Metabolism of L-canavanine and L-canaline in the tobacco budworm, Heliothis virescens [Noctuidae].

The metabolism of L-canavanine and L-canaline were investigated in larvae of the tobacco budworm, Heliothis virescens [Noctuidae]. H. virescens larvae were treated with L-[1,2,3,4-14C]canavanine or L-[U-14C]canaline with sufficient cold carrier to provide 5 mg g-1 canavanine or a molar equivalent of canaline (3.81 mg g-1). The preponderant catabolite in both canavanine- and canaline-treated larvae was [14C]homoserine. Other minor metabolites derived from canavanine included [14C]aspartate/asparagine, [14C]glutamate/glutamine, [14C]2-aminobutyrate, [14C]ornithine, [14C]proline, and [14C]isoleucine. Canaline yielded [14C]glutamate/glutamine, [14C]aspartate/asparagine, and [14C]-2-aminobutyrate. Our current studies support the belief that this destructive insect tolerates L-canavanine and L-canaline because of its ability to reductively cleave these potentially insecticidal natural products to L-homoserine and guanidine or ammonia, respectively.     

Studies on the biosynthesis of the antibiotic streptozotocin (streptozocin) by Streptomyces achromogenes var. streptozoticus. Feeding experiments with carbon-14 and tritium labelled precursors.

Feeding experiments and chemical degradations have shown that D-[1(-14)C,2(-3)H]-and-[1(-14)C,6(-3)H] glucosamine, L-[ureido-14C] citrulline, L-[guanidino-14C] arginine and L-[14CH3] methionine specifically label the glucosamine moiety, the urea carbonyl and the N-methyl group of the antibiotic streptozotocin, respectively. Feeding these precursors in amounts of 5 approximately 10 mumoles per 100 ml of culture medium under conditions where the fermentation yielded approximately 20 mumoles of streptozotocin in 24 hours gave incorporation rates which approached 40%. Upon feeding 100 mumoles of either D-[1(-14)C] glucosamine or L-[ureido-14C] citrulline they were incorporated into newly synthesized streptozotocin essentially without dilution by endogeneous precursors. D-[1(-14)C, 6(-3)H] Glucosamine was incorporated without change in T/C ratio while 20% of the tritium was lost from D-[1(-14)C,2(-3)H] glucosamine, suggesting the possibility that D-glucosamine can partially equilibrate with D-fructose prior to its incorporation.     

OF4949, new inhibitors of aminopeptidase B. IV. Effects of OF4949 and its derivatives on enzyme systems

OF4949-I and II inhibited aminopeptidase B from Ehrlich ascites carcinoma in a competitive way and the Ki value for both against L-arginine-beta-naphthylamide was 8 X 10(-9) M. Inhibition by I and II of various exopeptidases and endopeptidases was examined. OF4949-I and II both strongly inhibited leucine aminopeptidase and enkephalin-degrading aminopeptidase; I also inhibited enkephalinase B. The inhibitory effects of various derivatives of I and II on aminopeptidase B activity, showed that the terminal amino and carboxamide groups are essential for activity.     

OF4949, new inhibitors of aminopeptidase B. I. Taxonomy, fermentation, isolation and characterization

New aminopeptidase B inhibitors that we named OF4949-I, II, III and IV were isolated from the culture broth of a fungus, Penicillium rugulosum OF4949. The molecular formula of I was C23H26N4O8 and that of II, C22H24O8, judging from elemental analysis and secondary ion mass spectrometry. The concentrations of I, II, III and IV required for 50% inhibition of aminopeptidase, using Ehrlich ascites carcinoma cells as the source of the enzyme, were 0.0054, 0.0048, 3.4 and 1.7 micrograms/ml, respectively. Components I and II augmented delayed-type hypersensitivity in mice to sheep red blood cells.     

