Effects of track structure and cell inactivation on the calculation of heavy ion mutation rates in mammalian cells

NASA Technical Reports Server (NTRS)

Cucinotta, F. A.; Wilson, J. W.; Shavers, M. R.; Katz, R.

1996-01-01

It has long been suggested that inactivation severely effects the probability of mutation by heavy ions in mammalian cells. Heavy ions have observed cross sections of inactivation that approach and sometimes exceed the geometric size of the cell nucleus in mammalian cells. In the track structure model of Katz the inactivation cross section is found by summing an inactivation probability over all impact parameters from the ion to the sensitive sites within the cell nucleus. The inactivation probability is evaluated using the dose-response of the system to gamma-rays and the radial dose of the ions and may be equal to unity at small impact parameters for some ions. We show how the effects of inactivation may be taken into account in the evaluation of the mutation cross sections from heavy ions in the track structure model through correlation of sites for gene mutation and cell inactivation. The model is fit to available data for HPRT mutations in Chinese hamster cells and good agreement is found. The resulting calculations qualitatively show that mutation cross sections for heavy ions display minima at velocities where inactivation cross sections display maxima. Also, calculations show the high probability of mutation by relativistic heavy ions due to the radial extension of ions track from delta-rays in agreement with the microlesion concept. The effects of inactivation on mutations rates make it very unlikely that a single parameter such as LET or Z*2/beta(2) can be used to specify radiation quality for heavy ion bombardment.

Immunizations in solid organ and hematopoeitic stem cell transplant patients: A comprehensive review

PubMed Central

L'Huillier, Arnaud G; Kumar, Deepali

2015-01-01

The Solid Organ Transplantation (SOT) and Haematopoietic Stem Cell Transplantation (HSCT) population is continuously increasing as a result of broader indications for transplant and improved survival. Infectious diseases, including vaccine-preventable diseases, are a significant threat for this population, primarily after but also prior to transplantation. As a consequence, clinicians must ensure that patients are optimally immunized before transplantation, to provide the best protection during the early post-transplantation period, when immunosuppression is the strongest and vaccine responses are poor. After 3–6 months, inactivated vaccines immunization can be resumed. By contrast, live-attenuated vaccines are lifelong contraindicated in SOT patients, but can be considered in HSCT patients at least 2 years after transplantation, if there is no immunosuppression or graft-versus-host-disease. However, because of the advantages of live-attenuated over inactivated vaccines - and also sometimes the absence of an inactivated alternative - an increasing number of prospective studies on live vaccine immunization after transplantation are performed and give new insights about safety and immunogenicity in this population. PMID:26291740

Continuous Manufacturing of Recombinant Therapeutic Proteins: Upstream and Downstream Technologies.

PubMed

Patil, Rohan; Walther, Jason

2017-03-07

Continuous biomanufacturing of recombinant therapeutic proteins offers several potential advantages over conventional batch processing, including reduced cost of goods, more flexible and responsive manufacturing facilities, and improved and consistent product quality. Although continuous approaches to various upstream and downstream unit operations have been considered and studied for decades, in recent years interest and application have accelerated. Researchers have achieved increasingly higher levels of process intensification, and have also begun to integrate different continuous unit operations into larger, holistically continuous processes. This review first discusses approaches for continuous cell culture, with a focus on perfusion-enabling cell separation technologies including gravitational, centrifugal, and acoustic settling, as well as filtration-based techniques. We follow with a review of various continuous downstream unit operations, covering categories such as clarification, chromatography, formulation, and viral inactivation and filtration. The review ends by summarizing case studies of integrated and continuous processing as reported in the literature.

Numerical evaluation of lactoperoxidase inactivation during continuous pulsed electric field processing.

PubMed

Buckow, Roman; Semrau, Julius; Sui, Qian; Wan, Jason; Knoerzer, Kai

2012-01-01

A computational fluid dynamics (CFD) model describing the flow, electric field and temperature distribution of a laboratory-scale pulsed electric field (PEF) treatment chamber with co-field electrode configuration was developed. The predicted temperature increase was validated by means of integral temperature studies using thermocouples at the outlet of each flow cell for grape juice and salt solutions. Simulations of PEF treatments revealed intensity peaks of the electric field and laminar flow conditions in the treatment chamber causing local temperature hot spots near the chamber walls. Furthermore, thermal inactivation kinetics of lactoperoxidase (LPO) dissolved in simulated milk ultrafiltrate were determined with a glass capillary method at temperatures ranging from 65 to 80 °C. Temperature dependence of first order inactivation rate constants was accurately described by the Arrhenius equation yielding an activation energy of 597.1 kJ mol(-1). The thermal impact of different PEF processes on LPO activity was estimated by coupling the derived Arrhenius model with the CFD model and the predicted enzyme inactivation was compared to experimental measurements. Results indicated that LPO inactivation during combined PEF/thermal treatments was largely due to thermal effects, but 5-12% enzyme inactivation may be related to other electro-chemical effects occurring during PEF treatments. Copyright © 2012 American Institute of Chemical Engineers (AIChE).

Selective Destruction of Protein Function by Chromophore-Assisted Laser Inactivation

NASA Astrophysics Data System (ADS)

Jay, Daniel G.

1988-08-01

Chromophore-assisted laser inactivation of protein function has been achieved. After a protein binds a specific ligand or antibody conjugated with malachite green (C.I. 42000), it is selectively inactivated by laser irradiation at a wavelength of light absorbed by the dye but not significantly absorbed by cellular components. Ligand-bound proteins in solution and on the surfaces of cells can be denatured without other proteins in the same samples being affected. Chromophore-assisted laser inactivation can be used to study cell surface phenomena by inactivating the functions of single proteins on living cells, a molecular extension of cellular laser ablation. It has an advantage over genetics and the use of specific inhibitors in that the protein function of a single cell within the organism can be inactivated by focusing the laser beam.

Inactivation of Gating Currents of L-Type Calcium Channels

PubMed Central

Shirokov, Roman; Ferreira, Gonzalo; Yi, Jianxun; Ríos, Eduardo

1998-01-01

In studies of gating currents of rabbit cardiac Ca channels expressed as α1C/β2a or α1C/β2a/α2δ subunit combinations in tsA201 cells, we found that long-lasting depolarization shifted the distribution of mobile charge to very negative potentials. The phenomenon has been termed charge interconversion in native skeletal muscle (Brum, G., and E. Ríos. 1987. J. Physiol. (Camb.). 387:489–517) and cardiac Ca channels (Shirokov, R., R. Levis, N. Shirokova, and E. Ríos. 1992. J. Gen. Physiol. 99:863–895). Charge 1 (voltage of half-maximal transfer, V1/2 ≃ 0 mV) gates noninactivated channels, while charge 2 (V1/2 ≃ −90 mV) is generated in inactivated channels. In α1C/β2a cells, the available charge 1 decreased upon inactivating depolarization with a time constant τ ≃ 8, while the available charge 2 decreased upon recovery from inactivation (at −200 mV) with τ ≃ 0.3 s. These processes therefore are much slower than charge movement, which takes <50 ms. This separation between the time scale of measurable charge movement and that of changes in their availability, which was even wider in the presence of α2δ, implies that charges 1 and 2 originate from separate channel modes. Because clear modal separation characterizes slow (C-type) inactivation of Na and K channels, this observation establishes the nature of voltage-dependent inactivation of L-type Ca channels as slow or C-type. The presence of the α2δ subunit did not change the V1/2 of charge 2, but sped up the reduction of charge 1 upon inactivation at 40 mV (to τ ≃ 2 s), while slowing the reduction of charge 2 upon recovery (τ ≃ 2 s). The observations were well simulated with a model that describes activation as continuous electrodiffusion (Levitt, D. 1989. Biophys. J. 55:489–498) and inactivation as discrete modal change. The effects of α2δ are reproduced assuming that the subunit lowers the free energy of the inactivated mode. PMID:9607938

Inactivation of p53 by Human T-Cell Lymphotropic Virus Type 1 Tax Requires Activation of the NF-κB Pathway and Is Dependent on p53 Phosphorylation

PubMed Central

Pise-Masison, Cynthia A.; Mahieux, Renaud; Jiang, Hua; Ashcroft, Margaret; Radonovich, Michael; Duvall, Janet; Guillerm, Claire; Brady, John N.

2000-01-01

p53 plays a key role in guarding cells against DNA damage and transformation. We previously demonstrated that the human T-cell lymphotropic virus type 1 (HTLV-1) Tax can inactivate p53 transactivation function in lymphocytes. The present study demonstrates that in T cells, Tax-induced p53 inactivation is dependent upon NF-κB activation. Analysis of Tax mutants demonstrated that Tax inactivation of p53 function correlates with the ability of Tax to induce NF-κB but not p300 binding or CREB transactivation. The Tax-induced p53 inactivation can be overcome by overexpression of a dominant IκB mutant. Tax-NF-κB-induced p53 inactivation is not due to p300 squelching, since overexpression of p300 does not recover p53 activity in the presence of Tax. Further, using wild-type and p65 knockout mouse embryo fibroblasts (MEFs), we demonstrate that the p65 subunit of NF-κB is critical for Tax-induced p53 inactivation. While Tax can inactivate endogenous p53 function in wild-type MEFs, it fails to inactivate p53 function in p65 knockout MEFs. Importantly, Tax-induced p53 inactivation can be restored by expression of p65 in the knockout MEFs. Finally, we present evidence that phosphorylation of serines 15 and 392 correlates with inactivation of p53 by Tax in T cells. This study provides evidence that the divergent NF-κB proliferative and p53 cell cycle arrest pathways may be cross-regulated at several levels, including posttranslational modification of p53. PMID:10779327

High hydrostatic pressure inactivation of Lactobacillus plantarum cells in (O/W)-emulsions is independent from cell surface hydrophobicity and lipid phase parameters

NASA Astrophysics Data System (ADS)

Kafka, T. A.; Reitermayer, D.; Lenz, C. A.; Vogel, R. F.

2017-07-01

Inactivation efficiency of high hydrostatic pressure (HHP) processing of food is strongly affected by food matrix composition. We investigated effects of fat on HHP inactivation of spoilage-associated Lactobacillus (L.) plantarum strains using defined oil-in-water (O/W)-emulsion model systems. Since fat-mediated effects on HHP inactivation could be dependent on interactions between lipid phase and microbial cells, three major factors possibly influencing such interactions were considered, that is, cell surface hydrophobicity, presence and type of surfactants, and oil droplet size. Pressure tolerance varied noticeably among L. plantarum strains and was independent of cell surface hydrophobicity. We showed that HHP inactivation of all strains tended to be more effective in presence of fat. The observation in both, surfactant-stabilized and surfactant-free (O/W)-emulsion, indicates that cell surface hydrophobicity is no intrinsic pressure resistance factor. In contrast to the presence of fat per se, surfactant type and oil droplet size did not affect inactivation efficiency.

Control of the red tide dinoflagellate Cochlodinium polykrikoides by ozone in seawater.

PubMed

Shin, Minjung; Lee, Hye-Jin; Kim, Min Sik; Park, Noh-Back; Lee, Changha

2017-02-01

The inactivation of C. polykrikoides, a red tide dinoflagellate, by ozonation was investigated in seawater by monitoring numbers of viable and total cells. Parameters affecting the inactivation efficacy of C. polykrikoides such as the ozone dose, initial cell concentration, pH, and temperature were examined. The viable cell number rapidly decreased in the initial stage of the reaction (mostly in 1-2 min), whereas the decrease in total cell number was relatively slow and steady. Increasing ozone dose and decreasing initial cell concentration increased the inactivation efficacy of C. polykrikoides, while increasing pH and temperature decreased the cell inactivation efficacy. The addition of humic acid (a promoter for the ozone decomposition) inhibited the inactivation of C. polykrikoides, whereas bicarbonate ion (an inhibitor for the ozone decomposition) accelerated the C. polykrikoides inactivation. Observations regarding the effects of pH, temperature, humic acid, and bicarbonate ion collectively indicate that the inactivation of C. polykrikoides by ozonation is mainly attributed to oxidative cell damages by molecular ozone, rather than by hydroxyl radical, produced during the ozone decomposition. At high ozone dose (e.g., 5 mg/L), hypobromous acid formed by the reaction of bromide with ozone may partially contribute to cell inactivation. The use of ozone of less than 1 mg/L produced 0.75-2.03 μg/L bromate. Copyright © 2016 Elsevier Ltd. All rights reserved.

Green tea polyphenol, (-)-epigallocatechin-3-gallate, induces toxicity in human skin cancer cells by targeting β-catenin signaling.

PubMed

Singh, Tripti; Katiyar, Santosh K

2013-12-01

The green tea polyphenol, (-)-epigallocatechin-3-gallate (EGCG), has been shown to have anti-carcinogenic effects in several skin tumor models, and efforts are continued to investigate the molecular targets responsible for its cytotoxic effects to cancer cells. Our recent observation that β-catenin is upregulated in skin tumors suggested the possibility that the anti-skin carcinogenic effects of EGCG are mediated, at least in part, through its effects on β-catenin signaling. We have found that treatment of the A431 and SCC13 human skin cancer cell lines with EGCG resulted in reduced cell viability and increased cell death and that these cytotoxic effects were associated with inactivation of β-catenin signaling. Evidence of EGCG-induced inactivation of β-catenin included: (i) reduced accumulation of nuclear β-catenin; (ii) enhanced levels of casein kinase1α, reduced phosphorylation of glycogen synthase kinase-3β, and increased phosphorylation of β-catenin on critical serine(45,33/37) residues; and (iii) reduced levels of matrix metalloproteinase (MMP)-2 and MMP-9, which are down-stream targets of β-catenin. Treatment of cells with prostaglandin E2 (PGE2) enhanced the accumulation of β-catenin and enhanced β-catenin signaling. Treatment with either EGCG or an EP2 antagonist (AH6809) reduced the PGE2-enhanced levels of cAMP, an upstream regulator of β-catenin. Inactivation of β-catenin by EGCG resulted in suppression of cell survival signaling proteins. siRNA knockdown of β-catenin in A431 and SCC13 cells reduced cell viability. Collectively, these data suggest that induction of cytotoxicity in skin cancer cells by EGCG is mediated by targeting of β-catenin signaling and that the β-catenin signaling is upregulated by inflammatory mediators. © 2013.

Molecular Viability Testing of UV-Inactivated Bacteria.

PubMed

Weigel, Kris M; Nguyen, Felicia K; Kearney, Moira R; Meschke, John S; Cangelosi, Gerard A

2017-05-15

PCR is effective in detecting bacterial DNA in samples, but it is unable to differentiate viable bacteria from inactivated cells or free DNA fragments. New PCR-based analytical strategies have been developed to address this limitation. Molecular viability testing (MVT) correlates bacterial viability with the ability to rapidly synthesize species-specific rRNA precursors (pre-rRNA) in response to brief nutritional stimulation. Previous studies demonstrated that MVT can assess bacterial inactivation by chlorine, serum, and low-temperature pasteurization. Here, we demonstrate that MVT can detect inactivation of Escherichia coli , Aeromonas hydrophila , and Enterococcus faecalis cells by UV irradiation. Some UV-inactivated E. coli cells transiently retained the ability to synthesize pre-rRNA postirradiation (generating false-positive MVT results), but this activity ceased within 1 h following UV exposure. Viable but transiently undetectable (by culture) E. coli cells were consistently detected by MVT. An alternative viability testing method, viability PCR (vPCR), correlates viability with cell envelope integrity. This method did not distinguish viable bacteria from UV-inactivated bacteria under some conditions, indicating that the inactivated cells retained intact cell envelopes. MVT holds promise as a means to rapidly assess microbial inactivation by UV treatment. IMPORTANCE UV irradiation is increasingly being used to disinfect water, food, and other materials for human use. Confirming the effectiveness of UV disinfection remains a challenging task. In particular, microbiological methods that rely on rapid detection of microbial DNA can yield misleading results, due to the detection of remnant DNA associated with dead microbial cells. This report describes a novel method that rapidly distinguishes living microbial cells from dead microbial cells after UV disinfection. Copyright © 2017 American Society for Microbiology.

Transient removal of alkaline zones after excitation of Chara cells is associated with inactivation of high conductance in the plasmalemma

PubMed Central

2009-01-01

The action potential (AP) of excitable plant cells is a multifunctional physiological signal. Its generation in characean algae suppresses the pH banding for 15–30 min and enhances the heterogeneity of spatial distribution of photosynthetic activity. This suppression is largely due to the cessation of H+ influx (OH− efflux) in the alkaline cell regions. Measurements of local pH and membrane conductance in individual space-clamped alkaline zones (small cell areas bathed in an isolated pool of external medium) showed that the AP generation is followed by the transient disappearance of alkaline zone in parallel with a large decrease in membrane conductance. These changes, specific to alkaline zones, were only observed under continuous illumination following a relaxation period of at least 15 min after previous excitation. The excitation of dark-adapted cells produced no conductance changes in the post-excitation period. The results indicate that the origin of alkaline zones in characean cells is not due to operation of electroneutral H+/HCO3− symport or OH−/HCO3− antiport. It is concluded that the membrane excitation is associated with inactivation of plasmalemma high conductance in the alkaline cell regions. PMID:19820298

Factors affecting inactivation of Moraxell-Acinetobacter cells in an irradiation process. [/sup 137/Cs

DOE Office of Scientific and Technical Information (OSTI.GOV)

Firstenberg-Eden, R.; Rowley, D.B.; Shattuck, G.E.

1980-09-01

The effect of various stages of the irradiation processing of beef on the injury and inactivation of radiation-resistant Moraxella-Acinetobactor cells was studied. Moraxella-Acinetobacter cells were more resistant to heat inactivation and injury when heated in meat with salts (0.75% NaCl and 0.375% sodium tripolyphosphate) than in meat without salts. These salts had no effect on radiation resistance. Heated cells were more sensitive to radiation inactivation and injury than unheated cells. After repair, the cells regained their resistance to both NaCl and irradiation. Freezing and storage at -40/sup 0/C for 14 days had only a slight effect on either unstressed ormore » heat-stressed cells.« less

Development and Testing of a Method for Validating Chemical Inactivation of Ebola Virus.

PubMed

Alfson, Kendra J; Griffiths, Anthony

2018-03-13

Complete inactivation of infectious Ebola virus (EBOV) is required before a sample may be removed from a Biosafety Level 4 laboratory. The United States Federal Select Agent Program regulations require that procedures used to demonstrate chemical inactivation must be validated in-house to confirm complete inactivation. The objective of this study was to develop a method for validating chemical inactivation of EBOV and then demonstrate the effectiveness of several commonly-used inactivation methods. Samples containing infectious EBOV ( Zaire ebolavirus ) in different matrices were treated, and the sample was diluted to limit the cytopathic effect of the inactivant. The presence of infectious virus was determined by assessing the cytopathic effect in Vero E6 cells. Crucially, this method did not result in a loss of infectivity in control samples, and we were able to detect less than five infectious units of EBOV ( Zaire ebolavirus ). We found that TRIzol LS reagent and RNA-Bee inactivated EBOV in serum; TRIzol LS reagent inactivated EBOV in clarified cell culture media; TRIzol reagent inactivated EBOV in tissue and infected Vero E6 cells; 10% neutral buffered formalin inactivated EBOV in tissue; and osmium tetroxide vapors inactivated EBOV on transmission electron microscopy grids. The methods described herein are easily performed and can be adapted to validate inactivation of viruses in various matrices and by various chemical methods.

X-inactivation and X-reactivation: epigenetic hallmarks of mammalian reproduction and pluripotent stem cells.

PubMed

Payer, Bernhard; Lee, Jeannie T; Namekawa, Satoshi H

2011-08-01

X-chromosome inactivation is an epigenetic hallmark of mammalian development. Chromosome-wide regulation of the X-chromosome is essential in embryonic and germ cell development. In the male germline, the X-chromosome goes through meiotic sex chromosome inactivation, and the chromosome-wide silencing is maintained from meiosis into spermatids before the transmission to female embryos. In early female mouse embryos, X-inactivation is imprinted to occur on the paternal X-chromosome, representing the epigenetic programs acquired in both parental germlines. Recent advances revealed that the inactive X-chromosome in both females and males can be dissected into two elements: repeat elements versus unique coding genes. The inactive paternal X in female preimplantation embryos is reactivated in the inner cell mass of blastocysts in order to subsequently allow the random form of X-inactivation in the female embryo, by which both Xs have an equal chance of being inactivated. X-chromosome reactivation is regulated by pluripotency factors and also occurs in early female germ cells and in pluripotent stem cells, where X-reactivation is a stringent marker of naive ground state pluripotency. Here we summarize recent progress in the study of X-inactivation and X-reactivation during mammalian reproduction and development as well as in pluripotent stem cells.

Inactivation of Influenza A virus, Adenovirus, and Cytomegalovirus with PAXgene tissue fixative and formalin.

PubMed

Kap, Marcel; Arron, Georgina I; Loibner, M; Hausleitner, Anja; Siaulyte, Gintare; Zatloukal, Kurt; Murk, Jean-Luc; Riegman, Peter

2013-08-01

Formalin fixation is known to inactivate most viruses in a vaccine production context, but nothing is published about virus activity in tissues treated with alternative, non-crosslinking fixatives. We used a model assay based on cell culture to test formalin and PAXgene Tissue fixative for their virus-inactivating abilities. MDCK, A549, and MRC-5 cells were infected with Influenza A virus, Adenovirus, and Cytomegalovirus, respectively. When 75% of the cells showed a cytopathic effect (CPE), the cells were harvested and incubated for 15 min, or 1, 3, 6, or 24 hours, with PBS (positive control), 4% formalin, or PAXgene Tissue Fix. The cells were disrupted and the released virus was used to infect fresh MDCK, A549, and MRC-5 cells cultured on cover slips in 24-well plates. The viral cultures were monitored for CPE and by immunocytochemistry (ICC) to record viral replication and infectivity. Inactivation of Adenovirus by formalin occurred after 3 h, while Influenza A virus as well as Cytomegalovirus were inactivated by formalin after 15 min. All three virus strains were inactivated by PAXgene Tissue fixative after 15 min. We conclude that PAXgene Tissue fixative is at least as effective as formalin in inactivating infectivity of Influenza A virus, Adenovirus, and Cytomegalovirus.

Cell-Type Specific Inactivation of Hippocampal CA1 Disrupts Location-Dependent Object Recognition in the Mouse

ERIC Educational Resources Information Center

Haettig, Jakob; Sun, Yanjun; Wood, Marcelo A.; Xu, Xiangmin

2013-01-01

The allatostatin receptor (AlstR)/ligand inactivation system enables potent regulation of neuronal circuit activity. To examine how different cell types participate in memory formation, we have used this system through Cre-directed, cell-type specific expression in mouse hippocampal CA1 in vivo and examined functional effects of inactivation of…

Mammalian sex chromosomes. III. Activity of pseudoautosomal steroid sulfatase enzyme during spermatogenesis in Mus musculus.

PubMed

Raman, R; Das, P

1991-09-01

Parallel to the inactivation of the X chromosome in somatic cells of female, the male X in mammals is rendered inactive during spermatogenesis. Pseudoautosomal genes, those present on the X-Y meiotically pairable region of male, escape inactivation in female soma. It is suggested, but not demonstrated, that they may also be refractory to the inactivation signal in male germ cells. We have assayed activity of the enzyme steroid sulfatase, product of a pseudoautosomal gene, in testicular cells of the mouse and shown its presence in premeiotic, meiotic (pachytene), and postmeiotic (spermatid) cell types. It appears that, as in females, pseudoautosomal genes may escape inactivation in male germ cells also.

RB inactivation in keratin 18 positive thymic epithelial cells promotes non-cell autonomous T cell hyperproliferation in genetically engineered mice.

PubMed

Song, Yurong; Sullivan, Teresa; Klarmann, Kimberly; Gilbert, Debra; O'Sullivan, T Norene; Lu, Lucy; Wang, Sophie; Haines, Diana C; Van Dyke, Terry; Keller, Jonathan R

2017-01-01

Thymic epithelial cells (TEC), as part of thymic stroma, provide essential growth factors/cytokines and self-antigens to support T cell development and selection. Deletion of Rb family proteins in adult thymic stroma leads to T cell hyperplasia in vivo. To determine whether deletion of Rb specifically in keratin (K) 18 positive TEC was sufficient for thymocyte hyperplasia, we conditionally inactivated Rb and its family members p107 and p130 in K18+ TEC in genetically engineered mice (TgK18GT121; K18 mice). We found that thymocyte hyperproliferation was induced in mice with Rb inactivation in K18+ TEC, while normal T cell development was maintained; suggesting that inactivation of Rb specifically in K18+ TEC was sufficient and responsible for the phenotype. Transplantation of wild type bone marrow cells into mice with Rb inactivation in K18+ TEC resulted in donor T lymphocyte hyperplasia confirming the non-cell autonomous requirement for Rb proteins in K18+ TEC in regulating T cell proliferation. Our data suggests that thymic epithelial cells play an important role in regulating lymphoid proliferation and thymus size.

Calculation of Heavy Ion Inactivation and Mutation Rates in Radial Dose Model of Track Structure

NASA Technical Reports Server (NTRS)

Cucinotta, Francis A.; Wilson, John W.; Shavers, Mark R.; Katz, Robert

1997-01-01

In the track structure model, the inactivation cross section is found by summing an inactivation probability over all impact parameters from the ion to the sensitive sites within the cell nucleus. The inactivation probability is evaluated by using the dose response of the system to gamma rays and the radial dose of the ions and may be equal to unity at small impact parameters. We apply the track structure model to recent data with heavy ion beams irradiating biological samples of E. Coli, B. Subtilis spores, and Chinese hamster (V79) cells. Heavy ions have observed cross sections for inactivation that approach and sometimes exceed the geometric size of the cell nucleus. We show how the effects of inactivation may be taken into account in the evaluation of the mutation cross sections in the track structure model through correlation of sites for gene mutation and cell inactivation. The model is fit to available data for HPRT (hypoxanthine guanine phosphoribosyl transferase) mutations in V79 cells, and good agreement is found. Calculations show the high probability for mutation by relativistic ions due to the radial extension of ions track from delta rays. The effects of inactivation on mutation rates make it very unlikely that a single parameter such as LET (linear energy transfer) can be used to specify radiation quality for heavy ion bombardment.

Programmed Cell Death During Caenorhabditis elegans Development

PubMed Central

Conradt, Barbara; Wu, Yi-Chun; Xue, Ding

2016-01-01

Programmed cell death is an integral component of Caenorhabditis elegans development. Genetic and reverse genetic studies in C. elegans have led to the identification of many genes and conserved cell death pathways that are important for the specification of which cells should live or die, the activation of the suicide program, and the dismantling and removal of dying cells. Molecular, cell biological, and biochemical studies have revealed the underlying mechanisms that control these three phases of programmed cell death. In particular, the interplay of transcriptional regulatory cascades and networks involving multiple transcriptional regulators is crucial in activating the expression of the key death-inducing gene egl-1 and, in some cases, the ced-3 gene in cells destined to die. A protein interaction cascade involving EGL-1, CED-9, CED-4, and CED-3 results in the activation of the key cell death protease CED-3, which is tightly controlled by multiple positive and negative regulators. The activation of the CED-3 caspase then initiates the cell disassembly process by cleaving and activating or inactivating crucial CED-3 substrates; leading to activation of multiple cell death execution events, including nuclear DNA fragmentation, mitochondrial elimination, phosphatidylserine externalization, inactivation of survival signals, and clearance of apoptotic cells. Further studies of programmed cell death in C. elegans will continue to advance our understanding of how programmed cell death is regulated, activated, and executed in general. PMID:27516615

Cancer cell uptake behavior of Au nanoring and its localized surface plasmon resonance induced cell inactivation

NASA Astrophysics Data System (ADS)

Chu, Che-Kuan; Tu, Yi-Chou; Chang, Yu-Wei; Chu, Chih-Ken; Chen, Shih-Yang; Chi, Ting-Ta; Kiang, Yean-Woei; Yang, Chih-Chung

2015-02-01

Au nanorings (NRIs), which have the localized surface plasmon resonance (LSPR) wavelength around 1058 nm, either with or without linked antibodies, are applied to SAS oral cancer cells for cell inactivation through the LSPR-induced photothermal effect when they are illuminated by a laser of 1065 nm in wavelength. Different incubation times of cells with Au NRIs are considered for observing the variations of cell uptake efficiency of Au NRI and the threshold laser intensity for cell inactivation. In each case of incubation time, the cell sample is washed for evaluating the total Au NRI number per cell adsorbed and internalized by the cells based on inductively coupled plasma mass spectrometry measurement. Also, the Au NRIs remaining on cell membrane are etched with KI/I2 solution to evaluate the internalized Au NRI number per cell. The threshold laser intensities for cell inactivation before washout, after washout, and after KI/I2 etching are calibrated from the circular area sizes of inactivated cells around the illuminated laser spot center with various laser power levels. By using Au NRIs with antibodies, the internalized Au NRI number per cell increases monotonically with incubation time up to 24 h. However, the number of Au NRI remaining on cell membrane reaches a maximum at 12 h in incubation time. The cell uptake behavior of an Au NRI without antibodies is similar to that with antibodies except that the uptake NRI number is significantly smaller and the incubation time for the maximum NRI number remaining on cell membrane is delayed to 20 h. By comparing the threshold laser intensities before and after KI/I2 etching, it is found that the Au NRIs remaining on cell membrane cause more effective cancer cell inactivation, when compared with the internalized Au NRIs.

Proof-of-principle that a decoy virus protects oncolytic measles virus against neutralizing antibodies.

PubMed

Xu, Chun; Goß, Annika Verena; Dorneburg, Carmen; Debatin, Klaus-Michael; Wei, Jiwu; Beltinger, Christian

2018-01-01

Attenuated oncolytic measles virus (OMV) is a promising antitumor agent in early-phase clinical trials. However, pre-existing immunity against measles might be a hurdle for OMV therapy. OMV was inactivated with short-wavelength ultraviolet light (UV-C). Loss of replication and oncolytic activity of UV-inactivated OMV were confirmed by tissue culture infective dose 50 (TCID 50 ) assay using Vero cells and by flow cytometry using Jurkat cells. An enzyme-linked immunosorbent assay was performed to verify that UV-inactivated OMV remained antigenic. Different doses of UV-inactivated OMV were pre-cultured in media supplemented with measles immune serum. The mixture was transferred to Jurkat cells and active OMV was added. Active OMV-induced death of Jurkat cells was monitored by flow cytometry. UV-inactivation abrogates OMV replication while maintaining its antigenicity. UV-inactivated OMV sequesters pre-existing anti-MV antibodies in Jurkat cell culture, thereby protecting active OMV from neutralization and preserving oncolytic activity. We prove the principle that a non-replicating OMV can serve as a "decoy" for neutralizing anti-MV antibodies, thereby allowing antitumor activity of OMV.

Bmi1 represses Ink4a/Arf and Hox genes to regulate stem cells in the rodent incisor

PubMed Central

Biehs, Brian; Hu, Jimmy Kuang-Hsien; Strauli, Nicolas B.; Sangiorgi, Eugenio; Jung, Heekyung; Heber, Ralf-Peter; Ho, Sunita; Goodwin, Alice F.; Dasen, Jeremy S.; Capecchi, Mario R.; Klein, Ophir D.

2013-01-01

The polycomb group gene Bmi1 is required for maintenance of adult stem cells in many organs1, 2. Inactivation of Bmi1 leads to impaired stem cell self-renewal due to deregulated gene expression. One critical target of BMI1 is Ink4a/Arf, which encodes the cell cycle inhibitors p16ink4a and p19Arf3. However, deletion of Ink4a/Arf only partially rescues Bmi1 null phenotypes4, indicating that other important targets of BMI1 exist. Here, using the continuously-growing mouse incisor as a model system, we report that Bmi1 is expressed by incisor stem cells and that deletion of Bmi1 resulted in fewer stem cells, perturbed gene expression, and defective enamel production. Transcriptional profiling revealed that Hox expression is normally repressed by BMI1 in the adult, and functional assays demonstrated that BMI1-mediated repression of Hox genes preserves the undifferentiated state of stem cells. As Hox gene upregulation has also been reported in other systems when Bmi1 is inactivated1, 2, 5–7, our findings point to a general mechanism whereby BMI1-mediated repression of Hox genes is required for the maintenance of adult stem cells and for prevention of inappropriate differentiation. PMID:23728424

Membrane Permeabilization in Relation to Inactivation Kinetics of Lactobacillus Species due to Pulsed Electric Fields

PubMed Central

Wouters, Patrick C.; Bos, Ad P.; Ueckert, Joerg

2001-01-01

Membrane permeabilization due to pulsed electric field (PEF) treatment of gram-positive Lactobacillus cells was investigated by using propidium iodide uptake and single-cell analysis with flow cytometry. Electric field strength, energy input, treatment time, and growth phase affected membrane permeabilization of Lactobacillus plantarum during PEF treatment. A correlation between PEF inactivation and membrane permeabilization of L. plantarum cells was demonstrated, whereas no relationship was observed between membrane permeabilization and heat inactivation. The same results were obtained with a Lactobacillus fermentum strain, but the latter organism was more PEF resistant and exhibited less membrane permeabilization, indicating that various bacteria have different responses to PEF treatment. While membrane permeabilization was the main factor involved in the mechanism of inactivation, the growth phase and the acidity of the environment also influenced inactivation. By using flow cytometry it was possible to sort cells in the L. plantarum population based on different cell sizes and shapes, and the results were confirmed by image analysis. An apparent effect of morphology on membrane permeabilization was observed, and larger cells were more easily permeabilized than smaller cells. In conclusion, our results indicate that the ability of PEF treatment to cause membrane permeabilization is an important factor in determining inactivation. This finding should have an effect on the final choice of the processing parameters used so that all microorganisms can be inactivated and, consequently, on the use of PEF treatment as an alternative method for preserving food products. PMID:11425727

Eradication of high viable loads of Listeria monocytogenes contaminating food-contact surfaces

PubMed Central

de Candia, Silvia; Morea, Maria; Baruzzi, Federico

2015-01-01

This study demonstrates the efficacy of cold gaseous ozone treatments at low concentrations in the eradication of high Listeria monocytogenes viable cell loads from glass, polypropylene, stainless steel, and expanded polystyrene food-contact surfaces. Using a step by step approach, involving the selection of the most resistant strain-surface combinations, 11 Listeria sp. strains resulted inactivated by a continuous ozone flow at 1.07 mg m-3 after 24 or 48 h of cold incubation, depending on both strain and surface evaluated. Increasing the inoculum level to 9 log CFU coupon-1, the best inactivation rate was obtained after 48 h of treatment at 3.21 mg m-3 ozone concentration when cells were deposited onto stainless steel and expanded polystyrene coupons, resulted the most resistant food-contact surfaces in the previous assays. The addition of naturally contaminated meat extract to a high load of L. monocytogenes LMG 23775 cells, the most resistant strain out of the 11 assayed Listeria sp. strains, led to its complete inactivation after 4 days of treatment. To the best of our knowledge, this is the first report describing the survival of L. monocytogenes and the effect of ozone treatment under cold storage conditions on expanded polystyrene, a commonly used material in food packaging. The results of this study could be useful for reducing pathogen cross-contamination phenomena during cold food storage. PMID:26236306

Validation of the safety of MDCK cells as a substrate for the production of a cell-derived influenza vaccine.

PubMed

Onions, David; Egan, William; Jarrett, Ruth; Novicki, Deborah; Gregersen, Jens-Peter

2010-09-01

Cell culture-based production methods may assist in meeting increasing demand for seasonal influenza vaccines and developing production flexibility required for addressing influenza pandemics. MDCK-33016PF cells are used in propagation of a cell-based seasonal influenza vaccine (Optaflu); but, like most continuous cell lines, can grow in immunocompromised mice to produce tumors. It is, therefore, essential that no residual cells remain within the vaccine, that cell lysates or DNA are not oncogenic, and that the cell substrate does not contain oncogenic viruses or oncogenic DNA. Multiple, redundant processes ensure the safety of influenza vaccines produced in MDCK-33016PF cells. The probability of a residual cell being present in a dose of vaccine is approximately 1 in 10(34). Residual MDCK-DNA is < or =10 ng per dose and the ss-propiolactone used to inactivate influenza virus results in reduction of detectable DNA to less than 200 base pairs (bp). Degenerate PCR and specific PCR confirm exclusion of oncogenic viruses. The manufacturing process has been validated for its capacity to remove and inactivate viruses. We conclude that the theoretical risks arising from manufacturing seasonal influenza vaccine using MDCK-33016PF cells are reduced to levels that are effectively zero by the multiple, orthogonal processes used during production. Copyright 2010 The International Association for Biologicals. Published by Elsevier Ltd. All rights reserved.

Pathogen inactivation of human serum facilitates its clinical use for islet cell culture and subsequent transplantation.

PubMed

Ståhle, Magnus U; Brandhorst, Daniel; Korsgren, Olle; Knutson, Folke

2011-01-01

Serum is regarded as an essential supplement to promote survival and growth of cells during culture. However, the potential risk of transmitting diseases disqualifies the use of serum for clinical cell therapy in most countries. Hence, most clinical cell therapy programs have replaced human serum with human serum albumin, which can result in inferior quality of released cell products. Photochemical treatment of different blood products utilizing Intercept® technology has been shown to inactivate a broad variety of pathogens of RNA and DNA origin. The present study assesses the feasibility of using pathogen-inactivated, blood group-compatible serum for use in human pancreatic islet culture. Isolated human islets were cultured at 37°C for 3-4 days in CMRL 1066 supplemented with 10% of either pathogen-inactivated or nontreated human serum. Islet quality assessment included glucose-stimulated insulin release (perifusion), ADP/ATP ratio, cytokine expression, and posttransplant function in diabetic nude mice. No differences were found between islets cultured in pathogen-inactivated or control serum regarding stimulated insulin release, intracellular insulin content, and ADP/ATP ratio. Whether media was supplemented with treated or nontreated serum, islet expression of IL-6, IL-8, MCP-1, or tissue factor was not affected. The final diabetes-reversal rate of mice receiving islets cultured in pathogen-inactivated or nontreated serum was 78% and 87%, respectively (NS). As reported here, pathogen-inactivated human serum does not affect viability or functional integrity of cultured human islets. The implementation of this technology for RNA- and DNA-based pathogen inactivation should enable reintroduction of human serum for clinical cell therapy.

Single-hit mechanism of tumour cell killing by radiation.

PubMed

Chapman, J D

2003-02-01

To review the relative importance of the single-hit mechanism of radiation killing for tumour response to 1.8-2.0 Gy day(-1) fractions and to low dose-rate brachytherapy. Tumour cell killing by ionizing radiation is well described by the linear-quadratic equation that contains two independent components distinguished by dose kinetics. Analyses of tumour cell survival curves that contain six or more dose points usually provide good estimates of the alpha- and beta-inactivation coefficients. Superior estimates of tumour cell intrinsic radiosensitivity are obtained when synchronized populations are employed. The characteristics of single-hit inactivation of tumour cells are reviewed and compared with the characteristics of beta-inactivation. Potential molecular targets associated with single-hit inactivation are discussed along with strategies for potentiating cell killing by this mechanism. The single-hit mechanism of tumour cell killing shows no dependence on dose-rate and, consequently, no evidence of sublethal damage repair. It is uniquely potentiated by high linear-energy-transfer radiation, exhibits a smaller oxygen enhancement ratio and exhibits a larger indirect effect by hydroxyl radicals than the beta-mechanism. alpha-inactivation coefficients vary slightly throughout interphase but mitotic cells exhibit extremely high alpha-coefficients in the range of those observed for lymphocytes and some repair-deficient cells. Evidence is accumulating to suggest that chromatin in compacted form could be a radiation-hypersensitive target associated with single-hit radiation killing. Analyses of tumour cell survival curves demonstrate that it is the single-hit mechanism (alpha) that determines the majority of cell killing after doses of 2Gy and that this mechanism is highly variable between tumour cell lines. The characteristics of single-hit inactivation are qualitatively and quantitatively distinct from those of beta-inactivation. Compacted chromatin in tumour cells should be further investigated as a radiation-hypersensitive target that could be modulated for therapeutic advantage.

Inactivation Strategies for Clostridium perfringens Spores and Vegetative Cells

PubMed Central

Talukdar, Prabhat K.; Udompijitkul, Pathima; Hossain, Ashfaque

2016-01-01

ABSTRACT Clostridium perfringens is an important pathogen to human and animals and causes a wide array of diseases, including histotoxic and gastrointestinal illnesses. C. perfringens spores are crucial in terms of the pathogenicity of this bacterium because they can survive in a dormant state in the environment and return to being live bacteria when they come in contact with nutrients in food or the human body. Although the strategies to inactivate C. perfringens vegetative cells are effective, the inactivation of C. perfringens spores is still a great challenge. A number of studies have been conducted in the past decade or so toward developing efficient inactivation strategies for C. perfringens spores and vegetative cells, which include physical approaches and the use of chemical preservatives and naturally derived antimicrobial agents. In this review, different inactivation strategies applied to control C. perfringens cells and spores are summarized, and the potential limitations and challenges of these strategies are discussed. PMID:27795314

Proof-of-principle that a decoy virus protects oncolytic measles virus against neutralizing antibodies

PubMed Central

Dorneburg, Carmen; Debatin, Klaus-Michael; Wei, Jiwu; Beltinger, Christian

2018-01-01

Background Attenuated oncolytic measles virus (OMV) is a promising antitumor agent in early-phase clinical trials. However, pre-existing immunity against measles might be a hurdle for OMV therapy. Methods OMV was inactivated with short-wavelength ultraviolet light (UV-C). Loss of replication and oncolytic activity of UV-inactivated OMV were confirmed by tissue culture infective dose 50 (TCID50) assay using Vero cells and by flow cytometry using Jurkat cells. An enzyme-linked immunosorbent assay was performed to verify that UV-inactivated OMV remained antigenic. Different doses of UV-inactivated OMV were pre-cultured in media supplemented with measles immune serum. The mixture was transferred to Jurkat cells and active OMV was added. Active OMV-induced death of Jurkat cells was monitored by flow cytometry. Results UV-inactivation abrogates OMV replication while maintaining its antigenicity. UV-inactivated OMV sequesters pre-existing anti-MV antibodies in Jurkat cell culture, thereby protecting active OMV from neutralization and preserving oncolytic activity. Conclusion We prove the principle that a non-replicating OMV can serve as a “decoy” for neutralizing anti-MV antibodies, thereby allowing antitumor activity of OMV. PMID:29750140

Photosensitized inactivation of infectious blood-borne human parasites

NASA Astrophysics Data System (ADS)

Judy, Millard M.; Sogandares-Bernal, Franklin M.; Matthews, James Lester

1995-05-01

Blood-borne viruses and protozoan parasites that are infectious to humans pose risk world-wide of infection transmission through blood and blood product transfusion. Blood-borne infectious viruses include human immunodeficiency virus (HIV-I), which causes AIDS; hepatitis C virus, which can cause chronic hepatitis; and cytomegalovirus, which can be dangerous to immunocompromised patients, e.g., the newborn, transplant recipients, and AIDS patients. Infectious blood-borne protozoan parasites include Trypanosoma cruzi, which causes Chagas' disease, endemic throughout Central and South America; the Trypanosoma species causing African sleeping sickness endemic in Central Africa; and Plasmodium falciparum, which causes malignant and increasingly drug- resistant human malaria prevalent throughout the tropics. Some researchers have focused on using photosensitizers to inactivate HIV-I and other viruses in whole blood, packed red cells, and platelet concentrates without compromising blood product function. Our group previously has reported photosensitized in vitro inactivation of P. falciparum and the mouse malaria organism Plasmodium berghei in whole blood using hematoporphyrin derivative (HPD) and of T. cruzi using benzoporphyrin derivatives BPDMA and BPDDA, dihematoporphyrin ether (DHE), and hydroxyethylvinyldeuteroporphyrin (HEVD). These results suggest that continued investigation is warranted to evaluate the potential for photosensitized inactivation of blood-borne parasites in blood banking.

Differential Mechanism of Escherichia coli Inactivation by (+)-Limonene as a Function of Cell Physiological State and Drug's Concentration

PubMed Central

Chueca, Beatriz; Pagán, Rafael; García-Gonzalo, Diego

2014-01-01

(+)-limonene is a lipophilic antimicrobial compound, extracted from citrus fruits' essential oils, that is used as a flavouring agent and organic solvent by the food industry. A recent study has proposed a common and controversial mechanism of cell death for bactericidal antibiotics, in which hydroxyl radicals ultimately inactivated cells. Our objective was to determine whether the mechanism of Escherichia coli MG1655 inactivation by (+)-limonene follows that of bactericidal antibiotics. A treatment with 2,000 μL/L (+)-limonene inactivated 4 log10 cycles of exponentially growing E. coli cells in 3 hours. On one hand, an increase of cell survival in the ΔacnB mutant (deficient in a TCA cycle enzyme), or in the presence of 2,2′-dipyridyl (inhibitor of Fenton reaction by iron chelation), thiourea, or cysteamine (hydroxyl radical scavengers) was observed. Moreover, the ΔrecA mutant (deficient in an enzyme involved in SOS response to DNA damage) was more sensitive to (+)-limonene. Thus, this indirect evidence indicates that the mechanism of exponentially growing E. coli cells inactivation by 2,000 μL/L (+)-limonene is due to the TCA cycle and Fenton-mediated hydroxyl radical formation that caused oxidative DNA damage, as observed for bactericidal drugs. However, several differences have been observed between the proposed mechanism for bactericidal drugs and for (+)-limonene. In this regard, our results demonstrated that E. coli inactivation was influenced by its physiological state and the drug's concentration: experiments with stationary-phase cells or 4,000 μL/L (+)-limonene uncovered a different mechanism of cell death, likely unrelated to hydroxyl radicals. Our research has also shown that drug's concentration is an important factor influencing the mechanism of bacterial inactivation by antibiotics, such as kanamycin. These results might help in improving and spreading the use of (+)-limonene as an antimicrobial compound, and in clarifying the controversy about the mechanism of inactivation by bactericidal antibiotics. PMID:24705541

Differential mechanism of Escherichia coli Inactivation by (+)-limonene as a function of cell physiological state and drug's concentration.

PubMed

Chueca, Beatriz; Pagán, Rafael; García-Gonzalo, Diego

2014-01-01

(+)-limonene is a lipophilic antimicrobial compound, extracted from citrus fruits' essential oils, that is used as a flavouring agent and organic solvent by the food industry. A recent study has proposed a common and controversial mechanism of cell death for bactericidal antibiotics, in which hydroxyl radicals ultimately inactivated cells. Our objective was to determine whether the mechanism of Escherichia coli MG1655 inactivation by (+)-limonene follows that of bactericidal antibiotics. A treatment with 2,000 μL/L (+)-limonene inactivated 4 log10 cycles of exponentially growing E. coli cells in 3 hours. On one hand, an increase of cell survival in the ΔacnB mutant (deficient in a TCA cycle enzyme), or in the presence of 2,2'-dipyridyl (inhibitor of Fenton reaction by iron chelation), thiourea, or cysteamine (hydroxyl radical scavengers) was observed. Moreover, the ΔrecA mutant (deficient in an enzyme involved in SOS response to DNA damage) was more sensitive to (+)-limonene. Thus, this indirect evidence indicates that the mechanism of exponentially growing E. coli cells inactivation by 2,000 μL/L (+)-limonene is due to the TCA cycle and Fenton-mediated hydroxyl radical formation that caused oxidative DNA damage, as observed for bactericidal drugs. However, several differences have been observed between the proposed mechanism for bactericidal drugs and for (+)-limonene. In this regard, our results demonstrated that E. coli inactivation was influenced by its physiological state and the drug's concentration: experiments with stationary-phase cells or 4,000 μL/L (+)-limonene uncovered a different mechanism of cell death, likely unrelated to hydroxyl radicals. Our research has also shown that drug's concentration is an important factor influencing the mechanism of bacterial inactivation by antibiotics, such as kanamycin. These results might help in improving and spreading the use of (+)-limonene as an antimicrobial compound, and in clarifying the controversy about the mechanism of inactivation by bactericidal antibiotics.

Domain model for Ca2(+)-inactivation of Ca2+ channels at low channel density.

PubMed Central

Sherman, A; Keizer, J; Rinzel, J

1990-01-01

The "shell" model for Ca2(+)-inactivation of Ca2+ channels is based on the accumulation of Ca2+ in a macroscopic shell beneath the plasma membrane. The shell is filled by Ca2+ entering through open channels, with the elevated Ca2+ concentration inactivating both open and closed channels at a rate determined by how fast the shell is filled. In cells with low channel density, the high concentration Ca2+ "shell" degenerates into a collection of nonoverlapping "domains" localized near open channels. These domains form rapidly when channels open and disappear rapidly when channels close. We use this idea to develop a "domain" model for Ca2(+)-inactivation of Ca2+ channels. In this model the kinetics of formation of an inactivated state resulting from Ca2+ binding to open channels determines the inactivation rate, a mechanism identical with that which explains single-channel recordings on rabbit-mesenteric artery Ca2+ channels (Huang Y., J. M. Quayle, J. F. Worley, N. B. Standen, and M. T. Nelson. 1989. Biophys. J. 56:1023-1028). We show that the model correctly predicts five important features of the whole-cell Ca2(+)-inactivation for mouse pancreatic beta-cells (Plants, T. D. 1988. J. Physiol. 404:731-747) and that Ca2(+)-inactivation has only minor effects on the bursting electrical activity of these cells. PMID:2174274

Familial skewed X inactivation: a molecular trait associated with high spontaneous-abortion rate maps to Xq28.

PubMed Central

Pegoraro, E; Whitaker, J; Mowery-Rushton, P; Surti, U; Lanasa, M; Hoffman, E P

1997-01-01

We report a family ascertained for molecular diagnosis of muscular dystrophy in a young girl, in which preferential activation (> or = 95% of cells) of the paternal X chromosome was seen in both the proband and her mother. To determine the molecular basis for skewed X inactivation, we studied X-inactivation patterns in peripheral blood and/or oral mucosal cells from 50 members of this family and from a cohort of normal females. We found excellent concordance between X-inactivation patterns in blood and oral mucosal cell nuclei in all females. Of the 50 female pedigree members studied, 16 showed preferential use (> or = 95% cells) of the paternal X chromosome; none of 62 randomly selected females showed similarly skewed X inactivation was maternally inherited in this family. A linkage study using the molecular trait of skewed X inactivation as the scored phenotype localized this trait to Xq28 (DXS1108; maximum LOD score [Zmax] = 4.34, recombination fraction [theta] = 0). Both genotyping of additional markers and FISH of a YAC probe in Xq28 showed a deletion spanning from intron 22 of the factor VIII gene to DXS115-3. This deletion completely cosegregated with the trait (Zmax = 6.92, theta = 0). Comparison of clinical findings between affected and unaffected females in the 50-member pedigree showed a statistically significant increase in spontaneous-abortion rate in the females carrying the trait (P < .02). To our knowledge, this is the first gene-mapping study of abnormalities of X-inactivation patterns and is the first association of a specific locus for recurrent spontaneous abortion in a cytogenetically normal family. The involvement of this locus in cell lethality, cell-growth disadvantage, developmental abnormalities, or the X-inactivation process is discussed. Images Figure 4 Figure 7 PMID:9245997

Effects of heat-treatment on plasma rich in growth factors-derived autologous eye drop.

PubMed

Anitua, E; Muruzabal, F; De la Fuente, M; Merayo-Lloves, J; Orive, G

2014-02-01

We have developed and characterized a new type of plasma rich in growth factors (PRGF) derived eye-drop therapy for patients suffering from autoimmune diseases. To determine the concentration of several growth factors, proteins, immunoglobulins and complement activity of the heat-inactivated eye-drop and to study its biological effects on cell proliferation and migration of different ocular surface cells, blood from healthy donors was collected, centrifuged and PRGF was prepared avoiding the buffy coat. The half volume of the obtained plasma supernatant from each donor was heat-inactivated at 56 °C for 1 h (heat-inactivated PRGF). The concentration of several proteins involved on corneal wound healing, immunoglubolins G, M and E and functional integrity of the complement system assayed by CH50 test were determined. The proliferative and migratory potential of inactivated and non-inactivated PRGF eye drops were assayed on corneal epithelial cells (HCE), keratocytes (HK) and conjunctival fibroblasts (HConF). Heat-inactivated PRGF preserves the content of most of the proteins and morphogens involved in its wound healing effects while reduces drastically the content of IgE and complement activity. Heat-inactivated PRGF eye drops increased proliferation and migration potential of ocular surface cells with regard to PRGF showing significant differences on proliferation and migration rate of HCE and HConF respectively. In summary, heat-inactivation of PRGF eye drops completely reduced complement activity and deceased significantly the presence of IgE, maintaining the biological activity of PRGF on ocular surface cells. Copyright © 2013 Elsevier Ltd. All rights reserved.

New insight into the residual inactivation of Microcystis aeruginosa by dielectric barrier discharge

PubMed Central

Li, Lamei; Zhang, Hong; Huang, Qing

2015-01-01

We report the new insight into the dielectric barrier discharge (DBD) induced inactivation of Microcystis aeruginosa, the dominant algae which caused harmful cyanobacterial blooms in many developing countries. In contrast with the previous work, we employed flow cytometry to examine the algal cells, so that we could assess the dead and living cells with more accuracy, and distinguish an intermediate state of algal cells which were verified as apoptotic. Our results showed that the numbers of both dead and apoptotic cells increased with DBD treatment delay time, and hydrogen peroxide produced by DBD was the main reason for the time-delayed inactivation effect. However, apart from the influence of hydrogen peroxide, the DBD-induced initial injures on the algal cells during the discharge period also played a considerable role in the inactivation of the DBD treated cells, as indicated by the measurement of intracellular reactive oxygen species (ROS) inside the algal cells. We therefore propose an effective approach to utilization of non-thermal plasma technique that makes good use of the residual inactivation effect to optimize the experimental conditions in terms of discharge time and delay time, so that more efficient treatment of cyanobacterial blooms can be achieved. PMID:26347270

Systematic approach to in-depth understanding of photoelectrocatalytic bacterial inactivation mechanisms by tracking the decomposed building blocks.

PubMed

Sun, Hongwei; Li, Guiying; Nie, Xin; Shi, Huixian; Wong, Po-Keung; Zhao, Huijun; An, Taicheng

2014-08-19

A systematic approach was developed to understand, in-depth, the mechanisms involved during the inactivation of bacterial cells using photoelectrocatalytic (PEC) processes with Escherichia coli K-12 as the model microorganism. The bacterial cells were found to be inactivated and decomposed primarily due to attack from photogenerated H2O2. Extracellular reactive oxygen species (ROSs), such as H2O2, may penetrate into the bacterial cell and cause dramatically elevated intracellular ROSs levels, which would overwhelm the antioxidative capacity of bacterial protective enzymes such as superoxide dismutase and catalase. The activities of these two enzymes were found to decrease due to the ROSs attacks during PEC inactivation. Bacterial cell wall damage was then observed, including loss of cell membrane integrity and increased permeability, followed by the decomposition of cell envelope (demonstrated by scanning electronic microscope images). One of the bacterial building blocks, protein, was found to be oxidatively damaged due to the ROSs attacks, as well. Leakage of cytoplasm and biomolecules (bacterial building blocks such as proteins and nucleic acids) were evident during prolonged PEC inactivation process. The leaked cytoplasmic substances and cell debris could be further degraded and, ultimately, mineralized with prolonged PEC treatment.

Subcellular mechanism of Escherichia coli inactivation during electrochemical disinfection with boron-doped diamond anode: A comparative study of three electrolytes.

PubMed

Long, Yujiao; Ni, Jinren; Wang, Zuhui

2015-11-01

Although the identification of effective oxidant species has been extensively studied, yet the subcellular mechanism of bacterial inactivation has never been clearly elucidated in electrochemical disinfection processes. In this study, subcellular mechanism of Escherichia coli inactivation during electrochemical disinfection was revealed in terms of comprehensive factors such as cell morphology, total organic components, K(+) leakage, membrane permeability, lipid peroxidation, membrane potential, membrane proteins, intracellular enzyme, cellular ATP level and DNA. The electrolysis was conducted with boron-doped diamond anode in three electrolytes including chloride, sulfate and phosphate. Results demonstrated that cell inactivation was mainly attributed to damage to the intracellular enzymatic systems in chloride solution. In sulfate solution, certain essential membrane proteins like the K(+) ion transport systems were eliminated. Thus, the pronounced K(+) leakage from cytosol resulted in gradual collapse of the membrane potential, which would hinder the subcellular localization of cell division-related proteins as well as ATP synthesis and thereby lead to the bacterial inactivation. Remarkable lipid peroxidation was observed, while the intracellular damage was negligible. In phosphate solution, the cells sequentially underwent overall destruction as a whole cell with no captured intermediate state, during which the organic components of the cells were mostly subjected to mineralization. This study provided a thorough insight into the bacterial inactivation mechanism on the subcellular level. Copyright © 2015 Elsevier Ltd. All rights reserved.

The Use of Plant-Derived Ribosome Inactivating Proteins in Immunotoxin Development: Past, Present and Future Generations

PubMed Central

Rust, Aleksander; Partridge, Lynda J.; Davletov, Bazbek

2017-01-01

Ribosome inactivating proteins (RIPs) form a class of toxins that was identified over a century ago. They continue to fascinate scientists and the public due to their very high activity and long-term stability which might find useful applications in the therapeutic killing of unwanted cells but can also be used in acts of terror. We will focus our review on the canonical plant-derived RIPs which display ribosomal RNA N-glycosidase activity and irreversibly inhibit protein synthesis by cleaving the 28S ribosomal RNA of the large 60S subunit of eukaryotic ribosomes. We will place particular emphasis on therapeutic applications and the generation of immunotoxins by coupling antibodies to RIPs in an attempt to target specific cells. Several generations of immunotoxins have been developed and we will review their optimisation as well as their use and limitations in pre-clinical and clinical trials. Finally, we endeavour to provide a perspective on potential future developments for the therapeutic use of immunotoxins. PMID:29076988

Comparison of proteomic profiles of serum, plasma, and modified media supplements used for cell culture and expansion

PubMed Central

Ayache, Saleh; Panelli, Monica C; Byrne, Karen M; Slezak, Stefanie; Leitman, Susan F; Marincola, Francesco M; Stroncek, David F

2006-01-01

Background The culture and expansion of human cells for clinical use requires the presence of human serum or plasma in culture media. Although these supplements have been extensively characterized in their chemical composition, only recently it has been possible to provide by high throughput protein analysis, a comprehensive profile of the soluble factors contributing to cell survival. This study analyzed and compared the presence of 100 proteins including chemokines, cytokines and soluble factors in six different types of media supplements: serum, plasma, recalcified plasma, heat inactivated serum, heat inactivated plasma and heat inactivated recalcified plasma. Methods Serum, plasma, recalcified plasma, and heat inactivated supplements were prepared from ten healthy subjects. The levels of 100 soluble factors were measured in each sample using a multiplexed ELISA assay and compared by Eisen hierarchical clustering analysis. Results A comparison of serum and plasma levels of soluble factors found that 2 were greater in plasma but 18 factors were greater in serum including 11 chemokines. The levels of only four factors differed between recalcified plasma and plasma. Heat inactivation had the greatest effect on soluble factors. Supervised Eisen hierarchical clustering indicated that the differences between heat inactivated supplements and those that were not were greater than the differences within these two groups. The levels of 36 factors differed between heat inactivated plasma and plasma. Thirty one of these factors had a lower concentration in heat inactivated plasma including 12 chemokines, 4 growth factors, 4 matrix metalloproteases, and 3 adhesion molecules. Heat inactivated decalcified plasma is often used in place of heat inactivated serum and the levels of 19 soluble factors differed between these two supplements. Conclusion Our report provides a comprehensive protein profile of serum, plasma recalcified plasma, and heat inactivated supplements. This profile represents a qualitative and quantitative database that can aid in the selection of the appropriate blood derived supplement for human cell cultures with special requirements. PMID:17020621

Inactivation Strategies for Clostridium perfringens Spores and Vegetative Cells.

PubMed

Talukdar, Prabhat K; Udompijitkul, Pathima; Hossain, Ashfaque; Sarker, Mahfuzur R

2017-01-01

Clostridium perfringens is an important pathogen to human and animals and causes a wide array of diseases, including histotoxic and gastrointestinal illnesses. C. perfringens spores are crucial in terms of the pathogenicity of this bacterium because they can survive in a dormant state in the environment and return to being live bacteria when they come in contact with nutrients in food or the human body. Although the strategies to inactivate C. perfringens vegetative cells are effective, the inactivation of C. perfringens spores is still a great challenge. A number of studies have been conducted in the past decade or so toward developing efficient inactivation strategies for C. perfringens spores and vegetative cells, which include physical approaches and the use of chemical preservatives and naturally derived antimicrobial agents. In this review, different inactivation strategies applied to control C. perfringens cells and spores are summarized, and the potential limitations and challenges of these strategies are discussed. Copyright © 2016 American Society for Microbiology.

Chromophore-assisted laser inactivation of alpha- and gamma-tubulin SNAP-tag fusion proteins inside living cells.

PubMed

Keppler, Antje; Ellenberg, Jan

2009-02-20

Chromophore-assisted laser inactivation (CALI) can help to unravel localized activities of target proteins at defined times and locations within living cells. Covalent SNAP-tag labeling of fusion proteins with fluorophores such as fluorescein is a fast and highly specific tool to attach the photosensitizer to its target protein in vivo for selective inactivation of the fusion protein. Here, we demonstrate the effectiveness and specificity of SNAP-tag-based CALI by acute inactivation of alpha-tubulin and gamma-tubulin SNAP-tag fusions during live imaging assays of cell division. Singlet oxygen is confirmed as the reactive oxygen species that leads to loss of fusion protein function. The major advantage of SNAP-tag CALI is the ease, reliability, and high flexibility in labeling: the genetically encoded protein tag can be covalently labeled with various dyes matching the experimental requirements. This makes SNAP-tag CALI a very useful tool for rapid inactivation of tagged proteins in living cells.

HMG-CoA reductase is negatively associated with PCV2 infection and PCV2-induced apoptotic cell death.

PubMed

Yang, Xin; Ouyang, Hongsheng; Chen, Fuwang; Pang, Daxing; Dong, Meichen; Yang, Susu; Liu, Xiaoyun; Peng, Zhiyuan; Wang, Fei; Zhang, Xiao; Ren, Linzhu

2014-06-01

We examined the role of HMG-CoA reductase (HMGCR) during porcine circovirus 2 (PCV2) infection. The results demonstrated that levels of endogenous HMGCR were not significantly different in PCV2-infected cells and mock-infected cells. However, the level of phosphorylated HMGCR, an inactivated form of HMGCR, was increased in PCV2-infected cells. Furthermore, HMGCR was upregulated by overexpression, silenced by siRNA or inactivated using its dominant-negative form in PK-15 cells. The results showed that PCV2 infection was inhibited by HMGCR overexpression, whereas it was significantly increased in HMGCR-silenced cells and HMGCR inhibitor-treated cells. Moreover, there was a robust apoptotic response at 48 h post-infection (p.i.) in HMGCR-inactivated cells, and this response was significantly greater than that observed in PK-15 cells. A modest apoptotic response was also observed in HMGCR-silenced cells. Caspase-3 activity was also analysed in PCV2-infected cells at 48 h p.i. As expected, caspase-3 activity was significantly increased in HMGCR-inactivated and -silenced cells compared with PK-15 cells. PCV2 replication was dose-dependently increased in HMGCR-inactivated cells when treated with increasing amounts of caspase-3 inhibitor. Altogether, HMGCR was negatively associated with PCV2 infection and PCV2-induced apoptotic cell death. These data demonstrated that HMGCR can be used as a candidate target for PCV2 disease control and antivirus research. Furthermore, the cells generated in this study can be used to evaluate the potential effects of HMGCR on PCV2 replication. © 2014 The Authors.

Chromosomal inactivation of Bacillus subtilis exfusants: a prokaryotic model of epigenetic regulation.

PubMed

Grandjean, V; Hauck, Y; Beloin, C; Le Hégarat, F; Hirschbein, L

1998-01-01

Epigenetic mechanisms are not exclusively reserved to eukaryotic organisms. They are also observed in prokaryotes. As described first by Hotchkiss and Gabor, protoplast fusion between strains of Bacillus subtilis produces heterodiploid cells. Heterodiploidy is associated with the inactivation of one of the chromosomes. To study the physical structure of the fusion product and the molecular mechanisms of inactivation, we constructed heterodiploid clones containing two chromosomes labeled by a NotI restriction fragment length polymorphism. In the progeny, we identified haploid recombinant clones that contain a chromosome carrying large regions of inactivated DNA. Studies of both recombinants of the latter kind and heterodiploid cells indicated that chromosomal inactivation was not determined by alteration of the inactivated nucleotide sequence, but was probably due to a modification in the structure of the bacterial chromatin.

In situ real-time measurement of physical characteristics of airborne bacterial particles

NASA Astrophysics Data System (ADS)

Jung, Jae Hee; Lee, Jung Eun

2013-12-01

Bioaerosols, including aerosolized bacteria, viruses, and fungi, are associated with public health and environmental problems. One promising control method to reduce the harmful effects of bioaerosols is thermal inactivation via a continuous-flow high-temperature short-time (HTST) system. However, variations in bioaerosol physical characteristics - for example, the particle size and shape - during the continuous-flow inactivation process can change the transport properties in the air, which can affect particle deposition in the human respiratory system or the filtration efficiency of ventilation systems. Real-time particle monitoring techniques are a desirable alternative to the time-consuming process of microscopic analysis that is conventionally used in sampling and particle characterization. Here, we report in situ real-time optical scattering measurements of the physical characteristics of airborne bacteria particles following an HTST process in a continuous-flow system. Our results demonstrate that the aerodynamic diameter of bacterial aerosols decreases when exposed to a high-temperature environment, and that the shape of the bacterial cells is significantly altered. These variations in physical characteristics using optical scattering measurements were found to be in agreement with the results of scanning electron microscopy analysis.

Two pathogen reduction technologies--methylene blue plus light and shortwave ultraviolet light--effectively inactivate hepatitis C virus in blood products.

PubMed

Steinmann, Eike; Gravemann, Ute; Friesland, Martina; Doerrbecker, Juliane; Müller, Thomas H; Pietschmann, Thomas; Seltsam, Axel

2013-05-01

Contamination of blood products with hepatitis C virus (HCV) can cause infections resulting in acute and chronic liver diseases. Pathogen reduction methods such as photodynamic treatment with methylene blue (MB) plus visible light as well as irradiation with shortwave ultraviolet (UVC) light were developed to inactivate viruses and other pathogens in plasma and platelet concentrates (PCs), respectively. So far, their inactivation capacities for HCV have only been tested in inactivation studies using model viruses for HCV. Recently, a HCV infection system for the propagation of infectious HCV in cell culture was developed. Inactivation studies were performed with cell culture-derived HCV and bovine viral diarrhea virus (BVDV), a model for HCV. Plasma units or PCs were spiked with high titers of cell culture-grown viruses. After treatment of the blood units with MB plus light (Theraflex MB-Plasma system, MacoPharma) or UVC (Theraflex UV-Platelets system, MacoPharma), residual viral infectivity was assessed using sensitive cell culture systems. HCV was sensitive to inactivation by both pathogen reduction procedures. HCV in plasma was efficiently inactivated by MB plus light below the detection limit already by 1/12 of the full light dose. HCV in PCs was inactivated by UVC irradiation with a reduction factor of more than 5 log. BVDV was less sensitive to the two pathogen reduction methods. Functional assays with human HCV offer an efficient tool to directly assess the inactivation capacity of pathogen reduction procedures. Pathogen reduction technologies such as MB plus light treatment and UVC irradiation have the potential to significantly reduce transfusion-transmitted HCV infections. © 2012 American Association of Blood Banks.

21 CFR 866.5260 - Complement C3b inactivator immunological test system.

Code of Federal Regulations, 2010 CFR

2010-04-01

... HUMAN SERVICES (CONTINUED) MEDICAL DEVICES IMMUNOLOGY AND MICROBIOLOGY DEVICES Immunological Test Systems § 866.5260 Complement C3b inactivator immunological test system. (a) Identification. A complement... 21 Food and Drugs 8 2010-04-01 2010-04-01 false Complement C3b inactivator immunological test...

CDKN2A/p16 inactivation mechanisms and their relationship to smoke exposure and molecular features in non-small cell lung cancer

PubMed Central

Tam, Kit W.; Zhang, Wei; Soh, Junichi; Stastny, Victor; Chen, Min; Sun, Han; Thu, Kelsie; Rios, Jonathan J.; Yang, Chenchen; Marconett, Crystal N.; Selamat, Suhaida A.; Laird-Offringa, Ite A; Taguchi, Ayumu; Hanash, Samir; Shames, David; Ma, Xiaotu; Zhang, Michael Q; Lam, Wan L.; Gazdar, Adi

2013-01-01

Introduction CDKN2A(p16) inactivation is common in lung cancer and occurs via homozygous deletions (HD), methylation of promoter region, or point mutations. While p16 promoter methylation has been linked to KRAS mutation and smoking, the associations between p16 inactivation mechanisms and other common genetic mutations and smoking status are still controversial or unknown. Methods We determined all three p16 inactivation mechanisms using multiple methodologies for genomic status, methylation, RNA and protein expression, and correlated them with EGFR, KRAS, STK11 mutations and smoking status in 40 cell lines and 45 tumor samples of primary NSCLC. We also performed meta-analyses to investigate the impact of smoke exposure on p16 inactivation. Results p16 inactivation was the major mechanism of RB pathway perturbation in NSCLC, with HD being the most frequent method, followed by methylation and the rarer point mutations. Inactivating mechanisms were tightly correlated with loss of mRNA and protein expression. p16 inactivation occurred at comparable frequencies regardless of mutational status of EGFR, KRAS and STK11, however, the major inactivation mechanism of p16 varied. p16 methylation was linked to KRAS mutation but was mutually exclusive with EGFR mutation. Cell lines and tumor samples demonstrated similar results. Our meta-analyses confirmed a modest positive association between p16 promoter methylation and smoking. Conclusions Our results confirm that all of the inactivation mechanisms are truly associated with loss of gene product and identify specific associations between p16 inactivation mechanisms and other genetic changes and smoking status. PMID:24077454

Caspase-8 inactivation in T cells increases necroptosis and suppresses autoimmunity in Bim−/− mice

PubMed Central

Bohgaki, Toshiyuki; Mozo, Julien; Salmena, Leonardo; Matysiak-Zablocki, Elzbieta; Bohgaki, Miyuki; Sanchez, Otto; Strasser, Andreas

2011-01-01

Dysregulation of either the extrinsic or intrinsic apoptotic pathway can lead to various diseases including immune disorders and cancer. In addition to its role in the extrinsic apoptotic pathway, caspase-8 plays nonapoptotic functions and is essential for T cell homeostasis. The pro-apoptotic BH3-only Bcl-2 family member Bim is important for the intrinsic apoptotic pathway and its inactivation leads to autoimmunity that is further exacerbated by loss of function of the death receptor Fas. We report that inactivation of caspase-8 in T cells of Bim−/− mice restrained their autoimmunity and extended their life span. We show that, similar to caspase-8−/− T cells, Bim−/− T cells that also lack caspase-8 displayed elevated levels of necroptosis and that inhibition of this cell death process fully rescued the survival and proliferation of these cells. Collectively, our data demonstrate that inactivation of caspase-8 suppresses the survival and proliferative capacity of Bim−/− T cells and restrains autoimmunity in Bim−/− mice. PMID:22006951

An APC/C-Cdh1 Biosensor Reveals the Dynamics of Cdh1 Inactivation at the G1/S Transition.

PubMed

Ondracka, Andrej; Robbins, Jonathan A; Cross, Frederick R

2016-01-01

B-type cyclin-dependent kinase activity must be turned off for mitotic exit and G1 stabilization. B-type cyclin degradation is mediated by the anaphase-promoting complex/cyclosome (APC/C); during and after mitotic exit, APC/C is dependent on Cdh1. Cdh1 is in turn phosphorylated and inactivated by cyclin-CDK at the Start transition of the new cell cycle. We developed a biosensor to assess the cell cycle dynamics of APC/C-Cdh1. Nuclear exit of the G1 transcriptional repressor Whi5 is a known marker of Start; APC/C-Cdh1 is inactivated 12 min after Whi5 nuclear exit with little measurable cell-to-cell timing variability. Multiple phosphorylation sites on Cdh1 act in a redundant manner to repress its activity. Reducing the number of phosphorylation sites on Cdh1 can to some extent be tolerated for cell viability, but it increases variability in timing of APC/C-Cdh1 inactivation. Mutants with minimal subsets of phosphorylation sites required for viability exhibit striking stochasticity in multiple responses including budding, nuclear division, and APC/C-Cdh1 activity itself. Multiple cyclin-CDK complexes, as well as the stoichiometric inhibitor Acm1, contribute to APC/C-Cdh1 inactivation; this redundant control is likely to promote rapid and reliable APC/C-Cdh1 inactivation immediately following the Start transition.

Silver nanoparticle-E. coli colloidal interaction in water and effect on E. coli survival.

PubMed

Dror-Ehre, A; Mamane, H; Belenkova, T; Markovich, G; Adin, A

2009-11-15

Silver nanoparticles exhibit antibacterial properties via bacterial inactivation and growth inhibition. The mechanism is not yet completely understood. This work was aimed at elucidating the effect of silver nanoparticles on inactivation of Escherichia coli, by studying particle-particle interactions in aqueous suspensions. Stable, molecularly capped, positively or negatively charged silver nanoparticles were mixed at 1 to 60microgmL(-1) with suspended E. coli cells to examine their effect on inactivation of the bacteria. Gold nanoparticles with the same surfactant were used as a control, being of similar size but made up of a presumably inert metal. Log reduction of 5log(10) and complete inactivation were obtained with the silver nanoparticles while the gold nanoparticles did not show any inactivation ability. The effect of molecularly capped nanoparticles on E. coli survival was dependent on particle number. Log reduction of E. coli was associated with the ratio between the number of nanoparticles and the initial bacterial cell count. Electrostatic attraction or repulsion mechanisms in silver nanoparticle-E. coli cell interactions did not contribute to the inactivation process.

Oxygenated monoterpenes citral and carvacrol cause oxidative damage in Escherichia coli without the involvement of tricarboxylic acid cycle and Fenton reaction.

PubMed

Chueca, Beatriz; Pagán, Rafael; García-Gonzalo, Diego

2014-10-17

Oxygenated monoterpenes citral and carvacrol are common constituents of many essential oils (EOs) that have been extensively studied as antimicrobial agents but whose mechanisms of microbial inactivation have not been totally elucidated. A recent study described a mechanism of Escherichia coli death for (+)-limonene, a hydrocarbon monoterpene also frequently present in EOs, similar to the common mechanism proposed for bactericidal antibiotics. This mechanism involves the formation of Fenton-mediated hydroxyl radical, a reactive oxygen species (ROS), via tricarboxylic acid (TCA) cycle, which would ultimately inactivate cells. Our objective was to determine whether E. coli MG1655 inactivation by citral and carvacrol follows a similar mechanism of cell death. Challenging experiments with 300μL/L citral and 100μL/L carvacrol inactivated at least 2.5log10cycles of exponentially growing cells in 3h under aerobic conditions. The presence of thiourea (an ROS scavenger) reduced cell inactivation in 2log10cycles, demonstrating the role of ROS in cell death. Decreased resistance of a ΔrecA mutant (deficient in an enzyme involved in SOS response to DNA damage) indicated that citral and carvacrol caused oxidative damage to DNA. Although the mechanism of E. coli inactivation by carvacrol and citral was similarly mediated by ROS, their formation did not follow the same pathways described for (+)-limonene and bactericidal drugs because neither Fenton reaction nor NADH production via the TCA cycle was involved in cell death. Moreover, further experiments demonstrated antimicrobial activity of citral and carvacrol in anaerobic environments without the involvement of ROS. As a consequence, cell death by carvacrol and citral in anaerobiosis follows a different mechanism than that observed under aerobic conditions. These results demonstrated a different mechanism of inactivation by citral and carvacrol with regard to (+)-limonene and bactericidal antibiotics, indicating the complexity of the mechanisms of bacterial inactivation among EO constituents. Advancements in the description of these mechanisms will help in extending and improving the use of these compounds as natural antimicrobials. Copyright © 2014 Elsevier B.V. All rights reserved.

Role of chromosome 3p12-p21 tumour suppressor genes in clear cell renal cell carcinoma: analysis of VHL dependent and VHL independent pathways of tumorigenesis.

PubMed

Martinez, A; Fullwood, P; Kondo, K; Kishida, T; Yao, M; Maher, E R; Latif, F

2000-06-01

Chromosome 3p deletions and loss of heterozygosity (LOH) for 3p markers are features of clear cell renal cell carcinoma but are rare in non-clear cell renal cell carcinoma. The VHL tumour suppressor gene, which maps to 3p25, is a major gatekeeper gene for clear cell renal cell carcinoma and is inactivated in most sporadic cases of this disease. However, it has been suggested that inactivation of other 3p tumour suppressor genes might be crucial for clear cell renal cell carcinoma tumorigenesis, with inactivation (VHL negative) and without inactivation (VHL positive) of the VHL tumour suppressor gene. This study set out to investigate the role of non-VHL tumour suppressor genes in VHL negative and VHL positive clear cell renal cell carcinoma. Eighty two clear cell renal cell carcinomas of known VHL inactivation status were analysed for LOH at polymorphic loci within the candidate crucial regions for chromosome 3p tumour suppressor genes (3p25, LCTSGR1 at 3p21.3, LCTSGR2 at 3p12 and at 3p14.2). Chromosome 3p12-p21 LOH was frequent both in VHL negative and VHL positive clear cell renal cell carcinoma. However, although the frequency of 3p25 LOH in VHL negative clear cell renal cell carcinoma was similar to that at 3p12-p21, VHL positive tumours demonstrated significantly less LOH at 3p25 than at 3p12-p21. Although there was evidence of LOH for clear cell renal cell carcinoma tumour suppressor genes at 3p21, 3p14.2, and 3p12, both in VHL negative and VHL positive tumours, the major clear cell renal cell carcinoma LOH region mapped to 3p21.3, close to the lung cancer tumour suppressor gene region 1 (LCTSGR1). There was no association between tumour VHL status and tumour grade and stage. These findings further indicate that VHL inactivation is not sufficient to initiate clear cell renal cell carcinoma and that loss of a gatekeeper 3p21 tumour suppressor gene is a crucial event for renal cell carcinoma development in both VHL negative and VHL positive clear cell renal cell carcinoma.

The Activation and Inactivation of Mature CD4 T cells: A Case for Peripheral Self–Nonself Discrimination

PubMed Central

Bretscher, P A

2014-01-01

The establishment of central tolerance to most self-antigens results in a repertoire of mature peripheral lymphocytes specific for foreign and peripheral self-antigens. The framework that single, mature lymphocytes are inactivated by antigen, whereas their activation requires lymphocyte cooperation, accounts for diverse observations and incorporates a mechanism of peripheral tolerance. This framework accounts for the generalizations that the sustained activation by antigen of most B cells and CD8 T cells requires CD4 T helper cells; in the absence of CD4 T cells, antigen can inactivate these B cells and CD8 T cells. In this sense, CD4 T cells are the guardians of the fate of most B cells and CD8 T cells when they encounter antigen. I argue here that the single-lymphocyte/multiple-lymphocyte framework for the inactivation/activation of lymphocytes also applies to CD4 T cells. I consider within this framework a model for the activation/inactivation of CD4 T cells that is consistent with the large majority of contemporary observations, including significant clinical observations. I outline the grounds why I feel this model is more plausible than the contemporary and predominant pathogen-associated molecular pattern (PAMP) and Danger Models for CD4 T cell activation. These models are based upon what I consider the radical premise that self–nonself discrimination does not exist at the level of mature CD4 T cells. I explain why I feel this feature renders the PAMP and Danger Models somewhat implausible. The model I propose, in contrast, is conservative in that it embodies such a process of self–nonself discrimination. PMID:24684567

Inactivation of Escherichia coli by citral.

PubMed

Somolinos, M; García, D; Condón, S; Mackey, B; Pagán, R

2010-06-01

The aim was to evaluate (i) the resistance of Escherichia coli BJ4 to citral in a buffer system as a function of citral concentration, treatment medium pH, storage time and initial inoculum size, (ii) the role of the sigma factor RpoS on citral resistance of E. coli, (iii) the role of the cell envelope damage in the mechanism of microbial inactivation by citral and (iiii) possible synergistic effects of mild heat treatment and pulsed electric fields (PEF) treatment combined with citral. The initial inoculum size greatly affected the efficacy of citral against E. coli cells. Exposure to 200 microl l(-1) of citral at pH 4.0 for 24 h at 20 degrees C caused the inactivation of more than 5 log(10) cycles of cells starting at an inoculum size of 10(6) or 10(7) CFU ml(-1), whereas increasing the cell concentration to 10(9) CFU ml(-1) caused <1 log(10) cycle of inactivation. Escherichia coli showed higher resistance to citral at pH 4.0 than pH 7.0. The rpoS null mutant strain E. coli BJ4L1 was less resistant to citral than the wild-type strain. Occurrence of sublethal injury to both the cytoplasmic and outer membranes was demonstrated by adding sodium chloride or bile salts to the recovery media. The majority of sublethally injured cells by citral required energy and lipid synthesis for repair. A strongly synergistic lethal effect was shown by mild heat treatment combined with citral but the presence of citral during the application of a PEF treatment did not show any advantage. This work confirms that cell envelope damage is an important event in citral inactivation of bacteria, and it describes the key factors on the inactivation of E. coli cells by citral. Knowledge about the mechanism of microbial inactivation by citral helps establish successful combined preservation treatments.

The Transport and Inactivation Kinetics of Bacterial Lipopolysaccharide Influence its Immunological Potency in vivo1

PubMed Central

Lu, Mingfang; Munford, Robert S.

2011-01-01

The extraordinary potency and pathological relevance of Gram-negative bacterial lipopolysaccharides (LPSs) have made them very popular experimental agonists, yet little is known about what happens to these stimulatory molecules within animal tissues. We tracked fluorescent and radiolabeled-LPS from a subcutaneous inoculation site to its draining lymph nodes (DLN), blood and liver. Although we found FITC-labeled LPS in DLN within minutes of injection, drainage of radiolabeled LPS continued for more than six weeks. Within the DLN, most of the LPS was found in the subcapsular sinus or medulla, near or within lymphatic endothelial cells and CD169+ macrophages. Whereas most of the LPS seemed to pass through the DLN without entering B cell follicles, by 24 hrs after injection a small amount of LPS was found in the paracortex. In wildtype mice, ≥70% of the injected radiolabeled-LPS underwent inactivation by deacylation before it left the footpad; in animals that lacked acyloxyacyl hydrolase, the LPS-deacylating enzyme, prolonged drainage of fully acylated (active) LPS boosted polyclonal IgM and IgG3 antibody titers. LPS egress from a subcutaneous injection site thus occurred over many weeks and was mainly via lymphatic channels. Its immunological potency, as measured by its ability to stimulate polyclonal antibody production, was greatly influenced by the kinetics of both lymphatic drainage and enzymatic inactivation. PMID:21849675

Antimicrobial effects of gold/copper sulphide (Gold/Copper monosulfide) core/shell nanoparticles on Bacillus anthracis spores and cells

NASA Astrophysics Data System (ADS)

Addae, Ebenezer

Bacillus anthracis is a gram positive, rod shaped and spore forming bacteria. It causes anthrax, a deadly human and animal disease that can kill its victims in three days. The spores of B. anthracis can survive extreme environmental conditions for decades and germinate when exposed to proper conditions. Due to its potential as a bio-weapon, effective disinfectants that pose less harm to the environment and animals are urgently needed. Metal nanoparticles have the potential of killing microbial cells and spores. We present here the effect of Gold/Copper Sulphide core/shell (Au/CuS) nanoparticles on B. anthracis cells and spores. The results indicated that the continuous presence of 0.83 microM during the spore growth in nutrient medium completely inhibited spore outgrowth. Au/CuS nanoparticles at concentration of 4.15 μM completely inactivated B. anthracis cells (x 107) after 30 min of pre-treatment in any of the three buffers including water, PBS, and nutrient broth. However, the same and even higher concentrations of nanoparticles produce no significant spore (x 105) killing after 24 h of pre-treatment. SEM imaging, EDS analysis, and DNA extrusion experiments revealed that nanoparticles damaged the cell membrane causing DNA and cytosolic content efflux and eventually cell death. The study demonstrated the strong antimicrobial activity of Au/CuS nanoparticles to B. anthracis cells and revealed that Au/CuS NPs showed more effective inactivation effect against the cells than they did against the spores.

Live-cell imaging combined with immunofluorescence, RNA, or DNA FISH to study the nuclear dynamics and expression of the X-inactivation center.

PubMed

Pollex, Tim; Piolot, Tristan; Heard, Edith

2013-01-01

Differentiation of embryonic stem cells is accompanied by changes of gene expression and chromatin and chromosome dynamics. One of the most impressive examples for these changes is inactivation of one of the two X chromosomes occurring upon differentiation of mouse female embryonic stem cells. With a few exceptions, these events have been mainly studied in fixed cells. In order to better understand the dynamics, kinetics, and order of events during differentiation, one needs to employ live-cell imaging techniques. Here, we describe a combination of live-cell imaging with techniques that can be used in fixed cells (e.g., RNA FISH) to correlate locus dynamics or subnuclear localization with, e.g., gene expression. To study locus dynamics in female ES cells, we generated cell lines containing TetO arrays in the X-inactivation center, the locus on the X chromosome regulating X-inactivation, which can be visualized upon expression of TetR fused to fluorescent proteins. We will use this system to elaborate on how to generate ES cell lines for live-cell imaging of locus dynamics, how to culture ES cells prior to live-cell imaging, and to describe typical live-cell imaging conditions for ES cells using different microscopes. Furthermore, we will explain how RNA, DNA FISH, or immunofluorescence can be applied following live-cell imaging to correlate gene expression with locus dynamics.

Cold plasma technology: bactericidal effects on Geobacillus stearothermophilus and Bacillus cereus microorganisms.

PubMed

Morris, Angela D; McCombs, Gayle B; Akan, Tamer; Hynes, Wayne; Laroussi, Mounir; Tolle, Susan L

2009-01-01

Cold plasma, also known as Low Temperature Atmospheric Pressure Plasma (LTAPP) is a novel technology consisting of neutral and charged particles, including free radicals, which can be used to destroy or inactivate microorganisms. Research has been conducted regarding the effect of cold plasma on gram-positive bacteria; however, there is limited research regarding its ability to inactivate the spore-formers Geobacillus stearothermophilus and Bacillus cereus. The purpose of this study was to determine if cold plasma inactivates G. stearothermophilus and B. cereus vegetative cells and spores. Nine hundred eighty-one samples were included in this study (762 experimental and 219 controls). Experimental samples were exposed indirectly or directly to cold plasma, before plating and incubating for 16 hours. Control samples were not exposed to cold plasma. The percentage-kill and cell number reductions were calculated from Colony Forming Units (CFU). Data were statistically analyzed at the .05 level using one-way ANOVA, Kruskal Wallis and Tukey's tests. There was a statistically significant difference in the inactivation of G. stearothermophilus vegetative cells receiving indirect and direct exposure (p=0.0001 and p=0.0013, respectively), as well as for B. cereus vegetative cells and spores (p=0.0001 for direct and indirect). There was no statistically significant difference in the inactivation of G. stearothermophilus spores receiving indirect exposure (p=0.7208) or direct exposure (p=0.0835). Results demonstrate that cold plasma exposure effectively kills G. stearothermophilus vegetative cells and B. cereus vegetative cells and spores; however, G. stearothermophilus spores were not significantly inactivated.

Safety and immunogenicity of two inactivated poliovirus vaccines in combination with an acellular pertussis vaccine and diphtheria and tetanus toxoids in seventeen- to nineteen-month-old infants.

PubMed

Halperin, S A; Davies, H D; Barreto, L; Guasparini, R; Meekison, W; Humphreys, G; Eastwood, B J

1997-04-01

To compare the safety and immunity of an acellular pertussis vaccine containing pertussis toxoid, filamentous hemagglutinin, 69 kd protein, fimbriae 2 and 3 combined with diphtheria and tetanus toxoids given as single or separate injection with inactivated poliovirus vaccine (MRC-5-or Vero cell-derived) or live attenuated polio vaccine. A total of 425 healthy children between 17 and 19 months of age who were receiving the fourth dose of their routine immunization series were randomly allocated to receive either the acellular pertussis vaccine and oral poliovirus vaccine or one of two inactivated poliovirus vaccines as a combined injection or separate injections. Although minor adverse events were commonly reported, differences between the groups were few. Fever and decreased feeding were less common in recipients of live attenuated poliovirus vaccine than the combination vaccine containing MRC-5 cell-derived inactivated poliovirus vaccine. A significant antibody response was demonstrated in all groups against all the antigens contained in the vaccines. Antibodies against poliovirus were higher in the groups immunized with the inactivated poliovirus vaccine than the live attenuated vaccine. Anti-69 kd protein antibodies were higher in the group given the MRC-5 cell-derived inactivated poliovirus vaccine as a combined injection than in the group given the separate injection or the group immunized with the live attenuated poliovirus vaccine. The five-component acellular pertussis vaccine combined with diphtherid and tetanus toxoids is safe and immunogenic when combined with either MRC-5- or Vero cell-derived inactivated poliovirus vaccine. This will facilitate the implementation of acellular pertussis vaccine and the movement to inactivated poliovirus vaccine programs.

Distinct CD4+-T-cell responses to live and heat-inactivated Aspergillus fumigatus conidia.

