Science.gov

Sample records for lysis

  1. Argon endolaser suture lysis

    NASA Astrophysics Data System (ADS)

    Cameron, Bruce D.; Joos, Karen M.; Shen, Jin-Hui

    1996-05-01

    Purpose: To develop a simple suture lysis technique for post-trabeculectomy examinations under anesthesia since slit lamp laser suture lysis in the clinic cannot be performed on infants and young children. Methods: An argon endolaser probe lysed 10-0 nylon suture through conjunctiva harvested from human cadaver eyes. Since suture lysis failed with the thick Hoskins lens, clear plastic from the suture package compressed the conjunctiva. The conjunctiva was examined histologically. Results: Argon laser suture lysis (250 mW, 0.1 sec, 488 - 514 nm) was achieved without conjunctival damage. Conclusion: The argon endolaser probe is effective for suture lysis when the slit lamp cannot be used.

  2. Lysis from without

    PubMed Central

    2011-01-01

    In this commentary I consider use of the term “lysis from without” (LO) along with the phenomenon's biological relevance. LO originally described an early bacterial lysis induced by high-multiplicity virion adsorption and that occurs without phage production (here indicated as LOV). Notably, this is more than just high phage multiplicities of adsorption leading to bacterial killing. The action on bacteria of exogenously supplied phage lysin, too, has been described as a form of LO (here, LOL). LOV has been somewhat worked out mechanistically for T4 phages, has been used to elucidate various phage-associated phenomena including discovery of the phage eclipse, may be relevant to phage ecology, and, with resistance to LO (LOR), is blocked by certain phage gene products. Speculation as to the impact of LOV on phage therapy also is fairly common. Since LOV assays are relatively easily performed and not all phages are able to induce LOV, a phage's potential to lyse bacteria without first infecting should be subject to at least in vitro experimental confirmation before the LOV label is applied. The term “abortive infection” may be used more generally to describe non-productive phage infections that kill bacteria. PMID:21687534

  3. Inducible Lysis in Clostridium tetani

    PubMed Central

    Prescott, Lawrence M.; Altenbern, Robert A.

    1967-01-01

    Lysis was induced in seven strains of Clostridium tetani by exposure to mitomycin C. The search for a suitable indicator strain to detect bacteriophage in lysates has, so far, been unsuccessful. Inhibition studies on macromolecular synthesis during induction have shown that deoxyribonucleic acid, ribonucleic acid, and protein syntheses are all involved in the lysis induced by mitomycin C. In experiments comparing toxin and protein content in induced and uninduced cells of C. tetani, the toxin-protein ratio proved to be the same in both systems up to the point of lysis. Several possible hypotheses deduced from these results are discussed. PMID:4226682

  4. Bacteriophage lysis: mechanism and regulation.

    PubMed Central

    Young, R

    1992-01-01

    Bacteriophage lysis involves at least two fundamentally different strategies. Most phages elaborate at least two proteins, one of which is a murein hydrolase, or lysin, and the other is a membrane protein, which is given the designation holin in this review. The function of the holin is to create a lesion in the cytoplasmic membrane through which the murein hydrolase passes to gain access to the murein layer. This is necessary because phage-encoded lysins never have secretory signal sequences and are thus incapable of unassisted escape from the cytoplasm. The holins, whose prototype is the lambda S protein, share a common organization in terms of the arrangement of charged and hydrophobic residues, and they may all contain at least two transmembrane helical domains. The available evidence suggests that holins oligomerize to form nonspecific holes and that this hole-forming step is the regulated step in phage lysis. The correct scheduling of the lysis event is as much an essential feature of holin function as is the hole formation itself. In the second strategy of lysis, used by the small single-stranded DNA phage phi X174 and the single-stranded RNA phage MS2, no murein hydrolase activity is synthesized. Instead, there is a single species of small membrane protein, unlike the holins in primary structure, which somehow causes disruption of the envelope. These lysis proteins function by activation of cellular autolysins. A host locus is required for the lytic function of the phi X174 lysis gene E. Images PMID:1406491

  5. Membrane fusion during phage lysis.

    PubMed

    Rajaure, Manoj; Berry, Joel; Kongari, Rohit; Cahill, Jesse; Young, Ry

    2015-04-28

    In general, phages cause lysis of the bacterial host to effect release of the progeny virions. Until recently, it was thought that degradation of the peptidoglycan (PG) was necessary and sufficient for osmotic bursting of the cell. Recently, we have shown that in Gram-negative hosts, phage lysis also requires the disruption of the outer membrane (OM). This is accomplished by spanins, which are phage-encoded proteins that connect the cytoplasmic membrane (inner membrane, IM) and the OM. The mechanism by which the spanins destroy the OM is unknown. Here we show that the spanins of the paradigm coliphage lambda mediate efficient membrane fusion. This supports the notion that the last step of lysis is the fusion of the IM and OM. Moreover, data are provided indicating that spanin-mediated fusion is regulated by the meshwork of the PG, thus coupling fusion to murein degradation by the phage endolysin. Because endolysin function requires the formation of μm-scale holes by the phage holin, the lysis pathway is seen to require dramatic dynamics on the part of the OM and IM, as well as destruction of the PG.

  6. Arthroscopic lysis in knee arthrofibrosis.

    PubMed

    Vaquero, J; Vidal, C; Medina, E; Baena, J

    1993-01-01

    Arthrofibrosis continues to be a difficult complication in articular surgery on the knee. We present our experience in 21 cases of arthroscopic lysis as an alternative to mobilization under anesthesia. The technique begins with the liberation of the adhesions of the suprapatellar pouch, continues down both gutters, and ends with a cleaning of the notch where necessary. When a restriction of patellar mobility persists, we make a retinacular release. The results obtained are very satisfactory, with an average increase in the arc of mobility of 68 degrees. The average gain in mobility at 6 months is significantly greater than that achieved in the immediate postoperative period. Longer follow-up showed no improvement in range of motion after 6 months.

  7. Current techniques for single-cell lysis.

    PubMed

    Brown, Robert B; Audet, Julie

    2008-10-06

    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.

  8. Tumor lysis syndrome: A clinical review

    PubMed Central

    Mirrakhimov, Aibek E; Voore, Prakruthi; Khan, Maliha; Ali, Alaa M

    2015-01-01

    Tumor lysis syndrome is an oncometabolic emergency resulting from rapid cell death. Tumor lysis syndrome can occur as a consequence of tumor targeted therapy or spontaneously. Clinicians should stratify every hospitalized cancer patient and especially those receiving chemotherapy for the risk of tumor lysis syndrome. Several aspects of prevention include adequate hydration, use of uric acid lowering therapies, use of phosphate binders and minimization of potassium intake. Patients at high risk for the development of tumor lysis syndrome should be monitored in the intensive care unit. Established tumor lysis syndrome should be treated in the intensive care unit by aggressive hydration, possible use of loop diuretics, possible use of phosphate binders, use of uric acid lowering agents and dialysis in refractory cases. PMID:25938028

  9. Methadone induced lysis of mammalian cells.

    PubMed

    Will, P C; Noteboom, W D

    1978-08-01

    Methadone induced lysis of human erythrocytes and mouse leukemic cells was studied. The cells lyse without prior swelling that is a necessary step of colloid osmotic lysis. Methadone is accumulated by both cell types, and is widely distributed intracellurly in mouse leukemic cells. The maximum lytic rate is roughly proportional to the amount of methadone uptake and the Q10 for lysis is equal to the Q10 for methadone partitioning between octanol and water. It is concluded that the cells lyse as a result of a non-specific disruption of the plasma membrane.

  10. Genetic Dissection of T4 Lysis

    PubMed Central

    Moussa, Samir H.; Lawler, Jessica L.

    2014-01-01

    t is the holin gene for coliphage T4, encoding a 218-amino-acid (aa) protein essential for the inner membrane hole formation that initiates lysis and terminates the phage infection cycle. T is predicted to be an integral membrane protein that adopts an Nin-Cout topology with a single transmembrane domain (TMD). This holin topology is different from those of the well-studied holins S105 (3 TMDs; Nout-Cin) of the coliphage lambda and S68 (2 TMDs; Nin-Cin) of the lambdoid phage 21. Here, we used random mutagenesis to construct a library of lysis-defective alleles of t to discern residues and domains important for holin function and for the inhibition of lysis by the T4 antiholin, RI. The results show that mutations in all 3 topological domains (N-terminal cytoplasmic, TMD, and C-terminal periplasmic) can abrogate holin function. Additionally, several lysis-defective alleles in the C-terminal domain are no longer competent in binding RI. Taken together, these results shed light on the roles of the previously uncharacterized N-terminal and C-terminal domains in lysis and its real-time regulation. PMID:24706740

  11. Microfluidic device for acoustic cell lysis

    SciTech Connect

    Branch, Darren W.; Cooley, Erika Jane; Smith, Gennifer Tanabe; James, Conrad D.; McClain, Jaime L.

    2015-08-04

    A microfluidic acoustic-based cell lysing device that can be integrated with on-chip nucleic acid extraction. Using a bulk acoustic wave (BAW) transducer array, acoustic waves can be coupled into microfluidic cartridges resulting in the lysis of cells contained therein by localized acoustic pressure. Cellular materials can then be extracted from the lysed cells. For example, nucleic acids can be extracted from the lysate using silica-based sol-gel filled microchannels, nucleic acid binding magnetic beads, or Nafion-coated electrodes. Integration of cell lysis and nucleic acid extraction on-chip enables a small, portable system that allows for rapid analysis in the field.

  12. Micro-sonicator for spore lysis

    DOEpatents

    Miles, Robin R.; Belgrader, Phillip; Nasarabadi, Shanavaz L.

    2000-01-01

    A micro-sonicator for spore lysis. Using micromachining technology, the micro-sonicator uses ultrasonic excitation of spores to perform spore and cell lysis. The micro-sonicator comprises a container with a cavity therein for retaining the sample in an ultrasonic transmission medium, the cavity being closed by a silicon membrane to which an electrode and piezoelectric material are attached, with the electrode and piezoelectric material being electrically connected to an AC signal generator which causes the membrane to flex and vibrate at the frequency of the applied voltage.

  13. Detergent induced lysis of erythrocytes in kwashiorkor.

    PubMed

    Rao, A; Onuora, C U; Cherian, A

    1987-09-15

    The effect of the non-ionic detergent Nonidet P40 on lysis of erythrocytes in children suffering from kwashiorkor was studied. The concentration of the detergent causing 50% haemolysis was significantly reduced in these patients. Detergent haemolysis was more sensitive than osmotic fragility (which was reduced). The abnormality was only slight in marasmic children.

  14. [Guideline for management of tumor lysis syndrome].

    PubMed

    2011-02-01

    A right management of the tumor lysis syndrome is essential for the ongoing improvement in survival and treatment of patients with hematological malignancies. This guide establishes clinical and laboratory criteria for confirming diagnosis, states initial laboratory tests, enumerates risk factors-based stratification criteria, and develops guidelines for prevention and treatment of the syndrome and its complications.

  15. Factors influencing lysis time stochasticity in bacteriophage λ.

    PubMed

    Dennehy, John J; Wang, Ing-Nang

    2011-08-02

    Despite identical genotypes and seemingly uniform environments, stochastic gene expression and other dynamic intracellular processes can produce considerable phenotypic diversity within clonal microbes. One trait that provides a good model to explore the molecular basis of stochastic variation is the timing of host lysis by bacteriophage (phage). Individual lysis events of thermally-inducible λ lysogens were observed using a temperature-controlled perfusion chamber mounted on an inverted microscope. Both mean lysis time (MLT) and its associated standard deviation (SD) were estimated. Using the SD as a measure of lysis time stochasticity, we showed that lysogenic cells in controlled environments varied widely in lysis times, and that the level of lysis time stochasticity depended on allelic variation in the holin sequence, late promoter (pR') activity, and host growth rate. In general, the MLT was positively correlated with the SD. Both lower pR' activities and lower host growth rates resulted in larger SDs. Results from premature lysis, induced by adding KCN at different time points after lysogen induction, showed a negative correlation between the timing of KCN addition and lysis time stochasticity. Taken together with results published by others, we conclude that a large fraction of λ lysis time stochasticity is the result of random events following the expression and diffusion of the holin protein. Consequently, factors influencing the timing of reaching critical holin concentrations in the cell membrane, such as holin production rate, strongly influence the mean lysis time and the lysis time stochasticity.

  16. Factors influencing lysis time stochasticity in bacteriophage λ

    PubMed Central

    2011-01-01

    Background Despite identical genotypes and seemingly uniform environments, stochastic gene expression and other dynamic intracellular processes can produce considerable phenotypic diversity within clonal microbes. One trait that provides a good model to explore the molecular basis of stochastic variation is the timing of host lysis by bacteriophage (phage). Results Individual lysis events of thermally-inducible λ lysogens were observed using a temperature-controlled perfusion chamber mounted on an inverted microscope. Both mean lysis time (MLT) and its associated standard deviation (SD) were estimated. Using the SD as a measure of lysis time stochasticity, we showed that lysogenic cells in controlled environments varied widely in lysis times, and that the level of lysis time stochasticity depended on allelic variation in the holin sequence, late promoter (pR') activity, and host growth rate. In general, the MLT was positively correlated with the SD. Both lower pR' activities and lower host growth rates resulted in larger SDs. Results from premature lysis, induced by adding KCN at different time points after lysogen induction, showed a negative correlation between the timing of KCN addition and lysis time stochasticity. Conclusions Taken together with results published by others, we conclude that a large fraction of λ lysis time stochasticity is the result of random events following the expression and diffusion of the holin protein. Consequently, factors influencing the timing of reaching critical holin concentrations in the cell membrane, such as holin production rate, strongly influence the mean lysis time and the lysis time stochasticity. PMID:21810267

  17. A new device for measurement of fibrin clot lysis: application to the Euglobulin Clot Lysis Time

    PubMed Central

    Boudjeltia, K Zouaoui; Cauchie, Ph; Remacle, Cl; Guillaume, M; Brohée, D; Hubert, JL; Vanhaeverbeek, M

    2002-01-01

    Background Determination of clot lysis times on whole blood, diluted whole blood, plasma or plasma fraction has been used for many years to assess the overall activity of the fibrinolytic system. We designed a completely computerised semi-automatic 8-channel device for measurement and determination of fibrin clot lysis. The lysis time is evaluated by a mathematical analysis of the lysis curve and the results are expressed in minute (range: 5 to 9999). We have used this new device for Euglobulin Clot Lysis Time (ECLT) determination, which is the most common test used in laboratories to estimate plasma fibrinolytic capacity. Results The correlation between ECLT and manual method is very tight : R = 0,99; p < 10-6. The efficiency scores of the method are <4% in intra-assay and <7% in inter-assay. It allows to achieve the tests on hyperlipaemic samples. This new device has been easily integrated in laboratory routine and allows to achieve several ECLT every day without disturbance of laboratory workflow. Conclusions The routine use of this new device could be useful in various situations such as assessment in atherosclerosis and arteriosclerosis associated diseases, coagulation survey of liver transplantations, cardiovascular surgery or pharmacological research. It has already provided highly promising results in preliminary studies on the relation between fibrinolysis and cardiovascular risk factors. PMID:11985782

  18. Solubilization of proteins: the importance of lysis buffer choice.

    PubMed

    Peach, Mandy; Marsh, Noelle; Miskiewicz, Ewa I; MacPhee, Daniel J

    2015-01-01

    The efficient extraction of proteins of interest from cells and tissues is not always straightforward. Here we demonstrate the differences in extraction of the focal adhesion protein Kindlin-2 from choriocarcinoma cells using NP-40 and RIPA lysis buffer. Furthermore, we demonstrate the use of a more denaturing urea/thiourea lysis buffer for solubilization, by comparing its effectiveness for solubilization of small heat-shock proteins from smooth muscle with the often utilized RIPA lysis buffer. Overall, the results demonstrate the importance of establishing the optimal lysis buffer for specific protein solubilization within the experimental workflow.

  19. Electrical lysis of cells for detergent-free droplet assays

    PubMed Central

    Tran, T. M.; Abate, A. R.

    2016-01-01

    Efficient lysis is critical when analyzing single cells in microfluidic droplets, but existing methods utilize detergents that can interfere with the assays to be performed. We demonstrate robust cell lysis without the use of detergents or other chemicals. In our method, cells are exposed to electric field immediately before encapsulation in droplets, resulting in cell lysis. We characterize lysis efficiency as a function of control parameters and demonstrate compatibility with enzymatic assays by measuring the catalysis of β-glucosidase, an important cellulase used in the conversion of biomass to biofuel. Our method enables assays in microfluidic droplets that are incompatible with detergents. PMID:27051471

  20. Microscopic lysis of lumbar adhesive arachnoiditis.

    PubMed

    Johnston, J D; Matheny, J B

    1978-03-01

    The results of a long-term study of 28 patients operated on for adhesive lumbar arachnoiditis are presented. The technique involved was microscopic lysis of adhesions. The first case of surgery was performed in 1966 and the last, in 1970, with followup through 1976. Numerous observations are made regarding the clinical picture and the appearance of arachnoiditis at the time of surgery. Some conclusions are drawn regarding the causes of this condition with some emphasis on the role of Pantopaque, multiple surgeries, and other trauma. The conclusion is that surgical attack on arachnoiditis is a straightforward surgical exercise that, when carried out with appropriate caution, produces no further neurologic deficits and some short-term improvement. However, the authors feel that this procedure should not be performed at the present time because there does not appear to be a method for preventing the reaccumulation of the scar tissue and subsequent recurrence of the symptoms.

  1. 21 CFR 864.7275 - Euglobulin lysis time tests.

    Code of Federal Regulations, 2012 CFR

    2012-04-01

    ... 21 Food and Drugs 8 2012-04-01 2012-04-01 false Euglobulin lysis time tests. 864.7275 Section 864.7275 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) MEDICAL DEVICES HEMATOLOGY AND PATHOLOGY DEVICES Hematology Kits and Packages § 864.7275 Euglobulin lysis...

  2. 21 CFR 864.7275 - Euglobulin lysis time tests.

    Code of Federal Regulations, 2013 CFR

    2013-04-01

    ... 21 Food and Drugs 8 2013-04-01 2013-04-01 false Euglobulin lysis time tests. 864.7275 Section 864.7275 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) MEDICAL DEVICES HEMATOLOGY AND PATHOLOGY DEVICES Hematology Kits and Packages § 864.7275 Euglobulin lysis...

  3. 21 CFR 864.7275 - Euglobulin lysis time tests.

    Code of Federal Regulations, 2014 CFR

    2014-04-01

    ... 21 Food and Drugs 8 2014-04-01 2014-04-01 false Euglobulin lysis time tests. 864.7275 Section 864.7275 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) MEDICAL DEVICES HEMATOLOGY AND PATHOLOGY DEVICES Hematology Kits and Packages § 864.7275 Euglobulin lysis...

  4. An integratable microfluidic cartridge for forensic swab samples lysis.

    PubMed

    Yang, Jianing; Brooks, Carla; Estes, Matthew D; Hurth, Cedric M; Zenhausern, Frederic

    2014-01-01

    Fully automated rapid forensic DNA analysis requires integrating several multistep processes onto a single microfluidic platform, including substrate lysis, extraction of DNA from the released lysate solution, multiplexed PCR amplification of STR loci, separation of PCR products by capillary electrophoresis, and analysis for allelic peak calling. Over the past several years, most of the rapid DNA analysis systems developed started with the reference swab sample lysate and involved an off-chip lysis of collected substrates. As a result of advancement in technology and chemistry, addition of a microfluidic module for swab sample lysis has been achieved in a few of the rapid DNA analysis systems. However, recent reports on integrated rapid DNA analysis systems with swab-in and answer-out capability lack any quantitative and qualitative characterization of the swab-in sample lysis module, which is important for downstream forensic sample processing. Maximal collection and subsequent recovery of the biological material from the crime scene is one of the first and critical steps in forensic DNA technology. Herein we present the design, fabrication and characterization of an integratable swab lysis cartridge module and the test results obtained from different types of commonly used forensic swab samples, including buccal, saliva, and blood swab samples, demonstrating the compatibility with different downstream DNA extraction chemistries. This swab lysis cartridge module is easy to operate, compatible with both forensic and microfluidic requirements, and ready to be integrated with our existing automated rapid forensic DNA analysis system. Following the characterization of the swab lysis module, an integrated run from buccal swab sample-in to the microchip CE electropherogram-out was demonstrated on the integrated prototype instrument. Therefore, in this study, we demonstrate that this swab lysis cartridge module is: (1) functionally, comparable with routine benchtop lysis

  5. Changes in plasma membrane phospholipids inhibit antibody-mediated lysis.

    PubMed

    Harris, David T

    2012-01-06

    A variety of mechanisms have been proposed to explain how tumors evade immune destruction. This work has identified one such mechanism that determines susceptibility to immune lysis; membrane phospholipid composition altered susceptibility to antibody plus complement (Ab+C)-mediated lysis. Effects on antibody plus complement-mediated lysis were correlated with levels of major histocompatibility complex (MHC) molecules but not inherent resistance to complement damage. This cellular mechanism could be a means by which tumor cells escape immune detection and destruction. Copyright © 2011 Elsevier Inc. All rights reserved.

  6. Tumor Lysis Syndrome in a Retroperitoneal Sarcoma.

    PubMed

    Zakharia, Yousef; Mansour, Joshua; Vasireddi, Srinivasa; Zakharia, Kais; Fatakhov, Eduard; Koch, Christopher; Hrinczenko, Borys

    2014-01-01

    In the present case, a 49-year-old white female presented to the clinic with a 2-month history of nausea, vomiting, and right upper quadrant pain. On examination a 3-cm mass on the right anterior scalene muscle was noted. A computed tomography scan was performed revealing a 8.7 × 7.7 × 6.1 cm retroperitoneal mass with possible invasion of the inferior vena cava and right renal and left common iliac veins. An excisional biopsy was performed with pathology compatible with spindle cell sarcoma. The patient was then sent for follow-up at the sarcoma clinic as an outpatient. However, before chemotherapy was to be started the patient would be admitted to the hospital with progressively worse nausea and vomiting. At that time the patient's lab work showed lactic acidosis, acute renal failure, hyperuricemia, hyperphosphatemia, and hypocalcemia, which met the Cairo-Bishop criteria for tumor lysis syndrome (TLS). The patient was admitted to the intensive care unit and kidney dialysis initiated. The patient would become progressively obtunded at which time the family opted for hospice care. The patient eventually succumbed peacefully 3 days after her last admission. In this case report, we briefly review the literature on TLS in solid tumors, and we present a rare case of spontaneous TLS in a retroperitoneal sarcoma.

  7. Lateral lid lysis for medial lid and medial canthus lacerations.

    PubMed

    Hecht, S D; Dijkstal, D

    1985-02-01

    Techniques are now being used to provide adequate healing time for medial lid and medial canthal full-thickness lacerations. Lateral upper and lower lid lysis allows the needed extended period of healing.

  8. Nickel-inducible lysis system in Synechocystis sp. PCC 6803

    PubMed Central

    Liu, Xinyao; Curtiss, Roy

    2009-01-01

    We designed and constructed a controllable inducing lysis system in Synechocystis sp. PCC 6803 to facilitate extracting lipids for biofuel production. Several bacteriophage-derived lysis genes were integrated into the genome and placed downstream of a nickel-inducible signal transduction system. We applied 3 strategies: (i) directly using the phage lysis cassette, (ii) constitutively expressing endolysin genes while restricting holin genes, and (iii) combining lysis genes from different phages. Significant autolysis was induced in the Synechocystis sp. PCC 6803 cells with this system by the addition of NiSO4. Our inducible cyanobacterial lysing system eliminates the need for mechanical or chemical cell breakage and could facilitate recovery of biofuel from cyanobacteria. PMID:19995962

  9. Arthroscopic Lysis of Arthrofibrosis of the Fifth Tarsometatarsal Joint.

    PubMed

    Lui, Tun Hing

    2015-12-01

    Loss of motion of the fifth tarsometatarsal joint can be a cause of lateral foot pain after Lisfranc fracture-dislocation or fracture of the fifth metatarsal tubercle. Arthroscopic lysis of the joint can be an effective surgical treatment with the advantage of minimal soft-tissue trauma and early vigorous mobilization of the joint. The lysis can be extended to the fourth tarsometatarsal joint and the adjacent tendons if indicated.

  10. Urea enhances cell lysis of Schizosaccharomyces pombe ura4 mutants.

    PubMed

    Nishino, Kohei; Kushima, Misaki; Kaino, Tomohiro; Matsuo, Yasuhiro; Kawamukai, Makoto

    2017-07-01

    Cell lysis is induced in Schizosaccharomyces pombe ∆ura4 cells grown in YPD medium, which contains yeast extract, polypeptone, and glucose. To identify the medium components that induce cell lysis, we first tested various kinds of yeast extracts from different suppliers. Cell lysis of ∆ura4 cells on YE medium was observed when yeast extracts from OXOID, BD, Oriental, and Difco were used, but not when using yeast extract from Kyokuto. To determine which compounds induced cell lysis, we subjected yeast extract and polypeptone to GC-MS analysis. Ten kinds of compounds were detected in OXOID and BD yeast extracts, but not in Kyokuto yeast extract. Among them was urea, which was also present in polypeptone, and it clearly induced cell lysis. Deletion of the ure2 gene, which is responsible for utilizing urea, abolished the lytic effect of urea. The effect of urea was suppressed by deletion of pub1, and a similar phenotype was observed in the presence of polypeptone. Thus, urea is an inducer of cell lysis in S. pombe ∆ura4 cells.

  11. Delayed Lysis with Salmonella Bacteriophage P22: Induction of Lysis by Addition of Cysteine or Histidine to the Growth Medium

    PubMed Central

    Cohen, Larry W.

    1969-01-01

    A mutant (Lys−) of Salmonella bacteriophage P22 showed a delay in lysis of more than 3 hr in infections in unsupplemented M9 medium. The infected cells were induced to lyse during that interval by addition of histidine or sulfhydryl compounds cysteine, mercaptoethanol, glutathione, or ergothioneine. Urocanic acid, the first intermediate in the catabolic histidine pathway, did not induce lysis, nor did histamine, imidazolelactate, or carnosine. None of the other amino acids common to protein had any inductive effect. Both the d and l forms of histidine were effective in inducing lysis, suggesting that the incorporation of the histidine into protein is not involved. Chloramphenicol inhibited lysis when added at 60 min with or without histidine, but did not inhibit the induction of lysis when added with cysteine. Bacterial cells infected with Lys+ phage were induced to lyse prematurely when cysteine was added at 30 min but not at 20 min of infection. Iodoacetate inhibited lysis of Lys+-infected cells when added at 20 min but not at 30 min. PMID:16789095

  12. Carfilzomib-associated tumor lysis syndrome.

    PubMed

    Shely, Ryan N; Ratliff, Patrick D

    2014-05-01

    Multiple myeloma is the second most common type of hematologic malignancy. It is a B-cell malignancy that affects the bone marrow and often results in thrombocytopenia as well as renal dysfunction. Treatment options range from oral and intravenous chemotherapy to bone marrow transplantation and supportive care. Carfilzomib was approved by the U.S. Food and Drug Administration in 2012 as a treatment option for patients with refractory multiple myeloma who have received at least two previous therapies and have demonstrated recent disease progression. According to the product labeling, the frequency of tumor lysis syndrome (TLS) is less than 1% in patients treated with carfilzomib. To our knowledge, no postmarketing events of TLS have been reported or published. We describe a 55-year-old man with relapsed multiple myeloma who developed a case of TLS that occurred after he received his first two doses of carfilzomib therapy on days 1 and 2; he also had chronic kidney disease secondary to his neoplastic disease. Beginning on day 4, his uric acid levels spiked to critical levels, prompting the use of rasburicase, which returned the levels to within normal limits. His phosphorus and creatinine levels increased during days 5 and 6. On day 8, the patient died, likely due to a combination of disease progression and the adverse effects of treatment. Use of the Naranjo adverse drug reaction probability scale indicated a probable relationship (score of 6) between the patient's development of TLS and carfilzomib therapy. The Hill criteria were used as a secondary measure to ensure causality, which also suggested a link between the patient's development of TLS and the administration of carfilzomib. This case report shows that even the most unlikely of adverse events may occur with medications, especially in the case of a new or recently approved medication. Caution must be taken when deciding to treat and when choosing hydration and premedications with regard to biologic and

  13. Primary lysis of eosinophils in severe desquamative asthma.

    PubMed

    Persson, C

    2014-02-01

    Primary lysis of eosinophils liberates free eosinophil granules (FEGs) releasing toxic proteins in association with bronchial epithelial injury repair. Eosinophil lysis may be significantly pathogenic. Bronchial mucosal FEGs are associated with uncontrolled asthma, severe asthma, aspirin-sensitive asthma, and lethal asthma. FEGs in the bronchial wall may characterize severe asthma without sputum eosinophilia. Excessive numbers of sputum FEGs occur in severe exacerbations of asthma and are reduced along with clinical improvement. Occurrence of FEGs affects interpretation of other sputum biomarkers including numbers of eosinophils, ECP, and eosinophil-stained macrophages. Thus, eosinophil lysis produces FEGs as bronchial biomarkers of severe asthma. Blood eosinophils in severe asthma seem primed exhibiting a propensity to lyse that is greater the more severe the asthma. Proclivity of blood eosinophils to lyse also distinguished three levels of severity among children with exacerbations of asthma. Numerous FEGs releasing toxic proteins occur in association with grave derangement and shedding of epithelium in severe asthma. Subepithelial FEGs correlate negatively with intact bronchial epithelium in clinically uncontrolled asthma. Significant correlations between sputum ECP, Creola bodies, and severity of asthma exacerbations have also been demonstrated. Hence, eosinophil lysis apparently causes epithelial desquamation in severe asthma. Exaggerated epithelial repair in turn would contribute to inflammatory and remodelling features of severe asthma. Perseverance of FEGs together with maintained disease activity, despite treatment with 'eosinophil-depleting' steroids and anti-IL5 biologicals, agrees with the possibility that eosinophil lysis is worthy target for novel anti-asthma drugs. Priming and lysis of eosinophils, and protein release from FEGs, are regulated and can be targeted. Eosinophil lysis and FEGs belong to the disease picture of severe asthma and need

  14. Fixation probability for lytic viruses: the attachment-lysis model.

    PubMed

    Patwa, Z; Wahl, L M

    2008-09-01

    The fixation probability of a beneficial mutation is extremely sensitive to assumptions regarding the organism's life history. In this article we compute the fixation probability using a life-history model for lytic viruses, a key model organism in experimental studies of adaptation. The model assumes that attachment times are exponentially distributed, but that the lysis time, the time between attachment and host cell lysis, is constant. We assume that the growth of the wild-type viral population is controlled by periodic sampling (population bottlenecks) and also include the possibility that clearance may occur at a constant rate, for example, through washout in a chemostat. We then compute the fixation probability for mutations that increase the attachment rate, decrease the lysis time, increase the burst size, or reduce the probability of clearance. The fixation probability of these four types of beneficial mutations can be vastly different and depends critically on the time between population bottlenecks. We also explore mutations that affect lysis time, assuming that the burst size is constrained by the lysis time, for experimental protocols that sample either free phage or free phage and artificially lysed infected cells. In all cases we predict that the fixation probability of beneficial alleles is remarkably sensitive to the time between population bottlenecks.

  15. Spontaneous Tumor Lysis Syndrome in Small Cell Lung Cancer

    PubMed Central

    Saladi, Swetha; Patolia, Setu; Stoeckel, David

    2017-01-01

    Tumor lysis syndrome (TLS) is a life-threatening oncologic complication caused by the lysis of a vast number of malignant cells resulting in metabolic derangements and organ dysfunction. TLS can occur spontaneously before initiation of any therapies often referred to as spontaneous tumor lysis syndrome (STLS), or shortly after the induction of chemotherapy, radiotherapy, or cytolytic antibody therapy. TLS is vastly seen in patients with hematological malignancies with high rapid cell turnover rates such as Burkitt lymphoma, acute myelogenous leukemia, and acute lymphocytic leukemia, and is rarely observed in solid tumors. However, TLS can occur in solid tumors, and there are multiple reports in the literature on the occurrence of TLS in various solid tumors. In this article, we report a case of STLS in small cell lung cancer followed by a brief review of the occurrence of TLS and STLS in small cell lung cancer. PMID:28344911

  16. The Spanin Complex Is Essential for Lambda Lysis

    PubMed Central

    Berry, Joel; Rajaure, Manoj; Pang, Ting

    2012-01-01

    Phage lysis is a ubiquitous biological process, the most frequent cytocidal event in the biosphere. Lysis of Gram-negative hosts has been shown to require holins and endolysins, which attack the cytoplasmic membrane and peptidoglycan, respectively. Recently, a third class of lysis proteins, the spanins, was identified. The first spanins to be characterized were λ Rz and Rz1, an integral cytoplasmic membrane protein and an outer membrane lipoprotein, respectively. Previous work has shown that Rz and Rz1 form complexes that span the entire periplasm. Phase-contrast video microscopy was used to record the morphological changes involved in the lysis of induced λ lysogens carrying prophages with either the λ canonical holin-endolysin system or the phage 21 pinholin-signal anchor release (SAR) endolysin system. In the former, rod morphology persisted until the instant of an explosive polar rupture, immediately emptying the cell of its contents. In contrast, in pinholin-SAR endolysin lysis, the cell began to shorten and thicken uniformly, with the resultant rounded cell finally bursting. In both cases, lysis failed to occur in inductions of isogenic prophages carrying null mutations in the spanin genes. In both systems, instead of an envelope rupture, the induced cells were converted from a rod shape to a spherical form. A functional GFPΦRz chimera was shown to exhibit a punctate distribution when coexpressed with Rz1, despite the absence of endolysin function. A model is proposed in which the spanins carry out the essential step of disrupting the outer membrane, in a manner regulated by the state of the peptidoglycan layer. PMID:22904283

  17. 21 CFR 864.7275 - Euglobulin lysis time tests.

    Code of Federal Regulations, 2010 CFR

    2010-04-01

    ... 21 Food and Drugs 8 2010-04-01 2010-04-01 false Euglobulin lysis time tests. 864.7275 Section 864.7275 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) MEDICAL DEVICES HEMATOLOGY AND PATHOLOGY DEVICES Hematology Kits and Packages § 864.7275 Euglobulin...

  18. Sandia Text ANaLysis Extensible librarY Server

    SciTech Connect

    2006-05-11

    This is a server wrapper for STANLEY (Sandia Text ANaLysis Extensible librarY). STANLEY provides capabilities for analyzing, indexing and searching through text. STANLEY Server exposes this capability through a TCP/IP interface allowing third party applications and remote clients to access it.

  19. 21 CFR 864.7275 - Euglobulin lysis time tests.

    Code of Federal Regulations, 2011 CFR

    2011-04-01

    ... 21 Food and Drugs 8 2011-04-01 2011-04-01 false Euglobulin lysis time tests. 864.7275 Section 864.7275 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) MEDICAL DEVICES HEMATOLOGY AND PATHOLOGY DEVICES Hematology Kits and Packages § 864.7275 Euglobulin...

  20. A novel toolbox for E. coli lysis monitoring.

    PubMed

    Rajamanickam, Vignesh; Wurm, David; Slouka, Christoph; Herwig, Christoph; Spadiut, Oliver

    2017-01-01

    The bacterium Escherichia coli is a well-studied recombinant host organism with a plethora of applications in biotechnology. Highly valuable biopharmaceuticals, such as antibody fragments and growth factors, are currently being produced in E. coli. However, the high metabolic burden during recombinant protein production can lead to cell death, consequent lysis, and undesired product loss. Thus, fast and precise analyzers to monitor E. coli bioprocesses and to retrieve key process information, such as the optimal time point of harvest, are needed. However, such reliable monitoring tools are still scarce to date. In this study, we cultivated an E. coli strain producing a recombinant single-chain antibody fragment in the cytoplasm. In bioreactor cultivations, we purposely triggered cell lysis by pH ramps. We developed a novel toolbox using UV chromatograms as fingerprints and chemometric techniques to monitor these lysis events and used flow cytometry (FCM) as reference method to quantify viability offline. Summarizing, we were able to show that a novel toolbox comprising HPLC chromatogram fingerprinting and data science tools allowed the identification of E. coli lysis in a fast and reliable manner. We are convinced that this toolbox will not only facilitate E. coli bioprocess monitoring but will also allow enhanced process control in the future.

  1. Lysis matters: red cell lysis with FACS Lyse affects the flow cytometric enumeration of circulating leukemic blasts.

    PubMed

    Einwallner, Elisa; Subasic, Almira; Strasser, Andrea; Augustin, Dorothea; Thalhammer, Renate; Steiner, Irene; Schwarzinger, Ilse

    2013-04-30

    The whole blood lysis method has become a standard procedure to remove red cells prior to immunophenotypic analysis of leukocytes. In the present study we investigated the influence of four different lysis protocols on the flow cytometric recovery of leukemic blasts. 32 blast cells containing blood samples were stained with anti-CD45 and anti-CD34 monoclonal antibody combinations. Red cell lysis was performed with FACS Lysing Solution and BD PharmLyse™ (Becton Dickinson and Company BD Biosciences, San Jose, CA; n=32) as well as Optilyse C and IOTest 3 (Immunotech SAS, Marseille; n=15 out of 32). Flow cytometric enumeration of blasts was performed on a FACS-Canto flow cytometer. The percentage of blasts after treatment with FACS Lyse was significantly smaller than after PharmLyse™ (p<0.0001), Optilyse C (p<0.0001), or IOTest 3 (p<0.0001), respectively. The difference between PharmLyse™ and Optilyse C (p=0.93), PharmLyse™ and IOTest 3 (p=0.31), and Optilyse C and IOTest 3 (p=0.34) was not significant. These results emphasize the importance of harmonization of red cell lysis protocols for the application of flow cytometry in hematological neoplasms. Copyright © 2013 Elsevier B.V. All rights reserved.

  2. Comparison of human red cell lysis by hypochlorous and hypobromous acids: insights into the mechanism of lysis.

    PubMed Central

    Vissers, M C; Carr, A C; Chapman, A L

    1998-01-01

    Human red blood cells are lysed by the neutrophil-derived oxidant hypochlorous acid (HOCl), although the mechanism of lysis is unknown. Hypobromous acid (HOBr), a similarly reactive oxidant, lysed red cells approx. 10-fold faster than HOCl. Therefore we compared the effects of these oxidants on thiols, membrane lipids and proteins to determine which reactions are associated with lysis. There was no difference in the loss of reduced glutathione or membrane thiols with either oxidant, but HOBr reacted more readily with membrane lipids and proteins. Bromohydrin derivatives of phospholipids and cholesterol were seen at approx. one-tenth the level of oxidant than chlorohydrins were. However, these products were detected only with high concentrations of HOCl or HOBr, which caused instant haemolysis. Membrane protein modification occurred at much lower doses of oxidant and was more closely correlated with lysis. SDS/PAGE analysis showed that band 3, the anion transport protein, was lost at the lowest dose of HOBr and at the higher concentrations of HOCl. Labelling the red cells with eosin 5-maleimide, a fluorescent label for band 3, suggested possible clustering of this protein in oxidant-exposed cells. There was also irreversible cross-linking of all the major membrane proteins; this reaction occurred more readily with HOBr. The results indicate that membrane protein modification is the reaction responsible for HOCl-mediated lysis. These effects, and particularly cross-link formation, might result in clustering of band 3 and other membrane and cytoskeletal proteins to form haemolytic pores. PMID:9461501

  3. Proximal bacterial lysis and detection in nanoliter wells using electrochemistry.

    PubMed

    Besant, Justin D; Das, Jagotamoy; Sargent, Edward H; Kelley, Shana O

    2013-09-24

    Rapid and direct genetic analysis of low numbers of bacteria using chip-based sensors is limited by the slow diffusion of mRNA molecules. Long incubation times are required in dilute solutions in order to collect a sufficient number of molecules at the sensor surface to generate a detectable signal. To overcome this barrier here we present an integrated device that leverages electrochemistry-driven lysis less than 50 μm away from electrochemical nucleic acid sensors to overcome this barrier. Released intracellular mRNA can diffuse the short distance to the sensors within minutes, enabling rapid and sensitive detection. We validate this strategy through direct lysis and detection of E. coli mRNA at concentrations as low as 0.4 CFU/μL in 2 min, a clinically relevant combination of speed and sensitivity for a sample-to-answer molecular analysis approach.

  4. Direct Cellular Lysis/Protein Extraction Protocol for Soil Metaproteomics

    SciTech Connect

    Chourey, Karuna; Jansson, Janet; Verberkmoes, Nathan C; Shah, Manesh B; Chavarria, Krystle L.; Tom, Lauren M; Brodie, Eoin L.; Hettich, Robert {Bob} L

    2010-01-01

    We present a novel direct protocol for deep proteome characterization of microorganisms in soil. The method employs thermally assisted detergent-based cellular lysis (SDS) of soil samples, followed by TCA precipitation for proteome extraction/cleanup prior to liquid chromatography-mass spectrometric characterization. This approach was developed and optimized using different soils inoculated with genome-sequenced bacteria (Gram-negative Pseudomonas putida or Gram-positive Arthrobacter chlorophenolicus). Direct soil protein extraction was compared to protein extraction from cells isolated from the soil matrix prior to lysis (indirect method). Each approach resulted in identification of greater than 500 unique proteins, with a wide range in molecular mass and functional categories. To our knowledge, this SDS-TCA approach enables the deepest proteome characterizations of microbes in soil to date, without significant biases in protein size, localization, or functional category compared to pure cultures. This protocol should provide a powerful tool for ecological studies of soil microbial communities.

  5. Spontaneous Tumour Lysis Syndrome in a Multiple Myeloma

    PubMed Central

    Eliacik, Eylem; Saglam, Mustafa; Doner, Baris

    2016-01-01

    The tumor lysis syndrome (TLS) is a collection of metabolic abnormalities that occur in consequence of the release of intracellular contents following lysis of tumor cells. TLS occurs spontaneously or after chemotherapy. Spontaneous TLS is uncommon occurrence in multiple myeloma (MM). We define a case of a 70-year-old woman patient who was found to have MM with spontaneous TLS, following a compression fracture of the T-12 vertebrae. While serum uric acid and phosphorous levels were high, low calcium levels were identified. There were also acute kidney injury and metabolic acidosis. Upon the diagnosis of TLS, she was treated with hydration, allopurinol, sodium bicarbonate, and calcium gluconate. The improvement of her laboratory data was observed. We submitted this case in order to draw attention to the presentation of MM with spontaneous TLS. PMID:27956901

  6. Direct cellular lysis/protein extraction protocol for soil metaproteomics.

    PubMed

    Chourey, Karuna; Jansson, Janet; VerBerkmoes, Nathan; Shah, Manesh; Chavarria, Krystle L; Tom, Lauren M; Brodie, Eoin L; Hettich, Robert L

    2010-12-03

    We present a novel direct protocol for deep proteome characterization of microorganisms in soil. The method employs thermally assisted detergent-based cellular lysis (SDS) of soil samples, followed by TCA precipitation for proteome extraction/cleanup prior to liquid chromatography-mass spectrometric characterization. This approach was developed and optimized using different soils inoculated with genome-sequenced bacteria (Gram-negative Pseudomonas putida or Gram-positive Arthrobacter chlorophenolicus). Direct soil protein extraction was compared to protein extraction from cells isolated from the soil matrix prior to lysis (indirect method). Each approach resulted in identification of greater than 500 unique proteins, with a wide range in molecular mass and functional categories. To our knowledge, this SDS-TCA approach enables the deepest proteome characterizations of microbes in soil to date, without significant biases in protein size, localization, or functional category compared to pure cultures. This protocol should provide a powerful tool for ecological studies of soil microbial communities.

  7. Enhanced Spore Biomarker Detection Following Laser Induced Lysis

    SciTech Connect

    Wunschel, David S.; Beck, Kenneth M.; Wahl, Karen L.

    2002-12-01

    Matrix assisted laser desorption/ionization (MALDI) has grown in popularity as a means to rapidly analyze proteins directly from bacterial cells. This method provides identifying information by generating protein ?fingerprints? for each organism. However, generating rich protein fingerprints from spores, such as from the genus Bacillus, has proven difficult. We have examined the use of laser energy to induce spore lysis and increase the protein signature complexity. As a measure of lysis, the ions from calcium and dipicolinic acid (DPA) were monitored along with the higher m/z protein ions. DPA is a known marker of eubacterial spores usually as a complex with calcium. This is in contrast to the abundant geogenic calcium complexes with carbonate among other forms. A combination of general bacterial markers, DPA and calcium, and protein fingerprints can be used to provide complementary biomarkers from a single sample preparation.

  8. Radiation-Induced Tumor Lysis Syndrome in Chronic Lymphocytic Leukemia

    PubMed Central

    Alkan, Ali; Kütük, Tuğçe; Karcı, Ebru; Yaşar, Arzu; Hiçsönmez, Ayşe; Utkan, Güngör

    2016-01-01

    Tumor lysis syndrome (TLS) is an important oncological emergency that is usually observed with hematological malignancies and rarely with solid tumors. It can be induced either by therapy or spontaneously. Radiotherapy-induced TLS has been rarely reported in the literature. Here we present a patient with a diagnosis of metastatic prostate cancer and chronic lymphocytic leukemia complicated with TLS during palliative radiotherapy. PMID:27093891

  9. Synchronized cycles of bacterial lysis for in vivo delivery

    PubMed Central

    Prindle, Arthur; Skalak, Matt; Selimkhanov, Jangir; Allen, Kaitlin; Julio, Ellixis; Atolia, Eta; Tsimring, Lev S.; Bhatia, Sangeeta N.; Hasty, Jeff

    2016-01-01

    The pervasive view of bacteria as strictly pathogenic has given way to an appreciation of the widespread prevalence of beneficial microbes within the human body1–3. Given this milieu, it is perhaps inevitable that some bacteria would evolve to preferentially grow in environments that harbor disease and thus provide a natural platform for the development of engineered therapies4–6. Such therapies could benefit from bacteria that are programmed to limit bacterial growth while continually producing and releasing cytotoxic agents in situ7–10. Here, we engineer a clinically relevant bacterium to lyse synchronously at a threshold population density and to release genetically encoded cargo. Following quorum lysis, a small number of surviving bacteria reseed the growing population, thus leading to pulsatile delivery cycles. We use microfluidic devices to characterize the engineered lysis strain and we demonstrate its potential as a drug delivery platform via co-culture with human cancer cells in vitro. As a proof of principle, we track the bacterial population dynamics in ectopic syngeneic colorectal tumors in mice. The lysis strain exhibits pulsatile population dynamics in vivo, with mean bacterial luminescence that remained two orders of magnitude lower than an unmodified strain. Finally, guided by previous findings that certain bacteria can enhance the efficacy of standard therapies11, we orally administer the lysis strain, alone or in combination with a clinical chemotherapeutic, to a syngeneic transplantation model of hepatic colorectal metastases. We find that the combination of both circuit-engineered bacteria and chemotherapy leads to a notable reduction of tumor activity along with a marked survival benefit over either therapy alone. Our approach establishes a methodology for leveraging the tools of synthetic biology to exploit the natural propensity for certain bacteria to colonize disease sites. PMID:27437587

  10. An Interesting Case of Intramuscular Myxoma with Scapular Bone Lysis

    PubMed Central

    Tirefort, Jérôme; Kolo, Frank C.

    2017-01-01

    Introduction. Intramuscular myxoma is a rare benign primitive tumor of the mesenchyme founded at the skeletal muscle level; it presents itself like an unpainful, slow-growing mass. Myxomas with bone lysis are even more rare; only 7 cases have been reported in the English literature, but never at the shoulder level. Case Presentation. We describe an 83-year-old patient with a growing mass in the deltoid muscle with unique scapular lysis, without any symptom. Magnetic resonance imaging (MRI) and a biopsy were performed and the diagnosis of intramuscular myxoma has been retained. In front of this diagnosis of nonmalignant lesion, the decision of a simple follow-up was taken. One year after this decision, the patient was still asymptomatic. Conclusion. In the presence of an intramuscular growing mass with associated bone lysis, intramuscular myxoma as well as malignant tumor should be evoked. MRI has to be part of the initial radiologic appraisal but biopsy is essential to confirm the diagnosis. By consensus, the standard treatment is surgical excision but conservative treatment with simple follow-up can be an option. PMID:28194289

  11. Transcranial Clot Lysis Using High Intensity Focused Ultrasound

    NASA Astrophysics Data System (ADS)

    Hölscher, Thilo; Zadicario, Eyal; Fisher, David J.; Bradley, William G.

    2010-03-01

    Stroke is the third common cause of death worldwide. The majority of strokes are caused by sudden vessel occlusion, due to a blood clot. Vessel recanalization is the primary goal of all acute stroke treatment strategies. Initial data using ultrasound in combination with a therapeutic agent for clot lysis in stroke are promising. However, sound absorption and defocusing of the ultrasound beam occur during transskull insonation, limiting the efficiency of this approach to high extent. Using a transskull High Intensity Focused Ultrasound (HIFU) head system we were able to lyse blood clots within seconds and in absence of further lytic agents. We could show that any correction for the distortion might be negligible to focus the ultrasound beam after transskull insonation. The use of transskull HIFU for immediate clot lysis in the human brain without the need of further drugs and disregarding individual skull bone characteristics could become a successful strategy in early stroke treatment. Using magnetic resonance tomography for neuronavigation MRI Guided High Intensity Focused Ultrasound has the potential to open new avenues for therapeutic applications in the brain including Stroke, Intracranial Hemorrhages, Braintumors, Neurodegenerative Diseases, Thalamic Pain, BBB opening, and local drug delivery. First results in transcranial clot lysis will be presented in this paper.

  12. Lysis of Blue-Green Algae by Myxobacter

    PubMed Central

    Shilo, Miriam

    1970-01-01

    Enrichment from local fishponds led to the isolation of a bacterium capable of lysing many species of unicellular and filamentous blue-green algae, as well as certain bacteria. The isolate is an aflagellate, motile rod which moves in a gliding, flexuous manner; the organism is capable of digesting starch and agar, but not cellulose and gelatin. Its deoxyribonucleic acid base pair composition (per cent guanine plus cytosine ∼70) shows a close resemblance to that of the fruiting myxobacteria. Algae in lawns on agar plates were lysed rapidly by the myxobacter, but only limited and slow lysis occurred in liquid media, and no lysis took place when liquid cultures were shaken. No diffusible lytic factors would be demonstrated. Continuous observation of the lytic process under a phase-contrast microscope suggested that a close contact between the polar tip of the myxobacter and the alga is necessary for lysis. The lytic action is limited to the vegetative cells of the algae, whereas heterocysts are not affected. The gas vacuoles of the algal host are the only remnant visible after completion of digestion by the myxobacter. Images PMID:4990764

  13. Results of surgical lysis of lumbar adhesive arachnoiditis.

    PubMed

    Wilkinson, H A; Schuman, N

    1979-05-01

    From a series of 681 patients with lumbar disc disease treated between 1966 and 1978, 17 patients required surgical lysis of lumbar adhesive arachnoiditis, 8 having initially been operated upon by another surgeon. All patients had severe pain as a predominant feature, with pain being bilateral in 9 patients. Pain was the only major symptom in 3; the other 14 exhibited varying combinations of progressive neurological dysfunction. Three patients developed late symptoms after trauma, 8 to 21 years after back surgery. At operation, multisegmental arachnoiditis was found in 5 patients and anular or subtotal adhesions were found in 12. Complete lysis could not be obtained in 4 patients. Fourteen patients were treated with steroids at the time of operation. Follow-up after lysis was less than 1 year for 5 patients but averaged 4.8 years for the remaining 12. During the 1st year after operation, 76% experienced improvement in pain (35%, good to excellent), 71% experienced improvement in neurological status. Follow-up after at least 1 year revealed 50% still enjoying pain relief (25%, good to excellent) and 45% experiencing neurological improvement. Pain relief persisted in 4 of 5 patients followed 5 years or more. The etiological role of myelograpy and lumbar disc surgery in arachnoiditis has probably been over-rated. Arachnoiditis may be symptomatic or asymptomatic and may mask other, treatable lumbar lesions. More frequent intradural exploration for discrepancies between operative and myelographic findings might reveal, and benefit, more cases of spontaneous arachnoiditis mimicking lumbar disc disease.

  14. Wireless induction heating in a microfluidic device for cell lysis.

    PubMed

    Baek, Seung-ki; Min, Junghong; Park, Jung-Hwan

    2010-04-07

    A wireless induction heating system in a microfluidic device was devised for cell lysis to extract DNA and RNA from Escherichia coli. The thermal responses of nickel, iron and copper heating units were studied by applying an alternating magnetic field as a function of geometry of unit, strength of magnetic field, and kind of metal. Heating units were prepared by cutting metal film using a fiber laser, and the units were integrated into a microchannel system using a soft lithographic process. Variation and distribution of temperature on the surface of the heating units was observed using a thermographic camera and temperature labels. The amount of protein released from E. coli by thermal lysis was determined by protein concentration measurement. Hemoglobin released from red blood cells was observed using colorimetric intensity measurement. Extracted DNA was quantified by real-time polymerase chain reaction, and the profile was compared with that of a positive control of ultrasonically disrupted E. coli. The stability of RNA extracted by induction heating was quantified by the measurement of 23S/16S rRNA ratio and comparison with that by normal RNA extraction kit as a gold standard. A solid-shaped nickel structure was selected as the induction heating element in the microfluidic device because of the relatively small influence of geometries and faster thermal response.The amount of protein extracted from E. coli and hemoglobin released from red blood cells by induction heating of the nickel unit in the microfluidic device was proportional to the strength of the applied magnetic field. The lysis of E. coli by induction heating was as effective as lysis of DNA by the ultrasonication method because the threshold cycle values of the sample were compatible with those of the positive control as measured by ultrasonication. Thermal lysis of E. coli by induction heating represents a reasonable alternative to a commercial RNA extraction method as shown by the comparative

  15. Effect of von Willebrand factor on clot structure and lysis.

    PubMed

    Marchi, Rita; Rojas, Héctor

    2015-07-01

    Von Willebrand Factor (vWF) is constitutively secreted by the endothelium and incorporated in the fibrin clots under slow clotting conditions. The aim of the present work was to study the effect of vWF on clot structure and lysis. Purified fibrinogen was mixed with vWF or Tris-buffered saline and clotted with thrombin - activated factor XIII. Fibrin polymerization was followed by turbidity at 350 nm during 2.5 h. After this time, plasmin was added on the top of the clots, and the optical density (OD) was read until baseline values. vWF effect on network[Combining Acute Accent]s porosity was evaluated by permeation using the same clotting conditions as for fibrin polymerization. Clot structure was visualized and analyzed by laser scanning confocal microscopy (LSCM). The rate of fibrin polymerization was 1.47 mOD/s in the presence of vWF and 0.5 mOD/s when vWF was not added (P < 0.05). The fibrin lysis rate was approximately four times faster when vWF was added to fibrinogen. The fibrin network porosity was (20.4 ± 1.6) × 10 cm with vWF and (8.3 ± 1.2) × 10 cm without external vWF (P < 0.05). The analysis of LSCM images showed that vWF increased fibrin fibers diameter and the networks[Combining Acute Accent] pores size. In conclusion, vWF covalently crosslinked to fibrin modify its structure (increases fibrin diameter and the pores filling space of the meshwork) that accelerates the fibrin lysis rate.

  16. Phage lysis: three steps, three choices, one outcome.

    PubMed

    Young, Ryland

    2014-03-01

    The lysis of bacterial hosts by double-strand DNA bacteriophages, once thought to reflect merely the accumulation of sufficient lysozyme activity during the infection cycle, has been revealed to recently been revealed to be a carefully regulated and temporally scheduled process. For phages of Gramnegative hosts, there are three steps, corresponding to subversion of each of the three layers of the cell envelope: inner membrane, peptidoglycan, and outer membrane. The pathway is controlled at the level of the cytoplasmic membrane. In canonical lysis, a phage encoded protein, the holin, accumulates harmlessly in the cytoplasmic membrane until triggering at an allele-specific time to form micron-scale holes. This allows the soluble endolysin to escape from the cytoplasm to degrade the peptidoglycan. Recently a parallel pathway has been elucidated in which a different type of holin, the pinholin, which, instead of triggering to form large holes, triggers to form small, heptameric channels that serve to depolarize the membrane. Pinholins are associated with SAR endolysins, which accumulate in the periplasm as inactive, membrane-tethered enzymes. Pinholin triggering collapses the proton motive force, allowing the SAR endolysins to refold to an active form and attack the peptidoglycan. Surprisingly, a third step, the disruption of the outer membrane is also required. This is usually achieved by a spanin complex, consisting of a small outer membrane lipoprotein and an integral cytoplasmic membrane protein, designated as o-spanin and i-spanin, respectively. Without spanin function, lysis is blocked and progeny virions are trapped in dead spherical cells, suggesting that the outer membrane has considerable tensile strength. In addition to two-component spanins, there are some single-component spanins, or u-spanins, that have an N-terminal outer-membrane lipoprotein signal and a C-terminal transmembrane domain. A possible mechanism for spanin function to disrupt the outer

  17. Phage lysis: three steps, three choices, one outcome

    PubMed Central

    Young, Ry

    2014-01-01

    The lysis of bacterial hosts by double-strand DNA bacteriophages, once thought to reflect merely the accumulation of sufficient lysozyme activity during the infection cycle, has been revealed to recently been revealed to be a carefully regulated and temporally scheduled process. For phages of Gram-negative hosts, there are three steps, corresponding to subversion of each of the three layers of the cell envelope: inner membrane, peptidoglycan, and outer membrane. The pathway is controlled at the level of the cytoplasmic membrane. In canonical lysis, a phage encoded protein, the holin, accumulates harmlessly in the cytoplasmic membrane until triggering at an allele-specific time to form micron-scale holes. This allows the soluble endolysin to escape from the cytoplasm to degrade the peptidoglycan. Recently a parallel pathway has been elucidated in which a different type of holin, the pinholin, which, instead of triggering to form large holes, instead triggers to form small, heptameric channels that serve to depolarize the membrane. Pinholins are associated with SAR endolysins, which accumulate in the periplasm as inactive, membrane-tethered enzymes. Pinholin triggering collapses the proton motive force, allowing the SAR endolysins to refold to an active form and attack the peptidoglycan. Surprisingly, a third step, the disruption of the outer membrane is also required. This is usually achieved by a spanin complex, consisting of a small outer membrane lipoprotein and an integral cytoplasmic membrane protein, designated as o-spanins and i-spanins, respectively. Without spanin function, lysis is blocked and progeny virions are trapped in dead spherical cells, suggesting that the outer membrane has considerable tensile strength. In addition to two-component spanins, there are some single-component spanins, or u-spanins, that have an N-terminal outer-membrane lipoprotein signal and a C-terminal transmembrane domain. A possible mechanism for spanin function to disrupt the

  18. Miniature acoustic wave lysis system and uses thereof

    DOEpatents

    Branch, Darren W.; Vreeland, Erika Cooley; Smith, Gennifer Tanabe

    2016-12-06

    The present invention relates to an acoustic lysis system including a disposable cartridge that can be reversibly coupled to a platform having a small, high-frequency piezoelectric transducer array. In particular, the system releases viable DNA, RNA, and proteins from human or bacterial cells, without chemicals or additional processing, to enable high-speed sample preparation for clinical point-of-care medical diagnostics and use with nano/microfluidic cartridges. Also described herein are methods of making and using the system of the invention.

  19. Electroporation-induced cell lysis in SWLA-2 hybridomas.

    PubMed

    Oakley, Barbara A; Gross, Brooks A; Martens, Sara G; Hanna, Darrin M; Stryker, Gabrielle A

    2006-01-01

    This paper describes experimental results involving the percentage cell lysis in SWLA-2 murine hybridomas produced by square wave electric field pulses of 100, 200, and 300 V across a 1 mm gap width in a standard cuvette. Pulse lengths were of 0.2 and 0.6 ms duration; 1, 2, or 3 pulses were applied with 100 ms time interval between pulses. Cells were cultured and separate samples examined at 48 hours to determine cell mortality. Nearly 90% cell mortality was produced by applying 3 pulses at of 0.6 ms duration at 300 V.

  20. Quantitative turbidimetric assay of enzymatic gram-negative bacteria lysis.

    PubMed

    Levashov, Pavel A; Sedov, Sergey A; Shipovskov, Stepan; Belogurova, Natalia G; Levashov, Andrey V

    2010-03-01

    In this Technical Note, the quantitative turbidimetric assay for determination of the bacteriolytic activity of enzymes with gram-negative bacteria is proposed. The reactivity of hen white-egg lysozyme toward gram-negative E. coli intact cells was studied. It was found that the highest lysis rate occurred at pH 8.9 in the system containing 0.03 M NaCl. The mechanism of the reaction is discussed and applied for the quantitative evaluation of the reaction rate. The proposed method enables fast, reliable, and reproducible analysis of bacteriolytic activity of lysozyme with gram-negative bacteria.

  1. Germination-Lysis for Wide-Area Decontamination of Bacillus anthracis Spores

    DTIC Science & Technology

    2011-11-01

    30%EtOH u S0lo pH -adjusted bleach was 20 -M-70%EtOH • effective within 0 0 30 60 1S min ( 1 00°lo lysis) Time (min) • 3°lo H20 2 was...effective after 30 min ( 1 00°lo lysis) • Salt up to 30°lo was only partially effective at cell lysis • S0lo acetic acid ( vinegar ) was ineffective

  2. Evaluation of Lysis Methods for the Extraction of Bacterial DNA for Analysis of the Vaginal Microbiota.

    PubMed

    Gill, Christina; van de Wijgert, Janneke H H M; Blow, Frances; Darby, Alistair C

    2016-01-01

    Recent studies on the vaginal microbiota have employed molecular techniques such as 16S rRNA gene sequencing to describe the bacterial community as a whole. These techniques require the lysis of bacterial cells to release DNA before purification and PCR amplification of the 16S rRNA gene. Currently, methods for the lysis of bacterial cells are not standardised and there is potential for introducing bias into the results if some bacterial species are lysed less efficiently than others. This study aimed to compare the results of vaginal microbiota profiling using four different pretreatment methods for the lysis of bacterial samples (30 min of lysis with lysozyme, 16 hours of lysis with lysozyme, 60 min of lysis with a mixture of lysozyme, mutanolysin and lysostaphin and 30 min of lysis with lysozyme followed by bead beating) prior to chemical and enzyme-based DNA extraction with a commercial kit. After extraction, DNA yield did not significantly differ between methods with the exception of lysis with lysozyme combined with bead beating which produced significantly lower yields when compared to lysis with the enzyme cocktail or 30 min lysis with lysozyme only. However, this did not result in a statistically significant difference in the observed alpha diversity of samples. The beta diversity (Bray-Curtis dissimilarity) between different lysis methods was statistically significantly different, but this difference was small compared to differences between samples, and did not affect the grouping of samples with similar vaginal bacterial community structure by hierarchical clustering. An understanding of how laboratory methods affect the results of microbiota studies is vital in order to accurately interpret the results and make valid comparisons between studies. Our results indicate that the choice of lysis method does not prevent the detection of effects relating to the type of vaginal bacterial community one of the main outcome measures of epidemiological studies

  3. Requirement of Autolytic Activity for Bacteriocin-Induced Lysis

    PubMed Central

    Martínez-Cuesta, M. Carmen; Kok, Jan; Herranz, Elisabet; Peláez, Carmen; Requena, Teresa; Buist, Girbe

    2000-01-01

    The bacteriocin produced by Lactococcus lactis IFPL105 is bactericidal against several Lactococcus and Lactobacillus strains. Addition of the bacteriocin to exponential-growth-phase cells resulted in all cases in bacteriolysis. The bacteriolytic response of the strains was not related to differences in sensitivity to the bacteriocin and was strongly reduced in the presence of autolysin inhibitors (Co2+ and sodium dodecyl sulfate). When L. lactis MG1363 and its derivative deficient in the production of the major autolysin AcmA (MG1363acmAΔ1) were incubated with the bacteriocin, the latter did not lyse and no intracellular proteins were released into the medium. Incubation of cell wall fragments of L. lactis MG1363, or of L. lactis MG1363acmAΔ1 to which extracellular AcmA was added, in the presence or absence of the bacteriocin had no effect on the speed of cell wall degradation. This result indicates that the bacteriocin does not degrade cell walls, nor does it directly activate the autolysin AcmA. The autolysin was also responsible for the observed lysis of L. lactis MG1363 cells during incubation with nisin or the mixture of lactococcins A, B, and M. The results presented here show that lysis of L. lactis after addition of the bacteriocins is caused by the resulting cell damage, which promotes uncontrolled degradation of the cell walls by AcmA. PMID:10919766

  4. Weight reduction is associated with increased plasma fibrin clot lysis.

    PubMed

    Brzezińska-Kolarz, Beata; Kolarz, Marek; Wałach, Angelika; Undas, Anetta

    2014-11-01

    Obesity is associated with an increased risk of vascular thrombotic events. We sought to investigate how obesity and weight loss affect plasma fibrin clot properties. A total of 29 obese patients were studied before and after 3-month low-fat diet. Plasma fibrin clot parameters, including fibrin clot permeation coefficient (Ks), the lag phase of the turbidity curve, clot lysis time (t 50%), maximum rate of increase in D-dimer levels, and maximum D-dimer concentrations, were determined. Low-fat diet resulted in the reduction of body weight (P < .0001), body mass index (P < .0001), fat mass (P < .0001), total cholesterol (P < .0001), low-density lipoprotein cholesterol (P = .0005), triglycerides (P = .008), and plasminogen activator inhibitor 1 (P = .02), but not in fibrinogen or C-reactive protein. The only change in fibrin clot variables was shorter t 50% (P = .02). Baseline t 50%, but not posttreatment, correlated with waist circumference (r = .44, p = .02). This study demonstrates that weight loss in obese people can increase the efficiency of fibrin clot lysis.

  5. Fluorescent Method for Monitoring Cheese Starter Permeabilization and Lysis

    PubMed Central

    Bunthof, Christine J.; van Schalkwijk, Saskia; Meijer, Wilco; Abee, Tjakko; Hugenholtz, Jeroen

    2001-01-01

    A fluorescence method to monitor lysis of cheese starter bacteria using dual staining with the LIVE/DEAD BacLight bacterial viability kit is described. This kit combines membrane-permeant green fluorescent nucleic acid dye SYTO 9 and membrane-impermeant red fluorescent nucleic acid dye propidium iodide (PI), staining damaged membrane cells fluorescent red and intact cells fluorescent green. For evaluation of the fluorescence method, cells of Lactococcus lactis MG1363 were incubated under different conditions and subsequently labeled with SYTO 9 and PI and analyzed by flow cytometry and epifluorescence microscopy. Lysis was induced by treatment with cell wall-hydrolyzing enzyme mutanolysin. Cheese conditions were mimicked by incubating cells in a buffer with high protein, potassium, and magnesium, which stabilizes the cells. Under nonstabilizing conditions a high concentration of mutanolysin caused complete disruption of the cells. This resulted in a decrease in the total number of cells and release of cytoplasmic enzyme lactate dehydrogenase. In the stabilizing buffer, mutanolysin caused membrane damage as well but the cells disintegrated at a much lower rate. Stabilizing buffer supported permeabilized cells, as indicated by a high number of PI-labeled cells. In addition, permeable cells did not release intracellular aminopeptidase N, but increased enzyme activity was observed with the externally added and nonpermeable peptide substrate lysyl-p-nitroanilide. Finally, with these stains and confocal scanning laser microscopy the permeabilization of starter cells in cheese could be analyzed. PMID:11526032

  6. Silicon Nitride Bioceramics Induce Chemically Driven Lysis in Porphyromonas gingivalis.

    PubMed

    Pezzotti, Giuseppe; Bock, Ryan M; McEntire, Bryan J; Jones, Erin; Boffelli, Marco; Zhu, Wenliang; Baggio, Greta; Boschetto, Francesco; Puppulin, Leonardo; Adachi, Tetsuya; Yamamoto, Toshiro; Kanamura, Narisato; Marunaka, Yoshinori; Bal, B Sonny

    2016-03-29

    Organisms of Gram-negative phylum bacteroidetes, Porphyromonas gingivalis, underwent lysis on polished surfaces of silicon nitride (Si3N4) bioceramics. The antibacterial activity of Si3N4 was mainly the result of chemically driven principles. The lytic activity, although not osmotic in nature, was related to the peculiar pH-dependent surface chemistry of Si3N4. A buffering effect via the formation of ammonium ions (NH4(+)) (and their modifications) was experimentally observed by pH microscopy. Lysis was confirmed by conventional fluorescence spectroscopy, and the bacteria's metabolism was traced with the aid of in situ Raman microprobe spectroscopy. This latter technique revealed the formation of peroxynitrite within the bacterium itself. Degradation of the bacteria's nucleic acid, drastic reduction in phenilalanine, and reduction of lipid concentration were observed due to short-term exposure (6 days) to Si3N4. Altering the surface chemistry of Si3N4 by either chemical etching or thermal oxidation influenced peroxynitrite formation and affected bacteria metabolism in different ways. Exploiting the peculiar surface chemistry of Si3N4 bioceramics could be helpful in counteracting Porphyromonas gingivalis in an alkaline pH environment.

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

    PubMed

    Leuko, S; Goh, F; Ibáñez-Peral, R; Burns, B P; Walter, M R; Neilan, B A

    2008-03-01

    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.

  8. Does the duration of lysis affect the sensitivity of the in vitro alkaline comet assay?

    PubMed

    Enciso, José Manuel; Sánchez, Oscar; López de Cerain, Adela; Azqueta, Amaya

    2015-01-01

    The alkaline comet assay is now the method of choice for measuring different kinds of DNA damage in cells. Several attempts have been made to identify and evaluate the critical points affecting the comet assay outcome, highlighting the requirement of arriving at a standardised protocol in order to be able to compare the results obtained in different laboratories. However, reports on the effect of modifying the time of lysis are lacking. Here we tested different times of lysis (from no lysis to 1 week) in control HeLa cells and HeLa cells treated with different concentrations of methyl methanesulfonate (MMS) or H2O2. We also tested different times of lysis in the comet assay combined with formamidopyrimidine DNA glycosylase (FPG) in untreated and Ro 19-8022 plus light-treated HeLa cells. The same DNA damage levels were detected in the absence of lysis or after 1h of lysis when the standard comet assay was used to detect the MMS- and H2O2-induced lesions; the response increased when longer lysis was used, up to at least 1 week. When FPG was used, a minimum lysis period of 5 min was necessary to allow the enzyme to reach the DNA; the same DNA damage levels were detected after 5 min or 1h of lysis and the response increased up to 24h. In conclusion, the time of lysis can be varied depending on the sensitivity needed in both versions of the assay, and a constant time of lysis should be used if results from different experiments or laboratories are to be compared.

  9. LYSIS OF ESCHERICHIA COLI BY SULFHYDRYL-BINDING REAGENTS

    PubMed Central

    Schaechter, M.; Santomassino, Katherine A.

    1962-01-01

    Schaechter, M. (College of Medicine, University of Florida, Gainesville) and K. Santomassino. Lysis of Escherichia coli by sulfhydryl-binding reagents. J. Bacteriol. 84:318–325. 1962—Washed suspensions of gram-negative rods were lysed by low concentrations of some sulfhydryl-binding and oxidizing reagents, but not by reducing agents. Some kinetic aspects of this phenomenon were studied with p-chloromercuribenzoate and Escherichia coli B/r. Structures resulting from the action of this reagent consisted of impure cell walls. These could be purified by treatment with trypsin. Cell walls prepared mechanically and cell membranes obtained by lysing protoplasts were not overtly affected by this chemical. Images PMID:14497913

  10. Antimicrobial actions of hexachlorophene: lysis and fixation of bacterial protoplasts.

    PubMed

    Corner, T R; Joswick, H L; Silvernale, J N; Gerhardt, P

    1971-10-01

    Hexachlorophene was found to be both a lytic and a fixative agent for protoplasts isolated from Bacillus megaterium. Concentrations of 50 to 100 mug of drug per mg of original cell dry weight were required to lyse 4.4 x 10(9) protoplasts (2 mg of original cell dry weight). At higher drug concentrations, protoplasts became fixed against osmotic stress and reduced in sensitivity to disruption by n-butanol. Lower drug concentrations caused proportionate lysis in the protoplast population. Intact cells lost the ability to become plasmolyzed at these same hexachlorophene concentrations. Nonplasmolyzed, drug-treated cells were resistant to the action of lysozyme, whereas plasmolyzed, drug-treated cells were sensitive. But the sensitivity of isolated cell walls to lysozyme digestion was not markedly altered by hexachlorophene treatment. These effects appeared to be secondary in the killing of cells by hexachlorophene because they occurred at concentrations higher than the minimum lethal concentration.

  11. Kinetics of plasma coagulation and lysis I: Basic kinetic model for time course of coagulation-lysis systems and its potential application to clinical studies.

    PubMed

    Garrett, E R; Daver, J; Desnoyers, P; Kosser, M; Vincent, M

    1978-02-01

    The time courses of coagulation and coagulation-lysis were spectrophotometrically monitored after the addition of thrombin or thrombin-streptokinase to plasma, diluted 1:5 with normal saline, obtained from normal and presumably abnormal subjects. The kinetics of clotting, after an initial lag period of 0.5-1.5 min, demonstrated essentially first-order dependence on the amount of fibrinogen available to form the clot, and the asymptotic absorbance was independent of thrombin concentration. The rate of clotting was a function of added thrombin, and the ratios of the rate constants at 2.5 and 1.25 units of thrombin/ml of undiluted plasma were 1.65 +/- 0.03 SEM. At early times, the coagulation-lysis curve with thrombin-streptokinase could be superimposed on the clotting curve with thrombin alone for a given plasma with minor compensation for variable lag times. Subsequently, the curves diverged; lysis was monitored by the decrease in absorbance of the coagulation-lysis system. The rate of fibrinolysis increased with streptokinase concentration and was a function of the extent of lysis, and it permitted the description of the kinetics of lysis by a pseudoautocatalytic mechanism where the bimolecular rate constant appears proportional to streptokinase concentration. Ranges of clotting and lytic parameters for the plasma of normal subjects are given, and their potential use in diagnosing abnormalities is described.

  12. Electron Microscopy of Staphylococcus aureus Cell Wall Lysis

    PubMed Central

    Virgilio, R.; González, C.; Muñoz, Nubia; Mendoza, Silvia

    1966-01-01

    Virgilio, Rafael (Escuela de Química y Farmacia, Universidad de Chile, Santiago, Chile), C. González, Nubia Muñoz, and Silvia Mendoza. Electron microscopy of Staphylococcus aureus cell wall lysis. J. Bacteriol. 91:2018–2024. 1966.—A crude suspension of Staphylococcus aureus cell walls (strain Cowan III) in buffer solution was shown by electron microscopy to lyse slightly after 16 hr, probably owing to the action of autolysin. The lysis was considerably faster and more intense after the addition of lysozyme. A remarkable reduction in thickness and rigidity of the cell walls, together with the appearance of many irregular protrusions in their outlines, was observed after 2 hr; after 16 hr, there remained only a few recognizable cell wall fragments but many residual particulate remnants. When autolysin was previously inactivated by trypsin, there was a complete inhibition of the lytic action of lysozyme; on the other hand, when autolysin was inactivated by heat and lysozyme was added, a distinct decrease in the thickness of the cell walls was observed, but there was no destruction of the walls. The lytic action of lysozyme, after treatment with hot 5% trichloroacetic acid, gave rise to a marked dissolution of the structure of the cell walls, which became lost against the background, without, however, showing ostensible alteration of wall outlines. From a morphological point of view, the lytic action of autolysin plus lysozyme was quite different from that of trichloroacetic acid plus lysozyme, as shown by electron micrographs, but in both cases it was very intense. This would suggest different mechanisms of action for these agents. Images PMID:5939482

  13. Cyanobacterial blue color formation during lysis under natural conditions.

    PubMed

    Arii, Suzue; Tsuji, Kiyomi; Tomita, Koji; Hasegawa, Masateru; Bober, Beata; Harada, Ken-ichi

    2015-04-01

    Cyanobacteria produce numerous volatile organic compounds (VOCs), such as β-cyclocitral, geosmin, and 2-methylisoborneol, which show lytic activity against cyanobacteria. Among these compounds, only β-cyclocitral causes a characteristic color change from green to blue (blue color formation) in the culture broth during the lysis process. In August 2008 and September 2010, the lysis of cyanobacteria involving blue color formation was observed at Lake Tsukui in northern Kanagawa Prefecture, Japan. We collected lake water containing the cyanobacteria and investigated the VOCs, such as β-cyclocitral, β-ionone, 1-propanol, 3-methyl-1-butanol, and 2-phenylethanol, as well as the number of cyanobacterial cells and their damage and pH changes. As a result, the following results were confirmed: the detection of several VOCs, including β-cyclocitral and its oxidation product, 2,2,6-trimethylcyclohexene-1-carboxylic acid; the identification of phycocyanin based on its visible spectrum; the lower pH (6.7 and 5.4) of the lysed samples; and characteristic morphological change in the damaged cyanobacterial cells. We also encountered the same phenomenon on 6 September 2013 in Lake Sagami in northern Kanagawa Prefecture and obtained almost the same results, such as blue color formation, decreasing pH, damaged cells, and detection of VOCs, including the oxidation products of β-cyclocitral. β-Cyclocitral derived from Microcystis has lytic activity against Microcystis itself but has stronger inhibitory activity against other cyanobacteria and algae, suggesting that the VOCs play an important role in the ecology of aquatic environments.

  14. Cyanobacterial Blue Color Formation during Lysis under Natural Conditions

    PubMed Central

    Tsuji, Kiyomi; Tomita, Koji; Hasegawa, Masateru; Bober, Beata; Harada, Ken-Ichi

    2015-01-01

    Cyanobacteria produce numerous volatile organic compounds (VOCs), such as β-cyclocitral, geosmin, and 2-methylisoborneol, which show lytic activity against cyanobacteria. Among these compounds, only β-cyclocitral causes a characteristic color change from green to blue (blue color formation) in the culture broth during the lysis process. In August 2008 and September 2010, the lysis of cyanobacteria involving blue color formation was observed at Lake Tsukui in northern Kanagawa Prefecture, Japan. We collected lake water containing the cyanobacteria and investigated the VOCs, such as β-cyclocitral, β-ionone, 1-propanol, 3-methyl-1-butanol, and 2-phenylethanol, as well as the number of cyanobacterial cells and their damage and pH changes. As a result, the following results were confirmed: the detection of several VOCs, including β-cyclocitral and its oxidation product, 2,2,6-trimethylcyclohexene-1-carboxylic acid; the identification of phycocyanin based on its visible spectrum; the lower pH (6.7 and 5.4) of the lysed samples; and characteristic morphological change in the damaged cyanobacterial cells. We also encountered the same phenomenon on 6 September 2013 in Lake Sagami in northern Kanagawa Prefecture and obtained almost the same results, such as blue color formation, decreasing pH, damaged cells, and detection of VOCs, including the oxidation products of β-cyclocitral. β-Cyclocitral derived from Microcystis has lytic activity against Microcystis itself but has stronger inhibitory activity against other cyanobacteria and algae, suggesting that the VOCs play an important role in the ecology of aquatic environments. PMID:25662969

  15. Electron microscopy of Staphylococcus aureus cell wall lysis.

    PubMed

    Virgilio, R; González, C; Muñoz, N; Mendoza, S

    1966-05-01

    Virgilio, Rafael (Escuela de Química y Farmacia, Universidad de Chile, Santiago, Chile), C. González, Nubia Muñoz, and Silvia Mendoza. Electron microscopy of Staphylococcus aureus cell wall lysis. J. Bacteriol. 91:2018-2024. 1966.-A crude suspension of Staphylococcus aureus cell walls (strain Cowan III) in buffer solution was shown by electron microscopy to lyse slightly after 16 hr, probably owing to the action of autolysin. The lysis was considerably faster and more intense after the addition of lysozyme. A remarkable reduction in thickness and rigidity of the cell walls, together with the appearance of many irregular protrusions in their outlines, was observed after 2 hr; after 16 hr, there remained only a few recognizable cell wall fragments but many residual particulate remnants. When autolysin was previously inactivated by trypsin, there was a complete inhibition of the lytic action of lysozyme; on the other hand, when autolysin was inactivated by heat and lysozyme was added, a distinct decrease in the thickness of the cell walls was observed, but there was no destruction of the walls. The lytic action of lysozyme, after treatment with hot 5% trichloroacetic acid, gave rise to a marked dissolution of the structure of the cell walls, which became lost against the background, without, however, showing ostensible alteration of wall outlines. From a morphological point of view, the lytic action of autolysin plus lysozyme was quite different from that of trichloroacetic acid plus lysozyme, as shown by electron micrographs, but in both cases it was very intense. This would suggest different mechanisms of action for these agents.

  16. Education for Epiphany: The Case of Plato's "Lysis"

    ERIC Educational Resources Information Center

    Jonas, Mark E.

    2015-01-01

    While a great deal has been written on Plato's "Lysis" in philosophy and philology journals over the last thirty years, nothing has been published on "Lysis" in the major Anglo-American philosophy of education journals during that time. Nevertheless, this dialogue deserves attention from educators. In this essay, Mark…

  17. Education for Epiphany: The Case of Plato's "Lysis"

    ERIC Educational Resources Information Center

    Jonas, Mark E.

    2015-01-01

    While a great deal has been written on Plato's "Lysis" in philosophy and philology journals over the last thirty years, nothing has been published on "Lysis" in the major Anglo-American philosophy of education journals during that time. Nevertheless, this dialogue deserves attention from educators. In this essay, Mark…

  18. A Method for Purifying Obligate Intracellular Coxiella burnetii that Employs Digitonin Lysis of Host Cells

    PubMed Central

    Cockrell, Diane C.; Beare, Paul A.; Fischer, Elizabeth R.; Howe, Dale; Heinzen, Robert. A.

