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Biomedical subjects

Hans Lehrach

Publications and source records attributed to Hans Lehrach.

At least 19 recordsLinked to original sources
PubMed2007

Quantitative PCR based expression analysis on a nanoliter scale using polymer nano-well chips.

The analysis of gene expression is an essential element of functional genomics. Expression analysis is mainly based on DNA microarrays due to highly parallel readout and high throughput. Quantitative PCR (qPCR) based expression profiling is the gold standard for the precise monitoring of selected genes, and therefore used for validation of microarray data. Doing qPCR-based expression analysis in an array-like format can combine the higher sensitivity and accuracy of the qPCR methodology with a high data density at relatively low costs. This paper describes the development of an open-well based miniaturized platform for liquid PCR-based assays on the nanoliter scale using cost-effective polypropylene micro reactors (microPCR Chip). We show the quantification ability and reliability of qPCR in 200 nl with the microPCR chip down to 5 starting target molecules using TaqMan chemistry. An RNA expression analysis of four genes in mouse brain, liver and kidney tissues showed similar results in 200 nl as compared to standard 10 microl assays. The high sensitivity and quantification capability of the microPCR chip platform developed herein makes it a promising technology for performing high-throughput qPCR-based analysis in the nanoliter volume range.

Animals↗
PubMed2006

Triose phosphate isomerase deficiency is caused by altered dimerization--not catalytic inactivity--of the mutant enzymes.

Triosephosphate isomerase (TPI) deficiency is an autosomal recessive disorder caused by various mutations in the gene encoding the key glycolytic enzyme TPI. A drastic decrease in TPI activity and an increased level of its substrate, dihydroxyacetone phosphate, have been measured in unpurified cell extracts of affected individuals. These observations allowed concluding that the different mutations in the TPI alleles result in catalytically inactive enzymes. However, despite a high occurrence of TPI null alleles within several human populations, the frequency of this disorder is exceptionally rare. In order to address this apparent discrepancy, we generated a yeast model allowing us to perform comparative in vivo analyses of the enzymatic and functional properties of the different enzyme variants. We discovered that the majority of these variants exhibit no reduced catalytic activity per se. Instead, we observed, the dimerization behavior of TPI is influenced by the particular mutations investigated, and by the use of a potential alternative translation initiation site in the TPI gene. Additionally, we demonstrated that the overexpression of the most frequent TPI variant, Glu104Asp, which displays altered dimerization features, results in diminished endogenous TPI levels in mammalian cells. Thus, our results reveal that enzyme deregulation attributable to aberrant dimerization of TPI, rather than direct catalytic inactivation of the enzyme, underlies the pathogenesis of TPI deficiency. Finally, we discovered that yeast cells expressing a TPI variant exhibiting reduced catalytic activity are more resistant against oxidative stress caused by the thiol-oxidizing reagent diamide. This observed advantage might serve to explain the high allelic frequency of TPI null alleles detected among human populations.

Amino Acid Substitution↗
PubMed2006

A centrosome-independent role for gamma-TuRC proteins in the spindle assembly checkpoint.

The spindle assembly checkpoint guards the fidelity of chromosome segregation. It requires the close cooperation of cell cycle regulatory proteins and cytoskeletal elements to sense spindle integrity. The role of the centrosome, the organizing center of the microtubule cytoskeleton, in the spindle checkpoint is unclear. We found that the molecular requirements for a functional spindle checkpoint included components of the large gamma-tubulin ring complex (gamma-TuRC). However, their localization at the centrosome and centrosome integrity were not essential for this function. Thus, the spindle checkpoint can be activated at the level of microtubule nucleation.

Animals↗
PubMed2006

Analysis of Oct4-dependent transcriptional networks regulating self-renewal and pluripotency in human embryonic stem cells.

