Ascidian phenoloxidase: its release from hemocytes, isolation, characterization and physiological roles

Hemocytes of the solitary ascidian Halocynthia roretzi released phenoloxidase in response to sheep red blood cells and yeast cells but not to latex beads. Phenoloxidase was also released from the hemocytes by treatments with zymosan and lipopolysaccharides but not with β1–3 glucan. EDTA scarcely inhibited the activity of phenoloxidase but inhibited the release of the enzyme. Phenoloxidase was purified from H. roretzi hemocytes by SP-Sephadex chromatography and Sephadex G-100 gel filtration. The molecular weight of the purified enzyme was estimated to be 62 000. Phenoloxidase activity was strongly inhibited by diethyldithiocarbamate, phenylthiourea and reducing agents. H. roretzi phenoloxidase was characterized as a metalloenzyme that required copper ions for the expression of full activity. The phenoloxidase showed antibacterial activity in the presence of -(3,4-dihydroxy)-phenylalanine and H. roretzi plasma. Thus, it can be concluded that phenoloxidase released from H. roretzi hemocytes functions as a humoral factor in the defense system of H. roretzi.     

A fungal phenoloxidase (tyrosinase) involved in pentachlorophenol degradation

Amylomyces rouxii eliminated 85% of initial pentachlorophenol (PCP) at 12.5 mg l^–1 when grown with 0.1 g tyrosine l^–1, but only 55% without tyrosine. Addition of tyrosine in the culture medium increased the monophenolase activity by 1.8-fold. Tyrosinase is thus indicated to be the phenoloxidase involved in PCP degradation by A. rouxii.     

Induction of phenoloxidase in cell suspension cultures of Mucuna pruriens L.

The effects of limitating nitrogen-containing compounds in the medium and of adding the amino-acid analogues p-fluorophenylalanine and ethionine on both phenoloxidase activity and the accumulation of L-3,4-dihydroxyphenylalanine (L-DOPA) are reported for cell suspension cultures of Mucuna pruriens. Nitrogen limitation of the cultures, or the addition of p-fluorophenylalanine or ethionine to the culture medium resulted in an increased phenoloxidase activity. There appeared to be an inverse relationship between phenoloxidase activity and the acccumulation of L-tyrosine into L-DOPA by alginate-entrapped cells occurred at a higher rate when phenoloxidase activity was increased.Abbreviations pFPA p-fluorophenylalanine - L-DOPA L-3,4-dihydroxyphenylalanine     

Electrophoretic study of the enzyme phenoloxidase from the enzyme gland in the foot of Perna viridis Linnaeus

The enzyme phenoloxidase from the enzyme gland in the foot of Perna viridis Linnaeus 1758 has been characterized electrophoretically. After fractionation, the gels were incubated in various phenolic substrates: catechol, Dopa, dopamine, hydroquinone, and tyrosine. The behavioural differences exhibited by phenoloxidase on incubation in different substrates have been discussed and compared with similar observations from insects. The occurrence of phenoloxidase in multiple forms has also been discussed.     

Defense mechanisms in insects: Certain integumental proteins and tyrosinase are responsible for nonself-recognition and immobilization of Escherichia coli in the cuticle of developing Ceratitis capitata

A defense mechanism in the cuticle of developing C. capitata was demonstrated using an in vitro system consisting of isolated cuticular tyrosinase from C. capitata, cuticular tyrosinase-free proteins, tyrosine, and E. coli. The simultaneous presence of the above components resulted in the formation of large immobilized E. coli aggregates. By contrast, omission of any of the above components failed to produce such aggregates. In other words, E. coli retained their mobility and viability. The results indicate that certain cuticular proteins are responsible for the nonself-recognition, since they are able to bind to the E. coli surface in vitro, and a reactive tyrosine derivative is generated by the action of cuticular tyrosinase for the immobilization and probably killing of E. coli. Based on these studies the most likely explanation for the nonself-recognition and immobilization and/or killing of bacteria is the production of E. coli-protein complexes and their crosslinking through quinone intermediate. © 1993 Wiley-Liss, Inc.     

