Antimicrobial agent triclosan disrupts mitochondrial structure, revealed by super-resolution microscopy, and inhibits mast cell signaling via calcium modulation.

PubMed

Weatherly, Lisa M; Nelson, Andrew J; Shim, Juyoung; Riitano, Abigail M; Gerson, Erik D; Hart, Andrew J; de Juan-Sanz, Jaime; Ryan, Timothy A; Sher, Roger; Hess, Samuel T; Gosse, Julie A

2018-06-15

The antimicrobial agent triclosan (TCS) is used in products such as toothpaste and surgical soaps and is readily absorbed into oral mucosa and human skin. These and many other tissues contain mast cells, which are involved in numerous physiologies and diseases. Mast cells release chemical mediators through a process termed degranulation, which is inhibited by TCS. Investigation into the underlying mechanisms led to the finding that TCS is a mitochondrial uncoupler at non-cytotoxic, low-micromolar doses in several cell types and live zebrafish. Our aim was to determine the mechanisms underlying TCS disruption of mitochondrial function and of mast cell signaling. We combined super-resolution (fluorescence photoactivation localization) microscopy and multiple fluorescence-based assays to detail triclosan's effects in living mast cells, fibroblasts, and primary human keratinocytes. TCS disrupts mitochondrial nanostructure, causing mitochondria to undergo fission and to form a toroidal, "donut" shape. TCS increases reactive oxygen species production, decreases mitochondrial membrane potential, and disrupts ER and mitochondrial Ca 2+ levels, processes that cause mitochondrial fission. TCS is 60 × more potent than the banned uncoupler 2,4-dinitrophenol. TCS inhibits mast cell degranulation by decreasing mitochondrial membrane potential, disrupting microtubule polymerization, and inhibiting mitochondrial translocation, which reduces Ca 2+ influx into the cell. Our findings provide mechanisms for both triclosan's inhibition of mast cell signaling and its universal disruption of mitochondria. These mechanisms provide partial explanations for triclosan's adverse effects on human reproduction, immunology, and development. This study is the first to utilize super-resolution microscopy in the field of toxicology. Copyright © 2018 Elsevier Inc. All rights reserved.

Does Enzymatic Hydrolysis of Glycosidically Bound Volatile Compounds Really Contribute to the Formation of Volatile Compounds During the Oolong Tea Manufacturing Process?

PubMed

Gui, Jiadong; Fu, Xiumin; Zhou, Ying; Katsuno, Tsuyoshi; Mei, Xin; Deng, Rufang; Xu, Xinlan; Zhang, Linyun; Dong, Fang; Watanabe, Naoharu; Yang, Ziyin

2015-08-12

It was generally thought that aroma of oolong tea resulted from hydrolysis of glycosidically bound volatiles (GBVs). In this study, most GBVs showed no reduction during the oolong tea manufacturing process. β-Glycosidases either at protein or gene level were not activated during the manufacturing process. Subcellular localization of β-primeverosidase provided evidence that β-primeverosidase was located in the leaf cell wall. The cell wall remained intact during the enzyme-active manufacturing process. After the leaf cell disruption, GBV content was reduced. These findings reveal that, during the enzyme-active process of oolong tea, nondisruption of the leaf cell walls resulted in impossibility of interaction of GBVs and β-glycosidases. Indole, jasmine lactone, and trans-nerolidol were characteristic volatiles produced from the manufacturing process. Interestingly, the contents of the three volatiles was reduced after the leaf cell disruption, suggesting that mechanical damage with the cell disruption, which is similar to black tea manufacturing, did not induce accumulation of the three volatiles. In addition, 11 volatiles with flavor dilution factor ≥4(4) were identified as relatively potent odorants in the oolong tea. These results suggest that enzymatic hydrolysis of GBVs was not involved in the formation of volatiles of oolong tea, and some characteristic volatiles with potent odorants were produced from the manufacturing process.

Sequential pretreatment for cell disintegration of municipal sludge in a neutral Bio-electro-Fenton system.

PubMed

Yu, Qilin; Jin, Xiaochen; Zhang, Yaobin

2018-05-15

Sludge cell disruption was generally considered as the rate-limiting step for the anaerobic digestion of waste activated sludge (WAS). Advanced oxidation processes and bio-electro-chemical systems were recently reported to enhance the hydrolysis of WAS and sludge cell disruption, while the cell-breaking processes of these systems remain unclear yet. In this study, an innovative Bio-electro-Fenton system was developed to pretreat the WAS sequentially with cathode Fenton process and anode anaerobic digestion. Significant cell disruption and dissolution intracellular organics were founded after the treatment. X-ray photoelectron spectroscopy (XPS) analysis and fourier transform infrared spectroscopy (FT-IR) spectra indicated that Gram-negative bacteria were more sensitive to free radicals yielded in cathode to induce a chain reaction that destroyed the lipid-contained outer membrane, while Gram-positive bacteria with thick peptidoglycan layer were liable to be biologically decomposed in the anode. Compared with the oxidation of organic matters in the cathode Fenton, the secretion of enzyme increased in the anode which was beneficial to break down the complex matters (peptidoglycans) into simples that were available for anode oxidation by exoelectrogens. The results also showed a possible prospect for the application of this sequential pretreatment in bio-electro-Fenton systems to disrupt sludge cells and enhance the anaerobic digestion. Copyright © 2018 Elsevier Ltd. All rights reserved.

Influence of the extent of disruption of Bakers' yeast on protein adsorption in expanded beds.

PubMed

Balasundaram, B; Harrison, S T L

2008-02-01

Expanded bed adsorption chromatography is used to capture the protein product of interest from a crude biological suspension directly, thereby eliminating the need for the removal of the cell debris. While this technique may replace three or four unit operations in a typical downstream process for biological product recovery, the adsorption process is influenced by the interaction between the microbial cells or cell debris and the adsorbent as well as the presence of contaminating solutes. The influence of the extent and nature of disruption of Bakers' yeast on the adsorption of the total soluble protein and alpha-glucosidase was investigated in this study. Two different techniques were used for cell disruption: high pressure homogenisation and hydrodynamic cavitation. Two different adsorbents were chosen: anionic Streamline DEAE and cationic Streamline SP. The settled bed height and the superficial velocity were constant across all experiments. The feedstock was characterised in terms of viscosity, pH, conductivity, particle size distribution of the cell debris and the extent of protein and alpha-glucosidase released. The performance of the adsorption process was found to be influenced by the electrostatic interactions of cell debris with the anionic adsorbent Streamline DEAE and the intraparticle diffusional resistance inside the pores of the adsorbent matrix. The increase in the intensity of disruption resulted in an increase in the dynamic binding capacity (10% feed) of both the total soluble protein and the alpha-glucosidase. However, the increase in the DBC of protein and alpha-glucosidase were not proportional. The amount of protein that could be adsorbed per ml of adsorbent from the samples subjected to a lower intensity of disruption was found to exceed that obtained at a higher disruption intensity on increasing the volume of feed suggesting multilayer adsorption. In this case, selective adsorption of the model protein alpha-glucosidase was reduced, illustrating the compromise of maximising protein recovery through non-specific binding. The study illustrates the need for an interrogation of the intensity of disruption needed and a rigorous understanding of the influence of cell debris and adsorbent-protein interaction, in optimising the selective recovery of intracellular products by EBA.

Disruption of Brewers' yeast by hydrodynamic cavitation: Process variables and their influence on selective release.

PubMed

Balasundaram, B; Harrison, S T L

2006-06-05

Intracellular products, not secreted from the microbial cell, are released by breaking the cell envelope consisting of cytoplasmic membrane and an outer cell wall. Hydrodynamic cavitation has been reported to cause microbial cell disruption. By manipulating the operating variables involved, a wide range of intensity of cavitation can be achieved resulting in a varying extent of disruption. The effect of the process variables including cavitation number, initial cell concentration of the suspension and the number of passes across the cavitation zone on the release of enzymes from various locations of the Brewers' yeast was studied. The release profile of the enzymes studied include alpha-glucosidase (periplasmic), invertase (cell wall bound), alcohol dehydrogenase (ADH; cytoplasmic) and glucose-6-phosphate dehydrogenase (G6PDH; cytoplasmic). An optimum cavitation number Cv of 0.13 for maximum disruption was observed across the range Cv 0.09-0.99. The optimum cell concentration was found to be 0.5% (w/v, wet wt) when varying over the range 0.1%-5%. The sustained effect of cavitation on the yeast cell wall when re-circulating the suspension across the cavitation zone was found to release the cell wall bound enzyme invertase (86%) to a greater extent than the enzymes from other locations of the cell (e.g. periplasmic alpha-glucosidase at 17%). Localised damage to the cell wall could be observed using transmission electron microscopy (TEM) of cells subjected to less intense cavitation conditions. Absence of the release of cytoplasmic enzymes to a significant extent, absence of micronisation as observed by TEM and presence of a lower number of proteins bands in the culture supernatant on SDS-PAGE analysis following hydrodynamic cavitation compared to disruption by high-pressure homogenisation confirmed the selective release offered by hydrodynamic cavitation. Copyright 2006 Wiley Periodicals, Inc.

Technical difficulties and solutions of direct transesterification process of microbial oil for biodiesel synthesis.

PubMed

Yousuf, Abu; Khan, Maksudur Rahman; Islam, M Amirul; Wahid, Zularisam Ab; Pirozzi, Domenico

2017-01-01

Microbial oils are considered as alternative to vegetable oils or animal fats as biodiesel feedstock. Microalgae and oleaginous yeast are the main candidates of microbial oil producers' community. However, biodiesel synthesis from these sources is associated with high cost and process complexity. The traditional transesterification method includes several steps such as biomass drying, cell disruption, oil extraction and solvent recovery. Therefore, direct transesterification or in situ transesterification, which combines all the steps in a single reactor, has been suggested to make the process cost effective. Nevertheless, the process is not applicable for large-scale biodiesel production having some difficulties such as high water content of biomass that makes the reaction rate slower and hurdles of cell disruption makes the efficiency of oil extraction lower. Additionally, it requires high heating energy in the solvent extraction and recovery stage. To resolve these difficulties, this review suggests the application of antimicrobial peptides and high electric fields to foster the microbial cell wall disruption.

Substantial decrease in cell wall α-1,3-glucan caused by disruption of the kexB gene encoding a subtilisin-like processing protease in Aspergillus oryzae.

PubMed

Mizutani, Osamu; Shiina, Matsuko; Yoshimi, Akira; Sano, Motoaki; Watanabe, Takeshi; Yamagata, Youhei; Nakajima, Tasuku; Gomi, Katsuya; Abe, Keietsu

2016-09-01

Disruption of the kexB encoding a subtilisin-like processing protease in Aspergillus oryzae (ΔkexB) leads to substantial morphological defects when the cells are grown on Czapek-Dox agar plates. We previously found that the disruption of kexB causes a constitutive activation of the cell wall integrity pathway. To understand how the disruption of the kexB affects cell wall organization and components, we analyzed the cell wall of ΔkexB grown on the plates. The results revealed that both total N-acetylglucosamine content, which constitutes chitin, and chitin synthase activities were increased. Whereas total glucose content, which constitutes β-1,3-glucan and α-1,3-glucan, was decreased; this decrease was attributed to a remarkable decrease in α-1,3-glucan. Additionally, the β-1,3-glucan in the alkali-insoluble fraction of the ΔkexB showed a high degree of polymerization. These results suggested that the loss of α-1,3-glucan in the ΔkexB was compensated by increases in the chitin content and the average degree of β-1,3-glucan polymerization.

Disruption of the Crithidia fasciculata RNH1 gene results in the loss of two active forms of ribonuclease H.

PubMed Central

Ray, D S; Hines, J C

1995-01-01

Both prokaryotic and eukaryotic cells contain multiple forms of ribonuclease H, a ribonuclease that specifically degrades the RNA strand of RNA-DNA hybrids and which has been implicated in the processing of initiator RNAs and in the removal of RNA primers from Okazaki fragments. The Crithidia fasciculata RNH1 gene encodes an RNase H and was shown to be a single-copy gene in this diploid trypanosomatid. The RNH1 gene has been disrupted by targeted gene disruption using hygromycin or G418 drug-resistance cassettes. Major active forms of RNase H (38 and 45 kDa) were observed on activity gels of extracts of wild-type cells or cells in which one allele of RNH1 was disrupted. Both the 38 and 45 kDa activities were absent in extracts of cells in which both alleles of RNH1 were disrupted indicating that both forms of the C.fasciculata RNase H are encoded by the RNH1 gene. Images PMID:7630731

Optimization of bead milling parameters for the cell disruption of microalgae: process modeling and application to Porphyridium cruentum and Nannochloropsis oculata.

PubMed

Montalescot, V; Rinaldi, T; Touchard, R; Jubeau, S; Frappart, M; Jaouen, P; Bourseau, P; Marchal, L

2015-11-01

A study of cell disruption by bead milling for two microalgae, Nannochloropsis oculata and Porphyridium cruentum, was performed. Strains robustness was quantified by high-pressure disruption assays. The hydrodynamics in the bead mill grinding chamber was studied by Residence Time Distribution modeling. Operating parameters effects were analyzed and modeled in terms of stress intensities and stress number. RTD corresponded to a 2 CSTR in series model. First order kinetics cell disruption was modeled in consequence. Continuous bead milling was efficient for both strains disruption. SI-SN modeling was successfully adapted to microalgae. As predicted by high pressure assays, N. oculata was more resistant than P. cruentum. The critical stress intensity was twice more important for N. oculata than for P. cruentum. SI-SN modeling allows the determination of operating parameters minimizing energy consumption and gives a scalable approach to develop and optimize microalgal disruption by bead milling. Copyright © 2015 Elsevier Ltd. All rights reserved.

Contribution of cytoskeletal elements to the axonal mechanical properties

PubMed Central

2013-01-01

Background Microtubules, microfilaments, and neurofilaments are cytoskeletal elements that affect cell morphology, cellular processes, and mechanical structures in neural cells. The objective of the current study was to investigate the contribution of each type of cytoskeletal element to the mechanical properties of axons of dorsal root and sympathetic ganglia cells in chick embryos. Results Microtubules, microfilaments, and neurofilaments in axons were disrupted by nocodazole, cytochalasin D, and acrylamide, respectively, or a combination of the three. An atomic force microscope (AFM) was then used to compress the treated axons, and the resulting corresponding force-deformation information was analyzed to estimate the mechanical properties of axons that were partially or fully disrupted. Conclusion We have found that the mechanical stiffness was most reduced in microtubules-disrupted-axons, followed by neurofilaments-disrupted- and microfilaments-disrupted-axons. This suggests that microtubules contribute the most of the mechanical stiffness to axons. PMID:24007256

Validating the disruption of proliferating cell nuclear antigen interactions in the development of targeted cancer therapeutics.

PubMed

Smith, Shanna J; Hickey, Robert J; Malkas, Linda H

2016-01-01

Human DNA replication and repair is a highly coordinated process involving the specifically timed actions of numerous proteins and enzymes. Many of these proteins require interaction with proliferating cell nuclear antigen (PCNA) for activation within the process. The interdomain connector loop (IDCL) of PCNA provides a docking site for many of those proteins, suggesting that this region is critically important in the regulation of cellular function. Previous work in this laboratory has demonstrated that a peptide mimicking a specific region of the IDCL (caPeptide) has the ability to disrupt key protein-protein interactions between PCNA and its binding partners, thereby inhibiting DNA replication within the cells. In this study, we confirm the ability of the caPeptide to disrupt DNA replication function using both intact cell and in vitro DNA replication assays. Further, we were able to demonstrate that treatment with caPeptide results in a decrease of polymerase δ activity that correlates with the observed decrease in DNA replication. We have also successfully developed a surface plasmon resonance (SPR) assay to validate the disruption of the PCNA-pol δ interaction with caPeptide.

Regulated Assembly of Vacuolar ATPase Is Increased during Cluster Disruption-induced Maturation of Dendritic Cells through a Phosphatidylinositol 3-Kinase/mTOR-dependent Pathway*

PubMed Central

Liberman, Rachel; Bond, Sarah; Shainheit, Mara G.; Stadecker, Miguel J.; Forgac, Michael

2014-01-01

The vacuolar (H+)-ATPases (V-ATPases) are ATP-driven proton pumps composed of a peripheral V1 domain and a membrane-embedded V0 domain. Regulated assembly of V1 and V0 represents an important regulatory mechanism for controlling V-ATPase activity in vivo. Previous work has shown that V-ATPase assembly increases during maturation of bone marrow-derived dendritic cells induced by activation of Toll-like receptors. This increased assembly is essential for antigen processing, which is dependent upon an acidic lysosomal pH. Cluster disruption of dendritic cells induces a semi-mature phenotype associated with immune tolerance. Thus, semi-mature dendritic cells are able to process and present self-peptides to suppress autoimmune responses. We have investigated V-ATPase assembly in bone marrow-derived, murine dendritic cells and observed an increase in assembly following cluster disruption. This increased assembly is not dependent upon new protein synthesis and is associated with an increase in concanamycin A-sensitive proton transport in FITC-loaded lysosomes. Inhibition of phosphatidylinositol 3-kinase with wortmannin or mTORC1 with rapamycin effectively inhibits the increased assembly observed upon cluster disruption. These results suggest that the phosphatidylinositol 3-kinase/mTOR pathway is involved in controlling V-ATPase assembly during dendritic cell maturation. PMID:24273170

Promotion of malignant phenotype after disruption of the three-dimensional structure of cultured spheroids from colorectal cancer.

PubMed

Piulats, Jose M; Kondo, Jumpei; Endo, Hiroko; Ono, Hiromasa; Hagihara, Takeshi; Okuyama, Hiroaki; Nishizawa, Yasuko; Tomita, Yasuhiko; Ohue, Masayuki; Okita, Kouki; Oyama, Hidejiro; Bono, Hidemasa; Masuko, Takashi; Inoue, Masahiro

2018-03-23

Individual and small clusters of cancer cells may detach from the edges of a main tumor and invade vessels, which can act as the origin of metastasis; however, the mechanism for this phenomenon is not well understood. Using cancer tissue-originated spheroids, we studied whether disturbing the 3D architecture of cancer spheroids can provoke the reformation process and progression of malignancy. We developed a mechanical disruption method to achieve homogenous disruption of the spheroids while maintaining cell-cell contact. After the disruption, 9 spheroid lines from 9 patient samples reformed within a few hours, and 3 of the 9 lines exhibited accelerated spheroid growth. Marker expression, spheroid forming capacity, and tumorigenesis indicated that stemness increased after spheroid disruption. In addition, the spheroid forming capacity increased in 6 of 11 spheroid lines. The disruption signature determined by gene expression profiling supported the incidence of remodeling and predicted the prognosis of patients with colorectal cancer. Furthermore, WNT and HER3 signaling were increased in the reformed spheroids, and suppression of these signaling pathways attenuated the increased proliferation and stemness after the disruption. Overall, the disruption and subsequent reformation of cancer spheroids promoted malignancy-related phenotypes through the activation of the WNT and ERBB pathways.

Use of focused acoustics for cell disruption to provide ultra scale-down insights of microbial homogenization and its bioprocess impact--recovery of antibody fragments from rec E. coli.

PubMed

Li, Qiang; Aucamp, Jean P; Tang, Alison; Chatel, Alex; Hoare, Mike

2012-08-01

An ultra scale-down (USD) device that provides insight of how industrial homogenization impacts bioprocess performance is desirable in the biopharmaceutical industry, especially at the early stage of process development where only a small quantity of material is available. In this work, we assess the effectiveness of focused acoustics as the basis of an USD cell disruption method to mimic and study high-pressure, step-wise homogenization of rec Escherichia coli cells for the recovery of an intracellular protein, antibody fragment (Fab'). The release of both Fab' and of overall protein follows first-order reaction kinetics with respect to time of exposure to focused acoustics. The rate constant is directly proportional to applied electrical power input per unit volume. For nearly total protein or Fab' release (>99%), the key physical properties of the disruptate produced by focused acoustics, such as cell debris particle size distribution and apparent viscosity show good agreement with those for homogenates produced by high-pressure homogenization operated to give the same fractional release. The only key difference is observed for partial disruption of cells where focused acoustics yields a disruptate of lower viscosity than homogenization, evidently due to a greater extent of polynucleic acids degradation. Verification of this USD approach to cell disruption by high-pressure homogenization is achieved using USD centrifugation to demonstrate the same sedimentation characteristics of disruptates prepared using both the scaled-down focused acoustic and the pilot-scale homogenization methods for the same fraction of protein release. Copyright © 2012 Wiley Periodicals, Inc.

Modeling bubble dynamics and radical kinetics in ultrasound induced microalgal cell disruption.

PubMed

Wang, Meng; Yuan, Wenqiao

2016-01-01

Microalgal cell disruption induced by acoustic cavitation was simulated through solving the bubble dynamics in an acoustical field and their radial kinetics (chemical kinetics of radical species) occurring in the bubble during its oscillation, as well as calculating the bubble wall pressure at the collapse point. Modeling results indicated that increasing ultrasonic intensity led to a substantial increase in the number of bubbles formed during acoustic cavitation, however, the pressure generated when the bubbles collapsed decreased. Therefore, cumulative collapse pressure (CCP) of bubbles was used to quantify acoustic disruption of a freshwater alga, Scenedesmus dimorphus, and a marine alga, Nannochloropsis oculata and compare with experimental results. The strong correlations between CCP and the intracellular lipid fluorescence density, chlorophyll-a fluorescence density, and cell particle/debris concentration were found, which suggests that the developed models could accurately predict acoustic cell disruption, and can be utilized in the scale up and optimization of the process. Copyright © 2015 Elsevier B.V. All rights reserved.

Cell Cycle Control by PTEN.

PubMed

Brandmaier, Andrew; Hou, Sheng-Qi; Shen, Wen H

2017-07-21

Continuous and error-free chromosome inheritance through the cell cycle is essential for genomic stability and tumor suppression. However, accumulation of aberrant genetic materials often causes the cell cycle to go awry, leading to malignant transformation. In response to genotoxic stress, cells employ diverse adaptive mechanisms to halt or exit the cell cycle temporarily or permanently. The intrinsic machinery of cycling, resting, and exiting shapes the cellular response to extrinsic stimuli, whereas prevalent disruption of the cell cycle machinery in tumor cells often confers resistance to anticancer therapy. Phosphatase and tensin homolog (PTEN) is a tumor suppressor and a guardian of the genome that is frequently mutated or deleted in human cancer. Moreover, it is increasingly evident that PTEN deficiency disrupts the fundamental processes of genetic transmission. Cells lacking PTEN exhibit cell cycle deregulation and cell fate reprogramming. Here, we review the role of PTEN in regulating the key processes in and out of cell cycle to optimize genomic integrity. Copyright © 2017 Elsevier Ltd. All rights reserved.

Processing of Microalgae: Acoustic Cavitation and Hydrothermal Conversion

NASA Astrophysics Data System (ADS)

Greenly, Justin Michael

The production of energy dense fuels from renewable algal biomass feedstocks -- if sustainably developed at a sufficiently large scale -- may reduce the consumption of petroleum from fossil fuels and provide many environmental benefits. Achieving economic feasibility has several technical engineering challenges that arise from dilute concentration of growing algae in aqueous media, small cell sizes, and durable cell walls. For microalgae to be a sustainable source of biofuels and co-products, efficient fractionation and conversion of the cellular contents is necessary. Research was carried out to address two processing options for efficient microalgae biofuel production: 1. Ultrasonic cavitation for cell disruption and 2. Hydrothermal conversion of a model algal triglyceride. 1. Ultrasonic cell disruption, which relies on cavitating bubbles in the suspension to produce damaging shock waves, was investigated experimentally over a range of concentrations and species types. A few seconds of high intensity sonication at fixed frequency yielded significant cell disruption, even for the more durable cells. At longer exposure times, effectiveness was seen to decline and was attributed, using acoustic measurements, to ultrasonic power attenuation in the ensuing cloud of cavitating bubbles. Processing at higher cell concentrations slowed cell disintegration marginally, but increased the effectiveness of dissipating ultrasonic energy. A theoretical study effectively predicted optimal conditions for a variety of parameters that were inaccessible in this experimental investigation. In that study, single bubble collapse was modeled to identify operating conditions that would increase cavitation, and thus cell disruption. Simulations were conducted by varying frequency and pressure amplitude of the ultrasound wave, and initial bubble size. The simulation results indicated that low frequency, high sound wave amplitudes, and small initial bubble size generate the highest shock wave pressures. 2. Hydrolysis of a pure model triglyceride compound was experimentally examined for the first time at hydrothermal conditions -- from 225 to 300°C. Lipid product composition assessed by GC-FID was compared to previous studies with mixed vegetable oils and used to develop a kinetic model for this oil phase reaction.

APC binds the Miro/Milton motor complex to stimulate transport of mitochondria to the plasma membrane

PubMed Central

Mills, Kate M.; Brocardo, Mariana G.; Henderson, Beric R.

2016-01-01

Mutations in adenomatous polyposis coli (APC) disrupt regulation of Wnt signaling, mitosis, and the cytoskeleton. We describe a new role for APC in the transport of mitochondria. Silencing of wild-type APC by small interfering RNA caused mitochondria to redistribute from the cell periphery to the perinuclear region. We identified novel APC interactions with the mitochondrial kinesin-motor complex Miro/Milton that were mediated by the APC C-terminus. Truncating mutations in APC abolished its ability to bind Miro/Milton and reduced formation of the Miro/Milton complex, correlating with disrupted mitochondrial distribution in colorectal cancer cells that could be recovered by reconstitution of wild-type APC. Using proximity ligation assays, we identified endogenous APC-Miro/Milton complexes at mitochondria, and live-cell imaging showed that loss of APC slowed the frequency of anterograde mitochondrial transport to the membrane. We propose that APC helps drive mitochondria to the membrane to supply energy for cellular processes such as directed cell migration, a process disrupted by cancer mutations. PMID:26658612

Downstream Processing of Synechocystis for Biofuel Production

NASA Astrophysics Data System (ADS)

Sheng, Jie

Lipids and free fatty acids (FFA) from cyanobacterium Synechocystis can be used for biofuel (e.g. biodiesel or renewable diesel) production. In order to utilize and scale up this technique, downstream processes including culturing and harvest, cell disruption, and extraction were studied. Several solvents/solvent systems were screened for lipid extraction from Synechocystis. Chloroform + methanol-based Folch and Bligh & Dyer methods were proved to be "gold standard" for small-scale analysis due to their highest lipid recoveries that were confirmed by their penetration of the cell membranes, higher polarity, and stronger interaction with hydrogen bonds. Less toxic solvents, such as methanol and MTBE, or direct transesterification of biomass (without preextraction step) gave only slightly lower lipid-extraction yields and can be considered for large-scale application. Sustained exposure to high and low temperature extremes severely lowered the biomass and lipid productivity. Temperature stress also triggered changes of lipid quality such as the degree of unsaturation; thus, it affected the productivities and quality of Synechocystis-derived biofuel. Pulsed electric field (PEF) was evaluated for cell disruption prior to lipid extraction. A treatment intensity > 35 kWh/m3 caused significant damage to the plasma membrane, cell wall, and thylakoid membrane, and it even led to complete disruption of some cells into fragments. Treatment by PEF enhanced the potential for the low-toxicity solvent isopropanol to access lipid molecules during subsequent solvent extraction, leading to lower usage of isopropanol for the same extraction efficiency. Other cell-disruption methods also were tested. Distinct disruption effects to the cell envelope, plasma membrane, and thylakoid membranes were observed that were related to extraction efficiency. Microwave and ultrasound had significant enhancement of lipid extraction. Autoclaving, ultrasound, and French press caused significant release of lipid into the medium, which may increase solvent usage and make medium recycling difficult. Production of excreted FFA by mutant Synechocystis has the potential of reducing the complexity of downstream processing. Major problems, such as FFA precipitation and biodegradation by scavengers, account for FFA loss in operation. Even a low concentration of FFA scavengers could consume FFA at a high rate that outpaced FFA production rate. Potential strategies to overcome FFA loss include high pH, adsorptive resin, and sterilization techniques.

HopW1 from Pseudomonas syringae disrupts the actin cytoskeleton to promote virulence in Arabidopsis.

PubMed

Kang, Yongsung; Jelenska, Joanna; Cecchini, Nicolas M; Li, Yujie; Lee, Min Woo; Kovar, David R; Greenberg, Jean T

2014-06-01

A central mechanism of virulence of extracellular bacterial pathogens is the injection into host cells of effector proteins that modify host cellular functions. HopW1 is an effector injected by the type III secretion system that increases the growth of the plant pathogen Pseudomonas syringae on the Columbia accession of Arabidopsis. When delivered by P. syringae into plant cells, HopW1 causes a reduction in the filamentous actin (F-actin) network and the inhibition of endocytosis, a known actin-dependent process. When directly produced in plants, HopW1 forms complexes with actin, disrupts the actin cytoskeleton and inhibits endocytosis as well as the trafficking of certain proteins to vacuoles. The C-terminal region of HopW1 can reduce the length of actin filaments and therefore solubilize F-actin in vitro. Thus, HopW1 acts by disrupting the actin cytoskeleton and the cell biological processes that depend on actin, which in turn are needed for restricting P. syringae growth in Arabidopsis.

Adaxial cell migration in the zebrafish embryo is an active cell autonomous property that requires the Prdm1a transcription factor.

PubMed

Ono, Yosuke; Yu, Weimiao; Jackson, Harriet E; Parkin, Caroline A; Ingham, Philip W

2015-01-01

Adaxial cells, the progenitors of slow-twitch muscle fibres in zebrafish, exhibit a stereotypic migratory behaviour during somitogenesis. Although this process is known to be disrupted in various mutants, its precise nature has remained unclear. Here, using in vivo imaging and chimera analysis, we show that adaxial cell migration is a cell autonomous process, during which cells become polarised and extend filopodia at their leading edge. Loss of function of the Prdm1a transcription factor disrupts the polarisation and migration of adaxial cells, reflecting a role that is independent of its repression of sox6 expression. Expression of the M- and N-cadherins, previously implicated in driving adaxial cell migration, is largely unaffected by loss of Prdm1a function, suggesting that differential cadherin expression is not sufficient for adaxial cell migration. Copyright © 2015 International Society of Differentiation. Published by Elsevier B.V. All rights reserved.

Targeted CRISPR disruption reveals a role for RNase MRP RNA in human preribosomal RNA processing

PubMed Central

Goldfarb, Katherine C.; Cech, Thomas R.

2017-01-01

MRP RNA is an abundant, essential noncoding RNA whose functions have been proposed in yeast but are incompletely understood in humans. Mutations in the genomic locus for MRP RNA cause pleiotropic human diseases, including cartilage hair hypoplasia (CHH). Here we applied CRISPR–Cas9 genome editing to disrupt the endogenous human MRP RNA locus, thereby attaining what has eluded RNAi and RNase H experiments: elimination of MRP RNA in the majority of cells. The resulting accumulation of ribosomal RNA (rRNA) precursor—analyzed by RNA fluorescent in situ hybridization (FISH), Northern blots, and RNA sequencing—implicates MRP RNA in pre-rRNA processing. Amelioration of pre-rRNA imbalance is achieved through rescue of MRP RNA levels by ectopic expression. Furthermore, affinity-purified MRP ribonucleoprotein (RNP) from HeLa cells cleaves the human pre-rRNA in vitro at at least one site used in cells, while RNP isolated from cells with CRISPR-edited MRP loci loses this activity, and ectopic MRP RNA expression restores cleavage activity. Thus, a role for RNase MRP in human pre-rRNA processing is established. As demonstrated here, targeted CRISPR disruption is a valuable tool for functional studies of essential noncoding RNAs that are resistant to RNAi and RNase H-based degradation. PMID:28115465

Disruptive environmental chemicals and cellular mechanisms that confer resistance to cell death

PubMed Central

Narayanan, Kannan Badri; Ali, Manaf; Barclay, Barry J.; Cheng, Qiang (Shawn); D’Abronzo, Leandro; Dornetshuber-Fleiss, Rita; Ghosh, Paramita M.; Gonzalez Guzman, Michael J.; Lee, Tae-Jin; Leung, Po Sing; Li, Lin; Luanpitpong, Suidjit; Ratovitski, Edward; Rojanasakul, Yon; Romano, Maria Fiammetta; Romano, Simona; Sinha, Ranjeet K.; Yedjou, Clement; Al-Mulla, Fahd; Al-Temaimi, Rabeah; Amedei, Amedeo; Brown, Dustin G.; Ryan, Elizabeth P.; Colacci, Anna Maria; Hamid, Roslida A.; Mondello, Chiara; Raju, Jayadev; Salem, Hosni K.; Woodrick, Jordan; Scovassi, A.Ivana; Singh, Neetu; Vaccari, Monica; Roy, Rabindra; Forte, Stefano; Memeo, Lorenzo; Kim, Seo Yun; Bisson, William H.; Lowe, Leroy; Park, Hyun Ho

2015-01-01

Cell death is a process of dying within biological cells that are ceasing to function. This process is essential in regulating organism development, tissue homeostasis, and to eliminate cells in the body that are irreparably damaged. In general, dysfunction in normal cellular death is tightly linked to cancer progression. Specifically, the up-regulation of pro-survival factors, including oncogenic factors and antiapoptotic signaling pathways, and the down-regulation of pro-apoptotic factors, including tumor suppressive factors, confers resistance to cell death in tumor cells, which supports the emergence of a fully immortalized cellular phenotype. This review considers the potential relevance of ubiquitous environmental chemical exposures that have been shown to disrupt key pathways and mechanisms associated with this sort of dysfunction. Specifically, bisphenol A, chlorothalonil, dibutyl phthalate, dichlorvos, lindane, linuron, methoxychlor and oxyfluorfen are discussed as prototypical chemical disruptors; as their effects relate to resistance to cell death, as constituents within environmental mixtures and as potential contributors to environmental carcinogenesis. PMID:26106145

Combined effect of attrition and ultrasound on the disruption of Pseudomonas putida for the efficient release of arginine deiminase.

PubMed

Patil, Mahesh D; Shinde, Ashok S; Dev, Manoj J; Patel, Gopal; Bhilare, Kiran D; Banerjee, Uttam Chand

2018-06-08

Disruption of Pseudomonas putida KT2440 by ultrasound treatment in a bath sonicator, in presence of the glass beads, was carried out for the release of arginine deiminase (ADI) and the results were compared with that of by Dyno-mill. The release of ADI depended mainly on the bead size and cellmass concentration being disrupted in bead mill. Nearly 23 U/mL ADI was released when slurry with a cell-mass concentration of 250 g/L was disintegrated for 9 min with 80% bead loading (0.25 mm) in Dyno-mill. Marginally higher amount of ADI (24.1 U/mL) was released by the bath sonication of 250 g/L cellmass slurry for 30 min with the beads (0.1 mm) and a sonication power of 170 W. The glass beads, suspended along with the cellmass slurry in bath sonicator, efficiently disrupted the microbial cells to release ADI. Variation in the kinetic constants for the performance parameters implied that ADI release and cell disruption kinetics is a function of disruption technique used and the process variables thereof. Estimation of location factor suggested that selective release of ADI can be achieved. This article is protected by copyright. All rights reserved. © 2018 American Institute of Chemical Engineers.

MLKL activation triggers NLRP3-mediated processing and release of IL-1β independent of gasdermin-D

PubMed Central

Gutierrez, Kimberley D.; Davis, Michael A.; Daniels, Brian P.; Olsen, Tayla M.; Ralli-Jain, Pooja; Tait, Stephen W.G.; Gale, Michael; Oberst, Andrew

2017-01-01

Necroptosis is a form of programmed cell death defined by activation of the kinase RIPK3 and its downstream effector, the pseudokinase MLKL. Activated MLKL translocates to the cell membrane and disrupts it, leading to loss of cellular ion homeostasis. Here, we use a system in which this event can be specifically triggered by a small-molecule ligand to show that MLKL activation is sufficient to induce the processing and release of bioactive IL-1β. MLKL activation triggers potassium efflux and assembly of the NLRP3 inflammasome, which is required for the processing and activity of IL-1β released during necroptosis. Notably, MLKL activation also causes cell membrane disruption, which allows efficient release of IL-1β independent of the recently described pyroptotic effector gasdermin-D. Together, our findings indicate that MLKL is an endogenous activator of the NLRP3 inflammasome, and that MLKL activation provides a mechanism for concurrent processing and release of IL-1β independent of gasdermin-D. PMID:28130493

Inhibition of human copper trafficking by a small molecule significantly attenuates cancer cell proliferation.

PubMed

Wang, Jing; Luo, Cheng; Shan, Changliang; You, Qiancheng; Lu, Junyan; Elf, Shannon; Zhou, Yu; Wen, Yi; Vinkenborg, Jan L; Fan, Jun; Kang, Heebum; Lin, Ruiting; Han, Dali; Xie, Yuxin; Karpus, Jason; Chen, Shijie; Ouyang, Shisheng; Luan, Chihao; Zhang, Naixia; Ding, Hong; Merkx, Maarten; Liu, Hong; Chen, Jing; Jiang, Hualiang; He, Chuan

2015-12-01

Copper is a transition metal that plays critical roles in many life processes. Controlling the cellular concentration and trafficking of copper offers a route to disrupt these processes. Here we report small molecules that inhibit the human copper-trafficking proteins Atox1 and CCS, and so provide a selective approach to disrupt cellular copper transport. The knockdown of Atox1 and CCS or their inhibition leads to a significantly reduced proliferation of cancer cells, but not of normal cells, as well as to attenuated tumour growth in mouse models. We show that blocking copper trafficking induces cellular oxidative stress and reduces levels of cellular ATP. The reduced level of ATP results in activation of the AMP-activated protein kinase that leads to reduced lipogenesis. Both effects contribute to the inhibition of cancer cell proliferation. Our results establish copper chaperones as new targets for future developments in anticancer therapies.

Inhibition of human copper trafficking by a small molecule significantly attenuates cancer cell proliferation

PubMed Central

Wang, Jing; Luo, Cheng; Shan, Changliang; You, Qiancheng; Lu, Junyan; Elf, Shannon; Zhou, Yu; Wen, Yi; Vinkenborg, Jan L.; Fan, Jun; Kang, Heebum; Lin, Ruiting; Han, Dali; Xie, Yuxin; Karpus, Jason; Chen, Shijie; Ouyang, Shisheng; Luan, Chihao; Zhang, Naixia; Ding, Hong; Merkx, Maarten; Liu, Hong; Chen, Jing; Jiang, Hualiang; He, Chuan

2016-01-01

Copper is a transition metal that plays critical roles in many life processes. Controlling the cellular concentration and trafficking of copper offers a route to disrupt these processes. Here we report small molecules that inhibit the human copper-trafficking proteins Atox1 and CCS, and so provide a selective approach to disrupt cellular copper transport. The knockdown of Atox1 and CCS or their inhibition leads to a significantly reduced proliferation of cancer cells, but not of normal cells, as well as to attenuated tumour growth in mouse models. We show that blocking copper trafficking induces cellular oxidative stress and reduces levels of cellular ATP. The reduced level of ATP results in activation of the AMP-activated protein kinase that leads to reduced lipogenesis. Both effects contribute to the inhibition of cancer cell proliferation. Our results establish copper chaperones as new targets for future developments in anticancer therapies. PMID:26587712

Inhibition of human copper trafficking by a small molecule significantly attenuates cancer cell proliferation

NASA Astrophysics Data System (ADS)

Wang, Jing; Luo, Cheng; Shan, Changliang; You, Qiancheng; Lu, Junyan; Elf, Shannon; Zhou, Yu; Wen, Yi; Vinkenborg, Jan L.; Fan, Jun; Kang, Heebum; Lin, Ruiting; Han, Dali; Xie, Yuxin; Karpus, Jason; Chen, Shijie; Ouyang, Shisheng; Luan, Chihao; Zhang, Naixia; Ding, Hong; Merkx, Maarten; Liu, Hong; Chen, Jing; Jiang, Hualiang; He, Chuan

2015-12-01

Copper is a transition metal that plays critical roles in many life processes. Controlling the cellular concentration and trafficking of copper offers a route to disrupt these processes. Here we report small molecules that inhibit the human copper-trafficking proteins Atox1 and CCS, and so provide a selective approach to disrupt cellular copper transport. The knockdown of Atox1 and CCS or their inhibition leads to a significantly reduced proliferation of cancer cells, but not of normal cells, as well as to attenuated tumour growth in mouse models. We show that blocking copper trafficking induces cellular oxidative stress and reduces levels of cellular ATP. The reduced level of ATP results in activation of the AMP-activated protein kinase that leads to reduced lipogenesis. Both effects contribute to the inhibition of cancer cell proliferation. Our results establish copper chaperones as new targets for future developments in anticancer therapies.

A synthetic ion transporter that disrupts autophagy and induces apoptosis by perturbing cellular chloride concentrations

NASA Astrophysics Data System (ADS)

Busschaert, Nathalie; Park, Seong-Hyun; Baek, Kyung-Hwa; Choi, Yoon Pyo; Park, Jinhong; Howe, Ethan N. W.; Hiscock, Jennifer R.; Karagiannidis, Louise E.; Marques, Igor; Félix, Vítor; Namkung, Wan; Sessler, Jonathan L.; Gale, Philip A.; Shin, Injae

2017-07-01

Perturbations in cellular chloride concentrations can affect cellular pH and autophagy and lead to the onset of apoptosis. With this in mind, synthetic ion transporters have been used to disturb cellular ion homeostasis and thereby induce cell death; however, it is not clear whether synthetic ion transporters can also be used to disrupt autophagy. Here, we show that squaramide-based ion transporters enhance the transport of chloride anions in liposomal models and promote sodium chloride influx into the cytosol. Liposomal and cellular transport activity of the squaramides is shown to correlate with cell death activity, which is attributed to caspase-dependent apoptosis. One ion transporter was also shown to cause additional changes in lysosomal pH, which leads to impairment of lysosomal enzyme activity and disruption of autophagic processes. This disruption is independent of the initiation of apoptosis by the ion transporter. This study provides the first experimental evidence that synthetic ion transporters can disrupt both autophagy and induce apoptosis.

Targeted CRISPR disruption reveals a role for RNase MRP RNA in human preribosomal RNA processing.

PubMed

Goldfarb, Katherine C; Cech, Thomas R

2017-01-01

MRP RNA is an abundant, essential noncoding RNA whose functions have been proposed in yeast but are incompletely understood in humans. Mutations in the genomic locus for MRP RNA cause pleiotropic human diseases, including cartilage hair hypoplasia (CHH). Here we applied CRISPR-Cas9 genome editing to disrupt the endogenous human MRP RNA locus, thereby attaining what has eluded RNAi and RNase H experiments: elimination of MRP RNA in the majority of cells. The resulting accumulation of ribosomal RNA (rRNA) precursor-analyzed by RNA fluorescent in situ hybridization (FISH), Northern blots, and RNA sequencing-implicates MRP RNA in pre-rRNA processing. Amelioration of pre-rRNA imbalance is achieved through rescue of MRP RNA levels by ectopic expression. Furthermore, affinity-purified MRP ribonucleoprotein (RNP) from HeLa cells cleaves the human pre-rRNA in vitro at at least one site used in cells, while RNP isolated from cells with CRISPR-edited MRP loci loses this activity, and ectopic MRP RNA expression restores cleavage activity. Thus, a role for RNase MRP in human pre-rRNA processing is established. As demonstrated here, targeted CRISPR disruption is a valuable tool for functional studies of essential noncoding RNAs that are resistant to RNAi and RNase H-based degradation. © 2017 Goldfarb and Cech; Published by Cold Spring Harbor Laboratory Press.

METHYLMERCURY BUT NOT MERCURIC CHLORIDE INDUCES APOPTOTIC CELL DEATH IN PC12 CELLS.

EPA Science Inventory

Normal development of the nervous system requires the process of apoptosis, a form of programmed cell death, to remove superfluous neurons. Abnormal patterns of apoptosis may be a consequence of exposure to environmental neurotoxicants leading to a disruption in the tightly regul...

Promotion of malignant phenotype after disruption of the three-dimensional structure of cultured spheroids from colorectal cancer

PubMed Central

Endo, Hiroko; Ono, Hiromasa; Hagihara, Takeshi; Okuyama, Hiroaki; Nishizawa, Yasuko; Tomita, Yasuhiko; Ohue, Masayuki; Okita, Kouki; Oyama, Hidejiro; Bono, Hidemasa; Masuko, Takashi; Inoue, Masahiro

2018-01-01

Individual and small clusters of cancer cells may detach from the edges of a main tumor and invade vessels, which can act as the origin of metastasis; however, the mechanism for this phenomenon is not well understood. Using cancer tissue-originated spheroids, we studied whether disturbing the 3D architecture of cancer spheroids can provoke the reformation process and progression of malignancy. We developed a mechanical disruption method to achieve homogenous disruption of the spheroids while maintaining cell–cell contact. After the disruption, 9 spheroid lines from 9 patient samples reformed within a few hours, and 3 of the 9 lines exhibited accelerated spheroid growth. Marker expression, spheroid forming capacity, and tumorigenesis indicated that stemness increased after spheroid disruption. In addition, the spheroid forming capacity increased in 6 of 11 spheroid lines. The disruption signature determined by gene expression profiling supported the incidence of remodeling and predicted the prognosis of patients with colorectal cancer. Furthermore, WNT and HER3 signaling were increased in the reformed spheroids, and suppression of these signaling pathways attenuated the increased proliferation and stemness after the disruption. Overall, the disruption and subsequent reformation of cancer spheroids promoted malignancy-related phenotypes through the activation of the WNT and ERBB pathways. PMID:29662620

Efficient gene disruption in cultured primary human endothelial cells by CRISPR/Cas9.

PubMed

Abrahimi, Parwiz; Chang, William G; Kluger, Martin S; Qyang, Yibing; Tellides, George; Saltzman, W Mark; Pober, Jordan S

2015-07-03

The participation of endothelial cells (EC) in many physiological and pathological processes is widely modeled using human EC cultures, but genetic manipulation of these untransformed cells has been technically challenging. Clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 nuclease (Cas9) technology offers a promising new approach. However, mutagenized cultured cells require cloning to yield homogeneous populations, and the limited replicative lifespan of well-differentiated human EC presents a barrier for doing so. To create a simple but highly efficient method using CRISPR/Cas9 to generate biallelic gene disruption in untransformed human EC. To demonstrate proof-of-principle, we used CRISPR/Cas9 to disrupt the gene for the class II transactivator. We used endothelial colony forming cell-derived EC and lentiviral vectors to deliver CRISPR/Cas9 elements to ablate EC expression of class II major histocompatibility complex molecules and with it, the capacity to activate allogeneic CD4(+) T cells. We show the observed loss-of-function arises from biallelic gene disruption in class II transactivator that leaves other essential properties of the cells intact, including self-assembly into blood vessels in vivo, and that the altered phenotype can be rescued by reintroduction of class II transactivator expression. CRISPR/Cas9-modified human EC provides a powerful platform for vascular research and for regenerative medicine/tissue engineering. © 2015 American Heart Association, Inc.

Disruption of the processing alpha-mannosidase gene does not prevent outer chain synthesis in Saccharomyces cerevisiae.

PubMed

Puccia, R; Grondin, B; Herscovics, A

1993-02-15

Processing of N-linked oligosaccharides in Saccharomyces cerevisiae begins with the removal of glucose and mannose residues from Glc3Man9GlcNAc2 to form a single isomer of Man8GlcNAc2. The importance of mannose removal for subsequent outer chain synthesis was examined in strains of S. cerevisiae disrupted in the MNS1 gene encoding a specific alpha 1,2-mannosidase responsible for Man8GlcNAc2 synthesis [Camirand, Heysen, Grondin and Herscovics (1991) J. Biol. Chem. 266, 15120-15127]. Both MNS1 transcripts of 1.85 kb and 1.7 kb were not observed in Northern blots of mns1 cells (i.e. cells containing the disrupted gene). Analysis on Bio-Gel P-6 of endo-beta-N-acetylglucosaminidase-H-sensitive oligosaccharides following a 10 min pulse with [2-3H]mannose revealed similar amounts of labelled outer chains excluded from the gel in both control and mns1 cells. H.p.l.c. of the included oligosaccharides showed that a Man9GlcNAc, rather than a Man8GlcNAc, intermediate was formed in mns1 cells. Analysis of [3H]mannose-labelled core oligosaccharides from immunoprecipitated CPY and invertase by h.p.l.c. showed a similar size distribution in mns1 and control cells. Invertase immunoprecipitated from [35S]methionine-labelled mns1 cells was highly glycosylated, but migrated slightly faster than that from control cells on denaturing PAGE, indicating a small difference in glycosylation. A similar difference in mobility was observed for invertase activity stain following non-denaturing gel electrophoresis. It is concluded that the alpha-mannosidase encoded by MNS1 is the only enzyme responsible for mannose removal in vivo, and that this processing step is not essential for outer chain synthesis.

A mechanical cell disruption microfluidic platform based on an on-chip micropump.

PubMed

Cheng, Yinuo; Wang, Yue; Wang, Zhiyuan; Huang, Liang; Bi, Mingzhao; Xu, Wenxiao; Wang, Wenhui; Ye, Xiongying

2017-03-01

Cell disruption plays a vital role in detection of intracellular components which contain information about genetic and disease characteristics. In this paper, we demonstrate a novel microfluidic platform based on an on-chip micropump for mechanical cell disruption and sample transport. A 50  μ l cell sample can be effectively lysed through on-chip multi-disruption in 36 s without introducing any chemical agent and suffering from clogging by cellular debris. After 30 cycles of circulating disruption, 80.6% and 90.5% cell disruption rates were achieved for the HEK293 cell sample and human natural killer cell sample, respectively. Profiting from the feature of pump-on-chip, the highly integrated platform enables more convenient and cost-effective cell disruption for the analysis of intracellular components.

A mechanical cell disruption microfluidic platform based on an on-chip micropump

PubMed Central

Cheng, Yinuo; Wang, Yue; Wang, Zhiyuan; Bi, Mingzhao; Xu, Wenxiao; Ye, Xiongying

2017-01-01

Cell disruption plays a vital role in detection of intracellular components which contain information about genetic and disease characteristics. In this paper, we demonstrate a novel microfluidic platform based on an on-chip micropump for mechanical cell disruption and sample transport. A 50 μl cell sample can be effectively lysed through on-chip multi-disruption in 36 s without introducing any chemical agent and suffering from clogging by cellular debris. After 30 cycles of circulating disruption, 80.6% and 90.5% cell disruption rates were achieved for the HEK293 cell sample and human natural killer cell sample, respectively. Profiting from the feature of pump-on-chip, the highly integrated platform enables more convenient and cost-effective cell disruption for the analysis of intracellular components. PMID:28798848

Disruptive environmental chemicals and cellular mechanisms that confer resistance to cell death.

PubMed

Narayanan, Kannan Badri; Ali, Manaf; Barclay, Barry J; Cheng, Qiang Shawn; D'Abronzo, Leandro; Dornetshuber-Fleiss, Rita; Ghosh, Paramita M; Gonzalez Guzman, Michael J; Lee, Tae-Jin; Leung, Po Sing; Li, Lin; Luanpitpong, Suidjit; Ratovitski, Edward; Rojanasakul, Yon; Romano, Maria Fiammetta; Romano, Simona; Sinha, Ranjeet K; Yedjou, Clement; Al-Mulla, Fahd; Al-Temaimi, Rabeah; Amedei, Amedeo; Brown, Dustin G; Ryan, Elizabeth P; Colacci, Annamaria; Hamid, Roslida A; Mondello, Chiara; Raju, Jayadev; Salem, Hosni K; Woodrick, Jordan; Scovassi, A Ivana; Singh, Neetu; Vaccari, Monica; Roy, Rabindra; Forte, Stefano; Memeo, Lorenzo; Kim, Seo Yun; Bisson, William H; Lowe, Leroy; Park, Hyun Ho

2015-06-01

Cell death is a process of dying within biological cells that are ceasing to function. This process is essential in regulating organism development, tissue homeostasis, and to eliminate cells in the body that are irreparably damaged. In general, dysfunction in normal cellular death is tightly linked to cancer progression. Specifically, the up-regulation of pro-survival factors, including oncogenic factors and antiapoptotic signaling pathways, and the down-regulation of pro-apoptotic factors, including tumor suppressive factors, confers resistance to cell death in tumor cells, which supports the emergence of a fully immortalized cellular phenotype. This review considers the potential relevance of ubiquitous environmental chemical exposures that have been shown to disrupt key pathways and mechanisms associated with this sort of dysfunction. Specifically, bisphenol A, chlorothalonil, dibutyl phthalate, dichlorvos, lindane, linuron, methoxychlor and oxyfluorfen are discussed as prototypical chemical disruptors; as their effects relate to resistance to cell death, as constituents within environmental mixtures and as potential contributors to environmental carcinogenesis. © The Author 2015. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com.

Improved cell disruption of Pichia pastoris utilizing aminopropyl magnesium phyllosilicate (AMP) clay.

PubMed

Kim, Sun-Il; Wu, Yuanzheng; Kim, Ka-Lyun; Kim, Geun-Joong; Shin, Hyun-Jae

2013-06-01

An efficient method for Pichia cell disruption that employs an aminopropyl magnesium phyllosilicate (AMP) clay-assisted glass beads mill is presented. AMP clay is functionalized nanocomposite resembling the talc parent structure Si8Mg6O20(OH)4 that has been proven to permeate the bacterial membrane and cause cell lysis. The recombinant capsid protein of cowpea chlorotic mottle virus (CCMV) expressed in Pichia pastoris GS115 was used as demonstration system for their ability of self-assembly into icosahedral virus-like particles (VLPs). The total protein concentration reached 4.24 mg/ml after 4 min treatment by glass beads mill combined with 0.2 % AMP clay, which was 11.2 % higher compared to glass beads mill only and the time was half shortened. The stability of purified CCMV VLPs illustrated AMP clay had no influence on virus assembly process. Considering the tiny amount added and simple approach of AMP clay, it could be a reliable method for yeast cell disruption.

In Vitro Bioluminescence Assay to Characterize Circadian Rhythm in Mammary Epithelial Cells.

PubMed

Fang, Mingzhu; Kang, Hwan-Goo; Park, Youngil; Estrella, Brian; Zarbl, Helmut

2017-09-28

The circadian rhythm is a fundamental physiological process present in all organisms that regulates biological processes ranging from gene expression to sleep behavior. In vertebrates, circadian rhythm is controlled by a molecular oscillator that functions in both the suprachiasmatic nucleus (SCN; central pacemaker) and individual cells comprising most peripheral tissues. More importantly, disruption of circadian rhythm by exposure to light-at-night, environmental stressors and/or toxicants is associated with increased risk of chronic diseases and aging. The ability to identify agents that can disrupt central and/or peripheral biological clocks, and agents that can prevent or mitigate the effects of circadian disruption, has significant implications for prevention of chronic diseases. Although rodent models can be used to identify exposures and agents that induce or prevent/mitigate circadian disruption, these experiments require large numbers of animals. In vivo studies also require significant resources and infrastructure, and require researchers to work all night. Thus, there is an urgent need for a cell-type appropriate in vitro system to screen for environmental circadian disruptors and enhancers in cell types from different organs and disease states. We constructed a vector that drives transcription of the destabilized luciferase in eukaryotic cells under the control of the human PERIOD 2 gene promoter. This circadian reporter construct was stably transfected into human mammary epithelial cells, and circadian responsive reporter cells were selected to develop the in vitro bioluminescence assay. Here, we present a detailed protocol to establish and validate the assay. We further provide details for proof of concept experiments demonstrating the ability of our in vitro assay to recapitulate the in vivo effects of various chemicals on the cellular biological clock. The results indicate that the assay can be adapted to a variety of cell types to screen for both environmental disruptors and chemopreventive enhancers of circadian clocks.

SUMO5, a Novel Poly-SUMO Isoform, Regulates PML Nuclear Bodies

PubMed Central

Liang, Ya-Chen; Lee, Chia-Chin; Yao, Ya-Li; Lai, Chien-Chen; Schmitz, M. Lienhard; Yang, Wen-Ming

2016-01-01

Promyelocytic leukemia nuclear bodies (PML-NBs) are PML-based nuclear structures that regulate various cellular processes. SUMOylation, the process of covalently conjugating small ubiquitin-like modifiers (SUMOs), is required for both the formation and the disruption of PML-NBs. However, detailed mechanisms of how SUMOylation regulates these processes remain unknown. Here we report that SUMO5, a novel SUMO variant, mediates the growth and disruption of PML-NBs. PolySUMO5 conjugation of PML at lysine 160 facilitates recruitment of PML-NB components, which enlarges PML-NBs. SUMO5 also increases polySUMO2/3 conjugation of PML, resulting in RNF4-mediated disruption of PML-NBs. The acute promyelocytic leukemia oncoprotein PML-RARα blocks SUMO5 conjugation of PML, causing cytoplasmic displacement of PML and disruption of PML-NBs. Our work not only identifies a new member of the SUMO family but also reveals the mechanistic basis of the PML-NB life cycle in human cells. PMID:27211601

Process for biological material carbon-carbon bond formation

DOEpatents

Hollingsworth, R.I.; Jung, S.; Mindock, C.A.

1998-12-22

A process for providing vicinal dimethyl long chain between alkyl groups of organic compounds is described. The process uses intact or disrupted cells of various species of bacteria, particularly Thermoanaerobacter sp., Sarcina sp. and Butyrivibrio sp. The process can be conducted in an aqueous reaction mixture at room temperatures. 8 figs.

Process for biological material carbon-carbon bond formation

DOEpatents

Hollingsworth, Rawle I.; Jung, Seunho; Mindock, Carol A.

1998-01-01

A process for providing vicinal dimethyl long chain between alkyl groups of organic compounds is described. The process uses intact or disrupted cells of various species of bacteria, particularly Thermoanaerobacter sp., Sarcina sp. and Butyrivibrio sp. The process can be conducted in an aqueous reaction mixture at room temperatures.

Disruptions in follicle cell functions in the ovaries of rhesus monkeys during summer

PubMed Central

VandeVoort, Catherine A.; Mtango, Namdori R.; Midic, Uros

2015-01-01

Oocytes isolated from female rhesus monkeys following standard ovarian stimulation protocols during the summer months displayed a reduced capacity to mature compared with stimulation during the normal breeding season. Because the gene expression profiles of oocyte-associated cumulus cells and mural granulosa cells (CCs and GCs) are indicative of altered oocyte quality and can provide insight into intrafollicular processes that may be disrupted during oogenesis, we performed array-based transcriptome comparisons of CCs and GCs from summer and normal breeding season stimulation cycles. Summer CCs and GCs both display deficiencies in expression of mRNAs related to cell proliferation, angiogenesis, and endocrine signaling, as well as reduced expression of glycogen phosphorylase. Additionally, CCs display deficiencies in expression of mRNAs related to stress response. These results provide the first insight into the specific molecular pathways and processes that are disrupted in the follicles of rhesus macaque females during the summer season. Some of the changes seen in summer GCs and CCs have been reported in humans and in other model mammalian species. This suggests that the seasonal effects seen in the rhesus monkey may help us to understand better the mechanisms that contribute to reduced oocyte quality and fertility in humans. PMID:25586978

APC binds the Miro/Milton motor complex to stimulate transport of mitochondria to the plasma membrane.

PubMed

Mills, Kate M; Brocardo, Mariana G; Henderson, Beric R

2016-02-01

Mutations in adenomatous polyposis coli (APC) disrupt regulation of Wnt signaling, mitosis, and the cytoskeleton. We describe a new role for APC in the transport of mitochondria. Silencing of wild-type APC by small interfering RNA caused mitochondria to redistribute from the cell periphery to the perinuclear region. We identified novel APC interactions with the mitochondrial kinesin-motor complex Miro/Milton that were mediated by the APC C-terminus. Truncating mutations in APC abolished its ability to bind Miro/Milton and reduced formation of the Miro/Milton complex, correlating with disrupted mitochondrial distribution in colorectal cancer cells that could be recovered by reconstitution of wild-type APC. Using proximity ligation assays, we identified endogenous APC-Miro/Milton complexes at mitochondria, and live-cell imaging showed that loss of APC slowed the frequency of anterograde mitochondrial transport to the membrane. We propose that APC helps drive mitochondria to the membrane to supply energy for cellular processes such as directed cell migration, a process disrupted by cancer mutations. © 2016 Mills et al. This article is distributed by The American Society for Cell Biology under license from the author(s). Two months after publication it is available to the public under an Attribution–Noncommercial–Share Alike 3.0 Unported Creative Commons License (http://creativecommons.org/licenses/by-nc-sa/3.0).

Severe blood-brain barrier disruption and surrounding tissue injury.

PubMed

Chen, Bo; Friedman, Beth; Cheng, Qun; Tsai, Phil; Schim, Erica; Kleinfeld, David; Lyden, Patrick D

2009-12-01

Blood-brain barrier opening during ischemia follows a biphasic time course, may be partially reversible, and allows plasma constituents to enter brain and possibly damage cells. In contrast, severe vascular disruption after ischemia is unlikely to be reversible and allows even further extravasation of potentially harmful plasma constituents. We sought to use simple fluorescent tracers to allow wide-scale visualization of severely damaged vessels and determine whether such vascular disruption colocalized with regions of severe parenchymal injury. Severe vascular disruption and ischemic injury was produced in adult Sprague Dawley rats by transient occlusion of the middle cerebral artery for 1, 2, 4, or 8 hours, followed by 30 minutes of reperfusion. Fluorescein isothiocyanate-dextran (2 MDa) was injected intravenously before occlusion. After perfusion-fixation, brain sections were processed for ultrastructure or fluorescence imaging. We identified early evidence of tissue damage with Fluoro-Jade staining of dying cells. With increasing ischemia duration, greater quantities of high molecular weight dextran-fluorescein isothiocyanate invaded and marked ischemic regions in a characteristic pattern, appearing first in the medial striatum, spreading to the lateral striatum, and finally involving cortex; maximal injury was seen in the mid-parietal areas, consistent with the known ischemic zone in this model. The regional distribution of the severe vascular disruption correlated with the distribution of 24-hour 2,3,5-triphenyltetrazolium chloride pallor (r=0.75; P<0.05) and the cell death marker Fluoro-Jade (r=0.86; P<0.05). Ultrastructural examination showed significantly increased areas of swollen astrocytic foot process and swollen mitochondria in regions of high compared to low leakage, and compared to contralateral homologous regions (ANOVA P<0.01). Dextran extravasation into the basement membrane and surrounding tissue increased significantly from 2 to 8 hours of occlusion duration (Independent samples t test, P<0.05). Severe vascular disruption, as labeled with high-molecular-weight dextran-fluorescein isothiocyanate leakage, is associated with severe tissue injury. This marker of severe vascular disruption may be useful in further studies of the pathoanatomic mechanisms of vascular disruption-mediated tissue injury.

Lipid disequilibrium disrupts ER proteostasis by impairing ERAD substrate glycan trimming and dislocation

PubMed Central

To, Milton; Peterson, Clark W. H.; Roberts, Melissa A.; Counihan, Jessica L.; Wu, Tiffany T.; Forster, Mercedes S.; Nomura, Daniel K.; Olzmann, James A.

2017-01-01

The endoplasmic reticulum (ER) mediates the folding, maturation, and deployment of the secretory proteome. Proteins that fail to achieve their native conformation are retained in the ER and targeted for clearance by ER-associated degradation (ERAD), a sophisticated process that mediates the ubiquitin-dependent delivery of substrates to the 26S proteasome for proteolysis. Recent findings indicate that inhibition of long-chain acyl-CoA synthetases with triacsin C, a fatty acid analogue, impairs lipid droplet (LD) biogenesis and ERAD, suggesting a role for LDs in ERAD. However, whether LDs are involved in the ERAD process remains an outstanding question. Using chemical and genetic approaches to disrupt diacylglycerol acyltransferase (DGAT)–dependent LD biogenesis, we provide evidence that LDs are dispensable for ERAD in mammalian cells. Instead, our results suggest that triacsin C causes global alterations in the cellular lipid landscape that disrupt ER proteostasis by interfering with the glycan trimming and dislocation steps of ERAD. Prolonged triacsin C treatment activates both the IRE1 and PERK branches of the unfolded protein response and ultimately leads to IRE1-dependent cell death. These findings identify an intimate relationship between fatty acid metabolism and ER proteostasis that influences cell viability. PMID:27881664

Cellular level robotic surgery: Nanodissection of intermediate filaments in live keratinocytes.

PubMed

Yang, Ruiguo; Song, Bo; Sun, Zhiyong; Lai, King Wai Chiu; Fung, Carmen Kar Man; Patterson, Kevin C; Seiffert-Sinha, Kristina; Sinha, Animesh A; Xi, Ning

2015-01-01

We present the nanosurgery on the cytoskeleton of live cells using AFM based nanorobotics to achieve adhesiolysis and mimic the effect of pathophysiological modulation of intercellular adhesion. Nanosurgery successfully severs the intermediate filament bundles and disrupts cell-cell adhesion similar to the desmosomal protein disassembly in autoimmune disease, or the cationic modulation of desmosome formation. Our nanomechanical analysis revealed that adhesion loss results in a decrease in cellular stiffness in both cases of biochemical modulation of the desmosome junctions and mechanical disruption of intercellular adhesion, supporting the notion that intercellular adhesion through intermediate filaments anchors the cell structure as focal adhesion does and that intermediate filaments are integral components in cell mechanical integrity. The surgical process could potentially help reveal the mechanism of autoimmune pathology-induced cell-cell adhesion loss as well as its related pathways that lead to cell apoptosis. Copyright © 2015 Elsevier Inc. All rights reserved.

The cell proliferation antigen Ki-67 organises heterochromatin

PubMed Central

Sobecki, Michal; Mrouj, Karim; Camasses, Alain; Parisis, Nikolaos; Nicolas, Emilien; Llères, David; Gerbe, François; Prieto, Susana; Krasinska, Liliana; David, Alexandre; Eguren, Manuel; Birling, Marie-Christine; Urbach, Serge; Hem, Sonia; Déjardin, Jérôme; Malumbres, Marcos; Jay, Philippe; Dulic, Vjekoslav; Lafontaine, Denis LJ; Feil, Robert; Fisher, Daniel

2016-01-01

Antigen Ki-67 is a nuclear protein expressed in proliferating mammalian cells. It is widely used in cancer histopathology but its functions remain unclear. Here, we show that Ki-67 controls heterochromatin organisation. Altering Ki-67 expression levels did not significantly affect cell proliferation in vivo. Ki-67 mutant mice developed normally and cells lacking Ki-67 proliferated efficiently. Conversely, upregulation of Ki-67 expression in differentiated tissues did not prevent cell cycle arrest. Ki-67 interactors included proteins involved in nucleolar processes and chromatin regulators. Ki-67 depletion disrupted nucleologenesis but did not inhibit pre-rRNA processing. In contrast, it altered gene expression. Ki-67 silencing also had wide-ranging effects on chromatin organisation, disrupting heterochromatin compaction and long-range genomic interactions. Trimethylation of histone H3K9 and H4K20 was relocalised within the nucleus. Finally, overexpression of human or Xenopus Ki-67 induced ectopic heterochromatin formation. Altogether, our results suggest that Ki-67 expression in proliferating cells spatially organises heterochromatin, thereby controlling gene expression. DOI: http://dx.doi.org/10.7554/eLife.13722.001 PMID:26949251

Soybean agglutinin binding to corneal endothelial cell surfaces disrupts in situ monolayer integrity and actin organization and interferes with wound repair.

PubMed

Gordon, Sheldon R; Wood, Meredith

2009-03-01

Rat corneal endothelium demonstrates cell-surface soybean agglutinin (SBA) binding during organ-culture or injury. When organ-cultured in medium containing SBA, the endothelial monolayer is disrupted because of cell-cell and cell-matrix alterations. SBA binding disorganizes the circumferential microfilament bundles (CMBs), an effect that is partially prevented by phallacidin preincubation. This disruption is reversible if tissues are returned to standard culture medium. Serum heightens SBA binding, whereas puromycin prevents it. Neither actinomycin D nor alpha-amanitin inhibits SBA binding, suggesting that SBA-binding protein(s) may be post-transcriptionally regulated. During injury-induced cell migration in the presence of SBA, cellular processes are blunted and fail to extend significantly outward. By 72 h post-injury, cells of SBA-treated tissues repopulate the wound but demonstrate little association with neighboring cells. Cells migrating in the presence of N-acetylgalactosamine appear normal but also fail to reassociate with other cells in the jury zone. Immunofluorescent staining for ZO-1 reveals punctuate patterns in cells of control tissues, whereas neither SBA- nor N-acetylgalactosamine-treated tissues exhibit ZO-1 staining. Terminal N-acetylgalactosamine removal fails to affect cell morphology, actin organization, or migration but prevents lectin binding. Our results suggest that SBA binding reflects the synthesis of a stress-induced protein(s) that may play a role in reestablishing cell-cell relationships during monolayer reorganization following injury.

Microalgal cell disruption via ultrasonic nozzle spraying.

PubMed

Wang, M; Yuan, W

2015-01-01

The objective of this study was to understand the effect of operating parameters, including ultrasound amplitude, spraying pressure, nozzle orifice diameter, and initial cell concentration on microalgal cell disruption and lipid extraction in an ultrasonic nozzle spraying system (UNSS). Two algal species including Scenedesmus dimorphus and Nannochloropsis oculata were evaluated. Experimental results demonstrated that the UNSS was effective in the disruption of microalgal cells indicated by significant changes in cell concentration and Nile red-stained lipid fluorescence density between all treatments and the control. It was found that increasing ultrasound amplitude generally enhanced cell disruption and lipid recovery although excessive input energy was not necessary for best results. The effect of spraying pressure and nozzle orifice diameter on cell disruption and lipid recovery was believed to be dependent on the competition between ultrasound-induced cavitation and spraying-generated shear forces. Optimal cell disruption was not always achieved at the highest spraying pressure or biggest nozzle orifice diameter; instead, they appeared at moderate levels depending on the algal strain and specific settings. Increasing initial algal cell concentration significantly reduced cell disruption efficiency. In all UNSS treatments, the effectiveness of cell disruption and lipid recovery was found to be dependent on the algal species treated.

Cell-wall disruption and lipid/astaxanthin extraction from microalgae: Chlorella and Haematococcus.

PubMed

Kim, Dong-Yeon; Vijayan, Durairaj; Praveenkumar, Ramasamy; Han, Jong-In; Lee, Kyubock; Park, Ji-Yeon; Chang, Won-Seok; Lee, Jin-Suk; Oh, You-Kwan

2016-01-01

Recently, biofuels and nutraceuticals produced from microalgae have emerged as major interests, resulting in intensive research of the microalgal biorefinery process. In this paper, recent developments in cell-wall disruption and extraction methods are reviewed, focusing on lipid and astaxanthin production from the biotechnologically important microalgae Chlorella and Haematococcus, respectively. As a common, critical bottleneck for recovery of intracellular components such as lipid and astaxanthin from these microalgae, the composition and structure of rigid, thick cell-walls were analyzed. Various chemical, physical, physico-chemical, and biological methods applied for cell-wall breakage and lipid/astaxanthin extraction from Chlorella and Haematococcus are discussed in detail and compared based on efficiency, energy consumption, type and dosage of solvent, biomass concentration and status (wet/dried), toxicity, scalability, and synergistic combinations. This report could serve as a useful guide to the implementation of practical downstream processes for recovery of valuable products from microalgae including Chlorella and Haematococcus. Copyright © 2015 Elsevier Ltd. All rights reserved.

Human Cytomegalovirus UL50 and UL53 Recruit Viral Protein Kinase UL97, Not Protein Kinase C, for Disruption of Nuclear Lamina and Nuclear Egress in Infected Cells

PubMed Central

Sharma, Mayuri; Kamil, Jeremy P.; Coughlin, Margaret; Reim, Natalia I.

2014-01-01

Herpesvirus nucleocapsids traverse the nuclear envelope into the cytoplasm in a process called nuclear egress that includes disruption of the nuclear lamina. In several herpesviruses, a key player in nuclear egress is a complex of two proteins, whose homologs in human cytomegalovirus (HCMV) are UL50 and UL53. However, their roles in nuclear egress during HCMV infection have not been shown. Based largely on transfection studies, UL50 and UL53 have been proposed to facilitate disruption of the nuclear lamina by recruiting cellular protein kinase C (PKC), as occurs with certain other herpesviruses, and/or the viral protein kinase UL97 to phosphorylate lamins. To investigate these issues during HCMV infection, we generated viral mutants null for UL50 or UL53. Correlative light electron microscopic analysis of null mutant-infected cells showed the presence of intranuclear nucleocapsids and the absence of cytoplasmic nucleocapsids. Confocal immunofluorescence microscopy revealed that UL50 and UL53 are required for disruption of the nuclear lamina. A subpopulation of UL97 colocalized with the nuclear rim, and this was dependent on UL50 and, to a lesser extent, UL53. However, PKC was not recruited to the nuclear rim, and its localization was not affected by the absence of UL50 or UL53. Immunoprecipitation from cells infected with HCMV expressing tagged UL53 detected UL97 but not PKC. In summary, HCMV UL50 and UL53 are required for nuclear egress and disruption of nuclear lamina during HCMV infection, and they recruit UL97, not PKC, for these processes. Thus, despite the strong conservation of herpesvirus nuclear egress complexes, a key function can differ among them. PMID:24155370

Human cytomegalovirus UL50 and UL53 recruit viral protein kinase UL97, not protein kinase C, for disruption of nuclear lamina and nuclear egress in infected cells.

PubMed

Sharma, Mayuri; Kamil, Jeremy P; Coughlin, Margaret; Reim, Natalia I; Coen, Donald M

2014-01-01

Herpesvirus nucleocapsids traverse the nuclear envelope into the cytoplasm in a process called nuclear egress that includes disruption of the nuclear lamina. In several herpesviruses, a key player in nuclear egress is a complex of two proteins, whose homologs in human cytomegalovirus (HCMV) are UL50 and UL53. However, their roles in nuclear egress during HCMV infection have not been shown. Based largely on transfection studies, UL50 and UL53 have been proposed to facilitate disruption of the nuclear lamina by recruiting cellular protein kinase C (PKC), as occurs with certain other herpesviruses, and/or the viral protein kinase UL97 to phosphorylate lamins. To investigate these issues during HCMV infection, we generated viral mutants null for UL50 or UL53. Correlative light electron microscopic analysis of null mutant-infected cells showed the presence of intranuclear nucleocapsids and the absence of cytoplasmic nucleocapsids. Confocal immunofluorescence microscopy revealed that UL50 and UL53 are required for disruption of the nuclear lamina. A subpopulation of UL97 colocalized with the nuclear rim, and this was dependent on UL50 and, to a lesser extent, UL53. However, PKC was not recruited to the nuclear rim, and its localization was not affected by the absence of UL50 or UL53. Immunoprecipitation from cells infected with HCMV expressing tagged UL53 detected UL97 but not PKC. In summary, HCMV UL50 and UL53 are required for nuclear egress and disruption of nuclear lamina during HCMV infection, and they recruit UL97, not PKC, for these processes. Thus, despite the strong conservation of herpesvirus nuclear egress complexes, a key function can differ among them.

Disruption of TGF-β signaling in smooth muscle cell prevents flow-induced vascular remodeling

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

Gao, Fu; Chambon, Pierre; Tellides, George

Highlights: • TGF-β signaling in SMC contributes to the flow-induced vascular remodeling. • Disruption of TGF-β signaling in SMC can prevent this process. • Targeting SM-specific Tgfbr2 could be a novel therapeutic strategy for vascular remodeling. - Abstract: Transforming growth factor-β (TGF-β) signaling has been prominently implicated in the pathogenesis of vascular remodeling, especially the initiation and progression of flow-induced vascular remodeling. Smooth muscle cells (SMCs) are the principal resident cells in arterial wall and are critical for arterial remodeling. However, the role of TGF-β signaling in SMC for flow-induced vascular remodeling remains unknown. Therefore, the goal of our studymore » was to determine the effect of TGF-β pathway in SMC for vascular remodeling, by using a genetical smooth muscle-specific (SM-specific) TGF-β type II receptor (Tgfbr2) deletion mice model. Mice deficient in the expression of Tgfbr2 (MyhCre.Tgfbr2{sup f/f}) and their corresponding wild-type background mice (MyhCre.Tgfbr2{sup WT/WT}) underwent partial ligation of left common carotid artery for 1, 2, or 4 weeks. Then the carotid arteries were harvested and indicated that the disruption of Tgfbr2 in SMC provided prominent inhibition of vascular remodeling. And the thickening of carotid media, proliferation of SMC, infiltration of macrophage, and expression of matrix metalloproteinase (MMP) were all significantly attenuated in Tgfbr2 disruption mice. Our study demonstrated, for the first time, that the TGF-β signaling in SMC plays an essential role in flow-induced vascular remodeling and disruption can prevent this process.« less

Altered Middle Lamella Homogalacturonan and Disrupted Deposition of (1→5)-α-l-Arabinan in the Pericarp of Cnr, a Ripening Mutant of Tomato1

PubMed Central

Orfila, Caroline; Seymour, Graham B.; Willats, William G.T.; Huxham, I. Max; Jarvis, Michael C.; Dover, Colin J.; Thompson, Andrew J.; Knox, J. Paul

2001-01-01

Cnr (colorless non-ripening) is a pleiotropic tomato (Lycopersicon esculentum) fruit ripening mutant with altered tissue properties including weaker cell-to-cell contacts in the pericarp (A.J. Thompson, M. Tor, C.S. Barry, J. Vrebalov, C. Orfila, M.C. Jarvis, J.J. Giovannoni, D. Grierson, G.B. Seymour [1999] Plant Physiol 120: 383–390). Whereas the genetic basis of the Cnr mutation is being identified by molecular analyses, here we report the identification of cell biological factors underlying the Cnr texture phenotype. In comparison with wild type, ripe-stage Cnr fruits have stronger, non-swollen cell walls (CW) throughout the pericarp and extensive intercellular space in the inner pericarp. Using electron energy loss spectroscopy imaging of calcium-binding capacity and anti-homogalacturonan (HG) antibody probes (PAM1 and JIM5) we demonstrate that maturation processes involving middle lamella HG are altered in Cnr fruit, resulting in the absence or a low level of HG-/calcium-based cell adhesion. We also demonstrate that the deposition of (1→5)-α-l-arabinan is disrupted in Cnr pericarp CW and that this disruption occurs prior to fruit ripening. The relationship between the disruption of (1→5)-α-l-arabinan deposition in pericarp CW and the Cnr phenotype is discussed. PMID:11351084

Circadian Rhythm Disruption Promotes Lung Tumorigenesis.

