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Biomedical subjects

G Hagens

Publications and source records attributed to G Hagens.

11 recordsLinked to original sources
PubMed1999

Calcium-binding protein S100A7 and epidermal-type fatty acid-binding protein are associated in the cytosol of human keratinocytes.

Expression of epidermal-type fatty acid-binding protein (E-FABP) and S100A7 has previously been shown to be elevated in psoriatic skin, a disease characterized by abnormal keratinocyte differentiation. However, no causal relationship between the up-regulation of these proteins and the disease has been shown. E-FABP is thought to be involved in cytosolic fatty acid (FA) transport, whereas the role of S100A7 is still unknown. In this report, we show by overlay assays that E-FABP, immobilized on nitrocellulose, is able to capture S100A7 from cytosolic psoriatic protein extracts and vice versa, suggesting the formation of a complex between the two proteins. Using purified E-FABP and S100A7, the complex can be reconstituted only in presence of EDTA. Moreover, we show that increased EDTA concentrations in psoriatic cytosolic protein extracts enhance complex formation. Partial complex disruption was obtained by the addition of physiological concentrations of Zn2+ (0.1 mM), whereas Ca2+ at 5 mM and Mg2+ at 30 mM had no effect. On the other hand, high Ca2+ concentrations (30 mM) resulted in partial complex disruption. Oleic acid-binding properties were observed for free E-FABP and the complex E-FABP-S100A7, but not for free S100A7. By using confocal microscopy we show that S100A7 and E-FABP are co-localized in the cytoplasm of differentiating keratinocytes from lesional psoriatic skin. These data indicate that formation of the E-FABP-S100A7 complex and its FA-binding function might be regulated at least by bivalent cations.

Calcium-Binding Proteins↗
PubMed1999

The fatty acid-binding heterocomplex FA-p34 formed by S100A8 and S100A9 is the major fatty acid carrier in neutrophils and translocates from the cytosol to the membrane upon stimulation.

Since no data are available concerning fatty acid (FA) transport in neutrophils we studied the presence of possible FA carriers. The kFA-p34 complex, composed of S100A8 and S100A9, has been implicated in the intracellular transport of arachidonic acid and its precursors in human keratinocytes. Here, we show that FA-p34 is the major FA carrier in human neutrophils (nFA-p34). The complex is highly expressed in resting neutrophils (2.65% of cytosolic proteins) and translocates to the membrane fraction upon stimulation with opsonized zymosan. Comparison of purified nFA-p34 with kFA-p34 shows that both complexes are composed of nearly the same subunits and possess similar binding properties for oleic acid. Densitometrical analyses of 2D gels show that n and kFA-p34 contain twice as much S100A8 and S100A9 suggesting an estimated stoichiometry of (S100A8)2S100A9. A method is described allowing to distinguish n and kFA-p34 from S100A8/S100A9 homo- and heteromer complexes that are devoid of FA-binding properties. After solvent extraction, we find by GC analysis linoleic acid as major endogenous ligand of purified kFA-p34. Our results suggest that nFA-p34, might be involved in the shuttling of unsaturated FA between the cytosol and the plasma membrane of neutrophils.

Animals↗
PubMed1999

Probable interaction between S100A7 and E-FABP in the cytosol of human keratinocytes from psoriatic scales.

The overexpression of E-FABP and S100A7 in lesional psoriatic skin suggests a possible link with this hyperproliferative skin disease. In order to investigate a role for the proteins in this disease, the purifications for both proteins were re-analyzed. Moreover, a specific antiserum directed against purified human S100A7 was generated. By SDS-PAGE immunoblotting we show that E-FABP and S100A7 are expressed in cultured human differentiating keratinocytes and confirm their overexpression in psoriatic scales. Gel filtration and non-denaturing PAGE revealed that S100A7 co-purified with E-FABP, indicating an association between the two proteins. Ion-exchange chromatography resulted in the dissociation of the complex. Finally, immunoprecipitations using antiserum against E-FABP revealed that S100A7 co-immunoprecipitated with E-FABP from protein extracts of psoriatic scales. These data indicate that E-FABP and S100A7 might form a complex in the cytosol of human keratinocytes.

