Biomedical subjects

Takashi Manabe

Publications and source records attributed to Takashi Manabe.

18 recordsLinked to original sources

PubMed2007

Alkaline extraction of human plasma proteins from nondenaturing micro-2-D gels for protein/polypeptide mass measurement and peptide mass fingerprinting using MALDI-TOF MS.

Previously, we have reported a high-efficiency method of protein extraction from CBB-stained polyacrylamide gels for molecular mass measurement with MALDI-TOF MS [1]. In the present work, the alkaline extraction method was applied to CBB-stained 2-DE gels on which human plasma proteins were separated in the absence of denaturant. In order to examine the performance of the method, ten spots with apparent molecular masses (MMapp) in the range of 65 to 1000 kDa were selected and the proteins were extracted from the gel pieces. The extracts were subjected to whole-mass measurement by MALDI-TOF MS, with and without DTT treatment. In addition, the extracts were subjected to in-solution trypsin digestion followed by MALDI-TOF MS and PMF analysis. Successful extraction of proteins from the ten spots, up to MMapp 1000 kDa, has been ascertained by the significant PMF assignment (MASCOT) with high sequence coverage of the respective proteins or polypeptides. When direct mass measurement of the extracted proteins was attempted, three spots in MMapp range 65-100 kDa provided mass peaks. Five spots in MMapp range 150-400 kDa did not give mass peaks of the intact proteins, but showed those of the constituent polypeptides after the DTT treatment. Extraction of proteins prior to trypsin digestion enabled the procedure of PMF analysis to be much simpler than the conventional in-gel digestion method, providing comparable protein scores and sequence coverage. The technique presented here suggests a new strategy for the characterization of proteins separated by nondenaturing 2-DE.

Acrylic Resins↗

PubMed2006

Regulation of IL-27p28 gene by lipopolysaccharide in dendritic DC2.4 cells.

To elucidate the regulation of IL-27p28 gene, we analyzed the promoter region of the gene in DC2.4 cells with or without lipopolysaccharide (LPS)-treatment. The results indicate that a region (-648 to -364) of p28 promoter was responsible for LPS-induction. EMSA with DNA probes within the region reveals that binding of GATA motif bound proteins was decreased by LPS-treatment. We identified one of the proteins as non-POU domain-containing octamer binding protein (NonO). Taken together, LPS-induced activation of IL-27p28 gene can be accounted for by the displacement of bound NonO protein from the IL-27p28 promoter.

Animals↗

PubMed2006

Analysis of hydrolytic activity of phospholipase Calpha from porcine retina on retinyl ester and phosphatidylcholine using non-denaturing two-dimensional electrophoresis and mass spectrometry.

Hydrolysis of retinyl esters and phospholipids is important for visual functions of the animal retina. This study aimed to examine hydrolytic activity of an enzyme with native substrates such as retinyl esters and phospholipids responsible for this function in porcine retina. After cytosolic proteins were extracted from porcine retina, the proteins were separated using non-denaturing two-dimensional electrophoresis (2DE). Some major proteins and phospholipase Calpha were identified by matrix-assisted laser desorption ionisation-time of flight-mass spectrometry (MALDI-TOF-MS) or electrospray ionisation-tandem mass spectrometry (ESI-MS/MS). The phospholipase Calpha showed hydrolytic activities with not only alpha-naphtyl acetate but also with retinyl palmitate and phosphatidylcholine when effects of different substrates were investigated using enzyme activity staining on 2DE or MALDI-TOF-MS. Results indicated that hydrolytic activity of the enzyme with non-native and native substrates could be examined using a combination of non-denaturing 2DE and MALDI-TOF-MS.

Amino Acid Sequence↗

PubMed2005

Glucose oxidase assisted homogeneous electrochemical receptor binding assay for drug screening.

