Top-down cortical input during NREM sleep consolidates perceptual memory.

During tactile perception, long-range intracortical top-down axonal projections are essential for processing sensory information. Whether these projections regulate sleep-dependent long-term memory consolidation is unknown. We altered top-down inputs from higher-order cortex to sensory cortex during sleep and examined the consolidation of memories acquired earlier during awake texture perception. Mice learned novel textures and consolidated them during sleep. Within the first hour of non-rapid eye movement (NREM) sleep, optogenetic inhibition of top-down projecting axons from secondary motor cortex (M2) to primary somatosensory cortex (S1) impaired sleep-dependent reactivation of S1 neurons and memory consolidation. In NREM sleep and sleep-deprivation states, closed-loop asynchronous or synchronous M2-S1 coactivation, respectively, reduced or prolonged memory retention. Top-down cortical information flow in NREM sleep is thus required for perceptual memory consolidation.

An interaction between human colon carcinoma cells and hepatocytes activates transforming growth factor-beta1 in vitro.

We established in vitro heterotypic co-cultures of a human colon carcinoma cell line, HT-29, and a human hepatocyte line, tPH5CH, using transwells to investigate possible interactions between colon carcinoma cells and hepatocytes during the metastatic process. Co-culture, but not HT-29 conditioned medium, inhibited tPH5CH cell proliferation, and this inhibition was blocked by an anti-TGF-beta1 antibody. Significantly more activated TGF-beta1 was released by co-culture than by each cell line cultured alone, whereas there were no significant differences between the total TGF-beta1 released by the individual cultured cell lines and that released by co-culture. These data indicate that an interaction between human colon carcinoma cells and hepatocytes activates TGF-beta1 in vitro.

Different role of serum components and cytokines on alveolar macrophage activation by soluble fungal (1-->3)-beta-D-glucan.

In this study, we investigated the mechanism of alveolar macrophage activation by systemic administration of SSG, a soluble highly branched (1-->3)-beta-D-glucan obtained from a fungus Sclerotinia sclerotiorum IFO 9395. Multiple i.v. administration (10 mg/kg; once daily for 10 consecutive days) of SSG enhanced some functions of alveolar macrophages, such as lysosomal enzyme activity and nitric oxide secretion, on day 1 after the last administration, and it also elevated the concentrations of serum protein, interferon gamma and SSG in bronchoalveolar lavage fluid on the same day. On the in vitro assay system, stimulation by SSG alone (500 microg/ml) slightly augmented the lysosomal enzyme activity of alveolar macrophages, but it had no effect on nitric oxide production of cells. Stimulation by serum (1 or 10% mouse serum) or serum components, such as fibronectin (25 microg/ml) and albumin (500 microg/ml), alone strongly augmented only the lysosomal enzyme activity of alveolar macrophages, but it had no effect on nitric oxide secretion from cells, and no synergism or additive-like effect was observed between serum components and SSG. In contrast, stimulation by crude lymphokine (5%) or recombinant murine interferon gamma (100 U/ml) alone did not induce augmentation of lysosomal enzyme activity and nitric oxide production of alveolar macrophages in vitro, but when cells were incubated together with crude lymphokine or recombinant murine interferon gamma and SSG (500 microg/ml), a significant combined effect was observed on both functions of alveolar macrophages. In addition, pretreatment of crude lymphokine or recombinant murine interferon gamma enhanced the expression of beta-D-glucan specific binding sites on the alveolar macrophage surface in vitro though pretreatment by serum components had no effect. Based on these findings, the enhancement of alveolar macrophage functions by systemic administration of SSG appears to be mediated, at least in part, by both the simple effect of serum components including fibronectin and albumin leaked from pulmonary peripheral blood into the alveoli and the synergistic effect between lymphokines released from activated pulmonary T cells and SSG itself entering the alveoli after SSG injection via the priming effect of lymphokines which enhances the expression of beta-D-glucan specific binding sites on the alveolar macrophage surface.

Study of in vitro cytotoxicity of a water soluble organic arsenic compound, arsenosugar, in seaweed.

In the present study, we demonstrated the cytotoxic effect of a dimethylarsenic compound in seaweed, (R)-(2',3'-dihydroxypropyl) 5- deoxy-5-dimethylarsinoyl-beta-D-riboside, namely arsenosugar (AsSug), on mammalian cells, murine macrophages, in comparison with that of an inorganic arsenical, arsenite, in vitro. More than 99.5% pure AsSug was synthesized. Arsenite was strongly and equally toxic to both peritoneal macrophages (PMs) and alveolar macrophages (AMs), and the concentration of arsenite that inhibited the viability of cells by 50% compared to the viability of control cells (50% inhibitory concentration; IC50) was 5 microM. In contrast, AsSug showed no cytotoxicity to both PMs and AMs at the microM concentration level; however, it induced different and interesting cellular responses in both macrophages at high concentrations, 1-10 mM. AsSug enhanced the viability of PMs at an optimal dose of 5 mM; conversely, it showed weak but significant cytotoxicity to AMs (IC50 = 8 mM).

