Photodissociation spectroscopy of N2O+ in the ion storage ring RICE.

The A2Σ+-X2Π electronic transition of the nitrous oxide cation, N2O+, was measured via photodissociation spectroscopy in a cryogenic electrostatic ion storage ring. Rotationally resolved spectra of the N-O stretching vibrational sequence were obtained by detecting neutral N fragments produced via N2O+ → NO+ + N predissociation channels. A new set of molecular constants was determined for the high-lying vibrational levels of the A2Σ+ state.

Optical imaging of the propagation patterns of neural responses in the rat sensory cortex: comparison under two different anesthetic conditions.

Although many studies have reported the influence of anesthetics on the shape of somatic evoked potential, none has evaluated the influence on the spatio-temporal pattern of neural activity in detail. It is practically impossible to analyze neural activities spatially, using conventional electrophysiological methods. Applying our multiple-site optical recording technique for measuring membrane potential from multiple-sites with a high time resolution, we compared the spatio-temporal pattern of the evoked activity under two different anesthetic conditions induced by urethane or α-chloralose. The somatic cortical response was evoked by electrical stimulation of the hindlimb, and the optical signals were recorded from the rat sensorimotor cortex stained with a voltage-sensitive dye (RH414). The evoked activity emerged in a restricted area and propagated in a concentric manner. The spatio-temporal pattern of the evoked activity was analyzed using isochrone maps. There were significant differences in the latency and propagation velocity of the evoked activity, as well as the full width at half maximum of optical signal between the two anesthetic conditions. Differences in the amplitude and the slope of the rising phase were not significant.

Effect of flow rate and temperature on transmembrane blood pressure drop in an extracorporeal artificial lung.

INTRODUCTION: Transmembrane pressure drop reflects the resistance of an artificial lung system to blood transit. Decreased resistance (low transmembrane pressure drop) enhances blood flow through the oxygenator, thereby, enhancing gas exchange efficiency. This study is part of a previous one where we observed the behaviour and the modulation of blood pressure drop during the passage of blood through artificial lung membranes. METHODS: Before and after the induction of multi-organ dysfunction, the animals were instrumented and analysed for venous-venous extracorporeal membrane oxygenation, using a pre-defined sequence of blood flows. RESULTS: Blood flow and revolutions per minute (RPM) of the centrifugal pump varied in a linear fashion. At a blood flow of 5.5 L/min, pre- and post-pump blood pressures reached -120 and 450 mmHg, respectively. Transmembrane pressures showed a significant spread, particularly at blood flows above 2 L/min; over the entire range of blood flow rates, there was a positive association of pressure drop with blood flow (0.005 mmHg/mL/minute of blood flow) and a negative association of pressure drop with temperature (-4.828 mmHg/(°Celsius). These associations were similar when blood flows of below and above 2000 mL/minute were examined. CONCLUSIONS: During its passage through the extracorporeal system, blood is exposed to pressure variations from -120 to 450 mmHg. At high blood flows (above 2 L/min), the drop in transmembrane pressure becomes unpredictable and highly variable. Over the entire range of blood flows investigated (0-5500 mL/min), the drop in transmembrane pressure was positively associated with blood flow and negatively associated with body temperature.

Simultaneous determination of delta(15)N and delta(18)O of N2O and delta(13)C of CH4 in nanomolar quantities from a single water sample.

We have developed a rapid, sensitive, and automated analytical system to simultaneously determine the concentrations and stable isotopic compositions (delta(15)N, delta(18)O, and delta(13)C) of nanomolar quantities of nitrous oxide (N(2)O) and methane (CH(4)) in water, by combining continuous-flow isotope-ratio mass spectrometry and a helium-sparging system to extract and purify the dissolved gases. Our system, which is composed of cold traps and a capillary gas chromatograph that use ultra-pure helium as the carrier gas, achieves complete extraction of N(2)O and CH(4) in a water sample and separation among N(2)O, CH(4), and the other component gases. The flow path following exit from the gas chromatograph was periodically changed to pass the gases through the combustion furnace to convert CH(4) and the other hydrocarbons into CO(2), or to bypass the combustion furnace for the direct introduction of eluted N(2)O into the mass spectrometer, for determining the stable isotopic compositions through monitoring the ions of m/z 44, 45, and 46 of CO(2) (+) and N(2)O(+). The analytical system can be operated automatically with sequential software programmed on a personal computer. Analytical precisions better than 0.2 per thousand and 0.3 per thousand and better than 1.4 per thousand and 2.6 per thousand were obtained for the delta(15)N and delta(18)O of N(2)O, respectively, when more than 6.7 nmol and 0.2 nmol of N(2)O, respectively, were injected. Simultaneously, analytical precisions better than 0.07 per thousand and 2.1 per thousand were obtained for the delta(13)C of CH(4) when more than 5.5 nmol and 0.02 nmol of CH(4), respectively, were injected. In this manner, we can simultaneously determine stable isotopic compositions of a 120 mL water sample with concentrations as low as 1.7 nmol/kg for N(2)O and 0.2 nmol/kg for CH(4).

