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K Lingelbach

Publications and source records attributed to K Lingelbach.

34 records · Page 2Linked to original sources
PubMed1993

Decreased resistance to N,N-dimethylated anthracyclines in multidrug-resistant Friend erythroleukemia cells.

Doxorubicin-resistant Friend erythroleukemia cells, line F4-6 ADM2R, were selected by exposure of wild-type F4-6 cells to doxorubicin concentrations of up to 1 microgram/ml. In these cells, increased expression of multidrug resistance (MDR) genes was demonstrated by Northern blot analysis. The growth-inhibitory effect of doxorubicin, daunorubicin, N,N-dimethyldoxorubicin, N,N-dimethyldaunorubicin, morpholinodoxorubicin, and pyrromycin was comparatively investigated in resistant and wild-type cells. The doxorubicin-resistant F4-6 cells showed approx. 200-fold resistance to doxorubicin and about 100-fold resistance to daunorubicin with respect to the drug-sensitive counterpart. A dramatic decrease in resistance was observed for the N,N-dimethylated derivatives of doxorubicin and daunorubicin as well as for the N,N-dimethylated natural anthracycline pyrromycin and for morpholinodoxorubicin. Uptake studies using [14C]-daunorubicin and [14C]-N,N-dimethyldaunorubicin in resistant F4-6 cells showed a decreased accumulation of daunorubicin but no significant reduction in N,N-dimethyldaunorubicin accumulation as compared with the wild-type cells. Treatment with verapamil led to increased intracellular levels of daunorubicin in resistant cells, whereas an excess of N,N-dimethyldaunorubicin did not have this effect. Thus, the decreased resistance of the doxorubicin-resistant F4-6 cells to the N-alkylated anthracyclines may at least in part be due to a reduced affinity of these compounds for the efflux pump. The results indicate that the dimethylation of the amino group of the anthracycline sugar moiety and its incorporation within a morpholinyl ring may overcome MDR by similar mechanisms.

Antibiotics, Antineoplastic↗
PubMed1992

Localisation of hypoxanthine phosphoribosyl transferase in the malaria parasite Plasmodium falciparum.

The enzyme hypoxanthine phosphoribosyl transferase of the human malaria parasite Plasmodium falciparum has been located in parasites and parasite-infected erythrocytes by antibody probing. The probe was a polyclonal rabbit antiserum made against the parasite enzyme made in Escherichia coli. The enzyme is associated with membrane-bound compartments in merozoites and asexual blood parasites. In particular, indirect immunofluorescence studies reveal the enzyme localized in vesicle-like structures within the cytoplasm of the infected erythrocyte. This is the first time a P. falciparum protein of defined metabolic function has been tracked to a site outside the parasite cytosol. Studies on the targeting of the enzyme using a cell-free system suggests that the protein reaches its destination via a route different from the normal secretory pathway.

Amino Acid Sequence↗
PubMed1992

Induction of differentiation in Friend-erythroleukemia cells by aclacinomycin A: early transient decrease in c-myc and c-myb mRNA levels.

Chemical inducers of the differentiation are known to cause an early transient decrease in c-myc and c-myb mRNA levels in Friend erythroleukemia cells preceding the down-regulation of c-myc and c-myb expression in the course of irreversible terminal differentiation. We therefore investigated the early effect of the potent differentiation-inducing anthracycline antitumor antibiotic, aclacinomycin A, on the c-myc and c-myb mRNA levels in the Friend cell line, F4-6, using Northern blot analysis. Aclacinomycin A induced a rapid decrease in the levels of c-myc and c-myb transcripts within 0.5-1 h and 2-3 h, respectively. The time course of decline in c-myc and c-myb expression was similar to that observed with dimethylsulfoxide or after transcription blockage brought about by a high concentration of actinomycin D. By 12 to 18 h after aclacinomycin A exposure, the c-myc and c-myb mRNA levels had returned to about pretreatment levels. When the cells were treated with adriamycin, an anthracycline that reduces cell proliferation in F4-6 cells without increasing differentiation, an early decrease in c-myc and c-myb expression was not observed. These results suggest that the transient decrease in c-myc and c-myb mRNA levels in F4-6 cells may be an early differentiation-related biochemical effect of aclacinomycin A.

