Use of algae in the study of essential cell processes

Maintenance of genomic stability is of crucial importance for all living organisms. It is no surprise that during evolution, a series of highly selective and efficient systems to detect DNA damage and control its repair have evolved. To this end, signal transduction pathways are involved in pausing the cell division cycle to provide time for repair, and ultimately releasing the cell cycle from arrest. Genetic components of the damage and replication checkpoints have been identified and a working model is beginning to emerge. This area of biological inquiry has received a great deal of attention in the past decade with the realization that the underlying regulatory mechanisms controlling the cell cycle are conserved throughout eukaryotic evolution. Many of the key players in this response have structural and functional counterparts in species as diverse as yeast and human. In recent years attention has also been paid to the plant kingdom suggesting that checkpoint controls have been highly conserved during evolution. The unicellular green alga Chlamydomonas reinhardtii is a suitable model organism for the study of basic cellular processes including cell cycle regulation and DNA repair. To investigate how algal cells accomplish these tasks, we have isolated mutants in the recognition and repair of DNA damage or in the response to DNA damage. Presented at the International Symposium Biology and Taxonomy of Green Algae V, Smolenice, June 26–29, 2007, Slovakia.     

Type 2 diabetes susceptibility loci in the Ashkenazi Jewish population

Until last year, type 2 diabetes (T2D) susceptibility loci have hardly been identified, despite great effort. Recently, however, several whole-genome association (WGA) studies jointly uncovered 10 robustly replicated loci. Here, we examine these loci in the Ashkenazi Jewish (AJ) population in a sample of 1,131 cases versus 1,147 controls. Genetic predisposition to T2D in the AJ population was found similar to that established in the previous studies. One SNP, rs7754840 in the CDKAL1 gene, presented a significantly stronger effect in the AJ population as compared to the general Caucasian population. This may possibly be due to the increased homogeneity of the AJ population. The use of the SNPs considered in this study, to identify individuals at high (or low) risk to develop T2D, was found of limited value. Our study, however, strongly supports the robustness of WGA studies for the identification of genes affecting complex traits in general and T2D in particular.     

20-hydroxyeicosatetraenoic acid causes endothelial dysfunction via eNOS uncoupling

Nitric oxide (NO), generated from L-arginine by endothelial nitric oxide synthase (eNOS), is a key endothelial-derived factor whose bioavailability is essential to the normal function of the endothelium. Endothelium dysfunction is characterized by loss of NO bioavailability because of either reduced formation or accelerated degradation of NO. We have recently reported that overexpression of vascular cytochrome P-450 (CYP) 4A in rats caused hypertension and endothelial dysfunction driven by increased production of 20-hydroxyeicosatetraenoic acid (20-HETE), a major vasoconstrictor eicosanoid in the microcirculation. To further explore cellular mechanisms underlying CYP4A-20-HETE-driven endothelial dysfunction, the interactions between 20-HETE and the eNOS-NO system were examined in vitro. Addition of 20-HETE to endothelial cells at concentrations as low as 1 nM reduced calcium ionophore-stimulated NO release by 50%. This reduction was associated with a significant increase in superoxide production. The increase in superoxide in response to 20-HETE was prevented by N(G)-nitro-L-arginine methyl ester, suggesting that uncoupled eNOS is a source of this superoxide. The response to 20-HETE was specific in that 19-HETE did not affect NO or superoxide production, and, in fact, the response to 20-HETE could be competitively antagonized by 19(R)-HETE. 20-HETE had no effect on phosphorylation of eNOS protein at serine-1179 or threonine-497 following addition of calcium ionophore; however, 20-HETE inhibited association of eNOS with 90-kDa heat shock protein (HSP90). In vivo, impaired acetylcholine-induced relaxation in arteries overexpressing CYP4A was associated with a marked reduction in the levels of phosphorylated vasodilator-stimulated phosphoprotein, an indicator of bioactive NO, that was reversed by inhibition of 20-HETE synthesis or action. Because association of HSP90 with eNOS is critical for eNOS activation and coupled enzyme activity, inhibition of this association by 20-HETE may underlie the mechanism, at least in part, by which increased CYP4A expression and activity cause endothelial dysfunction.     