PubMed

Rivera, Amariliz; Van Epps, Heather L; Hohl, Tobias M; Rizzuto, Gabrielle; Pamer, Eric G

2005-11-01

Aspergillus fumigatus is an important fungal pathogen that causes invasive pulmonary disease in immunocompromised hosts. Respiratory exposure to A. fumigatus spores also causes allergic bronchopulmonary aspergillosis, a Th2 CD4+-T-cell-mediated disease that accompanies asthma. The microbial factors that influence the differentiation of A. fumigatus-specific CD4+ T lymphocytes into Th1 versus Th2 cells remain incompletely defined. We therefore examined CD4+-T-cell responses of immunologically intact mice to intratracheal challenge with live or heat-inactivated A. fumigatus spores. Live but not heat-inactivated fungal spores resulted in recruitment of gamma interferon (IFN-gamma)-producing, fungus-specific CD4+ T cells to lung airways, achieving A. fumigatus-specific frequencies exceeding 5% of total CD4+ T cells. While heat-inactivated spores did not induce detectable levels of IFN-gamma-producing, A. fumigatus-specific CD4+ T cells in the airways, they did prime CD4+ T-cell responses in draining lymph nodes that produced greater amounts of interleukin 4 (IL-4) and IL-13 than T cells responding to live conidia. While immunization with live fungal spores induced antibody responses, we found a marked decrease in isotype-switched, A. fumigatus-specific antibodies in sera of mice following immunization with heat-inactivated spores. Our studies demonstrate that robust Th1 T-cell and humoral responses are restricted to challenge with fungal spores that have the potential to germinate and cause invasive infection. How the adaptive immune system distinguishes between metabolically active and inactive fungal spores remains an important question.

Coadministration of Recombinant Adenovirus Expressing GM-CSF with Inactivated H5N1 Avian Influenza Vaccine Increased the Immune Responses and Protective Efficacy Against a Wild Bird Source of H5N1 Challenge.

PubMed

Wang, Xiangwei; Wang, Xinglong; Jia, Yanqing; Wang, Chongyang; Tang, Qiuxia; Han, Qingsong; Xiao, Sa; Yang, Zengqi

2017-10-01

Wild birds play a key role in the spread of avian influenza virus (AIV). There is a continual urgent requirement for AIV vaccines to address the ongoing genetic changes of AIV. In the current study, we trialed a novel AIV vaccine against the wild bird source of H5N1 type AIV with recombinant adenovirus expressing granulocyte monocyte colony-stimulating factor (GM-CSF) as an adjuvant. A total of 150-day-old commercial chicks, with AIV-maternal-derived antibody, were divided into 6 groups. The primary vaccination was performed at day 14 followed by a subsequent boosting and intramuscular challenge on day 28 and 42, respectively. Recombinant GM-CSF (rGM-CSF) expressed by adenovirus, named as rAd-GM-CSF, raised the hemagglutination inhibition (HI) titers (log 2 ) against AIV from 7.0 (vaccinate with inactivated vaccine alone) to 8.4 after booster immunization. Moreover, the rGM-CSF addition markedly increased the expression of interferon-γ, interleukin-4, and major histocompatibility complex-II in the lungs, compared with those immunized with inactivated vaccine alone on day 29, that is, 18 h post booster immunization. Following challenge, chicks inoculated with the inactivated AIV vaccine and rAd-GM-CSF together exhibited mild clinical signs and 62% survivals compared to 33% in the group immunized with inactivated AIV vaccine alone. Higher level of HI titers, immune related molecule expressions, and protection ratio demonstrates a good potential of rGM-CSF in improving humoral and cell mediated immune responses of inactivated AIV vaccines.

Effects of Background Fluid on the Efficiency of Inactivating Yeast with Non-Thermal Atmospheric Pressure Plasma

PubMed Central

Ryu, Young-Hyo; Kim, Yong-Hee; Lee, Jin-Young; Shim, Gun-Bo; Uhm, Han-Sup; Park, Gyungsoon; Choi, Eun Ha

2013-01-01

Non-thermal plasma at atmospheric pressure has been actively applied to sterilization. However, its efficiency for inactivating microorganisms often varies depending on microbial species and environments surrounding the microorganisms. We investigated the influence of environmental factors (surrounding media) on the efficiency of microbial inactivation by plasma using an eukaryotic model microbe, Saccharomyces cerevisiae, to elucidate the mechanisms for differential efficiency of sterilization by plasma. Yeast cells treated with plasma in water showed the most severe damage in viability and cell morphology as well as damage to membrane lipids, and genomic DNA. Cells in saline were less damaged compared to those in water, and those in YPD (Yeast extract, Peptone, Dextrose) were least impaired. HOG1 mitogen activated protein kinase was activated in cells exposed to plasma in water and saline. Inactivation of yeast cells in water and saline was due to the acidification of the solutions by plasma, but higher survival of yeast cells treated in saline may have resulted from the additional effect related to salt strength. Levels of hydroxyl radical (OH.) produced by plasma were the highest in water and the lowest in YPD. This may have resulted in differential inactivation of yeast cells in water, saline, and YPD by plasma. Taken together, our data suggest that the surrounding media (environment) can crucially affect the outcomes of yeast cell plasma treatment because plasma modulates vital properties of media, and the toxic nature of plasma can also be altered by the surrounding media. PMID:23799081

Inactivated probiotic Bacillus coagulans GBI-30 induces complex immune activating, anti-inflammatory, and regenerative markers in vitro

PubMed Central

Jensen, Gitte S; Cash, Howard A; Farmer, Sean; Keller, David

2017-01-01

Objective The aim of this study was to document the immune activating and anti-inflammatory effects of inactivated probiotic Bacillus coagulans GBI-30, 6086 (Staimune™) cells on human immune cells in vitro. Methods In vitro cultures of human peripheral blood mononuclear cells (PBMC) from healthy blood donors were treated with inactivated B. coagulans GBI-30, 6086 cells for 24 hours. After incubation, the PBMC were stained with fluorochrome-labeled monoclonal antibodies for CD3, CD56, and CD69 to monitor cellular activation by flow cytometry. The culture supernatants were tested for cytokine profile using a 27-plex Luminex array, including pro- and anti-inflammatory cytokines, chemokines, and growth factors. Results Inactivated B. coagulans GBI-30, 6086 cells induced the CD69 early activation marker on CD3+ CD56− T lymphocytes, CD3+ CD56+ NKT cells, CD3−CD56+ NK cells, and also some cells within the CD3−CD56− non-T non-NK cell subset. Culture supernatants showed robust increases in the immune-activating cytokines IL-1β, IL-6, IL-17A, and TNF-α. IFN-γ levels were increased, along with three chemokines, MCP-1, MIP-1α, and MIP-1β. The two anti-inflammatory cytokines IL-1ra and IL-10 showed increases, as well as the G-CSF growth factor involved in repair and stem cell biology. In contrast, GM-CSF levels showed a mild decrease, showing a highly selective growth factor response. Conclusion The inactivated B. coagulans GBI-30, 6086 cells activated human immune cells and altered the production of both immune activating and anti-inflammatory cytokines and chemokines. Of special importance is the novel demonstration of a selective upregulation of the G-CSF growth factor involved in postinjury and postinflammation repair and regeneration. This suggests that important immunogenic cell wall components, such as lipoteichoic acid, are undamaged after the inactivation and retain the complex beneficial biological activities previously demonstrated for the cell walls from live B. coagulans GBI-30, 6086 (GanedenBC30) probiotic bacteria. PMID:28848360

Inactivated probiotic Bacillus coagulans GBI-30 induces complex immune activating, anti-inflammatory, and regenerative markers in vitro.

PubMed

Jensen, Gitte S; Cash, Howard A; Farmer, Sean; Keller, David

2017-01-01

The aim of this study was to document the immune activating and anti-inflammatory effects of inactivated probiotic Bacillus coagulans GBI-30, 6086 (Staimune™) cells on human immune cells in vitro. In vitro cultures of human peripheral blood mononuclear cells (PBMC) from healthy blood donors were treated with inactivated B. coagulans GBI-30, 6086 cells for 24 hours. After incubation, the PBMC were stained with fluorochrome-labeled monoclonal antibodies for CD3, CD56, and CD69 to monitor cellular activation by flow cytometry. The culture supernatants were tested for cytokine profile using a 27-plex Luminex array, including pro- and anti-inflammatory cytokines, chemokines, and growth factors. Inactivated B. coagulans GBI-30, 6086 cells induced the CD69 early activation marker on CD3 + CD56 - T lymphocytes, CD3 + CD56 + NKT cells, CD3 - CD56 + NK cells, and also some cells within the CD3 - CD56 - non-T non-NK cell subset. Culture supernatants showed robust increases in the immune-activating cytokines IL-1β, IL-6, IL-17A, and TNF-α. IFN-γ levels were increased, along with three chemokines, MCP-1, MIP-1α, and MIP-1β. The two anti-inflammatory cytokines IL-1ra and IL-10 showed increases, as well as the G-CSF growth factor involved in repair and stem cell biology. In contrast, GM-CSF levels showed a mild decrease, showing a highly selective growth factor response. The inactivated B. coagulans GBI-30, 6086 cells activated human immune cells and altered the production of both immune activating and anti-inflammatory cytokines and chemokines. Of special importance is the novel demonstration of a selective upregulation of the G-CSF growth factor involved in postinjury and postinflammation repair and regeneration. This suggests that important immunogenic cell wall components, such as lipoteichoic acid, are undamaged after the inactivation and retain the complex beneficial biological activities previously demonstrated for the cell walls from live B. coagulans GBI-30, 6086 (GanedenBC30) probiotic bacteria.

Photodynamic inactivation of Candida albicans sensitized by tri- and tetra-cationic porphyrin derivatives.

PubMed

Cormick, M Paula; Alvarez, M Gabriela; Rovera, Marisa; Durantini, Edgardo N

2009-04-01

The photodynamic action of 5-(4-trifluorophenyl)-10,15,20-tris(4-trimethylammoniumphenyl)porphyrin iodide (TFAP(3+)) and 5,10,15,20-tetra(4-N,N,N-trimethylammonium phenyl)porphyrin p-tosylate (TMAP(4+)) has been studied in vitro on Candida albicans. The results of these cationic porphyrins were compared with those of 5,10,15,20-tetra(4-sulphonatophenyl)porphyrin (TPPS(4-)), which characterizes an anionic sensitizer. In vitro investigations show that these cationic porphyrins are rapidly bound to C. albicans cells, reaching a value of approximately 1.4 nmol/10(6) cells, when the cellular suspensions were incubated with 5 microM sensitizer for 30 min. In contrast, TPPS(4-) is poorly uptaken by yeast cells. The fluorescence spectra of these sensitizers into the cells confirm this behaviour. The amount of porphyrin binds to cells is dependent on both sensitizer concentrations (1-5 microM) and cells densities (10(6)-10(8) cells/mL). Photosensitized inactivation of C. albicans cellular suspensions increases with sensitizer concentration, causing a approximately 5 log decrease of cell survival, when the cultures are treated with 5 microM of cationic porphyrin and irradiated for 30 min. However, the photocytotoxicity decreases with an increase in the cell density, according to its low binding to cells. Under these conditions, the photodynamic activity of TFAP(3+) is quite similar to that produced by TMAP(4+), whereas no important inactivation effect was found for TPPS(4)(-). The high photodynamic activity of cationic porphyrins was confirmed by growth delay experiments. Thus, C. albicans cell growth was not detected in the presence of 5 microM TFAP(3+). Photodynamic inactivation capacities of these sensitizers were also evaluated on C. albicans cells growing in colonies on agar surfaces. Cationic porphyrins produce a growth delay of C. albicans colonies and viability of cells was not observed after 3 h irradiation, indicating a complete inactivation of yeast cells. Therefore, these results indicate that these cationic porphyrins are interesting sensitizers for photodynamic inactivation of yeasts in liquid suspensions or in localized foci of infection.

Flow cytometric evaluation of physico-chemical impact on Gram-positive and Gram-negative bacteria

PubMed Central

Fröhling, Antje; Schlüter, Oliver

2015-01-01

Since heat sensitivity of fruits and vegetables limits the application of thermal inactivation processes, new emerging inactivation technologies have to be established to fulfill the requirements of food safety without affecting the produce quality. The efficiency of inactivation treatments has to be ensured and monitored. Monitoring of inactivation effects is commonly performed using traditional cultivation methods which have the disadvantage of the time span needed to obtain results. The aim of this study was to compare the inactivation effects of peracetic acid (PAA), ozonated water (O3), and cold atmospheric pressure plasma (CAPP) on Gram-positive and Gram-negative bacteria using flow cytometric methods. E. coli cells were completely depolarized after treatment (15 s) with 0.25% PAA at 10°C, and after treatment (10 s) with 3.8 mg l−1 O3 at 12°C. The membrane potential of CAPP treated cells remained almost constant at an operating power of 20 W over a time period of 3 min, and subsequently decreased within 30 s of further treatment. Complete membrane permeabilization was observed after 10 s O3 treatment, but treatment with PAA and CAPP did not completely permeabilize the cells within 2 and 4 min, respectively. Similar results were obtained for esterase activity. O3 inactivates cellular esterase but esterase activity was detected after 4 min CAPP treatment and 2 min PAA treatment. L. innocua cells and P. carotovorum cells were also permeabilized instantaneously by O3 treatment at concentrations of 3.8 ± 1 mg l−1. However, higher membrane permeabilization of L. innocua and P. carotovorum than of E. coli was observed at CAPP treatment of 20 W. The degree of bacterial damage due to the inactivation processes is highly dependent on treatment parameters as well as on treated bacteria. Important information regarding the inactivation mechanisms can be obtained by flow cytometric measurements and this enables the definition of critical process parameters. PMID:26441874

v-Src-driven transformation is due to chromosome abnormalities but not Src-mediated growth signaling.

PubMed

Honda, Takuya; Morii, Mariko; Nakayama, Yuji; Suzuki, Ko; Yamaguchi, Noritaka; Yamaguchi, Naoto

2018-01-18

v-Src is the first identified oncogene product and has a strong tyrosine kinase activity. Much of the literature indicates that v-Src expression induces anchorage-independent and infinite cell proliferation through continuous stimulation of growth signaling by v-Src activity. Although all of v-Src-expressing cells are supposed to form transformed colonies, low frequencies of v-Src-induced colony formation have been observed so far. Using cells that exhibit high expression efficiencies of inducible v-Src, we show that v-Src expression causes cell-cycle arrest through p21 up-regulation despite ERK activation. v-Src expression also induces chromosome abnormalities and unexpected suppression of v-Src expression, leading to p21 down-regulation and ERK inactivation. Importantly, among v-Src-suppressed cells, only a limited number of cells gain the ability to re-proliferate and form transformed colonies. Our findings provide the first evidence that v-Src-driven transformation is attributed to chromosome abnormalities, but not continuous stimulation of growth signaling, possibly through stochastic genetic alterations.

Protective efficacy of heat-inactivated B. thailandensis, B. mallei or B. pseudomallei against experimental melioidosis and glanders.

PubMed

Sarkar-Tyson, Mitali; Smither, Sophie J; Harding, S V; Atkins, Timothy P; Titball, Richard W

2009-07-16

Burkholderia pseudomallei and Burkholderia mallei are gram-negative bacilli that are the causative agents of melioidosis and glanders, respectively. Both humans and animals are susceptible to both diseases. There is currently no vaccine available for the prevention of disease. We report the protective efficacy of heat-inactivated Burkholderia thailandensis, B. mallei or B. pseudomallei cells as vaccines against murine melioidosis and glanders. Immunisation with heat-inactivated B. pseudomallei cells provided the highest levels of protection against either melioidosis or glanders. These studies indicate the longer term potential for heat-inactivated bacteria to be developed as vaccines against melioidosis and glanders.

Inactivation of Vibrio parahaemolyticus by antimicrobial photodynamic technology using methylene blue.

PubMed

Deng, Xi; Tang, Shuze; Wu, Qian; Tian, Juan; Riley, William W; Chen, Zhenqiang

2016-03-30

Vibrio parahaemolyticus is the leading causative pathogen of gastroenteritis often related to contaminated seafood. Photodynamic inactivation has been recently proposed as a strategy for killing cells and viruses. The objective of this study was to verify the bactericidal effects caused by photodynamic inactivation using methylene blue (MB) over V. parahaemolyticus via flow cytometry, agarose gel electrophoresis and sodium dodecyl sulfate polyacrylamide gel electrophoresis. Vibrio parahaemolyticus counts were determined using the most probable number method. A scanning electron microscope and a transmission electron microscope were employed to intuitively analyze internal and external cell structure. Combination of MB and laser treatment significantly inhibited the growth of V. parahaemolyticus. The inactivation rate of V. parahaemolyticus was >99.99% and its counts were reduced by 5 log10 in the presence of 0.05 mg mL(-1) MB when illuminated with visible light (power density 200 mW cm(-2)) for 25 min. All inactivated cells showed morphological changes, leakage of cytoplasm and degradation of protein and DNA. Results from this study indicated that photodynamic technology using MB produced significant inactivation of V. parahaemolyticus mainly brought about by the degradation of protein and DNA. © 2015 Society of Chemical Industry.

Pertussis toxins, other antigens become likely targets for genetic engineering

DOE Office of Scientific and Technical Information (OSTI.GOV)

Marwick, C.

1990-11-14

Genetically engineered pertussis vaccines have yet to be fully tested clinically. But early human, animal, and in vitro studies indicate effectiveness in reducing toxic effects due to Bordetella pertussis. The licensed pertussis vaccines consists of inactivated whole cells of the organism. Although highly effective, they have been associated with neurologic complications. While the evidence continues to mount that these complications are extremely rare, if they occur at all, it has affected the public's acceptance of pertussis immunization.

The phosphatase JKAP/DUSP22 inhibits T-cell receptor signalling and autoimmunity by inactivating Lck.

PubMed

Li, Ju-Pi; Yang, Chia-Yu; Chuang, Huai-Chia; Lan, Joung-Liang; Chen, Der-Yuan; Chen, Yi-Ming; Wang, Xiaohong; Chen, Alice J; Belmont, John W; Tan, Tse-Hua

2014-04-09

JNK pathway-associated phosphatase (JKAP, also known as DUSP22 or JSP-1) is a JNK activator. The in vivo role of JKAP in immune regulation remains unclear. Here we report that JKAP directly inactivates Lck by dephosphorylating tyrosine-394 residue during T-cell receptor (TCR) signalling. JKAP-knockout T cells display enhanced cell proliferation and cytokine production. JKAP-knockout mice show enhanced T-cell-mediated immune responses and are more susceptible to experimental autoimmune encephalomyelitis (EAE). In addition, the recipient mice that are adoptively transferred with JKAP-knockout T cells show exacerbated EAE symptoms. Aged JKAP-knockout mice spontaneously develop inflammation and autoimmunity. Thus, our results indicate that JKAP is an important phosphatase that inactivates Lck in the TCR signalling turn-off stage, leading to suppression of T-cell-mediated immunity and autoimmunity.

Adjuvanticity of a CTLA-4 3' UTR complementary oligonucleotide for emulsion formulated recombinant subunit and inactivated vaccines.

PubMed

Li, Xin; Yang, Lei; Zhao, Peiyan; Yao, Yun; Lu, Fangjie; Tu, Liqun; Liu, Jiwei; Li, Zhiqin; Yu, Yongli; Wang, Liying

2017-04-25

Cytotoxic T-lymphocyte antigen 4 (CTLA-4) is recognized as a critical inhibitory regulator of T-cell proliferation and activation, opposing the action of CD28-mediated co-stimulation. Interfering or blocking CTLA-4 can result in continuous T-cell activation required for the full immune response to pathogenic microbes and vaccines. To test if nucleic acid-based CTLA-4 inhibitors could be developed into a novel adjuvant, we designed two oligonucleotides, CMD-1 and CMD-2, with the sequences complementary to the conserve regions identical between human and mouse CTLA-4 mRNA 3' untranslated region (3' UTR), and tested their in vitro effects on CTLA-4 production and their adjuvanticity for vaccines in mice. We found that CMD-1 inhibited the antigen-induced CTLA-4 up-regulation on the CD4 + T cells by interfering its mRNA expression, maintained higher levels of CD80 and CD86 on the CD11c + cells and promoted the recalled proliferation of the CD4 + T cells and CD19 + B cells, and that the CMD-1 enhanced the antibody response against recombinant PCV2b capsid protein or inactivated foot-and-mouth disease virus in both ICR and BALB/c mice. These data suggest that the CMD-1 could be used as a novel vaccine adjuvant capable of inhibiting inhibitory signals rather than inducing stimulatory signals of immune cells. Copyright © 2017 Elsevier Ltd. All rights reserved.

The DNA damage response during mitosis.

PubMed

Heijink, Anne Margriet; Krajewska, Małgorzata; van Vugt, Marcel A T M

2013-10-01

Cells are equipped with a cell-intrinsic signaling network called the DNA damage response (DDR). This signaling network recognizes DNA lesions and initiates various downstream pathways to coordinate a cell cycle arrest with the repair of the damaged DNA. Alternatively, the DDR can mediate clearance of affected cells that are beyond repair through apoptosis or senescence. The DDR can be activated in response to DNA damage throughout the cell cycle, although the extent of DDR signaling is different in each cell cycle phase. Especially in response to DNA double strand breaks, only a very marginal response was observed during mitosis. Early on it was recognized that cells which are irradiated during mitosis continued division without repairing broken chromosomes. Although these initial observations indicated diminished DNA repair and lack of an acute DNA damage-induced cell cycle arrest, insight into the mechanistic re-wiring of DDR signaling during mitosis was only recently provided. Different mechanisms appear to be at play to inactivate specific signaling axes of the DDR network in mitosis. Importantly, mitotic cells not simply inactivate the entire DDR, but appear to mark their DNA damage for repair after mitotic exit. Since the treatment of cancer frequently involves agents that induce DNA damage as well as agents that block mitotic progression, it is clinically relevant to obtain a better understanding of how cancer cells deal with DNA damage during interphase versus mitosis. In this review, the molecular details concerning DDR signaling during mitosis as well as the consequences of encountering DNA damage during mitosis for cellular fate are discussed. Copyright © 2013 Elsevier B.V. All rights reserved.

Inactivation of lipoprotein lipase occurs on the surface of THP-1 macrophages where oligomers of angiopoietin-like protein 4 are formed

DOE Office of Scientific and Technical Information (OSTI.GOV)

Makoveichuk, Elena; Sukonina, Valentina; Kroupa, Olessia

2012-08-24

Highlights: Black-Right-Pointing-Pointer Lipoprotein lipase (LPL) activity is controlled by ANGPTL4 in THP-1 macrophages. Black-Right-Pointing-Pointer Both LPL and ANGPTL4 bind to THP-1 macrophages in a heparin-releasable fashion. Black-Right-Pointing-Pointer Only monomers of ANGPTL4 are present within THP-1 macrophages. Black-Right-Pointing-Pointer Covalent oligomers of ANGPTL4 appear on cell surface and in medium. Black-Right-Pointing-Pointer Inactivation of LPL coincide with ANGPTL4 oligomer formation on cell surfaces. -- Abstract: Lipoprotein lipase (LPL) hydrolyzes triglycerides in plasma lipoproteins causing release of fatty acids for metabolic purposes in muscles and adipose tissue. LPL in macrophages in the artery wall may, however, promote foam cell formation and atherosclerosis. Angiopoietin-like proteinmore » (ANGPTL) 4 inactivates LPL and ANGPTL4 expression is controlled by peroxisome proliferator-activated receptors (PPAR). The mechanisms for inactivation of LPL by ANGPTL4 was studied in THP-1 macrophages where active LPL is associated with cell surfaces in a heparin-releasable form, while LPL in the culture medium is mostly inactive. The PPAR{delta} agonist GW501516 had no effect on LPL mRNA, but increased ANGPTL4 mRNA and caused a marked reduction of the heparin-releasable LPL activity concomitantly with accumulation of inactive, monomeric LPL in the medium. Intracellular ANGPTL4 was monomeric, while dimers and tetramers of ANGPTL4 were present in the heparin-releasable fraction and medium. GW501516 caused an increase in the amount of ANGPTL4 oligomers on the cell surface that paralleled the decrease in LPL activity. Actinomycin D blocked the effects of GW501516 on ANGPTL4 oligomer formation and prevented the inactivation of LPL. Antibodies against ANGPTL4 interfered with the inactivation of LPL. We conclude that inactivation of LPL in THP-1 macrophages primarily occurs on the cell surface where oligomers of ANGPTL4 are formed.« less

Infectivity of RNA from inactivated poliovirus.

PubMed

Nuanualsuwan, Suphachai; Cliver, Dean O

2003-03-01

During inactivation of poliovirus type 1 (PV-1) by exposure to UV, hypochlorite, and heat (72 degrees C), the infectivity of the virus was compared with that of its RNA. DEAE-dextran (1-mg/ml concentration in Dulbecco's modified Eagle medium buffered with 0.05 M Tris, pH 7.4) was used to facilitate transfecting PV-1 RNA into FRhK-4 host cells. After interaction of PV-1 RNA with cell monolayer at room temperature (21 to 22 degrees C) for 20 min, the monolayers were washed with 5 ml of Hanks balanced salt solution. The remainder of the procedure was the same as that for the conventional plaque technique, which was also used for quantifying the PV-1 whole-particle infectivity. Plaque formation by extracted RNA was approximately 100,000-fold less efficient than that by whole virions. The slopes of best-fit regression lines of inactivation curves for virion infectivity and RNA infectivity were compared to determine the target of inactivation. For UV and hypochlorite inactivation the slopes of inactivation curves of virion infectivity and RNA infectivity were not statistically different. However, the difference of slopes of inactivation curves of virion infectivity and RNA infectivity was statistically significant for thermal inactivation. The results of these experiments indicate that viral RNA is a primary target of UV and hypochlorite inactivations but that the sole target of thermal inactivation is the viral capsid.

Infectivity of RNA from Inactivated Poliovirus

PubMed Central

Nuanualsuwan, Suphachai; Cliver, Dean O.

2003-01-01

During inactivation of poliovirus type 1 (PV-1) by exposure to UV, hypochlorite, and heat (72°C), the infectivity of the virus was compared with that of its RNA. DEAE-dextran (1-mg/ml concentration in Dulbecco's modified Eagle medium buffered with 0.05 M Tris, pH 7.4) was used to facilitate transfecting PV-1 RNA into FRhK-4 host cells. After interaction of PV-1 RNA with cell monolayer at room temperature (21 to 22°C) for 20 min, the monolayers were washed with 5 ml of Hanks balanced salt solution. The remainder of the procedure was the same as that for the conventional plaque technique, which was also used for quantifying the PV-1 whole-particle infectivity. Plaque formation by extracted RNA was approximately 100,000-fold less efficient than that by whole virions. The slopes of best-fit regression lines of inactivation curves for virion infectivity and RNA infectivity were compared to determine the target of inactivation. For UV and hypochlorite inactivation the slopes of inactivation curves of virion infectivity and RNA infectivity were not statistically different. However, the difference of slopes of inactivation curves of virion infectivity and RNA infectivity was statistically significant for thermal inactivation. The results of these experiments indicate that viral RNA is a primary target of UV and hypochlorite inactivations but that the sole target of thermal inactivation is the viral capsid. PMID:12620852

Qualitation and Quantitation on Microplasma Jet for Bacteria Inactivation.

PubMed

Du, ChangMing; Liu, Ya; Huang, YaNi; Li, ZiMing; Men, Rui; Men, Yue; Tang, Jun

2016-01-06

In this work, a self-made microplasma jet system was used to conduct the qualitation and quantitation of inactivation with Escherichia coli as the target bacteria. The logarithmic concentration and the size of antimicrobial rings served as the evaluation parameters, respectively. The effect of various parameters on inactivation effect was studied. The results showed that the majority of bacteria had been inactivated in 30 s. The inactivation effect enhanced and then weakened with the increase of air flow rate, and receded as the extension of treatment distance. The effect with different carrier gases showed as follows: oxygen > air > nitrogen > argon. Meanwhile, the effect of different components of microplasma was studied in the optimum conditions (The flow rate was 5 L/min; inactivation distance was 2 cm). The results showed that electrically neutral active species was the main factor of inactivation rather than heating effect, ultraviolet radiation and charged particles. Finally the experiments of thallus change proved that microplasma jet had etching effect on cell membrane. It also found that microplasma could degrade organic material like protein. Furthermore, the images of scanning electron microscope (SEM) revealed the change of cell morphology step by step in the whole process of inactivation.

Qualitation and Quantitation on Microplasma Jet for Bacteria Inactivation

NASA Astrophysics Data System (ADS)

Du, Changming; Liu, Ya; Huang, Yani; Li, Ziming; Men, Rui; Men, Yue; Tang, Jun

2016-01-01

In this work, a self-made microplasma jet system was used to conduct the qualitation and quantitation of inactivation with Escherichia coli as the target bacteria. The logarithmic concentration and the size of antimicrobial rings served as the evaluation parameters, respectively. The effect of various parameters on inactivation effect was studied. The results showed that the majority of bacteria had been inactivated in 30 s. The inactivation effect enhanced and then weakened with the increase of air flow rate, and receded as the extension of treatment distance. The effect with different carrier gases showed as follows: oxygen > air > nitrogen > argon. Meanwhile, the effect of different components of microplasma was studied in the optimum conditions (The flow rate was 5 L/min inactivation distance was 2 cm). The results showed that electrically neutral active species was the main factor of inactivation rather than heating effect, ultraviolet radiation and charged particles. Finally the experiments of thallus change proved that microplasma jet had etching effect on cell membrane. It also found that microplasma could degrade organic material like protein. Furthermore, the images of scanning electron microscope (SEM) revealed the change of cell morphology step by step in the whole process of inactivation.

Qualitation and Quantitation on Microplasma Jet for Bacteria Inactivation

PubMed Central

Du, ChangMing; Liu, Ya; Huang, YaNi; Li, ZiMing; Men, Rui; Men, Yue; Tang, Jun

2016-01-01

In this work, a self-made microplasma jet system was used to conduct the qualitation and quantitation of inactivation with Escherichia coli as the target bacteria. The logarithmic concentration and the size of antimicrobial rings served as the evaluation parameters, respectively. The effect of various parameters on inactivation effect was studied. The results showed that the majority of bacteria had been inactivated in 30 s. The inactivation effect enhanced and then weakened with the increase of air flow rate, and receded as the extension of treatment distance. The effect with different carrier gases showed as follows: oxygen > air > nitrogen > argon. Meanwhile, the effect of different components of microplasma was studied in the optimum conditions (The flow rate was 5 L/min; inactivation distance was 2 cm). The results showed that electrically neutral active species was the main factor of inactivation rather than heating effect, ultraviolet radiation and charged particles. Finally the experiments of thallus change proved that microplasma jet had etching effect on cell membrane. It also found that microplasma could degrade organic material like protein. Furthermore, the images of scanning electron microscope (SEM) revealed the change of cell morphology step by step in the whole process of inactivation. PMID:26732987

Area 21a of cat visual cortex strongly modulates neuronal activities in the superior colliculus

PubMed Central

Hashemi-Nezhad, M; Wang, C; Burke, W; Dreher, B

2003-01-01

We have examined the influence of cortico-tectal projections from one of the pattern-processing extrastriate visual cortical areas, area 21a, on the responses to visual stimuli of single neurones in the superior colliculi of adult cats. For this purpose area 21a was briefly inactivated by cooling to 10 °C using a Peltier device. Responses to visual stimuli before and during cooling as well as after rewarming ipsilateral area 21a were compared. In addition, in a subpopulation of collicular neurones we have studied the effects of reversible inactivation of ipsilateral striate cortex (area 17, area V1). When area 21a was cooled, the temperature of area 17 was kept at 36 °C and vice versa. In the majority of cases (41/65; 63 %), irrespective of the velocity response profiles of collicular neurones, inactivation of area 21a resulted in a significant decrease in magnitude of responses of neurones in the ipsilateral colliculus and only in a small proportion of cells (2/65; 3.1 %) was there a significant increase in the magnitude of responses. Inactivation of area 21a resulted in significant changes in the magnitude of responses of collicular cells located not only in the retino-recipient layers but also in the stratum griseum intermediale. In most cases, reversible inactivation of area 17 resulted in a greater reduction in the magnitude of responses of collicular cells than inactivation of area 21a. Reversible inactivation of area 21a also affected the direction selectivity indices and length tuning of most collicular cells tested. PMID:12794178

Electron Transport Controls Glutamine Synthetase Activity in the Facultative Heterotrophic Cyanobacterium Synechocystis sp. PCC 6803.

PubMed Central

Reyes, J. C.; Crespo, J. L.; Garcia-Dominguez, M.; Florencio, F. J.

1995-01-01

Glutamine synthetase (GS) from Synechocystis sp. PCC 6803 was inactivated in vivo by transferring cells from light to darkness or by incubation with the photosynthetic inhibitor 3-(3,4-dichlorophenyl)-1,1-dimethylurea but not with 2,5-dibromo-3-methyl-6-isopropyl-p-benzoquinone. Addition of glucose prevented both dark and 3-(3,4-dichlorophenyl)-1,1-dimethylurea GS inactivation. In a Synechocystis psbE-psbF mutant (T1297) lacking photosystem II, glucose was required to maintain active GS, even in the light. However, in nitrogen-starved T1297 cells the removal of glucose did not affect GS activity. The fact that dark-inactivated GS was reactivated in vitro by the same treatments that reactivate the ammonium-inactivated GS points out that both nitrogen metabolism and redox state of the cells lead to the same molecular regulatory mechanism in the control of GS activity. Using GS antibodies we detected that dark-inactivated GS displayed a different electrophoretic migration with respect to the active form in nondenaturing polyacrylamide gel electrophoresis but not in sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The possible pathway to modulate GS activity by the electron transport flow in Synechocystis cells is discussed. PMID:12228640

Light-driven photosensitizer uptake increases Candida albicans photodynamic inactivation.

PubMed

Romano, Renan A; Pratavieira, Sebastião; Silva, Ana P da; Kurachi, Cristina; Guimarães, Francisco E G

2017-11-01

Photodynamic Inactivation (PDI) is based on the use of a photosensitizer (PS) and light that results mainly in the production of reactive oxygen species, aiming to produce microorganism cell death. PS incubation time and light dose are key protocol parameters that influence PDI response; the correct choice of them can increase the efficiency of inactivation. The results of this study show that a minor change in the PDI protocol, namely light-driven incubation leads to a higher photosensitizer and more uniform cell uptake inside the irradiated zone. Furthermore, as the uptake increases, the damage caused by PDI also increases. The proposed light-driven incubation prior to the inactivation illumination dose has advantages when compared to the traditional PDI treatments since it can be more selective and effective. Using a violet light as pre-illumination (light-driven incubation) source and a red-light system as PDI source, it was possible to demonstrate that when compared to the traditional protocol of dark incubation, the pre-illuminated cell culture showed an inactivation increase of 7 log units. These in vitro results performed in Candida albicans cells may result in the introduction of a new protocol for PDI. © 2017 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.

Role of chromosome 3p12–p21 tumour suppressor genes in clear cell renal cell carcinoma: analysis of VHL dependent and VHL independent pathways of tumorigenesis

PubMed Central

Martinez, A; Fullwood, P; Kondo, K; Kishida, T; Yao, M; Maher, E R; Latif, F

2000-01-01

Aims—Chromosome 3p deletions and loss of heterozygosity (LOH) for 3p markers are features of clear cell renal cell carcinoma but are rare in non-clear cell renal cell carcinoma. The VHL tumour suppressor gene, which maps to 3p25, is a major gatekeeper gene for clear cell renal cell carcinoma and is inactivated in most sporadic cases of this disease. However, it has been suggested that inactivation of other 3p tumour suppressor genes might be crucial for clear cell renal cell carcinoma tumorigenesis, with inactivation (VHL negative) and without inactivation (VHL positive) of the VHL tumour suppressor gene. This study set out to investigate the role of non-VHL tumour suppressor genes in VHL negative and VHL positive clear cell renal cell carcinoma. Methods—Eighty two clear cell renal cell carcinomas of known VHL inactivation status were analysed for LOH at polymorphic loci within the candidate crucial regions for chromosome 3p tumour suppressor genes (3p25, LCTSGR1 at 3p21.3, LCTSGR2 at 3p12 and at 3p14.2). Results—Chromosome 3p12–p21 LOH was frequent both in VHL negative and VHL positive clear cell renal cell carcinoma. However, although the frequency of 3p25 LOH in VHL negative clear cell renal cell carcinoma was similar to that at 3p12–p21, VHL positive tumours demonstrated significantly less LOH at 3p25 than at 3p12–p21. Although there was evidence of LOH for clear cell renal cell carcinoma tumour suppressor genes at 3p21, 3p14.2, and 3p12, both in VHL negative and VHL positive tumours, the major clear cell renal cell carcinoma LOH region mapped to 3p21.3, close to the lung cancer tumour suppressor gene region 1 (LCTSGR1). There was no association between tumour VHL status and tumour grade and stage. Conclusions—These findings further indicate that VHL inactivation is not sufficient to initiate clear cell renal cell carcinoma and that loss of a gatekeeper 3p21 tumour suppressor gene is a crucial event for renal cell carcinoma development in both VHL negative and VHL positive clear cell renal cell carcinoma. PMID:10897333

Expression pattern of X-linked genes in sex chromosome aneuploid bovine cells.

PubMed

Basrur, Parvathi K; Farazmand, Ali; Stranzinger, Gerald; Graphodatskaya, Daria; Reyes, Ed R; King, W Allan

2004-01-01

Expression of the X-inactive specific transcript (XIST) gene is a prerequisite step for dosage compensation in mammals, accomplished by silencing one of the two X chromosomes in normal female diploid cells or all X chromosomes in excess of one in sex chromosome aneuploids. Our previous studies showing that XIST expression does not eventuate the inactivation of X-linked genes in fetal bovine testis had suggested that XIST expression may not be an indicator of X inactivation in this species. In this study, we used a semi-quantitative reverse transcription polymerase chain reaction (RT-PCR) approach on cultures of bovine cells with varying sex chromosome constitution (XY, XX, XXY and XXX) to test whether the levels of XIST expressed conform to the number of late replicating (inactive) X chromosomes displayed by proliferating cells in these cultures. Expression patterns of four X-linked genes, including hypoxanthine phosphorybosyl transferase (HPRT), glucose-6-phosphate dehydrogenase (G6PD), zinc finger protein locus on the X (ZFX). and 'selected mouse cDNA on the X' (SMCX), in all these cells were also tested. Results showed that XIST expression was significantly higher (p < 0.05) in XXX cells compared to XX and XXY cells and that G6PD. HPRT, and SMCX loci are subject to X inactivation. The significantly higher levels of ZFX expressed in XXX cells compared to XX and XXY cells (p < 0.05) confirmed that this bovine locus, as human ZFX, escapes X inactivation. However, the levels of XIST and ZFX expressed were not proportional to the X chromosome load in these cells suggesting that X-linked loci escaping inactivation may be regulated at transcription (or post-transcription) level by mechanisms that prevent gene-specific product accumulation beyond certain levels in sex chromosome aneuploids.

Sex-specific silencing of X-linked genes by Xist RNA

PubMed Central

Gayen, Srimonta; Maclary, Emily; Hinten, Michael; Kalantry, Sundeep

2016-01-01

X-inactive specific transcript (Xist) long noncoding RNA (lncRNA) is thought to catalyze silencing of X-linked genes in cis during X-chromosome inactivation, which equalizes X-linked gene dosage between male and female mammals. To test the impact of Xist RNA on X-linked gene silencing, we ectopically induced endogenous Xist by ablating the antisense repressor Tsix in mice. We find that ectopic Xist RNA induction and subsequent X-linked gene silencing is sex specific in embryos and in differentiating embryonic stem cells (ESCs) and epiblast stem cells (EpiSCs). A higher frequency of XΔTsixY male cells displayed ectopic Xist RNA coating compared with XΔTsixX female cells. This increase reflected the inability of XΔTsixY cells to efficiently silence X-linked genes compared with XΔTsixX cells, despite equivalent Xist RNA induction and coating. Silencing of genes on both Xs resulted in significantly reduced proliferation and increased cell death in XΔTsixX female cells relative to XΔTsixY male cells. Thus, whereas Xist RNA can inactivate the X chromosome in females it may not do so in males. We further found comparable silencing in differentiating XΔTsixY and 39,XΔTsix (XΔTsixO) ESCs, excluding the Y chromosome and instead implicating the X-chromosome dose as the source of the sex-specific differences. Because XΔTsixX female embryonic epiblast cells and EpiSCs harbor an inactivated X chromosome prior to ectopic inactivation of the active XΔTsix X chromosome, we propose that the increased expression of one or more X-inactivation escapees activates Xist and, separately, helps trigger X-linked gene silencing. PMID:26739568

Eslicarbazepine and the enhancement of slow inactivation of voltage-gated sodium channels: a comparison with carbamazepine, oxcarbazepine and lacosamide.

PubMed

Hebeisen, Simon; Pires, Nuno; Loureiro, Ana I; Bonifácio, Maria João; Palma, Nuno; Whyment, Andrew; Spanswick, David; Soares-da-Silva, Patrício

2015-02-01

This study aimed at evaluating the effects of eslicarbazepine, carbamazepine (CBZ), oxcarbazepine (OXC) and lacosamide (LCM) on the fast and slow inactivated states of voltage-gated sodium channels (VGSC). The anti-epileptiform activity was evaluated in mouse isolated hippocampal slices. The anticonvulsant effects were evaluated in MES and the 6-Hz psychomotor tests. The whole-cell patch-clamp technique was used to investigate the effects of eslicarbazepine, CBZ, OXC and LCM on sodium channels endogenously expressed in N1E-115 mouse neuroblastoma cells. CBZ and eslicarbazepine exhibit similar concentration dependent suppression of epileptiform activity in hippocampal slices. In N1E-115 mouse neuroblastoma cells, at a concentration of 250 μM, the voltage dependence of the fast inactivation was not influenced by eslicarbazepine, whereas LCM, CBZ and OXC shifted the V0.5 value (mV) by -4.8, -12.0 and -16.6, respectively. Eslicarbazepine- and LCM-treated fast-inactivated channels recovered similarly to control conditions, whereas CBZ- and OXC-treated channels required longer pulses to recover. CBZ, eslicarbazepine and LCM shifted the voltage dependence of the slow inactivation (V0.5, mV) by -4.6, -31.2 and -53.3, respectively. For eslicarbazepine, LCM, CBZ and OXC, the affinity to the slow inactivated state was 5.9, 10.4, 1.7 and 1.8 times higher than to the channels in the resting state, respectively. In conclusion, eslicarbazepine did not share with CBZ and OXC the ability to alter fast inactivation of VGSC. Both eslicarbazepine and LCM reduce VGSC availability through enhancement of slow inactivation, but LCM demonstrated higher interaction with VGSC in the resting state and with fast inactivation gating. Copyright © 2014 Elsevier Ltd. All rights reserved.

CD4+ T-cells Contribute to the Remodeling of the Microenvironment Required for Sustained Tumor Regression upon Oncogene Inactivation

PubMed Central

Rakhra, Kavya; Bachireddy, Pavan; Zabuawala, Tahera; Zeiser, Robert; Xu, Liwen; Kopelman, Andrew; Fan, Alice C.; Yang, Qiwei; Braunstein, Lior; Crosby, Erika; Ryeom, Sandra; Felsher, Dean W.

2010-01-01

Summary Oncogene addiction is thought to occur cell autonomously. Immune effectors are implicated in the induction and restraint of tumorigenesis, but their role in oncogene inactivation mediated tumor regression is unclear. Here, we show that an intact immune system, specifically CD4+ T-cells, is required for the induction of cellular senescence, shut down of angiogenesis and chemokine expression resulting in sustained tumor regression upon inactivation of the MYC or BCR-ABL oncogenes in mouse models of T-cell acute lymphoblastic lymphoma and pro-B-cell leukemia, respectively. Moreover, immune effectors knocked out for thrombospondins failed to induce sustained tumor regression. Hence, CD4+ T-cells are required for the remodeling of the tumor microenvironment through the expression of chemokines, such as thrombospondins, in order to elicit oncogene addiction. PMID:21035406

[Methods for increasing the immunogenicity of vaccines].

PubMed

Kündig, T M

2000-09-14

In the past years, enormous efforts have been undertaken to develop vaccine strategies against cancer. The aim is to have the immune system generate what are called killer cells that can specifically recognize the tumor. The surface of tumor cells contains MHC/HLA antigens which present short-chain peptides of tumor specific antigens. A large number of these oligopeptide antigens have been characterized in recent years. They are now available for use as tumor-specific vaccines. The problem is, however, that the immune response of producing T killer cells is very inefficient when these oligopeptide antigens are injected. As the physiological function of these killer cells virus-infected cells, a process associated with substantial tissue damage, the immune system has learned to use these killer cells with reticence over the course of evolution, in other words, when the life of the host is threatened. This does not happen until pathogens start to spread via lymphogenous or hematogenous pathways. And then it takes a certain amount of time after the invader is present for replication to take place. Since the oligopeptide antigens used as vaccines have a very short half-life in the tissue, not enough of them get to the lymph nodes and stay there for enough time to efficiently induce an immune response. Using a mouse model, we were able to show that the efficiency of the vaccine can be increased a million-fold by directly injecting the vaccine into a lymph node or the spleen which imitates lymphogenous or hematogenous spread. The efficiency of the "inactivated vaccine" can be enhanced even more by continuous administration of the vaccine over several days, simulating an especially dangerous virus replication. The evidence gathered in this mouse model was transferred to a clinical trial. The melanoma-specific inactivated vaccine is infused directly into a lymph node of tumor patients. The infusion is continued for several days. Booster vaccines are given every two weeks.

Involvement of Atm and Trp53 in neural cell loss due to Terf2 inactivation during mouse brain development.

PubMed

Kim, Jusik; Choi, Inseo; Lee, Youngsoo

2017-11-01

Maintenance of genomic integrity is one of the critical features for proper neurodevelopment and inhibition of neurological diseases. The signals from both ATM and ATR to TP53 are well-known mechanisms to remove neural cells with DNA damage during neurogenesis. Here we examined the involvement of Atm and Atr in genomic instability due to Terf2 inactivation during mouse brain development. Selective inactivation of Terf2 in neural progenitors induced apoptosis, resulting in a complete loss of the brain structure. This neural loss was rescued partially in both Atm and Trp53 deficiency, but not in an Atr-deficient background in the mouse. Atm inactivation resulted in incomplete brain structures, whereas p53 deficiency led to the formation of multinucleated giant neural cells and the disruption of the brain structure. These giant neural cells disappeared in Lig4 deficiency. These data demonstrate ATM and TP53 are important for the maintenance of telomere homeostasis and the surveillance of telomere dysfunction during neurogenesis.

Enhanced EGFP-chromophore-assisted laser inactivation using deficient cells rescued with functional EGFP-fusion proteins.

PubMed

Vitriol, Eric A; Uetrecht, Andrea C; Shen, Feimo; Jacobson, Ken; Bear, James E

2007-04-17

Chromophore-assisted laser inactivation (CALI) is a light-mediated technique that offers precise spatiotemporal control of protein inactivation, enabling better understanding of the protein's role in cell function. EGFP has been used effectively as a CALI chromophore, and its cotranslational attachment to the target protein avoids having to use exogenously added labeling reagents. A potential drawback to EGFP-CALI is that the CALI phenotype can be obscured by the endogenous, unlabeled protein that is not susceptible to light inactivation. Performing EGFP-CALI experiments in deficient cells rescued with functional EGFP-fusion proteins permits more complete loss of function to be achieved. Here, we present a modified lentiviral system for rapid and efficient generation of knockdown cell lines complemented with physiological levels of EGFP-fusion proteins. We demonstrate that CALI of EGFP-CapZbeta increases uncapped actin filaments, resulting in enhanced filament growth and the formation of numerous protrusive structures. We show that these effects are completely dependent upon knocking down the endogenous protein. We also demonstrate that CALI of EGFP-Mena in Mena/VASP-deficient cells stabilizes lamellipodial protrusions.

Low pH inactivation for xenotropic gamma retrovirus in recombinant human TNF-α receptor immunoglobulin G and mechanism of inactivation.

PubMed

Ma, Rong; Cui, Xiaolan

2014-01-01

CHO-derived recombinant proteins for human therapeutic are used commonly. There are noninfectious endogenous retroviruses in CHO cells. Validation study for inactivation process is required. Murine xenotropic gamma retrovirus (X-MulV) is a model virus in validation study. In our previous study, optimum conditions for X-MulV inactivation were sifted. In this study, we performed a further research on low pH inactivation for evaluation of X-MulV clearance in manufacturing of recombinant human TNF-α receptor immunoglobulin G fusion proteins (rhTNF-α) for injection. Cell-based infectivity assay was used for the evaluation of X-MulV clearance. RhTNF-α were spiked with X-MulV and were inactivated at pH 3.60 ∼ 3.90, 25 ± 2 °C, and 0 ∼ 240 min, respectively. Samples incubated at the conditions for 15 ∼ 180 min were not inactivated effectively. For 4 h incubation, log10 reductions were achieved 5.0 log10. Biological activity of rhTNF-α incubated at pH 3.60, 25 °C for 4 h, which was assayed on murine L929 fibroblasts cells, was not affected by low pH. Env gene of X-MulV, which was detected by conventional PCR method for the first time, was not detected after incubation at pH 3.60, and it may be the mechanism of low pH inactivation. Copyright © 2013. Published by Elsevier Ltd.

Mechanistic Aspects of Adenovirus Serotype 2 Inactivation with Free Chlorine ▿ †

PubMed Central

Page, Martin A.; Shisler, Joanna L.; Mariñas, Benito J.

2010-01-01

Free chlorine is an effective disinfectant for controlling adenoviruses in drinking water, but little is known about the underlying inactivation mechanisms. The objective of this study was to elucidate the molecular components of adenovirus type 2 (Ad2) targeted by free chlorine during the inactivation process. The effects of free chlorine treatment on several Ad2 molecular components and associated life cycle events were compared to its effect on the ability of adenovirus to complete its life cycle, i.e., viability. Free chlorine treatment of Ad2 virions did not impair their ability to interact with monoclonal antibodies specific for hexon and fiber proteins of the Ad2 capsid, as measured by enzyme-linked immunosorbent assays, nor did it impair their interaction with recombinant, purified Coxsackie-adenovirus receptor (CAR) proteins in vitro. Free chlorine-treated Ad2 virions also retained their ability to bind to CAR receptors on A549 cell monolayers, despite being unable to form plaques, suggesting that free chlorine inactivates Ad2 by inhibiting a postbinding event of the Ad2 life cycle. DNA isolated from Ad2 virions that had been inactivated by free chlorine was able to be amplified by PCR, indicating that genome damage was not the cause of inactivation. However, inactivated Ad2 virions were unable to express E1A viral proteins during infection of A549 host cells, as measured by using immunoblotting. Collectively, these results indicate that free chlorine inactivates adenovirus by damaging proteins that govern life cycle processes occurring after host cell attachment, such as endocytosis, endosomal lysis, or nuclear delivery. PMID:20305026

Photonic approach to the selective inactivation of viruses with a near-infrared ultrashort pulsed laser

NASA Astrophysics Data System (ADS)

Tsen, K. T.; Tsen, Shaw-Wei D.; Fu, Q.; Lindsay, S. M.; Kibler, K.; Jacobs, B.; Wu, T. C.; Li, Zhe; Yan, Hao; Cope, Stephanie; Vaiana, Sara; Kiang, Juliann G.

2010-02-01

We report a photonic approach for selective inactivation of viruses with a near-infrared ultrashort pulsed (USP) laser. We demonstrate that this method can selectively inactivate viral particles ranging from nonpathogenic viruses such as M13 bacteriophage, tobacco mosaic virus (TMV) to pathogenic viruses like human papillomavirus (HPV) and human immunodeficiency virus (HIV). At the same time sensitive materials like human Jurkat T cells, human red blood cells, and mouse dendritic cells remain unharmed. Our photonic approach could be used for the disinfection of viral pathogens in blood products and for the treatment of blood-borne viral diseases in the clinic.

One pyrimidine dimer inactivates expression of a transfected gene in xeroderma pigmentosum cells

DOE Office of Scientific and Technical Information (OSTI.GOV)

Protic-Sabljic, M.; Kraemer, K.H.

1985-10-01

The authors have developed a host cell reactivation assay of DNA repair utilizing UV-treated plasmid vectors. The assay primarily reflects cellular repair of transcriptional activity of damaged DNA measured indirectly as enzyme activity of the transfected genes. They studied three plasmids (pSV2cat, 5020 base pairs; pSV2catSVgpt, 7268 base pairs; and pRSVcat, 5027 base pairs) with different sizes and promoters carrying the bacterial cat gene (CAT, chloramphenicol acetyltransferase) in a construction that permits cat expression in human cells. All human simian virus 40-transformed cells studied expressed high levels of the transfected cat gene. UV treatment of the plasmids prior to transfectionmore » resulted in differential decrease in CAT activity in different cell lines. With pSV2catSVgpt, UV inactivation of CAT expression was greater in the xeroderma pigmentosum group A and D lines than in the other human cell lines tested. The D0 of the CAT inactivation curve was 50 J X m-2 for pSV2cat and for pRSVcat in the xeroderma pigmentosum group A cells. The similarity of the D0 data in the xeroderma pigmentosum group A cells for three plasmids of different size and promoters implies they all have similar UV-inactivation target size. UV-induced pyrimidine dimer formation in the plasmids was quantified by assay of the number of UV-induced T4 endonuclease V-sensitive sites. In the most sensitive xeroderma pigmentosum cells, with all three plasmids, one UV-induced pyrimidine dimer inactivates a target of about 2 kilobases, close to the size of the putative CAT mRNA.« less

DNA aptamer functionalized gold nanostructures for molecular recognition and photothermal inactivation of methicillin-Resistant Staphylococcus aureus.

PubMed

Ocsoy, Ismail; Yusufbeyoglu, Sadi; Yılmaz, Vedat; McLamore, Eric S; Ildız, Nilay; Ülgen, Ahmet

2017-11-01

In this work, we report the development of DNA aptamer-functionalized gold nanoparticles (Apt@Au NPs) and gold nanorods (Apt@Au NRs) for inactivation of Methicillin-resistant Staphylococcus aureus (MRSA) with targeted photothermal therapy (PTT). Although both Apt@Au NPs and Apt@Au NRs specifically bind to MRSA cells, Apt@Au NPs and Apt@Au NRs inactivated ∼5% and over 95% of the cells,respectively through PTT. This difference in inactivation was based on the relatively high longitudinal absorption of near-infrared (NIR) radiation and strong photothermal conversion capability for the Apt@Au NRs compared to the Apt@Au NPs. The Au NRs served as a nanoplatform for the loading of thiolated aptamer and also provided multivalent effects for increasing binding strength and affinity to MRSA. Our results indicate that the type of aptamer and the degree of multivalent effect(s) are important factors for MRSA inactivation efficiency in PTT. We show that the Apt@Au NRs are a very effective and promising nanosystem for specific cell recognition and in vitro PTT. Copyright © 2017 Elsevier B.V. All rights reserved.

Inactivation of high-risk human papillomaviruses by Holder pasteurization: implications for donor human milk banking.

PubMed

Donalisio, Manuela; Cagno, Valeria; Vallino, Marta; Moro, Guido E; Arslanoglu, Sertac; Tonetto, Paola; Bertino, Enrico; Lembo, David

2014-01-01

Several studies have recently reported the detection of oncogenic human papillomaviruses (HPV) in human milk of a minority of lactating mothers. These findings raised safety concerns in the context of human donor milk banking given the potential risk of HPV transmission to recipient infants. The aim of this study was to investigate whether the Holder pasteurization, a procedure currently in use in human donor milk banks for milk pasteurization, completely inactivates high-risk and low-risk HPV. HPV pseudoviruses (PsV) were generated, spiked into cell culture medium or donor human milk and subjected to thermal inactivation. HPV PsV infectivity and morphological integrity was analyzed by cell-based assay and by electron microscopy, respectively. The Holder pasteurization completely inactivated the infectivity of high-risk (types 16 and 18) and low-risk (type 6) HPV both in cell culture medium and in human milk causing PsV particle disassembly. The results presented here indicate that the Holder pasteurization is an efficient procedure to inactivate high-risk and low-risk HPV thus preventing the potential risk of their transmission through human donor milk.

The radiation hypersensitivity of cells at mitosis.

PubMed

Stobbe, C C; Park, S J; Chapman, J D

2002-12-01

Mitotic cells are hypersensitive to ionizing radiation, exhibiting single-hit inactivation coefficients near to those of repair deficient cell lines and lymphocytes. To elucidate possible mechanisms for this hypersensitivity, the kinetics of oxygen radiosensitization, the proportion of indirect effect by OH radicals and the kinetics of radiation-induced DNA strand breakage in the chromatin of mitotic cells were investigated. Synchronized populations of >90% mitotic HT-29 cells were obtained by the mitotic shake-off method. Cells were irradiated at < or =4 degrees C with (137)Cs gamma-rays. Cellular oxygen concentration was varied by gassing cell suspensions prior to and during irradiation with mixtures of pure N(2) that contained 5% CO(2) and measured quantities of O(2). The indirect effect of OH radicals was investigated with the radical scavenger, DMSO. DNA strand breakage was measured by the comet assay. Mitotic HT-29 cell inactivation is well described by a single-hit inactivation coefficient (alpha) of 1.14 +/- 0.06 Gy(-1). The oxygen enhancement ratio of mitotic cells (at 10% survival) was found to be approximately 2.0, significantly lower than the value of 2.8 measured for interphase (asynchronous) cells. More than 60% of mitotic cell killing was eliminated when the media contained 2 M DMSO, indicating that indirect effect is as important in the killing of mitotic cells as it is for interphase cells. The chromatin in mitotic cells was found to be ~2.8 times more sensitive to radiation-induced DNA single-strand breakage than the chromatin of interphase cells. The alpha-inactivation coefficient of mitotic HT-29 cells was ~30 times larger than that of interphase cells. Mitotic cell chromatin appears to contain intrinsic DNA breaks that are not lethal. In addition, chromatin in mitotic cells was found to be more susceptible to radiation-induced DNA strand-breakage than the dispersed chromatin of interphase cells. How the enhanced production of these simple DNA lesions (that are usually reparable) translates into the lethal (non-reparable) events associated with alpha-inactivation is not known. The compaction/dispersion status of DNA throughout the cell cycle appears to be an important factor for determining intrinsic cell radiosensitivity and might be manipulated for radiotherapeutic advantage.

Epigenetic inactivation of CHFR in human tumors

PubMed Central

Toyota, Minoru; Sasaki, Yasushi; Satoh, Ayumi; Ogi, Kazuhiro; Kikuchi, Takefumi; Suzuki, Hiromu; Mita, Hiroaki; Tanaka, Nobuyuki; Itoh, Fumio; Issa, Jean-Pierre J.; Jair, Kam-Wing; Schuebel, Kornel E.; Imai, Kohzoh; Tokino, Takashi

2003-01-01

Cell-cycle checkpoints controlling the orderly progression through mitosis are frequently disrupted in human cancers. One such checkpoint, entry into metaphase, is regulated by the CHFR gene encoding a protein possessing forkhead-associated and RING finger domains as well as ubiquitin–ligase activity. Although defects in this checkpoint have been described, the molecular basis and prevalence of CHFR inactivation in human tumors are still not fully understood. To address this question, we analyzed the pattern of CHFR expression in a number of human cancer cell lines and primary tumors. We found CpG methylation-dependent silencing of CHFR expression in 45% of cancer cell lines, 40% of primary colorectal cancers, 53% of colorectal adenomas, and 30% of primary head and neck cancers. Expression of CHFR was precisely correlated with both CpG methylation and deacetylation of histones H3 and H4 in the CpG-rich regulatory region. Moreover, CpG methylation and thus silencing of CHFR depended on the activities of two DNA methyltransferases, DNMT1 and DNMT3b, as their genetic inactivation restored CHFR expression. Finally, cells with CHFR methylation had an intrinsically high mitotic index when treated with microtubule inhibitor. This means that cells in which CHFR was epigenetically inactivated constitute loss-of-function alleles for mitotic checkpoint control. Taken together, these findings shed light on a pathway by which mitotic checkpoint is bypassed in cancer cells and suggest that inactivation of checkpoint genes is much more widespread than previously suspected. PMID:12810945

Formalin-inactivated Venezuelan Equine Encephalomyelitis (Trinidad Strain) Vaccine Produced in Rolling-Bottle Cultures of Chicken Embryo Cells

PubMed Central

Cole, Francis E.; May, Stephen W.; Robinson, David M.

1973-01-01

Formalin-inactivated Venezuelan equine encephalomyelitis vaccine was prepared from virus grown in rolling-bottle cultures of chicken embryo cells. Trinidad strain virus was propagated in these cultures with a maintenance medium consisting of serum-free medium 199 containing 0.25% human serum albumin (USP) and antibiotics. Manipulation of multiplicity of inoculum (0.06 to 0.00006) and maintenance medium volume (100 to 300 ml) resulted in high-titered virus yields and only moderate cell destruction when fluids from infected cultures were harvested at 18 to 24 hr. The virus was inactivated at 37 C by 0.05% Formalin within 8 to 10 hr and with 0.1% Formalin within 6 to 8 hr. Single dose, antigen extinction tests in mice performed with 30 small-scale vaccine lots showed excellent potency at either Formalin concentration with inactivation periods ranging from 24 to 96 hr. PMID:4694345

Inactivation of Bacteria in Oil Field Injected Water by a Pulsed Plasma Discharge Process

NASA Astrophysics Data System (ADS)

Xin, Qing; Li, Zhongjian; Lei, Lecheng; Yang, Bin

2016-09-01

Pulsed plasma discharge was employed to inactivate bacteria in the injection water for an oil field. The effects of water conductivity and initial concentration of bacteria on elimination efficiency were investigated in the batch and continuous flow modes. It was demonstrated that Fe2+ contained in injection water could enhance the elimination efficiency greatly. The addition of reducing agent glutathione (GSH) indicated that active radicals generated by pulsed plasma discharges played an important role in the inactivation of bacteria. Moreover, it was found that the microbial inactivation process for both batch and continuous flow mode well fitted the model based on the Weibull's survival function. supported by Zhejiang Province Welfare Technology Applied Research Project of China (No. 2014C31137), National Natural Science Foundation of China (Nos. 21436007 and U1462201), and the Fundamental Research Funds for the Central Universities of China (No. 2015QNA4032)

Amustaline (S-303) treatment inactivates high levels of Zika virus in red blood cell components.

PubMed

Laughhunn, Andrew; Santa Maria, Felicia; Broult, Julien; Lanteri, Marion C; Stassinopoulos, Adonis; Musso, Didier; Aubry, Maite

2017-03-01

The potential for Zika virus (ZIKV) transfusion-transmission (TT) has been demonstrated in French Polynesia and Brazil. Pathogen inactivation (PI) of blood products is a proactive strategy to inactivate TT pathogens including arboviruses. Inactivation of West Nile, dengue, Zika, and chikungunya viruses was previously demonstrated by photochemical treatment with amotosalen and ultraviolet A (UVA) illumination. In this study, we evaluated ZIKV inactivation in red blood cell (RBC) components by a chemical approach that uses amustaline (S-303) and glutathione (GSH). RBC components were spiked with a high titer of ZIKV. Viral titers (infectivity) and ZIKV RNA loads (reverse transcription-polymerase chain reaction) were measured in spiked RBCs before and after S-303 and GSH treatment and confirmed using repetitive passages in cell culture. A mock-treated arm validated the approach by demonstrating stability of the virus (infectivity and RNA load) during the process. The mean ZIKV infectivity titer and RNA load in RBCs were 5.99 ± 0.2 log 50% tissue culture infectious dose (TCID 50 )/mL and 7.75 ± 0.16 log genomic equivalents/mL before inactivation. No infectivity was detected immediately after S-303 and GSH treatment and after five serial passages in cell culture. Complete ZIKV inactivation of more than 5.99 log TCID 50 /mL in RBCs was achieved using S-303 and GSH at levels higher than those found in asymptomatic ZIKV-infected blood donors. Therefore, the S-303 and GSH PI system is promising for mitigating the risk of ZIKV TT. © 2016 The Authors Transfusion published by Wiley Periodicals, Inc. on behalf of AABB.

Inactivation of lipoprotein lipase occurs on the surface of THP-1 macrophages where oligomers of angiopoietin-like protein 4 are formed.

PubMed

Makoveichuk, Elena; Sukonina, Valentina; Kroupa, Olessia; Thulin, Petra; Ehrenborg, Ewa; Olivecrona, Thomas; Olivecrona, Gunilla

2012-08-24

Lipoprotein lipase (LPL) hydrolyzes triglycerides in plasma lipoproteins causing release of fatty acids for metabolic purposes in muscles and adipose tissue. LPL in macrophages in the artery wall may, however, promote foam cell formation and atherosclerosis. Angiopoietin-like protein (ANGPTL) 4 inactivates LPL and ANGPTL4 expression is controlled by peroxisome proliferator-activated receptors (PPAR). The mechanisms for inactivation of LPL by ANGPTL4 was studied in THP-1 macrophages where active LPL is associated with cell surfaces in a heparin-releasable form, while LPL in the culture medium is mostly inactive. The PPARδ agonist GW501516 had no effect on LPL mRNA, but increased ANGPTL4 mRNA and caused a marked reduction of the heparin-releasable LPL activity concomitantly with accumulation of inactive, monomeric LPL in the medium. Intracellular ANGPTL4 was monomeric, while dimers and tetramers of ANGPTL4 were present in the heparin-releasable fraction and medium. GW501516 caused an increase in the amount of ANGPTL4 oligomers on the cell surface that paralleled the decrease in LPL activity. Actinomycin D blocked the effects of GW501516 on ANGPTL4 oligomer formation and prevented the inactivation of LPL. Antibodies against ANGPTL4 interfered with the inactivation of LPL. We conclude that inactivation of LPL in THP-1 macrophages primarily occurs on the cell surface where oligomers of ANGPTL4 are formed. Copyright © 2012 Elsevier Inc. All rights reserved.

Reverse Genetics Approaches for the Development of Influenza Vaccines

PubMed Central

Nogales, Aitor; Martínez-Sobrido, Luis

2016-01-01

Influenza viruses cause annual seasonal epidemics and occasional pandemics of human respiratory disease. Influenza virus infections represent a serious public health and economic problem, which are most effectively prevented through vaccination. However, influenza viruses undergo continual antigenic variation, which requires either the annual reformulation of seasonal influenza vaccines or the rapid generation of vaccines against potential pandemic virus strains. The segmented nature of influenza virus allows for the reassortment between two or more viruses within a co-infected cell, and this characteristic has also been harnessed in the laboratory to generate reassortant viruses for their use as either inactivated or live-attenuated influenza vaccines. With the implementation of plasmid-based reverse genetics techniques, it is now possible to engineer recombinant influenza viruses entirely from full-length complementary DNA copies of the viral genome by transfection of susceptible cells. These reverse genetics systems have provided investigators with novel and powerful approaches to answer important questions about the biology of influenza viruses, including the function of viral proteins, their interaction with cellular host factors and the mechanisms of influenza virus transmission and pathogenesis. In addition, reverse genetics techniques have allowed the generation of recombinant influenza viruses, providing a powerful technology to develop both inactivated and live-attenuated influenza vaccines. In this review, we will summarize the current knowledge of state-of-the-art, plasmid-based, influenza reverse genetics approaches and their implementation to provide rapid, convenient, safe and more effective influenza inactivated or live-attenuated vaccines. PMID:28025504

Ternary Kv4.2 channels recapitulate voltage-dependent inactivation kinetics of A-type K+ channels in cerebellar granule neurons.

PubMed

Amarillo, Yimy; De Santiago-Castillo, Jose A; Dougherty, Kevin; Maffie, Jonathon; Kwon, Elaine; Covarrubias, Manuel; Rudy, Bernardo

2008-04-15

Kv4 channels mediate most of the somatodendritic subthreshold operating A-type current (I(SA)) in neurons. This current plays essential roles in the regulation of spike timing, repetitive firing, dendritic integration and plasticity. Neuronal Kv4 channels are thought to be ternary complexes of Kv4 pore-forming subunits and two types of accessory proteins, Kv channel interacting proteins (KChIPs) and the dipeptidyl-peptidase-like proteins (DPPLs) DPPX (DPP6) and DPP10. In heterologous cells, ternary Kv4 channels exhibit inactivation that slows down with increasing depolarization. Here, we compared the voltage dependence of the inactivation rate of channels expressed in heterologous mammalian cells by Kv4.2 proteins with that of channels containing Kv4.2 and KChIP1, Kv4.2 and DPPX-S, or Kv4.2, KChIP1 and DPPX-S, and found that the relation between inactivation rate and membrane potential is distinct for these four conditions. Moreover, recordings from native neurons showed that the inactivation kinetics of the I(SA) in cerebellar granule neurons has voltage dependence that is remarkably similar to that of ternary Kv4 channels containing KChIP1 and DPPX-S proteins in heterologous cells. The fact that this complex and unique behaviour (among A-type K(+) currents) is observed in both the native current and the current expressed in heterologous cells by the ternary complex containing Kv4, DPPX and KChIP proteins supports the hypothesis that somatically recorded native Kv4 channels in neurons include both types of accessory protein. Furthermore, quantitative global kinetic modelling showed that preferential closed-state inactivation and a weakly voltage-dependent opening step can explain the slowing of the inactivation rate with increasing depolarization. Therefore, it is likely that preferential closed-state inactivation is the physiological mechanism that regulates the activity of both ternary Kv4 channel complexes and native I(SA)-mediating channels.

Isoform-specific tethering links the Golgi ribbon to maintain compartmentalization

PubMed Central

Jarvela, Timothy; Linstedt, Adam D.

2014-01-01

Homotypic membrane tethering by the Golgi reassembly and stacking proteins (GRASPs) is required for the lateral linkage of mammalian Golgi ministacks into a ribbon-like membrane network. Although GRASP65 and GRASP55 are specifically localized to cis and medial/trans cisternae, respectively, it is unknown whether each GRASP mediates cisternae-specific tethering and whether such specificity is necessary for Golgi compartmentalization. Here each GRASP was tagged with KillerRed (KR), expressed in HeLa cells, and inhibited by 1-min exposure to light. Significantly, inactivation of either GRASP unlinked the Golgi ribbon, and the immediate effect of GRASP65-KR inactivation was a loss of cis- rather than trans-Golgi integrity, whereas inactivation of GRASP55-KR first affected the trans- and not the cis-Golgi. Thus each GRASP appears to play a direct and cisternae-specific role in linking ministacks into a continuous membrane network. To test the consequence of loss of cisternae-specific tethering, we generated Golgi membranes with a single GRASP on all cisternae. Remarkably, the membranes exhibited the full connectivity of wild-type Golgi ribbons but were decompartmentalized and defective in glycan processing. Thus the GRASP isoforms specifically link analogous cisternae to ensure Golgi compartmentalization and proper processing. PMID:24227884

Modulation of basal cell fate during productive and transforming HPV-16 infection is mediated by progressive E6-driven depletion of Notch.

PubMed

Kranjec, Christian; Holleywood, Christina; Libert, Diane; Griffin, Heather; Mahmood, Radma; Isaacson, Erin; Doorbar, John

2017-08-01

In stratified epithelia such as the epidermis, homeostasis is maintained by the proliferation of cells in the lower epithelial layers and the concomitant loss of differentiated cells from the epithelial surface. These differentiating keratinocytes progressively stratify and form a self-regenerating multi-layered barrier that protects the underlying dermis. In such tissue, the continual loss and replacement of differentiated cells also limits the accumulation of oncogenic mutations within the tissue. Inactivating mutations in key driver genes, such as TP53 and NOTCH1, reduce the proportion of differentiating cells allowing for the long-term persistence of expanding mutant clones in the tissue. Here we show that through the expression of E6, HPV-16 prevents the early fate commitment of human keratinocytes towards differentiation and confers a strong growth advantage to human keratinocytes. When E6 is expressed either alone or with E7, it promotes keratinocyte proliferation at high cell densities, through the combined inactivation of p53 and Notch1. In organotypic raft culture, the activity of E6 is restricted to the basal layer of the epithelium and is enhanced during the progression from productive to abortive or transforming HPV-16 infection. Consistent with this, the expression of p53 and cleaved Notch1 becomes progressively more disrupted, and is associated with increased basal cell density and reduced commitment to differentiation. The expression of cleaved Notch1 is similarly disrupted also in HPV-16-positive cervical lesions, depending on neoplastic grade. When taken together, these data depict an important role of high-risk E6 in promoting the persistence of infected keratinocytes in the basal and parabasal layers through the inactivation of gene products that are commonly mutated in non-HPV-associated neoplastic squamous epithelia. © 2017 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of Pathological Society of Great Britain and Ireland. © 2017 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of Pathological Society of Great Britain and Ireland.

Thermal inactivation of infectious hematopoietic necrosis and infectious pancreatic necrosis virus

USGS Publications Warehouse

Gosting, L.; Gould, R.W.

1981-01-01

A plaque assay was used to follow the inactivation kinetics of infectious hematopoietic necrosis virus and infectious pancreatic necrosis virus in cell culture media at various temperatures. Inactivation of infectious hematopoietic necrosis virus in a visceral organ slurry was compared with that in culture media.

Subcutaneous immunization with inactivated bacterial components and purified protein of Escherichia coli, Fusobacterium necrophorum and Trueperella pyogenes prevents puerperal metritis in Holstein dairy cows.

PubMed

Machado, Vinícius Silva; Bicalho, Marcela Luccas de Souza; Meira Junior, Enoch Brandão de Souza; Rossi, Rodolfo; Ribeiro, Bruno Leonardo; Lima, Svetlana; Santos, Thiago; Kussler, Arieli; Foditsch, Carla; Ganda, Erika Korzune; Oikonomou, Georgios; Cheong, Soon Hon; Gilbert, Robert Owen; Bicalho, Rodrigo Carvalho

2014-01-01

In this study we evaluate the efficacy of five vaccine formulations containing different combinations of proteins (FimH; leukotoxin, LKT; and pyolysin, PLO) and/or inactivated whole cells (Escherichia coli, Fusobacterium necrophorum, and Trueperella pyogenes) in preventing postpartum uterine diseases. Inactivated whole cells were produced using two genetically distinct strains of each bacterial species (E. coli, F. necrophorum, and T. pyogenes). FimH and PLO subunits were produced using recombinant protein expression, and LKT was recovered from culturing a wild F. necrophorum strain. Three subcutaneous vaccines were formulated: Vaccine 1 was composed of inactivated bacterial whole cells and proteins; Vaccine 2 was composed of proteins only; and Vaccine 3 was composed of inactivated bacterial whole cells only. Two intravaginal vaccines were formulated: Vaccine 4 was composed of inactivated bacterial whole cells and proteins; and Vaccine 5 was composed of PLO and LKT. To evaluate vaccine efficacy, a randomized clinical trial was conducted at a commercial dairy farm; 371 spring heifers were allocated randomly into one of six different treatments groups: control, Vaccine 1, Vaccine 2, Vaccine 3, Vaccine 4 and Vaccine 5. Late pregnant heifers assigned to one of the vaccine groups were each vaccinated twice: at 230 and 260 days of pregnancy. When vaccines were evaluated grouped as subcutaneous and intravaginal, the subcutaneous ones were found to significantly reduce the incidence of puerperal metritis. Additionally, subcutaneous vaccination significantly reduced rectal temperature at 6±1 days in milk. Reproduction was improved for cows that received subcutaneous vaccines. In general, vaccination induced a significant increase in serum IgG titers against all antigens, with subcutaneous vaccination again being more effective. In conclusion, subcutaneous vaccination with inactivated bacterial components and/or protein subunits of E. coli, F. necrophorum and T. pyogenes can prevent puerperal metritis during the first lactation of dairy cows, leading to improved reproduction.

Characterization of slowly inactivating KV{alpha} current in rabbit pulmonary neuroepithelial bodies: effects of hypoxia and nicotine.

PubMed

Fu, Xiao Wen; Nurse, Colin; Cutz, Ernest

2007-10-01

Pulmonary neuroepithelial bodies (NEB) form innervated cell clusters that express voltage-activated currents and function as airway O(2) sensors. We investigated A-type K(+) currents in NEB cells using neonatal rabbit lung slice preparation. The whole cell K(+) current was slowly inactivating with activation threshold of approximately -30 mV. This current was blocked approximately 27% by blood-depressing substance I (BDS-I; 3 microM), a selective blocker of Kv3.4 subunit, and reduced approximately 20% by tetraethylammonium (TEA; 100 microM). The BDS-I-sensitive component had an average peak value of 189 +/- 14 pA and showed fast inactivation kinetics that could be fitted by one-component exponential function with a time constant of (tau1) 77 +/- 10 ms. This Kv slowly inactivating current was also blocked by heteropodatoxin-2 (HpTx-2; 0.2 microM), a blocker of Kv4 subunit. The HpTx-2-sensitive current had an average peak value of 234 +/- 23 pA with a time constant (tau) 82 +/- 11 ms. Hypoxia (Po(2) = 15-20 mmHg) inhibited the slowly inactivating K(+) current by approximately 47%, during voltage steps from -30 to +30 mV, and no further inhibition occurred when TEA was combined with hypoxia. Nicotine at concentrations of 50 and 100 microM suppressed the slowly inactivating K(+) current by approximately 24 and approximately 40%, respectively. This suppression was not reversed by mecamylamine suggesting a direct effect of nicotine on these K(+) channels. In situ hybridization experiments detected expression of mRNAs for Kv3.4 and Kv4.3 subunits, while double-label immunofluorescence confirmed membrane localization of respective channel proteins in NEB cells. These studies suggest that the hypoxia-sensitive current in NEB cells is carried by slowly inactivating A-type K(+) channels, which underlie their oxygen-sensitive potassium currents, and that exposure to nicotine may directly affect their function, contributing to smoking-related lung disease.

Hypothiocyanite produced by human and rat respiratory epithelial cells inactivates extracellular H1N2 influenza A virus.

PubMed

Gingerich, Aaron; Pang, Lan; Hanson, Jarod; Dlugolenski, Daniel; Streich, Rebecca; Lafontaine, Eric R; Nagy, Tamás; Tripp, Ralph A; Rada, Balázs

2016-01-01

Our aim was to study whether an extracellular, oxidative antimicrobial mechanism inherent to tracheal epithelial cells is capable of inactivating influenza H1N2 virus. Epithelial cells were isolated from tracheas of male Sprague-Dawley rats. Both primary human and rat tracheobronchial epithelial cells were differentiated in air-liquid interface cultures. A/swine/Illinois/02860/09 (swH1N2) influenza A virions were added to the apical side of airway cells for 1 h in the presence or absence of lactoperoxidase or thiocyanate. Characterization of rat epithelial cells (morphology, Duox expression) occurred via western blotting, PCR, hydrogen peroxide production measurement and histology. The number of viable virions was determined by plaque assays. Statistical difference of the results was analyzed by ANOVA and Tukey's test. Our data show that rat tracheobronchial epithelial cells develop a differentiated, polarized monolayer with high transepithelial electrical resistance, mucin production and expression of dual oxidases. Influenza A virions are inactivated by human and rat epithelial cells via a dual oxidase-, lactoperoxidase- and thiocyanate-dependent mechanism. Differentiated air-liquid interface cultures of rat tracheal epithelial cells provide a novel model to study airway epithelium-influenza interactions. The dual oxidase/lactoperoxidase/thiocyanate extracellular oxidative system producing hypothiocyanite is a fast and potent anti-influenza mechanism inactivating H1N2 viruses prior to infection of the epithelium.

Recovery from the DNA Replication Checkpoint

PubMed Central

Chaudhury, Indrajit; Koepp, Deanna M.

2016-01-01

Checkpoint recovery is integral to a successful checkpoint response. Checkpoint pathways monitor progress during cell division so that in the event of an error, the checkpoint is activated to block the cell cycle and activate repair pathways. Intrinsic to this process is that once repair has been achieved, the checkpoint signaling pathway is inactivated and cell cycle progression resumes. We use the term “checkpoint recovery” to describe the pathways responsible for the inactivation of checkpoint signaling and cell cycle re-entry after the initial stress has been alleviated. The DNA replication or S-phase checkpoint monitors the integrity of DNA synthesis. When replication stress is encountered, replication forks are stalled, and the checkpoint signaling pathway is activated. Central to recovery from the S-phase checkpoint is the restart of stalled replication forks. If checkpoint recovery fails, stalled forks may become unstable and lead to DNA breaks or unusual DNA structures that are difficult to resolve, causing genomic instability. Alternatively, if cell cycle resumption mechanisms become uncoupled from checkpoint inactivation, cells with under-replicated DNA might proceed through the cell cycle, also diminishing genomic stability. In this review, we discuss the molecular mechanisms that contribute to inactivation of the S-phase checkpoint signaling pathway and the restart of replication forks during recovery from replication stress. PMID:27801838

Gene Editing and Genetic Lung Disease. Basic Research Meets Therapeutic Application.

PubMed

Alapati, Deepthi; Morrisey, Edward E

2017-03-01

Although our understanding of the genetics and pathology of congenital lung diseases such as surfactant protein deficiency, cystic fibrosis, and alpha-1 antitrypsin deficiency is extensive, treatment options are lacking. Because the lung is a barrier organ in direct communication with the external environment, targeted delivery of gene corrective technologies to the respiratory system via intratracheal or intranasal routes is an attractive option for therapy. CRISPR/Cas9 gene-editing technology is a promising approach to repairing or inactivating disease-causing mutations. Recent reports have provided proof of concept by using CRISPR/Cas9 to successfully repair or inactivate mutations in animal models of monogenic human diseases. Potential pulmonary applications of CRISPR/Cas9 gene editing include gene correction of monogenic diseases in pre- or postnatal lungs and ex vivo gene editing of patient-specific airway stem cells followed by autologous cell transplant. Strategies to enhance gene-editing efficiency and eliminate off-target effects by targeting pulmonary stem/progenitor cells and the assessment of short-term and long-term effects of gene editing are important considerations as the field advances. If methods continue to advance rapidly, CRISPR/Cas9-mediated gene editing may provide a novel opportunity to correct monogenic diseases of the respiratory system.

Revealing mechanisms of selective, concentration-dependent potentials of 4-hydroxy-2-nonenal to induce apoptosis in cancer cells through inactivation of membrane-associated catalase.

PubMed

Bauer, Georg; Zarkovic, Neven

2015-04-01

Tumor cells generate extracellular superoxide anions and are protected against superoxide anion-mediated intercellular apoptosis-inducing signaling by the expression of membrane-associated catalase. 4-Hydroxy-2-nonenal (4-HNE), a versatile second messenger generated during lipid peroxidation, has been shown to induce apoptosis selectively in malignant cells. The findings described in this paper reveal the strong, concentration-dependent potential of 4-HNE to specifically inactivate extracellular catalase of tumor cells both indirectly and directly and to consequently trigger apoptosis in malignant cells through superoxide anion-mediated intercellular apoptosis-inducing signaling. Namely, 4-HNE caused apoptosis selectively in NOX1-expressing tumor cells through inactivation of their membrane-associated catalase, thus reactivating subsequent intercellular signaling through the NO/peroxynitrite and HOCl pathways, followed by the mitochondrial pathway of apoptosis. Concentrations of 4-HNE of 1.2 µM and higher directly inactivated membrane-associated catalase of tumor cells, whereas at lower concentrations, 4-HNE triggered a complex amplificatory pathway based on initial singlet oxygen formation through H2O2 and peroxynitrite interaction. Singlet-oxygen-dependent activation of the FAS receptor and caspase-8 increased superoxide anion generation by NOX1 and amplification of singlet oxygen generation, which allowed singlet-oxygen-dependent inactivation of catalase. 4-HNE and singlet oxygen cooperate in complex autoamplificatory loops during this process. The finding of these novel anticancer pathways may be useful for understanding the role of 4-HNE in the control of malignant cells and for the optimization of ROS-dependent therapeutic approaches including antioxidant treatments. Copyright © 2015 Elsevier Inc. All rights reserved.

Comparison of Heat Inactivation and Cell Disruption Protocols for Identification of Mycobacteria from Solid Culture Media by Use of Vitek Matrix-Assisted Laser Desorption Ionization–Time of Flight Mass Spectrometry

PubMed Central

Machen, Alexandra; Kobayashi, Miwako; Connelly, Mary Robin

2013-01-01

Two novel protocols for inactivation and extraction were developed and used to identify 107 Mycobacterium clinical isolates, including Mycobacterium tuberculosis complex, from solid cultures using Vitek matrix-assisted laser desorption ionization–time of flight (MALDI-TOF) mass spectrometry. The protocol using heat inactivation with sonication and cell disruption with glass beads resulted in 82.2% and 88.8% species and genus level identifications, respectively. PMID:24068013

Resolution of Conflicting Signals at the Single-Cell Level in the Regulation of Cyanobacterial Photosynthesis and Nitrogen Fixation.

PubMed

Mohr, Wiebke; Vagner, Tomas; Kuypers, Marcel M M; Ackermann, Martin; Laroche, Julie

2013-01-01

Unicellular, diazotrophic cyanobacteria temporally separate dinitrogen (N2) fixation and photosynthesis to prevent inactivation of the nitrogenase by oxygen. This temporal segregation is regulated by a circadian clock with oscillating activities of N2 fixation in the dark and photosynthesis in the light. On the population level, this separation is not always complete, since the two processes can overlap during transitions from dark to light. How do single cells avoid inactivation of nitrogenase during these periods? One possibility is that phenotypic heterogeneity in populations leads to segregation of the two processes. Here, we measured N2 fixation and photosynthesis of individual cells using nanometer-scale secondary ion mass spectrometry (nanoSIMS) to assess both processes in a culture of the unicellular, diazotrophic cyanobacterium Crocosphaera watsonii during a dark-light and a continuous light phase. We compared single-cell rates with bulk rates and gene expression profiles. During the regular dark and light phases, C. watsonii exhibited the temporal segregation of N2 fixation and photosynthesis commonly observed. However, N2 fixation and photosynthesis were concurrently measurable at the population level during the subjective dark phase in which cells were kept in the light rather than returned to the expected dark phase. At the single-cell level, though, cells discriminated against either one of the two processes. Cells that showed high levels of photosynthesis had low nitrogen fixing activities, and vice versa. These results suggest that, under ambiguous environmental signals, single cells discriminate against either photosynthesis or nitrogen fixation, and thereby might reduce costs associated with running incompatible processes in the same cell.

Resolution of Conflicting Signals at the Single-Cell Level in the Regulation of Cyanobacterial Photosynthesis and Nitrogen Fixation

PubMed Central

Mohr, Wiebke; Vagner, Tomas; Kuypers, Marcel M. M.; Ackermann, Martin; LaRoche, Julie

2013-01-01

Unicellular, diazotrophic cyanobacteria temporally separate dinitrogen (N2) fixation and photosynthesis to prevent inactivation of the nitrogenase by oxygen. This temporal segregation is regulated by a circadian clock with oscillating activities of N2 fixation in the dark and photosynthesis in the light. On the population level, this separation is not always complete, since the two processes can overlap during transitions from dark to light. How do single cells avoid inactivation of nitrogenase during these periods? One possibility is that phenotypic heterogeneity in populations leads to segregation of the two processes. Here, we measured N2 fixation and photosynthesis of individual cells using nanometer-scale secondary ion mass spectrometry (nanoSIMS) to assess both processes in a culture of the unicellular, diazotrophic cyanobacterium Crocosphaera watsonii during a dark-light and a continuous light phase. We compared single-cell rates with bulk rates and gene expression profiles. During the regular dark and light phases, C. watsonii exhibited the temporal segregation of N2 fixation and photosynthesis commonly observed. However, N2 fixation and photosynthesis were concurrently measurable at the population level during the subjective dark phase in which cells were kept in the light rather than returned to the expected dark phase. At the single-cell level, though, cells discriminated against either one of the two processes. Cells that showed high levels of photosynthesis had low nitrogen fixing activities, and vice versa. These results suggest that, under ambiguous environmental signals, single cells discriminate against either photosynthesis or nitrogen fixation, and thereby might reduce costs associated with running incompatible processes in the same cell. PMID:23805199

Application of carrier testing to genetic counseling for X-linked agammaglobulinemia

DOE Office of Scientific and Technical Information (OSTI.GOV)

Allen, R.C.; Nachtman, R.G.; Belmont, J.W.

Bruton X-linked agammaglobulinemia (XLA) is a phenotypically recessive genetic disorder of B lymphocyte development. Female carriers of XLA, although asymptomatic, have a characteristic B cell lineage-specific skewing of the pattern of X inactivation. Skewing apparently results from defective growth and maturation of B cell precursors bearing a mutant active X chromosome. In this study, carrier status was tested in 58 women from 22 families referred with a history of agammaglobulinemia. Primary carrier analysis to examine patterns of X inactivation in CD19[sup +] peripheral blood cells (B lymphocytes) was conducted using quantitative PCR at the androgen-receptor locus. Obligate carriers of XLAmore » demonstrated >95% skewing of X inactivation in peripheral blood CD19[sup +] cells but not in CD19[sup [minus

Study of Rpl22 in MDS and AML

DTIC Science & Technology

2014-10-01

Previously I had determined that Rpl22 functions as a haploinsufficient tumor suppressor in mouse T - cell lymphoma model by activating the NF B and...preclinical animal models of T cell malignancy as well as in the manipulation of development of primary hematopoietic stem cells in vitro and in vivo...allelic inactivation can accelerate the development of T - cell lymphoma in a mouse model where disease is driven by a MyrAkt2-transgene. Rpl22 inactivation

High pressure inactivation of human norovirus-like particles: evidence that the capsid of human norovirus is highly pressure resistant

USDA-ARS?s Scientific Manuscript database

High pressure processing (HPP) is a promising non-thermal technology to inactivate foodborne viruses. However, the effectiveness of HPP on inactivating human norovirus (HuNoV), the leading cause of acute gastroenteritis, is unknown because it cannot be propagated in cell culture. Therefore, developi...

