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Masafumi Tanaka

Publications and source records attributed to Masafumi Tanaka.

15 recordsLinked to original sources
PubMed2006

Contributions of the N- and C-terminal helical segments to the lipid-free structure and lipid interaction of apolipoprotein A-I.

The tertiary structure of lipid-free apolipoprotein (apo) A-I in the monomeric state comprises two domains: a N-terminal alpha-helix bundle and a less organized C-terminal domain. This study examined how the N- and C-terminal segments of apoA-I (residues 1-43 and 223-243), which contain the most hydrophobic regions in the molecule and are located in opposite structural domains, contribute to the lipid-free conformation and lipid interaction. Measurements of circular dichroism in conjunction with tryptophan and 8-anilino-1-naphthalenesulfonic acid fluorescence data demonstrated that single (L230P) or triple (L230P/L233P/Y236P) proline insertions into the C-terminal alpha helix disrupted the organization of the C-terminal domain without affecting the stability of the N-terminal helix bundle. In contrast, proline insertion into the N terminus (Y18P) disrupted the bundle structure in the N-terminal domain, indicating that the alpha-helical segment in this region is part of the helix bundle. Calorimetric and gel-filtration measurements showed that disruption of the C-terminal alpha helix significantly reduced the enthalpy and free energy of binding of apoA-I to lipids, whereas disruption of the N-terminal alpha helix had only a small effect on lipid binding. Significantly, the presence of the Y18P mutation offset the negative effects of disruption/removal of the C-terminal helical domain on lipid binding, suggesting that the alpha helix around Y18 concealed a potential lipid-binding region in the N-terminal domain, which was exposed by the disruption of the helix-bundle structure. When these results are taken together, they indicate that the alpha-helical segment in the N terminus of apoA-I modulates the lipid-free structure and lipid interaction in concert with the C-terminal domain.

Amino Acid Substitution↗
PubMed2006

Effect of carboxyl-terminal truncation on structure and lipid interaction of human apolipoprotein E4.

Apolipoprotein (apo) E4 has been identified as a major risk factor for Alzheimer's disease. Recently, apoE4 was found to undergo proteolytic cleavage in Alzheimer's disease brains, resulting in neurotoxic C-terminal-truncated fragments. In this study, we examined the effect of progressive truncation of the C-terminal domain in apoE4 on its lipid-free structure and lipid binding properties. Circular dichroism measurements demonstrated that deletion of residues 273-299 or 261-299 significantly decreased the number of helical residues, suggesting that the C-terminal residues 261-299 have alpha-helical structure. Although the progressive deletions in the C-terminal domain appear to somewhat increase thermal stability, apoE4 (delta273-299) and apoE4 (delta261-299) showed stability similar to that of the apoE4 22-kDa fragment (residues 1-191) when denatured with guanidine-HCl, indicating that residues 192-272 have a negligible effect on the stability of the C-terminal-truncated apoE4. Comparison of Trp-264 fluorescence in single Trp mutants of full-length and C-terminal-truncated apoE4 (delta273-299) indicated that the C-terminal domain structure in the latter is both less organized and cooperative. In addition, comparison of the binding of the C-terminal-truncated mutants to a hydrophobic fluorescent dye and to lipid emulsions revealed that residues 261-272 create a hydrophobic site which is critical for lipid binding. These results suggest that removal of a hydrophobic C-terminal alpha-helical segment (residues 273-299) to create C-terminal-truncated apoE4 forms found in brain leads to less organized C-terminal structure while still retaining a second alpha-helical lipid-binding region (residues 261-272) that is available for interaction with cell membranes and other proteins such as amyloid beta peptide.

Amino Acid Sequence↗
PubMed2005

Specific sequences in the N and C termini of apolipoprotein A-IV modulate its conformation and lipid association.