Effect of different K+ concentrations on cryptococcus neoformans phenoloxidase activity

Melanin synthesis in Cryptococcus neoformans, catalyzed by phenoloxidase activity, is one of the oldest virulence factors known. However, until now, the relationship between melanin production in C. neoformans and its virulence has been poorly understood. Among different chemical compounds only Fe³⁺ and Cu²⁺ cations enhance the phenoloxidase activity in C. neoformans. A few reports in the literature describe the influence of different cations on C. neoformans phenoloxidase activity, excluding iron [1–3]. In this study, 13 C. neoformans strains isolated from AIDS patients and 7 from bird droppings (B.D.), were examined in order to clarify the effect of different K⁺ concentrations on phenoloxidase activity. A new solid and liquid caffeic acid minimal synthetic medium (MSM-CAF) containing only caffeic acid and ferric citrate with different potassium concentrations was used to evaluate C. neoformans phenoloxidase activity. In the MSM-CAF solid medium the degree of brown pigmentation on the agar plates was read on days 1, 2 and 3 of incubation, and the pigmentation of the C. neoformans strains was classed into 5 categories. The brown pigment of the liquid MSM-CAF test tubes were checked after 24 hours of incubation by measuring the optical density (O.D.) at 480 nm. Three C. neoformans AIDS and B.D. strains, randomlychosen, were tested for phenoloxidase activity, according to the modified protocols of Polachecket al., Torres-Guerrero et al. and Rhodes [2–4]. According to the results obtained, it has been observed that K⁺ does not activate the phenoloxidase activity in the C. neoformans AIDS and B.D. strains. In particular, with an increase in potassium concentrations in the MSM-CAF solid and liquid medium, there was a corresponding inhibition of the phenoloxidase activity on both the C. neoformans AIDS and B.D. strains.This revised version was published online in October 2005 with corrections to the Cover Date.     

Effect of associated fungi on the immunocompetence of red turpentine beetle larvae,Dendroctonus valens (Coleoptera: Curculionidae: Scolytinae)

Abstract Dendroctonus–fungus symbioses are often considered as the ideal model systems to study the development and maintenance of ectosymbioses, and diverse interactions, including antagonism, commensalism and mutualism, have been documented between these organisms. The red turpentine beetle, Dendroctonus valens LeConte (Coleoptera: Curculionidae: Scolytinae) is a pine‐killing invasive beetle in northern China. Fungi species Ophiostoma minus, Leptographium sinoprocerum, L. terebrantis and L. procerum were associated with this bark beetle. Antagonistic interactions between D. valens and its associated fungi, such as O. minus and L. sinoprocerum, have been demonstrated, but the underlying causes of this phenomenon are unknown. Here, we first found the two tested fungi species retarded the net weight gain of D. valens larvae after completing 3‐day feeding on their media. Furthermore, we provide direct evidence indicating the effect of associated fungi on the immunocompetence of D. valens larvae to explain the documented antagonism. Our results showed that the activity of phenoloxidase and total phenoloxidase in D. valens larvae were significantly upregulated by two strains of associated fungi, O. minus and L. sinoprocerum as compared with the controls. The phenoloxidase ratio increased significantly in the larvae which had fed for 3 days on media inoculated with O. minus. Because insect immune defenses are costly to be deployed, these results could be explored as one of the underlying mechanisms of the documented antagonism.     

Purification and characterization of prophenoloxidase from cotton bollworm,Helicoverpa armigera

Phenoloxidases are oxidative enzymes, which play an important role in both cell mediated and humoral immunity. Purification and biochemical characterization of prophenoloxidase from cotton bollworm, Helicoverpa armigera (Hübner) were carried out to study its biochemical properties. Prophenoloxidase consists of a single polypeptide chain with a relative molecular weight of 85 kDa as determined by SDS–PAGE, MALDI–TOF MS and LC–ESI MS. After the final step, the enzyme showed 71.7 fold of purification with a recovery of 49.2%. Purified prophenoloxidase showed high specific activity and homology with phenoloxidase subunit‐1 of Bombyx mori and the conserved regions of copper binding (B) site of phenoloxidase. Purified prophenoloxidase has pH optima of 6.8 and has high catalytic efficiency towards the dopamine as a substrate in comparison to catechol and L‐Dopa. The PO activity was strongly inhibited by phenylthiourea, thiourea, dithiothreitol and kojic acid.     

