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M Otto

Publications and source records attributed to M Otto.

At least 55 records · Page 3Linked to original sources
PubMed2003

Metformin inhibits glycogen synthesis and gluconeogenesis in cultured rat hepatocytes.

AIM: Glycogen synthesis, and glucose and lactate production were examined in cultured rat hepatocytes preincubated with metformin (0-500 micro m) for 24 h. METHODS: Cells incubated with[1-13C]-glucose and [1-13C]-lactate allowed us to study the effect of metformin on glucose production from glycogenolysis and gluconeogenesis in a detailed manner using NMR spectroscopy. 1H and 13C-filtered 1H-NMR spectra were recorded by using flow-injection technique. RESULTS: Metformin decreased glycogen synthesis in a dose-dependent manner with an IC50 value of 196.5 micro m. This effect could not be reversed by the presence of the glycogen phosphorylase inhibitor DAB, suggesting that glycogenolysis was not affected. A clear correlation between glucose production and glycogen content (0.97 < R < 0.99; p < 0.001) and lactate production and glycogen content (0.97 < R < 0.99; p < 0.001) was observed. Moreover, a strong inhibition (62%, p < 0.001) of glucose produced from lactate/pyruvate (3 mm/0.3 mm) was observed in cells treated with 350 micro m metformin. CONCLUSION: Hepatocytes preincubated for 24 h in the presence of metformin at clinically relevant concentrations showed impaired glycogenesis as well as gluconeogenesis.

Animals↗
PubMed2003

Purification of human natural killer cells using a clinical-scale immunomagnetic method.

BACKGROUND: Infection, graft failure, disease relapse, and GvHD are significant adverse events associated with allogeneic BMT. Although donor leukocyte infusion has been used to prevent or to treat infection, graft failure, and relapse, the potential clinical benefits are often outweighed by the risk of T cell-mediated GvHD. Results from animal studies suggest that donor natural killer (NK) cells may be an ideal cell type for prevention or treatment of these adverse events. We have therefore sought to develop an automated, efficient, and clinical-scale human NK cell-purification method. METHODS: Twelve leukopheresis products were purified for NK cells using a two-step immunomagnetic method. CD3(+) cells were first depleted from the apheresis products. CD56(+) cells were then enriched from the CD3(+) cell-depleted products. RESULTS: The median percentage of CD3(-)CD56(+) NK cells in the final products was 91.0%, and the median recovery was 48.7%. The median depletion for CD3(+)CD56(-) T cells was 5.3 log. Natural cytotoxicity of the purified cells was approximately five-fold higher than that of unpurified mononuclear cells, and it could be further increased by stimulation of the purified cell with IL2. DISCUSSION: We described a large-scale purification method for automated, efficient, and rapid isolation of human NK cells that yielded minimal contamination with T cells or B cells. These purified NK cells may be expedient for preclinical and clinical uses.

Antigens, CD19↗
PubMed2002

Tau protein and 14-3-3 protein in the differential diagnosis of Creutzfeldt-Jakob disease.

BACKGROUND: Diagnosis of Creutzfeldt-Jakob disease (CJD) is made according to the typical clinical picture and can be supported by a positive 14-3-3 CSF immunoblot. Promising results for the diagnostic sensitivity and specificity of tau-protein measurement in CSF already have been described in a smaller group of patients. Both tests in a larger group of patients with the differential diagnosis of CJD were evaluated. METHODS: CSF of 297 patients under the differential diagnosis of CJD (109 definite, 55 probable, 39 possible; 85 others, 1 iatrogenic, 8 genetic), 23 nondemented control subjects, and 15 non-CJD patients with positive 14-3-3 immunoblots were analyzed. The 14-3-3 immunoblot bands were semiquantitatively rated as strong, medium, and weak. Tau-protein was analyzed using a commercially available ELISA. In addition, patients were neuropathologically classified according to prion protein type and polymorphism at codon 129. RESULTS: A diagnostic sensitivity of 94%, a diagnostic specificity of 90%, and a positive predictive value of 92% were achieved for tau-protein at a cut-off of 1,300 pg/mL. These results are comparable with those of the 14-3-3 immunoblot. For patients with type II prion protein and methionine/valine or valine/valine polymorphism at codon 129, tau-protein has a higher diagnostic sensitivity than 14-3-3 protein. Tau-protein levels were significantly higher in patients with higher-rated 14-3-3 immunoblot bands. CONCLUSION: The differential diagnostic significance of the 14-3-3 immunoblot is similar to that of the tau-protein ELISA. The advantage of the tau-protein ELISA is that it is easy to use in routine laboratories. Patients with a negative 14-3-3 immunoblot already have measurable tau-protein levels. This increases information on 14-3-3-negative patients with CJD and especially on patients with other diseases.

