The translational self-diffusion coefficients of supercooled water at atmospheric pressure were examined using pulsed-gradient spin−echo NMR diffusion measurements down to 238 K. As the temperature decreased, the diffusion behavior became distinctly non-Arrhenius. It was found that the diffusion behavior when plotted in an Arrhenius form was well-described by a Vogel−Tamman−Fulcher-type relationship in the temperature range from 298 to about 242 K. However, a fractional power-law-type equation was found to provide a better fit that extended over the entire measured temperature range. Below this temperature range, the diffusion coefficient decreased rather steeply, and at 238 K, the diffusion coefficient was 1.58 × 10-10 m2 s-1, the lowest value of the water diffusion coefficient so far determined. At this temperature the activation energy for the diffusion was found to be of the order of 44.4 kJ mol-1. The data presented here should allow theoretical models of water to be more stringently tested.

Xenopus laevis can regenerate an amputated limb completely at early limb bud stages, but the metamorphosed froglet gradually loses this capacity and can regenerate only a spike-like structure. We show that the spike formation in a Xenopus froglet is nerve dependent as is limb regeneration in urodeles, since denervation concomitant with amputation is sufficient to inhibit the initiation of blastema formation and fgf8 expression in the epidermis. Furthermore, in order to determine the cause of the reduction in regenerative capacity, we examined the expression patterns of several key genes for limb patterning during the spike-like structure formation, and we compared them with those in developing and regenerating limb buds that produce a complete limb structure. We cloned Xenopus HoxA13, a marker of the prospective autopodium region, and the expression pattern suggested that the spike-like structure in froglets is accompanied by elongation and patterning along the proximodistal (PD) axis. On the other hand, shh expression was not detected in the froglet blastema, which expresses fgf8 and msx1. Thus, although the wound epidermis probably induces outgrowth of the froglet blastema, the polarizing activity that organizes the anteroposterior (AP) axis formation is likely to be absent there. Our results demonstrate that the lost region in froglet limbs is regenerated along the PD axis and that the failure of organization of the AP pattern gives rise to a spike-like incomplete structure in the froglet, suggesting a relationship between regenerative capacity and AP patterning. These findings lead us to conclude that the spike formation in postometamorphic Xenopus limbs is epimorphic regeneration.

Blastema formation, the initial stage of epimorphic limb regeneration in amphibians, is an essential process to produce regenerates. In our study on nerve dependency of blastema formation, we used forelimb of Xenopus laevis froglets as a system and applied some histological and molecular approaches in order to determine early events during blastema formation. We also investigated the lateral wound healing in comparison to blastema formation in limb regeneration. Our study confirmed at the molecular level that there are nerve-dependent and -independent events during blastema formation after limb amputation, Tbx5 and Prx1, reliable markers of initiation of limb regeneration, that start to be expressed independently of nerve supply, although their expressions cannot be maintained without nerve supply. We also found that cell proliferation activity, cell survival and expression of Fgf8, Fgf10 and Msx1 in the blastema were affected by denervation, suggesting that these events specific for blastema outgrowth are controlled by the nerve supply. Wound healing, which is thought to be categorized into tissue regeneration, shares some nerve-independent events with epimorphic limb regeneration, although the healing process results in simple restoration of wounded tissue. Overall, our results demonstrate that dedifferentiated blastemal cells formed at the initial phase of limb regeneration must enter the nerve-dependent epimorphic phase for further processes, including blastema outgrowth, and that failure of entry results in a simple redifferentiation as tissue regeneration.

The Xenopus adult limb has very limited regeneration ability, and only a simple cartilaginous spike structure without digits is formed after limb amputation. We found that expression of Shh and its downstream genes is absent from the regenerating blastema of the Xenopus froglet limb. Moreover, we found that a limb enhancer region of the Shh gene is highly methylated in the froglet, although the sequence is hypomethylated in the Xenopus tadpole, which has complete limb regeneration ability. These findings, together with the fact that the promoter region of Shh is hardly methylated in Xenopus, suggest that regenerative failure (deficiency in repatterning) in the Xenopus adult limb is associated with methylation status of the enhancer region of Shh and that a target-specific epigenetic regulation is involved in gene re-activation for repatterning during the Xenopus limb regeneration process. Because the methylation level of the enhancer region was low in other amphibians that have Shh expression in the blastemas, a low methylation status may be the basic condition under which transcriptional regulation of Shh expression can progress during the limb regeneration process. These findings provide the first evidence for a relationship between epigenetic regulation and pattern formation during organ regeneration in vertebrates.

