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E Schulze

Publications and source records attributed to E Schulze.

At least 55 records · Page 3Linked to original sources
PubMed1996

Developmentally imprinted genes as markers for bladder tumor progression.

PURPOSE: Developmentally imprinted genes, such as H19 and insulin-like growth factor-II (IGF-II), play an important role during human embryogenesis and also have been implicated in the pathogenesis of embryonal tumors of childhood. Since H19 is expressed in human fetal bladder, we evaluated 35 bladder carcinomas for H19 expression by in situ hybridization analysis and correlated expression with tumor grade. As a prelude to gene transfer studies to determine if H19 is a bladder tumor oncogene, we also evaluated bladder cell lines for expression of H19, IGF-II, IGF-I and the type I IGF receptor. MATERIALS AND METHODS: H19 expression was evaluated by in situ hybridization analysis in bladder tumor specimens. Northern analysis was used to evaluate the expression of H19, IGF-II, IGF-I and the type I IGF receptor in bladder cell lines. RESULTS: H19 was expressed preferentially in advanced stage tumors: 2 of 12 grade I tumors were H19 positive, whereas 9 of 11 grade II and 7 of 10 grade III tumors expressed H19 (p = 0.004). Additionally, 6 of 6 carcinoma in situ tumors were H19 positive, whereas normal bladder mucosa cells were H19 negative. We found that 3 of 11 cell lines (HT-1376, HT-1197 and 5637) express high levels of H19 mRNA, and each of these cell lines and J82 also express IGF-II. All cell lines examined expressed the type I IGF receptor, whereas there was no detectable IGF-I mRNA. CONCLUSIONS: These data demonstrate that H19 is an oncodevelopmental marker of bladder tumor progression and raise the possibility that H19 may have oncogenic properties in bladder cancer.

Biomarkers, Tumor↗
PubMed1995

The expression of the imprinted H19 and IGF-2 genes in human bladder carcinoma.

The imprinted H19 gene is highly expressed in human embryos, fetal tissues and is nearly completely shut off in adults. However, it is reexpressed in a number of tumors including bladder carcinoma, demonstrating that H19 RNA is an oncofetal RNA. Tumors induced by injection of bladder carcinoma cell lines express H19 in contrast to the cells before injection. These observations support the notion of a positive correlation between H19 expression and bladder carcinoma. Loss of imprinting of H19 and IGF-2 was observed in samples of human bladder carcinoma.

Animals↗
PubMed1995

The vertebrate linker histones H1 zero, H5, and H1M are descendants of invertebrate "orphon" histone H1 genes.

We investigated the evolutionary history of the divergent vertebrate linker histones H1 zero, H5, and H1M. We observed that the sequence of the central conserved domain of these vertebrate proteins shares characteristic features with histone H1 proteins of plants and invertebrate animals which otherwise never appear in any vertebrate histone H1 protein. A quantitative analysis of 58 linker histone sequences also reveals that these proteins are more similar to invertebrate and plant histone H1 than to histone H1 of vertebrates. A phylogenetic tree deduced from an alignment of the central domain of all known linker histones places H1 zero, H5, and H1M in close vicinity to invertebrate sperm histone H1 proteins and to invertebrate histone H1 proteins encoded by polyadenylated mRNAs. We therefore conclude that the ancestors of the vertebrate linker histones H1 zero, H5, and H1M diverged from the main group of histone H1 proteins before the vertebrate type of histone H1 was established in evolution. We discuss this observation in the general context of linker histone evolution.

Amino Acid Sequence↗
PubMed1995

Cellular uptake and trafficking of a prototypical magnetic iron oxide label in vitro.

