Hormonal and ion regulatory response in three freshwater fish species following waterborne copper exposure

We evaluated effects of sublethal copper exposure in 3 different freshwater fish: rainbow trout (Oncorhynchus mykiss), common carp (Cyprinus carpio) and gibel carp (Carassius auratus gibelio). In a first experiment we exposed these fishes to an equally toxic Cu dose, a Cu level 10 times lower than their 96 h LC₅₀ value: 20, 65, and 150 µg/L Cu. In a second series we exposed them to the same Cu concentration (50 µg/L). Na⁺/K⁺-ATPase activity in gill tissue was disturbed differently in rainbow trout then in common and gibel carp. Rainbow trout showed a thorough disruption of plasma ion levels at the beginning of both exposures, whereas common carp and gibel carp displayed effects only after 3 days. Rainbow trout and common carp thyroid hormones experienced adverse effects in the beginning of the exposure. The involvement of prolactin in handling metal stress was reflected in changes of mRNA prolactin receptor concentrations in gill tissue, with an up regulation of this mRNA in rainbow trout and a down regulation in gibel carp, which was more pronounced in the latter. Overall, rainbow trout appeared more sensitive in the beginning of the exposure, however, when it overcame this first challenge, it handled copper exposure in a better manner then common and gibel carp as they showed more long term impacts of Cu exposure.     

Dynamics of yeast Myosin I: evidence for a possible role in scission of endocytic vesicles

Cortical actin patches are dynamic structures required for endocytosis in yeast. Recent studies have shown that components of cortical patches localize to the plasma membrane in a precisely orchestrated manner, and their movements at and away from the plasma membrane may define the endocytic membrane invagination and vesicle scission events, respectively. Here, through live-cell imaging, we analyze the dynamics of the highly conserved class I unconventional myosin, Myo5, which also localizes to cortical patches and is known to be involved in endocytosis and actin nucleation. Myo5 exhibits a pattern of dynamic localization different from all cortical patch components analyzed to date. Myo5 associates with cortical patches only transiently and remains stationary during its brief cortical lifespan. The peak of Myo5 association with cortical patches immediately precedes the fast movement of Arp2/3 complex-associated structures away from the plasma membrane, thus correlating precisely with the proposed vesicle scission event. To further test the role of Myo5, we generated a temperature-sensitive mutant myo5 allele. In the myo5 mutant cells, Myo5 exhibits a significantly extended cortical lifespan as a result of a general impairment of Myo5 function, and Arp2 patches exhibit an extended slow-movement phase prior to the fast movement toward the cell interior. The myo5 mutant cells are defective in fluid-phase endocytosis and exhibit an increased number of invaginations on the membrane. Based on these results, we hypothesize that the myosin I motor protein facilitates the membrane fusion/vesicle scission event of endocytosis.     

Determination of carbohydrates in medicinal plants--comparison between TLC, mf-MELDI-MS and GC-MS

Introduction – Quality control in the pharmaceutical and phytopharmaceutical industries requires fast and reliable methods for the analysis of raw materials and final products. Objective – This study evaluates different analytical approaches in order to recognise the most suitable technique for the analysis of carbohydrates in herbal drug preparations. Methodology – The specific focus of the study is on thin‐layer chromatography (TLC), gas chromatography (GC), and a newly developed mass spectrometric method, i.e. matrix free material enhanced laser desorption/ionisation time of flight mass spectrometry (mf‐MELDI‐MS). Samples employed in the study were standards and microwave‐assisted water extracts from Quercus. Results – TLC analysis proved the presence of mono‐, di‐ and trisaccharides within the biological sample and hinted at the existence of an unknown carbohydrate of higher oligomerisation degree. After evaluation of different derivatisation techniques, GC‐MS confirmed data obtained via TLC for mono‐ to trisaccharides, delivering additionally quantified values under a considerable amount of time. A carbohydrate of higher oligomerisation degree could not be found. The application of mf‐MELDI‐MS further confirmed the presence of carbohydrates up to trisaccharides, also hinting at the presence of a form of tetrasaccharide. Besides this information, mf‐MELDI‐MS delivered further data about other substances present in the extract. Quantitative determination resulted in 1.750, 1.736 and 0.336 mg/mL for glucose, sucrose and raffinose respectively. Conclusion – Evaluation of all three techniques employed, clearly proved the heightened performance of mf‐MELDI‐MS for the qualitative analysis of complex mixtures, as targets do not need modification and analysis requires only a few minutes. In addition, GC‐MS is suitable for quantitative analysis. Copyright © 2011 John Wiley & Sons, Ltd.     

