Role of keratinocyte-derived factors involved in regulating the proliferation and differentiation of mammalian epidermal melanocytes

Melanocytes characterized by the activities of tyrosinase, tyrosinase-related protein (TRP)-1 and TRP-2 as well as by melanosomes and dendrites are located mainly in the epidermis, dermis and hair bulb of the mammalian skin. Melanocytes differentiate from melanoblasts, undifferentiated precursors, derived from embryonic neural crest cells. Because hair bulb melanocytes are derived from epidermal melanoblasts and melanocytes, the mechanism of the regulation of the proliferation and differentiation of epidermal melanocytes should be clarified. The regulation by the tissue environment, especially by keratinocytes is indispensable in addition to the regulation by genetic factors in melanocytes. Recent advances in the techniques of tissue culture and biochemistry have enabled us to clarify factors derived from keratinocytes. Alpha-melanocyte-stimulating hormone, adrenocorticotrophic hormone, basic fibroblast growth factor, nerve growth factor, endothelins, granulocyte-macrophage colony-stimulating factor, steel factor, leukemia inhibitory factor and hepatocyte growth factor have been suggested to be the keratinocyte-derived factors and to regulate the proliferation and/or differentiation of mammalian epidermal melanocytes. Numerous factors may be produced in and released from keratinocytes and be involved in regulating the proliferation and differentiation of mammalian epidermal melanocytes through receptor-mediated signaling pathways.     

Role of keratinocyte‐derived factors involved in regulating the proliferation and differentiation of mammalian epidermal melanocytes

Melanocytes characterized by the activities of tyrosinase, tyrosinase‐related protein (TRP)‐1 and TRP‐2 as well as by melanosomes and dendrites are located mainly in the epidermis, dermis and hair bulb of the mammalian skin. Melanocytes differentiate from melanoblasts, undifferentiated precursors, derived from embryonic neural crest cells. Because hair bulb melanocytes are derived from epidermal melanoblasts and melanocytes, the mechanism of the regulation of the proliferation and differentiation of epidermal melanocytes should be clarified. The regulation by the tissue environment, especially by keratinocytes is indispensable in addition to the regulation by genetic factors in melanocytes. Recent advances in the techniques of tissue culture and biochemistry have enabled us to clarify factors derived from keratinocytes. Alpha‐melanocyte‐stimulating hormone, adrenocorticotrophic hormone, basic fibroblast growth factor, nerve growth factor, endothelins, granulocyte‐macrophage colony‐stimulating factor, steel factor, leukemia inhibitory factor and hepatocyte growth factor have been suggested to be the keratinocyte‐derived factors and to regulate the proliferation and/or differentiation of mammalian epidermal melanocytes. Numerous factors may be produced in and released from keratinocytes and be involved in regulating the proliferation and differentiation of mammalian epidermal melanocytes through receptor‐mediated signaling pathways.     

Cloning and expression of the Na+/H+ exchanger from Amphiuma RBCs: resemblance to mammalian NHE1

The cDNAencoding theNa⁺/H⁺exchanger (NHE) from Amphiumaerythrocytes was cloned, sequenced, and found to be highly homologous to the human NHE1 isoform (hNHE1), with 79% identity and 89%similarity at the amino acid level. Sequence comparisons with otherNHEs indicate that the Amphiumatridactylum NHE isoform 1 (atNHE1) islikely to be a phylogenetic progenitor of mammalian NHE1. The atNHE1protein, when stably transfected into the NHE-deficient AP-1 cell line(37), demonstrates robustNa⁺-dependent proton transportthat is sensitive to amiloride but not to the potent NHE1 inhibitorHOE-694. Interestingly, chimeric NHE proteins constructed by exchangingthe amino and carboxy termini between atNHE1 and hNHE1 exhibited drugsensitivities similar to atNHE1. Based on kinetic, sequence, andfunctional similarities between atNHE1 and mammalian NHE1, we proposethat the Amphiuma exchanger shouldprove to be a valuable model for studying the control of pH and volumeregulation of mammalian NHE1. However, low sensitivity of atNHE1 to theNHE inhibitor HOE-694 in both nativeAmphiuma red blood cells (RBCs) and intransfected mammalian cells distinguishes this transporter from itsmammalian homologue.     

