Biomedical subjects

T Demberg

Publications and source records attributed to T Demberg.

6 recordsLinked to original sources

PubMed2006

Recombinant generation of two fragments of the rat complement inhibitory factor H [FH(SCR1-7) and FH(SCR1-4)] and their structural and functional characterization in comparison to FH isolated from rat serum.

Factor H (FH) is the predominant soluble inhibitor of the complement system. With a concentration of 200-800 microg/ml in human and rat plasma it acts as a cofactor for the soluble factor I (FI)-mediated cleavage of the component C3b to iC3b. Furthermore it competes with factor B for binding to C3b and C3(H2O) and promotes the dissociation of the C3bBb complex. FH is a monomer of about 155 kDa which comprises 20 short consensus repeats (SCR), each of which is composed of approximately 60 amino acid (aa) residues. Two functional fragments of FH comprising the SCR1-4 or SCR1-7 were generated using either the Baculovirus system or stably transfected human embryonal kidney cells, respectively. These fragments, as well as FH purified from rat serum, were first analyzed for their relative molecular weights (Mr) using non-reducing or reducing SDS-PAGE. The Mr of the FH variants differed by about 20% depending on the experimental conditions employed. Only the Mr of proteins separated under reducing conditions were in accordance with the MW calculated from the aa sequence. Analyses of the glycosylation patterns using PAS-staining showed a lack of staining of the recombinant variants (SCR1-4 and SCR1-7) in contrast to FH(SCR1-20) from serum. Using a complement hemolysis assay (CH50-assay) all three variants exhibited a molar complement inhibitory activity of FH(1-20)/FH(1-7)/FH(1-4) of about 3/1/1. These data support the postulated model of FH bearing three binding sites for its ligand C3b, from which one is located in the SCR1-4, whereas the other two are located in the SCR8-20.

Amino Acid Sequence↗

PubMed2004

Upregulation of fibronectin but not of entactin, collagen IV and smooth muscle actin by anaphylatoxin C5a in rat hepatic stellate cells.

Rat Kupffer cells (KC), hepatic stellate cells (HSC) and sinusoidal endothelial cells (SEC) all express the C5a receptor (C5aR) constitutively in contrast to hepatocytes (HC). HSC showed an unexpectedly high level of expression of the C5aR. As these cells are known to play a key role in the induction of liver fibrosis we hypothesized that C5a may possibly induce fibrogenetic proteins in these cells. HSC are known to express the extracellular matrix (ECM) proteins collagen IV, fibronectin, entactin and the structure protein smooth muscle actin (SMA) which is regarded as a marker for the fibrotic conversion of HSC to myofibroblast-like cells. We investigated the effect of recombinant rat C5a (rrC5a) on the upregulation of these ECM-proteins and of SMA, all of which are known to be expressed by HSC. The profibrotic cytokine TGF-beta1 (2 ng/ml), which was used as a control, clearly upregulated the three matrix proteins but not SMA. In the absence of any stimulus HSC upregulated the three ECM-proteins as well as SMA during their conversion into myofibroblast-like cells. This resulted in a high stimulus-independent plateau of the mRNA expressions for all four proteins after four to five days of culture. Readouts were therefore taken at 72 h after the isolation of the HSC when the investigated mRNA levels had not yet reached their maxima due to the conversion of the cells. The first 24 h of culture were performed without stimulus and the following 48 h in the presence of 100 nM rrC5a (1 micro g/ml) or TGF-beta1 (2 ng/ml). Only fibronectin-specific mRNA was clearly upregulated by C5a whereas entactin, collagen IV and SMA were not affected by C5a. By competitive-quantitative PCR the upregulation of fibronectin-specific mRNA was determined to be about five-fold. As TGF-beta1 upregulated all of the three investigated ECM-proteins but not SMA it was checked as to whether C5a might act indirectly by upregulating the expression of TGF-beta1 in KC and HSC, as both cell types are known to be sources of this profibrotic cytokine. However, using RT-PCR, such an effect was not detectable in either cell type after 3, 10 or 24 h.

Actins↗

PubMed2003

Temperature optima of enzyme-catalysed reactions in microemulsion systems.

Ternary phase systems (water/surfactant/organic solvent) were utilised to increase and broaden the temperature optima of enzyme-catalysed reactions. Alcohol dehydrogenases from yeast and Thermoanaerobium brockii (EC 1.1.1.1 and EC 1.1.1.2), lactate dehydrogenase from Lactobacillus delbrueckii (EC 1.1.1.28) and the particulate hydrogenase from Ralstonia eutropha (EC 1.18.99.1) were used as model enzymes in microemulsions, consisting of the surfactant Aerosol OT, and various alkane solvent and aqueous phases. All enzymes exhibited, besides an increase in specific activity, an upshift of the temperature optimum of the catalysed reaction. The temperature optimum could be further shifted by variation of the chain length of the solvent used and/or the addition of compatible solutes to the aqueous phase. Under optimised conditions, catalytic reactions of enzymes from mesophilic microorganisms had temperature optima in the range generally obtained with enzymes from thermophilic organisms.

Alcohol Dehydrogenase↗

PubMed2002

Rat complement factor H: molecular cloning, sequencing and quantification with a newly established ELISA.

