Effect of tweens on the production of ergot alkaloids byAspergillus fumigatus

Tween 80, which caused increased biomass formation, also produced the highest increase in the uptake rate of all components of the medium. The fatty acid components of the respective Tweens,i.e. palmitic acid (Tween 40), stearic acid (Tween 60), and oleic acid (Tween 80), have no effect either on alkaloid production or on substrate uptake. The fatty acid composition was different in the cell membrane of the culture supplemented with Tween 60 and facilitated the transport of metabolites into the cells.     

Metabolism of tween-Fatty Acid esters by cultured soybean cells : kinetics of incorporation into lipids, subsequent turnover, and associated changes in endogenous Fatty Acid synthesis

Uptake of Tween-fatty acid esters and incorporation of the fatty acids into lipids by soybean (Glycine max [L.] Merr.) suspension cultures was investigated, together with subsequent turnover of the incorporated fatty acids and associated changes in endogenous fatty acid synthesis. Tween uptake was saturable, and fatty acids were rapidly transferred from Tweens to all acylated lipids. Patterns of incorporation into glycerolipids were similar in cells treated with Tweens carrying [1-¹⁴C]-fatty acids and in cells treated with [1-¹⁴C]acetate, indicating that exogenous fatty acids were used for glycerolipid synthesis essentially as if they had been made by the cell. In Tween-treated cells neutral lipids (which include Tweens) initially accounted for the majority of lipid radioactivity. Radioactivity was then rapidly transferred to glycerolipids. A transient pool of free fatty acids accounting for up to 10% of lipid radioactivity was observed. This was consistent with the hypothesis that fatty acids are transferred from Tweens to lipids by deacylation of the Tweens, creating a pool of free fatty acids which are then used for lipid synthesis. Sterols were only slightly labeled in cells treated with Tweens, but accounted for nearly 50% of lipid radioactivity in cells treated with acetate. This suggested very little degradation and reutilization of the radioactive fatty acids in cells treated with Tweens. In cells treated with either [1-¹⁴C]acetate or Tween-[1-¹⁴C]-18:1, 70% of the initial fatty acid radioactivity remained in fatty acids after a 100 hour chase. By contrast, fatty acids not normally present disappeared more rapidly, suggesting differential treatment of such fatty acids compared with those normally present. Cells which had incorporated large amounts of exogenous fatty acids altered fatty acid synthesis in three distinct ways: (a) amounts of [1-¹⁴C]acetate incorporated into fatty acids were reduced; (b) cells incorporating exogenous unsaturated fatty acids increased the proportion of [1-¹⁴C]acetate partitioned into saturated fatty acids, while the converse was true of cells which had incorporated exogenous saturated fatty acids; (c) desaturation of 18:1 to 18:2 and 18:3 was reduced in cells which had incorporated unsaturated fatty acids. These results suggest that Tween-fatty acid esters will be useful for supplying fatty acids to cells for a variety of studies related to fatty acid or membrane metabolism.     

The Fungus <Emphasis Type="Italic">Penicillium citrinum</Emphasis>, Isolated from Permafrost Sediments,as a Producer of Ergot Alkaloids and New Quinoline Alkaloids Quinocitrinines

Quinocitrinines and ergot alkaloids were synthesized by the strain Penicillium citrinum VKM FW-800 as the culture grews. The major part of these secondary metabolites was secreted into the medium. In the phase of growth deceleration, these metabolites were partly absorbed by the producer cells. Zinc ions stimulated both the primary and secondary metabolic processes. Addition of this microelement into the culture medium stimulated biomass accumulation and the synthesis of clavine alkaloids and quinocitrinines.     

Polyoxyethylene stimulates lignin peroxidase production inPhanerochœte chrysosporium

In shaken cultures ofPhanerochœte chrysosporium, different Tweens gave rise to similar and high lignin peroxidase (LiP) activities. The polyoxyethylene-sorbitan (POE-S) moieties isolated from Tweens gave rise to somewhat lower LiP activities, whereas fatty acids isolated from Tweens gave rise to much lower LiP activities than parent Tweens. LiP activity appeared 3 d after addition of Tween 80 if this was added within the first 4 d after inoculation. Of the three chemical moieties contained in Tweens,i.e., fatty acids, sorbitan, and polyoxyethylene (POE), only the latter one significantly stimulated the LiP activity of the culture. The stimulatory effect of POE on the LiP activity increased till its molar mass of approx. 1 kDa, then it levelled off. The quantity of POE in the culture decreased with time. Tween 80, its POE-S moiety and POEs seem to enhance LiP production and not only their release.     

