Marcus Klein

Detection of Chlamydia pneumoniae-Specific Antibodies Binding to the VD2 and VD3 Regions of the Major Outer Membrane Protein

ABSTRACT Although Chlamydia pneumoniae is an important human pathogen, the antigens eliciting a specific humoral immune response remain elusive. We scrutinized several recombinant chlamydial surface proteins for species-specific recognition by a panel of human sera previously tested for the presence of anti-C. pneumoniae and anti-C. trachomatis antibodies by microimmunofluorescence and enzyme-linked immunosorbent assay. The 15-kDa cysteine-rich protein (CrpA), porin-b (PorB), 9-kDa outer membrane protein (OMP3), 60-kDa outer membrane protein (OMP2), and four fragments of the major outer membrane protein (MOMP) representing each variable domain (VD) were overexpressed in Escherichia coli, affinity purified, and employed for Western blot analysis. None of the sera tested contained antibodies recognizing PorB and OMP3 of C. pneumoniae. Sera from C. pneumoniae-immune patients cross-reacted with OMP2 of C. trachomatis, and sera from C. trachomatis-immune patients cross-reacted with CrpA of C. pneumoniae, indicating that some of chlamydial surface molecules share antigenic epitopes. In contrast, the VD2, as well as the VD3, regions of the MOMP of C. pneumoniae were only recognized by C. pneumoniae-positive sera, suggesting the existence of species-specific epitopes. The identification of such epitopes of cell surface molecules provides new insights into C. pneumoniae-specific immune responses and may be of value for the improvement of C. pneumoniae-specific diagnostic assay systems based on defined recombinant antigens.

Isolation of a novel, phosphate-activated glutaminase from Bacillus pasteurii

In Bacillus pasteurii glutamine is being taken up efficiently by a sodium-dependent uptake system and subsequently hydrolysed to ammonium and glutamate. Concerning the latter process, a catabolic L-glutamine amidohydrolase (glutaminase) was isolated from the cytoplasm of this alkaliphilic bacterium and purified to homogeneity using liquid chromatography. Biochemical and physical parameters of the pure enzyme were examined in detail. Interestingly, analysis of the glutaminase revealed a marked increase in catalytic activity in the presence of phosphate, a property yet restricted to animal glutaminases. This is the first report on the presence of a phosphate-activated glutaminase in bacteria.

The picornavirus replication inhibitors HBB and guanidine in the echovirus-9 system: the significance of viral protein 2C.

HBB [2-(alpha-hydroxybenzyl)-benzimidazole] and guanidine are potent inhibitors of picornavirus replication. Among other evidence, limited cross-resistance and a synergistic effect of both inhibitors suggest similar but not identical mechanisms of antiviral action. Echovirus-9 variants resistant to each of these drugs were characterized and sequenced. Complete resistance to HBB or guanidine was shown to be due to single but different point mutations in the non-structural protein 2C. Protein 2C was expressed as GST fusion and His-tagged proteins for the wild-type and various mutants. Although three mutations were located in or near conserved NTP binding motifs, NTPase activity was not altered in the presence of HBB or guanidine.

Echovirus 9 strain Barty non-structural protein 2C has NTPase activity

Non-structural protein 2C is known to play a fundamental role in the replication of picornaviruses. Sequence analyses revealed that 2C belongs to a rapidly expanding group of proteins containing a consensus sequence for nucleotide binding (NTB). We report that echovirus 9 polypeptide 2C displays NTPase activity in vitro. In our experiments, several P2 genes were expressed in Escherichia coli as fusion proteins linked to glutathione S-transferase (GST) prior to purification close to homogeneity. In contrast to GST-2B, both GST-2C and GST-2BC showed ATPase as well as GTPase activity indicating that the site for NTB binding and splitting is located in 2C.

