Induction of sexual reproduction in Opalina sudafricana by injecting its host Bufo regularis with gibberellic acid

Induction of sexual reproduction in Opalina sudafricana by injecting its host Bufo regularis with gibberellic acid. International Journal for Parasitology4, 203–206. Opalina sudafricana parasitic in the rectum of Bufo regularis was induced to reproduce sexually when its host was injected subcutaneously with 0·3 mg of gibberellin-A₃. This plant growth substance had no effect on the induction of encystation in the parasites in vitro. Urine of toads injected with gibberellin-A₃ induced sexual reproduction (encystation) in the opalinids in vitro. It is speculated that the plant hormone must either be broken down into an active substance by the toad or cause the toad to excrete its own gonadal hormones (or other hormones) into the urine. This active substance or the excreted hormones may induce division in the parasites resulting in the formation of small forms which encyst.     

The effect of fresh toad bile on the induction of encystation in Opalina sudafricana parasitic in Bufo regularis

Opalina sudafricana reacted by encystation to small doses (0.04 ml) of fresh toad bile injected subcutaneously into its host Bufo regularis. It is speculated that androgens present in the injected bile of male toads, and oestrogens found in injected bile of female hosts reach the parasites in the recta of the treated animals and induce them to encyst. High doses of bile killed the parasites and their hosts. Small doses of bile (0·04 ml) added to in vitro cultures induced encystation in the opalinids. High doses killed the opalinids in vitro. There is also the possibility that bufodeoxycholic acid found in the toad bile may play a role in inducing the parasites to divide and encyst.     

The mechanism of action of adrenaline in the induction of sexual reproduction (encystation) in Opalina sudafricana parasitic in Bufo regularis

El Mofty M. M. and Sadek I. A. 1973. The mechanism of action of adrenaline in the induction of sexual reproduction (encystation) in Opalina sudafricana parasitic in Bufo regularis. International Journal for Parasitology3: 425–431. Adrenaline injections induced encystation in the opalinids of normal, hypophysectomized, and gonadectomized toads in the pre-breeding and post-breeding seasons. In normal and gonadectomized male and female toads, adrenaline injections may stimulate the release of ACTH of the anterior pituitary which in turn induces the secretion of androgenic steroids from the interrenal tissue. It is speculated that the breakdown products of these androgenic steroids cause the encystation of the parasites. In hypophysectomized male and female hosts, the injected adrenaline may stimulate the interstitial tissue of the testes and ovaries to secrete androgens, the breakdown products of which induce encystation of the opalinids. Adrenaline or ACTH gave negative results in vitro. Urine of male or female toad injected with adrenaline, ACTH, or testosterone propionate induced encystation in Opalina sudafricana m vitro.     

Nyctotheroides puytoraci: stimulation of encystment in toads, Bufo regularis, injected with rheumatoid arthritis patients' urine.

Nyctotheroides puytoraci, a ciliated protozoan parasite first described by Essawy (M.Sc. thesis, Alexandria University, Alexandria, Egypt, 1978), reacted by encystation in toad hosts Bufo regularis that had been injected subcutaneously with urine of patients with rheumatoid arthritis. It is speculated that carcinogenic tryptophan metabolites present in the injected urine reach parasites in the recta of treated host animals and are the inducers of encystment. Increased encystment was obtained when hosts were injected with the urine of rheumatoid arthritis patients who had been given 2 g l-tryptophan orally. On the other hand, injection of B. regularis with the urine of rheumatoid arthritis patients who had been given 100 mg of pyridoxine HCl did not induce increased cyst formation in the parasite. The abnormality of tryptophan metabolism in rheumatoid arthritis patients was readily corrected by the administration of pyridoxine.     

The use of Opalina sudafricana in a biological test for diagnosing disordered metabolism of tryptophan in human subjects

Injections of urine of patients with bladder cancer linked with bilharziasis, simple urinary bilharziasis, ascariasis or ancylostomiasis, induced cyst formation found in Opalina sudafricana when injected into its host Bufo regularis. It is suggested that the carcinogenic tryptophan metabolites present in the injected urine reach the parasites in the recta of the experimental toads and stimulate them to divide mitotically to form small forms which eventually encyst. This test may be of a diagnostic help in detecting any abnormality in tryptophan metabolism in some human patients.     

