Hemolymph phenol oxidases in Drosophila melanogaster, Locusta migratoria, and Austropotamobius pallipes

The early enzyme-mediated reaction sequence in the biosynthesis of melanin from L-tyrosine involves an initial hydroxylation (monophenol oxidase activity, MPO) of the aromatic amino acid precursor to form L-dopa (3,4-dihydroxyphenylalanine), and the ensuing oxidation (diphenol oxidase activity, DPO) of the resultant diphenol to form dopaquinone. By means of high pressure liquid chromatography with electrochemical detection (HPLC-ED) both phenol oxidase activities were observed in the blood (hemolymph) of two species of insect, third-stage larvae of Drosophila melanogaster and adult Locusta migratoria, and in an adult fresh-water crayfish, Austropotamobius pallipes. These results establish that in each species MPO and DPO can be detected readily without the use of exogenous activators.     

Identification of 3,4-dihydroxyphenylalanine, 5,6-dihydroxyindole, and N-acetylarterenone during eumelanin formation in immune reactive larvae of Drosophila melanogaster.

3,4-Dihydroxyphenylalanine, 5-6-dihydroxyindole, and N-acetylarterenone were detected by electrochemical methods in the hemolymph of immune reactive larvae of Drosophila melanogaster following parasitization by the wasp Leptopilina boulardi. Determinations of the catechols were made after separation by reverse phase, ion-pairing high pressure liquid chromatography with electrochemical detection. The presence of 5,6-dihydroxyindole unequivocally establishes the eumelanin pathway in the defense response of Drosophila, and confirms previous investigations which have implicated certain catecholamine metabolizing enzymes in insect immunity. The occurrence of N-acetylarterenone, a derivative of the principal sclerotizing agent N-acetyldopamine, verifies the existence and proposed involvement of quinone methide isomerase in the regulation of catecholamine metabolism, and suggests that the cellular capsule formed by Drosophila in immune reactions against parasites is most likely a composite of both eumelanin and sclerotin. The absence of 3,4-dihydroxyphenylacetic acid in hemolymph samples from immune reactive hosts suggests that during parasitization certain catecholamines and metabolic precursors may be re-employed in alternate pathways, some of which may be used in defense reactions.     

Parasite suppression of the oxidations of eumelanin precursors in Drosophila melanogaster

In insects, eukaryotic endoparasites encounter a series of innate immune effector responses mediated in large part by circulating blood cells (hemocytes) that rapidly form multilayer capsules around foreign organisms. Critical components of the encapsulation response are chemical and enzyme-catalyzed oxidations involving phenolic and catecholic substrates that lead to synthesis of eumelanin. These responses are initiated immediately upon infection and are very site-specific, provoking no undesirable systemic responses in the host. In this study, we were interested to learn if the principal oxidation pathways leading to the synthesis of eumelanin in larvae of Drosophila melanogaster were targets for inhibition by immune suppressive factors (ISF) derived from a virulent strain of the endoparasitic wasp Leptopilina boulardi. Comparative in vitro assays monitored by sensitive electrochemical detection methods showed that ISF derived from female reproductive tissues significantly diminished the oxidations of the two diphenol eumelanin precursors, dopamine and 5,6-dihydroxyindole (DHI). The oxidations of the monophenol tyrosine, and two other related diphenols, dopa and 5,6-dihydroxyindole-2-carboxylic acid (DHICA), were not significantly inhibited by ISF. The data suggest that melanogenesis represents at least one of the host responses suppressed by L. boulardi ISF, and that the oxidation pathways selectively targeted for inhibition are those synthesizing decarboxylated pigment precursors derived from DHI. These observations, together with previous reports of adverse effects of ISF on the ability of hemocytes to adhere to foreign surfaces, suggest a multifaceted approach by the parasitoid to circumvent the innate immune response of D. melanogaster.     