RATIONALE AND OBJECTIVES: Target-specific magnetic resonance (MR) contrast agents are being developed to improve the accuracy of MR imaging. The purpose of this study was to determine the mechanism of cell uptake, and modes of intracellular trafficking of a prototypical iron oxide label (RMA) used in the synthesis of some target-specific MR contrast agents. METHODS: The prototypical agent (RMA) consisted of a dextran-coated monocrystalline iron oxide that was modified with rhodamine (fluorescent label) and opsonized with albumin. Fluorescence microscopy was performed in a phagocytic C6 cell line and in murine bone marrow macrophages. Immunohistochemistry against lysosomal markers was used to confirm the intracellular location of the label. RESULTS: RMA was identified inside cells after incubation at concentrations as low as 4.0 x 10(-10) M Fe, typically observed with receptor mediated endocytosis and several orders of magnitude lower than that expected with fluid phase pinocytosis. Cell uptake could be inhibited by excess protein but not by dextran. RMA localized initially to tubular and to round intracellular organelles and co-localized with an antibody against a murine lysosomal glycoprotein antibodies (LGP-A) in macrophages. Three days after incubation, RMA was concentrated in perinuclear vesicles, which most likely represent terminal lysosomes where final breakdown appears to occur. CONCLUSIONS: The mechanism of cellular uptake of a prototypical opsonized iron oxide label is consistent with receptor-mediated endocytosis. Immediately after cell contact, RMA localizes to the lysosomal compartment and at long time points reside in vesicles that by morphology and distribution appear to be terminal lysosomes. Iron oxides therefore demonstrate metabolism via the lysosomal pathway.

Albumins↗
PubMed1995

Detection of bcr-abl mRNA in single progenitor colonies from patients with chronic myeloid leukemia by PCR: comparison with cytogenetics and PCR from uncultured cells.

Bone marrow and/or peripheral blood of patients with chronic myeloid leukemia (CML) was investigated by the following three parameters: Ph' chromosome, bcr-abl expression in fresh blood and/or bone marrow, and bcr-abl expression in single hematopoietic progenitor colonies generated from blood and/or bone marrow. Expression of bcr-abl was proven by a reverse "nested primer" polymerase chain reaction (PCR) that is able to detect 1 pg of hybrid mRNA. We performed 108 investigations on 68 patients containing all three parameters: 12 on untreated patients, seven after interferon-alpha (IFN-alpha), seven after low-dose cytosine arabinoside (Ara-C), 22 after cyclic high-dose hydroxyurea (HU), 49 after allogeneic BMT, five before and three after stem cell mobilization, and three after autologous stem cell transplantation (ASCT). In 53 cases (49%), cytogenetics and PCR gave identical results. In 40 cases (37%), PCR from single colonies gave additional information compared to cytogenetics (e.g., mosaic in colonies when all metaphases were positive or negative). Most interesting were the results of one patient after IFN, one patient after ASCT, and 10 patients after BMT (14 investigations = 13%), showing only Ph'-negative mitoses accompanied by a negative nested primer PCR from fresh blood/bone marrow but single bcr-abl-positive progenitor colonies. False-positive results could be widely excluded by repeated insertion of negative controls into the experiments. One explanation for these results could be that CML, progenitors survive in the patient's body by being inactive and not proliferating. These cells express no or very little RNA and bcr-abl is not detectable by reverse PCR. When stimulated ex vivo in a colony assay by external growth factors, cells proliferate and produce detectable amounts of hybrid mRNA. The value of these observations is not clear. A follow-up of the patients will show if such sleeping progenitors can be activated in vivo. Concluding our observations, we can say that in special cases (therapy follow-up, detection of minimal residual disease) it could be useful to perform a PCR analysis of single progenitors in parallel with the routine investigations.

Base Sequence↗
PubMed1994

Insect proteins homologous to mammalian high mobility group proteins I/Y (HMG I/Y). Characterization and binding to linear and four-way junction DNA.

A chromosomal high mobility group (HMG) protein from an epithelial cell line of Chironomus tentans (Diptera) was purified to homogeneity and chemically characterized. cDNA clones encoding this protein were isolated and sequenced. The deduced amino acid sequence revealed a high similarity to HMG protein I of mammalia. This insect protein has therefore been designated cHMGI. It has a deduced molecular mass of 10,371 kDa and appears to be a product of a single gene copy. Similarly to mammalian HMGI/Y proteins the insect cHMGI protein has three putative DNA-binding motifs with a sequence K/RXRGRP that are each encoded by one exon of the gene. Using synthetic peptides we have shown that the first and the second motif are necessary for high-affinity binding of the protein to DNA. Protein cHMGI binds preferentially to AT-rich DNA with a half-saturation value of 1.1 nM. Both cHMGI and human HMGI proteins recognize specifically a four-way junction DNA. We have also purified a related protein with similar physico-chemical properties from Drosophila melanogaster Kc cells. The identification and characterization of HMGI proteins in insects with polytene chromosomes and with the cytologic and genetic potential of Chironomus and Drosophila opens new possibilities for studying function(s) of this group of chromosomal proteins.

