Late boosting phenomenon in TST conversion among health care workers.

BACKGROUND: Available information is insufficient to guide determination of whether tuberculin skin test (TST) conversions of health care workers (HCWs) within 2 years of two-step testing are related to occupational exposures or to other causes, including late boosting. AIMS: To describe the epidemiologic factors of TST conversion in HCWs, comparing early TST conversion (≤2 years after two-step testing) with late conversion to possibly distinguish late boosting phenomenon from occupational TST conversion. METHODS: Retrospective analysis of a database of TSTs of HCWs from 1 January 1998, through 31 May 2014, in the United States Midwest. RESULTS: In total, 40142 HCWs had 197932 tests over the 16 years, with 123 conversions (conversion rate: 0.3%; 95% CI 0.3-0.4%). Among 61 HCWs with a negative two-step TST, 30 (49%) were found to have early TST conversion within 2 years; 31 (51%) had late conversion, with likely occupational exposure but no identifiable community risks. Persons with early conversion were more likely to be born outside the USA (89% versus 57%; P < 0.05), had a higher rate of prior bacille Calmette-Guérin (BCG) vaccination (89% versus 52%; P < 0.05) and had no identifiable risk factors for conversion (63% versus 58%; P < 0.05). CONCLUSIONS: Early conversions among HCWs after negative two-step TST are associated with various nonoccupational factors, including international birth and BCG vaccination history. Therefore, conversion is not a reliable indicator of recent tuberculosis contact in this population, and two-step TST is insufficient to discount a delayed boosting response for HCWs.

Screening for depression in the occupational health setting.

BACKGROUND: The cost of workplace absenteeism and presenteeism due to depression in the USA is substantial. AIMS: To assess the frequency of depression and its impact at the point of care in an occupational health (OH) practice. METHODS: Patients presenting to an OH practice completed a standardized depression screening tool and were compared to an unscreened group in the same clinic. Respondents with a nine-item Patient Health Questionnaire (PHQ-9) score >15 and untreated for depression were referred for further evaluation per usual practice. A comparison group of unscreened patients were selected from the same clinic from 1 year prior and records were reviewed for evidence of prior depression, treatment and outcomes. After 1 year, frequency of depression, PHQ-9 scoring for screened patients, days absent from work, days on restricted duties and permanent restrictions were recorded for both groups. RESULTS: Two hundred and five patients were screened for depression. Screening was associated with increased frequency of a diagnosis of current depression (30 versus 4%; P < 0.05). Screening was associated with similar rates of absenteeism but lower number of days on restricted duties (97 versus 159 days; P < 0.001). After adjusting for age, sex, history of and treatment for depression, screening was associated with lower odds of being on work restrictions [odds ratio (OR) 0.55; 95% confidence interval (CI) 0.38-0.78] or permanent restrictions (OR 0.35; 95% CI 0.23-0.52). CONCLUSIONS: Depression was common in this OH practice. Screening for depression, with appropriate recognition and referral, may reduce time for employed patients on restricted duties and permanent restrictions.

A survey of physicians' perceptions of their health care needs.

BACKGROUND: Physicians may face unique challenges in accessing health care and managing their own health. AIMS: To evaluate physicians' perceptions of their health care needs and desired services. METHODS: A written survey, distributed and collected anonymously among attendees at a large primary care continuing medical education conference. RESULTS: The survey was given to 346 physicians and 141 (41%) responded. The majority of physicians (53%) reported having difficulty accessing health care and reverting to self-diagnosis and treatment (63%). Over 83% reported having or knowing a colleague who had a career-threatening illness and 42% had experienced concern about a colleague's ability to practise safely. CONCLUSIONS: Physicians as an occupational group have challenges in accessing health care, very commonly suffer career-limiting illnesses and revert to self-diagnosis and treatment. Programmes tailored to providing health care to physicians are needed.

Cancer and thrombosis: new insights to an old problem.

Venous thromboembolism (VTE) is a common complication in patients with cancer and portends a poor prognosis. Our understanding of the underlying pathophysiology of VTE in cancer has advanced since Trousseau first described hypercoagulability in patients with malignancy and Virchow described his famous triad of thrombosis formation. Malignancy itself induces a thrombophilic state by increasing the risk of venous stasis, endothelial injury and an imbalance of pro and anti-thrombotic factors leading to a hypercoaguable state. Additional insults to this thrombotic balance are introduced by patient-specific, treatment related and tumor-specific factors. The importance of understanding the factors associated with increased thrombosis in cancer is paramount in order to adequately identify patients who will benefit from thromboprophylaxis.

