Glyphosate is an herbicide that is used worldwide with potential environmental risks to nontarget organisms. We applied an age-stage, two-sex life table approach to assess the sublethal effects of short-term oral exposure to a glyphosate-based herbicide on the life table parameters and biocontrol potential of Harmonia axyridis (Pallas) (Coleoptera: Coccinellidae). Aphids (Metopolophium dirhodum (Walker) (Sternorrhyncha: Aphididae)) treated with herbicide (an isopropylamine-salt of glyphosate) at low recommended, maximum recommended, and double the maximum recommended concentration for agricultural situations, and untreated controls were offered to the fourth instar of H. axyridis for 24 h. Development, consumption, and fecundity were measured daily until death. We detected minor differences in the hatching rate and mean generation time, whereas the longevity, fecundity, net reproductive rate, intrinsic rate of increase, finite rate of increase, and consumption were unaffected across treatments. We conclude that biocontrol potential of H. axyridis was not affected by acute oral intoxication by a glyphosate-based herbicide during the larval stage for 24 h under the study design.
- MeSH
- biologická kontrola škůdců MeSH
- brouci účinky léků růst a vývoj fyziologie MeSH
- glycin analogy a deriváty toxicita MeSH
- insekticidy toxicita MeSH
- kukla účinky léků růst a vývoj fyziologie MeSH
- larva účinky léků růst a vývoj fyziologie MeSH
- mšice chemie růst a vývoj MeSH
- nymfa chemie růst a vývoj MeSH
- potravní řetězec * MeSH
- predátorské chování účinky léků MeSH
- zvířata MeSH
- zvláštnosti životní historie * MeSH
- Check Tag
- mužské pohlaví MeSH
- ženské pohlaví MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
Acetyl-CoA:α-glucosaminide N-acetyltransferase (N-acetyltransferase) is a lysosomal membrane enzyme that catalyzes a key step in the lysosomal degradation of heparan sulfate. Its deficiency causes Sanfilippo syndrome type IIIC (Mucopolysaccharidosis type IIIC, MPS IIIC). Here we characterize the promoter region of HGSNAT, the gene encoding N-acetyltransferase, which is located in the pericentromeric region of chromosome 8. We show that HGSNAT transcription is driven by a TATA-less promoter whose key elements are contained within the 1054bp region upstream of exon 1. About 400 bases of the region's 3'-prime end overlap with an unmethylated CpG island. Reduced reporter activities from promoter serial deletion constructs suggested strong regulatory elements at positions -101 to -20bp and -1073 to -716bp of the downstream initiation codon (DS-ATG). Targeted mutagenesis of the first Specificity protein 1-A (Sp1-A) of the six in silico-predicted Sp1 sites in the region flanking the major transcription start sites (TSSs, +50/-101) led to a 55% decrease of reporter activity, while inactivation of each of Sp1-B and Sp1-C resulted in its almost two-fold increase. The binding of Sp1 to the region was confirmed by chromatin immunoprecipitation (ChIP). Overall, this confirms that Sp1 is important for regulation of the HGSNAT promoter. Promoter fragments in antisense orientation (constructs pGL4 -20/-1305 and pGL4 +50/-1305) led to reporter activities of about 50% of the pGL4 -1305/-20 activity, implying divergent initiation of transcription at the promoter. We identified two main TSSs at positions +1 and -15 from DS-ATG using Rapid amplification of cDNA ends (5'RACE). Transcripts initiating at the TSSs thus contain only DS-ATG. Five patients from our MPS IIIC cohort (n=23) carried the rs4523300 promoter variant and one the rs149596192 promoter variant. Both variants lowered the expression of the reporter down to 68% and 59%, respectively. However, white blood cell (WBC) N-acetyltransferase activities in individuals carrying the variants did not significantly differ from homozygotes for the wild-type alleles, suggesting only a partial impact of transcriptional regulation on N-acetyltransferase activities in vivo.
- MeSH
- acetyltransferasy genetika MeSH
- buňky Hep G2 MeSH
- jednonukleotidový polymorfismus * MeSH
- lidé MeSH
- mukopolysacharidóza III genetika MeSH
- počátek transkripce * MeSH
- studie případů a kontrol MeSH
- TATA box * MeSH
- transkripční faktor Sp1 metabolismus MeSH
- vazba proteinů MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
Glyphosate is the number one herbicide in the world. We investigated the sub-lethal effects of this herbicide on the aphid Metopolophium dirhodum (Walker), using an age-stage, two-sex life table approach. Three concentrations of the herbicide (low - 33.5, medium - 66.9 and high - 133.8 mmol dm(-3) of active ingredient) and distilled water as the control were used. The LC50 of the IPA salt of glyphosate on M. dirhodum was equivalent to 174.9 mmol dm(-3) of the active ingredient (CI95: 153.0, 199.0). The population parameters were significantly negatively affected by herbicide application, and this negative effect was progressive with the increasing concentration of the herbicide. A difference of two orders of magnitude existed in the predicted population development of M. dirhodum between the high concentration of the herbicide and the control. This is the first study that comprehensively documents such a negative effect on the population of an herbivorous insect.
