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Biomedical subjects

Hartmut Schmidt

Publications and source records attributed to Hartmut Schmidt.

At least 19 recordsLinked to original sources
PubMed2006

Mechanisms of egfr gene transcription modulation: relationship to cancer risk and therapy response.

The epidermal growth factor receptor (EGFR) plays a crucial role in growth, differentiation, and motility of normal as well as cancer cells. For predictive cancer diagnostics and therapeutic targeting of EGFR, it is important to know how the expression level of EGFR is controlled and related to receptor signaling. A novel transcriptional regulation mechanism has been described that depends on the length of a CA repeat in intron 1 [CA simple sequence repeat 1 (CA SSR I)] of the EGFR gene. Thereby, the number of CA repeats is inversely correlated to pre-mRNA synthesis. Indirect evidence for the importance of this mechanism includes the preferential occurrence of amplifications in cancer tissue harboring short CA repeats in this sequence and the discovery of distinct alleles in young breast cancer patients with a family history of the disease and in Japanese breast cancer patients. It can be postulated that the length of the CA repeat influences DNA bendability and, in consequence, the binding of repressor proteins. In summary, it seems that the CA SSR I represents an inherited variable for response to anti-EGFR therapies that could be determined before therapy. Moreover, the potential for synergistic effects with other polymorphism [e.g., EGFR R497K (HER-1 497K) and CCND1 A870G] leading to a simultaneous increase of EGFR signaling activity and expression should be investigated. From a practical perspective, assessment of the CA SSR I number of CA dinucleotide repeats as a predictor for clinical outcome is very attractive because it is a constant feature that does not change over time and can be easily measured in normal and cancer tissues (blood cells, skin, and tumor biopsies) in an assay that is technically simple, objective, and even quantitative.

Breast Neoplasms↗
PubMed2006

Keratin 8 sequence variants in patients with pancreatitis and pancreatic cancer.

Keratin 8 (KRT8) is one of the major intermediate filament proteins expressed in single-layered epithelia of the gastrointestinal tract. Transgenic mice over-expressing human KRT8 display pancreatic mononuclear infiltration, interstitial fibrosis and dysplasia of acinar cells resulting in exocrine pancreatic insufficiency. These experimental data are in accordance with a recent report describing an association between KRT8 variations and chronic pancreatitis. This prompted us to investigate KRT8 polymorphisms in patients with pancreatic disorders. The KRT8 Y54H and G62C polymorphisms were assessed in a cohort of patients with acute and chronic pancreatitis of various aetiologies or pancreatic cancer originating from Austria (n=16), the Czech Republic (n=90), Germany (n=1698), Great Britain (n=36), India (n=60), Italy (n=143), the Netherlands (n=128), Romania (n=3), Spain (n=133), and Switzerland (n=129). We also studied 4,234 control subjects from these countries and 1,492 control subjects originating from Benin, Cameroon, Ethiopia, Ecuador, and Turkey. Polymorphisms were analysed by melting curve analysis with fluorescence resonance energy transfer probes. The frequency of G62C did not differ between patients with acute or chronic pancreatitis, pancreatic adenocarcinoma and control individuals. The frequency of G62C varied in European populations from 0.4 to 3.8%, showing a northwest to southeast decline. The Y54H alteration was not detected in any of the 2,436 patients. Only 3/4,580 (0.07%) European, Turkish and Indian control subjects were heterozygous for Y54H in contrast to 34/951 (3.6%) control subjects of African descent. Our data suggest that the KRT8 alterations, Y54H and G62C, do not predispose patients to the development of pancreatitis or pancreatic cancer.

Acute Disease↗
PubMed2006

Asynchronous growth of prostate cancer is reflected by circulating tumor cells delivered from distinct, even small foci, harboring loss of heterozygosity of the PTEN gene.

