Coreplication of the major genotype group members of porcine circovirus type 2 as a prerequisite to coevolution may explain the variable disease manifestations

A member of the family Circoviridae, porcine circovirus type 2 (PCV2), is associated with postweaning multisystemic wasting syndrome (PMWS), a recent emerging disease worldwide. PCV2 is also found in clinically asymptomatic animals. This paradoxical finding makes the syndrome etiology challenging. We developed new assays to study PCV2 with links to syndrome etiology. For analysis, we used PCV2-infected tissues from subclinically infected and diseased piglets. We compared antigen- and PCV2 DNA-derived signals for tissue localization and intensity. Oligonucleotides were designed to the signature motif of the PCV2 capsid open reading frame to discriminate experimentally between PCV2 genotype groups by PCR, in situ hybridization (ISH), and fluorescence in situ hybridization (FISH). Unexpectedly, all PCV2-infected animals carried both PCV2a and PCV2b genotype group members. Using confocal microscopy, genotype single-cell infections and cell superinfections were visible. Additionally, we discriminated replicative DNA from total PCV2 DNA isoforms with FISH. This aided in our inquiry into cellular genotype-specific replication. Importantly, single-genotype-group replication was not observed. In infected cells with replicating virus, both genotype groups were equally present. These findings suggest PCV2 genotype group members relaxed replication regulation requirements and may even point to PCV2 replication cooperativity in vivo. These observations explain the readily seen PCV2 DNA recombinations and the high overall PCV2 genome plasticity. Hence, we suggest a novel mechanism of syndrome etiology that consists of a continuously changing PCV2 genome pool in hosts and pig herds, posing a constant challenge to the individual maturing immune system.     

The Importance of Synchrony and Temporal Order of Visual and Tactile Input for Illusory Limb Ownership Experiences – An fMRI Study Applying Virtual Reality

In the so-called rubber hand illusion, synchronous visuotactile stimulation of a visible rubber hand together with one''s own hidden hand elicits ownership experiences for the artificial limb. Recently, advanced virtual reality setups were developed to induce a virtual hand illusion (VHI). Here, we present functional imaging data from a sample of 25 healthy participants using a new device to induce the VHI in the environment of a magnetic resonance imaging (MRI) system. In order to evaluate the neuronal robustness of the illusion, we varied the degree of synchrony between visual and tactile events in five steps: in two conditions, the tactile stimulation was applied prior to visual stimulation (asynchrony of −300 ms or −600 ms), whereas in another two conditions, the tactile stimulation was applied after visual stimulation (asynchrony of +300 ms or +600 ms). In the fifth condition, tactile and visual stimulation was applied synchronously. On a subjective level, the VHI was successfully induced by synchronous visuotactile stimulation. Asynchronies between visual and tactile input of ±300 ms did not significantly diminish the vividness of illusion, whereas asynchronies of ±600 ms did. The temporal order of visual and tactile stimulation had no effect on VHI vividness. Conjunction analyses of functional MRI data across all conditions revealed significant activation in bilateral ventral premotor cortex (PMv). Further characteristic activation patterns included bilateral activity in the motion-sensitive medial superior temporal area as well as in the bilateral Rolandic operculum, suggesting their involvement in the processing of bodily awareness through the integration of visual and tactile events. A comparison of the VHI-inducing conditions with asynchronous control conditions of ±600 ms yielded significant PMv activity only contralateral to the stimulation site. These results underline the temporal limits of the induction of limb ownership related to multisensory body-related input.     

