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Groundwater is one of our most important resources, however groundwater ecosystems are among the most understudied habitats of the planet earth. Studies on groundwater organisms are hampered by the difficult accessibility of species, the lack of morphological differentiation and the limitation for laboratory cultures. One important approach to overcome these shortcomings is to provide sensitive genetic methods to unravel patterns of biodiversity, population structure and gene flow in natural populations. In this study we present five sets of microsatellite markers developed for the isopods Asellus aquaticus and Proasellus slavus, the cyclopoides Paracyclops fimbriatus and Acanthocyclops sensitivus and the harpacticoide Bryocamptus echinatus (Crustacea). Two of these species were subjected to detailed population genetic analyses: We studied 501 specimens of Asellus aquaticus from four different regions in Northern Germany using nine microsatellite markers and 70 specimens of Bryocamptus echinatus using nine microsatellite markers from three different sampling sites in South-Western Germany. Our results show that genetic diversity is high (A. aquaticus: 10 to 20 and B. echinatus: 4 to 18 alleles per locus) among populations of aquatic invertebrates, populations are highly differentiated (F

Inhaltlich stellt der Text zunächst die aktuelle neurobiologische und paarpsychologische Forschung zum Thema Liebesbeziehungen vor, ergänzt durch eine Übersicht der Entwicklung von Partnerschaften aus soziologischer und juristischer Perspektive. Da zwei Krankenhausserien im Mittelpunkt der Filmanalyse stehen, schließt der theoretische Teil mit einem historischen Abriss zu Serie und Krankenhausserie ab. Im Ergebnis der Filmanalyse erlaubt die interdisziplinäre Herangehensweise eine differenziertere Darstellung und Interpretation serialer Liebesbeziehungen jenseits der bisherigen Studien und den ihnen enthaltenen, eher pauschalisierend wirkenden Beschreibungen von Romantik und Idealisierung, aber auch entgegen den in den Kulturwissenschaften verbreiteten Vorstellungen von Liebe als Phänomen im Rahmen von Zuschreibungen oder Inszenierung. Kurz gefasst lassen sich die psychologischen und neurobiologischen Konstrukte der Verliebtheit und Bindung eingebettet in den narrativen seriellen Prozesscharakter gut einordnen, werden jedoch in Abhängigkeit von der Storyline und Filmästhetik der beiden Serien unterschiedlich konstruiert. Legt man weiterhin das psychologische Konstrukt der Paarqualität zugrunde, so lässt sich der Vorwurf der „Traumpaare und Traumhochzeiten“ nicht halten, vielmehr befinden sich die Serienpaare in desolaten, wenig wünschenswerten Verbindungen, denen es an Sicherheit, Vertrauen, Offenheit, Respekt sowie gegenseitiger Zuwendung und wesentlichen Gesprächen ganz offensichtlich mangelt. Im Ausblick werden mögliche Zuschauermotivationen und auch potentielle Auswirkungen auf die Vorstellung der Rezipienten über Paarbeziehungen reflektiert.

A prerequisite for structural genomics and related projects is to standardize the process of gene overexpression and protein solubility screening to enable automation for higher throughput. We have tested a methodology to rapidly subclone a large number of human genes and screen these for expression and protein solubility in Escherichia coli. The methodology, which can be partly automated, was used to compare the effect of six different N-terminal fusion proteins and an N-terminal 6*His tag. As a realistic test set we selected 32 potentially interesting human proteins with unknown structures and sizes suitable for NMR studies. The genes were transferred from cDNA to expression vectors using subcloning by recombination. The subcloning yield was 100% for 27 (of 32) genes for which a PCR fragment of correct size could be obtained. Of these, 26 genes (96%) could be overexpressed at detectable levels and 23 (85%) are detected in the soluble fraction with at least one fusion tag. We find large differences in the effects of fusion protein or tag on expression and solubility. In short, four of seven fusions perform very well, and much better than the 6*His tag, but individual differences motivate the inclusion of several fusions in expression and solubility screening. We also conclude that our methodology and expression vectors can be used for screening of genes for structural studies, and that it should be possible to obtain a large fraction of all NMR-sized and nonmembrane human proteins as soluble fusion proteins in E. coli.

