Extending coherence time of macro-scale diamond magnetometer by dynamical decoupling with coplanar waveguide resonator.

Ultimate sensitivity for quantum magnetometry using nitrogen-vacancy (NV) centers in a diamond is limited by a number of NV centers and coherence time. Microwave irradiation with a high and homogeneous power density for a large detection volume is necessary to achieve a highly sensitive magnetometer. Here, we demonstrate a microwave resonator to enhance the power density of the microwave field and an optical system with a detection volume of 1.4 × 10-3 mm3. The strong microwave field enables us to achieve 48 ns Rabi oscillation which is sufficiently faster than the phase relaxation time of NV centers. This system combined with a decoupling pulse sequence, XY16, extends the spin coherence time (T 2) up to 27 times longer than that with a spin echo method. Consequently, we obtained an AC magnetic field sensitivity of 10.8 pt/ Hz using the dynamical decoupling pulse sequence.

Novel antagonists for proteinase-activated receptor 2: inhibition of cellular and vascular responses in vitro and in vivo.

BACKGROUND AND PURPOSE: Proteinase-activated receptor 2 (PAR(2)) is a G-protein coupled receptor associated with many pathophysiological functions. To date, the development of PAR(2) antagonists has been limited. Here, we identify a number of novel peptide-mimetic PAR(2) antagonists and demonstrate inhibitory effects on PAR(2)-mediated intracellular signalling pathways and vascular responses. EXPERIMENTAL APPROACH: The peptide-mimetic compound library based on the structures of PAR(2) agonist peptides were screened for inhibition of PAR(2)-induced calcium mobilisation in human keratinocytes. Representative compounds were further evaluated by radioligand binding and inhibition of NFkappaB transcriptional activity and IL-8 production. The vascular effects of the antagonists were assessed using in vitro and in vivo models. KEY RESULTS: Two compounds, K-12940 and K-14585, significantly reduced SLIGKV-induced Ca(2+) mobilisation in primary human keratinocytes. Both K-12940 and K-14585 exhibited competitive inhibition for the binding of a high-affinity radiolabelled PAR(2)-ligand, [(3)H]-2-furoyl-LIGRL-NH(2), to human PAR(2) with K(i) values of 1.94 and 0.627 microM respectively. NFkappaB reporter activity and IL-8 production were also significantly reduced. Furthermore, relaxation of rat-isolated aorta induced by SLIGRL-NH(2) was inhibited competitively by K-14585. K-14585 also significantly lowered plasma extravasation in the dorsal skin of guinea pigs and reduced salivation in mice. CONCLUSIONS AND IMPLICATIONS: K-12940 and K-14585 antagonized PAR(2) competitively, resulting in inhibition of PAR(2)-mediated signalling and physiological responses both in vitro and in vivo. These peptide-mimetic PAR(2) antagonists could be useful in evaluating PAR(2)-mediated biological events and might lead to a new generation of therapeutically useful antagonists.

Suppressing actions of butyrate on growth hormone (GH) secretion induced by GH-releasing hormone in rat anterior pituitary cells.

We assessed the inhibitory effects of butyrate on the growth hormone (GH) secretion in order to investigate the cellular mechanisms in rat somatotrophs. Isolated anterior pituitary cells were cultured in DMEM for several hours, either in the presence (1, 3, or 10mM) or absence of butyrate, and then stimulated with 10(-7)M GHRH for 30 min, in the presence of butyrate at the concentrations used for the previous culture. The increase in GHRH-induced GH release was significantly reduced in a time-dependent and concentration-dependent manner in the cells previously cultured with butyrate. GH content (the sum of GH released into the medium induced by GHRH stimulation and the GH remaining in the cells after stimulation) was reduced by the culture of cells in the presence of butyrate, which was also inversely dependent on the concentrations used for the culture. Simultaneous addition of an L-type Ca(2+) channel blocker, nifedipine (10 pM), to the medium during 10(-9)M GHRH stimulation significantly reduced the stimulated GH release, which was further significantly decreased by a simultaneous addition of 10 mM butyrate. Butyrate blunted the GHRH (10(-9)M)-induced increase in cellular cyclic AMP and calcium ion concentrations, the activity of protein kinases (A and C), and GHmRNA expression. The expression of mRNA for GPR 41 and 43, known as receptors for short-chain fatty acids, was confirmed in the anterior pituitary cells. These findings suggest that butyrate inhibits GHRH-induced GH release as well as GH production, and the cellular inhibitory actions of butyrate occur in diverse cellular signaling pathways of rat somatotrophs.

