Two types of metabolic regulation of phenol oxidase activity in ontogenesis of house fly

Changes of the tyrosinase activity in ontogenesis of the house fly Musca domestica were shown to be phase-specific and ontogenetic changes of tyrosinase and dihydroxyphenylalanine oxidase activities proved to be coordinated. Ascorbic acid stimulated some ontogenetic stages of the house fly and physiological indices, such as fertility, survival at different stages, and weight of puparia. Also, ascorbic acid modulated the tyrosinase activity.     

Stabilization of mineralized and sclerotized puparial cuticle of muscid flies

Calcium, magnesium and phosphorus are the major mineral elements in puparial exuviae of the face fly, Musca autumnalis, house fly, M. domestica and stable fly, Stomoxys calcitrans, but they are 20–50 times more prevalent in face fly than in the other two species that sclerotize the puparium. Carbon and nitrogen are approx. 5 times more abundant in house fly puparia than in face fly puparia. Face fly puparia contain two and three-fold less total amino acids than the house fly and stable fly, respectively. β-Alanine is a major amino acid in puparial cuticle of the house fly and stable fly, but it is absent in the face fly. There is no significant difference in glucosamine (chitin) content between the three species. Dopamine is the major catechol detected in face fly puparial cuticle while N-β-alanyldopamine (NBAD) is 10 to 15 times more prevalent than other catechols such as dopamine, N-acetyldopamine (NADA), 3,4-dihydroxyphenylalanine (DOPA) and 3,4-dihydroxyphenylacetic acid (DOPAC) in house fly and stable fly puparial cuticles. The latter two species have 75 to nearly 200 times higher levels of extractable catechols than the face fly. At the onset of pupariation, dopamine and NBAD attain nearly equivalent titres in puparial cuticles of face fly and house fly, respectively. Dopamine subsequently decreases more than 40-fold in the face fly as the cuticle becomes stabilized, while NBAD continues to accumulate in the house fly. The house fly covalently incorporates about 150 times more catechols in the puparium than does the face fly. The force required to fracture house fly and stable fly puparia is about three-fold greater than that required to fracture face fly puparia of comparable thickness. However, the face fly puparium attains a strength comparable to those of house fly and stable fly puparia by significantly increasing its thickness. These results demonstrate that dipterans use both catecholamines and minerals for stabilization of puparial cuticle with the house fly and stable fly relying primarily on sclerotization and the face fly on mineralization.     

Effect of p-aminophenols on tyrosinase activity

Tyrosinase is involved in the synthesis of melanin in the skin and hair as well as neuromelanin in the brain. This rate limiting enzyme catalyzes two critical steps (reactions) in melanogenesis; the hydroxylation of tyrosine to form DOPA and the subsequent oxidation of DOPA into dopaquinone. Several new aminophenol derivatives have been synthesized based on structure–activity relationship studies of N-(4-hydroxyphenyl)retinamide (1), a derivative of retinoic acid. In order to find new tyrosinase inhibitors, we investigated the effects of these p-aminophenols, including p-decylaminophenol (3), on the activity of mushroom tyrosinase. Compound 3 was the most potent agent, showing significant inhibition as compared with control. The inhibitory effects of 3 on tyrosinase activities were greater than seen with kojic acid, a well-known potent inhibitor of tyrosinase activity, which also causes adverse effects, including rash and dermatitis. A Lineweaver–Burk kinetic analysis of inhibition showed that 3 suppresses tyrosinase activity in a non-competitive fashion for both substrates, tyrosine and DOPA. These results suggest that 3 might be a useful alternative to kojic acid as a tyrosinase inhibitor.     

