axis dimension, 33.4 +/- 7.5 mm) than that coagulated by standard RFA (greatest long-axis dimension, 35.3 +/- 4.7 mm; greatest short-axis dimension, 25.9 +/- 3.7 mm; P = 0.002 for greatest long-axis dimension; P = 0.041 for greatest short-axis dimension). However, there was significant difference only in the greatest short-axis dimension of the area coagulated comparing balloon-microcatheter-occluded RFA and standard RFA. CONCLUSIONS: We consider balloon-occluded RFA using a cool RF electrode to be superior to standard RFA for the treatment of HCC, especially when larger coagulation volumes are required.

Aged↗
PubMed2005

Human homologue of maid is a useful marker protein in hepatocarcinogenesis.

BACKGROUND & AIMS: Human homologue of maid (HHM) is a helix-loop-helix (HLH) transcriptional regulatory protein that is involved in the hepatic stem cell development and differentiation. We analyzed the potential involvement of HHM in hepatocarcinogenesis. METHODS: We analyzed HHM expression in the choline-deficient L-amino acid defined (CDAA) diet model of rat hepatocarcinogenesis and in human adenomatous hyperplasia (AH) and hepatocellular carcinoma (HCC) biopsy samples. We assessed the effects of HHM on cell proliferation. We screened proteins that bind to HHM protein using a yeast 2-hybrid screen. RESULTS: High HHM expression was seen in foci and HCC induced in the rat CDAA diet model. HHM protein was expressed in 23 of 32 AH samples (72%), 19 of 28 well-differentiated HCC samples (68%), and 9 of 18 poorly-moderately differentiated HCC samples (50%). Over-expressed HHM enhanced the S phase. HHM interference RNA significantly inhibited cell proliferation. A yeast 2-hybrid screen identified Jun activation domain-binding protein 1 (Jab1) as a binding partner for HHM. We confirmed HHM and Jab1 binding by immunoprecipitation and immunofluorescent histochemistry. The expression of Jab1 was found in human AH and HCC samples. We found an association between levels of expression of HHM and those of Jab1 in AH and HCC tissues examined (P = .027 by chi2 test). CONCLUSIONS: High-level HHM expression was found from the very early stages of hepatocarcinogenesis, suggesting that HHM may be a useful marker protein to detect.

Adenoma↗
PubMed2004

Two-metal ion, Ni(II) and Cu(II), binding alpha-helical coiled coil peptide.

Metalloproteins are an attractive target for de novo design. Usually, natural proteins incorporate two or more (hetero- or homo-) metal ions into their frameworks to perform their functions, but the design of multiple metal-binding sites is usually difficult to achieve. Here, we undertook the de novo engineering of heterometal-binding sites, Ni(II) and Cu(II), into a designed coiled coil structure based on an isoleucine zipper (IZ) peptide. Previously, we described two peptides, IZ-3adH and IZ-3aH. The former has two His residues and forms a triple-stranded coiled coil after binding Ni(II), Zn(II), or Cu(II). The latter has one His residue, which allowed binding with Cu(II) and Zn(II), but not with Ni(II). On the basis of these properties, we newly designed IZ(5)-2a3adH as a heterometal-binding peptide. This peptide can bind Cu(II) and Ni(II) simultaneously in the hydrophobic core of the triple-stranded coiled coil. The first metal ion binding induced the folding of the peptide into the triple-stranded coiled coil, thereby promoting the second metal ion binding. This is the first example of a peptide that can bind two different metal ions. This construction should provide valuable insights for the de novo design of metalloproteins.

Amino Acid Sequence↗
PubMed2004

Selective formation of AAB- and ABC-type heterotrimeric alpha-helical coiled coils.

