Functional characterization of CYP4G11-a highly conserved enzyme in the western honey bee Apis mellifera.

Determining the functionality of CYP4G11, the only CYP4G in the genome of the western honey bee Apis mellifera, can provide insight into its reduced CYP4 inventory. Toward this objective, CYP4G11 transcripts were quantified, and CYP4G11 was expressed as a fusion protein with housefly CPR in Sf9 cells. Transcript levels varied with age, task, and tissue type in a manner consistent with the need for cuticular hydrocarbon production to prevent desiccation or with comb wax production. Young larvae, with minimal need for desiccation protection, expressed CYP4G11 at very low levels. Higher levels were observed in nurses, and even higher levels in wax producers and foragers, the latter of which risk desiccation upon leaving the hive. Recombinant CYP4G11 readily converted octadecanal to n-heptadecane in a time-dependent manner, demonstrating its functions as an oxidative decarbonylase. CYP4G11 expression levels are high in antennae; heterologously expressed CYP4G11 converted tetradecanal to n-tridecane, demonstrating that it metabolizes shorter-chain aldehydes. Together, these findings confirm the involvement of CYP4G11 in cuticular hydrocarbon production and suggest a possible role in clearing pheromonal and phytochemical compounds from antennae. This possible dual functionality of CYP4G11, i.e., cuticular hydrocarbon and comb wax production and antennal odorant clearance, may explain how honey bees function with a reduced CYP4G inventory.

Two-year cortical and trabecular bone loss in CKD-5D: biochemical and clinical predictors.

This prospective two-year study of patients on chronic dialysis measured changes in bone mineral density (BMD). Patients with higher baseline BMD and shorter dialysis vintage lost more bone. Treatment with anti-hypertensives acting on the central nervous system was protective against bone loss. Baseline serum levels of sclerostin and bone-specific alkaline phosphatase predicted bone loss. INTRODUCTION: This prospective 2-year study of chronic kidney disease on dialysis (CKD-5D) patients assessed trabecular and cortical bone loss at the hip and spine and examined potential demographic, clinical, and serum biochemical predictors of bone loss. METHODS: Eighty-nine CKD-5D patients had baseline, year 1, and year 2 bone mineral density (BMD) measurements using dual X-ray absorptiometry (DXA) and quantitative computed tomography (QCT); concurrent blood samples were drawn and clinical variables recorded. No study treatments occurred. RESULTS: The 2-year total hip BMD change was - 5.9% by QCT and - 3.1% by DXA (p < 0.001). Spinal BMD was unchanged. QCT total hip cortical mass and volume decreased (- 7.3 and - 10.0%); trabecular volume increased by 5.9% (ps < 0.001). BMD changes did not vary with age, BMI, race, diabetes, smoking, or exercise. Patients with higher baseline BMD and shorter dialysis vintage lost more bone (p < 0.05). Vitamin D analogs and phosphate binders were not protective against bone loss; cinacalcet was protective by univariate but not by multivariable analysis. CNS-affecting antihypertensives were protective against loss of BMD, cortical mass, cortical volume (ps < 0.05) and trabecular mass (p = 0.007). These effects remained after adjustment. BSAP correlated with changes in BMD, cortical mass, and volume (p < 0.01) as did sclerostin (inversely). CONCLUSIONS: There was severe cortical bone loss at the hip best recognized by QCT. Patients with shorter dialysis vintage and less pre-existing bone loss lost more bone, while treatment with CNS-acting antihypertensives was protective. BSAP and sclerostin were useful markers of bone loss.

Comparative transcriptomics of mountain pine beetle pheromone-biosynthetic tissues and functional analysis of CYP6DE3.

