Microtiter assay for acetylcholinesterase

A microtiter plate adaptation of the classical Ellman colorimetric procedure for measurement of acetylcholinesterase activity is described. This method permits use of an enzyme-linked immunosorbent assay plate reader for rapid analysis of multiple samples and is particularly suitable for analysis of acetylcholinesterase activity on sucrose gradients. The novel procedure is rapid and sensitive and does not require use of radioactive material.     

Deletion analysis of the FliM flagellar switch protein of Salmonella typhimurium.

The flagellar switch of Salmonella typhimurium and Escherichia coli is composed of three proteins, FliG, FliM, and FliN. The switch complex modulates the direction of flagellar motor rotation in response to information about the environment received through the chemotaxis signal transduction pathway. In particular, chemotaxis protein CheY is believed to bind to switch protein FliM, inducing clockwise filament rotation and tumbling. To investigate the function of FliM and its interactions with FliG and FliN, we engineered a series of 34 FliM deletion mutant proteins, each lacking a different 10-amino-acid segment. We have determined the phenotype associated with each mutant protein, the ability of each mutant protein to interfere with the motility of wild-type cells, and the effect of additional FliG and FliN on the function of selected FliM mutant proteins. Overall, deletions at the N terminus produced a counterclockwise switch bias, deletions in the central region of the protein produced poorly motile or nonflagellate cells, and deletions near the C terminus produced only nonflagellate cells. On the basis of this evidence and the results of a previous study of spontaneous FliM mutants (H. Sockett, S. Yamaguchi, M. Kihara, V. M. Irikura, and R. M. Macnab, J. Bacteriol. 174:793-806, 1992), we propose a division of the FliM protein into four functional regions: an N-terminal region primarily involved in switching, an extended N-terminal region involved in switching and assembly, a middle region involved in switching and motor rotation, and a C-terminal region primarily involved in flagellar assembly.     

Prolific shoot regeneration from immature embryo explants of sainfoin (Onobrychis viciifolia Scop.)

Summary Immature cotyledons and embryo axes of sainfoin were cultured on Murashige and Skoog (MS) media supplemented with various concentrations of 6-benzylaminopurine (BAP) and a-naphthaleneacetic acid (NAA) to induce adventitious shoot regeneration. The highest frequency of shoot regeneration occurred following an initial callus growth on a MS medium containing 0.5 mg/l BAP and 2 mg/l NAA. Immature embryo axes showed higher regeneration capacity than immature cotyledons, however, shoot elongation was best achieved on immature cotyledons. Regenerated shoots were excised and rooted in half strength MS medium with 1 mg/l indole-butyric acid (IBA) or 1 mg/l NAA. The rooted plantlets were finally transferred to compost.     

Comparative bioinformatics analysis and abiotic stress responses of expansin proteins in Cucurbitaceae members: watermelon and melon

Protoplasma - Watermelon and melon are members of the Cucurbitaceae family including economically significant crops in the world. The expansin protein family, which is one of the members of the...     

First report of Garella musculana (Erschov, 1874) (Lepidoptera: Nolidae) in Italy with insights into its identity

The Asian walnut moth, Garella musculana (Erschov, 1874) (Lepidoptera: Nolidae), is a major pest of walnut. Native to Central Asia, it was found to be invasive in 2008 in Sevastopol (Crimea) and nowadays widespread in Turkey, Bulgaria, Romania and Russia. Here, we account for the finding of G. musculana in NE Italy (Veneto region) in 2021, where adults were found in a light lamp, representing the first record of the Asian walnut moth for this country and Western Europe. Adult specimens were identified morphologically on both external characters and genitalia features. G. musculana larvae and damage were also observed on a plantation of Juglans regia L. (Fagales: Juglandaceae) located in Veneto in October 2021. A COI-barcoding analysis was performed to attain a molecular characterization of our specimens and probate our morphological identification. However, because no sequence of G. musculana was present in major gene databases and the similarity of our sequences with those attributed to Garella ruficirra (Hampson, 1905) (Lepidoptera: Nolidae) made clear that these taxa deserved further scrutiny regarding their specific distinction. Some subtle differences in the male terminalia could be found between them and their vast geographic distributions, but the strong similarity in most features calls for further morphological and genetical insights on a broad set of samples to assess whether they represent two closely related, substantially parapatric species, or a unique, geographically varying entity. Solving this issue may turn out crucial in the identification and proper management of walnut moths of the genus Garella.     

