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Takeshi Nakanishi

Publications and source records attributed to Takeshi Nakanishi.

At least 19 recordsLinked to original sources
PubMed2006

Structural consequences of mutations in interfacial Tyr residues of a protein antigen-antibody complex. The case of HyHEL-10-HEL.

Tyrosine is an important amino acid in protein-protein interaction hot spots. In particular, many Tyr residues are located in the antigen-binding sites of antibodies and endow high affinity and high specificity to these antibodies. To investigate the role of interfacial Tyr residues in protein-protein interactions, we performed crystallographic studies and thermodynamic analyses of the interaction between hen egg lysozyme (HEL) and the anti-HEL antibody HyHEL-10 Fv fragment. HyHEL-10 has six Tyr residues in its antigen-binding site, which were systematically mutated to Phe and Ala using site-directed mutagenesis. The crystal structures revealed several critical roles for these Tyr residues in the interaction between HEL and HyHEL-10 as follows: 1) the aromatic ring of Tyr-50 in the light chain (LTyr-50) was important for the correct ternary structure of variable regions of the immunoglobulin light chain and heavy chain and of HEL; 2) deletion of the hydroxyl group of Tyr-50 in the heavy chain (HTyr-50) resulted in structural changes in the antigen-antibody interface; and 3) the side chains of HTyr-33 and HTyr-53 may help induce fitting of the antibody to the antigen. Hot spot Tyr residues may contribute to the high affinity and high specificity of the antigen-antibody interaction through a diverse set of structural and thermodynamic interactions.

Animals↗
PubMed2005

Tumor necrosis factor-alpha-induced iron sequestration and oxidative stress in human endothelial cells.

OBJECTIVE: Tumor necrosis factor (TNF)-alpha-induced endothelial injury, which is associated with atherosclerosis, is mediated by intracellular reactive oxygen species. Iron is essential for the amplification of oxidative stress. We tested whether TNF-alpha accelerated iron accumulation in vascular endothelium, favoring synthesis of hydroxyl radical. METHODS AND RESULTS: Diverse iron transporters, including iron import proteins (transferrin receptor [TfR] and divalent metal transporter 1 [DMT1]) and an iron export protein (ferroportin 1 [FP1]) coexist in human umbilical endothelial cells (HUVECs). TNF-alpha caused upregulation of TfR and DMT1 and downregulation of FP1, which were demonstrated in mRNA as well as protein levels. These changes in iron transporters were accompanied by accumulation of iron that was both transferrin-dependent and transferrin-independent. Modifications of these mRNAs were regulated post-transcriptionally, and were coordinated with activation of binding activity of iron regulatory protein 1 to the iron responsive element on transporter mRNAs. Using a salicylate trap method, we observed that only simultaneous exposure of endothelial cells to iron and TNF-alpha accelerated hydroxyl radical production. CONCLUSIONS: TNF-alpha could cause intracellular iron sequestration, which may participate importantly in the pathophysiology of atherosclerosis and cardiovascular disease.

Aconitate Hydratase↗
PubMed2005

Comparison of cytotoxicity of cysteine and homocysteine for renal epithelial cells.

BACKGROUND: Although the cytotoxic effects of cysteine (Cys) on renal cells have been established, the effects of homocysteine (Hcy), which causes endothelial cell dysfunction, have not been well tested. We compared the direct toxicity of Hcy on renal tubular cells to that of Cys and examined the mechanism of cell toxicity. METHODS: LLC-PK1 cells were incubated with test media containing 500 microM Cys or Hcy in the presence or absence of 100 microM copper. Lactate dehydrogenase release and thiobarbituric acid reactive substance were measured for estimating cytolysis and lipid peroxidation, respectively. The generation of hydrogen peroxide and hydroxyl radical, and the cell redox state were analyzed using the scopoletin method, salicylate-trap method, and glutathione (GSH) content, respectively. Superoxide dismutase, catalase, and vitamin E also were used for clarifying the mechanism of toxicity. RESULTS: In the presence of copper (+ Cu), cytolysis at 16 h was more prominent in cells exposed to Cys than Hcy. In accordance with cytotoxicity, lipid peroxidation at 4 h of incubation, as well as hydrogen peroxide and hydroxyl radical formation in a shorter incubation, were remarkably greater in Cys + Cu than Hcy + Cu. The addition of Hcy, but not Cys, decreased GSH content significantly. CONCLUSION: In the presence of copper, Cys was extraordinarily more cytotoxic to renal cells than Hcy. Cytotoxicity from Hcy may be dependent upon depletion of cellular GSH, while Cys cytotoxicity is primarily dependent upon the generation of reactive oxygen species and lipid peroxidation.