The clinical value of prostate-specific antigen (PSA)-positive circulating tumor cells (CTCs) is still a matter of debate and it is also still unclear if these CTCs actually represent the primary tumor. Therefore, we isolated PSA-positive CTCs from the peripheral blood of patients suffering from multifocal cancers and did genetic profiling of each cancer focus by a multiplex PCR-based microsatellite analysis (D7S522, D8S522, NEFL, D10S541, D13S153, D16S400, D16S402, D16S422, and D17S855). In 17 of 20 prostate cancer cases, the loss of heterozygosity (LOH) pattern of the CTCs was identical with only one focus of the primary tumor. Moreover, in six cases, the LOH pattern suggested that smaller foci, down to 0.2 cm3, might deliver CTCs. Interestingly, the highest number of LOHs was observed at the marker D10S541 (85%), the PTEN gene, which was observed much less frequently in unifocal prostate cancer (48%). Furthermore, the infrequently occurring LOH in the BRCA1 gene (38%) was found in four of the five cases where a biochemical recurrence was seen within 3 years after prostatectomy. Therefore, the data might support the assumption that CTCs in prostate cancer are derived from distinct foci of a primary tumor. The size of the tumor focus is not related to the delivery of cells. Although the number of cases that were investigated in this study was small, it might be suggested that the LOH at distinct markers such as D10S541 and D17S855 represent the genes PTEN and BRCA1, which might be associated with the occurrence of CTCs in the peripheral blood of patients as well as an early biochemical recurrence.

Humans↗
PubMed2006

A degradation-sensitive anionic trypsinogen (PRSS2) variant protects against chronic pancreatitis.

Chronic pancreatitis is a common inflammatory disease of the pancreas. Mutations in the genes encoding cationic trypsinogen (PRSS1) and the pancreatic secretory trypsin inhibitor (SPINK1) are associated with chronic pancreatitis. Because increased proteolytic activity owing to mutated PRSS1 enhances the risk for chronic pancreatitis, mutations in the gene encoding anionic trypsinogen (PRSS2) may also predispose to disease. Here we analyzed PRSS2 in individuals with chronic pancreatitis and controls and found, to our surprise, that a variant of codon 191 (G191R) is overrepresented in control subjects: G191R was present in 220/6,459 (3.4%) controls but in only 32/2,466 (1.3%) affected individuals (odds ratio 0.37; P = 1.1 x 10(-8)). Upon activation by enterokinase or trypsin, purified recombinant G191R protein showed a complete loss of trypsin activity owing to the introduction of a new tryptic cleavage site that renders the enzyme hypersensitive to autocatalytic proteolysis. In conclusion, the G191R variant of PRSS2 mitigates intrapancreatic trypsin activity and thereby protects against chronic pancreatitis.

Base Sequence↗
PubMed2006

Quantitative two-photon Ca2+ imaging via fluorescence lifetime analysis.

Two-photon microscopy (TPM) revolutionized Ca2+ imaging by allowing recordings in the depth of intact tissue and live organisms. A serious limitation in TPM, however, is the lack of an accurate and straightforward approach for the quantification of Ca2+ signals, an ability that became an invaluable tool in fluorescence microscopy. Here, we present time-correlated fluorescence lifetime imaging (tcFLIM) as a ratiometric method for the quantification of Ca2+ signals in TPM. The fluorescence lifetime of the Ca2+-indicator dye Oregon Green BAPTA-1 (OGB-1) can be recorded using the approximately 80 MHz excitation pulses utilized in TPM. It shows a Ca2+ dependence that can be explained by the Ca2+-affinity, spectral properties and purity of the dye. Pixel-wise lifetime recordings, controlled by a laser-scanning microscope, allowed quantitative Ca2+ imaging in full-frame and linescan mode. Although we focused on the high-affinity Ca2+ indicator OGB-1, our tcFLIM-based quantification is applicable to other Ca2+ dyes and to fluorescence indicators in general.

Calcium↗
PubMed2006

Chromosomal losses of regions on 5q and lack of high-level amplifications at 8q24 are associated with favorable prognosis for ovarian serous carcinoma.

