Identification of novel modifiers of Aß toxicity by transcriptomic analysis in the fruitfly.

The strongest risk factor for developing Alzheimer's Disease (AD) is age. Here, we study the relationship between ageing and AD using a systems biology approach that employs a Drosophila (fruitfly) model of AD in which the flies overexpress the human Aß42 peptide. We identified 712 genes that are differentially expressed between control and Aß-expressing flies. We further divided these genes according to how they change over the animal's lifetime and discovered that the AD-related gene expression signature is age-independent. We have identified a number of differentially expressed pathways that are likely to play an important role in the disease, including oxidative stress and innate immunity. In particular, we uncovered two new modifiers of the Aß phenotype, namely Sod3 and PGRP-SC1b.

TRPM channel function in Caenorhabditis elegans.

The nematode Caenorhabditis elegans contains over 20 genes for TRP (transient receptor potential) channels which include members of all of the subclasses identified in mammalian cells. These proteins include three members of the TRPM (TRP melastatin) family: gon-2 (abnormal gonad development), gtl-1 (gon-2-like 1) and gtl-2. Although studies of these genes are at an early stage, we are beginning to understand their functions in the life of C. elegans. Mutations in gon-2 have defective gonad formation because of failures in the cell division of the somatic gonad precursor cells. gon-2 and gtl-1 are both expressed in the intestine of the animal. Experiments on gon-2,gtl-1 double mutants show that they have a severe growth defect that is ameliorated by the addition of high levels of Mg(2+) to the growth medium. gon-2,gtl-1 double mutants have defective magnesium homoeostasis and also have altered sensitivity to toxic levels of Ni(2+). Furthermore gon-2 mutants have reduced levels of I(ORCa) (outwardly rectifying calcium current) in the intestinal cells. Thus these two channels appear to play an important role in cation homoeostasis in C. elegans. In addition, perturbing the function of gon-2 and gtl-1 disrupts the ultradian defecation rhythm in C. elegans, suggesting that these channels play an important role in regulating this calcium-dependent rhythmic process. The tractability of C. elegans as an experimental animal and its amenability to techniques such as RNAi (RNA interference) and in vivo imaging make it an excellent system for an integrative analysis of TRPM function.

Dissection of the promoter region of the inositol 1,4,5-trisphosphate receptor gene, itr-1, in C. elegans: a molecular basis for cell-specific expression of IP3R isoforms.

Inositol 1,4,5-trisphosphate receptors in Caenorhabditis elegans are encoded by a single gene, itr-1. This provides a powerful system in which to dissect the mechanisms that control the tissue-specific expression of molecules that determine the specificity of calcium signalling. We first identified the Caenorhabditis briggsae orthologue of itr-1, Cbitr-1. Comparison of the two itr-1 genes revealed that the chromosomal organisation, gene structure and predicted cDNA and protein sequences were all conserved. The conserved gene structure supports the hypothesis that the itr-1 gene has three promoters, each of which gives rise to an alternative mRNA and hence unique protein. To test this and to identify the roles of the three putative promoters (pA, pB and pC) in regulating itr-1 expression we fused each promoter to the green fluorescent protein gene and identified their expression patterns. Introduction of these transgenes into C. elegans identified unique and defined patterns of green fluorescent protein expression directed by each promoter: pA directs expression in the pharyngeal terminal bulb, the rectal epithelial cells and vulva; pB directs expression in the motor neurone PDA, the amphid socket cells and the spermatheca; pC directs expression in the spermathecal valve, uterine sheath cells, pharyngeal isthmus and intestine. Thus tissue-specific expression of itr-1 variants is directed by three promoters and this results in adjacent cells in the same tissue containing different inositol trisphosphate receptor isoforms. Within pA, four short regions (pA-A to pA-D) of sequence conservation between C. elegans and C. briggsae were identified. Deletion analysis demonstrated that the region containing pA-C is required for expression in the terminal bulb and rectal epithelial cells and the region containing pA-D is required for expression in the vulva. pA-C includes sequences similar to the binding sites for transcription factors that have been demonstrated to be important in pharyngeal development and gene expression.

