Biomedical subjects

Jörg D Hoheisel

Publications and source records attributed to Jörg D Hoheisel.

At least 37 records · Page 2Linked to original sources

PubMed2005

Multiplex approaches in protein microarray technology.

The success of genome sequencing projects has provided the basis for systematic analysis of protein function and has led to a shift from the description of single molecules to the characterization of complex samples. Such a task would not be possible without the provision of appropriate high-throughput technologies, such as protein microarray technology. In addition, the increasing number of samples necessitates the adaptation of such technologies to a multiplex format. This review will discuss protein microarray technology in the context of multiplex analysis and highlight its current prospects and limitations.

Animals↗

PubMed2004

DNA microarray analysis of pancreatic malignancies.

Pancreatic ductal adenocarcinoma (PDAC) has an extremely poor prognosis. To improve the prognosis, novel molecular markers and targets for earlier diagnosis and adjuvant and/or neoadjuvant treatment are needed. Recent advances in human genome research and high-throughput molecular technologies make it possible to cope with the molecular complexity of malignant tumors. With DNA array technology, mRNA expression levels of thousand of genes can be measured simultaneously in a single assay. As several studies using microarrays in PDAC have already been published, this review attempts to compare the published data and therefore to validate the results. In addition, the applied techniques are discussed in the context of pancreatic malignancies.

Adenocarcinoma↗

PubMed2004

Microcystic tubulopapillary carcinoma of the pancreas: a new tumor entity?

An unusual pancreatic tumor with microcystic and tubulopapillary features was observed in a 53-year-old woman. The tumor presented as a large, focally cystic mass in the head of the pancreas, which compressed the surrounding structures. The histological and immunohistochemical analysis revealed a neoplasm that could not be assigned to any of the known pancreatic tumor types. At the molecular level, the tumor showed inactivation of the DPC4/SMAD4 gene, deletion of exon 1 of the p16(INK4A) gene and a point mutation at codon 34 (GGA>AGA) of beta-catenin. Transcriptional profiling analyses and subsequent correspondence cluster analysis demonstrated that the transcriptional profile of the tumor differed distinctly from that of ductal adenocarcinomas, pancreatic cystic tumors and normal pancreatic tissues. These data suggest that the neoplasm most likely represents a new pancreatic tumor entity, which we would like to refer to as microcystic tubulopapillary tumor.

Adenocarcinoma↗

PubMed2004

Peptide nucleic acids on microarrays and other biosensors.

The analysis of biomolecules using microarrays and other biosensors has a significant role in molecular biotechnology, and will become even more important in the future as a versatile tool for research and diagnostics. For many applications, the synthetic DNA mimic peptide nucleic acid (PNA) could be advantageous as a probe molecule, owing to its unique physicochemical and biochemical properties. PNA exhibits superior hybridization characteristics and improved chemical and enzymatic stability relative to nucleic acids. Furthermore, its different molecular structure enables new modes of detection, especially procedures that avoid the introduction of a label. In our opinion, all of these factors contribute significantly toward the establishment of faster and more reliable analytical processes and opens new fields of application.

Algorithms↗

PubMed2004

Sed1p and Srl1p are required to compensate for cell wall instability in Saccharomyces cerevisiae mutants defective in multiple GPI-anchored mannoproteins.

The covalently linked cell wall protein Ccw12p of Saccharomyces cerevisiae is a GPI-anchored protein (V. Mrsa et al., 1999, J Bacteriol 181: 3076-3086). Although only 121 amino acids long, the haemagglutinin-tagged protein released by laminarinase from the cell wall possesses an apparent molecular mass of > 300 kDa. A membrane-bound form with an apparent molecular mass of 58 kDa is highly O- and N-glycosylated and contains the GPI anchor. With a half-life of 2 min, the membrane form is transformed to the > 300 kDa form. The deletion mutant ccw12Delta grows slower than the wild type, is highly sensitive to Calcofluor white and contains 2.5 times more chitin. Further, compared with wild-type yeast, significantly more proteins are released from intact cells when treated with dithiothreitol. Interestingly, these defects become less pronounced when further GPI-anchored cell wall proteins are deleted. Mutant DeltaGPI (simultaneous deletion of CCW12, CCW13/DAN1, CCW14, TIP1 and CWP1) is similar in many respects to wild-type yeast. To find out how the cell wall is stabilized in mutant DeltaGPI, a genome-wide transcription analysis was performed. Of 159 significantly regulated genes, 14 encode either known or suspected cell wall-associated proteins. Analysis of genes affected in transcription revealed that SED1 and SRL1 in particular are required to reconstruct cell wall stability in the absence of multiple GPI-anchored mannoproteins.

