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Biomedical subjects

A J Bitonti

Publications and source records attributed to A J Bitonti.

67 records · Page 4Linked to original sources
PubMed1985

Assaying ornithine and arginine decarboxylases in some plant species.

A release of (14)CO(2) not related to ornithine decarboxylase activity was found in crude leaf extracts from Lycopersicon esculentum, Avena sativa, and especially from the pyrrolizidine alkaloid-bearing Heliotropium angiospermum when incubated with [1-(14)C]- or [U-(14)C]ornithine. The total (14)CO(2) produced was about 5- to 100-fold higher than that due to ornithine decarboxylase activities calculated from labeled putrescine (Put) found by thin-layer electrophoresis in the incubation mixtures. Partial purification with (NH(4))(2)SO(4) did not eliminate completely the interfering decarboxylation. When incubated with labeled arginine, a very significant (14)CO(2) release not related to arginine decarboxylase activity was observed only in extracts from H. angiospermum leaves, especially in Tris.HCl buffer. Under the assay conditions, these extracts exhibited oxidative degradation of added Put and agmatine (Agm) and also revealed a high arginase activity. Amino-guanidine at 0.1 to 0.2 millimolar prevented Put degradation and greatly decreased oxidative degradation of Agm; ornithine at 15 to 20 millimolar significantly inhibited arginase activity. A verification of the reliability of the standard (14)CO(2)-based method by assessing labeled Put and/or Agm-formed in the presence of added aminoguanidine and/or ornithine when needed-is recommended especially when crude or semicrude plant extracts are assayed.When based on Put and/or Agm formed at 1.0 to 2.5 millimolar of substrate, the activities of ornithine decarboxylase and arginine decarboxylase in the youngest leaves of the tested species ranged between 1.1 and 3.6 and 1 and 1600 nanomoles per hour per gram fresh weight, respectively. The enzyme activities are discussed in relation to the biosynthesis of pyrrolizidine alkaloids.

Journal Article↗
PubMed1985

Activities of arginine and ornithine decarboxylases in various plant species.

In extracts from the youngest leaves of Avena sativa, Hordeum vulgare, Zea Mays, Pisum sativum, Phaseolus vulgaris, Lactuca sativa, and four pyrrolizidine alkaloid-bearing species of Heliotropium, the activities of ornithine decarboxylase, close to V(max), ranged between traces and 1.5 nanomoles per hour per gram fresh weight when based on putrescine formed during incubation with labeled ornithine. The arginine decarboxylase activities in the same extracts ranged between 8 and 8000 nanomoles per hour per gram fresh weight being lowest in the borages and highest in oat and barley. alpha-Difluoromethylornithine and alpha-difluoromethylarginine inhibited ornithine and arginine decarboxylases, respectively, in all species. Agmatine, putrescine, spermidine, and spermine were found in all, diaminopropane in eight, and cadaverine in three species.No correlation was observed between arginine or ornithine decarboxylase level and the levels of total polyamines. The in vitro decarboxylase activities found in the borages cannot explain the high accumulation of putrescine-derived pyrrolizidines in their youngest leaves if the pyrrolizidines are produced in situ from arginine and/or ornithine as precursors; other possibilities are discussed.In assays of ornithine decarboxylase, an interference of decarboxylation not due to this enzyme was observed in extracts from all species. In arginine decarboxylase assays, the interfering decarboxylation as well as the interference of arginase were apparent in two species. Addition of aminoguanidine was needed to suppress oxidative degradation of putrescine and agmatine during incubation of extracts from pea, bean, lettuce, Heliotropium angiospermum, and Heliotropium indicum.

Journal Article↗
PubMed1984

Inhibition of ADP-ribosyltransferase activity of cholera toxin by MDL 12330A and chlorpromazine.

ADP-ribosylation by cholera toxin of the guanine nucleotide binding regulatory protein (Gs) of rat liver membrane adenylate cyclase was inhibited by 0.1-1 mM MDL 12330A or 0.1-1 mM chlorpromazine. Basal as well as cholera toxin activated adenylate cyclase activity in liver membranes was also inhibited by the two drugs. NAD glycohydrolase activity and self-ADP-ribosylation of cholera toxin were also inhibited by MDL 12330A and chlorpromazine. These effects of MDL 12330A and chlorpromazine may be related to their effects on cholera toxin-induced fluid secretion in vivo.

Adenosine Diphosphate Ribose↗
PubMed1984

Regulation of growth and macromolecular synthesis by putrescine and spermidine in Pseudomonas aeruginosa.

