PubMed Health⌕ Search

Biomedical subjects

Dieter C Gruenert

Publications and source records attributed to Dieter C Gruenert.

13 recordsLinked to original sources
PubMed2006

Promoter hypomethylation of Toll-like receptor-2 gene is associated with increased proinflammatory response toward bacterial peptidoglycan in cystic fibrosis bronchial epithelial cells.

Cystic fibrosis (CF) is the most common lethal inherited disorder caused by mutation in the gene encoding CF transmembrane regulator (CFTR). The clinical course of CF is characterized by recurrent pulmonary infections and chronic inflammation. Here, we show that toll-like receptor-2 (TLR2) expression and response were strongly enhanced in the human CF bronchial epithelial cell line, CFBE41o-. Treatment of the cells with 5-azacytidine decreased the promoter methylation within TLR2 proximal promoter and increased endogenous expression of TLR2 in non-CF 16HBE14o- cells, suggesting that TLR2 expression is epigenetically regulated by CpG methylation. Moreover, bisulfite sequence analysis revealed that TLR2 promoters were highly demethylated in CFBE41o- cells, implying that decreased methylation of the TLR2 promoter is responsible for CF-related up-regulation of TLR2. Finally, stable expression of WT-CFTR in CFBE41o- cells (CFBE41o-/WT-CFTR cells) reduced TLR2 expression and the response to its ligand peptidoglycan (PGN), implying a causal relationship between CFTR dysfunction and TLR2 up-regulation. Consistent with reduced expression of TLR2 in CFBE41o-/WT-CFTR cells, CpG methylation was increased in CFBE41o-/WT-CFTR cells. Taken together, our results demonstrate that TLR2 expression is epigenetically up-regulated in CF bronchial epithelial cells and suggest that TLR2 overexpression or prolonged activation of TLR2 signaling might be critical in CF pathogenesis.

Azacitidine↗
PubMed2006

NDPK-A (but not NDPK-B) and AMPK alpha1 (but not AMPK alpha2) bind the cystic fibrosis transmembrane conductance regulator in epithelial cell membranes.

Cystic fibrosis (CF) results from mutations within the cystic fibrosis transmembrane-conductance regulator (CFTR) protein. The AMP-activated protein kinase (AMPK) is a heterotrimer composed of different isoforms of the alphabetagamma subunits, where the alpha1 catalytic subunit binds CFTR. Nucleoside diphosphate kinase (NDPK, NM23/awd) converts nucleoside diphosphates to nucleoside triphosphates but also acts as a protein kinase. We recently showed that AMPK alpha1 binds NDPK-A in lung epithelial cytosol. Here we report that in the plasma membrane of human airway epithelial cells, NDPK-A and AMPK alpha1 associate with the plasma membrane via CFTR. We show that the regulatory domain of CFTR binds NDPK-A whereas AMPK gamma1 or gamma2 bind the first nucleotide binding domain (NBD1) and AMPK alpha1 binds the second (NBD2) of CFTR. We also show that NDPK-A specifically binds AMPK alpha1 and AMPK gamma2 subunits, thereby specifying the isozyme of AMPK heterotrimer that associates with CFTR at the membrane. Thus, the combined data provide novel insight into the subunit composition of the epithelial CFTR/AMPK/NDPK complex, such that: CFTR interacts specifically with AMPK alpha1, gamma2 and NDPK-A and not NDPK-B or AMPK gamma1.

AMP-Activated Protein Kinases↗
PubMed2006

Small fragment homologous replacement-mediated modification of genomic beta-globin sequences in human hematopoietic stem/progenitor cells.

An ultimate goal of gene therapy is the development of a means to correct mutant genomic sequences in the cells that give rise to pathology. A number of oligonucleotide-based gene-targeting strategies have been developed to achieve this goal. One approach, small fragment homologous replacement (SFHR), has previously demonstrated disease-specific genotypic and phenotypic modification after introduction of small DNA fragments (SDFs) into somatic cells. To validate whether the gene responsible for sickle cell anemia (beta-globin) can be modified by SFHR, a series of studies were undertaken to introduce sickle globin sequences at the appropriate locus of human hematopoietic stem/progenitor cells (HSPCs). The characteristic A two head right arrow T transversion in codon 6 of the beta-globin gene was indicated by restriction fragment length polymorphic (RFLP) analysis of polymerase chain reaction (PCR) products generated by amplification of DNA and RNA. At the time of harvest, it was determined that the cells generally contained </=1 fragment per cell. Control studies mixing genomic DNA from nontransfected cells with varying amounts of the targeting SDFs did not indicate any PCR amplification artifacts due to the presence of residual SDF during amplification. RNA was analyzed after DNase treatment, thus eliminating the potential for SDF contamination. Stable SFHRmediated conversion of normal (beta (A)) to sickle (beta (S)) globin was detected at frequencies up to 13% in cells harvested 30-45 days posttransfection. The minimum conversion efficiency ranged from 0.2 to 3%, assuming modification of at least one cell per experiment showing conversion. Conversion of sickle (beta (S)) to normal (beta (A)) globin was detected up to 10 days posttransfection in lymphoblastoid cells from a sickle cell patient. These studies suggest that SFHR may be effective for ex vivo gene therapy of sickle cells in a patient's HSPCs before autologous transplantation.

Cells, Cultured↗
PubMed2006

Gel purification of genomic DNA removes contaminating small DNA fragments interfering with polymerase chain reaction analysis of small fragment homologous replacement.

