Kiyonori Hirota

Expression pattern of a newt Notch homologue in regenerating newt retina.

We isolated part of a newt Notch homologue, N-Notch, from regenerating newt retina. The spatio-temporal pattern of N-Notch expression was studied by in situ hybridization at different stages of newt retinal regeneration. Proliferating cells were confirmed by the injection of bromodeoxyuridine (BrdU). In the early stage of regeneration, when the retina was one to two cells thick, all proliferating retinal progenitors expressed N-Notch. As the thickness of the retina increased with regeneration, N-Notch expression decreased in BrdU-positive cells on the vitreal side of the retina. Subsequently, presumptive retinal ganglion cells that were BrdU-negative cells appeared at the vitreal edge of the regenerating retina. These differentiating cells did not express N-Notch. Later, N-Notch expression decreased in the BrdU-positive cells on the scleral surface of the retina. Subsequently, presumptive photoreceptor cells that were BrdU-negative cells appeared in this region. These differentiating cells also did not express N-Notch. The proliferating retinal progenitors ceased expressing N-Notch and then stopped dividing during the differentiation of ganglion cells and photoreceptor cells. It was found that retinal regeneration involves the expression of an important developmental signaling molecule, Notch, in retinal progenitors and the expression of Notch ceased as cell differentiation proceeded during retinal regeneration.

CLONING AND DISTRIBUTION OF A PUTATIVE TETRODOTOXIN-RESISTANT NA+ CHANNEL IN NEWT RETINA

Abstract Overlapping cDNA clones spanning the entire coding region of a Na+ channel were isolated from newt retina. The coding region predicts a 2,007 amino acid protein, designated nRNaCh (newt retina sodium channel), which is homologous to other Na+ channels. In situ hybridization indicated that nRNaCh is expressed exclusively in spiking neurons, where a tetrodotoxin (TTX)-resistant Na+ current has been recorded. Therefore, nRNaCh cDNA is sure to encode the TTX-resistant Na+ channel of newt retina. Sequence comparisons show that nRNaCh is more homologous to TTX-sensitive Na+ channels than to TTX-resistant Na+ channels. The length of the S5-S6 loop of repeat I of nRNaCh is similar to that of TTX-sensitive channels, whereas TTX-resistant Na+ channels have a deletion. The 3rd position in the SS2 region of repeat I of nRNaCh is a non-aromatic amino acid (Ala), which is a common feature of TTX-resistant channels. These findings suggest that whether the amino acid at the 3rd position in the SS2 region of repeat I is aromatic or non-aromatic determines the TTX sensitivity of Na+ channels, not the overall structure of the channel.

Site-specific chemical conjugation of human Fas ligand extracellular domain using trans-cyclooctene – methyltetrazine reactions

BackgroundFas ligand plays a key role in the human immune system as a major cell death inducing protein. The extracellular domain of human Fas ligand (hFasLECD) triggers apoptosis of malignant cells, and therefore is expected to have substantial potentials in medical biotechnology. However, the current application of this protein to clinical medicine is hampered by a shortage of the benefits relative to the drawbacks including the side-effects in systemic administration. Effective procedures for the engineering of the protein by attaching useful additional functions are required to overcome the problem.ResultsA procedure for the site-specific chemical conjugation of hFasLECD with a fluorochrome and functional proteins was devised using an inverse-electron-demand Diels-Alder reaction between trans-cyclooctene group and methyltetrazine group. The conjugations in the present study were attained by using much less molar excess amounts of the compounds to be attached as compared with the conventional chemical modification reactions using maleimide derivatives in the previous study. The isolated conjugates of hFasLECD with sulfo-Cy3, avidin and rabbit IgG Fab’ domain presented the functional and the structural integrities of the attached molecules without impairing the specific binding activity toward human Fas receptor extracellular domain.ConclusionsThe present study provided a new fundamental strategy for the production of the engineered hFasLECDs with additional beneficial functions, which will lead to the developments of the improved diagnostic systems and the effective treatment methods of serious diseases by using this protein as a component of novel molecular tools.

Cadherin Expression during Retinal Regeneration in the Adult Newt

Abstract The present study used an immunohistochemical technique to examine the expression of cadherins in the regenerating retina of adult newts. After surgical removal of the neural retina, retinal pigment epithelial (RPE) cells proliferate into retinal precursor cells and regenerate a fully functional retina. At the beginning of retinal regeneration, retinal cells originating from RPE cells are undifferentiated precursor cells. Both E-cadherin and R-cadherin are expressed at the surface of these precursor cells. As regeneration proceeds, precursor cells differentiate into retinal neurons. R-cadherin is expressed at the surface of the differentiated neurons, but E-cadherin is lost to the differentiated neurons. The difference in expression pattern suggests that each cadherin plays distinctive roles in retinal regeneration. And our finding that Ecadherin is expressed transiently by undifferentiated precursor cells implies the importance of cell-cell adhesions by E-cadherin for differentiation.

