Elucidation of the working principle of a gene-directed caged HDAC inhibitor with cell-type selectivity.

Histone deacetylases (HDACs) play crucial roles in the epigenetic regulation of gene expression. Here, we report CM-Bhc-SAHA, a novel caged HDAC inhibitor, genetically targeting cells of interest. Mammalian cells expressing porcine liver esterase led to the optochemical inhibition of endogenous HDAC activity when treated with CM-Bhc-SAHA and irradiated with 405 nm light.

Activation of aryl hydrocarbon receptor mediates suppression of hypoxia-inducible factor-dependent erythropoietin expression by indoxyl sulfate.

Indoxyl sulfate (IS) is a representative uremic toxin that accumulates in the blood of patients with chronic kidney disease (CKD). In addition to the involvement in the progression of CKD, a recent report indicates that IS suppresses hypoxia-inducible factor (HIF)-dependent erythropoietin (EPO) production, suggesting that IS may also contribute to the progression of renal anemia. In this report, we provide evidence that aryl hydrocarbon receptor (AhR) mediates IS-induced suppression of HIF activation and subsequent EPO production. In HepG2 cells, IS at concentrations similar to the blood levels in CKD patients suppressed hypoxia- or cobalt chloride-induced EPO mRNA expression and transcriptional activation of HIF. IS also induced AhR activation, and AhR blockade resulted in abolishment of IS-induced suppression of HIF activation. The HIF transcription factor is a heterodimeric complex composed of HIF-α subunits (HIF-1α and HIF-2α) and AhR nuclear translocator (ARNT). IS suppressed nuclear accumulation of the HIF-α-ARNT complex accompanied by an increase of the AhR-ARNT complex in the nucleus, implying the involvement of interactions among AhR, HIF-α, and ARNT in the suppression mechanism. In rats, oral administration of indole, a metabolic precursor of IS, inhibited bleeding-induced elevation of renal EPO mRNA expression and plasma EPO concentration and strongly induced AhR activation in the liver and renal cortex tissues. Collectively, this study is the first to elucidate the detailed mechanism by which AhR plays an indispensable role in the suppression of HIF activation by IS. Hence, IS-induced activation of AhR may be a potential therapeutic target for treating renal anemia.

In situ observation of a cell adhesion and metabolism using surface infrared spectroscopy.

In this study, we report on an in situ monitoring system of living cultured cells using infrared absorption spectroscopy in the geometry of multiple internal reflections (MIR-IRAS). In order to observe living cultured cells, the temperature in the sample chamber of a FT-IR spectrometer was maintained at 37 degrees C and a humidified gas mixture containing 5% CO(2) was introduced into the sample chamber. Human breast cell line MCF-7 cultured on Si MIR prisms were placed in the sample chamber and infrared spectra of MCF-7 cells were collected for 5 h. It was found that the adhesion and metabolism of MCF-7 cells could be monitored by the absorption intensity of amide-II protein band (1,545 cm(-1)) and also by the absorption intensities of CH( x ) bands (2,700-3,100 cm(-1)). These results suggest that our system is useful for a nondestructive and non-label monitoring of cell viability. Our method based on infrared absorption spectroscopy has a potential for bioscreening application.

Receptors and enzymes for medical sensing of L-glutamate.

Medical sensing systems using isolated or intact glutamate receptor (GluR) ion channels and glutamate oxidase (GluOx) are discussed for L-glutamate, one of the principal neurotransmitter in the central nervous systems of mammalian brain, and related agonists. The GluR-based sensing system used for the evaluation of signal transduction ability of GluR channels demonstrate that the agonist selectivity based on the signal transduction ability is not parallel to that of the binding assay. On the other hand, the appropriate design of the enzyme system, namely glutamate oxidase (GluOx), in combination with horseradish peroxidase (HRP), enables to real-time monitoring of L-glutamate in vivo and in vitro and also to visualize its release in submerged, acute mouse hippocampal slices.

Autophosphorylation of alphaCaMKII is not a general requirement for NMDA receptor-dependent LTP in the adult mouse.

