Epigenetic and pharmacological control of pigmentation via Bromodomain Protein 9 (BRD9)

Lineage-specific differentiation programs are activated by epigenetic changes in chromatin structure. Melanin-producing melanocytes maintain a gene expression program ensuring appropriate enzymatic conversion of metabolites into the pigment, melanin, and transfer to surrounding cells. During neuroectodermal development, SMARCA4 (BRG1), the catalytic subunit of SWItch/Sucrose Non-Fermentable (SWI/SNF) chromatin remodeling complexes, is essential for lineage specification. SMARCA4 is also required for development of multipotent neural crest precursors into melanoblasts, which differentiate into pigment-producing melanocytes. In addition to the catalytic domain, SMARCA4 and several SWI/SNF subunits contain bromodomains which are amenable to pharmacological inhibition. We investigated the effects of pharmacological inhibitors of SWI/SNF bromodomains on melanocyte differentiation. Strikingly, treatment of murine melanoblasts and human neonatal epidermal melanocytes with selected bromodomain inhibitors abrogated melanin synthesis and visible pigmentation. Using functional genomics, iBRD9, a small molecule selective for the bromodomain of BRD9 was found to repress pigmentation-specific gene expression. Depletion of BRD9 confirmed a requirement for expression of pigmentation genes in the differentiation program from melanoblasts into pigmented melanocytes and in melanoma cells. Chromatin immunoprecipitation assays showed that iBRD9 disrupts the occupancy of BRD9 and the catalytic subunit SMARCA4 at melanocyte-specific loci. These data indicate that BRD9 promotes melanocyte pigmentation whereas pharmacological inhibition of BRD9 is repressive.     

Mitochondrial DNA sequences of various species of the genus Equus with special reference to the phylogenetic relationship between Przewalskii's wild horse and domestic horse

The noncoding region between tRNA^Pro and the large conserved sequence block is the most variable region in the mammalian mitochondrial DNA D-loop region. This variable region (ca. 270 bp) of four species of Equus, including Mongolian and Japanese native domestic horses as well as Przewalskii's (or Mongolian) wild horse, were sequenced. These data were compared with our recently published Thoroughbred horse mitochondrial DNA sequences. The evolutionary rate of this region among the four species of Equus was estimated to be 2–4 × 10^–8 per site per year. Phylogenetic trees of Equus species demonstrate that Przewalskii's wild horse is within the genetic variation among the domestic horse. This suggests that the chromosome number change (probably increase) of the Przewalskii's wild horse occurred rather recently.Correspondence to: N. Ishida     

X-ray structure of recombinant horse L-chain apoferritin at 2.0 Å resolution: implications for stability and function

The X-ray structure of recombinant horse L-chain (rL) apoferritin, solved at 2.0?Å resolution with a final R factor of 17.9%, gives evidence that the residue at position 93 in the sequence is a proline and not a leucine, as found in earlier sequencing studies. The structure is isomorphous with other apoferritin structures, and we thus draw particular attention to those structural features which can be related to the stability and function of the protein. Analysis of hydrogen bonding and salt bridge interactions shows that dimers and tetramers are the most stable molecular entities within the protein shell: a result confirming earlier biophysical experiments. The stability of horse rL apoferritin to both dissociation into subunits at acidic pH values and to complete unfolding in guanidine chloride solutions is compared with that of other apoferritins. This emphasizes the role played by the salt bridge in the stability of this protein family. The horse rL apoferritin is significantly more resistant to denaturation than horse spleen ferritin, which in turn is more resistant than any human rH apoferritins, even those for which a salt bridge is restored. Finally, this structure determination not only establishes that a preformed pocket exists in L-chain apoferritin, at a site known to be able to bind porphyrin, but also underlines the particular function of a cluster of glutamic acids (E53, E56, E57 and E60) located at the entrance of this porphyrin-binding pocket.     

Directional and anteroposterior asymmetry of common white markings in the legs of the Arabian horse: response to selection

Arabian bay horses manifest, on the average, more common white markings in their hind legs than their forelegs (anteroposterior asymmetry) and more common white markings in their left legs than their right legs (directional asymmetry). To determine if genetic variation exists for these types of asymmetry, the phenotypic response was studied in bay foals when their dams and sires were selected for the directions of fore-hind and left-right differences. In the fore-hind studies, the quantitative shifts in the bay foals were in the direction specified by the selection scheme and the observed deviations were all statistically significant. The shifts were also consistently in the direction favored by selection in the left-right studies, but only two of six observed deviations were statistically significant using a one-tailed test of significance. Thus, only marginal statistical evidence is available to support the observed consistent responses to selection in the left-right studies. These differential responses are reflected in the magnitudes of the heritability estimates. Based on the overall results, it is concluded that both types of asymmetry have a genetic basis in the Arabian horse, but much more genetic variation is present for anteroposterior asymmetry than for directional asymmetry. This revised version was published online in July 2006 with corrections to the Cover Date.     

