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Biomedical subjects

Eiji Kinoshita

Publications and source records attributed to Eiji Kinoshita.

16 recordsLinked to original sources
PubMed2026

Selective Enrichment of Newly Synthesized Proteins Using Phos-Tag Click Tip Enables Nascent Proteome Analysis in Influenza A Virus Infection.

Profiling of newly synthesized proteins (NSPs) provides access to dynamic changes in protein production that accompany acute cellular responses. Bioorthogonal noncanonical amino acid tagging (BONCAT)-based approaches enable selective labeling of NSPs; however, their broader application remains constrained by labor-intensive enrichment workflows and limited sensitivity for direct peptide-level analysis. Here, we developed a workflow termed "Phos-tag Click Tip" by integrating a phosphorylated variant of bicyclononyne (pBCN) with Phos-tag affinity purification to selectively capture azidohomoalanine (AHA)-labeled peptides for newly synthesized proteome analysis (NSProteomics). This approach overcomes key limitations of conventional proteomics and BONCAT-based strategies by enabling efficient enrichment and sensitive detection of NSP-derived peptides. Using this workflow, we performed comprehensive NSP profiling of host cells during influenza A virus infection. We identified dynamic changes in distinct NSP profiles associated with viral replication, host restriction, and immune responses, many of which were not readily detected with conventional whole-cell- or phospho-proteomic analyses. Overall, the Phos-tag Click Tip workflow provides a complementary approach for stimulus-responsive NSP profiling, offering functionally relevant insights into host-virus interactions and cellular response mechanisms.

Proteome↗
PubMed2007

Non-SCN5A related Brugada syndromes: verification of normal splicing and trafficking of SCN5A without exonic mutations.

Recently, it has been reported that under 20% of Brugada syndrome cases are linked to SCN5A mutations. The purpose of this study was to clarify whether abnormalities other than exonic mutations, such as splicing disorders, decreased mRNA expression levels, or membrane transport abnormalities of SCN5A, play a role in the pathogenesis of Brugada syndrome. We analyzed all SCN5A exons and splice sites using genomic DNA from 23 Brugada syndrome patients. We also analyzed the mRNA obtained from RV cardiomyocytes using real time PCR and sequencing, to study the expression levels and splicing patterns of SCN5A. The localization of SCN5A was examined by immunofluorescence analysis. A de novo heterozygous G to A transversion in a 5' splice junction of the intron between exons 21 and 22 was detected in 1 patient. In the mRNA analysis of Brugada syndrome patients without a mutation of SCN5A no splicing abnormalities were detected, and the SCN5A mRNA levels were similar to those of normal controls. Immunofluorescence analyses revealed that SCN5A is located on the surface membrane not only in the RV cardiomyocytes of normal controls but also in those with Brugada syndrome. We can confirm that some Brugada syndrome patients without exonic mutations in SCN5A had no other SCN5A abnormalities, including any involving the location of the SCN5A protein. These results suggest the involvement of other proteins in the pathogenesis in Brugada syndrome.

Adult↗
PubMed2006

A single nucleotide polymorphism genotyping method using phosphate-affinity polyacrylamide gel electrophoresis.

To date, various methods have been developed to facilitate the genotyping of a single nucleotide polymorphism (SNP) for aiding in the diagnosis and treatment of inherited diseases. The most commonly used method for SNP genotyping is an allele-specific hybridization procedure using an expensive fluorochrome-labeled oligonucleotide probe and a specialized fluorescence analyzer. Here, we introduce a simple and reliable genotyping method using a 1:1 mixture of 5'-phosphate-labeled and nonlabeled allele-specific polymerase chain reaction (PCR) primers. The method is based on the difference in mobility of the phosphorylated and nonphosphorylated PCR products (in the same number of basepairs) on phosphate-affinity polyacrylamide gel electrophoresis. The phosphate-affinity site is a polyacrylamide-bound dinuclear zinc(II) complex, which preferentially captures the 5'-phosphate-labeled allele-specific product compared with the corresponding nonlabeled product. The obtained DNA migration bands can be visualized by ethidium bromide staining. We demonstrate the genotyping of a SNP reported in a human cardiac sodium channel gene, SCN5A, using this novel procedure.

