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Biomedical subjects

J A Schmid

Publications and source records attributed to J A Schmid.

14 recordsLinked to original sources
PubMed2004

A highly conserved proapoptotic gene, IKIP, located next to the APAF1 gene locus, is regulated by p53.

We describe here the identification and initial characterization of a novel human gene termed IKIP (I kappa B kinase interacting protein) that is located on chromosome 12 in close proximity to APAF1 (apoptotic protease-activating factor-1). IKIP and APAF1 share a common 488 bp promoter from which the two genes are transcribed in opposite directions. Three IKIP transcripts are generated by differential splicing and alternative exon usage that do not show significant homology to other genes in the databases. Similar to APAF1, expression of IKIP is enhanced by X-irradiation, and both genes are dependent on p53. Moreover, IKIP promotes apoptosis when transfected into endothelial cells. We conclude that IKIP is a novel p53 target gene with proapoptotic function.

Alternative Splicing↗
PubMed2001

The Ras-like GTPase Gem is involved in cell shape remodelling and interacts with the novel kinesin-like protein KIF9.

Gem belongs to the Rad/Gem/Kir (RGK) subfamily of Ras-related GTPases, which also comprises Rem, Rem2 and Ges. The RGK family members Ges and Rem have been shown to produce endothelial cell sprouting and reorganization of the actin cytoskeleton upon overexpression. Here we show that high intracellular Gem levels promote profound changes in cell morphology and we investigate how this phenotype arises dynamically. We also show that this effect requires intact microtubules and microfilaments, and that Gem is associated with both cytoskeletal components. In order to investigate the mechanisms of Gem recruitment to the cytoskeleton, we performed a yeast two-hybrid screen and identified a novel kinesin-like protein, termed KIF9, as a new Gem interacting partner. We further show that Gem and KIF9 interact by co-immunoprecipitation. Furthermore, Gem and KIF9 display identical patterns of gene expression in different tissues and developmental stages. The Gem- KIF9 interaction reported here is the first molecular link between RGK family members and the microtubule cytoskeleton.

Actin Cytoskeleton↗
PubMed2001

Adenovirus-mediated expression of a mutant IkappaB kinase 2 inhibits the response of endothelial cells to inflammatory stimuli.

In a variety of cell types, the transcription factor nuclear factor kappaB (NF-kappaB) functions as a mediator of stress and immune responses. In endothelial cells (ECs), it controls the expression of genes encoding, eg, cytokines, cell adhesion molecules, and procoagulatory proteins. This study investigates the effect of NF-kappaB suppression on several pathophysiologic functions of ECs, including inflammation, coagulation, and angiogenesis. A recombinant adenovirus was generated for expression of a dominant negative (dn) mutant of IkappaB kinase 2 (IKK2), a kinase that acts as an upstream activator of NF-kappaB. dnIKK2 inhibited NF-kappaB, resulting in strongly reduced nuclear translocation and DNA binding activity of the transcription factor and lack of expression of several proinflammatory markers, including E-selectin, intercellular adhesion molecule 1, vascular cell adhesion molecule 1, and interleukin-8. Concomitantly, inhibition of leukocyte binding to dnIKK2-expressing ECs could be demonstrated in a cell adhesion assay. Furthermore, expression of tissue factor as well as the ability to form capillary tubes in a matrigel assay was impaired in dnIKK2-expressing ECs. These data demonstrate that NF-kappaB is of central importance not only for the inflammatory response but also for a number of other EC functions. Therefore, this transcription factor as well as its upstream regulatory signaling molecules may represent favorable targets for therapeutic interference.

Adenoviridae↗
PubMed2001

Impact of oligomerization on the function of the human serotonin transporter.

