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M Bittner

Publications and source records attributed to M Bittner.

At least 19 recordsLinked to original sources
PubMed2000

Multivariate measurement of gene expression relationships.

The operational activities of cells are based on an awareness of their current state, coupled to a programmed response to internal and external cues in a context-dependent manner. One key goal of functional genomics is to develop analytical methods for delineating the ways in which the individual actions of genes are integrated into our understanding of the increasingly complex systems of organelle, cell, organ, and organism. This paper describes a novel approach to assess the codetermination of gene transcriptional states based upon statistical evaluation of reliably informative subsets of data derived from large-scale simultaneous gene expression measurements with cDNA microarrays. The method finds associations between the expression patterns of individual genes by determining whether knowledge of the transcriptional levels of a small gene set can be used to predict the associated transcriptional state of another gene. To test this approach for identification of the relevant contextual elements of cellular response, we have modeled our approach using data from known gene response pathways including ionizing radiation and downstream targets of inactivating gene mutations. This approach strongly suggests that evaluation of the transcriptional status of a given gene(s) can be combined with data from global expression analyses to predict the expression level of another gene. With data sets of the size currently available, this approach should be useful in finding sets of genes that participate in particular biological processes. As larger data sets and more computing power become available, the method can be extended to validating and ultimately identifying biologic (transcriptional) pathways based upon large-scale gene expression analysis.

Cyclin-Dependent Kinase Inhibitor p21↗
PubMed2000

Differentially painting human chromosome arms with combined binary ratio-labeling fluorescence in situ hybridization.

Recently we developed a novel strategy for differentially painting all 24 human chromosomes. It is termed COBRA-FISH, short for combined binary ratio labeling-fluorescence in situ hybridization. COBRA-FISH is distinct from the pure combinatorial approach in that only 4 instead of 5 fluorophores are needed to achieve color discrimination of 24 targets. Furthermore, multiplicity can be increased to 48 by introduction of a fifth fluorophore. Here we show that color identification by COBRA-FISH of all of the p and q arms of human chromosomes is feasible, and we apply the technique for detecting and elucidating intra- and interchromosomal rearrangements. Compared with 24-color whole chromosome painting FISH, PQ-COBRA-FISH considerably enhances the ability to determine the composition of rearranged chromosomes as demonstrated by the identification of pericentric inversions and isochromosomes as well as the elucidation of the arm identity of chromosomal material involved in complex translocations that occur in solid tumors.

Chromosome Painting↗
PubMed2000

The rfaE gene from Escherichia coli encodes a bifunctional protein involved in biosynthesis of the lipopolysaccharide core precursor ADP-L-glycero-D-manno-heptose.

The intermediate steps in the biosynthesis of the ADP-L-glycero-D-manno-heptose precursor of inner core lipopolysaccharide (LPS) are not yet elucidated. We isolated a mini-Tn10 insertion that confers a heptoseless LPS phenotype in the chromosome of Escherichia coli K-12. The mutation was in a gene homologous to the previously reported rfaE gene from Haemophilus influenzae. The E. coli rfaE gene was cloned into an expression vector, and an in vitro transcription-translation experiment revealed a polypeptide of approximately 55 kDa in mass. Comparisons of the predicted amino acid sequence with other proteins in the database showed the presence of two clearly separate domains. Domain I (amino acids 1 to 318) shared structural features with members of the ribokinase family, while Domain II (amino acids 344 to 477) had conserved features of the cytidylyltransferase superfamily that includes the aut gene product of Ralstonia eutrophus. Each domain was expressed individually, demonstrating that only Domain I could complement the rfaE::Tn10 mutation in E. coli, as well as the rfaE543 mutation of Salmonella enterica SL1102. DNA sequencing of the rfaE543 gene revealed that Domain I had one amino acid substitution and a 12-bp in-frame deletion resulting in the loss of four amino acids, while Domain II remained intact. We also demonstrated that the aut::Tn5 mutation in R. eutrophus is associated with heptoseless LPS, and this phenotype was restored following the introduction of a plasmid expressing the E. coli Domain II. Thus, both domains of rfaE are functionally different and genetically separable confirming that the encoded protein is bifunctional. We propose that Domain I is involved in the synthesis of D-glycero-D-manno-heptose 1-phosphate, whereas Domain II catalyzes the ADP transfer to form ADP-D-glycero-D-manno-heptose.