The associative behavior of aqueous methanol, ethanol, and tert-butyl alcohol solutions at mole fractions ranging from 0 to 1 at 273, 283, and 298 K was examined using PGSE NMR measurements of the self-diffusion coefficients of the alkyl group, water and, depending on the exchange rate, hydroxyl protons. The results show that tert-butyl alcohol has the greatest ability to stabilize water through hydrophobic hydration than methanol or ethanol due to the more ideal fit of the tert-butyl group to the structure of water. However, at higher concentrations tert-butyl alcohol is the least able to cohesively interact with water through hydrogen bonding. The results provide compelling evidence for alcohol self-association (methanol < ethanol < tert-butyl alcohol) in very dilute solution. The alcohol molecules can be likened to very short lipid molecules undergoing complicated solution interactions due to their amphiphilic nature.

By reciprocal transplantation experiments with regenerative and nonregenerative Xenopus limbs, we recently demonstrated that the regenerative capacity of a Xenopus limb depends on mesenchymal tissue and we suggested that fgf-10 is likely to be involved in this capacity (Yokoyama et al., 2000, Dev. Biol. 219, 18-29). However, the data obtained in that study are not conclusive evidence that FGF-10 is responsible for the regenerative capacity. We therefore investigated the role of FGF-10 in regenerative capacity by directly introducing FGF-10 protein into nonregenerative Xenopus limb stumps. Exogenously applied FGF-10 successfully stimulated the regenerative capacity, resulting in the reinduction of all gene expressions (including shh, msx-1, and fgf-10) that we examined and the regeneration of well-patterned limb structures. We report here for the first time that a certain molecule activates the regenerative capacity of Xenopus limb, and this finding suggests that FGF-10 could be a key molecule in possible regeneration of nonregenerative limbs in higher vertebrates.

Retinoic acid is a putative morphogen in limb formation in the chick and other vertebrates. In chick limb formation, it is thought that retinoic acid is released from the zone of polarizing activity (ZPA) and the concentration gradient of retinoic acid formed from the posterior to the anterior provides positional cues for digit formation. Implantation of a bead containing retinoic acid at the anterior margin of the limb bud induces a mirror-image symmetrical duplication of the digit pattern similar to that observed when the ZPA is grafted into the anterior margin of the host limb bud. Also, the level of endogenous retinoic acid (25 nM on average) is higher in the posterior one third of the limb bud. We found that when the bead containing either retinoic acid or an analogue but not the ZPA, was implanted in the anterior margin of the chick limb bud, expression of the retinoic acid receptor type-beta gene was induced around the bead within 4 h. These results indicate that exogenous retinoic acid is not identical with the ZPA morphogen. As the anterior tissue exposed to retinoic acid has polarizing activity, we conclude that the primary function of exogenous retinoic acid is to induce polarizing activity in the limb bud.

Members of the heat-shock protein (HSP)40 regulate the protein folding activity of HSP70 proteins and help the functional specialization of this molecular chaperone system in various types of cellular events. We have recently identified Hsp40 as a component of flagellar axoneme in the ascidian Ciona intestinalis, suggesting a correlation between Hsp40 related chaperone system and flagellar function. In this study, we have found that Ciona 37-kDa Hsp40 is extracted from KCl-treated axonemes with 0.5 M KI solution and comigrates with radial spoke protein (RSP)3 along with several proteins as a complex through gel filtration and ion exchange columns. Peptide mass fingerprinting with matrix-assisted laser desorption ionization/time of flight/mass spectrometry revealed that other proteins in the complex include a homolog of sea urchin spokehead protein (homolog of RSP4/6), a membrane occupation and recognition nexus repeat protein with sequence similarity with meichroacidin, and a functionally unknown 33-kDa protein. A spoke head protein, LRR37, is not included in the complex, suggesting that the complex constructs the stalk of radial spoke. Immunoelectron microscopy indicates that Hsp40 is localized in the distal portion of spoke stalk, possibly at the junction between spoke head and the stalk.