The first potassium channel toxin from the venom of the Iranian scorpion Odonthobuthus doriae

The very first member of K(+) channels toxins from the venom of the Iranian scorpion Odonthobuthus doriae (OdK1) was purified, sequenced and characterized physiologically. OdK1 has 29 amino acids, six conserved cysteines and a pI value of 4.95. Based on multiple sequence alignments, OdK1 was classified as alpha-KTx 8.5. The pharmacological effects of OdK1 were studied on six different cloned K(+) channels (vertebrate Kv1.1-Kv1.5 and Shaker IR) expressed in Xenopus laevis oocytes. Interestingly, OdK1 selectively inhibited the currents through Kv1.2 channels with an IC50 value of 183+/-3 nM but did not affect any of the other channels.     

Extracellular production and affinity purification of recombinant proteins with Escherichia coli using the versatility of the maltose binding protein

Recombinant proteins are essential products of today's industrial biotechnology. In this study we address two crucial factors in recombinant protein production: (i) product accessibility and (ii) product recovery. Escherichia coli, one of the most frequently used hosts for recombinant protein expression, does not inherently secrete proteins into the extracellular environment. The major drawback of this expression system is, therefore, to be found in the intracellular protein accumulation and hampered product accessibility. We have constructed a set of expression vectors in order to facilitate extracellular protein production and purification. The maltose binding protein from E. coli is used as fusion partner for several proteins of interest allowing an export to the bacteria's periplasm via both the Sec and the Tat pathway. Upon coexpression of a modified Cloacin DF13 bacteriocin release protein, the hybrid proteins are released into the culture medium. This essentially applies to a distinguished reporter molecule, the green fluorescent protein, for which an extracellular production was not reported so far. The sequestered proteins can be purified to approximate homogeneity by a simple, rapid and cheap procedure which utilizes the affinity of the maltose binding protein to α-1,4-glucans.     

A new genus and species of freshwater monostiliferous hoplonemertean (Nemertea, Enopla) from the People's Republic of China

A new genus and species of freshwater monostiliferous hoplonemertean, Limnemertes poyangensis gen. et sp. nov., from Poyang Lake, People's Republic of China, is described and illustrated. The taxon is compared and contrasted with previously described freshwater hoplonemerteans. This is the fourth species of freshwater nemertean to be described from China and the first recorded from Poyang Lake.     

The Rothmund-Thomson gene product RECQL4 localizes to the nucleolus in response to oxidative stress

Mutations in the RECQL4 helicase gene have been linked to Rothmund-Thomson syndrome (RTS), which is characterized by poikiloderma, growth deficiency, and a predisposition to cancer. Examination of RECQL4 subcellular localization in live cells demonstrated a nucleoplasmic pattern and, to a lesser degree, staining in nucleoli. Analysis of RECQL4-GFP deletion mutants revealed two nuclear localization regions in the N-terminal region of RECQL4 and a nucleolar localization signal at amino acids 376-386. RECQL4 localization did not change after treatment with the DNA-damaging agents bleomycin, etoposide, UV irradiation and gamma irradiation, in contrast to the Bloom and Werner syndrome helicases that relocate to distinct nuclear foci after damage. However, in a significant number of cells exposed to hydrogen peroxide or streptonigrin, RECQL4 accumulated in nucleoli. Using a T7 phage display screen, we determined that RECQL4 interacts with poly(ADP-ribose) polymerase-1 (PARP-1), a nuclear enzyme that promotes genomic integrity through its involvement in DNA repair and signaling pathways. The RECQL4 nucleolar localization was inhibited by pretreatment with a PARP-1 inhibitor. The C-terminal portion of RECQL4 was found to be an in vitro substrate for PARP-1. These results demonstrate changes in the intracellular localization of RECQL4 in response to oxidative stress and identify an interaction between RECQL4 and PARP-1.     

Developmentally regulated thyroid hormone distributor proteins in marsupials, a reptile, and fish

Thyroid hormones are essential for vertebrate development. There is a characteristic rise in thyroid hormone levels in blood during critical periods of thyroid hormone-regulated development. Thyroid hormones are lipophilic compounds, which readily partition from an aqueous environment into a lipid environment. Thyroid hormone distributor proteins are required to ensure adequate distribution of thyroid hormones, throughout the aqueous environment of the blood, and to counteract the avid partitioning of thyroid hormones into the lipid environment of cell membranes. In human blood, these proteins are albumin, transthyretin and thyroxine-binding globulin. We analyzed the developmental profile of thyroid hormone distributor proteins in serum from a representative of each order of marsupials (M. eugenii; S.crassicaudata), a reptile (C. porosus), in two species of salmonoid fishes (S. salar; O. tshawytsch), and throughout a calendar year for sea bream (S. aurata). We demonstrated that during development, these animals have a thyroid hormone distributor protein present in their blood which is not present in the adult blood. At least in mammals, this additional protein has higher affinity for thyroid hormones than the thyroid hormone distributor proteins in the blood of the adult. In fish, reptile and polyprotodont marsupial, this protein was transthyretin. In a diprotodont marsupial, it was thyroxine-binding globulin. We propose an hypothesis that an augmented thyroid hormone distributor protein network contributes to the rise in total thyroid hormone levels in the blood during development.     

