The effects of denatured major bovine whey proteins on the digestive tract, assessed by Caco-2 cell differentiation and on viability of suckling mice.

Alpha-lactalbumin (α-LA) and ß-lactoglobulin (ß-LG) are contained in bovine milk whey. Chemical and physical treatments are known to alter the conformation of these proteins and we have previously reported that α-LA denatured with trifluoroethanol (TFE) and isolated from sterilized market milk inhibited the growth of rat crypt IEC-6 cells. In the present study, we aimed to evaluate the effects of TFE-treated α-LA and ß-LG on cell growth using cultured intestinal cells and on their safety using a suckling mouse model. First, we investigated the effect of the TFE-treated whey proteins on human colonic Caco-2 cells at various differentiation stages. In the undifferentiated stage, we assessed cell growth by a water-soluble tetrazolium-1 method. The native whey proteins enhanced cell proliferation, whereas the TFE-treated whey proteins strongly inhibited cell growth. We investigated cell barrier function in the post-differentiated stage by measuring transepithelial electrical resistance (TER). Not only native but also the TFE-treated whey proteins increased TER. Next, we evaluated whether the TFE-treated α-LA and ß-LG have adverse effects on healthy suckling mice. No mice given by the TFE-treated samples showed any adverse symptoms. We also performed a safety test using a human rotavirus infected gastrointestinal disease suckling mice model. Even the TFE-treated whey proteins appeared to prevent the development of diarrheal symptoms without any adverse effects. Although we cannot know the effect of long-term ingestion of denatured whey proteins, these results suggest that they have no adverse effects on differentiated intestinal cells and digestive tract, at least in short-term ingestion.

Roles of Lys191 and Lys179 in regulating thermodynamic binding forces of ligands to determine their binding affinity for human histamine H1 receptors.

Docking simulations based on the crystal structure of human histamine H1 receptors have predicted crucial roles of Lys1915.39 and Lys179ECL2, which exist at the entrance of the ligand-binding pocket, in increasing the H1-receptor selectivity for carboxylated second-generation antihistamines via electrostatic interaction. In this study, we evaluated the roles of Lys1915.39 and Lys179ECL2 in regulating the thermodynamic binding forces of non-carboxylated and carboxylated antihistamines that determine their binding affinity for human H1 receptors. The binding enthalpy and entropy of the 3 sets of non-carboxylated and corresponding carboxylated antihistamines (doxepin and olopatadine, desloratadine and loratadine, and terfenadine and fexofenadine, respectively) were estimated using the van't Hoff equation with the dissociation constants obtained from the displacement curves of the non-carboxylated and carboxylated antihistamines against the binding of [3H]mepyramine to the membrane preparations of Chinese hamster ovary cells expressing human H1 receptors at various temperatures, ranging from 4 °C to 37 °C. We found that the affinity for carboxylated antihistamines was lower than that for the corresponding non-carboxylated compounds due to lower enthalpy-dependent electrostatic binding forces and/or entropy-dependent hydrophobic binding forces. Mutations of Lys1915.39 and/or Lys179ECL2 to alanine mostly increased the binding affinity for antihistamines due to a variety of changes in both enthalpy- and entropy-dependent binding forces. These results suggest that Lys1915.39 and Lys179ECL2 may not contribute to selectively increasing the binding affinity for carboxylated antihistamines via electrostatic interaction, but that they can negatively modulate the binding affinity for non-carboxylated and carboxylated antihistamines non-selectively by affecting their electrostatic as well as hydrophobic binding forces.

Differential Regulation of Thermodynamic Binding Forces of Levocetirizine and (S)-Cetirizine by Lys191 in Human Histamine H1 Receptors.

