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Biomedical subjects

R Sinervirta

Publications and source records attributed to R Sinervirta.

30 records · Page 2Linked to original sources
PubMed1987

Mouse and human ornithine decarboxylase genes. Methylation polymorphism and amplification.

With the use of the isoschizomeric restriction endonucleases HpaII and MspI, we found that mouse tumour ornithine decarboxylase (ODC; EC 4.1.1.17) genes are extensively methylated. ODC genes in L1210 mouse leukaemia cells were apparently more methylated than in Ehrlich ascites carcinoma, as revealed by the use of HpaII endonuclease, yet the digestion of genomic DNA isolated from these two murine tumour cell lines with MspI, which cleaves at a CCGG sequence, also with internally methylated cytosine, resulted in an apparently identical restriction pattern. It is possible that the amplification of ODC genes in Ehrlich ascites-carcinoma cells in response to 2-difluoromethylornithine (DFMO) was associated with hypomethylation, or that less-methylated genes were amplified. A human myeloma (Sultan) cell line only revealed three separate hybridization signals when cleaved with HpaII. One of these signals was amplified under the pressure of DFMO. When cleaved with MspI, these three HpaII fragments disappeared and were replaced by a double signal of 2.3-2.4 kilobase-pairs (kbp) in size. The amplified ODC sequences in the Sultan myeloma cell line apparently originated from chromosome 2, as indicated by a unique hybridization signal in a 5.8 kbp HindIII fragment specific for the human ODC locus on chromosome 2. A comparison of different human cells, the Sultan myeloma, a lymphocytic B-cell leukaemia (Ball), normal mononuclear leucocytes and leucocytes obtained from leukaemia patients, revealed interesting differences in the methylation of ODC genes. The use of two restriction endonucleases (HpaII and CfoI), the cleavage site for both of which contains a CG sequence and which only cleave when cytosine is unmethylated, indicated that ODC genes in the lymphocytic leukaemia cells were much less methylated than those in the normal leucocytes or in the Sultan cells.

Animals↗
PubMed1986

Studies on structure-activity relationship of gossypol, gossypol ethers and three naphthaldehydes in the inhibition of spermatozoal metabolism.

Gossypol tetramethyl ether [C30 H24 O2(OCH3)4] and gossypol hexamethyl ether [C30 H24 O2(OCH3)6], which in contrast to gossypol are stable compounds, were tested for their ability to depress fructose degradation in fresh human sperm cells. Both ethers inhibited spermatozoal fructolysis, yet less effectively than did the parent compound. A synthetic compound, O-hydroxylnaphthaldehyde, and two commercially available preparations, 1- and 2-naphthaldehydes, were also tested under the same experimental conditions. These preparations represent about half of the gossypol molecule and possess a reactive aldehyde group in their molecules. Their inhibitory effect on fructose degradation in fresh human sperm cells, however, was considerably smaller than that of gossypol itself. It thus appears that the whole ring structure of gossypol rather than the intact aldehyde group is required for an effective inhibition of spermatozoal energy metabolism.

Aldehydes↗
PubMed1985

Gene expression of ornithine decarboxylase in L1210 leukaemia cells exposed to DL-2-difluoromethylornithine in the presence of cadaverine.

Cultured mouse L1210 leukaemia cells treated with DL-2-difluoromethylornithine, an irreversible inhibitor of ornithine decarboxylase (EC 4.1.1.17), in the presence of micromolar concentrations of cadaverine, started to overproduce ornithine decarboxylase after an exposure of several weeks. The more than 60-fold excess of the enzyme protein in the drug-treated cells apparently resulted from a strikingly enhanced accumulation of mRNA for the enzyme associated with only a modest (about 2-fold) gene amplification.

Animals↗
PubMed1985

Tumourigenicity, cell-surface glycoprotein changes and ornithine decarboxylase gene pattern in Ehrlich ascites-carcinoma cells.