Factor H (FH) is the predominant soluble regulatory protein of the complement system. With a concentration of 300-600 microg/ml in human plasma it acts as a cofactor for the FI-mediated cleavage of the component C3b to iC3b. Furthermore, it competes with factor B for binding to C3b and C3(H2O) and promotes the dissociation of the C3bBb complex (i.e. it has decay accelerating activity). FH is a monomer of about 155 kDa which comprises 20 short consensus repeats (SCR), each of which is composed of nearly 60 amino acid residues. For the screening of a rat liver cDNA library, we used two hybridization probes which had been produced by polymerase chain reaction (PCR). The probes were generated using degenerated primers which corresponded to conserved parts of the human and the murine factor H nucleotide sequences. The entire rat sequence spanned 4240 nucleotides with an open reading frame of 3708 nucleotides. These were preceded by 23 nucleotides of the 5' untranslated region, followed by a stop codon and a 3' untranslated region of 478 nucleotides including the polyadenylation-signal up to the beginning of the poly A tail. Comparison of the rat cDNA-derived coding sequence revealed identities of 74% to the human and 87% to the mouse FH nucleotide sequence. The translation product of rat FH mRNA was 1236 aa in length (leader sequence included) with an identity of 63% to the human and 81.5% to the murine protein. The degree of glycosylation of rat FH-Mr is about 9.5%. To quantitate FH in rat serum and supernatants of primary cultures of rat hepatocytes (HC), a reliable and sensitive sandwich-enzyme-linked immunosorbent assay (ELISA) was established. The concentration of FH in rat serum was calculated to be 238 microg +/- 21 microg/ml (mean +/- SD). Its concentration in the culture supernatants of HC was upregulated about three-fold by interferon (IFN)-gamma (100 U/ml).

Amino Acid Sequence↗

PubMed2001

Expression and regulation of complement factors H and I in rat and human cells: some critical notes.

The complement factors I (FI) and H (FH) are complement regulatory proteins. FI, a highly glycosylated serine protease of 88 kDa cleaves the alpha-chains of both complement components C3b and C4b, thereby inactivating them. Complement FH, a glycoprotein of 150 kDa which is composed of 20 short consensus repeats synergizes with FI by increasing the affinity of FI for C3b in the C3b/FH complex by about 15-fold as compared to free C3b. Furthermore, FH prevents factor B from binding to C3b and promotes the dissociation of the C3bBb complex. Both, FI and FH are mainly synthesized in the liver. According to the quantification of specific mRNA of both factors, various amounts are produced by different liver cell types, i.e. hepatocytes (HC) and Kupffer cells (KC). Investigations of cultured primary HC and KC from rat liver showed that FI is exclusively synthesized and secreted by HC whereas FH is synthesized by both HC and KC. Using quantitative-competitive PCR for the quantification of FH-specific mRNA, its constitutive rate of synthesis was found to be nearly ten times higher in KC than in HC. An extrahepatic source of both proteins are human umbilical vein endothelial cells (HUVEC) in which the synthesis of FI is upregulated by IL-6 which is in accord with the upregulation observed in rat HC and two rat hepatoma cell lines (FAO and H4IIE). Three other proinflammatory cytokines, IL-1beta, IFN-gamma and TNF-alpha, were alone or in combination, without any effect on the regulation of FI. This demonstrates that the regulation of FI is similar in HUVEC and HC. These results are in contrast to a previously described IFN-gamma-mediated upregulation of FI in HUVEC and suggest, in accordance with other investigations on extrahepatic sources of FI (e.g. myoblasts), that IFN-gamma has probably no prominent role in the regulation of FI. Instead, IL-6 appears to be the main upregulating cytokine of FI mRNA and of FI protein synthesis in HC as well as in rat and human hepatoma cells and in HUVEC. Of note are experiments by others and us who could not identify FI-specific mRNA in peripheral blood-derived monocytes, granulocytes, or B- and T-cells of man or rat and in rat peritoneal macrophages. FI-specific mRNA could also not be detected in B- or T-cell lymphoma cells, whereas FH-specific mRNA was easily detectable in both human and rat monocytes, and in rat peritoneal macrophages. These data support the notion that FI in contrast to FH is not expressed by cells of the monocyte-macrophage lineage or by other leukocytes of peripheral blood, at least in the absence of additional stimulants.

Animals↗

PubMed2001

Complement factor I is upregulated in rat hepatocytes by interleukin-6 but not by interferon-gamma, interleukin-1beta, or tumor necrosis factor-alpha.

Complement factor I (FI) is a regulatory serine protease of the complement system which cleaves three peptide bonds in the alpha-chain of C3b and two bonds in the alpha-chain of C4b and thus prevents the assembly of the C3 and C5 convertases. We have investigated the proinflammatory cytokines IL-6, IL-1beta, TNF-alpha and IFN-gamma for their potential role in the regulation of FI expression. Of the investigated cytokines, only IL-6 increased the FI-specific RT-PCR signal in isolated hepatocytes, in the two rat hepatoma-derived cell lines FAO and H4IIE or in HUVECs. Quantitative competitive RT-PCR showed an IL-6 induced upregulation of FI-specific mRNA by about ten-fold. These data are in accord with Northern blot analyses in which the FI-mRNA was upregulated by IL-6 between five- and seven-fold. IL-6, but not IL-1beta, TNF-alpha or IFN-gamma also increased FI-protein levels in cell culture supernatants by about five-fold as determined by a semiquantitative immunoblot using a novel monoclonal antibody specific for rat FI.

Amino Acid Sequence↗