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The encapsulation of emulsion oil droplets by amorphous sugar matrices, formed by freeze-drying, was investigated, with a focus on the influence of the type of sugar. An oil-in-water emulsion, comprised of linoleic acid methyl ester (LME) and sucrose monolaurate (SML) as an oil phase and surfactant, respectively, were freeze-dried in the presence of different types of sugars. LME-droplet encapsulation during and after freeze-drying were evaluated by FTIR analysis. The loss of LME largely occurred in the early stage of freeze-drying. The size distribution of the encapsulated LME droplets remained unchanged before and after freeze-drying in most cases. The encapsulated fractions of LME droplets could be correlated with the glass transition temperature of the sugars in the fully hydrated state ( T g *), and the existence of an optimum T g * value for the sugar matrix was predicted. The encapsulation ability of an amorphous sugar matrix was maximized when mono- and polysaccharide were combined so as to give a value for T g * of approximately − 50 °C, although, individually, mono- and polysaccharides were quite poor for oil droplet encapsulation. These findings suggest that the structural flexibility of the amorphous sugar matrix is a major determinant in oil droplet encapsulation by an amorphous sugar matrix during freeze-drying.

We report on a novel fabrication approach of amperometric biosensor based on multilayer films containing carbon nanotubes (CNT), a nano-thin plasma-polymerized film (PPF), electron transfer mediator phenothiazine (PT), and enzyme glucose dehydrogenase (GDH). The configuration of the electrochemical electrode is sequentially composed of sputtered gold, acetonitrile PPF, PT, GDH, and acetonitrile PPF (denoted as PPF/GDH/PT/CNT/PPF/Au). First PPF deposited on Au acts as a permselective membrane and as a scaffold for CNT layer formation. Second PPF directly deposited on GDH acts as a matrix for enzyme immobilization. To facilitate the electrochemical communication between the CNT layer and GDH, CNT was treated with nitrogen plasma. The electron transfer mediator PT plays a role as the mediator in which the electron caused by enzymatic reaction transports to the electrode. The synergy between the mediator and CNT provides benefits in terms of lowering the operational potential and enhancing the sensitivity (current). The optimized glucose biosensor revealed a sensitivity of 5.1 ± 0.9 μA mM− 1 cm− 2 at + 0.2 V vs. Ag/AgCl, linear dynamic range of 4.9–19 mM, and a response time of 5 ± 1 s. Unlike conventional wet-chemical processes that are incompatible with mass production techniques, this dry-chemistry procedure has great potential for enabling high-throughput production of bioelectronic devices. Furthermore, those devices can be applied and expands for the cell biological functional field as a useful, helpful, or indispensable tool.

It is now believed that cancer stem cells (CSCs) that are resistant to chemotherapy due to their undifferentiated nature drive tumor growth, metastasis and relapse, so development of drugs that induce differentiation of CSCs should have a profound impact on cancer eradication. In this study, we screened medicines that are already in clinical use for drugs that induce differentiation of CSCs. We used MDA-MB-231, a human breast cancer cell line that contains cancer stem cell-like cells. We found that acetaminophen, an anti-inflammatory, antipyretic and analgesic drug, induces differentiation of MDA-MB-231 cells. Differentiation was assessed by observing alterations in cell shape and expression of differentiated and undifferentiated cell markers, a decrease in cell invasion activity and an increase in susceptibility to anti-tumor drugs. This increased susceptibility seems to involve suppression of expression of multidrug efflux pumps. We also suggest that this induction of differentiation is mediated by inhibition of a Wnt/β-catenin canonical signaling pathway. Treatment of MDA-MB-231 cells with acetaminophen in vitro resulted in the loss of their tumorigenic ability in nude mice. Furthermore, administration of acetaminophen inhibited the growth of tumor xenografts of MDA-MB-231 cells in both the presence and absence of simultaneous administration of doxorubicine, a typical anti-tumor drug for breast cancer. Analysis with various acetaminophen derivatives revealed that o-acetamidophenol has a similar differentiation-inducing activity and a similar inhibitory effect on tumor xenograft growth. These results suggest that acetaminophen may be beneficial for breast cancer chemotherapy by inducing the differentiation of CSCs.

