2-(Trimethylammonium) Ethyl (R)-3-Methoxy-3-oxo-2-Stearamidopropyl Phosphate Suppresses Osteoclast Maturation and Bone Resorption by Targeting Macrophage-Colony Stimulating Factor Signaling

2-(Trimethylammonium) ethyl (R)-3-methoxy-3-oxo-2-stearamidopropyl phosphate [(R)-TEMOSPho], a derivative of an organic chemical identified from a natural product library, promotes highly efficient megakaryopoiesis. Here, we show that (R)-TEMOSPho blocks osteoclast maturation from progenitor cells of hematopoietic origin, as well as blocking the resorptive function of mature osteoclasts. The inhibitory effect of (R)-TEMOSPho on osteoclasts was due to a disruption of the actin cytoskeleton, resulting from impaired downstream signaling of c-Fms, a receptor for macrophage-colony stimulating factor linked to c-Cbl, phosphoinositol-3-kinase (PI3K), Vav3, and Rac1. In addition, (R)-TEMOSPho blocked inflammation-induced bone destruction by reducing the numbers of osteoclasts produced in mice. Thus, (R)-TEMOSPho may represent a promising new class of antiresorptive drugs for the treatment of bone loss associated with increased osteoclast maturation and activity.     

Radio frequency radiation causes no nonthermal damage in enzymes and living cells

The ability of radio frequency radiation (RFR) to exert irreversible nonthermal (i.e., not caused by accompanying heat) effects on biologics has been widely debated due to a relative paucity of comprehensive critical details in published reports dealing with this issue. In this study, we used rigorous control over experimental conditions to determine whether continuous RFR nonthermally affects commercially important enzymes and live bacterial and human cells using three most commonly used frequencies in current RF identification technology, namely 2.45 GHz, 915 MHz, and 13.56 MHz. Diverse biological samples were exposed to RFR under deliberately harsh conditions to increase the likelihood of observing such effects should they exist. Enzymatic activities of horseradish peroxidase and β‐galactosidase in aqueous solution exhibited no statistically discernable consequences of even very intense RFR. Likewise, with putative thermal effects excluded, the viabilities of bacteria (both gram‐positive and gram‐negative) and of human cells were not detectably compromised by such an RFR exposure. © 2010 American Institute of Chemical Engineers Biotechnol. Prog., 2010     

Raf-independent, PP2A-dependent MEK activation in response to ERK silencing

Biological roles of ERK and MEK in signal transduction have been controversial. The aim of the current study was to determine the role of ERK1/2 in signaling through the ERK-MAPK cascade by using RNAi methodology. Transient transfection of erk1 or erk2 siRNA decreased the respective protein level to 3-8% in human lung fibroblasts. Interestingly, individual ERK isoform silencing resulted in a 2-fold reciprocal increase in phosphorylation of the alternate ERK isoform, with no change in respective total protein expression. Moreover, MEK was hyperphosphorylated as a result of combined ERK1 and ERK2 silencing, but was unaffected in individual ERK1 or ERK2 silenced cells. This hyperactivation of MEK was not due to activation of Raf family members, but rather was associated with PP2A downregulation. These data highlight the existence of a feedback loop in normal cells whereby ERK silencing is associated with decreased PP2A activity and consequent MEK activation.     

Effects of auditory stimuli on the lifespan of Drosophila melanogaster

It is well known that music can have calming effects on humans, other mammals and birds. Reducing environmental stress or enhancing the resistance to certain stressors has been shown to extend lifespan in several organisms. Evidence also suggests that mild temporary stress may also enhance stress resistance and ultimately slow the aging process. This study explored the possibility that music may influence the lifespan of the fruit fly Drosophila melanogaster, possibly by affecting responses to stress. Flies received either background sounds (control), or background sounds supplemented with music (experimental). The experimental group had classical music playing constantly at an average of 20 dB above background sound. The median lifespan of females receiving music was 42 days compared to the median of 45 days without music, but the difference was not significant. For males median lifespans were 42 days with music exposure, and 47 days without music and the difference was significant. These results suggest that exposure to classical music decreased the lifespan of male Drosophila. Both experimental and control populations showed age-dependent increases in mortality, indicating that music affects the normal aging process rather than showing overt toxicity. These results suggest that certain auditory stimuli may be stressful and can be used as insect management.     

Fast and sensitive delineation of brain tumor with clinically compatible moxifloxacin labeling and confocal microscopy

Delineation of brain tumor margins during surgery is critical to maximize tumor removal while preserving normal brain tissue to obtain optimal clinical outcomes. Although various imaging methods have been developed, they have limitations to be used in clinical practice. We developed a high‐speed cellular imaging method by using clinically compatible moxifloxacin and confocal microscopy for sensitive brain tumor detection and delineation. Moxifloxacin is a Food and Drug Administration (FDA) approved antibiotic and was used as a cell labeling agent through topical administration. Its strong fluorescence at short visible excitation wavelengths allowed video‐rate cellular imaging. Moxifloxacin‐based confocal microscopy (MBCM) was characterized in normal mouse brain specimens and visualized their cytoarchitecture clearly. Then, MBCM was applied to both brain tumor murine models and two malignant human brain tumors of glioblastoma and metastatic cancer. MBCM detected tumors in all the specimens by visualizing dense and irregular cell distributions, and tumor margins were easily delineated based on the cytoarchitecture. An image analysis method was developed for automated detection and delineation. MBCM demonstrated sensitive delineation of brain tumors through cytoarchitecture visualization and would have potentials for human applications, such as a surgery‐guiding method for tumor removal.      

