Potentiation by 2'-deoxycoformycin of the inhibitory effect of xylosyladenine on nuclear RNA synthesis in L1210 cells in vitro

The effect of the adenosine deaminase inhibitor, 2'-deoxycoformycin (dCF), on the inhibitory effect of 9-β-d-xylofuranosyladenine (XA) on nuclear RNA synthesis was examined in L1210 cells in vitro. Pretreatment of cells for 15 min with a 100 per cent inhibitory dose (1 × 10^?6 M) of dCF resulted in approximately a 3- to 8-fold reduction in the 50 per cent inhibitory dose (id₅₀) of XA for [³H]uridine and [³H]thymidine incorporation into RNA and DNA respectively. The id₅₀ for XA for RNA synthesis vs DNA synthesis was 5-fold lower in the absence of dCF and 20-fold lower in the presence of dCF, indicating the greater sensitivity of RNA synthesis to this inhibitor. Fractionation of nuclear RNA into rRNA, non-poly(A) heterogeneous RNA and poly(A)heterogeneous RNA revealed the latter species of RNA to be less sensitive to XA in the absence of dCF; however, in the presence of dCF, all three species of nuclear RNA showed similar sensitivities. Nuclear polyadenylic acid synthesis was among the most sensitive RNA fractions to XA, and was also inhibited to a greater degree by pretreatment of cells with dCF. These results indicate that XA is potentiated markedly by inhibition of adenosine deaminase, and that deamination serves as a major catabolic route for this drug.     

Enhancement of 5-fluorouracil incorporation into human lymphoblast ribonucleic acid

Various drug combinations have been explored as a means of enhancing the incorporation of 5-fluorouracil (5-FU) into RNA from the human T-lymphoblast CEM line. The relative incorporation of [³H]FU into newly synthesized alkali-sensitive RNA was determined by concurrent ³²P-incubation and calculation of the ratio of [³H]FU/³²P.$\text{N-(}\text{Phosphonacetyl}\text{)-}\text{L}\text{-}\text{aspartate}$ (PALA) was employed at concentrations of 0.1 mg/ml for 16 hr prior to the addition of [³H]FU; it enhanced the formation of (5-FU) RNA by 3- to 5-fold. The effects of methotrexate (MTX) and 6-methylmercaptopurine riboside (MMPR) on the formation of (5-FU)RNA and phosphoribosyl-l-pyrophosphate (PRPP) were also monitored. Increases of 10- to 15-fold in PRPP occurred following a 6-hr exposure to either MTX or MMPR at concentrations of 1 μM and resulted in up to a 5-fold enhancement in the [³H]FU/³²P ratio. Exposure of cells to combinations of PALA with MTX or MMPR resulted in up to 20-fold increases in (5-FU)RNA formation. These drug combinations were more than additive with 5-FU in their inhibitory effects as determined by assays monitoring cell growth. These studies should be applicable in expanding ongoing clinical trials of combination chemotherapy with PALA and 5-FU.     

Effect of sangivamycin and xylosyladenine on the synthesis and methylation of polysomal ribonucleic acid in Ehrlich ascites cells in vitro

The pyrrolopyrimidine, sangivamycin, and the adenosine analog, xylosyladenine, were examined for their effects on the synthesis and methylation of polysomal RNA in Ehrlich ascites tumor cells in vitro. The synthesis of non-polyriboadenylic acid (non-poly (A) ?) and poly(A)-containing RNA was inhibited 50 per cent at concentrations of 7 × 10^?6 M and 3 × 10^?6 M xylosyladenine, respectively, when adenosine deaminase was inhibited with 2'-deoxycoformycin. Sangivamycin inhibited the synthesis of non-poly(A)- and poly(A)RNA by 50 per cent at concentrations of 5 × 10^?5 M and 2 × 10^?5 M respectively. Electrophoretic separation of non-poly(A)RNA into rRNA and tRNA indicated that the inhibitory effects of both drugs were more pronounced on 28S than on 18S rRNA, and that xylosyladenine but not sangivamycin inhibited the synthesis of tRNA. Assessment of the effects of both analogs on the methylation of polysomal RNA revealed that xylosyladenine inhibited the methylation of nonpoly(A)-and poly(A)RNA, while sangivamycin only weakly affected the latter species of RNA. Base methylation of the affected species of RNA was inhibited slightly more than 2'-O-methylation by both drugs. These results indicate that sangivamycin is a more selective inhibitor of polysomal RNA in comparison to xylosyladenine under conditions where adenosine demainase is not a limiting factor.     