OF4949, new inhibitors of aminopeptidase B. II. Elucidation of structure

The structures of OF4949-I, II, III and IV were identified by analysis of the products of their chemical degradation and by 1H NMR, 13C NMR, and mass spectrometry. These compounds were new cyclic peptides containing diphenyl ether as a chromophore. OF4949-I had two amino acids, beta-hydroxy-L-asparagine and 4-methylisodityrosine. The structural differences between I and II and between III and IV lay solely in the diphenyl ether moiety; the phenolic hydroxyl group in II and IV was methylated in I and III. OF4949-III and IV contained L-asparagine instead of the beta-hydroxy-L-asparagine moiety of I and II.     

Biosynthesis of quinolactacin A, a TNF production inhibitor

Quinolactacins, which inhibit tumor necrosis factor production, contain a quinolone skeleton conjugated with a y-lactam. The biosynthesis of quinolactacin was investigated by feeding experiments using 13C single-labeled precursors (sodium [1-13C]acetate, DL-[1-13C]-isoleucine, L-[methyl-13C]methionine, and sodium [1-13C]-anthranilate) and D-[U-13C]glucose.     

Aspects on the Biosynthesis of the Cyanogenic Glucoside Triglochinin in Triglochin maritima1

The incorporation of L-[U- (14)C]phenylalanine, L-[U- (14)C]tyrosine and [U- (14)C]4-hydroxyphenylacetonitrile into triglochinin and taxiphyllin, the latter a possible precursor of the former, was studied in seedlings of TRIGLOCHIN MARITIMA L. The nitrile was by far the best substrate; incorporation of the amino acids was poor. Environmental factors such as light and humidity act differently on production of both compounds which also show a wide variation in individual seedlings. Quantitative evaluation of the specific activities indicate that taxiphyllin cannot be regarded as a precursor of triglochinin and that the two compounds are probably synthesized by two, at least mainly independent, pathways.     

Growth inhibitory effect of L-canavanine against MIA PaCa-2 pancreatic cancer cells is not due to conversion to its toxic metabolite canaline

L-Canavanine (CAV) is an arginine (ARG) analog isolated from the jack bean, Canavalia ensiformis. CAV becomes incorporated into cellular proteins of MIA PaCa-2 human pancreatic cancer cells and the aberrant, canavanyl proteins are not preferentially degraded. Hydrolytic cleavage of CAV to canaline (CAN) and urea is mediated by arginase. CAN is a potent metabolite that inactivates vitamin B6-containing enzymes and may inhibit cell growth. To determine the presence of arginase and its specificity for ARG and CAV in MIA PaCa-2 cells, a radiometric assay, which quantifies the 14C released from the hydrolytic cleavage of L-[guanidino-14C]ARG or L-[guanidinooxy-14C]CAV mediated by arginase, was employed. Insignificant amounts of 14CO2 were released when cells were exposed to [14C]CAV or to [14C]ARG. Pancreatic cancer cells secrete a negligible amount of arginase. Cytotoxic effects of CAN and CAV were compared on cells exposed to varying concentrations of ARG. These studies provide evidence that CAV's cytotoxic effects on MIA PaCa-2 cells cannot be attributed to conversion to the active metabolite CAN. A slower and decreased hydrolysis of CAV by arginase allows CAV to persist and increases its chances of incorporating into proteins in these cells. Lack of considerable amounts of arginase in the pancreas makes CAV a worthy candidate for further studies in pancreatic cancer.     

L-365,260 inhibits in vitro acid secretion by interacting with a PKA pathway.

The aim of this study was to analyse the antisecretory mechanism of L-365,260 in vitro in isolated rabbit gastric glands. We showed that compound L-365,260, described as a non-peptide specific competitive CCK-B receptor antagonist, was able to dose-dependently inhibit [14C]-aminopyrine accumulation induced by histamine (10(-4) M), carbachol (5x10(-5) M), 3-isobutyl-1-methyl-xanthine (IBMX) (5x10(-6) M) and forskolin (5x10(-7) M) with similar IC50 values respectively of 1.1+/-0.6x10(-7) M, 1.9+/-1.2x10(-7) M, 4.2+/-2.0x10(-7) M and 4.0+/-2.8x10(-7) M. We showed that L-365,260 acted beyond receptor activation and production of intracellular second messengers and that it had no action on the H+/K+ -ATPase. We found that L-365,260 inhibited cyclic AMP-induced [14C]-aminopyrine accumulation in digitonin-permeabilized rabbit gastric glands, suggesting that this compound acted, at least in part, as an inhibitor of the cyclic AMP-dependent protein kinase (PKA) pathway.     