Relationship between Acid-catalyzed Cyclization of Citral and Deterioration of Lemon Flavor

Phenoloxidase was purified from pupae of the housefly, Musca domestica L. The purification procedures included ammonium sulfate precipitation, affinity chromatography and Sephadex G-200 gel filtration. The final preparations appear to be homogeneous based on results obtained from polyac-rylamide gel electrophoresis. The molecular weight of phenoloxidase was estimated to be 330,000, as determined by gel filtration. The kinetic properties of phenoloxidase were studied using six catecholamines as substrates. The preferred order of substrates for phenoloxidase was found to be N-β-alanyldopamine > dopamine > TV-acetyldopamine > norepinephrine > epinephrine > DOPA.     

Mutations in the sigma subunit of E. coli RNA polymerase which affect positive control of transcription

Summary An electrophoretic mobility variant of phenoloxidase in a lz stock of Drosophila melanogaster was identified as the A₃ component of the phenoloxidase complex by using two different activators to study enzyme activity — natural activator isolated from pupae and 50% 2-propanol. The structural gene for the A₃ proenzyme, Dox-3, was not associated with lz on the X chromosome; it mapped to the right of rdo (53.1) and left of M(2)m in the second linkage group.The lz locus affects the differentiation of the crystal cell, the type of hemocyte that carries prophenoloxidase(s) in paracrystalline form. Alleles of lz lacking paracrystalline inclusions in their hemocytes do not have phenoloxidase activity whereas alleles with paracrystalline inclusions have enzyme activity. The presence of proenzyme in the paracrystalline inclusions was demonstrated by in situ activation with natural activator or propanol followed by incubation in buffered dopa.     

Effect of Various Elicitors on Lignan Biosynthesis in Callus Cultures of Linum austriacum

Effects of elicitors (mannan, -1,3-glucan, and ancymidol) on the activity of several key enzymes participating in lignan biosynthesis were studied in Linum austriacum L. cell cultures. The activities of L-phenylalanine ammonia-lyase, polyphenoloxydase, tyrosine ammonia-lyase, soluble phenoloxidase, and membrane-bound and soluble oxidases were assayed. The elicitors under study affected various steps in the metabolic pathway of lignan biosynthesis. Elevated enzyme activity accompanied an elicitor-enhanced synthesis of podophyllotoxins and peltatins.     

An isolate of Rhizopus nigricans capable of tolerating and removing pentachlorophenol

Rhizopus nigricans, isolated from an industrial effluent (paper mill), was resistant to pentachlorophenol (PCP) in Petri dishes and in submerged cultures (100 and 25 mg l^–1 respectively). It was shown that this strain of R. nigricans can remove PCP in submerged culture. When 12.5 mg of PCPl^–1 were added at 48 h, this compound had been completely removed by 144h. Results indicated that the fungus did not produce extracellular lignin peroxidase (LiP) and laccase, but extracellular phenoloxidase production was observed. The synthesis of the latter enzyme was stimulated by the presence of PCP and/or tyrosine. These results indicate that this fungus, and probably other filamentous fungi, have an interesting potential to be used in processes for chlorophenol biodegradation.     

Temperature and Ca2+ ion as modulators in cellular immunity of the Sunn pest Eurygaster integriceps Puton (Heteroptera: Scutelleridae)

Environmental conditions in addition to divalent cations may affect the interactions between pathogens and insects. Elucidation of factors which modulate insect immune responses could be a significant part of investigations in this area. In this study, adults of Eurygaster integriceps, as the destructive pest of wheat, were kept at different temperatures in addition to injection with different concentrations of Ca²⁺ to find the effect on cellular immune reactions against Beauveria bassiana. Results showed that total and differentiate hemocyte numbers, nodule formation and phenoloxidase activity increased with elevation of temperature so that the higher values were obtained at 30 and 40°C at various intervals. Higher concentrations of Ca²⁺ ion (5 mM) caused an increase in plasmatocyte length and width especially after 60 min. Similar results were observed for nodule formation and phenoloxidase activity of E. integriceps adults after injection by B. bassiana spores and phenoloxidase activity. It is clear from the current study that thermoregulation and Ca²⁺ ion can positively affect the hemocyte numbers especially plasmatocytes and granulocytes, nodule formation and phenoloxidase activity in E. integriceps. The understanding of modulators of the insect immune response may directly influence novel approaches to obtain safe and effective biological control agents.     

Soluble tyrosinases from pharate pupal integument of the tobacco hornworm,Manduca sexta (L.)

