Showing papers on "Lysis published in 2005"

Composition and Distribution of Extracellular Polymeric Substances in Aerobic Flocs and Granular Sludge

Abstract: Extracellular polymeric substances (EPS) were quantified in flocculent and aerobic granular sludge developed in two sequencing batch reactors with the same shear force but different settling times. Several EPS extraction methods were compared to investigate how different methods affect EPS chemical characterization, and fluorescent stains were used to visualize EPS in intact samples and 20-mum cryosections. Reactor 1 (operated with a 10-min settle) enriched predominantly flocculent sludge with a sludge volume index (SVI) of 120 +/- 12 ml g(-1), and reactor 2 (2-min settle time) formed compact aerobic granules with an SVI of 50 +/- 2 ml g(-1). EPS extraction by using a cation-exchange resin showed that proteins were more dominant than polysaccharides in all samples, and the protein content was 50% more in granular EPS than flocculent EPS. NaOH and heat extraction produced a higher protein and polysaccharide content from cell lysis. In situ EPS staining of granules showed that cells and polysaccharides were localized to the outer edge of granules, whereas the center was comprised mostly of proteins. These observations confirm the chemical extraction data and indicate that granule formation and stability are dependent on a noncellular, protein core. The comparison of EPS methods explains how significant cell lysis and contamination by dead biomass leads to different and opposing conclusions.

A microfluidic electroporation device for cell lysis

Abstract: We demonstrate a micro-electroporation device for cell lysis prior to subcellular analysis. Simple circuit models show that electrical lysis method is advantageous because it is selective towards plasma membrane while leaving organelle membrane undamaged. In addition, miniaturization of this concept leads to negligible heat generation and bubble formation. The designed microdevices were fabricated using a combination of photolithography, metal-film deposition, and electroplating. We demonstrate the electro-lysis of human carcinoma cells in these devices to release the subcellular materials.

Comparison of Six DNA Extraction Methods for Recovery of Fungal DNA as Assessed by Quantitative PCR

Abstract: The detection of fungal pathogens in clinical samples by PCR requires the use of extraction methods that efficiently lyse fungal cells and recover DNA suitable for amplification. We used quantitative PCR assays to measure the recovery of DNA from two important fungal pathogens subjected to six DNA extraction methods. Aspergillus fumigatus conidia or Candida albicans yeast cells were added to bronchoalveolar lavage fluid and subjected to DNA extraction in order to assess the recovery of DNA from a defined number of fungal propagules. In order to simulate hyphal growth in tissue, Aspergillus fumigatus conidia were allowed to form mycelia in tissue culture media and then harvested for DNA extraction. Differences among the DNA yields from the six extraction methods were highly significant (P<0.0001) in each of the three experimental systems. An extraction method based on enzymatic lysis of fungal cell walls (yeast cell lysis plus the use of GNOME kits) produced high levels of fungal DNA with Candida albicans but low levels of fungal DNA with Aspergillus fumigatus conidia or hyphae. Extraction methods employing mechanical agitation with beads produced the highest yields with Aspergillus hyphae. The Master Pure yeast method produced high levels of DNA from C. albicans but only moderate yields from A. fumigatus. A reagent from one extraction method was contaminated with fungal DNA, including DNA from Aspergillus and Candida species. In conclusion, the six extraction methods produce markedly differing yields of fungal DNA and thus can significantly affect the results of fungal PCR assays. No single extraction method was optimal for all organisms.