Cetirizine is a zwitterionic second-generation antihistamine containing R- and S-enantiomers, levocetirizine, and (S)-cetirizine. Levocetirizine is known to have a higher affinity for the histamine H1 receptors than (S)-cetirizine; ligand-receptor docking simulations have suggested the importance of the formation of a salt bridge (electrostatic interaction) between the carboxylic group of levocetirizine and the Lys191 residue at the fifth transmembrane domain of human histamine H1 receptors. In this study, we evaluated the roles of Lys191 in the regulation of the thermodynamic binding forces of levocetirizine in comparison with (S)-cetirizine. The binding enthalpy and entropy of these compounds were estimated from the van 't Hoff equation, by using the dissociation constants obtained from their displacement curves against the binding of [³H]mepyramine to the membrane preparations of Chinese hamster ovary cells expressing wild-type human H1 receptors and their Lys191 mutants to alanine at various temperatures. We found that the higher binding affinity of wild-type H1 receptors for levocetirizine than (S)-cetirizine was achieved by stronger forces of entropy-dependent hydrophobic binding of levocetirizine. The mutation of Lys191 to alanine reduced the affinities for levocetirizine and (S)-cetirizine, through a reduction in the entropy-dependent hydrophobic binding forces of levocetirizine and the enthalpy-dependent electrostatic binding forces of (S)-cetirizine. These results suggested that Lys191 differentially regulates the binding enthalpy and entropy of these enantiomers, and that Lys191 negatively regulates the enthalpy-dependent electrostatic binding forces of levocetirizine, contrary to the predictions derived from the ligand-receptor docking simulations.

In vitro measurement of platelet adhesion to intact endothelial cells under low shear conditions.

BACKGROUND: Prediction of thrombus formation at intact arterial walls under low shear flow conditions is clinically important particularly for better prognoses of embolisation in cerebral aneurysms. Although a new mathematical model for this purpose is necessary, little quantitative information has been known about platelet adhesion to intact endothelial cells. OBJECTIVE: The objective of this study is to measure the number of platelets adhering to intact endothelial cells with a focus upon the influence of the shear rate. METHODS: Endothelial cells disseminated in µ-slides were exposed to swine whole blood at different shear rates. Adenosine diphosphate (ADP) was used as an agonist. Adherent platelets were counted by means of scanning electron microscopy. RESULTS: At an ADP concentration of 1 µM, 20.8 ± 3.1 platelets per 900 µm2 were observed after 30-minute perfusion at a shear rate of 0.8 s-1 whereas only 3.0 ± 1.4 per 900 µm2 at 16.8 s-1. CONCLUSIONS: The number of adherent platelets is determined by a balance between the shear and the degree of stimulation by the agonist. At an ADP concentration of 1 µM, a limit to the shear rate at which platelets can adhere to intact endothelial cells is considered to be slightly higher than 16.8 s-1.

A Reactive Oxygen/Nitrogen Species Sensor Fabricated from an Electrode Modified with a Polymerized Iron Porphyrin and a Polymer Electrolyte Membrane.

We have developed an electrochemical reactive oxygen/nitrogen species sensor that can detect superoxide anion radicals (O2-•) and nitric oxide (NO). The reactive oxygen/nitrogen species sensor was fabricated by surface modification of an electrode with polymerized iron tetrakis(3-thienyl)porphyrin (FeT3ThP), and it can detect either O2-• or NO by switching the applied potential. Furthermore, we fabricated a sensor with improved selectivity by coating a Nafion® film onto the poly(FeT3ThP)-modified electrode. An interference current caused by NO2- was seen for the poly(FeT3ThP)-modified electrode, while the interference current was significantly reduced at the Nafion®/poly(FeT3ThP)-modified electrode, leading to improved selectivity for NO detection. The current response at the Nafion®/poly(FeT3ThP)-modified electrode exhibited good linearity in the O2-• and NO concentration ranges 1.3 - 4.1, and 0.5 - 10 µM, respectively. The Nafion®/poly(FeT3ThP)-modified and poly(FeT3ThP)-modified electrodes are highly versatile, because these electrodes can detect either O2-• or NO by switching the applied potential. Since the Nafion®/poly(FeT3ThP)-modified and poly(FeT3ThP)-modified electrodes contain no bio-derived compounds, the reactive oxygen/nitrogen species sensor should be safe even when it is used in vivo.

Effect of Laser Energy and Tip Insertion Depth on the Pressure Generated Outside the Apical Foramen During Er:YAG Laser-Activated Root Canal Irrigation.