Branching mode in complex-type triantennary N-glycans as regulatory element of their ligand properties

We address the question whether the two natural types of branching in complex-type triantennary N-glycans differ in ligand properties. Toward this end, we prepared the set of pergalactosylated undecasaccharides and derivatives with alpha2,3/6-sialylation by chemoenzymatic synthesis. Conjugation resulted in neoglycoproteins which were tested in assays with lectins/antibodies, cultured cells and animals. Solid-phase assays with galactoside-specific proteins (a plant toxin, galectins and an antibody fraction) disclosed that the branching mode did not significantly affect affinity. However, compared to previous studies under identical conditions increase in antennae number and presence of substitutions in biantennary N-glycans altered KD-values with differences between receptors. Neoglycoprotein binding to cells of eight human tumor lines was sensitive to N-glycan branching. Staining intensity revealed pronounced branch-mode-dependent differences in four cases. Biodistribution profiles in mice uncovered dramatic changes in clearance rates with prolonged serum presence associated with type II branching of sialylated N-glycans and markedly increased uptake of neoglycoproteins with type I-branched N-glycans into liver, spleen, heart and lungs. This part of the study is relevant for rational glycoengineering of pharmaproteins. In general, our study supports the concept to view details of N-glycan structure, here branching, as a means to modulate ligand properties.     

Helicobacter pylori Type IV Secretion Apparatus Exploits β1 Integrin in a Novel RGD-Independent Manner

Translocation of the Helicobacter pylori (Hp) cytotoxin-associated gene A (CagA) effector protein via the cag-Type IV Secretion System (T4SS) into host cells is a major risk factor for severe gastric diseases, including gastric cancer. However, the mechanism of translocation and the requirements from the host cell for that event are not well understood. The T4SS consists of inner- and outer membrane-spanning Cag protein complexes and a surface-located pilus. Previously an arginine-glycine-aspartate (RGD)-dependent typical integrin/ligand type interaction of CagL with α5β1 integrin was reported to be essential for CagA translocation. Here we report a specific binding of the T4SS-pilus-associated components CagY and the effector protein CagA to the host cell β1 Integrin receptor. Surface plasmon resonance measurements revealed that CagA binding to α5β1 integrin is rather strong (dissociation constant, K_D of 0.15 nM), in comparison to the reported RGD-dependent integrin/fibronectin interaction (K_D of 15 nM). For CagA translocation the extracellular part of the β1 integrin subunit is necessary, but not its cytoplasmic domain, nor downstream signalling via integrin-linked kinase. A set of β1 integrin-specific monoclonal antibodies directed against various defined β1 integrin epitopes, such as the PSI, the I-like, the EGF or the β-tail domain, were unable to interfere with CagA translocation. However, a specific antibody (9EG7), which stabilises the open active conformation of β1 integrin heterodimers, efficiently blocked CagA translocation. Our data support a novel model in which the cag-T4SS exploits the β1 integrin receptor by an RGD-independent interaction that involves a conformational switch from the open (extended) to the closed (bent) conformation, to initiate effector protein translocation.     

Integrin subunit CD18 Is the T-lymphocyte receptor for the Helicobacter pylori vacuolating cytotoxin

Helicobacter pylori infection is associated with gastritis, ulcerations, and gastric adenocarcinoma. H. pylori secretes the vacuolating cytotoxin (VacA), a major pathogenicity factor. VacA has immunosuppressive effects, inhibiting interleukin-2 (IL-2) secretion by interference with the T cell receptor/IL-2 signaling pathway at the level of calcineurin, the Ca2+-calmodulin-dependent phosphatase. Here, we show that VacA efficiently enters activated, migrating primary human T lymphocytes by binding to the beta2 (CD18) integrin receptor subunit and exploiting the recycling of lymphocyte function-associated antigen (LFA)-1. LFA-1-deficient Jurkat T cells were resistant to vacuolation and IL-2 modulation, and genetic complementation restored sensitivity to VacA. VacA targeted human, but not murine, CD18 for cell entry, consistent with the species-specific adaptation of H. pylori. Furthermore, expression of human integrin receptors (LFA-1 or Mac-1) in murine T cells resulted in VacA-mediated cellular vacuolation. Thus, H. pylori co-opts CD18 as a VacA receptor on human T lymphocytes to subvert the host immune response.     

