User satisfaction with rehabilitation services delivered using Internet video.

Rehabilitation services to four remote sites in New Brunswick were delivered via PC-based videoconferencing equipment, using ADSL connections to the Internet. Approximately 40 people used the equipment over 18 months. There were 32 videoconference sessions. A total of 60 questionnaires were returned (a 94% response rate). In 31 of the 32 videoconferences, a connection was successfully established between the computers. The videoconferences lasted on average 20 min. The most frequent applications were viewing of rehabilitative equipment and video communication. The technology was found to be useful and provided an enhanced form of communication from the video component. There were some problems with the stability and reliability of the equipment.

Estrogen agonist and antagonist action on the human estrogen receptor in Drosophila.

The estrogen receptor (ER) regulates the expression of genes involved in the growth, proliferation and differentiation of skeletal, cardiovascular, neural and reproductive tissues. A basic scheme for the mechanism for ER action has been developed, but precise details on the interactions between ER and the cellular signaling and transcription machinery required for receptor-mediated regulation of specific target genes are still lacking. We have developed a genetic approach to explore the functional interactions of ER. In this work, we describe the development of an estrogen responsive system in the fruit fly, Drosophila melanogaster. Transgenic flies carrying the human ER alpha and an estrogen responsive green fluorescent protein (GFP) reporter gene were constructed. In vivo expression of the GFP reporter gene was observed when larvae were grown on a food source containing steroidal or nonsteroidal estrogens. The induction of the reporter gene by estrogens was blocked upon treatment with tamoxifen, an estrogen antagonist. However, we failed to recapitulate ligand-independent activation of the receptor in vivo or in cultured Drosophila cells. An estrogen responsive Drosophila system could be used to identify and characterize the complex functional interactions between ER and the other components of the cellular transcriptional apparatus.

The ciD mutation encodes a chimeric protein whose activity is regulated by Wingless signaling.

The Drosophila cubitus interruptus (ci) gene encodes a sequence-specific DNA-binding protein that regulates transcription of Hedgehog (Hh) target genes. Activity of the Ci protein is posttranslationally regulated by Hh signaling. In animals homozygous for the ciD mutation, however, transcription of Hh target genes is regulated by Wingless (Wg) signaling rather than by Hh signaling. We show that ciD encodes a chimeric protein composed of the regulatory domain of dTCF/Pangolin (Pan) and the DNA binding domain of Ci. Pan is a Wg-regulated transcription factor that is activated by binding of Armadillo (Arm) to its regulatory domain. Arm is thought to activate Pan by contributing a transactivation domain. We find that a constitutively active form of Arm potentiates activity of a CiD transgene and coimmunoprecipitates with CiD protein. The Wg-responsive activity of CiD could be explained by recruitment of the Arm transactivation function to the promoters of Hh-target genes. We suggest that wild-type Ci also recruits a protein with a transactivation domain as part of its normal mechanism of activation.

Expression of sonic hedgehog, patched, and Gli1 in developing taste papillae of the mouse.

Lingual taste buds form within taste papillae, which are specialized structures that develop in a characteristic spatial and temporal pattern. To investigate the signaling events responsible for patterning and morphogenesis of taste papillae, the authors examined the time course and distribution of expression of several related developmental signaling genes as well as the time course of innervation of taste papillae in mouse embryos from embryonic day 12 (E12) to E18. Lingual expression of the signaling molecule Sonic hedgehog (Shh), its receptor Patched (Ptc), and the Shh-activated transcription factor Gli1 were assayed by using in situ hybridization. Shh is expressed broadly in the lingual epithelium at E12 but becomes progressively restricted to developing circumvallate and fungiform papillary epithelia. Shh is expressed specifically within the central cells of the papillary epithelium starting at E13.5 and persisting through E18. Ptc and Gli1 expression follow a pattern similar to that of Shh. Compared with Shh, Ptc is expressed in larger regions surrounding the central papillary cells and also in the mesenchyme underlying Shh-expressing epithelium. Innervation of taste papillae was examined by using the panneuronal antibody to ubiquitin carboxyl terminal hydrolase (protein gene product 9.5). Nerves reach the basal lamina of developing taste papillae at E14 to densely innervate the papillary epithelium by E16. Thus, the pattern of Shh expression within developing taste papillae is established prior to innervation, ruling out neuronal induction of papillae. The results suggest that the Shh signaling pathway may be involved in: 1) establishing papillary boundaries in taste papilla morphogenesis, 2) papillary epithelial-mesenchymal interactions, and/or 3) specifying the location or development of taste buds within taste papillae.