Selective serotonin reuptake inhibitor treatment of early postnatal mice reverses their prenatal stress-induced brain dysfunction.

Prenatal stress has long-lasting effects on cognitive function and on the hypothalamic-pituitary-adrenal response to stress. We previously reported that the serotonin concentration and synaptic density in the hippocampus were reduced following prenatal stress [Int J Dev Neurosci 16 (1998) 209]. Since serotonin plays a role in the formation and maintenance of synapses, we hypothesized that a neonatal reduction in hippocampal serotonin levels may lead to learning disabilities in prenatally stressed mice. To test this hypothesis, we treated prenatally stressed mice with a selective serotonin reuptake inhibitor in order to normalize their postnatal serotonin turnover levels. What we found was that the oral administration of a selective serotonin reuptake inhibitor to prenatally stressed mice during postnatal weeks 1-3 but not 6-8 normalized their corticosterone response to stress, serotonin turnover in the hippocampus, and density of dendritic spines and synapses in the hippocampal CA3 region. Concomitantly, such treatment partially restored their ability to learn spatial information.

Suppressing effects of bisphenol A on the secretory function of ovine anterior pituitary cells.

We investigated the action of bisphenol A (BPA) on cellular GH release and content, cell number, GHmRNA expression, and concentrations of cellular cyclic AMP ([cAMP]c) and calcium ion ([Ca2+]c) in primary cultured ovine anterior pituitary cells. The following results were found: (1) BPA as well as nonylphenol (NP) at 10(-6) to 10(-3) M significantly and concentration-dependently suppressed basal and GHRH-stimulated GH release, and the cellular GH content, (2) BPA suppressed the cell number in a time- and concentration-dependent manner, (3) 10(-4)M BPA suppressed GHmRNA expression to 68% of control (BPA-free), and abolished GHRH (10(-8) M)-induced increases in [cAMP]c and [Ca2+]c. From these findings we conclude that BPA possesses a suppressing action on GH synthesis and release, and this suppressing action is probably related to impairment of cellular signal transduction systems in ovine anterior pituitary cells.

Rapid suppressing action of insulin-like growth factor-I (IGF-I) on GH release from anterior pituitary cells of goats.

Goat anterior pituitary cells were cultured to investigate the effects of insulin-like growth factor-I (IGF-I), insulin, and growth hormone (GH) on basal and GH-releasing hormone (GHRH)-induced GH release. Changes in cellular Ca2+ concentrations were also assessed to enable discussion of the cellular mechanisms of IGF-I. The cells were cultured for 48 h, and then stimulated with GHRH (10 nmol/l) for 30 min, with or without each test substance. In the control cells, IGF-I (10 and 100 ng/ml) significantly raised the basal, but did not change GHRH-induced GH release, resulting in the abolishment of GH release induced by GHRH in the presence of 100 ng/ml IGF-I. However, there was no significant effect of insulin (10, 100, and 1000 microU/ml) on basal and GHRH-induced GH release. In the cells cultured for 48 h with each test substance but stimulated for 30 min without the test substance, no significant change in the basal and GHRH-stimulated GH release was observed. Regardless of treatment, there was no significant effect on intra-cellular GH content. Analysis with a confocal laser microscope revealed that IGF-I (100 ng/ml) significantly increased the basal, but significantly reduced GHRH (10 nmol/l)-induced increase in cellular Ca2+ concentrations. From these findings we conclude that IGF-I exerts an acute suppressing action on the GHRH-induced GH release, which partly involves changes in cellular Ca2+ metabolism in goat somatotrophs.

Dynamics of microbial populations and strong selection for Cycloclasticus pugetii following the Nakhodka oil spill.