Differentiation-dependent Requirement of Tsix long non-coding RNA in Imprinted X-chromosome Inactivation

PubMed Central

Maclary, Emily; Buttigieg, Emily; Hinten, Michael; Gayen, Srimonta; Harris, Clair; Sarkar, Mrinal Kumar; Purushothaman, Sonya; Kalantry, Sundeep

2014-01-01

Imprinted X-inactivation is a paradigm of mammalian transgenerational epigenetic regulation resulting in silencing of genes on the paternally-inherited X-chromosome. The pre-programmed fate of the X-chromosomes is thought to be controlled in cis by the parent-of-origin-specific expression of two long non-coding RNAs, Tsix and Xist, in mice. Exclusive expression of Tsix from the maternal–X has implicated it as the instrument through which the maternal germline prevents inactivation of the maternal–X in the offspring. Here, we show that Tsix is dispensable for inhibiting Xist and X-inactivation in the early embryo and in cultured stem cells of extra-embryonic lineages. Tsix is instead required to prevent Xist expression as trophectodermal progenitor cells differentiate. Despite induction of wild-type Xist RNA and accumulation of histone H3-K27me3, many Tsix-mutant X-chromosomes fail to undergo ectopic X-inactivation. We propose a novel model of lncRNA function in imprinted X-inactivation that may also apply to other genomically imprinted loci. PMID:24979243

Intracellular Cs+ activates the PKA pathway, revealing a fast, reversible, Ca2+-dependent inactivation of L-type Ca2+ current.

PubMed

Brette, Fabien; Lacampagne, Alain; Sallé, Laurent; Findlay, Ian; Le Guennec, Jean-Yves

2003-08-01

Inactivation of the L-type Ca2+ current (ICaL) was studied in isolated guinea pig ventricular myocytes with different ionic solutions. Under basal conditions, ICaL of 82% of cells infused with Cs+-based intracellular solutions showed enhanced amplitude with multiphasic decay and diastolic depolarization-induced facilitation. The characteristics of ICaL in this population of cells were not due to contamination by other currents or an artifact. These phenomena were reduced by ryanodine, caffeine, cyclopiazonic acid, the protein kinase A inhibitor H-89, and the cAMP-dependent protein kinase inhibitor. Forskolin and isoproterenol increased ICaL by only approximately 60% in these cells. Cells infused with either N-methyl-d-glucamine or K+-based intracellular solutions did not show multiphasic decay or facilitation under basal conditions. Isoproterenol increased ICaL by approximately 200% in these cells. In conclusion, we show that multiphasic inactivation of ICaL is due to Ca2+-dependent inactivation that is reversible on a time scale of tens of milliseconds. Cs+ seems to activate the cAMP-dependent protein kinase pathway when used as a substitute for K+ in the pipette solution.

Intracellular Fixation Buffer Inactivates Newcastle Disease Virus in Chicken Allantoic Fluid, Macrophages and Splenocytes for Immune Assessment During Infection

USDA-ARS?s Scientific Manuscript database

Inactivation of Newcastle disease virus (NDV) has been routinely achieved with heat, ß-propiolactone, binary ethylenimine, ultraviolet light and formalin, however these strategies have not been validated for cell surface ligand or receptor phenotype in viral-infected chicken immune cells. To study ...

Na/K pump inactivation, subsarcolemmal Na measurements, and cytoplasmic ion turnover kinetics contradict restricted Na spaces in murine cardiac myocytes

PubMed Central

Lu, Fang-Min

2017-01-01

Decades ago, it was proposed that Na transport in cardiac myocytes is modulated by large changes in cytoplasmic Na concentration within restricted subsarcolemmal spaces. Here, we probe this hypothesis for Na/K pumps by generating constitutive transsarcolemmal Na flux with the Na channel opener veratridine in whole-cell patch-clamp recordings. Using 25 mM Na in the patch pipette, pump currents decay strongly during continuous activation by extracellular K (τ, ∼2 s). In contradiction to depletion hypotheses, the decay becomes stronger when pump currents are decreased by hyperpolarization. Na channel currents are nearly unchanged by pump activity in these conditions, and conversely, continuous Na currents up to 0.5 nA in magnitude have negligible effects on pump currents. These outcomes are even more pronounced using 50 mM Li as a cytoplasmic Na congener. Thus, the Na/K pump current decay reflects mostly an inactivation mechanism that immobilizes Na/K pump charge movements, not cytoplasmic Na depletion. When channel currents are increased beyond 1 nA, models with unrestricted subsarcolemmal diffusion accurately predict current decay (τ ∼15 s) and reversal potential shifts observed for Na, Li, and K currents through Na channels opened by veratridine, as well as for Na, K, Cs, Li, and Cl currents recorded in nystatin-permeabilized myocytes. Ion concentrations in the pipette tip (i.e., access conductance) track without appreciable delay the current changes caused by sarcolemmal ion flux. Importantly, cytoplasmic mixing volumes, calculated from current decay kinetics, increase and decrease as expected with osmolarity changes (τ >30 s). Na/K pump current run-down over 20 min reflects a failure of pumps to recover from inactivation. Simulations reveal that pump inactivation coupled with Na-activated recovery enhances the rapidity and effectivity of Na homeostasis in cardiac myocytes. In conclusion, an autoregulatory mechanism enhances cardiac Na/K pump activity when cytoplasmic Na rises and suppresses pump activity when cytoplasmic Na declines. PMID:28606910

Factors affecting UV/H2O2 inactivation of Bacillus atrophaeus spores in drinking water.

PubMed

Zhang, Yongji; Zhang, Yiqing; Zhou, Lingling; Tan, Chaoqun

2014-05-05

This study aims at estimating the performance of the Bacillus atrophaeus spores inactivation by the UV treatment with addition of H2O2. The effect of factors affecting the inactivation was investigated, including initial H2O2 dose, UV irradiance, initial cell density, initial solution pH and various inorganic anions. Under the experimental conditions, the B. atrophaeus spores inactivation followed both the modified Hom Model and the Chick's Model. The results revealed that the H2O2 played dual roles in the reactions, while the optimum reduction of 5.88lg was received at 0.5mM H2O2 for 10min. The inactivation effect was affected by the UV irradiance, while better inactivation effect was achieved at higher irradiance. An increase in the initial cell density slowed down the inactivation process. A slight acid condition at pH 5 was considered as the optimal pH value. The inactivation effect within 10min followed the order of pH 5>pH 7>pH 9>pH 3>pH 11. The effects of three added inorganic anions were investigated and compared, including sulfate (SO4(2)(-)), nitrate (NO3(-)) and carbonate (CO3(2)(-)). The sequence of inactivation effect within 10min followed the order of control group>SO4(2)(-)>NO3(-)>CO3(2)(-). Copyright © 2014 Elsevier B.V. All rights reserved.

PKCδ phosphorylation is an upstream event of GSK3 inactivation-mediated ROS generation in TGF-β1-induced senescence.

PubMed

Byun, H-O; Jung, H-J; Kim, M-J; Yoon, G

2014-09-01

Transforming growth factor β1 (TGF-β1) induces Mv1Lu cell senescence through inactivating glycogen synthase kinase 3 (GSK3), thereby inactivating complex IV and increasing intracellular ROS. In the present study, we identified protein kinase C delta (PKCδ) as an upstream regulator of GSK3 inactivation in this mechanism of TGF-β1-induced senescence. When Mv1Lu cells were exposed to TGF-β1, PKCδ phosphorylation simultaneously increased with GSK3 phosphorylation, and then AKT and ERK were phosphorylated. AKT phosphorylation and Smad signaling were independent of GSK3 phosphorylation, but ERK phosphorylation was downstream of GSK3 inactivation. TGF-β1-triggered GSK3 phosphorylation was blocked by inhibition of PKCδ, using its pharmacological inhibitor, Rottlerin, or overexpression of a dominant negative PKCδ mutant, but GSK3 inhibition with SB415286 did not alter PKCδ phosphorylation. Activation of PKCδ by PMA delayed cell growth and increased intracellular ROS level, but did not induce senescent phenotypes. In addition, overexpression of wild type or a constitutively active PKCδ mutant was enough to delay cell growth and decrease the mitochondrial oxygen consumption rate and complex IV activity, but weakly induce senescence. However, PMA treatment on Mv1Lu cells, which overexpress wild type and constitutively active PKCδ mutants, effectively induced senescence. These results indicate that PKCδ plays a key role in TGF-β1-induced senescence of Mv1Lu cells through the phosphorylation of GSK3, thereby triggering mitochondrial complex IV dysfunction and intracellular ROS generation.

P-body-induced inactivation of let-7a miRNP prevents the death of growth factor-deprived neuronal cells.

PubMed

Patranabis, Somi; Bhattacharyya, Suvendra Nath

2018-03-01

RNA processing bodies (P-bodies) are cytoplasmic RNA granules in eukaryotic cells that regulate gene expression by executing the translation suppression and degradation of mRNAs that are targeted to these bodies. P-bodies can also serve as storage sites for translationally repressed mRNAs both in mammalian cells and yeast cells. In this report, a unique role of mammalian P-bodies is documented. Depletion of P-body components dedifferentiate nerve growth factor-treated PC12 cells, whereas ectopic expression of P-body components induces the neuronal differentiation of precursor cells. Trophic factor withdrawal from differentiated cells induces a decrease in cellular P-body size and numbers that are coupled with dedifferentiation and cell death. Here, we report how the expression of P-body proteins-by ensuring the phosphorylation of argonaute protein 2 and the subsequent inactivation let-7a miRNPs-prevents the apoptotic death of growth factor-depleted neuronal cells.-Patranabis, S., Bhattacharyya, S. N. P-body-induced inactivation of let-7a miRNP prevents the death of growth factor-deprived neuronal cells.

A cell cycle-independent, conditional gene inactivation strategy for differentially tagging wild-type and mutant cells.

PubMed

Nagarkar-Jaiswal, Sonal; Manivannan, Sathiya N; Zuo, Zhongyuan; Bellen, Hugo J

2017-05-31

Here, we describe a novel method based on intronic MiMIC insertions described in Nagarkar-Jaiswal et al. (2015) to perform conditional gene inactivation in Drosophila . Mosaic analysis in Drosophila cannot be easily performed in post-mitotic cells. We therefore, therefore, developed Flip-Flop, a flippase -dependent in vivo cassette-inversion method that marks wild-type cells with the endogenous EGFP-tagged protein, whereas mutant cells are marked with mCherry upon inversion. We document the ease and usefulness of this strategy in differential tagging of wild-type and mutant cells in mosaics. We use this approach to phenotypically characterize the loss of SNF4Aγ , encoding the γ subunit of the AMP Kinase complex. The Flip-Flop method is efficient and reliable, and permits conditional gene inactivation based on both spatial and temporal cues, in a cell cycle-, and developmental stage-independent fashion, creating a platform for systematic screens of gene function in developing and adult flies with unprecedented detail.

beta subunits influence the biophysical and pharmacological differences between P- and Q-type calcium currents expressed in a mammalian cell line.

PubMed

Moreno, H; Rudy, B; Llinás, R

1997-12-09

Human epithelial kidney cells (HEK) were prepared to coexpress alpha1A, alpha2delta with different beta calcium channel subunits and green fluorescence protein. To compare the calcium currents observed in these cells with the native neuronal currents, electrophysiological and pharmacological tools were used conjointly. Whole-cell current recordings of human epithelial kidney alpha1A-transfected cells showed small inactivating currents in 80 mM Ba2+ that were relatively insensitive to calcium blockers. Coexpression of alpha1A, betaIb, and alpha2delta produced a robust inactivating current detected in 10 mM Ba2+, reversibly blockable with low concentration of omega-agatoxin IVA (omega-Aga IVA) or synthetic funnel-web spider toxin (sFTX). Barium currents were also supported by alpha1A, beta2a, alpha2delta subunits, which demonstrated the slowest inactivation and were relatively insensitive to omega-Aga IVA and sFTX. Coexpression of beta3 with the same combination as above produced inactivating currents also insensitive to low concentration of omega-Aga IVA and sFTX. These data indicate that the combination alpha1A, betaIb, alpha2delta best resembles P-type channels given the rate of inactivation and the high sensitivity to omega-Aga IVA and sFTX. More importantly, the specificity of the channel blocker is highly influenced by the beta subunit associated with the alpha1A subunit.

Addition of ethanol to supercritical carbon dioxide enhances the inactivation of bacterial spores in the biofilm of Bacillus cereus.

PubMed

Park, Hyong Seok; Choi, Hee Jung; Kim, Myoung-Dong; Kim, Kyoung Heon

2013-09-02

Supercritical carbon dioxide (SC-CO2) was used to inactivate Bacillus cereus spores inside biofilms, which were grown on stainless steel. SC-CO2 treatment was tested using various conditions, such as pressure treatment (10-30 MPa), temperature (35-60°C), and time (10-120 min). B. cereus vegetative cells in the biofilm were completely inactivated by treatment with SC-CO2 at 10 MPa and at 35°C for 5 min. However, SC-CO2 alone did not inactivate spores in biofilm even after the treatment time was extended to 120 min. When ethanol was used as a cosolvent with SC-CO2 in the SC-CO2 treatment using only 2-10 ml of ethanol in 100ml of SC-CO2 vessel for 60-90 min of treatment time at 10 MPa and 60°C, B. cereus spores in the biofilm were found to be completely inactivated in the colony-forming test. We also assessed the viability of SC-CO2-treated bacterial spores and vegetative cells in the biofilm by staining with SYTO 9 and propidium iodide. The membrane integrity of the vegetative cells was completely lost, while the integrity of the membrane was still maintained in most spores. However, when SC-CO2 along with ethanol was used, both vegetative cells and spores lost their membrane integrity, indicating that the use of ethanol as a cosolvent with SC-CO2 is efficient in inactivating the bacterial spores in the biofilm. © 2013.

Reactive oxygen species in plasma against E. coli cells survival rate

NASA Astrophysics Data System (ADS)

Zhou, Ren-Wu; Zhang, Xian-Hui; Zong, Zi-Chao; Li, Jun-Xiong; Yang, Zhou-Bin; Liu, Dong-Ping; Yang, Si-Ze

2015-08-01

In this paper, we report on the contrastive analysis of inactivation efficiency of E. coli cells in solution with different disinfection methods. Compared with the hydrogen peroxide solution and the ozone gas, the atmospheric-pressure He plasma can completely kill the E. coli cells in the shortest time. The inactivation efficiency of E. coli cells in solution can be well described by using the chemical reaction rate model. X-ray photoelectron spectroscopy (XPS) analysis shows that the C-O or C=O content of the inactivated E. coli cell surface by plasma is predominantly increased, indicating the quantity of oxygen-containing species in plasma is more than those of two other methods, and then the C-C or C-H bonds can be broken, leading to the etching of organic compounds. Analysis also indicates that plasma-generated species can play a crucial role in the inactivation process by their direct reactions or the decompositions of reactive species, such as ozone into OH radicals in water, then reacting with E. coli cells. Project supported by the Natural Science Foundation of Fujian Province, China (Grant No. 2014J01025), the National Natural Science Foundation of China (Grant No. 11275261), and the Funds from the Fujian Provincial Key Laboratory for Plasma and Magnetic Resonance, China.

Inactivation of the inhA-Encoded Fatty Acid Synthase II (FASII) Enoyl-Acyl Carrier Protein Reductase Induces Accumulation of the FASI End Products and Cell Lysis of Mycobacterium smegmatis

PubMed Central

Vilchèze, Catherine; Morbidoni, Hector R.; Weisbrod, Torin R.; Iwamoto, Hiroyuki; Kuo, Mack; Sacchettini, James C.; Jacobs, William R.

2000-01-01

The mechanism of action of isoniazid (INH), a first-line antituberculosis drug, is complex, as mutations in at least five different genes (katG, inhA, ahpC, kasA, and ndh) have been found to correlate with isoniazid resistance. Despite this complexity, a preponderance of evidence implicates inhA, which codes for an enoyl-acyl carrier protein reductase of the fatty acid synthase II (FASII), as the primary target of INH. However, INH treatment of Mycobacterium tuberculosis causes the accumulation of hexacosanoic acid (C26:0), a result unexpected for the blocking of an enoyl-reductase. To test whether inactivation of InhA is identical to INH treatment of mycobacteria, we isolated a temperature-sensitive mutation in the inhA gene of Mycobacterium smegmatis that rendered InhA inactive at 42°C. Thermal inactivation of InhA in M. smegmatis resulted in the inhibition of mycolic acid biosynthesis, a decrease in hexadecanoic acid (C16:0) and a concomitant increase of tetracosanoic acid (C24:0) in a manner equivalent to that seen in INH-treated cells. Similarly, INH treatment of Mycobacterium bovis BCG caused an inhibition of mycolic acid biosynthesis, a decrease in C16:0, and a concomitant accumulation of C26:0. Moreover, the InhA-inactivated cells, like INH-treated cells, underwent a drastic morphological change, leading to cell lysis. These data show that InhA inactivation, alone, is sufficient to induce the accumulation of saturated fatty acids, cell wall alterations, and cell lysis and are consistent with InhA being a primary target of INH. PMID:10869086

The comprehensive electrophysiological study of curcuminoids on delayed-rectifier K+ currents in insulin-secreting cells.

PubMed

Kuo, Ping-Chung; Yang, Chia-Jung; Lee, Yu-Chi; Chen, Pei-Chun; Liu, Yen-Chin; Wu, Sheng-Nan

2018-01-15

Curcumin (CUR) has been demonstrated to induce insulin release from pancreatic β-cells; however, how curcuminoids (including demethoxycurcumin (DMC) and bisdemethoxycurcumin (BDMC)) exert any possible effects on membrane ion currents inherently in insulin-secreting cells remains largely unclear. The effects of CUR and other structurally similar curcuminoids on ion currents in rat insulin-secreting (INS-1) insulinoma cells were therefore investigated in this study. The effects of these compounds on ionic currents and membrane potential were studied by patch-clamp technique. CUR suppressed the amplitude of delayed-rectifier K + current (I K(DR) ) in a time-, state- and concentration-dependent manner in these cells and the inhibition was not reversed by diazoxide, nicorandil or chlorotoxin. The value of dissociation constant for CUR-induced suppression of I K(DR) in INS-1 cells was 1.26μM. Despite the inability of CUR to alter the activation rate of I K(DR) , it accelerated current inactivation elicited by membrane depolarization. Increasing CUR concentrations shifted the inactivation curve of I K(DR) to hyperpolarized potential and slowed the recovery of I K(DR) inactivation. CUR, DMC, and BDMC all exerted depressant actions on I K(DR) amplitude to a similar magnitude, although DMC and BDMC did not increase current inactivation clearly. CUR slightly suppressed the peak amplitude of voltage-gated Na + current. CUR, DMC and BDMC depolarized the resting potential and increased firing frequency of action potentials. The CUR-mediated decrease of I K(DR) and the increase of current inactivation also occurred in βTC-6 INS-1 cells. Taken these results together, these effects may be one of the possible mechanisms contributing their insulin-releasing effect. Copyright © 2017 Elsevier B.V. All rights reserved.

Inactivation, DNA double strand break induction and their rejoining in bacterial cells irradiated with heavy ions

NASA Technical Reports Server (NTRS)

Schaefer, M.; Zimmermann, H.; Schmitz, C.

1994-01-01

Besides inactivation one of the major interests in our experiments is to study the primary damage in the DNA double strand breaks (DSB) after heavy ion irradiation. These damages lead not only to cell death but also under repair activities to mutations. In further experiments we have investigated the inactivation with two different strains of Deinococcus radiodurans (R1, Rec 30) and the induction of DSB as well as the rejoining of DSB in stationary cells of E. coli (strain B/r) irradiated with radiations of different quality. In the latter case irradiations were done so that the cell survival was roughly at the same level. We measured the DSB using the pulse field gelelectrophoresis which allows to separate between intact (circular) and damaged (linear) DNA. The irradiated cells were transferred to NB medium and incubated for different times to allow rejoining.

Ftx is a non-coding RNA which affects Xist expression and chromatin structure within the X-inactivation center region.

PubMed

Chureau, Corinne; Chantalat, Sophie; Romito, Antonio; Galvani, Angélique; Duret, Laurent; Avner, Philip; Rougeulle, Claire

2011-02-15

X chromosome inactivation (XCI) is an essential epigenetic process which involves several non-coding RNAs (ncRNAs), including Xist, the master regulator of X-inactivation initiation. Xist is flanked in its 5' region by a large heterochromatic hotspot, which contains several transcription units including a gene of unknown function, Ftx (five prime to Xist). In this article, we describe the characterization and functional analysis of murine Ftx. We present evidence that Ftx produces a conserved functional long ncRNA, and additionally hosts microRNAs (miR) in its introns. Strikingly, Ftx partially escapes X-inactivation and is upregulated specifically in female ES cells at the onset of X-inactivation, an expression profile which closely follows that of Xist. We generated Ftx null ES cells to address the function of this gene. In these cells, only local changes in chromatin marks are detected within the hotspot, indicating that Ftx is not involved in the global maintenance of the heterochromatic structure of this region. The Ftx mutation, however, results in widespread alteration of transcript levels within the X-inactivation center (Xic) and particularly important decreases in Xist RNA levels, which were correlated with increased DNA methylation at the Xist CpG island. Altogether our results indicate that Ftx is a positive regulator of Xist and lead us to propose that Ftx is a novel ncRNA involved in XCI.

Preserved function of the plasma membrane calcium pump of red blood cells from diabetic subjects with high levels of glycated haemoglobin.

PubMed

Bookchin, Robert M; Etzion, Zipora; Lew, Virgilio L; Tiffert, Teresa

2009-03-01

The activity of the plasma membrane Ca(2+)-pump decreases steeply throughout the 120 days lifespan of normal human red blood cells. Experiments with isolated membrane preparations showed that glycation of a lysine residue near the catalytic site of the pump ATPase had a powerful inhibitory effect. This prompted the question of whether glycation is the mechanism of age-related decline in pump activity in vivo. It is important to investigate this mechanism because the Ca(2+) pump is a major regulator of Ca(2+) homeostasis in all cells. Its impaired activity in diabetic patients, continuously exposed to high glycation rates, may thus contribute to varied tissue pathology in this disease. We measured Ca(2+)-pump activity as a function of red cell age in red cells from diabetics continuously exposed to high glucose concentrations, as documented by their high mean levels of glycated haemoglobin. The distribution of Ca(2+)-pump activities was indistinguishable from that in non-diabetics, and the pattern of activity decline with cell age in the diabetics' red cells was identical to that observed in red cells from non-diabetics. These results indicate that in intact cells the Ca(2+) pump is protected from glycation-induced inactivation.

Base-Resolution Analysis of DNA Methylation Patterns Downstream of Dnmt3a in Mouse Naïve B Cells.

PubMed

Duncan, Christopher G; Kondilis-Mangum, Hrisavgi D; Grimm, Sara A; Bushel, Pierre R; Chrysovergis, Kaliopi; Roberts, John D; Tyson, Frederick L; Merrick, B Alex; Wade, Paul A

2018-03-02

The DNA methyltransferase, Dnmt3a , is dynamically regulated throughout mammalian B cell development and upon activation by antigenic stimulation. Dnmt3a inactivation in hematopoietic stem cells has been shown to drive B cell-related malignancies, including chronic lymphocytic leukemia, and associates with specific DNA methylation patterns in transformed cells. However, while it is clear that inactivation of Dnmt3a in hematopoietic stem cells has profound functional effects, the consequences of Dnmt3a inactivation in cells of the B lineage are unclear. To assess whether loss of Dnmt3a at the earliest stages of B cell development lead to DNA methylation defects that might impair function, we selectively inactivated Dnmt3a early in mouse B cell development and then utilized whole genome bisulfite sequencing to generate base-resolution profiles of Dnmt3a +/+ and Dnmt3a -/- naïve splenic B cells. Overall, we find that global methylation patterns are largely consistent between Dnmt3a +/+ and Dnmt3a -/- naïve B cells, indicating a minimal functional effect of DNMT3A in mature B cells. However, loss of Dnmt3a induced 449 focal DNA methylation changes, dominated by loss-of-methylation events. Regions found to be hypomethylated in Dnmt3a -/- naïve splenic B cells were enriched in gene bodies of transcripts expressed in B cells, a fraction of which are implicated in B cell-related disease. Overall, the results from this study suggest that factors other than Dnmt3a are the major drivers for methylome maintenance in B cell development. Copyright © 2018 Duncan et al.

Light based technologies for microbial inactivation of liquids, bead surfaces and powdered infant formula.

PubMed

Arroyo, Cristina; Dorozko, Anna; Gaston, Edurne; O'Sullivan, Michael; Whyte, Paul; Lyng, James G

2017-10-01

This study evaluates the potential of continuous wave Ultraviolet C light (UV-C) and broad-spectrum intense pulsed light (in this study referred to as High Intensity Light Pulses, HILP) for the inactivation of pathogens of public concern in powdered infant formula (PIF) producers. To achieve this goal a sequential set of experiments were performed, firstly in clear liquid media, secondly on the surface of spherical beads under agitation and, finally in PIF. L. innocua was the most sensitive microorganism to both technologies under all conditions studied with reductions exceeding 4 log 10 cycles in PIF. In the clear liquid medium, the maximum tolerance to light was observed for C. sakazakii against UV-C light and for B. subtilis spores against HILP, with a fluence of approximately 17 mJ/cm 2 required for a 1 log 10 cycle inactivation (D value) of each species. In PIF it was possible to inactivate >99% of the vegetative cell populations by HILP with a fluence of 199 mJ/cm 2 and of B. subtilis spores by doubling the fluence. By contrast, for UV-C treatments a fluence of 2853 mJ/cm 2 was needed for 99.9% reduction of C. sakazakii, which was the most light-resistant microorganism to UV-C. Results here obtained clearly show the potential for light-based interventions to improve PIF microbiological safety. Copyright © 2017 Elsevier Ltd. All rights reserved.

TNF-α Decreases VEGF Secretion in Highly Polarized RPE Cells but Increases It in Non-Polarized RPE Cells Related to Crosstalk between JNK and NF-κB Pathways

PubMed Central

Terasaki, Hiroto; Kase, Satoru; Shirasawa, Makoto; Otsuka, Hiroki; Hisatomi, Toshio; Sonoda, Shozo; Ishida, Susumu; Ishibashi, Tatsuro; Sakamoto, Taiji

2013-01-01

Asymmetrical secretion of vascular endothelial growth factor (VEGF) by retinal pigment epithelial (RPE) cells in situ is critical for maintaining the homeostasis of the retina and choroid. VEGF is also involved in the development and progression of age-related macular degeneration (AMD). We studied the effect of tumor necrosis factor-α (TNF-α) on the secretion of VEGF in polarized and non-polarized RPE cells (P-RPE cells and N-RPE cells, respectively) in culture and in situ in rats. A subretinal injection of TNF-α caused a decrease in VEGF expression and choroidal atrophy. Porcine RPE cells were seeded on Transwell™ filters, and their maturation and polarization were confirmed by the asymmetrical VEGF secretion and trans electrical resistance. Exposure to TNF-α decreased the VEGF secretion in P-RPE cells but increased it in N-RPE cells in culture. TNF-α inactivated JNK in P-RPE cells but activated it in N-RPE cells, and TNF-α activated NF-κB in P-RPE cells but not in N-RPE cells. Inhibition of NF-κB activated JNK in both types of RPE cells indicating crosstalk between JNK and NF-κB. TNF-α induced the inhibitory effects of NF-κB on JNK in P-RPE cells because NF-κB is continuously inactivated. In N-RPE cells, however, it was not evident because NF-κB was already activated. The basic activation pattern of JNK and NF-κB and their crosstalk led to opposing responses of RPE cells to TNF-α. These results suggest that VEGF secretion under inflammatory conditions depends on cellular polarization, and the TNF-α-induced VEGF down-regulation may result in choroidal atrophy in polarized physiological RPE cells. TNF-α-induced VEGF up-regulation may cause neovascularization by non-polarized or non-physiological RPE cells. PMID:23922887

Biochemical transformation of mouse cells by herpes simplex virus type 2: enhancement by means of low-level photodynamic treatment.

PubMed Central

Verwoerd, D W; Rapp, F

1978-01-01

The biochemical transformation of thymidine kinase-deficient cells by UV-inactivated herpes simplex virus is enhanced by low-level photodynamic treatment of the infected cells. At the concentration of proflavine used, the virus was not inactivated and both virus and cellular DNA syntheses were only marginally inhibited. The observed enhancement of the transfer of a virus gene to the cell genome suggests a possible cocarcinogenic role for photodynamically active dyes at very low concentrations. PMID:206727

Singlet oxygen treatment of tumor cells triggers extracellular singlet oxygen generation, catalase inactivation and reactivation of intercellular apoptosis-inducing signaling☆

PubMed Central

Riethmüller, Michaela; Burger, Nils; Bauer, Georg

2015-01-01

Intracellular singlet oxygen generation in photofrin-loaded cells caused cell death without discrimination between nonmalignant and malignant cells. In contrast, extracellular singlet oxygen generation caused apoptosis induction selectively in tumor cells through singlet oxygen-mediated inactivation of tumor cell protective catalase and subsequent reactivation of intercellular ROS-mediated apoptosis signaling through the HOCl and the NO/peroxynitrite signaling pathway. Singlet oxygen generation by extracellular photofrin alone was, however, not sufficient for optimal direct inactivation of catalase, but needed to trigger the generation of cell-derived extracellular singlet oxygen through the interaction between H2O2 and peroxynitrite. Thereby, formation of peroxynitrous acid, generation of hydroxyl radicals and formation of perhydroxyl radicals (HO2.) through hydroxyl radical/H2O2 interaction seemed to be required as intermediate steps. This amplificatory mechanism led to the formation of singlet oxygen at a sufficiently high concentration for optimal inactivation of membrane-associated catalase. At low initial concentrations of singlet oxygen, an additional amplification step needed to be activated. It depended on singlet oxygen-dependent activation of the FAS receptor and caspase-8, followed by caspase-8-mediated enhancement of NOX activity. The biochemical mechanisms described here might be considered as promising principle for the development of novel approaches in tumor therapy that specifically direct membrane-associated catalase of tumor cells and thus utilize tumor cell-specific apoptosis-inducing ROS signaling. PMID:26225731

Singlet oxygen treatment of tumor cells triggers extracellular singlet oxygen generation, catalase inactivation and reactivation of intercellular apoptosis-inducing signaling.

PubMed

Riethmüller, Michaela; Burger, Nils; Bauer, Georg

2015-12-01

Intracellular singlet oxygen generation in photofrin-loaded cells caused cell death without discrimination between nonmalignant and malignant cells. In contrast, extracellular singlet oxygen generation caused apoptosis induction selectively in tumor cells through singlet oxygen-mediated inactivation of tumor cell protective catalase and subsequent reactivation of intercellular ROS-mediated apoptosis signaling through the HOCl and the NO/peroxynitrite signaling pathway. Singlet oxygen generation by extracellular photofrin alone was, however, not sufficient for optimal direct inactivation of catalase, but needed to trigger the generation of cell-derived extracellular singlet oxygen through the interaction between H2O2 and peroxynitrite. Thereby, formation of peroxynitrous acid, generation of hydroxyl radicals and formation of perhydroxyl radicals (HO2(.)) through hydroxyl radical/H2O2 interaction seemed to be required as intermediate steps. This amplificatory mechanism led to the formation of singlet oxygen at a sufficiently high concentration for optimal inactivation of membrane-associated catalase. At low initial concentrations of singlet oxygen, an additional amplification step needed to be activated. It depended on singlet oxygen-dependent activation of the FAS receptor and caspase-8, followed by caspase-8-mediated enhancement of NOX activity. The biochemical mechanisms described here might be considered as promising principle for the development of novel approaches in tumor therapy that specifically direct membrane-associated catalase of tumor cells and thus utilize tumor cell-specific apoptosis-inducing ROS signaling. Copyright © 2015 The Authors. Published by Elsevier B.V. All rights reserved.

Stabilization and localization of Xist RNA are controlled by separate mechanisms and are not sufficient for X inactivation.

PubMed

Clemson, C M; Chow, J C; Brown, C J; Lawrence, J B

1998-07-13

These studies address whether XIST RNA is properly localized to the X chromosome in somatic cells where human XIST expression is reactivated, but fails to result in X inactivation (Tinker, A.V., and C.J. Brown. 1998. Nucl. Acids Res. 26:2935-2940). Despite a nuclear RNA accumulation of normal abundance and stability, XIST RNA does not localize in reactivants or in naturally inactive human X chromosomes in mouse/ human hybrid cells. The XIST transcripts are fully stabilized despite their inability to localize, and hence XIST RNA localization can be uncoupled from stabilization, indicating that these are separate steps controlled by distinct mechanisms. Mouse Xist RNA tightly localized to an active X chromosome, demonstrating for the first time that the active X chromosome in somatic cells is competent to associate with Xist RNA. These results imply that species-specific factors, present even in mature, somatic cells that do not normally express Xist, are necessary for localization. When Xist RNA is properly localized to an active mouse X chromosome, X inactivation does not result. Therefore, there is not a strict correlation between Xist localization and chromatin inactivation. Moreover, expression, stabilization, and localization of Xist RNA are not sufficient for X inactivation. We hypothesize that chromosomal association of XIST RNA may initiate subsequent developmental events required to enact transcriptional silencing.

Genetic Inactivation of ATRX Leads to a Decrease in the Amount of Telomeric Cohesin and Level of Telomere Transcription in Human Glioma Cells

PubMed Central

Eid, Rita; Demattei, Marie-Véronique; Episkopou, Harikleia; Augé-Gouillou, Corinne; Decottignies, Anabelle; Grandin, Nathalie

2015-01-01

Mutations in ATRX (alpha thalassemia/mental retardation syndrome X-linked), a chromatin-remodeling protein, are associated with the telomerase-independent ALT (alternative lengthening of telomeres) pathway of telomere maintenance in several types of cancer, including human gliomas. In telomerase-positive glioma cells, we found by immunofluorescence that ATRX localized not far from the chromosome ends but not exactly at the telomere termini. Chromatin immunoprecipitation (ChIP) experiments confirmed a subtelomeric localization for ATRX, yet short hairpin RNA (shRNA)-mediated genetic inactivation of ATRX failed to trigger the ALT pathway. Cohesin has been recently shown to be part of telomeric chromatin. Here, using ChIP, we showed that genetic inactivation of ATRX provoked diminution in the amount of cohesin in subtelomeric regions of telomerase-positive glioma cells. Inactivation of ATRX also led to diminution in the amount of TERRAs, noncoding RNAs resulting from transcription of telomeric DNA, as well as to a decrease in RNA polymerase II (RNAP II) levels at the telomeres. Our data suggest that ATRX might establish functional interactions with cohesin on telomeric chromatin in order to control TERRA levels and that one or the other or both of these events might be relevant to the triggering of the ALT pathway in cancer cells that exhibit genetic inactivation of ATRX. PMID:26055325

Genetic Inactivation of ATRX Leads to a Decrease in the Amount of Telomeric Cohesin and Level of Telomere Transcription in Human Glioma Cells.

PubMed

Eid, Rita; Demattei, Marie-Véronique; Episkopou, Harikleia; Augé-Gouillou, Corinne; Decottignies, Anabelle; Grandin, Nathalie; Charbonneau, Michel

2015-08-01

Mutations in ATRX (alpha thalassemia/mental retardation syndrome X-linked), a chromatin-remodeling protein, are associated with the telomerase-independent ALT (alternative lengthening of telomeres) pathway of telomere maintenance in several types of cancer, including human gliomas. In telomerase-positive glioma cells, we found by immunofluorescence that ATRX localized not far from the chromosome ends but not exactly at the telomere termini. Chromatin immunoprecipitation (ChIP) experiments confirmed a subtelomeric localization for ATRX, yet short hairpin RNA (shRNA)-mediated genetic inactivation of ATRX failed to trigger the ALT pathway. Cohesin has been recently shown to be part of telomeric chromatin. Here, using ChIP, we showed that genetic inactivation of ATRX provoked diminution in the amount of cohesin in subtelomeric regions of telomerase-positive glioma cells. Inactivation of ATRX also led to diminution in the amount of TERRAs, noncoding RNAs resulting from transcription of telomeric DNA, as well as to a decrease in RNA polymerase II (RNAP II) levels at the telomeres. Our data suggest that ATRX might establish functional interactions with cohesin on telomeric chromatin in order to control TERRA levels and that one or the other or both of these events might be relevant to the triggering of the ALT pathway in cancer cells that exhibit genetic inactivation of ATRX. Copyright © 2015, American Society for Microbiology. All Rights Reserved.

Synergistic Effect of Atmospheric-pressure Plasma and TiO2 Photocatalysis on Inactivation of Escherichia coli Cells in Aqueous Media

NASA Astrophysics Data System (ADS)

Zhou, Renwu; Zhou, Rusen; Zhang, Xianhui; Li, Jiangwei; Wang, Xingquan; Chen, Qiang; Yang, Size; Chen, Zhong; Bazaka, Kateryna; (Ken) Ostrikov, Kostya

2016-12-01

Atmospheric-pressure plasma and TiO2 photocatalysis have been widely investigated separately for the management and reduction of microorganisms in aqueous solutions. In this paper, the two methods were combined in order to achieve a more profound understanding of their interactions in disinfection of water contaminated by Escherichia coli. Under water discharges carried out by microplasma jet arrays can result in a rapid inactivation of E. coli cells. The inactivation efficiency is largely dependent on the feed gases used, the plasma treatment time, and the discharge power. Compared to atmospheric-pressure N2, He and air microplasma arrays, O2 microplasma had the highest activity against E. coli cells in aqueous solution, and showed >99.9% bacterial inactivation efficiency within 4 min. Addition of TiO2 photocatalytic film to the plasma discharge reactor significantly enhanced the inactivation efficiency of the O2 microplasma system, decreasing the time required to achieve 99.9% killing of E. coli cells to 1 min. This may be attributed to the enhancement of ROS generation due to high catalytic activity and stability of the TiO2 photocatalyst in the combined plasma-TiO2 systems. Present work demonstrated the synergistic effect of the two agents, which can be correlated in order to maximize treatment efficiency.

Synergistic Effect of Atmospheric-pressure Plasma and TiO2 Photocatalysis on Inactivation of Escherichia coli Cells in Aqueous Media

PubMed Central

Zhou, Renwu; Zhou, Rusen; Zhang, Xianhui; Li, Jiangwei; Wang, Xingquan; Chen, Qiang; Yang, Size; Chen, Zhong; Bazaka, Kateryna; (Ken) Ostrikov, Kostya

2016-01-01

Atmospheric-pressure plasma and TiO2 photocatalysis have been widely investigated separately for the management and reduction of microorganisms in aqueous solutions. In this paper, the two methods were combined in order to achieve a more profound understanding of their interactions in disinfection of water contaminated by Escherichia coli. Under water discharges carried out by microplasma jet arrays can result in a rapid inactivation of E. coli cells. The inactivation efficiency is largely dependent on the feed gases used, the plasma treatment time, and the discharge power. Compared to atmospheric-pressure N2, He and air microplasma arrays, O2 microplasma had the highest activity against E. coli cells in aqueous solution, and showed >99.9% bacterial inactivation efficiency within 4 min. Addition of TiO2 photocatalytic film to the plasma discharge reactor significantly enhanced the inactivation efficiency of the O2 microplasma system, decreasing the time required to achieve 99.9% killing of E. coli cells to 1 min. This may be attributed to the enhancement of ROS generation due to high catalytic activity and stability of the TiO2 photocatalyst in the combined plasma-TiO2 systems. Present work demonstrated the synergistic effect of the two agents, which can be correlated in order to maximize treatment efficiency. PMID:28004829

Newer Vaccines against Mosquito-borne Diseases.

PubMed

Aggarwal, Anju; Garg, Neha

2018-02-01

Mosquitos are responsible for a number of protozoal and viral diseases. Malaria, dengue, Japanese encephalitis (JE) and chikungunya epidemics occur commonly all over the world, leading to marked mortality and morbidity in children. Zika, Yellow fever and West Nile fever are others requiring prevention. Environmental control and mosquito bite prevention are useful in decreasing the burden of disease but vaccination has been found to be most cost-effective and is the need of the hour. RTS,S/AS01 vaccine is the first malaria vaccine being licensed for use against P. falciparum malaria. Dengvaxia (CYD-TDV) against dengue was licensed first in Mexico in 2015. A Vero-cell derived, inactivated and alum-adjuvanted JE vaccine based on the SA14-14-2 strain was approved in 2009 in North America, Australia and various European countries. It can be used from 2 mo of age. In India, immunization is carried out in endemic regions at 1 y of age. Another inactivated Vero-cell culture derived Kolar strain, 821564XY, JE vaccine is being used in India. Candidate vaccines against dengue, chikungunya and West Nile fever are been discussed. A continued research and development of new vaccines are required for controlling these mosquito-borne diseases.

Mycobacteria inactivation using Engineered Water Nanostructures (EWNS).

PubMed

Pyrgiotakis, Georgios; McDevitt, James; Gao, Ya; Branco, Alan; Eleftheriadou, Mary; Lemos, Bernardo; Nardell, Edward; Demokritou, Philip

2014-08-01

Airborne transmitted pathogens such as Mycobacterium tuberculosis (Mtb) cause serious, often fatal infectious disease with enormous global health implications. Due to their unique cell wall and slow growth, mycobacteria are among the most resilient microbial forms. Herein we evaluate the ability of an emerging, chemical-free, nanotechnology-based method to inactivate M. parafortuitum (Mtb surrogate). This method is based on the transformation of atmospheric water vapor into engineered water nano-structures (EWNS) via electrospray. We demonstrate that the EWNS can interact with and inactivate airborne mycobacteria, reducing their concentration levels significantly. Additionally, EWNS can inactivate M. parafortuitum on surfaces eight times faster than the control. The mechanism of mycobacteria inactivation was also investigated in this study. It was demonstrated that the EWNS effectively deliver the reactive oxygen species, encapsulated during the electrospray process, to the bacteria oxidizing their cell membrane resulting into inactivation. Overall, this is a method with the potential to become an effective intervention technology in the battle against airborne infections. This study demonstrates the feasibility of mycobacterium inactivation in airborne form or on contact surfaces using electrospray activated water nano-structures. Given that the method is free of toxic chemicals, this might become an important tool in the prevention of mycobacterial infections, which are notoriously hard to treat. Copyright © 2014 Elsevier Inc. All rights reserved.

Mycoplasmal Infections Prevent Apoptosis and Induce Malignant Transformation of Interleukin-3-Dependent 32D Hematopoietic Cells

PubMed Central

Feng, Shaw-Huey; Tsai, Shien; Rodriguez, Jose; Lo, Shyh-Ching

1999-01-01

32D cells, a murine myeloid cell line, rapidly undergo apoptosis upon withdrawal of interleukin-3 (IL-3) supplement in culture. We found that 32D cells, if infected by several species of human mycoplasmas that rapidly activated NF-κB, would live and continue to grow in IL-3-depleted culture. Mycoplasma-infected cells showed no evidence of autocrine production of IL-3. Pyrrolidine dithiocarbamate (PDTC) blocked activation of NF-κB and led to prominent cell death. Heat-killed mycoplasmas or mycoplasmal membrane preparations alone could support continued growth of 32D cells in culture without IL-3 supplement for a substantial period of time. However, upon removal of heat-inactivated mycoplasmas, 32D cells quickly became apoptotic. In comparison, live Mycoplasma fermentans or M. penetrans infection for 4 to 5 weeks induced malignant transformation of 32D cells. Transformed 32D cells grew autonomously and no longer required support of growth-stimulating factors including IL-3 and mycoplasmas. The transformed 32D cells quickly formed tumors when injected into nude mice. Karyotyping showed that development of chromosomal changes and trisomy 19 was often associated with malignant transformation and tumorigenicity of 32D cells. Mycoplasmal infections apparently affected the fidelity of genomic transmission in cell division as well as checkpoints coordinating the progression of cell cycle events. PMID:10567525

The phosphatase JKAP/DUSP22 inhibits t-cell receptor signalling and autoimmunity by inactivating Lck

USDA-ARS?s Scientific Manuscript database

JNK pathway-associated phosphatase (JKAP, also known as DUSP22 or JSP-1) is a JNK activator. The in vivo role of JKAP in immune regulation remains unclear. Here we report that JKAP directly inactivates Lck by dephosphorylating tyrosine-394 residue during T-cell receptor (TCR) signalling. JKAP-knocko...

Modeling Bacteriocin Resistance and Inactivation of Listeria innocua LMG 13568 by Lactobacillus sakei CTC 494 under Sausage Fermentation Conditions

PubMed Central

Leroy, Frédéric; Lievens, Kristoff; De Vuyst, Luc

2005-01-01

In mixed cultures, bacteriocin production by the sausage isolate Lactobacillus sakei CTC 494 rapidly inactivated sensitive Listeria innocua LMG 13568 cells, even at low bacteriocin activity levels. A small fraction of the listerial population was bacteriocin resistant. However, sausage fermentation conditions inhibited regrowth of resistant cells. PMID:16269805

Mechanisms of the effect of VUV radiation on the microfungi

NASA Astrophysics Data System (ADS)

Zvereva, Galina; Kirtsideli, Irina; Machs, Eduard; Vangonen, Albert

2018-04-01

The mechanisms of the effect of vacuum ultraviolet (VUV) radiation (λ = 172 nm) on various types of microfungi spores were investigated. It is found that there are several parallel direct and indirect mechanisms, which lead to spores inactivation, including destruction of the cell wall and DNA by means of direct absorption of VUV radiation and by VUV photolysis reactive products. IR transmission spectra indicate the etching of the spore cell wall material with the predominant degradation of the polysaccharides. Electrophoresis of irradiated spores DNA shows heavy (about 20 000 pairs of nucleotides) and light fragments appearance. Experiments using an antioxidant (iodine) indicate the participation of reactive radicals in inactivation, which provide not less than 10% of inactivated cells

Inactivation of the microbiota and effect on the quality attributes of pineapple juice using a continuous flow ultrasound-assisted supercritical carbon dioxide system.

PubMed

Paniagua-Martínez, I; Mulet, A; García-Alvarado, M A; Benedito, J

2018-01-01

Supercritical carbon dioxide inactivation technology represents a promising nonthermal processing method, as it causes minimum impact on the nutritional food properties. The aim of this study was to analyze the combined effect of supercritical carbon dioxide and high-power ultrasound on the inactivation of natural microbiota and the quality attributes of pineapple juice treated in a continuous flow system. Different juice residence times (3.06-4.6 min), at 100 bar and 31.5 ℃, were used. The results indicated that the microbiota inactivation was complete and the differences obtained in the quality attributes (2.2% for pH, 4.8% for °Brix, 2% for vitamin C) were minimal. During storage, microorganisms were not able to recover and the vitamin C decrease could be limited to 8.2% after four weeks. The results demonstrated that the supercritical carbon dioxide-high-power ultrasound technique could be an excellent alternative for the cold pasteurization of pineapple juice.

Selective control of cortical axonal spikes by a slowly inactivating K+ current

PubMed Central

Shu, Yousheng; Yu, Yuguo; Yang, Jing; McCormick, David A.

2007-01-01

Neurons are flexible electrophysiological entities in which the distribution and properties of ionic channels control their behaviors. Through simultaneous somatic and axonal whole-cell recording of layer 5 pyramidal cells, we demonstrate a remarkable differential expression of slowly inactivating K+ currents. Depolarizing the axon, but not the soma, rapidly activated a low-threshold, slowly inactivating, outward current that was potently blocked by low doses of 4-aminopyridine, α-dendrotoxin, and rTityustoxin-Kα. Block of this slowly inactivating current caused a large increase in spike duration in the axon but only a small increase in the soma and could result in distal axons generating repetitive discharge in response to local current injection. Importantly, this current was also responsible for slow changes in the axonal spike duration that are observed after somatic membrane potential change. These data indicate that low-threshold, slowly inactivating K+ currents, containing Kv1.2 α subunits, play a key role in the flexible properties of intracortical axons and may contribute significantly to intracortical processing. PMID:17581873

In vitro induction of apoptosis in tumor cells by inactivated NDV and IAV.

PubMed

Yang, ShuYan; Liu, WeiQuan; Cui, HuanXian; Sun, ShaoGuang; Wang, JiGui

2007-04-01

We examined how Newcastle disease virus (NDV) and influenza A virus (IAV) inactivated by 5% formaldehyde, used either alone or in combination, can induce apoptosis in both HeLa and SP2/0 cells. Inactive NDV and IAV demonstrated enhanced rates of lysis in apoptotic tumor cells and greater antitumor effects when combined. Our study supports the argument that viral replication does not cause virally induced apoptosis.

Calcium-dependent inactivation of calcium channels in cochlear hair cells of the chicken.

PubMed

Lee, Seunghwan; Briklin, Olga; Hiel, Hakim; Fuchs, Paul

2007-09-15

Voltage-gated calcium channels support both spontaneous and sound-evoked neurotransmitter release from ribbon synapses of cochlear hair cells. A variety of regulatory mechanisms must cooperate to ensure the appropriate level of activity in the restricted pool of synaptic calcium channels ( approximately 100) available to each synaptic ribbon. One potential feedback mechanism, calcium-dependent inactivation (CDI) of voltage-gated, L-type calcium channels, can be modulated by calmodulin-like calcium-binding proteins. CDI of voltage-gated calcium current was studied in hair cells of the chicken's basilar papilla (analogous to the mammalian cochlea) after blocking the predominant potassium conductances. For inactivating currents produced by 2.5 s steps to the peak of the current-voltage relation (1 mm EGTA internal calcium buffer), single exponential fits yielded an average decay time constant of 1.92 +/- 0.18 s (mean +/- s.e.m., n = 12) at 20-22 degrees C, while recovery occurred with a half-time of approximately 10 s. Inactivation produced no change in reversal potential, arguing that the observed relaxation did not result from alternative processes such as calcium accumulation or activation of residual potassium currents. Substitution of external calcium with barium greatly reduced inactivation, while inhibition of endoplasmic calcium pumps with t-benzohydroquinone (BHQ) or thapsigargin made inactivation occur faster and to a greater extent. Raising external calcium 10-fold (from 2 to 20 mm) increased peak current 3-fold, but did not alter the extent or time course of CDI. However, increasing levels of internal calcium buffer consistently reduced the rate and extent of inactivation. With 1 mm EGTA buffering and in 2 mm external calcium, the available pool of calcium channels was half-inactivated near the resting membrane potential (-50 mV). CDI may be further regulated by calmodulin-like calcium-binding proteins (CaBPs). mRNAs for several CaBPs are expressed in chicken cochlear tissue, and antibodies to CaBP4 label hair cells, but not supporting cells, equivalent to the pattern seen in mammalian cochlea. Thus, molecular mechanisms that underlie CDI appeared to be conserved across vertebrate species, may provide a means to adjust calcium channel open probability, and could serve to maintain the set-point for spontaneous release from the ribbon synapse.

Calcium-dependent inactivation of calcium channels in cochlear hair cells of the chicken

PubMed Central

Lee, Seunghwan; Briklin, Olga; Hiel, Hakim; Fuchs, Paul

2007-01-01

Voltage-gated calcium channels support both spontaneous and sound-evoked neurotransmitter release from ribbon synapses of cochlear hair cells. A variety of regulatory mechanisms must cooperate to ensure the appropriate level of activity in the restricted pool of synaptic calcium channels (∼100) available to each synaptic ribbon. One potential feedback mechanism, calcium-dependent inactivation (CDI) of voltage-gated, L-type calcium channels, can be modulated by calmodulin-like calcium-binding proteins. CDI of voltage-gated calcium current was studied in hair cells of the chicken's basilar papilla (analogous to the mammalian cochlea) after blocking the predominant potassium conductances. For inactivating currents produced by 2.5 s steps to the peak of the current–voltage relation (1 mm EGTA internal calcium buffer), single exponential fits yielded an average decay time constant of 1.92 ± 0.18 s (mean ±s.e.m., n = 12) at 20–22°C, while recovery occurred with a half-time of ∼10 s. Inactivation produced no change in reversal potential, arguing that the observed relaxation did not result from alternative processes such as calcium accumulation or activation of residual potassium currents. Substitution of external calcium with barium greatly reduced inactivation, while inhibition of endoplasmic calcium pumps with t-benzohydroquinone (BHQ) or thapsigargin made inactivation occur faster and to a greater extent. Raising external calcium 10-fold (from 2 to 20 mm) increased peak current 3-fold, but did not alter the extent or time course of CDI. However, increasing levels of internal calcium buffer consistently reduced the rate and extent of inactivation. With 1 mm EGTA buffering and in 2 mm external calcium, the available pool of calcium channels was half-inactivated near the resting membrane potential (−50 mV). CDI may be further regulated by calmodulin-like calcium-binding proteins (CaBPs). mRNAs for several CaBPs are expressed in chicken cochlear tissue, and antibodies to CaBP4 label hair cells, but not supporting cells, equivalent to the pattern seen in mammalian cochlea. Thus, molecular mechanisms that underlie CDI appeared to be conserved across vertebrate species, may provide a means to adjust calcium channel open probability, and could serve to maintain the set-point for spontaneous release from the ribbon synapse. PMID:17656437

Quasi-Instantaneous Bacterial Inactivation on Cu-Ag Nanoparticulate 3D Catheters in the Dark and Under Light: Mechanism and Dynamics.

PubMed

Rtimi, Sami; Sanjines, Rosendo; Pulgarin, Cesar; Kiwi, John

2016-01-13

The first evidence for Cu-Ag (50%/50%) nanoparticulate hybrid coatings is presented leading to a complete and almost instantaneous bacterial inactivation in the dark (≤5 min). Dark bacterial inactivation times on Cu-Ag (50%/50%) were observed to coincide with the times required by actinic light irradiation. This provides the evidence that the bimetal Cu-Ag driven inactivation predominates over a CuO/Cu2O and Ag2O oxides inducing a semiconductor driven behavior. Cu- or Ag-coated polyurethane (PU) catheters led to bacterial inactivation needing about ∼30 min. The accelerated bacterial inactivation by Cu-Ag coated on 3D catheters sputtered was investigated in a detailed way. The release of Cu/Ag ions during bacterial inactivation was followed by inductively coupled plasma mass-spectrometry (ICP-MS) and the amount of Cu and Ag-ions released were below the cytotoxicity levels permitted by the sanitary regulations. By stereomicroscopy the amount of live/dead cells were followed during the bacterial inactivation time. By Fourier transform infrared spectroscopy (FTIR), the systematic shift of the -(CH2) band stretching of the outer lipo-polysaccharide bilayer (LPS) was followed to monitor the changes leading to cell lysis. A hydrophobic to hydrophilic transformation of the Cu-Ag PU catheter surface under light was observed within 30 min followed concomitantly to a longer back transformation to the hydrophobic initial state in the dark. Physical insight is provided for the superior performance of Cu-Ag films compared to Cu or Ag films in view of the drastic acceleration of the bacterial inactivation observed on bimetal Cu-Ag films coating PU catheters. A mechanism of bacterial inactivation is suggested that is consistent with the findings reported in this study.

Lithium Chloride Dependent Glycogen Synthase Kinase 3 Inactivation Links Oxidative DNA Damage, Hypertrophy and Senescence in Human Articular Chondrocytes and Reproduces Chondrocyte Phenotype of Obese Osteoarthritis Patients.

PubMed

Guidotti, Serena; Minguzzi, Manuela; Platano, Daniela; Cattini, Luca; Trisolino, Giovanni; Mariani, Erminia; Borzì, Rosa Maria

2015-01-01

Recent evidence suggests that GSK3 activity is chondroprotective in osteoarthritis (OA), but at the same time, its inactivation has been proposed as an anti-inflammatory therapeutic option. Here we evaluated the extent of GSK3β inactivation in vivo in OA knee cartilage and the molecular events downstream GSK3β inactivation in vitro to assess their contribution to cell senescence and hypertrophy. In vivo level of phosphorylated GSK3β was analyzed in cartilage and oxidative damage was assessed by 8-oxo-deoxyguanosine staining. The in vitro effects of GSK3β inactivation (using either LiCl or SB216763) were evaluated on proliferating primary human chondrocytes by combined confocal microscopy analysis of Mitotracker staining and reactive oxygen species (ROS) production (2',7'-dichlorofluorescin diacetate staining). Downstream effects on DNA damage and senescence were investigated by western blot (γH2AX, GADD45β and p21), flow cytometric analysis of cell cycle and light scattering properties, quantitative assessment of senescence associated β galactosidase activity, and PAS staining. In vivo chondrocytes from obese OA patients showed higher levels of phosphorylated GSK3β, oxidative damage and expression of GADD45β and p21, in comparison with chondrocytes of nonobese OA patients. LiCl mediated GSK3β inactivation in vitro resulted in increased mitochondrial ROS production, responsible for reduced cell proliferation, S phase transient arrest, and increase in cell senescence, size and granularity. Collectively, western blot data supported the occurrence of a DNA damage response leading to cellular senescence with increase in γH2AX, GADD45β and p21. Moreover, LiCl boosted 8-oxo-dG staining, expression of IKKα and MMP-10. In articular chondrocytes, GSK3β activity is required for the maintenance of proliferative potential and phenotype. Conversely, GSK3β inactivation, although preserving chondrocyte survival, results in functional impairment via induction of hypertrophy and senescence. Indeed, GSK3β inactivation is responsible for ROS production, triggering oxidative stress and DNA damage response.

Germination and Inactivation of Alicyclobacillus acidoterrestris Spores Induced by Moderate Hydrostatic Pressure.

PubMed

Sokołowska, Barbara; Skapska, Sylwia; Fonberg-Broczek, Monika; Niezgoda, Jolanta; Porebska, Izabela; Dekowska, Agnieszka; Rzoska, Sylwester J

2015-01-01

Given the importance of spoilage caused by Alicyclobacillus acidoterrestris for the fruit juice industry, the objective of this work was to study the germination and inactivation of A. acidoterrestris spores induced by moderate hydrostatic pressure. Hydrostatic pressure treatment can induce the germination and inactivation of A. acidoterrestris spores. At low pH, spore germination of up to 3.59-3.75 log and inactivation of 1.85-2.04 log was observed in a low pressure window (200-300 MPa) applied at 50 degrees C for 20 min. Neutral pH suppressed inactivation, the number of spores inactivated at pH 7.0 was only 0.24-1.06 log. The pressurization temperature significantly affected spore germination and inactivation. The degree of germination in apple juice after pressurization for 30 min with 200 MPa at 20 degrees C was 2.04 log, with only 0.61 log of spores being inactivated, while at 70 degrees C spore germination was 5.94 log and inactivation 4.72 log. This temperature strongly stimulated germination and inactivation under higher (500 MPa) than lower (200 MPa) pressure. When the oscillatory mode was used, the degree of germination and inactivation was slightly higher than at continuous mode. The degree of germination and inactivation was inversely proportional to the soluble solids content and was lowest in concentrated apple juice.

Two distinct groups within the Bacillus subtilis group display significantly different spore heat resistance properties.

PubMed

Berendsen, Erwin M; Zwietering, Marcel H; Kuipers, Oscar P; Wells-Bennik, Marjon H J

2015-02-01

The survival of bacterial spores after heat treatment and the subsequent germination and outgrowth in a food product can lead to spoilage of the food product and economical losses. Prediction of time-temperature conditions that lead to sufficient inactivation requires access to detailed spore thermal inactivation kinetics of relevant model strains. In this study, the thermal inactivation kinetics of spores of fourteen strains belonging to the Bacillus subtilis group were determined in detail, using both batch heating in capillary tubes and continuous flow heating in a micro heater. The inactivation data were fitted using a log linear model. Based on the spore heat resistance data, two distinct groups (p < 0.001) within the B. subtilis group could be identified. One group of strains had spores with an average D120 °C of 0.33 s, while the spores of the other group displayed significantly higher heat resistances, with an average D120 °C of 45.7 s. When comparing spore inactivation data obtained using batch- and continuous flow heating, the z-values were significantly different, hence extrapolation from one system to the other was not justified. This study clearly shows that heat resistances of spores from different strains in the B. subtilis group can vary greatly. Strains can be separated into two groups, to which different spore heat inactivation kinetics apply. Copyright © 2014 Elsevier Ltd. All rights reserved.

Estrogen-mediated inactivation of FOXO3a by the G protein-coupled estrogen receptor GPER.

PubMed

Zekas, Erin; Prossnitz, Eric R

2015-10-15

Estrogen (17β-estradiol) promotes the survival and proliferation of breast cancer cells and its receptors represent important therapeutic targets. The cellular actions of estrogen are mediated by the nuclear estrogen receptors ERα and ERβ as well as the 7-transmembrane spanning G protein-coupled estrogen receptor (GPER). We previously reported that estrogen activates the phosphoinositide 3-kinase (PI3Kinase) pathway via GPER, resulting in phosphatidylinositol (3,4,5)-trisphosphate (PIP3) production within the nucleus of breast cancer cells; however, the mechanisms and consequences of this activity remained unclear. MCF7 breast cancer cells were transfected with GFP-fused Forkhead box O3 (FOXO3) as a reporter to assess localization in response to estrogen stimulation. Inhibitors of PI3Kinases and EGFR were employed to determine the mechanisms of estrogen-mediated FOXO3a inactivation. Receptor knockdown with siRNA and the selective GPER agonist G-1 elucidated the estrogen receptor(s) responsible for estrogen-mediated FOXO3a inactivation. The effects of selective estrogen receptor modulators and downregulators (SERMs and SERDs) on FOXO3a in MCF7 cells were also determined. Cell survival (inhibition of apoptosis) was assessed by caspase activation. In the estrogen-responsive breast cancer cell line MCF7, FOXO3a inactivation occurs on a rapid time scale as a result of GPER, but not ERα, stimulation by estrogen, established by the GPER-selective agonist G-1 and knockdown of GPER and ERα. GPER-mediated inactivation of FOXO3a is effected by the p110α catalytic subunit of PI3Kinase as a result of transactivation of the EGFR. The SERMs tamoxifen and raloxifene, as well as the SERD ICI182,780, were active in mediating FOXO3a inactivation in a GPER-dependent manner. Additionally, estrogen-and G-1-mediated stimulation of MCF7 cells results in a decrease in caspase activation under proapoptotic conditions. Our results suggest that non-genomic signaling by GPER contributes, at least in part, to the survival of breast cancer cells, particularly in the presence of ER-targeted therapies involving SERMs and SERDs. Our results further suggest that GPER expression and FOXO3a localization could be utilized as prognostic markers in breast cancer therapy and that GPER antagonists could promote apoptosis in GPER-positive breast cancers, particularly in combination with chemotherapeutic and ER-targeted drugs, by antagonizing estrogen-mediated FOXO3a inactivation.

Susceptibility of Glucokinase-MODY Mutants to Inactivation by Oxidative Stress in Pancreatic β-Cells

PubMed Central

Cullen, Kirsty S.; Matschinsky, Franz M.; Agius, Loranne; Arden, Catherine

2011-01-01

OBJECTIVE The posttranslational regulation of glucokinase (GK) differs in hepatocytes and pancreatic β-cells. We tested the hypothesis that GK mutants that cause maturity-onset diabetes of the young (GK-MODY) show compromised activity and posttranslational regulation in β-cells. RESEARCH DESIGN AND METHODS Activity and protein expression of GK-MODY and persistent hyperinsulinemic hypoglycemia of infancy (PHHI) mutants were studied in β-cell (MIN6) and non–β-cell (H4IIE) models. Binding of GK to phosphofructo-2-kinase, fructose-2,6-bisphosphatase (PFK2/FBPase2) was studied by bimolecular fluorescence complementation in cell-based models. RESULTS Nine of 11 GK-MODY mutants that have minimal effect on enzyme kinetics in vitro showed decreased specific activity relative to wild type when expressed in β-cells. A subset of these were stable in non–β-cells but showed increased inactivation in conditions of oxidative stress and partial reversal of inactivation by dithiothreitol. Unlike the GK-MODY mutants, four of five GK-PHHI mutants had similar specific activity to wild type and Y214C had higher activity than wild type. The GK-binding protein PFK2/FBPase2 protected wild-type GK from oxidative inactivation and the decreased stability of GK-MODY mutants correlated with decreased interaction with PFK2/FBPase2. CONCLUSIONS Several GK-MODY mutants show posttranslational defects in β-cells characterized by increased susceptibility to oxidative stress and/or protein instability. Regulation of GK activity through modulation of thiol status may be a physiological regulatory mechanism for the control of GK activity in β-cells. PMID:22028181

Susceptibility of glucokinase-MODY mutants to inactivation by oxidative stress in pancreatic β-cells.

PubMed

Cullen, Kirsty S; Matschinsky, Franz M; Agius, Loranne; Arden, Catherine

2011-12-01

The posttranslational regulation of glucokinase (GK) differs in hepatocytes and pancreatic β-cells. We tested the hypothesis that GK mutants that cause maturity-onset diabetes of the young (GK-MODY) show compromised activity and posttranslational regulation in β-cells. Activity and protein expression of GK-MODY and persistent hyperinsulinemic hypoglycemia of infancy (PHHI) mutants were studied in β-cell (MIN6) and non-β-cell (H4IIE) models. Binding of GK to phosphofructo-2-kinase, fructose-2,6-bisphosphatase (PFK2/FBPase2) was studied by bimolecular fluorescence complementation in cell-based models. Nine of 11 GK-MODY mutants that have minimal effect on enzyme kinetics in vitro showed decreased specific activity relative to wild type when expressed in β-cells. A subset of these were stable in non-β-cells but showed increased inactivation in conditions of oxidative stress and partial reversal of inactivation by dithiothreitol. Unlike the GK-MODY mutants, four of five GK-PHHI mutants had similar specific activity to wild type and Y214C had higher activity than wild type. The GK-binding protein PFK2/FBPase2 protected wild-type GK from oxidative inactivation and the decreased stability of GK-MODY mutants correlated with decreased interaction with PFK2/FBPase2. Several GK-MODY mutants show posttranslational defects in β-cells characterized by increased susceptibility to oxidative stress and/or protein instability. Regulation of GK activity through modulation of thiol status may be a physiological regulatory mechanism for the control of GK activity in β-cells.

Promotion of initiated cells by radiation-induced cell inactivation.

PubMed

Heidenreich, W F; Paretzke, H G

2008-11-01

Cells on the way to carcinogenesis can have a growth advantage relative to normal cells. It has been hypothesized that a radiation-induced growth advantage of these initiated cells might be induced by an increased cell replacement probability of initiated cells after inactivation of neighboring cells by radiation. Here Monte Carlo simulations extend this hypothesis for larger clones: The effective clonal expansion rate decreases with clone size. This effect is stronger for the two-dimensional than for the three-dimensional situation. The clones are irregular, far from a circular shape. An exposure-rate dependence of the effective clonal expansion rate could come in part from a minimal recovery time of the initiated cells for symmetric cell division.

Protection against murine potomac horse fever by an inactivated Ehrlichia risticii vaccine.

PubMed

Rikihisa, Y

1991-05-01

Ehrlichia risticii propagated in a murine macrophage cell line were freed from the host cell by hypotonic lysis of the infected cells. The cell-free ehrlichiae were inactivated with beta-propiolactone and combined or not combined with polymyxin-B. The vaccines were administered to mice with Quil-A (saponin) as an adjuvant twice at 2 to 3 week intervals and the mice were challenged with live E. risticii 2 to 3 weeks after the last vaccination. With or without the addition of polymyxin-B, the vaccine preparations protected mice from developing clinical signs and gross pathologic changes such as thymic atrophy, splenomegaly, and increase in whole intestinal weight. Mice vaccinated with or without polymyxin-B developed high titer IgG antibody against E. risticii before and after the challenge with live E. risticii. Spleen lymphocyte proliferative response assay at 11 days post challenge revealed that with polymyxin-B a higher lymphocyte proliferation occurred as compared with that of the mice which received polymyxin-B-free vaccine. Spleen lymphocytes of the placebo (polymyxin-B and Quil-A) pretreated/challenged mice showed no proliferative activity. Western blot analysis revealed that vaccinated mice reacted mainly with 110, 57 and 33 kDa antigen bands before and after challenge. The placebo (polymyxin-B and Quil-A)/challenged mice showed a very weak response to ehrlichial antigens at day 10 to 11 post challenge. Comparison with inactivated Renografin-purified E. risticii or 0.25% SDS-insoluble fraction of E. risticii with the inactivated host cell-free vaccine revealed no increased protection. These results indicate that inactivated host cell-free E. risticii can protect mice from murine Potomac horse fever. The presence of polymyxin-B appeared to be not harmful but rather beneficial for lymphocyte proliferation response upon challenge with live E. risticii.

Insulin increases excitability via a dose-dependent dual inhibition of voltage-activated K+ currents in differentiated N1E-115 neuroblastoma cells.

PubMed

Lima, Pedro A; Vicente, M Inês; Alves, Frederico M; Dionísio, José C; Costa, Pedro F

2008-04-01

A role in the control of excitability has been attributed to insulin via modulation of potassium (K(+)) currents. To investigate insulin modulatory effects on voltage-activated potassium currents in a neuronal cell line with origin in the sympathetic system, we performed whole-cell voltage-clamp recordings in differentiated N1E-115 neuroblastoma cells. Two main voltage-activated K(+) currents were identified: (a) a relatively fast inactivating current (I(fast) - time constant 50-300 ms); (b) a slow delayed rectifying K(+) current (I(slow) - time constant 1-4 s). The kinetics of inactivation of I(fast), rather than I(slow), showed clear voltage dependence. I(fast) and I(slow) exhibited different activation and inactivation dependence for voltage, and have different but nevertheless high sensitivities to tetraethylammonium, 4-aminopyridine and quinidine. In differentiated cells - rather than in non-differentiated cells - application of up to 300 nm insulin reduced I(slow) only (IC(50) = 6.7 nm), whereas at higher concentrations I(fast) was also affected (IC(50) = 7.7 microm). The insulin inhibitory effect is not due to a change in the activation or inactivation current-voltage profiles, and the time-dependent inactivation is also not altered; this is not likely to be a result of activation of the insulin-growth-factor-1 (IGF1) receptors, as application of IGF1 did not result in significant current alteration. Results suggest that the current sensitive to low concentrations of insulin is mediated by erg-like channels. Similar observations concerning the insulin inhibitory effect on slow voltage-activated K(+) currents were also made in isolated rat hippocampal pyramidal neurons, suggesting a widespread neuromodulator role of insulin on K(+) channels.

Impact of bacteria and bacterial components on osteogenic and adipogenic differentiation of adipose-derived mesenchymal stem cells

DOE Office of Scientific and Technical Information (OSTI.GOV)

Fiedler, Tomas, E-mail: tomas.fiedler@med.uni-rostock.de; Salamon, Achim; Adam, Stefanie

Adult mesenchymal stem cells (MSC) are present in several tissues, e.g. bone marrow, heart muscle, brain and subcutaneous adipose tissue. In invasive infections MSC get in contact with bacteria and bacterial components. Not much is known about how bacterial pathogens interact with MSC and how contact to bacteria influences MSC viability and differentiation potential. In this study we investigated the impact of three different wound infection relevant bacteria, Escherichia coli, Staphylococcus aureus, and Streptococcus pyogenes, and the cell wall components lipopolysaccharide (LPS; Gram-negative bacteria) and lipoteichoic acid (LTA; Gram-positive bacteria) on viability, proliferation, and osteogenic as well as adipogenic differentiationmore » of human adipose tissue-derived mesenchymal stem cells (adMSC). We show that all three tested species were able to attach to and internalize into adMSC. The heat-inactivated Gram-negative E. coli as well as LPS were able to induce proliferation and osteogenic differentiation but reduce adipogenic differentiation of adMSC. Conspicuously, the heat-inactivated Gram-positive species showed the same effects on proliferation and adipogenic differentiation, while its cell wall component LTA exhibited no significant impact on adMSC. Therefore, our data demonstrate that osteogenic and adipogenic differentiation of adMSC is influenced in an oppositional fashion by bacterial antigens and that MSC-governed regeneration is not necessarily reduced under infectious conditions. - Highlights: • Staphylococcus aureus, Streptococcus pyogenes and Escherichia coli bind to and internalize into adMSC. • Heat-inactivated cells of these bacterial species trigger proliferation of adMSC. • Heat-inactivated E. coli and LPS induce osteogenic differentiation of adMSC. • Heat-inactivated E. coli and LPS reduce adipogenic differentiation of adMSC. • LTA does not influence adipogenic or osteogenic differentiation of adMSC.« less

Gene Inactivation by CRISPR-Cas9 in Nicotiana tabacum BY-2 Suspension Cells.

PubMed

Mercx, Sébastien; Tollet, Jérémie; Magy, Bertrand; Navarre, Catherine; Boutry, Marc

2016-01-01

Plant suspension cells are interesting hosts for the heterologous production of pharmacological proteins such as antibodies. They have the advantage to facilitate the containment and the application of good manufacturing practices. Furthermore, antibodies can be secreted to the extracellular medium, which makes the purification steps much simpler. However, improvements are still to be made regarding the quality and the production yield. For instance, the inactivation of proteases and the humanization of glycosylation are both important targets which require either gene silencing or gene inactivation. To this purpose, CRISPR-Cas9 is a very promising technique which has been used recently in a series of plant species, but not yet in plant suspension cells. Here, we sought to use the CRISPR-Cas9 system for gene inactivation in Nicotiana tabacum BY-2 suspension cells. We transformed a transgenic line expressing a red fluorescent protein (mCherry) with a binary vector containing genes coding for Cas9 and three guide RNAs targeting mCherry restriction sites, as well as a bialaphos-resistant (bar) gene for selection. To demonstrate gene inactivation in the transgenic lines, the mCherry gene was PCR-amplified and analyzed by electrophoresis. Seven out of 20 transformants displayed a shortened fragment, indicating that a deletion occurred between two target sites. We also analyzed the transformants by restriction fragment length polymorphism and observed that the three targeted restriction sites were hit. DNA sequencing of the PCR fragments confirmed either deletion between two target sites or single nucleotide deletion. We therefore conclude that CRISPR-Cas9 can be used in N. tabacum BY2 cells.

Irreversible blockade of sigma-1 receptors by haloperidol and its metabolites in guinea pig brain and SH-SY5Y human neuroblastoma cells.

PubMed

Cobos, Enrique J; del Pozo, Esperanza; Baeyens, José M

2007-08-01

We evaluated the effect of haloperidol (HP) and its metabolites on [(3)H](+)-pentazocine binding to sigma(1) receptors in SH-SY5Y human neuroblastoma cells and guinea pig brain P(1), P(2) and P(3) subcellular fractions. Three days after a single i.p. injection in guinea pigs of HP (but not of other sigma(1) antagonists or (-)-sulpiride), [(3)H](+)-pentazocine binding to brain membranes was markedly decreased. Recovery of sigma(1) receptor density to steady state after HP-induced inactivation required more than 30 days. HP-metabolite II (reduced HP, 4-(4-chlorophenyl)-alpha-(4-fluorophenyl)-4-hydroxy-1-piperidinebutanol), but not HP-metabolite I (4-(4-chlorophenyl)-4-hydroxypiperidine), irreversibly blocked sigma(1) receptors in guinea pig brain homogenate and P(2) fraction in vitro. We found similar results in SH-SY5Y cells, which suggests that this process may also take place in humans. HP irreversibly inactivated sigma(1) receptors when it was incubated with brain homogenate and SH-SY5Y cells, but not when incubated with P(2) fraction membranes, which suggests that HP is metabolized to inactivate sigma(1) receptors. Menadione, an inhibitor of the ketone reductase activity that leads to the production of HP-metabolite II, completely prevented HP-induced inactivation of sigma(1) receptors in brain homogenates. These results suggest that HP may irreversibly inactivate sigma(1) receptors in guinea pig and human cells, probably after metabolism to reduced HP.

Feasibility of utilizing bioindicators for testing microbial inactivation in sweetpotato purees processed with a continuous-flow microwave system.

PubMed

Brinley, T A; Dock, C N; Truong, V-D; Coronel, P; Kumar, P; Simunovic, J; Sandeep, K P; Cartwright, G D; Swartzel, K R; Jaykus, L-A

2007-06-01

Continuous-flow microwave heating has potential in aseptic processing of various food products, including purees from sweetpotatoes and other vegetables. Establishing the feasibility of a new processing technology for achieving commercial sterility requires evaluating microbial inactivation. This study aimed to assess the feasibility of using commercially available plastic pouches of bioindicators containing spores of Geobacillius stearothermophilus ATCC 7953 and Bacillus subtilis ATCC 35021 for evaluating the degree of microbial inactivation achieved in vegetable purees processed in a continuous-flow microwave heating unit. Sweetpotato puree seeded with the bioindicators was subjected to 3 levels of processing based on the fastest particles: undertarget process (F(0) approximately 0.65), target process (F(0) approximately 2.8), and overtarget process (F(0) approximately 10.10). After initial experiments, we found it was necessary to engineer a setup with 2 removable tubes connected to the continuous-flow microwave system to facilitate the injection of indicators into the unit without interrupting the puree flow. Using this approach, 60% of the indicators injected into the system could be recovered postprocess. Spore survival after processing, as evaluated by use of growth indicator dyes and standard plating methods, verified inactivation of the spores in sweetpotato puree. The log reduction results for B. subtilis were equivalent to the predesigned degrees of sterilization (F(0)). This study presents the first report suggesting that bioindicators such as the flexible, food-grade plastic pouches can be used for microbial validation of commercial sterilization in aseptic processing of foods using a continuous-flow microwave system.

Complete inactivation of HIV-1 using photo-labeled non-nucleoside reverse transcriptase inhibitors.

PubMed

Rios, Adan; Quesada, Jorge; Anderson, Dallas; Goldstein, Allan; Fossum, Theresa; Colby-Germinario, Susan; Wainberg, Mark A

2011-01-01

We demonstrate that a photo-labeled derivative of the non-nucleoside reverse transcriptase inhibitor (NNRTI) dapivirine termed DAPY, when used together with exposure to ultraviolet light, was able to completely and irreversibly inactivate both HIV-1 RT activity as well as infectiousness in each of a T cell line and peripheral blood mononuclear cells. Control experiments using various concentrations of DAPY revealed that a combination of exposure to ultraviolet light together with use of the specific, high affinity photo-labeled compound was necessary for complete inactivation to occur. This method of HIV RT inactivation may have applicability toward preservation of an intact viral structure and warrants further investigation in regard to the potential of this approach to elicit a durable, broad protective immune response. Copyright © 2010 Elsevier B.V. All rights reserved.

A helix-breaking mutation in the epithelial Ca2+ channel TRPV5 leads to reduced Ca2+-dependent inactivation

PubMed Central

Lee, Kyu Pil; Nair, Anil V.; Grimm, Christian; van Zeeland, Femke; Heller, Stefan; Bindels, René J.M.; Hoenderop, Joost G.J.

2013-01-01

TRPV5, a member of transient receptor potential (TRP) superfamily of ion channels, plays a crucial role in epithelial calcium transport in the kidney. This channel has a high selectivity for Ca2+ and is tightly regulated by intracellular Ca2+ concentrations. Recently it was shown that the molecular basis of deafness in varitint-waddler mouse is the result of hair cell death caused by the constitutive activity of transient receptor potential mucolipin 3 (TRPML3) channel carrying a helix breaking mutation, A419P, at the intracellular proximity of the fifth transmembrane domain (TM5). This mutation significantly elevates intracellular Ca2+ concentration and causes rapid cell death. Here we show that substituting the equivalent location in TRPV5, the M490, to proline significantly modulates Ca2+-dependent inactivation of TRPV5. The single channel conductance, time constant of inactivation (τ) and half maximal inhibition constant (IC50) of TRPV5(M490P) were increased compared to TRPV5(WT). Moreover TRPV5(M490P) showed lower Ca2+ permeability. Out of different point mutations created to characterize the importance of M490 in Ca2+-dependent inactivation, only TRPV5(M490P)-expressing cells showed apoptosis and extremely altered Ca2+-dependent inactivation. In conclusion, the TRPV5 channel is susceptible for helix breaking mutations and the proximal intracellular region of TM5 of this channel plays an important role in Ca2+-dependent inactivation. PMID:21035851

Optimizing supercritical carbon dioxide in the inactivation of bacteria in clinical solid waste by using response surface methodology.

PubMed

Hossain, Md Sohrab; Nik Ab Rahman, Nik Norulaini; Balakrishnan, Venugopal; Alkarkhi, Abbas F M; Ahmad Rajion, Zainul; Ab Kadir, Mohd Omar

2015-04-01

Clinical solid waste (CSW) poses a challenge to health care facilities because of the presence of pathogenic microorganisms, leading to concerns in the effective sterilization of the CSW for safe handling and elimination of infectious disease transmission. In the present study, supercritical carbon dioxide (SC-CO2) was applied to inactivate gram-positive Staphylococcus aureus, Enterococcus faecalis, Bacillus subtilis, and gram-negative Escherichia coli in CSW. The effects of SC-CO2 sterilization parameters such as pressure, temperature, and time were investigated and optimized by response surface methodology (RSM). Results showed that the data were adequately fitted into the second-order polynomial model. The linear quadratic terms and interaction between pressure and temperature had significant effects on the inactivation of S. aureus, E. coli, E. faecalis, and B. subtilis in CSW. Optimum conditions for the complete inactivation of bacteria within the experimental range of the studied variables were 20 MPa, 60 °C, and 60 min. The SC-CO2-treated bacterial cells, observed under a scanning electron microscope, showed morphological changes, including cell breakage and dislodged cell walls, which could have caused the inactivation. This espouses the inference that SC-CO2 exerts strong inactivating effects on the bacteria present in CSW, and has the potential to be used in CSW management for the safe handling and recycling-reuse of CSW materials. Copyright © 2015 Elsevier Ltd. All rights reserved.

Thermal inactivation and sublethal injury kinetics of Salmonella enterica and Listeria monocytogenes in broth versus agar surface.

PubMed

Wang, Xiang; Devlieghere, Frank; Geeraerd, Annemie; Uyttendaele, Mieke

2017-02-21

The objective of the present study was to compare the thermal inactivation and sublethal injury kinetics of Salmonella enterica and Listeria monocytogenes in broth (suspended cells) and on solid surface (agar-seeded cells). A 3-strain cocktail of S. enterica or L. monocytogenes inoculated in broth or on agar was subjected to heating in a water bath at various set temperatures (55.0, 57.5 and 60.0°C for S. enterica and 60.0, 62.5 and 65°C for L. monocytogenes). The occurrence of sublethally injured cells was determined by comparing enumerations on nonselective (TSAYE) and selective (XLD or ALOA) media. Results showed that the inactivation curves obtained from selective media were log-linear, and significant shoulders (p<0.05) were observed on some of the inactivation curves from TSAYE media. The D-values derived from the total population were higher than those from the uninjured cells. Generally, cells on agar surface exhibited higher heat resistance than those in broth. For S. enterica, cell injury increased with the exposure time, no difference was observed when treated at temperatures from 55.0 to 60.0°C, while for L. monocytogenes, cell injury increased significantly with heating time and treatment temperature (from 60.0 to 65°C). Moreover, the degree of sublethal injury affected by thermal treatment in broth or on agar surface depended upon the target microorganism. Higher proportions of injured S. enterica cells were observed for treatment in broth than on agar surface, while the opposite was found for L. monocytogenes. The provided information may be used to assess the efficacy of thermal treatment processes on surfaces for inactivation of S. enterica and L. monocytogenes, and it provides insight into the sublethally injured survival state of S. enterica and L. monocytogenes treated in liquid or on solid food. Copyright © 2016 Elsevier B.V. All rights reserved.

Impact of food model (micro)structure on the microbial inactivation efficacy of cold atmospheric plasma.

PubMed

Smet, C; Noriega, E; Rosier, F; Walsh, J L; Valdramidis, V P; Van Impe, J F

2017-01-02

The large potential of cold atmospheric plasma (CAP) for food decontamination has recently been recognized. Room-temperature gas plasmas can decontaminate foods without causing undesired changes. This innovative technology is a promising alternative for treating fresh produce. However, more fundamental studies are needed before its application in the food industry. The impact of the food structure on CAP decontamination efficacy of Salmonella Typhimurium and Listeria monocytogenes was studied. Cells were grown planktonically or as surface colonies in/on model systems. Both microorganisms were grown in lab culture media in petri dishes at 20°C until cells reached the stationary phase. Before CAP treatment, cells were deposited in a liquid carrier, on a solid(like) surface or on a filter. A dielectric barrier discharge reactor generated helium-oxygen plasma, which was used to treat samples up to 10min. Although L. monocytogenes is more resistant to CAP treatment, similar trends in inactivation behavior as for S. Typhimurium are observed, with log reductions in the range [1.0-2.9] for S. Typhimurium and [0.2-2.2] for L. monocytogenes. For both microorganisms, cells grown planktonically are easily inactivated, as compared to surface colonies. More stressing growth conditions, due to cell immobilization, result in more resistant cells during CAP treatment. The main difference between the inactivation support systems is the absence or presence of a shoulder phase. For experiments in the liquid carrier, which exhibit a long shoulder, the plasma components need to diffuse and penetrate through the medium. This explains the higher efficacies of CAP treatment on cells deposited on a solid(like) surface or on a filter. This research demonstrates that the food structure influences the cell inactivation behavior and efficacy of CAP, and indicates that food intrinsic factors need to be accounted when designing plasma treatment. Copyright © 2016. Published by Elsevier B.V.

Epigenetic inactivation of TCF2 in ovarian cancer and various cancer cell lines

PubMed Central

Terasawa, K; Toyota, M; Sagae, S; Ogi, K; Suzuki, H; Sonoda, T; Akino, K; Maruyama, R; Nishikawa, N; Imai, K; Shinomura, Y; Saito, T; Tokino, T

2006-01-01

Transcription factor 2 gene (TCF2) encodes hepatocyte nuclear factor 1β (HNF1β), a transcription factor associated with development and metabolism. Mutation of TCF2 has been observed in renal cell cancer, and by screening aberrantly methylated genes, we have now identified TCF2 as a target for epigenetic inactivation in ovarian cancer. TCF2 was methylated in 53% of ovarian cancer cell lines and 26% of primary ovarian cancers, resulting in loss of the gene's expression. TCF2 expression was restored by treating cells with a methyltransferase inhibitor, 5-aza-2′deoxycitidine (5-aza-dC). In addition, chromatin immunoprecipitation showed deacetylation of histone H3 in methylated cells and, when combined with 5-aza-dC, the histone deacetylase inhibitor trichostatin A synergistically induced TCF2 expression. Epigenetic inactivation of TCF2 was also seen in colorectal, gastric and pancreatic cell lines, suggesting general involvement of epigenetic inactivation of TCF2 in tumorigenesis. Restoration of TCF2 expression induced expression of HNF4α, a transcriptional target of HNF1β, indicating that epigenetic silencing of TCF2 leads to alteration of the hepatocyte nuclear factor network in tumours. These results suggest that TCF2 is involved in the development of ovarian cancers and may represent a useful target for their detection and treatment. PMID:16479257

Efficient Inactivation of Burkholderia pseudomallei or Francisella tularensis in infected Cells for Safe Removal from Biosafety Level 3 Containment Laboratories

PubMed Central

Emery, Felicia D.; Stabenow, Jennifer M.; Miller, Mark A.

2014-01-01

Working with infectious agents that require BSL-3 level containment agents offers many challenges for researchers. BSL-3 containment laboratories are usually not equipped with expensive specialty equipment that is needed for studies such as flow cytometric analysis, microscopy, and proteomic analyses. Therefore, for most researchers that are working with BSL-3 level infectious agents, removal of samples from BSL-3 labs for these types of studies is necessary, and methods for complete and dependable inactivation of the samples are required. In this report we have done a thorough characterization of the effectiveness of paraformaldehyde fixation for inactivation of cell samples infected with the intracellular bacterial agents Burkholderia pseudomallei (Bp) and Francisella tularensis (Ft), both of which are Tier 1 select agent pathogens that require BSL-3 containment. We have demonstrated that cells infected with these pathogens are completely inactivated via 5-minute treatment with 4% paraformaldehyde. Moreover, a 15-minute treatment with 2% paraformaldehyde completely sterilized both Bp- and Ft-infected cells. These studies also revealed that Bp is significantly more sensitive to paraformaldehyde treatment than Ft. Our findings have clearly demonstrated that a 15-minute treatment of Bp- or Ft-infected cells with 4% paraformaldehyde solution will allow for safe removal of the cell samples from BSL-3 labs for downstream studies. PMID:24449562

Assessment of immunogenic potential of Vero adapted formalin inactivated vaccine derived from novel ECSA genotype of Chikungunya virus.

PubMed

Tiwari, Mugdha; Parida, Manmohan; Santhosh, S R; Khan, Mohsin; Dash, Paban Kumar; Rao, P V Lakshmana

2009-04-21

The recent resurgence of Chikungunya virus (CHIKV) in India and Indian Ocean Islands with unusual clinical severity is a matter of great public health concern. Despite the fact that CHIKV resurgence is associated with epidemic of unprecedented magnitude, no approved licensed vaccine is currently available. In the present study, a Vero cell adapted purified formalin inactivated prototype vaccine candidate was prepared using a current Indian strain implicated with the explosive epidemic during 2006. The bulk preparation of the vaccine candidate was undertaken in microcarrier based spinner culture using cytodex-1 in virus production serum free medium. The inactivation of the virus was accomplished through standard formalin inactivation protocol. The mice were immunized subcutaneously with alhydrogel gel formulation of inactivated virus preparation. The assessment of both humoral and cell-mediated immune response was accomplished through ELISA, plaque reduction neutralization test (PRNT), microcytotoxicity assay and cytokine production assay. The results revealed that formalin inactivated vaccine candidate induced both high titered ELISA (1:51,200) and plaque reduction neutralizing antibodies (1:6400) with peak antibody titer being observed during 6 -- 8 weeks of post-vaccination. In the absence of suitable murine challenge model, the protective efficacy was established by both in vitro and in vivo neutralization tests. Further assessment of cellular immunity through in vitro stimulation of spleenocytes from immunized mice revealed augmentation of high levels of both pro- and anti-inflammatory cytokines, indicating a mixed balance of Th1 and Th2 response. These findings suggest that the formalin inactivated Chikungunya vaccine candidate reported in this study has very good immunogenic potential to neutralize the virus infectivity by augmenting both humoral and cell-mediated immune response.

