RNA interference and functional characterization of a tergal gland alpha amylase in the German cockroach, Blattella germanica L.

German cockroach males possess tergal glands that secrete a combination of oligosaccharides, lipids and proteins. Four major proteins occur in the secretion, with one being the 63 kDa alpha-amylase Blattella germanica Tergal Gland protein-1 (BGTG-1). Denaturing and starch gel electrophoresis coupled with peptide sequencing verified amylase activity for the BGTG-1 protein. BGTG-1 gene expression profiles were determined by using quantitative real-time PCR to compare messenger RNA abundance among isolated tissues of males, females and gravid females. Differences in BGTG-1 gene expression occurred among male tissues, with tergal gland tissue showing the highest expression. Tissues of nongravid and gravid females had significantly lower expression in comparison with male tergal glands (gravid females lowest). RNA interference (RNAi) was used to silence BGTG-1 gene expression by injecting BGTG-1 homologous double-stranded RNA (dsRNA) into male cockroaches. Groups injected with BGTG-1 dsRNA showed ∼90% lower BGTG-1 gene and protein expression compared to controls, which correlated with lower amylase activity in colorimetric assays. However, behavioural assays comparing precopulatory behaviour and mating success between RNAi and control males did not reveal differences. These results connect amylase gene expression and activity in tergal gland tissue but suggest other factors, such as other tergal gland components, may contribute more strongly to mating success.

Diagnostic procedures employed by dental practitioners in Australia with a focus on endodontic diagnostic procedures.

BACKGROUND: An accurate diagnosis is the foundation for determining prognosis and appropriate management. This study adds to pre-existing (albeit limited) evidence by exploring the use of diagnostic techniques amongst dental practitioners. The main aim of the study was to identify the availability, usage and clinician preference for specific diagnostic tests. A secondary aim was to investigate the use of diagnostic tests for common clinical scenarios. METHODS: A cross-sectional survey was distributed online to dental practitioners registered with the Australian Dental Association. Quantitative data on clinician demography, and the availability and preference of diagnostic tests was summarized with Stata 13 software. Pearson's chi-squared test was used to determine associations. RESULTS: General dental practitioners (GDP) and specialists comprised 86% and 14% of the 433 respondents, respectively. Unlike light transillumination, most GDP had radiography, biting tests and pulp sensibility tests available. The electric pulp test and ethyl chloride were first choices of most practitioners despite markedly lower availability relative to cold spray. Symptoms and endodontic assessments generally attracted wider usage of pulp testing. CONCLUSIONS: More dental practitioners should utilize diagnostic testing in order to arrive at accurate diagnoses. The availability of diagnostic tests did not completely translate to usage and none of the scenarios presented warranted pulp sensibility testing from all respondents.

A neurotensin agonist and antagonist decrease and increase activity, respectively, but do not preclude discrete cue conditioning.

There is evidence to suggest that neurotensin (NT) may enhance cognitive function. For example, in aversive trace conditioning, the NT agonist PD149163 selectively increased trace conditioning (Grimond-Billa, et al., 2008). The present study, therefore, examined the role of NT in associative learning, tested using an appetitive trace conditioning procedure (0-s or 10-s inter-stimulus-interval [ISI]) with a mixed frequency noise as a conditioned stimulus (CS) and food delivery as the unconditioned stimulus (UCS). The effects of an NT agonist (PD149163, 0.125 and 0.25 mg/kg, Experiment 1) and an NT antagonist (SR142948A, 0.01 and 0.1 mg/kg, Experiment 2) were compared. To take nonspecific effects of these compounds into account, conditioning to the CS was measured as a percentage of total responding, during UCS deliveries and in the inter-trial-interval (ITI). In both experiments, associative learning to the contiguously (0-s) presented CS was demonstrated, although there was a relative reduction in this learning under 0.125 mg/kg PD149163. Counter to prediction, the only effect on trace conditioning was some overall reduction in responding to the CS in the 10-s group conditioned under 0.25 mg/kg PD149163. The NT antagonist was without any effect on appetitive conditioning. However, these NT compounds were not ineffective: decreases and increases in responding in the ITI, ISI and during UCS deliveries seen under PD149163 and SR142948A were dissociable from effects on discrete cue conditioning.