In this study, we describe characteristic chromosomal alterations in a consecutive series of 96 serous ovarian tumors by comparative genomic hybridization. We analyze their association with different pathways of progression, histological grade, and clinical outcome. The most striking difference between low-grade and high-grade serous carcinomas was seen in a higher incidence of chromosomal gains at 3q and 20q and losses of 13q in the high-grade carcinomas. In addition, high-level amplifications were significantly more frequent in high-grade carcinomas, specifically involving regions on 3q and 8q. Chromosomal amplifications of 19p and 19q and losses of 4q and 5q were among the most frequent changes found in both low-grade and high-grade carcinomas, distinguishing them from borderline tumors, which had very few recurrent alterations. The most significant impact on survival of patients with invasive carcinomas Stage II-IV was observed for high-level amplifications of regions on 8q (mean overall survival (OS) 69 versus 27 months, P = 0.0006). Interestingly, low-level gains on 8q do not show any impact compared to cases with no alteration. Surprisingly, chromosomal losses on 5q had a protective impact (mean OS 36 versus 76 months, P = 0.0007). Combination of both parameters resulted in two risk groups. Low risk: loss on 5q, no amplification on 8q (mean OS 84 months); high risk: no loss on 5q, amplification on 8q (mean OS 26 months). This difference is even more pronounced, if only cases with residual tumor of less than 2 cm are included, resulting in a 5-year survival of 100% and 0% (P = 0.0005).

Chromosome Aberrations↗
PubMed2006

Amplifications of the epidermal growth factor receptor gene (egfr) are common in phyllodes tumors of the breast and are associated with tumor progression.

Phyllodes tumors of the breast are rare biphasic tumors with the potential for invasion and metastatic spread. An important role of the epidermal growth factor receptor (EGFR) in phyllodes tumors has been proposed. However, detailed pathogenetic mechanisms remained unclear. We investigated 58 phyllodes tumors of the breast (40 benign, 10 borderline and eight malignant) by means of egfr fluorescence in situ hybridization (FISH) and gene dosage PCR for a regulatory sequence within intron 1 of egfr. Immunohistochemical staining was performed for EGFR, p16, p21, p27, p53, c-myc, Cyclin A, Cyclin D1, Cyclin E, c-kit and Ki67. Immunopositivity for EGFR was detected in 19% of phyllodes tumors (75% of all malignant tumors) in stromal tumor cells but not in the epithelial component. Whole-gene amplifications were seen by FISH in 15.8% (in stromal cells only) and intron 1 amplifications by gene dosage PCR in as much as 41.8% of all phyllodes tumors. Significant correlations were seen between tumor grade on the one hand and EGFR overexpression (P=0.001) and intron 1 amplifications (P<0.05) on the other. EGFR overexpression further correlated positively with immunohistochemical staining for p53, p16, Cyclin A, Cyclin E, Ki67 and c-kit. Presence of intron 1 amplifications correlated with p16 (P<0.01), p21 (P=0.009) and p53 immunoreactivity (P<0.001). Neither EGFR overexpression nor whole-gene amplification was observed in a control series of 167 fibroadenomas and only one of 43 (2.3%) exhibited intron 1 amplification in gene dosage PCR. In conclusion, our results show for the first time that activating mutations in and overexpression of egfr are associated with the progression in grade of phyllodes tumors of the breast. The observed association between intron 1 amplification and overexpression of EGFR provides further insight into regulation mechanisms of EGFR overexpression.

Base Sequence↗
PubMed2006

Improvements in the analysis strategy make single nucleotide polymorphism analysis a powerful tool in the detection and characterization of amplified chromosomal regions in human tumors.

