MUTANT GENES REGULATING THE INDUCIBILITY OF KYNURENINE SYNTHESIS

Alterations in the cellular synthesis of kynurenine in the larval fatbody of Drosophila melanogaster may be obtained by feeding the precursor tryptophan or by changing the genotype. In the wild type Ore-R strain, autofluorescent kynurenine globules normally occur in the cells in the anterior regions of the fatbody designated as regions 1, 2, and 3. When tryptophan is included in the larval diet, kynurenine will develop throughout the entire fatbody, thus extending to the cells in regions 4, 5, and 6. In the fatbodies of both the sepia mutant strain and the mutant combinations of the suppressible vermilion alleles with the suppressor gene (su²-s, v¹ and su²-s, v²), kynurenine is found in the cells from region 1 through region 4. This involvement of additional cells in the synthesis of kynurenine occurs under the usual culture conditions for Drosophila. When sepia larvae are fed tryptophan, kynurenine appears in all of the cells of the fatbody. However, dietary tryptophan does not induce kynurenine production in cells in regions 5 and 6 in the mutant combination su²-s, v¹ or su²-s, v². In the latter strains, an increase in the quantity of kynurenine in the fatbody is detected, but this increase remains limited to the same cells in which kynurenine production is found under normal feeding conditions. When the v^36f allele is combined with the su²-s allele, an extremely faint autofluorescence characteristic of kynurenine is found in some of the anteriormost fat cells of regions 1 and 2. This autofluorescence becomes intensified when tryptophan is fed to su²-s, v^36f larvae. The genetic control of kynurenine synthesis in the cells of the fatbody of Drosophila melanogaster has been previously demonstrated. The present observations establish genetic regulation of the ability to induce kynurenine production within a cell through the administration of the inducer tryptophan. Kynurenine production has been considered as a unit function of the cell as a whole rather than of the enzyme alone, and it has been concluded that even though cells in different parts of the body perform this same function (kynurenine production), the gene loci regulating this function may be different for cells in different regions of the body. A phenomenon of overlapping domains of gene actions at the cellular level offers a genetic and cellular basis for developmental and physiological homeostasis.     

GENETIC CONTROL OF CYTODIFFERENTIATION

The cells of the anterior region of the larval fatbody of Drosophila melanogaster accumulate kynurenine at the end of the third larval instar, whereas the cells of the posterior region are involved in pteridine metabolism. Through a series of transplantation experiments it has been demonstrated that the anterior fat cells synthesize kynurenine. The mutant vermilion lacks kynurenine, and the anterior fat cells of this mutant strain lack the autofluorescence characteristic of kynurenine. When the non-allelic suppressor gene is combined with vermilion, the synthesis of kynurenine is restored in the anterior fat cells, and some of the cells of the posterior region contain kynurenine as well. A similar extension in the number of cells containing kynurenine can be induced in the normal Ore-R strain by feeding the precursor tryptophan. It has been concluded that the absence of a physiological process in a differentiated cell does not necessarily represent a loss of the genetic potential for that process. The normal allele at the suppressor locus inhibits the occurrence of kynurenine in the posterior fat cells, whereas the mutant allele su²-s allows the expression of this potential. An inducer such as tryptophan can overcome this inhibition in the normal strain, and as a result the cells which are normally differentiated as "isoxanthopterin cells" may produce kynurenine as well.     

Substrate Oxidation by Indoleamine 2,3-Dioxygenase: EVIDENCE FOR A COMMON REACTION MECHANISM*

The kynurenine pathway is the major route of l-tryptophan (l-Trp) catabolism in biology, leading ultimately to the formation of NAD⁺. The initial and rate-limiting step of the kynurenine pathway involves oxidation of l-Trp to N-formylkynurenine. This is an O₂-dependent process and catalyzed by indoleamine 2,3-dioxygenase and tryptophan 2,3-dioxygenase. More than 60 years after these dioxygenase enzymes were first isolated (Kotake, Y., and Masayama, I. (1936) Z. Physiol. Chem. 243, 237–244), the mechanism of the reaction is not established. We examined the mechanism of substrate oxidation for a series of substituted tryptophan analogues by indoleamine 2,3-dioxygenase. We observed formation of a transient intermediate, assigned as a Compound II (ferryl) species, during oxidation of l-Trp, 1-methyl-l-Trp, and a number of other substrate analogues. The data are consistent with a common reaction mechanism for indoleamine 2,3-dioxygenase-catalyzed oxidation of tryptophan and other tryptophan analogues.     

