Homologous Overexpression of Tyrosinase in Trichoderma reesei and Its Application in Glycinin Cross-Linking.

Tyrosinase is capable of oxidizing tyrosine residues in proteins, leading to intermolecular protein cross-linking, which could modify the protein network of food and improve the texture of food. To obtain the recombinant tyrosinase with microbial cell factory instead of isolation tyrosinase from the mushroom Agaricus bisporus, a TYR expression cassette was constructed in this study. The expression cassette was electroporated into Trichoderma reesei Rut-C30 and integrated into its genome, resulting in a recombinant strain C30-TYR. After induction with microcrystalline cellulose for 7 days, recombinant tyrosinase could be successfully expressed and secreted by C30-TYR, corresponding to approximately 2.16 g/L tyrosinase in shake-flask cultures. The recombinant TYR was purified by ammonium sulfate precipitation and gel filtration, and the biological activity of purified TYR was 45.6 U/mL. The purified TYR could catalyze the cross-linking of glycinin, and the emulsion stability index of TYR-treated glycinin emulsion was increased by 30.6% compared with the untreated one. The cross-linking of soy glycinin by TYR resulted in altered properties of oil-in-water emulsions compared to emulsions stabilized by native glycinin. Therefore, cross-linking with this recombinant tyrosinase is a feasible approach to improve the properties of protein-stabilized emulsions and gels.

Cost-Effective Production of L-DOPA by Tyrosinase-Immobilized Polyhydroxyalkanoate Nanogranules in Engineered Halomonas bluephagenesis TD01.

3,4-dihydroxyphenyl-L-alanine (L-DOPA) is a preferred drug for Parkinson's disease, with an increasing demand worldwide that mainly relies on costly and environmentally problematic chemical synthesis. Yet, biological L-DOPA production is unfeasible at the industrial scale due to its low L-DOPA yield and high production cost. In this study, low-cost Halomonas bluephagenesis TD01 was engineered to produce tyrosinase TyrVs-immobilized polyhydroxyalkanoate (PHA) nanogranules in vivo, with the improved PHA content and increased immobilization efficiency of TyrVs accounting for 6.85% on the surface of PHA. A higher L-DOPA-forming monophenolase activity of 518.87 U/g PHA granules and an L-DOPA concentration of 974.36 mg/L in 3 h catalysis were achieved, compared to those of E. coli. Together with the result of L-DOPA production directly by cell lysates containing PHA-TyrVs nanogranules, our study demonstrated the robust and cost-effective production of L-DOPA by H. bluephagenesis, further contributing to its low-cost industrial production based on next-generation industrial biotechnology (NGIB).

Molecular and Biochemical Basis of Minocycline-Induced Hyperpigmentation-The Study on Normal Human Melanocytes Exposed to UVA and UVB Radiation.

Minocycline is a drug which induces skin hyperpigmentation. Its frequency reaches up to 50% of treated patients. The adverse effect diminishes the great therapeutic potential of minocycline, including antibacterial, neuroprotective, anti-inflammatory and anti-cancer actions. It is supposed that an elevated melanin level and drug accumulation in melanin-containing cells are related to skin hyperpigmentation. This study aimed to evaluate molecular and biochemical mechanism of minocycline-induced hyperpigmentation in human normal melanocytes, as well as the contribution of UV radiation to this side effect. The experiments involved the evaluation of cyto- and phototoxic potential of the drug using cell imaging with light and confocal microscopes as well as biochemical and molecular analysis of melanogenesis. We showed that minocycline induced melanin synthesis in epidermal melanocytes. The action was intensified by UV irradiation, especially with the UVB spectrum. Minocycline stimulated the expression of microphthalmia-associated transcription factor (MITF) and tyrosinase (TYR) gene. Higher levels of melanin and increased activity of tyrosinase were also observed in treated cells. Moreover, minocycline triggered the supranuclear accumulation of tyrosinase, similar to UV radiation. The decreased level of premelanosome protein PMEL17 observed in all minocycline-treated cultures suggests disorder of the formation, maturation or distribution of melanosomes. The study revealed that minocycline itself was able to enhance melanin synthesis. The action was intensified by irradiation, especially with the UVB spectrum. Demonstrated results confirmed the potential role of melanin and UV radiation minocycline-induced skin hyperpigmentation.

