Depigmenting Action of Phenylhydroquinone,an o‐Phenylphenol Metabolite,on the Skin of JY‐4 Black Guinea‐Pigs

The effects of o‐phenylphenol (OPP) and its metabolite, phenylhydroquinone (PHQ) on the skin of JY‐4 black guinea‐pigs were studied. Topical application of 1 or 5% PHQ on the black skin of the back caused marked depigmentation and hypopigmentation of the skin after 5 weeks, whereas OPP applied at the same concentrations had little effect. Depigmented skin had an increased L* (lightness) value in the CIE‐L*a*b* color system. This corresponded with a decreased number of melanocytes and melanosomes in the melanocytes and keratinocytes, the disruption of melanosomes in the melanocytes, and destruction of the membranous organelles of the melanocytes. These morphological and numerical changes in epidermal melanocytes indicate that selective melanocyte toxicity occurred. Furthermore, application of PHQ to the skin of white guinea‐pigs caused skin irritation, as shown by a colorimetric increase in a* value (redness) and by histological observation of inflammation. This study confirmed that OPP, which is a reported depigmenter, has little depigmenting action, while its metabolite, PHQ, is a potent depigmenter preferentially affecting melanocytes.     

Mutational Analysis of the Modulation of Tyrosinase by Tyrosinase‐Related Proteins 1 and 2 In Vitro

The albino (tyrosinase, Tyr^c), brown (tyrosinase‐related protein 1, Tyrp1^b) and slaty (tyrosinase‐related protein 2, tyrp2^slt) loci are all involved in the regulation of melanogenesis. Phenotypes of inbred mice mutant at two or more of these loci are not always explicable by simple summation of the established or suspected catalytic functions of the gene products. These phenotypes suggest that relationships among the proteins extend beyond the obvious fact that they catalyze different steps in the same melanogenic pathway, and that they may also interact intimately in such a way that a mutation in one impacts the function of the other(s). Previous studies have attributed catalytic activities to each member of this trio; however, it has been difficult to study the proteins individually, either in vivo or in tissues or cells. Therefore, we undertook to transfect the genes, in revealing combinations, into COS‐7 cells (which have no melanogenic apparatus of their own) to clarify the interacting functions of their encoded proteins. Specifically, we attempted to evaluate the effects of Tyrp1 and Tyrp2 proteins on tyrosinase protein. We report evidence that Tyrp1 stabilizes tyrosinase, confirming previous observations, and, in addition, demonstrate that Tyrp1 decreases tyrosinase activity. By contrast, Tyrp2 increases tyrosinase activity by stabilizing the protein. We conclude that both Tyrp1 and Tyrp2, in addition to other catalytic functions they may possess, act together to modulate tyrosinase activity.     

Melanosome Capping of Keratinocytes in Pigmented Reconstructed Epidermis – Effect of Ultraviolet Radiation and 3‐Isobutyl‐1‐Methyl‐Xanthine on Melanogenesis

Reconstructed pigmented epidermis was established by co‐seeding autologous melanocytes and keratinocytes onto a dermal substrate and culturing for up to 6 weeks at the air–liquid interface. Inspection of the tissue architecture revealed that melanocytes are regularly interspersed only in the basal layer and transfer melanosomes to the keratinocytes. We report for the first time, the in vitro formation of supranuclear melanin caps above the keratinocyte nuclei. The formation and abundance of these melanin caps could be enhanced by pigment modifiers such as ultraviolet light and 3‐isobutyl‐1‐methyl‐xanthine (IBMX). In untreated cultures, the capping was observed in the spinous layers after 6 weeks of culture, whereas after irradiation or supplementation of the culture medium with IBMX, the capping occurred already in the basal layer 2 weeks after initiation of the stimulus. In this study, we show that IBMX and ultraviolet irradiation stimulate pigmentation via different mechanisms. After supplementation of the culture medium with IBMX the increase in pigmentation was entirely due to the increase in melanocyte activity as observed by increased dendrite formation, melanin production and transport to the keratinocytes and was not due to an increase in melanocyte proliferation. In contrast, after UV irradiation, the increase in pigmentation was also accompanied with an increase in melanocyte proliferation as well as an increase in melanocyte activity. In conclusion, we describe the establishment of pigmented reconstructed epidermis with autologous keratinocytes and melanocytes that can be kept in culture for a period of at least 6 weeks. The complete program of melanogenesis occurs: melanosome synthesis, melanosome transport to keratinocytes, supranuclear capping of keratinocyte nuclei and tanning of the epidermis. This enables sustained application of pigment stimulators over a prolonged period of time and also repeated application of pigment stimulators to be studied.     

