Melanogenesis in oocytes of wild-type and mutant albino axolotls

Melanogenesis during oogenesis in the wild-type and albino (a/a) axolotl was compared. Tyrosine-dopa oxidase activity, melanin accumulation, and melanosome development were correlated and the effect of the a gene on these biochemical and morphological events was examined. Studies of wild-type oocytes at the electron and light microscope level revealed that premelanosomes first appear in stage 2 oocytes. Mature melanosomes are present in stage 3 oocytes and steadily increase in number, reaching a maximum level in stage 6 oocytes. Melanosomes were detected in the albino. No obvious structural abnormalities were observed in these organelles, although they fail to accumulate melanin. Tyrosine-dopa oxidase (TDO) activity assayed radiometrically is at a very low level in stages 1 and 2 oocytes, reaches a maximum level in stage 3 oocytes, and declines to zero activity in stage 6 oocytes. In contrast to the finding with albino skin homogenates (Harsa-King, 1978), TDO activity was detected in albino oocytes. This activity never declined from its maximal stage 3 level. The addition of an inhibitor of proteolytic enzymes, phenylmethyl sulfonyl fluoride (PMSF), to the oocyte homogenization buffer completely blocks TDO activity in albino samples and reduces it somewhat in wild-type samples. It is suggested that TDO activity eliminated by PMSF represents TDO existing in an inactive form in vivo which is activated by proteolytic enzymes released upon homogenization. These results suggest that TDO is found only in an inactive state in albinos, a conclusion in agreement with the earlier work on albino skin melanocytes (Harsa-King, 1978). There is an inverse relationship between TDO activity and melanization in the wild type. The greater the amount of melanin deposited within the premelanosomes, the less enzyme activity is present. It is suggested, as it has been by others, that as melanin is synthesized within the confines of the oocyte melanosome, the active sites of the enzyme are covered up, resulting in its inactivation. The findings with the albino mutant support this hypothesis. No melanin deposition occurs in the albino, and TDO activity in PMSF-untreated samples does not decline from its maximal stage 3 level.     

L-Dopa and the Albino Riddle: Content of L-Dopa in the Developing Retina of Pigmented and Albino Mice

Background

The absence or deficiency of melanin as in albinos, has detrimental effects on retinal development that include aberrant axonal projections from eye to brain and impaired vision. In pigmented retinal pigment epithelium (RPE), dihydroxyphenalanine (L-Dopa), an intermediate in the synthetic path for melanin, has been hypothesized to regulate the tempo of neurogenesis. The time course of expression of retinal L-Dopa, whether it is harbored exclusively in the RPE, the extent of deficiency in albinos compared to isogenic controls, and whether L-Dopa can be restored if exogenously delivered to the albino have been unknown.

Methodology/Principal Findings

L-Dopa and catecholamines including dopamine extracted from retinas of pigmented (C57BL/6J) and congenic albino (C57BL/6J-tyr^c2j) mice, were measured throughout development beginning at E10.5 and at maturity. L-Dopa, but not dopamine nor any other catecholamine, appears in pigmented retina as soon as tyrosinase is expressed in RPE at E10.5. In pigmented retina, L-Dopa content increases throughout pre- and postnatal development until the end of the first postnatal month after which it declines sharply. This time course reflects the onset and completion of retinal development. L-Dopa is absent from embryonic albino retina and is greatly reduced in postnatal albino retina compared to pigmented retina. Dopamine is undetectable in both albino and pigmented retinas until after the postnatal expression of the neuronal enzyme tyrosine hydroxylase. If provided to pregnant albino mothers, L-Dopa accumulates in the RPE of the fetuses.

Conclusions

L-Dopa in pigmented RPE is most abundant during development after which content declines. This L-Dopa is not converted to dopamine. L-Dopa is absent or at low levels in albino retina and can be restored to the RPE by administration in utero. These findings further implicate L-Dopa as a factor in the RPE that could influence development, and demonstrate that administration of L-Dopa could be a means to rescue developmental abnormalities characteristic of albinos.     

