Biomedical subjects

M Rohde

Publications and source records attributed to M Rohde.

At least 55 records · Page 3Linked to original sources

PubMed1999

Fc-mediated nonspecific binding between fibronectin-binding protein I of Streptococcus pyogenes and human immunoglobulins.

Fibronectin-binding protein I (SfbI) from Streptococcus pyogenes plays a key role in bacterial adhesion to, and invasion of, eukaryotic cells. In addition, SfbI exhibits a considerable potential as mucosal adjuvant and can trigger polyclonal activation of B cells. Here, we report that SfbI is also capable of binding human IgG in a nonimmune fashion. SfbI was reactive with IgG1, IgG2, IgG3, and IgG4 isotypes (type IIo IgG-binding profile). The affinity constant (Kd) of the SfbI-IgG interaction was in the range of 1-2 x 10(-5) M. Further studies demonstrated that the SfbI binding was mediated by the Fc component of the IgG molecule. Experiments performed using purified recombinant proteins spanning different domains of SfbI showed that the IgG-binding activity was restricted to the fibronectin-binding domains, and in particular to the fibronectin-binding repeats. Finally, the presence of recombinant SfbI resulted in an impairment of both phagocytosis of IgG-coated RBCs and Ab-dependent cell cytotoxicity by macrophages. These results demonstrated for the first time that, in addition to its major role during the colonization process, SfbI may also favor bacterial immune evasion after the onset of the infection by interfering with host clearance mechanisms.

Adhesins, Bacterial↗

PubMed1999

Isolation of a temperate bacteriophage encoding the type III effector protein SopE from an epidemic Salmonella typhimurium strain.

Salmonella typhimurium employs the specialized type III secretion system encoded in pathogenicity island 1 (SPI1) to translocate effector proteins into host cells and to modulate host cell signal transduction. The SPI1 type III system and the effector proteins are conserved among all salmonellae and are thought to be acquired by horizontal gene transfer. The genetic mechanisms mediating this horizontal transfer are unknown. Here, we describe that SopE, a SPI1-dependent translocated effector protein, is present in relatively few S. typhimurium isolates. We have isolated a temperate phage that encodes SopE. Phage morphology and DNA hybridization, as well as partial sequence information, suggest that this phage (SopEPhi) is a new member of the P2 family of bacteriophages. By lysogenic conversion this phage can horizontally transfer genes between different S. typhimurium strains. Strikingly, most of the isolates harboring SopEPhi belong to the small group of epidemic strains of S. typhimurium that have been responsible for a large percentage of human and animal salmonellosis and have persisted for a long period of time. Our data suggest that horizontal transfer of type III dependent effector proteins by lysogenic infection with bacteriophages (lysogenic conversion) may provide an efficient mechanism for fine-tuning the interaction of Salmonella spp. with their hosts.

Bacterial Proteins↗

PubMed1999

Heat-inactivation of plasmid-encoded CI857 repressor induces gene expression from Ind- lambda prophage in recombinant Escherichia coli.

We have observed significant cell lysis upon temperature up-shift of recombinant Escherichia coli cultures harboring CI857-repressed lambda-based expression vectors. This event, that becomes evident about 30-40 min after the heat shock, takes place when using the lambda promoter system in Ind- lysogenic strains, but not in others commonly employed for recombinant gene expression. These results strongly suggest that the thermosensitive CI857 repressor, encoded by the expression vector, competes with CI Ind- molecules for binding to the prophage operator region, allowing for expression of lytic genes from the integrated Ind- viral genome upon temperature up-shift. Transcription of viral lytic genes does not include unspecific expression of a reporter sulA::lacZ gene fusion carried in the prophage genome. These results prompt, however, to carefully evaluate the limitations of expression systems based on pL/pR-CI857 in bacterial strains modified through lambda Ind- gene transfer vehicles.

Bacteriolysis↗

PubMed1999

The actin-based motility of intracellular Listeria monocytogenes is not controlled by small GTP-binding proteins of the Rho- and Ras-subfamilies.

In this study, we analyzed whether the actin-based motility of intracellular Listeria monocytogenes is controlled by the small GTP-binding proteins of the Rho- and Ras-subfamilies. These signalling proteins are key regulatory elements in the control of actin dynamics and their activity is essential for the maintenance of most cellular microfilament structures. We used the Clostridium difficile toxins TcdB-10463 and TcdB-1470 to specifically inactivate these GTP-binding proteins. Treatment of eukaryotic cells with either of these toxins led to a dramatic breakdown of the normal actin cytoskeleton, but did not abrogate the invasion of epithelial cells by L. monocytogenes and had no effect on the actin-based motility of this bacterial parasite. Our data indicate that intracellular Listeria reorganize the actin cytoskeleton in a way that circumvents the control mechanisms mediated by the members of the Rho- and Ras-subfamilies that can be inactivated by the TcdB-10463 and TcdB-1470 toxins.

