Low Tyrosinase Activity Research Articles

Dual Antimelanogenic Effect of Nicotinamide-Stabilized Phloretin Nanocrystals in Larval Zebrafish.

Melanin is a kind of dark insoluble pigment that can cause pigmentation and free-radical clearance, inducing melasma, freckles, and chloasma, affecting the quality of life of patients. Due to poor water solubility and low safety, the absorption of poorly water-soluble drugs is limited by the hinderance of a skin barrier. Therefore, it is necessary to develop new, safe, and highly efficient drugs to improve their transdermal absorption efficiency and thus to inhibit the production of melanin. To address these issues, we developed a new nicotinamide (NIC)-stabilized phloretin nanocrystals (PHL-NCs). First, NC technology significantly increased the solubility of PHL. The in vitro release results indicated that at 6 h, the dissolution of the PHL-NIC-NCs was 101.39% ± 2.40% and of the PHL-NCs was 84.92% ± 4.30%, while that of the physical mixture of the two drugs was only 64.43% ± 0.02%. Second, NIC acted not only as a stabilizer to enlarge the storage time of PHL-NIC-NCs (improved to 10-day in vitro stability) but also as a melanin transfer inhibitor to inhibit melanin production. Finally, we verified the melanin inhibition effect of PHL-NIC-NCs evaluated by the zebrafish model. It showed that 0.38 mM/L PHL-NIC-NCs have a lower tyrosinase activity at 62.97% ± 0.52% and have less melanin at 36.57% ± 0.44%. The inhibition effect of PHL-NCs and PHL-NIC-NCs was stronger compared to the positive control arbutin. In conclusion, the combination of NIC and PHL achieves better inhibition of tyrosinase and inhibition of melanin production through synergism. This will provide a direction to the subsequent development of melanin-inhibiting drugs and the combined use of pharmaceutical agents.

127 - Thioredoxin reductase 1 knockdown disrupts pigment synthesis in melanocytes

Our labs have shown that levels of the antioxidant enzyme thioredoxin reductase 1 (TR1) is increased in human malignant melanomas relative to benign nevi (moles), and that TR1 knockdown affects mitochondrial metabolism in human melanomas. Here we report our examination of the role(s) of TR1 in normal melanocytes, the cells from which melanomas arise. Cellular processes affected by the TR1/thioredoxin (Trx) system in melanocytes are not well characterized. We created an immortalized human melanocyte cell line that stably expresses an siRNA directed against the TR1 transcript. As has been reported in other cell types, TR1 knockdown resulted minor effects on cell growth, likely due to a reciprocal upregulation of the synthesis of reduced glutathione. However we also found that TR1 knock out resulted in a significant decrease in melanin levels as well as reduced activity of tyrosinase, the rate limiting enzyme involved in melanin synthesis. Chemical inhibition of Trx using a small molecule inhibitor had a similar phenotype. Knockdown of TR1 caused a delay in upregulation of the melanocyte master transcriptional regulator, Mitf, after elevation of cAMP levels by treatment with forskolin, a common method of promoting melanin synthesis. We examined the effects of forskolin treatment on the canonical targets of Mitf regulation, but only Tyrp1 was affected, suggesting TR1/Txn affects another transcriptional regulator specific to Tyrp1. Given the role of Tyrp1 in stabilizing tyrosinase, this likely explains the low tyrosinase activity in TR1 knockout cells. Our preliminary data from a mouse model where TR1 is knocked out specifically in melanocytes shows a phenotype that is consistent with our cell culture data. These studies provide new insight into the redox signaling that controls melanogenesis, and may lead to novel targets for the diagnosis and treatment of human diseases involving melanocytes such as melanoma and vitiligo.