Autophosphorylation of alpha-Ca2+/calmodulin kinase II (alphaCaMKII) at Thr286 is thought to be a general effector mechanism for sustaining transcription-independent long-term potentiation (LTP) at pathways where LTP is NMDA receptor-dependent. We have compared LTP at two such hippocampal pathways in mutant mice with a disabling point mutation at the Thr286 autophosphorylation site. We find that autophosphorylation of alphaCaMKII is essential for induction of LTP at Schaffer commissural-CA1 synapses in vivo, but is not required for LTP that can be sustained over days at medial perforant path-granule cell synapses in awake mice. At these latter synapses LTP is supported by cyclic AMP-dependent signalling in the absence of alphaCaMKII signalling. Thus, the autophosphorylation of alphaCaMKII is not a general requirement for NMDA receptor-dependent LTP in the adult mouse.

Real-time monitoring of L-glutamate release from mouse brain slices under ischemia with a glass capillary-based enzyme electrode.

Real-time monitoring of L-glutamate release from various neuronal regions of mouse hippocampal slices under ischemia (a glucose-free hypoxia condition) is described. A glass capillary microelectrode with a tip size of approximately 10 microm containing a very small volume ( approximately 2 microL) of a solution of glutamate oxidase (GluOx) and ascorbate oxidase was used. First, the amperometric response behavior of the electrode at 0 V versus Ag/AgCl was characterized with a standard glutamate solution in terms of continuous measurements, effect of oxygen, viscosity of solution and concentration dependence. The electrode was applied to the real-time monitoring of L-glutamate released from different neuronal regions of acute hippocampal slices submerged in a hypoxia solution. The time-resolved amounts of L-glutamate released at various neuronal regions (CA1, CA3 and DG) of mouse hippocampal slices were quantified and compared with the reported L-glutamate fluxes using difference-image analysis during ischemia.

Induction and activation of the aryl hydrocarbon receptor by IL-4 in B cells.

It is widely known that IL-4 and IL-13 act on various kinds of cells, including B cells, resulting in enhancement of proliferation, class switching to IgE and expression of several surface proteins. These functions are important for the recognition of the various antigens in B cells and are known to be involved in the pathogenesis of allergic diseases. However, it has not been known whether IL-4/IL-13 is involved in the metabolism of various kinds of xenobiotics including 2,3,7,8-tetra-chlorodibenzo-p-dioxin (TCDD), and it remains undetermined whether TCDD, an environmental pollutant, influences IgE production in B cells, exaggerating allergic reactions. We identified IL-4- or IL-13-inducible genes in a human Burkitt lymphoma cell line, DND-39, using microarray technology, in which the AHR gene was included. The AHR gene product, the aryl hydrocarbon receptor (AhR), was induced by IL-4 in both mouse and human B cells in a STAT6-dependent manner. IL-4 alone had the ability to translocate the induced AhR to the nuclei. TCDD, a ligand for AhR, rapidly degraded the induced AhR by the proteasomal pathway, although IL-4-activated AhR sustained its expression. AhR activated by IL-4 caused expression of a xenobiotic-metabolizing gene, CYP1A1, and TCDD synergistically acted on the induction of this gene by IL-4. However, the induction of AhR had no effect on IgE synthesis or CD23 expression. These results indicate that the metabolism of xenobiotics would be a novel biological function of IL-4 and IL-13 in B cells, whereas TCDD is not involved in IgE synthesis in B cells.

Acute effect of corticosterone on N-methyl-D-aspartate receptor-mediated Ca2+ elevation in mouse hippocampal slices.

We examined the rapid effects of corticosterone (CORT) on N-methyl-D-aspartate (NMDA) receptor-mediated Ca2+ signals in adult mouse hippocampal slices by using Ca2+ imaging technique. Application of NMDA caused a transient elevation of intracellular Ca2+ concentration followed by a decay to a plateau within 150s. The 30min preincubation of CORT induced a significant decrease of the peak amplitude of NMDA-induced Ca2+ elevation in the CA1 region. The rapid effect of CORT was induced at a stress-induced level (0.4-10microM). Because the membrane non-permeable bovine serum albumin-conjugated CORT also induced a similar rapid effect, the rapid effect of CORT might be induced via putative surface CORT receptors. In contrast, CORT induced no significant effects on NMDA-induced Ca2+ elevation in the dentate gyrus. In the CA3 region, CORT effects were not evaluated, because the marked elevation of NMDA-induced Ca2+ signals was not observed there.

Single-channel recordings of gramicidin at agarose-supported bilayer lipid membranes formed by the tip-dip and painting methods.