BACKGROUND: The mountain pine beetle (MPB, Dendroctonus ponderosae Hopkins) is a highly destructive pest of pine forests in western North America. During flight to a new host tree and initiation of feeding, mountain pine beetles release aggregation pheromones. The biosynthetic pathways of these pheromones are sex-specific and localized in the midgut and fat body, but the enzymes involved have not all been identified or characterized. RESULTS: We used a comparative RNA-Seq analysis between fed and unfed male and female MPB midguts and fat bodies to identify candidate genes involved in pheromone biosynthesis. The 13,407 potentially unique transcripts showed clear separation based on feeding state and gender. Gene co-expression network construction and examination using petal identified gene groups that were tightly connected. This, as well as other co-expression and gene ontology analyses, identified all four known pheromone biosynthetic genes, confirmed the tentative identification of four others from a previous study, and suggested nine novel candidates. One cytochrome P450 monooxygenase, CYP6DE3, identified as a possible exo-brevicomin-biosynthetic enzyme in this study, was functionally characterized and likely is involved in resin detoxification rather than pheromone biosynthesis. CONCLUSIONS: Our analysis supported previously characterized pheromone-biosynthetic genes involved in exo-brevicomin and frontalin biosynthesis and identified a number of candidate cytochrome P450 monooxygenases and a putative cyclase for further studies. Functional analyses of CYP6DE3 suggest its role in resin detoxification and underscore the limitation of using high-throughput data to tentatively identify candidate genes. Further functional analyses of candidate genes found in this study should lead to the full characterization of MPB pheromone biosynthetic pathways and the identification of molecular targets for possible pest management strategies.

Quantitative genetics of costly neonatal sexual size dimorphism in squirrel monkeys (Saimiri boliviensis).

Offspring size is often an intimate link between the fitness of parents and offspring. Among mammals, neonate mass is also related to adult levels of dimorphism and intrasexual competitive mating. We describe the sex-specific genetic architecture of neonate mass in captive squirrel monkeys (Saimiri boliviensis), a small Neotropical primate. Best fitting quantitative genetic models show strong maternal genetic effects with little difference between sexes offering limited opportunity for neonatal dimorphism to respond to observed or hypothetical selection. Heritabilities that are approximately zero also imply it is unlikely that neonatal dimorphism can evolve as a correlated response to selection on adult size. However, male mass is also more dependent on maternal condition (age and parity) making dimorphism plastic. Finally, we hypothesize that large maternal genetic effects reflect income breeding and tightly synchronized seasonal reproduction in squirrel monkeys, both of which require strong maternal control of offspring growth and timing of birth.

Isolation, endocrine regulation and transcript distribution of a putative primary JH-responsive gene from the pine engraver, Ips pini (Coleoptera: Scolytidae).

We isolated a cDNA of unknown function from a juvenile hormone III (JH III)-treated male midgut cDNA library prepared from the pine engraver beetle, Ips pini, and examined its genomic structure. The gene, tentatively named "Ipi10G08", encoded a 410 amino acid translation product that shared 26-37% identity with unannotated matches from several insects. Semi-quantitative RT-PCR analysis of Ipi10G08 following application of a 10 microg dose of JH III demonstrated an early induction for both male and female beetles, with transcripts being detectable after 45 min. An expression profile of male midgut tissue indicated Ipi10G08 transcript levels reach a maximum induction of approximately 22.5-fold control levels at 4h post-treatment. Tissue distribution studies displayed a large induction of Ipi10G08 mRNA in the alimentary canal of JH III-treated beetles, especially in males. A dose curve from both sexes suggested there may be a difference in the ability to respond to lower levels of JH III and immunoblot analysis indicated that although JH III highly induces transcript levels in females, protein levels are not similarly induced, while protein levels are induced in males. Ipi10G08 is likely a primary JH response gene and may provide insight into how this hormone exerts its actions.

Inhibition of acetylcholinesterase in CSF versus brain assessed by 11C-PMP PET in AD patients treated with galantamine.

The relationship between acetylcholinesterase (AChE) activity in the CSF and brain of patients with Alzheimer's disease (AD) was investigated in 18 mild AD patients following galantamine treatment. The first 3 months of the study had a randomized double-blind placebo-controlled design, during which 12 patients received galantamine (16-24 mg/day) and six patients placebo. This was followed by 9 months galantamine treatment in all patients. Activities and protein levels of both the "read-through" AChE (AChE-R) and the synaptic (AChE-S) variants in CSF were assessed in parallel together with the regional brain AChE activity by (11)C-PMP and PET. The AChE-S inhibition was 30-36% in CSF, which correlated well with the in vivo AChE inhibition in the brain. No significant AChE inhibition was observed in the placebo group. The increased level of the AChE-R protein was 16% higher than that of AChE-S. Both the AChE inhibition and the increased level of AChE-R protein positively correlated with the patient's performance in cognitive tests associated with visuospatial ability and attention. In conclusion, AChE levels in CSF closely mirror in vivo brain AChE levels prior to and after treatment with the cholinesterase inhibitors. A positive cognitive response seems to dependent on the AChE inhibition level, which is balanced by an increased protein level of the AChE-R variant in the patients.