Simultaneous Recovery of Extracellular and Intracellular DNA Suitable for Molecular Studies from Marine Sediments

Abstract: The occurrence of high extracellular DNA concentrations in aquatic sediments (concentrations that are 3 to 4 orders of magnitude greater than those in the water column) might play an important role in biogeochemical cycling, as well as in horizontal gene transfer through natural transformation. Since isolation of extracellular DNA from sediments is a difficult and unsolved task, in this study we developed an efficient procedure to recover simultaneously DNA associated with microbial cells and extracellular DNA from the same sediment sample. This procedure is specifically suitable for studying extracellular DNA because it avoids any contamination with DNA released by cell lysis during handling and extraction. Applying this procedure to different sediment types, we obtained extracellular DNA concentrations that were about 10 to 70 times higher than the intracellular DNA concentrations. Using specific targeted prokaryotic primers, we obtained evidence that extracellular DNA recovered from different sediments did not contain amplifiable 16S rRNA genes. By contrast, using DNA extracted from microbial cells as the template, we always amplified 16S rRNA genes. Although 16S rRNA genes were not detected in extracellular DNA, analyses of the sizes of extracellular DNA indicated the presence of high-molecular-weight fragments that might have contained other gene sequences. This protocol allows investigation of extracellular DNA and its possible participation in natural transformation processes.

Dynamics of E. coli membrane cell peroxidation during TiO2 photocatalysis studied by ATR-FTIR spectroscopy and AFM microscopy

Abstract: Escherichia coli (E. coli) photokilling due to the TiO2 under light irradiation in a batch reactor was studied by using of attenuated total reflection Fourier transform infrared spectroscopy (ATR-FTIR) and atomic force microscopy (AFM). The ATR-FTIR spectral features and AFM images were analyzed in relation to E. coli viability data. ATR-FTIR is shown to be a suitable technique to follow the structural changes of the E. coli cell membranes during TiO2 photocatalysis. Formation of the peroxidation products due to the photocatalysis of E. coli cell is reported by this technique. Time dependent ATR-FTIR experiments provides the evidence for the changes in the E. coli cell wall membranes as the precursor events leading to bacterial lysis. Under the same experimental conditions used by ATR-FTIR spectroscopy, AFM microscopy was carried out to provide direct evidence for the E. coli lysis taking place under light irradiation after about 1 h in the presence of TiO2. By transmission electron microscopy (TEM), the aggregated TiO2 Degussa P-25 in aqueous solution is shown to interact with the bacteria surface and partly to remain in the aqueous solution at the concentration of 1 mg/ml.

Extraction of extracellular polymeric substances from the photosynthetic bacterium Rhodopseudomonas acidophila.

Abstract: Among the four methods for extracting extracellular polymeric substances (EPS) from Rhodopseudomonas acidophila (EDTA, NaOH, H(2)SO(4), heating/centrifugation), EDTA extraction was found to be the most effective. The contents of the major components of EPS from R. acidophila, i.e., carbohydrate, protein and nucleic acid, were 6.5, 58.4 and 5.4 mg g(-1) dry cells, respectively. The optimum extraction time was 1-3 h and the EDTA dosage was approximately 2.8 g g(-1) dry cells. Under these conditions, no cell lysis was observed. The EPS content and the percentage of the three main components were greatly dependent on the extraction method. The intensity of absorption peaks for photosynthetic pigments in the UV-visible spectrum of bacteria remained unchanged prior to and after EDTA extraction; and no pigment peaks appeared in the EPS spectrum. This suggests that few cells were destroyed and lysis did not occur. UV-visible spectrum analysis, an easy and rapid technique, could be used to monitor cell lysis during EPS extraction from R. acidophila.

Yapsins are a family of aspartyl proteases required for cell wall integrity in Saccharomyces cerevisiae.