Animals↗
PubMed2005

A case of ileoileal intussusception caused by metastatic pedunculated tumor of cutaneous angiosarcoma.

Cutaneous angiosarcoma is a rare aggressive vascular tumor that occurs in elderly patients and is usually located on the head and face. Metastases often develop in the cervical lymph nodes, lungs, bone, liver and spleen. There have been no reports of ileoileal intussusception due to metastatic tumor from cutaneous angiosarcoma. We reported a case of cutaneous angiosarcoma in a 67-year-old Japanese male accompanied with ileoileal intussusception due to metastatic angiosarcoma. We assume that the metastatic tumor in the small intestine was metastasized hematogeneously from cutaneous angiosarcoma, resulting in the formation of nodules and the rapid growth of a pedunculated tumor as a forerunner of the ileoileal intessusception.

Aged↗
PubMed2004

Structural evidence for guanidine-protein side chain interactions: crystal structure of CutA from Pyrococcus horikoshii in 3 M guanidine hydrochloride.

This study was carried out to investigate the structural perturbation of the protein's local structure by the denaturants under non-denaturing conditions. Crystal structure of CutA from an archaeon Pyrococcus horikosii (PhoCutA), a heavy-metal binding protein, was determined at 1.6-angstroms resolution in the presence of 3 M guanidine HCl (GdnHCl). Native PhoCutA has a large number of short intramolecular hydrogen bonds and salt bridges on the protein surface, of which greater than 90% of hydrogen bonds and all salt bridges were retained in 3 M GdnHCl. Hydrogen bonds that disappeared in the GdnHCl crystal structure were mainly located on the protein surface, especially around the structurally perturbed loop, suggesting interactions between peptide groups and GdnHCl. Only a few GdnH+ ions were observed in the crystal structure, although none at the surface, of the protein. Two GdnH+ ions were observed in the center of the trimeric structure, replacing water molecules, and were hydrogen bonded with Asp84 and Asp86 of each chain. The exterior loop from Tyr39 to Lys44, including Trp40-Trp41, was perturbed structurally. Decreases in temperature factors were observed in beta strand 5 and the N terminus of helix 3. These results suggest the specific bindings of GdnH+ with some acidic residues and the non-specific bindings around Trp residues and peptide groups on the protein surface and that binding of GdnHCl to the native protein is limited, resulting in local structural perturbation.

Archaeal Proteins↗
PubMed2004

Accumulation of cyanide and thiocyanate in haemodialysis patients.

BACKGROUND: Cyanide is a toxic agent, and its detoxification product, thiocyanate, may be a major pathogenetic substance in uraemia. Recent studies examining the myeloperoxidase(MPO)/thiocyanate system have suggested a link between thiocyanate and atherosclerosis. However, inaccuracies in conventional assays for cyanide and thiocyanate have limited the understanding of their metabolism in haemodialysis (HD) patients. METHODS: We used high-performance liquid chromatography to measure cyanide in erythrocytes and thiocyanate in plasma in 43 HD patients and in a group of 46 healthy controls that included 15 current smokers. To clarify the metabolic conversion of cyanide to thiocyanate in uraemic patients, we also measured cysteine and sulfate. We then used stepwise regression analysis to analyse factors that determine erythrocyte cyanide and plasma thiocyanate. RESULTS: Mean cyanide and thiocyanate were significantly greater in HD patients than in non-smoking controls. However, cyanide was far below lethal concentrations in dialysis patients. Thiocyanate was six to seven times greater in HD patients than in non-smoking controls, and decreases in thiocyanate following dialysis were only 19.3+/-3.5%. Multiple regression analysis showed a positive correlation between cyanide and thiocyanate in controls, but a negative correlation in HD patients. In patients, an inverse relationship between thiocyanate and BUN was also observed. CONCLUSIONS: The elevation of thiocyanate in patients undergoing dialysis probably is secondary to both limited efficiency of HD and deranged metabolism of cyanide and thiocyanate. Because thiocyanate is a preferred substrate for MPO, it may play a role in uraemic complications including cardiovascular events.

Adult↗
PubMed2004

Structural implications for heavy metal-induced reversible assembly and aggregation of a protein: the case of Pyrococcus horikoshii CutA.