DEXD/H-box RNA helicases couple ATP hydrolysis to RNA remodeling by an unknown mechanism. We used x-ray crystallography and biochemical analysis of the human DEXD/H-box protein DDX19 to investigate its regulatory mechanism. The crystal structures of DDX19, in its RNA-bound prehydrolysis and free posthydrolysis state, reveal an alpha-helix that inserts between the conserved domains of the free protein to negatively regulate ATPase activity. This finding was corroborated by biochemical data that confirm an autoregulatory function of the N-terminal region of the protein. This is the first study describing crystal structures of a DEXD/H-box protein in its open and closed cleft conformations.

Impairment of the formation or action of hydrogen sulfide (H(2)S), an endogenous gasotransmitter, is associated with various diseases, such as hypertension, diabetes mellitus, septic and hemorrhagic shock, and pancreatitis. Cystathionine beta-synthase and cystathionine gamma-lyase (CSE) are two pyridoxal-5'-phosphate (PLP)-dependent enzymes largely responsible for the production of H(2)S in mammals. Inhibition of CSE by DL-propargylglycine (PAG) has been shown to alleviate disease symptoms. Here we report crystal structures of human CSE (hCSE), in apo form, and in complex with PLP and PLP.PAG. Structural characterization, combined with biophysical and biochemical studies, provides new insights into the inhibition mechanism of hCSE-mediated production of H(2)S. Transition from the open form of apo-hCSE to the closed PLP-bound form reveals large conformational changes hitherto not reported. In addition, PAG binds hCSE via a unique binding mode, not observed in PAG-enzyme complexes previously. The interaction of PAG-hCSE was not predicted based on existing information from known PAG complexes. The structure of hCSE.PLP.PAG complex highlights the particular importance of Tyr(114) in hCSE and the mechanism of PAG-dependent inhibition of hCSE. These results provide significant insights, which will facilitate the structure-based design of novel inhibitors of hCSE to aid in the development of therapies for diseases involving disorders of sulfur metabolism.

Tankyrases are recently discovered proteins implicated in many important functions in the cell including telomere homeostasis and mitosis. Tankyrase modulates the activity of target proteins through poly(ADP-ribosyl)ation, and here we report the structure of the catalytic poly(ADP-ribose) polymerase (PARP) domain of human tankyrase 1. This is the first structure of a PARP domain from the tankyrase subfamily. The present structure reveals that tankyrases contain a short zinc-binding motif, which has not been predicted. Tankyrase activity contributes to telomere elongation observed in various cancer cells and tankyrase inhibition has been suggested as a potential route for cancer therapy. In comparison with other PARPs, significant structural differences are observed in the regions lining the substrate-binding site of tankyrase 1. These findings will be of great value to facilitate structure-based design of selective PARP inhibitors, in general, and tankyrase inhibitors, in particular.

Argininosuccinate synthetase catalyzes the transformation of citrulline and aspartate into argininosuccinate and pyrophos­phate using the hydrolysis of ATP to AMP and pyrophos­phate. This enzymatic process constitutes the rate-limiting step in both the urea and arginine–citrulline cycles. Previous studies have investigated the crystal structures of argininosuccinate synthetase from bacterial species. In this work, the first crystal structure of human argininosuccinate synthetase in complex with the substrates citrulline and aspartate is presented. The human enzyme is compared with structures of argininosuccinate synthetase from bacteria. In addition, the structure also provides new insights into the function of the numerous clinical mutations identified in patients with type I citrullinaemia (also known as classic citrullinaemia).