INTRACELLULAR LOCALIZATION OF KYNURENINE IN THE FATBODY OF DROSOPHILA

Light blue fluorescent globules accumulate in the cells of the anterior region of the fatbody of Drosophila larvae near the time of pupation. This fluorescent material appears in the Ore-R wild type strain as well as mutant strains in which the synthesis of both the red and brown eye pigments is affected. The vermilion mutant, which is characterized by the absence of the brown pigment component in the eye, was the only strain among those examined which did not develop the light blue fluorescent globules. Utilizing chromatographic techniques together with the information gained by examination of the mutant strains, the fluorescent material has been identified as kynurenine. Of particular interest is the manner of appearance of the fluorescent material in the vicinity of the nuclear membrane of the fat cells.     

Stable Isotope Labeling, in Vivo, of d- and l-Tryptophan Pools in Lemna gibba and the Low Incorporation of Label into Indole-3-Acetic Acid

We present evidence that the role of tryptophan and other potential intermediates in the pathways that could lead to indole derivatives needs to be reexamined. Two lines of Lemna gibba were tested for uptake of [¹⁵N-indole]-labeled tryptophan isomers and incorporation of that label into free indole-3-acetic acid (IAA). Both lines required levels of l-[¹⁵N]tryptophan 2 to 3 orders of magnitude over endogenous levels in order to obtain measurable incorporation of label into IAA. Labeled l-tryptophan was extractable from plant tissue after feeding and showed no measurable isomerization into d-tryptophan. d-[¹⁵N]tryptophan supplied to Lemna at rates of approximately 400 times excess of endogenous d-tryptophan levels (to yield an isotopic enrichment equal to that which allowed detection of the incorporation of l-tryptophan into IAA), did not result in measurable incorporation of label into free IAA. These results demonstrate that l-tryptophan is a more direct precursor to IAA than the d isomer and suggest (a) that the availability of tryptophan in vivo is not a limiting factor in the biosynthesis of IAA, thus implying that other regulatory mechanisms are in operation and (b) that l-tryptophan also may not be a primary precursor to IAA in plants.     

N-Malonyl-d-tryptophan in Apple Fruits Treated with Succinic Acid 2,2-Dimethylhydrazide

Fruit from Red Delicious apple trees treated with the growth retardant succinic acid 2,2-dimethylhydrazide contained more N-malonyl-d-tryptophan than control fruit. When succinic acid 2,2-dimethylhydrazide and tryptophan were injected into immature fruits, more N-methyl-d-tryptophan was produced than when dl-tryptophan was injected alone. Our results suggest that succinic acid 2,2-dimethylhydrazide may control fruit and vegetative growth by interfering with auxin production.     

Xanthommatin biosynthesis in wild-type and mutant strains of the Australian sheep blowfly Lucilia cuprina

The synthesis of eye pigments has been studied in the seven eye color mutants of the Australian sheep blowfly, Lucilia cuprina. Six appear to be affected primarily in the synthesis of xanthommatin. In wild type, the onset of xanthommatin biosynthesis occurs midway through metamorphosis. Developmental patterns of accumulation of the xanthommatin precursors tryptophan, kynurenine, and 3-hydroxykynurenine have also been established for wild type. By determining the levels of these precursors in late pupae of the mutants, it has been shown that the mutant yellowish accumulates excess tryptophan and the mutant yellow accumulates excess kynurenine. The implications of these results—that yellowish lacks tryptophan oxygenase, thus failing to convert tryptophan to kynurenine, and that yellow lacks kynurenine hydroxylase (blocked in the conversion of kynurenine to 3-hydroxykynurenine)—have been confirmed. This has involved in vitro assays of tryphophan oxygenase and precursor feeding experiments. The precursor accumulation patterns are less clear for the other mutants.     

