Multimeric CREB-binding sites in the promoter regions of a family of G-protein-coupled receptors related to the vertebrate galanin and nociceptin/orphanin-FQ receptor families.

Four related genes encoding a family of G-protein-coupled receptors (GPCRs) have been isolated from the mollusc Lymnaea stagnalis. The coding regions of this family of receptors share 97-99% sequence similarity at both the protein and nucleotide level, and they also share high sequence identity with vertebrate galanin and orphanin-FQ/nociceptin GPCR families. Analysis of the promoter regions reveals shared domains, some of which encode highly conserved repeating units. One 27-bp repeating unit, which encodes a c-AMP response element (CRE) and binds CREB protein, is repeated 14 times in one promoter. In situ hybridization showed expression of these receptors in identified neurons of several behaviourly important networks including those involved in feeding and ion and water regulation. These Lymnaea receptors are likely to represent members of a novel family of invertebrate neuropeptide receptors extensively regulated in response to intracellular signalling cascades.

Obesity in North America. An overview.

The terms "obesity" and "overweight" mean different things to different people. This article discusses such issues as prevalence, morbidity, mortality, and psychosocial effects. Definitions and various classifications of obesity are discussed also.

Maternal and child health and health care reform.

Women and children, because of particular characteristics and vulnerabilities, should be afforded special attention in health care reform. Health care reform provides an opportunity to positively affect the maternal and child health (MCH) status. Universal coverage of a comprehensive benefit package must be combined with a strong public health system of population-based services if benefits are to be realized. There must be an accountable public locus of responsibility for promoting MCH, including clear authority and resources for four primary activities: (1) core public health, (2) systems development, (3) coordinated services for children with special health care needs, and (4) access to care.

Genomic organization of the FMRFamide gene in Lymnaea: multiple exons encoding novel neuropeptides.

Based on the sequencing of genomic and cDNA clones, we were able to determine that the FMRFamide gene consists of five exons covering at least 20 kb and predict the presence of further novel peptides. The exons are alternatively spliced: exon I (hydrophobic leader sequence) to exon II (tetrapeptides) and exon I to exons III (heptapeptides), IV, and V. A cDNA clone encoding the heptapeptides is described and has also been shown to encode further novel peptides SKPYMRFamide, HDYMRFamide, and SSFPRYamide. Analysis of the right internal parietal nerve using mass spectrometry showed that the novel peptide SKPYMRFamide was cleaved from the precursor. This peptide excites neurons, suggesting a physiological function in the CNS.

Drosophila integrins and their ligands.

The major advance during the past year was the identification of ligands for two of the previously known position-specific integrins in Drosophila. At the same time, two new Drosophila integrin subunits (one alpha and one beta) were discovered, and significant progress was made on developmental genetic analyses of integrin functions, shedding light on the roles of integrins in Drosophila development.

Drosophila syndecan: conservation of a cell-surface heparan sulfate proteoglycan.

In mammals, cell-surface heparan sulfate is required for the action of basic fibroblast growth factor, fibronectin, antithrombin III, as well as other effectors. The syndecans, a gene family of four transmembrane proteoglycans that participates in these interactions, are the major source of this heparan sulfate. Based on the conserved transmembrane and cytoplasmic domains of the mammalian syndecans, a single syndecan-like gene was detected and localized in the Drosophila genome. As in mammals, Drosophila syndecan is a heparan sulfate proteoglycan expressed at the cell surface that can be shed from cultured cells. The single Drosophila syndecan is expressed in embryonic tissues that correspond with those tissues in mammals that express distinct members of the syndecan family predominantly. Conservation of this class of molecules suggests that Drosophila, like mammals, uses cell-surface heparan sulfate as a receptor or coreceptor for extracellular effector molecules.

Is there lead in the suburbs? Risk assessment in Chicago suburban pediatric practices. Pediatric Practice Research Group.

