Biomedical subjects

Jan Buer

Publications and source records attributed to Jan Buer.

At least 55 records · Page 3Linked to original sources

PubMed2004

An extended transcriptional regulatory network of Escherichia coli and analysis of its hierarchical structure and network motifs.

Recent studies of genome-wide transcriptional regulatory network (TRN) revealed several intriguing structural and dynamic features of gene expression at a system level. Unfortunately, the network under study is often far from complete. A critical question is thus how much the network is incomplete and to what extent this would affect the results of analysis. Here we compare the Escherichia coli TRN built by Shen-Orr et al. (Nature Genet., 31, 64-68) with two TRNs reconstructed from RegulonDB and Ecocyc respectively and present an extended E.coli TRN by integrating information from these databases and literature. The scale of the extended TRN is about twice as large as the previous ones. The new network preserves the multi-layer hierarchical structure which we recently reported but has more layers. More global regulators are inferred. While the feed forward loop (FFL) is confirmed to be highly representative in the network, the distribution of the different types of FFLs is different from that based on the incomplete network. In contrast to the notion of motif aggregation and formation of homologous motif clusters, we found that most FFLs interact and form a giant motif cluster. Furthermore, we show that only a small portion of the genes is solely regulated by only one FFL. Many genes are regulated by two or more interacting FFLs or other more complicated network motifs together with transcriptional factors not belonging to any network motifs, thereby forming complex regulatory circuits. Overall, the extended TRN represents a more solid basis for structural and functional analysis of genome-wide gene regulation in E.coli.

Databases, Genetic↗

PubMed2004

Hierarchical structure and modules in the Escherichia coli transcriptional regulatory network revealed by a new top-down approach.

BACKGROUND: Cellular functions are coordinately carried out by groups of genes forming functional modules. Identifying such modules in the transcriptional regulatory network (TRN) of organisms is important for understanding the structure and function of these fundamental cellular networks and essential for the emerging modular biology. So far, the global connectivity structure of TRN has not been well studied and consequently not applied for the identification of functional modules. Moreover, network motifs such as feed forward loop are recently proposed to be basic building blocks of TRN. However, their relationship to functional modules is not clear. RESULTS: In this work we proposed a top-down approach to identify modules in the TRN of E. coli. By studying the global connectivity structure of the regulatory network, we first revealed a five-layer hierarchical structure in which all the regulatory relationships are downward. Based on this regulatory hierarchy, we developed a new method to decompose the regulatory network into functional modules and to identify global regulators governing multiple modules. As a result, 10 global regulators and 39 modules were identified and shown to have well defined functions. We then investigated the distribution and composition of the two basic network motifs (feed forward loop and bi-fan motif) in the hierarchical structure of TRN. We found that most of these network motifs include global regulators, indicating that these motifs are not basic building blocks of modules since modules should not contain global regulators. CONCLUSION: The transcriptional regulatory network of E. coli possesses a multi-layer hierarchical modular structure without feedback regulation at transcription level. This hierarchical structure builds the basis for a new and simple decomposition method which is suitable for the identification of functional modules and global regulators in the transcriptional regulatory network of E. coli. Analysis of the distribution of feed forward loops and bi-fan motifs in the hierarchical structure suggests that these network motifs are not elementary building blocks of functional modules in the transcriptional regulatory network of E. coli.

Amino Acid Motifs↗

PubMed2004

Gene expression profile at the G1/S transition of liver regeneration after partial hepatectomy in mice.

Liver is a quiescent organ with >90% of the cells present in the G(0) stage of the cell cycle. However, adult hepatocytes have enormous ability to proliferate in response to liver injury. After 70% liver resection hepatocytes enter the cell cycle in a highly synchronized manner and undergo 1 to 2 rounds of cell division to restore the lost organ mass, thus, representing one of the most reliable model systems to study cell cycle progression in vivo. Using high density oligonucleotide micro-array we analyzed the expression patterns of genes at the G(1)/S transition of liver regeneration in comparison to quiescent livers. The G(1)/S boundary was identified by in vivo BrdU pulse labeling and we observed 199 genes/ESTs which were either up/down regulated at this time point. These differentially regulated genes have a wide range of functions including transcriptional regulation, signal transduction, cell cycle regulation, chromatin reorganization, protein targeting, metabolism, transport, surface receptors, circadian rhythms, xenobiotic metabolism, inflammation and acute phase response. The functions of most of the genes identified in this screen are not known in the process of liver regeneration and cell cycle control at G(1)/S transition.