Amino Acid Sequence↗
PubMed1994

DNA binding and nuclear translocation of insect high-mobility-group- protein-1 (HMG1) proteins are inhibited by phosphorylation.

The cells of the dipteran insects Chironomus and Drosophila contain high-mobility-group proteins (HMG) that are homologous to the HMG1 proteins of mammals, but contain only one HMG1 box instead of two. The C-terminal portions of both mammalian and insect HMG1 proteins comprise many charged residues that modulate the DNA-binding affinity of the HMG1 boxes and were found in Chironomus to be substrates for protein kinase C (PKC) in vitro and in vivo. Phosphorylation of Chironomus HMG1 proteins cHMG1a and cHMG1b by PKC resulted in a tenfold and fivefold reduction, respectively, of the DNA-binding strength. Phosphorylated and unphosphorylated cHMG1a protein was labelled with fluoresceine isothiocyanate and microinjected into the cytoplasm of Chironomus salivary gland cells. The translocation of phosphorylated cHMG1a into the nuclei was found to be remarkably delayed as compared to that of the unmodified form. The distribution of HMG1 proteins between nucleus and cytoplasm is known to vary according to the cell type and the state of differentiation. Our results suggest that this distribution may be regulated by changing the efficiency of nuclear translocation and the affinity for DNA via phosphorylation and dephosphorylation.

Amino Acid Sequence↗
PubMed1994

High affinity interaction of dipteran high mobility group (HMG) proteins 1 with DNA is modulated by COOH-terminal regions flanking the HMG box domain.

The cells of the dipteran insects Chironomus and Drosophila contain high mobility group (HMG) 1 proteins that are homologous to the HMG1 protein of mammals but comprise one instead of two DNA-binding HMG boxes. Mobility shift assays have revealed that Chironomus cHMG1a and cHMG1b bind double strand and four-way junction DNA in a similar way at apparent dissociation constants in the range of 7.5-20 x 10(-9) M. Both proteins are monomeric and highly asymmetric molecules in solution. cHMG1a and cHMG1b exhibit Stokes' radii of 2.4 and 2.3 nm, respectively, and both show a frictional ratio of 1.5. Despite these similarities in their hydrodynamic properties, the binding site of cHMG1a on DNA is approximately 1.5 of the size found for the cHMG1b. Enzymatically and chemically prepared peptides of cHMG1a as well as bacterially expressed cHMG1a with terminal deletions and point substitutions showed that sequences flanking the folded domain that constitutes the HMG box are essential for the interaction of the HMG box with DNA. In particular, changes in the number of positive and negative charges, respectively, within basic and acidic domains modulated the DNA binding affinity of the cHMG1a protein. The alteration of fluorescence of the Trp residues suggest that this modulation is due to interaction of the acidic domain with the positively charged HMG box.

Amino Acid Sequence↗
PubMed1994

Purification and characterization of an adenotin-like adenosine binding protein from human platelets.

A low-affinity adenosine binding protein (adenotin) was purified from human platelet membranes by a four-step procedure. Purification was achieved after extraction from human platelet membranes with 0.3% 3-[3-(cholamidopropyl)dimethylammonio]-1-propanesulfonate (CHAPS). Further purification included Sepharose CL6B gel filtration, DEAE-Sepharose CL6B, and hydroxylapatite chromatography. The protein was purified 884-fold to homogeneity with a 25% yield of binding activity from the membranes. 5'-[8(n)-3H]-N-ethylcarboxamidoadenosine ([3H]NECA) binds to the purified protein with a KD of 155 (144-167) nmol/l and a Bmax of 1.85 +/- 0.10 nmol/mg of protein. Sodium dodecylsulfate polyacrylamide gel electrophoresis of purified protein revealed a single band at 98 kDa. The 2-chloro-substituted adenosine analogs 2-chloro-5'-N-methylcarboxamidoadenosine (CIMECA) and 2-chloro-5'-N-ethylcarboxamidoadenosine (CINECA) were identified as new high affinity ligands of the purified protein showing Ki values of 18 nmol/l and 28 nmol/l, respectively. The low-affinity adenosine binding protein showed a pharmacological profile as follows: CIMECA > 5'-N-ethylcarboxamidoadenosine (NECA) > 2-chloroadenosine (CIA) > 2-[4-(2-carboxyethyl)phenethylamino]-5'-N-ethylcarboxamidoadenosin e (CGS 21,680) > R-N6-phenylisopropyl-adenosine (R-PIA). Amino-terminal sequence analysis revealed homologies to endoplasmin, glucose regulated protein (GRP94), tumor rejection antigen precursor (GP96), and some stress related proteins.