Characterization of NADPH-cytochrome P-450 reductase from house flies (Musca domestica,L.) susceptible and resistant to insecticides,and the blow fly (Phormia regina,meigen)

NADPH-cytochrome $\text{c}$ reductase was purified by affinity chromatographic techniques from microsomes prepared from the abdomens of insecticide-resistant (R) and -susceptible (S) house flies Musca domestica and of the black blow fly Phormia regina. Data are presented which describe (1) the ability of the purified enzymes to support an in vitro reconstitution of mono-oxygenase activity, (2) the changes in activity of these preparations observed in buffers of varying ionic strength, (3) comparative kinetic behaviour between microsomal and purified forms of the enzymes, (4) the immunochemical characteristics of these preparations, and (5) their amino acid composition. The reductases from the three sources were found to be very similar in all of these tests. Substrate binding constants were 5 μM for NADPH, 12 μM for cytochrome $\text{c}$, and the catalytic mechanism was interpreted as ordered Bi Bi. The inhibitory constant of the reductase from the resistant fly for 2′-AMP, an analogue of NADP⁺, was 187 μM; whereas no assciation of the inhibitor was observed below concentrations of 400 μM for the enzyme of either the susceptible house fly or the blow fly. However, the data are insufficient to suggest that the reductase is a significant factor in insecticide resistance. Compared to the same enzymes from rat and rabbit liver, the insect reductases show a different ionic strength optimum (0.14), have distinct antigenic determinants, and have different levels of acidic and basic amino acids in the membrane-binding peptide.     

Design,synthesis and anti-melanogenic effect of cinnamamide derivatives

Pigmentation disorders are attributed to excessive melanin which can be produced by tyrosinase. Therefore, tyrosinase is supposed to be a vital target for the treatment of disorders associated with overpigmentation. Based on our previous findings that an (E)-β-phenyl-α,β-unsaturated carbonyl scaffold can play a key role in the inhibition of tyrosinase activity, and the fact that cinnamic acid is a safe natural substance with a scaffolded structure, it was speculated that appropriate cinnamic acid derivatives may exhibit potent tyrosinase inhibitory activity. Thus, ten cinnamamides were designed, and synthesized by using a Horner-Emmons olefination as the key step. Cinnamamides 4 (93.72% inhibition), 9 (78.97% inhibition), and 10 (59.09% inhibition) with either a 2,4-dihydroxyphenyl, or 4-hydroxy-3-methoxyphenyl substituent showed much higher mushroom tyrosinase inhibition at 25?µM than kojic acid (18.81% inhibition), used as a positive control. Especially, the two cinnamamides 4 and 9 having a 2,4-dihydroxyphenyl group showed the strongest inhibition. Docking simulation with tyrosinase revealed that these three cinnamamides, 4, 9, and 10, bind to the active site of tyrosinase more strongly than kojic acid. Cell-based experiments carried out using B16F10 murine skin melanoma cells demonstrated that all three cinnamamides effectively inhibited cellular tyrosinase activity and melanin production in the cells without cytotoxicity. There was a close correlation between cellular tyrosinase activity and melanin content, indicating that the inhibitory effect of the three cinnamamides on melanin production is mainly attributed to their capability for cellular tyrosinase inhibition. These results imply that cinnamamides having the (E)-β-phenyl-α,β-unsaturated carbonyl scaffolds are promising candidates for skin-lighting agents.     

Tyrosinase inhibition and anti-melanin generation effect of cinnamamide analogues

Abnormal melanogenesis results in excessive production of melanin, leading to pigmentation disorders. As a key and rate-limiting enzyme for melanogenesis, tyrosinase has been considered an important target for developing therapeutic agents of pigment disorders. Despite having an (E)-β-phenyl-α,β-unsaturated carbonyl scaffold, which plays an important role in the potent inhibition of tyrosinase activity, cinnamic acids have not attracted attention as potential tyrosinase inhibitors, due to their low tyrosinase inhibitory activity and relatively high hydrophilicity. Given that cinnamic acids’ structure intrinsically features this (E)-scaffold and following our experience that minute changes in the chemical structure can powerfully affect tyrosinase activity, twenty less hydrophilic cinnamamide derivatives were designed as potential tyrosinase inhibitors and synthesised using a Horner-Wadsworth-Emmons reaction. Four of these cinnmamides (4, 9, 14, and 19) exhibited much stronger mushroom tyrosinase inhibition (over 90% inhibition) at 25 µM compared to kojic acid (20.57% inhibition); crucially, all four have a 2,4-dihydroxy group on the β-phenyl ring of the scaffold. A docking simulation using tyrosinase indicated that the four cinnamamides exceeded the binding affinity of kojic acid, and bound more strongly to the active site of tyrosinase. Based on the strength of their tyrosinase inhibition, these four cinnamamides were further evaluated in B16F10 melanoma cells. All four cinnamamides, without cytotoxicity, exhibited higher tyrosinase inhibitory activity (67.33 – 79.67% inhibition) at 25 μM than kojic acid (38.11% inhibition), with the following increasing inhibitory order: morpholino (9) = cyclopentylamino (14) < cyclohexylamino (19) < N-methylpiperazino (4) cinnamamides. Analysis of tyrosinase activity and melanin content in B16F10 cells showed that the four cinnamamides dose-dependently inhibited both cellular tyrosinase activity and melanin content and that their inhibitory activity at 25 μM was much better than that of kojic acid. The results of melanin content analysis well matched those of the cellular tyrosinase activity analysis, indicating that tyrosinase inhibition by the four cinnamamides is a major factor in the reduction of melanin production. These results imply that these four cinnamamides with a 2,4-dihydroxyphenyl group can act as excellent anti-melanogenic agents in the treatment of pigmentation disorders.     

