Yoshiro Hanyu

Chaos and phase locking in normal squid axons

Abstract By using periodic current stimulation, chaotic potential responses could be evoked in squid axons immersed in normal seawater. the occurence of intermittent chaos arose through a subcritical period-doubling bifurcation, indicating some similarity of the dynamical structures between the Rayleigh-Benard convection and the squid axon systems.

Global bifurcation structure in periodically stimulated giant axons of squid

Abstract The responses of a squid giant axon fell into three categories; phase-locked, quasi-periodic and chaotic. A bifurcation structure of the response patterns was obtained as a function parameters, I/It (the current pulse intensity) normalized by threshold current) and T (current pulse intervals). The periodic responses appeared through either tangent bifurcation or type III bifurcation, parameters of I/It and T. The characteristics of the response patterns and the bifurcation structure could be qualitatively explained by considering the two effects of periodic current input on normal axons; one is to act as a periodic external force and the other is to induce self-oscillation in the axon. Both the response and bifurcation characteristics could be ascribed to a specific nonlinear interaction between the induced self-oscillation and the periodic input.

Functional diversity of protein C-termini: more than zipcoding?

The carboxylated (C)-terminus of proteins, which includes the single terminal alpha-carboxyl group and preceding residues, is uniquely positioned to serve as a recognition signature for a variety of cell-biological processes, including protein targeting, subcellular anchoring and the static and dynamic formation of macromolecular complexes. The terminal sequence motifs can be processed by posttranslational modifications, thereby providing a means to increase sequence diversity and to regulate interactions. Several classes of protein domains have been identified that are either designed for or are capable of interacting with protein C-termini - these include PDZ and TPR domains. The interactions between these protein domains and various terminal epitopes play an important role in specifying cell-biological functions. The combination of diversity and the plasticity of the chemistry of C-termini provides mechanisms for spatial and temporal specificity that are exploited by a variety of biological processes, ranging from specifying prokaryotic protein degradation to nucleating mammalian neuronal signaling complexes. Understanding the diverse functions of protein C-termini might also provide an important indexing criterion for functional proteomics.

An FGF1:FGF2 chimeric growth factor exhibits universal FGF receptor specificity, enhanced stability and augmented activity useful for epithelial proliferation and radioprotection.

Structural instability of wild-type fibroblast growth factor (FGF)-1 and its dependence on exogenous heparin for optimal activity diminishes its potential utility as a therapeutic agent. Here we evaluated FGFC, an FGF1:FGF2 chimeric protein, for its receptor affinity, absolute heparin-dependence, stability and potential clinical applicability. Using BaF3 transfectants overexpressing each FGF receptor (FGFR) subtype, we found that, like FGF1, FGFC activates all of the FGFR subtypes (i.e., FGFR1c, FGFR1b, FGFR2c, FGFR2b, FGFR3c, FGFR3b and FGFR4) in the presence of heparin. Moreover, FGFC activates FGFRs even in the absence of heparin. FGFC stimulated keratinocytes proliferation much more strongly than FGF2, as would be expected from its ability to activate FGFR2b. FGFC showed greater structural stability, biological activity and resistance to trypsinization, and less loss in solution than FGF1 or FGF2. When FGFC was intraperitoneally administered to BALB/c mice prior to whole body gamma-irradiation, survival of small intestine crypts was significantly enhanced, as compared to control mice. These results suggest that FGFC could be useful in a variety of clinical applications, including promotion of wound healing and protection against radiation-induced damage.

Pax‐6 expression during retinal regeneration in the adult newt

The present study examined the expression of Pax‐6 during retinal regeneration in adult newts using in situ hybridization. In a normal retina, Pax‐6 is expressed in the ciliary marginal zone, the inner part of the inner nuclear layer, and the ganglion cell layer. After surgical removal of the neural retina, retinal pigment epithelial cells proliferate into retinal precursor cells and regenerate a fully functional retina. At the beginning of retinal regeneration, Pax‐6 was expressed in all retinal precursor cells. As regeneration proceeded, differentiating cells appeared at the scleral and vitreal margins of the regenerating retina, which had no distinct plexiform layers. In this stage, the expression of Pax‐6 was localized in a strip of cells along the vitreal margin of the regenerating retina. In the late stage of regeneration, when the layer structure was completed, the expression pattern of Pax‐6 became similar to that of a normal retina. It was found that Pax‐6 is expressed in the retinal precursor cells in the early regenerating retina and that the expression pattern of Pax‐6 changed as cell differentiation proceeded during retinal regeneration.

