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AbstractAnimals subject to environmental stress often exhibit higher levels of developmental instability, frequently measured as Fluctuating Asymmetry (FA), small, random deviations from perfect left-right symmetry in bilaterally symmetrical animals. We used FA measurement as a means of determining the impact of a catastrophic ecosystem disturbance (a major Class F3 tornado) on populations of Schizocosa ocreata, a common forest-dwelling wolf spider. As FA in sexually selected ornamental traits in males has been shown in some (but not all) species studied to be a sensitive indicator of environmental stress, we measured FA of foreleg tufts in male S. ocreata.Spiders from the first post-disturbance generation were collected by pitfall trapping in the spring of 2000, and preserved specimens were photographed and measured using digital imaging. Spiders from disturbed and undisturbed areas within the forest did not differ significantly in body size parameters, although body condition varied significantly between sites. Signed (R—L) FA of male tuft area was normally distributed with a mean of zero, indicating “ideal” or “true” FA, a reflection of Developmental Instability (DI). Although tuft area was not significantly different between sites, tuft area FA in male S. ocreatawas significantly higher in the disturbed site, suggesting that ecosystem-level stress may produce higher levels of developmental instability in arthropod populations. Results of our study indicate that FA in this secondary sexual character (male leg tufts) is a more sensitive indicator of putative environmental stress than a non-sexual trait (femur length). To our knowledge, this is the first study of FA in invertebrates associated with environmental stress from a catastrophic natural disturbance.

Yeast two-hybrid (Y2H) screening methods are an effective means for the detection of protein–protein interactions. Optimisation and automation has increased the throughput of the method to an extent that allows the systematic mapping of protein–protein interactions on a proteome-wide scale. Since two-hybrid screens fail to detect a great number of interactions, parallel high-throughput approaches are needed for proteome-wide interaction screens. In this review, we discuss and compare different approaches for adaptation of Y2H screening to high-throughput, the limits of the method and possible alternative approaches to complement the mapping of organism-wide protein–protein interactions.

Tracing the history of Saint Mark's Basilica in Venice: Historians have carried out painstaking research into the changes made to this elaborate building through the centuries.

Simple eukaryotic cells such as yeast could contain around 800 protein complexes, as two new comprehensive studies show. But slightly different approaches resulted in surprising differences between the two datasets, showing that more work is required to get a complete picture of the yeast interactome.

Den Ursprüngen der Markuskirche in Venedig auf der Spur: Wie das kunstvolle Bauwerk im Laufe der Jahrhunderte umgestaltet wurde, haben Bauforscher detailgenau untersucht.

Male Schizocosa ocreata wolf spiders court females using displays that involve dramatic waving motions of their ornamented forelegs. In nature males are often found missing one, and sometimes both, of these legs. We here find that, when exposed to female draglines, males missing these signalling appendages perform a high intensity courtship element ('jerky tapping') at a greater rate than intact males. Loss of forelegs was also associated with changes in chemoexploration activity, tendency to stand stationary, and performance of a low intensity courtship element ('tapping'). The age of the virgin female from which draglines were collected also had an important influence on male courtship behaviour; males on the draglines of old females were less likely to court and, if they did court, did so at a lower rate. These data indicate that courtship behaviour of male S. ocreata is flexible, varying both with the state of the male himself and the state of the female whose draglines he has encountered. Such individual courtship flexibility raises challenging questions of what processes and trade-offs underlie male courtship decision-making, and whether autotomy-induced changes in courtship activity represent adaptive compensation.

The phox homology (PX) domain is a phosphoinositide-binding domain that is conserved from yeast to human. Here we show for the first time by genome-wide two-hybrid screens and in vitrobinding assays that the PX domain is a bona fide protein interaction domain. The yeast PX domain-only proteins Grd19p (YOR357C) and Ypt35p (YHR105W), as well as the isolated PX domains from Mvp1p (YMR004W), Snx42p/Cvt20p/Atg20p (YDL113C), Vam7p (YGL212W), and Vps17p (YOR132W), yielded a total of 40 reproducible two-hybrid interactions. Thirty-five interactions were found for the full-length proteins of Bem1p (YBR200W), Snx42p, Snx4p/Cvt13p (YJL036W), Vam7p, Vps5p (YOR069W), and Vps17p, but these appear not to require the PX domain, because these interactions could not be reproduced with PX-only baits. Interactions of Grd19p, Vam7p, Vps5p, Vps17p, and Ypt35p with members of the Yip1p family of proteins were detected consistently and were verified by in vitrobinding assays. The N-terminal cytoplasmic domain of Yip1p and Yif1p mediates these interactions with PX domains. A mutation in the lipid-binding pocket of Ypt35p that reduces lipid binding markedly does not affect these PX domain protein interactions, arguing that lipid binding uses a different interaction surface than protein binding.