UP256 Inhibits Hyperpigmentation by Tyrosinase Expression/Dendrite Formation via Rho-Dependent Signaling and by Primary Cilium Formation in Melanocytes.

Skin hyperpigmentation is generally characterized by increased synthesis and deposition of melanin in the skin. UP256, containing bakuchiol, is a well-known medication for acne vulgaris. Acne sometimes leaves dark spots on the skin, and we hypothesized that UP256 may be effective against hyperpigmentation-associated diseases. UP256 was treated for anti-melanogenesis and melanocyte dendrite formation in cultured normal human epidermal melanocytes as well as in the reconstituted skin and zebrafish models. Western blot analysis and glutathione S-transferase (GST)-pull down assays were used to evaluate the expression and interaction of enzymes related in melanin synthesis and transportation. The cellular tyrosinase activity and melanin content assay revealed that UP256 decreased melanin synthesis by regulating the expression of proteins related on melanogenesis including tyrosinase, TRP-1 and -2, and SOX9. UP256 also decreased dendrite formation in melanocytes via regulating the Rac/Cdc42/α-PAK signaling proteins, without cytotoxic effects. UP256 also inhibited ciliogenesis-dependent melanogenesis in normal human epidermal melanocytes. Furthermore, UP256 suppressed melanin contents in the zebrafish and the 3D human skin tissue model. All things taken together, UP256 inhibits melanin synthesis, dendrite formation, and primary cilium formation leading to the inhibition of melanogenesis.

Pigmentation formation and expression analysis of tyrosinase in Siniperca chuatsi.

Animal pigmentation primarily depends on the presence and mixing ratio of chromatophores, functioning in animal survival and communication. For the benthic and carnivorous Siniperca chuatsi, pigmentation pattern is key to concealment and predation. In this study, the formation, distribution, and main pattern of chromatophores were observed in the embryos, larvae, skins, and visceral tissues from S. chuatsi. Melanophores were firstly visualized in the yolk sac at segmentation stage, and then they were migrated to the whole body and further clustered into the black stripes, bands, and patches. In adult S. chuatsi, the head, black band, and body side skins mainly contained melanophores, showing as deep or light black. The abdomen skin mainly contained iridophores, showing as silvery. In the eye, the pigment layers were located in the epithelial layers of iris and retina and shown as black. Then, the pigmentation-related gene, tyrosinase gene from S. chuatsi (Sc-tyr) was analyzed by bioinformatics and quantitative methods. The Sc-tyr gene encoded a protein with 540 amino acids (Sc-TYR). The Sc-TYR contained two copper ion binding sites, which were coordinated by six conserved histidines (H182, H205, H214, H366, H370, H393) and necessary for catalytic activity. The Sc-TYR was well conserved compared with TYR of various species with higher degree of sequence similarity with other fishes (77.6-98.3%). The qRT-PCR test showed that the Sc-tyr mRNA reached the peak value at segmentation stage in the embryo development, the black skins displayed a higher expression level than that in silvery skin, and the eye had the highest expression level compared with other tissues. Further research on enzyme activity showed that the expression patterns of tyrosinase activity were similar to that of the Sc-tyr mRNA. Comparing with the results of molecular and phenotype, it was found that the temporal and spatial distributions of tyrosinase corresponded well with changes in pigmentation patterns and the intensity of skin melanization. This study initially explored the pigmentation formation and tyrosinase expression, which served as a foundation for further insight into the genetics mechanism of body color formation in S. chuatsi.

Effect of sulfur mustard on melanogenesis in vitro.

