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Biomedical subjects

Stefan Lorkowski

Publications and source records attributed to Stefan Lorkowski.

At least 19 recordsLinked to original sources
PubMed2006

Butyrophilin controls milk fat globule secretion.

The molecular mechanism underlying milk fat globule secretion in mammary epithelial cells ostensibly involves the formation of complexes between plasma membrane butyrophilin and cytosolic xanthine oxidoreductase. These complexes bind adipophilin in the phospholipid monolayer of milk secretory granules, the precursors of milk fat globules, enveloping the nascent fat globules in a layer of plasma membrane and pinching them off the cell. However, using freeze-fracture immunocytochemistry, we find these proteins in locations other than those previously inferred. Significantly, butyrophilin in the residual plasma membrane of the fat globule envelope is concentrated in a network of ridges that are tightly apposed to the monolayer derived from the secretory granule, and the ridges coincide with butyrophilin labeling in the globule monolayer. Therefore, we propose that milk fat globule secretion is controlled by interactions between plasma membrane butyrophilin and butyrophilin in the secretory granule phospholipid monolayer rather than binding of butyrophilin-xanthine oxidoreductase complexes to secretory granule adipophilin.

Antibodies↗
PubMed2006

Unsaturated fatty acids suppress the expression of the ATP-binding cassette transporter G1 (ABCG1) and ABCA1 genes via an LXR/RXR responsive element.

ATP-binding cassette transporters (ABC) G1 and ABCA1 are membrane cholesterol transporters and have been implicated to mediate cholesterol efflux from cells in the presence of high density lipoproteins and its major protein constituent apolipoprotein A-I, respectively. We previously demonstrated that unsaturated fatty acids suppress the stimulatory effects of oxysterols and retinoids on ABCA1 gene transcription. We here demonstrate that unsaturated fatty acids significantly suppress the stimulatory effects of oxysterols and retinoids on the expression of ABCG1 mRNA and protein and the activity of the wild-type human ABCG1 promoter as well as ABCA1. Mutation or deletion of the DR4 element within the ABCG1 or ABCA1 promoters, to which the transcriptional inducers LXR and RXR bind, abolished the suppressive effects of unsaturated fatty acids. Our observations provide the first evidence that unsaturated fatty acids suppress ABCG1 gene expression by a mechanism which involves the binding of LXR/RXR to the promoters. Suppression of both the ABCA1 and ABCG1 genes may indicate that unsaturated fatty acids suppress not only cholesterol efflux to apoA-I and thereby nascent HDL formation but also HDL-dependent cholesterol efflux from vascular cells.

ATP Binding Cassette Transporter 1↗
PubMed2006

Simplifying gene trees for easier comprehension.

BACKGROUND: In the genomic age, gene trees may contain large amounts of data making them hard to read and understand. Therefore, an automated simplification is important. RESULTS: We present a simplification tool for gene trees called TreeSimplifier. Based on species tree information and HUGO gene names, it summarizes "monophyla". These monophyla correspond to subtrees of the gene tree where the evolution of a gene follows species phylogeny, and they are simplified to single leaves in the gene tree. Such a simplification may fail, for example, due to genes in the gene tree that are misplaced. In this way, misplaced genes can be identified. Optionally, our tool glosses over a limited degree of "paraphyly" in a further simplification step. In both simplification steps, species can be summarized into groups and treated as equivalent. In the present study we used our tool to derive a simplified tree of 397 leaves from a tree of 1138 leaves. Comparing the simplified tree to a "cartoon tree" created manually, we note that both agree to a high degree. CONCLUSION: Our automatic simplification tool for gene trees is fast, accurate, and effective. It yields results of similar quality as manual simplification. It should be valuable in phylogenetic studies of large protein families. The software is available at http://www.uni-muenster.de/Bioinformatics/services/treesim/.

Algorithms↗
PubMed2006

IsoSVM--distinguishing isoforms and paralogs on the protein level.

