Local Controlled Release of Polyphenol Conjugated with Gelatin Facilitates Bone Formation.

Catechins are extensively used in health care treatments. Nevertheless, there is scarce information about the feasibility of local administration with polyphenols for bone regeneration therapy, possibly due to lack of effective delivery systems. Here we demonstrated that the epigallocatechin-3-gallate-conjugated gelatin (EGCG/Gel) prepared by an aqueous chemical synthesis using 4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-morpholinium chloride (DMT-MM) gradually disintegrated with time and facilitated bone formation in a critical size defect of a mouse calvaria. Conjugation of EGCG with the Gel generated cross-linking between the two molecules, thereby leading to a retardation of the degradation of the EGCG/Gel and to a delayed release of EGCG. The prepared EGCG/Gels represented significant osteogenic capability compared with that of the uncross-linked Gel and the cross-linked Gel with uncombined-EGCG. In vitro experiments disclosed that the EGCG/Gel induced osteoblastogenesis of a mouse mesenchymal stem cell line (D1 cells) within 14 days. Using fluorescently-labeled EGCG/Gel, we found that the fraction of EGCG/Gel adsorbed onto the cell membrane of the D1 cells possibly via a Gel-cell interaction. The interaction might confer the long-term effects of EGCG on the cells, resulting in a potent osteogenic capability of the EGCG/Gel in vivo. These results should provide insight into local controlled release of polyphenols for bone therapy.

Severe aconite poisoning successfully treated with veno-arterial extracorporeal membrane oxygenation: A case report.

BACKGROUND: Most species of aconite contain highly toxic aconitines, the oral ingestion of which can be fatal, primarily because they cause ventricular arrhythmias. We describe a case of severe aconite poisoning that was successfully treated through veno-arterial extracorporeal membrane oxygenation (VA-ECMO) and in which detailed toxicological analyses of the aconite roots and biological samples were performed using liquid chromatography-tandem mass spectrometry (LC-MS/MS). CASE SUMMARY: A 23-year-old male presented to the emergency room with circulatory collapse and ventricular arrhythmia after ingesting approximately half of a root labeled, "Aconitum japonicum Thunb". Two hours after arrival, VA-ECMO was initiated as circulatory collapse became refractory to antiarrhythmics and vasopressors. Nine hours after arrival, an electrocardiogram revealed a return to sinus rhythm. The patient was weaned off VA-ECMO and the ventilator on hospital days 3 and 5, respectively. On hospital day 15, he was transferred to a psychiatric hospital. The other half of the root and his biological samples were toxicologically analyzed using LC-MS/MS, revealing 244.3 mg/kg of aconitine and 24.7 mg/kg of mesaconitine in the root. Serum on admission contained 1.50 ng/mL of aconitine. Beyond hospital day 2, neither were detected. Urine on admission showed 149.09 ng/mL of aconitine and 3.59 ng/mL of mesaconitine, but these rapidly decreased after hospital day 3. CONCLUSION: The key to saving the life of a patient with severe aconite poisoning is to introduce VA-ECMO as soon as possible.

Amatoxin poisoning caused by Galerina sulciceps, a species with no prior record of identification in Japan: a case report.

A 60-year-old man presented with acute gastroenteritis, hypovolemic shock, acute renal failure (BUN/Cr, 56.7/4.24 mg/dl), and aspiration pneumonia. The previous day, he ingested 30 caps of mushrooms of an unknown species. The patient was treated with a massive intravenous infusion, renal replacement therapy, and antimicrobial agents. Late-onset mild liver injury peaked on day 11 (AST/ALT, 62/67 IU/l). Acute renal failure improved once before worsening, with the worst symptoms on day 19 (BUN/Cr, 99/6.61 mg/dl). Thereafter, the patient showed gradual improvement, and renal replacement therapy was discontinued on day 23. His general condition improved fully and he was transferred to another hospital for rehabilitation on day 47. The mushrooms were later identified as Galerina sulciceps by the Basic Local Alignment Search Tool, and toxicologic analysis using liquid chromatography-tandem mass spectrometry revealed an average of 85 ppm α-amanitin and 330 ppm ß-amanitin in the tissue of the mushrooms brought in by the patient's family. Galerina sulciceps is distributed mainly in tropical and subtropical regions of Southeast Asia and had never been identified before in Japan. The heat of fermentation generated by the thick layer of wood chips on the ground or global warming may have contributed to its growth in Japan. Interestingly, our patient did not have liver dysfunction, which is one main and typical amatoxin poisoning symptom. Variation in clinical presentation may be attributed to the different ratios of α-amanitin to ß-amanitin in different mushroom species.