OBJECTIVE: This study was designed to determine: (1) the prevalence of elevated blood lead (BPb) levels (BPb > or = 10 micrograms/dL) in Chicago suburban children attending Pediatric Practice Research Group practices at 12 and 24 months of age, and (2) the efficacy of the Centers for Disease Control and Prevention (CDC) and Illinois lead exposure risk assessment questions. METHODS: Parents bringing their 1- and 2-year-old children for health supervision visits at pediatric practices completed questionnaires. BPb levels were drawn on children. Both questionnaire and an analyzable BPb level were obtained on 1393 subjects (79.2%). RESULTS: Only 2.1% of our sample had a venous BPb level > or = 10 micrograms/dL (0.48 mumol/L); no subjects had a level > or = 30 micrograms/dL (1.45 mumol/L). The CDC risk assessment questions had a sensitivity of .69 and specificity of .70. Due to the low prevalence of elevated BPb levels in this sample, CDC and Illinois screening strategies had high negative predictive values (.99) and low positive predictive values (.05 and .04, respectively). However, some of the subjects with BPb levels > or = 10 micrograms/dL were not at high risk by CDC and Illinois screening questions; 9 of 29 subjects with elevated lead levels (31%) did not respond affirmatively to any CDC risk assessment questions. The question best predicting an elevated BPb was the determination that the house the child lives in was built before 1960 (sensitivity = .83, specificity = .67). This question is not currently included in CDC or Illinois screening strategies. Screening based on the single question "Was your house built before 1960?" would have missed only five (17%) of the children with BPb levels > or = 10 micrograms/dL. Three of these five children were among the 17.1% of 1-year-olds and 26.3% of 2-year-olds in our sample who had moved. CONCLUSIONS: In this sample, children living in houses built before 1960 should be considered at high risk for high-dose lead exposure. Due to the high mobility of our sample, phrasing the question to include lifetime exposure (ie, Has your child ever lived in a house built before 1960?) should also be considered. Selective BPb testing of high-risk children in low-prevalence suburban areas using this question would miss few children with elevated BPb. Useful risk assessment questions in other areas and other populations may differ.

Mutually exclusive expression of alternatively spliced FMRFamide transcripts in identified neuronal systems of the snail Lymnaea.

The FMRFamide gene of the snail Lymnaea encodes tetrapeptides (FMRFamide/FLRFamide) and heptapeptides (GDPFLRFamide/SDPFLRFamide) on separate exons. In situ hybridization probes specific to these exons were used to map the expression of the two exons in identified neuronal systems of the CNS. Analysis of more than 200 preparations showed that cytoplasmic expression of mRNA was exclusively of one type, with individual neurons expressing either the tetrapeptide or heptapeptide exon. Of the approximately 340 neurons expressing the two exons, the majority (80%) expressed the tetrapeptide exon. The tetrapeptide exon was more widespread, occurring in neurons from all 11 ganglia of the CNS. The heptapeptide was mainly confined to two ganglia (visceral and right parietal), with a small number of cells in three other ganglia. Mapping studies combined with dye marking of identified neurons showed the presence of the tetrapeptide exon in several behaviorally important networks: heart motoneurons, whole body withdrawal response motoneurons, and probably penis motoneurons as well as giant identified neurons (LP1, RPD1). The heptapeptides were prominent in two main clusters of cells (Bgp and Fgp) together with a smaller number of tetrapeptide-expressing cells.

Cell-specific alternative RNA splicing of an FMRFamide gene transcript in the brain.

Individual neurons synthesize different peptide neurotransmitters and neuromodulators. In general, specificity is achieved by transcriptional regulation of neuropeptide-encoding genes. In Lymnaea, the FMRFamide and GDP/SDPFLRFamide neuropeptides are encoded by separate exons. Here we provide evidence that the two exons are part of the same gene and that in neurons expressing the gene the two exons are spliced onto a common upstream exon encoding a hydrophobic leader sequence. In addition, in situ hybridization data show that there is mutually exclusive cytoplasmic expression of each of the neuropeptide-encoding exons. Thus, differential neuropeptide synthesis is likely to be regulated by an alternative splicing mechanism. The cellular specificity of these splicing events is remarkable and suggests that cell-specific alternative splicing may be of major importance in establishing neuronal diversity in this system.