Background Early detection of vasospasm is essential for the treatment of delayed ischemic neurological deficits in subarachnoid hemorrhage (SAH). We evaluated cerebral blood oxygenation (CBO) changes after SAH employing quantitative time-resolved near-infrared spectroscopy (TR-NIRS) for this purpose. Methods We investigated 11 age-matched controls and 14 aneurysmal SAH patients, including 10 patients with WFNS grade V and 4 patients with grade II. Employing TR-NIRS, we measured the cortical oxygen saturation (CoSO2) and baseline hemoglobin concentrations in the middle cerebral artery territory. Measurements of TR-NIRS and transcranial Doppler sonography (TCD) were performed repeatedly after SAH. Results In six patients, the CoSO2 and hemoglobin concentrations remained stable after SAH; digital subtraction angiography (DSA) did not reveal vasospasm in these patients. In eight patients, however, CoSO2 and total hemoglobin decreased abruptly between 5 and 9 days after SAH. DSA revealed diffuse vasospasms in six of eight patients. The reduction of CoSO2 predicted occurrence of vasospasm at a cutoff value of 3.9%-6.4% with 100% of sensitivity and 85.7% of specificity. TCD failed to detect the vasospasm in four cases, which TR-NIRS could detect. Finally, TR-NIRS performed on Day 1 after SAH revealed significantly higher CoSO2 than that of controls (p = .048), but there was no significant difference in total hemoglobin. Conclusion TR-NIRS detected vasospasm by evaluating the CBO in the cortex and may be more sensitive than TCD, which assesses the blood flow velocity in the M1 portion. The cerebral oxygen metabolism in SAH might be reduced by brain damage due to aneurysmal rupture.

Skin hyperpigmentation disorders due to abnormal melanin production induced by ultraviolet (UV) irradiation are both a clinical and cosmetic problem. UV irradiation stimulates melanin production in melanocytes by increasing intracellular cAMP. Expression of heat shock proteins (HSPs), especially HSP70, is induced by various stressors, including UV irradiation, to provide cellular resistance to such stressors. In this study we examined the effect of expression of HSP70 on melanin production both in vitro and in vivo. 3-Isobutyl-1-methylxanthine (IBMX), a cAMP-elevating agent, stimulated melanin production in cultured mouse melanoma cells, and this stimulation was suppressed in cells overexpressing HSP70. IBMX-dependent transcriptional activation of the tyrosinase gene was also suppressed in HSP70-overexpressing cells. Expression of microphthalmia-associated transcription factor (MITF), which positively regulates transcription of the tyrosinase gene, was up-regulated by IBMX; however, this up-regulation was not suppressed in HSP70-overexpressing cells. On the other hand, immunoprecipitation and immunostaining analyses revealed a physical interaction between and co-localization of MITF and HSP70, respectively. Furthermore, the transcription of tyrosinase gene in nuclear extract was inhibited by HSP70. In vivo, UV irradiation of wild-type mice increased the amount of melanin in the basal layer of the epidermis, and this increase was suppressed in transgenic mice expressing HSP70. This study provides the first evidence of an inhibitory effect of HSP70 on melanin production both in vitro and in vivo. This effect seems to be mediated by modulation of MITF activity through a direct interaction between HSP70 and MITF.

Irradiation with UV light, especially UVB, causes epidermal damage via the induction of apoptosis, inflammatory responses, and DNA damage. Various stressors, including UV light, induce heat shock proteins (HSPs) and the induction, particularly that of HSP70, provides cellular resistance to such stressors. The anti-inflammatory activity of HSP70, such as its inhibition of nuclear factor kappa B (NF-kappaB), was recently revealed. These in vitro results suggest that HSP70 protects against UVB-induced epidermal damage. Here we tested this idea by using transgenic mice expressing HSP70 and cultured keratinocytes. Irradiation of wild-type mice with UVB caused epidermal damage such as induction of apoptosis, which was suppressed in transgenic mice expressing HSP70. UVB-induced apoptosis in cultured keratinocytes was suppressed by overexpression of HSP70. Irradiation of wild-type mice with UVB decreased the cutaneous level of IkappaB-alpha (an inhibitor of NF-kappaB) and increased the infiltration of leukocytes and levels of pro-inflammatory cytokines and chemokines in the epidermis. These inflammatory responses were suppressed in transgenic mice expressing HSP70. In vitro, the overexpression of HSP70 suppressed the expression of pro-inflammatory cytokines and chemokines and increased the level of IkappaB-alpha in keratinocytes irradiated with UVB. UVB induced an increase in cutaneous levels of cyclobutane pyrimidine dimers and 8-hydroxy-2'-deoxyguanosine, both of which were suppressed in transgenic mice expressing HSP70. This study provides genetic evidence that HSP70 protects the epidermis from UVB-induced radiation damage. The findings here also suggest that the protective action of HSP70 is mediated by anti-apoptotic, anti-inflammatory, and anti-DNA damage effects.

