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E Martens

Publications and source records attributed to E Martens.

30 records · Page 2Linked to original sources
PubMed1993

Gene sequence, cDNA construction, expression in Escherichia coli and genetically approached purification of porcine interleukin-1 beta.

A genomic clone (PIL3) covering the 8.8-kb prointerleukin-1 beta ('catabolin') gene of the domesticated swine (Sus scrofa domestica) was isolated from a genomic library and characterized by nucleotide sequencing. Typical features of the gene include a seven-exon structure, with the highest degree of nucleotide and amino acid conservation among human and porcine genes being found in the receptor-binding portion encoded by exons six and seven. Three 250-bp repetitive elements with a > 75% similarity to the pig repetitive element-1 family sequence are located in untranslated gene segments. Southern-hybridization experiments disclosed extensive genomic heterogeneity of the porcine interleukin-1 beta (IL-1 beta) gene region, suggesting a duplication of at least the 3' half of the gene in the porcine genome. Since similar hybridization patterns were observed for wild boar (Sus scrofa) genomic DNA, it was concluded that this gene rearrangement had preceded domestication of the wild swine. In addition, the cDNA for processed porcine IL-1 beta was constructed through polymerase-chain-reaction-mediated exon fusion by overlap extension starting from the genomic template. Recombinant IL-1 beta was expressed in Escherichia coli as a fusion protein containing an N-terminal hexahistidine tag followed by a factor-Xa-cleavage site. The protein was efficiently purified through adoption of a scheme that consisted of four alternating cycles of immobilized metal-ion-affinity chromatography and size-exclusion chromatography. 13.8 mg highly purified recombinant porcine IL-1 beta was obtained starting from a 900-ml thermo-induced E. coli culture (final endotoxin concentration < 0.22 ng/ml). The protein behaved homogeneously as a monomeric species, which was reactive in Western-blot experiments with an anti-(human-IL-1 beta) serum and which appeared to induce gelatinase B in MDBK cells in a dose-dependent fashion.

Amino Acid Sequence↗
PubMed1993

Refolding and single-step purification of porcine interferon-gamma from Escherichia coli inclusion bodies. Conditions for reconstitution of dimeric IFN-gamma.

Recombinant porcine interferon-gamma, overexpressed in Escherichia coli, was found to accumulate in cytoplasmic inclusion bodies. The influence of various physicochemical parameters on refolding was investigated using 6 M guanidine/HCl-solubilised inclusion bodies which had been purified by ultracentrifugation on a sucrose step gradient. It appeared that the yield of reconstitution of denatured protein reached 60-70% under optimum conditions, i.e. at an intermediary guanidine/HCl concentration of 0.5 M and at a protein concentration of 10-20 microM (0 degrees C). Since intermediary guanidine/HCl concentrations at 0.5-1.65 M increasingly promoted off-pathway formation of soluble aggregates and at 0.5-0.2 M progressively promoted precipitation, maximal recovery of biologically active protein required a twofold transition in the surrounding guanidine/HCl concentration (6 M-->0.5 M-->0 M). A single additional size-exclusion chromatographic step yielded a final product that was > 99.5% pure, had specific antiviral activity > 10(7) U/mg protein and contained < or = 25 pg/ml endotoxin. Cross-linking by means of disulfosuccinimidyl tartarate revealed that the refolded protein possessed a dimeric structure. Furthermore, we have characterized three different molecular species of recombinant porcine interferon-gamma that are formed under non-optimal refolding conditions (1 M guanidine/HCl) and that differ from each other in specific activity, size and stability. One of these converts irreversibly into dimeric interferon-gamma in a temperature-dependent manner and is therefore considered as a productive folding intermediate.

Animals↗
PubMed1993

Increased circulating interleukin-6 (IL-6) activity in endotoxin-challenged mice pretreated with anti-IL-6 antibody is due to IL-6 accumulated in antigen-antibody complexes.

Mice pretreated with monoclonal anti-interleukin-6 (IL-6) antibody and then challenged with lipopolysaccharide (LPS), paradoxically develop higher levels of circulating biological IL-6 activity, as measured by the hybridoma growth promotion assay, than mice similarly challenged but not pretreated with antibody. Here we provide evidence that this increased biological activity was entirely accounted for by the presence of increased amounts of IL-6 protein, which could be isolated by immunoaffinity chromatography and subsequently visualized after gel electrophoresis. Chromatography on a protein G matrix and a sandwich ELISA allowed to demonstrate that all IL-6 present in the serum was in the form of antigen-antibody complexes. Serum samples of antibody-treated animals which contained the highest biological activity typically contained near equimolar concentrations of IL-6 and antibody. In vitro neutralization tests with pure antibody and IL-6 demonstrated that, with both antibodies tested, more than 1000-fold molar excess of antibody is needed for neutralization in the hybridoma growth assay. It is concluded that increased biological activity in serum of the anti-IL-6 antibody-treated mice is due to sequestration of the endogenous IL-6 in the form of antigen-antibody complexes which, due to the lack of sufficient antibody excess, produce nearly full activity in the hybridoma growth assay.