Optimizing supercritical carbon dioxide in the inactivation of bacteria in clinical solid waste by using response surface methodology

DOE Office of Scientific and Technical Information (OSTI.GOV)

Hossain, Md. Sohrab; Nik Ab Rahman, Nik Norulaini; Balakrishnan, Venugopal

2015-04-15

Highlights: • Supercritical carbon dioxide sterilization of clinical solid waste. • Inactivation of bacteria in clinical solid waste using supercritical carbon dioxide. • Reduction of the hazardous exposure of clinical solid waste. • Optimization of the supercritical carbon dioxide experimental conditions. - Abstract: Clinical solid waste (CSW) poses a challenge to health care facilities because of the presence of pathogenic microorganisms, leading to concerns in the effective sterilization of the CSW for safe handling and elimination of infectious disease transmission. In the present study, supercritical carbon dioxide (SC-CO{sub 2}) was applied to inactivate gram-positive Staphylococcus aureus, Enterococcus faecalis, Bacillus subtilis,more » and gram-negative Escherichia coli in CSW. The effects of SC-CO{sub 2} sterilization parameters such as pressure, temperature, and time were investigated and optimized by response surface methodology (RSM). Results showed that the data were adequately fitted into the second-order polynomial model. The linear quadratic terms and interaction between pressure and temperature had significant effects on the inactivation of S. aureus, E. coli, E. faecalis, and B. subtilis in CSW. Optimum conditions for the complete inactivation of bacteria within the experimental range of the studied variables were 20 MPa, 60 °C, and 60 min. The SC-CO{sub 2}-treated bacterial cells, observed under a scanning electron microscope, showed morphological changes, including cell breakage and dislodged cell walls, which could have caused the inactivation. This espouses the inference that SC-CO{sub 2} exerts strong inactivating effects on the bacteria present in CSW, and has the potential to be used in CSW management for the safe handling and recycling-reuse of CSW materials.« less

[Inactivated poliovirus vaccines: an inevitable choice for eliminating poliomyelitis].

PubMed

Vidor, J D; Jean-Denis, Shu

2016-12-06

The inactivated poliovirus vaccine (IPV) is a very old tool in the fight against poliomyelitis. Though supplanted by oral poliovirus vaccine (OPV) in the 1960s and 1970s, the IPV has now become an inevitable choice because of the increasingly recognized risks associated with continuous use of OPVs. Following the pioneering work of Jonas Salk, who established key principles for the IPV, considerable experience has accumulated over the years. This work has led to modern Salk IPV-containing vaccines, based on the use of inactivated wildtype polioviruses, which have been deployed for routine use in many countries. Very good protection against paralysis is achieved with IPV through the presence of circulating antibodies able to neutralize virus infectivity toward motor neurons. In addition, with IPV, a variable degree of protection against mucosal infection (and therefore transmission) through mucosal antibodies and immune cells is achieved, depending on previous exposure of subjects to wildtype or vaccine polioviruses. The use of an IPV-followed-by-OPV sequential immunization schedule has the potential advantage of eliminating the vaccine-associated paralytic poliomyelitis (VAPP) risk, while limiting the risks of vaccine-derived poliovirus (VDPVs). Sabin strain-derived IPVs are new tools, only recently beginning to be deployed, and data are being generated to document their performance. IPVs will play an irreplaceable role in global eradication of polio.

Vero cell technology for rapid development of inactivated whole virus vaccines for emerging viral diseases.

PubMed

Barrett, P Noel; Terpening, Sara J; Snow, Doris; Cobb, Ronald R; Kistner, Otfried

2017-09-01

Rapid development and production of vaccines against emerging diseases requires well established, validated, robust technologies to allow industrial scale production and accelerated licensure of products. Areas covered: A versatile Vero cell platform has been developed and utilized to deliver a wide range of candidate and licensed vaccines against emerging viral diseases. This platform builds on the 35 years' experience and safety record with inactivated whole virus vaccines such as polio vaccine. The current platform has been optimized to include a novel double inactivation procedure in order to ensure a highly robust inactivation procedure for novel emerging viruses. The utility of this platform in rapidly developing inactivated whole virus vaccines against pandemic (-like) influenza viruses and other emerging viruses such as West Nile, Chikungunya, Ross River and SARS is reviewed. The potential of the platform for development of vaccines against other emerging viruses such as Zika virus is described. Expert commentary: Use of this platform can substantially accelerate process development and facilitate licensure because of the substantial existing data set available for the cell matrix. However, programs to provide vaccines against emerging diseases must allow alternative clinical development paths to licensure, without the requirement to carry out large scale field efficacy studies.

Unusual X-chromosome inactivation pattern in patients with Xp11.23-p11.22 duplication: Report and review.

PubMed

Di-Battista, Adriana; Meloni, Vera Ayres; da Silva, Magnus Dias; Moysés-Oliveira, Mariana; Melaragno, Maria Isabel

2016-12-01

In females carrying structural rearrangements of an X-chromosome, cells with the best dosage balance are preferentially selected, frequently resulting in a skewed inactivation pattern and amelioration of the phenotype. The Xp11.23-p11.22 region is involved in a recently described microduplication syndrome associated with severe clinical consequences in males and females, causing intellectual disability, behavior problems, epilepsy with electroencephalogram anomalies, minor facial anomalies, and early onset of puberty. Female carriers usually present an unusual X-chromosome inactivation pattern in favor of the aberrant chromosome, resulting in functional disomy of the duplicated segment. Here, we describe a girl carrying a de novo ∼9.7 Mb Xp11.3-p11.22 duplication of paternal origin and skewed X-chromosome inactivation pattern of the normal X-chromosome. We reviewed other cases previously reported and determined the minimal critical region possibly responsible for this unusual inactivation pattern. The critical region encompasses 36 RefSeq genes, including at least 10 oncogenes and/or genes related to the cell cycle control. We discuss the molecular mechanisms that underlie the positive selection of the cells with the active duplicated chromosome. © 2016 Wiley Periodicals, Inc. © 2016 Wiley Periodicals, Inc.

Latexin Inactivation Enhances Survival and Long-Term Engraftment of Hematopoietic Stem Cells and Expands the Entire Hematopoietic System in Mice.

PubMed

Liu, Yi; Zhang, Cuiping; Li, Zhenyu; Wang, Chi; Jia, Jianhang; Gao, Tianyan; Hildebrandt, Gerhard; Zhou, Daohong; Bondada, Subbarao; Ji, Peng; St Clair, Daret; Liu, Jinze; Zhan, Changguo; Geiger, Hartmut; Wang, Shuxia; Liang, Ying

2017-04-11

Natural genetic diversity offers an important yet largely untapped resource to decipher the molecular mechanisms regulating hematopoietic stem cell (HSC) function. Latexin (Lxn) is a negative stem cell regulatory gene identified on the basis of genetic diversity. By using an Lxn knockout mouse model, we found that Lxn inactivation in vivo led to the physiological expansion of the entire hematopoietic hierarchy. Loss of Lxn enhanced the competitive repopulation capacity and survival of HSCs in a cell-intrinsic manner. Gene profiling of Lxn-null HSCs showed altered expression of genes enriched in cell-matrix and cell-cell interactions. Thrombospondin 1 (Thbs1) was a potential downstream target with a dramatic downregulation in Lxn-null HSCs. Enforced expression of Thbs1 restored the Lxn inactivation-mediated HSC phenotypes. This study reveals that Lxn plays an important role in the maintenance of homeostatic hematopoiesis, and it may lead to development of safe and effective approaches to manipulate HSCs for clinical benefit. Copyright © 2017 The Author(s). Published by Elsevier Inc. All rights reserved.

Importance of cell damage causing growth delay for high pressure inactivation of Saccharomyces cerevisiae

NASA Astrophysics Data System (ADS)

Nanba, Masaru; Nomura, Kazuki; Nasuhara, Yusuke; Hayashi, Manabu; Kido, Miyuki; Hayashi, Mayumi; Iguchi, Akinori; Shigematsu, Toru; Hirayama, Masao; Ueno, Shigeaki; Fujii, Tomoyuki

2013-06-01

A high pressure (HP) tolerant (barotolerant) mutant a2568D8 and a variably barotolerant mutant a1210H12 were generated from Saccharomyces cerevisiae using ultra-violet mutagenesis. The two mutants, a barosensitive mutant a924E1 and the wild-type strain, were pressurized (225 MPa), and pressure inactivation behavior was analyzed. In the wild-type strain, a proportion of the growth-delayed cells were detected after exposure to HP. In a924E1, the proportion of growth-delayed cells significantly decreased compared with the wild-type. In a2568D8, the proportion of growth-delayed cells increased and the proportion of inactivated cells decreased compared with the wild-type. In a1210H12, the growth-delayed cells could not be detected within 120 s of exposure to HP. The proportion of growth-delayed cells, which incurred the damage, would affect the survival ratio by HP. These results suggested that cellular changes in barotolerance caused by mutations are remarkably affected by the ability to recover from cellular damage, which results in a growth delay.

X-chromosome inactivation in development and cancer.

PubMed

Chaligné, Ronan; Heard, Edith

2014-08-01

X-chromosome inactivation represents an epigenetics paradigm and a powerful model system of facultative heterochromatin formation triggered by a non-coding RNA, Xist, during development. Once established, the inactive state of the Xi is highly stable in somatic cells, thanks to a combination of chromatin associated proteins, DNA methylation and nuclear organization. However, sporadic reactivation of X-linked genes has been reported during ageing and in transformed cells and disappearance of the Barr body is frequently observed in cancer cells. In this review we summarise current knowledge on the epigenetic changes that accompany X inactivation and discuss the extent to which the inactive X chromosome may be epigenetically or genetically perturbed in breast cancer. Copyright © 2014 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.

Synergistic Inhibition of Delayed Rectifier K+ and Voltage-Gated Na+ Currents by Artemisinin in Pituitary Tumor (GH3) Cells.

PubMed

So, Edmund Cheung; Wu, Sheng-Nan; Wu, Ping-Ching; Chen, Hui-Zhen; Yang, Chia-Jung

2017-01-01

Artemisinin (ART) is an anti-malarial agent reported to influence endocrine function. Effects of ART on ionic currents and action potentials (APs) in pituitary tumor (GH3) cells were evaluated by patch clamp techniques. ART inhibited the amplitude of delayed-rectifier K+ current (IK(DR)) in response to membrane depolarization and accelerated the process of current inactivation. It exerted an inhibitory effect on IK(DR) with an IC50 value of 11.2 µM and enhanced IK(DR) inactivation with a KD value of 14.7 µM. The steady-state inactivation curve of IK(DR) was shifted to hyperpolarization by 10 mV. Pretreatment of chlorotoxin (1 µM) or iloprost (100 nM) did not alter the magnitude of ART-induced inhibition of IK(DR) in GH3 cells. ART also decreased the peak amplitude of voltage-gated Na+ current (INa) with a concentration-dependent slowing in inactivation rate. Application of KMUP-1, an inhibitor of late INa, was effective at reversing ART-induced prolongation in inactivation time constant of INa. Under current-clamp recordings, ART alone reduced the amplitude of APs and prolonged the duration of APs. Under ART exposure, the inhibitory actions on both IK(DR) and INa could be a potential mechanisms through which this drug influences membrane excitability of endocrine or neuroendocrine cells appearing in vivo. © 2017 The Author(s). Published by S. Karger AG, Basel.

Effects of several immunostimulants on phenoloxidase and hemocytes of the crab Charybdis japonica

NASA Astrophysics Data System (ADS)

Fan, Tingjun; Yu, Miaomiao; Yang, Lingling; Shi, Zhenping; Sun, Wenjie; Cong, Rishan; Yang, Xiuxia; Jiang, Guojian

2009-09-01

To investigate the stimulating effects of immunostimulants on the autogenous immunocompetence of crabs and the possible mechanisms involved, the immunostimulating effects of β-1,3-glucan, lipopolysaccharide (LPS), inactivated Vibrio harveyi and Vibrio anguillarum on phenoloxidase (PO) and hemocytes of Charybdis japonica were investigated in this study. It was found that the yields and the enzymatic activities of purified PO in C. japonica increased significantly after the crabs were treated with immunostimulants, while the unit enzymatic activities remained almost the same. After treatment with β-1,3-glucan and LPS, the amount of rough endoplasmic reticulum (RER) and the number of mitochondria in both semigranular cells and granular cells increased greatly, and the number of cytoplasmic granules decreased but with enlarged volume. However, the corresponding characteristics of hyaline cells remained almost the same. On the other hand, the number of granules in semigranular cells decreased greatly, and the number of mitochondria of hyaline cells increased greatly, after treatment with inactivated vibrios. It may be concluded that the effect of polysaccharide immunostimulants on the innate immune system of C. japonica is different from that of inactivated vibrio immunostimulants. The immunity-enhancing mechanism of polysaccharides in crab autogenous immunocompetence is probably accomplished by the increased yields of PO and total PO activities, while that of inactivated vibrios is probably accomplished by the partially increased yields of PO and total PO activities as well as the significantly improved phagocytotic abilities of semigranular cells and hyaline cells.

CHLORINE INACTIVATION OF CATEGORY "A" BIO-TERRORISM AGENTS

EPA Science Inventory

This poster presents information on the inactivation of select bioterrorist agents. Information will be presented on chlorine disinfection of vegetative cells of Brucella suis, Brucella melitensis, Burkholderia mallei, Burkholderia pseudomallei, Francisella tularensis and endos...

Solar and Temperature Treatments Affect the Ability of Human Rotavirus Wa To Bind to Host Cells and Synthesize Viral RNA

PubMed Central

Shisler, Joanna L.

2015-01-01

Rotavirus, the leading cause of diarrheal diseases in children under the age of five, is often resistant to conventional wastewater treatment and thus can remain infectious once released into the aquatic environment. Solar and heat treatments can inactivate rotavirus, but it is unknown how these treatments inactivate the virus on a molecular level. To answer this question, our approach was to correlate rotavirus inactivation with the inhibition of portions of the virus life cycle as a means to identify the mechanisms of solar or heat inactivation. Specifically, the integrity of the rotavirus NSP3 gene, virus-host cell interaction, and viral RNA synthesis were examined after heat (57°C) or solar treatment of rotavirus. Only the inhibition of viral RNA synthesis positively correlated with a loss of rotavirus infectivity; 57°C treatment of rotavirus resulted in a decrease of rotavirus RNA synthesis at the same rate as rotavirus infectivity. These data suggest that heat treatment neutralized rotaviruses primarily by targeting viral transcription functions. In contrast, when using solar disinfection, the decrease in RNA synthesis was responsible for approximately one-half of the decrease in infectivity, suggesting that other mechanisms, including posttranslational, contribute to inactivation. Nevertheless, both solar and heat inactivation of rotaviruses disrupted viral RNA synthesis as a mechanism for inactivation. PMID:25862222

Non-random X chromosome inactivation in an affected twin in a monozygotic twin pair discordant for Wiedemann-Beckwith syndrome

DOE Office of Scientific and Technical Information (OSTI.GOV)

Oestavik, R.E.; Eiklid, K.; Oerstavik, K.H.

1995-03-27

Wiedemann-Beckwith syndrome (WBS) is a syndrome including exomphalos, macroglossia, and generalized overgrowth. The locus has been assigned to 11p15, and genomic imprinting may play a part in the expression of one or more genes involved. Most cases are sporadic. An excess of female monozygotic twins discordant for WBS have been reported, and it has been proposed that this excess could be related to the process of X chromosome inactivation. We have therefore studied X chromosome inactivation in 13-year-old monozygotic twin girls who were discordant for WBS. In addition, both twins had Tourette syndrome. The twins were monochorionic and therefore themore » result of a late twinning process. This has also been the case in previously reported discordant twin pairs with information on placentation. X chromosome inactivation was determined in DNA from peripheral blood cells by PCR analysis at the androgen receptor locus. The affected twin had a completely skewed X inactivation, where the paternal allele was on the active X chromosome in all cells. The unaffected twin had a moderately skewed X inactivation in the same direction, whereas the mother had a random pattern. Further studies are necessary to establish a possible association between the expression of WBS and X chromosome inactivation. 18 refs., 2 figs., 1 tab.« less

High hydrostatic pressure-induced cell death in human chondrocytes and chondrosarcoma cells.

PubMed

Naal, Florian-Dominique; Mengele, Karin; Schauwecker, Johannes; Gollwitzer, Hans; Gerdesmeyer, Ludger; Reuning, Ute; Mittelmeier, Wolfram; Gradinger, Reiner; Schmitt, Manfred; Diehl, Peter

2005-01-01

In orthopedic surgery, sterilization of bone used for reconstruction of osteoarticular defects caused by malignant tumors is carried out in different ways. At present, to devitalize tumor-bearing osteochondral segments, extracorporal irradiation or autoclaving is mainly used, although both methods have substantial disadvantages, e.g. loss of biomechanical and/or biological integrity of the bone and destabilization of the articular surface. In this regard, high hydrostatic pressure (HHP) treatment of bone is a new, advancing technology, now being used in preclinical testing to inactivate tumor cells. To find out if this technique is also suited for extracorporal inactivation of chondrocytes and chondral tumor cells, the effect of HHP on cell viability and morphology of human chondrocytes / chondrosarcoma cells was investigated in the present study. SW1353 chondrosarcoma cells and chondrocytes were subjected to HHP in the range of 50 to 350 MPa (10 min, 37 degrees C) and, subsequently, cell viability and cell morphology assessed. After exposure at 350 MPa, all HHP-treated chondral cells showed explicit morphological changes, evident by membrane ruffling and bleb formation; chondrosarcoma cells treated this way were irreversibly damaged and not alive. We anticipate that, in orthopedic surgery, HHP eventually can serve as a novel, promising technical approach for cell inactivation (including tumor cells) and allow subsequent reimplantation of the osteoarticular autograft.

Integrated oxide graphene based device for laser inactivation of pathogenic microorganisms

NASA Astrophysics Data System (ADS)

Grishkanich, Alexsandr; Ruzankina, Julia; Afanasyev, Mikhail; Paklinov, Nikita; Hafizov, Nail

2018-02-01

We develop device for virus disinfection of pathogenic microorganisms. Viral decontamination can be carried out due to hard ultraviolet irradiation and singlet oxygen destroying the genetic material of a virus capsid. UV rays can destroy DNA, leading to the formation of dimers of nucleic acids. This practically does not occur in tissues, tk. UV rays penetrate badly through them, however, the viral particles are small and UV can destroy their genetic material, RNA / DNA and the virus can not replicate. It is with the construction of the ultraviolet laser water disinfection system (UFLOV) based on the continuous and periodic pulsed ultraviolet laser sources (pump) binds to solve sterility and depyrogenation of water. It has been established that small doses of UV irradiation stimulate reproduction, and large doses cause the death of pathogenic microorganisms. The effect of a dose of ultraviolet is the result of photochemical action on the substance of a living bacterial cell or virion. Also complex photodynamic laser inactivation on graphene oxide is realized.

Lipid-linked cell wall precursors regulate membrane association of bacterial actin MreB

PubMed Central

Schirner, Kathrin; Eun, Ye-Jin; Dion, Mike; Luo, Yun; Helmann, John D.; Garner, Ethan C.; Walker, Suzanne

2014-01-01

Summary The bacterial actin homolog MreB, which is critical for rod shape determination, forms filaments that rotate around the cell width on the inner surface of the cytoplasmic membrane. What determines filament association with the membranes or with other cell wall elongation proteins is not known. Using specific chemical and genetic perturbations while following MreB filament motion, we find that MreB membrane association is an actively regulated process that depends on the presence of lipid-linked peptidoglycan precursors. When precursors are depleted, MreB filaments disassemble into the cytoplasm and peptidoglycan synthesis becomes disorganized. In cells that lack wall teichoic acids, but continue to make peptidoglycan, dynamic MreB filaments are observed, although their presence is not sufficient to establish a rod shape. We propose that the cell regulates MreB filament association with the membrane, allowing rapid and reversible inactivation of cell wall enzyme complexes in response to the inhibition of cell wall synthesis. PMID:25402772

Lipid-linked cell wall precursors regulate membrane association of bacterial actin MreB.

PubMed

Schirner, Kathrin; Eun, Ye-Jin; Dion, Mike; Luo, Yun; Helmann, John D; Garner, Ethan C; Walker, Suzanne

2015-01-01

The bacterial actin homolog MreB, which is crucial for rod shape determination, forms filaments that rotate around the cell width on the inner surface of the cytoplasmic membrane. What determines filament association with the membranes or with other cell wall elongation proteins is not known. Using specific chemical and genetic perturbations while following MreB filament motion, we find that MreB membrane association is an actively regulated process that depends on the presence of lipid-linked peptidoglycan precursors. When precursors are depleted, MreB filaments disassemble into the cytoplasm, and peptidoglycan synthesis becomes disorganized. In cells that lack wall teichoic acids but continue to make peptidoglycan, dynamic MreB filaments are observed, although their presence is not sufficient to establish a rod shape. We propose that the cell regulates MreB filament association with the membrane, allowing rapid and reversible inactivation of cell wall enzyme complexes in response to the inhibition of cell wall synthesis.

Characterization of two distinct depolarization-activated K+ currents in isolated adult rat ventricular myocytes

PubMed Central

1991-01-01

Depolarization-activated outward K+ currents in isolated adult rat ventricular myocytes were characterized using the whole-cell variation of the patch-clamp recording technique. During brief depolarizations to potentials positive to -40 mV, Ca(2+)-independent outward K+ currents in these cells rise to a transient peak, followed by a slower decay to an apparent plateau. The analyses completed here reveal that the observed outward current waveforms result from the activation of two kinetically distinct voltage-dependent K+ currents: one that activates and inactivates rapidly, and one that activates and inactivates slowly, on membrane depolarization. These currents are referred to here as Ito (transient outward) and IK (delayed rectifier), respectively, because their properties are similar (although not identical) to these K+ current types in other cells. Although the voltage dependences of Ito and IK activation are similar, Ito activates approximately 10-fold and inactivates approximately 30-fold more rapidly than IK at all test potentials. In the composite current waveforms measured during brief depolarizations, therefore, the peak current predominantly reflects Ito, whereas IK is the primary determinant of the plateau. There are also marked differences in the voltage dependences of steady-state inactivation of these two K+ currents: IK undergoes steady-state inactivation at all potentials positive to -120 mV, and is 50% inactivated at -69 mV; Ito, in contrast, is insensitive to steady-state inactivation at membrane potentials negative to -50 mV. In addition, Ito recovers from steady-state inactivation faster than IK: at -90 mV, for example, approximately 70% recovery from the inactivation produced at -20 mV is observed within 20 ms for Ito; IK recovers approximately 25-fold more slowly. The pharmacological properties of Ito and IK are also distinct: 4-aminopyridine preferentially attenuates Ito, and tetraethylammonium suppresses predominantly IK. The voltage- and time- dependent properties of these currents are interpreted here in terms of a model in which Ito underlies the initial, rapid repolarization phase of the action potential (AP), and IK is responsible for the slower phase of AP repolarization back to the resting membrane potential, in adult rat ventricular myocytes. PMID:1865177

Heme impairs the ball-and-chain inactivation of potassium channels.

PubMed

Sahoo, Nirakar; Goradia, Nishit; Ohlenschläger, Oliver; Schönherr, Roland; Friedrich, Manfred; Plass, Winfried; Kappl, Reinhard; Hoshi, Toshinori; Heinemann, Stefan H

2013-10-15

Fine-tuned regulation of K(+) channel inactivation enables excitable cells to adjust action potential firing. Fast inactivation present in some K(+) channels is mediated by the distal N-terminal structure (ball) occluding the ion permeation pathway. Here we show that Kv1.4 K(+) channels are potently regulated by intracellular free heme; heme binds to the N-terminal inactivation domain and thereby impairs the inactivation process, thus enhancing the K(+) current with an apparent EC50 value of ∼20 nM. Functional studies on channel mutants and structural investigations on recombinant inactivation ball domain peptides encompassing the first 61 residues of Kv1.4 revealed a heme-responsive binding motif involving Cys13:His16 and a secondary histidine at position 35. Heme binding to the N-terminal inactivation domain induces a conformational constraint that prevents it from reaching its receptor site at the vestibule of the channel pore.

Distinct Responses of Stem Cells to Telomere Uncapping-A Potential Strategy to Improve the Safety of Cell Therapy.

PubMed

Liu, Chang Ching; Ma, Dong Liang; Yan, Ting-Dong; Fan, XiuBo; Poon, Zhiyong; Poon, Lai-Fong; Goh, Su-Ann; Rozen, Steve G; Hwang, William Ying Khee; Tergaonkar, Vinay; Tan, Patrick; Ghosh, Sujoy; Virshup, David M; Goh, Eyleen L K; Li, Shang

2016-10-01

In most human somatic cells, the lack of telomerase activity results in progressive telomere shortening during each cell division. Eventually, DNA damage responses triggered by critically short telomeres induce an irreversible cell cycle arrest termed replicative senescence. However, the cellular responses of human pluripotent stem cells to telomere uncapping remain unknown. We generated telomerase knockout human embryonic stem (ES) cells through gene targeting. Telomerase inactivation in ES cells results in progressive telomere shortening. Telomere DNA damage in ES cells and neural progenitor cells induces rapid apoptosis when telomeres are uncapped, in contrast to fibroblast cells that enter a state of replicative senescence. Significantly, telomerase inactivation limits the proliferation capacity of human ES cells without affecting their pluripotency. By targeting telomerase activity, we can functionally separate the two unique properties of human pluripotent stem cells, namely unlimited self-renewal and pluripotency. We show that the potential of ES cells to form teratomas in vivo is dictated by their telomere length. By controlling telomere length of ES cells through telomerase inactivation, we can inhibit teratoma formation and potentially improve the safety of cell therapies involving terminally differentiated cells as well as specific progenitor cells that do not require sustained cellular proliferation in vivo, and thus sustained telomerase activity. Stem Cells 2016;34:2471-2484. © 2016 AlphaMed Press.

Inactivation of Caliciviruses

PubMed Central

Nims, Raymond; Plavsic, Mark

2013-01-01

The Caliciviridae family of viruses contains clinically important human and animal pathogens, as well as vesivirus 2117, a known contaminant of biopharmaceutical manufacturing processes employing Chinese hamster cells. An extensive literature exists for inactivation of various animal caliciviruses, especially feline calicivirus and murine norovirus. The caliciviruses are susceptible to wet heat inactivation at temperatures in excess of 60 °C with contact times of 30 min or greater, to UV-C inactivation at fluence ≥30 mJ/cm2, to high pressure processing >200 MPa for >5 min at 4 °C, and to certain photodynamic inactivation approaches. The enteric caliciviruses (e.g.; noroviruses) display resistance to inactivation by low pH, while the non-enteric species (e.g.; feline calicivirus) are much more susceptible. The caliciviruses are inactivated by a variety of chemicals, including alcohols, oxidizing agents, aldehydes, and β-propiolactone. As with inactivation of viruses in general, inactivation of caliciviruses by the various approaches may be matrix-, temperature-, and/or contact time-dependent. The susceptibilities of the caliciviruses to the various physical and chemical inactivation approaches are generally similar to those displayed by other small, non-enveloped viruses, with the exception that the parvoviruses and circoviruses may require higher temperatures for inactivation, while these families appear to be more susceptible to UV-C inactivation than are the caliciviruses. PMID:24276023

Merging Structural Motifs of Functionalized Amino Acids and α-Aminoamides Results in Novel Anticonvulsant Compounds with Significant Effects on Slow and Fast Inactivation of Voltage-Gated Sodium Channels and in the Treatment of Neuropathic Pain

PubMed Central

2011-01-01

We recently reported that merging key structural pharmacophores of the anticonvulsant drugs lacosamide (a functionalized amino acid) with safinamide (an α-aminoamide) resulted in novel compounds with anticonvulsant activities superior to that of either drug alone. Here, we examined the effects of six such chimeric compounds on Na+-channel function in central nervous system catecholaminergic (CAD) cells. Using whole-cell patch clamp electrophysiology, we demonstrated that these compounds affected Na+ channel fast and slow inactivation processes. Detailed electrophysiological characterization of two of these chimeric compounds that contained either an oxymethylene ((R)-7) or a chemical bond ((R)-11) between the two aromatic rings showed comparable effects on slow inactivation, use-dependence of block, development of slow inactivation, and recovery of Na+ channels from inactivation. Both compounds were equally effective at inducing slow inactivation; (R)-7 shifted the fast inactivation curve in the hyperpolarizing direction greater than (R)-11, suggesting that in the presence of (R)-7 a larger fraction of the channels are in an inactivated state. None of the chimeric compounds affected veratridine- or KCl-induced glutamate release in neonatal cortical neurons. There was modest inhibition of KCl-induced calcium influx in cortical neurons. Finally, a single intraperitoneal administration of (R)-7, but not (R)-11, completely reversed mechanical hypersensitivity in a tibial-nerve injury model of neuropathic pain. The strong effects of (R)-7 on slow and fast inactivation of Na+ channels may contribute to its efficacy and provide a promising novel therapy for neuropathic pain, in addition to its antiepileptic potential. PMID:21765969

Effect of microwave radiation on inactivation of Clostridium sporogenes (PA 3679) spores.

PubMed Central

Welt, B A; Tong, C H; Rossen, J L; Lund, D B

1994-01-01

Three techniques for studying effects of microwave radiation on microorganisms were introduced. Spores of Clostridium sporogenes (PA 3679) were chosen as a test organism because the kinetic parameters for thermal inactivation are well known and because of the importance of the genus Clostridium to the food industry. For the first technique, a specially designed kinetics vessel was used to compare inactivation rates of microwave-heated and conventionally heated spores at steady-state temperatures of 90, 100, and 110 degrees C. Rates were found to be similar at the 95% confidence level. The second and third techniques were designed to study the effect of relatively high power microwave exposure at sublethal temperatures. In the second approach, the suspension was continuously cooled via direct contact with a copper cooling coil in a well-mixed vessel, outside the microwave oven. The suspension was pumped through a Teflon loop in the oven, where it continuously absorbed approximately 400 W of microwave power. Inactivation occurred in both irradiated and unirradiated samples. It was suspected that copper ions entered the suspension from the copper coil and were toxic to the spores. The fact that the results were similar, however, implied the absence of nonthermal microwave effects. In the third approach, the copper coil was replaced with a silicone tubing loop in a microwave transparent vessel. The suspension was continuously irradiated at 150 W of microwave power. No detectable inactivation occurred. Results indicated that the effect of microwave energy on viability of spores was indistinguishable from the effect of conventional heating. PMID:8135512

Viologen-modified electrodes for protection of hydrogenases from high potential inactivation while performing H2 oxidation at low overpotential.

PubMed

Oughli, Alaa A; Vélez, Marisela; Birrell, James A; Schuhmann, Wolfgang; Lubitz, Wolfgang; Plumeré, Nicolas; Rüdiger, Olaf

2018-06-08

In this work we present a viologen-modified electrode providing protection for hydrogenases against high potential inactivation. Hydrogenases, including O2-tolerant classes, suffer from reversible inactivation upon applying high potentials, which limits their use in biofuel cells to certain conditions. Our previously reported protection strategy based on the integration of hydrogenase into redox matrices enabled the use of these biocatalysts in biofuel cells even under anode limiting conditions. However, mediated catalysis required application of an overpotential to drive the reaction, and this translates into a power loss in a biofuel cell. In the present work, the enzyme is adsorbed on top of a covalently-attached viologen layer which leads to mixed, direct and mediated, electron transfer processes; at low overpotentials, the direct electron transfer process generates a catalytic current, while the mediated electron transfer through the viologens at higher potentials generates a redox buffer that prevents oxidative inactivation of the enzyme. Consequently, the enzyme starts the catalysis at no overpotential with viologen self-activated protection at high potentials.

Population Density and Moment-based Approaches to Modeling Domain Calcium-mediated Inactivation of L-type Calcium Channels.

PubMed

Wang, Xiao; Hardcastle, Kiah; Weinberg, Seth H; Smith, Gregory D

2016-03-01

We present a population density and moment-based description of the stochastic dynamics of domain [Formula: see text]-mediated inactivation of L-type [Formula: see text] channels. Our approach accounts for the effect of heterogeneity of local [Formula: see text] signals on whole cell [Formula: see text] currents; however, in contrast with prior work, e.g., Sherman et al. (Biophys J 58(4):985-995, 1990), we do not assume that [Formula: see text] domain formation and collapse are fast compared to channel gating. We demonstrate the population density and moment-based modeling approaches using a 12-state Markov chain model of an L-type [Formula: see text] channel introduced by Greenstein and Winslow (Biophys J 83(6):2918-2945, 2002). Simulated whole cell voltage clamp responses yield an inactivation function for the whole cell [Formula: see text] current that agrees with the traditional approach when domain dynamics are fast. We analyze the voltage-dependence of [Formula: see text] inactivation that may occur via slow heterogeneous domain [[Formula: see text

Inactivation of Bmp4 from the Tbx1 Expression Domain Causes Abnormal Pharyngeal Arch Artery and Cardiac Outflow Tract Remodeling

PubMed Central

Nie, Xuguang; Brown, Christopher B.; Wang, Qin; Jiao, Kai

2011-01-01

Maldevelopment of outflow tract and aortic arch arteries is among the most common forms of human congenital heart diseases. Both Bmp4 and Tbx1 are known to play critical roles during cardiovascular development. Expression of these two genes partially overlaps in pharyngeal arch areas in mouse embryos. In this study, we applied a conditional gene inactivation approach to test the hypothesis that Bmp4 expressed from the Tbx1 expression domain plays a critical role for normal development of outflow tract and pharyngeal arch arteries. We showed that inactivation of Bmp4 from Tbx1-expressing cells leads to the spectrum of deformities resembling the cardiovascular defects observed in human DiGeorge syndrome patients. Inactivation of Bmp4 from the Tbx1 expression domain did not cause patterning defects, but affected remodeling of outflow tract and pharyngeal arch arteries. Our further examination revealed that Bmp4 is required for normal recruitment/differentiation of smooth muscle cells surrounding the PAA4 and survival of outflow tract cushion mesenchymal cells. PMID:21123999

The effect of deep frying or conventional oven cooking on inactivation of Shiga toxin-producing cells of Escherichia coli (STEC) in meatballs

USDA-ARS?s Scientific Manuscript database

We investigated the effects deep frying or oven cooking on inactivation of Shiga toxin-producing cells of Escherichia coli (STEC) in meatballs. A finely-ground veal and/or a beef-pork-veal mixture were inoculated (ca. 7.0 log CFU/g) with an eight-strain, genetically-marked cocktail of rifampicin-res...

Controlled initiation of chromosomal replication in Escherichia coli requires functional Hda protein.

PubMed

Camara, Johanna Eltz; Skarstad, Kirsten; Crooke, Elliott

2003-05-01

Regulatory inactivation of DnaA helps ensure that the Escherichia coli chromosome is replicated only once per cell cycle, through accelerated hydrolysis of active replication initiator ATP-DnaA to inactive ADP-DnaA. Analysis of deltahda strains revealed that the regulatory inactivation of DnaA component Hda is necessary for maintaining controlled initiation but not for cell growth or viability.

Mesenchymal stromal cells having inactivated RB1 survive following low irradiation and accumulate damaged DNA: Hints for side effects following radiotherapy.

PubMed

Alessio, Nicola; Capasso, Stefania; Di Bernardo, Giovanni; Cappabianca, Salvatore; Casale, Fiorina; Calarco, Anna; Cipollaro, Marilena; Peluso, Gianfranco; Galderisi, Umberto

2017-02-01

Following radiotherapy, bone sarcomas account for a significant percentage of recurring tumors. This risk is further increased in patients with hereditary retinoblastoma that undergo radiotherapy. We analyzed the effect of low and medium dose radiation on mesenchymal stromal cells (MSCs) with inactivated RB1 gene to gain insights on the molecular mechanisms that can induce second malignant neoplasm in cancer survivors. MSC cultures contain subpopulations of mesenchymal stem cells and committed progenitors that can differentiate into mesodermal derivatives: adipocytes, chondrocytes, and osteocytes. These stem cells and committed osteoblast precursors are the cell of origin in osteosarcoma, and RB1 gene mutations have a strong role in its pathogenesis. Following 40 and 2000 mGy X-ray exposure, MSCs with inactivated RB1 do not proliferate and accumulate high levels of unrepaired DNA as detected by persistence of gamma-H2AX foci. In samples with inactivated RB1 the radiation treatment did not increase apoptosis, necrosis or senescence versus untreated cells. Following radiation, CFU analysis showed a discrete number of cells with clonogenic capacity in cultures with silenced RB1. We extended our analysis to the other members of retinoblastoma gene family: RB2/P130 and P107. Also in the MSCs with silenced RB2/P130 and P107 we detected the presence of cells with unrepaired DNA following X-ray irradiation. Cells with unrepaired DNA may represent a reservoir of cells that may undergo neoplastic transformation. Our study suggests that, following radiotherapy, cancer patients with mutations of retinoblastoma genes may be under strict controls to evaluate onset of secondary neoplasms following radiotherapy.

Bacteria, mould and yeast spore inactivation studies by scanning electron microscope observations.

PubMed

Rozali, Siti N M; Milani, Elham A; Deed, Rebecca C; Silva, Filipa V M

2017-12-18

Spores are the most resistant form of microbial cells, thus difficult to inactivate. The pathogenic or food spoilage effects of certain spore-forming microorganisms have been the primary basis of sterilization and pasteurization processes. Thermal sterilization is the most common method to inactivate spores present on medical equipment and foods. High pressure processing (HPP) is an emerging and commercial non-thermal food pasteurization technique. Although previous studies demonstrated the effectiveness of thermal and non-thermal spore inactivation, the in-depth mechanisms of spore inactivation are as yet unclear. Live and dead forms of two food spoilage bacteria, a mould and a yeast were examined using scanning electron microscopy before and after the inactivation treatment. Alicyclobacillus acidoterrestris and Geobacillus stearothermophilus bacteria are indicators of acidic foods pasteurization and sterilization processes, respectively. Neosartorya fischeri is a phyto-pathogenic mould attacking fruits. Saccharomyces cerevisiae is a yeast with various applications for winemaking, brewing, baking and the production of biofuel from crops (e.g. sugar cane). Spores of the four microbial species were thermally inactivated. Spores of S. cerevisiae were observed in the ascus and free form after thermal and HPP treatments. Different forms of damage and cell destruction were observed for each microbial spore. Thermal treatment inactivated bacterial spores of A. acidoterrestris and G. stearothermophilus by attacking the inner core of the spore. The heat first altered the membrane permeability allowing the release of intracellular components. Subsequently, hydration of spores, physicochemical modifications of proteins, flattening and formation of indentations occurred, with subsequent spore death. Regarding N. fischeri, thermal inactivation caused cell destruction and leakage of intracellular components. Both thermal and HPP treatments of S. cerevisiae free spores attacked the inner membrane, altering its permeability, and allowing in final stages the transfer of intracellular components to the outside. The spore destruction caused by thermal treatment was more severe than HPP, as HPP had less effect on the spore core. All injured spores have undergone irreversible volume and shape changes. While some of the leakage of spore contents is visible around the deformed but fully shaped spore, other spores exhibited large indentations and were completely deformed, apparently without any contents inside. This current study contributed to the understanding of spore inactivation by thermal and non-thermal processes. Copyright © 2017 Elsevier B.V. All rights reserved.

Inactivation of Salmonella enterica by UV-C Light Alone and in Combination with Mild Temperatures

PubMed Central

Gayán, E.; Serrano, M. J.; Raso, J.; Álvarez, I.

2012-01-01

The aim of this investigation was to study the efficacy of the combined processes of UV light and mild temperatures for the inactivation of Salmonella enterica subsp. enterica and to explore the mechanism of inactivation. The doses to inactivate the 99.99% (4D) of the initial population ranged from 18.03 (Salmonella enterica serovar Typhimurium STCC 878) to 12.75 J ml−1 (Salmonella enterica serovar Enteritidis ATCC 13076). The pH and water activity of the treatment medium did not change the UV tolerance, but it decreased exponentially by increasing the absorption coefficient. An inactivating synergistic effect was observed by applying simultaneous UV light and heat treatment (UV-H). A less synergistic effect was observed by applying UV light first and heat subsequently. UV did not damage cell envelopes, but the number of injured cells was higher after a UV-H treatment than after heating. The synergistic effect observed by combining simultaneous UV and heat treatment opens the possibility to design combined treatments for pasteurization of liquid food with high UV absorptivity, such as fruit juices. PMID:23001665

Structural inhibition and reactivation of Escherichia coli septation by elements of the SOS and TER pathways

DOE Office of Scientific and Technical Information (OSTI.GOV)

Dopazo, A.; Tormo, A.; Aldea, M.

1987-04-01

The inhibition of cell division caused by induction of the SOS pathway in Escherichia coli structurally blocks septation, as deduced from two sets of results. Potential septation sites active at the time of SOS induction became inactivated, while those initiated during the following doubling time were active. Penicillin resistance increased in wild-type UV light-irradiated cells, a behavior similar to that observed in mutants in which structural blocks were introduced by inactivation of FtsA. Potential septation sites that have been structurally blocked by either the SOS division inhibitor, furazlocillin inhibition of PBP3, or inactivation of a TER pathway component, FtsA3, couldmore » be reactivated one doubling time after removal of the inhibitory agent in the presence of an active lon gene product. Reactivation of potential septation sites blocked by the presence of an inactivated FtsA3 was significantly lower when the lon protease was not active, suggesting that Lon plays a role in the removal of inactivated TER pathway products from the blocked potential septation sites.« less

Susceptibility of mouse minute virus to inactivation by heat in two cell culture media types.

PubMed

Schleh, Marc; Romanowski, Peter; Bhebe, Prince; Zhang, Li; Chinniah, Shivanthi; Lawrence, Bill; Bashiri, Houman; Gaduh, Asri; Rajurs, Viveka; Rasmussen, Brian; Chuck, Alice; Dehghani, Houman

2009-01-01

Viral contaminations of biopharmaceutical manufacturing cell culture facilities are a significant threat and one for which having a risk mitigation strategy is highly desirable. High temperature, short time (HTST) mammalian cell media treatment may potentially safeguard manufacturing facilities from such contaminations. HTST is thought to inactivate virions by denaturing proteins of the viral capsid, and there is evidence that HTST provides ample virucidal efficacy against nonenveloped or naked viruses such as mouse minute virus (MMV), a parvovirus. The aim of the studies presented herein was to further delineate the susceptibility of MMV, known to have contaminated mammalian cell manufacturing facilities, to heat by exposing virus-spiked cell culture media to a broad range of temperatures and for various times of exposure. The results of these studies show that HTST is capable of inactivating MMV by three orders of magnitude or more. Thus, we believe that HTST is a useful technology for the purposes of providing a barrier to adventitious contamination of mammalian cell culture processes in the biopharmaceutical industry. 2009 American Institute of Chemical Engineers

Inactivation of Lactobacillus rhamnosus GG by fixation modifies its probiotic properties.

PubMed

Markowicz, C; Kubiak, P; Grajek, W; Schmidt, M T

2016-01-01

Probiotics are microorganisms that have beneficial effects on the host and are safe for oral intake in a suitable dose. However, there are situations in which the administration of living microorganisms poses a risk for immunocompromised host. The objective of this study was to evaluate the influence of several fixation methods on selected biological properties of Lactobacillus rhamnosus GG that are relevant to its probiotic action. Fixation of the bacterial cells with ethanol, 2-propanol, glutaraldehyde, paraformaldehyde, and heat treatment resulted in a significant decrease of alkaline phosphatase, peroxidase, and β-galactosidase activities. Most of the fixation procedures reduced bacterial cell hydrophobicity and increased adhesion capacity. The fixation procedures resulted in a different perception of the bacterial cells by enterocytes, which was shown as changes in gene expression in enterocytes. The results show that some procedures of inactivation allow a fraction of the enzymatic activity to be maintained. The adhesion properties of the bacterial cells were enhanced, but the response of enterocytes to fixed cells was different than to live bacteria. Inactivation allows maintenance and modification of some of the properties of the bacterial cells.

DOE Office of Scientific and Technical Information (OSTI.GOV)

Chen, Pei-Jie; Cai, Shuang-Peng; Yang, Yang

Liver fibrosis is a reversible wound-healing response to chronic hepatic injuries. Activation of hepatic stellate cells (HSCs) plays a pivotal role in the development of hepatic fibrosis. The currently accepted mechanism for the resolution of liver fibrosis is the apoptosis and inactivation of activated HSCs. Protein tyrosine phosphatase 1B (PTP1B), a prototype of non-receptor protein tyrosine phosphatase, is proved to be a vital modulator in cardiac fibrogenesis. However, the precise role of PTP1B on liver fibrosis and HSC activation is still unclear. Our study showed that the expression of PTP1B was elevated in fibrotic liver but reduced after spontaneous recovery.more » Moreover, stimulation of HSC-T6 cells with transforming growth factor-β1 (TGF-β1) resulted in a dose/time-dependent increase of PTP1B mRNA and protein. Co-incubation of HSC-T6 cells with PTP1B-siRNA inhibited the cell proliferation and activation induced by TGF-β1. Additionally, both mRNA and protein of PTP1B were dramatically decreased in inactivated HSCs after treated with adipogenic differentiation mixture (MDI). Over-expression of PTP1B hindered the inactivation of HSC-T6 cells induced by MDI. These observations revealed a regulatory role of PTP1B in liver fibrosis and implied PTP1B as a potential therapeutic target. - Highlights: • The expression of PTP1B in the fibrotic livers and recovery livers • The expression of PTP1B in activated and inactivated HSCs • Blockade of PTP1B inhibited the TGF-β1-induced proliferation and activation of HSCs. • Over-expression of PTP1B abolished the inactivation of HSCs induced by MDI.« less

Human T-Cell Leukemia Virus Type 1 (HTLV-1) Tax Requires CADM1/TSLC1 for Inactivation of the NF-κB Inhibitor A20 and Constitutive NF-κB Signaling

PubMed Central

Thomas, Remy; van der Weyden, Louise; Rauch, Dan; Ratner, Lee; Nyborg, Jennifer K.; Ramos, Juan Carlos; Takai, Yoshimi; Shembade, Noula

2015-01-01

Persistent activation of NF-κB by the Human T-cell leukemia virus type 1 (HTLV-1) oncoprotein, Tax, is vital for the development and pathogenesis of adult T-cell leukemia (ATL) and HTLV-1-associated myelopathy/tropical spastic paraparesis (HAM/TSP). K63-linked polyubiquitinated Tax activates the IKK complex in the plasma membrane-associated lipid raft microdomain. Tax also interacts with TAX1BP1 to inactivate the NF-κB negative regulatory ubiquitin-editing A20 enzyme complex. However, the molecular mechanisms of Tax-mediated IKK activation and A20 protein complex inactivation are poorly understood. Here, we demonstrated that membrane associated CADM1 (Cell adhesion molecule1) recruits Ubc13 to Tax, causing K63-linked polyubiquitination of Tax, and IKK complex activation in the membrane lipid raft. The c-terminal cytoplasmic tail containing PDZ binding motif of CADM1 is critical for Tax to maintain persistent NF-κB activation. Finally, Tax failed to inactivate the NF-κB negative regulator ubiquitin-editing enzyme A20 complex, and activate the IKK complex in the lipid raft in absence of CADM1. Our results thus indicate that CADM1 functions as a critical scaffold molecule for Tax and Ubc13 to form a cellular complex with NEMO, TAX1BP1 and NRP, to activate the IKK complex in the plasma membrane-associated lipid rafts, to inactivate NF-κB negative regulators, and maintain persistent NF-κB activation in HTLV-1 infected cells. PMID:25774694

Novel Role for p110β PI 3-Kinase in Male Fertility through Regulation of Androgen Receptor Activity in Sertoli Cells

PubMed Central

Guillermet-Guibert, Julie; Smith, Lee B.; Halet, Guillaume; Whitehead, Maria A.; Pearce, Wayne; Rebourcet, Diane; León, Kelly; Crépieux, Pascale; Nock, Gemma; Strömstedt, Maria; Enerback, Malin; Chelala, Claude; Graupera, Mariona; Carroll, John; Cosulich, Sabina; Saunders, Philippa T. K.; Huhtaniemi, Ilpo; Vanhaesebroeck, Bart

2015-01-01

The organismal roles of the ubiquitously expressed class I PI3K isoform p110β remain largely unknown. Using a new kinase-dead knockin mouse model that mimics constitutive pharmacological inactivation of p110β, we document that full inactivation of p110β leads to embryonic lethality in a substantial fraction of mice. Interestingly, the homozygous p110β kinase-dead mice that survive into adulthood (maximum ~26% on a mixed genetic background) have no apparent phenotypes, other than subfertility in females and complete infertility in males. Systemic inhibition of p110β results in a highly specific blockade in the maturation of spermatogonia to spermatocytes. p110β was previously suggested to signal downstream of the c-kit tyrosine kinase receptor in germ cells to regulate their proliferation and survival. We now report that p110β also plays a germ cell-extrinsic role in the Sertoli cells (SCs) that support the developing sperm, with p110β inactivation dampening expression of the SC-specific Androgen Receptor (AR) target gene Rhox5, a homeobox gene critical for spermatogenesis. All extragonadal androgen-dependent functions remain unaffected by global p110β inactivation. In line with a crucial role for p110β in SCs, selective inactivation of p110β in these cells results in male infertility. Our study is the first documentation of the involvement of a signalling enzyme, PI3K, in the regulation of AR activity during spermatogenesis. This developmental pathway may become active in prostate cancer where p110β and AR have previously been reported to functionally interact. PMID:26132308

Tributyltin induces a G2/M cell cycle arrest in human amniotic cells via PP2A inhibition-mediated inactivation of the ERK1/2 cascades.

PubMed

Zhang, Yali; Guo, Zonglou; Xu, Lihong

2014-03-01

The molecular mechanisms underlying the cell cycle alterations induced by tributyltin (TBT), a highly toxic environmental contaminant, remain elusive. In this study, cell cycle progression and some key regulators in G2/M phase were investigated in human amniotic cells treated with TBT. Furthermore, protein phosphatase (PP) 2A and the ERK cascades were examined. The results showed that TBT caused a G2/M cell cycle arrest that was accompanied by a decrease in the total cdc25C protein level and an increase in the p-cdc2 level in the nucleus. TBT caused a decrease in PP2A activity and inhibited the ERK cascade by inactivating Raf-1, resulting in the dephosphorylation of MEK1/2, ERK1/2, and c-Myc. Taken together, TBT leads to a G2/M cell cycle arrest in FL cells, an increase in p-cdc2 and a decrease in the levels of total cdc25C protein, which may be caused by the PP2A inhibition-mediated inactivation of the ERK1/2 cascades. Copyright © 2014 Elsevier B.V. All rights reserved.

Differences in antiproliferative effect of STAT3 inhibition in HCC cells with versus without HBV expression

DOE Office of Scientific and Technical Information (OSTI.GOV)

Hong, Yun; Zhou, Lin; Xie, Haiyang

2015-06-05

Chronic infection with hepatitis B virus (HBV) plays an important role in the etiology of hepatocellular carcinoma (HCC). Signal transducer and activator of transcription 3 (STAT3) inactivation could inhibit the tumor growth of HCC. In this study, differential antiproliferative effect of STAT3 inhibition was observed with HBV-related HCC cells being more resistant than non-HBV-related HCC cells. Resistance of HBV-related HCC cells to STAT3 inhibition was positively correlated to the expression of HBV. Enhanced ERK activation after STAT3 blockade was detected in HBV-related HCC cells but not in non-HBV-related HCC cells. Combined ERK and STAT3 inhibition eliminates the discrepancy between themore » two types of HCC cells. Moderate reduced HBV expression was found after STAT3 inhibition. These findings disclose a discrepancy in cellular response to STAT3 inhibition between non-HBV-related and HBV-related HCC cells and underscore the complexity of antiproliferative effect of STAT3 inactivation in HBV-related HCC cells. - Highlights: • HBV endows HCC cells with resistance to STAT3 inactivation on proliferation. • Abnormal ERK activation after STAT3 inhibition in HBV-related HCC cells. • Combined ERK and STAT3 inhibition eliminates the discrepancy. • STAT3 inhibition moderately reduces HBV expression.« less

Lidocaine reduces the transition to slow inactivation in Nav1.7 voltage-gated sodium channels

PubMed Central

Sheets, Patrick L; Jarecki, Brian W; Cummins, Theodore R

2011-01-01

BACKGROUND AND PURPOSE The primary use of local anaesthetics is to prevent or relieve pain by reversibly preventing action potential propagation through the inhibition of voltage-gated sodium channels. The tetrodotoxin-sensitive voltage-gated sodium channel subtype Nav1.7, abundantly expressed in pain-sensing neurons, plays a crucial role in perception and transmission of painful stimuli and in inherited chronic pain syndromes. Understanding the interaction of lidocaine with Nav1.7 channels could provide valuable insight into the drug's action in alleviating pain in distinct patient populations. The aim of this study was to determine how lidocaine interacts with multiple inactivated conformations of Nav1.7 channels. EXPERIMENTAL APPROACH We investigated the interactions of lidocaine with wild-type Nav1.7 channels and a paroxysmal extreme pain disorder mutation (I1461T) that destabilizes fast inactivation. Whole cell patch clamp recordings were used to examine the activity of channels expressed in human embryonic kidney 293 cells. KEY RESULTS Depolarizing pulses that increased slow inactivation of Nav1.7 channels also reduced lidocaine inhibition. Lidocaine enhanced recovery of Nav1.7 channels from prolonged depolarizing pulses by decreasing slow inactivation. A paroxysmal extreme pain disorder mutation that destabilizes fast inactivation of Nav1.7 channels decreased lidocaine inhibition. CONCLUSIONS AND IMPLICATIONS Lidocaine decreased the transition of Nav1.7 channels to the slow inactivated state. The fast inactivation gate (domain III–IV linker) is important for potentiating the interaction of lidocaine with the Nav1.7 channel. PMID:21232038

Comparison between the effects of ultrasound and gamma-rays on the inactivation of Saccharomyces cerevisiae: analyses of cell membrane permeability and DNA or RNA synthesis by flow cytometry.

PubMed

Oyane, Ikuko; Takeda, Tomo; Oda, Yasunori; Sakata, Takashi; Furuta, Masakazu; Okitsu, Kenji; Maeda, Yasuaki; Nishimura, Rokuro

2009-04-01

The effects of 200 kHz ultrasonic irradiation on DNA or RNA formation and membrane permeability of yeast cells were investigated by flow cytometry and compared with those of (60)Co gamma-ray radiation. Colony counting analyses were also performed for comparison. It was observed that the colony-forming activity of yeast cells was not affected by small doses of ultrasonic irradiation, but was closely related to the amounts of sonolytically formed hydrogen peroxide at concentrations of more than 80 microM. On the other hand, gamma-rays directly retarded colony-forming ability in addition to the effects of radiolytically formed hydrogen peroxide. The results obtained by flow cytometry also indicated that the amounts of DNA or RNA formed decreased with an increase in ultrasonic irradiation time without any threshold. These results indicated that flow cytometry can show early growth activities, but that colony counting analyses are insufficient to evaluate continuous and quantitative changes in these activities. In addition, by analyzing the amounts of DNA or RNA formed in the presence of the same amount of hydrogen peroxide, it was found that DNA or RNA formation behavior in the presence of hydrogen peroxide with no irradiation was similar to that following ultrasonic irradiation. These results suggested that similar chemical effects due to the formation of hydrogen peroxide were produced during ultrasonic irradiation. In addition, physical effects of ultrasound, such as shock wave, hardly contributed to cell inactivation and cell membrane damage, because relatively high frequency ultrasound was used here. In the case of gamma-ray radiation, direct physical effects on the cells were clearly observed.

Pulsed pressure treatment for inactivation of escherichia coli and listeria innocua in whole milk

NASA Astrophysics Data System (ADS)

Buzrul, S.; Largeteau, A.; Alpas, H.; Demazeau, G.

2008-07-01

E. coli and L. innocua in whole milk were subjected to continuous pressure treatments (300, 350, 400, 450, 500, 550 and 600 MPa) at ambient temperature for 5, 10, 15 and 20 min. These treatments underlined that at moderate pressure values (300, 350 and 400 MPa), increasing the pressurization time from 5 to 20 min did not improve cell death to a great extent. Therefore, pulsed pressure treatments (at 300, 350 and 400 MPa) for 5 min (2.5 min × 2 pulses, 1 min × 5 pulses and 0.5 min × 10 pulses), 10 min (5 min × 2 pulses, 2 min × 5 pulses and 1 min × 10 pulses), 15 min (5 min × 3 pulses, 3 min × 5 pulses and 1.5 min × 10 pulses) and 20 min (10 min × 2 pulses, 5 min × 4 pulses, 4 min × 5 pulses and 2 min × 10 pulses) were applied. As already observed in continuous pressure experiments, in pulsed pressure treatments the inactivation level is improved with increasing pressure level and in addition with the number of applied pulses; however, the effect of pulse number is not additive. Results obtained in this study indicated that pulsed pressure treatments could be used to pasteurize the whole milk at lower pressure values than the continuous pressure treatments. Nevertheless, an optimization appears definetely necessary between the number of pulses and pressure levels to reach the desirable number of log-reduction of microorganisms.

Evidence for Compression of Escherichia coli K12 Cells under the Effect of TiO₂ Nanoparticles.

PubMed

Zhukova, Lyudmila V

2015-12-16

It has been shown that treatment with titanium dioxide nanoparticles (TiO2 NPs) combined with near-ultraviolet (UV-A) irradiation or in certain dark conditions reduced the numbers of various microorganisms, but the mechanism of this effect remains unclear. In this study to further clarify the mechanism of the antibacterial effect of TiO2 NPs the physiological state of E. coli K12 cells was estimated after incubation with the NPs (0.2 g/L) for different periods of time, with or without UV-A irradiation. Cell incubation with TiO2 NPs, combined or not combined with UV-A irradiation, showed that inactive cells were located only within cell aggregates formed after incubation with TiO2 NPs and that the larger the aggregate, the greater the number of such cells. When the formation of large aggregates was prevented, exposure to NPs under UV-A irradiation failed to result in cell inactivation. A comparative analysis of fluorescence and optical microscopic images of the same aggregates showed that the location of inactivated cells coincided with the zone of increased optical density within the aggregate. After treatment with TiO2 NPs under UV-A for 30, 60, or 120 min cells within the aggregates were the first to be inactivated. Cells on which NPs irradiated more strongly (at the periphery of large aggregates and single) remained active for a longer time than cells within the aggregates. As the time of treatment increased, so did the degree of cell compaction, with some zones of the aggregates eventually transforming into an acellular mass. After UV-A irradiation the cell aggregates spontaneously moved toward each other and gradually fused into larger structures, indicating that such exposure enhanced mutual attraction of cells treated with the NPs. Present study provides evidence for hypothesis that bacterial cells covered with TiO2 NPs are inactivated due to their mutual attraction and consequent compression.

Spatio-temporal radiation biology with conventionally or laser-accelerated particles for ELIMED

NASA Astrophysics Data System (ADS)

Ristić-Fira, A.; Bulat, T.; Keta, O.; Romano, F.; Cirrone, P.; Cuttone, G.; Petrović, I.

2013-07-01

The aim of this study is to investigate the behavior of radio-resistant human malignant cells, thus enabling better understanding of radiobiological effects of ions in such a case. Radiation sources such as accelerated continuous ion beams and laser technology-based ultra short radiation sources with energy of around 10 MeV will be used. The HTB140 melanoma cells are chosen since it has been shown that they represent the limit case of cellular radio-resistance among the studied tumor cell lines. These cells are particularly interesting as they provide data on the very edge of inactivation capacity of each beam line that is tested. After exposing the cell monolayers to continuous radiations of low (γ-rays) and high (protons) linear energy transfer, the kinetics of disappearance of the phosphorylated histone H2AX (γ-H2AX) foci per cell will be determined. The same procedure will be performed with the pulsed high dose rate protons. Detection and quantification of γ-H2AX foci will be performed by immunohistochemical 3D time-dependent imaging analyses using laser scanning confocal microscopy. Immunoblotting will enable the follow-up of the relation between γ-H2AX and cell cycle arrest via the p53/p21 pathway. In such a way the spatio-temporal changes on sub-cellular level will be visualized, quantified and compared. These results will show whether there is a difference in the effects on cells between continuous and pulsed irradiation mode. Therefore, they will contribute to the data base that might promote pulsed sources for medical treatments of malignant growths.

Efficacy assessment of an inactivated Tembusu virus vaccine candidate in ducks.

PubMed

Zhang, Lijiao; Li, Zhanhong; Zhang, Qingshui; Sun, Mengxu; Li, Shuang; Su, Wenliang; Hu, Xueying; He, Weiyong; Su, Jingliang

2017-02-01

Duck Tembusu virus (TMUV) is a recently identified pathogen that causes severe egg drop and neurological disease in domestic duck and goose flocks. The infection has spread across the China mainland since its outbreak in 2010. Effective vaccines are needed to fight the disease. In this work, we describe the development and laboratory assessment of a cell culture-derived, inactivated duck TMUV vaccine. The TMUV-JXSP strain was successfully propagated on a baby hamster kidney cell line (BHK-21), inactivated with beta-propiolactone (BPL) and emulsified with mineral oil. The efficacy of different vaccination schedules was assessed in laying ducks and table ducks using virus challenge experiments. Two doses of vaccine provided efficient protection against the virus challenge to avoid the egg production drop in laying ducks. An ELISA demonstrated that 97% (39/40) of ducks seroconverted on day 21 after one dose of the inactivated vaccine and that significant increases in antibody titers against the virus were induced after the second immunization. For table ducks, a single dose of vaccine immunization resulted in a protection index of 87% and significant reduction of viral loads in tissues. Sterilizing immunity can be attained after second immunization. Our results demonstrate that BHK-21 cell culture is suitable for duck TMUV propagation and that BPL-inactivated TMUV vaccine can provide a high level of protection from virus challenge in laying ducks and table ducks. These data provide a scientific basis for the development of an inactivated vaccine for the prevention of duck TMUV infection. Copyright © 2016 Elsevier Ltd. All rights reserved.

Effect of slightly acidic electrolyzed water for inactivating Escherichia coli O157:H7 and Staphylococcus aureus analyzed by transmission electron microscopy.

PubMed

Nan, Songjian; Yongyu, L I; Baoming, L I; Wang, Chaoyuan; Cui, Xiaodong; Cao, Wei

2010-12-01

The use of different available chlorine concentrations (ACCs) of slightly acidic electrolyzed water (SAEW; 0.5 to 30 mg/liter), different treatment times, and different temperatures for inactivating Escherichia coli O157:H7 and Staphylococcus aureus was evaluated. The morphology of both pathogens also was analyzed with transmission electron microscopy. A 3-min treatment with SAEW (pH 6.0 to 6.5) at ACCs of 2 mg/liter for E. coli O157:H7 and 8 mg/liter for S. aureus resulted in 100% inactivation of two cultures (7.92- to 8.75-log reduction) at 25°C. The bactericidal activity of SAEW was independent of the treatment time and temperature at a higher ACC (P > 0.05). E. coli O157:H7 was much more sensitive than S. aureus to SAEW. The morphological damage to E. coli O157:H7 cells by SAEW was significantly greater than that to S. aureus cells. At an ACC as high as 30 mg/liter, E. coli O157:H7 cells were damaged, but S. aureus cells retained their structure and no cell wall damage or shrinkage was observed. SAEW with a near neutral pH may be a promising disinfectant for inactivation of foodborne pathogens.

The relative contribution of antibody and CD8+ T cells to vaccine immunity against West Nile encephalitis virus.

PubMed

Shrestha, Bimmi; Ng, Terry; Chu, Hsien-Jue; Noll, Michelle; Diamond, Michael S

2008-04-07

West Nile virus (WNV) is a mosquito borne, neurotropic flavivirus that causes a severe central nervous system (CNS) infection in humans and animals. Although commercial vaccines are available for horses, none is currently approved for human use. In this study, we evaluated the efficacy and mechanism of immune protection of two candidate WNV vaccines in mice. A formalin-inactivated WNV vaccine induced higher levels of specific and neutralizing antibodies compared to a DNA plasmid vaccine that produces virus-like particles. Accordingly, partial and almost complete protection against a highly stringent lethal intracranial WNV challenge were observed in mice 60 days after single dose immunization with the DNA plasmid and inactivated virus vaccines, respectively. In mice immunized with a single dose of DNA plasmid or inactivated vaccine, antigen-specific CD8(+) T cells were induced and contributed to protective immunity as acquired or genetic deficiencies of CD8(+) T cells lowered the survival rates. In contrast, in boosted animals, WNV-specific antibody titers were higher, survival rates after challenge were greater, and an absence of CD8(+) T cells did not appreciably affect mortality. Overall, our experiments suggest that in mice, both inactivated WNV and DNA plasmid vaccines are protective after two doses, and the specific contribution of antibody and CD8(+) T cells to vaccine immunity against WNV is modulated by the prime-boost strategy.

Cytotoxic cells induced after Chlamydia psittaci infection in mice

DOE Office of Scientific and Technical Information (OSTI.GOV)

Lammert, J.K.

1982-03-01

The ability of spleen cells from Chlamydia psittaci-infected mice to lyse C. psittaci-infected and uninfected target cell monolayers was studied. The cytotoxicity assay used was a terminal label method in which the number of adherent target cells surviving the interaction with effector cells was determined by measuring the uptake of (3H)uridine by such cells. It was observed that in the first few days postinfection (3 to 5), spleens contained cells that lysed infected and uninfected targets with equal efficiency. Subsequently, infected targets were killed primarily. The activity of effector spleen cells for infected targets continued, although at a reduced level,more » beyond 21 days postinfection. Intact effector cells were required since a disruption by sonication resulted in a loss of cytotoxicity. The enhanced killing observed with infected targets was also observed when target cells were sensitized with heat- or UV-inactivated C. psittaci. This study suggests that the induction of cytotoxic cells after C. psittaci infection may contribute to the ability of the host to control multiplication of the microorganism.« less

Generation of an allelic series of knock-in mice using recombinase-mediated cassette exchange (RMCE).

PubMed

Roebroek, Anton J M; Van Gool, Bart

2014-01-01

Molecular genetic strategies applying embryonic stem cell (ES cell) technologies to study the function of a gene in mice or to generate a mouse model for a human disease are continuously under development. Next to (conditional) inactivation of genes the application and importance of approaches to generate knock-in mutations are increasing. In this chapter the principle and application of recombinase-mediated cassette exchange (RMCE) are discussed as being a new emerging knock-in strategy, which enables easy generation of a series of different knock-in mutations within one gene. An RMCE protocol, which was used to generate a series of different knock-in mutations in the Lrp1 gene of ES cells, is described in detail as an example of how RMCE can be used to generate highly efficiently an allelic series of differently modified ES cell clones from a parental modified ES cell clone. Subsequently the differently modified ES cell clones can be used to generate an allelic series of mutant knock-in mice.

Mildly Acidic pH Triggers an Irreversible Conformational Change in the Fusion Domain of Herpes Simplex Virus 1 Glycoprotein B and Inactivation of Viral Entry.

PubMed

Weed, Darin J; Pritchard, Suzanne M; Gonzalez, Floricel; Aguilar, Hector C; Nicola, Anthony V

2017-03-01

Herpes simplex virus (HSV) entry into a subset of cells requires endocytosis and endosomal low pH. Preexposure of isolated virions to mildly acidic pH of 5 to 6 partially inactivates HSV infectivity in an irreversible manner. Acid inactivation is a hallmark of viruses that enter via low-pH pathways; this occurs by pretriggering conformational changes essential for fusion. The target and mechanism(s) of low-pH inactivation of HSV are unclear. Here, low-pH-treated HSV-1 was defective in fusion activity and yet retained normal levels of attachment to cell surface heparan sulfate and binding to nectin-1 receptor. Low-pH-triggered conformational changes in gB reported to date are reversible, despite irreversible low-pH inactivation. gB conformational changes and their reversibility were measured by antigenic analysis with a panel of monoclonal antibodies and by detecting changes in oligomeric conformation. Three-hour treatment of HSV-1 virions with pH 5 or multiple sequential treatments at pH 5 followed by neutral pH caused an irreversible >2.5 log infectivity reduction. While changes in several gB antigenic sites were reversible, alteration of the H126 epitope was irreversible. gB oligomeric conformational change remained reversible under all conditions tested. Altogether, our results reveal that oligomeric alterations and fusion domain changes represent distinct conformational changes in gB, and the latter correlates with irreversible low-pH inactivation of HSV. We propose that conformational change in the gB fusion domain is important for activation of membrane fusion during viral entry and that in the absence of a host target membrane, this change results in irreversible inactivation of virions. IMPORTANCE HSV-1 is an important pathogen with a high seroprevalence throughout the human population. HSV infects cells via multiple pathways, including a low-pH route into epithelial cells, the primary portal into the host. HSV is inactivated by low-pH preexposure, and gB, a class III fusion protein, undergoes reversible conformational changes in response to low-pH exposure. Here, we show that low-pH inactivation of HSV is irreversible and due to a defect in virion fusion activity. We identified an irreversible change in the fusion domain of gB following multiple sequential low-pH exposures or following prolonged low-pH treatment. This change appears to be separable from the alteration in gB quaternary structure. Together, the results are consistent with a model by which low pH can have an activating or inactivating effect on HSV depending on the presence of a target membrane. Copyright © 2017 American Society for Microbiology.

Inactivation of Dengue and Yellow Fever viruses by heme, cobalt-protoporphyrin IX and tin-protoporphyrin IX.

PubMed

Assunção-Miranda, I; Cruz-Oliveira, C; Neris, R L S; Figueiredo, C M; Pereira, L P S; Rodrigues, D; Araujo, D F F; Da Poian, A T; Bozza, M T

2016-03-01

To investigate the effect of heme, cobalt-protoporphyrin IX and tin-protoporphyrin IX (CoPPIX and SnPPIX), macrocyclic structures composed by a tetrapyrrole ring with a central metallic ion, on Dengue Virus (DENV) and Yellow Fever Virus (YFV) infection. Treatment of HepG2 cells with heme, CoPPIX and SnPPIX after DENV infection reduced infectious particles without affecting viral RNA contents in infected cells. The reduction of viral load occurs only with the direct contact of DENV with porphyrins, suggesting a direct effect on viral particles. Previously incubation of DENV and YFV with heme, CoPPIX and SnPPIX resulted in viral particles inactivation in a dose-dependent manner. Biliverdin, a noncyclical porphyrin, was unable to inactivate the viruses tested. Infection of HepG2 cells with porphyrin-pretreated DENV2 results in a reduced or abolished viral protein synthesis, RNA replication and cell death. Treatment of HepG2 or THP-1 cell lineage with heme or CoPPIX after DENV infection with a very low MOI resulted in a decreased DENV replication and protection from death. Heme, CoPPIX and SnPPIX possess a marked ability to inactivate DENV and YFV, impairing its ability to infect and induce cytopathic effects on target cells. These results open the possibility of therapeutic application of porphyrins or their use as models to design new antiviral drugs against DENV and YFV. © 2016 The Society for Applied Microbiology.

Distinct Mutations Led to Inactivation of Type 1 Fimbriae Expression in Shigella spp.

PubMed Central

Bravo, Verónica; Puhar, Andrea; Sansonetti, Philippe; Parsot, Claude; Toro, Cecilia S.

2015-01-01

Shigella spp. are responsible for bacillary dysentery in humans. The acquisition or the modification of the virulence plasmid encoding factors promoting entry of bacteria into and dissemination within epithelial cells was a critical step in the evolution of these bacteria from their Escherichia coli ancestor(s). Incorporation of genomic islands (GI) and gene inactivation also shaped interactions between these pathogens and their human host. Sequence analysis of the GI inserted next to the leuX tRNA gene in S. boydii, S. dysenteriae, S. flexneri, S. sonnei and enteroinvasive E. coli (EIEC) suggests that this region initially carried the fec, yjhATS and fim gene clusters. The fim cluster encoding type I fimbriae is systematically inactivated in both reference strains and clinical isolates and distinct mutations are responsible for this inactivation in at least three phylogenetic groups. To investigate consequences of the presence of fimbriae on the outcome of the interaction of Shigella with host cells, we used a S. flexneri strain harboring a plasmid encoding the E. coli fim operon. Production of fimbriae by this recombinant strain increased the ability of bacteria to adhere to and enter into epithelial cells and had no effect on their ability to disseminate from cell to cell. The observations that production of type I fimbriae increases invasion of epithelial cells and that independent mutations abolish fimbriae production in Shigella suggest that these mutations correspond to pathoadaptive events. PMID:25811616

Involvement of multiple stressors induced by non-thermal plasma-charged aerosols during inactivation of airborne bacteria

PubMed Central

Vaze, Nachiket D.; Park, Sin; Brooks, Ari D.; Fridman, Alexander; Joshi, Suresh G.

2017-01-01

A lab-scale, tunable, single-filament, point-to-point nonthermal dieletric-barrier discharge (DBD) plasma device was built to study the mechanisms of inactivation of aerosolized bacterial pathogens. The system inactivates airborne antibiotic-resistant pathogens efficiently. Nebulization mediated pre-optimized (4 log and 7 log) bacterial loads were challenged to plasma-charged aerosols, and lethal and sublethal doses determined using colony assay, and cell viability assay; and the loss of membrane potential and cellular respiration were determined using cell membrane potential assay and XTT assay. Using the strategies of Escherichia coli wildtype, over-expression mutant, deletion mutants, and peroxide and heat stress scavenging, we analyzed activation of intracellular reactive oxygen species (ROS) and heat shock protein (hsp) chaperons. Superoxide dismutase deletion mutants (ΔsodA, ΔsodB, ΔsodAΔsodB) and catalase mutants ΔkatG and ΔkatEΔkatG did not show significant difference from wildtype strain, and ΔkatE and ΔahpC was found significantly more susceptible to cell death than wildtype. The oxyR regulon was found to mediate plasma-charged aerosol-induced oxidative stress in bacteria. Hsp deficient E. coli (ΔhtpG, ΔgroEL, ΔclpX, ΔgrpE) showed complete inactivation of cells at ambient temperature, and the treatment at cold temperature (4°C) significantly protected hsp deletion mutants and wildtype cells, and indicate a direct involvement of hsp in plasma-charged aerosol mediated E. coli cell death. PMID:28166240

Differentiation of lymphoid cells: evidence for a B-cell specific serum suppressor.

PubMed Central

Kern, M

1978-01-01

The induction of immunoglobulin production by rabbit spleen cells is markedly inhibited by the presence of normal rabbit serum during cell culture. A similar inhibition is observed when spleen cell populations in which T cells have been inactivated are temporarily incubated with normal rabbit serum before being reconstituted with T cells by adding thymocytes. In contrast, no inhibition was observed upon temporary incubation of thymocytes with normal serum prior to addition of T cell-inactivated spleen cell populations. Removal of adherent cells did not affect the induction of immunoglobulin production or its inhibition by normal serum. Lipopolysaccharide-enhanced immunoglobin production was also inhibited by normal serum, thereby providing additional confidence that bone-marrow derived (B) cells are the target of the normal serum inhibitor. PMID:308042

Cellular Injuries in Cronobacter sakazakii CIP 103183T and Salmonella enterica Exposed to Drying and Subsequent Heat Treatment in Milk Powder.

PubMed

Lang, Emilie; Guyot, Stéphane; Peltier, Caroline; Alvarez-Martin, Pablo; Perrier-Cornet, Jean-Marie; Gervais, Patrick

2018-01-01

Because of the ability of foodborne pathogens to survive in low-moisture foods, their decontamination is an important issue in food protection. This study aimed to clarify some of the cellular mechanisms involved in inactivation of foodborne pathogens after drying and subsequent heating. Individual strains of Salmonella Typhimurium, Salmonella Senftenberg, and Cronobacter sakazakii were mixed into whole milk powder and dried to different water activity levels (0.25 and 0.58); the number of surviving cells was determined after drying and subsequent thermal treatments in closed vessels at 90 and 100°C, for 30 and 120 s. For each condition, the percentage of unculturable cells was estimated and, in parallel, membrane permeability and respiratory activity were estimated by flow cytometry using fluorescent probes. After drying, it was clearly observable that the percentage of unculturable cells was correlated with the percentage of permeabilized cells (responsible for 20-40% of the total inactivated bacteria after drying), and to a lesser degree with the percentage of cells presenting with loss of respiratory activity. In contrast, the percentages of unculturable cells observed after heat treatment were strongly correlated with the loss of respiratory activity and weakly with membrane permeability (for 70-80% of the total inactivated bacteria after heat treatment). We conclude that cell inactivation during drying is closely linked to membrane permeabilization and that heat treatment of dried cells affects principally their respiratory activity. These results legitimize the use of time-temperature scales and allow better understanding of the cellular mechanisms of bacterial death during drying and subsequent heat treatment. These results may also allow better optimization of the decontamination process to ensure food safety by targeting the most deleterious conditions for bacterial cells without denaturing the food product.

Plasminogen-induced aggregation of PANC-1 cells requires conversion to plasmin and is inhibited by endogenous plasminogen activator inhibitor-1.

PubMed

Deshet, Naamit; Lupu-Meiri, Monica; Espinoza, Ingrid; Fili, Oded; Shapira, Yuval; Lupu, Ruth; Gershengorn, Marvin C; Oron, Yoram

2008-09-01

PANC-1 cells express proteinase-activated receptors (PARs)-1, -2, and respond to their activation by transient elevation of cytosolic [Ca(2+)] and accelerated aggregation (Wei et al., 2006, J Cell Physiol 206:322-328). We studied the effect of plasminogen (PGN), an inactive precursor of the PAR-1-activating protease, plasmin (PN) on aggregation of pancreatic adenocarcinoma (PDAC) cells. A single dose of PGN time- and dose-dependently promoted PANC-1 cells aggregation in serum-free medium, while PN did not. PANC-1 cells express urokinase plasminogen activator (uPA), which continuously converted PGN to PN. This activity and PGN-induced aggregation were inhibited by the uPA inhibitor amiloride. PGN-induced aggregation was also inhibited by alpha-antiplasmin and by the PN inhibitor epsilon-aminocaproic acid (EACA). Direct assay of uPA activity revealed very low rate, markedly enhanced in the presence of PGN. Moreover, in PGN activator inhibitor 1-deficient PANC-1 cells, uPA activity and PGN-induced aggregation were markedly potentiated. Two additional human PDAC cell lines, MiaPaCa and Colo347, were assayed for PGN-induced aggregation. Both cell lines responded by aggregation and exhibited PGN-enhanced uPA activity. We hypothesized that the continuous conversion of PGN to PN by endogenous uPA is limited by PN's degradation and negatively controlled by endogenously produced PAI-1. Indeed, we found that PANC-1 cells inactivate PN with t1/2 of approximately 7 h, while the continuous addition of PN promoted aggregation. Our data suggest that PANC-1 cells possess intrinsic, PAI-1-sensitive mechanism for promotion of aggregation and differentiation by prolonged exposure to PGN and, possibly, additional precursors of PARs agonists.

Atypical properties of a conventional calcium channel beta subunit from the platyhelminth Schistosoma mansoni.

PubMed

Salvador-Recatalà, Vicenta; Schneider, Toni; Greenberg, Robert M

2008-03-26

The function of voltage-gated calcium (Cav) channels greatly depends on coupling to cytoplasmic accessory beta subunits, which not only promote surface expression, but also modulate gating and kinetic properties of the alpha1 subunit. Schistosomes, parasitic platyhelminths that cause schistosomiasis, express two beta subunit subtypes: a structurally conventional beta subunit and a variant beta subunit with unusual functional properties. We have previously characterized the functional properties of the variant Cavbeta subunit. Here, we focus on the modulatory phenotype of the conventional Cavbeta subunit (SmCavbeta) using the human Cav2.3 channel as the substrate for SmCavbeta and the whole-cell patch-clamp technique. The conventional Schistosoma mansoni Cavbeta subunit markedly increases Cav2.3 currents, slows macroscopic inactivation and shifts steady state inactivation in the hyperpolarizing direction. However, currents produced by Cav2.3 in the presence of SmCavbeta run-down to approximately 75% of their initial amplitudes within two minutes of establishing the whole-cell configuration. This suppressive effect was independent of Ca2+, but dependent on intracellular Mg2+-ATP. Additional experiments revealed that SmCavbeta lends the Cav2.3/SmCavbeta complex sensitivity to Na+ ions. A mutant version of the Cavbeta subunit lacking the first forty-six amino acids, including a string of twenty-two acidic residues, no longer conferred sensitivity to intracellular Mg2+-ATP and Na+ ions, while continuing to show wild type modulation of current amplitude and inactivation of Cav2.3. The data presented in this article provide insights into novel mechanisms employed by platyhelminth Cavbeta subunits to modulate voltage-gated Ca2+ currents that indicate interactions between the Ca2+ channel complex and chelated forms of ATP as well as Na+ ions. These results have potentially important implications for understanding previously unknown mechanisms by which platyhelminths and perhaps other organisms modulate Ca2+ currents in excitable cells.

A dielectric barrier discharge terminally inactivates RNase A by oxidizing sulfur-containing amino acids and breaking structural disulfide bonds

NASA Astrophysics Data System (ADS)

Lackmann, J.-W.; Baldus, S.; Steinborn, E.; Edengeiser, E.; Kogelheide, F.; Langklotz, S.; Schneider, S.; Leichert, L. I. O.; Benedikt, J.; Awakowicz, P.; Bandow, J. E.

2015-12-01

RNases are among the most stable proteins in nature. They even refold spontaneously after heat inactivation, regaining full activity. Due to their stability and universal presence, they often pose a problem when experimenting with RNA. We investigated the capabilities of nonthermal atmospheric-pressure plasmas to inactivate RNase A and studied the inactivation mechanism on a molecular level. While prolonged heating above 90 °C is required for heat inactivating RNase A, direct plasma treatment with a dielectric barrier discharge (DBD) source caused permanent inactivation within minutes. Circular dichroism spectroscopy showed that DBD-treated RNase A unfolds rapidly. Raman spectroscopy indicated methionine modifications and formation of sulfonic acid. A mass spectrometry-based analysis of the protein modifications that occur during plasma treatment over time revealed that methionine sulfoxide formation coincides with protein inactivation. Chemical reduction of methionine sulfoxides partially restored RNase A activity confirming that sulfoxidation is causal and sufficient for RNase A inactivation. Continued plasma exposure led to over-oxidation of structural disulfide bonds. Using antibodies, disulfide bond over-oxidation was shown to be a general protein inactivation mechanism of the DBD. The antibody’s heavy and light chains linked by disulfide bonds dissociated after plasma exposure. Based on their ability to inactivate proteins by oxidation of sulfur-containing amino acids and over-oxidation of disulfide bonds, DBD devices present a viable option for inactivating undesired or hazardous proteins on heat or solvent-sensitive surfaces.

Systems Biology of Immune Response to Live and Inactivated Dengue Virus Vaccines

DTIC Science & Technology

2017-09-01

transcriptomics; innate immunity; adaptive immunity; correlates of immunity; live-attenuated; purified inactivated; biomarkers; T- cell; B-cell; epitope. 5...original copies of journal articles, reprints of manuscripts and abstracts, a curriculum vitae, patent applications, study questionnaires, and surveys ...of DENV and capable of secreting IgG were detected in all arms at 6 moths post-vaccination. Of note is that little correlation with contemporaneous

Sputtered Gum metal thin films showing bacterial inactivation and biocompatibility.

PubMed

Achache, S; Alhussein, A; Lamri, S; François, M; Sanchette, F; Pulgarin, C; Kiwi, J; Rtimi, S

2016-10-01

Super-elastic Titanium based thin films Ti-23Nb-0.7Ta-2Zr-(O) (TNTZ-O) and Ti-24Nb-(N) (TN-N) (at.%) were deposited by direct current magnetron sputtering (DCMS) in different reactive atmospheres. The effects of oxygen doping (TNTZ-O) and/or nitrogen doping (TN-N) on the microstructure, mechanical properties and biocompatibility of the as-deposited coatings were investigated. Nano-indentation measurements show that, in both cases, 1sccm of reactive gas in the mixture is necessary to reach acceptable values of hardness and Young's modulus. Mechanical properties are considered in relation to the films compactness, the compressive stress and the changes in the grain size. Data on Bacterial inactivation and biocompatibility are reported in this study. The biocompatibility tests showed that O-containing samples led to higher cells proliferation. Bacterial inactivation was concomitant with the observed pH and surface potential changes under light and in the dark. The increased cell fluidity leading to bacterial lysis was followed during the bacterial inactivation time. The increasing cell wall fluidity was attributed to the damage of the bacterial outer cell which losing its capacity to regulate the ions exchange in and out of the bacteria. Copyright © 2016 Elsevier B.V. All rights reserved.

Inactivation disinfection property of Moringa Oleifera seed extract: optimization and kinetic studies

NASA Astrophysics Data System (ADS)

Idris, M. A.; Jami, M. S.; Hammed, A. M.

2017-05-01

This paper presents the statistical optimization study of disinfection inactivation parameters of defatted Moringa oleifera seed extract on Pseudomonas aeruginosa bacterial cells. Three level factorial design was used to estimate the optimum range and the kinetics of the inactivation process was also carried. The inactivation process involved comparing different disinfection models of Chicks-Watson, Collins-Selleck and Homs models. The results from analysis of variance (ANOVA) of the statistical optimization process revealed that only contact time was significant. The optimum disinfection range of the seed extract was 125 mg/L, 30 minutes and 120rpm agitation. At the optimum dose, the inactivation kinetics followed the Collin-Selleck model with coefficient of determination (R2) of 0.6320. This study is the first of its kind in determining the inactivation kinetics of pseudomonas aeruginosa using the defatted seed extract.

Co-Inactivation of GlnR and CodY Regulators Impacts Pneumococcal Cell Wall Physiology.

PubMed

Johnston, Calum; Bootsma, Hester J; Aldridge, Christine; Manuse, Sylvie; Gisch, Nicolas; Schwudke, Dominik; Hermans, Peter W M; Grangeasse, Christophe; Polard, Patrice; Vollmer, Waldemar; Claverys, Jean-Pierre

2015-01-01

CodY, a nutritional regulator highly conserved in low G+C Gram-positive bacteria, is essential in Streptococcus pneumoniae (the pneumococcus). A published codY mutant possessed suppressing mutations inactivating the fatC and amiC genes, respectively belonging to iron (Fat/Fec) and oligopeptide (Ami) ABC permease operons, which are directly repressed by CodY. Here we analyzed two additional published codY mutants to further explore the essentiality of CodY. We show that one, in which the regulator of glutamine/glutamate metabolism glnR had been inactivated by design, had only a suppressor in fecE (a gene in the fat/fec operon), while the other possessed both fecE and amiC mutations. Independent isolation of three different fat/fec suppressors thus establishes that reduction of iron import is crucial for survival without CodY. We refer to these as primary suppressors, while inactivation of ami, which is not essential for survival of codY mutants and acquired after initial fat/fec inactivation, can be regarded as a secondary suppressor. The availability of codY- ami+ cells allowed us to establish that CodY activates competence for genetic transformation indirectly, presumably by repressing ami which is known to antagonize competence. The glnR codY fecE mutant was then found to be only partially viable on solid medium and hypersensitive to peptidoglycan (PG) targeting agents such as the antibiotic cefotaxime and the muramidase lysozyme. While analysis of PG and teichoic acid composition uncovered no alteration in the glnR codY fecE mutant compared to wildtype, electron microscopy revealed altered ultrastructure of the cell wall in the mutant, establishing that co-inactivation of GlnR and CodY regulators impacts pneumococcal cell wall physiology. In light of rising levels of resistance to PG-targeting antibiotics of natural pneumococcal isolates, GlnR and CodY constitute potential alternative therapeutic targets to combat this debilitating pathogen, as co-inactivation of these regulators renders pneumococci sensitive to iron and PG-targeting agents.

Inhibition of oxidative metabolism leads to p53 genetic inactivation and transformation in neural stem cells

PubMed Central

Bartesaghi, Stefano; Graziano, Vincenzo; Galavotti, Sara; Henriquez, Nick V.; Betts, Joanne; Saxena, Jayeta; Minieri, Valentina; A, Deli; Karlsson, Anna; Martins, L. Miguel; Capasso, Melania; Nicotera, Pierluigi; Brandner, Sebastian; De Laurenzi, Vincenzo; Salomoni, Paolo

2015-01-01

Alterations of mitochondrial metabolism and genomic instability have been implicated in tumorigenesis in multiple tissues. High-grade glioma (HGG), one of the most lethal human neoplasms, displays genetic modifications of Krebs cycle components as well as electron transport chain (ETC) alterations. Furthermore, the p53 tumor suppressor, which has emerged as a key regulator of mitochondrial respiration at the expense of glycolysis, is genetically inactivated in a large proportion of HGG cases. Therefore, it is becoming evident that genetic modifications can affect cell metabolism in HGG; however, it is currently unclear whether mitochondrial metabolism alterations could vice versa promote genomic instability as a mechanism for neoplastic transformation. Here, we show that, in neural progenitor/stem cells (NPCs), which can act as HGG cell of origin, inhibition of mitochondrial metabolism leads to p53 genetic inactivation. Impairment of respiration via inhibition of complex I or decreased mitochondrial DNA copy number leads to p53 genetic loss and a glycolytic switch. p53 genetic inactivation in ETC-impaired neural stem cells is caused by increased reactive oxygen species and associated oxidative DNA damage. ETC-impaired cells display a marked growth advantage in the presence or absence of oncogenic RAS, and form undifferentiated tumors when transplanted into the mouse brain. Finally, p53 mutations correlated with alterations in ETC subunit composition and activity in primary glioma-initiating neural stem cells. Together, these findings provide previously unidentified insights into the relationship between mitochondria, genomic stability, and tumor suppressive control, with implications for our understanding of brain cancer pathogenesis. PMID:25583481

New evidence for Cu-decorated binary-oxides mediating bacterial inactivation/mineralization in aerobic media.