The chemical warfare agent sulfur mustard (SM) affects all cells in the epidermis including melanocytes which are responsible for melanin synthesis. After exposure to SM, pigment abnormalities like hypo- and hyperpigmentation can occur. The underlying molecular pathomechanisms of SM exposure on human melanogenesis have not been elucidated so far. In our study, we investigated the effect of SM on human melanocytes and melanogenesis. Normal human epidermal melanocytes (NHEM) were used as in vitro model and they were exposed to different concentrations of SM (4.5 µM-100 µM). Melanin production was analyzed by absorption measurements at 405 nm. In addition, quantitative real-time PCR (qPCR) and Western blot experiments were performed to determine the expression of essential melanogenesis-related proteins including tyrosinase (TYR), tyrosinase-related protein (TRP) 1 and 2 and microphthalmia transcription factor (MITF). Our findings demonstrated that exposure to low SM concentrations increased melanin synthesis accompanied with an increase in protein expression. In contrast, high SM concentrations led to decreased melanin content and a downregulation in expression of all investigated melanogenesis-associated proteins. We concluded that low SM concentrations may cause hyperpigmentation while high SM concentrations decreased melanin content which may explain hypopigmented skin areas in SM exposed patients.

Rhododenol Activates Melanocytes and Induces Morphological Alteration at Sub-Cytotoxic Levels.

Rhododenol (RD), a whitening cosmetic ingredient, was withdrawn from the market due to RD-induced leukoderma (RIL). While many attempts have been made to clarify the mechanism underlying RIL, RIL has not been fully understood yet. Indeed, affected subjects showed uneven skin pigmentation, but the features are different from vitiligo, a skin hypopigmentary disorder, alluding to events more complex than simple melanocyte cytotoxicity. Here, we discovered that rhododenol treatment reduced the number of melanocytes in a pigmented 3D human skin model, Melanoderm™, confirming the melanocyte toxicity of RD. Of note, melanocytes that survived in the RD treated tissues exhibited altered morphology, such as extended dendrites and increased cell sizes. Consistently with this, sub-cytotoxic level of RD increased cell size and elongated dendrites in B16 melanoma cells. Morphological changes of B16 cells were further confirmed in the immunocytochemistry of treated cells for actin and tubulin. Even more provoking, RD up-regulated the expression of tyrosinase and TRP1 in the survived B16 cells. Evaluation of mRNA expression of cytoskeletal proteins suggests that RD altered the cytoskeletal dynamic favoring cell size expansion and melanosome maturation. Collectively, these results suggest that RD not only induces cytotoxicity in melanocytes but also can lead to a profound perturbation of melanocyte integrity even at sub-cytotoxic levels.

The effect of antioxidant and whitening action on Plantago asiatica L. leaf ethanol extract for health care.

BACKGROUND: The Plantago asiatica L. is easy to cultivate and has been used as a folk remedy since ancient times because of various pharmacological actions such as anti-inflammation and antioxidation. It also contains a variety of flavonoids such as aucubin, which is thought to be excellent for whitening, antioxidant and anti-inflammatory action. OBJECTIVE: We investigated the effect of P. asiatica L. leaf ethanol extracts containing various active ingredients on antioxidative, anti-inflammation and whitening action and investigated its potential as a health care material. P. asiatica L. has been widely used in folk remedies. RESULTS: The cell toxicity test using RAW264.7 cells showed a high cell survival rate of over 75%, thus demonstrating the safety of the sample. In order to study the antioxidant activity of P. asiatica L. leaf ethanol extracts, we studied a sample which showed radical scavenging activity in a dose-dependent manner. To observe the antioxidant activity at the cell level, RAW 264.7 cells were used and inhibition of ROS production was measured. The ROS production was suppressed in a dose-dependent manner and the scavenging activity was stronger than the sample's own radical scavenging ability. To observe the anti-inflammatory effect of P. asiatica L. leaf ethanol extracts, inhibition of NO generation was observed using LPS-induced RAW 264.7 cells. NO generation was inhibited in a dose-dependent manner and was strongly inhibited by 31% at 100 µg/mL. In vitro, L-DOPA and L-tyrosine were used to inhibit tyrosinase action in a dose-dependent manner. The concentration of melanin at 1, 10, and 100 µg/mL was suppressed in B16 F10 melanin cells supplemented with α-MSH in the cells, and the inhibition was suppressed to 29% at 100 µg/mL. In the B16 F10 melanin cell stimulated with MSH, the P. asiatica L. leaf ethanol extracts inhibited melanin formation in a dose-dependent manner. CONCLUSION: P. asiatica L. leaf ethanol extracts are expected to be developed as whitening cosmeceutical ingredients and as health care ingredients with antioxidant and anti-inflammatory properties.