Although the idea of homogeneous electrochemical immunoassay using antibody and an electroactive modified antigen as a probe looks to be very useful for high-throughput drug screening, there have been few reports. One reason for this is the difficulty experienced making an electroactive probe, because the introduction of electroactive compounds to antigens often interferes with the antigen-antibody interaction. To apply a homogeneous electrochemical assay to drug screening, we have designed new probes referring to the information of immobilization on beads which could identify the drug receptor. FK506 (also called Tacrolimus), immunosuppressive agent is modified with ferrocene derivatives as an electron mediator between glucose oxidase and an electrode, at a non-obstructing part. One of the probes still indicated the electrochemical activity as a mediator and had the specific binding capability for FKBP12 (FK506 binding protein). The current decrease in response to the additional FKBP12, detected with constant voltage amperometry using the probe, was observed within 5 min. Then, free FK506 as a leader drug, rapamycin and cyclosporine A as unknown drugs were used as a model for drug screening. Since the order of response currents at the same concentration of each drug reflected their binding constants, it was shown that binding capacity of an unknown drug candidate could be estimated by comparison of response currents between the leader drug and the unknown drug candidate. Thus, this glucose oxidase assisted homogeneous electrochemical drug-receptor binding assay has been proved to be a useful tool for drug screening.

Biosensing Techniques↗

PubMed2005

Detection of activity and mass spectrometric identification of mouse liver carboxylesterase and aldehyde dehydrogenase separated by non-denaturing two-dimensional electrophoresis after extraction with detergents.

To examine the activities and identity of enzymes associated with organelles such as microsomes and mitochondria, proteins from mouse liver were extracted using the non-ionic detergents Nonidet P-40 (NP-40), polyoxyethylene sorbitan monooleate (Tween 80), polyoxyethylene isooctylphenyl ester (Triton X), n-octyl beta-D-glucoside (octyl glycoside) or anionic detergent sodium dodecylsulfate (SDS) after the removal of cytosolic proteins. The proteins extracted by detergents were separated by non-denaturing two-dimensional electrophoresis (2-DE). The activities of esterase and aldehyde dehydrogenase were retained by non-denaturing 2-DE after treatment with each non-ionic detergent, but the activities were reduced or lost when the proteins were extracted with more than 0.5% SDS. For proteomic analysis of the organelle-associated proteins in mouse liver, proteins were separated by non-denaturing 2-DE and were identified using electrospray ionization tandem mass spectrometry (ESI-MS/MS) after the proteins were solubilized by octyl glycoside, NP-40 and 0.1% SDS. Several organelle-associated proteins such as carboxylesterase, aldehyde dehydrogenase, glucose regulated protein and HSP60 were identified. These results indicate that the activities and identity of detergent-soluble enzymes can be examined by this non-denaturing 2-DE and mass spectrometry.

Aldehyde Dehydrogenase↗

PubMed2005

Selective ligand purification using high-performance affinity beads.

Since the development of affinity chromatography, affinity purification technology has been applied to many aspects of biological research, becoming an indispensable tool. Efficient strategies for the identification of biologically active compounds based on biochemical specificity have not yet been established, despite widespread interest in identifying chemicals that directly alter biomolecular functions. Here, we report a novel method for purifying chemicals that specifically interact with a target biomolecule using reverse affinity beads, a receptor-immobilized high-performance solid-phase matrix. When FK506-binding protein 12 (FKBP12) immobilized beads were used in this process, FK506 was efficiently purified in one step either from a mixture of chemical compounds or from fermented broth extract. The reverse affinity beads facilitated identification of drug/receptor complex binding proteins by reconstitution of immobilized ligand/receptor complexes on the beads. When FKBP12/FK506 and FKBP12/rapamycin complexes were immobilized, calcineurin and FKBP/rapamycin-associated protein were purified from a crude cell extract, respectively. These data indicate that reverse affinity beads are powerful tools for identification of both specific ligands and proteins that interact with receptor/ligand complexes.

Animals↗

PubMed2005

Alkaline cleavage of covalent bonds in chicken insulin and bovine alpha-lactalbumin analyzed by matrix-assisted laser desorption/ionization-mass spectrometry.

In the course of searching methods to extract proteins from Coomassie blue-stained polyacrylamide gels, we found proteins are extracted in relatively high recovery when the gel pieces are soaked in alkaline solutions. However, alkaline conditions are known to cause decomposition of proteins, especially peptide bond cleavage and disulfide degradation. We studied the effects of alkaline on two purified proteins, chicken insulin and bovine alpha-lactalbumin, both containing four disulfide bonds in their structure. The process of covalent bond cleavage was traced by analyzing the mass spectra of the proteins using matrix-assisted laser desorption/ionization-mass spectrometry (MALDI-MS). When the proteins are kept at pH 13 in the presence of 0.1% dithithreitol (DTT), peptide bonds at the C-terminal side of asparaginyl residues are preferably cleaved producing succinimides, whereas cysteinyl residues are not decomposed. In the absence of DTT, the disulfide bonds of the proteins are decomposed by alkaline and the cleavage of the peptide bonds are less obvious, possibly because the conformation of the proteins are partially retained until the full decomposition of disulfide bonds. These results identified for the first time the cleavage sites of proteins under alkaline treatment and further suggested the general tendency of the reactions, both in the presence and absence of DTT.