Leptopilina heterotoma and L. boulardi: strategies to avoid cellular defense responses of Drosophila melanogaster

Eggs of three strains of the cynipid parasitoid Leptopilina heterotoma and a Tunisian strain (G317) of L. boulardi are not encapsulated by hemocytes of Drosophila melanogaster hosts, but the eggs of a Congolese strain (L104) of L. boulardi are encapsulated. To determine the reason for the difference in host response against the parasitoid eggs, lamellocytes (hemocytes that encapsulate foreign objects and form capsules around endogenous tissues in melanotic tumor mutants) were examined in host larvae parasitized by the five Leptopilina strains. Parasitization by the three L. heterotoma strains affected the morphology of host lamellocytes and suppressed endogenous melanotic capsule formation in melanotic tumor hosts. L104 did not alter the morphology of host lamellocytes nor block tumor formation in melanotic tumor mutant hosts. The morphology of some lamellocytes was affected by G317 parasitization but host lamellocytes were still capable of forming melanotic tumors and encapsulating dead supernumerary parasitoid larvae. Therefore, the eggs of strains affecting lamellocyte morphology are protected from encapsulation by the host's blood cells. L. heterotoma eggs float freely in the host hemocoel but L. boulardi eggs are attached to host tissue surfaces. Lamellocytes cannot infiltrate the attachment site so the capsule around the L104 egg remains incomplete. The wasp larva uses this gap in the capsule as an escape hatch for emergence.     

Aldehyde oxidase activity in the tumorous-head strain of Drosophila melanogaster

Gross aldehyde oxidase activity from the egg-stage through 10-day-old adults and distribution of the enzyme in eye-antennal imaginal discs in third instar larvae were determined for the tumorous-head strain of Drosophila melanogaster. Aldehyde oxidase activity of several laboratory strains was measured for comparative purposes. Aldehyde oxidase activity was 100% higher during embryogenesis in tuh(ASU) eggs than in Oregon-R-C eggs. A second period of elevated aldehyde oxidase activity was observed during metamorphosis where tuh(ASU) pupae averaged 65% more enzyme activity than Oregon-R-C. Therefore, during determination and differentiation of the eye-antennal imaginal disc, the tuh(ASU) strain possesses a high aldehyde oxidase activity. Wild-type Drosophila melanogaster antennal imaginal discs are aldehyde oxidase positive, whereas attached eye imaginal discs are apparently aldehyde oxidase negative. A sample of eye-antennal imaginal discs from tuh(ASU) third instar larvae revealed that either one or both eye discs of 64% of the larvae were aldehyde oxidase positive. Aldehyde oxidase activity may be correlated with the homoeotic transformation in parts of the eye disc.     

The effects of parasite-derived immune-suppressive factors on the cellular innate immune and autoimmune responses of Drosophila melanogaster

Immune-suppressive factors (ISFs) introduced into larvae of Drosophila melanogaster during infection by virulent endoparasitic wasps effectively block the innate immune response mediated by blood cells (hemocytes) but have little influence on the autoimmune response made by a tumor strain in which the blood cells manifest a similar response but instead target and destroy endogenous tissues. Quantitative hemocyte analyses indicate that ISFs interfere with the immune effector responses downstream of nonself recognition, hemocyte activation and differentiation, because these responses were manifested by tumor hosts, in which the parasitoids developed. The data suggest that once activated to encapsulate aberrant tissues, the target specificity of the autoimmune-activated hemocytes, and the genetic program underlying tumor formation, cannot be blocked by parasitoid-derived ISFs, which effectively inhibit identical hemocyte-mediated responses during parasitization.     

High hemocyte load is associated with increased resistance against parasitoids in Drosophila suzukii, a relative of D. melanogaster

Among the most common parasites of Drosophila in nature are parasitoid wasps, which lay their eggs in fly larvae and pupae. D. melanogaster larvae can mount a cellular immune response against wasp eggs, but female wasps inject venom along with their eggs to block this immune response. Genetic variation in flies for immune resistance against wasps and genetic variation in wasps for virulence against flies largely determines the outcome of any fly-wasp interaction. Interestingly, up to 90% of the variation in fly resistance against wasp parasitism has been linked to a very simple mechanism: flies with increased constitutive blood cell (hemocyte) production are more resistant. However, this relationship has not been tested for Drosophila hosts outside of the melanogaster subgroup, nor has it been tested across a diversity of parasitoid wasp species and strains. We compared hemocyte levels in two fly species from different subgroups, D. melanogaster and D. suzukii, and found that D. suzukii constitutively produces up to five times more hemocytes than D. melanogaster. Using a panel of 24 parasitoid wasp strains representing fifteen species, four families, and multiple virulence strategies, we found that D. suzukii was significantly more resistant to wasp parasitism than D. melanogaster. Thus, our data suggest that the relationship between hemocyte production and wasp resistance is general. However, at least one sympatric wasp species was a highly successful infector of D. suzukii, suggesting specialists can overcome the general resistance afforded to hosts by excessive hemocyte production. Given that D. suzukii is an emerging agricultural pest, identification of the few parasitoid wasps that successfully infect D. suzukii may have value for biocontrol.     

