Biomedical subjects

Alfred Boettcher

Publications and source records attributed to Alfred Boettcher.

10 recordsLinked to original sources

PubMed2006

Alteration of the pulmonary surfactant system in full-term infants with hereditary ABCA3 deficiency.

RATIONALE: ABCA3 mutations are known to cause fatal surfactant deficiency. OBJECTIVE: We studied ABCA3 protein expression in full-term newborns with unexplained respiratory distress syndrome (URDS) as well as the relevance of ABCA3 mutations for surfactant homeostasis. METHODS: Lung tissue of infants with URDS was analyzed for the expression of ABCA3 in type II pneumocytes. Coding exons of the ABCA3 gene were sequenced. Surfactant protein expression was studied by immunohistochemistry, immunoelectron microscopy, and Western blotting. RESULTS: ABCA3 protein expression was found to be greatly reduced or absent in 10 of 14 infants with URDS. Direct sequencing revealed distinct ABCA3 mutations clustering within vulnerable domains of the ABCA3 protein. A strong expression of precursors of surfactant protein B (pro-SP-B) but only low levels and aggregates of mature surfactant protein B (SP-B) within electron-dense bodies in type II pneumocytes were found. Within the matrix of electron-dense bodies, we detected precursors of SP-C (pro-SP-C) and cathepsin D. SP-A was localized in small intracellular vesicles, but not in electron-dense bodies. SP-A and pro-SP-B were shown to accumulate in the intraalveolar space, whereas mature SP-B and SP-C were reduced or absent, respectively. CONCLUSION: Our data provide evidence that ABCA3 mutations are associated not only with a deficiency of ABCA3 but also with an abnormal processing and routing of SP-B and SP-C, leading to severe alterations of surfactant homeostasis and respiratory distress syndrome. To identify infants with hereditary ABCA3 deficiency, we suggest a combined diagnostic approach including immunohistochemical, ultrastructural, and mutation analysis.

ATP-Binding Cassette Transporters↗

PubMed2006

Induction of Raf kinase inhibitor protein contributes to macrophage differentiation.

Differential gene expression analysis of human blood monocytes has identified the Raf kinase inhibitor protein (RKIP) as a continuously upregulated gene in macrophage and dendritic cell maturation. Using realtime RT-PCR and Western blot analysis we were able to confirm the initial DNA-microarray findings of RKIP induction on mRNA and protein levels. RKIP upregulation in primary cells and overexpression in THP-1 cells did not alter ERK activity but strongly reduced the amount of the NFkappaB subunit p65 in the nucleus. mRNA levels and cell surface expression of maturation markers including the integrin CD11c and the scavenger receptor CD36 were significantly increased in RKIP transfected THP-1 cells. Our data show for the first time that RKIP is upregulated during macrophage and dendritic cell differentiation on mRNA and protein levels and we conclude that RKIP contributes to the monocytic differentiation process via inhibition of the NFkappaB signaling cascade independent from the canonical Ras/Raf/MEK/ERK pathway.

Androgen-Binding Protein↗

PubMed2006

Alterations in intestinal fatty acid metabolism in inflammatory bowel disease.

Inflammatory bowel disease (IBD) constitutes a severe intestinal disorder in developed countries with increasing incidence worldwide. Upcoming evidence indicates an important role of intestinal epithelial barrier function in the development of IBD. Fatty acids exert nutritional and protective effects on enterocytes, serve as activators of transcription and constitute precursors of inflammatory mediators. The aim of this study was to investigate differential regulation of genes involved in fatty acid uptake and endogenous fatty acid biosynthesis in IBD. Mucosal biopsy specimens from non-affected regions of the intestine were subjected to DNA microarray analysis. Gene array analysis revealed a variety of genes involved in fatty acid uptake and synthesis to be differentially expressed in ileum and colon of selected IBD patients. To verify these results, real-time RT-PCR was performed for selected regulated candidate genes in larger IBD sample numbers. In single biopsy analysis long chain acyl-CoA synthetase (ACSL) 1 and 4 were upregulated in IBD (P<0.05), while a significant decrease in fatty acid synthase expression was found in ileum and colon of ulcerative colitis patients (P<0.001). Expression of the transcription factor liver X receptor (LXR) which was previously shown to induce fatty acid synthase gene expression was not altered on mRNA level in IBD. However, in cell culture experiments using the human intestinal cell line LS174T induction of fatty acid synthase by the LXR ligand T0901317 was inhibited by TNFalpha. Moreover, these experiments indicated a decrease of LXR protein levels by TNFalpha treatment. These data suggest that the decrease of fatty acid synthase expression in ulcerative colitis patients could be at least partially due to a loss of LXR expression and function in the presence of pro-inflammatory cytokines. Observed alterations in expression of genes of fatty acid metabolism may contribute to the pathophysiology of ulcerative colitis.