The effects of colchicine and vincristine on the concentrations of glucose and related metabolites in goat's milk

The effects of colchicine and vincristine on the concentration of glucose and its metabolites in milk were studied following intramammary injection of the alkaloids into one mammary gland of lactating goats. Both alkaloids decreased the rate of milk secretion from the treated gland and produced similar changes in the concentrations of various metabolites in milk. The concentrations of glucose, UDP-galactose, galactose, pyruvate and lactate increased, while those of glucose 6-phosphate and phosphoenolpyruvate decreased in milk from treated glands. The rate of milk secretion from the untreated gland increased, along with the concentrations of glucose in the milk. The changes in the concentrations of the metabolites in milk are discussed in relation to possible biochemical changes occurring in the mammary gland during the suppression of milk secretion by the alkaloids. It is suggested that, before alkaloid treatment, the rate of milk secretion was limited by intracellular glucose supply.     

Pyrrolic and N-oxide metabolites formed from pyrrolizidine alkaloids by hepatic microsomes in vitro: Relevance to in vivo hepatotoxicity

An analytical method of improved sensitivity has enabled measurements to be made of N-oxide as well as pyrrolic metabolites formed from a range of unsaturated pyrrolizidine alkaloids in hepatic microsome preparations. Using microsomes from livers of phenobarbitone-pretreated male Fischer rats, all 13 alkaloids tested were metabolised to both N-oxides and pyrroles. The most lipophilic alkaloids gave enhanced rates of metabolism. No consistent relationship existed between rates of N-oxide and of pyrrole formation. The two pathways appeared to be independent. The ratio of N-oxide to pyrrolic metabolites varied, depending on the type of ester: it was highest for ‘open’ diester alkaloids, lowest for 12 membered macrocyclic diesters and for monoesters. Steric hindrance by the acid moiety could account for these differences, by affecting the balance between microsomal oxidation of the amino alcohol moiety at the nitrogen and C8 positions respectively and could explain the high pyrrole yields given by some macrocyclic diesters. The levels of pyrrolic metabolites bound to liver tissues and responsible for hepatotoxicity in rats given pyrrolizidine alkaloids, did not necessarily reflect the rates of formation of such metabolites measured in vitro. In the animal additional factors could influence the formation and tissue binding of pyrrolic metabolites, including the detoxication of alkaloids by hydrolysis and the chemical reactivity and stability of the toxic metabolites. A comparison of heliotridine esters with retronecine esters showed that the 7-hydroxyl or -ester configuration had a relatively small influence on the balance between formation of pyrrolic metabolites and detoxication by N-oxidation. The results did not support any hypothesis that heliotridine esters should generally be more hepatotoxic than analogous retronecine esters. The structure of the acid moiety was likely to have at least as much influence on toxicity as the base configuration.     

Fungus Penicillium citrinum, isolated from permafrost sediments, as a producer of ergot alkaloids and new quinoline alkaloids quinocitrinines

Quinocitrinines and ergot alkaloids are synthesized by the strain Penicillium citrinum VKM FW-800 as the culture grows. The major part of these secondary metabolites are secreted into the medium. In the phase of growth deceleration, these metabolites were partly absorbed by the producer cells. Zinc ions stimulated both the primary and secondary metabolic processes. Addition of this microelement into the culture medium stimulated biomass accumulation and the synthesis of clavine alkaloids and quinocitrinines.     

Metabolism studies of the Kratom alkaloids mitraciliatine and isopaynantheine, diastereomers of the main alkaloids mitragynine and paynantheine, in rat and human urine using liquid chromatography-linear ion trap-mass spectrometry

