Characterization of trehalose metabolic genes and corresponding enzymatic activities during diapause of Sitodiplosis mosellana.

Trehalose plays crucial roles in energy metabolism and stress tolerance in various organisms. The orange wheat blossom midge Sitodiplosis mosellana, a serious pest of wheat worldwide, undergoes long obligatory diapause as a larva to survive harsh temperature extremes in summer and winter. To gain an insight into trehalose function and metabolic mechanism in this process, we measured the content of trehalose and glucose, as well as enzymatic activities of trehalose-6-phosphate synthase (TPS), trehalose-6-phosphate phosphatase (TPP) and soluble trehalase (Treh1) at pre-diapause, diapause and post-diapause larvae of S. mosellana. Trehalose levels greatly increased upon entry into diapause, peaked in low-temperature quiescence phase, and significantly dropped after resumption of development, highly consistent with activity changes of trehalose-synthetic enzymes SmTPS and SmTPP. In marked contrast, the activity of trehalose-degrading SmTreh1 exhibited a completely reversed profile. This profile was in agreement with contents of its product i.e. glucose. Furthermore, deduced amino acid sequences of cloned SmTPS, SmTPPB, SmTPPC, SmTreh1-1 and SmTreh1-2 genes contained all conserved functional domains, motifs and active sites. Expression patterns of these genes were closely correlated with their enzyme activities. These results suggested that coordination of trehalose synthetic and degradation pathways is responsible for diapause-related trehalose accumulation, which may serve as an energy reserve for post-diapause development and a cryoprotectant against cold stress in winter.

What types of enzyme activities are useful biomarkers of bifenthrin exposure on Chironomus sp. (Diptera, Chironomidae) larvae under laboratory and field-based microcosm conditions?

Bifenthrin is a second generation synthetic pyrethroid insecticide that is widely used in Australia and worldwide. It is frequently found in urban freshwater sediments at concentrations likely to impact biota as it is highly toxic to fish and macroinvertebrates, such as chironomids. Our main goal was to evaluate if oxidative stress and hydrolase enzymes are useful biomarkers of effect of synthetic pyrethroids exposure under different scenarios. Chironomus tepperi larvae (5 days old) were exposed to sub-lethal sediment concentrations of bifenthrin for 5 days under controlled laboratory conditions. A field-based microcosm exposure with bifenthrin-spiked sediments (using the same concentrations as the laboratory exposure) was carried out at a clean field site for four weeks to allow for colonization and development of resident chironomid larvae. At the end of both experiments, Chironomus larvae (C. tepperi in the laboratory exposures and C. oppositus in the microcosm exposures) were collected and oxidative stress enzymes (Glutathione-s-Transferase, Glutathione Reductase and Glutathione Peroxidase) and hydrolase enzymes (Acetylcholinesterase and Carboxylesterase) were measured. Only the Glutathione Peroxidase activity was significantly impacted in larvae from the laboratory exposure. On the contrary, significant changes were observed in all the measured enzymes from the field-based microcosm exposure. This is likely because exposure was throughout the whole life cycle, from egg mass to fourth instar, showing a more realistic exposure scenario. Furthermore, this is the first time that changes in oxidative stress and hydrolase enzymes have been shown to occur in Australian non-biting midges exposed under field-based microcosm conditions. Thus, this study demonstrated the usefulness of these enzymes as biomarkers of effect following bifenthrin exposure in microcosms. It also highlights the importance of using a range of different biochemical endpoints to get a more holistic understanding of pesticide effects and the pathways involved.

Knockdown of α-enolase (Eno1) genes by RNAi does not increase the sensitivity of Propsilocerus akamusi to cadmium stress.