Electrophoresis, Polyacrylamide Gel↗
PubMed2006

Label-free kinase profiling using phosphate affinity polyacrylamide gel electrophoresis.

Herein we describe three applications of label-free kinase profiling using a novel type of phosphate affinity polyacrylamide gel electrophoresis. The phosphate affinity site is a polyacrylamide-bound dinuclear Mn2+ complex that enables the mobility shift detection of phosphorylated proteins from their nonphosphorylated counterpart. The first application is in vitro kinase activity profiling for the analysis of varied phosphoprotein isotypes in phosphorylation status. The activity profiles of six kinds of kinases, glycogen synthase kinase-3beta, cyclin-dependent kinase 5/p35, protein kinase A, mitogen-activated protein kinase (MAPK), casein kinase II, and calmodulin-dependent protein kinase II, were determined using a substrate protein, Tau, which has a number of phosphorylation sites. Each kinase demonstrated characteristic multiple electrophoresis migration bands up-shifted from the nonphosphorylated Tau due to differences in the phosphorylation sites and stoichiometry. The second application is in vivo kinase activity profiling for the analysis of protein phosphorylation involved in intracellular signal transduction. The time course changes in the epidermal growth factor-induced phosphorylation levels of Shc and MAPK in A431 cells were visualized as highly up-shifted migration bands by subsequent immunoblotting with anti-Shc and anti-MAPK antibodies. The third application is in vitro kinase inhibition profiling for the quantitative screening of kinase-specific inhibitors. The inhibition profile of a tyrosine kinase, Abl (a histidine-tagged recombinant mouse Abl kinase), was determined using the substrate Abltide-GST (a fusion protein consisting of a specific substrate peptide for Abl and glutathione S-transferase) and the approved drug Glivec (an ATP competitor). In the kinase assay, the slower migration band, monophosphorylated Abltide-GST, increased time-dependently, whereas the faster migration band, nonphosphorylated Abltide-GST, decreased. The dose-dependent inhibition of Glivec was determined by a change in the ratio of the faster and slower migration bands, which showed an IC50 value of 1.6 microM in the presence of 0.10 mM ATP.

Cell Line, Tumor↗
PubMed2006

Enrichment of phosphorylated proteins from cell lysate using a novel phosphate-affinity chromatography at physiological pH.

While phosphoproteins have attracted great interest toward the post-genome research (e.g. clinical diagnosis and drug design), there have been few procedures for the specific enrichment of native phosphoproteins from cells or tissues. Here, we describe a simple and efficient protocol to enrich phosphoproteins comprehensively from a complex mixture containing solubilized cellular proteins. This method is based on immobilized metal affinity chromatography using a phosphate-binding tag molecule (i.e. a dinuclear zinc(II) complex) attached on a highly cross-linked agarose. The binding, washing, and elution processes were all conducted without a detergent or a reducing agent at pH 7.5 and room temperature. An additive, 1.0 M CH3COONa, was necessary in the binding and washing buffers (0.10 M Tris-CH3COOH, pH 7.5) to prevent the nonphosphorylated protein from binding. The absorbed phosphoproteins were eluted using a mixed buffer solution (pH 7.5) consisting of 0.10 M Tris-CH3COOH, 10 mM NaH2PO4-NaOH, and 1.0 M NaCl. In this study, we demonstrate a typical example of phosphate-affinity chromatography using an epidermal growth factor-stimulated A431 cell lysate. The total time for the column chromatography (1 mL gel scale) was less than 1 h. The strong enrichment of the phosphoproteins into the elution fraction was evaluated using SDS-PAGE followed by Western blotting analysis.

