A genomewide survey of developmentally relevant genes in Ciona intestinalis - V. Genes for receptor tyrosine kinase pathway and Notch signaling pathway

In the present survey, we identified most of the genes involved in the receptor tyrosine kinase (RTK), mitogen activated protein kinase (MAPK) and Notch signaling pathways in the draft genome sequence of Ciona intestinalis, a basal chordate. Compared to vertebrates, most of the genes found in the Ciona genome had fewer paralogues, although several genes including ephrin, Eph and fringe appeared to have multiplied or duplicated independently in the ascidian genome. In contrast, some genes including kit/flt, PDGF and Trk receptor tyrosine kinases were not found in the present survey, suggesting that these genes are innovations in the vertebrate lineage or lost in the ascidian lineage. The gene set identified in the present analysis provides an insight into genes for the RTK, MAPK and Notch signaling pathways in the ancient chordate genome and thereby how chordates evolved these signaling pathway.

Caveolin interacts with the angiotensin II type 1 receptor during exocytic transport but not at the plasma membrane

The mechanisms involved in angiotensin II type 1 receptor (AT(1)-R) trafficking and membrane localization are largely unknown. In this study, we examined the role of caveolin in these processes. Electron microscopy of plasma membrane sheets shows that the AT(1)-R is not concentrated in caveolae but is clustered in cholesterol-independent microdomains; upon activation, it partially redistributes to lipid rafts. Despite the lack of AT(1)-R in caveolae, AT(1)-R. caveolin complexes are readily detectable in cells co-expressing both proteins. This interaction requires an intact caveolin scaffolding domain because mutant caveolins that lack a functional caveolin scaffolding domain do not interact with AT(1)-R. Expression of an N-terminally truncated caveolin-3, CavDGV, that localizes to lipid bodies, or a point mutant, Cav3-P104L, that accumulates in the Golgi mislocalizes AT(1)-R to lipid bodies and Golgi, respectively. Mislocalization results in aberrant maturation and surface expression of AT(1)-R, effects that are not reversed by supplementing cells with cholesterol. Similarly mutation of aromatic residues in the caveolin-binding site abrogates AT(1)-R cell surface expression. In cells lacking caveolin-1 or caveolin-3, AT(1)-R does not traffic to the cell surface unless caveolin is ectopically expressed. This observation is recapitulated in caveolin-1 null mice that have a 55% reduction in renal AT(1)-R levels compared with controls. Taken together our results indicate that a direct interaction with caveolin is required to traffic the AT(1)-R through the exocytic pathway, but this does not result in AT(1)-R sequestration in caveolae. Caveolin therefore acts as a molecular chaperone rather than a plasma membrane scaffold for AT(1)-R.

The role of melanocortin-1 receptor polymorphism in skin cancer risk phenotypes

We have examined melanocortin-1 receptor (MC1R) variant allele frequencies in the general population and in a collection of adolescent dizygotic and monozygotic twins to determine statistical associations of pigmentation phenotypes with increased skin cancer risk. This included hair and skin color, freckling, mole count and sun exposed skin reflectance. Nine variants were studied and designated as either strong R (OR = 63; 95% CI 32-140) or weak r (OR = 5; 95% CI 3-11) red hair alleles. Penetrance of each MC1R variant allele was consistent with an allelic model where effects were multiplicative for red hair but additive for skin reflectance. To assess the interaction of the brown eye color gene BEY2/OCA2 on the phenotypic effects of variant MC1R alleles we imputed OCA2 genotype in the twin collection. A modifying effect of OCA2 on MC1R variant alleles was seen on constitutive skin color, freckling and mole count. In order to study the individual effects of these variants on pigmentation phenotype we have established a series of human primary melanocyte strains genotyped for the MC1R receptor. These include strains which are MC1R wild-type consensus, variant heterozygotes, and homozygotes for strong R alleles Arg151Cys and Arg160Trp. Ultrastructural analysis demonstrated that only consensus strains contained stage III and IV melanosomes in their terminal dendrites whereas Arg151Cys and Arg160Trp homozygous strains contained only immature stage I and II melanosomes. Such genetic association studies combined with the functional analysis of MC1R variant alleles in melanocytic cells should provide a link in understanding the association between pigmentary phototypes and skin cancer risk.