Acute-Phase Proteins↗

PubMed2004

Skp2-dependent degradation of p27kip1 is essential for cell cycle progression.

The activity of the SCF(skp2) E3 ligase is required for the proteolytic turnover of several proteins involved in cell cycle control and transcriptional regulation. Loss of skp2 in the mouse leads to a complex phenotype including changes in cell size and DNA content as well as severe proliferation defects. Here we show that the loss of a single skp2 substrate, namely, the cyclin kinase inhibitor p27kip1, reverts the phenotype of skp2 knockout hepatocytes to normal. By comparing the kinetics of p27 turnover and cell cycle progression in skp2 knockout and p27T187A knock-in mice, we define a short period in G1 in which p27 is able to block the cell cycle after the exit from quiescence. Loss of p27 turnover during this period prevents mitotic division and instead leads to compensatory cell growth.

Animals↗

PubMed2004

CD4 T Lymphocyte-mediated lung disease: steady state between pathological and tolerogenic immune reactions.

Although considerable evidence indicates a role for CD4(+) T lymphocytes (T cells) in airway inflammation, little data exist regarding the mechanisms underlying the induction and regulation of CD4(+) T cell reactivity to lung-specific antigens. To dissect the immunologic and molecular mechanisms of CD4(+) T cell dysregulation, reactivity to a self-antigen expressed in the lung of mice bearing a major histocompatibility complex class-II-restricted T cell receptor specific for this antigen was studied. Transgenic mice developed a progressive interstitial pneumonitis characterized by massive lymphocytic and plasmacytic infiltration of interalveolar septa, a clinical picture closely resembling some of the interstitial lung diseases. Pulmonary inflammation reached a plateau state in older mice with prominent formation of lymphoid follicles but reduced interstitial infiltration. Extensive immunologic characterization of self-reactive CD4(+) T cells isolated from the inflamed lung suggested the induction of regulatory T cells in the site of inflammation. Moreover, inflammation was accompanied by broad changes in the gene expression pattern toward a profile partially resembling that of activated, but strikingly, also that of regulatory CD4(+) T cells. Together our data provide important insights into functional and molecular alterations being associated with the induction and/or regulation of T cell-mediated pulmonary inflammation.

Animals↗

PubMed2004

Developmental stage, phenotype, and migration distinguish naive- and effector/memory-like CD4+ regulatory T cells.

Regulatory T cells (Tregs) fulfill a central role in immune regulation. We reported previously that the integrin alphaEbeta7 discriminates distinct subsets of murine CD4+ regulatory T cells. Use of this marker has now helped to unravel a fundamental dichotomy among regulatory T cells. alphaE-CD25+ cells expressed L-selectin and CCR7, enabling recirculation through lymphoid tissues. In contrast, alphaE -positive subsets (CD25+ and CD25-) displayed an effector/memory phenotype expressing high levels of E/P-selectin-binding ligands, multiple adhesion molecules as well as receptors for inflammatory chemokines, allowing efficient migration into inflamed sites. Accordingly, alphaE -expressing cells were found to be the most potent suppressors of inflammatory processes in disease models such as antigen-induced arthritis.

Animals↗

PubMed2004

Neuropilin-1: a surface marker of regulatory T cells.

CD4+CD25+ regulatory T cells (Treg cells) control immune responsiveness to a large variety of antigens. The isolation and therapeutic manipulation of Treg cells requires the use of reliable surface receptors that are selectively up-regulated in Treg cells. On the basis of global gene expression studies, we identified neuropilin-1 (Nrp1) as a specific surface marker for CD4+CD25+ Treg cells. Nrp1, a receptor involved in axon guidance, angiogenesis, and the activation of T cells, is constitutively expressed on the surface of CD4+CD25+ Treg cells independently of their activation status. In contrast, Nrp1 expression is down-regulated in naive CD4+CD25- T cells after TCR stimulation. Furthermore, CD4+Nrp1(high) T cells express high levels of Foxp3 and suppress CD4+CD25- T cells. Thus, Nrp1 constitutes a useful surface marker to distinguish Treg cells from both naive and recently activated CD4+CD25+ non-regulatory T cells.