OBJECTIVE: The risk of extrusion of irritative irrigants into the extraradicular tissue should be minimized during root canal irrigation. This study aimed to examine the pressure generated outside the apex during Er:YAG laser-activated irrigation (LAI) in comparison with passive ultrasonic irrigation (PUI) and conventional syringe irrigation (SI). BACKGROUND DATA: LAI is reported to be efficient for root canal debridement, whereas the apical pressure generated during LAI has not been investigated in detail. METHODS: Plastic root canal models (apical diameter = 0.40 mm, taper = 6%, and length = 20 mm) were irrigated with water using either LAI with an Er:YAG laser (Erwin AdvEr; 30, 50, or 70 mJ, 10 pps), PUI, or SI. The tip insertion depth was set at 2, 5, or 10 mm from the apex. The pressure generated outside the apical foramen was measured with a pressure sensor connected to the apex of the model. RESULTS: When the tip was positioned at 2 mm and the power setting was changed, the mean maximum pressure values were LAI (30 mJ) = PUI < LAI (70 mJ) < SI (p < 0.05). When LAI was performed at 70 mJ and the tip was positioned at 5 or 10 mm, the pressure values were PUI < LAI = SI (p < 0.05). CONCLUSIONS: LAI generated similar or lower pressure outside the apical foramen compared with SI, irrespective of the laser energy and tip insertion depth examined. However, LAI generated higher pressure compared with PUI.

Soleal vein dilatation assessed by ultrasonography is an independent predictor for deep vein thrombosis after major orthopedic surgery.

BACKGROUND: Deep vein thrombosis (DVT) develops after major orthopedic surgery despite the current use of prophylaxis. DVT frequently develops in the soleal vein (SV) and might develop easily at the site of SV dilatation because of blood flow stasis. However, whether preoperative SV dilatation detected by ultrasonography predicts DVT after major orthopedic surgery remains unknown. OBJECTIVE: We examined whether SV dilatation detected by preoperative ultrasonography predicts DVT after major orthopedic surgery. METHODS: Ultrasonography was performed preoperatively and postoperatively in 243 patients with orthopedic diseases (mean age of 67±13 years, 77% women) who underwent total hip arthroplasty (THA, n=180) or total knee arthroplasty (TKA, n=63). Presence of DVT was diagnosed by ultrasonography and SV diameter ≥10mm was defined as SV dilatation. Patients with preoperative DVT were excluded. RESULTS: Sixty-nine patients (28%) developed postoperative DVT. SV dilatation was found in 24 patients (10%), and 16 (67%) of those patients had postoperative DVT. Multivariate logistic regression analysis showed that female gender [odds ratio (OR): 4.09, p=0.004], TKA (OR: 2.52, p=0.011), and SV dilatation (OR: 6.67, p<0.001), but not presence of comorbidities, medications, or plasma d-dimer value, independently predict postoperative DVT. Subgroup analyses according to the operation site showed that female gender (OR: 3.27, p=0.043) and SV dilatation (OR: 3.72, p=0.022) were independent predictors of postoperative DVT in the THA group. SV dilatation (OR: 12.0, p=0.027) was an independent predictor of postoperative DVT also in the TKA group. CONCLUSIONS: In addition to gender and TKA, SV dilatation detected by ultrasonography is an independent predictor of DVT after major orthopedic surgery. Determination of SV diameter by ultrasonography before major orthopedic surgery is useful for assessing the risk of postoperative DVT.

A comparison between two negative pressure irrigation techniques in simulated immature tooth: an ex vivo study.