Tumor 5-fluorodeoxyuridylate concentration as a determinant of 5-fluorouracil response

The antitumor activity of 5-fluoruoracil (5-FU) for two murine colonie adenocarcinomas was correlated with the concentration and the clearance of the active antimetabolite, 5-fluorodeoxyuridylate (FdUMP). Mice inoculated with a cell suspension of murine colonic adenocarcinomas 38 and 51 were treated with 5-FU (100 mg/kg i.p.) on 3 day post-transplantation. For mice bearing adenocarconoma 38, treatment with 5-FU was associated with a 97 per cent reduction in mean tumor weight a day 30 and a 77 per cent reduction at day 37 of tumor growth. In contrast, mice bearing colonic adenocarcinoma 51, treated with the same dose schedule of 5-FU did not demonstrate a reduction in the rate of tumor growth in vivo. Two hr after i.p. injection of 5-FU (100 mg/kg) the intracellular concentration of free FdUMP in the sensitive tumor 38 was 560 fmoles/μg of DNA. The active antimetabolite was maintained at a concentration in excess of 100 fmoles/μg of DNA for 72 hr. In contrast, the 2-hr free FdUMP concentration in the resistant tumor line 51 was 240 fmoles μg of DNA(P < 0.005), and a concentration in excess of 100 fmoles/μg of DNA was maintained for only 24 hr. There was no difference in the rate of progressive accumulation of the competitive metabolite, deoxyuridine monophosphate (dUMP), during the first 24 hr of the study. Two hr after i.p. injection of 5-FU (100 mh/kg), [³H] deoxyuridine ([³H]Udr) incorporation into DNA was reduced in both tumor lines to below 3 per cent of control. However, in the sensitive tumor, adeno-carcinoma 38, DNA synthesis was maximally inhibited for 72 hr, compared to 24 hr in the resistant adenocarcinoma 51. The reinitiation of DNA synthesis corresponded to the reduction of free FdUMP concentration to less than 100 fmoles/μg of DNA. There was no linear relationship between the FdUMP/ dUMP ratio and [³H]UdR incorporation into DNA in either tumor line. These data demonstrate that the peak tumor FdUMP concentration and the kinetics of its clearance correlated with the responsiveness of the two specific murine tumors to 5-FU. The measure of peak FdUMP level should be tested for its potential clinical application as a means of selecting patients with gastrointestinal and breast cancer to be treated with this agent.     

Studies on accumulation and metabolic fate of (N-Me3h)choline in human term placenta fragments.

Fragments from human term placenta accumulated [N-Me³H]choline against a concentration gradient. Uptake was linearly related to incubation time and temperature. Analysis of the kinetics of choline accumulation revealed the concurrent existence of a diffusional component occuring both at low temperature as well as high (50 mM) choline concentration and a carrier-mediated transport which had characteristics predicted by the Michaelis-Menten equation showing a K_m = 3.46 × 10^?4 M and a V_max of 75 nmoles/ml intracellular water × min^?1. Net concentration ratios, corrected for diffusion and extracellular water, were larger than 1.0 within 5 min and about 4.0 after 15 min. Hemicholinium-3 was a competitive inhibitor of choline uptake with a K_i of 0.45 mM. [³H]Choline accumulation was decreased by conditions known to lower intracellular ATP levels. Thus, 2,4-dimitrophenol (1 mM), sodium cyanide (5 mM) and anaerobic incubation reduced [³H]choline accumulation 36, 54 and 33 per cent respectively. Ouabain (0.1 mM) also decreased the concentration ratios by 50 per cent. Modification of the ionic environment led to an increase of 36 per cent in the amount of tritium in intracellular water when Na⁺ was reduced to one half of the usual 145 mM or 150 per cent when it was completely omitted and replaced by an osmotically equivalent amount of sucrose. Li⁺ was without effect, while high K⁺ (> 25mM), Rb⁺ and Cs⁺ (145 mM) depressed [³H]choline accumulation. The metabolic fate of [³H]choline was studied. Following a 5-min incubation with 5 μM [³H]choline 95 per cent of the radioactivity was acid-soluble and 5 per cent remained in the acid-insoluble fraction. After 30 min the distribution was 88 and 12 per cent, respectively. Paper high voltage electrophoretic analysis of the acid-soluble material showed that after 5 min 55 per cent of the ³H had a mobility equal to authentic choline, 35 per cent equal to acetylcholine, 6 per cent to phosphorylcholine and 1 per cent to betaine. After 20 min it was 25 per cent in choline, 60 per cent in acetylcholine, 10 per cent in phosphorylcholine and 2 per cent in betaine. A chloroform-methanol extract from the acid-insoluble residue revealed a linear increase of ³H-content suggesting incorporation of [³H]choline into phospholipids.     