PubMed

Papagiannakopoulos, Thales; Bauer, Matthew R; Davidson, Shawn M; Heimann, Megan; Subbaraj, Lakshmipriya; Bhutkar, Arjun; Bartlebaugh, Jordan; Vander Heiden, Matthew G; Jacks, Tyler

2016-08-09

Circadian rhythms are 24-hr oscillations that control a variety of biological processes in living systems, including two hallmarks of cancer, cell division and metabolism. Circadian rhythm disruption by shift work is associated with greater risk for cancer development and poor prognosis, suggesting a putative tumor-suppressive role for circadian rhythm homeostasis. Using a genetically engineered mouse model of lung adenocarcinoma, we have characterized the effects of circadian rhythm disruption on lung tumorigenesis. We demonstrate that both physiologic perturbation (jet lag) and genetic mutation of the central circadian clock components decreased survival and promoted lung tumor growth and progression. The core circadian genes Per2 and Bmal1 were shown to have cell-autonomous tumor-suppressive roles in transformation and lung tumor progression. Loss of the central clock components led to increased c-Myc expression, enhanced proliferation, and metabolic dysregulation. Our findings demonstrate that both systemic and somatic disruption of circadian rhythms contribute to cancer progression. Copyright © 2016 Elsevier Inc. All rights reserved.

Mechanical injury and repair of cells

NASA Technical Reports Server (NTRS)

Miyake, Katsuya; McNeil, Paul L.

2003-01-01

OBJECTIVE: To concisely review the field of cell plasma membrane disruption (torn cell surface) and repair. MAIN POINTS: Plasma membrane disruption is a common form of cell injury under physiologic conditions, after trauma, in certain muscular dystrophies, and during certain forms of clinical intervention. Rapid repair of a disruption is essential to cell survival and involves a complex and active cell response that includes membrane fusion and cytoskeletal activation. Tissues, such as cardiac and skeletal muscle, adapt to a disruption injury by hypertrophying. Cells adapt by increasing the efficiency of their resealing response. CONCLUSION: Plasma membrane disruption is an important cellular event in both health and disease. The disruption repair mechanism is now well understood at the cellular level, but much remains to be learned at the molecular level. Cell and tissue level adaptational responses to the disruption either prevent its further occurrence or facilitate future repairs. Therapeutically useful drugs might result if, using this accumulating knowledge, chemical agents can be developed that can enhance repair or adaptive responses.

Deciphering the Adaptive Immune Response to Ovarian Cancer

DTIC Science & Technology

2013-10-01

Additionally, anti-cancer drugs such as 2-phenyl benzimidazole (2- PB) disrupt the Golgi apparatus, which leads to reduced cell proliferation and tumour...of Golgi processing by 2-phenyl benzimidazole analogs blocks cell proliferation and slows tumor growth. Cancer Chemother Pharmacol 2008; 61: 1045–1058

Analysis of the Effects of Cell Stress and Cytotoxicity on In Vitro Assay Activity Across a Diverse Chemical and Assay Space

EPA Science Inventory

Chemical toxicity can arise from disruption of specific biomolecular functions or through more generalized cell stress and cytotoxicity-mediated processes. Here, concentration-dependent responses of 1063 chemicals including pharmaceuticals, natural products, pesticidals, consumer...

Exogenous retinoic acid induces digit reduction in opossums (Monodelphis domestica) by disrupting cell death and proliferation, and apical ectodermal ridge and zone of polarizing activity function.

PubMed

Molineaux, Anna C; Maier, Jennifer A; Schecker, Teresa; Sears, Karen E

2015-03-01

Retinoic acid (RA) is a vitamin A derivative. Exposure to exogenous RA generates congenital limb malformations (CLMs) in species from frogs to humans. These CLMs include but are not limited to oligodactyly and long-bone hypoplasia. The processes by which exogenous RA induces CLMs in mammals have been best studied in mouse, but as of yet remain unresolved. We investigated the impact of exogenous RA on the cellular and molecular development of the limbs of a nonrodent model mammal, the opossum Monodelphis domestica. Opossums exposed to exogenous retinoic acid display CLMs including oligodactly, and results are consistent with opossum development being more susceptible to RA-induced disruptions than mouse development. Exposure of developing opossums to exogenous RA leads to an increase in cell death in the limb mesenchyme that is most pronounced in the zone of polarizing activity, and a reduction in cell proliferation throughout the limb mesenchyme. Exogenous RA also disrupts the expression of Shh in the zone of polarizing activity, and Fgf8 in the apical ectodermal ridge, and other genes with roles in the regulation of limb development and cell death. Results are consistent with RA inducing CLMs in opossum limbs by disrupting the functions of the apical ectodermal ridge and zone of polarizing activity, and driving an increase in cell death and reduction of cell proliferation in the mesenchyme of the developing limb. © 2015 Wiley Periodicals, Inc.

Targeting pH regulating proteins for cancer therapy-Progress and limitations.

PubMed

Parks, Scott K; Pouysségur, Jacques

2017-04-01

Tumour acidity induced by metabolic alterations and incomplete vascularisation sets cancer cells apart from normal cellular physiology. This distinguishing tumour characteristic has been an area of intense study, as cellular pH (pH i ) disturbances disrupt protein function and therefore multiple cellular processes. Tumour cells effectively utilise pH i regulating machinery present in normal cells with enhancements provided by additional oncogenic or hypoxia induced protein modifications. This overall improvement of pH regulation enables maintenance of an alkaline pH i in the continued presence of external acidification (pH e ). Considerable experimentation has revealed targets that successfully disrupt tumour pH i regulation in efforts to develop novel means to weaken or kill tumour cells. However, redundancy in these pH-regulating proteins, which include Na + /H + exchangers (NHEs), carbonic anhydrases (CAs), Na + /HCO 3 - co-transporters (NBCs) and monocarboxylate transporters (MCTs) has prevented effective disruption of tumour pH i when individual protein targeting is performed. Here we synthesise recent advances in understanding both normoxic and hypoxic pH regulating mechanisms in tumour cells with an ultimate focus on the disruption of tumour growth, survival and metastasis. Interactions between tumour acidity and other cell types are also proving to be important in understanding therapeutic applications such as immune therapy. Promising therapeutic developments regarding pH manipulation along with current limitations are highlighted to provide a framework for future research directives. Copyright © 2017 Elsevier Ltd. All rights reserved.

Fatigue failure of osteocyte cellular processes: implications for the repair of bone.

PubMed

Dooley, C; Cafferky, D; Lee, T C; Taylor, D

2014-01-25

The physical effects of fatigue failure caused by cyclic strain are important and for most materials well understood. However, nothing is known about this mode of failure in living cells. We developed a novel method that allowed us to apply controlled levels of cyclic displacement to networks of osteocytes in bone. We showed that under cyclic loading, fatigue failure takes place in the dendritic processes of osteocytes at cyclic strain levels as low as one tenth of the strain needed for instantaneous rupture. The number of cycles to failure was inversely correlated with the strain level. Further experiments demonstrated that these failures were not artefacts of our methods of sample preparation and testing, and that fatigue failure of cell processes also occurs in vivo. This work is significant as it is the first time it has been possible to conduct fatigue testing on cellular material of any kind. Many types of cells experience repetitive loading which may cause failure or damage requiring repair. It is clinically important to determine how cyclic strain affects cells and how they respond in order to gain a deeper understanding of the physiological processes stimulated in this manner. The more we understand about the natural repair process in bone the more targeted the intervention methods may become if disruption of the repair process occurred. Our results will help to understand how the osteocyte cell network is disrupted in the vicinity of matrix damage, a crucial step in bone remodelling.

Microwave-Assisted Extraction for Microalgae: From Biofuels to Biorefinery

PubMed Central

Pandhal, Jagroop

2018-01-01

The commercial reality of bioactive compounds and oil production from microalgal species is constrained by the high cost of production. Downstream processing, which includes harvesting and extraction, can account for 70–80% of the total cost of production. Consequently, from an economic perspective extraction technologies need to be improved. Microalgal cells are difficult to disrupt due to polymers within their cell wall such as algaenan and sporopollenin. Consequently, solvents and disruption devices are required to obtain products of interest from within the cells. Conventional techniques used for cell disruption and extraction are expensive and are often hindered by low efficiencies. Microwave-assisted extraction offers a possibility for extraction of biochemical components including lipids, pigments, carbohydrates, vitamins and proteins, individually and as part of a biorefinery. Microwave technology has advanced since its use in the 1970s. It can cut down working times and result in higher yields and purity of products. In this review, the ability and challenges in using microwave technology are discussed for the extraction of bioactive products individually and as part of a biorefinery approach. PMID:29462888

Use of Non-Conventional Cell Disruption Method for Extraction of Proteins from Black Yeasts

PubMed Central

Čolnik, Maja; Primožič, Mateja; Knez, Željko; Leitgeb, Maja

2016-01-01

The influence of pressure and treatment time on cells disruption of different black yeasts and on activities of extracted proteins using supercritical carbon dioxide process was studied. The cells of three different black yeasts Phaeotheca triangularis, Trimatostroma salinum, and Wallemia ichthyophaga were exposed to supercritical carbon dioxide (SC CO2) by varying pressure at fixed temperature (35°C). The black yeasts cell walls were disrupted, and the content of the cells was spilled into the liquid medium. The impact of SC CO2 conditions on secretion of enzymes and proteins from black yeast cells suspension was studied. The residual activity of the enzymes cellulase, β-glucosidase, α-amylase, and protease was studied by enzymatic assay. The viability of black yeast cells was determined by measuring the optical density of the cell suspension at 600 nm. The total protein concentration in the suspension was determined on UV–Vis spectrophotometer at 595 nm. The release of intracellular and extracellular products from black yeast cells was achieved. Also, the observation by an environmental scanning electron microscopy shows major morphological changes with SC CO2-treated cells. The advantages of the proposed method are in a simple use, which is also possible for heat-sensitive materials on one hand and on the other hand integration of the extraction of enzymes and their use in biocatalytical reactions. PMID:27148527

Disruption of lysosome function promotes tumor growth and metastasis in Drosophila.

PubMed

Chi, Congwu; Zhu, Huanhu; Han, Min; Zhuang, Yuan; Wu, Xiaohui; Xu, Tian

2010-07-09

Lysosome function is essential to many physiological processes. It has been suggested that deregulation of lysosome function could contribute to cancer. Through a genetic screen in Drosophila, we have discovered that mutations disrupting lysosomal degradation pathway components contribute to tumor development and progression. Loss-of-function mutations in the Class C vacuolar protein sorting (VPS) gene, deep orange (dor), dramatically promote tumor overgrowth and invasion of the Ras(V12) cells. Knocking down either of the two other components of the Class C VPS complex, carnation (car) and vps16A, also renders Ras(V12) cells capable for uncontrolled growth and metastatic behavior. Finally, chemical disruption of the lysosomal function by feeding animals with antimalarial drugs, chloroquine or monensin, leads to malignant tumor growth of the Ras(V12) cells. Taken together, our data provide evidence for a causative role of lysosome dysfunction in tumor growth and invasion and indicate that members of the Class C VPS complex behave as tumor suppressors.

Effect of blueberries and insulin on glucose induced neurotoxicity in brain cells in vitro

USDA-ARS?s Scientific Manuscript database

Introduction Literature had shown that disruption in glucose metabolism seen in metabolic syndrome maybe responsible for neuronal cell-death. Oxidative stress (OS) and inflammation (INF) triggered by the impaired metabolic process are considered to be the primary factors for the toxic neuronal atmos...

Genotoxicity of multi-walled carbon nanotubes at occupationally relevant doses

PubMed Central

2014-01-01

Carbon nanotubes are commercially-important products of nanotechnology; however, their low density and small size makes carbon nanotube respiratory exposures likely during their production or processing. We have previously shown mitotic spindle aberrations in cultured primary and immortalized human airway epithelial cells exposed to single-walled carbon nanotubes (SWCNT). In this study, we examined whether multi-walled carbon nanotubes (MWCNT) cause mitotic spindle damage in cultured cells at doses equivalent to 34 years of exposure at the NIOSH Recommended Exposure Limit (REL). MWCNT induced a dose responsive increase in disrupted centrosomes, abnormal mitotic spindles and aneuploid chromosome number 24 hours after exposure to 0.024, 0.24, 2.4 and 24 μg/cm2 MWCNT. Monopolar mitotic spindles comprised 95% of disrupted mitoses. Three-dimensional reconstructions of 0.1 μm optical sections showed carbon nanotubes integrated with microtubules, DNA and within the centrosome structure. Cell cycle analysis demonstrated a greater number of cells in S-phase and fewer cells in the G2 phase in MWCNT-treated compared to diluent control, indicating a G1/S block in the cell cycle. The monopolar phenotype of the disrupted mitotic spindles and the G1/S block in the cell cycle is in sharp contrast to the multi-polar spindle and G2 block in the cell cycle previously observed following exposure to SWCNT. One month following exposure to MWCNT there was a dramatic increase in both size and number of colonies compared to diluent control cultures, indicating a potential to pass the genetic damage to daughter cells. Our results demonstrate significant disruption of the mitotic spindle by MWCNT at occupationally relevant exposure levels. PMID:24479647

AFM of the ultrastructural and mechanical properties of lipid-raft-disrupted and/or cold-treated endothelial cells.

PubMed

Wu, Li; Huang, Jie; Yu, Xiaoxue; Zhou, Xiaoqing; Gan, Chaoye; Li, Ming; Chen, Yong

2014-02-01

The nonionic detergent extraction at 4 °C and the cholesterol-depletion-induced lipid raft disruption are the two widely used experimental strategies for lipid raft research. However, the effects of raft disruption and/or cold treatment on the ultrastructural and mechanical properties of cells are still unclear. Here, we evaluated the effects of raft disruption and/or cold (4 °C) treatment on these properties of living human umbilical vein endothelial cells (HUVECs). At first, the cholesterol-depletion-induced raft disruption was visualized by confocal microscopy and atomic force microscopy (AFM) in combination with fluorescent quantum dots. Next, the cold-induced cell contraction and the formation of end-branched filopodia were observed by confocal microscopy and AFM. Then, the cell-surface ultrastructures were imaged by AFM, and the data showed that raft disruption and cold treatment induced opposite effects on cell-surface roughness (a significant decrease and a significant increase, respectively). Moreover, the cell-surface mechanical properties (stiffness and adhesion force) of raft-disrupted- and/or cold-treated HUVECs were measured by the force measurement function of AFM. We found that raft disruption and cold treatment induced parallel effects on cell stiffness (increase) or adhesion force (decrease) and that the combination of the two treatments caused dramatically strengthened effects. Finally, raft disruption was found to significantly impair cell migration as previously reported, whereas temporary cold treatment only caused a slight but nonsignificant decrease in cell migration performed at physiological temperature. Although the mechanisms for causing these results might be complicated and more in-depth studies will be needed, our data may provide important information for better understanding the effects of raft disruption or cold treatment on cells and the two strategies for lipid raft research.

Growth and differentiation of embryonic stem cells that lack an intact c-fos gene.

PubMed Central

Field, S J; Johnson, R S; Mortensen, R M; Papaioannou, V E; Spiegelman, B M; Greenberg, M E

1992-01-01

The c-fos protooncogene encodes a transcription factor that is thought to play a critical role in proliferation and differentiation as well as in the physiological response of mature cells to their environment. To test directly the role of c-fos in growth and differentiation, we generated mouse embryonic stem cell lines in which both copies of the c-fos gene were specifically disrupted by homologous recombination. Remarkably, the disruption of both copies of c-fos in these cells has no detectable effect on embryonic stem cell viability, growth rate, or differentiation potential. Embryonic stem cells lacking c-fos can differentiate into a wide range of cell types in tissue culture and also in chimeric mice. We conclude that despite a large body of literature suggesting an important role for c-fos in cell growth and differentiation, in at least some cell types this gene is not essential for these processes. Images PMID:1329091

Extraction of nucleic acids from yeast cells and plant tissues using ethanol as medium for sample preservation and cell disruption.

PubMed

Linke, Bettina; Schröder, Kersten; Arter, Juliane; Gasperazzo, Tatiana; Woehlecke, Holger; Ehwald, Rudolf

2010-09-01

Here we report that dehydrated ethanol is an excellent medium for both in situ preservation of nucleic acids and cell disruption of plant and yeast cells. Cell disruption was strongly facilitated by prior dehydration of the ethanol using dehydrated zeolite. Following removal of ethanol, nucleic acids were extracted from the homogenate pellet using denaturing buffers. The method provided DNA and RNA of high yield and integrity. Whereas cell wall disruption was essential for extraction of DNA and large RNA molecules, smaller molecules such as tRNAs could be selectively extracted from undisrupted, ethanol-treated yeast cells. Our results demonstrate the utility of absolute ethanol for sample fixation, cell membrane and cell wall disruption, as well as preservation of nucleic acids during sample storage.

Disruption of p53 function sensitizes breast cancer MCF-7 cells to cisplatin and pentoxifylline.

PubMed

Fan, S; Smith, M L; Rivet, D J; Duba, D; Zhan, Q; Kohn, K W; Fornace, A J; O'Connor, P M

1995-04-15

The possibility that appropriately designed chemotherapy could act selectively against p53-defective tumor cells was explored in MCF-7 human breast cancer cells. These cells were chosen because they have normal p53 function but are representative of a tumor cell type that does not readily undergo p53-dependent apoptosis. Two sublines (MCF-7/E6 and MCF-7/mu-p53) were established in which p53 function was disrupted by transfection with either the human papillomavirus type-16 E6 gene or a dominant-negative mutant p53 gene. p53 function in MCF-7/E6 and MCF-7/mu-p53 cells was defective relative to control cells in that there were no increases in p53 or p21Waf1/Cip1 protein levels and no G1 arrest following exposure to ionizing radiation. Survival assays showed that p53 disruption sensitized MCF-7 cells to cisplatin (CDDP) but not to several other DNA-damaging agents. CDDP sensitization was not limited to MCF-7 cells since p53 disruption in human colon carcinoma RKO cells also enhanced sensitivity to CDDP. Contrary to the other DNA-damaging agents tested, CDDP-induced DNA lesions are repaired extensively by nucleotide excision, and in agreement with a defect in this process, MCF-7/E6 and MCF-7/mu-p53 cells exhibited a reduced ability to repair a CDDP-damaged chloramphenicol acetyltransferase-reporter plasmid transfected into the cells. Therefore, we attributed the increased CDDP sensitivity of MCF-7 cells with disrupted p53 to defects in G1 checkpoint control, nucleotide excision repair, or both. The G2 checkpoint inhibitor pentoxifylline exhibited synergism with CDDP in killing MCF-7/E6 cells but did not affect sensitivity of the control cells. Moreover, pentoxifylline inhibited G2 checkpoint function to a greater extent in MCF-7/E6 than in the parental cells. These results suggested that, in the absence of p53 function, cancer cells are more vulnerable to G2 checkpoint abrogators. Our results show that a combination of CDDP and pentoxifylline is capable of synergistic and preferential killing of p53-defective tumor cells that do not readily undergo apoptosis.

Ultrastructural Characterization of the Glomerulopathy in Alport Mice by Helium Ion Scanning Microscopy (HIM).

PubMed

Tsuji, Kenji; Suleiman, Hani; Miner, Jeffrey H; Daley, James M; Capen, Diane E; Păunescu, Teodor G; Lu, Hua A Jenny

2017-09-15

The glomerulus exercises its filtration barrier function by establishing a complex filtration apparatus consisting of podocyte foot processes, glomerular basement membrane and endothelial cells. Disruption of any component of the glomerular filtration barrier leads to glomerular dysfunction, frequently manifested as proteinuria. Ultrastructural studies of the glomerulus by transmission electron microscopy (TEM) and conventional scanning electron microscopy (SEM) have been routinely used to identify and classify various glomerular diseases. Here we report the application of newly developed helium ion scanning microscopy (HIM) to examine the glomerulopathy in a Col4a3 mutant/Alport syndrome mouse model. Our study revealed unprecedented details of glomerular abnormalities in Col4a3 mutants including distorted podocyte cell bodies and disorganized primary processes. Strikingly, we observed abundant filamentous microprojections arising from podocyte cell bodies and processes, and presence of unique bridging processes that connect the primary processes and foot processes in Alport mice. Furthermore, we detected an altered glomerular endothelium with disrupted sub-endothelial integrity. More importantly, we were able to clearly visualize the complex, three-dimensional podocyte and endothelial interface by HIM. Our study demonstrates that HIM provides nanometer resolution to uncover and rediscover critical ultrastructural characteristics of the glomerulopathy in Col4a3 mutant mice.

Disruption of the processing alpha-mannosidase gene does not prevent outer chain synthesis in Saccharomyces cerevisiae.

PubMed Central

Puccia, R; Grondin, B; Herscovics, A

1993-01-01

Processing of N-linked oligosaccharides in Saccharomyces cerevisiae begins with the removal of glucose and mannose residues from Glc3Man9GlcNAc2 to form a single isomer of Man8GlcNAc2. The importance of mannose removal for subsequent outer chain synthesis was examined in strains of S. cerevisiae disrupted in the MNS1 gene encoding a specific alpha 1,2-mannosidase responsible for Man8GlcNAc2 synthesis [Camirand, Heysen, Grondin and Herscovics (1991) J. Biol. Chem. 266, 15120-15127]. Both MNS1 transcripts of 1.85 kb and 1.7 kb were not observed in Northern blots of mns1 cells (i.e. cells containing the disrupted gene). Analysis on Bio-Gel P-6 of endo-beta-N-acetylglucosaminidase-H-sensitive oligosaccharides following a 10 min pulse with [2-3H]mannose revealed similar amounts of labelled outer chains excluded from the gel in both control and mns1 cells. H.p.l.c. of the included oligosaccharides showed that a Man9GlcNAc, rather than a Man8GlcNAc, intermediate was formed in mns1 cells. Analysis of [3H]mannose-labelled core oligosaccharides from immunoprecipitated CPY and invertase by h.p.l.c. showed a similar size distribution in mns1 and control cells. Invertase immunoprecipitated from [35S]methionine-labelled mns1 cells was highly glycosylated, but migrated slightly faster than that from control cells on denaturing PAGE, indicating a small difference in glycosylation. A similar difference in mobility was observed for invertase activity stain following non-denaturing gel electrophoresis. It is concluded that the alpha-mannosidase encoded by MNS1 is the only enzyme responsible for mannose removal in vivo, and that this processing step is not essential for outer chain synthesis. Images Figure 1 Figure 4 PMID:8439291

Dystroglycan loss disrupts polarity and beta-casein induction inmammary epithelial cells by perturbing laminin anchoring

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

Weir, M. Lynn; Oppizzi, Maria Luisa; Henry, Michael D.

2006-02-17

Precise contact between epithelial cells and their underlying basement membrane is critical to the maintenance of tissue architecture and function. To understand the role that the laminin receptor dystroglycan (DG) plays in these processes, we assayed cell responses to laminin-111 following conditional ablation of DG expression in cultured mammary epithelial cells (MECs). Strikingly, DG loss disrupted laminin-111-induced polarity and {beta}-casein production, and abolished laminin assembly at the step of laminin binding to the cell surface. DG re-expression restored these deficiencies. Investigations of mechanism revealed that DG cytoplasmic sequences were not necessary for laminin assembly and signaling, and only when themore » entire mucin domain of extracellular DG was deleted did laminin assembly not occur. These results demonstrate that DG is essential as a laminin-111 co-receptor in MECs that functions by mediating laminin anchoring to the cell surface, a process that allows laminin polymerization, tissue polarity, and {beta}-casein induction. The observed loss of laminin-111 assembly and signaling in DG-/-MECs provides insights into the signaling changes occurring in breast carcinomas and other cancers, where DG's laminin-binding function is frequently defective.« less

Disintegration of Nannochloropsis sp. cells in an improved turbine bead mill.

PubMed

Pan, Zhidong; Huang, Ying; Wang, Yanmin; Wu, Zhiwei

2017-12-01

The Nannochloropsis sp. cells in aqueous solution were disintegrated in an improved bead mill with turbine agitator. The disintegration rates of cell samples disrupted under various operating parameters (i.e., circumferential speed, bead size, disintegration time, and cell concentration) were analyzed. An experimental strategy to optimize the parameters affecting the cell disintegration process was proposed. The results show that Nannochloropsis sp. cells can be effectively disintegrated in the turbine stirred bead mill under the optimum condition (i.e., circumferential speed of 2.3m/s, concentration of 15vol.%, disintegration time of 40min and bead size of 0.3-0.4mm). The disintegration mechanism was discussed via the selection and breakage functions from population balance modelling. It is revealed that the impact and compression effects of stirring beads are more effective for the disruption of coarser fraction of cells, and the shear effect dominates the production of finer fractions of disintegrated cells. Copyright © 2017 Elsevier Ltd. All rights reserved.

MLKL Activation Triggers NLRP3-Mediated Processing and Release of IL-1β Independently of Gasdermin-D.

PubMed

Gutierrez, Kimberley D; Davis, Michael A; Daniels, Brian P; Olsen, Tayla M; Ralli-Jain, Pooja; Tait, Stephen W G; Gale, Michael; Oberst, Andrew

2017-03-01

Necroptosis is a form of programmed cell death defined by activation of the kinase receptor interacting protein kinase 3 and its downstream effector, the pseudokinase mixed lineage kinase domain-like (MLKL). Activated MLKL translocates to the cell membrane and disrupts it, leading to loss of cellular ion homeostasis. In this study, we use a system in which this event can be specifically triggered by a small-molecule ligand to show that MLKL activation is sufficient to induce the processing and release of bioactive IL-1β. MLKL activation triggers potassium efflux and assembly of the NLRP3 inflammasome, which is required for the processing and activity of IL-1β released during necroptosis. Notably, MLKL activation also causes cell membrane disruption, which allows efficient release of IL-1β independently of the recently described pyroptotic effector gasdermin-D. Taken together, our findings indicate that MLKL is an endogenous activator of the NLRP3 inflammasome, and that MLKL activation provides a mechanism for concurrent processing and release of IL-1β independently of gasdermin-D. Copyright © 2017 by The American Association of Immunologists, Inc.

Poloxamer 188 (p188) as a membrane resealing reagent in biomedical applications.

PubMed

Moloughney, Joseph G; Weisleder, Noah

2012-12-01

Maintenance of the integrity of the plasma membrane is essential for maintenance of cellular function and prevention of cell death. Since the plasma membrane is frequently exposed to a variety of mechanical and chemical insults the cell has evolved active processes to defend against these injuries by resealing disruptions in the plasma membrane. Cell membrane repair is a conserved process observed in nearly every cell type where intracellular vesicles are recruited to sites of membrane disruption where they can fuse with themselves or the plasma membrane to create a repair patch. When disruptions are extensive or there is an underlying pathology that reduces the membrane repair capacity of a cell this defense mechanism may prove insufficient and the cell could die due to breakdown of the plasma membrane. Extensive loss of cells can compromise the integrity and function of tissues and leading to disease. Thus, methods to increase membrane resealing capacity could have broad utility in a number of disease states. Efforts to find reagents that can modulate plasma membrane reseal found that specific tri-block copolymers, such as poloxamer 188 (P188, or Pluronic F68), can increase the structural stability and resealing of the plasma membrane. Here we review several current patents and patent applications that present inventions making use of P188 and other copolymers to treat specific disease states such as muscular dystrophy, heart failure, neurodegenerative disorders and electrical injuries, or to facilitate biomedical applications such as transplantation. There appears to be promise for the application of poloxamers in the treatment of various diseases, however there are potential concerns with toxicity with long term application and bioavailability in some cases.

Disruption of Autolysis in Bacillus subtilis using TiO2 Nanoparticles

PubMed Central

McGivney, Eric; Han, Linchen; Avellan, Astrid; VanBriesen, Jeanne; Gregory, Kelvin B.

2017-01-01

In contrast to many nanotoxicity studies where nanoparticles (NPs) are observed to be toxic or reduce viable cells in a population of bacteria, we observed that increasing concentration of TiO2 NPs increased the cell survival of Bacillus subtilis in autolysis-inducing buffer by 0.5 to 5 orders of magnitude over an 8 hour exposure. Molecular investigations revealed that TiO2 NPs prevent or delay cell autolysis, an important survival and growth-regulating process in bacterial populations. Overall, the results suggest two potential mechanisms for the disruption of autolysis by TiO2 NPs in a concentration dependent manner: (i) directly, through TiO2 NP deposition on the cell wall, delaying the collapse of the protonmotive-force and preventing the onset of autolysis; and (ii) indirectly, through adsorption of autolysins on TiO2 NP, limiting the activity of released autolysins and preventing further lytic activity. Enhanced darkfield microscopy coupled to hyperspectral analysis was used to map TiO2 deposition on B. subtilis cell walls and released enzymes, supporting both mechanisms of autolysis interference. The disruption of autolysis in B. subtilis cultures by TiO2 NPs suggests the mechanisms and kinetics of cell death may be influenced by nano-scale metal oxide materials, which are abundant in natural systems. PMID:28303908

Semaphorin 6A knockout mice display abnormalities across ethologically-based topographies of exploration and in motor learning.

PubMed

Håkansson, Kerstin; Runker, Annette E; O'Sullivan, Gerard J; Mitchell, Kevin J; Waddington, John L; O'Tuathaigh, Colm M P

2017-02-22

Semaphorins are secreted or membrane-bound proteins implicated in neurodevelopmental processes of axon guidance and cell migration. Exploratory behaviour and motor learning was examined ethologically in Semaphorin 6A (Sema6A) mutant mice. The ethogram of initial exploration in Sema6A knockout mice was characterised by increased rearing to wall with decreased sifting; over subsequent habituation, locomotion, sniffing and rearing to wall were increased, with reduced habituation of rearing seated. Rotarod analysis indicated delayed motor learning in Sema6A heterozygous mutants. Disruption to the axonal guidance and cell migration processes regulated by Sema6A is associated with topographically specific disruption to fundamental aspects of behaviour, namely the ethogram of initial exploration and subsequent habituation to the environment, and motor learning. Copyright © 2017 Elsevier B.V. All rights reserved.

A High-Throughput Screening Method for Small-Molecule Inhibitors of the Aberrant Mutant SOD1 and Dynein Complex Interaction

PubMed Central

Tang, Xiaohu; Seyb, Kathleen I.; Huang, Mickey; Schuman, Eli R.; Shi, Ping; Zhu, Haining; Glicksman, Marcie A.

2013-01-01

Aberrant protein-protein interactions are attractive drug targets in a variety of neurodegenerative diseases due to the common pathology of accumulation of protein aggregates. In amyotrophic lateral sclerosis, mutations in SOD1 cause the formation of aggregates and inclusions that may sequester other proteins and disrupt cellular processes. It has been demonstrated that mutant SOD1, but not wild-type SOD1, interacts with the axonal transport motor dynein and that this interaction contributes to motor neuron cell death, suggesting that disrupting this interaction may be a potential therapeutic target. However, it can be challenging to configure a high-throughput screening (HTS)–compatible assay to detect inhibitors of a protein-protein interaction. Here we describe the development and challenges of an HTS for small-molecule inhibitors of the mutant SOD1-dynein interaction. We demonstrate that the interaction can be formed by coexpressing the A4V mutant SOD1 and dynein intermediate complex in cells and that this interaction can be disrupted by compounds added to the cell lysates. Finally, we show that some of the compounds identified from a pilot screen to inhibit the protein-protein interaction with this method specifically disrupt the interaction between the dynein complex and mtSOD1 but not the dynein complex itself when applied to live cells. PMID:22140121

Rho-GTPase effector ROCK phosphorylates cofilin in actin-meditated cytokinesis during mouse oocyte meiosis.

PubMed

Duan, Xing; Liu, Jun; Dai, Xiao-Xin; Liu, Hong-Lin; Cui, Xiang-Shun; Kim, Nam-Hyung; Wang, Zhen-Bo; Wang, Qiang; Sun, Shao-Chen

2014-02-01

During oocyte meiosis, a spindle forms in the central cytoplasm and migrates to the cortex. Subsequently, the oocyte extrudes a small body and forms a highly polarized egg; this process is regulated primarily by actin. ROCK is a Rho-GTPase effector that is involved in various cellular functions, such as stress fiber formation, cell migration, tumor cell invasion, and cell motility. In this study, we investigated possible roles for ROCK in mouse oocyte meiosis. ROCK was localized around spindles after germinal vesicle breakdown and was colocalized with cytoplasmic actin and mitochondria. Disrupting ROCK activity by RNAi or an inhibitor resulted in cell cycle progression and polar body extrusion failure. Time-lapse microscopy showed that this may have been due to spindle migration and cytokinesis defects, as chromosomes segregated but failed to extrude a polar body and then realigned. Actin expression at oocyte membranes and in cytoplasm was significantly decreased after these treatments. Actin caps were also disrupted, which was confirmed by a failure to form cortical granule-free domains. The mitochondrial distribution was also disrupted, which indicated that mitochondria were involved in the ROCK-mediated actin assembly. In addition, the phosphorylation levels of Cofilin, a downstream molecule of ROCK, decreased after disrupting ROCK activity. Thus, our results indicated that a ROCK-Cofilin-actin pathway regulated meiotic spindle migration and cytokinesis during mouse oocyte maturation.

Disruption of microtubules in plants suppresses macroautophagy and triggers starch excess-associated chloroplast autophagy

PubMed Central

Wang, Yan; Zheng, Xiyin; Yu, Bingjie; Han, Shaojie; Guo, Jiangbo; Tang, Haiping; Yu, Alice Yunzi L; Deng, Haiteng; Hong, Yiguo; Liu, Yule

2015-01-01

Microtubules, the major components of cytoskeleton, are involved in various fundamental biological processes in plants. Recent studies in mammalian cells have revealed the importance of microtubule cytoskeleton in autophagy. However, little is known about the roles of microtubules in plant autophagy. Here, we found that ATG6 interacts with TUB8/β-tubulin 8 and colocalizes with microtubules in Nicotiana benthamiana. Disruption of microtubules by either silencing of tubulin genes or treatment with microtubule-depolymerizing agents in N. benthamiana reduces autophagosome formation during upregulation of nocturnal or oxidation-induced macroautophagy. Furthermore, a blockage of leaf starch degradation occurred in microtubule-disrupted cells and triggered a distinct ATG6-, ATG5- and ATG7-independent autophagic pathway termed starch excess-associated chloroplast autophagy (SEX chlorophagy) for clearance of dysfunctional chloroplasts. Our findings reveal that an intact microtubule network is important for efficient macroautophagy and leaf starch degradation. PMID:26566764

Investigation of the Antimetastatic Effects of Agents that Inhibit Cell Adhesion or Protein Glycosylation

PubMed Central

Humphries, Martin J.; Matsumoto, Kazue; White, Sandra L.; Olden, Kenneth

1987-01-01

In this overview the authors describe their recent attempts to specifically interfere with the metastatic spread of B16-F10 melanoma cells. Using the experimental metastasis model system, inhibitory effects of (1) coinjection of cells with synthetic peptides derived from the glycoprotein fibronectin, which possess the ability to disrupt cell adhesion, and (2) treatment of cells with inhibitors of protein glycosylation and oligosaccharide processing have been examined. PMID:3295262

Disorganized Cortical Patches Suggest Prenatal Origin of Autism

MedlinePlus

... 2014 Disorganized cortical patches suggest prenatal origin of autism NIH-funded study shows disrupted cell layering process ... study suggests that brain irregularities in children with autism can be traced back to prenatal development. “While ...

LINE-1 Mediated Insertion into Poc1a (Protein of Centriole 1 A) Causes Growth Insufficiency and Male Infertility in Mice

PubMed Central

Geister, Krista A.; Brinkmeier, Michelle L.; Cheung, Leonard Y.; Wendt, Jennifer; Oatley, Melissa J.; Burgess, Daniel L.; Kozloff, Kenneth M.; Cavalcoli, James D.; Oatley, Jon M.; Camper, Sally A.

2015-01-01

Skeletal dysplasias are a common, genetically heterogeneous cause of short stature that can result from disruptions in many cellular processes. We report the identification of the lesion responsible for skeletal dysplasia and male infertility in the spontaneous, recessive mouse mutant chagun. We determined that Poc1a, encoding protein of the centriole 1a, is disrupted by the insertion of a processed Cenpw cDNA, which is flanked by target site duplications, suggestive of a LINE-1 retrotransposon-mediated event. Mutant fibroblasts have impaired cilia formation and multipolar spindles. Male infertility is caused by defective spermatogenesis early in meiosis and progressive germ cell loss. Spermatogonial stem cell transplantation studies revealed that Poc1a is essential for normal function of both Sertoli cells and germ cells. The proliferative zone of the growth plate is small and disorganized because chondrocytes fail to re-align after cell division and undergo increased apoptosis. Poc1a and several other genes associated with centrosome function can affect the skeleton and lead to skeletal dysplasias and primordial dwarfisms. This mouse mutant reveals how centrosome dysfunction contributes to defects in skeletal growth and male infertility. PMID:26496357

LINE-1 Mediated Insertion into Poc1a (Protein of Centriole 1 A) Causes Growth Insufficiency and Male Infertility in Mice.

PubMed

Geister, Krista A; Brinkmeier, Michelle L; Cheung, Leonard Y; Wendt, Jennifer; Oatley, Melissa J; Burgess, Daniel L; Kozloff, Kenneth M; Cavalcoli, James D; Oatley, Jon M; Camper, Sally A

2015-10-01

Skeletal dysplasias are a common, genetically heterogeneous cause of short stature that can result from disruptions in many cellular processes. We report the identification of the lesion responsible for skeletal dysplasia and male infertility in the spontaneous, recessive mouse mutant chagun. We determined that Poc1a, encoding protein of the centriole 1a, is disrupted by the insertion of a processed Cenpw cDNA, which is flanked by target site duplications, suggestive of a LINE-1 retrotransposon-mediated event. Mutant fibroblasts have impaired cilia formation and multipolar spindles. Male infertility is caused by defective spermatogenesis early in meiosis and progressive germ cell loss. Spermatogonial stem cell transplantation studies revealed that Poc1a is essential for normal function of both Sertoli cells and germ cells. The proliferative zone of the growth plate is small and disorganized because chondrocytes fail to re-align after cell division and undergo increased apoptosis. Poc1a and several other genes associated with centrosome function can affect the skeleton and lead to skeletal dysplasias and primordial dwarfisms. This mouse mutant reveals how centrosome dysfunction contributes to defects in skeletal growth and male infertility.

Plasma membrane disruption: repair, prevention, adaptation

NASA Technical Reports Server (NTRS)

McNeil, Paul L.; Steinhardt, Richard A.

2003-01-01

Many metazoan cells inhabit mechanically stressful environments and, consequently, their plasma membranes are frequently disrupted. Survival requires that the cell rapidly repair or reseal the disruption. Rapid resealing is an active and complex structural modification that employs endomembrane as its primary building block, and cytoskeletal and membrane fusion proteins as its catalysts. Endomembrane is delivered to the damaged plasma membrane through exocytosis, a ubiquitous Ca2+-triggered response to disruption. Tissue and cell level architecture prevent disruptions from occurring, either by shielding cells from damaging levels of force, or, when this is not possible, by promoting safe force transmission through the plasma membrane via protein-based cables and linkages. Prevention of disruption also can be a dynamic cell or tissue level adaptation triggered when a damaging level of mechanical stress is imposed. Disease results from failure of either the preventive or resealing mechanisms.

RNA disruption is associated with response to multiple classes of chemotherapy drugs in tumor cell lines.

PubMed

Narendrula, Rashmi; Mispel-Beyer, Kyle; Guo, Baoqing; Parissenti, Amadeo M; Pritzker, Laura B; Pritzker, Ken; Masilamani, Twinkle; Wang, Xiaohui; Lannér, Carita

2016-02-24

Cellular stressors and apoptosis-inducing agents have been shown to induce ribosomal RNA (rRNA) degradation in eukaryotic cells. Recently, RNA degradation in vivo was observed in patients with locally advanced breast cancer, where mid-treatment tumor RNA degradation was associated with complete tumor destruction and enhanced patient survival. However, it is not clear how widespread chemotherapy induced "RNA disruption" is, the extent to which it is associated with drug response or what the underlying mechanisms are. Ovarian (A2780, CaOV3) and breast (MDA-MB-231, MCF-7, BT474, SKBR3) cancer cell lines were treated with several cytotoxic chemotherapy drugs and total RNA was isolated. RNA was also prepared from docetaxel resistant A2780DXL and carboplatin resistant A2780CBN cells following drug exposure. Disruption of RNA was analyzed by capillary electrophoresis. Northern blotting was performed using probes complementary to the 28S and 18S rRNA to determine the origins of degradation bands. Apoptosis activation was assessed by flow cytometric monitoring of annexin-V and propidium iodide (PI) binding to cells and by measuring caspase-3 activation. The link between apoptosis and RNA degradation (disruption) was investigated using a caspase-3 inhibitor. All chemotherapy drugs tested were capable of inducing similar RNA disruption patterns. Docetaxel treatment of the resistant A2780DXL cells and carboplatin treatment of the A2780CBN cells did not result in RNA disruption. Northern blotting indicated that two RNA disruption bands were derived from the 3'-end of the 28S rRNA. Annexin-V and PI staining of docetaxel treated cells, along with assessment of caspase-3 activation, showed concurrent initiation of apoptosis and RNA disruption, while inhibition of caspase-3 activity significantly reduced RNA disruption. Supporting the in vivo evidence, our results demonstrate that RNA disruption is induced by multiple chemotherapy agents in cell lines from different tissues and is associated with drug response. Although present, the link between apoptosis and RNA disruption is not completely understood. Evaluation of RNA disruption is thus proposed as a novel and effective biomarker to assess response to chemotherapy drugs in vitro and in vivo.

Controlled permeation of cell membrane by single bubble acoustic cavitation

PubMed Central

Zhou, Y.; Yang, K.; Cui, J.; Ye, J. Y.; Deng, C. X.

2011-01-01

Sonoporation is the membrane disruption generated by ultrasound and has been exploited as a non-viral strategy for drug and gene delivery. Acoustic cavitation of microbubbles has been recognized to play an important role in sonoporation. However, due to the lack of adequate techniques for precise control of cavitation activities and real-time assessment of the resulting sub-micron process of sonoporation, limited knowledge has been available regarding the detail processes and correlation of cavitation with membrane disruption at the single cell level. In the current study, we developed a combined approach including optical, acoustic, and electrophysiological techniques to enable synchronized manipulation, imaging, and measurement of cavitation of single bubbles and the resulting cell membrane disruption in real-time. Using a self-focused femtosecond laser and high frequency (7.44 MHz) pulses, a single microbubble was generated and positioned at a desired distance from the membrane of a Xenopus oocyte. Cavitation of the bubble was achieved by applying a low frequency (1.5 MHz) ultrasound pulse (duration 13.3 or 40 µs) to induce bubble collapse. Disruption of the cell membrane was assessed by the increase in the transmembrane current (TMC) of the cell under voltage clamp. Simultaneous high-speed bright field imaging of cavitation and measurements of the TMC were obtained to correlate the ultrasound-generated bubble activities with the cell membrane poration. The change in membrane permeability was directly associated with the formation of a sub-micrometer pore from a local membrane rupture generated by bubble collapse or bubble compression depending on ultrasound amplitude and duration. The impact of the bubble collapse on membrane permeation decreased rapidly with increasing distance (D) between the bubble (diameter d) and the cell membrane. The effective range of cavitation impact on membrane poration was determined to be D/d = 0.75. The maximum mean radius of the pores was estimated from the measured TMC to be 0.106 ± 0.032 µm (n = 70) for acoustic pressure of 1.5 MPa (duration 13.3 µs), and increased to 0.171 ± 0.030 µm (n = 125) for acoustic pressure of 1.7 MPa and to 0.182 ± 0.052 µm (n=112) for a pulse duration of 40 µs (1.5 MPa). These results from controlled cell membrane permeation by cavitation of single bubbles revealed insights and key factors affecting sonoporation at the single cell level. PMID:21945682

Lipid rafts regulate PCB153-induced disruption of occludin and brain endothelial barrier function through protein phosphatase 2A and matrix metalloproteinase-2

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

Eum, Sung Yong, E-mail: seum@miami.edu; Jaraki, Dima; András, Ibolya E.

Occludin is an essential integral transmembrane protein regulating tight junction (TJ) integrity in brain endothelial cells. Phosphorylation of occludin is associated with its localization to TJ sites and incorporation into intact TJ assembly. The present study is focused on the role of lipid rafts in polychlorinated biphenyl (PCB)-induced disruption of occludin and endothelial barrier function. Exposure of human brain endothelial cells to 2,2′,4,4′,5,5′-hexachlorobiphenyl (PCB153) induced dephosphorylation of threonine residues of occludin and displacement of occludin from detergent-resistant membrane (DRM)/lipid raft fractions within 1 h. Moreover, lipid rafts modulated the reduction of occludin level through activation of matrix metalloproteinase 2 (MMP-2)more » after 24 h PCB153 treatment. Inhibition of protein phosphatase 2A (PP2A) activity by okadaic acid or fostriecin markedly protected against PCB153-induced displacement of occludin and increased permeability of endothelial cells. The implication of lipid rafts and PP2A signaling in these processes was further defined by co-immunoprecipitation of occludin with PP2A and caveolin-1, a marker protein of lipid rafts. Indeed, a significant MMP-2 activity was observed in lipid rafts and was increased by exposure to PCB153. The pretreatment of MMP-2 inhibitors protected against PCB153-induced loss of occludin and disruption of lipid raft structure prevented the increase of endothelial permeability. Overall, these results indicate that lipid raft-associated processes, such as PP2A and MMP-2 activation, participate in PCB153-induced disruption of occludin function in brain endothelial barrier. This study contributes to a better understanding of the mechanisms leading to brain endothelial barrier dysfunction in response to exposure to environmental pollutants, such as ortho-substituted PCBs. - Highlights: • PCB153 disturbed human brain endothelial barrier through disruption of occludin. • Lipid raft-associated PP2A/MMP-2 induced PCB153-induced dysfunction of occludin. • Disrupted lipid rafts modulated PCB153-induced increase of permeability. • Lipid rafts act as a signaling platform for PCB153-induced dysfunction of occludin.« less

Low solvent, low temperature method for extracting biodiesel lipids from concentrated microalgal biomass.

PubMed

Olmstead, Ian L D; Kentish, Sandra E; Scales, Peter J; Martin, Gregory J O

2013-11-01

An industrially relevant method for disrupting microalgal cells and preferentially extracting neutral lipids for large-scale biodiesel production was demonstrated on pastes (20-25% solids) of Nannochloropsis sp. The highly resistant Nannochloropsis sp. cells. were disrupted by incubation for 15 h at 37°C followed by high pressure homogenization at 1200 ± 100 bar. Lipid extraction was performed by twice contacting concentrated algal paste with minimal hexane (solvent:biomass ratios (w/w) of <2:1 and <1.3:1) in a stirred vessel at 35°C. Cell disruption prior to extraction increased lipid recovery 100-fold, with yields of 30-50% w/w obtained in the first hexane contact, and a further 6.5-20% in the second contact. The hexane preferentially extracted neutral lipids over glyco- and phospholipids, with up to 86% w/w of the neutral lipids recovered. The process was effective on wet concentrated paste, required minimal solvent and moderate temperature, and did not require difficult to recover polar solvents. Copyright © 2013 Elsevier Ltd. All rights reserved.

Cell membrane disruption stimulates cAMP and Ca2+ signaling to potentiate cell membrane resealing in neighboring cells.

PubMed

Togo, Tatsuru

2017-12-15

Disruption of cellular plasma membranes is a common event in many animal tissues, and the membranes are usually rapidly resealed. Moreover, repeated membrane disruptions within a single cell reseal faster than the initial wound in a protein kinase A (PKA)- and protein kinase C (PKC)-dependent manner. In addition to wounded cells, recent studies have demonstrated that wounding of Madin-Darby canine kidney (MDCK) cells potentiates membrane resealing in neighboring cells in the short-term by purinergic signaling, and in the long-term by nitric oxide/protein kinase G signaling. In the present study, real-time imaging showed that cell membrane disruption stimulated cAMP synthesis and Ca 2+ mobilization from intracellular stores by purinergic signaling in neighboring MDCK cells. Furthermore, inhibition of PKA and PKC suppressed the ATP-mediated short-term potentiation of membrane resealing in neighboring cells. These results suggest that cell membrane disruption stimulates PKA and PKC via purinergic signaling to potentiate cell membrane resealing in neighboring MDCK cells. © 2017. Published by The Company of Biologists Ltd.

Disruption of Canonical TGFβ-signaling in Murine Coronary Progenitor Cells by Low Level Arsenic

PubMed Central

Allison, Patrick; Huang, Tianfang; Broka, Derrick; Parker, Patti; Barnett, Joey V.; Camenisch, Todd D.

2013-01-01

Exposure to arsenic results in several types of cancers as well as heart disease. A major contributor to ischemic heart pathologies is coronary artery disease, however the influences by environmental arsenic in this disease process are not known. Similarly, the impact of toxicants on blood vessel formation and function during development has not been studied. During embryogenesis, the epicardium undergoes proliferation, migration, and differentiation into several cardiac cell types including smooth muscle cells which contribute to the coronary vessels. The TGFβ family of ligands and receptors are essential for developmental cardiac epithelial to mesenchymal transition (EMT) and differentiation into coronary smooth muscle cells. In this in vitrostudy, 18 hour exposure to 1.34 μMarsenite disrupted developmental EMT programming in murine epicardial cells causing a deficit in cardiac mesenchyme. The expression of EMT genes including TGFβ2, TGFβ receptor-3, Snail, and Has-2 are decreased in a dose-dependent manner following exposure to arsenite. TGFβ2 cell signaling is abrogated as detected by decreases in phosphorylated Smad2/3 when cells are exposed to 1.34 μMarsenite. There is also loss of nuclear accumulation pSmad due to arsenite exposure. These observations coincide with a decrease invimentinpositive mesenchymal cells invading three-dimensional collagen gels. However, arsenite does not block TGFβ2 mediated smooth muscle cell differentiation by epicardial cells. Overall these results show that arsenic exposure blocks developmental EMT gene programming in murine coronary progenitor cells by disrupting TGFβ2 signals and Smad activation, and that smooth muscle cell differentiation is refractory to this arsenic toxicity. PMID:23732083

PROLIFERATION AS A KEY EVENT IN DEVELOPMENTAL TOXICITY: "CHEMICAL SCREENING IN HUMAN NEURAL STEM CELLS USING HIGH CONTENT IMAGING

EPA Science Inventory

New toxicity testing approaches will rely on in vitro assays to assess chemical effects at the cellular and molecular level. Cell proliferation is imperative to normal development, and chemical disruption of this process can be detrimental to the organism. As part of an effort to...

Membrane oxidation in cell delivery and cell killing applications

PubMed Central

Wang, Ting-Yi; Libardo, M. Daben J.; Angeles-Boza, Alfredo M.; Pellois, Jean-Philippe

2018-01-01

Cell delivery or cell killing processes often involve the crossing or disruption of cellular membranes. We review how, by modifying the composition and properties of membranes, membrane oxidation can be exploited to enhance the delivery of macromolecular cargos into live human cells. We also describe how membrane oxidation can be utilized to achieve efficient killing of bacteria by antimicrobial peptides. Finally, we present recent evidence highlighting how membrane oxidation is intimately engaged in natural biological processes such as antigen delivery in dendritic cells and in the killing of bacteria by human macrophages. Overall, the insights that have been recently gained in this area should facilitate the development of more effective delivery technologies and antimicrobial therapeutic approaches. PMID:28355059

The multi-faceted role of allergen exposure to the local airway mucosa.

PubMed

Golebski, K; Röschmann, K I L; Toppila-Salmi, S; Hammad, H; Lambrecht, B N; Renkonen, R; Fokkens, W J; van Drunen, C M

2013-02-01

Airway epithelial cells are the first to encounter aeroallergens and therefore have recently become an interesting target of many studies investigating their involvement in the modulation of allergic inflammatory responses. Disruption of a passive structural barrier composed of epithelial cells by intrinsic proteolytic activity of allergens may facilitate allergen penetration into local tissues and additionally affect chronic and ongoing inflammatory processes in respiratory tissues. Furthermore, the ability of rhinoviruses to disrupt and interfere with epithelial tight junctions may alter the barrier integrity and enable a passive passage of inhaled allergens through the airway epithelium. On the other hand, epithelial cells are no longer considered to act only as a physical barrier toward inhaled allergens, but also to actively contribute to airway inflammation by detecting and responding to environmental factors. Epithelial cells can produce mediators, which may affect the recruitment and activation of more specialized immune cells to the local tissue and also create a microenvironment in which these activated immune cells may function and propagate the inflammatory processes. This review presents the dual role of epithelium acting as a passive and active barrier when encountering an inhaled allergen and how this double role contributes to the start of local immune responses. © 2012 John Wiley & Sons A/S. Published by Blackwell Publishing Ltd.

Perceptual consequences of disrupted auditory nerve activity.

PubMed

Zeng, Fan-Gang; Kong, Ying-Yee; Michalewski, Henry J; Starr, Arnold

2005-06-01

Perceptual consequences of disrupted auditory nerve activity were systematically studied in 21 subjects who had been clinically diagnosed with auditory neuropathy (AN), a recently defined disorder characterized by normal outer hair cell function but disrupted auditory nerve function. Neurological and electrophysical evidence suggests that disrupted auditory nerve activity is due to desynchronized or reduced neural activity or both. Psychophysical measures showed that the disrupted neural activity has minimal effects on intensity-related perception, such as loudness discrimination, pitch discrimination at high frequencies, and sound localization using interaural level differences. In contrast, the disrupted neural activity significantly impairs timing related perception, such as pitch discrimination at low frequencies, temporal integration, gap detection, temporal modulation detection, backward and forward masking, signal detection in noise, binaural beats, and sound localization using interaural time differences. These perceptual consequences are the opposite of what is typically observed in cochlear-impaired subjects who have impaired intensity perception but relatively normal temporal processing after taking their impaired intensity perception into account. These differences in perceptual consequences between auditory neuropathy and cochlear damage suggest the use of different neural codes in auditory perception: a suboptimal spike count code for intensity processing, a synchronized spike code for temporal processing, and a duplex code for frequency processing. We also proposed two underlying physiological models based on desynchronized and reduced discharge in the auditory nerve to successfully account for the observed neurological and behavioral data. These methods and measures cannot differentiate between these two AN models, but future studies using electric stimulation of the auditory nerve via a cochlear implant might. These results not only show the unique contribution of neural synchrony to sensory perception but also provide guidance for translational research in terms of better diagnosis and management of human communication disorders.

Cationic peptide exposure enhances pulsed-electric-field-mediated membrane disruption.

PubMed

Kennedy, Stephen M; Aiken, Erik J; Beres, Kaytlyn A; Hahn, Adam R; Kamin, Samantha J; Hagness, Susan C; Booske, John H; Murphy, William L

2014-01-01

The use of pulsed electric fields (PEFs) to irreversibly electroporate cells is a promising approach for destroying undesirable cells. This approach may gain enhanced applicability if the intensity of the PEF required to electrically disrupt cell membranes can be reduced via exposure to a molecular deliverable. This will be particularly impactful if that reduced PEF minimally influences cells that are not exposed to the deliverable. We hypothesized that the introduction of charged molecules to the cell surfaces would create regions of enhanced transmembrane electric potential in the vicinity of each charged molecule, thereby lowering the PEF intensity required to disrupt the plasma membranes. This study will therefore examine if exposure to cationic peptides can enhance a PEF's ability to disrupt plasma membranes. We exposed leukemia cells to 40 μs PEFs in media containing varying concentrations of a cationic peptide, polyarginine. We observed the internalization of a membrane integrity indicator, propidium iodide (PI), in real time. Based on an individual cell's PI fluorescence versus time signature, we were able to determine the relative degree of membrane disruption. When using 1-2 kV/cm, exposure to >50 μg/ml of polyarginine resulted in immediate and high levels of PI uptake, indicating severe membrane disruption, whereas in the absence of peptide, cells predominantly exhibited signatures indicative of no membrane disruption. Additionally, PI entered cells through the anode-facing membrane when exposed to cationic peptide, which was theoretically expected. Exposure to cationic peptides reduced the PEF intensity required to induce rapid and irreversible membrane disruption. Critically, peptide exposure reduced the PEF intensities required to elicit irreversible membrane disruption at normally sub-electroporation intensities. We believe that these cationic peptides, when coupled with current advancements in cell targeting techniques will be useful tools in applications where targeted destruction of unwanted cell populations is desired.

Coordination of Actin- and Microtubule-Based Cytoskeletons Supports Transport of Spermatids and Residual Bodies/Phagosomes During Spermatogenesis in the Rat Testis

PubMed Central

Tang, Elizabeth I.; Lee, Will M.

2016-01-01

Germ cell transport across the seminiferous epithelium during spermatogenesis requires the intricate coordination of cell junctions, signaling proteins, and both actin- and microtubule (MT)-based cytoskeletons. Although the involvement of cytoskeletons in germ cell transport has been suggested, the precise mechanism(s) remains elusive. Based on growing evidence that actin and MT interactions underlie fundamental cellular processes, such as cell motility, it is unlikely that actin- and MT-based cytoskeletons work independently to regulate germ cell transport in the testis. Using rats treated with adjudin, a potential male contraceptive that disrupts spermatid adhesion and transport in the testis, as a study model, we show herein that actin- and MT-based cytoskeletons are both necessary for transport of spermatids and residual bodies/phagosomes across the seminiferous epithelium in adult rat testes. Analysis of intratubular expression of F-actin and tubulin revealed disruption of both actin and MT networks, concomitant with misdirected spermatids and phagosomes in rats treated with adjudin. Actin regulatory proteins, epidermal growth factor receptor pathway substrate 8 and actin-related protein 3, were mislocalized and down-regulated at the actin-rich anchoring junction between germ and Sertoli cells (apical ectoplasmic specialization) after adjudin treatment. Nonreceptor tyrosine kinase p-FAK-Tyr407, known to regulate F-actin nucleation via actin-related protein 3, was also mislocalized and down-regulated at the apical ectoplasmic specialization, corroborating the observation of actin cytoskeleton disruption. Additionally, spatiotemporal expression of MT regulatory protein end-binding protein 1, shown to be involved in MT-actin cross talk herein, was also disrupted after adjudin treatment. In summary, spermatid/phagosome transport across the epithelium during spermatogenesis requires the coordination between actin- and MT-based cytoskeletons. PMID:26894662

Elongated Nanoparticle Aggregates in Cancer Cells for Mechanical Destruction with Low Frequency Rotating Magnetic Field.

PubMed

Shen, Yajing; Wu, Congyu; Uyeda, Taro Q P; Plaza, Gustavo R; Liu, Bin; Han, Yu; Lesniak, Maciej S; Cheng, Yu

2017-01-01

Magnetic nanoparticles (MNPs) functionalized with targeting moieties can recognize specific cell components and induce mechanical actuation under magnetic field. Their size is adequate for reaching tumors and targeting cancer cells. However, due to the nanometric size, the force generated by MNPs is smaller than the force required for largely disrupting key components of cells. Here, we show the magnetic assembly process of the nanoparticles inside the cells, to form elongated aggregates with the size required to produce elevated mechanical forces. We synthesized iron oxide nanoparticles doped with zinc, to obtain high magnetization, and functionalized with the epidermal growth factor (EGF) peptide for targeting cancer cells. Under a low frequency rotating magnetic field at 15 Hz and 40 mT, the internalized EGF-MNPs formed elongated aggregates and generated hundreds of pN to dramatically damage the plasma and lysosomal membranes. The physical disruption, including leakage of lysosomal hydrolases into the cytosol, led to programmed cell death and necrosis. Our work provides a novel strategy of designing magnetic nanomedicines for mechanical destruction of cancer cells.

Elongated Nanoparticle Aggregates in Cancer Cells for Mechanical Destruction with Low Frequency Rotating Magnetic Field

PubMed Central

Shen, Yajing; Wu, Congyu; Uyeda, Taro Q. P.; Plaza, Gustavo R.; Liu, Bin; Han, Yu; Lesniak, Maciej S.; Cheng, Yu

2017-01-01

Magnetic nanoparticles (MNPs) functionalized with targeting moieties can recognize specific cell components and induce mechanical actuation under magnetic field. Their size is adequate for reaching tumors and targeting cancer cells. However, due to the nanometric size, the force generated by MNPs is smaller than the force required for largely disrupting key components of cells. Here, we show the magnetic assembly process of the nanoparticles inside the cells, to form elongated aggregates with the size required to produce elevated mechanical forces. We synthesized iron oxide nanoparticles doped with zinc, to obtain high magnetization, and functionalized with the epidermal growth factor (EGF) peptide for targeting cancer cells. Under a low frequency rotating magnetic field at 15 Hz and 40 mT, the internalized EGF-MNPs formed elongated aggregates and generated hundreds of pN to dramatically damage the plasma and lysosomal membranes. The physical disruption, including leakage of lysosomal hydrolases into the cytosol, led to programmed cell death and necrosis. Our work provides a novel strategy of designing magnetic nanomedicines for mechanical destruction of cancer cells. PMID:28529648

Gene regulatory networks and the underlying biology of developmental toxicity

EPA Science Inventory

Embryonic cells are specified by large-scale networks of functionally linked regulatory genes. Knowledge of the relevant gene regulatory networks is essential for understanding phenotypic heterogeneity that emerges from disruption of molecular functions, cellular processes or sig...

The Factory of the Future

NASA Technical Reports Server (NTRS)

Byman, J. E.

1985-01-01

A brief history of aircraft production techniques is given. A flexible machining cell is then described. It is a computer controlled system capable of performing 4-axis machining part cleaning, dimensional inspection and materials handling functions in an unmanned environment. The cell was designed to: allow processing of similar and dissimilar parts in random order without disrupting production; allow serial (one-shipset-at-a-time) manufacturing; reduce work-in-process inventory; maximize machine utilization through remote set-up; maximize throughput and minimize labor.

Cationic Peptide Exposure Enhances Pulsed-Electric-Field-Mediated Membrane Disruption

PubMed Central

Kennedy, Stephen M.; Aiken, Erik J.; Beres, Kaytlyn A.; Hahn, Adam R.; Kamin, Samantha J.; Hagness, Susan C.; Booske, John H.; Murphy, William L.

2014-01-01

Background The use of pulsed electric fields (PEFs) to irreversibly electroporate cells is a promising approach for destroying undesirable cells. This approach may gain enhanced applicability if the intensity of the PEF required to electrically disrupt cell membranes can be reduced via exposure to a molecular deliverable. This will be particularly impactful if that reduced PEF minimally influences cells that are not exposed to the deliverable. We hypothesized that the introduction of charged molecules to the cell surfaces would create regions of enhanced transmembrane electric potential in the vicinity of each charged molecule, thereby lowering the PEF intensity required to disrupt the plasma membranes. This study will therefore examine if exposure to cationic peptides can enhance a PEF’s ability to disrupt plasma membranes. Methodology/Principal Findings We exposed leukemia cells to 40 μs PEFs in media containing varying concentrations of a cationic peptide, polyarginine. We observed the internalization of a membrane integrity indicator, propidium iodide (PI), in real time. Based on an individual cell’s PI fluorescence versus time signature, we were able to determine the relative degree of membrane disruption. When using 1–2 kV/cm, exposure to >50 μg/ml of polyarginine resulted in immediate and high levels of PI uptake, indicating severe membrane disruption, whereas in the absence of peptide, cells predominantly exhibited signatures indicative of no membrane disruption. Additionally, PI entered cells through the anode-facing membrane when exposed to cationic peptide, which was theoretically expected. Conclusions/Significance Exposure to cationic peptides reduced the PEF intensity required to induce rapid and irreversible membrane disruption. Critically, peptide exposure reduced the PEF intensities required to elicit irreversible membrane disruption at normally sub-electroporation intensities. We believe that these cationic peptides, when coupled with current advancements in cell targeting techniques will be useful tools in applications where targeted destruction of unwanted cell populations is desired. PMID:24671150

Anthrax edema toxin disrupts distinct steps in Rab11-dependent junctional transport

PubMed Central

Guichard, Annabel; Jain, Prashant; Moayeri, Mahtab; Cruz-Moreno, Beatriz; Leppla, Stephen H.; Nizet, Victor

2017-01-01

Various bacterial toxins circumvent host defenses through overproduction of cAMP. In a previous study, we showed that edema factor (EF), an adenylate cyclase from Bacillus anthracis, disrupts endocytic recycling mediated by the small GTPase Rab11. As a result, cargo proteins such as cadherins fail to reach inter-cellular junctions. In the present study, we provide further mechanistic dissection of Rab11 inhibition by EF using a combination of Drosophila and mammalian systems. EF blocks Rab11 trafficking after the GTP-loading step, preventing a constitutively active form of Rab11 from delivering cargo vesicles to the plasma membrane. Both of the primary cAMP effector pathways -PKA and Epac/Rap1- contribute to inhibition of Rab11-mediated trafficking, but act at distinct steps of the delivery process. PKA acts early, preventing Rab11 from associating with its effectors Rip11 and Sec15. In contrast, Epac functions subsequently via the small GTPase Rap1 to block fusion of recycling endosomes with the plasma membrane, and appears to be the primary effector of EF toxicity in this process. Similarly, experiments conducted in mammalian systems reveal that Epac, but not PKA, mediates the activity of EF both in cell culture and in vivo. The small GTPase Arf6, which initiates endocytic retrieval of cell adhesion components, also contributes to junctional homeostasis by counteracting Rab11-dependent delivery of cargo proteins at sites of cell-cell contact. These studies have potentially significant practical implications, since chemical inhibition of either Arf6 or Epac blocks the effect of EF in cell culture and in vivo, opening new potential therapeutic avenues for treating symptoms caused by cAMP-inducing toxins or related barrier-disrupting pathologies. PMID:28945820

Anthrax edema toxin disrupts distinct steps in Rab11-dependent junctional transport.

PubMed

Guichard, Annabel; Jain, Prashant; Moayeri, Mahtab; Schwartz, Ruth; Chin, Stephen; Zhu, Lin; Cruz-Moreno, Beatriz; Liu, Janet Z; Aguilar, Bernice; Hollands, Andrew; Leppla, Stephen H; Nizet, Victor; Bier, Ethan

2017-09-01

Various bacterial toxins circumvent host defenses through overproduction of cAMP. In a previous study, we showed that edema factor (EF), an adenylate cyclase from Bacillus anthracis, disrupts endocytic recycling mediated by the small GTPase Rab11. As a result, cargo proteins such as cadherins fail to reach inter-cellular junctions. In the present study, we provide further mechanistic dissection of Rab11 inhibition by EF using a combination of Drosophila and mammalian systems. EF blocks Rab11 trafficking after the GTP-loading step, preventing a constitutively active form of Rab11 from delivering cargo vesicles to the plasma membrane. Both of the primary cAMP effector pathways -PKA and Epac/Rap1- contribute to inhibition of Rab11-mediated trafficking, but act at distinct steps of the delivery process. PKA acts early, preventing Rab11 from associating with its effectors Rip11 and Sec15. In contrast, Epac functions subsequently via the small GTPase Rap1 to block fusion of recycling endosomes with the plasma membrane, and appears to be the primary effector of EF toxicity in this process. Similarly, experiments conducted in mammalian systems reveal that Epac, but not PKA, mediates the activity of EF both in cell culture and in vivo. The small GTPase Arf6, which initiates endocytic retrieval of cell adhesion components, also contributes to junctional homeostasis by counteracting Rab11-dependent delivery of cargo proteins at sites of cell-cell contact. These studies have potentially significant practical implications, since chemical inhibition of either Arf6 or Epac blocks the effect of EF in cell culture and in vivo, opening new potential therapeutic avenues for treating symptoms caused by cAMP-inducing toxins or related barrier-disrupting pathologies.

Enhanced cell-surface display of a heterologous protein using SED1 anchoring system in SED1-disrupted Saccharomyces cerevisiae strain.

PubMed

Bamba, Takahiro; Inokuma, Kentaro; Hasunuma, Tomohisa; Kondo, Akihiko

2018-03-01

Yeast displaying enzymes on the cell surface are used for developing whole-cell biocatalysts. High enzyme activity on the cell surface is required in certain applications such as direct ethanol production from lignocellulosic materials. However, the cell surface enzyme activity is limited by several factors, one of which is the protein amount of the yeast cell wall. In this study, we attempted to improve the incorporation capacity of a displayed heterologous enzyme by disrupting a native cell-wall protein. β-Glucosidase (BGL1) from Aspergillus aculeatus was fused with Saccharomyces cerevisiae Sed1 and displayed on the cell surface of S. cerevisiae BY4741 strain and its SED1 disruptant. Sed1 is one of the most abundant stationary phase yeast cell wall protein. A time course analysis revealed that BGL1 activity of the control strain reached saturation after 48 h of cultivation. In contrast, the BGL1 activity of the SED1 disruptant increased until 72 h of cultivation and was 22% higher than that of the control strain. We also performed relative quantification of cell wall proteins of these strains by nanoscale ultra pressure liquid chromatography electrospray ionization quadrupole time-of-flight tandem mass spectrometry (nano-UPLC-MS E ). The amount of the cell wall-associated BGL1 per unit dry cell-weight of the SED1 disruptant was 19% higher than that of the control strain. These results suggested that the incorporation capacity of the cell wall for BGL1 was increased by disruption of SED1. Disruption of SED1 would be a promising approach for improving display efficiency of heterologous protein fused with Sed1. Copyright © 2017 The Society for Biotechnology, Japan. Published by Elsevier B.V. All rights reserved.

Micromilling enhances iron bioaccessibility from wholegrain wheat.

PubMed

Latunde-Dada, G O; Li, X; Parodi, A; Edwards, C H; Ellis, P R; Sharp, P A

2014-11-19

Cereals constitute important sources of iron in human diet; however, much of the iron in wheat is lost during processing for the production of white flour. This study employed novel food processing techniques to increase the bioaccessibility of naturally occurring iron in wheat. Iron was localized in wheat by Perl's Prussian blue staining. Soluble iron from digested wheat flour was measured by a ferrozine spectrophotometric assay. Iron bioaccessibility was determined using an in vitro simulated peptic-pancreatic digestion, followed by measurement of ferritin (a surrogate marker for iron absorption) in Caco-2 cells. Light microscopy revealed that iron in wheat was encapsulated in cells of the aleurone layer and remained intact after in vivo digestion and passage through the gastrointestinal tract. The solubility of iron in wholegrain wheat and in purified wheat aleurone increased significantly after enzymatic digestion with Driselase, and following mechanical disruption using micromilling. Furthermore, following in vitro simulated peptic-pancreatic digestion, iron bioaccessibility, measured as ferritin formation in Caco-2 cells, from micromilled aleurone flour was significantly higher (52%) than from whole aleurone flour. Taken together our data show that disruption of aleurone cell walls could increase iron bioaccessibility. Micromilled aleurone could provide an alternative strategy for iron fortification of cereal products.

Thyroid hormone increases fibroblast growth factor receptor expression and disrupts cell mechanics in the developing organ of corti

PubMed Central

2013-01-01

Background Thyroid hormones regulate growth and development. However, the molecular mechanisms by which thyroid hormone regulates cell structural development are not fully understood. The mammalian cochlea is an intriguing system to examine these mechanisms, as cellular structure plays a key role in tissue development, and thyroid hormone is required for the maturation of the cochlea in the first postnatal week. Results In hypothyroid conditions, we found disruptions in sensory outer hair cell morphology and fewer microtubules in non-sensory supporting pillar cells. To test the functional consequences of these cytoskeletal defects on cell mechanics, we combined atomic force microscopy with live cell imaging. Hypothyroidism stiffened outer hair cells and supporting pillar cells, but pillar cells ultimately showed reduced cell stiffness, in part from a lack of microtubules. Analyses of changes in transcription and protein phosphorylation suggest that hypothyroidism prolonged expression of fibroblast growth factor receptors, and decreased phosphorylated Cofilin. Conclusions These findings demonstrate that thyroid hormones may be involved in coordinating the processes that regulate cytoskeletal dynamics and suggest that manipulating thyroid hormone sensitivity might provide insight into the relationship between cytoskeletal formation and developing cell mechanical properties. PMID:23394545

Self-organisation after embryonic kidney dissociation is driven via selective adhesion of ureteric epithelial cells.

PubMed

Lefevre, James G; Chiu, Han S; Combes, Alexander N; Vanslambrouck, Jessica M; Ju, Ali; Hamilton, Nicholas A; Little, Melissa H

2017-03-15

Human pluripotent stem cells, after directed differentiation in vitro , can spontaneously generate complex tissues via self-organisation of the component cells. Self-organisation can also reform embryonic organ structure after tissue disruption. It has previously been demonstrated that dissociated embryonic kidneys can recreate component epithelial and mesenchymal relationships sufficient to allow continued kidney morphogenesis. Here, we investigate the timing and underlying mechanisms driving self-organisation after dissociation of the embryonic kidney using time-lapse imaging, high-resolution confocal analyses and mathematical modelling. Organotypic self-organisation sufficient for nephron initiation was observed within a 24 h period. This involved cell movement, with structure emerging after the clustering of ureteric epithelial cells, a process consistent with models of random cell movement with preferential cell adhesion. Ureteric epithelialisation rapidly followed the formation of ureteric cell clusters with the reformation of nephron-forming niches representing a later event. Disruption of P-cadherin interactions was seen to impair this ureteric epithelial cell clustering without affecting epithelial maturation. This understanding could facilitate improved regulation of patterning within organoids and facilitate kidney engineering approaches guided by cell-cell self-organisation. © 2017. Published by The Company of Biologists Ltd.

Endocrine Disruptors (Chapter 14) in Mammalian Toxicology Book

EPA Science Inventory

Endocrine disrupting chemicals (EDCs) are exogenous substances that alter endocrine system function(s) and consequently cause adverse health effects in intact organisms or its progeny. The endocrine system is important for a wide range of biological processes, from normal cell si...

Putting tumours in context

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

Bissell, Mina J.; Radisky, Derek

2001-10-01

The interactions between cancer cells and their micro- and macroenvironment create a context that promotes tumor growth and protects it from immune attack. The functional association of cancer cells with their surrounding tissues forms a new 'organ' that changes as malignancy progresses. Investigation of this process might provide new insights into the mechanisms of tumorigenesis and could also lead to new therapeutic targets. Under normal conditions, ORGANS are made up of TISSUES that exchange information with other cell types via cell-cell contact, cytokines and the EXTRACELLULAR MATRIX (ECM). The ECM, which is produced by collaboration between STROMAL fibroblasts and EPITHELIALmore » cells, provides structural scaffolding for cells, as well as contextual information. The endothelial vasculature provides nutrients and oxygen, and cells of the immune system combat pathogens and remove apoptotic cells. Epithelial cells associate into intact, polarized sheets. These tissues communicate through a complex network of interactions: physically, through direct contact or through the intervening ECM, and biochemically, through both soluble and insoluble signalling molecules. In combination, these interactions provide the information that is necessary to maintain cellular differentiation and to create complex tissue structures. Occasionally, the intercellular signals that define the normal context become disrupted. Alterations in epithelial tissues can lead to movement of epithelial sheets and proliferation - for example, after activation of mesenchymal fibroblasts due to wounding.Normally, these conditions are temporary and reversible, but when inflammation is sustained, an escalating feedback loop ensues.Under persistent inflammatory conditions, continual upregulation of enzymes such as matrix metalloproteinases (MMPs) by stromal fibroblasts can disrupt the ECM, and invading immune cells can overproduce factors that promote abnormal proliferation. As this process progresses, the normal organization of the organ is replaced by a functional disorder. If there are pre-existing epithelial cells within this changing context that possess tumorigenic potential, they can start to proliferate. Alternatively, the abnormal interactions might lead to genomic instability within the epithelial cells and the acquisition of tumorigenic potential. The proliferating cancer cells can then interact with their microenvironment and enhance the abnormal interactions. At this point, the tumor has become its own organ, with a distinct context that now defines all its cellular responses. Here, we will examine how the mechanisms that contribute to the normal context also act to suppress developing tumors, how disruption of this context initiates and supports the process of tumorigenicity, and how some cells with a tumorigenic genotype can become phenotypically normal if the context is appropriately manipulated.« less

Disruption of SF3B1 results in deregulated expression and splicing of key genes and pathways in myelodysplastic syndrome hematopoietic stem and progenitor cells.

PubMed

Dolatshad, H; Pellagatti, A; Fernandez-Mercado, M; Yip, B H; Malcovati, L; Attwood, M; Przychodzen, B; Sahgal, N; Kanapin, A A; Lockstone, H; Scifo, L; Vandenberghe, P; Papaemmanuil, E; Smith, C W J; Campbell, P J; Ogawa, S; Maciejewski, J P; Cazzola, M; Savage, K I; Boultwood, J

2015-05-01

The splicing factor SF3B1 is the most commonly mutated gene in the myelodysplastic syndrome (MDS), particularly in patients with refractory anemia with ring sideroblasts (RARS). We investigated the functional effects of SF3B1 disruption in myeloid cell lines: SF3B1 knockdown resulted in growth inhibition, cell cycle arrest and impaired erythroid differentiation and deregulation of many genes and pathways, including cell cycle regulation and RNA processing. MDS is a disorder of the hematopoietic stem cell and we thus studied the transcriptome of CD34(+) cells from MDS patients with SF3B1 mutations using RNA sequencing. Genes significantly differentially expressed at the transcript and/or exon level in SF3B1 mutant compared with wild-type cases include genes that are involved in MDS pathogenesis (ASXL1 and CBL), iron homeostasis and mitochondrial metabolism (ALAS2, ABCB7 and SLC25A37) and RNA splicing/processing (PRPF8 and HNRNPD). Many genes regulated by a DNA damage-induced BRCA1-BCLAF1-SF3B1 protein complex showed differential expression/splicing in SF3B1 mutant cases. This is the first study to determine the target genes of SF3B1 mutation in MDS CD34(+) cells. Our data indicate that SF3B1 has a critical role in MDS by affecting the expression and splicing of genes involved in specific cellular processes/pathways, many of which are relevant to the known RARS pathophysiology, suggesting a causal link.