PET imaging of the in vivo brain acetylcholinesterase activity and nicotine binding in galantamine-treated patients with AD.

The effect of galantamine treatment on cortical acetylcholinesterase (AChE) activity and nicotinic receptor binding was investigated by positron emission tomography (PET) in 18 patients with mild Alzheimer's disease (AD) in relation to galantamine concentration and the patients' cognitive performances. The first 3 months of the study was of a randomized double-blind placebo-controlled design, during which 12 patients received galantamine (16-24mg/day) and 6 patients the placebo, and this was followed by 9 months' galantamine treatment in all patients. The patients underwent PET examinations to measure cortical AChE activity ((11)C-PMP) and (11)C-nicotine binding. Neuropsychological tests were performed throughout the study. Inhibition (30-40%) of cortical AChE activity was observed after 3 weeks to 12 months of galantamine treatment. No significant change in mean cortical (11)C-nicotine binding was observed during the study. (11)C-Nicotine binding, however, positively correlated with plasma galantamine concentration. Both the changes of AChE activity and (11)C-nicotine binding correlated positively with the results of a cognitive test of attention. In conclusion, galantamine caused sustained AChE inhibition for up to 12 months. At the individual level, the in vivo cortical AChE inhibition and (11)C-nicotine binding were associated with changes in the attention domain of cognition rather than episodic memory.

Isolation, endocrine regulation and mRNA distribution of the 3-hydroxy-3-methylglutaryl coenzyme A synthase (HMG-S) gene from the pine engraver, Ips pini (Coleoptera: Scolytidae).

We isolated a full-length cDNA encoding 3-hydroxy-3-methylglutaryl coenzyme A synthase (HMG-S) from the pine engraver beetle, Ips pini (Say), and examined its genomic structure. The intron-less gene has a predicted 460 amino acid cytosolic protein product with 73% identity to HMG-S from Dendroctonus jeffreyi, and high identity (58-64%) with other insect HMG-Ss. Topically applied juvenile hormone (JH) III induced HMG-S mRNA levels up to 6.5-fold in both sexes, mostly in the anterior midgut, though there were differences between males and females in the timing, sensitivity to JH III dose and tissue distribution of HMG-S mRNA. These data further validate the coordinate regulation of mevalonate pathway genes for de novo isoprenoid pheromone production in bark beetles.

Effects of juvenile hormone on gene expression in the pheromone-producing midgut of the pine engraver beetle, Ips pini.

Juvenile hormone III (JH III) stimulates biosynthesis of the monoterpenoid aggregation pheromone component, ipsdienol, in the anterior midgut of the male pine engraver beetle, Ips pini (Say). To understand better the hormonal regulation of pheromone biosynthesis in this forest pest, and identify JH III-responsive genes, microarrays were prepared and hybridized to cDNA from midguts of JH III-treated beetles. Expression patterns were confirmed by quantitative real-time RT-PCR. JH III co-ordinately regulated mevalonate pathway genes and many other genes implicated in pheromone biosynthesis. Sex differences in basal levels of mevalonate pathway genes were consistent with their role in male-specific pheromone biosynthesis. This is the first microarray-based study of the developmental and hormonal regulation of insect pheromone biosynthesis.

Isolation and molecular characterization of Musca domestica delta-9 desaturase sequences.

