Apyrase immobilized on paramagnetic beads used to improve detection limits in bioluminometric ATP monitoring

A novel technique that is useful for the control of the luminescence output in a bioluminometric assay has been developed. The method relies on the immobilization on paramagnetic beads of an enzyme that is capable of catalysing the hydrolysis or oxidation of the substrate for the luminescence reaction. This technique has been used for control of the level of ATP in the luciferin-luciferase system. Biotinylated apyrase was bound to streptavidin coated paramagnetic beads and added to the assay mixture. The level of ATP in the reaction mixture was then conveniently controlled using an external magnet. The possible use of this approach in other systems, such as the bacterial luciferase–oxidoreductase is discussed.     

A simple method of DNA extraction from whole tissues and blood using glass powder for detection of transgenic animals by PCR

A very simple and reliable method to extract DNA directly from mouse tail, rabbit ear and blood is described. Tissue was homogenized in a solution of NaI and the DNA was extracted using glass powder. The extracted DNA was obtained in sufficient quantity and purity to allow direct detection of transgene by PCR.     

海底玄武岩玻璃中蚀变微结构:探索海洋深地生物圈的新材料

玄武岩玻璃中的生物蚀变微结构为微生物摄取玄武岩玻璃中营养成分,通过新陈代谢产生有机酸溶解玄武岩玻璃而形成的微米级孔洞或钻穴.生物蚀变微结构在现代海洋洋壳及代表古老洋壳残片的蛇绿岩和绿岩带中广泛存在.研究玄武岩玻璃中蚀变微结构的形态特征、形成机制及其时空分布,不仅对探索地球早期生命起源和演化具有重要启示意义,也为研究海洋...     

Novel heterobifunctionalized polystyrene-polyethylene glycol resin for simultaneous preparation of free and immobilized peptides and biological activity detected by confocal microscopy

Summary Fast and convenient binding assays using synthetic peptides are of utmost and increasing importance, especially in the search for lead structures or in the field of diagnostics. A polymeric support suitable for solid-phase peptide synthesis was functionalized with two different anchor groups. The interior part of the aminomethylated polystyrene-1%-divinylbenzene resin beads, comprising about 98% of the total loading capacity, was modified by the acid-labile ADPV anchor whereas the 2% outer surface of the polymer was covalently coated with a PEG 10 000 derivative which renders the resin surface hydrophilic and biocompatible. The novel resin was characterized by introducing marker amino acids and by infrared spectroscopy. Employing this bifunctionalized resin for peptide synthesis, free as well as polymer-bound peptides were obtained which were tested for recognition by antibody. The resin-bound peptides proved to be suitable for ELISA and fluorescence assays, as shown by confocal laser microscopic investigations. Peptides from the interior part were obtained in high yield and purity as analyzed by HPLC, electrospray mass spectrometry and Edman degradation.     

Sustained Food Vacuole Formation by Axenic Paramecium tetraurelia and the Inhibition of Membrane Recycling by Alcian Blue

It is believed that the uptake mechanism of some nutrients by Paramecium tetraurelia primarily involves transport through the cell surface, whereas the uptake of other compounds appears to be restricted to bulk transport during food vacuole (phagosome) formation. In this study, we established that, in axenically grown cells, food vacuole formation occurred at continuous rates over long periods. This information allows quantitation of the volume of media taken up by bulk transport. India ink and latex beads were shown to be inert food vacuole markers and carmine was found to have an initial stimulatory effect on phagosome formation rates. Cultures grown for 3.5 h or longer with the glycocalyx stain Alcian Blue, contained only three phagosomes/cell, whereas cells cultured with the other markers contained 15 phagosomes/cell. Electron microscopy of fecal material that accumulated at the bottom of Alcian Blue-grown cells demonstrated the presence of membranes, suggesting that the vacuolar membrane was eliminated during defecation. Neither cell lysis nor the formation of autophagous vacuoles was detected in Alcian Blue-grown cells, indicating that the stain was not cytotoxic at the concentrations used. Thus it appeared that the binding of Alcian Blue to the digestive vacuole membrane resulted in a loss of the vacuole membranes from the cell which reduced the amount of membranes retrieved and recycled and hence eventually reduced the rate of phagosome formation. Alcian Blue-treated cultures exhibited decreased rate of growth and final density, which is consistent with a decrease in bulk transport of nutrients resulting from reduced membranes of digestive cycle organelles available in the cell.     