Repeated-batch fermentation using flocculent hybrid, <Emphasis Type="Italic">Saccharomyces cerevisiae</Emphasis> CHFY0321 for efficient production of bioethanol

A new tyrosinase was isolated from Aeromonas media strain WS and purified to homogeneity. The purified tyrosinase, termed TyrA, had a molecular mass of 58 kDa and an isoelectric point of 4.90. It exhibited optimal monophenol and diphenol oxidase activities under basic conditions (pH > 8.0). TyrA had a relatively higher affinity to diphenol substrate l-dihydroxyphenylalanine (l-dopa) than many other tyrosinases. EDTA or glutathione notably inhibited the enzymatic activities of TyrA, whereas Triton X-100 and SDS activated them. The full-length TyrA gene was cloned, and it encodes a 518 amino acid protein with little similarities to other reported tyrosinases. However, the purified recombinant TyrA expressed in Escherichia coli demonstrated tyrosinase activity. These results suggest that TyrA is the first reported distinct tyrosinase involved in melanin production in the genus Aeromonas.     

Haemocyte changes in resistant and susceptible strains of D. melanogaster caused by virulent and avirulent strains of the parasitic wasp Leptopilina boulardi

Two strains of Drosophila melanogaster (resistant and susceptible) were parasitized by a virulent or avirulent strain of the parasitoid wasp Leptopilina boulardi. The success of encapsulation depends on both the genetic status of the host strain and the genetic status of the parasitoid strain: the immune cellular reaction (capsule) is observed only with the resistant strain-avirulent strain combination. The total numbers of host haemocytes increased in all 4 combinations, suggesting that an immune reaction was triggered in all hosts. Resistant host larvae infected with the virulent or avirulent strains of parasitoid wasp had slightly more haemocytes per mm(3) than did susceptible host larvae at the beginning of the reaction (less than 15 h post-parasitization). This difference disappeared later. Only the virulent parasitoid strain caused the production of a high percentage of altered lamellocytes (from a discoid shape to a bipolar shape), half the total number of lamellocytes are altered. This suggests that the alteration of lamellocyte shape alone is not sufficient to explain the lack of capsule formation seen in resistant hosts parasitized by the virulent strain. Lastly, there were very few altered lamellocytes in resistant or susceptible hosts parasitized by the avirulent parasitoid strain, two combinations in which no capsule was formed. As is now established for Drosophila-parasitoid interactions, virus-like particles contained in the long gland of the female wasp affect the morphology of the lamellocytes. The results presented here are further proof of the action (direct or indirect) of virus like particles of the virulent strain on lamellocytes.     

Hemocyte load and immune resistance to Asobara tabida are correlated in species of the Drosophila melanogaster subgroup

Larvae from six Drosophila species of the melanogaster subgroup were compared for both the hemolymph concentration of hemocytes and the ability to encapsulate the eggs of the parasitoid Asobara tabida (Hymenoptera; Braconidae). Results showed a high correlation between the parasitized hosts' concentration of circulating hemocytes and their aptitude to form a hemocytic capsule around the parasitic eggs. Two conditions seem to be required for the encapsulation of A. tabida eggs to succeed: one condition, which may relate to the recognition of the parasite by the host defense system, is the occurrence of a primary hemocytic response, which gives rise to the amplification of the hemocyte population; the other condition is the presence in the parasitized hosts of a hemocyte load large enough for the cellular capsule to be completed before the parasitic egg becomes protected by embedment within the host tissues. Since the concentration in hemocytes of the parasitized hosts is partially related to the concentration in hemocytes before parasitization, Drosophila species carrying a high hemocyte load could be better predisposed to resist A. tabida. Results are discussed in regard to the importance of a non-specific, quantitative character, such as the host hemocyte load, for the co-evolutionary immune interactions between A. tabida and its Drosophila hosts.     