Transcaruncular approach to the medial orbit and orbital apex.

OBJECTIVE: To present a versatile approach to the medial orbit and orbital apex through the caruncle. DESIGN: Retrospective, noncomparative, case series with description of surgical technique. PARTICIPANTS: Twenty-five consecutive patients underwent orbital surgery by use of a transcaruncular approach. INTERVENTION: Inferior and medial wall fracture repair or orbital decompression by means of a transcaruncular or combined transfornix-transcaruncular approach. MAIN OUTCOME MEASURES: The surgical indications and complications were recorded for each patient. RESULTS: Ten patients (10 orbits) underwent combined inferior and medial orbital wall fracture repair through a combined transfornix-transcaruncular approach. In 8 of 10 (80%) orbits, the inferior oblique muscle was disinserted during surgery. Fifteen patients (24 orbits) underwent orbital decompression surgery for dysthyroid orbitopathy. An isolated transcaruncular approach was used in 5 of 24 orbits, and a combined transfornix-transcaruncular approach was used in 19 of 24 orbits. There were no complications related to either approach. CONCLUSIONS: Orbital bone removal and fracture reduction may be safely completed through a combined transfornix-transcaruncular approach. The transcaruncular approach provides excellent and safe exposure of the medial orbital wall, and it avoids scarring associated with the Lynch approach.

Frontalis muscle flap advancement for correction of blepharoptosis.

PURPOSE: To describe a technique of frontalis muscle flap advancement to repair myogenic ptosis in lieu of a graft or suture material. METHODS: Ten ptotic eyelids in eight patients were repaired using the frontalis flap technique. Patients were selected at random by two separate surgeons; all patients had eyelid excursion measured as poor (or less than 6 mm). RESULTS: Nine of 10 ptotic eyelids were adequately corrected by the frontalis flap technique, with follow-up intervals ranging from 18 to 42 months. Adequate correction was defined as ptosis corrected within 1 mm of the fellow eyelid. Complications of frontalis advancement were few and primarily transient. CONCLUSIONS: Frontalis flap advancement is a technically simple, safe, and effective technique for the repair of myogenic ptosis. The primary advantage of frontalis muscle flap advancement over a graft or suture material that it elevates the eyelid directly by moving the insertion of the frontalis muscle into the eyelid, rather than by graft or suture material.

The deep plane facelift: a 20-year evolution of technique.

PURPOSE: This report reviews the unique technical and conceptual oculoplastic innovations in the discipline of facelift surgery by analyzing the evolution of facelift technique at a university-based oculoplastic program. DESIGNED: Retrospective, noncomparative case series. PARTICIPANTS: We analyzed 313 patients undergoing a facelift from 1980 through 1997. Most procedures were performed by the senior author. METHODS: Three primary eras of surgical technique were identified: limited skin flap with superficial musculo-aponeurotic system plication (25 patients), extended skin flap with neck dissection and superficial musculo-aponeurotic system plication (210 patients), and deep plane facelift with robust superficial musculo-aponeurotic system flap (78 patients). RESULTS: The steps in the evolution were designed to improve the results of the surgery regarding rejuvenation of the neck, jowls, and nasolabial fold, and to reduce the "tattletale signs" of facelift surgery including postauricular scarring, change in the position of the sideburn and temporal hairline, and unnatural results caused by pulling the tissues posteriorly, rather than repositioning them vertically. There were no complications in the skin flap only group. In the extended skin flap and superficial musculo-aponeurotic system plication group, there was one mandibular paresis which partially resolved. In the deep plane facelift (n = 78), there was one laceration of the parotid duct, successfully stented during surgery. CONCLUSIONS: The deep plane facelift, with vertical elevation of the midface, jowls, and neck, is a logical extension of the mid-facelifting techniques that have been used by oculoplastic surgeons. Compared with cutaneous undermining with superficial musculo-aponeurotic system plication, we found patient and physician acceptance higher using the deep plane technique.

Inositol 1,4,5-trisphosphate receptors are strongly expressed in the nervous system, pharynx, intestine, gonad and excretory cell of Caenorhabditis elegans and are encoded by a single gene (itr-1).