In Vitro Methods for Predicting Chemical Leukoderma Caused by Quasi-Drug Cosmetics

Skin care cosmetics frequently contain whitening or lightening agents. The present study aimed to establish in vitro methods for predicting chemical leukoderma caused by whitening agents in cosmetics. The risks of chemical leukoderma were predicted based on percutaneous absorption rates, toxic concentrations, and toxicity mechanisms. Thus, in vitro skin permeation rate and cytotoxic concentrations of whitening agents were studied using excised skin and cultured B16 melanoma cells. Pigment cell toxicity was observed using transmission electron microscopy (TEM). The levels of hydroxyl radical (∙OH) were measured and the location of ∙OH generation sites were determined in cultured B16 melanoma cells. Pigment cells cultured under conditions with high tyrosinase activity developed cytotoxicity when exposed to compounds known to cause leukoderma, while those cultured under conditions with low tyrosinase activity did not. Phenolic compounds that cause leukoderma were applied to the pigment cells at the concentration absorbed percutaneously under conditions with high tyrosinase activity. Cells that were observed using TEM demonstrated a large number of vacuolar degenerations in intracellular melanosomes after treatment with phenolic compounds that are known to cause leukoderma. Hydroxyl radical generation during the tyrosinase reaction was examined, as the whitening agents that inhibit tyrosinase activity serve as tyrosinase substrates. Only phenolic compounds that cause leukoderma generated high amounts of hydroxyl radicals. Thus, the hydroxyl radical is a melanocyte-specific toxin that disrupts tyrosinase-containing melanosomes. Whitening agents that generate high amounts of hydroxyl radicals may cause leukoderma. The in vitro method being reported here can predict the potential of a drug to cause leukoderma and whether the use of a specific whitening agent poses a risk.

Depigmentation caused by application of the active brightening material, rhododendrol, is related to tyrosinase activity at a certain threshold

BackgroundTyrosinase, the rate-limiting enzyme required for melanin production, has been targeted to develop active brightening/lightening materials for skin products. Unexpected depigmentation of the skin characterized with the diverse symptoms was reported in some subjects who used a tyrosinase-competitive inhibiting quasi-drug, rhododendrol. ObjectiveTo investigate the mechanism underlying the depigmentation caused by rhododendrol-containing cosmetics, this study was performed. MethodsThe mechanism above was examined using more than dozen of melanocytes derived from donors of different ethnic backgrounds. The RNAi technology was utilized to confirm the effect of tyrosinase to induce the cytotoxicity of rhododendrol and liquid chromatography-tandem mass spectrometry was introduced to detect rhododendrol and its metabolites in the presence of tyrosinase. ResultsMelanocyte damage was related to tyrosinase activity at a certain threshold. Treatment with a tyrosinase-specific siRNA was shown to dramatically rescue the rhododendrol-induced melanocyte impairment. Hydroxyl-rhododendrol was detected only in melanocytes with higher tyrosinase activity. When an equivalent amount of hydroxyl-rhododendrol was administered, cell viability was almost equally suppressed even in melanocytes with lower tyrosinase activity. ConclusionThe generation of a tyrosinase-catalyzed hydroxyl-metabolite is one of the causes for the diminishment of the melanocyte viability by rhododendrol.

Photodermatoses in pigmented skin

Photodermatoses are a group of skin diseases primarily caused by, or exacerbated by exposure to ultraviolet and or visible radiation. The effect of sunlight on skin depends on a number of factors including skin colour, skin phototype and the content and type of melanin in the skin. There are only a few studies describing photodermatoses in populations with dark skin. A PubMed search was conducted to summarize currently available information on differences in biology of melanin in dark and light skin and photodermatoses in dark skin. Dark skin is characterised by higher content of melanin, higher eumelanin to pheomelanin ratio, lower tyrosinase activity, and more effective distribution of melanin for protection against ultraviolet light. Photodermatoses are common in dark skinned patients with some variation in the spectrum of photodermatoses. Polymorphous light eruption (PMLE) is the commonest, followed by chronic actinic dermatitis. Pin-point papular and lichenoid variants of PMLE and actinic lichen planus are more frequent in dark skin whereas actinic prurigo, solar urticaria and hydroa vacciniforme are uncommon. Photodermatoses are common in dark skinned patients despite better natural photoprotection. It is proposed that lichenoid photodermatoses may be added to the classification of photodermatoses in dark skin.