We have isolated fatty acyl-CoA desaturase cDNA (Mdomd9) and genomic sequences from the housefly, Musca domestica. Two approximately 1.66 kb cDNAs were recovered. They had identical coding regions and 3' untranslated regions (UTRs), but differed in their 5' UTRs. The open reading frame encodes a 380 amino acid (aa) protein with 82% identity to Drosophila melanogaster desat1, and significant (> 50%) identity with other insect delta-9 desaturases. Functional analyses in a yeast expression system confirmed the cDNA encodes a delta9 desaturase. Northern analysis indicated two transcripts of 1.7 and 2.9 kb that hybridized specifically to the open reading frame. PCR amplification of genomic templates revealed three intron sites that are conserved among other insect species. Southern analysis of genomic DNA indicated at least two desaturase gene copies per haploid genome. There is a high degree of polymorphism, most of which appears to be due to variable intron sequences; curiously, individual flies had varying morphs of intron II and intron III. Together, the data suggest that there are more delta9 desaturase alleles within the population studied than there are loci within the genome, and support other studies suggesting that insect fatty acyl-CoA desaturases are a dynamically evolving gene family.

Biochemical and molecular characterizaton of house cricket (Acheta domesticus, Orthoptera: Gryllidae) Delta9 desaturase.

Studies of insect fatty acyl-CoA desaturases have heretofore concentrated on the Diptera and Lepidoptera. We report here the isolation and characterization of a fatty acyl-CoA Delta9 desaturase from the house cricket, Acheta domesticus (Orthoptera). Two desaturase cDNAs were isolated from a library, one of which contained two intron sequences. The clones were identical in their respective coding regions, but had divergent 5' and 3' untranslated regions. The cDNAs encode a 359 amino acid desaturase enzyme that could rescue a fatty acyl-CoA desaturase auxotrophic phenotype when expressed in yeast. Biochemical analysis of lipids from transformed yeast cells confirmed that the enzyme is a Delta9 desaturase with activity on both palmitic and stearic acid substrates. Southern blotting indicated multiple Delta9 desaturase genes within the genome. A single message that was up-regulated in fed insects vs. starved insects was observed on northern blots, indicating transcriptional regulation in response to diet.

Specialization of midgut cells for synthesis of male isoprenoid pheromone components in two scolytid beetles, Dendroctonus jeffreyi and Ips pini.

Endodermal or midgut cells have only recently been recognized as the site of pheromone synthesis in bark beetles. Midgut cells are not only specialized for digestion, but they have also been recruited to form isoprenoid compounds that function as pheromone components in Ips pini and Dendroctonus jeffreyi. Male bark beetle midgut cells are competent to produce isoprenoid pheromones after feeding or stimulation by juvenile hormone (JH) III. Competent midgut cells share many ultrastructural features with cells that do not secrete isoprenoid pheromone, but they are distinguished from these by abundant and highly ordered arrays of smooth endoplasmic reticula. During secretion, both midgut cells that produce pheromone and cells that do not are characterized by the presence of apical extrusions (apocrine secretion) rather than the presence of vesicles that fuse with the apical membrane and undergo exocytosis (eccrine secretion). Pheromone-producing cells of the midgut do not represent a population of cells that are distinct from cells involved in digestion. All, or most, midgut cells of male I. pini and D. jeffreyi can secrete pheromones as well as digestive enzymes.

Male jeffrey pine beetle, Dendroctonus jeffreyi, synthesizes the pheromone component frontalin in anterior midgut tissue.

The male Jeffrey pine beetle, Dendroctonus jeffreyi Hopkins (Coleoptera: Scolytidae), produces the bicyclic ketal frontalin as part of a complex semiochemical blend. A key regulated enzyme in the mevalonate pathway, 3-hydroxy-3-methylglutaryl-CoA reductase (HMG-R), showed high transcript levels in the anterior midgut of male Jeffrey pine beetles by in situ hybridization. HMG-R expression in this area of the alimentary canal was related to male emergence, where emerged males demonstrated significant up-regulation of HMG-R transcript and pre-emerged males showed only basal levels. Pre-emerged males were induced to express high levels of HMG-R transcript by treatment with juvenile hormone (JH) III. Additionally, isolated anterior midgut tissue from JH III-treated males converted radiolabeled acetate to frontalin, as assayed by radio-HPLC, providing strong evidence that this is the site of frontalin production in male beetles.

Recognition errors in the quantification of micro-organisms by fluorescence microscopy.

