The secondary structure of bovine serum albumin (BSA) was first examined in the thermal denaturation up to 130 degrees C. The helicity (66%) of the protein decreased with rise of temperature. Half of the original helicity was lost at 80 degrees C, but the helicity of 16% was still maintained even at 130 degrees C. When the BSA solution was cooled down to 25 degrees C after heating at temperatures above 50 degrees C, the helicity was not completely recovered. The higher the thermal denaturation temperature was, the lower was the recovered helicity. On the other hand, upon the addition of sodium dodecyl sulfate (SDS), the secondary structure of BSA was partially protected against the thermal denaturation above 50 degrees C where the structural change became irreversible. A particular protective effect was observed below 85 degrees C upon the coexistence of SDS of extremely low concentrations. For example, the helicity was 34% at 80 degrees C in the absence of SDS, but it was maintained at 58% at the same temperature upon the coexistence of 0.75 mM SDS. Upon cooling down from 80 to 25 degrees C, the helicity of BSA was recovered to 62% in the presence of 0.75 mM SDS. Such a protective effect of SDS was not observed above 95 degrees C. In the interaction with the surfactant, this protein structure appeared likely to have a critical temperature between 90 and 100 degrees C in addition to the critical temperature in the vicinity of 50 degrees C. This protective effect of SDS, characterized by the specific amphiphilic nature of this anionic surfactant, is considered to be attained by building cross-linking bridges between particular nonpolar residues and particular positively charged residues in the protein molecule.

NXF, a member of the basic helix-loop-helix-PAS transcription factor family, is thought to be involved in functional regulation of neurons, because significant expression is found in the mature brain. To elucidate functions of NXF in vivo, here we generated mice lacking NXF using homologous recombination with embryonic stem cells. NXF(-/-) mice were morphologically indistinguishable (with no growth retardation) from their littermates (wild type) at birth. However, they started to die at a rate of 1 death/20-30 animals per week under specific pathogen-free grade breeding conditions when over 3 months old. Histological analyses revealed age-dependent neurodegeneration in brain, and only 20-30% of the NXF(-/-) mice survived for 16 months. To clarify the role of NXF in protection against neurodegeneration in normal cells, we analyzed gene expression under several conditions in vitro and in vivo. The NXF gene was up-regulated by several neurodegenerative cell-stress inducers such as thapsigargin (endoplasmic reticulum stress), SIN-1 (oxidative stress), and sorbitol (osmotic stress) in cultured cells. Furthermore, elevated NXF gene expression was apparent with in vivo stroke models featuring kainate-induced hippocampal injury and transient global ischemia. When NXF(-/-) mice were evaluated in the glutamate excitotoxicity model, they proved more susceptible to hippocampal injury at 15 weeks after birth. The findings in this study suggest that the NXF gene could be induced in response to several neurodegenerative stimuli/excitations for the cell protection, and thus provide an "on demand" cell-protection system in nervous tissue.

We provided an intervention to chronic post-stroke aphasic patients using low-frequency repetitive transcranial magnetic stimulation (LF-rTMS) guided by a functional magnetic resonance imaging (fMRI) evaluation of language laterality, combined with intensive speech therapy (ST). We performed a single photon emission-computed tomography (SPECT) scan pre- and post-intervention and investigated the relationship between cerebral blood flow (CBF) and language function. Fifty right-handed chronic post-stroke aphasic patients were enrolled in the study. During their 11-day hospital admission, the patients received a 40-min session of 1-Hz LF-rTMS on the left or right hemisphere, according to language localization identified by the fMRI evaluation, and intensive ST daily for 10 days, except for Sunday. A SPECT scan and language evaluation by the Standard Language Test of Aphasia (SLTA) were performed at the time of admission and at 3 months following discharge. We calculated laterality indices (LIs) of regional CBF (rCBF) in 13 language-related Brodmann area (BA) regions of interest. In patients who received LF-rTMS to the intact right hemisphere (RH-LF-rTMS), the improvement in the total SLTA score was significantly correlated with the pre- and post-intervention change of LI (ΔLI) in BA44. In patients who received LF-rTMS to the lesional left hemisphere (LH-LF-rTMS), this association was not observed. Analyses of the SLTA subscales and rCBF ΔLI demonstrated that in the RH-LF-rTMS group, the SLTA Speaking subscale scores were significantly correlated with ΔLIs in BA11, 20, and 21, and the SLTA Writing subscale scores were significantly correlated with ΔLIs in BA6 and 39. Conversely, in the LH-LF-rTMS group, the SLTA Speaking subscale scores were correlated with ΔLI in BA10, and the SLTA Reading subscale scores were significantly correlated with ΔLIs in BA13, 20, 22, and 44. Our results suggest the possibility that fMRI-guided LF-rTMS combined with intensive ST may affect CBF and contribute to the improvement of language function of post-stroke aphasic patients. LF-rTMS to the non-lesional and lesional hemispheres showed a difference in the associations between language performance and CBF. The results indicate that more effective rTMS intervention needs to be explored for patients who show right hemisphere language activation in an fMRI language evaluation.

