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Koji Nakamura

Publications and source records attributed to Koji Nakamura.

13 recordsLinked to original sources
PubMed2003

Effect of chromatographic conditions on resolution in high-performance ion-exchange chromatography of proteins on nonporous support.

We explored chromatographic conditions to obtain high resolution in protein separations by ion-exchange chromatography (IEC) on a nonporous anion-exchange resin of 2.5 microm in particle diameter. We studied the effects of gradient time (steepness of salt concentration gradient), flow-rate and column length on resolution in much wider ranges than had been studied before. It was found that two distinct conditions exist that provide high resolution. The first is a condition which has widely been employed in current high-performance IEC, namely, a combination of short gradient time, high flow-rate and comparatively short column. Separation times are usually 5-30 min, and even more rapid (1-2 min) separations are possible. The second is the condition which has rarely been employed in high-performance IEC. It is a combination of long gradient time, low flow-rate and long column. Although it takes several hours for one separation, very high resolution is attainable.

Chromatography, High Pressure Liquid↗
PubMed2003

Characterization of p-aminobenzamidine-based sorbent and its use for high-performance affinity chromatography of trypsin-like proteases.

An affinity sorbent, hydrophilic polymer-based carrier of different pore size (Toyopearl) with immobilized p-aminobenzamidine (ABA), has been prepared. Its basic properties and some applications for protein purification were studied. ABA, which is a synthetic inhibitor for trypsin-like proteases, was covalently immobilized to Toyopearl by reductive amination. The ligand density and binding capacity for porcine trypsin varied depending on the pore size of Toyopearl. The maximum binding capacity of the immobilized p-aminobenzamidine Toyopearl (ABA-Toyopearl) for trypsin was more than 40 mg/ml gel. ABA-Toyopearl thus obtained was very stable below pH 8 and was successfully used for high-performance affinity chromatography of trypsin-like proteases such as trypsin, thrombin, tissue-type plasminogen activator or urokinase in a single step at 25 degrees C.

Benzamidines↗
PubMed2003

Hydrophobicity gradient columns for the separation of trypsin inhibitor by hydrophobic interaction chromatography at low salt concentration.

We investigated hydrophobicity gradient columns composed of two columns packed with supports of different hydrophobicities in order to save time in protein separation by hydrophobic interaction chromatography at low salt concentration using a crude sample of trypsin inhibitor as a model sample. One of the two hydrophobicity gradient columns was packed with a support whose hydrophobicity was critically controlled for target protein (trypsin inhibitor) and the other was packed with a support which was less hydrophobic than the critically controlled hydrophobicity support. It was found that the hydrophobicity gradient columns are useful to separate samples containing impurities of a wide range of hydrophobicities within a reasonable time.

Chromatography, Liquid↗
PubMed2003

Regional intestinal absorption of FITC-dextran 4,400 with nanoparticles based on beta-sitosterol beta-D-glucoside in rats.

Nanoparticles (NP) are potential carriers for drug delivery to the targeted intestine. NP based on beta-sitosterol beta-D-glucoside (Sit-G) enhanced the colon-specific absorption of FITC-dextran 4,400 (FD-4), because the concentration-dependent increase of bioavailability appeared in only the colon. In a permeation study, the absorption enhancement in the colon was suppressed in the following conditions: (1) the addition of Sit-G NP to serosa; (2) a permeation study at 4 degrees C; (3) the addition of endocytosis inhibitor, cytochalasin B. NP based on sitosterol, the aglycon of Sit-G, did not increase the FD-4 colonic permeation. The addition of Sit-G NP to the mucosal side induced a decrease of transepithelial resistance (TEER), but this phenomenon was suppressed by an inhibitor of Na(+)-dependent specific glucose transporter, phrolidzin, which did not affect FD-4 permeation. These findings suggested that absorption enhancement by Sit-G NP may not be due to opening of a tight junction, but might be related to endocytosis via glucose residue of Sit-G.

Algorithms↗
PubMed2003

Structuring parameters for assessment of bone quality using a morphological filter and star volume analysis: structuring property in the cancellous bone of the human femoral head.

