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Biomedical subjects

Gerhard Schwarz

Publications and source records attributed to Gerhard Schwarz.

14 recordsLinked to original sources
PubMed2007

Validation of a real-time PCR for the quantitative estimation of a G143A mutation in the cytochrome bc1 gene of Pyrenophora teres.

A single nucleotide polymorphism (SNP) in the cytochrome b gene confers resistance to strobilurin fungicides for several fungal pathogens. Therefore, on the basis of a change at amino acid position 143 from glycine to alanine, a real-time PCR assay was established for the quantitative detection of the analogous SNP in the cytochrome b sequence of Pyrenophora teres Drechsler, which causes barley net blotch. Allelic discrimination was achieved by using allele specific primers with artificially mismatched nucleic acid bases and minor groove binding probes. Validation parameters for the lower limits of the working range, namely limits of detection (LOD) and limits of quantification (LOQ), were statistically determined by the variance of calibration data, as well as by the variance of the 100% non-strobilurin-resistant allele DNA sample (blank values). It was found that the detection was limited by the variance of blank values (five in 801 458 copies; 0.0006%), whereas the quantification was limited by the variance of calibration data (37 in 801 458 copies; 0.0046%). The real-time PCR assay was finally used to monitor strobilurin-resistant cytochrome b alleles in barley net blotch field samples, which were already classified in in vivo biotests to be fully sensitive to strobilurins. All signals for strobilurin-resistant cytochrome b alleles were below the LOD, and therefore the results are in total agreement with the phenotypes revealed by biotests.

Alleles↗
PubMed2006

Comparative expression profiling in meristems of inbred-hybrid triplets of maize based on morphological investigations of heterosis for plant height.

Heterosis, the superior performance of hybrids as compared to their parental mean is an agronomically important phenomenon well-described morphologically. However, little is known about its molecular basis. We investigated four genetically unrelated maize (Zea mays L.) inbred lines and their F(1) crosses both at the phenotype and transcriptome level, focusing on plant height (PHT) component traits. Substantial mid-parent heterosis (MPH) was found for all parent-hybrid triplets for PHT in the range of 37.9-56.4% in the field and 11.1-39.5% under controlled greenhouse conditions. Analyses of heterosis for number and length of internodes showed two to three times higher MPH in the field as compared to the greenhouse. All three traits exhibited high heritabilities, highest for PHT 95-98%. Two methods for gene expression quantification were applied. High-density cDNA uni-gene microarrays containing 11,827 ESTs were utilized for the selection of differentially expressed genes related to heterosis for PHT. For the four triplets with eight possible parent-hybrid comparisons we identified 434 consistently differentially expressed genes with a p < or = 0.05. Microarray results were used to verify the dominance/overdominance hypothesis. In our study, more than 50% genes showed overdominance, 26% partial dominance, 12.6% complete dominance and 10.2% additive gene action. Moreover, more consistently differentially expressed genes were detected in related triplets, sharing one parent, than in unrelated triplets. Quantitative RT-PCR was applied in order to validate microarray results. The role of the differentially expressed genes in relation to heterosis for PHT is discussed.

Gene Expression Profiling↗
PubMed2005

[Increases of intracranial pressure and changes of blood flow velocity due to acupressure, needle and laser needle acupuncture?].

BACKGROUND: Pressure on the acupoints St.7 and SJ.22 can lead to significant, reversible increases in intracranial pressure (ICP) in patients with elevated ICP. OBJECTIVE: In this study, we investigated whether changes in cerebral parameters in healthy volunteers can also be registered, when the mentioned acupoints associated with ICP, are stimulated. SUBJECTS AND METHODS: We investigated a total of 34 volunteers (24 females, 10 males) and a 15-year-old intensive care patient after severe head injury. The mean age of volunteers was 25.2 +/- 3.4 years (range 20-35). Stimulation was performed using acupressure techniques, manual needle and laser needle methods. We evaluated the main parameter of mean blood flow velocity in the middle cerebral artery (left and right) as well as the pulsatility index. In addition, near infrared spectroscopy and blood pressure parameters were registered. RESULTS: Acupressure, manual needle acupuncture and laser needle acupuncture partially led to significant changes in the main goal values. CONCLUSION: Acupressure as well as acupuncture (needle and laser needle) can evoke reproducible functional changes in the brain. These accompanying effects are not to be ignored, in particular in patients with increased ICP.