α-enolase (Eno1) is a multifunctional enzyme which can as a stress protein under various environmental stresses. Recent researches also reported that Eno1 appears to have Cd2+ stress-related functions in cadmium tolerant plants. Our previous study inferred that the Eno1 gene might play an important role in the response of Propsilocerus akamusi to exogenous Cd2+. However, reports on the role of the Eno1 gene in coping with cadmium stress are still limited. In this study, we evaluated the roles of PaEno1 in the tolerance of P. akamusi to Cd2+ using RNAi technology and the response of recombinant proteins of PaEno1 in an E. coli expression system under Cd2+ stress. Our results showed that knockdown of PaEno1 did not increase but reduce the sensitivity of P. akamusi larvae to Cd2+ stress. However, bioassays showed the expression of recombinant PaEno1 protein in Rosetta cells enhanced the growth ability of E. coli under Cd2+ stress. These results suggested that overexpression of PaEno1 can significantly enhance the tolerance to heavy metal cadmium stresses in E. coli cells. However, knockdown of PaEno1 genes by RNAi does not increase the sensitivity of P. akamusi to cadmium stress.

Cadmium-induced developmental alteration and upregulation of serine-type endopeptidase transcripts in wild freshwater populations of Chironomus plumosus.

Cadmium, a toxic heavy metal, is a persistent environmental contaminant with irreversible toxicity to aquatic organisms. Chironomus plumosus, a natural species, is the largest sediment-burrowing aquatic midge in freshwater environments. In this study, we evaluated developmental defects in C. plumosus resulting from Cd exposure. In C. plumosus larvae, Cd exposure induced decreased survival and growth rates, reduction of emergence rate and sex ratio, and delayed emergence, as well as elevating the incidence of split tooth deformities. To identify potential biomarker genes to assess environmental pollutants such as Cd, we identified differentially expressed genes (DEGs) in C. plumosus exposed to various Cd concentrations. Among fourteen characterized DEGs, serine-type endopeptidase (SP) and heat shock protein 70 (HSP70) genes exhibited significant upregulation in C. plumosus larvae after Cd exposure. Therefore, we evaluated SP and HSP70 responses in natural C. plumosus populations collected from three sites of a Korean river and analyzed their correlations with eighteen environmental quality characteristics using principal component analysis. The highest expression of SP and HSP70 transcripts was observed in C. plumosus populations from Yeosu in Korea, which has high concentrations of polluting heavy metals. SP transcript expression was positively correlated with concentrations of Cd, Pb, Al, Fe, NO2, and NO3. These results suggested that environmental pollutants such as Cd can impair proteolytic activity in the digestive system of C. plumosus and may ultimately induce developmental alterations. We therefore suggest SP as a potential biomarker to assess the effects of environmental pollutants in aquatic ecosystems.

A comparative study of acetylcholinesterase and general-esterase activity assays using different substrates, in vitro and in vivo exposures and model organisms.

Acetylcholinesterase (AChE) and general-esterase (GE) activities are important to understand detoxification processes of xenobiotics. The assays to quantify them have employed different substrates, inhibitors, types of experiments (in vitro and in vivo) and model organisms. The aim of this work was to give a systematic overview of the effect of the above factors on the outcome of AChE and GE activity measurements. We showed that AChE activity could be measured with the substrate acetylthiocholine iodide (AChI) but not with acetylcholine bromide (AChB) and only in in vitro assays. For GE activity, Michaelis-Menten kinetics differed between the substrates 4-methylumbellifery butyrate (4-MUB) and 1-naphtyl acetate (1-NA) in the measurements of in vitro activity, but their inhibition curves and IC50 values for the general inhibitor tetraisopropyl pyrophosphoramide (iso-OMPA) were similar, confirming that both substrates targeted the same group of enzymes. The GE substrate 4-MUB was applicable both in vitro and in vivo, while 1-NA was only applicable in vitro due to its high acute toxicity. When comparing the zooplankton crustacean Daphnia magna and the sediment dwelling Chironomus riparius, the latter had a four-fold higher maximal AChE activity (Vmax) and a higher susceptibility to the AChE inhibitor BW284c51 (four-fold lower 50% inhibitory concentration, IC50), but a lower maximal GE activity and lower susceptibility to iso-OMPA (higher IC50), indicating significant species differences between in C. riparius and D. magna. We conclude that both choice of substrate and exposure method matters for the outcome of esterase assays and that esterase compositions between species may vary significantly.

Effects of phenol on glutathione S-transferase expression and enzyme activity in Chironomus kiiensis larvae.