Urdamycins, new angucycline antibiotics from Streptomyces fradiae. IV. Biosynthetic studies of urdamycins A-D

The biogenetic origin of the angucycline antibiotics urdamycins A-D was studied by feeding experiments with isotope labeled precursors and by NMR analysis. Feeding experiments with [1-13C]acetate and [1,2-13C2]acetate show that the chromophores of urdamycins A and B and the angucycline 4-ring skeleton of the urdamycins C and D chromophores are formed from a single decapolyketide chain. The chromophores of the urdamycins C and D contain additional structural elements which derived from the amino acids tyrosine and tryptophan, respectively. The latter was shown by feeding deuterium-labeled tyrosine and 13C-labeled tryptophan derivatives. Feeding of [1-13C]glucose and of [U-13C3]glycerol proved that the C-glycosidic moiety and the three sugars (2 x L-rhodinose, 1 x D-olivose each) of the urdamycins arise from glucose. Experiments with 14C-labeled urdamycin A, obtained by biosynthesis from [14C]acetate, showed this compound to be a late precursor of the urdamycins C and D.     

In vivo meal model for the evaluation of agents which affect the absorption of triglyceride and cholesterol

A meal model in which rats were trained to consume within 2 hr a high fat meal containing glycerol tri[1-¹⁴C]oleate and [³H]cholesterol was compared to the corn oil bolus model. In the meal model, dietary triglyceride was absorbed from the small intestine faster during the first 6–8 hr and more completely than intubated corn oil, as determined by analysis of intestinal contents, serum radioactivity, serum triglycerides, adipose tissue and liver lipids. The effects of Cholestyramine, Colestipol and Pluronic L-101 (1% dietary admixes) on these variables were evaluated for 5 days in the meal model. Lipid absorption during a 72-hr period was reduced by all compounds. The per cent excretion of glycerol tri[1-¹⁴C]oleate was increased significantly by Pluronic L-101 (10-fold), Cholestyramine (5.7-fold) and Colestipol (2.7-fold). The excretion of [³ H]cholesterol was enhanced significantly by Cholestyramine (1.6-fold) and Colestipol (1.3-fold). The following observations were made 4hr after the initiation of the meal. Pluronic L-101 increased significantly the retention of glycerol tri[1-¹⁴C]oleate and [³H]cholesterol in stomach (380 and 375 per cent, respectively), and of glycerol tri[1-¹⁴C]oleate in the small intestine (1100 per cent). The percent of intestinal lipid remaining as triglyce-ride from the intestinal lumen was increased significantly by Pluronic L-101 (160 per cent). Pluronic L-101 reduced significantly [¹⁴C]lipid and [³H]cholesterol in liver; Cholestyramine and Colestipol suppressed only [³H]cholesterol. In adipose tissue, Pluronic L-101 treatment reduced significantly [¹⁴C]lipid content only; Cholestyramine and Colestipol suppressed selectively [³H]cholesterol. After 5 days of treatment, only Pluronic L-101 treatment resulted in significantly reduced serum triglycerides (32 per cent), cholesterol (21 per cent) and glucose (15 per cent). These data suggest that this in vivo meal-feeding model provides a physiological technique for evaluating agents affecting lipid absorption.     

Effect of the hypocholesterolemic agent YM-16638 on cholesterol biosynthesis activity and apolipoprotein B secretion in HepG2 and monkey liver

