Influence of temperature and humidity on insecticidal effect of three diatomaceous earth formulations against larger grain borer (Coleoptera: Bostrychidae)

Laboratory experiments were carried out to evaluated three diatomaceous earth (DE) formulations--Protect-It, PyriSec (at dose rates 500, 1000, and 1500 ppm), and DEA-P (at dose rates 75, 150, and 500 ppm)--against the larger grain borer, Prostephanus truncatus (Horn) (Coleoptera: Bostrychidae), adults in stored maize, Zea mays L., at three temperatures (20, 25, and 30 degrees C) and two relative humidity (RH) levels (55 and 75%). At these conditions, the capability of progeny production in the treated substrate also was assessed. Adult survival was high, at all doses of Protect-It and PyriSec. Progeny production was also high. In contrast with the other two DEs, DEA-P was highly effective and caused complete mortality to the exposed P. truncatus adults, even at the lowest dose rate (75 ppm). In addition, progeny production was completely suppressed. Generally, Protect-it and PyriSec were more effective at 20 degrees C than at 30 degrees C. In contrast, the efficacy of DEA-P was continuously high in all temperatures and relative humidities examined.     

Road crossing in bank voles and yellow-necked mice

Roads and highways represent one of the most important anthropogenic impacts on natural areas and contribute to habitat fragmentation, because they are linear features that can inhibit animal movement, thereby causing barrier effects subdividing the populations adjacent to the roads. The paper examines to what extent a narrow (2-lane) and a wide (4-lane) highways represent barriers for two small mammal species: bank volesClethrionomys glareolus Schreber, 1780 and yellow-necked miceApodemus flavicollis Melchior, 1834, and whether displaced rodents are able to return across roads of different widths. The study was performed at four sites in the Czech Republic. The capture-mark-recapture method was used to determine crossing rates. At two sites, the animals captured close to the road were transferred to the other side and released, to compare return movements across the roads with the movements made by the non-transferred animals. We found that the narrow highway did not prevent movement of neither of the species, although voles crossed only after they had been transferred. Wide highways, on the other hand, completely prevened crossing of both species. While the narrow highways acted at individuals level, the wide highways affected the population subvision.     

Limitation of the Ellman method: cholinesterase activity measurement in the presence of oximes

The Ellman method for assaying thiols is widely used for cholinesterase activity measurement. Cholinesterase activity is measured indirectly by quantifying the concentration of 5-thio-2-nitrobenzoic acid (TNB) ion formed in the reaction between the thiol reagent 5,5'-dithiobis-2-nitrobenzoic acid (DTNB) and thiocholine, a product of substrate (i.e., acetylthiocholine [ATCh]) hydrolysis by the cholinesterase. Oximes, reactivators of inhibited cholinesterase, are nucleophiles that also react with ATCh (oximolysis), producing thiocholine and (indirectly) TNB ion. The aim of this study was to characterize ATCh oximolysis. Therefore, we measured the oximolysis between oximes (K027 and HI-6) and ATCh in the presence of DTNB at different pH values, taking into account the final concentration of a product that is thiocholine. To confirm oximate ion involvement in the nucleophilic attack, we also determined the reaction rate between the oximes and ATCh, without DTNB, at different pH values by measuring the decrease in oximate ion absorption over time. The oximate ion of K027 reacted 14 times faster with ATCh (306M(-1)min(-1)) than the oximate ion of HI-6 (22M(-1)min(-1)). However, the rate constants obtained with the Ellman method were 84M(-1)min(-1) for K027 and 22M(-1)min(-1) for HI-6. Our results confirmed that the rate obtained with K027 using the Ellman method is actually the rate of the Ellman reaction itself. This suggests that the Ellman method cannot be used uncritically to evaluate oxime reaction with choline esters, in particular when oximolysis is faster than the Ellman reaction itself at a given pH.     

A quantitative morphometric study of rectal mucosa in adult and aged healthy subjects

Rectal mucosa is relatively susceptible to pathological processes and frequently it is affected by various diseases. However, there is a notable lack of quantitative data regarding normal rectal mucosa, which would provide a reference for histoquantitative studies of the pathologically changed tissue. Therefore, we obtained the tissue from 27 healthy patients subjected to diagnostic rectoscopy during active screening for asymptomatic cancer of the large intestine, in which no disease was found. Using computer-aided morphometric analysis, we studied all structural elements of the rectal mucosa. The patients were divided into four groups according to the age and sex: adult males, elderly males, adult females and elderly females. The patients under 60 years of age were grouped as adult and those older than 60 years as aged subjects. A decreased height of surface epithelium was registered in both elderly male and female groups. This finding, however, was significant only when adult and elderly male groups were compared. The tendency towards reduction of the mucosal height was also registered comparing male adult and elderly groups. The number of crypts per 0.1 mm2 of tissue increased with aging in both males and females, whereby the crypts were always more numerous in males than in females. The increase in number of crypts in male subjects was accompanied by a decrease in their diameter and perimeter. The changes associated with ageing were discrete and affected only the male subjects.     