PubMed

Rtimi, S; Pulgarin, C; Bensimon, M; Kiwi, J

2016-08-01

Binary oxide semiconductors TiO2-ZrO2 and Cu-decorated TiO2-ZrO2 (TiO2-ZrO2-Cu) uniform films were sputtered on polyester (PES). These films were irradiated under low intensity solar simulated light and led to bacterial inactivation in aerobic and anaerobic media as evaluated by CFU-plate counting. But bacterial mineralization was only induced by TiO2-ZrO2-Cu in aerobic media. The highly oxidative radicals generated on the films surface under light were identified by the use of appropriate scavengers. The hole generated on the TiO2-ZrO2 films is shown to be the main specie leading to bacterial inactivation. TiO2-ZrO2 and Cu-decorated TiO2-ZrO2 films release Zr and Ti <1ppb and Cu 4.6ppb/cm(2) as determined by inductively coupled plasma mass spectrometry (ICP-MS) This level is far below the citotoxicity permitted level allowed for mammalian cells suggesting that bacterial disinfection proceeds through an oligodynamic effect. By Fourier transform attenuated infrared spectroscopy (ATR-FTIR) the systematic shift of the predominating νs(CH2) vibrational-rotational peak making up most of the bacterial cell-wall content in C was monitored. Based on this evidence a mechanism suggested leading to CH bond stretching followed by cell lysis and cell death. Bacterial inactivation cycling was observed on TiO2-ZrO2-Cu showing the stability of these films leading to bacterial inactivation. Copyright © 2016 Elsevier B.V. All rights reserved.

Pseudomonas aeruginosa inactivation mechanism is affected by capsular extracellular polymeric substances reactivity with chlorine and monochloramine.

PubMed

Xue, Zheng; Hessler, Christopher M; Panmanee, Warunya; Hassett, Daniel J; Seo, Youngwoo

2013-01-01

The reactivity of capsular extracellular polymeric substances (EPS) to chlorine and monochloramine was assessed and compared in this study. The impact of capsular EPS on Gram-negative bacteria Pseudomonas aeruginosa inactivation mechanisms was investigated both qualitatively and quantitatively using a combination of batch experiments, viability tests with LIVE/DEAD staining, and Fourier transform infrared spectroscopy (FTIR). Both wild-type and isogenic mutant strains with different alginate EPS production capabilities were used to evaluate their susceptibility to chlorine and monochloramine. The mucA22 mutant strain, which overproduces the EPS composed largely of acidic polysaccharide alginate, exhibited high resistance and prolonged inactivation time to both chlorine and monochloramine relative to PAO1 (wild-type) and algT(U) mutant strains (alginate EPS deficient). Multiple analyses were combined to better understand the mechanistic role of EPS against chlorine-based disinfectants. The extracted EPS exhibited high reactivity with chlorine and very low reactivity with monochloramine, suggesting different mechanism of protection against disinfectants. Moreover, capsular EPS on cell membrane appeared to reduce membrane permeabilization by disinfectants as suggested by deformation of key functional groups in EPS and cell membrane (the C-O-C stretching of carbohydrate and the C=O stretching of ester group). The combined results supported that capsular EPS, acting either as a disinfectant consumer (for chlorine inactivation) or limiting access to reactive sites on cell membrane (for monochloramine inactivation), provide a protective role for bacterial cells against regulatory residual disinfectants by reducing membrane permeabilization. © 2012 Federation of European Microbiological Societies. Published by Blackwell Publishing Ltd. All rights reserved.

Antagonism of Lidocaine Inhibition by Open-Channel Blockers That Generate Resurgent Na Current

PubMed Central

Bant, Jason S.; Aman, Teresa K.; Raman, Indira M.

2013-01-01

Na channels that generate resurgent current express an intracellular endogenous open-channel blocking protein, whose rapid binding upon depolarization and unbinding upon repolarization minimizes fast and slow inactivation. Na channels also bind exogenous compounds, such as lidocaine, which functionally stabilize inactivation. Like the endogenous blocking protein, these use-dependent inhibitors bind most effectively at depolarized potentials, raising the question of how lidocaine-like compounds affect neurons with resurgent Na current. We therefore recorded lidocaine inhibition of voltage-clamped, tetrodotoxin-sensitive Na currents in mouse Purkinje neurons, which express a native blocking protein, and in mouse hippocampal CA3 pyramidal neurons with and without a peptide from the cytoplasmic tail of NaVβ4 (the β4 peptide), which mimics endogenous open-channel block. To control channel states during drug exposure, lidocaine was applied with rapid-solution exchange techniques during steps to specific voltages. Inhibition of Na currents by lidocaine was diminished by either the β4 peptide or the native blocking protein. In peptide-free CA3 cells, prolonging channel opening with a site-3 toxin, anemone toxin II, reduced lidocaine inhibition; this effect was largely occluded by open-channel blockers, suggesting that lidocaine binding is favored by inactivation but prevented by open-channel block. In constant 100 μM lidocaine, current-clamped Purkinje cells continued to fire spontaneously. Similarly, the β4 peptide reduced lidocaine-dependent suppression of spiking in CA3 neurons in slices. Thus, the open-channel blocking protein responsible for resurgent current acts as a natural antagonist of lidocaine. Neurons with resurgent current may therefore be less susceptible to use-dependent Na channel inhibitors used as local anesthetic, antiarrhythmic, and anticonvulsant drugs. PMID:23486968

In vitro granulocyte adherence and in vivo margination: two associated complement-dependent functions. Studies based on the acute neutropenia of filtration leukophoresis.

PubMed

Fehr, J; Jacob, H S

1977-09-01

To study mechanisms and mediators regulating the distribution of intravascular granulocytes between circulating and marginated pools, a human model with extreme transient margination, the neutropenia of continuous flow filtration leukophoresis, was analyzed. Studies in animals demonstrated the existence of a complement (C)-derived granulocytopenia-inducing factor. Thus, autologous plasma, exposed to nylon fibers (NF) of the filtration system, produced an acute selective decrement of circulating granulocytes and monocytes. This phenomenon was blocked by decomplementing plasma, by pretreatment of plasma with EDTA or hydrazine, and by preheating at 56 degrees C, but did occur after recombination of heat-inactivated and hydrazine-treated plasma before NF exposure. Preheating plasma at 50 degrees C did not inhibit the neutropenic response, suggesting involvement of the classical pathway of C activation. Ultrafiltration studies indicated that the NF-provoked neutropenia-inducing factor has a mol wt in the range of 10,000-30,000, and is heat stable (56 degrees C). To analyze the hypothesis that C- induced neutrophil margination might be consequent to increased cell adhesiveness to endothelial surfaces, the role of C in promoting granulocyte adherence was evaluated in vitro. Measured with a plastic Petridish assay, granulocyte adherence was significantly reduced in heat- inactivated (56 degrees C) and hydrazine-treated plasma, but adherence promoting capacity was restored by mixing the two plasmas, or by adding purified C3 to hydrazine-treated plasma. After exposure to activated C, neutrophils showed significantly increased adhesiveness which was maintained when cells were resuspended in heat-inactivated plasma, but progressively lost when resuspended in fresh plasma. On the basis of these results we conclude that granulocyte adhesiveness in vitro and margination in vivo are closely associated, C-dependent phenomena.

An inactivated yellow fever 17DD vaccine cultivated in Vero cell cultures.

PubMed

Pereira, Renata C; Silva, Andrea N M R; Souza, Marta Cristina O; Silva, Marlon V; Neves, Patrícia P C C; Silva, Andrea A M V; Matos, Denise D C S; Herrera, Miguel A O; Yamamura, Anna M Y; Freire, Marcos S; Gaspar, Luciane P; Caride, Elena

2015-08-20

Yellow fever is an acute infectious disease caused by prototype virus of the genus Flavivirus. It is endemic in Africa and South America where it represents a serious public health problem causing epidemics of hemorrhagic fever with mortality rates ranging from 20% to 50%. There is no available antiviral therapy and vaccination is the primary method of disease control. Although the attenuated vaccines for yellow fever show safety and efficacy it became necessary to develop a new yellow fever vaccine due to the occurrence of rare serious adverse events, which include visceral and neurotropic diseases. The new inactivated vaccine should be safer and effective as the existing attenuated one. In the present study, the immunogenicity of an inactivated 17DD vaccine in C57BL/6 mice was evaluated. The yellow fever virus was produced by cultivation of Vero cells in bioreactors, inactivated with β-propiolactone, and adsorbed to aluminum hydroxide (alum). Mice were inoculated with inactivated 17DD vaccine containing alum adjuvant and followed by intracerebral challenge with 17DD virus. The results showed that animals receiving 3 doses of the inactivated vaccine (2 μg/dose) with alum adjuvant had neutralizing antibody titers above the cut-off of PRNT50 (Plaque Reduction Neutralization Test). In addition, animals immunized with inactivated vaccine showed survival rate of 100% after the challenge as well as animals immunized with commercial attenuated 17DD vaccine. Copyright © 2015 Elsevier Ltd. All rights reserved.

Enhancing immune responses to inactivated porcine parvovirus oil emulsion vaccine by co-inoculating porcine transfer factor in mice.

PubMed

Wang, Rui-ning; Wang, Ya-bin; Geng, Jing-wei; Guo, Dong-hui; Liu, Fang; Chen, Hong-ying; Zhang, Hong-ying; Cui, Bao-an; Wei, Zhan-yong

2012-07-27

Inactivated porcine parvovirus (PPV) vaccines are available commercially and widely used in the breeding herds. However, inactivated PPV vaccines have deficiencies in induction of specific cellular immune response. Transfer factor (TF) is a material that obtained from the leukocytes, and is a novel immune-stimulatory reagent that as a modulator of the immune system. In this study, the immunogenicity of PPV oil emulsion vaccine and the immuno-regulatory activities of TF were investigated. The inactivated PPV oil emulsion vaccines with or without TF were inoculated into BALB/c mice by subcutaneous injection. Then humoral and cellular immune responses were evaluated by indirect enzyme-linked immunosorbent assays (ELISA), fluorescence-activated cell sorter analyses (FACS). The results showed that the PPV specific immune responses could be evoked in mice by inoculating with PPV oil emulsion vaccine alone or by co-inoculation with TF. The cellular immune response levels in the co-inoculation groups were higher than those groups receiving the PPV oil emulsion vaccine alone, with the phenomena of higher level of IFN-γ, a little IL-6 and a trace of IL-4 in serum, and a vigorous T-cell response. However, there was no significant difference in antibody titers between TF synergy inactivated vaccine and the inactivated vaccine group (P>0.05). In conclusion, these results suggest that TF possess better cellular immune-enhancing capability and would be exploited into an effective immune-adjuvant for inactivated vaccines. Copyright © 2012 Elsevier Ltd. All rights reserved.

Functional conversion between A-type and delayed rectifier K+ channels by membrane lipids.

PubMed

Oliver, Dominik; Lien, Cheng-Chang; Soom, Malle; Baukrowitz, Thomas; Jonas, Peter; Fakler, Bernd

2004-04-09

Voltage-gated potassium (Kv) channels control action potential repolarization, interspike membrane potential, and action potential frequency in excitable cells. It is thought that the combinatorial association between distinct alpha and beta subunits determines whether Kv channels function as non-inactivating delayed rectifiers or as rapidly inactivating A-type channels. We show that membrane lipids can convert A-type channels into delayed rectifiers and vice versa. Phosphoinositides remove N-type inactivation from A-type channels by immobilizing the inactivation domains. Conversely, arachidonic acid and its amide anandamide endow delayed rectifiers with rapid voltage-dependent inactivation. The bidirectional control of Kv channel gating by lipids may provide a mechanism for the dynamic regulation of electrical signaling in the nervous system.

Inactivation of the srtA Gene Affects Localization of Surface Proteins and Decreases Adhesion of Streptococcus pneumoniae to Human Pharyngeal Cells In Vitro

PubMed Central

Kharat, Arun S.; Tomasz, Alexander

2003-01-01

Inactivation of sortase gene srtA in Streptococcus pneumoniae strain R6 caused the release of β-galactosidase and neuraminidase A (NanA) from the cell wall into the surrounding medium. Both of these surface proteins contain the LPXTG motif in the C-terminal domain. Complementation with plasmid-borne srtA reversed protein release. Deletion of murM, a gene involved in the branching of pneumococcal peptidoglycan, also caused partial release of β-galactosidase, suggesting preferential attachment of the protein to branched muropeptides in the cell wall. Inactivation of srtA caused decreased adherence to human pharyngeal cells in vitro but had no effect on the virulence of a capsular type III strain of S. pneumoniae in the mouse intraperitoneal model. The observations suggest that—as in other gram-positive bacteria—sortase-dependent display of proteins occurs in S. pneumoniae and that some of these proteins may be involved in colonization of the human host. PMID:12704150

Inactivation of Gram-positive biofilms by low-temperature plasma jet at atmospheric pressure

NASA Astrophysics Data System (ADS)

Marchal, F.; Robert, H.; Merbahi, N.; Fontagné-Faucher, C.; Yousfi, M.; Romain, C. E.; Eichwald, O.; Rondel, C.; Gabriel, B.

2012-08-01

This work is devoted to the evaluation of the efficiency of a new low-temperature plasma jet driven in ambient air by a dc-corona discharge to inactivate adherent cells and biofilms of Gram-positive bacteria. The selected microorganisms were lactic acid bacteria, a Weissella confusa strain which has the particularity to excrete a polysaccharide polymer (dextran) when sucrose is present. Both adherent cells and biofilms were treated with the low-temperature plasma jet for different exposure times. The antimicrobial efficiency of the plasma was tested against adherent cells and 48 h-old biofilms grown with or without sucrose. Bacterial survival was estimated using both colony-forming unit counts and fluorescence-based assays for bacterial cell viability. The experiments show the ability of the low-temperature plasma jet at atmospheric pressure to inactivate the bacteria. An increased resistance of bacteria embedded within biofilms is clearly observed. The resistance is also significantly higher with biofilm in the presence of sucrose, which indicates that dextran could play a protective role.

Delayed rectifier K channels contribute to contrast adaptation in mammalian retinal ganglion cells

PubMed Central

Weick, Michael; Demb, Jonathan B.

2011-01-01

SUMMARY Retinal ganglion cells adapt by reducing their sensitivity during periods of high contrast. Contrast adaptation in the firing response depends on both presynaptic and intrinsic mechanisms. Here, we investigated intrinsic mechanisms for contrast adaptation in OFF Alpha ganglion cells in the in vitro guinea pig retina. Using either visual stimulation or current injection, we show that brief depolarization evoked spiking and suppressed firing during subsequent depolarization. The suppression could be explained by Na channel inactivation, as shown in salamander cells. However, brief hyperpolarization in the physiological range (5–10 mV) also suppressed firing during subsequent depolarization. This suppression was sensitive selectively to blockers of delayed-rectifier K channels (KDR). Somatic membrane patches showed TEA-sensitive KDR currents with activation near −25 mV and removal of inactivation at voltages negative to Vrest. Brief periods of hyperpolarization apparently remove KDR inactivation and thereby increase the channel pool available to suppress excitability during subsequent depolarization. PMID:21745646

Delayed-rectifier K channels contribute to contrast adaptation in mammalian retinal ganglion cells.

PubMed

Weick, Michael; Demb, Jonathan B

2011-07-14

Retinal ganglion cells adapt by reducing their sensitivity during periods of high contrast. Contrast adaptation in the firing response depends on both presynaptic and intrinsic mechanisms. Here, we investigated intrinsic mechanisms for contrast adaptation in OFF Alpha ganglion cells in the in vitro guinea pig retina. Using either visual stimulation or current injection, we show that brief depolarization evoked spiking and suppressed firing during subsequent depolarization. The suppression could be explained by Na channel inactivation, as shown in salamander cells. However, brief hyperpolarization in the physiological range (5-10 mV) also suppressed firing during subsequent depolarization. This suppression was selectively sensitive to blockers of delayed-rectifier K channels (K(DR)). In somatic membrane patches, we observed tetraethylammonium-sensitive K(DR) currents that activated near -25 mV. Recovery from inactivation occurred at potentials hyperpolarized to V(rest). Brief periods of hyperpolarization apparently remove K(DR) inactivation and thereby increase the channel pool available to suppress excitability during subsequent depolarization. Copyright © 2011 Elsevier Inc. All rights reserved.

Autologous aldrithiol-2-inactivated HIV-1 combined with polyinosinic-polycytidylic acid-poly-L-lysine carboxymethylcellulose as a vaccine platform for therapeutic dendritic cell immunotherapy.

PubMed

Miller, Elizabeth; Spadaccia, Meredith; Sabado, Rachel; Chertova, Elena; Bess, Julian; Trubey, Charles Mac; Holman, Rose Marie; Salazar, Andres; Lifson, Jeffrey; Bhardwaj, Nina

2015-01-03

Therapeutic interventions for HIV-1 that successfully augment adaptive immunity to promote killing of infected cells may be a requisite component of strategies to reduce latent cellular reservoirs. Adoptive immunotherapies utilizing autologous monocyte-derived dendritic cells (DCs) that have been activated and antigen loaded ex vivo may serve to circumvent defects in DC function that are present during HIV infection in order to enhance adaptive immune responses. Here we detail the clinical preparation of DCs loaded with autologous aldrithiol-2 (AT-2)-inactivated HIV that have been potently activated with the viral mimic, Polyinosinic-polycytidylic acid-poly-l-lysine carboxymethylcellulose (Poly-ICLC). HIV is first propagated from CD4+ T cells from HIV-infected donors and then rendered non-replicative by chemical inactivation with aldrithiol-2 (AT-2), purified, and quantified. Viral inactivation is confirmed through measurement of Tat-regulated β-galactosidase reporter gene expression following infection of TZM-bl cells. In-process testing for sterility, mycoplasma, LPS, adventitious agents, and removal of AT-2 is performed on viral preparations. Autologous DCs are generated and pulsed with autologous AT-2-inactivated virus and simultaneously stimulated with Poly-ICLC to constitute the final DC vaccine product. Phenotypic identity, maturation, and induction of HIV-specific adaptive immune responses are confirmed via flow cytometric analysis of DCs and cocultured autologous CD4+ and CD8+ T cells. Lot release criteria for the DC vaccine have been defined in accordance with Good Manufacturing Practice (GMP) guidelines. The demonstrated feasibility of this approach has resulted in approval by the FDA for investigational use in antiretroviral (ART) suppressed individuals. We discuss how this optimized DC formulation may enhance the quality of anti-HIV adaptive responses beyond what has been previously observed during DC immunotherapy trials for HIV infection. Copyright © 2014 Elsevier Ltd. All rights reserved.

Differential response of human melanoma and Ehrlich ascites cells in vitro to the ribosome-inactivating protein luffin.

PubMed

Poma, A; Miranda, M; Spanò, L

1998-10-01

The cytotoxicity and inhibitory effect on proliferation of the type 1 ribosome-inactivating protein luffin purified from the seeds of Luffa aegyptiaca were investigated both in human metastatic melanoma cells and in murine Ehrlich ascites tumour cells. Results indicate that luffin from the seeds of Luffa aegyptiaca is cytotoxic to the cell lines tested, with approximately 10 times greater potency in Ehrlich cells. Luffin was found to induce an increase in cytosolic oligonucleosome-bound DNA in both melanoma and Ehrlich ascites tumour cells, the level of DNA fragmentation in the former cell line being higher than in the latter. Experiments with melanoma cells indicate that an increase in cytosolic nucleosomes could be supportive of apoptosis as the type of cell death induced by luffin.

Galantamine inhibits slowly inactivating K+ currents with a dual dose-response relationship in differentiated N1E-115 cells and in CA1 neurones.

PubMed

Vicente, M Inês; Costa, Pedro F; Lima, Pedro A

2010-05-25

Galantamine, one of the major drugs used in Alzheimer's disease therapy, is a relatively weak acetylcholinesterase inhibitor and an allosteric potentiating ligand of nicotinic acetylcholine receptors. However, a role in the control of excitability has also been attributed to galantamine via modulation of K+ currents in central neurones. To further investigate the effect of galantamine on voltage-activated K+ currents, we performed whole-cell voltage-clamp recordings in differentiated neuroblastoma N1E-115 cells and in dissociated rat CA1 neurones. In both cell models, one can identify two main voltage-activated K+ current components: a relatively fast inactivating component (Ifast; time constant approximately hundred milliseconds) and a slowly inactivating one (Islow; time constant approximately 1 s). We show that galantamine (1 pM-300 microM) inhibits selectively Islow, exhibiting a dual dose-response relationship, in both differentiated N1E-115 cells and CA1 neurones. We also demonstrate that, in contrast with what was previously reported, galantamine-induced inhibition is not due to a shift on the steady-state inactivation and activation curves. Additionally, we characterized a methodological artefact that affects voltage-dependence as a function of time in whole-cell configuration, observed in both cell models. By resolving an inhibitory role on K+ currents in a non-central neuronal system and in hippocampal neurones, we are attributing a widespread role of galantamine on the modulation of cell excitability. The present results are relevant in the clinical context, since the effects at low dosages suggest that galantamine-induced K+ current inhibition may contribute to the efficiency of galantamine in the treatment of Alzheimer's disease. Copyright 2010 Elsevier B.V. All rights reserved.

Effects of Bacterial Inactivation Methods on Downstream Proteomic Analysis

DOE Office of Scientific and Technical Information (OSTI.GOV)

Lin, Andy; Merkley, Eric D.; Clowers, Brian H.

2015-05-01

Inactivation of pathogenic microbial samples is often necessary for the protection of researchers and to comply with local and federal regulations. By its nature, biological inactivation causes changes to microbial samples, potentially affecting observed experimental results. While inactivation induced damage to materials such as DNA has been evaluated, the effect of various inactivation strategies on proteomic data, to our knowledge, has not been discussed. To this end, we inactivated samples of Yersinia pestis and Escherichia coli by autoclave, ethanol, or irradiation treatment to determine how inactivation changes liquid chromatography tandem mass spectrometry data quality as well as apparent protein contentmore » of cells. Proteomic datasets obtained from aliquots of samples inactivated by different methods were highly similar, with Pearson correlation coefficients ranging from 0.822 to 0.985 and 0.816 to 0.985 for E. coli and Y. pestis, respectively, suggesting that inactivation had only slight impacts on the set of proteins identified. In addition, spectral quality metrics such as distributions of various database search algorithm scores remained constant across inactivation methods, indicating that inactivation does not appreciably degrade spectral quality. Though overall changes resulting from inactivation were small, there were detectable trends. For example, one-sided Fischer exact tests determined that periplasmic proteins decrease in observed abundance after sample inactivation by autoclaving (α = 1.71x10-2 for E. coli, α = 4.97x10-4 for Y. pestis) and irradiation (α = 9.43x10-7 for E. coli, α = 1.21x10-5 for Y. pestis) when compared to controls that were not inactivated. Based on our data, if sample inactivation is necessary, we recommend inactivation with ethanol treatment with secondary preference given to irradiation.« less

In situ studies of microbial inactivation during high pressure processing

NASA Astrophysics Data System (ADS)

Maldonado, Jose Antonio; Schaffner, Donald W.; Cuitiño, Alberto M.; Karwe, Mukund V.

2016-01-01

High pressure processing (HPP) has been shown to reduce microbial concentration in foods. The mechanisms of microbial inactivation by HPP have been associated with damage to cell membranes. The real-time response of bacteria to HPP was measured to elucidate the mechanisms of inactivation, which can aid in designing more effective processes. Different pressure cycling conditions were used to expose Enterobacter aerogenes cells to HPP. Propidium iodide (PI) was used as a probe, which fluoresces after penetrating cells with damaged membranes and binding with nucleic acids. A HPP vessel with sapphire windows was used for measuring fluorescence in situ. Membrane damage was detected during pressurization and hold time, but not during depressurization. The drop in fluorescence was larger than expected after pressure cycles at higher pressure and longer times. This indicated possible reversible disassociation of ribosomes resulting in additional binding of PI to exposed RNA under pressure and its release after depressurization.

Effect of Alkali Metal Cations on Slow Inactivation of Cardiac Na+ Channels

PubMed Central

Townsend, Claire; Horn, Richard

1997-01-01

Human heart Na+ channels were expressed transiently in both mammalian cells and Xenopus oocytes, and Na+ currents measured using 150 mM intracellular Na+. The kinetics of decaying outward Na+ current in response to 1-s depolarizations in the F1485Q mutant depends on the predominant cation in the extracellular solution, suggesting an effect on slow inactivation. The decay rate is lower for the alkali metal cations Li+, Na+, K+, Rb+, and Cs+ than for the organic cations Tris, tetramethylammonium, N-methylglucamine, and choline. In whole cell recordings, raising [Na+]o from 10 to 150 mM increases the rate of recovery from slow inactivation at −140 mV, decreases the rate of slow inactivation at relatively depolarized voltages, and shifts steady-state slow inactivation in a depolarized direction. Single channel recordings of F1485Q show a decrease in the number of blank (i.e., null) records when [Na+]o is increased. Significant clustering of blank records when depolarizing at a frequency of 0.5 Hz suggests that periods of inactivity represent the sojourn of a channel in a slow-inactivated state. Examination of the single channel kinetics at +60 mV during 90-ms depolarizations shows that neither open time, closed time, nor first latency is significantly affected by [Na+]o. However raising [Na+]o decreases the duration of the last closed interval terminated by the end of the depolarization, leading to an increased number of openings at the depolarized voltage. Analysis of single channel data indicates that at a depolarized voltage a single rate constant for entry into a slow-inactivated state is reduced in high [Na+]o, suggesting that the binding of an alkali metal cation, perhaps in the ion-conducting pore, inhibits the closing of the slow inactivation gate. PMID:9234168

Mitochondrial Cardiomyopathy Caused by Elevated Reactive Oxygen Species and Impaired Cardiomyocyte Proliferation.

PubMed

Zhang, Donghui; Li, Yifei; Heims-Waldron, Danielle; Bezzerides, Vassilios; Guatimosim, Silvia; Guo, Yuxuan; Gu, Fei; Zhou, Pingzhu; Lin, Zhiqiang; Ma, Qing; Liu, Jianming; Wang, Da-Zhi; Pu, William T

2018-01-05

Although mitochondrial diseases often cause abnormal myocardial development, the mechanisms by which mitochondria influence heart growth and function are poorly understood. To investigate these disease mechanisms, we studied a genetic model of mitochondrial dysfunction caused by inactivation of Tfam (transcription factor A, mitochondrial), a nuclear-encoded gene that is essential for mitochondrial gene transcription and mitochondrial DNA replication. Tfam inactivation by Nkx2.5 Cre caused mitochondrial dysfunction and embryonic lethal myocardial hypoplasia. Tfam inactivation was accompanied by elevated production of reactive oxygen species (ROS) and reduced cardiomyocyte proliferation. Mosaic embryonic Tfam inactivation confirmed that the block to cardiomyocyte proliferation was cell autonomous. Transcriptional profiling by RNA-seq demonstrated the activation of the DNA damage pathway. Pharmacological inhibition of ROS or the DNA damage response pathway restored cardiomyocyte proliferation in cultured fetal cardiomyocytes. Neonatal Tfam inactivation by AAV9-cTnT-Cre caused progressive, lethal dilated cardiomyopathy. Remarkably, postnatal Tfam inactivation and disruption of mitochondrial function did not impair cardiomyocyte maturation. Rather, it elevated ROS production, activated the DNA damage response pathway, and decreased cardiomyocyte proliferation. We identified a transient window during the first postnatal week when inhibition of ROS or the DNA damage response pathway ameliorated the detrimental effect of Tfam inactivation. Mitochondrial dysfunction caused by Tfam inactivation induced ROS production, activated the DNA damage response, and caused cardiomyocyte cell cycle arrest, ultimately resulting in lethal cardiomyopathy. Normal mitochondrial function was not required for cardiomyocyte maturation. Pharmacological inhibition of ROS or DNA damage response pathways is a potential strategy to prevent cardiac dysfunction caused by some forms of mitochondrial dysfunction. © 2017 American Heart Association, Inc.

cAMP-induced activation of protein kinase A and p190B RhoGAP mediates down-regulation of TC10 activity at the plasma membrane and neurite outgrowth.

PubMed

Koinuma, Shingo; Takeuchi, Kohei; Wada, Naoyuki; Nakamura, Takeshi

2017-11-01

Cyclic AMP plays a pivotal role in neurite growth. During outgrowth, a trafficking system supplies membrane at growth cones. However, the cAMP-induced signaling leading to the regulation of membrane trafficking remains unknown. TC10 is a Rho family GTPase that is essential for specific types of vesicular trafficking. Recent studies have shown a role of TC10 in neurite growth in NGF-treated PC12 cells. Here, we investigated a mechanical linkage between cAMP and TC10 in neuritogenesis. Plasmalemmal TC10 activity decreased abruptly after cAMP addition in neuronal cells. TC10 was locally inactivated at extending neurite tips in cAMP-treated PC12 cells. TC10 depletion led to a decrease in cAMP-induced neurite outgrowth. Constitutively active TC10 could not rescue this growth reduction, supporting our model for a role of GTP hydrolysis of TC10 in neuritogenesis by accelerating vesicle fusion. The cAMP-induced TC10 inactivation was mediated by PKA. Considering cAMP-induced RhoA inactivation, we found that p190B, but not p190A, mediated inactivation of TC10 and RhoA. Upon cAMP treatment, p190B was recruited to the plasma membrane. STEF depletion and Rac1-N17 expression reduced cAMP-induced TC10 inactivation. Together, the PKA-STEF-Rac1-p190B pathway leading to inactivation of TC10 and RhoA at the plasma membrane plays an important role in cAMP-induced neurite outgrowth. © 2017 Molecular Biology Society of Japan and John Wiley & Sons Australia, Ltd.

Activation of persulfates by natural magnetic pyrrhotite for water disinfection: Efficiency, mechanisms, and stability.

PubMed

Xia, Dehua; Li, Yan; Huang, Guocheng; Yin, Ran; An, Taicheng; Li, Guiying; Zhao, Huijun; Lu, Anhuai; Wong, Po Keung

2017-04-01

This study introduces natural occurring magnetic pyrrhotite (NP) as an environmentally friendly, easy available, and cost-effective alternative catalyst to activate persulfate (PS) of controlling microbial water contaminants. The E. coli K-12 inactivation kinetics observed in batch experiments was well described with first-order reaction. The optimum inactivation rate (k = 0.47 log/min) attained at a NP dose of 1 g/L and a PS dose of 1 mM, corresponding to total inactivation of 7 log 10  cfu/mL cells within 15 min. Measured k increased > 2-fold when temperature increased from 20 to 50 °C; and > 4-fold when pH decreased from 9 to 3. Aerobic conditions were more beneficial to cell inactivation than anaerobic conditions due to more reactive oxygen species (ROS) generated. ROS responsible for the inactivation were identified to be SO 4 -  > OH > H 2 O 2 based on a positive scavenging test and in situ ROS determination. In situ characterization suggested that PS effectively bind to NP surface was likely to form charge transfer complex (≡Fe(II)⋯O 3 SOOSO 3 - ), which mediated ROS generation and E. coli K-12 oxidation. The increased cell-envelope lesions consequently aggravated intracellular protein depletion and genome damage to cause definite bacterial death. The NP still maintained good physiochemical structure and stable activity even after 4 cycle. Moreover, NP/PS system also exhibited good E. coli K-12 inactivation efficiency in authentic water matrices like surface water and effluents of secondary wastewater. Copyright © 2017 Elsevier Ltd. All rights reserved.

Potassium channels cloned from neuroblastoma cells display slowly inactivating outward currents in Xenopus oocytes.

PubMed

Ito, Y; Yokoyama, S; Higashida, H

1992-05-22

Messenger RNAs (mRNAs) specific for NGK1 and NGK2 potassium channels were synthesized from complementary DNAs (cDNAs) that had been cloned from mouse neuroblastoma x rat glioma hybrid NG108-15 cells. Outward pottasium currents were evoked by 5 s depolarizing voltage commands in Xenopus oocytes injected with NGK1- or NGK2-specific mRNAs. The NGK1 or NGK2 currents showed different activation and inactivation kinetics, and different pharmacological sensitivities. The threshold potential for activation of the NGK2 current (-14 mV) was more positive than that for the NGK1 (-36 mV). The NGK2 current showed faster inactivation during a 5 s depolarizing pulse than did the NGK1 current. Inactivation was best fit by time constants of 0.37, 1.5 and 19 s for the NGK2 current and 4.4 and 19 s for NGK1. Extracellularly applied tetraethylammonium chloride (TEA) was 1000 times more potent on the NGK2 current than the NGK1 current. Furthermore we examined outward current following co-injection of an equal amount of mRNAs for NGK1 and NGK2. The timecourse of inactivation differed from either alone or from a simple sum of the two individual currents. TEA sensitivity could not be explained by summation of the two homomultimeric channels. These findings suggest that both NGK1 and NGK2 proteins assemble to form heteromultimeric K+ channels in addition to homomultimeric K+ channels. NGK2 channels and the heteromultimeric channels may be responsible for the native transient outward current with slow inactivation in NG108-15 hybrid cells.

X chromosome inactivation in a female carrier of a 1.28 Mb deletion encompassing the human X inactivation centre.

PubMed

de Hoon, B; Splinter, Erik; Eussen, B; Douben, J C W; Rentmeester, E; van de Heijning, M; Laven, J S E; de Klein, J E M M; Liebelt, J; Gribnau, J

2017-11-05

X chromosome inactivation (XCI) is a mechanism specifically initiated in female cells to silence one X chromosome, thereby equalizing the dose of X-linked gene products between male and female cells. XCI is regulated by a locus on the X chromosome termed the X-inactivation centre (XIC). Located within the XIC is XIST , which acts as a master regulator of XCI. During XCI, XIST is upregulated on the inactive X chromosome and chromosome-wide cis spreading of XIST leads to inactivation. In mouse, the Xic comprises Xist and all cis -regulatory elements and genes involved in Xist regulation. The activity of the XIC is regulated by trans -acting factors located elsewhere in the genome: X-encoded XCI activators positively regulating XCI, and autosomally encoded XCI inhibitors providing the threshold for XCI initiation. Whether human XCI is regulated through a similar mechanism, involving trans -regulatory factors acting on the XIC has remained elusive so far. Here, we describe a female individual with ovarian dysgenesis and a small X chromosomal deletion of the XIC. SNP-array and targeted locus amplification (TLA) analysis defined the deletion to a 1.28 megabase region, including XIST and all elements and genes that perform cis -regulatory functions in mouse XCI. Cells carrying this deletion still initiate XCI on the unaffected X chromosome, indicating that XCI can be initiated in the presence of only one XIC. Our results indicate that the trans -acting factors required for XCI initiation are located outside the deletion, providing evidence that the regulatory mechanisms of XCI are conserved between mouse and human.This article is part of the themed issue 'X-chromosome inactivation: a tribute to Mary Lyon'. © 2017 The Authors.

Hematopoietic stem cells are acutely sensitive to Acd shelterin gene inactivation

PubMed Central

Jones, Morgan; Osawa, Gail; Regal, Joshua A.; Weinberg, Daniel N.; Taggart, James; Kocak, Hande; Friedman, Ann; Ferguson, David O.; Keegan, Catherine E.; Maillard, Ivan

2013-01-01

The shelterin complex plays dual functions in telomere homeostasis by recruiting telomerase and preventing the activation of a DNA damage response at telomeric ends. Somatic stem cells require telomerase activity, as evidenced by progressive stem cell loss leading to bone marrow failure in hereditary dyskeratosis congenita. Recent work demonstrates that dyskeratosis congenita can also arise from mutations in specific shelterin genes, although little is known about shelterin functions in somatic stem cells. We found that mouse hematopoietic stem cells (HSCs) are acutely sensitive to inactivation of the shelterin gene Acd, encoding TPP1. Homozygosity for a hypomorphic acd allele preserved the emergence and expansion of fetal HSCs but led to profoundly defective function in transplantation assays. Upon complete Acd inactivation, HSCs expressed p53 target genes, underwent cell cycle arrest, and were severely depleted within days, leading to hematopoietic failure. TPP1 loss induced increased telomeric fusion events in bone marrow progenitors. However, unlike in epidermal stem cells, p53 deficiency did not rescue TPP1-deficient HSCs, indicating that shelterin dysfunction has unique effects in different stem cell populations. Because the consequences of telomere shortening are progressive and unsynchronized, acute loss of shelterin function represents an attractive alternative for studying telomere crisis in hematopoietic progenitors. PMID:24316971

Effector CD4 cell tolerization is mediated through functional inactivation and involves preferential impairment of TNF-α and IFN-γ expression potentials

PubMed Central

Long, Meixiao; Higgins, Amy D.; Mihalyo, Marianne A.; Adler, Adam J.

2010-01-01

It has recently been shown that effector/memory T cells can undergo peripheral tolerization in response to self-antigen. In the present study, we found that within 24 h self-antigen profoundly impairs the ability of CD4 effectors to express TNF-α (and to a lesser extent IFN-γ); however, several days of self-antigen exposure is required to impair non-effector functions such as IL-2 expression and proliferation. Since only half of the initial effector CD4 cell population expresses effector cytokines following brief antigenic stimulation, tolerization might have been mediated either through functional inactivation of effector-competent cells, or alternatively by the selective deletion of competent and expansion of non-competent cells. When briefly stimulated effectors were fractionated based on their expression of IFN-γ, the IFN-γ− sub-population was able to express IFN-γ following secondary stimulation, indicating that all effector CD4 cells are functionally competent. Furthermore, both IFN-γ+ and IFN-γ− sub-populations underwent tolerization in response to self-HA (although the former was slightly more prone to deletion at later time points). Thus, effector CD4 cell tolerization is mediated primarily through the functional inactivation of effector-competent cells. PMID:14609577

Characterization and inhibition of a cholecystokinin-inactivating serine peptidase.

PubMed

Rose, C; Vargas, F; Facchinetti, P; Bourgeat, P; Bambal, R B; Bishop, P B; Chan, S M; Moore, A N; Ganellin, C R; Schwartz, J C

1996-04-04

A cholecystokinin (CCK)-inactivating peptidase was purified and identified as a membrane-bound isoform of tripeptidyl peptidase II (EC 3.4.14.10), a cytosolic subtilisin-like peptidase of previously unknown functions. The peptidase was found in neurons responding to cholecystokinin, as well as in non-neuronal cells. Butabindide, a potent and specific inhibitor, was designed and shown to protect endogenous cholecystokinin from inactivation and to display pro-satiating effects mediated by the CCKA receptor.

Combination of cupric ion with hydroxylamine and hydrogen peroxide for the control of bacterial biofilms on RO membranes.

PubMed

Lee, Hye-Jin; Kim, Hyung-Eun; Lee, Changha

2017-03-01

Combinations of Cu(II) with hydroxylamine (HA) and hydrogen peroxide (H 2 O 2 ) (i.e., Cu(II)/HA, Cu(II)/H 2 O 2 , and Cu(II)/HA/H 2 O 2 systems) were investigated for the control of P. aeruginosa biofilms on reverse osmosis (RO) membranes. These Cu(II)-based disinfection systems effectively inactivated P. aeruginosa cells, exhibiting different behaviors depending on the state of bacterial cells (planktonic or biofilm) and the condition of biofilm growth and treatment (normal or pressurized condition). The Cu(II)/HA and Cu(II)/HA/H 2 O 2 systems were the most effective reagents for the inactivation of planktonic cells. However, these systems were not effective in inactivating cells in biofilms on the RO membranes possibly due to the interactions of Cu(I) with extracellular polymeric substances (EPS), where biofilms were grown and treated in center for disease control (CDC) reactors. Different from the results using CDC reactors, in a pressurized cross-flow RO filtration unit, the Cu(II)/HA/H 2 O 2 treatment significantly inactivated biofilm cells formed on the RO membranes, successfully recovering the permeate flux reduced by the biofouling. The pretreatment of feed solutions by Cu(II)/HA and Cu(II)/HA/H 2 O 2 systems (applied before the biofilm formation) effectively mitigated the permeate flux decline by preventing the biofilm growth on the RO membranes. Copyright © 2016 Elsevier Ltd. All rights reserved.

Inactivation of Msx1 and Msx2 in neural crest reveals an unexpected role in suppressing heterotopic bone formation in the head

PubMed Central

Roybal, Paul G.; Wu, Nancy L.; Sun, Jingjing; Ting, Man-chun; Schaefer, Christopher; Maxson, Robert E.

2011-01-01

In an effort to understand the morphogenetic forces that shape the bones of the skull, we inactivated Msx1 and Msx2 conditionally in neural crest. We show that Wnt1-Cre inactivation of up to three Msx1/2 alleles results in a progressively larger defect in the neural crest-derived frontal bone. Unexpectedly, in embryos lacking all four Msx1/2 alleles, the large defect is filled in with mispatterned bone consisting of ectopic islands of bone between the reduced frontal bones, just anterior to the parietal bones. The bone is derived from neural crest, not mesoderm, and, from DiI cell marking experiments, originates in a normally non-osteogenic layer of cells through which the rudiment elongates apically. Associated with the heterotopic osteogeneis is an upregulation of Bmp signaling in this cell layer. Prevention of this upregulation by implantation of noggin-soaked beads in head explants also prevented heterotopic bone formation. These results suggest that Msx genes have a dual role in calvarial development: They are required for the differentiation and proliferation of osteogenic cells within rudiments, and they are also required to suppress an osteogenic program in a cell layer within which the rudiments grow. We suggest that the inactivation of this repressive activity may be one cause of Wormian bones, ectopic bones that are a feature of a variety of pathological conditions in which calvarial bone development is compromised. PMID:20398647

Monitoring ultraviolet (UV) radiation inactivation of Cronobacter sakazakii in dry infant formula using Fourier transform infrared spectroscopy.

PubMed

Liu, Qian; Lu, Xiaonan; Swanson, Barry G; Rasco, Barbara A; Kang, Dong-Hyun

2012-01-01

Cronobacter sakazakii is an opportunistic pathogen associated with dry infant formula presenting a high risk to low birth weight neonates. The inactivation of C. sakazakii in dry infant formula by ultraviolet (UV) radiation alone and combined with hot water treatment at temperatures of 55, 60, and 65 °C were applied in this study. UV radiation with doses in a range from 12.1 ± 0.30 kJ/m² to 72.8 ± 1.83 kJ/m² at room temperature demonstrated significant inactivation of C. sakazakii in dry infant formula (P < 0.05). UV radiation combining 60 °C hot water treatment increased inactivation of C. sakazakii cells significantly (P < 0.05) in reconstituted infant formula. Significant effects of UV radiation on C. sakazakii inactivation kinetics (D value) were not observed in infant formula reconstituted in 55 and 65 °C water (P > 0.05). The inactivation mechanism was investigated using vibrational spectroscopy. Infrared spectroscopy detected significant stretching mode changes of macromolecules on the basis of spectral features, such as DNA, proteins, and lipids. Minor changes on cell membrane composition of C. sakazakii under UV radiation could be accurately and correctly monitored by infrared spectroscopy coupled with 2nd derivative transformation and principal component analysis. © 2011 Institute of Food Technologists®

Disruption of TTDA Results in Complete Nucleotide Excision Repair Deficiency and Embryonic Lethality

PubMed Central

Theil, Arjan F.; Nonnekens, Julie; Steurer, Barbara; Mari, Pierre-Olivier; de Wit, Jan; Lemaitre, Charlène; Marteijn, Jurgen A.; Raams, Anja; Maas, Alex; Vermeij, Marcel; Essers, Jeroen; Hoeijmakers, Jan H. J.; Giglia-Mari, Giuseppina; Vermeulen, Wim

2013-01-01

The ten-subunit transcription factor IIH (TFIIH) plays a crucial role in transcription and nucleotide excision repair (NER). Inactivating mutations in the smallest 8-kDa TFB5/TTDA subunit cause the neurodevelopmental progeroid repair syndrome trichothiodystrophy A (TTD-A). Previous studies have shown that TTDA is the only TFIIH subunit that appears not to be essential for NER, transcription, or viability. We studied the consequences of TTDA inactivation by generating a Ttda knock-out (Ttda−/−) mouse-model resembling TTD-A patients. Unexpectedly, Ttda−/− mice were embryonic lethal. However, in contrast to full disruption of all other TFIIH subunits, viability of Ttda−/− cells was not affected. Surprisingly, Ttda−/− cells were completely NER deficient, contrary to the incomplete NER deficiency of TTD-A patient-derived cells. We further showed that TTD-A patient mutations only partially inactivate TTDA function, explaining the relatively mild repair phenotype of TTD-A cells. Moreover, Ttda−/− cells were also highly sensitive to oxidizing agents. These findings reveal an essential role of TTDA for life, nucleotide excision repair, and oxidative DNA damage repair and identify Ttda−/− cells as a unique class of TFIIH mutants. PMID:23637614

Differential Adsorption of Ochratoxin A and Anthocyanins by Inactivated Yeasts and Yeast Cell Walls during Simulation of Wine Aging

PubMed Central

Petruzzi, Leonardo; Baiano, Antonietta; De Gianni, Antonio; Sinigaglia, Milena; Corbo, Maria Rosaria; Bevilacqua, Antonio

2015-01-01

The adsorption of ochratoxin A (OTA) by yeasts is a promising approach for the decontamination of musts and wines, but some potential competitive or interactive phenomena between mycotoxin, yeast cells, and anthocyanins might modify the intensity of the phenomenon. The aim of this study was to examine OTA adsorption by two strains of Saccharomyces cerevisiae (the wild strain W13, and the commercial isolate BM45), previously inactivated by heat, and a yeast cell wall preparation. Experiments were conducted using Nero di Troia red wine contaminated with 2 μg/L OTA and supplemented with yeast biomass (20 g/L). The samples were analyzed periodically to assess mycotoxin concentration, chromatic characteristics, and total anthocyanins over 84 days of aging. Yeast cell walls revealed the highest OTA-adsorption in comparison to thermally-inactivated cells (50% vs. 43% toxin reduction), whilst no significant differences were found for the amount of adsorbed anthocyanins in OTA-contaminated and control wines. OTA and anthocyanins adsorption were not competitive phenomena. Unfortunately, the addition of yeast cells to wine could cause color loss; therefore, yeast selection should also focus on this trait to select the best strain. PMID:26516913

Forskolin and protein kinase inhibitors differentially affect hair cell potassium currents and transmitter release at the cytoneural junction in the isolated frog labyrinth.

PubMed

Rossi, Maria Lisa; Rubbini, Gemma; Martini, Marta; Canella, Rita; Fesce, Riccardo

2017-08-15

The post-transductional elaboration of sensory input at the frog semicircular canal has been studied by correlating the effects of drugs that interfere with phosphorylation processes on: (i) potassium conductances in isolated hair cell and (ii) transmitter release at the cytoneural junction in the intact labyrinth. At hair cells, delayed potassium currents (IKD) undergo voltage- and time-dependent inactivation; inactivation removal requires ATP, is sensitive to kinase blockade, but is unaffected by exogenous application of cyclic nucleotides. We report here that forskolin, an activator of endogenous adenylyl cyclase, enhances IKD inactivation removal in isolated hair cells, but produces an overall decrease in IKD amplitude consistent with the direct blocking action of the drug on several families of K channels. In the intact labyrinth, forskolin enhances transmitter release, consistent with such depression of K conductances. Kinase blockers - H-89 and KT5823 - have been shown to reduce IKD inactivation removal and IKD amplitude at isolated hair cells. In the labyrinth, the effects of these drugs on junctional activity are quite variable, with predominant inhibition of transmitter release, rather than the enhancement expected from the impairment of K currents. The overall action of forskolin and kinase inhibitors on K conductances is similar (depression), but they have opposite effects on transmitter release: this indicates that some intermediate steps between the bioelectric control of hair cell membrane potential and transmitter release are affected in opposite ways and therefore are presumably regulated by protein phosphorylation. Copyright © 2017 The Author(s). Published by Elsevier Ltd.. All rights reserved.

Noncomplementing diploidy resulting from spontaneous zygogenesis in Escherichia coli.

PubMed

Gratia, Jean-Pierre

2005-09-01

With the aim of understanding sexual reproduction and phenotypic expression, a novel type of mating recently discovered in Escherichia coli was investigated. Termed spontaneous zygogenesis (or Z-mating), it differs from F-mediated conjugation. Its products proved phenotypically unstable, losing part of the phenotype for which they were selected. Inactivation of a parental chromosome in the zygote is strongly suggested by fluctuation tests, respreading experiments, analysis of reisolates, and segregation of non-viable cells detected by epifluorescence staining. Some phenotypically haploid subclones were interpreted as stable noncomplementing diploids carrying an inactivated co-replicating chromosome. Pedigree analysis indicated that the genetic composition of such cells consisted of parental genomes or one parental plus a recombinant genome. Inactivation of a chromosome carrying a prophage resulted in the disappearance of both the ability to produce phage particles and the immunity to superinfection. Phage production signalled transient reactivation of such a chromosome and constituted a sensitive test for stable noncomplementing diploidy. Chromosome inactivation thus appears to be a spontaneous event in bacteria.

Rhynchophylline from Uncaria rhynchophylla functionally turns delayed rectifiers into A-Type K+ channels.

PubMed

Chou, Chun-Hsiao; Gong, Chi-Li; Chao, Chia-Chia; Lin, Chia-Huei; Kwan, Chiu-Yin; Hsieh, Ching-Liang; Leung, Yuk-Man

2009-05-22

Rhynchophylline (1), a neuroprotective agent isolated from the traditional Chinese medicinal herb Uncaria rhynchophylla, was shown to affect voltage-gated K(+) (Kv) channel slow inactivation in mouse neuroblastoma N2A cells. Extracellular 1 (30 microM) accelerated the slow decay of Kv currents and shifted the steady-state inactivation curve to the left. Intracellular dialysis of 1 did not accelerate the slow current decay, suggesting that this compound acts extracellularly. In addition, the percent blockage of Kv currents by this substance was independent of the degree of depolarization and the intracellular K(+) concentration. Therefore, 1 did not appear to directly block the outer channel pore, with the results obtained suggesting that it drastically accelerated Kv channel slow inactivation. Interestingly, 1 also shifted the activation curve to the left. This alkaloid also strongly accelerated slow inactivation and caused a left shift of the activation curve of Kv1.2 channels heterologously expressed in HEK293 cells. Thus, this compound functionally turned delayed rectifiers into A-type K(+) channels.

Effects of Toll-Like Receptor Stimulation on Eosinophilic Infiltration in Lungs of BALB/c Mice Immunized with UV-Inactivated Severe Acute Respiratory Syndrome-Related Coronavirus Vaccine

PubMed Central

Iwata-Yoshikawa, Naoko; Uda, Akihiko; Suzuki, Tadaki; Tsunetsugu-Yokota, Yasuko; Sato, Yuko; Morikawa, Shigeru; Tashiro, Masato; Sata, Tetsutaro; Hasegawa, Hideki

2014-01-01

ABSTRACT Severe acute respiratory syndrome-related coronavirus (SARS-CoV) is an emerging pathogen that causes severe respiratory illness. Whole UV-inactivated SARS-CoV (UV-V), bearing multiple epitopes and proteins, is a candidate vaccine against this virus. However, whole inactivated SARS vaccine that includes nucleocapsid protein is reported to induce eosinophilic infiltration in mouse lungs after challenge with live SARS-CoV. In this study, an ability of Toll-like receptor (TLR) agonists to reduce the side effects of UV-V vaccination in a 6-month-old adult BALB/c mouse model was investigated, using the mouse-passaged Frankfurt 1 isolate of SARS-CoV. Immunization of adult mice with UV-V, with or without alum, resulted in partial protection from lethal doses of SARS-CoV challenge, but extensive eosinophil infiltration in the lungs was observed. In contrast, TLR agonists added to UV-V vaccine, including lipopolysaccharide, poly(U), and poly(I·C) (UV-V+TLR), strikingly reduced excess eosinophilic infiltration in the lungs and induced lower levels of interleukin-4 and -13 and eotaxin in the lungs than UV-V-immunization alone. Additionally, microarray analysis showed that genes associated with chemotaxis, eosinophil migration, eosinophilia, and cell movement and the polarization of Th2 cells were upregulated in UV-V-immunized but not in UV-V+TLR-immunized mice. In particular, CD11b+ cells in the lungs of UV-V-immunized mice showed the upregulation of genes associated with the induction of eosinophils after challenge. These findings suggest that vaccine-induced eosinophil immunopathology in the lungs upon SARS-CoV infection could be avoided by the TLR agonist adjuvants. IMPORTANCE Inactivated whole severe acute respiratory syndrome-related coronavirus (SARS-CoV) vaccines induce neutralizing antibodies in mouse models; however, they also cause increased eosinophilic immunopathology in the lungs upon SARS-CoV challenge. In this study, the ability of adjuvant Toll-like receptor (TLR) agonists to reduce the side effects of UV-inactivated SARS-CoV vaccination in a BALB/c mouse model was tested, using the mouse-passaged Frankfurt 1 isolate of SARS-CoV. We found that TLR stimulation reduced the high level of eosinophilic infiltration that occurred in the lungs of mice immunized with UV-inactivated SARS-CoV. Microarray analysis revealed that genes associated with chemotaxis, eosinophil migration, eosinophilia, and cell movement and the polarization of Th2 cells were upregulated in UV-inactivated SARS-CoV-immunized mice. This study may be helpful for elucidating the pathogenesis underlying eosinophilic infiltration resulting from immunization with inactivated vaccine. PMID:24850731

Role of N-terminal domain and accessory subunits in controlling deactivation-inactivation coupling of Kv4.2 channels.

PubMed

Barghaan, Jan; Tozakidou, Magdalini; Ehmke, Heimo; Bähring, Robert

2008-02-15

We examined the relationship between deactivation and inactivation in Kv4.2 channels. In particular, we were interested in the role of a Kv4.2 N-terminal domain and accessory subunits in controlling macroscopic gating kinetics and asked if the effects of N-terminal deletion and accessory subunit coexpression conform to a kinetic coupling of deactivation and inactivation. We expressed Kv4.2 wild-type channels and N-terminal deletion mutants in the absence and presence of Kv channel interacting proteins (KChIPs) and dipeptidyl aminopeptidase-like proteins (DPPs) in human embryonic kidney 293 cells. Kv4.2-mediated A-type currents at positive and deactivation tail currents at negative membrane potentials were recorded under whole-cell voltage-clamp and analyzed by multi-exponential fitting. The observed changes in Kv4.2 macroscopic inactivation kinetics caused by N-terminal deletion, accessory subunit coexpression, or a combination of the two maneuvers were compared with respective changes in deactivation kinetics. Extensive correlation analyses indicated that modulatory effects on deactivation closely parallel respective effects on inactivation, including both onset and recovery kinetics. Searching for the structural determinants, which control deactivation and inactivation, we found that in a Kv4.2 Delta 2-10 N-terminal deletion mutant both the initial rapid phase of macroscopic inactivation and tail current deactivation were slowed. On the other hand, the intermediate and slow phase of A-type current decay, recovery from inactivation, and tail current decay kinetics were accelerated in Kv4.2 Delta 2-10 by KChIP2 and DPPX. Thus, a Kv4.2 N-terminal domain, which may control both inactivation and deactivation, is not necessary for active modulation of current kinetics by accessory subunits. Our results further suggest distinct mechanisms for Kv4.2 gating modulation by KChIPs and DPPs.

Inactivation of A currents and A channels on rat nodose neurons in culture

PubMed Central

1989-01-01

Cultured sensory neurons from nodose ganglia were investigated with whole-cell patch-clamp techniques and single-channel recordings to characterize the A current. Membrane depolarization from -40 mV holding potential activated the delayed rectifier current (IK) at potentials positive to -30 mV; this current had a sigmoidal time course and showed little or no inactivation. In most neurons, the A current was completely inactivated at the -40 mV holding potential and required hyperpolarization to remove the inactivation; the A current was isolated by subtracting the IK evoked by depolarizations from -40 mV from the total outward current evoked by depolarizations from -90 mV. The decay of the A current on several neurons had complex kinetics and was fit by the sum of three exponentials whose time constants were 10- 40 ms, 100-350 ms, and 1-3 s. At the single-channel level we found that one class of channel underlies the A current. The conductance of A channels varied with the square root of the external K concentration: it was 22 pS when exposed to 5.4 mM K externally, the increased to 40 pS when exposed to 140 mM K externally. A channels activated rapidly upon depolarization and the latency to first opening decreased with depolarization. The open time distributions followed a single exponential and the mean open time increased with depolarization. A channels inactivate in three different modes: some A channels inactivated with little reopening and gave rise to ensemble averages that decayed in 10-40 ms; other A channels opened and closed three to four times before inactivating and gave rise to ensemble averages that decayed in 100-350 ms; still other A channels opened and closed several hundred times and required seconds to inactivate. Channels gating in all three modes contributed to the macroscopic A current from the whole cell, but their relative contribution differed among neurons. In addition, A channels could go directly from the closed, or resting, state to the inactivated state without opening, and the probability for channels inactivating in this way was greater at less depolarized voltages. In addition, a few A channels appeared to go reversibly from a mode where inactivation occurred rapidly to a slow mode of inactivation. PMID:2592953

Comparison of fungicidal properties of non-thermal plasma produced by corona discharge and dielectric barrier discharge.

PubMed

Julák, J; Soušková, H; Scholtz, V; Kvasničková, E; Savická, D; Kříha, V

2018-01-01

The inactivation of four micromycete species by action of non-thermal plasma was followed. Two sources of plasma were compared, namely, positive corona discharge and dielectric barrier discharge. The corona discharge appeared as suitable for fungal spore inactivation in water suspension, whereas the barrier discharge inactivated spores on the surface of cultivation agar. Cladosporium sphaerospermum was the most sensitive, being inactivated within 10 min of exposure to plasma, whereas Aspergillus oryzae displayed decrease in viable cell count only, the complete inactivation was not achieved even after 40 min of exposure. Intermediate sensitivity was found for Alternaria sp. and Byssochlamys nivea. The significant delay of growth was observed for all fungi after exposure to sublethal dose of plasma, but we failed to express this effect quantitatively.

Viral capsid mobility: a dynamic conduit for inactivation.

PubMed

Broo, K; Wei, J; Marshall, D; Brown, F; Smith, T J; Johnson, J E; Schneemann, A; Siuzdak, G

2001-02-27

Mass spectrometry and fluorescent probes have provided direct evidence that alkylating agents permeate the protein capsid of naked viruses and chemically inactivate the nucleic acid. N-acetyl-aziridine and a fluorescent alkylating agent, dansyl sulfonate aziridine, inactivated three different viruses, flock house virus, human rhinovirus-14, and foot and mouth disease virus. Mass spectral studies as well as fluorescent probes showed that alkylation of the genome was the mechanism of inactivation. Because particle integrity was not affected by selective alkylation (as shown by electron microscopy and sucrose gradient experiments), it was reasoned that the dynamic nature of the viral capsid acts as a conduit to the interior of the particle. Potential applications include fluorescent labeling for imaging viral genomes in living cells, the sterilization of blood products, vaccine development, and viral inactivation in vivo.

A general approach for chemical labeling and rapid, spatially controlled protein inactivation

PubMed Central

Marks, Kevin M.; Braun, Patrick D.; Nolan, Garry P.

2004-01-01

Chemical labeling of proteins inside of living cells can enable studies of the location, movement, and function of proteins in vivo. Here we demonstrate an approach for chemical labeling of proteins that uses the high-affinity interaction between an FKBP12 mutant (F36V) and a synthetic, engineered ligand (SLF′). A fluorescein conjugate to the engineered ligand (FL-SLF′) retained binding to FKBP12(F36V) and possessed similar fluorescence properties as parental fluorescein. FL-SLF′ labeled FKBP12(F36V) fusion proteins in live mammalian cells, and was used to monitor the subcellular localization of a membrane targeted FKBP12(F36V) construct. Chemical labeling of FKBP12(F36V) fusion proteins with FL-SLF′ was readily detectable at low expression levels of the FKBP12(F36V) fusion, and the level of fluorescent staining with FL-SLF′ was proportional to the FKBP12(F36V) expression level. This FL-SLF′-FKBP12(F36V) labeling technique was tested in fluorophore assisted laser inactivation (FALI), a light-mediated technique to rapidly inactivate fluorophore-labeled target proteins. FL-SLF′ mediated FALI of a β-galactosidase-FKBP12(F36V) fusion protein, causing rapid inactivation of >90% of enzyme activity upon irradiation in vitro. FL-SLF′ also mediated FALI of a β-galactosidase fusion expressed in living NIH 3T3 cells, where β-galactosidase activity was reduced in 15 s. Thus, FL-SLF′ can be used to monitor proteins in vivo and to target rapid, spatially and temporally defined inactivation of target proteins in living cells in a process that we call FK-FALI. PMID:15218100

Cometabolic degradation of trichloroethene by Rhodococcus sp. strain L4 immobilized on plant materials rich in essential oils.

PubMed

Suttinun, Oramas; Müller, Rudolf; Luepromchai, Ekawan

2010-07-01

The cometabolic degradation of trichloroethene (TCE) by Rhodococcus sp. L4 was limited by the loss of enzyme activity during TCE transformation. This problem was overcome by repeated addition of inducing substrates, such as cumene, limonene, or cumin aldehyde, to the cells. Alternatively, Rhodococcus sp. L4 was immobilized on plant materials which contain those inducers in their essential oils. Cumin seeds were the most suitable immobilizing material, and the immobilized cells tolerated up to 68 muM TCE and degraded TCE continuously. The activity of immobilized cells, which had been inactivated partially during TCE degradation, could be reactivated by incubation in mineral salts medium without TCE. These findings demonstrate that immobilization of Rhodococcus sp. L4 on plant materials rich in essential oils is a promising method for efficient cometabolic degradation of TCE.

Cometabolic Degradation of Trichloroethene by Rhodococcus sp. Strain L4 Immobilized on Plant Materials Rich in Essential Oils▿ †

PubMed Central

Suttinun, Oramas; Müller, Rudolf; Luepromchai, Ekawan

2010-01-01

The cometabolic degradation of trichloroethene (TCE) by Rhodococcus sp. L4 was limited by the loss of enzyme activity during TCE transformation. This problem was overcome by repeated addition of inducing substrates, such as cumene, limonene, or cumin aldehyde, to the cells. Alternatively, Rhodococcus sp. L4 was immobilized on plant materials which contain those inducers in their essential oils. Cumin seeds were the most suitable immobilizing material, and the immobilized cells tolerated up to 68 μM TCE and degraded TCE continuously. The activity of immobilized cells, which had been inactivated partially during TCE degradation, could be reactivated by incubation in mineral salts medium without TCE. These findings demonstrate that immobilization of Rhodococcus sp. L4 on plant materials rich in essential oils is a promising method for efficient cometabolic degradation of TCE. PMID:20472723

Cardiac transient outward potassium current: a pulse chemistry model of frequency-dependent properties.

PubMed

Liu, L; Krinsky, V I; Grant, A O; Starmer, C F

1996-01-01

Recent voltage-clamp studies of isolated myocytes have demonstrated widespread occurrence of a transient outward current (I(to)) carried by potassium ions. In the canine ventricle, this current is well developed in epicardial cells but not in endocardial cells. The resultant spatial dispersion of refractoriness is potentially proarrhythmic and may be amplified by channel blockade. The inactivation and recovery time constants of this channel are in excess of several hundred milliseconds, and consequently channel availability is frequency dependent at physiological stimulation rates. When the time constants associated with transitions between different channel conformations are rapid relative to drug binding kinetics, the interactions between drugs and an ion channel can be approximated by a sequence of first-order reactions, in which binding occurs in pulses in response to pulse train stimulation (pulse chemistry). When channel conformation transition time constants do not meet this constraint, analytical characterizations of the drug-channel interaction must then be modified to reflect the channel time-dependent properties. Here we report that the rate and steady-state amount of frequency-dependent inactivation of I(to) are consistent with a generalization of the channel blockade model: channel availability is reduced in a pulsatile exponential pattern as the stimulation frequency is increased, and the rate of reduction is a linear function of the pulse train depolarizing and recovery intervals. I(to) was reduced in the presence of quinidine. After accounting for the use-dependent availability of I(to) channels, we found little evidence of an additional use-dependent component of block after exposure to quinidine, suggesting that quinidine reacts with both open and closed I(to) channels as though the binding site is continuously accessible. The model provides a useful tool for assessing drug-channel interactions when the reaction cannot be continuously monitored.

Independent Evolution of Transcriptional Inactivation on Sex Chromosomes in Birds and Mammals

PubMed Central

Livernois, Alexandra M.; Waters, Shafagh A.; Deakin, Janine E.; Marshall Graves, Jennifer A.; Waters, Paul D.

2013-01-01

X chromosome inactivation in eutherian mammals has been thought to be tightly controlled, as expected from a mechanism that compensates for the different dosage of X-borne genes in XX females and XY males. However, many X genes escape inactivation in humans, inactivation of the X in marsupials is partial, and the unrelated sex chromosomes of monotreme mammals have incomplete and gene-specific inactivation of X-linked genes. The bird ZW sex chromosome system represents a third independently evolved amniote sex chromosome system with dosage compensation, albeit partial and gene-specific, via an unknown mechanism (i.e. upregulation of the single Z in females, down regulation of one or both Zs in males, or a combination). We used RNA-fluorescent in situ hybridization (RNA-FISH) to demonstrate, on individual fibroblast cells, inactivation of 11 genes on the chicken Z and 28 genes on the X chromosomes of platypus. Each gene displayed a reproducible frequency of 1Z/1X-active and 2Z/2X-active cells in the homogametic sex. Our results indicate that the probability of inactivation is controlled on a gene-by-gene basis (or small domains) on the chicken Z and platypus X chromosomes. This regulatory mechanism must have been exapted independently to the non-homologous sex chromosomes in birds and mammals in response to an over-expressed Z or X in the homogametic sex, highlighting the universal importance that (at least partial) silencing plays in the evolution on amniote dosage compensation and, therefore, the differentiation of sex chromosomes. PMID:23874231

In Vitro System for the Assessment of the Potential Antifertility Affect of Chemicals.

DTIC Science & Technology

1992-07-01

inactivated rabbit, calf, or horse serum; but cell viability was poor. Cell viability was improved in DN alone or in Biggers, Vhittinghaa, and Vhitten...constitute an official endorsement of any commer- cial products. This report may not be cited for purposes of advertisement. Reproduction of this...heat inactivated rabbit, horse , or calf serum and incubated in an atmosphere of 5% C02, 95% air at 37 OC, and sperm motion was recorded on videotape

A thin layer electrochemical cell for disinfection of water contaminated with Staphylococcus aureus

PubMed Central

Gusmão, Isabel C. P.; Moraes, Peterson B.; Bidoia, Ederio D.

2009-01-01

A thin layer electrochemical cell was tested and developed for disinfection treatment of water artificially contaminated with Staphylococcus aureus. Electrolysis was performed with a low-voltage DC power source applying current densities of 75 mA cm-2 (3 A) or 25 mA cm-2 (1 A). A dimensionally stable anode (DSA) of titanium coated with an oxide layer of 70%TiO2 plus 30%RuO2 (w/w) and a 3 mm from a stainless-steel 304 cathode was used in the thin layer cell. The experiments were carried out using a bacteria suspension containing 0.08 M sodium sulphate with chloride-free to determine the bacterial inactivation efficacy of the thin layer cell without the generation of chlorine. The chlorine can promote the formation of trihalomethanes (THM) that are carcinogenic. S. aureus inactivation increased with electrolysis time and lower flow rate. The flow rates used were 200 or 500 L h-1. At 500 L h-1 and 75 mA cm-2 the inactivation after 60 min was about three logs of decreasing for colony forming units by mL. However, 100% inactivation for S. aureus was observed at 5.6 V and 75 mA cm-2 after 30 min. Thus, significant disinfection levels can be achieved without adding oxidant substances or generation of chlorine in the water. PMID:24031410

Evaluation of Energy Dose and Output Power Optimum of Diode’s Laser of 450 nm and 650 nm in Photoantimicrobial Mechanisms Against Inhibition of C. Albicans Biofilm Cells

NASA Astrophysics Data System (ADS)

Dewi-Astuty, S.; Suhariningsih; Dyah-Astuti, S.; Baktir, A.

2018-03-01

Photoantimicrobial as a pathogenic microbial inhibitory therapy system such as C. albicans in biofilms forms has been studied in vitro. Mechanisms of inhibiting called inactivating used the absorb principles of a dye agents such as chlorophyll against the photon energy of diode laser which any number of ROS product depend on energy doses of a laser, time of irradiation, concentration and time of incubation the dyes agent. The inactivation profile of C. albicans biofilm cells was observed based on cell viability reduction after photoantimicrobial treatment with or without oxygenation by XTT assay test. Results show that the inhibiting significantly with the time incubation of the dye agents and the oxygen degree inside the sample. The inhibition for oxygenation biofilm’s group 10% lower than without oxygenation biofilm’s group at the maximum of reduction of cell viability occurred in the 3hour incubation group. Optimum of inactivation are 89.6% (without oxygenation) and 94.8% (with oxygenation) after irradiation with 450 nm laser (power output 128.73 at energy dose 86.09 J/cm2), While, by 650 nm laser (power output 164.53 mW at energy dose 92.52 J/cm2) irradiation treatment obtained optimum of inactivation are 89.5% (without oxygenation) and 92.3% (with oxygenation).

Photodynamic inactivation of Escherichia coli - Correlation of singlet oxygen kinetics and phototoxicity.

PubMed

Müller, Alexander; Preuß, Annegret; Röder, Beate

2018-01-01

Photodynamic inactivation (PDI) of bacteria may play a major role in facing the challenge of the ever expanding antibiotic resistances. Here we report about the direct correlation of singlet oxygen luminescence kinetics and phototoxicity in E. coli cell suspension under PDI using the widely applied cationic photosensitizer TMPyP. Through direct access to the microenvironment, the time resolved investigation of singlet oxygen luminescence plays a key role in understanding the photosensitization mechanism and inactivation pathway. Using the homemade set-up for highly sensitive time resolved singlet oxygen luminescence detection, we show that the cationic TMPyP is localized predominantly outside the bacterial cells but in their immediate vicinity prior to photodynamic inactivation. Throughout following light exposure, a clear change in singlet oxygen kinetics indicates a redistribution of photosensitizer molecules to at least one additional microenvironment. We found the signal kinetics mirrored in cell viability measurements of equally treated samples from same overnight cultures conducted in parallel: A significant drop in cell viability of the illuminated samples and stationary viability of dark controls. Thus, for the system investigated in this work - a Gram-negative model bacteria and a well-known PS for its PDI - singlet oxygen kinetics correlates with phototoxicity. This finding suggests that it is well possible to evaluate PDI efficiency directly via time resolved singlet oxygen detection. Copyright © 2017 Elsevier B.V. All rights reserved.

TolC plays a crucial role in immune protection conferred by Edwardsiella tarda whole-cell vaccines.

PubMed

Wang, Chao; Peng, Bo; Li, Hui; Peng, Xuan-Xian

2016-07-12

Although vaccines developed from live organisms have better efficacy than those developed from dead organisms, the mechanisms underlying this differential efficacy remain unexplored. In this study, we combined sub-immunoproteomics with immune challenge to investigate the action of the outer membrane proteome in the immune protection conferred by four Edwardsiella tarda whole-cell vaccines prepared via different treatments and to identify protective immunogens that play a key role in this immune protection. Thirteen spots representing five outer membrane proteins and one cytoplasmic protein were identified, and it was found that their abundance was altered in relation with the immune protective abilities of the four vaccines. Among these proteins, TolC and OmpA were found to be the key immunogens conferring the first and second highest degrees of protection, respectively. TolC was detected in the two effective vaccines (live and inactivated-30-F). The total antiserum and anti-OmpA titers were higher for the two effective vaccines than for the two ineffective vaccines (inactivated-80-F and inactivated-100). Further evidence demonstrated that the live and inactivated-30-F vaccines demonstrated stronger abilities to induce CD8+ and CD4+ T cell differentiation than the other two evaluated vaccines. Our results indicate that the outer membrane proteome changes dramatically following different treatments, which contributes to the effectiveness of whole-cell vaccines.

Capsid functions of inactivated human picornaviruses and feline calicivirus.

PubMed

Nuanualsuwan, Suphachai; Cliver, Dean O

2003-01-01

The exceptional stability of enteric viruses probably resides in their capsids. The capsid functions of inactivated human picornaviruses and feline calicivirus (FCV) were determined. Viruses were inactivated by UV, hypochlorite, high temperature (72 degrees C), and physiological temperature (37 degrees C), all of which are pertinent to transmission via food and water. Poliovirus (PV) and hepatitis A virus (HAV) are transmissible via water and food, and FCV is the best available surrogate for the Norwalk-like viruses, which are leading causes of food-borne and waterborne disease in the United States. The capsids of all 37 degrees C-inactivated viruses still protected the viral RNA against RNase, even in the presence of proteinase K, which contrasted with findings with viruses inactivated at 72 degrees C. The loss of ability of the virus to attach to homologous cell receptors was universal, regardless of virus type and inactivation method, except for UV-inactivated HAV, and so virus inactivation was almost always accompanied by the loss of virus attachment. Inactivated HAV and FCV were captured by homologous antibodies. However, inactivated PV type 1 (PV-1) was not captured by homologous antibody and 37 degrees C-inactivated PV-1 was only partially captured. The epitopes on the capsids of HAV and FCV are evidently discrete from the receptor attachment sites, unlike those of PV-1. These findings indicate that the primary target of UV, hypochlorite, and 72 degrees C inactivation is the capsid and that the target of thermal inactivation (37 degrees C versus 72 degrees C) is temperature dependent.

Electron-beam-inactivated vaccine against Salmonella enteritidis colonization in molting hens

USDA-ARS?s Scientific Manuscript database

Electron Beam (eBeam) ionization technology has a variety of applications in modern society. The underlying hypothesis was that electron beam (eBeam) inactivated Salmonella enterica serovar Enteritidis (SE) cells can serve as a vaccine to control Salmonella colonization and Salmonella shedding in c...

Inactivation of Staphylococcus aureus and Salmonella enteritidis in tryptic soy broth and caviar samples by high pressure processing.

PubMed

Fioretto, F; Cruz, C; Largeteau, A; Sarli, T A; Demazeau, G; El Moueffak, A

2005-08-01

We studied the action of high pressure processing on the inactivation of two foodborne pathogens, Staphylococcus aureus ATCC 6538 and Salmonella enteritidis ATCC 13076, suspended in a culture medium and inoculated into caviar samples. The baroresistance of the two pathogens in a tryptic soy broth suspension at a concentration of 10(8)-10(9) colony-forming units/ml was tested for continuous and cycled pressurization in the 150- to 550-MPa range and for 15-min treatments at room temperature. The increase of cycle number permitted the reduction of the pressure level able to totally inactivate both microorganisms in the tryptic soy broth suspension, whereas the effect of different procedure times on complete inactivation of the microorganisms inoculated into caviar was similar.

In Vitro Cytotoxicity of Low-Dose-Rate Radioimmunotherapy by the Alpha-Emitting Radioimmunoconjugate Thorium-227-DOTA-Rituximab

DOE Office of Scientific and Technical Information (OSTI.GOV)

Dahle, Jostein, E-mail: jostein.dahle@rr-research.n; Krogh, Cecilie; Melhus, Katrine B.

2009-11-01

Purpose: To determine whether the low-dose-rate alpha-particle-emitting radioimmunoconjugate {sup 227}Th-1,4,7,10-p-isothiocyanato-benzyl-tetraazacyclododecane-1,4,7, 10-tetraacetic acid (DOTA)-rituximab can be used to inactivate lymphoma cells growing as single cells and small colonies. Methods and Materials: CD20-positive lymphoma cell lines were treated with {sup 227}Th-DOTA-rituximab for 1-5 weeks. To simulate the in vivo situation with continuous but decreasing supply of radioimmunoconjugates from the blood pool, the cells were not washed after incubation with {sup 227}Th-DOTA-rituximab, but half of the medium was replaced with fresh medium, and cell concentration and cell-bound activity were determined every other day after start of incubation. A microdosimetric model was established tomore » estimate the average number of hits in the nucleus for different localizations of activity. Results: There was a specific targeted effect on cell growth of the {sup 227}Th-DOTA-rituximab treatment. Although the cells were not washed after incubation with {sup 227}Th-DOTA-rituximab, the average contribution of activity in the medium to the mean dose was only 6%, whereas the average contribution from activity on the cells' own surface was 78%. The mean dose rates after incubation with 800 Bq/mL {sup 227}Th-DOTA-rituximab varied from 0.01 to 0.03 cGy/min. The average delay in growing from 10{sup 5} to 10{sup 7} cells/mL was 15 days when the cells were treated with a mean absorbed radiation dose of 2 Gy alpha-particle radiation from {sup 227}Th-DOTA-rituximab, whereas it was 11 days when the cells were irradiated with 6 Gy of X-radiation. The relative biologic effect of the treatment was estimated to be 2.9-3.4. Conclusions: The low-dose-rate radioimmunoconjugate {sup 227}Th-DOTA-rituximab is suitable for inactivation of single lymphoma cells and small colonies of lymphoma cells.« less

An Environmentally Friendly Method for Testing Photocatalytic Inactivation of Cyanobacterial Propagation on a Hybrid Ag-TiO2 Photocatalyst under Solar Illumination

PubMed Central

Chang, Shu-Yu; Huang, Winn-Jung; Lu, Ben-Ren; Fang, Guor-Cheng; Chen, Yeah; Chen, Hsiu-Lin; Chang, Ming-Chin; Hsu, Cheng-Feng

2015-01-01

Cyanobacteria were inactivated under sunlight using mixed phase silver (Ag) and deposited titanium dioxide (TiO2) coated on the surface of diatomite (DM) as a hybrid photocatalyst (Ag-TiO2/DM). The endpoints of dose-response experiments were chlorophyll a, photosynthetic efficiency, and flow cytometry measurements. In vitro experiments revealed that axenic cultures of planktonic cyanobacteria lost their photosynthetic activity following photocatalyzed exposure to sunlight for more than 24 h. Nearly 92% of Microcystis aeruginosa cells lost their photosynthetic activity, and their cell morphology was severely damaged within 24 h of the reaction. Preliminary carbon-14 (14CO3−2) results suggest that the complete inactivation of cyanobacteria arises from damage to cell wall components (peroxidation). A small concomitant increase in cell wall disorder and a consequent decrease in cell wall functional groups increase the cell wall fluidity prior to cell lysis. A high dosage of Ag-TiO2/DM during photocatalysis increased the concentration of extracellular polymeric substances (EPSs) in the Microcystis aeruginosa suspension by up to approximately 260%. However, photocatalytic treatment had a small effect on the disinfection by-product (DBP) precursor, as revealed by only a slight increase in the formation of trihalomethanes (THMs) and haloacetic acids (HAAs). PMID:26690465

Inactivation and mutation induction in Saccharomyces cerevisiae exposed to simulated sunlight: evaluation of action spectra.

PubMed

Schenk-Meuser, K; Pawlowsky, K; Kiefer, J

1992-07-15

The effectiveness of polychromatic light irradiation was investigated for haploid yeast cells. Inactivation and mutation induction were measured in both a RAD-wildtype strain and an excision-repair defective strain. The behaviour of vegetative "wet" cells was compared to that of dehydrated cells. The aim of the study was to assess the interaction of UVC with other wavelengths in cells of different states of humidity. The irradiation procedure was therefore carried out using a solar simulator either with full spectrum or with a UVC-blocking filter (modified sunlight) added. The results were analysed on the basis of separately determined action spectra. The summation of the efficiency of individual wavelengths was compared to the values obtained from polychromatic irradiation. It is shown that the effects caused by the whole-spectrum irradiation in wet cells can be predicted sufficiently from the calculation, while dried wildtype cells exhibit higher mutation rates. Thus it can be assumed that drying-specific damage leads to lethal and mutagenic lesions which are processed in different ways, causing a synergistic behaviour in mutation induction. Irradiation of vegetative cells with modified sunlight (UVC-) results in less inactivation and lower mutation rates than were calculated. From these results it can be concluded that this antagonistic behaviour is caused by the interaction of near-UV photoproducts.

Immunogenicity of an inactivated oil-emulsion canine distemper vaccine in African wild dogs.

PubMed

Cirone, Francesco; Elia, Gabriella; Campolo, Marco; Friedrich, Klaus; Martella, Vito; Pratelli, Annamaria; Buonavoglia, Canio

2004-04-01

The immunogenicity of an inactivated oil-emulsion vaccine against canine distemper virus was evaluated in nine captive African wild dogs (Lycaon pictus). Antibody levels were determined by neutralization test in Vero cells. No significant local or systemic adverse reactions were observed in the animals. Virus neutralizing antibody levels >1:20 were detected, especially in animals that were vaccinated twice. The use of oil adjuvants is suggested as a good way to enhance the immune response to inactivated canine distemper vaccine.

Design, construction, and optimization of a novel, modular, and scalable incubation chamber for continuous viral inactivation.

PubMed

Orozco, Raquel; Godfrey, Scott; Coffman, Jon; Amarikwa, Linus; Parker, Stephanie; Hernandez, Lindsay; Wachuku, Chinenye; Mai, Ben; Song, Brian; Hoskatti, Shashidhar; Asong, Jinkeng; Shamlou, Parviz; Bardliving, Cameron; Fiadeiro, Marcus

2017-07-01

We designed, built or 3D printed, and screened tubular reactors that minimize axial dispersion to serve as incubation chambers for continuous virus inactivation of biological products. Empirical residence time distribution data were used to derive each tubular design's volume equivalent to a theoretical plate (VETP) values at a various process flow rates. One design, the Jig in a Box (JIB), yielded the lowest VETP, indicating optimal radial mixing and minimal axial dispersion. A minimum residence time (MRT) approach was employed, where the MRT is the minimum time the product spends in the tubular reactor. This incubation time is typically 60 minutes in a batch process. We provide recommendations for combinations of flow rates and device dimensions for operation of the JIB connected in series that will meet a 60-min MRT. The results show that under a wide range of flow rates and corresponding volumes, it takes 75 ± 3 min for 99% of the product to exit the reactor while meeting the 60-min MRT criterion and fulfilling the constraint of keeping a differential pressure drop under 5 psi. Under these conditions, the VETP increases slightly from 3 to 5 mL though the number of theoretical plates stays constant at about 1326 ± 88. We also demonstrated that the final design volume was only 6% ± 1% larger than the ideal plug flow volume. Using such a device would enable continuous viral inactivation in a truly continuous process or in the effluent of a batch chromatography column. Viral inactivation studies would be required to validate such a design. © 2017 American Institute of Chemical Engineers Biotechnol. Prog., 33:954-965, 2017. © 2017 American Institute of Chemical Engineers.

Silica Gel for Enhanced Activity and Hypochlorite Protection of Cyanuric Acid Hydrolase in Recombinant Escherichia coli.

PubMed

Radian, Adi; Aukema, Kelly G; Aksan, Alptekin; Wackett, Lawrence P

2015-11-03

Chlorinated isocyanuric acids are widely used water disinfectants that generate hypochlorite, but with repeated application, they build up cyanuric acid (CYA) that must be removed to maintain disinfection. 3-Aminopropyltriethoxysilane (APTES)-treated Escherichia coli cells expressing cyanuric acid hydrolase (CAH) from Moorella thermoacetica exhibited significantly high CYA degradation rates and provided protection against enzyme inactivation by hypochlorite (chlorine). APTES coating or encapsulation of cells had two benefits: (i) overcoming diffusion limitations imposed by the cell wall and (ii) protecting against hypochlorite inactivation of CAH activity. Cells encapsulated in APTES gels degraded CYA three times faster than nonfunctionalized tetraethoxysilane (TEOS) gels, and cells coated with APTES degraded CYA at a rate of 29 µmol/min per mg of CAH protein, similar to the rate with purified enzyme. UV spectroscopy, fluorescence spectroscopy, and scanning electron microscopy showed that the higher rates were due to APTES increasing membrane permeability and enhancing cyanuric acid diffusion into the cytoplasm to reach the CAH enzyme. Purified CAH enzyme was shown to be rapidly inactivated by hypochlorite. APTES aggregates surrounding cells protected via the amine groups reacting with hypochlorite as shown by pH changes, zeta potential measurements, and infrared spectroscopy. APTES-encapsulated E. coli cells expressing CAH degraded cyanuric acid at high rates in the presence of 1 to 10 ppm hypochlorite, showing effectiveness under swimming pool conditions. In contrast, CAH activity in TEOS gels or free cells was completely inactivated by hypochlorite. These studies show that commercially available silica materials can selectively enhance, protect, and immobilize whole-cell biocatalysts for specialized applications. Hypochlorite is used in vast quantities for water disinfection, killing bacteria on surfaces, and washing and whitening. In pools, spas, and other waters, hypochlorite is frequently delivered as chlorinated isocyanuric acids that release hypochlorite and cyanuric acid. Over time, cyanuric acid accumulates and impairs disinfection and must be removed. The microbial enzyme cyanuric acid hydrolase can potentially remove cyanuric acid to restore disinfection and protect swimmers. Whole bacterial cells expressing cyanuric acid hydrolase were encapsulated in an inert silica matrix containing an amine group. The amine group serves to permeabilize the cell membrane and accelerate cyanuric acid degradation, and it also reacts with hypochlorite to protect against inactivation of cyanuric acid hydrolase. Methods for promoting whole-cell biocatalysis are important in biotechnology, and the present work illustrates approaches to enhance rates and protect against an inhibitory substance. Copyright © 2015 Radian et al.

Arginine inactivates human herpesvirus 2 and inhibits genital herpesvirus infection.

PubMed

Ikeda, Keiko; Yamasaki, Hisashi; Minami, Sawako; Suzuki, Yukiko; Tsujimoto, Kazuko; Sekino, Yoshihisa; Irie, Hiroshi; Arakawa, Tsutomu; Koyama, A Hajime

2012-12-01

Arginine, among the amino acids, has demonstrated unique properties, including suppression of protein-protein interactions and virus inactivation. We investigated the effects of arginine on the infectivity of human herpesvirus 2 (HHV-2) and the potential application of arginine as a chemotherapeutic agent against genital herpes. Arginine directly inactivated HHV-2 and characterization of the inactivation demonstrated that 1 M arginine at pH 4.3 inactivated the virus more efficiently compared to 0.1 M citrate or 1 M sodium chloride, indicating that neither acidic pH nor ionic strength alone is sufficient for virus inactivation. The effect of arginine was rapid and concentration-dependent. Although virus inactivation was efficient at an acidic pH, arginine inactivated the virus even at a neutral pH, provided that a higher arginine concentration and prolonged incubation time were used. In addition, arginine suppressed the multiplication of HHV-2 under the conditions at which its effect on cell viability was insignificant. Pilot mouse model studies revealed a marked suppression of death by arginine when the mice were infected with HHV-2 through the vaginal route, followed by an intermittent application of acidic arginine by vaginal instillation.

Domain IV voltage-sensor movement is both sufficient and rate limiting for fast inactivation in sodium channels.

PubMed

Capes, Deborah L; Goldschen-Ohm, Marcel P; Arcisio-Miranda, Manoel; Bezanilla, Francisco; Chanda, Baron

2013-08-01

Voltage-gated sodium channels are critical for the generation and propagation of electrical signals in most excitable cells. Activation of Na(+) channels initiates an action potential, and fast inactivation facilitates repolarization of the membrane by the outward K(+) current. Fast inactivation is also the main determinant of the refractory period between successive electrical impulses. Although the voltage sensor of domain IV (DIV) has been implicated in fast inactivation, it remains unclear whether the activation of DIV alone is sufficient for fast inactivation to occur. Here, we functionally neutralize each specific voltage sensor by mutating several critical arginines in the S4 segment to glutamines. We assess the individual role of each voltage-sensing domain in the voltage dependence and kinetics of fast inactivation upon its specific inhibition. We show that movement of the DIV voltage sensor is the rate-limiting step for both development and recovery from fast inactivation. Our data suggest that activation of the DIV voltage sensor alone is sufficient for fast inactivation to occur, and that activation of DIV before channel opening is the molecular mechanism for closed-state inactivation. We propose a kinetic model of sodium channel gating that can account for our major findings over a wide voltage range by postulating that DIV movement is both necessary and sufficient for fast inactivation.

Antimicrobial and cold plasma treatments for inactivation of listeria monocytogenes on whole apple surface

USDA-ARS?s Scientific Manuscript database

Introduction: Produce and bacterial cell surface structure play an important role as to where and how bacteria attach to produce surfaces. The efficacy of a novel antimicrobial solution developed in our laboratory was investigated in combination with cold plasma treatments for inactivation of Liste...

The prognostic relevance of p16 inactivation in head and neck cancer.

PubMed

Koscielny, Sven; Dahse, Regine; Ernst, Gunther; von Eggeling, Ferdinand

2007-01-01

The inactivation of the tumor suppressor gene p16 plays an important role in the development of malignant tumors. p16 loss can result from point mutations, loss of heterozygosity (LOH) or methylation of the promoter region. A total of 67 samples of tumor tissue from squamous cell carcinoma of the oral cavity, the pharynx and the larynx were analyzed for an inactivation of p16. The results of the molecular-biological investigations were correlated with the known clinical prognostic parameters after a follow-up period of approximately 3 years. Methylation of the promoter region and LOH were the main mechanisms of p16 inactivation. Point mutations presented as rare events. An inactivation of p16 did not have any statistical influence on tumor prognosis. Patients with a p16 gene inactivated by promoter methylation appeared to have a slightly lower tendency for local and regional recurrences. The inactivation of the tumor suppressor gene p16 plays a role in the carcinogenesis of head and neck cancer. Copyright (c) 2007 S. Karger AG, Basel.