Tyrosinase and nestin immunohistochemical expression in melanocytic nevi as a histopathologic pattern to trace melanocyte differentiation and nevogenesis.

While histological analysis represents a powerful tool for the classification of melanocytic lesions as benign or malignant, a clear-cut distinction between a nevus and a melanoma is sometimes a challenging step of the diagnostic process. The immunohistochemical detection of tyrosinase, cardinal melanogenic enzyme during melanocytic maturation, has often been helpful in formulating a differential diagnosis due to the peculiar staining pattern in nevocytes compared with melanoma cells. Tyrosinase distribution in nevi appears to overlap with the cytoarchitectural changes observable within these lesions, that result in epidermal or superficial dermal nevocytes being larger and strongly expressing melanocytic differentiation antigens, such as tyrosinase, compared with deeper dermal nevus cells. Our study aimed to evaluate the immunohistochemical expression pattern of tyrosinase in different histological types of acquired dysplastic melanocytic nevi, including junctional, compound, and intradermal nevi. Moreover, to estimate whether in nevocytes the expression of tyrosinase was associated with their differentiation state, we investigated the expression of two recognized markers of pluripotency, CD34 and nestin. In all examined nevi, our analysis revealed a remarkable immunoreactivity for tyrosinase in junctional and superficial dermal nevocytes and a decreasing gradient of staining in dermal nevocytes, up to become negative in deeper dermis. Meanwhile, junctional and dermal nevocytes were lacking in CD34 protein. Furthermore, nestin immunostaining showed an opposite distribution compared with tyrosinase, leading us to look into the tyrosinase/nestin expression pattern in melanocytic nevus as a tool to better understand the final stages of differentiation of melanocyte precursors toward their ultimate anatomical site into the epidermis.

Streptomyces swartbergensis sp. nov., a novel tyrosinase and antibiotic producing actinobacterium.

As part of an antibiotic screening program, an actinobacterium, strain HMC13T, was isolated from soil collected from the banks of the Gamka River, Western Cape Province, South Africa. The isolate was found to produce branched mycelia that differentiated into spiral spore chains with spiny spores. 16S rRNA gene sequence analysis showed the strain to be closely related to Streptomyces caelestis NRRL 2418T (99.72%) and Streptomyces azureus NBRC 12744T (99.51%). Chemotaxonomic analyses confirmed the classification of the strain as a member of the genus Streptomyces: LL-DAP in the peptidoglycan, no diagnostic sugars in the whole cell sugar pattern, dominant menaquinones including MK9(H8), MK9(H6), and the polar lipids detected included phosphatidylethanolamine. The fatty acid profile revealed the presence of mostly branched, saturated fatty acids: iso-C15:0 (14.4%), anteiso-C15:0 (21.1%), iso-C16:0 (16.8%), C16:1ω7c/2-OH iso-C15:0 (5.8%), C16:0 (6.2%), iso-C17:1ω9c (5.8%), iso-C17:0 (5.9%), and anteiso-C17:0 (9.6%). Strain HMC13T is a tyrosinase producer and exhibits very strong antibiosis against Mycobacterium aurum A+ and Staphylococcus aureus subsp. aureus ATCC 33591 (methicillin resistant), while only weak activity was observed against Bacillus cereus ATCC 10876, Enterococcus faecium VanA (vancomycin resistant), Enterococcus faecalis ATCC 51299 (vancomycin resistant) and Candida tropicalis ATCC 750T. Strain HMC13T (= LMG 28849T = NRRL B-65294T) is proposed as the type strain of a new species, to be named Streptomyces swartbergensis sp. nov.