Animals↗
PubMed1993

Synergistic toxicity of IFN-gamma-producing Escherichia coli K12 cells.

Genetically modified microorganisms (GMMs) are frequently used as producers of mammalian immunomodulatory proteins, e.g. interferons and interleukins. Here we have examined the question of whether such GMMs interact in a way different from that of their non-modified parent micro-organisms with mammalian antimicrobial defence systems. As a typical GMM host micro-organism we used Escherichia coli K12, and as a typical immunomodulatory protein produced by a GMM we used mouse interferon-gamma (MuIFN-gamma). Two experimental systems are described in which synergistic "toxic" biological effects are induced by a combined treatment with E. coli and MuIFN-gamma but not, or less so, by the parental strain and the recombinant protein separately. First, it is shown that the IFN-gamma-producing GMM, or mixtures of E. coli cells and IFN-gamma, are cytolytic for mouse embryo fibroblastoid cells (MEF), whereas no cell killing occurs in MEF cultures treated with control E. coli cells or in those treated with bacteria-free recombinant IFN-gamma. Second, it is demonstrated that intraperitoneal injection in mice of high but not low numbers of control E. coli K12 cells induces a shock-like mortality, whereas co-injection with IFN-gamma induces killing at low numbers. IFN-gamma-producing E. coli cells cause a mortality rate that does not differ from that of control E. coli cells, probably because in these experimental conditions the level of recombinant MuIFN-gamma per cell is insufficiently high. Taken together, these data indicate that synergistic toxic effects induced by bacteria and their recombinant products can occur and may in certain situations enhance the intrinsic toxic capacity of the GMM. Synergistic toxic effects may thus be of relevance for identifying the safety level that should be employed when working with GMMs.

Animals↗
PubMed1991

Murine interferon-gamma/interleukin-1 fusion proteins used as antigens for the generation of hybridomas producing monoclonal anti-interleukin-1 antibodies.

In several biological systems interferon-gamma (IFN-gamma) and interleukin-1 (IL-1) act synergistically. We therefore examined whether it would be possible to construct IFN-gamma/IL-1 hybrid proteins that would be more active than the individual components. Hybrid proteins were examined that consisted of the amino-terminal 118 residues of mouse IFN-gamma and the 156 or 152 carboxyl-terminal residues of mouse IL-1 alpha or IL-1 beta, respectively. They were obtained by ligation of the respective coding sequences and expression of the fused genes under control of the PL promotor in Escherichia coli. Both the IFN-gamma/IL-1 alpha and the IFN-gamma/IL-1 beta fusion proteins were purified by affinity chromatography on an anti-IFN-gamma monoclonal antibody column. Analysis of biological activities showed that these fusion proteins were less active than the individual cytokines. Specific antiviral activity of the IFN-gamma/IL-1 beta hybrids was less than 0.1% that of IFN-gamma and D10.G4.1 T-cell proliferative (IL-1) activity amounted to 0.1% that of mouse IL-1. Affinity-purified preparations of the IFN-gamma/IL-1 alpha hybrid were found to contain variable proportions of a Mr 14,000 degradation product possessing IFN-gamma activity, whereas the undegraded Mr 30,000 fusion protein, while being devoid of detectable IFN-gamma activity, did possess IL-1 activity (1%). Serum from rats immunized with the IFN-gamma/IL-1 alpha hybrid contained high levels of IL-1 alpha-binding and -neutralizing antibodies and IFN-gamma-binding antibodies, but no detectable levels of IFN-gamma-neutralizing antibodies.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1991

[Bronchoalveolar lavage--a diagnostic method in chronic nonspecific bronchopulmonary diseases in childhood? 2. Studies of cellular and humoral parameters in BAL irrigation fluid].

