Biomedical subjects

Hideaki Takata

Publications and source records attributed to Hideaki Takata.

3 recordsLinked to original sources

PubMed2005

Generation of monoclonal antibodies against chromosomal antigens that have a high sequence similarity between human and mouse.

We raised monoclonal antibodies by immunizing mice with total chromosome proteins extracted from isolated human metaphase chromosomes. The indirect immunofluorescence screening of hybridoma cell lines provided 15 monoclonal antibodies against the chromosomal antigens. The antigen proteins of the mAbs were identified by immunoblotting as core histones or by immunoprecipitation followed by a peptide mass fingerprinting method as nuclear mitotic apparatus protein, heterogeneous nuclear ribonucleoprotein A2/B1, ribosomal protein S4, linker histone and beta-actin. During mitosis, localizations of these proteins on chromosomes were clearly observed using the obtained antibodies. These results indicate that the current strategy is effective for obtaining monoclonal antibodies useful for immunoblotting and/or immunofluorescent staining of human proteins, using the antigens with high homology to mouse proteins.

Animals↗

PubMed2005

Proteome analysis of human metaphase chromosomes.

DNA is packaged as chromatin in the interphase nucleus. During mitosis, chromatin fibers are highly condensed to form metaphase chromosomes, which ensure equal segregation of replicated chromosomal DNA into the daughter cells. Despite >1 century of research on metaphase chromosomes, information regarding the higher order structure of metaphase chromosomes is limited, and it is still not clear which proteins are involved in further folding of the chromatin fiber into metaphase chromosomes. To obtain a global view of the chromosomal proteins, we performed proteome analyses on three types of isolated human metaphase chromosomes. We first show the results from comparative proteome analyses of two types of isolated human metaphase chromosomes that have been frequently used in biochemical and morphological analyses. 209 proteins were quantitatively identified and classified into six groups on the basis of their known interphase localization. Furthermore, a list of 107 proteins was obtained from the proteome analyses of highly purified metaphase chromosomes, the majority of which are essential for chromosome structure and function. Based on the information obtained on these proteins and on their localizations during mitosis as assessed by immunostaining, we present a four-layer model of metaphase chromosomes. According to this model, the chromosomal proteins have been newly classified into each of four groups: chromosome coating proteins, chromosome peripheral proteins, chromosome structural proteins, and chromosome fibrous proteins. This analysis represents the first compositional view of human metaphase chromosomes and provides a protein framework for future research on this topic.

Chromatin↗

PubMed2002

Changes in chromosomal surface structure by different isolation conditions.

The human cell cycle was synchronized and the chromosomes were isolated by a centrifugation method using two representative solutions for chromosome isolation (a polyamine buffer, PAB and citric acid solution, CAS) and fixatives. The centrifugation method yielded sufficient amounts of human metaphase chromosomes. Observation of the isolated chromosomes by scanning electron microscopy (SEM) revealed two types of surface structure which have been repeatedly reported to date: the human chromosomes in the PAB were relatively smooth but covered irregularly with scaly structures, while the surface of the chromosomes in the CAS exhibited a dense fibrous structure with a uniform diameter of 50-70 nm. Comparison of proteins extracted from chromosomes isolated with the PAB and CAS clearly indicated the removal of linker histones, H1, from chromosomes isolated with the CAS. These findings imply that the two different images of human chromosomes frequently observed by SEM are due to the removal of peripheral chromosomal materials including linker histones and/or the depletion of linker histones which prevent the surface chromatin fibers from scattering.

Cell Cycle↗