We selected a 2-difluoromethylornithine-resistant Ehrlich ascites-carcinoma cell line that grows in the presence of 20 mM-difluoromethylornithine. These cells contain 10-20 times the normal amount of hybridizable sequences for ornithine decarboxylase (EC 4.1.1.17) in their genomic DNA. We used these gene-amplified cells, their revertant counterparts (grown in the absence of the drug after an established gene amplification) and tumour cells grown in the presence of putrescine to investigate the changes of ornithine decarboxylase gene pattern and simultaneously occurring phenotypic changes, such as tumourigenicity and the expression of cell-surface glycoproteins. In the tumour cells reverted back to the normal gene frequency, not only did the amplified sequences disappear, but there were also signs of gene re-arrangements seen as a "gene jump', when a signal evidently moved to a heavier restriction fragment. Similar gene re-arrangement likewise occurred in cells exposed to putrescine. Although the wild-type tumour cells and the gene-amplified cells readily grew in the peritoneal cavity of mice, the revertant cells and the putrescine-treated cells had lost their tumourigenicity in mice. Gene-amplified tumour cells and the revertant cells showed distinct changes in their surface glycoprotein pattern in comparison with the parental cell line. These findings indicate that alterations of ornithine decarboxylase gene pattern/dosage may be associated with phenotypic changes possibly related to the tumourigenicity of these carcinoma cells.

Animals↗
PubMed1985

Difluoromethylornithine-induced amplification of ornithine decarboxylase genes in Ehrlich ascites carcinoma cells.

Stepwise increments of the concentration of 2-difluoromethylornithine, a mechanism-based irreversible inhibitor of mammalian ornithine decarboxylase (EC 4.1.1.17), resulted in a selection of cultured Ehrlich ascites carcinoma cells capable of growing in the presence of up to 50 mM difluoromethylornithine. Dialyzed extracts of drug-resistant tumor cells exhibited a very high ornithine decarboxylase activity and contained large excess of immunoreactive ornithine decarboxylase protein. Hybridization analyses with cloned complementary DNA revealed that the difluoromethylornithine-resistant tumor cells also expressed mRNA of the enzyme at greatly enhanced rate. The overproduction of ornithine decarboxylase by the tumor cells grown under the pressure of difluoromethylornithine was at least partly attributable to a 10 to 20-fold increase in the total gene dosage of ornithine decarboxylase involving an amplification of several genes of the gene family. The gene amplification developed appeared to be stable, as the gene dosage only slowly (during a period of several months) returned towards the normal level upon the removal of difluoromethylornithine. The overproduction of ornithine decarboxylase was accompanied by an enhanced resistance of the enzyme towards difluoromethylornithine in vitro.

Animals↗
PubMed1985

Effects of inhibitors of polyamine biosynthesis on the growth and melanogenesis of murine melanoma cells.

Both 2-difluoromethylornithine (DFMO), an irreversible inhibitor of ornithine decarboxylase (EC 4.1.1.17), and methylglyoxal bis(guanylhydrazone) (MGBG), a competitive inhibitor of S-adenosylmethionine decarboxylase (EC 4.1.1.50), strikingly stimulated melanotic expression of murine Cloudman S91 melanoma cells. The stimulation of tyrosinase (EC 1.10.3.1) activity and melanin formation by DFMO was closely associated with intracellular depletion of putrescine and spermidine developed in response to the drug. However, little or no evidence was obtained indicating that enhanced melanogenesis in response to MGBG was mediated through an inhibition of polyamine biosynthesis. Indirect inhibitors of ornithine decarboxylase, such as 1,3-diaminopropane and 1,3-diaminopropan-2-ol, but not putrescine, likewise inhibited the growth of the melanoma cells and stimulated their melanin production. The stimulation of melanogenesis by polyamine antimetabolites was not mediated by cyclic adenosine 3':5'-monophosphate, in contrast to the effect elicited by alpha-melanotropin. It is also unlikely that MGBG or the diamines acted as lysosomotropic agents capable of stimulating tyrosinase activity in situ, since the enzyme activity was stimulated by the drugs irrespective of whether assayed in cultured cells or using cell-free homogenates. None of the agents stimulated tyrosinase activity in vitro. The effect of DFMO and MGBG on melanoma cell proliferation was reversible, but the restoration of normal growth and melanin formation, especially in cells exposed to DFMO, was remarkably slow. The present results represent a further experimental model, in which the inhibition of polyamine accumulation is accompanied by signs of terminal differentiation.

Animals↗
PubMed1983

Effect of gossypol on the motility and metabolism of human spermatozoa.

Gossypol, a polycyclic compound isolated from cotton seeds, had a dose-dependent inhibitory effect on human sperm motility. The drug also inhibited powerfully fructolysis and glycolysis by human spermatozoa. Both lactate and CO2 formation from the 14C-labelled sugars was inhibited, and the prevention of CO2 formation from [1-14C]pyruvate and [2-14C]pyruvate by gossypol indicated a direct effect on the tricarboxylic acid cycle. Repeated washing of the sperm cells after gossypol pretreatment failed to abolish the inhibitory effect on CO2 production. The profound disturbances of the sperm energy metabolism induced by gossypol were also reflected by a striking fall of the sperm ATP content. Gossypol had little effect on glucose utilization by minces of human vaginal mucosa, indicating the specificity of gossypol.