Microbial population changes were monitored immediately after the Nakhodka oil spill accident in January 1997 at the heavily oil-contaminated Mikuni coast along the Sea of Japan. The total cell number was almost stable for one year at 2-5 x 10(5) cells mL(-1), while the relative occurrence of culturable heterotrophs and degraders of oil components such as C-heavy oil, kerosene, and n-tetradecane varied, showing a maximum (>50% of the total) immediately following the accident. Gene amplification and phylogenetic analysis of a dilution culture using C-heavy oil as the sole carbon and energy source revealed that one of the predominant oil degraders at the oil-contaminated coast in 2 weeks after the accident closely resembled the aromatic hydrocarbon decomposer Cycloclasticus pugetii. Microbial community composition in oil-contaminated seawater was estimated at the molecular level using newly developed oligonucleotide probes, probe wash-off curve estimation, and quantitative fluorescence dot-blot hybridization techniques. At two different oil-polluted sites, harbor and intertidal regions, the C. pugetii group was estimated to make up 23-25% of the total Bacteria population, followed by the aliphatic hydrocarbon decomposer Alcanivorax borkumensis, which formed 4-7% of the Bacteria. In incubation experiments using floated oil slick and indigenous microbes collected at the harbor, oil degradation activities were enhanced by the addition of both organic and inorganic nutrients. Significant decreases were found in aromatic and aliphatic hydrocarbon fractions: 54-60% and 22-24% in 2 weeks to 68-77% and 23-32% in 2 months, respectively.

[Determination method of polysorbates in powdered soup by HPLC].

A method for qualitative and quantitative analyses of polysorbates in powdered soup by HPLC was studied. Polysorbates in samples were extracted with acetonitrile after rinsing with n-hexane to remove fats and oils. The extract was cleaned up using a Bond Elut silica gel cartridge (500 mg). The cartridge was washed with ethyl acetate and polysorbates were eluted with a small amount of acetonitrile-methanol (1:2) mixture. The eluate was treated with cobalt thiocyanate solution to form a blue complex with polysorbate. In order to determine polysorbate, the complex was subjected to HPLC with a GPC column, using a mixture of acetonitrile-water (95:5) as a mobile phase, with a detection wavelength of 620 nm. The recoveries of polysorbate 80 added to powdered soups were more than 75% and the determination limit was 0.04 mg/g. When the proposed method was applied to the determination of polysorbates in 16 commercial samples of powdered soup for instant noodles and seasoning consomme, no polysorbates were detected in any sample.

Effects of adenosine 5'-triphosphate and growth hormone on cellular H+ transport and calcium ion concentrations in cloned bovine mammary epithelial cells.

The present experiment was carried out to investigate the effects of exogenous adenosine 5'-triphosphate (ATP) and growth hormone (GH) on cellular H(+) efflux rate (extracellular acidification rate) and Ca(2+) concentration ([Ca(2+)](c)) in cloned bovine mammary epithelial cells (bMEC) raised from the mammary gland of a 26-day-pregnant Holstein heifer. Perifusion of 2-day cultured cells with a medium containing ATP (10, 100 and 1000 micromol/l) for 30 min caused a significant and concentration-dependent increase in the cellular H(+) efflux rate. ATP application (100 micromol/l) caused a transient and large increase in [Ca(2+)](c) in all cells. In contrast, perifusion with a medium containing bovine GH at 10, 50 and 250 ng/ml for 30 min caused a significant decrease in the cellular H(+) efflux rate in a concentration-dependent manner. However, bovine GH application (50 ng/ml) caused a small decrease followed by an increase, in some cases, in [Ca(2+)](c). In bMEC treated with lactogenic hormones (1 microgram/l prolactin, 1 nmol/ml dexamethasone and 5 microgram/ml insulin) for 2 days, the increased H(+) efflux rate induced by ATP was significantly reduced, whereas the negative response induced by GH was inversely and significantly changed to the positive. Treatment of the cells with lactogenic hormones reduced the increase in [Ca(2+)](c) induced by ATP stimulation, while it enhanced the increase in [Ca(2+)](c) induced by GH stimulation. Application of ATP or GH did not cause any significant changes in [pH](c). Treatment with lactogenic hormones enhanced GH receptor (GHR) transcription that was determined by RT-PCR. From these results, we conclude that exogenous application of ATP and GH causes prompt and significant responses in H(+) transport and [Ca(2+)](c) that were significantly changed in the opposite direction by the treatment with lactogenic hormones. The lactogenic hormone treatment also enhanced GHR transcription, which may change post-receptor signal transduction systems for both agents in the bMEC.