Sticky socks: Helicobacter pylori VacA takes shape

Several receptors have been described for the Helicobacter pylori vacuolating toxin VacA, which exerts different effects on epithelial cells and on immune cells. The crystal structure of the putative receptor-binding domain of VacA (p55) has now been solved. It consists of a parallel beta-helix with a C-terminal globular domain. A comparison between allelic variants of p55 and docking of the p55 domain into the quaternary structure, as shown by electron microscopy, revealed structural features that might be important for elucidating the molecular details of receptor interaction and channel formation.     

Isolation and localization of N4-methylasparagine in phycobiliproteins from the cyanobacterium Mastigocladus laminosus

The occurrence of post-translationally methylated asparagine residues in beta AP from Anabaena variabilis, Synechococcus PCC 6301 and Porphyridium cruentum has recently been reported (Klotz, A.V., Leary, J.A. & Glazer, A.N. (1986) J. Biol. Chem. 261, 15891-15894). We reinvestigated the amino-acid compositions of all phycobiliproteins from Mastigocladus laminosus. During total hydrolysis of beta AP, beta 16.2 and beta PC one mol methylamine per mol protein was released. These proteins were chemically and enzymatically fragmented and the sequences of the fragments containing the modified asparagine residue were determined by automated Edman degradation. Residues beta AP72, beta 16.2 72 and beta PC 72 were identified as N4-methylasparagine. This derivative of asparagine was also found at a homologous position in beta PE of Calothrix. In the x-ray structure model of C-phycocyanin (PC) the residue beta PC 72 points towards the chromophore beta 84, presumably having an effect on the spectroscopic characteristics of this light harvesting pigment protein complex.     

Crystallization of phycoerythrocyanin from the cyanobacterium Mastigocladus laminosus and preliminary characterization of two crystal forms

The light-harvesting protein phycoerythrocyanin from the cyanobacterium Mastigocladus laminosus Cohn has been crystallized in two different crystal forms by vapour diffusion. In 5% (w/v) polyethylene glycol at pH 8.5, hexagonal crystals of space group P63 with cell constants a = b = 158 A, c = 40.6 A were obtained, which turned out to be almost isomorphous with the hexagonal crystals of C-phycocyanin from the same organism. Consequently, the conformation of both phycobiliproteins must be very similar. From 1.5 M-ammonium sulfate (pH 8.5), orthorhombic crystals of space group P2221 with cell constants a = 60.5 A, b = 105 A, c = 188 A could be grown. Density measurements of these crystals indicate that the unit cell contains 18 (alpha beta)-units. A detailed packing scheme is proposed that is consistent with the observed pseudo-hexagonal X-ray intensity pattern and with the known size and shape of (alpha beta)3-trimers of C-phycocyanin. Accordingly, disc-like (alpha beta)3-trimers are associated face-to-face and stacked one upon another in rods with a period of 60.5 A, corresponding to the cell dimension a.     

Isolation procedures for thermostable neutral proteinases produced by Bacillus stearothermophilus

Summary Purification procedures for extracting and concentrating thermostable neutral proteinases using vacuum evaporation and ammonium sulfate precipitation, or adsorption chromatography on amberlite XAD-7 resin were compared. Adsorption chromatography proved to be the most effective method to concentrate, extract and partially purify the thermostable neutral proteases produced by Bacillus stearothermophilus NCIB 8924 and NRRL B-3880. Proteases can also be extracted from large volumes of culture media containing only weak proteolytic activity and a low protein concentration. Final purification of the thermostable neutral proteases was performed with an established affinity chromatography method. The method seems to be suitable for scaling up.     

Method for Microbiological Testing of Nonsterile Pharmaceuticals

A method for testing nonsterile pharmaceutical preparations for their microbial content is described. As far as possible, only solid culture media were used to obtain quantitative results. Aqueous and water-soluble products were tested with membrane-filter techniques. Nonfilterable products were first emulsified or suspended and the homogenate was used for examination. In both procedures, the total number of colonies is determined for aerobic bacteria and fungi. Tests for certain undesirable microbial groups were conducted with selected media. The method described is applicable for finished products, bulk products, raw materials, and active ingredients.