The formation of oligomeric structures has been proposed for a large number of membrane proteins, including G-protein-coupled receptors and ion channels. Biochemical studies employing gel filtration, cross-linking or co-immunoprecipitation techniques showed that the serotonin [5-hydroxytryptamine (5-HT)] transporter is also capable of forming oligomers. We investigated whether the human serotonin transporter (hSERT) can be visualized as an oligomer in the plasma membrane of intact cells. To test this working hypothesis, we generated fusion proteins of hSERT and spectral variants of green fluorescent protein [cyan and yellow fluorescent proteins (CFP and YFP, respectively)]. When expressed in HeLa or HEK-293 cells, the resulting fusion proteins (CFP-hSERT and YFP-hSERT) were inserted into the plasma membrane and were indistinguishable from wild-type hSERT on functional testing (5-HT uptake assays, inhibition of 5-HT uptake by blockers such as imipramine). Oligomers were visualized by fluorescence resonance energy transfer (FRET) microscopy in living cells using complementary methods. Interestingly, oligomerization was not confined to hSERT; FRET was also observed between CFP-and YFP-labelled rat gamma-aminobutyric acid transporter. Gel filtration experiments showed that most of the protein was recovered as higher molecular weight complexes; almost no monomeric form was detected. This indicates that the homo-oligomeric form is the favoured state of hSERT in living cells. The formation of oligomers was not significantly affected by co-incubation with transporter substrates or blockers. Based on our observations, oligomer formation might not be essential for the physiological function of the transporter protein, the re-uptake of substrates. Furthermore, we conclude that constitutive oligomer formation might be a general property of Na(+)/Cl(-)-dependent neurotransmitter transporters.

Antidepressive Agents↗
PubMed2000

Oligomerization of the human serotonin transporter and of the rat GABA transporter 1 visualized by fluorescence resonance energy transfer microscopy in living cells.

Recent biochemical studies indicate that the serotonin transporter can form oligomers. We investigated whether the human serotonin transporter (hSERT) can be visualized as an oligomer in the plasma membrane of intact cells. For this purpose, we generated fusion proteins of hSERT and spectral variants of the green fluorescent protein (cyan and yellow fluorescent proteins, CFP and YFP, respectively). When expressed in human embryonic kidney 293 cells, the resulting fusion proteins (CFP-hSERT and YFP-hSERT) were efficiently inserted into the plasma membrane and were functionally indistinguishable from wild-type hSERT. Oligomers were visualized by fluorescence resonance energy transfer microscopy in living cells using two complementary methods, i.e. ratio imaging and donor photobleaching. Interestingly, oligomerization was not confined to hSERT; fluorescence resonance energy transfer was also observed between CFP- and YFP-labeled rat gamma-aminobutyric acid transporter. The bulk of serotonin transporters was recovered as high molecular weight complexes upon gel filtration in detergent solution. In contrast, the monomers of CFP-hSERT and YFP-hSERT were essentially undetectable. This indicates that the homo-oligomeric form is the favored state of hSERT in living cells, which is not significantly affected by coincubation with transporter substrates or blockers. Based on our observations, we conclude that constitutive oligomer formation might be a general property of Na(+)/Cl(-)-dependent neurotransmitter transporters.

Animals↗
PubMed2000

Activation of NF-kappa B by XIAP, the X chromosome-linked inhibitor of apoptosis, in endothelial cells involves TAK1.

Exposure of endothelial and many other cell types to tumor necrosis factor alpha generates both apoptotic and anti-apoptotic signals. The anti-apoptotic pathway leads to activation of the transcription factor NF-kappaB that regulates the expression of genes such as A20 or members of the IAP gene family that protect cells from tumor necrosis factor alpha-mediated apoptosis. In turn, some anti-apoptotic genes have been shown to modulate NF-kappaB activity. Here we demonstrate that XIAP, a NF-kappaB-dependent member of the IAP gene family, is a strong stimulator of NF-kappaB. Expression of XIAP leads to increased nuclear translocation of the p65 subunit of NF-kappaB via a novel signaling pathway that involves the mitogen-activated protein kinase kinase kinase TAK1. We show that TAK1 physically interacts with NIK and with IKK2, and both XIAP or active TAK1 can stimulate IKK2 kinase activity. Thus, XIAP may be part of a system of regulatory loops that balance a cell's response to environmental stimuli.