Estimation of preservative concentrations in foods and their daily intake based on official inspection results in Japan in fiscal year 1998.

The mean concentration and daily intake of five preservatives were estimated based on the results of an analysis of 89,927 samples of food obtained in official inspections by Japanese local governments in fiscal year 1998. The mean concentration of benzoic acid was 9.5% of the allowable limit, and those of dehydroacetic acid, p-hydroxybenzoic acid, propionic acid, and sorbic acid were 1.5%, 5.7%, 1.7%, and 23.9%, respectively. Daily intake levels of these preservatives per person estimated from the concentration and daily consumption of foods were 6.23 mg, 0.0303 mg, 1.02 mg, 8.10 mg, and 25.0 mg, respectively, and assuming a body weight of 50 kg, the amounts of benzoic acid, p-hydroxybenzoic acid, and sorbic acid consumed were 2.5%, 0.2%, and 2.0% of their acceptable daily intakes, respectively. These values were similar to those obtained based on the results of the official inspections in fiscal years 1994 and 1996.

Characteristics and biodistribution of cationic liposomes and their DNA complexes.

We have developed some novel liposome formulations for gene transfection. The formulations consisting of O,O'-ditetradecanoyl-N-(alpha-trimethyl ammonio acetyl) diethanolamine chloride (DC-6-14) as a cationic lipid, phospholipid and cholesterol showed effective gene transfection activity in cultured cells with serum and in vivo, i.e., intraperitoneal injection in mice. In this report, the physicochemical characteristics and biodistribution of the liposomes containing DC-6-14 (DC-6-14 liposomes) as a drug (gene) carrier for gene therapy were investigated in vitro and in vivo. DC-6-14 liposome-DNA complexes were usually thought to have positive surface charge. However, depending on the ratio of DNA to liposomes, zeta-potential of the complexes became negative. The diameter of the complexes also depended on the DNA-liposome ratio, and showed a maximum when their surface potential was neutral. When biodistribution of the complexes was determined after intravenous injection, positively charged complexes showed an immediate lung accumulation. On the other hand, negatively charged complexes did not show lung accumulation. These results have suggested that biodistribution of the DNA-liposome complexes, prepared with DC-6-14 liposomes, depends on their surface charge. Therefore, some surface modification of DC-6-14 liposomes may improve the biodistribution and hence the targetability of their DNA complexes.

Short-chain fatty acids inhibit the release and content of growth hormone in anterior pituitary cells of the goat.

The effects of short-chain fatty acids (SCFA: acetate, propionate, and butyrate) on growth hormone (GH)-releasing hormone (GHRH)-induced GH secretion from pituitary somatotrophs were assessed on isolated anterior pituitary cells of goats. Cells were cultured in Dulbecco's modified Eagle's medium for 3 days, either in the presence (1, 3, or 10 mM) or in the absence of each SCFA, and then stimulated with GHRH (10(-12) to 10(-7) M) for 30 min, again in the presence of and at the concentration of SCFA used over the previous 3 days. In the cells cultured in the absence of SCFA, the addition of SCFA to the medium during the GHRH stimulation period did not significantly change GHRH-induced GH release. However, in cells cultured in the presence of either propionate (3 or 10 mM) or butyrate (1, 3, or 10 mM), the addition of SCFA to the medium during GHRH stimulation significantly reduced the GHRH-induced GH release. The inhibitory effects of SCFA were dependent on the concentrations of SCFA and were greater for butyrate than for propionate. In the cells cultured in the presence of butyrate, but not in the absence, the total GH production (the sum of the released GH and the remaining GH after stimulation) was also significantly reduced. The GHmRNA expression was reduced in the cells cultured with 10 mM butyrate, whereas it was enhanced by the stimulation with 10(-7) M GHRH. These findings suggest that propionate and butyrate may inhibit GHRH-induced GH release and GH production by caprine anterior pituitary cells.

Renal carcinogenicity of concurrently administered fish meal and sodium nitrite in F344 rats.