Growth hormone receptor and insulin-like growth factor-I immunoreactivity in osteoclast-like cells during tooth eruption in the toothless (Osteopetrotic) rat following treatment with colony-stimulating factor-1

In the toothless (tl/tl) osteopetrotic rat, teeth form but fail to erupt. Treatment of tl/tl rats with colony-stimulating factor-1 (CSF-1) activates bone resorption by osteoclasts, permits tooth eruption, and up-regulates the immunoreactivity of bone marrow mononuclear cells to growth hormone receptor (GHr) and insulin-like growth factor (IGF)-I. This study examined the distribution of tartrate-resistant acid phosphatase (TRAP) and immunoreactivity for GHr and IGF-I in osteoclast-like cells located on the alveolar bone margin, adjacent to the lower first molar crown, in 14-day-old normal and tl/tl rats, following treatment with CSF-1. Osteoclast-like cells demonstrated a positive reaction for TRAP, GHr, and IGF-I in all groups. However, in tl/tl tissue, osteoclast-like cells were generally negative for GHr. There was no significant difference in the total number of TRAP, GHr, and IGF-I-positive osteoclast-like cells on the adjacent bone margin in normal, normal treated with CSF-1, and tl/tl rats. CSF-1 treatment of the tl/tl rat significantly increased the total number of osteoclast-like cells, which were positive for TRAP (p < 0.001), GHr (p < 0.05) and IGF-I (P < 0.01).

The receptor tyrosine kinase regulator sprouty1 is a target of the tumor suppressor WT1 and important for kidney development

WT1 encodes a transcription factor involved in kidney development and tumorigenesis. Using representational difference analysis, we identified a new set of WT1 targets, including a homologue of the Drosophila receptor tyrosine kinase regulator, sprouty. Sprouty1 was up-regulated in cell lines expressing wild-type but not mutant WT1. WT1 bound to the endogenous sprouty1 promoter in vivo and directly regulated sprouty1 through an early growth response gene-1 binding site. Expression of Sprouty1 and WT1 overlapped in the developing metanephric mesenchyme, and Sprouty1, like WT1, plays a key role in the early steps of glomerulus formation. Disruption of Sprouty1 expression in embryonic kidney explants by antisense oligonucleotides reduced condensation of the metanephric mesenchyme, leading to a decreased number of glomeruli. In addition, sprouty1 was expressed in the ureteric tree and antisense-treated ureteric trees had cystic lumens. Therefore, sprouty1 represents a physiologically relevant target gene of WT1 during kidney development.

Screening of human primary melanocytes of defined melanocortin-1 receptor genotype: Pigmentation marker, ultrastructural and UV-survival studies

Recent population studies have demonstrated an association with the red-hair and fair-skin phenotype with variant alleles of the melanocortin-1 receptor (MC1R) which result in amino acid substitutions within the coding region leading to an altered receptor activity. In particular, Arg151Cys, Arg160Trp and Asp294His were the most commonly associated variants seen in the south-east Queensland population with at least one of these alleles found in 93% of those with red hair. In order to study the individual effects of these variants on melanocyte biology and melanocytic pigmentation, we established a series of human melanocyte strains genotyped for the MC1R receptor which included wild-type consensus, variant heterozygotes, compound heterozygotes and homozygotes for Arg151Cys, Arg160Trp, Val60Leu and Val92Met alleles. These strains ranged from darkly pigmented to amelanotic, with all strains of consensus sequence having dark pigmentation. UV sensitivity was found not to be associated with either MC1R genotype or the level of pigmentation with a range of sensitivities seen across all genotypes. Ultrastructural analysis demonstrated that while consensus strains contained stage IV melanosomes in their terminal dendrites, Arg151Cys and Arg160Trp homozygote strains contained only stage II melanosomes. This was despite being able to show expression of tyrosinase and tyrosinase-related protein-1 markers, although at reduced levels and an ability to convert exogenous 3,4-dihydroxyphenyl-alanine (DOPA) to melanin in these strains.

