Oligoribonucleotides: improvement in chromatographic separation.

The separation of mixed oligoribonucleotides obtained from enzymatic digestion of soluble RNA into fractions containing mixed oligonucleotides of identical chain length is improved by absence of terminal phosphate. Each fraction may be separated into individual components by ionexchange (Dowex) chromatography. Combination of these two chromatographic procedures yields maximum information on primary structure of the oligonucleotides.

Affective disorders and S-adenosylmethionine: a new hypothesis.

S-Adenosyl-L-methionine (AdoMet) is a safe and probably effective antidepressant agent in certain forms of clinical depression. This article presents a new hypothesis to account for the mechanism of action of S-adenosylmethionine in such illnesses, based upon the known biochemistry of this compound, and upon current knowledge of clinical and genetic aspects of affective disorders. Giulio Cantoni, S. Harvey Mudd and V. Andreoli postulate that at least some major mood disorders are due to abnormalities affecting the AdoMet-dependent methylation of a substance in the CNS. For convenience and without prejudging the chemical structure of this substance, they call it 'barinine'. The model requires that barinine be subject to AdoMet-dependent methylation and that methylbarinine be subject to metabolic demethylation to regenerate the original barinine. Methylbarinine should be mood elevating, whereas barinine itself should not be. Depression is a result of abnormalities lowering the normal steady-state concentration of methylbarinine, whereas mania results from an abnormal elevation of methylbarinine.

Inactivation of S-adenosylhomocysteine hydrolase by nucleosides.

The irreversible inactivation of S-adenosylhomocysteine hydrolase purified from hamster and bovine liver by adenosine analogs substituted in the 5' and 2 positions has been investigated in detail. 5'-Cyano-5'-deoxyadenosine inactivates as potently as 9-beta-D-arabinofuranosyladenine (Ara-A). Substitution of the Ara-A at the 2 position by halogens or deleting N at the 3 position decreases its potency. Although weak, 2',3'-dideoxyadenosine can also inactivate the enzyme. The irreversible inactivation of the hydrolase in rat hepatocytes incubated with 2-chloroadenosine or 3-deaza-Ara-A could be demonstrated, concomitant with increases in 35S-labeled S-adenosylhomocysteine and S-adenosylmethionine in the hepatocytes.

Carbocyclic analogue of 3-deazaadenosine: a novel antiviral agent using S-adenosylhomocysteine hydrolase as a pharmacological target.

The carbocyclic analogue of 3-deazaadenosine (3-deaza-C-Ado) has been synthesized and found to have antiviral activity in cell culture against herpes simplex virus type 1, vaccinia virus, and HL-23 C-type virus. It is relatively noncytotoxic at effective antiviral concentrations and is not subject to deamination or phosphorylation. It acts as a competitive inhibitor of S-adenosyl-L-homocysteine hydrolase, is at best a poor substrate, and does not inactivate the enzyme significantly. 3-Deaza-C-Ado may cause a selective inhibition of the methylation of the polynucleotide 5' cap of viral mRNA via higher cellular concentrations of S-adenosyl-L-homocysteine, resulting from the inhibition of S-adenosylhomocysteine hydrolase in infected cells, since increases in the intracellular level of S-adenosylhomocysteine, but no effects on DNA or RNA synthesis, were observed after incubation of these cells with it.

The hypotensive action of 3-deaza-adenosine.

The effects of 3-deaza-adenosine on the cardiovascular system was investigated in normotensive (WKY) and spontaneously hypertensive rate (SHR). This compound (50 mg/kg i.p. 2X) significantly lowers blood pressure in both groups of animals, an effect which lasts for up to 2 h. The hypotensive effect is not affected by ganglion blockade, and appears to be due in part to a direct effect on blood vessels. While the response is similar to that seem with adenosine, it is more long-lasting. The results of this study suggest a potential role for this compound or analogues of its as possible antihypertensive agents.

Effects of S-adenosyl-methionine on plasma norepinephrine, blood pressure, and heart rate in healthy volunteers.

