Showing papers on "Lysis published in 2008"

Insights into the Mode of Action of Chitosan as an Antibacterial Compound

Abstract: Chitosan is a polysaccharide biopolymer that combines a unique set of versatile physicochemical and biological characteristics which allow for a wide range of applications. Although its antimicrobial activity is well documented, its mode of action has hitherto remained only vaguely defined. In this work we investigated the antimicrobial mode of action of chitosan using a combination of approaches, including in vitro assays, killing kinetics, cellular leakage measurements, membrane potential estimations, and electron microscopy, in addition to transcriptional response analysis. Chitosan, whose antimicrobial activity was influenced by several factors, exhibited a dose-dependent growth-inhibitory effect. A simultaneous permeabilization of the cell membrane to small cellular components, coupled to a significant membrane depolarization, was detected. A concomitant interference with cell wall biosynthesis was not observed. Chitosan treatment of Staphylococcus simulans 22 cells did not give rise to cell wall lysis; the cell membrane also remained intact. Analysis of transcriptional response data revealed that chitosan treatment leads to multiple changes in the expression profiles of Staphylococcus aureus SG511 genes involved in the regulation of stress and autolysis, as well as genes associated with energy metabolism. Finally, a possible mechanism for chitosan's activity is postulated. Although we contend that there might not be a single classical target that would explain chitosan's antimicrobial action, we speculate that binding of chitosan to teichoic acids, coupled with a potential extraction of membrane lipids (predominantly lipoteichoic acid) results in a sequence of events, ultimately leading to bacterial death.

Extracellular DNA Chelates Cations and Induces Antibiotic Resistance in Pseudomonas aeruginosa Biofilms

Abstract: Biofilms are surface-adhered bacterial communities encased in an extracellular matrix composed of DNA, bacterial polysaccharides and proteins, which are up to 1000-fold more antibiotic resistant than planktonic cultures. To date, extracellular DNA has been shown to function as a structural support to maintain Pseudomonas aeruginosa biofilm architecture. Here we show that DNA is a multifaceted component of P. aeruginosa biofilms. At physiologically relevant concentrations, extracellular DNA has antimicrobial activity, causing cell lysis by chelating cations that stabilize lipopolysaccharide (LPS) and the outer membrane (OM). DNA-mediated killing occurred within minutes, as a result of perturbation of both the outer and inner membrane (IM) and the release of cytoplasmic contents, including genomic DNA. Sub-inhibitory concentrations of DNA created a cation-limited environment that resulted in induction of the PhoPQ- and PmrAB-regulated cationic antimicrobial peptide resistance operon PA3552-PA3559 in P. aeruginosa. Furthermore, DNA-induced expression of this operon resulted in up to 2560-fold increased resistance to cationic antimicrobial peptides and 640-fold increased resistance to aminoglycosides, but had no effect on beta-lactam and fluoroquinolone resistance. Thus, the presence of extracellular DNA in the biofilm matrix contributes to cation gradients, genomic DNA release and inducible antibiotic resistance. DNA-rich environments, including biofilms and other infection sites like the CF lung, are likely the in vivo environments where extracellular pathogens such as P. aeruginosa encounter cation limitation.

Molecular Control of Bacterial Death and Lysis

Abstract: Summary: Although the phenomenon of bacterial cell death and lysis has been studied for over 100 years, the contribution of these important processes to bacterial physiology and development has only recently been recognized. Contemporary study of cell death and lysis in a number of different bacteria has revealed that these processes, once thought of as being passive and unregulated, are actually governed by highly complex regulatory systems. An emerging paradigm in this field suggests that, analogous to programmed cell death in eukaryotes, regulated cell death and lysis in bacteria play an important role in both developmental processes, such as competence and biofilm development, and the elimination of damaged cells, such as those irreversibly injured by environmental or antibiotic stress. Further study in this exciting field of bacterial research may provide new insight into the potential evolutionary link between control of cell death in bacteria and programmed cell death (apoptosis) in eukaryotes.

Current techniques for single-cell lysis

Abstract: Owing to the small quantities of analytes and small volumes involved in single-cell analysis techniques, manipulation strategies must be chosen carefully. The lysis of single cells for downstream chemical analysis in capillaries and lab-on-a-chip devices can be achieved by optical, acoustic, mechanical, electrical or chemical means, each having their respective strengths and weaknesses. Selection of the most appropriate lysis method will depend on the particulars of the downstream cell lysate processing. Ultrafast lysis techniques such as the use of highly focused laser pulses or pulses of high voltage are suitable for applications requiring high temporal resolution. Other factors, such as whether the cells are adherent or in suspension and whether the proteins to be collected are desired to be native or denatured, will determine the suitability of detergent-based lysis methods. Therefore, careful selection of the proper lysis technique is essential for gathering accurate data from single cells.