Apolipoprotein (apoA-IV) is a 376-residue exchangeable apolipoprotein that may play a number of important roles in lipid metabolism, including chylomicron assembly, reverse cholesterol transport, and appetite regulation. In vivo, apoA-IV exists in both lipid-poor and lipid-associated forms, and the balance between these states may determine its function. We examined the structural elements that modulate apoA-IV lipid binding by producing a series of deletion mutants and determining their ability to interact with phospholipid liposomes. We found that the deletion of residues 333-343 strongly increased the lipid association rate versus native apoA-IV. Additional mutagenesis revealed that two phenylalanine residues at positions 334 and 335 mediated this lipid binding inhibitory effect. We also observed that residues 11-20 in the N terminus were required for the enhanced lipid affinity induced by deletion of the C-terminal sequence. We propose a structural model in which these sequences can modulate the conformation and lipid affinity of apoA-IV.

Amino Acid Sequence↗
PubMed2005

Effects of the core lipid on the energetics of binding of ApoA-I to model lipoprotein particles of different sizes.

Interaction of apolipoproteins (apo) with lipid surfaces plays crucial roles in lipoprotein metabolism and cholesterol homeostasis. To elucidate the thermodynamics of binding of apoA-I to lipid, we used lipid emulsions composed of triolein (TO) and egg phosphatidylcholine (PC) as lipoprotein models. Determination of the level of binding of wild-type (WT) apoA-I and some deletion mutants to large (120 nm diameter; LEM) and small (35 nm diameter; SEM) emulsions indicated that N-terminal (residues 44-65) and C-terminal (residues 190-243 and 223-243) deletions have large effects on lipid interaction, whereas deletion of the central region (residues 123-166) has little effect. Substitution of amino acids at either L230 or L230, L233, and Y236 with proline residues also decreases the level of binding, indicating that an alpha-helix conformation in this C-terminal region is required for efficient lipid binding. Calorimetry showed that binding of WT apoA-I to SEM generates endothermic heat (DeltaH approximately 30 kcal/mol) in contrast to the exothermic heat (ca. -85 kcal/mol) generated upon binding to LEM and egg PC small unilamellar vesicles (SUV). This exothermic heat arises from an approximately 25% increase in alpha-helix content, and it drives the binding of apoA-I to LEM and SUV. There is a similar increase in alpha-helix content of apoA-I upon binding to either SEM or SUV, but the binding of apoA-I to SEM is an entropy-driven process. These results suggest that the presence of a core triglyceride modifies the highly curved SEM surface packing and thereby the thermodynamics of apoA-I binding in a manner that compensates for the exothermic heat generated by alpha-helix formation.

Apolipoprotein A-I↗
PubMed2005

An EYFP insertion mutant containing a modified lox sequence for potential use as a recombination indicator.

The Cre-loxP system has been used for genetic engineering in many organisms ranging from E. coli to mouse. It is known that intermolecular recombination between two loxP sites is substantially less frequent than intramolecular recombination between the same sites. Therefore, for the efficient isolation of intermolecular loxP recombination products, it is essential to develop a powerful screening method. Here, we describe the construction of an EYFP variant (EYFP157) that could become a vital tool in such a screening method. EYFP157 contains, between Gln157 and Lys158, the JT15:JTZ17 mutant loxP sequence that is not cleaved by the Cre protein. When EYFP157 was expressed at 37 degrees C in E. coli and mouse cells, fluorescence of the protein was readily detectable under a standard microscope. EYFP157 possessed 1-3% of the fluorescence from the wild-type EYFP. Interestingly, at room temperature EYFP157 fluoresced more efficiently in E. coli cells and thus the difference in fluorescence intensity between EYFP157 and the wild-type EYFP became negligible. The potential application of EYFP157 as a recombination indicator in both E. coli and mouse cells is further discussed.

Amino Acid Sequence↗
PubMed2004

Multiplex real-time RT-PCR for prospective evaluation of WT1 and fusion gene transcripts in newly diagnosed de novo acute myeloid leukemia.

