Lysis Solution Research Articles

Microplate live cell assay system for early events in mechanotransduction

For studying mechanotransduction in cultured cells, we developed a microplate assay using a fluorescence/luminescence plate reader equipped with software-controlled injectors to deliver a reproducible mechanical stimulus (adjustable for both timing and force) and immediately measure adenosine 5 ′-triphosphate (ATP) release and calcium mobilization. Suspension or adherent chondrocyte cultures in 96-well plates were incubated with firefly luciferase and luciferin for the ATP assay or loaded with Fluo-3-acetoxy methylester for intracellular calcium measurement. Steady state ATP release was measured in resting cells; then mechanical stimulation was delivered by injection of an equal volume of buffer into the wells. Serial integrations of 20 to 500 ms allowed real-time analysis of the time course of ATP release. Luminescence increased within 500 ms indicating the rapidity of ATP release in chondrocyte mechanotransduction. Subsequent injection of a cell lysis solution allowed quantitation of total cellular ATP as an internal control of cell viability and number. Intracellular calcium was also elevated within 500 ms of fluid injection. This assay is easily adapted for changes in intracellular pH or other ions by use of different commercially available fluorescent indicators. The live-cell assay using fluid injection as a mechanical stimulus is a valuable tool for dissecting the role of signaling pathways in mechanotransduction.

Expression, purification, and PC1-mediated processing of (H10D, P28K, and K29P)-human proinsulin

Our previous methods for the generation of recombinant human proinsulin were inadequate in terms of reproducibility and yield. In addition, it was difficult to perform structure/function studies on proinsulin because of its tendency to form hexamers. We have developed an improved procedure, which overcomes many of the technical purification problems, and results in a potentially monomeric version of modified proinsulin. Inclusion bodies were prepared using a commercial bacterial lysis solution. The inclusion bodies were solubilized and the fusion protein’s affinity tag was removed by chemical cleavage. The polypeptide was then reduced and transferred into a refolding buffer. Following an overnight incubation, only a single form of proinsulin was detected using analytical reversed-phase high-performance liquid chromatography. The refolded (H10D, P28K, and K29P)-human proinsulin (DKP-hPI) was subjected to a final purification step using reversed-phase chromatography. The method is reproducible and produces milligram quantities of purified DKP-hPI from a single liter of bacterial culture. The final product is greater than 95% pure and is suitable for use as a substrate for the propeptide convertase PC1.

Monitoring for Heterosigma akashiwo using a sandwich hybridization assay

Field testing of a ribosomal RNA (rRNA)-targeted sandwich hybridization assay (SHA) for Heterosigma akashiwo (Raphidophyceae) in Puget Sound, WA, USA, has showed that the lower limit of detection is well below the level at which cells pose a danger to fish. Moreover, the assay has proven to be both rapid and easy-to-use. Isolates of H. akashiwo from Australia, Japan, New Zealand, South Korea, Spain and USA were correctly identified using the SHA, indicating that this diagnostic tool could be deployed globally. Samples containing H. akashiwo can be preserved for subsequent SHA analysis using several methods: fixation with acidic Lugol’s iodine followed by room temperature storage, collection onto Durapore filters followed by storage at −70 °C or, alternatively, the filters are mixed with a lysis solution buffer and the sample lysate stored at −70 °C. Additionally, we sought to determine whether the SHA could successfully detect H. akashiwo in the presence of clay that might some day be used to mitigate the impacts of natural H. akashiwo blooms. Results from preliminary laboratory trials indicate that clay at the maximum proposed dosage rate does not interfere with the assay. Thus, it may be possible to use the SHA as a simple means of following the fate of H. akashiwo cells during larger-scale clay mitigation trials.

New Insights on How Metals Disrupt Amyloid β-Aggregation and Their Effects on Amyloid-β Cytotoxicity

