Mouse LSECtin as a model for a human Ebola virus receptor

The biochemical properties of mouse LSECtin, a glycan-binding receptor that is a member of the C-type lectin family found on sinusoidal endothelial cells, have been investigated. The C-type carbohydrate-recognition domain of mouse LSECtin, expressed in bacteria, has been used in solid-phase binding assays, and a tetramerized form has been used to probe a glycan array. In spite of sequence differences near the glycan-binding sites, the mouse receptor closely mimics the properties of the human receptor, showing high affinity binding to glycans bearing terminal GlcNAcβ1-2Man motifs. Site-directed mutagenesis has been used to confirm that residues near the binding site that differ between the human and the mouse proteins do not affect this binding specificity. Mouse and human LSECtin have been shown to bind Ebola virus glycoprotein with equivalent affinities, and the GlcNAcβ1-2Man disaccharide has been demonstrated to be an effective inhibitor of this interaction. These studies provide a basis for using mouse LSECtin, and knockout mice lacking this receptor, to model the biological properties of the human receptor.     

Genome size and chromosomes in marine sponges [Suberites domuncula,Geodia cydonium]

The genome size of the marine sponges Suberites domuncula and Geodia cydonium has been determined by flow cytofluorometric analysis using diamidino-phenylindole [DAPI]. Using human lymphocytes as reference the amount of DNA in cells from S. domuncula has been determined to be 3.7 pg and that of G. cydonium 3.3 pg. While no chromosomes could be identified in G. cydonium, the karyotype of the Suberites domuncula is 32 chromsomes in the diploid state. The size of the chromosomes was between 0.25 and 1.0 μm. No pronounced banding pattern was visible.     

Sleep-related hippocampo-cortical interplay during emotional memory recollection

Emotional events are usually better remembered than neutral ones. This effect is mediated in part by a modulation of the hippocampus by the amygdala. Sleep plays a role in the consolidation of declarative memory. We examined the impact of sleep and lack of sleep on the consolidation of emotional (negative and positive) memories at the macroscopic systems level. Using functional MRI (fMRI), we compared the neural correlates of successful recollection by humans of emotional and neutral stimuli, 72 h after encoding, with or without total sleep deprivation during the first post-encoding night. In contrast to recollection of neutral and positive stimuli, which was deteriorated by sleep deprivation, similar recollection levels were achieved for negative stimuli in both groups. Successful recollection of emotional stimuli elicited larger responses in the hippocampus and various cortical areas, including the medial prefrontal cortex, in the sleep group than in the sleep deprived group. This effect was consistent across subjects for negative items but depended linearly on individual memory performance for positive items. In addition, the hippocampus and medial prefrontal cortex were functionally more connected during recollection of either negative or positive than neutral items, and more so in sleeping than in sleep-deprived subjects. In the sleep-deprived group, recollection of negative items elicited larger responses in the amygdala and an occipital area than in the sleep group. In contrast, no such difference in brain responses between groups was associated with recollection of positive stimuli. The results suggest that the emotional significance of memories influences their sleep-dependent systems-level consolidation. The recruitment of hippocampo-neocortical networks during recollection is enhanced after sleep and is hindered by sleep deprivation. After sleep deprivation, recollection of negative, potentially dangerous, memories recruits an alternate amygdalo-cortical network, which would keep track of emotional information despite sleep deprivation.     

Candida albicans Sun41p, a putative glycosidase, is involved in morphogenesis, cell wall biogenesis, and biofilm formation

The SUN gene family has been defined in Saccharomyces cerevisiae and comprises a fungus-specific family of proteins which show high similarity in their C-terminal domains. Genes of this family are involved in different cellular processes, like DNA replication, aging, mitochondrial biogenesis, and cytokinesis. In Candida albicans the SUN family comprises two genes, SUN41 and SIM1. We demonstrate that C. albicans mutants lacking SUN41 show similar defects as found for S. cerevisiae, including defects in cytokinesis. In addition, the SUN41 mutant showed a higher sensitivity towards the cell wall-disturbing agent Congo red, whereas no difference was observed in the presence of calcofluor white. Compared to the wild type, SUN41 deletion strains exhibited a defect in biofilm formation, a reduced adherence on a Caco-2 cell monolayer, and were unable to form hyphae on solid medium under the conditions tested. Interestingly, Sun41p was found to be secreted in the medium of cells growing as blastospores as well as those forming hyphae. Our results support a function of SUN41p as a glycosidase involved in cytokinesis, cell wall biogenesis, adhesion to host tissue, and biofilm formation, indicating an important role in the host-pathogen interaction.     