S-adenosyl-methionine (SAMe) is currently undergoing trials as a possible antidepressant. Because SAMe's mechanism of action is obscure and norepinephrine (NE) is often implicated in affective disorders, we studied the effects of SAMe on this neurotransmitter in volunteers. Plasma NE and 3-methoxy-4-hydroxyphenylglycol (MHPG) in the supine and standing position were studied before and after acute placebo or a single 400 mg dose of SAMe and following seven daily administrations; concomitant measures were heart rate (HR) and blood pressure. Subjects were unable to distinguish acute drug from placebo, and although chronic SAMe administration was open, they reported no behavioral effects. Standing HR and plasma NE were reduced following chronic SAMe. Qualitatively similar changes are obtained following chronic treatment with monoamine oxidase inhibitors (MAOIs). However, unlike MAOIs, chronic SAMe treatment was not associated with changes in plasma MHPG. Exaggerated standing NE is found in depressed patients; SAMe may reduce this abnormal response, providing a clue for its mechanism of action in depression.

Inhibition of promoter activity by methylation: possible involvement of protein mediators.

To study the relationship between DNA methylation and promoter activity we have methylated in vitro the promoters of the mouse metallothionein I gene and the herpes simplex virus thymidine kinase gene. We have transiently transfected these promoters fused to the human growth hormone in their methylated or unmethylated state into mouse L or F9 cells. Promoters methylated by methylase (M.) Hpa II and M.Hha I caused inhibition of reporter gene expression in L cells but not in F9 cells, while methylation of all CpGs by M.Sss I caused inhibition in both cell lines. Repression of promoter activity by M.Hpa II and M.Hha I methylation, but not by M.Sss I methylation, could be alleviated by cotransfection with an excess of untranscribable DNA methylated with M.Sss I. The methylated sites in nuclei isolated from the transfected L cells, but not F9 cells, were found to be protected from Msp I digestion. Taken together these results suggest that a factor present in L cells and missing in F9 cells mediates the methylation-directed inhibition of promoter activity. The ability of methylated DNA to overcome the inhibition seems to reflect competition for the mediator factor. Interestingly, treatment with Zn2+ ions brought about activation of the methylated promoter of the metallothionein gene. Similarly, butyrate could override the repression of the thymidine kinase methylated promoter. These activations were not accompanied by demethylation of the promoter or displacement of the mediator factor.

Adenosylhomocysteine hydrolase. Crystallization of the purified enzyme and its properties.

Adenosylhomocysteine hydrolase (EC 3.3.1.1) from calf liver was purified to homogeneity by crystallization. The purified enzyme exhibited one single component in polyacrylamide gel electrophoresis. But by Ampholine gel electrophoresis, two isoelectric focusing variants were observed, with pI values at 5.8 and 6.0. when subjected to polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate, one major subunit with a molecular weight of 60,000 was found; five other minor subunit variants were also observed, with molecular weights ranging between 50,000 and 57,000. These minor subunit variants comprised approximately 15% of the total protein applied. The molecular weight of the native enzyme was estimated to be 237,500 by gradient gel electrophoresis. The native enzyme is probably composed of four subunits, each with a molecular weight of not more than 60,000. Amino acid analyses of the purified enzyme revealed the presence of 1.2 residues of glucosamine/mol of enzyme, in addition to all of the common amino acids. The presence of enzyme-bound NAD was confirmed, probably 1 NAD molecule bound/enzyme subunit. In addition to adenosine, 3-deazaadenosine was found to be an effective substrate as well in the direction of synthesis.

5'-Methylthioadenosine nucleosidase. Purification and characterization of the enzyme from Lupinus luteus seeds.