Construction and Characterization of Human Mammary Epithelial Cell Lines Containing Mutations in the p53 or BRCA1 Genes

DTIC Science & Technology

1999-01-01

development of breast cancers. To study the effects of inactivating mutations in these tumor suppressor genes early in the breast-cancer pathway, we have...the effects of inactivating mutations in these tumor suppressor genes early in the breast-cancer pathway. The consequences of transduction of these...proposed three approaches for constructing p53-deficient cells; i.e., by mutating the p53 gene directly, by abrogating the protein’s normal cellular

Cellular Injuries in Cronobacter sakazakii CIP 103183T and Salmonella enterica Exposed to Drying and Subsequent Heat Treatment in Milk Powder

PubMed Central

Lang, Emilie; Guyot, Stéphane; Peltier, Caroline; Alvarez-Martin, Pablo; Perrier-Cornet, Jean-Marie; Gervais, Patrick

2018-01-01

Because of the ability of foodborne pathogens to survive in low-moisture foods, their decontamination is an important issue in food protection. This study aimed to clarify some of the cellular mechanisms involved in inactivation of foodborne pathogens after drying and subsequent heating. Individual strains of Salmonella Typhimurium, Salmonella Senftenberg, and Cronobacter sakazakii were mixed into whole milk powder and dried to different water activity levels (0.25 and 0.58); the number of surviving cells was determined after drying and subsequent thermal treatments in closed vessels at 90 and 100°C, for 30 and 120 s. For each condition, the percentage of unculturable cells was estimated and, in parallel, membrane permeability and respiratory activity were estimated by flow cytometry using fluorescent probes. After drying, it was clearly observable that the percentage of unculturable cells was correlated with the percentage of permeabilized cells (responsible for 20–40% of the total inactivated bacteria after drying), and to a lesser degree with the percentage of cells presenting with loss of respiratory activity. In contrast, the percentages of unculturable cells observed after heat treatment were strongly correlated with the loss of respiratory activity and weakly with membrane permeability (for 70–80% of the total inactivated bacteria after heat treatment). We conclude that cell inactivation during drying is closely linked to membrane permeabilization and that heat treatment of dried cells affects principally their respiratory activity. These results legitimize the use of time–temperature scales and allow better understanding of the cellular mechanisms of bacterial death during drying and subsequent heat treatment. These results may also allow better optimization of the decontamination process to ensure food safety by targeting the most deleterious conditions for bacterial cells without denaturing the food product. PMID:29593704

A Comprehensive Tutorial on In Vitro Characterization of New Photosensitizers for Photodynamic Antitumor Therapy and Photodynamic Inactivation of Microorganisms

PubMed Central

Maisch, Tim; Berneburg, Mark; Plaetzer, Kristjan

2013-01-01

In vitro research performed on eukaryotic or prokaryotic cell cultures usually represents the initial step for characterization of a novel photosensitizer (PS) intended for application in photodynamic therapy (PDT) of cancer or photodynamic inactivation (PDI) of microorganisms. Although many experimental steps of PS testing make use of the wide spectrum of methods readily employed in cell biology, special aspects of working with photoactive substances, such as the autofluorescence of the PS molecule or the requirement of light protection, need to be considered when performing in vitro experiments in PDT/PDI. This tutorial represents a comprehensive collection of operative instructions, by which, based on photochemical and photophysical properties of a PS, its uptake into cells, the intracellular localization and photodynamic action in both tumor cells and microorganisms novel photoactive molecules may be characterized for their suitability for PDT/PDI. Furthermore, it shall stimulate the efforts to expand the convincing benefits of photodynamic therapy and photodynamic inactivation within both established and new fields of applications and motivate scientists of all disciplines to get involved in photodynamic research. PMID:23762860

Apigenin induces apoptosis in human leukemia cells and exhibits anti-leukemic activity in vivo via inactivation of Akt and activation of JNK

PubMed Central

Budhraja, Amit; Gao, Ning; Zhang, Zhuo; Son, Young-Ok; Cheng, Senping; Wang, Xin; Ding, Songze; Hitron, Andrew; Chen, Gang; Luo, Jia; Shi, Xianglin

2015-01-01

In this study, we investigated the functional role of Akt and JNK signaling cascades in apigenin-induced apoptosis in U937 human leukemia cells and anti-leukemic activity of apigenin in vivo. Apigenin-induced apoptosis by inactivation of Akt with a concomitant activation of JNK, Mcl-1 and Bcl-2 down-regulation, cytochrome c release from mitochondria and activation of caspases. Constitutively active myristolated Akt prevented apigenin-induced JNK, caspases activation, and apoptosis. Conversely, LY294002 and a dominant negative construct of Akt potentiated apigenin-induced apoptosis in leukemia cells. Interruption of JNK pathway showed marked reduction in apigenin-induced caspases activation and apoptosis in leukemia cells. Furthermore, in vivo administration of apigenin resulted in attenuation of tumor growth in U937 xenografts accompanied inactivation of Akt and activation of JNK. Attenuation of tumor growth in U937 xenografts by apigenin raises the possibility that apigenin may have clinical implications and can be further tested for incorporating in leukemia treatment regimens. PMID:22084167

Inactivation of virus in intravenous immunoglobulin G using solvent/detergent treatment and pasteurization.

PubMed

Aghaie, A; Pourfatollah, A A; Bathaie, S Z; Moazzeni, S M; Khorsand Mohammad Pour, H; Sharifi, Z

2008-01-01

The safety of plasma derived medicinal products, such as immunoglobulin, depends on viral inactivation steps that are incorporated into the production process. Several attempts have been made to validate the effectiveness of these inactivation methods against a range of physio-chemically diverse viruses. Treatment with solvent/detergent (S/D) and pasteurization (P) has been continuously used in our IgG production and these methods were analysed in this study as models of viral inactivation. Bovine Viral Diarrhoea Virus (BVDV), Herpes Simplex Virus (HSV) and Vesicular Stomatitis Virus (VSV) were employed as models of HCV, HBV and HIV respectively. Polio and Reo viruses also were used as stable viruses to chemical substances. The infectivity of a range of viruses before and after treatment with two methods of viral inactivation was measured by end point titration and their effectiveness expressed as Logarithmic Reduction Factors (LRF). Solvent/detergent treatment reduced the amount of enveloped viruses by 5-6 logs. The reduction factor was between 5-6 logs for all viruses used in the pasteurization process. A final log reduction factor was obtained as the sum of the two individual methods. Both inactivation methods have advantages and disadvantages with respect to their ability to inactivate viruses. Thus,combination of two robust virus inactivation steps, solvent/detergent and pasteurization, increases the safety margin of immunoglobulin preparations.

Infectious Causes of Cholesteatoma and Treatment of Infected Ossicles prior to Reimplantation by Hydrostatic High-Pressure Inactivation

PubMed Central

Hinz, Rebecca

2015-01-01

Chronic inflammation, which is caused by recurrent infections, is one of the factors contributing to the pathogenesis of cholesteatoma. If reimplantation of autologous ossicles after a surgical intervention is intended, inactivation of planktonic bacteria and biofilms is desirable. High hydrostatic pressure treatment is a procedure, which has been used to inactivate cholesteatoma cells on ossicles. Here we discuss the potential inactivating effect of high hydrostatic pressure on microbial pathogens including biofilms. Recent experimental data suggest an incomplete inactivation at a pressure level, which is tolerable for the bone substance of ossicles and results at least in a considerable reduction of pathogen load. Further studies are necessary to access how far this quantitative reduction of pathogens is sufficient to prevent ongoing chronic infections, for example, due to forming of biofilms. PMID:25705686

In Vitro and In Vivo Gene Delivery by Recombinant Baculoviruses

PubMed Central

Tani, Hideki; Limn, Chang Kwang; Yap, Chan Choo; Onishi, Masayoshi; Nozaki, Masami; Nishimune, Yoshitake; Okahashi, Nobuo; Kitagawa, Yoshinori; Watanabe, Rie; Mochizuki, Rika; Moriishi, Kohji; Matsuura, Yoshiharu

2003-01-01

Although recombinant baculovirus vectors can be an efficient tool for gene transfer into mammalian cells in vitro, gene transduction in vivo has been hampered by the inactivation of baculoviruses by serum complement. Recombinant baculoviruses possessing excess envelope protein gp64 or other viral envelope proteins on the virion surface deliver foreign genes into a variety of mammalian cell lines more efficiently than the unmodified baculovirus. In this study, we examined the efficiency of gene transfer both in vitro and in vivo by recombinant baculoviruses possessing envelope proteins derived from either vesicular stomatitis virus (VSVG) or rabies virus. These recombinant viruses efficiently transferred reporter genes into neural cell lines, primary rat neural cells, and primary mouse osteal cells in vitro. The VSVG-modified baculovirus exhibited greater resistance to inactivation by animal sera than the unmodified baculovirus. A synthetic inhibitor of the complement activation pathway circumvented the serum inactivation of the unmodified baculovirus. Furthermore, the VSVG-modified baculovirus could transduce a reporter gene into the cerebral cortex and testis of mice by direct inoculation in vivo. These results suggest the possible use of the recombinant baculovirus vectors in combination with the administration of complement inhibitors for in vivo gene therapy. PMID:12941888

Malt1 protease inactivation efficiently dampens immune responses but causes spontaneous autoimmunity

PubMed Central

Jaworski, Maike; Marsland, Ben J; Gehrig, Jasmine; Held, Werner; Favre, Stéphanie; Luther, Sanjiv A; Perroud, Mai; Golshayan, Déla; Gaide, Olivier; Thome, Margot

2014-01-01

The protease activity of the paracaspase Malt1 has recently gained interest as a drug target for immunomodulation and the treatment of diffuse large B-cell lymphomas. To address the consequences of Malt1 protease inactivation on the immune response in vivo, we generated knock-in mice expressing a catalytically inactive C472A mutant of Malt1 that conserves its scaffold function. Like Malt1-deficient mice, knock-in mice had strong defects in the activation of lymphocytes, NK and dendritic cells, and the development of B1 and marginal zone B cells and were completely protected against the induction of autoimmune encephalomyelitis. Malt1 inactivation also protected the mice from experimental induction of colitis. However, Malt1 knock-in mice but not Malt1-deficient mice spontaneously developed signs of autoimmune gastritis that correlated with an absence of Treg cells, an accumulation of T cells with an activated phenotype and high serum levels of IgE and IgG1. Thus, removal of the enzymatic activity of Malt1 efficiently dampens the immune response, but favors autoimmunity through impaired Treg development, which could be relevant for therapeutic Malt1-targeting strategies. PMID:25319413

Genistein induces apoptosis by the inactivation of the IGF-1R/p-Akt signaling pathway in MCF-7 human breast cancer cells.

PubMed

Chen, Jun; Duan, Yuxin; Zhang, Xing; Ye, Yu; Ge, Bo; Chen, Jian

2015-03-01

Genistein is an estrogenic soy-derived compound belonging to the isoflavone class and shows anti-cancer effects. However, the specific cell apoptosis mechanisms of genistein have not been fully understood. In this study, we investigated the specific cell apoptosis mechanisms of genistein and the potential involvement of the IGF1R-Akt-Bcl-2 and Bax-mediated pathways in human breast cancer cells in vitro. MCF-7 human breast cancer cells were treated with various concentrations of genistein, and cell proliferation was evaluated by the MTT assay. Morphological changes in treated cells were examined by Hoechst 33258 staining, and treated cells were examined by flow cytometry. The levels of IGF-1R, p-Akt, Bcl-2, and Bax protein expression and Bcl-2 and Bax mRNA expression were evaluated by western blot and RT-PCR, respectively. Genistein inhibited the proliferation of MCF-7 cells and induced cell apoptosis, as determined by Hoechst staining and flow cytometry analysis. Furthermore, genistein induced the inactivation of IGF-1R and p-Akt and downregulated the Bcl-2/Bax protein ratio. These results suggest that genistein inhibited cell proliferation by inactivating the IGF-1R-PI3 K/Akt pathway and decreasing the Bcl-2/Bax mRNA and protein expressions. Our findings help elucidate the mechanisms by which genistein may contribute to the prevention of breast cancer carcinogenesis.

UV-inactivated HSV-1 potently activates NK cell killing of leukemic cells

PubMed Central

Samudio, Ismael; Rezvani, Katayoun; Shaim, Hila; Hofs, Elyse; Ngom, Mor; Bu, Luke; Liu, Guoyu; Lee, Jason T. C.; Imren, Suzan; Lam, Vivian; Poon, Grace F. T.; Ghaedi, Maryam; Takei, Fumio; Humphries, Keith; Jia, William

2016-01-01

Herein we demonstrate that oncolytic herpes simplex virus-1 (HSV-1) potently activates human peripheral blood mononuclear cells (PBMCs) to lyse leukemic cell lines and primary acute myeloid leukemia samples, but not healthy allogeneic lymphocytes. Intriguingly, we found that UV light–inactivated HSV-1 (UV-HSV-1) is equally effective in promoting PBMC cytolysis of leukemic cells and is 1000- to 10 000-fold more potent at stimulating innate antileukemic responses than UV-inactivated cytomegalovirus, vesicular stomatitis virus, reovirus, or adenovirus. Mechanistically, UV-HSV-1 stimulates PBMC cytolysis of leukemic cells, partly via Toll-like receptor-2/protein kinase C/nuclear factor-κB signaling, and potently stimulates expression of CD69, degranulation, migration, and cytokine production in natural killer (NK) cells, suggesting that surface components of UV-HSV-1 directly activate NK cells. Importantly, UV-HSV-1 synergizes with interleukin-15 (IL-15) and IL-2 in inducing activation and cytolytic activity of NK cells. Additionally, UV-HSV-1 stimulates glycolysis and fatty acid oxidation–dependent oxygen consumption in NK cells, but only glycolysis is required for their enhanced antileukemic activity. Last, we demonstrate that T cell–depleted human PBMCs exposed to UV-HSV-1 provide a survival benefit in a murine xenograft model of human acute myeloid leukemia (AML). Taken together, our results support the preclinical development of UV-HSV-1 as an adjuvant, alone or in combination with IL-15, for allogeneic donor mononuclear cell infusions to treat AML. PMID:26941401

Chemical Inactivation of the Cinnamate 4-Hydroxylase Allows for the Accumulation of Salicylic Acid in Elicited Cells1

PubMed Central

Schoch, Guillaume A.; Nikov, Georgi N.; Alworth, William L.; Werck-Reichhart, Danièle

2002-01-01

The cinnamate (CA) 4-hydroxylase (C4H) is a cytochrome P450 that catalyzes the second step of the main phenylpropanoid pathway, leading to the synthesis of lignin, pigments, and many defense molecules. Salicylic acid (SA) is an essential trigger of plant disease resistance. Some plant species can synthesize SA from CA by a mechanism not yet understood. A set of specific inhibitors of the C4H, including competitive, tight-binding, mechanism-based irreversible, and quasi-irreversible inhibitors have been developed with the main objective to redirect cinnamic acid to the synthesis of SA. Competitive inhibitors such as 2-hydroxy-1-naphthoic acid and the heme-coordinating compound 3-(4-pyridyl)-acrylic acid allowed strong inhibition of C4H activity in a tobacco (Nicotiana tabacum cv Bright Yellow [BY]) cell suspension culture. This inhibition was however rapidly relieved either because of substrate accumulation or because of inhibitor metabolism. Substrate analogs bearing a methylenedioxo function such as piperonylic acid (PIP) or a terminal acetylene such as 4-propynyloxybenzoic acid (4PB), 3-propynyloxybenzoic acid, and 4-propynyloxymethylbenzoic acid are potent mechanism-based inactivators of the C4H. PIP and 4PB, the best inactivators in vitro, were also efficient inhibitors of the enzyme in BY cells. Inhibition was not reversed 46 h after cell treatment. Cotreatment of BY cells with the fungal elicitor β-megaspermin and PIP or 4PB led to a dramatic increase in SA accumulation. PIP and 4PB do not trigger SA accumulation in nonelicited cells in which the SA biosynthetic pathway is not activated. Mechanism-based C4H inactivators, thus, are promising tools for the elucidation of the CA-derived SA biosynthetic pathway and for the potentiation of plant defense. PMID:12376665

Indocyanine green as effective antibody conjugate for intracellular molecular targeted photodynamic therapy

NASA Astrophysics Data System (ADS)

Wang, Sijia; Hüttmann, Gereon; Rudnitzki, Florian; Diddens-Tschoeke, Heyke; Zhang, Zhenxi; Rahmanzadeh, Ramtin

2016-07-01

The fluorescent dye indocyanine green (ICG) is clinically approved and has been applied for ophthalmic and intraoperative angiography, measurement of cardiac output and liver function, or as contrast agent in cancer surgery. Though ICG is known for its photochemical effects, it has played a minor role so far in photodynamic therapy or techniques for targeted protein-inactivation. Here, we investigated ICG as an antibody-conjugate for the selective inactivation of the protein Ki-67 in the nucleus of cells. Conjugates of the Ki-67 antibody TuBB-9 with different amounts of ICG were synthesized and delivered into HeLa and OVCAR-5 cells through conjugation to the nuclear localization sequence. Endosomal escape of the macromolecular antibodies into the cytoplasm was optically triggered by photochemical internalization with the photosensitizer BPD. The second light irradiation at 690 nm inactivated Ki-67 and subsequently caused cell death. Here, we show that ICG as an antibody-conjugate can be an effective photosensitizing agent. Best effects were achieved with 1.8 ICG molecules per antibody. Conjugated to antibodies, the ICG absorption peaks vary proportionally with concentration. The absorption of ICG above 650 nm within the optical window of tissue opens the possibility of selective Ki-67 inactivation deep inside of tissues.

Inactivation of adipose angiotensinogen reduces adipose tissue macrophages and increases metabolic activity.

PubMed

LeMieux, Monique J; Ramalingam, Latha; Mynatt, Randall L; Kalupahana, Nishan S; Kim, Jung Han; Moustaïd-Moussa, Naïma

2016-02-01

The adipose renin-angiotensin system (RAS) has been linked to obesity-induced inflammation, though mechanisms are not completely understood. In this study, adipose-specific angiotensinogen knockout mice (Agt-KO) were generated to determine whether Agt inactivation reduces inflammation and alters the metabolic profile of the Agt-KO mice compared to wild-type (WT) littermates. Adipose tissue-specific Agt-KO mice were created using the Cre-LoxP system with both Agt-KO and WT littermates fed either a low-fat or high-fat diet to assess metabolic changes. White adipose tissue was used for gene/protein expression analyses and WAT stromal vascular cells for metabolic extracellular flux assays. No significant differences were observed in body weight or fat mass between both genotypes on either diet. However, improved glucose clearance was observed in Agt-KO compared to WT littermates, consistent with higher expression of genes involved in insulin signaling, glucose transport, and fatty acid metabolism. Furthermore, Agt inactivation reduced total macrophage infiltration in Agt-KO mice fed both diets. Lastly, stroma vascular cells from Agt-KO mice revealed higher metabolic activity compared to WT mice. These findings indicate that adipose-specific Agt inactivation leads to reduced adipose inflammation and increased glucose tolerance mediated in part via increased metabolic activity of adipose cells. © 2015 The Obesity Society.

Growth Inhibition by Bupivacaine Is Associated with Inactivation of Ribosomal Protein S6 Kinase 1

PubMed Central

Beigh, Mushtaq Ahmad; Showkat, Mehvish; Bashir, Basharat; Bashir, Asma; Hussain, Mahboob ul; Andrabi, Khurshid Iqbal

2014-01-01

Bupivacaine is an amide type long acting local anesthetic used for epidural anesthesia and nerve blockade in patients. Use of bupivacaine is associated with severe cytotoxicity and apoptosis along with inhibition of cell growth and proliferation. Although inhibition of Erk, Akt, and AMPK seemingly appears to mediate some of the bupivacaine effects, potential downstream targets that mediate its effect remain unknown. S6 kinase 1 is a common downstream effector of several growth regulatory pathways involved in cell growth and proliferation known to be affected by bupivacaine. We have accordingly attempted to relate the growth inhibitory effects of bupivacaine with the status of S6K1 activity and we present evidence that decrease in cell growth and proliferation by bupivacaine is mediated through inactivation of S6 kinase 1 in a concentration and time dependent manner. We also show that ectopic expression of constitutively active S6 kinase 1 imparts substantial protection from bupivacaine induced cytotoxicity. Inactivation of S6K1 though associated with loss of putative mTOR mediated phosphorylation did not correspond with loss of similar phosphorylations in 4EBP1 indicating that S6K1 inhibition was not mediated through inactivation of mTORC1 signaling pathway or its down regulation. PMID:24605337

Reduced chondrocyte proliferation and chondrodysplasia in mice lacking the integrin-linked kinase in chondrocytes.

PubMed

Terpstra, Leonieke; Prud'homme, Josée; Arabian, Alice; Takeda, Shu; Karsenty, Gérard; Dedhar, Shoukat; St-Arnaud, René

2003-07-07

Chondrocyte proliferation and differentiation requires their attachment to the collagen type II-rich matrix of developing bone. This interaction is mediated by integrins and their cytoplasmic effectors, such as the integrin-linked kinase (ILK). To elucidate the molecular mechanisms whereby integrins control these processes, we have specifically inactivated the ILK gene in growth plate chondrocytes using the Cre-lox methodology. Mice carrying an ILK allele flanked by loxP sites (ILK-fl) were crossed to transgenic mice expressing the Cre recombinase under the control of the collagen type II promoter. Inactivation of both copies of the ILK-fl allele lead to a chondrodysplasia characterized by a disorganized growth plate and to dwarfism. Expression of chondrocyte differentiation markers such as collagen type II, collagen type X, Indian hedgehog and the PTH-PTHrP receptor was normal in ILK-deficient growth plates. In contrast, chondrocyte proliferation, assessed by BrdU or proliferating cell nuclear antigen labeling, was markedly reduced in the mutant growth plates. Cell-based assays showed that integrin-mediated adhesion of primary cultures of chondrocytes from mutant animals to collagen type II was impaired. ILK inactivation in chondrocytes resulted in reduced cyclin D1 expression, and this most likely explains the defect in chondrocyte proliferation observed when ILK is inactivated in growth plate cells.

Antimicrobial Blue Light Therapy for Multidrug-Resistant Acinetobacter baumannii Infection in a Mouse Burn Model: Implications for Prophylaxis and Treatment of Combat-related Wound Infections

PubMed Central

Zhang, Yunsong; Zhu, Yingbo; Gupta, Asheesh; Huang, Yingying; Murray, Clinton K.; Vrahas, Mark S.; Sherwood, Margaret E.; Baer, David G.; Hamblin, Michael R.; Dai, Tianhong

2014-01-01

In this study, we investigated the utility of antimicrobial blue light therapy for multidrug-resistant Acinetobacter baumannii infection in a mouse burn model. A bioluminescent clinical isolate of multidrug-resistant A. baumannii was obtained. The susceptibility of A. baumannii to blue light (415 nm)–inactivation was compared in vitro to that of human keratinocytes. Repeated cycles of sublethal inactivation of bacterial by blue light were performed to investigate the potential resistance development of A. baumannii to blue light. A mouse model of third degree burn infected with A. baumannii was developed. A single exposure of blue light was initiated 30 minutes after bacterial inoculation to inactivate A. baumannii in mouse burns. It was found that the multidrug-resistant A. baumannii strain was significantly more susceptible than keratinocytes to blue light inactivation. Transmission electron microscopy revealed blue light–induced ultrastructural damage in A. baumannii cells. Fluorescence spectroscopy suggested that endogenous porphyrins exist in A. baumannii cells. Blue light at an exposure of 55.8 J/cm2 significantly reduced the bacterial burden in mouse burns. No resistance development to blue light inactivation was observed in A. baumannii after 10 cycles of sublethal inactivation of bacteria. No significant DNA damage was detected in mouse skin by means of a skin TUNEL assay after a blue light exposure of 195 J/cm2. PMID:24381206

Effects of haloperidol on Kv4.3 potassium channels.

PubMed

Lee, Hong Joon; Sung, Ki-Wug; Hahn, Sang June

2014-10-05

Haloperidol is commonly used in clinical practice to treat acute and chronic psychosis, but it also has been associated with adverse cardiovascular events. We investigated the effects of haloperidol on Kv4.3 currents stably expressed in CHO cells using a whole-cell patch-clamp technique. Haloperidol did not significantly inhibit the peak amplitude of Kv4.3, but accelerated the decay rate of inactivation of Kv4.3 in a concentration-dependent manner. Thus, the effects of haloperidol on Kv4.3 were estimated from the integral of the Kv4.3 currents during the depolarization pulse. The Kv4.3 was decreased by haloperidol in a concentration-dependent manner with an IC50 value of 3.6 μM. Haloperidol accelerated the decay rate of Kv4.3 inactivation and activation kinetics in a concentration-dependent manner, thereby decreasing the time-to-peak. Haloperidol shifted the voltage dependence of the steady-state activation and inactivation of Kv4.3 in a hyperpolarizing direction. Haloperidol also caused an acceleration of the closed-state inactivation of Kv4.3. Haloperidol produced a use-dependent block of Kv4.3, which was accompanied by a slowing of recovery from the inactivation of Kv4.3. These results suggest that haloperidol blocks Kv4.3 by both interacting with the open state of Kv4.3 channels during depolarization and accelerating the closed-state inactivation at subthreshold membrane potentials. Copyright © 2014 Elsevier B.V. All rights reserved.

Photofrin binds to procaspase-3 and mediates photodynamic treatment-triggered methionine oxidation and inactivation of procaspase-3

PubMed Central

Hsieh, Y-J; Chien, K-Y; Lin, S-Y; Sabu, S; Hsu, R-M; Chi, L-M; Lyu, P-C; Yu, J-S

2012-01-01

Diverse death phenotypes of cancer cells can be induced by Photofrin-mediated photodynamic therapy (PDT), which has a decisive role in eliciting a tumor-specific immunity for long-term tumor control. However, the mechanism(s) underlying this diversity remain elusive. Caspase-3 is a critical factor in determining cell death phenotypes in many physiological settings. Here, we report that Photofrin-PDT can modify and inactivate procaspase-3 in cancer cells. In cells exposed to an external apoptotic trigger, high-dose Photofrin-PDT pretreatment blocked the proteolytic activation of procaspase-3 by its upstream caspase. We generated and purified recombinant procaspase-3-D3A (a mutant without autolysis/autoactivation activity) to explore the underlying mechanism(s). Photofrin could bind directly to procaspase-3-D3A, and Photofrin-PDT-triggered inactivation and modification of procaspase-3-D3A was seen in vitro. Mass spectrometry-based quantitative analysis for post-translational modifications using both 16O/18O- and 14N/15N-labeling strategies revealed that Photofrin-PDT triggered a significant oxidation of procaspase-3-D3A (mainly on Met-27, -39 and -44) in a Photofrin dose-dependent manner, whereas the active site Cys-163 remained largely unmodified. Site-directed mutagenesis experiments further showed that Met-44 has an important role in procaspase-3 activation. Collectively, our results reveal that Met oxidation is a novel mechanism for the Photofrin-PDT-mediated inactivation of procaspase-3, potentially explaining at least some of the complicated cell death phenotypes triggered by PDT. PMID:22785533

Spontaneous and radiation-induced genomic instability in human cell lines differing in cellular TP53 status.

PubMed

Moore, Stephen R; Ritter, Linda E; Gibbons, Catherine F; Grosovsky, Andrew J

2005-10-01

Structural chromosomal rearrangements are commonly observed in tumor karyotypes and in radiation-induced genomic instability. Here we report the effects of TP53 deficiency on karyotypic stability before and after irradiation using related cells and clones differing in cellular TP53 status. The parental cell line, TK6, is a TP53 wild-type human B-lymphoblastoid line with a highly stable karyotype. In the two TK6 derivatives used here, TP53 has been inactivated by biochemical means (expression of HPV16 E6; TK6-5E) or genetic means (allelic inactivation; NH32). Biochemical inactivation of TP53 (TK6-5E) had little effect on the spontaneous karyotype, whereas allelic inactivation of TP53 (NH32) resulted in a modest increase in spontaneous karyotypic instability. After 2 Gy gamma irradiation, the number of unstable clones derived from TP53-deficient cells was significantly elevated compared to the TP53 wild-type counterpart. Extensively destabilized clones were common after irradiation in the set of clones derived from NH32 cells, and one was observed in the set of TK6-5E clones; however, they were never observed in TK6-derived clones. In two of the irradiated NH32 clones, whole chromosomes or chromosome bands were preferentially involved in alterations. These results suggest that genomic instability may differ both quantitatively and qualitatively as a consequence of altered TP53 expression. Some of the results showing repeated and preferential chromosome involvement in aberrations support a model in which instability may be driven by cis mechanisms.

Bacterial spore inactivation induced by cold plasma.

PubMed

Liao, Xinyu; Muhammad, Aliyu Idris; Chen, Shiguo; Hu, Yaqin; Ye, Xingqian; Liu, Donghong; Ding, Tian

2018-04-05

Cold plasma has emerged as a non-thermal technology for microbial inactivation in the food industry over the last decade. Spore-forming microorganisms pose challenges for microbiological safety and for the prevention of food spoilage. Inactivation of spores induced by cold plasma has been reported by several studies. However, the exact mechanism of spore deactivation by cold plasma is poorly understood; therefore, it is difficult to control this process and to optimize cold plasma processing for efficient spore inactivation. In this review, we summarize the factors that affect the resistance of spores to cold plasma, including processing parameters, environmental elements, and spore properties. We then describe possible inactivation targets in spore cells (e.g., outer structure, DNA, and metabolic proteins) that associated with inactivation by cold plasma according to previous studies. Kinetic models of the sporicidal activity of cold plasma have also been described here. A better understanding of the interaction between spores and cold plasma is essential for the development and optimization of cold plasma technology in food the industry.

Formononetin-induced apoptosis of human prostate cancer cells through ERK1/2 mitogen-activated protein kinase inactivation.

PubMed

Ye, Y; Hou, R; Chen, J; Mo, L; Zhang, J; Huang, Y; Mo, Z

2012-04-01

Formononetin is a main active component of red clover plants (Trifolium pratense L.), and is considered as a phytoestrogen. Our previous studies demonstrated that formononetin caused cell cycle arrest at the G0/G1 phase by inactivating insulin-like growth factor 1(IGF1)/IGF1R-phosphatidylinositol 3-kinase (PI3K)/protein kinase B (Akt) pathway in MCF-7 cells. In the present study, we investigated the molecular mechanisms involved in the effect of formononetin on prostate cancer cells. Our results suggested that higher concentrations of formononetin inhibited the proliferation of prostate cancer cells (LNCaP and PC-3), while the most striking effect was observed in LNCaP cells. We further found that formononetin inactivated extracellular signal-regulated kinase1/2 (ERK1/2) mitogen-activated protein kinase (MAPK) signaling pathway in a dose-dependent manner, which resulted in increased the expression levels of BCL2-associated X (Bax) mRNA and protein, and induced apoptosis in LNCaP cells. Thus, we concluded that the induced apoptosis effect of formononetin on human prostate cancer cells was related to ERK1/2 MAPK-Bax pathway. Considering that red clover plants were widely used clinically, our results provided the foundation for future development of different concentrations formononetin for treatment of prostate cancer. © Georg Thieme Verlag KG Stuttgart · New York.

Mechanisms of poliovirus inactivation by the direct and indirect effects of ionizing radiation

DOE Office of Scientific and Technical Information (OSTI.GOV)

Ward, R.L.

1980-08-01

This study was designed to measure the effects of ionizing radiation on poliovirus particles when given under conditions where either direct (in broth) or indirect (in water) effects were predominant. Under direct conditions, inactivation of poliovirus was found to be due primarily to RNA damage, although capsid damage could account for about one-third of the viral inactivation. RNA damage did not appear to be due to strand breakage and therefore was probably caused primarily by base damage or crosslink formation. Capsid damage under direct irradiation conditions did not result in significant alterations of either the sedimentation coefficients or the isoelectricmore » points of the poliovirus particles or detectable modification of the sizes of the viral proteins. It did, however, cause loss of availability to bind to host cells. Under indirect conditions no more than 25% of viral inactivation appeared to be due to RNA damage. However, the sedimentation coefficients and isoelectric points of the viral particles were greatly altered, and their abilities to bind to cells were lost at about three-fourths the rate of loss of infectivity. Capsid damage in this case did result in changes in the sizes of capsid proteins. Therefore, the majority of the radiation inactivation under indirect conditions appeared to be due to protein damage.« less

Bioprocessing development: Immune/cellular applications: Anti-Ig autoantibody and complement-mediated destruction of neoplastic cells

NASA Technical Reports Server (NTRS)

Twomey, J. J.

1976-01-01

This space bioprocessing contract effort was comprised of four general objectives. These were: (1) the evaluation of current separation processes, (2) the identification of problems relevant to the separation of important biologicals, (3) the identification of ground-based assay methods needed for pre- and postflight analysis of space bioprocessing separation technology; and (4) the establishment of methods to determine the efficiency of space bioprocessing separation procedures. Immunology was deemed advantageous to study the diversity of cells and cell products involved and the extensive interest being given to their separation. Upon recognition of a cellular or molecular agent as foreign to the body, the immune system becomes activated to produce cells whose function is to destroy that agent and cell products whose function is to inactivate the agent and assist in its destruction. Long after the agent is removed from the body, some cells remain in a state of readiness to continue these destructive actions specifically against that agent should further exposure to it occur. This is the basis of acquired immunity to disease.

Voltage inactivation of Ca2+ entry and secretion associated with N- and P/Q-type but not L-type Ca2+ channels of bovine chromaffin cells

PubMed Central

Villarroya, Mercedes; Olivares, Román; Ruíz, Ana; Cano-Abad, María F; de Pascual, Ricardo; Lomax, Richard B; López, Manuela G; Mayorgas, Inés; Gandía, Luis; García, Antonio G

1999-01-01

In this study we pose the question of why the bovine adrenal medullary chromaffin cell needs various subtypes (L, N, P, Q) of the neuronal high-voltage activated Ca2+ channels to control a given physiological function, i.e. the exocytotic release of catecholamines. One plausible hypothesis is that Ca2+ channel subtypes undergo different patterns of inactivation during cell depolarization. The net Ca2+ uptake (measured using 45Ca2+) into hyperpolarized cells (bathed in a nominally Ca2+-free solution containing 1·2 mM K+) after application of a Ca2+ pulse (5 s exposure to 100 mM K+ and 2 mM Ca2+), amounted to 0·65 ± 0·02 fmol cell−1; in depolarized cells (bathed in nominally Ca2+-free solution containing 100 mM K+) the net Ca2+ uptake was 0·16 ± 0·01 fmol cell−1. This was paralleled by a dramatic reduction of the increase in the cytosolic Ca2+ concentration, [Ca2+]i, caused by Ca2+ pulses applied to fura-2-loaded single cells, from 1181 ± 104 nM in hyperpolarized cells to 115 ± 9 nM in depolarized cells. A similar decrease was observed when studying catecholamine release. Secretion was decreased when K+ concentration was increased from 1·2 to 100 mM; the Ca2+ pulse caused, when comparing the extreme conditions, the secretion of 807 ± 35 nA of catecholamines in hyperpolarized cells and 220 ± 19 nA in depolarized cells. The inactivation by depolarization of Ca2+ entry and secretion occluded the blocking effects of combined ω-conotoxin GVIA (1 μM) and ω-agatoxin IVA (2 μM), thus suggesting that depolarization caused a selective inactivation of the N- and P/Q-type Ca2+ channels. This was strengthened by two additional findings: (i) nifedipine (3 μM), an L-type Ca2+ channel blocker, suppressed the fraction of Ca2+ entry (24 %) and secretion (27 %) left unblocked by depolarization; (ii) FPL64176 (3 μM), an L-type Ca2+ channel ‘activator’, dramatically enhanced the entry of Ca2+ and the secretory response in depolarized cells. In voltage-clamped cells, switching the holding potential from -80 to -40 mV promoted the loss of 80 % of the whole-cell inward Ca2+ channel current carried by 10 mM Ba2+ (IBa). The residual current was blocked by 80 % upon addition of 3 μM nifedipine and dramatically enhanced by 3 μM FPL64176. Thus, it seems that the N- and P/Q-subtypes of calcium channels are more prone to inactivation at depolarizing voltages than the L-subtype. We propose that this different inactivation might occur physiologically during different patterns of action potential firing, triggered by endogenously released acetylcholine under various stressful conditions. PMID:10087342

Inactivation of infectious hematopoietic necrosis virus by low levels of iodine

USGS Publications Warehouse

Batts, William N.; Landolt, Marsha L.; Winton, James R.

1991-01-01

The fish rhabdovirus infectious hematopoietic necrosis virus (IHNV) was rapidly inactivated by extremely low concentrations of iodine in water. A 99.9% virus reduction was obtained in 7.5 s when virus (105PFU/ml) and iodine (0.1 mg/liter, final concentration) were combined in distilled-deionized or hatchery water. Iodine efficacy decreased at pHs greater than 7.5 or when proteinaceous material was added to the water. Bovine serum albumin blocked iodine inactivation of the virus more effectively than did equal concentrations of fetal bovine serum or river sediment. Sodium thiosulfate effectively neutralized free iodine. Powder, iodophor, and crystalline iodine solutions inactivated IHNV equally. Iodine rapidly inactivated IHNV isolates representing each of the five electropherotypes. Under the conditions used in this study, inactivation was not affected by temperature, salinity, or water hardness. When Dworshak National Fish Hatchery water was continuously treated to provide a free iodine concentration of 0.14 mg/liter, a 7.5-s exposure to iodine was sufficient to inactivate 99.9% of the IHNV. Iodine added to water that contained IHNV prevented infection of rainbow trout (Oncorhynchus mykiss) fry. These results suggest that the waterborne route of IHNV transmission can be blocked by adding low iodine concentrations to the water supplies of hatcheries.

Block of Inactivation-deficient Na+ Channels by Local Anesthetics in Stably Transfected Mammalian Cells

PubMed Central

Wang, Sho-Ya; Mitchell, Jane; Moczydlowski, Edward; Wang, Ging Kuo

2004-01-01

According to the classic modulated receptor hypothesis, local anesthetics (LAs) such as benzocaine and lidocaine bind preferentially to fast-inactivated Na+ channels with higher affinities. However, an alternative view suggests that activation of Na+ channels plays a crucial role in promoting high-affinity LA binding and that fast inactivation per se is not a prerequisite for LA preferential binding. We investigated the role of activation in LA action in inactivation-deficient rat muscle Na+ channels (rNav1.4-L435W/L437C/A438W) expressed in stably transfected Hek293 cells. The 50% inhibitory concentrations (IC50) for the open-channel block at +30 mV by lidocaine and benzocaine were 20.9 ± 3.3 μM (n = 5) and 81.7 ± 10.6 μM (n = 5), respectively; both were comparable to inactivated-channel affinities. In comparison, IC50 values for resting-channel block at −140 mV were >12-fold higher than those for open-channel block. With 300 μM benzocaine, rapid time-dependent block (τ ≈ 0.8 ms) of inactivation-deficient Na+ currents occurred at +30 mV, but such a rapid time-dependent block was not evident at −30 mV. The peak current at −30 mV, however, was reduced more severely than that at +30 mV. This phenomenon suggested that the LA block of intermediate closed states took place notably when channel activation was slow. Such closed-channel block also readily accounted for the LA-induced hyperpolarizing shift in the conventional steady-state inactivation measurement. Our data together illustrate that the Na+ channel activation pathway, including most, if not all, transient intermediate closed states and the final open state, promotes high-affinity LA binding. PMID:15545401

Endotoxin inactivation via steam-heat treatment in dilute simethicone emulsions used in biopharmaceutical processes.

PubMed

Britt, Keith A; Galvin, Jeffrey; Gammell, Patrick; Nti-Gyabaah, Joseph; Boras, George; Kolwyck, David; Ramirez, José G; Presente, Esther; Naugle, Gregory

2014-01-01

Simethicone emulsion is used to regulate foaming in cell culture operations in biopharmaceutical processes. It is also a potential source of endotoxin contamination. The inactivation of endotoxins in dilute simethicone emulsions was assessed as a function of time at different steam temperatures using a Limulus amebocyte lysate kinetic chromogenic technique. Endotoxin inactivation from steam-heat treatment was fit to a four-parameter double exponential decay model, which indicated that endotoxin inactivation was biphasic, consisting of fast and slow regimes. In the fast regime, temperature-related effects were dominant. Transitioning into the slow regime, the observed temperature dependence diminished, and concentration-related effects became increasingly significant. The change in the Gibbs free energy moving through the transition state indicated that a large energy barrier must be overcome for endotoxin inactivation to occur. The corresponding Arrhenius pre-exponential factor was >10(12) s(-1) suggesting that endotoxins in aqueous solution exist as aggregates. The disorder associated with the endotoxin inactivation reaction pathway was assessed via the change in entropy moving through the transition state. This quantity was positive indicating that endotoxin inactivation may result from hydrolysis of individual endotoxin molecules, which perturbs the conformation of endotoxin aggregates, thereby modulating the biological activity observed. Steam-heat treatment decreased endotoxin levels by 1-2 logarithm (log) reduction (LRV), which may be practically relevant depending on incoming raw material endotoxin levels. Antifoam efficiency and cell culture performance were negligibly impacted following steam-heat treatment. The results from this study show that steam-heat treatment is a viable endotoxin control strategy that can be implemented to support large-scale biopharmaceutical manufacturing. © 2014 American Institute of Chemical Engineers.

Influenza Virus Inactivation for Studies of Antigenicity and Phenotypic Neuraminidase Inhibitor Resistance Profiling ▿

PubMed Central

Jonges, Marcel; Liu, Wai Ming; van der Vries, Erhard; Jacobi, Ronald; Pronk, Inge; Boog, Claire; Koopmans, Marion; Meijer, Adam; Soethout, Ernst

2010-01-01

Introduction of a new influenza virus in humans urges quick analysis of its virological and immunological characteristics to determine the impact on public health and to develop protective measures for the human population. At present, however, the necessity of executing pandemic influenza virus research under biosafety level 3 (BSL-3) high-containment conditions severely hampers timely characterization of such viruses. We tested heat, formalin, Triton X-100, and β-propiolactone treatments for their potencies in inactivating human influenza A(H3N2) and avian A(H7N3) viruses, as well as seasonal and pandemic A(H1N1) virus isolates, while allowing the specimens to retain their virological and immunological properties. Successful heat inactivation coincided with the loss of hemagglutinin (HA) and neuraminidase (NA) characteristics, and β-propiolactone inactivation reduced the hemagglutination titer and NA activity of the human influenza virus 10-fold or more. Although Triton X-100 treatment resulted in inconsistent HA activity, the NA activities in culture supernatants were enhanced consistently. Nonetheless, formalin treatment permitted the best retention of HA and NA properties. Triton X-100 treatment proved to be the easiest-to-use influenza virus inactivation protocol for application in combination with phenotypic NA inhibitor susceptibility assays, while formalin treatment preserved B-cell and T-cell epitope antigenicity, allowing the detection of both humoral and cellular immune responses. In conclusion, we demonstrated successful influenza virus characterization using formalin- and Triton X-100-inactivated virus samples. Application of these inactivation protocols limits work under BSL-3 conditions to virus culture, thus enabling more timely determination of public health impact and development of protective measures when a new influenza virus, e.g., pandemic A(H1N1)v virus, is introduced in humans. PMID:20089763

Chlorine Dioxide Inactivation of Cryptosporidium parvum Oocysts and Bacterial Spore Indicators

PubMed Central

Chauret, Christian P.; Radziminski, Chris Z.; Lepuil, Michael; Creason, Robin; Andrews, Robert C.

2001-01-01

Cryptosporidium parvum, which is resistant to chlorine concentrations typically used in water treatment, is recognized as a significant waterborne pathogen. Recent studies have demonstrated that chlorine dioxide is a more efficient disinfectant than free chlorine against Cryptosporidium oocysts. It is not known, however, if oocysts from different suppliers are equally sensitive to chlorine dioxide. This study used both a most-probable-number–cell culture infectivity assay and in vitro excystation to evaluate chlorine dioxide inactivation kinetics in laboratory water at pH 8 and 21°C. The two viability methods produced significantly different results (P < 0.05). Products of disinfectant concentration and contact time (Ct values) of 1,000 mg · min/liter were needed to inactivate approximately 0.5 log10 and 2.0 log10 units (99% inactivation) of C. parvum as measured by in vitro excystation and cell infectivity, respectively, suggesting that excystation is not an adequate viability assay. Purified oocysts originating from three different suppliers were evaluated and showed marked differences with respect to their resistance to inactivation when using chlorine dioxide. Ct values of 75, 550, and 1,000 mg · min/liter were required to achieve approximately 2.0 log10 units of inactivation with oocysts from different sources. Finally, the study compared the relationship between easily measured indicators, including Bacillus subtilis (aerobic) spores and Clostridium sporogenes (anaerobic) spores, and C. parvum oocysts. The bacterial spores were found to be more sensitive to chlorine dioxide than C. parvum oocysts and therefore could not be used as direct indicators of C. parvum inactivation for this disinfectant. In conclusion, it is suggested that future studies address issues such as oocyst purification protocols and the genetic diversity of C. parvum, since these factors might affect oocyst disinfection sensitivity. PMID:11425712

Studying the Role of the Mitotic Exit Network in Cytokinesis.

PubMed

Foltman, Magdalena; Sanchez-Diaz, Alberto

2017-01-01

In budding yeast cells, cytokinesis is achieved by the successful division of the cytoplasm into two daughter cells, but the precise mechanisms of cell division and its regulation are still rather poorly understood. The Mitotic Exit Network (MEN) is the signaling cascade that is responsible for the release of Cdc14 phosphatase leading to the inactivation of the kinase activity associated to cyclin-dependent kinases (CDK), which drives exit from mitosis and a rapid and efficient cytokinesis. Mitotic CDK impairs the activation of MEN before anaphase, and activation of MEN in anaphase leads to the inactivation of CDK, which presents a challenge to determine the contribution that each pathway makes to the successful onset of cytokinesis. To determine CDK and MEN contribution to cytokinesis irrespectively of each other, here we present methods to induce cytokinesis after the inactivation of CDK activity in temperature sensitive mutants of the MEN pathway. An array of methods to monitor the cellular events associated with the successful cytokinesis is included.

Ethanol Inactivated Mouse Embryonic Fibroblasts Maintain the Self-Renew and Proliferation of Human Embryonic Stem Cells.

PubMed

Huang, Boxian; Ning, Song; Zhuang, Lili; Jiang, Chunyan; Cui, Yugui; Fan, Guoping; Qin, Lianju; Liu, Jiayin

2015-01-01

Conventionally, mouse embryonic fibroblasts (MEFs) inactivated by mitomycin C or irradiation were applied to support the self-renew and proliferation of human embryonic stem cells (hESCs). To avoid the disadvangtages of mitomycin C and irradiation, here MEFs were treated by ethanol (ET). Our data showed that 10% ET-inactivated MEFs (eiMEFs) could well maintain the self-renew and proliferation of hESCs. hESCs grown on eiMEFs expressed stem cell markers of NANOG, octamer-binding protein 4 (OCT4), stage-specific embryonic antigen-4 (SSEA4) and tumour related antigen-1-81 (TRA-1-81), meanwhile maintained normal karyotype after long time culture. Also, hESCs cocultured with eiMEFs were able to form embryoid body (EB) in vitro and develop teratoma in vivo. Moreover, eiMEFs could keep their nutrient functions after long time cryopreservation. Our results indicate that the application of eiMEF in hESCs culture is safe, economical and convenient, thus is a better choice.

A New Adjuvant Combined with Inactivated Influenza Enhances Specific CD8 T Cell Response in Mice and Decreases Symptoms in Swine Upon Challenge.

PubMed

Bouguyon, Edwige; Goncalves, Elodie; Shevtsov, Alexander; Maisonnasse, Pauline; Remyga, Stepan; Goryushev, Oleg; Deville, Sebastien; Bertho, Nicolas; Ben Arous, Juliette

2015-11-01

Vaccination is the most effective way to control swine influenza virus (SIV) in the field. Classical vaccines are based on inactivated antigens formulated with an oil emulsion or a polymeric adjuvant. Standard adjuvants enhance the humoral response and orient the immune response toward a Th2 response. An important issue is that current vaccines do not protect against new strains. One approach to improve cross-protection is to enhance Th1 and cytotoxic responses. The development of adjuvants orienting the immune response of inactivated vaccines toward Th1/Cytotoxic responses would be highly beneficial. This study shows that the water in oil in water emulsion adjuvant Montanide™ ISA 201 VG allows the induction of anti-influenza CD8 T cell in mice and induces homologous protection against an H1N1 challenge in swine. Such adjuvants that induce both humoral and cell-mediated immunity could improve the protection conferred by SIV vaccines in the field.

The effect of a non-denaturing detergent and a guanidinium-based inactivation agent on the viability of Ebola virus in mock clinical serum samples.

PubMed

Burton, J E; Easterbrook, L; Pitman, J; Anderson, D; Roddy, S; Bailey, D; Vipond, R; Bruce, C B; Roberts, A D

2017-12-01

The 2014 Ebola outbreak in West Africa required the rapid testing of clinical material for the presence of potentially high titre Ebola virus (EBOV). Safe, fast and effective methods for the inactivation of such clinical samples are required so that rapid diagnostic tests including downstream analysis by RT-qPCR or nucleotide sequencing can be carried out. One of the most commonly used guanidinium - based denaturing agents, AVL (Qiagen) has been shown to fully inactivate EBOV once ethanol is added, however this is not compatible with the use of automated nucleic acid extraction systems. Additional inactivation agents need to be identified that can be used in automated systems. A candidate inactivation agent is Triton X-100, a non-denaturing detergent that is frequently used in clinical nucleic acid extraction procedures and has previously been used for inactivation of EBOV. In this study the effect of 0.1% and 1.0% Triton X-100 (final concentration 0.08% and 0.8% respectively) alone and in combination with AVL on the viability of EBOV (10 6 TCID 50 /ml) spiked into commercially available pooled negative human serum was tested. The presence of viable EBOV in the treated samples was assessed by carrying out three serial passages of the samples in Vero E6 cells (37°C, 5% CO 2 , 1 week for each passage). At the end of each passage the cells were observed for evidence of cytopathic effect and samples were taken for rRT-PCR analysis for the presence of EBOV RNA. Before cell culture cytotoxic components of AVL and Triton X-100 were removed from the samples using size exclusion spin column technology or a hydrophobic adsorbent resin. The results of this study showed that EBOV spiked into human serum was not fully inactivated when treated with either 0.1% (v/v) Triton X-100 for 10 mins or 1.0% (v/v) Triton X-100 for 20 mins (final concentrations 0.08% and 0.8% Triton X-100 respectively). AVL alone also did not consistently provide complete inactivation. Samples treated with both AVL and 0.1% Triton X-100 for 10 or 20 mins were shown to be completely inactivated. This treatment is compatible with downstream analysis by RT-qPCR and next generation sequencing. Crown Copyright © 2017. Published by Elsevier B.V. All rights reserved.

Impaired hair follicle morphogenesis and polarized keratinocyte movement upon conditional inactivation of integrin-linked kinase in the epidermis.

PubMed

Nakrieko, Kerry-Ann; Welch, Ian; Dupuis, Holly; Bryce, Dawn; Pajak, Agnieszka; St Arnaud, René; Dedhar, Shoukat; D'Souza, Sudhir J A; Dagnino, Lina

2008-04-01

Integrin-linked kinase (ILK) is key for cell survival, migration, and adhesion, but little is known about its role in epidermal development and homeostasis in vivo. We generated mice with conditional inactivation of the Ilk gene in squamous epithelia. These mice die perinatally and exhibit skin blistering and severe defects in hair follicle morphogenesis, including greatly reduced follicle numbers, failure to progress beyond very early developmental stages, and pronounced defects in follicular keratinocyte proliferation. ILK-deficient epidermis shows abnormalities in adhesion to the basement membrane and in differentiation. ILK-deficient cultured keratinocytes fail to attach and spread efficiently and exhibit multiple abnormalities in actin cytoskeletal organization. Ilk gene inactivation in cultured keratinocytes causes impaired ability to form stable lamellipodia, to directionally migrate, and to polarize. These defects are accompanied by abnormal distribution of active Cdc42 to cell protrusions, as well as reduced activation of Rac1 upon induction of cell migration in scraped keratinocyte monolayers. Significantly, alterations in cell spreading and forward movement in single cells can be rescued by expression of constitutively active Rac1 or RhoG. Our studies underscore a central and distinct role for ILK in hair follicle development and in polarized cell movements, two key aspects of epithelial morphogenesis and function.

Impaired Hair Follicle Morphogenesis and Polarized Keratinocyte Movement upon Conditional Inactivation of Integrin-linked Kinase in the Epidermis

PubMed Central

Nakrieko, Kerry-Ann; Welch, Ian; Dupuis, Holly; Bryce, Dawn; Pajak, Agnieszka; St. Arnaud, René; Dedhar, Shoukat

2008-01-01

Integrin-linked kinase (ILK) is key for cell survival, migration, and adhesion, but little is known about its role in epidermal development and homeostasis in vivo. We generated mice with conditional inactivation of the Ilk gene in squamous epithelia. These mice die perinatally and exhibit skin blistering and severe defects in hair follicle morphogenesis, including greatly reduced follicle numbers, failure to progress beyond very early developmental stages, and pronounced defects in follicular keratinocyte proliferation. ILK-deficient epidermis shows abnormalities in adhesion to the basement membrane and in differentiation. ILK-deficient cultured keratinocytes fail to attach and spread efficiently and exhibit multiple abnormalities in actin cytoskeletal organization. Ilk gene inactivation in cultured keratinocytes causes impaired ability to form stable lamellipodia, to directionally migrate, and to polarize. These defects are accompanied by abnormal distribution of active Cdc42 to cell protrusions, as well as reduced activation of Rac1 upon induction of cell migration in scraped keratinocyte monolayers. Significantly, alterations in cell spreading and forward movement in single cells can be rescued by expression of constitutively active Rac1 or RhoG. Our studies underscore a central and distinct role for ILK in hair follicle development and in polarized cell movements, two key aspects of epithelial morphogenesis and function. PMID:18234842

Integrated viral clearance strategies-reflecting on the present, projecting to the future.

PubMed

Roush, David J

2018-01-20

Viral clearance and inactivation are critical steps in ensuring the safety of biological products derived from mammalian cell culture and are a component of an adventitious agent control strategy which spans both upstream and downstream processes. Although these approaches have been sufficient to support the development of biologics to date, the empirical and semi-quantitative nature of the approach leaves some potential gaps. For example, the concept of performing a quantitative risk assessment for the downstream components of virus safety was introduced in ICH Q5A for XMuLV. An ideal future state would be to perform a similar quantitative risk assessment for a range of viruses based on an assessment of potential virus risk in both upstream and downstream processes. This assessment combined with an integrated control strategy (including monitoring) would be extremely beneficial in minimizing potential adventitious agent risks. Significant progress has been achieved towards this goal in the last several years including recent advances in quantification of virus sequences in cell banks (ADVTIG), development of truly modular or generic viral clearance claims for specific unit operations, enhanced controls of upstream media (HTST/nanofiltration) and the use of RVLP for in-process monitoring. The recent shift towards continuous processing has the potential to enhance the criticality of in-line monitoring and the complexity of viral clearance and inactivation (owing to a wide range of potential 'worst case' viral clearance scenarios). However, gaps exist in, firstly, the ability to quantify potential virus risk levels in process streams in real-time, secondly, mechanistic understanding of virus/chromatography media interactions, and thirdly, mechanistic understanding of virus/filter interactions. Some new technologies may also need to be developed to allow for real-time confirmation of virus inactivation and clearance to support process development (both batch and continuous) and assessment of the impact of process deviations during manufacturing. This review paper provides an overview of the current state of an overall integrated control strategy for upstream and downstream processing and highlights the investments that could be pursued to achieve the future state of a quantitative virus risk assessment for a range of viruses. One potential approach to address these gaps is the use of data mining from large, comprehensive and diverse data sets to establish heuristic rules for virus detection, clearance and inactivation followed by specific hypothesis-driven experiments for cases that fall outside of the normal paradigm. Once this approach reaches a mature state suitable for implementation, there is an opportunity to update regulatory guidance (e.g. ICH Q5A) accordingly. Copyright © 2018 Elsevier Ltd. All rights reserved.

Adapting viral safety assurance strategies to continuous processing of biological products.

PubMed

Johnson, Sarah A; Brown, Matthew R; Lute, Scott C; Brorson, Kurt A

2017-01-01

There has been a recent drive in commercial large-scale production of biotechnology products to convert current batch mode processing to continuous processing manufacturing. There have been reports of model systems capable of adapting and linking upstream and downstream technologies into a continuous manufacturing pipeline. However, in many of these proposed continuous processing model systems, viral safety has not been comprehensively addressed. Viral safety and detection is a highly important and often expensive regulatory requirement for any new biological product. To ensure success in the adaption of continuous processing to large-scale production, there is a need to consider the development of approaches that allow for seamless incorporation of viral testing and clearance/inactivation methods. In this review, we outline potential strategies to apply current viral testing and clearance/inactivation technologies to continuous processing, as well as modifications of existing unit operations to ensure the successful integration of viral clearance into the continuous processing of biological products. Biotechnol. Bioeng. 2017;114: 21-32. © 2016 Wiley Periodicals, Inc. © 2016 Wiley Periodicals, Inc.

Difunctional bacteriophage conjugated with photosensitizers for Candida albicans-targeting photodynamic inactivation.

PubMed

Dong, Shuai; Shi, Hongxi; Zhang, Xintong; Chen, Xi; Cao, Donghui; Mao, Chuanbin; Gao, Xiang; Wang, Li

2018-01-01

Candida albicans is the most prevalent fungal pathogen of the human microbiota, causing infections ranging from superficial infections of the skin to life-threatening systemic infections. Due to the increasing occurrence of antibiotic-resistant C. albicans strains, new approaches to control this pathogen are needed. Photodynamic inactivation is an emerging alternative to treat infections based on the interactions between visible light and photosensitisers, in which pheophorbide a (PPA) is a chlorophyll-based photosensitizer that could induce cell death after light irradiation. Due to PPA's phototoxicity and low efficiency, the main challenge is to implement photosensitizer cell targeting and attacking. In this study, PPA was conjugated with JM-phage by EDC/NHS crosslinking. UV-Vis spectra was used to determine the optimum conjugation percentages of PPA and JM-phage complex for photodynamic inactivation. After photodynamic inactivation, the efficacy of PPA-JM-phage was assessed by performing in vitro experiments, such as MTS assay, scanning electron microscopy, measurement of dysfunctional mitochondria, ROS accumulation, S cell arrest and apoptotic pathway. A single-chain variable-fragment phage (JM) with high affinity to MP65 was screened from human single-fold single-chain variable-fragment libraries and designed as a binding target for C. albicans cells. Subsequently, PPa was integrated into JM phage to generate a combined nanoscale material, which was called PPA-JM-phage. After photodynamic inactivation, the growth of C. albicans was inhibited by PPA-JM-phage and apoptosis was observed. Scanning electron microscopy analysis revealed shrinking and rupturing of C. albicans . We also found that depolarization of mitochondrial membrane potential was decreased and intracellular reactive oxygen species levels were elevated significantly in C. albicans inhibited by PPA-JM-phage. Additionally, PPA-JM-phage also lead to S-phase arrest, and metacaspase activation resulting from mitochondrial dysfunction was also found to be involved in C. albicans apoptosis. PPa-JM-phage may induce C. albicans apoptosis through a caspase-dependent pathway and the results herein shed light on the potential application of phtototherapeutic nanostructures in fungal inactivation.

Difunctional bacteriophage conjugated with photosensitizers for Candida albicans-targeting photodynamic inactivation

PubMed Central

Dong, Shuai; Shi, Hongxi; Zhang, Xintong; Chen, Xi; Cao, Donghui; Mao, Chuanbin; Gao, Xiang; Wang, Li

2018-01-01

Background Candida albicans is the most prevalent fungal pathogen of the human microbiota, causing infections ranging from superficial infections of the skin to life-threatening systemic infections. Due to the increasing occurrence of antibiotic-resistant C. albicans strains, new approaches to control this pathogen are needed. Photodynamic inactivation is an emerging alternative to treat infections based on the interactions between visible light and photosensitisers, in which pheophorbide a (PPA) is a chlorophyll-based photosensitizer that could induce cell death after light irradiation. Due to PPA’s phototoxicity and low efficiency, the main challenge is to implement photosensitizer cell targeting and attacking. Methods In this study, PPA was conjugated with JM-phage by EDC/NHS crosslinking. UV-Vis spectra was used to determine the optimum conjugation percentages of PPA and JM-phage complex for photodynamic inactivation. After photodynamic inactivation, the efficacy of PPA-JM-phage was assessed by performing in vitro experiments, such as MTS assay, scanning electron microscopy, measurement of dysfunctional mitochondria, ROS accumulation, S cell arrest and apoptotic pathway. Results A single-chain variable-fragment phage (JM) with high affinity to MP65 was screened from human single-fold single-chain variable-fragment libraries and designed as a binding target for C. albicans cells. Subsequently, PPa was integrated into JM phage to generate a combined nanoscale material, which was called PPA-JM-phage. After photodynamic inactivation, the growth of C. albicans was inhibited by PPA-JM-phage and apoptosis was observed. Scanning electron microscopy analysis revealed shrinking and rupturing of C. albicans. We also found that depolarization of mitochondrial membrane potential was decreased and intracellular reactive oxygen species levels were elevated significantly in C. albicans inhibited by PPA-JM-phage. Additionally, PPA-JM-phage also lead to S-phase arrest, and metacaspase activation resulting from mitochondrial dysfunction was also found to be involved in C. albicans apoptosis. Conclusion PPa-JM-phage may induce C. albicans apoptosis through a caspase-dependent pathway and the results herein shed light on the potential application of phtototherapeutic nanostructures in fungal inactivation. PMID:29692614

Knockout of the BK β2 subunit abolishes inactivation of BK currents in mouse adrenal chromaffin cells and results in slow-wave burst activity

PubMed Central

Martinez-Espinosa, Pedro L.; Yang, Chengtao; Gonzalez-Perez, Vivian; Xia, Xiao-Ming

2014-01-01

Rat and mouse adrenal medullary chromaffin cells (CCs) express an inactivating BK current. This inactivation is thought to arise from the assembly of up to four β2 auxiliary subunits (encoded by the kcnmb2 gene) with a tetramer of pore-forming Slo1 α subunits. Although the physiological consequences of inactivation remain unclear, differences in depolarization-evoked firing among CCs have been proposed to arise from the ability of β2 subunits to shift the range of BK channel activation. To investigate the role of BK channels containing β2 subunits, we generated mice in which the gene encoding β2 was deleted (β2 knockout [KO]). Comparison of proteins from wild-type (WT) and β2 KO mice allowed unambiguous demonstration of the presence of β2 subunit in various tissues and its coassembly with the Slo1 α subunit. We compared current properties and cell firing properties of WT and β2 KO CCs in slices and found that β2 KO abolished inactivation, slowed action potential (AP) repolarization, and, during constant current injection, decreased AP firing. These results support the idea that the β2-mediated shift of the BK channel activation range affects repetitive firing and AP properties. Unexpectedly, CCs from β2 KO mice show an increased tendency toward spontaneous burst firing, suggesting that the particular properties of BK channels in the absence of β2 subunits may predispose to burst firing. PMID:25267913

Patulin reduction in apple juice by inactivated Alicyclobacillus spp.

PubMed

Yuan, Y; Wang, X; Hatab, S; Wang, Z; Wang, Y; Luo, Y; Yue, T

2014-12-01

This study aimed to investigate the reduction of patulin (PAT) in apple juice by 12 inactivated Alicyclobacillus strains. The reduction rate of PAT by each strain was determined by high-performance liquid chromatography (HPLC). The results indicated that the removal of PAT was strain specific. Alicyclobacillus acidoterrestris 92 and A. acidoterrestris 96 were the most effective ones among the 12 tested strains in the removal of PAT. Therefore, these two strains were selected to study the effects of incubation time, initial PAT concentration and bacteria powder amount on PAT removal abilities of Alicyclobacillus. The highest PAT reduction rates of 88·8 and 81·6% were achieved after 24-h incubation with initial PAT concentration of 100 μg l(-1) and bacteria powder amount of 40 g l(-1) , respectively. Moreover, it was found that the treatment by these 12 inactivated Alicyclobacillus strains had no negative effect on the quality parameters of apple juice. Similar assays were performed in supermarket apple juice, where inactivated Alicyclobacillus cells could efficiently reduce PAT content. Taken together, these data suggest the possible application of this strategy as a means to detoxify PAT-contaminated juices. Inactivated Alicyclobacillus cells can efficiently reduce patulin concentration in apple juice. It provides a theoretical foundation for recycling of Alicyclobacillus cells from spoiled apple juice to reduce the source of pollution and the cost of juice industry. This is the first report on the use of Alicyclobacillus to remove patulin from apple juice. © 2014 The Society for Applied Microbiology.

Trivalent inactivated influenza vaccine is not associated with sickle cell crises in children.

PubMed

Hambidge, Simon J; Ross, Colleen; Glanz, Jason; McClure, David; Daley, Matthew F; Xu, Stan; Shoup, Jo Ann; Narwaney, Komal; Baggs, James; Weintraub, Eric

2012-01-01

Children with sickle cell disease are considered at high risk for complications from influenza infection and are recommended to receive annual influenza vaccination. However, data on the safety of influenza vaccination in children with sickle cell anemia are sparse. Using a retrospective cohort of children aged 6 months to 17 years in 8 managed care organizations that comprise the Vaccine Safety Datalink and who had a diagnosis of sickle cell anemia from 1999 to 2006, we conducted matched case-control and self-controlled case series studies to examine the association of trivalent inactivated influenza vaccination with hospitalization for sickle cell crisis in the 2 weeks after vaccination. From an original pool of 1085 pediatric subjects with a diagnosis of sickle cell anemia, we identified 179 children with at least 1 sickle cell crisis during any influenza season (October 1-March 31). In the matched case-control study (matching on age category, gender, Vaccine Safety Datalink site, and season), the odds ratio of hospitalization for a crisis in vaccinated compared with unvaccinated children was not significant: 1.3 (95% confidence interval 0.8-2.2). In the self-controlled case series study of hospitalized cases, the incident rate ratio for hospitalization with sickle cell crisis in the 2 weeks after trivalent inactivated influenza vaccination was also not significant: 1.2 (95% confidence interval 0.75-1.95). This large cohort study did not find an association of influenza vaccination and hospitalization for sickle cell crises in children with sickle cell anemia.

Design and operation of a continuous integrated monoclonal antibody production process.

PubMed

Steinebach, Fabian; Ulmer, Nicole; Wolf, Moritz; Decker, Lara; Schneider, Veronika; Wälchli, Ruben; Karst, Daniel; Souquet, Jonathan; Morbidelli, Massimo

2017-09-01

The realization of an end-to-end integrated continuous lab-scale process for monoclonal antibody manufacturing is described. For this, a continuous cultivation with filter-based cell-retention, a continuous two column capture process, a virus inactivation step, a semi-continuous polishing step (twin-column MCSGP), and a batch-wise flow-through polishing step were integrated and operated together. In each unit, the implementation of internal recycle loops allows to improve the performance: (a) in the bioreactor, to simultaneously increase the cell density and volumetric productivity, (b) in the capture process, to achieve improved capacity utilization at high productivity and yield, and (c) in the MCSGP process, to overcome the purity-yield trade-off of classical batch-wise bind-elute polishing steps. Furthermore, the design principles, which allow the direct connection of these steps, some at steady state and some at cyclic steady state, as well as straight-through processing, are discussed. The setup was operated for the continuous production of a commercial monoclonal antibody, resulting in stable operation and uniform product quality over the 17 cycles of the end-to-end integration. The steady-state operation was fully characterized by analyzing at the outlet of each unit at steady state the product titer as well as the process (HCP, DNA, leached Protein A) and product (aggregates, fragments) related impurities. © 2017 American Institute of Chemical Engineers Biotechnol. Prog., 33:1303-1313, 2017. © 2017 American Institute of Chemical Engineers.

Adjuvant effects of invariant NKT cell ligand potentiates the innate and adaptive immunity to an inactivated H1N1 swine influenza virus vaccine in pigs.

PubMed

Dwivedi, Varun; Manickam, Cordelia; Dhakal, Santosh; Binjawadagi, Basavaraj; Ouyang, Kang; Hiremath, Jagadish; Khatri, Mahesh; Hague, Jacquelyn Gervay; Lee, Chang Won; Renukaradhya, Gourapura J

2016-04-15

Pigs are considered as the source of some of the emerging human flu viruses. Inactivated swine influenza virus (SwIV) vaccine has been in use in the US swine herds, but it failed to control the flu outbreaks. The main reason has been attributed to lack of induction of strong local mucosal immunity in the respiratory tract. Invariant natural killer T (iNKT) cell is a unique T cell subset, and activation of iNKT cell using its ligand α-Galactosylceramide (α-GalCer) has been shown to potentiate the cross-protective immunity to inactivated influenza virus vaccine candidates in mice. Recently, we discovered iNKT cell in pig and demonstrated its activation using α-GalCer. In this study, we evaluated the efficacy of an inactivated H1N1 SwIV coadministered with α-GalCer intranasally against a homologous viral challenge. Our results demonstrated the potent adjuvant effects of α-GalCer in potentiating both innate and adaptive immune responses to SwIV Ags in the lungs of pigs, which resulted in reduction in the lung viral load by 3 logs compared to without adjuvant. Immunologically, in the lungs of pigs vaccinated with α-GalCer an increased virus specific IgA response, IFN-α secretion and NK cell-cytotoxicity was observed. In addition, iNKT cell-stimulation enhanced the secretion of Th1 cytokines (IFN-γ and IL-12) and reduced the production of immunosuppressive cytokines (IL-10 and TGF-β) in the lungs of pigs⋅ In conclusion, we demonstrated for the first time iNKT cell adjuvant effects in pigs to SwIV Ags through augmenting the innate and adaptive immune responses in the respiratory tract. Copyright © 2016 Elsevier B.V. All rights reserved.

Inactivation of the cloned potassium channel mouse Kv1.1 by the human Kv3.4 'ball' peptide and its chemical modification.

PubMed Central

Stephens, G J; Robertson, B

1995-01-01

1. This study used the whole-cell patch clamp technique to investigate the action of a 28-mer 'inactivation peptide' based on part of the N-terminal sequence of the human Kv3.4 K+ channel (hKv3.4 peptide) on the cloned mouse brain K+ channel mKv1.1 expressed in Chinese hamster ovary (CHO) cells, and compared this with the inactivation produced by Shaker B inactivation peptide (ShB peptide). 2. Inclusion of the hKv3.4 peptide in the patch electrode (320 microM) transformed non-inactivating mKv1.1 into a rapidly inactivating current. The voltage dependence of time constants of decay and steady-state inactivation induced by hKv3.4 peptide were characteristic of an 'A-type' K+ current. 3. The hKv3.4 peptide had no effect on the voltage dependence of activation of mKv1.1, with a mid-point of activation of -8 mV, and a slope factor of 15 mV. Steady-state inactivation curves had a mid-point of inactivation of -36 mV and a slope factor of -7 mV; the time constant of recovery from inactivation at -90 mV was 1.3 s. 4. The chemical modification reagents N-bromoacetamide (NBA, 100 microM) and chloramine-T (CL-T, 500 microM) had no effect on the fast inactivation of mKv1.1 induced by ShB peptide. In contrast, the inactivation caused by hKv3.4 peptide was removed by brief exposure to NBA and CL-T. 5. Chemical modification resulted in a hyperpolarizing shift of -8 mV (CL-T) and -11 mV (NBA) in the voltage dependence of activation of mKv1.1 in the presence of hKv3.4 peptide. 6. Chemical modification was critically dependent on the presence of a cysteine residue at position 6, and not position 24, of hKv3.4 peptide. 7. NBA and CL-T caused only a slight inhibition of unmodified mKv1.1 current with no significant effect on the voltage dependence of mKv1.1 activation, and also had no effect on channel deactivation at -90 mV. 8. Chemical modification experiments were consistent with a selective action on the hKv3.4 peptide itself, specifically at the cysteine residue at position 6. PMID:7602512

A Caprine Herpesvirus 1 Vaccine Adjuvanted with MF59™ Protects against Vaginal Infection and Interferes with the Establishment of Latency in Goats

PubMed Central

Marinaro, Mariarosaria; Rezza, Giovanni; Del Giudice, Giuseppe; Colao, Valeriana; Tarsitano, Elvira; Camero, Michele; Losurdo, Michele; Buonavoglia, Canio; Tempesta, Maria

2012-01-01

The immunogenicity and the efficacy of a beta-propiolactone-inactivated caprine herpesvirus 1 (CpHV-1) vaccine adjuvanted with MF59™ were tested in goats. Following two subcutaneous immunizations, goats developed high titers of CpHV-1-specific serum and vaginal IgG and high serum virus neutralization (VN) titers. Peripheral blood mononuclear cells (PBMC) stimulated in vitro with inactivated CpHV-1 produced high levels of soluble IFN-gamma and exhibited high frequencies of IFN-gamma producing cells while soluble IL-4 was undetectable. On the other hand, control goats receiving the inactivated CpHV-1 vaccine without adjuvant produced only low serum antibody responses. A vaginal challenge with virulent CpHV-1 was performed in all vaccinated goats and in naïve goats to assess the efficacy of the two vaccines. Vaginal disease was not detected in goats vaccinated with inactivated CpHV-1 plus MF59™ and these animals had undetectable levels of infectious challenge virus in their vaginal washes. Goats vaccinated with inactivated CpHV-1 in the absence of adjuvant exhibited a less severe disease when compared to naïve goats but shed titers of challenge virus that were similar to those of naïve goats. Detection and quantitation of latent CpHV-1 DNA in sacral ganglia in challenged goats revealed that the inactivated CpHV-1 plus MF59™ vaccine was able to significantly reduce the latent viral load when compared either to the naïve goats or to the goats vaccinated with inactivated CpHV-1 in the absence of adjuvant. Thus, a vaccine composed of inactivated CpHV-1 plus MF59™ as adjuvant was strongly immunogenic and induced effective immunity against vaginal CpHV-1 infection in goats. PMID:22511971

TnBP⁄Triton X-45 Treatment of Plasma for Transfusion Efficiently Inactivates Hepatitis C Virus

PubMed Central

Chou, Ming-Li; Burnouf, Thierry; Chang, Shun-Pang; Hung, Ting-Chun; Lin, Chun-Ching; Richardson, Christopher D.; Lin, Liang-Tzung

2015-01-01

Risk of transmission of hepatitis C virus (HCV) by clinical plasma remains high in countries with a high prevalence of hepatitis C, justifying the implementation of viral inactivation treatments. In this study, we assessed the extent of inactivation of HCV during minipool solvent/detergent (SD; 1% TnBP / 1% Triton X-45) treatment of human plasma. Luciferase-tagged infectious cell culture-derived HCV (HCVcc) particles were used to spike human plasma prior to treatment by SD at 31 ± 0.5°C for 30 min. Samples were taken before and after SD treatment and filtered on a Sep-Pak Plus C18 cartridge to remove the SD agents. Risk of cytotoxicity was assessed by XTT cell viability assay. Viral infectivity was analyzed based on the luciferase signals, 50% tissue culture infectious dose viral titer, and immunofluorescence staining for HCV NS5A protein. Total protein, cholesterol, and triglyceride contents were determined before and after SD treatment and C18 cartridge filtration. Binding analysis, using patient-derived HCV clinical isolates, was also examined to validate the efficacy of the inactivation by SD. SD treatment effectively inactivated HCVcc within 30 min, as demonstrated by the baseline level of reporter signals, total loss of viral infectivity, and absence of viral protein NS5A. SD specifically targeted HCV particles to render them inactive, with essentially no effect on plasma protein content and hemostatic function. More importantly, the efficacy of the SD inactivation method was confirmed against various genotypes of patient-derived HCV clinical isolates and against HCVcc infection of primary human hepatocytes. Therefore, treatment by 1% TnBP / 1% Triton X-45 at 31°C is highly efficient to inactivate HCV in plasma for transfusion, showing its capacity to enhance the safety of therapeutic plasma products. We propose that the methodology used here to study HCV infectivity can be valuable in the validation of viral inactivation and removal processes of human plasma-derived products. PMID:25658612

A novel CD4-conjugated ultraviolet light-activated photocatalyst inactivates HIV-1 and SIV efficiently.

PubMed

Yamaguchi, Koushi; Sugiyama, Takahiro; Kato, Shinji; Kondo, Yoichi; Ageyama, Naohide; Kanekiyo, Masaru; Iwata, Misao; Koyanagi, Yoshio; Yamamoto, Naoki; Honda, Mitsuo

2008-08-01

In this study, we found that the electric potential derived from the redox reaction of ultraviolet (UV)-illuminated CD4-conjugated titanium dioxide (TiO2) inactivated a wide range of high-titered primary HIV-1 isolates, regardless of virus co-receptor usage or genetic clade. In vitro incubation of HIV-1 isolates with CD4-conjugated TiO2 (CD4-TiO2) followed by UV illumination led to inhibition of viral infectivity in both H9 cells and peripheral blood mononuclear cells as well as to the complete inactivation of plasma virions from HIV-1-infected individuals. Treatment with a newly established extra-corporeal circulation system with the photocatalyst in rhesus macaques completely inactivated plasma virus in the system and effectively reduced the infectious plasma viral load. Furthermore, plasma viremia and infectious viral loads were controlled following a second therapeutic photocatalyst treatment during primary SIV(mac239) infection of macaques. Our findings suggest that this therapeutic immunophysical strategy may help control human immunodeficiency viral infection in vivo.

TAK1 modulates satellite stem cell homeostasis and skeletal muscle repair

PubMed Central

Ogura, Yuji; Hindi, Sajedah M.; Sato, Shuichi; Xiong, Guangyan; Akira, Shizuo; Kumar, Ashok

2015-01-01

Satellite cells are resident adult stem cells that are required for regeneration of skeletal muscle. However, signalling mechanisms that regulate satellite cell function are less understood. Here we demonstrate that transforming growth factor-β-activated kinase 1 (TAK1) is important in satellite stem cell homeostasis and function. Inactivation of TAK1 in satellite cells inhibits muscle regeneration in adult mice. TAK1 is essential for satellite cell proliferation and its inactivation causes precocious differentiation. Moreover, TAK1-deficient satellite cells exhibit increased oxidative stress and undergo spontaneous cell death, primarily through necroptosis. TAK1 is required for the activation of NF-κB and JNK in satellite cells. Forced activation of NF-κB improves survival and proliferation of TAK1-deficient satellite cells. Furthermore, TAK1-mediated activation of JNK is essential to prevent oxidative stress and precocious differentiation of satellite cells. Collectively, our study suggests that TAK1 is required for maintaining the pool of satellite stem cells and for regenerative myogenesis. PMID:26648529

Tomato Fruit Cell Wall 1

PubMed Central

Koch, James L.; Nevins, Donald J.

1989-01-01

Cell wall isolation procedures were evaluated to determine their effect on the total pectin content and the degree of methylesterification of tomato (Lycopersicon esculentum L.) fruit cell walls. Water homogenates liberate substantial amounts of buffer soluble uronic acid, 5.2 milligrams uronic acid/100 milligrams wall. Solubilization appears to be a consequence of autohydrolysis mediated by polygalacturonase II, isoenzymes A and B, since the uronic acid release from the wall residue can be suppressed by homogenization in the presence of 50% ethanol followed by heating. The extent of methylesterification in heat-inactivated cell walls, 94 mole%, was significantly greater than with water homogenates, 56 mole%. The results suggest that autohydrolysis, mediated by cell wall-associated enzymes, accounts for the solubilization of tomato fruit pectin in vitro. Endogenous enzymes also account for a decrease in the methylesterification during the cell wall preparation. The heat-inactivated cell wall preparation was superior to the other methods studied since it reduces β-elimination during heating and inactivates constitutive enzymes that may modify pectin structure. This heat-inactivated cell wall preparation was used in subsequent enzymatic analysis of the pectin structure. Purified tomato fruit polygalacturonase and partially purified pectinmethylesterase were used to assess changes in constitutive substrates during tomato fruit ripening. Polygalacturonase treatment of heat-inactivated cell walls from mature green and breaker stages released 14% of the uronic acid. The extent of the release of polyuronides by polygalacturonase was fruit development stage dependent. At the turning stage, 21% of the pectin fraction was released, a value which increased to a maximum of 28% of the uronides at the red ripe stage. Pretreatment of the walls with purified tomato pectinesterase rendered walls from all ripening stages equally susceptible to polygalacturonase. Quantitatively, the release of uronides by polygalacturonase from all pectinesterase treated cell walls was equivalent to polygalacturonase treatment of walls at the ripe stage. Uronide polymers released by polygalacturonase contain galacturonic acid, rhamnose, galactose, arabinose, xylose, and glucose. As a function of development, an increase in the release of galacturonic acid and rhamnose was observed (40 and 6% of these polymers at the mature green stage to 54 and 15% at the red ripe stage, respectively). The amount of galactose and arabinose released by exogenous polygalacturonase decreased during development (41 and 11% from walls of mature green fruit to 11 and 6% at the red ripe stage, respectively). Minor amounts of glucose and xylose released from the wall by exogenous polygalacturonase (4-7%) remained constant throughout fruit development. PMID:16667142

Mechanism of poliovirus inactivation by ammonia

DOE Office of Scientific and Technical Information (OSTI.GOV)

Ward, R.L.

1978-05-01

Poliovirus inactivation by ammonia causes a slight reduction in the sedimentation coefficients of viral particles, but has no detectable effect on either the electrophoretic pattern of viral capsid proteins or the isoelectric points of inactivated particles. These virions still attach to cells, but are unable to repress host translation or stimulate the synthesis of detectable amounts of viral RNA. Although ammonia has no detectable effect on naked poliovirus RNA, it causes cleavage of this RNA when still within viral particles. Therefore, the RNA genome appears to be the only component of poliovirus significantly affected by ammonia.

Inactivation of Herpes Simplex Viruses by Nonionic Surfactants

PubMed Central

Asculai, Samuel S.; Weis, Margaret T.; Rancourt, Martha W.; Kupferberg, A. B.

1978-01-01

Nonionic surface-active agents possessing ether or amide linkages between the hydrophillic and hydrophobic portions of the molecule rapidly inactivated the infectivity of herpes simplex viruses. The activity stemmed from the ability of nonionic surfactants to dissolve lipid-containing membranes. This was confirmed by observing surfactant destruction of mammalian cell plasma membranes and herpes simplex virus envelopes. Proprietary vaginal contraceptive formulations containing nonionic surfactants also inactivated herpes simplex virus infectivity. This observation suggests that nonionic surfactants in appropriate formulation could effectively prevent herpes simplex virus transmission. Images PMID:208460

Increased skewing of X chromosome inactivation in Rett syndrome patients and their mothers.

PubMed

Knudsen, Gun Peggy S; Neilson, Tracey C S; Pedersen, June; Kerr, Alison; Schwartz, Marianne; Hulten, Maj; Bailey, Mark E S; Orstavik, Karen Helene

2006-11-01

Rett syndrome is a largely sporadic, X-linked neurological disorder with a characteristic phenotype, but which exhibits substantial phenotypic variability. This variability has been partly attributed to an effect of X chromosome inactivation (XCI). There have been conflicting reports regarding incidence of skewed X inactivation in Rett syndrome. In rare familial cases of Rett syndrome, favourably skewed X inactivation has been found in phenotypically normal carrier mothers. We have investigated the X inactivation pattern in DNA from blood and buccal cells of sporadic Rett patients (n=96) and their mothers (n=84). The mean degree of skewing in blood was higher in patients (70.7%) than controls (64.9%). Unexpectedly, the mothers of these patients also had a higher mean degree of skewing in blood (70.8%) than controls. In accordance with these findings, the frequency of skewed (XCI > or =80%) X inactivation in blood was also higher in both patients (25%) and mothers (30%) than in controls (11%). To test whether the Rett patients with skewed X inactivation were daughters of skewed mothers, 49 mother-daughter pairs were analysed. Of 14 patients with skewed X inactivation, only three had a mother with skewed X inactivation. Among patients, mildly affected cases were shown to be more skewed than more severely affected cases, and there was a trend towards preferential inactivation of the paternally inherited X chromosome in skewed cases. These findings, particularly the greater degree of X inactivation skewing in Rett syndrome patients, are of potential significance in the analysis of genotype-phenotype correlations in Rett syndrome.

Activity map of the tammar X chromosome shows that marsupial X inactivation is incomplete and escape is stochastic

PubMed Central

2010-01-01

Background X chromosome inactivation is a spectacular example of epigenetic silencing. In order to deduce how this complex system evolved, we examined X inactivation in a model marsupial, the tammar wallaby (Macropus eugenii). In marsupials, X inactivation is known to be paternal, incomplete and tissue-specific, and occurs in the absence of an XIST orthologue. Results We examined expression of X-borne genes using quantitative PCR, revealing a range of dosage compensation for different loci. To assess the frequency of 1X- or 2X-active fibroblasts, we investigated expression of 32 X-borne genes at the cellular level using RNA-FISH. In female fibroblasts, two-color RNA-FISH showed that genes were coordinately expressed from the same X (active X) in nuclei in which both loci were inactivated. However, loci on the other X escape inactivation independently, with each locus showing a characteristic frequency of 1X-active and 2X-active nuclei, equivalent to stochastic escape. We constructed an activity map of the tammar wallaby inactive X chromosome, which identified no relationship between gene location and extent of inactivation, nor any correlation with the presence or absence of a Y-borne paralog. Conclusions In the tammar wallaby, one X (presumed to be maternal) is expressed in all cells, but genes on the other (paternal) X escape inactivation independently and at characteristic frequencies. The paternal and incomplete X chromosome inactivation in marsupials, with stochastic escape, appears to be quite distinct from the X chromosome inactivation process in eutherians. We find no evidence for a polar spread of inactivation from an X inactivation center. PMID:21182760

A novel combined solar pasteurizer/TiO2 continuous-flow reactor for decontamination and disinfection of drinking water.

PubMed

Monteagudo, José María; Durán, Antonio; Martín, Israel San; Acevedo, Alba María

2017-02-01

A new combined solar plant including an annular continuous-flow compound parabolic collector (CPC) reactor and a pasteurization system was designed, built, and tested for simultaneous drinking water disinfection and chemical decontamination. The plant did not use pumps and had no electricity costs. First, water continuously flowed through the CPC reactor and then entered the pasteurizer. The temperature and water flow from the plant effluent were controlled by a thermostatic valve located at the pasteurizer outlet that opened at 80 °C. The pasteurization process was simulated by studying the effect of heat treatment on the death kinetic parameters (D and z values) of Escherichia coli K12 (CECT 4624). 99.1% bacteria photo-inactivation was reached in the TiO 2 -CPC system (0.60 mg cm -2 TiO 2 ), and chemical decontamination in terms of antipyrine degradation increased with increasing residence time in the TiO 2 -CPC system, reaching 70% degradation. The generation of hydroxyl radicals (between 100 and 400 nmol L -1 ) was a key factor in the CPC system efficiency. Total thermal bacteria inactivation was attained after pasteurization in all cases. Chemical degradation and bacterial photo-inactivation in the TiO 2 -CPC system were improved with the addition of 150 mg L -1 of H 2 O 2 , which generated approximately 2000-2300 nmol L -1 of HO ● radicals. Finally, chemical degradation and bacterial photo-inactivation kinetic modelling in the annular CPC photoreactor were evaluated. The effect of the superficial liquid velocity on the overall rate constant was also studied. Both antipyrine degradation and E. coli photo-inactivation were found to be controlled by the catalyst surface reaction rate. Copyright © 2016 Elsevier Ltd. All rights reserved.

Molecular physiology and modulation of somatodendritic A-type potassium channels.

PubMed

Jerng, Henry H; Pfaffinger, Paul J; Covarrubias, Manuel

2004-12-01

The somatodendritic subthreshold A-type K+ current (ISA) in nerve cells is a critical component of the ensemble of voltage-gated ionic currents that determine somatodendritic signal integration. The underlying K+ channel belongs to the Shal subfamily of voltage-gated K+ channels. Most Shal channels across the animal kingdom share a high degree of structural conservation, operate in the subthreshold range of membrane potentials, and exhibit relatively fast inactivation and recovery from inactivation. Mammalian Shal K+ channels (Kv4) undergo preferential closed-state inactivation with features that are generally inconsistent with the classical mechanisms of inactivation typical of Shaker K+ channels. Here, we review (1) the physiological and genetic properties of ISA, 2 the molecular mechanisms of Kv4 inactivation and its remodeling by a family of soluble calcium-binding proteins (KChIPs) and a membrane-bound dipeptidase-like protein (DPPX), and (3) the modulation of Kv4 channels by protein phosphorylation.

The Sleep-inducing Lipid Oleamide Deconvolutes Gap Junction Communication and Calcium Wave Transmission in Glial Cells

PubMed Central

Guan, Xiaojun; Cravatt, Benjamin F.; Ehring, George R.; Hall, James E.; Boger, Dale L.; Lerner, Richard A.; Gilula, Norton B.