Mitragyna speciosa (Kratom in Thai), native in Southeast Asia, is increasingly misused as a herbal drug of abuse. During metabolism studies on the Kratom alkaloids mitragynine, its diastereomers speciogynine and speciociliatine as well as paynantheine in rats and humans, further isomeric compounds were detected in Kratom users' urine. The question arose whether these compounds were formed from the low abundant, isomeric alkaloids mitraciliatine (MC) and isopaynantheine (ISO-PAY). Therefore, the aim of the presented study was to identify using liquid chromatography-linear ion trap-mass spectrometry their phase I and II metabolites in rat urine after administration of pure MC or ISO-PAY, to confirm their formation in humans, and finally to confirm whether the above-mentioned isomeric compounds in human urine represent MC and ISO-PAY and/or their metabolites. The metabolic pathways of both alkaloids in rats were found to be comparable to those of their corresponding diastereomers. In the human urines tested, not all metabolites found in rats could be detected because of the much lower amounts of MC and ISO-PAY in Kratom. However, all the above-mentioned so far unknown isomeric compounds could be identified in the human urine samples as MC, ISO-PAY and/or their metabolites. The used LC separation was also suitable for the differentiation of all other Kratom alkaloids and their metabolites in human urine.     

The effects of colchicine and vincristine on the concentrations of glucose and related metabolites in goat's milk

The effects of colchicine and vincristine on the concentration of glucose and its metabolites in milk were studied following intramammary injection of the alkaloids into one mammary gland of lactating goats. Both alkaloids decreased the rate of milk secretion from the treated gland and produced similar changes in the concentrations of various metabolites in milk. The concentrations of glucose, UDP-galactose, galactose, pyruvate and lactate increased, while those of glucose 6-phosphate and phosphoenolpyruvate decreased in milk from treated glands. The rate of milk secretion from the untreated gland increased, along with the concentrations of glucose in the milk, The changes in the concentrations of the metabolites in milk are discussed in relation to possible biochemical occuring in the mammary gland during the suppression of milk secretion by the alkaloids. It is suggested that, before alkaloid treatment, the rate of milk secretion was limited by intracellular glucose supply.     

Studies on Mucor Lipases

Lipases produced by Mucor lipolyticus Aac-0102 were separated into three different fractions (F-1, F-2 and F-3) by CM-Sephadex column chromatography.

Molecular weights of them were estimated to be higher in the order of F-1, F-2, and F-3 by gel filtration on a Sephadex G-100 column. F-1 and F-2 could hydrolyze water soluble substrate such as Tweens. F-3 showed a strong hydrolytic activity toward triglycerides but the activity toward Tweens was almost negligible.

Sodium lauryl sulfate inhibited the olive oil hydrolyzing activity of F-3. However, Tween hydrolyzing activity of F-2 was not affected with it. These results suggested that they are different with each other with respect to their molecular weights, substrate specificities and sensitivities to some inhibitors.     

Utilization of Photosynthetically Fixed 14CO2 into Alkaloids in Relation to Primary Metabolites in Developing Leaves of Catharanthus roseus

Partitioning of current photosynthates towards primary metabolites and its simultaneous incorporation in leaf alkaloids was investigated in developing leaves of medicinally important Catharanthus roseus. Of the total ¹⁴CO₂ assimilated, the leaves at positions 1–6 fixed 8, 22, 25, 19, 13, and 8 %, respectively, and stem 3 %. Leaf fresh mass, chlorophyll content, and CO₂ exchange rate increased up to the third leaf. The total alkaloid content was highest in young actively growing leaves, which declined with age. Total ¹⁴C fixed and its content in ethanol soluble fraction increased up to the third leaf and then declined. The ¹⁴C content in primary metabolites such as sugars and organic acids was also highest in the 3^rd leaf. The utilization of ¹⁴C assimilates into alkaloids was maximum in youngest leaf which declined with leaf age. Hence the capacity to synthesize alkaloids was highest in young growing leaves and metabolites from photosynthetic pathway were most efficiently utilized and incorporated into alkaloid biosynthetic pathway by young growing leaves.     

Effect of tweens on the lipase activity of Oospora lactis

Oospora lactis was found to use Tweens 20, 40, 60 and 80 as a source of carbon in a nutrient medium for lipase biosynthesis. The highest production of lipase was registered in the medium with Tween-80. The lipase activity of the culture increased if even small quantities of Tweens 40, 60 and, particularly, 80 (0.05--1.0%) were added to the medium containing cottonseed oil. All the tested Tweens stimulating the biosynthesis of lipase were also, at a particular concentration, the inhibitors of this enzyme.     

