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S Diekmann

Publications and source records attributed to S Diekmann.

At least 19 recordsLinked to original sources
PubMed2003

Two-step purification of the outer membrane transporter and activator protein ShlB from Escherichia coli using internally His6-tagged constructs.

ShlB from Serratia marcescens was isolated and purified from a porin-deficient Escherichia coli BL21 strain using a combination of detergent extraction, affinity and ion-exchange chromatography. An internal histidine affinity tag was introduced that did not interfere with activity. At each stage of the purification scheme biological activity of the ShlB protein was assessed. Using this scheme, several His(6)-tagged mutants of ShlB were purified to electrophoretic homogeneity.

Affinity Labels↗
PubMed2003

Use of iodine-based contrast media in digital full-field mammography--initial experience.

AIM: To investigate the use of iodine-based contrast media in digital full-field mammography. METHODS: After performing initial phantom studies, seven patients underwent digital mammography (Senographe 2000D, GE Medical Systems, Milwaukee, USA) using a specially filtered beam before as well as 60, 120, and 180 seconds after injection of 80 ml of iodine contrast medium (Ultravist 370, Schering AG, Germany). The precontrast mammograms were then subtracted from the postcontrast mammograms and the resulting images compared with a contrast-enhanced dynamic MRI study, performed on all women. RESULTS: Contrast medium accumulation within the tumors was visualized with a good quality in all cases. The conditions under which successful contrast-enhanced digital mammography can be performed were determined in phantom studies. CONCLUSIONS: Contrast-enhanced digital mammography has a potential for improving the visualization of breast tumors in mammography using special beam filtering, adjusted x-ray parameters, proper timing, and suitable subtraction software.

Breast Neoplasms↗
PubMed2003

[Dose reduction through gridless technique in digital full-field mammography].

PURPOSE: To determine the role of the scatter grid in digital full-field mammography with respect to image quality and dose and to compare the experimental results with initial clinical experience. MATERIALS AND METHODS: A phantom consisting of 205 fields that enclose gold dots of different thickness and size (CD-Mam phantom, Medical Department, Nijmegen, Netherlands) was used for digital full-field mammography with the conventional grid module and a special gridless module. Four different breast thicknesses were simulated using Plexiglas as scatter material. First, the phantom was exposed at the parameter and dose settings automatically selected in each experimental setup (with and without grid). Subsequently, the phantom was exposed at the different simulated breast thicknesses using the gridless module in combination with the parameters automatically selected for the grid module. This was followed by a series of phantom mammograms obtained with the experimental setup reversed. The 16 mammograms were evaluated by 3 readers and the results compared considering breast thickness, radiation dose, and quality. The gridless module was used for preoperative labeling in 16 patients for comparison of mammograms obtained with and without a grid. RESULTS: For the same entrance dose used in routine mammography, digital mammography without grid is superior to digital mammography with grid when performed on simulated thin breasts (Plexiglas less than 3 cm), with no difference found when performed on simulated large breasts. The advantages of gridless mammography are more pronounced at a markedly reduced entrance dose (identical parenchymal dose without and with grid using the dose automatically selected for the gridless module). This tendency is confirmed by the initial clinical comparison. CONCLUSION: Gridless digital full-field mammography has the potential to reduce the dose further, in addition to already known mechanisms of dose reduction. However, the gridless system must be adjusted (markedly higher "switch-off dose" at the detector) to achieve an overall dose reduction since gridless mammography is inferior to grid mammography for identical doses at the detector plane.

Artifacts↗
PubMed2003

[Visualization of microcalcifications on mammographies obtained by digital full-field mammography in comparison to conventional film-screen mammography].