    2008-01-01

    Purification of the obligate intracellular bacterium Coxiella burnetii requires physical disruption of infected cells. Here we describe a gentle and safe digitonin lysis procedure to release C. burnetii from infected cells. The purity, yield, and infectivity of digitonin-prepped organisms are comparable to that of organisms purified using cell lysis by sonication. PMID:18242746

  19. Increased sensitivity of early apoptotic cells to complement-mediated lysis.

    PubMed

    Attali, Gitit; Gancz, Dana; Fishelson, Zvi

    2004-11-01

    Opsonization of apoptotic cells with complement proteins contributes to their clearance by phagocytes. Little is known about the lytic effects of complement on apoptotic cells. Sensitivity of cells treated with anti-Fas antibody (Jurkat cells), staurosporine or etoposide (Raji cells) to lysis by complement was examined. As shown here, early apoptotic cells are more sensitive to lysis by antibody and complement than control cells. More complement C3 and C9 bound to apoptotic than to control cells, even though antibody binding was similar. Enhanced killing and C3/C9 deposition were blocked by benzyloxy-Val-Ala-Asp-fluoromethylketone, a pan-caspase inhibitor. Complement-mediated lysis of early apoptotic cells was also prevented by inhibitors of caspases 6, 8, 9 or 10. In contrast, caspase inhibitors had no effect on the lysis of non-apoptotic Jurkat and Raji cells. Early apoptotic Jurkat cells were also more sensitive to lysis by the pore formers streptolysin O and melittin. Sensitivity of Jurkat Bcl-2 transfectants to lysis by complement was analyzed. Enhanced Bcl-2 expression was associated with reduced C3 deposition and lower sensitivity to complement-mediated lysis. These results demonstrate that at an early stage in apoptosis, following caspase activation, cells become sensitive to necrotic-type death by complement and other pore formers. Furthermore, they suggest that Bcl-2 is actively protecting Jurkat cells from complement-mediated lysis.

  20. Portable lysis apparatus for rapid single-step DNA extraction of Bacillus subtilis.

    PubMed

    Lim, H J; Lee, E-H; Yoon, Y; Chua, B; Son, A

    2016-02-01

    To demonstrate and characterize a portable lysis apparatus for rapid single-step bacterial DNA extraction. Our portable lysis apparatus employed a novel design consisting of an annular piezo-element with perforated diaphragm. Using Bacillus subtilis as target bacteria, our portable lysis apparatus was able to achieve a normalized percent lysis as high as 66% within 30 s. This is comparable to that by microprobe ultrasonication and almost 7 times higher than that by conventional bead beating. The effect from adding glass beads was predictable. However, the results from the addition of sodium dodecyl sulphate (SDS) were counter-intuitive because a further increase from 0·5 to 1% concentration reduced the lysis performance. The portable lysis apparatus is also at least 1·5-5 times more power efficient than microprobe ultrasonication. Our portable lysis apparatus is capable of rapidly extracting bacterial DNA and is more power efficient than microprobe ultrasonication. The addition of glass beads or SDS concentration (up to 0·5%) improves its performance. The portable lysis apparatus provides a standalone, rapid, low cost and power efficient way of obtaining genomic constituents prior to a variety of bioassays used in the field of environmental, biomedical and other applied microbiology. © 2015 The Society for Applied Microbiology.

  1. Chemical Cell Lysis System Applicable to Lab-on-a-Disc.

    PubMed

    Lim, Dayeseul; Yoo, Jae Chern

    2017-02-09

    The design and fabrication of a heating system has been a significant challenge in implementing chemical lysis on a lab-on-a-disc (LOD). The proposed system contains a sample inlet, phase change material (PCM) array, heating chamber, and valve in a single disc, providing cost-effective, rapid, and fully automated chemical cell lysis. Compared to the conventional cell lysis system, our cell lysis system has many advantages, such as a compact structure that is easily integrated into the LOD and reduced processing time and labor. The experiments are conducted with Salmonella typhimurium strains to demonstrate the performance. The experimental results show that the proposed approach is greatly effective in realizing a chemical cell lysis system on an LOD with higher throughput in terms of purity and yield of DNA.

  2. All electronic approach for high-throughput cell trapping and lysis with electrical impedance monitoring.

    PubMed

    Ameri, Shideh Kabiri; Singh, Pramod K; Dokmeci, Mehmet R; Khademhosseini, Ali; Xu, Qiaobing; Sonkusale, Sameer R

    2014-04-15

    We present a portable lab-on-chip device for high-throughput trapping and lysis of single cells with in-situ impedance monitoring in an all-electronic approach. The lab-on-chip device consists of microwell arrays between transparent conducting electrodes within a microfluidic channel to deliver and extract cells using alternating current (AC) dielectrophoresis. Cells are lysed with high efficiency using direct current (DC) electric fields between the electrodes. Results are presented for trapping and lysis of human red blood cells. Impedance spectroscopy is used to estimate the percentage of filled wells with cells and to monitor lysis. The results show impedance between electrodes decreases with increase in the percentage of filled wells with cells and drops to a minimum after lysis. Impedance monitoring provides a reasonably accurate measurement of cell trapping and lysis. Utilizing an all-electronic approach eliminates the need for bulky optical components and cameras for monitoring.

  3. Trypanosome Lytic Factor-1 Initiates Oxidation-stimulated Osmotic Lysis of Trypanosoma brucei brucei*

    PubMed Central

    Greene, Amy Styer; Hajduk, Stephen L.

    2016-01-01

    Human innate immunity against the veterinary pathogen Trypanosoma brucei brucei is conferred by trypanosome lytic factors (TLFs), against which human-infective T. brucei gambiense and T. brucei rhodesiense have evolved resistance. TLF-1 is a subclass of high density lipoprotein particles defined by two primate-specific apolipoproteins: the ion channel-forming toxin ApoL1 (apolipoprotein L1) and the hemoglobin (Hb) scavenger Hpr (haptoglobin-related protein). The role of oxidative stress in the TLF-1 lytic mechanism has been controversial. Here we show that oxidative processes are involved in TLF-1 killing of T. brucei brucei. The lipophilic antioxidant N,N′-diphenyl-p-phenylenediamine protected TLF-1-treated T. brucei brucei from lysis. Conversely, lysis of TLF-1-treated T. brucei brucei was increased by the addition of peroxides or thiol-conjugating agents. Previously, the Hpr-Hb complex was postulated to be a source of free radicals during TLF-1 lysis. However, we found that the iron-containing heme of the Hpr-Hb complex was not involved in TLF-1 lysis. Furthermore, neither high concentrations of transferrin nor knock-out of cytosolic lipid peroxidases prevented TLF-1 lysis. Instead, purified ApoL1 was sufficient to induce lysis, and ApoL1 lysis was inhibited by the antioxidant DPPD. Swelling of TLF-1-treated T. brucei brucei was reminiscent of swelling under hypotonic stress. Moreover, TLF-1-treated T. brucei brucei became rapidly susceptible to hypotonic lysis. T. brucei brucei cells exposed to peroxides or thiol-binding agents were also sensitized to hypotonic lysis in the absence of TLF-1. We postulate that ApoL1 initiates osmotic stress at the plasma membrane, which sensitizes T. brucei brucei to oxidation-stimulated osmotic lysis. PMID:26645690

  4. Enzyme-mediated Nutrient Regeneration Following Lysis of Synechococcus WH7803

    NASA Astrophysics Data System (ADS)

    Mine, A. H.; Coleman, M.; Colman, A. S.

    2016-02-01

    Phosphate availability plays a pivotal role in limiting primary production in large regions of the oceans. In order to meet their metabolic needs, microbes use a variety of strategies to overcome phosphate stress. Expression of enzymes such as alkaline phosphatase (APase) allows cells to hydrolyze and use certain ambient dissolved organic phosphorus (DOP) compounds to meet their P demand. Cell lysis releases a range of nutrient forms and enzymes into the ambient environment and is an essential component of the microbial loop. Yet very few studies have attempted to characterize both the immediate and sustained nutrient remineralization linked to the milieu of organophosphorus compounds and enzymatic activity in lysate. We conducted experiments using Synechococcus WH7803 grown under nutrient replete and starved conditions to quantify the release of phosphate during viral lysis and lysis by lysozyme treatment. Dissolved inorganic and organic phosphorus concentrations and APase activity were monitored over time following lysis. We observed a significant initial release of orthophosphate that accompanies lysis. Following lysis, phosphate concentrations continue to rise for a period of hours to days as organophosphorus compounds continue to hydrolyze. Our observations suggest this is due to a combination of direct hydrolysis of DOP released during lysis, solubilization of POP followed by hydrolysis, and possibly polyphosphate decomposition. Size fractionated enzymatic assays suggest cellular debris associated enzymes and dissolved fractions are both important in DOP hydrolysis in the viral lysate, whereas particle associated APase activity dominates in the lysozyme treatments. Moreover, nutrient status prior to lysis has important controls on the initial nutrient release and subsequent regenerative flux. These findings underscore the significance of lysis and subsequent enzyme-mediated hydrolysis in nutrient regeneration and biogeochemical dynamics in marine ecosystems.

  5. Nucleation of holin domains and holes optimizes lysis timing of E. coli by phage λ

    NASA Astrophysics Data System (ADS)

    Ryan, Gillian; Rutenberg, Andrew

    2007-03-01

    Holin proteins regulate the precise scheduling of Escherichia coli lysis during infection by bacteriophage λ. Inserted into the host bacterium's inner membrane during infection, holins aggregate to form rafts and then holes within those rafts. We present a two-stage nucleation model of holin action, with the nucleation of condensed holin domains followed by the nucleation of holes within these domains. Late nucleation of holin rafts leads to a weak dependence of lysis timing on host cell size, though both nucleation events contribute equally to timing errors. Our simulations recover the accurate scheduling observed experimentally, and also suggest that phage-λ lysis of E.coli is optimized.

  6. Lysis of Escherichia coli by Glycine Is Potentiated by Pyridoxine Starvation

    PubMed Central

    Dempsey, Walter B.

    1973-01-01

    Pyridoxineless mutants of Escherichia coli are lysed in a few hours when starved for pyridoxine in a glucose minimal medium containing glycine at 10 mM. The lysis is prevented equally well by l-alanine and by d-alanine when either is present at 0.1 mM. The lysis is potentiated by 0.5 mM l-methionine. The peculiar susceptibility of E. coli B to glycine-mediated lysis during starvation for pyridoxine suggests that the starvation reduces the availability of some normal antagonist of glycine, presumably alanine. PMID:4583221

  7. Tumor lysis syndrome in the emergency department: challenges and solutions

    PubMed Central

    Ñamendys-Silva, Silvio A; Arredondo-Armenta, Juan M; Plata-Menchaca, Erika P; Guevara-García, Humberto; García-Guillén, Francisco J; Rivero-Sigarroa, Eduardo; Herrera-Gómez, Angel

    2015-01-01

    Tumor lysis syndrome (TLS) is the most common oncologic emergency. It is caused by rapid tumor cell destruction and the resulting nucleic acid degradation during or days after initiation of cytotoxic therapy. Also, a spontaneous form exists. The metabolic abnormalities associated with this syndrome include hyperkalemia, hyperphosphatemia, hypocalcemia, hyperuricemia, and acute kidney injury. These abnormalities can lead to life-threatening complications, such as heart rhythm abnormalities and neurologic manifestations. The emergency management of overt TLS involves proper fluid resuscitation with crystalloids in order to improve the intravascular volume and the urinary output and to increase the renal excretion of potassium, phosphorus, and uric acid. With this therapeutic strategy, prevention of calcium phosphate and uric acid crystal deposition within renal tubules is achieved. Other measures in the management of overt TLS are prescription of hypouricemic agents, renal replacement therapy, and correction of electrolyte imbalances. Hyperkalemia should be treated quickly and aggressively as its presence is the most hazardous acute complication that can cause sudden death from cardiac arrhythmias. Treatment of hypocalcemia is reserved for patients with electrocardiographic changes or symptoms of neuromuscular irritability. In patients who are refractory to medical management of electrolyte abnormalities or with severe cardiac and neurologic manifestations, early dialysis is recommended. PMID:27147889

  8. Evaluation of the Sterifil lysis-filtration blood culture system.

    PubMed

    Farmer, S G; Komorowski, R A

    1972-03-01

    This paper describes the comparison of the Sterifil lysis-filtration (SLF) blood culture procedure with a standard Trypticase soy broth (TSB) technique. The lysing solutions employed in the SLF system, Triton X-100 (alkyl phenoxy polyethoxy ethanol) and sodium carbonate, were deleterious to most bacteria commonly encountered in bacteremia except staphylococci and enterococci. Candida was not adversely affected. There was a positive correlation between the tolerance of the microbial isolants to the lysing solutions and their recovery by the SLF technique. A total of 3,554 cultures were run in parallel and 398 isolants were obtained. Of 201 gram-positive isolants, 135 were recovered by both techniques, 43 were detected by the TSB technique only, and 23 were recovered only with the SLF method. In sharp contrast, of 168 gram-negative isolants, 28 were recovered in common, 130 were isolated only by TSB, and 10 were recovered only with the SLF method. The SLF method detected all cases of candidemia detected by the TSB method plus an additional 12 for a total of 29 cases. The SLF method, as currently described, is generally too toxic to bacteria for routine use in a clinical laboratory.

  9. [Acute renal failure in patients with tumour lysis sindrome].

    PubMed

    Poskurica, Mileta; Petrović, Dejan; Poskurica, Mina

    2016-01-01

    `Hematologic malignancies (leukemia, lymphoma, multiple myeloma, et al.), as well as solid tumours (renal, liver, lung, ovarian, etc.), can lead to acute or chronic renal failure.The most common clinical manifestation is acute renal failure within the tumour lysis syndrome (TLS). It is characterized by specific laboratory and clinical criteria in order to prove that kidney disorders result from cytolysis of tumour cells after chemotherapy regimen given, although on significantly fewer occasions it is likely to occur spontaneously or after radiotherapy. Essentially, failure is the disorder of functionally conserved kidney or of kidney with varying degrees of renal insufficiency, which render the kidney impaired and unable to effectively eliminate the end products of massive cytolysis and to correct the resulting disorders: hyperuricemia, hyperkalemia, hypocalcaemia, hyperphosphatemia, and others. The risk of TLS depends on tumour size, proliferative potential of malignant cells, renal function and the presence of accompanying diseases and disorders. Hydration providing adequate diuresis and administration of urinary suppressants (allopurinol, febuxostat) significantly reduce the risk of developing TLS. If prevention of renal impairment isn't possible, the treatment should be supplemented with hemodynamic monitoring and pharmacological support, with the possible application of recombinant urate-oxidase enzyme (rasburicase). Depending on the severity of azotemia and hydroelectrolytic disorders, application of some of the methods of renal replacement therapy may be considered.

  10. Phosphoinositide-mediated oligomerization of a defensin induces cell lysis

    PubMed Central

    Poon, Ivan KH; Baxter, Amy A; Lay, Fung T; Mills, Grant D; Adda, Christopher G; Payne, Jennifer AE; Phan, Thanh Kha; Ryan, Gemma F; White, Julie A; Veneer, Prem K; van der Weerden, Nicole L; Anderson, Marilyn A; Kvansakul, Marc; Hulett, Mark D

    2014-01-01

    Cationic antimicrobial peptides (CAPs) such as defensins are ubiquitously found innate immune molecules that often exhibit broad activity against microbial pathogens and mammalian tumor cells. Many CAPs act at the plasma membrane of cells leading to membrane destabilization and permeabilization. In this study, we describe a novel cell lysis mechanism for fungal and tumor cells by the plant defensin NaD1 that acts via direct binding to the plasma membrane phospholipid phosphatidylinositol 4,5-bisphosphate (PIP2). We determined the crystal structure of a NaD1:PIP2 complex, revealing a striking oligomeric arrangement comprising seven dimers of NaD1 that cooperatively bind the anionic headgroups of 14 PIP2 molecules through a unique ‘cationic grip’ configuration. Site-directed mutagenesis of NaD1 confirms that PIP2-mediated oligomerization is important for fungal and tumor cell permeabilization. These observations identify an innate recognition system by NaD1 for direct binding of PIP2 that permeabilizes cells via a novel membrane disrupting mechanism. DOI: http://dx.doi.org/10.7554/eLife.01808.001 PMID:24692446

  11. Lysis of primary hepatic tumours by lymphokine activated killer cells.

    PubMed Central

    Hsieh, K H; Shu, S Y; Lee, C S; Chu, C T; Yang, C S; Chang, K J

    1987-01-01

    Lymphokine activated killer cell is a newly described lytic system against a variety of solid tumours and is distinct in several respects from the classic cytolytic T cell and the natural killer systems. This study was conducted to evaluate the lytic activity of lymphokine activated killer cells against fresh autologous and allogeneic, as well as cultured hepatocellular carcinoma cells. Lymphokine activated killer cell was generated by incubating peripheral blood mononuclear cells with various concentrations of recombinant IL-2 (rIL-2, Cetus, USA) for various periods of time. A four hour 51Cr release assay was used to measure cytotoxicity. The results show that fresh and cultured hepatocellular carcinoma cells were only slightly susceptible to natural killer cells. Normal hepatocytes were resistant to lymphokine activated killer-mediated lysis. Lymphokine activated killer cells could be generated from mononuclear cells of hepatocellular carcinoma patients and normal subjects with lytic activity against fresh autologous and allogeneic and cultured hepatocellular carcinoma cells, but lymphokine activated killer cells from the former was less efficient than that from the latter. It is concluded that the adoptive immunotherapy with combined rIL-2 and lymphokine activated killer may be worth trying in early cases of primary hepatocellular carcinoma. PMID:3030899

  12. Communication between viruses guides lysis-lysogeny decisions.

    PubMed

    Erez, Zohar; Steinberger-Levy, Ida; Shamir, Maya; Doron, Shany; Stokar-Avihail, Avigail; Peleg, Yoav; Melamed, Sarah; Leavitt, Azita; Savidor, Alon; Albeck, Shira; Amitai, Gil; Sorek, Rotem

    2017-01-26

    Temperate viruses can become dormant in their host cells, a process called lysogeny. In every infection, such viruses decide between the lytic and the lysogenic cycles, that is, whether to replicate and lyse their host or to lysogenize and keep the host viable. Here we show that viruses (phages) of the SPbeta group use a small-molecule communication system to coordinate lysis-lysogeny decisions. During infection of its Bacillus host cell, the phage produces a six amino-acids-long communication peptide that is released into the medium. In subsequent infections, progeny phages measure the concentration of this peptide and lysogenize if the concentration is sufficiently high. We found that different phages encode different versions of the communication peptide, demonstrating a phage-specific peptide communication code for lysogeny decisions. We term this communication system the 'arbitrium' system, and further show that it is encoded by three phage genes: aimP, which produces the peptide; aimR, the intracellular peptide receptor; and aimX, a negative regulator of lysogeny. The arbitrium system enables a descendant phage to 'communicate' with its predecessors, that is, to estimate the amount of recent previous infections and hence decide whether to employ the lytic or lysogenic cycle.

  13. Protein aggregation induced during glass bead lysis of yeast

    PubMed Central

    Papanayotou, Irene; Sun, Beimeng; Roth, Amy F.; Davis, Nicholas G.

    2013-01-01

    Yeast cell lysates produced by mechanical glass bead disruption are widely used in a variety of applications, including for the analysis of native function, e.g. protein–protein interaction, enzyme assays and membrane fractionations. Below, we report a striking case of protein denaturation and aggregation that is induced by this lysis protocol. Most of this analysis focuses on the type 1 casein kinase Yck2, which normally tethers to the plasma membrane through C-terminal palmitoylation. Surprisingly, when cells are subjected to glass bead disruption, non-palmitoylated, cytosolic forms of the kinase denature and aggregate, while membrane-associated forms, whether attached through their native palmitoyl tethers or through a variety of artificial membrane-tethering sequences, are wholly protected from denaturation and aggregation. A wider look at the yeast proteome finds that, while the majority of proteins resist glass bead-induced aggregation, a significant subset does, in fact, succumb to such denaturation. Thus, yeast researchers should be aware of this potential artifact when embarking on biochemical analyses that employ glass bead lysates to look at native protein function. Finally, we demonstrate an experimental utility for glass bead-induced aggregation, using its fine discrimination of membrane-associated from non-associated Yck2 forms to discern fractional palmitoylation states of Yck2 mutants that are partially defective for palmitoylation. PMID:20641011

  14. Fungal lysis by a soil bacterium fermenting cellulose.

    PubMed

    Tolonen, Andrew C; Cerisy, Tristan; El-Sayyed, Hafez; Boutard, Magali; Salanoubat, Marcel; Church, George M

    2015-08-01

    Recycling of plant biomass by a community of bacteria and fungi is fundamental to carbon flow in terrestrial ecosystems. Here we report how the plant fermenting, soil bacterium Clostridium phytofermentans enhances growth on cellulose by simultaneously lysing and consuming model fungi from soil. We investigate the mechanism of fungal lysis to show that among the dozens of different glycoside hydrolases C. phytofermentans secretes on cellulose, the most highly expressed enzymes degrade fungi rather than plant substrates. These enzymes, the GH18 Cphy1799 and Cphy1800, synergize to hydrolyse chitin, a main component of the fungal cell wall. Purified enzymes inhibit fungal growth and mutants lacking either GH18 grow normally on cellulose and other plant substrates, but have a reduced ability to hydrolyse chitinous substrates and fungal hyphae. Thus, C. phytofermentans boosts growth on cellulose by lysing fungi with its most highly expressed hydrolases, highlighting the importance of fungal interactions to the ecology of cellulolytic bacteria. © 2014 Society for Applied Microbiology and John Wiley & Sons Ltd.

  15. Spontaneous tumour lysis syndrome in hepatocellular carcinoma presenting with hypocalcemic tetany: An unusual case and systematic literature review.

    PubMed

    Agarwala, Roshan; Batta, Akash; Suryadevera, Varun; Kumar, Vivek; Sharma, Vishal; Rana, Surinder Singh

    2017-06-01

    Tumour lysis syndrome is an oncological emergency which is usually seen following chemotherapy for rapidly proliferating haematological malignancies. Spontaneous tumour lysis syndrome is rare in solid tumour and even rarer with hepatocellular carcinoma (HCC). Tumour lysis syndrome in the setting of HCC is usually reported as a consequence of therapeutic interventions like sorafenib administration or trans-arterial chemoembolization. We report about a case of a young lady with chronic hepatitis B related HCC who developed spontaneous tumour lysis syndrome and presented with hypocalcemic tetany. We also compare this case with the previously reported cases of spontaneous tumour lysis syndrome in hepatocellular carcinoma. Copyright © 2016 Elsevier Masson SAS. All rights reserved.

  16. Catheter placement for lysis of spontaneous intracerebral hematomas: does a catheter position in the core of the hematoma allow more effective and faster hematoma lysis?

    PubMed

    Malinova, Vesna; Schlegel, Anna; Rohde, Veit; Mielke, Dorothee

    2017-07-01

    For the fibrinolytic therapy of intracerebral hematomas (ICH) using recombinant tissue plasminogen activator (rtPA), a catheter position in the core of the hematoma along the largest clot diameter was assumed to be optimal for an effective clot lysis. However, it never had been proven that core position indeed enhances clot lysis if compared with less optimal catheter positions. In this study, the impact of the catheter position on the effectiveness and on the time course of clot lysis was evaluated. We analyzed the catheter position using a relative error calculating the distance perpendicular to the catheter's center in relation to hematoma's diameter and evaluated the relative hematoma volume reduction (RVR). The correlation of the RVR with the catheter position was evaluated. Additionally, we tried to identify patterns of clot lysis with different catheter positions. The patient's outcome at discharge was evaluated using the Glasgow outcome score. A total of 105 patients were included in the study. The mean hematoma volume was 56 ml. The overall RVR was 62.7 %. In 69 patients, a catheter position in the core of the clot was achieved. We found no significant correlation between catheter position and hematoma RVR (linear regression, p = 0.14). Core catheter position leads to more symmetrical hematoma RVR. Faster clot lysis happens in the vicinity of the catheter openings. We found no significant difference in the patient's outcome dependent on the catheter position (linear regression, p = 0.90). The catheter position in the core of the hematoma along its largest diameter does not significantly influence the effectiveness of clot lysis after rtPA application.

  17. Loss of susceptibility to complement lysis in Entamoeba histolytica HM1 by treatment with human serum.

    PubMed Central

    Calderon, J; Tovar, R

    1986-01-01

    Entamoeba histolytica HM1, exposed to a series of treatment with normal human serum (NHS), progressively lost susceptibility to complement lysis. Trophozoites were incubated daily with unheated or heat-inactivated NHS for 2 hr at 36 degrees, starting with 10% v/v serum and increasing by 5% every 3 days up to 40% NHS. Resistance to complement lysis was also obtained with two different HM1 clones but not with the low virulent strain HK9. Induction of resistance dependent on the number of NHS treatments, with a maximal 50% reduction occurring after 11 treatments. Susceptibility to complement-dependent lysis was regained 6 weeks after serum treatments were terminated, suggesting that resistance to lysis was an acquired rather than a genetic property. PMID:2874111

  18. Explosive cell lysis as a mechanism for the biogenesis of bacterial membrane vesicles and biofilms.

    PubMed

    Turnbull, Lynne; Toyofuku, Masanori; Hynen, Amelia L; Kurosawa, Masaharu; Pessi, Gabriella; Petty, Nicola K; Osvath, Sarah R; Cárcamo-Oyarce, Gerardo; Gloag, Erin S; Shimoni, Raz; Omasits, Ulrich; Ito, Satoshi; Yap, Xinhui; Monahan, Leigh G; Cavaliere, Rosalia; Ahrens, Christian H; Charles, Ian G; Nomura, Nobuhiko; Eberl, Leo; Whitchurch, Cynthia B

    2016-04-14

    Many bacteria produce extracellular and surface-associated components such as membrane vesicles (MVs), extracellular DNA and moonlighting cytosolic proteins for which the biogenesis and export pathways are not fully understood. Here we show that the explosive cell lysis of a sub-population of cells accounts for the liberation of cytosolic content in Pseudomonas aeruginosa biofilms. Super-resolution microscopy reveals that explosive cell lysis also produces shattered membrane fragments that rapidly form MVs. A prophage endolysin encoded within the R- and F-pyocin gene cluster is essential for explosive cell lysis. Endolysin-deficient mutants are defective in MV production and biofilm development, consistent with a crucial role in the biogenesis of MVs and liberation of extracellular DNA and other biofilm matrix components. Our findings reveal that explosive cell lysis, mediated through the activity of a cryptic prophage endolysin, acts as a mechanism for the production of bacterial MVs.

  19. Lysis of Escherichia coli cells by lysozyme: discrimination between adsorption and enzyme action.

    PubMed

    Sedov, S A; Belogurova, N G; Shipovskov, S; Levashov, A V; Levashov, P A

    2011-11-01

    The key factors of enzymatic lysis of cells are the interaction between the enzyme and the cell - catalytic and non-catalytic adsorption of enzyme on cell surface. Here, the studies of lysis of intact Escherichia coli cells by chicken egg white lysozyme were performed. It was found that the ionic strength has a dual effect onto the system. On the one hand, the desorption constant of the enzyme increases with the increase of the solution ionic strength, which results in a better enzyme performance. On the other hand, due to the higher osmosis, the cell lysis rate decreases with the increasing of ionic strength of the system. It was found that pH 8.6 and 30 mM NaCl are optimal conditions for lysis of E. coli cells by lysozyme. Copyright © 2011 Elsevier B.V. All rights reserved.

  20. Explosive cell lysis as a mechanism for the biogenesis of bacterial membrane vesicles and biofilms

    PubMed Central

    Turnbull, Lynne; Toyofuku, Masanori; Hynen, Amelia L.; Kurosawa, Masaharu; Pessi, Gabriella; Petty, Nicola K.; Osvath, Sarah R.; Cárcamo-Oyarce, Gerardo; Gloag, Erin S.; Shimoni, Raz; Omasits, Ulrich; Ito, Satoshi; Yap, Xinhui; Monahan, Leigh G.; Cavaliere, Rosalia; Ahrens, Christian H.; Charles, Ian G.; Nomura, Nobuhiko; Eberl, Leo; Whitchurch, Cynthia B.

    2016-01-01

    Many bacteria produce extracellular and surface-associated components such as membrane vesicles (MVs), extracellular DNA and moonlighting cytosolic proteins for which the biogenesis and export pathways are not fully understood. Here we show that the explosive cell lysis of a sub-population of cells accounts for the liberation of cytosolic content in Pseudomonas aeruginosa biofilms. Super-resolution microscopy reveals that explosive cell lysis also produces shattered membrane fragments that rapidly form MVs. A prophage endolysin encoded within the R- and F-pyocin gene cluster is essential for explosive cell lysis. Endolysin-deficient mutants are defective in MV production and biofilm development, consistent with a crucial role in the biogenesis of MVs and liberation of extracellular DNA and other biofilm matrix components. Our findings reveal that explosive cell lysis, mediated through the activity of a cryptic prophage endolysin, acts as a mechanism for the production of bacterial MVs. PMID:27075392

  1. Tumor lysis associated with sudden onset of syndrome of inappropriate antidiuretic hormone secretion.

    PubMed

    Saintigny, Pierre; Chouahnia, Kader; Cohen, Régis; Pailler, Marie-Christine; Brechot, Jeanne-Marie; Morere, Jean-François; Breau, Jean-Luc

    2007-01-01

    Syndrome of inappropriate antidiuretic hormone secretion is frequent in small-cell lung carcinomas. We report on a case of syndrome of inappropriate antidiuretic hormone secretion after each of the first 2 cycles of chemotherapy for small-cell lung cancer. The association with chemotherapy-induced tumor lysis is proposed, particularly based on the course of antidiuretic hormone levels, and a review of the literature is presented. Syndrome of inappropriate antidiuretic hormone secretion can occur during tumor lysis syndrome.

  2. Genetic evidence that the bacteriophage φX174 lysis protein inhibits cell wall synthesis

    PubMed Central

    Bernhardt, Thomas G.; Roof, William D.; Young, Ry

    2000-01-01

    Protein E, a 91-residue membrane protein of φX174, causes lysis of the host in a growth-dependent manner reminiscent of cell wall antibiotics, suggesting E acts by inhibiting peptidoglycan synthesis. In a search for the cellular target of E, we previously have isolated recessive mutations in the host gene slyD (sensitivity to lysis) that block the lytic effects of E. The role of slyD, which encodes a FK506 binding protein-type peptidyl-prolyl cis-trans isomerase, is not fully understood. However, E mutants referred to as Epos (plates on slyD) lack a slyD requirement, indicating that slyD is not crucial for lysis. To identify the gene encoding the cellular target, we selected for survivors of Epos. In this study, we describe the isolation of dominant mutations in the essential host gene mraY that result in a general lysis-defective phenotype. mraY encodes translocase I, which catalyzes the formation of the first lipid-linked intermediate in cell wall biosynthesis. The isolation of these lysis-defective mutants supports a model in which translocase I is the cellular target of E and that inhibition of cell wall synthesis is the mechanism of lysis. PMID:10760296

  3. Charge injection through nanocomposite electrode in microfluidic channel for electrical lysis of biological cells

    NASA Astrophysics Data System (ADS)

    Mishra, Madhusmita; Krishna, Anil; Chandra, Aman; Shenoy, B. M.; Hegde, G. M.; Mahapatra, D. Roy

    2013-03-01

    Several concepts have been developed in the recent years for nanomaterial based integrated MEMS platform in order to accelerate the process of biological sample preparation followed by selective screening and identification of target molecules. In this context, there exist several challenges which need to be addressed in the process of electrical lysis of biological cells. These are due to (i) low resource settings while achieving maximal lysis (ii) high throughput of target molecules to be detected (iii) automated extraction and purification of relevant molecules such as DNA and protein from extremely small volume of sample (iv) requirement of fast, accurate and yet scalable methods (v) multifunctionality toward process monitoring and (vi) downward compatibility with already existing diagnostic protocols. This paper reports on the optimization of electrical lysis process based on various different nanocomposite coated electrodes placed in a microfluidic channel. The nanocomposites are synthesized using different nanomaterials like Zinc nanorod dispersion in polymer. The efficiency of electrical lysis with various different electrode coatings has been experimentally verified in terms of DNA concentration, amplification and protein yield. The influence of the coating thickness on the injection current densities has been analyzed. We further correlate experimentally the current density vs. voltage relationship with the extent of bacterial cell lysis. A coupled multiphysics based simulation model is used to predict the cell trajectories and lysis efficiencies under various electrode boundary conditions as estimated from experimental results. Detailed in-situ fluorescence imaging and spectroscopy studies are performed to validate various hypotheses.

  4. Variable sensitivity to complement-mediated lysis among Trypanosoma rangeli reference strains.

    PubMed

    de Sousa, Maria Auxiliadora; Dos Santos Pereira, Sheila Medeiros; Dos Santos Faissal, Barbara Neves

    2012-02-01

    Six reference strains of Trypanosoma rangeli from different days of growth in axenic cultures were assayed for susceptibility to complement-mediated lysis by non-immune guinea-pig serum. Their authenticity was also confirmed by isoenzyme analyses. Parasites were incubated with 25% active or 68°C-inactivated serum (37°C, 30 min) for all tests; thereafter the lysis rates were determined. Highly variable lysis percentages were observed among T. rangeli strains and in the same stock at different growing days. In a few assays, three strains (Macias, R-1625 and Choachi) presented total or very high resistance. The others (H-14, San Agustín and SC-58) were generally most susceptible, and could reach lysis rates as high as Trypanosoma cruzi. After incubation with active sera, the epimastigotes were usually the predominant stages, being followed by spheromastigotes and/or transitional forms. Those stages and trypomastigotes could also be partially susceptible. In four strains, the short epimastigotes were more resistant to lysis than the long ones. Experiments with C3-deficient serum displayed total or partial participation of the alternative-complement pathway in T. rangeli lysis. This study confirmed the variable complement sensitivity of T. rangeli, which can be related to its intraspecific heterogeneity, to the remarkable complexity of its life-cycle stages, and to the methodology employed.

  5. Retrospective analysis of double-strand break rejoining data collected using warm-lysis PFGE protocols.

    PubMed

    Ratnayake, R K; Semenenko, V A; Stewart, R D

    2005-06-01

    Sample preparation procedures for the pulsed-field gel electrophoresis (PFGE) assay usually involve a lysis step at temperatures as high as 50 degrees C. During this warm-lysis procedure, multiply damaged sites containing heat-labile sites (HLS) can be converted into double-strand breaks (DSB). Once formed, these DSB cannot be distinguished from the DSB formed directly by ionizing radiation. This paper develops a method to correct DSB estimates for the effects of HLS in warm-lysis protocols. A first-order repair model is used to predict the number of HLS available for conversion into DSB as a function of the time available for repair before initiating warm-lysis. A mathematical expression is derived to separate prompt DSB from those formed through the artefactual conversion of HLS into DSB. The proposed formalism only requires the specification of two adjustable parameters, both of which can be estimated from measured data. Estimates of prompt DSB yields obtained by correcting warm-lysis data are in good agreement with estimates obtained using cold-lysis protocols, which do not include the effect of HLS. The retrospective analyses of two published datasets suggest that corrections for HLS have a substantial impact on DSB yields within the first 20-30 min after irradiation. Bi-exponential fits to the DSB data for Chinese hamster ovary cells suggest that corrections for HLS reduce the half-time for fast DSB rejoining by about 15%, whereas the half-time for the slow DSB rejoining only decreases by 4%. The total DSB yield and the fraction of fast-rejoining DSB decrease by 24 and 38%, respectively, when the correction is applied. The proposed formalism can be used to characterize trends and uncertainties in DSB rejoining kinetics associated with the artefactual conversion of HLS into DSB. The retrospective application of the methodology to warm-lysis data enhances their relevance and usefulness for studies of DSB rejoining kinetics.

  6. Micro Corona Ionizer as an Ozone Source for Bacterial Cell Lysis

    NASA Astrophysics Data System (ADS)

    Lee, Eun-Hee; Lim, Hyun Jeong; Chua, Beelee; Son, Ahjeong

    2015-04-01

    DNA extraction is a critical process of DNA assays including polymerase chain reaction (PCR), microarrays, molecular cloning, and DNA hybridization which has been well established and can be implemented by commercial kits. DNA extraction involves cell lysis, precipitation, and purification through the combination of physical and chemical processes. Cell lysis is essential to high DNA recovery yield which can be achieved via a variety of physical, chemical, and enzymatic methods. However, these methods were originally developed for bioassays that were labor intensive, time consuming, and vulnerable to contamination and inhibition. Here, we proposed to employ a micro corona ionizer as an ozone source to lyse bacterial cells. Ozone has been well known and used as a disinfectant which allows cell lysis and DNA extraction. Previously, we have shown that a micro corona ionizer is capable of generating a significant amount of ozone. In this study, we employed the micro corona ionizer for the bacterial cell lysis which consists of a 50 μm diameter cantilever wire as the discharge cathode and a 50 μm thick copper foil as anode. Applied voltages varied from 1900 to 2200 V with corresponding corona currents from 16 to 28 μA. The resultant ozone (concentration > 0.14 ppm) generated from the micro corona ionizer was bubbled into the sample via a miniature pump. We demonstrated the cell lysis of Pseudomonas putida as the target bacterium using the micro corona ionizer. At a flow rate of 38 ml/min and applied corona voltage of 2000 V, 98.5 ± 0.2% lysis (normalized to sonication result) was achieved after 10 min. In comparison, untreated and air-treated samples showed normalized % lysis of 11.9 ± 2.4 and 36.1 ± 1.7%, respectively. We also showed that the cell lysis efficiency could be significantly increased by increasing the flow rate and the applied corona voltage. By comparing the experimental results for continuous and pulsed treatment, we verified that the percentage of

  7. Ribosomes in the sea: a window on taxon-specific lysis

    NASA Astrophysics Data System (ADS)

    Suttle, C.; Zhong, X.; Wirth, J.

    2016-02-01

    Microbes are estimated to comprise more than 90% of the biomass in the world's oceans, are major drivers of biogeochemical cycles, and have turnover rates ranging from hours to days. Despite the central role that microbes play in marine ecosystems, there is no robust method to evaluate taxon-specific mortality rates. Here, we report a method that employs extracellular free-ribosomes as a proxy to evaluate taxon-specific microbial lysis. The method was validated with laboratory cultures of the marine heterotrophic bacterium Vibrio natriegens strain PWH3a and the photoautotroph Synechococcus strain DC2, with and without grazers or viruses, to identify the origin and fate of the extracellular free-ribosomes. Our results showed both viral lysis and programmed-cell-death (PCD) contribute to free-ribosome production. Ribosomes were not released when cells were grazed, but grazers could consume free-ribosomes. We show that extracellular free-ribosomes can be used to evaluate microbial mortality caused by viral lysis and PCD. This approach was applied to environmental samples by examining the taxonomic composition and relative abundance of free 16S-ribosomes in seawater samples collected from the Strait of Georgia and Saanich Inlet, British Columbia, Canada. Based on the presence of free ribosomes, lysis was detected in 2198 out of 4013 prokaryotic taxa, representing 22 bacterial and three archaeal phyla. Of these, lysis of 140 taxa could be detected in all nine samples. Based on the ratio of free ribosomes to cellular ribosomes, some taxa associated with specific ecological niches appeared to be subject to high rates of lysis, including the genera Achromobacter, Chryseobacterium, Clostridium, Delftia, Ferruginibacter, Lactobacillus, Marinomonas, Massilia, Microbacterium, Ochrobactrum, Paenibacillus, Phyllobacterium, Pseudomonas, Rhodobacter, and Stenotrophomonas. Our results showed high-lysis coupled with low-abundance, suggesting that taxa in lower abundance are subject

  8. Involvement of AQP6 in the Mercury-sensitive osmotic lysis of rat parotid secretory granules.

    PubMed

    Matsuki-Fukushima, Miwako; Fujita-Yoshigaki, Junko; Murakami, Masataka; Katsumata-Kato, Osamu; Yokoyama, Megumi; Sugiya, Hiroshi

    2013-03-01

    In secretory granules and vesicles, membrane transporters have been predicted to permeate water molecules, ions and/or small solutes to swell the granules and promote membrane fusion. We have previously demonstrated that aquaporin-6 (AQP6), a water channel protein, which permeates anions, is localized in rat parotid secretory granules (Matsuki-Fukushima et al., Cell Tissue Res 332:73-80, 2008). Because the localization of AQP6 in other organs is restricted to cytosolic vesicles, the native function or functions of AQP6 in vivo has not been well determined. To characterize the channel property in granule membranes, the solute permeation-induced lysis of purified secretory granules is a useful marker. To analyze the role of AQP6 in secretory granule membranes, we used Hg²⁺, which is known to activate AQP6, and investigated the characteristics of solute permeability in rat parotid secretory granule lysis induced by Hg²⁺ (Hg lysis). The kinetics of osmotic secretory granule lysis in an iso-osmotic KCl solution was monitored by the decay of optical density at 540 nm using a spectrophotometer. Osmotic secretory granule lysis was markedly facilitated in the presence of 0.5-2.0 μM Hg²⁺, concentrations that activate AQP6. The Hg lysis was completely blocked by β-mercaptoethanol which disrupts Hg²⁺-binding, or by removal of chloride ions from the reaction medium. An anion channel blocker, DIDS, which does not affect AQP6, discriminated between DIDS-insensitive and sensitive components in Hg lysis. These results suggest that Hg lysis is required for anion permeability through the protein transporter. Hg lysis depended on anion conductance with a sequence of NO(3) (-) > Br⁻ > I⁻ > Cl⁻ and was facilitated by acidic pH. The anion selectivity for NO(3) (-) and the acidic pH sensitivity were similar to the channel properties of AQP6. Taken together, it is likely that AQP6 permeates halide group anions as a Hg²⁺-sensitive anion channel in rat parotid

  9. Significance of Viral Lysis and Flagellate Grazing as Factors Controlling Bacterioplankton Production in a Eutrophic Lake

    PubMed Central

    Weinbauer, Markus G.; Höfle, Manfred G.

    1998-01-01

    The effects of viral lysis and heterotrophic nanoflagellate (HNF) grazing on bacterial mortality were estimated in a eutrophic lake (Lake Plußsee in northern Germany) which was separated by a steep temperature and oxygen gradient into a warm and oxic epilimnion and a cold and anoxic hypolimnion. Two transmission electron microscopy-based methods (whole-cell examination and thin sections) were used to determine the frequency of visibly infected cells, and a model was used to estimate bacterial mortality due to viral lysis. Examination of thin sections also showed that between 20.2 and 29.2% (average, 26.1%) of the bacterial cells were empty (ghosts) and thus could not contribute to viral production. The most important finding was that the mechanism for regulating bacterial production shifted with depth from grazing control in the epilimnion to control due to viral lysis in the hypolimnion. We estimated that in the epilimnion viral lysis accounted on average for 8.4 to 41.8% of the summed mortality (calculated by determining the sum of the mortalities due to lysis and grazing), compared to 51.3 to 91.0% of the summed mortality in the metalimninon and 88.5 to 94.2% of the summed mortality in the hypolimnion. Estimates of summed mortality values indicated that bacterial production was controlled completely or almost completely in the epilimnion (summed mortality, 66.6 to 128.5%) and the hypolimnion (summed mortality, 43.4 to 103.3%), whereas in the metalimnion viral lysis and HNF grazing were not sufficient to control bacterial production (summed mortality, 22.4 to 56.7%). The estimated contribution of organic matter released by viral lysis of cells into the pool of dissolved organic matter (DOM) was low; however, since cell lysis products are very likely labile compared to the bulk DOM, they might stimulate bacterial production. The high mortality of bacterioplankton due to viral lysis in anoxic water indicates that a significant portion of bacterial production in

  10. Natural killer cell-mediated lysis of autologous cells modified by gene therapy.

    PubMed

    Liberatore, C; Capanni, M; Albi, N; Volpi, I; Urbani, E; Ruggeri, L; Mencarelli, A; Grignani, F; Velardi, A

    1999-06-21

    This study investigated the role of natural killer (NK) cells as effectors of an immune response against autologous cells modified by gene therapy. T lymphocytes were transduced with LXSN, a retroviral vector adopted for human gene therapy that carries the selectable marker gene neo, and the autologous NK response was evaluated. We found that (i) infection with LXSN makes cells susceptible to autologous NK cell-mediated lysis; (ii) expression of the neo gene is responsible for conferring susceptibility to lysis; (iii) lysis of neo-expressing cells is clonally distributed and mediated only by NK clones that exhibit human histocompatibility leukocyte antigen (HLA)-Bw4 specificity and bear KIR3DL1, a Bw4-specific NK inhibitory receptor; and (iv) the targets are cells from HLA-Bw4(+) individuals. Finally, neo peptides anchoring to the Bw4 allele HLA-B27 interfered with KIR3DL1-mediated recognition of HLA-B27, i.e., they triggered NK lysis. Moreover, neo gene mutations preventing translation of two of the four potentially nonprotective peptides reduced KIR3DL1(+) NK clone-mediated autologous lysis. Thus, individuals expressing Bw4 alleles possess an NK repertoire with the potential to eliminate autologous cells modified by gene therapy. By demonstrating that NK cells can selectively detect the expression of heterologous genes, these observations provide a general model of the NK cell-mediated control of viral infections.

  11. [Tumor lysis syndrome in the course of neoplastic disease of hematopoietic system in children].

    PubMed

    Bubała, H; Sońta-Jakimczyk, D; Kałmuk, A; Janik-Moszant, A

    1998-01-01

    Tumor lysis syndrome is characterized by hyperuricemia, hyperkalemia and hyperphosphatemia followed by hypocalcemia. Sometimes they are accompanied by hyperglycemia. It can lead to acute renal insufficiency, neurological and cardiological disorders. In the Clinic of Paediatric Haematology and Chemotherapy in Zabrze, in the period from Jan.01, 1990 to Dec.31, 1997, 305 children were treated due to neoplastic diseases of haematopoietic system: 169 with ALL, 34--ANLL, 48--NHL and 54--HD. Tumor lysis syndrome was found in 11 children, what was 3.6% of all patients. They included 3 children with ALL/1 from group SRG and 2 with HRG/, 2 with ANLL, 6 with NHL/2 with NB-NHL and 4 with B-NHL/. Tumor lysis syndrome did not occur in any patient with HD. Two symptoms of tumor lysis syndrome were found in 13 children (4.26%) including 3 with ALL (1 SRG and 2 HRG), 2 with ANLL and 8 NHL/3-NB-NHL and 5-B-NHL/. Diabetes occurred in two children with ALL. Acute renal insufficiency occurred in 6 children with ALL, 2 with ANLL and 8 with NHL. Two patients with B-NHL required dialysis. One renal insufficiency treated with hemodialyses was the first symptom of lymphoma. To prevent the consequences of tumor lysis syndrome hydration with alkalization, constant monitoring of the above mentioned biochemical parameters are necessary. Acute renal insufficiency, not responding to conservative treatment, should be immediately treated with hemodialyses and chemotherapy should be introduced under its 'shield'.

  12. Low-dose steroid-induced tumor lysis syndrome in a hepatocellular carcinoma patient.

    PubMed

    Kim, Jin Ok; Jun, Dae Won; Tae, Hye Jin; Lee, Kang Nyeong; Lee, Hang Lak; Lee, Oh Young; Choi, Ho Soon; Yoon, Byung Chul; Hahm, Joon Soo

    2015-03-01

    Tumor lysis syndrome is rare in hepatocellular carcinoma (HCC), but it has been reported more frequently recently in response to treatments such as transcatheter arterial chemoembolization (TACE), radiofrequency thermal ablation (RFTA), and sorafenib. Tumor lysis syndrome induced by low-dose steroid appears to be very unusual in HCC. We report a patient with hepatitis-C-related liver cirrhosis and HCC in whom tumor lysis syndrome occurred due to low-dose steroid (10 mg of prednisolone). The patient was a 90-year-old male who presented at the emergency room of our hospital with general weakness and poor oral intake. He had started to take prednisolone to treat adrenal insufficiency 2 days previously. Laboratory results revealed hyperuricemia, hyperphosphatemia, and increased creatinine. These abnormalities fulfilled the criteria in the Cairo-Bishop definition of tumor lysis syndrome. Although the patient received adequate hydration, severe metabolic acidosis and acute kidney injury progressed unabated. He finally developed multiple organ failure, and died 3 days after admission. This was a case of tumor lysis syndrome caused by administration of low-dose steroid in a patient with HCC.

  13. Low-dose steroid-induced tumor lysis syndrome in a hepatocellular carcinoma patient

    PubMed Central

    Kim, Jin Ok; Tae, Hye Jin; Lee, Kang Nyeong; Lee, Hang Lak; Lee, Oh Young; Choi, Ho Soon; Yoon, Byung Chul; Hahm, Joon Soo

    2015-01-01

    Tumor lysis syndrome is rare in hepatocellular carcinoma (HCC), but it has been reported more frequently recently in response to treatments such as transcatheter arterial chemoembolization (TACE), radiofrequency thermal ablation (RFTA), and sorafenib. Tumor lysis syndrome induced by low-dose steroid appears to be very unusual in HCC. We report a patient with hepatitis-C-related liver cirrhosis and HCC in whom tumor lysis syndrome occurred due to low-dose steroid (10 mg of prednisolone). The patient was a 90-year-old male who presented at the emergency room of our hospital with general weakness and poor oral intake. He had started to take prednisolone to treat adrenal insufficiency 2 days previously. Laboratory results revealed hyperuricemia, hyperphosphatemia, and increased creatinine. These abnormalities fulfilled the criteria in the Cairo-Bishop definition of tumor lysis syndrome. Although the patient received adequate hydration, severe metabolic acidosis and acute kidney injury progressed unabated. He finally developed multiple organ failure, and died 3 days after admission. This was a case of tumor lysis syndrome caused by administration of low-dose steroid in a patient with HCC. PMID:25834806

  14. Stochastic holin expression can account for lysis time variation in the bacteriophage λ.

    PubMed

    Singh, Abhyudai; Dennehy, John J

    2014-06-06

    The inherent stochastic nature of biochemical processes can drive differences in gene expression between otherwise identical cells. While cell-to-cell variability in gene expression has received much attention, randomness in timing of events has been less studied. We investigate event timing at the single-cell level in a simple system, the lytic pathway of the bacterial virus phage λ. In individual cells, lysis occurs on average at 65 min, with an s.d. of 3.5 min. Interestingly, mutations in the lysis protein, holin, alter both the lysis time (LT) mean and variance. In our analysis, LT is formulated as the first-passage time (FPT) for cellular holin levels to cross a critical threshold. Exact analytical formulae for the FPT moments are derived for stochastic gene expression models. These formulae reveal how holin transcription and translation efficiencies independently modulate the LT mean and variation. Analytical expressions for the LT moments are used to evaluate previously published single-cell LT data for λ phages with mutations in the holin sequence or its promoter. Our results show that stochastic holin expression is sufficient to account for the intercellular LT differences in both wild-type phages, and phage variants where holin transcription and the threshold for lysis have been experimentally altered. Finally, our analysis reveals regulatory motifs that enhance the robustness of lysis timing to cellular noise.

  15. A colorimetric assay for measuring the lysis of a plasma clot

    SciTech Connect

    Mao, S.J.; Tucci, M.A. )

    1991-01-01

    The present study describes a simple, quantitative assay for measuring the lysis of a plasma clot. The principle of the assay is based on the release of Coomassie brilliant blue R-250 dye from the clot. Thirty microliters of freshly prepared Coomassie brilliant blue R-250 (1 mg/ml) was added to 200 microliters of diluted human plasma (1:5). After mixing, 100 microliters of thrombin (2.5 NIH units/ml) were added to mediate a plasma clot. One milliliter of streptokinase (0.1 mg/ml) was used as a plasminogen activator to initiate clot lysis. During the course of lysis, 100 microliters of soluble material were transferred to microtiter wells and the absorbance at 540 nm was determined as a measure of clot lysis. This assay was used to measure clot lysis in 18 human plasma samples. The colorimetric method (x) developed in this report correlated well with that determined using a conventional 125I-fibrinogen method (Y): Y = 0.83x + 7.98 (r = 0.91).

  16. Ibrutinib-associated tumor lysis syndrome in a patient with mantle cell lymphoma: A case report.

    PubMed

    Kaur, Varinder; Swami, Arjun

    2016-03-11

    Mantle cell lymphoma accounts for 5-7% of all non-Hodgkin's lymphomas. Under the current WHO classification, it is categorized as an indolent B cell lymphoma, but has an aggressive clinical course. New insights into leukemogenic molecular pathways of mantle cell lymphoma have uncovered unique therapeutic targets. Ibrutinib, a Bruton's tyrosine kinase inhibitor, is the newest drug in the arsenal that has shown promising efficacy in relapsed mantle cell lymphoma. Long-term studies have shown that grade 3 or 4 adverse events are infrequent. Asymptomatic lymphocytosis is frequently seen with ibrutinib use in mantle cell lymphoma; however, tumor lysis syndrome is an extremely rare complication. To date, only two patients with ibrutinib-associated tumor lysis syndrome in mantle cell lymphoma have been described in a long-term follow-up study. Both patients met laboratory criteria for tumor lysis syndrome, however, but did not develop clinical tumor lysis syndrome. We, here describe a patient with relapsed mantle cell lymphoma who developed clinical tumor lysis syndrome with ibrutinib monotherapy.

  17. Effective Prevention and Management of Tumor Lysis Syndrome in Children With Cancer: The Important Contributions of Pediatric Oncology Nurses.

    PubMed

    Li, Ho Cheung William; Chung, Oi Kwan Joyce; Tam, Ching Janice; Chiu, Sau Ying

    2015-01-01

    The practice guidelines aimed to identify appropriate nursing management for the prevention and treatment of tumor lysis syndrome, in line with the current evidence-based medical guidelines. Using a systematic approach, 15 relevant articles were identified for the review. The evaluation of patient risk factors for tumor lysis syndrome and the appropriate medical and nursing assessment were identified. The treatment algorithms for the prevention of tumor lysis syndrome from both the medical and nursing perspectives have been established. In particular, the guidelines highlight the importance of pediatric oncology nurses in contributing to the prevention and management of tumor lysis syndrome.

  18. The euglobulin clot lysis time to assess the impact of nanoparticles on fibrinolysis

    NASA Astrophysics Data System (ADS)

    Minet, Valentine; Alpan, Lutfiye; Mullier, François; Toussaint, Olivier; Lucas, Stéphane; Dogné, Jean-Michel; Laloy, Julie

    2015-07-01

    Nanoparticles (NPs) are developed for many applications in various fields, including nanomedicine. The NPs used in nanomedicine may disturb homeostasis in blood. Secondary hemostasis (blood coagulation) and fibrinolysis are complex physiological processes regulated by activators and inhibitors. An imbalance of this system can either lead to the development of hemorrhages or thrombosis. No data are currently available on the impact of NPs on fibrinolysis. The objectives of this study are (1) to select a screening test to study ex vivo the impact of NPs on fibrinolysis and (2) to test NPs with different physicochemical properties. Euglobulin clot lysis time test was selected to screen the impact of some NPs on fibrinolysis using normal pooled plasma. A dose-dependent decrease in the lysis time was observed with silicon dioxide and silver NPs without disturbing the fibrin network. Carbon black, silicon carbide, and copper oxide did not affect the lysis time at the tested concentrations.

  19. [Rapid extraction of DNA from Chinese medicinal products by alkaline lysis].

    PubMed

    Zheng, Qi; Jiang, Chao; Huang, Lu-Qi; Zhang, Zhi-Jie; Li, Rao-Rao; Chen, Kang; Yuan, Yuan; Jin, Yan

    2014-10-01

    The study is aimed to explore a rapid method to extract DNA from fried Chinese medicinal products. The alkaline lysis buffer was made of sodium hydroxide, 1% PVP and 1% TritonX-100 and Tris-HCl solution was neutralized, through heat cracking and neutralization two step to extract DNA from processed and prepared products of traditional Chinese medicine. Then universal primes were used to amplify PCR products for fired Chinese medicinal materials. The results indicated the optimized alkaline lysis method for extracting DNA is quick and easy. Extracting of the different processed Sophora japonica of DNA concentration was (420.61 ± 123.91) g x L(-1). Using 5% Chelex-100 resin purification can improve the DNA concentration. Our results showed that the optimized alkaline lysis method is suitable for Chinese medicinal materials for quickly DNA extraction.

  20. Leishmania (Leishmania) chagasi interactions with Serratia marcescens: ultrastructural studies, lysis and carbohydrate effects.

    PubMed

    Moraes, Caroline S; Seabra, Sergio H; Castro, Daniele P; Brazil, Reginaldo P; de Souza, Wanderley; Garcia, Eloi S; Azambuja, Patrícia

    2008-04-01

    Studies on the lysis of L. chagasi caused by the bacteria Serratia marcescens were carried out. In vitro experiments demonstrated that S. marcescens variant SM 365, a prodigiosin pigment producer, lysed this species of Leishmania but variant DB11, a nonpigmented bacteria, was unable to lyse the parasite. High concentrations of d-mannose were found to protect L. chagasi markedly diminishing the lysis by S. marcescens SM 365. Promastigotes of L. chagasi bound the lectin Concanavalin A conjugated with FITC, the fluorescence was intensely found at the base of the flagellum (flagellar pocket). Scanning electron microscopy revealed that the bacteria adherence occurred mainly in the flagellar pocket. S. marcescens SM 365 formed filamentous structures, identified as biofilms, which connect the protozoan to the developing bacterial clusters, in low concentrations of bacteria after 30 min incubation time. We suggest that bacterial mannose-sensitive (MS) fimbriae are relevant to S. marcescens SM 365 in the lysis of L. chagasi.

  1. Acute Respiratory Distress Syndrome Associated with Tumor Lysis Syndrome in a Child with Acute Lymphoblastic Leukemia

    PubMed Central

    Macaluso, Alessandra; Genova, Selene; Maringhini, Silvio; Coffaro, Giancarlo; Ziino, Ottavio; D’Angelo, Paolo

    2015-01-01

    Tumor lysis syndrome is a serious and dangerous complication usually associated with antiblastic treatment in some malignancies characterized by high cell turn-over. Mild or severe electrolyte abnormalities including high serum levels of uric acid, potassium, phosphorus, creatinine, bun and reduction of calcium can be responsible for multi-organ failure, involving mostly kidneys, heart and central nervous system. Renal damage can be followed by acute renal failure, weight gain, progressive liver impairment, overproduction of cytokines, and subsequent maintenance of multi-organ damage. Life-threatening acute respiratory failure associated with tumor lysis syndrome is rare. We describe a child with T-cell acute lymphoblastic leukemia, who developed an unusually dramatic tumor lysis syndrome, after administration of the first low doses of steroid, that was rapidly associated with severe acute respiratory distress syndrome. Subsequent clinical course and treatment modalities that resulted in the gradual and full recovery of the child are also described. PMID:25918625

  2. Selective laser suture lysis with a compact, low-cost, red diode laser.

    PubMed

    Shein, Petr; Cilip, Christopher M; Quinto, Guilherme; Behrens, Ashley; Fried, Nathaniel M

    2008-01-01

    The argon (blue-green) laser is currently used for vaporization of sutures during ophthalmic surgery. However, previous studies have reported more effective laser suture lysis and a lower rate of complications using the krypton (red) laser. Red wavelengths are selectively absorbed by the nylon sutures, but are minimally absorbed by adjacent tissue, and not absorbed by hemoglobin, unlike the argon laser wavelengths. More compact and less expensive red diode lasers have recently become commercially available for surgical applications. This study explores the use of a compact, lowpower, red diode laser for selective laser suture lysis. A 225 mW, 660-nm diode laser was used to vaporize 10-0 nylon sutures in human cornea samples with a single laser pulse, pulse energy of 150 mJ, pulse duration of 100 ms, and spot diameter of 55 mum. The red diode laser may represent an inexpensive, compact, and safer alternative laser for use in laser suture lysis during ophthalmic surgery.

  3. Characterizing osmotic lysis kinetics under microfluidic hydrodynamic focusing for erythrocyte fragility studies.

    PubMed

    Zhan, Yihong; Loufakis, Despina Nelie; Bao, Ning; Lu, Chang

    2012-12-07

    The biomechanics of erythrocytes, determined by the membrane integrity and cytoskeletal structure, provides critical information on diseases such as diabetes mellitus, myocardial infarction, hypertension, and sickle cell anemia. Here we demonstrate a simple microfluidic tool for examining erythrocyte fragility based on characterizing osmotic lysis kinetics. Hydrodynamic focusing is used for generating rapid dilution of the buffer and producing lysis of erythrocytes during their flow. The lysis kinetics are tracked by monitoring the release of intracellular contents from cells via recording the light intensity of erythrocytes at various locations in the channel. Such release profile reflects sensitively the changes in erythrocyte fragility induced by chemical, heating, and glucose treatment. Our tool provides a simple approach for probing red blood cell fragility in both basic research and clinical settings.

  4. Cell lysis induced by membrane-damaging detergent saponins from Quillaja saponaria.

    PubMed

    Berlowska, Joanna; Dudkiewicz, Marta; Kregiel, Dorota; Czyzowska, Agata; Witonska, Izabela

    2015-01-01

    This paper presents the results of a study to determine the effect of Quillaja saponaria saponins on the lysis of industrial yeast strains. Cell lysis induced by saponin from Q. saponaria combined with the plasmolysing effect of 5% NaCl for Saccharomyces cerevisiae, Kluyveromyces marxianus yeasts biomass was conducted at 50 °C for 24-48 h. Membrane permeability and integrity of the yeast cells were monitored using fluorescent techniques and concentrations of proteins, free amino nitrogen (FAN) and free amino acids in resulting lysates were analyzed. Protein release was significantly higher in the case of yeast cell lysis promoted with 0.008% Q. saponaria and 5% NaCl in comparison to plasmolysis triggered by NaCl only. Copyright © 2015 Elsevier Inc. All rights reserved.

  5. Utilization of graphene electrode in transparent microwell arrays for high throughput cell trapping and lysis.

    PubMed

    Ameri, S Kabiri; Singh, P K; Sonkusale, S

    2014-11-15

    Here we present a high-throughput, transparent microfluidic device with embedded microwell arrays sandwiched between transparent electrodes made from graphene (at the bottom) and indium tin oxide (at the top) for dielectrophoretic cell trapping and electrical lysis. Graphene suppresses unwanted faradaic reaction effects on the cells and the medium that is typically observed in ITO based electrodes from application of DC field for electrical lysis. This is because graphene is more electrochemically inert than indium tin oxide (ITO) where ITO undergoes reduction-oxidation (redox) reaction in the presence of electrolyte in most standard cell media. This redox process also compromises ITO's electrical properties and optical transparency over multiple use. The presented microfluidic device shows high efficiency for cell trapping and lysis and an electrochemically stable behavior for long operational life.

  6. Singlet oxygen enhances intrinsic thrombolysis: the intrinsic oxidative clot lysis assay (INOXCLA).

    PubMed

    Stief, Thomas W

    2007-10-01

    Granulocytes are important cells of inflammation and cellular thrombolysis. They produce urokinase (u-PA) and chloramines. In this study, u-PA/chloramine-mediated fibrinolysis is imitated in a microtiter-plate. Seventy-five microliters plasma are incubated with 50 microL 50% Pathromtin SL, 6% BSA, and 38 mM CaCl2 for 30 minutes (37 degrees C). Then, 50 microL 10 mM chloramine-T in PBS are added. After 30 minutes (37 degrees C), 50 microL 0, 100, or 10 IU/mL u-PA in 6% BSA-PBS are added and the turbidity is determined at 405 nm after 0, 3, or 16 hours. Clot lysis was increased more than tenfold by 0.5 to 1 micromoles chloramine (ED50 after 3h = about 0.25 micromoles = 2 mM final concentration). The normal range for the present intrinsic oxidative clot lysis assay (INOXCLA) is 100% +/- 25% (MV +/- SD; 100 relative % of norm; the normal lysis being 60 absolute %; CVs < 10%). Fifty percent lysis of adherent microclots occurred after 0.75 hours, 2 hours, 14 hours, 13 days, or 17 days when using 1000, 100, 10, 1, or 0 IU/mL u-PA reagent. If the u-PA activity is quenched by PAI-2, no clot lysis appears. Chloramines are important physiologic generators of nonradical excited singlet oxygen and enhance u-PA-mediated lysis of plasma clots. Based on the u-PA/chloramines coaction, a new global fibrinolysis assay has been derived.

  7. OPTIMIZATION OF A FILTER-LYSIS PROTOCOL TO PURIFY RAT TESTICULAR HOMOGENATES FOR AUTOMATED SPERMATID COUNTING

    PubMed Central

    Pacheco, Sara E.; Anderson, Linnea M.; Boekelheide, Kim

    2013-01-01

    Objectives Quantifying testicular homogenization resistant spermatid heads (HRSH) is a powerful indicator of spermatogenesis. These counts have traditionally been performed manually using a hemocytometer, but this method can be time consuming and biased. We aimed to develop a protocol to reduce debris for the application of automated counting, which would allow for efficient and unbiased quantification of rat HRSH. Findings We developed a filter-lysis protocol that effectively removes debris from rat testicular homogenates. After filtering and lysing the homogenates, we found no statistical differences between manual (classic and filter-lysis) and automated (filter-lysis) counts using one-way ANOVA with Bonferroni’s multiple comparison test. In addition, Pearson’s correlation coefficients were calculated to compare the counting methods and there was a strong correlation between the classic manual counts and the filter-lysis manual (r = 0.85, p = 0.002) and the filter-lysis automated (r = 0.89, p = 0.0005) counts. We also tested the utility of the automated method in a low dose exposure model known to decrease HRSH. Adult Fischer 344 rats exposed to 0.33% 2,5-hexanedione (HD) in the drinking water for 12 weeks demonstrated decreased body (p = 0.02) and testes (p = 0.002) weights. In addition, there was a significant reduction in the number of HRSH per testis (p = 0.002) when compared to control. Conclusions A filter-lysis protocol was optimized to purify rat testicular homogenates for automated HRSH counts. Automated counting systems yield unbiased data and can be applied to detect changes in the testis after low dose toxicant exposure. PMID:22240558

  8. Sensitivity of simian virus 40-transformed C57BL/6 mouse embryo fibroblasts to lysis by murine natural killer cells.

    PubMed

    Fresa, K L; Karjalainen, H E; Tevethia, S S

    1987-02-15

    The susceptibility of mouse cells expressing full-length or truncated transforming protein (T antigen) of simian virus 40 (SV40) to lysis by murine natural killer (NK) cells was assessed. For these studies, C57BL/6 mouse embryo fibroblasts (B6/MEF) were transformed by transfection with SV40 DNA encoding the entire T antigen. The transformed cell lines were tested for susceptibility to lysis by nonimmune CBA splenocytes as a source of NK cells and to lysis by C57BL/6, SV40-specific cytolytic T cells (CTL). It was found that 13 of 15 clonally derived, SV40-transformed H-2b cell lines were susceptible to lysis by NK cells. However, there was some variation in their susceptibility to lysis by NK cells. There was no correlation between susceptibility to lysis by SV40-specific CTL and to lysis by NK cells. Cells transfected with a plasmid which encodes only the N-terminal half of the SV40 T antigen were consistently less susceptible to lysis by NK cells, suggesting that expression of only the N-terminus of the T antigen was insufficient for optimal susceptibility to lysis by NK cells. Primary mouse embryo fibroblasts transformed by human adenovirus type 5 E1 region DNA were also found to be susceptible to NK cell-mediated lysis. Lysis of SV40-transformed cells by nonimmune CBA splenocytes was mediated by NK cells because: lysis was augmented when the effector cells were treated with interferon before assay; and lysis was abrogated when the effector cells were obtained from mice that had been depleted of NK activity by treatment with antiserum against the asialo GM1 surface marker. These results indicate that primary mouse cells which are transformed by SV40 and which express the native T antigen are susceptible to lysis by mouse NK cells. Conversely, cells transformed by a plasmid encoding only the N-terminal half of the T antigen express reduced susceptibility to lysis by NK cells.

  9. [Detection of Yersinia Enterocolitica Bacteriophage PhiYe-F10 Lysis Spectrum and Analysis of the Relationship between Lysis Ability and Virulence Gene of Yersinia Enterocolitica].

    PubMed

    Zha, Tao; Liang, Junrong; Xiao, Yuchun; Jing, Huaiqi

    2016-03-01

    To determine the lysis spectrum of Yersinia enterocolitica bacteriophage phiYe-F10 and to analyze the relationship between the lysis ability of phiYe-F10 and the virulence gene of Yersinia enterocolitica. To observe the lysis ability of the phage phiYe-F10 to the different Yersinia strains with the double-layer technique. The strains used in this study including 213 of Yersinia enterocolitica and 36 of Yersinia pseudotuberculosis and 1 of Yersinia pestis. The virulence genes of these Yersinia enterocolitica (attachment invasion locus (ail) and enterotoxin (ystA, ystB) and yersinia adhesin A (yadA), virulence factor (virF), specific gene for lipopolysaccharide O-side chain of serotype O : 3 (rfbc) were all detected. Among the 213 Yersinia enterocolitica, 84 strains were O : 3 serotype (78 strains with rfbc gene), 10 were serotype O : 5, 13 were serotype O : 8, 34 were serotype O : 9 and 72 were other serotypes. Of these, 77 were typical pathogenic Yersinia enterocolitica harboring with virulence plasmid (ail+, ystA+, ystB-, yadA+, virF+), and 15 were pathogenic bacterial strains deficiency virulence plasmid (ail+, ystA+, ystB-, yadA-, virF-) and the rest 121 were non pathogenic genotype strains. PhiYe-F10 lysed the 71 serotype O : 3 Yersinia enterocolitica strains which were all carried with rfbc+, including 52 pathogenic Yersinia enterocolitica, 19 nonpathogenic Y. enterocolitica. The phiYe-F10 can not lysed serotype O : 5, O : 9 and other serotype Y. enterocolitica, the lysis rate of serotype O : 3 was as high as 84.5%. The phiYe-F10 can not lysed Yersinia pseudotuberculosis and Yersinia pestis. Yersinia phage phiYe-F10 is highly specific for serotype O : 3 Yersinia enterocolitic at 25 degrees C, which showed a typical narrow lysis spectrum. Phage phiYe-F10 can lysed much more pathogenic Y. enterocolitica than nonpathogenic Y. enterocolitica.

  10. Fabrication of rigid microstructures with thiol-ene-based soft lithography for continuous-flow cell lysis.

    PubMed

    Burke, Jeffrey M; Pandit, Kunal R; Goertz, John P; White, Ian M

    2014-09-01

    In this work, we introduce a method for the soft-lithography-based fabrication of rigid microstructures and a new, simple bonding technique for use as a continuous-flow cell lysis device. While on-chip cell lysis techniques have been reported previously, these techniques generally require a long on-chip residence time, and thus cannot be performed in a rapid, continuous-flow manner. Microstructured microfluidic devices can perform mechanical lysis of cells, enabling continuous-flow lysis; however, rigid silicon-based devices require complex and expensive fabrication of each device, while polydimethylsiloxane (PMDS), the most common material used for soft lithography fabrication, is not rigid and expands under the pressures required, resulting in poor lysis performance. Here, we demonstrate the fabrication of microfluidic microstructures from off-stoichiometry thiol-ene (OSTE) polymer using soft-lithography replica molding combined with a post-assembly cure for easy bonding. With finite element simulations, we show that the rigid microstructures generate an energy dissipation rate of nearly 10(7), which is sufficient for continuous-flow cell lysis. Correspondingly, with the OSTE device we achieve lysis of highly deformable MDA-MB-231 breast cancer cells at a rate of 85%, while a comparable PDMS device leads to a lysis rate of only 40%.

  11. Use of pressure cycling technology for cell lysis and recovery of bacterial and fungal communities from soil.

    PubMed

    Bruner, Emily A; Okubara, Patricia A; Abi-Ghanem, Rita; Brown, David J; Reardon, Catherine L

    2015-04-01

    Selection of cell lysis methodology is critical to microbial community analyses due to the inability of any single extraction technology to recover the absolute genetic structure from environmental samples. Numerous methodologies are currently applied to interrogate soil communities, each with its own inherent bias. Here we compared the efficacy and bias of three physical cell lysis methods in conjunction with the PowerLyzer PowerSoil DNA Isolation Kit (MO BIO) for direct DNA extraction from soil: bead-beating, vortex disruption, and hydrostatic pressure cycling technology (PCT). PCT lysis, which is relatively new to soil DNA extraction, was optimized for soils of two different textures prior to comparison with traditional bead-beating and vortex disruption lysis. All cell lysis methods successfully recovered DNA. Although the two traditional mechanical lysis methods yielded greater genomic, bacterial, and fungal DNA per gram soil than the PCT method, the latter resulted in a greater number of unique terminal restriction fragments by terminal RFLP (T-RFLP) analysis. These findings indicate the importance of diversity and quantity measures when assessing DNA extraction bias, as soil DNA retrieved by PCT lysis represented populations not found using traditional mechanical lysis methods.

  12. Cytolysis by Ca-permeable transmembrane channels. Pore formation causes extensive DNA degradation and cell lysis

    PubMed Central

    1989-01-01

    This study investigates the effect of the purified membrane pore formers, staphylococcal alpha-toxin and CTL perforin, on target cell lysis as measured by 51Cr release and on nuclear damage as measured by DNA degradation and 125IUdR release. Both pore formers cause dose- dependent cell lysis, which is accompanied by DNA release. The ratio of DNA/Cr release depends on the nature of target cell and shows the same pattern as the ratio of release of the two markers reported for CTL- mediated lysis of the same targets. DNA degradation is dependent on the presence of intracellular Ca in the target cell and is totally blocked if Ca is chelated by Quin 2 intracellularly and EGTA extracellularly. DNA degradation, in addition, is inhibited by the lysosomotropic agents NH4Cl, chloroquine, and monensin. rTNF doubles the degree of DNA degradation mediated by alpha-toxin in 3-h assays. We conclude that pore formers alone can mediate DNA degradation. In addition, they may promote the uptake of other factors and thereby accelerate their time course of action. DNA degradation by pore formers requires active target participation in a pathway that is dependent on intracellular Ca and lysosomes. These aspects of target lysis resemble CTL- and NK cell- mediated cytolysis. PMID:2538546

  13. Microfluidic systems and methods of transport and lysis of cells and analysis of cell lysate

    DOEpatents

    Culbertson, Christopher T.; Jacobson, Stephen C.; McClain, Maxine A.; Ramsey, J. Michael

    2004-08-31

    Microfluidic systems and methods are disclosed which are adapted to transport and lyse cellular components of a test sample for analysis. The disclosed microfluidic systems and methods, which employ an electric field to rupture the cell membrane, cause unusually rapid lysis, thereby minimizing continued cellular activity and resulting in greater accuracy of analysis of cell processes.

  14. Microfluidic systems and methods for transport and lysis of cells and analysis of cell lysate

    DOEpatents

    Culbertson, Christopher T [Oak Ridge, TN; Jacobson, Stephen C [Knoxville, TN; McClain, Maxine A [Knoxville, TN; Ramsey, J Michael [Knoxville, TN

    2008-09-02

    Microfluidic systems and methods are disclosed which are adapted to transport and lyse cellular components of a test sample for analysis. The disclosed microfluidic systems and methods, which employ an electric field to rupture the cell membrane, cause unusually rapid lysis, thereby minimizing continued cellular activity and resulting in greater accuracy of analysis of cell processes.

  15. Genetically Determined Variation in Lysis Time Variance in the Bacteriophage φX174.

    PubMed

    Baker, Christopher W; Miller, Craig R; Thaweethai, Tanayott; Yuan, Jeffrey; Baker, Meghan Hollibaugh; Joyce, Paul; Weinreich, Daniel M

    2016-04-07

    Researchers in evolutionary genetics recently have recognized an exciting opportunity in decomposing beneficial mutations into their proximal, mechanistic determinants. The application of methods and concepts from molecular biology and life history theory to studies of lytic bacteriophages (phages) has allowed them to understand how natural selection sees mutations influencing life history. This work motivated the research presented here, in which we explored whether, under consistent experimental conditions, small differences in the genome of bacteriophage φX174 could lead to altered life history phenotypes among a panel of eight genetically distinct clones. We assessed the clones' phenotypes by applying a novel statistical framework to the results of a serially sampled parallel infection assay, in which we simultaneously inoculated each of a large number of replicate host volumes with ∼1 phage particle. We sequentially plated the volumes over the course of infection and counted the plaques that formed after incubation. These counts served as a proxy for the number of phage particles in a single volume as a function of time. From repeated assays, we inferred significant, genetically determined heterogeneity in lysis time and burst size, including lysis time variance. These findings are interesting in light of the genetic and phenotypic constraints on the single-protein lysis mechanism of φX174. We speculate briefly on the mechanisms underlying our results, and we discuss the potential importance of lysis time variance in viral evolution.

  16. Genetically Determined Variation in Lysis Time Variance in the Bacteriophage φX174

    PubMed Central

    Baker, Christopher W.; Miller, Craig R.; Thaweethai, Tanayott; Yuan, Jeffrey; Baker, Meghan Hollibaugh; Joyce, Paul; Weinreich, Daniel M.

    2016-01-01

    Researchers in evolutionary genetics recently have recognized an exciting opportunity in decomposing beneficial mutations into their proximal, mechanistic determinants. The application of methods and concepts from molecular biology and life history theory to studies of lytic bacteriophages (phages) has allowed them to understand how natural selection sees mutations influencing life history. This work motivated the research presented here, in which we explored whether, under consistent experimental conditions, small differences in the genome of bacteriophage φX174 could lead to altered life history phenotypes among a panel of eight genetically distinct clones. We assessed the clones’ phenotypes by applying a novel statistical framework to the results of a serially sampled parallel infection assay, in which we simultaneously inoculated each of a large number of replicate host volumes with ∼1 phage particle. We sequentially plated the volumes over the course of infection and counted the plaques that formed after incubation. These counts served as a proxy for the number of phage particles in a single volume as a function of time. From repeated assays, we inferred significant, genetically determined heterogeneity in lysis time and burst size, including lysis time variance. These findings are interesting in light of the genetic and phenotypic constraints on the single-protein lysis mechanism of φX174. We speculate briefly on the mechanisms underlying our results, and we discuss the potential importance of lysis time variance in viral evolution. PMID:26921293

  17. An optimised direct lysis method for gene expression studies on low cell numbers.

    PubMed

    Le, Anh Viet-Phuong; Huang, Dexing; Blick, Tony; Thompson, Erik W; Dobrovic, Alexander

    2015-08-05

    There is increasing interest in gene expression analysis of either single cells or limited numbers of cells. One such application is the analysis of harvested circulating tumour cells (CTCs), which are often present in very low numbers. A highly efficient protocol for RNA extraction, which involves a minimal number of steps to avoid RNA loss, is essential for low input cell numbers. We compared several lysis solutions that enable reverse transcription (RT) to be performed directly on the cell lysate, offering a simple rapid approach to minimise RNA loss for RT. The lysis solutions were assessed by reverse transcription quantitative polymerase chain reaction (RT-qPCR) in low cell numbers isolated from four breast cancer cell lines. We found that a lysis solution containing both the non-ionic detergent (IGEPAL CA-630, chemically equivalent to Nonidet P-40 or NP-40) and bovine serum albumin (BSA) gave the best RT-qPCR yield. This direct lysis to reverse transcription protocol outperformed a column-based extraction method using a commercial kit. This study demonstrates a simple, reliable, time- and cost-effective method that can be widely used in any situation where RNA needs to be prepared from low to very low cell numbers.

  18. A simple and novel modification of comet assay for determination of bacteriophage mediated bacterial cell lysis.

    PubMed

    Khairnar, Krishna; Sanmukh, Swapnil; Chandekar, Rajshree; Paunikar, Waman

    2014-07-01

    The comet assay is the widely used method for in vitro toxicity testing which is also an alternative to the use of animal models for in vivo testing. Since, its inception in 1984 by Ostling and Johansson, it is being modified frequently for a wide range of application. In spite of its wide applicability, unfortunately there is no report of its application in bacteriophages research. In this study, a novel application of comet assay for the detection of bacteriophage mediated bacterial cell lysis was described. The conventional methods in bacteriophage research for studying bacterial lysis by bacteriophages are plaque assay method. It is time consuming, laborious and costly. The lytic activity of bacteriophage devours the bacterial cell which results in the release of bacterial genomic material that gets detected by ethidium bromide staining method by the comet assay protocol. The objective of this study was to compare efficacy of comet assay with different assay used to study phage mediated bacterial lysis. The assay was performed on culture isolates (N=80 studies), modified comet assay appear to have relatively higher sensitivity and specificity than other assay. The results of the study showed that the application of comet assay can be an economical, time saving and less laborious alternative to conventional plaque assay for the detection of bacteriophage mediated bacterial cell lysis.

  19. Resistance of cytotoxic T lymphocytes to lysis by a clone of cytotoxic T lymphocytes

    SciTech Connect

    Kranz, D.M.; Eisen, H.N.

    1987-05-01

    To investigate how cytotoxic T lymphocytes (CTL) avoid killing themselves when they destroy target cells, the authors compared 20 different cell lines as target cells, including several CTL cell lines, for their susceptibility to lysis by CTL, measured by a standard /sup 51/Cr-release assay. Variations in recognition of this diverse set of target cells was circumvented by attaching to all of them a monoclonal antibody to the antigen-specific receptor of a cloned CTL cell line (clone 2C) and using the 2C cell line as the standard aggressor or effector cell. All of the nine tumor cell lines and the four noncytolytic T-helper cell lines tested as targets were highly susceptible to lysis by the aggressor CTL, but seven cytotoxic T-cell lines (six CTL and one T-helper cell line with cytotoxic activity) were largely resistant. These results, and the use of the lectin Con A as an alternative means for triggering CTL activity, point clearly to a level of resistance that could enable CTL to avoid their own destruction when they lyse target cells. The resistance of the cytolytic T cells did not appear to be accompanied by a similar resistance to complement-mediated lysis, indicating that mechanisms of CTL-mediated and complement-mediated lysis are not identical.

  20. Integration of nanoparticle cell lysis and microchip PCR for one-step rapid detection of bacteria.

    PubMed

    Wan, Weijie; Yeow, John T W

    2012-04-01

    This paper describes an integrated microchip system as an efficient and cost-effective solution involving Nanotechnology and Lab-on-a-Chip technology for the rapid detection of bacteria. The system is based on using surface-modified gold nanoparticles for efficient cell lysis followed by microchip PCR without having to remove the nanoparticles from the PCR solution. Poly(quaternary ammonium) modified gold nanoparticles are used to provide a novel and efficient cell lysis method without the need to go through time-consuming, expensive and complicated microfabrication processes as most of current cell lysis methods for Lab-on-a-Chip applications do. It also facilitates the integration of cell lysis and PCR by sharing the same reaction chamber as PCR uses. It is integrated with a prototype microchip PCR system consisting of a physical microchip PCR device and an automated temperature control mechanism. The research work explores solutions for the problem of PCR inhibition caused by gold nanoparticles as well as for the problem of non-specific PCR amplification in the integrated microchip system. It also explores the possibility of greatly reducing PCR cycling time to achieve the same result compared to the protocol for a regular PCR machine. The simplicity of the setup makes it easy to be integrated with other Lab-on-a-Chip functional modules to create customized solutions for target applications.