Synthesis and Biological Activity of New 4‐tert‐Butylcyclohexanone Derivatives

In the synthesis performed in this study, derivatives of 4‐tert‐butylcyclohexanone 1 were obtained using typical reactions of organic synthesis. The bioactivity of the selected compounds was evaluated. 1‐(Bromomethyl)‐8‐tert‐butyl‐2‐oxaspiro[4.5]decan‐3‐one ( 5 ) was characterized by attractant properties against larvae and a weak feeding deterrent activity against adults of Alphitobius diaperinus Panzer . This bromolactone was a moderate antifeedant towards Myzus persicae Sulzer . In addition, ethyl (4‐tert‐butylcyclohexylidene)acetate ( 2 ) and bromolactone 5 displayed antibacterial activity. The strongest bacteriostatic effect was observed against Gram‐positive strains: Bacillus subtilis and Staphylococcus aureus. The bromolactone 5 also limited the growth of Escherichia coli strain.     

Mechanism of action of 4‐substituted phenols to induce vitiligo and antimelanoma immunity

Monobenzone is a 4‐substituted phenol that can induce vitiligo and antimelanoma immunity. We investigated the influence of the chemical structure on the biological activity of a series of structurally related 4‐substituted phenols. All phenols inhibited cellular melanin synthesis, and eight of ten phenols inhibited tyrosinase activity, using the MBTH assay. These phenols also induced glutathione (GSH) depletion, indicative of quinone formation and protein thiol binding, which can increase the immunogenicity of melanosomal proteins. Specific T‐cell activation was found upon stimulation with phenol‐exposed pigmented cells, which also reacted with unexposed cells. In contrast, 4‐tertbutylphenol induced immune activation was not restricted to pigment cells, analogous to contact sensitization. We conclude that 4‐substituted phenols can induce specific T‐cell responses against melanocytes and melanoma cells, also acting at distant, unexposed body sites, and may confer a risk of chemical vitiligo. Conversely, these phenols may be applicable to induce specific antimelanoma immunity.     

Tyrosinase‐catalyzed oxidation of rhododendrol produces 2‐methylchromane‐6,7‐dione,the putative ultimate toxic metabolite: implications for melanocyte toxicity

RS‐4‐(4‐Hydroxyphenyl)‐2‐butanol (rhododendrol, RD) was used as a skin‐whitening agent until it was reported to induce leukoderma in July 2013. To explore the mechanism underlying its melanocyte toxicity, we characterized the tyrosinase‐catalyzed oxidation of RD using spectrophotometry and HPLC. Oxidation of RD with mushroom tyrosinase rapidly produced RD‐quinone, which was quickly converted to 2‐methylchromane‐6,7‐dione (RD‐cyclic quinone) and RD‐hydroxy‐p‐quinone through cyclization and addition of water molecule, respectively. RD‐quinone and RD‐cyclic quinone were identified as RD‐catechol and RD‐cyclic catechol after NaBH₄ reduction. Autoxidation of RD‐cyclic catechol produced superoxide radical. RD‐quinone and RD‐cyclic quinone quantitatively bound to thiols such as cysteine and GSH. These results suggest that the melanocyte toxicity of RD is caused by its tyrosinase‐catalyzed oxidation through production of RD‐cyclic quinone which depletes cytosolic GSH and then binds to essential cellular proteins through their sulfhydryl groups. The production of ROS through autoxidation of RD‐cyclic catechol may augment the toxicity.     