Aclarubicin↗
PubMed1991

In vitro translation of Plasmodium falciparum aldolase is not initiated at an unusual site.

It has been proposed recently that translation of fructose-1,6-diphosphate aldolase of the malaria parasite Plasmodium falciparum is initiated at a UAG codon, both in the parasite and in a rabbit reticulocyte cell-free translation system. We have introduced mutations around that UAG codon and find that cell-free expression of a construct encoding an AUG in this position results in a slightly larger translation product. The translation product of the construct encoding the UAG codon is of the same apparent molecular weight as the products obtained from two other constructs; one in which the UAG is replaced by AAG, and one in which nucleotides upstream from a second AUG codon are deleted. Thus we show that translation is not initiated at the UAG and conclude that synthesis of aldolase in the parasite starts at an AUG, provided after splicing of pre-mRNA.

Animals↗
PubMed1991

An exported protein of Plasmodium falciparum is synthesized as an integral membrane protein.

Exp-1 is an antigen of Plasmodium falciparum which is transported from the parasite cell to the membrane of the parasitophorous vacuole and to membranous compartments in the erythrocyte. To investigate how this protein is transported, we studied the synthesis and membrane translocation of exp-1 in a cell-free system. The protein was translocated into canine pancreatic microsomes. Its N-terminal half was thus protected from proteinase K digestion, suggesting that exp-1 is an integral membrane protein with its N-terminus facing the lumen of the microsomes. This conclusion has been confirmed in vivo. In parasitized erythrocytes, exp-1 is membrane-associated and resistant to extraction with alkali, as would be expected for an integral membrane protein. Moreover, using segment-specific monoclonal antibodies, we have shown that here again the N-terminus of exp-1 faces the inside of vesicles, inaccessible to proteases, whereas the C-terminus is degraded. We conclude that exp-1 is an integral membrane protein and infer that it is transported by vesicles from the parasite to a compartment in the host cell cytoplasm.

Animals↗
PubMed1990

Deletion mutagenesis in M13 by polymerase chain reaction using universal sequencing primers.

A simple procedure is described for the efficient deletion of large DNA sequences. The method involves a combination of oligonucleotide-directed mutagenesis in bacteriophage M13 and amplification of the mutagenized product by polymerase chain reaction. In contrast to other protocols employing polymerase chain reaction, synthesis of only one specific primer is required. The efficiency of heteroduplex formation between mutagenic primers directing large deletions and single-stranded template is discussed.

Bacteriophages↗
PubMed1990

In vitro biosynthesis and membrane translocation of the serine rich protein of Plasmodium falciparum.

The serine-rich protein (SERP) of Plasmodium falciparum is found within the parasitophorous vacuole. Exons 1 and 2 of the SERP gene were combined to a continuous open reading frame and expressed in a cell free translation/translocation system to study translocation of the protein across membranes. The protein was found to be translocated co-translationally across canine pancreatic microsomes. This process required the presence of the signal recognition particle, and it was accompanied by cleavage of a signal peptide. We conclude that the authentic SERP is exported from the parasite cell via the endoplasmic reticulum.

Animals↗
PubMed1990

The 54-kD protein of signal recognition particle contains a methionine-rich RNA binding domain.

Signal recognition particle (SRP) plays the key role in targeting secretory proteins to the membrane of the endoplasmic reticulum (Walter, P., and V. R. Lingappa. 1986. Annu. Rev. Cell Biol. 2:499-516). It consists of SRP7S RNA and six proteins. The 54-kD protein of SRP (SRP54) recognizes the signal sequence of nascent polypeptides. The 19-kD protein of SRP (SRP19) binds to SRP7S RNA directly and is required for the binding of SRP54 to the particle. We used deletion mutants of SRP19 and SRP54 and an in vitro assembly assay in the presence of SRP7S RNA to define the regions in both proteins which are required to form a ribonucleoprotein particle. Deletion of the 21 COOH-terminal amino acids of SRP19 does not interfere with its binding to SRP7S RNA. Further deletions abolish SRP19 binding to SRP7S RNA. The COOH-terminal 207 amino acids of SRP54 (M domain) were found to be necessary and sufficient for binding to the SRP19/7S RNA complex in vitro. Limited protease digestion of purified SRP confirmed our results for SRP54 from the in vitro binding assay. The SRP54M domain could also bind to Escherichia coli 4.5S RNA that is homologous to part of SRP7S RNA. We suggest that the methionine-rich COOH terminus of SRP54 is a RNA binding domain and that SRP19 serves to establish a binding site for SRP54 on the SRP7S RNA.