Inositol 1,4,5-trisphosphate (InsP3) activates receptors (InsP3Rs) that mediate intracellular Ca(2+ )release, thereby modulating intracellular calcium signals and regulating important aspects of cellular physiology and gene expression. To further our understanding of InsP3Rs we have characterised InsP3Rs and the InsP3R gene, itr-1, from the model organism Caenorhabditis elegans. cDNAs encoding InsP3Rs were cloned enabling us to: (a) identify three putative transcription start sites that result in alternative mRNA 5' ends: (b) detect alternative splicing at three sites and: (c) determine the full genomic organisation of the itr-1 gene. The InsP3R protein (ITR-1) is approximately 42 % identical with known InsP3Rs and possesses conserved structural features. When the putative InsP3 binding domain was expressed in Escherichia coli, specific binding of InsP3 was detected. Using antibodies against ITR-1 we detected a protein of 220 kDa in C. elegans membranes. These antibodies and itr-1::GFP (green fluorescent protein) reporter constructs were used to determine the expression pattern of itr-1 in C. elegans. Strong expression was observed in the intestine, pharynx, nerve ring, excretory cell and gonad. These results demonstrate the high degree of structural and functional conservation of InsP3Rs from nematodes to mammals and the utility of C. elegans as a system for studies on InsP3R mediated signalling.

The Caenorhabditis elegans orthologue of the human gene responsible for spinal muscular atrophy is a maternal product critical for germline maturation and embryonic viability.

Spinal muscular atrophy (SMA) is a common disorder characterized by loss of lower motor neurones of the spinal cord. The disease is caused by mutations in the survival motor neurone ( SMN ) gene. SMN is ubiquitously expressed and evolutionarily conserved, and its role in RNA processing has been well established. However, these properties do not explain the observed specificity of motor neurone death. To gain further insight into the function of SMN, we have isolated and characterized the Caenorhabditis elegans orthologue of the SMN gene ( CeSMN ). Here we show that CeSMN is transmitted maternally as a predominantly nuclear factor, which remains present in all the blastomeres throughout embryonic development and onwards into adulthood. In adult nematodes, a CeSMN-green fluorescent protein fusion protein is expressed in a number of cell types including the germline. Both disruption of the endogenous CeSMN function and overexpression of the gene result in a severe decrease in the number of progeny and in locomotive defects. In addition, its transient knockdown leads to sterility caused by a defect in germ cell maturation. The expression pattern and functional properties so far observed for CeSMN, together with its unusual behaviour in the germline, indicate that SMN may be involved in specific gene expression events at these very early developmental stages. We have also identified a deletion in the CeSMN promoter region in egl-32. This mutant may become a useful genetic tool with which to explore regulation of CeSMN and hence provide possible clues for novel therapeutic strategies for SMA.

Management of relative proptosis with a porous polyethylene orbital rim onlay implant.

PURPOSE: The position of the globe relative to the orbital rim plays a significant role in the relationship between the eyelids and the cornea. A prominent globe (relative proptosis) may cause eyelid retraction and exposure keratopathy. Simple horizontal lower eyelid tightening exacerbates eyelid retraction. Optimal correction with an orbital decompression or advancement of the orbital rim entails considerable risk. A technically simpler alternative, placement of an orbital rim onlay implant, was evaluated. METHODS: Fourteen patients with symptomatic relative proptosis underwent placement of a porous polyethylene orbital rim onlay implant. RESULTS: Lower eyelid position, exposure keratopathy, and ocular discomfort were improved in all patients. Two patients required minor surgical revisions. CONCLUSIONS: Porous polyethylene orbital rim onlay implants are a satisfactory option to treat the sequelae of relative proptosis.

Functional characterization of a mutated chicken alpha7 nicotinic acetylcholine receptor subunit with a leucine residue inserted in transmembrane domain 2.