1997-01-01

Oleamide is a sleep-inducing lipid originally isolated from the cerebrospinal fluid of sleep-deprived cats. Oleamide was found to potently and selectively inactivate gap junction–mediated communication between rat glial cells. In contrast, oleamide had no effect on mechanically stimulated calcium wave transmission in this same cell type. Other chemical compounds traditionally used as inhibitors of gap junctional communication, like heptanol and 18β-glycyrrhetinic acid, blocked not only gap junctional communication but also intercellular calcium signaling. Given the central role for intercellular small molecule and electrical signaling in central nervous system function, oleamide- induced inactivation of glial cell gap junction channels may serve to regulate communication between brain cells, and in doing so, may influence higher order neuronal events like sleep induction. PMID:9412472

Apoptosis in neural crest cells by functional loss of APC tumor suppressor gene

PubMed Central

Hasegawa, Sumitaka; Sato, Tomoyuki; Akazawa, Hiroshi; Okada, Hitoshi; Maeno, Akiteru; Ito, Masaki; Sugitani, Yoshinobu; Shibata, Hiroyuki; Miyazaki, Jun-ichi; Katsuki, Motoya; Yamauchi, Yasutaka; Yamamura, Ken-ichi; Katamine, Shigeru; Noda, Tetsuo

2002-01-01

Apc is a gene associated with familial adenomatous polyposis coli (FAP) and its inactivation is a critical step in colorectal tumor formation. The protein product, adenomatous polyposis coli (APC), acts to down-regulate intracellular levels of β-catenin, a key signal transducer in the Wnt signaling. Conditional targeting of Apc in the neural crest of mice caused massive apoptosis of cephalic and cardiac neural crest cells at about 11.5 days post coitum, resulting in craniofacial and cardiac anomalies at birth. Notably, the apoptotic cells localized in the regions where β-catenin had accumulated. In contrast to its role in colorectal epithelial cells, inactivation of APC leads to dysregulation of β-catenin/Wnt signaling with resultant apoptosis in certain tissues including neural crest cells. PMID:11756652

Regulation of Chloride Channels by Protein Kinase C in Normal and Cystic Fibrosis Airway Epithelia

NASA Astrophysics Data System (ADS)

Li, Ming; McCann, John D.; Anderson, Matthew P.; Clancy, John P.; Liedtke, Carole M.; Nairn, Angus C.; Greengard, Paul; Welsh, Michael J.

1989-06-01

Apical membrane chloride channels control chloride secretion by airway epithelial cells. Defective regulation of these channels is a prominent characteristic of cystic fibrosis. In normal intact cells, activation of protein kinase C (PKC) by phorbol ester either stimulated or inhibited chloride secretion, depending on the physiological status of the cell. In cell-free membrane patches, PKC also had a dual effect: at a high calcium concentration, PKC inactivated chloride channels; at a low calcium concentration, PKC activated chloride channels. In cystic fibrosis cells, PKC-dependent channel inactivation was normal, but activation was defective. Thus it appears that PKC phosphorylates and regulates two different sites on the channel or on an associated membrane protein, one of which is defective in cystic fibrosis.

Autoamplificatory singlet oxygen generation sensitizes tumor cells for intercellular apoptosis-inducing signaling.

PubMed

Bauer, Georg

2018-06-01

Tumor cells express NADPH oxidase-1 (NOX1) in their membrane and control NOX1-based intercellular reactive oxygen and nitrogen species (ROS/RNS)-dependent apoptosis-inducing signaling through membrane-associated catalase and superoxide dismutase. of tumor cells with high concentrations of H 2 O 2 , peroxnitrite, HOCl, or increasing the concentration of cell-derived NO causes initial generation of singlet oxygen and local inactivation of membrane-associated catalase. As a result, free peroxynitrite and H 2 O 2 interact and generate secondary singlet oxygen. Inactivation of further catalase molecules by secondary singlet oxygen leads to auto-amplification of singlet oxygen generation and catalase inactivation. This allows reactivation of intercellular ROS/RNS-signaling and selective apoptosis induction in tumor cells. The initial singlet oxygen generation seems to be the critical point in this complex biochemical multistep mechanism. Initial singlet oxygen generation requires the interaction between distinct tumor cell-derived ROS and RNS and may also depend on either the induction of NO synthase expression or NOX1 activation through the FAS receptor. FAS receptor activation can be achieved by singlet oxygen. Autoamplificatory generation of singlet oxygen through the interaction between peroxynitrite and hydrogen peroxide inherits a rich potential for the establishment of synergistic effects that may be instrumental for novel approaches of tumor therapy with high selectivity towards malignant cells. Copyright © 2017 Elsevier B.V. All rights reserved.

Dendritic cells pulsed with Pythium insidiosum (1,3)(1,6)-β-glucan, Heat-inactivated zoospores and immunotherapy prime naïve T cells to Th1 differentiation in vitro.

PubMed

Ledur, Pauline C; Tondolo, Juliana S M; Jesus, Francielli P K; Verdi, Camila M; Loreto, Érico S; Alves, Sydney H; Santurio, Janio M

2018-03-01

Pythiosis is a life-threatening disease caused by the fungus-like microorganism Pythium insidiosum that can lead to death if not treated. Since P. insidiosum has particular cell wall characteristics, pythiosis is difficult to treat, as it does not respond well to traditional antifungal drugs. In our study, we investigated a new immunotherapeutic approach with potential use in treatment and in the acquisition of immunity against pythiosis. Dendritic cells from both human and mouse, pulsed with P. insidiosum heat-inactivated zoospore, (1,3)(1,6)-β-glucan and the immunotherapeutic PitiumVac ® efficiently induced naïve T cell differentiation in a Th1 phenotype by the activation of specific Th1 cytokine production in vitro. Heat-inactivated zoospores showed the greatest Th1 response among the tested groups, with a significant increase in IL-6 and IFN-γ production in human cells. In mice cells, we also observed a Th17 pathway induction, with an increase on the IL-17A levels in lymphocytes cultured with β-glucan pulsed DCs. These results suggest a potential use of DCs pulsed with P. insidiosum antigens as a new therapeutic strategy in the treatment and acquisition of immunity against pythiosis. Copyright © 2017 Elsevier GmbH. All rights reserved.

3, 3′-Diindolylmethane Exhibits Antileukemic Activity In Vitro and In Vivo through a Akt-Dependent Process

PubMed Central

Gao, Ning; Cheng, Senping; Budhraja, Amit; Liu, E-Hu; Chen, Jieping; Chen, Deying; Yang, Zailin; Luo, Jia; Shi, Xianglin; Zhang, Zhuo

2012-01-01

3,3′-diindolylmethane (DIM), one of the active products derived from Brassica plants, is a promising antitumor agent. The present study indicated that DIM significantly induced apoptosis in U937 human leukemia cells in dose- and time-dependent manners. These events were also noted in other human leukemia cells (Jurkat and HL-60) and primary human leukemia cells (AML) but not in normal bone marrow mononuclear cells. We also found that DIM-induced lethality is associated with caspases activation, myeloid cell leukemia-1 (Mcl-1) down-regulation, p21cip1/waf1 up-regulation, and Akt inactivation accompanied by c-jun NH2-terminal kinase (JNK) activation. Enforced activation of Akt by a constitutively active Akt construct prevented DIM-mediated caspase activation, Mcl-1 down-regulation, JNK activation, and apoptosis. Conversely, DIM lethality was potentiated by the PI3K inhibitor LY294002. Interruption of the JNK pathway by pharmacologic or genetic approaches attenuated DIM-induced caspases activation, Mcl-1 down-regulation, and apoptosis. Lastly, DIM inhibits tumor growth of mouse U937 xenograft, which was related to induction of apoptosis and inactivation of Akt, as well as activation of JNK. Collectively, these findings suggest that DIM induces apoptosis in human leukemia cell lines and primary human leukemia cells, and exhibits antileukemic activity in vivo through Akt inactivation and JNK activation. PMID:22363731

The Inactivation of Arx in Pancreatic α-Cells Triggers Their Neogenesis and Conversion into Functional β-Like Cells

PubMed Central

Courtney, Monica; Gjernes, Elisabet; Druelle, Noémie; Ravaud, Christophe; Vieira, Andhira; Ben-Othman, Nouha; Pfeifer, Anja; Avolio, Fabio; Leuckx, Gunter; Lacas-Gervais, Sandra; Burel-Vandenbos, Fanny; Ambrosetti, Damien; Hecksher-Sorensen, Jacob; Ravassard, Philippe; Heimberg, Harry; Mansouri, Ahmed; Collombat, Patrick

2013-01-01

Recently, it was demonstrated that pancreatic new-born glucagon-producing cells can regenerate and convert into insulin-producing β-like cells through the ectopic expression of a single gene, Pax4. Here, combining conditional loss-of-function and lineage tracing approaches, we show that the selective inhibition of the Arx gene in α-cells is sufficient to promote the conversion of adult α-cells into β-like cells at any age. Interestingly, this conversion induces the continuous mobilization of duct-lining precursor cells to adopt an endocrine cell fate, the glucagon+ cells thereby generated being subsequently converted into β-like cells upon Arx inhibition. Of interest, through the generation and analysis of Arx and Pax4 conditional double-mutants, we provide evidence that Pax4 is dispensable for these regeneration processes, indicating that Arx represents the main trigger of α-cell-mediated β-like cell neogenesis. Importantly, the loss of Arx in α-cells is sufficient to regenerate a functional β-cell mass and thereby reverse diabetes following toxin-induced β-cell depletion. Our data therefore suggest that strategies aiming at inhibiting the expression of Arx, or its molecular targets/co-factors, may pave new avenues for the treatment of diabetes. PMID:24204325

Buffer AVL Alone Does Not Inactivate Ebola Virus in a Representative Clinical Sample Type.

PubMed

Smither, Sophie J; Weller, Simon A; Phelps, Amanda; Eastaugh, Lin; Ngugi, Sarah; O'Brien, Lyn M; Steward, Jackie; Lonsdale, Steve G; Lever, Mark S

2015-10-01

Rapid inactivation of Ebola virus (EBOV) is crucial for high-throughput testing of clinical samples in low-resource, outbreak scenarios. The EBOV inactivation efficacy of Buffer AVL (Qiagen) was tested against marmoset serum (EBOV concentration of 1 × 10(8) 50% tissue culture infective dose per milliliter [TCID50 · ml(-1)]) and murine blood (EBOV concentration of 1 × 10(7) TCID50 · ml(-1)) at 4:1 vol/vol buffer/sample ratios. Posttreatment cell culture and enzyme-linked immunosorbent assay (ELISA) analysis indicated that treatment with Buffer AVL did not inactivate EBOV in 67% of samples, indicating that Buffer AVL, which is designed for RNA extraction and not virus inactivation, cannot be guaranteed to inactivate EBOV in diagnostic samples. Murine blood samples treated with ethanol (4:1 [vol/vol] ethanol/sample) or heat (60°C for 15 min) also showed no viral inactivation in 67% or 100% of samples, respectively. However, combined Buffer AVL and ethanol or Buffer AVL and heat treatments showed total viral inactivation in 100% of samples tested. The Buffer AVL plus ethanol and Buffer AVL plus heat treatments were also shown not to affect the extraction of PCR quality RNA from EBOV-spiked murine blood samples. © Crown copyright 2015.

Inactivation of pathogenic bacteria in food matrices: high pressure processing, photodynamic inactivation and pressure-assisted photodynamic inactivation

NASA Astrophysics Data System (ADS)

Cunha, A.; Couceiro, J.; Bonifácio, D.; Martins, C.; Almeida, A.; Neves, M. G. P. M. S.; Faustino, M. A. F.; Saraiva, J. A.

2017-09-01

Traditional food processing methods frequently depend on the application of high temperature. However, heat may cause undesirable changes in food properties and often has a negative impact on nutritional value and organoleptic characteristics. Therefore, reducing the microbial load without compromising the desirable properties of food products is still a technological challenge. High-pressure processing (HPP) can be classified as a cold pasteurization technique, since it is a non-thermal food preservation method that uses hydrostatic pressure to inactivate spoilage microorganisms. At the same time, it increases shelf life and retains the original features of food. Photodynamic inactivation (PDI) is also regarded as promising approach for the decontamination of food matrices. In this case, the inactivation of bacterial cells is achieved by the cytotoxic effects of reactive oxygens species (ROS) produced from the combined interaction of a photosensitizer molecule, light and oxygen. This short review examines some recent developments on the application of HPP and PDI with food-grade photosensitizers for the inactivation of listeriae, taken as a food pathogen model. The results of a proof-of-concept trial of the use of high-pressure as a coadjutant to increase the efficiency of photodynamic inactivation of bacterial endospores is also addressed.

Demonstration of a novel Xp22.2 microdeletion as the cause of familial extreme skewing of X-inactivation utilizing case-parent trio SNP microarray analysis.

PubMed

Mason, Jane A; Aung, Hnin T; Nandini, Adayapalam; Woods, Rickie G; Fairbairn, David J; Rowell, John A; Young, David; Susman, Rachel D; Brown, Simon A; Hyland, Valentine J; Robertson, Jeremy D

2018-05-01

We report a kindred referred for molecular investigation of severe hemophilia A in a young female in which extremely skewed X-inactivation was observed in both the proband and her clinically normal mother. Bidirectional Sanger sequencing of all F8 gene coding regions and exon/intron boundaries was undertaken. Methylation-sensitive restriction enzymes were utilized to investigate skewed X-inactivation using both a classical human androgen receptor (HUMARA) assay, and a novel method targeting differential methylation patterns in multiple informative X-chromosome SNPs. Illumina Whole-Genome Infinium microarray analysis was performed in the case-parent trio (proband and both parents), and the proband's maternal grandmother. The proband was a cytogenetically normal female with severe hemophilia A resulting from a heterozygous F8 pathogenic variant inherited from her similarly affected father. No F8 mutation was identified in the proband's mother, however, both the proband and her mother both demonstrated completely skewed X-chromosome inactivation (100%) in association with a previously unreported 2.3 Mb deletion at Xp22.2. At least three disease-associated genes (FANCB, AP1S2, and PIGA) were contained within the deleted region. We hypothesize that true "extreme" skewing of X-inactivation (≥95%) is a rare occurrence, but when defined correctly there is a high probability of finding an X-chromosome disease-causing variant or larger deletion resulting in X-inactivation through a survival disadvantage or cell lethal mechanism. We postulate that the 2.3 Mb Xp22.2 deletion identified in our kindred arose de novo in the proband's mother (on the grandfather's homolog), and produced extreme skewing of X-inactivation via a "cell lethal" mechanism. We introduce a novel multitarget approach for X-inactivation analysis using multiple informative differentially methylated SNPs, as an alternative to the classical single locus (HUMARA) method. We propose that for females with unexplained severe phenotypic expression of an X-linked recessive disorder trio-SNP microarray should be undertaken in combination with X-inactivation analysis. © 2018 The Authors. Molecular Genetics & Genomic Medicine published by Wiley Periodicals, Inc.

Blockade by N-3 polyunsaturated fatty acid of the Kv4.3 current stably expressed in Chinese hamster ovary cells

PubMed Central

Singleton, C B; Valenzuela, S M; Walker, B D; Tie, H; Wyse, K R; Bursill, J A; Qiu, M R; Breit, S N; Campbell, T J

1999-01-01

The Kv4.3 gene is believed to encode a large proportion of the transient outward current (Ito), responsible for the early phase of repolarization of the human cardiac action potential. There is evidence that this current is involved in the dispersion of refractoriness which develops during myocardial ischaemia and which predisposes to the development of potentially fatal ventricular tachyarrhythmias. Epidemiological, clinical, animal, and cellular studies indicate that these arrhythmias may be ameliorated in myocardial ischaemia by n-3 polyunsaturated fatty acids (n-3 PUFA) present in fish oils. We describe stable transfection of the Kv4.3 gene into a mammalian cell line (Chinese hamster ovary cells), and using patch clamp techniques have shown that the resulting current closely resembles human Ito. The current is rapidly activating and inactivating, with both processes being well fit by double exponential functions (time constants of 3.8±0.2 and 5.3±0.4 ms for activation and 20.0±1.2 and 96.6±6.7 ms for inactivation at +45 mV at 23°C). Activation and steady state inactivation both show voltage dependence (V1/2 of activation=−6.7±2.5 mV, V1/2 of steady state inactivation=−51.3±0.2 mV at 23°C). Current inactivation and recovery from inactivation are faster at physiologic temperature (37°C) compared to room temperature (23°C). The n-3 PUFA docosahexaenoic acid blocks the Kv4.3 current with an IC50 of 3.6 μmol L−1. Blockade of the transient outward current may be an important mechanism by which n-3 PUFA provide protection against the development of ventricular fibrillation during myocardial ischaemia. PMID:10433502

Ultraviolet-C irradiation for inactivation of viruses in foetal bovine serum.

PubMed

Vaidya, Vivek; Dhere, Rajeev; Agnihotri, Snehal; Muley, Ravindra; Patil, Sanjay; Pawar, Amit

2018-07-05

Foetal Bovine Serum (FBS) and porcine trypsin are one of the essential raw materials used in the manufacturing of cell culture based viral vaccines. Being from animal origin, these raw materials can potentially contaminate the final product by known or unknown adventitious agents. The issue is more serious in case of live attenuated viral vaccines, where there is no inactivation step which can take care of such adventitious agents. It is essential to design production processes which can offer maximum viral clearance potential for animal origin products. Ultraviolet-C irradiation is known to inactivate various adventitious viral agents; however there are limited studies on ultraviolet inactivation of viruses in liquid media. We obtained a recently developed UVivatec ultraviolet-C (UV-C) irradiation based viral clearance system for evaluating its efficacy to inactivate selected model viruses. This system has a unique design with spiral path of liquid allowing maximum exposure to UV-C light of a short wavelength of 254 nm. Five live attenuated vaccine viruses and four other model viruses were spiked in tissue culture media and exposed to UV-C irradiation. The pre and post UV-C irradiation samples were analyzed for virus content to find out the extent of inactivation of various viruses. These experiments showed substantial log reduction for the majority of the viruses with few exceptions based on the characteristics of these viruses. Having known the effect of UV irradiation on protein structure, we also evaluated the post irradiation samples of culture media for growth promoting properties using one of the most fastidious human diploid cells (MRC-5). UV-C exposure did not show any notable impact on the nutritional properties of culture media. The use of an UV-C irradiation based system is considered to be promising approach to mitigate the risk of adventitious agents in cell culture media arising through animal derived products. Copyright © 2018 Elsevier Ltd. All rights reserved.

Antimicrobial activity of Bacillus amyloliquefaciens LBM 5006 is enhanced in the presence of Escherichia coli.

PubMed

Benitez, Lisianne; Correa, AnaPaula; Daroit, Daniel; Brandelli, Adriano

2011-03-01

Increased antimicrobial activity was observed when Bacillus amyloliquefaciens LBM 5006 strain was cultivated in the presence of thermally inactivated cells of Escherichia coli, but not with Staphylococcus aureus, Listeria monocytogenes, or Bacillus cereus. E. coli also enhanced the antimicrobial activity when it was added to the medium in the form of living cells or as cell debris after cellular fractionation. No inducing activity was observed with addition of cell-free supernatant of E. coli cultures, suggesting that inducing factor is associated to the cells. Polyacrylamide gel electrophoresis revealed that additional peptide bands are secreted when B. amyloliquefaciens was cultivated in the presence of cell debris of E. coli. These results suggest that the presence of intact or inactivated E. coli enhanced the synthesis of antimicrobial peptides by B. amyloliquefaciens LBM 5006.

Distinct subpopulations of FOXD1 stroma-derived cells regulate renal erythropoietin

PubMed Central

Liu, Qingdu; Binns, Thomas C.; Davidoff, Olena; Kapitsinou, Pinelopi P.; Pfaff, Andrew S.; Olauson, Hannes; Fogo, Agnes B.; Fong, Guo-Hua; Gross, Kenneth W.

2016-01-01

Renal peritubular interstitial fibroblast-like cells are critical for adult erythropoiesis, as they are the main source of erythropoietin (EPO). Hypoxia-inducible factor 2 (HIF-2) controls EPO synthesis in the kidney and liver and is regulated by prolyl-4-hydroxylase domain (PHD) dioxygenases PHD1, PHD2, and PHD3, which function as cellular oxygen sensors. Renal interstitial cells with EPO-producing capacity are poorly characterized, and the role of the PHD/HIF-2 axis in renal EPO-producing cell (REPC) plasticity is unclear. Here we targeted the PHD/HIF-2/EPO axis in FOXD1 stroma-derived renal interstitial cells and examined the role of individual PHDs in REPC pool size regulation and renal EPO output. Renal interstitial cells with EPO-producing capacity were entirely derived from FOXD1-expressing stroma, and Phd2 inactivation alone induced renal Epo in a limited number of renal interstitial cells. EPO induction was submaximal, as hypoxia or pharmacologic PHD inhibition further increased the REPC fraction among Phd2–/– renal interstitial cells. Moreover, Phd1 and Phd3 were differentially expressed in renal interstitium, and heterozygous deficiency for Phd1 and Phd3 increased REPC numbers in Phd2–/– mice. We propose that FOXD1 lineage renal interstitial cells consist of distinct subpopulations that differ in their responsiveness to Phd2 inactivation and thus regulation of HIF-2 activity and EPO production under hypoxia or conditions of pharmacologic or genetic PHD inactivation. PMID:27088801

Human X chromosome inactivation and reactivation: implications for cell reprogramming and disease.

PubMed

Cantone, Irene; Fisher, Amanda G

2017-11-05

X-chromosome inactivation (XCI) is an exemplar of epigenetic regulation that is set up as pluripotent cells differentiate. Once established, XCI is stably propagated, but can be reversed in vivo or by pluripotent reprogramming in vitro Although reprogramming provides a useful model for inactive X (Xi) reactivation in mouse, the relative instability and heterogeneity of human embryonic stem (ES) cells and induced pluripotent stem cells hampers comparable progress in human. Here we review studies aimed at reactivating the human Xi using different reprogramming strategies. We outline our recent results using mouse ES cells to reprogramme female human fibroblasts by cell-cell fusion. We show that pluripotent reprogramming induces widespread and rapid chromatin remodelling in which the human Xi loses XIST and H3K27m3 enrichment and selected Xi genes become reactivated, ahead of mitotic division. Using RNA sequencing to map the extent of human Xi reactivation, and chromatin-modifying drugs to potentiate reactivation, we outline how this approach could be used to better design strategies to re-express human X-linked loci. As cell fusion induces the expression of human pluripotency genes that represent both the 'primed' and 'naive' states, this approach may also offer a fresh opportunity to segregate human pluripotent states with distinct Xi expression profiles, using single-cell-based approaches.This article is part of the themed issue 'X-chromosome inactivation: a tribute to Mary Lyon'. © 2017 The Author(s).

Phasic Firing in Vasopressin Cells: Understanding Its Functional Significance through Computational Models

PubMed Central

MacGregor, Duncan J.; Leng, Gareth

2012-01-01

Vasopressin neurons, responding to input generated by osmotic pressure, use an intrinsic mechanism to shift from slow irregular firing to a distinct phasic pattern, consisting of long bursts and silences lasting tens of seconds. With increased input, bursts lengthen, eventually shifting to continuous firing. The phasic activity remains asynchronous across the cells and is not reflected in the population output signal. Here we have used a computational vasopressin neuron model to investigate the functional significance of the phasic firing pattern. We generated a concise model of the synaptic input driven spike firing mechanism that gives a close quantitative match to vasopressin neuron spike activity recorded in vivo, tested against endogenous activity and experimental interventions. The integrate-and-fire based model provides a simple physiological explanation of the phasic firing mechanism involving an activity-dependent slow depolarising afterpotential (DAP) generated by a calcium-inactivated potassium leak current. This is modulated by the slower, opposing, action of activity-dependent dendritic dynorphin release, which inactivates the DAP, the opposing effects generating successive periods of bursting and silence. Model cells are not spontaneously active, but fire when perturbed by random perturbations mimicking synaptic input. We constructed one population of such phasic neurons, and another population of similar cells but which lacked the ability to fire phasically. We then studied how these two populations differed in the way that they encoded changes in afferent inputs. By comparison with the non-phasic population, the phasic population responds linearly to increases in tonic synaptic input. Non-phasic cells respond to transient elevations in synaptic input in a way that strongly depends on background activity levels, phasic cells in a way that is independent of background levels, and show a similar strong linearization of the response. These findings show large differences in information coding between the populations, and apparent functional advantages of asynchronous phasic firing. PMID:23093929

PT2385 for the Treatment of Von Hippel-Lindau Disease-Associated Clear Cell Renal Cell Carcinoma

ClinicalTrials.gov

2017-08-23

VHL Gene Mutation; VHL; VHL Syndrome; VHL Gene Inactivation; Von Hippel; Von Hippel-Lindau Disease; Von Hippel's Disease; Von Hippel-Lindau Syndrome, Modifiers of; Clear Cell Renal Cell Carcinoma; Clear Cell RCC; ccRCC

Photodynamic inactivation of antigenic determinants of single-stranded DNA bacteriophage phiX174

DOE Office of Scientific and Technical Information (OSTI.GOV)

Khan, N.C.; Poddar, R.K.

1974-05-01

Bacteriophage phi X174 when photodynamically inactivated (i.e., when rendered unable to produce plaques as a result of exposure to visible light in air in the presence of proflavine) progressively lost their capacity to bind efficiently with homologous antiserum. Such loss of serum-blocking power was evident with heat-inactivated but not with uv-irradiated phage. The ability of the phages to adsorb to host cells, however, remained practically unaltered even after photodynamic inactivation. It thus appears that photodynamic damages in the so-called ''jacket'' component of the phi X174 coat proteins are partly responsible for the loss of plaque-forming ability, whereas the ''spikes'' aremore » either poor antigens or insensitive to photodynamic treatment. (auth)« less

Cefoperazone Prevents the Inactivation of α1-Antitrypsin by Activated Neutrophils

PubMed Central

Dallegri, Franco; Dapino, Patrizia; Arduino, Nicoletta; Bertolotto, Maria; Ottonello, Luciano

1999-01-01

At sites of neutrophilic inflammation, tissue injury by neutrophil elastase is favored by phagocyte-induced hypochlorous acid-dependent inactivation of the natural elastase inhibitor α1-antitrypsin. In the present study, cefoperazone prevented α1-antitrypsin inactivation by neutrophils and reduced the recovery of hypochlorous acid from these cells. Moreover, the antibiotic reduced the free elastase activity in a neutrophil suspension supplemented with α1-antitrypsin without affecting the cells’ ability to release elastase. These data suggest that the drug inactivates hypochlorous acid before its reaction with α1-antitrypsin, thereby permitting the antiprotease-mediated blockade of released elastase. In conclusion, cefoperazone appears to have the potential for limiting elastase-antielastase imbalances, attenuating the related tissue injury at sites of inflammation. PMID:10471586

Long Non-coding RNAs in the X-inactivation Center

PubMed Central

Kalantry, Sundeep

2014-01-01

The X-inactivation center is a hotbed of functional long non-coding RNAs in eutherian mammals. These RNAs are thought to help orchestrate the epigenetic transcriptional states of the two X-chromosomes in females as well as of the single X-chromosome in males. To balance X-linked gene expression between the sexes, females undergo transcriptional silencing of most genes on one of the two X-chromosomes in a process termed X-chromosome inactivation. While one X-chromosome is inactivated, the other X-chromosome remains active. Moreover, with a few notable exceptions, the originally established epigenetic transcriptional profiles of the two is maintained as such through many rounds of cell division, essentially for the life of the organism. The stable divergent transcriptional fates of the two X-chromosomes, despite residing in a shared nucleoplasm, make X-inactivation a paradigm of epigenetic transcriptional regulation. Originally proposed in 1961 by Mary Lyon, the X-inactivation hypothesis has been validated through much experimentation over the last fifty years. In the last 25 years, the discovery and functional characterization has firmly established X-linked long non-coding RNAs as key players in choreographing X-chromosome inactivation. PMID:24297756

An inactivated vaccine made from a U.S. field isolate of porcine epidemic disease virus is immunogenic in pigs as demonstrated by a dose-titration.

PubMed

Collin, Emily A; Anbalagan, Srivishnupriya; Okda, Faten; Batman, Ron; Nelson, Eric; Hause, Ben M

2015-03-15

Porcine epidemic diarrhea virus (PEDV), a highly pathogenic and transmissible virus in swine, was first detected in the U.S. in May, 2013, and has caused tremendous losses to the swine industry. Due to the difficulty in isolating and growing this virus in cell culture, few vaccine studies using cell culture propagated PEDV have been performed on U.S. strains in pigs. Therefore, the objective of this study was to evaluate the humoral immune response to the selected inactivated PEDV vaccine candidate in a dose-titration manner. PEDV was isolated from a pig with diarrhea and complete genome sequencing found >99% nucleotide identity to other U.S. PEDV. Inactivated adjuvanted monovalent vaccines were administered intramuscularly to five week old pigs in a dose titration experimental design, ranging from 6.0-8.0 log10 tissue culture infective dose (TCID50/mL), to evaluate immunogenicity using a fluorescent foci neutralization assay (FFN), fluorescent microsphere immunoassay (FMIA), and enzyme-linked immunosorbent assay (ELISA) on sera. Pigs vaccinated with 8.0 log10 TCID50/mL inactivated virus showed significantly higher FFN titers as well as FMIA and ELISA values than 6.0 log10 TCID50/mL vaccinates and the negative controls. These results demonstrate the immunogenicity of a PEDV inactivated viral vaccine with a U.S. strain via dose-titration. A future vaccination-challenge study would illustrate the efficacy of an inactivated vaccine and help evaluate protective FFN titers and ELISA and FMIA responses.

Antimicrobial blue light therapy for multidrug-resistant Acinetobacter baumannii infection in a mouse burn model: implications for prophylaxis and treatment of combat-related wound infections.

PubMed

Zhang, Yunsong; Zhu, Yingbo; Gupta, Asheesh; Huang, Yingying; Murray, Clinton K; Vrahas, Mark S; Sherwood, Margaret E; Baer, David G; Hamblin, Michael R; Dai, Tianhong

2014-06-15

In this study, we investigated the utility of antimicrobial blue light therapy for multidrug-resistant Acinetobacter baumannii infection in a mouse burn model. A bioluminescent clinical isolate of multidrug-resistant A. baumannii was obtained. The susceptibility of A. baumannii to blue light (415 nm)-inactivation was compared in vitro to that of human keratinocytes. Repeated cycles of sublethal inactivation of bacterial by blue light were performed to investigate the potential resistance development of A. baumannii to blue light. A mouse model of third degree burn infected with A. baumannii was developed. A single exposure of blue light was initiated 30 minutes after bacterial inoculation to inactivate A. baumannii in mouse burns. It was found that the multidrug-resistant A. baumannii strain was significantly more susceptible than keratinocytes to blue light inactivation. Transmission electron microscopy revealed blue light-induced ultrastructural damage in A. baumannii cells. Fluorescence spectroscopy suggested that endogenous porphyrins exist in A. baumannii cells. Blue light at an exposure of 55.8 J/cm(2) significantly reduced the bacterial burden in mouse burns. No resistance development to blue light inactivation was observed in A. baumannii after 10 cycles of sublethal inactivation of bacteria. No significant DNA damage was detected in mouse skin by means of a skin TUNEL assay after a blue light exposure of 195 J/cm(2). © The Author 2014. Published by Oxford University Press on behalf of the Infectious Diseases Society of America. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com.

UVA Causes Dual Inactivation of Cathepsin B and L Underlying Lysosomal Dysfunction in Human Dermal Fibroblasts

PubMed Central

Lamore, Sarah D.; Wondrak, Georg T.

2013-01-01

Cutaneous exposure to chronic solar UVA-radiation is a causative factor in photocarcinogenesis and photoaging. Recently, we have identified the thiol-dependent cysteine-protease cathepsin B as a novel UVA-target undergoing photo-oxidative inactivation upstream of autophagic-lysosomal dysfunction in fibroblasts. In this study, we examined UVA effects on a wider range of cathepsins and explored the occurrence of UVA-induced cathepsin inactivation in other cultured skin cell types. In dermal fibroblasts, chronic exposure to non-cytotoxic doses of UVA caused pronounced inactivation of the lysosomal cysteine-proteases cathepsin B and L, effects not observed in primary keratinocytes and occurring only to a minor extent in primary melanocytes. In order to determine if UVA-induced lysosomal impairment requires single or dual inactivation of cathepsin B and/or L, we used a genetic approach (siRNA) to selectively downregulate enzymatic activity of these target cathepsins. Monitoring an established set of protein markers (including LAMP1, LC3-II, and p62) and cell ultrastructural changes detected by electron microscopy, we observed that only dual genetic antagonism (targeting both CTSB and CTSL expression) could mimic UVA-induced autophagic-lysosomal alterations, whereas single knockdown (targeting CTSB or CTSL only) did not display ‘UVA-mimetic’ effects failing to reproduce the UVA-induced phenotype. Taken together, our data demonstrate that chronic UVA inhibits both cathepsin B and L enzymatic activity and that dual inactivation of both enzymes is a causative factor underlying UVA-induced impairment of lysosomal function in dermal fibroblasts. PMID:23603447

Inhibition of Focal Adhesion Kinase Signaling by Integrin α6β1 Supports Human Pluripotent Stem Cell Self-Renewal.

PubMed

Villa-Diaz, Luis G; Kim, Jin Koo; Laperle, Alex; Palecek, Sean P; Krebsbach, Paul H

2016-07-01

Self-renewal of human embryonic stem cells and human induced pluripotent stem cells (hiPSCs)-known as pluripotent stem cells (PSC)-is influenced by culture conditions, including the substrate on which they are grown. However, details of the molecular mechanisms interconnecting the substrate and self-renewal of these cells remain unclear. We describe a signaling pathway in hPSCs linking self-renewal and expression of pluripotency transcription factors to integrin α6β1 and inactivation of focal adhesion kinase (FAK). Disruption of this pathway results in hPSC differentiation. In hPSCs, α6β1 is the dominant integrin and FAK is not phosphorylated at Y397, and thus, it is inactive. During differentiation, integrin α6 levels diminish and Y397 FAK is phosphorylated and activated. During reprogramming of fibroblasts into iPSCs, integrin α6 is upregulated and FAK is inactivated. Knockdown of integrin α6 and activation of β1 integrin lead to FAK phosphorylation and reduction of Nanog, Oct4, and Sox2, suggesting that integrin α6 functions in inactivation of integrin β1 and FAK signaling and prevention of hPSC differentiation. The N-terminal domain of FAK, where Y397 is localized, is in the nuclei of hPSCs interacting with Oct4 and Sox2, and this immunolocalization is regulated by Oct4. hPSCs remodel the extracellular microenvironment and deposit laminin α5, the primary ligand of integrin α6β1. Knockdown of laminin α5 resulted in reduction of integrin α6 expression, phosphorylation of FAK and decreased Oct4. In conclusion, hPSCs promote the expression of integrin α6β1, and nuclear localization and inactivation of FAK to supports stem cell self-renewal. Stem Cells 2016;34:1753-1764. © 2016 AlphaMed Press.

PTEN loss represses glioblastoma tumor initiating cell differentiation via inactivation of Lgl1.

PubMed

Gont, Alexander; Hanson, Jennifer E L; Lavictoire, Sylvie J; Parolin, Doris A; Daneshmand, Manijeh; Restall, Ian J; Soucie, Mathieu; Nicholas, Garth; Woulfe, John; Kassam, Amin; Da Silva, Vasco F; Lorimer, Ian A J

2013-08-01

Glioblastoma multiforme is an aggressive and incurable type of brain tumor. A subset of undifferentiated glioblastoma cells, known as glioblastoma tumor initiating cells (GTICs), has an essential role in the malignancy of this disease and also appears to mediate resistance to radiation therapy and chemotherapy. GTICs retain the ability to differentiate into cells with reduced malignant potential, but the signaling pathways controlling differentiation are not fully understood at this time. PTEN loss is a very common in glioblastoma multiforme and leads to aberrant activation of the phosphoinositide 3-kinase pathway. Increased signalling through this pathway leads to activation of multiple protein kinases, including atypical protein kinase C. In Drosophila, active atypical protein kinase C has been shown to promote the self-renewal of neuroblasts, inhibiting their differentiation along a neuronal lineage. This effect is mediated by atypical protein kinase c-mediated phosphorylation and inactivation of Lgl, a protein that was first characterized as a tumour suppressor in Drosophila. The effects of the atypical protein kinase C/Lgl pathway on the differentiation status of GTICs, and its potential link to PTEN loss, have not been assessed previously. Here we show that PTEN loss leads to the phosphorylation and inactivation of Lgl by atypical protein kinase C in glioblastoma cells. Re-expression of PTEN in GTICs promoted their differentiation along a neuronal lineage. This effect was also seen when atypical protein kinase C was knocked down using RNA interference, and when a non-phosphorylatable, constitutively active form of Lgl was expressed in GTICs. Thus PTEN loss, acting via atypical protein kinase C activation and Lgl inactivation, helps to maintain GTICs in an undifferentiated state.

Investigation of antiviral state mediated by interferon-inducible transmembrane protein 1 induced by H9N2 virus and inactivated viral particle in human endothelial cells.

PubMed

Feng, Bo; Zhao, Lihong; Wang, Wei; Wang, Jianfang; Wang, Hongyan; Duan, Huiqin; Zhang, Jianjun; Qiao, Jian

2017-11-03

Endothelial cells are believed to play an important role in response to virus infection. Our previous microarray analysis showed that H9N2 virus infection and inactivated viral particle inoculation increased the expression of interferon-inducible transmembrane protein 1 (IFITM1) in human umbilical vein endothelial cells (HUVECs). In present study, we deeply investigated the expression patterns of IFITM1 and IFITM1-mediated antiviral response induced by H9N2 virus infection and inactivated viral particle inoculation in HUVECs. Epithelial cells that are considered target cells of the influenza virus were selected as a reference control. First, we quantified the expression levels of IFITM1 in HUVECs induced by H9N2 virus infection or viral particle inoculation using quantitative real-time PCR and western blot. Second, we observed whether hemagglutinin or neuraminidase affected IFITM1 expression in HUVECs. Finally, we investigated the effect of induced-IFITM1 on the antiviral state in HUVECs by siRNA and activation plasmid transfection. Both H9N2 virus infection and viral particle inoculation increased the expression of IFITM1 without elevating the levels of interferon-ɑ/β in HUVECs. HA or NA protein binding alone is not sufficient to increase the levels of IFITM1 and interferon-ɑ/β in HUVECs. IFITM1 induced by viral particle inoculation significantly decreased the virus titers in culture supernatants of HUVECs. Our results showed that inactivated viral particle inoculation increased the expression of IFITM1 at mRNA and protein levels. Moreover, the induction of IFITM1 expression mediated the antiviral state in HUVECs.

Inactivation of Gram-negative and Gram-positive bacteria in red cell concentrates using INACTINE PEN110 chemistry.

PubMed

Zavizion, B; Serebryanik, D; Chapman, J; Alford, B; Purmal, A

2004-10-01

The risk of transfusion-transmitted bacterial infections as a result of the presence of bacteria in blood is one of the major concerns in transfusion medicine. The purpose of this study was to investigate whether bacteria inoculated into red blood cell concentrates can be inactivated by the INACTINE PEN110 pathogen-reduction process. Four bacterial species were chosen for the study: anaerobic Gram-positive Clostridium perfringens and Propionibacterium acnes, known to be transfusion-transmitted; and two Gram-negative species, Acinetobacter johnsonii and Acinetobacter lwoffii, recently reported to be a common cause of transfusion-associated infections in Europe. Identical units of leucoreduced red cell concentrates were inoculated with A. johnsonii, A. lwoffii, C. perfringens, or P. acnes. The 4 degrees C control units were put on storage immediately after receiving the spike. The test units were subjected to PEN110 treatment and then stored. The bacterial titre in all units was monitored during a 6-week storage period. The PEN110 inactivation of all tested bacterial strains was time- and titre-dependent. For A. johnsonii and A. lwoffii, no viable bacteria were detected in the units spiked with up to 10(4) colony-forming units (CFU)/ml and treated with PEN110. For red cell units spiked with 10(4)-10(5) CFU/ml of C. perfringens and P. acnes, no viable bacteria were detected in the units treated with PEN110. In control units, there was a gradual decrease in A. johnsonii, A. lwoffii and C. perfringens titres during cold storage, while P. acnes titres remained stable. The PEN110 pathogen-reduction process was demonstrated to inactivate high titres of A. johnsonii, A. lwoffii, C. perfringens and P. acnes in red cell concentrates.

Inhibitory effects of hesperetin on Kv1.5 potassium channels stably expressed in HEK 293 cells and ultra-rapid delayed rectifier K(+) current in human atrial myocytes.

PubMed

Wang, Huan; Wang, Hong-Fei; Wang, Chen; Chen, Yu-Fang; Ma, Rong; Xiang, Ji-Zhou; Du, Xin-Ling; Tang, Qiang

2016-10-15

In the present study, the inhibitory effects of hesperetin (HSP) on human cardiac Kv1.5 channels expressed in HEK 293 cells and the ultra-rapid delayed rectifier K(+) current (Ikur) in human atrial myocytes were examined by using the whole-cell configuration of the patch-clamp techniques. We found that hesperetin rapidly and reversibly suppressed human Kv1.5 current in a concentration dependent manner with a half-maximal inhibition (IC50) of 23.15 μΜ with a Hill coefficient of 0.89. The current was maximally diminished about 71.36% at a concentration of 300μM hesperetin. Hesperetin significantly positive shifted the steady-state activation curve of Kv1.5, while negative shifted the steady-state inactivation curve. Hesperetin also accelerated the inactivation and markedly slowed the recovery from the inactivation of Kv1.5 currents. Block of Kv1.5 currents by hesperetin was in a frequency dependent manner. However, inclusion of 30μM hesperetin in pipette solution produced no effect on Kv1.5 channel current, while the current were remarkable and reversibly inhibited by extracellular application of 30μM hesperetin. We also found that hesperetin potently and reversibly inhibited the ultra-repaid delayed K(+) current (Ikur) in human atrial myocytes, which is in consistent with the effects of hesperetin on Kv1.5 currents in HEK 293 cells. In conclusion, hesperetin is a potent inhibitor of Ikur (which is encoded by Kv1.5), with blockade probably due to blocking of both open state and inactivated state channels from outside of the cell. Copyright © 2016 Elsevier B.V. All rights reserved.

Thermal inactivation of foot-and-mouth disease virus in milk using high-temperature, short-time pasteurization.

PubMed

Tomasula, P M; Kozempel, M F; Konstance, R P; Gregg, D; Boettcher, S; Baxt, B; Rodriguez, L L

2007-07-01

Previous studies of laboratory simulation of high temperature, short time pasteurization (HTST) to eliminate foot-and-mouth disease virus (FMDV) in milk have shown that the virus is not completely inactivated at the legal pasteurization minimum (71.7 degrees C/15 s) but is inactivated in a flow apparatus at 148 degrees C with holding times of 2 to 3 s. It was the intent of this study to determine whether HTST pasteurization conducted in a continuous-flow pasteurizer that simulates commercial operation would enhance FMDV inactivation in milk. Cows were inoculated in the mammary gland with the field strain of FMDV (01/UK). Infected raw whole milk and 2% milk were then pasteurized using an Arm-field pilot-scale, continuous-flow HTST pasteurizer equipped with a plate-and-frame heat exchanger and a holding tube. The milk samples, containing FMDV at levels of up to 10(4) plaque-forming units/mL, were pasteurized at temperatures ranging from 72 to 95 degrees C at holding times of either 18.6 or 36 s. Pasteurization decreased virus infectivity by 4 log10 to undetectable levels in tissue culture. However, residual infectivity was still detectable for selected pasteurized milk samples, as shown by intramuscular and intradermal inoculation of milk into naïve steers. Although HTST pasteurization did not completely inactivate viral infectivity in whole and 2% milk, possibly because a fraction of the virus was protected by the milk fat and the casein proteins, it greatly reduced the risk of natural transmission of FMDV by milk.

High pressure inactivation of human norovirus virus-like particles: evidence that the capsid of human norovirus is highly pressure resistant

USDA-ARS?s Scientific Manuscript database

Human norovirus (NoV) is the leading cause of non-bacterial acute gastroenteritis epidemics worldwide. High pressure processing (HPP) has been considered a promising non-thermal processing technology to inactivate food- and water-borne viral pathogens. Due to the lack of an effective cell culture fo...

Chemical Changes in Nonthermal Plasma-Treated N-Acetylcysteine (NAC) Solution and Their Contribution to Bacterial Inactivation.

PubMed

Ercan, Utku K; Smith, Josh; Ji, Hai-Feng; Brooks, Ari D; Joshi, Suresh G

2016-02-02

In continuation of our previous reports on the broad-spectrum antimicrobial activity of atmospheric non-thermal dielectric barrier discharge (DBD) plasma treated N-Acetylcysteine (NAC) solution against planktonic and biofilm forms of different multidrug resistant microorganisms, we present here the chemical changes that mediate inactivation of Escherichia coli. In this study, the mechanism and products of the chemical reactions in plasma-treated NAC solution are shown. UV-visible spectrometry, FT-IR, NMR, and colorimetric assays were utilized for chemical characterization of plasma treated NAC solution. The characterization results were correlated with the antimicrobial assays using determined chemical species in solution in order to confirm the major species that are responsible for antimicrobial inactivation. Our results have revealed that plasma treatment of NAC solution creates predominantly reactive nitrogen species versus reactive oxygen species, and the generated peroxynitrite is responsible for significant bacterial inactivation.

Chemical Changes in Nonthermal Plasma-Treated N-Acetylcysteine (NAC) Solution and Their Contribution to Bacterial Inactivation

PubMed Central

Ercan, Utku K.; Smith, Josh; Ji, Hai-Feng; Brooks, Ari D.; Joshi, Suresh G.

2016-01-01

In continuation of our previous reports on the broad-spectrum antimicrobial activity of atmospheric non-thermal dielectric barrier discharge (DBD) plasma treated N-Acetylcysteine (NAC) solution against planktonic and biofilm forms of different multidrug resistant microorganisms, we present here the chemical changes that mediate inactivation of Escherichia coli. In this study, the mechanism and products of the chemical reactions in plasma-treated NAC solution are shown. UV-visible spectrometry, FT-IR, NMR, and colorimetric assays were utilized for chemical characterization of plasma treated NAC solution. The characterization results were correlated with the antimicrobial assays using determined chemical species in solution in order to confirm the major species that are responsible for antimicrobial inactivation. Our results have revealed that plasma treatment of NAC solution creates predominantly reactive nitrogen species versus reactive oxygen species, and the generated peroxynitrite is responsible for significant bacterial inactivation. PMID:26832829

Inactivation of nuclear factor κB by MIP-based drug combinations augments cell death of breast cancer cells.

PubMed

Subramaniam, Menaga; Liew, Su Ki; In, Lionel LA; Awang, Khalijah; Ahmed, Niyaz; Nagoor, Noor Hasima

2018-01-01

Drug combination therapy to treat cancer is a strategic approach to increase successful treatment rate. Optimizing combination regimens is vital to increase therapeutic efficacy with minimal side effects. In the present study, we evaluated the in vitro cytotoxicity of double and triple combinations consisting of 1'S-1'-acetoxychavicol acetate (ACA), Mycobacterium indicus pranii (MIP) and cisplatin (CDDP) against 14 various human cancer cell lines to address the need for more effective therapy. Our data show synergistic effects in MCF-7 cells treated with MIP:ACA, MIP:CDDP and MIP:ACA:CDDP combinations. The type of interaction between MIP, ACA and CDDP was evaluated based on combination index being <0.8 for synergistic effect. Identifying the mechanism of cell death based on previous studies involved intrinsic apoptosis and nuclear factor kappa B (NF-κB) and tested in Western blot analysis. Inactivation of NF-κB was confirmed by p65 and IκBα, while intrinsic apoptosis pathway activation was confirmed by caspase-9 and Apaf-1 expression. All combinations confirmed intrinsic apoptosis activation and NF-κB inactivation. Double and triple combination regimens that target induction of the same death mechanism with reduced dosage of each drug could potentially be clinically beneficial in reducing dose-related toxicities.

Collagen Accumulation in Osteosarcoma Cells lacking GLT25D1 Collagen Galactosyltransferase.

PubMed

Baumann, Stephan; Hennet, Thierry

2016-08-26

Collagen is post-translationally modified by prolyl and lysyl hydroxylation and subsequently by glycosylation of hydroxylysine. Despite the widespread occurrence of the glycan structure Glc(α1-2)Gal linked to hydroxylysine in animals, the functional significance of collagen glycosylation remains elusive. To address the role of glycosylation in collagen expression, folding, and secretion, we used the CRISPR/Cas9 system to inactivate the collagen galactosyltransferase GLT25D1 and GLT25D2 genes in osteosarcoma cells. Loss of GLT25D1 led to increased expression and intracellular accumulation of collagen type I, whereas loss of GLT25D2 had no effect on collagen secretion. Inactivation of the GLT25D1 gene resulted in a compensatory induction of GLT25D2 expression. Loss of GLT25D1 decreased collagen glycosylation by up to 60% but did not alter collagen folding and thermal stability. Whereas cells harboring individually inactivated GLT25D1 and GLT25D2 genes could be recovered and maintained in culture, cell clones with simultaneously inactive GLT25D1 and GLT25D2 genes could be not grown and studied, suggesting that a complete loss of collagen glycosylation impairs osteosarcoma cell proliferation and viability. © 2016 by The American Society for Biochemistry and Molecular Biology, Inc.

Collagen Accumulation in Osteosarcoma Cells lacking GLT25D1 Collagen Galactosyltransferase*

PubMed Central

Baumann, Stephan

2016-01-01

Collagen is post-translationally modified by prolyl and lysyl hydroxylation and subsequently by glycosylation of hydroxylysine. Despite the widespread occurrence of the glycan structure Glc(α1–2)Gal linked to hydroxylysine in animals, the functional significance of collagen glycosylation remains elusive. To address the role of glycosylation in collagen expression, folding, and secretion, we used the CRISPR/Cas9 system to inactivate the collagen galactosyltransferase GLT25D1 and GLT25D2 genes in osteosarcoma cells. Loss of GLT25D1 led to increased expression and intracellular accumulation of collagen type I, whereas loss of GLT25D2 had no effect on collagen secretion. Inactivation of the GLT25D1 gene resulted in a compensatory induction of GLT25D2 expression. Loss of GLT25D1 decreased collagen glycosylation by up to 60% but did not alter collagen folding and thermal stability. Whereas cells harboring individually inactivated GLT25D1 and GLT25D2 genes could be recovered and maintained in culture, cell clones with simultaneously inactive GLT25D1 and GLT25D2 genes could be not grown and studied, suggesting that a complete loss of collagen glycosylation impairs osteosarcoma cell proliferation and viability. PMID:27402836

Inactivating UBE2M impacts the DNA damage response and genome integrity involving multiple cullin ligases.

PubMed

Cukras, Scott; Morffy, Nicholas; Ohn, Takbum; Kee, Younghoon

2014-01-01

Protein neddylation is involved in a wide variety of cellular processes. Here we show that the DNA damage response is perturbed in cells inactivated with an E2 Nedd8 conjugating enzyme UBE2M, measured by RAD51 foci formation kinetics and cell based DNA repair assays. UBE2M knockdown increases DNA breakages and cellular sensitivity to DNA damaging agents, further suggesting heightened genomic instability and defective DNA repair activity. Investigating the downstream Cullin targets of UBE2M revealed that silencing of Cullin 1, 2, and 4 ligases incurred significant DNA damage. In particular, UBE2M knockdown, or defective neddylation of Cullin 2, leads to a blockade in the G1 to S progression and is associated with delayed S-phase dependent DNA damage response. Cullin 4 inactivation leads to an aberrantly high DNA damage response that is associated with increased DNA breakages and sensitivity of cells to DNA damaging agents, suggesting a DNA repair defect is associated. siRNA interrogation of key Cullin substrates show that CDT1, p21, and Claspin are involved in elevated DNA damage in the UBE2M knockdown cells. Therefore, UBE2M is required to maintain genome integrity by activating multiple Cullin ligases throughout the cell cycle.

An Xp11.2 translocation renal cell carcinoma with SMARCB1 (INI1) inactivation in adult end-stage renal disease: a case report.

PubMed

Yu, Lu; Li, Jun; Xu, Sanpeng; Navia Miranda, Mariajose; Wang, Guoping; Duan, Yaqi

2016-10-12

Xp11.2 translocation/transcription factor E3 (TFE3) rearrangement renal cell carcinoma (RCC) is a rare subtype of RCC with limited clinical and pathological data. Here we present an unusual high-grade Xp11.2 translocation RCC with a rhabdoid feature and SMARCB1 (INI1) inactivation in a 40-year-old man with end-stage kidney disease. The histological examination of the dissected left renal tumor showed an organoid architecture of the eosinophilic or clear neoplastic cells with necrosis and high mitotic activity. In some areas, non-adhesive tumor cells with eccentric nuclei were observed. Immunohistochemically (IHC), the tumor cells are positive for TFE3 and the renal tubular markers (PAX2 and PAX8), and completely negative for SMARCB1, an oncosuppressor protein. Break-apart florescence in situ hybridization and reverse transcription polymerase chain reaction confirmed TFE3 rearrangement on Xp11.2 and the presence of ASPSCR1-TFE3 fusion gene. DNA sequencing revealed a frameshift mutation in exon 4 of SMARCB1 gene. It is important to recognize this rare RCC with both TFE3 rearrangement and SMARCB1 inactivation, as the prognosis and therapeutic strategies, particularly targeted therapies for such tumors, might be different.

Biallelic inactivation of REV7 is associated with Fanconi anemia.

PubMed

Bluteau, Dominique; Masliah-Planchon, Julien; Clairmont, Connor; Rousseau, Alix; Ceccaldi, Raphael; Dubois d'Enghien, Catherine; Bluteau, Olivier; Cuccuini, Wendy; Gachet, Stéphanie; Peffault de Latour, Régis; Leblanc, Thierry; Socié, Gérard; Baruchel, André; Stoppa-Lyonnet, Dominique; D'Andrea, Alan D; Soulier, Jean

2016-09-01

Fanconi anemia (FA) is a recessive genetic disease characterized by congenital abnormalities, chromosome instability, progressive bone marrow failure (BMF), and a strong predisposition to cancer. Twenty FA genes have been identified, and the FANC proteins they encode cooperate in a common pathway that regulates DNA crosslink repair and replication fork stability. We identified a child with severe BMF who harbored biallelic inactivating mutations of the translesion DNA synthesis (TLS) gene REV7 (also known as MAD2L2), which encodes the mutant REV7 protein REV7-V85E. Patient-derived cells demonstrated an extended FA phenotype, which included increased chromosome breaks and G2/M accumulation upon exposure to DNA crosslinking agents, γH2AX and 53BP1 foci accumulation, and enhanced p53/p21 activation relative to cells derived from healthy patients. Expression of WT REV7 restored normal cellular and functional phenotypes in the patient's cells, and CRISPR/Cas9 inactivation of REV7 in a non-FA human cell line produced an FA phenotype. Finally, silencing Rev7 in primary hematopoietic cells impaired progenitor function, suggesting that the DNA repair defect underlies the development of BMF in FA. Taken together, our genetic and functional analyses identified REV7 as a previously undescribed FA gene, which we term FANCV.

Biallelic inactivation of REV7 is associated with Fanconi anemia

PubMed Central

Masliah-Planchon, Julien; Clairmont, Connor; Rousseau, Alix; Ceccaldi, Raphael; Dubois d’Enghien, Catherine; Bluteau, Olivier; Cuccuini, Wendy; Gachet, Stéphanie; Peffault de Latour, Régis; Leblanc, Thierry; Socié, Gérard; Baruchel, André; Stoppa-Lyonnet, Dominique; D’Andrea, Alan D.

2016-01-01

Fanconi anemia (FA) is a recessive genetic disease characterized by congenital abnormalities, chromosome instability, progressive bone marrow failure (BMF), and a strong predisposition to cancer. Twenty FA genes have been identified, and the FANC proteins they encode cooperate in a common pathway that regulates DNA crosslink repair and replication fork stability. We identified a child with severe BMF who harbored biallelic inactivating mutations of the translesion DNA synthesis (TLS) gene REV7 (also known as MAD2L2), which encodes the mutant REV7 protein REV7-V85E. Patient-derived cells demonstrated an extended FA phenotype, which included increased chromosome breaks and G2/M accumulation upon exposure to DNA crosslinking agents, γH2AX and 53BP1 foci accumulation, and enhanced p53/p21 activation relative to cells derived from healthy patients. Expression of WT REV7 restored normal cellular and functional phenotypes in the patient’s cells, and CRISPR/Cas9 inactivation of REV7 in a non-FA human cell line produced an FA phenotype. Finally, silencing Rev7 in primary hematopoietic cells impaired progenitor function, suggesting that the DNA repair defect underlies the development of BMF in FA. Taken together, our genetic and functional analyses identified REV7 as a previously undescribed FA gene, which we term FANCV. PMID:27500492

Inactivation of the Fanconi anemia/BRCA pathway in lung and oral cancers: implications for treatment and survival.

PubMed

Marsit, Carmen J; Liu, Mei; Nelson, Heather H; Posner, Marshall; Suzuki, Makoto; Kelsey, Karl T

2004-01-29

Inactivation of the FANC-BRCA pathway via promoter methylation of the FANCF gene renders cells sensitive to DNA crosslinking agents, and has been identified in ovarian cancer cell lines and sporadic primary tumor tissues. We investigated epigenetic alterations in the FANC-BRCA pathway in head and neck squamous cell carcinomas (HNSCC) and non-small-cell lung cancers (NSCLC) using methylation-specific PCR. Promoter methylation of FANCF occurred in 15% (13/89) of HNSCCs and 14% (22/158) of NSCLCs. Methylation of BRCA1 occurred only in 6/158 NSCLC, and was limited to adenocarcinomas and large-cell carcinomas of the lung. No methylation of BRCA2 was detected. FANCF methylation was associated with a shorter duration of tobacco use (P=0.03) and a younger age of starting smoking (P=0.06) in NSCLC, and with a greater number of years of alcohol drinking (P=0.02) in HNSCC. In adenocarcinomas of the lung, FANCF promoter methylation was a significant predictor of poor survival with a hazard ratio of 3.1 (95% CI 1.2-7.9). This study demonstrates that inactivation of the FANC-BRCA pathway is relatively common in solid tumors and may be related to tobacco and alcohol exposure and survival of these patients.

Variation in cisplatinum sensitivity is not associated with Fanconi Anemia/BRCA pathway inactivation in head and neck squamous cell carcinoma cell lines.

PubMed

Snyder, Eric R; Ricker, Justin L; Chen, Zhong; Waes, Carter Van

2007-01-08

Fanconi Anemia has recently been associated with a high risk of head and neck squamous cell carcinoma (HNSCC). Inactivation of the Fanconi Anemia (FANC-BRCA) pathway via promoter methylation of the FANCF gene has been proposed to be responsible for variation in cisplatinum (CDDP) sensitivity seen in ovarian and HNSCCs. Promoter methylation of the FANCF gene has been observed in 15% of HNSCC specimens, but the relationship to FANC pathway activation and CDDP sensitivity has not been reported. In the present study, 10 HNSCC cell lines were examined for expression of nine genes involved in the FANC-BRCA pathway by RT-PCR: FANCA, FANCC, FANCD2, FANCE, FANCF, FANCG, FANCL, BRCA1 and BRCA2. FANC pathway function was evaluated by western blotting for FANCD2 mono-ubiquitination. All of the cell lines were also analyzed for variation in CDDP cytotoxicity. While significant differences were found in CDDP cytotoxicity, Fanconi pathway defects are an infrequent cause, as no evidence of transcriptional down-regulation of FANCF or other FANC mRNAs, or functional FANC-BRCA pathway defects were observed. These findings suggest that the variation in CDDP sensitivity of many HNSCCs is most frequently due to factors other than FANC-BRCA pathway inactivation.

Inositol synthesis regulates activation of GSK-3α in neuronal cells

PubMed Central

Ye, Cunqi; Greenberg, Miriam L.

2015-01-01

The synthesis of inositol provides precursors of inositol lipids and inositol phosphates that are pivotal for cell signaling. Mood-stabilizers lithium and valproic acid (VPA), used for treating bipolar disorder, cause cellular inositol depletion, which has been proposed as a therapeutic mechanism of action of both drugs. Despite the importance of inositol, the requirement for inositol synthesis in neuronal cells is not well understood. Here, we examined inositol effects on proliferation of SK-N-SH neuroblastoma cells. The essential role of inositol synthesis in proliferation is underscored by the findings that exogenous inositol was dispensable for proliferation, and inhibition of inositol synthesis decreased proliferation. Interestingly, the inhibition of inositol synthesis by knocking down INO1, which encodes inositol-3-phosphate synthase, the rate-limiting enzyme of inositol synthesis, led to inactivation of GSK-3α by increasing the inhibitory phosphorylation of this kinase. Similarly, the mood-stabilizer VPA effected transient decreases in intracellular inositol, leading to inactivation of GSK-3α. As GSK-3 inhibition has been proposed as a likely therapeutic mechanism of action, the finding that inhibition of inositol synthesis results in inactivation of GSK-3α suggests a unifying hypothesis for mechanism of mood-stabilizing drugs. PMID:25345501

Efficacy of atmospheric pressure dielectric barrier discharge for inactivating airborne pathogens

DOE PAGES

Romero-Mangado, Jaione; Dey, Avishek; Diaz-Cartagena, Diana C.; ...

2017-07-05

Atmospheric pressure plasmas have gained attention in recent years for several environmental applications. This technology could potentially be used to deactivate airborne microorganisms, surface-bound microorganisms, and biofilms. Here, the authors explore the efficacy of the atmospheric pressure dielectric barrier discharge (DBD) to inactivate airborne Staphylococcus epidermidis and Aspergillus niger that are opportunistic pathogens associated with nosocomial infections. This technology uses air as the source of gas and does not require any process gas such as helium, argon, nitrogen, or hydrogen. Moreover, the effect of DBD was studied on aerosolized S. epidermidis and aerosolized A. niger spores via scanning electron microscopymore » (SEM). The morphology observed on the SEM micrographs showed deformations in the cellular structure of both microorganisms. Cell structure damage upon interaction with the DBD suggests leakage of vital cellular materials, which is a key mechanism for microbial inactivation. The chemical structure of the cell surface of S. epidermidis was also analyzed by near edge x-ray absorption fine structure spectroscopy before and after DBD exposure. Our results from surface analysis revealed that reactive oxygen species from the DBD discharge contributed to alterations on the chemistry of the cell membrane/cell wall of S. epidermidis.« less

Biosynthetic requirements for the repair of sublethally injured Saccharomyces cerevisiae cells after pulsed electric fields.

PubMed

Somolinos, M; García, D; Condón, S; Mañas, P; Pagán, R

2008-07-01

The aim was to evaluate the biosynthetic requirements for the repair of sublethal membrane damages in Saccharomyces cerevisiae cells after exposure to pulsed electric fields (PEF). The partial loss of the integrity and functionality of the cytoplasmic membrane was assessed by adding sodium chloride to the recovery medium. More than 2 log(10) cycles of survivors were sublethally injured after PEF. Repair of sublethal membrane damages occurred when survivors to PEF were incubated in Sabouraud Broth for 4 h at room temperature. The addition of inhibitors, such as chloramphenicol, rifampicin, 5-fluorocytosine, nalidixic acid, cycloheximide, cerulenin, miconazol and sodium azide to the liquid repair medium showed that the repair of PEF-injured cells required energy and protein synthesis. The extent of the sublethal damages was greater in PEF-treated cells at pH 4.0 than at pH 7.0. This work confirms that membrane damage is an important event in the PEF-inactivation of yeast. The mechanism of yeast inactivation by PEF seems to differ from that of bacteria, as the repair of sublethal damages requires protein synthesis. Knowledge about the damages inflicted by PEF leads to a better description of the mechanism of yeast inactivation.

Cycloheximide- and puromycin-induced heat resistance: different effects on cytoplasmic and nuclear luciferases

PubMed Central

Michels, Annemieke A; Kanon, Bart; Konings, Antonius W.T; Bensaude, Olivier; Kampinga, Harm H

2000-01-01

Inhibition of translation can result in cytoprotection against heat shock. The mechanism of this protection has remained elusive so far. Here, the thermoprotective effects of the translation inhibitor cycloheximide (CHX) and puromycin were investigated, using as reporter firefly luciferase localized either in the nucleus or in the cytoplasm. A short preincubation of O23 cells with either translation inhibitor was found to attenuate the heat inactivation of a luciferase directed into the cytoplasm, whereas the heat sensitivity of a nuclear-targeted luciferase remained unaffected. After a long-term CHX pretreatment, both luciferases were more heat resistant. Both the cytoplasmic and the nuclear luciferase are protected against heat-induced inactivation in thermotolerant cells and in cells overexpressing heat shock protein (Hsp)70. CHX incubations further attenuated cytoplasmic luciferase inactivation in thermotolerant and in Hsp70 overexpressing cells, even when Hsp70-mediated protection was saturated. It is concluded that protection by translation inhibition is unlikely due to an increase in the pool of free Hsps normally engaged in translation and released from the nascent polypeptide chains on the ribosomes. Rather, a decrease in nascent chains and thermolabile polypeptides may account for the heat resistance promoted by inhibitors of translation. PMID:11005376

Efficacy of atmospheric pressure dielectric barrier discharge for inactivating airborne pathogens

DOE Office of Scientific and Technical Information (OSTI.GOV)

Romero-Mangado, Jaione; Dey, Avishek; Diaz-Cartagena, Diana C.

Atmospheric pressure plasmas have gained attention in recent years for several environmental applications. This technology could potentially be used to deactivate airborne microorganisms, surface-bound microorganisms, and biofilms. Here, the authors explore the efficacy of the atmospheric pressure dielectric barrier discharge (DBD) to inactivate airborne Staphylococcus epidermidis and Aspergillus niger that are opportunistic pathogens associated with nosocomial infections. This technology uses air as the source of gas and does not require any process gas such as helium, argon, nitrogen, or hydrogen. Moreover, the effect of DBD was studied on aerosolized S. epidermidis and aerosolized A. niger spores via scanning electron microscopymore » (SEM). The morphology observed on the SEM micrographs showed deformations in the cellular structure of both microorganisms. Cell structure damage upon interaction with the DBD suggests leakage of vital cellular materials, which is a key mechanism for microbial inactivation. The chemical structure of the cell surface of S. epidermidis was also analyzed by near edge x-ray absorption fine structure spectroscopy before and after DBD exposure. Our results from surface analysis revealed that reactive oxygen species from the DBD discharge contributed to alterations on the chemistry of the cell membrane/cell wall of S. epidermidis.« less

Activating PTEN by COX-2 inhibitors antagonizes radiation-induced AKT activation contributing to radiosensitization

DOE Office of Scientific and Technical Information (OSTI.GOV)

Meng, Zhen; Department of Oral & Maxillofacial Surgery, Peking University School and Hospital of Stomatology, 22 Zhongguancun Avenue South, Haidian District, Beijing 100081; Gan, Ye-Hua, E-mail: kqyehuagan@bjmu.edu.cn

2015-05-01

Radiotherapy is still one of the most effective nonsurgical treatments for many tumors. However, radioresistance remains a major impediment to radiotherapy. Although COX-2 inhibitors can induce radiosensitization, the underlying mechanism is not fully understood. In this study, we showed that COX-2 selective inhibitor celecoxib enhanced the radiation-induced inhibition of cell proliferation and apoptosis in HeLa and SACC-83 cells. Treatment with celecoxib alone dephosphorylated phosphatase and tensin homolog deleted on chromosome ten (PTEN), promoted PTEN membrane translocation or activation, and correspondingly dephosphorylated or inactivated protein kinase B (AKT). By contrast, treatment with radiation alone increased PTEN phosphorylation, inhibited PTEN membrane translocationmore » and correspondingly activated AKT in the two cell lines. However, treatment with celecoxib or another COX-2 selective inhibitor (valdecoxib) completely blocked radiation-induced increase of PTEN phosphorylation, rescued radiation-induced decrease in PTEN membrane translocation, and correspondingly inactivated AKT. Moreover, celecoxib could also upregulate PTEN protein expression by downregulating Sp1 expression, thereby leading to the activation of PTEN transcription. Our results suggested that COX-2 inhibitors could enhance radiosensitization at least partially by activating PTEN to antagonize radiation-induced AKT activation. - Highlights: • COX-2 inhibitor, celecoxib, could enhance radiosensitization. • Radiation induced PTEN inactivation (phosphorylation) and AKT activation. • COX-2 inhibitor induced PTEN expression and activation, and inactivated AKT. • COX-2 inhibitor enhanced radiosensitization through activating PTEN.« less

Photodynamic inactivation of Listeria innocua biofilms with food-grade photosensitizers: a curcumin-rich extract of Curcuma longa vs commercial curcumin.

PubMed

Bonifácio, D; Martins, C; David, B; Lemos, C; Neves, M G P M S; Almeida, A; Pinto, D C G A; Faustino, M A F; Cunha, Â

2018-03-22

The aim of this work is to assess the potential of curcumin in the photosensitization of biofilms of Listeria. Biofilms of Listeria innocua, were irradiated with blue light in the presence of a curcumin-rich extract of Curcuma longa or commercial curcumin. Similar experiments were conducted with planktonic cells, for comparison. A reduction of 4·9 log in the concentration of viable biofilm cells was obtained with 3·7 mg l -1 of commercial curcumin. Planktonic cells were much more susceptible (6·1 log reduction). A tetracationic porphyrin, used as a reference photosensitizer (PS), caused a very modest inactivation of the biofilm (1·1 log) and complete inactivation of the planktonic form (>8 log). Curcumin is an effective PS for the photodynamic control of Listeria biofilms and the inactivation efficiency attained with this natural compound is higher than with the porphyrin. This result may point to a better performance of type I PSs against bacterial biofilms by circumventing the limitations to singlet-oxygen diffusion imposed by the extracellular matrix. Curcumin represents a promising alternative to the control of bacteria and bacterial biofilms in food products particularly in the case of meat products in which turmeric is used as spice. © 2018 The Society for Applied Microbiology.

Immunolocalization of tripeptidyl peptidase II, a cholecystokinin-inactivating enzyme, in rat brain.

PubMed

Facchinetti, P; Rose, C; Rostaing, P; Triller, A; Schwartz, J C

1999-01-01

Tripeptidyl peptidase II (EC 3.4.14.10) is a serine peptidase apparently involved in the inactivation of cholecystokinin octapeptide [Rose C. et al. (1996) Nature 380, 403-409]. We have compared its distribution with that of cholecystokinin in rat brain, using a polyclonal antibody raised against a highly purified preparation for immunohistochemistry at the photon and electron microscope levels. Tripeptidyl peptidase II-like immunoreactivity was mostly detected in neurons, and also in ependymal cells and choroid plexuses, localizations consistent with a possible participation of the peptidase in the inactivation of cholecystokinin circulating in the cerebrospinal fluid. Immunoreactivity was mostly detected in cell bodies, large processes and, to a lesser extent, axons of various neuronal populations. Their localization, relative to that of cholecystokinin terminals, appears to define three distinct situations. The first corresponds to neurons with high immunoreactivity in areas containing cholecystokinin terminals, as in the cerebral cortex or hippocampal formation, where pyramidal cell bodies and processes surrounded by cholecystokinin axons were immunoreactive. A similar situation was encountered in many other areas, namely along the pathways through which cholecystokinin controls satiety, i.e. in sensory vagal neurons, the nucleus tractus solitarius and hypothalamic nuclei. The second situation corresponds to cholecystokinin neuronal populations containing tripeptidyl peptidase II-like immunoreactivity, as in neurons of the supraoptic or paraventricular nuclei, axons in the median eminence or nigral neurons. In both situations, localization of tripeptidyl peptidase II-like immunoreactivity is consistent with a role in cholecystokinin inactivation. The third situation corresponds to areas with mismatches, such as the cerebellum, a region devoid of cholecystokinin, but in which Purkinje cells displayed high tripeptidyl peptidase II-like immunoreactivity, possibly related to a role in the inactivation of neuropeptides other than cholecystokinin. Also, some areas with cholecystokinin terminals, e.g., the molecular layer of the cerebral cortex, were devoid of tripeptidyl peptidase II-like immunoreactivity, suggesting that processes other than cleavage by tripeptidyl peptidase II may be involved in cholecystokinin inactivation. Tripeptidyl peptidase II-like immunoreactivity was also detected at the ultrastructural level in the cerebral cortex and hypothalamus using either immunoperoxidase or silver-enhanced immunogold detection. It was mainly associated with the cytoplasm of neuronal somata and dendrites, often in the vicinity of reticulum cisternae, Golgi apparatus or vesicles, and with the inner side of the dendritic plasma membrane. Hence, whereas a fraction of tripeptidyl peptidase II-like immunoreactivity localization at the cellular level is consistent with its alleged function in cholecystokinin octapeptide inactivation, its association with the outside plasma membrane of neurons remains to be confirmed.

DOE Office of Scientific and Technical Information (OSTI.GOV)

Liu, Ying, E-mail: yingliu@doheny.org; Sun Yet-sen University, Zhongshan Ophthalmic Center, State Key Ophthalmic Laboratory, Guangzhou 510060; Kawai, Kirio

Research highlights: {yields} Inactivation of Smad4 caused disruption in the development of the anterior segment. {yields} Inactivation of Smad4 failed to disrupt early lens development. {yields} Smad4 controlled lens cell cycle and cell death processes. {yields} Smad4 may regulate actin stress fiber assembly and eyelid epithelial movement. -- Abstract: Purpose: Signaling by members of the TGF{beta} superfamily of molecules is essential for embryonic development and homeostasis. Smad4, a key intracellular mediator in TGF{beta} signaling, forms transcriptional activator complexes with Activin-, BMP-, and TGF{beta}-restricted Smad proteins. However, the functional role of Smad4 in controlling different visual system compartments has not beenmore » fully investigated. Methods: Using the Pax6 promoter-driven Cre transgenic, smad4 was conditionally inactivated in the lens, cornea and ectoderm of the eyelids. Standard histological and molecular analytical approaches were employed to reveal morphological and cellular changes. Results: Inactivation of Smad4 in the lens led to microphthalmia and cataract formation in addition to the persistent adhesion of the retina to the lens and the iris to the cornea. Inactivation of Smad4 from the ectoderm of the eyelid and cornea caused disruption to eyelid fusion and proper development of the corneal epithelium and corneal stroma. Conclusions: Smad4 is required for the development and maintenance of the lens in addition to the proper development of the cornea, eyelids, and retina.« less

Pathogen inactivation treatment of plasma and platelet concentrates and their predicted functionality in massive transfusion protocols.

PubMed

Arbaeen, Ahmad F; Schubert, Peter; Serrano, Katherine; Carter, Cedric J; Culibrk, Brankica; Devine, Dana V

2017-05-01

Trauma transfusion packages for hemorrhage control consist of red blood cells, plasma, and platelets at a set ratio. Although pathogen reduction improves the transfusion safety of platelet and plasma units, there is an associated reduction in quality. This study aimed to investigate the impact of riboflavin/ultraviolet light-treated plasma or platelets in transfusion trauma packages composed of red blood cell, plasma, and platelet units in a ratio of 1:1:1 in vitro by modeling transfusion scenarios for trauma patients and assessing function by rotational thromboelastometry. Pathogen-reduced or untreated plasma and buffy coat platelet concentrate units produced in plasma were used in different combinations with red blood cells in trauma transfusion packages. After reconstitution of these packages with hemodiluted blood, the hemostatic functionality was analyzed by rotational thromboelastometry. Hemostatic profiles of pathogen-inactivated buffy coat platelet concentrate and plasma indicated decreased activity compared with their respective controls. Reconstitution of hemodiluted blood (hematocrit = 20%) with packages that contained treated or nontreated components resulted in increased alpha and maximum clot firmness and enhanced clot-formation time. Simulating transfusion scenarios based on 30% blood replacement with a transfusion trauma package resulted in a nonsignificant difference in rotational thromboelastometry parameters between packages containing treated and nontreated blood components (p ≥ 0.05). Effects of pathogen inactivation treatment were evident when the trauma package percentage was 50% or greater and contained both pathogen inactivation-treated plasma and buffy coat platelet concentrate. Rotational thromboelastometry investigations suggest that there is relatively little impact of pathogen inactivation treatment on whole blood clot formation unless large amounts of treated components are used. © 2017 AABB.

Effect of tyrphostin AG879 on Kv4.2 and Kv4.3 potassium channels

PubMed Central

Yu, Haibo; Zou, Beiyan; Wang, Xiaoliang; Li, Min

2015-01-01

Background and Purpose A-type potassium channels (IA) are important proteins for modulating neuronal membrane excitability. The expression and activity of Kv4.2 channels are critical for neurological functions and pharmacological inhibitors of Kv4.2 channels may have therapeutic potential for Fragile X syndrome. While screening various compounds, we identified tyrphostin AG879, a tyrosine kinase inhibitor, as a Kv4.2 inhibitor from. In the present study we characterized the effect of AG879 on cloned Kv4.2/Kv channel-interacting protein 2 (KChIP2) channels. Experimental Approach To screen the library of pharmacologically active compounds, the thallium flux assay was performed on HEK-293 cells transiently-transfected with Kv4.2 cDNA using the Maxcyte transfection system. The effects of AG879 were further examined on CHO-K1 cells expressing Kv4.2/KChIP2 channels using a whole-cell patch-clamp technique. Key Results Tyrphostin AG879 selectively and dose-dependently inhibited Kv4.2 and Kv4.3 channels. In Kv4.2/KChIP2 channels, AG879 induced prominent acceleration of the inactivation rate, use-dependent block and slowed the recovery from inactivation. AG879 induced a hyperpolarizing shift in the voltage-dependence of the steady-state inactivation of Kv4.2 channels without apparent effect on the V1/2 of the voltage-dependent activation. The blocking effect of AG879 was enhanced as channel inactivation increased. Furthermore, AG879 significantly inhibited the A-type potassium currents in the cultured hippocampus neurons. Conclusion and Implications AG879 was identified as a selective and potent inhibitor the Kv4.2 channel. AG879 inhibited Kv4.2 channels by preferentially interacting with the open state and further accelerating their inactivation. PMID:25752739

Effect of tyrphostin AG879 on Kv 4.2 and Kv 4.3 potassium channels.

PubMed

Yu, Haibo; Zou, Beiyan; Wang, Xiaoliang; Li, Min

2015-07-01

A-type potassium channels (IA) are important proteins for modulating neuronal membrane excitability. The expression and activity of Kv 4.2 channels are critical for neurological functions and pharmacological inhibitors of Kv 4.2 channels may have therapeutic potential for Fragile X syndrome. While screening various compounds, we identified tyrphostin AG879, a tyrosine kinase inhibitor, as a Kv 4.2 inhibitor from. In the present study we characterized the effect of AG879 on cloned Kv 4.2/Kv channel-interacting protein 2 (KChIP2) channels. To screen the library of pharmacologically active compounds, the thallium flux assay was performed on HEK-293 cells transiently-transfected with Kv 4.2 cDNA using the Maxcyte transfection system. The effects of AG879 were further examined on CHO-K1 cells expressing Kv 4.2/KChIP2 channels using a whole-cell patch-clamp technique. Tyrphostin AG879 selectively and dose-dependently inhibited Kv 4.2 and Kv 4.3 channels. In Kv 4.2/KChIP2 channels, AG879 induced prominent acceleration of the inactivation rate, use-dependent block and slowed the recovery from inactivation. AG879 induced a hyperpolarizing shift in the voltage-dependence of the steady-state inactivation of Kv 4.2 channels without apparent effect on the V1/2 of the voltage-dependent activation. The blocking effect of AG879 was enhanced as channel inactivation increased. Furthermore, AG879 significantly inhibited the A-type potassium currents in the cultured hippocampus neurons. AG879 was identified as a selective and potent inhibitor the Kv 4.2 channel. AG879 inhibited Kv 4.2 channels by preferentially interacting with the open state and further accelerating their inactivation. © 2015 The British Pharmacological Society.

Optimization of the synthesis process of an iron oxide nanocatalyst supported on activated carbon for the inactivation of Ascaris eggs in water using the heterogeneous Fenton-like reaction.

PubMed

Morales-Pérez, Ariadna A; Maravilla, Pablo; Solís-López, Myriam; Schouwenaars, Rafael; Durán-Moreno, Alfonso; Ramírez-Zamora, Rosa-María

2016-01-01

An experimental design methodology was used to optimize the synthesis of an iron-supported nanocatalyst as well as the inactivation process of Ascaris eggs (Ae) using this material. A factor screening design was used for identifying the significant experimental factors for nanocatalyst support (supported %Fe, (w/w), temperature and time of calcination) and for the inactivation process called the heterogeneous Fenton-like reaction (H2O2 dose, mass ratio Fe/H2O2, pH and reaction time). The optimization of the significant factors was carried out using a face-centered central composite design. The optimal operating conditions for both processes were estimated with a statistical model and implemented experimentally with five replicates. The predicted value of the Ae inactivation rate was close to the laboratory results. At the optimal operating conditions of the nanocatalyst production and Ae inactivation process, the Ascaris ova showed genomic damage to the point that no cell reparation was possible showing that this advanced oxidation process was highly efficient for inactivating this pathogen.

Lack of X inactivation associated with maternal X isodisomy: evidence for a counting mechanism prior to X inactivation during human embryogenesis.

PubMed

Migeon, B R; Jeppesen, P; Torchia, B S; Fu, S; Dunn, M A; Axelman, J; Schmeckpeper, B J; Fantes, J; Zori, R T; Driscoll, D J

1996-01-01

We have previously reported functional disomy for X-linked genes in females with tiny ring X chromosomes and a phenotype significantly more abnormal than Turner syndrome. In such cases the disomy results from failure of these X chromosomes to inactivate because they lack DNA sequences essential for cis X inactivation. Here we describe a novel molecular mechanism for functional X disomy that is associated with maternal isodisomy. In this case, the severe mental retardation and multiple congenital abnormalities in a female with a mosaic 45,X/ 46,X,del(X)(q21.3-qter)/ 46X,r(X) karyotype are associated with overexpression of the genes within Xpter to Xq21.31 in many of her cells. Her normal X, ring X, and deleted linear X chromosomes originate from the same maternal X chromosome, and all are transcriptionally active. None expresses X inactive specific transcript (XIST), although the locus and region of the putative X inactivation center (XIC) are present on both normal and linear deleted X chromosomes. To our knowledge, this is the first report of a functional maternal X isodisomy, and the largest X chromosome to escape inactivation. In addition, these results (1) show that cis inactivation does not invariably occur in human females with two X chromosomes, even when the XIC region is present on both of them; (2) provide evidence for a critical time prior to the visible onset of X inactivation in the embryo when decisions about X inactivation are made; and (3) support the hypothesis that the X chromosome counting mechanism involves chromosomal imprinting, occurs prior to the onset of random inactivation, and is required for subsequent inactivation of the chromosome.

Neoplastic transformation induced by carbon ions.

PubMed

Bettega, Daniela; Calzolari, Paola; Hessel, Petra; Stucchi, Claudio G; Weyrather, Wilma K

2009-03-01

The objective of this experiment was to compare the oncogenic potential of carbon ion beams and conventional photon beams for use in radiotherapy. The HeLa X human skin fibroblast cell line CGL1 was irradiated with carbon ions of three different energies (270, 100, and 11.4 MeV/u). Inactivation and transformation data were compared with those for 15 MeV photons. Inactivation and transformation frequencies for the 270 MeV/u carbon ions were similar to those for 15-MeV photons. The maximal relative biologic effectiveness (RBE(alpha)) values for 100MeV/u and 11.4 MeV/u carbon ions, respectively, were as follows: inactivation, 1.6 +/- 0.2 and 6.7 +/- 0.7; and transformation per surviving cell, 2.5 +/- 0.6 and 12 +/- 3. The curve for dose-transformation per cell at risk exhibited a maximum that was shifted toward lower doses at lower energies. Transformation induction per cell at risk for carbon ions in the entrance channel was comparable to that for photons, whereas for the lower energies, 100 MeV/u and 11 MeV/u, which are representative of the energies delivered to the tumor margins and volume, respectively, the probability of transformation in a single cell was greater than it was for photons. In addition, at isoeffective doses with respect to cell killing, the 11.4-MeV/u beam was more oncogenic than were photons.

Enhanced visible-light-driven photocatalytic bacteria disinfection by g-C3N4-AgBr.

PubMed

Deng, Jun; Liang, Jialiang; Li, Mian; Tong, Meiping

2017-04-01

g-C 3 N 4 -AgBr was synthesized by depositing AgBr nanoparticles onto g-C 3 N 4 . Scanning electron microscopy (SEM), Transmission electron microscope (TEM), X-ray diffraction (XRD), X-ray photoelectron spectroscopy (XPS), UV-vis diffuse reflectance spectra (DRS) and Photoluminescence (PL) spectra were employed to characterize the as-synthesized photocatalysts. The disinfection activities towards representative Gram-negative strain E. coli and Gram-positive strain S. aureus were examined under visible light irradiation. Complete inactivation of 3×10 6 CFU/mL viable cell density was reached in 60min for E. coli and 150min for S. aureus, respectively. Ag + released from the photocatalysts did not contribute to the photocatalytic disinfection process. Direct contact of g-C 3 N 4 -AgBr composites and bacterial cells, as well as the presence of O 2 was indispensable for the cell inactivation. Photo-generated holes, surface bounded OH, and indirect generation of intracellular active species played important roles in disinfection process of g-C 3 N 4 -AgBr under visible light irradiation. The disruption of outside structure of cells as well as inner cell injury led to the inactivation. High pH condition led to increasing the cell disinfection due to the generation of surface bounded OH. Copyright © 2017 Elsevier B.V. All rights reserved.

Experimental and numerical study of heterogeneous pressure-temperature-induced lethal and sublethal injury of Lactococcus lactis in a medium scale high-pressure autoclave.

PubMed

Kilimann, K V; Kitsubun, P; Delgado, A; Gänzle, M G; Chapleau, N; Le Bail, A; Hartmann, C

2006-07-05

The present contribution is dedicated to experimental and theoretical assessment of microbiological process heterogeneities of the high-pressure (HP) inactivation of Lactococcus lactis ssp. cremoris MG 1363. The inactivation kinetics are determined in dependence of pressure, process time, temperature and absence or presence of co-solutes in the buffer system namely 4 M sodium chloride and 1.5 M sucrose. The kinetic analysis is carried out in a 0.1-L autoclave in order to minimise thermal and convective effects. Upon these data, a deterministic inactivation model is formulated with the logistic equation. Its independent variables represent the counts of viable cells (viable but injured) and of the stress-resistant cells (viable and not injured). This model is then coupled to a thermo-fluiddynamical simulation method, high-pressure computer fluid dynamics technique (HP-CFD), which yields spatiotemporal temperature and flow fields occurring during the HP application inside any considered autoclave. Besides the thermo-fluiddynamic quantities, the coupled model predicts also the spatiotemporal distribution of both viable (VC) and stress-resistant cell counts (SRC). In order to assess the process non-uniformity of the microbial inactivation in a 3.3-L autoclave experimentally, microbial samples are placed at two distinct locations and are exposed to various process conditions. It can be shown with both, experimental and theoretical models that thermal heterogeneities induce process non-uniformities of more than one decimal power in the counts of the viable cells at the end of the treatment. (c) 2006 Wiley Periodicals, Inc.

Kinetics and mechanism of bacterial inactivation by ultrasound waves and sonoprotective effect of milk components.

PubMed

Gera, N; Doores, S

2011-03-01

Inactivation of Escherichia coli and Listeria monocytogenes were investigated in buffer and milk upon treatment with ultrasound waves (USW). In addition, sonoprotective effect of milk components and ultrasound-induced changes in bacterial cells were investigated using scanning electron microscopy (SEM). Bacterial cells were added to phosphate buffer, whole milk, skim milk, or simulated milk ultrafiltrate (SMUF). To determine the sonoprotective effect of milk components, lactose (5%), casein (3%), or β lactoglobulin (0.3%) was added to SMUF. Samples were sonicated with 24 kHz pulse USW while maintaining the system temperature between 30 to 35 °C. Aliquots were drawn at set times during sonication and bacteria were enumerated by surface plating appropriate dilutions on selective and nonselective media plates. Escherichia coli exhibited significantly higher D values in whole (2.43 min) and skim milk (2.41 min) than phosphate buffer (2.19 min). Listeria monocytogenes also showed higher D values in whole (9.31 min) and skim milk (8.61 min) compared to phosphate buffer (7.63 min). Data suggest that milk exerts a sonoprotective effect on these bacteria. Escherichia coli exhibited a log-linear inactivation kinetics followed by tailing whereas L. monocytogenes showed 1st-order kinetics throughout. Among the milk components tested, presence of lactose in SMUF resulted in significantly higher D values than SMUF for both organisms suggesting that lactose was exerting a protective effect on bacteria. SEM images showed that USW caused mechanical damage to the cell wall and cell membrane of bacteria leading to their inactivation.

Ability of polymorphonuclear leukocytes to orient in gradients of chemotactic factors

PubMed Central

1977-01-01

Polymorphonuclear leukocyte (PMN) chemotaxis has been examined under conditions which allow phase microscope observations of cells responding to controlled gradients of chemotactic factors. With this visual assay, PMNs can be seen to orient rapidly and reversibly to gradients of N-formylmethionyl peptides. The level of orientation depends upon the mean concentration of peptide present as well as the concentration gradient. The response allows an estimation of the binding constant of the peptide to the cell. In optimal gradients, PMNs can detect a 1% difference in the concentration of peptide. At high cell densities, PMNs incubated with active peptides orient their locomotion away from the center of the cell population. This orientation appears to be due to inactivation of the peptides by the cells. Such inactivation in vivo could help to limit an inflammatory response. PMID:264125

Targeted inactivation of fh1 causes proliferative renal cyst development and activation of the hypoxia pathway.