Vibrio trachuri Sp. Nov., a New Species Isolated from Diseased Japanese Horse Mackerel

A new species, Vibrio trachuri sp. nov., was isolated from the cultured Japanese horse mackerel (Trachurus japonicus). These Vibrio were Gram negative, motile rods and formed yellow colonies on BTB teepol and TCBS plate, turned TSI medium to yellow and was sensitive to 150 μM O/129 (2,4-diamino-6,7-diisopropyl pteridine phosphate) like Listonella anguillarum which has been described as Vibrio anguillarum. However, the results of VP test and decarboxylation of lysine or dihydrolation of arginine suggested that these Vibrio are rather closely related to V. parahaemolyticus. DNA similarity determined by the microplate hybridization technique revealed that these Vibrio are genetically quite distant from Listonella anguillarum or V. parahaemolyticus and rather close to V. harveyi, although there was no Vibrio species which had more than 70% similarity value. From these results we propose to nominate Vibrio trachuri sp. nov. for this new Vibrio species.     

Abdominal pigmentation in Drosophila melanogaster females from natural Indian populations

Females of Drosophila melanogaster collected from five geographically distant populations in India were analysed for the intensity of pigmentation in the 5th, 6th and 7th segments of the abdomen. In all three segments, this intensity was found to vary among individuals of any given population, and, furthermore, different populations differ with respect to this phenotypic trait. Statistical analysis revealed significant intra- and interpopulational variation. A clinical pattern was detected: females from populations closer to the equator tended to have lighter cuticle, in which case differences between the three segments could not be detected and all three segments responded both independently and jointly to latitudinal variation, as indicated by a statistically significant positive correlation between latitude and pigmentation score. This is the first report on abdominal pigmentation analysis in natural populations of D. melanogaster that provides evidence that phenotypic flexibility reflects temperature differences, as a result of which abdominal pigmentation shows geographic differentiation.     

Effects of forskolin and cholera toxin on cyclic AMP release in a neurotensin-secreting rat C-cell line

The effects of forskolin and cholera toxin on the regulation of cAMP release were studied in a neurotensin-secreting rat C-cell line. The interaction of these agents with norepinephrine, a potent neurotensin secretagogue, was also investigated. Forskolin stimulated cAMP release 10(2)-10(3) fold while it increased neurotensin release 2-3 fold. Cholera toxin caused a 10(2)-10(3) fold increase in cAMP release and had no effect on neurotensin release. We conclude that the 44-2 C-cells provide a new model for studying the regulation of the concomitant (via forskolin) or independent (via cholera toxin) secretion of cyclic AMP and/or neurotensin.     

Migration of Gasterophilus intestinalis larvae (Diptera: Gasterophilidae) in the equine oral cavity

Cogley T. P., Anderson J. R. and Cooley L. J. 1982. Migration of Gasterophilus intestinalis larvae (Diptera: Gasterophilidae) in the equine oral cavity. International Journal for Parasitology12: 473–480. Larvae of G. intestinalis pursued a specific migratory pathway within the equine oral cavity en route to the stomach. The larval migration included the following sequence: burrowing in the tongue mucosa, invasion of the interdental spaces, transitory attachment at the root of the tongue and movement to the stomach. The molt from first to second instar did not occur in the tongue, as commonly believed, but between the interdental spaces. Ninety five percent of the larvae invading the interdental spaces were associated with gingiva of the upper molars. SEM analysis revealed further details of the oral migration: (1) air holes excavated in the epithelium which connect with deeper burrows; (2) an intimate association between air holes and posterior spiracles of larvae; (3) precise impressions of larvae in tissue immediately surrounding the most recently formed burrows; and (4) initial larval entry into the tongue through the use of natural disruptions or healing lesions. Factors influencing the development of the oral migration are discussed.     

Primary structure of horse erythrocyte glycophorin HA. its amino acid sequence has a unique homology with those of human and porcine erythrocyte glycophorins

Summary The complete amino acid sequence of the major sialoglycoproteins of horse erythrocyte membranes, glycophorin HA, was determined by manual sequencing methods, using tryptic, chymotryptic, and cyanogen bromide fragments. Glycophorin HA is a polypeptide chain of 120 amino acid residues and contains 10 oligosaccharide units attached to the amino-terminal side of the molecule. Its amino terminus is pyroglutamic acid. All of the oligosaccharides are linked O-glycosidically to threonine or serine residues. The amino acid sequence is consistent with the transmembrane orientation of glycophorins.There is no significant homology between the glycosylated domains of horse, human, and porcine glycophorins, but there is a considerable homology between the hydrophobic domains of the three glycophorins, which interact with the lipid bilayer of the erythrocyte membrane.