Ist Deutschland reif für eine Kanzlerin? Eine experimentelle Untersuchung aus Anlass der Bundestagswahl 2005

„Deutschland ist reif fur eine Bundeskanzlerin“. Dies auserte Volker Kauder, zum damaligen Zeitpunkt Generalsekretar der CDU, im Februar 2005 in einem Gesprach mit der Redaktion des Hamburger Abendblatts (Ausgabe vom 3. Februar 2005). Es kann mit einiger Berechtigung bezweifelt werden, ob er diese Aussage acht Monate spater so noch einmal wiederholt hatte. War die CDU/CSU doch bei der vorgezogenen Bundestagswahl im September 2005 mit ihrer Spitzenkandidatin Angela Merkel am Wahltag deutlich eingebrochen und mit nur 35,2 Prozent der Zweitstimmen weit hinter den Umfrageergebnissen der vorangegangenen beiden Wochen zuruckgeblieben, die bei mindestens 41 Prozent lagen (http://www.wahlrecht.de/umfragen/archiv/2005.htm).

Echovirus-9 protein 2C binds single-stranded RNA unspecifically.

Polypeptide 2C is essential for picornavirus replication. Although many data on multiple functions of this highly conserved protein are available, the mechanism of RNA binding is still obscure. In this work, protein 2C of echovirus-9 strain Barty was expressed as a histidine-tagged protein in E. coli followed by nondenaturing purification to homogeneity. After incubation of 2C protein with different kinds of RNA fragments, binding was shown in gel retardation assays. Competition experiments revealed that 2C targets linear RNA unspecifically; however, single-stranded linear DNA does not react with this protein. In contrast to poliovirus, protein 2C of echovirus-9 only recognizes RNA with a low content of secondary structures. This may be a first hint of a different binding specificity of 2C in echo- and polioviruses.

Determinants of pathogenicity of echovirus 9 in men: significance of a functional RGD-Motif

In this study, we investigated nine independent echovirus 9 isolates obtained from sick children in 1995. It is discovered that these isolates differ in respect to their pathogenicity for newborn mice indicating that the degree of human pathogenicity of an echovirus 9 variant does not necessarily correlate with mouse pathogenicity. Nevertheless, all virus variants are found to code for an RGD-motif within their VP1 protein. Hence, the RGD-motif and its highly conserved flanking regions are the conditio sine qua non, but, as expected, not sufficient for the mouse-pathogenic character.

Expression of lysostaphin in HeLa cells protects from host cell killing by intracellular Staphylococcus aureus

The Staphylococcus aureus-specific cell wall endopeptidase lysostaphin was used as a model for an intracellular acting bactericidal antibiotic. HeLa cells were transfected with an expression vector directing the heterologous expression of lysostaphin in the cytoplasm. Expression, subcellular localization and enzymatic activity of lysostaphin were investigated by immunoblotting, fluorescent microscopy and agar diffusion assays. Both transiently and stably transfected HeLa cells showed a strong expression of active lysostaphin. After infection with S. aureus, the intracellular number of S. aureus and the host cell viability were determined. This staphylolytic activity resulted in a strong reduction of intracellular S. aureus in a time- and dose-dependent manner. Furthermore, host cells expressing lysostaphin became protected from S. aureus-induced cell death. Our data demonstrate the potential of intracellularly acting cell-wall active drugs or antibiotics that kill S. aureus without causing harm to the infected host cells.

Lateinamerika, die USA und der 11. September 2001: interamerikanische Beziehungen und nationale Entwicklungen im Zeichen des „Krieges gegen den Terror“

Gegenstand dieses Beitrags sind die Beziehungen zwischen den USA und Lateinamerika nach dem 11. September. Gepragt wurden sie vom „Krieg gegen den Terror“, auch wenn der Subkontinent selbst nur ein Nebenschauplatz in der globalen Konfrontation war. Auswirkungen zeigten sich zum einen in der Debatte und der Gesetzgebung uber die Einwanderung in die Vereinigten Staaten, von der Lateinamerikaner im Allgemeinen und Mexikaner im Besonderen betroffen waren. Konsequenzen lassen sich zum anderen im „Krieg gegen die Drogen“ feststellen, den die USA in Kolumbien fuhrten und der in zunehmendem Mase Teil der Auseinandersetzung mit dem internationalen Terrorismus wurde.