Micelle-mediated extraction for concentrating hydrophobic organic compounds.

An extraction method based on polymer-induced phase separation of aqueous micellar solutions of octyl-beta-D-thioglucoside (OTG) was assessed for concentrating hydrophobic analytes. Various hydrophobic compounds such as polycyclicaromatic hydrocarbons, alkylbenzenes, alkylphenols, chlorobenzenes, chlorophenols, phthalic esters, pesticides, and steroid hormones could be efficiently concentrated into a small volume of surfactant-rich phase, while hydrophilic matrix components remained in the bulk aqueous phase. The surfactant-rich phase containing concentrated OTG could be directly introduced into the hygro-organic mobile phase of high-performance liquid chromatography with ultra-violet photometric detection. The application of this method greatly enhanced the signal intensity in the chromatogram while reducing the interference of matrix components.

Concentration of nonylphenol and its polyethoxylated derivatives by polymer-mediated extraction.

Polymer-mediated extraction based on thermoresponsive precipitation of poly(N-isopropylacrylamide) [PNIPAAm] was applied to the concentration of amphiphilic compounds, nonylphenyl polyethoxylates (NPnEOs), in water. Among these nonionic surfactants, NPnEOs possessing the number, n, of ethoxy unit less than 5 were quantitatively recovered in polymer precipitates when a 0.100-g portion of PNIPAAm was used for a 10 ml sample solution. Torelance limit (30 ppm) against a typical industrial anionic surfactant, sodium dodecylsulfate, strongly suggests the potential to use the present method for practical purposes. After preconcentration, trace nonylphenol and mono-ethoxylated nonylphenol (ppb-level) in a river water sample were successfully determined by high-performance liquid chromatography with ultra-violet photometric detection.

Polymer-mediated extraction of 8-quinolinolato metal chelates for the determination of metal ions in water with graphite furnace atomic absorption spectrometry.

Water-insoluble 8-quinolinolato metal chelates were formed and were stably solubilized in the aqueous solution of a water-soluble polymer, poly (N-isopropylacrylamide)(PNIPAAm), at room temperature. When the solution was heated at 50 degrees C, PNIPAAm precipitated and then formed a gum-like aggregate (polymer phase) having a very small volume. Accompanying the polymer precipitation, hydrophobic 8-quinolinolato chelates with cobalt(II), iron(III), nickel(II), and copper(II) ions were efficiently incorporated into the polymer phase. At 0.5% (w/v) of PNIPAAm and 8.0 mM of 8-quinolinol, the recoveries in the incorporation of four metal chelates were quantitative. The fluorescence spectra of a probe suggests that the hydrated polymer in the aqueous solution provides hydrophobic portions which can incorporate hydrophobic metal chelates. The polymer phase was easily taken out from the solution and was dissolved with a small amount of acetonitrile. The resulting solution could be directly introduced into a graphite furnace of atomic absorption spectrometry. The signal intensities for the absorbance of cobalt after concentrating the chelate were 100-fold greater than those before the concentration.

[Densitometric quantitation of platelet activating factor and other phospholipids in human saliva using enzyme reaction on a silica plate].

A sensitive quantitative analysis by thin layer chromatography was developed for the determination of platelet activating factor (PAF) and other phospholipids in human saliva. The saliva sample (0.6 ml) was pretreated by diatomite column extraction with chloroform-methanol (95:5, v/v). The extract (20 microliters) was spotted on a TLC plate. The mobile phase was chloroform-methanol-water (65:35:7, v/v). The development proceeded until the mobile phase front reached 8 cm from the spotted point, this process usually required 30 min. After development, phospholipase C and alkaline phosphatase solutions were sprayed on the TLC plate at 45 degrees C to hydrolyze phospholipids. By spraying a mixture of ammonium molybdate and Malachite Green, the produced phosphate was changed to molybdophosphate-Malachite Green aggregate, which gave a blue green spot. The colored spots were scanned at 620 nm by chromatoscanner. A linear relationship was obtained between peak area and PAF concentration in the range from 2 to 100 pmol/spot with a relative standard deviation of 2% (n = 7). By this procedure, lysophosphatidylcholine, phosphatidylcholine and phosphatidylethanolamine in human saliva were also determined sensitively. PAF levels in the range from 40 to 300 ng/ml were found in normal human salivas. Although differences in the total amounts of phospholipids in saliva were found for each healthy volunteer and sampling time, the composition of phospholipids was proved to be virtually constant.

