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G Schumann

Publications and source records attributed to G Schumann.

At least 55 records · Page 3Linked to original sources
PubMed1998

Potential link between interleukin-6 and arachidonic acid metabolism in Alzheimer's disease.

Prostaglandins (PGs) and cytokines, such as interleukin-1 (IL-1) and interleukin-6 (IL-6), have been implicated in the etiopathology of various inflammatory and degenerative disorders, including Alzheimer's disease (AD). Previously, we detected the presence of IL-6 in cortices of AD patients. On the other hand, non-steroidal antiinflammatory drugs (NSAIDs), potent inhibitors of prostaglandin synthesis, have been shown to be beneficial in the treatment of AD. Until now, it remained unclear whether and how these two observations were functionally connected. Here, we show that PGs are able to induce IL-6 synthesis in a human astrocytoma cell line. PGE1 and PGE2, but not PGD2 and PGF2 alpha, led to a rapid and transient induction of astrocytic IL-6 mRNA, followed by IL-6 protein synthesis. Furthermore, PGE2 potentiated IL-1 beta-induced IL-6 mRNA synthesis. These results suggest a possible link between the release of PGs from activated microglia and the astrocytic synthesis of IL-6, which itself may affect neuronal cells, as hypothesized for Alzheimer's disease. Finally we demonstrate that microglia are a strong source of PGE2 synthesis indicating that these cells may act as the origin of the pathogenic cascade.

Alprostadil↗
PubMed1997

Antiretroviral effect of a gag-RNase HI fusion gene.

We have previously shown that a molecule consisting of a fusion of a Ca(2+)-dependent nuclease (from Staphylococcus aureus) to a retroviral coat protein specifies a potent antiviral specific for that retrovirus. Genes specifying such fusion proteins can be delivered to virus-susceptible cells, providing an antiviral gene therapy aimed at limiting virus spread. We report here the results of experiments to vary the nuclease moiety of such fusion proteins. We found that one nuclease. Serratia marcescens nuclease, was extremely toxic to host cells and hence not likely to be useful for therapeutic purposes. A second nuclease, Escherichia coli RNase Hl was found to be nontoxic and highly effective against a murine leukemia virus when it was fused to the leukemia virus coat protein. The fusion protein was enzymatically active and stably expressed, without apparent toxicity to host cells. Reduction in infectious virus output was as high as 97-99%. These studies provide a model system for the development of gene therapeutic agents aimed at combating retroviral infections in vivo.

Animals↗
PubMed1996

Activation and analysis of cryptic crt genes for carotenoid biosynthesis from Streptomyces griseus.

Genes encoding enzymes with sequence similarity to carotenoid biosynthetic enzymes of other organisms were cloned from Streptomyces griseus JA3933 and transformed into the colourless (non-daunorubicin producing) mutant Streptomyces griseus IMET JA3933/956/2. Cells harbouring these genes showed an orange-red pigmentation, caused by the strongly hydrophobic, membrane-bound lycopene. The cloned fragment (9 kb) contained seven genes, four transcribed in one direction (crtEIBV) and three (crtYTU) transcribed convergently to them. Three of these genes encode polypeptides that resemble geranylgeranyl-pyrophosphate (GGPP) synthases (CrtE), phytoene synthases (PS) (CrtB) and phytoene dehydrogenases (PDH) (CrtI), respectively, of various bacteria. These enzymes are sufficient for the formation of lycopene. crtE alone was sufficient to induce zeaxanthin formation in an Escherichia coli clone containing the crt gene cluster from Erwinia herbicola deleted for crtE. The combination of crtE and crtB led to formation of phytoene in S. griseus. The putative crtEp promoter region was cloned and mapped by primer extension analysis. In a gel retardation experiment, this fragment was specifically shifted by an unknown protein. CrtY shows similarity to lycopene cyclases that convert lycopene into beta-carotene, CrtT resembles various methyltransferases and CrtU a dehydrogenase. We conclude that these genes are functionally intact, but not expressed (cryptic) in the wild-type S. griseus strain.

Bacterial Proteins↗
PubMed1996

Therapeutic effect of Gag-nuclease fusion protein on retrovirus-infected cell cultures.