Prognostic assessment is crucial for the management of AML. Although the use of karyotype analysis for risk-stratification is widely accepted, prognosis of AML remains ambiguous, particularly for patients categorized into the intermediate cytogenetic risk group and additional markers are required for an accurate prediction of outcome. For this study, we used multiplex real-time RT-PCR, which can simultaneously quantify WT1 and 10 distinct fusion gene transcripts, to prospectively evaluate the pre-treatment bone marrow findings of 53 de novo AML patients. Five patients with normal karyotype or insufficient metaphases detected by conventional karyotype analysis proved to have AML1-MTG8, CBFbeta-MYH11 or PML-RARalpha fusion transcripts. WT1 overexpression was observed in 92% of the patients, and the levels were significantly higher in the cytogenetic favorable risk group, especially patients with PML-RARalpha. WT1 levels also correlated with the percentage of blasts in bone marrow, especially in cases of core-binding factor leukemia. There was no association between initial WT1 levels and outcome in terms of event-free survival or overall survival. These results suggest that multiplex real-time RT-PCR is rapid and useful for the precise cytogenetic stratification of AML, and that WT1 levels at presentation correlate with several biologic features of leukemia, but have no prognostic significance.

Adolescent↗
PubMed2004

Effect of physical training on corrected QT dispersion in patients with nonischemic heart failure.

BACKGROUND: The effect of physical training (PT) on QTc dispersion and ventricular tachycardia (VT) remains unclear in patients with nonischemic heart failure. METHODS AND RESULTS: Eight patients with nonischemic heart failure performed PT using a bicycle ergometer and their exercise tolerance increased (4.9+/-1.8 to 7.0+/-2.5 METs, p<0.05) and QTc dispersion decreased (71+/-22 to 48+/-24 ms, p<0.05). However, PT did not change the frequency of VT. CONCLUSION: Physical training could improve QTc dispersion in patients with nonischemic heart failure, possibly by improving the autonomic nerve system.

Adult↗
PubMed2004

[A case of colon carcinoma with unresectable multiple liver metastases responding to various combined chemotherapies centering on hepatic arterial infusion therapy].

We have experienced a case of colon carcinoma with unresectable multiple liver metastases responding to various combined chemotherapies centering on hepatic arterial infusion therapy. A 42-year-old female with descending colon carcinoma and synchronous unresectable multiple liver metastases underwent left hemicolectomy in September 2001. She was treated with chemotherapy centering on hepatic arterial infusion (HAI) following implantation of a reservoir in the outpatient setting. Pharmacokinetic modulating therapy (PMC), and HAI with Leucovorin/CDDP/5-FU and Levoforinate/CDDP/5-FU were conducted sequentially. Alternative HAI of 5-FU with intravenous infusion of Levoforinate and systemic irinotecan/CDDP were continued biweekly after release of the hepatic artery occlusion. Liver metastases shrunk after 6 months of treatment and were remarkably decreased in size in May 2003. Although 3 metastatic lesions existed in both lungs, good quality of life has been maintained in the 2 years following surgery.

Adenocarcinoma↗
PubMed2004

[A case of hepatocellular carcinoma with bone metastasis responding to concurrent TS-1/low-dose cisplatin (CDDP) therapy and radiotherapy].

A standard treatment for hepatocellular carcinoma with extrahepatic metastasis is not established and chemotherapy is ineffective. We experienced a case of hepatocellular carcinoma with bone metastasis that responded to concurrent TS-1/low-dose cisplatin (CDDP) therapy and radiotherapy. A 58-year-old male patient with left iliac bone metastasis after 2 hepatectomies was admitted to our hospital. The titer of serum AFP and PIVKA-II showed an extremely high levels, 12,350.5 ng/ml and 993 mAU/ml, respectively. The uptake area was found at the left iliac bone by scintigraphy with 99mTc-HMDP. Treatment with TS-1/low-dose CDDP therapy and radiotherapy (36 Gy) was started concurrently. The chemotherapy regimen comprised daily oral administration of 100 mg of TS-1 for 21 days and CDDP 10 mg/body infusion (day 1-5, 8-12). An additional 2 courses of TS-1/low-dose CDDP therapy were repeated. After that, severe pain diminished and the titer of serum showed AFP and PIVKA-II had improved to within normal ranges. The uptake at the left iliac bone was found to have decreased by scintigraphy. Adverse events were grade 1 nausea and leucopenia. TS-1/low-dose CDDP therapy seems to be applicable for the treatment of hepatocellular carcinoma with bone metastasis.