Alkalies↗

PubMed2005

High-efficiency protein extraction from polyacrylamide gels for molecular mass measurement by matrix-assisted laser desorption/ionization-time of flight-mass spectrometry.

A simple and fast method of protein extraction from Coomassie Brilliant Blue (CBB)-stained polyacrylamide gels suited for molecular mass measurement of proteins by matrix-assisted laser desorption/ionization-time of flight-mass spectrometry (MALDI-TOF-MS) is reported. Proteins in CBB-stained gel pieces were extracted by a 10-min soaking in 0.1 M NaOH at 25 degrees C. The recovery of this one-step extraction method was 34-73% for proteins <67 kDa. CBB adduction to proteins during mass spectrometric analysis was avoided by a destaining step before the alkaline extraction. The molecular mass values of the extracted proteins coincided with those of purified proteins within +/-0.01-0.10% deviation for all the proteins <36 kDa. Because of the high extraction recovery, mass measurement was possible for the proteins extracted from CBB-stained gels with loaded protein quantities as little as 34 ng for cytochrome c, alpha-lactalbumin, myoglobin, beta-lactoglobulin, trypsinogen, and carbonic anhydrase (12.4-29.0 kDa), 340 ng for glyceraldehyde-3-phosphate dehydrogenase (35.6 kDa) and albumin (66.3 kDa). This method provides a highly efficient approach to utilize CBB-stained one- or two-dimensional gels for whole protein analysis using MALDI-TOF-MS.

Carbonic Anhydrases↗

PubMed2005

Direct targeting of human plasma for matrix-assisted laser desorption/ionization and analysis of plasma proteins by time of flight-mass spectrometry.

A method to analyze human plasma proteins without fractionation, directly applying a plasma-matrix mixture on the target plate of a matrix-assisted laser desorption/ionization-time of flight-mass spectrometer (MALDI-TOF-MS), has been described. Peaks of ionized plasma proteins could not be detected applying a mixture of an undiluted plasma sample and a matrix solution, but they appeared when the plasma was diluted before mixing with the matrix. Tenfold diluted plasma provided well-resolved protein peaks in the m/z range from 4000 to 30,000. The addition of a simple post-crystallization washing procedure performed on the target plate further improved the quality of mass spectra. We numbered 58 peaks in the range of 4-160 kDa and 32 out of which were assigned to the plasma protein species which have been reported. Especially high sensitivity and resolution were obtained in the region < 30 kDa, where multiple isoforms of apolipoprotein A-I, apolipoprotein A-II, apolipoprotein C-I, apolipoprotein C-II, apolipoprotein C-III, and transthyretin could be assigned. Various post-translational modifications are involved in the isoforms, e.g., proteolytic cleavage, glycosylation and chemical modifications. This method will become complementary with the present electrophoretic techniques, especially for the analysis of low-molecular-mass proteins.

Apolipoproteins↗

PubMed2005

Rapid separation of microorganisms by quartz microchip capillary electrophoresis.

We developed and optimized a system coupling microchip capillary electrophoresis (MCE) and laser-induced fluorescence (LIF) detection for the analysis of microorganisms. The MCE-LIF system successfully separated pure cultures of lactic acid bacteria and Saccharomyces cerevisiae within 200 s. The results indicate that the MCE system can be conveniently used for the rapid and highly sensitive detection of microorganisms. Thus, MCE can provide a cheap and simple method for the on-line detection of microbial contamination.

Bacteria↗

PubMed2004

Simultaneous analysis of esterase and transferase activities in cytosol proteins from the bovine retina by using microscale non-denaturing two-dimensional electrophoresis.

