A strategy for generating aryl radicals from arylborates through organic photoredox catalysis: photo-Meerwein type arylation of electron-deficient alkenes.

Photoinduced reactions of arylboronic acids with electron deficient alkenes under mild organic photoredox catalysis conditions lead to the formation of Meerwein arylation type adducts via the generation of aryl radicals.

The C-terminus of amelogenin enhances osteogenic differentiation of human cementoblast lineage cells.

BACKGROUND AND OBJECTIVES: Amelogenin proteins are the major constituent of developing extracellular enamel matrix and are believed to have an exclusively epithelial origin. Recent studies have suggested that amelogenins might induce the differentiation and maturation of various cells, including cementoblast lineage cells. However, the residues comprising the active site of amelogenin remain unclear. The purpose of this study was to identify the active site region of amelogenin by studying the effects of amelogenin fragments on the osteogenic differentiation of cementoblasts. MATERIAL AND METHODS: Amelogenin fragments lacking the C-terminus (rh163) and N-terminus (rh128) and a fragment consisting of the C-terminal region of rh174 (C11 peptide) were synthesized and purified. Human cementoblast lineage cells were cultured in osteogenic differentiation medium and treated with 0, 10, 100 or 1000 ng/mL of rh163, rh128 or C11 peptide. The mRNA levels of bone markers were examined by real-time polymerase chain reaction analysis. Alkaline phosphatase activity and calcium deposition were also determined. Mineralization was evaluated by alizarin red staining. RESULTS: The osteogenic differentiation of human cementoblast lineage cells was significantly enhanced by treatment with rh128 or C11 peptide, whereas rh163 had no significant effect as compared with untreated controls. CONCLUSIONS: The C-terminus of amelogenin promotes the osteogenic differentiation of human cementoblast lineage cells, indicating the possible utility of C11 peptide in periodontal tissue regeneration.

Amelogenin enhances the osteogenic differentiation of mesenchymal stem cells derived from bone marrow.

Amelogenins are the major constituent of developing extracellular enamel matrix proteins and are understood to have an exclusively epithelial origin. Recent studies have demonstrated that amelogenins can be detected in other tissues, including bone marrow mesenchymal stem cells (MSCs), but the role of amelogenins in MSCs remains unclear. The purpose of this study was to examine the effect of recombinant human full-length amelogenin (rh174) on the osteogenic differentiation of cultured human MSCs. MSCs isolated from human bone marrow were cultured in osteoblastic differentiation medium with 0, 10 or 100 ng/ml rh174. The mRNA levels of bone markers were examined by real-time PCR analysis. Alkaline phosphatase (ALP) activity and calcium concentration were determined. Mineralization was evaluated by alizarin red staining. The mRNA levels of ALP, type I collagen, osteopontin and bone sialoprotein in the MSCs treated with rh174 became significantly higher than those in non-treated controls. Treatment of MSCs with rh174 also enhanced ALP activity and calcium concentration, resulting in enhanced mineralization, as denoted by high intensity of alizarin red staining. In conclusion, the present study showed that rh174 enhances the mineralization accompanied by the upregulation of bone markers in human bone marrow MSCs during osteogenic differentiation, suggesting a certain role of amelogenin in the modulation of osteogenic differentiation of MSCs.

Potentiation of allergic bronchoconstriction by repeated exposure to formaldehyde in guinea-pigs in vivo.

BACKGROUND: Indoor formaldehyde (FA) might worsen allergies and be an underlying factor for the increasing incidence and severity of asthma; the exact mechanism, however, remains unclear. OBJECTIVE: The present study examined the effects of repeated exposure to FA on methacholine- and antigen-induced bronchoconstriction in guinea-pigs in vivo. METHODS: First, non-sensitized guinea-pigs were transnasally treated with 0.1 or 1.0% FA or saline three times a week for 6 weeks, and increasing concentrations of methacholine (50, 100, and 200 microg/mL) were inhaled at 5-min intervals. Second, guinea-pigs pre-treated with transnasal administration of FA or saline using the same protocol were passively sensitized with anti-ovalbumin (OA) serum 7 days before antigen challenge. Third, guinea-pigs were actively sensitized with OA and pre-treated with transnasal administration of FA or saline using the same protocol. The lateral pressure of the tracheal tube (Pao) was measured under anesthesia and artificial ventilation. RESULTS: The antigen-induced increase in Pao in actively sensitized guinea-pigs was significantly potentiated by FA exposure in a dose-dependent manner. The dose-response curve of the methacholine-induced increase in Pao in non-sensitized guinea-pigs or of the antigen-induced increase in Pao in passively sensitized guinea-pigs was not altered by FA exposure. Transnasal administration of FA significantly increased the serum anti-OA homocytotropic antibody titre (IgG) as measured by the passive cutaneous anaphylaxis reaction in actively sensitized guinea-pigs. CONCLUSION: The results suggest that repeated exposure to FA worsens allergic bronchoconstriction through enhancing antigen sensitization.

