Amatoxin and phallotoxin concentration in Amanita phalloides spores and tissues.

Most of the fatal cases of mushroom poisoning are caused by Amanita phalloides. The amount of toxin in mushroom varies according to climate and environmental conditions. The aim of this study is to measure α-, ß-, and γ-amanitin with phalloidin and phallacidin toxin concentrations. Six pieces of A. phalloides mushrooms were gathered from a wooded area of Düzce, Turkey, on November 23, 2011. The mushrooms were broken into pieces as spores, mycelium, pileus, gills, stipe, and volva. α-, ß-, and γ-Amanitin with phalloidin and phallacidin were analyzed using reversed-phase high-performance liquid chromatography. As a mobile phase, 50 mM ammonium acetate + acetonitrile (90 + 10, v/v) was used with a flow rate of 1 mL/min. C18 reverse phase column (150 × 4.6 mm; 5 µm particle) was used. The least amount of γ-amanitin toxins was found at the mycelium. The other toxins found to be in the least amount turned out to be the ones at the spores. The maximum amounts of amatoxins and phallotoxin were found at gills and pileus, respectively. In this study, the amount of toxin in the spores of A. phalloides was published for the first time, and this study is pioneering to deal with the amount of toxin in mushrooms grown in Turkey.

Efficacy of magnetic resonance urography in detecting renal scars in children with vesicoureteral reflux.

BACKGROUND: The detection of renal scars is of paramount importance for optimal clinical management of patients with urinary tract infection (UTI) and vesicoureteral reflux (VUR). The aim of our study was to compare the efficacy of unenhanced magnetic resonance urography (MRU) and Tc-99(m) dimercaptosuccinic acid (Tc-DMSA) scintigraphy to dectect renal scars. METHODS: Unenhanced MRU and Tc-DMSA scintigraphy were performed in 49 children (10 boys, 39 girls; mean age 7.4 ± 4.2 years, range 1-15 years) with documented VUR. MR imaging scans were obtained within 7 days after voiding cystourethrogram (VCUG) and Tc-DMSA scintigraphy. The diagnostic performance of MRU in renal scar detection was calculated relative to that of the Tc-DMSA scan. RESULTS: The renal scar detection rate of Tc-DMSA scintigraphy and unenhanced MRU in kidneys with VUR was 32.4 and 25.9%, respectively. The sensitivity and specificity of MRU in the detection of renal scars was 80 and 82.6% in kidneys with VUR, respectively. There was no statistically significant difference in lesion detection between MRU and Tc-DMSA scintigraphy (P > 0.05). MRU and Tc-DMSA scintigraphy showed good agreement (κ = 0.60). CONCLUSIONS: Unenhanced MRU is a robust technique for the morphologic assessment of the urinary system and detection of renal scars. The lack of radiation and contrast material makes this technique a much safer alternative to scintigraphy in children with VUR, particularly in those who require follow-up scanning and, consequently, considerable radiation exposure.

Apoptotic, antioxidant and antiradical effects of majdine and isomajdine from Vinca herbacea Waldst. and kit.

In the present study, apoptotic, antioxidant and antiradical effects of majdine and isomajdine from Vinca herbacea Waldst. and Kit were studied. For testing the possible apoptotic effects of majdine and isomajdine from V. herbacea, DNA fragmentation assay was conducted on the rat brain cortical tissue homogenates, in vitro. Also their possible effects on mitochondrial activity were tested by using the same tissue samples of rats. In addition, the antioxidant activity of isomajdine and majdine was determined using various in vitro antioxidant assays, including 2,2'-azino-bis(3-ethylbenzthiazoline-6-sulfonic acid) (ABTS(•+)) radical scavenging and N,N-dimethyl-p-phenylenediamine (DMPD(•+)) radical scavenging, ferric ions (Fe(3+)) and cupric ions (Cu(2+)) reducing abilities and ferrous ions (Fe(2+)) chelating activity. On the other hand, butylated hydroxyanisole (BHA), butylated hydroxytoluene (BHT), α-tocopherol and trolox (6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid) were used as reference antioxidants.

Erdosteine reduces cytotoxicity induced by alpha- and beta-amanitin, but not gamma-amanitin, in CA3 hepatocyte cultures.