OBJECTIVE: Single nucleotide polymorphism analysis (SNP) has recently been proposed as an alternative technique to comparative genomic hybridization (CGH) for defining loss of heterozygosity and gene copy number changes in a single experimental setup. In order to assess the potential of SNP analysis to complement or, ultimately, substitute CGH results, we applied both techniques to five primary tumor samples and two tumor cell lines. This was complemented by dilution experiments based on normal lymphocyte DNA to decipher the lower detection limit for genetic alterations. METHODS/RESULTS: Using an in-house software tool, we demonstrated that SNP analysis permits the generation of chromosomal alteration patterns that largely resemble conventional CGH ratio profiles of a given tumor/cell line. Moreover, compared to CGH, our SNP software tool allows a much more detailed definition of amplicon sizes and involved candidate genes. This advantage even persisted when the technique was applied to DNA with more than 60% of nontumor content. However, the detection of chromosomal losses is severely hampered by the presence of nontumor DNA so that the use of the SNP technique should be limited to tumor samples with more than 80% tumor DNA. CONCLUSION: SNP analysis is a very valuable tool for the detection and characterization of high-level chromosomal amplifications in the vast majority of primary tumor samples. Our software tool improves the analysis of SNP data and the presentation of the results, bridging the gap to existing CGH knowledge (http://bioinformatics.uni-muenster.de, 'Publications and Supplements').

Algorithms↗
PubMed2005

Introducing genetic testing for adult-type hypolactasia.

BACKGROUND AND AIMS: To evaluate genotyping for two DNA variants (c.1993+327C>T and c.1438+117G>A), recently found to be associated with adult-type hypolactasia, in the diagnosis of lactose intolerance. METHODS: In total, 166 consecutive patients with gastrointestinal symptoms mimicking hypolactasia admitted to the clinic between March 2002 and December 2002 were included. Genotyping for the two DNA variants (c.1993+327C>T and c.1438+117G>A) and standard H2 breath test was performed. RESULTS: Among 116 patients with positive H2 breath test, the c.1993+327C variant was detectable in 106 (91.4%) patients. Among 50 patients with negative H2 breath test, the c.1993+327C variant was seen in 2 patients. Sensitivity, specificity, positive and negative predictive values for the c.1993+327C variant were 91.4, 96.0, 98.1 and 82.8%, respectively. Genotyping for the c.1438+117G variant did not bring any additional information. Among 4 of the 10 patients with positive H2 breath test but negative for the c.1993+327C and the c.1438+117G variant,further evaluation revealed other diseases known to cause secondary hypolactasia such as celiac disease and short bowel syndrome. CONCLUSION: In symptomatic patients, genotyping for the DNA variant c.1993+327C is a reliable test for adult-type hypolactasia with high sensitivity and specificity and thus provides a new tool in the diagnostic workup of hypolactasia.

Blood Glucose↗
PubMed2005

Calbindin D28k targets myo-inositol monophosphatase in spines and dendrites of cerebellar Purkinje neurons.

The Ca(2+)-binding protein calbindin D28k (CB) is vital for the normal function of the central nervous system but its specific functional role is largely unclear. CB is typically described as a mobile Ca(2+)buffer that shapes the spatiotemporal extent of cellular Ca(2+)signals. Recent biochemical data, however, indicate that CB also has characteristics of a Ca(2+) sensor and activates myo-inositol monophosphatase (IMPase), a key enzyme of the inositol-1,4,5-trisphosphate signaling cascade and an assumed target of mood-stabilizing drugs in the treatment of bipolar disorder. Here, we show that CB interacts with IMPase in cerebellar Purkinje neurons, a cell type well known to rely on inositol-1,4,5-trisphosphate-dependent synaptic integration. Quantification of the mobility of dye-labeled CB with two-photon fluorescence recovery after photobleaching revealed that a substantial fraction of CB is immobilized in spines and dendrites, but not in axons. Immobilization occurs over several seconds, is increased by suprathreshold synaptic activity, and can be relieved by a synthetic peptide that resembles the putative CB-binding site of IMPase, indicating that CB binds to immobilized IMPase. Measurements of the apparent diffusion coefficients of CB imply that CB does not interact with cytosolic IMPase or that the latter is present only in minute amounts in the spiny dendrites of Purkinje neurons. Our results suggest that CB acts as an activity-dependent sensor that targets membrane/cytoskeleton-bound IMPase in central neurons.

