Assay of 6-mercaptopurine and its metabolites in patient plasma by high-performance liquid chromatography with diode-array detection

A reversed-phase high-performance liquid chromatography (HPLC) method was developed to determine 6-mercaptopurine (MP) and seven of its metabolites (6-thioguanine, 6-thioxanthine, 6-mercaptopurine riboside, 6-thioguanosine, 6-thioxanthine riboside, 6-methylmercaptopurine and 6-methylmercaptopurine riboside) simultaneously in human plasma. A volume of 100 μl of plasma was used. Protein was removed from the sample by a simple and easy ultrafiltration step and ultrafiltrate was directly injected onto the HPLC system. Analytes were detected and confirmed with a diode-array detector before quantitation at 295 and 330 nm. The limit of detection for the analytes ranged from 20 to 50 nM. For the majority of patients receiving a 1 g/m² MP intravenous infusion, MP and all metabolites except 6-thioguanine and 6-methylmercaptopurine riboside were present. This method serves as useful tool to characterize pharmacokinetics and pharmacodynamics of MP in oncology patients, and the small volume of plasma lends itself to pediatric studies.     

Accumulation of epidermal growth factor within cells does not depend on receptor recycling

Quiescent cells seemingly have a constant number of surface epidermal growth factor receptors. However, exposure of cells to agents which interfere with normal protein turnover suggests that these receptors are internalized and degraded with an apparent half-life of ～6 hours. We show that the time course of maximal accumulation of ligand-receptor complexes is not altered under conditions where degradation of the ligand is inhibited, indicating that no degradation occurs during its first hour of exposure to cells. We also conclusively demonstrate that epidermal growth factor receptors are not recycled during the initial uptake of the ligand, and that a component of pinocytosis of this growth factor is dependent on $\text{de}\text{novo}$ protein synthesis.     

Inhibition of hepatocytic protein degradation by methylaminopurines and inhibitors of protein synthesis

Nutritional control of protein degradation in isolated rat hepatocytes can take place in the absence of protein synthesis. Suppression of degradation by amino acids (step-up) is unaffected and the enhanced degradation seen upon amino acid deprivation (step-down) is only partially inhibited by cycloheximide at a concentration (10^?3 M) which inhibits protein synthesis virtually completely. Protein degradation per se is, however, inhibited by cycloheximide as well as by puromycin, apparently at least in part by mechanisms additional or unrelated to their effect on protein synthesis. Several puromycin analogues (methylaminopurines) are stronger inhibitors of protein degradation than of protein synthesis, most notably puromycin aminonucleoside and 6-dimethylaminopurine riboside (N⁶, N⁶-dimethyladenosine). The latter compounds appear to specifically inhibit cellular autophagy, since neither the degradation of endocytosed protein (asialofetuin) nor the extralysosoma (amino acid-, propylamine- and leupeptin-resistant) degradation are affected.     

Differential effects of cycloheximide on protein and RNA synthesis as a function of dose

The $\text{in}$$\text{vivo}$ dose response of rat liver protein and DNA synthesis to cycloheximide have been determined. Protein synthesis was quite sensitive to relatively low doses of cycloheximide being inhibited by more than 90% with 1.5 mg/kg. Maximal inhibition of 98% was achieved with 5 mg/kg. There was no inhibition of RNA synthesis with this dose of cycloheximide. Larger doses of cycloheximide did lead to quite marked inhibition of RNA synthesis without any change in the already maximally inhibited rate of protein synthesis. This differential effect of cycloheximide on protein and RNA synthesis as a function of dose indicates that the inhibition of RNA synthesis caused by the antibiotic is not a consequence of the inhibition of protein synthesis but related otherwise to the effects of large doses of cycloheximide.     