Adenosine Diphosphate↗
PubMed1999

Informatic selection of a neural crest-melanocyte cDNA set for microarray analysis.

With cDNA microarrays, it is now possible to compare the expression of many genes simultaneously. To maximize the likelihood of finding genes whose expression is altered under the experimental conditions, it would be advantageous to be able to select clones for tissue-appropriate cDNA sets. We have taken advantage of the extensive sequence information in the dbEST expressed sequence tag (EST) database to identify a neural crest-derived melanocyte cDNA set for microarray analysis. Analysis of characterized genes with dbEST identified one library that contained ESTs representing 21 neural crest-expressed genes (library 198). The distribution of the ESTs corresponding to these genes was biased toward being derived from library 198. This is in contrast to the EST distribution profile for a set of control genes, characterized to be more ubiquitously expressed in multiple tissues (P < 1 x 10(-9)). From library 198, a subset of 852 clustered ESTs were selected that have a library distribution profile similar to that of the 21 neural crest-expressed genes. Microarray analysis demonstrated the majority of the neural crest-selected 852 ESTs (Mel1 array) were differentially expressed in melanoma cell lines compared with a non-neural crest kidney epithelial cell line (P < 1 x 10(-8)). This was not observed with an array of 1,238 ESTs that was selected without library origin bias (P = 0.204). This study presents an approach for selecting tissue-appropriate cDNAs that can be used to examine the expression profiles of developmental processes and diseases.

Calibration↗
PubMed1999

Fluorescent cDNA microarray hybridization reveals complexity and heterogeneity of cellular genotoxic stress responses.

The fate of cells exposed to ionizing radiation (IR) may depend greatly on changes in gene expression, so that an improved view of gene induction profiles is important for understanding mechanisms of checkpoint control, repair and cell death following such exposures. We have used a quantitative fluorescent cDNA microarray hybridization approach to identify genes regulated in response to 7-irradiation in the p53 wild-type ML-1 human myeloid cell line. Hybridization of the array to fluorescently-labeled RNA from treated and untreated cells was followed by computer analysis to derive relative changes in expression levels of the genes present in the array, which agreed well with actual quantitative changes in expression. Forty-eight sequences, 30 not previously identified as IR-responsive, were significantly regulated by IR. Induction by IR and other stresses of a subset of these genes, including the previously characterized CIP1/ WAF1, MDM2 and BAX genes, as well as nine genes not previously reported to be IR-responsive, was examined in a panel of 12 human cell lines. Responses varied widely in cell lines with different tissues of origin and different genetic backgrounds, highlighting the importance of cellular context to genotoxic stress responses. Two of the newly identified IR-responsive genes, FRA-1 and ATF3, showed a p53-associated component to their IR-induction, and this was confirmed both in isogenic human cell lines and in mouse thymus. The majority of the IR-responsive genes, however, showed no indication of p53-dependent regulation, representing a potentially important class of stress-responsive genes in leukemic cells.

Animals↗
PubMed1999

Microarrays and toxicology: the advent of toxicogenomics.

The availability of genome-scale DNA sequence information and reagents has radically altered life-science research. This revolution has led to the development of a new scientific subdiscipline derived from a combination of the fields of toxicology and genomics. This subdiscipline, termed toxicogenomics, is concerned with the identification of potential human and environmental toxicants, and their putative mechanisms of action, through the use of genomics resources. One such resource is DNA microarrays or "chips," which allow the monitoring of the expression levels of thousands of genes simultaneously. Here we propose a general method by which gene expression, as measured by cDNA microarrays, can be used as a highly sensitive and informative marker for toxicity. Our purpose is to acquaint the reader with the development and current state of microarray technology and to present our view of the usefulness of microarrays to the field of toxicology.

Animal Testing Alternatives↗
PubMed1999

Spontaneous platelet aggregation as a predictive risk factor for vascular occlusions in healthy volunteers? Results of the HAPARG Study. Haemostatic parameters as risk factors in healthy volunteers.