OBJECTIVES: This ex vivo study evaluated the irrigation efficacy of a new apical negative pressure system (ANP) in canals with simulated immature teeth, by comparing it to EndoVac (EV) system in terms of smear layer (SL) removal and irrigation extrusion. MATERIALS AND METHODS: Three millimetres of the root end of 40 single canalled lower incisors were resected and decoronated to standardize root canal length. After instrumentation, the specimens were embedded in warm normal saline agar coloured with 1 % acid red and randomly divided into four groups; one control group and three experimental groups. Except in the control group where distilled water was used as irrigant using positive pressure irrigation needle, the canals were irrigated with 6 % NaOCl and 17 % EDTA using the intracanal negative pressure needle (iNP) system, the EV system or 27G open-ended needle under positive pressure (PP). NaOCl extrusion was determined by observing a discolouration of the agar surrounding the root. The SL was evaluated by observing scanning electron microscope images based on a four-level scoring system. RESULTS: Two specimens with irrigant extrusion were observed in the iNP group, which was significantly different (logistic regression, p < 0.05) to EV and PP. There were no significant differences (Kruskall-Wallis test, p > 0.05) among the experimental groups in terms of SL removal, but all were significantly different to the control group. CONCLUSIONS: Irrigation with the iNP could be a viable alternative to EV as an apical negative pressure irrigation technique especially while treating immature teeth. CLINICAL RELEVANCE: ANP in canal cleanliness is recommended to be utilized in treating immature teeth where periapical tissues should be saved and stimulated. The iNP system might have the potential to avoid irrigant extrusion while cleaning the canal till the apical end.

A comparison of head-out mist bathing, with or without facial fanning, with head-out half-body low-water level bathing in humans--a pilot study.

To reduce the risks of Japanese-style bathing, half-body bathing (HBLB) has been recommended in Japan, but discomfort due to the cold environment in winter prevents its widespread adoption. The development of the mist sauna, which causes a gradual core temperature rise with sufficient thermal comfort, has reduced the demerits of HBLB. We examined head-out 42 °C mist bathing with 38 °C HBLB up to the navel to see if it could improve thermal comfort without detracting from the merits of HBLB, with and without the effects of facial fanning (FF). The subjects were seven healthy males aged 22-25 years. The following bathing styles were provided: (1) HBLB-head-out half-body low bathing of 38 °C up to the navel (20 min); (2) HOMB-head-out mist bathing of 42 °C and HBLB of 38 °C (20 min); and (3) HOMBFF-HOMB with FF (20 min). HOMB raised the core temperature gradually. HOMBFF suppressed the core temperature rise in a similar fashion to HOMB. Increases in blood pressure and heart rate usually observed in Japanese traditional-style bathing were less marked in HOMBs with no significant difference with and without FF. The greatest body weight loss was observed after Japanese traditional-style bathing, with only one-third of this amount lost after mist bathing, and one-sixth after HBLB. HOMB increased thermal sensation, and FF also enhanced post-bathing invigoration. We conclude that HOMB reduces the risks of Japanese traditional style bathing by mitigating marked changes in the core temperature and hemodynamics, and FF provides thermal comfort and invigoration.

Deflecting load of nickel titanium rotary instruments during cyclic fatigue.

The dispersion of the lifetime of NiTi instruments, and their deflecting load (DL) changes during cyclic fatigue were investigated. A total of 120 ProFile NiTi rotary instruments were tested using a specially designed cyclic fatigue testing apparatus with three pins. Using these pins, the instrument was bent and rotated at 300 rpm to fracture. DL was recorded using a load cell attached to the central pin. For each sample, the working time was converted to number of cycles to fracture (NCF) and the mean DL (DL(m)) was calculated. The averages of NCF and DL(m) of 120 samples were 584.3±180.5 cycles and 6.44±0.91 N, respectively. All samples showed a sequential decrease in DL during rotation. Based on the present study, it is impossible to estimate the lifetime of a NiTi instrument from NCF. Thus, the change in DL could be an alternative criterion to determine the remaining lifetime.

Variation of telomeric DNA content in gingiva and dental pulp.