Search for Lorentz and CPT violation effects in Muon spin precession.

The spin precession frequency of muons stored in the (g-2) storage ring has been analyzed for evidence of Lorentz and CPT violation. Two Lorentz and CPT violation signatures were searched for a nonzero delta omega a(=omega a mu+ - omega a mu-) and a sidereal variation of omega a mu+/-). No significant effect is found, and the following limits on the standard-model extension parameters are obtained: bZ = -(1.0+/-1.1) x 10(-23) GeV; (m mu dZ0 + HXY)=(1.8+/-6.0) x 10(-23) GeV; and the 95% confidence level limits b perpendicular mu+ <1.4 x 10(-24) GeV and b perpendicular mu- <2.6 x 10(-24) GeV.

Effects of chronic infusion of neurotensin and a neurotensin NT1 selective analogue PD149163 on amphetamine-induced hyperlocomotion.

Neurotensin (NT) has been proposed as an endogenous antipsychotic based in part on the similarity in behavioural effects to antipsychotic drugs, for example, attenuation of both amphetamine-induced hyperlocomotion (AH) and amphetamine disrupted pre-pulse inhibition in the rat. However, there is some evidence that repeated administration of NT or an analogue produces behavioural tolerance to such effects. The present experiments sought to confirm and extend these findings by testing the effects on AH of 7 days central administration of NT and the NT1 selective analogue PD 149163 and the effects of 21 days central administration of NT. NT and PD149163 continuously administered for 7 days produced no effect on AH (in contrast to attenuation with a single injection here and previously reported), whereas 21 days of NT administration potentiated AH. Together, these studies report that the effects of NT or a NT analogue on AH depends on the duration of administration of peptide. The results are discussed in comparison with the reported antipsychotic properties of acute administration of NT and possible mechanisms involving NT1 receptors.

Thyrotrophin-releasing hormone decreases feeding and increases body temperature, activity and oxygen consumption in Siberian hamsters.

Thyrotrophin-releasing hormone (TRH) is known to play an important role in the control of food intake and energy metabolism in addition to its actions on the pituitary-thyroid axis. We have previously shown that central administration of TRH decreases food intake in Siberian hamsters. This species is being increasingly used as a physiological rodent model in which to understand hypothalamic control of long-term changes in energy balance because it accumulates fat reserves in long summer photoperiods, and decreases food intake and body weight when exposed to short winter photoperiods. The objectives of our study in Siberian hamsters were: (i) to investigate whether peripheral administration of TRH would mimic the effects of central administration of TRH on food intake and whether these effects would differ dependent upon the ambient photoperiod; (ii) to determine whether TRH would have an effect on energy expenditure; and (iii) to investigate the potential sites of action of TRH. Both peripheral (5-50 mg/kg body weight; i.p.) and central (0.5 microg/ml; i.c.v.) administration of TRH decreased food intake, and increased locomotor activity, body temperature and oxygen consumption in the Siberian hamster, with a rapid onset and short duration of action. Systemic treatment with TRH was equally effective in suppressing feeding regardless of ambient photoperiod. The acute effects of TRH are likely to be centrally mediated and independent of its role in the control of the production of thyroid hormones. We conclude that TRH functions to promote a catabolic energetic state by co-ordinating acute central and chronic peripheral (thyroid-mediated) function.

Characterization of BGTG-1, a tergal gland-secreted alpha-amylase, from the German cockroach, Blattella germanica (L.).