Cell viability of fine powders in hybrid resins and ceramic materials for CAD/CAM.

Resin blocks and ceramic blocks for CAD/CAM crowns were cut into powders and separated into three particle size groups. Oxidative stress and cell viability were measured in 3T3 and FRSK cells. The results of cytotoxicity tended to be slightly higher for resin than for ceramics. The values also increased as the particle size decreased in the powders. In addition, incorporation into cells was frequently observed under SEM, suggesting that the particle size of easily incorporated dust is different among cell types. Fluorescence-activated cell sorter (FACS) showed an increase in apoptosis and a decrease in cell viability in most of the sample groups compared to the control group. Hematoxylin and eosin staining of the cells showed deep staining of the nuclei in the sample groups. It was found that oxidative stress cell viability and apoptosis appeared differently depending on the size of the particles and the type of cells.

Comparison of cell viability between mouse-derived ES-D3 cells and Balb/c 3T3 cells using denture-base lining materials.

The study was done to compare cell viability between ES-D3 and Balb/c 3T3 cells, and evaluate the difference in cell viability between these cell lines using denture-base lining materials for prosthetic dentistry. To compare the cytotoxicity, three acrylic and three silicone dental materials were used. The cell viability was examined by MTT and lactate dehydrogenase (LDH) methods. The cell viability immediately after malaxation or light irradiation was lower only for the acrylic materials in 3T3 cells, and for both silicone and acrylic materials in ES-D3 cells. However, the cell viability determined 24 h after malaxation or light irradiation by the MTT and LDH methods did not significantly differ between samples. It was observed that ES-D3 cells are more sensitive depending on the type of material. The results suggest that ES-D3 cells can be used as in vitro systems for conducting biosafety assessment to predict embryotoxicity.

[Pulmonary Langerhans cell histiocytosis in identical twins].

Case 1 was a 22-year-old younger sister of identical twins who had smoked 10 cigarettes per day for 4 years since she was 18. She was admitted to our hospital due to dyspnea on exertion, dry cough and bilateral diffuse reticulonodular infiltrates on chest computed tomography (CT). Histological findings of transbronchial lung biopsy demonstrated nodular lesions consisting of spindle-shaped mononuclear cells. Immunohistochemical studies revealed that the cytoplasm of these mononuclear cells showed positive reactions to CD1a. Based on these findings, she was given a diagnosis of pulmonary Langerhans cell histiocytosis (LCH). She reduced the number of cigarettes smoked to 1 cigarette per week. After 6 months, her respiratory symptoms and radiographic abnormalities had improved. Case 2 was the elder twin sister of case 1. She had smoked 5 cigarettes per day for 4 years since she was 18. She presented no respiratory symptoms. However, as the diagnosis in case 1 was LCH, a chest CT was taken which revealed 3 small nodular lesions. After six months, there were no significant changes on chest CT. At the time of writing, she is 23-years-old and continues to smoke, however, has no respiratory symptoms.

[A case of diffuse alveolar hemorrhage occurring during the course of rheumatoid arthritis].

A 70-year-old woman, who underwent treatment with prednisolone and methotrexate for rheumatoid arthritis, was admitted to our hospital due to rapidly progressive dyspnea. A chest CT scan showed diffuse ground-glass opacities and reticulonodular shadows in both lungs. Intubation was performed due to severe hypoxia. The color of the bronchoalveolar lavage, using three sequential aliquots, became progressively more reddish, suggesting alveolar hemorrhage. Based on this, we made a diagnosis of diffuse alveolar hemorrhage. To the best of our knowledge, few studies have reported cases of diffuse alveolar hemorrhage associated with rheumatoid arthritis as an underlying disease. We consider that diffuse alveolar hemorrhage may occur as a complication of rheumatoid arthritis.

Disappearance of angina attacks after administration of mepolizumab.