Adenosine↗
PubMed1994

Structurally divergent histone H1 variants in chromosomes containing highly condensed interphase chromatin.

Condensed and late-replicating interphase chromatin in the Dipertan insect Chironomus contains a divergent type of histone H1 with an inserted KAP-KAP repeat that is conserved in single H1 variants of Caenorhabditis elegans and Volvox carteri. H1 peptides comprising the insertion interact specifically with DNA. The Chironomid Glyptotendipes exhibits a corresponding correlation between the presence of condensed chromosome sections and the appearance of a divergent H1 subtype. The centromere regions and other sections of Glyptotendipes barbipes chromosomes are inaccessible to immunodecoration by anti-H2B and anti-H1 antibodies one of which is known to recognize nine different epitopes in all domains of the H1 molecule. Microelectrophoresis of the histones from manually isolated unfixed centromeres revealed the presence of H1 and core histones. H1 genes of G. barpipes were sequenced and found to belong to two groups. H1 II and H1 III are rather similar but differ remarkably from H1 I. About 30% of the deduced amino acid residues were found to be unique to H1 I. Most conspicuous is the insertion, SPAKSPGR, in H1 I that is lacking in H1 II and H1 III and at its position gives rise to the sequence repeat SPAKSPAKSPGR. The homologous H1 I gene in Glyptotendipes salinus encodes the very similar repeat TPAKSPAKSPGR. Both sequences are structurally related to the KAPKAP repeat in H1 I-1 specific for condensed chromosome sites in Chironomus and to the SPKKSPKK repeat in sea urchin sperm H1, lie at almost the same distance from the central globular domain, and could interact with linker DNA in packaging condensed chromatin.

Amino Acid Sequence↗
PubMed1993

Structural and functional differences between histone H1 sequence variants with differential intranuclear distribution.

The chromatin of most cell types contains several different sequence variants of histone H1. The functional role of this heterogeneity is not known. In the larval tissues of the midge, Chironomus thummi, there are H1 variants of two types. H1 II-1, H1 II-2, and H1 III-1 have similar amino acid sequences and appear uniformly distributed in polytene interphase chromosomes. The total number of gene copies per genome for this type of H1 histones is about 40 in C. th. thummi and 50-60 in C. th. piger. In contrast, histone H1 I-1 is encoded by a single copy gene in C. th. thummi and by two to four genes in C. th. piger. It has a divergent structure and is found only in a limited number of condensed chromosome sites. The N-terminal domain of H1 I-1 contains an insertion that is lacking in the other H1 variants and that is part of a variant-specific bipartite sequence Lys-Ala-Pro-Lys-Ala-Pro-Xaa10-Lys-Val-Ala in front of the conserved central domain. N-terminal peptides of H1 I-1 including this motif, in contrast to the homologous peptide from H1 II-1, competed with the drug Hoechst 33258 for binding to the minor groove of the DNA double helix. Repeats of the sequence Lys-Ala-Pro are also present at the same distance from the conserved central domain, in a single H1 variant of a nematode and of a green alga. The motif could interact with linker DNA in intranuclear targeting or packaging a condensed subtype of chromatin, or both.

Amino Acid Sequence↗
PubMed1993

Structure/function relationships in the pyruvate dehydrogenase complex from Azotobacter vinelandii. Role of the linker region between the binding and catalytic domain of the dihydrolipoyl transacetylase component.