The POU domain transcription factor OCT4 is a key regulator of pluripotency in the early mammalian embryo and is highly expressed in the inner cell mass of the blastocyst. Consistent with its essential role in maintaining pluripotency, Oct4 expression is rapidly downregulated during formation of the trophoblast lineage. To enhance our understanding of the molecular basis of this differentiation event in humans, we used a functional genomics approach involving RNA interference-mediated suppression of OCT4 function in a human ESC line and analysis of the resulting transcriptional profiles to identify OCT4-dependent genes in human cells. We detected altered expression of >1,000 genes, including targets regulated directly by OCT4 either positively (NANOG, SOX2, REX1, LEFTB, LEFTA/EBAF DPPA4, THY1, and TDGF1) or negatively (CDX2, EOMES, BMP4, TBX18, Brachyury [T], DKK1, HLX1, GATA6, ID2, and DLX5), as well as targets for the OCT4-associated stem cell regulators SOX2 and NANOG. Our data set includes regulators of ACTIVIN, BMP, fibroblast growth factor, and WNT signaling. These pathways are implicated in regulating human ESC differentiation and therefore further validate the results of our analysis. In addition, we identified a number of differentially expressed genes that are involved in epigenetics, chromatin remodeling, apoptosis, and metabolism that may point to underlying molecular mechanisms that regulate pluripotency and trophoblast differentiation in humans. Significant concordance between this data set and previous comparisons between inner cell mass and trophectoderm in human embryos indicates that the study of human ESC differentiation in vitro represents a useful model of early embryonic differentiation in humans.

Apoptosis↗
PubMed2006

Fibroblast growth factor 2 modulates transforming growth factor beta signaling in mouse embryonic fibroblasts and human ESCs (hESCs) to support hESC self-renewal.

Fibroblast growth factor 2 (FGF2) is known to promote self-renewal of human embryonic stem cells (hESCs). In addition, it has been shown that transforming growth factor beta (TGFbeta) signaling is crucial in that the TGFbeta/Activin/Nodal branch of the pathway needs to be activated and the bone morphogenic protein (BMP)/GDF branch repressed to prevent differentiation. This holds particularly true for Serum Replacement-based medium containing BMP-like activity. We have reinvestigated a widely used protocol for conditioning hESC medium with mouse embryonic fibroblasts (MEFs). We show that FGF2 acts on MEFs to release supportive factors and reduce differentiation-inducing activity. FGF2 stimulation experiments with supportive and nonsupportive MEFs followed by genome-wide expression profiling revealed that FGF2 regulates the expression of key members of the TGFbeta pathway, with Inhba, Tgfb1, Grem1, and Bmp4 being the most likely candidates orchestrating the above activities. In addition, restimulation experiments in hESCs combined with global expression analysis revealed downstream targets of FGF2 signaling in these cells. Among these were the same factors previously identified in MEFs, thus suggesting that FGF2, at least in part, promotes self-renewal of hESCs by modulating the expression of TGFbeta ligands, which, in turn, act on hESCs in a concerted and autocrine manner.

Animals↗
PubMed2006

Analytical model of peptide mass cluster centres with applications.

BACKGROUND: The elemental composition of peptides results in formation of distinct, equidistantly spaced clusters across the mass range. The property of peptide mass clustering is used to calibrate peptide mass lists, to identify and remove non-peptide peaks and for data reduction. RESULTS: We developed an analytical model of the peptide mass cluster centres. Inputs to the model included, the amino acid frequencies in the sequence database, the average length of the proteins in the database, the cleavage specificity of the proteolytic enzyme used and the cleavage probability. We examined the accuracy of our model by comparing it with the model based on an in silico sequence database digest. To identify the crucial parameters we analysed how the cluster centre location depends on the inputs. The distance to the nearest cluster was used to calibrate mass spectrometric peptide peak-lists and to identify non-peptide peaks. CONCLUSION: The model introduced here enables us to predict the location of the peptide mass cluster centres. It explains how the location of the cluster centres depends on the input parameters. Fast and efficient calibration and filtering of non-peptide peaks is achieved by a distance measure suggested by Wool and Smilansky.

Journal Article↗
PubMed2006

An efficient and economic enhancer mix for PCR.