Effect of a cysteinyl leukotriene antagonist, pranlukast hydrate, on acetaldehyde-induced bronchoconstriction in asthmatic patients.

Acetaldehyde is a main factor of alcohol-induced asthma. We previously reported that the cysteinyl leukotriene (cys-LT) receptor antagonist, pranlukast hydrate, inhibits acetaldehyde-induced airway hyperresponsiveness in guinea pigs. The purpose of this study was to evaluate the involvement of cys-LT on bronchial responsiveness to acetaldehyde in asthmatic patients. We investigated the bronchial response to inhalation of acetaldehyde in 10 asthmatic patients, who were treated with placebo or pranlukast hydrate (225.5 mg), a cys-LT receptor antagonist, twice a day for 1 wk using a double-blind, randomized, placebo-controlled, cross-over design. Although a remarkable improvement of acetaldehyde bronchoconstriction was observed in 3 out of 10 subjects, PC(20)-AcCHO values were identical between placebo [12.0 (GSEM, 1.192) mg/ml] and pranlukast [14.7 (GSEM, 1.245) mg/ml] groups. The changes in bronchial responsiveness to acetaldehyde were similar in the six patients who had never experienced alcohol-induced asthma and the four who had. In conclusion, cys-LTs are not involved in acetaldehyde-induced bronchoconstriction.

Effect of thromboxane A2 (TXA2) synthase inhibitor and TXA2 receptor antagonist alone and in combination on antigen-induced bronchoconstriction in guinea pigs.

Thromboxane A2 (TXA2) causes bronchoconstriction and bronchial hyperresponsiveness. Two types of TXA2 modifiers, one synthase inhibitor and one receptor antagonist, are widely used for the treatment of asthma in Japan. Although the target of TXA2 modifiers is to inhibit bioactivity of TXA2, the pharmacological properties are somewhat different between these drugs. We studied the inhibitory effects of the TXA2 synthase inhibitor CS-518 and the TXA2 receptor antagonist S-1452 alone and in combination on antigen-induced bronchoconstriction in passively sensitized guinea pigs treated with diphenhydramine. Both CS-518 and S-1452 inhibited the antigen-induced bronchoconstriction dose-dependently with the plateau. The combination of these drugs at the maximal inhibitory doses did not have any more effect compared with each single dosing. The combination at the submaximal doses tended to show an additive effect, but the effect was not significant. These findings suggest that other prostanoids such as PGE2, PGI2, PGD2 and PGF2alpha may not take an important role in the antiasthmatic effects of TXA2 modifiers.

Sensory neuropeptides are not involved in acetaldehyde-induced bronchoconstriction in guinea-pigs.

1. Alcohol-induced asthma is characterized by worsening of asthmatic symptoms after alcohol ingestion. Acetaldehyde, a metabolite of ethanol, is thought to be a main factor of alcohol-induced asthma. Although airway sensory nerves are known to be activated in asthma, there have been no studies investigating the role of tachykinins in the airway response to acetaldehyde. The purpose of the present study was to evaluate the involvement of tachykinins on acetaldehyde-induced bronchoconstriction in guinea-pigs. 2. After capsaicin desensitization or intravenous administration of 10 mg kg(-1) FK224, a NK1 and NK2 dual antagonist, airway responses to ascending doses (2.5-20 mg ml(-1)) of inhaled acetaldehyde was examined using a modified Konzett-Rössler method in guinea-pigs. 3. Inhalation of acetaldehyde induced bronchoconstriction in a dose-dependent manner. The FK224 failed to reduce the acetaldehyde-induced bronchoconstriction. Pretreatment with capsaicin did not alter the bronchoconstriction induced by acetaldehyde at a dose of 2.5-10 mg ml(-1). Pretreatment with capsaicin slightly, but significantly, inhibited bronchoconstriction induced by 20 mg ml(-1) of acetaldehyde. 4. The present results suggest that tachykinins are not involved in acetaldehyde-induced bronchoconstriction in guinea-pigs.

