A multicomponent screen for feeding behaviour and nutritional status in Drosophila to interrogate mammalian appetite-related genes.

OBJECTIVE: More than 300 genetic variants have been robustly associated with measures of human adiposity. Highly penetrant mutations causing human obesity do so largely by disrupting satiety pathways in the brain and increasing food intake. Most of the common obesity-predisposing variants are in, or near, genes expressed highly in the brain, but little is known of their function. Exploring the biology of these genes at scale in mammalian systems is challenging. We sought to establish and validate the use of a multicomponent screen for feeding behaviour phenotypes, taking advantage of the tractable model organism Drosophila melanogaster. METHODS: We validated a screen for feeding behaviour in Drosophila by comparing results after disrupting the expression of centrally expressed genes that influence energy balance in flies to those of 10 control genes. We then used this screen to explore the effects of disrupted expression of genes either a) implicated in energy homeostasis through human genome-wide association studies (GWAS) or b) expressed and nutritionally responsive in specific populations of hypothalamic neurons with a known role in feeding/fasting. RESULTS: Using data from the validation study to classify responses, we studied 53 Drosophila orthologues of genes implicated by human GWAS in body mass index and found that 15 significantly influenced feeding behaviour or energy homeostasis in the Drosophila screen. We then studied 50 Drosophila homologues of 47 murine genes reciprocally nutritionally regulated in POMC and agouti-related peptide neurons. Seven of these 50 genes were found by our screen to influence feeding behaviour in flies. CONCLUSION: We demonstrated the utility of Drosophila as a tractable model organism in a high-throughput genetic screen for food intake phenotypes. This simple, cost-efficient strategy is ideal for high-throughput interrogation of genes implicated in feeding behaviour and obesity in mammals and will facilitate the process of reaching a functional understanding of obesity pathogenesis.

Intraoperative cell salvage in radical retropubic prostatectomy.

OBJECTIVES: To investigate the efficacy and safety of intraoperative cell salvage with autotransfusion using leukocyte reduction filters in patients undergoing radical retropubic prostatectomy (RRP). METHODS: Between September 1996 and March 1999, 62 patients (age range 48 to 70 years) with clinically localized prostate cancer underwent RRP with intraoperative cell salvage as the sole blood management technique. Salvaged blood was passed through a leukocyte reduction filter before autotransfusion. The 62 cell salvage patients were compared with a cohort who predonated 1 to 3 U autologous blood (n = 101). The estimated blood loss, preoperative and postoperative hematocrit, need for homologous transfusion, and biochemical recurrence rates were compared between the two groups. The progression-free survival rates were compared using the Kaplan-Meier method. RESULTS: No difference was found in preoperative prostate-specific antigen level, pathologic stage, or estimated blood loss between the cell salvage and autologous predonation groups. The preoperative and postoperative hematocrit levels were higher in the cell salvage group (42.7% versus 39.6% and 31.3% versus 27.9%, respectively; P <0.001 for each). The homologous transfusion rates were lower in the cell salvage group (3% versus 14%, P = 0.04). The incidence of progression-free survival (prostate-specific antigen level 0.4 ng/mL or greater) was no different between the groups (P = 0.41). CONCLUSIONS: Intraoperative cell salvage with autotransfusion using leukocyte reduction filters in RRP results in higher perioperative hematocrit levels and low homologous transfusion rates and eliminates the need for autologous predonation. Cell salvage does not appear to be associated with an increased risk of early biochemical progression after RRP.

Amphiphysin is necessary for organization of the excitation-contraction coupling machinery of muscles, but not for synaptic vesicle endocytosis in Drosophila.

Amphiphysins 1 and 2 are enriched in the mammalian brain and are proposed to recruit dynamin to sites of endocytosis. Shorter amphiphysin 2 splice variants are also found ubiquitously, with an enrichment in skeletal muscle. At the Drosophila larval neuromuscular junction, amphiphysin is localized postsynaptically and amphiphysin mutants have no major defects in neurotransmission; they are also viable, but flightless. Like mammalian amphiphysin 2 in muscles, Drosophila amphiphysin does not bind clathrin, but can tubulate lipids and is localized on T-tubules. Amphiphysin mutants have a novel phenotype, a severely disorganized T-tubule/sarcoplasmic reticulum system. We therefore propose that muscle amphiphysin is not involved in clathrin-mediated endocytosis, but in the structural organization of the membrane-bound compartments of the excitation-contraction coupling machinery of muscles.