Polymerase chain reaction (PCR) has become a fundamental technique in molecular biology. Nonetheless, further improvements of the existing protocols are required to broaden the applicability of PCR for routine diagnostic purposes, to enhance the specificity and the yield of PCRs as well as to reduce the costs for high-throughput applications. One known problem typically reported in PCR experiments is the poor amplification of GC-rich DNA sequences. Here we designed and tested a novel effective and low-cost PCR enhancer, a concentration-dependent combination of betaine, dithiothreitol, and dimethyl sulfoxide that broadly enhanced the quantitative and/or qualitative output of PCRs. Additionally, we showed that the performances of this enhancer mix are comparable to those of commercially available PCR additives and highly effective with different DNA polymerases. Thus, we propose the routine application of this PCR enhancer mix for low- and high-throughput experiments.

Enhancer Elements, Genetic↗
PubMed2006

Age-specific hormonal decline is accompanied by transcriptional changes in human sebocytes in vitro.

The importance of hormones in endogenous aging has been displayed by recent studies performed on animal models and humans. To decipher the molecular mechanisms involved in aging we maintained human sebocytes at defined hormone-substituted conditions that corresponded to average serum levels of females from 20 (f20) to 60 (f60) years of age. The corresponding hormone receptor expression was demonstrated by reverse transcription-polymerase chain reaction (RT-PCR), Western blotting and immunocytochemistry. Cells at f60 produced significantly lower lipids than at f20. Increased mRNA and protein levels of c-Myc and increased protein levels of FN1, which have been associated with aging, were detected in SZ95 sebocytes at f60 compared to those detected at f20 after 5 days of treatment. Expression profiling employing a cDNA microarray composed of 15 529 cDNAs identified 899 genes with altered expression levels at f20 vs. f60. Confirmation of gene regulation was performed by real-time RT-PCR. The functional annotation of these genes according to the Gene Ontology identified pathways related to mitochondrial function, oxidative stress, ubiquitin-mediated proteolysis, cell cycle, immune responses, steroid biosynthesis and phospholipid degradation - all hallmarks of aging. Twenty-five genes in common with those identified in aging kidneys and several genes involved in neurodegenerative diseases were also detected. This is the first report describing the transcriptome of human sebocytes and its modification by a cocktail of hormones administered in age-specific levels and provides an in vitro model system, which approximates some of the hormone-dependent changes in gene transcription that occur during aging in humans.

Aging↗
PubMed2006

Cell array-based intracellular localization screening reveals novel functional features of human chromosome 21 proteins.

BACKGROUND: Trisomy of human chromosome 21 (Chr21) results in Down's syndrome, a complex developmental and neurodegenerative disease. Molecular analysis of Down's syndrome, however, poses a particular challenge, because the aneuploid region of Chr21 contains many genes of unknown function. Subcellular localization of human Chr21 proteins may contribute to further understanding of the functions and regulatory mechanisms of the genes that code for these proteins. Following this idea, we used a transfected-cell array technique to perform a rapid and cost-effective analysis of the intracellular distribution of Chr 21 proteins. RESULTS: We chose 89 genes that were distributed over the majority of 21q, ranging from RBM11 (14.5 Mb) to MCM3AP (46.6 Mb), with part of them expressed aberrantly in the Down's syndrome mouse model. Open reading frames of these genes were cloned into a mammalian expression vector with an amino-terminal His6 tag. All of the constructs were arrayed on glass slides and reverse transfected into HEK293T cells for protein expression. Co-localization detection using a set of organelle markers was carried out for each Chr21 protein. Here, we report the subcellular localization properties of 52 proteins. For 34 of these proteins, their localization is described for the first time. Furthermore, the alteration in cell morphology and growth as a result of protein over-expression for claudin-8 and claudin-14 genes has been characterized. CONCLUSION: The cell array-based protein expression and detection approach is a cost-effective platform for large-scale functional analyses, including protein subcellular localization and cell phenotype screening. The results from this study reveal novel functional features of human Chr21 proteins, which should contribute to further understanding of the molecular pathology of Down's syndrome.

Cell Cycle↗
PubMed2006

Matrix-assisted laser desorption/ionization mass spectrometric analysis of DNA on microarrays.