Syndrome of inappropriate secretion of antidiuretic hormone associated with idiopathic normal pressure hydrocephalus.

A 79-year-old woman suffering from urinary incontinence and unsteady gait was diagnosed as having idiopathic normal pressure hydrocephalus (NPH) with hyponatremia due to the syndrome of inappropriate secretion of antidiuretic hormone (SIADH). The concentration of antidiuretic hormone was high while the plasma osmolality was low in the presence of concentrated urine during the episodes of hyponatremia. Magnetic resonance imaging (MRI) of the head showed enlargement of the third and lateral ventricles. After ventriculoperitoneal shunt surgery, the symptoms of NPH and hyponatremia improved. It may be possibly explained that mechanical pressure on the hypothalamus from the third ventricle is responsible for hyponatremia.

Development of a stopped flow reactor for the rapid, high sensitivity measurement of endotoxin concentration in dialysate.

Continuous measurement of endotoxin concentration in dialysate, using a separated endotoxin-specific limulus reagent, promises rapid measurement without the complex operating procedures of the limulus reagent. To achieve high sensitivity measurements in a short period of time, an improved system featuring stopped-flow operation was developed. To prevent dispersion of the limulus reagent and residence of reacting solution containing the limulus reagent in the system reactor, the circuit in the reactor was changed from a coil configuration to a straight line, and its length was reduced. An endotoxin test solution was supplied at 760 microl/min, into which 40 microl of limulus reagent was pulse-injected. Flow was stopped at the point where the test solution entered the reactor. After the completion of the reaction, the solution was passed through a spectrophotometer and the relationship between reaction time and absorbance was determined. Peak tailing was less than that obtained by the conventional technique, good correlation was obtained from the peak height, and a decrease in sensitivity caused by broadening of the peak was suppressed. The lower detection limit of dialysate was 100 endotoxin units (EU)/L at a reaction time of 20 minutes, and 60 EU/L at 30 minutes. Change from the monitoring system to stop-flow operation made high sensitivity monitoring of endotoxin concentration with a short reaction time possible.

Developmental changes in Asn-linked neutral oligosaccharides in murine cerebrum.

The changes in Asn-linked oligosaccharide composition in the murine cerebrum during development have been examined by high-performance liquid chromatography (HPLC) and electrospray ionization mass spectrometry (ESI-MS). The oligosaccharides, obtained from murine cerebrum in several developmental stages, were separated by HPLC on anion-exchange and reverse-phase columns. We found that two Asn-linked oligosaccharides, designated oligosaccharide I and oligosaccharide II, had their expression changed during postnatal development. Whereas oligosaccharide I was reduced during brain development, oligosaccharide II was increased. The structures of oligosaccharides I and II were analyzed by ESI-MS and sequential exoglycosidase digestions. Judging from the molecular and fragment ions in each oligosaccharide, the oligosaccharide I was composed of 5Hex+2HexNAc+ABOE (MW 1467.2) and the oligosaccharide II was 3Hex+4HexNAc+DoHex+ABOE (MW 1695.2). The results of sequential exoglycosidase digestion indicated that the oligosaccharide I was an oligomannose type saccharide and the oligosaccharide II was a biantennary complex type saccharide including fucose. The proposed structures are shown below. These results offer an important clue to the role of Asn-linked oligosaccharides associated with development of the central nervous system.

Development of a glucose sensor with on/off control of enzyme activity without the effects of protein adsorption.