Acupressure↗
PubMed2005

An NIRS matrix for detecting and correcting cerebral oxygen desaturation events during surgery and neuroendovascular procedures.

BACKGROUND: Transcranial cerebral oximetry was developed for early detection of cerebral hypoxia and to avoid cerebral dysfunctions. However, near infrared spectroscopy (NIRS) data obtained during surgery are subject to intrinsic and extrinsic influences that have to be accounted for when interpreting the recordings. METHODS: We developed an NIRS matrix to provide brief information for specific intervention to correct changes of cerebral oxygen saturation (COS). Selected vital data and the descriptors of cerebrovascular and neurofunctional status were linked to logistic chains. RESULTS: The matrix is horizontally and vertically grouped and contains five descriptors: 1. change of COS; 2. key variable (parameter related to the change of COS); 3. associated parameters (vital data that do not cause COS alterations); 4. interpretation of values or preconditions most probably due to COS changes; and 5. the intervention most likely to normalize the COS or return it to baseline. The descriptors are grouped horizontally to a logistics chain. CONCLUSION: The modular expandable NIRS matrix we describe has promise for clinical use in surgical, neurointerventional, and anaesthesiological contexts.

Adult↗
PubMed2004

Determination of detection and quantification limits for SNP allele frequency estimation in DNA pools using real time PCR.

The quantification of single nucleotide polymorphism (SNP) allele frequencies in pooled DNA samples using real time PCR is a promising approach for large-scale diagnostics and genotyping. The limits of detection (LOD) and limits of quantification (LOQ) for mutant SNP alleles are of particular importance for determination of the working range, which, in the case of allele-specific real time PCR, can be limited by the variance of calibration data from serially diluted mutant allele samples as well as by the variance of the 100% wild-type allele samples (blank values). In this study, 3sigma and 10sigma criteria were applied for the calculation of LOD and LOQ values. Alternatively, LOQ was derived from a 20% threshold for the relative standard deviation (%RSD) of measurements by fitting a curve for the relationship between %RSD and copy numbers of the mutant alleles. We found that detection and quantification of mutant alleles were exclusively limited by the variance of calibration data since the estimated LOD(calibration) (696 in 30 000 000 copies, 0.0023%), LOQ(20%RSD) (1470, 0.0049%) and LOQ(calibration) (2319, 0.0077) values were significantly higher than the LOD(blank) (130, 0.0004%) and LOQ(blank) (265, 0.0009%) values derived from measurements of wild-type allele samples. No significant matrix effects of the genomic background DNA on the estimation of LOD and LOQ were found. Furthermore, the impact of large genome sizes and the general application of the procedure for the estimation of LOD and LOQ in quantitative real time PCR diagnostics are discussed.

Calibration↗
PubMed2004

Pseudoparadoxical dissociation of cerebral oxygen saturation and cerebral blood flow velocity after acupuncture in a woman with cerebrovascular dementia: a case report.

Acupuncture can increase both cerebral oxygen saturation and cerebral blood flow velocity. We describe a 77-year-old woman with cerebrovascular dementia in whom acupuncture reproducibly induced an increase of blood flow velocity but a decrease of regional oxygen saturation. At four of 11 acupuncture sessions, blood flow velocity was measured in the middle cerebral artery with transcranial Doppler sonography and cerebral regional oxygen saturation (rSO(2)) with transcranial near infrared spectroscopy. Cerebral blood flow velocity increased by an average of 20% (range: 7-27%) at all four study points whereas rSO(2) consistently decreased by an average of 7% (range: 4-13%). Clinical status and cognitive function improved. These findings in a patient with vascular dementia may suggest increased oxygen extraction by activated neuronal structures.

