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Biomedical subjects

Oliver Müller

Publications and source records attributed to Oliver Müller.

At least 19 recordsLinked to original sources
PubMed2007

Latrunculin analogues with improved biological profiles by "diverted total synthesis": preparation, evaluation, and computational analysis.

Deliberate digression from the blueprint of the total syntheses of latrunculin A (1) and latrunculin B (2) reported in the accompanying paper allowed for the preparation of a focused library of "latrunculin-like" compounds, in which all characteristic structural elements of these macrolides were subject to pertinent molecular editing. Although all previously reported derivatives of 1 and 2 were essentially devoid of any actin-binding capacity, the synthetic compounds presented herein remain fully functional. One of the designer molecules with a relaxed macrocyclic backbone, that is compound 44, even surpasses latrunculin B in its effect on actin while being much easier to prepare. This favorable result highlights the power of "diverted total synthesis" as compared to the much more widely practiced chemical modification of a given lead compound by conventional functional group interconversion. A computational study was carried out to rationalize the observed effects. The analysis of the structure of the binding site occupied by the individual ligands on the G-actin host shows that latrunculin A and 44 both have similar hydrogen-bond network strengths and present similar ligand distortion. In contrast, the H-bond network is weaker for latrunculin B and the distortion of the ligand from its optimum geometry is larger. From this, one may expect that the binding ability follows the order 1 >/= 44 > 2, which is in accord with the experimental data. Furthermore, the biological results provide detailed insights into structure/activity relationships characteristic for the latrunculin family. Thus, it is demonstrated that the highly conserved thiazolidinone ring of the natural products can be replaced by an oxazolidinone moiety, and that inversion of the configuration at C16 (latrunculin B numbering) is also well accommodated. From a purely chemical perspective, this study attests to the maturity of ring-closing alkyne metathesis (RCAM) catalyzed by a molybdenum alkylidyne complex generated in situ, which constitutes a valuable tool for advanced organic synthesis and natural product chemistry.

Actins↗
PubMed2007

Total synthesis and evaluation of the actin-binding properties of microcarpalide and a focused library of analogues.

A comparative investigation shows that hydroxylated 10-membered lactones modeled around the fungal metabolites microcarpalide (1) and pinolidoxin (2) are endowed with selective actin-binding properties. Although less potent than the marine natural product latrunculin A, which represents the standard in the field, nonenolides of this type are significantly less toxic and accommodate substantial structural editing. Most notable is the fact that even an intramolecular transesterification with formation of a hydroxylated butanolide skeleton does not annihilate their microfilament disrupting capacity. This finding calls for a reinvestigation of the biological profile of other fungal metabolites that embody a similar motif. Microcarpalide (1) serving as the calibration point for this comparative study was prepared by total synthesis based on ring-closing metathesis (RCM) as the key step. The chosen route favorably compares to previous approaches to this target and provides further support for the notion that the (E,Z)-configuration of a medium-sized cycloalkene can be controlled by proper choice of the catalyst as previously outlined by our group. 9-epi-Microcarpalide 26 and furanone 27 as representative examples of the "natural productlike" compounds investigated herein have been characterized by crystal structure analysis.

4-Butyrolactone↗
PubMed2006

Hypermethylation of the APC promoter but lack of APC mutations in myxoid/round-cell liposarcoma.

The adenomatous polyposis coli (APC) protein is a key component of the WNT signalling pathway wherein it acts as a scaffolding protein in controlling the level of the proto-oncoprotein beta-catenin. Although APC has been shown to be genetically or epigenetically inactivated in a variety of carcinomas, little is known about its role in sarcoma. Liposarcomas (LPSs) are the second most common soft tissue sarcoma in adults. Despite different histology and malignancy, the myxoid and round-cell LPSs belong to one tumour entity characterized by a specific chromosomal translocation. We assessed the extent of genetic and epigenetic inactivation of the APC gene in myxoid/round-cell LPS. Sequencing of the mutation cluster region, the protein truncation test and a loss of heterozygosity (LOH) analysis did not reveal any genetic alterations of the APC gene in all of the liposarcoma samples. Methylation of the APC promoter was detected by methylation-specific PCR in 9 of 20 (45%) tumours. Analysis of APC expression by semiquantitative RT-PCR in a subset of the samples demonstrated that tumours with a methylated APC promoter showed a downregulation of the APC transcript. However, APC downregulation was not correlated with a stabilisation of the beta-catenin protein. Thus, the epigenetic regulation of the APC gene might play an important role in the pathogenesis of myxoid/round-cell LPS. However, the impact of APC methylation on liposarcoma development is quite likely not mediated through WNT signalling.

