Deciphering the ligand-binding sites in the Borrelia burgdorferi complement regulator-acquiring surface protein 2 required for interactions with the human immune regulators factor H and factor H-like protein 1

Borrelia burgdorferi, the etiologic agent of Lyme disease, employs sophisticated means to evade killing by its mammalian hosts. One important immune escape mechanism is the inhibition of complement activation mediated by interactions of the host-derived immune regulators factor H (CFH) and factor H-like protein 1 (CFHL1) with borrelial complement regulator-acquiring surface proteins (BbCRASPs). BbCRASP-2 is a distinctive CFH- and CFHL1-binding protein that is produced by serum-resistant B. burgdorferi strains. Here we show that binding of CFH by BbCRASP-2 is due to electrostatic as well as hydrophobic forces. In addition, 14 individual amino acid residues of BbCRASP-2 were identified as being involved in CFH and CFHL1 binding. Alanine substitutions of most of those residues significantly inhibited binding of CFH and/or CFHL1 by recombinant BbCRASP-2 proteins. To conclusively define the effects of BbCRASP-2 residue substitutions on serum sensitivity in the bacterial context, a serum-sensitive Borrelia garinii strain was transformed with plasmids that directed production of either wild-type or mutated BbCRASP-2 proteins. Critical amino acid residues within BbCRASP-2 were identified, with bacteria producing distinct mutant proteins being unable to bind either CFH or CFHL1, showing high levels of complement components C3, C6, and C5b-9 deposited on their surfaces and being highly sensitive to killing by normal serum. Collectively, we mapped a structurally sensitive CFH/CFHL1 binding site within borrelial BbCRASP-2 and identified single amino acid residues potentially involved in the interaction with both complement regulators.     

Acetate:succinate CoA-transferase in the hydrogenosomes of Trichomonas vaginalis: identification and characterization

Acetate:succinate CoA-transferases (ASCT) are acetate-producing enzymes in hydrogenosomes, anaerobically functioning mitochondria and in the aerobically functioning mitochondria of trypanosomatids. Although acetate is produced in the hydrogenosomes of a number of anaerobic microbial eukaryotes such as Trichomonas vaginalis, no acetate producing enzyme has ever been identified in these organelles. Acetate production is the last unidentified enzymatic reaction of hydrogenosomal carbohydrate metabolism. We identified a gene encoding an enzyme for acetate production in the genome of the hydrogenosome-containing protozoan parasite T. vaginalis. This gene shows high similarity to Saccharomyces cerevisiae acetyl-CoA hydrolase and Clostridium kluyveri succinyl-CoA:CoA-transferase. Here we demonstrate that this protein is expressed and is present in the hydrogenosomes where it functions as the T. vaginalis acetate:succinate CoA-transferase (TvASCT). Heterologous expression of TvASCT in CHO cells resulted in the expression of an active ASCT. Furthermore, homologous overexpression of the TvASCT gene in T. vaginalis resulted in an equivalent increase in ASCT activity. It was shown that the CoA transferase activity is succinate-dependent. These results demonstrate that this acetyl-CoA hydrolase/transferase homolog functions as the hydrogenosomal ASCT of T. vaginalis. This is the first hydrogenosomal acetate-producing enzyme to be identified. Interestingly, TvASCT does not share any similarity with the mitochondrial ASCT from Trypanosoma brucei, the only other eukaryotic succinate-dependent acetyl-CoA-transferase identified so far. The trichomonad enzyme clearly belongs to a distinct class of acetate:succinate CoA-transferases. Apparently, two completely different enzymes for succinate-dependent acetate production have evolved independently in ATP-generating organelles.     

Rac1 activation inhibits E-cadherin-mediated adherens junctions via binding to IQGAP1 in pancreatic carcinoma cells

