Isolation of melanosomes.

The methods used to purify early and late melanosomes are detailed. These methods include the use of highly pigmented cells to maximize recovery and the use of various sucrose density gradients to separate melanosome fractions based on their density (which is determined in large part by the amount of dense melanin pigment that they contain). Early melanosomes lacking pigment must be further purified using free-flow electrophoresis.

Proteomic analysis of immature murine melanocytes at different stages of maturation: a crucial role for calreticulin.

Background: We have established two immature melanocyte cell lines from murine neural crest cells. NCC-E3 cells have Stage II melanosomes and express tyrosinase while in NCCmelb4 cells, the melanosomes remain at Stage I and tyrosinase is not expressed. These cell lines may be useful in studying the differentiation of melanocyte precursors.

Oral antihistamine therapy influences plasma tryptase levels in adult atopic dermatitis.

Background: Atopic dermatitis (AD) is an allergic skin disease that follows a clinical course of 'flare-up' and remission. Histamine and tryptase are inducers of pruritus and non-sedating second-generation antihistamines, including fexofenadine, are widely used for treatment of allergic skin disorders.

Objective: We assessed the efficacy of a second-generation antihistamine in AD patients and examined its pharmacological effects on chemical mediators.

Establishment of a kit-negative cell line of melanocyte precursors from mouse neural crest cells.

We previously established a mouse neural crest cell line named NCCmelb4, which is positive for Kit and negative for tyrosinase. NCCmelb4 cells were useful to study the effects of extrinsic factors such as retinoic acids and vitamin D(3) on melanocyte differentiation, but in order to study the development of melanocytes from multipotent neural crest cells, cell lines of melanocyte progenitors in earlier developmental stages are needed. In the present study, we established an immortal cell line named NCCmelb4M5 that was derived from NCCmelb4 cells.

Bcl-2 reduced and fas activated by the inhibition of stem cell factor/KIT signaling in murine melanocyte precursors.

Stem cell factor (SCF) and its receptor, KIT, are essential to the migration and differentiation of melanocytes during embryogenesis. We previously demonstrated that apoptosis is induced by blocking survival function of the SCF/KIT interaction in a mouse neural crest cell (NCC) primary culture. Using the NCCmelb4 cell line, we investigated the occurrence of apoptosis in the cultured cells when KIT receptors were blocked by the monoclonal anti-KIT antibody (ACK2).

Epitope mapping of the melanosomal matrix protein gp100 (PMEL17): rapid processing in the endoplasmic reticulum and glycosylation in the early Golgi compartment.

Melanosomes, specific organelles produced only by melanocytes, undergo a unique maturation process that involves their transition form amorphous rounded vesicles to fibrillar ellipsoid organelles, during which they move from the perinuclear to the distal areas of the cells. This depends upon the trafficking and processing of gp100 (also known as Pmel17 and the silver protein), a protein of great interest, because it elicits immune responses in melanoma patients but in which specific function(s) remains elusive. In this study, we have used biochemical and immunochemical approaches to more critically assess the synthesis, processing, glycosylation, and trafficking of gp100.

Regulation of tyrosinase processing and trafficking by organellar pH and by proteasome activity.

Pigmentation of the hair, skin, and eyes of mammals results from a number of melanocyte-specific proteins that are required for the biosynthesis of melanin. Those proteins comprise the structural and enzymatic components of melanosomes, the membrane-bound organelles in which melanin is synthesized and deposited. Tyrosinase (TYR) is absolutely required for melanogenesis, but other melanosomal proteins, such as TYRP1, DCT, and gp100, also play important roles in regulating mammalian pigmentation.

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.

Proteomic analysis of early melanosomes: identification of novel melanosomal proteins.

Melanin is a heterogeneous biopolymer produced only by specific cells termed melanocytes, which synthesize and deposit the pigment in specialized membrane-bound organelles known as melanosomes. Although melanosomes have been suspected of being closely related to lysosomes and platelets, the total number of melanosomal proteins is still unknown. Thus far, six melanosome-specific proteins have been identified, and the challenge is to characterize the complete proteome of the melanosome to further understand its mechanism of biogenesis.

The etiology of oculocutaneous albinism (OCA) type II: the pink protein modulates the processing and transport of tyrosinase.

Oculocutaneous albinism (OCA) is caused by reduced or deficient melanin pigmentation in the skin, hair, and eyes. OCA has different phenotypes resulting from mutations in distinct pigmentation genes involved in melanogenesis. OCA type 2 (OCA2), the most common form of OCA, is an autosomal recessive disorder caused by mutations in the P gene, the function(s) of which is controversial.