The Conversion of d-Tryptophan to l-Tryptophan in Cell Cultures of Tobacco

d-Tryptophan was converted to l-tryptophan in tissue cultures of tobacco, in whole cells treated with dimethylsulfoxide, and in cell-free extracts treated by Sephadex G-25 filtration. Evidence was obtained that tryptophanase, tryptophan pyrrolase, and transaminase activities were not involved. The data were best explained by the presence of a tryptophan racemase as the enzyme catalyzing the reaction. The possible role of d-tryptophan in the biosynthesis of indoleacetic acid is discussed.     

Metabolism of l-Tryptophan to Kynurenate and Quinolinate in the Central Nervous System: Effects of 6-Chlorotryptophan and 4-Chloro-3-Hydroxyanthranilate

Abstract: The metabolism of l -tryptophan to the neuroactive kynurenine pathway metabolites, l -kynurenine, kynurenate and quinolinate, and the effects of two inhibitors of quinolinate synthesis (6-chlorotryptophan and 4-chloro-3-hydroxyanthranilate) were investigated by mass spectrometric assays in cultured cells and in vivo. Cell lines obtained from astrocytoma, neuroblastoma, macrophage/monocytes, lung, and liver metabolized l -[¹³C₆]-tryptophan to l -[¹³C₆]kynurenine and [¹³C₆]kynurenate, particularly after indoleamine-2,3-dioxygenase induction by interferon-γ. Kynurenine aminotransferase activity was measurable in all cell types examined but was unaffected by interferon-γ. These results suggest that many cell types can be sources of kynurenate following immune activation. In vivo synthesis of l -[¹³C₆]kynurenine and [¹³C₆]kynurenate from l -[¹³C₆]tryptophan was studied in the CSF of macaques infected with poliovirus, as a model of inflammatory neurologic disease. The effects of 6-chlorotryptophan and 4-chloro-3-hydroxyanthranilate on the synthesis of kynurenate were different. 6-Chlorotryptophan attenuated formation of l -[¹³C₆]kynurenine and [¹³C₆]kynurenate and was converted to 4-chlorokynurenine and 7-chlorokynurenate. It may be an effective prodrug for the delivery of 7-chlorokynurenate, which is a potent antagonist of NMDA receptors. In contrast, 4-chloro-3-hydroxyanthranilate did not reduce accumulation of l -[¹³C₆]kynurenine and [¹³C₆]kynurenate. 6-Chlorotryptophan and 4-chloro-3-hydroxyanthranilate are useful tools to manipulate concentrations of quinolinate and kynurenate in the animal models of neurologic disease to evaluate physiological roles of these neuroactive metabolites.     

TRYPTOPHAN LOADING: CONSEQUENT EFFECTS ON THE SYNTHESIS OF KYNURENINE AND 5-HYDROXYINDOLES IN RAT BRAIN

Abstract— Tryptophan loading of rats resulted in a continuous non-linear uptake of l -tryptophan from plasma into the brain. The optimum tryptophan load for increasing cerebral 5-hydroxytryptamine (5-HT) level was 25 mg/kg. Above this, there was a gradual decrease both in the levels and synthesis of 5-HT and 5-hydroxyindoleacetic acid (5-HIAA) as assessed from simultaneous intraperitoneal or intraventricular injections of l [¹⁴C]tryptophan. A 5–10 fold increase in cerebral tryptophan produced a limited stimulation of 5-HT synthesis. When the cerebral tryptophan level reached 1 ± 10 -4 , substrate inhibition in vivo of the tryptophan monooxygenase (tryptophan-5-hydroxylase) but not of the indoleamine-2,3-dioxygenase occurred. Cerebral synthesis of kynurenine increased linearly with increasing tryptophan load. At a plasma ratio of 50:1 tryptophan to kynurenine, tryptophan loading interfered with the entry of peripheral kynurenine. Tryptophan loading also increased the efflux of 5-hydroxyindoles from the brain. One hour after intraperitoneal injection of l -kynurenine sulfate (5 mg/kg) into rats, there was a shift in the plasma ratio of l -tryptophan to l -kynurenine to 4:1. In these rats, a 20% reduction of cerebral tryptophan was noted.     