PURPOSE: To evaluate the visualization of microcalcifications on mammographies obtained by full-field digital mammography (FFDM) in comparison to conventional film-screen mammography (FSM). MATERIAL AND METHODS: Forty-seven digital and film-screen mammographies depicting histologically proven lesions (27 benign, 20 malignant) were assessed by 4 readers. The images obtained with the different systems were comparable in terms of positioning. Maximum time interval between film-screen mammography and digital mammography was three months. Using a questionnaire, the readers evaluated the number of microcalcifications and their subjective conspicuity for FFDM (Senographe 2000D, GE Medical Systems, Milwaukee, USA) and FSM. A 7-point scale based on the BIRADS classification was used to characterize the calcifications by means of ROC analysis. RESULTS: No statistically significant differences were seen between the two types of mammography among the readers in assessing the number of microcalcifications. The subjective conspicuity of microcalcifications was found to be significantly better for digital mammographies. The diagnosis assigned by the readers did not show significant differences between the two systems. CONCLUSION: Although the subjective conspicuity of microcalcifications was found to be significantly better on digital mammography compared to film-screen mammography, there was no significant advantage of digital mammography resulting from the higher contrast resolution nor a disadvantage in terms of characterization of microcalcifications resulting from the lower spacial resolution. The advantages of digital mammography (e. g. CAD-systems, archiving, dose reduction) can be used without a loss of diagnostic quality.

Breast Diseases↗
PubMed2002

[Digital full field mammography after breast augmentation].

PURPOSE: The use of digital full field mammography for imaging augmented breasts. METHODS: Images of a sponge, as a tissue equivalent, partly covered with one of three different implants (silicone, silicone/saline, saline) were obtained using digital and film-screen technique. The visualisation of the sponge structure covered by the implant and next to the implant was evaluated by two radiologists. Results were compared to clinical results from 35 patients with implants. RESULTS: Two images with different parameter settings have to be obtained to visualize the covered sponge structure and the structure around the implant with film-screen technique. Digital mammography allows assessment of both areas using one image at different window settings. The assessability of covered structures depends on the type of implant. These results are in keeping with our clinical experiences. CONCLUSION: Digital mammography allows equal or better assessment of covered and non-covered tissue in augmented breasts without the need for additional exposures.

Breast Diseases↗
PubMed2002

Circumscribed numerical deficit of dorsal raphe neurons in mood disorders.

BACKGROUND: Neurocircuits comprising limbic, striato-pallidal and thalamo cortical brain areas are assumed to be involved in the pathophysiology of mood disorders. All these brain regions receive serotonergic afferents arising from the rostral raphe, mainly the dorsal raphe. Although serotonergic systems appear to be involved in the pathology of mood disorders, there is uncertainty as to whether structural alterations in raphe nuclei exist alongside a functional dysregulation of the serotonergic system. METHODS: In the brains of 12 patients with mood disorders (major depressive disorder N= 6, bipolar disorder N = 6) and 12 normal subjects we performed a morphometric post-mortem study on neuronal morphology in all subnuclei of the dorsal raphe nucleus using Nissl stained 20 microm axial serial sections of the brainstem. RESULTS: The number of neurones of the ventrolateral subnucleus of the dorsal raphe was reduced by 31 % in patients with mood disorders compared with non-psychiatric control subjects. Ventrally located subnuclei of the rostral dorsal raphe (ventrolateral, ventral, interfascicular) taken together also showed a smaller number of neurones. Neurone numbers of the dorsal and the caudal subnucleus and volumes of all single subnuclei appeared to be unchanged. Analysis of morphological neuronal types revealed a smaller number of triangular neurones in the ventrolateral subnucleus. Numbers of ovoid and round neurones in the ventrolateral subnucleus also showed a trend to reduction. No correlation was found between neurone numbers in any subnucleus of the dorsal raphe and duration of illness. Neurone numbers did not differ in any subnucleus between patients with unipolar and those with bipolar affective disorder. CONCLUSIONS: Results indicate that patients with primary mood disorders have a circumscribed numerical neuronal deficiency in the dorsal raphe. This structural deviation may contribute to impaired serotonergic innervation of brain regions which are involved in the pathology of mood disorders.

Adult↗
PubMed2002

[Comparing the visualization of microcalcifications with direct magnification in digital full-field mammography vs. film-screen mammography].

