Evaluation of maternal and embryotoxic effects following the treatment of chloral hydrate in Drosophila melanogaster

Chloral hydrate (CH) is commonly used as a sedative and a hypnotic in pediatric medicine. In this study, the effects of CH on various developmental stages of Drosophila melanogaster were investigated. Different concentrations of CH (0.1; 0.3; 0.5 and 1 mg/mL) were used during development of the flies. Maternal toxicity due to increasing the concentration of CH was observed as a large number of adult flies died. When the F₁ progeny of the control and application groups were compared, CH was found to extend the process of metamorphosis and to decrease the total number of offspring. The embryotoxic effects on the offspring and an increase in the number of malformed offspring was identified as depending on feeding. It was found that the difference between the groups was significantly important (p < 0.05).     

An <Emphasis Type="Italic">in vitro</Emphasis> model for the development of acquired tamoxifen resistance

The development of resistance to tamoxifen (Tam) remains a challenging clinical problem for ER+ breast cancer patients. To understand the mechanisms underlying of resistance, previous studies have driven the acquisition of Tam resistance by exposing cells to varying concentration of drug for varying lengths of time. However, a detailed protocol for the establishment of Tam-resistant cells remains to be clarified. In the present study, we aimed to determine and compare the effect of different in vitro protocols on the degree of resistance to 4-hydroxytamoxifen (4-OH Tam) for MCF7 cells. For this purpose, MCF7-Tam resistance (MCF7-TamR) cells were developed by treated with different concentrations (100, 200, 400, 600, 800 and 1000 nM) of 4-OH Tam over 3 months. The relative resistance was measured by WST-1 analysis. Studies characterizing of the 4-OH Tam resistance of MCF7-TamR cells were performed by 17 β-oestradiol (E2) and Annexin V/PI analysis. In addition, the expression levels of ABCC1, ABCG2 and ABCG1 were detected by RT-PCR, any changes in morphological of each resistance group were observed at the end of each month and compared with parental MCF7 cells. Consequently, exposure time and concentration can affect the degree of resistance to 4-OH Tam; thus, dose and treatment duration should be chosen according to the desired degree of resistance. This work presents a novel procedure for the generation of MCF7-TamR cells, thus enabling the identification and characterization of MCF7-TamR cells.     

The Performance of the Four Anaerobic Blood Culture Bottles BacT/ALERT-FN, -FN Plus,BACTEC-Plus and -Lytic in Detection of Anaerobic Bacteria and Identification by Direct MALDI-TOF MS

Detection and identification of anaerobic bacteria in blood cultures (BC) is a well-recognized challenge in clinical microbiology. We studied 100 clinical anaerobic BC isolates to evaluate the performance of BacT/ALERT-FN, -FN Plus (BioMérieux), BACTEC-Plus and -Lytic (Becton Dickinson BioSciences) BC bottles in detection and time to detection (TTD) of anaerobic bacteria. BACTEC Lytic had higher detection rate (94/100, 94%) than BacT/ALERT FN Plus (80/100, 80%) (p<0.01) in the studied material. There was no significant difference in detection of anaerobic bacteria among the remaining bottle types. The 67 anaerobic bacteria that signalled positive in all four bottle types were analyzed to compare the time to detection (TTD) and isolates were directly identified by MALDI-TOF MS. There was a significant difference in TTD among the four bottle types (p<0.0001). The shortest median TTD was 18 h in BACTEC Lytic followed by BacT/ALERT FN (23.5 h), BACTEC Plus (27 h) and finally BacT/ALERT FN Plus (38 h) bottles. In contrast, MALDI-TOF MS performed similarly in all bottle types with accurate identification in 51/67 (76%) BacT/ALERT FN, 51/67 (76%) BacT/ALERT FN Plus, 53/67 (79%) BACTEC Plus and 50/67 (75%) BACTEC Lytic bottles. In conclusion, BACTEC Lytic bottles have significantly better detection rates and shorter TTD compared to the three other bottle types. The anaerobic BC bottles are equally suitable for direct MALDI-TOF MS for rapid and reliable identification of common anaerobic bacteria. Further clinical studies are warranted to investigate the performance of anaerobic BC bottles in detection of anaerobic bacteria and identification by direct MALDI-TOF MS.     