Somatic mosaicism and allele complexity induced by CRISPR/Cas9 RNA injections in mouse zygotes

Tyrosinase is the rate-limiting enzyme for the production of melanin pigmentation. In the mouse and other animals, homozygous null mutations in the Tyrosinase gene (Tyr) result in the absence of pigmentation, i.e. albinism. Here we used the CRISPR/Cas9 system to generate mono- and bi-allelic null mutations in the Tyr locus by zygote injection of two single-guide and Cas9 RNAs. Injection into C57BL/6N wild-type embryos resulted in one completely albino founder carrying two different Tyr mutations. In addition, three pigmentation mosaics and fully pigmented littermates were obtained that transmitted new mutant Tyr alleles to progeny in test crosses with albinos. Injection into Tyr heterozygous (B6CBAF1/J×FVB/NJ) zygotes resulted in the generation of numerous albinos and also mice with a graded range of albino mosaicism. Deep sequencing revealed that the majority of the albinos and the mosaics had more than two new mutant alleles. These visual phenotypes and molecular genotypes highlight the somatic mosaicism and allele complexity in founders that occurs for targeted genes during CRISPR/Cas9-mediated mutagenesis by zygote injection in mice.     

Tyrosinase activity in the larva of the fleshfly, Sarcophaga barbarta

When plasma from third instar larvae of the fleshfly, Sarcophaga barbarta, was diluted tenfold with distilled water, lipoproteins precipitated out. After centrifuging, the water supernatant was rendered 30, 50, and 65% to ammonium sulphate, and it was found that the 50% fraction contained 95% of the tyrosinase activity in all the fractions, the enzyme being present in its inactive form or proenzyme. The proenzyme was activated by mixing it with activator isolated from the larval cuticle. After addition of activator there followed a lag period before the rapid phase of activation, the duration of the lag being dependent upon the concentration of both proenzyme and activator. The final activity attained was dependent upon the concentration of proenzyme but was independent of the activator concentration.The level of proenzyme in the plasma rose steadily throughout the third larval instar reaching a maximum in 7 day larvae, formation of the puparium commencing about 24 hr later, the rounded-off white stage (r.o.). At the r.o. and golden-brown stage (1 hr later) the level was still maximal, but 12 hr later at the dark-brown puparial stage no proenzyme was isolatable from the plasma, all the enzyme at this stage behaving as active enzyme.The vast majority (95%) of the proenzyme isolated from plasma in the larval stages and at the r.o. white stage was present in the 50% ammonium sulphate fraction, whereas 1 hr later at the golden-brown stage only 33% of the proenzyme was found in the 50% fraction, 62% now being found in the 65% fraction. At the dark-brown puparial stage 12 hr later, not only was there a further redistribution, but all the enzyme behaved as active enzyme. It is suggested that these changes in the distribution and behaviour of the proenzyme indicate that, in vivo, activation of the enzyme in the blood has taken place over the period r.o. white to the golden-brown to dark-brown puparial stage.     

Two cytosolic,Ca2+-dependent,neutral proteinases from rabbit liver: Purification and properties of the proenzymes

Two Ca²⁺-requiring proteinases have been purified from rabbit liver cytosol and shown to be present in isolated hepatocytes. They differ in relative molecular mass, with the major and minor forms, M_r = 150,000 and M_r = 200, 000, accounting for 75 and 18% of the total cytosolic neutral proteinase activity, respectively. Both are recovered as inactive proenzymes that can be converted to the active, low-Ca²⁺-requiring proteinases by incubation with Ca²⁺ and substrate [S. Pontremoli, E. Melloni, F. Salamino, B. Sparatore, M. Michetti, and B. L. Horecker (1984) Proc. Natl. Acad. Sci. USA81, 53–56. Each proenzyme is composed of two subunits, with molecular masses of 80 and 100 kDa, respectively. Activation of the proenzymes was found to correlate with their dissociation into subunits. The optimum pH for conversion of the proenzymes to the active proteinases in the presence of 5 mm Ca²⁺ and 2 mg/ml of denatured globin was approximately 7.5, and the same pH optimum was observed for the digestion of denatured globin by the activated proteinases. Following activation, each proteinase was observed to undergo autolytic inactivation at rates that were dependent on the concentration of both Ca²⁺ and the digestible substrate. A model is proposed for the activation of the proenzymes and the subsequent inactivation of the active proteinases.     