Amyloid-beta protein (A beta) aggregates in the brain to form senile plaques. By using thioflavin T, a dye that specifically binds to fibrillar structures, we found that metals such as Zn(II) and Cu(II) normally inhibit amyloid beta-aggregation. Another method for detecting A beta, which does not distinguish the types of aggregates, showed that these metals induce a non-beta-sheeted aggregation, as reported previously. Secondary structural analysis and microscopic studies revealed that metals induced A beta to make non-fibrillar aggregates by disrupting beta-sheet formation. These non-fibrillar A beta aggregates displayed much weaker Congo Red birefringence, and in separate cell culture experiments, were less toxic than self beta-aggregates, as demonstrated by 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide assay. The toxicity of soluble A beta was enhanced in the presence of Cu(II), which suggests the previously hypothesized role of A beta in generating oxidative stress. Finally, under an acidic condition, similar to that in the inflammation associated with senile plaques, beta-aggregation was robustly facilitated at one specific concentration of Zn(II) in the presence of heparin. However, because a higher concentration of Zn(II) virtually abolished this abnormal phenomenon, and at normal pH any concentrations strongly inhibit beta-aggregation and its associated cytotoxicity, including its anti-oxidative nature we suggest that Zn(II) has an overall protective effect against beta-amyloid toxicity.

Is the capacity of lead acetate and cadmium chloride to induce genotoxic damage due to direct DNA-metal interaction?

Even though the toxic effects of lead and cadmium compounds have been studied over many years, inconsistent results have been obtained about their mutagenic, clastogenic and carcinogenic properties. However, these metals are considered to be potential human carcinogens. The mechanism of metal-induced carcinogenesis is still unknown, but one possible pathway may involve the interaction of metals with DNA, either directly or indirectly. In this work we explore the capacity of lead, cadmium or a mixture of both metals to interact with acellular DNA, by employing a variant of the comet assay. Our results, using low non-cytotoxic metal concentrations (0.01, 0.1 and 1.0 microM) with the standard protocol for the acellular assay, showed an induction of DNA damage in cells of all organs studied; however, basal DNA damage was different in each organ. To confirm that we were working with pure DNA, proteinase K was added to the lysis solution. With this enriched-lysis solution we found a negative response in the induction of DNA damage in cells derived from the liver, kidney and lung of CD-1 male mice. To support the results obtained by the enriched-acellular assay, we studied the capacity of lead and cadmium (0.1 microM) to induce breaks in pooled genomic DNA in cells of the same organs, with negative results. Consistent with these findings, these metals do not induce DNA breaks in the plasmid pUSE amp+. On the whole, we did not detect direct induction of DNA strand breaks by lead acetate, cadmium chloride or a mixture of both metals, all at low non-cytotoxic concentrations. However, we found an induction of lipid peroxidation and an increase in free radical levels in the different organs of CD-1 male mice after inhalation of lead acetate (0.0068 microg/cc) or cadmium chloride (0.08 microg/cc) for 1 h, suggesting the induction of genotoxicity and carcinogenicity by indirect interactions, such as oxidative stress.

RELATIONSHIP BETWEEN PLASMID DNA TOPOLOGICAL FORMS AND IN VITRO TRANSFECTION

Topological isomers of plasmid DNA might be a factor affecting transfection efficiency. This study investigated the relationship between three major topoisomers of plasmid DNA, (supercoiled, open circular, and linear forms) and their biological activity. The three topoisomers of pDNA were transfected into CHO cells (Chinese Hamster Ovary) or SKnSH (human neuroblastoma) cells by (1) cationic liposomes, DOTAP/DOPE (1,2-dioleoyloxy-3-trimethylammonium propane/dioleolylphosphatidylethanolamine) or (2) electroporation. The open circular form of pCMV-luciferase demonstrated higher transfection efficiency (TE) than either supercoiled or linear while plasmid pGL3 showed no significant difference in TE for either delivery method. CHO and SKnSH cells showed similar patterns in transfection efficiency although the extent of expression was different. In order to test its stability in cell cytoplasmic lysate, pDNA was incubated at 37°C in NP40 lysate preparation. Surprisingly, supercoiled pDNA was the least stable in lysate solution (detected up to 4 min) whereas the open circular pDNA and linear form were degraded with a half-life value of 6 and 10 min respectively. To measure cellular associated pDNA, fluorescein labeled pDNA was complexed with DOTAP/DOPE liposome and transfected into CHO cells. The amount of pDNA associated with the cell was determined by flow cytometry. The flow cytometry studies demonstrated that the open circular form associated with the cell more than the linear forms when delivered with cationic liposomes. In conclusion, the three studied topoisomers of pDNA are transcriptionally active. The pattern of transgene production of the topoisoform of pDNA was independent of cell line and delivery method even though the level of expression was different.