The protein fraction of the German cockroach, Blattella germanica (L.), tergal gland secretion was examined. SDS-PAGE separation of proteins present in B. germanica tergal gland secretion revealed a tergal gland-secreted protein, BGTG-1, at approximately 63 kDa. BGTG-1 first appeared in tergal gland secretion at 2 days postimaginal moult and the amount of protein observed increased through day 5. A 2051 bp cDNA sequence, bgtg-1, was obtained by RACE polymerase chain reaction and contains a 1494 bp ORF encoding a predicted protein of 498 amino acids. In a Northern hybridization experiment using total RNA from B. germanica tergal gland tissue, a (32)P-labelled bgtg-1 probe hybridized to an RNA approximately 2000 bp and confirmed the 2051 bp cDNA size obtained by RACE PCR. Using the BLASTx sequence similarity search tool, the top match to the bgtg-1 ORF was found to be an alpha-amylase from Drosophila kikkawai (e-value = 1 x 10(-178)). Alignment of the bgtg-1 deduced protein sequence with alpha-amylases from fruit fly, Drosophila melanogaster, honey bee, Apis mellifera (L.) and yellow mealworm, Tenebrio molitor (L.), revealed conserved residues throughout the ORF and sequence identities ranging from 58.4 to 58.2%. Using a gel-based assay, degradation of starch by native BGTG-1 was demonstrated in vitro and we propose that BGTG-1 may be involved in processing phagostimulatory sugars present in B. germanica tergal gland secretion.

Genome-wide analysis of phenobarbital-inducible genes in Drosophila melanogaster.

An oligoarray analysis was conducted to determine the differential expression of genes due to phenobarbital exposure in Drosophila melanogaster (w(1118) strain) third instar larvae. Seventeen genes were observed to be induced with increased expression by a statistical analysis of microarrays approach with a q < or = 0.05. At q < or = 0.12, four more genes (Cyp12d1, DmGstd4, and two genes with unknown function) were found to be up-regulated, and 11 genes with unknown function were found to be down-regulated. Fifteen of these genes, Cyp4d14, Cyp6a2, Cyp6a8, Cyp12d1, Cyp6d5, Cyp6w1, CG2065, DmGstd6, DmGstd7, Amy-p/Amy-d, Ugt86Dd, GC5724, Jheh1, Jheh2 and CG11893, were verified using quantitative real time polymerase chain reaction. Some of these genes have been shown to be over-transcribed in metabolically DDT-resistant Drosophila strains.

Effects of the juvenile hormone analog pyriproxyfen on German cockroach, Blattella germanica (L.), tergal gland development and production of tergal gland secretion proteins.

Male German cockroaches possess secretory glands that secrete fluid into a pair of transverse depressions on the seventh and eighth abdominal tergites. We investigated the effects of altered juvenoid titer during the first part of the last instar on tergal gland secretory tissue development and the production of tergal gland secretion proteins. Male fifth (last) instar nymphs (1-3 days post-emergent) were topically treated with the JH analog pyriproxyfen. Light and transmission electron microscopy demonstrated that treatment with pyriproxyfen produced a visible decrease in the amount of tergal gland tissue present, a deformation of the overall shape of the gland located on tergite seven, and a less orderly arrangement of the secretory cells in the tissue. The protein fraction of tergal gland secretion was examined in pyriproxyfen-treated and control insects 1, 5, and 15 days after the insects molted to the adult stage. Amounts of all tergal secretion proteins were reduced in treated insects.

German cockroach proteases regulate matrix metalloproteinase-9 in human bronchial epithelial cells.