[Micelle-mediated extraction for concentrating conjugated bilirubin in urine].

An extraction method based on the phase separation of aqueous micellar solutions of n-octyl-beta-D-thioglucoside (OTG) was applied to the concentrating conjugated bilirubin in urine. The analyte in sample solutions could be efficiently concentrated into a small volume of surfactant-rich phase, while hydrophilic matrix components including urinary protein, ascorbic acid, and saccharide remained in the aqueous phase. The concentrated OTG negligibly affected the diazo reaction and the subsequent spectrophotometric detection. Conjugated bilirubin was successfully determined in the concentration range from 0.05 microgram/ml to 5 micrograms/ml with a 96-well microplate reader absorption spectrophotometer.

[Flow injection analysis for determination of choline-containing phospholipids by luminol chemiluminescence].

A sensitive flow injection analysis using luminol/peroxidase chemiluminescence was developed for the determination of choline-containing phospholipids in serum. Flow injection manifold was composed of two channel system with an enzyme column, in which phospholipase D was immobilized together with choline oxidase. The serum sample (5 microliters) was pretreated by Extrelut column (diatomite column) extraction with chloroform-methanol (95:5). The extract (20 microliters) was injected into a sample carrier at 38 degrees C and passed through the enzyme column, which converted phospholipid to choline and subsequently to hydrogen peroxide. Produced hydrogen peroxide was monitored by measuring the chemiluminescence intensity of luminol/peroxidase system at 5 degrees C. The response was linear against the amount of phospholipids ranging from 2 to 2000 pmol/test, and the relative standard deviation was less than 2%. In the determination of phospholipids in the serum, a correlation coefficient (r) between 4-aminoantipyrine/phenol and the proposed methods was found to be 0.983 (Y = 1.035X-6.2). The throughput rate was 15 samples/h.

[Spectrophotometric determination of uric acid in serum using a titanium (IV)-porphyrin complex].

Aqueous solution of oxo[5,10,15,20-tetra(4-pyridyl)porphyrinato]titanium (IV) complex, a Ti-TPyP reagent, was found to be very useful for the spectrophotometric determination of hydrogen peroxide. The reagent (lambda max 432 nm) reacts with hydrogen peroxide to form a monoperoxocomplex, resulting in a significant decrease of the absorbance at 432 nm. The decrease (delta A) in absorbance was proportional to the concentration of hydrogen peroxide. The Ti-TPyP reagent was successfully applied to the assay of uric acid in the serum, using uricase to produce hydrogen peroxide through enzymatic oxidation. Using only 5 microliters serum, a linear relationship was obtained between delta A and uric acid concentration in the serum ranging from 5 x 10(-6) to 1 x 10(-3) M. The apparent molar delta A of uric acid was 2.2 x 10(5) M-1 cm-1. The relative standard deviation of repeated runs (n = 8) was 2.8% at 3.77 x 10(-4) M uric acid. The analytical recovery of uric acid (5 x 10(-4) M) added to the serum was 96.8 to 105.0%. No pre-concentration and deproteinization were required to determine uric acid in the serum by the present method because of the high sensitivity and selectivity of the Ti-TPyP reagent for hydrogen peroxide.

[A case of breast cancer 5 cm in diameter treated with a breast preserving approach after preoperative intra-arterial chemotherapy].

The patient was a 59-year-old female with a mass in the right breast (area C). At the initial examination, the mass was 5.0 x 4.5 cm on palpation, aspiration cytology was Class V, and a diagnosis of T2aN1aM0, Stage II breast cancer was made. Since the patient strongly desired a breast preserving treatment, a reduction in the size of the mass was attempted by local intra-arterial chemotherapy. Docetaxel (TXT) was administered at 60 mg into the internal thoracic artery and lateral thoracic artery at a rate of once a month for a total of 5 times. After the fifth treatment, the mass was reduced in size to 2.8 x 2.5 cm on palpation, and breast-preserving resection was then carried out. On histopathological examination, cancer was observed in an area of 3.0 x 2.2 cm. Careful follow-up is still needed, but preoperative intra-arterial chemotherapy is considered to be significant as a step before breast preserving surgery for breast cancer with a diameter of 3-5 cm.

Inorganic and methylated arsenic compounds induce cell death in murine macrophages via different mechanisms.