Molecular cloning and heterologous expression of an alpha-adrenergic-like octopamine receptor from the silkworm Bombyx mori.

A cDNA encoding an octopamine (OA) receptor (BmOAR1) was isolated from the nerve tissue of silkworm (Bombyx mori) larvae. Comparison of amino acid sequences showed that BmOAR1 is highly identical to OA receptors isolated from Periplaneta americana (Pa oa(1)), Apis mellifera (AmOA1), and Drosophila melanogaster (OAMB or DmOA1A). BmOAR1 was stably expressed in HEK-293 cells. OA above 1 microM led to an increase in intracellular cyclic AMP concentration ([cAMP](i)). The synthetic OA-receptor agonist demethylchlordimeform also elevated [cAMP](i) to the same maximal level (approximately 5-fold over the basal level) as that induced by OA. However, other biogenic amines, tyramine and dopamine, and chlordimeform were without effects. The [cAMP](i) level raised by OA was lowered by antagonists; the rank order of antagonist activity was chlorpromazine > mianserin = yohimbine. Cyproheptadine and metoclopramide had little effect. OA above 100 nM induced a transient or sustained increase in intracellular Ca(2+) concentration ([Ca(2+)](i)), depending on the concentration of OA. Sequence homology and functional analysis data indicate that BmOAR1 is an alpha-adrenergic-like OA receptor of B. mori.

Ingestion of gelatin has differential effect on bone mineral density and body weight in protein undernutrition.

Malnutrition, particularly protein undernutrition, contributes to the occurrence of osteoporotic fracture by lowering bone mass. In this study, the effects of dietary protein on bone mineral density and body weight in protein undernutrition were compared between gelatin and milk casein. When mice were fed for 10 wk with a low protein diet containing 10(%) casein or 6% casein +4% gelatin, there was no significant difference in the final body weight between the 6% casein+4% gelatin group and the 10% casein group. In contrast, bone mineral content and bone mineral density of the femur were significantly higher in the 6% casein+4% gelatin group than in the 10% casein group. Bone mineral content and bone mineral density did not differ significantly in 14% protein groups between 14% casein and 6% casein +80% gelatin. These results suggest that gelatin has differential effects on bone mineral density and body weight in protein undernutrition.

Semiquantitative analysis of the expression of GABA-A receptor subunits in the developing embryonic chick brain stem.

The expression levels of seven types of gamma-aminobutyric acid-A (GABA-A) receptor subunits (alpha1, beta2, beta3, beta4, gamma1, gamma2, and gamma4) were quantified in the embryonic chick brain stem at 2 to 20 d of incubation (E2 to E20) and just after hatching. The expression level of mRNA was measured by using semiquantitative reverse transcription-polymerase chain reaction (RT-PCR). When property regions were amplified, two products were observed for each of the beta2, beta4, and gamma2 subunits because of alternative splicing. These were named beta2S and beta2L, beta4S and beta4L, and gamma2S and gamma2L for shorter and longer fragments, respectively. Transcripts of alpha1, beta2L, beta2S, beta3, beta4L, beta4S, gamma1, and gamma2S subunits were first detected from E2 to E5 brain stems. The expression level of each subunit increased gradually with development and reached a plateau at E9 to E12. In contrast, a delay occurred in the appearance of both the gamma4 and gamma2L subunits, which were not detected until E8 to E10. The absence of gamma4 and/or gamma2L subunits may explain differences in the pharmacological characteristics of GABA-A receptors at the early stages of development.