Abstract: The yeast cell wall is a crucial extracellular organelle that protects the cell from lysis during environmental stress and morphogenesis. Here, we demonstrate that the yapsin family of five glycosylphosphatidylinositol-linked aspartyl proteases is required for cell wall integrity in Saccharomyces cerevisiae. Yapsin null mutants show hypersensitivity to cell wall perturbation, and both the yps1Δ2Δ mutant and the quintuple yapsin mutant (5ypsΔ) undergo osmoremedial cell lysis at 37°C. The cell walls of both 5ypsΔ and yps1Δ2Δ mutants have decreased amounts of 1,3- and 1,6-β-glucan. Although there is decreased incorporation of both 1,3- and 1,6-β-glucan in the 5ypsΔ mutant in vivo, in vitro specific activity of both 1,3- and 1,6-β-glucan synthesis is similar to wild type, indicating that the yapsins affect processes downstream of glucan synthesis and that the yapsins may be involved in the incorporation or retention of cell wall glucan. Presumably as a response to the significant alterations in cell wall composition, the cell wall integrity mitogen-activated kinase signaling cascade (PKC1-MPK pathway) is basally active in 5ypsΔ. YPS1 expression is induced during cell wall stress and remodeling in a PKC1-MPK1-dependent manner, indicating that Yps1p is a direct, and important, output of the cell wall integrity response. The Candida albicans (SAP9) and Candida glabrata (CgYPS1) homologues of YPS1 complement the phenotypes of the yps1Δ mutant. Taken together, these data indicate that the yapsins play an important role in glucan homeostasis in S. cerevisiae and that yapsin homologues may play a similar role in the pathogenic yeasts C. albicans and C. glabrata.

Whole blood fixation and permeabilization protocol with red blood cell lysis for flow cytometry of intracellular phosphorylated epitopes in leukocyte subpopulations

Abstract: Background Previous studies of intracellular expression of phospho-epitopes in human leukocytes using flow cytometry have used erythrocyte removal or lysis before fixation. Because many of the phospho-epitopes of interest are part of signaling networks that respond to the environment and turn over rapidly, the interval and manipulations used to eliminate erythrocytes from samples have the potential to introduce artifacts. We report a procedure to fix samples containing red blood cells with formaldehyde and then remove erythrocytes by lysis. Detection of phospho-Thr 202/Tyr 204-p44/42 extracellular-regulated kinase (ERK) after phorbol ester acetate (PMA) stimulation was used as a model to measure phospho-epitopes in leukocyte populations in whole blood. Methods Normal blood samples were activated with PMA followed by formaldehyde fixation and subsequent treatments with detergents and protein denaturants. The effects of each treatment were monitored by light scatter, selected CD expression intensity, and phosphorylated ERK (pERK) expression. Results Red cells could be lysed using 0.1% Triton X-100 after brief fixation of whole blood with 2% or 4% formaldehyde. Light scatter improved as a function of formaldehyde concentration and inversely with MeOH concentration. CD3 signal intensity increased when MeOH concentration was reduced. The ratio of pERK immunofluorescence in PMA-stimulated versus nonstimulated (control) samples was highest with high MeOH (90%) and lowest without MeOH treatment. This pattern is consistent with epitope unmasking by alcohol. The pERK epitope could also be unmasked by treatment with high salt, urea, acid, or heat, but none of these produced the level of unmasking of MeOH and each of these was associated with degradation of light scatter and CD3 staining intensity. The final procedure employed 4% formaldehyde, 0.1% Triton X-100, followed by 50% methanol denaturation. Samples prepared in this way demonstrated good preservation of light scatter and surface immunophenotypic patterns, similar to those obtained using a commercial whole blood/red blood cell lysing system (Q-Prep) and an acceptable PMA-stimulated pERK signal (essentially 100% of CD3+ cells that are pERK positive). Conclusions Brief fixation of whole blood in 4% formaldehyde followed by treatment with Triton X-100 results in erythrocyte lysis and leukocyte light scatter and immunophenotypic features equivalent to those of other commercial lysis reagents. Intracellular pERK staining is significantly improved by treatment with methanol, but levels of MeOH above 50% degrade light scatter and CD3 expression. This protocol (formaldehyde/Triton X-100/MeOH) circumvents potential artifactual changes in phospho-epitopes due to removal of erythrocytes or erythrocyte lysis followed by fixation, and results in a pERK signal that resolves positive from negative cell populations. © 2005 Wiley-Liss, Inc.