The alpha-helical coiled coils have a representative amino acid sequence of (abcdefg)(n) heptad repeats. We previously reported that two peptides named IZ-2A and IZ-2W formed an (IZ-2A)(2)/IZ-2W heterotrimer with an Ala-Ala-Trp interaction in the hydrophobic core. In this paper, we describe the selective formation of AAB- and ABC-type heterotrimers. To increase the selectivity of the AAB-type heterotrimeric formation, Lys residues at the f position were mutated to either an Ala or a Gln residue to form IZ-2A(fA) or IZ-2W(fQ). Separately, both IZ-2A(fA) and IZ-2W(fQ) have a random structure at pH 7 and 20 degrees C. However, together IZ-2A(fA) and IZ-2W(fQ) form a 2:1 complex with a thermal transition midpoint (Tm) of 48 degrees C. This procedure was applied to prepare the ABC-type heterotrimer, in which two sets of Ala-Ala-Trp interactions were designed in the hydrophobic core. Interhelical interaction between the e and g positions and the alpha-helical propensity of the amino acid at the f position were also considered in the design. The resultant three peptides selectively formed the ABC-type heterotrimer with a Tm of 51 degrees C. Other peptide combinations had random coil properties.

Amino Acid Sequence↗
PubMed2004

Effect of phosphorothioate chirality on i-motif structure and stability.

The P-chiral stereo-defined phosphorothioate groups have been introduced into all of the four internucleotide positions of d(T(PS1)C(PS2)C(PS3)C(PS4)C) (PSn = phosphorothioate group), and among the 16 possible diastereomers of PS-d(TC(4)), 10 stereomers have been synthesized to investigate the effects of the sense of the P-chirality upon the structure and stability of the i-motif structure. The temperature dependence of circular dichroism spectra showed that the melting temperature (T(m)) of the [all R(p)]-PS-d(TC(4)) i-motifs was 31 degrees C, identical to that of the parent oligomer, PO-d(TC(4)), while that of the [all S(p)]-PS-d(TC(4)) i-motif was largely decreased by 11 degrees C. Single substitution of R(p) with S(p) caused a decrease of T(m) by 3-4 degrees C at positions of PS1, PS2, and PS3 and by 1 degrees C at that of PS4, showing the additive property of the T(m) suppression. The comparison of the NOESY spectra between [all R(p)]-PS-, [all S(p)]-PS-, and PO-d(TC(4)) showed that intraresidual H6-H3' and H2' '-H4' NOE cross-peaks of the all S(p) isomer are weaker than those of the all R(p) isomer and PO-d(TC(4)), indicating the change in the C3'-endo conformation and glycosidic bond angle. The structural alternation for the i-motif formed by [all S(p)]-PS-d(TC(4)) is also suggested by the chemical shift differences of C2/C3/C4 H2''and H4' protons from those of [all R(p)]-PS-d(TC(4)) and PO-d(TC(4)). These results suggest that the S(p) configuration at phosphorus of the phosphorothioate linkage changes the sugar-phosphorothioate conformation and intermolecular interaction in the narrow groove, leading to the destabilization of the i-motif structure.

Circular Dichroism↗
PubMed2004

One-step concentration of malarial parasite-infected red blood cells and removal of contaminating white blood cells.

BACKGROUND: Isolation of a concentrated, living preparation of malarial parasite-infected red blood cells (PRBCs) that have low contamination of white blood cells (WBCs) facilitates research on the molecular, biochemical and immunological aspects of malarial parasites. This is currently carried out by a two-step method, including the concentration of PRBCs using density gradient centrifugation through Percoll or Nycodenz, followed by the removal of host WBCs using a cellulose powder column or a commercially available filtration unit. These two-step methods can help isolate sufficient PRBCs, but they are laborious. In this study, a simplified one-step procedure that takes advantage of the difference between diamagnetic low-spin oxyhaemoglobin and paramagnetic haemozoin (haem polymer) was described. The paramagnetic polymer is deposited in the food vacuoles of the parasite, allowing the use of magnetic separation to efficiently and rapidly concentrate PRBCs while removing contaminating host WBCs. METHODS: The magnetic removal of WBCs using a commercial LD column (MACS) was evaluated as a new method for concentrating and purifying PRBCs. To compare this method with the two density gradient centrifugation methods using Percoll or Nycodenz, we analysed the quantities of enriched PRBCs and contaminating host WBCs as well as the viability of malarial parasites in the final preparations. RESULTS: The quantity of PRBCs and the viability of malarial parasites in the isolated PRBCs were similar between magnetic and centrifugation methods. However, 90-99% of the contaminating WBCs were removed from the starting material using a magnetic column, whereas WBC content did not change using the Percoll or Nycodenz methods. CONCLUSION: The use of a commercially available magnetic LD column is effective, safe and easy for the one-step purification of PRBCs. This simple method does not affect the viability of malarial parasites.