Tyrosinase Small Interfering RNA Effectively Suppresses Tyrosinase Gene Expression In Vitro and In Vivo

Tyrosinase is a bifunctional enzyme which oxidizes the initial step of melanin biosynthesis, that is, conversion of tyrosine to dopa and subsequently dopa to dopaquinone. It is a glycosylated protein and a major regulator of melanogenesis. To date, many approaches have been tried to regulate tyrosinase activity and melanin content. To that end, we screened small interfering RNA sequences for sequence-inhibited tyrosinase expression in B16 cells and in C57BL/6 mice. We analyzed tyrosinase mRNA levels by quantitative real-time PCR and determined tyrosinase activity and melanin content at 24, 48, and 72 hours after transfection. Results showed that siNM_011661_001 was the most efficient small interfering RNA sequence in suppressing tyrosinase mRNA expression, and cells transfected with this sequence showed lower tyrosinase activity. Moreover, intravitreous injection of siNM_011661_001 in C57BL/6 mice induced an efficient and stable gene-specific inhibition of expression at the posttranscriptional level.

Molecular cloning, sequence characteristics, and polymorphism analyses of the tyrosinase-related protein 2 / DOPAchrome tautomerase gene of black-boned sheep (Ovis aries)

Tyrosinase-related protein 2 (TYRP2) plays a pivotal role in the biosynthesis of eumelanin. Black-boned sheep have excessive melanin and eumelanin, resulting in dark (black) muscles and organs. This study was designed to investigate the effects of variants of the TYRP2 gene on black traits and coat colour of black-boned sheep. Melanin traits were measured in three populations of sheep (Nanping black-boned, Nanping normal, and Romney Marsh) and compared in this study. From the TYRP2 cDNA, all 8 exons and their flanking regions were amplified and characterized. Fifteen single nucleotide polymorphisms (SNPs) were identified in the exons and their flanking regions. Five exonic polymorphic sites, including two synonymous (c.93T>G and c.1140C>T) and three non-synonymous mutations (c.163C>T (p.R55W), c.605G>A (p.R202H), and c.1141A>G (p.T381A)), were retrieved. PCR-RFLP analysis of c.605G>A showed that the frequencies of allele G in the Nanping black-boned, Nanping normal, and Romney Marsh sheep were 0.632, 0.603, and 0.886, respectively. Sheep with the GG genotype had significantly (P < 0.05) lower tyrosinase activity, alkali-soluble melanin content, and ratio of eumelanin : total melanin than sheep with GA and AA genotypes when measured across all investigated samples but not when samples within each population of sheep were compared. However, there was no association of TYRP2 genotype at a single SNP position with coat colour across populations. Nonetheless, the two breeds with higher overall tyrosinase activity did produce darker and more varied coat colours than the breed with lower tyrosinase activity.

清酒麹菌における低褐変性グルコアミラーゼ高活性変異株の育種開発

In order to obtain new industrial koji molds with properties of both non-melanization and high glucoamylase activity, Aspergillus oryzae strains were mutated by irradiation of ultraviolet light. In the mutant strains, only one mutant, H-1 strain, with a high glucoamylase activity and a low tyrosinase activity like its parent strain was isolated. On the other hand, another mutant, G-4 strain, had higher activities of both enzymes than those of the parent strain. The amounts of glucoamylase produced by both mutant strains were more abundant than those of the parent strains. The specific activities and molecular weights of the glucoamylases of both mutant strains were almost equal to those of the parent strains.A fermentation test showed that the sake brewed by the H-1 strain had an excellent flavor and its sake cake did not turn brown even one month after Koji making and brewing at full-scale. The H-1 strain seems to be a practical, high quality strain for brewing sake with a sweet aroma, as it provided a high level of saccharification activity without browning of the sake cake.