Bacteriophage Adsorption Rate and Optimal Lysis Time

Abstract: The first step of bacteriophage (phage) infection is the attachment of the phage virion onto a susceptible host cell. This adsorption process is usually described by mass-action kinetics, which implicitly assume an equal influence of host density and adsorption rate on the adsorption process. Therefore, an environment with high host density can be considered as equivalent to a phage endowed with a high adsorption rate, and vice versa. On the basis of this assumption, the effect of adsorption rate on the evolution of phage optimal lysis time can be reinterpreted from previous optimality models on the evolution of optimal lysis time. That is, phage strains with a higher adsorption rate would have a shorter optimal lysis time and vice versa. Isogenic phage λ-strains with different combinations of six different lysis times (ranging from 29.3 to 68 min), two adsorption rates (9.9 × 10−9 and 1.3 × 10−9 phage−1 cell−1 ml−1 min−1), and two markers (resulting in “blue” or “white” plaques) were constructed. Various pairwise competitions among these strains were conducted to test the model prediction. As predicted by the reinterpreted model, the results showed that the optimal lysis time is shorter for phage strains with a high adsorption rate and vice versa. Competition between high- and low-adsorption strains also showed that, under current conditions and phenotype configurations, the adsorption rate has a much larger impact on phage relative fitness than the lysis time.

Cell division in Escherichia coli cultures monitored at single cell resolution

Abstract: A fundamental characteristic of cells is the ability to divide. To date, most parameters of bacterial cultures, including cell division, have been measured as cell population averages, assuming that all bacteria divide at a uniform rate. We monitored the division of individual cells in Escherichia coli cultures during different growth phases. Our experiments are based on the dilution of green fluorescent protein (GFP) upon cell division, monitored by flow cytometry. The results show that the vast majority of E. coli cells in exponentially growing cultures divided uniformly. In cultures that had been in stationary phase up to four days, no cell division was observed. However, upon dilution of stationary phase culture into fresh medium, two subpopulations of cells emerged: one that started dividing and another that did not. These populations were detectable by GFP dilution and displayed different side scatter parameters in flow cytometry. Further analysis showed that bacteria in the non-growing subpopulation were not dead, neither was the difference in growth capacity reducible to differences in stationary phase-specific gene expression since we observed uniform expression of several stress-related promoters. The presence of non-growing persisters, temporarily dormant bacteria that are tolerant to antibiotics, has previously been described within growing bacterial populations. Using the GFP dilution method combined with cell sorting, we showed that ampicillin lyses growing bacteria while non-growing bacteria retain viability and that some of them restart growth after the ampicillin is removed. Thus, our method enables persisters to be monitored even in liquid cultures of wild type strains in which persister formation has low frequency. In principle, the approaches developed here could be used to detect differences in cell division in response to different environmental conditions and in cultures of unicellular organisms other than E. coli.

Daptomycin Exerts Bactericidal Activity without Lysis of Staphylococcus aureus

Abstract: The ability of daptomycin to produce bactericidal activity against Staphylococcus aureus while causing negligible cell lysis has been demonstrated using electron microscopy and the membrane integrity probes calcein and ToPro3. The formation of aberrant septa on the cell wall, suggestive of impairment of the cell division machinery, was also observed.

Effect of shear stress on intrinsic CHO culture state and glycosylation of recombinant tissue-type plasminogen activator protein

Abstract: Shear stress in suspension culture was investigated as a possible manipulative parameter for the control of glycosylation of the recombinant tissue-type plasminogen activator protein (r-tPA) produced by recombinant Chinese hamster ovary (CHO) cell culture, grown in protein-free media. Resulting fractions of partially glycosylated, Type II, and fully glycosylated, Type I, r-tPA protein were monitored as a direct function of the shear characteristics of the culture environment. The shear-induced response of CHO culture to levels of low shear stress, where exponential growth was not obtained, and to higher levels of shear stress, which resulted in extensive cell death, were examined through manipulation of the bioreactor stirring velocity. Both apparent and intrinsic cell growth, metabolite consumption, byproduct and r-tPA production, and r-tPA glycosylation, from a variable site-occupancy standpoint, were monitored throughout. Kinetic analyses revealed a shear-stress-induced alteration of cellular homeostasis resulting in a nonlinear dependency of metabolic yield coefficients and an intrinsic cell lysis kinetic constant on shear stress. Damaging levels of shear stress were used to investigate the shear dependence of cell death and lysis, as well as the effects on the intrinsic growth rate of the culture. Kinetic models were also developed on the basis of the intrinsic state of the culture and compared to traditional models. Total r-tPA production was maximized under moderate shear conditions, as was the viable CHO cell density of the culture. However, Type II r-tPA production and the fraction of Type II glycoform production ratio was maximized under damaging levels of shear stress. Analyses of biomass production yield coefficients coupled with a plug-flow reactor model of glycan addition in the endoplasmic reticulum (ER) were used to propose an overall mechanism of decreased r-tPA protein site-occupancy glycosylation with increasing shear stress. Decreased residence time of r-tPA in the ER as a result of increased protein synthesis related to shear protection mechanisms is proposed to limit contact of site Asn184 with the membrane-bound oligosaccharyltransferase enzyme in the ER.