BACKGROUND: Recent progress in cDNA and EST sequencing is yielding a deluge of sequence data. Like database search results and proteome databases, this data gives rise to inferred protein sequences without ready access to the underlying genomic data. Analysis of this information (e.g. for EST clustering or phylogenetic reconstruction from proteome data) is hampered because it is not known if two protein sequences are isoforms (splice variants) or not (i.e. paralogs/orthologs). However, even without knowing the intron/exon structure, visual analysis of the pattern of similarity across the alignment of the two protein sequences is usually helpful since paralogs and orthologs feature substitutions with respect to each other, as opposed to isoforms, which do not. RESULTS: The IsoSVM tool introduces an automated approach to identifying isoforms on the protein level using a support vector machine (SVM) classifier. Based on three specific features used as input of the SVM classifier, it is possible to automatically identify isoforms with little effort and with an accuracy of more than 97%. We show that the SVM is superior to a radial basis function network and to a linear classifier. As an example application we use IsoSVM to estimate that a set of Xenopus laevis EST clusters consists of approximately 81% cases where sequences are each other's paralogs and 19% cases where sequences are each other's isoforms. The number of isoforms and paralogs in this allotetraploid species is of interest in the study of evolution. CONCLUSION: We developed an SVM classifier that can be used to distinguish isoforms from paralogs with high accuracy and without access to the genomic data. It can be used to analyze, for example, EST data and database search results. Our software is freely available on the Web, under the name IsoSVM.

Algorithms↗
PubMed2005

Correspondence of function and phylogeny of ABC proteins based on an automated analysis of 20 model protein data sets.

Using our BLAST-based procedure RiPE (Retrieval-induced Phylogeny Environment), which automates the evolutionary analysis of a protein family, we assembled a set of 1138 ABC protein components [adenosine triphosphate (ATP)-binding cassette and transmembrane domain] from the protein data sets of 20 model organisms and subjected them to phylogenetic and functional analysis. For maximum speed, we based the alignment directly on a homology search with a profile of all known human ABC proteins and used neighbor-joining tree estimation. All but 11 sequences from Homo sapiens, Arabidopsis thaliana, Drosophila melanogaster, and Saccharomyces cerevisiae were placed into the correct subtree/subfamily, reproducing published classifications of the individual organisms. By following a simple "function transfer rule", our comparative phylogenetic analysis successfully predicted the known function of human ABC proteins in 19 of 22 cases. Three functional predictions did not correspond, and 10 were novel. Predictions based on BLAST alone were inferior in five cases and superior in two. Bacterial sequences were placed close to the root of most subtrees. This placement coincides with domain architecture, suggesting an early diversification of the ABC family before the kingdoms split apart. Our approach can, in principle, be used to annotate any protein family of any organism included in the study.

ATP-Binding Cassette Transporters↗
PubMed2005

Cloning, cellular localization, genomic organization, and tissue-specific expression of the TGFbeta1-inducible SMAP-5 gene.

SMAP-5 is a member of the five-pass transmembrane protein family localizing in the Golgi apparatus and the endoplasmic reticulum. These proteins have been implicated in intracellular trafficking, in secretion and in vesicular transport. Phylogenetic analyses revealed that SMAP-5 is a member of a small Rab GTPase interacting factor protein family. The human SMAP-5 gene spans about 12.5 kb and comprises 6 exons on chromosomal locus 5q32. The proximal 5'-flanking region of the gene lacks a TATA box and is highly GC rich. Consistent with this, the SMAP-5 gene is expressed in all tissues. The highest level of expression was found in coronary smooth muscle cells, in which expression of the SMAP-5 gene was induced by transforming growth factor beta1, thus indicating that this protein may play an important role in inflammation.

Alternative Splicing↗
PubMed2005

Lipid droplets gain PAT family proteins by interaction with specialized plasma membrane domains.

Proteins of the PAT family, named after perilipin, adipophilin, and TIP47 (tail-interacting protein of 47 kDa), are associated with lipid droplets and have previously been localized by immunofluorescence microscopy exclusively to the droplet surface. These proteins are considered not to be present in any other subcellular compartment. By applying the high resolution technique of freeze-fracture electron microscopy combined with immunogold labeling, we now demonstrate that in macrophages and adipocytes PAT family proteins are, first, distributed not only in the surface but also throughout the lipid droplet core and, second, are integral components of the plasma membrane. Under normal culture conditions these proteins are dispersed in the cytoplasmic leaflet of the plasma membrane. Stimulation of lipid droplet formation by incubation of the cells with acetylated low density lipoprotein leads to clustering of the PAT family proteins in raised plasma membrane domains. Fractures penetrating beneath the plasma membrane demonstrate that lipid droplets are closely apposed to these domains. A similar distribution pattern of labeling in the form of linear aggregates within the clusters is apparent in the cytoplasmic monolayer of the plasma membrane and the immediately adjacent outer monolayer of the lipid droplet. The aggregation of the PAT family proteins into such assemblies may facilitate carrier-mediated lipid influx from the extracellular environment into the lipid droplet. Lipid droplets appear to acquire their PAT proteins by interaction with plasma membrane domains enriched in these proteins.