Temporal control of urate oxidase activity during development of the third-instar larva of Drosophila: The role of 20-hydroxyecdysone

The tissue-specific enzyme urate oxidase is confined exclusively to the Malpighian tubules of Drosophila melanogaster and expressed only in the third-instar larva and the adult. Shortly before pupariation urate oxidase activity declines precipitously and is not detectable 24 hours later. That 20-hydroxyecdysone is the factor that triggers the disappearance of urate oxidase activity in late third-instar larvae is demonstrated using the temperature sensitive mutant ecd¹ which at the nonpermissive temperature of 29°C fails to accumulate a sufficient concentration of 20-hydroxyecdysone necessary for puparium formation and thus remains a third-instar larva for 1 to 2 weeks before death. Both the life cycle and the temporal profile of urate oxidase activity in ecd¹ larvae at 19°C is identical to that of the wild type. However, at 29°C ecd¹ third-instar larvae retain high urate oxidase activity. A precipitous decline in urate oxidase activity is observed when ecd¹ larvae at 29°C are fed 20-hydroxyecdysone. These data implicate 20-hydroxyecdysone in the process that controls the rapid decline of urate oxidase activity at the time of puparium formation. In whole homogenates of Malpighian tubules, the urate oxidase polypeptide was identified in SDS-polyacrylamide gels by its Rf with respect to homogeneously pure Drosophila urate oxidase and also by immunoprecipitation with rabbit anti-Drosophila urate oxidase IgG. Throughout development the amount of the urate oxidase polypeptide is correlated with the magnitude of urate oxidase activity.     

Mutational mapping of the catalytic activities of human tyrosinase.

Tyrosinase (EC 1.14.18.1) is a copper-containing metalloglycoprotein that catalyzes several steps in the melanin pigment biosynthetic pathway; the hydroxylation of tyrosine to L-3,4-dihydroxyphenylalanine (dopa) and the subsequent oxidation of dopa to dopaquinone. It has been proposed that tyrosinase is also able to oxidize 5,6-dihydroxyindole (DHI), a later product in the melanogenic pathway, to indole-5,6-quinone. Tyrosinase enzymatic activity is deficient in patients with classic type I oculocutaneous albinism (OCA), and more than 50 distinct mutations have now been identified in the tyrosinase genes of such patients. To determine the effects of the various tyrosinase gene mutations on the catalytic activities of the enzyme, we carried out site-directed mutagenesis of human tyrosinase cDNA, transiently expressed the mutant cDNAs in transfected HeLa cells, and assayed the resultant encoded proteins for tyrosine hydroxylase, dopa, and DHI oxidase activities, and resulting melanin production. The tyrosine hydroxylase activity of normal tyrosinase is thermostable, whereas its dopa oxidase and DHI oxidase activities are temperature-sensitive. Although all amino acid substitutions tested generally affected the dopa oxidase and DHI oxidase activities in parallel, several exerted distinctly different effects on the tyrosine hydroxylase activities. Together, these results confirm the DHI oxidase activity of mammalian tyrosinase and suggest that the dopa oxidase and DHI oxidase activities of tyrosinase share a common catalytic site, whereas the tyrosine hydroxylase catalytic site is at least partially distinct in the tyrosinase polypeptide.     

Inhibitory effects of cupferron on the monophenolase and diphenolase activity of mushroom tyrosinase

Mushroom tyrosinase (EC 1.14.18.1) is a copper containing oxidase that catalyzes both the hydroxylation of tyrosine into o-diphenols and the oxidation of o-diphenols into o-quinones. In the present study, the kinetic assay was performed in air-saturated solutions and the kinetic behavior of this enzyme in the oxidation of L-tyrosine and L-DOPA has been studied. The effects of cupferron on the monophenolase and diphenolase activity of mushroom tyrosinase have been studied. The results show that cupferron can inhibit both monophenolase and diphenolase activity of mushroom tyrosinase. The lag phase of tyrosine oxidation catalyzed by the enzyme was obviously lengthened and the steady-state activity of the enzyme decreased sharply. Cupferron can lead to reversible inhibition of the enzyme, possibly by chelating copper at the active site of the enzyme. The IC(50) value was estimated as 0.52 microM for monophenolase and 0.84 microM for diphenolase. A kinetic analysis shows that the cupferron is a competitive inhibitor for both monophenolase and diphenolase. The apparent inhibition constant for cupferron binding with free enzyme has been determined to be 0.20 microM for monophenolase and 0.48 microM for diphenolase.     

Dexamethasone inhibition of the cellular immune response of Drosophila melanogaster against a parasitoid

Host larvae of Drosophila melanogaster injected with the eicosanoid biosynthesis inhibitor, dexamethasone, prior to parasitization by the wasp Leptopilina boulardi, exhibited significantly reduced rates of melanotic encapsulation in comparison with control and saline-injected larvae. The results of this investigation suggest that prostaglandins and other eicosanoids are involved as cell-signaling molecules in the hemocytic encapsulation reaction of D. melanogaster larvae.     