Capsid-targeted viral inactivation is a novel protein-based strategy for the treatment of viral infections. Virus particles are inactivated by targeting toxic fusion proteins to virions, where they destroy viral components from within. We have fused Staphylococcus nuclease (SN) to the C-terminal end of Moloney murine leukemia virus Gag and demonstrated that expression of this fusion protein in chronically infected chicken embryo fibroblasts resulted in its incorporation into virions and subsequent inactivation of the virus particles by degradation of viral RNA. Release of particles incorporating Gag-SN fusion proteins into the extracellular milieu activates the nuclease and results in destruction of the virion from within. By comparing the effects of incorporated SN and SN*, an enzymatically inactive missense mutant form of SN, on the infectivity of virus particles, we have clearly demonstrated that nucleolytic activity is the antiviral mechanism. Expression of Gag-SN fusion proteins as a therapeutic agent causes a stable reduction of infectious titers by 20- to 60-fold. The antiviral effect of capsid-targeted viral inactivation in our model system, using both prophylactic and therapeutic approaches, suggests that a similar anti-human immunodeficiency virus strategy might be successful.

Animals↗
PubMed1996

Consensus of a group of professional societies and diagnostic companies on guidelines for interim reference ranges for 14 proteins in serum based on the standardization against the IFCC/BCR/CAP Reference Material (CRM 470). International Federation of Clinical Chemistry. Community Bureau of Reference of the Commission of the European Communities. College of American Pathologists.

The release in 1993 of a new reference material for serum proteins, CRM 470/RPPHS 5 has given rise to a great improvement in the between-laboratory variability of serum protein measurements worldwide. Conversion to the new reference material results in significant changes in reference values for some proteins. The establishment of new reference ranges will take a considerable time, and in the interim several professional societies and diagnostic companies have agreed to use consensus reference ranges based on studies already undertaken.

Blood Proteins↗
PubMed1994

Specificity of signal transduction through CD16, TCR-CD3 and BCR receptor chains containing the tyrosine-associated activation motif.

FcR gamma III (CD16) is a member of a family of receptor proteins that bind to Fc regions of IgG molecules. Ligation of CD16 transduces signal and alters tyrosine phosphorylation of proteins in natural killer, B and T cells. However, the identity of protein tyrosine kinases (PTKs) involved in CD16 signaling and the mode of their interaction with the receptor are not completely understood. Here, we show that the transmembrane form of CD16 transfected in a CD3- Jurkat cell line associates with p56lck and ZAP-70 PTKs. Using chimeric proteins consisting of the extracellular domain of CD16 and the cytoplasmic portions of the zeta chain, we find that these PTKs interact with the 17-residue activation motif present in the zeta chain. Substitution of tyrosine residues in the motif with phenylalanine blocks the interaction of PTKs and abrogates signal transduction. Comparative studies demonstrate that similar to zeta, MB1 protein of the IgM receptor also binds to p56lck and ZAP-70 in T cells and induces strong activation responses, whereas CD3 gamma, delta and epsilon chains do not interact with ZAP-70 and transduce weak signals. Together, these results demonstrate that: (i) PTKs bound to CD16 mediate signal transduction, (ii) ZAP-70 plays a crucial role in signaling and (iii) apart from tyrosine residues in the activation motif, other structural constraints also regulate the interaction of PTKs with the receptor molecule.

CD3 Complex↗
PubMed1994

Internally located and oppositely oriented polymerase II promoters direct convergent transcription of a LINE-like retroelement, the Dictyostelium repetitive element, from Dictyostelium discoideum.

The Dictyostelium discoideum NC4 genome harbors approximately 150 individual copies of a retrotransposable element called the Dictyostelium repetitive element (DRE). This element contains nonidentical terminal repeats (TRs) consisting of conserved building blocks A and B in the left TR and B and C in the right TR. Seven different-sized classes of RNA transcripts from these elements were resolved by Northern (RNA) blot analysis, but their combined abundance was very low. When D. discoideum cells were grown in the presence of the respiratory chain blocker antimycin A, steady-state concentrations of these RNA species increased 10- to 20-fold. The D. discoideum genome contains two DRE subtypes, the full-length 5.7-kb DREa and the internally deleted 2.4-kb DREb. Both subtypes are transcribed, as confirmed by analysis of cloned cDNA. Primary transcripts from the sense strand originate at nucleotide +1 and terminate at two dominant sites, located 21 or 28 nucleotides upstream from the 3' end of the elements. The activity of a reasonably strong polymerase II promoter in the 5'-terminal A module is slightly upregulated by the tRNA gene located 50 +/- 4 nucleotides upstream and drastically reduced by the adjacent B module of the DRE. Transcripts from the opposite DNA strand (complementary-sense transcripts) were also detected, directed by an internally located polymerase II promoter residing within the C module. This latter transcription was initiated at multiple sites within the oligo(dA12) stretch which terminates DREs.