Randomised Double-blind Placebo Controlled Trial of Topical Autologous Platelet Lysate in Venous Ulcer Healing

Objectives: to assess the effect of topical autologous platelet lysate on the healing of chronic venous ulcers. Design: a randomised placebo controlled double-blind trial. Materials: all patients had blood taken for preparation of autologous platelet lysate. Methods: patients with proven chronic venous ulceration were randomised to the trial. Autologous platelet lysate or placebo buffer solution were applied twice per week for up to 9 months in combination with standardised compression bandaging. Results: a total of 86 patients (36 males and 50 females, median age 70 years) were entered into the study. The patient and treatment groups were equivalent for ulcer size, ulcer duration and other characteristics. Cox regression analysis of the time to ulcer healing did not show any difference in healing between platelet lysate and placebo application. Conclusions: platelet lysate prepared and delivered by the method used in this study had no influence on the healing of chronic venous ulceration.

Immunological Quantitation and Localization of ACAT-1 and ACAT-2 in Human Liver and Small Intestine

By using specific anti-ACAT-1 antibodies in immunodepletion studies, we previously found that ACAT-1, a 50-kDa protein, plays a major catalytic role in the adult human liver, adrenal glands, macrophages, and kidneys but not in the intestine. Acyl-coenzyme A:cholesterol acyltransferase (ACAT) activity in the intestine may be largely derived from a different ACAT protein. To test this hypothesis, we produced specific polyclonal anti-ACAT-2 antibodies that quantitatively immunodepleted human ACAT-2, a 46-kDa protein expressed in Chinese hamster ovary cells. In hepatocyte-like HepG2 cells, ACAT-1 comprises 85-90% of the total ACAT activity, with the remainder attributed to ACAT-2. In adult intestines, most of the ACAT activity can be immunodepleted by anti-ACAT-2. ACAT-1 and ACAT-2 do not form hetero-oligomeric complexes. In differentiating intestinal enterocyte-like Caco-2 cells, ACAT-2 protein content increases by 5-10-fold in 6 days, whereas ACAT-1 protein content remains relatively constant. In the small intestine, ACAT-2 is concentrated at the apices of the villi, whereas ACAT-1 is uniformly distributed along the villus-crypt axis. In the human liver, ACAT-1 is present in both fetal and adult hepatocytes. In contrast, ACAT-2 is evident in fetal but not adult hepatocytes. Our results collectively suggest that in humans, ACAT-2 performs significant catalytic roles in the fetal liver and in intestinal enterocytes.

Determination of Total Interleukin-8 in Whole Blood after Cell Lysis

It has been shown that a high percentage of interleukin-8 (IL-8) in blood is cell associated. Recently, a simple method for determination of cell-associated IL-8 in whole blood after cell lysis has been described. The purpose of this study was to evaluate this method, to examine the influence of preanalytic sample handling, and to establish the concentration range of total IL-8 and its relation to age and sex in healthy subjects. Total IL-8 content of whole blood was determined after lysing blood cells with Milenia((R)) cell lysis solution. IL-8 in the resulting blood lysate was measured with the IMMULITE((R)) IL-8 immunoassay. When freshly drawn blood was stored up to 48 h on ice, no significant changes in total IL-8 were measured in the subsequently prepared lysate, whereas with storage at room temperature, total IL-8 increased after 3 h from 94 +/- 13 ng/L to 114 +/- 16 ng/L (n = 10). In lysate stored for 48 h at 4 degrees C, marginal changes of the IL-8 concentration were noted, with storage at room temperature, only 76% +/- 5% (n = 12) of initial concentration was recovered. From lysate frozen at -20 and -80 degrees C, respectively, 84% +/- 4% and 93% +/- 2% of initial IL-8 was recovered after 70 days (n = 10). IL-8 was measured with comparable precision in plasma (CV, 3. 2-4.2%) and blood lysate (CV, 3.7-4.1%). When plasma was diluted with cell lysis solution, a slightly overestimated recovery (125% +/- 3%) was observed; for lysate specimens with a cell lysis solution content >/=75%, the recovery after dilution was 98% +/- 2%. In lysate prepared from 12 blood samples with exogenous IL-8 added, IL-8 recovery was 104% +/- 2% (recovery from plasma <35%). The median total IL-8 in blood lysates from 103 healthy subjects (22-61 years) was 83 ng/L of blood (2.5-97.5 percentile range, 49-202 ng/L of blood). In females but not in males, total IL-8 increased significantly with advancing age (P: <0.002). We found grossly increased total IL-8 in six pregnant women with amniotic infection syndrome. The evaluated method allows the assessment of total IL-8 in blood with good performance when appropriate conditions of sample pretreatment are considered. The values in healthy volunteers all were above the detection limit of the IL-8 assay; therefore, slight changes of total IL-8 could be noted. Thus, the present method is a suitable tool to study the diagnostic relevance of total IL-8 in blood.