Acupuncture↗
PubMed2003

Standardized determination of real-time PCR efficiency from a single reaction set-up.

We propose a computing method for the estimation of real-time PCR amplification efficiency. It is based on a statistic delimitation of the beginning of exponentially behaving observations in real-time PCR kinetics. PCR ground fluorescence phase, non-exponential and plateau phase were excluded from the calculation process by separate mathematical algorithms. We validated the method on experimental data on multiple targets obtained on the LightCycler platform. The developed method yields results of higher accuracy than the currently used method of serial dilutions for amplification efficiency estimation. The single reaction set-up estimation is sensitive to differences in starting concentrations of the target sequence in samples. Furthermore, it resists the subjective influence of researchers, and the estimation can therefore be fully instrumentalized.

Algorithms↗
PubMed2003

DHPLC scoring of a SNP between promoter sequences of HMW glutenin x-type alleles at the Glu-D1 locus in wheat.

The promoter regions of HMW glutenin x-type genes at the Glu-D1 locus were surveyed for SNPs within a subpopulation of German bread wheat cultivars. On the basis of the promoter sequences of HMW glutenin subunit genes Glu-A1-x1, Glu-A1-x2, Glu-B1-x1, Glu-B1-x7, Glu-D1-x2, and Glu-D1-x5, an amplification refractory mutation system assay was designed to selectively amplify Dx-specific PCR fragments. Comparative sequence analysis among seven Glu-D1-x2 and seven Glu-D1-x5 wheat cultivars only confirmed a G-A transition in the promoter sequence to be a true polymorphism. SNP scoring by DHPLC of 95 German bread wheat cultivars, with the exception of cv. Anemos, showed that the transition completely agreed with the presence of HMW glutenin subunits 1Dx5 + 1Dy10 in SDS-PAGE. Therefore, the developed DHPLC assay is suitable for high-throughput genotyping to assist the selection of HMW glutenin genes in wheat quality breeding programs.

Alleles↗
PubMed2003

Evaluation of Erysiphe graminis f sp tritici field isolates for resistance to strobilurin fungicides with different SNP detection systems.

A single nucleotide polymorphism (snp) in the cytochrome b gene confers resistance to strobilurin fungicides in Erysiphe graminis DC fsp tritici Marchal. On the basis of this point mutation three different types of molecular markers have been developed. Cleaved amplified polymorphic sequences and allele-specific PCR were used to score resistant and sensitive isolates from specifically selected regional populations across Europe. The results of molecular tests were in total agreement with the resistance phenotypes revealed by in vivo tests. Serial dilutions of mixed samples (resistant/sensitive) delimited the detection for strobilurin-resistant alleles to a range of 10-50% for both marker classes. Due to these detection limits no mixture of mitochondria within individual isolates was found. Denaturing high performance chromatography was used to increase the detection sensitivity for the mutant allele. Although the detection limit was lowered to 5-10%, there was no evidence for the existence of mixed mitochondrial genotypes.

Drug Resistance, Fungal↗
PubMed2003

On translocation through a membrane channel via an internal binding site: kinetics and voltage dependence.

Here we present a model for maltodextrin translocation through maltoporin channels. In a first step, our theoretical analysis does consider the case of a single binding site for a given substrate in a structurally unaffected channel with a possibly different entrance barrier on either side. It is shown how by means of conventional electrical conductance measurements (including current noise analysis) the basic equilibrium and rate constants can be determined as functions of the applied voltage. Then also the net translocation rate of the substrate becomes accessible quantitatively. This most simple model mechanism has been extended to include a voltage-dependent fast conformational change of the channel that prevents the binding process. The so developed approach has been tested with experimental data for a single maltoporin trimer being reconstituted in black lipid membranes when studied in the presence of maltohexaose as the substrate. The experimental results turned out to be clearly incompatible with binding alone. They are, however, very satisfactorily fitted by pertinent theoretical curves if also inhibition of binding by a conformational transition is taken into account. Accordingly, quantitative evaluations of the underlying parameters and eventually of the translocation rate have been carried out successfully. Our analysis reveals a set of parameters necessary for an optimal translocation that nicely corresponds to natural conditions.