A 75-year-old woman with bronchial asthma and angina presented with dyspnea. She was using nifedipine, nitroglycerin, pranlukast and salmeterol plus fluticasone propionate inhalations. We diagnosed her with severe bronchial asthma. Oral steroids prescribed for the asthma symptoms had only temporary effects. Therefore, we additionally prescribed mepolizumab; the bronchial asthma improved and the angina attacks disappeared. However, after the patient discontinued treatment, the bronchial asthma and angina attacks recurred, which were treated with nitroglycerin. When she returned 12 weeks later, we resumed mepolizumab administration; the bronchial asthma improved and the angina attacks disappeared again. Hence, we concluded that her angina was due to eosinophilia since the angina attacks disappeared with the mepolizumab therapy. Despite limitations such as lack of arterial blood gas data, perfusion scan, gastrointestinal fiberscopy and Aspergillus skin and precipitation tests, we believe that this is the first report to suggest mepolizumab treatment for angina attacks.

[A case of lung adenocarcinoma keeping complete remission by treatment with gefitinib].

A 74-year-old smoking man was admitted because of lung cancer with metastatic brain tumor. Examinations for lung cancer and brain tumor showed adenocarcinoma (clinical stage IV). Four courses of chemotherapy were not effective. Administration of gefitinib started from June 2003, but was halted after only two months because of skin rash. Lung tumor disappeared on chest computerized tomography in January 2004 and no recurrence has been detected as of March 2006. This is a rare male super-responder to gefitinib.

Verification of the airway securing capacity of a life jacket in a flood disaster.

OBJECTIVE: To verify the life jacket's airway-securing capacity to keep the nose and mouth out of the water in a flood disaster. DESIGN: Physical experiment. SETTING: University of Miyazaki. MATERIALS AND METHODS: We experimented with an open channel in which we created a wave and running water. In the channel, we observed a dummy attached to a balloon as a buoyant body. We used 170-, 220-, and 320-ml balloons. Experiment 1, we reproduced a tsunami. We created three sizes of wave. Experiment 2, we reproduced running water. When we installed an obstacle at the channel bottom, the current that submerged the dummy occurred downstream of the obstacle. We measured the submergence time of the dummy. RESULTS: For the medium-sized wave, the submergence time decreased as the balloon volume increased. For the largest wave, the submergence time was the shortest for a balloon of 220 ml, not 320 ml. Experiment 2, the submergence time decreased as the balloon volume increased. However, a dummy attached to a 320-ml balloon remained downstream of the obstacle. CONCLUSIONS: The biggest drawback is that the parameters of this study are not based on real-world experience. Therefore, if an evacuee with a life jacket is submerged in a flood disaster, we cannot easily decide whether the buoyancy is appropriate because in some situations, buoyancy adversely affected airway security. If we could decide buoyancy based on specific disaster conditions, the airway-securing capacity of a life jacket would improve.

Simultaneous determination of zonisamide, carbamazepine and carbamazepine-10,11-epoxide in infant serum by high-performance liquid chromatography.

This study developed a simple method for the simultaneous determination of zonisamide (ZNS), carbamazepine (CBZ) and its active metabolite, carbamazepine-10,11-epoxide (CBZE) in infant serum using reversed-phase high-performance liquid chromatograph (HPLC). The method involves a single-step protein precipitation procedure that uses no solid-phase or liquid-liquid extraction. The HPLC separation was carried out on a Cadenza CD-C18 column (3 microm, 4.6 mm x 150 mm) with potassium phosphate buffer (pH 4.6; 25 mM)-methanol-acetonitrile (65:20:15 (v/v/v)) as a mobile phase at a 1.0 ml/min flow rate: ZNS was detectable using a UV detector at 235 nm, and both CBZ and CBZE were at 215 nm. The quantification limits were established in accordance with each therapeutic range at 2.5 microg/ml for ZNS, 0.5 microg/ml for CBZ, and 0.25 microg/ml for CBZE. The respective coefficients of variation were 1.3-6.0% and 2.2-7.7% for the intra- and inter-assay.

In vitro embryotoxicity testing of metals for dental use by differentiation of embryonic stem cell test.