This study examines structuring parameters for the assessment of bone quality based on the relationship of bone strength to the trabecular structure or bone mineral density (BMD). Thirty-nine human cancellous bone blocks sampled from resected Ward's triangle area were investigated. The trabecular bone pattern of each bone block was binarized into its corresponding trabecular skeletal pattern using computed radiography with a morphological filter. Every binarized trabecular skeletal pattern was quantified to find the trabecular skeletal pixel percentage (SkP = volume parameter of trabecular skeletal signal component) and skeletal star volume (Vt = connection parameter of trabecular skeletal structure). After BMD and elasticity for each bone block was measured by dual X-ray absorptiometry and the breaking test, the correlations to SkP and Vt were determined. In addition, an attempt was made to detect changes of the trabecular structure in the Ward's triangle area of the femoral head after patients had received treatment for osteoporosis. This evaluation used the structuring parameters SkP and Vt. The results showed that the larger the BMD value, the greater the elasticity, although some samples with similar BMD values had considerably different elasticities. With increased Vt values the trabecular skeletal pattern that had horizontal skeletal elements orientated perpendicular to the loading direction showed higher elasticity. No correlation between SkP and either elasticity or BMD was observed in either the subset (sequential images of morphological filter) or sumset images (combined with subset images). On the other hand, the correlation coefficient between Vt and elasticity was similar to that between BMD and elasticity for subset image ( n = 6). For sumset images (3-6 and 4-7), the correlation coefficient of Vt was equal or higher than that of BMD and the connectivity of the trabecular skeletal structure was more closely linked to bone strength as compared to BMD. Therefore, it was concluded that because no high correlation could be detected between Vt and BMD, BMD and the connectivity of trabeculae are independently related to bone strength. In addition, this method was used to evaluate patients after treatment for osteoporosis, and noticeable changes were seen in the bone trabecular skeletal structure in the Ward's triangle area of the femoral head. In reference to these clinical images, variations in SkP and Vt were quantitatively assessed. These results suggest that the SkP and Vt obtained using computed radiography, morphological filter, and star volume analysis are useful structuring parameters for the assessment of bone quality.

Aged↗
PubMed2003

Orientation and interactions of an essential tryptophan (Trp-38) in the capsid subunit of Pf3 filamentous virus.

The filamentous bacteriophage Pf3 consists of a covalently closed DNA single strand of 5833 nucleotides sheathed by approximately 2500 copies of a 44-residue capsid subunit. The capsid subunit contains a single tryptophan residue (Trp-38), which is located within the basic C-terminal sequence (-RWIKAQFF) and is essential for virion assembly in vivo. Polarized Raman microspectroscopy has been employed to determine the orientation of the Trp-38 side chain in the native virus structure. The polarized Raman measurements show that the plane of the indolyl ring is tilted by 17 degrees from the virion axis and that the indolyl pseudo-twofold axis is inclined at 46 degrees to the virion axis. Using the presently determined orientation of the indolyl ring and side-chain torsion angles, chi(1) (N-C(alpha)-C(beta)-C(gamma)) and chi(2,1) (C(alpha)-C(beta)-C(gamma)-C(delta1)), we propose a detailed molecular model for the local structure of Trp-38 in the Pf3 virion. The present Pf3 model is consistent with previously reported Raman, ultraviolet-resonance Raman and fluorescence results suggesting an unusual environment for Trp-38 in the virion assembly, probably involving an intrasubunit cation-pi interaction between the guanidinium moiety of Arg-37 and the indolyl moiety of Trp-38. Such a C-terminal Trp-38/Arg-37 interaction may be important for the stabilization of a subunit conformation that is required for binding to the single-stranded DNA genome during virion assembly.

Capsid↗
PubMed2002

The Src homology 2 domain-containing inositol 5-phosphatase negatively regulates Fcgamma receptor-mediated phagocytosis through immunoreceptor tyrosine-based activation motif-bearing phagocytic receptors.

Molecular mechanisms by which the Src homology 2 domain-containing inositol 5-phosphatase (SHIP) negatively regulates phagocytosis in macrophages are unclear. We addressed the issue using bone marrow-derived macrophages from FcgammaR- or SHIP-deficient mice. Phagocytic activities of macrophages from FcgammaRII(b)(-/-) and SHIP(-/-) mice were enhanced to a similar extent, relative to those from wild type. However, calcium influx was only marginally affected in FcgammaRII(b)(-/-), but greatly enhanced in SHIP(-/-) macrophages. Furthermore, SHIP was phosphorylated on tyrosine residues upon FcgammaR aggregation even in macrophages from FcgammaRII(b)(-/-) mice or upon clustering of a chimeric receptor containing CD8 and the immunoreceptor tyrosine-based activation motif (ITAM)-bearing gamma-chain or human-restricted FcgammaRIIa. These findings indicate that, unlike B cells, SHIP is efficiently phosphorylated in the absence of an immunoreceptor tyrosine-based inhibition motif (ITIM)-bearing receptor. We further demonstrate that SHIP directly bound to phosphorylated peptides derived from FcgammaRIIa with a high affinity, comparable to that of FcgammaRII(b). Lastly, FcgammaRIIa-mediated phagocytosis was significantly enhanced in THP-1 cells overexpressing dominant-negative form of SHIP in the absence of FcgammaRII(b). These results indicate that SHIP negatively regulates FcgammaR-mediated phagocytosis through all ITAM-containing IgG receptors using a molecular mechanism distinct from that in B cells.

Amino Acid Motifs↗
PubMed2002

Evaluation and applications of a new dye affinity adsorbent.

The basic properties of a new dye affinity adsorbent Toyopearl AF-Blue HC-650M and its applications to the purification of proteins were studied. The binding capacity for human serum albumin (HSA) was greater than 18 mg per ml gel. The dye leakage from Toyopearl AF-Blue HC-650M in 0.5 M NaOH and 0.5 M HCI was less compared with an agarose adsorbent. Caustic stability study also demonstrated this material withstood exposure to 0.1 M NaOH for 1 month with no significant loss of binding capacity for HSA. We purified human albumin from human serum and lactate dehydrogenase (LDH) from rabbit muscle extract in a single step. Sodium dodecylsulfate-polyacrylamide gel electrophoresis indicates that human albumin and LDH were highly purified.

