The Seiji memorial lecture: the melanosome: an ideal model to study cellular differentiation

Melanosomes provide an intriguing model for study at many levels. In part this is due to their unique structure and function, but also in part to their involvement in pigmentary diseases and as a model to study basic cellular mechanisms of organelle biogenesis. Recent studies have elucidated the full proteome of the melanosome and the metabolic and molecular lesions involved in a number of pigmentary diseases have been resolved. This paper summarizes recent advances in the field in these areas.     

Human and mouse disorders of pigmentation

Disorders of pigmentation were among the first genetic diseases ever recognized because of their visually striking clinical phenotypes, resulting from defects of pigmentary melanocytes. Recent years have seen remarkable progress in understanding these diseases, largely as a result of the systematic parallel study of human patients and inbred mice with similar phenotypes. Our understanding of disorders of pigmentation indicates that these diseases may be most usefully considered as abnormalities of melanocyte development, function, or survival.     

Connective tissue responses in Negroes in realtion to disease

There is considerable evidence that Negroes have a tendency toward overgrowth of those connective-tissue components concerned with two functions—protection against infection (macrophages and plasma cells) and repair after injury (fibroblasts and their products). Thus, adaptation to the tropical environment in Africans may have involved a tendency toward connective-tissue overgrowth, as well as hypertrophy of the pigmentary apparatus. Both tendencies may have consequences in terms of: (1) susceptibility to certain chronic diseases; and (2) responses to disease processes or drug therapies. Some of these possible consequences are discussed.     

Involvement of inflammation, degradation, and apoptosis in a mouse model of glaucoma

Glaucoma is a common cause of blindness affecting at least 66 million people worldwide. Pigmentary glaucoma is one of the most common forms of secondary glaucoma, and its pathogenesis remains unclear. Interleukin-18 (IL-18) is an important regulator of innate and acquired immune responses and plays an important role in inflammatory/autoimmunity diseases. Using the DBA/2J mouse as an animal model of human pigmentary glaucoma, we demonstrated for the first time that the expression of the IL-18 protein and gene in the iris/ciliary body and level of IL-18 protein in the aqueous humor of DBA/2J mice are dramatically increased with age. This increase precedes the onset of clinical evidence of pigmentary glaucoma, implying a pathogenic role of inflammation/immunity in this disease. We also observed that activated NF-kappaB and phosphorylated MAPK are increased in the iris/ciliary body of DBA/2J mice, suggesting that both signaling pathways may be involved in IL-18 mediated pathogenesis of pigmentary glaucoma in the eyes of DBA/2J mice. In addition, matrix metalloproteinase-2 (MMP-2) expression in the iris/ciliary body and the activity of MMP-2 in the aqueous humor are increased whereas tissue inhibitor of matrix metalloproteinase-1 (TIMP-1) expression in the iris/ciliary body is decreased, indicating that the degradation process is involved in this mouse model of pigmentary glaucoma. Furthermore, the expressions of apoptosis-related genes, caspase-8, Fas, FADD, FAP, and FAF, and the activity of caspase-3 are increased in the iris/ciliary body of DBA/2J mice. Elucidation of biochemical and molecular mechanisms of IL-18 participation in the pathogenesis of pigmentary glaucoma should provide approaches for developing improved and targeted treatments to ameliorate this blinding disease. The possibility that altered IL-18 expression in the eye of DBA/2J mice initiates and/or amplifies the pathogenesis of pigmentary glaucoma requires further investigation.     

Are L-tyrosine and L-dopa hormone-like bioregulators?

Some amino acids have bioregulatory functions, which far exceed those of precursors for proteins or of substrates for specific enzymes. Two of these amino acids, L-tyrosine and L-dopa, are precursors to melanin and catecholamines. In vertebrates, they can act as inducers and regulators of the melanogenic apparatus and of MSH receptors--two quite complex functions that could hardly be performed by mere substrates. Focussing on the pigmentary system as a study model, we therefore explore the hypothesis that L-tyrosine and L-dopa act as hormone-like bioregulators in mammals, with melanocytes regulating tyrosine and dopa activity via their metabolic consumption.     