OBJECTIVES: An interlaboratory comparison of fluorescence microscopic counting of micro-organisms was carried out to assess counting errors in the measurement of micro-organisms in bioaerosols generated during handling of municipal waste. METHODS: Series of 27 replicate samples were collected in the work environment with a modified field exposure chamber. The preparation methods of three Scandinavian laboratories were compared. Four microscopists from these laboratories performed the counts which were also compared. Duplicate counting of identical microscopic fields allowed the assessment of recognition errors. RESULTS: The field exposure chamber collected replicate samples with a relative standard deviation of 5% when particles < or = 15 microm aerodynamic diameter were collected. Storage time of 40-200 days had no significant influence on the total micro-organism count. Differences between preparation methods were from 2 to 35% for bacteria, and from 15 to 35% for fungal spores when samples were analysed in Oslo; the results for fungal spore counts were significantly different (P < 0.01). These differences were not confirmed when samples were analysed in Umeå, Copenhagen and Oslo using those laboratories methods. These results can be explained by less efficient redispersion of aggregates when the Umeå and Copenhagen methods were recreated in Oslo yielding a greater number of innumerable aggregates. Differences between microscopists were minor for fungal spores (2-12%) but substantial for bacteria (4-53%). A major source of error was the recognition of bacteria which had a relative standard deviation (rsd) of 37% although a lower size limit of 0.75 microm was adopted for counting of bacteria. Fungal spores were recognised with much better precision (rsd 9%). CONCLUSIONS: Recognition errors of bacteria may be substantial and more specific fluorochromes are needed for fluorescence microscopic counting of micro-organisms.

Hydrocarbons of Rhodnius prolixus, a Chagas disease vector.

The surface hydrocarbons of the blood-sucking insect, Rhodnius prolixus, a major Chagas disease vector in Venezuela, Colombia and Central America, were characterized by capillary gas chromatography coupled to mass spectrometry (CGC-MS). A total of 54 single or multicomponent peaks of saturated, straight-chain and methyl-branched hydrocarbons were identified. Major n-alkanes were n-C27, n-C29, n-C31 and n-C33 hydrocarbons. In the branched fraction, methyl groups were at positions 3, 5, 7, 11, 13, 15 and 17- for monomethyl isomers, and separated by three or five methylene groups for the trimethyl or tetramethyl derivatives. For the higher molecular weight components of 37, 39 and 41 atoms in the carbon skeleton, the di-, tri- and tetramethyl branches were usually separated by three or five, and sometimes 7, 11 or 13, methylene groups. The internal hydrocarbon pool contained larger amounts of the higher molecular weight methyl-branched components. Qualitative differences among epicuticular and internal hydrocarbon compositions were detected, both in adult and nymphal stages. No significant sexual dimorphism was detected, but a significant shift in the major n-alkane components was evident from the nymphal to the adult stage, differing also in the relative amounts of the higher molecular weight methyl-branched chains. Comparison of the hydrocarbon components to that of other Chagas disease vectors is discussed.

Hydrocarbon-released nestmate aggression in the Argentine ant, Linepithema humile, following encounters with insect prey.

Argentine ants, Linepithema humile, were attacked by their nestmates following contact with a particular prey item, the brown-banded cockroach, Supella longipalpa. Contact with prey, as brief as 2 min, provoked nestmate aggression. Argentine ants contaminated with hydrocarbons extracted from S. longipalpa also released nestmate aggression behavior similar to that released by the whole prey item, confirming the involvement of hydrocarbons. In contrast to S. longipalpa, little or no nestmate aggression was induced by other ant prey from diverse taxa. A comparison of prey hydrocarbon profiles revealed that all hydrocarbons of S. longipalpa were very long chain components with 33 or more carbons, while other prey had either less, or none, of the very long chain hydrocarbons of 33 carbons or greater. We identified the hydrocarbons of S. longipalpa and some new groups of long chain hydrocarbons of L. humile. The majority of S. longipalpa hydrocarbons were 35 and 37 carbons in length with one to three methyl branches, and closely resembled two previously unidentified groups of compounds from L. humile of similar chain length. The hydrocarbons of S. longipalpa and L. humile were compared and their role in the Argentine ant nestmate recognition is discussed.

Dampness in buildings and health. Nordic interdisciplinary review of the scientific evidence on associations between exposure to "dampness" in buildings and health effects (NORDDAMP).