An aeropalynological survey in the city of Van,a high altitudinal region,East Anatolia-Turkey

Pollen concentrations in the atmosphere of Van city has been monitored for two consecutive years (2010–2011). This was the first detailed aeropalynological study for the elevated East Anatolia Region of Turkey. The sampling was performed by Hirst-type volumetric sampler, and pollen grains of 35 taxa were identified. The main pollen producers of the pollen flora were recorded as: Poaceae (20.94 %), Cupressaceae (10.53 %), Fraxinus (8.56 %), Chenopodiaceae/Amaranthaceae (7.77 %), Populus (7.75 %), Quercus (6.70 %), Platanus (6.68 %), Morus (5.57 %), Plantago (3.03 %). The pollen spectrum reflected the floristic diversity of the region, and the highest pollen concentration was recorded in April. There were a great percentage of allergenic taxa found in the city atmosphere, otherwise many of them scored under threshold values for risk of pollinosis. Statistical analyses were performed for correlating daily pollen concentrations of dominated pollen types concurrent with the data of meteorological parameters in MPS periods and number of significant correlations found. In addition, comparing 2-year data in terms of pollen concentrations and meteorological factors in MPS durations, many variables were found explanatory and concordant with the data. MPS starting dates of many plant taxa were found nearly a month later compared with western sites and lower altitudes of the country as well as Mediterranean countries; this case is mostly thought the ecological factors of the study area which directly affects the plant growth about the timing.     

Variations in the Bioactive Compounds Composition and Biological Activities of Loofah (Luffa cylindrica) Fruits in Relation to Maturation Stages

The present work aimed to determine the antioxidant and antiproliferative potential of Luffa cylindrica fruits collected at two different maturation stages and to identify and compare their functional components composition. The MeOH extracts of L. cylindrica fruits harvested at 60 – 65 days after seeding (S1) and 85 – 90 days after seeding (S2) were investigated for their antioxidant activity using various assays. Furthermore, the antiproliferative activity of the extracts against HeLa human cervical cancer cells was explored with xCELLigence real time cell analyzer, while the effect of the samples on the membrane integrity of the same cell line was assessed using LDH cytotoxicity leakage assay. Ultimately, the phytochemicals were analyzed using GC/MS and HPLC/TOF‐MS. The S1 sample had higher contents and more diversity in the phenolic compounds composition than S2. Furthermore, the S1 extract showed the highest antioxidant and antiproliferative activity, while the S2 extract had higher cytotoxicity towards HeLa cells. The findings revealed that the time of harvest has a big impact on the phytochemicals content and activity and that harvesting L. cylindrica at an early stage before the beginning of the development of the cellulose fibrous system is recommended for a rich phytochemical composition and efficient antioxidant and antiproliferative activities.     

Purification and biochemical characterization of a novel thermostable serine alkaline protease from Aeribacillus pallidus C10: a potential additive for detergents

An extracellular thermostable alkaline serine protease enzyme from Aeribacillus pallidus C10 (GenBank No: KC333049), was purified 4.85 and 17. 32-fold with a yield of 26.9 and 19.56%, respectively, through DE52 anion exchange and Probond affinity chromatography. The molecular mass of the enzyme was determined through sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), with approximately 38.35?kDa. The enzyme exhibited optimum activity at pH 9 and at temperature 60?°C. It was determined that the enzyme had remained stable at the range of pH 7.0–10.0, and that it had preserved more than 80% of its activity at a broad temperature range (20–80?°C). The enzyme activity was found to retain more than 70% and 55% in the presence of organic solvents and commercial detergents, respectively. In addition, it was observed that the enzyme activity had increased in the presence of 5% SDS. K_M and V_max values were calculated as 0.197?mg/mL and 7.29?μmol.mL^?1.min^?1, respectively.     

Total antioxidant capacity assay of human serum using copper(II)-neocuproine as chromogenic oxidant: the CUPRAC method