Cytotoxic T lymphocyte response against multiple simian immunodeficiency virusA (SIV) proteins in SIV-infected macaques.

To identify the target proteins of CD8+ T lymphocytes we have explored the cytolytic immune responses of 12 rhesus macaques experimentally infected with the simian immunodeficiency virus (SIVmac). Target cells were autologous B cell lines presenting SIVmac proteins after infection with recombinant vaccinia viruses. The eight following proteins were studied: ENV, POL, GAG, NEF, VIF, REV, TAT, and VPX. Macaque PBMC stimulated with Con A and expanded in T cell growth factor-containing medium produced cell lines with cytolytic activity in the majority of infected animals (9/12). The structural proteins ENV, POL, and GAG were recognized by cell lines derived from nine, eight, and six macaques, respectively. The small regulatory proteins also represented efficient CTL targets, a specific activity being detected against NEF (8/12), REV (7/12), VPX (7/12), TAT (6/12), and VIF (5/12). Most cytotoxic responses (except those directed against ENV) were mediated by CD8 cells and were MHC class I restricted. Limiting dilution analysis allowed us to quantify the frequency of CTL precursors and confirmed the high immunogenicity of multiple SIV proteins. Three different patterns of response could be defined: six animals were able to recognize at least six of the eight tested target proteins, two of them reacting with all eight target proteins. The other three responder macaques reacted only against a few SIV proteins, whereas no cytotoxic activity was detected in the three remaining infected macaques and in the nine negative controls. The six animals responding against multiple proteins were still healthy 12 to 22 mo after infection with two of them presenting a decrease in circulating CD4 cells concurrently to the disappearance of the CTL response. Conversely, three nonresponder or low responder macaques developed an overt disease after 4 to 12 mo, and two other presented a very low level of CD4 cells, suggesting that the pattern of response may be of prognostic value.     

Novel transmembrane protein 126A (TMEM126A) couples with CD137L reverse signals in myeloid cells

Members of the TNF family can promote signals in myeloid cells and both positively and negatively regulate the production of pro-inflammatory cytokines depending on the target myeloid cell type. Using the yeast-two hybrid system, we identified transmembrane protein 126A (TMEM126A) as a binding partner for CD137L (4-1BB ligand). We found that TMEM126A associated and co-localized with CD137L in a mouse macrophage cell line and knockdown of TMEM126A with siRNA abolished the CD137L-induced tyrosine phosphorylation as well as the up-regulation of M-CSF, IL-1β and TN-C expressions. Knockdown of TMEM126A also blocked the down-regulation of IL-1β and IL-6 expressions induced by CD137L in thioglycollate-elicited primary peritoneal macrophages. Knockdown of TMEM126A by stable retroviral TMEM126A shRNA transduction also abolished CD137L-induced tyrosine phosphorylation and cell adherence. These findings identify a novel molecule that bridges TNF family cytokines and pro-inflammatory cytokine secretion in myeloid cells.     

Regulation of c-Myc Expression by Ahnak Promotes Induced Pluripotent Stem Cell Generation

We have previously reported that Ahnak-mediated TGFβ signaling leads to down-regulation of c-Myc expression. Here, we show that inhibition of Ahnak can promote generation of induced pluripotent stem cells (iPSC) via up-regulation of endogenous c-Myc. Consistent with the c-Myc inhibitory role of Ahnak, mouse embryonic fibroblasts from Ahnak-deficient mouse (Ahnak^−/− MEF) show an increased level of c-Myc expression compared with wild type MEF. Generation of iPSC with just three of the four Yamanaka factors, Oct4, Sox2, and Klf4 (hereafter 3F), was significantly enhanced in Ahnak^−/− MEF. Similar results were obtained when Ahnak-specific shRNA was applied to wild type MEF. Of note, expressionof Ahnak was significantly induced during the formation of embryoid bodies from embryonic stem cells, suggesting that Ahnak-mediated c-Myc inhibition is involved in embryoid body formation and the initial differentiation of pluripotent stem cells. The iPSC from 3F-infected Ahnak^−/− MEF cells (Ahnak^−/−-iPSC-3F) showed expression of all stem cell markers examined and the capability to form three primary germ layers. Moreover, injection of Ahnak^−/−-iPSC-3F into athymic nude mice led to development of teratoma containing tissues from all three primary germ layers, indicating that iPSC from Ahnak^−/− MEF are bona fide pluripotent stem cells. Taken together, these data provide evidence for a new role for Ahnak in cell fate determination during development and suggest that manipulation of Ahnak and the associated signaling pathway may provide a means to regulate iPSC generation.