  1. Disposable on-chip microfluidic system for buccal cell lysis, DNA purification, and polymerase chain reaction.

    PubMed

    Cho, Woong; Maeng, Joon-Ho; Ahn, Yoomin; Hwang, Seung Yong

    2013-09-01

    This paper reports the development of a disposable, integrated biochip for DNA sample preparation and PCR. The hybrid biochip (25 × 45 mm) is composed of a disposable PDMS layer with a microchannel chamber and reusable glass substrate integrated with a microheater and thermal microsensor. Lysis, purification, and PCR can be performed sequentially on this microfluidic device. Cell lysis is achieved by heat and purification is performed by mechanical filtration. Passive check valves are integrated to enable sample preparation and PCR in a fixed sequence. Reactor temperature is needed to lysis and PCR reaction is controlled within ±1°C by PID controller of LabVIEW software. Buccal epithelial cell lysis, DNA purification, and SY158 gene PCR amplification were successfully performed on this novel chip. Our experiments confirm that the entire process, except the off-chip gel electrophoresis, requires only approximately 1 h for completion. This disposable microfluidic chip for sample preparation and PCR can be easily united with other technologies to realize a fully integrated DNA chip. © 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  2. Lysis of fresh human solid tumors by autologous lymphocytes activated in vitro with lectins

    SciTech Connect

    Mazumder, A.; Grimm, E.A.; Zhang, H.Z.; Rosenberg, S.A.

    1982-03-01

    Human peripheral blood lymphocytes (PBL), obtained from patients with a variety of cancers, were incubated in vitro with phytohemagglutinin, concanavalin A, and crude or lectin-free T-cell growth factors. The lectin-activated PBL of nine patients were capable of lysing fresh autologous tumor during a 4-hr 51Cr release assay. Multiple metastases from the same patient were equivalently lysed by these activated autologous PBL. No lysis of fresh PBL or lectin-induced lymphoblast cell targets was seen, although tumor, PBL, and lymphoblast cells were shown to be equally lysable using allosensitized cells. The activated cells could be expanded without loss of cytotoxicity in crude or lectin-free T-cell growth factors. The generation of cells lytic to fresh autologous tumor was dependent on the presence of adherent cells, although the lytic cell itself was not adherent. Proliferation was not involved in the induction of lytic cells since equal lysis was induced in irradiated and nonirradiated lymphocytes. Lectin was not required in the lytic assay, and the addition of alpha-methyl-D-mannoside to concanavalin A-activated lymphoid cells did not increase the lysis of fresh tumor cells. Activation by lectin for 3 days appears to be an efficient and convenient method for generating human cells lytic to fresh autologous tumor. These lytic cells may be of value for studies of the cell-mediated lysis of human tumor and possibly for tumor immunotherapy as well.

  3. Nonspecific cytotoxic cells in fish (Ictalurus punctatus). V. Metabolic requirements of lysis.

    PubMed

    Carlson, R L; Evans, D L; Graves, S S

    1985-01-01

    The mechanisms of lysis of nonspecific cytotoxic cells (NCC) from the channel catfish (Ictalurus punctatus) were studied by determining the effects of various inhibitors of cellular metabolism on cytolysis of NC-37 human lymphoma target cells. Inhibition of NCC-mediated lysis by dinitrophenol (DNP) and sodium azide (NaN3) indicated a requirement for cellular energy metabolism. Cytochalasin B, an inhibitor of microfilaments, and monensin, an inhibitor of cellular secretion, both prevented lysis by NCC. Three microtubule inhibitors, vinblastine sulfate, colchicine, and demecolcine, all inhibited target cell lysis. Two divalent cation chelators, EDTA and EGTA, blocked NCC activity. Elimination of both Ca2+ and Mg2+ by EDTA prevented target cell binding and killing. Selective removal of Ca2+ by EGTA prevented killing but did not block target cell binding. These results indicated that nonspecific cytotoxicity in fish is an active process which requires cell movement and an intact secretory apparatus. The mechanisms of cytolysis by NCC were found (except for the requirement of microtubules) to be analogous to those of mammalian NK cells. Combined with morphological studies, these data strongly suggest that a phylogenetic relationship exists between these effector cells.

  4. Lysis of typhus-group rickettsia-infected targets by lymphokine activated killers

    SciTech Connect

    Carl, M.; Dasch, G.A.

    1986-03-01

    The authors recently described a subset of OKT8, OKT3-positive lymphocytes from typhus-group rickettsia immune individuals which were capable of lysing autologous PHA-blasts or Epstein-Barr virus transformed B cells (LCL) infected with typhus-group rickettsiae. In order to determine if killing by these effectors was HLA-restricted, they stimulated peripheral blood mononuclear cells (PBMC) from typhus-group rickettsia immune individuals in vitro with typhus-group rickettsia-derived antigen for one week and then measured lysis of autologous LCL or HLA-mismatched LCL in a 4-6 hour Cr/sup 51/-release assay. There was significant lysis of both the autologous and the HLA-mismatched infected targets as compared to the corresponding uninfected targets. Since this suggested that the effectors were lymphokine activated killers (LAK) rather than cytotoxic T lymphocytes, they then tested this hypothesis by stimulating PBMC from both immune and non-immune individuals in vitro for one week with purified interleukin 2 and measuring lysis of infected, autologous LCL. PBMC thus treated, from both immune and non-immune individuals, were capable of significantly lysing autologous, infected LCL as compared to the non-infected control. They therefore conclude that targets infected with typhus-group rickettsiae are susceptible to lysis to LAK.

  5. Quantifying enzymatic lysis: estimating the combined effects of chemistry, physiology and physics

    NASA Astrophysics Data System (ADS)

    Mitchell, Gabriel J.; Nelson, Daniel C.; Weitz, Joshua S.

    2010-12-01

    The number of microbial pathogens resistant to antibiotics continues to increase even as the rate of discovery and approval of new antibiotic therapeutics steadily decreases. Many researchers have begun to investigate the therapeutic potential of naturally occurring lytic enzymes as an alternative to traditional antibiotics. However, direct characterization of lytic enzymes using techniques based on synthetic substrates is often difficult because lytic enzymes bind to the complex superstructure of intact cell walls. Here we present a new standard for the analysis of lytic enzymes based on turbidity assays which allow us to probe the dynamics of lysis without preparing a synthetic substrate. The challenge in the analysis of these assays is to infer the microscopic details of lysis from macroscopic turbidity data. We propose a model of enzymatic lysis that integrates the chemistry responsible for bond cleavage with the physical mechanisms leading to cell wall failure. We then present a solution to an inverse problem in which we estimate reaction rate constants and the heterogeneous susceptibility to lysis among target cells. We validate our model given simulated and experimental turbidity assays. The ability to estimate reaction rate constants for lytic enzymes will facilitate their biochemical characterization and development as antimicrobial therapeutics.

  6. NKG2D Signaling Leads to NK Cell Mediated Lysis of Childhood AML

    PubMed Central

    Ditthard, Kerstin; Lang, Peter; Mezger, Markus; Michaelis, Sebastian; Handgretinger, Rupert

    2015-01-01

    Natural killer cells have been shown to be relevant in the recognition and lysis of acute myeloid leukemia. In childhood acute lymphoblastic leukemia, it was shown that HLA I expression and KIR receptor-ligand mismatch significantly impact ALL cytolysis. We characterized 14 different primary childhood AML blasts by flow cytometry including NKG2D ligands. Further HLA I typing of blasts was performed and HLA I on the AML blasts was quantified. In two healthy volunteer NK cell donors HLA I typing and KIR genotyping were done. Blasts with high NKG2D ligand expression had significantly higher lysis by isolated NK cells. Grouping the blasts by NKG2D ligand expression led to a significant inverse correlation of HLA I expression and cytolysis in NKG2D low blasts. Furthermore, a significant positive correlation of NKG2D ligand expression and blast cytolysis was shown. No impact of KIR ligand-ligand mismatch was found but a significantly increased lysis of homozygous C2 blasts by KIR2DL1 negative NK cells (donor B) was revealed. In conclusion, NKG2D signaling leads to NK cell mediated lysis of childhood AML despite high HLA I expression. PMID:26236752

  7. Handheld mechanical cell lysis chip with ultra-sharp silicon nano-blade arrays for rapid intracellular protein extraction.

    PubMed

    Yun, Sung-Sik; Yoon, Sang Youl; Song, Min-Kyung; Im, Sin-Hyeog; Kim, Sohee; Lee, Jong-Hyun; Yang, Sung

    2010-06-07

    This paper presents a handheld mechanical cell lysis chip with ultra-sharp nano-blade arrays fabricated by simple and cost effective crystalline wet etching of (110) silicon. The ultra-sharp nano-blade array is simply formed by the undercutting of (110) silicon during the crystalline wet etching process. Cells can be easily disrupted by the silicon nano-blade array without the help of additional reagents or electrical sources. Based on the bench-top test of the proposed device, a handheld mechanical cell lysis chip with the nano-blade arrays is designed and fabricated for direct connection to a commercial syringe. The direct connection to a syringe provides rapid cell lysis, easy handling, and minimization of the lysate dead volume. The protein concentration in the cell lysate obtained by the proposed lysis chip is quantitatively comparable to the one prepared by a conventional chemical lysis method.

  8. LYSIS-FROM-WITHOUT OF STAPHYLOCOCCUS AUREUS STRAINS BY COMBINATIONS OF SPECIFIC PHAGES AND PHAGE-INDUCED LYTIC ENZYMES

    PubMed Central

    Ralston, Doris J.; McIvor, Mary

    1964-01-01

    Ralston, Doris J. (University of California, Berkeley) and Mary McIvor. Lysis-from-without of Staphylococcus aureus strains by combinations of specific phages and phage-induced lytic enzymes. J. Bacteriol. 88:676–681. 1964—Several typing phages, adsorbed in sufficient concentrations to their homologous propagating strains, altered the cell surface so as to render the cells sensitive to rapid and synergistic lysis by extra-cellular additions of wall lysins. Lysis was effected both by lysins induced by the individual phages and by phage K1 virolysin. Phage K1 also rendered cells sensitive to the lysins of the typing phages. With the exception of lysins from PS 53, 70, and 77, none of the lysins nor purified phages tested separately caused significant lysis of living cells. Lysis-from-without in Staphylococcus aureus appears to be a stepwise process: sensitization by phage followed by digestion of the wall by lysin. PMID:14208506

  9. Phytoplankton Cell Lysis Associated with Polyunsaturated Aldehyde Release in the Northern Adriatic Sea

    PubMed Central

    Ribalet, François; Bastianini, Mauro; Vidoudez, Charles; Acri, Francesco; Berges, John; Ianora, Adrianna; Miralto, Antonio; Pohnert, Georg; Romano, Giovanna; Wichard, Thomas; Casotti, Raffaella

    2014-01-01

    Diatoms are able to react to biotic and abiotic stress, such as competition, predation and unfavorable growth conditions, by producing bioactive compounds including polyunsaturated aldehydes (PUAs). PUAs have been shown to act against grazers and either enhance or inhibit the growth of different phytoplankton and bacteria both in culture and in the field. Presence of nanomolar concentrations of dissolved PUAs in seawater has been reported in the North Adriatic Sea (Mediterranean), suggesting that these compounds are released in seawater following diatom cell lysis. However, the origin of the PUAs and their effects on natural phytoplankton assemblages remain unclear. Here we present data from four oceanographic cruises that took place during diatom blooms in the northern Adriatic Sea where concentrations of particulate and dissolved PUAs were monitored along with phytoplankton cell lysis. Cell lysis was positively correlated with both concentrations of particulate and dissolved PUAs (R = 0.69 and R = 0.77, respectively), supporting the hypothesis that these compounds are released by cell lysis. However, the highest concentration of dissolved PUAs (2.53 nM) was measured when cell lysis was high (0.24 d−1) but no known PUA-producing diatoms were detected, suggesting either that other organisms can produce PUAs or that PUA-producing enzymes retain activity extracellularly after diatom cells have lysed. Although in situ concentrations of dissolved PUAs were one to three orders of magnitude lower than those typically used in laboratory culture experiments, we argue that concentrations produced in the field could induce similar effects to those observed in culture and therefore may help shape plankton community composition and function in the oceans. PMID:24497933

  10. Phytoplankton cell lysis associated with polyunsaturated aldehyde release in the Northern Adriatic Sea.

    PubMed

    Ribalet, François; Bastianini, Mauro; Vidoudez, Charles; Acri, Francesco; Berges, John; Ianora, Adrianna; Miralto, Antonio; Pohnert, Georg; Romano, Giovanna; Wichard, Thomas; Casotti, Raffaella

    2014-01-01

    Diatoms are able to react to biotic and abiotic stress, such as competition, predation and unfavorable growth conditions, by producing bioactive compounds including polyunsaturated aldehydes (PUAs). PUAs have been shown to act against grazers and either enhance or inhibit the growth of different phytoplankton and bacteria both in culture and in the field. Presence of nanomolar concentrations of dissolved PUAs in seawater has been reported in the North Adriatic Sea (Mediterranean), suggesting that these compounds are released in seawater following diatom cell lysis. However, the origin of the PUAs and their effects on natural phytoplankton assemblages remain unclear. Here we present data from four oceanographic cruises that took place during diatom blooms in the northern Adriatic Sea where concentrations of particulate and dissolved PUAs were monitored along with phytoplankton cell lysis. Cell lysis was positively correlated with both concentrations of particulate and dissolved PUAs (R = 0.69 and R = 0.77, respectively), supporting the hypothesis that these compounds are released by cell lysis. However, the highest concentration of dissolved PUAs (2.53 nM) was measured when cell lysis was high (0.24 d(-1)) but no known PUA-producing diatoms were detected, suggesting either that other organisms can produce PUAs or that PUA-producing enzymes retain activity extracellularly after diatom cells have lysed. Although in situ concentrations of dissolved PUAs were one to three orders of magnitude lower than those typically used in laboratory culture experiments, we argue that concentrations produced in the field could induce similar effects to those observed in culture and therefore may help shape plankton community composition and function in the oceans.

  11. On-chip cell lysis by antibacterial non-leaching reusable quaternary ammonium monolithic column.

    PubMed

    Aly Saad Aly, Mohamed; Gauthier, Mario; Yeow, John

    2016-02-01

    Reusable antibacterial non-leaching monolithic columns polymerized in microfluidic channels designed for on-chip cell lysis applications were obtained by the photoinitiated free radical copolymerization of diallyldimethylammonium chloride (DADMAC) and ethylene glycol diacrylate (EGDA) in the presence of a porogenic solvent. The microfluidic channels were fabricated in cross-linked poly(methyl methacrylate) (X-PMMA) substrates by laser micromachining. The monolithic columns have the ability to inhibit the growth of, kill and efficiently lyse Gram-positive Micrococcus luteus (Schroeter) (ATCC 4698) and Kocuria rosea (ATCC 186), and Gram-negative bacteria Pseudomonas putida (ATCC 12633) and Escherichia coli (ATCC 35218) by mechanically shearing the bacterial membrane when forcing the cells to pass through the narrow pores of the monolithic column, and simultaneously disintegrating the cell membrane by physical contact with the antibacterial surface of the column. Cell lysis was confirmed by off-chip PCR without the need for further purification. The influence of the cross-linking monomer on bacterial growth inhibition, leaching, lysis efficiency of the monolithic column and its mechanical stability within the microfluidic channel were investigated and analyzed for three different cross-linking monomers: ethylene glycol dimethacrylate (EGDA), ethylene glycol dimethacrylate (EGDMA) and 1,6-hexanediol dimethacrylate (1,6-HDDMA). Furthermore, the bonding efficiency of two X-PMMA substrates with different cross-linking levels was studied. The monolithic columns were shown to be stable, non-leaching, and reusable for over 30 lysis cycles without significant performance degradation or DNA carryover when they were back-flushed between lysis cycles.

  12. Dynamic monitoring of single cell lysis in an impedance-based microfluidic device.

    PubMed

    Zhou, Ying; Basu, Srinjan; Laue, Ernest D; Seshia, Ashwin A

    2016-08-01

    A microfluidic device that is capable of trapping and sensing dynamic variations in the electrical properties of individual cells is demonstrated. The device is applied to the real-time recording of impedance measurements of mouse embryonic stem cells (mESCs) during the process of membrane lysis, with the resulting changes in the electrical properties of cells during this process being quantitatively tracked over time. It is observed that the impedance magnitude decreases dramatically after cell membrane lysis. A significant shift in the phase spectrum is also observed during the time course of this process. By fitting experimental data to physical models, the electrical parameters of cells can be extracted and parameter variations quantified during the process. In the cell lysis experiments, the equivalent conductivity of the cell membrane is found to increase significantly due to pore formation in the membrane during lysis. An increase in the specific capacitance of the membrane is also observed. On the other hand, the conductivity of the cytoplasm is observed to decrease, which may be explained the fact that excess water enters the cell through the gradual permeabilization of the membrane during lysis. Cells can be trapped in the device for periods up to several days, and their electrical response can be monitored by real-time impedance measurements in a label-free and non-invasive manner. Furthermore, due to the highly efficient single cell trapping capacity of the device, a number of cells can be trapped and held in separate wells for concurrent parallel experiments, allowing for the possibility of stepped parametric experiments and studying cell heterogeneity by combining measurements across the array.

  13. Evidence that neurones accumulating amyloid can undergo lysis to form amyloid plaques in Alzheimer's disease.

    PubMed

    D'Andrea, M R; Nagele, R G; Wang, H Y; Peterson, P A; Lee, D H

    2001-02-01

    Amyloid has recently been shown to accumulate intracellularly in the brains of patients with Alzheimer's disease (AD), yet amyloid plaques are generally thought to arise from gradual extracellular amyloid deposition. We have investigated the possibility of a link between these two apparently conflicting observations. Immunohistochemistry and digital image analysis was used to examine the detailed localization of beta-amyloid(42) (A beta 42), a major component of amyloid plaques, in the entorhinal cortex and hippocampus of AD brains. A beta 42 first selectively accumulates in the perikaryon of pyramidal cells as discrete, granules that appear to be cathepsin D-positive, suggesting that they may represent lysosomes or lysosome-derived structures. AD brain regions abundantly populated with pyramidal neurones exhibiting excessive A beta 42 accumulations also contained evidence of neuronal lysis. Lysis of these A beta 42-burdened neurones apparently resulted in a local, radial dispersion of their cytoplasmic contents, including A beta 42 and lysosomal enzymes, into the surrounding extracellular space. A nuclear remnant was found at the dense core of many amyloid plaques, strengthening the idea that each amyloid plaque represents the end product of a single neuronal cell lysis. The inverse relationship between the amyloid plaque density and pyramidal cell density in the AD brain regions also supports this possibility, as does the close correlation between plaque size and the size of local pyramidal cells. Our findings suggest that excessive intracellular accumulation of A beta 42-positive material in pyramidal cells can result in cell lysis, and that cell lysis is an important source of amyloid plaques and neuronal loss in AD brains.

  14. Susceptibility of colorectal-carcinoma cells to natural-killer-mediated lysis: relationship to CEA expression and degree of differentiation.

    PubMed

    Prado, I B; Laudanna, A A; Carneiro, C R

    1995-06-09

    This study addresses the relevance of colorectal-carcinoma-cell (CRC) CEA expression and degree of differentiation in natural-killer(NK)-mediated lysis susceptibility. A 51Cr-release cytotoxicity assay performed with 5 human CRC lines demonstrated that CRC CEA expression was related to resistance to NK lysis. Moreover, the addition of anti-CEA Fab fragments to the assay led to a significant increase of lysability of high-CEA-producing and NK-resistant cells (LS 174-T), whereas purified CEA drastically reduced lysis of low-CEA-producing and NK-susceptible cells (LISP-I) in a dose-dependent manner. These results strongly suggest that CEA plays a causal role in CRC resistance to NK lysis. Nevertheless, our data did not demonstrate CEA binding to effector cell surface, suggesting that CEA expression can protect CRC, possibly by preventing NK-tumor-cell adhesion to occur. Our results also show that CRC susceptibility to NK lysis was related to a less differentiated phenotype. HCT-8, which are poorly differentiated and low-CEA-producing cells, were cultured in vitro in the presence of the differentiation agent sodium butyrate. Treated cells became less susceptible to NK lysis as they progressed towards a more differentiated phenotype. However, CEA production was not altered upon differentiation. Our study thus demonstrates that both features, CEA expression and degree of cellular differentiation, may individually influence CRC susceptibility to NK lysis.

  15. Gas vesicles isolated from Halobacterium cells by lysis in hypotonic solution are structurally weakened.

    PubMed

    Oren, Aharon; Pri-El, Nuphar; Shapiro, Orr; Siboni, Nachshon

    2005-11-15

    Analysis of pressure-collapse curves of Halobacterium cells containing gas vesicles and of gas vesicles released from such cells by hypotonic lysis shows that the isolated gas vesicles are considerably weaker than those present within the cells: their mean critical collapse pressure was around 0.049-0.058 MPa, as compared to 0.082-0.095 MPa for intact cells. The hypotonic lysis procedure, which is widely used for the isolation of gas vesicles from members of the Halobacteriaceae, thus damages the mechanical properties of the vesicles. The phenomenon can possibly be attributed to the loss of one or more structural gas vesicle proteins such as GvpC, the protein that strengthens the vesicles built of GvpA subunits: Halobacterium GvpC is a highly acidic, typically "halophilic" protein, expected to denature in the absence of molar concentrations of salt.

  16. Lysed BCG vaccines. 1. Observations on optimal conditions for BCG growth and lysis.

    PubMed

    Sato, H; Diena, B B; Greenberg, L

    1967-01-01

    As a first step towards the production of a lysed BCG vaccine that would have reduced toxicity and allergenicity and yet be immunogenic, the growth and lysis of BCG bacilli under strictly controlled conditions have been studied.BCG grown in both Dubos and Aldridge liquid media showed an arithmetic linear growth, preceded by a very short logarithmic phase. This suggested that some of the cell population became metabolically inactive at a very early stage, possibly owing to suboptimal conditions of growth.Glycine, lysozyme and lithium chloride initiated lysis of BCG growth in the aforementioned media 24-48 hours after inoculation. After 2 weeks' incubation approximately 50% of the original BCG culture was lysed and the viable count had fallen 1000-fold. The autolytic pattern of BCG in a nitrogen-deficient, chemically defined Aldridge medium is also described.

  17. One-step cell lysis suitable for quantitative bacteria detection in inhibitor-laden sands

    NASA Astrophysics Data System (ADS)

    Lim, Hyun Jeong; Choi, Jung-Hyun; Son, Ahjeong

    2015-04-01

    Complexity and heterogeneity of soils often hinder effective DNA extraction from the soil matrix. In particular, conventional DNA extraction techniques require extensive purification which makes DNA extraction time-consuming and labor-intensive. Other drawbacks include lower recovery yield, degradation, and damage of DNA, which are also caused by intensive purifications during DNA extraction. Therefore a rapid and simple and yet effective DNA pretreatment method is preferred for environmental monitoring and screening. This study has evaluated the feasibility of simple physical pretreatment for effective cell lysis of bacteria in sands. Bead beating method was selected as an effective physical cell lysis method in this study. We examined the capability of this physical lysis for Pseudomonas putida seeded sands without additional chemical purification steps. The lysate from the method was analysed by the quantitative polymerase chain reaction (qPCR) assay and subsequently compared to that by commercial DNA extraction kit. The best lysis condition (treatment with 0.1 mm glass beads at 3000 rpm for 3 minutes) was selected. The qPCR results of bead beating treated samples showed the better performance than that of conventional DNA extraction kit. Moreover, the qPCR assay was performed to the sands laden with qPCR inhibitors (humic acids, clay, and magnesium), which generally present in environmental samples. Further experiments with the sands containing less than 10 μg/g of humic acids and 70% of clay showed successful quantification results of qPCR assay. In conclusion, the bead beating method is useful for simplified DNA extraction prior to qPCR analysis for sand samples of particular composition. It is expected that this approach will be beneficial for environmental in-situ analysis or immediate pre-screening. It also provides the groundwork for future studies with real soil samples that have various physico-chemical properties.

  18. Peptidoglycan Loss During Hen Egg White Lysozyme-Inorganic Salt Lysis of Streptococcus mutans

    PubMed Central

    Goodman, Hannah; Pollock, Jerry J.; Iacono, Vincent J.; Wong, William; Shockman, Gerald D.

    1981-01-01

    Streptococcus mutans BHT was grown in Todd-Hewitt dialysate medium containing N-acetyl[14C]glucosamine for 6 to 11 generations. After treatment with cold and hot trichloroacetic acid and trypsin, 52 to 65% of the radioactivity remained present in insoluble peptidoglycan-containing residues. Hen egg white lysozyme or mutanolysin treatment of the peptidoglycan residues resulted in the release of 80 and 97%, respectively, of the 14C label to the supernatant fraction. Hydrochloric acid hydrolysates of such supernatants showed that essentially all of the radioactivity present in insoluble peptidoglycan fractions was present in compounds that comigrated on paper chromatography with glucosamine (∼60%) or muramic acid (∼30%). Treatment of whole cells with low and high concentrations of lysozyme alone resulted in losses of 45 and 70% of the insoluble peptidoglycan, respectively, yet release of deoxyribonucleic acid from cells was not detected. Sequential addition of appropriate concentrations of selected inorganic salts after lysozyme treatment did result in the liberation of deoxyribonucleic acid. Deoxyribonucleic acid release was correlated with a further release of peptidoglycan from the insoluble fraction. However, the total amount of peptidoglycan lost effected by the low concentration of lysozyme and NaSCN (lysis) was significantly less than the amount of peptidoglycan hydrolyzed by high concentrations of lysozyme alone (no lysis), suggesting that the overall amount of peptidoglycan lost did not correlate well with cellular lysis. The total amount of insoluble peptidoglycan lost at the highest salt concentrations tested was found to be greater than could be accounted for by lysozyme-sensitive linkages of the peptidoglycan, possibly implicating autolysins. The results obtained suggested that hydrolysis of peptidoglycan bonds in topologically localized, but strategically important, sites was a more significant factor in the sequence that results in loss of cellular

  19. Normal human serum (HS) prevents oxidant-induced lysis of cultured endothelial cells (ECs)

    SciTech Connect

    Callahan, K.S.; Harlan, J.M.

    1986-03-01

    Most studies demonstrating oxidant lysis of cultured ECs are performed in serum-free media or media containing low concentrations of bovine serum. The authors found that HS protects human and bovine ECs from lysis caused by reagent H/sub 2/O/sub 2/ or glucose/glucose oxidase (GO)-generated H/sub 2/O/sub 2/. EC injury was assessed by /sup 51/Cr release, cell detachment, or trypan blue dye exclusion. Protective HS activity was dose-dependent with concentrations greater than or equal to 25% preventing lethal injury. Cytotoxicity at 24 hrs, induced by 20 mU/ml GO, was 90.1 +/- 5.2% without HS vs 1.7 +/- 4.6% with 25% HS present (20 exp). Similar protection was observed with heparinized plasma. Of note, comparable concentrations of bovine serum were devoid of protective activity. Addition of fatty acid-free albumin to the media was also without protective effect. Preliminary characterization showed HS activity was stable to 60/sup 0/C for 30 min, non-dialyzable at 25,000 MW cutoff, and retained in delipidated serum. The HS protection was not merely due to scavenging of exogenous H/sub 2/O/sub 2/ as A23187-induced EC lysis was also prevented by HS. Protective activity was not reproduced by purified cerruloplasmin or transferrin. In conclusion, unidentified factor(s) present in HS protect cultured ECs from oxidant-induced lysis. Since endothelium is normally exposed to 100% plasma, the authors suggest that in vitro studies of oxidant-mediated injury be performed in the presence of HS. Factor(s) in HS may play an important role in modulating oxidant-induced vascular injury in vivo.

  20. Tumour lysis syndrome after treatment of chronic lymphocytic leukaemia with fludarabine.

    PubMed Central

    Montalban, C.; Liaño, F.; Aguilera, A.

    1994-01-01

    Fludarabine is one of the most recent and promising therapeutic agents for chronic lymphocytic leukaemia. We describe a patient who developed tumour lysis syndrome after the first course of treatment with fludarabine and call attention to this uncommon but potentially lethal complication that has not been previously taken into account in this neoplasia. It should always be anticipated when patients are treated with new and effective drugs. PMID:7971632

  1. Improved detection of Trypanosoma brucei by lysis of red blood cells, concentration and LED fluorescence microscopy.

    PubMed

    Biéler, Sylvain; Matovu, Enock; Mitashi, Patrick; Ssewannyana, Edward; Bi Shamamba, Stomy Karhemere; Bessell, Paul Richard; Ndung'u, Joseph Mathu

    2012-02-01

    Confirmatory diagnosis of African trypanosomiasis relies on demonstration of parasites in body fluids by bright field microscopy. The parasitaemia in infected patients and animals is usually low, and concentration methods are used to try and increase the chances of seeing parasites. Recently, fluorescence microscopes using light-emitting diodes (LED) have been developed. Since they emit strong light, their use does not require a dark room, making field application a possibility. We have combined LED fluorescence microscopy with lysis of red blood cells (RBC) to improve the sensitivity and speed of detecting trypanosomes. In studies conducted at four centers in Uganda and the Democratic Republic of the Congo, parasitaemic blood was serially diluted and the RBCs lysed using commercial buffer. Samples were then concentrated by centrifugation, and different volumes of the sediment used to make thin and thick smears. Next, these were stained with acridine orange or Giemsa, and examined using an LED microscope under fluorescence or bright light, respectively. Detection of parasites was significantly improved by RBC lysis and concentration, regardless of the staining and microscopy method used. Further improvements were made when smears were prepared using larger volumes of sediment. The best results were obtained with thin smears prepared using 20 μl of sediment and stained with acridine orange. The time taken to see the first parasite was dramatically reduced when smears were examined by LED fluorescence microscopy, compared to bright light. LED fluorescence microscopy was found to be easier and requiring less visual effort than bright field microscopy. These studies demonstrate the potential for incremental improvement in detection of Trypanosoma brucei by combining LED fluorescence microscopy with RBC lysis and concentration. The lysis and concentration method may also be useful in sample preparation for other diagnostic tests for trypanosomiasis. Copyright © 2011

  2. Erythrocyte Lysis and Xenopus laevis Oocyte Rupture by Recombinant Plasmodium falciparum Hemolysin III

    PubMed Central

    Moonah, Shannon; Sanders, Natalie G.; Persichetti, Jason K.

    2014-01-01

    Malaria kills more than 1 million people per year worldwide, with severe malaria anemia accounting for the majority of the deaths. Malaria anemia is multifactorial in etiology, including infected erythrocyte destruction and decrease in erythrocyte production, as well as destruction or clearance of noninfected erythrocytes. We identified a panspecies Plasmodium hemolysin type III related to bacterial hemolysins. The identification of a hemolysin III homologue in Plasmodium suggests a potential role in host erythrocyte lysis. Here, we report the first characterization of Plasmodium falciparum hemolysin III, showing that the soluble recombinant P. falciparum hemolysin III is a pore-forming protein capable of lysing human erythrocytes in a dose-, time-, and temperature-dependent fashion. The recombinant P. falciparum hemolysin III-induced hemolysis was partially inhibited by glibenclamide, a known channel antagonist. Studies with polyethylene glycol molecules of different molecular weights indicated a pore size of approximately 3.2 nm. Heterologous expression of recombinant P. falciparum hemolysin III in Xenopus oocytes demonstrated early hypotonic lysis similar to that of the pore-forming aquaporin control. Live fluorescence microscopy localized transfected recombinant green fluorescent protein (GFP)-tagged P. falciparum hemolysin III to the essential digestive vacuole of the P. falciparum parasite. These transfected trophozoites also possessed a swollen digestive vacuole phenotype. Native Plasmodium hemolysin III in the digestive vacuole may contribute to lysis of the parasitophorous vacuole membrane derived from the host erythrocyte. After merozoite egress from infected erythrocytes, remnant P. falciparum hemolysin III released from digestive vacuoles could potentially contribute to lysis of uninfected erythrocytes to contribute to severe life-threatening anemia. PMID:25148832

  3. Electrical lysis: dynamics revisited and advances in On-chip operation.

    PubMed

    Morshed, Bashir; Shams, Maitham; Mussivand, Tofy

    2013-01-01

    Electrical lysis (EL) is the process of breaking the cell membrane to expose the internal contents under an applied high electric field. Lysis is an important phenomenon for cellular analysis, medical treatment, and biofouling control. This paper aims to review, summarize, and analyze recent advancements on EL. Major databases including PubMed, Ei Engineering Village, IEEE Xplore, and Scholars Portal were searched using relevant keywords. More than 50 articles published in English since 1997 are cited in this article. EL has several key advantages compared to other lysis techniques such as chemical, mechanical, sonication, or laser, including rapid speed of operation, ability to control, miniaturization, low cost, and low power requirement. A variety of cell types have been investigated for including protoplasts, E. coli, yeasts, blood cells, and cancer cells. EL has been developed and applied for decontamination, cytology, genetics, single-cell analysis, cancer treatment, and other applications. On-chip EL is a promising technology for multiplexed automated implementation of cell-sample preparation and processing with micro- or nanoliter reagents.

  4. Rapid thermal lysis of cells using silicon-diamond microcantilever heaters.

    PubMed

    Privorotskaya, Natalya; Liu, Yi-Shao; Lee, Jungchul; Zeng, Hongjun; Carlisle, John A; Radadia, Adarsh; Millet, Larry; Bashir, Rashid; King, William P

    2010-05-07

    This paper presents the design and application of microcantilever heaters for biochemical applications. Thermal lysis of biological cells was demonstrated as a specific example. The microcantilever heaters, fabricated from selectively doped single crystal silicon, provide local resistive heating with highly uniform temperature distribution across the cantilevers. Very importantly, the microcantilever heaters were coated with a layer of 100 nm thick electrically insulating ultrananocrystalline diamond (UNCD) layer used for cell immobilization on the cantilever surface. Fibroblast cells or bacterial cells were immobilized on the UNCD/cantilever surfaces and thermal lysis was demonstrated via optical fluorescence microscopy. Upon electrical heating of the cantilever structures to 93 degrees C for 30 seconds, fibroblast cell and nuclear membrane were compromised and the cells were lysed. Over 90% of viable bacteria were also lysed after 15 seconds of heating at 93 degrees C. This work demonstrates the utility of silicon-UNCD heated microcantilevers for rapid cell lysis and forms the basis for other rapid and localized temperature-regulated microbiological experiments in cantilever-based lab on chip applications.

  5. Changes in Bacterial and Eukaryotic Community Structure after Mass Lysis of Filamentous Cyanobacteria Associated with Viruses†

    PubMed Central

    van Hannen, Erik J.; Zwart, Gabriel; van Agterveld, Miranda P.; Gons, Herman J.; Ebert, Jeannine; Laanbroek, Hendrikus J.

    1999-01-01

    During an experiment in two laboratory-scale enclosures filled with lake water (130 liters each) we noticed the almost-complete lysis of the cyanobacterial population. Based on electron microscopic observations of viral particles inside cyanobacterial filaments and counts of virus-like particles, we concluded that a viral lysis of the filamentous cyanobacteria had taken place. Denaturing gradient gel electrophoresis (DGGE) of 16S ribosomal DNA fragments qualitatively monitored the removal of the cyanobacterial species from the community and the appearance of newly emerging bacterial species. The majority of these bacteria were related to the Cytophagales and actinomycetes, bacterial divisions known to contain species capable of degrading complex organic molecules. A few days after the cyanobacteria started to lyse, a rotifer species became dominant in the DGGE profile of the eukaryotic community. Since rotifers play an important role in the carbon transfer between the microbial loop and higher trophic levels, these observations confirm the role of viruses in channeling carbon through food webs. Multidimensional scaling analysis of the DGGE profiles showed large changes in the structures of both the bacterial and eukaryotic communities at the time of lysis. These changes were remarkably similar in the two enclosures, indicating that such community structure changes are not random but occur according to a fixed pattern. Our findings strongly support the idea that viruses can structure microbial communities. PMID:9925618

  6. A volatile factor inducing transmissible lysis in Gaeumannomyces graminis (Sacc.) Arx and Olivier var. tritici Walker.

    PubMed

    Sivasithamparam, K; Stukely, M; Parker, C A

    1975-03-01

    Filtered water extract of Gabalong soil with a recent history of take-all in wheat caused lytic plaques to form in agar cultures of a virulent strain of Gaeumannomyces graminis var. tritici. The plaques resembled those produced by Bdellovibrio on plate seeded with bacteria. However, there was no evidence of the presence of bacteria, viruses, or mycoplasmas. The lytic factor was transmissible in culture filtrates to fresh subcultures of the fungus. Exposure of young healthy colonies to sublethal doses of ultraviolet light also induced transmissible lysis. The lytic factor was heat-stable, passed through a 25-nm filter, and was not affected by nuclease (enzymes) or severe irradiation with UV light. It also induced bysis in several other strains of G. graminis. Lysis was always preceded by a growth-stimulatory effect on the fungus. The lytic factor was active as a volatile chemical which induced transmissible lysis and continued to be formed, apparently as a self-perpetuating agent, in lysing cultures of the fungus.

  7. A novel, compact disk-like centrifugal microfluidics system for cell lysis and sample homogenization.

    PubMed

    Kido, Horacio; Micic, Miodrag; Smith, David; Zoval, Jim; Norton, Jim; Madou, Marc

    2007-07-01

    In this paper, we present the design and characterization of a novel platform for mechanical cell lysis of even the most difficult to lyse cell types on a micro or nanoscale (maximum 70 microL total volume). The system incorporates a machined plastic circular disk assembly, magnetic field actuated microfluidics, centrifugal cells and tissue homogenizer and centrifugation system. The mechanism of tissue disruption of this novel cell homogenization apparatus derives from the relative motion of ferromagnetic metal disks and grinding matrices in a liquid medium within individual chambers of the disk in the presence of an oscillating magnetic field. The oscillation of the ferromagnetic disks or blades produces mechanical impaction and shear forces capable of disrupting cells within the chamber both by direct action of the blade and by the motion of the surrounding lysis matrix, and by motion induced vortexing of buffer fluid. Glass beads or other grinding media are integrated into each lysis chamber within the disk to enhance the transfer of energy from the oscillating metal blade to the cells. The system also achieves the centrifugal elimination of solids from each liquid sample and allows the elution of clarified supernatants via siphoning into a collection chamber fabricated into the plastic disk assembly. This article describes system design, implementation and validation of proof of concept on two samples--Escherichia coli and Saccharomyces cerevisiae representing model systems for cells that are easy and difficult to lyse, respectively.

  8. Selective local lysis and sampling of live cells for nucleic acid analysis using a microfluidic probe

    PubMed Central

    Kashyap, Aditya; Autebert, Julien; Delamarche, Emmanuel; Kaigala, Govind V.

    2016-01-01

    Heterogeneity is inherent to biology, thus it is imperative to realize methods capable of obtaining spatially-resolved genomic and transcriptomic profiles of heterogeneous biological samples. Here, we present a new method for local lysis of live adherent cells for nucleic acid analyses. This method addresses bottlenecks in current approaches, such as dilution of analytes, one-sample-one-test, and incompatibility to adherent cells. We make use of a scanning probe technology - a microfluidic probe - and implement hierarchical hydrodynamic flow confinement (hHFC) to localize multiple biochemicals on a biological substrate in a non-contact, non-destructive manner. hHFC enables rapid recovery of nucleic acids by coupling cell lysis and lysate collection. We locally lysed ~300 cells with chemical systems adapted for DNA or RNA and obtained lysates of ~70 cells/μL for DNA analysis and ~15 cells/μL for mRNA analysis. The lysates were introduced into PCR-based workflows for genomic and transcriptomic analysis. This strategy further enabled selective local lysis of subpopulations in a co-culture of MCF7 and MDA-MB-231 cells, validated by characteristic E-cadherin gene expression in individually extracted cell types. The developed strategy can be applied to study cell-cell, cell-matrix interactions locally, with implications in understanding growth, progression and drug response of a tumor. PMID:27411740

  9. Endothelial Cells Organize Fibrin Clots into Structures That Are More Resistant to Lysis

    NASA Astrophysics Data System (ADS)

    Gray Jerome, W.; Handt, Stefan; Hantgan, Roy R.

    2005-06-01

    Acute myocardial infarction is a major cause of death and disability in the United States. Introducing thrombolytic agents into the clot to dissolve occlusive coronary artery thrombi is one method of treatment. However, despite advances in our knowledge of thrombosis and thrombolysis, survival rates following thrombolytic therapy have not improved substantially. This failure highlights the need for further study of the factors mediating clot stabilization. Using laser scanning confocal microscopy of clots formed from fluorescein-labeled fibrinogen, we investigated what effect binding of fibrin to the endothelial surface has on clot structure and resistance to lysis. Fluorescent fibrin clots were produced over human umbilical vein endothelial cells (HUVEC) and the clot structure analyzed. In the presence of HUVEC, fibrin near the endothelial surface was more organized and occurred in tighter bundles compared to fibrin just 50 [mu]m above. The HUVEC influence on fibrin architecture was blocked by inhibitory concentrations of antibodies to [alpha]V or [beta]3 integrin subunits. The regions of the clots associated with endothelial cells were more resistant to lysis than the more homogenous regions distal to endothelium. Thus, our data show that binding of fibrin to integrins on endothelial surfaces produces clots that are more resistant to lysis.

  10. Human lymph node lymphocytes fail to effect lysis of antibody-coated target cells.

    PubMed Central

    O'Toole, C; Saxon, A; Bohrer, R

    1977-01-01

    Human lymphocytes prepared from peripheral blood, lymph nodes, spleen and thymus were titrated for ability to mediate lysis of human target cells coated with rabbit anti target antibody. Lymphocytes from blood and spleen produced efficient lysis of targets in the presence of antibody. Lymph node cells and thymocytes were essentially non-reactive in this system. Lymph node preparations from non-cancer patients contained approximately 25% of non-T cells with receptors for Fc,C3 and/or Ig. Regional lymph nodes from patients with primary tumours contained 37-50% non-T cells by the same criteria. Failure of lymph node lymphocytes to effect lysis of antibody-coated targets did not therefore correlate with content of Fc or C3 bearing cells per se. The effector cell in antibody-dependent cytotoxicity in other systems has been shown to carry Fc and C3 receptors, but not surface Ig. This cell type appears to be absent or non-functional in human lymph nodes. PMID:300304

  11. Plasma nanotextured polymeric lab-on-a-chip for highly efficient bacteria capture and lysis.

    PubMed

    Tsougeni, K; Papadakis, G; Gianneli, M; Grammoustianou, A; Constantoudis, V; Dupuy, B; Petrou, P S; Kakabakos, S E; Tserepi, A; Gizeli, E; Gogolides, E

    2016-01-07

    We describe the design, fabrication, and successful demonstration of a sample preparation module comprising bacteria cell capture and thermal lysis on-chip with potential applications in food sample pathogen analysis. Plasma nanotexturing of the polymeric substrate allows increase of the surface area of the chip and the antibody binding capacity. Three different anti-Salmonella antibodies were directly and covalently linked to plasma treated chips without any additional linker chemistry or other treatment. Then, the Ab-modified chips were tested for their capacity to bind bacteria in the concentration range of 10(2)-10(8) cells per mL; the module exhibited 100% efficiency in Salmonella enterica serovar Typhimurium bacteria capture for cell suspensions below 10(5) cells per mL (10(4) cells injected with a 100 μL sample volume) and efficiency higher than 50% for 10(7) cells per mL. Moreover, thermal lysis achieved on-chip from as low as 10 captured cells was demonstrated and shown to compare well with off-chip lysis. Excellent selectivity (over 1 : 300) was obtained in a sample containing, in addition to S. Typhimurium and E. coli bacteria.

  12. Design and Modelling of a Microfluidic Electro-Lysis Device with Controlling Plates

    NASA Technical Reports Server (NTRS)

    Jenkins, A.; Chen, C. P.; Spearing, S.; Monaco, L. A.; Steele, A.; Flores, G.

    2006-01-01

    Many Lab-on-Chip applications require sample pre-treatment systems. Using electric fields to perform cell-lysis in bio-MEMS systems has provided a powerful tool which can be integrated into Lab-on-a-Chip platforms. The major design considerations for electro-lysis devices include optimal geometry and placement of micro-electrodes, cell concentration, flow rates, optimal electric field (e.g. pulsed DC vs. AC), etc. To avoid electrolysis of the flowing solution at the exposed electrode surfaces, magnitudes and the applied voltages and duration of the DC pulse, or the AC frequency of the AC, have to be optimized for a given configuration. Using simulation tools for calculation of electric fields has proved very useful, for exploring alternative configurations and operating conditions for achieving electro cell-lysis. To alleviate the problem associated with low electric fields within the microfluidics channel and the high voltage demand on the contact electrode strips, two "control plates" are added to the microfluidics configuration. The principle of placing the two controlling plate-electrodes is based on the electric fields generated by a combined insulator/dielectric (gladwater) media. Surface charges are established at the insulator/dielectric interface. This paper discusses the effects of this interface charge on the modification of the electric field of the flowing liquid/cell solution.

  13. Biophysical response to pulsed laser microbeam-induced cell lysis and molecular delivery.

    PubMed

    Hellman, Amy N; Rau, Kaustubh R; Yoon, Helen H; Venugopalan, Vasan

    2008-03-01

    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.

  14. Caspase-11 activation requires lysis of pathogen-containing vacuoles by IFN-induced GTPases.

    PubMed

    Meunier, Etienne; Dick, Mathias S; Dreier, Roland F; Schürmann, Nura; Kenzelmann Broz, Daniela; Warming, Søren; Roose-Girma, Merone; Bumann, Dirk; Kayagaki, Nobuhiko; Takeda, Kiyoshi; Yamamoto, Masahiro; Broz, Petr

    2014-05-15

    Lipopolysaccharide from Gram-negative bacteria is sensed in the host cell cytoplasm by a non-canonical inflammasome pathway that ultimately results in caspase-11 activation and cell death. In mouse macrophages, activation of this pathway requires the production of type-I interferons, indicating that interferon-induced genes have a critical role in initiating this pathway. Here we report that a cluster of small interferon-inducible GTPases, the so-called guanylate-binding proteins, is required for the full activity of the non-canonical caspase-11 inflammasome during infections with vacuolar Gram-negative bacteria. We show that guanylate-binding proteins are recruited to intracellular bacterial pathogens and are necessary to induce the lysis of the pathogen-containing vacuole. Lysis of the vacuole releases bacteria into the cytosol, thus allowing the detection of their lipopolysaccharide by a yet unknown lipopolysaccharide sensor. Moreover, recognition of the lysed vacuole by the danger sensor galectin-8 initiates the uptake of bacteria into autophagosomes, which results in a reduction of caspase-11 activation. These results indicate that host-mediated lysis of pathogen-containing vacuoles is an essential immune function and is necessary for efficient recognition of pathogens by inflammasome complexes in the cytosol.

  15. Critical cell wall hole size for lysis in Gram-positive bacteria

    NASA Astrophysics Data System (ADS)

    Mitchell, Gabriel; Wiesenfeld, Kurt; Nelson, Daniel; Weitz, Joshua

    2013-03-01

    Gram-positive bacteria transport molecules necessary for their survival through holes in their cell wall. The holes in cell walls need to be large enough to let critical nutrients pass through. However, the cell wall must also function to prevent the bacteria's membrane from protruding through a large hole into the environment and lysing the cell. As such, we hypothesize that there exists a range of cell wall hole sizes that allow for molecule transport but prevent membrane protrusion. Here we develop and analyze a biophysical theory of the response of a Gram-positive cell's membrane to the formation of a hole in the cell wall. We predict a critical hole size in the range 15-24nm beyond which lysis occurs. To test our theory, we measured hole sizes in Streptococcus pyogenes cells undergoing enzymatic lysis via transmission electron microscopy. The measured hole sizes are in strong agreement with our theoretical prediction. Together, the theory and experiments provide a means to quantify the mechanisms of death of Gram-positive cells via enzymatically mediated lysis and provides insight into the range of cell wall hole sizes compatible with bacterial homeostasis.

  16. Lysis gradient centrifugation: a flexible method for the isolation of nuclei from primary cells.

    PubMed

    Katholnig, Karl; Poglitsch, Marko; Hengstschläger, Markus; Weichhart, Thomas

    2015-01-01

    The isolation of nuclei from eukaryotic cells is essential for studying the composition and the dynamic changes of the nuclear proteome to gain insight into the mechanisms of gene expression and cell signalling. Primary cells are particularly challenging for standard nuclear isolation protocols due to low protein content, sample degradation, or nuclear clumping. Here, we describe a rapid and flexible protocol for the isolation of clean and intact nuclei, which results in the recovery of 90-95 % highly pure nuclei. The method, called lysis gradient centrifugation (LGC), is based on an iso-osmolar discontinuous iodixanol-based density gradient including a detergent-containing lysis layer. A single low g-force centrifugation step enables mild cell lysis and prevents extensive contact of the nuclei with the cytoplasmic environment. This fast method shows high reproducibility due to the relatively little cell manipulation required by the investigator. Further advantages are the low amount of starting material required, easy parallel processing of multiple samples, and isolation of nuclei and cytoplasm at the same time from the same sample.

  17. Eosinophil granule lysis in vitro induced by soluble antigen antibody complexes

    PubMed Central

    Archer, G. T.; Nelson, Margaret; Johnston, Jill

    1969-01-01

    A simple test system is described, for the demonstration of antigen—antibody reactions capable of causing eosinophil granule lysis in vitro. The antigen preparations used were extracts of the nematode Amplicaecum robertsi and body fluid of Ascaris suum. Antisera were obtained from rats infested with Amplicaecum. Eosinophils were obtained from the peritoneal cavity of normal rats. Centrifugation of the cells to form a cell button was an essential step in the procedure. Lysis of eosinophils occurred with antiserum obtained from the animals between the 12th and 32nd days of infestation with Amplicaecum, and was accompanied by vacuole formation in macrophages and mast cell disruption. The reaction was most pronounced during the 3rd week. Serum from adrenalectomized infested animals caused the most marked changes in eosinophils. Serum from cortisonetreated infested animals failed to cause eosinophil changes. Attempts at purification of the antigen in Ascaris body fluid resulted in two fractions with marked activity in the test system. The same two fractions were found to form precipitin lines on agarose gel diffusion against rat antiserum. It is postulated that antigen—antibody complexes soluble in low concentration were responsible for the changes observed in the eosinophils, macrophages and mast cells. One or more labile factors in the serum were found to be necessary for eosinophil granule lysis. The evidence, though incomplete, would favour the suggestion that both labile antibody and complement were necessary. ImagesFIG. 2 PMID:4982023

  18. Use of Surface Enhanced Blocking (SEB) Electrodes for Microbial Cell Lysis in Flow-Through Devices

    PubMed Central

    Talebpour, Abdossamad; Maaskant, Robert; Khine, Aye Aye; Alavie, Tino

    2014-01-01

    By simultaneously subjecting microbial cells to high amplitude pulsed electric fields and flash heating of the cell suspension fluid, effective release of intracellular contents was achieved. The synergistic effect of the applied electric field and elevated temperature on cell lysis in a flow-through device was demonstrated for Gram-negative and Gram-positive bacteria, and Mycobacterium species. The resulting lysate is suitable for downstream nucleic acid amplification and detection without requiring further preparation. The lysis chamber employs surface enhanced blocking electrodes which possess an etched micro-structured surface and a thin layer of dielectric metal oxide which provides a large effective area and blocks transmission of electrical current. The surface enhanced blocking electrodes enable simultaneous suppression of the rapid onset of electric field screening in the bulk of the cell suspension medium and avoidance of undesired electrochemical processes at the electrode-electrolyte interface. In addition the blocking layer ensures the robustness of the cell lysis device in applications involving prolonged flow-through processing of the microbial cells. PMID:25033080

  19. Hydrophobic ionic liquids for quantitative bacterial cell lysis with subsequent DNA quantification.

    PubMed

    Fuchs-Telka, Sabine; Fister, Susanne; Mester, Patrick-Julian; Wagner, Martin; Rossmanith, Peter

    2017-02-01

    DNA is one of the most frequently analyzed molecules in the life sciences. In this article we describe a simple and fast protocol for quantitative DNA isolation from bacteria based on hydrophobic ionic liquid supported cell lysis at elevated temperatures (120-150 °C) for subsequent PCR-based analysis. From a set of five hydrophobic ionic liquids, 1-butyl-1-methylpyrrolidinium bis(trifluoromethylsulfonyl)imide was identified as the most suitable for quantitative cell lysis and DNA extraction because of limited quantitative PCR inhibition by the aqueous eluate as well as no detectable DNA uptake. The newly developed method was able to efficiently lyse Gram-negative bacterial cells, whereas Gram-positive cells were protected by their thick cell wall. The performance of the final protocol resulted in quantitative DNA extraction efficiencies for Gram-negative bacteria similar to those obtained with a commercial kit, whereas the number of handling steps, and especially the time required, was dramatically reduced. Graphical Abstract After careful evaluation of five hydrophobic ionic liquids, 1-butyl-1-methylpyrrolidinium bis(trifluoromethylsulfonyl)imide ([BMPyr (+) ][Ntf 2(-) ]) was identified as the most suitable ionic liquid for quantitative cell lysis and DNA extraction. When used for Gram-negative bacteria, the protocol presented is simple and very fast and achieves DNA extraction efficiencies similar to those obtained with a commercial kit. ddH 2 O double-distilled water, qPCR quantitative PCR.

  20. A Flow-Through Ultrasonic Lysis System for the Disruption of Bacterial Spores

    SciTech Connect

    Warner, Cynthia L.; Bruckner-Lea, Cindy J.; Grate, Jay W.; Straub, Tim M.; Posakony, Gerald J.; Valentine, Nancy B.; Ozanich, Richard M.; Bond, Leonard J.; Matzke, Melissa M.; Dockendorff, Brian P.; Valdez, Catherine O.; Valdez, Patrick LJ; Owsley, Stanley L.

    2009-10-01

    An automated, flow-through spore lysis instrument that is capable of rapidly disrupting bacterial spores is described. The system utilizes a flow-through chamber that allows for direct injection of the sample without the need for a chemical or enzymatic pre-treatment step to soften the spore coat prior to lysis. Lysis of Bacillus subtilis spores, a benign simulant of Bacillus anthracis, is achieved by flowing the sample through a tube whose axis is parallel to the faces of two transducers that deliver 10 W cm-2 to the surface of the tube at 1.4 MHz frequency. Increases in amplifiable DNA were assessed by real-time PCR analysis, which showed at least a 25-fold increase in amplifiable DNA following ultrasonic treatment, and dilution-to-extinction PCR, which suggests up to a 100-1000-fold increase. The modular design of the ultrasonic system and integrated fluidics allow it to be incorporated into multi-step sample treatment and detection systems.

  1. Effects of amyloid beta-peptides on the lysis tension of lipid bilayer vesicles containing oxysterols.

    PubMed

    Kim, Dennis H; Frangos, John A

    2008-07-01

    Amyloid beta-peptides (Abeta) applied directly from solution to model lipid membranes produced dramatic changes in the material properties of the bilayer when certain oxysterols were present in the bilayer. These effects were dependent on both lipid and peptide composition, and occurred at peptide concentrations as low as 100 nM. Using micropipette manipulation of giant unilamellar vesicles, we directly measured the lysis tension of lipid bilayers of various compositions. The glycerophospholipid 1-stearoyl-2-oleoyl-sn-glycero-3-phosphocholine (SOPC) constituted the main lipid component at 70 mol %. The remaining 30 mol % was composed of the following pure or mixed sterols: cholesterol (CHOL), 7-ketocholesterol (KETO), or 7beta-hydroxycholesterol (OHCHOL). SOPC/CHOL bilayers did not exhibit significant changes in mechanical properties after exposure to either Abeta(1-42) or Abeta(1-40). Partial substitution of CHOL with KETO (5 mol %), however, caused a drastic reduction of the lysis tension after exposure to Abeta(1-42) but not to Abeta(1-40). Partial substitution of CHOL with OHCHOL (5 mol %) caused a drastic reduction of the lysis tension after exposure to Abeta(1-40) and to Abeta(1-42). We attribute these effects to the reduction in intermolecular cohesive interactions caused by the presence of the second dipole of oxysterols, which reduces the energetic barrier for Abeta insertion into the bilayer.

  2. Marking hypoxic cells for complement and cytotoxic T lymphocyte-mediated lysis: using pimonidazole.

    PubMed Central

    Chou, S. C.; Flood, P. M.; Raleigh, J. A.

    1996-01-01

    Artificial antigens are created when 2-nitroimidazoles bind to hypoxic cells. These antigens have been used in the immunodetection of tumour hypoxia but they might also serve to stimulate immune lysis of hypoxic tumour cells by complement- and cell-mediated processes. In order to test this hypothesis, lymphocytes isolated from the spleens of C3H/HeN mice that had been immunised with pimonidazole-labelled 3152-PRO cells were subcultured and tested for their ability to lyse chromium-51 loaded, pimonidazole-labelled 3152-PRO cells in an in vitro assay. In a parallel study, commercially available, rabbit complement was tested for its ability to lyse pimonidazole-labelled V79-4 cells in the presence of monoclonal antibodies which recognise protein adducts of reductively activated pimonidazole. Complement-mediated cell lysis was measured by means of an MTT assay. Complement-mediated and cell-mediated lysis was observed at pimonidazole concentrations which, in themselves, do not produce cell killing. PMID:8763883

  3. Laser tweezers Raman spectroscopy potential for studies of complex dynamic cellular processes: single cell bacterial lysis.

    PubMed

    Chen, De; Shelenkova, L; Li, Y; Kempf, C R; Sabelnikov, A

    2009-05-01

    The potential of laser tweezers Raman spectroscopy (LTRS) to study complex and dynamic cellular processes was investigated on the model of single E. coli cells lysed (1) from "outside" with egg white lysozyme and (2) from "within" by temperature-induced temperate bacteriophage lambdacI857. The two lysis processes differed in the final outcome (incomplete vs complete cell lysis) as revealed by the dynamic laser light scattering and exhibited distinctive dynamic Raman spectra changes. The technique enabled for the first time at the cellular level to observe and quantify real time interaction of lysozyme with E. coli cells, "visualize" a side effect of the process due to the presence of EDTA, and correlate the process of cell wall disruption, as evidenced by the onset and development of asymmetric speckle scattering patterns, with release/escape of intracellular material (ribosomes, nucleic acids, proteins, etc.) quantified by the intensity changes of Raman signatures. Raman spectra changes observed during the lysis from "within" suggest alleged production of heat shock proteins are consistent with the occurring synthesis of phage-related proteins and are in good agreement with the calculated potential contribution of the above proteins to the Raman spectra. It was also established and validated that the contribution of cellular DNA to the Raman spectra of bacterial cells is negligible compared to RNA. The results open new venues for LTRS research and strongly suggest that LTRS has a great potential especially in investigation of real-time processes.

  4. Prevention and treatment of tumor lysis syndrome, and the efficacy and role of rasburicase

    PubMed Central

    Alakel, Nael; Middeke, Jan Moritz; Schetelig, Johannes; Bornhäuser, Martin

    2017-01-01

    Tumor lysis syndrome (TLS) is a potentially life-threatening condition that occurs in oncologic and hematologic patients with large tumor burden, either due to cytotoxic therapy or, less commonly, spontaneously because of massive tumor cell lysis. TLS is clinically characterized by acute renal failure, hyperuricemia, hyperkalemia, hyperphosphatemia, and hypocalcemia. While limited options are available for treating TLS, identifying patients at high risk for developing TLS and prevention in high-risk patients remain an important aspect in the treatment of cancer patients. In general, treatment of TLS consists of intensive hydration, stimulation of diuresis, and, more specifically, in the use of allopurinol and rasburicase. Rasburicase, a recombinant urate oxidase, rapidly and effectively reduces hyperuricemia, which subsequently significantly decreases the risk of acute renal failure and other clinical manifestations of TLS. For this review, a comprehensive literature search using the term “tumor lysis syndrome” and/or “rasburicase” was performed considering articles listed in MEDLINE. Incidence, prevention, and therapy of TLS with a special focus on the role of rasburicase are discussed. We evaluated 120 relevant articles including 35 case reports, 32 clinical trials, and 14 meta-analyses. PMID:28203093

  5. Detecting cell lysis using viscosity monitoring in E. coli fermentation to prevent product loss

    PubMed Central

    Newton, Joseph M.; Schofield, Desmond; Vlahopoulou, Joanna

    2016-01-01

    Monitoring the physical or chemical properties of cell broths to infer cell status is often challenging due to the complex nature of the broth. Key factors indicative of cell status include cell density, cell viability, product leakage, and DNA release to the fermentation broth. The rapid and accurate prediction of cell status for hosts with intracellular protein products can minimise product loss due to leakage at the onset of cell lysis in fermentation. This article reports the rheological examination of an industrially relevant E. coli fermentation producing antibody fragments (Fab'). Viscosity monitoring showed an increase in viscosity during the exponential phase in relation to the cell density increase, a relatively flat profile in the stationary phase, followed by a rapid increase which correlated well with product loss, DNA release and loss of cell viability. This phenomenon was observed over several fermentations that a 25% increase in broth viscosity (using induction‐point viscosity as a reference) indicated 10% product loss. Our results suggest that viscosity can accurately detect cell lysis and product leakage in postinduction cell cultures, and can identify cell lysis earlier than several other common fermentation monitoring techniques. This work demonstrates the utility of rapidly monitoring the physical properties of fermentation broths, and that viscosity monitoring has the potential to be a tool for process development to determine the optimal harvest time and minimise product loss. © 2016 The Authors. Biotechnology Progress published by Wiley Periodicals, Inc. on behalf of American Institute of Chemical Engineers, 32:1069–1076, 2016 PMID:27111912

  6. Combining Genes from Multiple Phages for Improved Cell Lysis and DNA Transfer from Escherichia coli to Bacillus subtilis

    PubMed Central

    Juhas, Mario; Wong, Christine; Ajioka, James W.

    2016-01-01

    The ability to efficiently and reliably transfer genetic circuits between the key synthetic biology chassis, such as Escherichia coli and Bacillus subtilis, constitutes one of the major hurdles of the rational genome engineering. Using lambda Red recombineering we integrated the thermosensitive lambda repressor and the lysis genes of several bacteriophages into the E. coli chromosome. The lysis of the engineered autolytic cells is inducible by a simple temperature shift. We improved the lysis efficiency by introducing different combinations of lysis genes from bacteriophages lambda, ΦX174 and MS2 under the control of the thermosensitive lambda repressor into the E. coli chromosome. We tested the engineered autolytic cells by transferring plasmid and bacterial artificial chromosome (BAC)-borne genetic circuits from E. coli to B. subtilis. Our engineered system combines benefits of the two main synthetic biology chassis, E. coli and B. subtilis, and allows reliable and efficient transfer of DNA edited in E. coli into B. subtilis. PMID:27798678

  7. [Extraction of sperm DNA from mixed stain by the modified differential lysis method combined with silicon bead method].

    PubMed

    Han, Hai-Jun; Zhang, Yu-Hong; Yang, Min; Yi, Hai; Yang, Geng-Ye; Jia, Dong-Tao; Lu, Da-Ru

    2014-02-01

    To extract sperm DNA from mixed stain by the modified differential lysis method combined with silicon bead method and to evaluate its application value. Fifty-two mixed stains containing female STR genotypes detected by differential lysis method were collected. The sperm DNA was extracted by the modified method combined with silicon bead method, then genotyped with the Identifiler Kit, and compared with the results of genotyping by the conventional differential lysis method as control. Of the 52 samples, 38 samples with sole male STR genotypes in all loci were detected. The detection rate of male STR genotypes was 98.08% through the modified method combined with silicon bead method. The modified differential lysis method combined with silicon bead method can be used in extraction of sperm DNA from mixed stain.

  8. Adaptation of red blood cell lysis represents a fundamental breakthrough that improves the sensitivity of Salmonella detection in blood

    PubMed Central

    Boyd, MA; Tennant, SM; Melendez, JH; Toema, D; Galen, JE; Geddes, CD; Levine, MM

    2015-01-01

    Aims Isolation of Salmonella Typhi from blood culture is the standard diagnostic for confirming typhoid fever but it is unavailable in many developing countries. We previously described a Microwave Accelerated Metal Enhanced Fluorescence (MAMEF)-based assay to detect Salmonella in medium. Attempts to detect Salmonella in blood were unsuccessful, presumably due to the interference of erythrocytes. The objective of this study was to evaluate various blood treatment methods that could be used prior to PCR, real-time PCR or MAMEF to increase sensitivity of detection of Salmonella. Methods and Results We tested ammonium chloride and erythrocyte lysis buffer, water, Lymphocyte Separation Medium, BD Vacutainer® CPT™ Tubes and dextran. Erythrocyte lysis buffer was the best isolation method as it is fast, inexpensive and works with either fresh or stored blood. The sensitivity of PCR- and real-time PCR detection of Salmonella in spiked blood was improved when whole blood was first lysed using erythrocyte lysis buffer prior to DNA extraction. Removal of erythrocytes and clotting factors also enabled reproducible lysis of Salmonella and fragmentation of DNA, which are necessary for MAMEF sensing. Conclusions Use of the erythrocyte lysis procedure prior to DNA extraction has enabled improved sensitivity of Salmonella detection by PCR and real-time PCR and has allowed lysis and fragmentation of Salmonella using microwave radiation (for future detection by MAMEF). Significance and Impact of the Study Adaptation of the blood lysis method represents a fundamental breakthrough that improves the sensitivity of DNA-based detection of Salmonella in blood. PMID:25630831

  9. Reduced Plasminogen Binding and Delayed Activation Render γ′-Fibrin More Resistant to Lysis than γA-Fibrin*

    PubMed Central

    Kim, Paul Y.; Vu, Trang T.; Leslie, Beverly A.; Stafford, Alan R.; Fredenburgh, James C.; Weitz, Jeffrey I.

    2014-01-01

    Fibrin (Fn) clots formed from γ′-fibrinogen (γ′-Fg), a variant with an elongated γ-chain, are resistant to lysis when compared with clots formed from the predominant γA-Fg, a finding previously attributed to differences in clot structure due to delayed thrombin-mediated fibrinopeptide (FP) B release or impaired cross-linking by factor XIIIa. We investigated whether slower lysis of γ′-Fn reflects delayed plasminogen (Pg) binding and/or activation by tissue plasminogen activator (tPA), reduced plasmin-mediated proteolysis of γ′-Fn, and/or altered cross-linking. Clots formed from γ′-Fg lysed more slowly than those formed from γA-Fg when lysis was initiated with tPA/Pg when FPA and FPB were both released, but not when lysis was initiated with plasmin, or when only FPA was released. Pg bound to γ′-Fn with an association rate constant 22% lower than that to γA-Fn, and the lag time for initiation of Pg activation by tPA was longer with γ′-Fn than with γA-Fn. Once initiated, however, Pg activation kinetics were similar. Factor XIIIa had similar effects on clots formed from both Fg isoforms. Therefore, slower lysis of γ′-Fn clots reflects delayed FPB release, which results in delayed binding and activation of Pg. When clots were formed from Fg mixtures containing more than 20% γ′-Fg, the upper limit of the normal level, the delay in lysis was magnified. These data suggest that circulating levels of γ′-Fg modulate the susceptibility of clots to lysis by slowing Pg activation by tPA and provide another example of the intimate connections between coagulation and fibrinolysis. PMID:25128532

  10. Effect of anti plasminogen monoclonal antibodies on whole blood clot lysis.

    PubMed

    Ehrenreich, F; Hattey, E; Wojta, J; Binder, B R

    1988-01-01

    Several monoclonal antibodies (MABs) against specific parts of the plasminogen molecule were developed. One of them, MPW1PG, recognizes only the native form Glu-plasminogen, while the other one, MPW2PG, reacts equally well with Glu-plasminogen and its proteolytically degraded derivative Lys-plasminogen as confirmed by immunoblotting experiments. Both MABs do not alter the cleavage of the low molecular weight substrate S-2251 by plasmin in a purified system but rather increase the rate of plasmin formation by urokinase and t-PA in a system without fibrin. In the presence of fibrin MPW1PG has no effect on plasmin formation by t-PA, while MPW2PG exhibits mixed type inhibition. To investigate whether this effect can also be seen in a whole blood clot lysis system, the Chandler loop was used (PVC tubes, 4 mm inner diameter, 28 mm length), 125I-fibrinogen was added to citrated whole blood (0.36% sodium citrate) to about 5,000 cpm/10 microliter sample. 1.5 ml of the mixture were pipetted into each tube, the tubes were closed, put on a rotary plate, tilted to an angle of 23 degrees, and rotated at 16 rpm. 10 microliter samples were taken for radioactive counting before clotting by addition of 10 microliter 3.02M CaCl2 (100%-value), 45 min after clotting (0%-value), and 30, 60, 120, 180, 360 minutes after addition of activators. Lysis was started one hour after recalcification. Different amounts of MABs were added either 30 min before recalcification (MAB endogen) or together with the activator (MAB exogen) Percent lysis was calculated from the radioactivity released into the fluid phase. Double determinations were done in all experiments.(ABSTRACT TRUNCATED AT 250 WORDS)

  11. Leukocyte Lysis and Cytokine Induction by the Human Sexually Transmitted Parasite Trichomonas vaginalis

    PubMed Central

    Mercer, Frances; Diala, Fitz Gerald I.; Chen, Yi-Pei; Molgora, Brenda M.; Ng, Shek Hang; Johnson, Patricia J.

    2016-01-01

    Trichomonas vaginalis (Tv) is an extracellular protozoan parasite that causes the most common non-viral sexually transmitted infection: trichomoniasis. While acute symptoms in women may include vaginitis, infections are often asymptomatic, but can persist and are associated with medical complications including increased HIV susceptibility, infertility, pre-term labor, and higher incidence of cervical cancer. Heightened inflammation resulting from Tv infection could account for these complications. Effective cellular immune responses to Tv have not been characterized, and re-infection is common, suggesting a dysfunctional adaptive immune response. Using primary human leukocyte components, we have established an in vitro co-culture system to assess the interaction between Tv and the cells of the human immune system. We determined that in vitro, Tv is able to lyse T-cells and B-cells, showing a preference for B-cells. We also found that Tv lysis of lymphocytes was mediated by contact-dependent and soluble factors. Tv lysis of monocytes is far less efficient, and almost entirely contact-dependent. Interestingly, a common symbiont of Tv, Mycoplasma hominis, did not affect cytolytic activity of the parasite, but had a major impact on cytokine responses. M. hominis enabled more diverse inflammatory cytokine secretion in response to Tv and, of the cytokines tested, Tv strains cleared of M. hominis induced only IL-8 secretion from monocytes. The quality of the adaptive immune response to Tv is therefore likely influenced by Tv symbionts, commensals, and concomitant infections, and may be further complicated by direct parasite lysis of effector immune cells. PMID:27529696

  12. Leukocyte Lysis and Cytokine Induction by the Human Sexually Transmitted Parasite Trichomonas vaginalis.

    PubMed

    Mercer, Frances; Diala, Fitz Gerald I; Chen, Yi-Pei; Molgora, Brenda M; Ng, Shek Hang; Johnson, Patricia J

    2016-08-01

    Trichomonas vaginalis (Tv) is an extracellular protozoan parasite that causes the most common non-viral sexually transmitted infection: trichomoniasis. While acute symptoms in women may include vaginitis, infections are often asymptomatic, but can persist and are associated with medical complications including increased HIV susceptibility, infertility, pre-term labor, and higher incidence of cervical cancer. Heightened inflammation resulting from Tv infection could account for these complications. Effective cellular immune responses to Tv have not been characterized, and re-infection is common, suggesting a dysfunctional adaptive immune response. Using primary human leukocyte components, we have established an in vitro co-culture system to assess the interaction between Tv and the cells of the human immune system. We determined that in vitro, Tv is able to lyse T-cells and B-cells, showing a preference for B-cells. We also found that Tv lysis of lymphocytes was mediated by contact-dependent and soluble factors. Tv lysis of monocytes is far less efficient, and almost entirely contact-dependent. Interestingly, a common symbiont of Tv, Mycoplasma hominis, did not affect cytolytic activity of the parasite, but had a major impact on cytokine responses. M. hominis enabled more diverse inflammatory cytokine secretion in response to Tv and, of the cytokines tested, Tv strains cleared of M. hominis induced only IL-8 secretion from monocytes. The quality of the adaptive immune response to Tv is therefore likely influenced by Tv symbionts, commensals, and concomitant infections, and may be further complicated by direct parasite lysis of effector immune cells.

  13. Myeloma cells resistance to NK cell lysis mainly involves an HLA class I-dependent mechanism.

    PubMed

    Gao, Minjie; Gao, Lu; Yang, Guang; Tao, Yi; Hou, Jun; Xu, Hongwei; Hu, Xiaojing; Han, Ying; Zhang, Qianqiao; Zhan, Fenghuang; Wu, Xiaosong; Shi, Jumei

    2014-07-01

    The anti-multiple myeloma (MM) potential of natural killer (NK) cells has been of rising interest in recent years. However, the molecular mechanism of NK cell cytotoxicity to myeloma cells remains unclear. In the present study, we investigated the expressions of human leukocyte antigen (HLA) class I and HLA-G in patient myeloma cells, and determined their relevance in patient tumor-cell susceptibility to NK cell cytotoxicity. Our results showed that patient myeloma cells (n = 12) were relatively resistant to NK-92 cell lysis, compared with myeloma cell lines (n = 7, P < 0.01). Gene expression profiling and flow cytometry analysis showed that both mRNA and protein of HLA class I were highly expressed in 12 patient myeloma cells. Interestingly, no or low HLA-G surface expression was detected, although multiple HLA-G transcripts were detected in these myeloma cells. NK cell function assay showed that down-regulating HLA class I expression on patient cells by acid treatment significantly increased the susceptibility of MM cells to NK-mediated lysis. Furthermore, we found that the blocking of membrane-bound HLA class I rather than HLA-G using antibodies on myeloma samples markedly increased their susceptibility to NK-mediated killing. These results demonstrated that the resistance of patient MM cells to NK lysis mainly involves an HLA class I-dependent mechanism, suggesting that HLA class I may be involved in protecting MM cells from NK-mediated attack and contribute to their immune escape in vivo.

  14. Pars Plana Vitrectomy With in Vivo Cyst Lysis for Intraocular Cysticercosis.

    PubMed

    Azad, Shorya; Takkar, Brijesh; Roy, Sangeeta; Gangwe, Anil B; Kumar, Mahesh; Kumar, Atul

    2016-07-01

    To evaluate efficacy and safety of pars plana vitrectomy (PPV) and in vivo cyst lysis for intraocular cysticercosis. Retrospective analysis of the records of 15 patients undergoing PPV for intraocular cysticercosis at a tertiary eye care center was done. All patients had undergone in vivo cyst lysis. Of these 15 patients, one had intravitreal cysticercosis (IVC) with vitritis, two cases had IVC with vitritis with tractional retinal detachment (TRD), four cases had subretinal cysticercosis (SRC), four cases had SRC with extensive fibrosis without TRD, and five cases had SRC with fibrosis with TRD. Postoperative visual acuity at 3 months of follow-up was analyzed as the primary outcome measure. Mean age of patients was 26 years ± 12.27 years. Four out of 15 patients were female. Mean preoperative best-corrected visual acuity (BCVA) was 1.55 logMAR units ± 0.62 logMAR units, whereas mean postoperative BCVA at last follow-up (3 months) was 1.26 logMAR units ± 0.65 log-MAR units. Mean visual gain (0.29 logMAR units) post-surgery was statistically significant (P = .018). The final visual acuity correlated with preoperative BCVA with Pearson's coefficient being 0.78 (95% CI, 0.44-0.92; P = .001). Anatomical success was achieved in 13 of 15 (87%) cases. In one case the cyst was dead and calcified and could not be removed. TRD was associated with poor visual gain. Intravitreal cyst lysis is a safe and successful approach for managing intraocular cysticercosis. Visual results depend on preoperative condition. TRD implicates poor visual prognoses. [Ophthalmic Surg Lasers Imaging Retina. 2016;47:665-669.]. Copyright 2016, SLACK Incorporated.

  15. Role of DLP12 lysis genes in Escherichia coli biofilm formation

    PubMed Central

    Toba, Faustino A.; Thompson, Mitchell G.; Campbell, Bryan R.; Junker, Lauren M.; Rueggeberg, Karl-Gustav

    2011-01-01

    Phages have recently been implicated as important in biofilm development, although the mechanisms whereby phages impact biofilms remain unclear. One defective lambdoid phage carried by Escherichia coli K-12 is DLP12. Among the genes found in DLP12 are essD, ybcS and rzpD/rzoD, which are homologues of the Lambda phage genes encoding cell-lysis proteins (S, R and Rz/Rz1). The role that these DLP12 lysis genes play in biofilm formation was examined in deletion mutants of E. coli PHL628, a curli-overproducing, biofilm-forming K-12 derivative. Strains lacking essD, ybcS and rzpD/rzoD were unable to form wild-type biofilms. While all mutants were compromised in attachment to abiotic surfaces and aggregated less well than the wild-type, the effect of the essD knockout on biofilm formation was less dramatic than that of deleting ybcS or rzpD/rzoD. These results were consistent with electron micrographs of the mutants, which showed a decreased number of curli fibres on cell surfaces. Also consistent with this finding, we observed that expression from the promoter of csgB, which encodes the curli subunits, was downregulated in the mutants. As curli production is transcriptionally downregulated in response to cell wall stress, we challenged the mutants with SDS and found them to be more sensitive to the detergent than the wild-type. We also examined the release of 14C-labelled peptidoglycan from the mutants and found that they did not lose labelled peptidoglycan to the same extent as the wild-type. Given that curli production is known to be suppressed by N-acetylglucosamine 6-phosphate (NAG-6P), a metabolite produced during peptidoglycan recycling, we deleted nagK, the N-acetylglucosamine kinase gene, from the lysis mutants and found that this restored curli production. This suggested that deletion of the lysis genes affected cell wall status, which was transduced to the curli operon by NAG-6P via an as yet unknown mechanism. These observations provide evidence that the S, R

  16. Medical Management of Tumor Lysis Syndrome, Postprocedural Pain, and Venous Thromboembolism Following Interventional Radiology Procedures

    PubMed Central

    Faramarzalian, Ali; Armitage, Keith B.; Kapoor, Baljendra; Kalva, Sanjeeva P.

    2015-01-01

    The rapid expansion of minimally invasive image-guided procedures has led to their extensive use in the interdisciplinary management of patients with vascular, hepatobiliary, genitourinary, and oncologic diseases. Given the increased availability and breadth of these procedures, it is important for physicians to be aware of common complications and their management. In this article, the authors describe management of select common complications from interventional radiology procedures including tumor lysis syndrome, acute on chronic postprocedural pain, and venous thromboembolism. These complications are discussed in detail and their medical management is outlined according to generally accepted practice and evidence from the literature. PMID:26038627

  17. HIF-2α/ITPR1 axis: A new saboteur of NK-mediated lysis

    PubMed Central

    Messai, Yosra; Noman, Muhammad Zaeem; Hasmim, Meriem; Escudier, Bernard; Chouaib, Salem

    2015-01-01

    We recently investigated the role of von Hippel-Lindau (VHL) mutation and the subsequent induction of hypoxia-inducible factor 2α (HIF-2α) in the regulation of renal cell carcinoma (RCC) susceptibility to natural killer (NK) cell-mediated killing. We demonstrated that the resistance of VHL-mutated RCC cell line 786-0 to NK-mediated lysis requires HIF-2α and ITPR1, a direct novel target of HIF-2α, through the activation of autophagy in target cells by NK-derived signals. PMID:25949883

  18. Investigation of an optimal cell lysis method for the study of the zinc metalloproteome of Histoplasma capsulatum.

    PubMed

    Donnell, Anna M; Lewis, Stephanie; Abraham, Sami; Subramanian, Kavitha; Figueroa, Julio Landero; Deepe, George S; Vonderheide, Anne P

    2017-08-12

    This work sought to assess optimal extraction conditions in the study of the metalloproteome of the dimorphic fungus Histoplasma capsulatum. One of the body's responses to H. capsulatum infection is sequestration of zinc within host macrophage (MØ), as reported by Vignesh et al. (Immunity 39:697-710, 2013) and Vignesh et al. (PLOS Pathog 9:E1003815, 2013). Thus, metalloproteins containing zinc were of greatest interest as it plays a critical role in survival of the fungus. One challenge in metalloproteomics is the preservation of the native structure of proteins to retain non-covalently bound metals. Many of the conventional cell lysis, separation, and identification techniques in proteomics are carried out under conditions that could lead to protein denaturation. Various cell lysis techniques were investigated in an effort to both maintain the metalloproteins during lysis and subsequent analysis while, at the same time, serving to be strong enough to break the cell wall, allowing access to cytosolic metalloproteins. The addition of 1% Triton x-100, a non-ionic detergent, to the lysis buffer was also studied. Seven lysis methods were considered and these included: Glass Homogenizer (H), Bead Beater (BB), Sonication Probe (SP), Vortex with 1% Triton x-100 (V, T), Vortex with no Triton x-100 (V, NT), Sonication Bath, Vortex, and 1% Triton x-100 (SB, V, T) and Sonication Bath, Vortex, and no Triton x-100 (SB, V, NT). A Qubit® Assay was used to compare total protein concentration and inductively coupled plasma-mass spectrometry (ICP-MS) was utilized for total metal analysis of cell lysates. Size exclusion chromatography coupled to ICP-MS (SEC-HPLC-ICP-MS) was used for separation of the metalloproteins in the cell lysate and the concentration of Zn over a wide molecular weight range was examined. Additional factors such as potential contamination sources were also considered. A cell lysis method involving vortexing H. capsulatum yeast cells with 500 μm glass beads

  19. STAPHYLOCOCCAL SENSITIZATION: SPECIFIC BIOLOGICAL EFFECTS OF PHAGE K ON THE BACTERIAL CELL WALL IN LYSIS-FROM-WITHOUT.

    PubMed

    RALSTON, D J

    1963-06-01

    Ralston, Doris J. (University of California, Berkeley). Staphylococcal sensitization: specific biological effects of phage K on the bacterial cell wall in lysis-from-without. J. Bacteriol. 85:1185-1193. 1963.-Phage K, shown previously to sensitize staphylococcal-wall mucopeptide to the action of a phage-induced enzyme, virolysin, was found to act in a specific manner in that its sensitizing effects were restricted to chemical linkages affected by three staphylococcal lysins. These caused an immediate lysis, whereas egg-white lysozyme, which could also digest the wall mucopeptide, exerted variable effects, even when in the absence of phage it produced some lysis. Evidence was presented that the K(1) normal cell autolysin and the K phage virolysin could act synergistically with lysozyme on phage-sensitized cells, and that any effects observed with lysozyme were due to the simultaneous presence of trace amounts of these staphylococcal lysins. None of a series of lysozymelike agents from sea urchins, marine sepunculids, and from rabbit peritoneal histiocytes caused accelerated lysis of phage-sensitized cells, although like lysozyme they showed a slow lysis of phage-free living cells. Other enzymes which did not reduce the turbidity of sensitized cells included agents specific for intracellular components (proteins, lipids, nucleic acids), and enzymes, as decarboxylase, alkaline phosphatase, d-amino oxidase, and hyaluronidase. These results suggested that the main effects of the phage in sensitization were limited to areas of the cell wall involved in protection against the action of the staphylococcal lysins.