Bacterial Outer Membrane Proteins↗
PubMed2003

Proinflammatory role of fractalkine (CX3CL1) in rheumatoid arthritis.

OBJECTIVE: Fractalkine (CX3CL1) represents the sole member of the so-called CX3C chemokines. In rheumatoid arthritis (RA), functional studies suggest a role for this chemokine in monocyte chemotaxis and angiogenesis in the rheumatoid synovium. We analyzed the expression of fractalkine within different T cell subsets of the peripheral blood and expression of its receptor CX3CR1 within the rheumatoid synovium to further characterize its pathogenic role in RA. METHODS: Peripheral blood mononuclear cells (PBMC) were isolated from 17 patients with RA and analyzed by flow cytometry in comparison to healthy blood donors. To identify the T helper cell cytokine profile of fractalkine-expressing cells, flow cytometric analysis of PBMC was performed after stimulation with PMA and ionomycin. Expression of fractalkine and its receptor was characterized in RA synovium by immunohistochemistry and laser capture microdissection microscopy. RESULTS: Flow cytometric analysis of fractalkine-expressing T cell subsets revealed a low proportion of fractalkine-expressing CD4+ and CD8+ T cells in both RA patients and controls. In addition, fractalkine was predominantly expressed in CD4+ T cells with a Th1-type cytokine expression profile. In RA synovium, fractalkine was detected in synovial macrophages, dendritic cells, endothelial cells, and a small proportion of T cells. The fractalkine receptor CX3CR1 was found in synovial macrophages, dendritic cells, and T cells as well as in synovial fibroblasts. Fractalkine stimulation of cultured synovial fibroblasts resulted in a marked upregulation of matrix metalloproteinase-2 (MMP-2) production. CONCLUSION: The results suggest that fractalkine may represent a Th1-type chemokine. Upregulation of MMP-2 production in synovial fibroblasts upon fractalkine stimulation in vitro supports the hypothesis of a proinflammatory role of this chemokine in RA.

Adult↗
PubMed2002

Side-effects after inhalational anaesthesia for paediatric cerebral magnetic resonance imaging.

BACKGROUND: The aim of this study was to evaluate the type, incidence and duration of postprocedure side-effects in 168 children within the first 72 h after inhalational anaesthesia for magnetic resonance imaging (MRI). METHODS: Premedication and induction followed standardized routines. Maintenance of anaesthesia was performed with inhalational anaesthetics solely: isoflurane (n=60 of 112; 53%), sevoflurane (n=32 of 112; 29%), desflurane (n=12 of 112; 11%) or halothane (n= 8 of 112; 7%) using a strapped on face mask (FiO2=0.4; flow 5 l.min-1). When indicated, gadolinium was administered (n=45; OF 112; 40%). RESULTS: One hundred and twelve of 168 parents (67%) responded to questionnaires. In these 112 children, pathological MR findings were found supratentorially (n=31; 28%), infratentorially (n=9; 8%), extracerebrally (n=12; 11%) or combined (n=9; 8%). In 56 of these 112 children (50%), 14 different side-effects were reported. One hour after anaesthesia, 55 children suffered between one and four side-effects. Neurological side-effects were associated with age > or = 5 years (P < 0.01) or infratentorial pathophysiology (P < 0.01) and abdominal side-effects (P < 0.02), especially nausea (P < 0.001) with age > or = 5 years. CONCLUSIONS: Our findings indicate the need to inform parents of the incidence and variability of side-effects after inhalational anaesthesia for minimally invasive, diagnostic procedures, such as MRI.

Adolescent↗