The HAPARG Study (haemostatic parameters as risk factors in healthy volunteers) was performed in a subset of volunteers taking part in the MARISK Study (Mainzer Risikoindikatoren Studie für die koronare Herzkrankheit) sponsored by the German Ministry of Research and started in 1984. A previous study (Yamanishi et al., Thromb Haemostas 1985;54:539-543) had shown that spontaneously enhanced platelet aggregation as measured with the PAT-III-test and higher fibrinogen concentrations are significant risk factors for new vascular occlusions in diabetic patients. It was the aim of the HAPARG Study to establish whether spontaneous platelet aggregation and other hemostatic variables are independent risk factors for vascular occlusions in healthy volunteers. Employees of a chemical/pharmaceutical company aged 40-65 years and personnel of the University of Mainz, aged 30-60 years were included in this prospective study. Besides anamnestic data such as on smoking, hypertension and diabetes, blood pressure, the ankle/arm Doppler-index and an ECG were recorded and serum cholesterol, HDL, LDL, triglycerides, uric acid and glucose were measured. Men (1884) and women (989) entered the study and were followed for 4-6 years. In the age group of 30-50 years, more women than men were included. During the observation period 53 vascular occlusions occurred (36 coronary and nine cerebral events and eight peripheral vascular occlusions). Only three of these endpoints occurred in women. Besides age (odds ratio = 1.7, P = 0.02) and gender as expected risk factors, the multivariate logistic stepwise regression analysis revealed smoking (odds ratio = 2.2, P = 0.008), lower HDL-levels (odds ratio = 2.2, P = 0.013), elevated diastolic blood pressure (odds ratio = 1.4. P = 0.004) followed by spontaneous platelet aggregation (odds ratio = 1.1, P = 0.037), and slightly elevated blood glucose (P = 0.0047) as significant risk factors for men. Higher fibrinogen levels missed significance in this analysis (P = 0.059). None of the other hemostatic parameters showed a significant correlation with the vascular events. To our knowledge, this has been the first prospective trial in a large population of healthy individuals in which a platelet function parameter has been studied together with other possible risk factors. Spontaneously enhanced platelet aggregation is probably an independent risk factor and, like elevated fibrinogen and other haemostatic variables, an indicator of an ongoing active atherosclerotic process.

Adult↗
PubMed1999

Expression profiling using cDNA microarrays.

cDNA microarrays are capable of profiling gene expression patterns of tens of thousands of genes in a single experiment. DNA targets, in the form of 3' expressed sequence tags (ESTs), are arrayed onto glass slides (or membranes) and probed with fluorescent- or radioactively-labelled cDNAs. Here, we review technical aspects of cDNA microarrays, including the general principles, fabrication of the arrays, target labelling, image analysis and data extraction, management and mining.

Animals↗
PubMed1999

The complexity of radiation stress responses: analysis by informatics and functional genomics approaches.

Molecular responses to genotoxic stress are complex and are mediated by a variety of regulatory pathways. One key element in cellular response is the stress gene transcription factor p53, which can regulate nearly 100 genes that have already been identified. Although p53 plays a central role in the cellular response to DNA-damaging agents such as ionizing radiation (IR), other pathways can also have important roles. One example is the transcriptional responses associated with IR-induced apoptosis, where induction of some genes is limited to p53 wild-type (wt) cells that also have the ability to undergo rapid apoptosis after irradiation. In contrast, other genes are triggered after IR in lines undergoing rapid apoptosis regardless of p53 status. From this and other examples, it is apparent that the pattern of stress gene expression is cell type specific in both primary and transformed lines. The premise will be developed that such differences in stress gene responsiveness can be employed as molecular markers using a combination of informatics and functional genomics approaches. An example is given using the panel of lines of the NCI anticancer drug screen where both the p53 status and sensitivity to a large collection of cytotoxic agents have been determined. The utility of cDNA microarray hybridization to measure IR-stress gene responses has recently been demonstrated and a large number of additional IR-stress genes have been identified. The responses of some of these genes to IR and other DNA-damaging agents varied widely in cell lines from different tissues of origin and different genetic backgrounds, highlighting the importance of cellular context to genotoxic stress responses; this also highlights the need for informatics approaches to discover and prioritize hypotheses regarding the importance of particular cellular factors. The aim of this review is to demonstrate the utility of combining an informatics approach with functional genomics in the study of stress responses.

Animals↗
PubMed1998

Sequence motifs in the integrin alpha 4 cytoplasmic tail required for regulation of in vivo expansion of murine lymphoma cells.