Ammonium Transformation in a Nitrogen-Rich Tidal Freshwater Marsh

The fate and transport of watershed-derived ammonium in a tidal freshwater marsh fringing the nutrient rich Scheldt River, Belgium, was quantified in a whole ecosystem ¹⁵N labeling experiment. In late summer (September) we added ¹⁵N-NH₄⁺ to the flood water entering a 3477 m² tidal freshwater marsh area, and traced the ammonium processing and retention in four subsequent tide cycles. In this paper we present the results for the water-phase components of the marsh system and compare them to a similar experiment conducted in spring/early summer (May). Changes in concentration and isotopic enrichment of NO₃⁻ + NO₂⁻, N₂O, N₂, NH₄⁺ and suspended particulate nitrogen (SPN) were measured in concert with a mass balance study. All analyzed N-pools were labeled, and 49% of the added ¹⁵NH₄⁺ was retained or transformed. The most important pool for ¹⁵N was nitrate, accounting for 17% of ¹⁵N-transformation. N₂, N₂O and SPN accounted for 2.4, 0.02 and 1.4%, respectively. The temporal and spatial patterns of ¹⁵N transformation in the water phase component of the system were remarkably similar to those observed in May, indicating good reproducibility of the whole ecosystem labeling approach, but the absolute ammonium transformation rate was 3 times higher in May. While the marsh surface area was crucial for nitrification in May this was less pronounced in September. Denitrification, on the other hand, appeared more important in September compared to May.     

Patterns of diversifying selection in the phytotoxin-like scr74 gene family of Phytophthora infestans

Phytophthora infestans, the organism responsible for the Irish famine, causes late blight, a re-emerging disease of potato and tomato. Little is known about the molecular evolution of P. infestans genes. To identify candidate effector genes (virulence or avirulence genes) that may have co-evolved with the host, we mined expressed sequence tag (EST) data from infection stages of P. infestans for secreted and potentially polymorphic genes. This led to the identification of scr74, a gene that encodes a predicted 74-amino acid secreted cysteine-rich protein with similarity to the Phytophthora cactorum phytotoxin PcF. The expression of scr74 was upregulated approximately 60-fold 2 to 4 days after inoculation of tomato and was also significantly induced during early stages of colonization of potato. The scr74 gene was found to belong to a highly polymorphic gene family within P. infestans with 21 different sequences identified. Using the approximate and maximum likelihood (ML) methods, we found that diversifying selection likely caused the extensive polymorphism observed within the scr74 gene family. Pairwise comparisons of 17 scr74 sequences revealed elevated ratios of nonsynonymous to synonymous nucleotide-substitution rates, particularly in the mature region of the proteins. Using ML, all 21 polymorphic amino acid sites were identified to be under diversifying selection. Of these 21 amino acids, 19 are located in the mature protein region, suggesting that selection may have acted on the functional portions of the proteins. Further investigation of gene copy number and organization revealed that the scr74 gene family comprises at least three copies located in a region of no more than 300 kb of the P. infestans genome. We found evidence that recombination contributed to sequence divergence within at least one gene locus. These results led us to propose an evolutionary model that involves gene duplication and recombination, followed by functional divergence of scr74 genes. This study provides support for using diversifying selection as a criterion for identifying candidate effector genes from sequence databases.     

Automated analysis of vapor diffusion crystallization drops with an X-ray beam

Crystallogenesis, usually based on the vapor diffusion method, is currently considered one of the most difficult steps in macromolecular X-ray crystallography. Due to the increasing number of crystallization assays performed by protein crystallographers, several automated analysis methods are under development. Most of these methods are based on microscope images and shape recognition. We propose an alternative method of identifying protein crystals: by directly exposing the crystallization drops to an X-ray beam. The resulting diffraction provides far more information than classical microscope images. Not only is the presence of diffracting crystals revealed, but also a first estimation of the space group, cell parameters, and mosaicity is obtained. In certain cases, it is also possible to collect enough data to verify the presence of a specific substrate or a heavy atom. All these steps are performed without the sometimes tedious necessity of removing crystals from their crystallization drop.     

Ferredoxin-NADP+ reductase and ferredoxin of the protozoan parasite Toxoplasma gondii interact productively in Vitro and in Vivo

Toxoplasma gondii possesses an apicoplast-localized, plant-type ferredoxin-NADP(+) reductase. We have cloned a [2Fe-2S] ferredoxin from the same parasite to investigate the interplay of the two redox proteins. A detailed characterization of the two purified recombinant proteins, particularly as to their interaction, has been performed. The two-protein complex was able to catalyze electron transfer from NADPH to cytochrome c with high catalytic efficiency. The redox potential of the flavin cofactor (FAD/FADH(-)) of the reductase was shown to be more positive than that of the NADP(+)/NADPH couple, thus favoring electron transfer from NADPH to yield reduced ferredoxin. The complex formation between the reductase and ferredoxins from various sources was studied both in vitro by several approaches (enzymatic activity, cross-linking, protein fluorescence quenching, affinity chromatography) and in vivo by the yeast two-hybrid system. Our data show that the two proteins yield an active complex with high affinity, strongly suggesting that the two proteins of T. gondii form a physiological redox couple that transfers electrons from NADPH to ferredoxin, which in turn is used by some reductive biosynthetic pathway(s) of the apicoplast. These data provide the basis for the exploration of this redox couple as a drug target in apicomplexan parasites.