Generation of ubiquitin-based binder with an inserted active peptide

The grafting of active peptides onto structurally stable scaffold proteins is effective for the generation of functional proteins. In this study, we aimed to develop a grafting method using ubiquitin as a scaffold protein. Ubiquitin is a small protein consisting of 76 amino acid residues that is highly stable against heat and pH stress, which are desirable characteristics for a scaffold protein. Moreover, its structure is maintained even if it is split or additional residues are inserted. Therefore, we assumed that grafting of an active peptide into ubiquitin would result in a functional protein. As a proof of concept, we developed the ubiquitin-based binder (UbB), into which the p53 (17-28) peptide was inserted between Ile36 and Pro37. The p53 (17-28) peptide, utilized as a model active peptide in this work, is known to bind to mouse double minute 2 homolog (Mdm2). Size exclusion chromatography and circular dichroism indicated that UbB maintained a similar structure to that of ubiquitin. The affinity for Mdm2 measured by surface plasmon resonance was 292 times greater than that of the p53 (17-28) peptide. These observations indicate that ubiquitin is a robust scaffold for peptide grafting. We hope that this study will aid further development of ubiquitin-based protein engineering.

Method for Identification of Mutant Glutathione S-Transferases Conferring Enhanced Resistance to the Anti-Cancer Drug Chlorambucil.

We screened library of mutant glutathione S-transferases (GSTs) in Escherichia coli by successive treatments with anti-cancer drug chlorambucil and identified mutant GSTs that conferred enhanced resistance to host against chlorambucil compared with wild-type GST. This study provides a method to develop enzymes with improved efficiency of detoxification against cytotoxic substances.

117 Molecular cloning of a putative calcium channel from squid (Loligo bleekeri) optic lobe

The most widely used fluorescent Ca 2+ indicator, fira-2, has a high aflinity to Ca2+ (K~a=0.2pM). We found that fiua-2 was saturated during large rises in the cytosolic Ca2+ concentration ([Ca’+]i) in several cells examined, including cerebellar Purkinje cells and pancreatic acinar cells, The maximum rises in [Ca’+]r estimated with a low-aflinity Ca2+ indicator, BTC (KCa=lOuM), were larger than 100

119 Differential localization of the two putative sodium channel α-subunits in the optic lobe and the stellate ganglion of the squid Loligo breekeri

4 and 10 j.A4 in the Purkinje cells and acinar ceils, respectively. The micromolar rises in [Ca’+]i triggered exocytosis in the acinar cells, and likely cause significant functional consequences in other cells. In order to estimate [Ca’+]r in a large dynamic range, we have developed a method to use BTC and furasimultaneously, taking advantage of the fact that BTC has longer excitation wavelengths than fbra-2. Interestingly, we found that the acinar cell [Caz+]i estimated with BTC tended to be higher than that estimated with fUraat the onset of [Ca2+]i rises at the trigger zone. This discrepancy can be readily explained by the presence of CaZ+ domains due to CaZ+ release channels.

718 Cloning and sequence analysis of squid neural ankyrin

The effect of FK506, an inhibitor of calcineurin, was examined in rat hippocampal CA1 neurons. 2-4 weeks old Wister rats were used to record the membrane current in hippocampal CA1 neurons with the brain slice patch clamp techniques. The samemembrane currents were recorded in hippocampal neurons in primary culture prepared from fetal Wister rats. The cell-attached patch and inside-out patch clamp techniques were used to record the current. The open of channels, whose conductancewere about40pS and 5OpS, was observed attheextracellurer application of 100pMNMDA. When 10nMFK506 was applied to the neurons, the mean open time became about three times as long as before. The same prolongation of the mean open time occurred at application of cyclosporin A.

Detection And Analysis System For Protein Array

Ankyrin protems mediate the attachment of integral membrane proteins to spectrm. In neurons, the neural-specific ankyrins are candidates to participate in maintenance/targeting of ion channels and cell adhesion molecules to speclflc regions withm neurons. We ha1.e been cloning ankyrins of squid nervous system and succeeded in sequencing first half of an ankyrin gene. The conserved region within ankyrin-repeat domain was amplified with the PCR method. With this as a probe, the squid optic lobe cDNA hbrary was screened. Out of 250,000 clones, we found 29 posltlve clones, one of which covers the first half of the ankyrin gene, from N-terminal to the spectrin binding domain. So far we have sequenced only this region (about 3700bps), but comparison to mammalian counterparts shows that this squid ankyrm is more homologous to brain ankyrins than to erythrocyte ankyrins of mammals.