Pigmentation abnormalities in nucleotide excision repair disorders: Evidence and hypotheses

Skin pigmentation abnormalities are manifested in several disorders associated with deficient DNA repair mechanisms such as nucleotide excision repair (NER) and double‐strand break (DSB) diseases, a topic that has not received much attention up to now. Hereditary disorders associated with defective DNA repair are valuable models for understanding mechanisms that lead to hypo‐ and hyperpigmentation. Owing to the UV‐associated nature of abnormal pigmentary manifestations, the outcome of the activated DNA damage response (DDR) network could be the effector signal for alterations in pigmentation, ultimately manifesting as pigmentary abnormalities in repair‐deficient disorders. In this review, the role of the DDR network in the manifestation of pigmentary abnormalities in NER and DSB disorders is discussed with a special emphasis on NER disorders.     

The genetics of generalized vitiligo and associated autoimmune diseases

Vitiligo is an acquired disorder in which patches of depigmented skin and often overlying hair, and mucous membranes, are the result of progressive autoimmune loss of melanocytes from the involved areas. Considered the most common pigmentary disorder, vitiligo involves complex interaction of environmental and genetic factors that ultimately contribute to melanocyte destruction, resulting in the characteristic depigmented lesions. In the past few years, studies of the genetic epidemiology of vitiligo have led to the recognition that generalized vitiligo is part of a broader autoimmune disease diathesis. Attempts to identify genes involved in susceptibility to generalized vitiligo have involved gene expression studies, genetic association studies of candidate genes, and genome-wide linkage analyses to discover new genes. These studies have begun to yield results that shed light on the mechanisms of vitiligo pathogenesis. It is anticipated that the discovery of biological pathways of vitiligo pathogenesis will provide novel targets for future approaches to the treatment and prevention of vitiligo and its associated autoimmune diseases.     

Physiological role and potential clinical interest of mycobacterial pigments

The production of pigments by bacterial colonies has sparked interest among bacteriologists since the 19th century, whether for taxonomy or, in the case of carotenoids for their association with antibiotics resistance. Mycobacteria have gained a very special place in the bacterial world due to their clinical importance. Alone, Mycobacterium tuberculosis is responsible for about two million deaths annually worldwide making tuberculosis one of the most influential diseases in the history of mankind. Almost half of the Nontuberculous Mycobacteria species identified are associated with opportunistic infections in animals and humans. Mycobacterial pigmentary characteristics started to be documented about 80 years ago; but to date, their main use has been only for limited taxonomic and identification purposes. While mycobacterial pigments, especially carotenoids have been clearly associated with cellular photoprotection and survival, the regulation of their production and their physiological role have been largely unstudied. Recent advances in deciphering mycobacterial genomes and characterization of carotenoid synthesis genes, combined with an urgent need for innovative approaches to understand Mycobacterium tuberculosis pathogenic properties open new avenues for exciting research opportunities that might lead to new therapeutic strategies against a devastating secular disease.     

Immunization with Recombinant Prion Protein Leads to Partial Protection in a Murine Model of TSEs through a Novel Mechanism

Transmissible spongiform encephalopathies are neurodegenerative diseases, which despite fervent research remain incurable. Immunization approaches have shown great potential at providing protection, however tolerance effects hamper active immunization protocols. In this study we evaluated the antigenic potential of various forms of recombinant murine prion protein and estimated their protective efficacy in a mouse model of prion diseases. One of the forms tested provided a significant elongation of survival interval. The elongation was mediated via an acute depletion of mature follicular dendritic cells, which are associated with propagation of the prion infectious agent in the periphery and in part to the development of humoral immunity against prion protein. This unprecedented result could offer new strategies for protection against transmissible encephalopathies as well as other diseases associated with follicular dendritic cells.     