Synthesis,Biological Evaluation,and Molecular Docking Studies of Novel 4‐[4‐Arylpyridin‐1(4H)‐yl]benzoic Acid Derivatives as Anti‐HIV‐1 Agents

The structural similarities between N1 substituted 1,4‐dihydropyridines and the known gp41 inhibitors, NB ‐2 and NB ‐64 , were considered in the current research for the design of some novel anti‐HIV‐1 agents. A series of novel 4‐[4‐arylpyridin‐1(4H)‐yl]benzoic acid derivatives were synthesized and after a comprehensive structural elucidation were screened for in vitro anti‐HIV‐1 activity. Most of the tested compounds displayed moderate to good inhibitory activity against HIV‐1 growth and were evaluated for in vitro cytotoxic activity using XTT assay at the concentration of 100 μm . Among the tested compounds, 1c , 1d and 1e showed potent anti‐HIV‐1 activity against P24 expression at 100 μm with inhibition percentage of 84.00%, 76.42% and 80.50%, respectively. All the studied compounds possessed no significant cytotoxicity on MT‐2 cell line. The binding modes of these compounds to gp41 binding site were determined through molecular docking study. Docking studies proved 1a as the most potent compound and binding maps exhibited that the activities might be attributed to the electrostatic and hydrophobic interactions and additional H‐bonds with the gp41 binding site. The Lipinski's ‘rule of five’ and drug‐likeness criteria were also calculated for the studied compounds. All derivatives obeyed the Lipinski's ‘rule of five’ and had drug‐like features. The findings of this study suggest that novel 4‐[4‐arylpyridin‐1(4H)‐yl]benzoic acid might be a promising scaffold for the discovery and development of novel anti‐HIV‐1 agents.     

Regulation of the 5‐HT3A Receptor‐Mediated Current by Alkyl 4‐Hydroxybenzoates Isolated from the Seeds of Nelumbo nucifera

Four known alkyl 4‐hydroxybenzoates, i.e., methyl 4‐hydroxybenzoate ( 1 ), ethyl 4‐hydroxybenzoate ( 2 ), propyl 4‐hydroxybenzoate ( 3 ), and butyl 4‐hydroxybenzoate ( 4 ), were isolated from the seeds of Nelumbo nucifera Gaertner (Nymphaeaceae) for the first time. The structures of the isolates were identified by 1D‐ and 2D‐NMR spectroscopy and comparison with published values. The compounds were evaluated for their effects on the 5‐HT‐stimulated inward current (I_5‐HT) mediated by the human 5‐HT₃A receptors expressed in Xenopus oocytes. Compounds 1 and 2 enhanced the I_5‐HT, but 4 reduced it. These results indicate that 4 is an inhibitor of the 5‐HT₃A receptors expressed in Xenopus oocytes.     

Properties of Chlorogenic Acid Quinone: Relationship between Browning and the Formation of Hydrogen Peroxide from a Quinone Solution

Chlorogenic acid is the major polyphenol in foods derived from plants and is a good substrate for polyphenol oxidase. Chlorogenic acid quinone (CQA-Q), which is an oxidative product of chlorogenic acid by polyphenol oxidase, is an important intermediate compound in enzymatic browning. CQA-Q was prepared, and its properties and the relationship with browning were examined. The quinone solution was yellow or orange, and its molecular absorption coefficient was estimated to be 1.7×10³ for 325 nm and 9.7×10² for 400 nm in an acidic aqueous solution. Chlorogenic acid and H₂O₂ were spontaneously generated in the CQA-Q solution as the yellowish color of the solution gradually faded. A pale colored polymer was the major product in the reaction solution. Amino acids such as lysine and arginine added to CQA-Q solution did not repress the fading of the yellowish color of the solution. We concluded from these results that CQA-Q itself and a mixture of CQA-Q and amino acids did not form intensive brown pigments in the acidic aqueous solution. H₂O₂ spontaneously formed in the CQA-Q solution, and other polyphenols might have played an important role in the formation of the brown color by enzymatic browning.     