We demonstrate in this study the cytotoxic effects of inorganic arsenicals, arsenite and arsenate, and organic arsenic compounds, monomethylarsonic acid (MAA), dimethylarsinic acid (DMAA), and trimethylarsine oxide (TMAO), which are metabolites of inorganic arsenicals in human bodies, using murine macrophages in vitro. Inorganic arsenicals, both arsenite and arsenate, are strongly toxic to macrophages, and the concentration that decreased the number of surviving cells to 50% of that in untreated controls (IC50) was 5 or 500 microM, respectively. These inorganic arsenicals mainly caused necrotic cell death with partially apoptotic cell death; about 80% of dead cells were necrotic, and 20% were apoptotic. The inorganic arsenicals also induced marked release of an inflammatory cytokine, tumor necrosis factor alpha (TNF alpha), at cytotoxic doses. This strong cytotoxicity of an inorganic arsenical, arsenite, might be mediated via active oxygen and protease activation because it was inhibited by the addition of some antioxidant reagents, such as superoxide dismutase (SOD), catalase, and GSH, or by a peptide inhibitor of interleukin-1 beta-converting enzyme (ICE). It is likely that these immunotoxic effects of inorganic arsenicals may evoke both immunosuppression and inflammation, and they may be central factors causing carcinogenesis and severe inflammatory responses, such as hepatomegaly and splenomegaly, in chronic arsenicosis patients who daily ingested arsenic-contaminated well water. In contrast, the cytotoxic effects of methylated arsenic compounds were lower than those of inorganic arsenicals. The IC50 value of DMAA was about 5 mM, and MAA and TMAO had no toxicity even at concentrations over 10 mM. Additionally, these methylated chemicals suppressed the TNFalpha release from macrophages. DMAA induced mainly apoptotic cell death in macrophages as indicated by cellular morphological changes, condensed nuclei, terminal deoxynucleotidyltransferase-mediated dUTP-biotin nick end labeling (TUNEL), and DNA fragmentation. However, the cytotoxicity of DMAA might be induced via a different mechanism from that of inorganic arsenicals because it was not abolished by the additions of SOD, catalase, or ICE inhibitor. Conversely, GSH enhanced the toxicity of DMAA. These data suggest that methylation of inorganic arsenicals in mammals plays an important role in suppression of both severe immunosuppression and inflammatory responses caused by inorganic arsenicals.

Sensitive densitometry for the determination of platelet-activating factor and other phospholipids in human tears.

A sensitive TLC method is reported for the determination of the platelet-activating factor (PAF) and other phospholipids in human tears. Tear samples absorbed on filter-paper were subjected to diatomite column extraction with chloroform-methanol. The eluent containing phospholipids was spotted on a silica gel plate. After removing lipids other than phospholipids by pre-development with hexane-diethyl ether (4 + 1 v/v), individual phospholipid separation was carried out by development with chloroform-methanol-water (65 + 35 + 7 v/v). Phospholipase C and then alkaline phosphatase solutions were sprayed on the TLC plate at 45 degrees C to liberate phosphate from each phospholipid. By spray application of a mixture of ammonium molybdate and Malachite Green, the liberated phosphates appeared as blue-green spots of molybdophosphate-Malachite Green aggregate on a yellow-brown background. The absorbance of each spot on the plate was measured at 620 nm with a densitometer. The peak area was found to be linearly related to PAF content in the range 2-100 pmol per spot, the RSD being 2% (n = 7). Approximately 86% of standard PAF added to tears was recovered. By this method, lysophosphatidylcholine (lysoPC), phosphatidylcholine (PC) and phosphatidylethanolamine (PE) in human tears could also be detected with high sensitivity. Each phospholipid in healthy human tears showed a nearly constant content; PAF, lysoPC, PC and PE were present at levels of 26.2, 42.3, 10.0 and 19.7%, respectively.

A spectrophotometric method for the determination of free fatty acid in serum using acyl-coenzyme A synthetase and acyl-coenzyme A oxidase.

A mixture of Ti(IV) and 4-(2-pyridylazo)resorcinol was found to be useful in the spectrophotometric determination of trace amounts of hydrogen peroxide. The absorbance at 508 nm was proportional to the concentration of hydrogen peroxide added. The reagent was successfully applied to the assay of free fatty acid in serum through the combined use of acyl-CoA synthetase and acyl-CoA oxidase. The latter enzyme produces H2O2. As a result, hydrogen peroxide was produced through the enzymatic oxidation of free fatty acid. It was possible to determine free fatty acid in 50 microliters of serum at concentrations ranging from 0.02 to 1.5 mM. The coefficient of variation was less than 3% at concentrations ranging from 0.1 to 1.5 mM. In the present method, there is the advantage of minimal influence from reducible substances as well as greater simplicity and accuracy.