Albino midrib 1, encoding a putative potassium efflux antiporter,affects chloroplast development and drought tolerance in rice

Key message

Mutation of the AM1 gene causes an albino midrib phenotype and enhances tolerance to drought in rice

Abstract

K⁺ efflux antiporter (KEA) genes encode putative potassium efflux antiporters that are mainly located in plastid-containing organisms, ranging from lower green algae to higher flowering plants. However, little genetic evidence has been provided on the functions of KEA in chloroplast development. In this study, we isolated a rice mutant, albino midrib 1 (am1), with green- and white-variegation in the first few leaves, and albino midrib phenotype in older tissues. We found that AM1 encoded a putative KEA in chloroplast. AM1 was highly expressed in leaves, while lowly in roots. Chloroplast gene expression and proteins accumulation were affected during chlorophyll biosynthesis and photosynthesis in am1 mutants. Interestingly, AM1 was induced by salt and PEG, and am1 showed enhanced sensitivity to salinity in seed germination and increased tolerance to drought. Taken together, we concluded that KEAs were involved in chloroplast development and played important roles in drought tolerance.     

Tyrosinase activity of Greyia flanaganii (Bolus) constituents

Hyper-pigmentation of the skin is a common problem that is prevalent in middle aged and elderly people. It is caused by over production of melanin. Tyrosinase is known to be the key enzyme in melanin production. Ethanolic extract of Greyia flanaganii leaves showed significant (P < 0.05) antityrosinase activity exhibiting the IC₅₀ of 32.62 μg/ml. The total extract was further investigated for its toxicity and effect on melanin production by melanocytes cells, and showed significant inhibition (P < 0.05) (20%) of melanin production at 6.25 μg/ml and low levels of cytotoxicity (IC₅₀ < 400 μg/ml). The amount of antioxidants necessary to decrease the initial DPPH absorbance by 50% (EC₅₀) by the total ethanolic extract was found to be 22.01 μg/ml. The effect of G. flanaganii against acne causing bacteria, Propionibacterium acnes, was investigated using microdilution assay. The MIC of the extract of G. flanaganii was found to be 250 μg/ml. Bioassay-guided fractionation led to the isolation of (3S)-4-hydroxyphenethyl 3-hydroxy-5-phenylpentanoate (1), 2′,4′,6′-trihydroxydihydrochalcone (2), 2′,6′,4-trihydroxy-4′-methoxydihydrochalcone (3), 2′,6′-dihydroxy-4′-methoxydihydrochalcone (4), 5,7-dihydroxyflavanone [(2S)-pinocembrin] (5), 2′,6′-dihydroxy-4′,4-dimethoxy dihydrochalcone (6) and (2R,3R)-3,5,7-trihydroxy-3-O-acetylflavanone (7). The isolated compounds were tested for their antioxidant, cytotoxicity, tyrosinase inhibition and antibacterial activities. Compound 2 exhibited significant (P < 0.05) antityrosinase activity exhibiting the IC₅₀ of 69.15 μM. The isolated compounds showed low toxicity of the cells with reduction of melanin content of the cells. All compounds tested showed good radical scavenging activity. These data indicates that G. flanaganii extract and its isolated phenolic constituents could be possible skin lightening agents.     

Coloration affects heating and cooling in three color morphs of the Australian bluetongue lizard,Tiliqua scincoides

The color-mediated thermoregulation hypothesis predicts that dark body color (low reflectance) allows organisms to gain heat more efficiently than does pale coloration (high reflectance). This prediction is intuitive and widely assumed to be true, but has poor empirical support. We used rare, captive-bred, mutant melanistic, albino and wild-type Australian bluetongue lizards, Tiliqua scincoides to measure the effects of skin reflectance on the heating and cooling rates. We measured heating under an artificial radiant heat source and cooling rates in an ice-cooled box using live lizards in a room with still air. The effect of skin reflectance on heat transfer was clear, despite the substantial influence of body size. Melanistic T. scincoides showed low reflectance and gained heat faster than highly reflective albinos. Melanistic lizards also lost heat faster than albinos. Wild-type lizards were intermediate in reflectance, gained heat at rates indistinguishable from melanistic lizards, and lost heat at rates indistinguishable from albino lizards. This study system allowed us to control for variables that were confounded in other studies and may explain the inconsistent support for the color-mediated thermoregulation hypothesis. Our results provide clear evidence that skin reflectance influences the rate of heating and cooling in ectotherms.     