PURPOSE: To evaluate the conspicuity of microcalcifications in magnified mammographic views of preparations obtained with full field digital mammography (FFDM), film-screen mammography (FSM), and the DIMA technique. MATERIAL AND METHODS: Twelve preparations were examined by FFDM and FSM using 1.8 x magnification and DIMA using 7 x magnification. Parameter settings were identical for all three techniques. The number of visible microcalcifications was then determined for each modality by three radiologists. As far as possible, all preparations were X-rayed at 22 kV and 10 mAS. RESULTS: Altogether 9705 calcifications were counted (DIMA: 1609/1542/1534; FFDM: 1020/753/881; FSM: 901/643/822). The total number of microcalcifications identified with the DIMA technique was 4685 as compared to 2654 with FFDM and 2366 with FSM. The calcifications counted with FFDM and FSM thus corresponded to 56.6 % and 50.5 %, respectively, of those identified with DIMA. The differences between the groups were statistically significant (F-Test, p < 0.05). CONCLUSION: Significantly more calcifications are identified when magnified mammographic views of preparations containing microcalcifications are obtained with the DIMA technique compared to FFDM or FSM. FFDM depicts markedly more calcifications than FSM. This means one should increase spatial resolution. Digital mammography offers the potential for improved visualization of microcalcifications with advanced applications.

Breast Neoplasms↗
PubMed2002

Fewer beta-endorphin expressing arcuate nucleus neurons and reduced beta-endorphinergic innervation of paraventricular neurons in schizophrenics and patients with depression.

In order to elucidate whether the hypothalamic expression of beta-endorphin is altered in patients with mental disorders we studied the cellular localization of the peptide in arcuate nucleus neurons as well as the beta-endorphinergic innervation of paraventricular neurons in nine schizophrenics, six subjects with depression, and nine controls. A polyclonal antiserum against beta-endorphin was employed for the immunohistochemical detection of the peptide in sections of postmortem human brains. Quantitative analysis revealed that the number of beta-endorphin-containing arcuate neurons was statistically reduced in schizophrenics and depressives in comparison to controls. Moreover, the number of endorphinoceptive (i.e. beta-endorphin-innervated) paraventricular nerve cells was also lower in psychiatric patients than in control cases. Our results showing an altered endorphinergic system in human hypothalami of schizophrenics and depressives might contribute to a renewal of interest in this peptide as a possible factor of importance in psychiatric disorders.

Adult↗
PubMed2001

Determination of the binding constants of the centromere protein Cbf1 to all 16 centromere DNAs of Saccharomyces cerevisiae.

Cbf1p is a Saccharomyces cerevisiae chromatin protein belonging to the basic region helix-loop-helix leucine zipper (bHLHzip) family of DNA binding proteins. Cbf1p binds to a conserved element in the 5'-flanking region of methionine biosynthetic genes and to centromere DNA element I (CDEI) of S.cerevisiae centromeric DNA. We have determined the apparent equilibrium dissociation constants of Cbf1p binding to all 16 CDEI DNAs in gel retardation assays. Binding constants of full-length Cbf1p vary between 1.7 and 3.8 nM. However, the dissociation constants of a Cbf1p deletion variant that has been shown to be fully sufficient for Cbf1p function in vivo vary in a range between 3.2 and 12 nM. In addition, native polyacrylamide gel electrophoresis revealed distinct changes in the 3D structure of the Cbf1p/CEN complexes. We also show that the previously reported DNA binding stimulation activity of the centromere protein p64 functions on both the Cbf1 full-length protein and a deletion variant containing only the bHLHzip domain of Cbf1p. Our results suggest that centromeric DNA outside the consensus CDEI sequence and interaction of Cbf1p with adjacent centromere proteins contribute to the complex formation between Cbf1p and CEN DNA.

Basic Helix-Loop-Helix Leucine Zipper Transcriptio↗
PubMed2001

Quantitative distance information on protein-DNA complexes determined in polyacrylamide gels by fluorescence resonance energy transfer.

In polyacrylamide gels, we have quantitatively determined Forster transfer (fluorescense resonance energy transfer, FRET) between two fluorescent dyes attached to DNA in protein-DNA complexes. The donor-dye fluorescein labeled to DNA retains its free mobility in the polyacrylamide gel, however, its fluorescence properties change. The different quantum yield of fluorescein in the gel is found to be independent of the gel concentration and can thus be quantitatively taken into account by a reduced Forster distance R0 of 46 A compared to 50 A in solution. We have determined global structural properties of two proteins binding to double-labeled DNA using a novel gel-based fluorescence resonance energy transfer assay. In polyacrylamide gels we have analyzed the binding of integration host factor (IHF) and the high mobility group protein NHP6a to their substrate DNA. The measured Forster transfer efficiency allows us to deduce information on the overall shape and the DNA bending angle in the complex.