Calcium-Binding Proteins↗
PubMed1999

Differential regulation of tumor necrosing factor-alpha (TNF-alpha) and interleukin-10 (IL-10) secretion by protein kinase and phosphatase inhibitors in human alveolar macrophages.

IL-10, a cytokine first identified as a product of cloned Th2 lymphocytes, is also produced by monocytes/macrophages. By its ability to inhibit cytokine synthesis and the expression of surface antigens, IL-10 is able to temper inflammation. In contrast, TNF-alpha plays a key role in acute and chronic inflammation and has been implicated in several forms of lung injury. The objective of this study was to investigate whether activators or inhibitors of LPS-activated signalling pathways might be able to dissociate TNF-alpha from IL-10 secretion in alveolar macrophages (AM). The results show that PMA activates expression of TNF-alpha without inducing IL-10 expression. We further demonstrate that LPS-induced TNF-alpha secretion is independent of PKC activation and can be increased by inhibitors of the serine/threonine phosphatases PP1 and PP2A. In contrast, LPS-mediated IL-10 secretion is down-regulated by PMA and inhibitors of PP1 and PP2A. Addition of PKC inhibitors reverses the PMA-mediated down-regulation of LPS-induced IL-10 secretion, indicating that PKC, once activated in vivo, might play a prominent role in controlling the secretion of IL-10 by AM. This study provides evidence that the PKC activator PMA and the phosphatase inhibitor calyculin A are able to dissociate TNF-alpha from IL-10 secretion by AM. Our data further indicate that LPS-mediated activation of certain signalling molecules has different consequences on the secretion of TNF-alpha or IL-10 by AM, an observation which may be important for the modulation of immune and inflammatory processes.

Cells, Cultured↗
PubMed1998

Differentiation of human epidermal keratinocytes is accompanied by increased expression of CRABP-II and increased cellular concentration of retinoic acids: retention of newly synthesized retinoic acids by CRABP-II.

Keratinocytes differentiating in vitro exhibit greater cytosolic capacity for retinoic acid synthesis from retinol or retinaldehyde as compared to nondifferentiated cells (Siegenthaler et al. 1990. Biochem. J. 268: 371-378), and increased expression of CRABP-II (Siegenthaler et al. 1988. Exp. Cell Res. 178: 114-126). Based on these observations, the content and disposition of [3H]retinoic acids were determined in intact, nondifferentiated and differentiating keratinocytes incubated with [3H]retinaldehyde or [3H]retinol. Differentiating keratinocytes contained higher levels of [3H] retinoic acids compared to undifferentiated cells when either [3H]retinaldehyde or [3H]retinol was the substrate. The largest increases in [3H]retinoic acids were achieved with [3H]retinaldehyde. Differentiation-associated increases in [3H]retinoic acids correlated with cellular content of retinoid alcohol substrates in incubations with retinaldehyde but not in incubations with retinol. Consistent with previous observations, CRABP-II was significantly increased in differentiating cells. Moreover, newly synthesized [3H]retinoic acids were retained within cells bound to CRABP-II. The results suggest that increasing cellular concentration of retinoic acids in in vitro differentiating keratinocytes is achieved by a combination of increased activity of the retinoic acid synthesis pathway and increased cellular content of CRABP-II.

Biological Transport↗
PubMed1997

Jun NH2-terminal kinase is constitutively activated in T cells transformed by the intracellular parasite Theileria parva.