Although recent reports suggest that the endoplasmic reticulum (ER) stress response is induced in association with the development of inflammatory bowel disease, its role in the pathogenesis of inflammatory bowel disease remains unclear. The CCAAT/enhancer-binding protein (C/EBP) homologous protein (CHOP) is a transcription factor that is involved in the ER stress response, especially ER stress-induced apoptosis. In this study, we found that experimental colitis was ameliorated in CHOP-null mice, suggesting that CHOP exacerbates the development of colitis. The mRNA expression of Mac-1 (CD11b, a positive regulator of macrophage infiltration), Ero-1alpha, and Caspase-11 (a positive regulator of interleukin-1beta production) in the intestine was induced with the development of colitis, and this induction was suppressed in CHOP-null mice. ERO-1alpha is involved in the production of reactive oxygen species (ROS); an increase in ROS production, which is associated with the development of colitis in the intestine, was suppressed in CHOP-null mice. A greater number of apoptotic cells in the intestinal mucosa of wild-type mice were observed to accompany the development of colitis compared with CHOP-null mice, suggesting that up-regulation of CHOP expression exacerbates the development of colitis. Furthermore, this CHOP activity appears to involve various stimulatory mechanisms, such as macrophage infiltration via the induction of Mac-1, ROS production via the induction of ERO-1alpha, interleukin-1beta production via the induction of Caspase-11, and intestinal mucosal cell apoptosis.

Amyloid-beta peptides (Abeta), generated by proteolysis of the beta-amyloid precursor protein (APP) by beta- and gamma-secretases, play an important role in the pathogenesis of Alzheimer disease (AD). Inflammation is also believed to be integral to the pathogenesis of AD. Here we show that prostaglandin E(2) (PGE(2)), a strong inducer of inflammation, stimulates the production of Abeta in cultured human embryonic kidney (HEK) 293 or human neuroblastoma (SH-SY5Y) cells, both of which express a mutant type of APP. We have demonstrated using subtype-specific agonists that, of the four main subtypes of PGE(2) receptors (EP(1-4)), EP(4) receptors alone or EP(2) and EP(4) receptors together are responsible for this PGE(2)-stimulated production of Abeta in HEK293 or SH-SY5Y cells, respectively. An EP(4) receptor antagonist suppressed the PGE(2)-stimulated production of Abeta in HEK293 cells. This stimulation was accompanied by an increase in cellular cAMP levels, and an analogue of cAMP stimulated the production of Abeta, demonstrating that increases in the cellular level of cAMP are responsible for the PGE(2)-stimulated production of Abeta. Immunoblotting experiments and direct measurement of gamma-secretase activity suggested that PGE(2)-stimulated production of Abeta is mediated by activation ofgamma-secretase but not of beta-secretase. Transgenic mice expressing the mutant type of APP showed lower levels of Abeta in the brain, when they were crossed with mice lacking either EP(2) or EP(4) receptors, suggesting that PGE(2)-mediated activation of EP(2) and EP(4) receptors is involved in the production of Abeta in vivo and in the pathogenesis of AD.

NSAIDs such as celecoxib induce apoptosis in cancer cells. Although this apoptotic effect is involved in the anti-tumor activity associated with such drugs, the mechanism by which this occurs is not fully understood. We report here that various NSAIDs, including celecoxib, up-regulate PUMA, a Bcl-2 family protein with potent apoptosis-inducing activity, in human gastric carcinoma cell line, accompanying the induction of apoptosis. Experiments using siRNA and an intracellular Ca(2+) chelator revealed that Ca(2+)-dependent up-regulation of ATF4 and CHOP is involved in this up-regulation of PUMA. The siRNA for PUMA inhibited the celecoxib-induced activation and translocation of Bax, release of cytochrome c into the cytosol and induction of apoptosis, suggesting that PUMA plays an important role in celecoxib-induced mitochondrial dysfunction and the resulting apoptosis.