Antineoplastic Combined Chemotherapy Protocols↗
PubMed2003

Effect of cholesterol on bilayer location of the class A peptide Ac-18A-NH2 as revealed by fluorescence resonance energy transfer.

An amphipathic class A peptide, Ac-18A-NH(2), has been employed in modeling the alpha-helical lipid-binding site of apolipoprotein A-I (apoA-I). To gain insight into the nature of protein-lipid interactions responsible for the ability of apoA-I to promote the efflux of intracellular cholesterol, the peptide disposition in model membranes composed of phosphatidylcholine (PC) and its mixture with cholesterol (Chol) has been characterized. By examining resonance energy transfer between the peptide Trp as a donor and anthrylvinyl-labeled PC as an acceptor it was found that Chol inclusion is conducive to shallower bilayer location of the Ac-18A-NH(2) alpha-helix. The limits for the Trp distance from the membrane center were estimated to be 1.5-1.7 nm (PC) and 1.9-2.1 nm (PC:Chol), indicating that in the PC bilayer the Trp resides at the level of the glycerol backbone and carbonyl groups while the region of the phosphocholine moieties is preferable for Trp location in the PC:Chol bilayer. These findings suggest that Chol can modulate the interactions between apoA-I and membrane lipids via reducing the depth of alpha-helix bilayer penetration.

Apolipoproteins↗
PubMed2003

Regions of GAL4 critical for binding to a promoter in vivo revealed by a visual DNA-binding analysis.

Binding of transcriptional activators to specific sites on DNA, mediated by their DNA-binding domain, is a key regulatory point in transcriptional regulation. With a GFP-based microscopic assay, we investigated how the prototypical activator GAL4 effectively binds to a promoter in living yeast cells. We show that GAL4 relies on a previously unrevealed mechanism involving 'DNA-binding enhancers' (DBEs), the regions of GAL4 that assist DNA-binding domain association with DNA. GAL4 contains two DBEs, one, but not the other, physically overlapping the principal transcriptional activation domain. Either of the DBEs, however, can function independently of transcriptional activation, indicating a discrete mechanism responsible for DNA-binding enhancement. The effect of DBEs, while not limited to natural target promoters, is still not universal and can be profoundly affected by the binding-site context. The GAL4 DBEs can also enhance promoter binding of an unrelated DNA-binding domain, and possibly represent a new modular functional unit responsible for effective binding of diverse regulatory factors to DNA in vivo.

Amino Acid Sequence↗
PubMed2003

Tumor budding at the invasive margin can predict patients at high risk of recurrence after curative surgery for stage II, T3 colon cancer.