Actins↗

PubMed1999

Induction of cellular immunity to a mycobacterial antigen adsorbed on lamellar particles of lactide polymers.

Microspheres prepared from synthetic, biodegradable poly (L-lactide) [PLA] and copolymers of lactide and glycolide such as poly (DL lactide co-glycolide) [PLG] have been widely investigated for controlled delivery of encapsulated vaccine antigens. In this study we describe novel lamellar microparticles produced from PLA to which protein antigens can be adsorbed. These particles when administered to mice, induced strong Th1-type T cell responses to the adsorbed 38 kDa protein antigen from M. tuberculosis characterised by high levels of Interferon-gamma. In addition to proteins, we were also able to adsorb synthetic peptides resulting in specific T cell proliferation. Induction of strong cellular immunity together with the versatility of antigen adsorption to these particles should make such lamellae a useful tool to deliver protective antigens from intracellular pathogens.

Adsorption↗

PubMed1999

The effect of coenzyme Q10 administration on metabolic control in patients with type 2 diabetes mellitus.

A possible relationship between the pathogenesis of type 2 diabetes and coenzyme Q10 (CoQ10) deficiency has been proposed. The aim of this study was to assess the effect of CoQ10 on metabolic control in 23 type 2 diabetic patients in a randomized, placebo-controlled trial. Treatment with CoQ10 100 mg bid caused a more than 3-fold rise in serum CoQ10 concentration (p < 0.001). No correlation was observed between serum CoQ10 concentration and metabolic control. No significant changes in metabolic parameters were observed during CoQ10 supplementation. The treatment was well tolerated and did not interfere with glycemic control, therefore CoQ10 may be used as adjunctive therapy in patients with associated cardiovascular diseases.

Aged↗

PubMed1999

High serum coenzyme Q10, positively correlated with age, selenium and cholesterol, in Inuit of Greenland. A pilot study.

Greenlanders (Eskimos) have low prevalence of ischaemic heart disease, partly explained by a lower extent of atherosclerosis and a low n-6/n-3 ratio of polyunsaturated fatty acids. As atherosclerosis is also a result of oxidative stress, the total antioxidative readiness could have a substantial impact. From a health survey we chose the subpopulation from the most remote area, where the traditional Greenlandic diet with high intake of sea mammals and fish predominates. The mean (SD) of S-CoQ10 in males was 1.495 (0.529) nmol/ml and 1.421 (0.629) nmol/ml in females, significantly higher (p < 0.001) compared to a Danish population. In a linear multiple regression model the S-CoQ10 level is significantly positively associated with age and S-selenium in males, and S-total cholesterol in females. The high level of CoQ10 in Greenlanders probably reflects diet, since no bioaccumulation takes place, and it could probably be a substantial part of the antioxidative defense.

Adult↗

PubMed1999

Highly resistant Burkholderia pseudomallei small colony variants isolated in vitro and in experimental melioidosis.

Burkhloderia pseudomallei is the causative agent of melioidosis, a disease in which treatment failures and relapses are common. This study reports on slow growing B. pseudomallei 'small colony variants' (SCVs), isolated either in vitro after exposure to ceftazidime, ciprofloxacin or gentamicin or from the spleen and liver in a mouse model of melioidosis after treatment with ceftazidime. Interestingly, SCVs isolated by either method or antimicrobial agent showed a significant increase in the minimal inhibitory concentrations of various unrelated classes of antimicrobial agents. B. pseudomallei SCVs did not differ from their parental strains in standard biochemical profiles, nor by pulsed field gel electrophoresis or electron microscopy. Although the SCV phenotype was stable throughout numerous passages on antibiotic-free solid media, revertants with the parental colony morphology and, most importantly, with the parental susceptibility pattern occurred. These revertants led to rapid overgrowth of SCVs in liquid media without added antibiotics. Future studies will have to determine the clinical relevance of B. pseudomallei SCVs especially in treatment failure and relapse of infection.

Animals↗

PubMed1999

Cytoplasmic localization of the trypanothione peroxidase system in Crithidia fasciculata.