YM-16638 ([[5-[[3-(4-acetyl-3-hydroxy-2-propylphenoxy)propyl]thio]-1,3,4-++ +thiadiazol-2-yl] thio] acetic acid) showed a strong hypocholesterolemic effect in humans and monkeys. To clarify the mechanism of this hypocholesterolemic effect, the action of YM-16638 on cholesterol biosynthesis in the cultured human hepatoma cell line HepG2 and cynomolgus monkey liver was examined. Cholesterol biosynthesis activity derived from [14C]acetic acid, [3H/14C]mevalonic acid or [14C]isopentenyl pyrophosphate substrates was significantly decreased, but not that from [3H]farnesyl pyrophosphate or [3H]squalene substrates in HepG2 cells treated with YM-16638. Simultaneously, treatment of these cells with YM-16638 changed neither the rate of apolipoprotein B synthesis from [35S]methionine nor its secretion. In addition, the activities of hepatic cholesterol biosynthesis enzymes HMG-CoA reductase, mevalonate kinase (MK), isopentenyl pyrophosphate isomerase (IPPI), farnesyl pyrophosphate synthase (FPPS), squalene synthase and squalene epoxidase were measured in monkeys fed a diet supplemented with YM-16638. Among these enzymes, MK, IPPI and FPPS activities in the YM-16638-treated group significantly decreased by 38%, 56% and 30%, respectively, when compared to those from control animals receiving no drug treatment. These results indicate that YM-16638 has the characteristics of a cholesterol biosynthesis inhibitor.     

PAF and LTB4 biosynthesis in the human neutrophil: effects of putative inhibitors of phospholipase A2 and specific inhibitors of 5-lipoxygenase.

The effect of several putative phospholipase A2 (PLA2) inhibitors on [3H]-acetate incorporation into platelet-activating factor (PAF) upon calcium ionophore A23187 stimulation of purified human neutrophils (PMN) were evaluated in vitro. PLA2 inhibitors such as p-bromophenacyl bromide (pBPB), ellagic acid, aristolochic acid and gossypol were without effect or only weakly inhibited PAF biosynthesis. Luffariellolide, a potent PLA2 inhibitor isolated from the marine sponge Luffariella sp., dose-dependently inhibited PAF production (IC50 = 5 microM). Due to the relationship between PAF and LTB4 biosynthesis, the effect of inhibiting LTB4 production on PAF biosynthesis was investigated. At concentrations which reduce LTB4 production by greater than 95%, Wy-50,295 tromethamine and A-64,077, specific 5-lipoxygenase (5-LO) inhibitors, did not significantly effect PAF production. In contrast, L-663,536, the 5-LO translocation inhibitor, was a potent inhibitor of PAF production (IC50 = 1 microM). This activity of L-663,536 may contribute to its pharmacological profile at higher doses. These data also suggest that PAF biosynthesis in human PMNs is not dependent on the formation or continued presence of leukotrienes.     

5-Lipoxygenase-activating protein is the target of a novel hybrid of two classes of leukotriene biosynthesis inhibitors.

An 18-kDa leukocyte membrane protein, termed 5-lipoxygenase-activating protein (FLAP), has recently been shown to be the target of two structurally distinct classes of leukotriene biosynthesis inhibitors. These classes of inhibitors are based on indole and quinoline structures and are represented by MK-886 and L-674,573, respectively. A novel class of hybrid structure based on the indole and quinoline classes of inhibitors, termed quindoles, has recently been developed. These compounds, exemplified by L-689,037, are potent inhibitors of leukotriene biosynthesis, both in vitro and in vivo. In the present study, we have developed and characterized a potent radioiodinated photoaffinity analogue of L-689,037, termed [125I]L-691,678. This compound was used in immunoprecipitation studies with FLAP antisera to show that the quindole series of leukotriene biosynthesis inhibitors interact directly with FLAP. In addition, we show that MK-886, L-674,573, and L-689,037 specifically compete, in a concentration-dependent manner, with both [125I]L-691,678 and [125I]L-669,083, a photoaffinity analogue of MK-886, for binding to FLAP. These results suggest that these three classes of leukotriene biosynthesis inhibitors share a common binding site on FLAP, providing further evidence that FLAP represents a suitable target for structurally diverse classes of leukotriene biosynthesis inhibitors.     

Effect of hypocholesterolemic agents on central nervous system cholesterol biosynthesis. II. AY-9944 and AY-9944 plus zuclomiphene.

