Biomedical subjects

W Dansithong

Publications and source records attributed to W Dansithong.

3 recordsLinked to original sources

PubMed2001

A simple method for midkine purification by affinity chromatography with a heavy chain variable domain (VH) fragment of antibody.

A DNA fragment for a heavy chain variable domain (VH) was prepared from a hybridoma that produces a monoclonal antibody against human midkine (MK). The antibody fragment was produced in Escherichia coli and its affinity for chemically synthesized full length MK or recombinant midkine c-terminus (MKc-half) protein was confirmed by ELISA. An Escherichia coli cell lysate expressing MKc-half was applied to a VH fragment-coupled Sepharose 4B column and eluted with a buffer containing 0.5 M NaCl. SDS-PAGE analysis revealed a high degree of purity of the MKc-half protein in the eluent, showing the utility of a recombinant VH fragment in purification of proteins by affinity chromatography.

Amino Acid Sequence↗

PubMed2001

Production and characterization of a bacterial single-chain Fv fragment specific to human truncated midkine.

The production (and characterization) of a monoclonal antibody against human truncated midkine (tMK), and the detection of tMK in G401 cells, a Wilms' tumor cell line, as well as in Wilms' tumor patient specimens, have been reported (Paul et al., Cancer Lett. 163 (2001) 245-251). Here we report the molecular cloning and expression of this monoclonal antibody as a single-chain Fv fragment (scFv) in Escherichia coli. The scFv protein, purified by immobilized metal affinity chromatography, showed a specific affinity to recombinant tMK and native tMK in G401 cells as detected by enzyme-linked immunosorbent assay and immunofluorescence microscopy, respectively. The binding of this protein to recombinant tMK was competitive with the parental monoclonal antibody. These results suggest that this scFv can also be used for Wilms' tumor detection.

Amino Acid Sequence↗

PubMed1998

Induction of apoptosis in Neuro-2A cells by Zn2+ chelating.

Metal ions such as Ca2+, Mg2+, or Zn2+, are important for many cell functions, for example, signal transduction and the modulation of enzyme activity. The relationship between apoptosis and metal cations, especially Ca2+, has been described in many reports. We have investigated the role of metal cations in the regulation of apoptosis in the mouse neuroblastoma cell line, Neuro-2A. When Neuro-2A cells were treated with ethylene diaminetetraacetic acid (EDTA), apoptosis was detected as growth inhibition, DNA fragmentation with a ladder pattern in agarose gel electrophoresis, and nuclear decomposition. However, in case of the treatment with ethylene glycol bis- (beta-aminoethyl ether) N,N,N',N'-tetraacetic acid (EGTA), which has a higher chelating specificity for Ca2+ than EDTA, DNA fragmentation was not detected. Moreover, the apoptosis induced by EDTA was inhibited by exogenous Zn2+. The membrane permeable Zn2+ chelator N,N,N',N'-tetrakis (2-pyridylmethyl)ethylenediamine (TPEN) also induced apoptosis of the Neuro-2A cells, and addition of equimolar exogenous Zn2+ or Cu2+, but not Mn2+ or Fe2+, prevented TPEN-induced apoptosis. The results suggest that Zn2+ may be a key regulator of apoptosis in Neuro-2A cells.

Animals↗