BACKGROUND: Matrix metalloproteinases (MMPs) digest extracellular matrix proteins and may play a role in the pathogenesis of bronchial asthma. MMP-9 levels are increased in the bronchoalveolar lavage fluid and sputum of asthmatics compared with that of controls. As exposure to cockroaches is an environmental risk factor for asthma, we sought to investigate the role of German cockroach fecal remnants (frass) on MMP-9 expression. METHODS: Human bronchial epithelial cells (16HBE14o-) and primary normal human bronchial epithelial cells were treated with cockroach frass in the absence or presence of tumor necrosis factor (TNF)alpha. MMP-9 mRNA, protein levels and pro-MMP-9 activity were determined using real-time polymerase chain reaction (PCR), enzyme-linked immunosorbent assay (ELISA) and zymogram assays. Pretreatment of frass with aprotinin abolished protease activity. PD98059, a chemical inhibitor of extracellular signal regulated kinase (ERK), and SLIGKV, an activator of protease-activated receptor (PAR)-2 were also used. AP-1DNA binding was determined by electrophoretic mobility shift assay (EMSA) and ERK phosphorylation by Western blot analysis. RESULTS: Cockroach frass augmented TNFalpha-mediated MMP-9 mRNA and protein expression by a mechanism dependent on active serine proteases within frass and not on endogenous endotoxin. Frass increased ERK phosphorylation, and chemical inhibition of ERK attenuated cockroaches' effects on MMP-9. Serine proteases are known to activate the PAR-2 receptor. We found that selective activation of PAR-2 using the peptide SLIGKV augmented TNFalpha-induced MMP-9 protein levels and increased ERK phosphorylation. Frass and SLIGKV each increased AP-1 translocation and DNA binding. CONCLUSIONS: These data suggest that German cockroach frass contains active serine proteases which augment TNFalpha-induced MMP-9 expression by a mechanism involving PAR-2, ERK and AP-1.

Two hexamerin genes from the termite Reticulitermes flavipes: Sequence, expression, and proposed functions in caste regulation.

Previous molecular studies on the termite Reticulitermes flavipes have revealed that two hexamerin proteins serve an important status quo role in the regulation of juvenile hormone (JH)-dependent caste differentiation. Here, we report sequence data and other experimental evidence suggesting how these two hexamerins function in achieving caste regulation. The two hexamerin genes, named Hex-1 and Hex-2, encode highly unique sequence features relative to the 100+ other known insect hexamerins. These features include a long hydrophobic tail and prenylation motif in Hex-1, and a long hydrophilic insertion plus several putative protease cleavage sites in Hex-2. Both hexamerin genes are primarily expressed in fat body tissue, but only Hex-2 expression is substantially induced by JH. SDS-PAGE showed that the hexamerin proteins constitute a major proportion of total soluble termite protein. Also, although each protein occurs in both the membrane and soluble protein fractions, Hex-2 has stronger membrane affinity. Anti-JH antiserum specifically recognizes hemolymph-soluble Hex-1 protein, supporting that the unique prenylation site in Hex-1 facilitates covalent JH binding to the primary amino acid chain. Finally, increased ratios of Hex-2 to Hex-1 transcription occur in caste phenotypes and developmental stages that differentiate in response to rising JH titers. Two main conclusions can be taken from these studies. First, elevated ratios of Hex-2 to Hex-1 expression are associated with caste phenotypes that differentiate in response to rising JH titers (i.e., workers, presoldiers and soldiers). Second, due to their unique structural features and other observed characteristics, our findings support the hypothesis that the two hexamerins participate in the regulation of caste-differentiation by modulating JH availability.

Gene expression profiles among immature and adult reproductive castes of the termite Reticulitermes flavipes.

Array-based genomic studies were conducted with the goal of identifying immature (i.e. nymph) and adult reproductive caste-biased gene expression in the termite Reticulitermes flavipes. Using cDNA macro-arrays, we identified thirty-four nymph-biased genes falling into eight ontogenic categories. Based on gene expression profiles among diverse castes and developmental stages (determined by quantitative PCR), several important trends emerged. These findings highlight the importance of several developmental and survival-based factors among immature and adult termite reproductives, including: vitellogenesis, nutrient storage, juvenile hormone sequestration, ribosomal translational and filtering mechanisms, fatty acid biosynthesis, apoptosis inhibition, and both endogenous and symbiont cellulase-assisted nutrition. These findings are highly significant as they are the first to elucidate the molecular biology underlying termite reproductive caste differentiation and reproductive caste-specific biology. Other gene expression results are in agreement with previous findings that suggest roles for vitellogenin-like haemolymph proteins in soldier caste differentiation.