Cell Wall↗

PubMed2004

Fluorescent sample labeling for DNA microarray analyses.

Three fluorophor-labeling methods for gene expression profiling on deoxyribonucleic acid (DNA) microarrays are described. All three techniques start from total ribonucleic acid (RNA) samples. Two procedures are based on first-strand complementary DNA synthesis by reverse transcription. Label is introduced either by direct incorporation of fluorescently labeled nucleotides or indirectly by incorporation of aminoally-dUTP and subsequent coupling of fluorescent dyes. The third method is based on an amplification of antisense RNA by in vitro transcription subsequent to first- and second-strand complementary DNA synthesis. While the first two methods are applied mainly in analyses on microarrays made from spotted polymerase chain reaction products or long oligonucleotides, the last procedure is mostly used for experiments on in situ synthesized oligonucleotide arrays.

DNA, Complementary↗

PubMed2004

Peptide nucleic acid microarrays.

A fast and economical procedure for the production of peptide nucleic acid (PNA) microarrays is presented. PNA oligomers are synthesized in a fully automatic manner in 96-well plates using standard Fmoc chemistry. Subsequently, the oligomers are released from the support and spotted onto glass or silicone slides, which were activated by succinimidyl ester. This process allows for a concomitant purification of the oligomers directly on the chip surface. Although the terminal primary amino groups of the full-length products bind selectively to this surface, none of the byproducts of synthesis, such as truncated sequences or cleaved side chain protection groups, will bind and are therefore washed away. In this chapter, protocols are presented for the whole production process as well as sample hybridization.

Nucleic Acid Hybridization↗

PubMed2004

Creation of a minimal tiling path of genomic clones for Drosophila: provision of a common resource.

On the basis of shotgun subclone libraries used in the sequencing of the Drosophila melanogaster genome, a minimal tiling path of subclones across much of the genome was determined. About 320,000 shotgun clones for chromosomes X(12-20), 2R, 2L, 3R, and 4 were available from the Berkeley Drosophila Genome Project. The clone inserts have an average length of 3.4 kb and are amenable to standard PCR amplification. The resulting tiling path covers 86.2% of chromosome X(12-20), 86.2% of chromosomal arm 2R, 79.0% of 2L, 89.6% of 3R, and 80.5% of chromosome 4. In total, the 25,135 clones represent 76.7 Mb--equivalent to about 67% of the genome--and would be suitable for producing a microarray on a single slide.

Animals↗

PubMed2003

PNA microarrays for hybridisation of unlabelled DNA samples.

Several strategies have been developed for the production of peptide nucleic acid (PNA) microarrays by parallel probe synthesis and selective coupling of full-length molecules. Such microarrays were used for direct detection of the hybridisation of unlabelled DNA by time-of-flight secondary ion mass spectrometry. PNAs were synthesised by an automated process on filter-bottom microtitre plates. The resulting molecules were released from the solid support and attached without any purification to microarray surfaces via the terminal amino group itself or via modifications, which had been chemically introduced during synthesis. Thus, only full-length PNA oligomers were attached whereas truncated molecules, produced during synthesis because of incomplete condensation reactions, did not bind. Different surface chemistries and fitting modifications of the PNA terminus were tested. For an examination of coupling selectivity, bound PNAs were cleaved off microarray surfaces and analysed by MALDI-TOF mass spectrometry. Additionally, hybridisation experiments were performed to compare the attachment chemistries, with fully acetylated PNAs spotted as controls. Upon hybridisation of unlabelled DNA to such microarrays, binding events could be detected by visualisation of phosphates, which are an integral part of nucleic acids but missing entirely in PNA probes. Overall best results in terms of selectivity and sensitivity were obtained with thiol-modified PNAs on maleimide surfaces.

Base Sequence↗

PubMed2003

Late ischemic preconditioning of the myocardium alters the expression of genes involved in inflammatory response.

Myocardial ischemic preconditioning (IPC) is a potent endogenous mechanism of cardioprotection against ischemia-reperfusion injury. In this study we focused on the second phase of IPC as the most interesting in terms of therapeutic implementations. We aimed at the detection of genes, which are differentially expressed at 16 h after reperfusion. Preconditioning of canine myocardium was initiated by 5 min occlusion of the left anterior descending coronary artery with subsequent reperfusion. cDNA representational difference analysis in combination with microarray hybridization and reverse transcription polymerase chain reaction were used to reveal the changes in gene expression in canine hearts. We found that functionally related genes for tristetraproline (TTP), selectin E, matrix metalloproteinase 9, and tumor necrosis factor-alpha were highly upregulated at the late phase of IPC. The upregulation of TTP gene at the late phase of IPC, reported here for the first time, may represent a cardioprotective mechanism, which could be a promising perspective in clinical interventions against ischemia-reperfusion injuries of the heart.