Analysis of the reticulon gene family demonstrates the absence of the neurite growth inhibitor Nogo-A in fish

Reticulons (RTNs) are a family of evolutionary conserved proteinswith four RTN paralogs (RTN1, RTN2, RTN3, and RTN4) presentin land vertebrates. While the exact functions of RTN1 to RTN3are unknown, mammalian RTN4-A/Nogo-A was shown to inhibit theregeneration of severed axons in the mammalian central nervoussystem (CNS). This inhibitory function is exerted via two distinctregions, one within the Nogo-A–specific N-terminus andthe other in the conserved reticulon homology domain (RHD).In contrast to mammals, fish are capable of CNS axon regeneration.We performed detailed analyses of the fish rtn gene family todetermine whether this regeneration ability correlates withthe absence of the neurite growth inhibitory protein Nogo-A.A total of 7 rtn genes were identified in zebrafish, 6 in pufferfish,and 30 in eight additional fish species. Phylogenetic and syntenicrelationships indicate that the identified fish rtn genes areorthologs of mammalian RTN1, RTN2, RTN3, and RTN4 and that severalparalogous fish genes (e.g., rtn4 and rtn6) resulted from genomeduplication events early in actinopterygian evolution. Accordingly,sequences homologous to the conserved RTN4/Nogo RHD are presentin two fish genes, rtn4 and rtn6. However, sequences comparableto the first 1,000 amino acids of mammalian Nogo-A includinga major neurite growth inhibitory region are absent in zebrafish.This result is in accordance with functional data showing thataxon growth inhibitory molecules are less prominent in fisholigodendrocytes and CNS myelin compared to mammals.     

Trapping of Mammalian Promoters by Cre-lox Site-Specific Recombination

One of the challenges in human genome research is to identifythe promoter sequences which play a key role in the regulationof gene expression. We report here a new promoter trapping systemfor use with mammalian cells comprised of the following threesteps: 1) Cloning of DNA fragments into a promoter-trappingvector, 2) integration of the trapping vector into a designatedtarget in the mammalian genome using the Cre site-specific recombinase,and 3) screening of integrants for trapped promoter sequencesby activation of the luciferase gene. To assess the efficiencyof this system, lox trapping vectors containing sense tk promoter,antisense tk promoter, or a non-promoter sequence of the neogene were employed. The resulting levels of luciferase activityof the site-specific integrants were measured directly. Luciferaseactivity of the integrants can be assayed under conventionalculture conditions by simply replacing the culture medium withpotassium phosphate buffer containing luciferin. Only thoseG418^r colonies carrying the tk promoter in the normal orientationexhibited a 21- to 35-fold increase in luciferase activity overthat of the other integrants. These results indicate that thissystem is an effective means of trapping promoter sequencesfrom random mammalian genomic DNA fragments.     

Quantitative control of end products in the melanocyte lineage of the ascidian embryo.

Terminal amounts of tyrosinase (EC 1.10.3.1) activity and melanin pigment in the giant melanocytes of cleavage-arrestedCiona intestinalis (L.) embryos are regulated independently of cell size and number of nuclei in the cells. Embryos were cleavage-arrested in cytochalasin B at a time before the last two divisions of the melanocyte lineage took place. The resulting two giant melanocytes, one from each of the two bilateral melanocyte lineages, developed tyrosinase and melanin. The cells were about three times larger in volume than the normal larval melanocytes and each contained four nuclei instead of just one. Quantitative measurements of melanin synthesized and tyrosinase activity in embryos with the giant melanocytes revealed amounts identical to those found in normal embryos. This specification of exact quantities differs markedly from the situation in mammalian melanocytes where cell volume and gene dosage influence the extent of melanotic differentiation. Quantitative control of differentiation in ascidian melanocytes appears to be mediated by a cytoplasmic determinant segregated through the melanocyte lineage and inherited by one daughter at each division of the lineages.     

Evolutionary and Functional Aspects of Pituitary Gonadotropins in the Green Turtle, Chelonia Mydas

Information on the pituitary gonadotropins in Chelonia mydasrepresents some of the most complete data available for anyreptile and thus provides an important basis for evaluatingevolutionary processes in tetrapod endocrine physiology. Thetwo gonadotropins isolated from Chelonia pituitary glands showclear chemical and immunological homologies to mammalian follicle-stimulatinghormone (FSH) and luteinizing hormone (LH). However, receptorstudies and biological tests indicate that the functions ofthese hormones may be different in turtles and mammals. In particular,Chelonia LH shows an unusual ability to interact with FSH receptorsites and to stimulate physiological functions normally attributedto FSH. Results with Chelonia LH demonstrate that errors mayarise from using mammalian hormones to investigate reproductionin turtles. Measurements of endogenous gonadotropin levels inthe plasma of breeding and nesting Chelonia provide a differentperspective of the potential roles of the FSH and LH from thatobtained in physiological tests. In particular, FSH and LH secretionare markedly dissociated during the nesting cycle; FSH has onlya transient peak during oviposition, whereas LH, along withprogesterone, displays a pronounced "ovulatory" surge in theday following nesting. Preliminary studies with synthetic gonadotropicreleasing factors in Chelonia suggest that these may be usefulin inducing reproductive changes.     