Esterase and transferase activities were analyzed simultaneously after cytosol proteins in the bovine retina were separated by microscale non-denaturing two-dimensional electrophoresis (2-DE). Esterase activity was specifically inhibited by an esterase inhibitor, 9-amino-1,2,3,4-tetra hydroacridine (tacrine), and transferase activity was specifically inhibited by a glutathione S-transferase (GST) inhibitor, 2-phenyl-1,2-benziso selenazol-3(2H)-one (ebselen). Both esterase and transferase were precipitated when ammonium sulfate was added to the cytosol up to 50% saturation (50% AS fraction), and were detected in the 50% AS fraction by using the 2-DE. After the cytosol proteins in the 50% AS fraction were separated by using non-denaturing 2-DE, polypeptides of the separated proteins were identified by peptide mass fingerprinting and post-source decay analysis by using MALDI-MS, or by immunoreactivity by using a specific antibody. The spots of esterase and transferase activities in the 2-DE pattern were identified as phosphodiesterase and GST, respectively. This simultaneous analysis of enzyme activities can be applied to screen-specific or non-specific medicines which affect enzyme activities.

Animals↗

PubMed2004

Nondenaturing two-dimensional electrophoresis enzyme profile involving activity and sequence structure of cytosol proteins from mouse liver.

After cytosol proteins in the mouse liver were separated by nondenaturing two-dimensional electrophoresis (2-DE), activities of several enzymes, such as fructose bisphosphatase, sorbitol dehydrogenase and malate dehydrogenase, transferase and sorbitol dehydrogenase, or several dehydrogenases, were analyzed on the same 2-D gel. Further, peptidase (or protease) activity can be examined by matrix-assisted laser desorption/ionization-time of flight-mass spectrometry (MALDI-TOF-MS) when peptides such as angiotensin and adenocorticotropic hormone are incubated in the presence of the cytosol protein separated by nondenaturing 2-DE. Sequence structures of proteins on the 2-D gel were analyzed by peptide mass fingerprinting using MALDI-TOF-MS or by peptide sequencing using electrospray ionization-tandem mass spectrometry (ESI-MS/MS). The combination of activity and sequence structure accurately verified the position and activity range of the separated enzymes on the nondenaturing 2-D gel. From these results, we created a nondenaturing 2-D enzyme profile involving activities and sequence structure of cytosol proteins from mouse liver. This profile can be used for checking whether activities of enzymes were specifically or nonspecifically inhibited by inhibitors.

Adrenocorticotropic Hormone↗

PubMed2003

Positive relationship between bone mineral density and low back pain in middle-aged women.

There have been a large number of epidemiological studies demonstrating various primary factors that cause musculoskeletal disorders in middle-aged and older women. However, the relationship between low back pain and bone mineral density is not well documented, and no evidence for any direct relationship between the two has been found. To investigate the relationship, we conducted a cross-sectional study, on a population of 2,244 Japanese women aged 25-85 years who were participating in a regional health screening program. Information on lifestyle, reproductive characteristics and the presence of current low back pain was collected by self-administered questionnaires, and bone mineral density at the distal radius was measured. We found increasing bone mineral density to be significantly associated with low back pain in middle-aged women using a logistic regression analysis. Exercise and smoking were also significantly associated with low back pain. This association remained even after entering other lifestyle and reproductive factors into the final model. Accordingly, high bone mineral density would seem to be as important a public health problem as low bone mineral density and osteoporosis when considering the musculoskeletal symptoms and disabilities that appear in middle-aged women.

Adult↗

PubMed2003

Analysis of complex protein-polypeptide systems for proteomic studies.

Two-dimensional polyacrylamide gel electrophoresis (2-D PAGE), followed by protein extraction and characterization with chemical sequencing or mass spectrometry (MS), is the most commonly used method to analyze complex protein systems such as cells and organelles. However, it is claimed that 2-D PAGE is a slow and labor-intensive technique and also needs subsequent efforts for one-by-one identification of proteins. Recently, the combined methods of Fourier transform ion cyclotron resonance (FTICR) mass spectrometry, with preceding separation techniques such as capillary isoelectric focusing (CIEF) or liquid chromatography, have been demonstrated as high-throughput techniques suitable for proteomic analysis of protein systems. The studies which employ FTICR MS, aimed at the analysis of complex protein systems, have been reviewed, comparing their performance with that of 2-D PAGE. Also, the possibilities of combining 2-D PAGE and the FTICR MS method to analyze and reconstruct the structures and functions of complex systems are discussed.