Activation mechanism of melB tyrosinase from Aspergillus oryzae by acidic treatment

The pro form of recombinant tyrosinase from Aspergillus oryzae (melB) shows no catalytic activity, but acid treatment (around pH 3.5) of protyrosinase activates it to induce tyrosinase activity. Circular dichroism spectra, gel filtration analysis, and colorimetric assay have indicated that acid treatment around pH 3.5 induced the disruption of the conformation of the C-terminal domain covering the enzyme active site. These structural changes induced by the acid treatment may open the entrance to the enzyme active site for substrate incorporation. To compare the mechanism of hydroxylation by the acid-treated tyrosinase with that by trypsin-treated tyrosinase, a detailed steady-state kinetic analysis of the phenolase activity was performed by monitoring the O₂-consumption rate using a Clark-type oxygen electrode. The results clearly show that the phenolase activity (phenol hydroxylation) of the activated tyrosinase involves an electrophilic aromatic substitution mechanism as in the case of mushroom tyrosinase (Yamazaki and Itoh in J. Am. Chem. Soc. 125:13034–13035, 2003) and activated hemocyanin with urea (Morioka et al. in J. Am. Chem. Soc. 128:6788–6789, 2006).     

Transcriptional analysis of tyrosinase gene expression during Bufo bufo development

Tyrosinase (EC.1.14.18.1.) is a widespread enzyme, in the phylogenetic scale, that produces melanin, from bacteria to man, by using as substrates monophenols, o-diphenols and molecular oxygen. In this work we have confirmed and demonstrated that during Bufo bufo development tyrosinase activity and gene expression first occur at developmental stages 17–18 (tail bud-muscular response) as detected by a spectrophotometric assay and qRT-PCR. As expected, also during B. bufo development tyrosinase gene is expressed after the late gastrula (stage 12), differently from Rana pipiens development when tyrosinase mRNA appears at the neural plate stage and enzyme activity at stage 20 (gill circulation). We have cloned and sequenced the B. bufo tyrosinase cDNA in order to prepare B. bufo tyrosinase cDNA specific primers (forward and reverse). Tyrosinase mRNA cloning has been performed by using degenerate primers prepared according to the anuran tyrosinase gene sequence coding for the copper binding sites. The expressions of tyrosinase gene and enzymatic activity during B. bufo development support that until the developmental stage 17, embryo melanin is of maternal origin and at this stage can start embryo melanin synthesis. A correlation exists between tyrosinase expression and O₂ consumption during B. bufo development.     

Juvenile hormone activity of substituted aryl 3,7-dimethyl-6-octenyl ethers in the stable fly and house fly

Tests of substituted aryl 3,7-dimethyl-6-octenyl ethers and their epoxidized analogues produced a juvenile hormone (JH) mimic that was active against both the stable fly, Stomoxys calcitrans, and the house fly, Musca domestica. The criterion of activity in the JH bioassays was the formation of pupal-adult intermediates within the puparia.     