Neuropeptides Gly-Asp-Pro-Phe-Leu-Arg-Phe-amide (GDPFLRFamide) and Ser-Asp-Pro-Phe-Leu-Arg-Phe-amide (SDPFLRFamide) are encoded by an exon 3' to Phe-Met-Arg-Phe-NH2 (FMRFamide) in the snail Lymnaea stagnalis.

Biochemical analysis has shown the pond snail Lymnaea stagnalis to contain 2 main classes of Phe-Met-Arg-Phe-NH2 (FMRFamide)-like neuropeptides: the tetrapeptides FMRFamide and Phe-Leu-Arg-Phe-NH2 (FLRFamide), and the heptapeptides Gly-Asp-Pro-Phe-Leu-Arg-Phe-NH2 (GDP-FLRFamide) and Ser-Asp-Pro-Phe-Leu-Arg-Phe-NH2 (SDPFFRFamide). By genomic mapping and DNA sequencing, we show here that the GDP/SDPFLRFamide coding region lies 3' to the FMRFamide coding region. The absence of an initiating start methionine and the presence of good-concensus 3' and 5' splice sites suggests that the GDP/SDPFLRFamide coding region makes up 1 exon of a larger gene. In addition to 7 copies of GDPFLRFamide and 6 copies of SDPFLRFamide, the exon encoding the heptapeptides also encodes 3 novel peptides, Glu-Phe-Phe-Pro-Leu-NH2 (EFFPLamide), Ser-Asp-Pro-Tyr-Leu-Phe-Arg-NH2 (SDPYLFRamide), and Ser-Asp-Pro-Phe-Phe-Arg-Phe-NH2 (SDPFFRFamide). In contrast to the tetrapeptide FMRFamide precursor protein, the GDP/SDPFLRFamide peptides are encoded contiguously, being separated only by single basic amino acids.

High efficiency expression of transfected genes in a Drosophila melanogaster haploid (1182) cell line.

Drosophila tissue culture cells have been important in the study of homologous promoters and more recently in the study of mammalian transcriptional factors such as CTF and SP1 which bind and stimulate transcription from transfected genes. In this paper we show that a Drosophila melanogaster haploid cell line (1182-4), not previously used for transfection studies, is capable of taking up and expressing DNA without the use of a facilitating agent such as calcium phosphate. Furthermore expression from a variety of Drosophila promoters such as copia, heatshock and rudimentary as well as a mammalian promoter RSV-LTR, show between 20 and over 100 times more activity in 1182-4 cells than in D.hydei DH33 or D.melanogaster S3, or D1 cell lines. This cell line should prove to be particularly useful for the analysis of weak promoters and heterologous transcription factors.

EBNA-1: a virally induced nuclear antigen of primate lymphocytes and its expression in Drosophila cells.

EBNA-1 is a nuclear antigen of lymphocytes infected by Epstein-Barr virus and whose size polymorphism correlates only with the strain of infecting virus and the length of the glycine-alanine copolymer encoded by the third internal repeat of the viral genome. The major antigenic determinant(s) also appear to reside in this region. We have been able to obtain efficient expression of this nuclear antigen in cultured Drosophila cells transfected with a cosmid carrying the EBNA-1 coding region, indicating that insect mechanisms recognise control sequences and transcripts of the herpes virus. The association of a vimertin-like protein of mol. wt. 46,000 with Drosophila cell nuclei has been found to vary with culture conditions and heat shock. We now find that the level and nuclear association of this protein also increase after transfection with either EBNA-1 or yolk protein DNA.

Regulated expression of a Drosophila melanogaster heat shock locus after stable integration in a Drosophila hydei cell line.

DNA-mediated cotransformation has been used to transfer a Drosophila melanogaster heat shock locus into cultured Drosophila hydei cells by use of the copia-based selectable vector pCV2gpt and of pMH10A, a cloned 87A7 heat shock locus encoding a mutant heat shock protein (hsp). Transformed lines contain between 50 and 200 copies of both plasmids, each separately organized as a head-to-tail concatemer which is stably maintained in the transformed lines. Exposure of the cotransformants to heat shock temperatures induces the regulated expression of the hsp RNA and the mutant hsp in all the lines analyzed.