PubMed

Pollard, Patrick J; Spencer-Dene, Bradley; Shukla, Deepa; Howarth, Kimberley; Nye, Emma; El-Bahrawy, Mona; Deheragoda, Maesha; Joannou, Maria; McDonald, Stuart; Martin, Alison; Igarashi, Peter; Varsani-Brown, Sunita; Rosewell, Ian; Poulsom, Richard; Maxwell, Patrick; Stamp, Gordon W; Tomlinson, Ian P M

2007-04-01

Germline mutations in the fumarate hydratase (FH) tumor suppressor gene predispose to leiomyomatosis, renal cysts, and renal cell cancer (HLRCC). HLRCC tumors overexpress HIF1alpha and hypoxia pathway genes. We conditionally inactivated mouse Fh1 in the kidney. Fh1 mutants developed multiple clonal renal cysts that overexpressed Hif1alpha and Hif2alpha. Hif targets, such as Glut1 and Vegf, were upregulated. We found that Fh1-deficient murine embryonic stem cells and renal carcinomas from HLRCC showed similar overexpression of HIF and hypoxia pathway components to the mouse cysts. Our data have shown in vivo that pseudohypoxic drive, resulting from HIF1alpha (and HIF2alpha) overexpression, is a direct consequence of Fh1 inactivation. Our mouse may be useful for testing therapeutic interventions that target angiogenesis and HIF-prolyl hydroxylation.

Bacterial cheating limits antibiotic resistance

NASA Astrophysics Data System (ADS)

Xiao Chao, Hui; Yurtsev, Eugene; Datta, Manoshi; Artemova, Tanya; Gore, Jeff

2012-02-01

The widespread use of antibiotics has led to the evolution of resistance in bacteria. Bacteria can gain resistance to the antibiotic ampicillin by acquiring a plasmid carrying the gene beta-lactamase, which inactivates the antibiotic. This inactivation may represent a cooperative behavior, as the entire bacterial population benefits from removing the antibiotic. The cooperative nature of this growth suggests that a cheater strain---which does not contribute to breaking down the antibiotic---may be able to take advantage of cells cooperatively inactivating the antibiotic. Here we find experimentally that a ``sensitive'' bacterial strain lacking the plasmid conferring resistance can invade a population of resistant bacteria, even in antibiotic concentrations that should kill the sensitive strain. We observe stable coexistence between the two strains and find that a simple model successfully explains the behavior as a function of antibiotic concentration and cell density. We anticipate that our results will provide insight into the evolutionary origin of phenotypic diversity and cooperative behaviors.

Function of the Sex Chromosomes in Mammalian Fertility

PubMed Central

Heard, Edith; Turner, James

2011-01-01

The sex chromosomes play a highly specialized role in germ cell development in mammals, being enriched in genes expressed in the testis and ovary. Sex chromosome abnormalities (e.g., Klinefelter [XXY] and Turner [XO] syndrome) constitute the largest class of chromosome abnormalities and the commonest genetic cause of infertility in humans. Understanding how sex-gene expression is regulated is therefore critical to our understanding of human reproduction. Here, we describe how the expression of sex-linked genes varies during germ cell development; in females, the inactive X chromosome is reactivated before meiosis, whereas in males the X and Y chromosomes are inactivated at this stage. We discuss the epigenetics of sex chromosome inactivation and how this process has influenced the gene content of the mammalian X and Y chromosomes. We also present working models for how perturbations in sex chromosome inactivation or reactivation result in subfertility in the major classes of sex chromosome abnormalities. PMID:21730045

Early and late mammalian responses to heavy charged particles

NASA Technical Reports Server (NTRS)

Ainsworth, E. J.

1986-01-01

This overview summarizes murine results on acute lethality responses, inactivation of marrow CFU-S and intestinal microcolonies, testes weight loss, life span shortening, and posterior lens opacification in mice irradiated with heavy charged particles. RBE-LET relationships for these mammalian responses are compared with results from in vitro studies. The trend is that the maximum RBE for in vivo responses tends to be lower and occurs at a lower LET than for inactivation of V79 and T-1 cells in culture. Based on inactivation cross sections, the response of CFU-S in vivo conforms to expectations from earlier studies with prokaryotic systems and mammalian cells in culture. Effects of heavy ions are compared with fission spectrum neutrons, and the results are consistent with the interpretation that RBEs are lower than for fission neutrons at about the same LET, probably due to differences in track structure.

mTOR-dependent activation of the transcription factor TIF-IA links rRNA synthesis to nutrient availability.

PubMed

Mayer, Christine; Zhao, Jian; Yuan, Xuejun; Grummt, Ingrid

2004-02-15

In cycling cells, transcription of ribosomal RNA genes by RNA polymerase I (Pol I) is tightly coordinated with cell growth. Here, we show that the mammalian target of rapamycin (mTOR) regulates Pol I transcription by modulating the activity of TIF-IA, a regulatory factor that senses nutrient and growth-factor availability. Inhibition of mTOR signaling by rapamycin inactivates TIF-IA and impairs transcription-initiation complex formation. Moreover, rapamycin treatment leads to translocation of TIF-IA into the cytoplasm. Rapamycin-mediated inactivation of TIF-IA is caused by hypophosphorylation of Se 44 (S44) and hyperphosphorylation of Se 199 (S199). Phosphorylation at these sites affects TIF-IA activity in opposite ways, for example, phosphorylation of S44 activates and S199 inactivates TIF-IA. The results identify a new target formTOR-signaling pathways and elucidate the molecular mechanism underlying mTOR-dependent regulation of RNA synthesis.

(Biphenyl-4-yl)methylammonium chlorides: potent anticonvulsants that modulate Na+ currents.

PubMed

Lee, Hyosung; Park, Ki Duk; Yang, Xiao-Fang; Dustrude, Erik T; Wilson, Sarah M; Khanna, Rajesh; Kohn, Harold

2013-07-25

We have reported that compounds containing a biaryl linked unit (Ar-X-Ar') modulated Na(+) currents by promoting slow inactivation and fast inactivation processes and by inducing frequency (use)-dependent inhibition of Na(+) currents. These electrophysiological properties have been associated with the mode of action of several antiepileptic drugs. In this study, we demonstrate that the readily accessible (biphenyl-4-yl)methylammonium chlorides (compound class B) exhibited a broad range of anticonvulsant activities in animal models, and in the maximal electroshock seizure test the activity of (3'-trifluoromethoxybiphenyl-4-yl)methylammonium chloride (8) exceeded that of phenobarbital and phenytoin upon oral administration to rats. Electrophysiological studies of 8 using mouse catecholamine A-differentiated cells and rat embryonic cortical neurons confirmed that 8 promoted slow and fast inactivation in both cell types but did not affect the frequency (use)-dependent block of Na(+) currents.

Gene expression of Escherichia coli in continuous culture during adaptation to artificial sunlight.

PubMed

Berney, Michael; Weilenmann, Hans-Ulrich; Egli, Thomas

2006-09-01

Escherichia coli growing in continuous culture under continuous UVA irradiation exhibits growth inhibition with a subsequent adaptation to the stress. Transcriptome analysis was performed during transient growth inhibition and in the UVA light-adapted growth state. The results indicate that UVA light induces stringent response and an additional response that includes the upregulation of the synthesis of valine, isoleucine, leucine, phenylalanine, histidine and glutamate. The induction of several SOS response-genes strongly points to DNA damage as a result of UVA exposure. The involvement of oxidative stress was observed with the induction of ahpCF. Taken together it supports the hypothesis of the production of reactive oxygen species by UVA light. In the UVA-adapted cell population strong repression of the acid tolerance response was found. We identified the enzyme chorismate mutase as a possible chromophore for UVA light-inactivation and found strong repression of the pyrBI operon and the gene mgtA encoding for an ATP-dependent Mg2+ transporter. Furthermore, our results indicate that the role of RpoS may not be as important in the adaptation of E. coli to UVA light as it was implicated by previous results with starved cells, but that RpoS might be of crucial importance for the resistance under transient light exposure.

Pressure- and heat-induced inactivation of butyrylcholinesterase: evidence for multiple intermediates and the remnant inactivation process.

PubMed Central

Weingand-Ziade, A; Ribes, F; Renault, F; Masson, P

2001-01-01

The inactivation process of native (N) human butyrylcholinesterase (BuChE) by pressure and/or heat was found to be multi-step. It led to irreversible formation of an active intermediate (I) state and a denatured state. This series-inactivation process was described by expanding the Lumry-Eyring [Lumry, R. and Eyring, H. (1954) J. Phys. Chem. 58, 110-120] model. The intermediate state (I) was found to have a K(m) identical with that of the native state and a turnover rate (k(cat)) twofold higher than that of the native state with butyrylthiocholine as the substrate. The increased catalytic efficiency (k(cat)/K(m)) of I can be explained by a conformational change in the active-site gorge and/or restructuring of the water-molecule network in the active-site pocket, making the catalytic steps faster. However, a pressure/heat-induced covalent modification of native BuChE, affecting the catalytic machinery, cannot be ruled out. The inactivation process of BuChE induced by the combined action of pressure and heat was found to continue after interruption of pressure/temperature treatment. This secondary inactivation process was termed 'remnant inactivation'. We hypothesized that N and I were in equilibrium with populated metastable N' and I' states. The N' and I' states can either return to the active forms, N and I, or develop into inactive forms, N(')(in) and I(')(in). Both active N' and I' intermediate states displayed different rates of remnant inactivation depending on the pressure and temperature pretreatments and on the storage temperature. A first-order deactivation model describing the kinetics of the remnant inactivation of BuChE is proposed. PMID:11368776

The cell fate determinant Scribble is required for maintenance of hematopoietic stem cell function.

PubMed

Mohr, Juliane; Dash, Banaja P; Schnoeder, Tina M; Wolleschak, Denise; Herzog, Carolin; Tubio Santamaria, Nuria; Weinert, Sönke; Godavarthy, Sonika; Zanetti, Costanza; Naumann, Michael; Hartleben, Björn; Huber, Tobias B; Krause, Daniela S; Kähne, Thilo; Bullinger, Lars; Heidel, Florian H

2018-05-01

Cell fate determinants influence self-renewal potential of hematopoietic stem cells. Scribble and Llgl1 belong to the Scribble polarity complex and reveal tumor-suppressor function in drosophila. In hematopoietic cells, genetic inactivation of Llgl1 leads to expansion of the stem cell pool and increases self-renewal capacity without conferring malignant transformation. Here we show that genetic inactivation of its putative complex partner Scribble results in functional impairment of hematopoietic stem cells (HSC) over serial transplantation and during stress. Although loss of Scribble deregulates transcriptional downstream effectors involved in stem cell proliferation, cell signaling, and cell motility, these effectors do not overlap with transcriptional targets of Llgl1. Binding partner analysis of Scribble in hematopoietic cells using affinity purification followed by mass spectometry confirms its role in cell signaling and motility but not for binding to polarity modules described in drosophila. Finally, requirement of Scribble for self-renewal capacity also affects leukemia stem cell function. Thus, Scribble is a regulator of adult HSCs, essential for maintenance of HSCs during phases of cell stress.

Bacterial Imaging and Photodynamic Inactivation Using Zinc(II)-Dipicolylamine BODIPY Conjugates†

PubMed Central

Rice, Douglas R.; Gan, Haiying; Smith, Bradley D.

2015-01-01

Targeted imaging and antimicrobial photodynamic inactivation (PDI) are emerging methods for detecting and eradicating pathogenic microorganisms. This study describes two structurally related optical probes that are conjugates of a zinc(II)-dipicolylamine targeting unit and a BODIPY chromophore. One probe is a microbial targeted fluorescent imaging agent, mSeek, and the other is an oxygen photosensitizing analogue, mDestroy. The conjugates exhibited high fluorescence quantum yield and singlet oxygen production, respectively. Fluorescence imaging and detection studies examined four bacterial strains: E. coli, S. aureus, K. pneumonia, and B. thuringiensis vegetative cells and purified spores. The fluorescent probe, mSeek, is not phototoxic and enabled detection of all tested bacteria at concentrations of ~100 CFU/mL for B. thuringiensis spores, ~1000 CFU/mL for S. aureus and ~10,000 CFU/mL for E. coli. The photosensitizer analogue, mDestroy, inactivated 99–99.99% of bacterial samples and selectively killed bacterial cells in the presence of mammalian cells. However, mDestroy was ineffective against B. thuringiensis spores. Together, the results demonstrate a new two-probe strategy to optimize PDI of bacterial infection/contamination. PMID:26063101

Peroxiredoxins: Guardians Against Oxidative Stress and Modulators of Peroxide Signaling

PubMed Central

Perkins, Arden; Nelson, Kimberly J.; Parsonage, Derek; Poole, Leslie B.; Karplus, P. Andrew

2015-01-01

Peroxiredoxins (Prxs) are a ubiquitous family of cysteine-dependent peroxidase enzymes that play dominant roles in regulating peroxide levels within cells. These enzymes, often present at high levels and capable of rapidly clearing peroxides, display a remarkable array of variations in their oligomeric states and susceptibility to regulation by hyperoxidative inactivation and other post-translational modifications. Key conserved residues within the active site promote catalysis by stabilizing the transition state required for transferring the terminal oxygen of hydroperoxides to the active site (peroxidatic) cysteine residue. Extensive investigations continue to expand our understanding of the scope of their importance as well as the structures and forces at play within these critical defense and regulatory enzymes. PMID:26067716

Slow Inactivation in Shaker K Channels Is Delayed by Intracellular Tetraethylammonium

PubMed Central

González-Pérez, Vivian; Neely, Alan; Tapia, Christian; González-Gutiérrez, Giovanni; Contreras, Gustavo; Orio, Patricio; Lagos, Verónica; Rojas, Guillermo; Estévez, Tania; Stack, Katherine; Naranjo, David

2008-01-01

After removal of the fast N-type inactivation gate, voltage-sensitive Shaker (Shaker IR) K channels are still able to inactivate, albeit slowly, upon sustained depolarization. The classical mechanism proposed for the slow inactivation observed in cell-free membrane patches—the so called C inactivation—is a constriction of the external mouth of the channel pore that prevents K+ ion conduction. This constriction is antagonized by the external application of the pore blocker tetraethylammonium (TEA). In contrast to C inactivation, here we show that, when recorded in whole Xenopus oocytes, slow inactivation kinetics in Shaker IR K channels is poorly dependent on external TEA but severely delayed by internal TEA. Based on the antagonism with internally or externally added TEA, we used a two-pulse protocol to show that half of the channels inactivate by way of a gate sensitive to internal TEA. Such gate had a recovery time course in the tens of milliseconds range when the interpulse voltage was −90 mV, whereas C-inactivated channels took several seconds to recover. Internal TEA also reduced gating charge conversion associated to slow inactivation, suggesting that the closing of the internal TEA-sensitive inactivation gate could be associated with a significant amount of charge exchange of this type. We interpreted our data assuming that binding of internal TEA antagonized with U-type inactivation (Klemic, K.G., G.E. Kirsch, and S.W. Jones. 2001. Biophys. J. 81:814–826). Our results are consistent with a direct steric interference of internal TEA with an internally located slow inactivation gate as a “foot in the door” mechanism, implying a significant functional overlap between the gate of the internal TEA-sensitive slow inactivation and the primary activation gate. But, because U-type inactivation is reduced by channel opening, trapping the channel in the open conformation by TEA would also yield to an allosteric delay of slow inactivation. These results provide a framework to explain why constitutively C-inactivated channels exhibit gating charge conversion, and why mutations at the internal exit of the pore, such as those associated to episodic ataxia type I in hKv1.1, cause severe changes in inactivation kinetics. PMID:19029372

Loss of PHD3 allows tumours to overcome hypoxic growth inhibition and sustain proliferation through EGFR

PubMed Central

Henze, Anne-Theres; Garvalov, Boyan K.; Seidel, Sascha; Cuesta, Angel M.; Ritter, Mathias; Filatova, Alina; Foss, Franziska; Dopeso, Higinio; Essmann, Clara L.; Maxwell, Patrick H.; Reifenberger, Guido; Carmeliet, Peter; Acker-Palmer, Amparo; Acker, Till

2014-01-01

Solid tumours are exposed to microenvironmental factors such as hypoxia that normally inhibit cell growth. However, tumour cells are capable of counteracting these signals through mechanisms that are largely unknown. Here we show that the prolyl hydroxylase PHD3 restrains tumour growth in response to microenvironmental cues through the control of EGFR. PHD3 silencing in human gliomas or genetic deletion in a murine high-grade astrocytoma model markedly promotes tumour growth and the ability of tumours to continue growing under unfavourable conditions. The growth-suppressive function of PHD3 is independent of the established PHD3 targets HIF and NF-κB and its hydroxylase activity. Instead, loss of PHD3 results in hyperphosphorylation of epidermal growth factor receptor (EGFR). Importantly, epigenetic/genetic silencing of PHD3 preferentially occurs in gliomas without EGFR amplification. Our findings reveal that PHD3 inactivation provides an alternative route of EGFR activation through which tumour cells sustain proliferative signalling even under conditions of limited oxygen availability. PMID:25420773

The proapoptotic effect of formononetin in human osteosarcoma cells: involvement of inactivation of ERK and Akt pathways.

PubMed

Liu, Yun; He, JinJie; Chen, XiaoMing; Li, Jian; Shen, MaoRong; Yu, WenJun; Yang, Yuan; Xiao, ZengMing

2014-01-01

Previous studies have shown that some phytoestrogens inhibits proliferation and induces apoptosis in estrogen-dependent cancers via estrogen receptor (ER)-mediated signaling pathway. In view of the expression of ER in human osteosarcoma cells, the purpose of this study is to investigate whether formononetin and calycosin, two of the major isoflavones in Radix astragali, could also elicit anti-tumor activity against osteosarcoma, along with the underlying mechanism. Human osteosarcoma cells U2OS were respectively treated with various concentrations of formononetin or calycosin. Cell proliferation was determined by MTT assay, while apoptosis by flow cytometry. Next, the expression levels of apoptosis-related genes ERK, Akt, Bcl-2, Bax and caspase-3 were quantified by real-time PCR and Western blotting. Formononetin exhibited higher anti-proliferative activities toward human osteosarcoma cells U2OS, when compared with calycosin. Therefore, U2OS cells were then respectively treated with various concentrations of formononetin, in order to elucidate the isoflavones-related signaling pathway. It was found that formononetin dose-dependently triggered apoptosis of U2OS cells in vitro. Furthermore, treatment of formononetin led to significant inactivation of ERK and Akt, followed by downregulation of Bcl-2, upregulation of Bax and finally increased expression of caspase-3. Formononetin is more effective than calycosin at promoting cell death of U2OS cells by induction of apoptosis, which is mediated by inactivation of ERK and Akt signaling pathways. Thus isoflavones, especially formononetin, may be useful as anti-cancer drugs for osteosarcoma through their apoptosis-inducing effects. © 2014 S. Karger AG, Basel.

Ecology and thermal inactivation of microbes in and on interplanetary space vehicle components

NASA Technical Reports Server (NTRS)

Campbell, J. E.; Reyes, A. L.; Wehby, A. J.; Crawford, R. G.; Wimsatt, J. C.; Peeler, J. T.

1974-01-01

Dry heat sterilization of spacecraft was investigated by studying the production of spore crops, and thermal inactivation of the spores, and bacillus subtillus. Spore assays were made by conventional plate count methods, and survival curves for the spores are presented. The results indicate that the inherent resistance of spores from a parent cell can be maintained.

Acidosis Differentially Modulates Inactivation in NaV1.2, NaV1.4, and NaV1.5 Channels

PubMed Central

Vilin, Yury Y.; Peters, Colin H.; Ruben, Peter C.

2012-01-01

NaV channels play a crucial role in neuronal and muscle excitability. Using whole-cell recordings we studied effects of low extracellular pH on the biophysical properties of NaV1.2, NaV1.4, and NaV1.5, expressed in cultured mammalian cells. Low pH produced different effects on different channel subtypes. Whereas NaV1.4 exhibited very low sensitivity to acidosis, primarily limited to partial block of macroscopic currents, the effects of low pH on gating in NaV1.2 and NaV1.5 were profound. In NaV1.2 low pH reduced apparent valence of steady-state fast inactivation, shifted the τ(V) to depolarizing potentials and decreased channels availability during onset to slow and use-dependent inactivation (UDI). In contrast, low pH delayed open-state inactivation in NaV1.5, right-shifted the voltage-dependence of window current, and increased channel availability during onset to slow and UDI. These results suggest that protons affect channel availability in an isoform-specific manner. A computer model incorporating these results demonstrates their effects on membrane excitability. PMID:22701426

[Preparation of the vaccine with inactivated Feline Panleukopenia Virus isolated from tiger and the preliminary application].

PubMed

Yu, Yali; Zhou, Ming; Zhang, Jin; Hua, Yuping; Wang, Ligang; Liu, Yinan; Liu, Dan; Xia, Xianzhu

2009-05-01

To prepare a vaccine with inactivated Feline Panleukopenia Virus (FPV) isolated from Siberian tigers and to evaluate its immunological effect. FPV-HLJ, an FPV strain previously isolated from Siberian tiger in our lab was used to inoculate cat kidney cell line F81 with dose 1/10(v/v) using the synchronizing inoculation method. Inoculated F81 cell line was cultured at 37 degrees C and collected when cytopathic effect was up to 75%. Viral suspension was inactivated by using formaldehyde for 24 h and inactive vaccine preapred by adding aluminium hydroxide gel as adjuvant to the suspension. The inactive vaccine was applied to 2-month-old kitten tigers of hypodermically after protection effect was proved in 2-month-old nonimmunized cats by using the same vaccination procedure. Antibody level in vaccinated cats revealed an increasing trend that the valence of antibody reached 1:1024-2048 after three times of vaccination. All vaccinated cats survived challenges of virulent FPV virus. The valence of antibody reached 1:1024 in most vaccinated tigers 15 days after the third vaccination. The results indicated that the inactivated vaccine can produce immunoprotection for tigers.

Kinetics of Hydrothermal Inactivation of Endotoxins ▿

PubMed Central

Li, Lixiong; Wilbur, Chris L.; Mintz, Kathryn L.

2011-01-01

A kinetic model was established for the inactivation of endotoxins in water at temperatures ranging from 210°C to 270°C and a pressure of 6.2 × 106 Pa. Data were generated using a bench scale continuous-flow reactor system to process feed water spiked with endotoxin standard (Escherichia coli O113:H10). Product water samples were collected and quantified by the Limulus amebocyte lysate assay. At 250°C, 5-log endotoxin inactivation was achieved in about 1 s of exposure, followed by a lower inactivation rate. This non-log-linear pattern is similar to reported trends in microbial survival curves. Predictions and parameters of several non-log-linear models are presented. In the fast-reaction zone (3- to 5-log reduction), the Arrhenius rate constant fits well at temperatures ranging from 120°C to 250°C on the basis of data from this work and the literature. Both biphasic and modified Weibull models are comparable to account for both the high and low rates of inactivation in terms of prediction accuracy and the number of parameters used. A unified representation of thermal resistance curves for a 3-log reduction and a 3 D value associated with endotoxin inactivation and microbial survival, respectively, is presented. PMID:21193667

Incorporation of DPP6a and DPP6K variants in ternary Kv4 channel complex reconstitutes properties of A-type K current in rat cerebellar granule cells.

PubMed

Jerng, Henry H; Pfaffinger, Paul J

2012-01-01

Dipeptidyl peptidase-like protein 6 (DPP6) proteins co-assemble with Kv4 channel α-subunits and Kv channel-interacting proteins (KChIPs) to form channel protein complexes underlying neuronal somatodendritic A-type potassium current (I(SA)). DPP6 proteins are expressed as N-terminal variants (DPP6a, DPP6K, DPP6S, DPP6L) that result from alternative mRNA initiation and exhibit overlapping expression patterns. Here, we study the role DPP6 variants play in shaping the functional properties of I(SA) found in cerebellar granule (CG) cells using quantitative RT-PCR and voltage-clamp recordings of whole-cell currents from reconstituted channel complexes and native I(SA) channels. Differential expression of DPP6 variants was detected in rat CG cells, with DPP6K (41 ± 3%)>DPP6a (33 ± 3%)>DPP6S (18 ± 2%)>DPP6L (8 ± 3%). To better understand how DPP6 variants shape native neuronal I(SA), we focused on studying interactions between the two dominant variants, DPP6K and DPP6a. Although previous studies did not identify unique functional effects of DPP6K, we find that the unique N-terminus of DPP6K modulates the effects of KChIP proteins, slowing recovery and producing a negative shift in the steady-state inactivation curve. By contrast, DPP6a uses its distinct N-terminus to directly confer rapid N-type inactivation independently of KChIP3a. When DPP6a and DPP6K are co-expressed in ratios similar to those found in CG cells, their distinct effects compete in modulating channel function. The more rapid inactivation from DPP6a dominates during strong depolarization; however, DPP6K produces a negative shift in the steady-state inactivation curve and introduces a slow phase of recovery from inactivation. A direct comparison to the native CG cell I(SA) shows that these mixed effects are present in the native channels. Our results support the hypothesis that the precise expression and co-assembly of different auxiliary subunit variants are important factors in shaping the I(SA) functional properties in specific neuronal populations.

Antagonistic Microbial Interactions: Contributions and Potential Applications for Controlling Pathogens in the Aquatic Systems

PubMed Central

Feichtmayer, Judith; Deng, Li; Griebler, Christian

2017-01-01

Despite the active and intense treatment of wastewater, pathogenic microorganisms and viruses are frequently introduced into the aquatic environment. For most human pathogens, however, this is a rather hostile place, where starvation, continuous inactivation, and decay generally occur, rather than successful reproduction. Nevertheless, a great diversity of the pathogenic microorganisms can be detected, in particular, in the surface waters receiving wastewater. Pathogen survival depends majorly on abiotic factors such as irradiation, changes in water ionic strength, temperature, and redox state. In addition, inactivation is enhanced by the biotic interactions in the environment. Although knowledge of the antagonistic biotic interactions has been available since a long time, certain underlying processes and mechanisms still remain unclear. Others are well-appreciated and increasingly are applied to the present research. Our review compiles and discusses the presently known biotic interactions between autochthonous microbes and pathogens introduced into the aquatic environment, including protozoan grazing, virus-induced bacterial cell lysis, antimicrobial substances, and predatory bacteria. An overview is provided on the present knowledge, as well as on the obvious research gaps. Individual processes that appear promising for future applications in the aquatic environment are presented and discussed. PMID:29184541

Sonic hedgehog controls growth of external genitalia by regulating cell cycle kinetics

PubMed Central

Seifert, Ashley W.; Zheng, Zhengui; Ormerod, Brandi K.; Cohn, Martin J.

2010-01-01

During embryonic development, cells are instructed which position to occupy, they interpret these cues as differentiation programmes, and expand these patterns by growth. Sonic hedgehog (Shh) specifies positional identity in many organs; however, its role in growth is not well understood. In this study, we show that inactivation of Shh in external genitalia extends the cell cycle from 8.5 to 14.4 h, and genital growth is reduced by ∼75%. Transient Shh signalling establishes pattern in the genital tubercle; however, transcriptional levels of G1 cell cycle regulators are reduced. Consequently, G1 length is extended, leading to fewer progenitor cells entering S-phase. Cell cycle genes responded similarly to Shh inactivation in genitalia and limbs, suggesting that Shh may regulate growth by similar mechanisms in different organ systems. The finding that Shh regulates cell number by controlling the length of specific cell cycle phases identifies a novel mechanism by which Shh elaborates pattern during appendage development. PMID:20975695

Highly efficient gene inactivation by adenoviral CRISPR/Cas9 in human primary cells

PubMed Central

Tielen, Frans; Elstak, Edo; Benschop, Julian; Grimbergen, Max; Stallen, Jan; Janssen, Richard; van Marle, Andre; Essrich, Christian

2017-01-01

Phenotypic assays using human primary cells are highly valuable tools for target discovery and validation in drug discovery. Expression knockdown (KD) of such targets in these assays allows the investigation of their role in models of disease processes. Therefore, efficient and fast modes of protein KD in phenotypic assays are required. The CRISPR/Cas9 system has been shown to be a versatile and efficient means of gene inactivation in immortalized cell lines. Here we describe the use of adenoviral (AdV) CRISPR/Cas9 vectors for efficient gene inactivation in two human primary cell types, normal human lung fibroblasts and human bronchial epithelial cells. The effects of gene inactivation were studied in the TGF-β-induced fibroblast to myofibroblast transition assay (FMT) and the epithelial to mesenchymal transition assay (EMT), which are SMAD3 dependent and reflect pathogenic mechanisms observed in fibrosis. Co-transduction (co-TD) of AdV Cas9 with SMAD3-targeting guide RNAs (gRNAs) resulted in fast and efficient genome editing judged by insertion/deletion (indel) formation, as well as significant reduction of SMAD3 protein expression and nuclear translocation. This led to phenotypic changes downstream of SMAD3 inhibition, including substantially decreased alpha smooth muscle actin and fibronectin 1 expression, which are markers for FMT and EMT, respectively. A direct comparison between co-TD of separate Cas9 and gRNA AdV, versus TD with a single “all-in-one” Cas9/gRNA AdV, revealed that both methods achieve similar levels of indel formation. These data demonstrate that AdV CRISPR/Cas9 is a useful and efficient tool for protein KD in human primary cell phenotypic assays. The use of AdV CRISPR/Cas9 may offer significant advantages over the current existing tools and should enhance target discovery and validation opportunities. PMID:28800587

Downregulation of lncRNA H19 inhibits the migration and invasion of melanoma cells by inactivating the NF‑κB and PI3K/Akt signaling pathways.

PubMed

Liao, Zhichao; Zhao, Jun; Yang, Yun

2018-05-01

As the most aggressive type of skin cancer, melanoma seriously affects human health. Long noncoding (lncRNA) 19 has been demonstrated to be involved in the progression of a number of different types of human cancers. However, the involvement of lncRNA H19 in melanoma remains unknown. Therefore, the present study was performed to investigate the roles of H19 in the development and progression of melanoma. In the present study, 49 patients with melanoma were included. Expression of lncRNA H19 in tumor tissue, adjacent healthy tissue and various cell lines with different treatments was measured by reverse transcription‑quantitative polymerase chain reaction. The effects of H19 knockdown on melanoma cell proliferation, migration and invasion were detected by cell counting kit‑8, wound‑healing and transwell invasion assays, respectively. In addition, the effects of H19 knockdown on the expression of nuclear factor (NF)‑κB pathway‑associated proteins were investigated by western blotting. The results revealed that the expression level of H19 was significantly higher in tumor tissue than in the adjacent healthy tissue of 47 out of 49 patients. H19 knockdown significantly reduced the proliferation, migration and invasion ability of melanoma cells. H19 knockdown also inactivated the phosphoinositide 3‑kinase (PI3K)/protein kinase B (Akt) pathway, which in turn inhibited the activation of the NF‑κB signaling pathway. Thus, downregulation of lncRNA H19 may inhibit the migration and invasion of melanoma cells by inactivating the NF‑κB signaling pathway via the inactivation of the PI3K/Akt signaling pathway. The present study provided references for future studies on the pathogenesis of melanoma and the clinical treatment of this disease.

Silibinin inhibits migration and invasion of the rhabdoid tumor G401 cell line via inactivation of the PI3K/Akt signaling pathway.

PubMed

Li, Yumei; Zhang, Chunmei; Cai, Danfeng; Chen, Congde; Mu, Dongmei

2017-12-01

Rhabdoid tumors, which tend to occur prior to the age of 2 years, are one of the most aggressive malignancies and have a poor prognosis due to the frequency of metastasis. Silibinin, a natural extract, has been approved as a potential tumor suppressor in various studies, however, whether or not it also exerts its antitumor capacity in rhabdoid tumors, particularly with regards to tumor migration and invasion, is unclear. The rhabdoid tumor G401 cell line was used in the present in vitro study. An MTT assay was used to assess the cytotoxicity of silibinin on G401 cells, cell migration was studied using a wound healing assay and a Transwell migration assay, and cell invasion was determined using a Transwell invasion assay. The underlying mechanism in silibinin inhibited cell migration and invasion was investigated by western blot analysis and further confirmed using a specific inhibitor. Experimental results demonstrated that high doses of silibinin suppressed cell viability, and that low doses of silibinin inhibited cell migration and invasion without affecting cell proliferation. The phosphatidylinositol 3-kinase/protein kinase B (PI3K/Akt) signaling pathway was involved in the silibinin-induced inhibition of metastasis. Silibinin inactivated the PI3K/Akt pathway, and inhibited cell migration and invasion, an effect that was further enhanced when LY294002, a classic PI3K inhibitor, was used concurrently. In general, silibinin inhibits migration and invasion of the rhabdoid tumor G401 cell line via inactivation of the PI3K/Akt signaling pathway and may be a potential chemotherapeutic drug to combat rhabdoid tumors in the future.

Protection from oxidative inactivation of the 20S proteasome by heat-shock protein 90.

PubMed Central

Conconi, M; Petropoulos, I; Emod, I; Turlin, E; Biville, F; Friguet, B

1998-01-01

Heat-shock protein 90 (Hsp 90) has been implicated in both protection against oxidative inactivation and inhibition of the multicatalytic proteinase (MCP, also known as 20 S proteasome). We report here that the protective and inhibitory effects of Hsp 90 depend on the activation state of the proteasome. Hsp 90 (and also alpha-crystallin) inhibits the N-Cbz-Leu-Leu-Leu-MCA-hydrolysing activity (Cbz=benzyloxycarbonyl; MCA=7-amido-4-methylcoumarin) when the rat liver MCP is in its latent form, but no inhibitory effects are observed when the MCP is in its active form. Metal-catalysed oxidation of the active MCP inactivates the Ala-Ala-Phe-MCA-hydrolysing (chymotrypsin-like), N-Boc-Leu-Ser-Thr-Arg-MCA-hydrolysing (trypsin-like; Boc=t-butyloxycarbonyl), N-Cbz-Leu-Leu-Glu-beta-naphthylamine-hydrolysing (peptidylglutamyl-peptide hydrolase) and N-Cbz-Leu-Leu-Leu-MCA-hydrolysing activities, whereas these activities are actually increased when the MCP is in its latent form. Hsp 90 protects against oxidative inactivation of the trypsin-like and N-Cbz-Leu-Leu-Leu-MCA-hydrolysing activities of the MCP active form, and alpha-crystallin protects the trypsin-like activity. The specificity of the Hsp 90-mediated protection was assessed by a quantitative analysis of the two-dimensional electrophoretic pattern of MCP subunits before and after oxidation of the MCP, in the presence or absence of Hsp 90. Treatment of the FAO hepatoma cell line with iron and ascorbate was found to inactivate the MCP. Hsp 90 overexpression obtained by challenging the cells with iron was associated with a decreased susceptibility to oxidative inactivation of the MCP trypsin-like activity. Depletion of Hsp 90 by using antisense oligonucleotides resulted in an increased susceptibility to oxidative inactivation of the MCP trypsin-like activity, providing evidence for the physiological relevance of Hsp 90-mediated protection of the MCP. PMID:9657982

Protection from oxidative inactivation of the 20S proteasome by heat-shock protein 90.

PubMed

Conconi, M; Petropoulos, I; Emod, I; Turlin, E; Biville, F; Friguet, B

1998-07-15

Heat-shock protein 90 (Hsp 90) has been implicated in both protection against oxidative inactivation and inhibition of the multicatalytic proteinase (MCP, also known as 20 S proteasome). We report here that the protective and inhibitory effects of Hsp 90 depend on the activation state of the proteasome. Hsp 90 (and also alpha-crystallin) inhibits the N-Cbz-Leu-Leu-Leu-MCA-hydrolysing activity (Cbz=benzyloxycarbonyl; MCA=7-amido-4-methylcoumarin) when the rat liver MCP is in its latent form, but no inhibitory effects are observed when the MCP is in its active form. Metal-catalysed oxidation of the active MCP inactivates the Ala-Ala-Phe-MCA-hydrolysing (chymotrypsin-like), N-Boc-Leu-Ser-Thr-Arg-MCA-hydrolysing (trypsin-like; Boc=t-butyloxycarbonyl), N-Cbz-Leu-Leu-Glu-beta-naphthylamine-hydrolysing (peptidylglutamyl-peptide hydrolase) and N-Cbz-Leu-Leu-Leu-MCA-hydrolysing activities, whereas these activities are actually increased when the MCP is in its latent form. Hsp 90 protects against oxidative inactivation of the trypsin-like and N-Cbz-Leu-Leu-Leu-MCA-hydrolysing activities of the MCP active form, and alpha-crystallin protects the trypsin-like activity. The specificity of the Hsp 90-mediated protection was assessed by a quantitative analysis of the two-dimensional electrophoretic pattern of MCP subunits before and after oxidation of the MCP, in the presence or absence of Hsp 90. Treatment of the FAO hepatoma cell line with iron and ascorbate was found to inactivate the MCP. Hsp 90 overexpression obtained by challenging the cells with iron was associated with a decreased susceptibility to oxidative inactivation of the MCP trypsin-like activity. Depletion of Hsp 90 by using antisense oligonucleotides resulted in an increased susceptibility to oxidative inactivation of the MCP trypsin-like activity, providing evidence for the physiological relevance of Hsp 90-mediated protection of the MCP.

Cryo-gamma radiation inactivation of bovine herpesvirus type-1

NASA Astrophysics Data System (ADS)

Degiorgi, C. Fernández; Smolko, E. E.; Lombardo, J. H.

1999-07-01

The radioresistance of bovine herpesvirus-1 (BHV-1), commonly known as infectious bovine rhinotracheitis virus (IBRV), suspended in free serum Glasgow-MEM medium and frozen at -78°C was studied. The number of surviving virus at a given dose of gamma-radiation was determined by a plaque assay system. D 10 values were calculated before and after removal of cell debris. The D 10 values obtained were 4.72 kGy and 7.31 kGy before and after removal of cell debris, respectively. Our results indicate that the inactivated viral particles could be used for vaccine preparation or diagnostic reagents.

Bactericidal Effect of Zero-Valent Iron Nanoparticles on Escherichia coli

PubMed Central

Lee, Changha; Kim, Jee Yeon; Lee, Won Il; Nelson, Kara L.; Yoon, Jeyong; Sedlak, David L.

2008-01-01

Zero-valent iron nanoparticles (nano-Fe0) in aqueous solution rapidly inactivated Escherichia coli (E. coli). A strong bactericidal effect of nano-Fe0 was found under deaerated conditions, with a linear correlation between log inactivation and nano-Fe0 dose (0.82 log inactivation / mg/L nano-Fe0 · hr). The inactivation of E. coli under air saturation required much higher nano-Fe0 doses due to the corrosion and surface oxidation of nano-Fe0 by dissolved oxygen. Significant physical disruption of the cell membranes was observed in E. coli exposed to nano-Fe0, which may have caused the inactivation, or enhanced the biocidal effects of dissolved iron. The reaction of Fe(II) with intracellular oxygen or hydrogen peroxide also may have induced oxidative stress by producing reactive oxygen species. The bactericidal effect of nano-Fe0 was a unique property of nano-Fe0, which was not observed in other types of iron-based compounds. PMID:18678028

Protein Phosphatase 1 inactivates Mps1 to ensure efficient Spindle Assembly Checkpoint silencing.

PubMed

Moura, Margarida; Osswald, Mariana; Leça, Nelson; Barbosa, João; Pereira, António J; Maiato, Helder; Sunkel, Claudio E; Conde, Carlos

2017-05-02

Faithfull genome partitioning during cell division relies on the Spindle Assembly Checkpoint (SAC), a conserved signaling pathway that delays anaphase onset until all chromosomes are attached to spindle microtubules. Mps1 kinase is an upstream SAC regulator that promotes the assembly of an anaphase inhibitor through a sequential multi-target phosphorylation cascade. Thus, the SAC is highly responsive to Mps1, whose activity peaks in early mitosis as a result of its T-loop autophosphorylation. However, the mechanism controlling Mps1 inactivation once kinetochores attach to microtubules and the SAC is satisfied remains unknown. Here we show in vitro and in Drosophila that Protein Phosphatase 1 (PP1) inactivates Mps1 by dephosphorylating its T-loop. PP1-mediated dephosphorylation of Mps1 occurs at kinetochores and in the cytosol, and inactivation of both pools of Mps1 during metaphase is essential to ensure prompt and efficient SAC silencing. Overall, our findings uncover a mechanism of SAC inactivation required for timely mitotic exit.

Centromere inactivation on a neo-Y fusion chromosome in threespine stickleback fish

PubMed Central

Cech, Jennifer N.; Peichel, Catherine L.

2016-01-01

Having one and only one centromere per chromosome is essential for proper chromosome segregation during both mitosis and meiosis. Chromosomes containing two centromeres are known as dicentric and often mis-segregate during cell division, resulting in aneuploidy or chromosome breakage. Dicentric chromosome can be stabilized by centromere inactivation, a process which re-establishes monocentric chromosomes. However, little is known about this process in naturally occurring dicentric chromosomes. Using a combination of fluorescence in situ hybridization (FISH) and immunoflourescence combined with FISH (IF-FISH) on metaphase chromosome spreads, we demonstrate that centromere inactivation has evolved on a neo-Y chromosome fusion in the Japan Sea threespine stickleback fish (Gasterosteus nipponicus). We found that the centromere derived from the ancestral Y chromosome has been inactivated. Our data further suggest that there have been genetic changes to this centromere in the two million years since the formation of the neo-Y chromosome, but it remains unclear whether these genetic changes are a cause or consequence of centromere inactivation. PMID:27553478

Effective Thermal Inactivation of the Spores of Bacillus cereus Biofilms Using Microwave.

PubMed

Park, Hyong Seok; Yang, Jungwoo; Choi, Hee Jung; Kim, Kyoung Heon

2017-07-28

Microwave sterilization was performed to inactivate the spores of biofilms of Bacillus cereus involved in foodborne illness. The sterilization conditions, such as the amount of water and the operating temperature and treatment time, were optimized using statistical analysis based on 15 runs of experimental results designed by the Box-Behnken method. Statistical analysis showed that the optimal conditions for the inactivation of B. cereus biofilms were 14 ml of water, 108°C of temperature, and 15 min of treatment time. Interestingly, response surface plots showed that the amount of water is the most important factor for microwave sterilization under the present conditions. Complete inactivation by microwaves was achieved in 5 min, and the inactivation efficiency by microwave was obviously higher than that by conventional steam autoclave. Finally, confocal laser scanning microscopy images showed that the principal effect of microwave treatment was cell membrane disruption. Thus, this study can contribute to the development of a process to control food-associated pathogens.

Bacteria-instructed synthesis of polymers for self-selective microbial binding and labelling

PubMed Central

Magennis, E. Peter; Fernandez-Trillo, Francisco; Sui, Cheng; Spain, Sebastian G.; Bradshaw, David; Churchley, David; Mantovani, Giuseppe; Winzer, Klaus; Alexander, Cameron

2014-01-01

The detection and inactivation of pathogenic strains of bacteria continues to be an important therapeutic goal. Hence, there is a need for materials that can bind selectively to specific microorganisms, for diagnostic or anti-infective applications, but which can be formed from simple and inexpensive building blocks. Here, we exploit bacterial redox systems to induce a copper-mediated radical polymerisation of synthetic monomers at cell surfaces, generating polymers in situ that bind strongly to the microorganisms which produced them. This ‘bacteria-instructed synthesis’ can be carried out with a variety of microbial strains, and we show that the polymers produced are self-selective binding agents for the ‘instructing’ cell types. We further expand on the bacterial redox chemistries to ‘click’ fluorescent reporters onto polymers directly at the surfaces of a range of clinical isolate strains, allowing rapid, facile and simultaneous binding and visualisation of pathogens. PMID:24813421

Bacteria-instructed synthesis of polymers for self-selective microbial binding and labelling

NASA Astrophysics Data System (ADS)

Magennis, E. Peter; Fernandez-Trillo, Francisco; Sui, Cheng; Spain, Sebastian G.; Bradshaw, David J.; Churchley, David; Mantovani, Giuseppe; Winzer, Klaus; Alexander, Cameron

2014-07-01

The detection and inactivation of pathogenic strains of bacteria continues to be an important therapeutic goal. Hence, there is a need for materials that can bind selectively to specific microorganisms for diagnostic or anti-infective applications, but that can be formed from simple and inexpensive building blocks. Here, we exploit bacterial redox systems to induce a copper-mediated radical polymerization of synthetic monomers at cell surfaces, generating polymers in situ that bind strongly to the microorganisms that produced them. This ‘bacteria-instructed synthesis’ can be carried out with a variety of microbial strains, and we show that the polymers produced are self-selective binding agents for the ‘instructing’ cell types. We further expand on the bacterial redox chemistries to ‘click’ fluorescent reporters onto polymers directly at the surfaces of a range of clinical isolate strains, allowing rapid, facile and simultaneous binding and visualization of pathogens.

Artemin protects cells and proteins against oxidative and salt stress.

PubMed

Takalloo, Zeinab; Sajedi, Reza H; Hosseinkhani, Saman; Moazzenzade, Taghi

2017-02-01

Artemin is an abundant thermostable protein in Artemia encysted embryos under environmental stresses. It is confirmed that high regulatory expression of artemin is relevant to stress resistance in this crustacean. Here, the protective role of artemin from Artemia urmiana has been investigated on survival of bacterial cells under salt and oxidative shocks. Also, for continuous monitoring of the effect of artemin in prevention of proteins aggregation/inactivation, co-expression of artemin and luciferase (as an intracellular reporter) in bacterial cells was performed. According to the results, residual activity of luciferase in artemin expressing E. coli cells exposing to different concentrations of H 2 O 2 and NaCl was significantly higher than non-expressing cells. The luciferase activity was rapidly lost in control cells under salt treatments while in co-transformed cells, the activity was considerably retained at higher salt concentrations. Also, analysis from cell viability assays showed that artemin-expressing cells exhibited more resistance to both stress conditions. In the present study, we document for the first time that artemin can protect proteins and bacterial cells against oxidative and salt stress conditions. These results can declare the resistance property of this crustacean against harsh environmental conditions. Copyright © 2016 Elsevier B.V. All rights reserved.

Reduction of spiked porcine circovirus during the manufacture of a Vero cell-derived vaccine.

PubMed

Lackner, Cornelia; Leydold, Sandra M; Modrof, Jens; Farcet, Maria R; Grillberger, Leopold; Schäfer, Birgit; Anderle, Heinz; Kreil, Thomas R

2014-04-11

Porcine circovirus-1 (PCV1) was recently identified as a contaminant in live Rotavirus vaccines, which was likely caused by contaminated porcine trypsin. The event triggered the development of new regulatory guidance on the use of porcine trypsin which shall ensure that cell lines and porcine trypsin in use are free from PCV1. In addition, manufacturing processes of biologicals other than live vaccines include virus clearance steps that may prevent and mitigate any potential virus contamination of product. In this work, artificial spiking of down-scaled models for the manufacturing process of an inactivated pandemic influenza virus vaccine were used to investigate inactivation of PCV1 and the physico-chemically related porcine parvovirus (PPV) by formalin and ultraviolet-C (UV-C) treatment as well as removal by the purification step sucrose gradient ultracentrifugation. A PCV1 infectivity assay, using a real-time PCR infectivity readout was established. The formalin treatment (0.05% for 48h) showed substantial inactivation for both PCV1 and PPV with reduction factors of 3.0log10 and 6.8log10, respectively, whereas UV-C treatment resulted in complete PPV (≥5.9log10) inactivation already at a dose of 13mJ/cm but merely 1.7log10 at 24mJ/cm(2) for PCV1. The UV-C inactivation results with PPV were confirmed using minute virus of mice (MVM), indicating that parvoviruses are far more sensitive to UV-C than PCV1. The sucrose density gradient ultracentrifugation also contributed to PCV1 clearance with a reduction factor of 2log10. The low pH treatment during the production of procine trypsin was investigated and showed effective inactivation for both PCV1 (4.5log10) and PPV (6.4log10). In conclusion, PCV1 in general appears to be more resistant to virus inactivation than PPV. Still, the inactivated pandemic influenza vaccine manufacturing process provides for robust virus reduction, in addition to the already implemented testing for PCV1 to avoid any contaminations. Copyright © 2014 Elsevier Ltd. All rights reserved.

Towards pathogen inactivation of red blood cells and whole blood targeting viral DNA/RNA: design, technologies, and future prospects for developing countries.

PubMed

Drew, Victor J; Barro, Lassina; Seghatchian, Jerard; Burnouf, Thierry

2017-10-01

Over 110 million units of blood are collected yearly. The need for blood products is greater in developing countries, but so is the risk of contracting a transfusion-transmitted infection. Without efficient donor screening/viral testing and validated pathogen inactivation technology, the risk of transfusion-transmitted infections correlates with the infection rate of the donor population. The World Health Organization has published guidelines on good manufacturing practices in an effort to ensure a strong global standard of transfusion and blood product safety. Sub-Saharan Africa is a high-risk region for malaria, human immunodeficiency virus (HIV), hepatitis B virus and syphilis. Southeast Asia experiences high rates of hepatitis C virus. Areas with a tropical climate have an increased risk of Zika virus, Dengue virus, West Nile virus and Chikungunya, and impoverished countries face economical limitations which hinder efforts to acquire the most modern pathogen inactivation technology. These systems include Mirasol ® Pathogen Reduction Technology, INTERCEPT ® , and THERAFLEX ® . Their procedures use a chemical and ultraviolet or visible light for pathogen inactivation and significantly decrease the threat of pathogen transmission in plasma and platelets. They are licensed for use in Europe and are used in several other countries. The current interest in the blood industry is the development of pathogen inactivation technologies that can treat whole blood (WB) and red blood cell (RBC). The Mirasol system has recently undergone phase III clinical trials for treating WB in Ghana and has demonstrated some efficacy toward malaria inactivation and low risk of adverse effects. A 2 nd -generation of the INTERCEPT ® S-303 system for WB is currently undergoing a phase III clinical trial. Both methodologies are applicable for WB and components derived from virally reduced WB or RBC.

Defective Fast Inactivation Recovery of Nav1.4 in Congenital Myasthenic Syndrome

PubMed Central

Arnold, W. David; Feldman, Daniel H.; Ramirez, Sandra; He, Liuyuan; Kassar, Darine; Quick, Adam; Klassen, Tara L.; Lara, Marian; Nguyen, Joanna; Kissel, John T.; Lossin, Christoph; Maselli, Ricardo A.

2015-01-01

Objective To describe the unique phenotype and genetic findings in a 57-year-old female with a rare form of congenital myasthenic syndrome (CMS) associated with longstanding muscle fatigability, and to investigate the underlying pathophysiology. Methods We used whole-cell voltage clamping to compare the biophysical parameters of wild-type and Arg1457His-mutant Nav1.4. Results Clinical and neurophysiological evaluation revealed features consistent with CMS. Sequencing of candidate genes indicated no abnormalities. However, analysis of SCN4A, the gene encoding the skeletal muscle sodium channel Nav1.4, revealed a homozygous mutation predicting an arginine-to-histidine substitution at position 1457 (Arg1457His), which maps to the channel’s voltage sensor, specifically D4/S4. Whole-cell patch clamp studies revealed that the mutant required longer hyperpolarization to recover from fast inactivation, which produced a profound use-dependent current attenuation not seen in the wild type. The mutant channel also had a marked hyperpolarizing shift in its voltage dependence of inactivation as well as slowed inactivation kinetics. Interpretation We conclude that Arg1457His compromises muscle fiber excitability. The mutant fast-inactivates with significantly less depolarization, and it recovers only after extended hyperpolarization. The resulting enhancement in its use dependence reduces channel availability, which explains the patient’s muscle fatigability. Arg1457His offers molecular insight into a rare form of CMS precipitated by sodium channel inactivation defects. Given this channel’s involvement in other muscle disorders such as paramyotonia congenita and hyperkalemic periodic paralysis, our study exemplifies how variations within the same gene can give rise to multiple distinct dysfunctions and phenotypes, revealing residues important in basic channel function. PMID:25707578

Towards pathogen inactivation of red blood cells and whole blood targeting viral DNA/RNA: design, technologies, and future prospects for developing countries

PubMed Central

Drew, Victor J.; Barro, Lassina; Seghatchian, Jerard; Burnouf, Thierry

2017-01-01

Over 110 million units of blood are collected yearly. The need for blood products is greater in developing countries, but so is the risk of contracting a transfusion-transmitted infection. Without efficient donor screening/viral testing and validated pathogen inactivation technology, the risk of transfusion-transmitted infections correlates with the infection rate of the donor population. The World Health Organization has published guidelines on good manufacturing practices in an effort to ensure a strong global standard of transfusion and blood product safety. Sub-Saharan Africa is a high-risk region for malaria, human immunodeficiency virus (HIV), hepatitis B virus and syphilis. Southeast Asia experiences high rates of hepatitis C virus. Areas with a tropical climate have an increased risk of Zika virus, Dengue virus, West Nile virus and Chikungunya, and impoverished countries face economical limitations which hinder efforts to acquire the most modern pathogen inactivation technology. These systems include Mirasol® Pathogen Reduction Technology, INTERCEPT®, and THERAFLEX®. Their procedures use a chemical and ultraviolet or visible light for pathogen inactivation and significantly decrease the threat of pathogen transmission in plasma and platelets. They are licensed for use in Europe and are used in several other countries. The current interest in the blood industry is the development of pathogen inactivation technologies that can treat whole blood (WB) and red blood cell (RBC). The Mirasol system has recently undergone phase III clinical trials for treating WB in Ghana and has demonstrated some efficacy toward malaria inactivation and low risk of adverse effects. A 2nd-generation of the INTERCEPT® S-303 system for WB is currently undergoing a phase III clinical trial. Both methodologies are applicable for WB and components derived from virally reduced WB or RBC. PMID:28488960

The voltage sensor of excitation–contraction coupling in mammals: Inactivation and interaction with Ca2+

PubMed Central

2017-01-01

In skeletal muscle, the four-helix voltage-sensing modules (VSMs) of CaV1.1 calcium channels simultaneously gate two Ca2+ pathways: the CaV1.1 pore itself and the RyR1 calcium release channel in the sarcoplasmic reticulum. Here, to gain insight into the mechanism by which VSMs gate RyR1, we quantify intramembrane charge movement associated with VSM activation (sensing current) and gated Ca2+ release flux in single muscle cells of mice and rats. As found for most four-helix VSMs, upon sustained depolarization, rodent VSMs lose the ability to activate Ca2+ release channels opening; their properties change from a functionally capable mode, in which the mobile sensor charge is called charge 1, to an inactivated mode, charge 2, with a voltage dependence shifted toward more negative voltages. We find that charge 2 is promoted and Ca2+ release inactivated when resting, well-polarized muscle cells are exposed to low extracellular [Ca2+] and that the opposite occurs in high [Ca2+]. It follows that murine VSMs are partly inactivated at rest, which establishes the reduced availability of voltage sensing as a pathogenic mechanism in disorders of calcemia. We additionally find that the degree of resting inactivation is significantly different in two mouse strains, which underscores the variability of voltage sensor properties and their vulnerability to environmental conditions. Our studies reveal that the resting and activated states of VSMs are equally favored by extracellular Ca2+. Promotion by an extracellular species of two states of the VSM that differ in the conformation of the activation gate requires the existence of a second gate, inactivation, topologically extracellular and therefore accessible from outside regardless of the activation state. PMID:29021148

In Silico Docking and Electrophysiological Characterization of Lacosamide Binding Sites on Collapsin Response Mediator Protein-2 Identifies a Pocket Important in Modulating Sodium Channel Slow Inactivation*

PubMed Central

Wang, Yuying; Brittain, Joel M.; Jarecki, Brian W.; Park, Ki Duk; Wilson, Sarah M.; Wang, Bo; Hale, Rachel; Meroueh, Samy O.; Cummins, Theodore R.; Khanna, Rajesh

2010-01-01

The anti-epileptic drug (R)-lacosamide ((2R)-2-(acetylamino)-N-benzyl-3-methoxypropanamide (LCM)) modulates voltage-gated sodium channels (VGSCs) by preferentially interacting with slow inactivated sodium channels, but the observation that LCM binds to collapsin response mediator protein 2 (CRMP-2) suggests additional mechanisms of action for LCM. We postulated that CRMP-2 levels affects the actions of LCM on VGSCs. CRMP-2 labeling by LCM analogs was competitively displaced by excess LCM in rat brain lysates. Manipulation of CRMP-2 levels in the neuronal model system CAD cells affected slow inactivation of VGSCs without any effects on other voltage-dependent properties. In silico docking was performed to identify putative binding sites in CRMP-2 that may modulate the effects of LCM on VGSCs. These studies identified five cavities in CRMP-2 that can accommodate LCM. CRMP-2 alanine mutants of key residues within these cavities were functionally similar to wild-type CRMP-2 as assessed by similar levels of enhancement in dendritic complexity of cortical neurons. Next, we examined the effects of expression of wild-type and mutant CRMP-2 constructs on voltage-sensitive properties of VGSCs in CAD cells: 1) steady-state voltage-dependent activation and fast-inactivation properties were not affected by LCM, 2) CRMP-2 single alanine mutants reduced the LCM-mediated effects on the ability of endogenous Na+ channels to transition to a slow inactivated state, and 3) a quintuplicate CRMP-2 alanine mutant further decreased this slow inactivated fraction. Collectively, these results identify key CRMP-2 residues that can coordinate LCM binding thus making it more effective on its primary clinical target. PMID:20538611

Arid1a Inactivation in an Apc and Pten-defective Mouse Ovarian Cancer Model Enhances Epithelial Differentiation and Prolongs Survival

PubMed Central

Zhai, Yali; Kuick, Rork; Tipton, Courtney; Wu, Rong; Sessine, Michael; Wang, Zhong; Baker, Suzanne J.; Fearon, Eric R.; Cho, Kathleen R.

2015-01-01

Inactivation of the ARID1A tumor suppressor gene is frequent in ovarian endometrioid (OEC) and clear cell carcinomas (OCCC), often in conjunction with mutations activating the PI3K/AKT and/or canonical Wnt signaling pathways. Prior work has shown that conditional bi-allelic inactivation of the Apc and Pten tumor suppressor genes in the mouse ovarian surface epithelium (OSE) promotes outgrowth of tumors that reflect the biological behavior and gene expression profiles of human OECs harboring comparable Wnt and PI3K/AKT pathway defects, though the mouse tumors are more poorly differentiated than their human tumor counterparts. We found that conditional inactivation of one or both Arid1a alleles in OSE concurrently with Apc and Pten inactivation unexpectedly prolonged survival of tumor-bearing mice and promoted striking epithelial differentiation of the cancer cells, resulting in morphological features akin to those in human OECs. Enhanced epithelial differentiation was linked to reduced expression of mesenchymal markers N-cadherin and vimentin, and increased expression of epithelial markers Crb3 and E-cadherin. Global gene expression profiling showed enrichment for genes associated with mesenchymal-to-epithelial transition in the Arid1a-deficient tumors. We also found that an activating (E545K) Pik3ca mutation, unlike Pten inactivation or Pik3ca H1047R mutation, cannot cooperate with Arid1a loss to promote ovarian cancer development in the mouse. Our results indicate the Arid1a tumor suppressor gene has a key role in regulating OEC differentiation, and paradoxically the mouse cancers with more initiating tumor suppressor gene defects had a less aggressive phenotype than cancers arising from fewer gene alterations. PMID:26279473

A double tyrosine motif in the cardiac sodium channel domain III-IV linker couples calcium-dependent calmodulin binding to inactivation gating.

PubMed

Sarhan, Maen F; Van Petegem, Filip; Ahern, Christopher A

2009-11-27

Voltage-gated sodium channels maintain the electrical cadence and stability of neurons and muscle cells by selectively controlling the transmembrane passage of their namesake ion. The degree to which these channels contribute to cellular excitability can be managed therapeutically or fine-tuned by endogenous ligands. Intracellular calcium, for instance, modulates sodium channel inactivation, the process by which sodium conductance is negatively regulated. We explored the molecular basis for this effect by investigating the interaction between the ubiquitous calcium binding protein calmodulin (CaM) and the putative sodium channel inactivation gate composed of the cytosolic linker between homologous channel domains III and IV (DIII-IV). Experiments using isothermal titration calorimetry show that CaM binds to a novel double tyrosine motif in the center of the DIII-IV linker in a calcium-dependent manner, N-terminal to a region previously reported to be a CaM binding site. An alanine scan of aromatic residues in recombinant DIII-DIV linker peptides shows that whereas multiple side chains contribute to CaM binding, two tyrosines (Tyr(1494) and Tyr(1495)) play a crucial role in binding the CaM C-lobe. The functional relevance of these observations was then ascertained through electrophysiological measurement of sodium channel inactivation gating in the presence and absence of calcium. Experiments on patch-clamped transfected tsA201 cells show that only the Y1494A mutation of the five sites tested renders sodium channel steady-state inactivation insensitive to cytosolic calcium. The results demonstrate that calcium-dependent calmodulin binding to the sodium channel inactivation gate double tyrosine motif is required for calcium regulation of the cardiac sodium channel.

The mechanism of photosystem-II inactivation during sulphur deprivation-induced H2 production in Chlamydomonas reinhardtii.

PubMed

Nagy, Valéria; Vidal-Meireles, André; Podmaniczki, Anna; Szentmihályi, Klára; Rákhely, Gábor; Zsigmond, Laura; Kovács, László; Tóth, Szilvia Z

2018-05-01

Sulphur limitation may restrain cell growth and viability. In the green alga Chlamydomonas reinhardtii, sulphur limitation may induce H 2 production lasting for several days, which can be exploited as a renewable energy source. Sulphur limitation causes a large number of physiological changes, including the inactivation of photosystem II (PSII), leading to the establishment of hypoxia, essential for the increase in hydrogenase expression and activity. The inactivation of PSII has long been assumed to be caused by the sulphur-limited turnover of its reaction center protein PsbA. Here we reinvestigated this issue in detail and show that: (i) upon transferring Chlamydomonas cells to sulphur-free media, the cellular sulphur content decreases only by about 25%; (ii) as demonstrated by lincomycin treatments, PsbA has a significant turnover, and other photosynthetic subunits, namely RbcL and CP43, are degraded more rapidly than PsbA. On the other hand, sulphur limitation imposes oxidative stress early on, most probably involving the formation of singlet oxygen in PSII, which leads to an increase in the expression of GDP-L-galactose phosphorylase, playing an essential role in ascorbate biosynthesis. When accumulated to the millimolar concentration range, ascorbate may inactivate the oxygen-evolving complex and provide electrons to PSII, albeit at a low rate. In the absence of a functional donor side and sufficient electron transport, PSII reaction centers are inactivated and degraded. We therefore demonstrate that the inactivation of PSII is a complex and multistep process, which may serve to mitigate the damaging effects of sulphur limitation. © 2018 The Authors The Plant Journal © 2018 John Wiley & Sons Ltd.

Surface water disinfection by chlorination and advanced oxidation processes: Inactivation of an antibiotic resistant E. coli strain and cytotoxicity evaluation.

PubMed

Miranda, Andreza Costa; Lepretti, Marilena; Rizzo, Luigi; Caputo, Ivana; Vaiano, Vincenzo; Sacco, Olga; Lopes, Wilton Silva; Sannino, Diana

2016-06-01

The release of antibiotics into the environment can result in antibiotic resistance (AR) spread, which in turn can seriously affect human health. Antibiotic resistant bacteria have been detected in different aquatic environments used as drinking water source. Water disinfection may be a possible solution to minimize AR spread but conventional processes, such as chlorination, result in the formation of dangerous disinfection by-products. In this study advanced oxidation processes (AOPs), namely H2O2/UV, TiO2/UV and N-TiO2/UV, have been compared with chlorination in the inactivation of an AR Escherichia coli (E. coli) strain in surface water. TiO2 P25 and nitrogen doped TiO2 (N-TiO2), prepared by sol-gel method at two different synthesis temperatures (0 and -20°C), were investigated in heterogeneous photocatalysis experiments. Under the investigated conditions, chlorination (1.0 mg L(-1)) was the faster process (2.5 min) to achieve total inactivation (6 Log). Among AOPs, H2O2/UV resulted in the best inactivation rate: total inactivation (6 Log) was achieved in 45 min treatment. Total inactivation was not observed (4.5 Log), also after 120 min treatment, only for N-doped TiO2 synthesized at 0°C. Moreover, H2O2/UV and chlorination processes were evaluated in terms of cytotoxicity potential by means of 3-(4,5-dime-thylthiazol-2-yl)-2,5-diphenylte-trazolium colorimetric test on a human-derived cell line and they similarly affected HepG2 cells viability. Copyright © 2016 Elsevier B.V. All rights reserved.

Calmodulin and calcium differentially regulate the neuronal Nav1.1 voltage-dependent sodium channel

DOE Office of Scientific and Technical Information (OSTI.GOV)

Gaudioso, Christelle; Carlier, Edmond; Youssouf, Fahamoe

2011-07-29

Highlights: {yields} Both Ca{sup ++}-Calmodulin (CaM) and Ca{sup ++}-free CaM bind to the C-terminal region of Nav1.1. {yields} Ca{sup ++} and CaM have both opposite and convergent effects on I{sub Nav1.1}. {yields} Ca{sup ++}-CaM modulates I{sub Nav1.1} amplitude. {yields} CaM hyperpolarizes the voltage-dependence of activation, and increases the inactivation rate. {yields} Ca{sup ++} alone antagonizes CaM for both effects, and depolarizes the voltage-dependence of inactivation. -- Abstract: Mutations in the neuronal Nav1.1 voltage-gated sodium channel are responsible for mild to severe epileptic syndromes. The ubiquitous calcium sensor calmodulin (CaM) bound to rat brain Nav1.1 and to the human Nav1.1 channelmore » expressed by a stably transfected HEK-293 cell line. The C-terminal region of the channel, as a fusion protein or in the yeast two-hybrid system, interacted with CaM via a consensus C-terminal motif, the IQ domain. Patch clamp experiments on HEK1.1 cells showed that CaM overexpression increased peak current in a calcium-dependent way. CaM had no effect on the voltage-dependence of fast inactivation, and accelerated the inactivation kinetics. Elevating Ca{sup ++} depolarized the voltage-dependence of fast inactivation and slowed down the fast inactivation kinetics, and for high concentrations this effect competed with the acceleration induced by CaM alone. Similarly, the depolarizing action of calcium antagonized the hyperpolarizing shift of the voltage-dependence of activation due to CaM overexpression. Fluorescence spectroscopy measurements suggested that Ca{sup ++} could bind the Nav1.1 C-terminal region with micromolar affinity.« less

Effect of surface pretreatment of TiO2 films on interfacial processes leading to bacterial inactivation in the dark and under light irradiation

PubMed Central

Rtimi, Sami; Nesic, Jelena; Pulgarin, Cesar; Sanjines, Rosendo; Bensimon, Michael; Kiwi, John

2015-01-01

Evidence is presented for radio-frequency plasma pretreatment enhancing the amount and adhesion of TiO2 sputtered on polyester (PES) and on polyethylene (PE) films. Pretreatment is necessary to attain a suitable TiO2 loading leading to an acceptable Escherichia coli reduction kinetics in the dark or under light irradiation for PES–TiO2 and PE–TiO2 samples. The amount of TiO2 on the films was monitored by diffuse reflectance spectroscopy and X-ray fluorescence. X-ray electron spectroscopy shows the lack of accumulation of bacterial residues such as C, N and S during bacterial inactivation since they seem to be rapidly destroyed by TiO2 photocatalysis. Evidence was found for Ti4+/Ti3+ redox catalysis occurring on PES–TiO2 and PE–TiO2 during the bacterial inactivation process. On PE–TiO2 surfaces, Fourier transform infrared spectroscopy (ATR-FTIR) provides evidence for a systematic shift of the na(CH2) stretching vibrations preceding bacterial inactivation within 60 min. The discontinuous IR-peak shifts reflect the increase in the C–H inter-bond distance leading to bond scission. The mechanism leading to E. coli loss of viability on PES–TiO2 was investigated in the dark up to complete bacterial inactivation by monitoring the damage in the bacterial outer cell by transmission electron microscopy. After 30 min, the critical step during the E. coli inactivation commences for dark disinfection on 0.1–5% wt PES–TiO2 samples. The interactions between the TiO2 aggregates and the outer lipopolysaccharide cell wall involve electrostatic effects competing with the van der Waals forces. PMID:25657831

Effect of surface pretreatment of TiO2 films on interfacial processes leading to bacterial inactivation in the dark and under light irradiation.

PubMed

Rtimi, Sami; Nesic, Jelena; Pulgarin, Cesar; Sanjines, Rosendo; Bensimon, Michael; Kiwi, John

2015-02-06

Evidence is presented for radio-frequency plasma pretreatment enhancing the amount and adhesion of TiO2 sputtered on polyester (PES) and on polyethylene (PE) films. Pretreatment is necessary to attain a suitable TiO2 loading leading to an acceptable Escherichia coli reduction kinetics in the dark or under light irradiation for PES-TiO2 and PE-TiO2 samples. The amount of TiO2 on the films was monitored by diffuse reflectance spectroscopy and X-ray fluorescence. X-ray electron spectroscopy shows the lack of accumulation of bacterial residues such as C, N and S during bacterial inactivation since they seem to be rapidly destroyed by TiO2 photocatalysis. Evidence was found for Ti(4+)/Ti(3+) redox catalysis occurring on PES-TiO2 and PE-TiO2 during the bacterial inactivation process. On PE-TiO2 surfaces, Fourier transform infrared spectroscopy (ATR-FTIR) provides evidence for a systematic shift of the na(CH2) stretching vibrations preceding bacterial inactivation within 60 min. The discontinuous IR-peak shifts reflect the increase in the C-H inter-bond distance leading to bond scission. The mechanism leading to E. coli loss of viability on PES-TiO2 was investigated in the dark up to complete bacterial inactivation by monitoring the damage in the bacterial outer cell by transmission electron microscopy. After 30 min, the critical step during the E. coli inactivation commences for dark disinfection on 0.1-5% wt PES-TiO2 samples. The interactions between the TiO2 aggregates and the outer lipopolysaccharide cell wall involve electrostatic effects competing with the van der Waals forces.

Luteinizing hormone-releasing hormone inactivation by purified pituitary plasma membranes: effects of receptor-binding studies.

PubMed

Clayton, R N; Shakespear, R A; Duncan, J A; Marshall, J C

1979-05-01

Inactivation of LHRH by purified bovine pituitary plasma membranes was studied in vitro. After incubation of [125I]iodo-LHRH with plasma membranes, the amount of tracer bound to the pellet was measured, and the integrity of the unbound tracer in the supernatant was assessed. Reduction in ability to bind to anti-LHRH serum and to rebind to plasma membranes together with altered electrophoretic mobility on polyacrylamide gels showed that the unbound [125I]iodo-LHRH was inactivated. LHRH inactivation occurred rapidly and was dependent upon membrane concentration and incubation temperature. These results indicate that hormone inactivation must be taken into account in the interpretation of LHRH-receptor interactions. During 37 C incubations, the apparent absence of specific LHRH binding can be explained by inactivation of tracer hormone. Significant LHRH inactivation also occurred at 0 C, which in part explains the insensitivity of LHRH receptor assays. Assessment of LHRH inactivation by different particulate subcellular fractions of pituitary tissue showed that the inactivating enzyme was associated with the plasma membranes; other organelles did not alter LHRH. The enzyme appeared to be an integral part of the plasma membrane structure, since enzymic activity could not be removed by washing without reducing specific LHRH binding. Additionally, reduction of LHRH inactivation by the inhibitors Bacitracin and Trasylol and by magnesium was also accompanied by reduced LHRH binding. Previous studies have shown that the majority of LHRH binding to pituitary plasma membranes is to the low affinity site (approximately 10(-6) M), but the significance of this binding has been uncertain. Our findings indicate that low affinity binding probably represents binding of LHRH to the inactivating enzyme. The LHRH analog, D-Ser6(TBu), des Gly10, ethylamide, has greater biological activity than LHRH and is not inactivated to a significant extent by pituitary plasma membranes. The enhanced biological activity of the analog, therefore, may be due to its resistance to inactivation by enzymes on the pituitary cell surface. The membrane-associated inactivating enzyme could play an important role in vivo in determining the concentration of intact LHRH available at the receptor site which initiates gonadotropin release.

Use of 8-methoxypsoralen and long-wavelength ultraviolet radiation for decontamination of platelet concentrates

NASA Astrophysics Data System (ADS)

Corash, Laurence; Lin, Lily; Wiesehahn, Gary; Cimino, George

1992-06-01

Transmission of viral diseases through blood products remains a problem in transfusion medicine. A number of methods have been developed to inactivate viral pathogens in plasma and plasma fractions, including: dry heating, wet heating, solvent-detergent treatment, and immunoaffinity purification. While some of these methods successfully inactivate pathogenic viruses, inactivation may be incomplete or result in damage to labile plasma proteins and cells. We have developed a photochemical decontamination system (PCD) for platelet concentrates (PC) utilizing treatment with long wavelength ultraviolet radiation (UVA, 320 - 400 nm) and 8-methoxypsoralen (8-MOP). This system is capable of inactivating 25 - 30 logs/hr of bacteria E. coli or S. aureus, 6 logs/hr of bacteriophage fd, 0.9 log/hr of bacteriophage R17 and 1.1 logs/hr of feline leukemia virus (FeLV) in PC. Immediately following 6 hrs of PCD treatment, platelet integrity and function of PCD treated and control PC were equivalent. After overnight storage PCD treated and control PC platelet properties were equal, but there was a slight reduction in TXB-2 production of PCD treated PC compared to controls. Following PCD treatment, PC were stored for 48 to 96 hrs. Platelet counts, morphology scores, extracellular LDH levels, aggregation response, dense body (db) content, and alpha granule ((alpha) g) content of PCD treated and control PC were comparable. We assessed the ability of the PCD technique to inactivate intracellular and extracellular virus, quantified the degree of DNA adduct formation in contaminating lymphocytes, and measured the inhibition of polymerase chain reaction (PCR) mediated amplification of intracellular DNA. High titers of cell-free murine cytomegalovirus added to human platelet concentrates (final concentration 106) were inactivated by PCD within 30 min. Cat renal fibroblasts infected at high levels with feline rhinotracheitis virus (FeRTV) were seeded into PC followed by PCD treatment with inactivation of 4.8 logs of FeRTV within 10 minutes. Purified human lymphocytes were seeded into PC and treated with PCD in the presence of 3H 8-MOP. Six hours of PCD treatment resulted in the formation of 9.3 to 12.8 8-MOP adducts per 1000 base pairs (bp) of DNA. PCR amplification of a 242 bp segment at the HLA-DQ(alpha) locus was examined. Inhibition of PCR DNA amplification was dependent on the numbers of 8-MOP adducts formed, and no amplification was present when greater than 12 adducts per 1000 bp were formed. These studies indicate that PCD can effectively inactivate high titers of cell-associated and cell-free virus seeded into standard human PC. The efficiency of DNA adduct formation can be quantitated, and the level of 8-MOP adduct formation in lymphocytes contaminating PC is comparable to the level of adduct formation in cellular DNA reported in the absence of platelets.

Genes involved in nonpermissive temperature-induced cell differentiation in Sertoli TTE3 cells bearing temperature-sensitive simian virus 40 large T-antigen

DOE Office of Scientific and Technical Information (OSTI.GOV)

Tabuchi, Yoshiaki; Kondo, Takashi; Suzuki, Yoshihisa

2005-04-15

Sertoli TTE3 cells, derived from transgenic mice bearing temperature-sensitive simian virus 40 large T (tsSV40LT)-antigen, proliferated continuously at a permissive temperature (33 deg C) whereas inactivation of the large T-antigen by a nonpermissive temperature (39 deg C) led to differentiation as judged by elevation of transferrin. To clarify the detailed mechanisms of differentiation, we investigated the time course of changes in gene expression using cDNA microarrays. Of the 865 genes analyzed, 14 genes showed increased levels of expression. Real-time quantitative PCR revealed that the mRNA levels of p21{sup waf1}, milk fat globule membrane protein E8, heat-responsive protein 12, and selenoproteinmore » P were markedly elevated. Moreover, the differentiated condition induced by the nonpermissive temperature significantly increased mRNA levels of these four genes in several cell lines from the transgenic mice bearing the oncogene. The present results regarding changes in gene expression will provide a basis for a further understanding of molecular mechanisms of differentiation in both Sertoli cells and cell lines transformed by tsSV40LT-antigen.« less

Dynamical characterization of inactivation path in voltage-gated Na+ ion channel by non-equilibrium response spectroscopy

PubMed Central

Pal, Krishnendu; Gangopadhyay, Gautam

2016-01-01

ABSTRACT Inactivation path of voltage gated sodium channel has been studied here under various voltage protocols as it is the main governing factor for the periodic occurrence and shape of the action potential. These voltage protocols actually serve as non-equilibrium response spectroscopic tools to study the ion channel in non-equilibrium environment. In contrast to a lot of effort in finding the crystal structure based molecular mechanism of closed-state(CSI) and open-state inactivation(OSI); here our approach is to understand the dynamical characterization of inactivation. The kinetic flux as well as energetic contribution of the closed and open- state inactivation path is compared here for voltage protocols, namely constant, pulsed and oscillating. The non-equilibrium thermodynamic quantities used in response to these voltage protocols serve as improved characterization tools for theoretical understanding which not only agrees with the previously known kinetic measurements but also predict the energetically optimum processes to sustain the auto-regulatory mechanism of action potential and the consequent inactivation steps needed. The time dependent voltage pattern governs the population of the conformational states which when couple with characteristic rate parameters, the CSI and OSI selectivity arise dynamically to control the inactivation path. Using constant, pulsed and continuous oscillating voltage protocols we have shown that during depolarization the OSI path is more favored path of inactivation however, in the hyper-polarized situation the CSI is favored. It is also shown that the re-factorisation of inactivated sodium channel to resting state occurs via CSI path. Here we have shown how the subtle energetic and entropic cost due to the change in the depolarization magnitude determines the optimum path of inactivation. It is shown that an efficient CSI and OSI dynamical profile in principle can characterize the open-state drug blocking phenomena. PMID:27367642

Ionizing irradiation not only inactivates clonogenic potential in primary normal human diploid lens epithelial cells but also stimulates cell proliferation in a subset of this population.

PubMed

Fujimichi, Yuki; Hamada, Nobuyuki

2014-01-01

Over the past century, ionizing radiation has been known to induce cataracts in the crystalline lens of the eye, but its mechanistic underpinnings remain incompletely understood. This study is the first to report the clonogenic survival of irradiated primary normal human lens epithelial cells and stimulation of its proliferation. Here we used two primary normal human cell strains: HLEC1 lens epithelial cells and WI-38 lung fibroblasts. Both strains were diploid, and a replicative lifespan was shorter in HLEC1 cells. The colony formation assay demonstrated that the clonogenic survival of both strains decreases similarly with increasing doses of X-rays. A difference in the survival between two strains was actually insignificant, although HLEC1 cells had the lower plating efficiency. This indicates that the same dose inactivates the same fraction of clonogenic cells in both strains. Intriguingly, irradiation enlarged the size of clonogenic colonies arising from HLEC1 cells in marked contrast to those from WI-38 cells. Such enhanced proliferation of clonogenic HLEC1 cells was significant at ≥2 Gy, and manifested as increments of ≤2.6 population doublings besides sham-irradiated controls. These results suggest that irradiation of HLEC1 cells not only inactivates clonogenic potential but also stimulates proliferation of surviving uniactivated clonogenic cells. Given that the lens is a closed system, the stimulated proliferation of lens epithelial cells may not be a homeostatic mechanism to compensate for their cell loss, but rather should be regarded as abnormal. This is because these findings are consistent with the early in vivo evidence documenting that irradiation induces excessive proliferation of rabbit lens epithelial cells and that suppression of lens epithelial cell divisions inhibits radiation cataractogenesis in frogs and rats. Thus, our in vitro model will be useful to evaluate the excessive proliferation of primary normal human lens epithelial cells that may underlie radiation cataractogenesis, warranting further investigations.

Fiber type conversion alters inactivation of voltage-dependent sodium currents in murine C2C12 skeletal muscle cells.

PubMed

Zebedin, Eva; Sandtner, Walter; Galler, Stefan; Szendroedi, Julia; Just, Herwig; Todt, Hannes; Hilber, Karlheinz

2004-08-01

Each skeletal muscle of the body contains a unique composition of "fast" and "slow" muscle fibers, each of which is specialized for certain challenges. This composition is not static, and the muscle fibers are capable of adapting their molecular composition by altered gene expression (i.e., fiber type conversion). Whereas changes in the expression of contractile proteins and metabolic enzymes in the course of fiber type conversion are well described, little is known about possible adaptations in the electrophysiological properties of skeletal muscle cells. Such adaptations may involve changes in the expression and/or function of ion channels. In this study, we investigated the effects of fast-to-slow fiber type conversion on currents via voltage-gated Na+ channels in the C(2)C(12) murine skeletal muscle cell line. Prolonged treatment of cells with 25 nM of the Ca2+ ionophore A-23187 caused a significant shift in myosin heavy chain isoform expression from the fast toward the slow isoform, indicating fast-to-slow fiber type conversion. Moreover, Na+ current inactivation was significantly altered. Slow inactivation less strongly inhibited the Na+ currents of fast-to-slow fiber type-converted cells. Compared with control cells, the Na+ currents of converted cells were more resistant to block by tetrodotoxin, suggesting enhanced relative expression of the cardiac Na+ channel isoform Na(v)1.5 compared with the skeletal muscle isoform Na(v)1.4. These results imply that fast-to-slow fiber type conversion of skeletal muscle cells involves functional adaptation of their electrophysiological properties.

TRIM65 negatively regulates p53 through ubiquitination

DOE Office of Scientific and Technical Information (OSTI.GOV)

Li, Yang; Ma, Chengyuan; Zhou, Tong

2016-04-22

Tripartite-motif protein family member 65 (TRIM65) is an important protein involved in white matter lesion. However, the role of TRIM65 in human cancer remains less understood. Through the Cancer Genome Atlas (TCGA) gene alteration database, we found that TRIM65 is upregulated in a significant portion of non-small cell lung carcinoma (NSCLC) patients. Our cell growth assay revealed that TRIM65 overexpression promotes cell proliferation, while knockdown of TRIM65 displays opposite effect. Mechanistically, TRIM65 binds to p53, one of the most critical tumor suppressors, and serves as an E3 ligase toward p53. Consequently, TRIM65 inactivates p53 through facilitating p53 poly-ubiquitination and proteasome-mediatedmore » degradation. Notably, chemotherapeutic reagent cisplatin induction of p53 is markedly attenuated in response to ectopic expression of TRIM65. Cell growth inhibition by TRIM65 knockdown is more significant in p53 positive H460 than p53 negative H1299 cells, and knockdown of p53 in H460 cells also shows compromised cell growth inhibition by TRIM65 knockdown, indicating that p53 is required, at least in part, for TRIM65 function. Our findings demonstrate TRIM65 as a potential oncogenic protein, highly likely through p53 inactivation, and provide insight into development of novel approaches targeting TRIM65 for NSCLC treatment, and also overcoming chemotherapy resistance. - Highlights: • TRIM65 expression is elevated in NSCLC. • TRIM65 inactivates p53 through mediating p53 ubiquitination and degradation. • TRIM65 attenuates the response of NSCLC cells to cisplatin.« less

Ribosome-Inactivating Proteins: From Plant Defense to Tumor Attack

PubMed Central

de Virgilio, Maddalena; Lombardi, Alessio; Caliandro, Rocco; Fabbrini, Maria Serena

2010-01-01

Ribosome-inactivating proteins (RIPs) are EC3.2.32.22 N-glycosidases that recognize a universally conserved stem-loop structure in 23S/25S/28S rRNA, depurinating a single adenine (A4324 in rat) and irreversibly blocking protein translation, leading finally to cell death of intoxicated mammalian cells. Ricin, the plant RIP prototype that comprises a catalytic A subunit linked to a galactose-binding lectin B subunit to allow cell surface binding and toxin entry in most mammalian cells, shows a potency in the picomolar range. The most promising way to exploit plant RIPs as weapons against cancer cells is either by designing molecules in which the toxic domains are linked to selective tumor targeting domains or directly delivered as suicide genes for cancer gene therapy. Here, we will provide a comprehensive picture of plant RIPs and discuss successful designs and features of chimeric molecules having therapeutic potential. PMID:22069572

Oncogenes: The Passport for Viral Oncolysis Through PKR Inhibition.

PubMed

Fernandes, Janaina

2016-01-01

The transforming properties of oncogenes are derived from gain-of-function mutations, shifting cell signaling from highly regulated homeostatic to an uncontrolled oncogenic state, with the contribution of the inactivating mutations in tumor suppressor genes P53 and RB, leading to tumor resistance to conventional and target-directed therapy. On the other hand, this scenario fulfills two requirements for oncolytic virus infection in tumor cells: inactivation of tumor suppressors and presence of oncoproteins, also the requirements to engage malignancy. Several of these oncogenes have a negative impact on the main interferon antiviral defense, the double-stranded RNA-activated protein kinase (PKR), which helps viruses to spontaneously target tumor cells instead of normal cells. This review is focused on the negative impact of overexpression of oncogenes on conventional and targeted therapy and their positive impact on viral oncolysis due to their ability to inhibit PKR-induced translation blockage, allowing virion release and cell death.

Hda inactivation of DnaA is the predominant mechanism preventing hyperinitiation of Escherichia coli DNA replication.

PubMed

Camara, Johanna E; Breier, Adam M; Brendler, Therese; Austin, Stuart; Cozzarelli, Nicholas R; Crooke, Elliott

2005-08-01

Initiation of DNA replication from the Escherichia coli chromosomal origin is highly regulated, assuring that replication occurs precisely once per cell cycle. Three mechanisms for regulation of replication initiation have been proposed: titration of free DnaA initiator protein by the datA locus, sequestration of newly replicated origins by SeqA protein and regulatory inactivation of DnaA (RIDA), in which active ATP-DnaA is converted to the inactive ADP-bound form. DNA microarray analyses showed that the level of initiation in rapidly growing cells that lack datA was indistinguishable from that in wild-type cells, and that the absence of SeqA protein caused only a modest increase in initiation, in agreement with flow-cytometry data. In contrast, cells lacking Hda overinitiated replication twofold, implicating RIDA as the predominant mechanism preventing extra initiation events in a cell cycle.

Pulsed electric field processing of different fruit juices: impact of pH and temperature on inactivation of spoilage and pathogenic micro-organisms.

PubMed

Timmermans, R A H; Nierop Groot, M N; Nederhoff, A L; van Boekel, M A J S; Matser, A M; Mastwijk, H C

2014-03-03

Pulsed electrical field (PEF) technology can be used for the inactivation of micro-organisms and therefore for preservation of food products. It is a mild technology compared to thermal pasteurization because a lower temperature is used during processing, leading to a better retention of the quality. In this study, pathogenic and spoilage micro-organisms relevant in refrigerated fruit juices were studied to determine the impact of process parameters and juice composition on the effectiveness of the PEF process to inactivate the micro-organisms. Experiments were performed using a continuous-flow PEF system at an electrical field strength of 20 kV/cm with variable frequencies to evaluate the inactivation of Salmonella Panama, Escherichia coli, Listeria monocytogenes and Saccharomyces cerevisiae in apple, orange and watermelon juices. Kinetic data showed that under the same conditions, S. cerevisiae was the most sensitive micro-organism, followed by S. Panama and E. coli, which displayed comparable inactivation kinetics. L. monocytogenes was the most resistant micro-organism towards the treatment conditions tested. A synergistic effect between temperature and electric pulses was observed at inlet temperatures above 35 °C, hence less energy for inactivation was required at higher temperatures. Different juice matrices resulted in a different degree of inactivation, predominantly determined by pH. The survival curves were nonlinear and could satisfactorily be modeled with the Weibull model. Copyright © 2013 Elsevier B.V. All rights reserved.

Loss of diphthamide pre-activates NF-κB and death receptor pathways and renders MCF7 cells hypersensitive to tumor necrosis factor.

PubMed

Stahl, Sebastian; da Silva Mateus Seidl, Ana Rita; Ducret, Axel; Kux van Geijtenbeek, Sabine; Michel, Sven; Racek, Tomas; Birzele, Fabian; Haas, Alexander K; Rueger, Ruediger; Gerg, Michael; Niederfellner, Gerhard; Pastan, Ira; Brinkmann, Ulrich

2015-08-25

The diphthamide on human eukaryotic translation elongation factor 2 (eEF2) is the target of ADP ribosylating diphtheria toxin (DT) and Pseudomonas exotoxin A (PE). This modification is synthesized by seven dipthamide biosynthesis proteins (DPH1-DPH7) and is conserved among eukaryotes and archaea. We generated MCF7 breast cancer cell line-derived DPH gene knockout (ko) cells to assess the impact of complete or partial inactivation on diphthamide synthesis and toxin sensitivity, and to address the biological consequence of diphthamide deficiency. Cells with heterozygous gene inactivation still contained predominantly diphthamide-modified eEF2 and were as sensitive to PE and DT as parent cells. Thus, DPH gene copy number reduction does not affect overall diphthamide synthesis and toxin sensitivity. Complete inactivation of DPH1, DPH2, DPH4, and DPH5 generated viable cells without diphthamide. DPH1ko, DPH2ko, and DPH4ko harbored unmodified eEF2 and DPH5ko ACP- (diphthine-precursor) modified eEF2. Loss of diphthamide prevented ADP ribosylation of eEF2, rendered cells resistant to PE and DT, but does not affect sensitivity toward other protein synthesis inhibitors, such as saporin or cycloheximide. Surprisingly, cells without diphthamide (independent of which the DPH gene compromised) were presensitized toward nuclear factor of kappa light polypeptide gene enhancer in B cells (NF-κB) and death-receptor pathways without crossing lethal thresholds. In consequence, loss of diphthamide rendered cells hypersensitive toward TNF-mediated apoptosis. This finding suggests a role of diphthamide in modulating NF-κB, death receptor, or apoptosis pathways.