Simple and rapid detection of Streptococcus mutans and Streptococcus sobrinus in human saliva by polymerase chain reaction

Streptococcus mutans and Streptococcus sobrinus are major pathogens causing dental caries in humans. A simple and rapid method to detect these species in human saliva simultaneously was developed using the polymerase chain reaction (PCR). Chromosomal DNA was extracted by boiling bacterial cells in lysis solution containing 1% Triton X-100. Oligonucleotide primers specific for portions of the glucosyltransferase genes (gtfB of S. mutans and gtfI of S. sobrinus) were designed. After PCR using two sets of these primers, S. mutans and S. sobrinus were specifically identified. The method was capable of amplifying DNA fragments specific for these species from chromosomal DNA extracted from 1 x 10(3) cells, or from 10 microliters of clinical saliva samples containing 1 x 10(3) colony-forming units of either streptococcal species. A second PCR, using the first PCR product as a template with newly designed internal primers, made it possible to detect 1 x 10(2) colony-forming units of either streptococcal species in 10 microliters of saliva samples. These results indicate that the PCR method developed in this study is useful for detecting S. mutans and S. sobrinus in saliva and that it can be used in epidemiological studies to evaluate the prevalence level of these organisms.

Time-resolved fluorometric assay for leukocyte adhesion using a fluorescence enhancing ligand

A new 96-well microtiter plate, time-resolved fluorometric assay was developed to measure leukocyte adhesion in vitro. The assay is based on loading leukocytes with a fluorescence enhancing ligand 2,2′:6′,2″-terpyridine-6,6″-dicarboxylic acid (TDA), which in its acetoxymethyl ester form readily diffuses through the cell membrane. After hydrolysis by nonspecific intracellular esterases, the impermeable TDA accumulates inside the cells. When the TDA-labeled adherent leukocytes are lysed, the ligand is released and reacts with europium present in the lysis solution to produce a highly fluorescent and stable chelate. The fluorescence signal can be measured by time-resolved fluorometry and correlates directly with the number of adherent cells. In this study, we have optimized both the TDA-labeling and adhesion assay conditions in isolated human neutrophils. Furthermore, we have compared the assay with a traditional microscopic counting method. This time-resolved fluorometric assay provides a rapid, reproducible and convenient method for the routine analysis of leukocyte adhesion.

&lt;I&gt;Helicobacter pylori&lt;/I&gt;に対する電解機能水の殺菌効果

The bacteriocidal effect of electrolysed functioning water against Helicobacter pylori was examined by both the culture method (viable count on agar plate) and the bacterial ATP content analysis (biolumminescence assay). The culture method revealed the water's reduced bacteriocidal effect on organic substances such as horse serum and bovine serum albumin. The bacterial ATP content determined by the treatment with lysis solution correlated with the bacterial cell count. The ATP of bacteria treated with electrolysed functioning water, benzalkonium chloride, and chlorhexidine gluconate was still present after contact with these solutions for 30 minutes, while the bacterial growth was completely inhibited by the culture method. Therefore, it was suggested that the bacterial ATP was derived from the coccoid form which was observed morphologically due to the action of these solutions. These results demonstrate that these solutions are not effective bacteriocidally against the coccoid form. Consequently, when we utilize functioning water in the field, we should keep in mind its nonbacteriocidal effect against the coccoid form, as well as its reduced effect under the condition of the contamination by organic substances.

Immobiline-based two-dimensional gel electrophoresis: an optimised protocol for resolution of human colonic mucosal proteins.

