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Biomedical subjects

Akiko Mizutani

Publications and source records attributed to Akiko Mizutani.

6 recordsLinked to original sources
PubMed2005

Establishment of stable GH3 cell line expressing enhanced yellow fluorescein protein-growth hormone fusion protein.

To investigate, in real time, the transport and secretion of pituitary hormone, we have developed an experimental pituitary cell line, GH3 cell, which has secretory granules of growth hormone (GH) linked to enhanced yellow fluorescein protein (EYFP). This stable GH3 cell secretes secretory granules of GH linked to EYFP on stimulation by Ca2+ influx or Ca2 release from storage. This GH3 cell will be useful for the real-time visualization of the intracellular transport and secretion of GH.

Animals↗
PubMed2005

Sensitization to morphine withdrawal in guinea-pigs.

The aim of this study was to determine whether sensitization occurred to morphine withdrawal. Guinea-pigs were treated twice daily with increasing doses of morphine (10-100 mg/kg s.c.) for 3 days followed by injection of morphine 100 mg/kg on the fourth day. Sixty min after the last morphine injection, animals were withdrawn from morphine with naltrexone, 15 mg/kg s.c., and locomotor activity and all other behaviours scored over 90 min. Animals were then rested for 3 days. This procedure was repeated twice over the next 2 weeks. Control animals were treated with saline for the first two treatment cycles. Guinea-pigs subjected to three cycles of morphine withdrawal showed a significant increase in the total number of withdrawal behaviour counts over the 90-min observation period following the third cycle of withdrawal compared with the first and second withdrawal cycles. However, locomotor activity, a major sign of morphine withdrawal in guinea-pigs, was not significantly increased. Fos-LI was markedly increased in the repeatedly withdrawn animals in several brain regions, including amygdala, dorsal striatum, thalamus, ventral tegmental area, and ventrolateral periaqueductal gray area. It is concluded that sensitization to morphine withdrawal occurs in guinea-pigs.

Amygdala↗
PubMed2005

An EYFP insertion mutant containing a modified lox sequence for potential use as a recombination indicator.

The Cre-loxP system has been used for genetic engineering in many organisms ranging from E. coli to mouse. It is known that intermolecular recombination between two loxP sites is substantially less frequent than intramolecular recombination between the same sites. Therefore, for the efficient isolation of intermolecular loxP recombination products, it is essential to develop a powerful screening method. Here, we describe the construction of an EYFP variant (EYFP157) that could become a vital tool in such a screening method. EYFP157 contains, between Gln157 and Lys158, the JT15:JTZ17 mutant loxP sequence that is not cleaved by the Cre protein. When EYFP157 was expressed at 37 degrees C in E. coli and mouse cells, fluorescence of the protein was readily detectable under a standard microscope. EYFP157 possessed 1-3% of the fluorescence from the wild-type EYFP. Interestingly, at room temperature EYFP157 fluoresced more efficiently in E. coli cells and thus the difference in fluorescence intensity between EYFP157 and the wild-type EYFP became negligible. The potential application of EYFP157 as a recombination indicator in both E. coli and mouse cells is further discussed.

Amino Acid Sequence↗
PubMed2003

Regions of GAL4 critical for binding to a promoter in vivo revealed by a visual DNA-binding analysis.

Binding of transcriptional activators to specific sites on DNA, mediated by their DNA-binding domain, is a key regulatory point in transcriptional regulation. With a GFP-based microscopic assay, we investigated how the prototypical activator GAL4 effectively binds to a promoter in living yeast cells. We show that GAL4 relies on a previously unrevealed mechanism involving 'DNA-binding enhancers' (DBEs), the regions of GAL4 that assist DNA-binding domain association with DNA. GAL4 contains two DBEs, one, but not the other, physically overlapping the principal transcriptional activation domain. Either of the DBEs, however, can function independently of transcriptional activation, indicating a discrete mechanism responsible for DNA-binding enhancement. The effect of DBEs, while not limited to natural target promoters, is still not universal and can be profoundly affected by the binding-site context. The GAL4 DBEs can also enhance promoter binding of an unrelated DNA-binding domain, and possibly represent a new modular functional unit responsible for effective binding of diverse regulatory factors to DNA in vivo.

Amino Acid Sequence↗