Magnesium deficiency is suggested to contribute to many age-related diseases. Hypoxia-inducible factor 1␣ (HIF-1␣) is known to be a master regulator of hypoxic response. Here we show that hypomagnesemia suppresses reactive oxygen species (ROS)-induced HIF-1␣ activity in paraganglion cells of the adrenal medulla and carotid body. In PC12 cells cultured in the low magnesium medium and treated with cobalt chloride (CoCl 2 ) or exposed to intermittent hypoxia, ROS-mediated HIF-1␣ activity was suppressed. This suppression was due to up-regulation of inhibitory PAS (Per/Arnt/Sim) domain protein (IPAS) that was caused by NF-B activation, which resulted from ROS and calcium influx mainly through the T-type calcium channels. Induction of tyrosine hydroxylase, a target of HIF-1, by CoCl 2 injection was suppressed in the adrenal medulla of magnesium-deficient mice because of up-regulation of IPAS. Also in the carotid body of magnesium-deficient mice, CoCl 2 and chronic intermittent hypoxia failed to enhance the tyrosine hydroxylase expression. These results demonstrate that serum magnesium levels are a key determinant for ROS-induced hypoxic responses. Hypoxia-inducible factor 1␣ (HIF-1␣)2 and its family members are master regulators of hypoxic response (1-3). In hypoxia, the HIF-1, composed of HIF-1␣ and HIF-1␤/Arnt, binds to hypoxia response element (HRE) to induce the gene expression of hypoxia-responsive proteins, such as erythropoietin and vascular endothelial growth factor. In addition to these proteins, tyrosine hydroxylase (TH), the rate-limiting enzyme for catecholamine biosynthesis, is induced in rat pheochromocytoma-derived PC12 cells and paraganglion cells in the adrenal medulla (AM) and carotid body (CB) in response to hypoxia (4). The CB acts as the primary peripheral chemoreceptor (5), and glomus cells of the CB are responsible for monitoring oxygen levels in arterial blood (5, 6). Through the release of neurotransmitters, including dopamine, the CB delivers information to the respiratory and cardiovascular networks in the brainstem, resulting in increases of ventilatory frequency and volume and also raising cardiac output.HIF-dependent hypoxic response is also caused by chronic intermittent hypoxia (CIH), which is a common feature of obstructive sleep apnea (OSA). There is accumulating evidence that CIH is associated with an increased oxidative stress (7,8). Peng et al. (9) have shown that CIH induces reactive oxygen species (ROS) generation, thereby increasing HIF-1␣ expression, which is critical for eliciting CIH-induced cardiorespiratory responses by the CB. CIH also increases ROS generation and TH expression in the AM, although it is less sensitive than the CB (10).Recent studies have identified that IPAS, which is one of the alternatively spliced variants of HIF-3␣, acts as a dominant negative inhibitor of HIF-1␣ by a direct interaction with HIF-1␣ and prevents its DNA binding (11). IPAS is predominantly expressed in the Purkinje cells of the cerebellum and corneal epithelium. In addition, because the...

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