We examined embryotoxicity using the embryonic stem cell test (EST) protocol. Tests were conducted using standard reagents for the atomic absorption measurement of 11 metal ions, silver, cobalt, chromium, copper, mercury, nickel, palladium, antimony, tin, vanadium, and zinc from among metals comprising dental alloys. In addition, for four metals like silver, cobalt, chromium, and nickel, the tests were also conducted using a test solution extracted from powder in the cell culture medium. The embryotoxic potential was obtained from a biostatistics-based prediction model, which was calculated from three endpoints, the ID50, IC50ES and IC(50)3T3. Data with the standard reagents showed that chromium and mercury ions corresponded to class 3, that is, having a strong embryotoxicity, while antimony, tin, and vanadium ions exhibited a weak embryotoxicity. The other metal ions demonstrated no embryotoxicity. On the other hand, when extracts of metal powder in cell culture solutions were used, silver exhibited a weak embryotoxicity while all other metals exhibited no embryotoxicity. In the future, it will be important to clarify the embryotoxicity of the many dental materials that are in use today. In addition, it is necessary to develop substances to ensure they have no toxicity before use in dental applications.

Development of technique for in vitro embryotoxicity of dental biomaterials.

The Embryonic Stem Cell Test (EST) developed in Germany in 1997 is known as a screening test method capable of predicting the presence of unknown chemicals influencing normal human development. Firstly, we investigated the embryotoxicity of 24 types of monomer including dental monomers and dental alloy-component metal elements using this test. Monomers including Bis-GMA contained in base resin of composite resin exhibited weak embryotoxicity, and the toxicity level varied among dental alloy-component metal elements. It was clarified that metal ions eluted from currently sold dental alloys show no embryotoxicity. Then, we investigated a method that also considers human metabolic activity, which is not possible with the EST, in the results of embryotoxicity. In addition, an evaluation method using a hybrid culture system for hepatocytes and mouse ES cells and a method using oviduct or uterus cells for feeder cells were also investigated.

[Development of Determination Method of Ipfencarbazone in Agricultural Products, Livestock Products and Seafood by LC-MS/MS].

A method for the determination of ipfencarbazone in agricultural products, livestock products and seafood by LC-MS/MS was developed. Agricultural samples were extracted with acetone. An aliquot of crude extract was partitioned with n-hexane and sat. sodium chloride solution. Clean-up was performed using GC/PSA and C18 cartridges. In the case of livestock products and seafood, samples were extracted with a mixture of acetone and n-hexane, and the organic layer was collected. After acetonitrile-hexane partitioning, the extract was cleaned up using PAS and C18 cartridges. The gradient LC separation was performed on a C18 column with acetonitrile-water containing acetic acid as a mobile phase, and MS with positive ion electrospray ionization was used for detection. The average recoveries (n=5) of ipfencarbazone from 16 kinds of agricultural products, livestock products and seafood spiked at the MRLs or at the uniform limits (0.01 ppm) were 73-101%, and the relative standard deviations were 1.3-5.1%. The limit of quantitation of the developed method was 0.01 mg/kg for ipfencarbazone.

Effects of dehydroepiandrosterone on oleic acid accumulation in rat liver.

The purpose of the present study was to determine whether dehydroepiandrosterone (DHEA) affects de novo fatty acid synthesis, oleic acid formation, fatty acid oxidation, and very low density lipoprotein (VLDL) secretion, in relation to the accumulation of lipid containing oleic acid, in rat liver. The rates of hepatic de novo synthesis of both fatty acid and monounsaturated fatty acid, determined by incorporation of 3H from 3H(2)O into fatty acid, were increased markedly when rats were fed a diet containing 0.5% (w/w) DHEA for 14 days. The treatment of rats with DHEA also enhanced the conversion of [14C]stearic acid into oleic acid in the liver in vivo. DHEA did not suppress fatty acid degradation in the liver. Namely, mitochondrial palmitic acid oxidation in liver homogenates and isolated hepatocytes was increased approximately 1.9- and 5-fold, respectively, in DHEA-treated rats. Peroxisomal palmitic acid oxidation in isolated hepatocytes from rats treated with DHEA, however, was not significantly different from that of the control, despite the fact that peroxisomal degradation of palmitic acid in the liver homogenates was increased markedly. The rate of hepatic VLDL secretion in DHEA-treated rats was decreased markedly. These results indicate that the elevation of the hepatic fatty acid content, especially oleic acid, by DHEA feeding is due to an increase in both de novo fatty acid synthesis and the formation of oleic acid and to a decrease in the rate of hepatic VLDL secretion. Mitochondrial and peroxisomal fatty acid degradation does not appear to play a significant role in the accumulation of hepatic lipids.