Adult↗
PubMed2006

Access to lexical information in language comprehension: semantics before syntax.

The recognition of a word makes available its semantic and syntactic properties. Using electrophysiological recordings, we investigated whether one set of these properties is available earlier than the other set. Dutch participants saw nouns on a computer screen and performed push-button responses: In one task, grammatical gender determined response hand (left/right) and semantic category determined response execution (go/no-go). In the other task, response hand depended on semantic category, whereas response execution depended on gender. During the latter task, response preparation occurred on no-go trials, as measured by the lateralized readiness potential: Semantic information was used for response preparation before gender information inhibited this process. Furthermore, an inhibition-related N2 effect occurred earlier for inhibition by semantics than for inhibition by gender. In summary, electrophysiological measures of both response preparation and inhibition indicated that the semantic word property was available earlier than the syntactic word property when participants read single words.

Adolescent↗
PubMed2005

Solid-phase synthesis of lipidated peptides.

A new flexible and efficient methodology for the solid-phase synthesis of lipidated peptides has been developed. The approach is based on the use of previously synthesized building blocks and overcomes the limitations of previously reported methods, since long doubly lipidated peptides can be synthesized by using this route. Furthermore, it was thus possible to prepare a large number of N- and H-Ras peptides bearing a wide range of reporter and/or linking groups--efficient tools for the investigation of biological processes. In terms of efficiency and flexibility this solid-phase method is superior to the solution-phase synthesis. It gives pure peptides in multimilligram amounts within a much shorter time and with superior overall yield.

Amino Acid Sequence↗
PubMed2005

Diverted total synthesis: preparation of a focused library of latrunculin analogues and evaluation of their actin-binding properties.

Two largely catalysis-based and highly convergent total syntheses of latrunculin A (1) and B (2) were diverted to the preparation of a focused library of analogues of these potent actin-binding macrolides that enjoy widespread use in chemical biology. Because the chosen route allows for structural variations of all characteristic parts of the natural leads, it was possible to map the previously largely unknown structure/activity profile of this class of bioactive natural products. This led to the discovery that the removal of the methyl branches decorating the macrocycle in 2 engenders a significant increase in potency, while streamlining the synthesis to a considerable extent. Moreover, compelling evidence is provided that the conspicuous 2-thiazolidinone ring present in all naturally occurring latrunculins may be an optimal but not an essential structural motif for actin binding because it can be replaced by an oxazolidinone moiety with only slight loss in efficacy. Likewise, the inversion of the absolute configuration of the chiral center at C.16 is well accommodated. From the purely chemical perspective, this investigation attests to the maturity of alkyne metathesis, a method that has received attention as efficient means for the formation of macrocycles only recently.

Actin Cytoskeleton↗
PubMed2005

Feasibility of chemosensitivity testing in soft tissue sarcomas.

BACKGROUND: Soft tissue sarcomas comprise less than 1% of all solid malignancies. The presentation and behavior of these tumors differs depending on location and histological characteristics. Standard therapy consists of complete surgical resection in combination with adjuvant radiotherapy. The role of chemotherapy is not clearly defined and is largely restricted to clinical trials. Only a limited number of agents have proved to be effective in soft tissue sarcomas. The use of doxorubicin, epirubicin and ifosfamide allowed response rates of more than 20%. In addition, recent chemotherapy trials did not demonstrate any significant differences in efficacy for various histological subtypes. METHODS: The objective of this study was to gain additional information about the chemosensitivity of soft tissue sarcomas to seven 7 different chemotherapy agents as single drugs and 4 combinations. Therefore we used an established ATP based in-vitro testing system and examined 50 soft tissue sarcomas. Chemosensitivity was assessed using a luciferin-luciferase-based luminescence assay providing individual chemosensitivity indices for each agent tested. RESULTS: The sensitivity varied widely according to the histological subtypes. The tumors state of cellular dedifferentiation played a crucial role for the efficiency of the chemotherapeutic agents. The sensitivity also depended on the presentation of the sarcoma as a primary or recurrent tumor. The highest sensitivity was demonstrated for actinomycin D as a single agent, with 74% of the tumor samples exhibiting a high-grade sensitivity (20% low sensitivity, no resistance). The combination of actinomycin D and ifosfamide yielded a high sensitivity in 76% (2% resistance). Doxorubicin as a mono-therapy or in combination with ifosfamide achieved high sensitivity in 70% and 72%, respectively, and resistance in 6% of the samples. CONCLUSION: Chemosensitivity testing is feasible in soft tissue sarcomas. It can be used to create sensitivity and resistance profiles of established and new cytotoxic agents and their combinations in soft tissue sarcomas. Our data demonstrate measurable discrepancies of the drug efficiency in soft tissue sarcomas, sarcoma subtypes and tumor recurrencies. However, current therapeutic regime does not take this in consideration, yet.