Animals↗

PubMed2006

High density lipoprotein modulates platelet function.

BACKGROUND: Platelet activation by atherogenic lipoproteins can be antagonized by high density lipoprotein (HDL), probably via interaction with the ATP-binding cassette transporter A1 (ABCA1). METHODS: ABCA1 expression and its association with cholesterol rich membrane domains was analyzed by mRNA and Western blot analysis. HDL effects on platelet receptor clustering were analyzed by flow cytometric analysis of fluorescence resonance energy transfer between fluorochrome-labeled antibodies. RESULTS: ABCA1 expression increased upon megakaryocytic differentiation of human stem cells and ABCA1 protein partially associated to LubroIWX-resistant membrane domains. Plasma HDL-cholesterol in healthy donors negatively correlated to the platelet membrane cholesterol content. Receptor cluster analysis revealed a decrease in the association of Gplb and FcgammaRII upon incubation of platelets with HDL3. CONCLUSION: Our results suggest that HDL modulates platelet reactivity by altering lipid raft associated receptor clustering.

ATP Binding Cassette Transporter 1↗

PubMed2006

A flow cytometric screening test for detergent-resistant surface antigens in monocytes.

Rafts resemble cholesterol- and glycosphingolipid-enriched, liquid-ordered plasma membrane microdomains, showing resistance to nonionic detergents, and are involved in various cellular processes. In the present study, we have tested surface antigens on resting and lipopolysaccharide (LPS)-stimulated human peripheral blood monocytes for their detergent resistance (i.e. raft-association), by flow cytometry. Constitutive (CD14, CD32, CD55), or LPS-induced (CD81) raft-association, and detergent solubility (i.e. exclusion of rafts) (CD71) of monocyte antigens in the presence of 0.01% Triton X-100 are clearly demonstrated. Flow cytometric detergent insolubility is a powerful tool for rapid screening the raft-association of monocyte antigens in a whole-blood assay.

Antigens, CD↗

PubMed2004

Association of ABCA1 with syntaxin 13 and flotillin-1 and enhanced phagocytosis in tangier cells.

The ATP-binding cassette transporter A1 (ABCA1) facilitates the cellular release of cholesterol and choline-phospholipids to apolipoprotein A-I (apoA-I) and several studies indicate that vesicular transport is associated with ABCA1 function. Syntaxins play a major role in vesicular fusion and have also been demonstrated to interact with members of the ABC-transporter family. Therefore, we focused on the identification of syntaxins that directly interact with ABCA1. The expression of syntaxins and ABCA1 in cultured human monocytes during M-CSF differentiation and cholesterol loading was investigated and syntaxins 3, 6, and 13 were found induced in foam cells together with ABCA1. Immunoprecipitation experiments revealed a direct association of syntaxin 13 and full-length ABCA1, whereas syntaxin 3 and 6 failed to interact with ABCA1. The colocalization of ABCA1 and syntaxin 13 was also shown by immunofluorescence microscopy. Silencing of syntaxin 13 by small interfering RNA (siRNA) led to reduced ABCA1 protein levels and hence to a significant decrease in apoA-I-dependent choline-phospholipid efflux. ABCA1 is localized in Lubrol WX-insoluble raft microdomains in macrophages and syntaxin 13 and flotillin-1 were also detected in these detergent resistant microdomains along with ABCA1. Syntaxin 13, flotillin-1, and ABCA1 were identified as phagosomal proteins, indicating the involvement of the phagosomal compartment in ABCA1-mediated lipid efflux. In addition, the uptake of latex phagobeads by fibroblasts with mutated ABCA1 was enhanced when compared with control cells and the recombinant expression of functional ABCA1 normalized the phagocytosis rate in Tangier fibroblasts. It is concluded that ABCA1 forms a complex with syntaxin 13 and flotillin-1, residing at the plasma membrane and in phagosomes that are partially located in raft microdomains.

ATP Binding Cassette Transporter 1↗

PubMed2004

Apolipoprotein A-1 interaction with plasma membrane lipid rafts controls cholesterol export from macrophages.

Cholesterol efflux to apolipoprotein A-1 (apoA-1) from cholesterol-loaded macrophages is an important anti-atherosclerotic mechanism in reverse cholesterol transport. We recently provided kinetic evidence for two distinct pathways for cholesterol efflux to apoA-1 [Gaus et al. (2001) Biochemistry 40, 9363]. Cholesterol efflux from two membrane pools occurs sequentially with different kinetics; a small pool rapidly effluxed over the first hour, followed by progressive release from a major, slow efflux pool over several hours. In the present study, we propose that the rapid and slow cholesterol efflux pools represent cholesterol derived from lipid raft and nonraft domains of the plasma membrane, respectively. We provide direct evidence that apoA-1 binds to both lipid raft and nonraft domains of the macrophage plasma membrane. Conditions that selectively deplete plasma membrane lipid raft cholesterol, such as incorporation of 7-ketocholesterol or rapid exposure to cyclodextrins, block apoA-1 binding to these domains but also inhibit cholesterol efflux from the major, slow pool. We propose that cholesterol exported to apoA-1 from this major slow efflux pool derives from nonraft regions of the plasma membrane but that the interaction of apoA-1 with lipid rafts is necessary to stimulate this efflux.