The role of the hinge region between the binding domain and the catalytic domain in dihydrolipoyl transacetylase (E2p) from Azotobacter vinelandii was addressed by deletion mutagenesis. Mutated dihydrolipoyl transacetylase proteins were constructed with a deletion of 11 amino acids in the hinge region between the binding domain and the N-terminal part of the catalytic domain of E2p [E2p(pAPE1)] and with a further deletion of 9 amino acids into the N-terminal sequence protruding from the globular structure of the catalytic domain [E2p(pAPE2)] and found to take part in the intratrimer interaction. Both proteins behaved as wild-type E2p with respect to catalytic activity and quaternary structure. The interaction of the peripheral components pyruvate dehydrogenase (E1p) and lipoamide dehydrogenase (E3) with the mutated E2p proteins was studied. E2p(pAPE1) assembles to a trimeric pyruvate dehydrogenase complex (PDC) with 15% decreased complex activity. No difference in affinity towards the peripheral components was detected. Upon binding of E3, E2p(pAPE2) dissociates into trimers and monomers. At saturation, two dimers of E3 were bound/E2p monomer instead of one dimer/E2p chain in trimeric wild-type E2p or E2p (pAPE1). The monomeric E2p species was catalytically inactive. Upon binding of excess E1p, some monomer formation of the E2p mutant took place. E1p however can prevent monomerization by E3. It is concluded that E1p is bound between two different E2p chains in the trimer. The substrates CoA and acetyl-CoA also prevent monomerization because they are bound by amino acid residues of two different E2p chains. In the presence of CoA no difference in affinity with respect to E1p and E3 binding was observed. CoA (and acetylCoA) also prevent dissociation of the 24-subunit core structure of wild-type E2p when added before addition of E1p or E3. Therefore, it seems likely that in vivo A. vinelandii PDC is based on a 24-subunit E2p core, like Escherichia coli PDC. A functional difference between complexes based on a trimer or a 24-subunit core has not been observed. A role of the hinge region as a spacer to allow binding of E1p or E3 seems unlikely. The results are discussed on the basis of the three-dimensional structure of the catalytic domain.

Acetyltransferases↗
PubMed1993

The application of 13C-labelled short chain fatty acids to measure acetate and propionate production rates in the large intestines. Studies in a pig model.

The production rates of acetate and propionate were measured in the large intestine of pigs by applying the single injection technique of (1-13C)acetate and (1-13C)propionate. Both acids were injected individually through the caecal cannula and for both acids the experiments were performed during two diets with different crude fibre contents. For acetate the increase of dietary crude fibre from 5.1 to 18.3% of dry matter resulted in an increase of mean production rate from 27.4 to 56.2 mmol/h. The mean propionate production rate was raised from 3.6 to 7.0 mmol/h when the dietary crude fibre was increased from 4.4 to 24.3%. From both experimental series the contribution of hindgut fermentation to energy maintenance requirement were estimated to be in a range between 7 and 40% depending on the body weight of the animals and the percentage of dietary crude fibre.

Acetates↗
PubMed1992

Insect proteins homologous to mammalian high mobility group protein 1. Characterization and DNA-binding properties.

Two chromosomal high mobility group (HMG) proteins from larvae of Chironomus thummi (Diptera) and from an epithelial cell line of Chironomus tentans were purified to homogeneity and chemically characterized. cDNA clones encoding these proteins were isolated from an expression library using an immunoscreening approach and were sequenced. The deduced amino acid sequences revealed their homology to HMG protein 1 of vertebrates. These insect proteins have therefore been designated cHMG1a and cHMG1b. They have a molecular mass of 12,915 and 12,019 kDa, respectively, and preferentially bind to AT-rich DNA. Indirect immunofluorescence microscopy with a polyclonal antibody showed the presence of cHMG1a and cHMG1b in condensed chromomeres but not in puffs, nucleoli, and cytoplasm. The cHMG1a and cHMG1b genes were both localized to a single band in region 14 of chromosome 1 of C. tentans and appear to be single copy genes. An immunologically related protein was purified from Drosophila melanogaster Kc cells. Its size and amino acid composition indicate that it is an HMG1 of D. melanogaster. On the other hand, our antibody did not recognize calf HMG1. The identification and characterization of HMG1 proteins in insects with polytene chromosomes opens new possibilities for studying function(s) of this group of chromosomal proteins.