5'-Methylthioadenosine nucleosidase (EC 3.2.2.9), the enzyme which catalyzes hydrolytic cleavage of 5'-methylthioadenosine with the formation of adenine and 5'-methylthioribose, has been purified to homogeneity from Lupinus luteus seeds. The nucleosidase has a native molecular weight of 62 000 and consists of two identical subunits, as judged by gel filtration and dodecylsulfate/polyacrylamide gel electrophoresis. The nucleosidase exhibits highest specificity towards the natural substrate with a Km of 4.1 X 10(-7) M for 5'-methylthioadenosine. It does not cleave adenine from S-adenosylhomocysteine. Among the synthetic analogs of 5'-methylthioadenosine tested, eleven compounds appear to be able to substitute as substrates. Furthermore, the enzyme can liberate hypoxanthinine from six inosyl (deaminated) derivatives obtained by enzymatic deamination of 5'-methylthioadenosine and its synthetic analogs. The Km for 5'-methylthioinosine is 55 microM, and the maximal velocity about 50-times lower than for 5'-methylthioadenosine. The reaction catalyzed by the nucleosidase can be inhibited by adenine (Ki = 11 microM), 3-deazaadenine (Ki = 19 microM), and 9-erythro(2-hydroxyl-3-nonyl)adenine (ki = 37 microM).

Mechanism of the cytostatic activity of 3-deazaaristeromycin, an inhibitor of adenosylhomocysteine hydrolase.

3-Deazaaristeromycin (100 microM) is cytostatic for the RAW264 mouse macrophage cell line. In cells incubated with 3-deazaaristeromycin, Cell replication is effectively stimulated by 0.5 microM homocysteine thiolactone and no further stimulation occurs at concentrations of homocysteine thiolactone greater than 50 microM. Cell replication occurs when homocysteine thiolactone is administered either simultaneously with, or 4 days after, the administration of 3-deazaaristeromycin. In the presence of 3-deazaaristeromycin, cell replication also occurs when either folate (10 to 100 microM) or hypoxanthine and thymidine are included in the medium. Incorporation of [14C]formate into trichloroacetic acid-precipitable material is inhibited when cells are incubated with 3-deazaaristeromycin, and this inhibition is partially reversed by inclusion of either homocysteine thiolactone or folate in the medium. Experiments showed that adenosylhomocysteine hydrolase is inhibited to the same extent after the cells have been incubated for 4 days with either 3-deazaaristeromycin or 3-deazaaristeromycin and homocysteine thiolactone. The findings suggest that in cells incubated with 3-deazaaristeromycin, the formation of homocysteine from S-adenosylhomocysteine is insufficient for the regeneration of tetrahydrofolate for the synthesis of purines and pyrimidines.

Guanidoacetate methyltransferase. Purification and molecular properties.

Guanidoacetate methyltransferase has been purified about 140-fold from pig liver. Polyacrylamide gel electrophoresis of the purified enzyme showed four protein bands, each of which is associated with guanidoacetate methyltransferase activity. During gel electrophoresis at pH 3 in 8 M urea, guanidoacetate methyltransferase migrated as a single component. The molecular weight of the purified guanidoacetate methyltransferase was estimated to be 31,000 by sodium dodecyl sulfate-gel electrophoresis, which also showed only one protein component with guanidoacetate methyltransferase activity. This molecular weight is in agreement with that estimated by Sephadex G-75 chromatography. Guanidoacetate methyltransferase is inhibited by adenosylhomocysteine, 3-deazaadenosylhomocysteine, and sinefungin with Ki values of 16 microM, 39 microM, and 18 microM, respectively.

Effects of the S-adenosylhomocysteine hydrolase inhibitors 3-deazaadenosine and 3-deazaaristeromycin on RNA methylation and synthesis.

The effects of 3-deazaaristeromycin and 3-deazaadenosine on RNA methylation and synthesis were examined in the mouse macrophage cell line, RAW264. S-Adenosylhomocysteine accumulated in cells incubated with 3-deazaaristeromycin while S-3-deazaadenosylhomocysteine was the major product in cells incubated with 3-deazaadenosine and homocysteine thiolactone. RNA methylation was inhibited to a similar extent by the accumulation of either S-adenosylhomocysteine or S-3-deazaadenosylhomocysteine, with S-adenosylhomocysteine being a slightly better inhibitor. In mRNA, the synthesis of N6-methyladenosine and N6-methyl-2'-O-methyladenosine were inhibited to the greatest extent, while the synthesis of 7-methylguanosine and 2'-O-methyl nucleosides were inhibited to a lesser extent. Incubation of cells with 100 microM 3-deazaaristeromycin or with 10 microM 3-deazaadenosine and 50 microM homocysteine thiolactone produced little inhibition of mRNA synthesis, even though mRNA methylation was inhibited. In contrast, mRNA synthesis was greatly inhibited by treatment of cells with 100 microM 3-deazaadenosine and the inhibition of synthesis was not correlated with an inhibition of methylation.