Two soluble phenoloxidases were partially purified from pharate pupal integument of Manduca sexta by gel filtration or sucrose density gradient centrifugation. Thiourea was used to retard the formation of higher molecular weight aggregates. Subsequent analysis by gel filtration HPLC showed that the apparent molecular weights were about 300 kD and >700 kD. The smaller phenoloxidase was further purified by anion exchange HPLC. In comparative studies with mushroom tyrosinase and a fungal laccase, the Manduca phenoloxidases were identified as tyrosinases, since they exhibited monophenol mono-oxygenase activity (EC 1.14.18.1) and catechol oxidase activity (EC 1.10.3.1). Both enzyme preparations catalyzed ortho-hydroxylation of tyrosine at a relatively show rate, oxidized o-diphenols at a much faster rate than p-diphenols or monophenols, had broad pH optima from about pH 5.5–7.5, and were completely inhibited by 1 μM phenylthiourea, N-β-alanyldopamine (NBAD) and N-β-alanylnorepinephrine (NBANE), which are both abundant in pupal cuticle, were oxidized to o-quinones by the tyrosinases, with the former catecholamine oxidizing at a 10-fold higher rate than the latter. NBANE was synthesized from NBAD, apparently by spontaneous tautomerization of the o-quinone to a p-quinone methide, which then reacted with water to form the β-hydroxylated product. The possible role of tyrosinase in insect integument is discussed.     

The role of bacterial symbionts in suppressing the defence reaction in larval hemolymph of the cotton leafworm Spodoptera littoralis (Biosd.)

Abstract

Spodoptera littoralis hemolymph exhibited a decrease in phenoloxidase activity during the first hour of exposure to alive or dead Xenorhabdus nematophilus and Photorhabdus luminescens even in the presence or absence of laminarin and α-chymotrypsin. Also, as the bacterial numbers in the hemolymph of the larvae of S. littoralis increased, the suppression of the enzyme, phenoloxidase, activity in vivo increased. On the other hand, in the in vitro incubation of the infected hemolymph with X. nematophilus for 30 min with laminarin, the decrease in phenoloxidase activity reached 92% in the infection with the highest dose (1 × 10¹⁰ cells/ml) live bacteria, and reached 100% at the same dose in the infection with live bacteria in the absence of laminarin. A two-fold decrease in enzyme activity was recorded in the case of injection of (1 × 10⁶ cells/ml) dead X. nematophilus and the absence of laminarin compared with injection of the same dose in the presence of laminarin. The same trend was also observed by the end of incubation of X. nematophilus-treated hemolymph without α-chymotrypsin. The decrease in phenoloxidase activity was highly significantly different in injection of dead P. luminescens and the absence of laminarin during incubation. In the case of injection of (1 × 10⁸ cells/ml) live P. luminescens a higher degree in the reduction of enzyme activity was recorded in the absence of α-chymotrypsin, where it reached to nearly a two-fold decrease. The results of these studies indicated that both X. nematophilus and P. luminescens alive or dead suppress the phenoloxidase activity in the presence or absence of both laminarin and α-chymotrypsin but, the suppression in absence of both during the in vitro incubation was highly comparable.     

Variations des activités peroxydasique et phénoloxydasique au cours de la croissance de Coleus blumei Benth. var. Automne

Summary The specific peroxidase (1.11.1.7) and phenoloxidase (1.10.3.1) activities are quantitatively measured during the life of Coleus, from germination until flowering. In most organs investigated, the peroxidase activity increases rapidly with growth while the phenoloxidase activity remains low. The latter activity is higher in root apices than in more differentiated regions of roots. From the results obtained it may be concluded that the phenoloxidase activity accompanies cellular proliferation. It is suggested that the peroxidase activity plays an indirect role in root initiation through its role in cellular differentiation.

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Extrait d'une thèse de doctorat soutenue à la Faculté des Sciences de l'Université de Liège.     