Depigmentation of Melanocytes by the Treatment of Extracts from Traditional Chinese Herbs: a Cell Culture Assay

To obtain potential skin whitening agents from traditional Chinese herbs, we tested changes of melanin content in melanocyte cell lines after treatment with extracts of 90 traditional Chinese herbs. Mouse melanocyte cell lines were used. Depigmentation activity of the herb extracts were first screened in Mel-Ab cells, and then re-evaluated in melan-a cells and co-culture of melan-a and SP-1 cells. Melanin content and cell viability were the two indications for evaluation. Tyrosinase activity and the expression of melanin synthesis related enzymes in cells treated with the herb extracts were also tested. Nine herb extracts were proved to have depigmentation activity similar to or better than that of arbutin and low cytotoxicity to melanocytes. Two of them were more effective in co-cultured melan-a cells. Most of the effective herb extracts inhibited tyrosinase activity and the expression of tyrosinase. Some of them also inhibited tyrosinase related protein-1 and/or tyrosinase related protein-2 in cultured cells. We have found 9 herb extracts to be promising skin whitening agents. Among them, water extract of Galla Chinensis and ethanol extract of Radix Clematidis exhibited higher depigmentation activity and caused lower tyrosinase activity in cell culture assays and are worthy to be further studied.

Regional abnormalities in retinal development are associated with local ocular hypopigmentation

The retinal pigment epithelium (RPE) plays a key role in regulating retinal development. The critical enzyme in pigment production is tyrosinase. Transgenic mice with a tyrosinase construct where the locus control region was deleted (YRT4) display a variegated phenotype of tyrosinase expression. Their central retina is largely pigment free, whereas more peripheral regions are heavily pigmented. We have used this model to ask whether the influence of pigmented RPE over the retina during development is fundamentally governed by local interactions or is global. Our data show that YRT4 eyes have intermediate melanin content and relatively low tyrosinase activity compared with wild-type and albino animals. Rod counts are comparable to those in pigmented mice in peripheral regions but similar to those in albinos centrally. Anterograde labelling of retinal pathways demonstrates the presence of relatively normal ipsilateral chiasmatic projection in YRT4 mice, comparable with that in pigmented animals and consistent with the peripheral pigmented origin of this pathway. Examination of cellular proliferation levels during retinal development reveals that YRT4 mice display an extended period of mitosis, similar to that found in albinos. Hence, our results show that the regulatory influence of the RPE over the developing retina depends on localized interactions between these tissues.

The relationship between Na +/H + exchanger expression and tyrosinase activity in human melanocytes

The activity of melanosome-associated tyrosinase in human melanocytes differs based on racial skin type. In melanocytes from Black skin, tyrosinase activity is high while in White melanocytes the activity of the enzyme is low. Recent studies suggest that low tyrosinase activity in White melanocytes may be due to an acidic pH environment within the melanosome. Because sodium/hydrogen (Na +/H +) exchangers (NHEs) are known to regulate intracellular pH, melanocytes were treated with NHE inhibitors to determine what effect this inhibition might have on tyrosinase activity. Treatment of Black melanocytes with ethyl-isopropyl amiloride (EIPA) caused a rapid dose-dependent inhibition of tyrosinase activity. This inhibition was not due to either direct enzyme inhibition or to a decrease in tyrosinase abundance. In contrast, treatment of White melanocytes with EIPA, cimetidine, or clonidine resulted in little inhibition of tyrosinase activity. Reverse transcriptase-polymerase chain reaction (RT-PCR) and Western blot analysis showed that both Black and White melanocytes expressed mRNA and protein for NHE-1, NHE-3, NHE-5, NHE-6, and NHE-7. Immunohistochemical analysis showed that NHE-7 and NHE-3 co-localized with the melanosomal protein, Tyrosinase Related Protein-1 (TRP-1). In addition, the vesicular proton pump, vesicular ATPase (V-ATPase), was found to be present in both White and Black melanosomes, indicating that organelles from both racial skin types are capable of being acidified. The results suggest that one or more NHEs may help regulate melanosome pH and tyrosinase activity in human melanocytes.