Oligonucleotides can mediate sequence-specific gene modification that results in the correction and/or alteration of genomic DNA. There is evidence to suggest that the polymerase chain reaction (PCR)-based analytical methods usually used to analyze oligonucleotide-mediated modification can generate artifacts. To investigate the conditions under which a PCR artifact can be generated and eliminated when analyzing small fragment homologous replacement (SHFR)-mediated modification, cells homozygous for the DeltaF508 mutation (CFBE41o-) were mixed with small DNA fragments (SDFs) containing the wild-type CFTR (wt-CFTR) sequence. An artifact could be generated after wild-type allele-specific PCR (wtAS-PCR) if the genomic DNA was not gel purified. Without gel purification, the amount of SDF/cell required to generate the artifact was dependent to the AS primer pairs used. When the genomic DNA was gel purified, no artifact could be detected with any of the wtAS-PCR primers whether the SDF was mixed with the cells or transfected into the cells. Furthermore, treatment of cellular mRNA with DNase was sufficient to eliminate potential artifacts in the reverse transcriptase-polymerase chain reaction (RT-PCR) analysis. Thus, it is critical to gel purify genomic DNA and DNase treat mRNA when analyzing SFHR-mediated modification by PCR.

Base Sequence↗
PubMed2006

Modification of the pig CFTR gene mediated by small fragment homologous replacement.

The generation of a pig model of cystic fibrosis (CF) is a multistep process. Initial steps in this process involved the design and cloning of a small DNA fragment (SDF) or large oligodeoxynucleotide (LODN) that contains the F508del mutation and a silent restriction fragment length polymorphism causing mutation. This SDF/LODN was transfected into wild-type (WT) pig fetal fibroblast with the intention of modifying the pig genomic DNA by small fragment homologous replacement (SFHR). The targeted deletion (F508del) was detected in a subpopulation of transfected cells by allele-specific polymerase chain reaction (AS-PCR).

Animals↗
PubMed2005

Characterization of novel airway submucosal gland cell models for cystic fibrosis studies.

Cultured airway epithelial cells are widely used in cystic fibrosis (CF) research as in vitro models that mimic the in vivo manifestations of the disease and help to define a specific cellular phenotype. Recently, a number of in vitro studies have used an airway adenocarcinoma cell line, Calu-3 that expresses submucosal gland cell features and significant levels of the wild-type CFTR mRNA and protein. We further characterized previously described CF tracheobronchial gland cell lines, CFSMEo- and 6CFSMEo- and determined that these cell lines are compound heterozygotes for the F508del and Q2X mutations, produce vestigial amounts of CFTR mRNA, and do not express detectable CFTR protein. Electrophysiologically, both cell lines are characteristically CF in that they lack cAMP-induced Cl- currents. In this study the cell lines are evaluated in the context of their role as the CF correlate to the Calu-3 cells. Together these cell systems provide defined culture systems to study the biology and pathology of CF. These airway epithelial cell lines may also be a useful negative protein control for numerous studies involving gene therapy by cDNA complementation or gene targeting.

Base Sequence↗
PubMed2003

Construction and purification of pSABR 01, a pUC19-derived vector optimized for cloning full-length cDNA.

A novel pUC19-derived vector, pSABR 01, was constructed by sub-cloning a fragment of the pSPORT1 polylinker into PUC19. The insertion of the polylinker generated two inactivating mutations in the LacZ open reading frame. These were then repaired by a PCR-based Site Directed Mutagenesis strategy. The pSABR 01 plasmid has four sites that are recognized by 'rare-cutter' restriction endonucleases that will optimize the cloning of full-length cDNA and five dual restriction sites that increase the versatility of subcloning the inserted cDNA. Protocols were also defined for purification of pSABR 01 from residual pSPORT1, following pSABR 01 construction, and from another contaminating plasmid.

Cloning, Molecular↗
PubMed2003

Sequence-specific modification of genomic DNA by small DNA fragments.

Small DNA fragments have been used to modify endogenous genomic DNA in both human and mouse cells. This strategy for sequence-specific modification or genomic editing, known as small-fragment homologous replacement (SFHR), has yet to be characterized in terms of its underlying mechanisms. Genotypic and phenotypic analyses following SFHR have shown specific modification of disease-causing genetic loci associated with cystic fibrosis, beta-thalassemia, and Duchenne muscular dystrophy, suggesting that SFHR has potential as a therapeutic modality for the treatment of monogenic inherited disease.

Anemia, Sickle Cell↗
PubMed2002

In vitro correction of cystic fibrosis epithelial cell lines by small fragment homologous replacement (SFHR) technique.

BACKGROUND: SFHR (small fragment homologous replacement)-mediated targeting is a process that has been used to correct specific mutations in mammalian cells. This process involves both chemical and cellular factors that are not yet defined. To evaluate potential of this technique for gene therapy it is necessary to characterize gene transfer efficacy in terms of the transfection vehicle, the genetic target, and the cellular processing of the DNA and DNA-vehicle complex. METHODS: In this study, small fragments of genomic cystic fibrosis (CF) transmembrane conductance regulator (CFTR) DNA, that comprise the wild-type and DeltaF508 sequences, were transfected into immortalized CF and normal airway epithelial cells, respectively. Homologous replacement was evaluated using PCR and sequence-based analyses of cellular DNA and RNA. Individual stages of cationic lipid-facilitated SFHR in cultured cell lines were also examined using transmission electron microscopy (TEM). RESULTS: We demonstrated that the lipid/DNA (+/-) ratio influences the mode of entry into the cell and therefore affects the efficacy of SFHR-mediated gene targeting. Lipid/DNA complexes with more negative ratios entered the cell via a plasma membrane fusion pathway. Transfer of the DNA that relies on an endocytic pathway appeared more effective at mediating SFHR. In addition, it was also clear that there is a correlation between the specific cell line transfected and the optimal lipid/DNA ratio. CONCLUSIONS: These studies provide new insights into factors that underlie SFHR-mediated gene targeting efficacy and into the parameters that can be modulated for its optimization.

Journal Article↗