Bovine adrenocortical self-phosphorylating protein kinase,SPK 380: Purification and characterization

Recently, we described the partial purification and characterization of a novel adrenocortical cyclic nucleotide-independent protein kinase, PK 380, that catalyzes the phosphorylation of an endogenous peptide (120,000 daltons) and a serine residue(s) of the α subunit (38,000 daltons) of the eucaryotic initiation factor eIF-2 (Y. Kuroda, W. C. Merrick, and R. K. Sharma, 1982, Arch. Biochem. Biophys.213, 271–275). In the present communication we describe the purification to apparent homogeneity and characterization of this protein kinase (SPK 380). As shown by sucrose density sedimentation, the native enzyme has a molecular weight of 356,000. The protein is composed of three identical subunits of M_r 120,000. Polyacrylamide-gel isoelectric focusing electrophoresis revealed a single peak with pI 4.5. SPK 380 self-phosphorylated a histidine residue(s) of its 120,000-dalton peptide. This reaction utilized the terminal phosphate of ATP; GTP was inactive. Divalent cations (5 mm Mn²⁺ or 10 mm Mg²⁺) were essential for optimum activity. Thiol reagents (N-ethylmaleimide, p-chloromercuriphenylsulfonic acid) inhibited the kinase, indicating a sulfhydryl-group requirement for enzyme activity.     

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.     

The effect of nectar guides on pollinator preference: experimental studies with a montane herb

Summary Rare albino morphs of the montane larkspur Delphinium nelsonii differ from common blue-flowered morphs in overall flower color, and in the strength of a contrasting color pattern at the center of the flower that presumably guides pollinators to concealed nectar. Previous studies showed that bumblebees and hummingbirds discriminate against albinos when presented with mixtures of the 2 morphs, and that it takes these pollinators longer to fly between successive flowers on albino than on blue-flowered inflorescences. To explore the link between these observations, we measured pollinator preferences and flower-to-flower flight times (handling times) before and after painting flowers in 2 alternative ways that enhanced albino nectar guides. In all of 16 experimental replicates discrimination against albinos was reduced or eliminated after painting, and albino handling times declined toward values for blue-flowered inflorescences. This consistent result indicates that an inferior nectar guide increases the energetic cost of foraging at albinos. Increased cost in turn explains discrimination, under the reasonable assumption that hummingbirds and bumblebees are sensitive to foraging economics.     

Ommochrome Deficiency in an Albino Strain of a Terrestrial Isopod,Armadillidium vulgare

In order to clarify the cause of ommochrome deficiency in an albino strain of the terrestrial isopod, Armadillidium vulgare, levels of xanthom-matin, 3-hydroxykynurenine, 3-hydroxyanthranilic acid and tryptophan in whole body extracts of the albino and the wild type individuals were determined together with enzyme activities of kynurenine-3-hydroxylase, kynureninase and tryptophan-2,3-dioxygenase. Xanthommatin could not be detected in the albinos. The levels of 3-hydroxykynurenine and 3-hydroxyanthranilic acid were determined by high-performance liquid chro-matography (HPLC) with electrochemical detection and were markedly low in the albinos compared with the wild type individuals. In contrast to those, the tryptophan levels determined by HPLC with fluorescence detection did not differ significantly between the two phenotypes. In the albino A. vulgare, kynurenine-3-hydroxylase activity was lower and kynureninase activity was higher than in the wild type, although the differences were not statistically significant. Tryptophan-2,3-dioxygenase activity in the albinos was less than 10% that in the wild type. Thus, ommochrome deficiency in the albino A. vulgare is considered to be caused by the extremely low activity of tryptophan-2,3-dioxygenase.     