The binding of integrins to cognate ligands is tightly controlled by intracellular signals. Conversely, integrin occupancy generates biochemical signals inside the cell. The present study examined whether concepts of integrin function established by in vitro analysis apply to regulation of receptor function in complex biologic settings in vivo using a mouse model of tumor metastasis. Integrin alpha 4 subunits were truncated at amino acid Gln1014 (A4-1014), preserving the conserved GFFKR motif, and at position Glu1021 (A4-1021). In vitro adhesion assays revealed that cytoplasmic tail truncations did not affect constitutive ligand binding of alpha 4 integrins, while agonist-induced adhesion was abolished by the alpha 4-1014, but not by the alpha 4-1021, mutation. Inducible ligand binding of alpha 4 integrins was dependent on cytoskeletal function, whereas constitutive adhesion was not. In vivo metastasis formation assays demonstrated that expansion of murine T lymphoma cells in spleen is strongly inhibited by the wild-type alpha 4 subunit and the alpha 4-1021 mutant. In contrast, the in vivo phenotype of alpha 4 integrin expression in lymphoma cells was completely abrogated by the alpha 4-1014 mutation. Cross-linking of alpha 4 integrins in vitro inhibited proliferation and induced apoptosis of LB cells expressing wild-type alpha 4 subunits or the alpha 4-1021 mutant, but not of LB-A4-1014 cells. In summary, these results demonstrate that sequence motifs regulating cytoskeleton-dependent alpha 4 integrin activation in vitro are essential for the control of LB lymphoma cell expansion both in vitro and in vivo.

Amino Acid Sequence↗
PubMed1998

Gene expression profiling of alveolar rhabdomyosarcoma with cDNA microarrays.

Several forms of human sarcoma, lymphoma, and leukemia are characterized by somatically acquired chromosome translocations that result in fusion genes that encode chimeric transcription factors with oncogenic properties. We have used cDNA microarrays containing 1238 cDNAs to investigate the gene expression profile of a group of seven alveolar rhabdomyosarcoma (ARMS) cell lines characterized by the presence of the PAX3-FKHR fusion gene. Using the method of multidimensional scaling to represent the relationships among the cell lines in two-dimensional Euclidean space, we determined that ARMS cells show a consistent pattern of gene expression, which allows the cells to be clustered together. By searching across the seven ARMS cell lines, we found that 37 of 1238 genes were most consistently expressed in ARMS relative to a reference cell line. Only three of these genes have been previously reported to be expressed in ARMS. Among these 37 were genes related to both primary (PAX3-FKHR) and secondary (CDK4) genetic alterations in ARMS. These results in ARMS demonstrate the potential of cDNA microarray technology to elucidate tumor-specific gene expression profiles in human cancers.

Chromosomes, Human, Pair 13↗
PubMed1998

The human WD repeat protein WAIT-1 specifically interacts with the cytoplasmic tails of beta7-integrins.

Integrins of the beta7 subfamily, alpha4 beta7 and alphaE beta7, contribute to lymphocyte homing and to the development of protective or autoreactive immune responses at mucosal sites. The beta subunits of integrins are considered important for regulation of stimulated cell adhesion and adhesion-dependent signal transduction. Using a yeast interaction trap screen, a human WD repeat protein, termed WAIT-1, was isolated that interacts with the integrin beta7 cytoplasmic tail and is homologous to mouse EED and Drosophila ESC proteins. WAIT-1 also binds to the cytoplasmic domains of alpha4 and alphaE but not to those of integrin beta1, beta2, and alphaL subunits. Association of WAIT-1 and beta7-integrin was confirmed by coprecipitation from transiently transfected 293 cells. The binding site for WAIT-1 was mapped to a short membrane-proximal region of the beta7 cytoplasmic tail with Tyr-735 being of critical importance. Northern blot analysis revealed multiple WAIT-1-related transcripts with differential expression in circulating leukocytes, tissue-resident cells of diverse origin, and lymphoid malignancies. These results suggest that WAIT-1, together with the recently identified RACK1, may define a novel subfamily of WD repeat proteins that interact with distinct subsets of integrin cytoplasmic tails and may act as specific regulators of integrin function.

Amino Acid Sequence↗
PubMed1998

Comparative genomic hybridization: a comparison with molecular and cytogenetic analysis.