Methylation in the preinitiation domain suppresses gene transcription by an indirect mechanism.

Although the first observations of the inhibitory effect of methylation on gene activity were made almost a decade ago, the mechanism by which methyl groups affect transcription is still obscure. Here we use engineered promoters methylated in vitro in transient transfections to study the mechanism by which methylation mediates promoter repression. The results clearly show that the location of the methyl groups within the promoter region determines the extent of promoter repression. The most effective suppression was observed when methylation was in the preinitiation domain. The results also support a previous suggestion that a mediator protein is involved in the mechanism of promoter inhibition. The suppressor effect of methylation at sequences flanking the TATA box can be partially overcome in the presence of the simian virus 40 enhancer. In addition, results obtained by transient thymidine labeling of Ltk- cells that were transfected with a methylated thymidine kinase gene from herpes simplex virus, at the level of approximately one template per cell, further support the conclusion that methylation affects primarily transcription preinitiation.

The rat S-adenosylhomocysteine hydrolase promoter.

The 1.2-kb DNA sequence flanking the transcription start of the AdoHcy hydrolase gene was cloned into the luciferase reporter plasmid pGL3-basic, and promoter activity was measured in transiently transfected CHO cells. Deletion analysis showed that most promoter activity was located within a 153 bp fragment immediately upstream from the predominant transcription start. The 153 bp fragment includes sites for AP-2, glucocorticoid-responsive element, SP-1, and a TATA-like sequence TATTTAAA. Mutational analysis demonstrated that the SP-1 site nearest the start of transcription contributed significantly to promoter activity, whereas, the other elements, including the appropriately positioned TATTTAAA sequence, had little affect on promoter activity.

The differentiation of L5/A10 myoblast cell line (a subclone of L5 line) is controlled by changes of culture conditions.

We report here that it is possible to induce differentiation in a subline of L5 myoblast line (L5/A10) by manipulating the culture media. When L5/A10 myoblast are cultured in F14 supplemented with 10% fetal calf serum the cells grow with a division time of 12 h and reach confluency at a cell density of approximately 2.4 X 10(5) cells per cm2, without undergoing differentiation, characterized, morphologically, by formation of multinucleated fibers, and biochemically, by the synthesis of muscle specific proteins such as creatine phosphokinase or myokinase. However, cells, grown in F14 + 10% fetal calf serum, will undergo regular differentiation after a limited number of division when transferred to F14 medium supplemented with limiting concentrations (1-2%) of fetal calf serum. Investigations of the biochemistry of myoblast differentiation in cell culture will be facilitated by the availability of a cell line that can undergo differentiation under controlled conditions.

Cholera toxin inhibits chemotaxis by a cAMP-independent mechanism.

Cholera toxin inhibits chemotaxis of the RAW264 mouse macrophage cell line. The degree of inhibition by cholera toxin increases upon incubation with the cells, suggesting that the entry of the toxin is required for inhibition of chemotaxis. In the absence of guanine nucleotides, cholera toxin catalyzes the [32P]ADP-ribosylation of RAW264 cell membrane proteins of Mr 41,000, Mr 45,000, and a doublet of Mr 48,000-50,000. GTP increases the labeling of the Mr 45,000 protein and the Mr 48,000-50,000 doublet, and it decreases the labeling of the Mr 41,000 protein. Experiments with cholera toxin treatment of intact cells indicate that the Mr 45,000 protein is the major membrane protein ADP-ribosylated by the toxin in vivo. Cholera toxin increases cAMP levels in RAW264 cells, but increased cAMP levels do not correlate with inhibition of chemotaxis, because isoproterenol and forskolin, which also increase cAMP levels, have no effect on chemotaxis.