The biological significance of protein interactions, their method of generation and reliability is briefly reviewed. Protein interaction networks adopt a scale-free topology that explains their error tolerance or vulnerability, depending on whether hubs or peripheral proteins are attacked. Networks also allow the prediction of protein function from their interaction partners and therefore, the formulation of analytical hypotheses. Comparative network analysis predicts interactions for distantly related species based on conserved interactions, even if sequences are only weakly conserved. Finally, the medical relevance of protein interaction analysis is discussed and the necessity for data integration is emphasized.

All members of the regulator of G protein signaling (RGS) family contain a conserved core domain that can accelerate G protein GTPase activity. The RGS in yeast, Sst2, can inhibit a G protein signal leading to mating. In addition, some RGS proteins contain an N-terminal domain of unknown function. Here we use complementary whole genome analysis methods to investigate the function of the N-terminal Sst2 domain. To identify a signaling pathway regulated by N-Sst2, we performed genome-wide transcription profiling of cells expressing this fragment alone and found differences in 53 transcripts. Of these, 40 are induced by N-Sst2, and nearly all contain a stress response element (STRE) in the promoter region. To identify components of a signaling pathway leading from N-Sst2 to STREs, we performed a genome-wide two-hybrid analysis using N-Sst2 as bait and found 17 interacting proteins. To identify the functionally relevant interacting proteins, we analyzed all of the available gene deletion mutants and found three (vps36Δ, pep12Δ, and tlg2Δ) that induce STRE and also repress pheromone-dependent transcription. We selectedVPS36for further characterization. A vps36Δ mutation diminishes signaling by pheromone as well as by downstream components including the G protein, effector kinase (Ste11), and transcription factor (Ste12). Conversely, overexpression of Vps36 enhances the pheromone response in sst2Δ cells but not in wild type. These findings indicate that Vps36 and Sst2 have opposite and opposing effects on the pheromone and stress response pathways, with Vps36 acting downstream of the G protein and independently of Sst2 RGS activity.

A two-hybrid screen against an activation domain array of Saccharomyces cerevisiaeproteins was carried out for 31 yeast proteasome proteins. Fifty-five putative interactions were identified: 21 between components of the proteasome complex and 34 between proteasome proteins and other proteins. Many of these latter interactions involved either proteins of the ubiquitin pathway, cell cycle proteins, protein kinases or a translation initiation factor subunit. The role of eleven proteins associated with proteasome function by these screens was analyzed by examining the corresponding deletion strains for temperature sensitivity and canavanine sensitivity and for the stability of a ubiquitin-β-galactosidase fusion protein. These assays additionally implicated three proteins, Bim1, Ump1, and YKL171W, in proteasome function. This study demonstrates the utility of genome-wide two-hybrid assays as an entry point for the further analysis of a large protein complex.

A reduction of operating costs, investment and an improvement in the quality of hot strip are possible only by changing the technology of the production process. The new thin-slab cast-rolling technology realises the direct rolling of hot strip in one heat. For reaching a higher hot strip quality, besides the modern cast-rolling plant technology, also an efficient automation of the entire plant is necessary. The autowation of such modern cast-rolling plants will be outlined.

Abstract:Many studies have found that prey capture rates significantly affect how long foraging spiders remain in an area. Perceptual information on prey, even without prey capture, has also been shown to significantly influence spider foraging patch residence time. However, the values of each type of information (e.g., perception, capture, handling and ingestion) in the foraging decision rules have never been compared. This study examines the value of feeding experience versusperceiving food in the residence time decisions of wolf spiders. Spiders were exposed to four experimental treatments: 1) a foraging patch with a dead cricket to be consumed; 2) a patch with 12 live crickets behind a transparent screen, but no cricket for consumption; 3) a patch with both live crickets behind a screen and a single dead cricket for consumption; and 4) an empty patch serving as a control. Although feeding resulted in significantly longer patch residence times than the control, the perception of prey was the single most important factor influencing residence time. Results showed no significant difference in residence time between the sensory only treatment and the treatment with both prey consumption and the perception of prey. Analysis of residence times after the spider has fed on a single cricket indicates no significant effect of the experience of feeding in a patch and subsequent visits to that patch. These results suggest that potential energy value weighs more heavily than current or past energy consumed in a patch and that post-ingestive feedback information is less important than pre-ingestive sensory cues.