Choline-Binding Protein D (CbpD) in Streptococcus pneumoniae Is Essential for Competence-Induced Cell Lysis

Abstract: Streptococcus pneumoniae is an important human pathogen that is able to take up naked DNA from the environment by a quorum-sensing-regulated process called natural genetic transformation. This property enables members of this bacterial species to efficiently acquire new properties that may increase their ability to survive and multiply in the human host. We have previously reported that induction of the competent state in a liquid culture of Streptococcus pneumoniae triggers lysis of a subfraction of the bacterial population resulting in release of DNA. We have also proposed that such competence-induced DNA release is an integral part of natural genetic transformation that has evolved to increase the efficiency of gene transfer between pneumococci. In the present work, we have further elucidated the mechanism behind competence-induced cell lysis by identifying a putative murein hydrolase, choline-binding protein D (CbpD), as a key component of this process. By using real-time PCR to estimate the amount of extracellular DNA in competent relative to noncompetent cultures, we were able to show that competence-induced cell lysis and DNA release are strongly attenuated in a cbpD mutant. Ectopic expression of CbpD in the presence or absence of other competence proteins revealed that CbpD is essentially unable to cause cell lysis on its own but depends on at least one additional protein expressed during competence.

Cell wall modifications during osmotic stress in Lactobacillus casei

Abstract: Aims: To study the modification of the cell wall of Lactobacillus casei ATCC 393 grown in high salt conditions. Methods and Results: Differences in the overall structure of cell wall between growth in high salt (MRS + 1 mol l−1 NaCl; N condition) and control (MRS; C condition) conditions were determined by transmission electronic microscopy and analytical procedures. Lactobacillus casei cells grown in N condition were significantly larger than cells grown under unstressed C condition. Increased sensitivity to mutanolysin and antibiotics with target in the cell wall was observed in N condition. Purified cell wall also showed the increased sensitivity to lysis by mutanolysin. Analysis of peptidoglycan (PG) from stressed cells showed that modification was at the structural level in accordance with a decreased PG cross-link involving penicillin-binding proteins (PBP). Nine PBP were first described in this species and these proteins were expressed in low percentages or presented a modified pattern of saturation with penicillin G (Pen G) during growth in high salt. Three of the essential PBP were fully saturated in N condition at lower Pen G concentrations than in C condition, suggesting differences in functionality in vivo. Conclusions: The results show that growth in high salt modified the structural properties of the cell wall. Significance and Impact of Study: Advances in understanding the adaptation to high osmolarity, in particular those involving sensitivity to lysis of lactic acid bacteria.

Cell stimulus and lysis in a microfluidic device with segmented gas-liquid flow.

Abstract: We describe a microfluidic device with rapid stimulus and lysis of mammalian cells for resolving fast transient responses in cell signaling networks The device uses segmented gas−liquid flow to enhance mixing and has integrated thermoelectric heaters and coolers to control the temperature during cell stimulus and lysis Potential negative effects of segmented flow on cell responses are investigated in three different cell types, with no morphological changes and no activation of the cell stress-sensitive mitogen activated protein kinases observed Jurkat E6-1 cells are stimulated in the device using α-CD3, and the resulting activations of ERK and JNK are presented for different time points Stimulation of cells performed on chip results in pathway activation identical to that of conventionally treated cells under the same conditions

On-chip cell lysis by local hydroxide generation

Abstract: We present a novel method for on-chip cell lysis based on local hydroxide electro-generation. Hydroxide ions porate the cell membrane, leading to cell lysis. After lysis occurs, hydrogen ions, also generated on chip, react with excess hydroxide, creating a neutral pH lysate and eliminating the need for a wash step. Three different cell types are shown to be effectively lysed by this method: red blood cells, HeLa (human tumor line) and Chinese Hamster Ovary (CHO) cell lines. The release of cytoplasmic molecules from HeLa and CHO cells is demonstrated by monitoring the escape of a membrane impermeant dye from the cytoplasm. In the vicinity of the cathode, the hydroxide concentration is predicted by finite element simulations and shown to fit the lysis rates at different distances from the generating cathode. For flow-through experiments, a second device integrating a mechanical filter with hydroxide generation is fabricated and tested. The purpose of the filter is to trap whole cells and only allow lysate to pass through. The flow rate dependence of hydroxide concentration at the lysis filter is modeled and lysis efficiency is experimentally determined to be proportional to the hydroxide concentration for flow rates from 15 to 30 µl min−1.