Animals↗

PubMed2004

Resistance and susceptibility to tuberculosis analysed at the transcriptome level: lessons from mouse macrophages.

Gene expression patterns associated with resistance and susceptibility to tuberculosis (TB) were investigated at the macrophage level in the well-defined mouse model of infection. Oligonucleotide microarrays were used to analyse the regulation of gene expression in murine bone marrow-derived macrophages infected with Mycobacterium tuberculosis. Four mouse strains, known to differ in terms of growth permissiveness for M. tuberculosis in infected tissues, in the development of pulmonary pathology, and in the rate of premature death due to tuberculosis, were compared: C57BL/6 and BALB/c representing resistant, DBA/2 and CBA/J representing susceptible mouse strains. Genes (55) were regulated more than two-fold in macrophages of all strains investigated following M. tuberculosis infection. Importantly, 18 genes were commonly regulated only in macrophages of the two resistant strains upon infection, and 102 genes were commonly regulated exclusively in macrophages of the two susceptible strains. Using this approach, we have therefore identified more than 100 genes potentially associated with resistance and susceptibility, respectively, to TB at the macrophage level. A tentative interpretation of our microarray data suggests that macrophages from susceptible mice predominantly stimulate the recruitment of cells that contribute disproportionately to tissue damage rather than to microbial elimination. In conclusion, microarray gene chips are useful tools for generating new hypotheses about resistance and susceptibility to TB, and the mouse model can now be used to subject candidate genes identified by this approach to further functional analyses.

Animals↗

PubMed2004

Application of retinoids in the treatment of renal cell carcinoma--a futile effort?

The therapeutic benefit of adding retinoids such as all-trans retinoic acid (RA), 9-cis-RA or 13-cis-RA to established single-agent or combination immuno/chemotherapy regimens for the treatment of metastatic renal cell carcinoma (RCC) has been extensively investigated during the last decade. However, at present results are contradictory and their application controversial. Moreover, recent studies indicated a significantly higher incidence of toxic side-effects in patients treated with retinoids in addition to established bio/chemotherapy. This Commentary summarizes preclinical and clinical trials investigating efficacy and toxicity of retinoids in the treatment of RCC.

Animals↗

PubMed2004

The Mycoplasma-derived macrophage-activating 2-kilodalton lipopeptide triggers global immune activation on nasal mucosa-associated lymphoid tissues.

A better knowledge on how immune responses are initiated in mucosal tissues would facilitate the design of new mucosal vaccines, as well as improve our understanding on host defense against infection. We investigated the mechanisms of adjuvanticity of the Mycoplasma-derived macrophage-activating 2-kDa lipopeptide (MALP-2), which binds to the heterodimer formed by the Toll-like receptors 2 and 6 (TLR2 and -6), at the level of the murine nasal mucosa-associated lymphoid tissues (NALT). TLR2 expression analysis demonstrated that several cell types from the nasal cavity were able to overexpress this receptor, either constitutively (such as B cells) or after stimulation (i.e., T cells). MALP-2 stimulated a strong B-cell activation. In addition, the antigen presentation capacity of dendritic cells was improved after in vivo loading with antigen in the presence of MALP-2. We also observed an up-regulated expression of activation markers and adhesion molecules on T cells, suggesting that they have enhanced responsiveness and interaction potential. Quantitative reverse transcription-PCR analysis showed that MALP-2 administration resulted in the stimulation of a proinflammatory cascade. We observed an early up-regulated expression of IP-10, MCP-1, MCP-3, MIP-1alpha, MIP-2, and CCR-2 which was reversed within 36 h. The obtained results demonstrated that MALP-2 creates a reversible local microenvironment which promotes effective priming of T and B cells in the NALT.

Adjuvants, Immunologic↗

PubMed2004

Expression analysis of a highly adherent and cytotoxic small colony variant of Pseudomonas aeruginosa isolated from a lung of a patient with cystic fibrosis.