Ld proteins (formins) are encoded by the limb deformity (ld) gene and define a family of related gene products regulating establishment of embryonic polarity. In this study we establish that chicken and murine Ld proteins interact directly with Src family kinases (c-Src and c-Fyn). Specific binding is mediated by the proline-rich domain present in Ld proteins and the ligand binding surface of the Src SH3 domain. Co-immunoprecipitation of Ld and c-Src proteins from transfected cells shows that these proteins associate in vivo. Immunolocalization and biochemical fractionation of fibroblasts confirms the predominant nuclear localization of Ld proteins, but unexpectedly identifies a population of Ld proteins associated to cellular membranes. This population of Ld proteins co-localizes with membrane-associated c-Src proteins at both plasma and perinuclear membranes. These studies indicate that the morphoregulatory Ld proteins interact with signal transduction cascades by association to membrane-bound Src family kinases.

No abstract available for this article.

We have isolated the murine gene encoding the 5‐HT3receptor (5‐HT3R), a member of the ligand‐gated ion channels, that mediates a variety of physiological effects in central and peripheral neurons. DNA sequence analysis of the 5‐HT3R gene revealed its organisation in 9 exons distributed over approximately 12 kbp of DNA. Alternative use ofexon 9 splice acceptor sites generated two 5‐HT3R variants. The 5‐HT3R gene, whose structure is closely related to neuronal and muscle AChRα genes, as demonstrated by four common splice junctions, was localised on human chromosome 11.

An assay based on the consumption of nitrilotriacetate (NTA) was developed to measure the activity of NTA monooxygenase (NTA-Mo) in cell extracts of "Chelatobacter" strain ATCC 29600 and to purify a functional, NTA-hydroxylating enzyme complex. The complex consisted of two components that easily dissociated during purification and upon dilution. Both components were purified to more than 95% homogeneity, and it was possible to reconstitute the functional, NTA-hydroxylating enzyme complex from pure component A (cA) and component B (cB). cB exhibited NTA-stimulated NADH oxidation but was unable to hydroxylate NTA. It had a native molecular mass of 88 kDa and contained flavin mononucleotide (FMN). cA had a native molecular mass of 99 kDa. No catalytic activity has yet been shown for cA alone. Under unfavorable conditions, NADH oxidation was partly or completely uncoupled from hydroxylation, resulting in the formation of H2O2. Optimum hydroxylating activity was found to be dependent on the molar ratio of the two components, the absolute concentration of the enzyme complex, and the presence of FMN. Uncoupling of the reaction was favored in the presence of high salt concentrations and in the presence of flavin adenine dinucleotide. The NTA-Mo complex was sensitive to sulfhydryl reagents, but inhibition was reversible by addition of excess dithiothreitol. The Km values for Mg(2+)-NTA, FMN, and NADH were determined as 0.5 mM, 1.3 microM, and 0.35 mM, respectively. Of 26 tested compounds, NTA was the only substrate for NTA-Mo.

Rabbit cDNA of the α1 subunit of the skeletal muscle dihydropyridine (DHP) receptor was functionally expressed in a muscular dysgenesis mouse (mdg) cell line, GLT. L-type calcium currents and transients were recorded for the wild type and a mutant α1 subunit carrying an R528H substitution in the supposed voltage sensor of the second channel domain that is linked to a human disease, hypokalemic periodic paralysis. L-type channels expressed in GLT myotubes exhibited currents similar to those described for primary cultured mdg cells injected with rabbit wild type cDNA, indicating this system to be useful for functional studies of heterologous DHP receptors. Voltage dependence and kinetics of activation and inactivation of L-type calcium currents from mutant and wild type channels did not differ significantly. Intracellular calcium release activation measured by fura-2 microfluorimetry was not grossly altered by the mutation either. Analogous measurements on myotubes of three human R528H carriers revealed calcium transients comparable to controls while the voltage dependence of both activation and inactivation of the L-type current showed a shift to more negative potentials of approximately 6 mV. Similar effects on the voltage dependence of the fast T-type current and changes in the expression level of the third-type calcium current point to factors not primarily associated with the mutation perhaps participating in disease pathogenesis.

An extreme form of male dimorphism occurs in the jumping spider, Maevia inclemens, where two male morphs differ dramatically in both morphology and behavior. We examined the visual signals transmitted to females by the two different morphs during courtship display. Even though the display of each male is different, as a function of behavior and courtship distance, the males present females with images that are almost identical in height and visual target area. By substituting live males with computer-animated sequences of male courtship behavior, the male morphs were superimposed upon each other, and females were determined to have two distinct male-recognition templates. We hypothesize that the dimorphic males of this species evolved through sensory exploitation and selection for alternative male strategies that stimulate different fields of the female visual range.