Tetrahydrobiopterin Biosynthesis as a Potential Target of the Kynurenine Pathway Metabolite Xanthurenic Acid

Tryptophan metabolites in the kynurenine pathway are up-regulated by pro-inflammatory cytokines or glucocorticoids, and are linked to anti-inflammatory and immunosuppressive activities. In addition, they are up-regulated in pathologies such as cancer, autoimmune diseases, and psychiatric disorders. The molecular mechanisms of how kynurenine pathway metabolites cause these effects are incompletely understood. On the other hand, pro-inflammatory cytokines also up-regulate the amounts of tetrahydrobiopterin (BH₄), an enzyme cofactor essential for the synthesis of several neurotransmitter and nitric oxide species. Here we show that xanthurenic acid is a potent inhibitor of sepiapterin reductase (SPR), the final enzyme in de novo BH₄ synthesis. The crystal structure of xanthurenic acid bound to the active site of SPR reveals why among all kynurenine pathway metabolites xanthurenic acid is the most potent SPR inhibitor. Our findings suggest that increased xanthurenic acid levels resulting from up-regulation of the kynurenine pathway could attenuate BH₄ biosynthesis and BH₄-dependent enzymatic reactions, linking two major metabolic pathways known to be highly up-regulated in inflammation.     

A chromatographic technique for the analysis of oxidized metabolites: Application to carcinogenic N-hydroxyarylamines in urine

A new chromatographic detection method for oxidized metabolites has been developed based on the reaction of eluted compounds with an Fe⁺³-bathophenanthroline colorimetric reagent in a postcolumn reactor. The method is sensitive to N-hydroxyarylamines, aryldiamines, phenolic amines, and ascorbic acid. It has been applied to the analysis of toxic N-oxidized metabolites in rhesus monkey urine after the animals were dosed with the bladder carcinogens, 1- and 2-napthylamine. These compounds are oxidized to the corresponding N-hydroxyarylamines in the liver, conjugated as the N-glucuronide, and excreted in the urine. The N-glucuronide has been shown to undergo acidic hydrolysis in the urine to release the free N-hydroxyarylamine, an ultimate carcinogen for the induction of bladder tumors. In this study, the N-hydroxy-N-glucuronide of 2-naphthylamine was found to be excreted at a rate that was 6.8 times that of the 1-naphthylamine isomer. This is consistent with the much higher carcinogenic potency of 2-naphthylamine in a variety of species.     

3-Hydroxykynurenine, 3-hydroxyanthranilic acid, and o-aminophenol inhibit leucine-stimulated insulin release from rat pancreatic islets

Individual islets were isolated from rat pancreas to study the effects of tryptophan and its metabolites on leucine-stimulated release of insulin. 3-Hydroxykynurenine, 3-hydroxyanthranilic acid, and o-aminophenol were inhibitors at concentrations below 10 mM whereas tryptophan, kynurenine, kynurenic acid, xanthurenic acid, and anthranilic acid were ineffective inhibitors at concentrations up to 10 mM. A structure-activity analysis of these metabolites demonstrated that vicinal aromatic hydroxy and amino groups with their concomitant electron donating properties are required for inhibition of insulin release. Inhibition of islet insulin release by the three kynurenine metabolites may be involved in the depressed insulin levels found in vitamin B6-deficient rats by other workers.     

Uptake and metabolism of nicotine by the CNS of a nicotine-resistant insect,the tobacco hornworm (Manduca sexta)

Penetration of the CNS by nicotine occurred equally rapidly in the nicotine-insensitive Manduca cord and the nicotine-sensitive Periplaneta cord, ruling out the possibility that lowered permeability renders Manduca insensitive. Although a saturable concentrative component of nicotine uptake was found in the Manduca cord, it was difficult to examine this component rigorously, because, except at high concentrations, the CNS metabolises the bulk of the nicotine that is taken up. The CNS metabolites of nicotine are water-soluble compounds. They are special first by virtue of the fact that they are formed in the CNS itself and secondly because their chromatographic characteristics are different from mammalian nicotine metabolites (which are not formed by nervous tissue). When subjected to hydrolysis, the metabolites acted like conjugates. Periplaneta CNS also metabolised nicotine, but much less extensively than Manduca. It is speculated that enzymic detoxification of dietary neurotoxins may be a necessary function of the insect CNS, since insects have no anatomical equivalent of a hepatic-portal system for detoxifying ingested compounds before they reach the blood-brain interface.     