Acrylic Resins↗
PubMed2001

Recent advances in FRET: distance determination in protein-DNA complexes.

Fluorescence resonance energy transfer (FRET) provides information on the distance between a donor and an acceptor dye in the range 10 to 100 A. Knowledge of the exact positions of some dyes with respect to nucleic acids now enables us to translate these data into precise structural information using molecular modeling. Advances in the preparation of dye-labeled nucleic acid molecules and in new techniques, such as the measurement of FRET in polyacrylamide gels or in vivo, will lead to an increasingly important role of FRET in structural and molecular biology.

Bacterial Proteins↗
PubMed2001

Increased number of nitric oxide synthase immunoreactive Purkinje cells and dentate nucleus neurons in schizophrenia.

There is growing interest in the cerebellum as a site of neuropathological changes in schizophrenia. Reports showing that schizophrenics have higher nitric oxide synthase (NOS) activity and MAPKinase levels in the vermis, point to possible aberrations in the cerebellar signal transduction of schizophrenics. It has been speculated that Ca(2+)-dependent extracellular to intracellular signal transduction may be disrupted in the cerebellum of schizophrenics. We decided to test this hypothesis by studying the nitrergic system and markers of the Ca(2+)-triggered signal cascade in the cerebellum of schizophrenics, depressives and controls. The cellular distribution of two calcium sensor proteins (VILIP-1 and VILIP-3) and of neuronal NOS immunoreactivity was studied morphometrically in the flocculonodulus, the inferior vermis and the dentate nucleus of 9 schizophrenics, 7 depressive patients and 9 matched controls. In comparison to controls and depressed patients there were fewer Nissl-stained neurons in the dentate nucleus of schizophrenics. The number of NOS-expressing Purkinje neurons was however strongly increased. In the flocculonodulus and the vermis no differences between the groups were found with regard to the density of Nissl-stained Purkinje cells. The number of NOS-expressing Purkinje neurons was increased in schizophrenics, however. No differences between schizophrenics, depressives and controls were found in the number of VILIP-1 immunoreactive dentate nucleus neurons and VILIP-3 immunoreactive vermal and flocculonodular Purkinje cells. Our data provide further histochemical evidence in favor of structural abnormalities in discrete cerebellar regions of schizophrenics. They confirm and extend earlier reports of increased cerebellar NOS immunoreactivity in schizophrenia and point to possible neurodevelopmental disturbances. Our failure to show an altered expression of two calcium sensor proteins possibly points to a less important role of calcium signaling in cerebellar pathology of the disease.

Adult↗
PubMed2001

Multifunctional centromere binding factor 1 is essential for chromosome segregation in the human pathogenic yeast Candida glabrata.

The CBF1 (centromere binding factor 1) gene of Candida glabrata was cloned by functional complementation of the methionine biosynthesis defect of a Saccharomyces cerevisiae cbf1 deletion mutant. The C. glabrata-coded protein, CgCbf1, contains a basic-helix-loop-helix leucine zipper domain and has features similar to those of other budding yeast Cbf1 proteins. CgCbf1p binds in vitro to the centromere DNA element I (CDEI) sequence GTCACATG with high affinity (0.9 x 10(9) M(-1)). Bandshift experiments revealed a pattern of protein-DNA complexes on CgCEN DNA different from that known for S. cerevisiae. We examined the effect of altering the CDEI binding site on CEN plasmid segregation, using a newly developed colony-sectoring assay. Internal deletion of the CDEI binding site led only to a fivefold increase in rates of plasmid loss, indicating that direct binding of Cbf1p to the centromere DNA is not required for full function. Additional deletion of sequences to the left of CDEI, however, led to a 70-fold increase in plasmid loss rates. Deletion of the CBF1 gene proved to be lethal in C. glabrata. C. glabrata cells containing the CBF1 gene under the influence of a shutdown promoter (tetO-ScHOP) arrested their growth after 5 h of cultivation in the presence of the reactive drug doxycycline. DAPI (4',6'-diamidino-2-phenylindole) staining of the arrested cells revealed a significant increase in the number of large-budded cells with single nuclei, 2C DNA content, and short spindles, indicating a defect in the G(2)/M transition of the cell cycle. Thus, we conclude that Cbf1p is required for chromosome segregation in C. glabrata.