  20. Lead ions but not other metallic ions increase resistance to hypotonic lysis in prenatal hemopoiesis red blood cells.

    PubMed

    Corchs, J; Gioia, I A; Serrani, R E; Taborda, D

    2001-12-01

    Metals known to have toxic effects on exposed individuals (Aluminum (Al), Cadmium (Cd), Zinc (Zn) and lead (Pb)) were selected. Umbilical cord erythrocytes from normal newborns were incubated in isotonic media alone or with addition of Pb (20 microM), Cd, Zn or Al (concentration range: 20-250 microM). Red cells were then placed in media of diminishing tonicity, to measure cellular lysis and volume; the regression curves of percent lysis as a function of osmolarity were determined for each data set and the break points calculated. Resistance to lysis increased significantly in Pb treated cells whereas cells treated with the other metals did not differ from controls, even at concentrations ten times higher than that of Pb. Lead produced a reduction in cellular volume corrected by addition of quinidine (an inhibitor of potassium channels activation) to the cell suspension; on the other hand, quinidine did not modify the effect of lead on lysis sensitivity. These results suggest that the effect of lead on cell resistance to lysis might be mediated by changes in membrane structure. The other metals examined did not affect the variables studied.

  1. Mycobacterial ESX-1 secretion system mediates host cell lysis through bacterium contact-dependent gross membrane disruptions.

    PubMed

    Conrad, William H; Osman, Morwan M; Shanahan, Jonathan K; Chu, Frances; Takaki, Kevin K; Cameron, James; Hopkinson-Woolley, Digby; Brosch, Roland; Ramakrishnan, Lalita

    2017-02-07

    Mycobacterium tuberculosis and Mycobacterium marinum are thought to exert virulence, in part, through their ability to lyse host cell membranes. The type VII secretion system ESX-1 [6-kDa early secretory antigenic target (ESAT-6) secretion system 1] is required for both virulence and host cell membrane lysis. Both activities are attributed to the pore-forming activity of the ESX-1-secreted substrate ESAT-6 because multiple studies have reported that recombinant ESAT-6 lyses eukaryotic membranes. We too find ESX-1 of M. tuberculosis and M. marinum lyses host cell membranes. However, we find that recombinant ESAT-6 does not lyse cell membranes. The lytic activity previously attributed to ESAT-6 is due to residual detergent in the preparations. We report here that ESX-1-dependent cell membrane lysis is contact dependent and accompanied by gross membrane disruptions rather than discrete pores. ESX-1-mediated lysis is also morphologically distinct from the contact-dependent lysis of other bacterial secretion systems. Our findings suggest redirection of research to understand the mechanism of ESX-1-mediated lysis.

  2. Determination of growth and lysis kinetics in plant cell suspension cultures from the measurement of esterase release.

    PubMed

    Steward, N; Martin, R; Engasser, J M; Goergen, J L

    1999-01-01

    The death of Medicago sativa L. cells cultivated in a batch culture was investigated by measuring both the appearance of intact dead cells determined on the basis of the trypan blue (TB) dye exclusion, and the release of the cytoplasmic esterase activity into the culture medium upon cell death. Taking into account the strong instability of this released esterase activity, the total dead cell and lysed cell densities have been estimated. A mechanism for cell death and lysis is proposed and the specific rates of cell growth, death and lysis estimated. The specific rate of appearance of TB dead cells was low and essentially constant (0.25 day(-1)) during the first 8 days of the batch culture, and then increased above 1.5 day(-1) after 2 weeks of cultivation. Whereas no lysis occurred during the first seven days, this phenomenon occurred during the second period and accounted for about 20% of the total cell death by the end of the process. Thus, the viability determined by the trypan blue exclusion method appeared to be invalid after 7 days of culture. When lysis of viable cells is taken into consideration, the specific growth rate was significantly increased and growth was shown to continue for a further 8 days. Increased sensitivity of the cells to shear stresses and consequent cell lysis could be the result of a 35% increase in the cell size Copyright 1999 John Wiley & Sons, Inc.

  3. Mycobacterial ESX-1 secretion system mediates host cell lysis through bacterium contact-dependent gross membrane disruptions

    PubMed Central

    Conrad, William H.; Osman, Morwan M.; Shanahan, Jonathan K.; Chu, Frances; Takaki, Kevin K.; Cameron, James; Hopkinson-Woolley, Digby; Ramakrishnan, Lalita

    2017-01-01

    Mycobacterium tuberculosis and Mycobacterium marinum are thought to exert virulence, in part, through their ability to lyse host cell membranes. The type VII secretion system ESX-1 [6-kDa early secretory antigenic target (ESAT-6) secretion system 1] is required for both virulence and host cell membrane lysis. Both activities are attributed to the pore-forming activity of the ESX-1–secreted substrate ESAT-6 because multiple studies have reported that recombinant ESAT-6 lyses eukaryotic membranes. We too find ESX-1 of M. tuberculosis and M. marinum lyses host cell membranes. However, we find that recombinant ESAT-6 does not lyse cell membranes. The lytic activity previously attributed to ESAT-6 is due to residual detergent in the preparations. We report here that ESX-1–dependent cell membrane lysis is contact dependent and accompanied by gross membrane disruptions rather than discrete pores. ESX-1–mediated lysis is also morphologically distinct from the contact-dependent lysis of other bacterial secretion systems. Our findings suggest redirection of research to understand the mechanism of ESX-1–mediated lysis. PMID:28119503

  4. Excretion of cytoplasmic proteins in Staphylococcus is most likely not due to cell lysis.

    PubMed

    Ebner, Patrick; Rinker, Janina; Götz, Friedrich

    2016-02-01

    The excretion of cytoplasmic proteins (ECP) is a long-known phenomenon in bacteria and eukaryotes. So far, it was not possible to associate either a signal peptide-dependent or a signal peptide-independent pathway to ECP. Nevertheless 25% of the proteins found in Staphylococcus aureus supernatants were cytoplasmic proteins. Because the excreted proteins do not possess a common motive, the most widespread opinion is that ECP is due to cell lysis. This explanation seems to be too easy since several indications imply that there exists a yet unknown mechanism for ECP. Certainly, the up-regulation of autolysins as well as decreased peptidoglycan cross-linking increased ECP. However, in recent years, several evidences arose that cell lysis is not the only reason for ECP. It seems that ECP is a part of the normal cell cycle of S. aureus as it turned out that ECP with several model proteins occurs mainly during cell growth. It has common features as proteins secreted via the Sec translocon and finally the excretion site is the cross wall of dividing cells.

  5. Clotrimazole enhances lysis of human erythrocytes induced by t-BHP.

    PubMed

    Lisovskaya, Irene L; Shcherbachenko, Irina M; Volkova, Rimma I; Ataullakhanov, Fazoil I

    2009-08-14

    Clotrimazole (CLT) is an antifungal and antimalarial agent also effective as a Gardos channel inhibitor. In addition, CLT possesses antitumor properties. Recent data provide evidence that CLT forms a complex with heme (hemin), which produces a more potent lytic effect than heme alone. This study addressed the effect of CLT on the lysis of normal human erythrocytes induced by tert-butyl hydroperoxide (t-BHP). For the first time, it was shown that 10 microM CLT significantly enhanced the lytic effect of t-BHP on erythrocytes in both Ca(2+)-containing and Ca(2+)-free media, suggesting that the effect is not related to Gardos channels. CLT did not affect the rate of free radical generation, the kinetics of GSH degradation, methemoglobin formation and TBARS generation; therefore, we concluded that CLT does not cause additional oxidative damage to erythrocytes treated with t-BHP. It is tempted to speculate that CLT enhances t-BHP-induced changes in erythrocyte volume and lysis largely by forming a complex with hemin released during hemoglobin oxidation in erythrocytes: the CLT-hemin complex destabilizes the cell membrane more potently than hemin alone. If so, the effect of CLT on cell membrane damage during free-radical oxidation may be used to increase the efficacy of antitumor therapy.

  6. Impaired respiration elicits SrrAB-dependent programmed cell lysis and biofilm formation in Staphylococcus aureus

    PubMed Central

    Mashruwala, Ameya A; van de Guchte, Adriana; Boyd, Jeffrey M

    2017-01-01

    Biofilms are communities of microorganisms attached to a surface or each other. Biofilm-associated cells are the etiologic agents of recurrent Staphylococcus aureus infections. Infected human tissues are hypoxic or anoxic. S. aureus increases biofilm formation in response to hypoxia, but how this occurs is unknown. In the current study we report that oxygen influences biofilm formation in its capacity as a terminal electron acceptor for cellular respiration. Genetic, physiological, or chemical inhibition of respiratory processes elicited increased biofilm formation. Impaired respiration led to increased cell lysis via divergent regulation of two processes: increased expression of the AtlA murein hydrolase and decreased expression of wall-teichoic acids. The AltA-dependent release of cytosolic DNA contributed to increased biofilm formation. Further, cell lysis and biofilm formation were governed by the SrrAB two-component regulatory system. Data presented support a model wherein SrrAB-dependent biofilm formation occurs in response to the accumulation of reduced menaquinone. DOI: http://dx.doi.org/10.7554/eLife.23845.001 PMID:28221135

  7. Avidin acylation prevents the complement-dependent lysis of avidin-carrying erythrocytes.

    PubMed Central

    Muzykantov, V R; Smirnov, M D; Samokhin, G P

    1991-01-01

    Non-covalent binding of avidin to biotinylated erythrocytes results in complement-dependent haemolysis. Biotinylated erythrocytes, as well as native cells, are not lysed by complement. Complement activation requires a tight contact between avidin and the erythrocyte membrane, since avidin does not in itself activate complement and does not inhibit lysis of sensitized sheep erythrocytes. The efficiency of haemolysis depends on avidin's surface density. When the avidin concentration in the reaction mixture is less than 15 micrograms/ml, erythrocyte lysis is not induced. However, the attachment of biotinylated antibodies to avidin-carrying erythrocytes decreases dramatically. Acylation of avidin with succinic anhydride strongly decreases its ability to induce complement-dependent haemolysis. However, the ability of avidin to cross-link the biotin-containing structures decreases after acylation. A 50% modification of avidin by succinic anhydride (pI about 7.0) allows preparation of 'immunoerythrocytes', which retain their affinity to antigen and stability in the presence of complement. PMID:1991038

  8. Capacity of tumor necrosis factor to augment lymphocyte-mediated tumor cell lysis of malignant mesothelioma

    SciTech Connect

    Bowman, R.V.; Manning, L.S.; Davis, M.R.; Robinson, B.W. )

    1991-01-01

    Recombinant human tumor necrosis factor (rHuTNF) was evaluated both for direct anti-tumor action against human malignant mesothelioma and for its capacity to augment the generation and lytic phases of lymphocyte-mediated cytotoxicity against this tumor. rHuTNF was directly toxic by MTT assay to one of two mesothelioma cell lines evaluated, but had no effect on susceptibility to subsequent lymphocyte-mediated lysis of either line. TNF alone was incapable of generating anti-mesothelioma lymphokine-activated killer cell (LAK) activity. Furthermore, it did not augment the degree or LAK activity produced by submaximal interleukin-2 (IL-2) concentrations nor did it augment lysis of mesothelioma cells by natural killer (NK) or LAK effector cells during the 4-hr 51chromium release cytolytic reaction. The studies also suggest that mesothelioma targets are less responsive to TNF plus submaximal IL-2 concentrations than the standard LAK sensitive target Daudi, raising the possibility that intermediate LAK sensitive tumors such as mesothelioma may require separate and specific evaluation in immunomodulation studies. This in vitro study indicates that use of low-dose rHuTNF and IL-2 is unlikely to be an effective substitute for high-dose IL-2 in generation and maintenance of LAK activity in adoptive immunotherapy for mesothelioma.

  9. Kin cell lysis is a danger signal that activates antibacterial pathways of Pseudomonas aeruginosa

    PubMed Central

    LeRoux, Michele; Kirkpatrick, Robin L; Montauti, Elena I; Tran, Bao Q; Peterson, S Brook; Harding, Brittany N; Whitney, John C; Russell, Alistair B; Traxler, Beth; Goo, Young Ah; Goodlett, David R; Wiggins, Paul A; Mougous, Joseph D

    2015-01-01

    The perception and response to cellular death is an important aspect of multicellular eukaryotic life. For example, damage-associated molecular patterns activate an inflammatory cascade that leads to removal of cellular debris and promotion of healing. We demonstrate that lysis of Pseudomonas aeruginosa cells triggers a program in the remaining population that confers fitness in interspecies co-culture. We find that this program, termed P. aeruginosa response to antagonism (PARA), involves rapid deployment of antibacterial factors and is mediated by the Gac/Rsm global regulatory pathway. Type VI secretion, and, unexpectedly, conjugative type IV secretion within competing bacteria, induce P. aeruginosa lysis and activate PARA, thus providing a mechanism for the enhanced capacity of P. aeruginosa to target bacteria that elaborate these factors. Our finding that bacteria sense damaged kin and respond via a widely distributed pathway to mount a complex response raises the possibility that danger sensing is an evolutionarily conserved process. DOI: http://dx.doi.org/10.7554/eLife.05701.001 PMID:25643398

  10. Revisiting Bistability in the Lysis/Lysogeny Circuit of Bacteriophage Lambda

    PubMed Central

    Bednarz, Michael; Halliday, Jennifer A.; Herman, Christophe; Golding, Ido

    2014-01-01

    The lysis/lysogeny switch of bacteriophage lambda serves as a paradigm for binary cell fate decision, long-term maintenance of cellular state and stimulus-triggered switching between states. In the literature, the system is often referred to as “bistable.” However, it remains unclear whether this term provides an accurate description or is instead a misnomer. Here we address this question directly. We first quantify transcriptional regulation governing lysogenic maintenance using a single-cell fluorescence reporter. We then use the single-cell data to derive a stochastic theoretical model for the underlying regulatory network. We use the model to predict the steady states of the system and then validate these predictions experimentally. Specifically, a regime of bistability, and the resulting hysteretic behavior, are observed. Beyond the steady states, the theoretical model successfully predicts the kinetics of switching from lysogeny to lysis. Our results show how the physics-inspired concept of bistability can be reliably used to describe cellular phenotype, and how an experimentally-calibrated theoretical model can have accurate predictive power for cell-state switching. PMID:24963924

  11. Isolation and Expression of the Lysis Genes of Actinomyces naeslundii Phage Av-1

    PubMed Central

    Delisle, Allan L.; Barcak, Gerard J.; Guo, Ming

    2006-01-01

    Like most gram-positive oral bacteria, Actinomyces naeslundii is resistant to salivary lysozyme and to most other lytic enzymes. We are interested in studying the lysins of phages of this important oral bacterium as potential diagnostic and therapeutic agents. To identify the Actinomyces phage genes encoding these species-specific enzymes in Escherichia coli, we constructed a new cloning vector, pAD330, that can be used to enrich for and isolate phage holin genes, which are located adjacent to the lysin genes in most phage genomes. Cloned holin insert sequences were used to design sequencing primers to identify nearby lysin genes by using whole phage DNA as the template. From partial digestions of A. naeslundii phage Av-1 genomic DNA we were able to clone, in independent experiments, inserts that complemented the defective λ holin in pAD330, as evidenced by extensive lysis after thermal induction. The DNA sequence of the inserts in these plasmids revealed that both contained the complete lysis region of Av-1, which is comprised of two holin-like genes, designated holA and holB, and an endolysin gene, designated lysA. We were able to subclone and express these genes and determine some of the functional properties of their gene products. PMID:16461656

  12. Effects of cell lysis treatments on the yield of coenzyme Q10 following Agrobacterium tumefaciens fermentation.

    PubMed

    Yuting Tian; Tianli Yue; Jinjin Pei; Yahong Yuan; Juhai Li; Martin Lo, Y

    2010-04-01

    The yield of CoQ₁₀, an intracellular product extracted from Agrobacterium tumefaciens cells is dependent on the effectiveness of cell lysis post fermentation. Various cell lysis approaches are investigated, including ultrasound, repetitive freezing/thawing, grinding and acid-heat treatment. The acid-heat combination using hydrochloric acid is found the most effective in releasing CoQ₁₀, followed by lactic, sulfuric, phosphoric and oxalic acids. The most significant processing parameters, namely the ratio of acid solution volume and bacteria weight (A/B ratio), incubation temperature and reaction time, are optimized by using the central composite design with a quadratic regression model built by response surface methodology. The highest CoQ₁₀ yield at 1.518 mg/g dry cell is attained using hydrochloric acid (3 mol/L) under optimal A/B ratio, temperature and time at 10.8 mL/g, 84.2 °C and 35.3 min, respectively.

  13. Redirecting NK cells mediated tumor cell lysis by a new recombinant bifunctional protein

    PubMed Central

    Germain, Claire; Campigna, Emmanuelle; Salhi, Imed; Morisseau, Sébastien; Navarro-Teulon, Isabelle; Mach, Jean-Pierre; Pèlegrin, André; Robert, Bruno

    2008-01-01

    Natural killer (NK) cells are at the crossroad between innate and adaptive immunity and play a major role in cancer immunosurveillance. NK cell stimulation depends on a balance between inhibitory and activating receptors, such as the stimulatory lectinlike receptor NKG2D. To redirect NK cells against tumor cells we designed bifunctional proteins able to specifically bind tumor cells and to induce their lysis by NK cells, after NKG2D engagement. To this aim, we used the “knob into hole” heterodimerization strategy, in which “knob” and “hole” variants were generated by directed mutagenesis within the CH3 domain of human IgG1 Fc fragments fused to an anti-CEA or anti-HER2 scFv or to the H60 murine ligand of NKG2D, respectively. We demonstrated the capacity of the bifunctional proteins produced to specifically coat tumor cells surface with H60 ligand. Most importantly, we demonstrated that these bifunctional proteins were able to induce an NKG2D-dependent and antibody-specific tumor cell lysis by murine NK cells. Overall, the results show the possibility to redirect NK cytotoxicity to tumor cells by a new format of recombinant bispecific antibody, opening the way of potential NK cell-based cancer immunotherapies by specific activation of the NKG2D receptor at the tumor site. PMID:18790793

  14. Robust Oncolytic Virotherapy Induces Tumor Lysis Syndrome and Associated Toxicities in the MPC-11 Plasmacytoma Model.

    PubMed

    Zhang, Lianwen; Steele, Michael B; Jenks, Nathan; Grell, Jacquelyn; Behrens, Marshall; Nace, Rebecca; Naik, Shruthi; Federspiel, Mark J; Russell, Stephen J; Peng, Kah-Whye

    2016-12-01

    Tumor-selective oncolytic vesicular stomatitis viruses (VSVs) are being evaluated in clinical trials. Here, we report that the MPC-11 murine plasmacytoma model is so extraordinarily susceptible to oncolytic VSVs that a low dose of virus leads to extensive intratumoral viral replication, sustained viremia, intravascular coagulation, and a rapidly fatal tumor lysis syndrome (TLS). Rapid softening, shrinkage and hemorrhagic necrosis of flank tumors was noted within 1-2 days after virus administration, leading to hyperkalemia, hyperphosphatemia, hypocalcemia, hyperuricemia, increase in plasma cell free DNA, lymphopenia, consumptive coagulopathy, increase in fibrinogen degradation products, decreased liver function tests, dehydration, weight loss, and euthanasia or death after 5-8 days. Secondary viremia was observed but viral replication in normal host tissues was not detected. Toxicity could be mitigated by using VSVs with slowed replication kinetics, and was less marked in animals with smaller flank tumors. The MPC-11 tumor represents an interesting model to further study the complex interplay of robust intratumoral viral replication, tumor lysis, and associated toxicities in cases where tumors are highly responsive to oncolytic virotherapy.

  15. Treatment and prevention of tumor lysis syndrome in children. Experience of Associazione Italiana Ematologia Oncologia Pediatrica.

    PubMed

    Pession, Andrea; Barbieri, Eveline

    2005-01-01

    Hyperuricemia and tumor lysis syndrome (TLS) are complications that can arise from treatment of rapidly proliferating and drug-sensitive neoplasms. Clinical trials have shown rasburicase, a recombinant urate oxidase to be more effective than allopurinol for the prevention and treatment of malignancy-associated hyperuricemia. We investigated the safety and efficacy of rasburicase in the AIEOP centers' experience. We reviewed the data of 26 children with malignancy at risk for TLS, submitted to treatment (group 1) or prophylaxis (group 2) of acute hyperuricemia with rasburicase (0.20 mg/kg intravenously daily) for a median period of 4 days. Rasburicase produced a significant decrease in uric acid concentrations in all the patients. The control of uric acid levels was obtained in both the groups within 24 h of the first dose with a response rate of 100% (group 1) and 93% (group 2). Normalization of creatinine and phosphorus levels was obtained in 5 and 4 days respectively. Tolerance was excellent without toxicity. These data confirm that rasburicase is a safe, highly and rapidly effective agent in the treatment and prevention of malignancy-associated acute hyperuricemia and could be considered the treatment of choice to prevent tumor lysis syndrome in children at high risk for this metabolic complication.

  16. Streptococcus pneumoniae disrupts pulmonary immune defence via elastase release following pneumolysin-dependent neutrophil lysis

    PubMed Central

    Domon, Hisanori; Oda, Masataka; Maekawa, Tomoki; Nagai, Kosuke; Takeda, Wataru; Terao, Yutaka

    2016-01-01

    Streptococcus pneumoniae is a leading cause of bacterial pneumonia and is the principal cause of morbidity and mortality worldwide. Previous studies suggested that excessive activation of neutrophils results in the release of neutrophil elastase, which contributes to lung injury in severe pneumonia. Although both pneumococcal virulence factors and neutrophil elastase contribute to the development and progression of pneumonia, there are no studies analysing relationships between these factors. Here, we showed that pneumolysin, a pneumococcal pore-forming toxin, induced cell lysis in primary isolated human neutrophils, leading to the release of neutrophil elastase. Pneumolysin exerted minimal cytotoxicity against alveolar epithelial cells and macrophages, whereas neutrophil elastase induced detachment of alveolar epithelial cells and impaired phagocytic activity in macrophages. Additionally, activation of neutrophil elastase did not exert bactericidal activity against S. pneumoniae in vitro. P2X7 receptor, which belongs to a family of purinergic receptors, was involved in pneumolysin-induced cell lysis. These findings suggested that infiltrated neutrophils are the primary target cells of pneumolysin, and that S. pneumoniae exploits neutrophil-elastase leakage to induce the disruption of pulmonary immune defences, thereby causing lung injury. PMID:27892542

  17. Plasminogen associates with phosphatidylserine-exposing platelets and contributes to thrombus lysis under flow.

    PubMed

    Whyte, Claire S; Swieringa, Frauke; Mastenbroek, Tom G; Lionikiene, Ausra S; Lancé, Marcus D; van der Meijden, Paola E J; Heemskerk, Johan W M; Mutch, Nicola J

    2015-04-16

    The interaction of plasminogen with platelets and their localization during thrombus formation and fibrinolysis under flow are not defined. Using a novel model of whole blood thrombi, formed under flow, we examine dose-dependent fibrinolysis using fluorescence microscopy. Fibrinolysis was dependent upon flow and the balance between fibrin formation and plasminogen activation, with tissue plasminogen activator-mediated lysis being more efficient than urokinase plasminogen activator-mediated lysis. Fluorescently labeled plasminogen radiates from platelet aggregates at the base of thrombi, primarily in association with fibrin. Hirudin attenuates, but does not abolish plasminogen binding, denoting the importance of fibrin. Flow cytometry revealed that stimulation of platelets with thrombin/convulxin significantly increased the plasminogen signal associated with phosphatidylserine (PS)-exposing platelets. Binding was attenuated by tirofiban and Gly-Pro-Arg-Pro amide, confirming a role for fibrin in amplifying plasminogen binding to PS-exposing platelets. Confocal microscopy revealed direct binding of plasminogen and fibrinogen to different platelet subpopulations. Binding of plasminogen and fibrinogen co-localized with PAC-1 in the center of spread platelets. In contrast, PS-exposing platelets were PAC-1 negative, and bound plasminogen and fibrinogen in a protruding "cap." These data show that different subpopulations of platelets harbor plasminogen by diverse mechanisms and provide an essential scaffold for the accumulation of fibrinolytic proteins that mediate fibrinolysis under flow.

  18. Comparison of point-of-care-compatible lysis methods for bacteria and viruses.

    PubMed

    Heiniger, Erin K; Buser, Joshua R; Mireles, Lillian; Zhang, Xiaohong; Ladd, Paula D; Lutz, Barry R; Yager, Paul

    2016-09-01

    Nucleic acid sample preparation has been an especially challenging barrier to point-of-care nucleic acid amplification tests in low-resource settings. Here we provide a head-to-head comparison of methods for lysis of, and nucleic acid release from, several pathogenic bacteria and viruses-methods that are adaptable to point-of-care usage in low-resource settings. Digestion with achromopeptidase, a mixture of proteases and peptidoglycan-specific hydrolases, followed by thermal deactivation in a boiling water bath, effectively released amplifiable nucleic acid from Staphylococcus aureus, Bordetella pertussis, respiratory syncytial virus, and influenza virus. Achromopeptidase was functional after dehydration and reconstitution, even after eleven months of dry storage without refrigeration. Mechanical lysis methods proved to be effective against a hard-to-lyse Mycobacterium species, and a miniature bead-mill, the AudioLyse, is shown to be capable of releasing amplifiable DNA and RNA from this species. We conclude that point-of-care-compatible sample preparation methods for nucleic acid tests need not introduce amplification inhibitors, and can provide amplification-ready lysates from a wide range of bacterial and viral pathogens.

  19. Lytic action of cloned pneumococcal phage lysis genes in Streptococcus pneumoniae.

    PubMed

    Romero, A; López, R; García, P

    1993-03-15

    The genes hbl3, cpl1 and cpl7 coding for the pneumococcal phage lytic enzymes HBL3, CPL1 and CPL7, respectively, have been cloned into shuttle plasmids that can replicate in Streptococcus pneumoniae and Escherichia coli. All these genes were expressed in E. coli under the control of either the lytP promoter of the lytA gene, which codes for the major pneumococcal autolysin, or the promoter of the tetracycline-resistance gene (tetP). In contrast, cpl1 and cpl7 genes that code for lysozymes were expressed in pneumococcus only under the control of tetP, whereas the hbl3 gene that codes for an amidase can be expressed using either promoter. The phage lysozymes or amidase expressed in S. pneumoniae M31, a mutant deleted in the lytA gene coding for short chains, were placed under physiological control since these transformed bacteria grew as normal 'diplo' cells during the exponential phase and underwent autolysis only after long incubation at 37 degrees C. The lysis genes appear to be expressed constitutively in the transformed pneumococci, since sharply defined lysis of these cultures could be induced prematurely during the exponential phase of growth by addition of sodium deoxycholate.

  20. [Study of a lysis medium stabilizing microfilaments and microtubules in vitro and in vivo].

    PubMed

    Foucault, G; Raymond, M N; Coffe, G; Pudles, J

    1984-01-01

    Determination of experimental conditions which allow the evaluation of the variations in the ratio of non polymerized and polymerized forms of actin and tubulin during the reorganization of the cytoskeletal cell system is of most valuable importance. In order to prepare cell homogenates which would reflect the in vivo situation, we tested in vitro a lysis medium which stabilized both microfilaments and microtubules, which were determined by DNase inhibition assays and colchicine binding assays respectively. This lysis medium containing 10 mM potassium phosphate, 1mM magnesium chloride, 5 mM EGTA, 1 M hexylene glycol, 1% Triton X-100, pH 6.4, used at 4 degrees C a) diffused rapidly into the cells; b) did not denature actin and tubulin; c) did not displace the equilibrium between non polymerized and polymerized forms of actin and tubulin, allowing biochemical assays on cell homogenates; d) blocked the evolution of the cytoskeletal system and permitted structural studies; e) and allowed the decoration of microfilaments by heavy meromyosin.

  1. Clot Lysis and Antimitotic Study of Ficus glomerata Roxb Fruit Extracts

    PubMed Central

    Shivasharanappa, Kirankumar; Londonkar, Ramesh

    2014-01-01

    The present study was carried out to investigate the thrombolytic and antimitotic potentiality of various extracts of fruits of Ficus glomerata, a traditional medicinal plant, using an in vitro assay method. Three crude extracts such as petroleum ether (FGPE), chloroform (FGCE), and methanol (FGME) were used for the study, with a standard (streptokinase) and negative control (sterile distilled water) to validate the method. The thrombolytic nature of the plant was found significant with methanol extract and chloroform and petroleum ether extracts have recorded mild activity, when compared with the negative control (sterile distilled water). The extracts have shown mild clot lysis, that is, 2.16%, 23.06%, 27.60%, and 47.74% of sterile distilled water, FGPE, FGCE, and FGME, respectively, while the standard (streptokinase) has shown 74.22% clot lysis. FGME inhibited the root growth in number as well as length effectively, followed by FGPE, while FGCE exhibited moderate antimitotic activity and it was supported by mitotic index. Therefore, the obtained results suggest that among all the extracts of plant the methanolic extract has shown highest thrombolytic and antimitotic activity. PMID:25006495

  2. All-in-one nanowire-decorated multifunctional membrane for rapid cell lysis and direct DNA isolation.

    PubMed

    So, Hongyun; Lee, Kunwoo; Murthy, Niren; Pisano, Albert P

    2014-12-10

    This paper describes a handheld device that uses an all-in-one membrane for continuous mechanical cell lysis and rapid DNA isolation without the assistance of power sources, lysis reagents, and routine centrifugation. This nanowire-decorated multifunctional membrane was fabricated to isolate DNA by selective adsorption to silica surface immediately after disruption of nucleus membranes by ultrasharp tips of nanowires for a rapid cell lysis, and it can be directly assembled with commercial syringe filter holders. The membrane was fabricated by photoelectrochemical etching to create microchannel arrays followed by hydrothermal synthesis of nanowires and deposition of silica. The proposed membrane successfully purifies high-quality DNA within 5 min, whereas a commercial purification kit needs more than an hour.

  3. A disposable bacterial lysis cartridge (BLC) suitable for an in situ water-borne pathogen detection system.

    PubMed

    Lee, Eun-Hee; Lim, Hyun Jeong; Son, Ahjeong; Chua, Beelee

    2015-11-21

    We constructed a disposable bacterial lysis cartridge (BLC) suitable for an in situ pathogen detection system. It had an in-built micro corona discharge based ozone generator that provided ozone for cell lysis. Using a custom sample handling platform, its performance was evaluated with a Gram-positive bacterium of Bacillus subtilis. It was capable of achieving a similar degree of lysis as a commercial ultrasonic dismembrator with a P-1 microprobe in 10 min at an air pump flow rate of 29.4 ml min(-1) and an ozone generator operating voltage of 1600 V. The lysing duration could be significantly reduced to 5 min by increasing the air pump flow rate and the ozone generator operating voltage as well as by the addition of sodium dodecyl sulfate (SDS).

  4. Facile Alkaline Lysis of Escherichia coli Cells in High-Throughput Mode for Screening Enzyme Mutants: Arylsulfatase as an Example.

    PubMed

    Yuan, Mei; Yang, Xiaolan; Li, Yuwei; Liu, Hongbo; Pu, Jun; Zhan, Chang-Guo; Liao, Fei

    2016-06-01

    Facile alkaline lysis of Escherichia coli cells in high-throughput (HTP) mode for screening enzyme mutants was tested with Pseudomonas aeruginosa arylsulfatase (PAAS). The alkaline lysis buffer was 1.0 M Tris-HCl at pH 9.0 plus 0.1 % Tween-20 and 2.0 mM 4-aminobenzamidine, mixed with cell suspension at 8:1 to 12:1 ratio for continuous agitation of mixtures in 96-well plates under room temperature; enzymatic activity in lysates was measured with 96-well microplate. PAAS activity tolerated final 0.1 % Tween-20. Individual clones were amplified for 12 h in 0.50 mL TB medium with 48-well plates to enhance the repeatability of induced expression. During continuous agitation of the mixture of cells and the lysis buffer, PAAS activities in lysates were steady from 3 to 9 h and comparable to sonication treatment but better than freezing-thawing. Coefficients of variation of activities of PAAS/mutants in lysates after treatment for 7 h reached ∼22 %. The mutant M72Q had specific activity 2-fold of G138S. By HTP lysis of cells, M72Q was recognized as a positive mutant over G138S with the area under the curve of 0.873. Therefore, for enzymes tolerating concentrated alkaline buffers, the proposed alkaline lysis approach may be generally applicable for HTP lysis of host cells during directed evolution.

  5. Acute Tumor Lysis Syndrome Caused by Transcatheter Oily Chemoembolization in a Patient with a Large Hepatocellular Carcinoma

    SciTech Connect

    Sakamoto, Noriaki Monzawa, Shuichi; Nagano, Hidenobu; Nishizaki, Hogara; Arai, Yasuaki; Sugimura, Kazuro

    2007-06-15

    Acute tumor lysis syndrome results from a sudden and rapid release of products of cellular breakdown after anticancer therapy. Severe alterations of metabolic profile might occur and result in acute renal failure. We present a patient with a large hepatocellular carcinoma who received transcatheter oily chemoembolization and died subsequently of this syndrome. To our knowledge, there has been only one report of this syndrome induced by chemoembolization for hepatocellular carcinoma. This case illustrates the need to anticipate the development of acute tumor lysis syndrome when chemoembolization is planned for a large hepatocellular carcinoma.

  6. Programmed nuclear destruction in yeast: self-eating by vacuolar lysis.

    PubMed

    Eastwood, Michael D; Cheung, Sally W T; Meneghini, Marc D

    2013-02-01

    Studies of the budding yeast Saccharomyces cerevisiae have provided many of the most important insights into the mechanisms of autophagy, which are common to all eukaryotes. However, investigation of yeast self-destruction pathways, including autophagy and programmed cell death, has been almost exclusively restricted to cells undergoing vegetative growth, leaving very little exploration of their functions during developmental transitions in the yeast life cycle. We have recently discovered that whole nuclei are subject to programmed destruction during yeast gametogenesis. Programmed nuclear destruction (PND) possesses characteristics of apoptosis in the form of DNA cleavage by endonuclease G, and involves bulk protein turnover through an unusual autophagic pathway involving lysis of the vacuole rather than delivery of components to it through macroautophagy. We thus illuminate an example of developmentally programmed cellular "self-eating" in yeast, which is associated with the rupture of a lytic organelle, reminiscent of programmed cell death mechanisms in plants and animals.

  7. LYSIS OF BACTERIAL PROTOPLASTS AND SPHEROPLASTS BY STAPHYLOCOCCAL ALPHA-TOXIN AND STREPTOLYSIN S.

    PubMed

    BERNHEIMER, A W; SCHWARTZ, L L

    1965-05-01

    Bernheimer, Alan W. (New York University School of Medicine, New York, N.Y.), and Lois L. Schwartz. Lysis of bacterial protoplasts and spheroplasts by staphylococcal alpha-toxin and streptolysin S. J. Bacteriol. 89:1387-1392. 1965.-Protoplasts of Bacillus megaterium, Sarcina lutea, and Streptococcus pyogenes, and spheroplasts of Escherichia coli were lysed by staphylococcal alpha-toxin, whereas spheroplasts of Vibrio metschnikovii and V. comma were not. In the spectrum of its lytic action, streptolysin S qualitatively resembled staphylococcal alpha-toxin except for failure to lyse S. pyogenes protoplasts. In contrast to the two foregoing agents, streptolysin O did not lyse protoplasts and spheroplasts. The observations are interpreted in relation to similarities and differences in lipid composition of bacterial and mammalian cell membranes.

  8. Lysis of Escherichia coli by Ethylenediaminetetraacetate and Phospholipases as Measured by β-Galactosidase Activity

    PubMed Central

    Slein, Milton W.; Logan, Gerald F.

    1967-01-01

    A permeaseless mutant of Escherichia coli, which produces β-galactosidase constitutively, was treated briefly with ethylenediaminetetraacetate and then with the phospholipases of Bacillus cereus. Cell lysis occurred, as indicated by an increase in β-galactosidase activity and a decrease in absorbancy of the cell suspension. The susceptibility of the cells to attack by ethylenediaminetetraacetate and the phospholipases was markedly affected by the age of the cells when harvested. The results suggest that permeability changes may be associated with the activity of a phospholipase that specifically degrades phosphatidyl ethanolamine. A sonic-treatment method for determining the total β-galactosidase content of E. coli cells, which is independent of their age when harvested, is described. PMID:4168313

  9. Learning from the Jersey Turnpike:Cell Lysis, Labeling and Washing with Microfluidic Metamaterials

    NASA Astrophysics Data System (ADS)

    Loutherback, Kevin; Morton, Keith; Inglis, David; Tsui, Opheli; Sturm, James; Chou, Stephen; Austin, Robert

    2008-03-01

    Directing objects across functional streamlines at low Reynolds number is difficult but important since this motion can be used to label, lyse, and analyze complex biological objects on-chip without cross-contamination. Here we use an asymmeteric post array to move cells across coflowing reagents and show on-chip, immunofluorescent labeling of platelets with washing and E.Coli cell lysis with simultaneous separation of bacterial chromosome from the cell contents. Furthermore, we develop the concept of a microfluidic metamaterial by using the basic asymmetric post array as a building block for complex particle handling modes. These modular array elements could be of great use for developing robust techniques for on-chip, continuous flow manipulation and analysis of cells, large bio-particles, and functional beads.

  10. Learning from the Jersey Turnpike: Cell Lysis, Labeling and Washing with Microfluidic Metamaterials

    NASA Astrophysics Data System (ADS)

    Austin, Robert

    2008-03-01

    Directing objects across functional streamlines at low Reynolds number is difficult but important since this motion can be used to label, lyse, and analyze complex biological objects on-chip without cross-contamination. Here we use an asymmeteric post array to move cells across coflowing reagents and show on-chip, immunofluorescent labeling of platelets with washing and E.Coli cell lysis with simultaneous separation of bacterial chromosome from the cell contents. Furthermore, we develop the concept of a microfluidic metamaterial by using the basic asymmetric post array as a building block for complex particle handling modes. These modular array elements could be of great use for developing robust techniques for on-chip, continuous flow manipulation and analysis of cells, large bio-particles, and functional beads.

  11. Release of simian virus 40 virions from epithelial cells is polarized and occurs without cell lysis.

    PubMed Central

    Clayson, E T; Brando, L V; Compans, R W

    1989-01-01

    We have investigated the process of release of simian virus 40 (SV40) virions from several monkey kidney cell lines. High levels of virus release were observed prior to any significantly cytopathic effects in all cell lines examined, indicating that SV40 utilizes a mechanism for escape from the host cell which does not involve cell lysis. We demonstrate that SV40 release was polarized in two epithelial cell types (Vero C1008 and primary African green monkey kidney cells) grown on permeable supports; release of virus occurs almost exclusively at apical surfaces. In contrast, equivalent amounts of SV40 virions were recovered from apical and basal culture fluids of nonpolarized CV-1 cells. SV40 virions were observed in large numbers on apical surfaces of epithelial cells and in cytoplasmic smooth membrane vesicles. The sodium ionophore monensin, an inhibitor of vesicular transport, was found to inhibit SV40 release without altering viral protein synthesis or infectious virus production. Images PMID:2539518

  12. Dual Targeting of Cell Wall Precursors by Teixobactin Leads to Cell Lysis

    PubMed Central

    Homma, Tomoyuki; Nuxoll, Austin; Gandt, Autumn Brown; Ebner, Patrick; Engels, Ina; Schneider, Tanja; Götz, Friedrich; Lewis, Kim

    2016-01-01

    Teixobactin represents the first member of a newly discovered class of antibiotics that act through inhibition of cell wall synthesis. Teixobactin binds multiple bactoprenol-coupled cell wall precursors, inhibiting both peptidoglycan and teichoic acid synthesis. Here, we show that the impressive bactericidal activity of teixobactin is due to the synergistic inhibition of both targets, resulting in cell wall damage, delocalization of autolysins, and subsequent cell lysis. We also find that teixobactin does not bind mature peptidoglycan, further increasing its activity at high cell densities and against vancomycin-intermediate Staphylococcus aureus (VISA) isolates with thickened peptidoglycan layers. These findings add to the attractiveness of teixobactin as a potential therapeutic agent for the treatment of infection caused by antibiotic-resistant Gram-positive pathogens. PMID:27550357

  13. Hyphal Growth of Phagocytosed Fusarium oxysporum Causes Cell Lysis and Death of Murine Macrophages

    PubMed Central

    Schäfer, Katja; Bain, Judith M.

    2014-01-01

    Fusarium oxysporum is an important plant pathogen and an opportunistic pathogen of humans. Here we investigated phagocytosis of F. oxysporum by J774.1 murine cell line macrophages using live cell video microscopy. Macrophages avidly migrated towards F. oxysporum germlings and were rapidly engulfed after cell-cell contact was established. F. oxysporum germlings continued hyphal growth after engulfment by macrophages, leading to associated macrophage lysis and escape. Macrophage killing depended on the multiplicity of infection. After engulfment, F. oxysporum inhibited macrophages from completing mitosis, resulting in large daughter cells fused together by means of a F. oxysporum hypha. These results shed new light on the initial stages of Fusarium infection and the innate immune response of the mammalian host. PMID:25025395

  14. Hyphal growth of phagocytosed Fusarium oxysporum causes cell lysis and death of murine macrophages.

    PubMed

    Schäfer, Katja; Bain, Judith M; Di Pietro, Antonio; Gow, Neil A R; Erwig, Lars P

    2014-01-01

    Fusarium oxysporum is an important plant pathogen and an opportunistic pathogen of humans. Here we investigated phagocytosis of F. oxysporum by J774.1 murine cell line macrophages using live cell video microscopy. Macrophages avidly migrated towards F. oxysporum germlings and were rapidly engulfed after cell-cell contact was established. F. oxysporum germlings continued hyphal growth after engulfment by macrophages, leading to associated macrophage lysis and escape. Macrophage killing depended on the multiplicity of infection. After engulfment, F. oxysporum inhibited macrophages from completing mitosis, resulting in large daughter cells fused together by means of a F. oxysporum hypha. These results shed new light on the initial stages of Fusarium infection and the innate immune response of the mammalian host.

  15. Spontaneous tumour lysis syndrome associated with contrast dye iohexol use in mantle cell lymphoma.

    PubMed

    Yun, Seongseok; Vincelette, Nicole D; Phan, Tuan; Anwer, Faiz

    2014-07-15

    We describe a case of a 73-year-old man who presented with right-sided abdominal pain associated with palpable mass. Initial laboratory examination was normal except lactate dehydrogenase level. Subsequent CT image showed situs inversus and splenic mass with multiple lymph nodes enlargement. Biopsy taken from the splenic mass demonstrated mantle cell lymphoma. Staging CT examination was performed with intravenous contrast, and patient developed altered mental status, respiratory failure and acute kidney injury requiring intensive care unit care. Laboratory examination revealed hyperuricaemia, hyperphosphataemia, hyperkalaemia and hypocalcaemia, which are consistent with spontaneous tumour lysis syndrome. The patient was successfully treated with rasburicase and haemodialysis, and completed the first course of chemotherapy without further complications.

  16. Citrullination in Rheumatoid Arthritis—A Process Promoted by Neutrophil Lysis?

    PubMed Central

    Gazitt, Tal; Lood, Christian; Elkon, Keith B.

    2016-01-01

    Anti-citrullinated protein antibodies (ACPAs) are highly specific serologic markers for rheumatoid arthritis (RA) and can pre-date clinical disease onset by up to 10 years, also predicting erosive disease. The process of citrullination, the post-translational conversion of arginine to citrulline residues, is mediated by peptidylarginine deiminase (PAD) enzymes present in polymorphonuclear cells (PMNs). Calcium ions (Ca2+) are required for PAD activation, but the intracellular Ca2+ concentration in normal cells is much lower than the optimal Ca2+ concentration needed for PAD activation. For this reason, it has been proposed that PAD activation, and thus citrullination, occurs only during PMN cell death when PAD enzymes leak out of the cells into the extracellular matrix, or extracellular Ca2+ enters the cells, with the high Ca2+ concentration activating PAD. Recently, using artificial in vitro systems to corroborate their hypothesis, Romero et al. demonstrated that “hypercitrullination,” citrullination of multiple intracellular proteins, occurs within synovial fluid (SF) cells of RA patients, and that only modes of death leading to membranolysis such as perforin-granzyme pathway or complement membrane attack complex activation cause hypercitrullination. In order for Romero’s hypothesis to hold, it is reasonable to surmise that PMN-directed lysis should occur in the rheumatoid joint or the circulation of RA patients. Research conducted thus far has shown that immunoglobulin G (IgG) targeting PMNs are present in RA SF and mediate PMN activation. However, the role of anti-PMN IgG in mediating complement activation and subsequent PMN lysis and hypercitrullination has not been fully evaluated. PMID:27824546

  17. Sustainable microbial water quality monitoring programme design using phage-lysis and multivariate techniques.

    PubMed

    Nnane, Daniel Ekane

    2011-11-15

    Contamination of surface waters is a pervasive threat to human health, hence, the need to better understand the sources and spatio-temporal variations of contaminants within river catchments. River catchment managers are required to sustainably monitor and manage the quality of surface waters. Catchment managers therefore need cost-effective low-cost long-term sustainable water quality monitoring and management designs to proactively protect public health and aquatic ecosystems. Multivariate and phage-lysis techniques were used to investigate spatio-temporal variations of water quality, main polluting chemophysical and microbial parameters, faecal micro-organisms sources, and to establish 'sentry' sampling sites in the Ouse River catchment, southeast England, UK. 350 river water samples were analysed for fourteen chemophysical and microbial water quality parameters in conjunction with the novel human-specific phages of Bacteroides GB-124 (Bacteroides GB-124). Annual, autumn, spring, summer, and winter principal components (PCs) explained approximately 54%, 75%, 62%, 48%, and 60%, respectively, of the total variance present in the datasets. Significant loadings of Escherichia coli, intestinal enterococci, turbidity, and human-specific Bacteroides GB-124 were observed in all datasets. Cluster analysis successfully grouped sampling sites into five clusters. Importantly, multivariate and phage-lysis techniques were useful in determining the sources and spatial extent of water contamination in the catchment. Though human faecal contamination was significant during dry periods, the main source of contamination was non-human. Bacteroides GB-124 could potentially be used for catchment routine microbial water quality monitoring. For a cost-effective low-cost long-term sustainable water quality monitoring design, E. coli or intestinal enterococci, turbidity, and Bacteroides GB-124 should be monitored all-year round in this river catchment.

  18. Enhanced ultrasound-induced apoptosis and cell lysis by a hypotonic medium.

    PubMed

    Feril, L B; Kondo, T; Takaya, K; Riesz, P

    2004-02-01

    To test the hypothesis that non-lethal hypotonia will enhance ultrasound-induced cell killing in vitro and that the mechanism is mechanical in nature. Hypotonic RPMI medium (146 mOsm) was used to induce non-lethal osmotic swelling of human myelomonocytic leukaemia U937 cells. Hypotonia for 10 min was started just before exposure to 1 MHz ultrasound at 0.5 or 1.0 Wcm(-2) for 10 min, or 5 min before exposure to 2.0 Wcm(-2) for 1 min. Surviving intact cells were then determined by the trypan blue dye exclusion test immediately after treatment. After 6-h incubation of the treated cells, early apoptosis and secondary necrosis were measured using a flow cytometer. Intracellular free calcium ion imaging by Fura-2 fluorescence and cellular ion scanning using a secondary ion mass spectrometer were also performed. Enhancement of ultrasound-induced cell lysis was observed at all intensities, and most prominently at 2.0 Wcm(-2), while apoptosis induction was significantly enhanced at intensities of 0.5 and 1.0 Wcm(-2), but not at 2.0 Wcm(-2). The enhanced cell lysis is attributed to the increased susceptibility of the cells to mechanical damage. This is consistent with previous reports describing the effects of mechanical stresses on cell membranes. Cellular ion scanning images also suggest that hypotonia has an effect on the membrane damage-and-repair mechanism of the cells. The results support the hypothesis that non-lethal hypotonia can enhance ultrasound-induced cell killing. These findings also suggest the 'sonomechanical' nature of the effects on the cells.

  19. Effect of spermine on host-cell lysis and reproduction by a lactic streptococcal bacteriophage.

    PubMed

    Erskine, J M

    1970-04-01

    A method was tested for protecting a Streptococcus lactis strain, ML3, used as a starter in the manufacture of Cheddar cheese, against the lytic activity of its homologous phage, ml(3). At a concentration of 10(-2)m, a naturally occurring polyamine, spermine, in the form of its hydrochloride, protected ML3 against lysis-from-without and lysozyme activity and against lysis by the phage when added at the time of infection or up to 21 min after infection. It was found that the latter protective effect could be accounted for in terms of the spermine preventing the formation of mature particles rather than preventing the escape of viable phage. Single colonies selected from a culture of ML3 cells that had been previously infected with phage ml(3), in the presence of spermine, were all found to have acquired resistance to phage ml(3). They retained this resistance during a 3-month period of daily subculture in broth and, in the absence of spermine, could not be induced to liberate phage or phage components either by the techniques normally used for inducing lysogens or by artificial disruption of the cells. It is concluded that when spermine is added to ML3 cells before a certain critical stage of the phage infection cycle, the process of phage synthesis is irreversibly halted and the cells retain the infecting phage as a defective prophage that confers on the cells immunity to infection by the homologous phage. Phage-resistant cultures did not inherit reduced starter activity in association with their acquired resistance characteristic.

  20. Comparative analysis shows that bacterivory, not viral lysis, controls the abundance of heterotrophic prokaryotic plankton.

    PubMed

    Pedrós-Alió; Calderón-Paz; Gasol

    2000-04-01

    Empirical models derived from literature data were used to compare the factors controlling prokaryotic abundance (PN) and prokaryotic heterotrophic production (PHP) in solar salterns. These empirical relationships were generated as multiple linear regressions with PN or PHP as dependent variables, while the independent variables were chosen to reflect the likely sources of organic matter, inorganic nutrients and temperature. These variables were then measured in solar salterns and the predictions made by the general relationships were compared to actual saltern values of PN and PHP. Saltern ponds of salinity higher than 100 per thousand departed significantly from the general relationships, while the ponds of salinity lower than 100 per thousand fitted well within the range of values predicted by the general models. The most likely explanation for the discrepancy of the former was the absence of bacterivory. This hypothesis was tested with data from other very different aquatic systems: karstic lakes with anaerobic hypolimnia and two marine areas in the Mediterranean and the Southern Ocean. The anoxic regions of karstic lakes departed significantly from the predictions of the general model, while the oxic layers conformed to the predictions. As in the case of salterns, this difference could be explained by the presence of significant predation in the oxic, but not in the anoxic, layers of these lakes. Finally, two marine areas with similar predation pressure on prokaryotes but very different impacts of viral lysis were tested. In all cases, PN values conformed to the predictions, suggesting that lysis due to viruses is not the main factor controlling PN in aquatic systems, which is more likely to be determined by the balance between bacterivory and resource supply. The present work also demonstrates the usefulness of empirical comparative analyses to generate predictions and to draw inferences on the functioning of microbial communities.

  1. Perforin enhances the granulysin-induced lysis of Listeria innocua in human dendritic cells

    PubMed Central

    Walch, Michael; Latinovic-Golic, Sonja; Velic, Ana; Sundstrom, Hanna; Dumrese, Claudia; Wagner, Carsten A; Groscurth, Peter; Ziegler, Urs

    2007-01-01

    Background Cytotoxic T lymphocytes (CTL) and natural killer (NK) cells play an essential role in the host defence against intracellular pathogens such as Listeria, and Mycobacteria. The key mediator of bacteria-directed cytotoxicity is granulysin, a 9 kDa protein stored in cytolytic granules together with perforin and granzymes. Granulysin binds to cell membranes and is subsequently taken up via a lipid raft-associated mechanism. In dendritic cells (DC) granulysin is further transferred via early endosomes to L. innocua-containing phagosomes were bacteriolysis is induced. In the present study we analysed the role of perforin in granulysin-induced intracellular bacteriolysis in DC. Results We found granulysin-induced lysis of intracellular Listeria significantly increased when perforin was simultaneously present. In pulse-chase experiments enhanced bacteriolysis was observed when perforin was added up to 25 minutes after loading the cells with granulysin demonstrating no ultimate need for simultaneous uptake of granulysin and perforin. The perforin concentration sufficient to enhance granulysin-induced intracellular bacteriolysis did not cause permanent membrane pores in Listeria-challenged DC as shown by dye exclusion test and LDH release. This was in contrast to non challenged DC that were more susceptible to perforin lysis. For Listeria-challenged DC, there was clear evidence for an Ca2+ influx in response to sublytic perforin demonstrating a short-lived change in the plasma membrane permeability. Perforin treatment did not affect granulysin binding, initial uptake or intracellular trafficking to early endosomes. However, enhanced colocalization of granulysin with listerial DNA in presence of perforin was found by confocal laser scanning microscopy. Conclusion The results provide evidence that perforin increases granulysin-mediated killing of intracellular Listeria by enhanced phagosome-endosome fusion triggered by a transient Ca2+ flux. PMID:17705829

  2. A microfluidic device for continuous white blood cell separation and lysis from whole blood.

    PubMed

    Kim, Myounggon; Mo Jung, Seung; Lee, Kyeong-Hwan; Jun Kang, Yang; Yang, Sung

    2010-11-01

    A microfluidic device, which is composed of a blood inlet, a cell lysis solution inlet, a bifurcation outlet containing six microchannels, and a white blood cell (WBC)-lysed solution outlet, is proposed in this study to separate WBCs from whole blood and lyse the WBCs in a continuous and near real-time fashion. The geometry of the microfluidic device is determined based on the bifurcation law and a cell crossover method. The microflow patterns of blood cells in the microfluidic channels are simulated by computational fluid dynamics. The simulation results agree with the experiment results by considering the reduction of blood viscosity in the microfluidic channels. The performance of the microfluidic device is evaluated by investigating the WBC recovery efficiency and the ratio of spectrophotometric absorbance of the blood sample at 260 to that at 280nm. The WBC recovery efficiency at the main channel outlet is 97.2%. The measured spectrophotometric absorbance ratio of 1.82 indicates that the separated WBCs are completely lysed, leaving only pure DNA in the WBC-lysed solution. The continuous cell separation and lysis is completed within only 0.5s. Therefore, it is concluded that the proposed microfluidic device is promising for separating WBCs from whole blood without any pretreatment and lysing the WBCs in a continuous and near real-time fashion. The proposed microfluidic device may be applicable to a lab-on-a-chip for blood analysis. © 2010, Copyright the Authors. Artificial Organs © 2010, International Center for Artificial Organs and Transplantation and Wiley Periodicals, Inc.

  3. Continuous nucleus extraction by optically-induced cell lysis on a batch-type microfluidic platform.

    PubMed

    Huang, Shih-Hsuan; Hung, Lien-Yu; Lee, Gwo-Bin

    2016-04-21

    The extraction of a cell's nucleus is an essential technique required for a number of procedures, such as disease diagnosis, genetic replication, and animal cloning. However, existing nucleus extraction techniques are relatively inefficient and labor-intensive. Therefore, this study presents an innovative, microfluidics-based approach featuring optically-induced cell lysis (OICL) for nucleus extraction and collection in an automatic format. In comparison to previous micro-devices designed for nucleus extraction, the new OICL device designed herein is superior in terms of flexibility, selectivity, and efficiency. To facilitate this OICL module for continuous nucleus extraction, we further integrated an optically-induced dielectrophoresis (ODEP) module with the OICL device within the microfluidic chip. This on-chip integration circumvents the need for highly trained personnel and expensive, cumbersome equipment. Specifically, this microfluidic system automates four steps by 1) automatically focusing and transporting cells, 2) releasing the nuclei on the OICL module, 3) isolating the nuclei on the ODEP module, and 4) collecting the nuclei in the outlet chamber. The efficiency of cell membrane lysis and the ODEP nucleus separation was measured to be 78.04 ± 5.70% and 80.90 ± 5.98%, respectively, leading to an overall nucleus extraction efficiency of 58.21 ± 2.21%. These results demonstrate that this microfluidics-based system can successfully perform nucleus extraction, and the integrated platform is therefore promising in cell fusion technology with the goal of achieving genetic replication, or even animal cloning, in the near future.

  4. Stabilizing Additives Added during Cell Lysis Aid in the Solubilization of Recombinant Proteins

    PubMed Central

    Leibly, David J.; Nguyen, Trang Nhu; Kao, Louis T.; Hewitt, Stephen N.; Barrett, Lynn K.; Van Voorhis, Wesley C.

    2012-01-01

    Insoluble recombinant proteins are a major issue for both structural genomics and enzymology research. Greater than 30% of recombinant proteins expressed in Escherichia coli (E. coli) appear to be insoluble. The prevailing view is that insolubly expressed proteins cannot be easily solubilized, and are usually sequestered into inclusion bodies. However, we hypothesize that small molecules added during the cell lysis stage can yield soluble protein from insoluble protein previously screened without additives or ligands. We present a novel screening method that utilized 144 additive conditions to increase the solubility of recombinant proteins expressed in E. coli. These selected additives are natural ligands, detergents, salts, buffers, and chemicals that have been shown to increase the stability of proteins in vivo. We present the methods used for this additive solubility screen and detailed results for 41 potential drug target recombinant proteins from infectious organisms. Increased solubility was observed for 80% of the recombinant proteins during the primary and secondary screening of lysis with the additives; that is 33 of 41 target proteins had increased solubility compared with no additive controls. Eleven additives (trehalose, glycine betaine, mannitol, L-Arginine, potassium citrate, CuCl2, proline, xylitol, NDSB 201, CTAB and K2PO4) solubilized more than one of the 41 proteins; these additives can be easily screened to increase protein solubility. Large-scale purifications were attempted for 15 of the proteins using the additives identified and eight (40%) were prepared for crystallization trials during the first purification attempt. Thus, this protocol allowed us to recover about a third of seemingly insoluble proteins for crystallography and structure determination. If recombinant proteins are required in smaller quantities or less purity, the final success rate may be even higher. PMID:23285060

  5. A comparison of different pre-lysis methods and extraction kits for recovery of Streptococcus agalacticae (Lancefield group B Streptococcus) DNA from whole blood.

    PubMed

    Burke, Rachael M; McKenna, James P; Cox, Ciara; Coyle, Peter V; Shields, Michael D; Fairley, Derek J

    2016-10-01

    Sub-optimal recovery of bacterial DNA from whole blood samples can limit the sensitivity of molecular assays to detect pathogenic bacteria. We compared 3 different pre-lysis protocols (none, mechanical pre-lysis and achromopeptidase pre-lysis) and 5 commercially available DNA extraction platforms for direct detection of Group B Streptococcus (GBS) in spiked whole blood samples, without enrichment culture. DNA was extracted using the QIAamp Blood Mini kit (Qiagen), UCP Pathogen Mini kit (Qiagen), QuickGene DNA Whole Blood kit S (Fuji), Speed Xtract Nucleic Acid Kit 200 (Qiagen) and MagNA Pure Compact Nucleic Acid Isolation Kit I (Roche Diagnostics Corp). Mechanical pre-lysis increased yields of bacterial genomic DNA by 51.3 fold (95% confidence interval; 31.6-85.1, p<0.001) and pre-lysis with achromopeptidase by 6.1 fold (95% CI; 4.2-8.9, p<0.001), compared with no pre-lysis. Differences in yield due to pre-lysis were 2-3 fold larger than differences in yield between extraction methods. Including a pre-lysis step can improve the limits of detection of GBS using PCR or other molecular methods without need for culture.

  6. Hsp90 inhibition accelerates cell lysis. Anti-Hsp90 ribozyme reveals a complex mechanism of Hsp90 inhibitors involving both superoxide- and Hsp90-dependent events.

    PubMed

    Sreedhar, Amere Subbarao; Mihály, Katalin; Pató, Bálint; Schnaider, Tamás; Steták, Attila; Kis-Petik, Katalin; Fidy, Judit; Simonics, Tibor; Maraz, Anna; Csermely, Péter

    2003-09-12

    The 90 kDa heat shock protein, Hsp90, is an abundant molecular chaperone participating in the cytoprotection of eukaryotic cells. Here we analyzed the involvement of Hsp90 in the maintenance of cellular integrity using partial cell lysis as a measure. Inhibition of Hsp90 by geldanamycin, radicicol, cisplatin, and novobiocin induced a significant acceleration of detergent- and hypotonic shock-induced cell lysis. The concentration and time dependence of cell lysis acceleration was in agreement with the Hsp90 inhibition characteristics of the N-terminal inhibitors, geldanamycin and radicicol. Glutathione and other reducing agents partially blocked geldanamycin-induced acceleration of cell lysis but were largely ineffective with other inhibitors. Indeed, geldanamycin treatment led to superoxide production and a change in membrane fluidity. When Hsp90 content was diminished using anti-Hsp90 hammerhead ribozymes, an accelerated cell lysis was also observed. Hsp90 inhibition-induced cell lysis was more pronounced in eukaryotic (yeast, mouse red blood, and human T-lymphoma) cells than in bacteria. Our results indicate that besides the geldanamycin-induced superoxide production, and a consequent increase in cell lysis, inhibition or lack of Hsp90 alone can also compromise cellular integrity. Moreover, cell lysis after hypoxia and complement attack was also enhanced by any type of Hsp90 inhibition used, which shows that the maintenance of cellular integrity by Hsp90 is important in physiologically relevant lytic conditions of tumor cells.

  7. The dual role of paramagnetic particles for integrated lysis and measurement in a rapid immunoassay for intracellular proteins.

    PubMed

    Sharif, Elham; Kiely, Janice; Wraith, Patrick; Luxton, Richard

    2013-05-01

    A novel, integrated lysis and immunoassay methodology and system for intracellular protein measurement are described. The method uses paramagnetic particles both as a lysis agent and assay label resulting in a rapid test requiring minimal operator intervention, the test being homogeneous and completed in less than 10 min. A design study highlights the critical features of the magnetic detection system used to quantify the paramagnetic particles and a novel frequency-locked loop-based magnetometer is presented. A study of paramagnetic particle enhanced lysis demonstrates that the technique is more than twice as efficient at releasing intracellular protein as ultrasonic lysis alone. Results are presented for measurements of intracellular prostate specific antigen in an LNCAP cell line. This model was selected to demonstrate the rapidity and efficiency of intracellular protein quantification. It was shown that, on average, LNCAP cells contained 0.43 fg of prostate specific antigen. This system promises an attractive solution for applications that require a rapid determination of intracellular proteins.

  8. Minimal requirements for inhibition of MraY by lysis protein E from bacteriophage ΦX174.

    PubMed

    Tanaka, Shiho; Clemons, William M

    2012-09-01

    The DNA phage ΦX174 encodes the integral membrane protein E whose expression leads to host cell lysis by inhibition of the peptidoglycan synthesis enzyme MraY. Here we use mutagenesis to characterize the molecular details of the E lysis mechanism. We find that a minimal 18-residue region with the modified wild-type sequences of the conserved transmembrane helix of E is sufficient to lyse host cells and that specific residues within and at the boundaries of this helix are important for activity. This suggests that positioning of the helix in the membrane is critical for interactions with MraY. We further characterize the interaction site of the transmembrane helix with MraY demonstrating E forms a stable complex with MraY. Triggering cell lysis by peptidoglycan synthesis inhibition is a traditional route for antimicrobial strategies. Understanding the mechanism of bacterial cell lysis by E will provide insights into new antimicrobial strategies using re-engineered E peptides. © 2012 Blackwell Publishing Ltd.

  9. Minimal requirements for inhibition of MraY by lysis protein E from bacteriophage ΦX174

    PubMed Central

    Tanaka, Shiho; Clemons, William M.

    2012-01-01

    Summary The DNA phage ΦX174 encodes the integral membrane protein E whose expression leads to host cell lysis by inhibition of the peptidoglycan synthesis enzyme MraY. Here we use mutagenesis to characterize the molecular details of the E lysis mechanism. We find that a minimal 18-residue region with the modified wild-type sequences of the conserved transmembrane helix of E is sufficient to lyse host cells and that specific residues within and at the boundaries of this helix are important for activity. This suggests that positioning of the helix in the membrane is critical for interactions with MraY. We further characterize the interaction site of the transmembrane helix with MraY demonstrating E forms a stable complex with MraY. Triggering cell lysis by peptidoglycan synthesis inhibition is a traditional route for antimicrobial strategies. Understanding the mechanism of bacterial cell lysis by E will provide insights into new antimicrobial strategies using re-engineered E peptides. PMID:22742425

  10. Use of pressure cycling technology for cell lysis and recovery of bacterial and fungal communities from soil

    USDA-ARS?s Scientific Manuscript database

    Current molecular methodologies, specifically DNA-based approaches, provide access to previously hidden soil biodiversity and are routinely employed in environmental studies of microbial ecology. Selection of cell lysis methodology is critical to community analyses due to the inability of any singul...

  11. Rapid isolation of nuclei from living immune cells by a single centrifugation through a multifunctional lysis gradient.

    PubMed

    Poglitsch, Marko; Katholnig, Karl; Säemann, Marcus D; Weichhart, Thomas

    2011-10-28

    Due to their low protein content and limited nuclear detergent stability, primary human immune cells such as monocytes or T lymphocytes represent a great challenge for standard nuclear isolation protocols. Nuclei clumping during the multiple centrifugation steps or contamination of isolated nuclei with cytoplasmic proteins due to membrane lysis is a frequently observed problem. Here we describe a versatile and novel method for the isolation of clean and intact nuclei from primary human monocytes, which can be applied for virtually any cell type. Living cells were applied on an iso-osmolar discontinuous iodixanol-based density gradient including a detergent-containing lysis layer. Mild cell lysis as well as efficient washing of the nuclei was performed during the course of one single low g-force centrifugation step. The isolation procedure, which we call lysis gradient centrifugation (LGC), results in the recovery of 90-95% of highly pure nuclei. This easy and highly reproducible procedure allows an effective preparation of nuclei and the cytoplasm in only 15 min with the ability to handle as little as one million cells per sample and easy parallel processing of multiple samples.

  12. Reduction of excess sludge production in sequencing batch reactor (SBR) by lysis-cryptic growth using homogenization disruption.

    PubMed

    Lan, Wangcheng; Li, Yiyong; Bi, Qi; Hu, Yongyou

    2013-04-01

    A lysis-cryptic growth system, which combined high-pressure-homogenization (HPH) for sludge disruption, was proposed to reduce excess sludge production in SBR. Experimental data was analyzed with the aid of response surface models to determine the optimal HPH disruption pressure, which was 70 MPa. By combining a 5.4 m3/d pilot SBR with HPH disruption, the new system achieved a 42.4% sludge reduction rate over a 75 days operation. Based on measurement of oxygen uptake rate and activity of the dehydrogenase, the lysis-cryptic growth system resulted in negligible change of the sludge activity. However, an increase of 0.04 mg/L of total-phosphorus (TP) and 2.40 mg/L suspended-solids (SS) was observed in the effluent due to the process of lysis-cryptic growth. Except for above listed points, the new system demonstrated improved sludge reduction performance while the direct cost of pilot SBR lysis-cryptic growth was only 0.177US dollar per kilogram (dry sludge) according to estimation.

  13. [Effect of lysis of acupotomology on plasma vasoactive substance levels in rats with third lumbar vertebra transverse process syndrome].

    PubMed

    Guo, Chang-Qing; Liu, Nai-Gang; Li, Xiao-Hong; Sun, Hong-Mei; Lu, Jing; Ma, Hui-Fang; Chen, Zhan-Lu; Hu, Bo; Liu, Lin; Zhu, Han-Zhang

    2007-10-01

    To observe the effect of small needle-knife lysis on plasma calcitonin gene-related peptide (CGRP), endothelin (ET), 6-keto-prostaglandin F1alpha (6-keto-PGF1alpha), thromboxane A2 (TXA2) contents in rats with experimental third lumbar vertebra transverse process syndrome (TLVTPS) so as to explore its underlying mechanism in clinical treatment. Forty SD rats were randomly divided into normal control, model, lysis and EA groups. TLVTPS model was established by embedding a piece of gelatin sponge (0.5 cm x 0.5 cm) to the transverse process of the 3rd lumbar vertebra under anesthesia. EA (2/100 Hz, 1-2 mA) was applied to left "Shenshu" (BL23) -"Yaoyangguan" (GV3) for 20 min, once every other day, 6 times altogether. For animals of lysis group, the lysis was performed by using a small needle-knife in the induration spot or cord-like region near the incision, once a week and twice altogether. Four weeks later after modeling, plasma CGRP, ET, 6-keto-PGF1alpha and TXA2 contents were detected by using radioimmunoassay and enzyme linked immunosorbent assay (ELISA). Compared with normal control group, plasma CGRP, ET, TXA2 and 6-keto-PGF1alpha increased significantly in model group (P<0.01); in comparison with model group, plasma CGRP, TXA2 and 6-keto-PGF1alpha in both EA and lysis groups decreased considerably (P<0.05, 0.01). No significant differences were found between EA and lysis groups in plasma CGRP, ET and 6-keto-PGF1alpha levels (P>0.05). Both EA and lysis of acupotomology have an adjusting effect on vasoactive substances (CGRP, TXA2 and 6-keto-PGF1alpha) levels in TLVTPS rats, which may contribute to their effects in improving local blood circulation and relieving soft tissue injury in the treatment of third lumbar vertebra transverse process syndrome.

  14. A single lysis solution for the analysis of tissue samples by different proteomic technologies.

    PubMed

    Gromov, Pavel; Celis, Julio E; Gromova, Irina; Rank, Fritz; Timmermans-Wielenga, Vera; Moreira, José M A

    2008-12-01

    Cancer, being a major healthcare concern worldwide, is one of the main targets for the application of emerging proteomic technologies and these tools promise to revolutionize the way cancer will be diagnosed and treated in the near future. Today, as a result of the unprecedented advances that have taken place in molecular biology, cell biology and genomics there is a pressing need to accelerate the translation of basic discoveries into clinical applications. This need, compounded by mounting evidence that cellular model systems are unable to fully recapitulate all biological aspects of human dissease, is driving scientists to increasingly use clinically relevant samples for biomarker and target discovery. Tissues are heterogeneous and as a result optimization of sample preparation is critical for generating accurate, representative, and highly reproducible quantitative data. Although a large number of protocols for preparation of tissue lysates has been published, so far no single recipe is able to provide a "one-size fits all" solubilization procedure that can be used to analyse the same lysate using different proteomics technologies. Here we present evidence showing that cell lysis buffer 1 (CLB1), a lysis solution commercialized by Zeptosens [a division of Bayer (Schweiz) AG], provides excellent sample solubilization and very high 2D PAGE protein resolution both when using carrier ampholytes and immobilized pH gradient strips. Moreover, this buffer can also be used for array-based proteomics (reverse-phase lysate arrays or direct antibody arrays), allowing the direct comparison of qualitative and quantitative data yielded by these technologies when applied to the same samples. The usefulness of the CLB1 solution for gel-based proteomics was further established by 2D PAGE analysis of a number of technically demanding specimens such as breast carcinoma core needle biopsies and problematic tissues such as brain cortex, cerebellum, skeletal muscle, kidney cortex and

  15. Quorum-Sensing-Negative (lasR) Mutants of Pseudomonas aeruginosa Avoid Cell Lysis and Death

    PubMed Central

    Heurlier, Karin; Dénervaud, Valérie; Haenni, Marisa; Guy, Lionel; Krishnapillai, Viji; Haas, Dieter

    2005-01-01

    In Pseudomonas aeruginosa, N-acylhomoserine lactone signals regulate the expression of several hundreds of genes, via the transcriptional regulator LasR and, in part, also via the subordinate regulator RhlR. This regulatory network termed quorum sensing contributes to the virulence of P. aeruginosa as a pathogen. The fact that two supposed PAO1 wild-type strains from strain collections were found to be defective for LasR function because of independent point mutations in the lasR gene led to the hypothesis that loss of quorum sensing might confer a selective advantage on P. aeruginosa under certain environmental conditions. A convenient plate assay for LasR function was devised, based on the observation that lasR mutants did not grow on adenosine as the sole carbon source because a key degradative enzyme, nucleoside hydrolase (Nuh), is positively controlled by LasR. The wild-type PAO1 and lasR mutants showed similar growth rates when incubated in nutrient yeast broth at pH 6.8 and 37°C with good aeration. However, after termination of growth during 30 to 54 h of incubation, when the pH rose to ≥ 9, the lasR mutants were significantly more resistant to cell lysis and death than was the wild type. As a consequence, the lasR mutant-to-wild-type ratio increased about 10-fold in mixed cultures incubated for 54 h. In a PAO1 culture, five consecutive cycles of 48 h of incubation sufficed to enrich for about 10% of spontaneous mutants with a Nuh− phenotype, and five of these mutants, which were functionally complemented by lasR+, had mutations in lasR. The observation that, in buffered nutrient yeast broth, the wild type and lasR mutants exhibited similar low tendencies to undergo cell lysis and death suggests that alkaline stress may be a critical factor providing a selective survival advantage to lasR mutants. PMID:15995202

  16. Highly Efficient Microscale Purification of Glycerophospholipids by Microfluidic Cell Lysis and Lipid Extraction for Lipidomics Profiling

    PubMed Central

    Sun, Tao; Pawlowski, Sean; Johnson, Mitchell E.

    2011-01-01

    This article presents a novel method for small-scale lipidomics of bacterial cells by integrating extraction of glycerophospholipids on microchip with a nanoelectrospray ionization quadrupole time-of-flight tandem mass spectrometer (nanoESI-Q-TOF MS/MS). The standard starting point for typical macroscale lipid analysis is a multiphase liquid-liquid extraction. Working with small populations of cells (1 to about 1000) requires a scaled down process in order to minimize dilution and facilitate the interface with microscale separation methods for sample cleanup and introduction to mass spectrometry. We have developed a microfluidic system that allows for lysis of bacterial cells, capture of lipids, and elution of captured lipids from a solid phase for microscale purification of lipids. The best on-chip extraction efficiency for glycerophospholipids was as high as 83.3% by integrating silica beads as the packing material with methanol as the eluent. Ten successive measurements were evaluated indicating that the microchip packed with fresh silica beads is capable of being reused for four times without any loss in lipid extraction process. The initial screening based on high-resolution tandem mass spectrometry data along with discovery profiling approach revealed the presence of 173 identified phospholipid species from microfluidic cell extracts. This work demonstrates the potential of incorporating microchip-based lipid extraction into cellular lipidomics research. PMID:21766805

  17. Events leading to cell death and lysis of Neisseria meningitidis in low concentrations of penicillin G.

    PubMed Central

    Neirinck, L G; DeVoe, I W; Ingram, J M

    1980-01-01

    Neisseria meningitidis SD1C exhibited a low tolerance to penicillin G (0.03 microgram/ml). Loss of viability in the absence of polyvinylpyrrolidone-40 and horse serum was independent of the concentration of antibiotic above the minimum inhibitory concentration, whereas the rate of bacteriolysis was concentration dependent. Penicillin-induced lysis was a secondary event in this organism. At low levels of penicillin G, growth characteristics, i.e., absorbancy changes, respiratory rate, and uptake of Mg2+, appeared normal during the first 90 min in penicillin; however, viability dropped dramatically. Additionally, total cell numbers remained constant while cell mass continued to increase at a rate normal for the population. The increase in cellular mass in the absence of cell division could be observed microscopically. Only one ultrastructural change induced by penicillin correlated with the loss in viability: the loss in continuity of the outer membrane with the peptidoglycan but only at the site of septum formation. This lesion did not occur when cells were grown in media supplemented with the protective agents polyvinylpyrrolidone-40 and horse serum. Under these conditions of growth and with relatively high levels of penicillin, constant viability was maintained, but cell division no longer occurred. Cell populations treated with penicillin in the presence of the protective agents became increasingly more dependent on the presence of these agents for total viability even in the absence of penicillin in the culture. Images PMID:6772094

  18. Death and Lysis of Leptospirae When Cultured in Asbestos-Filtered Growth Media

    PubMed Central

    Ellinghausen, H. C.

    1973-01-01

    Death and lysis of leptospirae, when cultured in asbestos-filtered bovine albumin polysorbate 80 media, was quantitated. The pathogens (virulent and avirulent) required 2 × 106 cells/ml to initiate growth in such media, whereas inocula of 2 to 20 cells/ml grew in control medium. Saprophytic leptospirae initiated growth from 2 cells/ml in asbestos-filtered medium as well as control medium. The adverse action of asbestos-filtered medium was not removed by storage of medium for 2 years at 25 C and was not diminished when such medium was frozen at -80 C. Washing with water, HCl and NaHCO3-NaCl, citric acid, and medium components did not remove the lytic activity associated with asbestos-filtered culture medium. Continuous subculture in asbestos-filtered medium was possible from large inocula; however, upon subsequent dilution and reinoculation into asbestos-filtered media, there was no evidence of acquired resistance, and all pathogens failed to grow. Images PMID:4588200

  19. Rasburicase: an innovative new treatment for hyperuricemia associated with tumor lysis syndrome.

    PubMed

    Brant, Jeannine M

    2002-01-01

    Rasburicase is a new treatment for hyperuricemia, a metabolic manifestation of tumor lysis syndrome (TLS). Rasburicase has a unique mechanism of action that allows uric acid byproducts to be easily excreted in the urine. Clinical trials have shown that rasburicase has a rapid onset of action that allows chemotherapy to be delivered on time and prevents hyperuricemia-related complications, including renal compromise. The drug has been used successfully in adults and children. The main side effect of rasburicase is the potential for a hypersensitivity reaction. The drug is contraindicated in patients with glucose 6-phosphate dehydrogenase (G6PD) deficiency because this can precipitate hemolytic anemia. The drug has not been studied in patients with a history of allergies or asthma. Oncology nurses play a major role in the assessment and management of TLS-related complications. They must assess patients for G6PD deficiency and signs and symptoms of hypersensitivity reaction before and during chemotherapy or other therapeutic interventions. Nurses play a direct role in preventing complications related to TLS and contributing to the quality of life in this patient population.

  20. Large-scale clinical comparison of the lysis-centrifugation and radiometric systems for blood culture

    SciTech Connect

    Brannon, P.; Kiehn, T.E.

    1985-12-01

    The Isolator 10 lysis-centrifugation blood culture system (E. I. du Pont de Nemours and Co., Inc., Wilmington, Del.) was compared with the BACTEC radiometric method (Johnston Laboratories, Inc., Towson, Md.) with 6B and 7D broth media for the recovery of bacteria and yeasts. From 11,000 blood cultures, 1,174 clinically significant organisms were isolated. The Isolator system recovered significantly more total organisms, members of the family Enterobacteriaceae, Staphylococcus spp., and yeasts. The BACTEC system recovered significantly more Pseudomonas spp., Streptococcus spp., and anaerobes. Of the Isolator colony counts, 87% measured less than 11 CFU/ml of blood. Organisms, on an average, were detected the same day from each of the two culture systems. Only 13 of the 975 BACTEC isolates (0.01%) were recovered by subculture of growth-index-negative bottles, and 12 of the 13 were detected in another broth blood culture taken within 24 h. Contaminants were recovered from 4.8% of the Isolator 10 and 2.3% of the BACTEC cultures.

  1. Expression of blaA Underlies Unexpected Ampicillin-Induced Cell Lysis of Shewanella oneidensis

    PubMed Central

    Yin, Jianhua; Sun, Linlin; Dong, Yangyang; Chi, Xun; Zhu, Weiming; Qi, Shu-hua; Gao, Haichun

    2013-01-01

    Shewanella oneidensis is a facultative anaerobic γ-proteobacterium possessing remarkably diverse respiratory capacities for reducing various organic and inorganic substrates. As a veteran research model for investigating redox transformations of environmental contaminants the bacterium is well known to be a naturally ampicillin-resistant microorganism. However, in this study we discovered that ampicillin has a significant impact on growth of S. oneidensis. Particularly, cell lysis occurred only with ampicillin at levels ranging from 0.49 to 6.25 µg/ml but not at 50 µg/ml. This phenotype is attributable to insufficient expression of the β-lactamase BlaA. The subsequent analysis revealed that the blaA gene is strongly induced by ampicillin at high (50 µg/ml), but not at low levels (2.5 µg/ml). In addition, we demonstrated that penicillin binding protein 5 (PBP5), the most abundant low molecular weight PBP (LMW PBP), is the only one relevant to β-lactam resistance under the tested conditions. This nonessential PBP, largely resembling its Escherichia coli counterpart in functionality, mediates expression of the blaA gene. PMID:23555975

  2. Role of red blood cell lysis and iron in hydrocephalus after intraventricular hemorrhage.

    PubMed

    Gao, Chao; Du, Hanjian; Hua, Ya; Keep, Richard F; Strahle, Jennifer; Xi, Guohua

    2014-06-01

    Thrombin and iron are two major players in intracerebral hemorrhage-induced brain injury and our recent study found that thrombin contributes to hydrocephalus development in a rat model of intraventricular hemorrhage (IVH). This study investigated the role of red blood cell (RBC) lysis and iron in hydrocephalus after IVH. There were three parts to this study. First, male Sprague-Dawley rats received an injection of saline, packed, or lysed RBCs into the right lateral ventricle. Second, rats had an intraventricular injection of iron or saline. Third, the rats received intraventricular injection of lysed RBCs mixed with deferoxamine (0.5 mg in 5 μL saline) or saline. All rats underwent magnetic resonance imaging at 24 hours and were then euthanized for brain edema measurement, western blot analysis, or brain histology. We found that intraventricular injection of lysed RBCs, but not packed RBCs, resulted in ventricular enlargement and marked increases in brain heme oxygenase-1 and ferritin at 24 hours. Intraventricular injection of iron also resulted in ventricular enlargement and ventricular wall damage 24 hours later. Coinjection of deferoxamine reduced lysed RBC-induced ventricular enlargement (P<0.01). These results suggest that iron, a degradation product of hemoglobin, has an important role in hydrocephalus development after IVH.

  3. Prolonged clot lysis time increases the risk of a first but not recurrent venous thrombosis.

    PubMed

    Karasu, Alev; Baglin, Trevor P; Luddington, Roger; Baglin, Caroline A; van Hylckama Vlieg, Astrid

    2016-03-01

    The role of the fibrinolytic system in the development of venous thrombosis (VT) is unclear. We studied the risk of first and recurrent VT associated with reduced fibrinolysis, as measured by clot lysis time (CLT). We also studied the relationship between CLT and thrombin generation to determine if any relationship between CLT and VT was affected by thrombin generation. Analyses were performed in the Thrombophilia Hypercoagulability Environmental risk for Venous Thromboembolism Study, a two-centre population-based case-control study, including 579 patients and 338 controls, with patients followed from the event to determine incidence of recurrent VT. Hypofibrinolysis was associated with a 1·8-fold increased risk of a first VT [95% confidence interval (CI) 1·2-2·7]. Adjustment for sex, age, study location and Endogenous Thrombin Potential (ETP) did not change the result. The risk of VT was 2·9-fold increased when the 90th percentiles of prolonged CLT and high ETP were combined, with the highest risk for unprovoked first events (Odds Ratio = 4·2, 95% CI 1·3-13·5). In the follow-up study the Hazard Ratio for a recurrent VT associated with hypofibrinolysis was 1·5 (95% CI 0·9-2·6). A weak dose response effect was observed in relation to prolongation of CLT and recurrent VT. Although hypofibrinolysis constitutes a risk factor for a first VT, an association with recurrence is, at best, weak.

