Biomedical subjects

Aishan Han

Publications and source records attributed to Aishan Han.

5 recordsLinked to original sources

PubMed2006

Mass-tag technology responding to intracellular signals as a novel assay system for the diagnosis of tumor.

A novel mass spectrometry-based assay system for determining protein kinase activity employing mass-tagged substrate peptide probes was used for the diagnosis of tumors. Two peptide probes (H-type and D-type) were synthesized containing the same substrate peptide sequence for protein kinase C (PKC). The molecular weights of the two probes differ because of the incorporation of deuterium into the acetyl groups of the D-type probe. The lysates of the normal and tumor tissue were prepared and reacted with the H- and D-type peptide probes, respectively. The PKC activities of the normal and tumor tissues can be compared simply and directly by calculating the phosphorylated ratio to each peptide probe, obtained from the peak intensity of the mass spectrum after mixing of the two reaction solutions. The phosphorylation ratio for the reaction of the H-type peptide probe with the tumor tissue lysate (B16 melanoma) was more than three times higher than that of the D type peptide probe with the normal skin tissue lysate. These results show that the novel assay system for detecting protein kinase activity using mass-tag technology can be a simple and useful means to profile protein kinase activity for cell or tissue lysate samples, and can be applied to the diagnosis of tumors.

Animals↗

PubMed2006

Phosphorylation of Rho-associated kinase (Rho-kinase/ROCK/ROK) substrates by protein kinases A and C.

Rho-associated kinase (Rho-kinase/ROCK/ROK) is a serine/threonine kinase and plays an important role in various cellular functions. The cAMP-dependent protein kinase (protein kinase A/PKA) and protein kinase C (PKC) are also serine/threonine kinases, and directly and/or indirectly take part in the signal transduction pathways of Rho-kinase. They have similar phosphorylation site motifs, RXXS/T and RXS/T. The purpose of this study was to identify whether sites phosphorylated by Rho-kinase could be targets for PKA and PKC and to find peptide substrates that are specific to Rho-kinase, i.e., with no phosphorylation by PKA and PKC. A total of 18 substrates for Rho-kinase were tested for phosphorylation by PKA and PKC. Twelve of these sites were easily phosphorylated. These results mean that Rho-kinase substrates can be good substrates for PKA and/or PKC. On the other hand, six Rho-kinase substrates showing no or very low phosphorylation efficiency (<20%) for PKA and PKC were identified. Kinetic parameters (K(m) and k(cat)) showed that two of these peptides could be useful as substrates specific to Rho-kinase phosphorylation.

Amino Acid Motifs↗

PubMed2006

A MutS protein-immobilized au electrode for detecting single-base mismatch of DNA.

A novel electrochemical biosensor was developed to detect gene mutation by using a DNA-mismatch binding protein: MutS from Escherichia coli. The MutS protein was immobilized onto an Au-electrode surface via complex formation between a histidine tag of the MutS protein and a thiol-modified nitrilotriacetic acid chemically adsorbed on the Au-electrode surface. When a target double-stranded DNA having a single-base mismatch was captured by the MutS protein on the electrode, some electrostatic repulsion arose between polyanionic DNA strands and anionic redox couple ions. Consequently, their redox peak currents on a cyclic voltammogram with the Au electrode drastically decreased, depending on the concentration of the target DNA, according to the redox couple-mediated artificial ion-channel principle. By using this assay, one can detect all types of single-base mismatch and single-base deletion.

Base Pair Mismatch↗

PubMed2006

Development of a fluorescence peptide chip for the detection of caspase activity.

Proteases play a key role in cell functions, and it is very important to monitor their activities for drug screening and diagnosis of diseases. In the present study, a new class of fluorescence probe, into which a fluorophore and a quencher have been introduced, was developed and applied to the on-chip detection of caspase-3 activity. This probe is non fluorescent in the absence of caspase-3. However, when it is treated with active caspase-3, the fluorescence intensity increases dependent on the caspase-3 activity due to the cleavage of the quencher-containing moiety on a glass slide. This caspase-dependent increase in the fluorescence intensity was also detected when the glass slide immobilizing the probe peptide was treated with cell lysate stimulated by staurosporine (STP), which is an apoptosis-inducing agent. On the other hand, such an increase was not detected in the case of control cell lysate without STP-stimulation. The developed system is a rapid and sensitive method and is useful for the direct measurement of protease activity on a glass array.

Animals↗

PubMed2002

Gene mutation assay using a MutS protein-modified electrode.

A novel electrochemical biosensor for gene mutation detection was developed using a DNA mismatch recognizing protein MutS from E. coli. The MutS protein was immobilized onto an Au electrode by coordination of His-tag at its C-terminus to vacant sites of Ni(II)-nitrilotriacetato complex attached to the surface of electrode. When a target DNA duplex having a mismatch site was captured by the MutS protein on the electrode, the electrostatic repulsion arose between polyanionic DNA duplexes and negatively-charged ferrocyanide/ferricyanide redox couple ions. Consequently, their redox peak currents on a cyclic voltammogram with the Au electrode drastically decreased depending on the concentration of the target DNA according to the redox couple-mediated artificial ion-channel principle. Using this assay, we could detect GT mismatch and deletion mutation in the double-stranded DNA.

Adenosine Triphosphatases↗