Site-directed Mutagenesis Switching a Dimethylallyl Tryptophan Synthase to a Specific Tyrosine C3-Prenylating Enzyme

The tryptophan prenyltransferases FgaPT2 and 7-DMATS (7-dimethylallyl tryptophan synthase) from Aspergillus fumigatus catalyze C⁴- and C⁷-prenylation of the indole ring, respectively. 7-DMATS was found to accept l-tyrosine as substrate as well and converted it to an O-prenylated derivative. An acceptance of l-tyrosine by FgaPT2 was also observed in this study. Interestingly, isolation and structure elucidation revealed the identification of a C³-prenylated l-tyrosine as enzyme product. Molecular modeling and site-directed mutagenesis led to creation of a mutant FgaPT2_K174F, which showed much higher specificity toward l-tyrosine than l-tryptophan. Its catalytic efficiency toward l-tyrosine was found to be 4.9-fold in comparison with that of non-mutated FgaPT2, whereas the activity toward l-tryptophan was less than 0.4% of that of the wild-type. To the best of our knowledge, this is the first report on an enzymatic C-prenylation of l-tyrosine as free amino acid and altering the substrate preference of a prenyltransferase by mutagenesis.     

Ommochrome formation and kynurenine excretion in Pieris brassicae: Relation to tryptophan supply on an artificial diet

The brown-red pigment in the larval epidermis and in the testis of Pieris brassicae was identified as xanthommatin on the basis of solubility, redox behaviour, chromatography, degradation, visible and infrared spectra. In the epidermis, this pigment accumulates during the larval feeding period and disappears rapidly in the wandering stage. Larvae fed an artificial diet produce about half the amount of xanthommatin as larvae fed cabbage. This effect is caused by a lack of dietary tryptophan. Xanthommatin formation is increased by the addition of tryptophan which also increases body weight. At a tryptophan concentration of 0.2 mg per g, however, weight increase is lower than in controls and high mortality is observed. Pieris larvae excrete kynurenine in relation to dietary tryptophan. No measurable amounts are excreted in the last instar on the non-supplement diet. After feeding different quantities of tryptophan, different amounts of kynurenine are excreted only on the day following ecdysis.     

Endogenous Inactivators of Arginase, l-Arginine Decarboxylase, and Agmatine Amidinohydrolase in Evernia prunastri Thallus

Arginase (EC 3.5.3.1), l-arginine decarboxylase (EC 4.1.1.19), and agmatine amidinohydrolase (EC 3.5.3.11) activities spontaneously decay in Evernia prunastri thalli incubated on 40 millimolar l-arginine used as inducer of the three enzymes if dithiothreitol is not added to the media. Lichen thalli accumulate both chloroatranorin and evernic acid in parallel to the loss of activity. These substances behave as inactivators of the enzymes at a range of concentrations between 2 and 20 micromolar, whereas several concentrations of dithiothreitol reverse, to some extent, the in vitro inactivation.     

Auxin Biosynthesis during Seed Germination in Phaseolus vulgaris

The relative roles of de novo biosynthesis of indoleacetic acid (IAA) and IAA conjugates stored in mature seeds (Phaseolus vulgaris L.) in supplying auxin to germinating bean seedlings were studied. Using ²H oxide and 2,4,5,6,7-[²H]l-tryptophan as tracers of IAA synthesis, we have shown that de novo biosynthesis of IAA, primarily from tryptophan, is an important source of auxin for young bean seedlings. New synthesis of IAA was detected as early as the second day of germination, at which time the seedlings began to accumulate fresh weight intensively and the total content of free IAA began to increase steadily. IAA conjugates that accumulate in large amounts in cotyledons of mature seeds may thus be considered to be only one of the possible sources of IAA required for the growth of bean seedlings.     