Animals↗
PubMed2005

NOD2/CARD15 gene polymorphism in patients with inflammatory bowel disease: is Hungary different?

AIM: To analyse the impact of NOD2/CARD15 mutations on the clinical course of Crohn's disease patients from an eastern European country (Hungary). METHODS: We investigated the prevalence of the three common NOD2/CARD15 mutations (Arg702Trp, Gly908Arg, 1007finsC) in 148 patients with Crohn's disease, 128 patients with ulcerative colitis and 208 controls recruited from the University of Szeged, Hungary. In patients with Crohn's disease, the prevalence of NOD2/CARD15 mutations was correlated to the demographical and clinical parameters. RESULTS: In total, 32.4% of Crohn's disease patients carried at least one mutant allele within NOD2/CARD15 compared to 13.2% of patients with ulcerative colitis (P = 0.0002) and to 11.5% of controls (P<0.0001). In Crohn's disease patients, the allele frequencies for Arg702Trp, Gly908Arg and 1007finsC were 7.1%, 3.0% and 10.8% respectively. Interestingly, only the 1007finsC mutation was associated with a distinct clinical phenotype. The patients positive for the 1007finsC mutation suffered more frequently from stenotic disease behaviour (P = 0.008). Furthermore, 51.9% of patients positive for the 1007finsC mutation underwent a surgical resection within the ileum compared to only 17.4% of patients without the 1007finsC mutation (P = 0.001). With respect to the other two mutations (Arg702Trp and Gly908Arg), no associations were found with all investigated clinical parameters. CONCLUSION: NOD2/CARD15 mutations are frequently found in Crohn's disease patients from Hungary. The 1007finsC mutation is associated with stenotic disease behaviour and frequent ileal resections.

Adolescent↗
PubMed2005

Copper related toxic effects on cellular protein metabolism in human astrocytes.

INTRODUCTION: Copper overload due to a defect in the ATPase 7B mediated copper excretion within hepatocytes produces the phenotype of Wilson disease. The overload of hepatocytes with copper results in necrotic liver cells and is accompanied by a high concentration of blood copper levels. That occurs to be the reason for increasing neurological copper concentration. Although copper is linked to oxidation, there are no data on the direct copper related effects in human brain cells. AIM: To test the copper induced changes in protein oxidation in human astrocyte like cells. METHODS: We used U87 cells as model for human astrocytes. Cells were treated with increasing concentrations of copper(II)-chloride in Dulbeccos minimal essential medium. Subsequently, at different time points we investigated: cellular growth, cellular survival under copper treatment, the concentration of oxidized tryptophane in GADPH in vitro as well as the carbonyl concentration and the concentration of oxidized proteins in vivo in U87 glial cells. RESULTS: The viability of cells decreased with both increasing copper concentration and duration of treatment. The concentration of oxidized proteins was directly correlated to the increase of copper concentration and duration of exposure. CONCLUSION: These observations demonstrate the similarities between copper treatment and treatment with other commonly used oxidants, including hydrogen peroxide. Furthermore, the vulnerability of astrocytes towards copper exposure could be demonstrated. Therefore, these data give further insights into understanding of copper metabolism, which in turn is important to reveal the exact pathological mechanism in copper related diseases such as Wilson disease.

Astrocytes↗
PubMed2005

Cytogenetic changes and loss of heterozygosity in atypical adenomatous hyperplasia, in carcinoma of the prostate and in non-neoplastic prostate tissue using comparative genomic hybridization and multiplex-PCR.