Agarose-supported BLMs were prepared by the tip-dip and painting methods, and single-channel recordings of gramicidin were examined for the development of an ion-channel sensor. The supported BLMs formed by the tip-dip method had an electric resistance of >1.0 x 10(11) omega and a longer lifetime as compared with unsupported ones, which enabled single-channel recordings of gramicidin. The supported BLMs formed by the painting method also enabled single-channel recordings, but the lifetime was shorter than that of unsupported planar BLMs formed by the monolayer folding method.

A dot-blot method for quantification of apurinic/apyrimidinic sites in DNA using an avidin plate and liposomes encapsulating a fluorescence dye.

A dot-blot method for quantification of apurinic/apyrimidinic (AP) sites in genomic DNA (calf thymus DNA) is described using an avidin-modified glass slip and biotinylated liposomes containing sulforhodamine B as a fluorescence marker. Aldehyde reactive probe (ARP)-tagged DNA was found to be strongly adsorbed on an avidin slip, even if treated with ethanolamine and biotin, with an efficiency of 51% due to the positive surface charge of avidin, and unbound ARP was easily washed out of the surface with Milli-Q water. In the assay protocol, calf thymus DNA containing AP sites is reacted with ARP in solution and immobilized on an ethanolamine- and biotin-treated avidin slip (EAB-avidin slip), followed by incubation with streptavidin. The AP sites were finally quantified with biotinylated liposomes containing 1.5 mM sulforhodamine B as a fluorescence marker. The mean fluorescence intensity over the surface of the slip was an analytically relevant measure of the amount of AP sites in calf thymus DNA. By using the dot-blot assay, 1-5 AP sites per 10(4) nucleotides in 5 and 100 ng of DNA were quantified. The current dot-blot method has potential for quantification of AP sites in genomic DNA at a level of several nanograms.

An excised patch membrane sensor for arachidonic acid released in mouse hippocampal slices under stimulation of L-glutamate.

An excised patch membrane sensor for arachidonic acid (AA) is described, whose response stems from AA-induced channel-type transport of ions across the excised patch membrane. The patch membrane sensor was prepared in situ by excising mouse hippocampal cell membranes with patch pipets having a tip diameter of < 0.5 microm. The sensor responds to AA, giving rise to a channel-type current, and its magnitude (apparent conductance) increased with increasing AA concentration in the range from 10 to 30 nM. The detection limit was 2.1 nM (S/N = 3). The induction of channel-type currents was selective to AA over fatty acids such as palmitic acid, stearic acid, oleic acid, gamma-linolenic acid, and docosahexaenoic acid and AA metabolites such as 12-HETE, 5-HETE, and prostaglandin D(2). The sensor was applied to quantification of AA released from various neuronal regions (CA1, CA3, and DG) of mouse hippocampus under stimulation of 100 microM L-glutamate. The release of AA from each region was observed 1 min after the stimulation and the concentration of AA 5 min after the stimulation varied among the neuronal sites, i.e., 8+/-1 nM (n = 5) for CA1, 15+/-3 nM (n = 3) for CA3, and 6+/-2 nM (n = 9) for DG. The L-glutamate-evoked release of AA was partly inhibited by ionotropic glutamate receptor antagonists (APV and DNQX) and completely blocked by phospholipase A2 (PLA2) inhibitor (MAFP), suggesting that the release of AA occurred by glutamate receptor-mediated activation of PLA2. The potential use of the present sensor for detecting local concentration of AA at various neuronal sites is discussed.

Imaging of L-glutamate fluxes in mouse brain slices based on an enzyme-based membrane combined with a difference-image analysis.

A time-resolved imaging method for visualizing L-glutamate release in mammalian brain slices is proposed by using an enzyme membrane combined with a difference-image analysis. The enzyme membrane is composed of L-glutamate oxidase and horseradish peroxidase incorporated into a bovine serum albumin matrix. L-Glutamate triggers an enzyme-coupling reaction to convert a redox substrate (DA-64) to Bindschedler's Green, which gives a green color signal. The difference-image analysis is based on calculating slopes of a signal versus time (t) plot in the time range from (t - 40 s) to (t + 40 s) for visualizing L-glutamate release in terms of its flux (in mol min(-1) cm(-2)). The method was applied to a time-resolved imaging of hippocampal distribution of ischemia-induced L-glutamate release in mouse brain slices. The image of L-glutamate distribution showed that the level and time courses of L-glutamate fluxes were neuronal region-dependent. The maximum flux of L-glutamate at CA1 was observed at 7.7 min after ischemia. The flux at 7.7 min increased in the order of CA1 approximately CA3 > DG. The time course of the L-glutamate flux in the CA1 region was biphasic and that in the DG region was modestly biphasic. In the CA3 region, such biphasic release of L-glutamate was not seen. The ischemia-induced L-glutamate flux was accelerated when Mg2+ was omitted from an extracellular solution. The present enzyme membrane-based approach provides a useful method for visualizing distribution of L-glutamate release in the brain slices during ischemia.