Establishment and characterization of a normal melanocyte cell line derived from pig skin

Several minipig strains develop spontaneous malignant melanoma. As a first step toward the analysis of genes involved in the tumoral progression of melanoma in these animal models, we developed culture conditions for pig melanocytes whereby melanocytes from normal epidermis can be isolated directly onto mitotically inactivated keratinocytes in Eagle's minimal essential medium supplemented with fetal calf serum, tetradecanoyl phorbol acetate (TPA) and cholera toxin. We also derived an immortal line of pigmented melanocytes from the epidermis of a healthy Meishan pig. This cell line, designated PigMel, retains differentiation function in culture, dependence on TPA and cholera toxin and a diploid chromosome number. PigMel melanocytes exhibit morphological and molecular characteristics common to normal mammalian skin melanocytes.     

The Amphibian Melanization Inhibiting Factor (MIF) Blocks the α-MSH Effect on Mouse Malignant Melanocytes

We have found that a melanization inhibitory factor (MIF) extracted from the ventral skin of Rana forreri has a slight inhibitory effect on the activity levels of tyrosinase and dopachrome tautomerase in B16/F10 and Cloudman S-91 murine melanoma cell lines. Furthermore, this factor appears to block the effects of α-MSH on these enzymatic activities. However, MIF treatment does not affect the melanogenic action of theophylline on the same cells, suggesting that MIF acts proximal to MSH-mediated cAMP formation, possibly by interaction with the MSH receptor. In this way, we show that this amphibian factor has biological activity on mammalian melanocytes. This suggests the existence of mammalian counterparts of amphibian MIF in the mouse integument that might regulate epidermal melanocytes. These peptides might be related to the agouti protein, as they share similar mechanisms of action. The interaction of different peptides with the MSH receptor would be a complex but general mechanism responsible for many mammalian coat color variants.     

Free oligosaccharide regulation during mammalian protein N-glycosylation

During protein N-glycosylation in mammalian cells, free oligosaccharides(fOS) are generated from lipid-linked oligosaccharides by apyrophosphatase activity and oligosaccharyltransferase and frommisfolded glycoproteins by peptide:N-glycanase in both the ERand cytoplasm. Trafficking machinery comprising oligosaccharide-specificER and lysosomal transporters, an endo-β-N-acetyl-glucosaminidase,and the cytosolic M2C1 mannosidase drives a flux of fOS fromthe ER to cytoplasm and from the cytoplasm into lysosomes wherefOS are degraded. Transport of fOS out of the ER is normallyefficient and if inhibited causes fOS to be secreted via theGolgi apparatus. By contrast, fOS clearance from the cytosolinto lysosomes is less efficient resulting in low micromolarconcentrations of fOS in the cytoplasm. Structural analysisof cytosolic fOS reveals oligosaccharide families whose relativeabundance highlights the importance of different ER-associateddegradation (ERAD) pathways for misfolded glycoproteins andsuggests that in liver cells substantial amounts of glycoproteinsdestined for ERAD may transit early compartments of the Golgiapparatus. Glycoprotein quality control and ERAD are controlledby N-glycan/lectin interactions and the fOS trafficking pathwaywould seem to ensure that fOS do not interfere with these processeswhich occur in both the ER and cytoplasm. Although Saccharomycescerevisiae strains harbouring mutations in genes of the yeastfOS metabolic pathway do not display obvious phenotypes, mammalianfOS are quantitatively more important and the processes leadingto their regulation are more complex, raising the possibilitythat distinct phenotypes will be seen in mammalian cells oranimals in which fOS metabolism is modified.     

Hairless pigmented guinea pigs: a new model for the study of mammalian pigmentation

A stock of hairless pigmented guinea pigs was developed to facilitate studies of mammalian pigmentation. This stock combines the convenience of a hairless animal with a pigmentary system that is similar to human skin. In both human and guinea pig skin, active melanocytes are located in the basal layer of the interfollicular epidermis. Hairless albino guinea pigs on an outbred Hartley background (CrI:IAF/HA(hr/hr)BR; designated hr/hr) were mated with red-haired guinea pigs (designated Hr/Hr). Red-haired heterozygotes from the F1 generation (Hr/hr) were then mated with each other or with hairless albino guinea pigs. The F2 generation included hairless pigmented guinea pigs that retained their interfollicular epidermal melanocytes and whose skin was red-brown in color. Following UV irradiation, there was an increase in cutaneous pigmentation as well as an increase in the number of active epidermal melanocytes. An additional strain of black hairless guinea pigs was developed using black Hr/Hr animals and a similar breeding scheme. These two strains should serve as useful models for studies of the mammalian pigment system.     