The courtship behaviors of two morphologically similar spider species, Schizocosa ocreataand S. rovneri, are distinctive and prevent interbreeding. We used “forced” copulation between these species to investigate the mode of inheritance of the courtship behavior and to determine whether postmating isolating mechanisms exist. F1hybrids proved to be behaviorally sterile, but they were capable of producing viable offspring when forced to interbreed. Analysis of the courtship behaviors of F1, F2, and backcross progeny showed that the inheritance of some aspects of these behaviors is consistent with models involving single autosomal loci. The inheritance of secondary sexual characteristics in the males is also investigated. The genes for courtship behavior and secondary sexual characteristics do not assort independently. The origin of the premating isolating mechanisms may be explained by either an initial habitat separation between the two groups, or by a founding event with each group subsequently diverging in slightly different habitats. It is suggested that the differences in the microhabitats may have a profound effect on what type of signal (visual or vibratory) would be effective.

Abstract: Little information is known concerning hemodynamic parameters of bypass grafts after open heart surgery during the postoperative period. As there is no easy to use bedside-method for monitoring, we developed a new implantable Doppler probe to measure blood flow velocity with a 20 MHz pulsed Doppler system. The accuracy of this method was proved in a circulatory model and in animal studies. Our conclusion is that the implantable pulsed Doppler system is a sensitive method for monitoring small vessel hemodynamics.

No abstract available for this article.

Immunocytochemical labeling of thin sections of cryosubstituted, Lowicryl-embedded Escherichia coli cells with protein A-colloidal gold was used to study the structural organization of the bacterial nucleoid. We found that the histonelike protein HU was not associated with the bulk DNA in the nucleoid but was located in areas of the cell where metabolically active DNA is associated with ribosomes and where single-stranded DNA, RNA polymerase, and DNA topoisomerase I were also located. The resolution of the methods used did not allow us to decide whether HU was associated either with ribosomes or with transcriptionally active DNA, nor could we demonstrate interaction of HU with either.

Autosomal recessive polycystic kidney disease (ARPKD) is a severe disease of early childhood that is clinically characterized by fibrocystic changes of the kidneys and the liver. The main cause of ARPKD are variants in the PKHD1gene encoding the large transmembrane protein fibrocystin. The mechanisms underlying the observed clinical heterogeneity in ARPKD remain incompletely understood, partly due to the fact that genotype-phenotype correlations have been limited to the association of biallelic null variants in PKHD1with the most severe phenotypes. In this observational study we analyzed a deep clinical dataset of 304 patients with ARPKD from two independent cohorts and identified novel genotype-phenotype correlations during childhood and adolescence. Biallelic null variants frequently show severe courses. Additionally, our data suggest that the affected region in PKHD1is important in determining the phenotype. Patients with two missense variants affecting amino acids 709-1837 of fibrocystin or a missense variant in this region and a null variant less frequently developed chronic kidney failure and patients with missense variants affecting amino acids 1838-2624 showed better hepatic outcome. Variants affecting amino acids 2625-4074 of fibrocystin were associated with poorer hepatic outcome. Thus, our data expand the understanding of genotype-phenotype correlations in pediatric ARPKD patients and can lay the foundation for more precise and personalized counselling and treatment approaches.

ABSTRACTMore than 20% of all protein domains are currently annotated as “domains of unknown function” (DUFs). About 2,700 DUFs are found in bacteria compared with just over 1,500 in eukaryotes. Over 800 DUFs are shared between bacteria and eukaryotes, and about 300 of these are also present in archaea. A total of 2,786 bacterial Pfam domains even occur in animals, including 320 DUFs. Evolutionary conservation suggests that many of these DUFs are important. Here we show that 355 essential proteins in 16 model bacterial species contain 238 DUFs, most of which represent single-domain proteins, clearly establishing the biological essentiality of DUFs. We suggest that experimental research should focus on conserved and essential DUFs (eDUFs) for functional analysis given their important function and wide taxonomic distribution, including bacterial pathogens.IMPORTANCEThe functional units of proteins are domains. Typically, each domain has a distinct structure and function. Genomes encode thousands of domains, and many of the domains have no known function (domains of unknown function [DUFs]). They are often ignored as of little relevance, given that many of them are found in only a few genomes. Here we show that many DUFs are essential DUFs (eDUFs) based on their presence in essential proteins. We also show that eDUFs are often essential even if they are found in relatively few genomes. However, in general, more common DUFs are more often essential than rare DUFs.