Growth of P. aeruginosa, slowed by the addition of monofluoromethylornithine, difluoromethylarginine and dicyclohexylammonium sulfate, could be restored by addition of 0.1 mM putrescine plus 0.1 muM spermidine, or 0.1 mM spermidine or 5 mM putrescine by themselves. Lower concentrations of putrescine (0.1 mM - 1 mM) also partially reversed the growth inhibition. Conversion of putrescine to spermidine continued, although at a markedly reduced ratio, in the drug-inhibited cells, but intracellular spermidine concentrations remained depressed suggesting that reversal of inhibition by putrescine may be a direct effect. There was appreciable back-conversion of any added spermidine to putrescine with a demonstrable increase in total intracellular putrescine levels, making conclusions on the effects of spermidine ambiguous. Spermine (0.1 mM), a polyamine not present in bacteria, was also effective in reversing growth inhibition, probably because of its conversion into spermidine and putrescine. The effects of putrescine, spermidine and spermine were specific in that the non-physiological amines, 1,3-diaminopropane, 1,5-diaminopentane (cadaverine), 1,6-diaminohexane, or 1,7-diaminoheptane could not reverse the effects of the three drugs. Rates of total protein, RNA and DNA synthesis were all slowed to the same extent as growth rate and showed similar recovery with the addition of putrescine or spermidine. A role for putrescine in P. aeruginosa growth processes is suggested.

Bacterial Proteins↗
PubMed1984

Characterization of spermidine synthase from Trypanosoma brucei brucei.

Spermidine synthase from Trypanosoma brucei brucei was characterized and found to be similar to spermidine synthase from other sources. The Km for putrescine was found to be 0.2 mM and the Km for decarboxylated S-adenosylmethionine 0.1 microM. The approximate molecular weight of the enzyme was 74 000 as determined by a combination of molecular sieve chromatography and sucrose density gradient centrifugation. Spermidine synthase activity was markedly inhibited in vitro by dicyclohexylamine (50% inhibition at 3 microM) and cyclohexylamine (50% inhibition at 15 microM); both being competitive inhibitors with respect to putrescine. S-Adenosyl-1,8-diamino-3-thiooctane, a nucleoside bisubstrate analog, was also a potent inhibitor of enzyme activity (50% inhibition at 25 microM). Administration of dicyclohexylamine to mice with trypanosomiasis resulted in no increase in survival time probably due to the lack of effect on trypanosome spermidine concentrations. Other possible inhibitors remain to be tested in vivo.

Animals↗
PubMed1983

Inhibition of bacterial aminopropyltransferases by S-adenosyl-1,8-diamino-3-thiooctane and by dicyclohexylamine.

Bacterial aminopropyltransferases from Escherichia coli, Serratia marcescens and Pseudomonas aeruginosa were strongly inhibited by S-adenosyl-1,8-diamino-3-thiooctane (AdoDATO) and by dicyclohexylamine. The sensitivity to these drugs in vitro was comparable to that of mammalian spermidine synthase, but AdoDATO was much less potent in reducing spermidine content in the bacteria than in mammalian cells. Although AdoDATO was a stronger inhibitor than dicyclohexylamine in vitro, dicyclohexylamine was more active in reducing bacterial spermidine levels in vivo, suggesting that it is taken up better or is more stable in the cell and is the preferable compound for in vivo studies in microorganisms. The strong inhibition of spermidine synthases by AdoDATO which is a transition state analog supports the concept that these enzymes proceed by a single displacement reaction, rather than by a ping-pong mechanism.

Adenosine↗
PubMed1982

Restriction of bacterial growth by inhibition of polyamine biosynthesis by using monofluoromethylornithine, difluoromethylarginine and dicyclohexylammonium sulphate.

Bacterial growth was measurably slowed by a combination of drugs which inhibit polyamine-biosynthetic enzymes. Addition of DL-alpha-monofluoromethylornithine, which was shown to inactivate irreversibly ornithine decarboxylase extracted from Escherichia coli (Ki = 0.36 mM) and Pseudomonas aeruginosa (Ki = 0.30 mM), DL-alpha-difluoromethylarginine and dicyclohexylammonium sulphate to cultures of E. coli or P. aeruginosa resulted in a 40 and 70% increase in generation times (decreased growth rates) respectively, which was completely reversed by the addition of 0.1 mM-putrescine plus 0.1 mM-spermidine to the medium. Decreased intracellular polyamine concentrations correlated with increased generation times; putrescine concentration was decreased by 70% in E. coli and 80% in P. aeruginosa, while spermidine concentration was decreased by 50% in E. coli and 95% in P. aeruginosa. Subsequent investigation of the inactivation of the ornithine decarboxylase by monofluoromethylornithine indicated that it was active-site directed, as the normal substrate ornithine slowed the rate of inhibition. Specific interference with polyamine biosynthesis may be a viable approach to control of some bacterial infections.