Cell Extracts↗
PubMed2005

Phosphate-binding tag, a new tool to visualize phosphorylated proteins.

We introduce two methods for the visualization of phosphorylated proteins using alkoxide-bridged dinuclear metal (i.e. Zn(2+) or Mn(2+)) complexes as novel phosphate-binding tag (Phos-tag) molecules. Both Zn(2+)- and Mn(2+)-Phos-tag molecules preferentially capture phosphomonoester dianions bound to Ser, Thr, and Tyr residues. One method is based on an ECL system using biotin-pendant Zn(2+)-Phos-tag and horseradish peroxidase-conjugated streptavidin. We demonstrate the electroblotting analyses of protein phosphorylation status by the phosphate-selective ECL signals. Another method is based on the mobility shift of phosphorylated proteins in SDS-PAGE with polyacrylamide-bound Mn(2+)-Phos-tag. Phosphorylated proteins in the gel are visualized as slower migration bands compared with corresponding dephosphorylated proteins. We demonstrate the kinase and phosphatase assays by phosphate affinity electrophoresis (Mn(2+)-Phos-tag SDS-PAGE).

Blotting, Western↗
PubMed2005

Detection and quantification of on-chip phosphorylated peptides by surface plasmon resonance imaging techniques using a phosphate capture molecule.

We describe herein a detection and quantification system for on-chip phosphorylation of peptides by surface plasmon resonance (SPR) imaging techniques using a newly synthesized phosphate capture molecule (i.e., biotinylated zinc(II) complex). The biotinylated compound is a dinuclear zinc(II) complex that is suitable for accessing phosphate anions as a bridging ligand on the two zinc(II) ions. The compound was exposed on the peptide array and detected with streptavidin (SA) via a biotin-SA interaction by SPR imaging. In the conventional method using antibody, both anti-phosphoserine and anti-phosphotyrosine antibodies were required for phosphoserine and phosphotyrosine detection, respectively. Detection of the phosphate group by the zinc(II) complex, however, was independent of the phosphorylated amino acid residues. The calibration curve for the phosphorylation ratios was established with a calibration chip, on which phosphoserine-containing peptide probes were immobilized. The peptide probes, which were phosphorylated on the surface by protein kinase A, were detected and quantified by SPR imaging using the zinc(II) complex, SA, and anti-SA antibody. The reaction rate and the kinetics of on-chip phosphorylation were also evaluated with the peptide array. The phosphorylation ratio was saturated at approximately 20% in 2 h in this study.

Microchip Analytical Procedures↗
PubMed2005

A heteroduplex-preferential Tm depressor for the specificity-enhanced DNA polymerase chain reactions.

A macrocyclic tetraamine zinc(II) complex appended with two quinoline groups, Zn(2+)-1,7-bis(4-quinolylmethyl)-1,4,7,10- tetraazacyclododecane (Zn(2+)-Q2-cyclen), was successfully used as a novel additive to suppress nonspecific products in DNA polymerase chain reaction (PCR). In the presence of Zn(2+)-Q2-cyclen, the Tm drop of 20-bp heteroduplexes containing a noncomplementary basepair was greater than that of the corresponding homoduplex (i.e., primer DNA). Here, we applied such preferential DNA melting to a specificity-enhanced PCR using micromolar concentrations of Zn(2+)-Q2-cyclen. We demonstrated the selective amplification of target DNA fragments (i.e., the human heart sodium channel Nav1.5 gene) from genomic DNA or a cDNA library. The optimum condition for the specificity-enhanced PCR could be determined in the concentration range of 1-50muM of Zn(2+)-Q2-cyclen.

Animals↗
PubMed2005

Novel immobilized zinc(II) affinity chromatography for phosphopeptides and phosphorylated proteins.