Design, synthesis and biological evaluation of 2-(substituted phenyl)thiazolidine-4-carboxylic acid derivatives as novel tyrosinase inhibitors

Herein we describe the design, synthesis and biological activities of 2-(substituted phenyl)thiazolidine-4-carboxylic acid derivatives as novel tyrosinase inhibitors. The target compounds 2a–2j were designed and synthesized from the structural characteristics of N-phenylthiourea, tyrosinase inhibitor and tyrosine, and l-DOPA, the natural substrates of tyrosinase. Among them, (2R/S,4R)-2-(2,4-dimethoxyphenyl)thiazolidine-4-carboxylic acid (2g) caused the greatest inhibition 66.47% at 20 μM of l-DOPA oxidase activity of mushroom tyrosinase. Kinetic analysis of tyrosinase inhibition revealed that 2g is a competitive inhibitor. We predicted the tertiary structure of tyrosinase, and simulated the docking of mushroom tyrosinase with 2g. These results suggest that the binding affinity of 2g with tyrosinase is high. Also, 2g effectively inhibited tyrosinase activity and reduced melanin levels in B16 cells treated with α-MSH. These data strongly suggest that 2g can suppress the production of melanin via the inhibition of tyrosinase activity.     

Larvicidal activity of endectocides against pest flies in the dung of treated cattle

Cattle were treated with topical formulations of endectocides to assess the larvicidal activity of faecal residues against horn fly, Haematobia irritans (L.), house fly, Musca domestica L., and stable fly, Stomoxys calcitrans (L.) (Diptera: Muscidae). In laboratory bioassays, doramectin, eprinomectin and ivermectin suppressed horn fly in dung of cattle treated at least 4 weeks previously and suppressed house fly and stable fly in dung of cattle treated 1-5 weeks previously. Moxidectin suppressed horn fly in dung from cattle treated no more than one week previously and did not suppress house fly and stable fly. Results combined for the three species across two experiments suggested that, ranked in descending order of larvicidal activity, doramectin > ivermectin approximately = eprinomectin > moxidectin.     

Plants from Brazilian Cerrado with Potent Tyrosinase Inhibitory Activity

The increased amount of melanin leads to skin disorders such as age spots, freckles, melasma and malignant melanoma. Tyrosinase is known to be the key enzyme in melanin production. Plants and their extracts are inexpensive and rich resources of active compounds that can be utilized to inhibit tyrosinase as well as can be used for the treatment of dermatological disorders associated with melanin hyperpigmentation. Using in vitro tyrosinase inhibitory activity assay, extracts from 13 plant species from Brazilian Cerrado were evaluated. The results showed that Pouteria torta and Eugenia dysenterica extracts presented potent in vitro tyrosinase inhibition compared to positive control kojic acid. Ethanol extract of Eugenia dysenterica leaves showed significant (p<0.05) tyrosinase inhibitory activity exhibiting the IC₅₀ value of 11.88 µg/mL, compared to kojic acid (IC₅₀ value of 13.14 µg/mL). Pouteria torta aqueous extract leaves also showed significant inhibitory activity with IC₅₀ value of 30.01 µg/mL. These results indicate that Pouteria torta and Eugenia dysenterica extracts and their isolated constituents are promising agents for skin-whitening or antimelanogenesis formulations.     

Antioxidant,anti-tyrosinase and anti-melanogenic effects of (E)-2,3-diphenylacrylic acid derivatives