A single-channel sensor based on gramicidin controlled by molecular recognition at bilayer lipid membranes containing receptor.

A novel ion-channel sensor based on a membrane bound receptor and a single gramicidin channel is described, in which the binding of an analyte to the membrane bound receptor modulates the single-channel activity of gramicidin. The sensor is composed of a planar bilayer lipid membrane (BLM) containing biotin-labeled phosphatidylethanolamine as receptor for avidin and gramicidin as signal transducer. When the receptor catches an analyte (avidin or ferritin-labeled avidin (FA)) at the membrane surface, the bilayer structure is locally distorted and the gramicidin monomer/dimer kinetics is modulated in a manner that the fraction of channel opening with a short lifetime ( < or = 100 ms) to the total opening events increases. The fraction was found to increase with the concentration of avidin from 1.0 x 10(-9) to 1.0 x 10(-6) M and of FA from 1.0 x 10(-9) to 1.0 x 10(-8) M. With dinitrophenyl-labeled PE embedded as receptor in the BLM for monoclonal anti-dinitrophenyl antibody (anti-DNP), the fraction of channel openings ( < or = 100 ms) increased with the concentration of anti-DNP from 2.0 x 10(-9) to 2.0 x 10(-7) g/ml. Bovine serum albumin (BSA) and anti-BSA antibody caused no changes in the channel opening. The possible mechanism of analyte-induced modulation of single-channel activity of gramicidin is also discussed.

Arachidonic acid-induced channel- and carrier-type ion transport across planar bilayer lipid membranes.

Transmembrane ion transport by arachidonic acid (AA) through bilayer lipid membranes (BLMs) was investigated by means of electrochemical measurements to provide a basis for designing a sensor membrane. We found that AA induces a channel-type current, in addition to a carrier-type current, across planar BLMs. A linear relation between the logarithmic value of the AA concentration and the current responses (given as integrated currents) was observed for a carrier-type current, while a sigmoid relation was found for a channel-type current. Although AA transports Na+, Ca2+ and Mg2+ and exhibits ion selectivity between Na+ and Mg2+ for the carrier-type current, ion transport for the channel-type current was non-selective. It was found that ion transport via the channel mechanism occurs frequently for AA, while channel-type currents were only occasionally observed for y-linolenic acid and prostaglandin D2. No channel-type currents were induced by other fatty acids (oleic, linoleic, stearic, myristic, eicosapentanoic and docosahexanoic acids) and metabolites of AA (12-HETE and 5-HETE). The carrier-type ion transport occurs selectively to these compounds if the concentration is below 1.0 microM. These results suggest that AA selectively facilitates an ion flux through the BLMs, generating channel-type and/or carrier-type currents, which can be used as a measure of the AA concentration.

A glass capillary microelectrode based on capillarity and its application to the detection of L-glutamate release from mouse brain slices.

A new glass capillary microelectrode for L-glutamate is described using pulled glass capillaries (tip size, approximately 12.5 microm) with a very small volume (approximately 2 microl) of inner solution containing glutamate oxidase (GluOx) and ascorbate oxidase. The operation of the electrode is based on capillary action that samples L-glutamate into the inner solution. The enzyme reaction by GluOx generates hydrogen peroxide that is detected at an Os-gel-HRP polymer modified Pt electrode in a three-electrode configuration. The amperometric response behavior of the electrode was characterized in terms of the capillarity, response time, sensitivity and selectivity for measurements of L-glutamate. The currents at 0 V vs. Ag/AgCl increased linearly with the L-glutamate concentration from 10 to 150 microM for in vitro and in situ calibrations. The response was highly selective to L-glutamate over ascorbate, dopamine, serotonin and other amino acids. The detection of L-glutamate in the extracellular fluids of different regions of mouse hippocampal slices under stimulation of KCl was demonstrated.