Distribution of xanthurenic acid glucoside in species of the genus Drosophila

Xanthurenic acid 8-O-β-d-glucoside is a side metabolite of the tryptophan-xanthommatin pathway in Drosophila melanogaster, that is accumulated in some eye-color mutants. Since this compound had only been found in this species, we have analyzed 29 other Drosophila species for the occurrence of this compound using cellulose thin-layer chromatography. Xanthurenic acid glucoside was detected in 10 of them (8 of them belong to the Sophophora subgenus). In these species xanthurenic acid glucoside, as well as other metabolites of the final steps of the dihydroxanthommatin pathway (3-hydroxykynurenine, xanthurenic acid and dihydroxanthommatin) were quantitated in order to gain a better understanding of the relationships of the metabolites of this pathway.     

A rapid assay for peroxidase activity

1. Peroxidase has been assayed by a chronometric method involving the coupled reaction of ascorbic acid with the product of the enzymic action on benzidine. 2. Measurements of the activities of horseradish and tea peroxidase by this and two other methods, involving respectively pyrogallol and o-dianisidine, are compared. 3. It is claimed that the chronometric method is relatively simple, rapid and accurate. 4. The method can be used in the presence of polyphenol oxidases.     

Lack of correlation between the capability of inducing sister-chromatid exchanges in vivo and carcinogenic potency, for 16 aromatic amines and azo derivatives

16 aromatic amines and azo derivatives were studied. They were: benzidine; 2-acetylaminofluorene; 3'-methyl-p-dimethylaminoazobenzene; o-aminoazotoluene; p-dimethylaminoazobenzene; 2,4-diaminotoluene; 4,4'-oxydianiline; 2,4-diaminoanisole; 4,4'-methylenedianiline; 2-naphthylamine; auramine O; rhodamine B; ponceau MX; 1-naphthylamine; p-aminoazobenzene and aniline. Carcinogenic potency and potency in inducing sister-chromatid exchanges (SCEs) in vivo were compared. SCEs were absolutely not correlated with carcinogenic potency. A lack of correlation was also found with mutagenicity in the Ames test. On the contrary, a statistically significant correlation existed between DNA damage and SCEs.     

Effects of dietary pyrazinamide, an antituberculosis agent, on the metabolism of tryptophan to niacin and of tryptophan to serotonin in rats

The effects of pyrazinamide on the metabolism of tryptophan to niacin and of tryptophan to serotonin were investigated to elucidate the mechanism for pyrazinamide action against tuberculosis. Weanling rats were fed with a diet with or without 0.25% pyrazinamide for 61 days. Urine samples were periodically collected for measuring the tryptophan metabolites. The administration of pyrazinamide significantly increased the metabolites, 3-hydroxyanthranilic acid and beyond, especially quinolinic acid, nicotinamide, N'-methylnicotinamide, and N1-methyl-4-pyridone-3-carboxamide, and therefore significantly increased the conversion ratio of tryptophan to niacin and the blood NAD level . However, no difference in the upper metabolites of the tryptophan to niacin pathway such as anthranilic acid, kynurenic acid and xanthurenic acid was apparent between the two groups. No difference in the concentrations of trytptophan and serotonin in the blood were apparent either. It is suggested from these results that the action of pyrazinamide against tuberculosis is linked to the increase in turnover of NAD and to the increased content of NAD in the host cells.     