Elucidation of flow-mediated tumour cell-induced platelet aggregation using an ultrasound standing wave trap.

PubMed

Bazou, D; Santos-Martinez, M J; Medina, C; Radomski, M W

2011-04-01

Tumour cells activate and aggregate platelets [tumour cell-induced platelet aggregation (TCIPA)] and this process plays an important role in the successful metastasis of cancer cells. To date, most studies on TCIPA have been conducted under no-flow conditions. In this study, we have investigated TCIPA in real time under flow conditions, using an ultrasound standing wave trap that allows formation and levitation of cancer cell clusters in suspension, thus mimicking the conditions generated by flowing blood. Using 59M adenocarcinoma and HT1080 fibrosarcoma cells and human platelets, cancer cell cluster-platelet aggregates were imaged in real time using epi-fluorescence microscopy (F-actin) and investigated in detail using confocal microscopy (matrix metalloproteinase-2-GPIIb/IIIa co-localization) and scanning electron and helium-ion microscopy (<1 nm resolution). The release of gelatinases from aggregates was studied using zymography. We found that platelet activation and aggregation takes place on the surface of cancer cells (TCIPA), leading to time-dependent disruption of cancer cell clusters. Pharmacological modulation of TCIPA revealed that EDTA, prostacyclin, o-phenanthroline and apyrase significantly down-regulated TCIPA and, in turn, delayed cell cluster disruption, However, EGTA and aspirin were ineffective. Pharmacological inhibition of TCIPA correlated with the down-regulation of platelet activation as shown by flow-cytometry assay of platelet P-selectin. Our results show for the first time, that during TCIPA, platelet activation disrupts cancer cell clusters and this can contribute to metastasis. Thus, selective targeting of platelet aggregate-cancer cell clusters may be an important strategy to control metastasis. © 2011 The Authors. British Journal of Pharmacology © 2011 The British Pharmacological Society.

Elucidation of flow-mediated tumour cell-induced platelet aggregation using an ultrasound standing wave trap

PubMed Central

Bazou, D; Santos-Martinez, MJ; Medina, C; Radomski, MW

2011-01-01

BACKGROUND AND PURPOSE Tumour cells activate and aggregate platelets [tumour cell-induced platelet aggregation (TCIPA)] and this process plays an important role in the successful metastasis of cancer cells. To date, most studies on TCIPA have been conducted under no-flow conditions. In this study, we have investigated TCIPA in real time under flow conditions, using an ultrasound standing wave trap that allows formation and levitation of cancer cell clusters in suspension, thus mimicking the conditions generated by flowing blood. EXPERIMENTAL APPROACH Using 59M adenocarcinoma and HT1080 fibrosarcoma cells and human platelets, cancer cell cluster–platelet aggregates were imaged in real time using epi-fluorescence microscopy (F-actin) and investigated in detail using confocal microscopy (matrix metalloproteinase-2-GPIIb/IIIa co-localization) and scanning electron and helium-ion microscopy (<1 nm resolution). The release of gelatinases from aggregates was studied using zymography. KEY RESULTS We found that platelet activation and aggregation takes place on the surface of cancer cells (TCIPA), leading to time-dependent disruption of cancer cell clusters. Pharmacological modulation of TCIPA revealed that EDTA, prostacyclin, o-phenanthroline and apyrase significantly down-regulated TCIPA and, in turn, delayed cell cluster disruption, However, EGTA and aspirin were ineffective. Pharmacological inhibition of TCIPA correlated with the down-regulation of platelet activation as shown by flow-cytometry assay of platelet P-selectin. CONCLUSION AND IMPLICATIONS Our results show for the first time, that during TCIPA, platelet activation disrupts cancer cell clusters and this can contribute to metastasis. Thus, selective targeting of platelet aggregate–cancer cell clusters may be an important strategy to control metastasis. PMID:21182493

Dynamic interactions between Pit-1 and C/EBPalpha in the pituitary cell nucleus.

PubMed

Demarco, Ignacio A; Voss, Ty C; Booker, Cynthia F; Day, Richard N

2006-11-01

The homeodomain (HD) transcription factors are a structurally conserved family of proteins that, through networks of interactions with other nuclear proteins, control patterns of gene expression during development. For example, the network interactions of the pituitary-specific HD protein Pit-1 control the development of anterior pituitary cells and regulate the expression of the hormone products in the adult cells. Inactivating mutations in Pit-1 disrupt these processes, giving rise to the syndrome of combined pituitary hormone deficiency. Pit-1 interacts with CCAAT/enhancer-binding protein alpha (C/EBPalpha) to regulate prolactin transcription. Here, we used the combination of biochemical analysis and live-cell microscopy to show that two different point mutations in Pit-1, which disrupted distinct activities, affected the dynamic interactions between Pit-1 and C/EBPalpha in different ways. The results showed that the first alpha-helix of the POU-S domain is critical for the assembly of Pit-1 with C/EBPalpha, and they showed that DNA-binding activity conferred by the HD is critical for the final intranuclear positioning of the metastable complex. This likely reflects more general mechanisms that govern cell-type-specific transcriptional control, and the results from the analysis of the point mutations could indicate an important link between the mislocalization of transcriptional complexes and disease processes.

Endocrine disrupters--testing strategies to assess human hazard.

PubMed

Baker, V A

2001-01-01

During the last decade an hypothesis has been developed linking certain chemicals (natural and synthetic) to observed and suspected adverse effects on reproduction in both wildlife and humans. The issue of 'endocrine disruption' originally focused on chemicals that mimic the action of the natural hormone oestrogen. However, the concern is now encompassing effects on the whole endocrine system. In response to public awareness, regulatory agencies (including the US EPA) and the OECD are formulating potential testing strategies and have begun the process of validating defined tests to systematically assess chemicals for their endocrine-disrupting activities. In order to investigate chemicals that have the potential to cause endocrine disruption, a large number of in vitro and in vivo assays have been identified. In vitro test systems (particularly when used in combination) offer the possibility of providing an early screen for large numbers of chemicals and can be useful in characterising the mechanism of action and potency. In vitro assays in widespread use for the screening/characterisation of endocrine disrupting potential include hormone receptor ligand binding assays (determination of the ability of a chemical to bind to the hormone receptor), cell proliferation assays (analysis of the ability of a chemical to stimulate growth of oestrogen sensitive cells), reporter gene assays in yeast or mammalian cells (analysis of the ability of a chemical to stimulate the transcription of a reporter gene construct in cell culture), and the analysis of the regulation of endogenous oestrogen sensitive genes in cell lines. However, in vitro assays do not always reliably predict the outcome in vivo due to differences in metabolic capabilities of the test systems used and the diverse range of mechanisms by which endocrine disrupting chemicals may act. Therefore a complementary battery of short- and long-term in vitro and in vivo assays (that assess both receptor and non-receptor mediated mechanisms of action) seems the most appropriate way at present of assessing the potential endocrine disrupting activities of chemicals. At Unilever we have used a combination of in vitro assays (receptor binding, reporter gene and cell proliferation assays) together with short-term in vivo tests (uterotrophic assay in immature rodents) to examine the oestrogenic potential of a large number of chemicals. An evaluation of the advantages and limitations of these methods is provided. Finally, any potential test system needs to be validated and standardized before the information generated can be for the identification of hazard, and possibly for risk assessment purposes.

Leader cells regulate collective cell migration via Rac activation in the downstream signaling of integrin β1 and PI3K

PubMed Central

Yamaguchi, Naoya; Mizutani, Takeomi; Kawabata, Kazushige; Haga, Hisashi

2015-01-01

Collective cell migration plays a crucial role in several biological processes, such as embryonic development, wound healing, and cancer metastasis. Here, we focused on collectively migrating Madin-Darby Canine Kidney (MDCK) epithelial cells that follow a leader cell on a collagen gel to clarify the mechanism of collective cell migration. First, we removed a leader cell from the migrating collective with a micromanipulator. This then caused disruption of the cohesive migration of cells that followed in movement, called “follower” cells, which showed the importance of leader cells. Next, we observed localization of active Rac, integrin β1, and PI3K. These molecules were clearly localized in the leading edge of leader cells, but not in follower cells. Live cell imaging using active Rac and active PI3K indicators was performed to elucidate the relationship between Rac, integrin β1, and PI3K. Finally, we demonstrated that the inhibition of these molecules resulted in the disruption of collective migration. Our findings not only demonstrated the significance of a leader cell in collective cell migration, but also showed that Rac, integrin β1, and PI3K are upregulated in leader cells and drive collective cell migration. PMID:25563751

Leader cells regulate collective cell migration via Rac activation in the downstream signaling of integrin β1 and PI3K.

PubMed

Yamaguchi, Naoya; Mizutani, Takeomi; Kawabata, Kazushige; Haga, Hisashi

2015-01-07

Collective cell migration plays a crucial role in several biological processes, such as embryonic development, wound healing, and cancer metastasis. Here, we focused on collectively migrating Madin-Darby Canine Kidney (MDCK) epithelial cells that follow a leader cell on a collagen gel to clarify the mechanism of collective cell migration. First, we removed a leader cell from the migrating collective with a micromanipulator. This then caused disruption of the cohesive migration of cells that followed in movement, called "follower" cells, which showed the importance of leader cells. Next, we observed localization of active Rac, integrin β1, and PI3K. These molecules were clearly localized in the leading edge of leader cells, but not in follower cells. Live cell imaging using active Rac and active PI3K indicators was performed to elucidate the relationship between Rac, integrin β1, and PI3K. Finally, we demonstrated that the inhibition of these molecules resulted in the disruption of collective migration. Our findings not only demonstrated the significance of a leader cell in collective cell migration, but also showed that Rac, integrin β1, and PI3K are upregulated in leader cells and drive collective cell migration.

Cytoskeletal Reorganization Drives Mesenchymal Condensation and Regulates Downstream Molecular Signaling.

PubMed

Ray, Poulomi; Chapman, Susan C

2015-01-01

Skeletal condensation occurs when specified mesenchyme cells self-organize over several days to form a distinctive cartilage template. Here, we determine how and when specified mesenchyme cells integrate mechanical and molecular information from their environment, forming cartilage condensations in the pharyngeal arches of chick embryos. By disrupting cytoskeletal reorganization, we demonstrate that dynamic cell shape changes drive condensation and modulate the response of the condensing cells to Fibroblast Growth Factor (FGF), Bone Morphogenetic Protein (BMP) and Transforming Growth Factor beta (TGF-β) signaling pathways. Rho Kinase (ROCK)-driven actomyosin contractions and Myosin II-generated differential cell cortex tension regulate these cell shape changes. Disruption of the condensation process inhibits the differentiation of the mesenchyme cells into chondrocytes, demonstrating that condensation regulates the fate of the mesenchyme cells. We also find that dorsal and ventral condensations undergo distinct cell shape changes. BMP signaling is instructive for dorsal condensation-specific cell shape changes. Moreover, condensations exhibit ventral characteristics in the absence of BMP signaling, suggesting that in the pharyngeal arches ventral morphology is the ground pattern. Overall, this study characterizes the interplay between cytoskeletal dynamics and molecular signaling in a self-organizing system during tissue morphogenesis.

Cytoskeletal Reorganization Drives Mesenchymal Condensation and Regulates Downstream Molecular Signaling

PubMed Central

Ray, Poulomi; Chapman, Susan C.

2015-01-01

Skeletal condensation occurs when specified mesenchyme cells self-organize over several days to form a distinctive cartilage template. Here, we determine how and when specified mesenchyme cells integrate mechanical and molecular information from their environment, forming cartilage condensations in the pharyngeal arches of chick embryos. By disrupting cytoskeletal reorganization, we demonstrate that dynamic cell shape changes drive condensation and modulate the response of the condensing cells to Fibroblast Growth Factor (FGF), Bone Morphogenetic Protein (BMP) and Transforming Growth Factor beta (TGF-β) signaling pathways. Rho Kinase (ROCK)-driven actomyosin contractions and Myosin II-generated differential cell cortex tension regulate these cell shape changes. Disruption of the condensation process inhibits the differentiation of the mesenchyme cells into chondrocytes, demonstrating that condensation regulates the fate of the mesenchyme cells. We also find that dorsal and ventral condensations undergo distinct cell shape changes. BMP signaling is instructive for dorsal condensation-specific cell shape changes. Moreover, condensations exhibit ventral characteristics in the absence of BMP signaling, suggesting that in the pharyngeal arches ventral morphology is the ground pattern. Overall, this study characterizes the interplay between cytoskeletal dynamics and molecular signaling in a self-organizing system during tissue morphogenesis. PMID:26237312

Disruption of the actin cytoskeleton results in the promotion of gravitropism in inflorescence stems and hypocotyls of Arabidopsis

NASA Technical Reports Server (NTRS)

Yamamoto, Kazuyoshi; Kiss, John Z.

2002-01-01

The actin cytoskeleton is hypothesized to play a major role in gravity perception and transduction mechanisms in roots of plants. To determine whether actin microfilaments (MFs) are involved in these processes in stem-like organs, we studied gravitropism in Arabidopsis inflorescence stems and hypocotyls. Localization studies using Alexa Fluor-phalloidin in conjugation with confocal microscopy demonstrated a longitudinally and transversely oriented actin MF network in endodermal cells of stems and hypocotyls. Latrunculin B (Lat-B) treatment of hypocotyls caused depolymerization of actin MFs in endodermal cells and a significant reduction of hypocotyl growth rates. Actin MFs in Lat-B-treated inflorescence stems also were disrupted, but growth rates were not affected. Despite disruption of the actin cytoskeleton in these two organs, Lat-B-treated stems and hypocotyls exhibited a promotion of gravitropic curvature in response to reorientation. In contrast, Lat-B reduced gravitropic curvature in roots but also reduced the growth rate. Thus, in contrast to prevailing hypotheses, our results suggest that actin MFs are not a necessary component of gravitropism in inflorescence stems and hypocotyls. Furthermore, this is the first study to demonstrate a prominent actin MF network in endodermal cells in the putative gravity-perceiving cells in stems.

Disruption of the Actin Cytoskeleton Results in the Promotion of Gravitropism in Inflorescence Stems and Hypocotyls of Arabidopsis1

PubMed Central

Yamamoto, Kazuyoshi; Kiss, John Z.

2002-01-01

The actin cytoskeleton is hypothesized to play a major role in gravity perception and transduction mechanisms in roots of plants. To determine whether actin microfilaments (MFs) are involved in these processes in stem-like organs, we studied gravitropism in Arabidopsis inflorescence stems and hypocotyls. Localization studies using Alexa Fluor-phalloidin in conjugation with confocal microscopy demonstrated a longitudinally and transversely oriented actin MF network in endodermal cells of stems and hypocotyls. Latrunculin B (Lat-B) treatment of hypocotyls caused depolymerization of actin MFs in endodermal cells and a significant reduction of hypocotyl growth rates. Actin MFs in Lat-B-treated inflorescence stems also were disrupted, but growth rates were not affected. Despite disruption of the actin cytoskeleton in these two organs, Lat-B-treated stems and hypocotyls exhibited a promotion of gravitropic curvature in response to reorientation. In contrast, Lat-B reduced gravitropic curvature in roots but also reduced the growth rate. Thus, in contrast to prevailing hypotheses, our results suggest that actin MFs are not a necessary component of gravitropism in inflorescence stems and hypocotyls. Furthermore, this is the first study to demonstrate a prominent actin MF network in endodermal cells in the putative gravity-perceiving cells in stems. PMID:11842170

Effect of high pressure homogenization on aqueous phase solvent extraction of lipids from Nannochloris Oculata microalgae

DOE PAGES

Samarasinghe, Nalin; Fernando, Sandun; Faulkner, William B.

2012-12-01

The ability to extract lipids from high-moisture Nannochloris Oculata algal biomass disrupted with high pressure homogenization was investigated. During the first phase, the effect of high pressure homogenization (system pressure and number of passes) on disrupting aqueous algae (of different concentrations and degree of stress) was investigated. Secondly, the effect of degree of cell wall disruption on the amount of lipids extracted with three solvents, namely: hexane, dichloromethane and chloroform, were compared. Studies reveled that high pressure homogenization is effective on cell disruption while the amount of system pressure being the most significant factor affecting the degree of cell breakage.more » Although the number of passes had some impact, the level of disruption seemed to level-off after a certain number of passes. The study revealed that slightly polar solvents (such as chloroform and dichloromethane) performed better in aqueous-phase lipid extractions as compared to hexane. Also, it was revealed that it was not necessary to disrupt the algal cells completely to achieve appreciable levels of lipid yields. In fact, conditions that exerted only 20% of the cells to completely disrupt, allowed sufficient damage to liberate most of the lipids contained in the remainder of the cells.« less

Effect of high pressure homogenization on aqueous phase solvent extraction of lipids from Nannochloris Oculata microalgae

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

Samarasinghe, Nalin; Fernando, Sandun; Faulkner, William B.

The ability to extract lipids from high-moisture Nannochloris Oculata algal biomass disrupted with high pressure homogenization was investigated. During the first phase, the effect of high pressure homogenization (system pressure and number of passes) on disrupting aqueous algae (of different concentrations and degree of stress) was investigated. Secondly, the effect of degree of cell wall disruption on the amount of lipids extracted with three solvents, namely: hexane, dichloromethane and chloroform, were compared. Studies reveled that high pressure homogenization is effective on cell disruption while the amount of system pressure being the most significant factor affecting the degree of cell breakage.more » Although the number of passes had some impact, the level of disruption seemed to level-off after a certain number of passes. The study revealed that slightly polar solvents (such as chloroform and dichloromethane) performed better in aqueous-phase lipid extractions as compared to hexane. Also, it was revealed that it was not necessary to disrupt the algal cells completely to achieve appreciable levels of lipid yields. In fact, conditions that exerted only 20% of the cells to completely disrupt, allowed sufficient damage to liberate most of the lipids contained in the remainder of the cells.« less

Genomic deletion of GIT2 induces a premature age-related thymic dysfunction and systemic immune system disruption

PubMed Central

Siddiqui, Sana; Lustig, Ana; Carter, Arnell; Sankar, Mathavi; Daimon, Caitlin M.; Premont, Richard T.; Etienne, Harmonie; van Gastel, Jaana; Azmi, Abdelkrim; Janssens, Jonathan; Becker, Kevin G.; Zhang, Yongqing; Wood, William; Lehrmann, Elin; Martin, James G.; Martin, Bronwen; Taub, Dennis D.; Maudsley, Stuart

2017-01-01

Recent research has proposed that GIT2 (G protein-coupled receptor kinase interacting protein 2) acts as an integrator of the aging process through regulation of ‘neurometabolic’ integrity. One of the commonly accepted hallmarks of the aging process is thymic involution. At a relatively young age, 12 months old, GIT2−/− mice present a prematurely distorted thymic structure and dysfunction compared to age-matched 12 month-old wild-type control (C57BL/6) mice. Disruption of thymic structure in GIT2−/− (GIT2KO) mice was associated with a significant reduction in the expression of the cortical thymic marker, Troma-I (cytokeratin 8). Double positive (CD4+CD8+) and single positive CD4+ T cells were also markedly reduced in 12 month-old GIT2KO mice compared to age-matched control wild-type mice. Coincident with this premature thymic disruption in GIT2KO mice was the unique generation of a novel cervical ‘organ’, i.e. ‘parathymic lobes’. These novel organs did not exhibit classical peripheral lymph node-like characteristics but expressed high levels of T cell progenitors that were reflexively reduced in GIT2KO thymi. Using signaling pathway analysis of GIT2KO thymus and parathymic lobe transcriptomic data we found that the molecular signaling functions lost in the dysfunctional GIT2KO thymus were selectively reinstated in the novel parathymic lobe – suggestive of a compensatory effect for the premature thymic disruption. Broader inspection of high-dimensionality transcriptomic data from GIT2KO lymph nodes, spleen, thymus and parathymic lobes revealed a systemic alteration of multiple proteins (Dbp, Tef, Per1, Per2, Fbxl3, Ddit4, Sin3a) involved in the multidimensional control of cell cycle clock regulation, cell senescence, cellular metabolism and DNA damage. Altered cell clock regulation across both immune and non-immune tissues therefore may be responsible for the premature ‘aging’ phenotype of GIT2KO mice. PMID:28260693

Study of physical and biological factors involved in the disruption of E. coli by hydrodynamic cavitation.

PubMed

Balasundaram, B; Harrison, S T L

2006-01-01

Hydrodynamic cavitation results in flow restriction in a flow system causing rapid pressure fluctuations and significant fluid forces. These can be harnessed to mediate microbial cell damage. Hydrodynamic cavitation was studied for the partial disruption of E. coli and selective release of specific proteins relative to the total soluble protein. The effects of the cavitation number, the number of passes, and the specific growth rate of E. coli on the release of periplasmic and cytoplasmic proteins were studied. At the optimum cavitation number of 0.17 for this experimental configuration, 48% of the total soluble protein, 88% of acid phosphatase, and 67% of beta-galactosidase were released by hydrodynamic cavitation in comparison with the maximum release attained using multiple passes through the French Press. The higher release of the acid phosphatase over the total soluble protein suggested preferred release of periplasmic compounds. This was supported by SDS-PAGE analysis. The absence of micronization of cell material resulting in the potential for ease of solid-liquid separation downstream of the cell disruption operation was confirmed by TEM microscopy. E. coli cells cultivated at a higher specific growth rate (0.36 h(-1)) were more easily disrupted than slower grown cells (0.11 h(-1)). The specific activity of the enzyme of interest released by hydrodynamic cavitation, defined as the units of enzyme in solution per milligram of total soluble protein, was greater than that obtained on release by the French Press, high-pressure homogenization, osmotic shock, and EDTA treatment. The selectivity offered indicates the potential of enzyme release by hydrodynamic cavitation to ease the purification in the subsequent downstream processing.

Endocrine disrupting chemicals affect the adipogenic differentiation of mesenchymal stem cells in distinct ontogenetic windows

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

Biemann, Ronald, E-mail: ronald.biemann@medizin.uni-halle.de; Navarrete Santos, Anne; Navarrete Santos, Alexander

Highlights: Black-Right-Pointing-Pointer Endocrine disrupting chemicals affect adipogenesis in mesenchymal stem cells (MSC). Black-Right-Pointing-Pointer The adipogenic impact depends strongly on the window of exposure. Black-Right-Pointing-Pointer Bisphenol A reduces the potential of MSC to differentiate into adipocytes. Black-Right-Pointing-Pointer DEHP and TBT trigger the adipogenic differentiation of mesenchymal stem cells. Black-Right-Pointing-Pointer BPA, DEHP and TBT did not affect adipogenesis in embryonic stem cells. -- Abstract: Endocrine disrupting chemicals (EDC) like bisphenol A (BPA), bis(2-ethylhexyl)phthalate (DEHP) and tributyltin (TBT) are ubiquitously present in the environment and in human tissues. They bind to nuclear hormone receptors and affect cellular and developmental processes. In this study,more » we show that BPA, DEHP and TBT affect the adipogenic differentiation of murine mesenchymal stem cells (MSC, C3H/10T1/2) in a concentration-, stage- and compound-specific manner. C3H/10T1/2 cells and embryonic stem cells (CGR8) were exposed to BPA, DEHP or TBT at different stages of cell determination and differentiation (undifferentiated growth, adipogenic induction and terminal adipogenic differentiation). The final amount of differentiated adipocytes, cellular triglyceride content and mRNA expression of adipogenic marker genes (adiponectin, FABP4, PPAR{gamma}2, LPL) were quantified and compared with corresponding unexposed cells. BPA (10 {mu}M) decreased subsequent adipogenic differentiation of MSC, when cells were exposed during undifferentiated growth. In contrast, DEHP (100 {mu}M) during the hormonal induction period, and TBT (100 nM) in all investigated stages, enhanced adipogenesis. Importantly, exposure of undifferentiated murine embryonic stem cells did not show any effect of the investigated EDC on subsequent adipogenic differentiation.« less

Virtual Embryo: Cell-Agent Based Modeling of Developmental Processes and Toxicities (CSS BOSC)

EPA Science Inventory

Spatial regulation of cellular dynamics is fundamental to morphological development. As such, chemical disruption of spatial dynamics is a determinant of developmental toxicity. Incorporating spatial dynamics into AOPs for developmental toxicity is desired but constrained by the ...

From Molecular Circuit Dysfunction to Disease: Case Studies in Epilepsy, Traumatic Brain Injury, and Alzheimer’s Disease

PubMed Central

Dulla, Chris G.; Coulter, Douglas A.; Ziburkus, Jokubas

2015-01-01

Complex circuitry with feed-forward and feed-back systems regulate neuronal activity throughout the brain. Cell biological, electrical, and neurotransmitter systems enable neural networks to process and drive the entire spectrum of cognitive, behavioral, and motor functions. Simultaneous orchestration of distinct cells and interconnected neural circuits relies on hundreds, if not thousands, of unique molecular interactions. Even single molecule dysfunctions can be disrupting to neural circuit activity, leading to neurological pathology. Here, we sample our current understanding of how molecular aberrations lead to disruptions in networks using three neurological pathologies as exemplars: epilepsy, traumatic brain injury (TBI), and Alzheimer’s disease (AD). Epilepsy provides a window into how total destabilization of network balance can occur. TBI is an abrupt physical disruption that manifests in both acute and chronic neurological deficits. Last, in AD progressive cell loss leads to devastating cognitive consequences. Interestingly, all three of these neurological diseases are interrelated. The goal of this review, therefore, is to identify molecular changes that may lead to network dysfunction, elaborate on how altered network activity and circuit structure can contribute to neurological disease, and suggest common threads that may lie at the heart of molecular circuit dysfunction. PMID:25948650

Withaferin A disrupts ubiquitin-based NEMO reorganization induced by canonical NF-κB signaling

PubMed Central

Jackson, Shawn S.; Oberley, Christopher; Hooper, Christopher P.; Grindle, Kreg; Wuerzberger-Davis, Shelly; Wolff, Jared; McCool, Kevin; Rui, Lixin; Miyamoto, Shigeki

2014-01-01

The NF-κB family of transcription factors regulates numerous cellular processes, including cell proliferation and survival responses. The constitutive activation of NF-κB has also emerged as an important oncogenic driver in many malignancies, such as activated B-cell like diffuse large B cell lymphoma, among others. In this study, we investigated the impact and mechanisms of action of Withaferin A, a naturally produced steroidal lactone, against both signal-inducible as well as constitutive NF-κB activities. We found that Withaferin A is a robust inhibitor of canonical and constitutive NF-κB activities, leading to apoptosis of certain lymphoma lines. In the canonical pathway induced by TNF, Withaferin A did not disrupt RIP1 polyubiquitination or NEMO-IKKβ interaction and was a poor direct IKKβ inhibitor, but prevented the formation of TNF induced NEMO foci which colocalized with TNF ligand. While GFP-NEMO efficiently formed TNF-induced foci, a GFP-NEMOY308S mutant that is defective in binding to polyubiquitin chains did not form foci. Our study reveals that Withaferin A is a novel type of IKK inhibitor which acts by disrupting NEMO reorganization into ubiquitin-based signaling structures in vivo. PMID:25304104

Technological process for cell disruption, extraction and encapsulation of astaxanthin from Haematococcus pluvialis.

PubMed

Machado, Francisco R S; Trevisol, Thalles C; Boschetto, Daiane L; Burkert, Janaína F M; Ferreira, Sandra R S; Oliveira, J Vladimir; Burkert, Carlos André V

2016-01-20

In this work, the effectiveness of different enzymatic techniques for cell wall disruption of Haematococcus pluvialis for the extraction of carotenoids and subsequent encapsulation of extracts in the co-polymer poly(hydroxybutyrate-co-hydroxyvalerate) (PHBV) using the Solution Enhanced Dispersion by Supercritical fluids (SEDS) technique was investigated. Glucanex(®) performed best compared with Lyticase(®) and Driselase(®). The conditions for enzymatic lysis using this enzyme preparation were established as a pH of 4.5, a temperature of 55 °C, an initial activity of β-1,3-glucanase of 0.6 U mL(-1) and a reaction time of 30 min. Enzymatic lysis assisted by ultrasound without biomass freezing was shown to be a promising and simple one-step technique for cell wall disruption, reaching 83.90% extractability. In the co-precipitation experiments, the highest encapsulation efficiency (51.21%) was obtained when using a higher biomass to dichloromethane ratio (10 mg mL(-1)) at the carotenoid extraction step and a lower pressure of precipitation (80 bar). In these conditions, spherical particles in the micrometer range (0.228 μm) were obtained. Copyright © 2015 Elsevier B.V. All rights reserved.

From Molecular Circuit Dysfunction to Disease: Case Studies in Epilepsy, Traumatic Brain Injury, and Alzheimer's Disease.

PubMed

Dulla, Chris G; Coulter, Douglas A; Ziburkus, Jokubas

2016-06-01

Complex circuitry with feed-forward and feed-back systems regulate neuronal activity throughout the brain. Cell biological, electrical, and neurotransmitter systems enable neural networks to process and drive the entire spectrum of cognitive, behavioral, and motor functions. Simultaneous orchestration of distinct cells and interconnected neural circuits relies on hundreds, if not thousands, of unique molecular interactions. Even single molecule dysfunctions can be disrupting to neural circuit activity, leading to neurological pathology. Here, we sample our current understanding of how molecular aberrations lead to disruptions in networks using three neurological pathologies as exemplars: epilepsy, traumatic brain injury (TBI), and Alzheimer's disease (AD). Epilepsy provides a window into how total destabilization of network balance can occur. TBI is an abrupt physical disruption that manifests in both acute and chronic neurological deficits. Last, in AD progressive cell loss leads to devastating cognitive consequences. Interestingly, all three of these neurological diseases are interrelated. The goal of this review, therefore, is to identify molecular changes that may lead to network dysfunction, elaborate on how altered network activity and circuit structure can contribute to neurological disease, and suggest common threads that may lie at the heart of molecular circuit dysfunction. © The Author(s) 2015.

Withaferin A disrupts ubiquitin-based NEMO reorganization induced by canonical NF-κB signaling

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

Jackson, Shawn S.; Medical Scientist Training Program, University of Wisconsin-Madison, 1111 Highland Avenue, Madison, WI 53705; Cellular and Molecular Biology Program, University of Wisconsin-Madison, 1111 Highland Avenue, Madison, WI 53705

2015-02-01

The NF-κB family of transcription factors regulates numerous cellular processes, including cell proliferation and survival responses. The constitutive activation of NF-κB has also emerged as an important oncogenic driver in many malignancies, such as activated B-cell like diffuse large B cell lymphoma, among others. In this study, we investigated the impact and mechanisms of action of Withaferin A, a naturally produced steroidal lactone, against both signal-inducible as well as constitutive NF-κB activities. We found that Withaferin A is a robust inhibitor of canonical and constitutive NF-κB activities, leading to apoptosis of certain lymphoma lines. In the canonical pathway induced bymore » TNF, Withaferin A did not disrupt RIP1 polyubiquitination or NEMO–IKKβ interaction and was a poor direct IKKβ inhibitor, but prevented the formation of TNF-induced NEMO foci which colocalized with TNF ligand. While GFP-NEMO efficiently formed TNF-induced foci, a GFP-NEMO{sup Y308S} mutant that is defective in binding to polyubiquitin chains did not form foci. Our study reveals that Withaferin A is a novel type of IKK inhibitor which acts by disrupting NEMO reorganization into ubiquitin-based signaling structures in vivo. - Highlights: • Withaferin A, a NF-κB inhibitor, disrupts signaling induced NEMO localization, a novel point of inhibition. • NEMO can be localized to distinct signaling foci after treatment with TNF. • ABC-type DLCBL cells can be sensitized to apoptosis after treatment with Withaferin A.« less

Induction of lateral lumens through disruption of a monoleucine-based basolateral-sorting motif in betacellulin

PubMed Central

Singh, Bhuminder; Bogatcheva, Galina; Starchenko, Alina; Sinnaeve, Justine; Lapierre, Lynne A.; Williams, Janice A.; Goldenring, James R.; Coffey, Robert J.

2015-01-01

ABSTRACT Directed delivery of EGF receptor (EGFR) ligands to the apical or basolateral surface is a crucial regulatory step in the initiation of EGFR signaling in polarized epithelial cells. Herein, we show that the EGFR ligand betacellulin (BTC) is preferentially sorted to the basolateral surface of polarized MDCK cells. By using sequential truncations and site-directed mutagenesis within the BTC cytoplasmic domain, combined with selective cell-surface biotinylation and immunofluorescence, we have uncovered a monoleucine-based basolateral-sorting motif (EExxxL, specifically 156EEMETL161). Disruption of this sorting motif led to equivalent apical and basolateral localization of BTC. Unlike other EGFR ligands, BTC mistrafficking induced formation of lateral lumens in polarized MDCK cells, and this process was significantly attenuated by inhibition of EGFR. Additionally, expression of a cancer-associated somatic BTC mutation (E156K) led to BTC mistrafficking and induced lateral lumens in MDCK cells. Overexpression of BTC, especially mistrafficking forms, increased the growth of MDCK cells. These results uncover a unique role for BTC mistrafficking in promoting epithelial reorganization. PMID:26272915

Disruption of endolysosomal trafficking pathways in glioma cells by methuosis-inducing indole-based chalcones.

PubMed

Mbah, Nneka E; Overmeyer, Jean H; Maltese, William A

2017-06-01

Methuosis is a form of non-apoptotic cell death involving massive vacuolization of macropinosome-derived endocytic compartments, followed by a decline in metabolic activity and loss of membrane integrity. To explore the induction of methuosis as a potential therapeutic strategy for killing cancer cells, we have developed small molecules (indole-based chalcones) that trigger this form of cell death in glioblastoma and other cancer cell lines. Here, we report that in addition to causing fusion and expansion of macropinosome compartments, the lead compound, 3-(5-methoxy-2-methyl-1H-indol-3-yl)-1-(4-pyridinyl)-2-propen-1-one (MOMIPP), disrupts vesicular trafficking at the lysosomal nexus, manifested by impaired degradation of EGF and LDL receptors, defective processing of procathepsins, and accumulation of autophagosomes. In contrast, secretion of the ectodomain derived from a prototypical type-I membrane glycoprotein, β-amyloid precursor protein, is increased rather than diminished. A closely related MOMIPP analog, which causes substantial vacuolization without reducing cell viability, also impedes cathepsin processing and autophagic flux, but has more modest effects on receptor degradation. A third analog, which causes neither vacuolization nor loss of viability, has no effect on endolysosomal trafficking. The results suggest that differential cytotoxicity of structurally similar indole-based chalcones is related, at least in part, to the severity of their effects on endolysosomal trafficking pathways.

Inhibition of autophagy enhances DNA damage-induced apoptosis by disrupting CHK1-dependent S phase arrest

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

Liou, Jong-Shian; Wu, Yi-Chen; Yen, Wen-Yen

2014-08-01

DNA damage has been shown to induce autophagy, but the role of autophagy in the DNA damage response and cell fate is not fully understood. BO-1012, a bifunctional alkylating derivative of 3a-aza-cyclopenta[a]indene, is a potent DNA interstrand cross-linking agent with anticancer activity. In this study, BO-1012 was found to reduce DNA synthesis, inhibit S phase progression, and induce phosphorylation of histone H2AX on serine 139 (γH2AX) exclusively in S phase cells. Both CHK1 and CHK2 were phosphorylated in response to BO-1012 treatment, but only depletion of CHK1, but not CHK2, impaired BO-1012-induced S phase arrest and facilitated the entry ofmore » γH2AX-positive cells into G2 phase. CHK1 depletion also significantly enhanced BO-1012-induced cell death and apoptosis. These results indicate that BO-1012-induced S phase arrest is a CHK1-dependent pro-survival response. BO-1012 also resulted in marked induction of acidic vesicular organelle (AVO) formation and microtubule-associated protein 1 light chain 3 (LC3) processing and redistribution, features characteristic of autophagy. Depletion of ATG7 or co-treatment of cells with BO-1012 and either 3-methyladenine or bafilomycin A1, two inhibitors of autophagy, not only reduced CHK1 phosphorylation and disrupted S phase arrest, but also increased cleavage of caspase-9 and PARP, and cell death. These results suggest that cells initiate S phase arrest and autophagy as pro-survival responses to BO-1012-induced DNA damage, and that suppression of autophagy enhances BO-1012-induced apoptosis via disruption of CHK1-dependent S phase arrest. - Highlights: • Autophagy inhibitors enhanced the cytotoxicity of a DNA alkylating agent, BO-1012. • BO-1012-induced S phase arrest was a CHK1-dependent pro-survival response. • Autophagy inhibition enhanced BO-1012 cytotoxicity via disrupting the S phase arrest.« less

Causal evidence for retina dependent and independent visual motion computations in mouse cortex

PubMed Central

Hillier, Daniel; Fiscella, Michele; Drinnenberg, Antonia; Trenholm, Stuart; Rompani, Santiago B.; Raics, Zoltan; Katona, Gergely; Juettner, Josephine; Hierlemann, Andreas; Rozsa, Balazs; Roska, Botond

2017-01-01

How neuronal computations in the sensory periphery contribute to computations in the cortex is not well understood. We examined this question in the context of visual-motion processing in the retina and primary visual cortex (V1) of mice. We disrupted retinal direction selectivity – either exclusively along the horizontal axis using FRMD7 mutants or along all directions by ablating starburst amacrine cells – and monitored neuronal activity in layer 2/3 of V1 during stimulation with visual motion. In control mice, we found an overrepresentation of cortical cells preferring posterior visual motion, the dominant motion direction an animal experiences when it moves forward. In mice with disrupted retinal direction selectivity, the overrepresentation of posterior-motion-preferring cortical cells disappeared, and their response at higher stimulus speeds was reduced. This work reveals the existence of two functionally distinct, sensory-periphery-dependent and -independent computations of visual motion in the cortex. PMID:28530661

Observations on the effects of odours on the homeopathic response.

PubMed

McGuigan, Moira

2014-07-01

Samuel Hahnemann described incidences where the homeopathic response was disrupted by noxious smells in the environment. An earlier paper proposed that homeopathic medicines may be sensed by vomeronasal cells (VNCs) i.e. microvillus or brush cells in the vomeronasal organ (VNO), the taste buds and associated with the trigeminal nerve and nervus terminalis. This paper proposes an extension to the theory and suggests that a subset of solitary chemosensory cells (SCCs) in the diffuse chemosensory system (DCS) that is morphologically similar to VNCs might also be receptive to homeopathic medicines. The types of odours that may interfere with this process are described. Two clinical cases of disruption of the homeopathic response are given as examples, showing that successful re-establishment of remedy action can be produced by timely repetition of the medicine. The ramifications on clinical homeopathic practice are discussed. Copyright © 2014 The Faculty of Homeopathy. Published by Elsevier Ltd. All rights reserved.

An ultra scale-down approach to study the interaction of fermentation, homogenization, and centrifugation for antibody fragment recovery from rec E. coli.

PubMed

Li, Qiang; Mannall, Gareth J; Ali, Shaukat; Hoare, Mike

2013-08-01

Escherichia coli is frequently used as a microbial host to express recombinant proteins but it lacks the ability to secrete proteins into medium. One option for protein release is to use high-pressure homogenization followed by a centrifugation step to remove cell debris. While this does not give selective release of proteins in the periplasmic space, it does provide a robust process. An ultra scale-down (USD) approach based on focused acoustics is described to study rec E. coli cell disruption by high-pressure homogenization for recovery of an antibody fragment (Fab') and the impact of fermentation harvest time. This approach is followed by microwell-based USD centrifugation to study the removal of the resultant cell debris. Successful verification of this USD approach is achieved using pilot scale high-pressure homogenization and pilot scale, continuous flow, disc stack centrifugation comparing performance parameters such as the fraction of Fab' release, cell debris size distribution and the carryover of cell debris fine particles in the supernatant. The integration of fermentation and primary recovery stages is examined using USD monitoring of different phases of cell growth. Increasing susceptibility of the cells to disruption is observed with time following induction. For a given recovery process this results in a higher fraction of product release and a greater proportion of fine cell debris particles that are difficult to remove by centrifugation. Such observations are confirmed at pilot scale. Copyright © 2013 Wiley Periodicals, Inc.

Cigarette smoke disrupts the integrity of airway adherens junctions through the aberrant interaction of p120-catenin with the cytoplasmic tail of MUC1

PubMed Central

Zhang, Lili; Gallup, Marianne; Zlock, Lorna; Basbaum, Carol; Finkbeiner, Walter E.; McNamara, Nancy A.

2014-01-01

Adherens junctions (AJs) containing epithelial cadherin (E-cad) bound to p120-catenin (p120ctn) and β-catenin (β-ctn) play a crucial role in regulating cell–cell adhesion. Cigarette smoke abrogates cell–cell adhesion between epithelial cells by disrupting E-cad, a hallmark of epithelial–mesenchymal transition (EMT), yet the underlying mechanism remains unknown. We used an organotypic culture of primary human bronchial epithelial (HBE) cells treated with smoke-concentrated medium (Smk) to establish an essential role for the interaction between p120ctn and the cytoplasmic tail of MUC1 (MUC1-CT) in regulating E-cad disruption. Within the first 4 h of smoke exposure, apical MUC1-CT repositioned to the basolateral membrane of pseudo-stratified HBE cells, where it interacted with p120ctn. A time-dependent increase in MUC1-CT/p120ctn complexes occurred in conjunction with a time-dependent dissociation of p120ctn/E-cad/β-ctn complexes, as well as the coordinated degradation of p120ctn and E-cad. Interestingly, Smk induced a similar interaction between MUC1-CT and β-ctn, but this occurred 44 h after MUC1-CT’s initial interaction with p120ctn, and well after the AJs were destroyed. Blocking MUC1-CT’s interaction with p120ctn using a MUC1-CT dominant-negative peptide, PMIP, successfully abolished Smk’s disruptive effects on AJs and recovered apical-basolateral polarity of HBE cells. The MUC1-CT/p120ctn interaction was highly dependent on EGFR/Src/Jnk-mediated tyrosine phosphorylation (TyrP) of MUC1-CT. Accordingly, EGFR, Src or Jnk inhibitors (AG1478, PP2, SP600125, respectively) abrogated Smk-induced MUC1-CT-TyrP, MUC1-CT/p120ctn interaction, AJ disruption, and loss of cellular polarity. Our work identified MUC1-CT and p120ctn as important regulators of epithelial polarity and cell-cell adhesion during a smoke-induced EMT-like process. Novel therapeutics designed to inhibit MUC1-CT/p120ctn complex formation may prevent EMT in the smoker’s airway. PMID:22833523

Micro-tubular flame-assisted fuel cells for micro-combined heat and power systems

NASA Astrophysics Data System (ADS)

Milcarek, Ryan J.; Wang, Kang; Falkenstein-Smith, Ryan L.; Ahn, Jeongmin

2016-02-01

Currently the role of fuel cells in future power generation is being examined, tested and discussed. However, implementing systems is more difficult because of sealing challenges, slow start-up and complex thermal management and fuel processing. A novel furnace system with a flame-assisted fuel cell is proposed that combines the thermal management and fuel processing systems by utilizing fuel-rich combustion. In addition, the flame-assisted fuel cell furnace is a micro-combined heat and power system, which can produce electricity for homes or businesses, providing resilience during power disruption while still providing heat. A micro-tubular solid oxide fuel cell achieves a significant performance of 430 mW cm-2 operating in a model fuel-rich exhaust stream.

Plasma membrane repair in plants.

PubMed

Schapire, Arnaldo L; Valpuesta, Victoriano; Botella, Miguel A

2009-12-01

Resealing is the membrane-repair process that enables cells to survive disruption, preventing the loss of irreplaceable cell types and eliminating the cost of replacing injured cells. Given that failure in the resealing process in animal cells causes diverse types of muscular dystrophy, plasma membrane repair has been extensively studied in these systems. Animal proteins with Ca(2+)-binding domains such as synaptotagmins and dysferlin mediate Ca(2+)-dependent exocytosis to repair plasma membranes after mechanical damage. Until recently, no components or proof for membrane repair mechanisms have been discovered in plants. However, Arabidopsis SYT1 is now the first plant synaptotagmin demonstrated to participate in Ca(2+)-dependent repair of membranes. This suggests a conservation of membrane repair mechanisms between animal and plant cells.

Airway epithelial homeostasis and planar cell polarity signaling depend on multiciliated cell differentiation.

PubMed

Vladar, Eszter K; Nayak, Jayakar V; Milla, Carlos E; Axelrod, Jeffrey D

2016-08-18

Motile airway cilia that propel contaminants out of the lung are oriented in a common direction by planar cell polarity (PCP) signaling, which localizes PCP protein complexes to opposite cell sides throughout the epithelium to orient cytoskeletal remodeling. In airway epithelia, PCP is determined in a 2-phase process. First, cell-cell communication via PCP complexes polarizes all cells with respect to the proximal-distal tissue axis. Second, during ciliogenesis, multiciliated cells (MCCs) undergo cytoskeletal remodeling to orient their cilia in the proximal direction. The second phase not only directs cilium polarization, but also consolidates polarization across the epithelium. Here, we demonstrate that in airway epithelia, PCP depends on MCC differentiation. PCP mutant epithelia have misaligned cilia, and also display defective barrier function and regeneration, indicating that PCP regulates multiple aspects of airway epithelial homeostasis. In humans, MCCs are often sparse in chronic inflammatory diseases, and these airways exhibit PCP dysfunction. The presence of insufficient MCCs impairs mucociliary clearance in part by disrupting PCP-driven polarization of the epithelium. Consistent with defective PCP, barrier function and regeneration are also disrupted. Pharmacological stimulation of MCC differentiation restores PCP and reverses these defects, suggesting its potential for broad therapeutic benefit in chronic inflammatory disease.

Myeloma cell-induced disruption of bone remodelling compartments leads to osteolytic lesions and generation of osteoclast-myeloma hybrid cells.

PubMed

Andersen, Thomas L; Søe, Kent; Sondergaard, Teis E; Plesner, Torben; Delaisse, Jean-Marie

2010-02-01

Osteolytic lesions are a hallmark of multiple myeloma. They are due to the hyperactivity of bone resorbing osteoclasts and hypoactivity of bone forming osteoblasts, in response to neighbouring myeloma cells. This study identified a structure that deeply affects this response, because of its impact on the physical organisation of the myeloma cell microenvironment. The proximity between myeloma cells and osteoclasts or osteoblasts was shown to be conditioned by the recently discovered layer of flat cells that separates the osteoclasts and osteoblasts from the bone marrow, by forming a canopy over bone remodelling compartment (BRC). These canopies are frequently disrupted in myeloma, and this disruption correlates with increased proximity and density of myeloma cells. In vitro evidence indicates that this disruption may be due to direct contact between myeloma and BRC canopy cells. Importantly, this disruption and increased proximity and density of myeloma cells coincides with key myeloma-induced bone events, such as osteolytic lesions, impaired bone formation despite increased bone resorption, and fusion of myeloma cells with osteoclasts thereby forming myeloma-osteoclast hybrid cells. These findings strongly support a critical role of BRC canopies in myeloma-induced bone disease. BRC canopies could therefore be considered as a new therapeutic target.

PRODUCTION OF IMMUNOLOGICAL TOLERANCE IN MICE AFTER REPEATED INJECTIONS OF DISRUPTED SPLEEN CELLS

PubMed Central

Martinez, C.; Smith, J. M.; Blaese, M.; Good, R. A.

1963-01-01

1. Tolerance of male skin isografts has been regularly produced in female mice of the C57B1 strain sublines 1, 4, and 6 during adult life by repeated injection of completely disrupted spleen cells derived from male donors. The tolerant state is long-lasting since such grafts have remained in place more than 9 months. 2. Prolonged survival of homotransplants of skin has regularly been produced in DBA/2 mice during adult life by repeated injections of completely disrupted spleen cells from Balb/C donors. When injections of disrupted spleen cell material are continued over a sufficiently long period, permanent acceptance of the skin homografts may be obtained between these strains. 3. Immunological tolerance across even the strong H-2 histocompatibility barrier was obtained in the neonatal period and during adult life by repeated injection of disrupted spleen cell preparations. The tolerant state has been revealed by both mammary adenocarcinoma and skin homografting across this strong histocompatibility barrier. 4. In contradistinction to the tolerant state produced by injection of intact spleen cells in neonatal animals or during adult life or that produced by parabiotic union, the tolerance produced by repeated injection of disrupted spleen cell preparations cannot be transferred to syngenic neonatal mice with spleen cells of the tolerant animal. 5. The implications of these findings in transplantation biology and in consideration of the basic nature of tolerance are discussed. PMID:14087619

The burden of trisomy 21 disrupts the proteostasis network in Down syndrome

PubMed Central

Rauniyar, Abhishek K.; Jiang, Hua; Liggett, L. Alexander; Maclean, Kenneth N.

2017-01-01

Down syndrome (DS) is a genetic disorder caused by trisomy of chromosome 21. Abnormalities in chromosome number have the potential to lead to disruption of the proteostasis network (PN) and accumulation of misfolded proteins. DS individuals suffer from several comorbidities, and we hypothesized that disruption of proteostasis could contribute to the observed pathology and decreased cell viability in DS. Our results confirm the presence of a disrupted PN in DS, as several of its elements, including the unfolded protein response, chaperone system, and proteasomal degradation exhibited significant alterations compared to euploid controls in both cell and mouse models. Additionally, when cell models were treated with compounds that promote disrupted proteostasis, we observed diminished levels of cell viability in DS compared to controls. Collectively our findings provide a cellular-level characterization of PN dysfunction in DS and an improved understanding of the potential pathogenic mechanisms contributing to disrupted cellular physiology in DS. Lastly, this study highlights the future potential of designing therapeutic strategies that mitigate protein quality control dysfunction. PMID:28430800

Rapid Actions of Xenoestrogens Disrupt Normal Estrogenic Signaling

PubMed Central

Watson, Cheryl S.; Hu, Guangzhen; Paulucci-Holthauzen, Adriana A.

2014-01-01

Some chemicals used in consumer products or manufacturing (eg. plastics, surfactants, pesticides, resins) have estrogenic activities; these xenoestrogens (XEs) chemically resemble physiological estrogens and are one of the major categories of synthesized compounds that disrupt endocrine actions. Potent rapid actions of XEs via nongenomic mechanisms contribute significantly to their disruptive effects on functional endpoints (eg. cell proliferation/death, transport, peptide release). Membrane-initiated hormonal signaling in our pituitary cell model is predominantly driven by mERα with mERβ and GPR30 participation. We visualized ERα on plasma membranes using many techniques in the past (impeded ligands, antibodies to ERα ) and now add observations of epitope proximity with other membrane signaling proteins. We have demonstrated a range of rapid signals/protein activations by XEs including: calcium channels, cAMP/PKA, MAPKs, G proteins, caspases, and transcription factors. XEs can cause disruptions of the oscillating temporal patterns of nongenomic signaling elicited by endogenous estrogens. Concentration effects of XEs are nonmonotonic (a trait shared with natural hormones), making it difficult to design efficient (single concentration) toxicology tests to monitor their harmful effects. A plastics monomer, Bisphenol A, modified by waste treatment (chlorination) and other processes causes dephosphorylation of extracellular-regulated kinases, in contrast to having no effects as it does in genomic signaling. Mixtures of XEs, commonly found in contaminated environments, disrupt the signaling actions of physiological estrogens even more severely than do single XEs. Understanding the features of XEs that drive these disruptive mechanisms will allow us to redesign useful chemicals that exclude estrogenic or anti-estrogenic activities. PMID:24269739

Membrane raft organization is more sensitive to disruption by (n-3) PUFA than nonraft organization in EL4 and B cells.

PubMed

Rockett, Benjamin Drew; Franklin, Andrew; Harris, Mitchel; Teague, Heather; Rockett, Alexis; Shaikh, Saame Raza

2011-06-01

Model membrane and cellular detergent extraction studies show (n-3) PUFA predominately incorporate into nonrafts; thus, we hypothesized (n-3) PUFA could disrupt nonraft organization. The first objective of this study was to determine whether (n-3) PUFA disrupted nonrafts of EL4 cells, an extension of our previous work in which we discovered an (n-3) PUFA diminished raft clustering. EPA or DHA treatment of EL4 cells increased plasma membrane accumulation of the nonraft probe 1,1'-dilinoleyl-3,3,3',3'-tetramethylindocarbocyanine perchlorate by ~50-70% relative to a BSA control. Förster resonance energy transfer imaging showed EPA and DHA also disrupted EL4 nanometer scale nonraft organization by increasing the distance between nonraft molecules by ~25% compared with BSA. However, changes in nonrafts were due to an increase in cell size; under conditions where EPA or DHA did not increase cell size, nonraft organization was unaffected. We next translated findings on EL4 cells by testing if (n-3) PUFA administered to mice disrupted nonrafts and rafts. Imaging of B cells isolated from mice fed low- or high-fat (HF) (n-3) PUFA diets showed no change in nonraft organization compared with a control diet (CD). However, confocal microscopy revealed the HF (n-3) PUFA diet disrupted lipid raft clustering and size by ~40% relative to CD. Taken together, our data from 2 different model systems suggest (n-3) PUFA have limited effects on nonrafts. The ex vivo data, which confirm previous studies with EL4 cells, provide evidence that (n-3) PUFA consumed through the diet disrupt B cell lipid raft clustering.

Membrane Raft Organization Is More Sensitive to Disruption by (n-3) PUFA Than Nonraft Organization in EL4 and B Cells123

PubMed Central

Rockett, Benjamin Drew; Franklin, Andrew; Harris, Mitchel; Teague, Heather; Rockett, Alexis; Shaikh, Saame Raza

2011-01-01

Model membrane and cellular detergent extraction studies show (n-3) PUFA predominately incorporate into nonrafts; thus, we hypothesized (n-3) PUFA could disrupt nonraft organization. The first objective of this study was to determine whether (n-3) PUFA disrupted nonrafts of EL4 cells, an extension of our previous work in which we discovered an (n-3) PUFA diminished raft clustering. EPA or DHA treatment of EL4 cells increased plasma membrane accumulation of the nonraft probe 1,1′-dilinoleyl-3,3,3′,3′-tetramethylindocarbocyanine perchlorate by ~50–70% relative to a BSA control. Förster resonance energy transfer imaging showed EPA and DHA also disrupted EL4 nanometer scale nonraft organization by increasing the distance between nonraft molecules by ~25% compared with BSA. However, changes in nonrafts were due to an increase in cell size; under conditions where EPA or DHA did not increase cell size, nonraft organization was unaffected. We next translated findings on EL4 cells by testing if (n-3) PUFA administered to mice disrupted nonrafts and rafts. Imaging of B cells isolated from mice fed low- or high-fat (HF) (n-3) PUFA diets showed no change in nonraft organization compared with a control diet (CD). However, confocal microscopy revealed the HF (n-3) PUFA diet disrupted lipid raft clustering and size by ~40% relative to CD. Taken together, our data from 2 different model systems suggest (n-3) PUFA have limited effects on nonrafts. The ex vivo data, which confirm previous studies with EL4 cells, provide evidence that (n-3) PUFA consumed through the diet disrupt B cell lipid raft clustering. PMID:21525263

Nuclear envelope disruption involving host caspases plays a role in the parvovirus replication cycle.

PubMed

Cohen, Sarah; Marr, Alexandra K; Garcin, Pierre; Panté, Nelly

2011-05-01

Parvoviruses are small, nonenveloped, single-stranded DNA viruses which replicate in the nucleus of the host cell. We have previously found that early during infection the parvovirus minute virus of mice (MVM) causes small, transient disruptions of the nuclear envelope (NE). We have now investigated the mechanism used by MVM to disrupt the NE. Here we show that the viral phospholipase A2, the only known enzymatic domain on the parvovirus capsid, is not involved in causing NE disruption. Instead, the virus utilizes host cell caspases, which are proteases involved in causing NE breakdown during apoptosis, to facilitate these nuclear membrane disruptions. Studies with pharmacological inhibitors indicate that caspase-3 in particular is involved. A caspase-3 inhibitor prevents nuclear lamin cleavage and NE disruption in MVM-infected mouse fibroblast cells and reduces nuclear entry of MVM capsids and viral gene expression. Caspase-3 is, however, not activated above basal levels in MVM-infected cells, and other aspects of apoptosis are not triggered during early MVM infection. Instead, basally active caspase-3 is relocalized to the nuclei of infected cells. We propose that NE disruption involving caspases plays a role in (i) parvovirus entry into the nucleus and (ii) alteration of the compartmentalization of host proteins in a way that is favorable for the virus.

The Emerging Role of Zinc in the Pathogenesis of Multiple Sclerosis.

PubMed

Choi, Bo Young; Jung, Jong Won; Suh, Sang Won

2017-09-28

Our lab has previously demonstrated that multiple sclerosis-induced spinal cord white matter damage and motor deficits are mediated by the pathological disruption of zinc homeostasis. Abnormal vesicular zinc release and intracellular zinc accumulation may mediate several steps in the pathophysiological processes of multiple sclerosis (MS), such as matrix metallopeptidase 9 (MMP-9) activation, blood-brain barrier (BBB) disruption, and subsequent immune cell infiltration from peripheral systems. Oral administration of a zinc chelator decreased BBB disruption, immune cell infiltration, and spinal white matter myelin destruction. Therefore, we hypothesized that zinc released into the extracellular space during MS progression is involved in destruction of the myelin sheath in spinal cord white mater and in generation of motor deficits. To confirm our previous study, we employed zinc transporter 3 ( ZnT3 ) knockout mice to test whether vesicular zinc depletion shows protective effects on multiple sclerosis-induced white matter damage and motor deficits. ZnT3 gene deletion profoundly reduced the daily clinical score of experimental autoimmune encephalomyelitis (EAE) by suppression of inflammation and demyelination in the spinal cord. ZnT3 gene deletion also remarkably inhibited formation of multiple sclerosis-associated aberrant synaptic zinc patches, MMP-9 activation, and BBB disruption. These two studies strongly support our hypothesis that zinc release from presynaptic terminals may be involved in multiple sclerosis pathogenesis. Further studies will no doubt continue to add mechanistic detail to this process and with luck, clarify how these observations may lead to development of novel therapeutic approaches for the treatment of multiple sclerosis.

Skeletal Collagen Turnover by the Osteoblast

NASA Technical Reports Server (NTRS)

Partridge, Nicola C.

1997-01-01

Among the most overt negative changes experienced by man and experimental animals under conditions of weightlessness are the loss of skeletal mass and attendant hypercalciuria. These clearly result from some disruption in the balance between bone formation and bone resorption (i.e. remodelling) which appears to be due to a decrease in the functions of the osteoblast. In the studies funded by this project, the clonal osteoblastic cell line, UMR 106-01, has been used to investigate the regulation of collagenase and Tissue Inhibitors of MetalloProteases (TIMPs). This project has shed light on the comprehensive role of the osteoblast in the remodelling process, and, in so doing, provided some insight into how the process might be disrupted under conditions of microgravity.

Second International Conference on Accelerating Biopharmaceutical Development

PubMed Central

2009-01-01

The Second International Conference on Accelerating Biopharmaceutical Development was held in Coronado, California. The meeting was organized by the Society for Biological Engineering (SBE) and the American Institute of Chemical Engineers (AIChE); SBE is a technological community of the AIChE. Bob Adamson (Wyeth) and Chuck Goochee (Centocor) were co-chairs of the event, which had the theme “Delivering cost-effective, robust processes and methods quickly and efficiently.” The first day focused on emerging disruptive technologies and cutting-edge analytical techniques. Day two featured presentations on accelerated cell culture process development, critical quality attributes, specifications and comparability, and high throughput protein formulation development. The final day was dedicated to discussion of technology options and new analysis methods provided by emerging disruptive technologies; functional interaction, integration and synergy in platform development; and rapid and economic purification process development. PMID:20065637

Focused ultrasound-mediated bbb disruption is associated with an increase in activation of AKT: experimental study in rats

PubMed Central

2010-01-01

Background The Blood Brain Barrier (BBB) maintains the homeostasis of central nervous system by preventing the free passage of macromolecules from the systemic circulation into the brain. This normal physiological function of the BBB presents a challenge for delivery of therapeutic compounds into the brain. Recent studies have shown that the application of focused ultrasound together with ultrasound contrast agent (microbubbles) temporarily increases the permeability of the BBB. This effect is associated with breakdown of tight junctions, the structures that regulate the paracellular permeability of the endothelial cell layer. The influence of this ultrasound effect on the activation of intracellular signaling proteins is currently not well understood. Therefore, the aim of this study was to investigate the activation of cell survival signaling molecules in response to ultrasound-mediated BBB opening; Methods The BBB was disrupted in two four-spot lines (1-1.5 mm spacing) along the right hemisphere of rat brain with ultrasound beams (0.3 MPa, 120 s, 10 ms bursts, repetition frequency = 1 Hz) in the presence Definity microbubbles. Contrast-enhanced MRI images were acquired to assess the extent of BBB opening upon which the animals were sacrificed and the brains removed and processed for biochemical and immunohistochemical analyses; Results Immunoblotting of sonicated brain lysates resolved by SDS-PAGE demonstrated an increase in phosphorylation of Akt and its downstream signaling molecule, GSK3β, while the phosphorylation of MAPK remained unchanged. The elevated levels of pAkt and pGSK3β are still evident after 24 hours post-sonication, a time point where the integrity of the BBB is known to be re-established. Furthermore, immunofluoresence staining localized this increase in pAkt and pGSK3β levels to neuronal cells flanking the region of the disrupted BBB; Conclusions Our data demonstrates that ultrasound-mediated BBB disruption causes an activation of the Akt signaling pathway in neuronal cells surrounding the disrupted BBB. PMID:21078165

Keeping Signals Straight: How Cells Process Information and Make Decisions

PubMed Central

Laub, Michael T.

2016-01-01

As we become increasingly dependent on electronic information-processing systems at home and work, it’s easy to lose sight of the fact that our very survival depends on highly complex biological information-processing systems. Each of the trillions of cells that form the human body has the ability to detect and respond to a wide range of stimuli and inputs, using an extraordinary set of signaling proteins to process this information and make decisions accordingly. Indeed, cells in all organisms rely on these signaling proteins to survive and proliferate in unpredictable and sometimes rapidly changing environments. But how exactly do these proteins relay information within cells, and how do they keep a multitude of incoming signals straight? Here, I describe recent efforts to understand the fidelity of information flow inside cells. This work is providing fundamental insight into how cells function. Additionally, it may lead to the design of novel antibiotics that disrupt the signaling of pathogenic bacteria or it could help to guide the treatment of cancer, which often involves information-processing gone awry inside human cells. PMID:27427909

Disruption of endothelial adherens junction by invasive breast cancer cells is mediated by reactive oxygen species and is attenuated by AHCC.

PubMed

Haidari, Mehran; Zhang, Wei; Wakame, Koji

2013-12-18

The effect of antioxidants on treatment of cancer is still controversial. Previously, we demonstrated that interaction of breast cancer cells with endothelial cells leads to tyrosine phosphorylation of VE-cadherin and disruption of endothelial adherens junction (EAJ). The molecular mechanism underlying the anti-metastatic effects of mushroom-derived active hexode correlated compound (AHCC) remains elusive. Several cellular and biochemical techniques were used to determine the contribution of oxidative stress in the disruption of EAJ and to test this hypothesis that AHCC inhibits the breast cancer cell-induced disruption of EAJ. Interaction of breast cancer cells (MDA-MB-231 cells) with human umbilical vein endothelial cells (HUVECs) leads to an increase in generation of reactive oxygen species (ROS). Treatment of HUVECs with H2O2 or phorbol myristate acetate (PMA) led to tyrosine phosphorylation of VE-cadherin, dissociation of β-catenin from VE-cadherin complex and increased transendothelial migration (TEM) of MDA-MB-231 cells. Induction of VE-cadherin tyrosine phosphorylation by PMA or by interaction of MDA-MB-231 cells with HUVECs was mediated by HRas and protein kinase C-α signaling pathways. Disruption of EAJ and phosphorylation of VE-cadherin induced by interaction of MDA-MB-231 cells with HUVECs were attenuated when HUVECs were pretreated with an antioxidant, N-acetylcysteine (NAC) or AHCC. AHCC inhibited TEM of MDA-MB-231 cells and generation of ROS induced by interaction of MDA-MB-231 cells with HUVECs. Our studies suggest that ROS contributes to disruption of EAJ induced by interaction of MDA-MB-231 cells with HUVECs and AHCC attenuates this alteration. Copyright © 2013 Elsevier Inc. All rights reserved.

Arsenic as an endocrine disruptor: arsenic disrupts retinoic acid receptor-and thyroid hormone receptor-mediated gene regulation and thyroid hormone-mediated amphibian tail metamorphosis.

PubMed

Davey, Jennifer C; Nomikos, Athena P; Wungjiranirun, Manida; Sherman, Jenna R; Ingram, Liam; Batki, Cavus; Lariviere, Jean P; Hamilton, Joshua W

2008-02-01

Chronic exposure to excess arsenic in drinking water has been strongly associated with increased risks of multiple cancers, diabetes, heart disease, and reproductive and developmental problems in humans. We previously demonstrated that As, a potent endocrine disruptor at low, environmentally relevant levels, alters steroid signaling at the level of receptor-mediated gene regulation for all five steroid receptors. The goal of this study was to determine whether As can also disrupt gene regulation via the retinoic acid (RA) receptor (RAR) and/or the thyroid hormone (TH) receptor (TR) and whether these effects are similar to previously observed effects on steroid regulation. Human embryonic NT2 or rat pituitary GH3 cells were treated with 0.01-5 microM sodium arsenite for 24 hr, with or without RA or TH, respectively, to examine effects of As on receptor-mediated gene transcription. At low, noncytotoxic doses, As significantly altered RAR-dependent gene transcription of a transfected RAR response element-luciferase construct and the native RA-inducible cytochrome P450 CYP26A gene in NT2 cells. Likewise, low-dose As significantly altered expression of a transfected TR response element-luciferase construct and the endogenous TR-regulated type I deiodinase (DIO1) gene in a similar manner in GH3 cells. An amphibian ex vivo tail metamorphosis assay was used to examine whether endocrine disruption by low-dose As could have specific pathophysiologic consequences, because tail metamorphosis is tightly controlled by TH through TR. TH-dependent tail shrinkage was inhibited in a dose-dependent manner by 0.1- 4.0 microM As. As had similar effects on RAR- and TR-mediated gene regulation as those previously observed for the steroid receptors, suggesting a common mechanism or action. Arsenic also profoundly affected a TR-dependent developmental process in a model animal system at very low concentrations. Because RAR and TH are critical for both normal human development and adult function and their dysregulation is associated with many disease processes, disruption of these hormone receptor-dependent processes by As is also potentially relevant to human developmental problems and disease risk.

Epithelin/Granulin Precursor Expression in Human Breast Carcinoma

DTIC Science & Technology

1998-09-01

antisense RNA as an inhibitor of oncogenic protein production (13). The development of stable transfected clones with antisense cDNA is advantageous...in that it allows a continuous supply of antisense RNA to disrupt protein synthesis, and it is well suited for in vivo tumorigenic assays. Our...processed form epithelin 1 in normal mammary epithelial cells and mammary carcinoma cells. 3- Effect of inhibition of PCDGF expression ( antisense

Actin cables and the exocyst form two independent morphogenesis pathways in the fission yeast

PubMed Central

Bendezú, Felipe O.; Martin, Sophie G.

2011-01-01

Cell morphogenesis depends on polarized exocytosis. One widely held model posits that long-range transport and exocyst-dependent tethering of exocytic vesicles at the plasma membrane sequentially drive this process. Here, we describe that disruption of either actin-based long-range transport and microtubules or the exocyst did not abolish polarized growth in rod-shaped fission yeast cells. However, disruption of both actin cables and exocyst led to isotropic growth. Exocytic vesicles localized to cell tips in single mutants but were dispersed in double mutants. In contrast, a marker for active Cdc42, a major polarity landmark, localized to discreet cortical sites even in double mutants. Localization and photobleaching studies show that the exocyst subunits Sec6 and Sec8 localize to cell tips largely independently of the actin cytoskeleton, but in a cdc42 and phospholipid phosphatidylinositol 4,5-bisphosphate (PIP2)–dependent manner. Thus in fission yeast long-range cytoskeletal transport and PIP2-dependent exocyst represent parallel morphogenetic modules downstream of Cdc42, raising the possibility of similar mechanisms in other cell types. PMID:21148300

Copy number determination of genetically-modified hematopoietic stem cells.

PubMed

Schuesler, Todd; Reeves, Lilith; Kalle, Christof von; Grassman, Elke

2009-01-01

Human gene transfer with gammaretroviral, murine leukemia virus (MLV) based vectors has been shown to effectively insert and express transgene sequences at a level of therapeutic benefit. However, there are numerous reports of disruption of the normal cellular processes caused by the viral insertion, even of replication deficient gammaretroviral vectors. Current gammaretroviral and lentiviral vectors do not control the site of insertion into the genome, hence, the possibility of disruption of the target cell genome. Risk related to viral insertions is linked to the number of insertions of the transgene into the cellular DNA, as has been demonstrated for replication competent and replication deficient retroviruses in experiments. At high number of insertions per cell, cell transformation due to vector induced activation of proto-oncogenes is more likely to occur, in particular since more than one transforming event is needed for oncogenesis. Thus, determination of the vector copy number in bulk transduced populations, individual colony forming units, and tissue from the recipient of the transduced cells is an increasingly important safety assay and has become a standard, though not straightforward assay, since the inception of quantitative PCR.

Replication-mediated disassociation of replication protein A-XPA complex upon DNA damage: implications for RPA handing off.

PubMed

Jiang, Gaofeng; Zou, Yue; Wu, Xiaoming

2012-08-01

RPA (replication protein A), the eukaryotic ssDNA (single-stranded DNA)-binding protein, participates in most cellular processes in response to genotoxic insults, such as NER (nucleotide excision repair), DNA, DSB (double-strand break) repair and activation of cell cycle checkpoint signalling. RPA interacts with XPA (xeroderma pigmentosum A) and functions in early stage of NER. We have shown that in cells the RPA-XPA complex disassociated upon exposure of cells to high dose of UV irradiation. The dissociation required replication stress and was partially attributed to tRPA hyperphosphorylation. Treatment of cells with CPT (camptothecin) and HU (hydroxyurea), which cause DSB DNA damage and replication fork collapse respectively and also leads to the disruption of RPA-XPA complex. Purified RPA and XPA were unable to form complex in vitro in the presence of ssDNA. We propose that the competition-based RPA switch among different DNA metabolic pathways regulates the dissociation of RPA with XPA in cells after DNA damage. The biological significances of RPA-XPA complex disruption in relation with checkpoint activation, DSB repair and RPA hyperphosphorylation are discussed.

Bad seeds produce bad crops: a single stage-process of prostate tumor invasion

PubMed Central

Man, Yan-gao; Gardner, William A.

2008-01-01

It is a commonly held belief that prostate carcinogenesis is a multi-stage process and that tumor invasion is triggered by the overproduction of proteolytic enzymes. This belief is consistent with data from cell cultures and animal models, whereas is hard to interpret several critical facts, including the presence of cancer in “healthy” young men and cancer DNA phenotype in morphologically normal prostate tissues. These facts argue that alternative pathways may exist for prostate tumor invasion in some cases. Since degradation of the basal cell layer is the most distinct sign of invasion, our recent studies have attempted to identify pre-invasive lesions with focal basal cell layer alterations. Our studies revealed that about 30% of prostate cancer patients harbored normal appearing duct or acinar clusters with a high frequency of focal basal cell layer disruptions. These focally disrupted basal cell layers had significantly reduced cell proliferation and tumor suppressor expression, whereas significantly elevated degeneration, apoptosis, and infiltration of immunoreactive cells. In sharp contrast, associated epithelial cell had significantly elevated proliferation, expression of malignancy-signature markers, and physical continuity with invasive lesions. Based on these and other findings, we have proposed that these normal appearing duct or acinar clusters are derived from monoclonal proliferation of genetically damaged stem cells and could progress directly to invasion through two pathways: 1) clonal in situ transformation (CIST) and 2) multi-potential progenitor mediated “budding” (MPMB). These pathways may contribute to early onset of prostate cancer at young ages, and to clinically more aggressive prostate tumors. PMID:18725981

Extracellular Vesicles Produced by the Gram-positive Bacterium Bacillus subtilis are Disrupted by the Lipopeptide Surfactin

PubMed Central

Brown, Lisa; Kessler, Anne; Cabezas-Sanchez, Pablo; Luque-Garcia, Jose L.; Casadevall, Arturo

2014-01-01

Summary Previously, extracellular vesicle production in Gram-positive bacteria was dismissed due to the absence of an outer membrane, where Gram-negative vesicles originate, and the difficulty in envisioning how such a process could occur through the cell wall. However, recent work has shown that Gram-positive bacteria produce extracellular vesicles and that the vesicles are biologically active. In this study, we show that Bacillus subtilis produces extracellular vesicles similar in size and morphology to other bacteria, characterized vesicles using a variety of techniques, provide evidence that these vesicles are actively produced by cells, show differences in vesicle production between strains, and identified a mechanism for such differences based on vesicle disruption. We found that in wild strains of B. subtilis, surfactin disrupted vesicles while in laboratory strains harboring a mutation in the gene sfp, vesicles accumulated in the culture supernatant. Surfactin not only lysed B. subtilis vesicles, but also vesicles from Bacillus anthracis, indicating a mechanism that crossed species boundaries. To our knowledge, this is the first time a gene and a mechanism has been identified in the active disruption of extracellular vesicles and subsequent release of vesicular cargo in Gram-positive bacteria. We also identify a new mechanism of action for surfactin. PMID:24826903

The Dynamic Duo–Inflammatory M1 macrophages and Th17 cells in Rheumatic Diseases

PubMed Central

Li, Jun; Hsu, Hui-Chen; Mountz, John D.

2014-01-01

The synovial tissue of Rheumatoid Arthritis (RA) patients is enriched with macrophages and T lymphocytes which are two central players in the pathogenesis of RA. Interaction between myeloid cells and T cells are essential for the initiation and progression of the inflammatory processes in the synovium. With the rapid evolution of our understanding of how these two cell types are involved in the regulation of immune responses, RA is emerging as an ideal disease model for investigating the cell-cell interactions and consequently introducing novel biologic agents that are designed to disrupt these processes. This review will discuss the bidirectional interaction between the IL-23+ inflammatory macrophages and IL-17+ GM-CSF+ CD4 T cells in rheumatic diseases as well as potential antirheumatic strategies via apoptosis induction in this context. PMID:25309946

Disruption of canonical TGFβ-signaling in murine coronary progenitor cells by low level arsenic

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

Allison, Patrick; Huang, Tianfang; Broka, Derrick

2013-10-01

Exposure to arsenic results in several types of cancers as well as heart disease. A major contributor to ischemic heart pathologies is coronary artery disease, however the influences by environmental arsenic in this disease process are not known. Similarly, the impact of toxicants on blood vessel formation and function during development has not been studied. During embryogenesis, the epicardium undergoes proliferation, migration, and differentiation into several cardiac cell types including smooth muscle cells which contribute to the coronary vessels. The TGFβ family of ligands and receptors is essential for developmental cardiac epithelial to mesenchymal transition (EMT) and differentiation into coronarymore » smooth muscle cells. In this in vitro study, 18 hour exposure to 1.34 μM arsenite disrupted developmental EMT programming in murine epicardial cells causing a deficit in cardiac mesenchyme. The expression of EMT genes including TGFβ2, TGFβ receptor-3, Snail, and Has-2 are decreased in a dose-dependent manner following exposure to arsenite. TGFβ2 cell signaling is abrogated as detected by decreases in phosphorylated Smad2/3 when cells are exposed to 1.34 μM arsenite. There is also loss of nuclear accumulation pSmad due to arsenite exposure. These observations coincide with a decrease in vimentin positive mesenchymal cells invading three-dimensional collagen gels. However, arsenite does not block TGFβ2 mediated smooth muscle cell differentiation by epicardial cells. Overall these results show that arsenic exposure blocks developmental EMT gene programming in murine coronary progenitor cells by disrupting TGFβ2 signals and Smad activation, and that smooth muscle cell differentiation is refractory to this arsenic toxicity. - Highlights: • Arsenic blocks TGFβ2 induced expression of EMT genes. • Arsenic blocks TGFβ2 triggered Smad2/3 phosphorylation and nuclear translocation. • Arsenic blocks epicardial cell differentiation into cardiac mesenchyme. • Arsenic does not block TGFβ2 induced smooth muscle cell differentiation.« less

CD94/NKG2A inhibits NK cell activation by disrupting the actin network at the immunological synapse.

PubMed

Masilamani, Madhan; Nguyen, Connie; Kabat, Juraj; Borrego, Francisco; Coligan, John E

2006-09-15

An adequate immune response is the result of the fine balance between activation and inhibitory signals. The exact means by which inhibitory signals obviate activation signals in immune cells are not totally elucidated. Human CD94/NKG2A is an ITIM-containing inhibitory receptor expressed by NK cells and some CD8+ T cells that recognize HLA-E. We show that the engagement of this receptor prevents NK cell activation by disruption of the actin network and exclusion of lipid rafts at the point of contact with its ligand (inhibitory NK cell immunological synapse, iNKIS). CD94/NKG2A engagement leads to recruitment and activation of src homology 2 domain-bearing tyrosine phosphatase 1. This likely explains the observed dephosphorylation of guanine nucleotide exchange factor and regulator of actin, Vav1, as well as ezrin-radixin-moesin proteins that connect actin filaments to membrane structures. In contrast, NK cell activation by NKG2D induced Vav1 and ezrin-radixin-moesin phosphorylation. Thus, CD94/NKG2A prevents actin-dependent recruitment of raft-associated activation receptors complexes to the activating synapse. This was further substantiated by showing that inhibition of actin polymerization abolished lipid rafts exclusion at the iNKIS, whereas cholesterol depletion had no effect on actin disruption at the iNKIS. These data indicate that the lipid rafts exclusion at the iNKIS is an active process which requires an intact cytoskeleton to maintain lipid rafts outside the inhibitory synapse. The net effect is to maintain an inhibitory state in the proximity of the iNKIS, while allowing the formation of activation synapse at distal points within the same NK cell.

Method for facilitating the introduction of material into cells

DOEpatents

Holcomb, David E.; McKnight, Timothy E.