Multi-locus DNA metabarcoding of zooplankton communities and scat reveal trophic interactions of a generalist predator.

To understand the ecosystem dynamics that underpin the year-round presence of a large generalist consumer, the Bryde's whale (Balaenoptera edeni brydei), we use a DNA metabarcoding approach and systematic zooplankton surveys to investigate seasonal and regional changes in zooplankton communities and if whale diet reflects such changes. Twenty-four zooplankton community samples were collected from three regions throughout the Hauraki Gulf, New Zealand, over two temperature regimes (warm and cool seasons), as well as 20 samples of opportunistically collected Bryde's whale scat. Multi-locus DNA barcode libraries were constructed from 18S and COI gene fragments, representing a trade-off between identification and resolution of metazoan taxa. Zooplankton community OTU occurrence and relative read abundance showed regional and seasonal differences based on permutational analyses of variance in both DNA barcodes, with significant changes in biodiversity indices linked to season in COI only. In contrast, we did not find evidence that Bryde's whale diet shows seasonal or regional trends, but instead indicated clear prey preferences for krill-like crustaceans, copepods, salps and ray-finned fishes independent of prey availability. The year-round presence of Bryde's whales in the Hauraki Gulf is likely associated with the patterns of distribution and abundance of these key prey items.

Expressed sequence tags and proteomics of antennae from the tortricid moth, Epiphyas postvittana.

Genomic and proteomic analyses of the antennae of the light brown apple moth, Epiphyas postvittana (Walker) (Lepidoptera: Tortricidae) were undertaken to identify genes and proteins potentially involved in odorant and pheromone binding and turnover. An EST approach yielded 5739 sequences, comprising 808 contigs and 1545 singletons. InterPro and Blast analyses revealed members of families implicated in odorant and pheromone binding (PBPs, GOBPs, ABPXs and CSPs) and turnover (CXEs, GSTs, CYPs). Of the three pheromone binding proteins (PBPs) identified, two were more highly expressed at the RNA and protein levels in adult male antennae (EpPBP1, EpPBP3), while a third was more highly expressed in female antennae (EpPBP2). To identify proteins involved in the detection of sex-specific signals, differential 2D gel electrophoresis (pH 5-8) followed by mass spectrometry was conducted on antennal proteins from males versus females. Identified male-biased proteins included a pheromone binding protein, a porin, a short chain dehydrogenase/reductase, and a member of the takeout family.

Proteolytic degradation of exocrine and serum immunoglobulins.

The susceptibility of exocrine and serum immunoglobulins and antibodies to proteolytic degradation was assessed. Colostral and duodenal fluid exocrine 11S IgA, monomeric serum IgA, and IgG were digested with trypsin, chymotrypsin, or duodenal fluid. Exocrine IgA was more resistant to digestion than were the serum immunoglobulins. Under conditions of the experiments, most of colostral IgA retained its 11S quaternary structure, including the secretory piece; the portion degraded was reduced almost entirely to peptides. The superior resistance of exocrine IgA was also demonstrated by digestion of serum IgG and nasal exocrine IgA diphtheria antitoxins with trypsin or duodenal fluid. Selective precipitation of trypsin-digested antitoxins with antibodies to heavy chains, light chains, or secretory piece revealed that the differences in susceptibility to digestion were due to differences in lability of the Fc portions of the IgA and IgG antibody molecules. The Fc portions of IgG antibody molecules were degraded or cleaved from the Fab units of the molecules, whereas the Fc-like portions of IgA antibody molecules remained associated with their Fab-like units and the secretory piece. On the other hand, trypsin treatment did not affect the antigen binding ability of the Fab parts of either the exocrine IgA or IgG antibodies. The Fc-like portions of exocrine IgA may be protected from tryptic degradation by the quaternary structure of the 11S molecules, which includes a dimer of 7S IgA subunits and the secretory piece.

An R-type Ca(2+) current in neurohypophysial terminals preferentially regulates oxytocin secretion.

