Biomedical subjects

S Rose-John

Publications and source records attributed to S Rose-John.

157 records · Page 9Linked to original sources

PubMed1990

The three carboxy-terminal amino acids of human interleukin-6 are essential for its biological activity.

We have constructed on the cDNA level deletion mutants of human interleukin-6 lacking one, two, three or four amino acids from the carboxy-terminus of the molecule. After in vitro transcription and translation the biological activity of these deletion mutants was determined by two independent bioassays. Both, the mouse B9 cell proliferation assay and the fibrinogen induction assay with the human hepatoma cell line HepG2 led to the following result: already the removal of the last amino acid resulted in a five-fold loss of biological activity. An additional slight reduction was seen when two amino acids were removed from the carboxy-terminus. Interleukin-6 lacking three or four C-terminal amino acids were completely inactive. The presented results emphasize the extreme importance of the carboxy-terminus of interleukin-6 for its biological function.

Amino Acid Sequence↗

PubMed1990

Studies on the structure and regulation of the human hepatic interleukin-6 receptor.

Affinity cross-linking of 125I-labeled recombinant human interleukin-6 (IL-6) to human hepatoma cells (HepG2) allowed the detection of three IL-6-containing complexes with molecular masses of 100 kDa, 120 kDa and 200 kDa. Treatment of HepG2 cells with dexamethasone led to a time- and dose-dependent up-regulation of IL-6-receptor mRNA levels. By the use of cross-linking this effect was also seen at the protein level, where all three IL-6-binding complexes increased upon incubation of HepG2 cells with dexamethasone. Under conditions of IL-6-receptor up-regulation by dexamethasone, gamma-fibrinogen mRNA induction by IL-6 is stronger and occurs earlier than without dexamethasone. We propose therefore that the expression of the IL-6 receptor might be a rate-limiting step in acute-phase-protein induction.

Carcinoma, Hepatocellular↗

PubMed1990

Structure-function analysis of human interleukin-6. Evidence for the involvement of the carboxy-terminus in function.

C-terminally deleted analogs of human interleukin-6 (IL-6) have been constructed at the cDNA level, and after cell-free transcription and translation their biological activity was analyzed. Removal of only 4 amino acids resulted in complete loss of biological activity as determined by the B9 cell proliferation assay. Secondary structure prediction of human IL-6 resulted in 58% helix, 14% beta-structure, and 28% turn and coil (average of 3 independent methods). The circular dichroism of recombinant human IL-6 was measured in the near and far UV. Evaluation of the latter in terms of secondary structures gave 67% helix, 15% beta-structure, and 18% turn and coil.

Animals↗

PubMed1990

Microheterogeneity of human interleukin 6 synthesized by transfected NIH/3T3 cells: comparison with human monocytes, fibroblasts and endothelial cells.

A cDNA containing the entire coding region of human interleukin 6 (IL 6) was stably expressed in murine NIH/3T3 fibroblasts using a bovine papilloma virus-based expression vector with a metallothionein promoter. Expression of IL 6 in transfected cells was highly inducible by heavy metals like cadmium as measured at mRNA and protein levels. Cadmium-stimulated transfected NIH/3T3 cells synthesized and exported biologically active IL 6 (1.7 x 10(4) U/10(6) cells/24 h). IL 6 from the culture medium of transfected NIH/3T3 cells exhibited at least eight bands on Western blots after sodium dodecyl sulfate-polyacrylamide gel electrophoresis, indicating that human IL 6 expressed in NIH/3T3 cells shows a complex glycosylation pattern. Using glycosidases and the N-glycosylation inhibitor tunicamycin it was possible to discriminate between five species carrying N-linked carbohydrate side chains and two species carrying only O-linked side chains. In addition, a substantial amount of unglycosylated IL 5 was observed. IL 6 from transfected NIH/3T3 cells differed markedly in its glycosylation pattern from those of stimulated human monocytes, fibroblasts and endothelial cells.

Animals↗

PubMed1989

cDNA-cloning, sequencing and expression in glucocorticoid-stimulated quiescent Swiss 3T3 fibroblasts of mouse lipocortin I.