CutA is a small protein that appears to be involved in the mechanism of divalent metal cation tolerance in microorganisms. Here we report the crystal structure of Pyrococcus horikoshii CutA (PhoCutA), with and without Cu(2+), and its metal-binding properties. Crystallographic analyses revealed that PhoCutA forms a stable trimeric structure with intertwined antiparallel beta-strands. The crystal structure of the Cu(2+)-PhoCutA complex shows that the Cu(2+) is located at a trimer-trimer interface and is recognized by the side chains of one Asp(48) from each trimer. In an in vitro experiment, PhoCutA bound to several heavy metals, most of which led to reversible aggregation of the protein; i.e. the aggregates could be completely solubilized by addition of ethylenediamine tetraacetic acid (EDTA) or dialysis against metal free buffer. Substitution of Asp(48) with Ala led to a decrease in the amount of aggregates, suggesting the significant contribution of Asp(48) to the reversible aggregation. To the best of our knowledge, this is the first report which provides the structural evidence for heavy metal-induced multimerization of a protein.

Amino Acid Sequence↗
PubMed2004

Defective regulation of iron transporters leading to iron excess in the polymorphonuclear leukocytes of patients on maintenance hemodialysis.

BACKGROUND: Although hemodialysis (HD) patients are suspected of having defectively regulated iron metabolism, intracellular iron status has never been investigated thoroughly. To clarify the iron metabolism of HD patients, proteins involved in iron import (transferrin receptor [TfR]), as well as export (ferroportin 1), were investigated in polymorphonuclear leukocytes (PMNLs). Relations between iron status and several PMNL functions also were tested. METHODS: Seventeen HD patients and 17 controls were recruited. Relative quantitative polymerase chain reaction was used to measure ferroportin 1 and TfR messenger RNA (mRNA), and ferroportin 1 and TfR expression were semiquantified by means of Western blot analysis or immunohistochemistry. PMNL functions also were examined. RESULTS: Serum iron levels were significantly lower in HD patients than controls, and serum ferritin levels, as well as PMNL ferritin and iron content, were elevated in HD patients. Ferroportin 1 mRNA levels were substantially lower in PMNLs from HD patients, whereas TfR mRNA levels were higher. Western blot analysis and immunohistochemistry confirmed that expression of the corresponding proteins paralleled those of the mRNAs. PMNL phagocytic and bactericidal activity did not differ between HD patients and controls. Chemotactic peptide f-Met-Leu-Phe-stimulated degranulation activity of lactoferrin (Lf) was decreased significantly in HD patients, whereas those of myeloperoxidase and elastase were accelerated. Lf release correlated negatively with intracellular ferritin level. CONCLUSION: We show for the first time that increased iron levels in PMNLs of HD patients were associated with downregulation of ferroportin 1 and upregulation of TfR, which might be linked to hypercytokinemia.

Aged↗
PubMed2004

Application of cell-free translation systems to studies of cofactor binding proteins.

To develop applications of in vitro cell-free translation systems for production and characterization of cofactor binding proteins, we investigate the production of apo- or holo-forms of Flavin Mono Nucleotide (FMN)-binding protein from Desulfovibrio vulgaris (Miyazaki F) and purified them. The redox potential analysis and measurements of UV-, visible, and fluorescent spectra of reconstructed holo-protein showed that the FMN correctly bound to the FMN binding site. On the other hand, contrary to our expectation, we found that the apo-protein formed a dimer structure and the incorporation of the FMN led the conformational alterations of the protein. These studies demonstrate the utility of cell-free translation systems to analyses of cofactor-binding proteins.

Bacterial Proteins↗
PubMed2004

[Endothelin].

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Acute Kidney Injury↗
PubMed2004

[Pericarditis].

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Anti-Inflammatory Agents↗
PubMed2003

Free cysteine is increased in plasma from hemodialysis patients.

BACKGROUND: Although only the total thiol concentration, which includes bound and free forms, has been determined in most previous clinical studies, the free form may be a better predictor of cardiovascular risk. METHODS: We measured the apparent concentration of free homocysteine (Hcy) and cysteine (Cys) in filtered and acid-soluble fractions of plasma in healthy control subjects and in patients with chronic renal failure just before and after a hemodialysis session. RESULTS: In control, filtered Hcy and acid-soluble Hcy were similar, while filtered Cys was much smaller than in acid-soluble Cys. In prehemodialysis samples, filtered Cys was more than 60 times as abundant (259.2 +/- 26.2 micromol/L) as in control samples (4.1 +/- 0.7 micromol/L ). Free-to-total ratios for filtered Cys were 1.6 +/- 0.3% in controls, but 40.9 +/- 2.7% in prehemodialysis patients. CONCLUSIONS: The filtered fraction of thiols can be used to estimate solute transport across the dialysis membrane. In addition, the possible involvement of cysteine in the pathogenesis of atherosclerosis in hemodialysis patients should be reexamined.