Proteomic and bioinformatic characterization of the biogenesis and function of melanosomes

Melanin, which is responsible for virtually all visible skin, hair, and eye pigmentation in humans, is synthesized, deposited, and distributed in subcellular organelles termed melanosomes. A comprehensive determination of the protein composition of this organelle has been obstructed by the melanin present. Here, we report a novel method of removing melanin that includes in-solution digestion and immobilized metal affinity chromatography (IMAC). Together with in-gel digestion, this method has allowed us to characterize melanosome proteomes at various developmental stages by tandem mass spectrometry. Comparative profiling and functional characterization of the melanosome proteomes identified approximately 1500 proteins in melanosomes of all stages, with approximately 600 in any given stage. These proteins include 16 homologous to mouse coat color genes and many associated with human pigmentary diseases. Approximately 100 proteins shared by melanosomes from pigmented and nonpigmented melanocytes define the essential melanosome proteome. Proteins validated by confirming their intracellular localization include PEDF (pigment-epithelium derived factor) and SLC24A5 (sodium/potassium/calcium exchanger 5, NCKX5). The sharing of proteins between melanosomes and other lysosome-related organelles suggests a common evolutionary origin. This work represents a model for the study of the biogenesis of lysosome-related organelles.     

Optimal Timing of Disease Transmission in an Age-Structured Population

It is a common medical folk-practice for parents to encourage their children to contract certain infectious diseases while they are young. This folk-practice is controversial, in part, because it contradicts the long-term public health goal of minimizing disease incidence. We study an epidemiological model of infectious disease in an age-structured population where virulence is age-dependent and show that, in some cases, the optimal behavior will increase disease transmission. This provides a rigorous justification of the concept of “endemic stability,” and demonstrates that folk-practices may have been historically justified.     

Medical genetics study of the population of Kostroma Province. II. The diversity of hereditary pathology in 5 districts of the province

The diversity of hereditary pathology in 5 regions of Kostroma district was studied. 32 nosological forms of autosomal dominant, 30 autosomal recessive and 7 X-linked recessive disorders were found. The most frequent autosomal dominant disorders were: neurofibromatosis, pigmentary degeneration of retina, hypochondroplasia, ichtiosis, idiopathic scoliosis. The most frequent among the autosomal recessive disorders were: oligophrenia, pigmentary degeneration of retina, muscular atrophy of juvenile Kugelberg--Welander type, congenital cataract. The most frequent X-linked disorders were: muscular Duchenne type dystrophy and hemophilia A. Analysis of mutant gene distribution over the territory by the study of birthplaces of probands and their parents was carried out.     

An unusual human mosaic for skin pigmentation

Patterned pigmentary disturbances are seen in a large variety of human genetic disorders. Cytogenetic studies have provided evidence that such skin lesions often reflect chromosomal mosaicism. In addition to the well-known pattern of Blaschko's lines a classification of several distinct types was proposed by Happle. This report add the case of a boy with an unusual mosaic-like distribution of skin pigmentation and a further chromosomal anomaly which has not been described in pigmentary mosaicism previously. The proband was born after an uneventful pregnancy and delivery. Developmental milestones were delayed. A generalised hirsutism was noted with a facial dysmorphia: coarse facies. short philtrum, synophris, and large low set years. Hyperpigmentation followed a checkerboard pattern: alternating squares of pigmentary anomalies with a sharp midline separation. Cytogenetic findings Included a normal karyotype (peripheral blood) and a mosaicism 12q;14q translocation (70% of fibroblasts). The present case stresses the importance of careful chromosomal analysis of different tissues in patients with pigmentary anomalies.     