Amino Acid Sequence↗
PubMed2000

Interaction of yeast kinetochore proteins with centromere-protein/transcription factor Cbf1.

The centromere-kinetochore complex of Saccharomyces cerevisiae is a specialized chromosomal substructure that mediates attachment of duplicated chromosomes to the mitotic spindle by a regulated network of protein-DNA and protein-protein interactions. We have used in vitro assays to analyze putative molecular interactions between components of the yeast centromerekinetochore complex. Glutathione S-transferase pull-down experiments showed the direct interaction of in vitro translated p110, p64, and p58 of the essential CBF3 kinetochore protein complex with Cbf1p, a basic region helix-loop-helix zipper protein (bHLHzip) that specifically binds to the CDEI region on the centromere DNA. Furthermore, recombinant p64 and p23 each stimulated the in vitro DNA binding activity of Cbf1p. The N-terminal 70 amino acids of p23 were sufficient to mediate this effect. P64 could also promote the multimerization activity of Cbf1p in the presence of centromere DNA in vitro. These results show the direct physical interaction of Cbf1p and CBF3 subunits and provide evidence that CBF3 components can promote the binding of Cbf1p to its binding site in the yeast kinetochore. A functional comparison of the centromere binding proteins with transcription factors binding at MET16 promoters reveals the strong analogy between centromeres and the MET16 promoter.

Basic Helix-Loop-Helix Leucine Zipper Transcriptio↗
PubMed2000

Design of helical proteins for real-time endoprotease assays.

Proteases play a key role in cellular biology and have become priority targets for new pharmaceuticals. Thus, there is a high demand for specific, sensitive, and quick assays to monitor the activity of endoproteases. We designed affinity-tagged helical proteins with unique protease cleavage sites and thus constructed universal, molecularly defined, and uniform substrates for in vitro detection of IgA endoprotease. The substrate is a 10.5-kDa recombinant helical protein with a high-affinity (His)(6)-tag at the amino-terminal end. Further elements are a unique proteolytic recognition site and a C-terminal helical extension, which is cut off by the protease. Proteolytic action can be monitored in real time using surface plasmon resonance spectroscopy. Femtomole amounts of protease could be reliably and quantitatively detected within a few minutes after the start of the reaction. The detection signal changed linearly with the amount of protease and was independent of the applied sample flow rate. The biochip can be reversibly loaded with the recombinant protease substrate, so that the SPR assay is well-suited for automation. By substituting an HIV protease site for the recognition site of the IgAse, we also obtained a substrate for the quantitative and sensitive detection of HIV-1 endoprotease. Our substrate design is thus generally applicable.

Amino Acid Sequence↗
PubMed2000

Enzymatic microtiter plate-based nitrate detection in environmental and medical analysis.

Our microtiter plate assay is based on the enzymatic reduction of nitrate by dissimilatory nitrate reductase from Pseudomonas stutzeri [EC 1.7.99.4]. Exogenous redox mediators like methyl viologen, methylene blue, and cibachron blue were applied to reduce nitrate reductase. Concentrations of 0.02-0.9 mM nitrate can be detected with +/-6% standard deviation, by using a photometric Griess reaction for the formed nitrite. Nitrate reductase is stable in the pH range 6.5-9.0 and works in the temperature range 4-76 degrees C. The assay shows no interferences with salt content up to 1 M chloride or 11 mM chlorate, and serum albumin content up to 50 mg/ml. The time demand of our two-step procedure is 20 min/100 samples. Nitrate reductase could be conserved on site of the wells of microtiter plates for at least 6 months at room temperature. The nitrate assay was applied in environmental and consumer goods analysis, and for medical diagnostics in human plasma samples.

Autoanalysis↗