The movement of the prospective eye vesicles from the neural plate into the neural fold in Ambystoma mexicanum and Xenopus laevis

Excision experiments performed on amphibian neurulae by H. Spemann (1901, Verh. Anat. Ges.15, 61–79) and W. H. Lewis (1907, Amer. J. Anat.7, 259–276) have localized the eye primordia in the anterior neural plate. This was confirmed by the results of the classical vital dye mapping studies by E. Manchot (1929, Wilhelm Roux Arch. Entwicklungsmech.116, 689–709) and (M. W. Woerdeman, 1929, Wilhelm Roux Arch. Entwicklungsmech.116, 220–241). Spemann published a figure which might suggest that the prospective eye vesicles are still located in this position when the neural folds are present. This paper shows that the eye primordia move from the anterior neural plate into the forming neural folds. This result was obtained by time-lapse photography and excision experiments. Grafting experiments exchanging presumptive optic tissue between wild-type and albino embryos were also performed.     

Melanin biosynthesis and the metabolism of flaviolin and 2-hydroxyjuglone inWangiella dermatitidis

Melanin biosynthesis in the human pathogenWangiella dermatitidis was inhibited by tricyclazole, causing pentaketide melanin metabolites to accumulate in the cultures. One of these metabolites, scytalone, was racemic and thus different than the (+)-enantiomer fromVerticillium dahliae. An albino mutant ofW. dermatitidis metabolized scytalone to a pigment ultrastructurally identical to wild-type melanin. Cell-free homogenates of the wild type carried out typical reductive and dehydrative reactions with known melanin intermediates and the reductive reactions were inhibited by tricyclazole. Other reductive and dehydrative reactions that utilize flaviolin and 2-hydroxyjuglone were studied anaerobically with homogenates from both the wild type and the albino mutant. The homogenates converted flaviolin to 5-hydroxyscytalone and products identical to those obtained from 2-hydroxyjuglone. The albino, in culture, carried out the same reactions with 2-hydroxyjuglone but metabolized flaviolin to a number of unknown colored products apparently through oxidative reactions. Similarities between the melanin pathway and the flaviolin and 2-hydroxyjuglone branch pathways are discussed and tricyclazole is shown to inhibit reductive reactions with naphthols in the three pathways.Abbreviations DHN dihydroxynaphtalene - HJ hydroxyjuglone - THT trihydroxytetralone - THN trihydroxynaphthalene or tetrahydroxynaphthalene - DTT dithiothreitol - HS hydroxyscytalone - PHN pentahydroxynaphthalene     

Albino Weddell seal at Cape Shirreff,Livingston Island,Antarctica

To our knowledge, this paper is the first record/report of a juvenile light-coloured Weddell seal (Leptonychotes weddellii) at Cape Shirreff, Livingston Island in January 1998, determining that it was an albino individual. Based on available literature, three cases of albino seals have been reported exclusively for Harbour seal pups, and no albino has been reported for Antarctic pagophilic true seals. Therefore, this is the first confirmed case of albinism in Antarctic pagophilic true seals species, indicating that this phenomenon is indeed of a rare occurrence.

Novel conjugates with dual suppression of glutathione S-transferases and tryptophan-2,3-dioxygenase activities for improving hepatocellular carcinoma therapy

Tryptophan-2,3-dioxygenase (TDO) is an immune checkpoint enzyme expressed in human tumors and involved in immune evasion and tumor tolerance. While glutathione S-transferases (GSTs) are pharmacological targets for several cancer. Here we demonstrated the utility of NBDHEX (GSTs inhibitor) and TDO inhibitor by the combinatorial linker design. Two novel conjugates with different linkers were prepared to reverse tumor immune suppression. The conjugates displayed significant antitumor activity against TDO and GSTs expression of HepG2 cancer cells. Further study indicated that compound 4 could induce higher apoptotic effect than its mother compounds via a mitochondrial-dependent pathway, simultaneously more effective to inhibit TDO and GSTs protein expression. Further study indicated that 4 could decrease the production of kynurenine and deactivate aryl hydrocarbon receptor (AHR), leading to CD3⁺ T-cell activation and proliferation to involve in antitumor immune response.