Detoxifying enzyme mRNAs are potentially useful stress biomarkers. Glutathione S-transferase (GST) metabolises lipophilic organic contaminants and mitigates oxidative damage caused by environmental pollutants. Herein, 12 Chironomus kiiensis GSTs (CkGSTs1-6, CkGSTt1-2, CkGSTd1-2, CkGSTm1-2) were cloned and grouped into sigma, theta, delta and microsomal subclasses. Open reading frames (450-699 bp) encode 170-232 amino acid proteins with predicted molecular masses of 17.31-26.84 kDa and isoelectric points from 4.94 to 9.58. All 12 GSTs were expressed during all tested developmental stages, and 11 displayed higher expression in fourth-instar larvae than eggs. GST activity after 24 h of phenol exposure was used to estimate environmental phenol contamination. After exposure to sublethal concentrations of phenol for 48 h, expression and activity of CkGSTs were inhibited in C. kiiensis larvae. Expression of CkGSTd1-2 and CkGSTs1-2 varied with phenol concentration, indicating potential use as biomarkers for monitoring environmental phenol contamination.

The synergistic potential of azole fungicides does not directly correlate to the inhibition of cytochrome P450 activity in aquatic invertebrates.

The ability of azole fungicides to inhibit cytochrome P450 dependent metabolism is proposed to be the main mechanism for their synergizing effect on pyrethroid insecticide toxicity in aquatic invertebrates. This study investigates the correlation between inhibition strength and synergistic potential of azole fungicides in the crustacean Daphnia magna and the insect larvae Chironomus riparius. Inhibition strength was measured in vivo toward the cytochrome P450 catalysed conversion of 7-ethoxycoumarin to 7-hydroxycoumarin (ECOD). Synergistic potentials were determined as the ratio between predicted and observed toxicity of mixtures based on the model of concentration addition (CA) and independent action (IA). Azoles (n = 9-11) enhanced the toxicity of α-cypermethrin in D. magna (Synergy ratios CA: 0.8 - 16; IA: 1.1 - 22) and inhibited cytochrome P450 activity by different degrees (IC50: 0.0023 - 36 µM for D. magna and 0.08 - 24 µM for C. riparius). Inhibition strengths were strongly correlated in the two organisms (r: 0.937 p: 0.019 for triazoles and r: 0.903 p: 0.097 for imidazoles). Lipophilicity governed the inhibition strength of triazoles in both species (r > 0.9, p < 0.05). No correlation was observed between inhibition strengths and synergistic potentials. Several reasons for the apparent lack of correlation were discussed.

Bioaccumulation and Biomagnification of 2-Ethylhexyl-4-dimethylaminobenzoate in Aquatic Animals.

2-Ethylhexyl-4-dimethylaminobenzoate (EHDAB) is a commonly used organic ultraviolet filter. The bioaccumulation and biomagnification of EHDAB were investigated in two aquatic animals, the larvae of midge (Chironomus riparius) and crucian carp (Carassius carassius), and the metabolic enzyme responses in fish liver were determined. EHDAB in the larvae of midge reached a steady state within 10 days of sediment exposure. The biota-sediment accumulation factors ranged from 0.10 to 0.54, and were inversely proportional to the exposure concentrations. The EHDAB-contaminated larvae were used to feed the crucian carp. Within 28 days of feeding exposure, the EHDAB levels in fish tissues gradually increased with the increase of the exposure concentration, exhibiting an apparent concentration-dependence and time-dependence. The liver and kidneys were the main organs of accumulation, and the biomagnification factors of EHDAB ranged from 8.97 to 11.0 and 6.44 to 10.8, respectively. In addition, EHDAB significantly increased the activities of cytochrome P450 (CYP) 1A, CYP3A and glutathione S-transferase in the fish liver. Our results indicate that EHDAB may pose a risk of biomagnification in an aquatic environment and influence the biological processes of exposed organisms.

iTRAQ-based quantitative proteomic analysis identified Eno1 as a cadmium stress response gene in Propsilocerus akamusi (Tokunaga) hemolymph.