BACKGROUND: Matrix-assisted laser desorption/ionization (MALDI) mass spectrometry is a powerful tool in biomolecule analysis with a wide range of application possibilities, including genotyping of single-base variations (also known as single-nucleotide polymorphisms, or SNPs) for candidate gene studies and diagnostic typing of DNA markers. We tested a method that does not require stringent purification of the nucleic acids and/or the use of modification chemistry before mass spectrometry analysis. METHODS: We used an alternative direct analysis approach that allows MALDI analysis of crude DNA samples printed on microscope slides densely coated with primary amino groups that efficiently bind negatively charged DNA. After simple washing of the slides, we applied MALDI matrix and used a conventional MALDI mass spectrometer to detect DNA products. RESULTS: We analyzed crude oligonucleotide samples and performed automated genotyping of single-base variations in 72 DNA samples. CONCLUSION: This procedure offers an operational short-cut compared with standard MALDI procedures for preparation of oligonucleotides, including purification, and thus is an efficient tool for genotyping applications, particularly those requiring accurate, flexible, and rapid data generation and medium throughput.

Amines↗
PubMed2006

The bovine IGF2 gene is differentially methylated in oocyte and sperm DNA.

The insulin-like growth factor 2 gene (IGF2) encodes an essential growth factor and is imprinted in various mammalian species. Differentially methylated regions (DMRs) are often located within CpG islands and are critically involved in the regulation of monoallelic Igf2 expression in the mouse. Only partial sequence information is available for the bovine IGF2 gene and no DMR has currently been identified. The goal of this study was to identify a DMR within the bovine IGF2 gene as a prerequisite for further studies on gene-specific methylation patterns during preimplantation development. Here we describe the sequence analysis of a CpG-rich DNA fragment from the 5' untranslated region spanning exons and introns 4 and 5 and the identification of a previously unknown DMR in exon 10 of the bovine IGF2 gene. Bisulfite analysis revealed that this DMR is differentially methylated in mature oocytes and sperm. The identification of an intragenic DMR within a developmentally important gene such as the bovine IGF2 gene provides a useful tool to evaluate the methylation patterns of embryos derived in vivo and in vitro. Our study is the first report of a differentially methylated region in a bovine imprinted gene discovered by the analysis of female and male gametes.

5' Untranslated Regions↗
PubMed2006

Profiling humoral autoimmune repertoire of dilated cardiomyopathy (DCM) patients and development of a disease-associated protein chip.

Dilated cardiomyopathy (DCM) is a myocardial disease characterized by progressive depression of myocardial contractile function and ventricular dilatation. Thirty percent of DCM patients belong to the inherited genetic form; the rest may be idiopathic, viral, autoimmune, or immune-mediated associated with a viral infection. Disturbances in humoral and cellular immunity have been described in cases of myocarditis and DCM. A number of autoantibodies against cardiac cell proteins have been identified in DCM. In this study, we have profiled the autoantibody repertoire of plasma from DCM patients against a human protein array consisting of 37,200 redundant, recombinant human proteins and performed qualitative and quantitative validation of these putative autoantigens on protein microarrays to identify novel putative DCM specific autoantigens. In addition to analyzing the whole IgG autoantibody repertoire, we have also analyzed the IgG3 antibody repertoire in the plasma samples to study the characteristics of IgG3 subclass antibodies. By combining screening of a protein expression library with protein microarray technology, we have detected 26 proteins identified by the IgG antibody repertoire and 6 proteins bound by the IgG3 subclass. Several of these autoantibodies found in plasma of DCM patients, such as the autoantibody against the Kv channel-interacting protein, are associated with heart failure.

Amino Acid Sequence↗
PubMed2006

LNA-modified oligodeoxynucleotide hybridization with DNA microarrays printed on nanoporous membrane slides.