  4. Mechanical properties of vesicles. I. Coordinated analysis of osmotic swelling and lysis.

    PubMed Central

    Ertel, A; Marangoni, A G; Marsh, J; Hallett, F R; Wood, J M

    1993-01-01

    To determine how transmembrane osmotic gradients perturb the structure and dynamics of biological membranes, we examined the effects of medium dilution on the structures of osmolyte-loaded lipid vesicles. Our preparations were characterized by dynamic light scattering (DLS) and nuclear magnetic resonance (NMR) spectroscopies. Populations of Escherichia coli phosphatidylethanolamine (PE) or dioleoylphosphatidylglycerol (DOPG) vesicles prepared by the pH jump technique were variable and polymodal in size distribution. Complex and variable structural changes occurred when PE vesicles were diluted with hypotonic buffer. Such vesicles could not be used as model systems for the analysis of membrane mechanical properties. NaCl-loaded, DOPG vesicles prepared by extrusion through 100 nm (diameter) pores were reproducible and monomodal in size distribution and unilamellar, whereas those prepared by extrusion through 200-, 400-, or 600-nm pores were variable and polymodal in size distribution and/or multilamellar. Time and pressure regimes associated with osmotic lysis of extruded vesicles were defined by monitoring release of carboxyfluorescein, a self-quenching fluorescent dye. Corresponding effects of medium dilution on vesicle structure were assessed by DLS spectroscopy. These experiments and the accompanying analysis (Hallett, F.R., J. Marsh, B.G. Nickel, and J.M. Wood. 1993. Biophys. J. 64:000-000) revealed conditions under which vesicles are expected to reside in a consistently strained state. PMID:8457668

  5. Floor of the nose mucosa lysis and labial abscess caused by a bee sting.

    PubMed

    Alemán Navas, Ramón Manuel; Martínez Mendoza, María Guadalupe; Herrera, Henry; Herrera, Helen Piccolo de

    2009-01-01

    Hymenoptera order includes bees, which have a stinging apparatus at the tail capable of delivering venom to the affected tissues. Myocardial infarction, acute renal failure, Necrotizing fasciitis, fatal infection and hemifacial asymmetry, are some of the unusual reactions reported following hymenoptera stings. This paper reports a case of bee sting in the right floor of the nose that mimicked an odontogenic infection affecting the upper lip, canine space and nasal cavity such as in cases of infection secondary to pulpal or periodontal pathology of the anterior teeth. After a thorough clinical and radiographic examination, odontogenic infection was discarded and the diagnosis of floor of the nose mucosal lysis and lip abscess secondary to a bee sting was made. This case was successfully managed with adequate incision, drainage and antibiotics without any further complication. There are several reports of unusual reactions following hymenoptera stings. However, just a few of them referred to infections of local reactions and none of them related to the anatomic location affected in the patient of the present case. Early diagnosis and treatment prevented infection dissemination and the likelihood of tissue necrosis as in previously reported cases of Necrotizing fasciitis.

  6. Screening of plants acting against Heterometrus laoticus scorpion venom activity on fibroblast cell lysis.

    PubMed

    Uawonggul, Nunthawun; Chaveerach, Arunrat; Thammasirirak, Sompong; Arkaravichien, Tarinee; Chuachan, Chattong; Daduang, Sakda

    2006-01-16

    The aqueous extracts of 64 plant species, listed as animal- or insect-bite antidotes in old Thai drug recipes were screened for their activity against fibroblast cell lysis after Heterometrus laoticus scorpion venom treatment. The venom was preincubated with plant extract for 30 min and furthered treated to confluent fibroblast cells for 30 min. More than 40% efficiency (test/control) was obtained from cell treatment with venom preincubated with extracts of Andrographis paniculata Nees (Acanthaceae), Barringtonia acutangula (L.) Gaertn. (Lecythidaceae), Calamus sp. (Palmae), Clinacanthus nutans Lindau (Acanthaceae), Euphorbia neriifolia L. (Euphorbiaceae), Ipomoea aquatica Forssk (Convolvulaceae), Mesua ferrea L. (Guttiferae), Passiflora laurifolia L. (Passifloraceae), Plectranthus amboinicus (Lour.) Spreng. (Labiatae), Ricinus communis L. (Euphorbiaceae), Rumex sp. (Polygonaceae) and Sapindus rarak DC. (Sapindaceae), indicating that they had a tendency to be scorpion venom antidotes. However, only Andrographis paniculata and Barringtonia acutangula extracts provided around 50% viable cells from extract treatments without venom preincubation. These two plant extracts are expected to be scorpion venom antidotes with low cytotoxicity.

  7. On-Chip Single-Cell Lysis for Extracting Intracellular Material

    NASA Astrophysics Data System (ADS)

    Ikeda, Norifumi; Tanaka, Nobuaki; Yanagida, Yasuko; Hatsuzawa, Takeshi

    2007-09-01

    A newly designed microfluidic chip with a pinched-channel structure and two pairs of electrodes has been developed to enable easier single-cell capture and lysis. The function of the chip was evaluated by introducing zucchini protoplast cells into the channel. In the first experiment, we attempted to break a cell using the through force of a triangular pinched structure via electroosmotic flow generated by outer electrodes. The pinched structure appeared to break the cell without applying the electric field to the cell directly; however, in this case, the breakable size of the cell was limited by the width of the pinched structure. The next attempt was to break cells regardless of their sizes using a pair of inner electrodes located under the pinched structure. The inner electrodes generated a gradient electric field around the captured cell by applying an alternative voltage to the electrodes. Captured cells with a diameter from 40 to 85 μm could be broken using the inner electrodes with a trapezoidal pinched structure, and the cells were successfully broken at 10 Vpp or less at a frequency of 1 MHz.

  8. Estimates of bacterioplankton and Synechococcus spp. mortality from nanoflagellate grazing and viral lysis in the subtropical Danshui River estuary

    NASA Astrophysics Data System (ADS)

    Tsai, An-Yi; Gong, Gwo-Ching; Huang, Yu Wen; Chao, Chien Fu

    2015-02-01

    To better understand picoplankton dynamics in the surface waters of upriver the Danshui River and its estuary, we assessed nanoflagellate-induced and virus-induced mortality of bacteria and Synechococcus spp. during different seasons (October, 2012 and January, April and July, 2013) using a modified dilution technique. Bacteria and viruses were significantly higher in abundance upriver than at the estuary. The distribution of Synechococcus spp. did not follow this spatial pattern. Abundance of Synechococcus spp. was relatively low during the whole sampling period in the upriver region. Furthermore, bacterial mortality resulting from nanoflagellate grazing were generally higher than those resulting from viral lysis in the upriver region, while Synechococcus spp. losses appeared to be mainly due to viral lysis upriver and in the estuary. Our dilution experiments suggested that nanoflagellates largely depend on bacteria as an important energy source there.

  9. [Value of the technique of cellular lysis by thermic shock in the isolation of bacteria causing osteoarticular infections].

    PubMed

    Zannier, A; Drancourt, M; Franceschi, J P; Aubaniac, J M; Raoult, D

    1991-05-01

    The purpose of this work is to compare, a conventional technique, for bacterial isolation, with a lysis-centrifugation method using a rapid freezing in liquid nitrogen, followed by decongelation at 37 degrees C for bone and joint samples. The bone and joint specimens were biopsies and punctions (35 cases) or fistula (10 cases). The residual antibiotic activity of the sample was determined using a susceptible strain of Micrococcus luteus and of Staphylococcus epidermidis. Among the 45 samples, 20 were sterile with both methods among which 8 exhibited a residual antibiotic activity which may have contributed to isolation failure. In 12 cases, bacterial isolates were obtained with both methods. Cultures were obtained from 13 samples with the lysis centrifugation method alone. The isolates were 4 Staphylococcus aureus, 8 coagulase-negative Staphylococcus and 1 Acinetobacter sp. These results suggest that the intracellular location of bacteria may be responsible for the negative cultures from bone or joint specimens.

  10. "Non-toxic" cyclic peptides induce lysis of cyanobacteria-an effective cell population density control mechanism in cyanobacterial blooms.

    PubMed

    Sedmak, B; Carmeli, S; Elersek, Tina

    2008-08-01

    The presence of planktopeptin BL1125, anabaenopeptin B and anabaenopeptin F, two types of "non-toxic" cyclic peptide produced in bloom forming cyanobacteria, can provoke lysis of different non-axenic Microcystis aeruginosa cell lines via the induction of virus-like particles. The resulting particles are also able to infect the axenic M. aeruginosa cell line without lytic effects. Nevertheless, the presence of "non-toxic" cyclic peptides of cyanobacterial origin can induce lysis of these previously infected cells. This effect implies that a possible role of these peptides in the natural environment is the control of cyanobacterial population density. Lysogenic cyanobacteria can consequently act as hot-spots that, in the presence of cyanobacterial cyclic peptides, release numerous infectious particles. The process can be self-augmented with the simultaneous release of additional cyclic peptides from the producing lysogens, starting a forest fire effect that ends in collapse of cyanobacterial blooms.

  11. Acute spontaneous tumor lysis in anaplastic large T-cell lymphoma presenting with hyperuricemic acute renal failure.

    PubMed

    Hsu, Hsiang-Hao; Huang, Chiu-Ching

    2004-01-01

    Acute spontaneous tumor lysis (ASTL) syndrome, an extremely rare disease, requires prompt recognition and aggressive management because it is fulminant at its outset, associated with severe metabolic derangement, and potentially reversible. We describe an unusual case in which spontaneous tumor lysis occurred in anaplastic large T-cell lymphoma associated with acute uric acid nephropathy, persistent oliguria, and shock. This case contrasts markedly with previously reported cases of ASTL syndrome, which developed mainly in the pathologic type of Burkitt lymphoma. To our knowledge, this is the first reported occurrence of ASTL syndrome associated with anaplastic large T-cell type lymphoma. This report also chronicles our successful experience with continuous renal replacement therapy in the presence of compromised hemodynamic status.

  12. Naringenin enhances NK cell lysis activity by increasing the expression of NKG2D ligands on Burkitt's lymphoma cells.

    PubMed

    Kim, Jeong Hwa; Lee, Jae Kwon

    2015-11-01

    Natural killer (NK) cells are capable of identifying and killing tumor cells as well as virus infected cells without pre-sensitization. NK cells express activating and inhibitory receptors, and can distinguish between normal and tumor cells. The present study was designed to demonstrate the importance of the expression level of NKG2D ligands on the Burkitt's lymphoma cell line, Raji, in enhancing NK cell cytolytic activity. Various flavonoids were used as stimulants to enhance the expression of NKG2D ligands. NK cell lysis activity against Raji was not changed by pre-treatment of Raji with luteolin, kaempferol, taxifolin and hesperetin. However, treatment of Raji with naringenin showed increased sensitivity to NK cell lysis than untreated control cells. The activity of naringenin was due to enhanced NKG2D ligand expression. These results provide evidence that narigenin's antitumor activity may be due to targeting of NKG2D ligand expression and suggests a possible immunotherapeutic role for cancer treatment.

  13. Comparison of different methods of cell lysis and protein measurements in Clostridium perfringens: application to the cell volume determination.

    PubMed

    Guerlava, P; Izac, V; Tholozan, J L

    1998-03-01

    Four cell lysis methods (NaOH-SDS solubilization, French press treatment, sonication, mutanolysin treatment) and three methods of protein assays (Lowry, Bradford, Pierce) were studied for their applicability to determination of cell volume in Clostridium perfringens NCTC 8798 cell suspensions. Protein contents were higher after a mechanical disruption of the cells than with the other techniques of lysis. The lowest concentrations of protein were obtained with the Bradford procedure. With each of the three protein assay methods, Clostridium perfringens NCTC 8798 protein cell contents were 45% to 58% of protein. Other factors possibly involved in variations of the intracellular volume measurements were examined. A control of the level of protein concentration in the test sample and the type of silicone oil used for the centrifugation were of prime importance during sample preparation. Under our conditions, an intracellular volume of 4 microl/(mg of protein) was routinely found for Clostridium perfringens NCTC 8798.

  14. Sulfolobus turreted icosahedral virus c92 protein responsible for the formation of pyramid-like cellular lysis structures.

    PubMed

    Snyder, Jamie C; Brumfield, Susan K; Peng, Nan; She, Qunxin; Young, Mark J

    2011-07-01

    Host cells infected by Sulfolobus turreted icosahedral virus (STIV) have been shown to produce unusual pyramid-like structures on the cell surface. These structures represent a virus-induced lysis mechanism that is present in Archaea and appears to be distinct from the holin/endolysin system described for DNA bacteriophages. This study investigated the STIV gene products required for pyramid formation in its host Sulfolobus solfataricus. Overexpression of STIV open reading frame (ORF) c92 in S. solfataricus alone is sufficient to produce the pyramid-like lysis structures in cells. Gene disruption of c92 within STIV demonstrates that c92 is an essential protein for virus replication. Immunolocalization of c92 shows that the protein is localized to the cellular membranes forming the pyramid-like structures.

  15. Electromechanical cell lysis using a portable audio device: enabling challenging sample preparation at the point-of-care.

    PubMed

    Buser, J R; Wollen, A; Heiniger, E K; Byrnes, S A; Kauffman, P C; Ladd, P D; Yager, P

    2015-05-07

    Audio sources are ubiquitously available on portable electronic devices, including cell phones. Here we demonstrate lysis of Mycobacterium marinum and Staphylococcus epidermidis bacteria utilizing a portable audio device coupled with a simple and inexpensive electromagnetic coil. The resulting alternating magnetic field rotates a magnet in a tube with the sample and glass beads, lysing the cells and enabling sample preparation for these bacteria anywhere there is a cell phone, mp3 player, laptop, or other device with a headphone jack.

  16. Viral lysis of Phaeocystis pouchetii: implications for algal population dynamics and heterotrophic C, N and P cycling.

    PubMed

    Haaber, Jakob; Middelboe, Mathias

    2009-04-01

    A model ecosystem with two autotrophic flagellates, Phaeocystis pouchetii and Rhodomonas salina, a virus specific to P. pouchetii (PpV) and bacteria and heterotrophic nanoflagellates was used to investigate effects of viral lysis on algal population dynamics and heterotrophic nitrogen and phosphorus mineralization. Lysis of P. pouchetii by PpV had strong positive effects on bacterial and HNF abundance, and the mass balance of C, N and P suggested an efficient transfer of organic material from P. pouchetii to bacterial and HNF biomass through viral lysis. At the same time, the degradation of P. pouchetii lysates was associated with significant regeneration of inorganic N and P resulting in 148 microg N l(-1) and 7 microg P l(-1), corresponding to 78% and 26% of lysate N and P being mineralized to NH(4)(+) and PO(4)(3-), respectively. These results showed that the turnover of viral lysates in the microbial food web was associated with significant N and P mineralization, supporting the current view that viral lysates can be an important source of inorganic nutrients in marine systems. In the presence of R. salina, the generated NH(4)(+) supported 11% of the observed R. salina growth. Regrowth of virus-resistant P. pouchetii following cell lysis was observed in long-term incubations (150 days), and possibly influenced by nutrient availability and competition from R. salina. The observed impact of viral activity on autotrophic and heterotrophic processes provides direct experimental evidence for virus-driven nutrient generation and emphasizes the potential importance of the viral activity in supporting marine primary production.

  17. Detection of Mycobacterium tuberculosis DNA in clinical samples by using a simple lysis method and polymerase chain reaction.

    PubMed Central

    Folgueira, L; Delgado, R; Palenque, E; Noriega, A R

    1993-01-01

    We have evaluated the polymerase chain reaction for detection of Mycobacterium tuberculosis in clinical samples from patients with tuberculous infection. Two simple methods for mycobacterial DNA release have been compared: sonication and lysis with nonionic detergents and proteinase K. The more effective method was the enzymatic technique. By using this protocol with 75 specimens we detected M. tuberculosis DNA in all of the samples, whereas only 48 and 71 samples were positive by acid-fast staining and culture, respectively. Images PMID:8463383

  18. A super-family of transcriptional activators regulates bacteriophage packaging and lysis in Gram-positive bacteria

    PubMed Central

    Quiles-Puchalt, Nuria; Tormo-Más, María Ángeles; Campoy, Susana; Toledo-Arana, Alejandro; Monedero, Vicente; Lasa, Íñigo; Novick, Richard P.; Christie, Gail E.; Penadés, José R.

    2013-01-01

    The propagation of bacteriophages and other mobile genetic elements requires exploitation of the phage mechanisms involved in virion assembly and DNA packaging. Here, we identified and characterized four different families of phage-encoded proteins that function as activators required for transcription of the late operons (morphogenetic and lysis genes) in a large group of phages infecting Gram-positive bacteria. These regulators constitute a super-family of proteins, here named late transcriptional regulators (Ltr), which share common structural, biochemical and functional characteristics and are unique to this group of phages. They are all small basic proteins, encoded by genes present at the end of the early gene cluster in their respective phage genomes and expressed under cI repressor control. To control expression of the late operon, the Ltr proteins bind to a DNA repeat region situated upstream of the terS gene, activating its transcription. This involves the C-terminal part of the Ltr proteins, which control specificity for the DNA repeat region. Finally, we show that the Ltr proteins are the only phage-encoded proteins required for the activation of the packaging and lysis modules. In summary, we provide evidence that phage packaging and lysis is a conserved mechanism in Siphoviridae infecting a wide variety of Gram-positive bacteria. PMID:23771138

  19. Flow rate and duty cycle effects in lysis of Chlamydomonas reinhardtii using high-energy pulsed focused ultrasound.

    PubMed

    Riesberg, Grant; Bigelow, Timothy A; Stessman, Dan J; Spalding, Martin H; Yao, Linxing; Wang, Tong; Xu, Jin

    2014-06-01

    To consider microalgae lipid biofuel as a viable energy source, it is a necessity to maximize algal cell lysis, lipid harvest, and thus biofuel production versus the energy used to lyse the cells. Previous techniques have been to use energy consumptive ultrasound waves in the 10-40 kHz range in a stationary exposure environment. This study evaluated the potential of using 1.1 MHz ultrasound pulses in a new flow through type chamber on Chlamydomonas reinhardtii as a model organism for cell breakage. The ultrasound was generated using a spherically focused transducer with a focal length of 6.34 cm and an active diameter of 6.36 cm driven by 20 cycle sine-wave tone bursts at varied pulse repetition frequencies. First, variations in flow rate were examined at a constant duty cycle of 3.6%. After assessing flow rates, the duty cycle was varied to further explore the dependence on the tone burst parameters. Cell lysis was assessed by quantifying protein and chlorophyll release into the supernatant as well as by lipid extractability. Appropriate flow rates with higher duty cycles led to statistically significant increases in cell lysis relative to controls and other exposure conditions.

  20. Effects of complement depletion in experimental chagas disease: immune lysis of virulent blood forms of Trypanosoma cruzi.

    PubMed Central

    Budzko, D B; Pizzimenti, M C; Kierszenbaum, F

    1975-01-01

    In mice infected with virulent blood (trypomastigote) forms of Trypanosoma cruzi, complement depletion with cobra venom factor caused a marked exacerbation of the disease evidenced by significantly increased parasitemia levels and early mortality as compared with those of untreated infected animals. The effect was greater in mice receiving cobra venom factor on day 7 postinfection, i.e., at the time when the parasites had had time to localize and multiply in the tissues and appeared in the circulation in appreciable numbers. The possibility that complement participates in host defense against T. cruzi infection through a mechanism involving immune lysis was explored in vitro. T. cruzi trypomastigotes were found to undergo immune lysis in sera of patients with chronic Chagas' disease, in sera of immunized mice, and in solutions containing both immune mouse gamma globulin and a source of active complement. This phenomenon failed to take place either in the absence of complement or after complement inactivation by heat or utilizing complement inactivators. The lytic capacity of heated sera was restored by the addition of active complement to the system. During the immune lysis of T. cruzi blood forms, complement was activated in human sera via both the classical and the alternate pathways. In mouse sera, activation followed at least the alternate pathway. PMID:46840

  1. A super-family of transcriptional activators regulates bacteriophage packaging and lysis in Gram-positive bacteria.

    PubMed

    Quiles-Puchalt, Nuria; Tormo-Más, María Ángeles; Campoy, Susana; Toledo-Arana, Alejandro; Monedero, Vicente; Lasa, Iñigo; Novick, Richard P; Christie, Gail E; Penadés, José R

    2013-08-01

    The propagation of bacteriophages and other mobile genetic elements requires exploitation of the phage mechanisms involved in virion assembly and DNA packaging. Here, we identified and characterized four different families of phage-encoded proteins that function as activators required for transcription of the late operons (morphogenetic and lysis genes) in a large group of phages infecting Gram-positive bacteria. These regulators constitute a super-family of proteins, here named late transcriptional regulators (Ltr), which share common structural, biochemical and functional characteristics and are unique to this group of phages. They are all small basic proteins, encoded by genes present at the end of the early gene cluster in their respective phage genomes and expressed under cI repressor control. To control expression of the late operon, the Ltr proteins bind to a DNA repeat region situated upstream of the terS gene, activating its transcription. This involves the C-terminal part of the Ltr proteins, which control specificity for the DNA repeat region. Finally, we show that the Ltr proteins are the only phage-encoded proteins required for the activation of the packaging and lysis modules. In summary, we provide evidence that phage packaging and lysis is a conserved mechanism in Siphoviridae infecting a wide variety of Gram-positive bacteria.

  2. Protective effect of platelet activating factor antagonists on cultured endothelial cell lysis induced by elastase or activated neutrophils.

    PubMed Central

    Renesto, P.; Vicart, P.; Paulin, D.; Chignard, M.

    1996-01-01

    1. The mechanism(s) responsible for injury of endothelial cells induced by human leukocyte elastase (HLE) was investigated in an immortalized venous human endothelial cell line (IVEC). 2. First, the proteinase concentrations and incubation delays necessary to trigger a significant IVEC cytotoxicity were determined by chromium assays. Thus, exposure of IVEC for 6 h to 10 micrograms ml-1 HLE resulted in 22 +/- 2.8% lysis and 36.4 +/- 5.4% detachment (mean +/- s.e. mean; n = 4; P < 0.05). 3. WEB 2086, a specific platelet-activating factor (PAF) receptor antagonist, induced a significant concentration-dependent decrease of such a lysis (39.6 +/- 7.7% protection at 100 microM; n = 4). This potential role for PAF was confirmed with two other antagonists of this lipid mediator, i.e., BN 52021 and RP 48740. 4. Finally, we demonstrated that pretreatment of IVEC with WEB 2086 protected significantly against cell lysis induced by stimulated human neutrophils, an experimental model in which HLE participates. PMID:8851508

  3. Celecoxib increases lung cancer cell lysis by lymphokine-activated killer cells via upregulation of ICAM-1

    PubMed Central

    Frank, Marcus; Linnebacher, Michael; Hinz, Burkhard

    2015-01-01

    The antitumorigenic mechanism of the selective cyclooxygenase-2 (COX-2) inhibitor celecoxib is still a matter of debate. Using lung cancer cell lines (A549, H460) and metastatic cells derived from a lung cancer patient, the present study investigates the impact of celecoxib on the expression of intercellular adhesion molecule 1 (ICAM-1) and cancer cell lysis by lymphokine-activated killer (LAK) cells. Celecoxib, but not other structurally related selective COX-2 inhibitors (i.e., etoricoxib, rofecoxib, valdecoxib), was found to cause a substantial upregulation of ICAM-1 protein levels. Likewise, ICAM-1 mRNA expression was increased by celecoxib. Celecoxib enhanced the susceptibility of cancer cells to be lysed by LAK cells with the respective effect being reversed by a neutralizing ICAM-1 antibody. In addition, enhanced killing of celecoxib-treated cancer cells was reversed by preincubation of LAK cells with an antibody to lymphocyte function associated antigen 1 (LFA-1), suggesting intercellular ICAM-1/LFA-1 crosslink as crucial event within this process. Finally, celecoxib elicited no significant increase of LAK cell-mediated lysis of non-tumor bronchial epithelial cells, BEAS-2B, associated with a far less ICAM-1 induction as compared to cancer cells. Altogether, our data demonstrate celecoxib-induced upregulation of ICAM-1 on lung cancer cells to be responsible for intercellular ICAM-1/LFA-1 crosslink that confers increased cancer cell lysis by LAK cells. These findings provide proof for a novel antitumorigenic mechanism of celecoxib. PMID:26513172

  4. Malaria parasites co-opt human factor H to prevent complement-mediated lysis in the mosquito midgut.

    PubMed

    Simon, Nina; Lasonder, Edwin; Scheuermayer, Matthias; Kuehn, Andrea; Tews, Sabrina; Fischer, Rainer; Zipfel, Peter F; Skerka, Christine; Pradel, Gabriele

    2013-01-16

    Human complement is a first line defense against infection in which circulating proteins initiate an enzyme cascade on the microbial surface that leads to phagocytosis and lysis. Various pathogens evade complement recognition by binding to regulator proteins that protect host cells from complement activation. We show that emerging gametes of the malaria parasite Plasmodium falciparum bind the host complement regulator factor H (FH) following transmission to the mosquito to protect from complement-mediated lysis by the blood meal. Human complement is active in the mosquito midgut for approximately 1 hr postfeeding. During this period, the gamete surface protein PfGAP50 binds to FH and uses surface-bound FH to inactivate the complement protein C3b. Loss of FH-mediated protection, either through neutralization of FH or blockade of PfGAP50, significantly impairs gametogenesis and inhibits parasite transmission to the mosquito. Thus, Plasmodium co-opts the protective host protein FH to evade complement-mediated lysis within the mosquito midgut.

  5. Celecoxib increases lung cancer cell lysis by lymphokine-activated killer cells via upregulation of ICAM-1.

    PubMed

    Schellhorn, Melina; Haustein, Maria; Frank, Marcus; Linnebacher, Michael; Hinz, Burkhard

    2015-11-17

    The antitumorigenic mechanism of the selective cyclooxygenase-2 (COX-2) inhibitor celecoxib is still a matter of debate. Using lung cancer cell lines (A549, H460) and metastatic cells derived from a lung cancer patient, the present study investigates the impact of celecoxib on the expression of intercellular adhesion molecule 1 (ICAM-1) and cancer cell lysis by lymphokine-activated killer (LAK) cells. Celecoxib, but not other structurally related selective COX-2 inhibitors (i.e., etoricoxib, rofecoxib, valdecoxib), was found to cause a substantial upregulation of ICAM-1 protein levels. Likewise, ICAM-1 mRNA expression was increased by celecoxib. Celecoxib enhanced the susceptibility of cancer cells to be lysed by LAK cells with the respective effect being reversed by a neutralizing ICAM-1 antibody. In addition, enhanced killing of celecoxib-treated cancer cells was reversed by preincubation of LAK cells with an antibody to lymphocyte function associated antigen 1 (LFA-1), suggesting intercellular ICAM-1/LFA-1 crosslink as crucial event within this process. Finally, celecoxib elicited no significant increase of LAK cell-mediated lysis of non-tumor bronchial epithelial cells, BEAS-2B, associated with a far less ICAM-1 induction as compared to cancer cells. Altogether, our data demonstrate celecoxib-induced upregulation of ICAM-1 on lung cancer cells to be responsible for intercellular ICAM-1/LFA-1 crosslink that confers increased cancer cell lysis by LAK cells. These findings provide proof for a novel antitumorigenic mechanism of celecoxib.

  6. Myocardial lysis in a fetus induced by maternal paraphenylenediamine poisoning following an intentional ingestion to induce abortion.

    PubMed

    Abidi, K; Himdi, B; Cherradi, N; Lamalmi, N; Alhamany, Z; Zeggwagh, Aa; Abouqal, R

    2008-05-01

    The acute toxicity of paraphenylenediamine (PPD) has been associated with several histopathological changes. In humans, acute PPD poisoning is known to cause rhabdomyolisis and particularly myocardial lysis. However, its toxicity for the fetus has never been reported in the literature. We report a case of myocardial lysis in a fetus expelled by a 22-year-old mother after apparent ingestion of an unknown amount of PPD. The patient was admitted to our intensive care unit with acute onset of respiratory distress and rhabdomyolysis. The pelvic ultrasonography on admission showed a normally progressing pregnancy of 23-24 weeks. On day 9 post-ingestion, the patient spontaneously expelled a non-viable fetus. The fetal examination did not show any external or macroscopic abnormalities. However, the histopathological exam showed an important heart and lung congestion. There was also some interstitial edema and inflammation at the base of the lingua, in addition to a chorionic villus thrombosis and abruptio placentae. The histopathology of the myocardium showed lysis of the cardiac muscle. This observation suggests that the PPD was most likely responsible for the myocardial injury in the fetus.

  7. Susceptibility to cytotoxic T cell lysis of cancer stem cells derived from cervical and head and neck tumor cell lines.

    PubMed

    Liao, Tian; Kaufmann, Andreas M; Qian, Xu; Sangvatanakul, Voramon; Chen, Chao; Kube, Tina; Zhang, Guoyou; Albers, Andreas E

    2013-01-01

    To explore cancer stem cell susceptibility to a host's cytotoxic T lymphocyte (CTL)-mediated immune response. We compared the susceptibility of putative CSC generated from cancer cell lines to immunologic recognition and killing by alloantigen-specific CD8(+) CTL. CSC-enriched spheroid culture-derived cells (SDC) exhibited higher expression of ALDH, ICAM1 and of stem/progenitor cell markers on all 3 tumor cell lines investigated and lower MHC class I on the cervical cancer cell line as compared to their monolayer-derived cells (MDC). The expression of ICAM1 and MHCI was upregulated by IFN-γ treatment. CSC populations were less sensitive to MHC class I-restricted alloantigen-specific CD8(+) CTL lysis as compared to matched MDC. IFN-γ pretreatment resulted in over-proportionally enhanced lysis of SDC. Finally, the subset of ALDH(high) expressing SDC presented more sensitivity toward CD8(+) CTL killing than the ALDH(low) SDC. Tumor therapy resistance has been attributed to cancer stem cells (CSC). We show in vitro susceptibility of CSC to CTL-mediated lysis. Immunotherapy targeting of ALDH(+) CSC may therefore be a promising approach. Our results and method may be helpful for the development and optimization of adjuvants, as here exemplified for INF-γ, for CSC-targeted vaccines, independent of the availability of CSC-specific antigens.

  8. Isothermal single nucleotide polymorphism genotyping and direct PCR from whole blood using a novel whole-blood lysis buffer.

    PubMed

    Victor, Sylvia T; Lezhava, Alexander; Ishidao, Takefumi; Endo, Ryuta; Mitani, Yasumasa; Kawaoka, Yoshihiro; Hayashizaki, Yoshihide

    2009-12-01

    Cell lysis and subsequent release of genomic DNA is an ongoing dilemma for molecular biological techniques. In most cases, technologies such as PCR and other amplification techniques require DNA extraction and purification steps. The Smart Amplification Process Version 2 (SmartAmp2) is an isothermal and integrated amplification technology that eliminates the need for time-consuming sample preparation for the rapid detection of nucleic acids, including single nucleotide polymorphisms (SNPs), mutations, and other targets. In addition, DNA amplification directly from whole blood is beneficial and lessens the risk of cross-contamination. Traditional SmartAmp2 assays entail two steps and require an alkali pretreatment step at 98 degrees C prior to the 60 degrees C run. To make SmartAmp2 truly isothermal and to simplify DNA amplification, we hereby introduce the SmartAmp Isothermal Lysis Buffer (SIL-B), a newly developed chaotropic lysis buffer that enables the simultaneous recovery and denaturation of genomic material directly from whole blood at a uniform 60 degrees C. The improved method for isolating nucleic acids from whole blood is a critical milestone in making SmartAmp2 truly isothermal from start to finish at one temperature, increasing its potential to be routinely used in field point-of-care testing. Furthermore, pretreatment with SIL-B enables the PCR amplification of genomic material directly from whole blood.

  9. Sera from patients with colon, breast and lung cancer induce resistance to lysis mediated by NK cytotoxic factors (NKCF).

    PubMed Central

    Marubayashi, M.; Solana, R.; Ramirez, R.; Aranda, E.; Galan, F.; Peña, J.

    1991-01-01

    Natural killer (NK) cells are involved in the antitumoral immunologic mechanism. These cells act through the release of cytotoxic molecules defined as NK cytotoxic factors (NKCF). Inhibitory factors of NK and NKCF mediated lysis have been described in in vitro assays. This study evaluates the induction of resistance to NKCF cytotoxicity by sera from 27 patients with colon, breast and lung cancer. Addition of these sera to the cytolytic assay where K562 cells and concentrated NKCF were used, induced resistance to NKCF mediated cytotoxicity in 21 cases (77%). The sera from the group with metastasis blocked NKCF lysis more markedly than the group with local tumours. However, no differences were observed when the groups with colon, breast and lung cancers were compared. This blocking effect was not found to be related to gamma interferon (IFN) levels. In a previous study, we described a tumour factor (NK-RIF) produced by human cell lines derived from metastatic adenocarcinomas. This factor blocked lysis of tumour target cells by NK cells. Consequently, it is proposed that the release of similar tumour factors with a capacity to induce resistance to NKCF may be involved in tumour growth and metastatic spreading in in vivo. PMID:1906292

  10. Lysis of pig endothelium by IL-2 activated human natural killer cells is inhibited by swine and human major histocompatibility complex (MHC) class I gene products.

    PubMed

    Itescu, S; Artrip, J H; Kwiatkowski, P A; Wang, S F; Minanov, O P; Morgenthau, A S; Michler, R E

    1997-01-01

    We have previously described a form of xenograft rejection, mediated by natural killer (NK) cells, occurring in pig-to-primate organ transplants beyond the period of antibody-mediated hyperacute rejection. In this study, two distinct NK activation pathways were identified as mechanisms of pig aortic endotheliual cell (PAEC) lysis by human NK cells. Using an antibody-dependent cellular cytotoxicity (ADCC) assay, a progressive increase in human NK lysis of PAEC was observed following incubation with human IgG at increasing serum titer. In the absence of IgG, a second mechanism of PAEC lysis by human NK cells was observed following activation with IL-2. IL-2 activation of human NK cells increased lysis of PAEC by over 3-fold compared with ADCC. These results indicate that IL-2 activation of human NK cells induces significantly higher levels of lytic activity than does conventional ADCC involving IgG and FcRIII. We next investigated the role of MHC class I molecules in the regulation of NK lysis following IL-2 activation. PAEC expression of SLA class I molecules was increased by up to 75% by treatment with human TNFa. Following treatment with TNFa at 1 u/ml, IL-2 activated human NK lysis of PAEC was inhibited at every effector:target (E:T) ratio tested. Maximal effect occurred at an E:T ratio of 10:1, with TNFa inhibiting specific lysis by 59% (p < 0.01). Incubation with an anti-SLA class I Mab, but not IgG isotype control, abrogated the protective effects of TNFa on NK lysis of PAEC, suggesting direct inhibitory effects of SLA class I molecules on human NK function. To investigate whether human MHC class I molecules might have similar effects on human NK lysis of PAEC, further experiments were performed using a soluble peptide derived from the alpha-helical region of HLA-B7. Incubation with the HLA-B7 derived peptide significantly reduced the IL-2 activated NK lytic activity against PAEC in a dose-dependent fashion. Maximal effect occurred at a concentration of 10 mg

  11. Virus-specific HLA-restricted lysis of herpes simplex virus-infected human monocytes and macrophages mediated by cytotoxic T lymphocytes

    SciTech Connect

    Torpey, D.J. III

    1987-01-01

    Freshly-isolated peripheral blood human monocytes and 5 day in vitro cultured macrophages were infected with herpes simplex virus type 1 (HSV-1), labeled with /sup 51/Cr, and used as target cells in a 12-14 hour cell-mediated cytotoxicity assay. Mononuclear leukocytes (MNL) from HSV-1 non-immune individuals, whether unstimulated or stimulated with HSV-1 antigen, did not mediate significant lysis of either target cell. HSV-immune MNL, both freshly-isolated and cultured for 5 days without antigen, demonstrated only low levels of natural killer (NK) cell-mediate lysis. MNL from HSV-immune individuals incubated for 5 days in vitro with HSV-1 antigen mediated significant virus-specific lysis of both target cells. Mean virus-specific lysis of autologous monocytes was 8.5(/+-/2.0)% compared to a three-fold greater virus-specific lysis of autologous macrophages. Greater than 70% of this lytic activity was mediated by Leu-11-negative, T3-positive cytotoxic T lymphocytes (CTL). Allogeneic target cells lacking a common HLA determinant were not significantly lysed while T8-positive CTL mediated infrequent lysis of target cells sharing a common HLA-A and/or HLA-B determinant. T4-positive lymphocytes were demonstrated to be the predominant cell mediating lysis of autologous target cells and allogeneic target cells sharing both HLA-A and/or HLA-B plus HLA-DR determinants with the CTL; the T4-positive cell was the sole CTL mediator of lysis of allogeneic target cells having a common HLA-DR determinant.

  12. Artificial Intelligence vs. Statistical Modeling and Optimization of Continuous Bead Milling Process for Bacterial Cell Lysis.

    PubMed

    Haque, Shafiul; Khan, Saif; Wahid, Mohd; Dar, Sajad A; Soni, Nipunjot; Mandal, Raju K; Singh, Vineeta; Tiwari, Dileep; Lohani, Mohtashim; Areeshi, Mohammed Y; Govender, Thavendran; Kruger, Hendrik G; Jawed, Arshad

    2016-01-01

    For a commercially viable recombinant intracellular protein production process, efficient cell lysis and protein release is a major bottleneck. The recovery of recombinant protein, cholesterol oxidase (COD) was studied in a continuous bead milling process. A full factorial response surface methodology (RSM) design was employed and compared to artificial neural networks coupled with genetic algorithm (ANN-GA). Significant process variables, cell slurry feed rate (A), bead load (B), cell load (C), and run time (D), were investigated and optimized for maximizing COD recovery. RSM predicted an optimum of feed rate of 310.73 mL/h, bead loading of 79.9% (v/v), cell loading OD600nm of 74, and run time of 29.9 min with a recovery of ~3.2 g/L. ANN-GA predicted a maximum COD recovery of ~3.5 g/L at an optimum feed rate (mL/h): 258.08, bead loading (%, v/v): 80%, cell loading (OD600nm): 73.99, and run time of 32 min. An overall 3.7-fold increase in productivity is obtained when compared to a batch process. Optimization and comparison of statistical vs. artificial intelligence techniques in continuous bead milling process has been attempted for the very first time in our study. We were able to successfully represent the complex non-linear multivariable dependence of enzyme recovery on bead milling parameters. The quadratic second order response functions are not flexible enough to represent such complex non-linear dependence. ANN being a summation function of multiple layers are capable to represent complex non-linear dependence of variables in this case; enzyme recovery as a function of bead milling parameters. Since GA can even optimize discontinuous functions present study cites a perfect example of using machine learning (ANN) in combination with evolutionary optimization (GA) for representing undefined biological functions which is the case for common industrial processes involving biological moieties.

  13. Mingled Mortality: the Interplay Between Protist Grazing and Viral Lysis on Emiliania huxleyi Cell Fate

    NASA Astrophysics Data System (ADS)

    Harvey, E.; Bidle, K. D.; Johnson, M. D.

    2016-02-01

    The coccolithophore, Emiliania huxleyi plays a prominent role in global carbon cycling, as their calcite coccoliths account for a third of all oceanic calcite production. Mortality due to grazing by microzooplankton is the largest contributor to phytoplankton loss in the marine environment. However, viral infection of E. huxleyi is now thought to be as important as grazing pressure in contributing to its mortality. To understand the influence of viral infection on grazing dynamics, we examined the response of the dinoflagellate predator, Oxyrrhis marina to E. huxleyi infected with four different strains of the E. huxleyi virus (EhV). Grazing rate was significantly slower on E. huxleyi cultures that had been infected for 48 h compared to an uninfected control and this reduction in grazing rate was dependent on the strain identity of infecting EhVs. Additional experimentation indicated that grazing was the primary source of E. huxleyi loss ( 78-98%) during the first 24 h of exposure to both predator and virus. However, as viral infection progressed into the late lytic phase (48 h hour post infection), the relative contribution of grazing to total E. huxleyi mortality decreased ( 5-60%). These results suggest that mortality is partitioned along a gradient between predator-based consumption and virus-induced cell lysis, dependent on the timing of infection. Deciphering the relative importance and interactive nature of these alga-predator-viral interactions will help to elucidate the mechanisms that drive bulk measurements of phytoplankton loss, a necessary understanding to interpret and predict phytoplankton population dynamics and associated biogeochemical cycling.

  14. Purification and Functional Characterization of the ϕX174 Lysis Protein E†

    PubMed Central

    Zheng, Yi; Struck, Douglas K.; Young, Ry

    2011-01-01

    Two classes of bacteriophages, the single-stranded DNA Microviridae, and the single-stranded RNA Alloleviviridae, accomplish lysis by expressing “protein antibiotics”, or polypeptides that inhibit cell wall biosynthesis. Previously, we have provided genetic and physiological evidence that E, a 91 aa membrane protein encoded by the prototype microvirus, ϕX174, is a specific inhibitor of the translocase MraY, an essential membrane-embedded enzyme that catalyzes the formation of the murein precursor, Lipid I, from UDP-N-acetylmuramic acid-pentapeptide and the lipid carrier, undecaprenol phosphate. Here we report the first purification of E, which has been refractory to over-expression because of its lethality to E. coli. Moreover, using a fluorescently-labeled analog of the sugar-nucleotide substrate, we demonstrate that E acts as a non-competitive inhibitor of detergent-solubilized MraY, with respect to both soluble and lipid substrates. In addition, we show that the E-sensitivity of five MraY mutant proteins, produced from alleles selected for resistance to E, can be correlated to the apparent affinities determined by in vivo multicopy suppression experiments. These results are inconsistent with previous reports that E inhibited membrane-embedded MraY but not the detergent-solubilized enzyme, which led to a model in which E functions by binding MraY and blocking the formation of an essential hetero-multimeric complex involving MraY and other murein biosynthesis enzymes. We discuss a new model in which E binds to MraY at a site composed of the two transmembrane domains within which the E-resistance mutations map and that the result of this binding is a conformational change that inactivates the enzyme. PMID:19379010

  15. Purification and functional characterization of phiX174 lysis protein E.

    PubMed

    Zheng, Yi; Struck, Douglas K; Young, Ry

    2009-06-09

    Two classes of bacteriophages, the single-stranded DNA Microviridae and the single-stranded RNA Alloleviviridae, accomplish lysis by expressing "protein antibiotics", or polypeptides that inhibit cell wall biosynthesis. Previously, we have provided genetic and physiological evidence that E, a 91-amino acid membrane protein encoded by the prototype microvirus, varphiX174, is a specific inhibitor of the translocase MraY, an essential membrane-embedded enzyme that catalyzes the formation of the murein precursor, Lipid I, from UDP-N-acetylmuramic acid-pentapeptide and the lipid carrier, undecaprenol phosphate. Here we report the first purification of E, which has been refractory to overexpression because of its lethality to Escherichia coli. Moreover, using a fluorescently labeled analogue of the sugar-nucleotide substrate, we demonstrate that E acts as a noncompetitive inhibitor of detergent-solubilized MraY, with respect to both soluble and lipid substrates. In addition, we show that the E sensitivity of five MraY mutant proteins, produced from alleles selected for resistance to E, can be correlated to the apparent affinities determined by in vivo multicopy suppression experiments. These results are inconsistent with previous reports that E inhibited membrane-embedded MraY but not the detergent-solubilized enzyme, which led to a model in which E functions by binding MraY and blocking the formation of an essential heteromultimeric complex involving MraY and other murein biosynthesis enzymes. We discuss a new model in which E binds to MraY at a site composed of the two transmembrane domains within which the E resistance mutations map and the fact that the result of this binding is a conformational change that inactivates the enzyme.

  16. Haemoglobin-Triton X-100 conjugate as model system for red blood cell lysis

    NASA Astrophysics Data System (ADS)

    Pop, Simona-Florentina; Ion, Rodica-Mariana; Doncea, Sanda

    2010-11-01

    The action of detergents is thought to be connected primarily with micelle formation. However, detergent monomers can also affect biological systems. It was found that human red blood cells can be disintegrated with Triton X-100 non-ionic detergent at a concentration of 0.007%, lower than the critical micellar concentration (CMC). The lytic membrane of non-ionic detergent Triton X-100 (as a model), and its ability to lyse red blood cells in vitro used as an indicator of conjugate conformation at different pHs. The time dependent release of hemoglobin (Hb) and potassium from red blood cells was detected at 37 °C and both were sigmoid in character. Although Triton X-100 was highly lytic at pH 5.5, 7.4 and 8.0, the conjugate only show a lysis concentration-dependent of red blood cell at pH 5.5. Triton X-100 causes the Hb to aggregate, a condition that can be simulated when this non-ionic surfactant is incubated with Hb in vitro. The determination of Triton-X was done by HPLC, in accordance to characterize the surfactant. The increased stability in micellar medium can be attributed to deep penetration with the polar group -OH oriented towarded to the micelle surface. Thermal stability of hemoglobin has been investigated in order to evaluate the nature of thermal behavior of this compound. We studied the effects of surfactant Triton -X on the rate constants for the destroying of hemoglobin.

  17. The Salmonella virulence plasmid enhances Salmonella-induced lysis of macrophages and influences inflammatory responses.

    PubMed Central

    Guilloteau, L A; Wallis, T S; Gautier, A V; MacIntyre, S; Platt, D J; Lax, A J

    1996-01-01

    The Salmonella dublin virulence plasmid mediates systemic infection in mice and cattle. Here, we analyze the interaction between wild-type and plasmid-cured Salmonella strains with phagocytes in vitro and in vivo. The intracellular recovery of S. dublin from murine peritoneal and bovine alveolar macrophages cultured in the presence of gentamicin in vitro was not related to virulence plasmid carriage. However, the virulence plasmid increased the lytic activity of S. dublin, Salmonella typhimurium, and Salmonella choleraesuis for resident or activated mouse peritoneal macrophages. Lysis was not mediated by spv genes and was abolished by cytochalasin D treatment. Peritoneal and splenic macrophages were isolated from mice 4 days after intraperitoneal infection with wild-type or plasmid-cured S. dublin strains. The wild-type strain was recovered in significantly higher numbers than the plasmid-cured strain. However, the intracellular killing rates of such cells cultured in vitro for both S. dublin strains were not significantly different. Four days after infection, there was a lower increase of phagocyte numbers in the peritoneal cavities and spleens of mice infected with the wild-type strain compared with the plasmid-cured strain. The virulence plasmid influenced the survival of macrophages in vitro following infection in vivo as assessed by microscopy. Cells from mice infected with the plasmid-cured strain survived better than those from mice infected with the wild-type strain. This is the first report demonstrating an effect of the virulence plasmid on the interaction of Salmonella strains with macrophages. Plasmid-mediated macrophage dysfunction could influence the recruitment and/or the activation of phagocytic cells and consequently the net growth of Salmonella strains during infection. PMID:8757880

  18. Single Lysis-Salting Out Method of Genomic DNA Extraction From Dried Blood Spots.

    PubMed

    Shaik, Muntaj; Shivanna, Devaraju Kuramkote; Kamate, Mahesh; Ab, Vedamurthy; Tp, Kruthika-Vinod

    2016-11-01

    Dried blood spots (DBS) are an important form of bio-sampling and valuable approach for storing blood samples for genetic studies. This has necessitated in developing an effective protocol to isolate genomic DNA (gDNA) from DBS samples.In this study, we have elucidated a dependable and non-hazardous "single lysis-salting out" (SLSO) protocol of gDNA extraction from DBS and compared against the available commercial kits. For the purpose of this study, blood spots were collected on S&S 903 filter cards from 10 healthy volunteers and 30 patients with glutaric aciduria type I (GA-I). The gDNA was extracted from theseDBS samples by SLSO, QIAamp® gDNA Micro kit and innuPREP forensic kit methods. The quantity and quality of gDNA obtained from these methods were determined by measuring the absorbance using a Nanodrop spectrophotometer. The SLSO method showed four-fold and eight-fold increased yield of gDNA in healthy volunteers and patient samples, respectively, compared to commercial kits (p<0.0001). The protocol was also found to be cost efficient, reducing the per sample cost to almost half. The suitability of this method for genetic studies was confirmed by performing R402W genotyping by RFLP in GA-I patients. The genotyping results showed the presence of R402W mutation in 20% (6/30) of patients. The SLSO method was found to be inexpensive, non-hazardous and a suitable technique for isolating gDNA from DBS samples for genetic studies. © 2016 Wiley Periodicals, Inc.

  19. Artificial Intelligence vs. Statistical Modeling and Optimization of Continuous Bead Milling Process for Bacterial Cell Lysis

    PubMed Central

    Haque, Shafiul; Khan, Saif; Wahid, Mohd; Dar, Sajad A.; Soni, Nipunjot; Mandal, Raju K.; Singh, Vineeta; Tiwari, Dileep; Lohani, Mohtashim; Areeshi, Mohammed Y.; Govender, Thavendran; Kruger, Hendrik G.; Jawed, Arshad

    2016-01-01

    For a commercially viable recombinant intracellular protein production process, efficient cell lysis and protein release is a major bottleneck. The recovery of recombinant protein, cholesterol oxidase (COD) was studied in a continuous bead milling process. A full factorial response surface methodology (RSM) design was employed and compared to artificial neural networks coupled with genetic algorithm (ANN-GA). Significant process variables, cell slurry feed rate (A), bead load (B), cell load (C), and run time (D), were investigated and optimized for maximizing COD recovery. RSM predicted an optimum of feed rate of 310.73 mL/h, bead loading of 79.9% (v/v), cell loading OD600 nm of 74, and run time of 29.9 min with a recovery of ~3.2 g/L. ANN-GA predicted a maximum COD recovery of ~3.5 g/L at an optimum feed rate (mL/h): 258.08, bead loading (%, v/v): 80%, cell loading (OD600 nm): 73.99, and run time of 32 min. An overall 3.7-fold increase in productivity is obtained when compared to a batch process. Optimization and comparison of statistical vs. artificial intelligence techniques in continuous bead milling process has been attempted for the very first time in our study. We were able to successfully represent the complex non-linear multivariable dependence of enzyme recovery on bead milling parameters. The quadratic second order response functions are not flexible enough to represent such complex non-linear dependence. ANN being a summation function of multiple layers are capable to represent complex non-linear dependence of variables in this case; enzyme recovery as a function of bead milling parameters. Since GA can even optimize discontinuous functions present study cites a perfect example of using machine learning (ANN) in combination with evolutionary optimization (GA) for representing undefined biological functions which is the case for common industrial processes involving biological moieties. PMID:27920762

  20. A chloride-inducible gene expression cassette and its use in induced lysis of Lactococcus lactis.

    PubMed Central

    Sanders, J W; Venema, G; Kok, J

    1997-01-01

    A chloride-inducible promoter previously isolated from the chromosome of Lactococcus lactis (J. W. Sanders, G. Venema, J. Kok, and K. Leenhouts, Mol. Gen. Genet., in press) was exploited for the inducible expression of homologous and heterologous genes. An expression cassette consisting of the positive-regulator gene gadR, the chloride-inducible promoter Pgad, and the translation initiation signals of gadC was amplified by PCR. The cassette was cloned upstream of Escherichia coli lacZ, the holin-lysin cassette (lytPR) of the lactococcal bacteriophage r1t, and the autolysin gene of L. lactis, acmA. Basal activity of Pgad resulted in a low level of expression of all three proteins. Growth in the presence of 0.5 M NaCl of a strain containing the gadC::lacZ fusion resulted in a 1,500-fold increase of beta-galactosidase activity. The background activity levels of LytPR and AcmA had no deleterious effects on cell growth, but induction of lysin expression by addition of 0.5 M NaCl resulted in inhibition of growth. Lysis was monitored by following the release of the cytoplasmic marker enzyme PepX. Released PepX activity was maximal at 1 day after induction of lytPR expression with 0.1 M NaCl. Induction of acmA expression resulted in slower release of PepX from the cells. The presence of the inducing agent NaCl resulted in the stabilization of osmotically fragile cells. PMID:9406408

  1. Microphotographs of cyanobacteria documenting the effects of various cell-lysis techniques

    USGS Publications Warehouse

    Rosen, Barry H.; Loftin, Keith A.; Smith, Christopher E.; Lane, Rachael F.; Keydel, Susan P.

    2011-01-01

    Cyanotoxins are a group of organic compounds biosynthesized intracellularly by many species of cyanobacteria found in surface water. The United States Environmental Protection Agency has listed cyanotoxins on the Safe Drinking Water Act's Contaminant Candidate List 3 for consideration for future regulation to protect public health. Cyanotoxins also pose a risk to humans and other organisms in a variety of other exposure scenarios. Accurate and precise analytical measurements of cyanotoxins are critical to the evaluation of concentrations in surface water to address the human health and ecosystem effects. A common approach to total cyanotoxin measurement involves cell membrane disruption to release the cyanotoxins to the dissolved phase followed by filtration to remove cellular debris. Several methods have been used historically, however no standard protocols exist to ensure this process is consistent between laboratories before the dissolved phase is measured by an analytical technique for cyanotoxin identification and quantitation. No systematic evaluation has been conducted comparing the multiple laboratory sample processing techniques for physical disruption of cell membrane or cyanotoxins recovery. Surface water samples collected from lakes, reservoirs, and rivers containing mixed assemblages of organisms dominated by cyanobacteria, as well as laboratory cultures of species-specific cyanobacteria, were used as part of this study evaluating multiple laboratory cell-lysis techniques in partnership with the U.S. Environmental Protection Agency. Evaluated extraction techniques included boiling, autoclaving, sonication, chemical treatment, and freeze-thaw. Both treated and untreated samples were evaluated for cell membrane integrity microscopically via light, epifluorescence, and epifluorescence in the presence of a DNA stain. The DNA stain, which does not permeate live cells with intact membrane structures, was used as an indicator for cyanotoxin release into the

  2. Myxoma Virus Infection Promotes NK Lysis of Malignant Gliomas In Vitro and In Vivo

    PubMed Central

    Ogbomo, Henry; Zemp, Franz J.; Lun, Xueqing; Zhang, Jiqing; Stack, Danuta; Rahman, Masmudur M.; Mcfadden, Grant; Mody, Christopher H.; Forsyth, Peter A.

    2013-01-01

    Myxoma virus (MYXV) is a well-established oncolytic agent against different types of tumors. MYXV is also known for its immunomodulatory properties in down-regulating major histocompatibility complex (MHC) I surface expression (via the M153R gene product, a viral E3-ubiquitin ligase) and suppressing T cell killing of infected target cells. MHC I down-regulation, however, favors NK cell activation. Brain tumors including gliomas are characterized by high MHC I expression with impaired NK activity. We thus hypothesized that MYXV infection of glioma cells will promote NK cell-mediated recognition and killing of gliomas. We infected human gliomas with MYXV and evaluated their susceptibility to NK cell-mediated cytotoxicity. MYXV enhanced NK cell-mediated killing of glioma cells (U87 cells, MYXV vs. Mock: 51.73% vs. 28.63%, P = .0001, t test; U251 cells, MYXV vs. Mock: 40.4% vs. 20.03%, P .0007, t test). Using MYXV M153R targeted knockout (designated vMyx-M153KO) to infect gliomas, we demonstrate that M153R was responsible for reduced expression of MHC I on gliomas and enhanced NK cell-mediated antiglioma activity (U87 cells, MYXV vs. vMyx-M153KO: 51.73% vs. 25.17%, P = .0002, t test; U251 cells, MYXV vs. vMyx-M153KO: 40.4% vs. 19.27, P = .0013, t test). Consequently, NK cell-mediated lysis of established human glioma tumors in CB-17 SCID mice was accelerated with improved mouse survival (log-rank P = .0072). These results demonstrate the potential for combining MYXV with NK cells to effectively kill malignant gliomas. PMID:23762498

  3. Myxoma virus infection promotes NK lysis of malignant gliomas in vitro and in vivo.

    PubMed

    Ogbomo, Henry; Zemp, Franz J; Lun, Xueqing; Zhang, Jiqing; Stack, Danuta; Rahman, Masmudur M; McFadden, Grant; Mody, Christopher H; Forsyth, Peter A

    2013-01-01

    Myxoma virus (MYXV) is a well-established oncolytic agent against different types of tumors. MYXV is also known for its immunomodulatory properties in down-regulating major histocompatibility complex (MHC) I surface expression (via the M153R gene product, a viral E3-ubiquitin ligase) and suppressing T cell killing of infected target cells. MHC I down-regulation, however, favors NK cell activation. Brain tumors including gliomas are characterized by high MHC I expression with impaired NK activity. We thus hypothesized that MYXV infection of glioma cells will promote NK cell-mediated recognition and killing of gliomas. We infected human gliomas with MYXV and evaluated their susceptibility to NK cell-mediated cytotoxicity. MYXV enhanced NK cell-mediated killing of glioma cells (U87 cells, MYXV vs. Mock: 51.73% vs. 28.63%, P = .0001, t test; U251 cells, MYXV vs. Mock: 40.4% vs. 20.03%, P .0007, t test). Using MYXV M153R targeted knockout (designated vMyx-M153KO) to infect gliomas, we demonstrate that M153R was responsible for reduced expression of MHC I on gliomas and enhanced NK cell-mediated antiglioma activity (U87 cells, MYXV vs. vMyx-M153KO: 51.73% vs. 25.17%, P = .0002, t test; U251 cells, MYXV vs. vMyx-M153KO: 40.4% vs. 19.27, P = .0013, t test). Consequently, NK cell-mediated lysis of established human glioma tumors in CB-17 SCID mice was accelerated with improved mouse survival (log-rank P = .0072). These results demonstrate the potential for combining MYXV with NK cells to effectively kill malignant gliomas.

  4. Clinical laboratory comparison of lysis-centrifugation and BACTEC radiometric blood culture techniques.

    PubMed

    McLaughlin, J C; Hamilton, P; Scholes, J V; Bartlett, R C

    1983-11-01

    The lysis-centrifugation technique (ISOLATOR; E. I. du Pont de Nemours & Co., Wilmington, Del.) and the radiometric blood culture technique (BACTEC; Johnston Laboratories, Inc., Cockeysville, Md.) were compared on 1,000 blood cultures. A total of 16 ml of blood was distributed: 8 ml into an ISOLATOR 7.5 microbial tube and 4 ml each into BACTEC 7C and 8B bottles. The concentrate from the ISOLATOR tubes was inoculated under a laminar-flow hood onto two sheep blood agar plates (one incubated in CO2 and one incubated anaerobically), one chocolate agar plate, and one brain heart infusion agar plate. Of 91 blood specimens obtained that yielded clinically significant organisms, 52 were positive by both systems, 27 were positive by the ISOLATOR system only, and 12 were positive by the BACTEC system only. From the positive blood specimens, 97 clinically significant organisms were isolated: 57 by both systems, 27 by the ISOLATOR system only, and 13 by the BACTEC system only. Of the 57 organisms detected by both systems, 28 were detected simultaneously, 13 were detected earlier by the ISOLATOR system, and 16 were detected earlier by the BACTEC system. Isolated colonies were obtained earlier by the ISOLATOR system in 40 cases and by the BACTEC system in 5 cases. Organisms determined to be contaminants by thorough chart review were isolated from 138 ISOLATOR tubes. In 98 instances, these were represented by one colony of Staphylococcus epidermidis, alpha-hemolytic streptococci, or diphtheroids. The ability to determine CFU per milliliter with the ISOLATOR system did not help differentiate clinically significant organisms from contaminants.

  5. Fractalkine Expression Induces Endothelial Progenitor Cell Lysis by Natural Killer Cells

    PubMed Central

    Todorova, Dilyana; Sabatier, Florence; Doria, Evelyne; Lyonnet, Luc; Vacher Coponat, Henri; Robert, Stéphane; Despoix, Nicolas; Legris, Tristan; Moal, Valérie; Loundou, Anderson; Morange, Sophie; Berland, Yvon; George, Francoise Dignat; Burtey, Stéphane; Paul, Pascale

    2011-01-01

    Background Circulating CD34+ cells, a population that includes endothelial progenitors, participate in the maintenance of endothelial integrity. Better understanding of the mechanisms that regulate their survival is crucial to improve their regenerative activity in cardiovascular and renal diseases. Chemokine-receptor cross talk is critical in regulating cell homeostasis. We hypothesized that cell surface expression of the chemokine fractalkine (FKN) could target progenitor cell injury by Natural Killer (NK) cells, thereby limiting their availability for vascular repair. Methodology/Principal Findings We show that CD34+-derived Endothelial Colony Forming Cells (ECFC) can express FKN in response to TNF-α and IFN-γ inflammatory cytokines and that FKN expression by ECFC stimulates NK cell adhesion, NK cell-mediated ECFC lysis and microparticles release in vitro. The specific involvement of membrane FKN in these processes was demonstrated using FKN-transfected ECFC and anti-FKN blocking antibody. FKN expression was also evidenced on circulating CD34+ progenitor cells and was detected at higher frequency in kidney transplant recipients, when compared to healthy controls. The proportion of CD34+ cells expressing FKN was identified as an independent variable inversely correlated to CD34+ progenitor cell count. We further showed that treatment of CD34+ circulating cells isolated from adult blood donors with transplant serum or TNF-α/IFN-γ can induce FKN expression. Conclusions Our data highlights a novel mechanism by which FKN expression on CD34+ progenitor cells may target their NK cell mediated killing and participate to their immune depletion in transplant recipients. Considering the numerous diseased contexts shown to promote FKN expression, our data identify FKN as a hallmark of altered progenitor cell homeostasis with potential implications in better evaluation of vascular repair in patients. PMID:22039526

  6. Chitosan as coagulant on cyanobacteria in lake restoration management may cause rapid cell lysis.

    PubMed

    Mucci, Maíra; Noyma, Natalia Pessoa; de Magalhães, Leonardo; Miranda, Marcela; van Oosterhout, Frank; Guedes, Iamê Alves; Huszar, Vera L M; Marinho, Marcelo Manzi; Lürling, Miquel

    2017-07-01

    Combining coagulant and ballast to remove cyanobacteria from the water column is a promising restoration technique to mitigate cyanobacterial nuisance in surface waters. The organic, biodegradable polymer chitosan has been promoted as a coagulant and is viewed as non-toxic. In this study, we show that chitosan may rapidly compromise membrane integrity and kill certain cyanobacteria leading to release of cell contents in the water. A strain of Cylindrospermopsis raciborskii and one strain of Planktothrix agardhii were most sensitive. A 1.3 h exposure to a low dose of 0.5 mg l(-1) chitosan already almost completely killed these cultures resulting in release of cell contents. After 24 h, reductions in PSII efficiencies of all cyanobacteria tested were observed. EC50 values varied from around 0.5 mg l(-1) chitosan for the two sensitive strains, via about 5 mg l(-1) chitosan for an Aphanizomenon flos-aquae strain, a toxic P. agardhii strain and two Anabaena cylindrica cultures, to more than 8 mg l(-1) chitosan for a Microcystis aeruginosa strain and another A. flos-aquae strain. Differences in sensitivity to chitosan might be related to polymeric substances that surround cyanobacteria. Rapid lysis of toxic strains is likely and when chitosan flocking and sinking of cyanobacteria is considered in lake restoration, flocculation efficacy studies should be complemented with investigation on the effects of chitosan on the cyanobacteria assemblage being targeted. Copyright © 2017 The Authors. Published by Elsevier Ltd.. All rights reserved.

  7. Pseudomonas aeruginosa Pore-Forming Exolysin and Type IV Pili Cooperate To Induce Host Cell Lysis

    PubMed Central

    Basso, Pauline; Ragno, Michel; Elsen, Sylvie; Reboud, Emeline; Golovkine, Guillaume; Bouillot, Stephanie; Huber, Philippe; Lory, Stephen; Faudry, Eric

    2017-01-01

    ABSTRACT   Clinical strains of Pseudomonas aeruginosa lacking the type III secretion system genes employ a toxin, exolysin (ExlA), for host cell membrane disruption. Here, we demonstrated that ExlA export requires a predicted outer membrane protein, ExlB, showing that ExlA and ExlB define a new active two-partner secretion (TPS) system of P. aeruginosa. In addition to the TPS signals, ExlA harbors several distinct domains, which include one hemagglutinin domain, five arginine-glycine-aspartic acid (RGD) motifs, and a C-terminal region lacking any identifiable sequence motifs. However, this C-terminal region is important for the toxic activity, since its deletion abolishes host cell lysis. Using lipid vesicles and eukaryotic cells, including red blood cells, we demonstrated that ExlA has a pore-forming activity which precedes cell membrane disruption of nucleated cells. Finally, we developed a high-throughput cell-based live-dead assay and used it to screen a transposon mutant library of an ExlA-producing P. aeruginosa clinical strain for bacterial factors required for ExlA-mediated toxicity. The screen resulted in the identification of proteins involved in the formation of type IV pili as being required for ExlA to exert its cytotoxic activity by promoting close contact between bacteria and the host cell. These findings represent the first example of cooperation between a pore-forming toxin of the TPS family and surface appendages in host cell intoxication. PMID:28119472

  8. Pseudomonas aeruginosa Pore-Forming Exolysin and Type IV Pili Cooperate To Induce Host Cell Lysis.

    PubMed

    Basso, Pauline; Ragno, Michel; Elsen, Sylvie; Reboud, Emeline; Golovkine, Guillaume; Bouillot, Stephanie; Huber, Philippe; Lory, Stephen; Faudry, Eric; Attrée, Ina

    2017-01-24

    Clinical strains of Pseudomonas aeruginosa lacking the type III secretion system genes employ a toxin, exolysin (ExlA), for host cell membrane disruption. Here, we demonstrated that ExlA export requires a predicted outer membrane protein, ExlB, showing that ExlA and ExlB define a new active two-partner secretion (TPS) system of P. aeruginosa In addition to the TPS signals, ExlA harbors several distinct domains, which include one hemagglutinin domain, five arginine-glycine-aspartic acid (RGD) motifs, and a C-terminal region lacking any identifiable sequence motifs. However, this C-terminal region is important for the toxic activity, since its deletion abolishes host cell lysis. Using lipid vesicles and eukaryotic cells, including red blood cells, we demonstrated that ExlA has a pore-forming activity which precedes cell membrane disruption of nucleated cells. Finally, we developed a high-throughput cell-based live-dead assay and used it to screen a transposon mutant library of an ExlA-producing P. aeruginosa clinical strain for bacterial factors required for ExlA-mediated toxicity. The screen resulted in the identification of proteins involved in the formation of type IV pili as being required for ExlA to exert its cytotoxic activity by promoting close contact between bacteria and the host cell. These findings represent the first example of cooperation between a pore-forming toxin of the TPS family and surface appendages in host cell intoxication.

  9. Lysis of gram-positive and gram-negative bacteria by antibacterial porous polymeric monolith formed in microfluidic biochips for sample preparation.

    PubMed

    Aly, Mohamed Aly Saad; Gauthier, Mario; Yeow, John

    2014-09-01

    Bacterial cell lysis is demonstrated using polymeric microfluidic biochips operating via a hybrid mechanical shearing/contact killing mechanism. These biochips are fabricated from a cross-linked poly(methyl methacrylate) (X-PMMA) substrate by well-controlled, high-throughput laser micromachining. The unreacted double bonds at the surface of X-PMMA provide covalent bonding for the formation of a porous polymeric monolith (PPM), thus contributing to the mechanical stability of the biochip and eliminating the need for surface treatment. The lysis efficiency of these biochips was tested for gram-positive (Enterococcus saccharolyticus and Bacillus subtilis) and gram-negative bacteria (Escherichia coli and Pseudomonas fluorescens) and confirmed by off-chip PCR without further purification. The influence of the flow rate when pumping the bacterial suspension through the PPM, and of the hydrophobic-hydrophilic balance on the cell lysis efficiency was investigated at a cell concentration of 10(5) CFU/mL. It was shown that the contribution of contact killing to cell lysis was more important than that of mechanical shearing in the PPM. The biochip showed better lysis efficiency than the off-chip chemical, mechanical, and thermal lysis techniques used in this work. The biochip also acts as a filter that isolates cell debris and allows PCR-amplifiable DNA to pass through. The system performs more efficient lysis for gram-negative than for gram-positive bacteria. The biochip does not require chemical/enzymatic reagents, power consumption, or complicated design and fabrication processes, which makes it an attractive on-chip lysis device that can be used in sample preparation for genetics and point-of-care diagnostics. The biochips were reused for 20 lysis cycles without any evidence of physical damage to the PPM, significant performance degradation, or DNA carryover when they were back-flushed between cycles. The biochips efficiently lysed both gram-positive and gram

  10. Bacterial Lysis through Interference with Peptidoglycan Synthesis Increases Biofilm Formation by Nontypeable Haemophilus influenzae

    PubMed Central

    Puig, Carmen; Merlos, Alexandra; Viñas, Miguel; de Jonge, Marien I.; Liñares, Josefina; Ardanuy, Carmen

    2017-01-01

    ABSTRACT Nontypeable Haemophilus influenzae (NTHi) is an opportunistic pathogen that mainly causes otitis media in children and community-acquired pneumonia or exacerbations of chronic obstructive pulmonary disease in adults. A large variety of studies suggest that biofilm formation by NTHi may be an important step in the pathogenesis of this bacterium. However, the underlying mechanisms involved in this process are poorly elucidated. In this study, we used a transposon mutant library to identify bacterial genes involved in biofilm formation. The growth and biofilm formation of 4,172 transposon mutants were determined, and the involvement of the identified genes in biofilm formation was validated in in vitro experiments. Here, we present experimental data showing that increased bacterial lysis, through interference with peptidoglycan synthesis, results in elevated levels of extracellular DNA, which increased biofilm formation. Interestingly, similar results were obtained with subinhibitory concentrations of β-lactam antibiotics, known to interfere with peptidoglycan synthesis, but such an effect does not appear with other classes of antibiotics. These results indicate that treatment with β-lactam antibiotics, especially for β-lactam-resistant NTHi isolates, might increase resistance to antibiotics by increasing biofilm formation. IMPORTANCE Most, if not all, bacteria form a biofilm, a multicellular structure that protects them from antimicrobial actions of the host immune system and affords resistance to antibiotics. The latter is especially disturbing with the increase in multiresistant bacterial clones worldwide. Bacterial biofilm formation is a multistep process that starts with surface adhesion, after which attached bacteria divide and give rise to biomass. The actual steps required for Haemophilus influenzae biofilm formation are largely not known. We show that interference with peptidoglycan biosynthesis increases biofilm formation because of the release

  11. Host range and in vitro lysis of Listeria monocytogenes seafood isolates by bacteriophages.

    PubMed

    Arachchi, Geevika J Ganegama; Cruz, Cristina D; Dias-Wanigasekera, Beatrice M; McIntyre, Lynn; Billington, Craig; Hudson, Andrew; Flint, Steve H; Mutukumira, Anthony N

    2014-12-01

    Listeria-infecting bacteriophages (listeriaphages) can be used to control Listeria monocytogenes in the food industry. However, the sensitivity of many of seafood-borne Listeria strains to phages has not been reported. This research investigated the host ranges of three listeriaphages (FWLLm1, FWLLm3 and FWLLm5) by the formation of lytic zones and plaques on host lawns and in vitro lysis kinetics of listeriaphage FWLLm3. The study also predicted the phage titres required to lyse host cells. The host ranges of the phages were determined using 50 L. monocytogenes strains, of which 48 were isolated from the seafood industry and two from clinical cases. Of the 50 strains, 36 were tested at 25 and 30 ℃ and the remainder (14) at 15 and 25 ℃. Based on the formation of either discrete plaques or lytic zones (host kill zones), the host ranges of FWLLm1, FWLLm3 and FWLLm5 were about 87%, 81% and 87%, respectively, at 25 ℃. Six L. monocytogenes strains from the seafood environment were insensitive to all three phages, while the other seafood strains (42) were phage-sensitive. The adsorption rate constant (k value) of listeriaphage FWLLm3 was between 1.2 × 10(-9) and 1.6 × 10(-9 )ml/min across four host strains in tryptic soy broth at 25 ℃. The cultures (at 3-4 log colony-forming unit (CFU/ml) were completely lysed (<1 log CFU/ml) when cultures were infected with FWLLm3 at > 8.7 log phage-forming units (PFU/ml) for 30 min. Re-growth of phage-infected cultures was not detected after 24 h. The effective empirical phage titre was similar to the calculated titre using a kinetic model. Results indicate the potential use of the three phages for controlling L. monocytogenes strains in seafood processing environments. © The Author(s) 2013 Reprints and permissions: sagepub.co.uk/journalsPermissions.nav.

  12. Virioplankton dynamics and virally induced phytoplankton lysis versus microzooplankton grazing southeast of the Kerguelen (Southern Ocean)

    NASA Astrophysics Data System (ADS)

    Brussaard, C. P. D.; Timmermans, K. R.; Uitz, J.; Veldhuis, M. J. W.

    2008-03-01

    Viral dynamics, community structure, and the impact of viruses on phytoplankton mortality in comparison with microzooplankton grazing were determined in the natural iron-fertilized waters southeast of the Kerguelen Islands, Southern Ocean, during the austral summer (January-February 2005). The study area was characterized by a phytoplankton bloom above the Kerguelen Plateau and the high-nutrient low-chlorophyll waters surrounding it. During the Kerguelen Ocean and Plateau compared Study (KEOPS), viral abundance was relatively high (1-19×10 7 mL -1) as compared to the few other studies in the Southern Ocean, significantly correlating with depth and system productivity. Viral abundance showed a strong positive relationship with the numerically dominant bacterial hosts, which in turn were correlated to phytoplankton biomass. In total, 13 different viral genome sizes were detected, with the lower-sized genomes 34 and 68 kb dominating at all stations. The viral community at the low chlorophyll C-transect grouped apart from the more productive transects A and B. Potential algal viruses were recorded for all stations, but only at very low intensities. Virally induced lysis of the smaller-sized (<10 μm) phytoplankton was a minor loss factor as compared to microzooplankton grazing (up to 6% and 45% of total <30 μm algal standing stock per day, respectively). Grazing was phytoplankton population-specific, but was in all cases able to keep the standing stock of the small-sized phytoplankton low (net growth rates between -0.2 and 0.2 d -1). Microzooplankton regenerated on average 1.1 pM Fe d -1 (present study), which represented approximately 30% of the total regeneration rate and at least 15% of the total biogenic Fe demand as calculated by [Sarthou, G., Vincent, D., Christaki, U., Obernosterer, I., Timmermans, K.R., Brussaard, C.P.D., 2008. The fate of biogenic iron during a phytoplankton bloom induced by natural fertilization: impact of copepod grazing. Deep-Sea Research

  13. Viral Lysis of Photosynthesizing Microbes As a Mechanism for Calcium Carbonate Nucleation in Seawater

    PubMed Central

    Lisle, John T.; Robbins, Lisa L.

    2016-01-01

    Removal of carbon through the precipitation and burial of calcium carbonate in marine sediments constitutes over 70% of the total carbon on Earth and is partitioned between coastal and pelagic zones. The precipitation of authigenic calcium carbonate in seawater, however, has been hotly debated because despite being in a supersaturated state, there is an absence of persistent precipitation. One of the explanations for this paradox is the geochemical conditions in seawater cannot overcome the activation energy barrier for the first step in any precipitation reaction; nucleation. Here we show that virally induced rupturing of photosynthetic cyanobacterial cells releases cytoplasmic-associated bicarbonate at concentrations ~23-fold greater than in the surrounding seawater, thereby shifting the carbonate chemistry toward the homogenous nucleation of one or more of the calcium carbonate polymorphs. Using geochemical reaction energetics, we show the saturation states (Ω) in typical seawater for calcite (Ω = 4.3), aragonite (Ω = 3.1), and vaterite (Ω = 1.2) are significantly elevated following the release and diffusion of the cytoplasmic bicarbonate (Ωcalcite = 95.7; Ωaragonite = 68.5; Ωvaterite = 25.9). These increases in Ω significantly reduce the activation energy for nuclei formation thresholds for all three polymorphs, but only vaterite nucleation is energetically favored. In the post-lysis seawater, vaterite's nuclei formation activation energy is significantly reduced from 1.85 × 10−17 J to 3.85 × 10−20 J, which increases the nuclei formation rate from highly improbable (<<1.0 nuclei cm−3 s−1) to instantaneous (8.60 × 1025 nuclei cm−3 s−1). The proposed model for homogenous nucleation of calcium carbonate in seawater describes a mechanism through which the initial step in the production of carbonate sediments may proceed. It also presents an additional role of photosynthesizing microbes and their viruses in marine carbon cycles and reveals

  14. Viral Lysis of Photosynthesizing Microbes As a Mechanism for Calcium Carbonate Nucleation in Seawater.

    PubMed

    Lisle, John T; Robbins, Lisa L

    2016-01-01

    Removal of carbon through the precipitation and burial of calcium carbonate in marine sediments constitutes over 70% of the total carbon on Earth and is partitioned between coastal and pelagic zones. The precipitation of authigenic calcium carbonate in seawater, however, has been hotly debated because despite being in a supersaturated state, there is an absence of persistent precipitation. One of the explanations for this paradox is the geochemical conditions in seawater cannot overcome the activation energy barrier for the first step in any precipitation reaction; nucleation. Here we show that virally induced rupturing of photosynthetic cyanobacterial cells releases cytoplasmic-associated bicarbonate at concentrations ~23-fold greater than in the surrounding seawater, thereby shifting the carbonate chemistry toward the homogenous nucleation of one or more of the calcium carbonate polymorphs. Using geochemical reaction energetics, we show the saturation states (Ω) in typical seawater for calcite (Ω = 4.3), aragonite (Ω = 3.1), and vaterite (Ω = 1.2) are significantly elevated following the release and diffusion of the cytoplasmic bicarbonate (Ωcalcite = 95.7; Ωaragonite = 68.5; Ωvaterite = 25.9). These increases in Ω significantly reduce the activation energy for nuclei formation thresholds for all three polymorphs, but only vaterite nucleation is energetically favored. In the post-lysis seawater, vaterite's nuclei formation activation energy is significantly reduced from 1.85 × 10(-17) J to 3.85 × 10(-20) J, which increases the nuclei formation rate from highly improbable (<1.0 nuclei cm(-3) s(-1)) to instantaneous (8.60 × 10(25) nuclei cm(-3) s(-1)). The proposed model for homogenous nucleation of calcium carbonate in seawater describes a mechanism through which the initial step in the production of carbonate sediments may proceed. It also presents an additional role of photosynthesizing microbes and their viruses in marine carbon cycles and reveals

  15. A microchip electrophoresis-mass spectrometric platform with double cell lysis nano-electrodes for automated single cell analysis.

    PubMed

    Li, Xiangtang; Zhao, Shulin; Hu, Hankun; Liu, Yi-Ming

    2016-06-17

    Capillary electrophoresis-based single cell analysis has become an essential approach in researches at the cellular level. However, automation of single cell analysis has been a challenge due to the difficulty to control the number of cells injected and the irreproducibility associated with cell aggregation. Herein we report the development of a new microfluidic platform deploying the double nano-electrode cell lysis technique for automated analysis of single cells with mass spectrometric detection. The proposed microfluidic chip features integration of a cell-sized high voltage zone for quick single cell lysis, a microfluidic channel for electrophoretic separation, and a nanoelectrospray emitter for ionization in MS detection. Built upon this platform, a microchip electrophoresis-mass spectrometric method (MCE-MS) has been developed for automated single cell analysis. In the method, cell introduction, cell lysis, and MCE-MS separation are computer controlled and integrated as a cycle into consecutive assays. Analysis of large numbers of individual PC-12 neuronal cells (both intact and exposed to 25mM KCl) was carried out to determine intracellular levels of dopamine (DA) and glutamic acid (Glu). It was found that DA content in PC-12 cells was higher than Glu content, and both varied from cell to cell. The ratio of intracellular DA to Glu was 4.20±0.8 (n=150). Interestingly, the ratio drastically decreased to 0.38±0.20 (n=150) after the cells are exposed to 25mM KCl for 8min, suggesting the cells released DA promptly and heavily while they released Glu at a much slower pace in response to KCl-induced depolarization. These results indicate that the proposed MCE-MS analytical platform may have a great potential in researches at the cellular level. Copyright © 2016 Elsevier B.V. All rights reserved.

  16. A microchip electrophoresis-mass spectrometric platform with double cell lysis nano-electrodes for automated single cell analysis

    PubMed Central

    Li, Xiangtang; Zhao, Shulin; Hu, Hankun; Liu, Yi-Ming

    2016-01-01

    Capillary electrophoresis-based single cell analysis has become an essential approach in researches at the cellular level. However, automation of single cell analysis has been a challenge due to the difficulty to control the number of cells injected and the irreproducibility associated with cell aggregation. Herein we report the development of a new microfluidic platform deploying the double nano-electrode cell lysis technique for automated analysis of single cells with mass spectrometric detection. The proposed microfluidic chip features integration of a cell-sized high voltage zone for quick single cell lysis, a microfluidic channel for electrophoretic separation, and a nanoelectrospray emitter for ionization in MS detection. Built upon this platform, a microchip electrophoresis - mass spectrometric method (MCE-MS) has been developed for automated single cell analysis. In the method, cell introduction, cell lysis, and MCE-MS separation are computer controlled and integrated as a cycle into consecutive assays. Analysis of large numbers of individual PC-12 neuronal cells (both intact and exposed to 25 mM KCl) was carried out to determine intracellular levels of dopamine (DA) and glutamic acid (Glu). It was found that DA content in PC-12 cells was higher than Glu content, and both varied from cell to cell. The ratio of intracellular DA to Glu was 4.20 ± 0.8 (n=150). Interestingly, the ratio drastically decreased to 0.38 ± 0.20 (n= 150) after the cells are exposed to 25 mM KCl for 8 min, suggesting the cells released DA promptly and heavily while they released Glu at a much slower pace in response to KCl-induced depolarization. These results indicate that the proposed MCE-MS analytical platform may have a great potential in researches at the cellular level. PMID:27207575

  17. Cannabinoids increase lung cancer cell lysis by lymphokine-activated killer cells via upregulation of ICAM-1.

    PubMed

    Haustein, Maria; Ramer, Robert; Linnebacher, Michael; Manda, Katrin; Hinz, Burkhard

    2014-11-15

    Cannabinoids have been shown to promote the expression of the intercellular adhesion molecule 1 (ICAM-1) on lung cancer cells as part of their anti-invasive and antimetastatic action. Using lung cancer cell lines (A549, H460) and metastatic cells derived from a lung cancer patient, the present study addressed the impact of cannabinoid-induced ICAM-1 on cancer cell adhesion to lymphokine-activated killer (LAK) cells and LAK cell-mediated cytotoxicity. Cannabidiol (CBD), a non-psychoactive cannabinoid, enhanced the susceptibility of cancer cells to adhere to and subsequently be lysed by LAK cells, with both effects being reversed by a neutralizing ICAM-1 antibody. Increased cancer cell lysis by CBD was likewise abrogated when CBD-induced ICAM-1 expression was blocked by specific siRNA or by antagonists to cannabinoid receptors (CB1, CB2) and to transient receptor potential vanilloid 1. In addition, enhanced killing of CBD-treated cancer cells was reversed by preincubation of LAK cells with an antibody to lymphocyte function associated antigen-1 (LFA-1) suggesting intercellular ICAM-1/LFA-1 crosslink as crucial event within this process. ICAM-1-dependent pro-killing effects were further confirmed for the phytocannabinoid Δ(9)-tetrahydrocannabinol (THC) and R(+)-methanandamide (MA), a hydrolysis-stable endocannabinoid analogue. Finally, each cannabinoid elicited no significant increase of LAK cell-mediated lysis of non-tumor bronchial epithelial cells, BEAS-2B, associated with a far less pronounced (CBD, THC) or absent (MA) ICAM-1 induction as compared to cancer cells. Altogether, our data demonstrate cannabinoid-induced upregulation of ICAM-1 on lung cancer cells to be responsible for increased cancer cell lysis by LAK cells. These findings provide proof for a novel antitumorigenic mechanism of cannabinoids. Copyright © 2014 Elsevier Inc. All rights reserved.