Propsilocerus akamusi (Tokunaga) is a common species of midge in Siberia, Japan, and China and an important prey species for fish and aquatic birds. Furthermore, this species has been shown to have an extraordinary capacity to resist cadmium (Cd) toxicity. In this study, isobaric tags for relative and absolute quantitation (iTRAQ) coupled liquid chromatography tandem mass spectrometry (LC-MS/MS) was used to analyze relative changes in the P. akamusi hemolymph proteome following exposure to a sublethal concentration of Cd2+. The results showed that Cd2+ stress affects energy metabolism in P. akamusi. After examining the differentially expressed proteins (DEPs), only one up-regulated protein associated with metabolism, α-enolase (Eno1) was identified and further isolated and characterized. Sequence alignments showed that the deduced P. akamusi Eno1 amino acid sequence is highly conserved, with similarities of 77-95% noted when compared to other Dipteran Eno1 sequences. Furthermore, prolonged Cd2+ exposure impacted Eno1 transcription, protein expression and enzyme activity levels. These results suggest that Eno1 may play a role in the response to Cd2+ stress in P. akamusi.

Cytochrome P450 CYP6EV11 in Chironomus kiiensis Larvae Involved in Phenol Stress.

Phenol is one of the organic pollutants which can cause water environment pollution. It is not only enriched in aquatic organisms but is also a serious threat to human health. Chironomus kiiensis is very sensitive to the contaminants in water and its cytochrome P450s are usually chosen as biomarkers for water pollution. To examine whether CYP6EV11 plays a role in the oxidative metabolism of phenol, we measured the silencing efficiency of CYP6EV11 and evaluated larval susceptibility to sublethal phenol levels by RNA interference (RNAi) technology. The results showed that the transcription of CYP6EV11 was found significantly up-regulated when the 4th instar C.kiiensis larvae were exposed to three doses of phenol. However, the transcriptional levels of CYP6EV11 were significantly suppressed by 92.7% in the 4th instar C. kiiensis larvae soaked in dsCYP6EV11 compared with those soaked in dsGFP for 6 h. The CYP6EV11 expression and mortality of the 4th instar C. kiiensis larvae with CYP6EV11 silencing were mostly decreased under phenol stress. Therefore, the CYP6EV11 gene may be used as a molecular biomarker for earlier warning and monitoring for water pollution.

Cytochrome P450 genes from the aquatic midge Chironomus tentans: Atrazine-induced up-regulation of CtCYP6EX3 enhanced the toxicity of chlorpyrifos.

The open reading frames of 19 cytochrome P450 monooxygenase (CYP) genes were sequenced from Chironomus tentans, a commonly used freshwater invertebrate model. Phylogenetic analysis of the 19 CYPs along with a previously reported CYP (CtCYP4G33) revealed that they belong to three different clans, including 3 in CYP4, 15 in CYP3, and 2 in mitochondria clan. When third-instar larvae were exposed to atrazine at 5000 µg/L, the transcription of CtCYP6EX3, CtCYP6EV3, CtCYP9AT1 and CtCYPEX1 was significantly up-regulated. To examine whether CtCYP6EX3 played a role in oxidative activation of chlorpyrifos to chlorpyrifos-oxon, we evaluated larval susceptibility to chlorpyrifos after CtCYP6EX3 transcript was suppressed by RNAi. The larvae fed chitosan/dsCtCYP6EX3 nanoparticles showed a significantly decreased CtCYP6EX3 transcript (53.1%) as compared with the control larvae fed chitosan/dsGFP nanoparticles. When the CtCYP6EX3-silenced larvae were exposed to chlorpyrifos at 6 µg/L or its binary mixture with atrazine (chlorpyrifos at 3 µg/L and atrazine at 1000 µg/L), the larvae became less susceptible to the pesticides as their mortalities decreased by 24.1% and 20.5%, respectively. These results along with our previous findings suggested that the increased toxicity of chlorpyrifos was likely due to an enhanced oxidative process from chlorpyrifos to chlorpyrifos-oxon by CtCYP6EX3 as RNAi of CtCYP6EX3 led to decreased susceptibility of C. tentans larvae to chlorpyrifos alone and the binary mixture of atrazine and chlorpyrifos. However, further study would be necessary to validate our results by functional assays using heterologously expressed CtCYP6EX3 enzyme.

Toxicity of organic UV-filters to the aquatic midge Chironomus riparius.

