PubMed Health⌕ Search

Biomedical subjects

U Bachrach

Publications and source records attributed to U Bachrach.

At least 37 records · Page 2Linked to original sources
PubMed1990

Plasmodium falciparum: properties of an alpha-like DNA polymerase, the key enzyme in DNA synthesis.

An alpha-like DNA polymerase has been identified and characterized in the extracts from the malarial parasite Plasmodium falciparum. The enzyme is sensitive to the specific inhibitors of alpha-DNA polymerase, N-ethylmaleimide and aphidicolin, and is cell-cycle specific. High activity has been found in the schizont, is lower in trophozoites, and has only negligible activity in the ring form. The enzyme has a molecular weight of about Mr 100,000-103,000 estimated by detecting activity in SDS-polyacrylamide electrophoresis and by Bio-Gel filtration. Another active band of a molecular Mr 68,000 was detected by SDS electrophoresis when the enzyme was stored for 2 months at -20 degrees C. The catalytic activity of parasite enzyme was optimal between pH 8 and pH 9. The apparent Michaelis constant for dTTP was 4.3 microM.

Animals↗
PubMed1990

Effect of sinefungin on macromolecular biosynthesis and cell cycle of Plasmodium falciparum.

The growth of the malarial parasite P. falciparum was arrested by the adenine containing nucleoside sinefungin at the trophozoite stage. The synthesis of DNA, of polyamines and the specific proteins of the schizont stage were completely blocked by the drug. The inhibition of DNA synthesis was not due to a decrease in the amount of DNA polymerase, but to the depletion of polyamines which are required for DNA synthesis.

Adenosine↗
PubMed1989

The effect of substituted aminoalkylaminoanthraquinones on eukaryotic cells.

Nine aminoalkylaminoanthraquinones (I-IX) were evaluated for their cytotoxic potency against normal and malignant mammalian cells. The 1.8-di-(aminopropylamino) derivative (VIII) exhibited significant activity against several tumor cell systems and had some selectivity. The toxicity of this compound was also tested in growing chick embryos.

Animals↗
PubMed1988

Polyamine metabolism and interconversion in NIH 3T3 and ras-transfected NIH 3T3 cells.

The effect of transfection of NIH 3T3 cells by the human ras (c-Ha-ras-1) oncogene on uptake, interconversion, and excretion of polyamines was studied. Uptake and interconversion of spermidine were higher in the ras-transfected cells. Acetylpolyamines were excreted into the medium by the ras-transfected cells, whereas they were retained by NIH 3T3 cells. In addition to acetylpolyamines, some unknown polyamine conjugates occurred in the ras-transfected cells.

Acetylation↗
PubMed1988

Plasmodium falciparum: purification, properties, and immunochemical study of ornithine decarboxylase, the key enzyme in polyamine biosynthesis.

Ornithine decarboxylase, the rate-limiting enzyme in the polyamine biosynthetic pathway has been purified 7,600 fold from Plasmodium falciparum by affinity chromatography on a pyridoxamine phosphate column. The partially purified enzyme was specifically tagged with radioactive DL-alpha-difluoromethylornithine and subjected to polyacrylamide gel electrophoresis under denaturing conditions. A major protein band of 49 kilodalton was obtained while with the purified mouse enzyme, a typical 53 kilodalton band, was observed. The catalytic activity of parasite enzyme was dependent on pyridoxal 5'-phosphate and was optimal at pH 8.0. The apparent Michaelis constant for L-ornithine was 52 microM. DL-alpha-difluoromethylornithine efficiently and irreversibly inhibited ornithine decarboxylase activity from P. falciparum grown in vitro or Plasmodium berghei grown in vivo. The Ki of the human malarial enzyme for this inhibitor was 16 microM. Ornithine decarboxylase activity in P. falciparum cultures was rapidly lost upon exposure to the direct product, putrescine. Despite the profound inhibition of protein synthesis with cycloheximide in vitro, parasite enzyme activity was only slightly reduced by 75 min of treatment, suggesting a relatively long half-life for the malarial enzyme. Ornithine decarboxylase activity from P. falciparum and P. berghei was not eliminated by antiserum prepared against purified mouse enzyme. Furthermore, RNA or DNA extracted from P. falciparum failed to hybridize to a mouse ornithine decarboxylase cDNA probe. These results suggest that ODC from P. falciparum bears some structural differences as compared to the mammalian enzyme.

Animals↗
PubMed1987

Oxidation of N-alkyl and C-alkylputrescines by diamine oxidases.