Comparison of in vitro cytotoxicity of N-acetyl and N-propionyl derivatives of phenolic thioether amines in melanoma and neuroblastoma cells and the relationship to tyrosinase and tyrosine hydroxylase enzyme activity

Our laboratory has synthesized two new phenolic thioether amines, N-propionyl-4-S-cysteaminylphenol (N-Pr-4-S-CAP) and N[2-[(4-propionyloxyphenyl)thio]ethyl] propionamide (N,O-diPr-4-S-CAP). These compounds, along with the previously described phenolic thioether amine N-acetyl-4-S-cysteaminylphenol (N-Ac-4-S-CAP) and its acetyl form (N,O-diAc-4-S-CAP), are tyrosine-amine derivative analogues. The cytotoxicity of these compounds is thought to be tyrosinase dependent, which may make them suitable for targeted anti-melanoma therapy since only melanocytes and their malignant counterparts contain this active enzyme. To further investigate this hypothesis, we performed MTT [3-(4,5-dimethylthiazol-2-yl)2,5-diphenyltetrazolium bromide] assays to determine the cytotoxicity of these compounds in 10 different cell lines. Specifically, we examined to what extent cytotoxicity is related to tyrosinase and tyrosine hydroxylase activity using melanoma and neuroblastoma cells, which have a common metabolic pathway using tyrosinase and tyrosine hydroxylase, respectively. The most sensitive cell line was the highly pigmented SK-MEL-23 melanoma cell line, which shows a very high tyrosinase activity with the highest melanin pigmentation. KAN and SK-NSH (two neuroblastoma cell lines), which have no tyrosinase activity but high tyrosine hydroxylase, were also sensitive. However, C32 (a non-pigmented melanoma with a lower tyrosinase activity) was also sensitive, and MeWo (a moderately pigmented melanoma with a high tyrosinase activity) was less sensitive. This in vitro study may indicate that there is a non-tyrosinase-mediated mechanism of cytotoxicity for phenolic thioether amines in addition to the tyrosinase-mediated one described previously.

Comparison of antimelanoma effects of 4-S-cysteaminylphenol and its homologues.

4-S-Cysteaminylphenol (4-S-CAP), a phenolic thioether, has been evaluated for melanocytotoxicity. We have recently shown that dihydro-1,4-benzothiazine-6,7-dione (benzothiazine BQ) is the ultimate toxic metabolite produced by tyrosinase oxidation of 4-SCAP. In this study we compared the antimelanoma effects of 4-SCAP and its two homologues, alpha-methyl-4-S-cysteaminylphenol (alpha-Me-4-SCAP) and 4-S-homocysteaminylphenol (4-S-Homo-CAP). Biochemical experiments showed that upon tyrosinase oxidation alpha-Me-S-CAP and 4-S-Homo-CAP also produced homologues of BQ which reacted rapidly with reduced glutathione (GSH) and also inhibited alcohol dehydrogenase, an SH enzyme. In vitro experiments showed that 4-S-CAP and its two homologues were taken up into B16-F1 melanoma cells at comparable rates but that 4-S-Homo-CAP was least effective in GSH deprivation, which was reflected in the low cytotoxicity of this phenol, and that the cytotoxicity of the phenols was tyrosinase dependent, as proved by the negligible effects on B16-G4F cells which have a much lower tyrosinase activity. In vivo experiments showed that direct intratumoral administration of these phenols inhibited the subcutaneous growth of B16 melanoma, with 4-S-Homo-CAP being the least effective, and that indirect Intraperitoneal administration of 4-S-CAP inhibited melanoma growth much more effectively than the two homologues. These results indicate that 4-S-CAP is the most promising antimelanoma agent among the three phenols examined.

Autosomal recessive ocular albinism associated with a functionally significant tyrosinase gene polymorphism

Autosomal recessive ocular albinism (AROA) is a disorder characterized by reduced pigmentation of the retina and iris, hypoplastic fovea, variably reduced visual acuity and nystagmus. Pigmentation of the skin and hair is normal, but is usually slightly lighter than in unaffected sibs. We analysed 12 unrelated patients with AROA, and found that two had abnormalities of the tyrosinase (TYR) gene. These two patients were each a compound heterozygote for a different pathologic mutant allele and an allele containing a 'normal' polymorphism, Arg402Gln, which results in a tyrosinase polypeptide with reduced thermal stability. In these patients, AROA thus appears to represent a clinically mild form of OCA1, with a fixed visual deficit resulting from low tyrosinase activity during fetal development but with normal pigmentation of the skin and hair postnatally.