14-3-3 Proteins↗
PubMed2002

[Growth stimulation of human osteoblast-like cells by thrombocyte concentrates in vitro].

AIM OF THE STUDY: Thrombocytes contain different growth factors. Their potential use for increasing bone regeneration is currently under discussion. Basic data about the biologic effect of platelet concentrates (platelet-rich plasma, PRP) are still lacking. This study analyzes the in vitro effect of PRP on the proliferation rate of human osteoblast-like cells (OLC). MATERIAL AND METHOD: Human OLC were stimulated with thrombocytes in different dilutions (0-1,700,000 thrombocytes/microliter). Three days later a WST test (Roche Diagnostics) was performed to measure the mitochondrial syntheses and cellular proliferation rate. RESULTS: The proliferation rate of the OLC was (concentration-dependent) increased up to a certain plateau by adding thrombocytes. Further stimulation led to a slight decrease in the proliferation rate. The statistical analysis of the extinction measurements showed significant differences in the cell proliferation rates between mostly all thrombocyte concentration groups (p < 0.0001). Moreover, a statistically significant difference was evaluated for the time-dependent change in extinction (0-5 h) (p < 0.0001). CONCLUSION: This study shows that the proliferation rate of human OLC can be stimulated in vitro by concentration-dependent platelet concentrates. This in vitro result supports the currently discussed assumption that the clinical use of PRP might increase bone regeneration.

Bone Regeneration↗
PubMed2002

Tissue response and biomaterial integration: the efficacy of in vitro methods.

Implantation involves tissue trauma, which evokes an inflammatory response, coupled to a wound healing reaction, involving angiogenesis, fibroblast activation and matrix remodelling. Until now the type and extent of such reactions to give optimal integration of various biomaterials are practically unknown. Three principal fields of research can yield useful data to understand these phenomena better: studies on explanted biomaterials, animal models and relevant in vitro techniques. This paper will present examples of the latter field and the application of endothelial cell (EC) culture systems to study the effects of important tissue (e.g. pro-inflammatory cytokines, chemokines) and material (e.g. metal ions, particulate debris) factors on the regulation of the inflammatory and angiogenic response. A central feature is the use of microvascular endothelial cells (MEC), which can be used in both 2-and 3-dimensional (3-D) assays. We have also used genetic manipulation to develop a permanent MEC line from the human lung (HPMEC-ST1), which is being tested for its suitability to study cell-biomaterial interactions. In addition, suitable in vitro techniques are being developed in order to investigate drug delivery systems (DDS). Of particular interest is the targeting of the central nervous system, our approach being to establish a human model of the blood-brain barrier (BBB). A mainstay of our scientific philosophy is that such in vitro methods can make an important contribution to understanding biological reactions at the tissue-biomaterial interface and thus further a causal approach to tissue engineering (TE) and drug delivery applications.

Animals↗
PubMed2002

Specific 14-3-3 isoform detection and immunolocalization in prion diseases.