Inhibition of sterol synthesis in animal cells by 15-oxygenated sterols with the unnatural cis-C-D ring junction-"5alpha,14beta-cholest-7-en-15alpha-ol-3-one and 5alpha,14beta-cholest-7-en-15beta-ol-3-one.

5α,14β-Cholest-7-en-15α-ol-3-one was prepared in 72 per cent yield by selective oxidation of the 3β-hydroxyl function of 5α,14β-cholest-7-en-3β,15α-diol by cholesterol oxidase. 5α,14β-Cholest-7-en-15β-ol-3-one was prepared in a similar manner from 5α,14β-cholest-7-en-3β,15β-diol in 66 per cent yield. The new compounds were found to inhibit sterol synthesis in mouse fibroblasts in culture. The 15α-hydroxy-3-ketosterol and the 15β-hydroxy-3-ketosterol caused a 50 per cent inhibition of the incorporation of [1?¹⁴C]acetate into digitonin-precipitable sterols at concentrations of 2.0 × 10^?6 M and 2.5 × 10^?7M, respectively, and suppressed the level of activity of 3-hydroxy-3-methylglutaryl Coenzyme A reductase activity by 50 per cent at concentrations of 3.3 × 10^?7 M and 2.5 × 10^?7 M respectively.     

Regulation of cyclic AMP level and synthesis of DNA, RNA and protein by quercetin in Ehrlich ascites tumor cells.

Quercetin (3.3, 4′, 5.7-pentahydroxy flavone) at the concentration of 10^?4 M as well as 10^?2 M theophylline and 10^?3 M dibutyryl cyclic AMP caused at least 85 per cent inhibition of [³H]thymidine incorporation in Ehrlich ascites tumor cells. At the same concentrations, these drugs decreased [³H]uridine and [³H]-L-leucine incorporation by 50–60 per cent and 35–45 per cent respectively. Ouabain (10^?3 M), the specific inhibitor of Na⁺-K⁺ pump system, did not alter the incorporation of [³H]thymidine and [³]uridine, but decreased the incorporation of [³H]-L-leucine in these cells. Treatment of Ehrlich ascites tumor cells with the polyanion dextran sulfate did not change the inhibitory effect of quercetin, theophylline and dibutyrlyl cyclic AMP on [³H]thymidine incorporation. On the other hand, this polyanion decreased the inhibitory effect of these drugs on incorporation of [³H]uridine and abolished completely their effect on incorporation of [³H]-L-leucine.     

Inhibition of catecholamine uptake and retention in synaptosomal preparations by tetrahydroisoquinoline and tetrahydroprotoberberine alkaloids

The effects of racemic mixtures of representative tetrahydroisoquinoline and tetrahydroprotoberberine alkaloids on the mechanisms of catecholamine uptake and retention were studied in synaptosomal preparations from whole rat brain. The synaptosomes were incubated with $\text{(}{}^{14}\text{C}\text{)-d,l-}\text{norepinephrine}$ or (¹⁴C)-dopamine in the presence of various concentrations of salsolinol (SAL), tetrahydropapaveroline (THP), 2,3,10,11-tetrahydroxyberbine (THB), or 2,3,10,11-tetramethoxyberbine (TMB). Levels of radioactivity in synaptosomes preloaded with labeled norepinephrine were significantly diminished by the addition of 10^?4 M THP, THB or SAL to approximately 42.7 per cent (P < 0·001), 85.8 per cent (P < 0·02) and 85.9 per cent (P < 0·01), respectively, of control preparations. THP, 10^?5 M, also significantly decreased synaptosomal retention of the labeled neuroamine. (¹⁴C)-dopamine was used in an analysis of alkaloid effects on catecholamine uptake kinetics. K_i values obtained were: 0·7 × 10^?5 M (THP); 3·5 × 10^?5 M (THB); 1·25 × 10^?4 M (SAL); and 2·2 × 10^?4 M (TMB). These results have been interpreted to suggest that the affinity of these amine-derived alkaloids for the catecholaminergic uptake mechanisms, although not marked when compared to that of dopamine or norepinephrine, may be sufficient under conditions of highly localized formation and accumulation to have important physiological sequelae.     