Journal Article↗
PubMed2005

Modulation of MRP-1-mediated multidrug resistance by indomethacin analogues.

Multidrug resistance (MDR) is a major limiting factor in the development and application of drug candidates. MDR caused by MRP-1 is known to be modulated by the nonsteroidal antiinflammatory drug indomethacin. We have synthesized and biologically evaluated a library of indomethacin analogues. The indomethacin-derived compound library was synthesized employing the Fischer-indole synthesis as the key transformation and making use of a "resin-capture-release" strategy. Sixty representative members of the library were evaluated in a cell biological cytotoxicity assay employing the MRP-1 expressing human glioblastoma cell line T98G as a model system. Nine of the 60 tested derivatives increased the doxorubicin-mediated cytotoxicity at a comparable or higher level than indomethacin itself. Analysis of these derivatives revealed an interesting structure-function relationship. Most remarkably, two substances increased the toxicity, when doxorubicin was used at clinically relevant low concentrations, at a higher degree than indomethacin.

Anti-Inflammatory Agents, Non-Steroidal↗
PubMed2005

Non-invasive detection of colorectal tumours by the combined application of molecular diagnosis and the faecal occult blood test.

The treatment of early-stage tumours decreases the overall mortality of colorectal tumour patients. In this retrospective study we determined the sensitivity and the specificity of the faecal occult blood test (FOBT) and the molecular diagnosis (MD). We analysed 57 stool samples from patients with colorectal carcinomas for the presence of occult blood using a standard FOBT and for alterations in the three different tumour relevant markers APC, BAT26 and L-DNA. Stool samples from 44 control donors were analysed to determine the specificity of the applied methods. Twenty-nine (51%; 95% confidence interval (CI): 38-63%) stool samples of the cancer patients gave positive FOBT results. Thirty-seven (65%; CI: 52-76%) samples showed alterations in at least one DNA marker. Sixteen (28%) samples were positive only in the FOBT, and 24 (42%) samples showed a positive result exclusively in MD. The combined application of both methods resulted in a sensitivity of 93% (CI: 83-97%) and an overall specificity of 89% (CI: 76-95%). The combined application of FOBT and MD resulted in an overall sensitivity, which could not be achieved by any of the methods alone and which is in the range of invasive diagnostic methods.

Aged↗
PubMed2005

Novel target genes of the Wnt pathway and statistical insights into Wnt target promoter regulation.

The Wnt pathway controls biological processes via the regulation of target gene expression. The expression of direct Wnt target genes, e.g. cyclin D1 and MYC, is activated by the transcription factor TCF, which binds to specific sequence motifs in the promoter. Indirect target genes are regulated via transcription regulators, which are targets of the Wnt pathway. As an example, MYC regulates the MYC interacting zinc finger protein-1 (MIZ-1), which is able to inhibit the expression of the indirect target p21WAF1. We intended to identify new Wnt target genes and to get a deeper insight into the regulatory mechanisms of Wnt target gene expression. For this we analyzed the differential expression pattern of Wnt-1 activated cells by microarray analysis. We identified 43 sequences including eight expressed sequence tags (ESTs), which showed increased transcript levels, and 104 sequences including 19 ESTs with decreased RNA levels. Northern blot and real-time quantitative PCR analysis of the differential expression levels of 15 genes confirmed the differential expression trends of eight candidate genes. When the Wnt pathway was regulated at the lower level of glycogen synthase kinase-3 beta (GSK-3 beta) or adenomatous polyposis coli (APC), we detected discrepant expression trends. We compared the number of binding sites of transcription factors in the genomic regions of all candidate target genes with the number of sites in control genes. We found that the genomic regions of the down-regulated genes include an increased number of putative MIZ-1 binding sites. Our study introduces several new Wnt target genes and provides indications that the specific gene expression pattern depends on the type of the activation trigger or the level of interference with the Wnt pathway. Furthermore, our data indicate that a high proportion of Wnt target genes are regulated by indirect mechanisms.