Sorbicillinol, a key intermediate of bisorbicillinoid biosynthesis in Trichoderma sp. USF-2690.

In the course of our screening program for free radical scavengers from Trichoderma sp. USF-2690, we found an unidentified metabolite (1) that appeared by the method used for HPLC analysis. Metabolite 1 gradually decreased with the production of bisorbicillinoids and was easily missed during the general isolation procedure. The LC-ESI-MS (negative) analysis for 1 gave m/z 247 as the (M-1)- ion peak. The hydrolysis of synthetic 6-O-acetylsorbicillinol (+/- -2) by 0.05 M KOH and acetylation of product 1 in an aqueous solution indicated that the structure of 1 was (6S)-4-(2,4-hexadienoyl)-3,6-dihydroxy-2,6-dimethyl-2,4-cyclohexadien-1-one, designated sorbicillinol, a quinol that has been postulated to be important in bisorbicillinoid biosynthesis.

1,1-Diphenyl-2-picrylhydrazyl radical-scavenging compounds from soybean miso and antiproliferative activity of isoflavones from soybean miso toward the cancer cell lines.

Guided by their DPPH radical-scavenging activity, nine compounds were isolated from soybean miso. Of these, 8-hydroxydaidzein, 8-hydroxygenistein and syringic acid had as high DPPH radical-scavenging activity as that of alpha-tocopherol. The antiproliferative activity of four of the isolated isoflavones toward three cancer cell lines was examined. 8-Hydroxygenistein showed the highest activity (IC50=5.2 microM) toward human promyelocytic leukemia cells (HL-60).

Novel fungal metabolites, demethylsorbicillin and oxosorbicillinol, isolated from Trichoderma sp. USF-2690.

The novel compounds, demethylsorbicillin (1) and oxosorbicillinol (2), were isolated from a fermentation broth of Trichoderma sp. USF-2690. The structures of these compounds, which were determined from spectroscopic evidence, suggest the possibility that methylation at C-6 and oxidation at C-1 and C-6 of sorbicillin were controlled in the early polyketide stage before the formation of oxidized sorbicillin dimers. In a 1,1-diphenyl-2-picrylhydrazyl (DPPH) radical-scavenging assay, 2 gave an ED50 value of 87.7 microM.

Studies on the 1,1-diphenyl-2-picrylhydrazyl radical scavenging mechanism for a 2-pyrone compound.

The radical scavenging mechanisms for the 2-pyrone compound, 4-hydroxy-3,6-dimethyl-2H-pyrane-2-one (1), and the 1,1-diphenyl-2-picrylhydrazyl (DPPH) radical (4) in several solvent systems were evaluated by the quantitative change in compounds detected at 270 nm and subsequent HPLC analyses. The HPLC profile for each condition suggested that the reaction proceeded by a different mechanism in each solvent system. In organic solvents (CHCl3, iso-propanol, and EtOH), 1-[4-(3,4-dihydro-3,6-dimethyl-2,4-dioxo-2H-pyran-3-yl) phenyl]-1-phenyl-2-picrylhydrazine (2) was produced as an adduct of the DPPH radical and 1. On the other hand, the reaction in a buffer solution (an acetate buffer at pH 5.5) gave several degradation products with 1[4-(2,3-dihydro-2,5-dimethyl-3-oxo-fur-2-yl) phenyl]-1-phenyl-2-picrylhydrazine (5), this being structurally elucidated by spectroscopic analyses. The decrease of the DPPH radical in each reaction system suggests that compound 1 could scavenge about 1.5-1.8 equivalents of the radical in organic solvents and about 3.5-3.9 in the buffer solution.

[Effect of stellate ganglion block on human retinal blood flow].

PURPOSE: The effect of stellate ganglion block (SGB) on human retinal blood flow was evaluated. METHODS: We measured the diameter of the retinal artery and vein, and retinal venous flow rate by laser speckle retinal blood flow meter simultaneously in 11 eyes of 11 normal volunteers. RESULTS: The reliable data from 9 eyes of 9 person were used for analysis. SGB did not change the blood pressure, heart rate, retinal arterial diameter, or venous diameter. However SGB increased retinal blood velocity significantly from 9.9 +/- 1.6 (mean +/- standard deviation) mm/s to 11.1 +/- 1.5 mm/s (p < 0.01). Intraocular pressure decreased from 12.3 +/- 2.1 (mean +/- standard deviation)mmHg to 9.4 +/- 2.2 mmHg after SGB (p < 0.01). There was no relationship between the change of ocular perfusion pressure and that of retinal venous blood velocity. CONCLUSION: SGB increased the retinal venous blood velocity without changing the retinal vessel diameter.