1. Site-directed mutagenesis was used to create an altered form of the chicken alpha7 nicotinic acetylcholine (ACh) receptor subunit (alpha7x61) in which a leucine residue was inserted between residues Leu9' and Ser10' in transmembrane domain 2. The properties of alpha7x61 receptors are distinct from those of the wild-type receptor. 2. Oocytes expressing wild-type alpha7 receptors responded to 10 microM nicotine with rapid inward currents that desensitized with a time-constant of 710+/-409 ms (mean+/-s.e.mean, n=5). However in alpha7x61 receptors 10 microM nicotine resulted in slower onset inward currents that desensitized with a time-constant of 5684+/-3403 ms (mean+/-s.e.mean, n = 4). No significant difference in the apparent affinity of nicotine or acetylcholine between mutant and wild-type receptors was observed. Dihydro-beta-erythroidine (DHbetaE) acted as an antagonist on both receptors. 3. Molecular modelling of the alpha7x61 receptor channel pore formed by a bundle of M2 alpha-helices suggested that three of the channel lining residues would be altered by the leucine insertion i.e.; Ser10 would be replaced by the leucine insertion, Val13' and Phe14' would be replaced, by Thr and Val, respectively. 4 When present in the LEV-1 nicotinic ACh receptor subunit from Caenorhabditis elegans the same alteration conferred resistance to levamisole anthelmintic drug. Levamisole blocked responses to nicotine of wild-type and alpha7x61 receptors. However, block was more dependent on membrane potential for the alpha7x61 receptors. 5. We conclude that the leucine insertion in transmembrane domain 2 has the unusual effect of slowing desensitization without altering apparent agonist affinity.

The endoscopic forehead lift.

Endoscopic techniques are being successfully applied to address eyebrow and forehead ptosis. The methods rely on extensive subperiosteal and subgaleal release of the forehead and scalp flap, allowing the elevation of soft tissues. Ablation of the depressor supercilli and procerus can be performed to address skin folding in the glabellar region. The mobilized frontotemporal flap is then elevated to the desired level and fixated with microscrews to the outer table of the skull. Laterally, the flap is fixed to the deep temporalis fascia. The technique relies upon a solid knowledge of the regional anatomy and the use of specialized instruments now available for dissecting under the flap. The endoscopic forehead lift can achieve results comparable to those obtained by the open coronal forehead lift while minimizing the incidence and extent of postoperative cutaneous anesthesia and telogenic hair loss, which frequently follows open coronal forehead surgery. In addition, the endoscopic technique is able to address eyebrow ptosis in the balding male without causing disfiguring scarring.

Effects of the alpha subunit on imidacloprid sensitivity of recombinant nicotinic acetylcholine receptors.

1. Imidacloprid is a new insecticide with selective toxicity for insects over vertebrates. Recombinant (alpha4beta2) chicken neuronal nicotinic acetylcholine receptors (AChRs) and a hybrid nicotinic AChR formed by co-expression of a Drosophila melanogaster neuronal alpha subunit (SAD) with the chicken beta2 subunit were heterologously expressed in Xenopus oocytes by nuclear injection of cDNAs. The agonist actions of imidacloprid and other nicotinic AChR ligands ((+)-epibatidine, (-)-nicotine and acetylcholine) were compared on both recombinant nicotinic AChRs by use of two-electrode, voltage-clamp electrophysiology. 2. Imidacloprid alone of the 4 agonists behaved as a partial agonist on the alpha4beta2 receptor; (+)-epibatidine, (-)-nicotine and acetylcholine were all full, or near full, agonists. Imidacloprid was also a partial agonist of the hybrid Drosophila SAD chicken beta2 receptor, as was (-)-nicotine, whereas (+)-epibatidine and acetylcholine were full agonists. 3. The EC50 of imidacloprid was decreased by replacing the chicken alpha4 subunit with the Drosophila SAD alpha subunit. This alpha subunit substitution also resulted in an increase in the EC50 for (+)-epibatidine, (-)-nicotine and acetylcholine. Thus, the Drosophila (SAD) alpha subunit contributes to the greater apparent affinity of imidacloprid for recombinant insect/vertebrate nicotinic AChRs. 4. Imidacloprid acted as a weak antagonist of ACh-mediated responses mediated by SADbeta2 hybrid receptors and as a weak potentiator of ACh responses mediated by alpha4beta2 receptors. This suggests that imidacloprid has complex effects upon these recombinant receptors, determined at least in part by the alpha subunit.