Multiple types of voltage-dependent Ca(2+) channels are involved in the regulation of neurotransmitter release (Tsien et al., 1991; Dunlap et al., 1995). In the nerve terminals of the neurohypophysis, the roles of L-, N-, and P/Q-type Ca(2+) channels in neuropeptide release have been identified previously (Wang et al., 1997a). Although the L- and N-type Ca(2+) currents play equivalent roles in both vasopressin and oxytocin release, the P/Q-type Ca(2+) current only regulates vasopressin release. An oxytocin-release and Ca(2+) current component is resistant to the L-, N-, and P/Q-type Ca(2+) channel blockers but is inhibited by Ni(2+). A new polypeptide toxin, SNX-482, which is a specific alpha(1E)-type Ca(2+) channel blocker (Newcomb et al., 1998), was used to characterize the biophysical properties of this resistant Ca(2+) current component and its role in neuropeptide release. This resistant component was dose dependently inhibited by SNX-482, with an IC(50) of 4.1 nM. Furthermore, SNX-482 did not affect the other Ca(2+) current types in these CNS terminals. Like the N- and P/Q-type Ca(2+) currents, this SNX-482-sensitive transient Ca(2+) current is high-threshold activated and shows moderate steady-state inactivation. At the same concentrations, SNX-482 blocked the component of oxytocin, but not of vasopressin, release that was resistant to the other channel blockers, indicating a preferential role for this type of Ca(2+) current in oxytocin release from neurohypophysial terminals. Our results suggest that an alpha(1E) or "R"-type Ca(2+) channel exists in oxytocinergic nerve terminals and, thus, functions in controlling only oxytocin release from the rat neurohypophysis.

Fitness, training, and the growth hormone-->insulin-like growth factor I axis in prepubertal girls.

We recently demonstrated that a brief endurance type training program led to increases in thigh muscle mass and peak oxygen uptake (VO(2)) in prepubertal girls. In this study, we examined the effect of training on the GH-->insulin-like growth factor I (GH-->IGF-I) axis, a system known to be involved both in the process of growth and development and in the response to exercise. Healthy girls (mean age 9.17 +/- 0.10 yr old) volunteered for the study and were randomized to control (n = 20) and training groups (n = 19) for 5 weeks. Peak VO(2), thigh muscle volume, and blood samples [for IGF-I, IGF-binding proteins (IGFBP)-1 to -6, and GHBP] were measured. At baseline, IGF-I was significantly correlated with both peak VO(2) (r = 0.44, P < 0.02) and muscle volume (r = 0.58, P < 0.004). IGFBP-1 was negatively correlated with muscle volume (r = -0.71, P < 0.0001), as was IGFBP-2. IGFBP-4 and -5 were significantly correlated with muscle volume. We found a threshold value of body mass index percentile (by age) of about 71, above which systematic changes in GHBP, IGFBP-1, and peak VO(2) per kilogram were noted, suggesting decreases in the following: 1) GH function, 2) insulin sensitivity, and 3) fitness. Following the training intervention, IGF-I increased in control (19.4 +/- 9.6%, P < 0.05) but not trained subjects, and both IGFBP-3 and GHBP decreased in the training group (-4.2 +/- 3.1% and -9.9 +/- 3.8%, respectively, P < 0.05). Fitness in prepubertal girls is associated with an activated GH-->IGF-I axis, but, paradoxically, early in a training program, children first pass through what appears to be a neuroendocrine state more consistent with catabolism.

Secondary elevation of extracellular neurotransmitter amino acids in the reperfusion phase following focal cerebral ischemia.

The purpose of this study was to evaluate amino acid neurotransmitter dynamics in the reperfusion phase after transient cerebral ischemia. In vivo microdialysis was used to measure extracellular amino acid levels in a rabbit model of focal ischemia. During 30 min of transient ischemia (n = 5), small but significant (p < 0.05) increases in glutamate, aspartate, gamma-aminobutyric acid (GABA), and taurine were noted. These elevations rapidly returned to baseline levels upon recirculation and remained constant for up to 5.5 h of reperfusion. In rabbits subjected to 2 h of transient ischemia (n = 5), two phases of amino acid release were seen. During ischemia, large (5- to 50-fold) elevations in glutamate, aspartate, GABA, and taurine occurred, as expected. These elevations rapidly normalized upon unocclusion. However, significant (p < 0.05) secondary elevations in glutamate, aspartate, and GABA occurred after 2-4 h of reperfusion. Regression analysis demonstrated significant correlations between primary (ischemic) and secondary (reperfusion) efflux. In permanent ischemia (n = 5), amino acid levels remained elevated throughout the entire experiment. Secondary elevations in excitatory amino acids may further contribute to the excitotoxic cascade during reperfusion.