The final step in the phage infection cycle: the Rz and Rz1 lysis proteins link the inner and outer membranes

Abstract: Bacteriophage λ has four adjacent genes - S, R, Rz and Rz1 - dedicated to host cell lysis. While S, encoding the holin and antiholin, and R, encoding the endolysin, have been intensively studied, the products of Rz and Rz1 have not been characterized at either the structural or functional levels. Rz1 is an outer membrane lipoprotein and our results indicate that Rz is a type II signal anchor protein. Here we present evidence that an Rz-Rz1 complex that spans the periplasm carries out the final step in the process of host lysis. These results are discussed in terms of a model where endolysin-mediated degradation of the cell wall is a prerequisite for conformational changes in the Rz-Rz1 complex leading to the juxtaposition and fusion of the IM and OM. Fusion of the two membranes removes the last physical barrier to efficient release of progeny virions.

Rapid, continuous purification of proteins in a microfluidic device using genetically-engineered partition tags

Abstract: High-throughput screening assays of native and recombinant proteins are increasingly crucial in life science research, including fields such as drug screening and enzyme engineering. These assays are typically highly parallel, and require minute amounts of purified protein per assay. To address this need, we have developed a rapid, automated microscale process for isolating specific proteins from sub-microlitre volumes of E. Colicell lysate. Recombinant proteins are genetically tagged to drive partitioning into the PEG-rich phase of a flowing aqueous two-phase system, which removes ∼85% of contaminating proteins, as well as unwanted nucleic acids and cell debris, on a simple microfluidic device. Inclusion of the genetic tag roughly triples recovery of the autofluorescent protein AcGFP1, and also significantly improves recovery of the enzyme glutathione S-transferase (GST), from nearly zero recovery for the wild-type enzyme, up to 40% with genetic tagging. The extraction process operates continuously, with only a single step from cell lysate to purified protein, and does not require expensive affinity reagents or troublesome chromatographic steps. The two-phase system is mild and does not disrupt protein function, as evidenced by recovery of active enzymes and functional fluorescent protein from our microfluidic process. The microfluidic aqueous two-phase extraction forms the core component of an integrated lab-on-a-chip device comprising cell culture, lysis, purification and analysis on a single device.

Extracellular plasma RNA from colon cancer patients is confined in a vesicle-like structure and is mRNA-enriched

Abstract: Little is yet known about the origin and protective mechanism of free nucleic acids in plasma. We investigated the possibility of these free nucleic acids being particle associated. Plasma samples from colon cancer patients and cell culture media were subjected to various antibody incubations, ultracentrifugation, and RNA extraction protocols for total RNA, epithelial RNA, and mRNA. Flow cytometry using a Ber-EP4 antibody and confocal laser microscopy after staining with propidium iodide were also performed. mRNA levels of the LISCH7 and SDHA genes were determined in cells and in culture media. Ber-EP4 antibody and polystyrene beads coated with oligo dT sequences were employed. We observed that, after incubation, total RNA and mRNA were always detected after membrane digestion, and that epithelial RNA was detected before this procedure. In ultracentrifugation, mRNA was caught in the supernatant only if a former lysis mediated or in the pellet if there was no previous digestion. Flow cytometry determinations showed that antibody-coated microbeads keep acellular structures bearing epithelial antigens apart. Confocal laser microscopy made 1- to 2-μm-diameter particles perceptible in the vicinity of magnetic polystyrene beads. Relevant differences were observed between mRNA of cells and culture media, as there was a considerable difference in LISCH7 mRNA levels between HT29 and IMR90 cell co-cultures and their culture media. Our results support the view that extracellular RNA found in plasma from cancer patients circulates in association with or is protected in a multiparticle complex, and that an active release mechanism by tumor cells may be a possible origin.

Extracellular recombinant protein production from an Escherichia coli lpp deletion mutant.