  18. Polychromatic Light (480-3400 nm) Upregulates Sensitivity of Tumor Cells to Lysis by Natural Killers.

    PubMed

    Knyazev, Nickolay A; Samoilova, Kira A; Abrahamse, Heidi; Filatova, Natalia A

    2016-09-01

    This study evaluates the participation of immunological mechanisms of downregulation of murine hepatoma cells MH22a after direct exposure to polychromatic polarized light. Previous studies have shown that exposure to a combination of visible (VIS) and infrared (IR) light leads to decreased tumorigenicity of the murine hepatoma cells MH22a, which correlated with an increase in the amount of cells with reorganized cytoskeleton in the submembrane region. The mechanism of tumor inhibition and elimination has not been determined. Polychromatic light (480-3400 nm) has been used at doses of 4.8 and 9.6 J/cm(2) to determine the sensitivity of murine MH22a cells and human erythroleukemia cells K562 exposed to this light, to lysis by effector cells of innate immunity (NK cells), and enhancement of the glycocalyx of the studied tumor cells. This was determined using flow cytometry, the H(3)-uridine cytotoxic test followed by spectrophotometry. VIS-IR light increases the sensitivity of MH-22a cells at a dose 4.8 J/cm(2) and K562 cells at 9.6 J/cm(2). The enhancement of sensitivity of tumor cells to NK lysis changed their ability to absorb alcian blue, reflecting a change in the expression of the glycocalyx. Increasing the sensitivity of the murine tumor cells MH22a and human K562 irradiated VIS-IR light correlated with a change in the expression of their glycocalyx. The results of the present study demonstrate that the reduction of tumorigenicity of irradiated tumor cells is due to their sensitivity to lysis by NK cells of the immune system.

  19. Severe Tumor Lysis Syndrome and Acute Pulmonary Edema Requiring Extracorporeal Membrane Oxygenation Following Initiation of Chemotherapy for Metastatic Alveolar Rhabdomyosarcoma.

    PubMed

    Sanford, Ethan; Wolbrink, Traci; Mack, Jennifer; Rowe, R Grant

    2016-05-01

    We present an 8-year-old male with metastatic alveolar rhabdomyosarcoma (ARMS) who developed precipitous cardiopulmonary collapse with severe tumor lysis syndrome (TLS) 48 hr after initiation of chemotherapy. Despite no detectable pulmonary metastases, acute hypoxemic respiratory failure developed, requiring extracorporeal membrane oxygenation (ECMO). Although TLS has been reported in disseminated ARMS, this singular case of life-threatening respiratory deterioration developing after initiation of chemotherapy presented unique therapeutic dilemmas. We review the clinical aspects of this case, including possible mechanisms of respiratory failure, and discuss the role of ECMO utilization in pediatric oncology.

  20. The optimization of in vitro high-throughput chemical lysis of Escherichia coli. Application to ACP domain of the polyketide synthase ppsC from Mycobacterium tuberculosis.

    PubMed

    Listwan, Pawel; Pédelacq, Jean-Denis; Lockard, Meghan; Bell, Carolyn; Terwilliger, Thomas C; Waldo, Geoffrey S

    2010-03-01

    Protein production in Escherichia coli involves high-level expression in a culture, followed by harvesting of the cells and finally their disruption, or lysis, to release the expressed proteins. We compare three high-throughput chemical lysis methods to sonication, using a robotic platform and methodologies developed in our laboratory [1]. Under the same expression conditions, all lysis methods varied in the degree of released soluble proteins. With a set of 96 test proteins, we used our split GFP to quantify the soluble and insoluble protein fractions after lysis. Both the amount of soluble protein and the percentage recovered in the soluble fraction using SoluLyse were well correlated with sonication. Two other methods, Bugbuster and lysozyme, did not correlate well with sonication. Considering the effects of lysis methods on protein solubility is especially important when accurate protein solubility measurements are needed, for example, when testing adjuvants, growth media, temperature, or when establishing the effects of truncation or sequence variation on protein stability.

  1. Concerning the role of cell lysis-cryptic growth in anaerobic side-stream reactors: the single-cell analysis of viable, dead and lysed bacteria.

    PubMed

    Foladori, P; Velho, V F; Costa, R H R; Bruni, L; Quaranta, A; Andreottola, G

    2015-05-01

    In the Anaerobic Side-Stream Reactor (ASSR), part of the return sludge undergoes alternating aerobic and anaerobic conditions with the aim of reducing sludge production. In this paper, viability, enzymatic activity, death and lysis of bacterial cells exposed to aerobic and anaerobic conditions for 16 d were investigated at single-cell level by flow cytometry, with the objective of contributing to the understanding of the mechanisms of sludge reduction in the ASSR systems. Results indicated that total and viable bacteria did not decrease during the anaerobic phase, indicating that anaerobiosis at ambient temperature does not produce a significant cell lysis. Bacteria decay and lysis occurred principally under aerobic conditions. The aerobic decay rate of total bacteria (bTB) was considered as the rate of generation of lysed bacteria. Values of bTB of 0.07-0.11 d(-1) were measured in anaerobic + aerobic sequence. The enzymatic activity was not particularly affected by the transition from anaerobiosis to aerobiosis. Large solubilisation of COD and NH4(+) was observed only under anaerobic conditions, as a consequence of hydrolysis of organic matter, but not due to cell lysis. The observations supported the proposal of two independent mechanisms contributing equally to sludge reduction: (1) under anaerobic conditions: sludge hydrolysis of non-bacterial material, (2) under aerobic conditions: bacterial cell lysis and oxidation of released biodegradable compounds.

  2. The Combined Approach to Lysis Utilizing Eptifibatide and rt-PA in Acute Ischemic Stroke

    PubMed Central

    Pancioli, Arthur M.; Broderick, Joseph; Brott, Thomas; Tomsick, Thomas; Khoury, Jane; Bean, Judy; del Zoppo, Gregory; Kleindorfer, Dawn; Woo, Daniel; Khatri, Pooja; Castaldo, John; Frey, James; Gebel, James; Kasner, Scott; Kidwell, Chelsea; Kwiatkowski, Thomas; Libman, Richard; Mackenzie, Richard; Scott, Phillip; Starkman, Sidney; Thurman, R. Jason

    2008-01-01

    Background and Purpose Multiple approaches are being studied to enhance the rate of thrombolysis for acute ischemic stroke. Treatment of myocardial infarction with a combination of a reduced-dose fibrinolytic agent and a glycoprotein (GP) IIb/IIIa receptor antagonist has been shown to improve the rate of recanalization versus fibrinolysis alone. The combined approach to lysis utilizing eptifibatide and recombinant tissue-type plasminogen activator (rt-PA) (CLEAR) stroke trial assessed the safety of treating acute ischemic stroke patients within 3 hours of symptom onset with this combination. Methods The CLEAR trial was a National Institutes of Health/National Institute of Neurological Disorders and Stroke–funded multicenter, double-blind, randomized, dose-escalation and safety study. Patients were randomized 3:1 to either low-dose rt-PA (tier 1=0.3 mg/kg, tier 2=0.45 mg/kg) plus eptifibatide (75 μg/kg bolus followed by 0.75 μg/kg per min infusion for 2 hours) or standard-dose rt-PA (0.9 mg/kg). The primary safety end point was the incidence of symptomatic intracerebral hemorrhage within 36 hours. Secondary analyses were performed regarding clinical efficacy. Results Ninety-four patients (40 in tier 1 and 54 in tier 2) were enrolled. The combination group of the 2 dose tiers (n=69) had a median age of 71 years and a median baseline National Institutes of Health Stroke Scale (NIHSS) score of 14, and the standard-dose rt-PA group (n=25) had a median age of 61 years and a median baseline NIHSS score of 10 (P=0.01 for NIHSS score). Fifty-two (75%) of the combination treatment group and 24 (96%) of the standard treatment group had a baseline modified Rankin scale score of 0 (P=0.04). There was 1 (1.4%; 95% CI, 0% to 4.3%) symptomatic intracranial hemorrhage in the combination group and 2 (8.0%; 95% CI, 0% to 19.2%) in the rt-PA–only arm (P=0.17). During randomization in tier 2, a review by the independent data safety monitoring board demonstrated that the safety

  3. Pitfalls, prevention, and treatment of hyperuricemia during tumor lysis syndrome in the era of rasburicase (recombinant urate oxidase)

    PubMed Central

    Pession, Andrea; Melchionda, Fraia; Castellini, Claudia

    2008-01-01

    Along with hydration and urinary alkalinization, allopurinol has been the standard agent for the management of hyperuricemia in patients with a high tumor burden at risk of tumor lysis syndrome; however, this agent often fails to prevent and treat this complication effectively. Rasburicase (recombinant urate oxidase) has been shown to be effective in reducing uric acid and preventing uric acid accumulation in patients with hematologic malignancies with hyperuricemia or at high risk of developing it. Rasburicase acts at the end of the purine catabolic pathway and, unlike allopurinol, does not induce accumulation of xanthine or hypoxanthine. Its rapid onset of action and the ability to lower pre-existing elevated uric acid levels are the advantages of rasburicase over allopurinol. Rasburicase represents an effective alternative to allopurinol to promptly reduce uric acid levels, improve patient’s electrolyte status, and reverse renal insufficiency. The drug, initially studied in pediatric patients with acute lymphoblastic leukemia and aggressive non-Hodgkin lymphoma, seems to show comparable benefit in adults with similar lymphoid malignancies or at high risk of tumor lysis syndrome. Current and future trials will evaluate alternative doses and different schedules of rasburicase to maintain its efficacy while reducing its cost. The review provides a comprehensive and detailed review of pathogenesis, laboratory, and clinical presentation of TLS together with clinical studies already performed both in pediatric and adult patients. PMID:19707436

  4. Recyclable Photo-Thermal Nano-Aggregates of Magnetic Nanoparticle Conjugated Gold Nanorods for Effective Pathogenic Bacteria Lysis.

    PubMed

    Ramasamy, Mohankandhasamy; Kim, Sanghyo; Lee, Su Seong; Yi, Dong Kee

    2016-01-01

    We describe the nucleophilic hybridization technique for fabricating magnetic nanoparticle (MNP) around gold nanorod (AuNR) for desired photo-thermal lysis on pathogenic bacteria. From the electromagnetic energy conversion into heat to the surrounding medium, a significant and quicker temperature rise was noted after light absorption on nanohybrids, at a controlled laser light output and optimum nanoparticle concentration. We observed a similar photo-thermal pattern for more than three times for the same material up on repeated magnetic separation. Regardless of the cell wall nature, superior pathogenic cell lysis has been observed for the bacteria suspensions of individual and mixed samples of Salmonella typhi (S.typhi) and Bacillus subtilis (B.subtilis) by the photo-heated nanoparticles. The synthesis of short gold nanorod, conjugation with magnetic nanoparticle and its subsequent laser exposure provides a rapid and reiterated photo-thermal effect with enhanced magnetic separation for efficient bactericidal application in water samples. Resultant novel properties of the nano-aggregates makes them a candidate to be used for a rapid, effective, and re-iterated photo-thermal agent against a wide variety of pathogens to attain microbe free water.

  5. A filterable lytic agent obtained from a red tide bloom that caused lysis of Karenia brevis (Gymnodinum breve) cultures

    USGS Publications Warehouse

    2002-01-01

    A filterable lytic agent (FLA) was obtained from seawater in the southeastern Gulf of Mexico during a red tide bloom that caused lysis of Karenia brevis (formerly Gymnodinium breve) Piney Island. This agent was obtained from <0.2µ  filtrates that were concentrated by ultrafiltration using a 100 kDa filter. The FLA was propagated by passage on K. brevis cultures, and the filtered supernatants of such cultures resulted in K. brevis lysis when added to such cultures. The lytic activity was lost upon heating to 65°C or by 0.02 µm filtration. Epifluorescence and transmission electron microscopy (TEM) of supernatants of K. brevis cultures treated with the lytic agent indicated a high abundance of viral particles (4 × 109 to 7 × 109 virus-like particles [VLPs] ml–1) compared to control cultures (~107 ml–1). However, viral particles were seldom found in TEM photomicrograph thin sections of lysing K. brevis cells. Although a virus specific for K. brevis may have been the FLA, other explanations such as filterable bacteria or bacteriophages specific for bacteria associated with the K. brevis cultures cannot be discounted.

  6. A Simple Method for DNA Extraction from Mature Date Palm Leaves: Impact of Sand Grinding and Composition of Lysis Buffer

    PubMed Central

    Arif, Ibrahim A.; Bakir, Mohammad A.; Khan, Haseeb A.; Ahamed, Anis; Al Farhan, Ahmad H.; Al Homaidan, Ali A.; Al Sadoon, Mohammad; Bahkali, Ali H.; Shobrak, Mohammad

    2010-01-01

    Molecular marker techniques have been widely used for cultivar identification of inbred date palms (Phoenix dactylifera L.; Arecaceae) and biodiversity conservation. Isolation of highly pure DNA is the prerequisite for PCR amplification and subsequent use such as DNA fingerprinting and sequencing of genes that have recently been developed for barcoding. To avoid problems related to the preservation and use of liquid nitrogen, we examined sterile sand for grinding the date palm leaves. Individual and combined effects of sodium chloride (NaCl), polyvinylpyrrolidone (PVP) and lithium chloride (LiCl) with the cetyltrimethylammonium bromide (CTAB) method for a DNA yield of sufficient purity and PCR amplification were evaluated in this study. Presence of LiCl and PVP alone or together in the lysis buffer did not significantly improve the DNA yield and purity compared with the addition of NaCl. Our study suggested that grinding of date palm leaf with sterile sand and inclusion of NaCl (1.4 M) in the lysis buffer without the costly use of liquid nitrogen, PVP and LiCl, provides a DNA yield of sufficient purity, suitable for PCR amplification. PMID:20957085

  7. A simple method for DNA extraction from mature date palm leaves: impact of sand grinding and composition of lysis buffer.

    PubMed

    Arif, Ibrahim A; Bakir, Mohammad A; Khan, Haseeb A; Ahamed, Anis; Al Farhan, Ahmad H; Al Homaidan, Ali A; Al Sadoon, Mohammad; Bahkali, Ali H; Shobrak, Mohammad

    2010-09-08

    Molecular marker techniques have been widely used for cultivar identification of inbred date palms (Phoenix dactylifera L.; Arecaceae) and biodiversity conservation. Isolation of highly pure DNA is the prerequisite for PCR amplification and subsequent use such as DNA fingerprinting and sequencing of genes that have recently been developed for barcoding. To avoid problems related to the preservation and use of liquid nitrogen, we examined sterile sand for grinding the date palm leaves. Individual and combined effects of sodium chloride (NaCl), polyvinylpyrrolidone (PVP) and lithium chloride (LiCl) with the cetyltrimethylammonium bromide (CTAB) method for a DNA yield of sufficient purity and PCR amplification were evaluated in this study. Presence of LiCl and PVP alone or together in the lysis buffer did not significantly improve the DNA yield and purity compared with the addition of NaCl. Our study suggested that grinding of date palm leaf with sterile sand and inclusion of NaCl (1.4 M) in the lysis buffer without the costly use of liquid nitrogen, PVP and LiCl, provides a DNA yield of sufficient purity, suitable for PCR amplification.

  8. Monocytes and macrophages in malignant melanoma. II. Lysis of antibody-coated human erythrocytes as an assay of monocyte function.

    PubMed Central

    Nyholm, R. E.; Currie, G. A.

    1978-01-01

    Peripheral blood mononuclear cells will lyse antibody-treated human erythrocytes. Using Group A red cells and a hyperimmune anti-A1 serum, we have devised a microassay for the cytolytic capacity of mononuclear cell suspensions. The effector cells responsible for red-cell lysis are mononuclear, adherent and phagocytic, and their activity is blocked by aggregated IgG. Their presence correlates well with non-specific esterase-containing cells and we conclude that they are monocytes. Dose-response curves of red-cell lysis plotted against numbers of monocytes were used to derive a simple parameter expressing the number of monocytes needed to lyse 15% of the 51Cr-labelled red cells. The assay was applied to a group of 27 normal controls and 36 patients with a histologically proven diagnosis of malignant melanoma. The results indicate that monocytes from patients show significantly greater lytic activity than those from the controls. These data suggest that monocytes from cancer patients are in some way activated, and that other defects in monocyte function which have been detected in cancer patients (defective chemotaxis and maturation) may be associated with monocyte "activation". PMID:638013

  9. RNA interference by osmotic lysis of pinosomes: liposome-independent transfection of siRNAs into mammalian cells.

    PubMed

    Gruber, Jens; Boese, Guido; Tuschl, Thomas; Osborn, Mary; Weber, Klaus

    2004-07-01

    The osmotic lysis of pinosomes procedure has been adapted to deliver small interfering RNAs (siRNAs) into cells in culture. Under hypertonic conditions, siRNAs were internalized into pinosomes. A subsequent osmotic shock in hypotonic buffer disrupted the pinosomes and caused the release of siRNAs into the cell cytoplasm. Both steps could be demonstrated directly using fluorescein-labeled siRNAs and confocal laser-scanning microscopy. Uptake by the pinocytosis/osmotic lysis procedure is concentration- and time-dependent. At an siRNA concentration of 0.4 microM, treatment for 40 or 80 min results in silencing efficiencies of 60% and 90%, respectively, after 44 h. A double treatment resulted in approximately equal silencing efficiencies but in reduced viability. This method has been used on a variety of human and murine cell lines including HEK293, HeLa SS6, and SW3T3 cells. Targets such as lamin A/C and Eg5 were effectively silenced. Novel silencing data are provided for Ki67, one of the few reliable prognostic markers for tumor patients. The new procedure avoids certain technical problems encountered with commercial transfection reagents while yielding silencing efficiencies that are comparable to those obtained with liposome-mediated siRNA transfection.

  10. Fibrinolytic enzyme production by newly isolated Bacillus cereus SRM-001 with enhanced in-vitro blood clot lysis potential.

    PubMed

    Narasimhan, Manoj Kumar; Chandrasekaran, Muthukumaran; Rajesh, Mathur

    2015-01-01

    The discovery of plasmin-like microbial fibrinolytic enzymes having high specificity and negligible side effects is crucial for thrombolytic therapy. Herein, we report one such extra-cellular fibrinolytic enzyme producing Bacillus cereus SRM-001 isolated from the blood-laden soil of a chicken dump yard. The potency of the enzyme was established with fibrin plate assay and in-vitro blood clot lysis assay. The shake-flask operating parameters and media composition were optimized for maximizing the productivity of the enzyme. The operating parameters, pH 7, 37°C, 1% inoculum volume and 24 h inoculum age, were found to be the optimum. The levels of media components, corn flour (0.3% w/v), soyabean powder (1.9% w/v) and MnSO4 (11.5 mM) were optimized by statistical analysis using Box-Behnken design derived RSM. This resulted in an almost 1.8 fold increase in fibrinolytic enzyme productivity. The 3D response surface plots showed soyabean powder and MnSO4 to be the key ingredients for enhancing the enzyme productivity, whereas corn flour had a marginal effect. The in-vitro blood clot lysis assay conducted at near physiological pH 7 at 37°C showed the enzyme to be a potential therapeutic thrombolytic agent.

  11. Group B Streptococcus GAPDH Is Released upon Cell Lysis, Associates with Bacterial Surface, and Induces Apoptosis in Murine Macrophages

    PubMed Central

    Oliveira, Liliana; Madureira, Pedro; Andrade, Elva Bonifácio; Bouaboud, Abdelouhab; Morello, Eric; Ferreira, Paula; Poyart, Claire; Trieu-Cuot, Patrick; Dramsi, Shaynoor

    2012-01-01

    Glyceraldehyde 3-phosphate dehydrogenases (GAPDH) are cytoplasmic glycolytic enzymes that, despite lacking identifiable secretion signals, have been detected at the surface of several prokaryotic and eukaryotic organisms where they exhibit non-glycolytic functions including adhesion to host components. Group B Streptococcus (GBS) is a human commensal bacterium that has the capacity to cause life-threatening meningitis and septicemia in newborns. Electron microscopy and fluorescence-activated cell sorter (FACS) analysis demonstrated the surface localization of GAPDH in GBS. By addressing the question of GAPDH export to the cell surface of GBS strain NEM316 and isogenic mutant derivatives of our collection, we found that impaired GAPDH presence in the surface and supernatant of GBS was associated with a lower level of bacterial lysis. We also found that following GBS lysis, GAPDH can associate to the surface of many living bacteria. Finally, we provide evidence for a novel function of the secreted GAPDH as an inducer of apoptosis of murine macrophages. PMID:22291899

  12. Real-time Measurement of Membrane Conformational States Induced by Antimicrobial Peptides: Balance Between Recovery and Lysis

    PubMed Central

    Hall, Kristopher; Lee, Tzong-Hsien; Mechler, Adam I.; Swann, Marcus J.; Aguilar, Marie-Isabel

    2014-01-01

    The disruption of membranes by antimicrobial peptides is a multi-state process involving significant structural changes in the phospholipid bilayer. However, direct measurement of these membrane structural changes is lacking. We used a combination of dual polarisation interferometry (DPI), surface plasmon resonance spectroscopy (SPR) and atomic force microscopy (AFM) to measure the real-time changes in membrane structure through the measurement of birefringence during the binding of magainin 2 (Mag2) and a highly potent analogue in which Ser8, Gly13 and Gly18 has been replaced with alanine (Mag-A). We show that the membrane bilayer undergoes a series of structural changes upon peptide binding before a critical threshold concentration is reached which triggers a significant membrane disturbance. We also propose a detailed model for antimicrobial peptide action as a function of the degree of bilayer disruption to provide an unprecedented in-depth understanding of the membrane lysis in terms of the interconversion of different membrane conformational states in which there is a balance between recovery and lysis. PMID:24969959

  13. Erythrocyte-mediated delivery of pravastatin: in vitro study of effect of hypotonic lysis on biochemical parameters and loading efficiency.

    PubMed

    Harisa, Gamaleldin I; Ibrahim, Mohamed F; Alanazi, Fars K

    2012-08-01

    Exposure of erythrocytes to hypotonic lysis creates pores in the cell membrane, through which pravastatin can enter and become trapped, after resealing them with a suitable buffer. We investigated the effects of tonicity, incubation time and drug concentration on drug loading into erythrocytes. Furthermore, we investigate the effects of pravastatin on erythrocyte oxidative stress markers and osmotic fragility behavior. Encapsulation was achieved using buffer solutions of different tonicities (0.5, 0.6 and 0.7% NaCl) and different drug concentrations (2, 4, 8 and 10 mg/mL) for a range of incubation times (15, 30, 60 and 120 min). The results demonstrated that controlled hypotonic lysis could entrap pravastatin in human erythrocytes, with acceptable loading parameters. The highest loading (34%) was achieved at 0.6% NaCl and 10 mg/mL pravastatin for 60 min incubation. At this pravastatin concentration, oxidative stress markers were similar to those seen in controls, and fragility and hematological parameters were unaffected in drug-loaded erythrocytes. These results indicate that the loading process and pravastatin concentration had no deleterious effects on the structure of pravastatin-loaded erythrocytes, suggesting that they may therefore have a similar life span to normal cells. Pravastatin-loaded erythrocytes may thus provide an effective extended-release-delivery system for pravastatin.

  14. Temperature-regulated bleaching and lysis of the coral Pocillopora damicornis by the novel pathogen Vibrio coralliilyticus.

    PubMed

    Ben-Haim, Yael; Zicherman-Keren, Maya; Rosenberg, Eugene

    2003-07-01

    Coral bleaching is the disruption of symbioses between coral animals and their photosynthetic microalgal endosymbionts (zooxanthellae). It has been suggested that large-scale bleaching episodes are linked to global warming. The data presented here demonstrate that Vibrio coralliilyticus is an etiological agent of bleaching of the coral Pocillopora damicornis. This bacterium was present at high levels in bleached P. damicornis but absent from healthy corals. The bacterium was isolated in pure culture, characterized microbiologically, and shown to cause bleaching when it was inoculated onto healthy corals at 25 degrees C. The pathogen was reisolated from the diseased tissues of the infected corals. The zooxanthella concentration in the bacterium-bleached corals was less than 12% of the zooxanthella concentration in healthy corals. When P. damicornis was infected with V. coralliilyticus at higher temperatures (27 and 29 degrees C), the corals lysed within 2 weeks, indicating that the seawater temperature is a critical environmental parameter in determining the outcome of infection. A large increase in the level of the extracellular protease activity of V. coralliilyticus occurred at the same temperature range (24 to 28 degrees C) as the transition from bleaching to lysis of the corals. We suggest that bleaching of P. damicornis results from an attack on the algae, whereas bacterium-induced lysis and death are promoted by bacterial extracellular proteases. The data presented here support the bacterial hypothesis of coral bleaching.

  15. Association of alpha interferon production with natural killer cell lysis of U937 cells infected with human immunodeficiency virus.

    PubMed Central

    Rappocciolo, G; Toso, J F; Torpey, D J; Gupta, P; Rinaldo, C R

    1989-01-01

    Mononuclear leukocytes from human immunodeficiency virus (HIV)-seronegative and -seropositive homosexual men lysed HIV-infected U937 cells to a significantly greater degree than uninfected U937 cells. Depletion of cell subsets with monoclonal antibodies and complement indicated that the effector cells were primarily of the CD16+ phenotype. Acid-stable alpha interferon (IFN-alpha) production induced by the HIV-infected cells correlated with, although was not an absolute requisite for, preferential lysis of the infected targets. The activity of these CD16+, natural killer (NK) cells decreased in relation to the duration of HIV infection and the presence of acquired immunodeficiency syndrome. Pretreatment of peripheral blood mononuclear cells from HIV-seronegative subjects, but not HIV-seropositive men, with IFN-alpha or recombinant interleukin-2 enhanced lysis of both uninfected and HIV-infected U937 cells. These results suggest that IFN-alpha-associated, NK-like mechanisms are active in the cytotoxic response against HIV-infected cells and that HIV infection results in an early and progressive depression of such responses. Prospective investigations may be useful in determining the role of this NK cell response in the natural history and pathogenesis of HIV infection and the efficacy of therapeutic modalities. PMID:2913035

  16. Enhanced lysis of herpes simplex virus type 1-infected mouse cell lines by NC and NK effectors

    SciTech Connect

    Colmenares, C.; Lopez, C.

    1986-05-01

    Spontaneously cytotoxic murine lymphocytes lysed certain cell types infected by herpes simplex virus type 1 (HSV-1) better than uninfected cells. Although HSV-1 adsorbed to the surface of all the target cells, those in which the virus replicated more efficiently were lysed to a greater extent. As targets, the authors used cell lines that, when uninfected, were spontaneously lysed by NK cells (YAC-1) or by NC cells (WEHI-164). They also used a fibroblastoid cell line (M50) and a monocytic tumor line (PU51R), which were not spontaneously killed. NK cells lysed HSV-1-infected YAC cells better than uninfected cells, and an NC-like activity selectively lysed HSV-1-infected WEHI cells. These findings were consistent with the results of experiments performed to define the role of interferon in induction of virus-augmented cytolysis. Increased lysis of YAC-HSV and PU51R-HSV was entirely due to interferon activation and was completely abolished by performing the /sup 51/Cr-release assay in the presence of anti-interferon serum. The data show that HSV-1 infection of NK/NC targets induces increased cytotoxity, but the effector cell responsible for lysis is determined by the uninfected target, or by an interaction between the virus and target cell, rather than by a viral determinant alone.

  17. Arthroscopic lysis of adhesions improves knee range of motion after fixation of intra-articular fractures about the knee.

    PubMed

    Gittings, Daniel; Hesketh, Patrick; Dattilo, Jonathan; Zgonis, Miltiadis; Kelly, John; Mehta, Samir

    2016-12-01

    Post-traumatic stiffness after open reduction and internal fixation of fractures about the knee can have dramatic effects on function. Traditionally, open quadricepsplasty has been the treatment of choice, but is associated with significant morbidity. The purpose of this study is to examine the immediate and sustainable range of motion (ROM) changes after surgical arthroscopic lysis of knee adhesions (SALKA) for post-traumatic knee stiffness after open reduction internal fixation (ORIF). We retrospectively reviewed a consecutive series of patients at a single institution who underwent SALKA for knee stiffness after intra-articular fractures about the knee treated with ORIF from 2009 to 2015. Pre-operative and immediate post-operative total ROM was assessed while patients were sedated during the SALKA procedure. Total ROM was assessed in the office pre-operatively and compared to the latest post-operative follow-up visit. Immediate post-operative ROM was also compared to the latest post-operative follow-up visit. Two-tailed paired Student's t test was calculated for analysis. Of the 14 patients included in the study, 10 (71 %) had tibial plateau ORIF, 3 (21 %) had patella ORIF, and 1 (8 %) had intra-articular distal femur ORIF. The mean time between ORIF and SALKA was 244 days. The mean follow-up time after SALKA was 135 days. Under sedation during SALKA, the mean total ROM increased from 72° to 127° immediately post-operatively (p < 0.01). The mean pre-operative in-office total ROM was 73° and increased to 104° at the latest follow-up visit (p < 0.01). The mean immediate post-operative ROM was 127° and decreased to 104° at the latest follow-up visit (p = 0.01). Lysis of adhesions utilizing SALKA after ORIF about the knee improves knee ROM immediately post-operatively and in the short-term follow-up. However, there is a decrease in the gains in the range of motion over time. Patients should be counseled as such. Lysis of adhesions utilizing

  18. Immobilized lysozyme for the continuous lysis of lactic bacteria in wine: Bench-scale fluidized-bed reactor study.

    PubMed

    Cappannella, Elena; Benucci, Ilaria; Lombardelli, Claudio; Liburdi, Katia; Bavaro, Teodora; Esti, Marco

    2016-11-01

    Lysozyme from hen egg white (HEWL) was covalently immobilized on spherical supports based on microbial chitosan in order to develop a system for the continuous, efficient and food-grade enzymatic lysis of lactic bacteria (Oenococcus oeni) in white and red wine. The objective is to limit the sulfur dioxide dosage required to control malolactic fermentation, via a cell concentration typical during this process. The immobilization procedure was optimized in batch mode, evaluating the enzyme loading, the specific activity, and the kinetic parameters in model wine. Subsequently, a bench-scale fluidized-bed reactor was developed, applying the optimized process conditions. HEWL appeared more effective in the immobilized form than in the free one, when the reactor was applied in real white and red wine. This preliminary study suggests that covalent immobilization renders the enzyme less sensitive to the inhibitory effect of wine flavans. Copyright © 2016 Elsevier Ltd. All rights reserved.

  19. High-intensity focused ultrasound sonothrombolysis: the use of perfluorocarbon droplets to achieve clot lysis at reduced acoustic power.

    PubMed

    Pajek, Daniel; Burgess, Alison; Huang, Yuexi; Hynynen, Kullervo

    2014-09-01

    The purpose of this study was to evaluate use of intravascular perfluorocarbon droplets to reduce the sonication power required to achieve clot lysis with high-intensity focused ultrasound. High-intensity focused ultrasound with droplets was initially applied to blood clots in an in vitro flow apparatus, and inertial cavitation thresholds were determined. An embolic model for ischemic stroke was used to illustrate the feasibility of this technique in vivo. Recanalization with intravascular droplets was achieved in vivo at 24 ± 5% of the sonication power without droplets. Recanalization occurred in 71% of rabbits that received 1-ms pulsed sonications during continuous intravascular droplet infusion (p = 0.041 vs controls). Preliminary experiments indicated that damage was confined to the ultrasonic focus, suggesting that tolerable treatments would be possible with a more tightly focused hemispheric array that allows the whole focus to be placed inside of the main arteries in the human brain.

  20. Interactions between bacterial membranes and peptidolipids: lysis of micrococcus luteus protoplasts by derivatives of peptidolipidic antibiotics from bacillus subtilis.

    PubMed

    Besson, F; Peypoux, F; Michel, G

    1979-04-19

    The lysis of protoplasts of Micrococcus luteus has been tested with various derivatives of three peptidolipidic antibiotics: iturin A, mycosubtilin and bacillomycin L. The lytic activity is dependent to the nature of the substituting group and to the position of the substituted aminoacid residue. The acetylation of OH groups leads to a decrease of the lytic activity of the natural antibiotics. The methylation of aspartyl residues of bacillomycin L gives a strong lytic activity while natural bacillomycin L has no lytic activity. The methylation of the tyrosyl residue enhances the lytic activities of iturin A and of bacillomycin L-dimethyl ester and reduces that of mycosubtilin. Correlations between the structures of derivatives and their lytic action on M. luteus protoplasts are discussed.

  1. Continuous ethanol production and evaluation of yeast cell lysis and viability loss under very high gravity medium conditions.

    PubMed

    Bai, F W; Chen, L J; Zhang, Z; Anderson, W A; Moo-Young, M

    2004-06-10

    A combined bioreactor system, composed of a stirred tank and a three-stage tubular bioreactor in series and with a total working volume of 3260 ml, was established. Continuous ethanol production was carried out using Saccharomyces cerevisiae and a very high gravity (VHG) medium containing 280 g l(-1) glucose. An average ethanol concentration of 124.6 g l(-1) or 15.8% (v) was produced when the bioreactor system was operated at a dilution rate of 0.012 h(-1). The yield of ethanol to glucose consumed was calculated to be 0.484 or 94.7% of its theoretical value of 0.511 when ethanol entrapped in the exhaust gas was incorporated. Meanwhile, quasi-steady states and non-steady oscillations were observed for residual glucose, ethanol and biomass concentrations for all of these bioreactors during their operations. Models that can be used to predict yeast cell lysis and viability loss were developed.

  2. Amount of Colicin Release in Escherichia coli Is Regulated by Lysis Gene Expression of the Colicin E2 Operon

    PubMed Central

    Mader, Andreas; von Bronk, Benedikt; Ewald, Benedikt; Kesel, Sara; Schnetz, Karin; Frey, Erwin; Opitz, Madeleine

    2015-01-01

    The production of bacteriocins in response to worsening environmental conditions is one means of bacteria to outcompete other microorganisms. Colicins, one class of bacteriocins in Escherichia coli, are effective against closely related Enterobacteriaceae. Current research focuses on production, release and uptake of these toxins by bacteria. However, little is known about the quantitative aspects of these dynamic processes. Here, we quantitatively study expression dynamics of the Colicin E2 operon in E. coli on a single cell level using fluorescence time-lapse microscopy. DNA damage, triggering SOS response leads to the heterogeneous expression of this operon including the cea gene encoding the toxin, Colicin E2, and the cel gene coding for the induction of cell lysis and subsequent colicin release. Advancing previous whole population investigations, our time-lapse experiments reveal that at low exogenous stress levels all cells eventually respond after a given time (heterogeneous timing). This heterogeneous timing is lost at high stress levels, at which a synchronized stress response of all cells 60 min after induction via stress can be observed. We further demonstrate, that the amount of colicin released is dependent on cel (lysis) gene expression, independent of the applied exogenous stress level. A heterogeneous response in combination with heterogeneous timing can be biologically significant. It might enable a bacterial population to endure low stress levels, while at high stress levels an immediate and synchronized population wide response can give single surviving cells of the own species the chance to take over the bacterial community after the stress has ceased. PMID:25751274

  3. Contribution of virus-induced lysis and protozoan grazing to benthic bacterial mortality estimated simultaneously in microcosms.

    PubMed

    Fischer, Ulrike R; Wieltschnig, Claudia; Kirschner, Alexander K T; Velimirov, Branko

    2006-08-01

    In contrast to the water column, the fate of bacterial production in freshwater sediments is still a matter of debate. Thus, the importance of virus-induced lysis and protozoan grazing of bacteria was investigated for the first time simultaneously in a silty sediment layer of a mesotrophic oxbow lake. Microcosms were installed in the laboratory in order to study the dynamics of these processes over 15 days. All microbial and physicochemical parameters showed acceptable resemblance to field data observed during a concomitant in situ study, and similar conclusions can be drawn with respect to the quantitative impact of viruses and protozoa on the bacterial compartment. Viral decay rates ranged from undetectable to 0.078 h(-1) (average, 0.033 h(-1)), and the control of bacterial production from below the detection limit to 36% (average, 12%). The contribution of virus-induced lysis of bacteria to the dissolved organic matter pool as well as to benthic bacterial nutrition was low. Ingestion rates of protozoan grazers ranged from undetectable to 24.7 bacteria per heterotrophic nanoflagellate (HNF) per hour (average, 4.8 bacteria HNF(-1) h(-1)) and from undetectable to 73.3 bacteria per ciliate per hour (average, 11.2 bacteria ciliate(-1) h(-1)). Heterotrophic nanoflagellate and ciliates together cropped up to 5% (average, 1%) of bacterial production. The viral impact on bacteria prevailed over protozoan grazing by a factor of 2.5-19.9 (average, 9.5). In sum, these factors together removed up to 36% (average, 12%) of bacterial production. The high number of correlations between viral and protozoan parameters is discussed in view of a possible relationship between virus removal and the presence of protozoan grazers.

  4. CADM1/TSLC1 Identifies HTLV-1-Infected Cells and Determines Their Susceptibility to CTL-Mediated Lysis.

    PubMed

    Manivannan, Kiruthika; Rowan, Aileen G; Tanaka, Yuetsu; Taylor, Graham P; Bangham, Charles R M

    2016-04-01

    Human T cell lymphotropic virus-1 (HTLV-1) primarily infects CD4+ T cells, causing inflammatory disorders or a T cell malignancy in 5% to 10% of carriers. The cytotoxic T lymphocyte (CTL) response is a key factor that controls the viral load and thus the risk of disease. The ability to detect the viral protein Tax in primary cells has made it possible to estimate the rate at which Tax-expressing infected cells are eliminated by CTLs in persistently infected people. However, most HTLV-1-infected cells are Tax-at a given time, and their immunophenotype is poorly defined. Here, we aimed to identify a cell-surface molecule expressed by both Tax+ and Tax-HTLV-1-infected cells and use it to analyse the CTL response in fresh peripheral blood mononuclear cells. Cell adhesion molecule 1 (CADM1/TSLC1) was the best single marker of HTLV-1 infection, identifying HTLV-1-infected cells with greater sensitivity and specificity than CD25, CCR4 or ICAM-1. CADM1+CD4+ T cells carried a median of 65% of proviral copies in peripheral blood. In a cohort of 23 individuals, we quantified the rate of CTL-mediated killing of Tax+ and Tax-CADM1+ cells. We show that CADM1 expression is associated with enhanced susceptibility of infected cells to CTL lysis: despite the immunodominance of Tax in the CTL response, Tax+CADM1- cells were inefficiently lysed by CTLs. Upregulation of the CADM1 ligand CRTAM on CD8+ T cells correlated with efficient lysis of infected cells. Tax-CADM1+ cells were lysed at a very low rate by autologous CTLs, however, were efficiently killed when loaded with exogenous peptide antigen. High expression of CADM1 on most HTLV-1-infected cells in the face of enhanced CTL counterselection implies that CADM1 confers a strong benefit on the virus.

  5. CADM1/TSLC1 Identifies HTLV-1-Infected Cells and Determines Their Susceptibility to CTL-Mediated Lysis

    PubMed Central

    Tanaka, Yuetsu; Taylor, Graham P.; Bangham, Charles R. M.

    2016-01-01

    Human T cell lymphotropic virus-1 (HTLV-1) primarily infects CD4+ T cells, causing inflammatory disorders or a T cell malignancy in 5% to 10% of carriers. The cytotoxic T lymphocyte (CTL) response is a key factor that controls the viral load and thus the risk of disease. The ability to detect the viral protein Tax in primary cells has made it possible to estimate the rate at which Tax-expressing infected cells are eliminated by CTLs in persistently infected people. However, most HTLV-1-infected cells are Tax–at a given time, and their immunophenotype is poorly defined. Here, we aimed to identify a cell-surface molecule expressed by both Tax+ and Tax–HTLV-1-infected cells and use it to analyse the CTL response in fresh peripheral blood mononuclear cells. Cell adhesion molecule 1 (CADM1/TSLC1) was the best single marker of HTLV-1 infection, identifying HTLV-1-infected cells with greater sensitivity and specificity than CD25, CCR4 or ICAM-1. CADM1+CD4+ T cells carried a median of 65% of proviral copies in peripheral blood. In a cohort of 23 individuals, we quantified the rate of CTL-mediated killing of Tax+ and Tax−CADM1+ cells. We show that CADM1 expression is associated with enhanced susceptibility of infected cells to CTL lysis: despite the immunodominance of Tax in the CTL response, Tax+CADM1– cells were inefficiently lysed by CTLs. Upregulation of the CADM1 ligand CRTAM on CD8+ T cells correlated with efficient lysis of infected cells. Tax–CADM1+ cells were lysed at a very low rate by autologous CTLs, however, were efficiently killed when loaded with exogenous peptide antigen. High expression of CADM1 on most HTLV-1-infected cells in the face of enhanced CTL counterselection implies that CADM1 confers a strong benefit on the virus. PMID:27105228

  6. Provirus activation plus CD59 blockage triggers antibody-dependent complement-mediated lysis of latently HIV-1-infected cells.

    PubMed

    Lan, Jie; Yang, Kai; Byrd, Daniel; Hu, Ningjie; Amet, Tohti; Shepherd, Nicole; Desai, Mona; Gao, Jimin; Gupta, Samir; Sun, Yongtao; Yu, Qigui

    2014-10-01

    Latently HIV-1-infected cells are recognized as the last barrier toward viral eradication and cure. To purge these cells, we combined a provirus stimulant with a blocker of human CD59, a key member of the regulators of complement activation, to trigger Ab-dependent complement-mediated lysis. Provirus stimulants including prostratin and histone deacetylase inhibitors such as romidepsin and suberoylanilide hydroxamic acid activated proviruses in the latently HIV-1-infected T cell line ACH-2 as virion production and viral protein expression on the cell surface were induced. Romidepsin was the most attractive provirus stimulant as it effectively activated proviruses at nanomolar concentrations that can be achieved clinically. Antiretroviral drugs including two protease inhibitors (atazanavir and darunavir) and an RT inhibitor (emtricitabine) did not affect the activity of provirus stimulants in the activation of proviruses. However, saquinavir (a protease inhibitor) markedly suppressed virus production, although it did not affect the percentage of cells expressing viral Env on the cell surface. Provirus-activated ACH-2 cells expressed HIV-1 Env that colocalized with CD59 in lipid rafts on the cell surface, facilitating direct interaction between them. Blockage of CD59 rendered provirus-activated ACH-2 cells and primary human CD4(+) T cells that were latently infected with HIV-1 sensitive to Ab-dependent complement-mediated lysis by anti-HIV-1 polyclonal Abs or plasma from HIV-1-infected patients. Therefore, a combination of provirus stimulants with regulators of complement activation blockers represents a novel approach to eliminate HIV-1. Copyright © 2014 by The American Association of Immunologists, Inc.

  7. HLA-A11-mediated protection from NK cell-mediated lysis: role of HLA-A11-presented peptides.

    PubMed

    Gavioli, R; Zhang, Q J; Masucci, M G

    1996-08-01

    The capacity of MHC class I to protect target cells from NK is well established, but the mechanism by which these molecules influence NK recognition and the physical properties associated with this function remain poorly defined. We have examined this issue using as a model the HLA-A11 allele. HLA-A11 expression correlated with reduced susceptibility to NK and interferon-activated cytotoxicity in transfected sublines of the A11-defective Burkitt's lymphoma WW2-BL and the HLA class I A,B-null C1R cell line. Protection was also achieved by transfection of HLA-A11 in the peptide processing mutant T2 cells line (T2/A11), despite a very low expression of the transfected product at the cell surface. Induction of surface HLA-A11 by culture of T2/A11 cells at 26 degrees C or in the presence of beta 2m did not affect lysis, whereas NK sensitivity was restored by culture in the presence of HLA-All-binding synthetic peptides derived from viral or cellular proteins. Acid treatment rendered T2/A11 and C1R/A11 cells sensitive to lysis, but protection was restored after preincubation with peptide preparations derived from surface stripping of T2/A11 cells. Similar peptide preparations from T2 cells had no effect. The results suggest that NK protection is mediated by HLA-A11 molecules carrying a particular set of peptides that are translocated to the site of MHC class I assembly in the ER in a TAP-independent fashion.

  8. Semi-direct lysis of swabs and evaluation of their efficiencies to recover human noroviruses GI and GII from surfaces.

    PubMed

    De Keuckelaere, Ann; Stals, Ambroos; Uyttendaele, Mieke

    2014-06-01

    Enteric viruses such as noroviruses (NoVs) continue to be the cause of widespread viral outbreaks due to person-to-person transmission, contaminated food, and contaminated surfaces. In order to optimize swabbing methodology for the detection of viruses on (food) contact surfaces, three swab elution/extraction strategies were compared in part one of this study, out of which, one strategy was based on the recently launched ISO protocol (ISO/TS 15216-1) for the determination of hepatitis A virus and NoV in food using real-time RT-PCR (RT-qPCR). These three swab elution/extraction strategies were tested for the detection of GI.4 and GII.4 NoV on high-density polyethylene (HD-PE) surfaces with the use of cotton swabs. For detection of GI.4 and GII.4, the sample recovery efficiency (SRE) obtained with the direct lysis strategy (based on ISO/TS 15216-1) was significantly lower than the SRE obtained with both other strategies. The semi-direct lysis strategy was chosen to assess the SRE of two common swabs (cotton swab and polyester swab) versus the biowipe (Biomérieux, Lyon, France) on three surfaces (HD-PE, neoprene rubber (NR), and nitrile gloves (GL)). For both surfaces, HD-PE and GL, no significant differences in SREs of GI.4 and GII.4 NoVs were detected between the three different swabs. For the coarser NR, biowipes turned out to be the best option for detecting both GI.4 and GII.4 NoV.

  9. Urate oxidase for the prevention and treatment of tumour lysis syndrome in children with cancer.

    PubMed

    Cheuk, Daniel Kl; Chiang, Alan Ks; Chan, Godfrey Cf; Ha, Shau Yin

    2017-03-08

    Tumour lysis syndrome (TLS) is a serious complication of malignancies and can result in renal failure or death. Previous reviews did not find clear evidence of benefit of urate oxidase in children with cancer. This review is the second update of a previously published Cochrane review. To assess the effects and safety of urate oxidase for the prevention and treatment of TLS in children with malignancies. In March 2016 we searched CENTRAL, MEDLINE, Embase, and CINAHL. In addition, we searched the reference lists of all identified relevant papers, trials registers and other databases. We also screened conference proceedings and we contacted experts in the field and the manufacturer of rasburicase, Sanofi-aventis. Randomised controlled trials (RCT) and controlled clinical trials (CCT) of urate oxidase for the prevention or treatment of TLS in children under 18 years with any malignancy. Two review authors independently extracted trial data and assessed individual trial quality. We used risk ratios (RR) for dichotomous data and mean difference (MD) for continuous data. We included seven trials, involving 471 participants in the treatment groups and 603 participants in the control groups. No new studies were identified in the update. One RCT and five CCTs compared urate oxidase and allopurinol. Three trials tested Uricozyme, and three trials tested rasburicase for the prevention of TLS.The RCT did not evaluate the primary outcome (incidence of clinical TLS). It showed no clear evidence of a difference in mortality (both all-cause mortality (Fisher's exact test P = 0.23) and mortality due to TLS (no deaths in either group)), renal failure (Fisher's exact test P = 0.46), and adverse effects between the treatment and the control groups (Fisher's exact test P = 1.0). The frequency of normalisation of uric acid at four hours (10 out of 10 participants in the treatment group versus zero out of nine participants in the control group, Fisher's exact test P < 0.001) and area

  10. Improved lysis efficiency and immunogenicity of Salmonella ghosts mediated by co-expression of λ phage holin-endolysin and ɸX174 gene E

    PubMed Central

    Won, Gayeon; Hajam, Irshad Ahmed; Lee, John Hwa

    2017-01-01

    Bacterial ghosts (BGs) are empty cell envelopes derived from Gram-negative bacteria by bacteriophage ɸX174 gene E mediated lysis. They represent a novel inactivated vaccine platform; however, the practical application of BGs for human vaccines seems to be limited due to the safety concerns on the presence of viable cells in BGs. Therefore, to improve the lysis efficiency of the gene E, we exploited the peptidoglycan hydrolyzing ability of the λ phage holin-endolysins to expedite the process of current BG production system. In this report, we constructed a novel ghost plasmid encoding protein E and holin-endolysins in tandem. We observed that sequential expressions of the gene E and the holin-endolysins elicited rapid and highly efficient Salmonella lysis compared to the lysis mediated by gene E only. These lysed BGs displayed improved immunogenicity in mice compared to the gene E mediated BGs. Consequently, seventy percent of the mice immunized with these novel ghosts survived against a lethal challenge while all the mice vaccinated with gene E mediated ghosts died by day 9 post-infection. We conclude that this novel strategy has the potential to generate highly efficient inactivated candidate vaccines that could replace the currently available bacterial vaccines. PMID:28332591

  11. Comparison of Two Cell Lysis Procedures for Recovery of Microcystins in Water Samples from Silver Lake in Dover, Delaware, with Microcystin Producing Cyanobacterial Accumulations

    USGS Publications Warehouse

    Loftin, Keith A.; Meyer, Michael T.; Rubio, Fernando; Kamp, Lisa; Humphries, Edythe; Whereat, Ed

    2008-01-01

    A collaboration was developed between Abraxis, LLC, the State of Delaware Department of Natural Resources and Environmental Control Division of Water Resources Environmental Laboratory, the University of Delaware, and the United States Geological Survey to investigate the efficacy of the QuikLyse procedure developed by Abraxis, LLC as an alternative cell-lysis technique suitable for use with an existing liquid chromatography/tandem mass spectrometry research method developed at the United States Geological Survey Organic Geochemistry Research Laboratory to analyze cyanotoxins. A comparison of three sequential freeze/thaw cycles versus QuikLyse, a proprietary chemical lysis procedure was conducted on four water samples collected from Silver Lake in Dover, Delaware. Results from the Abraxis Microcystins-DM enzyme-linked immunosorbent assay and liquid chromatography/tandem mass spectrometry were tabulated as a function of the cell lysis technique. Stastical comparison of percent relative standard deviations showed no significant difference (alpha = 0.05) between both cell-lysis techniques when measured by enzyme-linked immunosorbent assay or liquid chromatography/tandem mass spectrometry for three of the four samples.

  12. Local thrombus formation in the site of intravenous injection of chlorpromazine: possible colloid-osmotic lysis of the local endothelial cells.

    PubMed

    Yabuki, M; Tani, N; Yoshioka, T; Nishibe, H; Kanamaru, H; Kaneko, H

    2000-08-01

    Since amphiphilic drugs are known to interact with biomembranes, we investigated local vessel damage and thrombosis which might be brought about by intravenous dosing using chlorpromazine (CPZ) as a representative compound. CPZ-induced hemolysis was suppressed by an increase in sucrose concentration in the medium, characterizing this hemolysis to be colloid-osmotic lysis, which includes the enhancement of membrane phospholipid fluidity and consequent small pore formation in the membranes. This was supported by the observation that hemolysis by filipin, not featuring the stage of small pore formation, was not affected by sucrose. [14C]Glucose-entrapping liposomes were degraded by CPZ, and this degradation was enhanced by an increase in the intravesicle glucose concentration. These results indicated that the compound could induce colloid-osmotic lysis in erythrocytes and artificial membrane vesicles. CPZ also injured cultured porcine aortic endothelial cells (PAEC), as evidenced by lactate dehydrogenase (LDH) leakage. This injury was also suppressed by increase in sucrose concentration in the medium, suggesting that colloid-osmotic lysis again occurred. When rats were intravenously injected with CPZ, local endothelial cell (EC) injury and associated thrombus formation were observed, suggesting that CPZ's action was also evident in vivo. To our knowledge, this is the first finding which suggests that an intravenously dosed amphiphilic drug can injure local ECs based on a colloid-osmotic lysis mechanism leading to thrombosis.

  13. Soil pretreatment and fast cell lysis for direct polymerase chain reaction from forest soils for terminal restriction fragment length polymorphism analysis of fungal communities

    Treesearch

    Fei Cheng; Lin Hou; Keith Woeste; Zhengchun Shang; Xiaobang Peng; Peng Zhao; Shuoxin Zhang

    2016-01-01

    Humic substances in soil DNA samples can influence the assessment of microbial diversity and community composition. Using multiple steps during or after cell lysis adds expenses, is time-consuming, and causes DNA loss. A pretreatment of soil samples and a single step DNA extraction may improve experimental results. In order to optimize a protocol for obtaining high...

  14. Reoperative surgery: a critical risk factor for complications inadequately captured by operative reporting and coding of lysis of adhesions.

    PubMed

    Aloia, Thomas A; Cooper, Amanda; Shi, Weiming; Vauthey, Jean-Nicolas; Lee, Jeffrey E

    2014-07-01

    Reoperative surgery is suspected, but not proven, to increase postoperative complication rates. In the absence of a specific definition for reoperative surgery, the American College of Surgeons NSQIP has proposed using procedural coding for lysis of adhesions (LOA) as a surrogate for reoperative surgery to risk adjust hospitals. We hypothesized that coding of reoperative surgery will be associated with worse 30-day outcomes and, for abdominal procedures, will be more accurate than operative dictation and coding of "lysis of adhesions." Reoperative surgery was categorized at the time of data abstraction from February 2012 to December 2012 for all NSQIP cases collected at a single institution by independent surgical clinical reviewers. Reoperative surgery classification and coding of LOA were compared with each other and with 30-day outcomes. The setting was a tertiary cancer center, multispecialty NSQIP model. During the study period, 1,289 operations were classified as nonreoperative (n = 793), regionally reoperative (n = 39; prior surgery in an adjacent area of current operation), or locally reoperative (n = 457; prior surgery at same site or organ). In the multispecialty cohort, the non-risk-adjusted rates of overall 30-day morbidity, serious morbidity, and mortality were 21.5%, 17.7%, and 0.5%. Compared with nonreoperative surgery (overall 30-day morbidity 16.8%, serious morbidity 13.9%, and mortality .38%), both regionally reoperative surgery (overall 30-day morbidity 30.8%, serious morbidity 28.2%, and mortality 2.5%) and locally reoperative surgery (overall 30-day morbidity 28.9%, serious morbidity 23.4%, and mortality .66%) were associated with worse outcomes (p < 0.001). One hundred ninety-nine of the 327 gastrointestinal/laparotomy cases were recorded as reoperative, but only of 20 of these were CPT coded as LOA (sensitivity = 10%). Reoperative surgery is frequent, increases the risk of complications, and can be captured. Operative LOA coding vastly

  15. Real-time monitoring of bioaerosols via cell-lysis by air ion and ATP bioluminescence detection.

    PubMed

    Park, Chul Woo; Park, Ji-Woon; Lee, Sung Hwa; Hwang, Jungho

    2014-02-15

    In this study, we introduce a methodology for disrupting cell membranes with air ions coupled with ATP bioluminescence detection for real-time monitoring of bioaerosol concentrations. A carbon fiber ionizer was used to extract ATP from bacterial cells for generating ATP bioluminescence. Our methodology was tested using Staphylococcus epidermidis and Escherichia coli, which were aerosolized with an atomizer, and then indoor bioaerosols were also used for testing the methodology. Bioaerosol concentrations were estimated without culturing which requires several days for colony formation. Correlation equations were obtained for results acquired using our methodology (Relative Luminescent Unit (RLU)/m(3)) and a culture-based (Colony Forming Unit (CFU)/m(3)) method; CFU/m(3)=1.8 × measured RLU/m(3) for S. epidermidis and E. coli, and CFU/m(3)=1.1 × measured RLU/m(3) for indoor bioaerosols under the experimental conditions. Our methodology is an affordable solution for rapidly monitoring bioaerosols due to rapid detection time (cell-lysis time: 3 min; bioluminescence detection time: <1 min) and easy operation.

  16. Preventing phage lysis of Lactococcus lactis in cheese production using a neutralizing heavy-chain antibody fragment from llama.

    PubMed

    Ledeboer, A M; Bezemer, S; de Hiaard, J J W; Schaffers, I M; Verrips, C T; van Vliet, C; Düsterhöft, E M; Zoon, P; Moineau, S; Frenken, L G J

    2002-06-01

    Bacteriophage infection is still a persistent problem in large dairy processes despite extensive studies over the last decades. Consequently, new methods are constantly sought to prevent phage infection. In this paper, we show that phage neutralizing heavy-chain antibody fragments, obtained from Camelidae and produced at a large scale in the generally regarded as safe microorganism Saccharomyces cerevisiae, can effectively be used to impede phage induced lysis during a cheese process. The growth inhibition of the cheese starter culture by 10(5) pfu/ml cheese-milk of the small isometric-headed 936-type phage p2 was prevented by the addition of only 0.1 microg/ml (7 nM) of the neutralizing antibody fragment. The use of such antibody fragments in cheese manufacturing are a realistic and interesting option because of the small amount of antibody fragments that are needed. Moreover the antibodies are produced in a food grade microorganism and can easily be isolated from the fermentation liquid in a pure and DNA free form.

  17. Mode of resistance to viral lysis affects host growth across multiple environments in the marine picoeukaryote Ostreococcus tauri.

    PubMed

    Heath, Sarah E; Collins, Sinead

    2016-12-01

    Viruses play important roles in population dynamics and as drivers of evolution in single-celled marine phytoplankton. Viral infection of Ostreococcus tauri often causes cell lysis, but two spontaneously arising resistance mechanisms occur: resistant cells that cannot become infected and resistant producer cells that are infected but not lysed, and which may slowly release viruses. As of yet, little is known about how consistent the effects of viruses on their hosts are across different environments. To measure the effect of host resistance on host growth, and to determine whether this effect is environmentally dependent, we compared the growth and survival of susceptible, resistant and resistant producer O. tauri cells under five environmental conditions with and without exposure to O. tauri virus. While the effects of exposure to virus on growth rates did not show a consistent pattern in populations of resistant cells, there were several cases where exposure to virus affected growth in resistant hosts, sometimes positively. In the absence of virus, there was no detectable cost of resistance in any environment, as measured by growth rate. In fact, the opposite was the case, with populations of resistant producer cells having the highest growth rates across four of the five environments. © 2016 Society for Applied Microbiology and John Wiley & Sons Ltd.

  18. Modified Atkins diet induces subacute selective ragged-red-fiber lysis in mitochondrial myopathy patients.

    PubMed

    Ahola, Sofia; Auranen, Mari; Isohanni, Pirjo; Niemisalo, Satu; Urho, Niina; Buzkova, Jana; Velagapudi, Vidya; Lundbom, Nina; Hakkarainen, Antti; Muurinen, Tiina; Piirilä, Päivi; Pietiläinen, Kirsi H; Suomalainen, Anu

    2016-11-01

    Mitochondrial myopathy (MM) with progressive external ophthalmoplegia (PEO) is a common manifestation of mitochondrial disease in adulthood, for which there is no curative therapy. In mice with MM, ketogenic diet significantly delayed progression of the disease. We asked in this pilot study what effects high-fat, low-carbohydrate "modified Atkins" diet (mAD) had for PEO/MM patients and control subjects and followed up the effects by clinical, morphological, transcriptomic, and metabolomic analyses. All of our five patients, irrespective of genotype, showed a subacute response after 1.5-2 weeks of diet, with progressive muscle pain and leakage of muscle enzymes, leading to premature discontinuation of the diet. Analysis of muscle ultrastructure revealed selective fiber damage, especially in the ragged-red-fibers (RRFs), a MM hallmark. Two years of follow-up showed improvement of muscle strength, suggesting activation of muscle regeneration. Our results indicate that (i) nutrition can modify mitochondrial disease progression, (ii) dietary counseling should be part of MM care, (iii) short mAD is a tool to induce targeted RRF lysis, and (iv) mAD, a common weight-loss method, may induce muscle damage in a population subgroup.

  19. Pilot-scale study of sludge pretreatment by microwave and sludge reduction based on lysis-cryptic growth.

    PubMed

    Wang, Yawei; Xiao, Qingcong; Liu, Jibao; Yan, Hong; Wei, Yuansong

    2015-08-01

    To evaluate the performance of microwave (MW)-chemical hybrid sludge treatment system, a pilot scale MW disintegration unit (treatment capacity of 500L/d) was constructed. The results showed that organic matter, nitrogen, and phosphorus were effectively released from the MW-pretreated sludge. The values of COD released were 15.91%, 15.07%, 13.83%, 19.35%, and 15.07% for the MW, MW-acid, MW-alkali, MW-H2O2, and MW-H2O2-alkali treatment processes, respectively. Additionally, for a wastewater treatment system with a capacity of 200m(3)/d, when coupled with a MW sludge pretreatment unit, the sludge production and sludge yield were greatly reduced by 38.60% and to 0.35kg VSS/kg CODconsumed, respectively. The total operating cost of the lysis-cryptic growth system was 13.64% lower than that of the CAS system without a MW unit. Copyright © 2015 Elsevier Ltd. All rights reserved.

  20. Investigation of low-voltage pulse parameters on electroporation and electrical lysis using a microfluidic device with interdigitated electrodes.

    PubMed

    Morshed, Bashir I; Shams, Maitham; Mussivand, Tofy

    2014-03-01

    Electroporation (EP) of biological cells leads to the exchange of materials through the permeabilized cell membrane, while electrical lysis (EL) irreversibly disrupts the cell membrane. We report a microfluidic device to study these two phenomena with low-voltage excitation for lab-on-a-chip (LOC) applications. For systematic study of EP, we have employed a quantification metric: flow Index (FI) of EP. Simulation and experimental results with the microfluidic device containing interdigitated, coplanar, integrated electrodes to electroporate, and rapidly lyse biological cells are presented. H&E stained human buccal cells were subjected to various pulse magnitudes, pulsewidths, and number of pulses. Simulations show that an electric field of 25 kV/cm with a 20 V applied potential produced 1.3 (°)C temperature rise for a 5 s of excitation. For a 20 V pulse-excitation with pulse-widths between 0.5 to 5 s, EL was observed, whereas for lower excitations, only EP was observed. FI of EP is found to be a direct function of pulse magnitudes, pulsewidths, and numbers of pulses. To release DNA from nucleus, excitation-pulses of 5 s were required. Quantification of EP would be useful for systematic study of EP toward optimization with various excitation pulses, while low-voltage requirement and high yield of EP and EL are critical to develop LOC for drug delivery and cell-sample preparation, respectively.

  1. The effect of grazing and viral lysis on the diel variations of Synechococcus spp. abundance in the East China Sea

    NASA Astrophysics Data System (ADS)

    Tsai, An Yi; Gong, Gwo-Ching; Hu, Shu Ling; Chao, Chien Fu

    2015-09-01

    Diel variations in the nanoflagellate grazing and viral-mediated mortality of Synechococcus spp. were simultaneously estimated using a dilution and size-fractionation approach in the inner (I-1 and I-2) and outer regions (O-1, O2 and O-3) of the Changjiang River plume in the East China Sea during summer 2014. Synechococcus spp. abundance generally tended to increase during the dark period, followed by a plateau until midnight at all sampling stations. Overall, gross growth rate of Synechococcus spp. ranged from 0.069 h-1 to 0.122 h-1 during the growth phase. Microzooplankton, nanoflagellate grazing, and viral lysis had no effect on the Synechococcus spp. abundance during this phase. Moreover, nanoflagellate grazing was the largest cause of Synechococcus spp. mortality during the loss phase at nighttime. Compared to the predators, viruses exerted only a minor impact on mortality at St. I-1, where we detected some of the effect that this community had on Synechococcus spp.. Little is known about the impact of nanoflagellates and viruses on the short-term dynamics of Synechococcus spp. in the East China Sea. Therefore, this study's characterization of the relative importance of nanoflagellates and viruses may help provide a better understanding of trophic structures and the energy flow within the microbial loop.

  2. Early postnatal diagnosis of hereditary spherocytosis by combining light microscopy, acidified glycerol lysis test and eosin-5'-maleimide binding assay.

    PubMed

    Andres, Oliver; Eber, Stefan; Speer, Christian P

    2015-12-01

    Exact diagnosis of hereditary spherocytosis (HS) is widely considered unreliable around birth. However, early postnatal diagnosis at the beginning of congenital hemolysis may be essential for managing neonatal anemia and hemolytic icterus, identifying those at high risk for severe hyperbilirubinemia, irreversible kernicterus, or sudden need for red cell transfusion. We analyzed 37 blood samples from neonates or infants up to six weeks of life that had been collected in-house or shipped to our laboratory due to suspected red cell membrane disorder. By combining assessment of red cell morphology, acidified glycerol lysis test (AGLT), and eosin-5'-maleimide (EMA) binding assay, we were able to clearly exclude HS in 22 and confirm HS in 10 patients, of which one had undergone red cell transfusion prior to blood sampling. Assessment of red cell morphology and normal test results allowed diagnosis of infantile pyknocytosis or Heinz body anemia in three neonates. Re-evaluation of five patients with inconsistent results of AGLT and EMA binding led to confirmation of HS in two cases. Automated analysis of hematologic parameters revealed elevated proportion of hyperdense cells to be a highly significant indicator for HS in neonatal infants. We showed that assessment of red cell morphology in combination with AGLT and EMA binding assay is a reliable basis for confirming or rejecting suspected diagnosis of HS even in neonates. Our data underline the necessity for blood sampling and laboratory exploration in suspected red cell membrane or enzyme defects at the earliest occasion.

  3. Mechanism of mammalian cell lysis mediated by peptide defensins. Evidence for an initial alteration of the plasma membrane.

    PubMed Central

    Lichtenstein, A

    1991-01-01

    Defensins induce ion channels in model lipid bilayers and permeabilize the membranes of Escherichia coli. We investigated whether similar membrane-active events occur during defensin-mediated cytolysis of tumor cells. Although defensin-treated K562 targets did not release chromium-labeled cytoplasmic components for 5-6 h, they experienced a rapid collapse (within minutes) of the membrane potential, efflux of rubidium, and influx of trypan blue. Defensin treatment also blunted the subsequent acidification response induced by nigericin, thereby further supporting the notion of enhanced transmembrane ion flow during exposure. These initial effects on the plasma membrane were not sufficient for subsequent lysis; a second phase of injury was required which involved the continued presence of defensin. The rapid membrane permeabilization phase was inhibited by azide/2-deoxyglucose, cytochalasin B, and increased concentrations of extracellular potassium and was unaffected by actinomycin-D, cycloheximide, and varying the calcium concentration. In contrast, the second phase was unaffected by cytochalasin B, inhibited by azide/2-deoxyglucose, enhanced by actinomycin D and cycloheximide, and varied with calcium concentration. These results indicate the initial adverse effect of defensins on mammalian cells occurs at the cell membrane. It is possible that the second phase of injury is mediated intracellularly by defensin that has been internalized through this leaky membrane. PMID:2056135

  4. Rendering of mycobacteria safe for molecular diagnostic studies and development of a lysis method for strand displacement amplification and PCR.

    PubMed Central

    Zwadyk, P; Down, J A; Myers, N; Dey, M S

    1994-01-01

    Two criteria must be met before mycobacterial specimens can be tested by DNA amplification methods: (i) the sample must be rendered noninfectious, and (ii) the organisms must be lysed to free the DNA. Previous publications reporting DNA amplification of mycobacteria have concentrated on lysis and amplification procedures and have not addressed the issue of sample safety. We have shown that heating of samples below 100 degrees C may not consistently kill mycobacteria; however, heating at 100 degrees C in a boiling-water bath or a forced-air oven for a minimum of 5 min kills mycobacteria, including Mycobacterium thermoresistibile. Furthermore, heating at 100 degrees C for 30 min consistently lyses mycobacteria to produce short fragments of DNA that are suitable for amplification by PCR and strand displacement amplification. This procedure works with clinical samples digested by the n-acetyl cysteine-NaOH method as well as with suspensions of organisms in phosphate buffer. This paper also demonstrates the feasibility of using strand displacement amplification with clinical specimens. Images PMID:7814537

  5. Induction of natural competence in Streptococcus pneumoniae triggers lysis and DNA release from a subfraction of the cell population

    PubMed Central

    Steinmoen, Hilde; Knutsen, Eivind; Håvarstein, Leiv Sigve

    2002-01-01

    Naturally competent bacteria have the ability to take up free DNA from the surrounding medium and incorporate this DNA into their genomes by homologous recombination. In naturally competent Streptococcus pneumoniae, and related streptococcal species from the mitis phylogenetic group, the competent state is not a constitutive property but is induced by a peptide pheromone through a quorum-sensing mechanism. Recent studies have shown that natural genetic transformation is an important mechanism for gene exchange between streptococci in nature. A prerequisite for effective gene exchange is the presence of streptococcal donor DNA in the environment. Despite decades of study of the transformation process we still do not know how this donor DNA is released from streptococcal cells to the external milieu. Traditionally, it has been assumed that donor DNA originates from cells that die and fall apart from natural causes. In this study we show that induction of the competent state initiates release of DNA from a subfraction of the bacterial population, probably by cell lysis. The majority of the cells induced to competence take up DNA and act as recipients, whereas the rest release DNA and act as donors. These findings show that natural transformation in streptococci provides a natural mechanism for genetic recombination that resembles sex in higher organisms. PMID:12032343

  6. Imaging secondary metabolism of Streptomyces sp. Mg1 during cellular lysis and colony degradation of competing Bacillus subtilis.

    PubMed

    Barger, Sarah R; Hoefler, B Chris; Cubillos-Ruiz, Andrés; Russell, William K; Russell, David H; Straight, Paul D

    2012-10-01

    Soil streptomycetes are saprotrophic bacteria that secrete numerous secondary metabolites and enzymes for extracellular functions. Many streptomycetes produce antibiotics thought to protect vegetative mycelia from competing organisms. Here we report that an organism isolated from soil, Streptomyces sp. Mg1, actively degrades colonies and causes cellular lysis of Bacillus subtilis when the organisms are cultured together. We predicted that the inhibition and degradation of B. subtilis colonies in this competition depends upon a combination of secreted factors, including small molecule metabolites and enzymes. To begin to unravel this complex competitive phenomenon, we use a MALDI imaging mass spectrometry strategy to map the positions of metabolites secreted by both organisms. In this report, we show that Streptomyces sp. Mg1 produces the macrolide antibiotic chalcomycin A, which contributes to inhibition of B. subtilis growth in combination with other, as yet unidentified factors. We suggest that efforts to understand competitive and cooperative interactions between bacterial species benefit from assays that pair living organisms and probe the complexity of metabolic exchanges between them.

  7. Differential ability to resist to complement lysis and invade host cells mediated by MBL in R4 and 860 strains of Trypanosoma cruzi.

    PubMed

    Evans-Osses, Ingrid; Mojoli, Andres; Beltrame, Marcia Holsbach; da Costa, Denise Endo; DaRocha, Wanderson Duarte; Velavan, Thirumalaisamy P; de Messias-Reason, Iara; Ramirez, Marcel Ivan

    2014-03-18

    To produce an infection Trypanosoma cruzi must evade lysis by the complement system. During early stages of infection, the lectin pathway plays an important role in host defense and can be activated by binding of mannan-binding lectin (MBL) to carbohydrates on the surface of pathogens. We hypothesized that MBL has a dual role during parasite-host cell interaction as lectin complement pathway activator and as binding molecule to invade the host cell. We used two polarized strains of T. cruzi, R4 (susceptible) and 860 (resistant) strains, to investigate the role of MBL in complement-mediated lysis. Interestingly R4, but not 860 metacyclic strain, markedly increases the invasion of host cells, suggesting that MBL drives the invasion process while the parasite deactivates the Lectin complement pathway. Copyright © 2014 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.

  8. Human trypanolytic factor APOL1 forms pH-gated cation-selective channels in planar lipid bilayers: Relevance to trypanosome lysis

    PubMed Central

    Thomson, Russell; Finkelstein, Alan

    2015-01-01

    Apolipoprotein L-1 (APOL1), the trypanolytic factor of human serum, can lyse several African trypanosome species including Trypanosoma brucei brucei, but not the human-infective pathogens T. brucei rhodesiense and T. brucei gambiense, which are resistant to lysis by human serum. Lysis follows the uptake of APOL1 into acidic endosomes and is apparently caused by colloid-osmotic swelling due to an increased ion permeability of the plasma membrane. Here we demonstrate that nanogram quantities of full-length recombinant APOL1 induce ideally cation-selective macroscopic conductances in planar lipid bilayers. The conductances were highly sensitive to pH: their induction required acidic pH (pH 5.3), but their magnitude could be increased 3,000-fold upon alkalinization of the milieu (pKa = 7.1). We show that this phenomenon can be attributed to the association of APOL1 with the bilayer at acidic pH, followed by the opening of APOL1-induced cation-selective channels upon pH neutralization. Furthermore, the conductance increase at neutral pH (but not membrane association at acidic pH) was prevented by the interaction of APOL1 with the serum resistance-associated protein, which is produced by T. brucei rhodesiense and prevents trypanosome lysis by APOL1. These data are consistent with a model of lysis that involves endocytic recycling of APOL1 and the formation of cation-selective channels, at neutral pH, in the parasite plasma membrane. PMID:25730870

  9. Soil pretreatment and fast cell lysis for direct polymerase chain reaction from forest soils for terminal restriction fragment length polymorphism analysis of fungal communities.

    PubMed

    Cheng, Fei; Hou, Lin; Woeste, Keith; Shang, Zhengchun; Peng, Xiaobang; Zhao, Peng; Zhang, Shuoxin

    Humic substances in soil DNA samples can influence the assessment of microbial diversity and community composition. Using multiple steps during or after cell lysis adds expenses, is time-consuming, and causes DNA loss. A pretreatment of soil samples and a single step DNA extraction may improve experimental results. In order to optimize a protocol for obtaining high purity DNA from soil microbiota, five prewashing agents were compared in terms of their efficiency and effectiveness in removing soil contaminants. Residual contaminants were precipitated by adding 0.6mL of 0.5M CaCl2. Four cell lysis methods were applied to test their compatibility with the pretreatment (prewashing+Ca(2+) flocculation) and to ultimately identify the optimal cell lysis method for analyzing fungal communities in forest soils. The results showed that pretreatment with TNP+Triton X-100+skim milk (100mM Tris, 100mM Na4P2O7, 1% polyvinylpyrrolidone, 100mM NaCl, 0.05% Triton X-100, 4% skim milk, pH 10.0) removed most soil humic contaminants. When the pretreatment was combined with Ca(2+) flocculation, the purity of all soil DNA samples was further improved. DNA samples obtained by the fast glass bead-beating method (MethodFGB) had the highest purity. The resulting DNA was successfully used, without further purification steps, as a template for polymerase chain reaction targeting fungal internal transcribed spacer regions. The results obtained by terminal restriction fragment length polymorphism analysis indicated that the MethodFGB revealed greater fungal diversity and more distinctive community structure compared with the other methods tested. Our study provides a protocol for fungal cell lysis in soil, which is fast, convenient, and effective for analyzing fungal communities in forest soils.

  10. Evaluation of the Check-Points Check MDR CT103 and CT103 XL Microarray Kits by Use of Preparatory Rapid Cell Lysis

    PubMed Central

    Cunningham, Scott A.; Vasoo, Shawn

    2016-01-01

    Using a rapid bacterial lysis method, the Check MDR CT103 and CT103 XL microarrays demonstrated accuracies of 98.1% and 94.2%, respectively, for detection of known resistance genes in 108 multidrug-resistant Gram-negative bacilli. In 45 isolates, 49 previously unrecognized extended-spectrum β-lactamase or plasmid AmpC targets were detected and confirmed by conventional PCR. PMID:26888905

  11. Resistance to Cytarabine Induces the Up-regulation of NKG2D Ligands and Enhances Natural Killer Cell Lysis of Leukemic Cells1

    PubMed Central

    Ogbomo, Henry; Michaelis, Martin; Klassert, Denise; Doerr, Hans Wilhelm; Cinatl, Jindrich

    2008-01-01

    Prolonged treatment of leukemic cells with chemotherapeutic agents frequently results in development of drug resistance. Moreover, selection of drug-resistant cell populations may be associated with changes in malignant properties such as proliferation rate, invasiveness, and immunogenicity. In the present study, the sensitivity of cytarabine (1-β-d-arabinofuranosylcytosine, araC)-resistant and parental human leukemic cell lines (T-lymphoid H9 and acute T-lymphoblastic leukemia Molt-4) to natural killer (NK) cell-mediated killing was investigated. The results obtained demonstrate that araC-resistant H9 and Molt-4 (H9rARAC100 and Molt-4rARAC100) cell lines are more sensitive to NK cell-mediated lysis than their respective parental cell lines. This increased sensitivity was associated with a higher surface expression of ligands for the NK cell-activating receptor NKG2D, notably UL16 binding protein-2 (ULBP-2) and ULBP-3 in H9rARAC100 and Molt-4rARAC100 cell lines. Blocking ULBP-2 and ULBP-3 or NKG2D with monoclonal antibody completely abrogated NK cell lysis. Constitutive phosphorylated extracellular signal-regulated kinase (ERK) but not pAKT was higher in araC-resistant cells than in parental cell lines. Inhibition of ERK using ERK inhibitor PD98059 decreased both ULBP-2/ULBP-3 expression and NK cell cytotoxicity. Furthermore, overexpression of constitutively active ERK in H9 parental cells resulted in increased ULBP-2/ULBP-3 expression and enhanced NK cell lysis. These results demonstrate that increased sensitivity of araC-resistant leukemic cells to NK cell lysis is caused by higher NKG2D ligand expression, resulting from more active ERK signaling pathway. PMID:19048119

  12. A novel integrated strategy for detection of human bocavirus based on a heminested PCR assay combined with boiling lysis method of samples in human specimens.

    PubMed

    Chen, Long; Yao, Qing; Ma, Jing; Li, Jianning; Zhang, Qian; Yang, Yi; Li, Fang; Sun, Yuning

    2014-07-01

    Human bocavirus (HBoV) has been shown to be associated with acute respiratory tract infection in children. The aim of the work was to develop a novel integrated strategy for human bocavirus detection: heminested PCR assay combined with boiling lysis method of samples. The detection limit of the heminested PCR assay was 1.2 copies of a recombinant DNA plasmid, and no cross-reaction with other respiratory viruses or bacteria was observed. By using the integrated strategy, a total of 202 secretions of the lower respiratory tract of children with acute respiratory diseases were collected and tested. The samples were treated and lysed in boiling lysis buffer rather than extracting viral DNA from secretions, then these sample lysates could be templates and tested by heminested PCR assay, and the amplification of HBoV DNA was detected by using agarose gel electrophoresis. The results showed that, only 7 samples were found to be positive by conventional single-round PCR; importantly, the other new 41 samples were positive by heminested PCR assay. Additionally, the genomic viral DNA was extracted from all positive and some negative specimens, amplified, and sequenced. The results were perfectly consistent with those of the integrated strategy. Taken together, these results suggest that the novel integrated strategy (heminested PCR assay combined with boiling lysis method of samples) is a convenient, sensitive, cost-effective and reliable detective method for HBoV detection and will have broad application prospects in clinical diagnosis.

  13. Production of lipase from Pseudomonas gessardii using blood tissue lipid and thereof for the hydrolysis of blood cholesterol and triglycerides and lysis of red blood cells.

    PubMed

    Ramani, K; Sekaran, G

    2012-08-01

    The study demonstrates the production of lipase (LIP) from Pseudomonas gessardii using blood tissue lipid as the substrate for the hydrolysis of blood cholesterol and triglycerides. The lipase was purified with the specific activity of 828 U/mg protein and the molecular weight of 56 kDa. The maximum lipase activity was observed at the pH 7.0 and the temperature 37 °C. The amino acid composition of purified lipase was determined by HPLC. The mesoporous activated carbon (MAC) was used for the immobilization of lipase for the repeated use of the enzyme catalyst. The K (m) value of immobilized lipase (MAC-LIP) and the free lipase (LIP) was 0.182 and 1.96 mM, respectively. The V (max) value of MAC-LIP and LIP was 1.33 and 1.26 mM/min, respectively. The MAC and MAC-LIP were characterized by scanning electron microscopy (SEM). The hydrolysis study showed 78 and 100% hydrolysis of triglycerides and cholesterol, respectively, for LIP and 84 and 100% hydrolysis of triglycerides and cholesterol, respectively, for MAC-LIP at the reaction time of 1 h. The effect of lipase on cell wall lysis was carried out on the RBCs of blood plasma. Interestingly, 99.9% lysis of RBCs was observed within 2 h. SEM images and phase contrast microscopy confirmed the lysis of RBCs. This work provides a potential biocatalyst for the hydrolysis of blood cholesterol and triglycerides.

  14. T cell-recruiting triplebody 19-3-19 mediates serial lysis of malignant B-lymphoid cells by a single T cell

    PubMed Central

    Roskopf, Claudia C.; Schiller, Christian B.; Braciak, Todd A.; Kobold, Sebastian; Schubert, Ingo A.; Fey, Georg H.; Hopfner, Karl-Peter; Oduncu, Fuat S.