Animals↗
PubMed1988

Isolation and characterization of a cDNA clone encoding the 19 kDa protein of signal recognition particle (SRP): expression and binding to 7SL RNA.

Signal recognition particle (SRP) consists of a 7SL RNA molecule and 6 protein subunits. We have isolated and characterized cDNA clones from human liver which encode the 19kDa protein subunit (SRP19). This subunit binds to the RNA directly and mediates binding of a second polypeptide, the 54kDa subunit which is involved in signal sequence recognition. Amino acid sequences deduced from the human cDNA sequence were identical to amino acid sequences of tryptic peptides from canine pancreatic SRP19. In vitro transcription and translation of the human cDNA resulted in a protein product the same size as canine SRP19 which could be immunoprecipitated by an antiserum raised against canine SRP19. SRP19 synthesized in a cell-free system specifically bound to 7SL RNA. The sequence of SRP19 is discussed with respect to its binding to 7SL RNA.

Amino Acid Sequence↗
PubMed1988

An extended RNA/RNA duplex structure within the coding region of mRNA does not block translational elongation.

RNA/RNA duplex formation involving the 5'untranslated region of a mRNA can efficiently block translation. Here we investigated the effect on translation of an RNA/RNA duplex between part of the coding region and sequences of the 3'untranslated region of lysozyme mRNA. A cDNA was constructed which contained 2 identical sequences of 150 nucleotides, one of which was an inverted repeat of the other. Cell-free transcription of this cDNA with T7 RNA polymerase resulted in a mRNA with an extended RNA/RNA duplex within the coding region. The presence of the double stranded structure was confirmed by the accessibility of complementary oligonucleotides to this region. mRNA was cleaved by RNaseH, endogenous to the wheat germ lysate, when hybridization of a complementary oligonucleotide occurred outside but not within the predicted double stranded structure. When this mRNA was translated in a cell-free wheat germ translation system, the translation product was found to be of the size of full-length prelysozyme and not arrested. We conclude that the extend of a secondary structure within the coding region of a mRNA does not restrict the ability of the ribosome to translate this mRNA efficiently. Our data are consistent with the presence of an activity unwinding RNA/RNA duplexes, which is associated with the translating ribosome.

DNA↗
PubMed1988

Mitochondrial import of rat pre-ornithine transcarbamylase: accurate processing of the precursor form is not required for uptake into mitochondria, nor assembly into catalytically active enzyme.

Mitochondrial uptake of the cytoplasmically synthesized precursor of the mammalian enzyme ornithine transcarbamylase is mediated by an N-terminal leader sequence of 32 amino acids. In the mitochondrial matrix, the precursor form is processed to the mature subunit by proteolytic removal of this pre-sequence and in the enzyme from rat liver it has been suggested that this occurs in a two-step process which involves an intermediate cleavage at residue 24. We show that deletion of residues 20-26 spanning this intermediate cleavage site prevents correct processing to the mature subunit but it does not prevent mitochondrial targeting and internalization or assembly of the incorrectly processed product into a catalytically active enzyme. The incorrectly processed enzyme, which is larger than the normal mature enzyme, is nevertheless more susceptible to proteolytic degradation in permanently transfected human cells than the correctly processed enzyme.

Absorption↗
PubMed1986

Primary structure of the (1-->3,1-->4)-beta-D-glucan 4-glucohydrolase from barley aleurone.

During germination of barley grains, the cell walls of the starchy endosperm are degraded by (1-->3,1-->4)-beta-glucanases (EC 3.2.1.73) secreted from the aleurone and scutellar tissues. The complete sequence of the aleurone (1-->3,1-->4)-beta-glucanase isoenzyme II comprises 306 amino acids and was determined by sequencing nine tryptic peptides (110 residues) and aligning them with the amino acid sequence deduced from a cDNA clone encoding the 291 NH(2)-terminal residues. Although no amino acid sequence homology with a bacterial (1-->3)(1-->4)-beta-glucanase is apparent, close to 50% homology is found with two large regions of a (1-->3)-beta-glucanase from tobacco pith tissue. The gene for barley (1-->3,1-->4)-beta-glucanase isoenzyme II shares with that for the alpha-amylase isoenzyme 1 a strongly preferred use of codons with G and C in the wobble position (94% and 90%, respectively). Both enzymes are secreted from the aleurone cells during germination. Such one-sided codon usage is not characteristic for the gene encoding the (1-->3)-beta-glucanase of tobacco pith tissue or the hor2-4 gene encoding the B(1) hordein storage protein in the endosperm.