- MeSH
- analýza přežití MeSH
- fertilita účinky léků MeSH
- glycin analogy a deriváty toxicita MeSH
- herbicidy toxicita MeSH
- mšice účinky léků růst a vývoj MeSH
- Rosa MeSH
- rozmnožování účinky léků MeSH
- stadia vývoje účinky léků MeSH
- stárnutí MeSH
- zvířata MeSH
- Check Tag
- ženské pohlaví MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
We present a simple method for enrichment of lysosomal membranes from HEK293 and HeLa cell lines taking advantage of selective disruption of lysosomes by methionine methyl ester. Organelle concentrate from postnuclear supernatant was treated with 20 mmol/l methionine methyl ester for 45 min to lyse the lysosomes. Subsequently, lysosomal membranes were resolved on a step sucrose gradient. An enriched lysosomal membrane fraction was collected from the 20%/35% sucrose interface. The washed lysosomal membrane fraction was enriched 30 times relative to the homogenate and gave the yield of more than 8%. These results are comparable to lysosomal membranes isolated by magnetic chromatography from cultured cells (Diettrich et al., 1998). The procedure effectively eliminated mitochondrial contamination and minimized contamination from other cell compartments. The enriched fractions retained the ability to acidify membrane vesicles through the activity of lysosomal vacuolar ATPase. The method avoids non-physiological overloading of cells with superparamagnetic particles and appears to be quite robust among the tested cell lines. We expect it may be of more general use, adaptable to other cell lines and tissues.
- MeSH
- adenosintrifosfát farmakologie MeSH
- centrifugace - gradient hustoty MeSH
- frakcionace buněk metody MeSH
- glukosylceramidasa metabolismus MeSH
- HEK293 buňky MeSH
- HeLa buňky MeSH
- intracelulární membrány účinky léků metabolismus MeSH
- kyseliny metabolismus MeSH
- lidé MeSH
- lyzozomy účinky léků metabolismus MeSH
- subcelulární frakce účinky léků metabolismus MeSH
- western blotting MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
Larvae of the greater waxmoth (Galleria mellonella) become paralysed by the venom of the braconid wasp (Habrobracon hebetor) a few minutes after intoxication. The profound neuromuscular paralysis, which may last for several weeks, includes all somatic muscles that are innervated through neuromuscular transmission. The peristaltic contractions of the heart and intestine, which are regulated by the depolarisation potentials of the myocardium or intestinal epithelial muscles, remain unaffected and fully functional. Heartbeat patterns and intestinal pulsations were monitored in the motionless, paralysed larvae by means of advanced electrocardiographic recording methods (contact thermography, pulse-light optocardiography). The records revealed more or less constant cardiac pulsations characterised by 20-25 systolic contractions per minute. The contractions were peristaltically propagated in the forward (anterograde) direction, with a more or less constant speed of 10mm per second (23-25°C). Additional electrocardiographic investigations on larvae immobilised by decapitation revealed the autonomic (brain independent) nature of heartbeat regulation. Sectioning performed in the middle of the heart (4th abdominal segment) seriously impaired the pacemaker rhythmicity and slowed down the rate of heartbeat in the anterior sections. By contrast, the functions of the posterior compartments of the disconnected heart remained unaffected. These results confirmed our previous conclusions about the existence of an autonomic, myogenic, pacemaker nodus in the terminal part of an insect heart. They show an analogy to the similar myogenic, sinoatrial or atrioventricular nodi regulating rhythmicity of the human heart. Peristaltic contractions of the intestine also represent a purely myogenic system, which is fully functional in larvae with complete neuromuscular paralysis. Unlike the constant anterograde direction of the heartbeat, intestinal peristaltic waves periodically reversed anterograde and retrograde directions. A possibility that the functional similarity between insect and human hearts may open new avenues in the field of comparative cardiology has been discussed.
- MeSH
- biologické hodiny účinky léků MeSH
- elektrokardiografie metody MeSH
- kousnutí a bodnutí hmyzem patofyziologie MeSH
- můry účinky léků fyziologie MeSH
- nervový přenos účinky léků MeSH
- peristaltika účinky léků MeSH
- srdeční frekvence MeSH
- sršňovití MeSH
- střeva fyziologie MeSH
- svaly patofyziologie MeSH
- vosí jedy farmakologie MeSH
- zvířata MeSH
- Check Tag
- ženské pohlaví MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
HIC1 (hypermethylated in cancer 1) is a tumor suppressor gene located on chromosome 17p13.3, a region frequently hypermethylated or deleted in human neoplasias. In mouse, Hic1 is essential for embryonic development and exerts an antitumor role in adult animals. Since Hic1-deficient mice die perinatally, we generated a conditional Hic1 null allele by flanking the Hic1-coding region by loxP sites. When crossed to animals expressing Cre recombinase in a cell-specific manner, the Hic1 conditional mice will provide new insights into the function of Hic1 in developing and mature tissues. Additionally, we used gene targeting to replace sequence-encoding amino acids 186-893 of Hic1 by citrine fluorescent protein cDNA. We demonstrate that the distribution of Hic1-citrine fusion polypeptide corresponds to the expression pattern of wild-type Hic1. Consequently, Hic1-citrine "reporter" mice can be used to monitor the activity of the Hic1 locus using citrine fluorescence.