When T cells become infected by the parasite Theileria parva, they acquire a transformed phenotype and no longer require antigen-specific stimulation or exogenous growth factors. This is accompanied by constitutive interleukin 2 (IL-2) and IL-2 receptor expression. Transformation can be reversed entirely by elimination of the parasites using the specific drug BW720c. Extracellular signal-regulated kinase and jun NH2-terminal kinase (JNK) are members of the mitogen-activated protein kinase family, which play a central role in the regulation of cellular differentiation and proliferation and also participate in the regulation of IL-2 and IL-2 receptor gene expression. T. parva was found to induce an unorthodox pattern of mitogen-activated protein kinase expression in infected T cells. JNK-1 and JNK-2 are constitutively active in a parasite-dependent manner, but have altered properties. In contrast, extracellular signal-regulated kinase-2 is not activated even though its activation pathway is functionally intact. Different components of the T cell receptor (TCR)-dependent signal transduction pathways also were examined. The TCRzeta or CD3epsilon chains were found not to be phosphorylated and T. parva-transformed T cells were resistant to inhibitors that block the early steps of T cell activation. Compounds that inhibit the progression of T cells to proliferation, however, were inhibitory. Our data provide the first example, to our knowledge, for parasite-mediated JNK activation, and our findings strongly suggest that T. parva not only lifts the requirement for antigenic stimulation but also entirely bypasses early TCR-dependent signal transduction pathways to induce continuous proliferation.

8-Bromo Cyclic Adenosine Monophosphate↗
PubMed1997

A heterocomplex formed by the calcium-binding proteins MRP8 (S100A8) and MRP14 (S100A9) binds unsaturated fatty acids with high affinity.

We show that unsaturated fatty acids (FAs) bind reversibly and with high affinity to a heterocomplex of 34 kDa (FA-p34) formed by the non-covalent association of two calcium-binding proteins of the S100 family: MRP8 (S100A8) and MRP14 (S100A9). Fatty acid-competition studies on the [3H]oleic acid.FA-p34-complex show that oleic, alpha-linoleic, gamma-linolenic, and arachidonic acids have IC50 values of about 1 microM, whereas palmitic and stearic acids are poor competitors. The binding of arachidonic acid is saturable with a single class of binding site per FA-p34, and a dissociation constant (Kd) of 0.13 microM is calculated. The individual subunits MRP8 and MRP14 show no binding properties for fatty acids, whereas a p34 complex reconstituted in vitro by the recombinant molecules exhibits binding properties, suggesting that the fatty acid-binding site of FA-p34 is created through heterocomplex formation. Furthermore, we demonstrate that lowering free Ca2+ levels to 16 nM results in a loss of the fatty acid-binding capacity of purified FA-p34. In calcium-induced differentiating keratinocytes, the amounts of FA-p34 are increased in the particulate (2.0 +/- 0.5 pmol of [3H]oleic acid/mg protein) and in the cytosolic (4.5 +/- 0.6 pmol of [3H]oleic acid/mg protein) fractions, whereas no FA-p34 can be detected in non-differentiated cultured keratinocytes. In abnormally differentiated keratinocytes (psoriasis) and in human polymorphonuclear leukocytes, FA-p34 is highly expressed (31.35 +/- 1.6 and 349.8 +/- 17.9 pmol of [3H]oleic acid/mg protein, respectively), pointing toward a role for this heteromer in mediating effects of unsaturated fatty acids in a calcium-dependent way during cell differentiation and/or inflammation.

Antigens, Differentiation↗
PubMed1997

Endothelial cells of the human microvasculature express epidermal fatty acid-binding protein.