Abstract: E. coli is one of the most commonly used host strains for recombinant protein production. However, recombinant proteins are usually found intracellularly, in either cytoplasm or periplasmic space. Inadequate secretion to the extracellular environment is one of its limitations. This study addresses the outer membrane barrier for the translocation of recombinant protein directed to the periplasmic space. Specifically, using recombinant maltose binding protein (MalE), xylanase, and cellulase as model proteins, we investigated whether the lpp deletion could render the outer membrane permeable enough to allow extracellular protein production. In each case, significantly higher excretion of recombinant protein was observed with the lpp deletion mutant. Up to 90% of the recombinant xylanase activity and 70% of recombinant cellulase activity were found in the culture medium with the deletion mutant, whereas only 40-50% of the xylanase and cellulase activities were extracellular for the control strain. Despite the weakened outer membrane in the mutant strain, cell lysis did not occur, and increased excretion of periplasmic protein was not due to cell lysis. The lpp deletion is a simple method to generate an E. coli strain to effect significant extracellular protein production. The phenotype of extracellular protein production without cell lysis is useful in many biotechnological applications, such as bioremediation and plant biomass conversion.

Biophysical Response to Pulsed Laser Microbeam-Induced Cell Lysis and Molecular Delivery

Abstract: Cell lysis and molecular delivery in confluent monolayers of PtK(2) cells are achieved by the delivery of 6 ns, lambda = 532 nm laser pulses via a 40x, 0.8 NA microscope objective. With increasing distance from the point of laser focus we find regions of (a) immediate cell lysis; (b) necrotic cells that detach during the fluorescence assays; (c) permeabilized cells sufficient to facilitate the uptake of small (3 kDa) FITC-conjugated Dextran molecules in viable cells; and (d) unaffected, viable cells. The spatial extent of cell lysis, cell detachment, and molecular delivery increased with laser pulse energy. Hydrodynamic analysis from time-resolved imaging studies reveal that the maximum wall shear stress associated with the pulsed laser microbeam-induced cavitation bubble expansion governs the location and spatial extent of each of these regions independent of laser pulse energy. Specifically, cells exposed to maximum wall shear stresses tau(w, max) > 190 +/- 20 kPa are immediately lysed while cells exposed to tau(w, max) > 18 +/- 2 kPa are necrotic and subsequently detach. Cells exposed to tau(w, max) in the range 8-18 kPa are viable and successfully optoporated with 3 kDa Dextran molecules. Cells exposed to tau(w, max) < 8 +/- 1 kPa remain viable without molecular delivery. These findings provide the first direct correlation between pulsed laser microbeam-induced shear stresses and subsequent cellular outcome.

A microscale yeast cell disruption technique for integrated process development strategies.

Abstract: Miniaturizing protein purification processes at the microliter scale (microscale) holds the promise of accelerating process development by enabling multi-parallel experimentation and automation. For intracellular proteins expressed in yeast, small-scale cell breakage methods capable of disrupting the rigid cell wall are needed that can match the protein release and contaminant profile of full-scale methods like homogenization, thereby enabling representative studies of subsequent downstream operations to be performed. In this study, a noncontact method known as adaptive focused acoustics (AFA) was optimized for the disruption of milligram quantities of yeast cells for the subsequent purification of recombinant human papillomavirus (HPV) virus-like particles (VLPs). AFA operates by delivering highly focused, computer-controlled acoustic radiation at frequencies significantly higher than those used in conventional sonication. With this method, the total soluble protein release was equivalent to that of laboratory-scale homogenization, and cell disruption was evident by light microscopy. The recovery of VLPs through a microscale chromatographic purification following AFA treatment was within 10% of that obtained using homogenization, with equivalent product purity. The addition of a yeast lytic enzyme prior to cell disruption reduced processing time by nearly 3-fold and further improved the comparability of the lysate to that of the laboratory-scale homogenate. In addition, unlike conventional sonication methods, sample heating was minimized (< =8 degrees C increase), even using the maximum power settings required for yeast cell disruption. This disruption technique in combination with microscale chromatographic methods for protein purification enables a strategy for the rapid process development of intracellularly expressed proteins.

Native conformation at specific residues in recombinant inclusion body protein in whole cells determined with solid-state NMR spectroscopy.