Measurement of the negative muon anomalous magnetic moment to 0.7 ppm.

The anomalous magnetic moment of the negative muon has been measured to a precision of 0.7 ppm (ppm) at the Brookhaven Alternating Gradient Synchrotron. This result is based on data collected in 2001, and is over an order of magnitude more precise than the previous measurement for the negative muon. The result a(mu(-))=11 659 214(8)(3) x 10(-10) (0.7 ppm), where the first uncertainty is statistical and the second is systematic, is consistent with previous measurements of the anomaly for the positive and the negative muon. The average of the measurements of the muon anomaly is a(mu)(exp)=11 659 208(6) x 10(-10) (0.5 ppm).

Measurement of the positive muon anomalous magnetic moment to 0.7 ppm.

A higher precision measurement of the anomalous g value, a(mu)=(g-2)/2, for the positive muon has been made at the Brookhaven Alternating Gradient Synchrotron, based on data collected in the year 2000. The result a(mu(+))=11 659 204(7)(5)x10(-10) (0.7 ppm) is in good agreement with previous measurements and has an error about one-half that of the combined previous data. The present world average experimental value is a(mu)(expt)=11 659 203(8)x10(-10) (0.7 ppm).

Determination of barakol extracted from Cassia siamea by HPLC with electrochemical detection.

Barakol is an active compound extracted from the leaves and flowers of the plant called Cassia siamea grown widely in Southeast Asia. There have been a number of reports on the chemical structure and properties of this compound since it was first extracted in 1969. The compound has been tested in various laboratories for its pharmacological properties with reference to the clinical use of the plant in traditional Thai medicines. For these studies it is necessary to establish the stability of the extracted barakol under various laboratory conditions. This report is the first to use high performance liquid chromatography with electrochemical detection to determine the extent of purity and stability of extracted barakol solution.

Stereological analysis of regional brain volumes and neuron numbers in rats displaying a spontaneous hydrocephalic condition.

Stereological methods were employed to investigate a novel spontaneously occurring brain mutation in an inbred colony of Wistar rats. These mutants displayed changes (enlarged cerebral ventricles and malformed hippocampi) similar to those seen in H-Tx hydrocephalic rats. Mutant and control rats were studied at three postnatal ages: 4, 7, and 13 weeks. Brain weight in the mutant animals was significantly (P < 0.05) increased when compared to age-matched controls. Using systematic random sampling and the Cavalieri principle we estimated the volumes of various brain compartments, including the cerebral ventricles, forebrain, and cerebral cortex. We found that ventricular volume (P < 0.001) and forebrain volume (P < 0.05) were significantly increased in mutant rats when compared to control rats. Total numbers of nucleoli, estimated using the physical fractionator, were obtained for neurons in the cerebral cortex and granule cells in the dentate gyrus. Numbers were not altered significantly in mutant rats. Nor were mean soma volumes as estimated from total volumes and numbers. The changes in brain and ventricle volumes provide quantitative evidence that these animals display a hydrocephalic condition. This condition appears not to compromise cell number or mean soma size in the brain regions examined.

Evaluation of control measures for black carpenter ant (Hymenoptera: Formicidae).

Current control methods for the black carpenter ant, Camponotus pennsylvanicus (De Geer), include the use of remedial and preventative residual sprays as well as toxic baits. We evaluated the acceptance of three baits (Maxforce, Niban, and Baygon) to field colonies of the black carpenter ant in the spring and fall. Maxforce bait granules were more readily accepted than either Niban or Baygon bait granules in the spring. A change in food preference from protein to sugar by the black carpenter ant appeared to reduce the number of Maxforce bait granules removed in the fall, resulting in no differences in bait acceptability. The longevity of Dursban 50W and Tempo 20WP were evaluated in the summer and fall on painted wood panels. Panels aged outside for 15 d under prevailing weather conditions exhibited increased LT50 values. For each sampling period, panels aged on the south face (in the sun) exhibited less insecticidal activity (i.e., large LT50 values) than panels on the north face (shaded; small LT50 values). At each sampling period, Tempo 20WP provided smaller LT50 values than Dursban 50W. Because of changing dietary preferences, our data highlight the importance of using various bait types for carpenter ant control. Moreover, the application of residual spays should be made to locations protected from direct sunlight.