With a view of the pathogenesis of chronic bronchopulmonary diseases the interrelations between infections and evolving defense system are of interest, they are perhaps detectable by means of diagnostic bronchoalveolar lavage. We carried out cytodifferentiation, investigated adenosine deaminase activities and interleukin 1 formation of macrophages, determined immunoglobulin concentrations (secretory IgA), lysozyme, alpha 2-macroglobulin, alpha 1-antitrypsin, albumin. Because the cytodifferentiation yields insight into topical inflammatory reactions, shows diagnostic useful informations in single cases and because it is simple to carry out we can recommend it for each bronchological examination. There were no results specific for any disease group for parameters mentioned above.

Adenosine Deaminase↗
PubMed1988

Assessment of insulin action in man: role of hyperglycemia.

The effect of hyperglycemia on insulin-induced glucose metabolism (M) was investigated in healthy subjects using sequential clamp protocols at constant insulin + somatostatin infusions and varying plasma glucose. During euglycemia (4.8 mmol/l) M increased from 5.6 to 12.5 mg.kg-1.min-1 with increasing plasma insulin (0.34-3.00 nmol/l). At increasing glucose (6.7 mmol/l), M further increased (9.7 to 19.2 mg.kg-1.min-1) with the plasma insulin level (0.41 to 2.99 nmol/l). At a plasma glucose level of 9.8 mmol/l insulin (0.42 to 3.17 nmol/l) was still effective to increase M (13.7 to 25.2 mg.kg-1.min-1). Regression analysis showed that hyperglycemia does not only increase the maximal insulin-stimulated M, but also decreases the insulin concentration causing a half maximum effect. During prolonged clamp studies M increased by about 10% per h, independent by the plasma glucose level. We conclude that hyperglycemia increases M by increasing insulin responsiveness as well as insulin sensitivity. Data derived from euglycemic clamp studies alone are of limited value with respect to the assessment of insulin action.

Adult↗
PubMed1988

[Bronchoalveolar lavage (BAL)--a diagnostic method in chronic nonspecific bronchopulmonary diseases in childhood. 1. Implementation and tolerance].

While the effectiveness of bronchoalveolar lavage (BAL) in pediatric therapy has been recognized for years, there are hardly any data on its usefulness in diagnosis, its performance and compatibility in childhood. After 123 bronchological investigations including BAL on 118 children in the age of three months through 16 years, with lavage fluid amounts between 40 and 160 ml, febrile reactions occurred in 17 cases (= 13.8%), i.e. no more frequent than in a group of 100 children bronchologically investigated but without BAL. After blood gas analyses, ECG examinations, blood pressure readings the compatibility of BAL turned out to be good in children of every age. Bronchography pictures after BAL were not reduced in quality, atelectasis formation occurred rarely.

Adolescent↗
PubMed1987

Excretion of 15N and incorporation into plasma proteins after high-dosage pulse labelling with various tracer substances in infants.

The suitability of a biosynthetically produced [15N] yeast-protein-thermitasehydrolysate ([15N] YPTH), [15N] yeast protein and [15N] glycine for use as 15N-tracers was tested in three groups of four infants each. The [15N] YPTH was obtained by hydrolysing 15N-labelled yeast protein with thermitase, a proteinase from Thermoactinomyces vulgaris. Following oral single-pulse labelling in a dosage of 10 mg 15N/kg body weight the 15N-excretion in stools and urine as well as the 15N-abundance in plasma proteins and in the TCA-soluble plasma fraction were determined. The [15N] YPTH differs from [15N] glycine in terms of the complete distribution of 15N among all 20 amino acids. This could be demonstrated by a distinctly lower [15N]ammonia (0.5 per cent) and a higher [15N2] urea excretion (5.0 per cent) compared with [15N] glycine (1.2 and 3.4 per cent respectively). The faecal loss of 15N from the [15N] YPTH was 3.7 per cent of the tracer dose, while the corresponding value after administration of [15N] yeast protein was found to be 7.4 per cent on average. There were no differences between the tracer substances in terms of the measured 15N-abundance in the plasma proteins (mean: 0.07 atom per cent excess) and in the TCA-soluble fraction (mean: 0.21 atom per cent excess).

Blood Proteins↗
PubMed1983

[Results of treatment of functional sterility with clomifen].

Our experiences in the treatment of functional sterility in 482 patients with clomiphene are reported. The dosage of clomiphene was 100 mg per day for 5 days. If treatment was without success after 3 cycles dosage was changed to 150 mg per day for 7 days. The ovulation induction rate detected by basal body temperature was 74.5%. Conceptions were obtained in 35.7%. Pregnancy was terminated by spontaneous abortion in 18.8%. In high weight patients there was the best success rate with clomiphene treatment.

Adult↗