Cyclic 3',5'-adenosine monophosphate stimulates trehalose degradation in baker's yeast

The levels of cyclic 3′,5′-AMP and trehalose, as well as the specific activity of the trehalase have been investigated in cells of baker's yeast ($\text{Saccharomyces cerevisiae}$) during the lag phase preceding growth. During the first few minutes a substantial increase in the intracellular concentration of cyclic 3′,5′-AMP was observed, followed by a 6–8 fold increase in trehalase activity concomitant with the rapid degradation of trehalose. Cell free extracts prepared from resting yeast were shown to contain a cryptic trehalase, which under physiological conditions could be activated by cyclic 3′,5′-AMP to the same degree as $\text{in vivo}$. These observations suggest that in the lag phase of growth, the level of trehalose in baker's yeast is under control of a system, regulated by the level of cyclic 3′,5′-AMP.     

Differential effect of Ca+2 on the translation of yeast mitochondrial and some viral RNA'S in an E.coli cell-free system.

Addition of 6mM CaCl₂ to an $\text{E. coli}$ cell-free system resulted in a several-fold enhancement of yeast mt RNA translation and in a severe inhibition of protein synthesis directed by MS2, Qβ and T₅ RNA's. CaCl₂ did not alter the Mg⁺² optimum or the time-course of protein synthesis and had no apparent effect on RNA degradation. Formaldehyde treatment of MS2 RNA markedly diminished the CaCl₂-mediated inhibition of its translation. Addition of equimolar amounts of EGTA, together with CaCl₂, abolished the effect of the latter on cell-free protein synthesis. FMet tRNA binding to ribosomes was enhanced by CaCl₂ in the presence of mt RNA, inhibited in the presence of MS2 RNA, and unaffected in the presence of formaldehyde-treated MS2 RNA. Maximal effect on initiation complex formation was observed with 0.1 mM CaCl₂.     

8,12-secoprostaglandins. 10-AZA and 8-OXA analogs

The synthesis of the 8,12-secoprostaglandin E₁ analogs $\text{6}$ and $\text{12}$ is reported.     

Induction of different species of cytochrome P-450 by coplanar and noncoplanar isomers of hexachlorobiphenyl

The effects of coplanar⁺ 3,4,5,3′,4′,5′-hexachlorobiphenyl (HCB) and noncoplanar 2,4,5,2′,4′,5′-HCB, 2,3,5,2′,3′,5′-HCB, phenobarbitone (PB) and 3-methylcholanthrene (3-MC) on drug metabolizing enzymes have been studied 72 hr after dosing in male rat liver. 3-MC and 3,4,5,3′,4′,5′-HCB induced the activity of ethoxyresorufin deethylase dramatically. NADPH cytochrome P-450 reductase and benzphetamine $\text{N}$-demethylase were induced by PB and noncoplanar isomers and not by 3-MC or 3,4,5,3′,4′,5′-HCB. Sodium dodecyl sulfate polyacrylamide gel electrophoresis of the microsomes obtained from various groups showed that 3-MC and 3,4,5,3′,4′,5′-HCB induced the synthesis of a polypeptide of approximate 54,500 daltons which was absent in the microsomes obtained from control, PB or noncoplanar isomer treated animals. Noncoplanar isomers and PB induced the synthesis of a polypeptide of approximate 51,000 daltons. These results, along with the reduced, CO difference spectra, demonstrate that 3,4,5,3′,4′,5′-HCB induces the synthesis of cytochrome P-448 and resembled 3-MC in its mechanism of action, while noncoplanar isomers induced the synthesis of cytochrome P-450 and resembled PB in its mechanism of action. Further administration of various doses of 3,4,5,3′,4′,5′-HCB to genetically responsive mice (C57BL/6J), induced cytochrome P-450, caused one nm shift in the difference spectrum of reduced microsomes and induced the activity of ethoxyresorufin deethylase, whereas it did not induce the activity of ethoxyresorufin deethylase in non-responsive mice (DBA/2J) even at the highest dose studied. These studies indicate the fact that coplanar and noncoplanar isomers have differential interaction with $\text{Ah locus}$.     

Cyclic AMP-dependent protein kinase of Neurospora crassa

$\text{Neurospora}\text{crassa}$ was surveyed for cyclic AMP-dependent protein kinase activity. Two peaks (I and II) of protein kinase activity were demonstrated by DEAE-cellulose chromatography of wild type $\text{Neurospora}$ extracts. Peak I was stimulated by cyclic AMP, eluted below 60 mM NaCl and had high activity using histone H2B as substrate. Peak II eluted at 200–250 mM NaCl; its activity was not cyclic AMP stimulated and was highest with dephosphorylated casein as a substrate. Cyclic AMP binding to a protein associated with the protein kinase is specifically inhibited by certain cyclic AMP analogs.     