Tryparedoxin I (TXNI) and tryparedoxin peroxidase (TXNPx), novel proteins isolated from Crithidia fasciculata, have been reported to reconstitute a trypanothione peroxidase activity in vitro (Nogoceke, E.; Gommel, D. U.; Kiess, M.; Kalisz, H. M.; Flohé, L. Biol. Chem. 378:827-836; 1997). Combined with trypanothione reductase, they may form an NADPH-fueled trypanothione-mediated defense system against hydroperoxides in the trypanosomatids. In situ confocal microscopy of antibody-stained TXNI and TXNPx and electron microscopy of the immunogold labeled proteins revealed their colocalization in the cytosol. Insignificant amounts of the enzymes were detected in the nucleus and vesicular structures, whereas the kinetoplast and the mitochondrion are virtually free of any label. Comparison of the PCR product sequences obtained with genomic and cDNA templates rules out any editing typical of kinetoplast mRNA. Sequence similarities with any of the established maxicircle genes of trypanosomatids were not detectable. It is concluded that both, TXNI as well as TXNPx are encoded by nuclear DNA and predominantly, if not exclusively localized in the cytosol. Working in concert with trypanothione reductase, they can function as an enzymatic system that reduces hydroperoxides at the expense of NADPH without any impairment of the flux of reduction equivalents by cellular compartmentation.

Animals↗

PubMed1999

No effect of antioxidant supplementation in triathletes on maximal oxygen uptake, 31P-NMRS detected muscle energy metabolism and muscle fatigue.

A double-blind placebo-controlled cross-over trial was undertaken to evaluate the effect of antioxidant supplementation on maximal oxygen uptake during bicycling, 31-phosphorus nuclear magnetic response spectroscopy (31P-NMRS) detected muscle energy metabolism during plantar flexion and muscle fatigue evaluated by 1-s electrical stimulation at low (10 Hz) and high (50 Hz) frequency. Seven male triathletes received daily oral antioxidant supplementation in capsule form including 100 mg coenzyme Q10 (CoQ10), 600 mg ascorbic acid and 270 mg alpha-tocopherol or placebo over a 6-week interval. Serum concentration of CoQ10 was significantly higher in the antioxidant phase (1.80+/-1 microg x ml(-1), mean +/- SD) than control (0.9+/-0.21 microg ml(-1)) or placebo phase (0.9+/-0.3 microg x ml(-1)) (P<0.01). Maximal oxygen uptake was 63.8+/-3.0 ml x min(-1) x kg(-1) in the control phase, and did not change significantly in the antioxidant (67.6+/-10.8 ml x min(-1) x kg(-1)) or the placebo phase (61.9+/-4.5 ml x min(-1) x kg(-1)). The combined 31P-NMRS/low frequency fatigue test (plantar flexion of the foot) did not show differences in the gastrocnemius muscle pH (6.77+/-0.14), phosphocreatine reduction at the end of exercise (23+/-14% of rest) and half-time for recovery of phosphocreatine (33+/-12 sec) between the placebo and the antioxidant trial. No difference in muscle fatigue at 10 Hz electrical stimulation was found between the three phases. In conclusion, the results demonstrate no effect of antioxidative vitamin supplementation on maximal oxygen uptake, muscle energy metabolism or muscle fatigue in triathletes.

Adult↗

PubMed1999

Small-colony variants of Pseudomonas aeruginosa in cystic fibrosis.

In the context of chronic lung infection due to Pseudomonas aeruginosa in cystic fibrosis (CF), attention has been focused on the presence of the most common mucoid phenotype. In this study, the presence of small-colony variants (SCVs) of P. aeruginosa in respiratory tract specimens from patients with CF was investigated, and the clinical conditions predisposing to SCVs were analyzed. P. aeruginosa SCVs were isolated from 33 of 86 P. aeruginosa-positive CF patients over a 2-year period. Fast-growing revertants with larger surface colonies could be isolated from SCV populations. Electron microscopy revealed no significant difference in cell size or morphology. MICs of a broad range of antipseudomonas agents for SCVs were two- to eightfold higher than values for revertants. Recovery of SCVs was correlated with parameters revealing poor lung function and was significantly associated with daily inhalation of tobramycin or colistin.

Adolescent↗

PubMed1999

Sulfitobacter mediterraneus sp. nov., a new sulfite-oxidizing member of the alpha-Proteobacteria.