Adenosine Triphosphate↗
PubMed1977

Mechanism of action of oxidized polyamines on the metabolism of human spermatozoa.

Oxidized spermine, an iminoaldehyde (N,N'-bis (3-propionaldehyde) 1,4-diaminobutane), is a non-competitive inhibitor of fructolysis by human spermatozoa. The inhibition constant is about 0.3 mM. In experiments with [U-14C]fructose the iminoaldehyde caused a more pronounced depression of the formation of CO2 than of lactate. The iminoaldehyde was without influence on the conversion of fructose to lactate by cell-free extracts of spermatozoa, but it markedly decreased the uptake of fructose and lactate by spermatozoa. These findings strongly suggest that inhibition of the fructose metabolism of intact spermatozoa was due to interaction of the iminoaldehyde with sperm membranes and not to inhibition of any enzyme of the glycolytic pathway. Several aliphatic and aromatic aldehydes were also tested for their ability to inhibit sugar utilization of human spermatozoa: only n-hexanal exerted an inhibitory effect, the extent of which approached that of oxidized spermine.

Aldehydes↗
PubMed1975

S-adenosylmethionine decarboxylase from baker's yeast.

1. S-Adenosyl-L-methionine decarboxylase (S-adenosyl-L-methionine carboxy-lyase, EC 4.1.1.50) was purified more than 1100-fold from extracts of Saccharomyces cerevisiae by affinity chromatography on columns of Sepharose containing covalently bound methylglyoxal bis(guanylhydrazone) (1,1'[(methylethanediylidene)dinitrilo]diguanidine) [Pegg, (1974) Biochem J. 141, 581-583]. The final preparation appeared to be homogeneous on polyacrylamide-gel electrophoresis at pH 8.4. 2. S-Adenosylmethionine decarboxylase activity was completely separated from spermidine synthase activity [5'-deoxyadenosyl-(5'),3-aminopropyl-(1),methylsulphonium-salt-putrescine 3-aminopropyltransferase, EC 2.5.1.16] during the purification procedure. 3. Adenosylmethionine decarboxylase activity from crude extracts of baker's yeast was stimulated by putrescine, 1,3-diamino-propane, cadaverine (1,5-diaminopentane) and spermidine; however, the purified enzyme, although still stimulated by the diamines, was completely insensitive to spermidine. 4. Adenosylmethionine decarboxylase has an apparent Km value of 0.09 mM for adenosylmethionine in the presence of saturating concentrations of putrescine. The omission of putrescine resulted in a five-fold increase in the apparent Km value for adenosylmethionine. 5. The apparent Ka value for putrescine, as the activator of the reaction, was 0.012 mM. 6. Methylglyoxal bis(guanylhydrazone) and S-methyladenosylhomocysteamine (decarboxylated adenosylmethionine) were powerful inhibitors of the enzyme. 7. Adenosylmethionine decarboxylase from baker's yeast was inhibited by a number of conventional carbonyl reagents, but in no case could the inhibition be reversed with exogenous pyridoxal 5'-phosphate.

Adenosylmethionine Decarboxylase↗
PubMed1975

Putrescine-insensitive S-adenosyl-L-methionine decarboxylase from Tetrahymena pyriformis.

Extracts of Tetrahymena pyriformis contain a soluble S-adenosyl-L-methionine decarboxylase which, in contrast to the enzyme from most eukaryotic organisms, is not stimulated by putrescine or spermidine. The protozoan adenosylmethionine decarboxylase, unlike the putrescine-insensitive enzyme form Escherichia coli, did not require any metal ions for catalytic activity either. Adenosylmethionine decarboxylase from Tetrahymena resembled the prokaryotic enzyme as far the inhibition by methylglyoxal bis(guanylhydrazone) was concerned, but behaved more like putrescine-activated enzyme in regard to the inhibition by 4-bromo-3-hydroxy benzyl-oxyamine. Adesylmethionine decarboxylase from rat liver, baker's yeast, E. coli and Tetrahymena were strongly inhibited by S-methyladenosylhomocysteamine (decarboxylated adenosylmethionine), the product of the reaction. The function of adenosylmethionine decarboxylase from Tetrahymena like that of the enzymes from other organisms appears to be closely connected to the synthesis of spermidine.

Adenosylmethionine Decarboxylase↗