Animals↗
PubMed1994

Kinetics of extracellular potassium concentration in irradiated red blood cells.

BACKGROUND: Irradiation of cellular blood components prevents onset of transfusion-associated graft-versus-host disease. The present study was designed to monitor the post-irradiation changes of the extracellular potassium concentration in red blood cells (RBC) stored in SAG-M (saline adenine glucose-mannitol), in order to estimate the right time for their prophylactic irradiation and the right span of post-irradiation storage. MATERIALS AND METHODS: Ten units each of fresh, 10 days and more than 20 days stored SAG-M RBCs were divided into 2 equal portions each. One portion was gamma-irradiated with 30 Gy (137Cs-source). Another 10 fresh RBC units were separated, and portions were irradiated with 15, 30, 60 or 120 Gy. The determination of potassium was performed simultaneously in corresponding irradiated and non-irradiated portions; immediately after irradiation, after 1 and 5 days, and every 10 days of additional storage (up to 30 days). All measurements were performed in the cell-free supernatant, using flame emission photometry. RESULTS: The extracellular potassium concentration increased permanently with the duration of storage both in non-irradiated and irradiated specimens. At the end of the period of storage this increase in the irradiated portions was approximately 2 times the potassium concentration of the non-irradiated portions (60 mmol/l vs. 38 mmol/l, p < 0.001). Thereby no significant differences of the potassium efflux rates were estimated at the end of the period of storage between fresh RBCs irradiated and stored RBCs irradiated. The mean 5 days post-irradiation potassium release from fresh RBC units was similar to that in 25-30 days stored non-irradiated portions (34.3 mmol/l vs. 38 mmol/l). The extracellular potassium increase was irradiation dose-dependent (r = 0.89, p < 0.001). CONCLUSIONS: SAG-M RBCs can be irradiated with 30 Gy immediately post-harvest and subsequently stored for a couple of days prior to transfusion without producing critically high extracellular potassium concentrations.

Blood Preservation↗
PubMed1994

Characterization of transcripts from the Dictyostelium discoideum retrotransposable genetic element DRE.

All of the approximately 150 copies of the Dictyostelium discoideum retrotransposable element DRE are integrated 50 +/- 4 nucleotides upstream from different transfer RNA genes. These genomic regions are generally devoid of informative DNA why integration of the retrotransposon at these position never causes phenotypic mutations. This property makes DRE attractive as a tool in gene therapy where vectors with predictable integration specificities are still not available. Due to the nature as retrotransposon transcription is a prerequisite for the element to integrate at a new genomic position. Here we describe some transcription properties of DRE based on Northern blot analyses and on the characterization of in vitro synthesized cDNAs.

Animals↗
PubMed1993

Different organization of the tRNA-gene-associated repetitive element, DRE, in NC4-derived strains and in other wild-type Dictyostelium discoideum strains.

The retrotransposon DRE (Dictyostelium repetitive element) was discovered in the course of an extensive study concerning the genomic organization of tRNA genes in the NC4-derived strains AX2 and AX3 of the cellular slime mold Dictyostelium discoideum. As a striking feature, DRE was found exclusively in a constant orientation and at a constant distance upstream from different tRNA genes. About 150-200 DRE with intact 5'-terminal-repeat structures are present in NC4-derived strains. These strains were termed high-copy DRE strains (HCD strains) as opposed to low-copy DRE strains (LCD strains) such as the wild-type D. discoideum isolates DD61, WS380B, OHIO and V12. LCD strains contain only 3-15 DRE with intact 5'-terminal-repeat-structures. However, in addition to these few intact elements, many 5'-truncated DRE elements are present in LCD strains. In HCD strains, most DRE show typical structural characteristics of retrotransposons containing terminal repeats at both ends, which seems to be one prerequisite for active transposition. In LCD strains, however, most DRE elements are 5'-truncated, which is a common feature of eukaryotic LINE elements. Despite their truncated 5'-ends, DRE in LCD strains retain unique integration specificities, i.e. they are always found position-specifically and orientation-specifically integrated in front of tRNA genes, flanked by a 12-16-bp target-site duplication.