2000-01-01

The present invention is a method for creating a localized disruption within a boundary of a cell or structure by exposing a boundary of a cell or structure to a set of energetically charged particles while regulating the energy of the charged particles so that the charged particles have an amount of kinetic energy sufficient to create a localized disruption within an area of the boundary of the cell or structure, then upon creation of the localized disruption, the amount of kinetic energy decreases to an amount insufficient to create further damage within the cell or structure beyond the boundary. The present invention is also a method for facilitating the introduction of a material into a cell or structure using the same methodology then further exciting the area of the boundary of the cell or structure where the localized disruption was created so to create a localized temporary opening within the boundary then further introducing the material through the temporary opening into the cell or structure.

The Anti-Inflammatory Activity of Curcumin Protects the Genital Mucosal Epithelial Barrier from Disruption and Blocks Replication of HIV-1 and HSV-2

PubMed Central

Ferreira, Victor H.; Mueller, Kristen; Kaushic, Charu

2015-01-01

Inflammation is a known mechanism that facilitates HIV acquisition and the spread of infection. In this study, we evaluated whether curcumin, a potent and safe anti-inflammatory compound, could be used to abrogate inflammatory processes that facilitate HIV-1 acquisition in the female genital tract (FGT) and contribute to HIV amplification. Primary, human genital epithelial cells (GECs) were pretreated with curcumin and exposed to HIV-1 or HIV glycoprotein 120 (gp120), both of which have been shown to disrupt epithelial tight junction proteins, including ZO-1 and occludin. Pre-treatment with curcumin prevented disruption of the mucosal barrier by maintaining ZO-1 and occludin expression and maintained trans-epithelial electric resistance across the genital epithelium. Curcumin pre-treatment also abrogated the gp120-mediated upregulation of the proinflammatory cytokines tumor necrosis factor-α and interleukin (IL)-6, which mediate barrier disruption, as well as the chemokines IL-8, RANTES and interferon gamma-induced protein-10 (IP-10), which are capable of recruiting HIV target cells to the FGT. GECs treated with curcumin and exposed to the sexually transmitted co-infecting microbes HSV-1, HSV-2 and Neisseria gonorrhoeae were unable to elicit innate inflammatory responses that indirectly induced activation of the HIV promoter and curcumin blocked Toll-like receptor (TLR)-mediated induction of HIV replication in chronically infected T-cells. Finally, curcumin treatment resulted in significantly decreased HIV-1 and HSV-2 replication in chronically infected T-cells and primary GECs, respectively. All together, our results suggest that the use of anti-inflammatory compounds such as curcumin may offer a viable alternative for the prevention and/or control of HIV replication in the FGT. PMID:25856395

Stationary organization of the actin cytoskeleton in Vallisneria: the role of stable microfilaments at the end walls.

PubMed

Ryu, J H; Takagi, S; Nagai, R

1995-04-01

In mesophyll cells of the aquatic angiosperm Vallisneria gigantea, bundles of microfilaments (MFs) serve as tracks for the rotational streaming of the cytoplasm, which occurs along the two longer side walls and the two shorter end walls. The stationary organization of these bundles has been shown to depend on the association of the bundles with the plasma membrane at the end walls. To identify the sites of such association, the effects of cytochalasin B (CB) on the configuration of the bundles of MFs were examined. In the case of the side walls, MFs were completely disrupted after treatment with CB at 100 micrograms/ml for 24 hours. By contrast, in the case of the end walls, a number of partially disrupted MFs remained even after 48 hours of treatment. After removal of CB, a completely normal arrangement of bundles of MFs was once again evident within 24 hours after a rather complicated process of reassembly. When reassembly had been completed, the direction of cytoplasmic streaming was reversed only in a small fraction of the treated cells, suggesting that bundles of MFs are anchored and stabilized at the end walls of each cell and that the polarity of reorganized bundles and, therefore, the direction of the cytoplasmic streaming is determined in a manner that depends on the original polarity of MFs that remained in spite of the disruptive action of CB. By contrast, the direction of reinitiated cytoplasmic streaming was reversed in 50% of cells in which the bundles of MFs had been completely disrupted by exogenously applied trypsin prior treatment with CB.(ABSTRACT TRUNCATED AT 250 WORDS)

SCHEMA computational design of virus capsid chimeras: calibrating how genome packaging, protection, and transduction correlate with calculated structural disruption.

PubMed

Ho, Michelle L; Adler, Benjamin A; Torre, Michael L; Silberg, Jonathan J; Suh, Junghae

2013-12-20

Adeno-associated virus (AAV) recombination can result in chimeric capsid protein subunits whose ability to assemble into an oligomeric capsid, package a genome, and transduce cells depends on the inheritance of sequence from different AAV parents. To develop quantitative design principles for guiding site-directed recombination of AAV capsids, we have examined how capsid structural perturbations predicted by the SCHEMA algorithm correlate with experimental measurements of disruption in seventeen chimeric capsid proteins. In our small chimera population, created by recombining AAV serotypes 2 and 4, we found that protection of viral genomes and cellular transduction were inversely related to calculated disruption of the capsid structure. Interestingly, however, we did not observe a correlation between genome packaging and calculated structural disruption; a majority of the chimeric capsid proteins formed at least partially assembled capsids and more than half packaged genomes, including those with the highest SCHEMA disruption. These results suggest that the sequence space accessed by recombination of divergent AAV serotypes is rich in capsid chimeras that assemble into 60-mer capsids and package viral genomes. Overall, the SCHEMA algorithm may be useful for delineating quantitative design principles to guide the creation of libraries enriched in genome-protecting virus nanoparticles that can effectively transduce cells. Such improvements to the virus design process may help advance not only gene therapy applications but also other bionanotechnologies dependent upon the development of viruses with new sequences and functions.

SCHEMA computational design of virus capsid chimeras: calibrating how genome packaging, protection, and transduction correlate with calculated structural disruption

PubMed Central

Ho, Michelle L.; Adler, Benjamin A.; Torre, Michael L.; Silberg, Jonathan J.; Suh, Junghae

2013-01-01

Adeno-associated virus (AAV) recombination can result in chimeric capsid protein subunits whose ability to assemble into an oligomeric capsid, package a genome, and transduce cells depends on the inheritance of sequence from different AAV parents. To develop quantitative design principles for guiding site-directed recombination of AAV capsids, we have examined how capsid structural perturbations predicted by the SCHEMA algorithm correlate with experimental measurements of disruption in seventeen chimeric capsid proteins. In our small chimera population, created by recombining AAV serotypes 2 and 4, we found that protection of viral genomes and cellular transduction were inversely related to calculated disruption of the capsid structure. Interestingly, however, we did not observe a correlation between genome packaging and calculated structural disruption; a majority of the chimeric capsid proteins formed at least partially assembled capsids and more than half packaged genomes, including those with the highest SCHEMA disruption. These results suggest that the sequence space accessed by recombination of divergent AAV serotypes is rich in capsid chimeras that assemble into 60-mer capsids and package viral genomes. Overall, the SCHEMA algorithm may be useful for delineating quantitative design principles to guide the creation of libraries enriched in genome-protecting virus nanoparticles that can effectively transduce cells. Such improvements to the virus design process may help advance not only gene therapy applications, but also other bionanotechnologies dependent upon the development of viruses with new sequences and functions. PMID:23899192

Mutations in Fis1 disrupt orderly disposal of defective mitochondria

PubMed Central

Shen, Qinfang; Yamano, Koji; Head, Brian P.; Kawajiri, Sumihiro; Cheung, Jesmine T. M.; Wang, Chunxin; Cho, Jeong-Hoon; Hattori, Nobutaka; Youle, Richard J.; van der Bliek, Alexander M.

2014-01-01

Mitochondrial fission is mediated by the dynamin-related protein Drp1 in metazoans. Drp1 is recruited from the cytosol to mitochondria by the mitochondrial outer membrane protein Mff. A second mitochondrial outer membrane protein, named Fis1, was previously proposed as recruitment factor, but Fis1−/− cells have mild or no mitochondrial fission defects. Here we show that Fis1 is nevertheless part of the mitochondrial fission complex in metazoan cells. During the fission cycle, Drp1 first binds to Mff on the surface of mitochondria, followed by entry into a complex that includes Fis1 and endoplasmic reticulum (ER) proteins at the ER–mitochondrial interface. Mutations in Fis1 do not normally affect fission, but they can disrupt downstream degradation events when specific mitochondrial toxins are used to induce fission. The disruptions caused by mutations in Fis1 lead to an accumulation of large LC3 aggregates. We conclude that Fis1 can act in sequence with Mff at the ER–mitochondrial interface to couple stress-induced mitochondrial fission with downstream degradation processes. PMID:24196833

The adherens junction is lost during normal pregnancy but not during ovarian hyperstimulated pregnancy.

PubMed

Dowland, Samson N; Madawala, Romanthi J; Lindsay, Laura A; Murphy, Christopher R

2016-03-01

During early pregnancy in the rat, the luminal uterine epithelial cells (UECs) must transform to a receptive state to permit blastocyst attachment and implantation. The implantation process involves penetration of the epithelial barrier, so it is expected that the transformation of UECs includes alterations in the lateral junctional complex. Previous studies have demonstrated a deepening of the tight junction (zonula occludens) and a reduction in the number of desmosomes (macula adherens) in UECs at the time of implantation. However, the adherens junction (zonula adherens), which is primarily responsible for cell-cell adhesion, has been little studied during early pregnancy. This study investigated the adherens junction in rat UECs during the early stages of normal pregnancy and ovarian hyperstimulated (OH) pregnancy using transmission electron microscopy. The adherens junction is present in UECs at the time of fertilisation, but is lost at the time of blastocyst implantation during normal pregnancy. Interestingly, at the time of implantation after OH, adherens junctions are retained and may impede blastocyst penetration of the epithelium. The adherens junction anchors the actin-based terminal web, which is known to be disrupted in UECs during early pregnancy. However, artificial disruption of the terminal web, using cytochalasin D, did not cause removal of the adherens junction in UECs. This study revealed that adherens junction disassembly occurs during early pregnancy, but that this process does not occur during OH pregnancy. Such disassembly does not appear to depend on the disruption of the terminal web. Copyright © 2015 Elsevier GmbH. All rights reserved.

Is There Evidence for Myelin Modeling by Astrocytes in the Normal Adult Brain?

PubMed Central

Varela-Echevarría, Alfredo; Vargas-Barroso, Víctor; Lozano-Flores, Carlos; Larriva-Sahd, Jorge

2017-01-01

A set of astrocytic process associated with altered myelinated axons is described in the forebrain of normal adult rodents with confocal, electron microscopy, and 3D reconstructions. Each process consists of a protuberance that contains secretory organelles including numerous lysosomes which polarize and open next to disrupted myelinated axons. Because of the distinctive asymmetric organelle distribution and ubiquity throughout the forebrain neuropil, this enlargement is named paraxial process (PAP). The myelin envelope contiguous to the PAP displays focal disruption or disintegration. In routine electron microscopy clusters of large, confluent, lysosomes proved to be an effective landmark for PAP identification. In 3D assemblies lysosomes organize a series of interconnected saccules that open up to the plasmalemma next to the disrupted myelin envelope(s). Activity for acid hydrolases was visualized in lysosomes, and extracellularly at the PAP-myelin interface and/or between the glial and neuronal outer aspects. Organelles in astrocytic processes involved in digesting pyknotic cells and debris resemble those encountered in PAPs supporting a likewise lytic function of the later. Conversely, processes entangling tripartite synapses and glomeruli were devoid of lysosomes. Both oligodendrocytic and microglial processes were not associated with altered myelin envelopes. The possible roles of the PAP in myelin remodeling in the context of the oligodendrocyte-astrocyte interactions and in the astrocyte's secretory pathways are discussed. PMID:28932188

Nuclear Envelope Disruption Involving Host Caspases Plays a Role in the Parvovirus Replication Cycle ▿

PubMed Central

Cohen, Sarah; Marr, Alexandra K.; Garcin, Pierre; Panté, Nelly

2011-01-01

Parvoviruses are small, nonenveloped, single-stranded DNA viruses which replicate in the nucleus of the host cell. We have previously found that early during infection the parvovirus minute virus of mice (MVM) causes small, transient disruptions of the nuclear envelope (NE). We have now investigated the mechanism used by MVM to disrupt the NE. Here we show that the viral phospholipase A2, the only known enzymatic domain on the parvovirus capsid, is not involved in causing NE disruption. Instead, the virus utilizes host cell caspases, which are proteases involved in causing NE breakdown during apoptosis, to facilitate these nuclear membrane disruptions. Studies with pharmacological inhibitors indicate that caspase-3 in particular is involved. A caspase-3 inhibitor prevents nuclear lamin cleavage and NE disruption in MVM-infected mouse fibroblast cells and reduces nuclear entry of MVM capsids and viral gene expression. Caspase-3 is, however, not activated above basal levels in MVM-infected cells, and other aspects of apoptosis are not triggered during early MVM infection. Instead, basally active caspase-3 is relocalized to the nuclei of infected cells. We propose that NE disruption involving caspases plays a role in (i) parvovirus entry into the nucleus and (ii) alteration of the compartmentalization of host proteins in a way that is favorable for the virus. PMID:21367902

Poly(2-alkylacrylic acid) polymers deliver molecules to the cytosol by pH-sensitive disruption of endosomal vesicles.

PubMed

Jones, Rachel A; Cheung, Charles Y; Black, Fiona E; Zia, Jasmine K; Stayton, Patrick S; Hoffman, Allan S; Wilson, Mark R

2003-05-15

The permeability barrier posed by cell membranes represents a challenge for the delivery of hydrophilic molecules into cells. We previously proposed that poly(2-alkylacrylic acid)s are endocytosed by cells into acidified vesicles and are there triggered by low pH to disrupt membranes and release the contents of endosomes/lysosomes to the cytosol. If this hypothesis is correct, these polymers could be valuable in drug-delivery applications. The present paper reports functional comparisons of a family of three poly(2-alkylacrylic acid)s. Poly(2-propylacrylic acid) (PPAA), poly(2-ethylacrylic acid) (PEAA) and poly(2-methylacrylic acid) (PMAA) were compared in red-blood-cell haemolysis assays and in a lipoplex (liposome-DNA complex) assay. We also directly examined the ability of these polymers to disrupt endosomes and lysosomes in cultured human cells. Our results show that: (i) unlike membrane-disruptive peptides, the endosomal-disruptive ability of poly(2-alkylacrylic acid)s cannot necessarily be predicted from their haemolytic activity at low pH, (ii) PPAA (but not PEAA or PMAA) potently facilitates gene transfection by cationic lipoplexes and (iii) endocytosed poly(2-alkylacrylic acid)s are triggered by luminal acidification to selectively disrupt endosomes (not lysosomes) and release their contents to the cytosol. These results will facilitate the rational design of future endosomal-disrupting polymers for drug delivery.

Withaferin A disrupts ubiquitin-based NEMO reorganization induced by canonical NF-κB signaling.

PubMed

Jackson, Shawn S; Oberley, Christopher; Hooper, Christopher P; Grindle, Kreg; Wuerzberger-Davis, Shelly; Wolff, Jared; McCool, Kevin; Rui, Lixin; Miyamoto, Shigeki

2015-02-01

The NF-κB family of transcription factors regulates numerous cellular processes, including cell proliferation and survival responses. The constitutive activation of NF-κB has also emerged as an important oncogenic driver in many malignancies, such as activated B-cell like diffuse large B cell lymphoma, among others. In this study, we investigated the impact and mechanisms of action of Withaferin A, a naturally produced steroidal lactone, against both signal-inducible as well as constitutive NF-κB activities. We found that Withaferin A is a robust inhibitor of canonical and constitutive NF-κB activities, leading to apoptosis of certain lymphoma lines. In the canonical pathway induced by TNF, Withaferin A did not disrupt RIP1 polyubiquitination or NEMO-IKKβ interaction and was a poor direct IKKβ inhibitor, but prevented the formation of TNF-induced NEMO foci which colocalized with TNF ligand. While GFP-NEMO efficiently formed TNF-induced foci, a GFP-NEMO(Y308S) mutant that is defective in binding to polyubiquitin chains did not form foci. Our study reveals that Withaferin A is a novel type of IKK inhibitor which acts by disrupting NEMO reorganization into ubiquitin-based signaling structures in vivo. Copyright © 2014 Elsevier Inc. All rights reserved.

Disruption of Mediator rescues the stunted growth of a lignin-deficient Arabidopsis mutant.

PubMed

Bonawitz, Nicholas D; Kim, Jeong Im; Tobimatsu, Yuki; Ciesielski, Peter N; Anderson, Nickolas A; Ximenes, Eduardo; Maeda, Junko; Ralph, John; Donohoe, Bryon S; Ladisch, Michael; Chapple, Clint

2014-05-15

Lignin is a phenylpropanoid-derived heteropolymer important for the strength and rigidity of the plant secondary cell wall. Genetic disruption of lignin biosynthesis has been proposed as a means to improve forage and bioenergy crops, but frequently results in stunted growth and developmental abnormalities, the mechanisms of which are poorly understood. Here we show that the phenotype of a lignin-deficient Arabidopsis mutant is dependent on the transcriptional co-regulatory complex, Mediator. Disruption of the Mediator complex subunits MED5a (also known as REF4) and MED5b (also known as RFR1) rescues the stunted growth, lignin deficiency and widespread changes in gene expression seen in the phenylpropanoid pathway mutant ref8, without restoring the synthesis of guaiacyl and syringyl lignin subunits. Cell walls of rescued med5a/5b ref8 plants instead contain a novel lignin consisting almost exclusively of p-hydroxyphenyl lignin subunits, and moreover exhibit substantially facilitated polysaccharide saccharification. These results demonstrate that guaiacyl and syringyl lignin subunits are largely dispensable for normal growth and development, implicate Mediator in an active transcriptional process responsible for dwarfing and inhibition of lignin biosynthesis, and suggest that the transcription machinery and signalling pathways responding to cell wall defects may be important targets to include in efforts to reduce biomass recalcitrance.

Cystic Fibrosis, Cystic Fibrosis Transmembrane Conductance Regulator and Drugs: Insights from Cellular Trafficking.

PubMed

Bridges, Robert J; Bradbury, Neil A

2018-01-01

The eukaryotic cell is organized into membrane-delineated compartments that are characterized by specific cadres of proteins sustaining biochemically distinct cellular processes. The appropriate subcellular localization of proteins is key to proper organelle function and provides a physiological context for cellular processes. Disruption of normal trafficking pathways for proteins is seen in several genetic diseases, where a protein's absence for a specific subcellular compartment leads to organelle disruption, and in the context of an individual, a disruption of normal physiology. Importantly, several drug therapies can also alter protein trafficking, causing unwanted side effects. Thus, a deeper understanding of trafficking pathways needs to be appreciated as novel therapeutic modalities are proposed. Despite the promising efficacy of novel therapeutic agents, the intracellular bioavailability of these compounds has proved to be a potential barrier, leading to failures in treatments for various diseases and disorders. While endocytosis of drug moieties provides an efficient means of getting material into cells, the subsequent release and endosomal escape of materials into the cytosol where they need to act has been a barrier. An understanding of cellular protein/lipid trafficking pathways has opened up strategies for increasing drug bioavailability. Approaches to enhance endosomal exit have greatly increased the cytosolic bioavailability of drugs and will provide a means of investigating previous drugs that may have been shelved due to their low cytosolic concentration.

The relative effect of citral on mitotic microtubules in wheat roots and BY2 cells.

PubMed

Chaimovitsh, D; Rogovoy Stelmakh, O; Altshuler, O; Belausov, E; Abu-Abied, M; Rubin, B; Sadot, E; Dudai, N

2012-03-01

The plant volatile monoterpene citral is a highly active compound with suggested allelopathic traits. Seed germination and seedling development are inhibited in the presence of citral, and it disrupts microtubules in both plant and animal cells in interphase. We addressed the following additional questions: can citral interfere with cell division; what is the relative effect of citral on mitotic microtubules compared to interphase cortical microtubules; what is its effect on newly formed cell plates; and how does it affect the association of microtubules with γ-tubulin? In wheat seedlings, citral led to inhibition of root elongation, curvature of newly formed cell walls and deformation of microtubule arrays. Citral's effect on microtubules was both dose- and time-dependent, with mitotic microtubules appearing to be more sensitive to citral than cortical microtubules. Association of γ-tubulin with microtubules was more sensitive to citral than were the microtubules themselves. To reveal the role of disrupted mitotic microtubules in dictating aberrations in cell plates in the presence of citral, we used tobacco BY2 cells expressing GFP-Tua6. Citral disrupted mitotic microtubules, inhibited the cell cycle and increased the frequency of asymmetric cell plates in these cells. The time scale of citral's effect in BY2 cells suggested a direct influence on cell plates during their formation. Taken together, we suggest that at lower concentrations, citral interferes with cell division by disrupting mitotic microtubules and cell plates, and at higher concentrations it inhibits cell elongation by disrupting cortical microtubules. © 2011 German Botanical Society and The Royal Botanical Society of the Netherlands.

Evaluation of automated cell disruptor methods for oomycetous and ascomycetous model organisms

USDA-ARS?s Scientific Manuscript database

Two automated cell disruptor-based methods for RNA extraction; disruption of thawed cells submerged in TRIzol Reagent (method QP), and direct disruption of frozen cells on dry ice (method CP), were optimized for a model oomycete, Phytophthora capsici, and compared with grinding in a mortar and pestl...

Advances of Stem Cell Therapeutics in Cutaneous Wound Healing and Regeneration.

PubMed

Kanji, Suman; Das, Hiranmoy

2017-01-01

Cutaneous wound healing is a complex multiple phase process, which overlaps each other, where several growth factors, cytokines, chemokines, and various cells interact in a well-orchestrated manner. However, an imbalance in any of these phases and factors may lead to disruption in harmony of normal wound healing process, resulting in transformation towards chronic nonhealing wounds and abnormal scar formation. Although various therapeutic interventions are available to treat chronic wounds, current wound-care has met with limited success. Progenitor stem cells possess potential therapeutic ability to overcome limitations of the present treatments as it offers accelerated wound repair with tissue regeneration. A substantial number of stem cell therapies for cutaneous wounds are currently under development as a result of encouraging preliminary findings in both preclinical and clinical studies. However, the mechanisms by which these stem cells contribute to the healing process have yet to be elucidated. In this review, we emphasize on the major treatment modalities currently available for the treatment of the wound, role of various interstitial stem cells and exogenous adult stem cells in cutaneous wound healing, and possible mechanisms involved in the healing process.

DNA methylation as a dynamic regulator of development and disease processes: spotlight on the prostate.

PubMed

Keil, Kimberly P; Vezina, Chad M

2015-01-01

Prostate development, benign hyperplasia and cancer involve androgen and growth factor signaling as well as stromal-epithelial interactions. We review how DNA methylation influences these and related processes in other organ systems such as how proliferation is restricted to specific cell populations during defined temporal windows, how androgens elicit their actions and how cells establish, maintain and remodel DNA methylation in a time and cell specific fashion. We also discuss mechanisms by which hormones and endocrine disrupting chemicals reprogram DNA methylation in the prostate and elsewhere and examine evidence for a reawakening of developmental epigenetic pathways as drivers of prostate cancer and benign prostate hyperplasia.

DNA methylation as a dynamic regulator of development and disease processes: spotlight on the prostate

PubMed Central

Keil, Kimberly P; Vezina, Chad M

2015-01-01

Prostate development, benign hyperplasia and cancer involve androgen and growth factor signaling as well as stromal–epithelial interactions. We review how DNA methylation influences these and related processes in other organ systems such as how proliferation is restricted to specific cell populations during defined temporal windows, how androgens elicit their actions and how cells establish, maintain and remodel DNA methylation in a time and cell specific fashion. We also discuss mechanisms by which hormones and endocrine disrupting chemicals reprogram DNA methylation in the prostate and elsewhere and examine evidence for a reawakening of developmental epigenetic pathways as drivers of prostate cancer and benign prostate hyperplasia. PMID:26077429

Second International Conference on Accelerating Biopharmaceutical Development: March 9-12, 2009, Coronado, CA USA.

PubMed

Reichert, Janice M; Jacob, Nitya; Amanullah, Ashraf

2009-01-01

The Second International Conference on Accelerating Biopharmaceutical Development was held in Coronado, California. The meeting was organized by the Society for Biological Engineering (SBE) and the American Institute of Chemical Engineers (AIChE); SBE is a technological community of the AIChE. Bob Adamson (Wyeth) and Chuck Goochee (Centocor) were co-chairs of the event, which had the theme "Delivering cost-effective, robust processes and methods quickly and efficiently." The first day focused on emerging disruptive technologies and cutting-edge analytical techniques. Day two featured presentations on accelerated cell culture process development, critical quality attributes, specifications and comparability, and high throughput protein formulation development. The final day was dedicated to discussion of technology options and new analysis methods provided by emerging disruptive technologies; functional interaction, integration and synergy in platform development; and rapid and economic purification process development.

Second International Conference on Accelerating Biopharmaceutical Development: March 9-12, 2009, Coronado, CA, USA.

PubMed

Reichert, Janice M; Jacob, Nitya M; Amanullah, Ashraf

2009-01-01

The Second International Conference on Accelerating Biopharmaceutical Development was held in Coronado, California. The meeting was organized by the Society for Biological Engineering (SBE) and the American Institute of Chemical Engineers (AIChE); SBE is a technological community of the AIChE. Bob Adamson (Wyeth) and Chuck Goochee (Centocor) were co-chairs of the event, which had the theme "Delivering cost-effective, robust processes and methods quickly and efficiently." The first day focused on emerging disruptive technologies and cutting-edge analytical techniques. Day two featured presentations on accelerated cell culture process development, critical quality attributes, specifications and comparability, and high throughput protein formulation development. The final day was dedicated to discussion of technology options and new analysis methods provided by emerging disruptive technologies; functional interaction, integration and synergy in platform development; and rapid and economic purification process development.

A statistical model describing combined irreversible electroporation and electroporation-induced blood-brain barrier disruption.

PubMed

Sharabi, Shirley; Kos, Bor; Last, David; Guez, David; Daniels, Dianne; Harnof, Sagi; Mardor, Yael; Miklavcic, Damijan

2016-03-01

Electroporation-based therapies such as electrochemotherapy (ECT) and irreversible electroporation (IRE) are emerging as promising tools for treatment of tumors. When applied to the brain, electroporation can also induce transient blood-brain-barrier (BBB) disruption in volumes extending beyond IRE, thus enabling efficient drug penetration. The main objective of this study was to develop a statistical model predicting cell death and BBB disruption induced by electroporation. This model can be used for individual treatment planning. Cell death and BBB disruption models were developed based on the Peleg-Fermi model in combination with numerical models of the electric field. The model calculates the electric field thresholds for cell kill and BBB disruption and describes the dependence on the number of treatment pulses. The model was validated using in vivo experimental data consisting of rats brains MRIs post electroporation treatments. Linear regression analysis confirmed that the model described the IRE and BBB disruption volumes as a function of treatment pulses number (r(2) = 0.79; p < 0.008, r(2) = 0.91; p < 0.001). The results presented a strong plateau effect as the pulse number increased. The ratio between complete cell death and no cell death thresholds was relatively narrow (between 0.88-0.91) even for small numbers of pulses and depended weakly on the number of pulses. For BBB disruption, the ratio increased with the number of pulses. BBB disruption radii were on average 67% ± 11% larger than IRE volumes. The statistical model can be used to describe the dependence of treatment-effects on the number of pulses independent of the experimental setup.

ATP synthase promotes germ cell differentiation independent of oxidative phosphorylation

PubMed Central

Teixeira, Felipe K.; Sanchez, Carlos G.; Hurd, Thomas R.; Seifert, Jessica R. K.; Czech, Benjamin; Preall, Jonathan B.; Hannon, Gregory J.; Lehmann, Ruth

2015-01-01

The differentiation of stem cells is a tightly regulated process essential for animal development and tissue homeostasis. Through this process, attainment of new identity and function is achieved by marked changes in cellular properties. Intrinsic cellular mechanisms governing stem cell differentiation remain largely unknown, in part because systematic forward genetic approaches to the problem have not been widely used1,2. Analysing genes required for germline stem cell differentiation in the Drosophila ovary, we find that the mitochondrial ATP synthase plays a critical role in this process. Unexpectedly, the ATP synthesizing function of this complex was not necessary for differentiation, as knockdown of other members of the oxidative phosphorylation system did not disrupt the process. Instead, the ATP synthase acted to promote the maturation of mitochondrial cristae during differentiation through dimerization and specific upregulation of the ATP synthase complex. Taken together, our results suggest that ATP synthase-dependent crista maturation is a key developmental process required for differentiation independent of oxidative phosphorylation. PMID:25915123

Analysis of the Effects of Cell Stress and Cytotoxicity on In ...

EPA Pesticide Factsheets

Chemical toxicity can arise from disruption of specific biomolecular functions or through more generalized cell stress and cytotoxicity-mediated processes. Here, concentration-dependent responses of 1063 chemicals including pharmaceuticals, natural products, pesticidals, consumer, and industrial chemicals across a diverse battery of 821 in vitro assay endpoints from 7 high-throughput assay technology platforms were analyzed in order to better distinguish between these types of activities. Both cell-based and cell-free assays showed a rapid increase in the frequency of responses at concentrations where cell stress / cytotoxicity responses were observed in cell-based assays. Chemicals that were positive on at least two viability/cytotoxicity assays within the concentration range tested (typically up to 100 M) activated a median of 12% of assay endpoints while those that were not cytotoxic in this concentration range activated 1.3% of the assays endpoints. The results suggest that activity can be broadly divided into: (1) specific biomolecular interactions against one or more targets (e.g., receptors or enzymes) at concentrations below which overt cytotoxicity-associated activity is observed; and (2) activity associated with cell stress or cytotoxicity, which may result from triggering of specific cell stress pathways, chemical reactivity, physico-chemical disruption of proteins or membranes, or broad low-affinity non-covalent interactions. Chemicals showing a g

Airway epithelial homeostasis and planar cell polarity signaling depend on multiciliated cell differentiation

PubMed Central

Vladar, Eszter K.; Nayak, Jayakar V.; Milla, Carlos E.; Axelrod, Jeffrey D.

2016-01-01

Motile airway cilia that propel contaminants out of the lung are oriented in a common direction by planar cell polarity (PCP) signaling, which localizes PCP protein complexes to opposite cell sides throughout the epithelium to orient cytoskeletal remodeling. In airway epithelia, PCP is determined in a 2-phase process. First, cell-cell communication via PCP complexes polarizes all cells with respect to the proximal-distal tissue axis. Second, during ciliogenesis, multiciliated cells (MCCs) undergo cytoskeletal remodeling to orient their cilia in the proximal direction. The second phase not only directs cilium polarization, but also consolidates polarization across the epithelium. Here, we demonstrate that in airway epithelia, PCP depends on MCC differentiation. PCP mutant epithelia have misaligned cilia, and also display defective barrier function and regeneration, indicating that PCP regulates multiple aspects of airway epithelial homeostasis. In humans, MCCs are often sparse in chronic inflammatory diseases, and these airways exhibit PCP dysfunction. The presence of insufficient MCCs impairs mucociliary clearance in part by disrupting PCP-driven polarization of the epithelium. Consistent with defective PCP, barrier function and regeneration are also disrupted. Pharmacological stimulation of MCC differentiation restores PCP and reverses these defects, suggesting its potential for broad therapeutic benefit in chronic inflammatory disease. PMID:27570836

Sleep Disruption in Hematopoietic Cell Transplant Recipients: Prevalence, Severity, and Clinical Management

PubMed Central

Jim, Heather S.L.; Evans, Bryan; Jeong, Jiyeon M.; Gonzalez, Brian D.; Johnston, Laura; Nelson, Ashley M.; Kesler, Shelli; Phillips, Kristin M.; Barata, Anna; Pidala, Joseph; Palesh, Oxana

2014-01-01

Sleep disruption is common among hematopoietic cell transplant (HCT) recipients, with over 50% of patients experiencing sleep disruption pre-transplant, up to 82% experiencing moderate to severe sleep disruption during hospitalization for transplant, and up to 43% in the post-transplant period. These rates of sleep disruption are substantially higher than the general population. Although sleep disruption can be distressing to patients and contribute to diminished quality of life, it is rarely discussed during clinical visits. The goal of the current review is to draw attention to sleep disruption as a clinical problem in HCT in order to facilitate patient education, intervention, and research. The review opens with a discussion of sleep disruption measurement and clinical diagnosis of sleep disorders. An overview of the prevalence, severity, and chronicity of sleep disruption and disorders in patients receiving HCT follows. Current evidence regarding sociodemographic and clinical predictors of sleep disruption and disorders is summarized. The review concludes with suggestions for behavioral and pharmacologic management of sleep disruption and disorders as well as directions for future research. PMID:24747335

Autophagy inhibitor 3-methyladenine protects against endothelial cell barrier dysfunction in acute lung injury.

PubMed

Slavin, Spencer A; Leonard, Antony; Grose, Valerie; Fazal, Fabeha; Rahman, Arshad

2018-03-01

Autophagy is an evolutionarily conserved cellular process that facilitates the continuous recycling of intracellular components (organelles and proteins) and provides an alternative source of energy when nutrients are scarce. Recent studies have implicated autophagy in many disorders, including pulmonary diseases. However, the role of autophagy in endothelial cell (EC) barrier dysfunction and its relevance in the context of acute lung injury (ALI) remain uncertain. Here, we provide evidence that autophagy is a critical component of EC barrier disruption in ALI. Using an aerosolized bacterial lipopolysaccharide (LPS) inhalation mouse model of ALI, we found that administration of the autophagy inhibitor 3-methyladenine (3-MA), either prophylactically or therapeutically, markedly reduced lung vascular leakage and tissue edema. 3-MA was also effective in reducing the levels of proinflammatory mediators and lung neutrophil sequestration induced by LPS. To test the possibility that autophagy in EC could contribute to lung vascular injury, we addressed its role in the mechanism of EC barrier disruption. Knockdown of ATG5, an essential regulator of autophagy, attenuated thrombin-induced EC barrier disruption, confirming the involvement of autophagy in the response. Similarly, exposure of cells to 3-MA, either before or after thrombin, protected against EC barrier dysfunction by inhibiting the cleavage and loss of vascular endothelial cadherin at adherens junctions, as well as formation of actin stress fibers. 3-MA also reversed LPS-induced EC barrier disruption. Together, these data imply a role of autophagy in lung vascular injury and reveal the protective and therapeutic utility of 3-MA against ALI.

Loss of MACF1 Abolishes Ciliogenesis and Disrupts Apicobasal Polarity Establishment in the Retina.

PubMed

May-Simera, Helen L; Gumerson, Jessica D; Gao, Chun; Campos, Maria; Cologna, Stephanie M; Beyer, Tina; Boldt, Karsten; Kaya, Koray D; Patel, Nisha; Kretschmer, Friedrich; Kelley, Matthew W; Petralia, Ronald S; Davey, Megan G; Li, Tiansen

2016-10-25

Microtubule actin crosslinking factor 1 (MACF1) plays a role in the coordination of microtubules and actin in multiple cellular processes. Here, we show that MACF1 is also critical for ciliogenesis in multiple cell types. Ablation of Macf1 in the developing retina abolishes ciliogenesis, and basal bodies fail to dock to ciliary vesicles or migrate apically. Photoreceptor polarity is randomized, while inner retinal cells laminate correctly, suggesting that photoreceptor maturation is guided by polarity cues provided by cilia. Deletion of MACF1 in adult photoreceptors causes reversal of basal body docking and loss of outer segments, reflecting a continuous requirement for MACF1 function. MACF1 also interacts with the ciliary proteins MKKS and TALPID3. We propose that a disruption of trafficking across microtubles to actin filaments underlies the ciliogenesis defect in cells lacking MACF1 and that MKKS and TALPID3 are involved in the coordination of microtubule and actin interactions. Published by Elsevier Inc.

Corruption of homeostatic mechanisms in the guanylyl cyclase C signaling pathway underlying colorectal tumorigenesis

PubMed Central

Waldman, Scott A

2010-01-01

Colon cancer, the second leading cause of cancer-related mortality worldwide, originates from the malignant transformation of intestinal epithelial cells. The intestinal epithelium undergoes a highly organized process of rapid regeneration along the crypt-villus axis, characterized by proliferation, migration, differentiation and apoptosis, whose coordination is essential to maintaining the mucosal barrier. Disruption of these homeostatic processes predisposes cells to mutations in tumor suppressors or oncogenes, whose dysfunction provides transformed cells an evolutionary growth advantage. While sequences of genetic mutations at different stages along the neoplastic continuum have been established, little is known of the events initiating tumorigenesis prior to adenomatous polyposis coli (APC) mutations. Here, we examine a role for the corruption of homeostasis induced by silencing novel tumor suppressors, including the intestine-specific transcription factor CDX2 and its gene target guanylyl cyclase C (GCC), as early events predisposing cells to mutations in APC and other sequential genes that initiate colorectal cancer. CDX2 and GCC maintain homeostatic regeneration in the intestine by restricting cell proliferation, promoting cell maturation and adhesion, regulating cell migration and defending the intestinal barrier and genomic integrity. Elimination of CDX2 or GCC promotes intestinal tumor initiation and growth in aged mice, mice carrying APC mutations or mice exposed to carcinogens. The roles of CDX2 and GCC in suppressing intestinal tumorigenesis, universal disruption in their signaling through silencing of hormones driving GCC, and the uniform overexpression of GCC by tumors underscore the potential value of oral replacement with GCC ligands as targeted prevention and therapy for colorectal cancer. PMID:20592492

Subcellular Biological Effects of Nanosecond Pulsed Electric Fields

NASA Astrophysics Data System (ADS)

Kolb, Juergen F.; Stacey, Michael

Membranes of biological cells can be charged by exposure to pulsed electric fields. After the potential difference across the barrier reaches critical values on the order of 1 V, pores will form. For moderate pulse parameters of duration and amplitude, the effect is limited to the outer cell membrane. With the exposure to nanosecond pulses of several tens of kilovolts per centimeter, a similar effect is also expected for subcellular membranes and structures. Cells will respond to the disruption by different biochemical processes. This offers possibilities for the development of novel medical therapies, the manipulation of cells and microbiological decontamination.

Tight junctions and the modulation of barrier function in disease

PubMed Central

2008-01-01

Tight junctions create a paracellular barrier in epithelial and endothelial cells protecting them from the external environment. Two different classes of integral membrane proteins constitute the tight junction strands in epithelial cells and endothelial cells, occludin and members of the claudin protein family. In addition, cytoplasmic scaffolding molecules associated with these junctions regulate diverse physiological processes like proliferation, cell polarity and regulated diffusion. In many diseases, disruption of this regulated barrier occurs. This review will briefly describe the molecular composition of the tight junctions and then present evidence of the link between tight junction dysfunction and disease. PMID:18415116

Image analysis supported moss cell disruption in photo-bioreactors.

PubMed

Lucumi, A; Posten, C; Pons, M-N

2005-05-01

Diverse methods for the disruption of cell entanglements and pellets of the moss Physcomitrella patens were tested in order to improve the homogeneity of suspension cultures. The morphological characterization of the moss was carried out by means of image analysis. Selected morphological parameters were defined and compared to the reduction of the carbon dioxide fixation, and the released pigments after cell disruption. The size control of the moss entanglements based on the rotor stator principle allowed a focused shear stress, avoiding a severe reduction in the photosynthesis. Batch cultures of P. patens in a 30.0-l pilot tubular photo-bioreactor with cell disruption showed no significant variation in growth rate and a delayed cell differentiation, when compared to undisrupted cultures. A highly controlled photoautotrophic culture of P. patens in a scalable photo-bioreactor was established, contributing to the development required for the future use of mosses as producers of relevant heterologous proteins.

Nanosecond pulsed electric field (nsPEF) enhance cytotoxicity of cisplatin to hepatocellular cells by microdomain disruption on plasma membrane.

PubMed

Yin, Shengyong; Chen, Xinhua; Xie, Haiyang; Zhou, Lin; Guo, Danjing; Xu, Yuning; Wu, Liming; Zheng, Shusen

2016-08-15

Previous studies showed nanosecond pulsed electric field (nsPEF) can ablate solid tumors including hepatocellular carcinoma (HCC) but its effect on cell membrane is not fully understood. We hypothesized nsPEF disrupt the microdomains on outer-cellular membrane with direct mechanical force and as a result the plasma membrane permeability increases to facilitate the small molecule intake. Three HCC cells were pulsed one pulse per minute, an interval longer than nanopore resealing time. The cationized ferritin was used to mark up the electronegative microdomains, propidium iodide (PI) for membrane permeabilization, energy dispersive X-ray spectroscopy (EDS) for the negative cell surface charge and cisplatin for inner-cellular cytotoxicity. We demonstrated that the ferritin marked-microdomain and negative cell surface charge were disrupted by nsPEF caused-mechanical force. The cell uptake of propidium and cytotoxicity of DNA-targeted cisplatin increased with a dose effect. Cisplatin gains its maximum inner-cellular cytotoxicity when combining with nsPEF stimulation. We conclude that nsPEF disrupt the microdomains on the outer cellular membrane directly and increase the membrane permeabilization for PI and cisplatin. The microdomain disruption and membrane infiltration changes are caused by the mechanical force from the changes of negative cell surface charge. Copyright © 2016 Elsevier Inc. All rights reserved.

Cadherin 11 Involved in Basement Membrane Damage and Dermal Changes in Melasma.

PubMed

Kim, Nan-Hyung; Choi, Soo-Hyun; Lee, Tae Ryong; Lee, Chang-Hoon; Lee, Ai-Young

2016-06-15

Basement membrane (BM) disruption and dermal changes (elastosis, collagenolysis, vascular ectasia) have been reported in melasma. Although ultraviolet (UV) irradiation can induce these changes, UV is not always necessary for melasma development. Cadherin 11 (CDH11), which is upregulated in some melasma patients, has previously been shown to stimulate melanogenesis. Because CDH11 action requires cell-cell adhesion between fibroblasts and melanocytes, BM disruption in vivo should facilitate this. The aim of this study was to examine whether CDH11 overexpression leads to BM disruption and dermal changes, independent of UV irradiation. Immunohistochemistry/immunofluorescence, real-time PCR, Western blotting, and zymography suggested that BM disruption/dermal changes and related factors were present in the hyperpigmented skin of CDH11-upregulated melasma patients and in CDH11-overexpressing fibroblasts/keratinocytes. The opposite was seen in CDH11-knockdown cells. UV irradiation of the cultured cells did not increase CDH11 expression. Collectively, these data demonstrate that CDH11 overexpression could induce BM disruption and dermal changes in melasma, regardless of UV exposure.

Structural basis of membrane disruption and cellular toxicity by α-synuclein oligomers.

PubMed

Fusco, Giuliana; Chen, Serene W; Williamson, Philip T F; Cascella, Roberta; Perni, Michele; Jarvis, James A; Cecchi, Cristina; Vendruscolo, Michele; Chiti, Fabrizio; Cremades, Nunilo; Ying, Liming; Dobson, Christopher M; De Simone, Alfonso

2017-12-15

Oligomeric species populated during the aggregation process of α-synuclein have been linked to neuronal impairment in Parkinson's disease and related neurodegenerative disorders. By using solution and solid-state nuclear magnetic resonance techniques in conjunction with other structural methods, we identified the fundamental characteristics that enable toxic α-synuclein oligomers to perturb biological membranes and disrupt cellular function; these include a highly lipophilic element that promotes strong membrane interactions and a structured region that inserts into lipid bilayers and disrupts their integrity. In support of these conclusions, mutations that target the region that promotes strong membrane interactions by α-synuclein oligomers suppressed their toxicity in neuroblastoma cells and primary cortical neurons. Copyright © 2017 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works.

Global Genetic Profiles of Gene Network Disruption in Bovine Peripheral Blood Mononuclear Cells Induced Bovine Leukemia Virus (BLV) Infection

USDA-ARS?s Scientific Manuscript database

Efficient nutrient assimilation into useful animal-derived products is the ultimate requirement for successful animal production. Infection in young growing animals can decrease energy and nutrient use required for growth rate by redirection of nutrients to support immune defense processes. Bovine l...

The Use of Protein Hydrolysates for Weed Control

NASA Astrophysics Data System (ADS)

Christians, Nick; Liu, Dianna; Unruh, Jay Bryan

Corn gluten meal, the protein fraction of corn (Zea mays L.) grain, is commercially used as a natural weed control agent and nitrogen source in horticultural crops and in the turf and ornamental markets. Corn gluten hydrolysate, a water soluble form of gluten meal, has also been proposed for the same purpose, although it could be sprayed on the soil rather than applied in the granular form. Five depeptides, glutaminyl-glutamine (Gln-Gln), glycinyl-alanine (Gly-Ala), alanyl-­glutamine (Ala-Glu), alanyl-asparagine (Ala-Asp), and alaninyl-alanine (Ala-Ala) and a pentapeptide leucine-serine-proline-alanine-glutamine (Leu-Ser-Pro-Ala-Gln) were identified as the active components of the hydrolysate. Microscopic analysis revealed that Ala-Ala acted on some metabolic process rather than directly on the mitotic apparatus. Similar to the chloracetamides and sulfonyl-urea hebicides, Ala-Ala inhibits cell division rather than disrupting of cell division processes. Cellular ultrastructure changes caused by exposure to Ala-Ala implicate Ala-Ala as having membrane-disrupting characteristics similar to several synthetic herbicides. The potential use of the hydrolysate and the peptides as weed controls is discussed.

Traffic jam hypothesis: Relationship between endocytic dysfunction and Alzheimer's disease.

PubMed

Kimura, Nobuyuki; Yanagisawa, Katsuhiko

2017-07-08

Membrane trafficking pathways, like the endocytic pathway, carry out fundamental cellular processes that are essential for normal functioning. One such process is regulation of cell surface receptor signaling. A growing body of evidence suggests that β-amyloid protein (Aβ) plays a key role in Alzheimer's disease (AD) pathogenesis. Cleavage of Aβ from its precursor, β-amyloid precursor protein (APP), occurs through the endocytic pathway in neuronal cells. In early-stage AD, intraneuronal accumulation of abnormally enlarged endosomes is common, indicating that endosome trafficking is disrupted. Strikingly, genome-wide association studies reveal that several endocytosis-related genes are associated with AD onset. Also, recent studies demonstrate that alteration in endocytosis induces not only Aβ pathology but also the propagation of tau protein pathology, another key pathological feature of AD. Endocytic dysfunction can disrupt neuronal physiological functions, such as synaptic vesicle transport and neurotransmitter release. Thus, "traffic jams" in the endocytic pathway may be involved in AD pathogenesis and may serve as a novel target for the development of new therapeutics. Copyright © 2017 Elsevier Ltd. All rights reserved.

Modulation of eukaryotic cell apoptosis by members of the bacterial order Actinomycetales.

PubMed

Barry, Daniel P; Beaman, Blaine L

2006-10-01

Apoptosis, or programmed cell death, is normally responsible for the orderly elimination of aged or damaged cells, and is a necessary part of the homeostasis and development of multicellular organisms. Some pathogenic bacteria can disrupt this process by triggering excess apoptosis or by preventing it when appropriate. Either event can lead to disease. There has been extensive research into the modulation of host cell death by microorganisms, and several reviews have been published on the phenomenon. Rather than covering the entire field, this review focuses on the dysregulation of host cell apoptosis by members of the order Actinomycetales, containing the genera Corynebacterium, Mycobacterium, Rhodococcus, and Nocardia.

Formation of Orthopoxvirus Cytoplasmic A-Type Inclusion Bodies and Embedding of Virions Are Dynamic Processes Requiring Microtubules

PubMed Central

Howard, Amanda R.

2012-01-01

In cells infected with some orthopoxviruses, numerous mature virions (MVs) become embedded within large, cytoplasmic A-type inclusions (ATIs) that can protect infectivity after cell lysis. ATIs are composed of an abundant viral protein called ATIp, which is truncated in orthopoxviruses such as vaccinia virus (VACV) that do not form ATIs. To study ATI formation and occlusion of MVs within ATIs, we used recombinant VACVs that express the cowpox full-length ATIp or we transfected plasmids encoding ATIp into cells infected with VACV, enabling ATI formation. ATI enlargement and MV embedment required continued protein synthesis and an intact microtubular network. For live imaging of ATIs and MVs, plasmids expressing mCherry fluorescent protein fused to ATIp were transfected into cells infected with VACV expressing the viral core protein A4 fused to yellow fluorescent protein. ATIs appeared as dynamic, mobile bodies that enlarged by multiple coalescence events, which could be prevented by disrupting microtubules. Coalescence of ATIs was confirmed in cells infected with cowpox virus. MVs were predominantly at the periphery of ATIs early in infection. We determined that coalescence contributed to the distribution of MVs within ATIs and that microtubule-disrupting drugs abrogated coalescence-mediated MV embedment. In addition, MVs were shown to move from viral factories at speeds consistent with microtubular transport to the peripheries of ATIs, whereas disruption of microtubules prevented such trafficking. The data indicate an important role for microtubules in the coalescence of ATIs into larger structures, transport of MVs to ATIs, and embedment of MVs within the ATI matrix. PMID:22438543

A statistical model describing combined irreversible electroporation and electroporation-induced blood-brain barrier disruption

PubMed Central

Sharabi, Shirley; Kos, Bor; Last, David; Guez, David; Daniels, Dianne; Harnof, Sagi; Miklavcic, Damijan

2016-01-01

Background Electroporation-based therapies such as electrochemotherapy (ECT) and irreversible electroporation (IRE) are emerging as promising tools for treatment of tumors. When applied to the brain, electroporation can also induce transient blood-brain-barrier (BBB) disruption in volumes extending beyond IRE, thus enabling efficient drug penetration. The main objective of this study was to develop a statistical model predicting cell death and BBB disruption induced by electroporation. This model can be used for individual treatment planning. Material and methods Cell death and BBB disruption models were developed based on the Peleg-Fermi model in combination with numerical models of the electric field. The model calculates the electric field thresholds for cell kill and BBB disruption and describes the dependence on the number of treatment pulses. The model was validated using in vivo experimental data consisting of rats brains MRIs post electroporation treatments. Results Linear regression analysis confirmed that the model described the IRE and BBB disruption volumes as a function of treatment pulses number (r2 = 0.79; p < 0.008, r2 = 0.91; p < 0.001). The results presented a strong plateau effect as the pulse number increased. The ratio between complete cell death and no cell death thresholds was relatively narrow (between 0.88-0.91) even for small numbers of pulses and depended weakly on the number of pulses. For BBB disruption, the ratio increased with the number of pulses. BBB disruption radii were on average 67% ± 11% larger than IRE volumes. Conclusions The statistical model can be used to describe the dependence of treatment-effects on the number of pulses independent of the experimental setup. PMID:27069447

Brg1 coordinates multiple processes during retinogenesis and is a tumor suppressor in retinoblastoma

DOE PAGES

Aldiri, Issam; Ajioka, Itsuki; Xu, Beisi; ...

2015-12-01

Retinal development requires precise temporal and spatial coordination of cell cycle exit, cell fate specification, cell migration and differentiation. When this process is disrupted, retinoblastoma, a developmental tumor of the retina, can form. Epigenetic modulators are central to precisely coordinating developmental events, and many epigenetic processes have been implicated in cancer. Studying epigenetic mechanisms in development is challenging because they often regulate multiple cellular processes; therefore, elucidating the primary molecular mechanisms involved can be difficult. Here we explore the role of Brg1 (Smarca4) in retinal development and retinoblastoma in mice using molecular and cellular approaches. Brg1 was found to regulatemore » retinal size by controlling cell cycle length, cell cycle exit and cell survival during development. Brg1 was not required for cell fate specification but was required for photoreceptor differentiation and cell adhesion/polarity programs that contribute to proper retinal lamination during development. The combination of defective cell differentiation and lamination led to retinal degeneration in Brg1-deficient retinae. Despite the hypocellularity, premature cell cycle exit, increased cell death and extended cell cycle length, retinal progenitor cells persisted in Brg1-deficient retinae, making them more susceptible to retinoblastoma. In conclusion, ChIP-Seq analysis suggests that Brg1 might regulate gene expression through multiple mechanisms.« less

Brg1 coordinates multiple processes during retinogenesis and is a tumor suppressor in retinoblastoma

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

Aldiri, Issam; Ajioka, Itsuki; Xu, Beisi

Retinal development requires precise temporal and spatial coordination of cell cycle exit, cell fate specification, cell migration and differentiation. When this process is disrupted, retinoblastoma, a developmental tumor of the retina, can form. Epigenetic modulators are central to precisely coordinating developmental events, and many epigenetic processes have been implicated in cancer. Studying epigenetic mechanisms in development is challenging because they often regulate multiple cellular processes; therefore, elucidating the primary molecular mechanisms involved can be difficult. Here we explore the role of Brg1 (Smarca4) in retinal development and retinoblastoma in mice using molecular and cellular approaches. Brg1 was found to regulatemore » retinal size by controlling cell cycle length, cell cycle exit and cell survival during development. Brg1 was not required for cell fate specification but was required for photoreceptor differentiation and cell adhesion/polarity programs that contribute to proper retinal lamination during development. The combination of defective cell differentiation and lamination led to retinal degeneration in Brg1-deficient retinae. Despite the hypocellularity, premature cell cycle exit, increased cell death and extended cell cycle length, retinal progenitor cells persisted in Brg1-deficient retinae, making them more susceptible to retinoblastoma. In conclusion, ChIP-Seq analysis suggests that Brg1 might regulate gene expression through multiple mechanisms.« less

Critical role for NHE1 in intracellular pH regulation in pancreatic acinar cells.

PubMed

Brown, David A; Melvin, James E; Yule, David I

2003-11-01

The primary function of pancreatic acinar cells is to secrete digestive enzymes together with a NaCl-rich primary fluid which is later greatly supplemented and modified by the pancreatic duct. A Na+/H+ exchanger(s) [NHE(s)] is proposed to be integral in the process of fluid secretion both in terms of the transcellular flux of Na+ and intracellular pH (pHi) regulation. Multiple NHE isoforms have been identified in pancreatic tissue, but little is known about their individual functions in acinar cells. The Na+/H+ exchange inhibitor 5-(N-ethyl-N-isopropyl) amiloride completely blocked pHi recovery after an NH4Cl-induced acid challenge, confirming a general role for NHE in pHi regulation. The targeted disruption of the Nhe1 gene also completely abolished pHi recovery from an acid load in pancreatic acini in both HCO3--containing and HCO3--free solutions. In contrast, the disruption of either Nhe2 or Nhe3 had no effect on pHi recovery. In addition, NHE1 activity was upregulated in response to muscarinic stimulation in wild-type mice but not in NHE1-deficient mice. Fluctuations in pHi could potentially have major effects on Ca2+ signaling following secretagogue stimulation; however, the targeted disruption of Nhe1 was found to have no significant effect on intracellular Ca2+ homeostasis. These data demonstrate that NHE1 is the major regulator of pHi in both resting and muscarinic agonist-stimulated pancreatic acinar cells.

HIV-1 gp120 Glycoprotein Interacting with Dendritic Cell-specific Intercellular Adhesion Molecule 3-grabbing Non-integrin (DC-SIGN) Down-Regulates Tight Junction Proteins to Disrupt the Blood Retinal Barrier and Increase Its Permeability.

PubMed

Qian, Yi-Wen; Li, Chuan; Jiang, Ai-Ping; Ge, Shengfang; Gu, Ping; Fan, Xianqun; Li, Tai-Sheng; Jin, Xia; Wang, Jian-Hua; Wang, Zhi-Liang

2016-10-28

Approximately 70% of HIV-1 infected patients acquire ocular opportunistic infections and manifest eye disorders during the course of their illness. The mechanisms by which pathogens invade the ocular site, however, are unclear. Under normal circumstances, vascular endothelium and retinal pigment epithelium (RPE), which possess a well developed tight junction complex, form the blood-retinal barrier (BRB) to prevent pathogen invasion. We hypothesize that disruption of the BRB allows pathogen entry into ocular sites. The hypothesis was tested using in vitro models. We discovered that human RPE cells could bind to either HIV-1 gp120 glycoproteins or HIV-1 viral particles. Furthermore, the binding was mediated by dendritic cell-specific intercellular adhesion molecule 3-grabbing non-integrin (DC-SIGN) expressed on RPE cells. Upon gp120 binding to DC-SIGN, cellular NF-κB signaling was triggered, leading to the induction of matrix metalloproteinases, which subsequently degraded tight junction proteins and disrupted the BRB integrity. DC-SIGN knockdown or prior blocking with a specific antibody abolished gp120-induced matrix metalloproteinase expression and reduced the degradation of tight junction proteins. This study elucidates a novel mechanism by which HIV, type 1 invades ocular tissues and provides additional insights into the translocation or invasion process of ocular complication-associated pathogens. © 2016 by The American Society for Biochemistry and Molecular Biology, Inc.

Roles of specific membrane lipid domains in EGF receptor activation and cell adhesion molecule stabilization in a developing olfactory system.

PubMed

Gibson, Nicholas J; Tolbert, Leslie P; Oland, Lynne A

2009-09-29

Reciprocal interactions between glial cells and olfactory receptor neurons (ORNs) cause ORN axons entering the brain to sort, to fasciculate into bundles destined for specific glomeruli, and to form stable protoglomeruli in the developing olfactory system of an experimentally advantageous animal species, the moth Manduca sexta. Epidermal growth factor receptors (EGFRs) and the cell adhesion molecules (IgCAMs) neuroglian and fasciclin II are known to be important players in these processes. We report in situ and cell-culture studies that suggest a role for glycosphingolipid-rich membrane subdomains in neuron-glia interactions. Disruption of these subdomains by the use of methyl-beta-cyclodextrin results in loss of EGFR activation, depletion of fasciclin II in ORN axons, and loss of neuroglian stabilization in the membrane. At the cellular level, disruption leads to aberrant ORN axon trajectories, small antennal lobes, abnormal arrays of olfactory glomerul, and loss of normal glial cell migration. We propose that glycosphingolipid-rich membrane subdomains (possible membrane rafts or platforms) are essential for IgCAM-mediated EGFR activation and for anchoring of neuroglian to the cytoskeleton, both required for normal extension and sorting of ORN axons.

Replication-mediated disassociation of replication protein A–XPA complex upon DNA damage: implications for RPA handing off

PubMed Central

Jiang, Gaofeng; Zou, Yue; Wu, Xiaoming

2013-01-01

RPA (replication protein A), the eukaryotic ssDNA (single-stranded DNA)-binding protein, participates in most cellular processes in response to genotoxic insults, such as NER (nucleotide excision repair), DNA, DSB (double-strand break) repair and activation of cell cycle checkpoint signalling. RPA interacts with XPA (xeroderma pigmentosum A) and functions in early stage of NER. We have shown that in cells the RPA–XPA complex disassociated upon exposure of cells to high dose of UV irradiation. The dissociation required replication stress and was partially attributed to tRPA hyperphosphorylation. Treatment of cells with CPT (camptothecin) and HU (hydroxyurea), which cause DSB DNA damage and replication fork collapse respectively and also leads to the disruption of RPA–XPA complex. Purified RPA and XPA were unable to form complex in vitro in the presence of ssDNA. We propose that the competition-based RPA switch among different DNA metabolic pathways regulates the dissociation of RPA with XPA in cells after DNA damage. The biological significances of RPA–XPA complex disruption in relation with checkpoint activation, DSB repair and RPA hyperphosphorylation are discussed. PMID:22578086

Correlated fluorescence microscopy and cryo-electron tomography of virus-infected or transfected mammalian cells

PubMed Central

Hampton, Cheri M; Strauss, Joshua D; Ke, Zunlong; Dillard, Rebecca S; Hammonds, Jason E; Alonas, Eric; Desai, Tanay M; Marin, Mariana; Storms, Rachel E; Leon, Fredrick; Melikyan, Gregory B; Santangelo, Philip J; Spearman, Paul W; Wright, Elizabeth R

2016-01-01

Correlative light and electron microscopy (CLEM) combines spatiotemporal information from fluorescence light microscopy (fLM) with high-resolution structural data from cryo-electron tomography (cryo-ET). These technologies provide opportunities to bridge knowledge gaps between cell and structural biology. Here we describe our protocol for correlated cryo-fLM, cryo-electron microscopy (cryo-EM), and cryo-ET (i.e., cryo-CLEM) of virus-infected or transfected mammalian cells. Mammalian-derived cells are cultured on EM substrates, using optimized conditions that ensure that the cells are spread thinly across the substrate and are not physically disrupted. The cells are then screened by fLM and vitrified before acquisition of cryo-fLM and cryo-ET images, which is followed by data processing. A complete session from grid preparation through data collection and processing takes 5–15 d for an individual experienced in cryo-EM. PMID:27977021

Smooth muscle cells healing atherosclerotic plaque disruptions are of local, not blood, origin in apolipoprotein E knockout mice.

PubMed

Bentzon, Jacob F; Sondergaard, Claus S; Kassem, Moustapha; Falk, Erling

2007-10-30

Signs of preceding episodes of plaque rupture and smooth muscle cell (SMC)-mediated healing are common in atherosclerotic plaques, but the source of the healing SMCs is unknown. Recent studies suggest that activated platelets adhering to sites of injury recruit neointimal SMCs from circulating bone marrow-derived progenitor cells. Here, we analyzed the contribution of this mechanism to plaque healing after spontaneous and mechanical plaque disruption in apolipoprotein E knockout (apoE-/-) mice. To determine the origin of SMCs after spontaneous plaque disruption, irradiated 18-month-old apoE-/- mice were reconstituted with bone marrow cells from enhanced green fluorescent protein (eGFP) transgenic apoE-/- mice and examined when they died up to 9 months later. Plaque hemorrhage, indicating previous plaque disruption, was widely present, but no bone marrow-derived eGFP+ SMCs were detected. To examine the origin of healing SMCs in a model that recapitulates more features of human plaque rupture and healing, we developed a mechanical technique that produced consistent plaque disruption, superimposed thrombosis, and SMC-mediated plaque healing in apoE-/- mice. Mechanical plaque disruption was produced in irradiated apoE-/- mice reconstituted with eGFP+ apoE-/- bone marrow cells and in carotid bifurcations cross-grafted between apoE-/- and eGFP+ apoE-/- mice. Apart from few non-graft-derived SMCs near the anastomosis site in 1 transplanted carotid bifurcation, no SMCs originating from outside the local arterial segment were detected in healed plaques. Healing SMCs after atherosclerotic plaque disruption are derived entirely from the local arterial wall and not circulating progenitor cells in apoE-/- mice.

Circadian rhythm disruption impairs tissue homeostasis and exacerbates chronic inflammation in the intestine.

PubMed

Pagel, René; Bär, Florian; Schröder, Torsten; Sünderhauf, Annika; Künstner, Axel; Ibrahim, Saleh M; Autenrieth, Stella E; Kalies, Kathrin; König, Peter; Tsang, Anthony H; Bettenworth, Dominik; Divanovic, Senad; Lehnert, Hendrik; Fellermann, Klaus; Oster, Henrik; Derer, Stefanie; Sina, Christian

2017-11-01

Endogenous circadian clocks regulate 24-h rhythms of physiology and behavior. Circadian rhythm disruption (CRD) is suggested as a risk factor for inflammatory bowel disease. However, the underlying molecular mechanisms remain unknown. Intestinal biopsies from Per1/2 mutant and wild-type (WT) mice were investigated by electron microscopy, immunohistochemistry, and bromodeoxyuridine pulse-chase experiments. TNF-α was injected intraperitoneally, with or without necrostatin-1, into Per1/2 mice or rhythmic and externally desynchronized WT mice to study intestinal epithelial cell death. Experimental chronic colitis was induced by oral administration of dextran sodium sulfate. In vitro , caspase activity was assayed in Per1/2-specific small interfering RNA-transfected cells. Wee1 was overexpressed to study antiapoptosis and the cell cycle. Genetic ablation of circadian clock function or environmental CRD in mice increased susceptibility to severe intestinal inflammation and epithelial dysregulation, accompanied by excessive necroptotic cell death and a reduced number of secretory epithelial cells. Receptor-interacting serine/threonine-protein kinase (RIP)-3-mediated intestinal necroptosis was linked to increased mitotic cell cycle arrest via Per1/2-controlled Wee1, resulting in increased antiapoptosis via cellular inhibitor of apoptosis-2. Together, our data suggest that circadian rhythm stability is pivotal for the maintenance of mucosal barrier function. CRD increases intestinal necroptosis, thus rendering the gut epithelium more susceptible to inflammatory processes.-Pagel, R., Bär, F., Schröder, T., Sünderhauf, A., Künstner, A., Ibrahim, S. M., Autenrieth, S. E., Kalies, K., König, P., Tsang, A. H., Bettenworth, D., Divanovic, S., Lehnert, H., Fellermann, K., Oster, H., Derer, S., Sina, C. Circadian rhythm disruption impairs tissue homeostasis and exacerbates chronic inflammation in the intestine. © FASEB.

Ras1 interacts with multiple new signaling and cytoskeletal loci in Drosophila eggshell patterning and morphogenesis.

PubMed Central

Schnorr, J D; Holdcraft, R; Chevalier, B; Berg, C A

2001-01-01

Little is known about the genes that interact with Ras signaling pathways to regulate morphogenesis. The synthesis of dorsal eggshell structures in Drosophila melanogaster requires multiple rounds of Ras signaling followed by dramatic epithelial sheet movements. We took advantage of this process to identify genes that link patterning and morphogenesis; we screened lethal mutations on the second chromosome for those that could enhance a weak Ras1 eggshell phenotype. Of 1618 lethal P-element mutations tested, 13 showed significant enhancement, resulting in forked and fused dorsal appendages. Our genetic and molecular analyses together with information from the Berkeley Drosophila Genome Project reveal that 11 of these lines carry mutations in previously characterized genes. Three mutations disrupt the known Ras1 cell signaling components Star, Egfr, and Blistered, while one mutation disrupts Sec61beta, implicated in ligand secretion. Seven lines represent cell signaling and cytoskeletal components that are new to the Ras1 pathway; these are Chickadee (Profilin), Tec29, Dreadlocks, POSH, Peanut, Smt3, and MESK2, a suppressor of dominant-negative Ksr. A twelfth insertion disrupts two genes, Nrk, a "neurospecific" receptor tyrosine kinase, and Tpp, which encodes a neuropeptidase. These results suggest that Ras1 signaling during oogenesis involves novel components that may be intimately associated with additional signaling processes and with the reorganization of the cytoskeleton. To determine whether these Ras1 Enhancers function upstream or downstream of the Egf receptor, four mutations were tested for their ability to suppress an activated Egfr construct (lambdatop) expressed in oogenesis exclusively in the follicle cells. Mutations in Star and l(2)43Bb had no significant effect upon the lambdatop eggshell defect whereas smt3 and dock alleles significantly suppressed the lambdatop phenotype. PMID:11606538

Ras1 interacts with multiple new signaling and cytoskeletal loci in Drosophila eggshell patterning and morphogenesis.

PubMed

Schnorr, J D; Holdcraft, R; Chevalier, B; Berg, C A

2001-10-01

Little is known about the genes that interact with Ras signaling pathways to regulate morphogenesis. The synthesis of dorsal eggshell structures in Drosophila melanogaster requires multiple rounds of Ras signaling followed by dramatic epithelial sheet movements. We took advantage of this process to identify genes that link patterning and morphogenesis; we screened lethal mutations on the second chromosome for those that could enhance a weak Ras1 eggshell phenotype. Of 1618 lethal P-element mutations tested, 13 showed significant enhancement, resulting in forked and fused dorsal appendages. Our genetic and molecular analyses together with information from the Berkeley Drosophila Genome Project reveal that 11 of these lines carry mutations in previously characterized genes. Three mutations disrupt the known Ras1 cell signaling components Star, Egfr, and Blistered, while one mutation disrupts Sec61beta, implicated in ligand secretion. Seven lines represent cell signaling and cytoskeletal components that are new to the Ras1 pathway; these are Chickadee (Profilin), Tec29, Dreadlocks, POSH, Peanut, Smt3, and MESK2, a suppressor of dominant-negative Ksr. A twelfth insertion disrupts two genes, Nrk, a "neurospecific" receptor tyrosine kinase, and Tpp, which encodes a neuropeptidase. These results suggest that Ras1 signaling during oogenesis involves novel components that may be intimately associated with additional signaling processes and with the reorganization of the cytoskeleton. To determine whether these Ras1 Enhancers function upstream or downstream of the Egf receptor, four mutations were tested for their ability to suppress an activated Egfr construct (lambdatop) expressed in oogenesis exclusively in the follicle cells. Mutations in Star and l(2)43Bb had no significant effect upon the lambdatop eggshell defect whereas smt3 and dock alleles significantly suppressed the lambdatop phenotype.

Impact of nano zero valent iron (NZVI) on methanogenic activity and population dynamics in anaerobic digestion.

PubMed

Yang, Yu; Guo, Jialiang; Hu, Zhiqiang

2013-11-01

Nano zero valent iron (NZVI), although being increasingly used for environmental remediation, has potential negative impact on methanogenesis in anaerobic digestion. In this study, NZVI (average size = 55 ± 11 nm) showed inhibition of methanogenesis due to its disruption of cell integrity. The inhibition was coincident with the fast hydrogen production and accumulation due to NZVI dissolution under anaerobic conditions. At the concentrations of 1 mM and above, NZVI reduced methane production by more than 20%. At the concentration of 30 mM, NZVI led to a significant increase in soluble COD (an indication of cell disruption) and volatile fatty acids in the mixed liquor along with an accumulation of H2, resulting in a reduction of methane production by 69% (±4% [standard deviation]). By adding a specific methanogenesis inhibitor-sodium 2-bromoethanesulfonate (BES) to the anaerobic sludge containing 30 mM NZVI, the amount of H2 produced was only 79% (±1%) of that with heat-killed sludge, indicating the occurrence of bacterially controlled hydrogen utilization processes. Quantitative PCR data was in accordance with the result of methanogenesis inhibition, as the level of methanogenic population (dominated by Methanosaeta) in the presence of 30 mM NZVI decreased significantly compared to that of the control. On the contrary, ZVI powder (average size <212 μm) at the same concentration (30 mM) increased methane production presumably due to hydrogenotrophic methanogenesis of hydrogen gas that was slowly released from the NZVI powder. While it is a known fact that NZVI disrupts cell membranes, which inhibited methanogenesis described herein, the results suggest that the rapid hydrogen production due to NZVI dissolution also contribute to methanogenesis inhibition and lead to bacterially controlled hydrogenotrophic processes. Copyright © 2013 Elsevier Ltd. All rights reserved.

Double-stranded RNA-dependent protein kinase (pkr) is essential for thermotolerance, accumulation of HSP70, and stabilization of ARE-containing HSP70 mRNA during stress.

PubMed

Zhao, Meijuan; Tang, Dan; Lechpammer, Stanislav; Hoffman, Alexander; Asea, Alexzander; Stevenson, Mary Ann; Calderwood, Stuart K

2002-11-15

We have investigated the role of the double-stranded RNA-dependent protein kinase gene (pkr) in the regulation of the heat shock response. We show that the pkr gene is essential for efficient activation of the heat shock response and that pkr disruption profoundly inhibits heat shock protein 70 (HSP70) synthesis and blocks the development of thermotolerance. Despite these profound effects, pkr disruption did not markedly affect the activation of heat shock factor 1 by heat and did not reduce the rate of transcription of the HSP70 gene after heat shock. However, despite the lack of effect of pkr disruption on HSP70 gene transcription, we found a significant decrease in the expression of HSP70 mRNA in pkr-/- cells after heat shock. Kinetic studies of mRNA turnover suggested a block in the thermal stabilization of HSP70 mRNA in pkr-/- cells. As the thermal stabilization of HSP70 mRNA is thought to involve cis-acting A+U rich (ARE) elements in the 3'-untranslated region (UTR), we examined a potential role for pkr in this process. We found that a reporter beta-galactosidase mRNA destabilized by introduction of a functional ARE into the 3'-UTR became stabilized by heat but only in cells containing an intact pkr gene. Our studies suggest therefore that pkr plays a significant role in the stabilization of mRNA species containing ARE destruction sequences in the 3'-UTR and through this mechanism, contributes to the regulation of the heat shock response and other processes.

The endomembrane requirement for cell surface repair

NASA Technical Reports Server (NTRS)

McNeil, Paul L.; Miyake, Katsuya; Vogel, Steven S.

2003-01-01

The capacity to reseal a plasma membrane disruption rapidly is required for cell survival in many physiological environments. Intracellular membrane (endomembrane) is thought to play a central role in the rapid resealing response. We here directly compare the resealing response of a cell that lacks endomembrane, the red blood cell, with that of several nucleated cells possessing an abundant endomembrane compartment. RBC membrane disruptions inflicted by a mode-locked Ti:sapphire laser, even those initially smaller than hemoglobin, failed to reseal rapidly. By contrast, much larger laser-induced disruptions made in sea urchin eggs, fibroblasts, and neurons exhibited rapid, Ca(2+)-dependent resealing. We conclude that rapid resealing is not mediated by simple physiochemical mechanisms; endomembrane is required.

Embryonic demise caused by targeted disruption of a cysteine protease Dub-2.

PubMed

Baek, Kwang-Hyun; Lee, Heyjin; Yang, Sunmee; Lim, Soo-Bin; Lee, Wonwoo; Lee, Jeoung Eun; Lim, Jung-Jin; Jun, Kisun; Lee, Dong-Ryul; Chung, Young

2012-01-01

A plethora of biological metabolisms are regulated by the mechanisms of ubiquitination, wherein this process is balanced with the action of deubiquitination system. Dub-2 is an IL-2-inducible, immediate-early gene that encodes a deubiquitinating enzyme with growth regulatory activity. DUB-2 presumably removes ubiquitin from ubiquitin-conjugated target proteins regulating ubiquitin-mediated proteolysis, but its specific target proteins are unknown yet. To elucidate the functional role of Dub-2, we generated genetically modified mice by introducing neo cassette into the second exon of Dub-2 and then homologous recombination was done to completely abrogate the activity of DUB-2 proteins. We generated Dub-2+/- heterozygous mice showing a normal phenotype and are fertile, whereas new born mouse of Dub-2-/- homozygous alleles could not survive. In addition, Dub-2-/- embryo could not be seen between E6.5 and E12.5 stages. Furthermore, the number of embryos showing normal embryonic development for further stages is decreased in heterozygotes. Even embryonic stem cells from inner cell mass of Dub-2-/- embryos could not be established. Our study suggests that the targeted disruption of Dub-2 may cause embryonic lethality during early gestation, possibly due to the failure of cell proliferation during hatching process.

Somatostatin-Positive Gamma-Aminobutyric Acid Interneuron Deficits in Depression: Cortical Microcircuit and Therapeutic Perspectives.

PubMed

Fee, Corey; Banasr, Mounira; Sibille, Etienne

2017-10-15

The functional integration of external and internal signals forms the basis of information processing and is essential for higher cognitive functions. This occurs in finely tuned cortical microcircuits whose functions are balanced at the cellular level by excitatory glutamatergic pyramidal neurons and inhibitory gamma-aminobutyric acidergic (GABAergic) interneurons. The balance of excitation and inhibition, from cellular processes to neural network activity, is characteristically disrupted in multiple neuropsychiatric disorders, including major depressive disorder (MDD), bipolar disorder, anxiety disorders, and schizophrenia. Specifically, nearly 3 decades of research demonstrate a role for reduced inhibitory GABA level and function across disorders. In MDD, recent evidence from human postmortem and animal studies suggests a selective vulnerability of GABAergic interneurons that coexpress the neuropeptide somatostatin (SST). Advances in cell type-specific molecular genetics have now helped to elucidate several important roles for SST interneurons in cortical processing (regulation of pyramidal cell excitatory input) and behavioral control (mood and cognition). Here, we review evidence for altered inhibitory function arising from GABAergic deficits across disorders and specifically in MDD. We then focus on properties of the cortical microcircuit, where SST-positive GABAergic interneuron deficits may disrupt functioning in several ways. Finally, we discuss the putative origins of SST cell deficits, as informed by recent research, and implications for therapeutic approaches. We conclude that deficits in SST interneurons represent a contributing cellular pathology and therefore a promising target for normalizing altered inhibitory function in MDD and other disorders with reduced SST cell and GABA functions. Copyright © 2017 Society of Biological Psychiatry. Published by Elsevier Inc. All rights reserved.

Circadian disruption promotes tumor growth by anabolic host metabolism; experimental evidence in a rat model.

PubMed

Guerrero-Vargas, Natalí N; Navarro-Espíndola, Raful; Guzmán-Ruíz, Mara A; Basualdo, María Del Carmen; Espitia-Bautista, Estefania; López-Bago, Ana; Lascurain, Ricardo; Córdoba-Manilla, Cinthya; Buijs, Ruud M; Escobar, Carolina

2017-09-06

Light at night creates a conflicting signal to the biological clock and disrupts circadian physiology. In rodents, light at night increases the risk to develop mood disorders, overweight, disrupted energy metabolism, immune dysfunction and cancer. We hypothesized that constant light (LL) in rats may facilitate tumor growth via disrupted metabolism and increased inflammatory response in the host, inducing a propitious microenvironment for tumor cells. Male Wistar rats were exposed to LL or a regular light-dark cycle (LD) for 5 weeks. Body weight gain, food consumption, triglycerides and glucose blood levels were evaluated; a glucose tolerance test was also performed. Inflammation and sickness behavior were evaluated after the administration of intravenous lipopolysaccharide. Tumors were induced by subcutaneous inoculation of glioma cells (C6). In tumor-bearing rats, the metabolic state and immune cells infiltration to the tumor was investigated by using immunohistochemistry and flow cytometry. The mRNA expression of genes involved metabolic, growth, angiogenes and inflammatory pathways was measured in the tumor microenvironment by qPCR. Tumor growth was also evaluated in animals fed with a high sugar diet. We found that LL induced overweight, high plasma triglycerides and glucose levels as well as reduced glucose clearance. In response to an LPS challenge, LL rats responded with higher pro-inflammatory cytokines and exacerbated sickness behavior. Tumor cell inoculation resulted in increased tumor volume in LL as compared with LD rats, associated with high blood glucose levels and decreased triglycerides levels in the host. More macrophages were recruited in the LL tumor and the microenvironment was characterized by upregulation of genes involved in lipogenesis (Acaca, Fasn, and Pparγ), glucose uptake (Glut-1), and tumor growth (Vegfα, Myc, Ir) suggesting that LL tumors rely on these processes in order to support their enhanced growth. Genes related with the inflammatory state in the tumor microenvironment were not different between LL and LD conditions. In rats fed a high caloric diet tumor growth was similar to LL conditions. Data indicates that circadian disruption by LL provides a favorable condition for tumor growth by promoting an anabolic metabolism in the host.