Purine control of mouse oocyte maturation: Evidence that nonmetabolized hypoxanthine maintains meiotic arrest

Hypoxanthine is present in preparations of follicular fluid and has been shown to suppress the spontaneous meiotic maturation of mammalian oocytes in vitro. The present experiments examined the possible role of hypoxanthine metabolism in mediating this meiotic arrest. Four putative inhibitors of the enzyme, hypoxanthine phosphoribosyltransferase (HPRT), which metabolizes hypoxanthine to inosine monophosphate, were tested on lysates of oocyte-cumulus cell complexes. At a concentration of 1 mM, 6-mercapto-9-(tetrahydro-2-furyl)-purine (MPTF) and 6-mercaptopurine (6-MP) suppressed enzymatic activity by 86% and 98%, respectively, while 6-azauridine and 2,6-bis-(hydroxyamino)-9-β-D-ribofuranosyl-purine had no effect. MPTF and 6-MP increased the inhibitory effect of hypoxanthine on germinal vesicle breakdown, but the other agents did not. The 2 active agents had similar effects on salvage activity and hypoxanthine-maintained meiotic arrest in denuded oocytes. Also, oocytes from XO mice were more sensitive to the meiosis-arresting action of hypoxanthine than oocytes from XX littermates, which have twice the HPRT activity. The actions of the HPRT inhibitors were not due to their conversion to nucleotides via HPRT and negative feedback on purine de novo synthesis, because azaserine and 6-methylmercaptopurine riboside, which are more potent inhibitors of de novo synthesis, had a stimulatory, rather than inhibitory, effect on hypoxanthine-arrested oocytes. Furthermore, several lines of evidence indicate that metabolism of hypoxanthine to xanthine and uric acid by xanthine oxidase does not mediate the inhibitory action of this purine base on meiotic maturation. The data therefore suggest that nonmetabolized hypoxanthine is responsible for the meiotic arrest observed, most likely through suppression of cAMP degradation. © 1993 Wiley-Liss, Inc.     

Substrate specificity of aromatic-L-amino acid decarboxylase

The benzo[b]thiophene and l-methylindole analogs of tryptophan have been tested for their ability to act as substrates for aromatic-L-amino acid decarboxylase $\text{in vitro}$. Neither of the analogs showed detectable decarboxylation under conditions that produced significant rates of decarboxylation of tryptophan. In addition, both analogs inhibited the decarboxylation of tryptophan and phenylalanine by the $\text{in vitro}$ system.     

Identification and characterization of a new methylated amino acid in ribosomal protein L33 of Escherichiacoli

Methylated amino acids from ribosomal protein L33 of various $\text{Escherichia}\text{coli}$ strains (Q13, B and MRE600) were analyzed. It was found that while protein L33 from $\text{E.}\text{coli}$ Q13 contains two methylated neutral amino acids (peaks I and II), only one methylated neutral amino acid (peak I) was found in protein L33 derived from both $\text{E.}\text{coli}$ strains B and MRE600. The methylated amino acid present in peak I was identified as N-monomethylalanine by ion-exchange column chromatography, high-voltage paper electrophoresis and descending paper chromatography using different solvent systems. This marks the first time that N-monomethylalanine was found in any ribosomal protein.     

Identification of a phi X174 coded protein involved in the inhibition of beta-galactosidase synthesis in Escherichia coli

The synthesis of β-galactosidase (EC 3.2.1.23: β-D-galactoside galactohydrolase) in $\text{Escherichia}\text{coli}$ is repressed as a result of infection with single-stranded DNA phage ØX174. An $\text{amber}$ mutant in ØX174 cistron A, which codes for two proteins, does not inhibit the enzyme synthesis while $\text{amber}$ mutants in all other genes do cause repression. A mutant near the amino-terminal end of cistron A, which produces the small 35,000 molecular weight cistron A polypeptide, also inhibits the synthesis of β-galactosidase. Inhibition is also observed in an $\text{Escherichia}\text{coli}\text{rep}$ mutant which does not support the replication of replicative-form DNA. Exogenous nucleotide bases and cyclic 3′,5′-adenosine monophosphate (cyclic AMP) do not have any effect on the degree of repression.     