Hedgehog signaling regulates transcription through cubitus interruptus, a sequence-specific DNA binding protein.

Hedgehog (Hh) is a member of a family of secreted proteins that direct patterning at multiple stages in both Drosophila and vertebrate development. During Drosophila embryogenesis, Hh protein is secreted by the cells of the posterior compartment of each segment. hh activates transcription of wingless (wg), gooseberry (gsb), and patched (ptc) in the cells immediately adjacent to Hh-secreting cells. Hh signaling is thought to involve the segment polarity gene cubitus interruptus (ci). ci encodes a zinc finger protein of the Gli family of sequence-specific DNA binding proteins. ci mRNA is expressed in all non-Hh expressing cells. Here we demonstrate ci activity is both necessary and sufficient to drive expression of Hh-responsive genes in the Drosophila embryos. We show that Ci is a sequence-specific DNA binding protein that drives transcription from the wg promoter in transiently transfected cells. We demonstrate that Ci binding sites in the wg promoter are necessary for this transcriptional activation. These data taken together provide strong evidence that Ci is a transcriptional effector of Hh signaling.

Hedgehog signaling regulates transcription through Gli/Ci binding sites in the wingless enhancer.

The segment polarity gene cubitus interruptus (ci) encodes a transcriptional effector of Hedgehog (Hh) signaling in Drosophila. The Ci gene product is a zinc finger protein belonging to the Gli family of sequence-specific DNA binding proteins. After gastrulation, segmental expression of the segment polarity gene wingless (wg) is maintained by Hh signaling in a pathway requiring Ci activity. In the absence of Hh or Ci activity, wg expression is initiated normally and then fades in the ectoderm after stage 10. We have previously identified a wingless enhancer region whose Ci binding sites mediate Ci-dependent transcriptional activation in transiently transfected cells. Here we demonstrate that Hh and Patched (Ptc) act through those Ci binding sites to modulate the level of Ci-dependent transcriptional activation in S2 cells. We demonstrate that this same wg enhancer region is Hh responsive in vivo and that its Ci binding sites are necessary for its activity. This provides strong evidence that Hh affects wg transcription through post-translational activation of Ci.

The tumour-suppressor gene patched encodes a candidate receptor for Sonic hedgehog.

The protein Sonic hedgehog (Shh) controls patterning and growth during vertebrate development. Here we demonstrate that it binds Patched (vPtc), which has been identified as a tumour-suppressor protein in basal cell carcinoma, with high affinity. We show that Ptc can form a physical complex with a newly cloned vertebrate homologue of the Drosophila protein Smoothened (vSmo), and that vSmo is coexpressed with vPtc in many tissues but does not bind Shh directly. These findings, combined with available genetic evidence from Drosophila, support the hypothesis that Ptc is a receptor for Shh, and that vSmo could be a signalling component that is linked to Ptc.

The Drosophila smoothened gene encodes a seven-pass membrane protein, a putative receptor for the hedgehog signal.

Smoothened (smo) is a segment polarity gene required for correct patterning of every segment in Drosophila. The earliest defect in smo mutant embryos is loss of expression of the Hedgehog-responsive gene wingless between 1 and 2 hr after gastrulation. Since smo mutant embryos cannot respond to exogenous Hedgehog (Hh) but can respond to exogenous Wingless, the smo product functions in Hh signaling. Smo acts downstream of or in parallel to Patched, an antagonist of the Hh signal. The smo gene encodes an integral membrane protein with characteristics of G protein-coupled receptors and shows homology to the Drosophila Frizzled protein. Based on its predicted physical characteristics and on its position in the Hh signaling pathway, we suggest that smo encodes a receptor for the Hh signal.

