Differentiation of ameloblasts from undifferentiated epithelial cells is controlled by diverse growth factors, as well as interactions between epithelium and mesenchyme. However, there is a considerable lack of knowledge regarding the precise mechanisms that control ameloblast differentiation and enamel biomineralization. We found that the expression level of carbonic anhydrase II (CAII) is strongly up-regulated in parallel with differentiation of enamel epithelium tissues, while the enzyme activity of CA was also increased along with differentiation in ameloblast primary cultures. The expression level of amelogenin, a marker of secretory-stage ameloblasts, was enhanced by ethoxzolamide (EZA), a CA inhibitor, as well as CAII antisense (CAIIAS), whereas the expression of enamel matrix serine proteinase-1 (EMSP-1), a marker for maturation-stage ameloblasts, was suppressed by both. These agents also promoted ameloblast proliferation. In addition, inhibition of ameloblast differentiation by EZA and CAIIAS was confirmed using tooth germ organ cultures. Furthermore, EZA and CAIIAS elevated intracellular pH in ameloblasts, while experimental decreases in intracellular pH abolished the effect of CAIIAS on ameloblasts and triggered the activation of c-Jun N-terminal kinase (JNK). SP600125, a JNK inhibitor, abrogated the response of ameloblasts to an experimental decrease in intracellular pH, while the inhibition of JNK also impaired ameloblast differentiation. These results suggest a novel role for CAII during amelogenesis, that is, controlling the differentiation of ameloblasts. Regulation of intracellular pH, followed by activation of the JNK signaling pathway, may be responsible for the effects of CAII on ameloblasts.

L ymph nodes are small organs occurring in series along the course of lymphatic vessels. The parenchyma consists of a highly organized accumulation of lymphoid tissue, which recognizes antigens in the lymph and builds up a specific immune reaction against them especially in lymphoid follicles. Afferent lymphatic vessels come from the relatively huge area in the body and enter the node at multiple sites over its convex surface. Therefore, in this study, we investigated whether the parenchyma of the node is restricted by the afferent vessels. Two different colored tracers, inks black and red or fluorescent dyes AlexaFluor ® 488/594 , were subcutaneously injected in the tongue and gingiva of mice. At the indicated time, submandibular lymph nodes were dissected and examined under light and/or fluorescent microscopy. Injection of 0.1 ml of a tracer at 1 h showed that the tracers entered into the whole area of the node whereas a 0.02 ml injection showed that traces localized some restricted area in the node. The lymph node at 1 h after the injection of two tracers showed that both traces penetrated into different parts of the node. Furthermore, at 48 h two different tracers were detected at the different follicles. These results indicated the regional restriction of lymphoid follicles in the node which might be regulated by the different site of afferent vessels to the node and suggest that each of the lymphoid follicles in a node respond to different antigens.

The skeletal system, characterized by a hard tissue component, is an extraordinarily dynamic system with disparate functions such as structural support, movement, soft-organ protection and the maintenance of calcium homeostasis. In addition, bone houses definitive hematopoiesis. During vertebrate ontogeny, hematopoiesis is established sequentially in different anatomical sites. Around birth time, hematopoietic site shifts from fetal liver to the forming bone marrow cavity which is the major site of hematopoiesis during adult life. 1～3) Vertebrate bones are formed by two different types of ossification systems, endochondral and intra membranous ossification. Intramembranous bone formation is mediated by osteoblasts differentiated from the periosteal osteogenic cells, which form bone without the mediation of cartilage, while, in the case of endochondral bone formation, osteoblasts form bone on a calcified cartilage scaffold after epiphyseal and physeal cartilage have shaped and elongated. 4～6) In both of ossification systems, hematopoietic tissues are located inside the forming bones.