Identification and relative quantification of membrane proteins by surface biotinylation and two-dimensional peptide mapping.

Abstract: Membrane proteins play a central role in biological processes, but their separation and quantification using two-dimensional gel electrophoresis is often limited by their poor solubility and relatively low abundance. We now present a method for the simultaneous recovery, separation, identification, and relative quantification of membrane proteins, following their selective covalent modification with a cleavable biotin derivative. After cell lysis, biotinylated proteins are purified on streptavidin-coated resin and proteolytically digested. The resulting peptides are analyzed by high-pressure liquid chromatography and mass spectrometry, thus yielding a two-dimensional peptide map. Matrix assisted laser desorption/ionization-time of flight signal intensity of peptides, in the presence of internal standards, is used to quantify the relative abundance of membrane proteins from cells treated in different experimental conditions. As experimental examples, we present (i) an analysis of a BSA-spiked human embryonic kidney membrane protein extract, and (ii) an analysis of membrane proteins of human umbilical vein endothelial cells cultured in normoxic and hypoxic conditions. This last study allowed the recovery of the vascular endothelial-cadherin/actin/catenin complex, revealing an increased accumulation of beta-catenin at 2% O(2) concentration.

Proteome analysis of membrane and cell wall associated proteins from Staphylococcus aureus.

Abstract: Pathogenesis of Staphylococcus aureus, an opportunistic human pathogen, is complex and involves many virulence factors including an array of surface proteins (adhesins) that promote bacterial interactions with extracellular matrix components A better understanding of these interactions can be achieved by studying the expression of membrane and cell wall associated proteins using a proteome analysis approach To accomplish this, our goal here was to construct a reference map of membrane and cell wall associated proteins for S aureus Various lytic and solubilization methods have been tested to identify a suitable methodology for detection of these proteins in two-dimensional electrophoresis (2DE) Results demonstrate that cell lysis with lysostaphin, which lyses staphylococcal peptidoglycan, followed by solubilization with urea, thiourea, amidosulfobetaine 14 (ASB 14) and dithiothreitol (DTT) is an effective method, yielding a sample comprising proteins of wide molecular ranges and isoelectric points with minimum contamination from cytosolic proteins Mass spectrometric analysis was employed to identify the membrane and cell surface proteins present in the sample and consequently an initial proteomic map of membrane and cell wall associated proteins for S aureus is presented

Microfluidic sonicator for real-time disruption of eukaryotic cells and bacterial spores for DNA analysis.

Abstract: Biologic agent screening is a three-step process: lysis of host cell membranes or walls to release their DNA, polymerase chain reaction to amplify the genetic material and screening for distinguishing genetic signatures. Macrofluidic devices commonly use sonication as a lysis method. Here, we present a piezoelectric microfluidic minisonicator and test its performance. Eukaryotic human leukemia HL-60 cells and Bacillus subtilis bacterial spores were lysed as they passed through a microfluidic channel at 50 microL/min and 5 microL/min, respectively, in the absence of any chemical denaturants, enzymes or microparticles. We used fluorescence-activated cell sorting and hematocytometry to measure 80% lysis of HL-60 cells after 3 s of sonication. Real-time polymerase chain reaction indicated 50% lysis of B. subtilis spores with 30 s of sonication. Advantages of the minisonicator over macrofluidic implementations include a small sample volume (2.5 microL), reduced energy consumption and compatibility with other microfluidic blocks. These features make this device an attractive option for "lab-on-a-chip" and portable applications.