Comparative genomic hybridization (CGH) is a powerful technique for detecting copy number changes throughout the genome. We describe the development of a versatile image analysis program for CGH studies. Several methods for the production of metaphases which give optimum hybridization signals have also been assessed. CGH analysis was performed on DNA samples from several different and clinically relevant specimens: amniotic fluid cells trisomic for a single chromosome, lymphoblastoid cell lines with abnormalities involving single chromosome bands, malignant cell lines and biopsy material from primary ovarian carcinomas. The results were compared with those derived from G-banding, chromosome painting, and molecular genetic techniques. Our data demonstrate that CGH was able to detect a wide range of quantitative genetic alterations including duplication or deletion of single chromosome bands. CGH analysis also indicated the presence of genetic abnormalities that were not detected by other cytogenetic or molecular approaches. Moreover, our CGH methodology allowed the ready comparison of CGH results from different tumors, a process which greatly facilitated identification of shared genetic changes.

Amniotic Fluid↗
PubMed1998

alpha 4 integrins and tumor metastasis.

Taken together, alpha 4 integrins may influence metastatic process at various stages (Fig. 1). The detachment of tumor cells from the primary tumor and the invasion of the surrounding tissue represent the onset of tumor metastasis. There is good experimental evidence that at the primary tumor site expression of alpha 4 integrins inhibits the ability of melanoma cells to break loose. This could be achieved either by strengthening of homotypic adhesion to adjacent tumor cells or by down regulation of matrix metalloproteases that are required for tumor cell migration through the extracellular matrix. After entering the blood circulation, alpha 4 integrins on tumor cells derived from melanomas, sarcomas or lymphomas rather promote than inhibit accumulation of disseminated cells in distant organs. The positive effects of alpha 4 integrins at this stage of metastasis formation appear to depend on alpha 4 integrin interactions with ligands expressed on the surface of endothelial cells. While VCAM-1 is expressed on endothelial cells exposed to inflammatory cytokines, MAdCAM-1 is constitutively expressed on mucosal endothelium. In addition, it is conceivable that tumor cell aggregates trapped in the microcirculation may trigger local inflammatory reactions that result in VCAM-1 up-regulation. Tumor cell-bound alpha 4 integrins may strengthen adhesion to endothelium and promote trans-endothelial migration (HAUZENBERGER et al. 1997; MEERSCHAERT and FURIE 1994). Successful formation of new tumor colonies in distant organs is the final step in the metastatic cascade. Interestingly, alpha 4 integrin dependent mechanisms may either promote or inhibit this process. Thus, it was observed that alpha 4 integrins may direct cancer cells like CHO and lymphoma cells to organ compartments, where ligands for alpha 4 integrins are expressed (e.g., bone marrow). Depending on the tumor type this event may result in enhanced metastasis formation. However, as was documented for murine lymphoma cells alpha 4 integrins may also inhibit tumor cell growth either by inducing apoptosis or by reducing the proliferation rate. Based on numerous studies on human cancers and experimental tumor models, alpha 4 integrins may represent attractive target molecules for therapeutic manipulation of tumor cell behavior. To this end, however, it will be of great importance to precisely define the molecular basis for the adverse effects of alpha 4 integrins on metastasis formation.

Animals↗
PubMed1998

Separate and variably shaped chromosome arm domains are disclosed by chromosome arm painting in human cell nuclei.

Fluorescence in situ hybridization (FISH) with microdissection probes from human chromosomes 3 and 6 was applied to visualize arm and subregional band domains in human amniotic fluid cell nuclei. Confocal laser scanning microscopy and quantitative three-dimensional image analysis showed a pronounced variability of p- and q-arm domain arrangements and shapes. Apparent intermingling of neighbouring arm domains was limited to the domain surface. Three-dimensional distance measurements with pter and qter probes supported a high variability of chromosome territory folding.

Amniotic Fluid↗
PubMed1997

The MEN frangible: study of a new bullet in gelatin. Metallwerk Elisenhütte Nassau.

Experimental gunshots were made with 9 x 19 mm (9-mm Luger) Frangible ammunition using skin, gelatin, and bones as targets. Direct gunshots penetrated 50 cm into gelatin blocks without mushrooming or fragmentation. Skull bone was easily perforated. Head models were penetrated by the bullets, which broke down to myriad fragments within or outside the head. Gunshots through windshields and steel plates led to dangerous bullet fragmentation with a high risk of injury. Gunshots perforating tibias produced comminuted fractures. In all soft tissues, except the direct gunshots into gelatin, high-resolution radiography revealed many metallic fragments along the bullet path. Clinical radiography can show the larger copper fragments, but the explosion-like distribution of metallic foreign bodies in soft tissue is a serious surgical problem.

Adult↗