Purification of Recombinant Human Tyrosinase from Insect Larvae Infected with the Baculovirus Vector.

The purification of an enzyme from insect larvae infected with a baculovirus vector is described. The enzyme tyrosinase is of biomedical importance and catalyzes the first rate-limiting steps in melanin production. Tyrosinase mutations can result in oculocutaneous albinism type 1 (OCA1), an inherited eye disease associated with decreased melanin pigment production and vision defects. To simplify expression and subsequent purification, the extracellular domain is expressed in insect cells, produced in Trichoplusia ni larvae, and purified using affinity and size-exclusion chromatography. The purified recombinant human tyrosinase is a soluble monomeric glycoprotein with an activity that mirrors the tyrosinase in vivo function. © 2017 by John Wiley & Sons, Inc.

Peroxisome proliferator-activated receptor α (PPARα) contributes to control of melanogenesis in B16 F10 melanoma cells.

Recent studies revealed the cooperation between peroxisome proliferator-activated receptor gamma (PPARγ) and α-MSH signaling, which results in enhanced melanogenesis in melanocytes and melanoma cells. However, the agonists of PPARα, such as fenofibrate, exert depigmenting effect. Therefore, we aimed to check how the PPARα expression level affects the antimelanogenic activity of fenofibrate and whether PPARα modulates melanogenesis independently of its agonist. To answer these questions, we used three B16 F10-derived cell lines, which varied in the PPARα expression level and were developed by stable transfection with plasmids driving shRNA-based PPARα silencing or overexpression of PPARα-emerald GFP fusion protein. Melanin contents were assessed with electron paramagnetic resonance spectroscopy along with color component image analysis-a novel approach to pigment content characteristics in melanoma cells. B16 F10 wt and Ctrl shRNA lines showed intermediate pigmentation, whereas the pigmentation of the B16 F10-derived cell lines was inversely correlated with the PPARα expression level. We observed that cells overexpressing PPARα were almost amelanotic and cells with reduced PPARα protein level were heavily melanized. Furthermore, fenofibrate down-regulated the melanogenic apparatus (MITF, tyrosinase, and tyrosinase-related proteins) in the cells with the regular PPARα expression level resulting in their visibly lower total melanin content in all the cell lines. From these observations, we conclude that fenofibrate works as a strong depigmenting agent, which acts independently of PPARα, but in an additive fashion. Our results also indicate that alterations in PGC-1a acetylation and expression level might contribute to the regulation of melanogenesis by PPARα and fenofibrate.

Aberrant tyrosinase expression in an atypical fibroxanthoma: A case report.

Atypical fibroxanthoma (AFX) is a histologic mimicker of a variety of spindle cell neoplasms, and careful microscopic and immunohistochemical evaluation is critical in establishing the correct diagnosis. Here we report the histologic and immunohistochemical work up of a 1 cm nodule involving the left dorsal hand of a 66-year-old patient. Light microscopy revealed fascicles of spindled and pleomorphic cells within the dermis showing increased mitotic activity occurring in the background of sun-damaged skin. There were numerous multinucleated cells with hyperchromatic nuclei and ample finely vacuolated or foamy cytoplasms. There was strong and diffuse CD10 and patchy CD68 expression among the spindled cells and multinucleated cells. The neoplastic cells did not show immunoreactivity against S100, p75-NGFR, HMB-45 or a panel of keratinocytic, vascular and smooth muscle markers. Tyrosinase and Melan-A were not expressed within the spindle cell component of this neoplasm; however, there was tyrosinase expression among numerous multinucleated giant cells. Melan-A expression was also observed among rare multinucleated giant cells. Tyrosinase expression has not previously been reported in AFX.