    2014-01-01

    Triplebody 19-3-19, an antibody-derived protein, carries three single chain fragment variable domains in tandem in a single polypeptide chain. 19-3-19 binds CD19-bearing lymphoid cells via its two distal domains and primary T cells via its CD3-targeting central domain in an antigen-specific manner. Here, malignant B-lymphoid cell lines and primary cells from patients with B cell malignancies were used as targets in cytotoxicity tests with pre-stimulated allogeneic T cells as effectors. 19-3-19 mediated up to 95% specific lysis of CD19-positive tumor cells and, at picomolar EC50 doses, had similar cytolytic potency as the clinically successful agent BlinatumomabTM. 19-3-19 activated resting T cells from healthy unrelated donors and mediated specific lysis of both autologous and allogeneic CD19-positive cells. 19-3-19 led to the elimination of 70% of CD19-positive target cells even with resting T cells as effectors at an effector-to-target cell ratio of 1 : 10. The molecule is therefore capable of mediating serial lysis of target cells by a single T cell. These results highlight that central domains capable of engaging different immune effectors can be incorporated into the triplebody format to provide more individualized therapy tailored to a patient’s specific immune status. PMID:25115385

  15. Enhancement of NK cell-mediated lysis of non-small lung cancer cells by nPKC activator, ingenol 3,20 dibenzoate.

    PubMed

    Gong, Chenyuan; Yao, Chao; Xu, Zihang; Ni, Zhongya; Zhu, Xiaowen; Wang, Lixin; Yan, Xuewei; Zhou, Wuxiong; Zhu, Shiguo

    2017-03-01

    The IFN-γ production is crucial for NK cell-mediated lysis of cancer cells. Thus increasing the IFN-γ production by NK cells may be an ideal strategy to improve their tumoricidal effect. Since the focus on new drug development has shifted towards natural products, limited information is out there about natural products that enhance the IFN-γ production by NK cells. In this study, through a high-throughput screening, we have identified a natural product ingenol 3,20 dibenzoate (IDB), an activator of tumor suppressor protein kinase C (PKC) isozymes, could increase the IFN-γ production and degranulation by NK cells, especially when NK cells were stimulated by non-small lung cancer (NSCLC) cells. IDB also significantly enhanced the NK cell-mediated lysis of NSCLC cells. Furthermore, PKC inhibitor, sotrastaurin abrogated IDB-induced IFN-γ production, degranulation and cytotoxicity, but did not affect IFN-γ production by NK cells without IDB treatment and NSCLC cell stimulation. The IFN-γ neutralization reversed the IDB-induced enhancement of NK cell mediated killing. In conclusion, our study indicated that IDB enhanced NK cell-mediated lysis of NSCLC cells is dependent on specific PKC mediated IFN-γ production and degranulation. Thus, IDB may have a promising application in clinic for NK cell-based cancer immunotherapy.

  16. Reduction of excess sludge in a sequencing batch reactor by lysis-cryptic growth using quick lime for disintegration under low temperature.

    PubMed

    Lv, Xiao-Mei; Song, Ju-Sheng; Li, Ji; Zhai, Kun

    2017-08-01

    In the present study, quick-lime-based thermal-alkaline sludge disintegration (SD) under low temperature was combined with cryptic growth to investigate the excess sludge reduction efficiency in the sequencing batch reactor (SBR). The optimized condition of SD was as follows: T = 80℃, pH = 11, t = 180 min, and the SD rate was about 42.1%. With 65.6% of excess sludge disintegrated and returned to the SBR, the system achieved sludge reduction rate of about 40.1%. The lysis-cryptic growth still obtained satisfactory sludge reduction efficiency despite the comparative low SD rate, which suggested that disintegration rate might not be the decisive factor for cryptic-growth-based sludge reduction. Lysis-cryptic growth did not impact the effluent quality, yet the phosphorus removal performance was enhanced, with effluent total phosphorus concentration decreased by 0.3 mg/L (33%). Crystal compounds of calcium phosphate precipitate were detected in the system by Fourier transform infrared spectroscopy and X-ray diffraction, which indicated the phosphorus removal potential of SD using lime. Moreover, endogenous dehydrogenase activity of activated sludge in the lysis-cryptic system was enhanced, which was beneficial for sludge reduction. SD and cryptic growth in the present study demonstrates an economical and effective approach for sludge reduction.

  17. CD19-specific triplebody SPM-1 engages NK and γδ T cells for rapid and efficient lysis of malignant B-lymphoid cells

    PubMed Central

    Schiller, Christian B.; Braciak, Todd A.; Fenn, Nadja C.; Seidel, Ursula J. E.; Roskopf, Claudia C.; Wildenhain, Sarah; Honegger, Annemarie; Schubert, Ingo A.; Schele, Alexandra; Lämmermann, Kerstin; Fey, Georg H.; Jacob, Uwe; Lang, Peter; Hopfner, Karl-Peter; Oduncu, Fuat S.

    2016-01-01

    Triplebodies are antibody-derived recombinant proteins carrying 3 antigen-binding domains in a single polypeptide chain. Triplebody SPM-1 was designed for lysis of CD19-bearing malignant B-lymphoid cells through the engagement of CD16-expressing cytolytic effectors, including NK and γδ T cells. SPM-1 is an optimized version of triplebody ds(19-16-19) and includes humanization, disulfide stabilization and the removal of potentially immunogenic sequences. A three-step chromatographic procedure yielded 1.7 - 5.5 mg of purified, monomeric protein per liter of culture medium. In cytolysis assays with NK cell effectors, SPM-1 mediated potent lysis of cancer-derived B cell lines and primary cells from patients with various B-lymphoid malignancies, which surpassed the ADCC activity of the therapeutic antibody Rituximab. EC50-values ranged from 3 to 86 pM. Finally, in an impedance-based assay, SPM-1 mediated a particularly rapid lysis of CD19-bearing target cells by engaging and activating both primary and expanded human γδ T cells from healthy donors as effectors. These data establish SPM-1 as a useful tool for a kinetic analysis of the cytolytic reactions mediated by γδ T and NK cells and as an agent deserving further development towards clinical use for the treatment of B-lymphoid malignancies. PMID:27825135

  18. Cyclosporin a, but not FK506, induces osmotic lysis of pancreas zymogen granules, intra-acinar enzyme release, and lysosome instability by activating K+ channel.

    PubMed

    Lee, Wing-Kee; Braun, Matthias; Langelüddecke, Christian; Thévenod, Frank

    2012-05-01

    The immunosuppressant tacrolimus (FK506) has improved pancreas allograft survival compared with cyclosporin A (CsA), possibly because of reduced acute pancreatitis following ischemia-reperfusion injury. Ion permeabilities in zymogen granule (ZG) membranes, including a KCNQ1 K channel, promote hormone-stimulated enzyme secretion. We investigated whether a differential modulation of ZG and lysosomal ion permeabilities and enzyme secretion by CsA/FK506 contributes to pancreatitis. Rat ZGs and lysosomes were isolated by gradient centrifugation, ion permeabilities assayed by osmotic lysis, and single-channel currents recorded in a planar lipid bilayer. Amylase release was measured in permeabilized acini and lysosomal cathepsin B release detected by immunoblotting. CsA (1-10 μM), but not FK506, enhanced ZGs osmotic lysis by selectively increasing K permeability up to 5-fold. Zymogen granule membrane K channels showed ∼2-fold increased single-channel open probability with CsA only. Cyclosporin A selectively increased basal (∼2-fold), but not cholecystokinin-octapeptide (1 nM)-induced amylase secretion in K medium only. Cyclosporin A (5 μM), but not FK506, increased cathepsin B release from lysosomes. Cyclosporin A selectively opens the ZG K channel and induces cathepsin B release from lysosomes, which cause increased in situ lysis of ZGs and may aggravate or fuel acute allograft pancreatitis following hypoxia-reperfusion injury.

  19. Membrane fusion catalyzed by a Rab, SNAREs, and SNARE chaperones is accompanied by enhanced permeability to small molecules and by lysis

    PubMed Central

    Zucchi, Paola C.; Zick, Michael

    2011-01-01

    The fusion of sealed biological membranes joins their enclosed aqueous compartments while mixing their membrane bilayers. Reconstituted fusion reactions are commonly assayed by lipid mixing, which can result from either true fusion or from lysis and its attendant reannealing of membranes. Fusion is also frequently assayed by the mixing of lumenal aqueous compartments, using probes of low molecular weight. With several probes (biotin, methylumbelliferyl-N-acetyl-α-d-neuraminic acid, and dithionite), we find that yeast vacuolar SNAREs (SNAP [Soluble NSF attachment protein] Receptors) increase the permeability of membranes to small molecules and that this permeabilization is enhanced by homotypic fusion and vacuole protein sorting complex (HOPS) and Sec17p/Sec18p, the vacuolar tethering and SNARE chaperone proteins. We now report the development of a novel assay that allows the parallel assessment of lipid mixing, the mixing of intact lumenal compartments, any lysis that occurs, and the membrane permeation of small molecules. Applying this assay to an all-purified reconstituted system consisting of vacuolar lipids, the four vacuolar SNAREs, the SNARE disassembly chaperones Sec17p and Sec18p, the Rab Ypt7p, and the Rab effector/SM protein complex HOPS, we show that true fusion is accompanied by strongly enhanced membrane permeability to small molecules and a measurable rate of lysis. PMID:21976702

  20. Disruption of Chlorella vulgaris cells for the release of biodiesel-producing lipids: a comparison of grinding, ultrasonication, bead milling, enzymatic lysis, and microwaves.

    PubMed

    Zheng, Hongli; Yin, Jilong; Gao, Zhen; Huang, He; Ji, Xiaojun; Dou, Chang

    2011-08-01

    A comparative evaluation of different cell disruption methods for the release of lipids from marine Chlorella vulgaris cells was investigated. The cell growth of C. vulgaris was observed. Lipid concentrations from different disruption methods were determined, and the fatty acid composition of the extracted lipids was analyzed. The results showed that average productivity of C. vulgaris biomass was 208 mg L⁻¹ day⁻¹. The lipid concentrations of C. vulgaris were 5%, 6%, 29%, 15%, 10%, 7%, 22%, 24%, and 18% when using grinding with quartz sand under wet condition, grinding with quartz sand under dehydrated condition, grinding in liquid nitrogen, ultrasonication, bead milling, enzymatic lysis by snailase, enzymatic lysis by lysozyme, enzymatic lysis by cellulose, and microwaves, respectively. The shortest disruption time was 2 min by grinding in liquid nitrogen. The unsaturated and saturated fatty acid contents of C. vulgaris were 71.76% and 28.24%, respectively. The extracted lipids displayed a suitable fatty acid profile for biodiesel [C16:0 (~23%), C16:1 (~23%), and C18:1 (~45%)]. Overall, grinding in liquid nitrogen was identified as the most effective method in terms of disruption efficiency and time.

  1. Involvement of HLA class I alleles in natural killer (NK) cell-specific functions: expression of HLA-Cw3 confers selective protection from lysis by alloreactive NK clones displaying a defined specificity (specificity 2)

    PubMed Central

    1992-01-01

    This study was designed to identify the target molecules of the natural killer (NK) cell-mediated recognition of normal allogeneic target cells. As previously shown, the gene(s) governing the first NK-defined allospecificity (specificity 1) were found to be localized in the major histocompatibility complex region between BF gene and HLA-A. In addition, the analysis of a previously described family revealed that a donor (donor 81) was heterozygous for three distinct NK-defined allospecificities (specificities 1, 2, and 5). HLA variants were derived from the B-Epstein-Barr virus cell line of donor 81 by gamma irradiation followed by negative selection using monoclonal antibodies specific for the appropriate HLA allele. Several variants were derived that lacked one or more class I antigen expressions. These variants were analyzed for the susceptibility to lysis by NK clones recognizing different allospecificities. The loss of HLA-A did not modify the phenotype (i.e., "resistance to lysis"). On the other hand, a variant lacking expression of all class I antigens became susceptible to lysis by all alloreactive clones. Variants characterized by the selective loss of class I antigens coded for by the maternal chromosome became susceptible to lysis by anti-2-specific clones. Conversely, variants selectively lacking class I antigens coded for by paternal chromosome became susceptible to lysis by anti-1 and anti-5 clones (but not by anti-2 clones). Since the Cw3 allele was lost in the variant that acquired susceptibility to lysis by anti-2 clones and, in informative families, it was found to cosegregate with the character "resistance to lysis" by anti-2 clones, we analyzed whether Cw3 could represent the element conferring selective resistance to lysis by anti-2 clones. To this end, murine P815 cells transfected with HLA Cw3 (or with other HLA class I genes) were used as target cells in a cytolytic assay in which effector cells were represented by alloreactive NK clones

  2. Clot lysis time in platelet-rich plasma: method assessment, comparison with assays in platelet-free and platelet-poor plasmas, and response to tranexamic acid.

    PubMed

    Panes, Olga; Padilla, Oslando; Matus, Valeria; Sáez, Claudia G; Berkovits, Alejandro; Pereira, Jaime; Mezzano, Diego

    2012-01-01

    Fibrinolysis dysfunctions cause bleeding or predisposition to thrombosis. Platelets contain several factors of the fibrinolytic system, which could up or down regulate this process. However, the temporal relationship and relative contributions of plasma and platelet components in clot lysis are mostly unknown. We developed a clot lysis time (CLT) assay in platelet-rich plasma (PRP-CLT, with and without stimulation) and compared it to a similar one in platelet-free plasma (PFP) and to another previously reported test in platelet-poor plasma (PPP). We also studied the differential effects of a single dose of tranexamic acid (TXA) on these tests in healthy subjects. PFP- and PPP-CLT were significantly shorter than PRP-CLT, and the three assays were highly correlated (p < 0.0001). PFP- and PPP-, but more significantly PRP-CLT, were positively correlated with age and plasma PAI-1, von Willebrand factor, fibrinogen, LDL-cholesterol, and triglycerides (p < 0.001). All these CLT assays had no significant correlations with platelet aggregation/secretion, platelet counts, and pro-coagulant tests to explore factor X activation by platelets, PRP clotting time, and thrombin generation in PRP. Among all the studied variables, PFP-CLT was independently associated with plasma PAI-1, LDL-cholesterol, and triglycerides and, additionally, stimulated PRP-CLT was also independently associated with plasma fibrinogen. A single 1 g dose of TXA strikingly prolonged all three CLTs, but in contrast to the results without the drug, the lysis times were substantially shorter in non-stimulated or stimulated PRP than in PFP and PPP. This standardized PRP-CLT may become a useful tool to study the role of platelets in clot resistance and lysis. Our results suggest that initially, the platelets enmeshed in the clot slow down the fibrinolysis process. However, the increased clot resistance to lysis induced by TXA is overcome earlier in platelet-rich clots than in PFP or PPP clots. This is

  3. Distribution and lysis of weakly and strongly immunogenic malignant lymphoid cells in normal and immune mice: Effects of prior exposure to heat or X-irradiation

    SciTech Connect

    Nawrocki, J.F.; Look, A.T.; Selwa, J.F.; Haggis, J.R.; Murphy, W.H.

    1984-05-01

    With the use of cells doubly labeled with /sup 51/Cr and /sup 125/I, an analysis was made of the distribution and lysis in normal and immune syngeneic C58 mice of weakly immunogenic malignant lymphocytes (Ib) and a highly immunogenic variant (IbN) derived from them. The results showed that /sup 51/Cr release was not a valid measure of tumor cell lysis in vivo. Prior exposure to 55 degrees C for 30 minutes caused Ib to lyse immediately after iv injection. Prior X-irradiation (10,000 R) enhanced the lysis of Ib in vivo but had only minor effects on IbN. In normal mice /sup 125/I-labeled Ib were entrapped in the lungs (approximately to 95%) immediately after iv injection, released in a diphasic manner, and then accumulated in the liver, spleen, bone marrow, and lymph nodes. In immune mice initial entrapment of Ib in the lungs was only about 42%, and label did not accumulate in the described organs. Heat-inactivated Ib were not retained to a significant degree in any of the organs of normal or immune mice. IbN were retained initially (2 hr) at values lower than Ib in the whole body, lungs, and livers of normal mice. /sup 125/I-labeled Ib were used to analyze how they were distributed and lysed in normal and immune mice during the first 6 hours after iv injection. To account for the loss of /sup 125/I from a tissue because of cell lysis (as distinct from cell redistribution), the amount of /sup 125/I associated with cells and blood plasma, and lost from the whole body, was quantified. A significant loss of /sup 125/I from the whole body began at 2 hours and continued at a linear rate thereafter. Non-cell-associated /sup 125/I occurred in the blood plasma at 1-2 hours and increased at a linear rate thereafter. These results made it possible to distinguish between loss of /sup 125/I from a tissue because of cell lysis as distinct from the redistribution of intact labeled cells.

  4. In-Hospital Outcomes of Tumor Lysis Syndrome: A Population-Based Study Using the National Inpatient Sample.

    PubMed

    Durani, Urshila; Shah, Nilay D; Go, Ronald S

    2017-09-13

    The epidemiology and outcomes of tumor lysis syndrome (TLS) are understudied. We used the National Inpatient Sample (NIS), a nationally representative weighted sample of all U.S. hospital discharges, to study outcomes and predictors of mortality in hospitalized patients with TLS. The NIS was queried for patients with a discharge diagnosis of TLS (ICD-9 code 277.88) from 2010-2013. Baseline characteristics and outcomes were analyzed. A multivariable logistic regression analysis was performed to identify predictors of mortality. From 2010-2013, 28,370 patients were discharged with a diagnosis of TLS. The most common malignancies were non-Hodgkin lymphoma (30%), solid tumors (20%), acute myeloid leukemia (19%), and acute lymphocytic leukemia (13%). Overall in-hospital mortality was 21%. The median length of stay was 10 days (IQR 5-22). Sixty-nine percent of patients experienced a severe complication, including sepsis (22%, 95% confidence interval [CI] 21-23), dialysis (15%, 95% CI 14-16), acute respiratory failure (23%, 95% CI 22-24), mechanical ventilation (16%, 95% CI 15-17), gastrointestinal hemorrhage (6%, 95% CI 5-7), cerebral hemorrhage (2%, 95% CI 2-3), seizures (1%, 95% CI 0.6-1), and cardiac arrest (2%, 95% CI 2-3). Predictors of mortality were derived from a multivariable logistic regression and included age, Elixhauser comorbidity score, insurance status, teaching versus nonteaching hospital, and cancer type. Predictors of increased length of stay included age, race, teaching versus nonteaching hospital, and cancer type. In the U.S., many patients with TLS develop life-threatening complications and a quarter die during hospitalization. As more cancer treatments become available, strategies to improve the supportive care of patients with TLS should be a priority. © 2017 AlphaMed Press.

  5. Adenylate kinase release as a high-throughput-screening-compatible reporter of bacterial lysis for identification of antibacterial agents.

    PubMed

    Jacobs, Anna C; Didone, Louis; Jobson, Jennielle; Sofia, Madeline K; Krysan, Damian; Dunman, Paul M

    2013-01-01

    Adenylate kinase (AK) is a ubiquitous intracellular enzyme that is released into the extracellular space upon cell lysis. We have shown that AK release serves as a useful reporter of bactericidal agent activity and can be exploited for antimicrobial screening purposes. The AK assay exhibits improved sensitivity over that of growth-based assays and can detect agents that are active against bacteria in clinically relevant growth states that are difficult to screen using conventional approaches, such as small colony variants (SCV) and bacteria within established biofilms. The usefulness of the AK assay was validated by screening a library of off-patent drugs for agents that exhibit antimicrobial properties toward a variety of bacterial species, including Escherichia coli and all members of the "ESKAPE" pathogens (Enterococcus faecium, Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa, and Enterobacter species). The assay detected antibiotics within the library that were expected to be active against the organism screened. Moreover, 38 drugs with no previously reported antibacterial activity elicited AK release. Four of these were acquired, and all were verified to exhibit antimicrobial activity by standard susceptibility testing. Two of these molecules were further characterized. The antihistamine, terfenadine, was active against S. aureus planktonic, SCV population, and biofilm-associated cells. Tamoxifen, an estrogen receptor antagonist, was active toward E. faecium in vitro and also reduced E. faecium pathogenesis in a Galleria mellonella infection model. Our data demonstrate that the AK assay provides an attractive screening approach for identifying new antimicrobial agents. Further, terfenadine and tamoxifen may represent novel antimicrobial drug development scaffolds.

  6. Adenylate Kinase Release as a High-Throughput-Screening-Compatible Reporter of Bacterial Lysis for Identification of Antibacterial Agents

    PubMed Central

    Jacobs, Anna C.; DiDone, Louis; Jobson, Jennielle; Sofia, Madeline K.

    2013-01-01

    Adenylate kinase (AK) is a ubiquitous intracellular enzyme that is released into the extracellular space upon cell lysis. We have shown that AK release serves as a useful reporter of bactericidal agent activity and can be exploited for antimicrobial screening purposes. The AK assay exhibits improved sensitivity over that of growth-based assays and can detect agents that are active against bacteria in clinically relevant growth states that are difficult to screen using conventional approaches, such as small colony variants (SCV) and bacteria within established biofilms. The usefulness of the AK assay was validated by screening a library of off-patent drugs for agents that exhibit antimicrobial properties toward a variety of bacterial species, including Escherichia coli and all members of the “ESKAPE” pathogens (Enterococcus faecium, Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa, and Enterobacter species). The assay detected antibiotics within the library that were expected to be active against the organism screened. Moreover, 38 drugs with no previously reported antibacterial activity elicited AK release. Four of these were acquired, and all were verified to exhibit antimicrobial activity by standard susceptibility testing. Two of these molecules were further characterized. The antihistamine, terfenadine, was active against S. aureus planktonic, SCV population, and biofilm-associated cells. Tamoxifen, an estrogen receptor antagonist, was active toward E. faecium in vitro and also reduced E. faecium pathogenesis in a Galleria mellonella infection model. Our data demonstrate that the AK assay provides an attractive screening approach for identifying new antimicrobial agents. Further, terfenadine and tamoxifen may represent novel antimicrobial drug development scaffolds. PMID:23027196

  7. Host lysozyme-mediated lysis of Lactococcus lactis facilitates delivery of colitis-attenuating superoxide dismutase to inflamed colons.

    PubMed

    Ballal, Sonia A; Veiga, Patrick; Fenn, Kathrin; Michaud, Monia; Kim, Jason H; Gallini, Carey Ann; Glickman, Jonathan N; Quéré, Gaëlle; Garault, Peggy; Béal, Chloé; Derrien, Muriel; Courtin, Pascal; Kulakauskas, Saulius; Chapot-Chartier, Marie-Pierre; van Hylckama Vlieg, Johan; Garrett, Wendy S

    2015-06-23

    Beneficial microbes that target molecules and pathways, such as oxidative stress, which can negatively affect both host and microbiota, may hold promise as an inflammatory bowel disease therapy. Prior work showed that a five-strain fermented milk product (FMP) improved colitis in T-bet(-/-) Rag2(-/-) mice. By varying the number of strains used in the FMP, we found that Lactococcus lactis I-1631 was sufficient to ameliorate colitis. Using comparative genomic analyses, we identified genes unique to L. lactis I-1631 involved in oxygen respiration. Respiration of oxygen results in reactive oxygen species (ROS) generation. Also, ROS are produced at high levels during intestinal inflammation and cause tissue damage. L. lactis I-1631 possesses genes encoding enzymes that detoxify ROS, such as superoxide dismutase (SodA). Thus, we hypothesized that lactococcal SodA played a role in attenuating colitis. Inactivation of the sodA gene abolished L. lactis I-1631's beneficial effect in the T-bet(-/-) Rag2(-/-) model. Similar effects were obtained in two additional colonic inflammation models, Il10(-/-) mice and dextran sulfate sodium-treated mice. Efforts to understand how a lipophobic superoxide anion (O2 (-)) can be detoxified by cytoplasmic lactoccocal SodA led to the finding that host antimicrobial-mediated lysis is a prerequisite for SodA release and SodA's extracytoplasmic O2 (-) scavenging. L. lactis I-1631 may represent a promising vehicle to deliver antioxidant, colitis-attenuating SodA to the inflamed intestinal mucosa, and host antimicrobials may play a critical role in mediating SodA's bioaccessibility.

  8. Tumor lysis syndrome in the era of novel and targeted agents in patients with hematologic malignancies: a systematic review.

    PubMed

    Howard, Scott C; Trifilio, Steven; Gregory, Tara K; Baxter, Nadine; McBride, Ali

    2016-03-01

    Effective new treatments are now available for patients with hematologic malignancies. However, their propensity to cause tumor lysis syndrome (TLS) has not been systematically examined. A literature search identified published Phase I-III clinical trials of monoclonal antibodies (otlertuzumab, brentuximab, obinutuzumab, ibritumomab, ofatumumab); tyrosine kinase inhibitors (alvocidib [flavopiridol], dinaciclib, ibrutinib, nilotinib, dasatinib, idelalisib, venetoclax [ABT-199]); proteasome inhibitors (oprozomib, carfilzomib); chimeric antigen receptor (CAR) T cells; and the proapoptotic agent lenalidomide. Abstracts from major congresses were also reviewed. Idelalisib and ofatumumab had no reported TLS. TLS incidence was ≤5 % with brentuximab vedotin (for anaplastic large-cell lymphoma), carfilzomib and lenalidomide (for multiple myeloma), dasatinib (for acute lymphoblastic leukemia), and oprozomib (for various hematologic malignancies). TLS incidences were 8.3 and 8.9 % in two trials of venetoclax (for chronic lymphocytic leukemia [CLL]) and 10 % in trials of CAR T cells (for B-cell malignancies) and obinutuzumab (for non-Hodgkin lymphoma). TLS rates of 15 % with dinaciclib and 42 and 53 % with alvocidib (with sequential cytarabine and mitoxantrone) were seen in trials of acute leukemias. TLS mitigation was employed routinely in clinical trials of alvocidib and lenalidomide. However, TLS mitigation strategies were not mentioned or stated only in general terms for many studies of other agents. The risk of TLS persists in the current era of novel and targeted therapy for hematologic malignancies and was seen to some extent with most agents. Our findings underscore the importance of continued awareness, risk assessment, and prevention to reduce this serious potential complication of effective anticancer therapy.

  9. Host lysozyme-mediated lysis of Lactococcus lactis facilitates delivery of colitis-attenuating superoxide dismutase to inflamed colons

    PubMed Central

    Ballal, Sonia A.; Veiga, Patrick; Fenn, Kathrin; Michaud, Monia; Kim, Jason H.; Gallini, Carey Ann; Glickman, Jonathan N.; Quéré, Gaëlle; Garault, Peggy; Béal, Chloé; Derrien, Muriel; Courtin, Pascal; Kulakauskas, Saulius; Chapot-Chartier, Marie-Pierre; van Hylckama Vlieg, Johan; Garrett, Wendy S.

    2015-01-01

    Beneficial microbes that target molecules and pathways, such as oxidative stress, which can negatively affect both host and microbiota, may hold promise as an inflammatory bowel disease therapy. Prior work showed that a five-strain fermented milk product (FMP) improved colitis in T-bet−/− Rag2−/− mice. By varying the number of strains used in the FMP, we found that Lactococcus lactis I-1631 was sufficient to ameliorate colitis. Using comparative genomic analyses, we identified genes unique to L. lactis I-1631 involved in oxygen respiration. Respiration of oxygen results in reactive oxygen species (ROS) generation. Also, ROS are produced at high levels during intestinal inflammation and cause tissue damage. L. lactis I-1631 possesses genes encoding enzymes that detoxify ROS, such as superoxide dismutase (SodA). Thus, we hypothesized that lactococcal SodA played a role in attenuating colitis. Inactivation of the sodA gene abolished L. lactis I-1631’s beneficial effect in the T-bet−/− Rag2−/− model. Similar effects were obtained in two additional colonic inflammation models, Il10−/− mice and dextran sulfate sodium-treated mice. Efforts to understand how a lipophobic superoxide anion (O2−) can be detoxified by cytoplasmic lactoccocal SodA led to the finding that host antimicrobial-mediated lysis is a prerequisite for SodA release and SodA’s extracytoplasmic O2− scavenging. L. lactis I-1631 may represent a promising vehicle to deliver antioxidant, colitis-attenuating SodA to the inflamed intestinal mucosa, and host antimicrobials may play a critical role in mediating SodA’s bioaccessibility. PMID:26056274

  10. CMV-Independent Lysis of Glioblastoma by Ex Vivo Expanded/Activated Vδ1+ γδ T Cells

    PubMed Central

    Knight, Andrea; Arnouk, Hilal; Britt, William; Gillespie, G. Yancey; Cloud, Gretchen A.; Harkins, Lualhati; Su, Yun

    2013-01-01

    Vδ2neg γδ T cells, of which Vδ1+ γδ T cells are by far the largest subset, are important effectors against CMV infection. Malignant gliomas often contain CMV genetic material and proteins, and evidence exists that CMV infection may be associated with initiation and/or progression of glioblastoma multiforme (GBM). We sought to determine if Vδ1+ γδ T cells were cytotoxic to GBM and the extent to which their cytotoxicity was CMV dependent. We examined the cytotoxic effect of ex vivo expanded/activated Vδ1+ γδ T cells from healthy CMV seropositive and CMV seronegative donors on unmanipulated and CMV-infected established GBM cell lines and cell lines developed from short- term culture of primary tumors. Expanded/activated Vδ1+ T cells killed CMV-negative U251, U87, and U373 GBM cell lines and two primary tumor explants regardless of the serologic status of the donor. Experimental CMV infection did not increase Vδ1+ T cell - mediated cytotoxicity and in some cases the cell lines were more resistant to lysis when infected with CMV. Flow cytometry analysis of CMV-infected cell lines revealed down-regulation of the NKG2D ligands ULBP-2, and ULBP-3 as well as MICA/B in CMV-infected cells. These studies show that ex vivo expanded/activated Vδ1+ γδ T cells readily recognize and kill established GBM cell lines and primary tumor-derived GBM cells regardless of whether CMV infection is present, however, CMV may enhance the resistance GBM cell lines to innate recognition possibly contributing to the poor immunogenicity of GBM. PMID:23950874

  11. Single 4.5 mg fixed-dose of rasburicase for hyperuricemia associated with tumor lysis syndrome.

    PubMed

    Patel, Kajal S; Lau, Jessica E; Zembillas, Anthony S; Gallagher, Erika M

    2017-07-01

    Rasburicase is a recombinant urate oxidase enzyme administered for treatment of hyperuricemia associated with tumor lysis syndrome. Studies demonstrate effectiveness of single fixed-dose rasburicase as compared to the FDA-approved dose of 0.2 mg/kg intravenously daily for up to five days. Doses in these studies range from 1.5 mg to 7.5 mg. Our study evaluated outcomes in patients who received single 4.5 mg fixed-dose rasburicase. This retrospective, IRB-approved chart review evaluated adult oncology subjects who received fixed-dose rasburicase between January 2007 and April 2014. The primary outcome was percentage of patients with normalization of uric acid (level <8 mg/dL within 24 h) after a single 4.5 mg fixed-dose of rasburicase. Secondary objectives were incidence of initial failure of fixed-dose rasburicase and normalization of uric acid in overweight (body mass index ≥25 kg/m(2)) versus non-overweight patients. Initial failure was defined as need for additional doses or progression to dialysis within one week of the initial fixed-dose. In the 128 patients included, the mean baseline uric acid level was 14.84 mg/dL. Of the 112 patients with a follow-up uric acid level, 68% achieved normalization within 24 h of rasburicase administration. Thirty-eight patients received additional treatment: 10 received additional dose(s) and 28 underwent dialysis. Normalization of uric acid in overweight versus non-overweight patients was 66% and 73%, respectively. Overall, a single 4.5 mg fixed-dose of rasburicase effectively normalized uric acid in 68% of patients within 24 h. Further studies are needed to determine the optimal single fixed-dose necessary for treatment response across all patients.

  12. Enterococcus faecalis Produces Abundant Extracellular Structures Containing DNA in the Absence of Cell Lysis during Early Biofilm Formation

    PubMed Central

    Barnes, Aaron M. T.; Ballering, Katie S.; Leibman, Rachel S.; Wells, Carol L.; Dunny, Gary M.

    2012-01-01

    ABSTRACT Enterococcus faecalis is a common Gram-positive commensal bacterium of the metazoan gastrointestinal tract capable of biofilm formation and an opportunistic pathogen of increasing clinical concern. Dogma has held that biofilms are slow-growing structures, often taking days to form mature microcolonies. Here we report that extracellular DNA (eDNA) is an integral structural component of early E. faecalis biofilms (≤4 h postinoculation). Combining cationic dye-based biofilm matrix stabilization techniques with correlative immuno-scanning electron microscopy (SEM) and fluorescent techniques, we demonstrate that—in early E. faecalis biofilms—eDNA localizes to previously undescribed intercellular filamentous structures, as well as to thick mats of extruded extracellular matrix material. Both of these results are consistent with previous reports that early biofilms are exquisitely sensitive to exogenous DNase treatment. High-resolution SEM demonstrates a punctate labeling pattern in both structures, suggesting the presence of an additional, non-DNA constituent. Notably, the previously described fratricidal or lytic mechanism reported as the source of eDNA in older (≥24 h) E. faecalis biofilms does not appear to be at work under these conditions; extensive visual examination by SEM revealed a striking lack of lysed cells, and bulk biochemical assays also support an absence of significant lysis at these early time points. In addition, some cells demonstrated eDNA labeling localized at the septum, suggesting the possibility of DNA secretion from metabolically active cells. Overall, these data are consistent with a model in which a subpopulation of viable E. faecalis cells secrete or extrude DNA into the extracellular matrix. PMID:22829679

  13. From cellular lysis to microarray detection, an integrated thermoplastic elastomer (TPE) point of care Lab on a Disc.

    PubMed

    Roy, Emmanuel; Stewart, Gale; Mounier, Maxence; Malic, Lidija; Peytavi, Régis; Clime, Liviu; Madou, Marc; Bossinot, Maurice; Bergeron, Michel G; Veres, Teodor

    2015-01-21

    We present an all-thermoplastic integrated sample-to-answer centrifugal microfluidic Lab-on-Disc system (LoD) for nucleic acid analysis. The proposed CD system and engineered platform were employed for analysis of Bacillus atrophaeus subsp. globigii spores. The complete assay comprised cellular lysis, polymerase chain reaction (PCR) amplification, amplicon digestion, and microarray hybridization on a plastic support. The fluidic robustness and operating efficiency of the assay were ensured through analytical optimization of microfluidic tools enabling beneficial implementation of capillary valves and accurate control of all flow timing procedures. The assay reliability was further improved through the development of two novel microfluidic strategies for reagents mixing and flow delay on the CD platform. In order to bridge the gap between the proof-of-concept LoD and production prototype demonstration, low-cost thermoplastic elastomer (TPE) was selected as the material for CD fabrication and assembly, allowing the use of both, high quality hot-embossing and injection molding processes. Additionally, the low-temperature and pressure-free assembly and bonding properties of TPE material offer a pertinent solution for simple and efficient loading and storage of reagents and other on-board components. This feature was demonstrated through integration and conditioning of microbeads, magnetic discs, dried DNA buffer reagents and spotted DNA array inserts. Furthermore, all microfluidic functions and plastic parts were designed according to the current injection mold-making knowledge for industrialization purposes. Therefore, the current work highlights a seamless strategy that promotes a feasible path for the transfer from prototype toward realistic industrialization. This work aims to establish the full potential for TPE-based centrifugal system as a mainstream microfluidic diagnostic platform for clinical diagnosis, water and food safety, and other molecular diagnostic

  14. 'Lysi-T-FACE

    NASA Astrophysics Data System (ADS)

    Herndl, Markus; Pötsch, Erich; Kandolf, Matthias; Bohner, Andreas; Schaumberger, Andreas; Resch, Reinhard; Graiss, Wilhelm; Krautzer, Bernhard; Buchgraber, Karl

    2010-05-01

    During the past century the average global surface air temperature has already increased by 1°C. A doubling of atmospheric concentration of CO2 near the end of the 21st century is predicted to result in a 3°C temperature increase. The Alpine region has experienced above average warming over the last century and is considered particularly vulnerable to global change. In Austria in some regions, grassland production suffered severe droughts during the last decade leading to serious damages and even temporal shortage in feed supply. Changes in temperature and precipitation have evident consequences for grassland vegetation. Increasing atmospheric CO2 concentration is a major driver of climate change. Photosynthesis and productivity of most grassland species might be stimulated by increasing CO2 when soil nutrients and water are not limiting. On dry grassland sites increasing CO2 also reduces plant water loss, thereby increasing plant water use efficiency. When plant production is limited by seasonal cold temperatures as e.g. in the inner Alpine parts of Austria and in high altitude or high latitude grasslands, combined warming and higher CO2 might continue to enhance plant production. However, it is still unknown to what extend a further increase of temperature and CO2 will result in higher biomass yield in different grassland communities. To study the effects of global warming on future grassland communities and management, the application of a heating treatment combined with free-air controlled enhancement of CO2 (T-FACE) to open-field plant canopies at lysimeters is an innovative approach which allows studying responses of the plant-soil-systems as well as carbon- water and nutrient fluxes under expected future climate. The experiment is scheduled to run in the first phase 6 years (2011-2017) and is located at the AREC Raumberg-Gumpenstein, Upper Enns Valley, Austria (47,49; 14,10). Heater arrays and miniFACE rings are installed in 1.6 m diameter plots and expose a grassland mixture to a factorial combination of two CO2 and two temperature regimes, with four replications each. To simulate water limitation a treatment with rainout shelters is planed. The results of the experiment should give answers to many aspects caused by global changes e.g. on plant productivity, nutrient and water dynamics and in the end shall give advice for farmers how future grassland require adjustments in management.

  15. Euglobulin lysis time

    MedlinePlus

    ... tendency. In: Bain BJ, Bates I, Laffan MA, Lewis SM, eds. Dacie and Lewis Practical Haematology . 11th ed. Philadelphia, PA: Elsevier Churchill ... provided by VeriMed Healthcare Network. Also reviewed by David Zieve, MD, MHA, Medical Director, Brenda Conaway, Editorial ...

  16. AnaLysis of Expression on human chromosome 21, ALE-HSA21: a pilot integrated web resource

    PubMed Central

    Scarpato, Margherita; Esposito, Roberta; Evangelista, Daniela; Aprile, Marianna; Ambrosio, Maria Rosaria; Angelini, Claudia; Ciccodicola, Alfredo; Costa, Valerio

    2014-01-01

    Transcriptome studies have shown the pervasive nature of transcription, demonstrating almost all the genes undergo alternative splicing. Accurately annotating all transcripts of a gene is crucial. It is needed to understand the impact of mutations on phenotypes, to shed light on genetic and epigenetic regulation of mRNAs and more generally to widen our knowledge about cell functionality and tissue diversity. RNA-sequencing (RNA-Seq), and the other applications of the next-generation sequencing, provides precious data to improve annotations' accuracy, simultaneously creating issues related to the variety, complexity and the size of produced data. In this ‘scenario’, the lack of user-friendly resources, easily accessible to researchers with low skills in bioinformatics, makes difficult to retrieve complete information about one or few genes without browsing a jungle of databases. Concordantly, the increasing amount of data from ‘omics’ technologies imposes to develop integrated databases merging different data formats coming from distinct but complementary sources. In light of these considerations, and given the wide interest in studying Down syndrome—a genetic condition due to the trisomy of human chromosome 21 (HSA21)—we developed an integrated relational database and a web interface, named ALE-HSA21 (AnaLysis of Expression on HSA21), accessible at http://bioinfo.na.iac.cnr.it/ALE-HSA21. This comprehensive and user-friendly web resource integrates—for all coding and noncoding transcripts of chromosome 21—existing gene annotations and transcripts identified de novo through RNA-Seq analysis with predictive computational analysis of regulatory sequences. Given the role of noncoding RNAs and untranslated regions of coding genes in key regulatory mechanisms, ALE-HSA21 is also an interesting web-based platform to investigate such processes. The ‘transcript-centric’ and easily-accessible nature of ALE-HSA21 makes this resource a valuable tool to

  17. Sevelamer is an Effective Drug in Treating Hyperphosphatemia Due to Tumor Lysis Syndrome in Children: A Developing World Experience.

    PubMed

    Kahlon, Dilraj Kaur; Dinand, Veronique; Yadav, Satya Prakash; Sachdeva, Anupam

    2016-03-01

    We report here a study on efficacy of sevelamer hydrochloride in treating hyperphosphatemia due to tumor lysis syndrome (TLS) in a developing world setting. Twenty one children with hyperphosphatemia due to TLS were included. All received hyper-hydration, allopurinol and sevelamer. Efficacy was assessed by decrease in serum phosphate level, calcium-phosphate product and TLS score as per Cairo Bishop definition. Four children who underwent dialysis were excluded from analysis. Among the remaining 17 patients with hyperphosphatemia, laboratory TLS was recorded in 15 patients and clinical TLS in five. Sevelamer was given according to weight, most often 400 mg twice to thrice daily. Mean phosphatemia decreased from 8.3 ± 3.0 to 6.7 ± 2.1 mg/dl within 24 h of starting sevelamer (p = 0.02), 6.0 ± 2.1 mg/dl at 48 h, 4.9 ± 1.5 mg/dl at 72 h and 4.39 ± 1.7 mg/dl at 96 h. TLS was corrected in 72 h in 14 patients, 96 h in 1 and 120 h in another patient. Mean calcium-phosphate product decreased from 63.0 ± 14.0 to 49.2 ± 9.7 mg/dl (p = 0.002) at 24 h, 46.1 ± 17.0 mg/dl at 48 h and 39.7 ± 13.5 mg/dl at 72 h. There was no mortality due to hyperphosphatemia. Sevelamer is efficacious in children with malignancy-associated hyperphosphatemia in the developing world.

  18. [Clinical analysis of 18 cases with acute tumor lysis syndrome in children with B-cell lymphoma].

    PubMed

    Huang, Shuang; Yang, Jing; Zhang, Rui; Duan, Yan-long; Zhang, Yong-hong

    2011-08-01

    To investigate risk factors associated with acute tumor lysis syndrome (ATLS) in children with B-cell lymphoma and to explore feasible means for the prophylaxis and treatment. Data from 18 children with ATLS in B-cell lymphoma were collected to assess their tumor burden at diagnosis and before chemotherapy. Evaluation was performed at the 8th day, 3 month, and the end of chemotherapy and follow up. The incidence of ATLS in B-cell lymphoma, and the relationship between the incidence of ATLS and whether the kidney was involved and large tumor burden were analyzed respectively. All patients received hydration, alkalinization and received allopurinol routinely. Urate oxidase and hemodialysis treatment were administered in some cases. Of the 103 children with B-cell lymphoma, 18 were diagnosed as having ATLS (17.5%). All the 18 cases with ATLS were histopathologically confirmed as having Burkitt's lymphoma. All the patients were at stage III or IV and all had large tumor sizes, and 7 were found to have blasts in the bone marrow>25% (38.9%). Lactate dehydrogenase (LDH) levels≥1000 U/L were found in 11 (61.1%) cases. All patients had developed metabolic abnormalities, including hyperuricemia, hyperphosphatemia, hypocalcemia, and uremia. In terms of clinical features and prognosis, all cases had nausea, vomiting, anorexia, oliguria, and anuria at different levels. One had gastrointestinal bleeding, 7 patients experienced seizures. The etiology in five was hypocalcemia and two had reversible posterior encephalopathy syndrome and all responded well to treatment. Nine cases of ATLS responded to supportive care, 4 required hemodialysis, and the other 4 responded to urate oxidase. Ten cases survived and 8 died. The major cause of death was severe complications and treatment was given up in 5 cases and recurrence occurred in 3 cases. ATLS was commonly seen in Burkitt's subtype of B-cell lymphoma. Higher LDH and large tumor sizes and kidney involvement were important risk

  19. A facile one-step method for cell lysis and DNA extraction of waterborne pathogens using a microchip.

    PubMed

    Kamat, Vivek; Pandey, Sulaxna; Paknikar, Kishore; Bodas, Dhananjay

    2018-01-15

    Globally, waterborne organisms are the primary causative agents for the transmission of various forms of diarrheal diseases. For accurate diagnosis, molecular tools have gained considerable attention in the recent past. Molecular tools require DNA as the starting material for diagnosis, and hence, a prerequisite is the quality and integrity of DNA. To obtain high quality DNA rapidly, we have fabricated a microchip in poly(dimethyl siloxane) (PDMS) by soft lithography process. The microchip facilitated in-flow coating of chitosan on the magnetic nanoparticles, which under external mechanical vibration caused cell lysis and released DNA in the supernatant. The released DNA was captured by the nanoparticles owing to its positive charge (chitosan coating). The magnetic nanoparticle-DNA complex was then isolated from the in-flow matrix using permanent magnet, Further, removal of the cell debris, proteins, and carbohydrates was done using wash buffer. DNA extracted using the microchip was pure with absorbance (260/280) ratio of 1.77±0.04, as compared to 1.79±0.03 obtained by TRIzol method. The complete isolation of the DNA using the microchip took ~ 15min as against>2h with a TRIzol method. Six gram-negative waterborne pathogens were used to demonstrate the efficacy of the microchip based DNA extraction process. The integrity of the isolated DNA was assessed by amplifying the 16S rRNA gene using Com1 and Com2 universal primers. The presence of a band at 407bp on gel electrophoresis confirmed the amplified product. Further, the gel image was used for quantification of the amplified product using ImageJ software. Higher regression values obtained using microchip confirmed better quality and integrity of the extracted DNA as opposed to the conventional method. The lower (<2%) relative standard deviation values obtained from the data suggested that the microchip process was reproducible. The quality and integrity of the obtained DNA proved the simplicity, rapidity, and

  20. Differences in the toxicity of six Gambierdiscus (Dinophyceae) species measured using an in vitro human erythrocyte lysis assay.

    PubMed

    Holland, William C; Litaker, R Wayne; Tomas, Carmelo R; Kibler, Steven R; Place, Allen R; Davenport, Erik D; Tester, Patricia A

    2013-04-01

    This study examined the toxicity of six Gambierdiscus species (Gambierdiscus belizeanus, Gambierdiscus caribaeus, Gambierdiscus carolinianus, Gambierdiscus carpenteri, Gambierdiscus ribotype 2 and Gambierdiscus ruetzleri) using a human erythrocyte lysis assay. In all, 56 isolates were tested. The results showed certain species were significantly more toxic than others. Depending on the species, hemolytic activity consistently increased by ∼7-40% from log phase growth to late log - early stationary growth phase and then declined in mid-stationary growth phase. Increasing growth temperatures from 20 to 31 °C for clones of G. caribaeus showed only a slight increase in hemolytic activity between 20 and 27 °C. Hemolytic activity in the G. carolinianus isolates from different regions grown over the same 20-31 °C range remained constant. These data suggest that growth temperature is not a significant factor in modulating the inter-isolate and interspecific differences in hemolytic activity. The hemolytic activity of various isolates measured repeatedly over a 2 year period remained constant, consistent with the hemolytic compounds being constitutively produced and under strong genetic control. Depending on species, greater than 60-90% of the total hemolytic activity was initially associated with the cell membranes but diffused into solution over a 24 h assay incubation period at 4 °C. These findings suggest that hemolytic compounds produced by Gambierdiscus isolates were held in membrane bound vesicles as reported for brevetoxins produced by Karenia brevis. Gambierdiscus isolates obtained from other parts of the world exhibited hemolytic activities comparable to those found in the Caribbean and Gulf of Mexico confirming the range of toxicities is similar among Gambierdiscus species worldwide. Experiments using specific inhibitors of the MTX pathway and purified MTX, Gambierdiscus whole cell extracts, and hydrophilic cell extracts containing MTX, were consistent with

  1. Influence of polymer architecture on antigens camouflage, CD47 protection and complement mediated lysis of surface grafted red blood cells.

    PubMed

    Chapanian, Rafi; Constantinescu, Iren; Rossi, Nicholas A A; Medvedev, Nadia; Brooks, Donald E; Scott, Mark D; Kizhakkedathu, Jayachandran N

    2012-11-01

    Hyperbranched polyglycerol (HPG) and polyethylene glycol (PEG) polymers with similar hydrodynamic sizes in solution were grafted to red blood cells (RBCs) to investigate the impact of polymer architecture on the cell structure and function. The hydrodynamic sizes of polymers were calculated from the diffusion coefficients measured by pulsed field gradient NMR. The hydration of the HPG and PEG was determined by differential scanning calorimetry analyses. RBCs grafted with linear PEG had different properties compared to the compact HPG grafted RBCs. HPG grafted RBCs showed much higher electrophoretic mobility values than PEG grafted RBCs at similar grafting concentrations and hydrodynamic sizes indicating differences in the structure of the polymer exclusion layer on the cell surface. PEG grafting impacted the deformation properties of the membrane to a greater degree than HPG. The complement mediated lysis of the grafted RBCs was dependent on the type of polymer, grafting concentration and molecular size of grafted chains. At higher molecular weights and graft concentrations both HPG and PEG triggered complement activation. The magnitude of activation was higher with HPG possibly due to the presence of many hydroxyl groups per molecule. HPG grafted RBCs showed significantly higher levels of CD47 self-protein accessibility than PEG grafted RBCs at all grafting concentrations and molecular sizes. PEG grafted polymers provided, in general, a better shielding and protection to ABO and minor antigens from antibody recognition than HPG polymers, however, the compact HPGs provided greater protection of certain antigens on the RBC surface. Our data showed that HPG 20 kDa and HPG 60 kDa grafted RBCs exhibited properties that are more comparable to the native RBC than PEG 5 kDa and PEG 10 kDa grafted RBCs of comparable hydrodynamic sizes. The study shows that small compact polymers such as HPG 20 kDa have a greater potential in the generation of functional RBC for therapeutic

  2. A rapid method for authentication of Buffalo (Bubalus bubalis) meat by Alkaline Lysis method of DNA extraction and species specific polymerase chain reaction.

    PubMed

    Girish, P S; Haunshi, S; Vaithiyanathan, S; Rajitha, R; Ramakrishna, C

    2013-02-01

    Buffalo (Bubalus bubalis) meat is a major item of export from India but export of beef i.e. meat from cattle (Bos indicus) is prohibited. Also, adulteration of buffalo meat with that of beef (meat from cattle) is a common fraudulent practice because of prohibition on cow slaughter in most states of India. Food analysts require precise identification techniques to implement such regulations. In the present study, a method of DNA extraction by Alkaline lysis from meat samples and speciation of buffalo meat using species specific Polymerase Chain Reaction (PCR) has been reported. Alkaline lysis technique is a rapid method which involves triturating meat with four volumes of 0.2N NaOH, dilution of resultant liquid extract with eight volumes of 0.2N NaOH, heating the mix 75 °C for 20 min followed by neutralization with eight volumes of 0.04N Tris HCl. Entire procedure of DNA extraction takes less than 30 min and it is economical as it involves less expensive chemicals. Method was successfully applied in animal byproducts also viz., liver, heart and kidney. For authentication of buffalo meat, pair of primers was designed based on mitochondrial D loop gene nucleotide sequence. PCR amplification using the designed primers gave amplicon of size 482 bp in buffalo and no amplification was detected in closely related species viz., cattle, sheep and goat meat samples. Results of the assay were highly repetitive and reliable. An export sample referred by export regulation authorities was also analyzed by using the Alkaline lysis method of DNA extraction and species specific PCR which enabled authentication of meat within 5 h.

  3. Understanding the Structure-Function Relationship of Lysozyme Resistance in Staphylococcus aureus by Peptidoglycan O-Acetylation Using Molecular Docking, Dynamics, and Lysis Assay.

    PubMed

    Pushkaran, Anju C; Nataraj, Namrata; Nair, Nisha; Götz, Friedrich; Biswas, Raja; Mohan, C Gopi

    2015-04-27

    Lysozyme is an important component of the host innate defense system. It cleaves the β-1,4 glycosidic bonds between N-acetylmuramic acid and N-acetylglucosamine of bacterial peptidoglycan and induce bacterial lysis. Staphylococcus aureus (S. aureus), an opportunistic commensal pathogen, is highly resistant to lysozyme, because of the O-acetylation of peptidoglycan by O-acetyl transferase (oatA). To understand the structure-function relationship of lysozyme resistance in S. aureus by peptidoglycan O-acetylation, we adapted an integrated approach to (i) understand the effect of lysozyme on the growth of S. aureus parental and the oatA mutant strain, (ii) study the lysozyme induced lysis of exponentially grown and stationary phase of both the S. aureus parental and oatA mutant strain, (iii) investigate the dynamic interaction mechanism between normal (de-O-acetylated) and O-acetylated peptidoglycan substrate in complex with lysozyme using molecular docking and molecular dynamics simulations, and (iv) quantify lysozyme resistance of S. aureus parental and the oatA mutant in different human biological fluids. The results indicated for the first time that the active site cleft of lysozyme binding with O-acetylated peptidoglycan in S. aureus was sterically hindered and the structural stability was higher for the lysozyme in complex with normal peptidoglycan. This could have conferred reduced survival of the S. aureus oatA mutant in different human biological fluids. Consistent with this computational analysis, the experimental data confirmed decrease in the growth, lysozyme induced lysis, and lysozyme resistance, due to peptidoglycan O-acetylation in S. aureus.

  4. The application of alkaline lysis and pressure cycling technology in the differential extraction of DNA from sperm and epithelial cells recovered from cotton swabs.

    PubMed

    Nori, Deepthi V; McCord, Bruce R

    2015-09-01

    This study reports the development of a two-step protocol using pressure cycling technology (PCT) and alkaline lysis for differential extraction of DNA from mixtures of sperm and vaginal epithelial cells recovered from cotton swabs. In controlled experiments, in which equal quantities of sperm and female epithelial cells were added to cotton swabs, 5 min of pressure pulsing in the presence of 0.4 M NaOH resulted in 104 ± 6% recovery of female epithelial DNA present on the swab. Following the pressure treatment, exposing the swabs to a second 5-min alkaline treatment at 95 °C without pressure resulted in the selective recovery of 69 ± 6% of the sperm DNA. The recovery of the vaginal epithelia and sperm DNA was optimized by examining the effect of sodium hydroxide concentration, incubation temperature, and time. Following the alkaline lysis steps, the samples were neutralized with 2 M Tris (pH 7.5) and purified with phenol-chloroform-isoamyl alcohol to permit downstream analysis. The total processing time to remove both fractions from the swab was less than 20 min. Short tandem repeat (STR) analysis of these fractions obtained from PCT treatment and alkaline lysis generated clean profiles of female epithelial DNA and male sperm DNA for 1:1 mixtures of female and male cells and predominant male profiles for mixtures up to 5:1 female to male cells. By reducing the time and increasing the recovery of DNA from cotton swabs, this new method presents a novel and potentially useful procedure for forensic differential extractions.

  5. Cytotoxic T lymphocyte lysis of HTLV-1 infected cells is limited by weak HBZ protein expression, but non-specifically enhanced on induction of Tax expression.

    PubMed

    Rowan, Aileen G; Suemori, Koichiro; Fujiwara, Hiroshi; Yasukawa, Masaki; Tanaka, Yuetsu; Taylor, Graham P; Bangham, Charles R M

    2014-12-14

    Immunogenetic evidence indicates that cytotoxic T lymphocytes (CTLs) specific for the weak CTL antigen HBZ limit HTLV-1 proviral load in vivo, whereas there is no clear relationship between the proviral load and the frequency of CTLs specific for the immunodominant antigen Tax. In vivo, circulating HTLV-1-infected cells express HBZ mRNA in contrast, Tax expression is typically low or undetectable. To elucidate the virus-suppressing potential of CTLs targeting HBZ, we compared the ability of HBZ- and Tax-specific CTLs to lyse naturally-infected cells, by co-incubating HBZ- and Tax-specific CTL clones with primary CD4(+) T cells from HLA-matched HTLV-1-infected donors. We quantified lysis of infected cells, and tested whether specific virus-induced host cell surface molecules determine the susceptibility of infected cells to CTL-mediated lysis. Primary infected cells upregulated HLA-A*02, ICAM-1, Fas and TRAIL-R1/2 in concert with Tax expression, forming efficient targets for both HTLV-1-specific CTLs and CTLs specific for an unrelated virus. We detected expression of HBZ mRNA (spliced isoform) in both Tax-expressing and non-expressing infected cells, and the HBZ26-34 epitope was processed and presented by cells transfected with an HBZ expression plasmid. However, when coincubated with primary cells, a high-avidity HBZ-specific CTL clone killed significantly fewer infected cells than were killed by a Tax-specific CTL clone. Finally, incubation with Tax- or HBZ-specific CTLs resulted in a significant decrease in the frequency of cells expressing high levels of HLA-A*02. HTLV-1 gene expression in primary CD4(+) T cells non-specifically increases susceptibility to CTL lysis. Despite the presence of HBZ spliced-isoform mRNA, HBZ epitope presentation by primary cells is significantly less efficient than that of Tax.

  6. Valproic Acid Upregulates NKG2D Ligand Expression through an ERK-dependent Mechanism and Potentially Enhances NK Cell-mediated Lysis of Myeloma1

    PubMed Central

    Wu, Xiaosong; Tao, Yi; Hou, Jun; Meng, Xiuqin; Shi, Jumei

    2012-01-01

    Modulation of the antitumor immune response through the engagement of NKG2D receptors with their ligands (L) on targets represents a promising therapeutic approach against cancer. In this study, we tested the effect of valproic acid (VPA), a histone deacetylase (HDAC) inhibitor, on the expression of NKG2D ligands in myeloma cells. We demonstrated that VPA was able to upregulate both protein and mRNA expression of major histocompatibility complex class I-related chain (MIC) A/B and UL16-binding protein (ULBP) 2 without any significant effect on the expression of ULBP1, ULBP3, and ULBP4 or induction of other natural killer (NK) cell ligands, such as NKp30-L, NKp44-L, and NKp46-L in myeloma cells. A 51Cr release assay and degranulation assay indicated that the induction of MICA/B and ULBP2 augmented NK cell-mediated lysis of myeloma cells, which was abolished by the addition of a blocking NKG2D antibody. Activation of constitutively phosphorylated extracellular signal-regulated kinase (ERK) by VPA is essential for the up-regulation of MICA/B and ULBP2 expressions. Inhibition of ERK using ERK inhibitor PD98059 decreased both MICA/B and ULBP2 expressions and NK cell cytotoxicity. Furthermore, overexpression of constitutively active ERK in ARK resulted in increased MICA/B and ULBP2 expressions and enhanced NK cell lysis. These data indicate that increased sensitivity of VPA-treated myeloma cells to NK cell lysis is caused by higher NKG2D ligand expression, resulting from more active ERK signaling pathway. Our results provide evidence that targeting ERK signaling pathway may be an additional mechanism supporting the antimyeloma activity of HDAC inhibitors and suggest its possible immunotherapeutic value for myeloma treatment. PMID:23308050

  7. Viral Lysis of Cells Influences The Concentration and Compostion of Dissolved Organic Matter and The Formation of Organic Aggregates (marine Snow)

    NASA Astrophysics Data System (ADS)

    Weinbauer, M. G.; Peduzzi, P.

    The effect of moderately (ca. 2.5 fold) increasing the concentration of the virus-size fraction (VSF) of seawater on the chemical composition of the dissolved organic mat- ter (DOM) pool during the formation of organic aggregates (marine snow) was tested experimentally with seawater samples collected in the Northern Adriatic Sea. The VSF enrichment did not significantly change the concentration of selected DOM com- pounds, whereas viral abundance was ca. 2-fold higher. During long-term experiments (40 - 200 hrs), bacterial abundance was on average 25% lower in the VSF amended than in the control incubations, and the frequency of visibly infected cells was stimu- lated by ca. 50%. VSF delayed the development of phytoplankton blooms (diatoms), but in the end of the experiments, Chl a concentrations in the VSF amended incuba- tions exceeded those in the control incubations. The VSF enrichment caused an enrich- ment of Serine and Threonine in the dissolved hydrolysable amino acid (AA) fraction indicative of viral lysis of diatoms. Bulk dissolved free AA acid and monomeric car- bohydrate (CHO) concentrations were repressed, whereas bulk dissolved hydrolysable AA and CHO concentrations were stimulated in the VSF enriched incubations. Viral lysis was likely the major reason for the stimulation of hydrolysable DOM. The for- mation of organic aggregates was repressed by the VSF enrichment, but the aggregates were larger and more persistent in the VSF amended than in the control incubations. Stimulation of hydrolysable DOM and sticky viral lysis products might be the reason for the larger and more persistent aggregates. This demonstrates that bioactive mate- rial in the VSF of seawater can have major implications for primary production and the cycling of organic carbon in the ocean.

  8. Identification of Bombyx mori nucleopolyhedrovirus bm58a as an auxiliary gene and its requirement for cell lysis and larval liquefaction.

    PubMed

    Yang, Rui; Zhang, Jianjia; Feng, Min; Wu, Xiaofeng

    2016-11-01

    Bombyx mori nucleopolyhedrovirus orf58a (bm58a) and its homologues are highly conserved in genomes of all sequenced group I alphabaculoviruses and its function is still unknown. Transcriptional analysis revealed that bm58a is a very late gene initiated from a late transcriptional start motif TAAG. To examine its role in the virus, a bm58a knockout virus (vBmbm-58a-KO-PH-GFP) was generated through homologous recombination in Escherichia coli. Analysis of fluorescence microscopy, titration assays and electron microscopy examination showed that the deletion of bm58a did not affect viral replication and occlusion bodies formation in vitro, indicating that bm58a is not required for viral propagation. However, vBmbm-58a-KO-PH-GFP did not result in cell lysis when wild-type virus infected cells began to lyse, and the vBmbm-58a-KO-PH-GFP infected cells remained intact until 2 weeks post-infection. Quantification of polyhedra release from cells confirmed this observation. Accordingly, though deletion of bm58a did not reduce Bombyx mori nucleopolyhedrovirus infectivity in vivo in bioassays, it did significantly disrupt the larval liquefaction, reducing the level of polyhedra release from infected host. Immunofluorescence analysis demonstrated that Bm58a was predominantly localized on the cellular membrane at the late stage of infection, which may contribute to its function of facilitating cell lysis and larval liquefaction. Our results suggest that although bm58a is not essential for viral propagation as an auxiliary gene, it is a key factor of virus-induced cell lysis and larval liquefaction in vitro and in vivo.

  9. Sublethal exposure to alpha radiation (223Ra dichloride) enhances various carcinomas’ sensitivity to lysis by antigen-specific cytotoxic T lymphocytes through calreticulin-mediated immunogenic modulation

    PubMed Central

    Malamas, Anthony S.; Gameiro, Sofia R.; Knudson, Karin M.; Hodge, James W.

    2016-01-01

    Radium-223 dichloride (Xofigo®; 223Ra) is an alpha-emitting radiopharmaceutical FDA-approved for the treatment of bone metastases in patients with advanced castration-resistant prostate cancer. It is also being examined clinically in patients with breast and lung carcinoma and patients with multiple myeloma. As with other forms of radiation, the aim of 223Ra is to reduce tumor burden by directly killing tumor cells. External beam (photon) and proton radiation have been shown to augment tumor sensitivity to antigen-specific CD8+ cytotoxic T lymphocytes (CTLs). However, little is known about whether treatment with 223Ra can also induce such immunogenic modulation in tumor cells that survive irradiation. We examined these effects in vitro by exposing human prostate, breast, and lung carcinoma cells to sublethal doses of 223Ra. 223Ra significantly enhanced T cell-mediated lysis of each tumor type by CD8+ CTLs specific for MUC-1, brachyury, and CEA tumor antigens. Immunofluorescence analysis revealed that the increase in CTL killing was accompanied by augmented protein expression of MHC-I and calreticulin in each tumor type, molecules that are essential for efficient antigen presentation. Enhanced tumor-cell lysis was facilitated by calreticulin surface translocation following 223Ra exposure. The phenotypic changes observed after treatment appear to be mediated by induction of the endoplasmic reticulum stress response pathway. By rendering tumor cells more susceptible to T cell-mediated lysis, 223Ra may potentially be effective in combination with various immunotherapies, particularly cancer vaccines that are designed to generate and expand patients’ endogenous antigen-specific T-cell populations against specific tumor antigens. PMID:27893426

  10. Sublethal exposure to alpha radiation (223Ra dichloride) enhances various carcinomas' sensitivity to lysis by antigen-specific cytotoxic T lymphocytes through calreticulin-mediated immunogenic modulation.

    PubMed

    Malamas, Anthony S; Gameiro, Sofia R; Knudson, Karin M; Hodge, James W

    2016-12-27

    Radium-223 dichloride (Xofigo®; 223Ra) is an alpha-emitting radiopharmaceutical FDA-approved for the treatment of bone metastases in patients with advanced castration-resistant prostate cancer. It is also being examined clinically in patients with breast and lung carcinoma and patients with multiple myeloma. As with other forms of radiation, the aim of 223Ra is to reduce tumor burden by directly killing tumor cells. External beam (photon) and proton radiation have been shown to augment tumor sensitivity to antigen-specific CD8+ cytotoxic T lymphocytes (CTLs). However, little is known about whether treatment with 223Ra can also induce such immunogenic modulation in tumor cells that survive irradiation. We examined these effects in vitro by exposing human prostate, breast, and lung carcinoma cells to sublethal doses of 223Ra. 223Ra significantly enhanced T cell-mediated lysis of each tumor type by CD8+ CTLs specific for MUC-1, brachyury, and CEA tumor antigens. Immunofluorescence analysis revealed that the increase in CTL killing was accompanied by augmented protein expression of MHC-I and calreticulin in each tumor type, molecules that are essential for efficient antigen presentation. Enhanced tumor-cell lysis was facilitated by calreticulin surface translocation following 223Ra exposure. The phenotypic changes observed after treatment appear to be mediated by induction of the endoplasmic reticulum stress response pathway. By rendering tumor cells more susceptible to T cell-mediated lysis, 223Ra may potentially be effective in combination with various immunotherapies, particularly cancer vaccines that are designed to generate and expand patients' endogenous antigen-specific T-cell populations against specific tumor antigens.

  11. Evaluation of minimally invasive therapies and rationale for a prospective randomized trial to evaluate selective intra-arterial lysis for clinically complete central retinal artery occlusion.

    PubMed

    Mueller, Arthur J; Neubauer, Aljoscha S; Schaller, Ulrich; Kampik, Anselm

    2003-10-01

    To determine the effect of commonly used minimally invasive treatments for clinically complete nonarteritic central retinal artery occlusion (CRAO) and design a prospective randomized trial to evaluate selective intra-arterial lysis for this condition. In this retrospective noncomparative case series, all medical records of patients with a diagnosis of CRAO treated at the Department of Ophthalmology, Ludwig-Maximilians-Universität, Munich, Germany, from 1994 through 1999 were reviewed for treatments administered and course of visual acuity. Best-corrected visual acuity (BCVA) at initial and last visit. We identified 102 patient medical records; 71 were suitable for further analysis. Forty-four (62%) of the 71 patients included were treated with oral acetylsalicylate; 44 (62%), with oral acetazolamide; 32 (45%), with ocular massage; 22 (31%), with isovolemic hemodilution; 19 (27%), with oral pentoxifylline; 8 (11%), with topical beta-blocker; 6 (8%), with paracentesis of the anterior chamber; 4 (6%), with subcutaneous heparin. A mean +/- SD number of treatments of 2.5 +/- 1.4 was administered per patient, and BCVA increased by a mean +/- SD number of Snellen lines of 0.7 +/- 2.8. The BCVA in 11 patients (15%) increased by 3 or more lines. Multivariate stepwise regression did not reveal any single or combination treatment as a significant factor for improvement in BCVA. Patient age and duration of visual impairment before initial examination were not significant predictors of final BCVA. Commonly used minimally invasive treatments of CRAO do not improve the natural course of the disease. A prospective trial by the European Assessment Group for Lysis in the Eye is under way to evaluate selective intra-arterial lysis, and in this trial some of these minimally invasive treatments are used in the control group.

  12. Transcatheter arterial embolization with trisacryl gelatin microspheres (Embosphere®) leads to life-threatening tumor lysis syndrome in a rectal carcinoid patient with hepatic metastases

    PubMed Central

    Lo, Yuan-Hao; Tsai, Ming-Tsun; Kuo, Chen-Yu; Liu, Wen-Sheng; Lee, Rheun-Chuan; Yeh, Yi-Chen; Li, Chung-Pin; Chen, Jinn-Yang; Chao, Yee

    2012-01-01

    The incidence of gastrointestinal carcinoids appears to be increasing, and the rectum is the third most common location. Transcatheter arterial embolization (TAE) with trisacryl gelatin microspheres (Embosphere®) has been reported as an effective method for hepatic metastases of rectal carcinoids. Complications are uncommon and usually of minor consequence. We report an unusual case of a 34-year-old man with tumor lysis syndrome following TAE with Embosphere® in a patient with multiple hepatic metastases of a rectal carcinoid. Early detection and effective treatment are essential for this rare but potentially catastrophic complication. PMID:23986828

  13. Inhibition of phospho-MurNAc-pentapeptide translocase (MraY) by nucleoside natural product antibiotics, bacteriophage ϕX174 lysis protein E, and cationic antibacterial peptides.

    PubMed

    Bugg, Timothy D H; Rodolis, Maria T; Mihalyi, Agnes; Jamshidi, Shirin

    2016-12-15

    This review covers recent developments in the inhibition of translocase MraY and related phospho-GlcNAc transferases WecA and TagO, and insight into the inhibition and catalytic mechanism of this class of integral membrane proteins from the structure of Aquifex aeolicus MraY. Recent studies have also identified a protein-protein interaction site in Escherichia coli MraY, that is targeted by bacteriophage ϕX174 lysis protein E, and also by cationic antimicrobial peptides containing Arg-Trp close to their N- or C-termini. Copyright © 2016 Elsevier Ltd. All rights reserved.

  14. Evaluation of cell lysis procedures and use of a micro fluidic system for an automated DNA-based cell identification in interplanetary missions

    NASA Astrophysics Data System (ADS)

    Hall, J. A.; Felnagle, E.; Fries, M.; Spearing, S.; Monaco, L.; Steele, A.

    2006-12-01

    A Modular Assay System for Solar System Exploration (MASSE) is being developed to include sample handling, pre-treatment, separation and analysis of biological target compounds by both DNA and protein microarrays. To better design sensitive and accurate initial upstream sample handling of the MASSE instrument, experiments investigating the sensitivity and potential extraction bias of commercially available DNA extraction kits between classes of environmentally relevant prokaryotes such as gram-negative bacteria ( Escherichia coli), gram-positive bacteria ( Bacillus megatarium), and Archaea ( Haloarcula marismortui) were performed. For extractions of both planktonic cultures and spiked Mars simulated regolith, FTA ® paper demonstrated the highest sensitivity, with detection as low as ˜1×10 1 cells and ˜3.3×10 2 cells, respectively. In addition to the highest sensitivity, custom modified application of FTA ® paper extraction protocol is the simplest in terms of incorporation into MASSE and displayed little bias in sensitivity with respect to prokaryotic cell type. The implementation of FTA paper for environmental microbiology investigations appears to be a viable and effective option potentially negating the need for other pre-concentration steps such as filtration and negating concerns regarding extraction efficiency of cells. In addition to investigations on useful technology for upstream sample handling in MASSE, we have also evaluated the potential for μTAS to be employed in the MASSE instrument by employing proprietary lab-on-a-chip development technology to investigate the potential for microfluidic cell lysis of different prokaryotic cells employing both chemical and biological lysis agents. Real-time bright-field microscopy and quantitative PMT detection indicated that that gram positive, gram negative and archaeal cells were effectively lyzed in a few seconds using the microfluidic chip protocol developed. This included employing a lysis buffer with

  15. Lysis of Lactococcus lactis subsp. cremoris SK110 and Its Nisin-Immune Transconjugant in Relation to Flavor Development in Cheese

    PubMed Central

    Meijer, Wilco; van de Bunt, Bert; Twigt, Marja; de Jonge, Boudewijn; Smit, Gerrit; Hugenholtz, Jeroen

    1998-01-01

    To develop a nisin-producing cheese starter, Lactococcus lactis subsp. cremoris SK110 was conjugated with transposon Tn5276-NI, which codes for nisin immunity but not for nisin production. Cheese made with transconjugant SK110::Tn5276-NI as the starter was bitter. The muropeptide of the transconjugant contained a significantly greater amount of tetrapeptides than the muropeptide of strain SK110, which could have decreased the susceptibility of the cells to lysis and thereby the release of intracellular debittering enzymes. PMID:9572979

  16. IMPAIRED NATURAL KILLER CELL LYSIS IN BREAST CANCER PATIENTS WITH HIGH LEVELS OF PSYCHOLOGICAL STRESS IS ASSOCIATED WITH ALTERED EXPRESSION OF KILLER IMMUNOGLOBULIN-LIKE RECEPTORS

    PubMed Central

    Varker, Kimberly A.; Terrell, Catherine E.; Welt, Marilyn; Suleiman, Samer; Thornton, Lisa; Andersen, Barbara L.; Carson, William E.

    2007-01-01

    Background We previously reported that cancer-related psychological stress is associated with reduced natural killer (NK) cell lysis. We hypothesized that reduced NK cell cytotoxicity in patients with increased levels of stress would correlate with alterations in the expression of inhibitory NK cell receptors (killer immunoglobulin-like receptors, or KIRs). The specific aim of this study was to examine KIR expression in patients with high or low levels of psychologic stress and correlate alterations in KIR expression with NK cell function. Materials and Methods 227 patients underwent baseline evaluation of cancer-related psychological stress and were randomized to psychosocial intervention versus observation. From this population, two groups were defined based on pre-treatment measurements of NK lytic activity, stress levels, and the availability of cryopreserved peripheral blood mononuclear cells (PBMC). Group I (n = 9) had low stress by the Impact of Events Scale (IES), and high NK cell lysis at the 50:1 effector: target ratio (NK50 = 52–89%). Group II (n = 8) had high stress and low NK50 (27–52%). Lymphokine activated killer (LAK) activity, antibody dependent cellular cytotoxicity (ADCC), and expression of cytokine receptors, adhesion molecules, and killer immunoglobulin-like receptors (KIRs) were assessed in PBMC. Results Incubation of PBMC with NK-stimulatory cytokines (IL-2, IL-12, or IL-15) led to significant increases in cytotoxic activity regardless of IES/NK50 scores. There were no significant group differences in NK cell surface expression of the IL-2 receptor components CD25 and CD122, antibody-dependent lysis of HER2/neu-positive SKBr3 cells treated with an anti-HER2/neu monoclonal antibody, expression of adhesion molecules (CD2, CD11a, CD18) and markers of activation (CD69), or expression of the KIRs CD158a, NKG2a, NKB1, and CD161. However, levels of CD158b were significantly higher in Group I after incubation in media alone or with IL-2, and CD94

  17. The combined approach to lysis utilizing eptifibatide and rt-PA in acute ischemic stroke: the CLEAR stroke trial.

    PubMed

    Pancioli, Arthur M; Broderick, Joseph; Brott, Thomas; Tomsick, Thomas; Khoury, Jane; Bean, Judy; del Zoppo, Gregory; Kleindorfer, Dawn; Woo, Daniel; Khatri, Pooja; Castaldo, John; Frey, James; Gebel, James; Kasner, Scott; Kidwell, Chelsea; Kwiatkowski, Thomas; Libman, Richard; Mackenzie, Richard; Scott, Phillip; Starkman, Sidney; Thurman, R Jason

    2008-12-01

    Multiple approaches are being studied to enhance the rate of thrombolysis for acute ischemic stroke. Treatment of myocardial infarction with a combination of a reduced-dose fibrinolytic agent and a glycoprotein (GP) IIb/IIIa receptor antagonist has been shown to improve the rate of recanalization versus fibrinolysis alone. The combined approach to lysis utilizing eptifibatide and recombinant tissue-type plasminogen activator (rt-PA) (CLEAR) stroke trial assessed the safety of treating acute ischemic stroke patients within 3 hours of symptom onset with this combination. The CLEAR trial was a National Institutes of Health/National Institute of Neurological Disorders and Stroke-funded multicenter, double-blind, randomized, dose-escalation and safety study. Patients were randomized 3:1 to either low-dose rt-PA (tier 1=0.3 mg/kg, tier 2=0.45 mg/kg) plus eptifibatide (75 microg/kg bolus followed by 0.75 microg/kg per min infusion for 2 hours) or standard-dose rt-PA (0.9 mg/kg). The primary safety end point was the incidence of symptomatic intracerebral hemorrhage within 36 hours. Secondary analyses were performed regarding clinical efficacy. Ninety-four patients (40 in tier 1 and 54 in tier 2) were enrolled. The combination group of the 2 dose tiers (n=69) had a median age of 71 years and a median baseline National Institutes of Health Stroke Scale (NIHSS) score of 14, and the standard-dose rt-PA group (n=25) had a median age of 61 years and a median baseline NIHSS score of 10 (P=0.01 for NIHSS score). Fifty-two (75%) of the combination treatment group and 24 (96%) of the standard treatment group had a baseline modified Rankin scale score of 0 (P=0.04). There was 1 (1.4%; 95% CI, 0% to 4.3%) symptomatic intracranial hemorrhage in the combination group and 2 (8.0%; 95% CI, 0% to 19.2%) in the rt-PA-only arm (P=0.17). During randomization in tier 2, a review by the independent data safety monitoring board demonstrated that the safety profile of combination therapy at the

  18. Phosphate Concentration and the Putative Sensor Kinase Protein CckA Modulate Cell Lysis and Release of the Rhodobacter capsulatus Gene Transfer Agent

    PubMed Central

    Westbye, A. B.; Leung, M. M.; Florizone, S. M.; Taylor, T. A.; Johnson, J. A.; Fogg, P. C.

    2013-01-01

    The gene transfer agent of Rhodobacter capsulatus (RcGTA) is a bacteriophage-like genetic element with the sole known function of horizontal gene transfer. Homologues of RcGTA genes are present in many members of the alphaproteobacteria and may serve an important role in microbial evolution. Transcription of RcGTA genes is induced as cultures enter the stationary phase; however, little is known about cis-active sequences. In this work, we identify the promoter of the first gene in the RcGTA structural gene cluster. Additionally, gene transduction frequency depends on the growth medium, and the reason for this is not known. We report that millimolar concentrations of phosphate posttranslationally inhibit the lysis-dependent release of RcGTA from cells in both a complex medium and a defined medium. Furthermore, we found that cell lysis requires the genes rcc00555 and rcc00556, which were expressed and studied in Escherichia coli to determine their predicted functions as an endolysin and holin, respectively. Production of RcGTA is regulated by host systems, including a putative histidine kinase, CckA, and we found that CckA is required for maximal expression of rcc00555 and for maturation of RcGTA to yield gene transduction-functional particles. PMID:23995641

  19. Spontaneous bacterial cell lysis and biofilm formation in the colon of the Cape Dune mole-rat and the laboratory rabbit

    PubMed Central

    Kotzé, Sanet H.; Holzknecht, Zoie E.; Thomas, Anitra D.; Everett, Mary Lou; Taylor, Shanna; Duckett, Larry D.; Whitesides, John; McDermott, Patrice; Lin, Shu S.

    2013-01-01

    A wide range of techniques, including highthroughput DNA sequencing methods, have been applied to the evaluation of the normal intestinal flora. However, the inability to grow many of those species in culture imposes substantial constraints on the techniques used to evaluate this important community. The presence of biofilms in the normal gut adds further complexity to the issue. In this study, a flow cytometric analysis was used to separate intact bacterial cells, cell debris, and other particulate matter based on bacteria-specific staining and particle size. In addition, an analysis of biofilm formation using fluorescent light microscopy was conducted. Using these approaches, the ratio of bacterial cell debris to intact bacterial cells as a measure of spontaneous lysis of bacterial cells in the gut of the Cape dune mole-rat (Bathyergus suillus) and the laboratory rabbit (Oryctolagus cuniculus) was examined, and the degree of biofilm formation was semi-quantitatively assessed. The results suggest that the degree of spontaneous cell lysis was greater in the appendix than in the cecum in both the mole-rat and the rabbit. Further, the results point toward extensive epithelial-associated biofilm formation in the proximal mole-rat and rabbit large bowel, although the biofilms may be less structured than those found in laboratory rodents and in humans. PMID:21538116

  20. Proteolytic enzymes in skeletal development: histochemical methods adapted to the study of matrix lysis during the transformation of a "cartilage model" into bone.

    PubMed

    Lee, Eunice Rosalind

    2006-09-01

    The replacement of a "cartilage model" by definitive bone is characterized by a series of localized excavations of the cartilage which are eventually followed by bone deposition. Each excavation requires lysis of cartilage components (defined here as the breakdown of a peptide bond) and their eventual resorption (defined here as microscopical visible cartilage loss). More precisely we have proposed that the lysis is affected by proteases capable of breaking down the main proteoglycan "aggrecan" and the main fibril element, "type II collagen". Four approaches combining biochemical, immunologic and microscopic techniques have been adapted to test this hypothesis. Each is applied to the rat tibial head's "cartilage model" where proteases have been shown to be major contributors to secondary ossification center formation. The approaches have been found both effective and distinct as cartilage resorbing enzymes have not only been identified but also detected in situ before and after activation. Achieved overall is an understanding of when, where and how specified proteases contribute to tissue component lyses. While the focus resides on the in situ proteolysis of cartilage, three of the approaches could be translated without change to other tissues, whereas one may require tissue specific adjustments before use.

  1. The lysis protein E of phi X174 is a specific inhibitor of the MraY-catalyzed step in peptidoglycan synthesis.

    PubMed

    Bernhardt, T G; Struck, D K; Young, R

    2001-03-02

    Coliphage phi X174 encodes a single lysis protein, E, a 91-amino acid membrane protein. Dominant mutations have been isolated in the host gene mraY that confer E resistance. mraY encodes translocase I, which catalyzes the formation of the first lipid intermediate in bacterial cell wall synthesis, suggesting a model in which E inhibits MraY and promotes cell lysis in a manner analogous to cell wall synthesis inhibitors like penicillin. To test this model biochemically, we monitored the effect of E on cell wall synthesis in vivo and in vitro. We find that expression of Emyc, encoding an epitope-tagged E protein, from a multicopy plasmid inhibits the incorporation of [(3)H]diaminopimelic acid into cell wall and leads to a profile of labeled precursors consistent with MraY inhibition. Moreover, we find that membranes isolated after Emyc expression are drastically reduced in MraY activity, whereas the activity of Rfe, an enzyme in the same superfamily, was unaffected. We therefore conclude that E is indeed a cell wall synthesis inhibitor and that this inhibition results from a specific block at the MraY-catalyzed step in the pathway.

  2. Targeted lysis of HIV-infected cells by natural killer cells armed and triggered by a recombinant immunoglobulin fusion protein: implications for immunotherapy

    SciTech Connect

    Gupta, Neil; Arthos, James . E-mail: jarthos@niaid.nih.gov; Khazanie, Prateeti; Steenbeke, Tavis D.; Censoplano, Nina M.; Chung, Eva A.; Cruz, Catherine C.; Chaikin, Margery A.; Daucher, Marybeth; Kottilil, Shyam; Mavilio, Domenico; Van Ryk, Donald; Cicala, Claudia; Fauci, Anthony S.; Schuck, Peter; Sun, Peter D.; Radaev, Sergei; Rabin, Ronald L.