Opalina ranarum: inhibitory effect of 13-cis-retinoic acid or retinyl palmitate on the induction of cyst formation

20-Methylcholanthrene induced the encystment of Opalina ranarum when injected into its host, Rana ridibunda. Also, urine of frogs injected with this hydrocarbon induced encystment of the parasites. It is speculated that methylcholanthrene or its metabolites reach the parasites in the recta of the frogs and stimulate the parasites to encyst. Injections of frogs with methylcholanthrene and 13-cis-retinoic acid failed to induce cyst formation in the opalinids. Moreover, encystment of the parasite was lessened when the host was injected with methylcholanthrene and retinyl palmitate. Urine of frogs injected with methylcholanthrene and 13-cis-retinoic acid failed to induce cyst formation in the parasites. Moreover, urine of frogs injected with this hydrocarbon and retinyl palmitate lessened the induction of cyst formation in the parasites in vitro. It is suggested that 13-cis-retinoic acid as well as retinyl palmitate inhibits methylcholanthrene-induced cyst formation of the opalinids.     

Fluorometric assay for intestinal peptidases

A fluorometric assay for intestinal peptidases has been developed. Amino acids liberated by hydrolysis are estimated by use of l-amino-acid oxidase, peroxidase, and the fluorogenic reagent p-hydroxyphenylacetic acid, which yields a highly fluorescent compound on oxidation. During development of fluorescence, continued hydrolysis of peptides by peptidases which contaminate available preparations of l-amino-acid oxidase is prevented by the use of two inhibitors, 1,10-phenanthroline and p-hydroxymercuribenzoate. The assay is at least 10 times more sensitive than comparable spectrophotometric methods which employ the potentially carcinogenic chromogen o-dianisidine for detection of amino acids.     

Identification of Atg8 Isoform in Encysting Acanthamoeba

Autophagy-related protein 8 (Atg8) is an essential component of autophagy formation and encystment of cyst-forming parasites, and some protozoa, such as, Acanthamoeba, Entamoeba, and Dictyostelium, have been reported to possess a type of Atg8. In this study, an isoform of Atg8 was identified and characterized in Acanthamoeba castellanii (AcAtg8b). AcAtg8b protein was found to encode 132 amino acids and to be longer than AcAtg8 protein, which encoded 117 amino acids. Real-time PCR analysis showed high expression levels of AcAtg8b and AcAtg8 during encystation. Fluorescence microscopy demonstrated that AcAtg8b is involved in the formation of the autophagosomal membrane. Chemically synthesized siRNA against AcAtg8b reduced the encystation efficiency of Acanthamoeba, confirming that AcAtg8b, like AcAtg8, is an essential component of cyst formation in Acanthamoeba. Our findings suggest that Acanthamoeba has doubled the number of Atg8 gene copies to ensure the successful encystation for survival when 1 copy is lost. These 2 types of Atg8 identified in Acanthamoeba provide important information regarding autophagy formation, encystation mechanism, and survival of primitive, cyst-forming protozoan parasites.     

Novel Interaction between Laccase and Cellobiose Dehydrogenase during Pigment Synthesis in the White Rot Fungus Pycnoporus cinnabarinus

When glucose is the carbon source, the white rot fungus Pycnoporus cinnabarinus produces a characteristic red pigment, cinnabarinic acid, which is formed by laccase-catalyzed oxidation of the precursor 3-hydroxyanthranilic acid. When P. cinnabarinus was grown on media containing cellobiose or cellulose as the carbon source, the amount of cinnabarinic acid that accumulated was reduced or, in the case of cellulose, no cinnabarinic acid accumulated. Cellobiose-dependent quinone reducing enzymes, the cellobiose dehydrogenases (CDHs), inhibited the redox interaction between laccase and 3-hydroxyanthranilic acid. Two distinct proteins were purified from cellulose-grown cultures of P. cinnabarinus; these proteins were designated CDH I and CDH II. CDH I and CDH II were both monomeric proteins and had apparent molecular weights of about 81,000 and 101,000, respectively, as determined by both gel filtration and sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The pI values were approximately 5.9 for CDH I and 3.8 for CDH II. Both CDHs used several known CDH substrates as electron acceptors and specifically adsorbed to cellulose. Only CDH II could reduce cytochrome c. The optimum pH values for CDH I and CDH II were 5.5 and 4.5, respectively. In in vitro experiments, both enzymes inhibited laccase-mediated formation of cinnabarinic acid. Oxidation intermediates of 3-hydroxyanthranilic acid served as endogenous electron acceptors for the two CDHs from P. cinnabarinus. These results demonstrated that in the presence of a suitable cellulose-derived electron donor, CDHs can regenerate fungal metabolites oxidized by laccase, and they also supported the hypothesis that CDHs act as links between cellulolytic and ligninolytic pathways.     