Calcium channel antagonist peptides define several components of transmitter release in the hippocampus.

The use of subtype-selective voltage-sensitive calcium channel (VSCC) antagonists has established that neurotransmitter release in mammalian brain is mediated by N-like and P-like VSCCs, and that other subtypes also contribute significantly. To determine the roles presynaptic VSCCs play in nervous system function and to evaluate the therapeutic potential of their selective inhibition, it is necessary to define further the contributions of VSCC subtypes to neurotransmitter release. The novel conopeptide, SNX-230 (omega-conopeptide MVIIC), has revealed a new VSCC subtype, the Q-type, in cerebellar granule cells. We have compared the effects of SNX-230 on release of tritiated D-aspartate ([3H]D-Asp; a non-metabolizable analog of glutamate), gamma-aminobutyric acid ([3H]GABA), and norepinephrine ([3H]NE) from rat hippocampal slices to those of the N-type VSCC blocker, SNX-111 (omega-conopeptide MVIIA), and the P-type blocker, omega-agatoxin-IVA (AgaIVA). SNX-230 blocks both [3H]D-Asp and [3H]GABA release completely, whereas AgaIVA blocks them potently but partially and SNX-111 has no effect. These results suggest that glutamate and GABA release are mediated by two VSCC subtypes, a P-type and another, perhaps Q-like. SNX-111 blocks [3H]NE release potently but partially, while SNX-230 blockade is complete, consisting of one very potent phase and one less potent phase. AgaIVA also blocks [3H]NE release potently but partially. These results suggest that at least two VSCC subtypes, an N-type and a novel non-N-type, mediate NE release. Pair-wise combinations of the three ligands indicate that at least three pharmacologically distinct components comprise [3H]NE release in the hippocampus.

Alterations in K+ evoked profiles of neurotransmitter and neuromodulator amino acids after focal ischemia-reperfusion.

Secondary elevations in extracellular amino acids occur during reperfusion after transient cerebral ischemia. The delayed accumulation of excitatory amino acids may contribute to the progressive development of neuronal injury. In this study, we explored the mechanisms that may be involved in this phenomenon. Microdialysis samples from probes located in rabbit cortex were analysed with a chiral amino acid procedure. Concentrations of neurotransmitters (L-Glu, GABA), N-methyl-D-aspartate receptor modulators (D-Ser, Gly), an inhibitory neuromodulator (Tau), the lipid component phosphoethanolamine, and L-Gln, L-Ser and L-Ala were measured. Depolarization via perfusion with potassium was used to assess the status of release/reuptake systems at 2 and 4 h reperfusion after 2 h transient focal ischemia. Background experiments classified potassium evoked responses as calcium dependent or calcium-independent by inclusion of 30 microM omega-conopeptide MVIIC or by inclusion of 20 mM magnesium and ommision of calcium. During ischemia, large elevations of almost all amino acids occurred. During reperfusion, secondary elevations in transmitter amino acids (L-Glu, GABA) and N-methyl-D-aspartate receptor modulators (D-Ser, Gly) occurred. Tau remained slightly elevated whereas the lipid component phosphoethanolamine remained high and stable during reperfusion. Reperfusion significantly potentiated the potassium response for amino acids with calcium-dependent responses (L-Glu and GABA). In contrast, calcium-independent responses (Tau, phosphoethanolamine, L-Gln) were significantly attenuated. Intermediate behavior was observed with Gly, while no potassium responses were observed for D-Ser, L-Ser or L-Ala. These data demonstrate that perturbations in evoked amino acid profiles after ischemia-reperfusion are selective. Reduction of calcium-independent responses implicate a general decline in efficacy of transporter mechanisms that restore transmembrane gradients of ions and transmitters. Decreased efficacy of transporter systems may reduce transmitter reuptake and account for the amplified release of L-Glu and GABA, thus contributing to progressive neural dysfunction after cerebral ischemia.

Binding of Bacillus thuringiensis delta-endotoxins Cry1Ac and Cry1Ba to a 120-kDa aminopeptidase-N of Epiphyas postvittana purified from both brush border membrane vesicles and baculovirus-infected Sf9 cells.