14-3-3 proteins are involved in signalling processes in neuronal cells. Using isoform-specific antibodies we have examined the variation in 14-3-3 isoform neurolocation in normal and scrapie-infected murine brain and show that in defined areas of the brain there are significant changes associated with the pathology of the disease process. The appearance of 14-3-3 proteins in the cerebrospinal fluid (CSF) is a consequence of neuronal disease and the detection of specific isoforms of the 14-3-3 proteins in the CSF is characteristic of some neurodegenerative diseases. In this study, monitoring specifically for the gamma 14-3-3 isoform in the CSF by both Western-blot analysis and ELISA we can show a level of correlation between the assays.

14-3-3 Proteins↗
PubMed2002

Highly conserved and disease-specific patterns of carboxyterminally truncated Abeta peptides 1-37/38/39 in addition to 1-40/42 in Alzheimer's disease and in patients with chronic neuroinflammation.

Human lumbar CSF patterns of Abeta peptides were analysed by urea-based beta-amyloid sodium dodecyl sulphate polyacrylamide gel electrophoresis with western immunoblot (Abeta-SDS-PAGE/immunoblot). A highly conserved pattern of carboxyterminally truncated Abeta1-37/38/39 was found in addition to Abeta1-40 and Abeta1-42. Remarkably, Abeta1-38 was present at a higher concentration than Abeta1-42, being the second prominent Abeta peptide species in CSF. Patients with Alzheimer's disease (AD, n = 12) and patients with chronic inflammatory CNS disease (CID, n = 10) were differentiated by unique CSF Abeta peptide patterns from patients with other neuropsychiatric diseases (OND, n = 37). This became evident only when we investigated the amount of Abeta peptides relative to their total Abeta peptide concentration (Abeta1-x%, fractional Abeta peptide pattern), which may reflect disease-specific gamma-secretase activities. Remarkably, patients with AD and CID shared elevated Abeta1-38% values, whereas otherwise the patterns were distinct, allowing separation of AD from CID or OND patients without overlap. The presence of one or two ApoE epsilon4 alleles resulted in an overall reduction of CSF Abeta peptides, which was pronounced for Abeta1-42. The severity of dementia was significantly correlated to the fractional Abeta peptide pattern but not to the absolute Abeta peptide concentrations.

Adult↗
PubMed2001

Expression of apoptosis-related proteins, p53, and DNA fragmentation in sarcomas of the pulmonary artery.

BACKGROUND: Apoptosis is a common feature in a variety of pathologic conditions. Induction of apoptosis through apoptotic stimuli such as, chemotherapy or radiation, presents new insights into tumor biology and therapy. In particular, members of the Bcl-2 family as well as the Fas system are known to be involved in the regulation of apoptosis in different tumor entities. METHODS: In the current study, the expression of the apoptosis-related molecules p53, Bax, Bcl-2, Fas (CD95), Fas-Ligand and perforin was examined in 7 patients with a sarcoma of the pulmonary artery. Furthermore, the TUNEL-method for the detection of apoptotic cells was applied as well as sequencing of the p53 gene. RESULTS: In the TUNEL assay, approximately 10% of the sarcoma cells displayed DNA fragmentation. In addition, Bax was expressed in tumor cells. Accumulation of p53 was evident in 4 of 7 patients (pAB 240 antibody), and 2 of them were positive for the pAB 1801 antibody. Only 1 case had a point mutation in Exon 5 of the p53 sequence. A few tumor cells showed a double labeling of Bax and p53. Bcl-2 could be detected only in tumor-associated lymphocytes. Finally, several lymphocytes could be stained with perforin, but none of the specimens showed a reactivity for Fas or Fas-Ligand. CONCLUSION: The expression of Bax indicated a possible role of this molecule in programmed cell death in pulmonary sarcomas. The limited coexpression of Bax and p53 suggested that induction of Bax can occur independently of p53. The detection of perforin in lymphocytes suggested a possible role for this molecule in apoptosis of the sarcoma cells. In contrast, the Fas system did not seem to play an essential role in sarcomas of the great vessels.

Adult↗
PubMed2001

Staphylococcus aureus resistance to human defensins and evasion of neutrophil killing via the novel virulence factor MprF is based on modification of membrane lipids with l-lysine.