Metabolism of [5-3H]Kynurenine in the Rat Brain In Vivo: Evidence for the Existence of a Functional Kynurenine Pathway

Abstract: The incorporation of tritium label into quinolinic acid (QUIN), kynurenic acid (KYNA), and other kynurenine (KYN) pathway metabolites was studied in normal and QUIN-lesioned rat striata after a focal injection of [5-³H]KYN in vivo. The time course of metabolite accumulation was examined 15 min to 4 h after injection of [5-³H]KYN, and the concentration dependence of KYN metabolism was studied in rats killed 2 h after injection of 1.5–1,500 µ M [5-³H]KYN. Labeled QUIN, KYNA, 3-hydroxykynurenine (3-HK), 3-hydroxyanthranilic acid, and xanthurenic acid (XA) were recovered from the striatum in every experiment. Following injection of 15 µ M [5-³H]KYN, a lesion-induced increase in KYN metabolism was noted. Thus, the proportional recoveries of [³H]KYNA (5.0 vs. 1.8%), [³H]3-HK (20.9 vs. 4.5%), [³H]XA (1.5 vs. 0.4%), and [³H]QUIN (3.6 vs. 0.6%) were markedly elevated in the lesioned striatum. Increases in KYN metabolism in lesioned tissue were evident at all time points and KYN concentrations used. Lesion-induced increases of the activities of kynurenine-3-hydroxylase (3.6-fold), kynureninase (7.6-fold), kynurenine aminotransferase (1.8-fold), and 3-hydroxyanthranilic acid oxygenase (4.2-fold) likely contributed to the enhanced flux through the pathway in the lesioned striatum. These data provide evidence for the existence of a functional KYN pathway in the normal rat brain and for a substantial increase in flux after neuronal ablation. This method should be of value for in vivo studies of cerebral KYN pathway function and dysfunction.     

Identification of Protein Arginine Methyltransferase 5 as a Regulator for Encystation of Acanthamoeba

Encystation is an essential process for Acanthamoeba survival under nutrient-limiting conditions and exposure to drugs. The expression of several genes has been observed to increase or decrease during encystation. Epigenetic processes involved in regulation of gene expression have been shown to play a role in several pathogenic parasites. In the present study, we identified the protein arginine methyltransferase 5 (PRMT5), a known epigenetic regulator, in Acanthamoeba castellanii. PRMT5 of A. castellanii (AcPRMT5) contained domains found in S-adenosylmethionine-dependent methyltransferases and in PRMT5 arginine-N-methyltransferase. Expression levels of AcPRMT5 were increased during encystation of A. castellanii. The EGFP-PRMT5 fusion protein was mainly localized in the nucleus of trophozoites. A. castellanii transfected with siRNA designed against AcPRMT5 failed to form mature cysts. The findings of this study lead to a better understanding of epigenetic mechanisms behind the regulation of encystation in cyst-forming pathogenic protozoa.     

Production of novel antioxidative phenolic amides through heterologous expression of the plant’s chlorogenic acid biosynthesis genes in yeast

Phenolic esters like chlorogenic acid play an important role in therapeutic properties of many plant extracts. We aimed to produce phenolic esters in baker’s yeast, by expressing tobacco 4CL and globe artichoke HCT. Indeed yeast produced phenolic esters. However, the primary product was identified as N-(E)-p-coumaroyl-3-hydroxyanthranilic acid by NMR. This compound is an amide condensation product of p-coumaric acid, which was supplied to the yeast, with 3-hydroxyanthranilic acid, which was unexpectedly recruited from the yeast metabolism by the HCT enzyme. N-(E)-p-coumaroyl-3-hydroxyanthranilic acid has not been described before, and it shows structural similarity to avenanthramides, a group of inflammation-inhibiting compounds present in oat. When applied to mouse fibroblasts, N-(E)-p-coumaroyl-3-hydroxyanthranilic acid induced a reduction of intracellular reactive oxygen species, indicating a potential therapeutic value for this novel compound.