Abstract: In recent years there have been many advances in the production of recombinant proteins in bacterial hosts. A common problem is the sequestration of recombinant protein by the bacterium in an inclusion body which is the general term for a large insoluble protein aggregate. Harsh denaturing conditions are typically needed to solubilize the inclusion body protein with a consequent requirement of refolding to obtain functional protein. Despite these difficulties, inclusion bodies have some attractive features for recombinant protein production including each cell having one inclusion body which contains high purity protein and as much as 50% of the total mass of cellular protein.1 Inclusion bodies are noncrystalline solids and there has been little high-resolution, residue-specific study to ascertain their molecular structure. The most common structural method has been infrared (IR) spectroscopy which provided information about the overall fractions of different types of regular secondary structure. These studies were typically carried out on dehydrated samples and suggested that there was more β sheet structure in the inclusion body protein than in the native fold. It was therefore proposed that the non-native β strands from different proteins associated together to form intermolecular β-sheets in a structure similar to amyloid protein.2–8 Some reports indicate that a fraction of inclusion body protein is natively folded and functional.3, 9 This paper describes application of solid-state NMR to determine residue-specific secondary structure in inclusion body protein. The methods are straightforward and inexpensive and should be broadly applicable to a wide variety of proteins in inclusion bodies. The methods were initially developed to study residue-specific conformation in membrane-reconstituted proteins and were based on the observation that in sequences of proteins of moderate size, a large fraction of the residues are in “unique sequential pairs”, for example, there would only be one instance of a Leu followed by a Leu. In addition, NMR methods such as “rotational-echo double-resonance” (REDOR) can selectively detect the signal of the 13C carbonyl (13CO) nuclei which are directly bonded to 15N nuclei, and there are well-known correlations between the backbone 13CO NMR chemical shift of a residue and its local conformation.10–12 Recombinant protein was therefore produced in E. coli cells in minimal media containing the 1-13C amino acid and 15N-amino acid of the respective N- and C-terminal residues of the unique sequential pair, and the filtered 13CO NMR signal of the N-terminal residue of the pair was used to determine its conformation.12 These methods were first applied to “FHA2” which is a N-terminal 185-residue functional domain of the 221-residue “HA2” subunit of the influenza virus hemagglutinin protein.12 FHA2 contains a ~20-residue N-terminal “fusion peptide” that binds to the host cell membrane and plays a key role in infection. FHA2 is a membrane protein because of the fusion peptide while shorter constructs which lack the fusion peptide are soluble in aqueous solution and have been crystallized.13 There is also a NMR structure of the HA2 fusion peptide in detergent micelles.14 One essential feature of the method was expression in minimal media containing labeled amino acids. However, growth only in minimal media provided <0.5 mg purified FHA2 per L culture and an alternate protocol was developed to first grow to high cell densities in rich media and to then switch to minimal media prior to expression.12, 15, 16 Using this method, the purified yield of FHA2 from the soluble cell lysate was ~10 mg/L culture. This was likely the fraction of FHA2 which was incorporated in cell membranes because the lysis buffer contained N-lauroylsarcosine detergent which is effective at solubilizing membrane proteins. There was a significant amount of FHA2 in inclusion bodies as evidenced by a dominant FHA2 band in the SDS-PAGE gels of the unlysed fully hydrated E. coli cells and in the hydrated pellet formed from centrifugation of the insoluble material in the cell lysate, Figure 1a. In addition, ~10 mg/L more FHA2 was purified after sonication of the aforementioned pellet in 8 M urea, and this denatured FHA2 could be refolded in detergent and reconstituted in membranes.16 The relative band intensities in Figure 1a suggest that FHA2 is at least 10–20% of the total protein mass in the insoluble fraction and in unlysed whole cells and in the present study, the labeling/solid-state NMR method was applied to FHA2 in these two hydrated materials.17, 18 The 13CO NMR signals were dominated by FHA2 as evidenced by comparison of the “S0” (black) and “S1” (red) REDOR NMR spectra of the pellet for which the labeling targeted the Leu-98/Leu-99 unique sequential pair, Figure 1b. The S0 spectrum was composed of all the 13CO signal in the sample whereas the S1 spectrum lacked signal from 13CO nuclei directly bonded to 15N nuclei. The experimental S1/S0 integrated intensity ratio was 0.89 and correlated with the 0.92 ratio expected for fully labeled FHA2 with 13 Leus in the sequence. The experimental and (expected) ratios for labeling which targeted Gly-4 and Ala-7 were 0.93 (0.94) and 0.93 (0.91), respectively and were generally consistent with labeled amino acids being in the media only during the expression period and with FHA2 being the primary protein produced during the expression period. Figure 1 (a) SDS-PAGE gel: lane 1, molecular weight standards in kDa; lane 2, whole E. coli cells without lysis; and lane 3, the solid pellet obtained after centrifugation of the cell lysate. Different amounts of total protein were loaded in each lane and the ... The S0 - S1 REDOR difference spectrum primarily represents the filtered 13CO signal of the N-terminal residue of the unique sequential pair and such spectra are presented in Figure 1 for (c, f, i) Gly-4, (d, g, j) Ala-7, or (e, h, k) Leu-98 and for FHA2 (c-e) purified and reconstituted in membranes, (f-h) in inclusion bodies in the insoluble cell lysate, or (i-k) in unlysed whole cells. There is general similarity of the spectra and peak 13CO chemical shifts of the membrane-reconstituted, inclusion body, and whole cell FHA2 protein, Figure 1c-k and Table 1. The similarity of the insoluble fraction and the whole cell spectra correlated with most of the FHA2 being in inclusion bodies. In the existing structures, a N-terminal helix extends to residue 10 in the fusion peptide and there is a helix that extends from residue 38 to residue 105. The peak 13CO shifts for Gly-1, Gly-4, Ala-7, and Leu-98 correlate with these helical conformations as evidenced by better agreement with distributions of backbone 13CO shifts in helical conformation (175.5 ± 1.2, 179.4 ± 1.3, and 178.5 ± 1.3 ppm for Gly, Ala, and Leu, respectively) than with characteristic shifts in β sheet/amyloid samples(170–172, 174–176, and 173–175 ppm, respectively).5, 6, 11, 19, 20 The Ala-7 spectra of the insoluble fraction and whole cell samples are broader than the other spectra which may indicate some conformational heterogeneity at this residue. Overall, the data support a model in which native helical conformation is retained at least for a significant fraction of inclusion body FHA2. It is interesting that dominant β sheet chemical shifts were not observed in the fusion peptide region because this region is hydrophobic and could potentially form amyloid aggregates in the absence of membranes. Although 13CO shifts are not absolutely definitive in determining conformation and are correlated with regions of the Ramachandran plot rather than precise dihedral angles, 13CO shifts do provide significant conformational information particularly when helical shifts at several nearby residues are obtained (as in the fusion peptide region) or when sharp signals (indicating structural order) with very similar helical shifts are obtained in both the natively folded and inclusion body protein (as with Leu-98).11 Table 1 Peak 13C Carbonyl Chemical Shifts of FHA2 in ppm Units The approach presented in this paper has two significant advantages over the more commonly used IR approach for study of inclusion body structure. First, conformation at individual residues is straightforwardly studied with the NMR method but only overall protein conformation is typically obtained with the IR method. It should therefore be possible to develop more detailed structural models with the NMR data. Second, the inclusion body and whole cell samples are fully hydrated with the NMR method rather than being dehydrated with the IR method. A large number of specifically labeled samples are required for a high-resolution NMR structural model of inclusion body protein but depending on linewidths, it may be possible to obtain structural data for samples with more significant labeling using methods previously applied to NMR structure determination of microcrystalline, amyloid, and membrane-associated proteins.5, 6, 19–21 In summary, this study describes a general approach for residue-specific structural analysis of recombinant protein in inclusion bodies including those in whole E. coli cells as well as evidence for native conformation at some specific residues of a particular inclusion body protein.