OBJECTIVE: The purpose of the present study was to examine the age- and tissue-related variations of the telomere length in gingiva and dental pulp of donor patients. DESIGN: We quantified the relative telomeric DNA content corresponding to the telomere length in gingiva or dental pulp from donor patients (male and female, aged at 19-68) by using genomic DNA of oral tissues in dot-blot hybridization with telomere-specific probe. RESULTS: Telomeric DNA content in the dental pulp showed a negative correlation with the age of donor patients, with smaller telomeric DNA content observed in the elders (p<0.05). In age-matched gingival samples, the average telomeric DNA content was not significantly different between male and female donors. In the age- and gender-matched samples, telomeric DNA content was significantly greater (p<0.001) in dental pulp than in gingiva. CONCLUSION: The telomere length is greater in the dental pulp than in the gingiva. In the dental pulp, but not in the gingiva, telomere length shortens with age.

Dentin strain induced by laser irradiation.

The purpose of this study was to investigate the strain and temperature change in dentin induced by laser irradiation of human root canals with or without water cooling. Twenty-eight palatal roots of extracted human maxillary first molars were used. The strain in dentin was measured using strain gauges fixed on the apical third of the buccal root surface. The temperature change of the root dentin was monitored using thermocouples embedded into dentin near the apex. The root canal was irradiated with Er:YAG or Nd:YAG laser at an output of 1 W (100 mJ, 10 pps) for 5 s. The tip of the irradiation fibre was located 2.0 mm from the root apex. With water cooling, the mean maximum strain induced by Er:YAG laser was significantly lower than that by Nd:YAG laser (P < 0.05). However, without water cooling, no significant difference between the two lasers (P > 0.05) was found. The results suggest that the strain induced by Er:YAG laser irradiation in dentin with water cooling may be minimal, but there still might be a risk of root micro-fracture if cooling is not sufficient.

Urinary trypsin inhibitor suppresses excessive superoxide anion radical generation in blood, oxidative stress, early inflammation, and endothelial injury in forebrain ischemia/reperfusion rats.

OBJECTIVES: To investigate the effects of ulinastatin, a urinary trypsin inhibitor (UTI), on jugular venous superoxide radical (O2⁻·) generation, oxidative stress, early inflammation, and endothelial activation in forebrain ischemia/reperfusion (FBI/R) rats. METHODS: Fourteen Wistar rats were allocated to a control group (n = 7) and a UTI group (n = 7). Throughout the experiments, O2⁻· in the jugular vein was measured by the produced current using a novel electrochemical O2⁻· sensor. Forebrain ischemia was induced by occlusion of the bilateral common caroti darteries with hemorrhagic hypotension for 20 min, followed by reperfusion. In the UTI group, UTI (5 U/g) was administered intravenously immediately after reperfusion. At 60 min after reperfusion, plasma and brain were harvested, and malondialdehyde, high-mobility group box 1 (HMGB1) protein, and intercellular adhesion molecule-1 (ICAM-1) were measured. RESULTS: O2⁻· current increased gradually during forebrain ischemia in both groups. The current increased markedly in the control group immediately after reperfusion but was significantly attenuated in the UTI group after reperfusion. Brain and plasma malondialdehyde, HMGB1, and ICAM-1 were significantly attenuated in the UTI group compared with those in the control group, except for brain HMGB1, which was associated with the amount of O2⁻· generated during FBI/R. DISCUSSION: UTI suppressed jugular venous O2⁻· generation, oxidative stress, early inflammation, and endothelial activation in FBI/R rats. Therefore, UTI might be a useful agent for the therapy of the cerebral ischemia/reperfusion pathophysiology.

Xanthine oxidase is one of the major sources of superoxide anion radicals in blood after reperfusion in rats with forebrain ischemia/reperfusion.

We recently reported that excessive superoxide anion radical (O(2)(-)) was generated in the jugular vein during reperfusion in rats with forebrain ischemia/reperfusion using a novel electrochemical sensor and excessive O(2)(-) generation was associated with oxidative stress, early inflammation, and endothelial injury. However, the source of O(2)(-) was still unclear. Therefore, we used allopurinol, a potent inhibitor of xanthine oxidase (XO), to clarify the source of O(2)(-) generated in rats with forebrain ischemia/reperfusion. The increased O(2)(-) current and the quantified partial value of electricity (Q), which was calculated by the integration of the current, were significantly attenuated after reperfusion by pretreatment with allopurinol. Malondialdehyde (MDA) in the brain and plasma, high-mobility group box 1 (HMGB1) in plasma, and intercellular adhesion molecule-1 (ICAM-1) in the brain and plasma were significantly attenuated in rats pretreated with allopurinol with dose-dependency in comparison to those in control rats. There were significant correlations between total Q and MDA, HMGB, or ICAM-1 in the brain and plasma. Allopurinol pretreatment suppressed O(2)(-) generation in the brain-perfused blood in the jugular vein, and oxidative stress, early inflammation, and endothelial injury in the acute phase of forebrain ischemia/reperfusion. Thus, XO is one of the major sources of O(2)(-)- in blood after reperfusion in rats with forebrain ischemia/reperfusion.