Product induction in Pseudomonas acidovorans of a permease system which transports L-tryptophan

Growth of Pseudomonas acidovorans in the presence of l-tryptophan resulted in the appearance of a tryptophan transport system which was extremely sensitive to sodium azide or 2,4-dinitrophenol. Asparagine-grown cells possessed no detectable tryptophan "permease" activity. Substitution of l-kynurenine for l-tryptophan in the growth medium also induced the tryptophan permease activity, along with tryptophan oxygenase and kynurenine formamidase. This is the first reported example of the product induction of a permease activity. Irrespective of whether Pseudomonas cells are grown in the presence of d- or l-tryptophan, the resulting induced tryptophan permease activity is specific for the l-isomer. In addition, the radioactive compounds l-leucine, l-phenylalanine, or dl-5-hydroxytryptophan are not transported. When dl-5-fluorotryptophan is a component of the inducing medium (with l-tryptophan), induction of tryptophan permease activity, as well as tryptophan oxygenase, is inhibited. In the permease assay system, using normally induced cells, the fluoroanalogue inhibited strikingly tryptophan transport. Therefore, this analogue may inhibit induction by blocking inducer transport into the cell. When added to the l-tryptophan-inducing medium, dl-7-azatryptophan markedly enhanced induction of tryptophan oxygenase, but the level of tryptophan permease activity was not further elevated. The mechanism of this analogue is unclear at present. Invariant tryptophan permease activity levels are found in cells grown with 5 or 15 mml-tryptophan or 5 mml-kynurenine, whereas the respective tryptophan oxygenase levels are greatly different. Together with other results, these results indicate that the synthesis of tryptophan permease activity is not coordinate with that of tryptophan oxygenase. Tryptophan transport is strongly inhibited by l-formylkynurenine and by l-kynurenine. These two metabolites were prepared in radioactive form, and they are actively transported following bacterial growth on l-tryptophan or l-kynurenine. Preliminary results suggest the tryptophan permease activity may be distinct from the permease(s) activity for l-formylkynurenine and l-kynurenine. Kynurenine, then, is capable of inducing tryptophan permease and kynurenine permease activities.     

l-Kynurenine Its synthesis and possible regulatory function in brain

One pathway by which tryptophan is metabolized in the brain as well as in the periphery is through cleavage of the indole ring to formylkynurenine and then kynurenine. Indoleamine-2,3-dioxygenase, the enzyme that catalyzes this clavage, and kynurenine are distributed all across the different anatomic regions of brain. Approximately 40% of the kynurenine in brain is synthesized there, the remainder having come from plasma. Tryptophan loading, which has been used both experimentally and therapeutically as a means of increasing tryptophan conversion to serotonin, also increases kynurenine formation in the brain and in the periphery. Because of the formation of kynurenine, which competes for cerebral transport and cellular uptake ofl-tryptophan, and because of substrate inhibition on tryptophan hydroxylase, excessively high doses of tryptophan may actually decrease the production of cerebral serotonin and 5-hydroxyindoleacetic acid.Some aspects of this paper were presented in a lecture at the meeting of the International Study Group for Tryptophan Research (ISTRY-77) on August 11, 1977 at the University of Wisconsin, Madison, Wisconsin.     

CYTODIFFERENTIATION IN THE ROSY MUTANT OF DROSOPHILA MELANOGASTER

In the rosy mutant of Drosophila melanogaster, two types of autofluorescent cytoplasmic inclusions are found in the cells of the posterior region of the fatbody at the prepupal stage. Bright yellow autofluorescent granules accumulating within larger inclusions clearly demarcate this area of the fatbody which also contains cobalt blue fluorescent globular material. Such inclusions were not noted in the normal Ore-R strain at this stage nor in the series of mutant strains examined other than the rosy² and maroon-like mutants. The pattern of biochemical deviation of the latter two mutants is known to be identical to that of the rosy mutant, and a portion of this mutant upset can be ascribed to the absence of xanthine dehydrogenase. These mutants lack the products of enzyme activity, uric acid and isoxanthopterin, and accumulate their precursors, hypoxanthine and 2-amino-4-hydroxypteridine. Chromatographic studies on the fatbody of rosy prepupae have shown that 2-amino-4-hydroxypteridine is limited to the posterior region; this correspondence in location as well as color of fluorescence indicates that the cobalt blue auto fluorescent globules in the fatbody contain 2-amino-4-hydroxypteridine. In the normal strain, isoxanthopterin was identified in the chromatograms of the posterior region of the fatbody, but it was not obtained from the anterior region of the fatbody. On the other hand, xanthine dehydrogenase activity could be demonstrated throughout the fatbody of the normal strain. The restriction of isoxanthopterin to a certain group of fat cells in the wild type strain and its absence from other fat cells can be explained by the differential distribution of its immediate precursor, 2-amino-4-hydroxypteridine, as displayed in the mutant rosy.     

