Skeletal development and bone homeostasis are dynamic but coordinated cellular process that involves proliferation, migration and differentiation. Molecular biology and genome science promoted this realm of biomedical science by elucidating cell lineages and essential molecules and their interactions. Fluorescence live imaging has made it possible to quantitatively analyze multi-cellular processes in 4 dimensions, thus providing coherent understanding of distinct levels of description from molecular levels to the tissue, organ and organism. Application of this approach has given further insight into and comprehension of the dynamic process of, not a mere description of the molecular hierarchy, skeletal biology. In this review paper, how fluorescent imaging has shed new light on the skeletal biology will be discussed. We introduce several current topics in the application of fluorescent imaging on skeletal patterning, morphometry of bone cells and cellular behavior in bone marrow.

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High resolution imaging of intracellular calcium (Ca²⁺) concentrations ( [Ca²⁺] _i) has revealed that Ca²⁺ signals show diverse responses, such as Ca²⁺ oscillations and Ca²⁺ waves; these signals are exploited to control diverse cellular processes. Ca²⁺ responses in non-excitable cells are primarily regulated by inositol 1,4,5-trisphosphate (IP₃). To elucidate the mechanisms of these diverse Ca²⁺ responses, we have developed a series of fluorescent IP₃ biosensors, the LIBRAv series, using cyan fluorescent protein (CFP) and yellow fluorescent protein (YFP) in combination with fluorescence resonance energy transfer (FRET) technologies. These fluorescent IP₃ biosensors are specific to IP₃ and permit the monitoring of IP₃ dynamics in single living cells. Studies conducted with IP₃ biosensors during agonist-induced Ca²⁺ oscillations revealed cell type-specific differences in IP₃ dynamics, non-fluctuating rises in cytosolic IP₃ concentrations ( [IP₃] _i) and repetitive IP₃ spikes. These results provide important experimental information for resolving long-standing questions regarding the mechanisms responsible for Ca²⁺ oscillations. IP₃ biosensors also demonstrated an intracellular gradient of IP₃ and its wave-like expansion during mechanical stimulation-induced Ca²⁺ waves. As such, continued improvements in IP₃ biosensors and the development of additional novel fluorescent biosensors are highly likely to provide a better understanding of the regulatory mechanisms of various forms of Ca²⁺ signals and clarification of their physiologic roles.

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Phosphoglycerate mutase (PGAM) family member 5 (PGAM5) is a member of the PGAM family, the prototypical members of which are enzymes of intermediary metabolism that convert 3-phosphoglycerate to 2-phosphoglycerate in glycolysis. It has recently been found that PGAM5 lacks authentic PGAM activity but possesses protein phosphatase activity highly specific to serine and threonine residues. Depending on its phosphatase activity, PGAM5 activates the stress-activated c-Jun N-terminal kinase (JNK) and p38 MAP kinase pathways through their upstream regulator ASK1. Furthermore, PGAM5 is localized to the mitochondria through its N-terminal transmembrane domain and appears to be involved in the regulation of mitochondrial functions. Here we introduce this novel type of protein phosphatase and discuss its roles as a signaling intermediate.

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Cathepsin E (CatE), an endolysosomal aspartic protease, is predominantly expressed in cells of the immune system, and CatE-deficient mice exhibit phenotypes affecting immune responses; however, the precise role of this enzyme remains speculative. In this review, some of the knowledge obtained from a study of CatE functions in host defense against tumor cells is highlighted. In vivo studies using three different genotypes of syngeneic mice (CatE-deficent, wild-type and CatE-overexpressing transgenic mice) revealed that endogenous CatE expression levels are positively associated with the extent of tumor suppression. The number of apoptotic tumor cells and tumor-infiltrating activated macrophages increased in proportion to the endogenous CatE levels. In vitro studies also demonstrated the growth arrest and apoptosis of tumor cells by CatE without affecting normal cells through the proteolytic release of soluble tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) from the cell surface. On the basis of these observations, the present review gives an account of the manifold functions of CatE in host defense against tumor cells.