Animals↗
PubMed2004

1H NMR study on equilibrium between parallel G-quartet structures.

Structural transition of parallel G-quartet formed by d(TG4T) are induced by monovalent cations. In the presence of K+ ion, G-quartet takes a single conformation, while in the presence of Na+ ion, the equilibrium between more than three conformations were observed.

Base Pairing↗
PubMed2003

Nitration of 2'-deoxyguanosine by a NO/O2 gas mixture: identification and characterization of N2-nitro-2'-deoxyguanosine.

[reaction: see text] A gas mixture of NO and O(2) was bubbled into 2'-deoxyguanosine solution at neutral pH and 37 degrees C. A novel nitrated nucleoside was generated in the reaction mixture in addition to 8-nitroguanine, 8-nitroxanthine, 2'-deoxyxanthosine, xanthine, and guanine. The novel nucleoside was identified as N(2)-nitro-2'-deoxyguanosine by spectrometric data.

Deoxyguanosine↗
PubMed2003

Effects of 6-formylpterin as an internal source of hydrogen peroxide on cell death of human peripheral blood leukocytes.

The intracellular generation of hydrogen peroxide (H(2)O(2)) by 6-formylpterin and its effects on the cell surface exposure of phosphatidylserine (PS) as a marker of cell death were examined in human peripheral blood leukocytes, and the effects were compared with those of exogenously administered H(2)O(2). Neutrophils, monocytes and lymphocytes were isolated from fresh blood, and cultured for 24 h in vitro. In neutrophils, the intracellular H(2)O(2) generation was observed when the cells were incubated with 100-500 microM 6-formylpterin, and the PS exposure due to spontaneous apoptosis was inhibited. The underlying mechanism of the inhibition was attributed to the suppression of both activation and activity of caspase-3. On the other hand, exogenously administered 100 microM H(2)O(2) did not affect the PS exposure. The intracellular H(2)O(2) generation was also observed in monocytes and lymphocytes. In monocytes, 500 microM 6-formylpterin induced more PS exposure than 100 microM H(2)O(2) did. In lymphocytes, up to 500 microM 6-formylpterin did not induce conspicuous PS exposure, while 100 microM H(2)O(2) induced severe PS exposure. These findings indicated that the resistance against an internal and external source of H(2)O(2) are different among leukocytes, for example, lymphocytes are poorly resistant against external H(2)O(2) but highly resistant against internal one.

Adult↗
PubMed2003

Neutralization of toxic heme by Plasmodium falciparum histidine-rich protein 2.

Plasmodium falciparum histidine-rich protein 2 (PfHRP2) has been suggested to be an initiator of the polymerization of heme, which is produced as by-product on the digestion of hemoglobin, and a promoter of the H(2)O(2)-induced degradation of heme in food vacuoles of the malarial parasite. In this work, we have designed PfHRP2 model peptides, R18 and R27 (18 and 27 residues, respectively), and used them for optical and electron spin resonance spectroscopic measurements to confirm that the axial ligands of the heme-PfHRP2 complex are the nitrogenous donors derived from the imidazole moieties of histidine residues of PfHRP2. In addition, we revealed that the affinities of R18 and R27 for heme (K(d) = 2.21 x 10(-6) M and 0.71 x 10(-6) M, respectively) might be as high as that of PfHRP2 (K(d) = 0.94 x 10(-6) M). The R27 peptide can remove heme from membrane-intercalated heme and inhibit heme-induced hemolysis. Therefore, we suggest another function of PfHRP2: it may play an important role in the neutralization of toxic heme in the parasite cytoplasm and infected erythrocytes by removing heme from heme-bound membranes or reducing heme-induced hemolysis.

Amino Acid Sequence↗
PubMed2002

Formation of a fairly stable diazoate intermediate of 5-methyl-2'-deoxycytidine by HNO2 and NO, and its implication to a novel mutation mechanism in CpG site.