Evidence against superoxide involvement in tyrosine hydroxylation by mushroom tyrosinase

The possible involvement of superoxide anions in the hydroxylation of tyrosine by mushroom tyrosinase was studied. Superoxide dismutase and scavengers of superoxide ions of smaller MW than superoxide dismutase, such as nitroblue tetrazolium and copper salicylate, had no direct effect on the monohydroxyphenolase activity of mushroom tyrosinase. The kinetics of tyrosine hydroxylation, but not of DOPA oxidation, by mushroom tyrosinase was atrected by the addition of a xanthine-xanthine oxidase system. In the presence of the xanthine-xanthine oxidase system, the lag period of tyrosine hydroxylation was shortened compared to the lag period in the absence of the xanthine-xanthine oxidase system. The xanthine- xanthine oxidase system alone (without mushroom tyrosinase) had no effect on tyrosine conversion to dopachrome. Superoxide dismutase, catalase and hydroxyl radical scavengers counteracted to some extent the shortening of the lag period of tyrosine hydroxylation by mushroom tyrosinase caused by the xanthin e-xanthine oxidase system. It is suggested that the shortening of the lag period is due mainly to hydroxyl radicals generated by the xanthine-xanthine oxidase system via interaction of O₂^?. and hydrogen paroxide (a Haber-Weiss type reaction). The data do not support the direct participation of superoxide anions in tyrosine hydroxylation by mushroom tyrosinase.     

Genetic control of phenoloxidase in the imago of Drosophila melanogaster]

The A1 component of Drosophila melanogaster phenol oxidase is controlled by an independent structural gene which has been located by the method of recombination mapping on the second chromosome at 67.3. The Dox-A1 is the only one from the group of genes coding for phenol oxidase in Drosophila which is expressed at the imago stage. The activity of phenol oxidase depends on the level of gene Dox-A1 expression and also on the presence in haemolymph of proenzyme activators. Males have a minimum level of phenol oxidase activity, which possibly protects them from mechanical damages of cuticle and toxic effects. Females are characterized by a higher level of A1-phenol oxidase activity than males. This may be caused by their reproduction function.     

Stress response in Drosophila melanogaster strain inactive with decreased tyramine and octopamine contents

Juvenile hormone hydrolysis, tyrosine decarboxylase activity, dopamine content and fitness (viability and fertility) were studied under normal and stress conditions in the adults of Drosophila melanogaster inactive strain carrying a mutation that decreases tyrosine decarboxylase activity and results in the lower contents of tyramine and octopamine. A sexual dimorphism of tyrosine decarboxylase activity, dopamine level and survival under heat stress in inactive flies was found. inactive adults showed higher dopamine levels and lower survival levels under heat stress as compared to wild type (Canton S) adults. Juvenile hormone degradation is decreased in young and increased in mature inactive females as compared to wild type. Fertility of the inactive strain did not differ from that of wild type strain under normal conditions, but after heat exposure the dynamics of its restoration was different. inactive females were found to develop the stress reaction, with juvenile hormone degradation, tyrosine decarboxylase activity, dopamine content and fertility levels used as the reaction indicators.     

绞股蓝皂苷-硒配合物对酪氨酸酶的活性影响及动力学分析

为考察绞股蓝皂苷及其硒配合物对酪氨酸酶的动力学参数和作用机理。本研究采用体外酶促反应,以L-酪氨酸和L-DOPA为底物,模拟了酪氨酸酶单酚和二酚酶的体外催化氧化过程。绞股蓝总皂苷在50%、70%乙醇洗脱段和50%、70%乙醇洗脱绞股蓝皂苷-硒配合物在酪氨酸酶上的Ki值分别为1. 533、1. 767、1. 312和1. 210 mmol/L。Ki值越低,对酪氨酸酶的抑制作用越强,单酚酶的氧化阶段越快,表明硒元素显著提高了绞股蓝皂苷对酪氨酸酶的抑制作用。酶反应动力学分析表明,四种绞股蓝皂苷及其硒配合物对酪氨酸的抑制作用均为混合竞争抑制。其独特的药理化学特性为绞股蓝及硒系美白化妆品的进一步研究开发提供了理论依据和参考。