Influence of maternal dexamethasone treatment on morphometric characteristics of pituitary GH cells and body weight in near-term rat fetuses

Growth hormone (GH) and glucocorticoids have a powerful influence on controlling fetal growth, differentiation and maturation of numerous tissues. In the present study, the effect of maternal dexamethasone (Dx) treatment on GH cells and body weight in 19- and 21-day-old rat fetuses was investigated using immunocytochemical and morphometric methods. Pregnant female rats received daily injections of 1.0-0.5-0.5 mg Dx/kg b.w. on days 16-18 of pregnancy (experimental group), while the control group received an equal volume of saline. Dx treatment of pregnant rats enhanced immunostaining intensity and significantly increased (p<0.05) GH nuclear and cell volume, as well as volume density and number of GH cells per square millimeter in 19-day-old fetuses compared to the controls. In 21-day-old fetuses after maternal Dx administration, immunoreactivity, volume density and number of GH cells remained significantly increased (p<0.05). Dx treatment of pregnant rats resulted in marked body weight reduction of 21-day-old but not 19 days old fetuses in comparison with the corresponding controls. The presented results demonstrate that maternal Dx application has pronounced effect on morphometric parameters of GH cells of 19- and 21-day-old fetuses. Also, in near-term rat fetuses body weight was largely independent of pituitary GH cell activity.     

Continuum simulations of acetylcholine diffusion with reaction-determined boundaries in neuromuscular junction models

The reaction-diffusion system of the neuromuscular junction has been modeled in 3D using the finite element package FEtk. The numerical solution of the dynamics of acetylcholine with the detailed reaction processes of acetylcholinesterases and nicotinic acetylcholine receptors has been discussed with the reaction-determined boundary conditions. The simulation results describe the detailed acetylcholine hydrolysis process, and reveal the time-dependent interconversion of the closed and open states of the acetylcholine receptors as well as the percentages of unliganded/monoliganded/diliganded states during the neuro-transmission. The finite element method has demonstrated its flexibility and robustness in modeling large biological systems.     

Synthetic esters recognized by glucuronoyl esterase from <Emphasis Type="Italic">Schizophyllum commune</Emphasis>

Glucuronoyl esterase is a novel carbohydrate esterase recently discovered in the cellulolytic system of the wood-rotting fungus Schizophyllum commune on the basis of its ability to hydrolyze methyl ester of 4-O-methyl-d-glucuronic acid. This substrate was not fully corresponding to the anticipated function of the enzyme to hydrolyze esters between xylan-bound 4-O-methyl-d-glucuronic acid and lignin alcohols occurring in plant cell walls. In this work we showed that the enzyme was capable of hydrolyzing two synthetic compounds that mimic the ester linkages described in lignin-carbohydrate complexes, esters of 4-O-methyl-d-glucuronic and d-glucuronic acid with 3-(4-methoxyphenyl)propyl alcohol. A comparison of kinetics of hydrolysis of methyl and 3-(4-methoxyphenyl)propyl esters indicated that the glucuronoyl esterase recognizes the uronic acid part of the substrates better than the alcohol type. The catalytic efficiency of the enzyme was much higher with the ester of 4-O-methyl-d-glucuronic acid than with that of d-glucuronic acid. Examination of the action of glucuronoyl esterase on a series of methyl esters of 4-O-methyl-d-glucopyranuronosyl residues α-1,2-linked to xylose and several xylooligosaccharides suggested that the rate of deesterification is independent of the character of the carbohydrate part glycosylated by the 4-O-methyl-d-glucuronic acid.     

Assisted large fragment insertion by Red/ET-recombination (ALFIRE)--an alternative and enhanced method for large fragment recombineering

Functional genomics require manipulation and modification of large fragments of the genome. Such manipulation has only recently become more efficient due to the discovery of different techniques based on homologous recombination. However, certain limitations of these strategies still exist since insertion of homology arms (HAs) is often based on amplification of DNA sequences with PCR. Large quantities of PCR products longer than 4-5 kb can be difficult to obtain and the risk of mutations or mismatches increases with the size of the template that is being amplified. This can be overcome by adding HAs by conventional cloning techniques, but with large fragments such as entire genes the procedure becomes time-consuming and tedious. Second, homologous recombination techniques often require addition of antibiotic selection genes, which may not be desired in the final construct. Here, we report a method to overcome the size and selection marker limitations by a two- or three-step procedure. The method can insert any fragment into small or large episomes, without the need of an antibiotic selection gene. We have humanized the mouse luteinizing hormone receptor gene (Lhcgr) by inserting a approximately 55 kb fragment from a BAC clone containing the human Lhcgr gene into a 170 kb BAC clone comprising the entire mouse orthologue. The methodology is based on the rationale to introduce a counter-selection cassette flanked by unique restriction sites and HAs for the insert, into the vector that is modified. Upon enzymatic digestion, in vitro or in Escherichia coli, double-strand breaks are generated leading to recombination between the vector and the insert. The procedure described here is thus an additional powerful tool for manipulating large and complex genomic fragments.     

Association of putative ammonium exporters Ato with detergent-resistant compartments of plasma membrane during yeast colony development: pH affects Ato1p localisation in patches

It was proposed that Ato1p, Ato2p and Ato3p have a role in ammonia production by Saccharomyces cerevisiae colonies (Palkova et al., Mol Biol Cell 13: 3901-3914, 2002). In this study, we show that all three Ato proteins localise to the plasma membrane and their appearance correlates with the beginning of ammonia release. The expression of ATO genes is controlled by ammonia. All three Ato-GFP proteins associate with detergent-resistant membranes; two of them, Ato1p-GFP and Ato3p-GFP, localise to patches visible under the fluorescence microscope. In contrast with Ato3p-GFP which forms stable patches, the formation of those of Ato1p-GFP is pH dependent. Ato1p-GFP patches form at pH above 6 and they disappear at pH 5 or lower. Both changes, Ato1p-GFP clustering and patches spreading are reversible. The Ato1p-GFP spreading at low pH is independent on endocytosis. These data suggest that besides the ammonia induction of Ato protein synthesis, pH may rapidly regulate Ato1p function.