Nck is a ubiquitously expressed adapter protein that is almost exclusively built of one SH2 domain and three SH3 domains. The two isoproteins of Nck are functionally redundant in many aspects and differ in only few amino acids that are mostly located in the linker regions between the interaction modules. Nck proteins connect receptor and non-receptor tyrosine kinases to the machinery of actin reorganisation. Thereby, Nck regulates activation-dependent processes during cell polarisation and migration and plays a crucial role in the signal transduction of a variety of receptors including for instance PDGF-, HGF-, VEGF- and Ephrin receptors. In most cases, the SH2 domain mediates binding to the phosphorylated receptor or associated phosphoproteins, while SH3 domain interactions lead to the formation of larger protein complexes. In T lymphocytes, Nck plays a pivotal role in the T cell receptor (TCR)-induced reorganisation of the actin cytoskeleton and the formation of the immunological synapse. However, in this context, two different mechanisms and adapter complexes are discussed. In the first scenario, dependent on an activation-induced conformational change in the CD3ε subunits, a direct binding of Nck to components of the TCR/CD3 complex was shown. In the second scenario, Nck is recruited to the TCR complex via phosphorylated Slp76, another central constituent of the membrane proximal activation complex. Over the past years, a large number of putative Nck interactors have been identified in different cellular systems that point to diverse additional functions of the adapter protein, e.g. in the control of gene expression and proliferation.     

Protein-primed and de novo initiation of RNA synthesis by norovirus 3Dpol

Noroviruses (Caliciviridae) are RNA viruses with a single-stranded, positive-oriented polyadenylated genome. To date, little is known about the replication strategy of norovirus, a so-far noncultivable virus. We have examined the initiation of replication of the norovirus genome in vitro, using the active norovirus RNA-dependent RNA polymerase (3D(pol)), homopolymeric templates, and synthetic subgenomic or antisubgenomic RNA. Initiation of RNA synthesis on homopolymeric templates as well as replication of subgenomic polyadenylated RNA was strictly primer dependent. In this context and as observed for other enteric RNA viruses, i.e., poliovirus, a protein-primed initiation of RNA synthesis after elongation of the VPg by norovirus 3D(pol) was postulated. To address this question, norovirus VPg was expressed in Escherichia coli and purified. Incubation of VPg with norovirus 3D(pol) generated VPg-poly(U), which primed the replication of subgenomic polyadenylated RNA. In contrast, replication of antisubgenomic RNA was not primer dependent, nor did it depend on a leader sequence, as evidenced by deletion analysis of the 3' termini of subgenomic and antisubgenomic RNA. On nonpolyadenylated RNA, i.e., antisubgenomic RNA, norovirus 3D(pol) initiated RNA synthesis de novo and terminated RNA synthesis by a poly(C) stretch. Interestingly, on poly(C) RNA templates, norovirus 3D(pol) initiated RNA synthesis de novo in the presence of high concentrations of GTP. We propose a novel model for initiation of replication of the norovirus genome by 3D(pol), with a VPg-protein-primed initiation of replication of polyadenylated genomic RNA and a de novo initiation of replication of antigenomic RNA.     

Investigations on reproductive physiology in the male Eurasian lynx (Lynx lynx)

This study characterized (in vivo) morphological and functional parameters of reproductive organs of adult male lynx (n = 3) prior to, during, and after the breeding season (n = 3). Size and morphology of the reproductive tract were monitored by transcutaneous (testes) and transrectal (accessory sex glands) ultrasonography. Semen was collected by electroejaculation. Ejaculate volume, sperm number, motility, and morphology of spermatozoa as well as testosterone concentrations in blood serum and feces were evaluated. The testes and prostate had seasonal changes in size and echotexture. The mean (+/- S.D.) maximum and minimum testicular volume were 2.8 +/- 0.8 cm3 and 1.5 +/- 0.3 cm3, respectively. Fecal testosterone concentrations were highest in February (1240 +/- 393 ng/g feces), with a second increase in May (971 +/- 202 ng/g feces), but concentrations were lowest in January (481 +/- 52.9 ng/g feces). Ejaculate volume, total sperm number and percentage of motile, and intact spermatozoa were maximal in March (the middle of the breeding season). In one of the eight litters, multiple paternity was proven; however, in the remaining seven litters, all 16 cubs were sired by the same male. This particular male had the most developed and active testes and best semen quality, which may be important for sperm competition.     

Adaptastat--a new method for optimising of bacterial growth conditions in continuous culture: Interactive substrate limitation based on dissolved oxygen measurement

A new method for continuous cultivation of microbes, called adaptastat, is described. It involves automatic adaptation of microbial cultures to their maximum feeding rates and avoids substrate accumulation. The state of the culture is estimated at intervals by briefly (and markedly) decreasing the substrate feeding rate and measuring, via the dissolved oxygen response, the time taken to exhaust the residual substrate. The method has been exemplified by cultivating Escherichia coli on single carbon sources (glucose, acetate, succinate, and fully deuteriated medium based on deuteriated succinate) and also by simultaneous limitation of two feeding channels (succinate/acetate and glucose/ammonium chloride). Several possible applications of the adaptastat technology are presented. The method provides an efficient means of labelling microbial components and products with stable isotopes. In particular, adaptastat technology can be used to adapt disabled bacterial strains to the use of simple, inexpensive substrates. It can also be used more generally in the study of microbial cell cultures, for example for the determination of maximum specific growth rates and the stoichiometric ratio of utilisation of two nutrients in conditions of simultaneous limitation.     