Apoptosis↗
PubMed2000

Dynamics of NF kappa B and Ikappa Balpha studied with green fluorescent protein (GFP) fusion proteins. Investigation of GFP-p65 binding to DNa by fluorescence resonance energy transfer.

We investigated the dynamics of nuclear transcription factor kappaB (NF-kappaB) by using fusion proteins of the p65 subunit with mutants of green fluorescent protein (GFP). GFP-NF-kappaB chimeras were functional both in vitro and in vivo, as demonstrated by electrophoretic mobility shift assays and reporter gene studies. GFP-p65 was regulated by IkappaBalpha similar to wild type p65 and associated with its inhibitor even if both proteins were linked to a GFP protein. This finding was also verified by fluorescence resonance energy transfer (FRET) microscopy and studies showing mutual regulation of the intracellular localization of both GFP chimerae. Incubation of GFP-p65 with fluorescently labeled NF-kappaB-binding oligonucleotides also resulted in FRET. This effect was DNA sequence-specific and exhibited saturation characteristics. Application of stopped-flow fluorometry to measure the kinetics of FRET between GFP-p65 and oligonucleotides revealed a fast increase of acceptor fluorescence with a plateau after about 10 ms. The observed initial binding rate showed a temperature-dependent linear correlation with the oligonucleotide concentration. The association constant calculated according to pre-steady state kinetics was 3 x 10(6) m(-1), although equilibrium binding studies implied significantly higher values. This observation suggests that the binding process involves a rapid association with a rather high off-rate followed by a conformational change resulting in an increase of the association constant.

Animals↗
PubMed2000

The transcription factor NF-kappa B and the regulation of vascular cell function.

A variety of pathophysiological situations that affect cells of the vasculature, including endothelial and smooth muscle cells, leads to the expression of genes such as adhesion molecules and chemokines that are dependent on members of the nuclear factor (NF)-kappaB family of transcription factors. The corresponding gene products mediate important biological functions such as immune and inflammatory reactions, smooth muscle cell proliferation, and angiogenesis. The beneficial and usually transient NF-kappaB-dependent gene expression may be exaggerated in pathological situations and results in damage to the vessel wall and impaired vascular cell function. In this review, we will capitalize on the favorable and adverse roles of NF-kappaB in the context of vascular disease, eg, chronic and localized inflammation, arteriosclerosis, and neoangiogenesis.

Animals↗
PubMed1999

Accumulation of sialic acid in endocytic compartments interferes with the formation of mature lysosomes. Impaired proteolytic processing of cathepsin B in fibroblasts of patients with lysosomal sialic acid storage disease.

The impact of an altered endocytic environment on the biogenesis of lysosomes was studied in fibroblasts of patients suffering from sialic acid storage disease (SASD). This inherited disorder is characterized by the accumulation of acidic monosaccharides in lysosomal compartments and a concomitant decrease of their buoyant density. We demonstrate that C-terminal trimming of the lysosomal cysteine proteinase cathepsin B is inhibited in SASD fibroblasts. This late event in the biosynthesis of cathepsin B normally takes place in mature lysosomes, suggesting an impaired biogenesis of these organelles in SASD cells. When normal fibroblasts are loaded with sucrose, which inhibits transport from late endosomes to lysosomes, C-terminal cathepsin B processing is prevented to the same extent. Further characterization of the terminal endocytic compartments of SASD cells revealed properties usually associated with late endosomes/prelysosomes. In addition to a decreased buoyant density, SASD "lysosomes" show a reduced acidification capacity and appear smaller than their normal counterparts. We conclude that the accumulation of small non-diffusible compounds within endocytic compartments interferes with the formation of mature lysosomes and that the acidic environment of the latter organelles is a prerequisite for C-terminal processing of lysosomal hydrolases.

Cathepsin B↗
PubMed1999

The NF-kappaB/Rel family of transcription factors in oncogenic transformation and apoptosis.