Identification of the site of human mannan-binding lectin involved in the interaction with its partner serine proteases: the essential role of Lys55

Mannan-binding lectin (MBL) is an oligomeric lectin that binds neutral carbohydrates on pathogens, forms complexes with MBL-associated serine proteases (MASP)-1, -2, and -3 and 19-kDa MBL-associated protein (MAp19), and triggers the complement lectin pathway through activation of MASP-2. To identify the MASP binding site(s) of human MBL, point mutants targeting residues C-terminal to the hinge region were produced and tested for their interaction with the MASPs and MAp19 using surface plasmon resonance and functional assays. Mutation Lys(55)Ala abolished interaction with the MASPs and MAp19 and prevented formation of functional MBL-MASP-2 complexes. Mutations Lys(55)Gln and Lys(55)Glu abolished binding to MASP-1 and -3 and strongly inhibited interaction with MAp19. Conversely, mutation Lys(55)Arg abolished interaction with MASP-2 and MAp19, but only weakened interaction with MASP-1 and -3. Mutation Arg(47)Glu inhibited interaction with MAp19 and decreased the ability of MBL to trigger the lectin pathway. Mutant Arg(47)Lys showed no interaction with the MASPs or MAp19, likely resulting from a defect in oligomerization. In contrast, mutation Arg(47)Ala had no impact on the interaction with the MASPs and MAp19, nor on the ability of MBL to trigger the lectin pathway. Mutation Pro(53)Ala only had a slight effect on the interaction with MASP-1 and -3, whereas mutations at residues Leu(49) and Leu(56) were ineffective. In conclusion, the MASP binding site of MBL involves a sequence stretch centered on residue Lys(55), which may form an ionic bond representing the major component of the MBL-MASP interaction. The binding sites for MASP-2/MAp19 and MASP-1/3 have common features but are not strictly identical.     

Impact of a new glucose utilization pathway in amino acid-producing Corynebacterium glutamicum

Corynebacterium glutamicum imports and phosphorylates glucose, fructose and sucrose by the phosphoenolpyruvate-dependent phosphotransferase carbohydrate uptake system (PTS). Recently, we have discovered how glucose can be utilized by C. glutamicum in a PTS-independent manner. PTS-independent glucose uptake is mediated by one of two inositol permeases (IolT1 or IolT2) and the second function of PTS, substrate phosphorylation, is catalyzed by one of two glucokinases (Glk or PpgK). PTS-deficient C. glutamicum strains exclusively utilizing glucose via this system grew comparably well on glucose minimal media as the parental strain. Furthermore, PTS-deficient L-lysine producing C. glutamicum strains overexpressing genes for inositol permease and glucokinase showed increased L-lysine production and reduced formation of by-products derived from pyruvate. Here, we discuss the impact of our findings on engineering strategies of C. glutamicum strains used in various biotechnological production processes.     

RNase Y in Bacillus subtilis: a Natively disordered protein that is the functional equivalent of RNase E from Escherichia coli

The control of mRNA stability is an important component of regulation in bacteria. Processing and degradation of mRNAs are initiated by an endonucleolytic attack, and the cleavage products are processively degraded by exoribonucleases. In many bacteria, these RNases, as well as RNA helicases and other proteins, are organized in a protein complex called the RNA degradosome. In Escherichia coli, the RNA degradosome is assembled around the essential endoribonuclease E. In Bacillus subtilis, the recently discovered essential endoribonuclease RNase Y is involved in the initiation of RNA degradation. Moreover, RNase Y interacts with other RNases, the RNA helicase CshA, and the glycolytic enzymes enolase and phosphofructokinase in a degradosome-like complex. In this work, we have studied the domain organization of RNase Y and the contribution of the domains to protein-protein interactions. We provide evidence for the physical interaction between RNase Y and the degradosome partners in vivo. We present experimental and bioinformatic data which indicate that the RNase Y contains significant regions of intrinsic disorder and discuss the possible functional implications of this finding. The localization of RNase Y in the membrane is essential both for the viability of B. subtilis and for all interactions that involve RNase Y. The results presented in this study provide novel evidence for the idea that RNase Y is the functional equivalent of RNase E, even though the two enzymes do not share any sequence similarity.     