Synthesis and bioactivity of novel isoxazole chalcone derivatives on tyrosinase and melanin synthesis in murine B16 cells for the treatment of vitiligo.

A new series of chalcone derivatives 1-18, bearing isoxazole moieties were designed and synthesized, and biologically evaluated for their activity on mushroom tyrosinase and melanin synthesis in murine B16 cells. The result indicated that most of prepared compounds 1-18 showed potent activating effect on tyrosinase, especially for 1-2, 4, 6-7, 9 and 15. Among them, compounds 2, 4 and 9 demonstrated the best activity with EC50=1.3, 2.5 and 3.0µmol·L-1 respectively, much better than the positive control 8-methoxypsoralan (8-MOP, EC50=14.8µmol·L-1); In B16 cells, all the tested compounds exhibited a stronger activity on melanogenesis than 8-MOP (with the value of 115%). It was interesting that derivatives substituted with halogen (1, 2, 4, 5, 7, 9) were generally more potent. Compounds 2 (463%) and 18 (438%) with 3 and 4-fold potency compared with 8-MOP respectively, were recognized as the most promising candidate hits for further pharmacological study of anti-vitiligo.

Transcriptome expression profiling of fur color formation in domestic rabbits using Solexa sequencing.

Fur color is an important, genetically determined characteristic of domestic rabbits, and rabbit furs are of great economic value. To investigate the molecular genetics associated with fur color determination in domestic rabbits, we used Solexa-sequencing technology to probe gene expression in dorsal skin tissues sampled from full-sibling Rex rabbits of different colors. The number of expressed genes in each sample was approximately 14,700. Among the top 30 genes and transcription factors with the highest reads per kilobase per million values, the elongation factor-alpha 1 gene was highly expressed in all samples, as were genes of the ribosomal protein and keratin gene families. Compared with the chinchilla (C) Rex rabbit control sample, the numbers of genes in the black (B) and white (W) rabbit samples were 1809 and 460, respectively, and the number of common differentially expressed genes was 257. Clustering analysis of these 257 genes revealed that 32 were up-regulated in sample B and down-regulated in sample W. Of these 32 genes, we identified some that are related to fur formation, including Tyrosinase-related protein 1 (TYRP1) and Tyrosinase (TYR), as well as genes with unknown functions. Quantitative real-time polymerase chain reaction was used to verify the expression patterns of those genes. The findings are expected to provide reference for the further study of fur color formation in rabbits.

Biological characteristics of mouse skin melanocytes.

The objective of this research was to evaluate the optimal passage number according to the biological characteristics of mouse skin melanocytes from different passages. Skin punch biopsies harvested from the dorsal region of 2-day old mice were used to establish melanocyte cultures. The cells from passage 4, 7, 10 and 13 were collected and evaluated for their melanogenic activity. Histochemical staining for tyrosinase (TYR) activity and immunostaining for the melanocyte specific markers including S-100 antigen, TYR, tyrosinase related protein 1 (TYRP1), tyrosinase related protein 2 (TYRP2) and micropthalmia associated transcription factor (MITF) confirmed purity and melanogenic capacity of melanocytes from different passages, with better melanogenic activity of passage 10 and 13 cells being observed. Treatment of passage 13 melanocytes with α-melanocyte stimulating hormone (α-MSH) showed increased expression of MITF, TYR and TYRP2 mRNA. However, considering the TYR mRNA dramatically high expression which is the characteristics of melanoma cells, melanocytes from passage 10 was the optimal passage number for the further research. Our results demonstrate culture of pure populations of mouse melanocytes to at least 10 passages and illustrate the potential utility of passage 10 cells for studies of intrinsic and extrinsic regulation of genes controlling pigmentation and coat color in mouse.

Hyperpigmentation mechanism of methyl 3,5-di-caffeoylquinate through activation of p38 and MITF induction of tyrosinase.