When the hypocholesterolemic agent AY-9944 was examined in vitro and in vivo for its influence on developing brain sterol biosynthesis, multiple effects were exhibited. Addition of AY-9944 to incubations of cell-free preparations resulted in a decrease in [¹⁴C]sterol biosynthesis from [2-¹⁴C]mevalonic acid. Pretreatment of animals with AY-9944 and subsequent incubation of their brain tissue in vitro resulted in greater labeled sterol synthesis than in control incubations. Pretreatment of animals with AY-9944 followed by intracerebral injection of [2-¹⁴C]mevalonic acid resulted in comparable labeled sterol biosynthesis in control and drug-treated brains, but reduced [¹⁴C]cholesterol formation by the AY-9944-treated animals. Examination of the labeled free sterol fractions by thin-layer chromatography and radioactivity-monitored gas chromatography indicated several labeled sterols present in the AY-9944-treated brain tissue that were not seen in controls. These [¹⁴C]sterols were: 4,4-dimethyl-5α-cholesta-8,14,24-trien-3β-ol; 5α-cholesta-7,24-dien-3β-ol; 5α-cholesta-8,14,24-trien-3β-ol; cholesta-5,7,24-trien-3β-ol; cholesta-5.7-dien-3β-ol; and an unknown 4α-methyl sterol. Another set of developing rats was pretreated with a combination of AY-9944 and zuclomiphene. another hypocholesterolemic agent. Intracerebral administration of [2-¹⁴C]mevalonic acid resulted in lower [¹⁴C]sterol and labeled cholesterol synthesis in the brain tissue of drug-treated animals than in controls. Thin-layer and radioactivity-monitored gas Chromatographie analysis again revealed [¹⁴C]sterols other than those present in controls. Two labeled Δ⁷ sterols, 5α-cholesta-7,24-dien-3β-ol and cholesta-5.7,24-trien-3β-ol. a 4α-methyl sterol of unknown structure, previously seen in the AY-9944-treated brain tissue, and a 4,4-dimethyl sterol, also of unknown structure, were present in the brains of the drug-treated animals. The present findings indicate not only that both drugs block central nervous system sterol synthesis at several points, but also that use of these hypocholesterolemic agents in various combinations may further elucidate the pathway(s) and control of neural sterol biosynthesis.     

Evidence for an inhibitory presynaptic component of neuroleptic drug action.

1 The action of five neuroleptic drugs (haloperidol, cis-flupenthixol, chlorpromazine, fluphenazine and thioridazine) was studied on the synthesis and release of dopamine from rat striatal synaptosomes. 2. In vitro application of the drugs induced an inhibition of synthesis of [14C]-dopamine from L-[U-14C]-tyrosine and a decrease in the tissue content of [14-C]-dopamine, with IC50 values for the latter effect ranging from 3.6 x 10(-7) to 5.9 x 10(-5) M. The rank of their potency was similar to the order of their clinical effectiveness: haloperidol greater than fluphenazine greater than cis-flupenthixol greater than chlorpromazine greater than thioridazine. Trans flupenthixol was without effect up to a concentration of 10(-4) M. 3 The tissue level and release of GABA were not affected by concentration of the neuroleptics up to 10(-4) M. 4 When the neuroleptics were administered in vivo, changes were also detected in the synthesis and release of [14C-]-dopamine from subsequently prepared synaptosomes. A marked inhibition of the K+-induced increase in [14C]-dopamine synthesis was seen following a dose of 2 mg/kg cis-flupenthixol and haloperidol. At this concentration, haloperidol also increased the control release of [14C]-dopamine and reduced the K+-induced increase in release of [14C]-dopamine. 5 Cis-flupenthixol at a dose of 20 mg/kg reduced the K+-induced release of [14C]-dopamine by 48% and to a lesser extent, that of gamma-aminobutyric acid (GABA, 25%). 6 An inhibitory mode of action is proposed for neuroleptics mediated through a presynaptic mechanism.     

9-Hydroxybenfluron: cytostatic effects and inhibition of macromolecular biosynthesis in Ehrlich ascites and P388 murine leukemia cells.