Animals↗
PubMed2004

Molecular analysis of APC promoter methylation and protein expression in colorectal cancer metastasis.

APC (adenomatous polyposis coli) promoter methylation has been linked to the early development of colorectal cancers. However, the role of APC methylation and its effect on protein expression in colon cancer metastasis is largely unknown. In this study, we investigated APC promoter methylation by Methylight analysis and analysed the APC protein levels by immunohistochemistry and western blot analysis in 24 liver metastasis and 39 primary colorectal cancers. Promoter methylation of the APC gene was found to be a frequent event in liver metastasis (10/24) and significantly more frequent compared with primary colorectal cancer (7/39, P = 0.047). APC methylation was not found in 14 matched normal colon tissues. APC protein was detected in the cytoplasm of primary and metastatic cancer cells and non-tumorous colon epithelium. By western blot analysis, APC protein levels were found to be decreased in primary tumour tissues compared with the normal colon mucosa. In contrast, APC protein levels were not decreased in the cancer cells that had metastasized to the liver. APC protein levels were independent of the presence of APC promoter methylation or gene mutations. In summary, APC promoter methylation is a frequent epigenetic alteration in colorectal cancer metastasis. However, we observed no significant association between APC promoter methylation or gene mutation and APC protein expression in colorectal metastasis. Therefore, metastatic cancer cells seem to harbour a heterogenous genetic and epigenetic background, in which cancer cells may exhibit APC promoter methylation that is independent of APC expression.

Adult↗
PubMed2004

Impaired regulation of sterol regulatory element binding protein 2 in cholesterol gallstone-susceptible mice.

Lipid synthesis is under tight transcriptional control involving sterol regulatory element binding proteins (SREBP1, SREBP2). Rising cellular cholesterol levels prevent SREBP2 from entering the nucleus to directly activate the expression of 3-hydroxy-3-methylglutaryl-coenzyme A reductase (Hmgcr), the key enzyme of cholesterol synthesis. The failure to down-regulate cholesterol synthesis in gallstone-susceptible C57L/J but not AKR/J mice prompted us to study the processing of SREBP2 in these mice. Male mice of each strain received a control or lithogenic diet for 28 days. Membrane and nuclear extracts of pooled livers were prepared for immunoblot analysis of SREBP. Steady-state mRNA levels of Hmgcr, Srebp1, Srebp2 and the SREBP cleavage activating protein (Scap) were measured from individual livers. There was no marked difference between the two strains with regard to processing of SREBP1 as well as steady-state mRNA levels of Srebp1, Srebp2 and Scap. However, a near-complete suppression of nuclear SREBP2 related to low Hmgcr mRNA levels was noticed only for gallstone-resistant AKR mice. Abnormal regulation of SREBP2 appears to be responsible for the failure to suppress cholesterol synthesis in genetically cholesterol gallstone-susceptible mice. This defect may contribute to cholesterol hypersecretion and gallstone formation.

Animals↗
PubMed2003

Beta-casein-derived peptides, produced by bacteria, stimulate cancer cell invasion and motility.

In colon cancer, enteric bacteria and dietary factors are major determinants of the microenvironment but their effect on cellular invasion is not known. We therefore incubated human HCT-8/E11 colon cancer cells with bacteria or bacterial conditioned medium on top of collagen type I gels. Listeria monocytogenes stimulate cellular invasion through the formation of a soluble motility-promoting factor, identified as a 13mer beta-casein-derived peptide (HKEMPFPKYPVEP). The peptide is formed through the combined action of Mpl, a Listeria thermolysin-like metalloprotease, and a collagen-associated trypsin-like serine protease. The 13mer peptide was also formed by tumour biopsies isolated from colon cancer patients and incubated with a beta-casein source. The pro- invasive 13mer peptide-signalling pathway implicates activation of Cdc42 and inactivation of RhoA, linked to each other through the serine/threonine p21- activated kinase 1. Since both changes are necessary but not sufficient, another pathway might branch upstream of Cdc42 at phosphatidylinositol 3-kinase. Delta opioid receptor (deltaOR) is a candidate receptor for the 13mer peptide since naloxone, an deltaOR antagonist, blocks both deltaOR serine phosphorylation and 13mer peptide-mediated invasion.

Bacterial Proteins↗