Pigment cell-related manifestations in neurofibromatosis type 1: an overview

Neurofibromatosis type 1 (NF1) is an autosomal dominant neurocutaneous disorder, affecting approximately 1 in 3500 individuals. The most commonly seen tumors in NF1 patients are the (sub)cutaneous neurofibromas. However, individuals with NF1 typically present in childhood with well-defined pigmentary defects, including cafe-au-lait macules (CALMs), intertriginous freckling and iris Lisch nodules. NF1 is considered a neurocristopathy, primarily affecting tissues derived from the neural crest. Since the pigment producing melanocyte originates in the neural crest, the presence of (hyper)pigmentary lesions in the NF1 phenotype because of changes in melanocyte cell growth and differentiation is to be expected. We want to discuss the pigmentary cutaneous manifestations of NF1 represented by CALMs and intertriginous freckles and the pigmentary non-cutaneous manifestations represented by iris Lisch nodules. Several hypotheses have been suggested in explaining the poorly understood etiopathogenesis of CALMs. Whether other pigmentary manifestations might share similar etiopathogenic mechanisms remains obscure. Additional attention will be drawn to a readily seen phenomenon in NF1: hyperpigmentation overlying (plexiform) neurofibromas, which could suggest common etiopathogenetic-environmental cues or mechanisms underlying CALMs and neurofibromas. Finally, we want to address the relationship between malignant melanoma and NF1.     

Pigment cell‐related manifestations in neurofibromatosis type 1: an overview

Neurofibromatosis type 1 (NF1) is an autosomal dominant neurocutaneous disorder, affecting approximately 1 in 3500 individuals. The most commonly seen tumors in NF1 patients are the (sub)cutaneous neurofibromas. However, individuals with NF1 typically present in childhood with well‐defined pigmentary defects, including café‐au‐lait macules (CALMs), intertriginous freckling and iris Lisch nodules. NF1 is considered a neurocristopathy, primarily affecting tissues derived from the neural crest. Since the pigment producing melanocyte originates in the neural crest, the presence of (hyper)pigmentary lesions in the NF1 phenotype because of changes in melanocyte cell growth and differentiation is to be expected. We want to discuss the pigmentary cutaneous manifestations of NF1 represented by CALMs and intertriginous freckles and the pigmentary non‐cutaneous manifestations represented by iris Lisch nodules. Several hypotheses have been suggested in explaining the poorly understood etiopathogenesis of CALMs. Whether other pigmentary manifestations might share similar etiopathogenic mechanisms remains obscure. Additional attention will be drawn to a readily seen phenomenon in NF1: hyperpigmentation overlying (plexiform) neurofibromas, which could suggest common etiopathogenetic‐environmental cues or mechanisms underlying CALMs and neurofibromas. Finally, we want to address the relationship between malignant melanoma and NF1.     

Melanotropic activity of gamma MSH peptides in melanoma cells.

We studied the pigmentary activity of the peptides gamma 1, gamma 2 and gamma 3 melanocyte stimulating hormone (MSH), which differ in the structure of their C-termini, using hamster and mouse melanoma cell lines responsive to beta-MSH by increasing tyrosinase activity. Gamma 1-MSH alone or in combination with beta-MSH had no effect on either cell line. Gamma 2-MSH alone was biologically inactive but potentiated beta-MSH stimulation of tyrosinase activity. Gamma 3-MSH at high concentration (10 microM) induced tyrosinase activity and dendrite formation in the hamster melanoma line. When added together with beta-MSH, gamma 3-MSH partially inhibited the tyrosinase activity response to beta-MSH. Thus, gamma-MSH peptides have low intrinsic melanotropic activity in mammalian melanoma cells; the specific pigmentary responses appear to be affected by the structure of the C-terminal portion.     

Morphological,immunohistochemical and ultrastructural characterization of the skin of turbot (Psetta maxima L.)