Anatomy and affinities of a new 535‐million‐year‐old medusozoan from the Kuanchuanpu Formation,South China

We describe here Sinaster petalon gen. et sp. nov., a new embryonic form from the c. 535 million‐year‐old Kuanchuanpu Formation of South China (Ningqiang, Shaanxi Province). The excellent three‐dimensional, phosphatic preservation of these microfossils allowed us to use x‐ray microtomographic techniques to make accurate reconstructions of their internal structures and to compare their anatomy point‐by‐point with that of extant cnidarians and other animal groups. Sinaster petalon has anatomical features typical of extant Medusozoa (Cnidaria), such as coronal muscles, perradial and adradial frenula, interradial septa, accessory septa, gonad‐lamellae, tentacle buds and perradial pockets. Although Sinaster cannot be straightforwardly assigned to any crown‐group within Medusozoa, the presence of marginal lappets and endodermal lamellae suggests that it is closer to Cubozoa and Scyphozoa than to any other group of modern cnidarians. The tentative placement of Sinaster within the stem‐group Cubozoa is justified by the presence of a velarium supported by a frenulum. The cubozoan affinities of Sinaster are also supported by cladistic analysis.     

Synthesis of 5‐tert‐butyl‐1‐(3‐tert‐butyldimethylsiloxy)phenyl‐4,4‐dimethyl‐2,6,7‐trioxabicyclo[3.2.0]heptanes and their fluoride‐induced chemiluminescent decomposition: effect of a phenolic electron donor on the CIEEL decay rate in aprotic polar solvent

Four bicyclic dioxetanes bearing a phenolic substituent, 3‐tert‐butyldimethylsiloxy‐4‐chlorophenyl ( 3a ), 5‐tert‐butyldimethylsiloxy‐4‐chloro‐2‐ethylphenyl ( 3b ), 5‐tert‐butyldimethylsiloxy‐2‐ethylphenyl ( 3c ), and 3‐tert‐butyldimethylsiloxy‐4‐ethylphenyl ( 3d ), were synthesized. All dioxetanes 3a – 3d gave intense blue light on treatment with tetrabutylammonium fluoride (TBAF) in DMSO or acetonitrile. Kinetic study on the fluoride‐induced CIEEL decay of these dioxetanes 3a – 3d and the parent dioxetane 2b revealed that the para‐substitution with chlorine on the phenolic moiety of dioxetane increases free energy of activation (ΔG?), while the para‐substitution with ethyl on the aryl decreases ΔG?. On the other hand, substitution with an ethyl at the ortho‐position instead of the para‐position was found to increase ΔG? and to suppress the CIEEL decay. This fact is attributed to the steric factor of the ortho‐ethyl group which would prevent the aromatic ring from rotating freely around the axis joined to the peroxide ring, and supports the suggestion for a CIEEL‐active dioxetane bearing a phenolic moiety that an intramolecular electron transfer occurs preferentially from the phenolic donor to O–O of the dioxetane ring, when the aromatic ring lies in a certain conformation(s). Copyright © 2002 John Wiley & Sons, Ltd.     