Amanitin poisoning still has no particular, effective antidote. Erdosteine has been shown to protect numerous tissues, particularly those in the liver. This study investigates the potential therapeutic effects of erdosteine on alpha-, beta- and gamma-amanitin-induced hepatotoxicity in in vitro models. Three hours after administering amatoxins at various concentrations (1-50 µg/mL) to the cells of the C3A human hepatocyte cell line, erdosteine was administered in different concentrations (i.e., 1, 10, 50, 100 and 250 µg/mL). The 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay was selected to determine cell viability. When concentrations of 1, 10, 50, 100 and 250 µg/mL of erdosteine were applied to cell lines, the following cell viability rates were obtained: 106%,99%,93%,86% and 86%, respectively, at a 10 µg/mL alpha-amanitin-induced toxicity; 43%,41%,41%,37% and 35%, respectively, at a 25 µg/mL alpha-amanitin-induced toxicity; 44%,42%,41%,39% and 41%, respectively, at a 50 µg/mL alpha-amanitin-induced toxicity; 136%,142%,143%,137% and 120%, respectively, at a 10 µg/mL beta-amanitin-induced toxicity; 113%,107%,107%,106% and 86%, respectively, at a 25 µg/mL beta-amanitin-induced toxicity; 78%,77%,77%,74% and 70%, respectively, at a 10 µg/mL gamma-amanitin-induced toxicity; and 39%,40%,39%,35% and 31%, respectively, at a 25 µg/mL gamma-amanitin-induced toxicity. This study was the first to evaluate the in vitro efficacy of erdosteine in cytotoxicity induced by alpha-, beta- and gamma-amanitin. Non-high (low and medium) doses of erdosteine are capable of nearly entirely preventing toxicity at mild hepatotoxic concentrations caused by amatoxin and partially preventing toxicity at moderate and severe concentrations. The beneficial effects of erdosteine, especially on the toxicity of alpha- and beta-amanitin, are promising.

Can Gastric Volume be Accurately Estimated by Ultrasound?

OBJECTIVE: Knowing the degree of gastric fullness is critical in determining the potential risk of pulmonary aspiration prior to urgent or elective intubation. This study aims to investigate the role of ultrasound in predicting the gastric volume accurately. METHODS: 176 patients who underwent upper gastric endoscopy after 12-hour fasting were examined by gastric US. The patients were ran- domly divided into 6 groups according to the volume of ingested semifluid meal: (1) empty stomach (no volume), (2) 50 mL, (3) 100 mL, (4) 200 mL, (5) 300 mL, and (6) 400 mL. Antral cross-sectional area (CSA) was measured by US after each ingestion. RESULTS: We found a strong linear correlation between antral CSA and gastric volume up to 200 mL. The diagnostic performance of ultra- sound was found to be more powerful in the supine position than in the right lateral position. A new mathematical model was established to predict gastric volume. The threshold value for antral cross-sectional area at risk of pulmonary aspiration was determined as 3.1 cm2 by sonographic measurement. CONCLUSION: Ultrasonography could be preferred to gastric endoscopy or scintigraphy in terms of non-invasiveness and easiness, although it still merits further investigation.

Effect of Breast Milk Calcium and Fluidity on Breast Cancer Cells: An In Vitro Cell Culture Study.

AIM: The aims of this study were to investigate the effects of calcium at the same concentration as that found in human milk on the viability, proliferation, and adhesion of MCF-7 human breast ductal carcinoma cells by exposing them to calcium at the same frequency as in breastfeeding. MATERIALS AND METHODS: High-concentration calcium was applied for 30 minutes every 4 hours for 24, 48, and 72 hours. Cell proliferation and viability were measured using a hemocytometer and the MTT cell viability assay. The effects of calcium treatment were evaluated by a comparison among a multiple-, single-dose calcium treatment, and a control group. RESULTS: We show that calcium at the same concentration as that in milk caused a decrease in the number of cells but did not affect cell viability. CONCLUSIONS: The results of this study suggest that calcium caused a lowering of the number of cells from the luminal surface of the breast by triggering proliferation under the condition of fluidity. Calcium and fluidity together serve to eliminate breast cancer stem cells during the lactation period. Effects of the other components of milk can be analyzed by the new method developed in this study.

Diffusion weighted magnetic resonance imaging of kidneys in children with vesicoureteral reflux.