Immobilized metal ion affinity chromatography (IMAC) is now a widely accepted technique for the separation of natural or artificial products that is beginning to find industrial applications. Here, we introduce a novel procedure for the separation of phosphopeptides and phosphorylated proteins by immobilized zinc(II) affinity chromatography. The phosphate-binding site of the affinity gel is an alkoxide-bridged dinuclear zinc(II) complex, the 1,3-bis[bis(pyridin-2-ylmethyl)amino]propan-2-olato dizinc(II) complex (Phos-tag), which is linked to a highly cross-linked 4% (w/v) agarose. The affinity gel (Phos-tag agarose) was prepared by the quantitative reaction of N-hydroxysuccinimide-activated Sepharose and a Phos-tag derivative having a 2-aminoethylcarbamoyl group in dry CH3CN. Phosphopeptides were retrieved in a quantitative and highly selective manner by a spin column method using Phos-tag agarose at room temperature. Furthermore, in this study, we demonstrate a simple, rapid, and reusable affinity column chromatography for the separation of phosphorylated proteins such as ovalbumin, alpha(s1)-casein, and beta-casein at physiological pH.

Animals↗
PubMed2004

Recognition of phosphate monoester dianion by an alkoxide-bridged dinuclear zinc(II) complex.

Recognition of phosphate monoester dianion by an alkoxide-bridged dinuclear zinc(II) complex (Zn2L3+) has been studied (L = alkoxide species of 1,3-bis[bis(pyridin-2-ylmethyl)amino]propan-2-ol). Potentiometric pH titration study disclosed a 1 : 1 phenyl phosphate complexation with Zn2L3+ in aqueous solution. The dissociation constant (= [Zn2L3+][PhOPO3(2-)]/[Zn2L3+-PhOPO3(2-)]) is an extremely small value of 2.5 x 10(-8) mol dm(-3) at 25 degrees C with I = 0.10 (NaNO3). The X-ray crystal analysis of the dizinc(II) complex with p-nitrophenyl phosphate showed that the phosphate dianion binds as a bridging ligand to the two zinc(II) ions.

Journal Article↗
PubMed2003

Distinct sites regulating grayanotoxin binding and unbinding to D4S6 of Na(v)1.4 sodium channel as revealed by improved estimation of toxin sensitivity.

Grayanotoxin (GTX) exerts selective effects on voltage-dependent sodium channels by eliminating fast sodium inactivation and causing a hyperpolarizing shift in voltage dependence of channel activation. In this study, we adopted a newly developed protocol that provides independent estimates of the binding and unbinding rate constants of GTX (k(on) and k(off)) to GTX sites on the sodium channel protein, important in the molecular analysis of channel modification. Novel GTX sites were determined in D2S6 (Asn-784) and D3S6 (Ser-1276) by means of site-directed mutagenesis; the results suggested that the GTX receptor consists of the S6 transmembrane segments of four homologous domains facing the ion-conducting pore. We systematically introduced at two sites in D4S6 (Na(v)1.4-Phe-1579 and Na(v)1.4-Tyr-1586) amino acid substituents with residues containing hydrophobic, aromatic, charged, or polar groups. Generally, substitutions at Phe-1579 increased both k(on) and k(off), resulting in no prominent change in dissociation constant (K(d)). It seems that the smaller the molecular size of the residue at Na(v)1.4-Phe-1579, the larger the rates of k(on) and k(off), indicating that this site acts as a gate regulating access of toxin molecules to a receptor site. Substitutions at Tyr-1586 selectively increased k(off) but had virtually no effect on k(on), thus causing a drastic increase in K(d). At position Tyr-1586, a hydrophobic or aromatic amino acid side chain was required to maintain normal sensitivity to GTX. These results suggest that the residue at position Tyr-1586 has a more critical role in mediating GTX binding than the one at position Phe-1579. Here, we propose that the affinity of GTX to Na(v)1.4 sodium channels might be regulated by two residues (Phe and Tyr) at positions Phe-1579 and Tyr-1586, which, respectively, control access and binding of GTX to its receptor.