The heterogeneous environment of the lung of the cystic fibrosis (CF) patient gives rise to Pseudomonas aeruginosa small colony variants (SCVs) with increased antibiotic resistance, autoaggregative growth behavior, and an enhanced ability to form biofilms. In this study, oligonucleotide DNA microarrays were used to perform a genome-wide expression study of autoaggregative and highly adherent P. aeruginosa SCV 20265 isolated from a CF patient's lung in comparison with its clonal wild type and a revertant generated in vitro from the SCV population. Most strikingly, SCV 20265 showed a pronounced upregulation of the type III protein secretion system (TTSS) and the respective effector proteins. This differential expression was shown to be biologically meaningful, as SCV 20265 and other hyperpiliated and autoaggregative SCVs with increased TTSS expression were significantly more cytotoxic for macrophages in vitro and were more virulent in a mouse model of respiratory tract infection than the wild type. The observed cytotoxicity and virulence of SCV 20265 required exsA, an important transcriptional activator of the TTSS. Thus, the prevailing assumption that P. aeruginosa is subject to selection towards reduced cytotoxicity and attenuated virulence during chronic CF lung infection might not apply to all clonal variants.

Animals↗

PubMed2004

Long-term anaerobic survival of the opportunistic pathogen Pseudomonas aeruginosa via pyruvate fermentation.

Denitrification and arginine fermentation are central metabolic processes performed by the opportunistic pathogen Pseudomonas aeruginosa during biofilm formation and infection of lungs of patients with cystic fibrosis. Genome-wide searches for additional components of the anaerobic metabolism identified potential genes for pyruvate-metabolizing NADH-dependent lactate dehydrogenase (ldhA), phosphotransacetylase (pta), and acetate kinase (ackA). While pyruvate fermentation alone does not sustain significant anaerobic growth of P. aeruginosa, it provides the bacterium with the metabolic capacity for long-term survival of up to 18 days. Detected conversion of pyruvate to lactate and acetate is dependent on the presence of intact ldhA and ackA-pta loci, respectively. DNA microarray studies in combination with reporter gene fusion analysis and enzyme activity measurements demonstrated the anr- and ihfA-dependent anaerobic induction of the ackA-pta promoter. Potential Anr and integration host factor binding sites were localized. Pyruvate-dependent anaerobic long-term survival was found to be significantly reduced in anr and ihfA mutants. No obvious ldhA regulation by oxygen tension was observed. Pyruvate fermentation is pH dependent. Nitrate respiration abolished pyruvate fermentation, while arginine fermentation occurs independently of pyruvate utilization.

Acetate Kinase↗

PubMed2004

Evidence for polyadenylated mRNA in Pseudomonas aeruginosa.

In this paper, we report the synthesis of Pseudomonas aeruginosa cDNA in the presence of oligo(dT) primers. Hybridization of oligonucleotide DNA microarrays indicates that under the experimental conditions used, at least 43.7% of the expressed genes from P. aeruginosa PAO1, representing many different functional classes, can be detected by using oligo(dT)-primed cDNAs.

Bacterial Proteins↗

PubMed2003

Molecular and functional analysis of Shiga toxin-induced response patterns in human vascular endothelial cells.

Enterohemorrhagic Escherichia coli (EHEC) is the major cause of hemolyticuremic syndrome (HUS) characterized by microangiopathic hemolytic anemia, thrombocytopenia, and acute renal failure. EHEC produces one or more Shiga toxins (Stx1 and Stx2), and it was assumed that Stx's only relevant biologic activity was cell destruction through inhibition of protein synthesis. However, recent data indicate that in vivo the cytokine milieu may determine whether endothelial cells survive or undergo apoptosis/necrosis when exposed to Stxs. In this study, we analyzed the genome-wide expression patterns of human endothelial cells stimulated with subinhibitory concentrations of Stxs in order to characterize the genomic expression program involved in the vascular pathology of HUS. We found that Stxs elicited few, but reproducible, changes in gene expression. The majority of genes reported in this study encodes for chemokines and cytokines, which might contribute to the multifaceted inflammatory response of host endothelial cells observed in patients suffering from EHEC disease. In addition, our data provide for the first time molecular insights into the epidemiologically well-established higher pathogenicity of Stx2 over Stx1.

Apoptosis↗

PubMed2003

G-CSF as immune regulator in T cells expressing the G-CSF receptor: implications for transplantation and autoimmune diseases.