Amino Acid Sequence↗
PubMed1992

Reconstitution of pyruvate dehydrogenase multienzyme complexes based on chimeric core structures from Azotobacter vinelandii and Escherichia coli.

Two unique restriction sites were introduced by site-directed mutagenesis at identical positions in the DNA encoding the dihydrolipoyltransacetylase (E2p) components of the pyruvate dehydrogenase complex from Azotobacter vinelandii and from Escherichia coli. In this manner each DNA chain could be cut into three parts, coding for the lipoyl domain, which consists of three lipoyl subdomains, the binding domain and the core-forming catalytic domain, respectively. Chimeric E2p components were constructed by exchanging the three domains between E2p from A. vinelandii and E. coli on gene level. The six chimeric E2p proteins were expressed and purified from E. coli TG2. All chimeras were catalytically active, 24-subunit E2p proteins. Interactions of the peripheral components E1p and E3 with the wild-type enzymes from A. vinelandii and E. coli and with the chimeric proteins were studied by gel-filtration experiments, analytical ultracentrifugation and reconstitution of the overall activity of the complex. A. vinelandii E3 interacts only with those chimeras that contain the A. vinelandii binding domain, whereas E. coli E3 interacts with all chimeras. Exchange of the lipoyl or catalytic domain did not influence the binding properties of E3. Recognition of E1p depends on the origin of both the binding domain and the catalytic domain. E. coli E1p interacts strongly with those chimeras in which both the binding domain and the catalytic domain were derived from E. coli E2p and weakly with chimeras that contained either the binding domain or the catalytic domain from E. coli E2p. No binding of E. coli E1p was observed when both domains were of A. vinelandii origin. A. vinelandii E1p recognizes E2p from A. vinelandii and E. coli, but strong interaction required that the binding and catalytic domain were of the same origin. Exchange of lipoyl domains had no effect on the binding properties of the E1p component. These observations confirm previous conclusions, based on site-directed mutagenesis of A. vinelandii E2p [Schulze, E., Westphal, A. H., Boumans, H., and de Kok, A. (1991) Eur. J. Biochem. 202, 841-848], that the binding site for E1p consists of amino acid residues derived from both the binding and the catalytic domain and extend these conclusions to E. coli E2p. Dissociation of the 24 subunit E2p core was only detected when the chimeric E2p proteins contained the catalytic domain from A. vinelandii E2p. Dissociation depends on the binding of peripheral components to the E1p-binding sites, pointing to differences in the inter-trimer contacts between the E2p proteins from both species.(ABSTRACT TRUNCATED AT 400 WORDS)

Azotobacter vinelandii↗
PubMed1992

Purification and cellular localization of wild type and mutated dihydrolipoyltransacetylases from Azotobacter vinelandii and Escherichia coli expressed in E. coli.

Wild type dihydrolipoyltransacetylase(E2p)-components from the pyruvate dehydrogenase complex of A. vinelandii or E. coli, and mutants of A. vinelandii E2p with stepwise deletions of the lipoyl domains or the alanine- and proline-rich region between the binding and the catalytic domain have been overexpressed in E. coli TG2. The high expression of A. vinelandii wild type E2p (20% of cellular protein) and of a mutant enzyme with two lipoyl domains changed the properties of the inner bacterial membrane. This resulted in a solubilization of A. vinelandii E2p after degradation of the outer membrane by lysozyme without any contamination by E. coli pyruvate dehydrogenase complex (PDC) or other high-molecular-weight contaminants. The same effect could be detected for A. vinelandii E2o, an E2 which contains only one lipoyl domain, whereas almost no solubilization of A. vinelandii E2p with one lipoyl domain or of E2p consisting only of the binding and catalytic domain was found. Partial or complete deletion of the alanine- and proline-rich sequence between the binding and the catalytic domain did also decrease the solubilization of the E2p-mutants after lysozyme treatment. Immunocytochemical experiments on E. coli TG2 cells expressing A. vinelandii wild type E2p indicated that the enzyme was present as a soluble protein in the cytoplasm. In contrast, overexpressed A. vinelandii E2p with deletion of all three lipoyl domains and E. coli wild type E2p aggregated intracellularly. The solubilization by lysozyme is therefore ascribed to excluded volume effects leading to changes in the properties of the inner bacterial membrane.

Acetyltransferases↗