A 120-kDa protein was purified from brush border membrane vesicles of the tortricid moth Epiphyas postvittana (Walker) based both on its activity as an aminopeptidase and the ability to bind the Bacillus thuringiensis delta-endotoxin Cry1Ac. The purified enzyme had a pI of 5.6 and was a leucine aminopeptidase, with some isoleucine, phenylalanine and tryptophan aminopeptidase activity. Further characterisation showed that the protein was also able to bind Cry1Ba. During purification, the molecular weight of the protein decreased from 120 to 115 kDa due to the loss of a glycophosphatidinyl anchor. The protein was N-terminally sequenced and, using this information and conserved regions within other insect aminopeptidase-N (APN) sequences, redundant primers were designed to amplify the aminopeptidase coding sequence from E. postvittana midgut cDNA. The predicted protein sequence from the full-length cDNA was most closely related to the APN protein sequence from Heliothis virescens (61% identity) and shared other features of insect APNs including a Zn(2+) binding site motif and four conserved cysteines. The E. postvittana was expressed in Sf9 cells using baculovirus, yielding a protein of molecular weight 130 kDa, but with unchanged N-terminal sequence. Purified recombinant protein bound both Cry1Ac and Cry1Ba by ligand blot assays. However, despite the protein being expressed on the external surface of the Sf9 cells, it bound neither Cry1Ac nor Cry1Ba in vivo.

The acetylcholinesterase gene and organophosphorus resistance in the Australian sheep blowfly, Lucilia cuprina.

Acetylcholinesterase (AChE), encoded by the Ace gene, is the primary target of organophosphorous (OP) and carbamate insecticides. Ace mutations have been identified in OP resistants strains of Drosophila melanogaster. However, in the Australian sheep blowfly, Lucilia cuprina, resistance in field and laboratory generated strains is determined by point mutations in the Rop-1 gene, which encodes a carboxylesterase, E3. To investigate the apparent bias for the Rop-1/E3 mechanism in the evolution of OP resistance in L. cuprina, we have cloned the Ace gene from this species and characterized its product. Southern hybridization indicates the existence of a single Ace gene in L. cuprina. The amino acid sequence of L. cuprina AChE shares 85.3% identity with D. melanogaster and 92.4% with Musca domestica AChE. Five point mutations in Ace associated with reduced sensitivity to OP insecticides have been previously detected in resistant strains of D. melanogaster. These residues are identical in susceptible strains of D. melanogaster and L. cuprina, although different codons are used. Each of the amino acid substitutions that confer OP resistance in D. melanogaster could also occur in L. cuprina by a single non-synonymous substitution. These data suggest that the resistance mechanism used in L. cuprina is determined by factors other than codon bias. The same point mutations, singly and in combination, were introduced into the Ace gene of L. cuprina by site-directed mutagenesis and the resulting AChE enzymes expressed using a baculovirus system to characterise their kinetic properties and interactions with OP insecticides. The K(m) of wild type AChE for acetylthiocholine (ASCh) is 23.13 microM and the point mutations change the affinity to the substrate. The turnover number of Lucilia AChE for ASCh was estimated to be 1.27x10(3) min(-1), similar to Drosophila or housefly AChE. The single amino acid replacements reduce the affinities of the AChE for OPs and give up to 8.7-fold OP insensitivity, while combined mutations give up to 35-fold insensitivity. However, other published studies indicate these same mutations yield higher levels of OP insensitivity in D. melanogaster and A. aegypti. The inhibition data indicate that the wild type form of AChE of L. cuprina is 12.4-fold less sensitive to OP inhibition than the susceptible form of E3, suggesting that the carboxylesterases may have a role in the protection of AChE via a sequestration mechanism. This provides a possible explanation for the bias towards the evolution of resistance via the Rop-1/E3 mechanism in L. cuprina.

Pheromone binding proteins of Epiphyas postvittana (Lepidoptera: Tortricidae) are encoded at a single locus.