Methyl 3,5-di-caffeoylquinate (3,5-diCQM) has been used for the treatment of various diseases in oriental medicine, but its effect on melanogenesis has not been reported yet. In this study, the molecular mechanism of 3,5-diCQM-induced melanogenesis was investigated. It was found that 3,5-diCQM induced synthesis of melanin pigments in murine B16F10 melanoma cells in a concentration-dependent manner. Treatment of cells with 3,5-diCQM for 48 h increased extracellular and intracellular melanin production and tyrosinase activity. The expressions of tyrosinase, tyrosinase-related protein 1 (TRP1), and TRP2 were up-regulated in a dose-dependent manner 48 h after 3,5-diCQM treatment. Western blot analysis showed that 3,5-diCQM increased the phosphorylation of p38 mitogen-activated protein kinase and cAMP responsive element binding as well as the expression of microphthalmia-associated transcription factor. In addition, 3,5-diCQM-stimulated cAMP production, and 3,5-diCQM-induced tyrosinase activity and melanin synthesis were attenuated by H89, a protein kinase A inhibitor. These results suggested that 3,5-diCQM-mediated activation of the p38 pathway may represent a novel approach for an effective therapy for vitiligo and hair graying.

The natural yeast extract isolated by ethanol precipitation inhibits melanin synthesis by modulating tyrosinase activity and downregulating melanosome transfer.

This study was conducted to examine the effects of EP-2, a natural yeast extract isolated by ethanol precipitation from Saccharomyces cerevisiae, on melanogenesis and to determine its underlying mechanism of action. Our results show that although EP-2 is not a direct tyrosinase inhibitor, when EP-2 was added to the culture media of B16F10 melanoma cells, intracellular tyrosinase activity was decreased. However, EP-2 had no effect on the expression of microphthalmia-associated transcription factor or tyrosinase. EP-2 was found to inhibit melanogenesis and melanosome transfer when it was added to melanocytes and keratinocytes in coculture. In addition, protease-activated receptor 2, a key protein associated with melanosome transfer from melanocytes to keratinocytes, was downregulated in the presence of EP-2. In conclusion, EP-2 is a potent inhibitor of melanogenesis and its hypomelanogenic effect is related to the inhibition of tyrosinase activity and transfer of melanosomes.

Molecular characterization of a peptidoglycan recognition protein from the cotton bollworm, Helicoverpa armigera and its role in the prophenoloxidase activation pathway.

Peptidoglycan recognition proteins (PGRPs), which are evolutionarily conserved from invertebrates to vertebrates, function as pattern-recognition and effector molecules in innate immunity. In this study, a PGRP (HaPGRP-A) from the cotton bollworm, Helicoverpa armigera was identified and characterized. Sequence analysis indicated that HaPGRP-A is not an amidase-type PGRP. Increased levels of HaPGRP-A mRNA were observed in the fat body and hemocytes of H. armigera larvae following the injection of microbes or Sephadex beads. Analysis using purified recombinant HaPGRP-A showed that it (i) could bind and agglutinate Gram-negative Escherichia coli and Gram-positive Staphylococcus aureus, (ii) enhanced prophenoloxidase activation in the presence of microbes, (iii) promoted the formation of melanotic nodules in vivo, and (iv) enhanced the melanization of Sephadex beads in vivo. RNA interference assays were performed to further confirm the function of HaPGRP-A. When the expression of HaPGRP-A in H. armigera larvae was inhibited by dsHaPGRP-A injection, the phenoloxidase activity in larval hemolymph was significantly decreased and RNAi-treated insects infected with bacteria showed higher bacterial growth in hemolymph compared with infected control larvae. These results indicated that HaPGRP-A acts as a pattern recognition receptor and binds to the invading organism to trigger the prophenoloxidase activation pathway of H. armigera, and the activated phenoloxidase may participate in the melanization process of nodulation and encapsulation responses.