This study was undertaken to identify the normal morphologic, immunohistochemical and ultrastructural features of skin of the turbot (Psetta maxima L.). In the turbot skin, three morphologically distinct layers were identified: epidermis, dermis and hypodermis. The epidermis was non-keratinizing, stratified squamous epithelium that varies in thickness from 5 to 14 cells and 60 to 100 μm in size. Goblet cells were seen randomly distributed between malpighian cells in the epidermal layer. These mucous cells were mainly located in the upper third of the epidermis and displayed a spherical to elongated morphology. Dermis was divided in two well-differentiated layers, the superficial stratum laxum and the deeper stratum compactum. Hypodermis was a loose layer mainly composed by adipocytes but we could observe variable amounts of fibroblast, collagen and blood vessels. In turbot two pigmentary layers could be identified: the pigmentary layer of dermis was located between basement membrane and dermis and the pigmentary layer of hypodermis immediately above the muscular layer. Three different types of chromatophores were present: melanophores, iridophores and xanthophores. The main differences observed between groups of fish with different colouration were in the amount of melanophores and xanthophores. The purpose of this article is to provide an overview of normal cutaneous biology prior to consideration of specific cutaneous alterations and diseases in turbot.     

The patterns of birthmarks suggest a novel population of melanocyte precursors arising around the time of gastrulation

Systematic work in the mouse and chicken has mapped out two neural crest‐derived pathways of melanocyte precursor migration. With these in mind, this study reappraises the patterns of congenital pigmentary disorders in humans and identifies three recurrent patterns consistent across genetically different diseases. Only two of these are seen in diseases known to be melanocyte cell‐autonomous. The segmental pattern correlates well with the classical dorsolateral population from animal studies, demonstrating respect of the midline, cranio‐caudal axial mixing, unilateral migration and involvement of key epidermally derived structures. Importantly however, the melanocyte precursors responsible for the non‐segmental pattern, which demonstrates circular, bilateral migration centred on the midline, and not involving key epidermally derived structures, have not been identified previously. We propose that this population originates around the time of gastrulation, most likely within the mesoderm, and ultimately resides within the dermis. Whether it contributes to mature melanocytes in non‐disease states is not known; however, parallels with the patterns of acquired vitiligo would suggest that it does. The third pattern, hypo‐ or hyperpigmented fine and whorled Blaschko's lines, is proposed to be non‐cell‐autonomous.     

Dietary fatty acids and arthritis

Musculoskeletal complaints are the second most frequent reason for medical treatments. Within these diseases rheumatoid arthritis (RA) and, especially, osteoarthritis (OA) are common. Although the causes of arthritis are multifactorial and not fully understood, clinical trials have generally shown benefit from dietary n-3 polyunsaturated fatty acids. This has usually been attributed to their anti-inflammatory properties. Recently we have used in vitro model systems to study the molecular mechanism(s) by which n-3 PUFAs may act to alleviate the symptoms of arthritis. These experiments showed that n-3 PUFAs reduce expression of cartilage-degrading proteinases, cyclooxygenase-2 and inflammatory cytokines. Eicosapentaenoic acid (EPA) was more effective than docosahexaenoic acid (DHA) or alpha-linolenic acid. The data provide a scientific rationale for the consumption of n-3 fatty acids as part of a healthy diet and perhaps in treating arthritis.     

Missing heritability of complex diseases: Enlightenment by genetic variants from intermediate phenotypes

Diseases of complex origin have a component of quantitative genetics that contributes to their susceptibility and phenotypic variability. However, after several studies, a major part of the genetic component of complex phenotypes has still not been found, a situation known as “missing heritability.” Although there have been many hypotheses put forward to explain the reasons for the missing heritability, its definitive causes remain unknown. Complex diseases are caused by multiple intermediate phenotypes involved in their pathogenesis and, very often, each one of these intermediate phenotypes also has a component of quantitative inheritance. Here we propose that at least part of the missing heritability can be explained by the genetic component of intermediate phenotypes that is not detectable at the level of the main complex trait. At the same time, the identification of the genetic component of intermediate phenotypes provides an opportunity to identify part of the missing heritability of complex diseases.