Animals↗
PubMed1993

The Dictyostelium discoideum 5S rDNA is organized in the same transcriptional orientation as the other rDNAs.

In eukaryotes 5S rRNA genes are transcribed by RNA polymerase III. These genes occur in D. discoideum on the ca. 90 copies of an extrachromosomal palindrom together with the other ribosomal RNAs, which are generally transcribed by RNA polymerase I. A 5S rRNA gene has been isolated and its nucleotide sequence as well as the organization of the gene relative to the RNA polymerase I operon has been determined. The sequence of the coding region corresponds exactly to an earlier published 5S rRNA sequence. The genes are located just downstream from the 26S RNA and transcription orientations of the pol I genes and the pol III gene point into the same direction, away from the centromer of the palindrom. The isolated gene appears to be functional since it serves as a specific target for a nuclear protein, most likely TFIIIA. A genomic copy of a 5S rRNA pseudogene has been isolated from the D. discoideum strain V12. This pseudocopy contains nucleotides 52 to 82 of a bona fide 5S rRNA gene with only three mismatches. It resides 78 nucleotides downstream from the glu13(UUC) tRNA gene which in the D. discoideum strain V12 is associated with the retrotransposable element DRE.

Animals↗
PubMed1993

Nucleotide sequence analysis of five putative Streptomyces griseus genes, one of which complements an early function in daunorubicin biosynthesis that is linked to a putative gene cluster involved in TDP-daunosamine formation.

Sequence analysis of the lkmB region of the daunorubicin biosynthetic gene cluster of Streptomyces griseus JA3933 revealed two contiguous open reading frames (ORF) in the same orientation, and three ORFs in the opposite orientation together extending over a 4.6 kb region adjacent to a homologue of the S. peucetius dnrJ gene. ORF1 complemented in trans the lkmB mutation, which seems to affect an early step in daunorubicin biosynthesis. Its deduced product showed no similarity to any known enzyme in the databases. The mutation in ORF1 was localised to a C-T transition at position 1172, leading to the change from a glycine to aspartic acid in the deduced protein. The lack of any homology to known polyketide synthesis enzymes indicates a regulatory role for the product of ORF1, despite the ability of lkmB mutants to further metabolise alkanoic acid. The genes of the oppositely oriented cluster seem to be involved in sugar metabolism. The putative ORF3 protein revealed strong homology to eukaryotic acyl CoA dehydrogenases and might encode an enzyme for the oxidoreduction preceding the introduction of the amino group into daunosamine, and the ORF4 protein is homologous to several epimerases, central enzymes in the formation of the L-2,3,6-trideoxy-3-aminohexoses from TDP-D-glucose. ORF5 seems also to be related to enzymes metabolising nucleotide-activated hexoses.

Amino Acid Sequence↗
PubMed1993

Monitoring cyclosporin A (ciclosporin, INN) concentrations in whole blood: evaluation of the EMIT assay in comparison with HPLC and RIA.

We report here on an evaluation of the enzyme-multiplied immunoassay technique (EMIT) from Syva for cyclosporin A (ciclosporin, INN) concentration measurements in whole blood. The assay incorporates a monoclonal antibody for specific determination of ciclosporin. Measurements by EMIT were performed on the Cobas Mira-S from Roche. A total of 197 blood specimens from heart-(n = 74), kidney-(n = 62) and liver-(n = 61) transplant recipients were analyzed. EMIT values correlated well with those obtained by HPLC, as well as with those obtained by a selective radioimmunoassay (INCStar). Ciclosporin concentrations determined by EMIT (y) agreed very well with those by RIA, and averaged 8% higher than those by HPLC (x) [n = 197, mean = 143 micrograms/l, y = 155 micrograms/l, y = 1.09x - 0.6, r = 0.969]. Within-series and between-days CVs ranged from 4.6% to 7.3% for ciclosporin concentrations > 100 micrograms/l, and from 5.5% to 10.5% for ciclosporin concentrations between 69.5 micrograms/l and 100 micrograms/l. The within-series CV for a concentration of 45.5 micrograms/l was 14.8%. Calibration employing a 2-point mode instead of a continuous mode of UV-signal evaluation improved the precision of the EMIT assay at low ciclosporin concentrations. Sample pretreatment required thorough and skillful performance to avoid false positive ciclosporin measurements. We conclude that the EMIT assay is specific, and rapid to perform. It can be effectively used in the monitoring of ciclosporin concentrations in whole blood.