Effects of 8-azaadenosine and formycin on cell lethality and the synthesis and methylation of nucleic acids in human colon carcinoma cells in culture

The cytocidal and biochemical effects of formycin and 8-azaadenosine in the presence and absence of the adenosine deaminase inhibitor, 2′-deoxycoformycin, were studied in human colon carcinoma (HT-29) cells in culture. Logarithmically growing cells were unaffected by 24-hr exposure to either 10^?6M formycin or 8-azaadenosine, but 1 to 1.4 log reductions in colony formation were produced by 10^?5M of each analog. In the presence of 10^?6M 2′-deoxycoformycin, a 3- and 30-fold potentiation of the cytocidal activity of 8-azaadenosine and formycin, respectively, was produced. Inhibition of DNA synthesis but not RNA synthesis by 8-azaadenosine paralleled its cytocidal activity; however, neither variable correlated closely with the cytotoxic effects of formycin. In addition, the methylation of nuclear RNA was unaffected by both drugs while the methylation of 5-methyl-deoxycytidine in DNA was inhibited to a lesser extent than DNA synthesis. Measurements of the incorporation of [³H]formycin and [³H]8-azaadenosine into nuclear RNA and DNA in the presence and absence of 2′-deoxycorformycin indicated that formycin substitution in RNA and DNA was enhanced 10- and 20-fold, respectively, while [³H]8-azaadenosine incorporation into both nucleic acids was increased 6- to 7-fold. These results suggest that the incorporation of formycin into nucleic acids, particularly DNA, correlates closely with its lethal effect on cell viability. On the other hand, the cytocidal activity of 8-azaadenosine more clearly parallels its inhibitory effect on DNA synthesis rather than its substitution into nucleic acids.     

Effect of diphenylhydantoin on the uptake and catabolism of l-[3H]norepinephrine in vitro in rat cerebral cortex tissue

The effect of diphenylhydantoin on the accumulation of [³H]norepinephrine in vitro was examined in brain slices prepared from rat cerebral cortex. High concentrations of diphenylhydantoin (10^?3 M) caused a significant reduction in the 5-min accumulation of [³H]norepinephrine. On the other hand, 10^?5–10^?4 M diphenylhydantoin facilitated the 20-min accumulation of [³H]norepinephrine. This facilitative action of diphenylhydantoin was (1) associated with a reduction in oxidative catabolism of [³H]norepinephrine and (2) abolished by the 2-hr pretreatment of rats with 100 mg/kg of nialamide (i.p.). The inhibitory action of diphenylhydantoin on the oxidative catabolism of [³H]norepinephrine was observed in both whole and lyzed crude synaptosomal preparations. When diphenylhydantoin and pargyline were compared, it was found that pargyline $\text{(}\text{id}{}_{50}\text{= 1.5 × 10}{}^{\mathrm{?6}}\text{M}\text{)}$ was 37 times more effective than diphenylhydantoin $\text{(}\text{id}{}_{50}\text{= 5.5 × 10}{}^{\mathrm{?5}}\text{M}\text{)}$ in inhibiting the oxidative deamination of [³H]norepinephrine. These results suggest that diphenylhydantoin alters norepinephrine metabolism in cerebral cortex slices by an inhibitory action on (1) monoamine oxidase activity and (2) the neuronal uptake system.     