[Farmer's lung cases of a farmer and his son with high BAL fluid beta-D glucan levels].

A farmer and his son, who treated straw in a cowshed, were admitted to our hospital because of severe dyspnea during summer time. Their chest X-ray films revealed bilateral reticulonodular shadows in the middle to lower lung fields. Bronchoalveolar lavage (BAL) fluid analyses showed a high proportion of lymphocytes and an increased CD4/8 ratio. They were diagnosed with farmer's lung and treated with pulse therapy with methylprednisolone and tapering of steroid. Hypoxemia and interstitial shadow improved, though the farmer relapsed one day after getting home. Immune precipitation showed positive reactions against Aspergillus fumigatus, Aspergillus terreus and Nocardiopsis alba. Their beta D-glucan levels in BAL fluid were higher than those of healthy normal volunteers, whereas their beta D-glucan levels in serum were below the detection levels. Ventilation of the cowshed and wearing a mask should prevent recurrence of the disease.

[Formation of trans-1,3-pentadiene in off-flavor food].

The purpose of the present study is to investigate the production of trans-1,3-pentadiene in a sorbic acid-containing food which was the subject of a complaint that it was off-flavor. Penicillium sp. was isolated from the off-flavor food. The isolated Penicillium sp. was identified as Penicillium chrysogenum by DNA sequencing of the internal transcribed spacer region and the D1/D2 region of the 28S subunit. When P. chrysogenum was cultured in the presence of potassium sorbate, trans-1,3-pentadiene was produced and detected by GC-MS after solid-phase micro extraction. The production of trans-1,3-pentadiene by P. chrysogenum in the culture solution was pH-dependent. These results suggest that the production of trans-1,3-pentadiene in the off-flavor food was mainly due to the decomposition of sorbic acid by P. chrysogenum.

Up-regulation of stearoyl-CoA desaturase 1 increases liver MUFA content in obese Zucker but not Goto-Kakizaki rats.

The Goto-Kakizaki (GK) rat is an animal model for spontaneous-onset, non-obese type 2 diabetes. Despite abundant evidence about disorders in metabolism, little information is available about fatty acid metabolism in the liver of GK rats. This study aimed to investigate the characteristics of the fatty acid profile, particularly MUFA, and the mechanism underlying the alterations in fatty acid profiles in the liver of GK rats. The activities of enzymes that participate in the biosynthesis of MUFA, expressions of genes encoding these enzymes, and the fatty acid profile in the liver were compared with those of obese Zucker (fa/fa) (ZF) rats, which are obese and non-diabetic. Stearoyl-CoA desaturase (SCD) activity and SCD1 gene expression were considerably up-regulated in GK rats, and these levels were largely comparable to those in ZF rats. However, the proportions and contents of oleic acid and palmitoleic acid were very low considering the highly elevated activity of SCD in the liver of GK rats, when compared with ZF rats. Palmitoyl-CoA chain elongation (PCE) activity and fatty acid elongase (Elovl6) gene expression were markedly up-regulated in ZF rats, whereas PCE activity was up-regulated much less and Elovl6 gene expression was unchanged in GK rats. These results suggest the possibility that up-regulation of gene expression of Elovl6 along with SCD1 is indispensable to elevate the proportions and contents of oleic acid in the liver.

A simple, sensitive determination of ganciclovir in infant plasma by high-performance liquid chromatography with fluorescence detection.

This study developed a simple and sensitive method using reversed-phase high-performance liquid chromatography (HPLC) for ganciclovir (GCV) plasma concentrations in cytomegalovirus infectious infants with hearing loss. The method involves a simple protein precipitation procedure that uses no solid-phase or liquid-liquid extraction. The HPLC separation was carried out on a Cadenza CD-C(18) column (3 microm, 4.6 mm x 150 mm) with phosphate buffer (pH 2.5, 25 mM) containing 1% methanol-acetonitrile mixture (4:3, v/v) as a mobile phase at a 0.7 mL/min flow rate. GCV was detected using a fluorescence detection (lambdaex/em: 265/380 nm). The quantification limit was 0.025 microg/mL for 100 microL of plasma sample at which good intra- and inter-assay coefficient of variation values (< 4.96%) and recoveries (94.9-96.5%) were established.