- MeSH
- alely MeSH
- delece genu MeSH
- DNA vazebné proteiny genetika MeSH
- embryo savčí metabolismus MeSH
- genový targeting MeSH
- integrasy genetika metabolismus MeSH
- myši transgenní MeSH
- myši MeSH
- reportérové geny MeSH
- transkripční faktory Krüppel-like genetika MeSH
- transkripční faktory genetika MeSH
- tumor supresorové geny MeSH
- vývojová regulace genové exprese MeSH
- zvířata MeSH
- Check Tag
- mužské pohlaví MeSH
- myši MeSH
- ženské pohlaví MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
The Wnt family of proteins is a group of extracellular signalling molecules that regulate cell-fate decisions in developing and adult tissues. It is presumed that all 19 mammalian Wnt family members contain two types of post-translational modification: the covalent attachment of fatty acids at two distinct positions, and the N-glycosylation of multiple asparagines. We examined how these modifications contribute to the secretion, extracellular movement and signalling activity of mouse Wnt1 and Wnt3a ligands. We revealed that O-linked acylation of serine is required for the subsequent S-palmitoylation of cysteine. As such, mutant proteins that lack the crucial serine residue are not lipidated. Interestingly, although double-acylation of Wnt1 was indispensable for signalling in mammalian cells, in Xenopus embryos the S-palmitoyl-deficient form retained the signalling activity. In the case of Wnt3a, the functional duality of the attached acyls was less prominent, since the ligand lacking S-linked palmitate was still capable of signalling in various cellular contexts. Finally, we show that the signalling competency of both Wnt1 and Wnt3a is related to their ability to associate with the extracellular matrix.
- MeSH
- buněčné linie MeSH
- cystein metabolismus MeSH
- embryonální vývoj MeSH
- krysa rodu rattus MeSH
- lidé MeSH
- lipoylace MeSH
- molekulární sekvence - údaje MeSH
- mutace MeSH
- myši MeSH
- protein Wnt1 genetika metabolismus MeSH
- protein Wnt3 MeSH
- protein Wnt3A MeSH
- proteiny Wnt genetika metabolismus MeSH
- proteiny Xenopus MeSH
- sekvence aminokyselin MeSH
- serin metabolismus MeSH
- substituce aminokyselin MeSH
- Xenopus embryologie metabolismus MeSH
- zvířata MeSH
- Check Tag
- krysa rodu rattus MeSH
- lidé MeSH
- myši MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
A major outcome of the canonical Wnt/beta-catenin-signalling pathway is the transcriptional activation of a specific set of target genes. A typical feature of the transcriptional response induced by Wnt signalling is the involvement of Tcf/Lef factors that function in the nucleus as the principal mediators of signalling. Vertebrate Tcf/Lef proteins perform two well-characterized functions: in association with beta-catenin they activate gene expression, and in the absence of Wnt ligands they bind TLE/Groucho proteins to act as transcriptional repressors. Although the general characteristics of Tcf/Lef factors are well understood, the mechanisms that control their specific roles in various cellular backgrounds are much less defined. In this report we reveal that the evolutionary conserved Dazap2 protein functions as a TCF-4 interacting partner. We demonstrate that a short region proximal to the TCF-4 HMG box mediates the interaction and that all Tcf/Lef family members associate with Dazap2. Interestingly, knockdown of Dazap2 not only reduced the activity of Wnt signalling as measured by Tcf/beta-catenin reporters but additionally altered the expression of Wnt-signalling target genes. Finally, chromatin immunoprecipitation studies indicate that Dazap2 modulates the affinity of TCF-4 for its DNA-recognition motif.
- MeSH
- beta-katenin metabolismus MeSH
- buněčné linie MeSH
- DNA vazebné proteiny chemie metabolismus MeSH
- genetická transkripce MeSH
- genový knockdown MeSH
- lidé MeSH
- myši MeSH
- promotorové oblasti (genetika) MeSH
- proteiny vázající RNA antagonisté a inhibitory genetika metabolismus MeSH
- proteiny Wnt metabolismus MeSH
- regulace genové exprese MeSH
- transkripční faktory BHLH-Zip MeSH
- transkripční faktory chemie metabolismus MeSH
- vazebná místa MeSH
- zvířata MeSH
- Check Tag
- lidé MeSH
- myši MeSH
- zvířata MeSH
- Publikační typ
- práce podpořená grantem MeSH