Several epidemiological investigations concerning indoor environments have indicated that "dampness" in buildings is associated to health effects such as respiratory symptoms, asthma and allergy. The aim of the present interdisciplinary review is to evaluate this association as shown in the epidemiological literature. A literature search identified 590 peer-reviewed articles of which 61 have been the foundation for this review. The review shows that "dampness" in buildings appears to increase the risk for health effects in the airways, such as cough, wheeze and asthma. Relative risks are in the range of OR 1.4-2.2. There also seems to be an association between "dampness" and other symptoms such as tiredness, headache and airways infections. It is concluded that the evidence for a causal association between "dampness" and health effects is strong. However, the mechanisms are unknown. Several definitions of dampness have been used in the studies, but all seems to be associated with health problems. Sensitisation to mites may be one but obviously not the only mechanism. Even if the mechanisms are unknown, there is sufficient evidence to take preventive measures against dampness in buildings.

Airborne chemical compounds on dairy farms.

The chemical environment that dairy farmers are exposed to during milking was investigated. Volatile organic compounds (VOCs) were analysed and identified, and the levels of formaldehyde, ammonia and carbon dioxide were measured in eight farms in northern Sweden. Both stationary and personal samples were taken. A total of 70 VOCs were identified from the adsorbent samples, with p-cresol, 2-butanone, ethyl acetate, alpha-pinene and delta 3-carene occurring at the highest levels. All monitored levels were significantly lower for compounds having a stated highest occupational exposure level (OEL). Using multivariate techniques some differences in the composition of the workplace air between and within the farms were found. No difference was found between personal exposure and the surrounding environment in the cowshed.

Effect of age and sex on the production of internal and external hydrocarbons and pheromones in the housefly, Musca domestica.

The epicuticular and internal waxes of male and female houseflies were examined by capillary gas chromatography-mass spectrometry at closely timed intervals from emergence until day-6 of adulthood. New components identified included tricosan-10-one, 9,10-epoxyheptacosane, heptacosen-12-one, a series of odd-carbon numbered dienes from C31 to C39, several positional isomers of monoenes including (Z)-9- and 7-pentacosene and a number of methyl- and dimethylalkanes. (Z)-9-tricosene appears in internal lipids prior to appearing on the surface of the insect, suggesting that it is transported in the hemolymph to its site of deposition on the epicuticle. The large increases in the amount of (Z)-9-tricosene in females from day-2 until day-6 is compensated for by a concomitant decrease in (Z)-9-heptacosene. The C23 epoxide and ketone only appear in females after the production of (Z)-9-tricosene is induced, and are only abundant in epicuticular waxes, suggesting they are formed after (Z)-9-tricosene is transported to the cells which are involved in taking them to the surface of the insect. Mathematical analysis indicated that the time shift between internal production and external accumulation in females is more than 24 h. The divergence between male and female lipid production occurs at an early stage, when insects are less than one day old.

Tissue distribution and lipophorin transport of hydrocarbons and sex pheromones in the house fly, Musca domestica.

We investigated the relationship between epicuticular and internal hydrocarbons in the adult house fly, Musca domestica and the distribution of hydrocarbons, including the female sex pheromone component, (Z)-9-tricosene, in tissues. Internal hydrocarbons increased dramatically in relation to sexual maturation and were found in the hemolymph, ovaries, digestive tract, and fat body. (Z)-9-Tricosene comprised a relatively large fraction of the hydrocarbons in the female carcass and hemolymph, and less so in other tissues, while other hydrocarbons were represented in greater amounts in the ovaries than in other tissues. It therefore appears that certain hydrocarbons were selectively provisioned to certain tissues such as the ovaries, from which pheromone was relatively excluded. Both KBr gradient ultracentrifugation and specific immunoprecipitation indicated that > 90% of hemolymph hydrocarbons were associated with a high-density lipophorin (density = 1.09 g ml(-1)), composed of two apoproteins under denaturing conditions, apolipophorin I (approximately 240 kD) and apolipophorin II (approximately 85 kD). Our results support a predicted model (Chino, 1985) that lipophorin is involved in the transport of sex pheromone in M. domestica. In addition to delivering hydrocarbons and sex pheromones to the cuticular surface, we suggest that lipophorin may play an important role in an active mechanism that selectively deposits certain subsets of hydrocarbons at specific tissues.