Chronic, but not acute, dosing of antipsychotic drugs alters neurotensin binding in rat brain regions.

The present study compared high affinity neurotensin (NT) binding in rat brain following acute or chronic treatment with the classical antipsychotic, haloperidol, and the newer antipsychotic drugs, clozapine and zotepine. Drugs were given orally, as an acute treatment (1 dose) or chronically (21 day dosing) and binding to the NT high affinity receptor was examined in three brain regions; striatum, nucleus accumbens/olfactory tubercle and frontal cortex. Acute dosing with either vehicle, haloperidol, clozapine or zotepine produced no significant changes in NT binding from controls (naïve rats). Chronic (21 day) dosing resulted in an increase in the K:(D:) and B(max) of high affinity receptors in the striatum following haloperidol, but not clozapine, zotepine or vehicles. In contrast, the newer antipsychotics, clozapine and zotepine but not haloperidol or vehicles, significantly altered NT binding in the nucleus accumbens/olfactory tubercle by decreasing the K:(D:) and B(max). Further differentiation between the two newer antipsychotic drugs occurred in the frontal cortex. Clozapine had no significant effect on NT binding, whereas zotepine significantly reduced the K:(D:) of the high affinity receptor with no alteration in B(max). The antipsychotic drugs tested did not interact directly with the NT high affinity receptor. Therefore, they must be acting indirectly via an alternative receptor mechanism to alter NT high affinity binding. In accordance with previously reported NT/dopamine receptor interactions, this would suggest cross-talk between these systems. Overall, these data demonstrate that chronic, but not acute, administration of antipsychotic drugs alters NT binding in the rat brain. In addition, anatomical differences in NT binding arise according to the antipsychotic drug under test. This may be predictive of drug side-effect profile, antipsychotic efficacy or atypicality.

A highly sensitive and selective radioimmunoassay for the measurement of neurotensin.

A highly selective and sensitive radioimmunoassay (RIA) for the detection of endogenous neurotensin (NT) has been developed. We have raised a C-terminally-directed antibody (CAb) that specifically binds 'biologically active' NT (NT and NT(8-13)) and that does not significantly cross-react with inactive NT metabolites or other bioactive peptides in the CNS. By reducing the volume of the assay to a low volume-RIA (30 microl), such that in vivo measurements can be made, we have increased the sensitivity (<0.3 fmol per tube), with inter- and intra-assay variations of 11.2 and 5.8%, respectively. Comparisons with similar methods of detecting NT have demonstrated that this RIA has a higher sensitivity than previously used RIA's and ELISA's. The data presented suggests that this sensitive RIA is a reliable method ideal for the detection of small quantities of biologically active NT.

Feeding inhibition and mortality in Reticulitermes flavipes (Isoptera: Rhinotermitidae) after exposure to imidacloprid-treated soils.

Feeding inhibition and mortality of Reticulitermes flavipes (Kollar) exposed to sand, sandy loam, loam, and silty clay loam soils treated with several concentrations of imidacloprid were studied using bioassay techniques under laboratory conditions. Termite workers stopped feeding after exposure to treated soils. Differences in feeding reduction varied among the soil types. Based on the magnitude of the F-statistics, the effect of imidacloprid on the reduction of termite feeding was greatest in sand followed by sandy loam, loam, and silty clay loam soils. Soil properties such as organic matter content, silt and clay proportions, pH, and cation exchange capacity were suggested to affect the bioavailability of imidacloprid. Similar soil effects on mortality were observed in termites continuously exposed to treated soil for 21 d. In three of four soils tested, susceptibility to imidacloprid was not affected by the source of the termites tested.