C9orf10/Ossa regulates the bone metastasis of established lung adenocarcinoma cell subline H322L-BO4 in a mouse model.

Lung cancer frequently metastasizes to the bones. An in vivo model is urgently required to identify potential therapeutic targets for the prevention and treatment of lung cancer with bone metastasis. We established a lung adenocarcinoma cell subline (H322L-BO4) that specifically showed metastasis to the leg bones and adrenal glands. This was achieved by repeated isolation of metastatic cells from the leg bones of mice. The cells were intracardially injected into nude mice. Survival was prolonged for mice that received H322L-BO4 cells versus original cells (H322L). H322L-BO4 cells did not exhibit obvious changes in general in vitro properties associated with the metastatic potential (e.g., cell growth, migration, and invasion) compared with H322L cells. However, the phosphorylation of chromosome 9 open reading frame 10/oxidative stress-associated Src activator (C9orf10/Ossa) was increased in H322L-BO4 cells. This result confirmed the increased anchorage independence through C9orf10/Ossa-mediated activation of Src family tyrosine kinase. Reduction of C9orf10/Ossa by shRNA reduced cells' metastasis to the leg bone and prolonged survival in mice. These findings indicate that H322L-BO4 cells can be used to evaluate the effect of candidate therapeutic targets against bone metastatic lung cancer cells. Moreover, C9orf10/Ossa may be a useful target for treatment of lung cancer with bone metastasis.

Ablation of Csk in neural crest lineages causes corneal anomaly by deregulating collagen fibril organization and cell motility.

Src family kinases (SFKs) have been implicated in the regulation of cell motility. To verify their in vivo roles during development, we generated mutant mice in which Csk, a negative regulator of SFKs, was inactivated in neural crest lineages using the Protein zero promoter in a Cre-loxP system. Inactivation of Csk caused deformities in various tissues of neural crest origins, including facial dysplasia and corneal opacity. In the cornea, the stromal collagen fibril was disorganized and there was an overproduction of collagen 1a1 and several metalloproteases. The corneal endothelium failed to overlie the central region of the eye and the peripheral endothelium displayed a disorganized cytoskeleton. Corneal mesenchymal cells cultured from mutant mice showed attenuated cell motility. In these cells, p130 Crk-associated substrate (Cas) was hyperphosphorylated and markedly downregulated. The expression of a dominant negative Cas (Cas Delta SD) could suppress the cell motility defects. Fluorescence resonance energy transfer analysis revealed that activation of Rac1 and Cdc42 was depolarized in Csk-inactivated cells, which was restored by the expression of either Csk or Cas Delta SD. These results demonstrate that the SFKs/Csk circuit plays crucial roles in corneal development by controlling stromal organization and by ensuring cell motility via the Cas-Rac/Cdc42 pathways.

Electroencephalogram characteristics during possession trances in healthy individuals.

Despite intensive studies on cerebral activity during trances involving tranquil arousal states, there are little data on physiological basis of naturally induced possession trances involving hyperarousal active states because of the difficulty of gathering data from participants within a natural cultural context in the field. We investigated the characteristics of electroencephalograms (EEGs) that were specific for naturally induced possession trances involving hyperarousal states in actual rituals. We measured the EEG signals of 12 healthy participants, seven with trance and five without trance, before, during, and after a dedicatory ritual drama in Bali, Indonesia, using a custom-modified field telemetry system. During trance, θ (4-7.5 Hz), α-1 (8-9.5 Hz), α-2 (10-12.5 Hz), and ß (13-30 Hz) signals were significantly increased compared with those during the control phases. Such findings were not observed in participants without trance when they performed similar movements in the rituals. The α-1 and α-2 signals tended to remain elevated for several minutes postritual compared with those recorded during the preritual resting state. These results suggest that spontaneous EEG patterns during possession trances may be related, at least in part, to the activation of the reward-generating neuronal system situated in deep-lying brain structures and deactivation of the cerebral cortex.

The role of biological system other than auditory air-conduction in the emergence of the hypersonic effect.