Distinct pathways for autocrine and paracrine Wingless signalling in Drosophila embryos.

Two secreted proteins, Wingless and Hedgehog, instruct cell fates within the segmented epidermis of Drosophila embryos (reviewed in ref. 5). Wingless (Wg) is expressed by the most posterior cells in each parasegment; Hedgehog (Hh) is expressed in the most anterior cells of the next parasegment. Immediately after gastrulation, the two cell types are mutually dependent. Local Wg signalling stabilizes Hh expression and local Hh signalling stabilizes Wg expression. Direct Wg autoregulation (autocrine signalling) is masked by its paracrine role in maintaining hh, which in turn maintains wg. I have used zeste-white3 (zw3) and patched (ptc) mutant backgrounds to uncouple genetically this positive-feedback loop and to study autocrine Wg signalling. I report here that direct Wg autoregulation differs from Wg signalling to adjacent cells in the importance of fused (fu), smoothened (smo) and cubitus interruptus (ci) relative to zw3 and armadillo (arm). I also find that Wg autoregulation during this early hh-dependent phase differs from later Wg autoregulation by lack of gooseberry (gsb) participation.

patched overexpression causes loss of wingless expression in Drosophila embryos.

The patched (ptc) segment polarity gene of Drosophila encodes a transmembrane protein involved in cell signaling that establishes pattern within the segment. In the posterior half of the parasegment Patched protein represses transcription of the wingless (wg) gene by an unknown mechanism. In the most posterior row of cells in each parasegment this repression is neutralized by a signal possibly carried by the product of the hedgehog gene, allowing wg expression. High levels of Patched expression might therefore overcome the repression and repress wg in all cells. Here we use a heat shock-inducible promoter to transiently express high levels of Patched in all cells. A single pulse of Patched transgene expression has little or no effect on the segmental pattern, as has been previously reported. Repeated pulses of Patched production drastically alter the segment pattern to mimic embryos lacking one of the wg class of segment polarity genes. We observe repression of wg and gooseberry (a wg class gene) transcription in the germband ectoderm but not in the head. Expression of two other segment polarity genes, engrailed and cubitus interruptus, is unaffected. Thus excess Patched is capable of overcoming the neutralizing signal.

Comparative studies of Drosophila Antennapedia genes.

The Antennapedia (Antp) homeotic gene of Drosophila melanogaster controls cell fates and pattern formation in the epidermis, nervous system and mesoderm of thoracic segments. Its expression is controlled at the levels of transcription, alternative RNA splicing, polyadenylation and translation. Two nested Antp transcription units extend over 103 kb and produce sixteen different transcripts. We have compared the Antp genes of Drosophila virilis, Drosophila subobscura and D. melanogaster to determine which structural features are conserved and therefore may be important to the gene's function. The overall gene structures are similar. There are many conserved sequence blocks throughout the large introns, at least 15 kb upstream of the first promoter, and at least 3 kb downstream of the last polyadenylation site. Intron and exon sequence conservation around alternative splice sites indicates that alternative protein coding forms may also be conserved. Protein coding potential is perfectly conserved around the C-terminal homeodomain, well conserved in the N-terminal region, and more variable in the middle. The large size of the Antp gene may reflect a large number of control elements necessary for appropriate Antp protein expression. The conservation of transcript complexity suggests functional requirements for the different protein forms.

The Drosophila patched gene encodes a putative membrane protein required for segmental patterning.

The patched (ptc) gene is one of several segment polarity genes required for correct patterning within every segment of Drosophila. The absence of ptc gene function causes a transformation of the fate of cells in the middle part of each segment so that they form pattern elements characteristic of cells positioned around the segment border. Analysis of the mutant phenotype demonstrates that both segment and parasegment borders are included in the duplicated pattern of ptc mutants. We have cloned the ptc gene and deduced that the product is a 1286 amino acid protein with at least seven putative transmembrane alpha helices. ptc RNA is expressed in embryos in broad stripes of segmental periodicity that later split into two stripes per segment primordium. The pattern of expression does not directly predict the transformation seen in ptc mutant embryos, suggesting that ptc participates in cell interactions that establish pattern within the segment.