3T3-L1 Cells↗
PubMed2005

Pharmacological regulation of cholesterol efflux in human monocyte-derived macrophages in the absence of exogenous cholesterol acceptors.

Cholesterol efflux from human monocyte-derived macrophages in the absence of exogenous acceptors has been described, but is unclear in mechanism. We investigated this process in relation to the expression of relevant genes, intracellular cholesterol storage and apoE secretion using drugs affecting different aspects of cholesterol metabolism. Both natural (22R-hydroxycholesterol/9-cis-retinoic acid) and synthetic (T0901317 and RO264456) LXR/RXR ligands increased ABCA1 and ABCG1 mRNAs in native macrophages and in cells loaded with acetylated LDL (acLDL). The ACAT inhibitor avasimibe increased only ABCG1 mRNA, whereas no treatment affected apoE mRNA. Avasimibe, progesterone, and natural but not synthetic LXR/RXR ligands prevented cholesterol esterification after acLDL-loading. Cholesterol efflux into acceptor-free medium was increased only by synthetic LXR/RXR ligands and avasimibe in acLDL-loaded cells. ApoE secretion was reduced by drugs affecting cholesterol trafficking but enhanced by LXR/RXR ligands. Incubation with an anti-apoE antibody virtually removed immunodetectable apoE from the medium, significantly increasing cholesterol storage and decreasing efflux. These findings indicate that in human macrophages spontaneous cholesterol efflux: (i) is not necessarily promoted by increasing intracellular free cholesterol, (ii) is increased by compounds that activate ABCA1 and, to a greater extent, ABCG1 and (iii) is only partially correlated with secretion of endogenous apoE, which acted as a cholesterol acceptor.

ATP Binding Cassette Transporter 1↗
PubMed2004

Spatial integration of TIP47 and adipophilin in macrophage lipid bodies.

We studied the distribution of the PAT family proteins TIP47 and adipophilin in lipid bodies of THP-1 cell-derived macrophages using freeze-fracture immunolabeling and other techniques. Lipid bodies in macrophages comprise lipid droplets and extensive, previously scantily characterized sheet-like organelles, which we descriptively call "lipid sails." TIP47 and adipophilin are components of many, but not all, the lipid droplets. Both proteins are not confined to the surface of lipid droplets, as supposed, but are also inside lipid droplet cores. They are not codistributed stoichiometrically in lipid droplets. How TIP47 and adipophilin, which are polar proteins, enter the lipid droplets and are packaged among the hydrophobic neutral lipids of the core is unclear. However, in the lipid layers of the core, these proteins are directed sometimes inward and sometimes outward. Because TIP47 and adipophilin also localize to lipid sails, lipid sails are intimately involved in intracellular lipid metabolism.

Animals↗
PubMed2004

Characterization of the synthetic compatible solute homoectoine as a potent PCR enhancer.

Different substances such as dimethyl sulfoxide, tetramethylene sulfoxide, 2-pyrollidone, and the naturally occurring compatible solute betaine enhance PCR amplification of GC-rich DNA templates with high melting temperatures. In particular, cyclic compatible solutes outperform traditional PCR enhancers. We therefore investigated the effects that cyclic naturally occurring ectoine-type compatible solutes and their synthetic derivatives have on melting temperature of double-stranded DNA (dsDNA) and on PCR amplification of different templates. L-ectoine, betaine, and derivatives of L-ectoine decreased, whereas beta-hydroxyectoine increased, the melting temperature of dsDNA. The ability to decrease the melting temperature was greatest for homoectoine, a new synthetic derivative of l-ectoine. Furthermore, compatible solutes, especially homoectoine, enhanced PCR amplification of GC-rich DNA (72.6% GC content; effective range: 0.1-0.5M).

Amino Acids, Diamino↗
PubMed2004

Cholesterol absorption inhibitor Ezetimibe blocks uptake of oxidized LDL in human macrophages.