Antibodies, Monoclonal↗
PubMed1992

Two distinct subforms of the retrotransposable DRE element in NC4 strains of Dictyostelium discoideum.

Approximately 2% of the Dictyostelium discoideum genome consists of multiple copies of a retrotransposable element termed DRE (Dictyostelium Repetitive Element). These elements have always been found integrated in a position and orientation-specific manner 50 +/- 4 nucleotides upstream of the coding region of tRNA genes (tDNAs). An intact DRE is 5.7 kb long. It carries an extensive coding region flanked by non-identical long terminal repeats (LTRs), composed of three distinct modules A, B and C. The left LTR proximal to the tRNA gene contains one or several A-modules followed by a single B-module (AnB). By contrast, the right LTR is composed of a B-module followed by a C-module (BC). Approximately 50% of the DRE elements in NC4 derivatives of D. discoideum are structurally different from the 5.7 kb DRE described above. They carry the following alterations: a) a 3.1 kb deletion in the coding region; b) two small deletions of 8 and 29 nucleotides in the B-module of the right LTR; c) a 72 bp deletion in the B-C junction; and d) three distinct point mutations within the A-module of the left LTR. The deletion in the open reading frame encompasses the putative coding regions for reverse transcriptase adn integrase. At least 60 copies of this smaller 2.4 kb DRE subtype are found in the genome of D. discoideum NC4 strains associated with tRNA genes. Thus, inspite of their lack in reverse transcriptase and integrase those 2.4 kb elements are presumably transposable and at least all isolated copies are found exclusively in the proximity of tRNA gene loci. The enzymes needed for their replication and transposition are likely to be provided by the intact 5.7 kb DREs.

Animals↗
PubMed1992

Localization of the cholesterol oxidase in Rhodococcus erythropolis IMET 7185 studied by immunoelectron microscopy.

Rhodococcus erythropolis IMET 7185 produces an inducible cholesterol oxidase (COD) which can easily be extracted by treatment of cells with 0.1% Triton X-100. The yield of the enzyme was 3.3 U/g wet wt from induced cells, which is about 5 times more than from non-induced cells. A study of the location of COD on intact cells and on ultrathin sections by means of immunogold electron microscopy revealed the following distribution, which corresponds with the biochemical results: (1) COD, which is extractable by the detergent, was localized in a distance up to 80 nm above the cell surface. It belongs to a surface layer, which only became visible after lysine/glutaraldehyde treatment and staining with ruthenium red, indicating a high carbohydrate content. (2) Non-extractable COD was found on the cell surface in a shorter distance to the cell wall as well as within the cell wall, in the cytoplasmic membrane and in the peripheral cytoplasm. In the latter clusters of gold particles on some places suggest the presence of larger amounts of insoluble enzyme.

Blotting, Western↗
PubMed1992

Structure of DRE, a retrotransposable element which integrates with position specificity upstream of Dictyostelium discoideum tRNA genes.

Different Dictyostelium discoideum strains contain between 2 and 200 copies of a retrotransposable element termed DRE (Dictyostelium repetitive element). From the analysis of more than 50 elements, it can be concluded that DRE elements always occur 50 +/- 3 nucleotides upstream of tRNA genes. All analyzed clones contain DRE in a constant orientation relative to the tRNA gene, implying orientation specificity as well as position specificity. DRE contains two open reading frames which are flanked by nonidentical terminal repeats. Long terminal repeats (LTRs) are composed of three distinct modules, called A, B, and C. The tRNA gene-proximal LTR is characterized by one or multiple A modules followed by a single B module (AnB). With respect to the distal LTR, two different subforms of DRE have been isolated. The majority of isolated clones contains a distal LTR composed of a B module followed by a C module (BC), whereas the distal LTR of the other subform contains a consecutive array of a B module, a C module, a slightly altered A module, another B module, and another C module (BC.ABC). Full-length as well as smaller transcripts from DRE elements have been detected, but in comparison with the high copy number in D. discoideum strains derived from the wild-type strain NC4, transcription is rather poor.

Amino Acid Sequence↗