Defensins, antimicrobial peptides of the innate immune system, protect human mucosal epithelia and skin against microbial infections and are produced in large amounts by neutrophils. The bacterial pathogen Staphylococcus aureus is insensitive to defensins by virtue of an unknown resistance mechanism. We describe a novel staphylococcal gene, mprF, which determines resistance to several host defense peptides such as defensins and protegrins. An mprF mutant strain was killed considerably faster by human neutrophils and exhibited attenuated virulence in mice, indicating a key role for defensin resistance in the pathogenicity of S. aureus. Analysis of membrane lipids demonstrated that the mprF mutant no longer modifies phosphatidylglycerol with l-lysine. As this unusual modification leads to a reduced negative charge of the membrane surface, MprF-mediated peptide resistance is most likely based on repulsion of the cationic peptides. Accordingly, inactivation of mprF led to increased binding of antimicrobial peptides by the bacteria. MprF has no similarity with genes of known function, but related genes were identified in the genomes of several pathogens including Mycobacterium tuberculosis, Pseudomonas aeruginosa, and Enterococcus faecalis. MprF thus constitutes a novel virulence factor, which may be of general relevance for bacterial pathogens and represents a new target for attacking multidrug resistant bacteria.

Amino Acid Sequence↗
PubMed2001

Large-scale, multicenter study of cerebrospinal fluid tau protein phosphorylated at serine 199 for the antemortem diagnosis of Alzheimer's disease.

We surveyed a total of 570 cerebrospinal fluid (CSF) samples from a variety of diseases, including Alzheimer's disease (AD; n = 236), non-AD-demented and nondemented diseases (n = 239), and normal controls (n = 95) to quantitate levels of tau protein phosphorylated at serine 199 (CSF/phospho-tau199) by a recently established sandwich ELISA. The CSF/phospho-tau199 levels in the AD group were significantly elevated compared to those in all the other non-AD groups. Receiver operating characteristics curves showed that the diagnostic sensitivity and specificity for the AD group vs all the other non-AD groups using the CSF/phospho-tau199 were 85.2% and 85.0%, respectively. Furthermore, there was a significant positive correlation between CSF/phospho-tau199 and CSF/total-tau levels in the AD group. Elevated CSF/phospho-tau199 in the AD group was noted irrespective of age, gender, dementia severity, and number of apolipoprotein E4 alleles. Thus, we suggest that CSF/phospho-tau199 may be a novel and logical biomarker in supporting antemortem diagnosis of AD.

Aged↗
PubMed2001

Structure-function relationships in the tryptophan-rich, antimicrobial peptide indolicidin.

Indolicidin is a cationic 13 amino acid peptide amide produced in the granules of bovine neutrophils with the sequence H-ILPWKWPWWPWRR-NH2. Indolicidin is both antimicrobial and, to a lesser extent, haemolytic. In order to systematically investigate structure-function relationships, the solid-phase synthesis of indolicidin and 48 distinct analogues are reported, as well as the characterization of their respective biological properties. Peptides synthesized and characterized include analogues with modified terminal functions, truncations from either terminus, an alanine scan to determine the role of each individual amino acid, specific amino acid exchanges of aromatic, charged and structural residues and several retro-, inverso- and retroinverso-analogues. Together, characterization of these analogues identifies specific residues involved in antimicrobial or haemolytic activity and suggests a core structure that may form a scaffold for the further development of peptidomimetic analogues of indolicidin.

Amino Acid Sequence↗
PubMed2001

Unaltered apoptotic behaviour of mononuclear cells from patients with sporadic Creutzfeldt-Jakob disease.