DExD-box helicases are involved in all aspects of cellular RNA metabolism. Conserved domains 1 and 2 contain nine signature motifs that are responsible for nucleotide binding, RNA binding and ATP hydrolysis. The human DEAD-box helicase DDX3X has been associated with several different cellular processes, such as cell-growth control, mRNA transport and translation, and is suggested to be essential for the export of unspliced/partially spliced HIV mRNAs from the nucleus to the cytoplasm. Here, the crystal structure of conserved domains 1 and 2 of DDX3X, including a DDX3-specific insertion that is not generally found in human DExD-box helicases, is presented. The N-terminal domain 1 and the C-terminal domain 2 both display RecA-like folds comprising a central beta-sheet flanked by alpha-helices. Interestingly, the DDX3X-specific insertion forms a helical element that extends a highly positively charged sequence in a loop, thus increasing the RNA-binding surface of the protein. Surprisingly, although DDX3X was crystallized in the presence of a large excess of ADP or the slowly hydrolyzable ATP analogue ATPgammaS the contaminant AMP was seen in the structure. A fluorescent-based stability assay showed that the thermal stability of DDX3X was increased by the mononucleotide AMP but not by ADP or ATPgammaS, suggesting that DDX3X is stabilized by AMP and elucidating why AMP was found in the nucleotide-binding pocket.

The efficiency and high specificity of tobacco etch virus (TEV) protease has made it widely used for cleavage of recombinant fusion proteins. However, the production of TEV protease in E. coli is hampered by low solubility. We have subjected the gene encoding TEV protease to directed evolution to improve the yield of soluble protein. Libraries of mutated genes obtained by error-prone PCR and gene shuffling were introduced into the Gateway cloning system for facilitated transfer between vectors for screening, purification, or other applications. Fluorescence based in vivo solubility screening was carried out by cloning the libraries into a plasmid encoding a C-terminal GFP fusion. Mutant genes giving rise to high GFP fluorescence intensity indicating high levels of soluble TEV-GFP were subsequently transferred to a vector providing a C-terminal histidine tag for expression, purification, and activity tests of mutated TEV. We identified a mutant, TEV(SH), in which three amino acid substitutions result in a five-fold increase in the yield of purified protease with retained activity.

Tobacco etch virus (TEV) protease is generally used to remove affinity tags from target proteins. It has been reported that some detergents inhibit the activity of this protease, and therefore should be avoided when removing affinity tags from membrane proteins. The aim of this study was to explore and evaluate this further. Hence, affinity tag removal with TEV protease was tested from three membrane proteins (a Pgp synthase and two CorA homologs) in the presence of 16 different detergents commonly used in membrane protein purification and crystallization. We observed that in the presence of the same detergent (Triton X-100), TEV protease could remove the affinity tag completely from one protein (CorA) but not from another protein (Pgp synthase). There was also a large variation in yield of cleaved membrane protein in different detergents, which probably depends on features of the protein-detergent complex. These observations show that, contrary to an earlier report, detergents do not inhibit the enzymatic activity of the TEV protease.

Selenium and sulfur are two closely related basic elements utilized in nature for a vast array of biochemical reactions. While toxic at higher concentrations, selenium is an essential trace element incorporated into selenoproteins as selenocysteine (Sec), the selenium analogue of cysteine (Cys). Sec lyases (SCLs) and Cys desulfurases (CDs) catalyze the removal of selenium or sulfur from Sec or Cys and generally act on both substrates. In contrast, human SCL (hSCL) is specific for Sec although the only difference between Sec and Cys is the identity of a single atom. The chemical basis of this selenium-over-sulfur discrimination is not understood. Here we describe the X-ray crystal structure of hSCL and identify Asp146 as the key residue that provides the Sec specificity. A D146K variant resulted in loss of Sec specificity and appearance of CD activity. A dynamic active site segment also provides the structural prerequisites for direct product delivery of selenide produced by Sec cleavage, thus avoiding release of reactive selenide species into the cell. We thus here define a molecular determinant for enzymatic specificity discrimination between a single selenium versus sulfur atom, elements with very similar chemical properties. Our findings thus provide molecular insights into a key level of control in human selenium and selenoprotein turnover and metabolism.