Nutritional factors modify the inhibition of CNS development by combined exposure to methadone and ethanol in neonatal rats.

The consequences resulting from the combined exposure to methadone and ethanol during a time period equivalent to the third trimester brain growth spurt was the purpose of this study. Rat pups were treated on postnatal days 6-10 and sacrificed on postnatal day 11. Body weight along with the heart, liver, kidneys, whole brain, cerebrum, cerebellum, and brain stem weights were measured. The impact of nutritional factors were identified by delivery of the drug solutions in one of two intubation vehicles differing in both caloric density and composition. Ethanol and methadone in combination result in significantly increased detrimental effects compared to methadone alone only when possible nutritional compromise was present. The combined effect of both drugs significantly inhibited body growth and the development of all brain regions studied. Neither drug alone, nor in combination, produced significant inhibition of growth in the liver, heart, or kidney. The nutritional status of the pup, as represented by vehicle composition, was able to modify the specific drug effects and suggests that nutritional status can mask or enhance the determination of specific drug effects.

Health care reform and rural mental health: severe mental illness.

Service needs of rural severely mentally ill and strengths of rural communities are addressed. Health care reform policy development at present appears to neglect the seriously mentally ill in general and rural services specifically. Examples of strategies to meet the needs for health care, psychiatric treatment, psychosocial rehabilitation and appropriate housing are described. The advantages and drawbacks of such efforts are considered.

Ionic control of beta cell function in nesidioblastosis. A possible therapeutic role for calcium channel blockade.

A preterm female infant presented with intractable hypoglycaemia within 10 minutes of delivery. Normoglycaemia could be maintained only by the intravenous infusion of glucose at a rate of 20-22 mg/kg/min. Persistent hyperinsulinaemic hypoglycaemia of infancy was diagnosed from an inappropriately raised plasma insulin concentration (33 mU/l) at the time of hypoglycaemia (blood glucose < 0.5 mmol/l). Medical treatment with glucagon, somatostatin, and diazoxide led to only a modest reduction in the intravenous glucose requirement; a 95% pancreatectomy was performed and histological 'nesidioblastosis' confirmed. In vitro electrophysiological studies using patch clamp techniques on isolated pancreatic beta cells characterised the ionic basis for insulin secretion in nesidioblastosis. The beta cells were depolarised in low ambient glucose concentrations with persistently firing action potentials; these were blocked reversibly by the calcium channel blocking agent verapamil. Persistent postoperative hyperinsulinaemic hypoglycaemia was treated with oral nifedipine. This increased median blood glucose concentrations from 3.5 to 4.8 mmol/l and increased in duration the child's tolerance to fasting from 3 to 10.5 hours. These data allude to an abnormality in the ionic control of insulin release in nesidioblastosis and offer a new logical approach to treatment which requires further evaluation.

Syntaxin and synaptobrevin function downstream of vesicle docking in Drosophila.

In synaptic transmission, vesicles are proposed to dock at presynaptic active zones by the association of synaptobrevin (v-SNARE) with syntaxin (t-SNARE). We test this hypothesis in Drosophila strains lacking neural synaptobrevin (n-synaptobrevin) or syntaxin. We showed previously that loss of either protein completely blocks synaptic transmission. Here, we attempt to establish the level of this blockade. Ultrastructurally, vesicles are still targeted to the presynaptic membrane and dock normally at specialized release sites. These vesicles are mature and functional since spontaneous vesicle fusion persists in the absence of n-synaptobrevin and since vesicle fusion is triggered by hyperosmotic saline in the absence of syntaxin. We conclude that the SNARE hypothesis cannot fully explain the role of these proteins in synaptic transmission. Instead, both proteins play distinct roles downstream of docking.

Methyl gallate, methyl-3,4,5-trihydroxybenzoate, is a potent and highly specific inhibitor of herpes simplex virus in vitro. I. Purification and characterization of methyl gallate from Sapium sebiferum.

A potent anti-herpetic compound was identified and purified to homogeneity from the leaves of Sapium sebiferum by plaque reduction assay using herpes simplex virus type 2. The chemical structure of the purified compound was determined by mass spectroscopy and proton and carbon-13 nuclear magnetic resonance as methyl gallate, methyl-3,4,5-trihydroxybenzoate. This is the first demonstration that methyl gallate is a potent anti-herpetic compound in vitro, present in high concentration in the leaves of S. sebiferum, a Chinese folk medicine for shingles.