A diagnostic system for carrying out a nucleic acid sequence amplification and detection process

Abstract: An integrated lab-on-a-chip diagnostic system for carrying out a sample preparation process on a fluid sample containing cells and/or particles, the system comprising: (a) an inlet for a fluid sample; (b) a lysis unit for lysis of cells and/or particles contained in the fluid sample; (c) a nucleic acid extraction unit for extraction of nucleic acids from the cells and/or particles contained in the fluid sample; (d) a reservoir containing a lysis fluid; (e) a reservoir containing an eluent for removing nucleic acids collected in the nucleic acid extraction unit; wherein the sample inlet is in fluid communication with the lysis unit, an optional valve being present to control the flow of fluid therebetween; wherein the lysis unit is in fluid communication with the nucleic acid extraction unit, an optional valve being present to control the flow of fluid therebetween; wherein the reservoir containing the lysis fluid is in fluid communication with the lysis unit, an optional valve being present to control the flow of fluid therebetween; and wherein the reservoir containing the eluent is in fluid communication with the nucleic acid extraction unit, an optional valve being present to control the flow of fluid therebetween.

Null mutation of myeloperoxidase in mice prevents mechanical activation of neutrophil lysis of muscle cell membranes in vitro and in vivo

Abstract: Membrane lysis is a common and early defect in muscles experiencing acute injuries or inflammation. Although increased mechanical loading of muscles can induce inflammation and membrane lysis, whether mechanical loads applied to muscle can promote the activation and cytolytic capacity of inflammatory cells and thereby increase muscle damage is unknown. We tested whether mechanical loads applied to mouse muscle cells in vitro can increase membrane lysis, and whether neutrophil-mediated lysis of muscle cells is promoted by mechanical loads applied in vitro and in vivo. Cyclic loads applied to muscle cells for 24 h in vitro produced little muscle cell lysis. Similarly, the addition of neutrophils to muscle cell cultures in the presence of superoxide dismutase (SOD) produced little muscle cell lysis. However, when cyclic mechanical loads were applied to neutrophil–muscle co-cultures in the presence of SOD, there was a synergistic effect on muscle cell lysis, suggesting that mechanical loading activates neutrophil cytotoxicity. However, application of mechanical loads to co-cultures of muscle cells and neutrophils that are null mutants for myeloperoxidase (MPO) showed no mechanical activation of neutrophil cytotoxicity. This indicates that loading promotes neutrophil cytotoxicity via MPO. Activity assays confirmed that mechanical loading of neutrophil–muscle co-cultures significantly increased MPO activity. We further tested whether muscle membrane lysis in vivo was mediated by neutrophils when muscle was subjected to modified loading by using a mouse model of muscle reloading following a period of unloading. We observed that MPO −/− soleus muscles showed a significant 52% reduction in membrane lysis compared to wild-type mice, although the mutation did not decrease inflammatory cell extravasation. Together, these in vitro and in vivo findings show that mechanical loading activates neutrophil-mediated lysis of muscle cells through an MPO-dependent pathway.

Pre-lysis washing improves DNA extraction from a forest soil

Abstract: A pre-lysis buffer washing procedure was introduced to DNA extraction from a forest soil with high organic matter and iron oxide contents. Sodium phosphate of 0.1 M (pH 7.5) was used as a buffer to wash soil samples when subsequent lysis buffer was phosphate, and 20 mM EDTA (pH 7.5) was used when subsequent lysis buffer included EDTA. Initial experiments were not successful because the DNA extracts could not be amplified by polymerase chain reaction (PCR). The consideration of introducing a pre-lysis washing procedure was based on the idea that the washing should promote soil dispersion and homogeneity, decrease DNA adsorption by soil components (e.g. iron oxides), and remove covalent cations and those easily-dissolving organic compounds from the soil samples. Results revealed that humic substance content decreased by 31%, but DNA yield increased by 24% in the DNA extracts of the pre-lysis washing procedures, compared to the non-washing procedures. DNA extracted by the pre-washing procedure needed less purification for subsequent 18S and 16S rDNA PCR amplifications. It was recommended that the pre-lysis buffer washing should be used for DNA extraction from those difficult environmental samples, such as the forest soil with high contents of organic matter and iron oxides.