A laccase-like phenoloxidase is correlated with lignin biosynthesis in Zinnia elegans stem tissues

Stem tissues from different internodes of 4–6 week-old Zinnia elegans cv. Envy plants were sectioned and stained with chromogenic substrates previously used in studies of laccases (p-diphenol:O₂ oxidoreductases) isolated from tree tissues. The pattern of color development found when stem sections were stained in the presence and absence of H₂O₂ suggested that p-diphenol:O₂ oxidoreductase activity was tightly correlated spatially and temporally with the lignification of secondary cell walls in developing primary xylem. The correlation between this laccase-like phenoloxidase activity and lignification appeared tighter than that between lignification and peroxidases stained using the same substrates. Zymogram analysis of the phenoloxidase activities catalyzed by enzymes that were not boiled prior to separation by SDS—PAGE suggested that a single enzyme was predominantly responsible for the laccase-like phenoloxidase activity in Zinnia stems. Some of this enzyme was released from cell wall residue by washing with high ionic strength buffer; however, substantial amounts of the enzyme could only be recovered after treatment of the residue with cell wall-degrading enzymes. This phenoloxidase appears to share significant characteristics with the coniferyl alcohol oxidase isolated from developing secondary xylem in pines, which suggests that such enzymes may be widespread in vascular plants.     

Teratocytes of the solitary endoparasitoid Meteorus gyrator (Hymenoptera: Braconidae): morphology, numbers and possible functions

Abstract. Laboratory studies investigated the development of teratocytes derived from the eggs of the parasitoid Meteous gyrator (Thun.) in its host, the tomato moth Lacanobia oleracea (L.). At hatching, each parasitoid egg produced an average of approximately 1000 teratocytes, but this number declined to approximately 400 during the course of parasitism. The teratocytes increased in size markedly, such that 7 days after egg hatch their mean diameter was approximately four times that of the cells immediately after dissociation. The haemolymph of parasitized hosts had reduced phenoloxidase activity, and teratocytes inhibited phenoloxidase activity when coincubated with plasma from nonparasitized hosts. The injection of teratocytes into nonparasitized fifth‐instar L. oleracea larvae suppressed growth and induced a supernumerary moult in some larvae. A number of parasitism‐specific proteins were detected in the haemolymph of parasitized hosts, and incubation of teratocytes in culture media indicated that these cells were a source of at least two of these proteins.     

CLP gene family,a new gene family of Cotesia vestalis bracovirus inhibits melanization of Plutella xylostella hemolymph

Polydnaviruses (PDVs) are obligatory symbionts of parasitoid wasps and play an important role in suppressing host immune defenses. Although PDV genes that inhibit host melanization are known in Microplitis bracovirus, the functional homologs in Cotesia bracoviruses remain unknown. Here, we find that Cotesia vestalis bracovirus (CvBV) can inhibit hemolymph melanization of its host, Plutella xylostella larvae, during the early stages of parasitization, and that overexpression of highly expressed CvBV genes reduced host phenoloxidase activity. Furthermore, CvBV-7-1 in particular reduced host phenoloxidase activity within 12 h, and the injection of anti-CvBV-7-1 antibody increased the melanization of parasitized host larvae. Further analyses showed that CvBV-7-1 and three homologs from other Cotesia bracoviruses possessed a C-terminal leucine/isoleucine-rich region and had a similar function in inhibiting melanization. Therefore, a new family of bracovirus genes was proposed and named as C -terminal L eucine/isoleucine-rich P rotein (CLP). Ectopic expression of CvBV-7-1 in Drosophila hemocytes increased susceptibility to bacterial repression of melanization and reduced the melanotic encapsulation of parasitized D. melanogaster by the parasitoid Leptopilina boulardi. The formation rate of wasp pupae and the eclosion rate of C. vestalis were affected when the function of CvBV-7-1 was blocked. Our findings suggest that CLP genes from Cotesia bracoviruses encoded proteins that contain a C-terminal leucine/isoleucine-rich region and function as melanization inhibitors during the early stage of parasitization, which is important for successful parasitization.     

Differentiation of Tyrosine Hydroxylase and Phenol Oxidase after Electrophoresis

Tyrosine hydroxylase and phe-noloxidase differ in that tyrosine hydroxylase (E.C. 1.14.16.2) can hydroxylate tyrosine into -o-diphenol, but cannot oxidize the -o-diphenol, whereas phenoloxidase (E.C.1.14.18.1) is capable of oxidizing -o-diphenol to quinone. This difference can be exploited by staining tyrosine hydroxylase activity with a substrate-PMS-NBT method and staining the phenoloxidase with a dopamine-MBTH method. Based on the staining properties of the bands separated after electrophoresis, tyrosine hydroxylase has been differentiated from phenoloxidase in the silkworm Bombyx mori and the Occurrence of tyrosine hydroxylase has been reported for the first time in this worm.