Biomedical subjects

Marcus Klein

Publications and source records attributed to Marcus Klein.

6 recordsLinked to original sources

PubMed2006

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.

Cytoplasm↗

PubMed2003

Detection of Chlamydia pneumoniae-specific antibodies binding to the VD2 and VD3 regions of the major outer membrane protein.

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.

Antibodies, Bacterial↗

PubMed2002

Radical trapping in glycogen storage disease 1a.

UNLABELLED: Oxidative mechanisms involving lipid peroxidation in the subendothelium of the arterial vessel wall play a key role in atherogenesis. Despite severe hyperlipidaemia, patients with glycogen storage disease type 1a (GSD1a) do not develop premature atherosclerosis. Therefore, we analysed parameters of antioxidative defence and oxidative stress in plasma and serum of patients with GSD1a ( n=17) and compared them with those of patients with type 1 diabetes mellitus ( n=17), familial hypercholesterolaemia ( n=18) and healthy controls ( n=20). We measured the total radical trapping ability parameter (TRAP), single plasma antioxidants (sulfhydryl-groups, uric acid, vitamin C, alpha-tocopherol, coenzyme-Q10), markers of lipid peroxidation, lipoprotein (a) and homocysteine. Patients with GSD1a showed an elevated TRAP ( P<0.01) compared to the three other groups. This can mainly be attributed to elevated uric acid levels ( P<0.05 versus control). Lipoprotein (a) was significantly lower in the GSD1a group compared to the three other groups ( P<0.05). CONCLUSION: Patients with glycogen storage disease type 1a show an increased antioxidative defence in plasma which may protect them against lipid peroxidation and thus against premature atherosclerosis. Our finding of low lipoprotein(a) levels in this small group of patients warrants further investigation in a greater number of patients before assessing its role in atherogenesis in glycogen storage disease type 1a.

Adolescent↗

PubMed2002

Plasma antioxidants in pediatric patients with glycogen storage disease, diabetes mellitus, and hypercholesterolemia.

Oxidative modification of lipoproteins in vessel walls plays a key role in atherogenesis. Patients with glycogen storage disease type Ia (GSD Ia) do not develop premature atherosclerosis despite severe hyperlipidemia. We analyzed antioxidative defense and oxidative stress in plasma and serum of patients with GSD Ia (n = 17) compared to patients with type I diabetes mellitus (DMI, n = 17), familial hypercholesterolemia (FH, n = 18), and healthy controls (n = 20). We measured the total radical-trapping antioxidant parameter (TRAP), single antioxidants (sulfhydryl groups, uric acid, vitamin C, alpha-tocopherol, coenzyme Q10), malondialdehyde, oxidized low density lipoprotein (LDL) antibodies, lipid profile [cholesterol, triglyceride, lipoprotein (a)], homocysteine, and hemoglobin (Hb)A(1C). TRAP levels were elevated in the GSD Ia group (p <.01) and correlated with elevated uric acid levels (r = 0.72, p =.001). None of the other plasma antioxidants correlated with TRAP levels. DMI patients showed decreased sulfhydryl groups (p <.01) and a reduced ubiquinol-10 fraction (p <.01). Malondialdehyde (p <.001) and oxidized LDL autoantibodies (p <.05) were increased in the diabetic group. In FH patients, parameters of oxidative stress and TRAP did not differ from controls. We conclude that in GSD Ia an increased antioxidative defense in plasma may protect against lipid peroxidation and thus against premature atherosclerosis. Furthermore, we demonstrated that in DMI increased oxidative mechanisms are already present in childhood.

Adolescent↗

PubMed2002

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.

Bacillus↗

PubMed2000

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.

Echovirus 9↗