srb: a Bacillus subtilis Gene Encoding a Homologue of the {alpha}-Subunit of the Mammalian Signal Recognition Particle Receptor

We cloned a Bacillus subtilis gene (srb) encoding a homologueof the mammalian signal recognition particle receptor -subunit(SR). The gene is 987 bp in length and encodes a 329-amino acidprotein. The deduced amino acid sequence of the protein shared26.6, 36.2 and 49.7% identity with those of mammalian SR, archaebacterialDP and Escherichia coli FtsY, respectively. The protein containsthree conserved GTP-binding elements like the other three SRPreceptor proteins, though the N-terminal portion of the putativeB. subtilis protein was shorter than the others. Secondary structureprediction showed that an amphipathic -helix is positioned inthe N-terminal region. A defect in srb inhibited cell growthand protein translocation.     

A variation of pigmentation in the glabrous skin of dogs

The usual pigmentation pattern in mammalian skin consists of fixed melanocytes in the basal layer of the epidermis, supplying keratinocytes with melanosomes. We observed that the glabrous skin (rhinaria and footpads) of dogs deviates from this pattern. In dogs, melanocytes are found in both the dermis and epidermis. The epidermal melanocytes are situated in the intercellular spaces of the basal and spinous layers. They are characterized by a quantity of cytoplasm containing a centriole, also developing melanosomes, and in some cases annulate lamellae. There is a high frequency of closely apposed melanocytes in the epidermis. Melanosomes in different stages of formation are also abundant. The morphology of the glabrous skin of dogs suggests transport of melanocytes from the dermis into the epidermis and formation of melanosomes in the epidermis. A distributed and intense pigment formation may be necessary to achieve the black noses of many dog breeds and wild canids, as well as dark footpads despite heavy abrasion and rapid skin renewal.     

Research in Mammalian Mastication

Ongoing research efforts in mammalian mastication have definedseveral broad areas of mutual interest to workers in the discipline.They are (1) interrelationships between masticatory movements,(2) actions of the masticatory muscles, (3) comparisons betweenmasticatory structures and functions, (4) developmental aspects,(5) comparisons between limbs and jaws, and (6) neurophysiologicconsiderations. The roles (potential and actual) of masticatorycentral pattern generators, cerebral "mastication areas," differentneural mechanisms between mammalian taxa, neurophysiologic/morphologicinteractions, and biochemical factors within the total milieuof mammalian mastication are discussed.     

Mammalian Conception, Sev Differentiation, and Hermaphroditism as Viewed in Historical Perspective

This paper surveys in broad perspective some of the steps inthe development of theones of mammalian sex differentiationfrom antiquity to the present. The questions of why and howmales and females differentnte and why there are intersexeshave been answered by conjectures, speculations, and theones.In antiquity, when the existence of mimimhan egg and sperm wasunknownsex determination was con]ec tuicd to depend upon such factorsas which testis contiibuted the semen or which sideof the uterusreceived it. These fanciful notions peisisted until the middleof the 18th centuiy. Discovery of mammalian sperm in the samecentury and the ovum in the 10th centuiy led to obseivationsof fertilization. With advances in genetics andthe identificationot sex chromosomes, the 20th century was prepared to constructtheories foi normal sev differentiation and inteisexuality basedon experimental observation. The bovine freemartin—a naturalexperiment—prompted the hounonal theory leseaich on frogsled to a coittco medullaiy inductor theory, cellular cmmerismin fteemaitin and co tvun was the basis for a cellular theory.Of these, the hormonal theory in particular, provided some verifiablespeculations. However, there is no completely acceptable theoryand new lines of research are badly needed     

Cloning and Identification of the hemG Gene Encoding Protoporphyrinogen Oxidase (PPO) of Escherichia coli K-12

Cells of the VSR751 strain, which was previously isolated asa photoresistant revertant of the visA-deleted (hemH-deleted)strain of Escherichia coli K-12, accumulated uroporphyrin (uro),coproporphyrin (copro) and protoporphyrin IX (proto), but didnot accumulate as much protoporphyrin as cells of the parentalstrain (hemH-deleted). Therefore, we concluded that strain VSR751must be defective in protoporphyrinogen oxidase (PPO), the productof the hemG gene. By complementation analysis using VSR751,we isolated and identified this gene. The hemG gene is locatedat 86 mim on the E. coli chromosome, just upstream of the rrnAoperon, and is transcribed clockwise in the same direction asthe rrnA operon. This gene encodes a 181-amino acid proteinwith a calculated molecular mass of about 21 kDa. Sequence analysisrevealed the presence of flavodoxin motif, suggesting that acofactor of this enzyme is flavin mononucleotide, which is consistentwith the previous report that the mammalian PPO had the flavincofactor.