Synthesis of 6‐[4‐(4‐Propoxyphenyl)piperazin‐1‐yl]‐9H‐purine Derivatives as Antimycobacterial and Antifungal Agents: In Vitro Evaluation and In Silico Study

A series of novel alkyl substituted purines were synthesized. 6‐[4‐(4‐Propoxyphenyl)piperazin‐1‐yl]‐9H‐purine was used as the key starting material, which was synthesized via a multistep protocol and finally subjected for N‐alkylation with various alkyl halides with an aim to get prospective antimicrobial agents. The structures of the novel compounds were established by substantiating them through spectral techniques like ¹H‐NMR, ¹³C‐NMR, FT‐IR and EI‐MS. They were explored for antitubercular activity against Mycobacterium tuberculosis H37RV. Furthermore, they were checked for their antimicrobial activity concerning bacterial and fungal strains. The title compounds exhibited considerable antimicrobial activity without any significant toxicity. In silico studies depicted their good binding profile against Mycobacterium tuberculosis enoyl reductase (InhA; PDB ID: 4TZK) and Candida albicans dihydrofolate reductase (PDB ID: 1AI9). The title compounds obeyed Lipinski's parameters and have exhibited good drug‐like properties.     

Si‐Doping Mediated Phase Control from β‐ to γ‐Form Li3VO4 toward Smoothing Li Insertion/Extraction

The γ phase Li₃VO₄ which possesses higher ionic conductivity is more preferable for lithium ion batteries, but it is only stable at high temperature and would convert to low temperature β phase spontaneously when cooling down. Here, the phase control of Li₃VO₄ to stabilize its γ phase in room temperature is successfully mediated by introducing proper Si‐doping, and for the first time the electrochemical performances of γ‐Li₃VO₄ is investigated. It is found that pure γ‐Li₃VO₄ can be obtained in a doping ratio of x = 0.05–0.15 in Li_3+xV_1?xSi_xO₄ with addition of excess Li source in synthesis design. The doping mechanism and the energy changes are investigated in detail by using the first principle calculations, it reveals that an interstitial Li⁺ is formed with doping of Si⁴⁺ in Li₃VO₄ to balance the system charge. When served as an anode, the Si‐doped γ‐Li₃VO₄ shows much smoothed Li⁺ insertion/extraction and enhanced cycle stability with only a single pair of redox peaks, which behaves much different with the complex multicouples of redox peaks in typical β‐Li₃VO₄. These changes in electrochemical performances implies that γ‐Li₃VO₄ can effectively accommodate Li⁺ in an easier and more facile way and relieved structure stress during the charge/discharge process.     

Design,Synthesis, and Biological Evaluation of 2‐(2‐Bromo‐3‐nitrophenyl)‐5‐phenyl‐1,3,4‐oxadiazole Derivatives as Possible Anti‐Breast Cancer Agents

Breast Cancer (BCa) is the most often diagnosed cancer among women who were in the late 1940’s. Breast cancer growth is largely dependent on the expression of estrogen and progesterone receptor. Breast cancer cells may have one, both, or none of these receptors. The treatment for breast cancer may involve surgery, hormonal therapy (Tamoxifen, an aromatase inhibitor, etc.) and oral chemotherapeutic drugs. The molecular docking technique reported the findings on the potential binding modes of the 2‐(2‐bromo‐3‐nitrophenyl)‐5‐phenyl‐1,3,4‐oxadiazole derivatives with the estrogen receptor (PDB ID: 3ERT). The 1,3,4‐oxadiazole derivatives 4a – 4j have been synthesized and described by spectroscopic method. 2‐(2‐Bromo‐6‐nitrophenyl)‐5‐(4‐bromophenyl)‐1,3,4‐oxadiazole ( 4c ) was reconfirmed by single‐crystal XRD. All the compounds have been tested in combination with generic Imatinib pharmaceutical drug against breast cancer cell lines isolated from Caucasian woman MCF‐7, MDA‐MB‐453 and MCF‐10A non‐cancer cell lines. The compounds with the methoxy (in 4c ) and methyl (in 4j ) substitution were shown to have significant cytotoxicity, with 4c showing dose‐dependent activation and decreased cell viability. The mechanism of action was reported by induced apoptosis and tested by a DNA enzyme inhibitor experiment (ELISA) for Methyl Transferase. Molecular dynamics simulations were made for hit molecule 4c to study the stability and interaction of the protein?ligand complex. The toxicity properties of ADME were calculated for all the compounds. All these results provide essential information for further clinical trials.     