Analysis of PCR products of 16S rDNA of 680 isolates from Mediterranean Sea mesocosm experiments with taxon-specific 16S rDNA oligonucleotides revealed that 262 isolates belonged to the alpha subclass of the class Proteobacteria. Partial 16S rDNA sequence analysis of selected isolates and oligonucleotide probing with a Sulfitobacter-specific 16S rDNA probe affiliated 33 strains to the genus Sulfitobacter. Analysis of the HaeIII digest pattern of 16S rDNA revealed the presence of two groups; while 30 strains showed a pattern identical with that obtained for Sulfitobacter pontiacus DSM 10014T, a second group of three strains had a unique pattern that was different from that of the type strain. Five isolates of group 1 and one isolates of group 2, strain CH-B427T, were selected for detailed taxonomic analysis. All six isolates closely resembled the type strain Sulfitobacter pontiacus DSM 10014T in physiological reactions. However, strain CH-B427T differed quantitatively in the composition of fatty acids from Sulfitobacter pontiacus DSM 10014T and showed only 98.2% 16S rDNA sequence similarity with strain DSM 10014T. DNA-DNA reassociation value obtained for strains DSM 10014T and CH-B427T revealed 46% similarity. Based on the results of DNA-DNA reassociation and discrete differences in the nucleotide composition of 16S rDNA, a new species of the genus Sulfitobacter is proposed, designated Sulfitobacter mediterraneus sp. nov., the type strain being strain CH-B427T (= DSM 12244T).

Bacterial Typing Techniques↗

PubMed1999

Bacillus silvestris sp. nov., a new member of the genus Bacillus that contains lysine in its cell wall.

A Gram-positive, aerobic, rod-shaped, peritrichously flagellated, round-endospore-forming bacterium was isolated from a forest soil near Braunschweig, Lower Saxony, Germany, and designated strain HR3-23T (T = type strain). Morphologically, strain HR3-23T shows the characteristics of a member of the genus Bacillus. The spore position is terminal in a swollen sporangium. Comparative analysis of the 16S rDNA sequence shows strain HR3-23T to be most closely related to Caryophanon tenue (95.8% 16S rRNA similarity) and to Bacillus sphaericus (95.4% 16S rRNA similarity). Phylogenetically, the isolate clusters among species of Bacillus RNA group 2. The DNA G + C content of isolate HR3-23T is 39.3 mol%, the peptidoglycan type is A4 alpha (L-Lys-D-Glu), the major respiratory lipoquinone is menaquinone MK-7 and the predominant fatty acid is of the iso-C15:0 type. Based on the morphological, chemotaxonomic, physiological and phylogenetic properties, a new species, Bacillus silvestris, is proposed; strain HR3-23T is the type strain (= DSM 12223T).

Bacillus↗

PubMed1999

Electron microscopy studies of cell-wall-anchored cellulose (Avicel)-binding protein (AbpS) from Streptomyces reticuli.

Streptomyces reticuli produces a 35-kDa cellulose (Avicel)-binding protein (AbpS) which interacts strongly with crystalline cellulose but not with soluble types of cellulose. Antibodies that were highly specific for the NH2-terminal part of AbpS were isolated by using truncated AbpS proteins that differed in the length of the NH2 terminus. Using these antibodies for immunolabelling and investigations in which fluorescence, transmission electron, or immunofield scanning electron microscopy was used showed that the NH2 terminus of AbpS protrudes from the murein layer of S. reticuli. Additionally, inspection of ultrathin sections of the cell wall, as well as biochemical experiments performed with isolated murein, revealed that AbpS is tightly and very likely covalently linked to the polyglucane layer. As AbpS has also been found to be associated with protoplasts, we predicted that a COOH-terminal stretch consisting of 17 hydrophobic amino acids anchors the protein to the membrane. Different amounts of AbpS homologues of several Streptomyces strains were synthesized.

Bacterial Proteins↗

PubMed1999

Characterization of a murine model of melioidosis: comparison of different strains of mice.