Phylogeny of the lice (Insecta, Phthiraptera) inferred from small subunit rRNA

There has been much argument about the phylogenetic relationships of the four suborders of lice (Insecta: Phthiraptera). Lyal's study of the morphology of lice indicated that chewing/biting lice (Mallophaga) are paraphyletic with respect to sucking lice (Anoplura). To test this hypothesis we inferred the phylogeny of 33 species of lice from small subunit (SSU) rRNA sequences (18S rRNA). Liposcelis sp. from the Liposcelididae (Psocoptera) was used for outgroup reference. Phylogenetic relationships among the four suborders of lice inferred from these sequences were the same as those inferred from morphology. The Amblycera is apparently the sister-group to all other lice whereas the Rhynchophthirina is apparently sister to the Anoplura; these two suborders are sister to the Ischnocera, i.e. (Amblycera (Ischnocera (Anoplura, Rhynchophthirina))). Thus, the Mallophaga (Amblycera, Ischnocera, Rhynchophthirina) is apparently paraphyletic with respect to the Anoplura. Our analyses also provide evidence that: (i) each of the three suborders of lice that are well represented in our study (the Amblycera, Ischnocera, and Anoplura) are monophyletic; (ii) the Boopiidae is monophyletic; (iii) the genera Heterodoxus and Latumcephalum (Boopiidae) are more closely related to one another than either is to the genus Boopia (also Boopiidae); (iv) the Ricinidae and Laemobothridae may be sister-taxa; (v) the Philopteridae may be paraphyletic with respect to the Trichodectidae; (vi) the genera Pediculus and Pthirus are more closely related to each other than either is to the genus Pedicinus ; and (vii) in contrast to published data for mitochondrial genes, the rates of nucleotide substitution in the SSU rRNA of lice are not higher than those of other insects, nor do substitution rates in the suborders differ substantially from one another.

Molecular and functional characterization of an octopamine receptor from honeybee (Apis mellifera) brain

Biogenic amines and their receptors regulate and modulate many physiological and behavioural processes in animals. In vertebrates, octopamine is only found in trace amounts and its function as a true neurotransmitter is unclear. In protostomes, however, octopamine can act as neurotransmitter, neuromodulator and neurohormone. In the honeybee, octopamine acts as a neuromodulator and is involved in learning and memory formation. The identification of potential octopamine receptors is decisive for an understanding of the cellular pathways involved in mediating the effects of octopamine. Here we report the cloning and functional characterization of the first octopamine receptor from the honeybee, Apis mellifera . The gene was isolated from a brain-specific cDNA library. It encodes a protein most closely related to octopamine receptors from Drosophila melanogaster and Lymnea stagnalis . Signalling properties of the cloned receptor were studied in transiently transfected human embryonic kidney (HEK) 293 cells. Nanomolar to micromolar concentrations of octopamine induced oscillatory increases in the intracellular Ca2+ concentration. In contrast to octopamine, tyramine only elicited Ca2+ responses at micromolar concentrations. The gene is abundantly expressed in many somata of the honeybee brain, suggesting that this octopamine receptor is involved in the processing of sensory inputs, antennal motor outputs and higher-order brain functions.

Invertebrate D2 type dopamine receptor exhibits age-based plasticity of expression in the mushroom bodies of the honeybee brain

We have isolated a cDNA clone from the honeybee brain encoding a dopamine receptor, AmDop2, which is positively coupled to adenylyl cyclase. The transmembrane domains of this receptor are 88% identical to the orthologous Drosophila D2 dopamine receptor, DmDop2, though phylogenetic analysis and sequence homology both indicate that invertebrate and vertebrate D2 receptors are quite distinct. In situ hybridization to mRNA in whole-mount preparations of honeybee brains reveals gene expression in the mushroom bodies, a primary site of associative learning. Furthermore, two anatomically distinct cell types in the mushroom bodies exhibit differential regulation of AmDop2 expression. In all nonreproductive females (worker caste) and reproductive males (drones) the receptor gene is strongly and constitutively expressed in all mushroom body interneurons with small cell bodies. In contrast, the large cell-bodied interneurons exhibit dramatic plasticity of AmDop2 gene expression. In newly emerged worker bees (cell-cleaning specialists) and newly emerged drones, no AmDop2 transcript is observed in the large interneurons whereas this transcript is abundant in these cells in the oldest worker bees (resource foragers) and older drones. Differentiation of the mushroom body interneurons into two distinct classes (i.e., plastic or nonplastic with respect to AmDop2 gene expression) indicates that this receptor contributes to the differential regulation of distinct neural circuits. Moreover, the plasticity of expression observed in the large cells implicates this receptor in the behavioral maturation of the bee.