DEAD-box RNA helicases play various, often critical, roles in all processes where RNAs are involved. Members of this family of proteins are linked to human disease, including cancer and viral infections. DEAD-box proteins contain two conserved domains that both contribute to RNA and ATP binding. Despite recent advances the molecular details of how these enzymes convert chemical energy into RNA remodeling is unknown. We present crystal structures of the isolated DEAD-domains of human DDX2A/eIF4A1, DDX2B/eIF4A2, DDX5, DDX10/DBP4, DDX18/myc-regulated DEAD-box protein, DDX20, DDX47, DDX52/ROK1, and DDX53/CAGE, and of the helicase domains of DDX25 and DDX41. Together with prior knowledge this enables a family-wide comparative structural analysis. We propose a general mechanism for opening of the RNA binding site. This analysis also provides insights into the diversity of DExD/H- proteins, with implications for understanding the functions of individual family members.

Insulin-like growth factor II (IGF-II) cDNA was isolated from adult guinea pig liver by polymerase chain reaction (PCR) screening. A cDNA sequence was obtained corresponding to part of the preproIGF-II, including the signal peptide, the mature IGF-II and 37 amino acids of the acid carboxy-terminal E-domain. Amino acid sequence prediction, based on the cDNA clone, showed that mature guinea pig IGF-II has a high homology with both human and rat IGF-II, 100 and 94% identity, respectively. Levels of IGF-II mRNA in guinea pigs of different ages were analyzed by solution hybridization/RNase protection assay using part of the isolated IGF-II cDNA as a probe. There is a marked developmental regulation of IGF-II after birth. IGF-II mRNA levels were high in fetal livers, and decreased 15- to 30-fold in adults. As in man, but in contrast to rats, adult guinea pigs have significant levels of IGF-II mRNA in the liver. In fetal guinea pigs, the expression of IGF-II mRNA was 5-, 2- and 70-fold lower in kidney, skeletal muscle and brain cortex, respectively, than in liver. IGF-II mRNA levels in kidney and skeletal muscle of fetal guinea pigs were 5- and 4-fold higher, respectively, compared with adults. Similar sizes of IGF-II mRNA transcripts could be observed on Northern blots in newborn rats and in fetal guinea pigs. Our conclusions are that the mature IGF-II peptide in the guinea pig is 100% identical to the mature peptide in the human. IGF-II mRNA is developmentally regulated in the guinea pig and this developmental regulation of IGF-II expression is different when compared with that of rats, guinea pigs having a higher adult IGF-II expression in liver, kidney and skeletal muscle. Finally, individual IGF-II transcripts may be both differentially and developmentally regulated in guinea pig tissues.

Paraplegin is an m-AAA protease of the mitochondrial inner membrane that is linked to hereditary spastic paraplegias. The gene encodes an FtsH-homology protease domain in tandem with an AAA+ homology ATPase domain. The protein is believed to form a hexamer that uses ATPase-driven conformational changes in its AAA-domain to deliver substrate peptides to its protease domain. We present the crystal structure of the AAA-domain of human paraplegin bound to ADP at 2.2 Å. This enables assignment of the roles of specific side chains within the catalytic cycle, and provides the structural basis for understanding the mechanism of disease mutations. Enhanced version This article can also be viewed as an enhanced version in which the text of the article is integrated with interactive 3D representations and animated transitions. Please note that a web plugin is required to access this enhanced functionality. Instructions for the installation and use of the web plugin are available in Text S1.