Despite the frequent detection of organic ultraviolet-filters (UV-filters) in freshwater sediments, there is a lack of ecotoxicological data undermining a correct risk assessment for these emerging contaminants. The present study assessed the effects of three of the most commonly used UV-filters (benzophenone-3 - BP3; 3-(4-methylbenzylidene)camphor - 4-MBC and octocrylene - OC) on Chironomus riparius life history and biochemical responses. Standard ecotoxicological assays confirmed that all compounds impaired growth of C. riparius larvae and induced developmental effects such as delayed emergence and a reduction of imagoes weight. Concerning the biochemical responses analysed no evidences of oxidative damage in lipids or neurotoxicity (tested assessing acetylcholinesterase activity) were observed for any of the tested compounds. However, 4-MBC exposure induced a decrease in catalase activity and an increase in glutathione-S-transferase activity at 14.13mg/Kg while OC exposure caused an increase in total glutathione levels at 0.23 and 18.23mg/Kg. Exposure to all UV-filters tested, increased energy consumption on C. riparius with significant differences above 1.00mg/Kg for BP3, 0.09mg/Kg for 4-MBC and 2.13mg/Kg for OC. These results suggest that environmental relevant concentrations of UV-filters can cause deleterious effects to aquatic benthic species, such as C. riparius, and call for further research concerning effects of organic UV-filters on natural invertebrate communities and ecosystem functioning.

Germline-specific labeling of the somatic chromosomes by protein phosphatase 2A and histone H3S28 phosphorylation in Acricotopus lucidus.

Additional chromosomes limited to the germline (=Ks) were established as a special form of germline-soma differentiation in the Orthocladiinae, a subfamily of the Chironomidae (Bauer and Beermann in Z Naturforsch 7b: 557-563, 1952). The Ks together with the somatic chromosomes (=Ss) pass through a complex chromosome cycle with elimination at mitosis and a monopolar migration of all Ks. The dissimilar behavior of Ks and Ss in these exceptional mitoses initiated the search for differential chromosome marks in the orthocladiid Acricotopus lucidus. The search, using immunofluorescence, revealed that in metaphases of male gonial mitoses, and both meiotic divisions, the Ss are fully labeled by protein phosphatase 2A (PP2A) and histone H3S28ph, while in metaphases of somatic cells both marks were detected only at the centromeres of the Ss. In another orthocladiid, Psectrocladius obvius, the same labeling pattern of the Ss as in A. lucidus was established for H3S28ph, but not for PP2A, which was localised solely at the centromeres. In Chironomus nuditaris, a species possessing no Ks, PP2A and H3S28ph signals were always restricted to the centromeres. High levels of H3K4me3, a marker of transcriptionally competent chromatin, were detected on the Ss in metaphases I of C. nuditaris, while in both orthocladiids, the Ss in metaphases I were devoid of H3K4me3 signals. This strongly supports an earlier idea of a silencing of the Ss in male meiosis of A. lucidus suggesting the possibility of extending this concept to the Orthocladiinae. The germline-soma differentiation in A. lucidus is not only made apparent by the occurrence of Ks but also by a germline-specific labeling of the Ss by PP2A and H3S28ph.

Polybrominated diphenyl ethers (PBDEs) effects on Chironomus sancticaroli larvae after short-term exposure.

In-vivo effects of polybrominated diphenyl ethers (PBDEs) containing 3, 4 and 5 bromine atoms were tested on fourth-instar larvae of Chironomus sancticaroli (Diptera: Chironomidae) after 48h of exposure, by measuring the activity of the acetyl cholinesterase, alpha and beta esterases and glutathione S-transferase. The PBDE congeners 2,2',4-triBDE (BDE-17), 2,2',4,4'-tetraBDE (BDE-47) and 2,2',4,4',5-pentaBDE (BDE-99) were evaluated at 0.5, 1.0, 2.0 and 3.0ngmL-1. Acetyl cholinesterase activity decreased significantly (p≤0.05) at all evaluated concentrations of the three PBDE congeners, except for larvae exposed to BDE-17 at 1.0 and 2.0ngmL-1. The significant inhibition of acetyl cholinesterase activity ranged from 18% (BDE-47 at 0.5ngmL-1) to 72% (BDE-47 at 2.0ngmL-1). The enzymes alpha and beta esterase were also affected by the three congeners, reducing their activity from 14% (BDE-99 at 1.0ngmL-1) to 52% (BDE-47 at 2.0ngmL-1) and from 7% (BDE-99 at 2.0ngmL-1) to 34% (BDE-47 at 3.0ngmL-1) respectively. Substantial increments in glutathione S-transferase activity were similarly observed, varying from 138% (BDE-99 2.0 at ng mL-1) to 346% (BDE-17 at 1.0ngmL-1). DNA strand breaks were detected exclusively in larvae exposed to BDE-99 at 2.0 and 3.0ngmL-1 (H=11.7, p=0.019). These results showed that C. sancticaroli larvae were sensitive to the PBDEs treatments under the experimental conditions.