Topoisomerase I‐Mediated Antiproliferative Activity of 10‐Substituted and 12‐Substituted Homocamptothecins

Homocamptothecin (hCPT) is an E‐ring modified camptothecin (CPT) analogue, which showed pronounced inhibitory activity of topoisomerase I. In search of novel hCPT‐type anticancer agents, two series of hCPT derivatives were synthesized and evaluated in vitro against three human tumor cell lines. The results indicated that the 10‐substituted hCPT derivatives had a considerably higher cytotoxic activity than the 12‐substituted ones. Among the 10‐substituted compounds, 8a, 8b, 9b , and 9i showed an equivalent or even more potent activity than the positive control drug topotecan against the lung cancer cell line A‐549. Moreover, the hCPT analogues 8a and 8b exhibited a higher topoisomerase I inhibitory activity than CPT at a concentration of 100 μM .     

Large‐scale synthesis of tert‐butyl (3R,5S)‐6‐chloro‐3,5‐dihydroxyhexanoate by a stereoselective carbonyl reductase with high substrate concentration and product yield

To biosynthesize the (3R,5S)‐CDHH in an industrial scale, a newly synthesized stereoselective short chain carbonyl reductase (SCR) was successfully cloned and expressed in Escherichia coli. The fermentation of recombinant E. coli harboring SCR was carried out in 500 L and 5000 L fermenters, with biomass and specific activity of 9.7 g DCW/L, 15749.95 U/g DCW, and 10.97 g DCW/L, 19210.12 U/g DCW, respectively. The recombinant SCR was successfully applied for efficient production of (3R,5S)‐CDHH. The scale‐up synthesis of (3R,5S)‐CDHH was performed in 5000 L bioreactor with 400 g/L of (S)‐CHOH at 30°C, resulting in a space‐time yield of 13.7 mM/h/g DCW, which was the highest ever reported. After isolation and purification, the yield and d.e. of (3R,5S)‐CDHH reached 97.5% and 99.5%, respectively. © 2017 American Institute of Chemical Engineers Biotechnol. Prog., 33:612–620, 2017     

Screening of Human Primary Melanocytes of Defined Melanocortin‐1 Receptor Genotype: Pigmentation Marker,Ultrastructural and UV‐Survival Studies

Recent population studies have demonstrated an association with the red‐hair and fair‐skin phenotype with variant alleles of the melanocortin‐1 receptor (MC1R) which result in amino acid substitutions within the coding region leading to an altered receptor activity. In particular, Arg151Cys, Arg160Trp and Asp294His were the most commonly associated variants seen in the south‐east Queensland population with at least one of these alleles found in 93% of those with red hair. In order to study the individual effects of these variants on melanocyte biology and melanocytic pigmentation, we established a series of human melanocyte strains genotyped for the MC1R receptor which included wild‐type consensus, variant heterozygotes, compound heterozygotes and homozygotes for Arg151Cys, Arg160Trp, Val60Leu and Val92Met alleles. These strains ranged from darkly pigmented to amelanotic, with all strains of consensus sequence having dark pigmentation. UV sensitivity was found not to be associated with either MC1R genotype or the level of pigmentation with a range of sensitivities seen across all genotypes. Ultrastructural analysis demonstrated that while consensus strains contained stage IV melanosomes in their terminal dendrites, Arg151Cys and Arg160Trp homozygote strains contained only stage II melanosomes. This was despite being able to show expression of tyrosinase and tyrosinase‐related protein‐1 markers, although at reduced levels and an ability to convert exogenous 3,4‐dihydroxyphenyl‐alanine (DOPA) to melanin in these strains.