Variations of the candidate SEZ6L2 gene on Chromosome 16p11.2 in patients with autism spectrum disorders and in human populations

Background

Autism spectrum disorders (ASD) are a group of severe childhood neurodevelopmental disorders with still unknown etiology. One of the most frequently reported associations is the presence of recurrent de novo or inherited microdeletions and microduplications on chromosome 16p11.2. The analysis of rare variations of 8 candidate genes among the 27 genes located in this region suggested SEZ6L2 as a compelling candidate.

Methodology/Principal Findings

We further explored the role of SEZ6L2 variations by screening its coding part in a group of 452 individuals, including 170 patients with ASD and 282 individuals from different ethnic backgrounds of the Human Genome Diversity Panel (HGDP), complementing the previously reported screening. We detected 7 previously unidentified non-synonymous variations of SEZ6L2 in ASD patients. We also identified 6 non-synonymous variations present only in HGDP. When we merged our results with the previously published, no enrichment of non-synonymous variation in SEZ6L2 was observed in the ASD group compared with controls.

Conclusions/Significance

Our results provide an extensive ascertainment of the genetic variability of SEZ6L2 in human populations and do not support a major role for SEZ6L2 sequence variations in the susceptibility to ASD.     

Are the characeae able to indicate the origin of groundwater in former river channels?

The macroscopic algae Characeae are usually assumed to occur in waterbodies supplied by groundwater with low phosphate content, but the indicative value of the species is seldom defined in bibliography. Former braided channels of the Rhône river are supplied with groundwater originating from the main channel (seepage) or from hillslope aquifer. The aim of the present paper was to determine if it possible to use the Characeae as indicators of physicochemical characteristies of water in order to assess the origin of groundwater supplying former river channels. Four former braided channels of the Rhône River colonized by Characeae were investigated, and the physico-chemical characteristics of i) the channels, ii) the groundwater and iii) the river were measured over a period of several months. Species are arranged along a gradient of conductivity, alkalinity, ammonium and phosphate content of the water. Charophyte species can indicate the origin of groundwater, either seepage or hillslope nutrient-poor aquifer, and integrate both the average value of the chemical parameter, and their variations. C. hispida occurs in a nutrient-poor channel mainly supplied by highly calcareous groundwater coming from hillslope aquifer. Chara major has requirements close to those of C. hispida, but is more tolerant to periodic inputs of nutrients. C. vulgaris and N. syncarpa both tolerate mesotrophic waters originating from both hillslope aquifer and seepage, and C. globularis is associated to a channel mainly supplied by mesotrophic to eutrophic river seepage.     

Repair of DNA damage induced by the mycotoxin alternariol involves tyrosyl-DNA phosphodiesterase 1

Alternariol (AOH) was reported recently to act as a topoisomerase poison. To underline the relevance of topoisomerase targeting for the genotoxic properties of AOH, we addressed the question whether human tyrosyl-DNA phosphodiesterase 1 (TDP1), an enzyme vital to the repair of covalent DNA-topoisomerase adducts, affects AOH-mediated genotoxicity. The relevance of TDP1 activity on AOH-induced genotoxicity was investigated by the comet assay in human cells overexpressing GFP chimera of TDP1 or the inactive mutant TDP1^H263A as well as in cells subjected to siRNA-mediated knock-down of endogenous TDP1. Cells overexpressing TDP1 exhibited significantly less DNA damage after treatment with AOH in comparison to cells expressing the inactive mutant TDP1^H263A. In accordance with these results, levels of AOH inducing DNA strand breaks were increased in TDP1-suppressed cells in comparison to cells transfected with control siRNA. The specific topoisomerase poisons camptothecin and etoposide caused comparable effects, underlining that TDP1 plays an important role in the repair of topoisomerase-mediated DNA damage. In summary, the repair enzyme TDP1 was identified as a factor for the modulation of AOH-mediated DNA damage in human cells.     

Mutational scanning of PCR products by subtractive oligonucleotide hybridization analysis

Here, we describe a new approach for mutational scanning of PCR products through hybridization analysis between complementary oligonucleotides. Sets of overlapping probe oligonucleotides complementary to wild-type (WT) sequence are hybridized to microbead-immobilized PCR products under solution-like conditions. Mismatch-hybridization situations between a mutant sample and probe oligonucleotides result in higher remaining concentrations in solution of involved probe oligonucleotides. Post-hybridization supernatants are subsequently analyzed for their probe oligonucleotide compositions using surface plasmon resonance-based biosensor technology. Relative remaining probe oligonucleotide concentrations are monitored in real-time through hybridization analysis between probe oligonucleotides and their corresponding sensor-chip immobilized complementary counterparts. This allows for the construction of composition diagrams revealing the existence and approximate location of a mutation within an investigated sample DNA sequence. Applied on PCR products derived from clinical samples of microdissected tumor biopsies, single mutations in exons 6 and 7 of the human p53 tumor-suppressor gene were successfully detected and approximately localized.