BACKGROUND: Tests measuring the combined antioxidant effect of the nonenzymatic defenses in biological fluids may be useful in providing an index of the organism's capability to counteract reactive species known as prooxidants, resist oxidative damage and combat oxidative stress-related diseases. The selected chromogenic redox reagent for the assay of human serum should be easily accessible, stable, selective, respond to all types of biologically important antioxidants such as ascorbic acid, alpha-tocopherol, beta-carotene, reduced glutathione (GSH), uric acid and bilirubin, regardless of chemical type or hydrophilicity. Currently, there is no rapid method for total antioxidant assay of human serum meeting the above criteria.METHODS: Our recently developed cupric reducing antioxidant capacity (CUPRAC) spectrophotometric method for a number of polyphenols and flavonoids using the copper(II)-neocuproine reagent in ammonium acetate buffer was now applied to a complete series of plasma antioxidants for the assay of total antioxidant capacity (TAC) of serum, and the resulting absorbance at 450 nm was recorded either directly (e.g. for ascorbic acid, alpha-tocopherol and glutathione) or after incubation at 50 degrees C for 20 min (e.g. for uric acid, bilirubin and albumin), quantitation being made by means of a calibration curve. The lipophilic antioxidants, alpha-tocopherol and beta-carotene, were assayed in dichloromethane (DCM). Lipophilic antioxidants of serum were extracted with n-hexane from an ethanolic solution of serum subjected to centrifugation. Hydrophilic antioxidants of serum were assayed after perchloric acid precipitation of proteins in the centrifugate.Results: The molar absorptivities, linear ranges and trolox equivalent antioxidant capacity (TEAC) coefficients of the serum antioxidants were established with respect to the CUPRAC spectrophotometric method, and the results (TEAC, or TEAC coefficients) were evaluated in comparison to the findings of the ABTS/TEAC reference method using persulfate as oxidant. As for hydrophilic phase, a linear correlation existed between the CUPRAC and ABTS findings (r=0.58), contrary to current literature reporting that either serum ORAC or serum ferric reducing antioxidant potency (FRAP) does not correlate at all with serum TEAC. The analytical responses of serum antioxidants were shown to be additive, enabling a TAC assay. The intra- and inter-assay CVs were 0.7 and 1.5%, respectively, for serum.Conclusions: The CUPRAC assay proved to be efficient for glutathione and thiol-type antioxidants, for which the FRAP test was nonresponsive. The findings of CUPRAC completely agreed with those of ABTS-persulfate for lipophilic phase. The additivity of absorbances of all the tested antioxidants confirmed that antioxidants in the CUPRAC test did not chemically interact among each other so as to cause an intensification or quenching of the theoretically expected absorbance. As a distinct advantage over other electron-transfer based assays (e.g. Folin, FRAP, ABTS, DPPH), CUPRAC is superior in regard to its realistic pH close to the physiological pH, favourable redox potential, accessibility and stability of reagents and applicability to lipophilic antioxidants as well as hydrophilic ones.     

Electron-conformational study for the structure-hallucinogenic activity relationships of phenylalkylamines

The structure-hallucinogenic activity relationships of a series of phenylethylamine and phenylisopropylamine derivatives have been investigated in the frameworks of electron-conformational method. The calculated geometry and electronic structure parameters accompanying to each atom and bond of each molecule in view were arranged as a matrix called electron-conformational matrix of contiguity (ECMC). The features that are responsible for strong and weak activity demonstrations have been found as submatrices of ECMCs belonging to some template compounds. Two electron-conformational features present in nonhallucinogenic compounds have been revealed. A quantitative model has been improved for predicting hallucinogenic activity numerically. A test series was used to verify the results obtained.     

Detection of ubiquitin-proteasome enzymatic activities in cells: Application of activity-based probes to inhibitor development

Background: Synthetic probes that mimic natural substrates can enable the detection of enzymatic activities in a cellular environment. One area where such activity-based probes have been applied is the ubiquitin-proteasome pathway, which is emerging as an important therapeutic target. A family of reagents has been developed that specifically label deubiquitylating enzymes (DUBs) and facilitate characterization of their inhibitors. Scope of review: Here we focus on the application of probes for intracellular DUBs, a group of specific proteases involved in the ubiquitin proteasome system. In particular, the functional characterization of the active subunits of this family of proteases that specifically recognize ubiquitin and ubiquitin-like proteins will be discussed. In addition we present the potential and design of activity-based probes targeting kinases and phosphatases to study phosphorylation. Major conclusions: Synthetic molecular probes have increased our understanding of the functional role of DUBs in living cells. In addition to the detection of enzymatic activities of known members, activity-based probes have contributed to a number of functional assignments of previously uncharacterized enzymes. This method enables cellular validation of the specificity of small molecule DUB inhibitors. General significance: Molecular probes combined with mass spectrometry-based proteomics and cellular assays represent a powerful approach for discovery and functional validation, a concept that can be expanded to other enzyme classes. This addresses a need for more informative cell-based assays that are required to accelerate the drug development process. This article is part of a Special Issue entitled: Ubiquitin Drug Discovery and Diagnostics.