Comparative Evaluation of Different Cell Lysis and Extraction Methods for Studying Benzo(a)pyrene Metabolism in HT-29 Colon Cancer Cell Cultures

PubMed Central

Myers, Jeremy N.; Rekhadevi, Perumalla V.; Ramesh, Aramandla

2011-01-01

Lysis and extraction of cells are essential sample processing steps for investigations pertaining to metabolism of xenobiotics in cell culture studies. Of particular importance to these procedures are maintaining high lysis efficiency and analyte integrity as they influence the qualitative and quantitative distribution of drug and toxicant metabolites in the intra- and extracellular milieus. In this study we have compared the efficiency of different procedures viz. homogenization, sonication, bead beating, and molecular grinding resin treatment for disruption of HT-29 colon cells exposed to benzo(a)pyrene (BaP), a polycyclic aromatic hydrocarbon (PAH) compound and a suspected colon carcinogen. Also, we have evaluated the efficiency of various procedures for extracting BaP parent compound/metabolites from colon cells and culture media prior to High Performance Liquid Chromatography (HPLC) analyses. The extraction procedures include solid phase extraction, solid-supported liquid- liquid extraction, liquid-liquid extraction, and homogeneous liquid- liquid extraction. Our findings showed that bead-beating in combination with detergent treatment of cell pellet coupled with liquid-liquid extraction yielded greater concentrations of BaP metabolites compared to the other methods employed. Our method optimization strategy revealed that disruption of HT-29 colon cells by a combination of mechanical and chemical lysis followed by liquid-liquid extraction is efficient and robust enough for analyzing BaP metabolites from cell culture studies. PMID:21865728

Circadian physiology of metabolism.

PubMed

Panda, Satchidananda

2016-11-25

A majority of mammalian genes exhibit daily fluctuations in expression levels, making circadian expression rhythms the largest known regulatory network in normal physiology. Cell-autonomous circadian clocks interact with daily light-dark and feeding-fasting cycles to generate approximately 24-hour oscillations in the function of thousands of genes. Circadian expression of secreted molecules and signaling components transmits timing information between cells and tissues. Such intra- and intercellular daily rhythms optimize physiology both by managing energy use and by temporally segregating incompatible processes. Experimental animal models and epidemiological data indicate that chronic circadian rhythm disruption increases the risk of metabolic diseases. Conversely, time-restricted feeding, which imposes daily cycles of feeding and fasting without caloric reduction, sustains robust diurnal rhythms and can alleviate metabolic diseases. These findings highlight an integrative role of circadian rhythms in physiology and offer a new perspective for treating chronic diseases in which metabolic disruption is a hallmark. Copyright © 2016, American Association for the Advancement of Science.

Spermatogenesis disruption by dioxins: Epigenetic reprograming and windows of susceptibility.

PubMed

Pilsner, J Richard; Parker, Mikhail; Sergeyev, Oleg; Suvorov, Alexander

2017-04-01

Dioxins are a group of highly persistent chemicals that are generated as by-products of industrial and natural processes. Reduction in sperm counts is among the most sensitive endpoints of dioxin toxicity. The exact mechanism by which dioxins reduce sperm counts is not known. Recent data implicate the role of epididymal factors rather than disruption of spermatogenesis. Studies reviewed here demonstrate that dioxins induce the transfer of environmental conditions to the next generation via male germline following exposures during the window of epigenetic reprogramming of primordial germ cells. Increased incidence of birth defects in offspring of male veterans exposed to dioxin containing, Agent Orange, suggest that dioxins may induce epigenomic changes in male germ cells of adults during spermatogenesis. This is supported by recent animal data that show that environmental conditions can cause epigenetic dysregulation in sperm in the context of specific windows of epigenetic reprogramming during spermatogenesis. Copyright © 2017 Elsevier Inc. All rights reserved.

Calcium-mediated oxidative stress: a common mechanism in tight junction disruption by different types of cellular stress.

PubMed

Gangwar, Ruchika; Meena, Avtar S; Shukla, Pradeep K; Nagaraja, Archana S; Dorniak, Piotr L; Pallikuth, Sandeep; Waters, Christopher M; Sood, Anil; Rao, RadhaKrishna

2017-02-20

The role of reactive oxygen species (ROS) in osmotic stress, dextran sulfate sodium (DSS) and cyclic stretch-induced tight junction (TJ) disruption was investigated in Caco-2 cell monolayers in vitro and restraint stress-induced barrier dysfunction in mouse colon in vivo Live cell imaging showed that osmotic stress, cyclic stretch and DSS triggered rapid production of ROS in Caco-2 cell monolayers, which was blocked by depletion of intracellular Ca 2+ by 1,2-bis-( o -aminophenoxy)ethane- N , N , N ', N '-tetraacetic acid. Knockdown of Ca V 1.3 or TRPV6 channels blocked osmotic stress and DSS-induced ROS production and attenuated TJ disruption and barrier dysfunction. N -Acetyl l-cysteine (NAC) and l- N G -Nitroarginine methyl ester (l-NAME) blocked stress-induced TJ disruption and barrier dysfunction. NAC and l-NAME also blocked stress-induced activation of c-Jun N -terminal kinase (JNK) and c-Src. ROS was colocalized with the mitochondrial marker in stressed cells. Cyclosporin A blocked osmotic stress and DSS-induced ROS production, barrier dysfunction, TJ disruption and JNK activation. Mitochondria-targeted Mito-TEMPO blocked osmotic stress and DSS-induced barrier dysfunction and TJ disruption. Chronic restraint stress in mice resulted in the elevation of intracellular Ca 2+ , activation of JNK and c-Src, and disruption of TJ in the colonic epithelium. Furthermore, corticosterone administration induced JNK and c-Src activation, TJ disruption and protein thiol oxidation in colonic mucosa. The present study demonstrates that oxidative stress is a common signal in the mechanism of TJ disruption in the intestinal epithelium by different types of cellular stress in vitro and bio behavioral stress in vivo . © 2017 The Author(s); published by Portland Press Limited on behalf of the Biochemical Society.

Calcium-Mediated Oxidative Stress: a Common Mechanism in Tight Junction Disruption by Different Types of Cellular Stress

PubMed Central

Gangwar, Ruchika; Meena, Avtar S.; Shukla, Pradeep K.; Nagaraja, Archana S.; Dorniak, Piotr L.; Pallikuth, Sandeep; Waters, Christopher M.; Sood, Anil; Rao, RadhaKrishna

2017-01-01

The role of reactive oxygen species (ROS) in osmotic stress, dextran sulfate sodium (DSS) and cyclic stretch-induced tight junction disruption was investigated in Caco-2 cell monolayers in vitro, and restraint stress-induced barrier dysfunction in mouse colon in vivo. Live cell imaging showed that osmotic stress, cyclic stretch and DSS triggered rapid production of ROS in Caco-2 cell monolayers, which was blocked by depletion of intracellular Ca2+ by BAPTA. Knockdown of CaV1.3 or TRPV6 channels blocked osmotic stress and DSS-induced ROS production and attenuated tight junction disruption and barrier dysfunction. N-acetyl L-cysteine (NAC) and L-nitroarginine methyl ester (L-NAME) blocked stress-induced tight junction disruption and barrier dysfunction. NAC and L-NAME also blocked stress-induced activation of JNK and c-Src. ROS was co-localized with the mitochondrial marker in stressed cells. Cyclosporin A blocked osmotic stress and DSS-induced ROS production, barrier dysfunction, tight junction disruption and JNK activation. Mitochondria-targeted Mito-TEMPO blocked osmotic stress and DSS-induced barrier dysfunction and tight junction disruption. Chronic restraint stress in mice resulted in the elevation of intracellular Ca2+, activation of JNK and c-Src, and disruption of tight junction in the colonic epithelium. Furthermore, corticosterone administration induced JNK and c-Src activation, tight junction disruption and protein thiol oxidation in colonic mucosa. This study demonstrates that oxidative stress is a common signal in the mechanism of tight junction disruption in the intestinal epithelium by different types of cellular stress in vitro and bio behavioral stress in vivo. PMID:28057718

VE-cadherin cleavage by ovarian cancer microparticles induces β-catenin phosphorylation in endothelial cells

PubMed Central

Thawadi, Hamda Al; Abu-Kaoud, Nadine; Farsi, Haleema Al; Hoarau-Véchot, Jessica; Rafii, Shahin; Rafii, Arash; Pasquier, Jennifer

2016-01-01

Microparticles (MPs) are increasingly recognized as important mediators of cell-cell communication in tumour growth and metastasis by facilitating angiogenesis-related processes. While the effects of the MPs on recipient cells are usually well described in the literature, the leading process remains unclear. Here we isolated MPs from ovarian cancer cells and investigated their effect on endothelial cells. First, we demonstrated that ovarian cancer MPs trigger β-catenin activation in endothelial cells, inducing the upregulation of Wnt/β-catenin target genes and an increase of angiogenic properties. We showed that this MPs mediated activation of β-catenin in ECs was Wnt/Frizzled independent; but dependent on VE-cadherin localization disruption, αVβ3 integrin activation and MMP activity. Finally, we revealed that Rac1 and AKT were responsible for β-catenin phosphorylation and translocation to the nucleus. Overall, our results indicate that MPs released from cancer cells could play a major role in neo-angiogenesis through activation of beta catenin pathway in endothelial cells. PMID:26700621

Production of Prnp-/- goats by gene targeting in adult fibroblasts.

PubMed

Zhu, Caihong; Li, Bei; Yu, Guohua; Chen, Jianquan; Yu, Huiqing; Chen, Juan; Xu, Xujun; Wu, Youbing; Zhang, Aimin; Cheng, Guoxiang

2009-04-01

Homozygous mice devoid of functional Prnp are resistant to scrapie and prion propagation, but heterozygous mice for Prnp disruption still suffer from prion disease and prion deposition. We have previously generated heterozygous cloned goats with one allele of Prnp functional disruption. To obtain goats with both alleles of Prnp be disrupted which would be resistant to scrapie completely, a second-round gene targeting was applied to disrupt the wild type allele of Prnp in the heterozygous goats. By second-round gene targeting, we successfully disrupted the wild type allele of Prnp in primary Prnp (+/-) goat skin fibroblasts and obtained a Prnp (-/-) cell line without Prnp expression. This is the first report on successful targeting modification in primary adult somatic cells of animals. These cells were used as nuclear donors for somatic cell cloning to produce Prnp (-/-) goats. A total of 57 morulae or blastocytes developed from the reconstructed embryos were transferred to 31 recipients, which produced 7 pregnancies at day 35. At 73 days of gestation, we obtained one cloned fetus with Prnp (-/-) genotype. Our research not only indicated that multiple genetic modifications could be accomplished by multi-round gene targeting in primary somatic cells, but also provided strong evidence that gene targeting in adult cells other than fetal cells could be applied to introduce precise genetic modifications in animals without destroying the embryos.

How the evolution of multicellularity set the stage for cancer

PubMed Central

Trigos, Anna S; Pearson, Richard B; Papenfuss, Anthony T; Goode, David L

2018-01-01

Neoplastic growth and many of the hallmark properties of cancer are driven by the disruption of molecular networks established during the emergence of multicellularity. Regulatory pathways and molecules that evolved to impose regulatory constraints upon networks established in earlier unicellular organisms enabled greater communication and coordination between the diverse cell types required for multicellularity, but also created liabilities in the form of points of vulnerability in the network that when mutated or dysregulated facilitate the development of cancer. These factors are usually overlooked in genomic analyses of cancer, but understanding where vulnerabilities to cancer lie in the networks of multicellular species would provide important new insights into how core molecular processes and gene regulation change during tumourigenesis. We describe how the evolutionary origins of genes influence their roles in cancer, and how connections formed between unicellular and multicellular genes that act as key regulatory hubs for normal tissue homeostasis can also contribute to malignant transformation when disrupted. Tumours in general are characterised by increased dependence on unicellular processes for survival, and major dysregulation of the control structures imposed on these processes during the evolution of multicellularity. Mounting molecular evidence suggests altered interactions at the interface between unicellular and multicellular genes play key roles in the initiation and progression of cancer. Furthermore, unicellular network regions activated in cancer show high degrees of robustness and plasticity, conferring increased adaptability to tumour cells by supporting effective responses to environmental pressures such as drug exposure. Examining how the links between multicellular and unicellular regions get disrupted in tumours has great potential to identify novel drivers of cancer, and to guide improvements to cancer treatment by identifying more effective therapeutic strategies. Recent successes in targeting unicellular processes by novel compounds underscore the logic of such approaches. Further gains could come from identifying genes at the interface between unicellular and multicellular processes and manipulating the communication between network regions of different evolutionary ages. PMID:29337961

Arsenic as an Endocrine Disruptor: Arsenic Disrupts Retinoic Acid Receptor–and Thyroid Hormone Receptor–Mediated Gene Regulation and Thyroid Hormone–Mediated Amphibian Tail Metamorphosis

PubMed Central

Davey, Jennifer C.; Nomikos, Athena P.; Wungjiranirun, Manida; Sherman, Jenna R.; Ingram, Liam; Batki, Cavus; Lariviere, Jean P.; Hamilton, Joshua W.

2008-01-01

Background Chronic exposure to excess arsenic in drinking water has been strongly associated with increased risks of multiple cancers, diabetes, heart disease, and reproductive and developmental problems in humans. We previously demonstrated that As, a potent endocrine disruptor at low, environmentally relevant levels, alters steroid signaling at the level of receptor-mediated gene regulation for all five steroid receptors. Objectives The goal of this study was to determine whether As can also disrupt gene regulation via the retinoic acid (RA) receptor (RAR) and/or the thyroid hormone (TH) receptor (TR) and whether these effects are similar to previously observed effects on steroid regulation. Methods and results Human embryonic NT2 or rat pituitary GH3 cells were treated with 0.01–5 μM sodium arsenite for 24 hr, with or without RA or TH, respectively, to examine effects of As on receptor-mediated gene transcription. At low, noncytotoxic doses, As significantly altered RAR-dependent gene transcription of a transfected RAR response element–luciferase construct and the native RA-inducible cytochrome P450 CYP26A gene in NT2 cells. Likewise, low-dose As significantly altered expression of a transfected TR response element–luciferase construct and the endogenous TR-regulated type I deiodinase (DIO1) gene in a similar manner in GH3 cells. An amphibian ex vivo tail metamorphosis assay was used to examine whether endocrine disruption by low-dose As could have specific pathophysiologic consequences, because tail metamorphosis is tightly controlled by TH through TR. TH-dependent tail shrinkage was inhibited in a dose-dependent manner by 0.1– 4.0 μM As. Conclusions As had similar effects on RAR- and TR-mediated gene regulation as those previously observed for the steroid receptors, suggesting a common mechanism or action. Arsenic also profoundly affected a TR-dependent developmental process in a model animal system at very low concentrations. Because RAR and TH are critical for both normal human development and adult function and their dysregulation is associated with many disease processes, disruption of these hormone receptor–dependent processes by As is also potentially relevant to human developmental problems and disease risk. PMID:18288313

Quantifying pulsed electric field-induced membrane nanoporation in single cells.

PubMed

Moen, Erick K; Ibey, Bennett L; Beier, Hope T; Armani, Andrea M

2016-11-01

Plasma membrane disruption can trigger a host of cellular activities. One commonly observed type of disruption is pore formation. Molecular dynamic (MD) simulations of simplified lipid membrane structures predict that controllably disrupting the membrane via nano-scale poration may be possible with nanosecond pulsed electric fields (nsPEF). Until recently, researchers hoping to verify this hypothesis experimentally have been limited to measuring the relatively slow process of fluorescent markers diffusing across the membrane, which is indirect evidence of nanoporation that could be channel-mediated. Leveraging recent advances in nonlinear optical microscopy, we elucidate the role of pulse parameters in nsPEF-induced membrane permeabilization in live cells. Unlike previous techniques, it is able to directly observe loss of membrane order at the onset of the pulse. We also develop a complementary theoretical model that relates increasing membrane permeabilization to membrane pore density. Due to the significantly improved spatial and temporal resolution possible with our imaging method, we are able to directly compare our experimental and theoretical results. Their agreement provides substantial evidence that nanoporation does occur and that its development is dictated by the electric field distribution. Copyright © 2016 Elsevier B.V. All rights reserved.

Low-molecular-weight hyaluronan (LMW-HA) accelerates lymph node metastasis of melanoma cells by inducing disruption of lymphatic intercellular adhesion.

PubMed

Du, Yan; Cao, Manlin; Liu, Yiwen; He, Yiqing; Yang, Cuixia; Wu, Man; Zhang, Guoliang; Gao, Feng

2016-01-01

Endothelial integrity defects initiate lymphatic metastasis of tumor cells. Low-molecular-weight hyaluronan (LMW-HA) derived from plasma and interstitial fluid was reported to be associated with tumor lymphatic metastasis. In addition, LMW-HA was proved to disrupt lymphatic vessel endothelium integrity, thus promoting lymphatic metastasis of tumor cells. Until now, there are few reports on how LMW-HA modulates lymphatic endothelial cells adhesion junctions and affects cancer cells metastasizing into lymph vessels. The aim of our study is to unravel the novel mechanism of LMW-HA in mediating tumor lymphatic metastasis. Here, we employed a melanoma metastasis model to investigate whether LMW-HA facilitates tumor cells transferring from foci to remote lymph nodes by disrupting the lymphatic endothelial integrity. Our data indicate that LMW-HA significantly induces metastasis of melanoma cells to lymph nodes and accelerates interstitial-lymphatic flow in vivo . Further experiments show that increased migration of melanoma cells across human dermal lymphatic endothelial cell (HDLEC) monolayers is accompanied by impaired lymphatic endothelial barrier function and increased permeability. The mechanism study reveals that VE-cadherin-β-catenin pathway and relevant signals are involved in modulating the interactions between endothelial cells and that a significant inhibition of lymphatic endothelium disruption is observed when antibodies to the LMW-HA receptor (LYVE-1) are present. Thus, our findings demonstrate a disruptive effect of LMW-HA on lymphatic endothelium continuity which leads to a promotion on melanoma lymphatic metastasis and also suggest a cellular signaling mechanism associated with VE-cadherin-mediated lymphatic intercellular junctions.

Designing Endocrine Disruption Out of the Next Generation of Chemicals

PubMed Central

Schug, T.T; Abagyan, R.; Blumberg, B.; Collins, T.J.; Crews, D.; DeFur, P.L.; Dickerson, S.M.; Edwards, T.M.; Gore, A.C.; Guillette, L.J.; Hayes, T.; Heindel, J.J.; Moores, A.; Patisaul, H.B.; Tal, T.L.; Thayer, K.A.; Vandenberg, L.N.; Warner, J.; Watson, C.S.; Saal, F.S. vom; Zoeller, R.T.; O’Brien, K.P.; Myers, J.P.

2013-01-01

A central goal of green chemistry is to avoid hazard in the design of new chemicals. This objective is best achieved when information about a chemical’s potential hazardous effects is obtained as early in the design process as feasible. Endocrine disruption is a type of hazard that to date has been inadequately addressed by both industrial and regulatory science. To aid chemists in avoiding this hazard, we propose an endocrine disruption testing protocol for use by chemists in the design of new chemicals. The Tiered Protocol for Endocrine Disruption (TiPED) has been created under the oversight of a scientific advisory committee composed of leading representatives from both green chemistry and the environmental health sciences. TiPED is conceived as a tool for new chemical design, thus it starts with a chemist theoretically at “the drawing board.” It consists of five testing tiers ranging from broad in silico evaluation up through specific cell- and whole organism-based assays. To be effective at detecting endocrine disruption, a testing protocol must be able to measure potential hormone-like or hormone-inhibiting effects of chemicals, as well as the many possible interactions and signaling sequellae such chemicals may have with cell-based receptors. Accordingly, we have designed this protocol to broadly interrogate the endocrine system. The proposed protocol will not detect all possible mechanisms of endocrine disruption, because scientific understanding of these phenomena is advancing rapidly. To ensure that the protocol remains current, we have established a plan for incorporating new assays into the protocol as the science advances. In this paper we present the principles that should guide the science of testing new chemicals for endocrine disruption, as well as principles by which to evaluate individual assays for applicability, and laboratories for reliability. In a ‘proof-of-principle’ test, we ran 6 endocrine disrupting chemicals (EDCs) that act via different endocrinological mechanisms through the protocol using published literature. Each was identified as endocrine active by one or more tiers. We believe that this voluntary testing protocol will be a dynamic tool to facilitate efficient and early identification of potentially problematic chemicals, while ultimately reducing the risks to public health. PMID:25110461

Designing Endocrine Disruption Out of the Next Generation of Chemicals.

PubMed

Schug, T T; Abagyan, R; Blumberg, B; Collins, T J; Crews, D; DeFur, P L; Dickerson, S M; Edwards, T M; Gore, A C; Guillette, L J; Hayes, T; Heindel, J J; Moores, A; Patisaul, H B; Tal, T L; Thayer, K A; Vandenberg, L N; Warner, J; Watson, C S; Saal, F S Vom; Zoeller, R T; O'Brien, K P; Myers, J P

2013-01-01

A central goal of green chemistry is to avoid hazard in the design of new chemicals. This objective is best achieved when information about a chemical's potential hazardous effects is obtained as early in the design process as feasible. Endocrine disruption is a type of hazard that to date has been inadequately addressed by both industrial and regulatory science. To aid chemists in avoiding this hazard, we propose an endocrine disruption testing protocol for use by chemists in the design of new chemicals. The Tiered Protocol for Endocrine Disruption (TiPED) has been created under the oversight of a scientific advisory committee composed of leading representatives from both green chemistry and the environmental health sciences. TiPED is conceived as a tool for new chemical design, thus it starts with a chemist theoretically at "the drawing board." It consists of five testing tiers ranging from broad in silico evaluation up through specific cell- and whole organism-based assays. To be effective at detecting endocrine disruption, a testing protocol must be able to measure potential hormone-like or hormone-inhibiting effects of chemicals, as well as the many possible interactions and signaling sequellae such chemicals may have with cell-based receptors. Accordingly, we have designed this protocol to broadly interrogate the endocrine system. The proposed protocol will not detect all possible mechanisms of endocrine disruption, because scientific understanding of these phenomena is advancing rapidly. To ensure that the protocol remains current, we have established a plan for incorporating new assays into the protocol as the science advances. In this paper we present the principles that should guide the science of testing new chemicals for endocrine disruption, as well as principles by which to evaluate individual assays for applicability, and laboratories for reliability. In a 'proof-of-principle' test, we ran 6 endocrine disrupting chemicals (EDCs) that act via different endocrinological mechanisms through the protocol using published literature. Each was identified as endocrine active by one or more tiers. We believe that this voluntary testing protocol will be a dynamic tool to facilitate efficient and early identification of potentially problematic chemicals, while ultimately reducing the risks to public health.

A Single Disulfide Bond Disruption in the β3 Integrin Subunit Promotes Thiol/Disulfide Exchange, a Molecular Dynamics Study

PubMed Central

Levin, Lihie; Zelzion, Ehud; Nachliel, Esther; Gutman, Menachem; Tsfadia, Yossi; Einav, Yulia

2013-01-01

The integrins are a family of membrane receptors that attach a cell to its surrounding and play a crucial function in cell signaling. The combination of internal and external stimuli alters a folded non-active state of these proteins to an extended active configuration. The β3 subunit of the platelet αIIbβ3 integrin is made of well-structured domains rich in disulfide bonds. During the activation process some of the disulfides are re-shuffled by a mechanism requiring partial reduction of some of these bonds; any disruption in this mechanism can lead to inherent blood clotting diseases. In the present study we employed Molecular Dynamics simulations for tracing the sequence of structural fluctuations initiated by a single cysteine mutation in the β3 subunit of the receptor. These simulations showed that in-silico protein mutants exhibit major conformational deformations leading to possible disulfide exchange reactions. We suggest that any mutation that prevents Cys560 from reacting with one of the Cys567–Cys581 bonded pair, thus disrupting its ability to participate in a disulfide exchange reaction, will damage the activation mechanism of the integrin. This suggestion is in full agreement with previously published experiments. Furthermore, we suggest that rearrangement of disulfide bonds could be a part of a natural cascade of thiol/disulfide exchange reactions in the αIIbβ3 integrin, which are essential for the native activation process. PMID:23527123

Impaired lysosomal activity mediated autophagic flux disruption by graphite carbon nanofibers induce apoptosis in human lung epithelial cells through oxidative stress and energetic impairment.

PubMed

Mittal, Sandeep; Sharma, Pradeep Kumar; Tiwari, Ratnakar; Rayavarapu, Raja Gopal; Shankar, Jai; Chauhan, Lalit Kumar Singh; Pandey, Alok Kumar

2017-04-28

Graphite carbon nanofibers (GCNF) have emerged as a potential alternative of carbon nanotubes (CNT) for various biomedical applications due to their superior physico-chemical properties. Therefore in-depth understanding of the GCNF induced toxic effects and underlying mechanisms in biological systems is of great interest. Currently, autophagy activation by nanomaterials is recognized as an emerging toxicity mechanism. However, the association of GCNF induced toxicity with this form of cell death is largely unknown. In this study, we have assessed the possible mechanism; especially the role of autophagy, underlying the GCNF induced toxicity. Human lung adenocarcinoma (A549) cells were exposed to a range of GCNF concentrations and various cellular parameters were analyzed (up to 48 h). Transmission electron microscopy, immunofluorescent staining, western blot and quantitative real time PCR were performed to detect apoptosis, autophagy induction, lysosomal destabilization and cytoskeleton disruption in GCNF exposed cells. DCFDA assay was used to evaluate the reactive oxygen species (ROS) production. Experiments with N-acetyl-L-cysteine (NAC), 3-methyladenine (3-MA) and LC3 siRNA was carried out to confirm the involvement of oxidative stress and autophagy in GCNF induced cell death. Comet assay and micronucleus (MN) assay was performed to assess the genotoxicity potential. In the present study, GCNF was found to induce nanotoxicity in human lung cells through autophagosomes accumulation followed by apoptosis via intracellular ROS generation. Mechanistically, impaired lysosomal function and cytoskeleton disruption mediated autophagic flux blockade was found to be the major cause of accumulation rather than autophagy induction which further activates apoptosis. The whole process was in line with the increased ROS level and their pharmacological inhibition leads to mitigation of GCNF induced cell death. Moreover the inhibition of autophagy attenuates apoptosis indicating the role of autophagy as cell death process. GCNF was also found to induce genomic instability. Our present study demonstrates that GCNF perturbs various interrelated signaling pathway and unveils the potential nanotoxicity mechanism of GCNF through targeting ROS-autophagy-apoptosis axis. The current study is significant to evaluate the safety and risk assessment of fibrous carbon nanomaterials prior to their potential use and suggests caution on their utilization for biomedical research.

Neutral sphingomyelinase 2 modulates cytotoxic effects of protopanaxadiol on different human cancer cells

PubMed Central

2013-01-01

Background Some of ginsenosides, root extracts from Panax ginseng, exert cytotoxicity against cancer cells through disruption of membrane subdomains called lipid rafts. Protopanaxadiol (PPD) exhibits the highest cytotoxic effect among 8 ginsenosides which we evaluated for anti-cancer activity. We investigated if PPD disrupts lipid rafts in its cytotoxic effects and also the possible mechanisms. Methods Eight ginsenosides were evaluated using different cancer cells and cell viability assays. The potent ginsenoside, PPD was investigated for its roles in lipid raft disruption and downstream pathways to apoptosis of cancer cells. Anti-cancer effects of PPD was also investigated in vivo using mouse xenograft model. Results PPD consistently exerts its potent cytotoxicity in 2 cell survival assays using 5 different cancer cell lines. PPD disrupts lipid rafts in different ways from methyl-β-cyclodextrin (MβCD) depleting cholesterol out of the subdomains, since lipid raft proteins were differentially modulated by the saponin. During disruption of lipid rafts, PPD activated neutral sphingomyelinase 2 (nSMase 2) hydrolyzing membrane sphingomyelins into pro-apoptotic intracellular ceramides. Furthermore, PPD demonstrated its anti-cancer activities against K562 tumor cells in mouse xenograft model, confirming its potential as an adjunct or chemotherapeutic agent by itself in vivo. Conclusions This study demonstrates that neutral sphingomyelinase 2 is responsible for the cytotoxicity of PPD through production of apoptotic ceramides from membrane sphingomyelins. Thus neutral sphingomyelinase 2 and its relevant mechanisms may potentially be employed in cancer chemotherapies. PMID:23889969

Histological assessments on the abnormalities of mouse epiphyseal chondrocytes with short term centrifugal loading.

PubMed

de Freitas, Paulo Henrique Luiz; Kojima, Taku; Ubaidus, Sobhan; Li, Minqi; Shang, Guangwei; Takagi, Ritsuo; Maeda, Takeyasu; Oda, Kimimitsu; Ozawa, Hidehiro; Amizuka, Norio

2007-08-01

We have examined the morphological changes in chondrocytes after exposure to experimental hypergravity. Tibial epiphyseal cartilages of 17-days-old mouse fetuses were exposed to centrifugation at 3G for 16 h mimicking hypergravitational environment (experimental group), or subjected to stationary cultures (control group). Centrifugation did not affect the sizes of epiphyseal cartilage, chondrocyte proliferation, type X collagen-positive hypertrophic zone, and the mRNA expressions of parathyroid hormone-related peptide and fibroblast growth factor receptor III. However, centrifuged chondrocytes showed abnormal morphology and aberrant spatial arrangements, resulting in disrupted chondrocytic columns. Through histochemical assessments, actin filaments were shown to distribute evenly along cell membranes of control proliferative chondrocytes, while chondrocytes subjected to centrifugal force developed a thicker layer of actin filaments. Transmission electron microscopic observations revealed spotty electron-dense materials underlying control chondrocytes' cell membranes, while experimental chondrocytes showed their thick layer. In the intracolumnar regions of the control cartilage, longitudinal electron-dense fibrils were associated with short cytoplasmic processes of normal chondrocytes, indicating assumed cell-tomatrix interactions. These extracellular fibrils were disrupted in the centrifuged samples. Summarizing, altered actin filaments associated with cell membranes, irregular cell shape and disappearance of intracolumnar extracellular fibrils suggest that hypergravity disturbs cell-to-matrix interactions in our cartilage model.

Elucidation of the Ebola virus VP24 cellular interactome and disruption of virus biology through targeted inhibition of host-cell protein function.

PubMed

García-Dorival, Isabel; Wu, Weining; Dowall, Stuart; Armstrong, Stuart; Touzelet, Olivier; Wastling, Jonathan; Barr, John N; Matthews, David; Carroll, Miles; Hewson, Roger; Hiscox, Julian A

2014-11-07

Viral pathogenesis in the infected cell is a balance between antiviral responses and subversion of host-cell processes. Many viral proteins specifically interact with host-cell proteins to promote virus biology. Understanding these interactions can lead to knowledge gains about infection and provide potential targets for antiviral therapy. One such virus is Ebola, which has profound consequences for human health and causes viral hemorrhagic fever where case fatality rates can approach 90%. The Ebola virus VP24 protein plays a critical role in the evasion of the host immune response and is likely to interact with multiple cellular proteins. To map these interactions and better understand the potential functions of VP24, label-free quantitative proteomics was used to identify cellular proteins that had a high probability of forming the VP24 cellular interactome. Several known interactions were confirmed, thus placing confidence in the technique, but new interactions were also discovered including one with ATP1A1, which is involved in osmoregulation and cell signaling. Disrupting the activity of ATP1A1 in Ebola-virus-infected cells with a small molecule inhibitor resulted in a decrease in progeny virus, thus illustrating how quantitative proteomics can be used to identify potential therapeutic targets.

Overexpression of Arabidopsis thaliana PTEN caused accumulation of autophagic bodies in pollen tubes by disrupting phosphatidylinositol 3-phosphate dynamics

USDA-ARS?s Scientific Manuscript database

Autophagy is a pathway in eukaryotes by which nutrient remobilization occurs through bulk protein and organelle turnover. Autophagy not only aides cells in coping with harsh environments but also plays a key role in many physiological processes that include pollen germination and tube growth. Most a...

On the energy budget in the current disruption region. [of geomagnetic tail

NASA Technical Reports Server (NTRS)

Hesse, Michael; Birn, Joachim

1993-01-01

This study investigates the energy budget in the current disruption region of the magnetotail, coincident with a pre-onset thin current sheet, around substorm onset time using published observational data and theoretical estimates. We find that the current disruption/dipolarization process typically requires energy inflow into the primary disruption region. The disruption dipolarization process is therefore endoenergetic, i.e., requires energy input to operate. Therefore we argue that some other simultaneously operating process, possibly a large scale magnetotail instability, is required to provide the necessary energy input into the current disruption region.

Advances of Stem Cell Therapeutics in Cutaneous Wound Healing and Regeneration

PubMed Central

Kanji, Suman

2017-01-01

Cutaneous wound healing is a complex multiple phase process, which overlaps each other, where several growth factors, cytokines, chemokines, and various cells interact in a well-orchestrated manner. However, an imbalance in any of these phases and factors may lead to disruption in harmony of normal wound healing process, resulting in transformation towards chronic nonhealing wounds and abnormal scar formation. Although various therapeutic interventions are available to treat chronic wounds, current wound-care has met with limited success. Progenitor stem cells possess potential therapeutic ability to overcome limitations of the present treatments as it offers accelerated wound repair with tissue regeneration. A substantial number of stem cell therapies for cutaneous wounds are currently under development as a result of encouraging preliminary findings in both preclinical and clinical studies. However, the mechanisms by which these stem cells contribute to the healing process have yet to be elucidated. In this review, we emphasize on the major treatment modalities currently available for the treatment of the wound, role of various interstitial stem cells and exogenous adult stem cells in cutaneous wound healing, and possible mechanisms involved in the healing process. PMID:29213192

Perinuclear Arp2/3-driven actin polymerization enables nuclear deformation to facilitate cell migration through complex environments

PubMed Central

Thiam, Hawa-Racine; Vargas, Pablo; Carpi, Nicolas; Crespo, Carolina Lage; Raab, Matthew; Terriac, Emmanuel; King, Megan C.; Jacobelli, Jordan; Alberts, Arthur S.; Stradal, Theresia; Lennon-Dumenil, Ana-Maria; Piel, Matthieu

2016-01-01

Cell migration has two opposite faces: although necessary for physiological processes such as immune responses, it can also have detrimental effects by enabling metastatic cells to invade new organs. In vivo, migration occurs in complex environments and often requires a high cellular deformability, a property limited by the cell nucleus. Here we show that dendritic cells, the sentinels of the immune system, possess a mechanism to pass through micrometric constrictions. This mechanism is based on a rapid Arp2/3-dependent actin nucleation around the nucleus that disrupts the nuclear lamina, the main structure limiting nuclear deformability. The cells' requirement for Arp2/3 to pass through constrictions can be relieved when nuclear stiffness is decreased by suppressing lamin A/C expression. We propose a new role for Arp2/3 in three-dimensional cell migration, allowing fast-moving cells such as leukocytes to rapidly and efficiently migrate through narrow gaps, a process probably important for their function. PMID:26975831

Endoplasmic Reticulum Stress, Calcium Dysregulation and Altered Protein Translation: Intersection of Processes That Contribute to Cancer Cachexia Induced Skeletal Muscle Wasting.

PubMed

Isaac, Stephanie T; Tan, Timothy C; Polly, Patsie

2016-01-01

Cancer cachexia is a debilitating paraneoplastic wasting syndrome characterized by skeletal muscle depletion and unintentional weight loss. It affects up to 50-80% of patients with cancer and directly accounts for one-quarter of cancer-related deaths due to cardio-respiratory failure. Muscle weakness, one of the hallmarks of this syndrome, has been postulated to be due to a combination of muscle breakdown, dysfunction and decrease in the ability to repair, with effective treatment strategies presently limited. Excessive inflammatory cytokine levels due to the host-tumor interaction, such as Interleukin (IL)-6 and Tumor Necrosis Factor (TNF)-α, are hypothesised to drive this pathological process but the specific mechanisms by which these cytokines produce skeletal muscle dysfunction in cancer cachexia remain undefined. Endoplasmic Reticulum (ER) stress and the associated disruptions in calcium signaling have been implicated in cytokine-mediated disruptions in skeletal muscle and function. Disrupted ER stress-related processes such as the Unfolded Protein Response (UPR), calcium homeostasis and altered muscle protein synthesis have been reported in clinical and experimental cachexia and other inflammation-driven muscle diseases such as myositis, potentially suggesting a link between increased IL-6 and TNF-α and ER stress in skeletal muscle cells. As the concept of upregulated ER stress in skeletal muscle cells due to elevated cytokines is novel and potentially very relevant to our understanding of cancer cachexia, this review aims to examine the potential relationship between inflammatory cytokine mediated muscle breakdown and ER stress, in the context of cancer cachexia, and to discuss the molecular signaling pathways underpinning this pathology.

Neutrophil proteinase 3 (PR3) acts on protease-activated receptor-2 (PAR-2) to enhance vascular endothelial cell barrier function

PubMed Central

Kuckleburg, Christopher J.; Newman, Peter J.

2013-01-01

The principle role of the vascular endothelium is to present a semi-impermeable barrier to soluble factors and circulating cells, while still permitting the passage of leukocytes from the bloodstream into the tissue. The process of diapedesis involves the selective disruption of endothelial cell junctions, an event that could in theory compromise vascular integrity. It is therefore somewhat surprising that neutrophil transmigration does not significantly impair endothelial barrier function. We examined whether neutrophils might secrete factors that promote vascular integrity during the latter stages of neutrophil transmigration, and found that neutrophil proteinase 3 (PR3) – a serine protease harbored in azurophilic granules – markedly enhanced barrier function in endothelial cells. PR3 functioned in this capacity both in its soluble form and in a complex with cell-surface NB1. PR3-mediated enhancement of endothelial cell junctional integrity required its proteolytic activity, as well as endothelial cell expression of the protease-activated receptor, PAR-2. Importantly, PR3 suppressed the vascular permeability changes and disruption of junctional proteins induced by the action of PAR-1 agonists. These findings establish the potential for neutrophil-derived PR3 to play a role in reestablishing vascular integrity following leukocyte transmigration, and in protecting endothelial cells from PAR-1-induced permeability changes that occur during thrombotic and inflammatory events. PMID:23202369

Enzymatic cell wall degradation of high-pressure-homogenized tomato puree and its effect on lycopene bioaccessibility.

PubMed

Palmero, Paola; Colle, Ines; Lemmens, Lien; Panozzo, Agnese; Nguyen, Tuyen Thi My; Hendrickx, Marc; Van Loey, Ann

2016-01-15

High-pressure homogenization disrupts cell structures, assisting carotenoid release from the matrix and subsequent micellarization. However, lycopene bioaccessibility of tomato puree upon high-pressure homogenization is limited by the formation of a process-induced barrier. In this context, cell wall-degrading enzymes were applied to hydrolyze the formed barrier and enhance lycopene bioaccessibility. The effectiveness of the enzymes in degrading their corresponding substrates was evaluated (consistency, amount of reducing sugars, molar mass distribution and immunolabeling). An in vitro digestion procedure was applied to evaluate the effect of the enzymatic treatments on lycopene bioaccessibility. Enzymatic treatments with pectinases and cellulase were proved to effectively degrade their corresponding cell wall polymers; however, no further significant increase in lycopene bioaccessibility was obtained. A process-induced barrier consisting of cell wall material is not the only factor governing lycopene bioaccessibility upon high-pressure homogenization. © 2015 Society of Chemical Industry.

The Parkinsonian mimetic, 6-OHDA, impairs axonal transport in dopaminergic axons

PubMed Central

2014-01-01

6-hydroxydopamine (6-OHDA) is one of the most commonly used toxins for modeling degeneration of dopaminergic (DA) neurons in Parkinson's disease. 6-OHDA also causes axonal degeneration, a process that appears to precede the death of DA neurons. To understand the processes involved in 6-OHDA-mediated axonal degeneration, a microdevice designed to isolate axons fluidically from cell bodies was used in conjunction with green fluorescent protein (GFP)-labeled DA neurons. Results showed that 6-OHDA quickly induced mitochondrial transport dysfunction in both DA and non-DA axons. This appeared to be a general effect on transport function since 6-OHDA also disrupted transport of synaptophysin-tagged vesicles. The effects of 6-OHDA on mitochondrial transport were blocked by the addition of the SOD1-mimetic, Mn(III)tetrakis(4-benzoic acid)porphyrin chloride (MnTBAP), as well as the anti-oxidant N-acetyl-cysteine (NAC) suggesting that free radical species played a role in this process. Temporally, microtubule disruption and autophagy occurred after transport dysfunction yet before DA cell death following 6-OHDA treatment. The results from the study suggest that ROS-mediated transport dysfunction occurs early and plays a significant role in inducing axonal degeneration in response to 6-OHDA treatment. PMID:24885281

Severity, course, and predictors of sleep disruption following hematopoietic cell transplantation: a secondary data analysis from the BMT CTN 0902 trial.

PubMed

Jim, Heather S L; Sutton, Steven; Majhail, Navneet S; Wood, William A; Jacobsen, Paul B; Wingard, John R; Wu, Juan; Knight, Jennifer M; Syrjala, Karen L; Lee, Stephanie J

2018-03-07

Sleep disruption has received little attention in hematopoietic cell transplantation (HCT). The goal of this study was to describe severity, course, and predictors of sleep disruption following HCT. A secondary data analysis was conducted of the Blood and Marrow Transplantation Clinical Trials Network (BMT CTN) 0902 study. Participants completed a modified version of the Pittsburgh Sleep Quality Index prior to transplant and 100 and 180 days posttransplant. Growth mixture models were used to characterize subgroups of patients based on baseline sleep disruption and change over time. A total of 570 patients (mean age 55 years, 42% female) were included in the current analyses. Patients could be grouped into four distinct classes based on sleep disruption: (1) clinically significant sleep disruption at baseline that did not improve over time (20%); (2) clinically significant sleep disruption at baseline that improved over time (22%); (3) sleep disruption that did not reach clinical significance at baseline and did not improve over time (45%); and (4) no sleep disruption at baseline or over time (13%). These data provide a more comprehensive understanding of sleep disruption that can be used to develop interventions to improve sleep in HCT recipients.

Release of cellular tension signals self-restorative ventral lamellipodia to heal barrier micro-wounds

PubMed Central

Martinelli, Roberta; Kamei, Masataka; Sage, Peter T.; Massol, Ramiro; Varghese, Laya; Sciuto, Tracey; Toporsian, Mourad; Dvorak, Ann M.; Kirchhausen, Tomas; Springer, Timothy A.

2013-01-01

Basic mechanisms by which cellular barriers sense and respond to integrity disruptions remain poorly understood. Despite its tenuous structure and constitutive exposure to disruptive strains, the vascular endothelium exhibits robust barrier function. We show that in response to micrometer-scale disruptions induced by transmigrating leukocytes, endothelial cells generate unique ventral lamellipodia that propagate via integrins toward and across these “micro-wounds” to close them. This novel actin remodeling activity progressively healed multiple micro-wounds in succession and changed direction during this process. Mechanical probe-induced micro-wounding of both endothelia and epithelia suggests that ventral lamellipodia formed as a response to force imbalance and specifically loss of isometric tension. Ventral lamellipodia were enriched in the Rac1 effectors cortactin, IQGAP, and p47Phox and exhibited localized production of hydrogen peroxide. Together with Apr2/3, these were functionally required for effective micro-wound healing. We propose that barrier disruptions are detected as local release of isometric tension/force unloading, which is directly coupled to reactive oxygen species–dependent self-restorative actin remodeling dynamics. PMID:23629967

Effects of advanced glycation end products on ezrin-dependent functions in LLC-PK1 proximal tubule cells.

PubMed

Bach, Leon A; Gallicchio, Marisa A; McRobert, E Anne; Tikoo, Anjali; Cooper, Mark E

2005-06-01

We have recently shown that advanced glycation products (AGEs) bind to the ERM (ezrin, radixin, moesin) family of proteins. ERM proteins act as cross-linkers between cell membrane proteins and the actin cytoskeleton. They are also involved in signal transduction pathways. They therefore have a critical role in normal cell processes, including modulation of cell shape, adhesion, and motility. We postulate that AGEs may contribute to diabetic complications by disrupting ERM function. In support of this hypothesis, AGEs inhibit ezrin-dependent tubulogenesis of proximal tubule cells. Phosphorylation is an important activating mechanism for ERM proteins, and AGEs inhibit ezrin phosphorylation mediated by the epidermal growth factor receptor.

Camouflage and misdirection: the full-on assault of ebola virus disease.

PubMed

Misasi, John; Sullivan, Nancy J

2014-10-23

Ebolaviruses cause a severe hemorrhagic fever syndrome that is rapidly fatal to humans and nonhuman primates. Ebola protein interactions with host cellular proteins disrupt type I and type II interferon responses, RNAi antiviral responses, antigen presentation, T-cell-dependent B cell responses, humoral antibodies, and cell-mediated immunity. This multifaceted approach to evasion and suppression of innate and adaptive immune responses in their target hosts leads to the severe immune dysregulation and "cytokine storm" that is characteristic of fatal ebolavirus infection. Here, we highlight some of the processes by which Ebola interacts with its mammalian hosts to evade antiviral defenses.

Glycan Remodeling with Processing Inhibitors and Lectin-Resistant Eukaryotic Cells.

PubMed

Chang, Veronica T; Spooner, Robert A; Crispin, Max; Davis, Simon J

2015-01-01

Some of the most important and interesting molecules in metazoan biology are glycoproteins. The importance of the carbohydrate component of these structures is often revealed by the disease phenotypes that manifest when the biosynthesis of particular glycoforms is disrupted. On the other hand, the presence of large amounts of carbohydrate can often hinder the structural and functional analysis of glycoproteins. There are often good reasons, therefore, for wanting to engineer and predefine the N-glycans present on glycoproteins, e.g., in order to characterize the functions of the glycans or facilitate their subsequent removal. Here, we describe in detail two distinct ways in which to usefully interfere with oligosaccharide processing, one involving the use of specific processing inhibitors, and the other the selection of cell lines mutated at gene loci that control oligosaccharide processing, using cytotoxic lectins. Both approaches have the capacity for controlled, radical alteration of oligosaccharide processing in eukaryotic cells used for heterologous protein expression, and have great utility in the structural analysis of glycoproteins.

Calcium Channels and Oxidative Stress Mediate a Synergistic Disruption of Tight Junctions by Ethanol and Acetaldehyde in Caco-2 Cell Monolayers.

PubMed

Samak, Geetha; Gangwar, Ruchika; Meena, Avtar S; Rao, Roshan G; Shukla, Pradeep K; Manda, Bhargavi; Narayanan, Damodaran; Jaggar, Jonathan H; Rao, RadhaKrishna

2016-12-13

Ethanol is metabolized into acetaldehyde in most tissues. In this study, we investigated the synergistic effect of ethanol and acetaldehyde on the tight junction integrity in Caco-2 cell monolayers. Expression of alcohol dehydrogenase sensitized Caco-2 cells to ethanol-induced tight junction disruption and barrier dysfunction, whereas aldehyde dehydrogenase attenuated acetaldehyde-induced tight junction disruption. Ethanol up to 150 mM did not affect tight junction integrity or barrier function, but it dose-dependently increased acetaldehyde-mediated tight junction disruption and barrier dysfunction. Src kinase and MLCK inhibitors blocked this synergistic effect of ethanol and acetaldehyde on tight junction. Ethanol and acetaldehyde caused a rapid and synergistic elevation of intracellular calcium. Calcium depletion by BAPTA or Ca 2+ -free medium blocked ethanol and acetaldehyde-induced barrier dysfunction and tight junction disruption. Diltiazem and selective knockdown of TRPV6 or Ca V 1.3 channels, by shRNA blocked ethanol and acetaldehyde-induced tight junction disruption and barrier dysfunction. Ethanol and acetaldehyde induced a rapid and synergistic increase in reactive oxygen species by a calcium-dependent mechanism. N-acetyl-L-cysteine and cyclosporine A, blocked ethanol and acetaldehyde-induced barrier dysfunction and tight junction disruption. These results demonstrate that ethanol and acetaldehyde synergistically disrupt tight junctions by a mechanism involving calcium, oxidative stress, Src kinase and MLCK.

Vav1 GEF activity is required for T cell mediated allograft rejection

PubMed Central

Haubert, Dirk; Li, Jianping; Saveliev, Alexander; Calzascia, Thomas; Sutter, Esther; Metzler, Barbara; Kaiser, Daniel; Tybulewicz, Victor L.J.; Weckbecker, Gisbert

2012-01-01

The GDP exchange factor (GEF) Vav1 is a central signal transducer downstream of the T cell receptor and has been identified as a key factor for T cell activation in the context of allograft rejection. Vav1 has been shown to transduce signals both dependent and independent of its GEF function. The most promising approach to disrupt Vav1 activity by pharmacological inhibition would be to target its GEF function. However, the contribution of Vav1 GEF activity for allogeneic T cell activation has not been clarified yet. To address this question, we used knock-in mice bearing a mutated Vav1 with disrupted GEF activity but intact GEF-independent functions. T cells from these mice showed strongly reduced proliferation and activation in response to allogeneic stimulation. Furthermore, lack of Vav1 GEF activity strongly abrogated the in vivo expansion of T cells in a systemic graft-versus-host model. In a cardiac transplantation model, mice with disrupted Vav1 GEF activity show prolonged allograft survival. These findings demonstrate a strong requirement for Vav1 GEF activity for allogeneic T cell activation and graft rejection suggesting that disruption of Vav1 GEF activity alone is sufficient to induce significant immunosuppression. PMID:22456277

Calcium oxalate crystals induces tight junction disruption in distal renal tubular epithelial cells by activating ROS/Akt/p38 MAPK signaling pathway.

PubMed

Yu, Lei; Gan, Xiuguo; Liu, Xukun; An, Ruihua

2017-11-01

Tight junction plays important roles in regulating paracellular transports and maintaining cell polarity. Calcium oxalate monohydrate (COM) crystals, the major crystalline composition of kidney stones, have been demonstrated to be able to cause tight junction disruption to accelerate renal cell injury. However, the cellular signaling involved in COM crystal-induced tight junction disruption remains largely to be investigated. In the present study, we proved that COM crystals induced tight junction disruption by activating ROS/Akt/p38 MAPK pathway. Treating Madin-Darby canine kidney (MDCK) cells with COM crystals induced a substantial increasing of ROS generation and activation of Akt that triggered subsequential activation of ASK1 and p38 mitogen-activated protein kinase (MAPK). Western blot revealed a significantly decreased expression of ZO-1 and occludin, two important structural proteins of tight junction. Besides, redistribution and dissociation of ZO-1 were observed by COM crystals treatment. Inhibition of ROS by N-acetyl-l-cysteine (NAC) attenuated the activation of Akt, ASK1, p38 MAPK, and down-regulation of ZO-1 and occludin. The redistribution and dissociation of ZO-1 were also alleviated by NAC treatment. These results indicated that ROS were involved in the regulation of tight junction disruption induced by COM crystals. In addition, the down-regulation of ZO-1 and occludin, the phosphorylation of ASK1 and p38 MAPK were also attenuated by MK-2206, an inhibitor of Akt kinase, implying Akt was involved in the disruption of tight junction upstream of p38 MAPK. Thus, these results suggested that ROS-Akt-p38 MAPK signaling pathway was activated in COM crystal-induced disruption of tight junction in MDCK cells.

Decoding the Heart through Next Generation Sequencing Approaches.

PubMed

Pawlak, Michal; Niescierowicz, Katarzyna; Winata, Cecilia Lanny

2018-06-07

: Vertebrate organs develop through a complex process which involves interaction between multiple signaling pathways at the molecular, cell, and tissue levels. Heart development is an example of such complex process which, when disrupted, results in congenital heart disease (CHD). This complexity necessitates a holistic approach which allows the visualization of genome-wide interaction networks, as opposed to assessment of limited subsets of factors. Genomics offers a powerful solution to address the problem of biological complexity by enabling the observation of molecular processes at a genome-wide scale. The emergence of next generation sequencing (NGS) technology has facilitated the expansion of genomics, increasing its output capacity and applicability in various biological disciplines. The application of NGS in various aspects of heart biology has resulted in new discoveries, generating novel insights into this field of study. Here we review the contributions of NGS technology into the understanding of heart development and its disruption reflected in CHD and discuss how emerging NGS based methodologies can contribute to the further understanding of heart repair.

High temperature induced disruption of the cell wall integrity and structure in Pleurotus ostreatus mycelia.

PubMed

Qiu, Zhiheng; Wu, Xiangli; Gao, Wei; Zhang, Jinxia; Huang, Chenyang

2018-05-30

Fungal cells are surrounded by a tight cell wall to protect them from harmful environmental conditions and to resist lysis. The synthesis and assembly determine the shape, structure, and integrity of the cell wall during the process of mycelial growth and development. High temperature is an important abiotic stress, which affects the synthesis and assembly of cell walls. In the present study, the chitin and β-1,3-glucan concentrations in the cell wall of Pleurotus ostreatus mycelia were changed after high-temperature treatment. Significantly higher chitin and β-1,3-glucan concentrations were detected at 36 °C than those incubated at 28 °C. With the increased temperature, many aberrant chitin deposition patches occurred, and the distribution of chitin in the cell wall was uneven. Moreover, high temperature disrupts the cell wall integrity, and P. ostreatus mycelia became hypersensitive to cell wall-perturbing agents at 36 °C. The cell wall structure tended to shrink or distorted after high temperature. The cell walls were observed to be thicker and looser by using transmission electron microscopy. High temperature can decrease the mannose content in the cell wall and increase the relative cell wall porosity. According to infrared absorption spectrum, high temperature broke or decreased the glycosidic linkages. Finally, P. ostreatus mycelial cell wall was easily degraded by lysing enzymes after high-temperature treatment. In other words, the cell wall destruction caused by high temperature may be a breakthrough for P. ostreatus to be easily infected by Trichoderma.

Bacteria permeabilization and disruption caused by sludge reduction technologies evaluated by flow cytometry.

PubMed

Foladori, P; Tamburini, S; Bruni, L

2010-09-01

Technologies proposed in the last decades for the reduction of the sludge production in wastewater treatment plants and based on the mechanism of cell lysis-cryptic growth (physical, mechanical, thermal, chemical, oxidative treatments) have been widely investigated at lab-, pilot- and, in some cases, at full-scale but the effects on cellular lysis have not always been demonstrated in depth. The research presented in this paper aims to investigate how these sludge reduction technologies affect the integrity and permeabilization of bacterial cells in sludge using flow cytometry (FCM), which permits the rapid and statistically accurate quantification of intact, permeabilised or disrupted bacteria in the sludge using a double fluorescent DNA-staining instead of using conventional methods like plate counts and microscope. Physical/mechanical treatments (ultrasonication and high pressure homogenisation) caused moderate effects on cell integrity and caused significant cell disruption only at high specific energy levels. Conversely, thermal treatment caused significant damage of bacterial membranes even at moderate temperatures (45-55 °C). Ozonation significantly affected cell integrity, even at low ozone dosages, below 10 mgO(3)/gTSS, causing an increase of permeabilised and disrupted cells. At higher ozone dosages the compounds solubilised after cell lysis act as scavengers in the competition between soluble compounds and (particulate) bacterial cells. An original aspect of this paper, not yet reported in the literature, is the comparison of the effects of these sludge reduction technologies on bacterial cell integrity and permeabilization by converting pressure, temperature and ozone dosage to an equivalent value of specific energy. Among these technologies, comparison of the applied specific energy demonstrates that achieving the complete disruption of bacterial cells is not always economically advantageous because excessive energy levels may be required. Copyright © 2010 Elsevier Ltd. All rights reserved.

Rapid, portable detection of endocrine disrupting chemicals through ligand-nuclear hormone receptor interactions.

PubMed

Hunt, J Porter; Schinn, Song-Min; Jones, Matthew D; Bundy, Bradley C

2017-12-04

Endocrine disrupting chemicals (EDC) are structurally diverse compounds that can interact with nuclear hormone receptors, posing significant risk to human and ecological health. Unfortunately, many conventional biosensors have been too structure-specific, labor-intensive or laboratory-oriented to detect broad ranges of EDC effectively. Recently, several technological advances are providing more rapid, portable, and affordable detection of endocrine-disrupting activity through ligand-nuclear hormone receptor interactions. Here, we overview these recent advances applied to EDC biosensors - including cell lyophilization, cell immobilization, cell-free systems, smartphone-based signal detection, and improved competitive binding assays.

Association of Plasmodium falciparum with Human Endothelial Cells in vitro

PubMed Central

Utter, Christopher; Serrano, Adelfa E.; Glod, John W.; Leibowitz, Michael J.

2017-01-01

Endothelial abnormalities play a critical role in the pathogenesis of malaria caused by the human pathogen, Plasmodium falciparum. In serious infections and especially in cerebral malaria, red blood cells infected with the parasite are sequestered in small venules in various organs, resulting in endothelial activation and vascular occlusion, which are believed to be largely responsible for the morbidity and mortality caused by this infection, especially in children. We demonstrate that after incubation with infected red blood cells (iRBCs), cultured human umbilical vein endothelial cells (HUVECs) contain parasite protein, genomic DNA, and RNA, as well as intracellular vacuoles with apparent parasite-derived material, but not engulfed or adherent iRBCs. The association of this material with the HUVECs is observed over 96 hours after removal of iRBCs. This phenomenon may occur in endothelial cells in vivo by the process of trogocytosis, in which transfer of material between cells depends on direct cell contact. This process may contribute to the endothelial activation and disruption involved in the pathogenesis of cerebral malaria. PMID:28656007

GPATCH3 negatively regulates RLR-mediated innate antiviral responses by disrupting the assembly of VISA signalosome.

PubMed

Nie, Ying; Ran, Yong; Zhang, Hong-Yan; Huang, Zhe-Fu; Pan, Zhao-Yi; Wang, Su-Yun; Wang, Yan-Yi

2017-04-01

Upon viral infection, retinoic acid-inducible gene I-like receptors (RLRs) recognize viral RNA and trigger a series of signaling events, leading to the induction of type I interferons (IFNs). These processes are delicately regulated to prevent excessive and harmful immune responses. In this study, we identified G patch domain-containing protein 3 (GPATCH3) as a negative regulator of RLR-mediated antiviral signaling pathways. Overexpression of GPATCH3 impaired RNA virus- triggered induction of downstream antiviral genes, whereas its knockdown had opposite effects and attenuated viral replication. In addition, GPATCH3-deficient cells had higher IFNB1 mRNA level compared with control cells after RNA virus infection. Mechanistically, GPATCH3 was recruited to VISA in a viral infection dependent manner and the assembly of VISA/TRAF6/TBK1 signalosome was impaired in GPATCH3-overexpressing cells. In contrast, upon viral infection, the recruitment of TRAF6 and TBK1 to VISA was enhanced in GPATCH3 deficient cells. Taking together, our findings demonstrate that GPATCH3 interacts with VISA and disrupts the assembly of virus-induced VISA signalosome therefore acts as a negative regulator of RLR-mediated innate antiviral immune responses.

GPATCH3 negatively regulates RLR-mediated innate antiviral responses by disrupting the assembly of VISA signalosome

PubMed Central

Nie, Ying; Ran, Yong; Zhang, Hong-Yan; Huang, Zhe-Fu; Wang, Su-Yun

2017-01-01

Upon viral infection, retinoic acid–inducible gene I–like receptors (RLRs) recognize viral RNA and trigger a series of signaling events, leading to the induction of type I interferons (IFNs). These processes are delicately regulated to prevent excessive and harmful immune responses. In this study, we identified G patch domain-containing protein 3 (GPATCH3) as a negative regulator of RLR-mediated antiviral signaling pathways. Overexpression of GPATCH3 impaired RNA virus- triggered induction of downstream antiviral genes, whereas its knockdown had opposite effects and attenuated viral replication. In addition, GPATCH3-deficient cells had higher IFNB1 mRNA level compared with control cells after RNA virus infection. Mechanistically, GPATCH3 was recruited to VISA in a viral infection dependent manner and the assembly of VISA/TRAF6/TBK1 signalosome was impaired in GPATCH3-overexpressing cells. In contrast, upon viral infection, the recruitment of TRAF6 and TBK1 to VISA was enhanced in GPATCH3 deficient cells. Taking together, our findings demonstrate that GPATCH3 interacts with VISA and disrupts the assembly of virus-induced VISA signalosome therefore acts as a negative regulator of RLR-mediated innate antiviral immune responses. PMID:28414768

Non-linear optical measurements using a scanned, Bessel beam

NASA Astrophysics Data System (ADS)

Collier, Bradley B.; Awasthi, Samir; Lieu, Deborah K.; Chan, James W.

2015-03-01

Oftentimes cells are removed from the body for disease diagnosis or cellular research. This typically requires fluorescent labeling followed by sorting with a flow cytometer; however, possible disruption of cellular function or even cell death due to the presence of the label can occur. This may be acceptable for ex vivo applications, but as cells are more frequently moving from the lab to the body, label-free methods of cell sorting are needed to eliminate these issues. This is especially true of the growing field of stem cell research where specialized cells are needed for treatments. Because differentiation processes are not completely efficient, cells must be sorted to eliminate any unwanted cells (i.e. un-differentiated or differentiated into an unwanted cell type). In order to perform label-free measurements, non-linear optics (NLO) have been increasingly utilized for single cell analysis because of their ability to not disrupt cellular function. An optical system was developed for the measurement of NLO in a microfluidic channel similar to a flow cytometer. In order to improve the excitation efficiency of NLO, a scanned Bessel beam was utilized to create a light-sheet across the channel. The system was tested by monitoring twophoton fluorescence from polystyrene microbeads of different sizes. Fluorescence intensity obtained from light-sheet measurements were significantly greater than measurements made using a static Gaussian beam. In addition, the increase in intensity from larger sized beads was more evident for the light-sheet system.

Disruption of vertical motility by shear triggers formation of thin phytoplankton layers.

PubMed

Durham, William M; Kessler, John O; Stocker, Roman

2009-02-20

Thin layers of phytoplankton are important hotspots of ecological activity that are found in the coastal ocean, meters beneath the surface, and contain cell concentrations up to two orders of magnitude above ambient concentrations. Current interpretations of their formation favor abiotic processes, yet many phytoplankton species found in these layers are motile. We demonstrated that layers formed when the vertical migration of phytoplankton was disrupted by hydrodynamic shear. This mechanism, which we call gyrotactic trapping, can be responsible for the thin layers of phytoplankton commonly observed in the ocean. These results reveal that the coupling between active microorganism motility and ambient fluid motion can shape the macroscopic features of the marine ecological landscape.

Microbial products activate monocytic cells through detergent-resistant membrane microdomains.

PubMed

Epelman, Slava; Berenger, Byron; Stack, Danuta; Neely, Graham G; Ma, Ling Ling; Mody, Christopher H

2008-12-01

Patients with cystic fibrosis suffer recurrent pulmonary infections that are characterized by an overactive yet ineffective and destructive inflammatory response that is associated with respiratory infections by Pseudomonas aeruginosa, a pathogen that produces a number of phlogistic molecules. To better understand this process, we used exoenzyme S (ExoS), one of the key P. aeruginosa-secreted exoproducts, which is known to stimulate cells via the Toll-like receptor (TLR) pathway. We found that ExoS induced proinflammatory cytokine production via the NF-kappaB, Erk1/2, and Src kinase pathways. Because Src kinases are concentrated within cholesterol-containing, detergent-resistant membrane microdomains (DRM) (also called lipid rafts) and DRM act as signaling platforms and amplifiers on the surface of cells, we addressed the role of DRM in ExoS signaling. ExoS bound directly to a subset of DRM and induced the phosphorylation of multiple proteins within DRM, including Src kinases. Disruption of DRM by cholesterol extraction prevented NF-kappaB and Erk 1/2 activation and TNF-alpha production in response to ExoS. Activation of monocytic cells by other TLR and Nod-like receptor agonists, such as lipoteichoic acid, lipopolysaccharide, and peptidoglycan, were also dependent on DRM, and disruption prevented TNF-alpha production. Disruption of DRM did not prevent ExoS binding but did release the Src kinase, Lyn, from the DRM fraction into the detergent-soluble fraction, a site in which Src kinases are not active. These studies show that ExoS, a TLR agonist, requires direct binding to DRM for optimal signaling, which suggests that DRM are possible therapeutic targets in cystic fibrosis.

Autophagy as an Emerging Common Pathomechanism in Inherited Peripheral Neuropathies

PubMed Central

Haidar, Mansour; Timmerman, Vincent

2017-01-01

The inherited peripheral neuropathies (IPNs) comprise a growing list of genetically heterogeneous diseases. With mutations in more than 80 genes being reported to cause IPNs, a wide spectrum of functional consequences is expected to follow this genotypic diversity. Hence, the search for a common pathomechanism among the different phenotypes has become the holy grail of functional research into IPNs. During the last decade, studies on several affected genes have shown a direct and/or indirect correlation with autophagy. Autophagy, a cellular homeostatic process, is required for the removal of cell aggregates, long-lived proteins and dead organelles from the cell in double-membraned vesicles destined for the lysosomes. As an evolutionarily highly conserved process, autophagy is essential for the survival and proper functioning of the cell. Recently, neuronal cells have been shown to be particularly vulnerable to disruption of the autophagic pathway. Furthermore, autophagy has been shown to be affected in various common neurodegenerative diseases of both the central and the peripheral nervous system including Alzheimer’s, Parkinson’s, and Huntington’s diseases. In this review we provide an overview of the genes involved in hereditary neuropathies which are linked to autophagy and we propose the disruption of the autophagic flux as an emerging common pathomechanism. We also shed light on the different steps of the autophagy pathway linked to these genes. Finally, we review the concept of autophagy being a therapeutic target in IPNs, and the possibilities and challenges of this pathway-specific targeting. PMID:28553203

Femtosecond laser nanosurgery of sub-cellular structures in HeLa cells by employing Third Harmonic Generation imaging modality as diagnostic tool.

PubMed

Tserevelakis, George J; Psycharakis, Stylianos; Resan, Bojan; Brunner, Felix; Gavgiotaki, Evagelia; Weingarten, Kurt; Filippidis, George

2012-02-01

Femtosecond laser assisted nanosurgery of microscopic biological specimens is a relatively new technique which allows the selective disruption of sub-cellular structures without causing any undesirable damage to the surrounding regions. The targeted structures have to be stained in order to be clearly visualized for the nanosurgery procedure. However, the validation of the final nanosurgery result is difficult, since the targeted structure could be simply photobleached rather than selectively destroyed. This fact comprises a main drawback of this technique. In our study we employed a multimodal system which integrates non-linear imaging modalities with nanosurgery capabilities, for the selective disruption of sub-cellular structures in HeLa cancer cells. Third Harmonic Generation (THG) imaging modality was used as a tool for the identification of structures that were subjected to nanosurgery experiments. No staining of the biological samples was required, since THG is an intrinsic property of matter. Furthermore, cells' viability after nanosurgery processing was verified via Two Photon Excitation Fluorescence (TPEF) measurements. Copyright © 2012 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Low level exposure to inorganic mercury interferes with B cell receptor signaling in transitional type 1 B cells.

PubMed

Gill, R; McCabe, M J; Rosenspire, A J

2017-09-01

Mercury (Hg) has been implicated as a factor contributing to autoimmune disease in animal models and humans. However the mechanism by which this occurs has remained elusive. Since the discovery of B cells it has been appreciated by immunologists that during the normal course of B cell development, some immature B cells must be generated that produce immunoglobulin reactive to self-antigens (auto-antibodies). However in the course of normal development, the vast majority of immature auto-reactive B cells are prevented from maturing by processes collectively known as tolerance. Autoimmune disease arises when these mechanisms of tolerance are disrupted. In the B cell compartment, it is firmly established that tolerance depends in part upon negative selection of self-reactive immature (transitional type 1) B cells. In these cells negative selection depends upon signals generated by the B Cell Receptor (BCR), in the sense that those T1 B cells who's BCRs most strongly bind to, and so generate the strongest signals to self-antigens are neutralized. In this report we have utilized multicolor phosphoflow cytometry to show that in immature T1 B cells Hg attenuates signal generation by the BCR through mechanisms that may involve Lyn, a key tyrosine kinase in the BCR signal transduction pathway. We suggest that exposure to low, environmentally relevant levels of Hg, disrupts tolerance by interfering with BCR signaling in immature B cells, potentially leading to the appearance of mature auto-reactive B cells which have the ability to contribute to auto-immune disease. Copyright © 2017 Elsevier Inc. All rights reserved.

Zinc oxide nanoparticles decrease the expression and activity of plasma membrane calcium ATPase, disrupt the intracellular calcium homeostasis in rat retinal ganglion cells.

PubMed

Guo, Dadong; Bi, Hongsheng; Wang, Daoguang; Wu, Qiuxin

2013-08-01

Zinc oxide nanoparticle is one of the most important materials with diverse applications. However, it has been reported that zinc oxide nanoparticles are toxic to organisms, and that oxidative stress is often hypothesized to be an important factor in cytotoxicity mediated by zinc oxide nanoparticles. Nevertheless, the mechanism of toxicity of zinc oxide nanoparticles has not been completely understood. In this study, we investigated the cytotoxic effect of zinc oxide nanoparticles and the possible molecular mechanism involved in calcium homeostasis mediated by plasma membrane calcium ATPase in rat retinal ganglion cells. Real-time cell electronic sensing assay showed that zinc oxide nanoparticles could exert cytotoxic effect on rat retinal ganglion cells in a concentration-dependent manner; flow cytometric analysis indicated that zinc oxide nanoparticles could lead to cell damage by inducing the overproduction of reactive oxygen species. Furthermore, zinc oxide nanoparticles could also apparently decrease the expression level and their activity of plasma membrane calcium ATPase, which finally disrupt the intracellular calcium homeostasis and result in cell death. Taken together, zinc oxide nanoparticles could apparently decrease the plasma membrane calcium ATPase expression, inhibit their activity, cause the elevated intracellular calcium ion level and disrupt the intracellular calcium homeostasis. Further, the disrupted calcium homeostasis will trigger mitochondrial dysfunction, generate excessive reactive oxygen species, and finally initiate cell death. Thus, the disrupted calcium homeostasis is involved in the zinc oxide nanoparticle-induced rat retinal ganglion cell death. Copyright © 2013 Elsevier Ltd. All rights reserved.

Molecular Programs Underlying Asymmetric Stem Cell Division and Their Disruption in Malignancy.

PubMed

Mukherjee, Subhas; Brat, Daniel J

2017-01-01

Asymmetric division of stem cells is a highly conserved and tightly regulated process by which a single stem cell produces two unequal daughter cells. One retains its stem cell identity while the other becomes specialized through a differentiation program and loses stem cell properties. Coordinating these events requires control over numerous intra- and extracellular biological processes and signaling networks. In the initial stages, critical events include the compartmentalization of fate determining proteins within the mother cell and their subsequent passage to the appropriate daughter cell in order to direct their destiny. Disturbance of these events results in an altered dynamic of self-renewing and differentiation within the cell population, which is highly relevant to the growth and progression of cancer. Other critical events include proper asymmetric spindle assembly, extrinsic regulation through micro-environmental cues, and non-canonical signaling networks that impact cell division and fate determination. In this review, we discuss mechanisms that maintain the delicate balance of asymmetric cell division in normal tissues and describe the current understanding how some of these mechanisms are deregulated in cancer.

Calmodulin protects cells from death under normal growth conditions and mitogenic starvation but plays a mediating role in cell death upon B-cell receptor stimulation

PubMed Central

Schmalzigaug, Robert; Ye, Qunrui; Berchtold, Martin W

2001-01-01

Calmodulin (CaM) is the main intracellular Ca2+ sensor protein responsible for mediating Ca2+ triggered processes. Chicken DT40 lymphoma B cells express CaM from the two genes, CaMI and CaMII. Here we report the phenotypes of DT40 cells with the CaMII gene knocked out. The disruption of the CaMII gene causes the intracellular CaM level to decrease by 60%. CaMII−/− cells grow more slowly and die more frequently as compared to wild type (wt) cells but do not exhibit significant differences in their cell cycle profile. Both phenotypes are more pronounced at reduced serum concentrations. Upon stimulation of the B-cell receptor (BCR), the resting Ca2+ levels remain elevated after the initial transient in CaMII−/− cells. Despite higher Ca2+ resting levels, the CaMII−/− cells are partially protected from BCR induced apoptosis indicating that CaM plays a dual role in apoptotic processes. PMID:11454062

Endothelial cell-initiated extravasation of cancer cells visualized in zebrafish

PubMed Central

Kanada, Masamitsu; Zhang, Jinyan; Yan, Libo; Sakurai, Takashi

2014-01-01

The extravasation of cancer cells, a key step for distant metastasis, is thought to be initiated by disruption of the endothelial barrier by malignant cancer cells. An endothelial covering-type extravasation of cancer cells in addition to conventional cancer cell invasion-type extravasation was dynamically visualized in a zebrafish hematogenous metastasis model. The inhibition of VEGF-signaling impaired the invasion-type extravasation via inhibition of cancer cell polarization and motility. Paradoxically, the anti-angiogenic treatment showed the promotion, rather than the inhibition, of the endothelial covering-type extravasation of cancer cells, with structural changes in the endothelial walls. These findings may be a set of clues to the full understanding of the metastatic process as well as the metastatic acceleration by anti-angiogenic reagents observed in preclinical studies. PMID:25551022

Endothelial cell-initiated extravasation of cancer cells visualized in zebrafish.

PubMed

Kanada, Masamitsu; Zhang, Jinyan; Yan, Libo; Sakurai, Takashi; Terakawa, Susumu

2014-01-01

The extravasation of cancer cells, a key step for distant metastasis, is thought to be initiated by disruption of the endothelial barrier by malignant cancer cells. An endothelial covering-type extravasation of cancer cells in addition to conventional cancer cell invasion-type extravasation was dynamically visualized in a zebrafish hematogenous metastasis model. The inhibition of VEGF-signaling impaired the invasion-type extravasation via inhibition of cancer cell polarization and motility. Paradoxically, the anti-angiogenic treatment showed the promotion, rather than the inhibition, of the endothelial covering-type extravasation of cancer cells, with structural changes in the endothelial walls. These findings may be a set of clues to the full understanding of the metastatic process as well as the metastatic acceleration by anti-angiogenic reagents observed in preclinical studies.

Disruption of centrifugal inhibition to olfactory bulb granule cells impairs olfactory discrimination.

PubMed

Nunez-Parra, Alexia; Maurer, Robert K; Krahe, Krista; Smith, Richard S; Araneda, Ricardo C

2013-09-03

Granule cells (GCs) are the most abundant inhibitory neuronal type in the olfactory bulb and play a critical role in olfactory processing. GCs regulate the activity of principal neurons, the mitral cells, through dendrodendritic synapses, shaping the olfactory bulb output to other brain regions. GC excitability is regulated precisely by intrinsic and extrinsic inputs, and this regulation is fundamental for odor discrimination. Here, we used channelrhodopsin to stimulate GABAergic axons from the basal forebrain selectively and show that this stimulation generates reliable inhibitory responses in GCs. Furthermore, selective in vivo inhibition of GABAergic neurons in the basal forebrain by targeted expression of designer receptors exclusively activated by designer drugs produced a reversible impairment in the discrimination of structurally similar odors, indicating an important role of these inhibitory afferents in olfactory processing.

PIF1 disruption or NBS1 hypomorphism does not affect chromosome healing or fusion resulting from double-strand breaks near telomeres in murine embryonic stem cells.

PubMed

Reynolds, Gloria E; Gao, Qing; Miller, Douglas; Snow, Bryan E; Harrington, Lea A; Murnane, John P

2011-11-10

Telomerase serves to maintain telomeric repeat sequences at the ends of chromosomes. However, telomerase can also add telomeric repeat sequences at DNA double-strand breaks (DSBs), a process called chromosome healing. Here, we employed a method of inducing DSBs near telomeres to query the role of two proteins, PIF1 and NBS1, in chromosome healing in mammalian cells. PIF1 was investigated because the PIF1 homolog in Saccharomyces cerevisiae inhibits chromosome healing, as shown by a 1000-fold increase in chromosome in PIF1-deficient cells. NBS1 was investigated because the functional homolog of NBS1 in S. cerevisiae, Xrs2, is part of the Mre11/Rad50/Xrs2 complex that is required for chromosome healing due to its role in the processing of DSBs and recruitment of telomerase. We found that disruption of mPif1 had no detectable effect on the frequency of chromosome healing at DSBs near telomeres in murine embryonic stem cells. Moreover, the Nbs1(ΔB) hypomorph, which is defective in the processing of DSBs, also had no detectable effect on the frequency of chromosome healing, DNA degradation, or gross chromosome rearrangements (GCRs) that result from telomeric DSBs. Although we cannot rule out small changes in chromosome healing using this system, it is clear from our results that knockout of PIF1 or the Nbs1(ΔB) hypomorph does not result in large differences in chromosome healing in murine cells. These results represent the first genetic assessment of the role of these proteins in chromosome healing in mammals, and suggest that murine cells have evolved mechanisms to ensure the functional redundancy of Pif1 or Nbs1 in the regulation of chromosome healing. Copyright © 2011 Elsevier B.V. All rights reserved.

BAG3 regulates formation of the SNARE complex and insulin secretion

PubMed Central

Iorio, V; Festa, M; Rosati, A; Hahne, M; Tiberti, C; Capunzo, M; De Laurenzi, V; Turco, M C

2015-01-01

Insulin release in response to glucose stimulation requires exocytosis of insulin-containing granules. Glucose stimulation of beta cells leads to focal adhesion kinase (FAK) phosphorylation, which acts on the Rho family proteins (Rho, Rac and Cdc42) that direct F-actin remodeling. This process requires docking and fusion of secretory vesicles to the release sites at the plasma membrane and is a complex mechanism that is mediated by SNAREs. This transiently disrupts the F-actin barrier and allows the redistribution of the insulin-containing granules to more peripheral regions of the β cell, hence facilitating insulin secretion. In this manuscript, we show for the first time that BAG3 plays an important role in this process. We show that BAG3 downregulation results in increased insulin secretion in response to glucose stimulation and in disruption of the F-actin network. Moreover, we show that BAG3 binds to SNAP-25 and syntaxin-1, two components of the t-SNARE complex preventing the interaction between SNAP-25 and syntaxin-1. Upon glucose stimulation BAG3 is phosphorylated by FAK and dissociates from SNAP-25 allowing the formation of the SNARE complex, destabilization of the F-actin network and insulin release. PMID:25766323

Roles of Specific Membrane Lipid Domains in EGF Receptor Activation and Cell Adhesion Molecule Stabilization in a Developing Olfactory System

PubMed Central

Gibson, Nicholas J.; Tolbert, Leslie P.; Oland, Lynne A.