Journal Article↗
PubMed1985

Comparison of ornithine transcarbamylase from rat liver and intestine. Evidence for differential regulation of enzyme levels.

Ornithine transcarbamylase (OTCase) was purified from the small intestine of rat and the properties of the gut enzyme were compared with those of the enzyme from liver. The enzymes from both sources bound to the transition-state analog inhibitor, delta-N-(phosphonoacetyl)-L-ornithine, immobilized on Sepharose and eluted with carbamyl phosphate as a homogeneous preparation. The specific activities of the pure enzymes were 966 mumol min-1 mg-1 and 928 mumol min-1 mg-1 from liver and gut respectively, and the molecular mass, based on electrophoretic mobility, was 38 000 Da. The isoelectric point of the enzymes from both sources was 7.3. The enzymes from both sources cross-react to the same extent with antibodies against the liver enzyme on Western transfers and the size of the mRNA was identical on Northern transfers probed with a cDNA for the liver enzyme. Although OTCase is apparently the same gene product in both liver and gut, the enzyme levels respond differently to alterations in the protein content of the diet. OTCase in liver increased from 0.76 mumol min-1 microgram-1 DNA on 15% casein to 1.3 mumol min-1 microgram-1 DNA on 60% casein (P less than 0.01) whereas in small intestine the level decreased from 8.8 nmol min-1 microgram DNA on 15% casein to 5.7 nmol min-1 microgram-1 DNA on 60% casein (P less than 0.05). When expressed on a fresh-weight basis, the enzyme activity in liver shows the characteristic increase with increasing protein, whereas the activity in gut does not. The connection between these differences in gene expression and the different physiological roles of OTCase in liver and gut is discussed.

Animals↗
PubMed1984

Echinococcus multilocularis: identification of proteins inducing antibody formation in metacestode infection.

An approach to the identification of parasite proteins which are immunogenic in natural infections is described, using the infection with the larval stage of Echinococcus multilocularis as a parasite model. Metacestode proteins were separated by SDS-polyacrylamide gel electrophoresis, and transferred electrophoretically to nitrocellulose sheets (Western blotting). Subsequently, immune recognition of the proteins was performed with various host sera and antigen-antibody complexes were detected enzymatically. Using homologous antisera, different patterns of immunogenic bands were revealed by sera of different host species. Cross-reactions with sera from individuals infected with unrelated helminths were analysed. Four proteins of E. multilocularis which failed to show any cross-reaction were identified.

Animals↗
PubMed1984

Differential antibody screening of cloned Plasmodium falciparum sequences expressed in Escherichia coli: procedure for isolation of defined antigens and analysis of human antisera.

We describe a procedure that uses polyspecific human sera for screening Escherichia coli colonies expressing cloned Plasmodium falciparum cDNA sequences in order to detect colonies that react differentially with different sera. This procedure can be used for two distinct purposes. First, it enables the isolation of clones encoding specified antigenic sequences present in the complex mixture, without purification of either antigens or antibodies by conventional procedures. This requires that the antigen can be expressed in E. coli and that antisera are available that differ substantially in their reactivities to the component of interest. To develop the procedure, we used two polyspecific sera that shared many anti-P. falciparum specificities but differed in that only one was reactive to the isolate-specific S antigen of P. falciparum strain FCQ27/PNG (called FC27). Differential screening with the two sera identified 30 cDNA clones, and colony hybridization confirmed that 25 of these express S-antigen sequences. Second, the procedure identifies defined antibody specificities within polyspecific human sera by virtue of their ability to react with any given cDNA clone. The procedure has been used here to identify antibody specificities that increase dramatically in titer between the acute and convalescent phases of malaria in certain individuals and, hence, to isolate clones encoding the corresponding antigens.

Animals↗