The higher plant ADP-glucose pyrophosphorylase is a heterotetramer consisting of two subunit types, which have evolved at different rates from a common ancestral gene. The potato tuber small subunit (SS) displays both catalytic and regulatory properties, whereas the exact role of the large subunit (LS), which contains substrate and effector binding sites, remains unresolved. We identified a mutation, S302N, which increased the solubility of the recombinant potato tuber LS and, in turn, enabling it to form a homotetrameric structure. The LS302N homotetramer possesses very little enzyme activity at a level 100-fold less than that seen for the unactivated SS homotetramer. Unlike the SS enzyme, however, the LS302N homotetramer enzyme is neither activated by the effector 3-phosphoglycerate nor inhibited by P i . When combined with the catalytically silenced SS, S D143N , however, the LS302N-containing enzyme shows significantly enhanced catalytic activity and restored 3-PGA activation. This unmasking of catalytic and regulatory potential of the LS is conspicuously evident when the activities of the resurrected L K41R⅐T51K⅐S302N homotetramer are compared with its heterotetrameric form assembled with S D143N . Overall, these results indicate that the LS possesses catalytic and regulatory properties only when assembled with SS and that the net properties of the heterotetrameric enzyme is a product of subunit synergy.

Cell-cell adhesion is fundamental in morphogenesis and is known to be mediated by several groups of cell adhesion molecules. Cadherins are a group of such molecules involved in the Ca2+-dependent cell-cell adhesion mechanism and are found in most kinds of tissue. In this study using indirect immunofluorescence microscopy, we analyzed the distribution of two kinds of cadherins, E- and P-cadherin, in developing tooth germs. In the molar tooth germs at the early bud stage, marginal cells of the epithelial tooth bud expressed both E- and P-cadherin, whereas central cells expressed only E-cadherin. At the cap stage, in addition to the cells of the inner and outer enamel epithelium, which outline the enamal organ, cells of the enamel knot, which is thought to control tooth morphogenesis, strongly expressed P-cadherin. The expression of P-cadherin was prominent in the inner enamel epithelium during the early to mid bell stage, and was also evident in the non-dividing cell masses at future cusp tips, which are the so-called secondary enamel knots. In the tooth germ at the late bell stage when the cells of the inner enamel epithelium began to polarize to differentiate into ameloblasts, the polarizing ameloblasts lost P-cadherin and strongly expressed E-cadherin. However, E-cadherin was also lost from polarized ameloblasts at later stages. The stratum intermedium and the stellate reticulum were E-cadherin positive from the bell stage onward even at the stages when the ameloblasts became E-cadherin negative again. These results suggest that the differential expression of E- and P-cadherin during morphogenetic stages plays a role in the regulation of tooth morphogenesis, whereas alteration of E-cadherin expression during later stages of tooth development is related to differentiation and function of the ameloblasts and other cells supporting amelogenesis.

Apoptotic cells in the taste buds of mouse circumvallate papillae after the sectioning of bilateral glossopharyngeal nerves were examined by the method of DNA nick-end labeling (TUNEL), together with standard electron microscopy. The taste buds decreased in number and size 3-11 days after denervation and disappeared at 11 days. The TUNEL method revealed only a few positively stained nuclei in normal taste buds but, in those of mice 1-5 days after denervation, the number of positive nuclei had increased to 3-5 times that of taste buds from normal mice. Electron-microscopic observation after denervation demonstrated taste bud cells containing condensed and fragmentary nuclei in a cytoplasm with increased density. The results show that taste bud cells under normal conditions die by apoptosis at the end of their life span, and that gustatory nerve sectioning causes apoptosis of taste bud cells with taste buds decreasing in number and ultimately disappearing.

We had previously demonstrated that expression of a cytoplasmic-localized ADPglucose pyrophosphorylase (AGPase) mutant gene from Escherichia coli in rice endosperm resulted in enhanced starch synthesis and, in turn, higher seed weights. In this study, the levels of the major primary carbon metabolites were assessed in wild type and four transgenic CS8 rice lines expressing 3- to 6-fold higher AGPase activity. Consistent with the increase in AGPase activity, all four transgenic CS8 lines showed elevated levels of ADPglucose (ADPglc) although the extent of increases in this metabolite was much higher than the extent of increases in starch as measured by seed weight. Surprisingly, the levels of several other key intermediates were significantly altered. Glucose 1-phosphate (Glc 1-P), a substrate of the AGPase reaction, as well as UDPglucose and Glc 6-P were also elevated to the same relative extent in the transgenic lines compared with the wild-type control. Analysis of metabolite ratios showed no significant differences between the wild type and transgenic lines, indicating that the reactions leading from sucrose metabolism to ADPglc formation were in near equilibrium. Moreover, glucose and fructose levels were also elevated in three transgenic lines that showed the largest differences in metabolites and seed weight over the wild type, suggesting the induction of invertase. Overall, the results indicate that the AGPase-catalyzed reaction is no longer limiting in the transgenic lines, and constraints on carbon flux into starch are downstream of ADPglc formation, resulting in an elevation of precursors upstream of ADPglc formation.

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