The intermediate produced from 5-methyl-2'-deoxycytidine ((5me)dCyd) by HNO2 and NO treatments was isolated and characterized. When 10mM (5me)dCyd was incubated with 100mM NaNO2 at pH 3.7 and 37 degrees C, a previously unidentified product was formed. The product was identified as a diazoate derivative of (5me)dCyd, 1-(beta-D-2'-deoxyribofuranosyl)-5-methyl-2-oxopyrimidine-4-diazoate ((5me)dCyd-diazoate), on the bases of several measurements including LC/MS. The time course of the concentration change of the diazoate showed a characteristic profile of a reaction intermediate, and the steady state concentration was 2.3 microM (0.023% yield). When an aqueous solution of 10mM (5me)dCyd (10 mL) was bubbled by NO at 37 degrees C under aerobic conditions holding the pH around 7.4, the diazoate was also generated. The yield of the diazoate was 0.041 micromol (0.041% yield) at 20 mmol of NO absorption. At physiological pH and temperature (pH 7.4, 37 degrees C), the diazoate was converted to dThd exclusively with a first order rate constant k=9.1x10(-6) x s(-1) (t(1/2)=21 h). These results show that the diazoate is generated as a relatively stable intermediate in the reactions of (5me)dCyd with HNO2 and NO and further suggest that the diazoate can be formed in cellular DNA with biologically relevant doses of HNO2 and NO.

Azo Compounds↗
PubMed2002

Effect of antifungal azoles on the heme detoxification system of malarial parasite.

The antimalarial activities of some antifungal azole agents (ketoconazole, miconazole, and clotrimazole) have been known for several years, however, their antimalarial mechanism remains equivocal. Our recent study showed that clotrimazole has a relative high affinity for heme, inhibits reduced glutathione-dependent heme catabolism, and enhances heme-induced hemolysis. In the present study, we have found that clotrimazole can remove heme from histidine rich peptide-heme complex, which initiates heme-polymerization in malaria. In addition, we show that two other azoles (ketoconazole and miconazole) behave similarly to clotrimazole in binding to heme: they bind to heme with similar affinities, remove heme from the histidine rich peptide-heme complex and from the reduced glutathione-heme complex to form stable heme-azole complexes with two nitrogenous ligands derived from the imidazole moieties of two azole molecules. We have also revealed that clotrimazole and miconazole have stronger promoting activities for heme-induced hemolysis than ketoconazole, implying that the stronger antimalarial activities of clotrimazole and miconazole might arise from their stronger ability to promote heme-induced hemolysis of clotrimazole and clotrimazole than that of ketoconazole. These results also suggest that ketoconazole and miconazole, like clotrimazole, might possess an antimalarial mechanism relating to their inhibition of heme polymerization and the degradation of reduced glutathione-dependent heme.

Animals↗
PubMed2001

Clotrimazole binds to heme and enhances heme-dependent hemolysis: proposed antimalarial mechanism of clotrimazole.

Two recent studies have demonstrated that clotrimazole, a potent antifungal agent, inhibits the growth of chloroquine-resistant strains of the malaria parasite, Plasmodium falciparum, in vitro. We explored the mechanism of antimalarial activity of clotrimazole in relation to hemoglobin catabolism in the malaria parasite. Because free heme produced from hemoglobin catabolism is highly toxic to the malaria parasite, the parasite protects itself by polymerizing heme into insoluble nontoxic hemozoin or by decomposing heme coupled to reduced glutathione. We have shown that clotrimazole has a high binding affinity for heme in aqueous 40% dimethyl sulfoxide solution (association equilibrium constant: K(a) = 6.54 x 10(8) m(-2)). Even in water, clotrimazole formed a stable and soluble complex with heme and suppressed its aggregation. The results of optical absorption spectroscopy and electron spin resonance spectroscopy revealed that the heme-clotrimazole complex assumes a ferric low spin state (S = 1/2), having two nitrogenous ligands derived from the imidazole moieties of two clotrimazole molecules. Furthermore, we found that the formation of heme-clotrimazole complexes protects heme from degradation by reduced glutathione, and the complex damages the cell membrane more than free heme. The results described herein indicate that the antimalarial activity of clotrimazole might be due to a disturbance of hemoglobin catabolism in the malaria parasite.

Animals↗