We report a robust method for the detection of hybridization events using a microarray-based assay on a nanoporous membrane platform. The technique is characterized by a hybridization time of only 1 hour and uses Cy5-labeled, 7-mer oligodeoxynucleotide probes modified with locked nucleic acid (LNA) nucleotides. We show that the volume of the DNA spotted onto a nanomembrane can be reduced to approximately 4 nL with detectable signal intensity. Moreover, the amount of the DNA target could be reduced to 4 fmol. The described approach could dramatically increase the throughput of techniques based on sequencing by hybridization, such as oligofingerprinting, by decreasing the total number of probes that are needed for analysis of large clone sets and reduction of the sample/reagent consumption. The method is particularly advantageous when numerous hybridization-based assays must be performed for characterization of sample sets of 100,000 or more.

Membranes, Artificial↗
PubMed2006

Expression profiling of drug response--from genes to pathways.

Understanding individual response to a drug-what determines its efficacy and tolerability-is the major bottleneck in current drug development and clinical trials. Intracellular response and metabolism, for example through cytochrome P-450 enzymes, may either enhance or decrease the effect of different drugs, dependent on the genetic variant. Microarrays offer the potential to screen the genetic composition of the individual patient. However, experiments are "noisy" and must be accompanied by solid and robust data analysis. Furthermore, recent research aims at the combination of high-throughput data with methods of mathematical modeling, enabling problem-oriented assistance in the drug discovery process. This article will discuss state-of-the-art DNA array technology platforms and the basic elements of data analysis and bioinformatics research in drug discovery. Enhancing single-gene analysis, we will present a new method for interpreting gene expression changes in the context of entire pathways. Furthermore, we will introduce the concept of systems biology as a new paradigm for drug development and highlight our recent research-the development of a modeling and simulation platform for biomedical applications. We discuss the potentials of systems biology for modeling the drug response of the individual patient.

Animals↗
PubMed2005

Recent advances of protein microarrays.

Technological innovations and novel applications have greatly advanced the field of protein microarrays. Over the past two years, different types of protein microarrays have been used for serum profiling, protein abundance determinations, and identification of proteins that bind DNA or small compounds. However, considerable development is still required to ensure common quality standards and to establish large content repertoires. Here, we summarize applications available to date and discuss recent technological achievements and efforts on standardization.

Animals↗
PubMed2005

Transformation and other factors of the peptide mass spectrometry pairwise peak-list comparison process.

BACKGROUND: Biological Mass Spectrometry is used to analyse peptides and proteins. A mass spectrum generates a list of measured mass to charge ratios and intensities of ionised peptides, which is called a peak-list. In order to classify the underlying amino acid sequence, the acquired spectra are usually compared with synthetic ones. Development of suitable methods of direct peak-list comparison may be advantageous for many applications. RESULTS: The pairwise peak-list comparison is a multistage process composed of matching of peaks embedded in two peak-lists, normalisation, scaling of peak intensities and dissimilarity measures. In our analysis, we focused on binary and intensity based measures. We have modified the measures in order to comprise the mass spectrometry specific properties of mass measurement accuracy and non-matching peaks. We compared the labelling of peak-list pairs, obtained using different factors of the pairwise peak-list comparison, as being the same or different to those determined by sequence database searches. In order to elucidate how these factors influence the peak-list comparison we adopted an analysis of variance type method with the partial area under the ROC curve as a dependent variable. CONCLUSION: The analysis of variance provides insight into the relevance of various factors influencing the outcome of the pairwise peak-list comparison. For large MS/MS and PMF data sets the outcome of ANOVA analysis was consistent, providing a strong indication that the results presented here might be valid for many various types of peptide mass measurements.

Algorithms↗
PubMed2005

Liquid-based hybridization assay with real-time detection in miniaturized array platforms.

An assay for the fluorescent detection of short oligonucleotide probe hybridization in miniaturized high-density array platforms is presented. It combines hybridization in solution with real-time fluorescent detection, which involves measurement of fluorescence increase by means of an induced fluorescence resonance energy transfer. The feasibility of this approach using DNA or RNA as a target, and short DNA- as well as LNA (locked nucleic acid)-modified oligonucleotides as probes is shown. The presented approach could potentially contribute to a significant increase in the throughput of large-scale genomic applications, such as oligofingerprinting and genotyping, and also reduce material consumption.

Computer Systems↗