Epidermal fatty acid-binding protein (E-FABP), previously characterized in human keratinocytes, is a cytoplasmic protein of 15 kD that specifically binds fatty acids (FAs). Previous PAGE-immunoblotting studies indicated that several human tissues display an immunoreactive band with an electrophoretic mobility identical to that of E-FABP. The aim of this study was to determine in which cells, other than keratinocytes, E-FABP might be expressed. By immunohistochemistry, we show that E-FABP is expressed in endothelial cells of the microvasculature of the placenta, heart, skeletal muscle, small intestine, lung, and renal medulla. Interestingly, in lung, a tissue of endodermal origin, E-FABP staining was also localized to secretory cells, ie, Clara cells, goblet cells, and probably a subpopulation of pneumocytes. RNA isolated from cultured human umbilical vein and normal human dermal microvascular endothelial cells was analyzed by reverse-transcriptase polymerase chain reaction (RT-PCR). Southern blotting and sequencing of the cloned RT-PCR products demonstrate that endothelial E-FABP is identical to keratinocyte E-FABP. These data suggest that E-FABP-mediated FA transport occurs at the level of the microvasculature in several FA target organs.

Base Sequence↗
PubMed1996

Cloning, sequencing and expression of the bovine CD3 epsilon and TCR-zeta chains, two invariant components of the T-cell receptor complex.

CD3 epsilon and the zeta-chain of the bovine T-cell receptor (TCR) are two invariant molecules with an important role in signal transduction via the TCR/CD3 complex. The nucleotide sequence of a bovine CD3 epsilon cDNA clone containing the complete coding sequence was determined and the deduced amino acid (aa) sequence compared to that of other species. The cytoplasmic domains of the different CD3 epsilon clearly show a higher degree of conservation than the extracellular domains. Bovine CD3 epsilon produced in Escherichia coli using different bacterial expression vectors was recognised by antibodies (Ab) directed against the intracytoplasmic domain of human CD3 epsilon. A partial bovine TCR zeta-chain cDNA was generated by the polymerase chain reaction (PCR) using primers that were based on sequences that are conserved between different species; 3' and 5' RACE-PCR were carried out to obtain the complete TCR zeta-chain cDNA sequence. A comparison of the predicted TCR zeta-chain aa sequence reveals that the GDP/GTP-binding motif, which is conserved in other species, shows marked differences in the bovine and ovine TCR zeta-chains. In contrast to CD3 epsilon, the short extracellular domain of the TCR zeta-chain is 100% conserved between the different species and the transmembrane domain also shows a high degree of identity. Ab were raised against the TCR zeta-chain, produced as a glutathione S-transferase fusion protein in E. coli, and were used in Western blot analysis to further characterise TCR zeta-chain expression in T-cells. The regents provide valuable tools for the study of signal transduction pathways in normal and transformed bovine T-cells.

Amino Acid Sequence↗
PubMed1994

Saccharomyces cerevisiae cells lacking the homologous pairing protein p175SEP1 arrest at pachytene during meiotic prophase.

Saccharomyces cerevisiae cells containing null mutations in the SEP1 gene, which encodes the homologous pairing and strand exchange protein p175SEP1, enter pachytene with a delay. They arrest uniformly at this stage of meiotic prophase, probably revealing a checkpoint in the transition from pachytene to meiosis I. At the arrest point, the cells remain largely viable and are cytologically characterized by the duplicated but unseparated spindle pole bodies of equal size and by the persistence of the synaptonemal complex, a cytological marker for pachytene. In addition, fluorescence in situ hybridization revealed that in arrested mutant cells maximal chromatin condensation and normal homolog pairing is achieved, typical for pachytene in wild type. A hallmark of meiosis is the high level of homologous recombination, which was analyzed both genetically and physically. Formation and processing of the double-strand break intermediate in meiotic recombination is achieved prior to arrest. Physical intragenic (conversion) and intergenic (crossover) products are formed just prior to, or directly at, the arrest point. Structural deficits in synaptonemal complex morphology, failure to separate spindle pole bodies, and/or defects in prophase DNA metabolism might be responsible for triggering the observed arrest. The pachytene arrest in sep1 cells is likely to be regulatory, but is clearly different from the RAD9 checkpoint in meiotic prophase, which occurs prior to the pachytene stage.

Chromosomes, Fungal↗