An optimised protocol for the production of two-dimensional protein patterns of human colonic mucosal cells using immobilised pH gradients in the first dimension is presented. We tested a wide variety of solubilising agents and electrophoretic parameters, separately and in combination. Protein solubilisation was found to be best using a lysis solution containing 9 M urea, 2% Triton X-100, 2% 2-mercaptoethanol, 0.8% Pharmalyte pH 3-10 and 8 mM phenylmethylsulfonyl fluoride (PMSF). Horizontal streaking of basic proteins in the first dimension was virtually eliminated by a combination of washing Immobiline strips in 100 mM asorbic acid, pH 4.5, for 24 h before use and isoelectric focusing samples at 40 degrees C. The presence of 2% glycerol in the first dimension resulted in tighter resolution throughout the entire pH range. The use of these conditions may prove to have broad applicability to the generation of optimally resolved Immobiline-based two-dimensional protein patterns of many other tissues.

Absolute quantitation of specific mRNAs in cell and tissue samples by comparative PCR.

A comparative PCR assay, for the absolute quantitation of specific mRNAs in cell and tissue samples, has been designed to overcome problems with previous techniques. cDNAs made from the RNAs are co-amplified with "competitor" plasmid templates under conditions in which reagents are not limiting at the equivalence point, thereby preventing competition between target and competitor templates and distinguishing the assay from competitive PCR assays. The cDNAs are serially diluted, and competitor templates concentrations are kept constant, rather than vice versa, as occurs in competitive PCR assays. Products from target and competitor templates are resolved by electrophoresis and measured by phosphorescent or fluorescent imagery. Both products are measured to minimize errors in the competitor:target ratio. A synthetic external standard RNA is included in the tissue lysis solution and co-purified with endogenous mRNAs, thereby being subjected to identical losses of yield during subsequent procedures. The determination of the number of copies of external standard cDNA allows inefficiencies of RNA extraction and cDNA synthesis to be taken into account. Standard concentrations of plasmids containing the endogenous target sequences are also measured, so that corrections can be made for discrepancies due to unequal amplification of target and competitor sequences. These corrections, together with the use of an external standard and the PCR conditions chosen, allow for the accurate, specific and sensitive determination of the absolute number of mRNA copies in a sample.

Short protocol for pulsed field gel electrophoresis of a variety of Clostridia species

While pulsed field gel electrophoresis has become an important tool for genotyping of bacteria, one of its drawbacks is that standard methods are rather time-consuming. In order to overcome this problem, shortened procedures for DNA preparation have been developed for some bacterial species. The aim of this study was to examine if a short procedure used for pulsed field gel electrophoresis of Clostridium botulinum could be applied to other Clostridia species. For this, the protocol was modified and used to prepare the DNA of 34 strains of 25 different Clostridia species. In contrast to a standard procedure, which takes at least 5 days from DNA extraction to completion of the electrophoresis, this protocol yielded results within 2 days. In order to directly compare the results of the short protocol with those of the standard, long procedure, parallel DNA preparations were performed using both methods and the two DNA samples thus obtained per strain were then run on the same gel. Briefly, the procedure was as follows. After embedding the bacterial cells in agarose, the agarose blocks were incubated for 1 h in lysis solution containing lysozyme, mutanolysin, lysostaphin and RNase. This was followed by a 1-h proteinase K treatment. Then, slices were cut from the agarose blocks and washed for 15 min in TE buffer, these washes were repeated four times with fresh TE. After a 2-h restriction with SmaI, electrophoresis was carried out overnight.

Short protocol for pulsed field gel electrophoresis of a variety of Clostridia species.

While pulsed field gel electrophoresis has become an important tool for genotyping of bacteria, one of its drawbacks is that standard methods are rather time-consuming. In order to overcome this problem, shortened procedures for DNA preparation have been developed for some bacterial species. The aim of this study was to examine if a short procedure used for pulsed field gel electrophoresis of Clostridium botulinum could be applied to other Clostridia species. For this, the protocol was modified and used to prepare the DNA of 34 strains of 25 different Clostridia species. In contrast to a standard procedure, which takes at least 5 days from DNA extraction to completion of the electrophoresis, this protocol yielded results within 2 days. In order to directly compare the results of the short protocol with those of the standard, long procedure, parallel DNA preparations were performed using both methods and the two DNA samples thus obtained per strain were then run on the same gel. Briefly, the procedure was as follows. After embedding the bacterial cells in agarose, the agarose blocks were incubated for 1 h in lysis solution containing lysozyme, mutanolysin, lysostaphin and RNase. This was followed by a 1-h proteinase K treatment. Then, slices were cut from the agarose blocks and washed for 15 min in TE buffer, these washes were repeated four times with fresh TE. After a 2-h restriction with SmaI, electrophoresis was carried out overnight.