The main factor mitigating against the realization of a hypodermically inserted glucose sensor for an artificial pancreas is the change in response current due to fibroblast adhesion and protein adsorption to the sensor surface. To overcome this problem, we have developed a method whereby the activity of glucose oxidase (GOD) fixed on the membrane of the sensor surface is switched on and off, and measurements are made during a transient state in which the glucose concentration gradient within the GOD membrane is small. Measuring in a transient state while GOD activity is being controlled, a correlation was observed between glucose concentration and response current in a phosphate buffer solution. Calibration curves of response current against glucose concentration in aqueous solutions of human serum albumin and in phosphate buffer solution were then compared using the transient method and a steady state method without control of GOD activity. In addition, glucose concentration was measured in bovine plasma for 480 min, and the time courses of the response currents for the transient and steady state measurements were compared. It was found that in both experiments the response current decreased greatly under steady state measurement as a result of protein adsorption, but during the transient measurement, response current was virtually unchanged. By measuring glucose concentration in the transient state while controlling GOD activity, it is possible to inhibit the effects of protein adsorption.

Development of a new endotoxin sensor with intermittent injection of limulus reagent for continuous monitoring of dialysate fluid.

This report describes a method of continuously, stably, and inexpensively measuring endotoxin (ET) concentrations in dialysate fluid using an ET sensor with intermittent injection of limulus reagent. An ET solution simulating dialysate fluid was sampled in a single tube at a flow rate of 260 microliters/min and mixed with 30 microliters of limulus reagent intermittently injected into the tube. The absorbance of the solution was measured after the limulus reaction at 313 or 318 degrees K at 26 min. A good linear relationship (r = 0.98) between peak area of absorbance and ET concentration at ET concentrations ranging from 0 to 0.12 endotoxin unit (EU)/ml was obtained, using a spectrophotometer with a cell volume of 8 microliter. The baseline rose after the measurements were taken because the cell volume was so small that the cell was stuffed with gel. A good linear relationship (r = 1.00) at ET concentration of 0.1-0.25 EU/ml was also obtained, and the baseline was unchanged after measurements, using a metal free spectrophotometer with a cell volume of 420 microliters. In conclusion, to measure ET concentrations below 0.1 EU/ml, the cell volume of a metal free spectrophotometer should be minimal.

A novel method for the continuous measurement of endotoxin concentration.

Conventional limulus amebocyte lysate tests involved procedures to prevent contamination by atmospheric endotoxins. To address this problem, the authors have proposed a technique in which the sampling, reagent mixing, and reaction steps are carried out consecutively in a single tube. Since reagents do not come in contact with the atmosphere, the new technique promises stable determination of the concentration of endotoxins in dialysate fluid. An aqueous solution of endotoxin simulating dialysate fluid was sampled in a silicone rubber tube from a sterile infusion bag, then mixed with an indicator in the same tube. After reaction at 310 K, measurements were made of light absorbance at 405 nm and its linearity with endotoxin concentration was determined. Results showed a high degree of linearity (correlation coefficient of not less than 0.99) at endotoxin concentrations of 0-15 pg/ml. The time for the reaction was shortened to 12 min, in which case the response time was 15 min. It is suggested that this new test for determining endotoxin concentration using limulus amebocyte lysate reagent, in which all three steps--sampling, mixing, and reaction--proceed continuously in a single tube, offers higher reliability, greater ease of operation, and shorter response time than conventional tests.

Microlunatus phosphovorus gen. nov., sp. nov., a new gram-positive polyphosphate-accumulating bacterium isolated from activated sludge.

Polyphosphate-accumulating bacteria that were previously isolated from activated sludge and exhibited high phosphate removal activity were studied taxonomically and phylogenetically. These organisms were gram-positive, coccus-shaped, aerobic chemoorganotrophs that had a strictly respiratory type of metabolism in which oxygen was a terminal electron acceptor. They accumulated large amounts of polyphosphate under aerobic conditions. The major quinone was menaquinone MK-9(H4). The cell wall peptidoglycan contained LL-diaminopimelic acid. The guanine-plus-cytosine content of the DNA was 67.9 mol%. Our isolates were similar phenotypically and chemotaxonomically to Luteococcus japonicus, which was proposed recently as a new genus and species. However, our isolates differed from L. japonicus in cellular fatty acid composition and some other traits. A phylogenetic analysis based on 16S rRNA sequences showed that our isolate differ from the genus Luteococcus and other genera belonging to the high-G+C-content gram-positive group. Accordingly, we concluded that our strain NM-1T (T = type strain) should be assigned to a new genus and species, for which we propose the name Microlunatus phosphovorus.