Although human beings cannot perceive elastic vibrations in the frequency range above 20 kHz, nonstationary sounds containing a wealth of inaudible high-frequency components (HFC) above the human audible range activate deep-lying brain structures, including the brainstem and thalamus and evoke various physiological, psychological, and behavioral responses. In the previous reports, we have called these phenomena collectively "the hypersonic effect." It remains unclear, however, if vibratory stimuli above the audible range are transduced and perceived solely via the conventional air-conducting auditory system or if other mechanisms also contribute to mediate transduction and perception. In the present study, we have examined the emergence of the hypersonic effect when inaudible HFC and audible low-frequency components (LFC) were presented selectively to the ears, the entrance of an air-conducting auditory system, or to the body surface including the head which might contain some unknown vibratory sensing mechanisms. We used two independent measurements based on differing principles; one physiological (alpha 2 frequency of spontaneous electroencephalogram [alpha-EEG]) and the other behavioral (the comfortable listening level [CLL]). Only when the listener's entire body surface was exposed to HFC, but not when HFC was presented exclusively to the air-conducting auditory system, did both the alpha-EEG and the CLL significantly increase compared to the presentation of LFC alone, that is to say, there was an evident emergence of the hypersonic effect. The present findings suggest that the conventional air-conducting auditory system alone does not bring about the hypersonic effect. We may need to consider the possible involvement of a biological system distinct from the conventional air-conducting auditory nervous system in sensing and transducing high-frequency elastic vibration above the human audible range.

Modulatory effect of inaudible high-frequency sounds on human acoustic perception.

We evaluated the effects of the intensity of an inaudible high-frequency component (HFC) of sound on human responses by employing a multi-parametric approach consisting of behavioral measurements of the comfortable listening level (CLL), psychological measurements of the subjective impression of sounds, and physiological measurements using electroencephalogram (EEG). Increasing the intensity of the inaudible HFC resulted in a significant increase in the CLL, the subjective impression of sounds, and the occipital alpha frequency component of the spontaneous EEG. These effects peaked with an increase of 6 dB in HFC intensity. The results of the present study suggest that the intensity of inaudible HFC non-linearly modulates human sound perception.

Frequencies of inaudible high-frequency sounds differentially affect brain activity: positive and negative hypersonic effects.

The hypersonic effect is a phenomenon in which sounds containing significant quantities of non-stationary high-frequency components (HFCs) above the human audible range (max. 20 kHz) activate the midbrain and diencephalon and evoke various physiological, psychological and behavioral responses. Yet important issues remain unverified, especially the relationship existing between the frequency of HFCs and the emergence of the hypersonic effect. In this study, to investigate the relationship between the hypersonic effect and HFC frequencies, we divided an HFC (above 16 kHz) of recorded gamelan music into 12 band components and applied them to subjects along with an audible component (below 16 kHz) to observe changes in the alpha2 frequency component (10-13 Hz) of spontaneous EEGs measured from centro-parieto-occipital regions (Alpha-2 EEG), which we previously reported as an index of the hypersonic effect. Our results showed reciprocal directional changes in Alpha-2 EEGs depending on the frequency of the HFCs presented with audible low-frequency component (LFC). When an HFC above approximately 32 kHz was applied, Alpha-2 EEG increased significantly compared to when only audible sound was applied (positive hypersonic effect), while, when an HFC below approximately 32 kHz was applied, the Alpha-2 EEG decreased (negative hypersonic effect). These findings suggest that the emergence of the hypersonic effect depends on the frequencies of inaudible HFC.

C-terminal Src kinase controls development and maintenance of mouse squamous epithelia.

Carboxy-terminal Src kinase (Csk) is a negative regulator of Src family kinases, which play pivotal roles in controlling cell adhesion, migration, and cancer progression. To elucidate the in vivo role of Csk in epithelial tissues, we conditionally inactivated Csk in squamous epithelia using the keratin-5 promoter/Cre-loxP system in mice. The mutant mice developed apparent defects in the skin, esophagus, and forestomach, with concomitant hyperplasia and chronic inflammation. Histology of the mutant epidermis revealed impaired cell-cell adhesion in basal cell layers. Analysis of primary keratinocytes showed that the defective cell-cell adhesion was caused by cytoskeletal remodeling via activation of the Rac1 pathway. Mutant keratinocytes also showed elevated expression of mesenchymal proteins, matrix metalloproteinases (MMPs), and the proinflammatory cytokine TNF-alpha. Inhibition of the expression of TNF-alpha and MMP9 by the anti-inflammatory reagent FK506 could cure the epidermal hyperplasia, suggesting a causal link between inflammation and epidermal hyperplasia. These observations demonstrate that the Src/Csk circuit plays crucial roles in development and maintenance of epithelia by controlling cytoskeletal organization as well as phenotypic conversion linked to inflammatory events.