Amino Acid Sequence↗
PubMed2002

A novel procedure for simple and efficient genotyping of single nucleotide polymorphisms by using the Zn2+-cyclen complex.

The analysis of single nucleotide polymorphisms (SNPs) is increasingly utilized in the study of various genetic determinants. Here, we introduce a simple, rapid, low-cost and accurate procedure for the detection of SNPs by polyacrylamide gel electrophoresis (PAGE) with a novel additive, the Zn2+- cyclen complex (cyclen = 1,4,7,10-tetraazacyclododecane). The method is based on the difference in mobility of mutant DNA (in the same length) in PAGE, which is due to Zn2+-cyclen binding to thymine bases accompanying a total charge decrease and a local conformation change of target DNA. Various nucleotide substitutions (e.g. AT to GC) in DNA fragments (up to 150 bp) can be visualized with ethidium bromide staining. Furthermore, heteroduplex and homoduplex DNAs are clearly separated as different bands in the gel. We demonstrate the analysis of single- and multiple-nucleotide substitutions in a voltage-dependent sodium channel gene by using this novel procedure (Zn2+-cyclen-PAGE).

Base Sequence↗
PubMed2002

Structural determinants for the action of grayanotoxin in D1 S4-S5 and D4 S4-S5 intracellular linkers of sodium channel alpha-subunits.

We located a novel binding site for grayanotoxin on the cytoplasmic linkers of voltage-dependent cardiac (rH1) or skeletal-muscle (mu 1) Na(+) channel isoforms (segments S4-S5 in domains D1 and D4), using the alanine scanning substitution method. GTX-modification of Na(+) channels, transiently expressed in HEK 293 cells, was evaluated under whole-cell voltage clamp, from the ratio of maximum chord conductance for modified and unmodified Na(+) channels. In mu 1, mutations K237A, L243A, S246A, K248A, K249A, L250A, S251A, or T1463A, caused a moderate, but statistically significant decrease in this ratio. On making corresponding mutations in rH1, only L244A dramatically reduced the ratio. Because in mu 1, the serine at position 251 is the only heterologous residue with respect to rH1 (Ala-252), we made a double mutant L243A&S251A to match the sequence of mu 1 and rH1 in S4-S5 linkers of both domains. This double mutation resulted in a significant decrease in the ratio, to the same extent as L244A substitution in rH1 did, indicating that the site at Leu-244 in rH1 or at Leu-243 in mu 1 is a novel one, exhibiting a synergistic effect of grayanotoxin.

Alanine↗
PubMed2002

Molecular basis for exaggerated sensitivity to mexiletine in the cardiac isoform of the fast Na channel.

Cardiac sodium channels have been shown to have a higher sensitivity to local anesthetic agents, such as lidocaine, than the sodium channels of other tissues. To examine if this is also true for mexiletine, we have systematically measured mexiletine sensitivity of the Na channel isoforms, rH1, (mu)1, and rBII, which were transiently expressed in human embryonic kidney (HEK) 293 cells. We confirmed that the cardiac isoform rH1 exhibited the highest sensitivity among the three tested channel isoforms. In rH1, (mu)1, and rBII, the respective IC(50) values were 62, 294, and 308 microM mexiletine, in regard to tonic block, and 18, 54, and 268 microM mexiletine, in relation to use (8 Hz)-dependent block. The relatively high drug sensitivity of rH1 was an invariant finding, irrespective of channel state or whether channels were subjected to infrequent or frequent depolarizing stimuli. Mutating specific amino acids in the skeletal muscle isoform (mu)1 (namely, (mu)1-I433V and (mu)1-S251A) to those of the cardiac isoform at putative binding sites for local anesthetic agents revealed that only one of the point mutations ((mu)1-S251A) has relevance to the high cardiac drug sensitivity, because mexiletine produced significantly more use-dependent and tonic block in (mu)1-S251A than wild-type (mu)1.

Anti-Arrhythmia Agents↗