A new procedure for stable quantification of endotoxin in dialysate fluid using Limulus reagent.

For reliable determination of endotoxin, the activation of enzymes in lysate before measurement should be prevented, and the authors have designed a new procedure to effect this by dissolving the enzymes in lysate in a buffer solution of low pH. A given amount of the enzymes in lysate was dissolved in a lower pH buffer solution (pH 6.1-6.3) and the substrate was dissolved in a higher pH buffer solution (pH 8.0). After standing for 0-24 hr, both solutions were mixed with the sample solution. Data on blank absorbance and calibration line slope obtained by the new procedure were compared with those obtained by the conventional procedure. In the conventional procedure, blank absorbance increased with standing time, reaching approximately seven times the initial value in 24 hr, whereas in the improved procedure, it increased by 1.5 times at a standing time of 3 hr, after which it was independent of standing time. The change in slope of the calibration line with standing time was more gradual in the improved procedure than in the conventional procedure. The authors conclude that the activation of enzymes in lysate can be prevented by dissolving the enzymes in a buffer solution of low pH, and that this procedure is effective for long-term monitoring of endotoxin concentration.

[Effects of self-efficacy and outcome expectation on observational learning of altruistic behavior].

The purpose of the present study was to examined the hypothesis that the effects of observational learning of altruistic behavior are related to personal viewpoint, self-efficacy, outcome expectation, and other factors. One hundred and fifty-five fourth-grade school children were assigned randomly to four conditions; distress viewpoint, outcome expectation, altruistic behavior, or non-observational control. After observational learning, the students were immediately administered the generalization test of altruistic behavior, empathy, reward and punishment expectations, and rating tests of self-efficacy belief. It was found that; (a) altruistic behavior and outcome expectation conditions had significant learning effects, (b) self-efficacy was able to predict altruistic response in the altruistic behavior viewpoint condition, and (c) from an outcome expectation viewpoint, altruistic behavior toward peers and adults related to punishment expectation, whereas social sharing was related to reward expectation. According to these findings, an observational learning model of altruistic behavior was proposed.

[The effects of the viewpoints and observers' traits on the observational learning of altruistic behavior].

This study was to examine the changes of altruistic responses through observational learning (OL) in elementary school children (4th graders) with the conditions of the different viewpoints and observers' traits. The OL was the symbolic modeling of altruistic behavior by presentation of pictures with narration. All the stories had the same situation, but differed whether the actor behaved himself in an altruistic fashion to the object, or not. Observers' viewpoints were determined by the instruction: they were told to observe the story as if they were one of the characters and to pay attention to emotional responses or behavior of the character. The emphasis of each story corresponded to the viewpoint. Out of these eight combined experimental conditions six were performed. Traits were concerned with observers' impression, empathy, and evaluation of the story. The response measure was the change of rating between pre- and post-questionnaires through observation. The effect of OL was higher significantly when the viewpoint directed to the characters' emotion. Observers who reacted strongly to empathy and impression items showed higher altruistic changes.

[Modeled events and observers' traits underlying observational learning of altruistic behavior].

The present experiments investigated the changes of altruistic responses through observational learning (OL) in elementary school children (ages 10 and 11) with relation to the characteristics of the modeled events and the observers' traits. The former included certain categories of observed materials and the methods of presentation. The latter included subject's impression, empathy for, and evaluation of these materials. The OL was the symbolic modeling of altruistic behavior by presentation of pictures with narration. The response measures were the rating of pre- and post-questionnaires on the altruistic stories. The main result of Experiment I showed that subjects responded selectively to each pretest questionnaire, and that the observational learning was most effective on the similar kind of behavior type. The generalization of OL to other items of categories was also observed. In Experiment II, the combined effect of a pair of observed materials (positive or negative altruistic content) was examined. The effect of OL was highest when the material presented was a P-N pair, lowest when it was a N-N pair. The observers' traits in empathy, impression, and evaluation on observed materials were positively correlated to the effect of OL.