Ophthalmic Features of Minimal Pigment Oculocutaneous Albinism

The purpose of this study is to describe the heterogeneous phenotype of individuals with an unusual type of albinism--minimal pigment oculocutaneous albinism. Nine patients with minimal pigment oculocutaneous albinism were identified and followed for up to 11 years. The criteria were the presence of oculocutaneous albinism in association with low hairbulb tyrosinase activity in the patient and disparate activity in the parents with one parent having normal activity and the other having low tyrosinase activity. Changes in skin, hair, and ocular pigment were followed as the patients matured. As a measure of ocular pigment, iris transillumination and macular transparency were graded according to a previously published scheme. Patients were born with white scalp hair and skin, and nystagmus developed. Visual acuity was reduced to 20/50 to 20/200 for the group, but in one patient vision improved with maturity. Irides were blue. In seven patients, iris pigment developed, which was detected by transillumination with slit-lamp biomicroscopy, including the one patient with improved visual acuity. All patients had foveal hypoplasia, and melanin pigment in the fundi could not be detected by clinical examination. Visual acuity in the group did not correlate directly with the presence or development of iris transillumination or macular transparency. The pedigrees were consistent with an autosomal recessive inheritance pattern. This unique type of oculocutaneous albinism has heterogeneous clinical features. Minimal pigment oculocutaneous albinism appears to represent a new type of tyrosinase-related oculocutaneous albinism (OCA1MP).

Role of Tyrosinase as the Determinant of Pigmentation in Cultured Human Melanocytes

Variations in human pigmentation among different racial groups are due to differences in the production and deposition of melanin in the skin. Although melanin synthesis is known to be controlled by the rate-limiting enzyme tyrosinase, the role of this enzyme as the principal determinant of skin pigmentation is unclear. Results from studies with human melanocyte cultures derived from different racial skin types reveal an excellent correlation between the melanin content of melanocyte cultures and the in situ activity of tyrosinase. Melanocytes derived from black skin have up to 10 times more tyrosinase activity and produce up to 10 times more melanin than melanocytes derived from white skin. However, the higher level of tyrosinase activity in melanocytes derived from black skin is not due to a greater abundance of tyrosinase. Results from immunotitration experiments and Western immunoblots reveal that the number of tyrosinase molecules present in white-skin melanocytes may equal the number found in highly pigmented black skin types. Moreover, approximately equivalent levels of tyrosinase mRNA are present in white and black skin cell strains. In contrast, melanocytes derived from red-haired neonates with low tyrosinase activity contain low numbers of tyrosinase molecules and low levels of tyrosinase mRNA. These results show that tyrosinase activity and melanin production in most light-skinned people is controlled primarily by a post-translational regulation of pre-existing enzyme and not by regulating tyrosinase gene activity. In contrast, melanocytes from red-haired (type I) people have low levels of tyrosinase protein and mRNA, suggesting that transcriptional activity of the tyrosinase gene is suppressed.

Differential Regulation of Tyrosinase Activity in Skin of White and Black Individuals In Vivo by Topical Retinoic Acid

Tyrosinase activity is a key determinant of melanin production in skin. Because retinoic acid regulates tyrosinase activity in melanoma cells, we analyzed modulation of pigmentation in vivo by retinoic acid. Black and white subjects were either not treated, or treated topically for 4 d under occlusion with vehicle, retinoic acid (0.1%), or the irritant sodium lauryl sulfate (2%). In untreated skin, tyrosinase activity and melanin content were significantly greater (2.3 times, and 3.2 times, respectively) in blacks versus whites. Four days of treatment with topical retinoic acid did not alter tyrosinase activity or melanin content in black skin. In contrast, retinoic acid treatment significantly induced (2.7 times, n = 8) tyrosinase activity, compared to vehicle treatment, in white skin. Melanin content, however, remained unchanged at 4 d. In separate experiments, tyrosinase activity in white subjects (n = 25) was increased 16% (p = 0.01) in sodium lauryl sulfate-treated skin, and 77% (p = 0.0005) in retinoic acid-treated skin, compared to vehicle-treated skin. The effect of retinoic acid on tyrosinase activity could be differentiated from non-specific irritation, because tyrosinase activity in retinoic acid-treated skin was significantly greater (52%, p = 0.004) than sodium lauryl sulfate-treated skin. Similar results were obtained with the dihydroxyphenylalanine reaction done on vehicle, sodium lauryl sulfate-, and retinoic acid-treated white skin. Northern analysis (n = 6) and semi-quantitative polymerase chain reaction (n = 6) demonstrated that retinoic acid treatment did not alter tyrosinase mRNA levels in white skin. Western analysis revealed that induction of tyrosinase activity by retinoic acid also was not associated with increased tyrosinase protein content (n = 9), indicating that regulation of tyrosinase activity by retinoic acid occurs through a post-translational mechanism. These data demonstrate that low tyrosinase activity in white skin in vivo is retinoic acid inducible and high tyrosinase activity in black skin in vivo is neither further induced nor reduced by retinoic acid.