Electrophoresis, Gel, Two-Dimensional↗

PubMed2003

Detection of protein-protein interactions and a group of immunoglobulin G-associated minor proteins in human plasma by nondenaturing and denaturing two-dimensional gel electrophoresis.

The dissociation of noncovalently associated protein-protein complexes in human plasma was examined by comparing two-dimensional gel electrophoresis (2-DE) patterns obtained in two different electrophoretic conditions. A type I 2-DE pattern was obtained running nondenaturing isoelectric focusing (IEF) followed by nondenaturing gel electrophoresis and a type II 2-DE pattern was nondenaturing IEF followed by sodium dodecyl sulfate gel electrophoresis. Micro-sized gels (internal diameter(id) 1.3 x 35 mm polyacrylamide IEF gels and 38 x 38 x 1 mm polyacryamide slab gels) were used to follow the dissociation processes of major plasma proteins. Larger gel sizes (id 3.4 x 160 mm agarose IEF gels and 160 x 120 x 2.8 mm polyacrylamide slab gels) were used to detect minor plasma proteins dissociated from major proteins. About 110 spots, which have not been detected on type I (nondenaturing) 2-D gels, newly appeared on type II large-sized 2-D gels at molecular masses smaller than 67 kDa. Some of these spots had been analyzed and identified, but about 70 minor spots (isoelectric point 5.5-7.5 and relative molecular mass 8-45 kDa) were detected for the first time by applying large volumes of human plasma samples to the large type II 2-D gels. These minor spots could be concentrated on type II 2-D gels by enriching the immunoglobulin G (IgG) fraction under nondenaturing conditions, and they disappeared when IgG was removed from the fraction. These results strongly suggest that many of the minor spots newly detected were bound to IgG in physiological conditions.

Blood Proteins↗

PubMed2003

Analysis of the activity and identification of enzymes after separation of cytosol proteins in mouse liver by microscale nondenaturing two-dimensional electrophoresis.

Enzyme activities such as of fructose bisphosphatase, malate dehydrogenase and carbonic anhydrase were analyzed after cytosol proteins in the mouse liver and were separated using nondenaturing two-dimensional electrophoresis (2-DE). The activities of both fructose bisphosphatase and malate dehydrogenase were inhibited by thyroxine, and fructose bisphosphatase activity was specifically inhibited by adenosine monophosphate in nondenaturing 2-DE. Furthermore, polypeptides of the separated proteins were analyzed by peptide mass fingerprinting using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry or by peptide sequencing using electrospray ionization-tandem mass spectrometry, or both. Proteins separated by 2-DE were identified. These results indicate that the function of proteins such as enzyme activity, and their sequence structure can be analyzed, for example by peptide mapping and peptide sequencing, after the proteins have been separated by nondenaturing 2-DE. Present results also indicate analysis of enzyme activity using nondenaturing 2-DE can be applied to screen substances which affect enzyme activity.

Adenosine Monophosphate↗

PubMed2002

Simple quantification of Cu,Zn-superoxide dismutase activity after separation by non-denaturing isoelectric focusing.

The Cu,Zn-superoxide dismutase (SOD) activity in bovine retina cytosol was separated from retinal pigment using short-length non-denaturing isoelectric focusing (IEF) (15-mm long x 1.3-mm i.d. column) and detected using non-denaturing two-dimensional electrophoresis (2-DE). After the SOD and pigment in the retina cytosol are separated, SOD activity can be quantified by water-soluble tetrazolium salt. We also found that SOD separated by this IEF retained its native function.

Animals↗

PubMed2002

Estimation of isoelectric points of human plasma proteins employing capillary isoelectric focusing and peptide isoelectric point markers.

Synthetic UV-detectable peptide pI markers were used to estimate isoelectric point (pI) values of proteins separated by capillary isoelectric focusing (CIEF) followed by cathodic mobilization in the absence of denaturing agents. The pI calculation and quantitative analysis of purified proteins showed the feasibility of these peptides as pI markers and internal standards in CIEF separation of proteins. Estimation of pI values of major proteins in human plasma was performed using the peptide pI markers, and the values were compared with those previously obtained by gel isoelectric focusing (IEF). Sera of immunoglobulin G (IgG) myeloma patients, which showed characteristic peaks of myeloma IgG in their CIEF patterns, were also subjected to the analysis and the pI values of the myeloma proteins have been estimated.

Blood Proteins↗