Dissolved Nitrogen, Phosphorus, and Sulfur forms in the Ecosystem Fluxes of a Montane Forest in Ecuador

The N, P, and S cycles in pristine forests are assumed to differ from those of anthropogenically impacted areas, but there are only a few studies to support this. Our objective was therefore to assess the controls of N, P, and S release, immobilization, and transport in a remote tropical montane forest. The study forest is located on steep slopes of the northern Andes in Ecuador. We determined the concentrations of NO₃-N, NH₄-N, dissolved organic N (DON), PO₄-P, dissolved organic P (DOP), SO₄-S, dissolved organic S (DOS), and dissolved organic C (DOC) in rainfall, throughfall, stemflow, lateral flow (in the organic layer), litter leachate, mineral soil solution, and stream water of three 8–13 ha catchments (1900–2200 m a.s.l.). The organic forms of N, P, and S contributed, on average, 55, 66, and 63% to the total N, P, and S concentrations in all ecosystem fluxes, respectively. The organic layer was the largest source of all N, P, and S species except for inorganic P and S. Most PO₄ was released in the canopy by leaching and most SO₄ in the mineral soil by weathering. The mineral soil was a sink for all studied compounds except for SO₄. Consequently, concentrations of dissolved inorganic and organic N and P were as low in stream water (TDN: 0.34–0.39 mg N l⁻¹, P not detectable) as in rainfall (TDN: 0.39–0.48 mg N l⁻¹, P not detectable), whereas total S concentrations were elevated (stream water: 0.04–0.15, rainfall: 0.01–0.07 mg S l⁻¹). Dissolved N, P, and S forms were positively correlated with pH at the scale of soil peda except inorganic S. Soil drying and rewetting promoted the release of dissolved inorganic N. High discharge levels following heavy rainstorms were associated with increased DOC, DON, NO₃-N and partly also NH₄-N concentrations in stream water. Nitrate-N concentrations in the stream water were positively correlated with stream discharge during the wetter period of the year. Our results demonstrate that the sources and sinks of N, P, and S were element-specific. More than half of the cycling N, P, and S was organic. Soil pH and moisture were important controls of N, P, and S solubility at the scale of individual soil peda whereas the flow regime influenced the export with stream water.     

Identification and isolation of Dictyostelium microtubule-associated protein interactors by tandem affinity purification

Tandem affinity purification (TAP) is a method originally established in yeast to isolate highly purified protein complexes in a very gentle and efficient way. In this work, we have modified TAP for Dictyostelium applications and have proved it as a useful method to specifically isolate and identify microtubule-associated protein (MAP) complexes. MAPs are known to interact with other proteins to fulfill their complex functions in balancing the dynamic instability of microtubules as well as anchoring microtubules at the cell cortex, controlling mitosis at the centrosome and guiding transport along them. DdEB1 and the Dictyostelium member of the XMAP215 protein family, DdCP224, are known to be part of complexes at the microtubule tips as well as at the centrosome. Employing TAP and mass spectrometry we were able to prove an interaction between EB1 and the DdCP224. Additionally, among other interactions that remain to be confirmed by other methods, an interaction between DdCP224 and a TACC-family protein could be shown for the first time in Dictyostelium and was confirmed by colocalization and co-immunoprecipitation analyses.     

Competitive dominance among sessile marine organisms in a high Arctic boulder community

In most hard substrate environments, space is a limiting resource for sessile organisms. Competition for space is often high and is a structuring force within the community. In the Beaufort Sea’s Boulder Patch, crustose coralline red algae are major space occupiers. This research determined if coralline algae were competitively dominant over other sessile organisms. To test this hypothesis, overgrowth was documented in terms of “winners” and “losers” on the contact borders between different species. Crustose corallines occurred in over 80% of the observed interactions but were only winners in approximately half of them. Most frequently, bryozoans, tunicates, and sponges were superior competitors over crustose corallines, while at the same time these invertebrate groups were among the least abundant space occupiers.