Reversible Adsorption and Nonreversible Insertion of Escherichia coli a-Hemolysin into Lipid Bilayers

Abstract: a-Hemolysin is an extracellular protein toxin (107 kDa) produced by some pathogenic strains of Escherichia coli. Although stable in aqueous medium, it can bind to lipid bilayers and produce membrane disruption in model and cell membranes. Previous studies had shown that toxin binding to the bilayer did not always lead to membrane lysis. In this paper, we find that a-hemolysin may bind the membranes in at least two ways, a reversible adsorption and an irreversible insertion. Reversibility is detected by the ability of liposome-bound toxin to induce hemolysis of added horse erythrocytes; insertion is accompanied by an increase in the protein intrinsic fluorescence. Toxin insertion does not necessarily lead to membrane lysis. Studies of a-hemolysin insertion into bilayers formed from a variety of single phospholipids, or binary mixtures of phospholipids, or of phospholipid and cholesterol, reveal that irreversible insertion is favored by fluid over gel states, by low over high cholesterol concentrations, by disordered liquid phases over gel or ordered liquid phases, and by gel over ordered liquid phases. These results are relevant to the mechanism of action of a-hemolysin and provide new insights into the membrane insertion of large proteins.

Scalable recovery of plasmid DNA based on aqueous two-phase separation.

Abstract: Future developments in gene therapy and DNA vaccination depend on cost-effective large-scale production of pharmaceutical-grade pDNA (plasmid DNA). Given the large amount of impurities present in the feedstock, purification processes that have high specificity and capacity at a moderate cost are required. In the present study, we describe a non-chromatographic procedure based on aqueous two-phase extraction allowing a fast and simply scalable capture step. PEG [poly(ethylene glycol)] in combination with potassium citrate or potassium phosphate was tested as phase component for extraction. By increasing either PEG or salt concentration, the partitioning of nucleic acids changed from bottom to top phase. Phase systems with a composition of 15% PEG 800 and 20% potassium phosphate at pH 7.0 showed a strong partitioning of pDNA to the bottom phase, linked to a clear decrease in open circular pDNA, while proteins, genomic DNA and RNA remain at the top or at the interphase. A great advantage of the current process is that the complete procedure of lysis, precipitation, clarification and extraction can be performed in a single vessel. The number of denatured and sheared genomic DNAs in a spiking experiment was found to be depleted by more than 99%.

Development of a novel adenovirus purification process utilizing selective precipitation of cellular DNA.

Abstract: The use of recombinant adenoviral vectors for vaccination and gene therapy requires the development of purification processes that are cost-effective, scalable, and capable of robust host cell DNA clearance. An adenovirus purification process was developed which incorporates selective precipitation of host cell DNA, enabling a reduction in the use of costly nucleases and chromatographic resins while substantially improving DNA and protein clearance capabilities. In this work, three cationic detergents were evaluated for their capacity to selectively precipitate DNA from adenovirus-containing cell lysate. Parameters including pH, sodium chloride concentration, nonionic surfactant concentration, and cell density were investigated during development of the precipitation step. In a novel application, the cationic detergent domiphen bromide was found to have superior selectivity for host cell DNA. In addition, domiphen bromide-induced precipitation of adenovirus was shown to be reversible, which reduces the importance of mixing. Precipitation of DNA in the cell lysate coupled with primary clarification resulted in 3 logs of DNA clearance and improved impurity clearance in the subsequent ultrafiltration step. As a result, nuclease treatment and/or anion exchange chromatography can be eliminated, or included exclusively to improve process robustness.