The SARS-coronavirus-host interactome: identification of cyclophilins as target for pan-coronavirus inhibitors

Coronaviruses (CoVs) are important human and animal pathogens that induce fatal respiratory, gastrointestinal and neurological disease. The outbreak of the severe acute respiratory syndrome (SARS) in 2002/2003 has demonstrated human vulnerability to (Coronavirus) CoV epidemics. Neither vaccines nor therapeutics are available against human and animal CoVs. Knowledge of host cell proteins that take part in pivotal virus-host interactions could define broad-spectrum antiviral targets. In this study, we used a systems biology approach employing a genome-wide yeast-two hybrid interaction screen to identify immunopilins (PPIA, PPIB, PPIH, PPIG, FKBP1A, FKBP1B) as interaction partners of the CoV non-structural protein 1 (Nsp1). These molecules modulate the Calcineurin/NFAT pathway that plays an important role in immune cell activation. Overexpression of NSP1 and infection with live SARS-CoV strongly increased signalling through the Calcineurin/NFAT pathway and enhanced the induction of interleukin 2, compatible with late-stage immunopathogenicity and long-term cytokine dysregulation as observed in severe SARS cases. Conversely, inhibition of cyclophilins by cyclosporine A (CspA) blocked the replication of CoVs of all genera, including SARS-CoV, human CoV-229E and -NL-63, feline CoV, as well as avian infectious bronchitis virus. Non-immunosuppressive derivatives of CspA might serve as broad-range CoV inhibitors applicable against emerging CoVs as well as ubiquitous pathogens of humans and livestock.     

Shoot regeneration of mesophyll protoplasts transformed by Agrobacterium tumefaciens,not achievable with untransformed protoplasts

Summary Alternative methods for shoot regeneration in protoplast derived cultures were developed in Nicotiana paniculata and Physalis minima. In both species protoplast derived callus is not regeneratable to shoots by conventional methods, e.g. hormone treatment. Leaf discs and stem segments of N. paniculata and P. minima were incubated with Agrobacterium tumefaciens shooter strains harbouring pGV 2215 or pGV 2298 or wildtype strain B6S3. After 36 h of co-incubation protoplasts were prepared. (Leaf disc and stem segment cloning). Co-cultivation experiments were also undertaken with protoplasts of both species. Transformed clones, characterized by their hormone independent growth and octopine production, could be isolated after about two months. Transformation frequencies of leaf disc and stem segment cloning and co-cultivation experiments varied from 5×10^–3 to 5×10^–5. After about one year of cultivation on hormone-free culture medium, shoots could be recovered from colonies of N. paniculata, transformed by the strain harbouring pGV 2298. In protoplast derived colonies of P. minima, shoot induction was obtained only after transformation by bacteria carrying pGV 2215. This demonstrates the importance of the particular shooter mutant, as well as the response of the host plant. Transformed shoots of P. minima produced octopine, whereas octopine production in transformed shoots and callus of N. paniculata was undetectable after one year of cultivation, though T-DNA was still present in the plant genome. Transformed shoots of N. paniculata and P. minima do not produce any roots. Shoots of N. paniculata have an especially tumerous phenotype. Shoots of both species were successfully grafted to normal donor plants of N. tabacum.Abbreviations B5-h Gamborg medium without hormones (Gamborg 1968) - V47 protoplast medium (Binding 1974) - D2a protoplast medium (Li et al. 1980) - MS-h Murashige and Skoog medium without hormones (Murashige and Skoog 1962) Dedicated to Professor Dr. G. Melchers in occasion of his 80th birthday     

Removal and mineralization of polycyclic aromatic hydrocarbons by litter-decomposing basidiomycetous fungi

Nine strains of litter-decomposing fungi, representing eight species of agaric basidiomycetes, were tested for their ability to remove a mixture of three polycyclic aromatic hydrocarbons (PAHs) (total 60 mg l(-1)) comprising anthracene, pyrene and benzo(a)pyrene (BaP) in liquid culture. All strains were able to convert this mixture to some extent, but considerable differences in degradative activity were observed depending on the species, the Mn(II) concentration, and the particular PAH. Stropharia rugosoannulata was the most efficient degrader, removing or transforming BaP almost completely and about 95% of anthracene and 85% of pyrene, in cultures supplemented with 200 micro M Mn(II), within 6 weeks. In contrast less than 40, 18, and 50% BaP, anthracene and pyrene, respectively, were degraded in the absence of supplemental Mn(II). In the case of Stropharia coronilla, the presence of Mn(II) led to a 20-fold increase of anthracene conversion. The effect of manganese could be attributed to the stimulation of manganese peroxidase (MnP). The maximum activity of MnP increased in S. rugosoannulata cultures from 10 U l(-1) in the absence of Mn(II) to 320 U l(-1) in Mn(II)-supplemented cultures. The latter degraded about 6% of a (14)C-labeled BaP into (14)CO(2) whereas only 0.7% was mineralized in the absence of Mn(II). In solid-state straw cultures, S. rugosoannulata, S. coronilla and Agrocybe praecox mineralized between 4 and 6% of (14)C-labeled BaP within 12 weeks.