We isolated and sequenced mouse lipocortin I cDNA clones from a lambda gt10 cDNA library prepared from Swiss 3T3 mRNA. The homology with human lipocortin I at the amino acid level is 86%. When confluent layers of Swiss 3T3 cells were stimulated with 10% fetal calf serum, expression of lipocortin I was strongly stimulated. In parallel, DNA synthesis was induced with a peak at 24 hours after glucocorticoid treatment indicating induction of cell proliferation. In the absence of serum glucocorticoid treatment provoked neither induction of DNA synthesis nor expression of lipocortin I. We conclude that serum contains an unidentified factor, which acts synergistically with glucocorticoids on cell proliferation and lipocortin I expression.

5,8,11,14-Eicosatetraynoic Acid↗

PubMed1989

Expression of the kinase domain of mouse protein kinase C in E. coli.

The kinase domain of mouse protein kinase C type alpha has been expressed at high levels in E. coli. The protein has been purified 500-fold taking advantage of the fact that highly expressed fusion proteins precipitate out in the bacterial cell and can be solubilized in 7 M urea. The purified protein can be detected with an antibody generated against a PKC alpha derived synthetic peptide. The purified kinase domain exhibits no measurable kinase activity in a protamin phosphorylation assay. This could be an indication that post-translational modifications of the protein kinase domain which do not happen in bacteria are a requirement for functional enzyme activity or that the regulatory domain of protein kinase C is indispensable for kinase function.

Animals↗

PubMed1988

Molecular cloning of mouse protein kinase C (PKC) cDNA from Swiss 3T3 fibroblasts.

A set of complementary DNAs (cDNAs) that encode the complete 672-amino acid sequence for mouse protein kinase C (PKC) type alpha have been isolated from a mouse Swiss 3T3 cDNA library. Extensive rescreening of this cDNA library only resulted in the isolation of clones of the same PKC species, indicating that Swiss 3T3 fibroblasts express exclusively PKC-alpha. This enzyme is encoded by two different mRNAs that exhibit a substantial heterogeneity in their 3'-noncoding regions. This heterogeneity could have been derived from alternative splicing of the pre-mRNAs or be due to differential usage of the polyadenylation motif. The expression of PKC mRNA in fibroblasts is very low and it is not influenced by treatment with the tumor promoter 12-O-tetradecanoyl-13-phorbol-acetate.

Animals↗

PubMed1988

Bryostatin 1, an activator of protein kinase C, mimics as well as inhibits biological effects of the phorbol ester TPA in vivo and in vitro.

The macrocyclic lactone bryostatin 1 activates protein kinase C as effectively as the phorbol ester 12-O-tetradecanoyl-phorbol-13-acetate (TPA). Nevertheless, there are only certain TPA-effects that can be induced by bryostatin 1. These include stimulation of epidermal DNA synthesis and alkaline phosphatase activity in vivo as well as activation of the Ca2+-independent, phospholipid-requiring phosphorylation of an epidermal protein in a cell-free system. Various other TPA-effects in vivo and in vitro, which are not mimicked by bryostatin 1 can be inhibited by applying bryostatin 1 30 min prior to TPA. TPA-effects suppressible by bryostatin 1 include the Ca2+-dependent stimulation of arachidonic acid and prostaglandin E2 release, of ornithine decarboxylase (ODC) activity and ODC-mRNA expression and of transglutaminase activity in keratinocytes in vivo and/or in vitro and, in addition, Epstein-Barr virus induction in Raji cells. The same is true for the conversion step (first stage of promotion) of multistage carcinogenesis. In contrast to the TPA induction of arachidonic acid and prostaglandin E2 release and of transglutaminase activity, induction by the Ca2+-ionophore and by high Ca2+-shift, respectively, are not significantly inhibited by bryostatin 1. We suggest that bryostatin 1 might inhibit a specific 'Ca2+-component' of TPA action.

Animals↗

PubMed1988

Differential effects of phorbol esters on c-fos and c-myc and ornithine decarboxylase gene expression in mouse skin in vivo.

Hyperplasiogenic and tumor-promoting phorbol esters such as 12-O-tetradecanoylphorbol-13-acetate or 12-O-retinoylphorbol-13-acetate induce the sequential transient expression of the proto-oncogenes c-fos and c-myc and the ornithine decarboxylase gene in mouse skin in vivo. This sequence of biochemical events probably depends on an activation of protein kinase C by these agents. The non-irritant skin mitogens 4-O-methyl-12-O-tetradecanoylphorbol-13-acetate and ethyl phenyl propiolate do not increase the expression of these genes to a comparable extent. Thus, 12-O-tetradecanoylphorbol-13-acetate and 12-O-retinoylphorbol-13-acetate induce epidermal hyperproliferation by different biochemical mechanisms as do 4-O-methyl-12-O-tetradecanoylphorbol-13-acetate and ethylphenylpropiolate.