Gastric mucosal cell death by non-steroidal anti-inflammatory drugs (NSAIDs) is suggested to be involved in NSAID-induced gastric lesions. Therefore, cellular factors that suppress this cell death are important for protection of the gastric mucosa from NSAIDs. Heme oxygenase-1 (HO-1) is up-regulated by various stressors and protects cells against stressors. Here, we have examined up-regulation of HO-1 by NSAIDs and the contribution of HO-1 to the protection of gastric mucosal cells against NSAIDs both in vitro and in vivo. In cultured gastric mucosal cells, all NSAIDs tested up-regulated HO-1. In rats, orally administered indomethacin up-regulated HO-1, induced apoptosis, and produced lesions at gastric mucosa. An inhibitor of HO-stimulated NSAID-induced apoptosis in vitro and in vivo and also stimulated NSAID-produced gastric lesions, suggesting that NSAID-induced up-regulation of HO-1 protects the gastric mucosa from NSAID-induced gastric lesions by inhibiting NSAID-induced apoptosis. Indomethacin activated the HO-1 promoter and caused nuclear accumulation of NF-E2-related factor 2 (Nrf2), a transcription factor for the HO-1 gene. Examination of phosphorylation of p38 mitogen-activated protein kinase (MAPK) and experiments with its inhibitor strongly suggest that the nuclear accumulation of Nrf2 and resulting up-regulation of HO-1 by NSAIDs is mediated through NSAID-dependent activation (phosphorylation) of p38 MAPK. This is the first report showing the protective role of HO-1 against irritant-induced gastric lesions.

We recently reported that nonsteroidal anti-inflammatory drug (NSAID)-induced gastric lesions involve NSAID-induced apoptosis of gastric mucosal cells, which in turn involves the endoplasmic reticulum stress response, in particular the up-regulation of CCAAT/enhancer-binding protein homologous transcription factor (CHOP). In this study, we have examined the molecular mechanism governing this NSAID-induced apoptosis in primary cultures of gastric mucosal cells. Various NSAIDs showed membrane permeabilization activity that correlated with their apoptosis-inducing activity. Various NSAIDs, particularly celecoxib, also increased intracellular Ca2+ levels. This increase was accompanied by K+ efflux from cells and was virtually absent when extracellular Ca2+ had been depleted. These data indicate that the increase in intracellular Ca2+ levels that is observed in the presence of NSAIDs is due to the stimulation of Ca2+ influx across the cytoplasmic membrane, which results from their membrane permeabilization activity. An intracellular Ca2+ chelator partially inhibited celecoxib-induced release of cytochrome c from mitochondria, reduced the magnitude of the celecoxib-induced decrease in mitochondrial membrane potential and inhibited celecoxib-induced apoptotic cell death. It is therefore likely that an increase in intracellular Ca2+ levels is involved in celecoxib-induced mitochondrial dysfunction and the resulting apoptosis. An inhibitor of calpain, a Ca2+-dependent cysteine protease, partially suppressed mitochondrial dysfunction and apoptosis in the presence of celecoxib. Celecoxib-dependent CHOP-induction was partially inhibited by the intracellular Ca2+ chelator but not by the calpain inhibitor. These results suggest that Ca2+-stimulated calpain activity and CHOP expression play important roles in celecoxib-induced apoptosis in gastric mucosal cells.

Background Cortical stimulation via a subdural grid electrode (SGE) is one of the most reliable methods for identifying eloquent areas before surgery. However, the physiological conditions of the cortex during stimulation cannot be monitored electrophysiologically because of electrical artifacts. In the present case, we tested whether or not multichannel near-infrared spectroscopy (NIRS) topography, a noninvasive optical imaging technique, is applicable for monitoring the physiological conditions of the stimulated cortex. Case description The patient (a 27-year-old right-handed man) suffered from glioma in the left frontal lobe. For preoperative cortical mapping, SGEs were implanted over the left motor cortex before tumor resection. Employing NIRS topography, we undertook 2 dimensional imaging of the changes in oxyhemoglobin (Oxy-Hb) and deoxyhemoglobin (Deoxy-Hb) concentration during electrical stimulation. Five-hertz stimulation with 5 mA at the left-hand area produced a localized increase in Oxy-Hb and a decrease in Deoxy-Hb, associated with slight twitching of the right hand. In contrast, 50-Hz stimulation produced significant increases in both Oxy-Hb and Deoxy-Hb at the stimulation site, and the area with such cerebral blood oxygenation (CBO) changes propagated beyond the hand area associated with prominent muscle contractions of the right upper extremity, suggesting that 50-Hz stimulation caused epileptic discharge. Conclusion Near-infrared spectroscopy topography may represent a useful tool for imaging the degree and extent of the physiological effects of electrical stimulation on the cortex, and permits safe and accurate cortical mapping.