2009-01-01

Background Reciprocal interactions between glial cells and olfactory receptor neurons (ORNs) cause ORN axons entering the brain to sort, to fasciculate into bundles destined for specific glomeruli, and to form stable protoglomeruli in the developing olfactory system of an experimentally advantageous animal species, the moth Manduca sexta. Epidermal growth factor receptors (EGFRs) and the cell adhesion molecules (IgCAMs) neuroglian and fasciclin II are known to be important players in these processes. Methodology/Principal Findings We report in situ and cell-culture studies that suggest a role for glycosphingolipid-rich membrane subdomains in neuron-glia interactions. Disruption of these subdomains by the use of methyl-β-cyclodextrin results in loss of EGFR activation, depletion of fasciclin II in ORN axons, and loss of neuroglian stabilization in the membrane. At the cellular level, disruption leads to aberrant ORN axon trajectories, small antennal lobes, abnormal arrays of olfactory glomerul, and loss of normal glial cell migration. Conclusions/Significance We propose that glycosphingolipid-rich membrane subdomains (possible membrane rafts or platforms) are essential for IgCAM-mediated EGFR activation and for anchoring of neuroglian to the cytoskeleton, both required for normal extension and sorting of ORN axons. PMID:19787046

Recent developments of downstream processing for microbial lipids and conversion to biodiesel.

PubMed

Yellapu, Sravan Kumar; Bharti; Kaur, Rajwinder; Kumar, Lalit R; Tiwari, Bhagyashree; Zhang, Xiaolei; Tyagi, Rajeshwar D

2018-05-01

With increasing global population and depleting resources, there is an apparent demand for radical unprecedented innovation to satisfy the basal needs of lives. Hence, non-conventional renewable energy resources like biodiesel have been worked out in past few decades. Biofuel (e.g. Biodiesel) serves to be the most sustainable answer to solve "food vs. fuel crisis". In biorefinery process, lipid extraction from oleaginous microbial lipids is an integral part as it facilitates the release of fatty acids. Direct lipid extraction from wet cell-biomass is favorable in comparison to dry-cell biomass because it eliminates the application of expensive dehydration. However, this process is not commercialized yet, instead, it requires intensive research and development in order to establish robust approaches for lipid extraction that can be practically applied on an industrial scale. This review aims for the critical presentation on cell disruption, lipid recovery and purification to support extraction from wet cell-biomass for an efficient transesterification. Copyright © 2018 Elsevier Ltd. All rights reserved.

Dose-Dependent Thresholds of 10-ns Electric Pulse Induced Plasma Membrane Disruption and Cytotoxicity in Multiple Cell Lines

DTIC Science & Technology

2011-01-01

normalized to parallel controls. Flow Cytometry and Confocal Microscopy Upon exposure to 10-ns EP, aliquots of the cellular suspension were added to a tube...Survival data was processed and plotted using GrapherH software (Golden Software, Golden, Colorado). Flow cytometry results were processed in C6 software...Accuri Cytometers, Inc., Ann Arbor, MI) and FCSExpress software (DeNovo Software, Los Angeles, CA). Final analysis and presentation of flow cytometry

Use of hydrodynamic disintegration to accelerate anaerobic digestion of surplus activated sludge.

PubMed

Grübel, Klaudiusz; Machnicka, Alicja

2009-12-01

Hydrodynamic disintegration of activated sludge resulted in organic matter and polymers transfer from the solid phase into the liquid phase. Disintegration by hydrodynamic cavitation had a positive effect on the degree and rate of excess sludge anaerobic digestion. Also, addition of a part of anaerobic digested sludge containing adapted microorganisms resulted in acceleration of the process. The disruption of cells of foam microorganisms and addition to the digestion process led to an increase of biogas production.

Cytochalasin E alters the cytoskeleton and decreases ENaC activity in Xenopus 2F3 cells.

PubMed

Reifenberger, Matthew S; Yu, Ling; Bao, Hui-Fang; Duke, Billie Jeanne; Liu, Bing-Chen; Ma, He-Ping; Alli, Ahmed A; Eaton, Douglas C; Alli, Abdel A

2014-07-01

Numerous reports have linked cytoskeleton-associated proteins with the regulation of epithelial Na(+) channel (ENaC) activity. The purpose of the present study was to determine the effect of actin cytoskeleton disruption by cytochalasin E on ENaC activity in Xenopus 2F3 cells. Here, we show that cytochalasin E treatment for 60 min can disrupt the integrity of the actin cytoskeleton in cultured Xenopus 2F3 cells. We show using single channel patch-clamp experiments and measurements of short-circuit current that ENaC activity, but not its density, is altered by cytochalasin E-induced disruption of the cytoskeleton. In nontreated cells, 8 of 33 patches (24%) had no measurable ENaC activity, whereas in cytochalasin E-treated cells, 17 of 32 patches (53%) had no activity. Analysis of those patches that did contain ENaC activity showed channel open probability significantly decreased from 0.081 ± 0.01 in nontreated cells to 0.043 ± 0.01 in cells treated with cytochalasin E. Transepithelial current from mpkCCD cells treated with cytochalasin E, cytochalasin D, or latrunculin B for 60 min was decreased compared with vehicle-treated cells. The subcellular expression of fodrin changed significantly, and several protein elements of the cytoskeleton decreased at least twofold after 60 min of cytochalasin E treatment. Cytochalasin E treatment disrupted the association between ENaC and myristoylated alanine-rich C-kinase substrate. The results presented here suggest disruption of the actin cytoskeleton by different compounds can attenuate ENaC activity through a mechanism involving changes in the subcellular expression of fodrin, several elements of the cytoskeleton, and destabilization of the ENaC-myristoylated alanine-rich C-kinase substrate complex. Copyright © 2014 the American Physiological Society.

Cytochalasin E alters the cytoskeleton and decreases ENaC activity in Xenopus 2F3 cells

PubMed Central

Reifenberger, Matthew S.; Yu, Ling; Bao, Hui-Fang; Duke, Billie Jeanne; Liu, Bing-Chen; Ma, He-Ping; Eaton, Douglas C.; Alli, Abdel A.

2014-01-01

Numerous reports have linked cytoskeleton-associated proteins with the regulation of epithelial Na+ channel (ENaC) activity. The purpose of the present study was to determine the effect of actin cytoskeleton disruption by cytochalasin E on ENaC activity in Xenopus 2F3 cells. Here, we show that cytochalasin E treatment for 60 min can disrupt the integrity of the actin cytoskeleton in cultured Xenopus 2F3 cells. We show using single channel patch-clamp experiments and measurements of short-circuit current that ENaC activity, but not its density, is altered by cytochalasin E-induced disruption of the cytoskeleton. In nontreated cells, 8 of 33 patches (24%) had no measurable ENaC activity, whereas in cytochalasin E-treated cells, 17 of 32 patches (53%) had no activity. Analysis of those patches that did contain ENaC activity showed channel open probability significantly decreased from 0.081 ± 0.01 in nontreated cells to 0.043 ± 0.01 in cells treated with cytochalasin E. Transepithelial current from mpkCCD cells treated with cytochalasin E, cytochalasin D, or latrunculin B for 60 min was decreased compared with vehicle-treated cells. The subcellular expression of fodrin changed significantly, and several protein elements of the cytoskeleton decreased at least twofold after 60 min of cytochalasin E treatment. Cytochalasin E treatment disrupted the association between ENaC and myristoylated alanine-rich C-kinase substrate. The results presented here suggest disruption of the actin cytoskeleton by different compounds can attenuate ENaC activity through a mechanism involving changes in the subcellular expression of fodrin, several elements of the cytoskeleton, and destabilization of the ENaC-myristoylated alanine-rich C-kinase substrate complex. PMID:24829507

Disruption of oxidative phosphorylation (OXPHOS) by hydroxylated polybrominated diphenyl ethers (OH-PBDEs) present in the marine environment.

PubMed

Legradi, Jessica; Dahlberg, Anna-Karin; Cenijn, Peter; Marsh, Göran; Asplund, Lillemor; Bergman, Åke; Legler, Juliette

2014-12-16

Hydroxylated polybrominated diphenyl ethers (OH-PBDEs) are of growing concern, as they have been detected in both humans and wildlife and have been shown to be toxic. Recent studies have indicated that OH-PBDEs can be more toxic than PBDEs, partly due to their ability to disrupt oxidative phosphorylation (OXPHOS), an essential process in energy metabolism. In this study, we determined the OXPHOS disruption potential of 18 OH-PBDE congeners reported in marine wildlife using two in vitro bioassays, namely the classic rat mitochondrial respiration assay, and a mitochondrial membrane potential assay using zebrafish PAC2 cells. Single OH-PBDE congeners as well as mixtures were tested to study potential additive or synergistic effects. An environmental mixture composed of seven OH-PBDE congeners mimicking the concentrations reported in Baltic blue mussels were also studied. We report that all OH-PBDEs tested were able to disrupt OXPHOS via either protonophoric uncoupling and/or inhibition of the electron transport chain. Additionally we show that OH-PBDEs tested in combinations as found in the environment have the potential to disrupt OXPHOS. Importantly, mixtures of OH-PBDEs may show very strong synergistic effects, stressing the importance of further research on the in vivo impacts of these compounds in the environment.

Coactosin accelerates cell dynamism by promoting actin polymerization.

PubMed

Hou, Xubin; Katahira, Tatsuya; Ohashi, Kazumasa; Mizuno, Kensaku; Sugiyama, Sayaka; Nakamura, Harukazu

2013-07-01

During development, cells dynamically move or extend their processes, which are achieved by actin dynamics. In the present study, we paid attention to Coactosin, an actin binding protein, and studied its role in actin dynamics. Coactosin was associated with actin and Capping protein in neural crest cells and N1E-115 neuroblastoma cells. Accumulation of Coactosin to cellular processes and its association with actin filaments prompted us to reveal the effect of Coactosin on cell migration. Coactosin overexpression induced cellular processes in cultured neural crest cells. In contrast, knock-down of Coactosin resulted in disruption of actin polymerization and of neural crest cell migration. Importantly, Coactosin was recruited to lamellipodia and filopodia in response to Rac signaling, and mutated Coactosin that cannot bind to F-actin did not react to Rac signaling, nor support neural crest cell migration. It was also shown that deprivation of Rac signaling from neural crest cells by dominant negative Rac1 (DN-Rac1) interfered with neural crest cell migration, and that co-transfection of DN-Rac1 and Coactosin restored neural crest cell migration. From these results we have concluded that Coactosin functions downstream of Rac signaling and that it is involved in neurite extension and neural crest cell migration by actively participating in actin polymerization. Copyright © 2013 Elsevier Inc. All rights reserved.

Lipid rafts are disrupted in mildly inflamed intestinal microenvironments without overt disruption of the epithelial barrier.

PubMed

Bowie, Rachel V; Donatello, Simona; Lyes, Clíona; Owens, Mark B; Babina, Irina S; Hudson, Lance; Walsh, Shaun V; O'Donoghue, Diarmuid P; Amu, Sylvie; Barry, Sean P; Fallon, Padraic G; Hopkins, Ann M

2012-04-15

Intestinal epithelial barrier disruption is a feature of inflammatory bowel disease (IBD), but whether barrier disruption precedes or merely accompanies inflammation remains controversial. Tight junction (TJ) adhesion complexes control epithelial barrier integrity. Since some TJ proteins reside in cholesterol-enriched regions of the cell membrane termed lipid rafts, we sought to elucidate the relationship between rafts and intestinal epithelial barrier function. Lipid rafts were isolated from Caco-2 intestinal epithelial cells primed with the proinflammatory cytokine interferon-γ (IFN-γ) or treated with methyl-β-cyclodextrin as a positive control for raft disruption. Rafts were also isolated from the ilea of mice in which colitis had been induced in conjunction with in vivo intestinal permeability measurements, and lastly from intestinal biopsies of ulcerative colitis (UC) patients with predominantly mild or quiescent disease. Raft distribution was analyzed by measuring activity of the raft-associated enzyme alkaline phosphatase and by performing Western blot analysis for flotillin-1. Epithelial barrier integrity was estimated by measuring transepithelial resistance in cytokine-treated cells or in vivo permeability to fluorescent dextran in colitic mice. Raft and nonraft fractions were analyzed by Western blotting for the TJ proteins occludin and zonula occludens-1 (ZO-1). Our results revealed that lipid rafts were disrupted in IFN-γ-treated cells, in the ilea of mice with subclinical colitis, and in UC patients with quiescent inflammation. This was not associated with a clear pattern of occludin or ZO-1 relocalization from raft to nonraft fractions. Significantly, a time-course study in colitic mice revealed that disruption of lipid rafts preceded the onset of increased intestinal permeability. Our data suggest for the first time that lipid raft disruption occurs early in the inflammatory cascade in murine and human colitis and, we speculate, may contribute to subsequent disruption of epithelial barrier function.

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

Yin, Shengyong; Chen, Xinhua; Xie, Haiyang

Previous studies showed nanosecond pulsed electric field (nsPEF) can ablate solid tumors including hepatocellular carcinoma (HCC) but its effect on cell membrane is not fully understood. We hypothesized nsPEF disrupt the microdomains on outer-cellular membrane with direct mechanical force and as a result the plasma membrane permeability increases to facilitate the small molecule intake. Three HCC cells were pulsed one pulse per minute, an interval longer than nanopore resealing time. The cationized ferritin was used to mark up the electronegative microdomains, propidium iodide (PI) for membrane permeabilization, energy dispersive X-ray spectroscopy (EDS) for the negative cell surface charge and cisplatinmore » for inner-cellular cytotoxicity. We demonstrated that the ferritin marked-microdomain and negative cell surface charge were disrupted by nsPEF caused-mechanical force. The cell uptake of propidium and cytotoxicity of DNA-targeted cisplatin increased with a dose effect. Cisplatin gains its maximum inner-cellular cytotoxicity when combining with nsPEF stimulation. We conclude that nsPEF disrupt the microdomains on the outer cellular membrane directly and increase the membrane permeabilization for PI and cisplatin. The microdomain disruption and membrane infiltration changes are caused by the mechanical force from the changes of negative cell surface charge.« less

Cysteine desulfurase Nfs1 and Pim1 protease control levels of Isu, the Fe-S cluster biogenesis scaffold.

PubMed

Song, Ji-Yoon; Marszalek, Jaroslaw; Craig, Elizabeth Anne

2012-06-26

Fe-S clusters are critical prosthetic groups for proteins involved in various critical biological processes. Before being transferred to recipient apo-proteins, Fe-S clusters are assembled on the highly conserved scaffold protein Isu, the abundance of which is regulated posttranslationally on disruption of the cluster biogenesis system. Here we report that Isu is degraded by the Lon-type AAA+ ATPase protease of the mitochondrial matrix, Pim1. Nfs1, the cysteine desulfurase responsible for providing sulfur for cluster formation, is required for the increased Isu stability occurring after disruption of cluster formation on or transfer from Isu. Physical interaction between the Isu and Nfs1 proteins, not the enzymatic activity of Nfs1, is the important factor in increased stability. Analysis of several conditions revealed that high Isu levels can be advantageous or disadvantageous, depending on the physiological condition. During the stationary phase, elevated Isu levels were advantageous, resulting in prolonged chronological lifespan. On the other hand, under iron-limiting conditions, high Isu levels were deleterious. Compared with cells expressing normal levels of Isu, such cells grew poorly and exhibited reduced activity of the heme-containing enzyme ferric reductase. Our results suggest that modulation of the degradation of Isu by the Pim1 protease is a regulatory mechanism serving to rapidly help balance the cell's need for critical iron-requiring processes under changing environmental conditions.

A guide to manufacturing CAR T cell therapies.

PubMed

Vormittag, Philipp; Gunn, Rebecca; Ghorashian, Sara; Veraitch, Farlan S

2018-02-17

In recent years, chimeric antigen receptor (CAR) modified T cells have been used as a treatment for haematological malignancies in several phase I and II trials and with Kymriah of Novartis and Yescarta of KITE Pharma, the first CAR T cell therapy products have been approved. Promising clinical outcomes have yet been tempered by the fact that many therapies may be prohibitively expensive to manufacture. The process is not yet defined, far from being standardised and often requires extensive manual handling steps. For academia, big pharma and contract manufacturers it is difficult to obtain an overview over the process strategies and their respective advantages and disadvantages. This review details current production processes being used for CAR T cells with a particular focus on efficacy, reproducibility, manufacturing costs and release testing. By undertaking a systematic analysis of the manufacture of CAR T cells from reported clinical trial data to date, we have been able to quantify recent trends and track the uptake of new process technology. Delivering new processing options will be key to the success of the CAR-T cells ensuring that excessive manufacturing costs do not disrupt the delivery of exciting new therapies to the wide possible patient cohort. Copyright © 2018 Elsevier Ltd. All rights reserved.

Role of HIF-1α in skeletal development

PubMed Central

Wan, Chao; Shao, Jin; Gilbert, Shawn R.; Riddle, Ryan C.; Long, Fanxin; Johnson, Randall S.; Schipani, Ernestina; Clemens, Thomas L.

2011-01-01

Angiogenesis and osteogenesis are tightly coupled during bone development and regeneration. Mesenchymal cells in the developing stroma elicit angiogenic signals to recruit new blood vessels into bone. Reciprocal signals, likely emanating from the incoming vascular endothelium, stimulate mesenchymal cell specification through additional interactions with cells within the vascular stem cell niche. The hypoxia-inducible factor-1 alpha (HIF-1) pathway has been identified as a key component in this process. We demonstrated that overexpression of HIF-1 in mature osteoblasts through disruption of the von Hippel-Lindau protein profoundly increases angiogenesis and osteogenesis; these processes appear to be coupled by cell nonautonomous mechanisms involving the action of vascular endothelial growth factor (VEGF) on the endothelial cells. The same occurred in the model of injury-mediated bone regeneration (distraction osteogenesis). Surprisingly, manipulation of HIF-1 does not influence angiogenesis of the skull bones, where earlier activation of HIF-1 in the condensing mesenchyme upregulates osterix during cranial bone formation. PMID:20392254

Design and use of an artificial capillary in the study of metastatic cell adhesion

NASA Astrophysics Data System (ADS)

Rafi, Adam; Peramo, Antonio; Boren, Rebecca; Heim, August; Matthews, William G.

2006-03-01

To improve the quality of life of patients with cancer, treatments will need to both minimize existing tumors and reduce the metastasis of cancer cells. The effectiveness of potential treatments on existing tumors can be directly probed, but anti-metastasis treatments are difficult to quantify. Therefore, a detailed understanding of the metastatic process is required for drug design. Details of the metastatic deposition of tumor cells in the circulatory system are not well understood. We are investigating the binding of tumor cells to an artificial endothelium. The model system allows for control over molecular composition at the interface, presenting the proteoglycans (PGs) found in the glycocalyx to tumor cells under shear flow conditions. Whether rolling or static adhesion is preferred, as well as what mechanical properties of the interaction between the cells and the PGs are important is to be determined. The outcomes of these experiments will help guide the search for pharmaceuticals that can disrupt the metastatic process at the endothelial adhesion step.

Mutagenesis Screen Identifies agtpbp1 and eps15L1 as Essential for T lymphocyte Development in Zebrafish.

PubMed

Seiler, Christoph; Gebhart, Nichole; Zhang, Yong; Shinton, Susan A; Li, Yue-sheng; Ross, Nicola L; Liu, Xingjun; Li, Qin; Bilbee, Alison N; Varshney, Gaurav K; LaFave, Matthew C; Burgess, Shawn M; Balciuniene, Jorune; Balciunas, Darius; Hardy, Richard R; Kappes, Dietmar J; Wiest, David L; Rhodes, Jennifer

2015-01-01

Genetic screens are a powerful tool to discover genes that are important in immune cell development and function. The evolutionarily conserved development of lymphoid cells paired with the genetic tractability of zebrafish make this a powerful model system for this purpose. We used a Tol2-based gene-breaking transposon to induce mutations in the zebrafish (Danio rerio, AB strain) genome, which served the dual purpose of fluorescently tagging cells and tissues that express the disrupted gene and provided a means of identifying the disrupted gene. We identified 12 lines in which hematopoietic tissues expressed green fluorescent protein (GFP) during embryonic development, as detected by microscopy. Subsequent analysis of young adult fish, using a novel approach in which single cell suspensions of whole fish were analyzed by flow cytometry, revealed that 8 of these lines also exhibited GFP expression in young adult cells. An additional 15 lines that did not have embryonic GFP+ hematopoietic tissue by microscopy, nevertheless exhibited GFP+ cells in young adults. RT-PCR analysis of purified GFP+ populations for expression of T and B cell-specific markers identified 18 lines in which T and/or B cells were fluorescently tagged at 6 weeks of age. As transposon insertion is expected to cause gene disruption, these lines can be used to assess the requirement for the disrupted genes in immune cell development. Focusing on the lines with embryonic GFP+ hematopoietic tissue, we identified three lines in which homozygous mutants exhibited impaired T cell development at 6 days of age. In two of the lines we identified the disrupted genes, agtpbp1 and eps15L1. Morpholino-mediated knockdown of these genes mimicked the T cell defects in the corresponding mutant embryos, demonstrating the previously unrecognized, essential roles of agtpbp1 and eps15L1 in T cell development.

In vivo cell biology in zebrafish - providing insights into vertebrate development and disease.

PubMed

Vacaru, Ana M; Unlu, Gokhan; Spitzner, Marie; Mione, Marina; Knapik, Ela W; Sadler, Kirsten C

2014-02-01

Over the past decades, studies using zebrafish have significantly advanced our understanding of the cellular basis for development and human diseases. Zebrafish have rapidly developing transparent embryos that allow comprehensive imaging of embryogenesis combined with powerful genetic approaches. However, forward genetic screens in zebrafish have generated unanticipated findings that are mirrored by human genetic studies: disruption of genes implicated in basic cellular processes, such as protein secretion or cytoskeletal dynamics, causes discrete developmental or disease phenotypes. This is surprising because many processes that were assumed to be fundamental to the function and survival of all cell types appear instead to be regulated by cell-specific mechanisms. Such discoveries are facilitated by experiments in whole animals, where zebrafish provides an ideal model for visualization and manipulation of organelles and cellular processes in a live vertebrate. Here, we review well-characterized mutants and newly developed tools that underscore this notion. We focus on the secretory pathway and microtubule-based trafficking as illustrative examples of how studying cell biology in vivo using zebrafish has broadened our understanding of the role fundamental cellular processes play in embryogenesis and disease.

Polarized Cell Division of Chlamydia trachomatis

PubMed Central

Abdelrahman, Yasser; Ouellette, Scot P.; Belland, Robert J.; Cox, John V.

2016-01-01

Bacterial cell division predominantly occurs by a highly conserved process, termed binary fission, that requires the bacterial homologue of tubulin, FtsZ. Other mechanisms of bacterial cell division that are independent of FtsZ are rare. Although the obligate intracellular human pathogen Chlamydia trachomatis, the leading bacterial cause of sexually transmitted infections and trachoma, lacks FtsZ, it has been assumed to divide by binary fission. We show here that Chlamydia divides by a polarized cell division process similar to the budding process of a subset of the Planctomycetes that also lack FtsZ. Prior to cell division, the major outer-membrane protein of Chlamydia is restricted to one pole of the cell, and the nascent daughter cell emerges from this pole by an asymmetric expansion of the membrane. Components of the chlamydial cell division machinery accumulate at the site of polar growth prior to the initiation of asymmetric membrane expansion and inhibitors that disrupt the polarity of C. trachomatis prevent cell division. The polarized cell division of C. trachomatis is the result of the unipolar growth and FtsZ-independent fission of this coccoid organism. This mechanism of cell division has not been documented in other human bacterial pathogens suggesting the potential for developing Chlamydia-specific therapeutic treatments. PMID:27505160

APOC3 induces endothelial dysfunction through TNF-α and JAM-1.

PubMed

Tao, Yun; Xiong, Yisong; Wang, Huimin; Chu, Shaopeng; Zhong, Renqian; Wang, Jianxin; Wang, Guihua; Ren, Xiumei; Yu, Juan

2016-09-13

The fatality rate for cardiovascular disease (CVD) has increased in recent years and higher levels of triglyceride have been shown to be an independent risk factor for atherosclerotic CVD. Dysfunction of endothelial cells (ECs) is also a key factor of CVD. APOC3 is an important molecule in lipid metabolism that is closely associated with hyperlipidemia and an increased risk of developing CVD. But the direct effects of APOC3 on ECs were still unknown. This study was aimed at determining the effects of APOC3 on inflammation, chemotaxis and exudation in ECs. ELISA, qRT-PCR, immunofluorescence, flow cytometry and transwell assays were used to investigate the effects of APOC3 on human umbilical vein endothelial cells (HUVECs). SiRNA-induced TNF-α and JAM-1 silencing were used to observe how APOC3 influenced the inflammatory process in the ECs. Our results showed that APOC3 was closely associated with the inflammatory process in ECs, and that this process was characterized by the increased expression of TNF-α. Inflammatory processes further disrupted the tight junctions (TJs) between HUVECs by causing increased expression of JAM-1. JAM-1 was involved in maintaining the integrity of TJs, and it promoted the assembly of platelets and the exudation of leukocytes. Changes in its expression promoted chemotaxis and the exudation of ECs, which contributed to atherosclerosis. While the integrity of the TJs was disrupted, the adhesion of THP-1 cells to HUVECs was also increased by APOC3. In this study, we describe the mechanism by which APOC3 causes inflammation, chemotaxis and the exudation of ECs, and we suggest that controlling the inflammatory reactions that are caused by APOC3 may be a new method to treat CVD.

Disruption of Annexin II /p11 Interaction Suppresses Leukemia Cell Binding, Homing and Engraftment, and Sensitizes the Leukemia Cells to Chemotherapy.

PubMed

Gopalakrishnapillai, Anilkumar; Kolb, E Anders; Dhanan, Priyanka; Mason, Robert W; Napper, Andrew; Barwe, Sonali P

2015-01-01

The bone marrow microenvironment plays an important role in acute lymphoblastic leukemia (ALL) cell proliferation, maintenance, and resistance to chemotherapy. Annexin II (ANX2) is abundantly expressed on bone marrow cells and complexes with p11 to form ANX2/p11-hetero-tetramer (ANX2T). We present evidence that p11 is upregulated in refractory ALL cell lines and patient samples. A small molecule inhibitor that disrupts ANX2/p11 interaction (ANX2T inhibitor), an anti-ANX2 antibody, and knockdown of p11, abrogated ALL cell adhesion to osteoblasts, indicating that ANX2/p11 interaction facilitates binding and retention of ALL cells in the bone marrow. Furthermore, ANX2T inhibitor increased the sensitivity of primary ALL cells co-cultured with osteoblasts to dexamethasone and vincristine induced cell death. Finally, in an orthotopic leukemia xenograft mouse model, the number of ALL cells homing to the bone marrow was reduced by 40-50% in mice injected with anti-ANX2 antibody, anti-p11 antibody or ANX2T inhibitor compared to respective controls. In a long-term engraftment assay, the percentage of ALL cells in mouse blood, bone marrow and spleen was reduced in mice treated with agents that disrupt ANX2/p11 interaction. These data show that disruption of ANX2/p11 interaction results in reduced ALL cell adhesion to osteoblasts, increased ALL cell sensitization to chemotherapy, and suppression of ALL cell homing and engraftment.

Oncogenicity of L-type amino-acid transporter 1 (LAT1) revealed by targeted gene disruption in chicken DT40 cells: LAT1 is a promising molecular target for human cancer therapy

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

Ohkawa, Mayumi; Ohno, Yoshiya; Masuko, Kazue

Highlights: {yields} We established LAT1 amino-acid transporter-disrupted DT40 cells. {yields} LAT1-disrupted cells showed slow growth and lost the oncogenicity. {yields} siRNA and mAb inhibited human tumor growth in vitro and in vivo. {yields} LAT1 is a promising target molecule for cancer therapy. -- Abstract: L-type amino-acid transporter 1 (LAT1) is the first identified light chain of CD98 molecule, disulfide-linked to a heavy chain of CD98. Following cDNA cloning of chicken full-length LAT1, we have constructed targeting vectors for the disruption of chicken LAT1 gene from genomic DNA of chicken LAT1 consisting of 5.4 kb. We established five homozygous LAT1-disrupted (LAT1{supmore » -/-}) cell clones, derived from a heterozygous LAT1{sup +/-} clone of DT40 chicken B cell line. Reactivity of anti-chicken CD98hc monoclonal antibody (mAb) with LAT1{sup -/-} DT40 cells was markedly decreased compared with that of wild-type DT40 cells. All LAT1{sup -/-} cells were deficient in L-type amino-acid transporting activity, although alternative-splice variant but not full-length mRNA of LAT1 was detected in these cells. LAT1{sup -/-} DT40 clones showed outstandingly slow growth in liquid culture and decreased colony-formation capacity in soft agar compared with wild-type DT40 cells. Cell-cycle analyses indicated that LAT1{sup -/-} DT40 clones have prolonged cell-cycle phases compared with wild-type or LAT1{sup +/-} DT40 cells. Knockdown of human LAT1 by small interfering RNAs resulted in marked in vitro cell-growth inhibition of human cancer cells, and in vivo tumor growth of HeLa cells in athymic mice was significantly inhibited by anti-human LAT1 mAb. All these results indicate essential roles of LAT1 in the cell proliferation and occurrence of malignant phenotypes and that LAT1 is a promising candidate as a molecular target of human cancer therapy.« less

Cinnamic Acid Analogs as Intervention Catalysts for Overcoming Antifungal Tolerance.

PubMed

Kim, Jong H; Chan, Kathleen L; Cheng, Luisa W

2017-10-21

Disruption of fungal cell wall should be an effective intervention strategy. However, the cell wall-disrupting echinocandin drugs, such as caspofungin (CAS), cannot exterminate filamentous fungal pathogens during treatment. For potency improvement of cell wall-disrupting agents (CAS, octyl gallate (OG)), antifungal efficacy of thirty-three cinnamic acid derivatives was investigated against Saccharomyces cerevisiae slt2 Δ, bck1 Δ, mutants of the mitogen-activated protein kinase (MAPK), and MAPK kinase kinase, respectively, in cell wall integrity system, and glr1 Δ, mutant of CAS-responsive glutathione reductase. Cell wall mutants were highly susceptible to four cinnamic acids (4-chloro-α-methyl-, 4-methoxy-, 4-methyl-, 3-methylcinnamic acids), where 4-chloro-α-methyl- and 4-methylcinnamic acids possessed the highest activity. Structure-activity relationship revealed that 4-methylcinnamic acid, the deoxygenated structure of 4-methoxycinnamic acid, overcame tolerance of glr1 Δ to 4-methoxycinnamic acid, indicating the significance of para substitution of methyl moiety for effective fungal control. The potential of compounds as chemosensitizers (intervention catalysts) to cell wall disruptants (viz., 4-chloro-α-methyl- or 4-methylcinnamic acids + CAS or OG) was assessed according to Clinical Laboratory Standards Institute M38-A. Synergistic chemosensitization greatly lowers minimum inhibitory concentrations of the co-administered drug/agents. 4-Chloro-α-methylcinnamic acid further overcame fludioxonil tolerance of Aspergillus fumigatus antioxidant MAPK mutants ( sakA Δ, mpkC Δ). Collectively, 4-chloro-α-methyl- and 4-methylcinnamic acids possess chemosensitizing capability to augment antifungal efficacy of conventional drug/agents, thus could be developed as target-based (i.e., cell wall disruption) intervention catalysts.

Vav1 GEF activity is required for T cell mediated allograft rejection.

PubMed

Haubert, Dirk; Li, Jianping; Saveliev, Alexander; Calzascia, Thomas; Sutter, Esther; Metzler, Barbara; Kaiser, Daniel; Tybulewicz, Victor L J; Weckbecker, Gisbert

2012-06-01

The GDP exchange factor (GEF) Vav1 is a central signal transducer downstream of the T cell receptor and has been identified as a key factor for T cell activation in the context of allograft rejection. Vav1 has been shown to transduce signals both dependent and independent of its GEF function. The most promising approach to disrupt Vav1 activity by pharmacological inhibition would be to target its GEF function. However, the contribution of Vav1 GEF activity for allogeneic T cell activation has not been clarified yet. To address this question, we used knock-in mice bearing a mutated Vav1 with disrupted GEF activity but intact GEF-independent functions. T cells from these mice showed strongly reduced proliferation and activation in response to allogeneic stimulation. Furthermore, lack of Vav1 GEF activity strongly abrogated the in vivo expansion of T cells in a systemic graft-versus-host model. In a cardiac transplantation model, mice with disrupted Vav1 GEF activity show prolonged allograft survival. These findings demonstrate a strong requirement for Vav1 GEF activity for allogeneic T cell activation and graft rejection suggesting that disruption of Vav1 GEF activity alone is sufficient to induce significant immunosuppression. Copyright © 2012 Elsevier B.V. All rights reserved.

Translational Control of Viral Gene Expression in Eukaryotes

PubMed Central

Gale, Michael; Tan, Seng-Lai; Katze, Michael G.

2000-01-01

As obligate intracellular parasites, viruses rely exclusively on the translational machinery of the host cell for the synthesis of viral proteins. This relationship has imposed numerous challenges on both the infecting virus and the host cell. Importantly, viruses must compete with the endogenous transcripts of the host cell for the translation of viral mRNA. Eukaryotic viruses have thus evolved diverse mechanisms to ensure translational efficiency of viral mRNA above and beyond that of cellular mRNA. Mechanisms that facilitate the efficient and selective translation of viral mRNA may be inherent in the structure of the viral nucleic acid itself and can involve the recruitment and/or modification of specific host factors. These processes serve to redirect the translation apparatus to favor viral transcripts, and they often come at the expense of the host cell. Accordingly, eukaryotic cells have developed antiviral countermeasures to target the translational machinery and disrupt protein synthesis during the course of virus infection. Not to be outdone, many viruses have answered these countermeasures with their own mechanisms to disrupt cellular antiviral pathways, thereby ensuring the uncompromised translation of virion proteins. Here we review the varied and complex translational programs employed by eukaryotic viruses. We discuss how these translational strategies have been incorporated into the virus life cycle and examine how such programming contributes to the pathogenesis of the host cell. PMID:10839817

Non-Saccharomyces yeasts protect against epithelial cell barrier disruption induced by Salmonella enterica subsp. enterica serovar Typhimurium.

PubMed

Smith, I M; Baker, A; Arneborg, N; Jespersen, L

2015-11-01

The human gastrointestinal epithelium makes up the largest barrier separating the body from the external environment. Whereas invasive pathogens cause epithelial barrier disruption, probiotic micro-organisms modulate tight junction regulation and improve epithelial barrier function. In addition, probiotic strains may be able to reduce epithelial barrier disruption caused by pathogenic species. The aim of this study was to explore non-Saccharomyces yeast modulation of epithelial cell barrier function in vitro. Benchmarking against established probiotic strains, we evaluated the ability of four nonpathogenic yeast species to modulate transepithelial electrical resistance (TER) across a monolayer of differentiated human colonocytes (Caco-2 cells). Further, we assessed yeast modulation of a Salmonella Typhimurium-induced epithelial cell barrier function insult. Our findings demonstrate distinct patterns of non-Saccharomyces yeast modulation of epithelial cell barrier function. While the established probiotic yeast Saccharomyces boulardii increased TER across a Caco-2 monolayer by 30%, Kluyveromyces marxianus exhibited significantly stronger properties of TER enhancement (50% TER increase). In addition, our data demonstrate significant yeast-mediated modulation of Salmonella-induced epithelial cell barrier disruption and identify K. marxianus and Metschnikowia gruessii as two non-Saccharomyces yeasts capable of protecting human epithelial cells from pathogen invasion. This study demonstrates distinct patterns of non-Saccharomyces yeast modulation of epithelial cell barrier function in vitro. Further, our data demonstrate significant yeast-mediated modulation of Salmonella Typhimurium-induced epithelial cell barrier disruption and identify Kluyveromyces marxianus and Metschnikowia gruessii as two non-Saccharomyces yeasts capable of protecting human epithelial cells from pathogen invasion. This study is the first to demonstrate significant non-Saccharomyces yeast-mediated epithelial cell barrier protection from Salmonella invasion, thus encouraging future efforts aimed at confirming the observed effects in vivo and driving further strain development towards novel yeast probiotics. © 2015 The Society for Applied Microbiology.

Correlated alterations in prostate basal cell layer and basement membrane

PubMed Central

Liu, Aijun; Wei, Lixin; Gardner, William A.; Deng, Chu-Xia; Man, Yan-Gao

2009-01-01

Our recent studies revealed that focal basal cell layer disruption (FBCLD) induced auto-immunoreactions represented a contributing factor for human prostate tumor progression and invasion. As the basement membrane surrounds and attaches to the basal cell layer, our current study assessed whether FBCLD would impact the physical integrity of the associated basement membrane. Paraffin sections from 25-human prostate tumors were subjected to double immunohistochemistry to simultaneously elucidate the basal cell layer and the basement membrane with corresponding biomarkers. The physical integrity of the basement membrane overlying FBCLD was examined to determine the extent of correlated alterations. Of a total of 89 FBCLD encountered, 76 (85 %) showed correlated alterations in the overlying basement membrane, which included distinct focal disruptions or fragmentations. In the remaining 13 (15%) FBCLD, the overlying basement membrane showed significant attenuation or reduction of the immunostaining intensity. The basement membrane in all or nearly all ducts or acini with p63 positive basal cells was substantially thicker and more uniform than that in ducts or acini without p63 positive basal cells, and also, a vast majority of the focal disruptions occurred near basal cells that lack p63 expression. These findings suggest that focal disruptions in the basal cell layer and alterations in the basement membrane are correlated events and that the physical and functional status of the basal cells could significantly impact the physical integrity of the overlying basement membrane. As the degradation of both the basal cell layer and the basement membrane is a pre-requisite for prostate tumor invasion or progression, ducts or acini with focally disrupted basal cell layer and basement membrane are likely at greater risk to develop invasive lesions. Thus, further elucidation of the specific molecules and mechanism associated with these events may lead to the development of a more effective alternative for repeat biopsy to monitor tumor progression and invasion. PMID:19343113

Usher syndrome: molecular links of pathogenesis, proteins and pathways.

PubMed

Kremer, Hannie; van Wijk, Erwin; Märker, Tina; Wolfrum, Uwe; Roepman, Ronald

2006-10-15

Usher syndrome is the most common form of deaf-blindness. The syndrome is both clinically and genetically heterogeneous, and to date, eight causative genes have been identified. The proteins encoded by these genes are part of a dynamic protein complex that is present in hair cells of the inner ear and in photoreceptor cells of the retina. The localization of the Usher proteins and the phenotype in animal models indicate that the Usher protein complex is essential in the morphogenesis of the stereocilia bundle in hair cells and in the calycal processes of photoreceptor cells. In addition, the Usher proteins are important in the synaptic processes of both cell types. The association of other proteins with the complex indicates functional links to a number of basic cell-biological processes. Prominently present is the connection to the dynamics of the actin cytoskeleton, involved in cellular morphology, cell polarity and cell-cell interactions. The Usher protein complex can also be linked to the cadherins/catenins in the adherens junction-associated protein complexes, suggesting a role in cell polarity and tissue organization. A third link can be established to the integrin transmembrane signaling network. The Usher interactome, as outlined in this review, participates in pathways common in inner ear and retina that are disrupted in the Usher syndrome.

Lectin-based food poisoning: a new mechanism of protein toxicity.

PubMed

Miyake, Katsuya; Tanaka, Toru; McNeil, Paul L

2007-08-01

Ingestion of the lectins present in certain improperly cooked vegetables can result in acute GI tract distress, but the mechanism of toxicity is unknown. In vivo, gut epithelial cells are constantly exposed to mechanical and other stresses and consequently individual cells frequently experience plasma membrane disruptions. Repair of these cell surface disruptions allows the wounded cell to survive: failure results in necrotic cell death. Plasma membrane repair is mediated, in part, by an exocytotic event that adds a patch of internal membrane to the defect site. Lectins are known to inhibit exocytosis. We therefore tested the novel hypothesis that lectin toxicity is due to an inhibitory effect on plasma membrane repair. Repair of plasma membrane disruptions and exocytosis of mucus was assessed after treatment of cultured cell models and excised segments of the GI tract with lectins. Plasma membrane disruptions were produced by focal irradiation of individual cells, using a microscope-based laser, or by mechanical abrasion of multiple cells, using a syringe needle. Repair was then assessed by monitoring the cytosolic penetration of dyes incapable of crossing the intact plasma membrane. We found that cell surface-bound lectins potently inhibited plasma membrane repair, and the exocytosis of mucus that normally accompanies the repair response. Lectins potently inhibit plasma membrane repair, and hence are toxic to wounded cells. This represents a novel form of protein-based toxicity, one that, we propose, is the basis of plant lectin food poisoning.

Triptolide disrupts the actin-based Sertoli-germ cells adherens junctions by inhibiting Rho GTPases expression

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

Wang, Xiang; Zhao, Fang

Triptolide (TP), derived from the medicinal plant Triterygium wilfordii Hook. f. (TWHF), is a diterpene triepoxide with variety biological and pharmacological activities. However, TP has been restricted in clinical application due to its narrow therapeutic window especially in reproductive system. During spermatogenesis, Sertoli cell cytoskeleton plays an essential role in facilitating germ cell movement and cell-cell actin-based adherens junctions (AJ). At Sertoli cell-spermatid interface, the anchoring device is a kind of AJ, known as ectoplasmic specializations (ES). In this study, we demonstrate that β-actin, an important component of cytoskeleton, has been significantly down-regulated after TP treatment. TP can inhibit themore » expression of Rho GTPase such as, RhoA, RhoB, Cdc42 and Rac1. Downstream of Rho GTPase, Rho-associated protein kinase (ROCKs) gene expressions were also suppressed by TP. F-actin immunofluorescence proved that TP disrupts Sertoli cells cytoskeleton network. As a result of β-actin down-regulation, TP treatment increased expression of testin, which indicating ES has been disassembled. In summary, this report illustrates that TP induces cytoskeleton dysfunction and disrupts cell-cell adherens junctions via inhibition of Rho GTPases. - Highlights: • Triptolide induced the disruption of Sertoli-germ cell adherens junction. • Rho GTPases expression and actin dynamics have been suppressed by triptolide. • Actin-based adherens junction is a potential antifertility target of triptolide. • Rho-Rock is involved in the regulation of actin dynamics.« less

Role of p53 in cdk Inhibitor VMY-1-103-induced Apoptosis in Prostate Cancer

DTIC Science & Technology

2013-11-01

DAOY medulloblastoma cells, which have a p53 mutation (6). In order to examine if this holds true in prostate cancer cell lines, I stably transfected...disrupts chromosome organization and delays metaphase progression in medulloblastoma cells. Cancer Biol Ther. 2011 Nov 1;12(9):818-26  Other...1-103 is a novel CDK inhibitor that disrupts chromosome organization and delays metaphase progression in medulloblastoma cells. Cancer Biol Ther

Non-coding RNAs in lung cancer

PubMed Central

Ricciuti, Biagio; Mecca, Carmen; Crinò, Lucio; Baglivo, Sara; Cenci, Matteo; Metro, Giulio

2014-01-01

The discovery that protein-coding genes represent less than 2% of all human genome, and the evidence that more than 90% of it is actively transcribed, changed the classical point of view of the central dogma of molecular biology, which was always based on the assumption that RNA functions mainly as an intermediate bridge between DNA sequences and protein synthesis machinery. Accumulating data indicates that non-coding RNAs are involved in different physiological processes, providing for the maintenance of cellular homeostasis. They are important regulators of gene expression, cellular differentiation, proliferation, migration, apoptosis, and stem cell maintenance. Alterations and disruptions of their expression or activity have increasingly been associated with pathological changes of cancer cells, this evidence and the prospect of using these molecules as diagnostic markers and therapeutic targets, make currently non-coding RNAs among the most relevant molecules in cancer research. In this paper we will provide an overview of non-coding RNA function and disruption in lung cancer biology, also focusing on their potential as diagnostic, prognostic and predictive biomarkers. PMID:25593996

Inactivation of the first nucleotide-binding fold of the sulfonylurea receptor, and familial persistent hyperinsulinemic hypoglycemia of infancy

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

Thomas, P.M.; Wohllk, N.; Huang, E.

1996-09-01

Familial persistent hyperinsulinemic hypoglycemia of infancy is a disorder of glucose homeostasis and is characterized by unregulated insulin secretion and profound hypoglycemia. Loss-of-function mutations in the second nucleotide-binding fold of the sulfonylurea receptor, a subunit of the pancreatic-islet {beta}-cell ATP-dependent potassium channel, has been demonstrated to be causative for persistent hyperinsulinemic hypoglycemia of infancy. We now describe three additional mutations in the first nucleotide-binding fold of the sulfonylurea-receptor gene. One point mutation disrupts the highly conserved Walker A motif of the first nucleotide-binding-fold region. The other two mutations occur in noncoding sequences required for RNA processing and are predicted tomore » disrupt the normal splicing pathway of the sulfonylurea-receptor mRNA precursor. These data suggest that both nucleotide-binding-fold regions of the sulfortylurea receptor are required for normal regulation of {beta}-cell ATP-dependent potassium channel activity and insulin secretion. 32 refs., 4 figs., 1 tab.« less

Gallium and its competing roles with iron in biological systems.

PubMed

Chitambar, Christopher R

2016-08-01

Gallium, a group IIIa metal, shares chemical properties with iron. Studies have shown that gallium-based compounds have potential therapeutic activity against certain cancers and infectious microorganisms. By functioning as an iron mimetic, gallium perturbs iron-dependent proliferation processes in tumor cells. Gallium's action on iron homeostasis leads to disruption of ribonucleotide reductase, mitochondrial function, and the regulation of transferrin receptor and ferritin. In addition, gallium nitrate stimulates an increase in mitochondrial reactive oxygen species in cells which triggers downstream upregulation of metallothionein and hemoxygenase-1. Gallium's anti-infective activity against bacteria and fungi results from disruption of microbial iron utilization through mechanisms which include gallium binding to siderophores and downregulation of bacterial iron uptake. Gallium compounds lack cross-resistance to conventional chemotherapeutic drugs and antibiotics thus making them attractive agents for drug development. This review will focus on the mechanisms of action of gallium with emphasis on its interaction with iron and iron proteins. Copyright © 2016 Elsevier B.V. All rights reserved.

Drosophila melanogaster auxilin regulates the internalization of Delta to control activity of the Notch signaling pathway

PubMed Central

Hagedorn, Elliott J.; Bayraktar, Jennifer L.; Kandachar, Vasundhara R.; Bai, Ting; Englert, Dane M.; Chang, Henry C.

2006-01-01

We have isolated mutations in the Drosophila melanogaster homologue of auxilin, a J-domain–containing protein known to cooperate with Hsc70 in the disassembly of clathrin coats from clathrin-coated vesicles in vitro. Consistent with this biochemical role, animals with reduced auxilin function exhibit genetic interactions with Hsc70 and clathrin. Interestingly, the auxilin mutations interact specifically with Notch and disrupt several Notch-mediated processes. Genetic evidence places auxilin function in the signal-sending cells, upstream of Notch receptor activation, suggesting that the relevant cargo for this auxilin-mediated endocytosis is the Notch ligand Delta. Indeed, the localization of Delta protein is disrupted in auxilin mutant tissues. Thus, our data suggest that auxilin is an integral component of the Notch signaling pathway, participating in the ubiquitin-dependent endocytosis of Delta. Furthermore, the fact that auxilin is required for Notch signaling suggests that ligand endocytosis in the signal-sending cells needs to proceed past coat disassembly to activate Notch. PMID:16682530

Circadian gating of epithelial-to-mesenchymal transition in breast cancer cells via melatonin-regulation of GSK3β.

PubMed

Mao, Lulu; Dauchy, Robert T; Blask, David E; Slakey, Lauren M; Xiang, Shulin; Yuan, Lin; Dauchy, Erin M; Shan, Bin; Brainard, George C; Hanifin, John P; Frasch, Tripp; Duplessis, Tamika T; Hill, Steven M

2012-11-01

Disturbed sleep-wake cycle and circadian rhythmicity are associated with cancer, but the underlying mechanisms are unknown. Employing a tissue-isolated human breast xenograft tumor nude rat model, we observed that glycogen synthase kinase 3β (GSK3β), an enzyme critical in metabolism and cell proliferation/survival, exhibits a circadian rhythm of phosphorylation in human breast tumors. Exposure to light-at-night suppresses the nocturnal pineal melatonin synthesis, disrupting the circadian rhythm of GSK3β phosphorylation. Melatonin activates GSK3β by inhibiting the serine-threonine kinase Akt phosphorylation, inducing β-catenin degradation and inhibiting epithelial-to-mesenchymal transition, a fundamental process underlying cancer metastasis. Thus, chronic circadian disruption by light-at-night via occupational exposure or age-related sleep disturbances may contribute to cancer incidence and the metastatic spread of breast cancer by inhibiting GSK3β activity and driving epithelial-to-mesenchymal transition in breast cancer patients.

Circadian Gating of Epithelial-to-Mesenchymal Transition in Breast Cancer Cells Via Melatonin-Regulation of GSK3β

PubMed Central

Mao, Lulu; Dauchy, Robert T.; Blask, David E.; Slakey, Lauren M.; Xiang, Shulin; Yuan, Lin; Dauchy, Erin M.; Shan, Bin; Brainard, George C.; Hanifin, John P.; Duplessis, Tamika T.; Hill, Steven M.

2012-01-01

Disturbed sleep-wake cycle and circadian rhythmicity are associated with cancer, but the underlying mechanisms are unknown. Employing a tissue-isolated human breast xenograft tumor nude rat model, we observed that glycogen synthase kinase 3β (GSK3β), an enzyme critical in metabolism and cell proliferation/survival, exhibits a circadian rhythm of phosphorylation in human breast tumors. Exposure to light-at-night suppresses the nocturnal pineal melatonin synthesis, disrupting the circadian rhythm of GSK3β phosphorylation. Melatonin activates GSK3β by inhibiting the serine-threonine kinase Akt phosphorylation, inducing β-catenin degradation and inhibiting epithelial-to-mesenchymal transition, a fundamental process underlying cancer metastasis. Thus, chronic circadian disruption by light-at-night via occupational exposure or age-related sleep disturbances may contribute to cancer incidence and the metastatic spread of breast cancer by inhibiting GSK3β activity and driving epithelial-to-mesenchymal transition in breast cancer patients. PMID:23002080

Environmental Enrichment Rescues Binocular Matching of Orientation Preference in Mice that Have a Precocious Critical Period

PubMed Central

Wang, Bor-Shuen; Feng, Liang; Liu, Mingna; Liu, Xiaorong; Cang, Jianhua

2013-01-01

SUMMARY Experience shapes neural circuits during critical periods in early life. The timing of critical periods is regulated by both genetics and the environment. Here we study the functional significance of such temporal regulations in the mouse primary visual cortex, where critical period plasticity drives binocular matching of orientation preference. We find that the binocular matching is permanently disrupted in mice that have a precocious critical period due to genetically enhanced inhibition. The disruption is specific to one type of neurons, the complex cells, which, as we reveal, normally match after the simple cells. Early environmental enrichment completely rescues the deficit by inducing histone acetylation and consequently advancing the matching process to coincide with the precocious plasticity. Our experiments thus demonstrate that the proper timing of the critical period is essential for establishing normal binocularity and the detrimental impact of its genetic misregulation can be ameliorated by environmental manipulations via epigenetic mechanisms. PMID:24012279

SCFSlimb ubiquitin ligase suppresses condensin II–mediated nuclear reorganization by degrading Cap-H2

PubMed Central

Buster, Daniel W.; Daniel, Scott G.; Nguyen, Huy Q.; Windler, Sarah L.; Skwarek, Lara C.; Peterson, Maureen; Roberts, Meredith; Meserve, Joy H.; Hartl, Tom; Klebba, Joseph E.; Bilder, David; Bosco, Giovanni

2013-01-01

Condensin complexes play vital roles in chromosome condensation during mitosis and meiosis. Condensin II uniquely localizes to chromatin throughout the cell cycle and, in addition to its mitotic duties, modulates chromosome organization and gene expression during interphase. Mitotic condensin activity is regulated by phosphorylation, but mechanisms that regulate condensin II during interphase are unclear. Here, we report that condensin II is inactivated when its subunit Cap-H2 is targeted for degradation by the SCFSlimb ubiquitin ligase complex and that disruption of this process dramatically changed interphase chromatin organization. Inhibition of SCFSlimb function reorganized interphase chromosomes into dense, compact domains and disrupted homologue pairing in both cultured Drosophila cells and in vivo, but these effects were rescued by condensin II inactivation. Furthermore, Cap-H2 stabilization distorted nuclear envelopes and dispersed Cid/CENP-A on interphase chromosomes. Therefore, SCFSlimb-mediated down-regulation of condensin II is required to maintain proper organization and morphology of the interphase nucleus. PMID:23530065

Exocyst-Dependent Membrane Addition Is Required for Anaphase Cell Elongation and Cytokinesis in Drosophila

PubMed Central

Giansanti, Maria Grazia; Vanderleest, Timothy E.; Jewett, Cayla E.; Sechi, Stefano; Frappaolo, Anna; Fabian, Lacramioara; Robinett, Carmen C.; Brill, Julie A.; Loerke, Dinah; Fuller, Margaret T.; Blankenship, J. Todd

2015-01-01

Mitotic and cytokinetic processes harness cell machinery to drive chromosomal segregation and the physical separation of dividing cells. Here, we investigate the functional requirements for exocyst complex function during cell division in vivo, and demonstrate a common mechanism that directs anaphase cell elongation and cleavage furrow progression during cell division. We show that onion rings (onr) and funnel cakes (fun) encode the Drosophila homologs of the Exo84 and Sec8 exocyst subunits, respectively. In onr and fun mutant cells, contractile ring proteins are recruited to the equatorial region of dividing spermatocytes. However, cytokinesis is disrupted early in furrow ingression, leading to cytokinesis failure. We use high temporal and spatial resolution confocal imaging with automated computational analysis to quantitatively compare wild-type versus onr and fun mutant cells. These results demonstrate that anaphase cell elongation is grossly disrupted in cells that are compromised in exocyst complex function. Additionally, we observe that the increase in cell surface area in wild type peaks a few minutes into cytokinesis, and that onr and fun mutant cells have a greatly reduced rate of surface area growth specifically during cell division. Analysis by transmission electron microscopy reveals a massive build-up of cytoplasmic astral membrane and loss of normal Golgi architecture in onr and fun spermatocytes, suggesting that exocyst complex is required for proper vesicular trafficking through these compartments. Moreover, recruitment of the small GTPase Rab11 and the PITP Giotto to the cleavage site depends on wild-type function of the exocyst subunits Exo84 and Sec8. Finally, we show that the exocyst subunit Sec5 coimmunoprecipitates with Rab11. Our results are consistent with the exocyst complex mediating an essential, coordinated increase in cell surface area that potentiates anaphase cell elongation and cleavage furrow ingression. PMID:26528720

Sleep disruption among cancer patients following autologous hematopoietic cell transplantation.

PubMed

Nelson, Ashley M; Jim, Heather S L; Small, Brent J; Nishihori, Taiga; Gonzalez, Brian D; Cessna, Julie M; Hyland, Kelly A; Rumble, Meredith E; Jacobsen, Paul B

2018-03-01

Despite a high prevalence of sleep disruption among hematopoietic cell transplant (HCT) recipients, relatively little research has investigated its relationships with modifiable cognitive or behavioral factors or used actigraphy to characterize sleep disruption in this population. Autologous HCT recipients who were 6-18 months post transplant completed self-report measures of cancer-related distress, fear of cancer recurrence, dysfunctional sleep cognitions, and inhibitory sleep behaviors upon enrollment. Patients then wore an actigraph for 7 days and completed a self-report measure of sleep disruption on day 7 of the study. Among the 84 participants (age M = 60, 45% female), 41% reported clinically relevant sleep disruption. Examination of actigraph data confirmed that, on average, sleep was disrupted (wake after sleep onset M = 66 min) and sleep efficiency was less than recommended (sleep efficiency M = 78%). Cancer-related distress, fear of recurrence, dysfunctional sleep cognitions, and inhibitory sleep behaviors were related to self-reported sleep disruption (p values<0.05) but not objective sleep indices. Results suggest that many HCT recipients experience sleep disruption after transplant. Cancer-related distress, fear of recurrence, dysfunctional sleep cognitions, and maladaptive sleep behaviors are related to self-reported sleep disruption and should be considered targets for cognitive behavioral intervention in this population.

Sleep disruption in hematopoietic cell transplantation recipients: prevalence, severity, and clinical management.

PubMed

Jim, Heather S L; Evans, Bryan; Jeong, Jiyeon M; Gonzalez, Brian D; Johnston, Laura; Nelson, Ashley M; Kesler, Shelli; Phillips, Kristin M; Barata, Anna; Pidala, Joseph; Palesh, Oxana

2014-10-01

Sleep disruption is common among hematopoietic cell transplant (HCT) recipients, with over 50% of recipients experiencing sleep disruption pre-transplant, with up to 82% of patients experiencing moderate to severe sleep disruption during hospitalization for transplant and up to 43% after transplant. These rates of sleep disruption are substantially higher than what we see in the general population. Although sleep disruption can be distressing to patients and contribute to diminished quality of life, it is rarely discussed during clinical visits. The goal of the current review is to draw attention to sleep disruption and disorders (ie, insomnia, obstructive sleep apnea, restless legs syndrome) as a clinical problem in HCT in order to facilitate patient education, intervention, and research. We identified 35 observational studies published in the past decade that examined sleep disruption or disorders in HCT. Most studies utilized a single item measure of sleep, had small sample size, and included heterogeneous samples of patients. Six studies of the effects of psychosocial and exercise interventions on sleep in HCT have reported no significant improvements. These results highlight the need for rigorous observational and interventional studies of sleep disruption and disorders in HCT recipients.. Copyright © 2014 American Society for Blood and Marrow Transplantation. All rights reserved.

Production Strategies and Applications of Microbial Single Cell Oils

PubMed Central

Ochsenreither, Katrin; Glück, Claudia; Stressler, Timo; Fischer, Lutz; Syldatk, Christoph

2016-01-01

Polyunsaturated fatty acids (PUFAs) of the ω-3 and ω-6 class (e.g., α-linolenic acid, linoleic acid) are essential for maintaining biofunctions in mammalians like humans. Due to the fact that humans cannot synthesize these essential fatty acids, they must be taken up from different food sources. Classical sources for these fatty acids are porcine liver and fish oil. However, microbial lipids or single cell oils, produced by oleaginous microorganisms such as algae, fungi and bacteria, are a promising source as well. These single cell oils can be used for many valuable chemicals with applications not only for nutrition but also for fuels and are therefore an ideal basis for a bio-based economy. A crucial point for the establishment of microbial lipids utilization is the cost-effective production and purification of fuels or products of higher value. The fermentative production can be realized by submerged (SmF) or solid state fermentation (SSF). The yield and the composition of the obtained microbial lipids depend on the type of fermentation and the particular conditions (e.g., medium, pH-value, temperature, aeration, nitrogen source). From an economical point of view, waste or by-product streams can be used as cheap and renewable carbon and nitrogen sources. In general, downstream processing costs are one of the major obstacles to be solved for full economic efficiency of microbial lipids. For the extraction of lipids from microbial biomass cell disruption is most important, because efficiency of cell disruption directly influences subsequent downstream operations and overall extraction efficiencies. A multitude of cell disruption and lipid extraction methods are available, conventional as well as newly emerging methods, which will be described and discussed in terms of large scale applicability, their potential in a modern biorefinery and their influence on product quality. Furthermore, an overview is given about applications of microbial lipids or derived fatty acids with emphasis on food applications. PMID:27761130

Effective oligonucleotide-mediated gene disruption in ES cells lacking the mismatch repair protein MSH3.

PubMed

Dekker, M; Brouwers, C; Aarts, M; van der Torre, J; de Vries, S; van de Vrugt, H; te Riele, H

2006-04-01

We have previously demonstrated that site-specific insertion, deletion or substitution of one or two nucleotides in mouse embryonic stem cells (ES cells) by single-stranded deoxyribo-oligonucleotides is several hundred-fold suppressed by DNA mismatch repair (MMR) activity. Here, we have investigated whether compound mismatches and larger insertions escape detection by the MMR machinery and can be effectively introduced in MMR-proficient cells. We identified several compound mismatches that escaped detection by the MMR machinery to some extent, but could not define general rules predicting the efficacy of complex base-pair substitutions. In contrast, we found that four-nucleotide insertions were largely subject to suppression by the MSH2/MSH3 branch of MMR and could be effectively introduced in Msh3-deficient cells. As these cells have no overt mutator phenotype and Msh3-deficient mice do not develop cancer, Msh3-deficient ES cells can be used for oligonucleotide-mediated gene disruption. As an example, we present disruption of the Fanconi anemia gene Fancf.

Emergence of an apical epithelial cell surface in vivo

PubMed Central

Sedzinski, Jakub; Hannezo, Edouard; Tu, Fan; Biro, Maté; Wallingford, John B.

2016-01-01

Epithelial sheets are crucial components of all metazoan animals, enclosing organs and protecting the animal from its environment. Epithelial homeostasis poses unique challenges, as addition of new cells and loss of old cells must be achieved without disrupting the fluid-tight barrier and apicobasal polarity of the epithelium. Several studies have identified cell biological mechanisms underlying extrusion of cells from epithelia, but far less is known of the converse mechanism by which new cells are added. Here, we combine molecular, pharmacological and laser-dissection experiments with theoretical modelling to characterize forces driving emergence of an apical surface as single nascent cells are added to a vertebrate epithelium in vivo. We find that this process involves the interplay between cell-autonomous actin-generated pushing forces in the emerging cell and mechanical properties of neighboring cells. Our findings define the forces driving this cell behavior, contributing to a more comprehensive understanding of epithelial homeostasis. PMID:26766441

Fibrosis and diseases of the eye

PubMed Central

Friedlander, Martin

2007-01-01

Most diseases that cause catastrophic loss of vision do so as a result of abnormal angiogenesis and wound healing, often in response to tissue ischemia or inflammation. Disruption of the highly ordered tissue architecture in the eye caused by vascular leakage, hemorrhage, and concomitant fibrosis can lead to mechanical disruption of the visual axis and/or biological malfunctioning. An increased understanding of inflammation, wound healing, and angiogenesis has led to the development of drugs effective in modulating these biological processes and, in certain circumstances, the preservation of vision. Unfortunately, such pharmacological interventions often are too little, too late, and progression of vision loss frequently occurs. The recent development of progenitor and/or stem cell technologies holds promise for the treatment of currently incurable ocular diseases. PMID:17332885

Numerical models of cell death in RF ablation with monopolar and bipolar probes

NASA Astrophysics Data System (ADS)

Bright, Benjamin M.; Pearce, John A.

2013-02-01

Radio frequency (RF) is used clinically to treat unresectible tumors. Finite element modeling has proven useful in treatment planning and applicator design. Typically isotherms in the middle 50s °C have been used as the parameter of assessment in these models. We compare and contrast isotherms for multiple known Arrhenius thermal damage predictors including collagen denaturation, vascular disruption, liver coagulation and cell death. Models for RITA probe geometries are included in the study. Comparison to isotherms is sensible when the activation time is held constant, but varies considerably when heating times vary. The purpose of this paper is to demonstrate the importance of looking at specific processes and keeping track of the methods used to derive the Arrhenius coefficients in order to study the extremely complex cell death processes due to thermal therapies.

The Pathophysiologic Role of Disrupted Circadian and Neuroendocrine Rhythms in Breast Carcinogenesis.

PubMed

Ball, Lonnele J; Palesh, Oxana; Kriegsfeld, Lance J

2016-10-01

Most physiological processes in the brain and body exhibit daily (circadian) rhythms coordinated by an endogenous master clock located in the suprachiasmatic nucleus of the hypothalamus that are essential for normal health and functioning. Exposure to sunlight during the day and darkness at night optimally entrains biological rhythms to promote homeostasis and human health. Unfortunately, a major consequence of the modern lifestyle is increased exposure to sun-free environments during the day and artificial lighting at night. Additionally, behavioral disruptions to circadian rhythms (ie, repeated transmeridian flights, night or rotating shift work, or sleep disturbances) have a profound influence on health and have been linked to a number of pathological conditions, including endocrine-dependent cancers. Specifically, night shift work has been identified as a significant risk factor for breast cancer in industrialized countries. Several mechanisms have been proposed by which shift work-induced circadian disruptions promote cancer. In this review, we examine the importance of the brain-body link through which circadian disruptions contribute to endocrine-dependent diseases, including breast carcinogenesis, by negatively impacting neuroendocrine and neuroimmune cells, and we consider preventive measures directed at maximizing circadian health.

The Pathophysiologic Role of Disrupted Circadian and Neuroendocrine Rhythms in Breast Carcinogenesis

PubMed Central

Ball, Lonnele J.; Palesh, Oxana

2016-01-01

Most physiological processes in the brain and body exhibit daily (circadian) rhythms coordinated by an endogenous master clock located in the suprachiasmatic nucleus of the hypothalamus that are essential for normal health and functioning. Exposure to sunlight during the day and darkness at night optimally entrains biological rhythms to promote homeostasis and human health. Unfortunately, a major consequence of the modern lifestyle is increased exposure to sun-free environments during the day and artificial lighting at night. Additionally, behavioral disruptions to circadian rhythms (ie, repeated transmeridian flights, night or rotating shift work, or sleep disturbances) have a profound influence on health and have been linked to a number of pathological conditions, including endocrine-dependent cancers. Specifically, night shift work has been identified as a significant risk factor for breast cancer in industrialized countries. Several mechanisms have been proposed by which shift work-induced circadian disruptions promote cancer. In this review, we examine the importance of the brain-body link through which circadian disruptions contribute to endocrine-dependent diseases, including breast carcinogenesis, by negatively impacting neuroendocrine and neuroimmune cells, and we consider preventive measures directed at maximizing circadian health. PMID:27712099

Mild myelin disruption elicits early alteration in behavior and proliferation in the subventricular zone.

PubMed

Gould, Elizabeth A; Busquet, Nicolas; Shepherd, Douglas; Dietz, Robert M; Herson, Paco S; Simoes de Souza, Fabio M; Li, Anan; George, Nicholas M; Restrepo, Diego; Macklin, Wendy B

2018-02-13

Myelin, the insulating sheath around axons, supports axon function. An important question is the impact of mild myelin disruption. In the absence of the myelin protein proteolipid protein (PLP1), myelin is generated but with age, axonal function/maintenance is disrupted. Axon disruption occurs in Plp1 -null mice as early as 2 months in cortical projection neurons. High-volume cellular quantification techniques revealed a region-specific increase in oligodendrocyte density in the olfactory bulb and rostral corpus callosum that increased during adulthood. A distinct proliferative response of progenitor cells was observed in the subventricular zone (SVZ), while the number and proliferation of parenchymal oligodendrocyte progenitor cells was unchanged. This SVZ proliferative response occurred prior to evidence of axonal disruption. Thus, a novel SVZ response contributes to the region-specific increase in oligodendrocytes in Plp1 -null mice. Young adult Plp1- null mice exhibited subtle but substantial behavioral alterations, indicative of an early impact of mild myelin disruption. © 2018, Gould et al.

Mild myelin disruption elicits early alteration in behavior and proliferation in the subventricular zone

PubMed Central

Gould, Elizabeth A; Busquet, Nicolas; Shepherd, Douglas; Dietz, Robert M; Herson, Paco S; Simoes de Souza, Fabio M; Li, Anan; George, Nicholas M

2018-01-01

Myelin, the insulating sheath around axons, supports axon function. An important question is the impact of mild myelin disruption. In the absence of the myelin protein proteolipid protein (PLP1), myelin is generated but with age, axonal function/maintenance is disrupted. Axon disruption occurs in Plp1-null mice as early as 2 months in cortical projection neurons. High-volume cellular quantification techniques revealed a region-specific increase in oligodendrocyte density in the olfactory bulb and rostral corpus callosum that increased during adulthood. A distinct proliferative response of progenitor cells was observed in the subventricular zone (SVZ), while the number and proliferation of parenchymal oligodendrocyte progenitor cells was unchanged. This SVZ proliferative response occurred prior to evidence of axonal disruption. Thus, a novel SVZ response contributes to the region-specific increase in oligodendrocytes in Plp1-null mice. Young adult Plp1-null mice exhibited subtle but substantial behavioral alterations, indicative of an early impact of mild myelin disruption. PMID:29436368

Effect of sonication frequency on the disruption of algae.

PubMed

Kurokawa, Masaki; King, Patrick M; Wu, Xiaoge; Joyce, Eadaoin M; Mason, Timothy J; Yamamoto, Ken

2016-07-01

In this study, the efficiency of ultrasonic disruption of Chaetoceros gracilis, Chaetoceros calcitrans, and Nannochloropsis sp. was investigated by applying ultrasonic waves of 0.02, 0.4, 1.0, 2.2, 3.3, and 4.3 MHz to algal suspensions. The results showed that reduction in the number of algae was frequency dependent and that the highest efficiency was achieved at 2.2, 3.3, and 4.3MHz for C. gracilis, C. calcitrans, and Nannochloropsis sp., respectively. A review of the literature suggested that cavitation, rather than direct effects of ultrasonication, are required for ultrasonic algae disruption, and that chemical effects are likely not the main mechanism for algal cell disruption. The mechanical resonance frequencies estimated by a shell model, taking into account elastic properties, demonstrated that suitable disruption frequencies for each alga were associated with the cell's mechanical properties. Taken together, we consider here that physical effects of ultrasonication were responsible for algae disruption. Copyright © 2015 Elsevier B.V. All rights reserved.

Important role of catalase in the cellular response of the budding yeast Saccharomyces cerevisiae exposed to ionizing radiation.

PubMed

Nishimoto, Takuto; Furuta, Masakazu; Kataoka, Michihiko; Kishida, Masao

2015-03-01

Ionizing radiation indirectly causes oxidative stress in cells via reactive oxygen species (ROS), such as hydroxyl radicals (OH(-)) generated by the radiolysis of water. We investigated how the catalase function was affected by ionizing radiation and analyzed the phenotype of mutants with a disrupted catalase gene in Saccharomyces cerevisiae exposed to radiation. The wild-type yeast strain and isogenic mutants with disrupted catalase genes were exposed to various doses of (60)Co gamma-rays. There was no difference between the wild-type strain and the cta1 disruption mutant following exposure to gamma-ray irradiation. In contrast, there was a significant decrease in the ctt1 disruption mutant, suggesting that this strain exhibited decreased survival on gamma-ray exposure compared with other strains. In all three strains, stationary phase cells were more tolerant to the exposure of gamma-rays than exponential phase cells, whereas the catalase activity in the wild-type strain and cta1 disruption mutant was higher in the stationary phase than in the exponential phase. These data suggest a correlation between catalase activity and survival following gamma-ray exposure. However, this correlation was not clear in the ctt1 disruption mutant, suggesting that other factors are involved in the tolerance to ROS induced by irradiation.

[Comparison between lymphocytic infiltration in early spontaneous abortions and in elective abortions with signs of disruption at the chorio-decidual interface].

PubMed

Caliendo, L

2007-12-01

The aim of the study was to compare lymphocytic infiltrations in early spontaneous abortions with those with signs of disruption at the chorio-decidual interface in elective abortions. Determinations were performed on preparations received at the Anatomy-Pathology Services of Ospedale San Paolo, Savona (Italy) in 2005. Immunohistochemistry studies were performed using antisera CD3, CD4 and CD14 with a DAB detection kit on a Ventana BenchMark automated slide staining system. The material was grouped into three classes: early spontaneous abortions (class 1); elective abortions with signs of disruption at the chorio-decidual interface (class 2); elective abortions without such signs (class 3). Preparations from classes 1 and 2 shared a similar picture of lymphocytic activation and the presence of macrophagic elements. The test results demonstrated that the proportion of the T cell population increased with the rise in CD8+ lymphocytes in both class 1 and class 2 preparations. The results indicate that T-cell-mediated immune activation may the cause or one of the causes of spontaneous abortion and that the effects of disruption at the chorio-decidual interface observed in elective abortion provide a clue to initial signs of loss of pregnancy. From the discovery of a population without evident signs of active abortion (elective abortion with a disturbed chorio-decidual interface) but with evidence of initial lymphocytic activation compared with that devoid of such signs (elective abortion) one can conjecture that lymphocytic activation is a major factor in the process leading to early spontaneous abortion.

Petri Net-Based Model of Helicobacter pylori Mediated Disruption of Tight Junction Proteins in Stomach Lining during Gastric Carcinoma

PubMed Central

Naz, Anam; Obaid, Ayesha; Awan, Faryal M.; Ikram, Aqsa; Ahmad, Jamil; Ali, Amjad

2017-01-01

Tight junctions help prevent the passage of digestive enzymes and microorganisms through the space between adjacent epithelial cells lining. However, Helicobacter pylori encoded virulence factors negatively regulate these tight junctions and contribute to dysfunction of gastric mucosa. Here, we have predicted the regulation of important tight junction proteins, such as Zonula occludens-1, Claudin-2 and Connexin32 in the presence of pathogenic proteins. Molecular events such as post translational modifications and crosstalk between phosphorylation, O-glycosylation, palmitoylation and methylation are explored which may compromise the integrity of these tight junction proteins. Furthermore, the signaling pathways disrupted by dysregulated kinases, proteins and post-translational modifications are reviewed to design an abstracted computational model showing the situation-dependent dynamic behaviors of these biological processes and entities. A qualitative hybrid Petri Net model is therefore constructed showing the altered host pathways in the presence of virulence factor cytotoxin-associated gene A, leading to the disruption of tight junction proteins. The model is qualitative logic-based, which does not depend on any kinetic parameter and quantitative data and depends on knowledge derived from experiments. The designed model provides insights into the tight junction disruption and disease progression. Model is then verified by the available experimental data, nevertheless formal in vitro experimentation is a promising way to ensure its validation. The major findings propose that H. pylori activated kinases are responsible to trigger specific post translational modifications within tight junction proteins, at specific sites. These modifications may favor alterations in gastric barrier and provide a route to bacterial invasion into host cells. PMID:28932213

Petri Net-Based Model of Helicobacter pylori Mediated Disruption of Tight Junction Proteins in Stomach Lining during Gastric Carcinoma.

PubMed

Naz, Anam; Obaid, Ayesha; Awan, Faryal M; Ikram, Aqsa; Ahmad, Jamil; Ali, Amjad

2017-01-01

Tight junctions help prevent the passage of digestive enzymes and microorganisms through the space between adjacent epithelial cells lining. However, Helicobacter pylori encoded virulence factors negatively regulate these tight junctions and contribute to dysfunction of gastric mucosa. Here, we have predicted the regulation of important tight junction proteins, such as Zonula occludens-1, Claudin-2 and Connexin32 in the presence of pathogenic proteins. Molecular events such as post translational modifications and crosstalk between phosphorylation, O-glycosylation, palmitoylation and methylation are explored which may compromise the integrity of these tight junction proteins. Furthermore, the signaling pathways disrupted by dysregulated kinases, proteins and post-translational modifications are reviewed to design an abstracted computational model showing the situation-dependent dynamic behaviors of these biological processes and entities. A qualitative hybrid Petri Net model is therefore constructed showing the altered host pathways in the presence of virulence factor cytotoxin-associated gene A, leading to the disruption of tight junction proteins. The model is qualitative logic-based, which does not depend on any kinetic parameter and quantitative data and depends on knowledge derived from experiments. The designed model provides insights into the tight junction disruption and disease progression. Model is then verified by the available experimental data, nevertheless formal in vitro experimentation is a promising way to ensure its validation. The major findings propose that H. pylori activated kinases are responsible to trigger specific post translational modifications within tight junction proteins, at specific sites. These modifications may favor alterations in gastric barrier and provide a route to bacterial invasion into host cells.

Deregulation of Mitochondria-Shaping Proteins Opa-1 and Drp-1 in Manganese-Induced Apoptosis

PubMed Central

Alaimo, Agustina; Gorojod, Roxana M.; Beauquis, Juan; Muñoz, Manuel J.; Saravia, Flavia; Kotler, Mónica L.

2014-01-01

Mitochondria are dynamic organelles that undergo fusion and fission processes. These events are regulated by mitochondria-shaping proteins. Changes in the expression and/or localization of these proteins lead to a mitochondrial dynamics impairment and may promote apoptosis. Increasing evidence correlates the mitochondrial dynamics disruption with the occurrence of neurodegenerative diseases. Therefore, we focused on this topic in Manganese (Mn)-induced Parkinsonism, a disorder associated with Mn accumulation preferentially in the basal ganglia where mitochondria from astrocytes represent an early target. Using MitoTracker Red staining we observed increased mitochondrial network fission in Mn-exposed rat astrocytoma C6 cells. Moreover, Mn induced a marked decrease in fusion protein Opa-1 levels as well as a dramatic increase in the expression of fission protein Drp-1. Additionally, Mn provoked a significant release of high MW Opa-1 isoforms from the mitochondria to the cytosol as well as an increased Drp-1 translocation to the mitochondria. Both Mdivi-1, a pharmacological Drp-1 inhibitor, and rat Drp-1 siRNA reduced the number of apoptotic nuclei, preserved the mitochondrial network integrity and prevented cell death. CsA, an MPTP opening inhibitor, prevented mitochondrial Δψm disruption, Opa-1 processing and Drp-1 translocation to the mitochondria therefore protecting Mn-exposed cells from mitochondrial disruption and apoptosis. The histological analysis and Hoechst 33258 staining of brain sections of Mn-injected rats in the striatum showed a decrease in cellular mass paralleled with an increase in the occurrence of apoptotic nuclei. Opa-1 and Drp-1 expression levels were also changed by Mn-treatment. Our results demonstrate for the first time that abnormal mitochondrial dynamics is implicated in both in vitro and in vivo Mn toxicity. In addition we show that the imbalance in fusion/fission equilibrium might be involved in Mn-induced apoptosis. This knowledge may provide new therapeutic tools for the treatment of Manganism and other neurodegenerative diseases. PMID:24632637

Deregulation of mitochondria-shaping proteins Opa-1 and Drp-1 in manganese-induced apoptosis.