Evaluation of the standards compliance of the queen bees reared in the Mediterranean region in Turkey

The influence of different commercial queen producers on the quality of Apis mellifera queens was assessed. It was aimed to determine the quality characteristics of queens reared by commercial queen producers located in the province of Antalya, which is an important region in queens production due to its climatic characteristics. For this purpose, the quality characteristics of a total of 105 queen bees obtained from 21 enterprises were determined. Differences between the enterprises in terms of the number of spermatozoa (P < 0.01) were determined. In terms of the diameter of spermatheca, spermatheca volume and live weight, statistical differences between the enterprises were also observed (P < 0.05). When the relationships between the measured characteristics were examined, significant values were obtained statistically between live weight and diameter of spermathecae (0.268) and spermatheca volume (0.258). It was also determined that there is a significant correlation between spermatheca diameter and spermatheca volume (0.995). The spermatheca diameter of a good quality queen bee should not be <1.2 mm, spermatheca volume 0.90 mm³ and live weight not <200 mg. Only live weight was found to be within the normal quality standard values when the average results of the quality criteria are taken into consideration. Other characters such as spermathecae diameter, spermathecae volume and number of spermatozoa in spermathecae seem to be below quality standard values.     

On-line preconcentration/determination of zinc from water,biological and food samples using synthesized chelating resin and flame atomic absorption spectrometry

An on-line flow injection pre-concentration-flame atomic absorption spectrometry method was developed to determine trace zinc in water (tap, dam, and well water), biological (hair and nail), and liver samples. As a solid phase extractant, a synthesized new chelating resin, poly(2-thiozylmethacrylamide-co-divinylbenzene-co-2-acrylamido-2-methyl-1-propane sulfonic acid) was used. The resin was characterized by Fourier transform infrared spectroscopy, elemental analysis, and surface area by nitrogen sorption. A pre-concentration factor of 40-fold for a sample volume of 12.6 mL was obtained by using the time-based technique. The detection limit for the pre-concentration method was found to be 2.2 μg L^?1. The precision (as RSD,%) for 10 replicate determinations at the 0.04 μg mL^?1 Zn concentration was 1.2%. The calibration graph using the pre-concentration system for zinc was linear with a correlation coefficient of 0.998 in the concentration range from 0.005 to 0.05 μg mL^?1. The applicability and accuracy of the developed method were estimated by the analysis spiked water, biological, liver samples (83–105%), and also certified reference material TMDA-70 (fortified lake water) and SPS-WW1 Batch 111-Wastewater. The results were in agreement with the certified values.     

Single-step purification of recombinant Thermus aquaticus DNA polymerase using DNA-aptamer immobilized novel affinity magnetic beads

A DNA aptamer specific for Thermus aquaticus DNA polymerase (Taq-polymerase) was immobilized on magnetic beads, which were prepared in the presented study. The effect of various parameters including pH, temperaturem and aptamer concentration on the immobilization of 5'-thiol labeled DNA-aptamer onto glutaric dialdhyde activated magnetic beads was evaluated. The binding conditions of Taq-polymerase on the aptamer immobilized magnetic beads were studied using commercial Taq-polymerase to characterize the surface complexation reaction. Efficiency of affinity magnetic beads in the purification of recombinant Taq-polymerase from crude extracts was also evaluated. For this case, the enzyme "recombinant Taq-DNA polymerase" was cloned and expressed using an Amersham E. coli GST-Gene Fusion Expression system. Crude extracts were in contact with affinity magnetic beads for 30 min and were collected by magnetic field application. The purity of the eluted Tag-polymerase from the affinity beads, as determined by HPLC, was 93% with a recovery of 89% in a one-step purification protocol. Apparently, the system was found highly effective as one step for the low-cost purification of Taq-polymerase in bacterial crude extract.     

K2P18.1 translates T cell receptor signals into thymic regulatory T cell development

It remains largely unclear how thymocytes translate relative differences in T cell receptor (TCR) signal strength into distinct developmental programs that driv...