Genotyping of individual fish oocytes based on polymerase chain reaction amplification of mitochondrial DNA

A major reason for the routine use of the polymerase chain reaction (PCR) in genetic projects is that it requires small amounts of imperfectly puri®ed DNA for the detection of polymorphic markers. Recently, a number of protocols have been proposed that permit PCR ampli®cation from a variety of tissues. They have been summarized brie y by Estoup, Largiader, Perrot & Chourrout (1996). Here, we propose a procedure of fast mitochondrial (mt) DNA extraction from individual salmonid eggs that allows further PCR ampli®cation and genotype studies. Fish eggs (in particular salmonid eggs) are the samples that require mechanical treatment before DNA extraction in order to allow contact between cytoplasm and lysis solution. Eggs of white®sh (Fig. 1A) and rainbow trout were tapwater activated (5 min), and then their chorion was pierced with a glass cannula under a stereomicroscope to allow the escape of cytoplasmic uid (Fig. 1B). The uid was transferred into a 0.5-mL Eppendorf tube, lysed in 3 mL of lysis buffer [200 mM KOH, 50 mM dithiothreitol (DTT)] for 30 min at 60 °C, and neutralized by 4 mL of neutralizing buffer (900 mM Tris-HCl, pH 8.3, 300 mM KCl, 200 mM HCl; modi®ed from Li, Cui & Arnheim 1991; Fig. 1C). Before use, samples were vortexed brie y for 3 s. An aliquot of 1±3 mL (usually 2 mL) was taken as a template for a 12-mL total volume of PCR reaction. Forty cycles of PCR (beginning with preliminary denaturation at 95 °C for 1 min) were carried out in a GeneAmp PCR system 2400 with a primer pair L-H (L: 5¢-CCACTAGCTCCCCAAAGCTA-3¢; H: 5¢-ACTTTCTAGGGTCCATC-3¢), which spans the entire control region of both white®sh and rainbow trout mtDNA (Brzuzan 1998). The cycle consisted of 92 °C for 1 min, 50 °C for 1 min and 72 °C for 1.5 min. Positive ampli®cations were observed regularly and allowed further sequence analysis with restriction enzymes (Fig. 1D). The extraction procedure presented is simple, rapid and ef®cient for several ampli®cations of mitochondrial sequences. The ability to analyse DNA sequence variation in single cells (oocytes) provides a unique tool for solving many important biological problems. For example, it permits revelation of the presence of both mutant and wild-type mtDNA (heteroplasmy) in the same ®sh egg, which is equal to an individual `®sh'. Investigating mature oocytes from heteroplastic females can determine the relationship between the levels of drift occurring during oogenesis and mutation rates, thus giving an insight into the evolution of ®sh mtDNA (Nesbu, Arab & Jakobsen 1998). It is possible that the `single-cell' PCR is applicable to the analysis of the polymorphic DNA regions in an embryo derived from an egg at the earliest stages of development. Finally, because of the type of tissue used (an egg), it R

Osmotically driven radial diffusion of single-stranded DNA fragments on an agarose bed as a convenient measure of DNA strand scission

The present study describes the development and characterization of a novel technique, the alkaline-halo assay, for the assessment of DNA single strand breakage in mammalian cells. This technique allows the measurement of DNA lesions at the single cell level and presents the additional advantages of being rapid, sensitive, virtually costless and environmentally friendly, because it does not require the use of isotopes. The alkaline halo assay involves a series of sequential steps in which the cells are first treated, then embedded in melted agarose and spread onto microscope slides that are incubated for 2 min at ice-bath temperature to allow complete geling. The slides are then incubated for 20 min in a high salt alkaline lysis solution, for an additional 15 min in a hypotonic alkaline solution and, finally, for 10 min in ethidium bromide. Under these conditions, single-stranded DNA fragments spread radially from the nuclear cage and generate a fluorescent image that resembles a halo concentric to the nucleus remnants. The area of the halos increased at increasing levels of DNA fragmentation and this process was associated with a progressive reduction of areas of the nuclear remnants. These events were conveniently monitored with a fluorescence microscope and quantified by image processing analysis. The sensitivity of the alkaline-halo assay, which is based on the osmotically driven radial diffusion of single-stranded DNA fragments through agarose pores, is remarkably similar to that of the widely used alkaline elution and comet assays.