PubMed

Alaimo, Agustina; Gorojod, Roxana M; Beauquis, Juan; Muñoz, Manuel J; Saravia, Flavia; Kotler, Mónica L

2014-01-01

Mitochondria are dynamic organelles that undergo fusion and fission processes. These events are regulated by mitochondria-shaping proteins. Changes in the expression and/or localization of these proteins lead to a mitochondrial dynamics impairment and may promote apoptosis. Increasing evidence correlates the mitochondrial dynamics disruption with the occurrence of neurodegenerative diseases. Therefore, we focused on this topic in Manganese (Mn)-induced Parkinsonism, a disorder associated with Mn accumulation preferentially in the basal ganglia where mitochondria from astrocytes represent an early target. Using MitoTracker Red staining we observed increased mitochondrial network fission in Mn-exposed rat astrocytoma C6 cells. Moreover, Mn induced a marked decrease in fusion protein Opa-1 levels as well as a dramatic increase in the expression of fission protein Drp-1. Additionally, Mn provoked a significant release of high MW Opa-1 isoforms from the mitochondria to the cytosol as well as an increased Drp-1 translocation to the mitochondria. Both Mdivi-1, a pharmacological Drp-1 inhibitor, and rat Drp-1 siRNA reduced the number of apoptotic nuclei, preserved the mitochondrial network integrity and prevented cell death. CsA, an MPTP opening inhibitor, prevented mitochondrial Δψm disruption, Opa-1 processing and Drp-1 translocation to the mitochondria therefore protecting Mn-exposed cells from mitochondrial disruption and apoptosis. The histological analysis and Hoechst 33258 staining of brain sections of Mn-injected rats in the striatum showed a decrease in cellular mass paralleled with an increase in the occurrence of apoptotic nuclei. Opa-1 and Drp-1 expression levels were also changed by Mn-treatment. Our results demonstrate for the first time that abnormal mitochondrial dynamics is implicated in both in vitro and in vivo Mn toxicity. In addition we show that the imbalance in fusion/fission equilibrium might be involved in Mn-induced apoptosis. This knowledge may provide new therapeutic tools for the treatment of Manganism and other neurodegenerative diseases.

Predicting Essential Components of Signal Transduction Networks: A Dynamic Model of Guard Cell Abscisic Acid Signaling

PubMed Central

Li, Song; Assmann, Sarah M; Albert, Réka

2006-01-01

Plants both lose water and take in carbon dioxide through microscopic stomatal pores, each of which is regulated by a surrounding pair of guard cells. During drought, the plant hormone abscisic acid (ABA) inhibits stomatal opening and promotes stomatal closure, thereby promoting water conservation. Dozens of cellular components have been identified to function in ABA regulation of guard cell volume and thus of stomatal aperture, but a dynamic description is still not available for this complex process. Here we synthesize experimental results into a consistent guard cell signal transduction network for ABA-induced stomatal closure, and develop a dynamic model of this process. Our model captures the regulation of more than 40 identified network components, and accords well with previous experimental results at both the pathway and whole-cell physiological level. By simulating gene disruptions and pharmacological interventions we find that the network is robust against a significant fraction of possible perturbations. Our analysis reveals the novel predictions that the disruption of membrane depolarizability, anion efflux, actin cytoskeleton reorganization, cytosolic pH increase, the phosphatidic acid pathway, or K+ efflux through slowly activating K+ channels at the plasma membrane lead to the strongest reduction in ABA responsiveness. Initial experimental analysis assessing ABA-induced stomatal closure in the presence of cytosolic pH clamp imposed by the weak acid butyrate is consistent with model prediction. Simulations of stomatal response as derived from our model provide an efficient tool for the identification of candidate manipulations that have the best chance of conferring increased drought stress tolerance and for the prioritization of future wet bench analyses. Our method can be readily applied to other biological signaling networks to identify key regulatory components in systems where quantitative information is limited. PMID:16968132

Progressive polarity loss and luminal collapse disrupt tissue organization in carcinoma

PubMed Central

Halaoui, Ruba; Rejon, Carlis; Chatterjee, Sudipa June; Szymborski, Joseph; Meterissian, Sarkis; Muller, William J.; Omeroglu, Atilla; McCaffrey, Luke

2017-01-01

Epithelial cancers (carcinoma) account for 80%–90% of all cancers. The development of carcinoma is associated with disrupted epithelial organization and solid ductal structures. The mechanisms underlying the morphological development of carcinoma are poorly understood, but it is thought that loss of cell polarity is an early event. Here we report the characterization of the development of human breast lesions leading to carcinoma. We identified a unique mechanism that generates solid ducts in carcinoma through progressive loss of polarity and collapse of the luminal architecture. This program initiates with asymmetric divisions of polarized cells that generate a stratified epithelium containing both polarized and depolarized cells. Stratified regions form cords that penetrate into the lumen, subdividing it into polarized secondary lumina. The secondary lumina then collapse with a concomitant decrease in RhoA and myosin II activity at the apical membrane and ultimately lose apical–basal polarity. By restoring RhoA activity in mice, ducts maintained lumen and cell polarity. Notably, disrupted tissue architecture through luminal collapse was reversible, and ducts with a lumen were re-established after oncogene suppression in vivo. This reveals a novel and common mechanism that contributes to carcinoma development by progressively disrupting cell and tissue organization. PMID:28887414

Disrupting CCT-β : β-tubulin selectively kills CCT-β overexpressed cancer cells through MAPKs activation

PubMed Central

Liu, Yan-Jin; Kumar, Vathan; Lin, Yuan-Feng; Liang, Po-Huang

2017-01-01

We have previously demonstrated the ability of I-Trp to disrupt the protein–protein interaction of β-tubulin with chaperonin-containing TCP-1β (CCT-β). This caused more severe apoptosis in multidrug-resistant MES-SA/Dx5, compared to MES-SA, due to its higher CCT-β overexpression. In this study, we screened a panel of cancer cell lines, finding CCT-β overexpression in the triple-negative breast cancer cell line MDA-MB-231, colorectal cancer cell lines Colo205 and HCT116, and a gastric cancer cell line MKN-45. Thus, I-Trp killed these cancers with sub- to low-μM EC50, whereas it was non-toxic to MCF-10A. We then synthesized analogs of I-Trp and evaluated their cytotoxicity. Furthermore, apoptotic mechanism investigations revealed the activation of both protein ubiquitination/degradation and ER-associated protein degradation pathways. These pathways proceeded through activation of MAPKs at the onset of CCT-β : β-tubulin complex disruption. We thus establish an effective strategy to treat CCT-β overexpressed cancers by disrupting the CCT-β : β-tubulin complex. PMID:28906489

Caspase-8 acts as a key upstream executor of mitochondria during justicidin A-induced apoptosis in human hepatoma cells.

PubMed

Su, Chun-Li; Huang, Lynn L H; Huang, Li-Min; Lee, Jenq-Chang; Lin, Chun-Nan; Won, Shen-Jeu

2006-05-29

Justicia procumbens is a traditional Taiwanese herbal remedy used to treat fever, pain, and cancer. Justicidin A, isolated from Justicia procumbens, has been reported to suppress in vitro growth of several tumor cell lines as well as hepatoma cells. In this study, justicidin A activated caspase-8 to increase tBid, disrupted mitochondrial membrane potential (Delta psi(m)), and caused the release of cytochrome c and Smac/DIABLO in Hep 3B and Hep G2 cells. Justicidin A also reduced Bcl-x(L) and increased Bax and Bak in mitochondria. Caspase-8 inhibitor (Z-IETD) attenuated the justicidin A-induced disruption of Delta psi(m). Growth of Hep 3B implanted in NOD-SCID mice was suppressed significantly by oral justicidin A (20 mg/kg/day). These results indicate that justicidin A-induced apoptosis in these cells proceeds via caspase-8 and is followed by mitochondrial disruption.

Functional Analysis of the α-1,3-Glucan Synthase Genes agsA and agsB in Aspergillus nidulans: AgsB Is the Major α-1,3-Glucan Synthase in This Fungus

PubMed Central

Yoshimi, Akira; Sano, Motoaki; Inaba, Azusa; Kokubun, Yuko; Fujioka, Tomonori; Mizutani, Osamu; Hagiwara, Daisuke; Fujikawa, Takashi; Nishimura, Marie; Yano, Shigekazu; Kasahara, Shin; Shimizu, Kiminori; Yamaguchi, Masashi; Kawakami, Kazuyoshi; Abe, Keietsu

2013-01-01

Although α-1,3-glucan is one of the major cell wall polysaccharides in filamentous fungi, the physiological roles of α-1,3-glucan remain unclear. The model fungus Aspergillus nidulans possesses two α-1,3-glucan synthase (AGS) genes, agsA and agsB. For functional analysis of these genes, we constructed several mutant strains in A. nidulans: agsA disruption, agsB disruption, and double-disruption strains. We also constructed several CagsB strains in which agsB expression was controlled by the inducible alcA promoter, with or without the agsA-disrupting mutation. The agsA disruption strains did not show markedly different phenotypes from those of the wild-type strain. The agsB disruption strains formed dispersed hyphal cells under liquid culture conditions, regardless of the agsA genetic background. Dispersed hyphal cells were also observed in liquid culture of the CagsB strains when agsB expression was repressed, whereas these strains grew normally in plate culture even under the agsB-repressed conditions. Fractionation of the cell wall based on the alkali solubility of its components, quantification of sugars, and 13C-NMR spectroscopic analysis revealed that α-1,3-glucan was the main component of the alkali-soluble fraction in the wild-type and agsA disruption strains, but almost no α-1,3-glucan was found in the alkali-soluble fraction derived from either the agsB disruption strain or the CagsB strain under the agsB-repressed conditions, regardless of the agsA genetic background. Taken together, our data demonstrate that the two AGS genes are dispensable in A. nidulans, but that AgsB is required for normal growth characteristics under liquid culture conditions and is the major AGS in this species. PMID:23365684

Serine/Threonine Kinase Unc-51-like Kinase-1 (Ulk1) Phosphorylates the Co-chaperone Cell Division Cycle Protein 37 (Cdc37) and Thereby Disrupts the Stability of Cdc37 Client Proteins.

PubMed

Li, Ran; Yuan, Fengjie; Fu, Wan; Zhang, Luyao; Zhang, Nan; Wang, Yanan; Ma, Ke; Li, Xue; Wang, Lina; Zhu, Wei-Guo; Zhao, Ying

2017-02-17

The serine/threonine kinase Unc-51-like kinase-1 (Ulk1) is thought to be essential for induction of autophagy, an intracellular bulk degradation process that is activated by various stresses. Although several proteins have been suggested as Ulk1 substrates during autophagic process, it still remains largely unknown about Ulk1's physiological substrates. Here, by performing in vitro and in vivo phosphorylation assay, we report that the co-chaperone cell division cycle protein 37 (Cdc37) is a Ulk1 substrate. Ulk1-mediated phosphorylation of Ser-339 in Cdc37 compromised the recruitment of client kinases to a complex comprising Cdc37 and heat shock protein 90 (Hsp90) but only modestly affected Cdc37 binding to Hsp90. Because the recruitment of protein kinase clients to the Hsp90 complex is essential for their stability and functions, Ser-339 phosphorylation of Cdc37 disrupts its ability as a co-chaperone to coordinate Hsp90. Hsp90 inhibitors are cancer chemotherapeutic agents by inducing depletion of clients, many of which are oncogenes. Upon treatment with an Hsp90 inhibitor in cancer cells, Ulk1 promoted the degradation of Hsp90-Cdc37 client kinases, resulting in increased cellular sensitivity to Hsp90 inhibitors. Thus, our study provides evidence for an anti-proliferative role of Ulk1 in response to Hsp90 inhibition in cancer cells. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

Methods of introducing nucleic acids into cellular DNA

DOEpatents

Lajoie, Marc J.; Gregg, Christopher J.; Mosberg, Joshua A.; Church, George M.

2017-06-27

A method of introducing a nucleic acid sequence into a cell is provided where the cell has impaired or inhibited or disrupted DnaG primase activity or impaired or inhibited or disrupted DnaB helicase activity, or larger or increased gaps or distance between Okazaki fragments or lowered or reduced frequency of Okazaki fragment initiation, or the cell has increased single stranded DNA (ssDNA) on the lagging strand of the replication fork including transforming the cell through recombination with a nucleic acid oligomer.

Genetically Engineered Materials for Biofuels Production

NASA Astrophysics Data System (ADS)

Raab, Michael

2012-02-01

Agrivida, Inc., is an agricultural biotechnology company developing industrial crop feedstocks for the fuel and chemical industries. Agrivida's crops have improved processing traits that enable efficient, low cost conversion of the crops' cellulosic components into fermentable sugars. Currently, pretreatment and enzymatic conversion of the major cell wall components, cellulose and hemicellulose, into fermentable sugars is the most expensive processing step that prevents widespread adoption of biomass in biofuels processes. To lower production costs we are consolidating pretreatment and enzyme production within the crop. In this strategy, transgenic plants express engineered cell wall degrading enzymes in an inactive form, which can be reactivated after harvest. We have engineered protein elements that disrupt enzyme activity during normal plant growth. Upon exposure to specific processing conditions, the engineered enzymes are converted into their active forms. This mechanism significantly lowers pretreatment costs and enzyme loadings (>75% reduction) below those currently available to the industry.

Digital Collaboration Tools in the Military: Their Historical and Current Status

DTIC Science & Technology

2006-02-16

Writer = online word processor that edits, stores and shares your documents from anywhere. February 16, 2006 31 Recent “ Disruptive ” Technologies Cell...Webcasts Wikis February 16, 2006 32 Now Consider: Disruptive Technologies (1997) becomes Disruptive Innovations in 2003. Military Transformation: Drivers...from http://www.sims.berkeley.edu/how-much-info-2003 Schneiderman, R. (2005). Preparing for the Disruptive Technologies of Tomorrow. http

Hot-Alkaline DNA Extraction Method for Deep-Subseafloor Archaeal Communities

PubMed Central

Terada, Takeshi; Hoshino, Tatsuhiko; Inagaki, Fumio

2014-01-01

A prerequisite for DNA-based microbial community analysis is even and effective cell disruption for DNA extraction. With a commonly used DNA extraction kit, roughly two-thirds of subseafloor sediment microbial cells remain intact on average (i.e., the cells are not disrupted), indicating that microbial community analyses may be biased at the DNA extraction step, prior to subsequent molecular analyses. To address this issue, we standardized a new DNA extraction method using alkaline treatment and heating. Upon treatment with 1 M NaOH at 98°C for 20 min, over 98% of microbial cells in subseafloor sediment samples collected at different depths were disrupted. However, DNA integrity tests showed that such strong alkaline and heat treatment also cleaved DNA molecules into short fragments that could not be amplified by PCR. Subsequently, we optimized the alkaline and temperature conditions to minimize DNA fragmentation and retain high cell disruption efficiency. The best conditions produced a cell disruption rate of 50 to 80% in subseafloor sediment samples from various depths and retained sufficient DNA integrity for amplification of the complete 16S rRNA gene (i.e., ∼1,500 bp). The optimized method also yielded higher DNA concentrations in all samples tested compared with extractions using a conventional kit-based approach. Comparative molecular analysis using real-time PCR and pyrosequencing of bacterial and archaeal 16S rRNA genes showed that the new method produced an increase in archaeal DNA and its diversity, suggesting that it provides better analytical coverage of subseafloor microbial communities than conventional methods. PMID:24441163

Disrupting circadian homeostatis of sympathetic signaling promotes tumor development in mice

USDA-ARS?s Scientific Manuscript database

Cell proliferation in all rapidly renewing mammalian tissues follows a circadian rhythm that is often disrupted in advanced-stage tumors. Epidemiologic studies have revealed a clear link between disruption of circadian rhythms and cancer development in humans. Mice lacking the circadian genes Perio...

Tissue specific characterisation of Lim-kinase 1 expression during mouse embryogenesis

PubMed Central

Lindström, Nils O.; Neves, Carlos; McIntosh, Rebecca; Miedzybrodzka, Zosia; Vargesson, Neil; Collinson, J. Martin

2012-01-01

The Lim-kinase (LIMK) proteins are important for the regulation of the actin cytoskeleton, in particular the control of actin nucleation and depolymerisation via regulation of cofilin, and hence may control a large number of processes during development, including cell tensegrity, migration, cell cycling, and axon guidance. LIMK1/LIMK2 knockouts disrupt spinal cord morphogenesis and synapse formation but other tissues and developmental processes that require LIMK are yet to be fully determined. To identify tissues and cell-types that may require LIMK, we characterised the pattern of LIMK1 protein during mouse embryogenesis. We showed that LIMK1 displays an expression pattern that is temporally dynamic and tissue-specific. In several tissues LIMK1 is detected in cell-types that also express Wilms’ tumour protein 1 and that undergo transitions between epithelial and mesenchymal states, including the pleura, epicardium, kidney nephrons, and gonads. LIMK1 was also found in a subset of cells in the dorsal retina, and in mesenchymal cells surrounding the peripheral nerves. This detailed study of the spatial and temporal expression of LIMK1 shows that LIMK1 expression is more dynamic than previously reported, in particular at sites of tissue–tissue interactions guiding multiple developmental processes. PMID:21167960

Re-engineering the Pancreas Tumor Microenvironment: A “Regenerative Program” Hacked

PubMed Central

Evan, Gerard I.; Hah, Nasun; Littlewood, Trevor D.; Sodir, Nicole M.; Vidal, Tania Campos; Downes, Michael; Evans, Ronald M.

2017-01-01

The “hallmarks” of pancreatic ductal adenocarcinoma (PDAC) include proliferative, invasive and metastatic tumor cells and an associated dense desmoplasia comprised of fibroblasts, pancreatic stellate cells, extracellular matrix and immune cells. The oncogenically-activated pancreatic epithelium and its associated stroma are obligatorily interdependent, with the resulting inflammatory and immune-suppressive microenvironment contributing greatly to the evolution and maintenance of PDAC. The peculiar pancreas-specific tumor phenotype is a consequence of oncogenes hacking the resident pancreas regenerative program, a tissue specific repair mechanism regulated by discrete super enhancer networks. Defined as genomic regions containing clusters of multiple enhancers, super enhancers play pivotal roles in cell/tissue specification, identity and maintenance. Hence, interfering with such super enhancer driven repair networks should exert a disproportionately disruptive effect on tumor versus normal pancreatic tissue. Novel drugs that directly or indirectly inhibit processes regulating epigenetic status and integrity, including those driven by histone deacetylases, histone methyltransferase and hydroxylases, DNA methyltransferases, various metabolic enzymes, and bromodomain and extra-terminal motif proteins (BETs) have shown the feasibility of disrupting super enhancer-dependent transcription in treating multiple tumor types, including PDAC. The idea that pancreatic adenocarcinomas rely on embedded super enhancer transcriptional mechanism suggests a vulnerability that can be potentially targeted as novel therapies for this intractable disease. PMID:28373363

Heparan sulfate proteoglycans regulate autophagy in Drosophila.

PubMed

Reynolds-Peterson, Claire E; Zhao, Na; Xu, Jie; Serman, Taryn M; Xu, Jielin; Selleck, Scott B

2017-08-03

Heparan sulfate-modified proteoglycans (HSPGs) are important regulators of signaling and molecular recognition at the cell surface and in the extracellular space. Disruption of HSPG core proteins, HS-synthesis, or HS-degradation can have profound effects on growth, patterning, and cell survival. The Drosophila neuromuscular junction provides a tractable model for understanding the activities of HSPGs at a synapse that displays developmental and activity-dependent plasticity. Muscle cell-specific knockdown of HS biosynthesis disrupted the organization of a specialized postsynaptic membrane, the subsynaptic reticulum (SSR), and affected the number and morphology of mitochondria. We provide evidence that these changes result from a dysregulation of macroautophagy (hereafter referred to as autophagy). Cellular and molecular markers of autophagy are all consistent with an increase in the levels of autophagy in the absence of normal HS-chain biosynthesis and modification. HS production is also required for normal levels of autophagy in the fat body, the central energy storage and nutritional sensing organ in Drosophila. Genetic mosaic analysis indicates that HS-dependent regulation of autophagy occurs non-cell autonomously, consistent with HSPGs influencing this cellular process via signaling in the extracellular space. These findings demonstrate that HS biosynthesis has important regulatory effects on autophagy and that autophagy is critical for normal assembly of postsynaptic membrane specializations.

6.0 K microarray reveals differential transcriptomic responses in the dinoflagellate Prorocentrum minimum exposed to polychlorinated biphenyl (PCB).

PubMed

Wang, Hui; Guo, Ruoyu; Ki, Jang-Seu

2018-03-01

Endocrine disrupting chemicals (EDCs) have toxic effects on algae; however, their molecular genomic responses have not been sufficiently elucidated. Here, we evaluated genome-scaled responses of the dinoflagellate alga Prorocentrum minimum exposed to an EDC, polychlorinated biphenyl (PCB), using a 6.0 K microarray. Based on two-fold change cut-off, we identified that 609 genes (∼10.2%) responded to the PCB treatment. KEGG pathway analysis showed that differentially expressed genes (DEGs) were related to ribosomes, biosynthesis of amino acids, spliceosomes, and cellular processes. Many DEGs were involved in cell cycle progression, apoptosis, signal transduction, ion binding, and cellular transportation. In contrast, only a few genes related to photosynthesis and oxidative stress were expressed in response to PCB exposure. This was supported by that fact that there were no obvious changes in the photosynthetic efficiency and reactive oxygen species (ROS) production. These results suggest that PCB might not cause chloroplast and oxidative damage, but could lead to cell cycle arrest and apoptosis. In addition, various signal transduction and transport pathways might be disrupted in the cells, which could further contribute to cell death. These results expand the genomic understanding of the effects of EDCs on this dinoflagellate protist. Copyright © 2017 Elsevier Ltd. All rights reserved.

An Educational Model for Disruption of Bacteria for Protein Studies.

ERIC Educational Resources Information Center

Bhaduri, Saumya; Demchick, Paul H.

1984-01-01

A simple, rapid, and safe method has been developed for disrupting bacterial cells for protein studies. The method involved stepwise treatment of cells with acetone and with sodium dodecyl sulfate solution to allow extraction of cellular proteins for analysis by polyacrylamide gel electrophoresis. Applications for instructional purposes are noted.…

Modeling Physiological Events in 2D vs. 3D Cell Culture

PubMed Central

Duval, Kayla; Grover, Hannah; Han, Li-Hsin; Mou, Yongchao; Pegoraro, Adrian F.; Fredberg, Jeffery

2017-01-01

Cell culture has become an indispensable tool to help uncover fundamental biophysical and biomolecular mechanisms by which cells assemble into tissues and organs, how these tissues function, and how that function becomes disrupted in disease. Cell culture is now widely used in biomedical research, tissue engineering, regenerative medicine, and industrial practices. Although flat, two-dimensional (2D) cell culture has predominated, recent research has shifted toward culture using three-dimensional (3D) structures, and more realistic biochemical and biomechanical microenvironments. Nevertheless, in 3D cell culture, many challenges remain, including the tissue-tissue interface, the mechanical microenvironment, and the spatiotemporal distributions of oxygen, nutrients, and metabolic wastes. Here, we review 2D and 3D cell culture methods, discuss advantages and limitations of these techniques in modeling physiologically and pathologically relevant processes, and suggest directions for future research. PMID:28615311

CELL SEGREGATION, MIXING, AND TISSUE PATTERN IN THE SPINAL CORD OF THE XENOPUS LAEVIS NEURULA

PubMed Central

Davidson, Lance A.; Keller, Raymond E.

2014-01-01

Background During Xenopus laevis neurulation, neural ectodermal cells of the spinal cord are patterned at the same time that they intercalate mediolaterally and radially, moving within and between two cell layers. Curious if these rearrangements disrupt early cell identities, we lineage-traced cells in each layer from neural plate stages to the closed neural tube, and used in situ hybridization to assay gene expression in the moving cells. Results Our biotin- and fluorescent labeling of deep and superficial cells reveals that mediolateral intercalation does not disrupt cell cohorts, in other words it is conservative. However, outside the midline notoplate, later radial intercalation does displace superficial cells dorsoventrally, radically disrupting cell cohorts. The tube roof is composed almost exclusively of superficial cells, including some displaced from ventral positions; gene expression in these displaced cells must now be surveyed further. Superficial cells also flank the tube’s floor, which is, itself, almost exclusively composed of deep cells. Conclusions Our data provide: 1) a fate map of superficial- and deep-cell positions within the Xenopus neural tube, 2) the paths taken to these positions, and 3) preliminary evidence of re-patterning in cells carried out of one environment and into another, during neural morphogenesis. PMID:23813905

Acetylcholine contributes to the integration of self-movement cues in head direction cells.

PubMed

Yoder, Ryan M; Chan, Jeremy H M; Taube, Jeffrey S

2017-08-01

Acetylcholine contributes to accurate performance on some navigational tasks, but details of its contribution to the underlying brain signals are not fully understood. The medial septal area provides widespread cholinergic input to various brain regions, but selective damage to medial septal cholinergic neurons generally has little effect on landmark-based navigation, or the underlying neural representations of location and directional heading in visual environments. In contrast, the loss of medial septal cholinergic neurons disrupts navigation based on path integration, but no studies have tested whether these path integration deficits are associated with disrupted head direction (HD) cell activity. Therefore, we evaluated HD cell responses to visual cue rotations in a familiar arena, and during navigation between familiar and novel arenas, after muscarinic receptor blockade with systemic atropine. Atropine treatment reduced the peak firing rate of HD cells, but failed to significantly affect other HD cell firing properties. Atropine also failed to significantly disrupt the dominant landmark control of the HD signal, even though we used a procedure that challenged this landmark control. In contrast, atropine disrupted HD cell stability during navigation between familiar and novel arenas, where path integration normally maintains a consistent HD cell signal across arenas. These results suggest that acetylcholine contributes to path integration, in part, by facilitating the use of idiothetic cues to maintain a consistent representation of directional heading. (PsycINFO Database Record (c) 2017 APA, all rights reserved).

7-Hydroxystaurosporine (UCN-01) preferentially sensitizes cells with a disrupted TP53 to gamma radiation in lung cancer cell lines.

PubMed

Xiao, Helen H; Makeyev, Yan; Butler, James; Vikram, Bhadrasain; Franklin, William A

2002-07-01

Mutations in TP53 occur in more than 50% of the lung cancer patients and are associated with an increased resistance to chemotherapy and radiotherapy. The human lung adenocarcinoma cell lines A549 and LXSN contain a wild-type TP53 and were growth arrested at both the G(1)- and G(2)-phase checkpoints after irradiation. However, a TP53-disrupted cell line, E6, was arrested only at the G(2)-phase checkpoint. UCN-01 (7-hydroxystaurosporine), a CHEK1 inhibitor that abrogates the G(2) block, has been reported to enhance radiation toxicity in human lymphoma and colon cancer cell lines. In this study, UCN-01 preferentially enhanced the radiosensitivity of the TP53-disrupted E6 cells compared to the TP53 wild-type cells. This effect was more pronounced in cells synchronized in early G(1) phase, where the E6 cells showed a higher resistance to radiation in the absence of drug. These results indicate that the combination of UCN-01 and radiation can more specifically target resistant TP53 mutated cancer cells and spare TP53 wild-type normal cells.

Optimization of interneuron function by direct coupling of cell migration and axonal targeting.

PubMed

Lim, Lynette; Pakan, Janelle M P; Selten, Martijn M; Marques-Smith, André; Llorca, Alfredo; Bae, Sung Eun; Rochefort, Nathalie L; Marín, Oscar

2018-06-18

Neural circuit assembly relies on the precise synchronization of developmental processes, such as cell migration and axon targeting, but the cell-autonomous mechanisms coordinating these events remain largely unknown. Here we found that different classes of interneurons use distinct routes of migration to reach the embryonic cerebral cortex. Somatostatin-expressing interneurons that migrate through the marginal zone develop into Martinotti cells, one of the most distinctive classes of cortical interneurons. For these cells, migration through the marginal zone is linked to the development of their characteristic layer 1 axonal arborization. Altering the normal migratory route of Martinotti cells by conditional deletion of Mafb-a gene that is preferentially expressed by these cells-cell-autonomously disrupts axonal development and impairs the function of these cells in vivo. Our results suggest that migration and axon targeting programs are coupled to optimize the assembly of inhibitory circuits in the cerebral cortex.

The new approaches to preservation of graft cell integrity in preservation for transplantation.

PubMed

Gewartowska, Magdalena; Olszewski, Waldemar L

2005-01-01

Restoration of cell plasma membrane integrity after injury is essential for the survival of animal cells. In case of graft preservation or during chemotherapy in cancer, cell membrane integrity and the process of its repair are disrupted. Cytoprotective substances are important in such cases, as well as in other diseases, for example in myocardial infarction, acute insults and in chronic neurodegenerative diseases. Hyperosmolarity is a condition in which cell membrane stability may be damaged in vivo but preserved in the in vitro conditions. Hypertonicity causes water leaving from cells by osmosis, decreasing cell volume and increasing of intracellular ionic strength. High intracellular ionic strength perturbs cellular function by decreasing the rates of biochemical reaction. We review the new experimentally studied cytoprotective substances and their application in cell membrane protection. Moreover, we present our data on the effects of hyperosmolarity and its protective effect on cell internal structure.

Disruption of glycolytic flux is a signal for inflammasome signaling and pyroptotic cell death

PubMed Central

Sanman, Laura E; Qian, Yu; Eisele, Nicholas A; Ng, Tessie M; van der Linden, Wouter A; Monack, Denise M; Weerapana, Eranthie; Bogyo, Matthew

2016-01-01

When innate immune cells such as macrophages are challenged with environmental stresses or infection by pathogens, they trigger the rapid assembly of multi-protein complexes called inflammasomes that are responsible for initiating pro-inflammatory responses and a form of cell death termed pyroptosis. We describe here the identification of an intracellular trigger of NLRP3-mediated inflammatory signaling, IL-1β production and pyroptosis in primed murine bone marrow-derived macrophages that is mediated by the disruption of glycolytic flux. This signal results from a drop of NADH levels and induction of mitochondrial ROS production and can be rescued by addition of products that restore NADH production. This signal is also important for host-cell response to the intracellular pathogen Salmonella typhimurium, which can disrupt metabolism by uptake of host-cell glucose. These results reveal an important inflammatory signaling network used by immune cells to sense metabolic dysfunction or infection by intracellular pathogens. DOI: http://dx.doi.org/10.7554/eLife.13663.001 PMID:27011353

Nanosphere-based one-step strategy for efficient and nondestructive detection of circulating tumor cells.

PubMed

Wu, Ling-Ling; Wen, Cong-Ying; Hu, Jiao; Tang, Man; Qi, Chu-Bo; Li, Na; Liu, Cui; Chen, Lan; Pang, Dai-Wen; Zhang, Zhi-Ling

2017-08-15

Detecting viable circulating tumor cells (CTCs) without disruption to their functions for in vitro culture and functional study could unravel the biology of metastasis and promote the development of personalized anti-tumor therapies. However, existing CTC detection approaches commonly include CTC isolation and subsequent destructive identification, which damages CTC viability and functions and generates substantial CTC loss. To address the challenge of efficiently detecting viable CTCs for functional study, we develop a nanosphere-based cell-friendly one-step strategy. Immunonanospheres with prominent magnetic/fluorescence properties and extraordinary stability in complex matrices enable simultaneous efficient magnetic capture and specific fluorescence labeling of tumor cells directly in whole blood. The collected cells with fluorescent tags can be reliably identified, free of the tedious and destructive manipulations from conventional CTC identification. Hence, as few as 5 tumor cells in ca. 1mL of whole blood can be efficiently detected via only 20min incubation, and this strategy also shows good reproducibility with the relative standard deviation (RSD) of 8.7%. Moreover, due to the time-saving and gentle processing and the minimum disruption of immunonanospheres to cells, 93.8±0.1% of detected tumor cells retain cell viability and proliferation ability with negligible changes of cell functions, capacitating functional study on cell migration, invasion and glucose uptake. Additionally, this strategy exhibits successful CTC detection in 10/10 peripheral blood samples of cancer patients. Therefore, this nanosphere-based cell-friendly one-step strategy enables viable CTC detection and further functional analyses, which will help to unravel tumor metastasis and guide treatment selection. Copyright © 2017 Elsevier B.V. All rights reserved.

Disruption of direct 3D telomere-TRF2 interaction through two molecularly disparate mechanisms is a hallmark of primary Hodgkin and Reed-Sternberg cells.

PubMed

Knecht, Hans; Johnson, Nathalie A; Haliotis, Tina; Lichtensztejn, Daniel; Mai, Sabine

2017-07-01

In classical Hodgkin's lymphoma (cHL), specific changes in the 3D telomere organization cause progression from mononuclear Hodgkin cells (H) to multinucleated Reed-Sternberg cells (RS). In a post-germinal center B-cell in vitro model, permanent latent membrane protein 1 (LMP1) expression, as observed in Epstein-Barr virus (EBV)-associated cHL, results in multinuclearity and complex chromosomal aberrations through downregulation of key element of the shelterin complex, the telomere repeat binding factor 2 (TRF2). Thus, we hypothesized that the three-dimensional (3D) telomere-TRF2 interaction was progressively disturbed during transition from H to RS cells. To this end, we developed and applied for the first time a combined quantitative 3D TRF2-telomere immune fluorescent in situ hybridization (3D TRF2/Telo-Q-FISH) technique to monolayers of primary H and RS cells, and adjacent benign internal control lymphocytes of lymph node biopsy suspensions from diagnostic lymph node biopsies of 14 patients with cHL. We show that H and RS cells are characterized by two distinct patterns of disruption of 3D telomere-TRF2 interaction. Disruption pattern A is defined by massive attrition of telomere signals and a considerable increase of TRF2 signals not associated with telomeres. This pattern is restricted to EBV-negative cHL. Disruption pattern B is defined by telomere de-protection due to an impressive loss of TRF2 signals, physically linked to telomeres. This pattern is typical of, but is not restricted to, LMP1+EBV-associated cHL. In the disruption pattern B group, so-called 'ghost' end-stage RS cells, void of both TRF2 and telomere signals, were identified, whether or not associated with EBV. Our findings demonstrate that two molecularly disparate mechanisms converge on the level of 3D telomere-TRF2 interaction in the formation of RS cells.

Understanding cell cycle and cell death regulation provides novel weapons against human diseases.

PubMed

Wiman, K G; Zhivotovsky, B

2017-05-01

Cell division, cell differentiation and cell death are the three principal physiological processes that regulate tissue homoeostasis in multicellular organisms. The growth and survival of cells as well as the integrity of the genome are regulated by a complex network of pathways, in which cell cycle checkpoints, DNA repair and programmed cell death have critical roles. Disruption of genomic integrity and impaired regulation of cell death may both lead to uncontrolled cell growth. Compromised cell death can also favour genomic instability. It is becoming increasingly clear that dysregulation of cell cycle and cell death processes plays an important role in the development of major disorders such as cancer, cardiovascular disease, infection, inflammation and neurodegenerative diseases. Research achievements in these fields have led to the development of novel approaches for treatment of various conditions associated with abnormalities in the regulation of cell cycle progression or cell death. A better understanding of how cellular life-and-death processes are regulated is essential for this development. To highlight these important advances, the Third Nobel Conference entitled 'The Cell Cycle and Cell Death in Disease' was organized at Karolinska Institutet in 2016. In this review we will summarize current understanding of cell cycle progression and cell death and discuss some of the recent advances in therapeutic applications in pathological conditions such as cancer, neurological disorders and inflammation. © 2017 The Association for the Publication of the Journal of Internal Medicine.

Multicolor Fluorescence Imaging of Traumatic Brain Injury in a Cryolesion Mouse Model

PubMed Central

2012-01-01

Traumatic brain injury is characterized by initial tissue damage, which then can lead to secondary processes such as cell death and blood-brain-barrier disruption. Clinical and preclinical studies of traumatic brain injury typically employ anatomical imaging techniques and there is a need for new molecular imaging methods that provide complementary biochemical information. Here, we assess the ability of a targeted, near-infrared fluorescent probe, named PSS-794, to detect cell death in a brain cryolesion mouse model that replicates certain features of traumatic brain injury. In short, the model involves brief contact of a cold rod to the head of a living, anesthetized mouse. Using noninvasive whole-body fluorescence imaging, PSS-794 permitted visualization of the cryolesion in the living animal. Ex vivo imaging and histological analysis confirmed PSS-794 localization to site of brain cell death. The nontargeted, deep-red Tracer-653 was validated as a tracer dye for monitoring blood-brain-barrier disruption, and a binary mixture of PSS-794 and Tracer-653 was employed for multicolor imaging of cell death and blood-brain-barrier permeability in a single animal. The imaging data indicates that at 3 days after brain cryoinjury the amount of cell death had decreased significantly, but the integrity of the blood-brain-barrier was still impaired; at 7 days, the blood-brain-barrier was still three times more permeable than before cryoinjury. PMID:22860222

The effect of acute microgravity on mechanically-induced membrane damage and membrane-membrane fusion events

NASA Technical Reports Server (NTRS)

Clarke, M. S.; Vanderburg, C. R.; Feeback, D. L.; McIntire, L. V. (Principal Investigator)

2001-01-01

Although it is unclear how a living cell senses gravitational forces there is no doubt that perturbation of the gravitational environment results in profound alterations in cellular function. In the present study, we have focused our attention on how acute microgravity exposure during parabolic flight affects the skeletal muscle cell plasma membrane (i.e. sarcolemma), with specific reference to a mechanically-reactive signaling mechanism known as mechanically-induced membrane disruption or "wounding". Both membrane rupture and membrane resealing events mediated by membrane-membrane fusion characterize this response. We here present experimental evidence that acute microgravity exposure can inhibit membrane-membrane fusion events essential for the resealing of sarcolemmal wounds in individual human myoblasts. Additional evidence to support this contention comes from experimental studies that demonstrate acute microgravity exposure also inhibits secretagogue-stimulated intracellular vesicle fusion with the plasma membrane in HL-60 cells. Based on our own observations and those of other investigators in a variety of ground-based models of membrane wounding and membrane-membrane fusion, we suggest that the disruption in the membrane resealing process observed during acute microgravity is consistent with a microgravity-induced decrease in membrane order.

The Effect of Acute Microgravity on Mechanically-Induced Membrane Damage and Membrane-Membrane Fusion Events

NASA Technical Reports Server (NTRS)

Clarke, Mark, S. F.; Vanderburg, Charles R.; Feedback, Daniel L.

2001-01-01

Although it is unclear how a living cell senses gravitational forces there is no doubt that perturbation of the gravitational environment results in profound alterations in cellular function. In the present study, we have focused our attention on how acute microgravity exposure during parabolic flight affects the skeletal muscle cell plasma membrane (i.e. sarcolemma), with specific reference to a mechanically-reactive signaling mechanism known as mechanically-induced membrane disruption or "wounding". This response is characterized by both membrane rupture and membrane resealing events mediated by membrane-membrane fusion. We here present experimental evidence that acute microgravity exposure can inhibit membrane-membrane fusion events essential for the resealing of sarcolemmal wounds in individual human myoblasts. Additional evidence to support this contention comes from experimental studies that demonstrate acute microgravity exposure also inhibits secretagogue-stimulated intracellular vesicle fusion with the plasma membrane in HL-60 cells. Based on our own observations and those of other investigators in a variety of ground-based models of membrane wounding and membrane-membrane fusion, we suggest that the disruption in the membrane resealing process observed during acute microgravity is consistent with a microgravity-induced decrease in membrane order.

Site-Directed Disruption of the fimA and fimF Fimbrial Genes of Xylella fastidiosa.

PubMed

Feil, Helene; Feil, William S; Detter, John C; Purcel, Alexander H; Lindow, Steven E

2003-06-01

ABSTRACT Xylella fastidiosa causes Pierce's disease, a serious disease of grape, citrus variegated chlorosis, almond and oleander leaf scorches, and many other similar diseases. Although the complete genome sequences of several strains of this organism are now available, the function of most genes in this organism, especially those conferring virulence, is lacking. Attachment of X. fastidiosa to xylem vessels and insect vectors may be required for virulence and transmission; therefore, we disrupted fimA and fimF, genes encoding the major fimbrial protein FimA and a homolog of the fimbrial adhesin MrkD, to determine their role in the attachment process. Disruption of the fimA and fimF genes in Temecula1 and STL grape strains of X. fastidiosa was obtained by homologous recombination using plasmids pFAK and pFFK, respectively. These vectors contained a kanamycin resistance gene cloned into either the fimA or fimF genes of X. fastidiosa grape strains Temecula1 or STL. Efficiency of transformation was sufficiently high ( approximately 600 transformants per mug of pFFK DNA) to enable selection of rare recombination events. Polymerase chain reaction and Southern blot analyses of the mutants indicated that a double crossover event had occurred exclusively within the fimA and fimF genes, replacing the chromosomal gene with the disrupted gene and abolishing production of the corresponding proteins, FimA or FimF. Scanning electron microscopy revealed that fimbriae size and number, cell aggregation, and cell size were reduced for the FimA or FimF mutants of X. fastidiosa when compared with the parental strain. FimA or FimF mutants of X. fastidiosa remained pathogenic to grapevines, with bacterial populations slightly reduced compared with those of the wild-type X. fastidiosa cells. These mutants maintained their resistance to kanamycin in planta for at least 6 months in the greenhouse.

Two-Step Mechanism of Membrane Disruption by Aβ through Membrane Fragmentation and Pore Formation

PubMed Central

Sciacca, Michele F.M.; Kotler, Samuel A.; Brender, Jeffrey R.; Chen, Jennifer; Lee, Dong-kuk; Ramamoorthy, Ayyalusamy

2012-01-01

Disruption of cell membranes by Aβ is believed to be one of the key components of Aβ toxicity. However, the mechanism by which this occurs is not fully understood. Here, we demonstrate that membrane disruption by Aβ occurs by a two-step process, with the initial formation of ion-selective pores followed by nonspecific fragmentation of the lipid membrane during amyloid fiber formation. Immediately after the addition of freshly dissolved Aβ1–40, defects form on the membrane that share many of the properties of Aβ channels originally reported from single-channel electrical recording, such as cation selectivity and the ability to be blockaded by zinc. By contrast, subsequent amyloid fiber formation on the surface of the membrane fragments the membrane in a way that is not cation selective and cannot be stopped by zinc ions. Moreover, we observed that the presence of ganglioside enhances both the initial pore formation and the fiber-dependent membrane fragmentation process. Whereas pore formation by freshly dissolved Aβ1–40 is weakly observed in the absence of gangliosides, fiber-dependent membrane fragmentation can only be observed in their presence. These results provide insights into the toxicity of Aβ and may aid in the design of specific compounds to alleviate the neurodegeneration of Alzheimer’s disease. PMID:22947931

A non-aggregating Surfactant Protein C mutant is misdirected to early endosomes and disrupts phospholipid recycling

PubMed Central

Beers, Michael F.; Hawkins, Arie; Maguire, Jean Ann; Kotorashvili, Adam; Zhao, Ming; Newitt, Jennifer L.; Ding, Wenge; Russo, Scott; Guttentag, Susan; Gonzales, Linda; Mulugeta, Surafel

2011-01-01

Interstitial lung disease in both children and adults has been linked to mutations in the lung-specific Surfactant protein C gene (SFTPC). Among these, the missense mutation (isoleucine to threonine at codon 73 = hSP-CI73T) accounts for ~30% of all described SFTPC mutations. We reported previously that unlike the BRICHOS misfolding SFTPC mutants, expression of hSP-CI73T induces lung remodeling and alveolar lipoproteinosis without a substantial ER stress response or ER-mediated intrinsic apoptosis. We show here that, in contrast to its wild type counterpart that is directly routed to lysosomal-like organelles for processing, SP-CI73T is misdirected to the plasma membrane and subsequently internalized to the endocytic pathway via early endosomes, leading to the accumulation of abnormally processed proSP-C isoforms. Functionally, cells expressing hSP-CI73T demonstrated both impaired uptake and degradation of surfactant phospholipid, thus providing a molecular mechanism for the observed lipid accumulation in patients expressing hSP-CI73T through the disruption of normal phospholipid recycling. Our data provide evidence for a novel cellular mechanism for conformational protein associated diseases, and suggest a paradigm for mistargeted proteins involved in the disruption of the endosomal/lysosomal sorting machinery. PMID:21707890

Endothelin and hepatic wound healing

PubMed Central

Khimji, Al-karim; Rockey, Don C.

2014-01-01

Liver wound healing is a coordinated response to injury caused by infections (hepatitis) or toxins (alcohol) or other processes where activation of hepatic stellate cells are a central component. During stellate cell activation, a major phenotypic transformation occurs which leads to increased production of increased extracellular matrix proteins and smooth muscle α-actin the results is organ dysfunction due to gross architectural disruption and impaired blood flow. Endothelin-1 (ET-1) is produced in increased amounts and the cellular source of ET-1 shifts from endothelial cells to stellate cells during liver injury thus setting a feedback loop which accentuates further activation, stellate cell proliferation, and production of extracellular matrix proteins. Therapy directed at intervening the ET-1 signaling pathway has significant therapeutic potential in patients with liver disease. PMID:21421048

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

USGS Publications Warehouse

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

2011-01-01

Cyanotoxins are a group of organic compounds biosynthesized intracellularly by many species of cyanobacteria found in surface water. The United States Environmental Protection Agency has listed cyanotoxins on the Safe Drinking Water Act's Contaminant Candidate List 3 for consideration for future regulation to protect public health. Cyanotoxins also pose a risk to humans and other organisms in a variety of other exposure scenarios. Accurate and precise analytical measurements of cyanotoxins are critical to the evaluation of concentrations in surface water to address the human health and ecosystem effects. A common approach to total cyanotoxin measurement involves cell membrane disruption to release the cyanotoxins to the dissolved phase followed by filtration to remove cellular debris. Several methods have been used historically, however no standard protocols exist to ensure this process is consistent between laboratories before the dissolved phase is measured by an analytical technique for cyanotoxin identification and quantitation. No systematic evaluation has been conducted comparing the multiple laboratory sample processing techniques for physical disruption of cell membrane or cyanotoxins recovery. Surface water samples collected from lakes, reservoirs, and rivers containing mixed assemblages of organisms dominated by cyanobacteria, as well as laboratory cultures of species-specific cyanobacteria, were used as part of this study evaluating multiple laboratory cell-lysis techniques in partnership with the U.S. Environmental Protection Agency. Evaluated extraction techniques included boiling, autoclaving, sonication, chemical treatment, and freeze-thaw. Both treated and untreated samples were evaluated for cell membrane integrity microscopically via light, epifluorescence, and epifluorescence in the presence of a DNA stain. The DNA stain, which does not permeate live cells with intact membrane structures, was used as an indicator for cyanotoxin release into the dissolved phase. Of the five techniques, sonication (at 70 percent) was most effective at complete cell destruction while QuikLyse (Trademarked) was least effective. Autoclaving, boiling, and sequential freeze-thaw were moderately effective in physical destruction of colonies and filaments.

Absence of a Universal Mechanism of Mitochondrial Toxicity by Nucleoside Analogs▿

PubMed Central

Lund, Kaleb C.; Peterson, LaRae L.; Wallace, Kendall B.

2007-01-01

Nucleoside analogs are associated with various mitochondrial toxicities, and it is becoming increasingly difficult to accommodate these differences solely in the context of DNA polymerase gamma inhibition. Therefore, we examined the toxicities of zidovudine (AZT) (10 and 50 μM; 2.7 and 13.4 μg/ml), didanosine (ddI) (10 and 50 μM; 2.4 and 11.8 μg/ml), and zalcitabine (ddC) (1 and 5 μM; 0.21 and 1.1 μg/ml) in HepG2 and H9c2 cells without the presumption of mitochondrial DNA (mtDNA) depletion. Ethidium bromide (EtBr) (0.5 μg/ml; 1.3 μM) was used as a positive control. AZT treatment resulted in metabolic disruption (increased lactate and superoxide) and increased cell mortality with decreased proliferation, while mtDNA remained unchanged or increased (HepG2 cells; 50 μM AZT). ddC caused pronounced mtDNA depletion in HepG2 cells but not in H9c2 cells and increased mortality in HepG2 cells, but no significant metabolic disruption in either cell type. ddI caused a moderate depletion of mtDNA in both cell types but showed no other effects. EtBr exposure resulted in metabolic disruption, increased cell mortality with decreased cell proliferation, and mtDNA depletion in both cell types. We conclude that nucleoside analogs display unique toxicities within and between culture models, and therefore, care should be taken when generalizing about the mechanisms of nucleoside reverse transcriptase inhibitor toxicity. Additionally, mtDNA abundance does not necessarily correlate with metabolic disruption, especially in cell culture; careful discernment is recommended in this regard. PMID:17470651

Absence of a universal mechanism of mitochondrial toxicity by nucleoside analogs.

PubMed

Lund, Kaleb C; Peterson, LaRae L; Wallace, Kendall B

2007-07-01

Nucleoside analogs are associated with various mitochondrial toxicities, and it is becoming increasingly difficult to accommodate these differences solely in the context of DNA polymerase gamma inhibition. Therefore, we examined the toxicities of zidovudine (AZT) (10 and 50 microM; 2.7 and 13.4 microg/ml), didanosine (ddI) (10 and 50 microM; 2.4 and 11.8 microg/ml), and zalcitabine (ddC) (1 and 5 microM; 0.21 and 1.1 microg/ml) in HepG2 and H9c2 cells without the presumption of mitochondrial DNA (mtDNA) depletion. Ethidium bromide (EtBr) (0.5 microg/ml; 1.3 microM) was used as a positive control. AZT treatment resulted in metabolic disruption (increased lactate and superoxide) and increased cell mortality with decreased proliferation, while mtDNA remained unchanged or increased (HepG2 cells; 50 microM AZT). ddC caused pronounced mtDNA depletion in HepG2 cells but not in H9c2 cells and increased mortality in HepG2 cells, but no significant metabolic disruption in either cell type. ddI caused a moderate depletion of mtDNA in both cell types but showed no other effects. EtBr exposure resulted in metabolic disruption, increased cell mortality with decreased cell proliferation, and mtDNA depletion in both cell types. We conclude that nucleoside analogs display unique toxicities within and between culture models, and therefore, care should be taken when generalizing about the mechanisms of nucleoside reverse transcriptase inhibitor toxicity. Additionally, mtDNA abundance does not necessarily correlate with metabolic disruption, especially in cell culture; careful discernment is recommended in this regard.

Process-Oriented Review of Bacterial Quorum Quenching for Membrane Biofouling Mitigation in Membrane Bioreactors (MBRs)

PubMed Central

Bouayed, Naila; Dietrich, Nicolas; Lafforgue, Christine; Lee, Chung-Hak; Guigui, Christelle

2016-01-01

Quorum Quenching (QQ) has been developed over the last few years to overcome practical issues related to membrane biofouling, which is currently the major difficulty thwarting the extensive development of membrane bioreactors (MBRs). QQ is the disruption of Quorum Sensing (QS), cell-to-cell communication enabling the bacteria to harmonize their behavior. The production of biofilm, which is recognized as a major part of the biocake formed on a membrane surface, and which leads to biofouling, has been found to be one of the bacterial behaviors controlled by QS. Since the enzymatic disruption of QS was reported to be efficient as a membrane biofouling mitigation technique in MBRs, the application of QQ to lab-scale MBRs has been the subject of much research using different approaches under different operating conditions. This paper gives an overview of the effectiveness of QQ in mitigating membrane biofouling in MBRs. It is based on the results of previous studies, using two microbial strains, Rhodococcus sp. BH4 and Pseudomonas sp. 1A1. The effect of bacterial QQ on the physical phenomena of the MBR process is analyzed, adopting an original multi-scale approach. Finally, the potential influence of the MBR operating conditions on QQ effectiveness is discussed. PMID:27983578

Inhibition of mammalian S6 kinase by resveratrol suppresses autophagy

PubMed Central

Armour, Sean M.; Baur, Joseph A.; Hsieh, Sherry N.; Land-Bracha, Abigail; Thomas, Sheila M.; Sinclair, David A.

2009-01-01

Resveratrol is a plant-derived polyphenol that promotes health and disease resistance in rodent models, and extends lifespan in lower organisms. A major challenge is to understand the biological processes and molecular pathways by which resveratrol induces these beneficial effects. Autophagy is a critical process by which cells turn over damaged components and maintain bioenergetic requirements. Disruption of the normal balance between pro- and anti-autophagic signals is linked to cancer, liver disease, and neurodegenerative disorders. Here we show that resveratrol attenuates autophagy in response to nutrient limitation or rapamycin in multiple cell lines through a pathway independent of a known target, SIRT1. In a large-scalein vitro kinase screen we identified p70 S6 kinase (S6K1) as a target of resveratrol. Blocking S6K1 activity by expression of a dominant-negative mutant or RNA interference is sufficient to disrupt autophagy to a similar extent as resveratrol. Furthermore, co-administration of resveratrol with S6K1 knockdown does not produce an additive effect. These data indicate that S6K1 is important for the full induction of autophagy in mammals and raise the possibility that some of the beneficial effects of resveratrol are due to modulation of S6K1 activity. PMID:20157535

Inhibition of mammalian S6 kinase by resveratrol suppresses autophagy.

PubMed

Armour, Sean M; Baur, Joseph A; Hsieh, Sherry N; Land-Bracha, Abigail; Thomas, Sheila M; Sinclair, David A

2009-06-03

Resveratrol is a plant-derived polyphenol that promotes health and disease resistance in rodent models, and extends lifespan in lower organisms. A major challenge is to understand the biological processes and molecular pathways by which resveratrol induces these beneficial effects. Autophagy is a critical process by which cells turn over damaged components and maintain bioenergetic requirements. Disruption of the normal balance between pro- and anti-autophagic signals is linked to cancer, liver disease, and neurodegenerative disorders. Here we show that resveratrol attenuates autophagy in response to nutrient limitation or rapamycin in multiple cell lines through a pathway independent of a known target, SIRT1. In a large-scalein vitro kinase screen we identified p70 S6 kinase (S6K1) as a target of resveratrol. Blocking S6K1 activity by expression of a dominant-negative mutant or RNA interference is sufficient to disrupt autophagy to a similar extent as resveratrol. Furthermore, co-administration of resveratrol with S6K1 knockdown does not produce an additive effect. These data indicate that S6K1 is important for the full induction of autophagy in mammals and raise the possibility that some of the beneficial effects of resveratrol are due to modulation of S6K1 activity.

Disrupted autophagy after spinal cord injury is associated with ER stress and neuronal cell death

PubMed Central

Liu, S; Sarkar, C; Dinizo, M; Faden, A I; Koh, E Y; Lipinski, M M; Wu, J

2015-01-01

Autophagy is a catabolic mechanism facilitating degradation of cytoplasmic proteins and organelles in a lysosome-dependent manner. Autophagy flux is necessary for normal neuronal homeostasis and its dysfunction contributes to neuronal cell death in several neurodegenerative diseases. Elevated autophagy has been reported after spinal cord injury (SCI); however, its mechanism, cell type specificity and relationship to cell death are unknown. Using a rat model of contusive SCI, we observed accumulation of LC3-II-positive autophagosomes starting at posttrauma day 1. This was accompanied by a pronounced accumulation of autophagy substrate protein p62, indicating that early elevation of autophagy markers reflected disrupted autophagosome degradation. Levels of lysosomal protease cathepsin D and numbers of cathepsin-D-positive lysosomes were also decreased at this time, suggesting that lysosomal damage may contribute to the observed defect in autophagy flux. Normalization of p62 levels started by day 7 after SCI, and was associated with increased cathepsin D levels. At day 1 after SCI, accumulation of autophagosomes was pronounced in ventral horn motor neurons and dorsal column oligodendrocytes and microglia. In motor neurons, disruption of autophagy strongly correlated with evidence of endoplasmic reticulum (ER) stress. As autophagy is thought to protect against ER stress, its disruption after SCI could contribute to ER-stress-induced neuronal apoptosis. Consistently, motor neurons showing disrupted autophagy co-expressed ER-stress-associated initiator caspase 12 and cleaved executioner caspase 3. Together, these findings indicate that SCI causes lysosomal dysfunction that contributes to autophagy disruption and associated ER-stress-induced neuronal apoptosis. PMID:25569099

Context-Dependent Sensitivity to Mutations Disrupting the Structural Integrity of Individual EGF Repeats in the Mouse Notch Ligand DLL1

PubMed Central

Schuster-Gossler, Karin; Cordes, Ralf; Müller, Julia; Geffers, Insa; Delany-Heiken, Patricia; Taft, Manuel; Preller, Matthias; Gossler, Achim

2016-01-01

The highly conserved Notch-signaling pathway mediates cell-to-cell communication and is pivotal for multiple developmental processes and tissue homeostasis in adult organisms. Notch receptors and their ligands are transmembrane proteins with multiple epidermal-growth-factor-like (EGF) repeats in their extracellular domains. In vitro the EGF repeats of mammalian ligands that are essential for Notch activation have been defined. However, in vivo the significance of the structural integrity of each EGF repeat in the ligand ectodomain for ligand function is still unclear. Here, we analyzed the mouse Notch ligand DLL1. We expressed DLL1 proteins with mutations disrupting disulfide bridges in each individual EGF repeat from single-copy transgenes in the HPRT locus of embryonic stem cells. In Notch transactivation assays all mutations impinged on DLL1 function and affected both NOTCH1 and NOTCH2 receptors similarly. An allelic series in mice that carried the same point mutations in endogenous Dll1, generated using a mini-gene strategy, showed that early developmental processes depending on DLL1-mediated NOTCH activation were differently sensitive to mutation of individual EGF repeats in DLL1. Notably, some mutations affected only somite patterning and resulted in vertebral column defects resembling spondylocostal dysostosis. In conclusion, the structural integrity of each individual EGF repeat in the extracellular domain of DLL1 is necessary for full DLL1 activity, and certain mutations in Dll1 might contribute to spondylocostal dysostosis in humans. PMID:26801181

Assessing the carcinogenic potential of low-dose exposures to chemical mixtures in the environment: focus on the cancer hallmark of tumor angiogenesis

PubMed Central

Hu, Zhiwei; Brooks, Samira A.; Dormoy, Valérian; Hsu, Chia-Wen; Hsu, Hsue-Yin; Lin, Liang-Tzung; Massfelder, Thierry; Rathmell, W. Kimryn; Xia, Menghang; Al-Mulla, Fahd; Al-Temaimi, Rabeah; Amedei, Amedeo; Brown, Dustin G.; Prudhomme, Kalan R.; Colacci, Annamaria; Hamid, Roslida A.; Mondello, Chiara; Raju, Jayadev; Ryan, Elizabeth P.; Woodrick, Jordan; Scovassi, A. Ivana; Singh, Neetu; Vaccari, Monica; Roy, Rabindra; Forte, Stefano; Memeo, Lorenzo; Salem, Hosni K.; Lowe, Leroy; Jensen, Lasse; Bisson, William H.; Kleinstreuer, Nicole

2015-01-01

One of the important ‘hallmarks’ of cancer is angiogenesis, which is the process of formation of new blood vessels that are necessary for tumor expansion, invasion and metastasis. Under normal physiological conditions, angiogenesis is well balanced and controlled by endogenous proangiogenic factors and antiangiogenic factors. However, factors produced by cancer cells, cancer stem cells and other cell types in the tumor stroma can disrupt the balance so that the tumor microenvironment favors tumor angiogenesis. These factors include vascular endothelial growth factor, endothelial tissue factor and other membrane bound receptors that mediate multiple intracellular signaling pathways that contribute to tumor angiogenesis. Though environmental exposures to certain chemicals have been found to initiate and promote tumor development, the role of these exposures (particularly to low doses of multiple substances), is largely unknown in relation to tumor angiogenesis. This review summarizes the evidence of the role of environmental chemical bioactivity and exposure in tumor angiogenesis and carcinogenesis. We identify a number of ubiquitous (prototypical) chemicals with disruptive potential that may warrant further investigation given their selectivity for high-throughput screening assay targets associated with proangiogenic pathways. We also consider the cross-hallmark relationships of a number of important angiogenic pathway targets with other cancer hallmarks and we make recommendations for future research. Understanding of the role of low-dose exposure of chemicals with disruptive potential could help us refine our approach to cancer risk assessment, and may ultimately aid in preventing cancer by reducing or eliminating exposures to synergistic mixtures of chemicals with carcinogenic potential. PMID:26106137

Vascularization, high-volume solution flow, and localized roles for enzymes of sucrose metabolism during tumorigenesis by Agrobacterium tumefaciens.

PubMed

Wächter, Rebecca; Langhans, Markus; Aloni, Roni; Götz, Simone; Weilmünster, Anke; Koops, Ariane; Temguia, Leopoldine; Mistrik, Igor; Pavlovkin, Jan; Rascher, Uwe; Schwalm, Katja; Koch, Karen E; Ullrich, Cornelia I

2003-11-01

Vascular differentiation and epidermal disruption are associated with establishment of tumors induced by Agrobacterium tumefaciens. Here, we address the relationship of these processes to the redirection of nutrient-bearing water flow and carbohydrate delivery for tumor growth within the castor bean (Ricinus communis) host. Treatment with aminoethoxyvinyl-glycine showed that vascular differentiation and epidermal disruption were central to ethylene-dependent tumor establishment. CO2 release paralleled tumor growth, but water flow increased dramatically during the first 3 weeks. However, tumor water loss contributed little to water flow to host shoots. Tumor water loss was followed by accumulation of the osmoprotectants, sucrose (Suc) and proline, in the tumor periphery, shifting hexose-to-Suc balance in favor of sugar signals for maturation and desiccation tolerance. Concurrent activities and sites of action for enzymes of Suc metabolism changed: Vacuolar invertase predominated during initial import of Suc into the symplastic continuum, corresponding to hexose concentrations in expanding tumors. Later, Suc synthase (SuSy) and cell wall invertase rose in the tumor periphery to modulate both Suc accumulation and descending turgor for import by metabolization. Sites of abscisic acid immunolocalization correlated with both central vacuolar invertase and peripheral cell wall invertase. Vascular roles were indicated by SuSy immunolocalization in xylem parenchyma for inorganic nutrient uptake and in phloem, where resolution allowed SuSy identification in sieve elements and companion cells, which has widespread implications for SuSy function in transport. Together, data indicate key roles for ethylene-dependent vascularization and cuticular disruption in the redirection of water flow and carbohydrate transport for successful tumor establishment.

Epigenetics and autoimmune diseases: the X chromosome-nucleolus nexus

PubMed Central

Brooks, Wesley H.; Renaudineau, Yves

2015-01-01

Autoimmune diseases occur more often in females, suggesting a key role for the X chromosome. X chromosome inactivation, a major epigenetic feature in female cells that provides dosage compensation of X-linked genes to avoid overexpression, presents special vulnerabilities that can contribute to the disease process. Disruption of X inactivation can result in loss of dosage compensation with expression from previously sequestered genes, imbalance of gene products, and altered endogenous material out of normal epigenetic context. In addition, the human X has significant differences compared to other species and these differences can contribute to the frequency and intensity of the autoimmune disease in humans as well as the types of autoantigens encountered. Here a link is demonstrated between autoimmune diseases, such as systemic lupus erythematosus, and the X chromosome by discussing cases in which typically non-autoimmune disorders complicated with X chromosome abnormalities also present lupus-like symptoms. The discussion is then extended to the reported spatial and temporal associations of the inactive X chromosome with the nucleolus. When frequent episodes of cellular stress occur, the inactive X chromosome may be disrupted and inadvertently become involved in the nucleolar stress response. Development of autoantigens, many of which are at least transiently components of the nucleolus, is then described. Polyamines, which aid in nucleoprotein complex assembly in the nucleolus, increase further during cell stress, and appear to have an important role in the autoimmune disease process. Autoantigenic endogenous material can potentially be stabilized by polyamines. This presents a new paradigm for autoimmune diseases: that many are antigen-driven and the autoantigens originate from altered endogenous material due to episodes of cellular stress that disrupt epigenetic control. This suggests that epigenetics and the X chromosome are important aspects of autoimmune diseases. PMID:25763008

Cellular mechanisms of cyclophosphamide-induced taste loss in mice

PubMed Central

Mukherjee, Nabanita; Pal Choudhuri, Shreoshi; Delay, Rona J.

2017-01-01

Many commonly prescribed chemotherapy drugs such as cyclophosphamide (CYP) have adverse side effects including disruptions in taste which can result in loss of appetite, malnutrition, poorer recovery and reduced quality of life. Previous studies in mice found evidence that CYP has a two-phase disturbance in taste behavior: a disturbance immediately following drug administration and a second which emerges several days later. In this study, we examined the processes by which CYP disturbs the taste system by examining the effects of the drug on taste buds and cells responsible for taste cell renewal using immunohistochemical assays. Data reported here suggest CYP has direct cytotoxic effects on lingual epithelium immediately following administration, causing an early loss of taste sensory cells. Types II and III cells in fungiform taste buds appear to be more susceptible to this effect than circumvallate cells. In addition, CYP disrupts the population of rapidly dividing cells in the basal layer of taste epithelium responsible for taste cell renewal, manifesting a disturbance days later. The loss of these cells temporarily retards the system’s capacity to replace Type II and Type III taste sensory cells that survived the cytotoxic effects of CYP and died at the end of their natural lifespan. The timing of an immediate, direct loss of taste cells and a delayed, indirect loss without replacement of taste sensory cells are broadly congruent with previously published behavioral data reporting two periods of elevated detection thresholds for umami and sucrose stimuli. These findings suggest that chemotherapeutic disturbances in the peripheral mechanisms of the taste system may cause dietary challenges at a time when the cancer patient has significant need for well balanced, high energy nutritional intake. PMID:28950008

Cellular mechanisms of cyclophosphamide-induced taste loss in mice.

PubMed

Mukherjee, Nabanita; Pal Choudhuri, Shreoshi; Delay, Rona J; Delay, Eugene R

2017-01-01

Many commonly prescribed chemotherapy drugs such as cyclophosphamide (CYP) have adverse side effects including disruptions in taste which can result in loss of appetite, malnutrition, poorer recovery and reduced quality of life. Previous studies in mice found evidence that CYP has a two-phase disturbance in taste behavior: a disturbance immediately following drug administration and a second which emerges several days later. In this study, we examined the processes by which CYP disturbs the taste system by examining the effects of the drug on taste buds and cells responsible for taste cell renewal using immunohistochemical assays. Data reported here suggest CYP has direct cytotoxic effects on lingual epithelium immediately following administration, causing an early loss of taste sensory cells. Types II and III cells in fungiform taste buds appear to be more susceptible to this effect than circumvallate cells. In addition, CYP disrupts the population of rapidly dividing cells in the basal layer of taste epithelium responsible for taste cell renewal, manifesting a disturbance days later. The loss of these cells temporarily retards the system's capacity to replace Type II and Type III taste sensory cells that survived the cytotoxic effects of CYP and died at the end of their natural lifespan. The timing of an immediate, direct loss of taste cells and a delayed, indirect loss without replacement of taste sensory cells are broadly congruent with previously published behavioral data reporting two periods of elevated detection thresholds for umami and sucrose stimuli. These findings suggest that chemotherapeutic disturbances in the peripheral mechanisms of the taste system may cause dietary challenges at a time when the cancer patient has significant need for well balanced, high energy nutritional intake.

Roles for herpes simplex virus type 1 U{sub L}34 and U{sub S}3 proteins in disrupting the nuclear lamina during herpes simplex virus type 1 egress

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

Bjerke, Susan L.; Roller, Richard J.

2006-04-10

Cells infected with wild type HSV-1 showed significant lamin A/C and lamin B rearrangement, while U{sub L}34-null virus-infected cells exhibited few changes in lamin localization, indicating that U{sub L}34 is necessary for lamin disruption. During HSV infection, U{sub S}3 limited the development of disruptions in the lamina, since cells infected with a U{sub S}3-null virus developed large perforations in the lamin layer. U{sub S}3 regulation of lamin disruption does not correlate with the induction of apoptosis. Expression of either U{sub L}34 or U{sub S}3 proteins alone disrupted lamin A/C and lamin B localization. Expression of U{sub L}34 and U{sub S}3more » together had little effect on lamin A/C localization, suggesting a regulatory interaction between the two proteins. The data presented in this paper argue for crucial roles for both U{sub L}34 and U{sub S}3 in regulating the state of the nuclear lamina during viral infection.« less

Rescue of perfluorooctanesulfonate (PFOS)-mediated Sertoli cell injury by overexpression of gap junction protein connexin 43

NASA Astrophysics Data System (ADS)

Li, Nan; Mruk, Dolores D.; Chen, Haiqi; Wong, Chris K. C.; Lee, Will M.; Cheng, C. Yan

2016-07-01

Perfluorooctanesulfonate (PFOS) is an environmental toxicant used in developing countries, including China, as a stain repellent for clothing, carpets and draperies, but it has been banned in the U.S. and Canada since the late 2000s. PFOS perturbed the Sertoli cell tight junction (TJ)-permeability barrier, causing disruption of actin microfilaments in cell cytosol, perturbing the localization of cell junction proteins (e.g., occluden-ZO-1, N-cadherin-ß-catenin). These changes destabilized Sertoli cell blood-testis barrier (BTB) integrity. These findings suggest that human exposure to PFOS might induce BTB dysfunction and infertility. Interestingly, PFOS-induced Sertoli cell injury associated with a down-regulation of the gap junction (GJ) protein connexin43 (Cx43). We next investigated if overexpression of Cx43 in Sertoli cells could rescue the PFOS-induced cell injury. Indeed, overexpression of Cx43 in Sertoli cells with an established TJ-barrier blocked the disruption in PFOS-induced GJ-intercellular communication, resulting in the re-organization of actin microfilaments, which rendered them similar to those in control cells. Furthermore, cell adhesion proteins that utilized F-actin for attachment became properly distributed at the cell-cell interface, resealing the disrupted TJ-barrier. In summary, Cx43 is a good target that might be used to manage PFOS-induced reproductive dysfunction.

On the role of PDZ domain-encoding genes in Drosophila border cell migration.

PubMed

Aranjuez, George; Kudlaty, Elizabeth; Longworth, Michelle S; McDonald, Jocelyn A

2012-11-01

Cells often move as collective groups during normal embryonic development and wound healing, although the mechanisms governing this type of migration are poorly understood. The Drosophila melanogaster border cells migrate as a cluster during late oogenesis and serve as a powerful in vivo genetic model for collective cell migration. To discover new genes that participate in border cell migration, 64 out of 66 genes that encode PDZ domain-containing proteins were systematically targeted by in vivo RNAi knockdown. The PDZ domain is one of the largest families of protein-protein interaction domains found in eukaryotes. Proteins that contain PDZ domains participate in a variety of biological processes, including signal transduction and establishment of epithelial apical-basal polarity. Targeting PDZ proteins effectively assesses a larger number of genes via the protein complexes and pathways through which these proteins function. par-6, a known regulator of border cell migration, was a positive hit and thus validated the approach. Knockdown of 14 PDZ domain genes disrupted migration with multiple RNAi lines. The candidate genes have diverse predicted cellular functions and are anticipated to provide new insights into the mechanisms that control border cell movement. As a test of this concept, two genes that disrupted migration were characterized in more detail: big bang and the Dlg5 homolog CG6509. We present evidence that Big bang regulates JAK/STAT signaling, whereas Dlg5/CG6509 maintains cluster cohesion. Moreover, these results demonstrate that targeting a selected class of genes by RNAi can uncover novel regulators of collective cell migration.

Angiotensin receptors and β-catenin regulate brain endothelial integrity in malaria

PubMed Central

Basu-Roy, Upal; Ty, Maureen; Alique, Matilde; Fernandez-Arias, Cristina; Movila, Alexandru; Gomes, Pollyanna; Edagha, Innocent; Wassmer, Samuel C.; Walther, Thomas

2016-01-01

Cerebral malaria is characterized by cytoadhesion of Plasmodium falciparum–infected red blood cells (Pf-iRBCs) to endothelial cells in the brain, disruption of the blood-brain barrier, and cerebral microhemorrhages. No available antimalarial drugs specifically target the endothelial disruptions underlying this complication, which is responsible for the majority of malaria-associated deaths. Here, we have demonstrated that ruptured Pf-iRBCs induce activation of β-catenin, leading to disruption of inter–endothelial cell junctions in human brain microvascular endothelial cells (HBMECs). Inhibition of β-catenin–induced TCF/LEF transcription in the nucleus of HBMECs prevented the disruption of endothelial junctions, confirming that β-catenin is a key mediator of P. falciparum adverse effects on endothelial integrity. Blockade of the angiotensin II type 1 receptor (AT1) or stimulation of the type 2 receptor (AT2) abrogated Pf-iRBC–induced activation of β-catenin and prevented the disruption of HBMEC monolayers. In a mouse model of cerebral malaria, modulation of angiotensin II receptors produced similar effects, leading to protection against cerebral malaria, reduced cerebral hemorrhages, and increased survival. In contrast, AT2-deficient mice were more susceptible to cerebral malaria. The interrelation of the β-catenin and the angiotensin II signaling pathways opens immediate host-targeted therapeutic possibilities for cerebral malaria and other diseases in which brain endothelial integrity is compromised. PMID:27643439

BEST: barcode enabled sequencing of tetrads.

PubMed

Scott, Adrian C; Ludlow, Catherine L; Cromie, Gareth A; Dudley, Aimée M

2014-05-01

Tetrad analysis is a valuable tool for yeast genetics, but the laborious manual nature of the process has hindered its application on large scales. Barcode Enabled Sequencing of Tetrads (BEST)1 replaces the manual processes of isolating, disrupting and spacing tetrads. BEST isolates tetrads by virtue of a sporulation-specific GFP fusion protein that permits fluorescence-activated cell sorting of tetrads directly onto agar plates, where the ascus is enzymatically digested and the spores are disrupted and randomly arrayed by glass bead plating. The haploid colonies are then assigned sister spore relationships, i.e. information about which spores originated from the same tetrad, using molecular barcodes read during genotyping. By removing the bottleneck of manual dissection, hundreds or even thousands of tetrads can be isolated in minutes. Here we present a detailed description of the experimental procedures required to perform BEST in the yeast Saccharomyces cerevisiae, starting with a heterozygous diploid strain through the isolation of colonies derived from the haploid meiotic progeny.

Responds of Bone Cells to Microgravity: Ground-Based Research

NASA Astrophysics Data System (ADS)

Zhang, Jian; Li, Jingbao; Xu, Huiyun; Yang, Pengfei; Xie, Li; Qian, Airong; Zhao, Yong; Shang, Peng

2015-11-01

Severe loss of bone occurs due to long-duration spaceflight. Mechanical loading stimulates bone formation, while bone degradation happens under mechanical unloading. Bone remodeling is a dynamic process in which bone formation and bone resorption are tightly coupled. Increased bone resorption and decreased bone formation caused by reduced mechanical loading, generally result in disrupted bone remodeling. Bone remodeling is orchestrated by multiple bone cells including osteoblast, osteocyte, osteoclast and mesenchymal stem cell. It is yet not clear that how these bone cells sense altered gravity, translate physical stimulus into biochemical signals, and then regulate themselves structurally and functionally. In this paper, studies elucidating the bioeffects of microgravity on bone cells (osteoblast, osteocyte, osteoclast, mesenchymal stem cell) using various platforms including spaceflight and ground-based simulated microgravity were summarized. Promising gravity-sensitive signaling pathways and protein molecules were proposed.

Plant meiosis: the means to 1N.

PubMed

Bhatt, A M; Canales, C; Dickinson, H G

2001-03-01

Meiosis is pivotal in the life history of plants. In addition to providing an opportunity for genetic reassortment, it marks the transition from diploid sporophyte to haploid gametophyte. Recent molecular data suggest that, like animals, plants possess a common set of genes (also conserved in eukaryotic microorganisms) responsible for meiotic recombination and chromosome segregation. However, unlike animals, plant meiocytes do not differentiate from a pool of primordial germ cells, but rather arise de novo from a germline formed from sub-epidermal cells in the anthers and ovules. Mutants defective in the specification of these reproductive cell lines and disrupted in different aspects of the meiotic process are beginning to reveal many features unique to plant meiosis.

Keratinocyte cytoskeletal roles in cell sheet engineering

PubMed Central

2013-01-01

Background There is an increasing need to understand cell-cell interactions for cell and tissue engineering purposes, such as optimizing cell sheet constructs, as well as for examining adhesion defect diseases. For cell-sheet engineering, one major obstacle to sheet function is that cell sheets in suspension are fragile and, over time, will contract. While the role of the cytoskeleton in maintaining the structure and adhesion of cells cultured on a rigid substrate is well-characterized, a systematic examination of the role played by different components of the cytoskeleton in regulating cell sheet contraction and cohesion in the absence of a substrate has been lacking. Results In this study, keratinocytes were cultured until confluent and cell sheets were generated using dispase to remove the influence of the substrate. The effects of disrupting actin, microtubules or intermediate filaments on cell-cell interactions were assessed by measuring cell sheet cohesion and contraction. Keratin intermediate filament disruption caused comparable effects on cell sheet cohesion and contraction, when compared to actin or microtubule disruption. Interfering with actomyosin contraction demonstrated that interfering with cell contraction can also diminish cell cohesion. Conclusions All components of the cytoskeleton are involved in maintaining cell sheet cohesion and contraction, although not to the same extent. These findings demonstrate that substrate-free cell sheet biomechanical properties are dependent on the integrity of the cytoskeleton network. PMID:23442760

In vivo cell biology in zebrafish – providing insights into vertebrate development and disease

PubMed Central

Vacaru, Ana M.; Unlu, Gokhan; Spitzner, Marie; Mione, Marina; Knapik, Ela W.; Sadler, Kirsten C.