Inhibitory effects of cordycepin on cyclic nucleotide-dependent and cyclic nucleotide-independent protein kinases

The in vitro effect of cordydepin was tested using various protein kinase preparations. These included cyclic AMP-dependent protein kinase (A-PK) from bovine heart, cyclic GMP-dependent protein kinase (G-PK) from fetal guinea pig lung, and two cyclic nucleotide-independent nuclear protein kinases (PK-I and PK-II) prepared from rat hepatoma 3924A and rat liver. The 50 per cent inhibitory concentrations (id₅₀) of cordycepin for A-PK and G-PK ranged from 1.5–5.0 × 10^?4M and 2.5–8.0 × 10^?4 M, respectively, depending on the presence or absence of cyclic AMP and cyclic GMP in the assay. The id₅₀ of cordycepin with either hepatoma 3924A or rat liver PK-I and PK-II was 4.5 × 10^?5 M and 1.0 × 10^?3 M. respectively. The inhibitory effect of cordycepin was competitive with respect to ATP in all cases. The K_{m} for ATP was increased 3-fold and 5-fold by 5 × 10^?4 M cordycepin for G-PK and A-PK, respectively, while the K_m for ATP was increased 10-fold and 4-fold by 1 × 10^?3 M cordycepin for PK-I and PK-II, respectively.     

Effects of haloperidol and apomorphine on catecholamine metabolism in brain slices: Reserpine-like effects of haloperidol

The accumulation, release and catabolism of [³H]dopamine (DA) and [³H]norepinephrine (NE) synthesized from [³H]tyrosine were measured in mouse striatal and substantia nigral slices. Apomorphine inhibited both [³H]NE and [³H]DA accumulation $\text{(}\text{ic}{}_{50}\text{< 10}{}^{\mathrm{?6}}\text{M}\text{)}$, presumably by acting on a presynaptic receptor. Haloperidol (10^?8 M) caused a small, but significant increase in [³H]DA accumulation from [³H]tyrosine in the presence of 26 mM K⁺, possibly reflecting blockade of presynaptic receptors activated by released DA. However, at higher concentrations (10^?6 to 10^?5 M), haloperidol inhibited [³H]DA and [³H]NE accumulation. Reserpine also potently inhibited catecholamine synthesis; chlorpromazine had only a weak effect, and fluphenazine was ineffective. Both haloperidol (10^?5 M) and reserpine (10^?7 M), but not chlorpromazine and fluphenazine, markedly increased the formation of labeled dihydroxyphenylacetic acid (DOPAC) and increased the spontaneous release of labeled DA from striatal slices preloaded with [³H]tyrosine or [¹⁴C]DA. These data suggest that haloperidol has some direct effects on DA metabolism that are unrelated to DA-receptor blockade. Because the effects of haloperidol are apparently independent of DA release, haloperidol may elevate cytoplasmic DA by altering its vesicular storage. This would, in turn, increase the spontaneous release of labeled DA by diffusion, the oxidation of DA to DOPAC by monoamine oxidase, and the end-product inhibition of tyrosine hydroxylase.     

Tight-binding inhibitors—IV. Inhibition of adenosine deaminases by various inhibitors

Three ADA (adenosine deaminase) inhibitors, DHMPR (1,6-dihydro-6-hydroxymethyl purine ribonucleoside); EHNA [erythro-9-(2-hydroxy-3 nonyl)adenine] ; and deoxycoformycin [(R)-3-(2-deoxy-β-d-erythro-pento-furanosyl)-3, 6,7,8-tetrahydroimidazo[4,5-d] [1,3-diazepin-8-ol] or Covidarabin, were compared with regard to their inhibitory behavior with ADAs from human erythrocytes and calf intestine. Marked differences in the times required for establishment of steady state between the enzyme and inhibitors were observed, e.g. DHMPR, virtually instantaneous; EHNA, 2–3 min; and deoxycoformycin, many hr. The parameters of the inhibition of human erythrocytic ADA by deoxycoformycin were as follows: the association rate constant (k₁) = 2.6 × 10⁶ M^?1 sec^?1 ; the dissociation rate constant of the enzyme-inhibitor complex (k₂) = 6.6 × 10^?6 sec^?1; K_i (from $\text{k}{}_2\text{k}{}_1\text{) = 2.5 × 10}{}^{\mathrm{?12}}\text{M}$ and K_i (from I₅₀) = 1.5 × 10^?11 M. The K_i values for EHNA and DHMPR, as determined by classical methods after attainment of steady state, were 1.6 × 10^?9 and 1.3 × 10^?6 M, respectively, for human erythrocytic ADA. The kinetic parameters for EHNA and calf intestinal ADA were as follows: K_i = 6.5 × 10^?9 M (by the method of I₅₀); k₁ = 0.7 × 10⁶ M^?1 sec^?1' and k₂ = 4.6 × 10^?3 sec^?1. On the basis of K_i values, the inhibitors. DHMPR, EHNA and deoxycoformycin (a transition state analog), were classified as readily reversible, semi-tight-binding and tight-binding inhibitors. The difficulties encountered in the kinetic analyses of different types of inhibitors and the methods for dealing with the problems of these inhibitors are discussed.     