Radiation-Induced DNA Damage in Tumors and Normal Tissues: V. Influence of pH and Nutrient Depletion on the Formation of DNA-Protein Crosslinks in Irradiated Partially and Fully Hypoxic Tumor Cells

It has been demonstrated that the yield of radiation-induced DNA strand breaks and DNA-protein crosslinks (DPCs) appears to depend predominantly on the oxygen concentration in the microenvironment around the DNA of mammalian cells (Radiat. Res. 142, 163-168, 1995). Consequently, these DNA lesions have the potential to be used to detect hypoxic cells or estimate the hypoxic fraction of solid tumors. Although it has been demonstrated that physiological factors (e.g. pH, temperature, nutrient depletion, etc.) have no influence on the induction and repair of both DNA strand breaks and DPCs in irradiated oxygenated cells (Radiat. Res. 140, 321-326, 1994), there are no data to suggest how these physiological factors influence the induction and repair of DNA strand breaks and DPCs in irradiated partially hypoxic or fully hypoxic cells. In the present study, the influence of pH (6.6-7.3) and nutrient depletion on the formation and/or repair of radiation-induced DNA strand breaks and DPCs in partially hypoxic (O2 concentration in the gas phase of 2.0-8.0%) or fully hypoxic (O2 concentration in the gas phase of 0.3-0.4%) populations of exponential (day 3) and plateau-phase (day 5) 9L rat brain tumor cells in culture was determined. Each population of cells was irradiated at 37 degrees C with 15 Gy and trypsinized at 4 degrees C after one half-time of strand break repair at 37 degrees C, and the DNA damage was measured using our alkaline elution technique, with or without proteinase K (PK) in the lysis solution. An analysis of the DNA-protein crosslink factor as a function of the oxygen concentration and a statistical comparison of the single-strand scission factor measured at eluted fraction 7 (SSSF7), with or without PK in the lysis solution, indicate that pH and nutrient depletion do not significantly influence the formation and/or repair of DNA strand breaks and DPCs in irradiated partially hypoxic or fully hypoxic 9L cells. The data also demonstrate that the DNA lesions produced in irradiated partially hypoxic cells will cause the hypoxic fraction or fractional hypoxic volume to be overestimated by this DNA damage assay, not underestimated. However, this potential overestimation of the hypoxic fraction or fractional hypoxic volume should not limit the usefulness of this DNA damage assay in most laboratory studies, because it will take the contribution of a large cohort of partially hypoxic cells to overcome the dominance of the signal from a relatively small cohort of fully hypoxic cells.

Effect of shear on plasmid DNA in solution

This study was designed to evaluate the effect of shear on the supercoiled circular (SC) form of plasmid DNA. The conditions chosen are representative of those occurring during the processing of plasmid-based genes for gene therapy and DNA vaccination. Controlled shear was generated using a capillary rheometer and a rotating disk shear device. Plasmid DNA was tested in a clarified alkaline lysate solution. This chemical environment is characteristic of the early stages of plasmid purification. Quantitative data is reported on shear degradation of three homologous recombinant plasmids of 13, 20 and 29 kb in size. Shear sensitivity increased dramatically with plasmid molecular weight. Ultrapure plasmid DNA redissolved in 10 mM Tris/HCl, 1 mM EDTA pH 8 (TE buffer) was subjected to shear using the capillary rheometer. The shear sensitivity of the three plasmids was similar to that observed for the same plasmids in the clarified alkaline lysate. Further experiments were carried out using the 20 kb plasmid and the rotating disk shear device. In contrast with the capillary rheometer data, ultrapure DNA redissolved in TE buffer was up to eight times more sensitive to shear compared to plasmid DNA in the clarified alkaline lysate. However, this enhanced sensitivity decreased when the ionic strength of the solution was raised by the addition of NaCl to 150 mM. In addition, shear damage was found to be independent of plasmid DNA concentration in the range from 0.2 μg/ml to 20 μg/ml. The combination of shear and air-liquid interfaces caused extensive degradation of the plasmid DNA. The damage was more evident at low ionic strength and low DNA concentration. These findings show that the tertiary structure of plasmid DNA can be severely affected by shear forces. The extent of damage was found to be critically dependent on plasmid size and the ionic strength of the environment. The interaction of shear with air-liquid interfaces shows the highest potential for damaging SC plasmid DNA during bioprocesses.