Characterization and use of laser-based lysis for cell analysis on-chip

Abstract: We demonstrate the use of a pulsed laser microbeam for cell lysis followed by electrophoretic separation of cellular analytes in a microfluidic device. The influence of pulse energy and laser focal point within the microchannel on the threshold for plasma formation was measured. The thickness of the poly(dimethylsiloxane) (PDMS) layer through which the beam travelled was a critical determinant of the threshold energy. An effective optical path length, Leff ,f or the laser beam can be used to predict the threshold for optical breakdown at different microchannel locations. A key benefit of laser-based cell lysis is the very limited zone (less than 5 mm) of lysis. A second asset is the rapid cell lysis times (approx. microseconds). These features enable two analytes, fluorescein and Oregon Green, from a cell to be electrophoretically separated in the channel in which cell lysis occurred. The resolution and efficiency of the separation of the cellular analytes are similar to those of standards demonstrating the feasibility of using a pulsed laser microbeam in single-cell analysis.

Specific DNA extraction through fluid channels with immobilization of layered double hydroxides on polycarbonate surface

Abstract: The purpose of this study was to immobilize inorganic layered double hydroxides (LDHs) on the polycarbonate (PC) substrate as the media to extract the specific DNA molecules through fluidic system and to enhance the extraction efficiency of specific DNA molecules from extreme low concentration in sample solution. LDH immobilized through solvent swelling and plasma treatment on the polymer surface captured the specific DNA molecules lysed from Escherichia coli (E. coli) cells as the target DNA molecules with 2 × 10−4 g/l of concentration in sample solution mixed biomacromolecules lysed from human blood. The encapsulated DNA molecules released through dissolve of LDHs by slight acid (pH 4–5) solution then amplified by polymerase chain reaction (PCR) process through the primers for E. coli cells. The DNA molecules amplified by PCR process were characterized by gel electrophoresis to recognize the existence of E. coli cells. The results show that immobilized LDHs could be regarded as the specific DNA detector in the sample solution mixed biomacromolecules for rapid disease diagnosis through fluidic system to approach the lab-on-a-chip (LOC).

Mechanisms of alpha-defensin bactericidal action: comparative membrane disruption by Cryptdin-4 and its disulfide-null analogue.