Cortisol effects on the glycosaminoglycan synthesis and molecular weight distribution in vitro

The effect of cortisol at different concentrations on the incorporation rate of [³H]glucosamine and [³⁵S]sulphate into glycosaminoglycans (GAGs) in human fibroblast culture medium was studied. The mol. wt distribution of the synthesized GAGs was determined by Sepharose 2B chromatography. Two sensitivity levels of GAGs to cortisol were observed: at a low cortisol concentration ( 1 × 10^?7 M) only the hyaluronic acid synthesis decreased and no changes were observed in the synthesis of sulphated GAGs or glycoproteins. At a high steroid concentration (1 × 10^?3 M) both the synthesis of hyaluronic acid and sulphated GAGs drastically decreased. The mol. wt distribution of medium GAGs did not change at cortisol concentrations 1 × 10^?9 M-1 × 10^?5 M. The possible role of cortisol in the metabolism of hyaluronic acid in vivo is discussed.     

Studies on the inhibition of poly(A) polymerase from rat liver and hepatoma 3924A by rifamycin SV derivatives

The inhibition of poly (A) polymerase activity from liver and hepatoma 3924A by several O-n-alky derivatives of rifamycin SV:3-formyloxime was studied. Only the O-n-pentyl (AF/012) and the O-n-octyl (AF/013) analogs were active at the maximum tested concentration of 1 × 10^?4 M. Equivalent concentrations of liver and hepatoma enzymes were inhibited to the same degree by both compounds. The 50 per cent inhibitory concentration (IC₅₀) for AF/013 and AF/012 was 1.2 × 10^?5M and 7.5 × 10^?5M respectively. Poly (A) polymerase activity was more sensitive to AF/013 if the enzyme was preincubated with the drug before the initiation of the reaction with poly (A) than if the reaction was initiated by the addition of the enzyme. Addition of AF/013 after initiation of the assay led to a rapid inhibition of poly (A) synthesis. The mechanism of inhibition by AF/012 and AF/013 of poly(A) polymerase was of the non-competitive type with respect to ATP.     

Human placental choline acetyltransferase: Nature and molecular aspects of the inhibition by iodo- and bromoacetylcholines

Human placental choline acetyltransferase (ChA) was assayed by the formation of acetyl[¹⁴C]choline (ACh) from choline and acetyl[¹⁴C]coenzyme A (ACoA). Among the four halogenoacetylcholines, iodo- and bromoacetylcholines were potent inhibitors (I₅₀: 2 × 10^?6 M) of ChA. They were studied in the following experiments: (1) variation of initial velocity as a function of choline concentration (5 × 10^?5 to 2 × 10^?4 M, ACoA 10^?5 M) at four levels of iodo- and bromoacetylcholines (5 × 10^?7 to 2.5 × 10^?6 M); (2) variation of initial velocity as a function of ACoA concentration (6 × 10^?6 to 2.5 × 10^?5 M, choline 10^?4 M) at four levels of iodo- and bromoacetylcholines; (3) dialysis of the inhibited ChA; and (4) capacities of iodo- and bromoacetates to inhibit ChA. In (I) and (2), primary plots were noncompetitive and secondary plots of slopes and intercepts were linear. In (3), only 70–80 per cent of the inhibition was reversible. In (4), high levels of halogeno-acetates (10^?2 M) inhibited ChA by 20–30 per cent. These investigations support the formation of ternary intermediates (CoA ChA · IACh or CoA · ChA · BrACh) during the reaction of placental ChA. The quaternary ammonium head and the positive carboxyl-carbon do seem to contribute to the ChA inhibition by IACh and BrACh. The irreversible component of the inhibition (20–30 per cent) with XACh or iodo- and bromoacetates can be explained by possible (1) formation of R-carboxymethylated ChA (where R = -O-, -S-or = N-) which is less active than the original ChA, (2) formation of S-carboxymethylated CoA which is slowly dialyzed free from the enzyme surface, or (3) conversion of the part of the enzyme to the —N—carboxymethylated form which is inactive.     