Calcium-dependent calmodulin binding to cholinergic synaptic vesicles.

Calmodulin is a major nerve terminal protein and a potential mediator of calcium-dependent nerve terminal functions. Calcium-dependent calmodulin binding has been reported in secretory membrane preparations including chromaffin granules and crude rat brain vesicles. Here we demonstrate a calcium-dependent calmodulin-binding site on cholinergic synaptic vesicles from electric organ. It is saturable with high affinity (KD = 10 nM; Bmax = 80 pmol/mg). The binding is inhibited by trifluoperazine (I50 = 8 microM) and is at least 1000-fold specific for calmodulin over troponin C. Association and dissociation rates (k = 3.1 X 10(6) M-1S-1; k-1 = 1.3 X 10(-2) S-1) are consistent with the dissociation constant measured at equilibrium. Intact synaptic vesicles bind to calmodulin immobilized on polyacrylamide matrix, suggesting that the binding site is cytoplasmically oriented in the vesicle population. Intact synaptic vesicles bind calmodulin up to 80-fold more effectively than do side fractions from the vesicle purification. The quantitative difference is largely due to latency of binding sites since it disappears when the binding is assayed in detergent. Binding of calmodulin to proteins separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis shows that a subset of nerve terminal and electric organ calmodulin-binding proteins are found in synaptic vesicles.

Calmodulin is tightly associated with synaptic vesicles independent of calcium.

A protein in highly purified synaptic vesicles from elasmobranch electric organ is recognized by two specific antisera that recognize different determinants of calmodulin. The protein is indistinguishable from authentic calmodulin by migration on sodium dodecyl sulfate-polyacrylamide gel electrophoresis in the presence or absence of calcium. It is tightly associated with the intact synaptic vesicle membrane even in the absence of calcium. It is on vesicles rather than membrane contaminants and cytoplasmically oriented since a calmodulin antibody (sheep anti-calmodulin antibody) immunoprecipitates at least 86% of intact synaptic vesicles. Surprisingly, another calmodulin antiserum (rabbit anti-calmodulin serum) specifically precipitates less than 20% of the intact vesicles. This antiserum (rabbit anti-calmodulin serum) also detects 4-15 times less calmodulin immunoreactivity than sheep anti-calmodulin antibody by radioimmunoassay of vesicles solubilized with nondenaturing detergents. The difference essentially disappears if the vesicle calmodulin is solubilized in sodium dodecyl sulfate. We suggest that the antigenic determinant recognized by rabbit anti-calmodulin serum is concealed in vesicle-associated calmodulin and may be involved in binding calmodulin to the vesicle.

Nerve terminal components from normal and denervated Narcine electric organ.

Exocytosis at the nerve terminal will only be fully understood when purified synaptic vesicles can be made to fuse with presynaptic plasma membrane in vitro. While the purification of synaptic vesicles from electric organ is now straightforward, isolation of the presynaptic plasma membrane presents a greater difficulty because of lack of specific markers. We have utilized pure synaptic vesicles in a novel way to overcome this difficulty. Antibodies raised to pure synaptic vesicles can be used to detect the presence of vesicle antigens. Thus, for example, we can show that synaptosome preparations isolated from electric organ by conventional procedures have about 5% of their protein in synaptic vesicles. The synaptic vesicle antigens and choline acetyltransferase both disappear from the synaptosome fraction after denervation of the electric organ, verifying that they are nerve terminal specific. Some of the synaptic vesicle antigens can be detected on the outside of the intact synaptosomes by binding rabbit anti-synaptic vesicle antibodies. Such antibody-coated synaptosomes will absorb specifically to goat anti-rabbit IgG attached to polyacrylamide beads. Lysis of such bead-bound synaptosomes leaves a plasma membrane fraction firmly attached to the beads. By raising antibodies to this membrane fraction an antiserum has been generated that binds to antigenic determinants in the electric organ. Some of these antibodies bind to the frog neuromuscular junction. The antigens recognized by this antiserum are, like synaptic vesicle antigens, lost on denervation. Unlike synaptic vesicle antigens, however, the antigens recognized by the anti-plasma membrane serum are present on the outside of resting frog nerve terminals.