Biochemical characterization of two mutants of human pyruvate dehydrogenase, F205L and T231A of the E1 alpha subunit

Mutations in the E1alpha subunit of the pyruvate dehydrogenase multienzyme complex may result in congenital lactic acidosis, but little is known about the consequences of these mutations at the enzymatic level. Here we characterize two mutants (F205L and T231A) of human pyruvate dehydrogenase in vitro, using the enzyme expressed in Escherichia coli. Wild-type and mutant proteins were purified successfully and their kinetic parameters were measured. F205L shows impaired binding of the thiamin diphosphate cofactor, which may explain why patients carrying this mutation respond to high-dose vitamin B-1 therapy. T231A has very low activity and a greatly elevated K-m for pyruvate, and this combination of effects would be expected to result in severe lactic acidosis. The results lead to a better understanding of the consequences of these mutations on the functional and structural properties of the enzyme, which may lead to improved therapies for patients carrying these mutations.

Autosomal dominant hypocalcemia: A novel activating mutation (E604K) in the cysteine-rich domain of the calcium-sensing receptor

We report a novel activating mutation (E604K) of the calcium-sensing receptor in a family with autosomal dominant hypocalcemia. Whereas all affected individuals exhibited marked hypocalcemia, some cases with untreated hypocalcemia exhibited seizures in infancy, whereas others were largely asymptomatic from birth into adulthood. The missense mutation E604K (G2182A, GenBank accession no. U20759), which affects an amino acid residue in the C terminus of the cysteine-rich domain of the extracellular head, co-segregated with hypocalcemia in all seven individuals for whom DNA was available. Two unaffected, normocalcemic members of the family did not exhibit the mutation. The molecular impact of the mutation on two key components of the signaling response was assessed in HEK-293 cells transiently transfected with cDNA corresponding to either the wild-type calcium-sensing receptor or the E604K mutation derived by site-directed mutagenesis. There was a significant leftward shift in the concentration response curves for the effects of extracellular Ca2+ on both intracellular Ca2+ mobilization (determined by aequorin luminescence) and MAPK activity (determined by luciferase expression). The C terminus of the cysteine-rich domain of the extracellular head may normally act to suppress receptor activity in the presence of low extracellular Ca2+ concentrations.

Bovine-serum-albumin-containing receptor phase better predicts transdermal absorption parameters for lipophilic compounds

Based on the hypothesis that limited receptor solubility of lipophilic compounds may result in lower observed permeability parameters, the aim of this study was to determine the in vitro human epidermal permeability coefficients and membrane retention of a series of aliphatic alcohols (C1-C10, log p -0.72 to 4.06) using two different receptor solutions (water and 4% bovine serum albumin in phosphate-buffered saline). Aqueous solutions of radiolabeled alcohols were dosed into the stratum corneum side of membranes mounted in side-by-side glass diffusion cells. Appearance of alcohol in the receptor compartment filled with either of the two solutions was monitored over a 7 h period when both stratum corneum (assessed by tape stripping) and the remaining epidermis levels of radioactivity were determined. In a separate study the degree of binding of alcohols to 4% bovine serum albumin was determined. The data showed increased receptor phase solubility in the bovine serum albumin solution and higher permeability coefficients for the more lipophilic alcohols in the series. No changes were seen in the partitioning of the alcohols from the vehicle into either the stratum corneum or tape-stripped epidermis with the two receptor phases; however, a decrease in the amount of the more lipophilic alcohols partitioning into the water receptor phase from the tape-stripped epidermis was observed. We conclude that bovine serum albumin receptor phase allows better estimation of real permeability parameters for lipophilic compounds due to its increased solubility capacity and we question whether permeability parameters for lipophilic solutes from older data sets based on aqueous receptor phases are completely reliable.