PURPOSE: The apparent diffusion coefficient (ADC) which obtain from diffusion-weighted magnetic resonance imaging (DWI), is a quantitative parameter representing the renal function and parenchymal damage in some renal disorders. The primary aim of this study was to investigate whether renal tissue alterations associated with vesicoureteral reflux (VUR) can be displayed by DWI. The secondary aim was to assess how ADC values change with age in kidneys with and without VUR. MATERIALS AND METHODS: This prospective study included 46 patients (8 boys, 38 girls; mean age 7.3 ± 4.2; range 1-15 years) with VUR and 54 control subjects (21 boys, 33 girls; mean age 7.7 ± 5.2; range 1-17 years). All subjects underwent DWI of the kidneys using b value of 600s/mm(2) in addition to MR urography. The ADC values of 71 kidneys with VUR were compared with those of 81 kidneys without VUR. RESULTS: The mean ADC values were (1.93 ± 0.36)×10(-3)mm(2)/s, (1.97 ± 0.24)× 10(-3)mm(2)/s, (1.83 ± 0.37)× 10(-3)mm(2)/s, (1.98 ± 0.20)×10(-3)mm(2)/s and (2.08 ± 0.42)× 10(-3)mm(2)/s in normal kidneys, and in those with grade 1, grade 2, grade 3 and grade 4 VUR, respectively. There was no significant difference in ADC values between kidneys with and without VUR. There was a significant positive correlation between the age and ADC values both in kidneys with and without VUR (r=0.79, p<0.001 and r=0.82; p<0.001, respectively). CONCLUSION: DWI does not reveal probable parenchymal alterations in reflux nephropathy. ADC values increase with age during childhood not only in normal kidneys but also in kidneys with VUR.

Extracorporeal shockwave increases the effectiveness of systemic antibiotic treatment in implant-related chronic osteomyelitis: experimental study in a rat model.

Implant-related chronic osteomyelitis is a serious complication of orthopedic surgery requiring implant removal and radical debridement. Extracorporeal shockwave (ESW) have demonstrated significant bactericidal effectiveness in vitro and effectiveness and safety were evaluated in an animal model of osteomyelitis. In this experimental study, we aimed to test our hypothesis that the use of ESW together with systemic antibiotic treatment will provide synergy for the treatment of implant-related chronic osteomyelitis caused by methicillin-susceptible Staphylococcus aureus (MSSA). The proximal tibia of 32 rats was contaminated with (10) 8 CFU/ml methicillin-sensitive S. aureus (MSSA-ATCC 29213) and Kirschner-wires were placed into the medulla of the tibia. After 4 weeks, Kirschner-wires were removed and the rats were randomly divided into four groups: group I, untreated contaminated control group; group II, receiving only ESW therapy; group III, receiving only systemic teicoplanin; group IV, treated with a combination of ESW and systemic teicoplanin. ESW was applied twice to the infected limbs and all rats were sacrificed at the end of 8th week. The degree of tibial osteomyelitis was assessed by quantitative culture analysis. Bacterial counts in groups III and IV were significantly reduced relative to the control (p=0.002 and 0.001, respectively). The decrease in bacterial counts was more pronounced and significant in group IV compared to group III (p=0.024). In group II, bacterial counts also decreased, but the differences were in significant (p=0.068). Our experimental model suggests that ESW provides significant synergy for systemic antibiotic treatment. However, further clinical trials are required in order to use this treatment modality safely in patients, even though our study demonstrated successful results in the treatment of implant-related chronic osteomyelitis in rats.

Clinical importance of toxin concentration in Amanita verna mushroom.

Poisoning from Amanita group of mushrooms comprises approximately 3% of all poisonings in our country and their being responsible for nearly the entire fatal mushroom poisonings makes them important. These mushrooms contain primarily two types of toxins, amatoxins and phallotoxins. Phallotoxins have a more limited toxicity potential and they primarily consist of phalloidin (PHN) and phallacidin (PCN). Amatoxins, on the other hand, are very toxic and they primarily consist of alpha-amanitin (AA), beta-amanitin (BA) and gamma-amanitin (GA). Toxin levels can vary among various species, even among varieties of the same species, of Amanita mushroom family. Revealing the differences between the toxin compositions of the Amanita species that grow in our region may contribute to the clinics of poisonings. Our study aims at showing in detail the toxin levels in various parts of Amanita verna mushroom. A. verna mushrooms needed for toxin analysis were collected from Kozak Plateau near Ayvalik county of Balikesir, Turkey in April 2013. The mushrooms were divided into their parts as pileus, gills, stripe and volva. Following the procedures required before the analysis, the AA, BA, GA, PHN and PCN levels were measured using the RP-HPLC method. While the lowest level of amatoxin was in the volva of the mushroom, the highest was measured in the gills. This was followed by pileus and stripe where the levels were close to each other. Similarly, the highest level of phallotoxin was measured in the gills. Gamma toxin and phalloidin were at lower amounts than the other toxins. A. verna is frequently confused with edible mushrooms with white caps due to its macroscopic similarity. If just one of them is eaten by mistake by an adult person with no mushroom experience, it can easily poison them. The amount of amatoxin is more as compared to Amanita phalloides and A. phalloides var. alba. Particularly, the AA and BA levels are approximately three times higher, whereas GA levels are lower. Similarly, the level of PCN is approximately four times higher as compared to A. phalloides and A. phalloides var. alba; by contrast, the level of PNH is about a half of theirs. In summary, it can be said that A. verna is a more toxic mushroom than A. phalloides and has a higher rate of mortality. With our study, the amatoxin and phallotoxin concentrations and distribution in A. verna mushrooms were shown in detail for the first time and it would be useful to carry out more similar studies with other members of Amanita family growing in various parts of the world.