An improved cryofixation method: cryoquenching of small tissue blocks during microwave irradiation

The metal contact method of rapid freezing is greatly improved by irradiating the specimen with microwaves at 2.45 GHz for a short period of time (50 ms), while pushing the specimen onto the surface of the copper block cooled by liquid N2. The microwave irradiation, together with two technical improvements (a light‐mass plunger and a recently developed β‐gel shock absorber) for preventing bounce, produces a good freezing zone for squid retina, with high reproducibility for each experimental trial, extending from the contact surface to a depth of about 15 μm, which is comparable to the depth obtained by the metal contact method using liquid He in the absence of microwave irradiation. A good freezing zone was also experimentally demonstrated in specimens of rat liver and heart muscle. Microwave irradiation does not have appreciable effects on the ultrastructure of squid retina. The mechanism underlying the improvement in the rapid freezing under the microwave irradiation is discussed.

Chaos, Phase Locking and Bifurcation in Normal Squid Axons

Membrane potential responses of squid giant axons to periodic trains of current pulses were experimentally studied in detail. The giant axon of squid (Doryteuthis bleekeri) was exposed under normal physiological conditions; the intact axon was immersed in natural sea water at 14 ± 0.01°C, and stimulated with periodic trains of current pulses with the pulse intensity I and the period T. The firing modes were determined as a function of I/Ith and T where Ith stood for the threshold current intensity: periodic and chaotic responses were obtained.

Cell activation by CpG ODN leads to improved electrofusion in hybridoma production

Hybridoma formation is an indispensable step in the production of monoclonal antibodies. Obtaining highly efficient fusion of an antibody-producing cell to the myeloma cell to form the hybridoma is an important step in this process. The electrofusion method is superior to chemical fusion methods such as the polyethylene glycol (PEG) method due to its high fusion efficiency. However, this method requires cell activation prior to electrofusion, a process that is time-consuming and tends to cause cell death. In this study, we achieved much higher fusion efficiency by stimulating B cells with CpG oligodeoxynucleotide (CpG ODN) over shorter periods. Splenocytes were isolated from immunized mice and cultured in the presence of a CpG ODN for 1 or 2 days. This CpG ODN stimulation evokes about one order of magnitude higher fusion efficiency than other stimulators. CpG ODN stimulation not only increases the fusion efficiency but also the number of antibody-producing cells. This leads to a substantial increase in the number of positive clones obtained. This highly efficient fusion method was used to produce a functional antibody against Gaussia luciferase. This method was found to produce greater numbers of hybridomas and to enable direct screening for antibodies with functional characteristics such as inhibition of the luminescence activity of an antigen. We were able to establish a functional antibody against Gaussia luciferase after a single fusion experiment using our electrofusion method.

Spatial long-range interactions in squid giant axons

Abstract Membrane states and their bifurcation characteristics are studied for the squid giant axon as a function of external Ca2+ concentration, temperature and externally applied current step. It is well known that the membrane states are divided into two states, resting (R) and spontaneous oscillation (O), according to the Ca2+ concentration contained in the solution surrounding the axon. The present experiments further clarified that each of these states was further subdivided into higher (H) and lower (L) temperature phases, according to temperature. The spatially unclamped axon in the higher-temperature phase, either in the R or O state, can bifurcate to produce limit-cycle oscillations of action potentials. The bifurcation parameters are external Ca2+ concentrations and externally applied current for the axon in the O and R states, respectively. Both the axon in the lower-temperature phase and the spatially clamped axon in the higher-temperature phase can bifurcate to produce intermittent oscillations of action potentials. The bifurcation characteristics at or between the higher- and lower-temperature phases are closely related to the spatial properties of the preoscillatory fluctuations along the axon, suggesting that a particular spatial interaction is responsible for the periodically oscillatory dynamics and the bifurcation to it. The molecular origin of the spatial interaction possibly originates from the specific distribution of Na channels, which may be regulated by subaxolemmal cytoskeletons. Electron microscopic experiments and other evidence to support this idea are described.

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.