The in vitro biosynthesis and secretion of glycerol by larval fat bodies of chilled Ostrinia nubilalis

Fat bodies from diapausing fifth-instar larvae of Ostrinia nubilalis were incubated in vitro at 5 or 23°C in Grace's medium and the glycerol contents of the organ and incubation medium determined. Fat bodies from diapausing larvae chilled 3 weeks at 5°C secreted glycerol into the medium at 5°C at a net rate of approx. 0.75 nmol/mg fat body dry wt/h for at least 96 h while the tissue levels remained essentially constant. Depending upon the experiment, from 6 to 15 times more glycerol was produced in 24 h at 5°C by these fat bodies than by those taken from diapausing unchilled larvae and incubated at either 5 or 23°C. A minimal chilling period of 10–12 days was recognized as necessary for chilled larval fat bodies to demonstrate rates of glycerol synthesis greater than those of unchilled larvae and the lag showed a temporal correlation with changes in haemolymph glycerol concentrations. These results suggest that this response to chilling by O. nubilalis is relatively slow. While incubation, at 23°C, of fat bodies from previously chilled larvae did not result in cessation of glycerol secretion, the rate of its appearance in the culture medium decreased during the 24-h incubation period. Although the ability of chilled fifth-instar larvae to accumulate glycerol is not dependent upon the diapause state results show that clearance of glycerol from the haemolymph by rewarmed O. nubilalis is related to diapause intensity.     

Accumulation of an Endogenous Tryptophan-Derived Metabolite in Colorectal and Breast Cancers

Tumor immune escape mechanisms are being regarded as suitable targets for tumor therapy. Among these, tryptophan catabolism plays a central role in creating an immunosuppressive environment, leading to tolerance to potentially immunogenic tumor antigens. Tryptophan catabolism is initiated by either indoleamine 2,3-dioxygenase (IDO-1/-2) or tryptophan 2,3-dioxygenase 2 (TDO2), resulting in biostatic tryptophan starvation and l-kynurenine production, which participates in shaping the dynamic relationship of the host’s immune system with tumor cells. Current immunotherapy strategies include blockade of IDO-1/-2 or TDO2, to restore efficient antitumor responses. Patients who might benefit from this approach are currently identified based on expression analyses of IDO-1/-2 or TDO2 in tumor tissue and/or enzymatic activity assessed by kynurenine/tryptophan ratios in the serum. We developed a monoclonal antibody targeting l-kynurenine as an in situ biomarker of IDO-1/-2 or TDO2 activity. Using Tissue Micro Array technology and immunostaining, colorectal and breast cancer patients were phenotyped based on l-kynurenine production. In colorectal cancer l-kynurenine was not unequivocally associated with IDO-1 expression, suggesting that the mere expression of tryptophan catabolic enzymes is not sufficiently informative for optimal immunotherapy.     

Synthesis of adipokinetic hormone (AKH-I) in the locust brain

Methanol extracts of vitellogenic female locust brains contain two factors that inhibit protein synthesis in fat body tissue excised from such individuals. One of these factors (BI) elicits lipid mobilization when injected into adult male locusts. The retention times of BI on an RP-18 column and on an RP-4 column are identical to those of synthetic locust adipokinetic hormone (AKH-I) on each of these columns. Half maximal inhibition of protein synthesis in excised adult locust fat bodies is exerted by 0.05 brain extract equivalents of BI, which is equivalent to activity elicited by 1.5 pmol of AKH-I, as previously determined by AKH-radioimmunoassay. Enzymatic hydrolysis of the N-terminal pyroglutamate, followed by amino acid sequence analysis, indicates that the structure of BI is similar to that of the decapeptide AKH-I synthesized in the glandular lobe of the locust corpora cardiaca (CC). Incorporation of [5-³H]tryptophan into BI of locust brains incubated in vitro indicates that the AKH-I present in the brain is synthesized in situ and is not transported from the CC. Similar incorporation of radiolabel into AKH-I is obtained when excised CC are incubated in vitro.