Effects of phenol on metabolic activities and transcription profiles of cytochrome P450 enzymes in Chironomus kiinensis larvae.

Phenol, also known as carbolic acid or phenic acid, is a priority pollutant in aquatic ecosystems. The present study has investigated metabolic activities and transcription profiles of cytochrome P450 enzymes in Chironomus kiinensis under phenol stress. Exposure of C. kiinensis larvae to three sublethal doses of phenol (1, 10 and 100 µM) inhibited cytochrome P450 enzyme activity during the 96 h exposure period. The P450 activity measured after the 24 h exposure to phenol stress could be used to assess the level (low or high) of phenol contamination in the environment. To investigate the potential of cytochrome P450 genes as molecular biomarkers to monitor phenol contamination, the cDNA of ten CYP6 genes from the transcriptome of C. kiinensis were identified and sequenced. The open reading frames of the CYP6 genes ranged from 1266 to 1587 bp, encoding deduced polypeptides composed of between 421 and 528 amino acids, with predicted molecular masses from 49.01 to 61.94 kDa and isoelectric points (PI) from 6.01 to 8.89. Among the CYP6 genes, the mRNA expression levels of the CYP6EW3, CYP6EV9, CYP6FV1 and CYP6FV2 genes significantly altered in response to phenol exposure; therefore, these genes could potentially serve as biomarkers in the environment. This study shows that P450 activity combined with one or multiple CYP6 genes could be used to monitor phenol pollution.

Exploring Genetic Divergence in a Species-Rich Insect Genus Using 2790 DNA Barcodes.

DNA barcoding using a fragment of the mitochondrial cytochrome c oxidase subunit 1 gene (COI) has proven to be successful for species-level identification in many animal groups. However, most studies have been focused on relatively small datasets or on large datasets of taxonomically high-ranked groups. We explore the quality of DNA barcodes to delimit species in the diverse chironomid genus Tanytarsus (Diptera: Chironomidae) by using different analytical tools. The genus Tanytarsus is the most species-rich taxon of tribe Tanytarsini (Diptera: Chironomidae) with more than 400 species worldwide, some of which can be notoriously difficult to identify to species-level using morphology. Our dataset, based on sequences generated from own material and publicly available data in BOLD, consist of 2790 DNA barcodes with a fragment length of at least 500 base pairs. A neighbor joining tree of this dataset comprises 131 well separated clusters representing 121 morphological species of Tanytarsus: 77 named, 16 unnamed and 28 unidentified theoretical species. For our geographically widespread dataset, DNA barcodes unambiguously discriminate 94.6% of the Tanytarsus species recognized through prior morphological study. Deep intraspecific divergences exist in some species complexes, and need further taxonomic studies using appropriate nuclear markers as well as morphological and ecological data to be resolved. The DNA barcodes cluster into 120-242 molecular operational taxonomic units (OTUs) depending on whether Objective Clustering, Automatic Barcode Gap Discovery (ABGD), Generalized Mixed Yule Coalescent model (GMYC), Poisson Tree Process (PTP), subjective evaluation of the neighbor joining tree or Barcode Index Numbers (BINs) are used. We suggest that a 4-5% threshold is appropriate to delineate species of Tanytarsus non-biting midges.

New synthesis and biological evaluation of uniflorine A derivatives: towards specific insect trehalase inhibitors.