N-Methyl-, N-ethyl-, N-propyl- and N-butylputrescine were assayed as substrates of diamine oxidase from pea seedling and pig kidney. With the exception of N-methylputrescine they were found to be oxidized to the corresponding aminoaldehydes. 1-Methyl-, 2-methyl-, 1-ethyl- and 1-propylputrescine were oxidized by the oxidases at lower rates than the N-alkylderivatives. 1,3-Dimethylputrescine had negligible oxidation rates while 1,4-dimethylputrescine (2,5-diaminohexane) was not a substrate. The oxidation of putrescine by the kidney oxidase was inhibited by 1,4-dimethylputrescine, while the pea oxidase was strongly inhibited by the former as well as by 2-methylputrescine and 1,3-dimethylputrescine. Serum amine oxidase did not oxidize the substituted putrescines although several of the latter inhibited spermidine oxidation by this oxidase.

Alkylation↗
PubMed1987

Effect of polyamine depletion on macromolecular synthesis of the malarial parasite, Plasmodium falciparum, cultured in human erythrocytes.

DL-alpha-Difluoromethylornithine (DFMO), an irreversible inhibitor of ornithine decarboxylase, prevented the increases in putrescine and spermidine, but not in spermine, in human erythrocytes infected with the malarial parasite Plasmodium falciparum. The addition of putrescine to these polyamine-depleted cultures restored the normal concentrations of spermidine, whereas that of putrescine even exceeded that of the control cultures. DFMO also inhibited the incorporation of radioactive amino acids into the proteins of parasitized erythrocytes. Electrophoresis on polyacrylamide gels revealed that the synthesis of some proteins was completely blocked by DFMO, but the synthesis of others was not affected. DFMO also caused a partial inhibition of RNA synthesis, and DNA synthesis was completely blocked in polyamine-depleted parasitized erythrocytes. It has been suggested that putrescine and/or spermidine are required for the synthesis of certain proteins in parasitized erythrocytes and that at least one of those proteins is related to the synthesis of DNA of the malarial parasite. It appears that polyamines regulate the schizogony process of P. falciparum.

Eflornithine↗
PubMed1987

Intracellular distribution of active and inactive transglutaminase in stimulated cultured C6 glioma cells.

The cellular distribution of active and inactive transglutaminase (TGase) was studied in C6 glioma cells before and during stimulation by a serum-containing medium. The activity of the enzyme was determined in the soluble and insoluble fractions obtained by freezing and thawing the cells, followed by centrifugation at 12,000g for 5 min. In the soluble fractions, the activity of TGase decreased 2.5 h post-stimulation and increased after 5 and 8 h. In the corresponding insoluble fractions, no significant changes in the activity of the enzyme were noted up to 8 h after stimulating the cells with fresh medium. An immunological approach was next used to determine the quantity of TGase antigen during the stimulation of the cultured glioma cells. In the soluble fraction, the quantity of the antigen decreases significantly at 2.5, 5, and 8 h. In contrast, in the insoluble fraction, a significant increase in TGase antigen was detected 8 h after the addition of fresh medium. Cycloheximide completely inhibited the increase in the quantity of TGase antigen in the insoluble fraction, 8 h post-stimulation, while actinomycin D caused a partial inhibition. Trypsin, neuraminidase, or Sendai viruses increased the activity of TGase significantly, when added to nonstimulated cells. Trypsin had no effect on TGase activity when added to the cells 2 h after stimulation with a serum-containing medium. These findings suggest that an inactive form of the enzyme is present in the insoluble cellular fraction. A model has been proposed to explain the variations in TGase activity, its distribution and translocation during cellular stimulation.

Animals↗
PubMed1987

Fusion-mediated microinjection of active amine and diamine oxidases into cultured cells: effect on protein and DNA synthesis in chick embryo fibroblasts and in glioma cells.

Serum amine oxidase and/or porcine kidney diamine oxidase were trapped within reconstituted Sendai virus envelopes, and retained their activity. The trapped enzymes that were detected by radioimmunoblots were microinjected into cultured cells by fusion. When diamine oxidase was microinjected into cultured fibroblasts of chick or rat embryos, a temporary arrest in protein and DNA synthesis was observed. The inhibitory effect was more significant when both serum amine oxidase and kidney diamine oxidase were microinjected into those cultured cells. Fibroblasts of either chick or rat embryos transformed by Rous sarcoma virus were more susceptible to the injected enzymes than the normal cultures, showing a complete arrest in protein and DNA synthesis within 4 hours. Similar results were obtained by microinjecting diamine oxidase into cultured glioma cells. The injected enzyme catalyzed the oxidation of intracellular polyamines. The resulting oxidation product (hydrogen peroxide and aminoaldehydes) apparently caused the arrest in the synthesis of macromolecules.

Amine Oxidase (Copper-Containing)↗
PubMed1987

Cytostatic effect of DL-alpha-difluoromethylornithine against Plasmodium falciparum and its reversal by diamines and spermidine.