Creutzfeldt-Jakob disease (CJD) belongs to the group of transmissible spongiform encephalopathies. It is suspected that a pathologically altered form of the prion protein (PrPSc) is the decisive trigger of the disease. Data from animal experiments suggest an involvement of the lymphatic system in the intracorporal transport of PrPSc. However, it has not so far been possible to detect PrPSc on mononuclear cells (MNCs) either in the sporadic form of CJD or in the new variant of CJD (vCJD). In order to determine a possible alteration of MNCs in CJD, we investigated the natural and induced apoptotic behaviour of these cells. MNCs from 19 patients with sporadic CJD and from 20 patients with other neurological disorders were used. The cells were analysed by fluorescence cytometry with and without apoptosis induction by xanthine oxidase and hypoxanthine. The apoptosis rate was quantified using the stain 7-amino-actinomycin D (7-AAD). In the morphological investigation of the cells before apoptosis induction, there were no significant differences between the groups with regard to cell size and granularity of the MNCs. After apoptosis induction, the typical significant decrease in cell size and increase in granularity of the cells occurred in both groups. Significant differences between the patient populations were not found. For the first time, our investigation has demonstrated that a functional impairment of MNCs with regard to their apoptotic behaviour does not occur in sporadic CJD. It remains open to question whether this mechanism plays an important role in forms of transmissible encephalopathy other than sporadic CJD, especially after oral transmission.

Adult↗
PubMed2001

Nonmetals in the argon-inductively coupled plasma-optical emission spectrometry: I. Phosphorus, sulfur and carbon.

The behavior of phosphorus, sulfur and carbon in the argon-ICP-OES was systematically investigated for a wide range of nebulizer gas flows and observation heights. Five lines of phosphorus, four lines of sulfur and three lines of carbon, which have analytical usable detection limits, were taken into consideration. The further parameter set was inspired by the needs of every-day-analysis in the laboratory, especially the low integration time that is necessary for analysis of large amounts of samples. For each element line a signal-to-noise plot was obtained with a method described earlier. The optimum conditions for the combined analysis were determined from signal-to-noise plots of those element lines with the lowest detection limits. The lowest detection limits for phosphorus (0.13 mg/L) and carbon (0.01 mg/L) are useful for solving many analytical problems. However, even the best detection limit for sulfur (2.97 mg/L) is only of limited use without further preconcentration. A major problem was the background of carbon, most probably from carbon dioxide, which increased the available detection limit to about 0.04 mg/L. Surprisingly, the best detection limits were obtained at very low observation heights, which were only a couple of millimeters above the load coil. Fortunately, all elements showed a similar behavior and so the detection limits at compromise conditions were only slightly higher compared with the single-element detection limits.

Journal Article↗
PubMed2001

Capillary zone electrophoresis of proteins with poly(2-hydroxyethyl methacrylate)-coated capillaries: fundamental and applications.

Fused silica capillaries have been modified by atom-transfer radical polymerization (ATRP) to generate covalently bonded polymer films of 2-hydroxyethyl methacrylate. Because the kinetics of ATRP have mainly been investigated in bulk solutions, a GC experiment was set up to examine monomer conversion inside narrow-bore capillaries. It was shown that after 1 to 4 h the reaction was nearly complete. The coating process was further optimized by monitoring EOF, because low EOF indicates high surface coverage. To deal with the very low EOF values, a new approach was used to dramatically reduce the measurement time by overlaying hydrodynamic flow on the electroosmotic flow. The corresponding equations are derived separately in detail. Capillaries were then coated under optimum conditions with linear or cross-linked polymer films. The EOF was reduced over a wide range of pH values. A long-term reproducibility test with both types of functionalization showed that the efficiency of the linear polymer coating decreased significantly over time. With cross-linked films, however, the efficiency even increased. Relative standard deviations for protein migration times were also much lower in cross-linked coated capillaries. Highly efficient separations could be performed for basic and acidic proteins in acidic media, and for the latter even in basic media.

Electrophoresis, Capillary↗
PubMed2001

Interpretation of analytical patterns from the output of chaotic dynamical memories.

A fundamental study has been carried out to use a nonlinear dynamic system, Freeman's KIII model, for pattern recognition in analytical chemistry. Filtering of the output of the neuron activity traces in the gamma range has been studied by applying a fast Fourier transform filter. Different possibilities for interpretation of the system output have been investigated. Preliminary results are demonstrated for classification of drugs based on their spectra in the mid infrared range.

Journal Article↗