The Female Brindled Mouse as a Model of Menkes' Disease: The Relationship of Fur Pattern to Behavioral and Neurochemical Abnormalities

The brindled mottled mutant mouse, a model of Menkes' disease, has alterations in copper homeostasis which cause, among other sequelae, neuronal degeneration in selected areas of brain. This work examined the neurochemical changes at postnatal days (PND) 15, 30 and 60 in females heterozygous for the sex-linked brindled mutation. These data were compared to behavioral alterations and to fur coat color at these same time points. The brindled heterozygotic females had lower concentrations of norepinephrine (NE) in the cingulate cortex, and higher levels of dopamine or dopamine metabolites in the cingulate cortex, thalamus and hypothalamus across all ages, although the difference was greatest at PND 15. The brindled females were much less active than their normal littermates at PND 15, but the differences were no longer evident at PND 30 and 60. Mottling of the fur is believed to result from low tyrosinase activity caused by abnormalities in copper metabolism. The fur pattern and behavior of the brindled mice were highly correlated with NE levels in the cingulate cortex and thalamus. These data show that female brindled mice have neurochemical abnormalities similar to (if less severe than) the male hemizygotes, that these abnormalities are regionally specific, are most apparent prior to 30 days of age, and are linked to behavioral deficits. These data also show that the extent of such deficits can be predicted by a quantitative analysis of the fur pattern of these females.

Minimal pigment: a new type of oculocutaneous albinism.

Minimal pigment, a new type of oculocutaneous albinism (OCA), is described. At birth, affected individuals had no skin or eye pigment, and white hair and blue irides, but minimal amounts of pigment developed in the iris during the first decade of life. They had no measurable hairbulb tyrosinase activity. A characteristic and unusual pattern of parental activity was found in each of three families studied, with one parent having normal and the other parent having abnormally low tyrosinase activity. The melanocyte ultrastructure was normal and variations in premelanosomal pigmentation correlated with tyrosinase activity. This clinical and biochemical pattern has not been seen in any of the previously described types of OCA. The biochemical defect in minimal pigment OCA is unknown.

Tyrosinase activity and abundance in Cloudman melanoma cells

Rabbit anti-tyrosinase antibodies were used to study the abundance, processing, and degradation of tyrosinase in murine (Cloudman) melanoma cells. The polyclonal antibodies precipitated low-molecular-weight (68,000 and 70,000) and high-molecular-weight (78,000 and 80,000) tyrosinases that had a precursor-product relationship. Cells with high basal tyrosinase activity had high levels of newly synthesized tyrosinase. Cells with low tyrosinase activity synthesized less tyrosinase and degraded the enzyme at a faster rate than cells with high tyrosinase activity. Melanotropin (melanocyte stimulating hormone), dibutyryl cyclic adenosine monophosphate, and isobutylmethylxanthine caused an increase in the abundance of newly synthesized tyrosinase that was directly proportional to the increase in enzyme activity. This enzyme was not a phosphoprotein. Other changes in the culture conditions that increased the level of tyrosinase activity increased the abundance of newly synthesized enzyme. It is thus concluded that the level of tyrosinase activity in Cloudman melanoma cells is a direct reflection of the abundance of enzyme protein.