During our continued search for strong skin whitening agents over the past ten years, we have investigated the efficacies of many tyrosinase inhibitors containing a common (E)-β-phenyl-α,β-unsaturated carbonyl scaffold, which we found to be essential for the effective inhibition of mushroom and mammalian tyrosinases. In this study, we explored the tyrosinase inhibitory effects of 2,3-diphenylacrylic acid (2,3-DPA) derivatives, which also possess the (E)-β-phenyl-α,β-unsaturated carbonyl motif. We synthesized fourteen (E)-2,3-DPA derivatives 1a–1n and one (Z)-2,3-DPA-derivative 1l′ using a Perkin reaction with phenylacetic acid and appropriate substituted benzaldehydes. In our mushroom tyrosinase assay, 1c showed higher tyrosinase inhibitory activity (76.43 ± 3.53%, IC₅₀ = 20.04 ± 1.91 µM) with than the other 2,3-DPA derivatives or kojic acid (21.56 ± 2.93%, IC₅₀ = 30.64 ± 1.27 μM). Our mushroom tyrosinase inhibitory results were supported by our docking study, which showed compound 1c (−7.2 kcal/mole) exhibited stronger binding affinity for mushroom tyrosinase than kojic acid (−5.7 kcal/mole). In B16F10 melanoma cells (a murine cell-line), 1c showed no cytotoxic effect up to a concentration of 25 μM and exhibited greater tyrosinase inhibitory activity (68.83%) than kojic acid (49.39%). In these cells, arbutin (a well-known tyrosinase inhibitor used as the positive control) only inhibited tyrosinase by 42.67% even at a concentration of 400 μM. Furthermore, at 25 µM, 1c reduced melanin contents in B16F10 melanoma cells by 24.3% more than kojic acid (62.77% vs. 38.52%). These results indicate 1c is a promising candidate treatment for pigmentation-related diseases and potential skin whitening agents.     

An insecticidal diacetylene from artemisia monosperma

The essential oil of Artemisia monosperma obtained by steam distillation of the aerial parts of the plant was shown to have insecticidal activity against house fly, cotton leaf worm and the rice weevil. The chemical structure of the active ingredient from the steam distillate was shown to be 3-methyl, 3-phenyl-1,4-pentadiyne.     

Expression of cytochrome P-450lpr Is developmentally regulated and limited to house fly

Expression of house fly cytochrome P-450_lpr was examined using immunoblotting in male and female adult LPR house flies, mixed sex adult house flies at 12 different ages, larvae, and pupae. P-450_lpr was expressed in both male and female adult house flies. P-450_lpr was clearly present in all adult stages examined, was barely detectable in pupae, and could not be detected in larvae. Thus, cytochrome P-450_lpr is developmentally regulated and present in both sexes of house fly. Expression of cytochrome P-450, immunologically homologous to house fly cytochrome P-450_lpr was examined in other species using immunoblot analysis. Eleven animal species were tested in the orders Diptera, Hymenoptera, Lepidoptera, Orthoptera, Acari, and Rodentia, using microsomes in some species from both induced and noninduced animals or insecticide-resistant and susceptible strains. P-450_lpr appears to be restricted to house flies, as none of these species contained cytochrome P-450 that reacted with antiserum to cytochrome P-450_lpr.     

LIFE IN ZERO MAGNETIC FIELD. IV. INVESTIGATION OF DEVELOPMENTAL EFFECTS ON FRUITFLY VISION

The fruitfly (Drosophila melanogaster) visual system was investigated electrophysiologically in vivo after exposure to a zero magnetic field (ZMF). Electroretinographic (ERG) recording of fly eye electrical activity was performed on adult insects raised from pupae maintained for 20 hr in zero magnetic field. A flickering excitation regime was applied to excite the visual system, since in this way, a quasistable hyperpolarization component of the electroretinogram can be obtained, containing information from the neural cells, which are the most sensitive to the action of external factors during early ontogenetic stages. Results of the investigation of two D. melanogaster populations, sample and control, were statistically compared.

We found a significant statistical increase of sensitivity in neural cells from the first optic ganglion in the fly population developed from pupae exposed to ZMF.     

Synthesis,characterization and tyrosinase inhibitory properties of benzimidazole derivatives

1-Alkylbenzimidazole and 1,3-dialkyl benzimidazolium salts were synthesized and characterized by the data of IR, ¹H NMR, ¹³C NMR spectra and elemental analyses. These compounds were investigated as tyrosinase inhibitors. Tyrosinase has been purified from banana by affinity chromatography on a Sepharose 4B gel conjugated with L-tyrosine-p-aminobenzoic acid. All the synthesized compounds inhibited the tyrosinase activity. Among the compounds studied, 1,4-di(1H-benzo[d]imidazol-1-yl)butane was found to be the most active tyrosinase inhibitor (IC₅₀ 0.31 mM).     