Melioidosis is an infectious disease caused by the saprophytic gram-negative rod Burkholderia pseudomallei. The aim of this study was to establish and characterize a murine model of melioidosis to provide a basis for further investigations on the pathogenesis of the disease. After intravenous infection with B. pseudomallei, C57BL/6 mice were found to be significantly more resistant than BALB/c mice. There was a marked organotropism of B. pseudomallei for the spleen and liver in both strains of mice, with the highest bacterial load in the spleen. Electron microscopic investigations of the spleen clearly demonstrated intracellular replication within membrane-bound phagosomes. Electron micrographs of the liver provided evidence that B. pseudomallei-containing phagosomes in hepatocytes fuse with lysosomes, leading to degradation of bacteria. In both strains of mice, the course of infection was highly dependent on the infective dose and the bacterial strain used, ranging from death within a few days to death after several weeks. In comparison with BALB/c mice, the bacterial counts in C57BL/6 mice were decreased 12 h after infection, which is suggestive of an innate immune mechanism against B. pseudomallei in this early phase of infection contributing to the lower susceptibility of C57BL/6 mice. BALB/c mice developed a more pronounced lymphopenia, granulocytosis, and splenomegaly at a lower infective dose compared to C57BL/6 mice. Analysis of the antibody response against B. pseudomallei 11 days after infection revealed a significantly higher immunoglobulin G2A (IgG2a)/IgG1 ratio in C57BL/6 mice than in BALB/c mice, indicating that a T helper type 1 immune response is associated with resistance to infection with B. pseudomallei.

Animals↗

PubMed1999

The EspD protein of enterohemorrhagic Escherichia coli is required for the formation of bacterial surface appendages and is incorporated in the cytoplasmic membranes of target cells.

The formation of EspA-containing surface appendages in pathogenic Escherichia coli strains, both enteropathogenic E. coli (EPEC) and Shiga toxin-producing E. coli strains, is essential for critical events in the infective process, e.g., localized bacterial adherence to host cells with formation of microcolonies and induction of attaching and effacing lesions. It has been reported that EPEC mutants deficient in the production of EspD, which is encoded by the esp operon, are unable to accumulate actin underneath adherent bacteria but exhibit an attachment similar to that of the wild type. Here, we report the construction and characterization of an in-frame espD deletion mutant of the enterohemorrhagic E. coli (EHEC) strain EDL933. In contrast to what was observed in EPEC mutants, the EDL933 espD mutant not only lacked the capacity to accumulate actin but also exhibited an impaired attachment to HeLa cells. The synthesis of the EspD protein was also essential for the formation of EspA-containing filaments. Finally, localization studies demonstrated that the EspD protein is transferred to the cytoplasm and integrated into the cytoplasmic membranes of infected cells. These results help to elucidate the underlying molecular events in infections caused by EHEC.

Bacterial Proteins↗

PubMed1999

A cytoplasmic structure resembling large protein aggregates induced by interferons.

IFP 35 is an interferon (IFN)-regulated leucine zipper protein, expression of which is observed in a variety of cell types including monocytes/macrophages, epithelial cells and fibroblasts. Using immunofluorescence studies, we demonstrate that IFP 35 is found in characteristic punctate cytoplasmic structures after IFN treatment. Co-localization experiments using double immunofluorescence and confocal laser scanning microscopy failed to show association of IFP 35 with known organelles (mitochondria, peroxisomes, endoplasmic reticulum, lysosomes, endosomes, Golgi complex), ribosomes, or actin filaments. Subcellular fractionation to separate membrane-associated from cytoplasmic proteins demonstrated that IFP 35 localizes to the cytoplasm. Separation of postnuclear supernatant from HeLa cells by gel filtration revealed that IFP 35 eluted at a molecular mass of 200-440 kD, suggesting that IFP 35 is part of protein complexes. Electron microscopic studies showed cytoplasmic clusters of a few aggregates of IFP 35 in IFN-treated cells which were neither associated with nor surrounded by a membrane. A combination of immunoprecipitation and immunofluorescence studies of cells transfected with a hemagglutinin epitope-tagged IFP 35 expression construct demonstrated complex formation and co-localization of endogenous and transfected IFP 35. Taken together, our studies demonstrate that IFP 35 associates with unique cytoplasmic structures that are distinct from known organelles and resemble large protein aggregates.

Blotting, Western↗

PubMed1998

Expression of a p16INK4a-specific ribozyme downmodulates p16INK4a abundance and accelerates cell proliferation.

The pl6INK4a tumor suppressor negatively regulates progression through the G1 phase of the mammalian cell cycle. To mimic the downmodulation of p16INK4a commonly seen in cancer, we designed and characterized a hammerhead ribozyme against exon E1alpha of the murine pl6INK4a transcript. Stable expression of the ribozyme in murine erythroleukemia (MEL) cells reduced the endogenous pl6INK4a protein by more than 70% and significantly accelerated cell cycle progression. The specificity and efficiency of our new ribozyme suggest its possible application in elucidating the role of p16INK4a in fundamental biological processes including homeostatic tissue renewal, protection against oncogenic transformation, and cellular senescence.

Animals↗