Animals↗

PubMed2003

What's in the genome of a filamentous fungus? Analysis of the Neurospora genome sequence.

The German Neurospora Genome Project has assembled sequences from ordered cosmid and BAC clones of linkage groups II and V of the genome of Neurospora crassa in 13 and 12 contigs, respectively. Including additional sequences located on other linkage groups a total of 12 Mb were subjected to a manual gene extraction and annotation process. The genome comprises a small number of repetitive elements, a low degree of segmental duplications and very few paralogous genes. The analysis of the 3218 identified open reading frames provides a first overview of the protein equipment of a filamentous fungus. Significantly, N.crassa possesses a large variety of metabolic enzymes including a substantial number of enzymes involved in the degradation of complex substrates as well as secondary metabolism. While several of these enzymes are specific for filamentous fungi many are shared exclusively with prokaryotes.

Chromosome Mapping↗

PubMed2003

Genome-wide analysis of the response to cell wall mutations in the yeast Saccharomyces cerevisiae.

Perturbations of the yeast cell wall trigger a repair mechanism that reconfigures its molecular structure to preserve cell integrity. To investigate this mechanism, we compared the global gene expression in five mutant strains, each bearing a mutation (i.e. fks1, kre6, mnn9, gas1, and knr4 mutants) that affects in a different manner the cell wall construction. Altogether, 300 responsive genes were kept based on high stringency criteria during data processing. Functional classification of these differentially expressed genes showed a substantial subset of induced genes involved in cell wall construction and an enrichment of metabolic, energy generation, and cell defense categories, whereas families of genes belonging to transcription, protein synthesis, and cellular growth were underrepresented. Clustering methods isolated a single group of approximately 80 up-regulated genes that could be considered as the stereotypical transcriptional response of the cell wall compensatory mechanism. The in silico analysis of the DNA upstream region of these co-regulated genes revealed pairwise combinations of DNA-binding sites for transcriptional factors implicated in stress and heat shock responses (Msn2/4p and Hsf1p) with Rlm1p and Swi4p, two PKC1-regulated transcription factors involved in the activation genes related to cell wall biogenesis and G1/S transition. Moreover, this computational analysis also uncovered the 6-bp 5'-AGCCTC-3' CDRE (calcineurin-dependent response element) motif in 40% of the co-regulated genes. This motif was recently shown to be the DNA binding site for Crz1p, the major effector of calcineurin-regulated gene expression in yeast. Taken altogether, the data presented here lead to the conclusion that the cell wall compensatory mechanism, as triggered by cell wall mutations, integrates three major regulatory systems: namely the PKC1-SLT2 mitogen-activated protein kinase-signaling module, the "global stress" response mediated by Msn2/4p, and the Ca2+/calcineurin-dependent pathway. The relative importance of these regulatory systems in the cell wall compensatory mechanism is discussed.

Base Sequence↗

PubMed2003

Antibody microarrays: an evaluation of production parameters.

Antibody microarrays could have an enormous impact on the functional analysis of cellular activity and regulation, especially at the level of protein expression and protein-protein interaction, and might become an invaluable tool in disease diagnostics. The array surface is bound to have a tremendous influence on the findings from such studies. Apart from the basic issue of how to attach antibodies optimally without affecting their function, it is also important that the cognate antigens, applied within a complex protein mixture, all bind to the arrayed antibodies irrespective of their enormous variety in structure. In this study, various factors in the production of antibody microarrays on glass support were analysed: the modification of the glass surface; kind and length of cross-linkers; composition and pH of the spotting buffer; blocking reagents; antibody concentration and storage procedures, in order to evaluate their effect on array performance. Altogether, data from more than 700 individual array experiments were taken into account. In addition to home-made slides, commercially available systems were also included in the analysis.

Absorption↗

PubMed2003

Analysis of nutrient-dependent transcript variations in Neurospora crassa.

Nutrient-dependent variations in transcript levels of the filamentous fungus Neurospora crassa were studied on a microarray containing some 4700 cDNAs. Cells were grown in minimal and acetate medium. The isolated RNA was analyzed in comparison to the results obtained upon the hybridization of samples prepared from the RNA of cells grown in full medium. Altogether, 160 cDNA clones exhibited significant variations, falling into five distinct subgroups of very similar transcription profiles. This is indicative of the occurrence of a high degree of co-regulation of genes in N. crassa. Especially the regulation of the expression of proteins involved in metabolic pathways was found to be strongly regulated at the RNA level.