The light brown apple moth, Epiphyas postvittana (Tortricidae: Lepidoptera) uses a blend of (E)-11-tetradecenyl acetate and (E,E)-9,11-tetradecadienyl acetate as its sex pheromone. Odorant binding proteins, abundant in the antennae of male and female E. postvittana, were separated by native PAGE to reveal four major proteins with distinct mobilities. Microsequencing of their N-terminal residues showed that two were general odorant binding proteins (GOBPs) while two were pheromone binding proteins (PBPs). Full length cDNAs encoding these proteins were amplified using a combination of PCR and RACE-PCR. Sequence of the GOBPs revealed two genes (EposGOBP1, EposGOBP2), similar to orthologues in other species of Lepidoptera. Eleven cDNAs of the PBP gene were amplified, cloned and sequenced revealing two major phylogenetic clusters of PBP sequences differing by six amino acid substitutions. The position of the six amino acid differences on the protein was predicted by mapping onto the three-dimensional structure of PBP of Bombyx mori. All six substitutions were predicted to fall on the outside of the protein away from the inner pheromone binding pocket. One substitution does fall close to the putative dimerisation region of the protein (Ser63Thr). Expression of three of the cDNAs in a baculovirus expression system revealed that one class encodes an electrophoretically slow form (EposPBP1-12) while the other encodes a fast form (EposPBP1-2, EposPBP1-3). A native Western of these expressed proteins compared with antennal protein extracts demonstrated that PBP is also expressed in female antennae and that PBP may be present as a dimer as well as a monomer in E. postvittana. The fast and slow forms of EposPBP1 are allelic. Westerns on single antennal pair protein extracts and allele-specific PCR from genomic DNA both show a segregating pattern of inheritance in laboratory and wild populations. Radio labelled (E)-11-tetradecenyl acetate binds to both fast and slow PBP forms in gel assays. Taken together, the genetic and biochemical data do not support the hypothesis that these PBPs are specific for each component of the E. postvittana pheromone. However, duplication of this PBP locus in the future might allow such diversification to evolve, as observed in the other species.

cDNA cloning, baculovirus-expression and kinetic properties of the esterase, E3, involved in organophosphorus resistance in Lucilia cuprina.

Resistance to organophosphorus insecticides (OPs) in the sheep blowfly, Lucilia cuprina, is associated with a non-staining phenotype of the carboxylesterase isozyme, E3 (E.C. 3.1.1.1). Here, we show that a member of alpha-esterase multigene family, Lc alpha E7, encodes E3. An Lc alpha E7 cDNA has been isolated from an OP-susceptible strain and expressed in a baculovirus. The expressed product is the same as E3 in its electrophoretic mobility and preference for alpha-over beta-naphthyl acetate as substrate. Its preference (kcat/K(m)) for a range of carboxylester substrates is alpha-naphthyl butyrate > alpha-naphthyl propionate > alpha-naphthyl acetate > methylthiobutyrate > p-nitrophenyl acetate. The enzyme is potently inhibited by OPs (ki [paraoxon] = 6.3 +/- 1.4 x 10(7)/M/min, ki [chlorfenvinphos] = 5.9 +/- 0.6 x 10(7)/M/min) and exhibits a high turnover of methylthiobutyrate (1009/s), consistent with its proposed homology to the ali-esterase that is thought to mutate to confer OP resistance in Musca domestica. E3 shares 64% amino acid identity with its Drosophila melanogaster homologue, Dm alpha E7, and is also closely related to other esterases involved in OP resistance such as the B1 esterase of Culex pipiens (38%) and E4 of Myzus persicae (30%).

Molecular cloning of an alpha-esterase gene cluster on chromosome 3r of Drosophila melanogaster.

All or part of the alpha-esterase gene cluster in Drosophila melanogaster has been isolated by screening a YAC clone that spans cytological region 84D3-10 with consensus carboxyl/cholinesterase oligonucleotides. The cluster encompasses 11 putative esterase genes within 65 kb of genomic DNA and is one of the largest clusters of related protein-coding genes yet reported in Drosophila. The cluster must include the gene encoding the major alpha-esterase isozyme, EST9, which has previously been mapped to 84D3-5. It probably also includes the genes encoding the EST23, MCE and ALI esterases that have previously been mapped to 84D3-E2. The latter three are homologs of genes involved in organophosphate insecticide resistance in the sheep blowfly, Lucilia cuprina and the housefly, Musca domestica. Sequencing of one of the putative esterase genes in the Drosophila cluster, alpha E1, shows that it would encode features characteristic of an active carboxyl/cholinesterase, including the so-called catalytic triad, the nucleophilic elbow and oxyanion hole. It also shows that the closest relative of alpha E1 amongst previously published esterase sequences is ESTB1, which confers organophosphate resistance in Culex mosquitoes. We argue that we have cloned the D. melanogaster version of a major cluster of esterase genes which have variously mutated to confer organophosphate resistance in diverse Diptera.