Use of an immobilized cell bioreactor for the continuous inoculation of milk in fresh cheese manufacturing

A system was developed to continuously acidify and inoculate skim milk for the production of fresh cheese. Four strains of mesophilic lactic acid bacteria were entrapped separately in κ -carrageenan/locust bean gum gel beads and used in a stirred bioreactor operated at 26°C with a 25% (v/v) gel load. The pH in the reactor was controlled at 6.0 by adding fresh milk using proportional integrated derived regulation. The bioreactor was operated during 8-h daily cycles for up to 7 weeks with different milks (heat treatment, dry matter content) and differing starting procedures. The heat treatment of the milk was an important factor for process performance: a dilution rate increase of 57% and an inoculation level decrease of 63% were observed with sterilized UHT skim milk (142°C – 7.5 s) compared with pasteurized skim milk (72°C – 15 s). The dry matter content of the milk (8–13% w/w) had no detectable effect on these parameters. A convenient starting procedure of the system was tested; steady-state was reached in less than 40 min following an interruption period of 16–60 h. These results combined with our published data on process performance show the feasibility of using an integrated immobilized cell bioreactor for milk prefermentation in cheese manufacture. Received 10 June 1996/ Accepted in revised form 15 October 1996     

Water flow induced transport of Pseudomonas fluorescens cells through soil columns as affected by inoculant treatment

Abstract Water flow induced transport of Pseudomonas fluorescens cells through soil columns was measured as affected by the inoculant treatment. Bacterial cells were introduced into the topsoil of columns, either encapsulated in alginate beads of different types or mixed with bentonite clay in concentrations ranging from 0.5 to 5.0% (w/v). Survival of bacterial cells was improved with the use of alginate or bentonite. Transport, as determined by destructive sampling of the columns, was reduced with the use of alginate encapsulation. Drying of the beads had no influence on transport. The presence of bentonite in the topsoil, either pre-mixed through the soil, or applied as a slurry together with the bacteria, also reduced transport, except when 0.5% was pre-mixed through the soil. P. fluorescens cells encapsulated in alginate beads prepared with water and supplemented with skim milk powder and bentonite showed the best survival during the time of the experiment and the most reduced transport compared to the control. Therefore, cells encapsulated in this way are suitable, due to their optimal survival and reduced spread, for use in a field experiment with genetically manipulated bacteria.     

Discrepancies between otoliths of larvae and juveniles of the American eel: is something fishy happening at metamorphosis?

The use of otoliths to interpret early life history in fishes depends upon the assumptions that otoliths record past events accurately and consistently and that records of events in otoliths are continuous. Both the number of growth microincrements ( I ), and the radii ( R ,μm) of otoliths of American eel Anguilla rostrata , leptocephali increased linearly and highly significantly with leptocephalus body length ( L , mm), as expected on the above assumptions ( I , =2·29 L , − 5·75 and R , =1·05 L , + 12·02, r ²,=0·938 and 0·931, n , =20). In contrast, the number of increments and the radii of the leptocephalus growth zones of otoliths of glass-phase American eels were not related to body length, and they were lower than predicted by the relationships developed for leptocephali. Thus, otoliths of American eels apparently violate one or both assumptions. Possibly, the margin of the otolith is resorbed during metamorphosis from leptocephalus to glass eel, perhaps as part of calcium metabolism as skeletal elements are being formed.     

Improving the suitability of glass fiber filters for use as culture supports

Summary Commercially available glass fiber filters are useful as physical supports for cultures; however, as received from the manufacturers, the filters frequently contain substances that render them unsuitable for some types of experimental studies. These substances contribute to the formation of precipitates in the culture media, alter the media pH, and repress synchronous development among embryo cultures of eastern white pine (Pinus strobus L.). A simplified technique was developed to remove the contaminants and to saturate the cation exchange sites on the glass fibers with specific ions. The use of trade, product, or corporation names in this publication is for the information and convenience of the reader. Such use does not constitute an official endorsement or approval by the U.S. Department of Agriculture or the Forest Service of any product or service to the exclusion of others that also may be suitable.