Genotoxic activity of a technical toxaphene mixture and its photodegradation products in SOS genotoxicity tests

Toxaphene (CAS No. 800-35-2) is a complex mixture of several hundred components that was used worldwide primarily as an agricultural pesticide with insecticide effects in the second half of the 20th century. In vitro investigations of the genotoxicity and mutagenicity of toxaphene were generally described in the literature, but they provided somewhat equivocal results. We re-evaluated the genotoxicity of technical toxaphene in two prokaryotic systems. The SOS Chromotest showed high sensitivity to toxaphene: three concentrations (40, 20 and 10 mg/l) were clearly positive and the dose–response effect was evident. In the umuC assay, a dose-dependent increase in genotoxic activity was observed at toxaphene concentrations from 2.5 to 40.0 mg/l, but these results were found to be not significant. The genotoxicity of toxaphene and its photodegradation products after UV-irradiation (3–6–9 h) at concentrations ranging from 7.5 to 60.0 mg/l was also examined in this study. An irradiated solution of technical toxaphene after 3 h showed no significant evidence of bacterial growth inhibition. However, exposure of Salmonella to 6 h UV-irradiated toxaphene showed a toxic effect compared with the negative control. After 9 h irradiation, a decrease of bacterial growth was observed. Activity of β-galactosidase in the presence of a toxaphene solution was significantly increased after 6 and 9 h irradiation, reaching values that were 2.4- and 3.1-fold higher, respectively, than the control, which exceeded the criteria of significant genotoxicity. These results show that while technical toxaphene is a weak, direct-acting mutagen in some bacterial tests, a dose-dependent toxicity and genotoxicity of its photoproducts could be conclusively demonstrated by the umuC test.     

Recording relative water table depth using PVC tape discolouration: Advantages and constraints in fens

Question: Recognizing water table dynamics in wetlands is crucial for understanding species‐environment relationships, ecosystem function and changes during restoration. The PVC tape discolouration method enables spatially and temporally extensive studies of reductive conditions associated with long‐term water table dynamics in peat soils. The reliability of the method has been verified only for ombrotrophic bogs, even though wide usage can be expected in minerotrophic fens. Location: T?reboň basin, Czech Republic. Methods: Using data from 49 plots in six poor and moderately rich fens, we correlated the directly measured lowest, highest and mean water table depths and the same variables indicated by discolouration of PVC tape attached to green bamboo stakes installed vertically in the soil profile. Results: The depth to the first sign of PVC discolouration was highly correlated with the directly measured position of the highest water table, the correlation between the depth of complete discolouration and the directly measured position of the lowest water table was poorer. The accuracy of the minimum water table measurement depended on the depth of the peat layer. Surprisingly, the depth at which the green bamboo stakes turned brown correlated highly with the minimum water table. Conclusions: The PVC tape discolouration method reliably indicates water table maxima in fens, but minima are not accurately indicated. The depth of the green bamboo discolouration is suggested as a new alternative indicator of the minimum water table, even in fens and mineral soils. Combining both methods enables efficient monitoring of water table dynamics at a large number of mire sites.     

Less input same output: simplified approach for population size assessment in Lepidoptera

With the aim of creating a simplified sampling scheme that would retain the accuracy of standard mark–release–recapture (MRR) sampling, but at a greatly reduced cost, we analysed 23 capture–recapture data sets from spatially closed populations of six Lepidoptera species according to the constrained Cormack–Jolly–Seber models. Subsequently the relationships between the estimates of population parameters were investigated in order to develop a regression equation that would enable us to calculate seasonal population size without sampling the population throughout the entire flight period. The proportion of individuals flying at peak population was highly variable (CV=0.39), but the variation decreased considerably (CV=0.14) after different life span and flight period length were accounted for. Over 90% of the variance of this proportion was explained by the life span:flight period length ratio. Simulations of hypothetical sampling schemes proved that schemes covering the second and third quarter of the flight period performed much better than those restricted to the second quarter only. The accuracy of seasonal population size estimated with the regression equation developed was comparable for intensive schemes (daily sampling) and non-intensive ones (sampling once in 2 or 3 days). We propose a simplified method of surveying butterfly populations that should be based on checking the presence of flying adults at the beginning and end of the flight period to assess its length, and MRR sampling covering its middle part, with intervals between capture days corresponding to the average life span of investigated butterflies.     

