Elimination of artifactual immunogold labeling from secondary walls of woody stem sections

Preliminary in situ transmission electron microscopy immunogold localization of a 24-kDa dehydrin-like protein in red-osier dogwood (Cornus sericea L.) stem cross-sections was contaminated with extensive background labeling of secondary cell walls in both positive and negative control samples. Alterations in antibody dilution, buffer salt concentration and stringency of the washing solutions failed to eliminate background cell-wall labeling. A procedure was developed in which lyophilized cold-acclimated C. sericea xylem tissue was pulverized and boiled with sodium dodecyl sulfate (SDS)-protein extraction buffer to remove soluble proteins and to inactivate proteases. Wood powder, treated with SDS-protein extraction buffer, was used to pre-absorb chicken immune serum specific for a 24-kDa dehydrin-like protein prior to immunolocalization assays. Pre-incubation of primary antibodies did not compromise the recognition of the 24-kDa protein, and this technique effectively eliminated background cell-wall labeling.     

Distribution of tritiated ecdysone in salivary glands and other tissues ofRhynchosciara andChironomus larvae an autoradiographic study

Summary The distribution of tritium-labelled ecdysone in salivary glands ofChironomus tentans and different tissues ofRhynchosciara spec, has been studied, following in vivo incubations and either of two modified ethanol-acetic acid fixation techniques. The results of autoradiographic analysis show maximum label over the cytoplasm or evenly labelled cytoplasm and nuclei, within a wide range of incubation times. Indications of distinct localization at chromosomal loci are not conclusive. There is a remarkably high amount of radioactivity in the lumen of the anterior intestine as soon as six minutes after hormone injection. The limitations of histoautoradiographic studies in attempts to localize the cellular receptor of ecdysone are discussed.

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Zusammenfassung Nach Injektion von tritiummarkiertem Ecdyson in die Larven wurde dessen Verteilung in Speicheldrüsen vonChironomus tentans und verschiedenen Geweben vonRhynchosciara spec. studiert. Die Gewebe wurden nach zwei verschiedenen modifizierten äthanol-Essigsäure-Fixierungsmethoden aufgearbeitet. über einen weiten Bereich verschiedener Inkubationszeiten ergab sich bei autoradiographischer Untersuchung entweder höhere Markierung des Cytoplasmas oder gleichmäßige Markierung von Cytoplasma und Kern. Es fanden sich keine signifikanten Anzeichen für spezifische Lokalisierung an chromosomalen Quersoheiben. Das Lumen des Vorderdarmes zeigt schon 6 min nach Hormoninjektion außerordentlich hohe Radioaktivität. Es wird diskutiert, wie weit histoautoradiographische Methoden zur Lokalisierung intracellulärer Ecdysonreceptoren geeignet sind.

     

Adsorption of ethylenediaminetetraacetic dianhydride modified oxalate decarboxylase on calcium oxalate

We used ethylenediaminetetraacetic acid dianhydride (EDTAD) to modify oxalate decarboxylase (OXDC) to improve its adsorption on calcium oxalate stones. The modified sites were identified by Ultra performance liquid chromatography-mass spectrometry (UPLC-MS) and the adsorption mechanism of the EDTAD-modified OXDC on calcium oxalate (CaOx) was investigated. We investigated adsorption time, initial enzyme concentration, temperature and solution pH on the adsorption process. Data were analyzed using kinetics, thermodynamics and isotherm adsorption models. UPLC-MS showed that EDTAD was attached to OXDC covalently and suggested that the chemical modification occurred at both the free amino of the side chain and the α-NH₂ of the peptide. The adsorption capacity of the EDTAD-OXDC on calcium oxalate was 53.37% greater than that of OXDC at the initial enzyme concentration of 5 mg/ml, pH = 7.0, at 37° C. The modified enzyme (EDTAD-OXDC) demonstrated improved oxalate degradation activity at pH 4.5?6.0. Kinetic data fitting analysis suggested a pseudo second order kinetic model. Estimates of the thermodynamic parameters including ΔG⁰, ΔH⁰ and ΔS⁰ of the adsorption process showed it to be feasible, spontaneous and endothermic. Isotherm data fitting analysis indicated that the adsorption process is reduced to monolayer adsorption at a low enzyme concentration and to multilayer adsorption at a high enzyme concentration. It may be possible to apply OXDC to degradation of calcium oxalate stones.     

Production of Specific Agglutinins for Mycobacteria in Peritoneal Fluid of Mice

Specific agglutinins were produced in ascitic fluid of mice by intraperitoneal injection of certain mycobacteria suspended in Freund's incomplete adjuvant.     

Regulation of gene activity by hormones

Summary Regulation of gene expression, i.e. the reading of the genetic message, is of great importance for many vital processes. According to Jacob and Monod, genes are regulated by a repressor (a protein) which may interact with an inducer (a small molecule). In higher organisms, hormones often function as inducers. Evidence stems from (i) the induction of puffs in giant chromosomes by the insect hormone ecdysone, (ii) the stimulation of nucleic acid synthesis by hormones in vivo and in vitro, (iii) the induction of enzymes by hormones, e.g. cortisol, and (iv) the action of hormones on isolated nuclei where the activity of the DNA-dependent RNA polymerase is stimulated. This is most probably due to an increased template activity of the chromatin. — The implication of these findings for developmental processes are discussed.     

The gut microbiota of the pine weevil is similar across Europe and resembles that of other conifer‐feeding beetles

The pine weevil (Hylobius abietis, Coleoptera: Curculionidae) is an important pest of conifer seedlings in Europe. Despite its economic importance, little is known about the composition of its gut microbial community and the role it plays in mediating the weevil's ability to utilize conifers as a food source. Here, we characterized the gut bacterial communities of different populations of H. abietis across Europe and compared them to those of other beetles that occupy similar ecological niches. We demonstrate that the microbial community of H. abietis is similar at higher taxonomic levels (family and genus) across locations in Europe, with Wolbachia as the dominant microbe, followed by Enterobacteria and Firmicutes. Despite this similarity, we observed consistent differences between countries and locations, but not sexes. Our meta‐analysis demonstrates that the gut bacterial community of the pine weevil is very similar to that of bark beetles that also exploit conifers as a food source. The Enterobacteriaceae symbionts of both host taxa are especially closely related phylogenetically. Conversely, the microbiota of H. abietis is distinct from that of closely related weevils feeding on nonconifer food sources, suggesting that the microbial community of the pine weevil is determined by the environment and may be relevant to host ecology. Furthermore, several H. abietis‐associated members of the Enterobacteriaceae family are known to contain genes involved in terpenoid degradation. As such, we hypothesize that the gut microbial community is important for the utilization of conifer seedlings as a food source, either through the detoxification of plant secondary metabolites or through the supplementation of essential nutrients.