The effects of long-term concurrent administration of powdered fish meal and sodium nitrite were examined in F344 rats. A total of 600, 6-week-old rats were divided into 6 male and 6 female groups, each consisting of 50 animals. Rats in groups 1-3 and 7-9 were respectively fed diets supplemented with 64%, 32% and 8% (basal diet) fish meal, and simultaneously given 0.12% sodium nitrite in their drinking water. Groups 4-6 and 10-12 were respectively given 64%, 32% and 8% fish meal and tap water. At the 104th week, all surviving animals were killed and examined histopathologically. Treatment with fish meal dose-dependently increased the incidences and multiplicities of atypical tubules, adenomas and renal cell carcinomas in sodium nitrite-treated males. Females were less susceptible than males for renal tumor induction. In males given the 64% fish meal diet alone, the incidence and multiplicity of atypical tubules were also significantly increased as compared with the 8% fish meal alone case. Nephropathy was apparent in fish meal-treated groups in a clear dose-dependent manner, irrespective of the sodium nitrite treatment, and was more prominent in males than in females. Dimethylnitrosamine was found in the stomach contents after 4-week treatment with 64% fish meal plus 0.12% sodium nitrite, at a level twice that in the 8% fish meal plus 0.12% sodium nitrite group. The results clearly indicate that concurrent administration of fish meal and sodium nitrite induces renal epithelial tumors. Further studies are required to elucidate how nephropathy and nitrosamines produced in stomach contents may contribute to the observed renal tumor induction.

Suppressing effects of short-chain fatty acids on growth hormone (GH)-releasing hormone-induced GH release in isolated anterior pituitary cells of goats.

The involvement of tetrodotoxin-sensitive Na+ channels and receptor-operated nonspecific Ca2+ channels, and the effects of short-chain fatty acids, on growth hormone (GH) release induced by GH-releasing hormone (GHRH) were investigated in cultured and freshly isolated caprine anterior pituitary cells. In 3-d cultured cells in Dulbecco's modified Eagle's medium, an increase in GH release induced by GHRH (10 nmol/l) was moderately, but significantly, reduced by a voltage-sensitive Na+ channel antagonist tetrodotoxin (1 micromol). The GHRH-induced GH increase, which was not affected by a simultaneous addition of a receptor-operated nonspecific Ca2+ channel antagonist tetramethrine (0.1 mmol/l), was significantly reduced by a voltage-sensitive L-type Ca2+ channel antagonist nifedipine (1 micromol/l). Propionate and butyrate at 10 mmol/l, however, not only suppressed basal GH release but also significantly reduced the GH increase induced by 10 nmol/l of GHRH. The inhibitory action of these acids was also reproduced by an addition of beta-hydroxy butyrate (10 mmol/l) and octanoate (10 mmol/l). In freshly isolated and perifused cells, butyrate (10 mmol/l) as well as somatostatin (100 nmol/l) significantly reduced the GH increase induced by GHRH. From these findings we conclude that tetrodotoxin-sensitive Na+ channels and voltage-dependent L-type Ca2+ channels are involved in the cellular mechanism for GHRH-induced GH release, and that short-chain fatty acids such as propionate and butyrate have a direct action on somatotrophs to reduce basal and GHRH-induced GH release, in caprine somatotrophs.

[Inspection of undesignated food color in imported chewing gum].

An unknown color in chewing gum imported from Canada was determined. The color was identified by TLC and HPLC as the trisodium salt of 3-hydroxy-4[(4-sulfopheny)azo]-2,7-naphthalenedisulfonic acid (RS-SA), one of the subsidiary colors of sunset yellow FCF. The concentration of RS-SA in sunset yellow FCF used in the chewing gum was 4.3%

[A qualitative analytical method for nonpermitted food colors by HPLC].

The Ministry of Health and Welfare has been proposed an analytical method for food colors by HPLC. Conditions in the method and modified conditions of the proposed method were applied for permitted and nonpermitted food colors, and relative retention times were obtained. The relative retention times would be a clue of the confirmation of these nonpermitted colors by other method.

Poly(ethylene glycol) derivative of cholesterol reduces binding step of liposome uptake by murine macrophage-like cell line J774 and human hepatoma cell line HepG2.