We investigated whether an acyclic polyisoprenoid antiulcer drug, geranylgeranylacetone (GGA), induces the expression of HSP70 in the rat cochlea. Immunoblotting revealed upregulation of HSP70 in the cochlea at 12 h after transtympanic (local) or oral (systemic) administration of GGA, and this increased at 24 h after administration. Positive immunohistochemical staining of HSP70 was observed in the hair cells, the spiral ganglion, the stria vascularis, the spiral ligament, and the perivascular portion of modiolar vessels. We therefore subsequently studied the effects of GGA as an HSP-inducer on inner ear trauma due to inflammation. Damage to the lateral wall due to inflammation induced by lipopolysaccharide inoculation was protected against by pretreatment with GGA, as assessed physiologically by measurement of cochlear blood flow and morphologically by electron microscopy. The results of the present study suggest that GGA can protect the cochlea against other injuries including those induced by noise, ototoxic drugs, and ischemia by upregulating HSP70.

Prostaglandin E(2) (PGE(2)) has a strong protective effect on the gastric mucosa in vivo; however, the molecular mechanism of a direct cytoprotective effect of PGE(2) on gastric mucosal cells has yet to be elucidated. Although we reported previously that PGE(2) inhibited gastric irritant-induced apoptotic DNA fragmentation in primary cultures of guinea pig gastric mucosal cells, we show here that PGE(2) inhibits the ethanol-dependent release of cytochrome c from mitochondria. Of the four main subtypes of PGE(2) receptors, we also demonstrated, using subtype-specific agonists, that EP(2) and EP(4) receptors are involved in the PGE(2)-mediated protection of gastric mucosal cells from ethanol-induced apoptosis. Activation of EP(2) and EP(4) receptors is coupled with an increase in cAMP, for which a cAMP analogue was found here to inhibit the ethanol-induced apoptosis. The increase in cAMP is known to activate both protein kinase A (PKA) and phosphatidylinositol 3-kinase pathways. An inhibitor of PKA but not of phosphatidylinositol 3-kinase blocked the PGE(2)-mediated protection of cells from ethanol-induced apoptosis, suggesting that a PKA pathway is mainly responsible for the PGE(2)-mediated inhibition of apoptosis. Based on these results, we considered that PGE(2) inhibited gastric irritant-induced apoptosis in gastric mucosal cells via induction of an increase in cAMP and activation of PKA, and that this effect was involved in the PGE(2)-mediated protection of the gastric mucosa from gastric irritants in vivo.

We evaluated cerebral blood oxygenation (CBO) changes after SAH employing quantitative time-resolved near infrared spectroscopy (TR-NIRS). We investigated 11 age-matched controls and 14 aneurysmal SAH patients. Employing TR-NIRS, we measured the cortical oxygen saturation (CoSO 2 ) and baseline hemoglobin concentrations in the middle cerebral artery territory. Measurements of TR-NIRS and transcranial Doppler sonography (TCD) were performed repeatedly after SAH. In six patients, the CoSO 2 and hemoglobin concentrations remained stable after SAH; DSA did not reveal vasospasm in these patients. In eight patients, however, CoSO 2 and total hemoglobin decreased abruptly between five and nine days after SAH. DSA revealed diffuse vasospasms in six out of eight patients. The reduction of CoSO 2 predicted occurrence of vasospasm at a cut of value of 3.9–6.4% with 100% of sensitivity and 85.7% of specificity. TCD failed to detect the vasospasm in four cases, which TR-NIRS could detect. Finally, TR-NIRS performed on day 1 after SAH revealed significantly higher CoSO 2 than that of controls; but there was no significant difference in total hemoglobin. TR-NIRS detected vasospasm by evaluating the CBO in the cortex, and may be more sensitive than TCD. The cerebral oxygen metabolism in SAH might be reduced by brain damage due to aneurysmal rupture.