PURPOSE: The aim of this study was to identify indicators that can predict patients at high risk of tumor recurrence in Stage II, T3 colon cancer. METHODS: A total of 138 patients classified as Stage II, T3 underwent curative resection of colon cancer between 1981 and 1993. Clinical variables included age, gender, bowel obstruction, tumor location, and emergency presentation. For each colon tumor specimen, the following histopathological variables were assessed: maximum tumor diameter (<5 vs. >or=5 cm), depth, tumor grade (well and moderate vs. other), lymphatic and venous invasion (absent vs. present), perineural invasion, tumor necrosis, and tumor margin (expanding vs. infiltrating). We also categorized tumor budding, defined as a single cancer cell or small clusters of undifferentiated cancer cells in the invasive frontal lesion, into two categories: none or minimal (BD-1), and moderate or severe (BD-2). Univariate analysis for factors regarding recurrence and disease-specific survival were performed with the logistic regression model and the log-rank test. RESULTS: Among the factors analyzed, tumor budding was the only factor that was significantly associated with recurrence and survival. The numbers of patients with BD-1 and BD-2 tumors were 111 and 27, respectively. Forty-eight percent of BD-2 tumor patients developed recurrence, compared with 4.5 percent of BD-1 tumor patients (P < 0.0001). The cumulative disease-specific survival rates at five years for patients with BD-1 and BD-2 tumors were 98 and 74 percent, respectively (P < 0.0001). CONCLUSION: The presence of moderate or severe budding at the invasive margin in Stage II, T3 colon cancer indicated a high risk of tumor recurrence after curative surgery, providing useful information for the decision regarding postoperative adjuvant chemotherapy.

Aged↗
PubMed2002

Cholesterol modulates interaction between an amphipathic class A peptide, Ac-18A-NH2, and phosphatidylcholine bilayers.

Cholesterol (Chol) in phosphatidylcholine large unilamellar vesicles (PC LUV) modulated interaction of the bilayers with a class A amphipathic peptide, Ac-18A-NH2: Chol increased the peptide binding capacity and reduced the affinity together with the peptide-induced leakage of calcein from LUV. Similar effects of Chol have been observed on the interaction of LUV with apoA-I [Saito, H., Miyako, Y., Handa, T., and Miyajima, K. (1997) J. Lipid Res. 38, 287-294]. Circular dichroism (CD) spectra of the peptide indicated a similar helical structure formation in LUV with and without Chol. The fluorescence spectral shift, quantum yield, anisotropy, and acrylamide-quenching of the peptide Trp indicated that in PC:Chol (3:2) LUV, Ac-18A-NH2 was located in a more polar membrane environment with increased motional freedom and greater accessibility to the aqueous medium. Fluorescence energy transfer from the Trp indole ring to acceptors situated at different depths in the bilayers revealed that the amphipathic peptide penetrated the hydrophobic interior of PC bilayers, while the peptide was located at the polar zwitterionic surface in PC:Chol LUV. The inclusion of Chol causes the headgroup separation of PC at the surface of LUV and increases the binding maximum of the wedge-shaped amphipathic peptide without disrupting the membrane structure. In addition, the rigidifying effect of Chol on PC acyl chains prevents the penetration of the peptide into the bilayer interior. These findings imply that Chol in membranes affects the binding and motional freedom of exchangeable plasma apolipoproteins containing class A amphipathic sequences, e.g., apoA-I and apoCs.

Cholesterol↗
PubMed2002

Depth profiling of ultra trace metal impurities in polytetrafluoroethylene wares by surface scraping and acid-vapor extraction followed by ICP-MS analysis.

This paper describes the development of the depth profiling method of ultra trace metal impurities in polytetrafluoroethylene (PTFE) wares based on contamination-free sampling followed by acid-vapor extraction and its application to evaluate the washing method for PTFE wares. A contamination-free sampling process was achieved by scraping the surface of PTFE wares with the cleft face of a silicon wafer followed by exposing the PTFE scraped to highly pure acid-vapor. The concentration of metal impurities in extractants was determined by ICP-MS equipped with an electrothermal vaporizer (ETV-ICP-MS). The blank values of Al, Cr, Fe, Ni and Cu by the depth profiling method were 0.006, 0.004, 0.005, 0.002 and 0.003 ng, respectively. By analyzing the depth profile of beakers, the distributions of ultra trace (ng g(-1) level) metal impurities were clarified. An examination of the washing methods by the depth profiling method also clarified that exposing to acid-vapor was more effective than the acid-dipping method for the elimination of metal impurities.

Journal Article↗