Population ecology of the endangered butterflies Maculinea teleius and M. nausithous and the implications for conservation

Butterflies of the genus Maculinea are highly endangered in Europe. The cuckoo species M. rebeli has been thoroughly investigated through both empirical and modelling studies, but less is known about the population ecology of predatory Maculinea. We present the findings of a 2-year research study on sympatric populations of two endangered butterflies: Maculinea teleius and M. nausithous in the Kraków region, southern Poland. The study comprised mark–release–recapture sampling and laboratory rearing of butterflies from larvae collected in the field. For both species the sex ratio was slightly, but consistently, female-biased and there was little year-to-year change in the seasonal population sizes. Daily numbers showed greater variation between the 2 years of the study due to the differences in daily survival rate. The average life span of laboratory-raised butterflies kept in ideal conditions was more than 6 days, compared to only 2–3 days in the field. The recruitment of both males and females consistently followed a bimodal pattern. A small proportion of individuals (maximum 25%) changed sites, in spite of relatively short distances of ca. 100 m separating them. The results indicate that populations of both species are typically stable within their sites, possibly due to larval polymorphism, but there is little inter-site mobility and thus landscape corridors seem necessary to enhance metapopulation viability. A further problem to be considered in the conservation of Maculinea butterflies is the fact that their very short life span in relation to flight-period length reduces the effective population size.     

Phase transitions and antiferromagnetism in copper(II) hexanoates: a new tetranuclear type of copper carboxylate paddle-wheel association

A series of compounds of formula [{Cu₂(OOCC_mH_2m + 1)₄(urea)}₂] (m=5-11) have been characterized. X-ray structure analysis for the hexanoate compound reveals a new type of tetranuclear dicopper(II) tetracarboxylate, where the central coordination sphere in [{Cu₂(OOCC₅H₁₁)₄(urea)}₂] is composed of two dinuclear dicopper tetracarboxylates, connected via two inter-dinuclear Cu-O coordination bonds at a distance 2.222(2) Å through the apical positions of two dimers. Urea molecules (Cu-O 2.114(2) Å) occupy both outside apical positions of the resulting tetranuclear units. A strong antiferromagnetic behaviour has been shown for [{Cu₂(OOCC₅H₁₁)₄(urea)}₂] (−2J=261.4(4) cm⁻¹), and compared with related isolated dinuclear and polymeric hexanoate compounds [Cu₂(OOCC₅H₁₁)₄(urea)₂], [Cu₂(OOCC₅H₁₁)₄]_n, respectively. Only small differences in the magnetic susceptibility have been found, while EPR spectroscopy showed significantly different results for all three hexanoate compounds, also with the dicopper tetracarboxylate central core and square-pyramidal CuO₄O chromophores. A solid-to-solid phase transition for [{Cu₂(OOCC₅H₁₁)₄(urea)}₂] was observed by magnetic measurement and analysed for the whole series [{Cu₂(OOCC_mH_2m + 1)₄(urea)}₂] by TG, DTA, and variable temperature XRD studies.     

Proteins involved in maturation pathways of plant mitochondrial and plastid c-type cytochromes

c-type cytochromes are characterized by the presence of two covalent bonds linking heme to apocytochrome and by the heme attachment motif in the apoprotein. Several molecular systems for the maturation of c-type cytochromes have evolved in different organisms. The best characterized are three of them: system I, system II and system III. Heme is synthesized in bacterial cytoplasm, in plastids, and in animal and fungal mitochondria. Therefore the maturation of bacterial and plastid c-type cytochromes involves the transport of heme and apocytochrome from the n-side to the p-side of the respective biological membranes and the formation of the covalent bond at the p-side. It should be underlined that the site of the c-type apocytochrome synthesis is also distinct from the site of its functioning. The aim of this review is to present the current state of knowledge concerning the structure and function of two systems - system I and system II - in the maturation of plant mitochondrial and plastid c-type cytochromes, respectively.