An extensive and diverse gene family of nicotinic acetylcholine receptor alpha subunits in Caenorhabditis elegans.

Using reverse transcription-polymerase chain reactions the transcription of eight novel candidate nicotinic acetylcholine receptor (nAChR) alpha subunit genes has been demonstrated in the nematode Caenorhabditis elegans. Together with five other alpha subunit genes described elsewhere by ourselves (unc-38) and other workers (deg-3, acr-4, Ce21 and acr-6), this is now the largest known family of nAChR alpha subunit genes in a single species. By homology we have identified four groups of alpha subunits: DEG-3-like; ACR-16[Ce21]-like; UNC-38-like and ACR-8-like. Five C. elegans nAChR alpha subunits contain a modification in loop C of the ACh binding site in which the normally conserved Tyr-x-Cys-Cys, is replaced by a distinct motif (Tyr-x-x-Cys-Cys). Variation is also found in the channel lining M2 regions, including the replacement in four subunits of the highly conserved leucine at the 9' position by valine and most notably, the replacement in all ACR-8-like subunits of the highly conserved glutamic acid at the -1' position by histidine. Restrained molecular dynamics simulations have been used to generate homo-pentameric M2 helix bundle models for alpha subunits and possible functional implications examined. The calculated electrostatic potential energy profile for the M2 region of ACR-8 differs strikingly from that of ACR-16[Ce21] largely due to the presence of histidine at the -1' position, suggesting a possible perturbation of nAChR channel action permeability in the presence of this subunit type.

Blepharoplasty and periorbital surgery.

Blepharoplasty is one of the most successful aesthetic surgical procedures. Careful preoperative planning and conservative tissue resections can help to minimize complications and optimize results. Although some young patients request blepharoplasty specifically because of age-related changes in the eyelid skin, the surgery is that of sculpture and contouring of the entire aesthetic unit. The aging process in the eyelid complex is characterized by skin texture changes with loss of elasticity and formation of wrinkles, fat redistribution, enophthalmos, and anterior displacement of fat with a lower eyelid orbital fat prolapse. Once the etiology of the deformity and the associated periorbital anatomy are recognized, a local assessment and surgical treatment plan can produce optimal results.

ACR-3, a Caenorhabditis elegans nicotinic acetylcholine receptor subunit. Molecular cloning and functional expression.

The molecular cloning and functional co-expression of a novel nicotinic acetylcholine receptor (nAChR) non-alpha subunit gene, acr-3, is described. Previously we determined the sequence and demonstrated the functional co-expression of acr-2, a nAChR non-alpha subunit gene from Caenorhabditis elegans. Analysis of the acr-2 genomic DNA revealed the existence of another potential nAChR subunit gene, acr-3, in the same orientation, only 281 bp downstream of acr-2. A cDNA containing the entire acr-3 coding sequence was isolated by RT-PCR and sequenced. The predicted protein contains the conserved features typical of nAChR non-alpha subunits and most closely resembles other invertebrate nAChR non-alpha polypeptides. Unusually, the highly conserved glycine residue (equivalent to residue 240 in the Torpedo alpha subunit) upstream of transmembrane domain 2 (m2) is replaced by a serine residue in ACR-3. When acr-3 cDNA was injected alone into Xenopus oocytes no levamisole-gated channel activity was observed. However when co-expressed with a C. elegans alpha subunit (UNC-38), ACR-3 contributed to the formation of levamisole-gated channels. The response of this hetero-oligomer to levamisole (100 microM) was reduced by the nAChR antagonists mecamylamine (1 microM) and d-tubocurarine (10 microM).

Polycyclic dinitriles: a novel class of potent GABAergic insecticides provides a new radioligand, [3H]BIDN.