    2005-02-20

    Natural killer (NK) cells play an important role in both innate and adaptive antiviral immune responses. The adaptive response typically requires that virus-specific antibodies decorate infected cells which then direct NK cell lysis through a CD16 mediated process termed antibody-dependent cellular cytotoxicity (ADCC). In this report, we employ a highly polymerized chimeric IgG1/IgA immunoglobulin (Ig) fusion protein that, by virtue of its capacity to extensively crosslink CD16, activates NK cells while directing the lysis of infected target cells. We employ HIV as a model system, and demonstrate that freshly isolated NK cells preloaded with an HIV gp120-specific chimeric IgG1/IgA fusion protein efficiently lyse HIV-infected target cells at picomolar concentrations. NK cells pre-armed in this manner retain the capacity to kill targets over an extended period of time. This strategy may have application to other disease states including various viral infections and cancers.

  3. Increased plasma clot permeability and susceptibility to lysis are associated with heavy menstrual bleeding of unknown cause: a case-control study.

    PubMed

    Szczepaniak, Piotr; Zabczyk, Michał; Undas, Anetta

    2015-01-01

    Formation of compact and poorly lysable clots has been reported in thromboembolic disorders. Little is known about clot properties in bleeding disorders. We hypothesized that more permeable and lysis-sensitive fibrin clots can be detected in women with heavy menstrual bleeding (HMB). We studied 52 women with HMB of unknown cause and 52 age-matched control women. Plasma clot permeability (Ks), turbidity and efficiency of fibrinolysis, together with coagulation factors, fibrinolysis proteins, and platelet aggregation were measured. Women with HMB formed looser plasma fibrin clots (+16% [95%CI 7-18%] Ks) that displayed lower maximum absorbancy (-7% [95%CI -9 - -1%] ΔAbsmax), and shorter clot lysis time (-17% [95%CI -23 - -11%] CLT). The HMB patients and controls did not differ with regard to coagulation factors, fibrinogen, von Willebrand antigen, thrombin generation markers and the proportion of subjects with defective platelet aggregation. The patients had lower platelet count (-12% [95%CI -19 - -2%]), tissue plasminogen activator antigen (-39% [95%CI -41 - -29%] tPA:Ag), and plasminogen activator inhibitor-1 antigen (-28% [95%CI -38 - -18%] PAI-1:Ag) compared with the controls. Multiple regression analysis upon adjustment for age, body mass index, glucose, and fibrinogen showed that decreased tPA:Ag and shortened CLT were the independent predictors of HMB. Increased clot permeability and susceptibility to fibrinolysis are associated with HMB, suggesting that altered plasma fibrin clot properties might contribute to bleeding disorders of unknown origin.

  4. Increased Plasma Clot Permeability and Susceptibility to Lysis Are Associated with Heavy Menstrual Bleeding of Unknown Cause: A Case-Control Study

    PubMed Central

    Szczepaniak, Piotr; Zabczyk, Michał; Undas, Anetta

    2015-01-01

    Background Formation of compact and poorly lysable clots has been reported in thromboembolic disorders. Little is known about clot properties in bleeding disorders. Objectives We hypothesized that more permeable and lysis-sensitive fibrin clots can be detected in women with heavy menstrual bleeding (HMB). Methods We studied 52 women with HMB of unknown cause and 52 age-matched control women. Plasma clot permeability (Ks), turbidity and efficiency of fibrinolysis, together with coagulation factors, fibrinolysis proteins, and platelet aggregation were measured. Results Women with HMB formed looser plasma fibrin clots (+16% [95%CI 7–18%] Ks) that displayed lower maximum absorbancy (-7% [95%CI -9 – -1%] ΔAbsmax), and shorter clot lysis time (-17% [95%CI -23 – -11%] CLT). The HMB patients and controls did not differ with regard to coagulation factors, fibrinogen, von Willebrand antigen, thrombin generation markers and the proportion of subjects with defective platelet aggregation. The patients had lower platelet count (-12% [95%CI -19 – -2%]), tissue plasminogen activator antigen (-39% [95%CI -41 – -29%] tPA:Ag), and plasminogen activator inhibitor-1 antigen (-28% [95%CI -38 – -18%] PAI-1:Ag) compared with the controls. Multiple regression analysis upon adjustment for age, body mass index, glucose, and fibrinogen showed that decreased tPA:Ag and shortened CLT were the independent predictors of HMB. Conclusions Increased clot permeability and susceptibility to fibrinolysis are associated with HMB, suggesting that altered plasma fibrin clot properties might contribute to bleeding disorders of unknown origin. PMID:25909989

  5. A novel BCMA/CD3 bispecific T-cell engager for the treatment of multiple myeloma induces selective lysis in vitro and in vivo.

    PubMed

    Hipp, S; Tai, Y-T; Blanset, D; Deegen, P; Wahl, J; Thomas, O; Rattel, B; Adam, P J; Anderson, K C; Friedrich, M

    2017-01-13

    B-cell maturation antigen (BCMA) is a highly plasma cell-selective protein that is expressed on malignant plasma cells of multiple myeloma (MM) patients and therefore is an ideal target for T-cell redirecting therapies. We developed a bispecific T-cell engager (BiTE) targeting BCMA and CD3ɛ (BI 836909) and studied its therapeutic impacts on MM. BI 836909 induced selective lysis of BCMA-positive MM cells, activation of T cells, release of cytokines and T-cell proliferation; whereas BCMA-negative cells were not affected. Activity of BI 836909 was not influenced by the presence of bone marrow stromal cells, soluble BCMA or a proliferation-inducing ligand (APRIL). In ex vivo assays, BI 836909 induced potent autologous MM cell lysis in both, newly diagnosed and relapsed/refractory patient samples. In mouse xenograft studies, BI 836909 induced tumor cell depletion in a subcutaneous NCI-H929 xenograft model and prolonged survival in an orthotopic L-363 xenograft model. In a cynomolgus monkey study, administration of BI 836909 led to depletion of BCMA-positive plasma cells in the bone marrow. Taken together, these results show that BI 836909 is a highly potent and efficacious approach to selectively deplete BCMA-positive MM cells and represents a novel immunotherapeutic for the treatment of MM.Leukemia advance online publication, 13 January 2017; doi:10.1038/leu.2016.388.

  6. Studies on nonidet P40 lysis of murine lymphoid cells. I. Use of cholera toxin and cell surface Ig to determine degree of dissociation of the plasma membrane.

    PubMed

    Hart, D A

    1975-09-01

    Lymphoid cells from A/J mice were iodinated (125I) by the lactoperoxidase lysed with the non-ionic detergent NP-40. The plasma membrane glycolipid receptor for cholera toxin and cell surface immunoglobulin were utilized in immune precipitation systems to characterize the degree of dissociation of the plasma membrane under various conditions. It was found that at 0.1% NP-40 and at cell concentration from 5 to 10 times 10(7) cells/ml, lipid-protein and protein-lipid-protein complexes formed in NP-40 which were soluble after centrifugation at 10(5) times G. Column chromatography of 125I-cell lysates on agarose A-0.5 M in 0.1% or 0.5% NP-40/PBS indicated that the majority of iodinated cell surface material existed as aggregates in detergent micelles. The availability of the oligosaccharide moiety of the glycolipid to interact with the cholera toxin was dependent on both the detergent concentration and the cell concentration used for cell lysis. However, the cell surface immunoglobulin was immunoprecipitable under all conditions of lysis tested.

  7. c-Myc regulates expression of NKG2D ligands ULBP1/2/3 in AML and modulates their susceptibility to NK-mediated lysis.

    PubMed

    Nanbakhsh, Arash; Pochon, Cécile; Mallavialle, Aude; Amsellem, Sophie; Bourhis, Jean Henri; Chouaib, Salem

    2014-06-05

    Cytarabine (cytosine arabinoside) is one of the most effective drugs for the treatment of patients diagnosed with acute myeloid leukemia (AML). Despite its efficiency against AML cells, the emergence of drug resistance due to prolonged chemotherapy in most patients is still a major obstacle. Several studies have shown that drug resistance mechanisms alter the sensitivity of leukemia cells to immune system effector cells. To investigate this phenomenon, parental acute myeloid cell lines, HL-60 and KG-1, were continuously exposed to increasing doses of cytarabine in order to establish equivalent resistant cell lines, HL-60(R) and KG-1(R). Our data indicate that cytarabine-resistant cells are more susceptible to natural killer (NK)-mediated cell lysis as compared with parental cytarabine-sensitive cells. The increased susceptibility correlates with the induction of UL-16 binding proteins (ULBP) 1/2/3 and NK group 2, member D (NKG2D) ligands on target cells by a mechanism involving c-Myc induction. More importantly, chromatin immunoprecipitation assay revealed that ULBP1/3 are direct targets of c-Myc. Using drug-resistant primary AML blasts as target cells, inhibition of c-Myc resulted in decreased expression of NKG2D ligands and the subsequent impairment of NK cell lysis. This study provides for the first time, the c-Myc dependent regulation of NKG2D ligands in AML. © 2014 by The American Society of Hematology.

  8. EspP, an Extracellular Serine Protease from Enterohemorrhagic E. coli, Reduces Coagulation Factor Activities, Reduces Clot Strength, and Promotes Clot Lysis.

    PubMed

    Kuo, Kevin H M; Khan, Shekeb; Rand, Margaret L; Mian, Hira S; Brnjac, Elena; Sandercock, Linda E; Akula, Indira; Julien, Jean-Philippe; Pai, Emil F; Chesney, Alden E

    2016-01-01

    EspP (E. coli secreted serine protease, large plasmid encoded) is an extracellular serine protease produced by enterohemorrhagic E. coli (EHEC) O157:H7, a causative agent of diarrhea-associated Hemolytic Uremic Syndrome (D+HUS). The mechanism by which EHEC induces D+HUS has not been fully elucidated. We investigated the effects of EspP on clot formation and lysis in human blood. Human whole blood and plasma were incubated with EspP(WT )at various concentrations and sampled at various time points. Thrombin time (TT), prothrombin time (PT), and activated partial thromboplastin time (aPTT), coagulation factor activities, and thrombelastgraphy (TEG) were measured. Human whole blood or plasma incubated with EspP(WT) was found to have prolonged PT, aPTT, and TT. Furthermore, human whole blood or plasma incubated with EspP(WT) had reduced activities of coagulation factors V, VII, VIII, and XII, as well as prothrombin. EspP did not alter the activities of coagulation factors IX, X, or XI. When analyzed by whole blood TEG, EspP decreased the maximum amplitude of the clot, and increased the clot lysis. Our results indicate that EspP alters hemostasis in vitro by decreasing the activities of coagulation factors V, VII, VIII, and XII, and of prothrombin, by reducing the clot strength and accelerating fibrinolysis, and provide further evidence of a functional role for this protease in the virulence of EHEC and the development of D+HUS.

  9. Delivery of woodchuck hepatitis virus-like particle presented influenza M2e by recombinant attenuated Salmonella displaying a delayed lysis phenotype.

    PubMed

    Ameiss, Keith; Ashraf, Shamaila; Kong, Wei; Pekosz, Andrew; Wu, Wai-Hong; Milich, David; Billaud, Jean-Noel; Curtiss, Roy

    2010-09-24

    The use of live recombinant attenuated Salmonella vaccines (RASV) is a promising approach for controlling infections by multiple pathogens. The highly conserved extracellular domain of the influenza M2 protein (M2e) has been shown to provide broad spectrum protection against multiple influenza subtypes sharing similar M2e sequences. An M2e epitope common to a number of avian influenza subtypes was inserted into the core antigen of woodchuck hepatitis virus and expressed in two different recombinant attenuated Salmonella Typhimurium strains. One strain was attenuated via deletion of the cya and crp genes. The second strain was engineered to exhibit a programmed delayed lysis phenotype. Both strains were able to produce both monomeric fusion proteins and fully assembled core particles. Mice orally immunized with the strain exhibiting delayed lysis induced significantly greater antibody titers than the Δcya Δcrp strain and provided moderate protection against weight loss to a low level challenge with the influenza strain A/WSN/33 modified to express the M2e sequence common to avian viruses. Further studies indicated that the Salmonella expressed core antigen induced comparable antibody levels to the purified core antigen injected with an alum adjuvant and that both are able to reduce viral replication in the lungs. To our knowledge this is the first report demonstrating Salmonella-mediated delivery of influenza virus M2e protein in a mammalian host to induce a protective immune response against viral challenge. Copyright © 2010 Elsevier Ltd. All rights reserved.

  10. Mechanism of action of Melaleuca alternifolia (tea tree) oil on Staphylococcus aureus determined by time-kill, lysis, leakage, and salt tolerance assays and electron microscopy.

    PubMed

    Carson, Christine F; Mee, Brian J; Riley, Thomas V

    2002-06-01

    The essential oil of Melaleuca alternifolia (tea tree) has broad-spectrum antimicrobial activity. The mechanisms of action of tea tree oil and three of its components, 1,8-cineole, terpinen-4-ol, and alpha-terpineol, against Staphylococcus aureus ATCC 9144 were investigated. Treatment with these agents at their MICs and two times their MICs, particularly treatment with terpinen-4-ol and alpha-terpineol, reduced the viability of S. aureus. None of the agents caused lysis, as determined by measurement of the optical density at 620 nm, although cells became disproportionately sensitive to subsequent autolysis. Loss of 260-nm-absorbing material occurred after treatment with concentrations equivalent to the MIC, particularly after treatment with 1,8-cineole and alpha-terpineol. S. aureus organisms treated with tea tree oil or its components at the MIC or two times the MIC showed a significant loss of tolerance to NaCl. When the agents were tested at one-half the MIC, only 1,8-cineole significantly reduced the tolerance of S. aureus to NaCl. Electron microscopy of terpinen-4-ol-treated cells showed the formation of mesosomes and the loss of cytoplasmic contents. The predisposition to lysis, the loss of 260-nm-absorbing material, the loss of tolerance to NaCl, and the altered morphology seen by electron microscopy all suggest that tea tree oil and its components compromise the cytoplasmic membrane.

  11. Rapid method for DNA extraction from the honey bee Apis mellifera and the parasitic bee mite Varroa destructor using lysis buffer and proteinase K.

    PubMed

    Issa, M R C; Figueiredo, V L C; De Jong, D; Sakamoto, C H; Simões, Z L P

    2013-10-22

    We developed a rapid method for extraction of DNA from honey bees, Apis mellifera, and from the parasitic bee mite, Varroa destructor. The advantages include fast processing and low toxicity of the substances that are utilized. We used lysis buffer with nonionic detergents to lyse cell walls and proteinase K to digest proteins. We tested whole thorax, thoracic muscle mass, legs, and antennae from individual bees; the mites were processed whole (1 mite/sample). Each thorax was incubated whole, without cutting, because exocuticle color pigment darkened the extraction solution, interfering with PCR results. The procedure was performed with autoclaved equipment and laboratory gloves. For each sample, we used 100 µL lysis buffer (2 mL stock solution of 0.5 M Tris/HCl, pH 8.5, 10 mL stock solution of 2 M KCl, 500 µL solution of 1 M MgCl2, 2 mL NP40, and 27.6 g sucrose, completed to 200 mL with bidistilled water and autoclaved) and 2 µL proteinase K (10 mg/mL in bidistilled water previously autoclaved, as proteinase K cannot be autoclaved). Tissues were incubated in the solutions for 1-2 h in a water bath (62°-68 °C) or overnight at 37 °C. After incubation, the tissues were removed from the extraction solution (lysis buffer + proteinase K) and the solution heated to 92 °C for 10 min, for proteinase K inactivation. Then, the solution with the extracted DNA was stored in a refrigerator (4°-8 °C) or a freezer (-20 °C). This method does not require centrifugation or phenol/chloroform extraction. The reduced number of steps allowed us to sample many individuals/day. Whole mites and bee antennae were the most rapidly processed. All bee tissues gave the same quality DNA. This method, even using a single bee antenna or a single mite, was adequate for extraction and analysis of bee genomic and mitochondrial DNA and mite genomic DNA.

  12. Lysis to Kill: Evaluation of the Lytic Abilities, and Genomics of Nine Bacteriophages Infective for Gordonia spp. and Their Potential Use in Activated Sludge Foam Biocontrol.

    PubMed

    Dyson, Zoe A; Tucci, Joseph; Seviour, Robert J; Petrovski, Steve

    2015-01-01

    Nine bacteriophages (phages) infective for members of the genus Gordonia were isolated from wastewater and other natural water environments using standard enrichment techniques. The majority were broad host range phages targeting more than one Gordonia species. When their genomes were sequenced, they all emerged as double stranded DNA Siphoviridae phages, ranging from 17,562 to 103,424 bp in size, and containing between 27 and 127 genes, many of which were detailed for the first time. Many of these phage genomes diverged from the expected modular genome architecture of other characterized Siphoviridae phages and contained unusual lysis gene arrangements. Whole genome sequencing also revealed that infection with lytic phages does not appear to prevent spontaneous prophage induction in Gordonia malaquae lysogen strain BEN700. TEM sample preparation techniques were developed to view both attachment and replication stages of phage infection.

  13. Lysis to Kill: Evaluation of the Lytic Abilities, and Genomics of Nine Bacteriophages Infective for Gordonia spp. and Their Potential Use in Activated Sludge Foam Biocontrol

    PubMed Central

    Dyson, Zoe A.; Tucci, Joseph; Seviour, Robert J.; Petrovski, Steve

    2015-01-01

    Nine bacteriophages (phages) infective for members of the genus Gordonia were isolated from wastewater and other natural water environments using standard enrichment techniques. The majority were broad host range phages targeting more than one Gordonia species. When their genomes were sequenced, they all emerged as double stranded DNA Siphoviridae phages, ranging from 17,562 to 103,424 bp in size, and containing between 27 and 127 genes, many of which were detailed for the first time. Many of these phage genomes diverged from the expected modular genome architecture of other characterized Siphoviridae phages and contained unusual lysis gene arrangements. Whole genome sequencing also revealed that infection with lytic phages does not appear to prevent spontaneous prophage induction in Gordonia malaquae lysogen strain BEN700. TEM sample preparation techniques were developed to view both attachment and replication stages of phage infection. PMID:26241321

  14. Re-directing bacterial microcompartment systems to enhance recombinant expression of lysis protein E from bacteriophage ΦX174 in Escherichia coli

    DOE PAGES

    Yung, Mimi C.; Bourguet, Feliza A.; Carpenter, Timothy S.; ...

    2017-04-26

    Recombinant expression of toxic proteins remains a challenging problem. Furthermore, one potential method to shield toxicity and thus improve expression of these proteins is to encapsulate them within protein compartments to sequester them away from their targets. Many bacteria naturally produce so-called bacterial microcompartments (BMCs) in which enzymes comprising a biosynthetic pathway are encapsulated in a proteinaeous shell, which is in part thought to shield the cells from the toxicity of reaction intermediates. As a proof-of-concept, we attempted to encapsulate toxic, lysis protein E (E) from bacteriophage ΦX174 inside recombinant BMCs to enhance its expression and achieve higher yields duringmore » downstream purification.« less

  15. Contaminating coagulase-negative staphylococci isolated in a lysis-centrifugation (Isolator) blood culture system. Application of different epidemiological markers for deduction of mode of contamination.

    PubMed

    Arpi, M; Gahrn-Hansen, B; Rosdahl, V T

    1988-07-01

    The lysis-centrifugation blood culture system, Isolator, is a promising system with respect to detection of many significant microorganisms, e.g. Staphylococcus aureus and Enterobacteriaceae, as compared with conventional systems. A drawback of the Isolator system is a disturbingly high rate of clinically insignificant, supposedly contaminating coagulase-negative staphylococci, which leads to considerable waste of time and materials in the laboratory. Several sources of these isolates have been proposed (viz. the patient, the ward environment, the laboratory handling, and the plate media). The aim of this study was to pinpoint the origin of these clinically doubtful coagulase-negative staphylococci, using different epidemiological markers, such as species identification, antibiotic susceptibility patterns, phage-types, and plasmid profiles. Plasmid profile analysis proved to be more discriminating than the other techniques and made it possible to conclude that the laboratory handling of the Isolator system was a major source of coagulase-negative staphylococci in this system.

  16. One for All or All for One: Heterogeneous Expression and Host Cell Lysis Are Key to Gene Transfer Agent Activity in Rhodobacter capsulatus

    PubMed Central

    Fogg, Paul C. M.; Westbye, Alexander B.; Beatty, J. Thomas

    2012-01-01

    The gene transfer agent (RcGTA) of Rhodobacter capsulatus is the model for a family of novel bacteriophage-related genetic elements that carry out lateral transfer of essentially random host DNA. Genuine and putative gene transfer agents have been discovered in diverse genera and are becoming recognized as potentially an important source of genetic exchange and microbial evolution in the oceans. Despite being discovered over 30 years ago, little is known about many essential aspects of RcGTA biology. Here, we validate the use of direct fluorescence reporter constructs, which express the red fluorescent protein mCherry in R. capsulatus. A construct containing the RcGTA promoter fused to mCherry was used to examine the single-cell expression profiles of wild type and RcGTA overproducer R. capsulatus populations, under different growth conditions and growth phases. The majority of RcGTA production clearly arises from a small, distinct sub-set of the population in the wild type strain and a larger sub-set in the overproducer. The most likely RcGTA release mechanism concomitant with this expression pattern is host cell lysis and we present direct evidence for the release of an intracellular enzyme accompanying RcGTA release. RcGTA ORF s is annotated as a ‘cell wall peptidase’ but we rule out a role in host lysis and propose an alternative function as a key contributor to RcGTA invasion of a target cell during infection. PMID:22916305

  17. [Lysis of the incus long process and incudostapedial rebridging ossiculoplasty: comparative study of titanium-gold angle prosthesis Plester-type versus Martin Incudo prosthesis hydroxylapatite].

    PubMed

    Faye, M B; Martin, C; Schmerber, S

    2013-01-01

    We report two surgical techniques devised to restore a disrupted incudostapedial joint. Thirty patients underwent rebridging of distal portion of incus long process in the ENT Department of University of Grenoble and Saint-Etienne, between October 1998 and September 2002. Two types of ossicular prostheses were used: A titanium-gold angle prosthesis according to Plester Winkel Kurz (n = 16 patients), and a hydroxylapatite prosthesis as Martin Incudo Prosthesis (n = 14 patients). The average hearing gain in short term is of 8.30 dB for the Martin-Incudo group. It is of 5.23 dB in the Winkel group. Seven and three cases of failures (Residual Rinne > 20 dB) were noticed respectively in the groups Martin-Incudo and Winkel. Seven and four cases of labyrinthisation were observed respectively in the groups Martin-Incudo and Winkel. The average hearing gain in long term is 3.43 dB in the Martin-Incudo group; and 2.85 dB among patients with Winkel Kurz prosthesis. Average residual Rinne is higher than 20 dB in the Winkel group. The hearing gain is not statistically significant between the two groups (p > 0.05). The titanium partial prosthesis did not give good functional results. In the case of a limited lysis (< 2 mm) of the distal portion of incus, we use the cement or cartilage interposition. When ossicular chain cannot be preserved entirely, we privilege incus transposition or a titanium PORP. The Martin-Incudo prosthesis seems interesting in the event of lysis of 2 mm of the long process of incus, nevertheless engineering changes are necessary in order to make rigid the incudostapedial joint.

  18. Inhibition of postbinding target cell lysis and of lymphokine-induced enhancement of human natural killer cell activity by in vitro exposure to ultraviolet B radiation

    SciTech Connect

    Elmets, C.A.; Larson, K.; Urda, G.A.; Schacter, B.

    1987-01-01

    In vitro exposure of human peripheral blood mononuclear cells (PBMC) to ultraviolet B (uvB) radiation has been shown to inhibit natural killer (NK) cell-mediated cytotoxicity in a dose-dependent fashion. The purpose of this study was to examine the manner by which uvB produced these deleterious effects. Inhibition of NK activity was not due to lethal injury to NK cells since the viability of cell populations enriched for NK activity was greater than 90% with the uvB doses employed. uvB appeared to directly affect NK cells since procedures which removed suppressor mechanisms, such as removal of monocytes and pharmacologic inhibition of the cyclooxygenase pathway, failed to reverse the response. Furthermore, no suppression of activity of unirradiated NK cells could be produced by coincubation of unirradiated NK cells with uv-irradiated NK cells. When the single cell assay for binding and killing was employed to determine at which stage in the lytic sequence inhibition occurred, it was found that binding was normal but lysis of bound targets and the recycling capacity of active NK cells were markedly reduced. At uvB doses above 50 J/m2, both interferon alpha (IFN-alpha) and interleukin 2 (IL-2) were ineffective in augmenting NK cell-mediated cytotoxic reactions after cells had been irradiated with uvB. Furthermore, incubation of NK cells with IFN-alpha prior to irradiation failed to protect against the inhibitory effects. These studies provide evidence that in vitro exposure of NK cells to uvB radiation inhibits their function by a direct nonlethal effect and that this inhibition occurs selectively at the postbinding stage of target cell lysis.

  19. Spontaneous acute tumor lysis syndrome as a cause of early deaths in short-term carcinogenicity studies using p53 +/- mice.

    PubMed

    Vogel, P; Pletcher, J M; Liang, Y

    2010-07-01

    Acute tumor lysis syndrome (ATLS) is a potentially lethal condition precipitated by the massive release of intracellular components such as nucleic acids, potassium, and phosphorus, following a rapid and widespread lysis of tumor cells. Herein, the authors describe the high incidence and characteristic histopathologic lesions of acute ATLS in p53-deficient mice used in 2 short-term carcinogenicity studies. ATLS was a frequent cause of early death in p53 (+/-) mice in these studies and was consistently associated with the presence of disseminated lymphoma and leukemia. Although a heavy tumor burden and leukemia were present in all affected mice, the absence of ATLS in other mice with equally severe lymphoma and leukemia indicates that these tumor burdens are necessary but insufficient to cause ATLS in mice. The hallmark histopathologic findings of ATLS in mice are the disseminated microemboli composed of nuclear and cytoplasmic debris derived from lysed tumor cells. The mechanical obstruction of capillary beds by these microemboli appeared to be the proximate cause of the early deaths of mice in these studies. Microemboli may contribute to the pathogenesis of acute renal failure and other clinical signs associated with ATLS in other species. Recognition of ATLS in laboratory animals is critical in studies intended to evaluate the efficacy and/or toxicity of anticancer treatments, where early deaths due to ATLS might otherwise be attributed to test article toxicity. Further studies on the role of microemboli in the pathogenesis of ATLS may elucidate pathogenetic mechanisms and lead to improved approaches to clinical management and treatment of this potentially lethal condition.

  20. A single-chain triplebody with specificity for CD19 and CD33 mediates effective lysis of mixed lineage leukemia cells by dual targeting.

    PubMed

    Schubert, Ingo; Kellner, Christian; Stein, Christoph; Kügler, Markus; Schwenkert, Michael; Saul, Domenica; Mentz, Kristin; Singer, Heiko; Stockmeyer, Bernhard; Hillen, Wolfgang; Mackensen, Andreas; Fey, Georg H

    2011-01-01

    A single-chain triplebody (sctb) 33-ds16-ds19 comprising two distal single-chain Fv fragments (scFvs) specific for the lymphoid antigen CD19 and the myeloid antigen CD33 flanking a central scFv specific for CD16, which is the low affinity Fc-receptor (FcγRIII) present on natural killer cells and macrophages, was produced and its properties were investigated. CD33 and CD19 in combination are present on acute leukemiablasts with mixed lineage phenotype, but not on normal human hematopoietic cells. For comparison, two bispecific scFvs (bsscFvs), ds19-ds16 and 33-ds16, with monovalent binding to CD19 and CD33, respectively, were also studied. The sctb 33-ds16-ds19 specifically interacted with all 3 antigens. On the antigen double-positive cell line BV-173, the sctb bound with 2-fold greater avidity than bsscFv ds19-ds16 (KD = 21 vs. 42 nM) and with 1.4-fold greater avidity than bsscFv 33-ds16 (KD = 29 nM). All 3 fusion proteins had similar affinity for CD16 and sufficient thermic stability in human serum. In antibody-dependent cellular cytotoxicity (ADCC) reactions with human mononuclear cells as effectors, the sctb promoted lysis of BV-173 cells at 23-fold lower concentrations than bsscFv ds19-ds16 and at 1.4-fold lower concentrations than bsscFv 33-ds16. The sctb also mediated potent ADCC of the antigen double-positive mixed lineage leukemia cell line SEM, and the half-maximal concentration EC50 for BV-173 cells was 7 pM. Therefore, CD19 and CD33 are present on the surface of these leukemic cell lines such that they can be connected by a single sctb molecule, permitting the recruitment of NK cells via CD16 and tumor cell lysis.

  1. A novel modified vaccination technique produces IgG antibodies that cause complement-mediated lysis of multiple myeloma cells carrying CD38 antigen.

    PubMed

    Barabas, Arpad Z; Cole, Chad D; Graeff, Richard M; Morcol, Tulin; Lafreniere, Rene

    2016-01-01

    Objectives were to: 1) induce a lytic IgG antibody (ab) response (via the so called `third vaccination method') against CD38 antigen (ag) residing on the extra-cellular domain of multiple myeloma (MM) cells in recipient rabbits, by combining the CD38 ag with donor-derived anti-CD38 ag lytic IgG ab into an immune complex (IC); and 2) determine whether abs produced would cause complement-mediated lysis (in vitro) of human MM cells containing CD38 ag. The vaccine was created in a two-step process. First, ab (rabbit anti-CD38 ag IgG ab) was raised in donor rabbits by injections of low molecular weight soluble CD38 ag in Freund's complete adjuvant (FCA) and aqueous solution. Second, transfer of pathogenic lytic IgG ab response into recipient rabbits was achieved by injections of ICs composed of CD38 ag and homologous anti-CD38 ag IgG ab. Consequently, recipient rabbits produced the same ab with the same specificity against the target ag as was present in the inoculum, namely agglutinating, precipitating and lytic (as demonstrated in vitro). In an in vitro study, in the presence of complement, donor and recipient rabbits' immune sera caused lysis of CD38 ag associated human MM cells. The most effective lytic ab response causing sera were those from donor rabbits injected with CD38 ag in FCA and those from rabbits injected with ICs, especially when they were administered in adjuvants. These results provided proof of concept that the third vaccination method has good potential as a stand-alone and efficacious method of controlling cancer.

  2. Lymphokine-activated killer cell phenomenon. Lysis of natural killer-resistant fresh solid tumor cells by interleukin 2-activated autologous human peripheral blood lymphocytes

    SciTech Connect

    Grimm, E.A.; Mazumder, A.; Zhang, H.Z.; Rosenberg, S.A.

    1982-06-01

    Activation in lectin-free interleukin 2 (IL-2) containing supernatants of peripheral blood mononuclear leukocytes (PBL) from cancer patients or normal individuals resulted in expression of cytotoxicity toward 20 of 21 natural killer (NK)-resistant fresh solid tumor cells tested. Fresh solid tumor cells were resistant to NK-mediated lysis in 10 autologous patients' PBL-tumor interactions, and from 17 normal individuals tested against 13 allogeneic fresh tumors. Culture of PBL in IL-2 for 2-3 d was required for the lymphokine activated killers (LAK) to be expressed, and lytic activity toward a variety of NK-resistant fresh and cultured tumor targets developed in parallel. Autologous IL-2 was functional in LAK activation, as well as interferon-depleted IL-2 preparations. Irradiation of responder PBL before culture in IL-2 prevented LAK development. Precursors of LAK were present in PBL depleted of adherent cells and in NK-void thoracic duct lymphocytes, suggesting that the precursor is neither a monocyte nor an NK cell. LAK effectors expressed the serologically defined T cell markers of OKT.3, Leu-1, and 4F2, but did not express the monocyte/NK marker OKM-1. Lysis of autologous fresh solid tumors by LAK from cancer patients' PBL was demonstrated in 85% of the patient-fresh tumor combinations. Our data present evidence that the LAK system is a phenomenon distinct from either NK or CTL systems that probably accounts for a large number of reported nonclassical cytotoxicities. The biological role of LAK cells is not yet known, although it is suggested that these cells may be functional in immune surveillance against human solid tumors.

  3. In black South Africans from rural and urban communities, the 4G/5G PAI-1 polymorphism influences PAI-1 activity, but not plasma clot lysis time.

    PubMed

    de Lange, Zelda; Rijken, Dingeman C; Hoekstra, Tiny; Conradie, Karin R; Jerling, Johann C; Pieters, Marlien

    2013-01-01

    Data on genetic and environmental factors influencing PAI-1 levels and their consequent effect on clot lysis in black African populations are limited. We identified polymorphisms in the promoter area of the PAI-1 gene and determined their influence on PAI-1act levels and plasma clot lysis time (CLT). We also describe gene-environment interactions and the effect of urbanisation. Data from 2010 apparently healthy urban and rural black participants from the South African arm of the PURE study were cross-sectionally analysed. The 5G allele frequency of the 4G/5G polymorphism was 0.85. PAI-1act increased across genotypes in the urban subgroup (p = 0.009) but not significantly in the rural subgroup, while CLT did not differ across genotypes. Significant interaction terms were found between the 4G/5G polymorphism and BMI, waist circumference and triglycerides in determining PAI-1act, and between the 4G/5G polymorphism and fibrinogen and fibrinogen gamma prime in determining CLT. The C428T and G429A polymorphisms did not show direct relationships with PAI-1act or CLT but they did influence the association of other environmental factors with PAI-1act and CLT. Several of these interactions differed significantly between rural and urban subgroups, particularly in individuals harbouring the mutant alleles. In conclusion, although the 4G/5G polymorphism significantly affected PAI-1act, it contributed less than 1% to the PAI-1act variance. (Central) obesity was the biggest contributor to PAI-1act variance (12.5%). Urbanisation significantly influenced the effect of the 4G/5G polymorphism on PAI-1act as well as gene-environment interactions for the C428T and G429A genotypes in determining PAI-1act and CLT.

  4. In Black South Africans from Rural and Urban Communities, the 4G/5G PAI-1 Polymorphism Influences PAI-1 Activity, but Not Plasma Clot Lysis Time

    PubMed Central

    de Lange, Zelda; Rijken, Dingeman C.; Hoekstra, Tiny; Conradie, Karin R.; Jerling, Johann C.; Pieters, Marlien

    2013-01-01

    Data on genetic and environmental factors influencing PAI-1 levels and their consequent effect on clot lysis in black African populations are limited. We identified polymorphisms in the promoter area of the PAI-1 gene and determined their influence on PAI-1act levels and plasma clot lysis time (CLT). We also describe gene-environment interactions and the effect of urbanisation. Data from 2010 apparently healthy urban and rural black participants from the South African arm of the PURE study were cross-sectionally analysed. The 5G allele frequency of the 4G/5G polymorphism was 0.85. PAI-1act increased across genotypes in the urban subgroup (p = 0.009) but not significantly in the rural subgroup, while CLT did not differ across genotypes. Significant interaction terms were found between the 4G/5G polymorphism and BMI, waist circumference and triglycerides in determining PAI-1act, and between the 4G/5G polymorphism and fibrinogen and fibrinogen gamma prime in determining CLT. The C428T and G429A polymorphisms did not show direct relationships with PAI-1act or CLT but they did influence the association of other environmental factors with PAI-1act and CLT. Several of these interactions differed significantly between rural and urban subgroups, particularly in individuals harbouring the mutant alleles. In conclusion, although the 4G/5G polymorphism significantly affected PAI-1act, it contributed less than 1% to the PAI-1act variance. (Central) obesity was the biggest contributor to PAI-1act variance (12.5%). Urbanisation significantly influenced the effect of the 4G/5G polymorphism on PAI-1act as well as gene-environment interactions for the C428T and G429A genotypes in determining PAI-1act and CLT. PMID:24386152

  5. Redirected lysis of human melanoma cells by a MCSP/CD3-bispecific BiTE antibody that engages patient-derived T cells.

    PubMed

    Torisu-Itakura, Hitoe; Schoellhammer, Hans F; Sim, Myung-Shin; Irie, Reiko F; Hausmann, Susanne; Raum, Tobias; Baeuerle, Patrick A; Morton, Donald L

    2011-10-01

    Melanoma-associated chondroitin sulfate proteoglycan (MCSP; also called HMW-MAA, CSPG4, NG2, MSK16, MCSPG, MEL-CSPG, or gp240) is a well characterized melanoma cell-surface antigen. In this study, a new bispecific T-cell engaging (BiTE) antibody that binds to MCSP and human CD3 (MCSP-BiTE) was tested for its cytotoxic activity against human melanoma cell lines. When unstimulated peripheral mononuclear blood cells (PBMCs) derived from healthy donors were cocultured with melanoma cells at effector:target ratios of 1:1, 1:5, or 1:10, and treated with MCSP-BiTE antibody at doses of 10, 100, or 1000 ng/mL, all MCSP-expressing melanoma cell lines (n=23) were lysed in a dose-dependent and effector:target ratio-dependent manner, whereas there was no cytotoxic activity against MCSP-negative melanoma cell lines (n=2). To investigate whether T cells from melanoma patients could act as effector cells, we cocultured unstimulated PBMCs with allogeneic melanoma cells from 13 patients (4 stage I/II, 3 stage III, and 6 stage IV) or with autologous melanoma cells from 2 patients (stage IV). Although cytotoxic activity varied, all 15 PBMC samples mediated significant redirected lysis by the BiTE antibody. When PBMC or CD8 T cells were prestimulated by anti-CD3 antibody OKT-3 and interleukin-2, the MCSP-BiTE concentrations needed for melanoma cell lysis decreased up to 1000-fold. As MCSP is expressed on most human melanomas, immunotherapy with MCSP/CD3-bispecific antibodies merits clinical investigation.

  6. Alterations in Receptor Binding Properties of Recent Human Influenza H3N2 Viruses Are Associated with Reduced Natural Killer Cell Lysis of Infected Cells▿

    PubMed Central

    Owen, Rachel E.; Yamada, Eriko; Thompson, Catherine I.; Phillipson, Louisa J.; Thompson, Clare; Taylor, Elizabeth; Zambon, Maria; Osborn, Helen M. I.; Barclay, Wendy S.; Borrow, Persephone

    2007-01-01

    Natural killer (NK) cell recognition of influenza virus-infected cells involves hemagglutinin (HA) binding to sialic acid (SA) on activating NK receptors. SA also acts as a receptor for the binding of influenza virus to its target host cells. The SA binding properties of H3N2 influenza viruses have been observed to change during circulation in humans: recent isolates are unable to agglutinate chicken red blood cells and show reduced affinity for synthetic glycopolymers representing SA-α-2,3-lactose (3′SL-PAA) and SA-α-2,6-N-acetyl lactosamine (6′SLN-PAA) carbohydrates. Here, NK lysis of cells infected with human H3N2 influenza viruses isolated between 1969 and 2003 was analyzed. Cells infected with recent isolates (1999 to 2003) were found to be lysed less effectively than cells infected with older isolates (1969 to 1996). This change occurred concurrently with the acquisition of two new potential glycosylation site motifs in HA. Deletion of the potential glycosylation site motif at 133 to 135 in HA1 from a recent isolate partially restored the agglutination phenotype to a recombinant virus, indicating that the HA-SA interaction is inhibited by the glycosylation modification. Deletion of either of the recently acquired potential glycosylation sites from HA led to increased NK lysis of cells infected with recombinant viruses carrying modified HA. These results indicate that alterations in HA glycosylation may affect NK cell recognition of influenza virus-infected cells in addition to virus binding to host cells. PMID:17670834

  7. EspP, an Extracellular Serine Protease from Enterohemorrhagic E. coli, Reduces Coagulation Factor Activities, Reduces Clot Strength, and Promotes Clot Lysis

    PubMed Central

    Rand, Margaret L.; Mian, Hira S.; Brnjac, Elena; Sandercock, Linda E.; Akula, Indira; Julien, Jean-Philippe; Pai, Emil F.; Chesney, Alden E.

    2016-01-01

    Background EspP (E. coli secreted serine protease, large plasmid encoded) is an extracellular serine protease produced by enterohemorrhagic E. coli (EHEC) O157:H7, a causative agent of diarrhea-associated Hemolytic Uremic Syndrome (D+HUS). The mechanism by which EHEC induces D+HUS has not been fully elucidated. Objectives We investigated the effects of EspP on clot formation and lysis in human blood. Methods Human whole blood and plasma were incubated with EspPWT at various concentrations and sampled at various time points. Thrombin time (TT), prothrombin time (PT), and activated partial thromboplastin time (aPTT), coagulation factor activities, and thrombelastgraphy (TEG) were measured. Results and Conclusions Human whole blood or plasma incubated with EspPWT was found to have prolonged PT, aPTT, and TT. Furthermore, human whole blood or plasma incubated with EspPWT had reduced activities of coagulation factors V, VII, VIII, and XII, as well as prothrombin. EspP did not alter the activities of coagulation factors IX, X, or XI. When analyzed by whole blood TEG, EspP decreased the maximum amplitude of the clot, and increased the clot lysis. Our results indicate that EspP alters hemostasis in vitro by decreasing the activities of coagulation factors V, VII, VIII, and XII, and of prothrombin, by reducing the clot strength and accelerating fibrinolysis, and provide further evidence of a functional role for this protease in the virulence of EHEC and the development of D+HUS. PMID:26934472

  8. Bacteriophage cell lysis of Shiga toxin-producing Escherichia coli for top-down proteomic identification of Shiga toxin 1 & 2 using matrix-assisted laser desorption/ionization tandem time-of-light mass spectrometry

    USDA-ARS?s Scientific Manuscript database

    RATIONALE: Analysis of bacteria by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS) often relies upon sample preparation methods that result in cell lysis, e.g. bead-beating. However, Shiga toxin-producing Escherichia coli (STEC) can undergo bacteriophage...

  9. Membrane lysis by the antibacterial peptides cecropins B1 and B3: A spin-label electron spin resonance study on phospholipid bilayers.

    PubMed Central

    Hung, S C; Wang, W; Chan, S I; Chen, H M

    1999-01-01

    Custom antibacterial peptides, cecropins B1 (CB1) and B3 (CB3), were synthesized. These peptides have particular sequence characteristics, with CB1 having two amphipathic alpha-helical segments and CB3 having two hydrophobic alpha-helical segments. These differences were exploited for a study of their efficacy in breaking up liposomes, which had different combinations of phosphatidic acid (PA) and phosphatidylcholine (PC), and a study of their lipid binding ability. Binding and nonbinding lysis actions of CB1 and CB3 on liposomes were examined further by electron spin resonance (ESR). The spin-labeled lipids 5'SL-PC, 7'SL-PC, 10'SL-PC, 12'SL-PC, and 16'SL-PC were used as probes. The ESR spectra revealed larger outer hyperfine splittings (2A(max)) for CB1 when the interactions of CB1 and CB3 with liposomes were compared. These observations indicate a larger restriction of the motion of the spin-labeled chains in the presence of CB1. Plots of the effective order parameter at the various probe positions (chain flexibility gradient) versus the peptide-lipid ratio further suggested that the lysis action of CB1 is related to its capacity to bind to the lipid bilayers. In contrast, there is no evidence of binding for CB3. To augment these findings, four spin-labeled peptides, C8SL-CB1, C32SL-CB1, C5SL-CB3, and C30SL-CB3, were also examined for their binding to and their state of aggregation within the lipid bilayers. Association isotherms of the peptides were measured for liposomes containing two molar fractions of PA (0.25 and 0.75). The membrane binding of the CB1 peptides exhibited a cooperative behavior, whereas the association isotherm of CB3 revealed binding to the lipid only for beta = 0.75 liposomes. To further identify the location of CB1 in the lipid bilayers, measurements of the collision rate with chromium oxalate in solution were conducted. Results from ESR power saturation measurements suggested that the NH(2)-terminal alpha-helix of CB1 is located on the

  10. Inhibition of T-lymphocyte-mediated tumor-specific lysis by alloantisera directed against the H-2 serological specificities of the tumor.

    PubMed

    Germain, R N; Dorf, M E; Benacerraf, B

    1975-10-01

    After appropriate in vivo or in vitro immunization, cytotoxic T lymphocytes (CTL) are generated which efficiently kill cells bearing particular membrane antigens in common with the immunizing cell (reviewed in reference 1). Such CTL have been most thoroughly studied in mice, employing alloimmunization with cells differing at the major histocompatibility locus, H-2. in such cases, the predominant cell surface antigens recognized by the CTL appear to be the molecules carrying the serologically defined H-2 specificities, coded for by the K and D regions of the H-2 complex (2). In other syngeneic models of cell-mediated specific cytolysis, involving lymphocyte chariomeningitis (LCM) virus- or ectromelia virus-infected cells or TNP-modified lymphoid cells, thymus-derived cells also constitute the main effector cell type. The CTL generated in these latter systems function most efficiently when virus-infected or TNP-modified target cells share identitites at the H-2K or H-2D loci with the effector CTL and stimulator cells (3-5). Another set of experimental systems in which CTL are generated and play a significant biological role is that of immunity to tumor-associated antigens (TAA) (6). The nature of the TAA which the CTL recognize is only beginning to be understood. Several recent reports indicated the existence of physiochemical and/or antigenic relationships between TAA and H-2 antigens (7,8). These relationships, together with the genetic restrictions cited above in the generation of CTL involving products of the H-2K or H-2D loci suggested the possibility that in certain tumor systems, the TAA which are able to most effectively stimulate CTL responses might be structurally similar to, or linked with, the H-2K or H- 2D molecules on the tumor surface. It has been previously demonstrated in allogenic models that antisera specific for the appropriate H-2K or H-2D products present on a target cell could specifically block CTL-mediated lysis (1,9). This report

  11. Inhibition of T-lymphocyte-mediated tumor-specific lysis by alloantisera directed against the H-2 serological specificities of the tumor

    PubMed Central

    Germain, RN; Dorf, ME; Benacerraf, B

    1975-01-01

    After appropriate in vivo or in vitro immunization, cytotoxic T lymphocytes (CTL) are generated which efficiently kill cells bearing particular membrane antigens in common with the immunizing cell (reviewed in reference 1). Such CTL have been most thoroughly studied in mice, employing alloimmunization with cells differing at the major histocompatibility locus, H-2. in such cases, the predominant cell surface antigens recognized by the CTL appear to be the molecules carrying the serologically defined H-2 specificities, coded for by the K and D regions of the H-2 complex (2). In other syngeneic models of cell-mediated specific cytolysis, involving lymphocyte chariomeningitis (LCM) virus- or ectromelia virus-infected cells or TNP-modified lymphoid cells, thymus-derived cells also constitute the main effector cell type. The CTL generated in these latter systems function most efficiently when virus-infected or TNP-modified target cells share identitites at the H-2K or H-2D loci with the effector CTL and stimulator cells (3-5). Another set of experimental systems in which CTL are generated and play a significant biological role is that of immunity to tumor-associated antigens (TAA) (6). The nature of the TAA which the CTL recognize is only beginning to be understood. Several recent reports indicated the existence of physiochemical and/or antigenic relationships between TAA and H-2 antigens (7,8). These relationships, together with the genetic restrictions cited above in the generation of CTL involving products of the H-2K or H-2D loci suggested the possibility that in certain tumor systems, the TAA which are able to most effectively stimulate CTL responses might be structurally similar to, or linked with, the H-2K or H- 2D molecules on the tumor surface. It has been previously demonstrated in allogenic models that antisera specific for the appropriate H-2K or H-2D products present on a target cell could specifically block CTL-mediated lysis (1,9). This report

  12. Attachment of an anti-receptor antibody to non-target cells renders them susceptible to lysis by a clone of cytotoxic T lymphocytes.

    PubMed

    Kranz, D M; Tonegawa, S; Eisen, H N

    1984-12-01

    The molecular basis for the dependence of antigen recognition by T cells on products of the major histocompatibility complex (MHC) is unknown, and the antigenic structures that are actually bound by T-cell receptors are ill-defined. In this study, we asked whether a monoclonal antibody (mAb) that reacts with the T-cell receptor of a clone of murine cytotoxic T lymphocytes (CTL) and not with the receptors of other CTL clones can substitute for that clone's natural ligand in specific cytolytic reactions. To answer the question, a mAb (1B2) to the receptor of a CTL clone (2C) was attached covalently to 51Cr-labeled cells that were not otherwise susceptible to lysis by clone 2C, and the cells thus modified were then tested as targets for clone 2C and other CTL clones of similar specificity. All labeled cells modified in this way, including a murine cell line that expresses no cell-surface MHC class I molecules and a human cell line, were lysed by clone 2C but not by other CTL clones. If, however, instead of attaching the mAb to the receptor of clone 2C, the cells were modified by attaching to them mAbs to other surface antigens on CTL [lymphocyte function-associated antigen (LFA-1), Thy-1.2], they were not lysed. In cytolytic titrations, the cells that had been converted by attachment of mAb 1B2 into specific targets for clone 2C were just as susceptible to lysis by that clone as the clone's natural H-2d targets (e.g., P815 cells). However, some accessory surface molecules (LFA-1, Lyt-2) that are required for clone 2C to lyse its natural H-2d targets seemed not to be required for this clone to lyse the mAb-converted target cells. By demonstrating that a variety of different cell types can be thus converted into target cells for CTL, the approach described in this study may provide opportunities to analyze further the mechanisms by which CTL destroy target cells.

  13. A single-chain triplebody with specificity for CD19 and CD33 mediates effective lysis of mixed lineage leukemia cells by dual targeting

    PubMed Central

    Kellner, Christian; Stein, Christoph; Kügler, Markus; Schwenkert, Michael; Saul, Domenica; Mentz, Kristin; Singer, Heiko; Stockmeyer, Bernhard; Hillen, Wolfgang; Mackensen, Andreas; Fey, Georg H

    2011-01-01

    A single-chain triplebody (sctb) 33-ds16-ds19 comprising two distal single-chain Fv fragments (scFvs) specific for the lymphoid antigen CD19 and the myeloid antigen CD33 flanking a central scFv specific for CD16, which is the low affinity Fc-receptor (FcγRIII) present on natural killer cells and macrophages, was produced and its properties were investigated. CD33 and CD19 in combination are present on acute leukemiablasts with mixed lineage phenotype, but not on normal human hematopoietic cells. For comparison, two bispecific scFvs (bsscFvs), ds19-ds16 and 33-ds16, with monovalent binding to CD19 and CD33, respectively, were also studied. The sctb 33-ds16-ds19 specifically interacted with all three antigens. On the antigen double-positive cell line BV-173, the sctb bound with 2-fold greater avidity than bsscFv ds19-ds16 (KD = 21 vs. 42 nM) and with 1.4-fold greater avidity than bsscFv 33-ds16 (KD = 29 nM). All three fusion proteins had similar affinity for CD16 and sufficient thermic stability in human serum. In antibody-dependent cellular cytotoxicity (ADCC) reactions with human mononuclear cells as effectors, the sctb promoted lysis of BV-173 cells at 23-fold lower concentrations than bsscFv ds19-ds16 and at 1.4-fold lower concentrations than bsscFv 33-ds16. The sctb also mediated potent ADCC of the antigen double-positive mixed lineage leukemia cell line SEM, and the half-maximal concentration EC50 for BV-173 cells was 7 pM. Therefore, CD19 and CD33 are present on the surface of these leukemic cell lines such that they can be connected by a single sctb molecule, permitting the recruitment of NK cells via CD16 and tumor cell lysis. PMID:21081841

  14. Fate and distribution of brevetoxin (PbTx) following lysis of Karenia brevis by algicidal bacteria, including analysis of open A-ring derivatives.

    PubMed

    Roth, Patricia B; Twiner, Michael J; Wang, Zhihong; Bottein Dechraoui, Marie-Yasmine; Doucette, Gregory J

    2007-12-15

    Flavobacteriaceae (strain S03) and Cytophaga sp. (strain 41-DBG2) are algicidal bacteria active against the brevetoxin (PbTx)-producing, red tide dinoflagellate, Karenia brevis. Little is known about the fate of PbTx associated with K. brevis cells following attack by such bacteria. The fate and distribution of PbTx in K. brevis cultures exposed to these algicidal strains were thus examined by receptor binding assay and liquid chromatography/mass spectrometry (LC/MS) in three size fractions (>5, 0.22-5, <0.22microm) over a 2-week time course. In control cultures, brevetoxin concentrations in the >5microm particulate size fraction correlated with changes in cell density, whereas significant increases in dissolved (i.e., <0.22microm) toxin were observed in the later stages of culture growth. Exposure of K. brevis to either of the two algicidal bacteria tested caused cell lysis, coinciding with a rapid decline in the >5microm PbTX size fraction and a simultaneous release of dissolved toxin into the growth medium. Upon cell lysis, dissolved brevetoxin accounted for ca. 60% of total toxin and consisted of 51-82% open A-ring derivatives. Open A-ring PbTx-2 and PbTx-3 derivatives bound with lower affinity (approximately 22- and 57-fold, respectively) to voltage-gated sodium channels and were considerably less cytotoxic (86- and 142-fold, respectively) to N2A cells than their individual parent toxins (i.e., PbTx-2 and PbTx-3). These novel findings of changes in PbTx size-fractioned distribution and overall reduction in K. brevis toxicity following attack by algicidal bacteria improve our understanding of potential trophic transfer routes and the fate of PbTx during red tide events. Moreover, this information will be important to consider when evaluating the potential role of algicidal bacteria in harmful algal bloom (HAB) management strategies involving control of bloom populations.

  15. Revision stapes surgery for lysis of the long process of the incus: comparing hydroxyapatite bone cement versus malleovestibulopexy and total ossicular replacement prosthesis.

    PubMed

    Pitiot, Vincent; Hermann, Ruben; Tringali, Stéphane; Dubreuil, Christian; Truy, Eric

    2016-09-01

    The objective of the study was to report audiological results in revision stapes surgery, comparing hydroxyapatite (HAP) bone cement, malleovestibular (MV) prosthesis, and total ossicular replacement prosthesis (TORP). The study is a retrospective case review conducted in a tertiary referral center. Patients treated for revision stapes surgery from 2010 to 2014, where a lysis of the long process of the incus (LPI) was observed with the use of HAP bone cement, MV prosthesis, or a TORP were included in the study. The main outcomes measured were pre- and postoperative bone conduction (BC) and air conduction (AC) pure-tone averages (PTA) (0.5, 1, 2, 3 kHz), including high frequencies BC (HFBC) (1, 2, 3, 4 kHz) and air-bone gap (ABG). 107 revision stapes surgery were performed in 96 ears. Main cause of failure was LPI lysis in 38 cases (39.6 %). 31 patients were analyzed: HAP bone cement was used in 11 patients (Group I), MV prosthesis in ten patients (Group II), and TORP in ten patients (Group III). The mean post-operative ABG was 10.7 dB (±7.4) (p = 0.003), 10.7 dB (±8.8) (p = 0.001), and 16.9 dB (±9.8) (p = 0.001), respectively. There were no significant differences between groups. In Group I, the mean change in HFBC revealed an improvement of 5.6 dB (±7.9) (p = 0.03), while in Group III there was a significant deterioration of the thresholds of 5.8 dB (±7.6) (p = 0.04). There were no cases of post-operative anacusis. In revision stapes surgery when LPI is eroded, we recommend to perform a cement ossiculoplasty for stabilizing a standard Teflon piston when LPI is still usable, the LPI lengthening with cement being not recommended. When LPI is too eroded, we prefer performing a malleovestibulopexy, and reserve TORP for cases with a bad anatomical presentation.

  16. Bacteriophage cell lysis of Shiga toxin-producing Escherichia coli for top-down proteomic identification of Shiga toxins 1 & 2 using matrix-assisted laser desorption/ionization tandem time-of-flight mass spectrometry.

    PubMed

    Fagerquist, Clifton K; Zaragoza, William J

    2016-03-30

    Analysis of bacteria by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOFMS) often relies upon sample preparation methods that result in cell lysis, e.g. bead-beating. However, Shiga toxin-producing Escherichia coli (STEC) can undergo bacteriophage-induced cell lysis triggered by antibiotic exposure that may allow greater selectivity of the proteins extracted. We have developed a sample preparation method for selective extraction of bacteriophage-encoded proteins and specifically Shiga toxins 1 and 2 (Stx1 & 2) expressed from STEC strains induced by DNA-damaging antibiotics. STEC strains were cultured overnight on agar supplemented with ciprofloxacin, mitomycin-C or an iron chelator to induce the bacteriophage lytic cycle with concomitant expression and release of Stx1 and/or Stx2. Sample preparation relied exclusively on bacteriophage lysis for release Stx into the extraction solution. Three clinical STEC strains were analyzed by matrix-assisted laser desorption/ionization tandem time-of-flight mass spectrometry (MALDI-TOF-TOF-MS/MS) and top-down proteomics analysis: E. coli O157:H7 strain EDL933, E. coli O91:H21 strain B2F1 and E. coli O26:H11 strain ECRC #05.2217. The B-subunit of Stx1a of EDL933 was detected and identified even though it was ~100-fold less abundant than the B-subunit of Stx2a that had been identified previously for this strain. Two bacteriophage-encoded proteins were also identified: L0117 and L0136. The B-subunits of Stx2d of strain B2F1 and Stx1a of strain ECRC #05.2217 were also detected and identified. Bacteriophage lysis appeared to enhance the detection sensitivity of Stx for these STEC strains compared to previous work using mechanical lysis. Detection/identification of other bacteriophage-encoded proteins (beyond Stx) tends to support the hypothesis of Stx release by bacteriophage cell lysis. Published in 2016. This article is a U.S. Government work and is in the public domain in the USA.

  17. Human Adipose-Derived Stem Cells Impair Natural Killer Cell Function and Exhibit Low Susceptibility to Natural Killer-Mediated Lysis

    PubMed Central

    DelaRosa, Olga; Sánchez-Correa, Beatriz; Morgado, Sara; Ramírez, Cristina; del Río, Borja; Menta, Ramón; Lombardo, Eleuterio

    2012-01-01

    Human adipose-derived stem cells (hASCs) have been successfully used in treating numerous diseases. However, several aspects need to be considered, particularly in the context of allogeneic cell therapy. To better understand hASCs-host interactions, we studied the phenotype of hASCs and their modulatory effect on natural killer (NK) cells by using bone marrow-mesenchymal stem cells (hBM-MSCs) as a reference. The hASCs displayed a lower susceptibility to NK cell-mediated lysis and a lower expression of ligands for DNAM-1 when compared with hBM-MSCs. Moreover, here we demonstrated that hASCs and hBM-MSCs can modulate NK cells through the action of soluble factors such as indoleamine 2,3-dioxygenase. Altogether, these results suggest that for an adoptive cell therapy based on the transfer of allogeneic hASCs, the NK-hASCs crosstalk will not result in an immediate recognition of the transferred cells. Thus, hASCs may remain in the tissue long enough to balance the immune response before being cleared. PMID:21867426

  18. Enhancement of activated sludge dewatering performance by combined composite enzymatic lysis and chemical re-flocculation with inorganic coagulants: Kinetics of enzymatic reaction and re-flocculation morphology.

    PubMed

    Chen, Zhan; Zhang, Weijun; Wang, Dongsheng; Ma, Teng; Bai, Runying

    2015-10-15

    The feasibility of combined process of composite enzymatic treatment and chemical flocculation with inorganic salt coagulants was investigated in this study. The evolution of extracellular polymeric substances (EPS) distribution, composition and morphological properties were analyzed to unravel the sludge conditioning mechanism. It was found that sludge filtration performance was deteriorated due to release of a large amount of biopolymers after enzymatic treatment. The change in EPS followed the pseudo-first-order kinetic equation well under enzymatic treatment. The feeding modes of enzymes had a significant influence on sludge lysis efficiency under compound enzymes treatment. Alpha amylase + protease was more effective in solubilization than other two addition modes (protease + α-amylase or simultaneous addition). The sludge floc re-formed and macromolecule biopolymers were effectively removed through coagulation process. At the same time, both of filtration rate and cake solid content of sludge treated with enzymes were improved with increasing dosage of coagulants, and ferric iron (FeCl3) had better performance in sludge dewaterability enhancement than polyaluminium chloride (PACl). In addition, sludge filtration property was slightly deteriorated, while the cake moisture reduction was favored at the optimal dosage of inorganic coagulants. Copyright © 2015 Elsevier Ltd. All rights reserved.

  19. Aberrant expression of the hematopoietic-restricted minor histocompatibility antigen LRH-1 on solid tumors results in efficient cytotoxic T cell-mediated lysis.

    PubMed

    Overes, Ingrid M; Levenga, T Henriëtte; Vos, Johanna C M; van Horssen-Zoetbrood, Agnes; van der Voort, Robbert; De Mulder, Pieter H; de Witte, Theo M; Dolstra, Harry

    2009-03-01

    CD8(+) T cells recognizing minor histocompatibility antigens (MiHA) on solid tumor cells may mediate effective graft-versus-tumor (GVT) reactivity after allogeneic stem cell transplantation (SCT). Previously, we identified LRH-1 as a hematopoietic-restricted MiHA encoded by the P2X5 gene. Here, we report that LRH-1 is aberrantly expressed on solid tumor cells. P2X5 mRNA expression is demonstrated in a significant portion of solid tumor cell lines, including renal cell carcinoma (RCC), melanoma, colorectal carcinoma, brain cancer and breast cancer. Importantly, P2X5 gene expression was also detected in a subset of primary solid tumor specimens derived from RCC, brain cancer and breast cancer patients. Furthermore, P2X5 expressing solid tumor cells can be effectively targeted by LRH-1-specific cytotoxic T lymphocytes under inflammatory conditions. The expression of HLA-B7 and CD54 on tumor cells increases upon cytokine stimulation resulting in improved T cell activation as observed by higher levels of degranulation and enhanced tumor cell lysis. Overall, hematopoietic-restricted MiHA LRH-1 is aberrantly expressed on solid tumor cells and may be used as target in GVT-specific immunotherapy after SCT.

  20. Staphylococcus haemolyticus prophage ΦSH2 endolysin relies on cysteine, histidine-dependent amidohydrolases/peptidases activity for lysis 'from without'.

    PubMed

    Schmelcher, Mathias; Korobova, Olga; Schischkova, Nina; Kiseleva, Natalia; Kopylov, Paul; Pryamchuk, Sergey; Donovan, David M; Abaev, Igor

    2012-12-31

    Staphylococcus aureus is an important pathogen, with methicillin-resistant (MRSA) and multi-drug resistant strains becoming increasingly prevalent in both human and veterinary clinics. S. aureus causing bovine mastitis yields high annual losses to the dairy industry. Conventional treatment of mastitis by broad range antibiotics is often not successful and may contribute to development of antibiotic resistance. Bacteriophage endolysins present a promising new source of antimicrobials. The endolysin of prophage ΦSH2 of Staphylococcus haemolyticus strain JCSC1435 (ΦSH2 lysin) is a peptidoglycan hydrolase consisting of two catalytic domains (CHAP and amidase) and an SH3b cell wall binding domain. In this work, we demonstrated its lytic activity against live staphylococcal cells and investigated the contribution of each functional module to bacterial lysis by testing a series of deletion constructs in zymograms and turbidity reduction assays. The CHAP domain exhibited three-fold higher activity than the full length protein and optimum activity in physiological saline. This activity was further enhanced by the presence of bivalent calcium ions. The SH3b domain was shown to be required for full activity of the complete ΦSH2 lysin. The full length enzyme and the CHAP domain showed activity against multiple staphylococcal strains, including MRSA strains, mastitis isolates, and CoNS.

  1. Peptidomic profiling of secreted products from pancreatic islet culture results in a higher yield of full-length peptide hormones than found using cell lysis procedures.

    PubMed

    Taylor, Steven W; Nikoulina, Svetlana E; Andon, Nancy L; Lowe, Carolyn

    2013-08-02

    Peptide Hormone Acquisition through Smart Sampling Technique-Mass Spectrometry (PHASST-MS) is a peptidomics platform that employs high resolution liquid chromatography-mass spectrometry (LC-MS) techniques to identify peptide hormones secreted from in vitro or ex vivo cultures enriched in endocrine cells. Application of the methodology to the study of murine pancreatic islets has permitted evaluation of the strengths and weaknesses of the approach, as well as comparison of our results with published islet studies that employed traditional cellular lysis procedures. We found that, while our PHASST-MS approach identified fewer peptides in total, we had greater representation of intact peptide hormones. The technique was further refined to improve coverage of hydrophilic as well as hydrophobic peptides and subsequently applied to human pancreatic islet cultures derived from normal donors or donors with type 2 diabetes. Interestingly, in addition to the expected islet hormones, we identified alpha-cell-derived bioactive GLP-1, consistent with recent reports of paracrine effects of this hormone on beta-cell function. We also identified many novel peptides derived from neurohormonal precursors and proteins related to the cell secretory system. Taken together, these results suggest the PHASST-MS strategy of focusing on cellular secreted products rather than the total tissue peptidome may improve the probability of discovering novel bioactive peptides and also has the potential to offer important new insights into the secretion and function of known hormones.

  2. Yeast on-target lysis (YOTL), a procedure for making auxiliary mass spectrum data sets for clinical routine identification of yeasts.

    PubMed

    Bernhard, Mareike; Weig, Michael; Zautner, Andreas E; Groß, Uwe; Bader, Oliver

    2014-12-01

    Matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS)-based species identification has become a reliable and fast tool for use in clinical diagnostics, including in mycology. To identify yeasts in the MALDI Biotyper system, a multistep extraction protocol, which is also used to generate the reference spectra, is recommended. Sample preparation by on-target lysis (OTL) requires significantly less hands-on time and is therefore highly desirable, but it results in too-low MALDI Biotyper log score values to allow automated species identification. To overcome this problem, we developed a procedure for generating and validating an OTL spectrum data set for the most relevant and frequently occurring yeast species in clinical specimens. The performance was evaluated against a set of OTL spectra derived during clinical routine procedures and from a set of closely related yeasts. In the diagnostic setting, the OTL procedure significantly decreased the workload but allowed species identification with high specificity and sensitivity. False identifications were not observed. The use of in-house-generated OTL reference spectra can highly accelerate MALDI-TOF MS-based yeast species identification using the MALDI Biotyper.

  3. Cell lysis and DNA extraction of gram-positive and gram-negative bacteria from whole blood in a disposable microfluidic chip.

    PubMed

    Mahalanabis, Madhumita; Al-Muayad, Hussam; Kulinski, M Dominika; Altman, Dave; Klapperich, Catherine M

    2009-10-07

    Sepsis caused by gram positive and gram negative bacteria is the leading cause of death in noncoronary ICUs and the tenth leading cause of death in the United States. We have developed a microfluidic sample preparation platform for rapid on-chip detection of infectious organisms for point-of-care diagnostics. The microfluidic chips are made of a robust thermoplastic and can be easily multiplexed for high throughput applications. Bacteria are lysed on-chip via hybrid chemical/mechanical method. Once lysed, the bacterial DNA is isolated using a microscale silica bead/polymer composite solid-phase-extraction (SPE) column. Lysis was confirmed using off-chip real time PCR. We isolated and detected both gram-negative (Escherichia coli) and gram-positive (Bacillussubtilis and Enterococcus faecalis) bacterial genomic DNA from microliter scale spiked whole human blood samples. The system performs better for gram-negative bacteria than it does for gram-positive bacteria, with limits of detection at 10(2) CFU/ml and 10(3)-10(4) CFU/ml, respectively. Total extraction times are less than one hour and can be further decreased by altering the channel geometry and pumping configuration.

  4. Modulation of CD112 by the alphaherpesvirus gD protein suppresses DNAM-1-dependent NK cell-mediated lysis of infected cells.

    PubMed

    Grauwet, Korneel; Cantoni, Claudia; Parodi, Monica; De Maria, Andrea; Devriendt, Bert; Pende, Daniela; Moretta, Lorenzo; Vitale, Massimo; Favoreel, Herman W

    2014-11-11

    Natural killer (NK) cells are key players in the innate response to viruses, including herpesviruses. In particular, the variety of viral strategies to modulate the recognition of certain herpesviruses witnesses the importance of NK cells in the control of this group of viruses. Still, NK evasion strategies have remained largely elusive for the largest herpesvirus subfamily, the alphaherpesviruses. Here, we report that the gD glycoprotein of the alphaherpesviruses pseudorabies virus (PRV) and herpes simplex virus 2 (HSV-2) displays previously uncharacterized immune evasion properties toward NK cells. Expression of gD during infection or transfection led to degradation and consequent down-regulation of CD112, a ligand for the activating NK receptor DNAX accessory molecule 1 (DNAM-1). CD112 downregulation resulted in a reduced ability of DNAM-1 to bind to the surface of both virus-infected and gD-transfected cells. Consequently, expression of gD suppressed NK cell degranulation and NK cell-mediated lysis of PRV- or HSV-2-infected cells. These data identify an alphaherpesvirus evasion strategy from NK cells and point out that interactions between viral envelope proteins and host cell receptors can have biological consequences that stretch beyond virus entry.

  5. Neutralization of (NK-cell-derived) B-cell activating factor by Belimumab restores sensitivity of chronic lymphoid leukemia cells to direct and Rituximab-induced NK lysis.

    PubMed

    Wild, J; Schmiedel, B J; Maurer, A; Raab, S; Prokop, L; Stevanović, S; Dörfel, D; Schneider, P; Salih, H R

    2015-08-01

    Natural killer (NK) cells are cytotoxic lymphocytes that substantially contribute to the therapeutic benefit of antitumor antibodies like Rituximab, a crucial component in the treatment of B-cell malignancies. In chronic lymphocytic leukemia (CLL), the ability of NK cells to lyse the malignant cells and to mediate antibody-dependent cellular cytotoxicity upon Fc receptor stimulation is compromised, but the underlying mechanisms are largely unclear. We report here that NK-cells activation-dependently produce the tumor necrosis factor family member 'B-cell activating factor' (BAFF) in soluble form with no detectable surface expression, also in response to Fc receptor triggering by therapeutic CD20-antibodies. BAFF in turn enhanced the metabolic activity of primary CLL cells and impaired direct and Rituximab-induced lysis of CLL cells without affecting NK reactivity per se. The neutralizing BAFF antibody Belimumab, which is approved for treatment of systemic lupus erythematosus, prevented the effects of BAFF on the metabolism of CLL cells and restored their susceptibility to direct and Rituximab-induced NK-cell killing in allogeneic and autologous experimental systems. Our findings unravel the involvement of BAFF in the resistance of CLL cells to NK-cell antitumor immunity and Rituximab treatment and point to a benefit of combinatory approaches employing BAFF-neutralizing drugs in B-cell malignancies.

  6. Continuous cell introduction and rapid dynamic lysis for high-throughput single-cell analysis on microfludic chips with hydrodynamic focusing.

    PubMed

    Xu, Chun-Xiu; Yin, Xue-Feng

    2011-02-04

    A chip-based microfluidic system for high-throughput single-cell analysis is described. The system was integrated with continuous introduction of individual cells, rapid dynamic lysis, capillary electrophoretic (CE) separation and laser induced fluorescence (LIF) detection. A cross microfluidic chip with one sheath-flow channel located on each side of the sampling channel was designed. The labeled cells were hydrodynamically focused by sheath-flow streams and sequentially introduced into the cross section of the microchip under hydrostatic pressure generated by adjusting liquid levels in the reservoirs. Combined with the electric field applied on the separation channel, the aligned cells were driven into the separation channel and rapidly lysed within 33ms at the entry of the separation channel by Triton X-100 added in the sheath-flow solution. The maximum rate for introducing individual cells into the separation channel was about 150cells/min. The introduction of sheath-flow streams also significantly reduced the concentration of phosphate-buffered saline (PBS) injected into the separation channel along with single cells, thus reducing Joule heating during electrophoretic separation. The performance of this microfluidic system was evaluated by analysis of reduced glutathione (GSH) and reactive oxygen species (ROS) in single erythrocytes. A throughput of 38cells/min was obtained. The proposed method is simple and robust for high-throughput single-cell analysis, allowing for analysis of cell population with considerable size to generate results with statistical significance.

  7. Intermedilysin-receptor interactions during assembly of the pore complex: assembly intermediates increase host cell susceptibility to complement-mediated lysis.

    PubMed

    LaChapelle, Stephanie; Tweten, Rodney K; Hotze, Eileen M

    2009-05-08

    Intermedilysin (ILY) is an unusual member of the family of cholesterol-dependent cytolysins because it binds to human CD59 (hCD59) rather than directly to cholesterol-rich membranes. Binding of ILY to hCD59 initiates a series of conformational changes within the toxin that result in the conversion of the soluble monomer into an oligomeric membrane-embedded pore complex. In this study the association of ILY with its membrane receptor has been examined throughout the assembly and formation of the pore complex. Using ILY mutants trapped at various stages of pore assembly, we show ILY remains engaged with hCD59 throughout the assembly of the prepore oligomer, but it disengages from the receptor upon the conversion to the pore complex. We further show that the assembly intermediates increase the sensitivity of the host cell to lysis by its complement membrane attack complex, apparently by blocking the hCD59-binding site for complement proteins C8alpha and C9.

  8. CD155 on HIV-Infected Cells Is Not Modulated by HIV-1 Vpu and Nef but Synergizes with NKG2D Ligands to Trigger NK Cell Lysis of Autologous Primary HIV-Infected Cells.

    PubMed

    Davis, Zachary B; Sowrirajan, Bharatwaj; Cogswell, Andrew; Ward, Jeffery P; Planelles, Vicente; Barker, Edward

    2017-02-01

    Activation of primary CD4(+) T cells induces the CD155, but not the CD112 ligands for the natural killer (NK) cell activation receptor (aNKR) CD226 [DNAX accessory molecule-1 (DNAM-1)]. We hypothesize that HIV productively infects activated CD4(+) T cells and makes itself vulnerable to NK cell-mediated lysis when CD155 on infected T cells engages DNAM-1. The primary objective of this study is to determine whether CD155 alone or together with NKG2D ligands triggers autologous NK cell lysis of HIV-infected T cells and whether HIV modulates CD155. To determine whether HIV modulates this activation ligand, we infected "activated" CD4(+) T cells with HIV in the absence or presence of Nef and/or Vpu and determined by flow cytometry whether they modulated CD155. To determine if CD155 alone, or together with NKG2D ligands, triggered NK cell lysis of autologous HIV-infected T cells, we treated purified NK cells with DNAM-1 and/or NKG2D blocking antibodies before the addition of purified autologous HIV-infected cells in cytolytic assays. Finally, we determined whether DNAM-1 works together with NKG2D as an NK cell coactivation receptor (caNKR) or whether they work independently as aNKRs to induce an NK cell lytic response. We demonstrate that HIV and specifically Nef and/or Vpu do not modulate CD155 on infected primary T cells; and both CD155 and NKG2D ligands synergize as aNKRs to trigger NK cell lysis of the infected cell.

  9. Characterization of the Staphylococcus aureus CidR regulon: elucidation of a novel role for acetoin metabolism in cell death and lysis.

    PubMed

    Yang, Soo-Jin; Dunman, Paul M; Projan, Steven J; Bayles, Kenneth W

    2006-04-01

    The Staphylococcus aureus cid and lrg operons encode a novel regulatory system that affects murein hydrolase activity, stationary-phase survival and antibiotic tolerance. Expression of the lrgAB operon is positively regulated by a two-component regulatory system encoded by the lytSR operon located immediately upstream to lrgAB. By comparison, the cidABC operon lies downstream from the cidR gene, encoding a protein homologous to the LysR-type family of transcriptional regulators. Transcription analysis of a cidR mutant revealed that CidR enhances cidABC expression in the presence of acetic acid generated by the metabolism of excess glucose. Microarray studies identified additional CidR-regulated operons including the IrgAB and alsSD encoding proteins involved in acetoin production. Surprisingly, Northern blot analyses revealed that cidABC and lrgAB transcription was uninducible in an alsSD mutant grown in the presence of excess glucose, suggesting that the CidR-mediated upregulation of cidABC and lrgAB transcription is dependent on the presence of intact alsSD genes. Zymographic and quantitative analyses of murein hydrolase activity also revealed that disruption of the alsSD genes results in significantly decreased extracellular murein hydrolase activity compared with that of the parental strain, UAMS-1. Furthermore, the alsSD mutant displayed decreased stationary-phase survival relative to UAMS-1, both in the presence and absence of glucose. The results of this study define the CidR regulon and demonstrate that the generation of acetoin is linked to the control of cell death and lysis in S. aureus.

  10. Characterization of a thermostable endo-1,3(4)-β-glucanase from Caldicellulosiruptor sp. strain F32 and its application for yeast lysis.

    PubMed

    Meng, Dong-Dong; Wang, Bing; Ma, Xiao-Qing; Ji, Shi-Qi; Lu, Ming; Li, Fu-Li

    2016-06-01

    β-1,3-Glucans, important structural components of cell wall or nutritional components of the endosperm, are extensively found in bacteria, fungi, yeast, algae, and plants. The structural complexity of β-1,3-glucans implies that the enzymatic depolymerization of polysaccharides needs combined activities of distinct enzymes. In this study, Lam16A-GH, the catalytic module of a putative glycoside hydrolase (GH) family 16 laminarinase/lichenase from thermophilic bacterium Caldicellulosiruptor sp. F32, was purified and characterized through heterologous expression in Escherichia coli. Lam16A-GH can hydrolyze both β-1,3-glucan (laminarin) and β-1,3-1,4-glucan (barley β-glucan) revealed by analysis of the products of polysaccharide degradation using thin-layer chromatography (TLC). The time required for the loss of 50 % of its activity is 45 h under the optimal condition of 75 °C and pH 6.5. Oligosaccharides degradation assay indicated that Lam16A-GH can catalyze endo-hydrolysis of the β-1,4 glycosidic linkage adjacent to a 3-O-substituted glucosyl residue in the mixed linked β-glucans, as well as the β-1,3 linkage. The survival rate of Saccharomyces cerevisiae cells depends on the addition of Lam16A-GH, and the cytoplasm protein was released from the apparently deconstructed yeast cells. These results indicate that the bi-functional thermostable Lam16A-GH exhibits unique enzymatic properties and potential for yeast lysis.

  11. A tetravalent bispecific TandAb (CD19/CD3), AFM11, efficiently recruits T cells for the potent lysis of CD19+ tumor cells

    PubMed Central

    Reusch, Uwe; Duell, Johannes; Ellwanger, Kristina; Herbrecht, Carmen; Knackmuss, Stefan HJ; Fucek, Ivica; Eser, Markus; McAleese, Fionnuala; Molkenthin, Vera; Le Gall, Fabrice; Topp, Max; Little, Melvyn; Zhukovsky, Eugene A

    2015-01-01

    To harness the potent tumor-killing capacity of T cells for the treatment of CD19+ malignancies, we constructed AFM11, a humanized tetravalent bispecific CD19/CD3 tandem diabody (TandAb) consisting solely of Fv domains. The molecule exhibits good manufacturability and stability properties. AFM11 has 2 binding sites for CD3 and 2 for CD19, an antigen that is expressed from early B cell development through differentiation into plasma cells, and is an attractive alternative to CD20 as a target for the development of therapeutic antibodies to treat B cell malignancies. Comparison of the binding and cytotoxicity of AFM11 with those of a tandem scFv bispecific T cell engager (BiTE) molecule targeting the same antigens revealed that AFM11 elicited more potent in vitro B cell lysis. Though possessing high affinity to CD3, the TandAb mediates serial-killing of CD19+ cells with little dependence of potency or efficacy upon effector:target ratio, unlike the BiTE. The advantage of the TandAb over the BiTE was most pronounced at lower effector:target ratios. AFM11 mediated strictly target-dependent T cell activation evidenced by CD25 and CD69 induction, proliferation, and cytokine release, notwithstanding bivalent CD3 engagement. In a NOD/scid xenograft model, AFM11 induced dose-dependent growth inhibition of Raji tumors in vivo, and radiolabeled TandAb exhibited excellent localization to tumor but not to normal tissue. After intravenous administration in mice, half-life ranged from 18.4 to 22.9 h. In a human ex vivo B-cell chronic lymphocytic leukemia study, AFM11 exhibited substantial cytotoxic activity in an autologous setting. Thus, AFM11 may represent a promising therapeutic for treatment of CD19+ malignancies with an advantageous safety risk profile and anticipated dosing regimen. PMID:25875246

  12. Dietary supplementation of fermented soybean, natto, suppresses intimal thickening and modulates the lysis of mural thrombi after endothelial injury in rat femoral artery.

    PubMed

    Suzuki, Yasuhiro; Kondo, Kazunao; Matsumoto, Yuji; Zhao, Bing-Qing; Otsuguro, Kenichi; Maeda, Tetsuya; Tsukamoto, Yoshinori; Urano, Tetsumei; Umemura, Kazuo

    2003-07-25

    We have previously demonstrated that natto-extracts containing nattokinase (NK) inactivates plasminogen activator inhibitor type 1 and then potentiates fibrinolytic activity. In the present study, we investigated the effects of dietary supplementation with natto-extracts on neointima formation and on thrombolysis at the site of endothelial injury. Endothelial damage in the rat femoral artery was induced by intravenous injection of rose bengal followed by focal irradiation by transluminal green light. Dietary natto-extracts supplementation containing NK of 50 or 100 CU/body was started 3 weeks before endothelial injury and then continued for another 3 weeks. Intimal thickening in animals given supplementation was significantly (P<0.01) suppressed compared with controls and the intima/media ratio in animals with 50 and 100 CU/body NK and control group was 0.09 +/- 0.03, 0.09 +/- 0.06 and 0.16 +/- 0.12, respectively. Although femoral arteries were reopened both in control animals and those treated with NK within 8 hours after endothelial injury, mural thrombi were histologically observed at the site of endothelial injury. In the control group, the center of vessel lumen was reopened and mural thrombi were attached on the surface of vessel walls. In contrast, in NK-treated groups, thrombi near the vessel wall showed lysis and most of them detached from the surface of vessel walls. In conclusion, dietary natto-extracts supplementation suppressed intimal thickening produced by endothelial injury in rat femoral artery. These effects may partially be attributable to NK, which showed enhanced thrombolysis near the vessel wall.

  13. An autosomal dominant locus, Nka, mapping to the Ly-49 region of a rat natural killer (NK) gene complex, controls NK cell lysis of allogeneic lymphocytes

    PubMed Central

    1996-01-01

    Natural Killer (NK) cells can recognize and kill MHC-incompatible normal bone marrow-derived cells. Presently characterized MHC-binding receptors on NK cells, including the Ly-49 family in the mouse, transmit inhibitory signals upon binding to cognate class I MHC ligands. Here we study in vivo NK-mediated lysis of normal allogeneic lymphocytes in crosses between alloreactivity-competent PVG rats and alloreactivity-deficient DA rats. NK cells from both strains are able to lyse standard tumor targets. We identify an aut