Acids↗
PubMed2003

Extra-renal erythropoietin secretion after bilateral nephrectomy.

Serum concentrations of erythropoietin and soluble transferrin receptor were examined immediately after removal of the remaining kidney in a patient with a history of unilateral radical nephrectomy. Unexpectedly, the erythropoietin concentration increased from 18.2 U/L to 42.5 within 6 h after the operation. Soluble transferrin receptor (TfR) concentration rapidly decreased from 2,725 microgram/L to 1,548 during the 12-h postoperative period. Extra-renal erythropoietin secretion could increase to more than twice as much as the renal secretion of erythropoietin immediately after the loss of renal tissue, and the rise in erythropoietin could accelerate the recycling of TfR, thus causing a decrease in its serum concentration.

Aged↗
PubMed2003

Cloning and expression of the superoxide dismutase gene from the obligate anaerobic bacterium Desulfovibrio vulgaris (Miyazaki F).

We identified the SOD gene in the obligate anaerobic bacterium Desulfovibrio vulgaris (Miyazaki F) and constructed a high-level expression system in Escherichia coli. A 2.6-kbp DNA fragment isolated from D. vulgaris (Miyazaki F) by double digestion with EcoRI and SmaI contained the SOD gene and part of another open reading frame. The amino acid sequence deduced from the SOD gene, which was composed of 238 amino acid residues, showed high homogeneity with iron-containing SOD (Fe-SOD) and predicted that the amino terminus of this protein would carry an export signal peptide. We produced the precursor form of SOD (PSOD) and the mature form of SOD (MSOD), which lacked the putative signal peptide. In E. coli, PSOD was present in insoluble inclusion bodies, and its putative signal peptide was not cleaved. In contrast, MSOD contained one iron per mononer and formed a dimer, which exhibited an SOD activity of 850 U/mg. Furthermore, D. vulgaris soluble extract showed a band of SOD activity on native polyacrylamide gel that migrated to the same point as MSOD. The intracellular localization of SOD and its role in D. vulgaris are also discussed.

Amino Acid Sequence↗
PubMed2003

A nitric oxide-generating beta-blocking agent prevents renal injury in the rat remnant kidney model. Comparative study of two beta-blocking drugs, nipradilol and propranolol.

BACKGROUND: The L-arginine-nitric oxide (NO) pathway plays an important role in the modulation of glomerular disease. We investigated whether beta-blocking agents, with and without an NO-generating function, had renoprotective effects in the 5/6 nephrectomized rats (Nx), an animal model of glomerulosclerosis. METHODS: Nipradilol, a beta-blocker with an ONO(2) group (5, 10 or 15 mg/kg/day) and propranolol, a beta-blocker without this group (50 mg/kg/day) were administered for 12 weeks to Nx together with and without nitro-L-arginine methyl ester (L-NAME). We evaluated the effects of both drugs on proteinuria, hypertension, renal function, glomerulosclerosis and urinary excretion of NO metabolites (U(NOx)) and cyclic GMP (U(cGMP)). RESULTS: Both drugs similarly attenuated the elevated blood pressure in Nx. However, nipradilol, at doses of 10 and 15 mg/kg/day, significantly decreased proteinuria and glomerulosclerosis, while propranolol did not. Nx showed reduced U(NOx) in comparison with the sham-operated rats. Nipradilol increased U(NOx) and U(cGMP) significantly and in a dose- dependent manner, whereas propranolol reduced them to levels lower than those in Nx. Nx receiving L-NAME reduced U(NOx). The addition of nipradilol increased U(NOx) and decreased urinary protein excretion and glomerulosclerosis, suggesting that the NO released from the drug contributed to its renoprotective effect. CONCLUSION: These findings indicate that nipradilol exerts its renoprotective effect through NO generation, and not by lowering blood pressure. The beta-adrenergic blocking action per se does not seem to be related to the renoprotective effect of these agents.

Adrenergic beta-Antagonists↗