Direct and Synergistic Influence of Tweens on Avena Coleoptile Section Elongation

The Avena cylinder test was used with Tweens at 3 different concentrations with and without the addition of varying concentrations of IAA. Tween 20 was a toxic substance for oat coleoptile sections at such concentration as 0.1 %. Tween 40 gave erratic results and Tween 80 has practically no direct effect on cell elongation. An inhibitory effect continues to increase with increasing HLB values. The second object of this experiment was to ascertain whether cell elongation promoted by IAA would be enhanced by osmotic substances such as Tweens, as consequence of a specific modification in the permeability when the membranes are lipid in nature. The hypothesis was supported by the experimental work when 0.05 % Tweens were assayed although the inverse was also true, with concentrations of Tweens as low as 0.01 %. Accordingly, evidence must be verified by further investigation.     

Occurrence of indole alkaloids among secondary metabolites of soil Aspergillus

The occurrence of indole alkaloids among secondary fungal metabolites was studied in species of the genus Aspergillus, isolated from soils that were sampled in various regions of Russia (a total of 102 isolates of the species A. niger, A. phoenicis, A. fumigatus, A. flavus, A. versicolor, A. ustus, A. clavatus, and A. ochraceus). Clavine alkaloids were represented by fumigaclavine, which was formed by A. fumigatus. alpha-Cyclopiazonic acid was formed by isolates of A. fumigatus, A. flavus, A. versicolor, A. phoenicis, and A. clavatus. The occurrence of indole-containing diketopiperazine alkaloids was documented for isolates of A. flavus, A. fumigatus, A. clavatus, and A. ochraceus. No indole-containing metabolites were found among the metabolites of A. ustus or A. niger.     

Identification of benzo[c]phenanthridine metabolites in human hepatocytes by liquid chromatography with electrospray ion-trap and quadrupole time-of-flight mass spectrometry

The metabolism of the benzo[c]phenanthridine alkaloids was studied using human hepatocytes which are an excellent model system for biotransformation studies. For analysis of the alkaloids and their metabolites, an electrospray quadrupole ion-trap mass spectrometry (ESI ion-trap MS) connected to a reversed phase chromatographic system based on cyanopropyl modified silica was used. The optimized experimental protocol allowed simultaneous analysis of the alkaloids and their metabolites and enabled study of their uptake into and interconversion in human hepatocytes. The results show that formation of the dihydro metabolite which may be followed by specific O-demethylenation/O-demethylation processes, is probably the main route of biotransformation (detoxification) of the benzo[c]phenanthridines in human hepatocytes. The structure of the main O-demethyl metabolite (2-methoxy-12-methyl-12,13-dihydro-[1,3]dioxolo[4',5':4,5]benzo[1,2-c]phenanthridin-1-ol; 336.1 m/z,) was proposed by the multi-stage MS and quadrupole time-of-flight MS methods using chemically synthesized standard.     

On the suitability of gelatin for plate cultures. II

Summary In continuation of our investigation into the factors which determine the suitability of gelatin for colony formation it was demonstrated that surface tension lowering substances added in small concentrations to plate media prepared with a poor gelatin have a decidedly favourable effect. Especially Tweens were examined. An addition of 0.01% Tween 60 or 80 to the gelatin media was sufficient to bring about growth of typical lobated colonies of bothBacterium coli andFlavobacterium aquatile. In none of the experiments made did the Tweens, in the low concentrations applied, exhibit any toxic effect. No direct method for the determination of the surface tension of gels being available, we resorted to the determination of the wettability of the various gelatin gels. With the aid of this method it was found that the contact angle of water droplets on 10% gelatin gels correlated satisfactory with colony appearance. It seems probable that gelatins of good quality contain some constituent which increases the wettability of the gel. Finally it was shown that addition of Tweens to agar gels produces analogous effects on colony growth.     

Secondary metabolites in taxonomy of the <Emphasis Type="Italic">Penicillium</Emphasis> fungi

A correlation was established between species specificity and synthesis of specific secondary metabolites by the Penicillium fungi. Strains of the subgenus Aspergilloides usually synthesize metabolites of polyketide nature. Most strains of the subgenus Furcatum produce clavine ergot alkaloids and metabolites of diketopiperazine nature. The only clavine ergot alkaloids and diketopiperazine alkaloids produced by strains of the subgenus Biverticillium are rugulovasines and rugulosuvines, respectively. Species designations of the strains of the subgenus Penicillium isolated from permafrost soil, the Mir orbital complex, and sites undergoing anthropogenic load were refined based on the marker secondary metabolites. Changes in the taxonomic position of some strains in the genus Penicillium are suggested.     

