Lysis

About: Lysis is a research topic. Over the lifetime, 6072 publications have been published within this topic receiving 216978 citations.

Rapid DNA extraction protocol from soil for polymerase chain reaction‐mediated amplification

Abstract: A simple and rapid method of DNA extraction from soil was developed and DNA was made suitable for subsequent efficient amplification by the polymerase chain reaction (PCR). Key features of the extraction and purification were cold lysozyme- and SDS-assisted lysis with either freezing-thawing or bead beating, cold phenol extraction of the resulting soil suspension, CsCl and KAc precipitation and, finally, spermine-HCl or glass milk purification of DNA. Crude DNA preparations contained 4–20 μg DNA per g of soil extracted, and at least 50% of this was recovered in the final purified DNA preparations. The resulting DNA was pure enough to be restricted by various enzymes, and was amplifiable at concentrations of up to 20 ng of soil-derived DNA per 50 μl reaction mix. Amplification of a 683 bp target sequence, pat, was performed with different Taq DNA polymerases. Application of the protocol enabled us to detect target DNA derived from roughly 103 introduced Pseudomonas fluorescens (RP4 :: pat) cfu per g of soil. The fate of an introduced population in the soil could be followed to this limit with PCR-assisted detection of target DNA. In addition, target DNA was detected in soil 5 months after release, when the introduced organism was no longer detectable on selective agar plates. The extraction and purification protocol applied to various different soil types resulted in DNA of sufficient purity to permit amplification by PCR.

Photometric determination of lysozyme activity.

Abstract: SummaryAn accurate photometric method has been developed using a Pfalz and Bauer Fluorophotometer for measuring the lysis of suspensions of M. lysodiekticus by lysozyme. This method has the distinct advantages of reproducing activities in 60 seconds after addition of the enzyme. Measurement of maximal rate of lysis in the early part of the time-course of reaction decreases the possibility of interference due to synergism by proteolytic enzymes or solution of cellular material by alkali reaction. This method has been extended to extracts of mammalian tissues.

Release and bioavailability of C, N, P Se, and Fe following viral lysis of a marine chrysophyte

Abstract: The potential importance of the viral lysis of phytoplankton for nutrient and carbon cycling has been acknowledged, but no quantitative assessments of this phenomenon exist. Radiotracer experiments examined the release and bioavailability of C, N, P, Fe, and Se following viral lysis of the "brown tide" chrysophyte Aureococcus anophagefferens. Photochemical effects on the dissolved-particulate partitioning and biological uptake of virally released elements were also investigated. Viral lysis of A. anophagefferens released 50% mon C and Se than uninfected control cells to the dissolved phase, while N, P, and Fe remained in the particulate phase. There was a significant inverse correlation between A. anophagefferens and bacterial densities, as well as an increase in particulate organic nitrogen levels in cultures during viral lysis. These observations indicate that released dissolved organic matter supported bacterial growth and may be a pathway by which various elements are diverted in microbial food webs. Dissolved nutrients released by viral lysis were accumulated to varying degrees by natural assemblages of marine bacteria and cultured diatoms, and vitally regenerated N and P relieved diatom nutrient limitation. During a 4-wk incubation, 80% of C and P within cell lysis debris was released to the dissolved phase, likely due to bacterial activity. Photochemical degradation of cell lysis debris enhanced dissolved levels of Se (100%) and Fe (50%) and reduced total dissolved C by 15%. Photochemistry doubled the bioavailability of virally released Se to diatoms, while decreasing the bioavailability of C to bacteria threefold. The viral lysis of an A. anophagefferens bloom in the field could release 40 mu M dissolved organic carbon and rapidly transfer other released elements to bacteria. Such occurrences may significantly affect water column chemistry, species composition, and succession within marine plankton communities.

Development of a fed-batch process for a recombinant Pichia pastoris Δoch1 strain expressing a plant peroxidase.

Abstract: Pichia pastoris is a prominent host for recombinant protein production, amongst other things due to its capability of glycosylation. However, N-linked glycans on recombinant proteins get hypermannosylated, causing problems in subsequent unit operations and medical applications. Hypermannosylation is triggered by an α-1,6-mannosyltransferase called OCH1. In a recent study, we knocked out OCH1 in a recombinant P. pastoris CBS7435 MutS strain (Δoch1) expressing the biopharmaceutically relevant enzyme horseradish peroxidase. We characterized the strain in the controlled environment of a bioreactor in dynamic batch cultivations and identified the strain to be physiologically impaired. We faced cell cluster formation, cell lysis and uncontrollable foam formation. In the present study, we investigated the effects of the 3 process parameters temperature, pH and dissolved oxygen concentration on 1) cell physiology, 2) cell morphology, 3) cell lysis, 4) productivity and 5) product purity of the recombinant Δoch1 strain in a multivariate manner. Cultivation at 30°C resulted in low specific methanol uptake during adaptation and the risk of methanol accumulation during cultivation. Cell cluster formation was a function of the C-source rather than process parameters and went along with cell lysis. In terms of productivity and product purity a temperature of 20°C was highly beneficial. In summary, we determined cultivation conditions for a recombinant P. pastoris Δoch1 strain allowing high productivity and product purity.

The preparation of, and studies on, free cell suspensions from mouse pancreatic islets.

Abstract: Pancreatic islets, isolated from the pancreas of obese-hyperglycemic mice, were used to prepare free islet cells in suspension. Batches of 100 islets were disrupted by mechanical shaking for 10 sec in a Ca2+-free HEPES-buffered Krebs-Ring'er medium containing 1 mM EGTA. From 200–500 islets, about 2.2×106 cells could be obtained in suspension, corresponding to a yield of roughly 55% as calculated from the content of DNA in cells and islets. The isolated islet cells appeared well-preserved in the light and the electron microscope and seemed to exhibit a high degree of viability as judged by viable cell counts, a radioactive assay for lysis and insulin release. Insulin release from islet cell suspensions, as calculated per cell, per content of DNA or insulin or per packed cell volume, was stimulated by glucose alone or in combination with theophylline. The glucose response was low compared with that of intact isolated islets.