A comparison of the inhibitory effects of new non-tricyclic amine uptake inhibitors on the uptake of norepinephrine and 5-hydroxytryptamine into synaptosomes of the rat brain

The effects of new non-tricyclic amine uptake inhibitors, FS32 and FS97, on the uptake of [³H]-norepinephrine (NE) into the hypothalamic synaptosomes and [³H]-5-hydroxytryptamine (5-HT) into whole brain synaptosomes were studied. Their effects were compared with those of tricyclic antidepressants. The uptake of [³H]-NE was inhibited competitively by FS32 and FS97 with a respective K_i value of 6.5 × 10^?7 M and 3.8 × 10^?7 M. The potency of FS32 and FS97 to inhibit this uptake was almost comparable to that of clomipramine and imipramine, respectively. In the case of [³H]-5-HT uptake, FS32 and FS97 also showed competitive inhibition with a respective K_i value of 2.9 × 10^?6 M and 5.9 × 10^?6M. The ability of FS32 to inhibit [³H]-5-HT uptake was almost equal to that of nortriptyline, while FS97 was two times more potent than iprindole in inhibiting this uptake.     

Effects of chlorpromazine and actinomycin D on uptake and incorporation of certain amino acids,hypoxanthine and thymidine in cultures of human skin epithelial cells

Chlorpromazine (CPZ) 1· × 10^?4 M inhibited the uptake and incorporation of alanine (25 and 3 per cent of controls respectively), the uptake of α-aminoisobutyric acid (AIB, 39 per cent of controls) and the uptake and incorporation of hypoxanthine (36 and 44 per cent of controls) into acid-soluble and insoluble fractions of human skin epithelial cells (HE cells, NCTC 2544) grown in culture. The uptake of phenylalanine and 1-aminocyclopentane-1-carboxylic acid (cycloleucine) was not inhibited by CPZ in the same dose range, but CPZ above 10^?5 M inhibited the incorporation of phenylalanine into acid-insoluble material with 50 per cent inhibition at 6·5 × 10^?5 M. Actinomycin D stimulated the uptake of thymidine into the acid-soluble fraction of the HE-cells, 5·0 μg/ml increased the uptake to 160 per cent of the controls. The uptake of hypoxanthine was inhibited by actinomycin D, 5·0 μg/ml reduced the uptake to 67 per cent of controls. Actinomycin D did not alter the uptake of AIB or cycloleucine.     

Inhibition of methylation of nuclear ribonucleic acid in L1210 cells by tubercidin, 8-azaadenosine and formycin

The adenosine analogs tubercidin (7-deazaadenosine), formycin (7-amino-3-[β-d-ribofuranosyl] pyrazolo[4,3-d]pyrimidine) and 8-azaadenosine were examined for their effects on the synthesis and methylation of nuclear RNA in L1210 cells in vitro. Total RNA and DNA synthesis was affected to the greatest extent by tubercidin (IC₅₀ = 7 × 10^?6M) and to an insignificant degree by 8-azaadenosine and formycin; however, the effects of the latter two drugs, but not of tubercidin, were potentiated by 2'-deoxycoformycin, an inhibitor of adenosine deaminase. In the presence of 2'-deoxycoformycin, RNA synthesis was inhibited by 40 per cent at 1 × 10^?4 M 8-azaadenosine and by 50 per cent at 2 × 10^?4 M formycin, while DNA synthesis was inhibited less extensively. Alkaline hydrolysis of nuclear RNA labeled with [¹⁴C]uridine and l-[methyl-³H]methionine showed preferential inhibition of base methylation in mononucleotides, but not of 2′-O-methylation in dinucleotides, for all three drugs. This differential effect persisted to varying degrees in ?18S and 4S nuclear RNA separated by electrophoresis. The reduction in base methylation in 4S RNA was associated with seven of the eight methylated nucleosides in 4S RNA separated by two-dimensional thin-layer chromatography. These results indicate that tubercidin, 8-azaadenosine and formycin can preferentially inhibit the base methylation of nuclear RNA relative to its synthesis.