Dehydration, deamination and enzymatic repair of cytosine glycols from oxidized poly(dG-dC) and poly(dI-dC)

Cytosine glycols (5,6-dihydroxy-5,6-dihydrocytosine) are initial products of cytosine oxidation. Because these products are not stable, virtually all biological studies have focused on the stable oxidation products of cytosine, including 5-hydroxycytosine, uracil glycols and 5-hydroxyuracil. Previously, we reported that the lifetime of cytosine glycols was greatly enhanced in double-stranded DNA, thus implicating these products in DNA repair and mutagenesis. In the present work, cytosine and uracil glycols were generated in double-stranded alternating co-polymers by oxidation with KMnO₄. The half-life of cytosine glycols in poly(dG-dC) was 6.5 h giving a ratio of dehydration to deamination of 5:1. At high substrate concentrations, the excision of cytosine glycols from poly(dG-dC) by purified endonuclease III was comparable to that of uracil glycols, whereas the excision of these substrates was 5-fold greater than that of 5-hydroxycytosine. Kinetic studies revealed that the V_max was several fold higher for the excision of cytosine glycols compared to 5-hydroxycytosine. In contrast to cytosine glycols, uracil glycols did not undergo detectable dehydration to 5-hydroxyuracil. Replacing poly(dG-dC) for poly(dI-dC) gave similar results with respect to the lifetime and excision of cytosine glycols. This work demonstrates the formation of cytosine glycols in DNA and their removal by base excision repair.     

Pyrrolizidine alkaloids in human diet.

Pyrrolizidine alkaloids are the leading plant toxins associated with disease in humans and animals. Upon ingestion, metabolic activation in liver converts the parent compounds into highly reactive electrophiles capable of reacting with cellular macromolecules forming adducts which may initiate acute or chronic toxicity. The pyrrolizidine alkaloids present a serious health risk to human populations that may be exposed to them through contamination of foodstuffs or when plants containing them are consumed as medicinal herbs. Some pyrrolizidine alkaloids (PA) adducts are persistent in animal tissue and the metabolites may be re-released and cause damage long after the initial period of ingestion. PAs are also known to act as teratogens and abortifacients. Chronic ingestion of plants containing PAs has also led to cancer in experimental animals and metabolites of several PAs have been shown to be mutagenic in the Salmonella typhimurium/mammalian microsome system. However, no clinical association has yet been found between human cancer and exposure to PAs. Based on the extensive reports on the outcome of human exposure available in the literature, we conclude that while humans face the risk of veno-occlusive disease and childhood cirrhosis PAs are not carcinogenic to humans.     

The biosynthesis of papaverine proceeds via (S)-reticuline

Papaverine is one of the earliest opium alkaloids for which a biosynthetic hypothesis was developed on theoretical grounds. Norlaudanosoline (=tetrahydropapaveroline) was claimed as the immediate precursor alkaloid for a multitude of nitrogen containing plant metabolites. This tetrahydroxylated compound was proposed to be fully O-methylated. The resulting tetrahydropapaverine should then aromatize to papaverine. In view of experimental data, this pathway has to be revised. Precursor administration to 8-day-old seedlings of Papaver followed by direct examination of the metabolic fate of the stable-isotope-labeled precursors in the total plant extract, without further purification of the metabolites, led to elucidation of the papaverine pathway in vivo. The central and earliest benzylisoquinoline alkaloid is not the tetraoxygenated norlaudanosoline, but instead the trihydroxylated norcoclaurine that is further converted into (S)-reticuline, the established precursor for poppy alkaloids. The papaverine pathway is opened by the methylation of (S)-reticuline to generate (S)-laudanine. A second methylation at the 3′ position of laudanine leads to laudanosine, both known alkaloids from the opium poppy. Subsequent N-demethylation of laudanosine yields the known precursor of papaverine: tetrahydropapaverine. Inspection of the subsequent aromatization reaction established the presence of an intermediate, 1,2-dihydropapaverine, which has been characterized. The final step to papaverine is dehydrogenation of the 1,2-bond, yielding the target compound papaverine. We conclusively show herein that the previously claimed norreticuline does not play a role in the biosynthesis of papaverine.