Spatiotemporal changes of fibronectin, tenascin-C, fibulin-1, and fibulin-2 in the skin during the development of chronic contact dermatitis.

In order to elucidate how chronic inflammation affects the organization of the extracellular matrix in the skin, a prolonged allergic contact dermatitis was induced in a mouse by repeated application to the ear of 2,4-dinitrofluorobenzene every 3 d for 66 d. Subsequently, the spatiotemporal changes of fibronectin, tenascin-C, fibulin-1, and fibulin-2 in the skin were examined. In the acute phase of inflammation (day 3-day 12), the amount of fibronectin and tenascin-C increased markedly and were degraded, whereas the amount of fibulin-2 changed slightly. Abundant deposition of tenascin-C was observed in the connective tissue. Fibulin-1 and fibulin-2 distributed as fine fibrils. In contrast, the amounts of fibronectin and tenascin-C decreased and their degradation was suppressed in the chronic phase (day 15-day 66), but the amount of fibulin-2 increased. Tenascin-C was observed mainly at and underneath the epidermal basement membrane. In the subepidermal region, many fibulin-2-positive microfibrils were distributed. The amount and distribution of fibulin-1 did not change markedly in either phase. MMP-like enzymes of 62 kDa, probably activated MMP-2, were upregulated in the chronic phase, whereas components of 92, 85, or 67 kDa were highly induced in the acute phase. These results suggest that chronic inflammation in allergic contact dermatitis is associated with temporal changes in the expression, deposition, and degradation of inducible extracellular matrix components.

Phosphate depletion enhances tissue-nonspecific alkaline phosphatase gene expression in a cultured mouse marrow stromal cell line ST2.

Alkaline phosphatases (ALP) are highly ubiquitous enzymes present in the majority of animals from bacteria to higher vertebrate. Although their wide distribution in nature has suggested that these enzymes should perform important biological functions, their detailed roles or natural substrates remain unknown. In Escherichia coli, the extracellular phosphate (Pi) limitation induces the ALP gene, indicating the role of extracellular Pi in ALP gene regulation. However, little is known about the similar mechanisms in mammalian cells. This study was designed to examine the effect of low Pi medium on the ALP activity and its expression in the mouse stromal cell line ST2. The enzymatic property was classified into tissue-nonspecific ALP (TNSALP). After treatment by Pi starvation for 3 days, there was a 2-fold increase in the specific activity of TNSALP. RT-PCR analysis revealed that the mRNA of the TNSALP gene was highly stimulated. These results indicated that the effect of Pi depletion on ALP activity was regulated at the TNSALP transcriptional level, suggesting that the possible role of the Pi sensing system for biological functions of ALP might have been conserved in evolution. Our findings also made it possible to discuss the physiological roles of ALP in vivo.

Molecular cloning and characterization of two cDNAs for Glycyrrhiza glabra squalene synthase.

Two cDNAs (GgSQS1 and GgSQS2) encoding squalene synthase have been isolated from licorice, Glycyrrhiza glabra L., and characterized. The deduced amino acid sequence of GgSQS1 was 88%, 81%, 78%, 45-44%, and 45-41% identical to those of GgSQS2, Nicotiana, Arabidopsis, mammal and yeast squalene synthases, respectively. Squalene synthase activity was found in the cell-free extracts of Escherichia coli transformed with the recombinant plasmids for GgSQS1 and GgSQS2, respectively. Genomic Southern blot hybridization indicated that there are three squalene synthase genes in the licorice genome. Northern blot analysis showed that GgSQS2 mRNA is mainly expressed during the exponential growth phase of the cultured licorice cells.

Skin blood flow responses during sleep by laser Doppler flowmetry.

Skin surface blood flow at the fingertips was measured in a 21-year-old woman during sleep using laser Doppler flowmetry. Skin blood flow responses (SBFR) are transient reductions in skin blood flow of about 25s in duration. In our subject, SBFR showed a delay time of 8-9s from the onset of skin potential responses (SPR) and appeared more consistently than SPR. The frequency of SBFR decreased during non-rapid eye movement sleep, especially when the sleep stage became deep. These findings indicate the validity of SBFR as a discrete index of autonomic function during sleep.