Abstract: Mammalian alpha-defensins all have a conserved triple-stranded beta-sheet structure that is constrained by an invariant tridisulfide array, and the peptides exert bactericidal effects by permeabilizing the target cell envelope. Curiously, the disordered, disulfide-null variant of mouse alpha-defensin cryptdin-4 (Crp4), termed (6C/A)-Crp4, has bactericidal activity equal to or greater than that of the native peptide, providing a rationale for comparing the mechanisms by which the peptides interact with and disrupt phospholipid vesicles of defined composition. For both live Escherichia coli ML35 cells and model membranes, disordered (6C/A)-Crp4 induced leakage in a manner similar to that of Crp4 but had less overall membrane permeabilizing activity. Crp4 induction of the leakage of the fluorophore from electronegative liposomes was strongly dependent on vesicle lipid charge and composition, and the incorporation of cardiolipin into liposomes of low electronegative charge to mimic bacterial membrane composition conferred sensitivity to Crp4- and (6C/A)-Crp4-mediated vesicle lysis. Membrane perturbation studies using biomimetic lipid/polydiacetylene vesicles showed that Crp4 inserts more pronouncedly into membranes containing a high fraction of electronegative lipids or cardiolipin than (6C/A)-Crp4 does, correlating directly with measurements of induced leakage. Fluorescence resonance energy transfer experiments provided evidence that Crp4 translocates across highly charged or cardiolipin-containing membranes, in a process coupled with membrane permeabilization, but (6C/A)-Crp4 did not translocate across lipid bilayers and consistently displayed membrane surface association. Thus, despite the greater in vitro bactericidal activity of (6C/A)-Crp4, native, beta-sheet-containing Crp4 induces membrane permeabilization more effectively than disulfide-null Crp4 by translocating and forming transient membrane defects. (6C/A)-Crp4, on the other hand, appears to induce greater membrane disintegration.

Large-Scale Production of Endotoxin-Free Plasmids for Transient Expression in Mammalian Cell Culture

Abstract: Transient expression of recombinant proteins in mammalian cell culture in a 100-L scale requires a large quantity of plasmid that is very labour intensive to achieve with shake flask cultures and commercially available plasmid purification kits. In this paper we describe a process for plasmid production in 100-mg scale. The fermentation is carried out in a 4-L fed-batch culture with a minimal medium. The detection of the end of batch and triggering the exponential (0.1 h(-1)) feed profile was unattended and controlled by Multi-fermenter Control System. A restricted specific growth rate in fed-batch culture increased the specific plasmid yield compared to batch cultures with minimal and rich media. This together with high biomass concentration (68-107 g L(-1) wet weight) achieves high volumetric yields of plasmid (95-277 mg L(-1) depending on the construct). The purification process consisted of alkaline lysis, lysate clarification and ultrafiltration, two-phase extraction with Triton X-114 for endotoxin removal, anion-exchange chromatography as a polishing step, ultrafiltration and sterile filtration. Both fermentation and purification processes were used without optimisation for production of four plasmids yielding from 39 to 163 mg of plasmids with endotoxin content of 2.5 EU mg(-1) or less.

Lysis efficiency of standard DNA extraction methods for Halococcus spp. in an organic rich environment

Abstract: The extraction of nucleic acids from a given environment marks a crucial and essential starting point in any molecular investigation. Members of Halococcus spp. are known for their rigid cell walls, and are thus difficult to lyse and could potentially be overlooked in an environment. Furthermore, the lack of a suitable lysis method hinders subsequent molecular analysis. The effects of six different DNA extraction methods were tested on Halococcus hamelinensis, Halococcus saccharolyticus and Halobacterium salinarum NRC-1 as well as on an organic rich, highly carbonated sediment from stromatolites spiked with Halococcus hamelinensis. The methods tested were based on physical disruption (boiling and freeze/thawing), chemical lysis (Triton X-100, potassium ethyl xanthogenate (XS) buffer and CTAB) and on enzymatic lysis (lysozyme). Results showed that boiling and freeze/thawing had little effect on the lysis of both Halococcus strains. Methods based on chemical lysis (Triton X-100, XS-buffer, and CTAB) showed the best results, however, Triton X-100 treatment failed to produce visible DNA fragments. Using a combination of bead beating, chemical lysis with lysozyme, and thermal shock, lysis of cells was achieved however DNA was badly sheared. Lysis of cells and DNA extraction of samples from spiked sediment proved to be difficult, with the XS-buffer method indicating the best results. This study provides an evaluation of six commonly used methods of cell lysis and DNA extraction of Halococcus spp., and the suitability of the resulting DNA for molecular analysis.

Natural killer cell-mediated lysis of T cell lines chronically infected with HIV-1.