Transient overexpression of ras, mos, or fos transcribed from various inducible promoters in NIH 3T3 cells causes significant increases in the frequency of chromosomal aberrations and, as shown for fos, in gene mutations. Under the experimental conditions of exponential growth and full serum supply, overexpression of the oncogenes does not increase the proliferation rate of cells. The generation of ras- and mos-induced chromosomal aberrations was suppressed in cells that had been deprived of fos protein by antisense c-fos oligodeoxynucleotides. The induction of chromosomal aberrations by ultraviolet irradiation is also suppressed by antisense c-fos oligodeoxynucleotides. The data suggest that fos protein alone, or a transcription factor that contains fos protein as a subunit, activates or induces the synthesis of one or several mutator functions. Oncogene-driven mutagenesis could account for the accumulation of additional mutations after the activation of an oncogene, which may furnish a mechanistic basis for tumor promotion and tumor progression.

We have compared four different vectors for expression of proteins with N- or C-terminal hexahistidine (His6) tags in Escherichia coli by testing these on 20 human proteins. We looked at total recombinant protein production levels per gram dry cell weight, solubility of the target proteins, and yield of soluble and total protein when purified by immobilized metal ion affinity purification. It was found that, in general, both N- and C-terminal His6 tags have a noticeable negative effect on protein solubility, but the effect is target protein specific. A solubilizing fusion tag was able to partly counteract this negative effect. Most target proteins could be purified under denaturing conditions and about half of the proteins could be purified under physiological conditions. The highest protein production levels and yield of purified protein were obtained from a construct with a C-terminal His tag. We also observe a large variation in cell growth rate, which we determined to be partly caused by the expression vectors and partly by the targets. This variation was found to be independent of the production level, solubility and tertiary structure content of the target proteins. abbreviations: BSA – bovine serum albumin; DBD – DNA binding domain; DCW – dry cell weight; EDTA – ethylenediaminetetraacetic acid; GFP – green fluorescent protein; IMAC – immobilized metal ion affinity chromatography; IPTG – isopropyl-β-d-thiogalactopyranoside; LB – Luria-Bertani; MES – 2-(N-morpholino) ethane sulfonic acid; OD – optical density; ORF – open reading frame; PCR – polymerase chain reaction; SDS-PAGE – sodium dodecyl sulfate polyacrylamide gel electrophoresis.

We examined the internal mobility of the estrogen receptor DNA-binding domain (ER DBD) using NMR15N relaxation measurements and compared it to that of the glucocorticoid receptor DNA-binding domain (GR DBD). The studied protein fragments consist of residues Arg183-His267 of the human ER and residues Lys438-Gln520 of the rat GR. The15N longitudinal (R1) and transverse (R2) relaxation rates and steady state {1H}-15N nuclear Overhauser enhancements (NOEs) were measured at 30 degrees C at1H NMR frequencies of 500 and 600 MHz. The NOE versus sequence profile and calculated order parameters for ER DBD backbone motions indicate enhanced internal dynamics on pico- to nanosecond time-scales in two regions of the core DBD. These are the extended strand which links the DNA recognition helix to the second zinc domain and the larger loop region of the second zinc domain. The mobility of the corresponding regions of the GR DBD, in particular that of the second zinc domain, is more limited. In addition, we find large differences between the ER and GR DBDs in the extent of conformational exchange mobility on micro- to millisecond time-scales. Based on measurements of R2as a function of the15N refocusing (CPMG) delay and quantitative (Lipari-Szabo-type) analysis, we conclude that conformational exchange occurs in the loop of the first zinc domain and throughout most of the second zinc domain of the ER DBD. The conformational exchange dynamics in GR DBD is less extensive and localized to two sites in the second zinc domain. The different dynamical features seen in the two proteins is consistent with previous studies of the free state structures in which the second zinc domain in the ER DBD was concluded to be disordered whereas the corresponding region of the GR DBD adopts a stable fold. Moreover, the regions of the ER DBD that undergo conformational dynamics on the micro- to millisecond time-scales in the free state are involved in intermolecular protein-DNA and protein-protein interactions in the dimeric bound state. Based on the present data and the previously published dynamical and DNA binding properties of a GR DBD triple mutant which recognize an ER binding site on DNA, we argue that the free state dynamical properties of the nuclear receptor DBDs is an important element in molecular recognition upon DNA binding.