Results from experimental models, in vitro studies, and clinical data indicate that granulocyte colony-stimulating factor (G-CSF) stimulation alters T-cell function and induces Th2 immune responses. The immune modulatory effect of G-CSF on T cells results in an unexpected low incidence of acute graft-versus-host disease in peripheral stem cell transplantation. However, the underlying mechanism for the reduced reactivity and/or alloreactivity of T cells upon G-CSF treatment is still unknown. In contrast to the general belief that G-CSF acts exclusively on T cells via monocytes and dendritic cells, our results clearly show the expression of the G-CSF receptor in class I- and II- restricted T cells at the single-cell level both in vivo and in vitro. Kinetic studies demonstrate the induction and functional activity of the G-CSF receptor in T cells upon G-CSF exposure. Expression profiling of T cells from G-CSF-treated stem cell donors allowed identification of several immune modulatory genes, which are regulated upon G-CSF administration in vivo (eg, LFA1-alpha, ISGF3-gamma) and that are likely responsible for the reduced reactivity and/or alloreactivity. Most importantly, the induction of GATA-3, the master transcription factor for a Th2 immune response, could be demonstrated in T cells upon G-CSF treatment in vivo accompanied by an increase of spontaneous interleukin-4 secretion. Hence, G-CSF is a strong immune regulator of T cells and a promising therapeutic tool in acute graft-versus-host disease as well as in conditions associated with Th1/Th2 imbalance, such as bone marrow failure syndromes and autoimmune diseases.

Autoimmune Diseases↗

PubMed2003

GATA-3 promotes maturation, IFN-gamma production, and liver-specific homing of NK cells.

The GATA-3 transcription factor has a determinant role in T cell specification and is an essential mediator of T helper 2-type polarized immune responses. While both committed NK precursors and mature NK cells express GATA-3, a role of this transcription factor in murine NK cell differentiation is not known. We found that NK cells, in contrast to T cells, can be generated in the absence of GATA-3. However, while GATA-3 antagonizes IFN-gamma production in differentiating T cells, GATA-3-deficient NK cells paradoxically produced less IFN-gamma compared to control NK cells and failed to provide early protection in vivo against infection with Listeria monocytogenes. Surprisingly, GATA-3 was essential for NK cell homing to the liver. Our results suggest that GATA-3 promotes NK cell maturation and acts in this lineage to specify distinct effector phenotypes.

Animals↗

PubMed2003

Mice, microbes and models of infection.

We urgently need animal models to study infectious disease. Mice are susceptible to a similar range of microbial infections as humans. Marked differences between inbred strains of mice in their response to pathogen infection can be exploited to analyse the genetic basis of infections. In addition, the genetic tools that are available in the laboratory mouse, and new techniques to monitor the expression of bacterial genes in vivo, make it the principal experimental animal model for studying mechanisms of infection and immunity.

Animals↗

PubMed2003

Application of real-time reverse transcriptase-polymerase chain reaction in urological oncology.

PURPOSE: During the last decade numerous different reverse transcriptase-polymerase chain reaction (RT-PCR) techniques have been described. However, the lack of highly sensitive, quantitative and reliable methodology has been responsible for its limited use in modern urology. Early semiquantitative RT-PCR techniques often proved not to produce consistent results and have a high failure rate due to complicated working models. In this article we provide a comprehensive and intelligible description of real-time PCR technology, which is a novel quantitative methodology to analyze gene expression. In addition, we report the first preclinical and clinical applications in molecular urology. MATERIALS AND METHODS: The current literature was reviewed in regard to different current real-time RT-PCR protocols and their use in modern urological oncology. RESULTS: Real-time RT-PCR is a reliable, rapid and relatively inexpensive technique that can be easily adapted for standardized preclinical and clinical applications at different centers. Its sensitivity equals at least that of conventional RT-PCR and the option of exact quantification of gene expressions allows proper differentiation among high, low and illegitimate RNA transcription. It eliminates post-PCR processing of PCR products, thereby, increasing throughput and decreasing the chance of carryover contamination. CONCLUSIONS: Although the application of real-time RT-PCR has gained wide acceptance in urological research, its routine clinical use is still in its infancy. However, due to its high sensitivity and exact quantitation real-time RT-PCR may be the method of choice for modern preclinical and clinical studies in the future.

Humans↗