Liposome uptake by HepG2 human hepatoma cells was investigated in comparison with the uptake by J774 murine macrophage-like cells. HepG2 cells accumulated liposomes (egg yolk phosphatidylcholine (EPC)/Chol; 75/25, diameter 0.2 micron) at 37 degrees C comparably to J774 macrophage-like cells. Confocal microscopic observations revealed that J774 cells internalized EPC/Chol liposomes efficiently but HepG2 cells kept most of the liposomes bound on their plasma membrane surfaces. Poly(ethylene glycol) (PEG)-coated liposomes (0.2 micron) containing poly(ethylene glycol) cholesteryl ether (PEG-Chol) avoided cellular uptake at 37 degrees C by either cell line. In both cell lines, binding of PEG-coated liposomes was lower than that of EPC/Chol liposomes when incubation was carried out at 4 degrees C. To analyze the binding process at 37 degrees C, surface-bound liposomes were removed from the cells by pronase treatment. A reduction of the amount of bound-liposomes on cell surfaces was observed in the case of PEG-coated liposomes. Therefore, PEG-coating reduces direct binding of liposomes to the cell surfaces. The presence of apolipoprotein E (apoE) increased the uptake to EPC/Chol liposomes via its receptor in both cell lines. In contrast, cellular uptake of PEG-coated liposomes was not enhanced by treatment with apoE. Therefore, while apoE-mediated liposome uptake occurs in the case of EPC/Chol liposomes, it does not occur for PEG-coated liposomes; PEG-coating also inhibits protein-mediated binding to the cells. These results further imply that elusion from liver clearance of PEG-coated liposomes is not only due to the reduction of uptake by Kupffer cells but also by hepatocytes when liposomes are small enough to go through the fenestrates of the endothelial lining.

Cholesterol derivative of poly(ethylene glycol) inhibits clathrin-independent, but not clathrin-dependent endocytosis.

The effect of poly(ethylene glycol) cholesteryl ethers (PEG(n)-Chols) with two different numbers of units (n = 50 and 200) in the hydrophilic PEG moiety on cellular endocytic activity was studied on HT-1080 cells. The amphipathic molecules were soluble in aqueous solution. When fluorescein derivatives of PEG-Chols (one fluorescein at the distal end of PEG) were incubated with the cells in culture, the cellular fluorescence was localized at the plasma membrane level and in intracellular vesicles. Fluorescence quantification indicated that for the same external concentration, twice more FPEG(50)-Chol than FPEG(200)-Chol was associated with the cells under the same conditions. Regardless of the length of PEG moiety, PEG-Chols' interaction with cells reduced the endocytic internalization of a fluid phase marker, horseradish peroxidase (HRP) depending on the cell-associated amount. In contrast, internalization of 125I-labeled epidermal growth factor (EGF) through receptor-mediated endocytosis did not change upon incubation with PEG(50)-Chol. The effect of PEG(200)-Chol was also small, since EGF internalization showed a reduction of 10-20%, while at the same concentration as much as 80% of HRP uptake was inhibited. PEG(50)-Chol did not influence the internalization of a larger ligand, 125I-transferrin (Tfn). However, in the presence of PEG(200)-Chol, the uptake of 125I-Tfn decreased remarkably, and yet, PEG(200)-Chol has no influence on the binding and internalization of a monoclonal antibody directed toward the ectodomain of the Tfn-receptor. These results suggested that incorporation of PEG-Chols in the outer monolayer of the plasma membrane specifically inhibited clathrin-independent, but not clathrin-dependent endocytosis.

Stability of the mandibular position in occlusion of mandibulectomy patients with lateral discontinuity defect.

In mandibulectomy patients with lateral discontinuity defect, the mandible is severely deviated and the occlusion is considered to be unstable. A thorough understanding of the mandibular occlusal position of these patients is important to achieve desirable results in their occlusal rehabilitation. This study compared the stability of the mandibular positions in occlusion, when the opening distance or the biting force was changed during mandibular movements, by simultaneously measuring four points on the mandible three-dimensionally. This study indicated that the mandibular positions in occlusion of these patients were extremely unstable as compared with those of the normal subjects and were considerably different from each other when the opening distance or the biting force was changed during mandibular movements.