Rapid, single-tube method for quantitative preparation and analysis of RNA and DNA in samples as small as one cell

Abstract: Current methods for accurate quantification of nucleic acids typically begin with a template preparation step in which DNA and/or RNA are freed of bound proteins and are then purified. Isolation of RNA is particularly challenging because this molecule is sensitive to elevated temperatures and is degraded by RNases, which therefore have to be immediately inactivated upon cell lysis. Many protocols for nucleic acids purification, reverse transcription of RNA and/or amplification of DNA require repeated transfers from tube to tube and other manipulations during which materials may be lost. This paper introduces a novel and highly reliable single-tube method for rapid cell lysis, followed by quantitative preparation and analysis of both RNA and/or DNA molecules in small samples. In contrast to previous approaches, this procedure allows all steps to be carried out by sequential dilution in a single tube, without chemical extraction or binding to a matrix. We demonstrate the utility of this method by quantification of four genes, Xist, Sry and the two heat-inducible hsp70i (hsp70.1 and hsp70.3), as well as their RNA transcripts in single mouse embryos and in isolated blastomeres. This method virtually eliminates losses of nucleic acids and is sensitive and accurate down to single molecules.

Cell wall remodeling at the fission yeast cell division site requires the Rho-GEF Rgf3p.

Abstract: Cytokinesis in Schizosaccharomyces pombe is accompanied by several stages of cell wall remodeling at the division site. Coincident with actomyosin ring constriction, primary and secondary septa are deposited and then the primary septum is degraded to release daughter cells from one another. These steps require the activities of glucan synthases and glucanases, respectively, which must be coordinated with one another to prevent cell lysis. The lad1-1 mutation undergoes cell lysis specifically at cell division owing to the absence of the Rgf3p Rho1-guanine nucleotide exchange factor (GEF) at the division site. Electron microscopic analysis indicates that lysis occurs only as the primary septum begins to be degraded. Overproduction of either Rho1p or the previously uncharacterized Rab-GTPase-activating protein (GAP) involved in secretion, Gyp10p, suppresses lad1-1 lethality. Rgf3p is periodically produced in an Ace2p-dependent manner and localizes to the medial region of the cell early in mitosis, a pattern of expression distinct from the highly related Rho-GEF, Rgf1p. Although rgf1+ is not an essential gene, it is synthetically lethal with rgf2-deleted cells whereas no negative genetic interactions were detected between rgf2-deleted cells and lad1-1. Our data suggest that the three closely related fission yeast Rho-GEF molecules perform two distinct essential functions. Rgf3p appears necessary to stimulate Rho1p-mediated activation of a glucan synthase crucial after septation for proper new cell-end formation.

The Holin protein of bacteriophage PRD1 forms a pore for small-molecule and endolysin translocation.

Abstract: PRD1 is a bacteriophage with an icosahedral outer protein layer surrounding the viral membrane, which encloses the linear double-stranded DNA genome. PRD1 infects gram-negative cells harboring a conjugative IncP plasmid. Here we studied the lytic functions of PRD1. Using infected cells and plasmid-borne lysis genes, we demonstrated that a two-component lysis system (holin-endolysin) operates to release progeny phage particles from the host cell. Monitoring of ion fluxes and the ATP content of the infected cells allowed us to build a model of the sequence of lysis-related physiological changes. A decrease in the intracellular level of ATP is the earliest indicator of cell lysis, followed by the leakage of K+ from the cytosol approximately 20 min prior to the decrease in culture turbidity. However, the K+ efflux does not immediately lead to the depolarization of the cytoplasmic membrane or leakage of the intracellular ATP. These effects are observed only ∼5 to 10 min prior to cell lysis. Similar results were obtained using cells expressing the holin and endolysin genes from plasmids.