Evaluation and comparison of alpha- and beta-amanitin toxicity on MCF-7 cell line.

BACKGROUND/AIM: Alpha- and beta-amanitins are the main toxins of the poisonous Amanita phalloides mushroom. Although there are many studies available concerning alpha-amanitin, there are limited data about beta-amanitin in the literature. Therefore, this study is aimed at comparing the toxic effects of alpha- and beta-amanitin on the MCF-7 cell line. MATERIALS AND METHODS: The alpha- and beta-amanitins used for this research were purified from Amanita phalloides by preparative high-performance liquid chromatography. The MCF-7 breast cancer cell line was used, and specific concentrations of the toxins (100, 10, 1, 0.1, and 0.01 µg/mL) were applied to the cells. The MTT test was performed to determine the level of toxicity, and the quantity of protein in the cell was measured using the biuret test. RESULTS: The aLpha-amanitin showed a higher toxicity at 36 h, while the highest inhibition of protein synthesis by the beta-amanitin was observed at 24 h. CONCLUSION: It was shown that the beta-amanitin may be responsible for toxicity, like alpha-amanitin, in Amanita phalloides mushroom poisoning. The early inhibition of protein synthesis for beta-amanitin might be useful for future experiments and research.

Amanitin and phallotoxin concentration in Amanita phalloides var. alba mushroom.

Although rarely seen, Amanita phalloides var. alba, a variety of A. phalloides type mushrooms, causes mushroom poisoning resulting in death. Since it is frequently confused with some edible mushrooms due to its white colored cap and macroscopic appearance, it becomes important in toxicological terms. Knowledge of the toxin amount contained in this mushroom type is invaluable in the treatment of cases involving poisoning. In this study, we examined the toxin levels of various parts of the A. phalloides var. alba mushroom growing Duzce region of Turkey. Toxin analyses were carried out for A. phalloides var. alba, which were collected from the forests Duzce region of Turkey in 2011, as a whole and also separately in its spore, pileus, gills, stipe and volva parts. The alpha amanitin, beta amanitin, gamma amanitin, phalloidin and phallacidine analyses of the mushrooms were carried out using the RP-HPLC method. A genetic analysis of the mushroom showed that it had similar genetic characteristics as A. phalloides and was a variety of it. The lowest toxins quantity was detected in spores, volva and stipe among all parts of the mushroom. The maximum amount of amatoxins was measured in the gills. The pileus also contained a high amount of amatoxins. Generally, amatoxins and phallotoxin concentrations were lower as compared to A. phalloides, but interestingly all toxins other than gamma toxin were higher in the spores of A. phalloides var. alba. The amount of toxin in all of its parts had sufficient concentrations to cause death. With this study, the amatoxin and phallotoxin concentrations in A. phalloides var. alba mushroom and in its parts have been revealed in detail for the first time.

Efficacy of daptomycin on experimental methicillin-resistant Staphylococcus aureus keratitis in rabbits.

PURPOSE: We aimed to compare the efficacy of topical daptomycin (DAP) with that of vancomycin (VA) in the treatment of keratitis caused by methicillin-resistant Staphylococcus aureus (MRSA). METHODS: One hundred colony-forming unit MRSA bacteria were injected intrastromally into both corneas of 28 rabbits. Sixteen hours after injection, the rabbits' eyes were treated with 1 drop of topical DAP (10 or 50 mg/mL), VA (50 mg/mL), or isotonic saline for 19 doses. Their eyes were examined for clinical severity before and after treatment. RESULTS: The minimum inhibitory concentration values of VA and DAP against the bacterial strain were found to be 2 and 0.5 µg/mL, respectively. The mean pre- and post-treatment clinical scores of the eyes did not differ significantly among the groups. However, the mean difference between the post- and pretreatment clinical scores was significantly lower in the 50 mg/mL DAP group than in the other groups (P=0.042). A marked decrease in bacterial load was detected in all treatment groups compared to the control group (P=0.002). Although there were no significant differences in bacterial load among the treatment groups, the 50 mg/mL DAP group showed the greatest decrease. The mean % epithelial erosion rate tended to be higher in the 50 mg/mL VA group than in the other groups (P=0.31). CONCLUSIONS: Topical DAP significantly reduced the bacterial load and showed activity against MRSA comparable to that of fortified VA in this experimental model.