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The number of water-induced swallows was decreased following capsaicin injection into the facial (whisker pad) skin, masseter or lingual muscle. The capsaicin-induced inhibitory effect on the swallowing reflex was depressed after intrathecal administration of mitogen-activated protein kinase (MAPK) kinase (MEK) inhibitor. The inhibitory effect on swallowing following capsaicin injection into the lingual muscle was diminished by paratrigeminal nucleus (Pa5) lesioning. Many phosphorylated extracellular signal-regulated kinase-like immunoreactive neurons in the nucleus tractus solitarii (NTS) showed gamma-aminobutyric acid (GABA) immunoreactivity and capsaicin-induced inhibition of the swallowing reflex was diminished by local microinjection of the GABA_A receptor antagonist into the NTS.
The present findings suggest that facial skin-NTS, masseter muscle-NTS, lingual muscle-NTS and lingual muscle-Pa5-NTS pathways are involved in the swallowing inhibition by facial, masseter and lingual pain, and that the activation of GABAergic NTS neurons may be involved in inhibition of the swallowing reflex.

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Mechanical stress is an important factor in bone homeostasis, which is impossible to visualize and therefore difficult to evaluate. Biomechanical analysis based on the three-dimensional finite element method (3D FEM) has become more accurate, and it is now possible to analyze load transfer pathways taking into consideration the trabecular structures. It has even become feasible to develop treatment plans based on mechanical factors using image data acquired from the patient. This review describes the usefulness and prospects of biomechanical analysis of the jaw bones, specifically finite element analysis (FEA) based upon the consideration of internal microstructures.

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To elucidate the effects of water on the mechanical properties of the food bolus, the texture of the bolus was assessed. Thirteen adult subjects were instructed to chew 6 g of two test foods, a biscuit and rice cake, with and without water (2 mL). In the early, middle and late stages of mastication, subjects were instructed to spit the food bolus into a cup. Texture parameters (hardness, adhesiveness and cohesiveness)of the bolus were identified by texture profile analysis. Addition of water significantly (p>0.001) decreased the number of chewing strokes (NCS) until swallowing for both test foods. Hardness of each of the two test food boluses decreased during mastication regardless of the presence or absence of water. Adhesiveness and cohesiveness of the biscuit bolus increased, whereas those of the rice cake bolus decreased regardless of the presence or absence of water. There were no significant differences in the three texture parameters immediately before swallowing for both food boluses with and without water, although the NCS until swallowing decreased during mastication with water. These results suggest that the presence of water and saliva accelerates bolus formation for swallowing the biscuits and rice cakes studied.

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Purpose: Detection of a precancerous or cancerous lesion when small is one of the most important factors to improve 5-year survival rates of oral cancer. Although surgical biopsy is the Gold Standard for diagnosing oral lesions, it is impractical to routinely subject large numbers of patients to biopsy. This study had been undertaken to evaluate quantitatively the cellular changes in exfoliated cells from different premalignant and malignant lesions in terms of cell diameter (CD), nuclear diameter (ND) and nucleocytoplasmic ratio and micronucleus frequency. Patients and Methods: Oral mucosal smears were obtained from patients presenting with precancer and cancer using a cytobrush immediately before biopsy. The study group consisted of Group I: lesions with no dysplasia, Group II: lesions with mild and moderate dysplasia, Group III: lesions with severe dysplasia and carcinoma in situ, Group IV: lesions with invasive carcinoma. Results: Comparison of study group and controls showed a highly significant decrease in mean cellular diameter, increase in the nuclear diameter and a larger nucleo cytoplasmic ratio of exfoliated cells. (p<0.0001) Also micronuclei (MN) frequencies were found higher in SCC patients and in precancer than in control subjects. Conclusions: Cytomorphometric analysis via oral brush biopsy is a valuable adjunct to biopsy & CD, ND and micronuclei evaluation serves as important diagnostic markers.

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Evaluation of the temporal correlation between two spike trains is an important issue when studying the dynamics of local neural networks. In the postcentral somatosensory cortex (SI), the temporal profile of neural activity during sustained stimulation is quite variable on the order of 100 msec, reflecting various thalamocortical inputs as well as various stages of the integrating process that might take place within local neural networks. Therefore, a numerical method is needed to evaluate the difference in the gross temporal profile over several hundreds of msec or more. To address this requirement, we tested a method using the dot product between a pair of spike trains. We applied the method to our own data, i. e., 88 pairs of SI neurons recorded simultaneously in one conscious monkey. Each discrete spike train was first smoothed by convolving with a Gaussian function, then the normalized dot product (similarity index) was taken between a pair of spike trains. We next conducted a simulation study using random spike trains (surrogate data set), in which the number of trials and the number of spikes of both units in each trial were set strictly to those of the original data set. The simulation was repeated 100 times for each pair, and the mean and standard deviation of the similarity index were taken. Finally, the difference in the similarity indexes was determined between the original and surrogate data set (z-score). The method successfully quantifies the gross temporal difference of activity in each pair of SI neurons.

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