Recent progress in the identification and functional analysis of protein kinases and adapter molecules that lead to activation of NF-kappaB family transcription factors has lead to a quite detailed understanding of one of the major signalling pathways that mediate a cell's response to environmental stress in a variety of host-defense situations. NF-kappaB is recognized as a key regulatory factor mediating the coordinate expression of genes which are part of the cellular machinery that functions to protect an organism against damage posed by physical, chemical or microbial noxae. In a wide variety of patho-physiological situations such as immune and inflammatory reactions, the expression of cytokines, interleukins and adhesion molecules in cells of the immune system including T and B cells, endothelial as well as phagocytic/antigen presenting cells is to a large extent regulated by NF-kappaB. Moreover, this transcription factor appears to play a central role in the regulation of apoptosis, an important cellular program that decides upon a cell's fate not only during embryonic development but also on its way from normal to the transformed phenotype. Thus, NF-kappaB has emerged also as an attractive target for therapeutic interference in a variety of pathological situations, including chronic inflammatory and autoimmune diseases, HIV infection and cancer.

Animals↗
PubMed1998

Nuclear factor (NF)-kappaB-regulated X-chromosome-linked iap gene expression protects endothelial cells from tumor necrosis factor alpha-induced apoptosis.

By differential screening of tumor necrosis factor alpha (TNF-alpha) and lipopolysaccharide (LPS)- activated endothelial cells (ECs), we have identified a cDNA clone that turned out to be a member of the inhibitor of apoptosis (iap) gene family. iap genes function to protect cells from undergoing apoptotic death in response to a variety of stimuli. These iap genes, hiap1, hiap2, and xiap were found to be strongly upregulated upon treatment of ECs with the inflammatory cytokines TNF-alpha, interleukin 1beta, and LPS, reagents that lead to activation of the nuclear transcription factor kappaB (NF-kappaB). Indeed, overexpression of IkappaBalpha, an inhibitor of NF-kappaB, suppresses the induced expression of iap genes and sensitizes ECs to TNF-alpha-induced apoptosis. Ectopic expression of one member of the human iap genes, human X-chromosome-linked iap (xiap), using recombinant adenovirus overrules the IkappaBalpha effect and protects ECs from TNF-alpha- induced apoptosis. We conclude that xiap represents one of the NF-kappaB-regulated genes that counteracts the apoptotic signals caused by TNF-alpha and thereby prevents ECs from undergoing apoptosis during inflammation.

Adenoviridae↗
PubMed1998

In vitro fusion of tissue-derived endosomes and lysosomes.

We investigated the in vitro fusion of different endocytic compartments derived from perfused rat liver, where the cells are assumed to be in their physiological state. Specifically labelled early, late and transcytotic endosomes, as well as lysosomes were tested for their fusion properties. In addition to the expected ATP-dependent fusion between early endosomes, we observed fusion between early and late endosomes with similar efficiency, kinetics and cytosol dependence. Fusion between early endosomes and transcytotic vesicles could not be detected. Prolonged incubation of complementary labelled early endosomes under fusion-supporting conditions followed by Percoll gradient centrifugation revealed the occurrence of fusion product at a dense position, indicating fusion events between light and dense compartments. Incubation of membrane preparations containing avidin-labelled endosomes and biotin-dextran-loaded lysosomes resulted in the formation of avidin-biotin complexes, indicating that fusion between early and late endosomes is followed by fusion with lysosomes. This was verified by colocalization of fluorescently labelled endosomes and lysosomes, as assessed by laser scanning microscopy. Endosome fusion, as well as content mixing between endosomes and lysosomes, were dependent on temperature and ATP, and could be inhibited by N-ethylmaleimide (NEM). The NEM-sensitivity was localised on endosomes and in the cytosol, but not on lysosomes. These observations indicate that early and late endosomes of rat liver exhibit a high fusion competence in vitro, promoting not only homotypic, but also heterotypic fusion.

Animals↗