High grade prostatic intraepithelial neoplasia (PIN) and atypical adenomatous hyperplasia (AAH) are discussed to be precursors of prostate cancer (PC). Unlike high grade PIN the relation between AAH and PC is however unclear. In the present study we analyzed AAH, accompanying prostate carcinomas and carcinomas of the transitional zone after microdissection using comparative genomic hybridization (CGH) and multipelx-PCR with 10 microsatellite polymorphic markers. In every case non-neoplastic prostatic tissue was investigated for the same allelic imbalances. Two AAH showed allelic imbalances in multiplex-PCR. These imbalances did not correlate with the corresponding tumours and furthermore were different to the LOH found in the investigated prostate tumours of the transitional zone. One AAH showed loss on chromosome 22q. We found allelic imbalances in over 50% of non-neoplastic tissue adjacent to prostatic carcinoma. Our findings support the idea that AAH does not seem to be linked closely to PC and should not be considered as an obligate premalignant lesion.

Aged↗
PubMed2005

Overexpression of Endothelin-A-receptor in breast cancer: regulation by estradiol and cobalt-chloride induced hypoxia.

Previous studies have demonstrated the potential significance of Endothelin (ET)-1 and its receptors, ETAR and ETBR, in the development and progression of breast cancer. The objective of this study was to assess the expression levels and potential regulation of the "ET axis" in human non-neoplastic and neoplastic breast tissue as well as in various human breast cancer cell lines. Expression of ET-1, ETAR and ETBR was evaluated in 31 neoplastic and 7 non-neoplastic breast tissue samples and in six human breast cancer cell lines using conventional and quantitative real-time RT-PCR, Western blotting and immunohistochemistry. The effects of 17beta-estradiol (E2) and cobalt-chloride (CoCl2) treatment on ET-1, ETAR and ETBR expression were studied in vitro. ETAR mRNA expression levels were found to be statistically significantly higher in breast cancer specimens than in non-neoplastic breast tissue (p<0.001). For ET-1 and ETBR mRNA expression, no significant difference was observed between the two groups. All cell lines exhibited expression of ET-1 and ETAR mRNA, whereas none showed significant ETBR mRNA expression. We observed a strong and reproducible induction of ETAR mRNA and protein expression by E2 and CoCl2 in MDA-MB-468 and BT-474 cells and in MDA-MB-453 and SK-BR-3 cells with a maximum increase after 8 and 16 h of treatment, respectively, while MCF-7 and HBL-100 cells showed a constitutive expression pattern. The present data suggest a novel mechanism in the regulation of ETAR expression in breast cancer. Based on these findings, a combination of ETAR-antagonists with adjuvant endocrine treatment seems to be a reasonable therapeutic strategy.

Adult↗
PubMed2004

A new culturing strategy optimises Drosophila primary cell cultures for structural and functional analyses.

Neurons in primary cell cultures provide important experimental possibilities complementing or substituting those in the nervous system. However, Drosophila primary cell cultures have unfortunate limitations: they lack either a range of naturally occurring cell types, or of mature physiological properties. Here, we demonstrate a strategy which supports both aspects integrated in one culture: Initial culturing in conventional serum-supplemented Schneider's medium (SM(20K)) guarantees acquisition of all properties known from 30 years of work on cell type-specific differentiation in this medium. Through subsequent shift to newly developed active Schneider's medium (SM(active)), neurons adopt additional mature properties like the ability to carry out plastic morphological changes, neurotransmitter expression and electrical activity. We introduce long-term FM-dye measurements as a tool for Drosophila primary cell cultures demonstrating the presence of increased, action potential-dependent synaptic activity in SM(active). This is confirmed by patch-clamp recordings, which in addition show that SM(active)-cultured neurons display different spiking patterns. Furthermore, we demonstrate that transmission can be evoked in SM(active) cultures, revealing the existence of synaptic plasticity. Thus, these culture conditions support developmental, structural and physiological properties known or expected from the nervous system, enhancing possibilities for future experiments complementing or substituting those in nervous systems of Drosophila.

Action Potentials↗