Astronauts experience osteoporosis‐like loss of bone mass because of microgravity conditions during space flight. To prevent bone loss, they need a riskless and antiresorptive drug. Melatonin is reported to suppress osteoclast function. However, no studies have examined the effects of melatonin on bone metabolism under microgravity conditions. We used goldfish scales as a bone model of coexisting osteoclasts and osteoblasts and demonstrated that mRNA expression level of acetylserotonin O‐methyltransferase, an enzyme essential for melatonin synthesis, decreased significantly under microgravity. During space flight, microgravity stimulated osteoclastic activity and significantly increased gene expression for osteoclast differentiation and activation. Melatonin treatment significantly stimulated Calcitonin (an osteoclast‐inhibiting hormone) mRNA expression and decreased the mRNA expression of receptor activator of nuclear factor κB ligand (a promoter of osteoclastogenesis), which coincided with suppressed gene expression levels for osteoclast functions. This is the first study to report the inhibitory effect of melatonin on osteoclastic activation by microgravity. We also observed a novel action pathway of melatonin on osteoclasts via an increase in CALCITONIN secretion. Melatonin could be the source of a potential novel drug to prevent bone loss during space flight.

In this study, to better understand the molecular mechanism underlying cellular responses to mild hyperthermia, we investigated gene expression patterns and genetic networks in human myelomonocytic lymphoma U937 cells using high-density oligonucleotide microarrays and computational gene expression analysis tools. The cells were incubated at 41 degrees C for 30 min (mild hyperthermia treatment) and then at 37 degrees C for 0-6 h. Although the mild hyperthermia treatment of the cells did not induce apoptosis, significant increases in the protein expression levels of heat shock proteins (HSPs), namely, Hsp27, Hsp40 and Hsp70, were observed following the activation of heat shock factor-1. Of the 22,283 probe sets analyzed, 423 probe sets were up-regulated and 515 probe sets were down-regulated by >1.5-fold in the cells 3 h post-treatment. Computational gene network analysis demonstrated that the significant genetic network A that contained many HSPs such as DNAJB1, HSPA1A, and HSPA1B was associated with cellular function and maintenance, post-transcriptional modification, or protein folding. Moreover, the significant genetic network B whose core contained v-myc myelocytomatosis viral oncogene homolog (MYC) was associated with cell morphology, cell cycle, and cellular development. The expression levels of nine selected genes were comparable to those determined by microarray analysis with real-time quantitative PCR assay. The present results indicate that mild hyperthermia affects the expression of a large number of genes and provides additional novel insights into the molecular basis of mild hyperthermia in cells.

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It is known that the teleost scale regenerates after being removed. We previously reported that the osteogenesis in regenerating scales was very similar to that in calvarial bone, which suggests that regenerating scale can be used as a model for osteogenesis to analyze the inte raction b e t we e n oste oblasts and osteoclasts. In the present study, we developed an assay system using regenerating scales by modifying our assay system with normal scales. The weight of one regenerating scale cannot be measured precisely because the rate of calcification in regenerating scales is lower than that in normal scales. In regenerating scales, thus, the respective marker enzyme activit y, i.e ., alkaline phosphatase (ALP) for osteoblasts and tartrate-resistant acid phosphatase (TRAP) for osteoclasts, was normalized by the surface area (mm 2) of each goldfish scale. With this modified method, we were able to detect ALP and TRAP activities with no variation in the lines of regenerating scales. In addition, we found that the ALP activity in the regenerating scales significantly increased under 3G acceleration loading by vibration, while the TRAP activity in the loaded regenerating scales significantly decreased. We strongly believe that the regenerating scale is a good material for the analysis of bone me tabolism unde r dif fe re nt gravit y environments.

Abstract. Local hyperthermia (HT) for various types of malignant tumors has shown promising antitumor effects. To confirm the detailed molecular mechanism underlying cell death induced by HT, gene expression patterns and gene networks in human oral squamous cell carcinoma (OSCC) cells were examined using a combination of DNA microarray and bioinformatics tools. OSCC HSC-3 cells were treated with HT at 44˚C for 90 min or mild hyperthermia (MHT) at 42˚C for 90 min, followed by culturing at 37˚C for 0-24 h. Treatment of cells with HT prevented cell proliferation (62%) and induced cell death (17%), whereas these alterations were not observed in cells treated with MHT. Microarray analysis revealed substantial differences with respect to gene expression patterns and biological function for the two different hyperthermic treatments. Moreover, we identified the temperature-specific gene networks D and H that were obtained from significantly up-regulated genes in the HT and MHT conditions, respectively, using Ingenuity pathway analysis tools. Gene network D, which contains 14 genes such as ATF3, DUSP1 and JUN, was associated with relevant biological functions including cell death and cellular movement. Gene network H, which contains 13 genes such as BAG3, DNAJB1 and HSPA1B, was associated with cellular function and maintenance and cellular assembly and organization. These findings provide a basis for understanding the detailed molecular mechanisms of cell death elicited by HT in human OSCC cells. IntroductionIn the treatment of oral squamous cell carcinoma (OSCC), treatment outcomes have improved in the past two decades due to progress in reconstructive techniques, stereotactic radiotherapy, new anticancer drugs such as taxanes, and combinations of these therapeutic modalities (1). Nonetheless, it is well known that patients with advanced tumors or tumor recurrence have a poor prognosis with a median survival period less than one year (2). Local hyperthermia (HT) for various malignant tumors including OSCC has been recognized as an effective and attractive tool with the advantages of relatively few side effects and slight damage to normal tissue. Combinations of HT with chemotherapy, radiotherapy or both have been clinically used for patients with cancer in various organs, and their antitumor effects have been verified by many clinical trials (3-7).In general, HT elicits a wide spectrum of stress responses such as induction of heat shock proteins (HSPs), protein aggregation, an imbalance of protein homeostasis, DNA and RNA damage, reactive oxygen species production, cell growth arrest and cell death in mammalian cells (8,9). In particular, HSPs, which are induced by heat, behave as molecular chaperones and exert strong cytoprotective effects that prevent cell death. The treatment of cells with HT induces numerous signal transduction pathways which contribute variously to cell death and survival (10-12). Although many biological processes are affected by HT, the overall responses to HT in mammalian cells remain unknown.Re...