The polycyclic dinitriles are a potent class of insecticides which are non-competitive GABA (gamma-aminobutyric acid) antagonists acting at the convulsant site. Comparison with other classes of GABA convulsant site ligands using molecular modelling has shown significant structural similarities. We have developed a pharmacophore model which unifies this class and some previous classes of GABA convulsants. Key pharmacophore elements are a polarizable functionality separated by a fixed distance from two H-bond accepting elements. This model is based on information from X-ray crystal structures and Sybyl using the Tripos force field. Using this pharmacophore model, numerous structural modifications were explored to enhance understanding of structure-activity relationships at the GABA receptor convulsant site of insects and mammals. A radiolabelled bicyclic dinitrile, [3H]BIDN [3H]3,3-bis-trifluoromethyl-bicyclo[2,2,1]heptane-2,2-dicarbonitrile+ ++), was prepared from this area of chemistry and was used as a probe for the interaction of polycyclic dinitriles at the target site.

Genetic analysis of cholinergic nerve terminal function in invertebrates.

Genetic analysis of nerve terminal function is proving fruitful and studies on invertebrates are making a substantial impact. In this survey, particular emphasis has been placed on cholinergic chemical synaptic transmission. The advanced genetics of Drosophila melanogaster and Caenorhabditis elegans with their rich diversity of behavioural and biochemical mutants is providing new insights into the functions of key molecular components of synapses. A 'space-invader' mutant of Periplaneta americana permits investigations of competition between neurons during synaptogenesis and its impact on neurotransmitter release. The growing importance of the C. elegans genome as a major research resource is emphasized in this survey.

pH-dependent actions of THIP and ZAPA on an ionotropic Drosophila melanogaster GABA receptor.

The actions of THIP (4,5,6,7-tetrahydroisoxazolo[5,4-c]pyridin-3-ol) and ZAPA (Z-3-[(aminoiminomethyl)thio]prop-2-enoic acid) were tested on an ionotropic homo-oligomeric GABA receptor of Drosophila melanogaster. The amplitude of currents activated by THIP and ZAPA declined rapidly during agonist application and a rebound response was observed on washout. By correcting the pH shift induced by these acid salts, responses more typical of GABA agonists were seen. Less striking pH-dependence was observed in the case of GABA responses.

Molecular cloning and in vitro expression of C. elegans and parasitic nematode ionotropic receptors.

The free living nematode, C. elegans is understood at a level of detail equalled by few other organisms, and much of the cell biology and sequence information is proving of considerable utility in the study of parasitic nematodes. Already, C. elegans provides a convenient vehicle for investigating anthelmintic drug action and resistance mechanisms. Among the ionotropic receptors, with their important roles in the behaviour and development of the organism, are targets for anthelmintics. The subunits of nicotinic acetylcholine receptors of C. elegans form a large and diverse multigene family. Members of this family are among the 11 genes associated with resistance to the anthelmintic drug levamisole.

Wild-type and insecticide-resistant homo-oligomeric GABA receptors of Drosophila melanogaster stably expressed in a Drosophila cell line.

RDL is an ionotropic GABA receptor subunit, a product of the Rdl gene, originally identified in the Maryland strain of Drosophila melanogaster. Here, we report the generation of a Drosophila melanogaster cell line (S2-RDLA302S) stably expressing a mutated, dieldrin-resistant (A302S) form of RDL. The properties of this dieldrin-resistant, homo-oligomeric receptor have been compared with those of the stably expressed, wild-type form (S2-RDL). Using these stable lines, a striking reduction in sensitivity to both picrotoxinin and dieldrin was observed for responses to GABA of S2-RDLA302S compared to S2-RDL. To determine if these stable insect cell lines generate results similar to those obtained by transient expression in Xenopus laevis oocytes, we have examined the actions of two widely used convulsants, EBOB and TBPS, and a recently developed convulsant BIDN, on RDL-mediated GABA responses in the two expression systems. In both oocytes and S2 cells, the three convulsants suppressed the amplitude of responses to GABA. Thus, in accord with earlier work on agonist and allosteric sites, the S2-RDL cell line is found to yield similar pharmacological results to those obtained in transient expression studies. Stable cell lines are now available expressing susceptible and resistant forms of an ionotropic receptor by GABAergic insecticides.