Quantitative HPLC analysis of rat neurophysin processing.

Neuropeptides exist in multiple bioactive forms and it is important to understand quantitative aspects of variations in the metabolism of the different forms. Towards this end a sensitive HPLC procedure, utilizing on-line reaction with fluorescamine, was developed for the quantitative analysis of the major neural lobe peptides of male Wistar rats. Peptide peaks were defined by amino acid analysis, carboxypeptidase digestion, gel electrophoresis and, in the case of the glycopeptide, gel filtration and Edman degradation. Control experiments were done to show that in vivo amounts of all of the peptides were being measured. Quantitation of the extent of conversion of the oxytocin neurophysin to the form missing the carboxyl-terminal glutamate residue provided an indirect measure of secretory granule age in (1) single neural lobes from animals of different ages; (2) single neural lobes from animals at various stages of dehydration and rehydration; (3) single neural lobes from animals subjected to chronic osmotic stress by providing 1.5% NaCl as drinking water; (4) high potassium perfusates from pooled isolated neural lobes; (5) isolated secretory granules of varying densities. Relative to homogenates of control neural lobes, the extent of conversion of the oxytocin neurophysin is increased in neural lobes from animals of larger size, in secretory granules of greater than the mean density, and in the neural lobes taken from animals during the early stage of dehydration. The extent of conversion is decreased in neural lobes from animals of smaller sizes, in the later stages of dehydration, after a chronic osmotic stress and in secretory granules of greater than mean density. No large change relative to control homogenates was seen in the high potassium perfusates. We argue from the data that the peptide content of axon terminals (putative release sites) and swellings (putative storage sites) is relatively homogeneous and that rates of secretory granule transfer between axonal compartments are, therefore, fast relative to rates of secretory granule turnover. Granule movements between release and storage sites will be of fundamental importance in determining the types of changes in secreted peptide content which will occur with changes in secretory demand.

Structure function analysis of an arthropod peptide hormone: proctolin and synthetic analogues compared on the cockroach hindgut receptor.

Proctolin is a pentapeptide (arg-tyr-leu-pro-thr) found in nervous tissues throughout the phylum Arthropoda. Initially described as a peptidergic neuromuscular transmitter, it now appears that proctolin is a major arthropod neurohormone modulating nervous activity, muscle tonus and contractile force. Structure-function studies with synthetic analogues demonstrate diverse peptides which retain agonistic activity, but few exhibit a high degree of affinity for the cockroach hindgut receptor compared with proctolin (Kdapp = 2 x 10(-8) M). High affinity agonists (Kdapp less than or equal to 10(-7) M) are limited to [phe2]-proctolin, [lys1]-proctolin and specific N-terminal additions. In this regard the hindgut receptor differs in its ligand specificity from that reported for the locust extensor tibia receptor. Using the analogue studies to predict sequences which may act as agonists, we have examined the known vertebrate peptide hormones for proctolin-like sequences. A possible relationship between vasoactive intestinal peptide, proctolin and erythrophore concentrating hormone is critically evaluated.

Structural and biosynthetic properties of peptides in cone snail venoms.

Venoms of the predatory cone snails Conus textile, Conus striatus, and Conus magus were subjected to comprehensive analysis of peptide content. With the fish-eating cone snails C. magus and C. striatus, the most abundant venom peptides were of > 30-50 residues, whereas the predominant peptides in the venom of the mollusc-eating snail, C. textile, were of 20-35 residues. Amino acid sequencing revealed an identical but unusual amino acid in a conserved position in four novel omega-type peptides from the C. textile venom. Two conserved amino acid sequences were obtained from the venoms of both C. magus and C. striatus. The amino acid compositions of the isolated C. textile peptides and the expected processing products of the propeptides (42) were compared. Despite the recovery in abundance of the carboxyl-terminal omega-type peptides, none of the isolated peptides had compositions expected from the propeptide amino-terminal fragments. We conclude that there are likely mechanisms for excluding the amino-terminal propeptide fragments from this venom, resulting in a venom with greater potency. Amounts of the different omega-type peptides in the venom vary widely, suggesting a distinct mechanism that results in the selective synthesis of different bioactive carboxyl-terminal propeptide fragments at elevated levels.