Culture Media↗

PubMed2003

Glucose triggers different global responses in yeast, depending on the strength of the signal, and transiently stabilizes ribosomal protein mRNAs.

Glucose exerts profound effects upon yeast physiology. In general, the effects of high glucose concentrations (>1%) upon Saccharomyces cerevisiae have been studied. In this paper, we have characterized the global responses of yeast cells to very low (0.01%), low (0.1%) and high glucose signals (1.0%) by transcript profiling. We show that yeast is more sensitive to very low glucose signals than was previously thought, and that yeast displays different responses to these different glucose signals. Genes involved in central metabolic pathways respond rapidly to very low glucose signals, whereas genes involved in the biogenesis of cytoplasmic ribosomes generally respond only to glucose concentrations of> 0.1%. We also show that cytoplasmic ribosomal protein mRNAs are transiently stabilized by glucose, indicating that both transcriptional and post-transcriptional mechanisms combine to accelerate the accumulation of ribosomal protein mRNAs. Presumably, this facilitates rapid ribosome biogenesis after exposure to glucose. However, our data indicate that yeast activates ribosome biogenesis only when sufficient glucose is available to make this metabolic investment worthwhile. In contrast, the regulation of metabolic functions in response to very low glucose signals presumably ensures that yeast can exploit even minute amounts of this preferred nutrient.

Carbon↗

PubMed2002

Analysis of stage-specific gene expression in the bloodstream and the procyclic form of Trypanosoma brucei using a genomic DNA-microarray.

A microarray comprising 21,024 different PCR products spotted on glass slides was constructed for gene expression studies on Trypanosoma brucei. The arrayed fragments were generated from a T. brucei shotgun clone library, which had been prepared from randomly sheared and size-fractionated genomic DNA. For the identification of stage-specific gene activity, total RNA from in vitro cultures of the human, long slender form and the insect, procyclic form of the parasite was labelled and hybridised to the microarray. Approximately 75% of the genomic fragments produced a signal and about 2% exhibited significant differences between the transcript levels in the bloodstream and procyclic forms. A few results were confirmed by Northern blot analysis or reverse-transcription and PCR. Three hundred differentially regulated clones have been selected for sequencing. So far, of 33 clones that showed about 2-fold or more over-expression in bloodstream forms, 15 contained sequences similar to those of VSG expression sites and at least six others appeared non-protein-coding. Of 29 procyclic-specific clones, at least eight appeared not to be protein-coding. A surprisingly large proportion of known regulated genes was already identified in this small sample, and some new ones were found, illustrating the utility of genomic arrays.

Animals↗

PubMed2002

Manufacturing DNA microarrays from unpurified PCR products.

For the production of DNA microarrays from PCR products, purification of the the DNA fragments prior to spotting is a major expense in cost and time. Also, a considerable amount of material is lost during this process and contamination might occur. Here, a protocol is presented that permits the manufacture of microarrays from unpurified PCR products on aminated surfaces such as glass slides coated with the widely used poly(L-lysine) or aminosilane. The presence of primer molecules in the PCR sample does not increase the non-specific signal upon hybridisation. Overall, signal intensity on arrays made of unpurified PCR products is 94% of the intensity obtained with the respective purified molecules. This slight loss in signal, however, is offset by a reduced variation in the amount of DNA present at the individual spot positions across an array, apart from the considerable savings in time and cost. In addition, a larger number of arrays can be made from one batch of amplification products.

Animals↗

PubMed2002

Cardiac ankyrin repeat protein, a negative regulator of cardiac gene expression, is augmented in human heart failure.

The technique of representational difference analysis of cDNA has been applied to screen for differentially expressed genes in a canine model of pacing-induced heart failure. We identified the canine homolog of the cardiac ankyrin repeat protein (CARP) which has been shown to be involved in the regulation of the transcription of cardiac genes. To confirm the significance for human heart failure, cardiac tissue specimens obtained from non-failing donor hearts and from explanted hearts from patients with end-stage heart failure were investigated. CARP mRNA and protein levels were markedly increased in failing left ventricles. Interestingly, alterations in CARP expression were restricted to ventricular tissue and were not observed in atria. Fractionation experiments revealed that CARP was expressed predominantly in the nuclei consistent with the proposed function of CARP as a modulator of transcription. Together, these findings raise the possibility that augmented ventricular CARP expression may play a role in the pathogenesis of human heart failure.

Adult↗