DL-alpha-difluoromethylornithine (DFMO) inhibited ornithine decarboxylase (ODC) activity and arrested the growth of Plasmodium falciparum at the early trophozoite stage. The inhibition of ODC activity did not result in the formation of an alternative diamine such as cadaverine. When putrescine or spermidine were added to the parasites grown in culture, the arrest was reversed, and normal schizogony was completed even in the presence of DFMO. Some reversal of the inhibition was achieved with cadaverine at high concentrations, while 1,3-diaminopropane and spermine failed to restore the development. Resumption of growth could be detected when putrescine was added even after 67 h of DFMO treatment. Electron microscopy did not reveal any changes in the morphology of parasites treated for 47 h, while 73 h of treatment with DFMO induced massive accumulation of pigment. Death was observed a few hours later. These results suggest that DFMO acts as a cytostatic rather than as a cytocidal agent. The four carbon diamine restored cell growth while the shorter or the longer homologous compounds showed little activity.

Animals↗
PubMed1987

Effect of microinjected amine and diamine oxidases on the ultrastructure of eukaryotic cultured cells.

Diamine oxide and serum amine oxidase, which catalyse the oxidation of diamines and polyamines, respectively, were trapped within reconstituted Sendai virus envelopes. These loaded envelopes were incubated with cultured normal chick fibroblasts or with fibroblasts transformed by Rous sarcoma viruses. The binding of the reconstituted envelopes to the cultured cells was confirmed by scanning electron microscopy. It has been shown that the reconstituted envelopes (1-3 microns diameter) were attached to the eukaryotic cells. No significant changes in the morphology of the normal chick embryo fibroblasts were noted upon treatment with enzyme-loaded envelopes. On the other hand, chick embryo fibroblasts transformed by Rous sarcoma virus were affected by the microinjected amine oxidases. Scanning electron microscopy demonstrated the formation of holes in the microinjected cells. Similar morphological changes were also observed when diamine oxidase was microinjected into cultured glioma cells. These holes may be the result of the ejection of the nucleus. These findings are in line with the observed effect of the injected amine oxidases on macromolecular synthesis in normal and transformed chick embryo fibroblasts.

Amine Oxidase (Copper-Containing)↗
PubMed1986

The effect of purified aminoaldehydes produced by polyamine oxidation on the development in vitro of Plasmodium falciparum in normal and glucose-6-phosphate-dehydrogenase-deficient erythrocytes.

Purified aminoaldehydes produced by polyamine oxidation were toxic to the malarial parasite, Plasmodium falciparum, cultured in human erythrocytes. There was a profound effect on young ring forms, and, during maturation, parasites became more sensitive to the aldehydes. Oxidation of the aldehydes abolished the lethal effect. The plasmodia within glucose-6-phosphate-dehydrogenase (G6PD)-deficient erythrocytes were more sensitive to mono- and di-aldehydes than were parasites in normal erythrocytes. G6PD-deficient erythrocytes were also more sensitive to pretreatment with the dialdehyde produced by the oxidation of spermine. Pretreatment prevented further invasion by the parasites.

Aldehydes↗
PubMed1986

A sensitive, rapid, chemiluminescence-based method for the determination of diamines and polyamines.

A new sensitive and rapid chemiluminescence-based method for the determination of diamines and polyamines is described. Phosphocellulose paper strips are used for the removal of neutral or negatively charged molecules from polyamine-containing fluid. The procedure is based on the determination of hydrogen peroxide, produced during the oxidation of polyamines, by a fairly specific serum amine oxidase. A plant diamine oxidase is used for the assay of diamines. This method permits the determination of diamines and polyamines in a range of 10 to 100 pmol and may be used for the assay of urinary polyamines.

Amine Oxidase (Copper-Containing)↗
PubMed1986

Plasmodium falciparum: synchronization of cultures with DL-alpha-difluoromethylornithine, an inhibitor of polyamine biosynthesis.

DL-alpha-Difluoromethylornithine, an inhibitor of polyamine biosynthesis, was tested for its ability to synchronize Plasmodium falciparum. Asynchronous cultures were pretreated with sorbitol and incubated for 28-30 hr. Then, when cultures consisted of mainly schizont stage parasites, DL-alpha-difluoromethylornithine was added to the growth medium for another 38-47 hr of incubation. Putrescine was added to parasites arrested at the early trophozoite stage. This resulted in a synchronous resumption of growth. After 19 hr, 83% of parasites were at the schizont stage. After 30 hr, more than 98% of the parasites were in the ring form stage. Furthermore, the transformation of early trophozoites to schizonts occurred within 3 hr, with a slight reduction in parasitemia. Synchrony was maintained for 4-5 biological cycles as confirmed also by flow fluorimetry. It appears that this new approach to synchronize P. falciparum cultures is simple, reproducible, and effective.

Animals↗