Optical imaging of the spatiotemporal patterning of neural responses in the embryonic chick superior cervical ganglion.

Multiple-site optical recording of transmembrane potential changes with a voltage-sensitive dye was used to reveal the functional expression and developmental changes of the postsynaptic potentials in the early embryonic chick superior cervical ganglion. The ganglia were isolated from five- to 12-day-old chick embryos with preganglionic nerve fibres (vertebral and/or cervical carotic nerves) attached. The preparations were stained with a voltage-sensitive merocyanine-rhodanine dye (NK2761). Voltage-related optical (absorbance) changes were recorded simultaneously from 127 contiguous loci in the preparation, using a 12 x 12-element photodiode array. Optical changes having two components were evoked by preganglionic nerve stimulation. One component was the fast spike-like signal and another the delayed slow signal. The amplitude of the slow signal was decreased by repetitive stimulation, reduced by low external calcium ion concentrations and eliminated in the presence of manganese or cadmium ions. The slow signals were also eliminated in the presence of D-tubocurarine. Accordingly, we concluded that the slow signal corresponds to cholinergic excitatory postsynaptic potentials. In the five- and six-day-old superior cervical ganglia, only the fast optical signals (referred to as the action potentials) were recorded. Slow optical signals (referred to as the excitatory postsynaptic potentials) were detected from preparations older than seven days. The amplitude of the slow optical signal gradually increased, together with an expansion of the response area, as the developmental stage proceeded from seven to 10 days. To compare the distribution patterns of the neural responses evoked by stimuli applied to the cervical carotic and vertebral nerves, we have mapped and imaged the spatial patterning of the synaptic responses. In the maps, the positions of the peak size regions of the slow signals were assessed, and we found that there were differences in the location of these areas for the cervical carotic vs vertebral nerves. From these experimental results, we conclude that synaptic function within the chick superior cervical ganglion is initiated at the seven-day-old embryonic stage, and reaches a maximum level at 10 days. Synaptic transmission at these stages is mediated solely by nicotinic acetylcholine receptors. The spatial mapping of the synaptic responses reveals that the neural populations related synaptically to the cervical carotic and vertebral nerves are located separately within the ganglion, even at an early developmental stage.

Isolation of a 2-Pyrone Compound as an Antioxidant from a Fungus and Its New Reaction Product with 1,1-Diphenyl-2-picrylhydrazyl Radical.

The indophenol-reducing compound, 4-hydroxy-3,6-dimethyl-2H-pyrane-2-one (I), was isolated from the culture filtrate of an unidentified fungus. I also reacted with the DPPH radical to form a reaction product IV which was determined to be 1-[4-(3,4-dihydro-3,6-dimethyl-2,4-dioxo- 2H-pyran-3-yl)phenyl]-1-phenyl-2-picrylhydrazine. This is the first report describing the formation of an adduct of the DPPH radical and its scavenger.

A regional gradient of cardiac intrinsic rhythmicity depicted in embryonic cultured multiple hearts.

We used optical methods to examine the spatial gradient of intrinsic rhythmicity in early-stage multiple-heart chick embryos. The latter were induced experimentally in whole-embryo culture. The embryos were cut microsurgically through the tissue of the anterior intestinal portal at the 5- to early 7-somite developmental stage. Spontaneous electrical activity in 4 to 6 segmented hearts, during the 7- to 10-somite stages of development, were monitored simultaneously by means of multiple-site optical recordings of membrane potential activity, using a voltage-sensitive merocyanine-rhodanine dye (NK2761). Each segment of the heart exhibited its own inherent rhythmicity. In quadruple hearts, the order of the rhythmicity was often left-caudal segment>right-caudal segment>left-cephalic segment>right-cephalic segment; the heart rate in the left-caudal segment was often faster than that in the other segments. An atypical pattern of "bursting" rhythm was observed in the cephalic segments suggesting that, in these segments, the development of rhythmicity is relatively poor. These findings strongly emphasize the concept that, in the early phases of cardiogenesis, the formation of a regional gradient of pacemaker activity (i.e. a spatial gradient of intrinsic rhythmicity) results in the functional self-organization of the pacemaking area.