Abstract: The susceptibility of HIV-1-infected CD4+ T cell lines to natural killer (NK) cell-mediated lysis was examined Non-adherent peripheral blood mononuclear cells (PBMC) of healthy adults lysed HUT cells chronically infected with the IIIB or WMJ1 strains of HIV-1 to a significantly greater extent than uninfected HUT cells In contrast, Sup-T1 cells chronically infected with these two strains of HIV-1 were not lysed to a greater extent than uninfected Sup-T1 cells Clone A125-infected Sup-T1 (A125/Sup-T1), derived from IIIB-infected Sup-T1 cells (IIIB/Sup-T1), were susceptible to non-adherent PBMC-mediated lysis, as were A125-infected HUT cells (A125/HUT) When non-adherent PBMC were depleted of CD16 (Leu-11b)+ NK cells by treatment with anti-Leu-11b plus C, lysis of HIV-1-infected HUT or Sup-T1 cells was reduced to low levels, indicating that the lysis was mediated by NK cells Expression of HIV antigens on these target cells did not correlate with their susceptibility to NK cell-mediated lysis Depletion of interferon-alpha (IFN-alpha) producing HLA-DR+ cells from non-adherent PBMC had no effect on the magnitude of NK cell-mediated lysis of IIIB or WMJ1-infected HUT cells In contrast, lysis of A125/Sup-T1 or A125/HUT cells required the presence of HLA-DR+ cells IFN-alpha production appeared to be required for NK cell-mediated lysis of A125/Sup-T1 or A125/HUT cells, while lysis of HUT cells infected with the WMJ1 or IIIB strains of HIV-1 was IFN-alpha independent These results indicate considerable variability in the susceptibility of different HIV-1 infected T cell lines to NK cell-mediated lysis and suggest the existence of alternative mechanisms of activation of NK cells for lysis of HIV-1-infected T cell lines

Identification and functional analysis of the Rz/Rz1-like accessory lysis genes in the membrane-containing bacteriophage PRD1.

Abstract: Summary Bacteriophage PRD1 is a tailless membrane-containing double-stranded (ds) DNA virus infecting a variety of Gram-negative bacteria. In order to affect cell lysis, like most dsDNA phages, PRD1 uses the holin-endolysin system. In this study, we identified two accessory lysis genes, XXXVI and XXXVII, coding for proteins P36 and P37, respectively. Using genetic complementation assays, we show that protein pair P36/P37 is a functional and interchangeable analogue of the Rz/Rz1 of bacteriophage λ. Utilizing molecular biology, electrochemical as well as various microscopic techniques, we characterized the lysis phenotypes of PRD1 host cells infected with mutant viruses. Our results indicate that proteins P36 and P37 confer a competitive advantage to the phage by securing the efficient disruption of the infected cell and consequent release of the phage progeny under less favourable growth conditions. In concordance with prior data and the results obtained in this study, we propose a model explaining the role of Rz/Rz1-like proteins in the lysis process: Rz/Rz1 complexes transform the mechanical stress caused by the holin lesion at the CM to the OM leading to its disintegration. Finally, identification of the Rz/Rz1-like genes in PRD1 suggests that tailless icosahedral phages are involved in genetic trade with tailed bacteriophages.

On-line cell lysis and DNA extraction on a microfluidic biochip fabricated by microelectromechanical system technology.

Abstract: Integrating cell lysis and DNA purification process into a micrototal analytical system (microTAS) is one critical step for the analysis of nucleic acids. On-chip cell lysis based on a chemical method is realized by sufficient blend of blood sample and the lyzing reagent. In this paper two mixing models, T-type mixing model and sandwich-type mixing model, are proposed and simulation of those models is conducted. Result of simulation shows that the sandwich-type mixing model with coiled channel performs best and this model is further used to construct the microfluidic biochip for on-line cell lysis and DNA extraction. The result of simulation is further verified by experiments. It asserts that more than 80% mixing of blood sample and lyzing reagent which guarantees that completed cell lysis can be achieved near the inlet location when the cell/buffer velocity ratio is less than 1:5. After cell lysis, DNA extraction by means of a solid-phase method is implemented by using porous silicon matrix which is integrated in the biochip. During continuous flow process in the microchip, rapid cell lysis and PCR-amplifiable genomic DNA purification can be achieved within 20 min. The potential of this microfluidic biochip is illustrated by pretreating a whole blood sample, which shows the possibility of integration of sample preparation, PCR, and separation on a single device to work as portable point-of-care medical diagnostic system.

Comparative evaluation of different cell disruption methods for the release of recombinant hepatitis B core antigen from Escherichia coli

Abstract: A comparative evaluation of five different cell-disruption methods for the release of recombinant hepatitis B core antigen (HBcAg) from Escherichia coli was investigated. The cell disruption techniques evaluated in this study were high-pressure homogenization, batch-mode bead milling, continuous-recycling bead milling, ultrasonication, and enzymatic lysis. Continuous-recycling bead milling was found to be the most effective method in terms of operating cost and time. However, the highest degree of cell disruption and amounts of HBcAg were obtained from the high-pressure homogenization process. The direct purification of HBcAg from the unclarified cell disruptate derived from high-pressure homogenization and bead milling techniques, using batch anion-exchange adsorption methods, showed that the conditions of cell disruption have a substantial effect on subsequent protein recovery steps.