Metabolic profiling, free-radical scavenging and tyrosinase inhibitory activities of Lemna minor whole plants cultivated in various concentrations of proline and sucrose

Metabolic profiling of Lemna minor whole plants cultivated in proline and sucrose at various concentrations was performed using gas chromatography-mass spectrometry coupled with multivariate statistical analysis. In total, 46 compounds including alkaloids, amino acids, fatty acids, organic acids, phenolics, phytosterols, purines, and sugars were assigned, and the relative levels of these metabolites were compared in the L. minor whole plant samples. Free-radical scavenging and tyrosinase inhibitory activities were also investigated in L. minor whole plants cultivated in proline and sucrose at various concentrations. The whole plants cultivated in 3% sucrose and 0.5 mM proline for 42 days exhibited the highest antioxidative and tyrosinase inhibitory activities, and biomass accumulation. Total phenolic content and cinnamic acid content were also highest under those conditions, which might have contributed to the free-radical scavenging and tyrosinase inhibitory activities. There were strong correlations between free-radical scavenging activity and total phenolic content, and between tyrosinase inhibition activity and cinnamic acid content in L. minor whole plants cultivated under various conditions.     

Impact of the Darkling Beetle Alphitobius diaperinus (Panzer) on Establishment of the Predaceous Beetle Carcinops pumilio (Erichson) for Musca domestica Control in Caged-Layer Poultry Houses

Understanding the insect natural history in a caged-layer poultry house is essential to developing Integrated Pest Management strategies. In this study we observed the interaction of three insects commonly found in poultry manure: a filth fly predator, Carcinops pumilio (Erichson) (Histeridae), and two poultry pests, the house fly, Musca domestica L. (Muscidae), and the darkling beetle, Alphitobius diaperinus (Panzer) (Tenebrionidae). Manure samples were collected weekly and the insects were extracted using Berlese–Tullgren funnels. Collected insects were identified to species and life stage. When C. pumilio populations equaled or exceeded those of the larval house fly, subsequent adult house fly populations were not considered pestiferous. C. pumilio adult and larval cohorts varied significantly among poultry houses. Few C. pumilio larvae were found in houses with abundant darkling beetle populations, suggesting a negative impact on the establishment of C. pumilio. Laboratory studies confirmed that larval darkling beetles significantly reduce the survival of C. pumilio eggs and larvae. Adult darkling beetles did not reduce C. pumilio egg or larval survival.     

Inhibitory effects of N-(acryloyl)benzamide derivatives on tyrosinase and melanogenesis

Targeting of tyrosinase has proven to be the best means of identifying safe, efficacious, and potent tyrosinase inhibitors for whitening skin. We designed and synthesized ten NAB (N-(acryloyl)benzamide) derivatives (1a–1j) using the Horner-Wadsworth-Emmons olefination of diethyl (2-benzamido-2-oxoethyl)phosphonate and appropriate benzaldehydes. A mushroom tyrosinase inhibitory assay showed compounds 1a (36.71 ± 2.14% inhibition) and 1j (25.99 ± 2.77% inhibition) inhibited tyrosinase more than the other eight NAB derivatives and kojic acid (21.56 ± 2.93% inhibition), and docking studies indicated 1a (−6.9 kcal/mole) and 1j (−7.5 kcal/mole) had stronger binding affinities for tyrosinase than kojic acid (−5.7 kcal/mole). At a concentration of 25 μM, 1a and 1j were nontoxic in B16F10 melanoma cells and exhibited stronger tyrosinase inhibition (59.70% and 76.77%, respectively) than kojic acid (50.30% inhibition) or arbutin (41.78% inhibition at 400 μM). Similarly, in B16F10 melanoma cells, compounds 1a and 1j at 25 μM decreased total melanin content by 47.97% and 61.77%, respectively (kojic acid; 38.98%). Similarities between inhibitions of tyrosinase activity and melanin contents suggested the anti-melanogenic effects of 1a and 1j were due to tyrosinase inhibition. The excellent DPPH scavenging activity of 1j suggests it might enhance in vivo effect on melanin contents. The study suggests compound 1j offers a potential starting point for the development of safe, potent tyrosinase inhibitors.