Ezetimibe belongs to a group of selective and very effective 2-azetidione cholesterol absorption inhibitors which act on the level of cholesterol entry into enterocytes. Recent data indicated that the drug prevents the formation of a heterocomplex consisting of annexin-2 and caveolin-l and leads to specific inhibition of an NPCILI-dependent cholesterol uptake pathway required for uptake of micellar cholesterol into enterocytes. Earlier studies have shown that caveolin-l and annexin-2 are also expressed in human macro-phages and we show in this study that human macrophages express NPC1L1. Moreover in human macrophages, Ezetimibe(SCH58235) and its analogue, SCH354909, are bound to specific cell surface receptors followed by endocytosis via the classical endocytic pathway. SCH58235 had no effect on uptake and/or processing of acetylated LDL (Ac-LDL). In contrast, the compound inhibited uptake of oxidized LDL (Ox-LDL) by -50% in a dose-dependent manner. SCH58235 blocked the lipid-induced induction of LXR/RXR target genes ABCAI, ABCGI, and apolipoprotein E distinctively more effectively in macrophages loaded with Ox-LDL than in those loaded with Ac-LDL. Based on these findings, we presume that the caveolin-l-, annexin-2-, and NPClLI-dependent cholesterol uptake system that is operating in enterocytes may also contribute to class B scavenger receptor-dependent uptake of Ox-LDL in human monocyte-derived macrophages.

Anticholesteremic Agents↗
PubMed2004

Proinflammatory cytokines regulate LOX-1 expression in vascular smooth muscle cells.

OBJECTIVE: Atherogenesis represents a type of chronic inflammation and involves elements of the immune response, eg, the expression of proinflammatory cytokines. In advanced atherosclerotic lesions, lectin-like oxidized low-density lipoprotein receptor-1 (LOX-1) is expressed in endothelial cells, macrophages, and smooth muscle cells (SMCs). In vitro, the expression of LOX-1 is induced by inflammatory cytokines like TNF-alpha and transforming growth factor (TGF)-beta. Therefore, LOX-1 is thought to be upregulated locally in response to cytokines in vivo. METHODS AND RESULTS: We determined by reverse-transcription polymerase chain reaction (PCR) and Western blot analysis whether the mediators of the acute phase response in inflammation, IL-1alpha, IL-1beta, and TNF-alpha, regulate LOX-1 expression in cultured SMC, and whether this regulation is influenced by peroxisome proliferator-activated receptor gamma (PPARgamma). We studied by immunohistochemistry whether these cytokines are spatially correlated with LOX-1 expression in advanced atherosclerotic lesions. We found upregulation of LOX-1 expression in SMC in a dose- and time-dependent manner after incubation with IL-1alpha, IL-1beta, and TNF-alpha. Simultaneous incubation with these cytokines at saturated concentrations had an additive effect on LOX-1 expression. The PPARgamma activator, 15d-PGJ(2), however, inhibited IL-1beta-induced upregulation of LOX-1. In the intima of atherosclerotic lesions regions of IL-1alpha, IL-1beta, and TNF-alpha expression corresponded to regions of LOX-1 expression. CONCLUSIONS: We suppose that upregulated LOX-1 expression in SMC of advanced atherosclerotic lesions is a response to these proinflammatory cytokines. Moreover, the proinflammatory effects of these cytokines can be decreased by the antiinflammatory effect of PPARgamma.

Aorta↗
PubMed2004

ADP-ribosylation factor (ARF)-like 7 (ARL7) is induced by cholesterol loading and participates in apolipoprotein AI-dependent cholesterol export.

Here, we identify ADP-ribosylation factor (ARF)-like 7 (ARL7) as the only ARF- and ARL-family member whose mRNA-expression is induced by liver X-receptor/retinoid X-receptor agonists or cholesterol loading in human macrophages. Moreover, subcellular distribution of mutant and wild type ARL7-enhanced green fluorescent protein (EGFP) supports that ARL7 may be involved in a vesicular transport step between a perinuclear compartment and the plasma membrane. Therefore, we investigated the effect of ARL7 over-expression on the cholesterol secretory pathway. We found that expression of wild type and dominant active ARL7-EGFP stimulated the rate of apolipoprotein AI-specific cholesterol efflux 1.7- and 2.8-fold. In contrast, expression of the dominant negative form of ARL7-EGFP led to approximately 50% inhibition of cholesterol efflux. This data is consistent with a model in which ARL7 is involved in transport between a perinuclear compartment and the plasma membrane apparently linked to the ABCA1-mediated cholesterol secretion pathway.

ADP-Ribosylation Factors↗
PubMed2004

VisCoSe: visualization and comparison of consensus sequences.

We introduce visualization and comparison of consensus sequences (VisCoSe) as a WWW service and a stand-alone command line Perl script for visualizing and comparing consensus sequences of protein and nucleotide sequences. VisCoSe is the only interface available that simultaneously calculates consensus sequences of multiple data sets and automatically compares these consensus sequences. Furthermore, VisCoSe allows visualization of chemical properties of amino acids.

Algorithms↗