In vitro methylation of rat liver tRNA-synthesis of 3-methylcytosine and 1-methylhypoxanthine

Rat liver methylating enzymes could methylate tRNA extracted from the livers of rats treated with 35 mg/100 g L-ethionine 19 h prior to sacrifice. 1-methylhypoxanthine and 3-methylcytosine were among the methylated bases synthesized $\text{in vitro}$. The synthesis of 3-methylcytosine was dependent on the presence of Mg⁺⁺ although this ion inhibited the overall methylation of the tRNA.     

Inhibition of nuclear protein kinases by adenosine analogues

Two cyclic AMP-independent protein kinases from rat liver nuclei were inhibited competitively by adenosine and a variety of its analogues: cardycepin, tubercidin, 6-mercaptopurine riboside, 6-methylaminopurine riboside, 6-dimethylaminopurine riboside, and 2'-deoxyadenosine. Neither enzyme was inhibited by 6-methoxypurine riboside. Kinase NI was more sensitive to cordycepin, tubercidin, 6-methylaminopurine riboside,, 2'-deoxyadenosine, and adenosine than was kinase NII. Although the effects of all analogues tested were reversed by increasing the concentration of ATP, kinases NII and NI exhibited marked differences in their affinities for adenosine. The results presented here suggest that 1) differences in the catalytic properties of nuclear protein kinases can be detected by inhibitor studies and 2) modifications in the purine ring and sugar moiety of an adenosine analogue can alter its ability to inhibit nuclear protein kinases.     

Leucine biosynthesis: effect of branched-chain amino acids and threonine on a-isopropylmalate synthase activity from aerobic and anaerobic microorganisms

$\text{a-}\text{Isopropylmalate}$ synthase activity was demonstrated in the Sephadex G 25 gel filtrated crude extracts of one yeast and 43 bacterial strains belonging to 14 families. The enzyme was inhibited by leucine from all strains Bacteroides fragilis, Clostridia and several phototropic bacteria. The enzyme was inhibited by leucine from all strains investigated. In crude extracts of 17 species (8 genera) the leucine-mediated inhibition could be relieved by the addition of valine or isoleucine , but not by the addition of threonine or alanine. The enzymes from 11 species (7 genera) were inhibited by 1 mM valine and isoleucine, whereas the enzyme activity from 5 bacteria (4genera) were not so affected. These results suggest that valine and isoleucine are specifically involved in the regulation of leucine biosynthesis in several bacteria. The affect of valine and isoleucine on the IPM-synthase activity from mycobacteria and Corynebacterium autotrophicum lends support to the reclassification of Mycobacterium flavum 301 to C. autotrophicum. The antagonism between 5′,5′,5′-trifluoroleucine and amino acids and $\text{a-}\text{ketoisovalerate}$ was $\text{a-}\text{isopropylmalate}$ synthase in the presence or abssence of leucine and the reversal of the 5′,5′,5′-trifluoroleucine-mediated growth inhibition by these amino acids.     

Effects of nuclear proteins on the activity of soybean DNA polymerase

The effects of nuclear proteins on DNA synthesis were investigated before and after incubation with radioactive ATP and a crude preparation of nuclear protein kinase. After partial purification by DEAE-cellulose chromatography, the major protein fractions were added separately to DNA polymerase assays. One of the seven protein fractions inhibited DNA synthesis by 50%, whereas three other fractions stimulated DNA polymerase activity 3 to 4-fold. After incubation with ATP, one fraction became inhibitory, and the three stimulatory fractions, which had high levels of radioactivity, were more effective. This stimulation of DNA polymerase activity was proportional to added nuclear protein and was maximum at $\text{6 μ}\text{g}\text{20 μ}\text{g}$ DNA.