Arginine↗
PubMed1982

Resolution and activity of adenylate cyclase components in a zwitterionic cholate derivative [3-[(3-cholamidopropyl) dimethylammonio]-1-propanesulfonate].

Bovine brain adenylate cyclase was solubilized with 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate (CHAPS), sodium cholate, sodium deoxycholate, or these detergents plus (NH4)2SO4. The specific activity of the extract obtained with 13 mM CHAPS alone was several times those of the other detergent extracts with or without (NH4)2SO4. After solubilization with 13 mM CHAPS, gel filtration completely separated the catalytic unit (C) from the guanine nucleotide binding protein (G/F). C activity when assayed with 5 mM Mn2+ was 5 times that assayed with 10 mM Mg2+ and was unresponsive to GPP(NH)P. C activity was increased approximately 150% by GPP(NH)P in the presence of G/F extracted from human erythrocyte ghosts and approximately 100% by Ca2+ plus calmodulin in assays with Mg2+. On gel filtration and/or density gradient centrifugation, the physical properties of C from brain or AC- cells and G/F from bovine or pig erythrocytes in CHAPs were similar to those observed in other detergents. It appears that the use of CHAPS for solubilization of adenylate cyclase and separation of C and G/F may well prove advantageous in studies of the molecular interactions between the protein subunits and activators of the enzyme as well as for the initial purification of C.

Adenylyl Cyclases↗
PubMed1980

Prostaglandins increase GTP hydrolysis by membranes from human mononuclear cells.

Both adenylate cyclase and GTPase activities in human mononuclear cell membranes were increased by prostaglandins. Adenylate cyclase activity, however, was enhanced by much lower concentrations of PGE1 (prostaglandin E1) than were required to increase GTPase. PGE2, PGA1, PGB1, and PGF1 alpha also stimulated GTPase activity. These same prostaglandins, with the notable exception of PGF1 alpha, increased adenylate cyclase activity (PGE2 greater than PGA1 greater than or equal to PGB1). Isoproterenol, 100 microM, doubled adenylate cyclase without altering GTPase activity. Choleragen activated adenylate cyclase in mononuclear cell membranes but had no effect on GTPase activity whether or not PGE1 was present. Mononuclear cells were separated into adherent and nonadherent populations by two different methods to examine the possibility that the prostaglandin-stimulated GTPase was confined to a specific type of mononuclear cell. Adenylate cyclase in membranes from both adherent and nonadherent cells was activated by PGE1, but neither PGE1 nor choleragen altered GTPase activity in these preparations. It appears that, although several prostaglandins can increase GTPase activity in mononuclear cell membranes, the increase in GTPase activity is not consistently associated with activation of adenylate cyclase by prostaglandins.

Animals↗
PubMedDate not supplied

Response of human colon and prostate tumor xenografts to (E)-2'-deoxy-2'-(fluoromethylene) cytidine, an inhibitor of ribonucleotide reductase.

Daily oral or intravenous administration of the ribonucleoside diphosphate reductase inhibitor, (E)-2'-deoxy-2'-(fluoromethylene)cytidine (MDL 101,731), to nude mice caused rapid regression of colon and prostate xenografts. Studies were performed to optimize dosing schedule and route of administration. MDL 101,731 was tested against colon (HT-29) and prostate (PC-3) xenografts using twice weekly oral and intravenous administration. PC-3 tumors regressed almost completely with doses of 20 mg/kg. HT-29 xenografts regressed during intravenous administration of 100 mg/kg MDL 101,731, whereas oral administration was less effective. Based on these data it seems that MDL 101,731 is effective when administered intravenously, twice weekly and is an excellent candidate for clinical development against solid tumors.

Animals↗
PubMedDate not supplied

Antitumor and chemopreventive effects of a clomiphene analog, MDL 103,323, in mammary carcinoma.

MDL 103,323, an enclomiphene analog, was tested for binding to the estrogen receptor, inhibition of human tumor xenografts and prevention of carcinogen-induced mammary tumors. MDL 103,323 had 5-6 fold greater affinity for the human estrogen receptor than did either tamoxifen or enclomiphene. Consistent with enhanced binding affinity, MDL 103,323 was more potent against MCF-7 cell proliferation and MCF-7 xenografts in nude mice were inhibited almost completely by MDL 103,323 doses > or = 0.02 mg/mouse/day (ED50 ł 0.01 mg/mouse/day). N-methylnitrosourea induced rat mammary carcinomas were inhibited by > or = 50% at 0.003 mg/kg daily and by 90% at 0.1 mg/kg/day. The antitumor potency and efficacy of MDL 103,323 are striking and further evaluation of the compound for potential clinical utility is warranted.

Animals↗