Animals↗

PubMed1987

The induction of ornithine decarboxylase by the tumor promoter TPA is controlled at the post-transcriptional level in murine Swiss 3T3 fibroblasts.

The expression of ornithine decarboxylase (ODC) mRNA after treatment of murine Swiss 3T3 cells with the tumor promoter TPA was studied. The induction of ODC mRNA was detectable after 20-40 min, peaked after 60-120 min and declined within 24 hrs. Using an in vitro nuclear transcription assay, we found that the polymerase II density on the ODC gene is not affected by TPA treatment. Additionally, we were able to detect stable ODC mRNAs in cycloheximide pretreated fibroblasts. These two different experimental approaches lead us to the interpretation that in Swiss 3T3 cells TPA controls ODC expression predominantly at the post transcriptional level by prolonging the half-life of ODC-mRNA.

Animals↗

PubMed1987

An evaluation of 2,5-norbornadiene as a reversible inhibitor of ethylene action in deepwater rice.

Partial submergence of deepwater rice (Oryza sativa L. cv Habiganj Aman II) elicits three responses: enhancement of internodal elongation, inhibition of leaf growth, and promotion of adventitious root formation. All three responses can be induced in isolated stem sections by treatment with ethylene. Dose-response curves indicate that the responses are linearly related to the logarithm of the ethylene concentration over two orders of magnitude. Application of the cyclic olefin 2,5-norbornadiene (NBD) to ethylene-treated sections results in a parallel shift in dose-response curves to higher ethylene concentrations, indicating that NBD behaves as a competitive inhibitor of ethylene action. Internodal elongation of stem sections is promoted by gibberellic acid (GA(3)) in the absence of exogenous ethylene. Endogenous ethylene levels do not increase in GA(3)-treated sections, and application of NBD does not prevent GA(3)-promoted elongation. To the contrary, NBD treatment results in increased growth at intermediate GA(3) concentrations. These results support the idea that ethylene acts through endogenous GA in promoting growth in deepwater rice. NBD applied to GA(3)-treated stem sections results in increased ethylene production. This enhancement of ethylene formation is reversed by application of either ethylene or propylene, indicating that ethylene biosynthesis in rice internodes is under negative feedback control.

Journal Article↗

PubMed1984

Effect of submergence on the cell wall composition of deep-water rice internodes.

The cell wall composition of internodes of deep-water rice plants (Oryza sativa L. cv Habiganj Aman II) which were induced to grow rapidly by submergence in water was compared to that of nonsubmerged plants which grew slowly. No differences could be detected in cellulose, uronic acid, and lignin content expressed on a dry weight basis. Cell wall preparations of rapidly growing, submerged internodes contained more hydroxyproline and had a higher hydration capacity than those of control internodes. The silicon content of submerged rice internodes was considerably lower than that of air-grown plants. The role of silicon as a structural component of the cell wall of grasses is discussed in relation to lodging of deep-water rice plants after the flood waters have receded.

Journal Article↗

PubMedDate not supplied

IL-6 type cytokine receptor complexes: hexamer, tetramer or both?

The typical protein fold of most cytokines is a bundle of four antiparallel helices. This 'four-helical bundle fold' seems to be unique to cytokines and has not been detected in other proteins. Cytokine receptors, however, can be classified as a subfamily of the immunoglobulin superfamily. Cytokines using the same receptor subunits are grouped into cytokine families. The interleukin-6 (IL-6) type cytokine family comprises six members. IL-6 type cytokines may interact with three receptor subunits instead of the usual two subunits. A tetramer would be the simplest model to describe such a receptor complex, but present orthodoxy describes the active complexes of IL-6 and ciliary neurotrophic factor (CNTF) as hexamers. Here, we summarize the structural and biochemical information on IL-6 type cytokines and discuss interactions between cytokine and individual receptor subunits at alternative positions. Contradictory results regarding the stoichiometry and assembly of signaling receptor complexes are rationalized by a new, unique model. The model stipulates that a ligand-induced transition from an active tetrameric to an inactive hexameric complex serves as a molecular switch that turns off cytokine signals in the presence of supraoptimal cytokine concentrations.

Biopolymers↗