7-Deoxy-uniflorine A (6), synthesized ex novo with a straightforward and simple strategy, and the analogues 4, 5 and 7, were evaluated as potential inhibitors of insect trehalase from Chironomus riparius and Spodoptera littoralis. All the compounds were tested against porcine trehalase as the mammalian counterpart and α-amylase from human saliva as a relevant glucolytic enzyme. The aim of this work is the identification of the simplest pyrrolizidine structure necessary to impart selective insect trehalase inhibition, in order to identify new specific inhibitors that can be easily synthesized compared to our previous reports with the potential to act as non-toxic insecticides and/or fungicides. All the derivatives 4­7 proved to be active (from low micromolar to high nanomolar range activity) towards insect trehalases, while no activity was observed against α-amylase. In particular, the natural compound uniflorine A and its 7-deoxy analogue were found to selectively inhibit insect trehalases, as they are inactive towards the mammalian enzyme. The effect of compound 6 was also analyzed in preliminary in vivo experiments. These new findings allow the identification of natural uniflorine A and its 7-deoxy analogue as the most promising inhibitors among a series of pyrrolizidine derivatives for future development in the agrochemical field, and the investigation also outlined the importance of the stereochemistry at C-6 of pyrrolizidine nucleus to confer such enzyme specificity.

Transcription regulation during stable elongation by a reversible halt of RNA polymerase II.

Regulation of RNA polymerase II (RNAPII) during transcription is essential for controlling gene expression. Here we report that the transcriptional activity of RNAPII at the Balbiani ring 2.1 gene could be halted during stable elongation in salivary gland cells of Chironomus tentans larvae for extended time periods in a regulated manner. The transcription halt was triggered by heat shock and affected all RNAPII independently of their position in the gene. During the halt, incomplete transcripts and RNAPII remained at the transcription site, the phosphorylation state of RNAPII was unaltered, and the transcription bubbles remained open. The transcription of halted transcripts was resumed upon relief of the heat shock. The observed mechanism allows cells to interrupt transcription for extended time periods and rapidly reactivate it without the need to reinitiate transcription of the complete gene. Our results suggest a so-far-unknown level of transcriptional control in eukaryotic cells.

N-Bridged 1-deoxynojirimycin dimers as selective insect trehalase inhibitors.

A small set of N-bridged 1-deoxynojirimycin dimers has been synthesized and evaluated as potential inhibitors of insect trehalase from midge larvae of Chironomus riparius, porcine trehalase as the mammalian counterpart and α-amylase from human saliva. All the tested compounds (2-4) proved to be active (micromolar range activity) against insect trehalase, showing selectivity toward the insect glycosidase. No activity was observed against α-amylase.

GTPase Ran strongly accumulates at the kinetochores of somatic chromosomes in the spermatogonial mitoses of Acricotopus lucidus (Diptera, Chironomidae).

Unequal chromosome segregation and spindle formation occurs in the last gonial mitosis in the germ line of the chironomid Acricotopus lucidus. During this differential mitosis, all germ line-limited chromosomes (=Ks) migrate undivided to only one pole of the cell, while the somatic chromosomes (=Ss) first remain in the metaphase plane, and with the arrival of the Ks at the pole, they then separate equally. The evolutionarily conserved GTPase Ran plays a crucial role in many cellular processes. This includes the regulation of microtubule nucleation and stabilisation at kinetochores and of spindle assembly during mitosis, which is promoted by a RanGTP concentration gradient that forms around the mitotic chromosomes (Kalab et al. in Science 295:2452-2456, 2002, Nature 440:697-701, 2006). In the present study, a strong accumulation of Ran was detected by immunofluorescence at the kinetochores of the Ss in normal gonial and differential gonial mitoses of males of A. lucidus. In contrast, no Ran accumulation was observed at the kinetochores of the Ss in the metaphases of brain ganglia mitoses or of aberrant spermatocytes or in metaphases I and II of spermatocyte meiotic divisions. Likewise, there was no accumulation at the kinetochores of Drosophila melanogaster mitotic chromosomes from larval brains. The specific accumulation of Ran at the kinetochores of the Ss in differential gonial mitoses of A. lucidus strongly suggests that Ran is involved in a mechanism acting in this exceptional mitosis, which retains the Ss at the metaphase plane and prevents a premature separation and unequal segregation of the Ss during monopolar migration of the Ks.