[Fabrication of sensor for reactive oxygen species using gold electrodes modified with electropolymerized porphyrins and application for detection of stress of plants].

Reactive Oxygen Spiecies (ROS) such as superoxide anion radical (.O(2)(-)) act as signals for the activation of stress-response and defense pathways. However, excess ROS generated by perturbing .O(2)(-) homeostasis stimulated many environmental stress, including intense light, drought, temperature stress, herbicides, induce high radical toxicity. Consequently, quantitative analysis of .O(2)(-) is a subject of intense research, since most of ROS are derived from .O(2)(-). Iron meso-tetrakis(3-thienyl)porphyrin complexes were electropolymerized onto a Au wire electrode. The modified Au electrode were applied to .O(2)(-) sensor to detect catalytic oxidation current of .O(2)(-) which was generated as an intermediate during the oxidation of xanthine by catalystic XOD. It was revealed that the sensor was quantitative to measure .O(2)(-). The modified Au electrode were applied to measure oxidation current of .O(2)(-) in mung beans under environmental stress condition. Plants were grown in atmosphere, 25 degrees C and in black darkness. The other plants were exposed to oxygen excess. The oxidation current of .O(2)(-) were increased plants were grown by high-oxygen environment compared to plants were grown at atmosphere. This experiment was indicated that environmental stress such as hyperoxia induced excess .O(2)(-) and Au wire sensor using iron porphyrin complexes is capable of .O(2)(-) detection in plants under environmental stresses.

Spreading of root canal irrigants on root dentine.

The purpose of this study was to evaluate the spreading of root canal irrigants on human root dentine. The spreading of various solutions on dentine surfaces was measured after pre-treatment with various conditional solutions. Seven experimental groups were set based on the combination of conditioning solution-test solution: none-distilled water; none-6% sodium hypochlorite (NaOCl); none-14.3% ethylenediaminetetraacetic acid containing surfactant 0.084% cetrimide (Morhonine); NaOCl-NaOCl; NaOCl-Morhonine; Morhonine-Morhonine and Morhonine-NaOCl. After application of 0.1 mL of conditioning solution for 60 s, 0.5 microL of test solution was placed on dentine surfaces. Spreading of test solution was measured for 480 s to examine the size of the contact area ratio. Those at 300 s were compared among groups using Fisher's Protected Least Significant Difference (P < 0.05). It was concluded that the contact area ratio of Morhonine-NaOCl increased the most significantly among all the experimental groups.

Evaluation of the efficiency of a new file removal system in comparison with two conventional systems.

A novel file-removal system (FRS) was designed to address weak points of conventional file-removal methods. The purpose of this study was to compare file-removal time and dentin removal rates among the FRS, the Masserann kit (Micro-Mega, Besancon, France), and an ultrasonic file-removal method. Ninety extracted mandibular incisors with separated nickel titanium files were divided into 3 groups of 30 teeth each. Groups 1, 2, and 3 had file-removal attempts made by using the Masserann kit, a CPR-7 titanium ultrasonic tip (Obtura-Spartan Corp., Fenton, MO), and the FRS, respectively. Each group had three operators removing the separated files. Pre-/postoperative digital radiographs were downloaded into image analyzing software that calculated the amount of dentin removed. The FRS needed less time and had less dentin loss than the others (p<0.05). There were statistical differences between the experienced operator and less experienced operators regarding the file-removal time and the dentin removal rates (p<0.05).