2-Hydroxypropyl-beta-cyclodextrin↗

PubMed2004

Expression pattern and raft association of NIPSNAP3 and NIPSNAP4, highly homologous proteins encoded by genes in close proximity to the ATP-binding cassette transporter A1.

The highly homologous genes NIPSNAP3 and NIPSNAP4, with 87% amino acid identity, are members of the NIPSNAP family with putative roles in vesicular trafficking. NIPSNAP3 mRNA and NIPSNAP4 mRNA and protein were detected in multiple tissues and cells at varying degrees. Interestingly, NIPSNAP3 is most highly expressed in skeletal muscle, where NIPSNAP4 has a low mRNA abundance. NIPSNAP4 was found associated with membranes and partly localized in rafts. The ubiquitous expression of the highly conserved NIPSNAPs and their association with membranes further support an important cellular function of these proteins probably linked to vesicular trafficking. The NIPSNAP3 and NIPSNAP4 genes are located in close proximity to the 3' end of the ATP-binding cassette transporter A1 (ABCA1), whose mutations cause familial high-density lipoprotein deficiency syndromes. The adjacent genomic location and the finding that ABCA1 is a regulator of vesicular trafficking may indicate a functional relation of these proteins, even though NIPSNAP4 does not interact directly with ABCA1 nor is its expression altered in cells with mutated ABCA1.

ATP Binding Cassette Transporter 1↗

PubMed2002

The carboxyterminus of the ATP-binding cassette transporter A1 interacts with a beta2-syntrophin/utrophin complex.

Recent work identified ABCA1 as the major regulator of plasma HDL-cholesterol responsible for the removal of excess choline-phospholipids and cholesterol from peripheral cells and tissues. ABCA1 function may depend on the association with heteromeric proteins and to identify these candidates a human liver yeast two-hybrid library was screened with the carboxyterminal 144 amino acids of ABCA1. Beta2-syntrophin was found to interact with ABCA1 and the C-terminal five amino acids of ABCA1 proned to represent a perfect tail for binding to syntrophin PDZ domains. Immunoprecipitation further confirmed the association of ABCA1 and beta2-syntrophin and in addition utrophin, known to couple beta2-syntrophin and its PDZ ligands to the F-actin cytoskeleton, was identified as a constituent of this complex. ABCA1 in the plasmamembrane of human macrophages was found to be partially associated with Lubrol rafts and effluxed choline-phospholipids involve these microdomains. Beta2-syntrophin does not colocalize in these rafts indicating that beta2-syntrophin may participate in the retaining of ABCA1 in cytoplasmic vesicles and for the targeting of ABCA1 to plasmamembrane microdomains when ABCA1 is released from beta2-syntrophin.

ATP Binding Cassette Transporter 1↗

PubMed2002

Cloning and characterization of a novel apolipoprotein A-I binding protein, AI-BP, secreted by cells of the kidney proximal tubules in response to HDL or ApoA-I.

Apolipoprotein A-I (apoA-I) is the major apolipoprotein of high-density lipoproteins (HDL) and has an important role in the regulation of the stability, lipid transport, and metabolism of HDL particles. To identify novel proteins that are involved in HDL metabolism, we used mature apoA-I (amino acids 25-267) as a bait for the screening of a human liver two-hybrid cDNA library. Among the identified genes, several encoded known proteins, including serum amyloid A(2a) (SAA(2a)), apoC-I, and phosphodiesterase HCAM1 (PDE1A), found to interact with apoA-I. In addition, we have cloned a novel 29 kDa apoA-I interacting protein, which we named AI-BP (apoA-I binding protein). The AI-BP encoding gene, APOA1BP, which is located on chromosome 1q21, is composed of six exons and five introns and spans 2.5 kb. Northern blot analysis demonstrated ubiquitous expression of the APOA1BP mRNA with the highest expression in kidney, heart, liver, thyroid gland, adrenal gland, and testis. AI-BP protein is not detectable in serum of healthy probands, but serum samples of patients with septic syndromes may contain elevated levels of AI-BP. Significant amounts of AI-BP protein are found in cerebrospinal fluid and urine of healthy probands. The stimulation of cells derived from the kidney proximal tubules with apoA-I or HDL induces a concentration-dependent secretion of AI-BP indicating an important role for AI-BP, in the renal tubular degradation or resorption of apoA-I.

Amino Acid Sequence↗