Adsorption↗
PubMed2002

Separation of proteins by hydrophobic interaction chromatography at low salt concentration.

We investigated protein separation by hydrophobic interaction chromatography (HIC) at low salt concentration on the supports of various hydrophobicities. Hydrophobic proteins could be successfully separated with more than 90% recovery by gradient elution of ammonium sulfate from 0.3-0.5 M to 0 in 50 mM phosphate buffer (pH 6.8) by using supports whose hydrophobicities were properly adjusted individually for each protein. Satisfactory results were also obtained by isocratic elution without ammonium sulfate and gradient elution of ethanol from 0 to 10%. HIC at low salt concentration was compatible with other modes of liquid chromatography like ion-exchange chromatography. On the other hand, it was not successful to separate hydrophilic proteins at low salt concentration. Recoveries of hydrophilic proteins decreased before they were retained enough as support hydrophobicity increased. Therefore, it is inevitable to use a higher concentration of salt, e.g., 1-2 M ammonium sulfate, on hydrophilic or moderately hydrophobic support in order to retain hydrophilic proteins without decrease in recovery.

Ammonium Sulfate↗
PubMed2002

How do inhibitory phosphatases work?

We present a hypothesis regarding the mode of induction of the inhibitory phosphatases SHP-1 and SHIP in hematopoietic cells. One mode is a general one in which the phosphatase regulates but does not abort signal transduction and biology. Regulator phosphatases are induced by directly or indirectly engaging the amino acid motifs present in the activating receptor, and act to control the biochemical and biological output. The other mode of induction is a specific one, which critically involves paired co-clustering of activating and inhibitory receptors. Phosphatases working in this way act only under conditions of paired co-clustering of activating and inhibitory receptors, and directly bind amino acid motifs present in the inhibitory receptor. However, this mode of induction is apparently more efficient, as cellular activation is completely aborted. This review presents several examples of each mode of inhibition and speculates on their mechanisms.

Amino Acid Motifs↗
PubMed2002

A novel low-molecular-mass dual-specificity phosphatase, LDP-2, with a naturally occurring substitution that affects substrate specificity.

We have identified a novel dual-specificity phosphatase (DSP), called LDP-2 (low-molecular-mass DSP-2), composed of 220 amino acid residues showing high sequence homology to VHR and LDP-1/TMDP, which belong to a family of DSPs with low molecular masses. The LDP-2 gene is ubiquitously expressed, and LDP-2 is localized in the cytoplasm. The main structural feature of LDP-2 is that the serine-156 residue located in the common active site sequence motif, HCXXGXXRS, for DSP is naturally substituted with an alanine residue. The recombinant LDP-2 protein showed extremely low phosphatase activity towards p-nitrophenyl phosphate (pNPP). Back-mutation of Ala-156 in LDP-2 to a serine (A156S mutation) conferred significant phosphatase activity towards pNPP. However, both LDP-2 and LDP-2 (A156S) exhibited substantial phosphatase activities towards both phospho-seryl/threonyl and -tyrosyl residues of myelin basic protein, with similar specific activities. Ala-156 of LDP-2 might be crucially involved in the recognition of a physiological substrate. We analyzed the effect of VHR and LDP-2 on mitogen-activated protein kinases (MAPKs) in vivo. We first found that VHR inhibits the activation of p38 as well as ERK and JNK, with similar efficiency. Under the conditions used, LDP-2 specifically suppressed JNK activation.

Amino Acid Sequence↗
PubMed2002

Strenuous exercise-induced change in redox state of human serum albumin during intensive kendo training.

A high-performance liquid chromatographic (HPLC) analysis of human serum albumin (HSA) using an ion-exchange (DEAE-form) column shows three components: The principal component corresponds to human mercaptalbumin (HMA); the secondary to nonmercaptalbumin (HNA), having mixed disulfide with cystine (HNA[Cys]), or oxidized glutathione (HNA[Glut]); and the tertiary to HNA, oxidized more highly than mixed disulfide. The purpose of the present study is to clarify the effects of strenuous exercise load on HMA--><--HNA conversion (i.e., dynamic change in redox state) of HSA from elite kendo athletes (n=30; 20.0+/-1.1 years old). They participated in an intensive kendo training camp for 5 d. The mean value for the HMA fraction (f[HMA]) of kendo athletes after camp (62.8+/-2.4%) was significantly lower than before camp (71.9+/-3.7%) (p<0.0005). In contrast, the mean value for f(HNA-1) (i.e., f[HNA(Cys) and HNA(Glut)]) after camp (34.2+/-2.1%) was significantly higher than before camp (25.7+/-3.7%) (p<0.0005). These results suggested that strenuous physical exercise markedly increased the oxidized albumin level in extracellular fluids during the intensive training camp.

Adult↗