Primary screening in vitro and study on the mode of action of 9-hydroxybenfluron (HBF) in both murine P388 leukemia and Ehrlich ascites carcinoma cells have been performed. Metabolite HBF is approximately twice as effective as a reference drug (benfluron). To elucidate the biochemical mode of action, the effect of HBF on the biosynthesis of macromolecules indicated by the incorporation rate of [14C]adenine (in DNA and RNA), [14C]thymidine (in DNA), [14C]uridine (in RNA) and [14C]valine (in protein) was studied in concentration and time dependence. HBF inhibited incorporation of all four precursors into the trichloroacetic acid-insoluble fraction of Ehrlich ascites cells. The fact that incorporation of these four precursors is inhibited suggests that the effect of HBF lies at an underlying level of energy generation or transfer rather than at specific reactions in the biosynthesis of DNA and proteins.     

Biosynthesis of the avermectins by Streptomyces avermitilis. Incorporation of labeled precursors

The biosynthesis of the avermectins, a group of 16 membered macrolides with potent anthelmintic and insecticidal activity produced by Streptomyces avermitilis, was studied by supplying cultures with 14C and 13C precursors. [1-14C] and [2-14C]acetate and propionate were poor precursors of the avermectins and were instead rapidly oxidized to 14CO2. The S-methyl of methionine in contrast was incorporated extensively and equally into the three methoxyl groups of the avermectins. The carbon backbone of methionine was not a precursor of the avermectins. Feeding of [1-13C]glucose yielded avermectins labeled specifically in the C1' and C1" of the oleandrose moiety and in the aglycone moiety in carbons known to be derived from the methyl of acetate. Feeding [U-13C]glucose showed that the entire avermectin molecule is derived from glucose carbons.     

Alteration of decreased plasma NO metabolites and platelet NO synthase activity by paroxetine in depressed patients.

Although major depression (MD) and cardiovascular disease (CVD) have been conclusively linked in the literature, the mechanism associating MD and CVD is yet undetermined. The purpose of this paper is to further investigate a potential mechanism involving nitric oxide (NO) and to examine the effect of the selective serotonin reuptake inhibitor paroxetine on NO production by both platelets and the endothelium. In total, 17 subjects with MD and 12 healthy controls (HCs) with no known history of cardiovascular illness completed the study. Paroxetine was administered to both the MD patients and HCs over an 8-week period, and then medication was discontinued. Blood samples were taken at various times throughout paroxetine treatment and after discontinuation. Plasma NO metabolite (NOx) levels were measured by a chemiluminescence method. Platelet endothelial NO synthase (eNOS) activity was examined through the conversion of L-[14C]arginine to L-[(14)C]citrulline. Data were analyzed using t-tests and a linear mixed effects model. Baseline levels of both plasma NOx and platelet NOS activity were significantly lower in subjects with MD compared to HCs. Throughout paroxetine treatment, plasma NOx levels increased in both HCs and MD patients. However, platelet eNOS activity decreased in HCs, while no statistically significant change was evidenced in MD patients. These data suggest that, in MD patients, decreased peripheral production of NO, a potential contributor to increased cardiovascular risk, is modified by administration of the antidepressant paroxetine.     

Repression of beta-lactam production in Cephalosporium acremonium by nitrogen sources

A variety of inorganic and organic nitrogen sources were added to fermentation media to determine their regulatory effects on the production of beta-lactam antibiotics by Cephalosporium acremonium. (NH4)2SO4 at concentrations higher than 100 mM (1.3%) strongly inhibited beta-lactam production. L-Asparagine and L-arginine proved to be the best nitrogen sources tested for beta-lactam production. The optimum concentration of asparagine was 1.2%. Higher concentrations led to NH3 accumulation, increase in pH, and lower growth rates. Addition of tribasic magnesium phosphate [Mg3(PO4)2 X 8H2O] to the (NH4)2SO4-containing medium stimulated beta-lactam production markedly and ammonium repression of the ring-expansion enzyme was reversed. It appears that the ring-expansion step is a very sensitive part of beta-lactam biosynthesis in C. acremonium with respect to nitrogen source repression. Other enzymes may also be sensitive in view of the fact that nitrogen source derepression not only led to increases in cephalosporin C but, to a lesser extent, penicillin N and total beta-lactam titers.