2014-01-01

ABSTRACT Over the past decades, studies using zebrafish have significantly advanced our understanding of the cellular basis for development and human diseases. Zebrafish have rapidly developing transparent embryos that allow comprehensive imaging of embryogenesis combined with powerful genetic approaches. However, forward genetic screens in zebrafish have generated unanticipated findings that are mirrored by human genetic studies: disruption of genes implicated in basic cellular processes, such as protein secretion or cytoskeletal dynamics, causes discrete developmental or disease phenotypes. This is surprising because many processes that were assumed to be fundamental to the function and survival of all cell types appear instead to be regulated by cell-specific mechanisms. Such discoveries are facilitated by experiments in whole animals, where zebrafish provides an ideal model for visualization and manipulation of organelles and cellular processes in a live vertebrate. Here, we review well-characterized mutants and newly developed tools that underscore this notion. We focus on the secretory pathway and microtubule-based trafficking as illustrative examples of how studying cell biology in vivo using zebrafish has broadened our understanding of the role fundamental cellular processes play in embryogenesis and disease. PMID:24481493

Disruption of Core Planar Cell Polarity Signaling Regulates Renal Tubule Morphogenesis but Is Not Cystogenic.

PubMed

Kunimoto, Koshi; Bayly, Roy D; Vladar, Eszter K; Vonderfecht, Tyson; Gallagher, Anna-Rachel; Axelrod, Jeffrey D

2017-10-23

Oriented cell division (OCD) and convergent extension (CE) shape developing renal tubules, and their disruption has been associated with polycystic kidney disease (PKD) genes, the majority of which encode proteins that localize to primary cilia. Core planar cell polarity (PCP) signaling controls OCD and CE in other contexts, leading to the hypothesis that disruption of PCP signaling interferes with CE and/or OCD to produce PKD. Nonetheless, the contribution of PCP to tubulogenesis and cystogenesis is uncertain, and two major questions remain unanswered. Specifically, the inference that mutation of PKD genes interferes with PCP signaling is untested, and the importance of PCP signaling for cystogenic PKD phenotypes has not been examined. We show that, during proliferative stages, PCP signaling polarizes renal tubules to control OCD. However, we find that, contrary to the prevailing model, PKD mutations do not disrupt PCP signaling but instead act independently and in parallel with PCP signaling to affect OCD. Indeed, PCP signaling that is normally downregulated once development is completed is retained in cystic adult kidneys. Disrupting PCP signaling results in inaccurate control of tubule diameter, a tightly regulated parameter with important physiological ramifications. However, we show that disruption of PCP signaling is not cystogenic. Our results suggest that regulating tubule diameter is a key function of PCP signaling but that loss of this control does not induce cysts. Copyright © 2017 Elsevier Ltd. All rights reserved.

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

Identification and Validation of Novel Small Molecule Disruptors of HuR-mRNA Interaction

PubMed Central

Wu, Xiaoqing; Lan, Lan; Wilson, David Michael; Marquez, Rebecca T.; Tsao, Wei-chung; Gao, Philip; Roy, Anuradha; Turner, Benjamin Andrew; McDonald, Peter; Tunge, Jon A; Rogers, Steven A; Dixon, Dan A.; Aubé, Jeffrey; Xu, Liang

2015-01-01

HuR, an RNA binding protein, binds to adenine- and uridine-rich elements (ARE) in the 3′-untranslated region (UTR) of target mRNAs, regulating their stability and translation. HuR is highly abundant in many types of cancer, and it promotes tumorigenesis by interacting with cancer-associated mRNAs, which encode proteins that are implicated in different tumor processes including cell proliferation, cell survival, angiogenesis, invasion, and metastasis. Drugs that disrupt the stabilizing effect of HuR upon mRNA targets could have dramatic effects on inhibiting cancer growth and persistence. In order to identify small molecules that directly disrupt the HuR–ARE interaction, we established a fluorescence polarization (FP) assay optimized for high throughput screening (HTS) using HuR protein and an ARE oligo from Musashi RNA-binding protein 1 (Msi1) mRNA, a HuR target. Following the performance of an HTS of ~6000 compounds, we discovered a cluster of potential disruptors, which were then validated by AlphaLISA (Amplified Luminescent Proximity Homogeneous Assay), surface plasmon resonance (SPR), ribonucleoprotein immunoprecipitation (RNP IP) assay, and luciferase reporter functional studies. These compounds disrupted HuR–ARE interactions at the nanomolar level and blocked HuR function by competitive binding to HuR. These results support future studies toward chemical probes for a HuR function study and possibly a novel therapy for HuR-overexpressing cancers. PMID:25750985

Electroporation transiently decreases GJB2 (connexin 26) expression in B16/BL6 melanoma cell line.

PubMed

Rangel, Marcelo Monte Mór; Chaible, Lucas Martins; Nagamine, Marcia Kazumi; Mennecier, Gregory; Cogliati, Bruno; de Oliveira, Krishna Duro; Fukumasu, Heidge; Sinhorini, Idércio Luiz; Mir, Lluis Maria; Dagli, Maria Lúcia Zaidan

2015-02-01

Connexins are proteins that form gap junctions. Perturbations in the cell membrane reportedly promote changes in the expression profile of connexins. Electroporation promotes destabilization by applying electrical pulses, and this procedure is used in electrochemotherapy and gene therapy, among others. This in vitro work aimed to study the interference of electroporation on the expression profile of GJB2 (Cx26 gene) and Connexin 26 in melanoma cell line B16/BL6. The techniques of immunocytochemistry, Western blot, and real-time PCR were used. After electroporation, cells showed a transient decrease in GJB2 mRNA. The immunostaining of Cx26 showed no noticeable change after electroporation at different time points. However, Western blot showed a significant reduction in Cx26 30 min after electroporation. Our results showed that electroporation interferes transiently in the expression of Connexin 26 in melanoma and are consistent with the idea that electroporation is a process of intense stress that promotes cell homeostatic imbalance and results in disruption of cell physiological processes such as transcription and translation.

Epstein-Barr virus and nasopharyngeal carcinoma

PubMed Central

Young, Lawrence S.; Dawson, Christopher W.

2014-01-01

Since its discovery 50 years ago, Epstein-Barr virus (EBV) has been linked to the development of cancers originating from both lymphoid and epithelial cells. Approximately 95% of the world's population sustains an asymptomatic, life-long infection with EBV. The virus persists in the memory B-cell pool of normal healthy individuals, and any disruption of this interaction results in virus-associated B-cell tumors. The association of EBV with epithelial cell tumors, specifically nasopharyngeal carcinoma (NPC) and EBV-positive gastric carcinoma (EBV-GC), is less clear and is currently thought to be caused by the aberrant establishment of virus latency in epithelial cells that display premalignant genetic changes. Although the precise role of EBV in the carcinogenic process is currently poorly understood, the presence of the virus in all tumor cells provides opportunities for developing novel therapeutic and diagnostic approaches. The study of EBV and its role in carcinomas continues to provide insight into the carcinogenic process that is relevant to a broader understanding of tumor pathogenesis and to the development of targeted cancer therapies. PMID:25418193

Hsp90 C-Terminal Inhibitors Exhibit Antimigratory Activity by Disrupting the Hsp90α/Aha1 Complex in PC3-MM2 Cells

PubMed Central

2015-01-01

Human Hsp90 isoforms are molecular chaperones that are often up-regulated in malignances and represent a primary target for Hsp90 inhibitors undergoing clinical evaluation. Hsp90α is a stress-inducible isoform of Hsp90 that plays a significant role in apoptosis and metastasis. Though Hsp90α is secreted into the extracellular space under metastatic conditions, its role in cancer biology is poorly understood. We report that Hsp90α associates with the Aha1 co-chaperone and found this complex to localize in secretory vesicles and at the leading edge of migrating cells. Knockdown of Hsp90α resulted in a defect in cell migration. The functional role of Hsp90α/Aha1 was studied by treating the cells with various novobiocin-based Hsp90 C-terminal inhibitors. These inhibitors disrupted the Hsp90α/Aha1 complex, caused a cytoplasmic redistribution of Hsp90α and Aha1, and decreased cell migration. Structure–function studies determined that disruption of Hsp90α/Aha1 association and inhibition of cell migration correlated with the presence of a benzamide side chain, since an acetamide substituted analog was less effective. Our results show that disruption of Hsp90α/Aha1 interactions with novobiocin-based Hsp90 C-terminal inhibitors may limit the metastatic potential of tumors. PMID:25402753

CRISPR-Cas9-mediated disruption of PD-1 on human T cells for adoptive cellular therapies of EBV positive gastric cancer.

PubMed

Su, Shu; Zou, Zhengyun; Chen, Fangjun; Ding, Naiqing; Du, Juan; Shao, Jie; Li, Lin; Fu, Yao; Hu, Bian; Yang, Yang; Sha, Huizi; Meng, Fanyan; Wei, Jia; Huang, Xingxu; Liu, Baorui

2017-01-01

The successful use of immune cell checkpoint inhibitors PD-1 and PD-L1, over the past 5 y has raised the concern of using immunotherapy to treat various cancers. Epstein-Barr virus-associated gastric cancer (EBVaGC) exhibits high infiltration of lymphocytes and high amplification of immune-related genes including PD-L1 as distinguished from Epstein-Barr virus-non-associated gastric cancer (EBVnGC). Here, we presume that this PD-1/PD-L1 pathway may hinder the efficacy of adoptive T cell therapy toward EBVaGC. These studies reveal possibility of generating PD-1-disrupted CTL by CRISPR-Cas9 system and demonstrate enhanced immune response of these PD-1-disrupted CTLs to the EBV-LMP2A antigen and superior cytotoxicity to the EBV-positive gastric cancer cell. In addition, when combined with low-dose radiotherapy, these PD-1-disrupted CTLs mediated an impressive antitumor effect in a xenograft mouse model of EBVaGC. Taken together, these studies illustrate PD-1/PD-L1-mediated immune tolerance of EBVaGC and provide a new strategy for targeting immune checkpoints to break the tolerance for the T cell-based adoptive therapy.

The Function of V-ATPases in Cancer

PubMed Central

Stransky, Laura; Cotter, Kristina

2016-01-01

The vacuolar ATPases (V-ATPases) are a family of proton pumps that couple ATP hydrolysis to proton transport into intracellular compartments and across the plasma membrane. They function in a wide array of normal cellular processes, including membrane traffic, protein processing and degradation, and the coupled transport of small molecules, as well as such physiological processes as urinary acidification and bone resorption. The V-ATPases have also been implicated in a number of disease processes, including viral infection, renal disease, and bone resorption defects. This review is focused on the growing evidence for the important role of V-ATPases in cancer. This includes functions in cellular signaling (particularly Wnt, Notch, and mTOR signaling), cancer cell survival in the highly acidic environment of tumors, aiding the development of drug resistance, as well as crucial roles in tumor cell invasion, migration, and metastasis. Of greatest excitement is evidence that at least some tumors express isoforms of V-ATPase subunits whose disruption is not lethal, leading to the possibility of developing anti-cancer therapeutics that selectively target V-ATPases that function in cancer cells. PMID:27335445

Investigation of cell cycle-associated structural reorganization in nucleolar FC/DFCs from mouse MFC cells by electron microscopy.

PubMed

Chen, Lingling; Jiao, Yang; Guan, Xin; Li, Xiliang; Feng, Yunpeng; Jiao, Mingda

2018-05-01

Nucleolus structure alters as the cell cycle is progressing. It is established in telophase, maintained throughout the entire interphase and disassembled in metaphase. Fibrillar centers (FCs), dense fibrillar components (DFCs) and granular components (GCs) are essential nucleolar organizations where rRNA transcription and processing and ribosome assembly take place. Hitherto, little is known about the cell cycle-dependent reorganization of these structures. In this study, we followed the nucleolus structure during the cell cycle by electron microscopy (EM). We found the nucleolus experienced multiple rounds of structural reorganization within a single cell cycle: (1) when nucleoli are formed during the transition from late M to G1 phase, FCs, DFCs and GCs are constructed, leading to the establishment of tripartite nucleolus; (2) as FC/DFCs are disrupted at mid-G1, tripartite nucleolus is gradually changed into a bipartite organization; (3) at late G1, the reassembly of FC/DFCs results in a structural transition from bipartite nucleolus towards tripartite nucleolus; (4) as cells enter S phase, FC/DFCs are disassembled again and tripartite nucleolus is thus changed into a bipartite organization. Of note, FC/DFCs were not observed until late S phase; (5) FC/DFCs experience structural disruption and restoration during G2 and (6) when cells are at mitotic stage, FC/DFCs disappear before nucleolus structure is disassembled. These results also suggest that bipartite nucleolus can exist in higher eukaryotes at certain period of the cell cycle. As structures are the fundamental basis of diverse cell activities, unveiling the structural reorganization of nucleolar FCs and DFCs may bring insights into the spatial-temporal compartmentalization of relevant cellular functions.

Septic disruption of lactiferous ducts with heterogeneous carcinoma of the breast in a lactating woman.

PubMed

Naim, Mohammed; John, Vanesa T; Gaur, Kavita; Anees, Afzal

2010-08-06

This report documents the diagnostic histopathological features of heterogeneous breast carcinoma following sepsis and disruption of the lactiferous ducts in a lactating woman and discusses the pathogenesis. Sections from the nipple revealed disrupted collecting lactiferous ducts presenting with intraduct precarcinoma and carcinoma of the epidermoid type, and attached reparative sprouts lined by lactiferous cells. Breast lobules showed generalised benign adenotic change with various foci of carcinoma microscopically identifiable as intraduct primitive lactiferal ectodermal carcinoma, lactating carcinoma, primitive neuroendocrine carcinoma and myoepithelioid granulomatous carcinoma. The findings led to the conclusion that the lactiferous ducts are susceptible to sepsis and disruption, which may predispose a patient to breast carcinoma. The pattern of carcinoma suggested that lactiferous epithelial cells behaved colonially, with different metaplastic changes, precarcinoma and carcinoma.

Reproductive isolation in hybrid mice due to spermatogenesis defects at three meiotic stages.

PubMed

Oka, Ayako; Mita, Akihiko; Takada, Yuki; Koseki, Haruhiko; Shiroishi, Toshihiko

2010-09-01

Early in the process of speciation, reproductive failures occur in hybrid animals between genetically diverged populations. The sterile hybrid animals are often males in mammals and they exhibit spermatogenic disruptions, resulting in decreased number and/or malformation of mature sperms. Despite the generality of this phenomenon, comparative study of phenotypes in hybrid males from various crosses has not been done, and therefore the comprehensive genetic basis of the disruption is still elusive. In this study, we characterized the spermatogenic phenotype especially during meiosis in four different cases of reproductive isolation: B6-ChrX(MSM), PGN-ChrX(MSM), (B6 × Mus musculus musculus-NJL/Ms) F(1), and (B6 × Mus spretus) F(1). The first two are consomic strains, both bearing the X chromosome of M. m. molossinus; in B6-ChrX(MSM), the genetic background is the laboratory strain C57BL/6J (predominantly M. m. domesticus), while in PGN-ChrX(MSM) the background is the PGN2/Ms strain purely derived from wild M. m. domesticus. The last two cases are F(1) hybrids between mouse subspecies or species. Each of the hybrid males exhibited cell-cycle arrest and/or apoptosis at either one or two of three distinct meiotic stages: premeiotic stage, zygotene-to-pachytene stage of prophase I, and metaphase I. This study shows that the sterility in hybrid males is caused by spermatogenic disruptions at multiple stages, suggesting that the responsible genes function in different cellular processes. Furthermore, the stages with disruptions are not correlated with the genetic distance between the respective parental strains.

Alcohol reversibly disrupts TNF-α/TACE interactions in the cell membrane

PubMed Central

Song, Kejing; Zhao, Xue-Jun; Marrero, Luis; Oliver, Peter; Nelson, Steve; Kolls, Jay K

2005-01-01

Background Alcohol abuse has long been known to adversely affect innate and adaptive immune responses and pre-dispose to infections. One cellular mechanism responsible for this effect is alcohol-induced suppression of TNF-α (TNF) by mononuclear phagocytes. We have previously shown that alcohol in part inhibits TNF-α processing by TNF converting enzyme (TACE) in human monocytes. We hypothesized that the chain length of the alcohol is critical for post-transcriptional suppression of TNF secretion. Methods Due to the complex transcriptional and post-transcriptional regulation of TNF in macrophages, to specifically study TNF processing at the cell membrane we performed transient transfections of A549 cells with the TNF cDNA driven by the heterologous CMV promoter. TNF/TACE interactions at the cell surface were assessed using fluorescent resonance energy transfer (FRET) microscopy. Results The single carbon alcohol, methanol suppressed neither TNF secretion nor FRET efficiency between TNF and TACE. However, 2, 3, and 4 carbon alcohols were potent suppressors of TNF processing and FRET efficiency. The effect of ethanol, a 2-carbon alcohol was reversible. Conclusion These data show that inhibition of TNF-α processing by acute ethanol is a direct affect of ethanol on the cell membrane and is reversible upon cessation or metabolism. PMID:16246259

Calcium/Ask1/MKK7/JNK2/c-Src signalling cascade mediates disruption of intestinal epithelial tight junctions by dextran sulfate sodium.

PubMed

Samak, Geetha; Chaudhry, Kamaljit K; Gangwar, Ruchika; Narayanan, Damodaran; Jaggar, Jonathan H; Rao, RadhaKrishna

2015-02-01

Disruption of intestinal epithelial tight junctions is an important event in the pathogenesis of ulcerative colitis. Dextran sodium sulfate (DSS) induces colitis in mice with symptoms similar to ulcerative colitis. However, the mechanism of DSS-induced colitis is unknown. We investigated the mechanism of DSS-induced disruption of intestinal epithelial tight junctions and barrier dysfunction in Caco-2 cell monolayers in vitro and mouse colon in vivo. DSS treatment resulted in disruption of tight junctions, adherens junctions and actin cytoskeleton leading to barrier dysfunction in Caco-2 cell monolayers. DSS induced a rapid activation of c-Jun N-terminal kinase (JNK), and the inhibition or knockdown of JNK2 attenuated DSS-induced tight junction disruption and barrier dysfunction. In mice, DSS administration for 4 days caused redistribution of tight junction and adherens junction proteins from the epithelial junctions, which was blocked by JNK inhibitor. In Caco-2 cell monolayers, DSS increased intracellular Ca(2+) concentration, and depletion of intracellular Ca(2+) by 1,2-bis-(o-aminophenoxy)ethane-N,N,N',N'-tetra-acetic acid tetrakis(acetoxymethyl ester) (BAPTA/AM) or thapsigargin attenuated DSS-induced JNK activation, tight junction disruption and barrier dysfunction. Knockdown of apoptosis signal-regulated kinase 1 (Ask1) or MKK7 blocked DSS-induced tight junction disruption and barrier dysfunction. DSS activated c-Src by a Ca2+ and JNK-dependent mechanism. Inhibition of Src kinase activity or knockdown of c-Src blocked DSS-induced tight junction disruption and barrier dysfunction. DSS increased tyrosine phosphorylation of occludin, zonula occludens-1 (ZO-1), E-cadherin and β-catenin. SP600125 abrogated DSS-induced tyrosine phosphorylation of junctional proteins. Recombinant JNK2 induced threonine phosphorylation and auto-phosphorylation of c-Src. The present study demonstrates that Ca(2+)/Ask1/MKK7/JNK2/cSrc signalling cascade mediates DSS-induced tight junction disruption and barrier dysfunction.

Functional Genomic Screening Reveals Splicing of the EWS-FLI1 Fusion Transcript as a Vulnerability in Ewing Sarcoma.

PubMed

Grohar, Patrick J; Kim, Suntae; Rangel Rivera, Guillermo O; Sen, Nirmalya; Haddock, Sara; Harlow, Matt L; Maloney, Nichole K; Zhu, Jack; O'Neill, Maura; Jones, Tamara L; Huppi, Konrad; Grandin, Magdalena; Gehlhaus, Kristen; Klumpp-Thomas, Carleen A; Buehler, Eugen; Helman, Lee J; Martin, Scott E; Caplen, Natasha J

2016-01-26

Ewing sarcoma cells depend on the EWS-FLI1 fusion transcription factor for cell survival. Using an assay of EWS-FLI1 activity and genome-wide RNAi screening, we have identified proteins required for the processing of the EWS-FLI1 pre-mRNA. We show that Ewing sarcoma cells harboring a genomic breakpoint that retains exon 8 of EWSR1 require the RNA-binding protein HNRNPH1 to express in-frame EWS-FLI1. We also demonstrate the sensitivity of EWS-FLI1 fusion transcripts to the loss of function of the U2 snRNP component, SF3B1. Disrupted splicing of the EWS-FLI1 transcript alters EWS-FLI1 protein expression and EWS-FLI1-driven expression. Our results show that the processing of the EWS-FLI1 fusion RNA is a potentially targetable vulnerability in Ewing sarcoma cells. Copyright © 2016 The Authors. Published by Elsevier Inc. All rights reserved.

A Morphological and Cytochemical Study of the Interaction between Paracoccidiodes brasiliensis and Neutrophils

NASA Astrophysics Data System (ADS)

Dias, Maria Fernanda R. G.; Filgueira, Absalom L.; de Souza, Wanderley

2004-04-01

Paracoccidioidomycosis is a systemic granulomatous disease caused by the dimorphic fungus Paracoccidioides brasiliensis. It is the most prevalent systemic mycosis of Latin America and 80% of the reported cases are from Brazil. Because of the great number of neutrophils found in the P. brasiliensis granuloma, studies have been done to evaluate the role of these cells during the development of the infection. Scanning and transmission electron microscopy of thin sections showed that the neutrophils ingest yeast cells through a typical phagocytic process with the formation of pseudopodes. The pseudopodes even disrupt the connection established between the mother and the bud cells. Neutrophils also associate to each other, forming a kind of extracellular vacuole where large yeast cells are encapsulated. Cytochemical studies showed that once P. brasiliensis attaches to the neutrophil surface, it triggers a respiratory burst with release of oxygen-derived products. Attachment also triggers neutrophils' degranulation, with release of endogenous peroxidase localized in cytoplasmic granules. Together, these processes lead to killing of both ingested and extracellular P. brasiliensis.

Mitochondrial dysfunction in choline deficiency-induced apoptosis in cultured rat hepatocytes.

PubMed

Guo, Wei-Xing; Pye, Quentin N; Williamson, Kelly S; Stewart, Charles A; Hensley, Kenneth L; Kotake, Yashige; Floyd, Robert A; Broyles, Robert H

2005-09-01

Our recent studies have demonstrated that generation of ROS is associated with choline deficiency (CD)-induced apoptosis in CWSV-1 cells, an immortalized rat hepatocyte that becomes tumorigenic by stepwise culturing in decreasing levels of choline. In the present study, we investigated the effect of CD on loss of mitochondrial membrane potential (MMP), using the JC-1 probe by FASCAN assay. Our data demonstrate that MMP in CD-cultured cells was decreased in a time- and dose-dependent manner and that significant disruption occurred at 24 h, relative to high choline (HC, 70 microM) cultured cells. In order to investigate further the relationship among the CD-induced ROS, MMP collapse, and apoptosis, we examined the effects of different inhibitors on ROS production, MMP disruption, and apoptosis in CD or HC-cultured CWSV-1 cells. These data indicate that the disruption of MMP is an upstream event in CD-induced apoptosis, and mitochondrial dysfunction plays a key role in mediating CD-induced apoptosis in CWSV-1 cells.

Targeted cellular ablation based on the morphology of malignant cells

NASA Astrophysics Data System (ADS)

Ivey, Jill W.; Latouche, Eduardo L.; Sano, Michael B.; Rossmeisl, John H.; Davalos, Rafael V.; Verbridge, Scott S.

2015-11-01

Treatment of glioblastoma multiforme (GBM) is especially challenging due to a shortage of methods to preferentially target diffuse infiltrative cells, and therapy-resistant glioma stem cell populations. Here we report a physical treatment method based on electrical disruption of cells, whose action depends strongly on cellular morphology. Interestingly, numerical modeling suggests that while outer lipid bilayer disruption induced by long pulses (~100 μs) is enhanced for larger cells, short pulses (~1 μs) preferentially result in high fields within the cell interior, which scale in magnitude with nucleus size. Because enlarged nuclei represent a reliable indicator of malignancy, this suggested a means of preferentially targeting malignant cells. While we demonstrate killing of both normal and malignant cells using pulsed electric fields (PEFs) to treat spontaneous canine GBM, we proposed that properly tuned PEFs might provide targeted ablation based on nuclear size. Using 3D hydrogel models of normal and malignant brain tissues, which permit high-resolution interrogation during treatment testing, we confirmed that PEFs could be tuned to preferentially kill cancerous cells. Finally, we estimated the nuclear envelope electric potential disruption needed for cell death from PEFs. Our results may be useful in safely targeting the therapy-resistant cell niches that cause recurrence of GBM tumors.

Targeted cellular ablation based on the morphology of malignant cells

PubMed Central

Ivey, Jill W.; Latouche, Eduardo L.; Sano, Michael B.; Rossmeisl, John H.; Davalos, Rafael V.; Verbridge, Scott S.

2015-01-01

Treatment of glioblastoma multiforme (GBM) is especially challenging due to a shortage of methods to preferentially target diffuse infiltrative cells, and therapy-resistant glioma stem cell populations. Here we report a physical treatment method based on electrical disruption of cells, whose action depends strongly on cellular morphology. Interestingly, numerical modeling suggests that while outer lipid bilayer disruption induced by long pulses (~100 μs) is enhanced for larger cells, short pulses (~1 μs) preferentially result in high fields within the cell interior, which scale in magnitude with nucleus size. Because enlarged nuclei represent a reliable indicator of malignancy, this suggested a means of preferentially targeting malignant cells. While we demonstrate killing of both normal and malignant cells using pulsed electric fields (PEFs) to treat spontaneous canine GBM, we proposed that properly tuned PEFs might provide targeted ablation based on nuclear size. Using 3D hydrogel models of normal and malignant brain tissues, which permit high-resolution interrogation during treatment testing, we confirmed that PEFs could be tuned to preferentially kill cancerous cells. Finally, we estimated the nuclear envelope electric potential disruption needed for cell death from PEFs. Our results may be useful in safely targeting the therapy-resistant cell niches that cause recurrence of GBM tumors. PMID:26596248

Microbial cell disruption for improving lipid recovery using pressurized CO2 : Role of CO2 solubility in cell suspension, sugar broth, and spent media.

PubMed

Howlader, Md Shamim; French, William Todd; Shields-Menard, Sara A; Amirsadeghi, Marta; Green, Magan; Rai, Neeraj

2017-05-01

The study of in situ gas explosion to lyse the triglyceride-rich cells involves the solubilization of gas (e.g., carbon dioxide, CO 2 ) in lipid-rich cells under pressure followed by a rapid decompression, which allows the gas inside the cell to rapidly expand and rupture the cell from inside out. The aim of this study was to perform the cell disruption using pressurized CO 2 as well as to determine the solubility of CO 2 in Rhodotorula glutinis cell suspension, sugar broth media, and spent media. Cell disruption of R. glutinis was performed at two pressures of 2,000 and 3,500 kPa, respectively, at 295.2 K, and it was found from both scanning electron microscopy (SEM) and plate count that a substantial amount of R. glutinis was disrupted due to the pressurized CO 2 . We also found a considerable portion of lipid present in the aqueous phase after the disruption at P = 3,500 kPa compared to control (no pressure) and P = 2,000 kPa, which implied that more intracellular lipid was released due to the pressurized CO 2 . Solubility of CO 2 in R. glutinis cell suspension was found to be higher than the solubility of CO 2 in both sugar broth media and spent media. Experimental solubility was correlated using the extended Henry's law, which showed a good agreement with the experimental data. Enthalpy and entropy of dissolution of CO 2 were found to be -14.22 kJ mol -1 and 48.10 kJ mol -1  K -1 , 9.64 kJ mol -1 and 32.52 kJ mol -1  K -1 , and 7.50 kJ mol -1 and 25.22 kJ mol -1  K -1 in R. glutinis, spent media, and sugar broth media, respectively. © 2017 American Institute of Chemical Engineers Biotechnol. Prog., 33:737-748, 2017. © 2017 American Institute of Chemical Engineers.

Asymmetric Distribution of Primary Cilia Allocates Satellite Cells for Self-Renewal.

PubMed

Jaafar Marican, Nur Hayati; Cruz-Migoni, Sara B; Borycki, Anne-Gaëlle

2016-06-14

Regeneration of vertebrate skeletal muscles requires satellite cells, a population of stem cells that are quiescent in normal conditions and divide, differentiate, and self-renew upon activation triggered by exercise, injury, and degenerative diseases. Satellite cell self-renewal is essential for long-term tissue homeostasis, and previous work has identified a number of external cues that control this process. However, little is known of the possible intrinsic control mechanisms of satellite cell self-renewal. Here, we show that quiescent satellite cells harbor a primary cilium, which is rapidly disassembled upon entry into the cell cycle. Contrasting with a commonly accepted belief, cilia reassembly does not occur uniformly in cells exiting the cell cycle. We found that primary cilia reassemble preferentially in cells committed to self-renew, and disruption of cilia reassembly causes a specific deficit in self-renewing satellite cells. These observations indicate that primary cilia provide an intrinsic cue essential for satellite cell self-renewal. Copyright © 2016 The Authors. Published by Elsevier Inc. All rights reserved.

Dysregulation of haematopoietic stem cell regulatory programs in acute myeloid leukaemia.

PubMed

Basilico, Silvia; Göttgens, Berthold

2017-07-01

Haematopoietic stem cells (HSC) are situated at the apex of the haematopoietic differentiation hierarchy, ensuring the life-long supply of mature haematopoietic cells and forming a reservoir to replenish the haematopoietic system in case of emergency such as acute blood loss. To maintain a balanced production of all mature lineages and at the same time secure a stem cell reservoir, intricate regulatory programs have evolved to control multi-lineage differentiation and self-renewal in haematopoietic stem and progenitor cells (HSPCs). Leukaemogenic mutations commonly disrupt these regulatory programs causing a block in differentiation with simultaneous enhancement of proliferation. Here, we briefly summarize key aspects of HSPC regulatory programs, and then focus on their disruption by leukaemogenic fusion genes containing the mixed lineage leukaemia (MLL) gene. Using MLL as an example, we explore important questions of wider significance that are still under debate, including the importance of cell of origin, to what extent leukaemia oncogenes impose specific regulatory programs and the relevance of leukaemia stem cells for disease development and prognosis. Finally, we suggest that disruption of stem cell regulatory programs is likely to play an important role in many other pathologies including ageing-associated regenerative failure.

Excess titanium dioxide nanoparticles on the cell surface induce cytotoxicity by hindering ion exchange and disrupting exocytosis processes.

PubMed

Wang, Yanli; Yao, Chenjie; Li, Chenchen; Ding, Lin; Liu, Jian; Dong, Peng; Fang, Haiping; Lei, Zhendong; Shi, Guosheng; Wu, Minghong

2015-08-14

To date, considerable effort has been devoted to determine the potential toxicity of nanoparticles to cells and organisms. However, determining the mechanism of cytotoxicity induced by different types of nanoparticles remains challenging. Herein, typically low toxicity nanomaterials were used as a model to investigate the mechanism of cytotoxicity induced by low toxicity nanomaterials. We studied the effect of nano-TiO2, nano-Al2O3 and nano-SiO2 deposition films on the ion concentration on a cell-free system simulating the cell membrane. The results showed that the ion concentration of K(+), Ca(2+), Na(+), Mg(2+) and SO4(2-) decreased significantly following filtration of the prepared deposition films. More specifically, at a high nano-TiO2 concentration (200 mg L(-1)) and a long nano-TiO2 deposition time (48 h), the concentration of Na(+) decreased from 2958.01 to 2775.72, 2749.86, 2757.36, and 2719.82 mg L(-1), respectively, for the four types of nano-TiO2 studied. Likewise, the concentration of SO4(2-) decreased from 38.83 to 35.00, 35.80, 35.40, and 35.27 mg L(-1), respectively. The other two kinds of typical low toxicity nanomaterials (nano-Al2O3 and nano-SiO2) have a similar impact on the ion concentration change trend. Adsorption of ions on nanoparticles and the hydrated shell around the ions strongly hindered the ions through the nanoparticle films. The endocytosed nanoparticles could be released from the cells without inducing cytotoxicity. Hindering the ion exchange and disrupting the exocytosis process are the main factors that induce cytotoxicity in the presence of excess nano-TiO2 on the cell surface. The current findings may offer a universal principle for understanding the mechanism of cytotoxicity induced by low toxicity nanomaterials.

Therapeutic Strategy for Targeting Aggressive Malignant Gliomas by Disrupting Their Energy Balance.

PubMed

Hegazy, Ahmed M; Yamada, Daisuke; Kobayashi, Masahiko; Kohno, Susumu; Ueno, Masaya; Ali, Mohamed A E; Ohta, Kumiko; Tadokoro, Yuko; Ino, Yasushi; Todo, Tomoki; Soga, Tomoyoshi; Takahashi, Chiaki; Hirao, Atsushi

2016-10-07

Although abnormal metabolic regulation is a critical determinant of cancer cell behavior, it is still unclear how an altered balance between ATP production and consumption contributes to malignancy. Here we show that disruption of this energy balance efficiently suppresses aggressive malignant gliomas driven by mammalian target of rapamycin complex 1 (mTORC1) hyperactivation. In a mouse glioma model, mTORC1 hyperactivation induced by conditional Tsc1 deletion increased numbers of glioma-initiating cells (GICs) in vitro and in vivo Metabolic analysis revealed that mTORC1 hyperactivation enhanced mitochondrial biogenesis, as evidenced by elevations in oxygen consumption rate and ATP production. Inhibition of mitochondrial ATP synthetase was more effective in repressing sphere formation by Tsc1-deficient glioma cells than that by Tsc1-competent glioma cells, indicating a crucial function for mitochondrial bioenergetic capacity in GIC expansion. To translate this observation into the development of novel therapeutics targeting malignant gliomas, we screened drug libraries for small molecule compounds showing greater efficacy in inhibiting the proliferation/survival of Tsc1-deficient cells compared with controls. We identified several compounds able to preferentially inhibit mitochondrial activity, dramatically reducing ATP levels and blocking glioma sphere formation. In human patient-derived glioma cells, nigericin, which reportedly suppresses cancer stem cell properties, induced AMPK phosphorylation that was associated with mTORC1 inactivation and induction of autophagy and led to a marked decrease in sphere formation with loss of GIC marker expression. Furthermore, malignant characteristics of human glioma cells were markedly suppressed by nigericin treatment in vivo Thus, targeting mTORC1-driven processes, particularly those involved in maintaining a cancer cell's energy balance, may be an effective therapeutic strategy for glioma patients. © 2016 by The American Society for Biochemistry and Molecular Biology, Inc.

Picornaviruses and nuclear functions: targeting a cellular compartment distinct from the replication site of a positive-strand RNA virus

PubMed Central

Flather, Dylan; Semler, Bert L.

2015-01-01

The compartmentalization of DNA replication and gene transcription in the nucleus and protein production in the cytoplasm is a defining feature of eukaryotic cells. The nucleus functions to maintain the integrity of the nuclear genome of the cell and to control gene expression based on intracellular and environmental signals received through the cytoplasm. The spatial separation of the major processes that lead to the expression of protein-coding genes establishes the necessity of a transport network to allow biomolecules to translocate between these two regions of the cell. The nucleocytoplasmic transport network is therefore essential for regulating normal cellular functioning. The Picornaviridae virus family is one of many viral families that disrupt the nucleocytoplasmic trafficking of cells to promote viral replication. Picornaviruses contain positive-sense, single-stranded RNA genomes and replicate in the cytoplasm of infected cells. As a result of the limited coding capacity of these viruses, cellular proteins are required by these intracellular parasites for both translation and genomic RNA replication. Being of messenger RNA polarity, a picornavirus genome can immediately be translated upon entering the cell cytoplasm. However, the replication of viral RNA requires the activity of RNA-binding proteins, many of which function in host gene expression, and are consequently localized to the nucleus. As a result, picornaviruses disrupt nucleocytoplasmic trafficking to exploit protein functions normally localized to a different cellular compartment from which they translate their genome to facilitate efficient replication. Furthermore, picornavirus proteins are also known to enter the nucleus of infected cells to limit host-cell transcription and down-regulate innate antiviral responses. The interactions of picornavirus proteins and host-cell nuclei are extensive, required for a productive infection, and are the focus of this review. PMID:26150805

Energy consumption and water-soluble protein release by cell wall disruption of Nannochloropsis gaditana.

PubMed

Safi, C; Cabas Rodriguez, L; Mulder, W J; Engelen-Smit, N; Spekking, W; van den Broek, L A M; Olivieri, G; Sijtsma, L

2017-09-01

Several cell disruption methods were tested on Nannochloropsis gaditana, to evaluate their efficiency in terms of cell disintegration, energy input and release of soluble proteins. High-pressure homogenization (HPH) and bead milling were the most efficient with >95% cell disintegration, ±50% (w/w) release of total proteins and low energy input (<0.5kWh.kg -1 biomass ). Enzymatic treatment required low energy input (<0.34kWh.kg -1 biomass ), but it only released ±35% protein (w/w). Pulsed Electric Field (PEF) was neither energy-efficient (10.44kWh.kg -1 biomass ) nor successful for protein release (only 10% proteins w/w) and cell disintegration. The release of proteins after applying HPH and bead milling always required less intensive operating conditions for cell disruption. The energy cost per unit of released protein ranged from 0.15-0.25 €.kg Protein -1 in case of HPH, and up to 2-20 €.kg Protein -1 in case of PEF. Copyright © 2017 Elsevier Ltd. All rights reserved.

Na+ influx via Orai1 inhibits intracellular ATP-induced mTORC2 signaling to disrupt CD4 T cell gene expression and differentiation.

PubMed

Miao, Yong; Bhushan, Jaya; Dani, Adish; Vig, Monika

2017-05-11

T cell effector functions require sustained calcium influx. However, the signaling and phenotypic consequences of non-specific sodium permeation via calcium channels remain unknown. α-SNAP is a crucial component of Orai1 channels, and its depletion disrupts the functional assembly of Orai1 multimers. Here we show that α-SNAP hypomorph, hydrocephalus with hopping gait, Napa hyh/hyh mice harbor significant defects in CD4 T cell gene expression and Foxp3 regulatory T cell (Treg) differentiation. Mechanistically, TCR stimulation induced rapid sodium influx in Napa hyh/hyh CD4 T cells, which reduced intracellular ATP, [ATP] i . Depletion of [ATP] i inhibited mTORC2 dependent NFκB activation in Napa hyh/hyh cells but ablation of Orai1 restored it. Remarkably, TCR stimulation in the presence of monensin phenocopied the defects in Napa hyh/hyh signaling and Treg differentiation, but not IL-2 expression. Thus, non-specific sodium influx via bonafide calcium channels disrupts unexpected signaling nodes and may provide mechanistic insights into some divergent phenotypes associated with Orai1 function.

Endothelial permeability is controlled by spatially defined cytoskeletal mechanics: atomic force microscopy force mapping of pulmonary endothelial monolayer.

PubMed

Birukova, Anna A; Arce, Fernando T; Moldobaeva, Nurgul; Dudek, Steven M; Garcia, Joe G N; Lal, Ratnesh; Birukov, Konstantin G

2009-03-01

Actomyosin contraction directly regulates endothelial cell (EC) permeability, but intracellular redistribution of cytoskeletal tension associated with EC permeability is poorly understood. We used atomic force microscopy (AFM), EC permeability assays, and fluorescence microscopy to link barrier regulation, cell remodeling, and cytoskeletal mechanical properties in EC treated with barrier-protective as well as barrier-disruptive agonists. Thrombin, vascular endothelial growth factor, and hydrogen peroxide increased EC permeability, disrupted cell junctions, and induced stress fiber formation. Oxidized 1-palmitoyl-2-arachidonoyl-sn-glycero-3-phosphocholine, hepatocyte growth factor, and iloprost tightened EC barriers, enhanced peripheral actin cytoskeleton and adherens junctions, and abolished thrombin-induced permeability and EC remodeling. AFM force mapping and imaging showed differential distribution of cell stiffness: barrier-disruptive agonists increased stiffness in the central region, and barrier-protective agents decreased stiffness in the center and increased it at the periphery. Attenuation of thrombin-induced permeability correlates well with stiffness changes from the cell center to periphery. These results directly link for the first time the patterns of cell stiffness with specific EC permeability responses.

Coordinated gene expression during gilthead sea bream skeletogenesis and its disruption by nutritional hypervitaminosis A.

PubMed

Fernández, Ignacio; Darias, Maria; Andree, Karl B; Mazurais, David; Zambonino-Infante, Jose Luís; Gisbert, Enric

2011-02-09

Vitamin A (VA) has a key role in vertebrate morphogenesis, determining body patterning and growth through the control of cell proliferation and differentiation processes. VA regulates primary molecular pathways of those processes by the binding of its active metabolite (retinoic acid) to two types of specific nuclear receptors: retinoic acid receptors (RARs) and retinoid X receptors (RXRs), which promote transcription of downstream target genes. This process is well known in most of higher vertebrates; however, scarce information is available regarding fishes. Therefore, in order to gain further knowledge of fish larval development and its disruption by nutritional VA imbalance, the relative expression of some RARs and RXRs, as well as several genes involved in morpho- and skeletogenesis such as peroxisome proliferator-activated receptors (PPARA, PPARB and PPARG); retinol-binding protein (RBP); insulin-like growth factors I and II (IGF1 and IGF2, respectively); bone morphogenetic protein 2 (Bmp2); transforming growth factor β-1 (TGFB1); and genes encoding different extracellular matrix (ECM) proteins such as matrix Gla protein (mgp), osteocalcin (bglap), osteopontin (SPP1), secreted protein acidic and rich in cysteine (SPARC) and type I collagen α1 chain (COL1A1) have been studied in gilthead sea bream. During gilthead sea bream larval development, specific expression profiles for each gene were tightly regulated during fish morphogenesis and correlated with specific morphogenetic events and tissue development. Dietary hypervitaminosis A during early larval development disrupted the normal gene expression profile for genes involved in RA signalling (RARA), VA homeostasis (RBP) and several genes encoding ECM proteins that are linked to skeletogenesis, such as bglap and mgp. Present data reflects the specific gene expression patterns of several genes involved in larval fish RA signalling and skeletogenesis; and how specific gene disruption induced by a nutritional VA imbalance underlie the skeletal deformities. Our results are of basic interest for fish VA signalling and point out some of the potential molecular players involved in fish skeletogenesis. Increased incidences of skeletal deformities in gilthead sea bream fed with hypervitaminosis A were the likely ultimate consequence of specific gene expression disruption at critical development stages.

Coordinated gene expression during gilthead sea bream skeletogenesis and its disruption by nutritional hypervitaminosis A

PubMed Central

2011-01-01

Background Vitamin A (VA) has a key role in vertebrate morphogenesis, determining body patterning and growth through the control of cell proliferation and differentiation processes. VA regulates primary molecular pathways of those processes by the binding of its active metabolite (retinoic acid) to two types of specific nuclear receptors: retinoic acid receptors (RARs) and retinoid X receptors (RXRs), which promote transcription of downstream target genes. This process is well known in most of higher vertebrates; however, scarce information is available regarding fishes. Therefore, in order to gain further knowledge of fish larval development and its disruption by nutritional VA imbalance, the relative expression of some RARs and RXRs, as well as several genes involved in morpho- and skeletogenesis such as peroxisome proliferator-activated receptors (PPARA, PPARB and PPARG); retinol-binding protein (RBP); insulin-like growth factors I and II (IGF1 and IGF2, respectively); bone morphogenetic protein 2 (Bmp2); transforming growth factor β-1 (TGFB1); and genes encoding different extracellular matrix (ECM) proteins such as matrix Gla protein (mgp), osteocalcin (bglap), osteopontin (SPP1), secreted protein acidic and rich in cysteine (SPARC) and type I collagen α1 chain (COL1A1) have been studied in gilthead sea bream. Results During gilthead sea bream larval development, specific expression profiles for each gene were tightly regulated during fish morphogenesis and correlated with specific morphogenetic events and tissue development. Dietary hypervitaminosis A during early larval development disrupted the normal gene expression profile for genes involved in RA signalling (RARA), VA homeostasis (RBP) and several genes encoding ECM proteins that are linked to skeletogenesis, such as bglap and mgp. Conclusions Present data reflects the specific gene expression patterns of several genes involved in larval fish RA signalling and skeletogenesis; and how specific gene disruption induced by a nutritional VA imbalance underlie the skeletal deformities. Our results are of basic interest for fish VA signalling and point out some of the potential molecular players involved in fish skeletogenesis. Increased incidences of skeletal deformities in gilthead sea bream fed with hypervitaminosis A were the likely ultimate consequence of specific gene expression disruption at critical development stages. PMID:21306609

Prenatal PCBs disrupt early neuroendocrine development of the rat hypothalamus

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

Dickerson, Sarah M.; Cunningham, Stephanie L.; Gore, Andrea C., E-mail: andrea.gore@mail.utexas.edu

Neonatal exposure to endocrine disrupting chemicals (EDCs) such as polychlorinated biphenyls (PCBs) can interfere with hormone-sensitive developmental processes, including brain sexual differentiation. We hypothesized that disruption of these processes by gestational PCB exposure would be detectable as early as the day after birth (postnatal day (P) 1) through alterations in hypothalamic gene and protein expression. Pregnant Sprague-Dawley rats were injected twice, once each on gestational days 16 and 18, with one of the following: DMSO vehicle; the industrial PCB mixture Aroclor 1221 (A1221); a reconstituted mixture of the three most prevalent congeners found in humans, PCB138, PCB153, and PCB180; ormore » estradiol benzoate (EB). On P1, litter composition, anogenital distance (AGD), and body weight were assessed. Pups were euthanized for immunohistochemistry of estrogen receptor {alpha} (ER{alpha}) or TUNEL labeling of apoptotic cells or quantitative PCR of 48 selected genes in the preoptic area (POA). We found that treatment with EB or A1221 had a sex-specific effect on developmental apoptosis in the neonatal anteroventral periventricular nucleus (AVPV), a sexually dimorphic hypothalamic region involved in the regulation of reproductive neuroendocrine function. In this region, exposed females had increased numbers of apoptotic nuclei, whereas there was no effect of treatment in males. For ER{alpha}, EB treatment increased immunoreactive cell numbers and density in the medial preoptic nucleus (MPN) of both males and females, while A1221 and the PCB mixture had no effect. PCR analysis of gene expression in the POA identified nine genes that were significantly altered by prenatal EDC exposure, in a manner that varied by sex and treatment. These genes included brain-derived neurotrophic factor, GABA{sub B} receptors-1 and -2, IGF-1, kisspeptin receptor, NMDA receptor subunits NR2b and NR2c, prodynorphin, and TGF{alpha}. Collectively, these results suggest that the disrupted sexual differentiation of the POA by prenatal EDC exposures is already evident as early as the day after birth, effects that may change the trajectory of postnatal development and compromise adult reproductive function.« less

Asymmetric leaves1 mediates leaf patterning and stem cell function in Arabidopsis.

PubMed

Byrne, M E; Barley, R; Curtis, M; Arroyo, J M; Dunham, M; Hudson, A; Martienssen, R A

Meristem function in plants requires both the maintenance of stem cells and the specification of founder cells from which lateral organs arise. Lateral organs are patterned along proximodistal, dorsoventral and mediolateral axes. Here we show that the Arabidopsis mutant asymmetric leaves1 (as1) disrupts this process. AS1 encodes a myb domain protein, closely related to PHANTASTICA in Antirrhinum and ROUGH SHEATH2 in maize, both of which negatively regulate knotted-class homeobox genes. AS1 negatively regulates the homeobox genes KNAT1 and KNAT2 and is, in turn, negatively regulated by the meristematic homeobox gene SHOOT MERISTEMLESS. This genetic pathway defines a mechanism for differentiating between stem cells and organ founder cells within the shoot apical meristem and demonstrates that genes expressed in organ primordia interact with meristematic genes to regulate shoot morphogenesis.

Ultra-localized single cell electroporation using silicon nanowires.

PubMed

Jokilaakso, Nima; Salm, Eric; Chen, Aaron; Millet, Larry; Guevara, Carlos Duarte; Dorvel, Brian; Reddy, Bobby; Karlstrom, Amelie Eriksson; Chen, Yu; Ji, Hongmiao; Chen, Yu; Sooryakumar, Ratnasingham; Bashir, Rashid

2013-02-07

Analysis of cell-to-cell variation can further the understanding of intracellular processes and the role of individual cell function within a larger cell population. The ability to precisely lyse single cells can be used to release cellular components to resolve cellular heterogeneity that might be obscured when whole populations are examined. We report a method to position and lyse individual cells on silicon nanowire and nanoribbon biological field effect transistors. In this study, HT-29 cancer cells were positioned on top of transistors by manipulating magnetic beads using external magnetic fields. Ultra-rapid cell lysis was subsequently performed by applying 600-900 mV(pp) at 10 MHz for as little as 2 ms across the transistor channel and the bulk substrate. We show that the fringing electric field at the device surface disrupts the cell membrane, leading to lysis from irreversible electroporation. This methodology allows rapid and simple single cell lysis and analysis with potential applications in medical diagnostics, proteome analysis and developmental biology studies.

Complex disruption effect of natural polyphenols on Bcl-2-Bax: molecular dynamics simulation and essential dynamics study.

PubMed

Verma, Sharad; Singh, Amit; Mishra, Abha

2015-01-01

Apoptosis (programmed cell death) is a process by which cells died after completing physiological function or after a severe genetic damage. Apoptosis is mainly regulated by the Bcl-2 family of proteins. Anti apoptotic protein Bcl-2 prevents the Bax activation/oligomerization to form heterodimer which is responsible for release of the cytochrome c from mitochondria to the cytosol in response to death signal. Quercetin and taxifolin (natural polyphenols) efficiently bound to hydrophobic groove of Bcl-2 and altered the structure by inducing conformational changes. Taxifolin was found more efficient when compared to quercetin in terms of interaction energy and collapse of hydrophobic groove. Taxifolin and quercetin were found to dissociate the Bcl-2-Bax complex during 12 ns MD simulation. The effect of taxifolin and quercetin was, further validated by the MD simulation of ligand-unbound Bcl-2-Bax which showed stability during the simulation. Obatoclax (an inhibitor of Bcl-2) had no significant dissociation effect on Bcl-2-Bax during simulation which favored the previous experimental results and disruption effect of taxifolin and quercetin.

Tumor Cell Invasion Can Be Blocked by Modulators of Collagen Fibril Alignment That Control Assembly of the Extracellular Matrix.

PubMed

Grossman, Moran; Ben-Chetrit, Nir; Zhuravlev, Alina; Afik, Ran; Bassat, Elad; Solomonov, Inna; Yarden, Yosef; Sagi, Irit

2016-07-15

Abnormal architectures of collagen fibers in the extracellular matrix (ECM) are hallmarks of many invasive diseases, including cancer. Targeting specific stages of collagen assembly in vivo presents a great challenge due to the involvement of various crosslinking enzymes in the multistep, hierarchical process of ECM build-up. Using advanced microscopic tools, we monitored stages of fibrillary collagen assembly in a native fibroblast-derived 3D matrix system and identified anti-lysyl oxidase-like 2 (LOXL2) antibodies that alter the natural alignment and width of endogenic fibrillary collagens without affecting ECM composition. The disrupted collagen morphologies interfered with the adhesion and invasion properties of human breast cancer cells. Treatment of mice bearing breast cancer xenografts with the inhibitory antibodies resulted in disruption of the tumorigenic collagen superstructure and in reduction of primary tumor growth. Our approach could serve as a general methodology to identify novel therapeutics targeting fibrillary protein organization to treat ECM-associated pathologies. Cancer Res; 76(14); 4249-58. ©2016 AACR. ©2016 American Association for Cancer Research.

Functional analysis of histone deacetylase and its role in stress response, drug resistance and solid-state cultivation in Aspergillus oryzae.

PubMed

Kawauchi, Moriyuki; Iwashita, Kazuhiro

2014-08-01

In the eukaryotic cell, histone deacetylases (HDACs) play key roles in the regulation of fundamental cellular process such as development regulation, stress response, secondary metabolism and genome integrity. Here, we provide a comprehensive phenotypic analysis using HDAC disruptants in Aspergillus oryzae. Our study revealed that four HDACs, hdaA/Aohda1, hdaB/Aorpd3, hdaD/Aohos2 and hst4/AohstD were involved in stress response, cell wall synthesis and chromatin integrity in A. oryzae. Osmotic stress sensitivity of HDAC disruptants differed between plate cultures and liquid cultures, suggesting that HDACs adapt to the difference environmental conditions. Using a common A. oryzae fermentation medium, rice-koji, we also characterized HDACs related to growth and enzyme production to investigate which HDACs will be required for adaptation to environmental conditions and stress resistances. Because HDACs are widely conserved, our study has broad applications and may inform work with filamentous fungi and other eukaryote. Copyright © 2014 The Society for Biotechnology, Japan. Published by Elsevier B.V. All rights reserved.

Extraction of Fucoxanthin from Raw Macroalgae excluding Drying and Cell Wall Disruption by Liquefied Dimethyl Ether

PubMed Central

Kanda, Hideki; Kamo, Yuichi; Machmudah, Siti; Wahyudiono; Goto, Motonobu

2014-01-01

Macroalgae are one of potential sources for carotenoids, such as fucoxanthin, which are consumed by humans and animals. This carotenoid has been applied in both the pharmaceutical and food industries. In this study, extraction of fucoxanthin from wet brown seaweed Undaria pinnatifida (water content was 93.2%) was carried out with a simple method using liquefied dimethyl ether (DME) as an extractant in semi-continuous flow-type system. The extraction temperature and absolute pressure were 25 °C and 0.59 MPa, respectively. The liquefied DME was passed through the extractor that filled by U. pinnatifida at different time intervals. The time of experiment was only 43 min. The amount of fucoxanthin could approach to 390 μg/g dry of wet U. pinnatifida when the amount of DME used was 286 g. Compared with ethanol Soxhlet and supercritical CO2 extraction, which includes drying and cell disruption, the result was quite high. Thus, DME extraction process appears to be a good method for fucoxanthin recovery from U. pinnatifida with improved yields. PMID:24796299

Cadmium-induced apoptosis of Siberian tiger fibroblasts via disrupted intracellular homeostasis.

PubMed

Wang, Hui; Liu, Zheng; Zhang, Wenxiu; Yuan, Ziao; Yuan, Hongyi; Liu, Xueting; Yang, Chunwen; Guan, Weijun

2016-10-24

Heavy metals can cause great harm to Siberian tigers in the natural environment. Cadmium (Cd 2+ ) is an environmental contaminant that affects multiple cellular processes, including cell proliferation, differentiation, and survival. It has been shown to induce apoptosis in a variety of cell types and tissues. We investigated the apoptotic effects of Cd 2+ on Siberian tiger fibroblasts in vitro. Our research revealed the typical signs of apoptosis after Cd 2+ exposure. Apoptosis was dose- (0-4.8 μM) and duration-dependent (12-48 h), and proliferation was strongly inhibited. Cd 2+ increased the activity of caspase-3, -8, and -9 and disrupted calcium homeostasis by causing oxidative stress and mitochondrial dysfunction. It also increased K + efflux and altered the mRNA levels of Bax, Bcl-2, caspase-3, caspase-8, Fas, and p53. Our results suggest that Cd 2+ triggers the apoptosis of Siberian tiger fibroblasts by disturbing intracellular homeostasis. These results will aid in our understanding of the effects of Cd 2+ on Siberian tigers and in developing interventions to treat and prevent cadmium poisoning.

The Cytoplasmic Zinc Finger Protein ZPR1 Accumulates in the Nucleolus of Proliferating Cells

PubMed Central

Galcheva-Gargova, Zoya; Gangwani, Laxman; Konstantinov, Konstantin N.; Mikrut, Monique; Theroux, Steven J.; Enoch, Tamar; Davis, Roger J.

1998-01-01

The zinc finger protein ZPR1 translocates from the cytoplasm to the nucleus after treatment of cells with mitogens. The function of nuclear ZPR1 has not been defined. Here we demonstrate that ZPR1 accumulates in the nucleolus of proliferating cells. The role of ZPR1 was examined using a gene disruption strategy. Cells lacking ZPR1 are not viable. Biochemical analysis demonstrated that the loss of ZPR1 caused disruption of nucleolar function, including preribosomal RNA expression. These data establish ZPR1 as an essential protein that is required for normal nucleolar function in proliferating cells. PMID:9763455

Expression Pattern of the Pro-apoptotic Gene PAR-4 During the Morphogenesis of MCF-10A Human Mammary Epithelial Cells.

PubMed

de Bessa Garcia, Simone A; Pereira, Michelly C; Nagai, Maria A

2010-12-21

The histological organization of the mammary gland involves a spatial interaction of epithelial and myoepithelial cells with the specialized basement membrane (BM), composed of extra-cellular matrix (ECM) proteins, which is disrupted during the tumorigenic process. The interactions between mammary epithelial cells and ECM components play a major role in mammary gland branching morphogenesis. Critical signals for mammary epithelial cell proliferation, differentiation, and survival are provided by the ECM proteins. Three-dimensional (3D) cell culture was developed to establish a system that simulates several features of the breast epithelium in vivo; 3D cell culture of the spontaneously immortalized cell line, MCF10A, is a well-established model system to study breast epithelial cell biology and morphogenesis. Mammary epithelial cells grown in 3D form spheroids, acquire apicobasal polarization, and form lumens that resemble acini structures, processes that involve cell death. Using this system, we evaluated the expression of the pro-apoptotic gene PAWR (PKC apoptosis WT1 regulator; also named PAR-4, prostate apoptosis response-4) by immunofluorescence and quantitative real time PCR (qPCR). A time-dependent increase in PAR-4 mRNA expression was found during the process of MCF10A acinar morphogenesis. Confocal microscopy analysis also showed that PAR-4 protein was highly expressed in the MCF10A cells inside the acini structure. During the morphogenesis of MCF10A cells in 3D cell culture, the cells within the lumen showed caspase-3 activation, indicating apoptotic activity. PAR-4 was only partially co-expressed with activated caspase-3 on these cells. Our results provide evidence, for the first time, that PAR-4 is differentially expressed during the process of MCF10A acinar morphogenesis.

Mechanisms of lung endothelial barrier disruption induced by cigarette smoke: role of oxidative stress and ceramides.

PubMed

Schweitzer, Kelly S; Hatoum, Hadi; Brown, Mary Beth; Gupta, Mehak; Justice, Matthew J; Beteck, Besem; Van Demark, Mary; Gu, Yuan; Presson, Robert G; Hubbard, Walter C; Petrache, Irina

2011-12-01

The epithelial and endothelial cells lining the alveolus form a barrier essential for the preservation of the lung respiratory function, which is, however, vulnerable to excessive oxidative, inflammatory, and apoptotic insults. Whereas profound breaches in this barrier function cause pulmonary edema, more subtle changes may contribute to inflammation. The mechanisms by which cigarette smoke (CS) exposure induce lung inflammation are not fully understood, but an early alteration in the epithelial barrier function has been documented. We sought to investigate the occurrence and mechanisms by which soluble components of mainstream CS disrupt the lung endothelial cell barrier function. Using cultured primary rat microvascular cell monolayers, we report that CS induces endothelial cell barrier disruption in a dose- and time-dependent manner of similar magnitude to that of the epithelial cell barrier. CS exposure triggered a mechanism of neutral sphingomyelinase-mediated ceramide upregulation and p38 MAPK and JNK activation that were oxidative stress dependent and that, along with Rho kinase activation, mediated the endothelial barrier dysfunction. The morphological changes in endothelial cell monolayers induced by CS included actin cytoskeletal rearrangement, junctional protein zonula occludens-1 loss, and intercellular gap formation, which were abolished by the glutathione modulator N-acetylcysteine and ameliorated by neutral sphingomyelinase inhibition. The direct application of ceramide recapitulated the effects of CS, by disrupting both endothelial and epithelial cells barrier, by a mechanism that was redox and apoptosis independent and required Rho kinase activation. Furthermore, ceramide induced dose-dependent alterations of alveolar microcirculatory barrier in vivo, measured by two-photon excitation microscopy in the intact rat. In conclusion, soluble components of CS have direct endothelial barrier-disruptive effects that could be ameliorated by glutathione modulators or by inhibitors of neutral sphingomyelinase, p38 MAPK, JNK, and Rho kinase. Amelioration of endothelial permeability may alleviate lung and systemic vascular dysfunction associated with smoking-related chronic obstructive lung diseases.

Rescue of perfluorooctanesulfonate (PFOS)-mediated Sertoli cell injury by overexpression of gap junction protein connexin 43

PubMed Central

Li, Nan; Mruk, Dolores D.; Chen, Haiqi; Wong, Chris K. C.; Lee, Will M.; Cheng, C. Yan

2016-01-01

Perfluorooctanesulfonate (PFOS) is an environmental toxicant used in developing countries, including China, as a stain repellent for clothing, carpets and draperies, but it has been banned in the U.S. and Canada since the late 2000s. PFOS perturbed the Sertoli cell tight junction (TJ)-permeability barrier, causing disruption of actin microfilaments in cell cytosol, perturbing the localization of cell junction proteins (e.g., occluden-ZO-1, N-cadherin-ß-catenin). These changes destabilized Sertoli cell blood-testis barrier (BTB) integrity. These findings suggest that human exposure to PFOS might induce BTB dysfunction and infertility. Interestingly, PFOS-induced Sertoli cell injury associated with a down-regulation of the gap junction (GJ) protein connexin43 (Cx43). We next investigated if overexpression of Cx43 in Sertoli cells could rescue the PFOS-induced cell injury. Indeed, overexpression of Cx43 in Sertoli cells with an established TJ-barrier blocked the disruption in PFOS-induced GJ-intercellular communication, resulting in the re-organization of actin microfilaments, which rendered them similar to those in control cells. Furthermore, cell adhesion proteins that utilized F-actin for attachment became properly distributed at the cell-cell interface, resealing the disrupted TJ-barrier. In summary, Cx43 is a good target that might be used to manage PFOS-induced reproductive dysfunction. PMID:27436542

The role of retinoic acid receptors and their cognate ligands in reproduction in a context of triorganotin based endocrine disrupting chemicals.

PubMed

Macejova, Dana; Toporova, L; Brtko, J

2016-07-01

Retinoic acid (RA), an active form of vitamin A, regulates the embryonic development, male and female reproduction and induces important effects on the cell development, proliferation, and differentiation. These effects are mediated by the retinoid (RAR) and rexinoid nuclear receptors (RXR), which are considered to be a ligand-activated, DNA-binding, trans-acting, and transcription-modulating proteins, involved in a general molecular mechanism responsible for the transcriptional responses in target genes. Organotin compounds are typical environmental contaminants and suspected endocrine disrupting substances. They may affect processes of reproductive system in mammals, predominantly via nuclear receptor signaling pathways. Triorganotins, such as tributyltin chloride (TBTCl) and triphenyltin chloride (TPTCl), are capable to bind to RXR molecules, and thus represent potent agonists of RXR subtypes of nuclear receptors not sharing any structural characteristics with endogenous ligands of nuclear receptors. Th is article summarizes selected effects of biologically active retinoids and rexinoids on both male and female reproduction and also deals with the effects of organotin compounds evoking endocrine disrupting actions in reproduction.

Disruptive chemicals, senescence and immortality

PubMed Central

Carnero, Amancio; Blanco-Aparicio, Carmen; Kondoh, Hiroshi; Lleonart, Matilde E.; Martinez-Leal, Juan Fernando; Mondello, Chiara; Ivana Scovassi, A.; Bisson, William H.; Amedei, Amedeo; Roy, Rabindra; Woodrick, Jordan; Colacci, Annamaria; Vaccari, Monica; Raju, Jayadev; Al-Mulla, Fahd; Al-Temaimi, Rabeah; Salem, Hosni K.; Memeo, Lorenzo; Forte, Stefano; Singh, Neetu; Hamid, Roslida A.; Ryan, Elizabeth P.; Brown, Dustin G.; Wise, John Pierce; Wise, Sandra S.; Yasaei, Hemad

2015-01-01

Carcinogenesis is thought to be a multistep process, with clonal evolution playing a central role in the process. Clonal evolution involves the repeated ‘selection and succession’ of rare variant cells that acquire a growth advantage over the remaining cell population through the acquisition of ‘driver mutations’ enabling a selective advantage in a particular micro-environment. Clonal selection is the driving force behind tumorigenesis and possesses three basic requirements: (i) effective competitive proliferation of the variant clone when compared with its neighboring cells, (ii) acquisition of an indefinite capacity for self-renewal, and (iii) establishment of sufficiently high levels of genetic and epigenetic variability to permit the emergence of rare variants. However, several questions regarding the process of clonal evolution remain. Which cellular processes initiate carcinogenesis in the first place? To what extent are environmental carcinogens responsible for the initiation of clonal evolution? What are the roles of genotoxic and non-genotoxic carcinogens in carcinogenesis? What are the underlying mechanisms responsible for chemical carcinogen-induced cellular immortality? Here, we explore the possible mechanisms of cellular immortalization, the contribution of immortalization to tumorigenesis and the mechanisms by which chemical carcinogens may contribute to these processes. PMID:26106138

Re-engineering the Pancreas Tumor Microenvironment: A "Regenerative Program" Hacked.

PubMed

Evan, Gerard I; Hah, Nasun; Littlewood, Trevor D; Sodir, Nicole M; Campos, Tania; Downes, Michael; Evans, Ronald M

2017-04-01

The "hallmarks" of pancreatic ductal adenocarcinoma (PDAC) include proliferative, invasive, and metastatic tumor cells and an associated dense desmoplasia comprised of fibroblasts, pancreatic stellate cells, extracellular matrix, and immune cells. The oncogenically activated pancreatic epithelium and its associated stroma are obligatorily interdependent, with the resulting inflammatory and immunosuppressive microenvironment contributing greatly to the evolution and maintenance of PDAC. The peculiar pancreas-specific tumor phenotype is a consequence of oncogenes hacking the resident pancreas regenerative program, a tissue-specific repair mechanism regulated by discrete super enhancer networks. Defined as genomic regions containing clusters of multiple enhancers, super enhancers play pivotal roles in cell/tissue specification, identity, and maintenance. Hence, interfering with such super enhancer-driven repair networks should exert a disproportionately disruptive effect on tumor versus normal pancreatic tissue. Novel drugs that directly or indirectly inhibit processes regulating epigenetic status and integrity, including those driven by histone deacetylases, histone methyltransferase and hydroxylases, DNA methyltransferases, various metabolic enzymes, and bromodomain and extraterminal motif proteins, have shown the feasibility of disrupting super enhancer-dependent transcription in treating multiple tumor types, including PDAC. The idea that pancreatic adenocarcinomas rely on embedded super enhancer transcriptional mechanisms suggests a vulnerability that can be potentially targeted as novel therapies for this intractable disease. Clin Cancer Res; 23(7); 1647-55. ©2017 AACR See all articles in this CCR Focus section, "Pancreatic Cancer: Challenge and Inspiration." ©2017 American Association for Cancer Research.

Automated cell disruption is a reliable and effective method of isolating RNA from fresh snap-frozen normal and malignant oral mucosa samples.

PubMed

Van der Vorst, Sébastien; Dekairelle, Anne-France; Irenge, Léonid; Hamoir, Marc; Robert, Annie; Gala, Jean-Luc

2009-01-01

This study compared automated vs. manual tissue grinding in terms of RNA yield obtained from oral mucosa biopsies. A total of 20 patients undergoing uvulectomy for sleep-related disorders and 10 patients undergoing biopsy for head and neck squamous cell carcinoma were enrolled in the study. Samples were collected, snap-frozen in liquid nitrogen, and divided into two parts of similar weight. Sample grinding was performed on one sample from each pair, either manually or using an automated cell disruptor. The performance and efficacy of each homogenization approach was compared in terms of total RNA yield (spectrophotometry, fluorometry), mRNA quantity [densitometry of specific TP53 amplicons and TP53 quantitative reverse-transcribed real-time PCR (qRT-PCR)], and mRNA quality (functional analysis of separated alleles in yeast). Although spectrophotometry and fluorometry results were comparable for both homogenization methods, TP53 expression values obtained by amplicon densitometry and qRT-PCR were significantly and consistently better after automated homogenization (p<0.005) for both uvula and tumor samples. Functional analysis of separated alleles in yeast results was better with the automated technique for tumor samples. Automated tissue homogenization appears to be a versatile, quick, and reliable method of cell disruption and is especially useful in the case of small malignant samples, which show unreliable results when processed by manual homogenization.

Imaging of activated caspase-3 in living cell by fluorescence resonance energy transfer during photosensitization-induced apoptosis

NASA Astrophysics Data System (ADS)

Wu, Yunxia; Xing, Da; Chen, Qun; Tang, Yonghong

2005-01-01

Photodynamic therapy (PDT) is a novel and promising cancer treatment that employs a combination of a photosensitizing chemical and visible light, induces apoptosis in cell, and activation of caspase-3 is considered to be the final step in many apoptosis pathways. The changes of caspase-3 activation in cell during TNFα- and photodynamic therapy-induced apoptosis was measured by fluorescence resonance energy transfer (FRET) analysis. FRET probe consisting of fusions of an enhanced cyan fluorescent protein (ECFP), Venus and a linker peptide containing the caspase-3 cleavage sequence DEVD was utilized. Therefore, activated caspase-3 cleaved the linker peptide of FRET probe and disrupted the FRET signal. Human lung adenocarcinoma cell line (ASTC-a-1) were stably transfected with the plasmid (ECFP-DEVD-Venus) and then were treated by TNF-α and PDT, respectively. Experimental results indicated that caspase-3 activation resulted in cleavage of linker peptide and subsequent disruption of the FRET signal during TNFα- and photodynamic therapy-induced apoptosis, and that the activation of caspase-3 induced by photodynamic therapy was faster than that induce by TNF-α. The study supports that using FRET technique and different recombinant substrates as FRET probes could be used to detect the process of PDT-induced apoptosis and provide a new means to investigate apoptotic mechanism of PDT.

Intestine-specific Disruption of Hypoxia-inducible Factor (HIF)-2α Improves Anemia in Sickle Cell Disease.

PubMed

Das, Nupur; Xie, Liwei; Ramakrishnan, Sadeesh K; Campbell, Andrew; Rivella, Stefano; Shah, Yatrik M

2015-09-25

Sickle cell disease (SCD) is caused by genetic defects in the β-globin chain. SCD is a frequently inherited blood disorder, and sickle cell anemia is a common type of hemoglobinopathy. During anemia, the hypoxic response via the transcription factor hypoxia-inducible factor (HIF)-2α is highly activated in the intestine and is essential in iron absorption. Intestinal disruption of HIF-2α protects against tissue iron accumulation in iron overload anemias. However, the role of intestinal HIF-2α in regulating anemia in SCD is currently not known. Here we show that in mouse models of SCD, disruption of intestinal HIF-2α significantly decreased tissue iron accumulation. This was attributed to a decrease in intestinal iron absorptive genes, which were highly induced in a mouse model of SCD. Interestingly, disruption of intestinal HIF-2α led to a robust improvement in anemia with an increase in RBC, hemoglobin, and hematocrit. This was attributed to improvement in RBC survival, hemolysis, and insufficient erythropoiesis, which is evident from a significant decrease in serum bilirubin, reticulocyte counts, and serum erythropoietin following intestinal HIF-2α disruption. These data suggest that targeting intestinal HIF-2α has a significant therapeutic potential in SCD pathophysiology. © 2015 by The American Society for Biochemistry and Molecular Biology, Inc.

Aspirin suppresses the abnormal lipid metabolism in liver cancer cells via disrupting an NFκB-ACSL1 signaling

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

Yang, Guang; Wang, Yuan; Feng, Jinyan

Abnormal lipid metabolism is a hallmark of tumorigenesis. Hence, the alterations of metabolism enhance the development of hepatocellular carcinoma (HCC). Aspirin is able to inhibit the growth of cancers through targeting nuclear factor κB (NF-κB). However, the role of aspirin in disrupting abnormal lipid metabolism in HCC remains poorly understood. In this study, we report that aspirin can suppress the abnormal lipid metabolism of HCC cells through inhibiting acyl-CoA synthetase long-chain family member 1 (ACSL1), a lipid metabolism-related enzyme. Interestingly, oil red O staining showed that aspirin suppressed lipogenesis in HepG2 cells and Huh7 cells in a dose-dependent manner. Inmore » addition, aspirin attenuated the levels of triglyceride and cholesterol in the cells, respectively. Strikingly, we identified that aspirin was able to down-regulate ACSL1 at the levels of mRNA and protein. Moreover, we validated that aspirin decreased the nuclear levels of NF-κB in HepG2 cells. Mechanically, PDTC, an inhibitor of NF-κB, could down-regulate ACSL1 at the levels of mRNA and protein in the cells. Functionally, PDTC reduced the levels of lipid droplets, triglyceride and cholesterol in HepG2 cells. Thus, we conclude that aspirin suppresses the abnormal lipid metabolism in HCC cells via disrupting an NFκB-ACSL1 signaling. Our finding provides new insights into the mechanism by which aspirin inhibits abnormal lipid metabolism of HCC. Therapeutically, aspirin is potentially available for HCC through controlling abnormal lipid metabolism. - Highlights: • Aspirin inhibits the levels of liquid droplets, triglyceride and cholesterol in HCC cells. • Aspirin is able to down-regulate ACSL1 in HCC cells. • NF-κB inhibitor PDTC can down-regulate ACSL1 and reduces lipogenesis in HCC cells. • Aspirin suppresses the abnormal lipid metabolism in HCC cells via disrupting an NFκB-ACSL1 signaling.« less

A Rat α-Fetoprotein Binding Activity Prediction Model to Facilitate Assessment of the Endocrine Disruption Potential of Environmental Chemicals.

PubMed

Hong, Huixiao; Shen, Jie; Ng, Hui Wen; Sakkiah, Sugunadevi; Ye, Hao; Ge, Weigong; Gong, Ping; Xiao, Wenming; Tong, Weida

2016-03-25

Endocrine disruptors such as polychlorinated biphenyls (PCBs), diethylstilbestrol (DES) and dichlorodiphenyltrichloroethane (DDT) are agents that interfere with the endocrine system and cause adverse health effects. Huge public health concern about endocrine disruptors has arisen. One of the mechanisms of endocrine disruption is through binding of endocrine disruptors with the hormone receptors in the target cells. Entrance of endocrine disruptors into target cells is the precondition of endocrine disruption. The binding capability of a chemical with proteins in the blood affects its entrance into the target cells and, thus, is very informative for the assessment of potential endocrine disruption of chemicals. α-fetoprotein is one of the major serum proteins that binds to a variety of chemicals such as estrogens. To better facilitate assessment of endocrine disruption of environmental chemicals, we developed a model for α-fetoprotein binding activity prediction using the novel pattern recognition method (Decision Forest) and the molecular descriptors calculated from two-dimensional structures by Mold² software. The predictive capability of the model has been evaluated through internal validation using 125 training chemicals (average balanced accuracy of 69%) and external validations using 22 chemicals (balanced accuracy of 71%). Prediction confidence analysis revealed the model performed much better at high prediction confidence. Our results indicate that the model is useful (when predictions are in high confidence) in endocrine disruption risk assessment of environmental chemicals though improvement by increasing number of training chemicals is needed.

Pathophysiological consequences of VEGF-induced vascular permeability

NASA Astrophysics Data System (ADS)

Weis, Sara M.; Cheresh, David A.

2005-09-01

Although vascular endothelial growth factor (VEGF) induces angiogenesis, it also disrupts vascular barrier function in diseased tissues. Accordingly, VEGF expression in cancer and ischaemic disease has unexpected pathophysiological consequences. By uncoupling endothelial cell-cell junctions VEGF causes vascular permeability and oedema, resulting in extensive injury to ischaemic tissues after stroke or myocardial infarction. In cancer, VEGF-mediated disruption of the vascular barrier may potentiate tumour cell extravasation, leading to widespread metastatic disease. Therefore, by blocking the vascular permeability promoting effects of VEGF it may be feasible to reduce tissue injury after ischaemic disease and minimize the invasive properties of circulating tumour cells.

Generating hypoimmunogenic human embryonic stem cells by the disruption of beta 2-microglobulin.

PubMed

Lu, Pengfei; Chen, Jijun; He, Lixiazi; Ren, Jiangtao; Chen, Haide; Rao, Lingjun; Zhuang, Qinggang; Li, Hui; Li, Lei; Bao, Lei; He, Ji; Zhang, Wei; Zhu, Faming; Cui, Chun; Xiao, Lei

2013-12-01

Immune rejection hinders the application of human embryonic stem cells (hESCs) in transplantation therapy. Human leukocyte antigens (HLAs) on the cell surface are the major cause of graft rejection. In this study, we generated HLA class I-deficient hESCs via disruption of beta 2-microglobulin (β2m), the light chain of HLA Class I. We found that HLA class I proteins were not present on the cell surface of β2m-null hESCs. These cells showed the same pluripotency as wildtype hESCs and demonstrated hypoimmunogenicity. Thus, HLA class I-deficient hESCs might serve as an unlimited cell source for the generation of universally compatible "off-the-shelf" cell grafts, tissues or organs in the future.

Lung Regeneration: Endogenous and Exogenous Stem Cell Mediated Therapeutic Approaches.

PubMed

Akram, Khondoker M; Patel, Neil; Spiteri, Monica A; Forsyth, Nicholas R

2016-01-19

The tissue turnover of unperturbed adult lung is remarkably slow. However, after injury or insult, a specialised group of facultative lung progenitors become activated to replenish damaged tissue through a reparative process called regeneration. Disruption in this process results in healing by fibrosis causing aberrant lung remodelling and organ dysfunction. Post-insult failure of regeneration leads to various incurable lung diseases including chronic obstructive pulmonary disease (COPD) and idiopathic pulmonary fibrosis. Therefore, identification of true endogenous lung progenitors/stem cells, and their regenerative pathway are crucial for next-generation therapeutic development. Recent studies provide exciting and novel insights into postnatal lung development and post-injury lung regeneration by native lung progenitors. Furthermore, exogenous application of bone marrow stem cells, embryonic stem cells and inducible pluripotent stem cells (iPSC) show evidences of their regenerative capacity in the repair of injured and diseased lungs. With the advent of modern tissue engineering techniques, whole lung regeneration in the lab using de-cellularised tissue scaffold and stem cells is now becoming reality. In this review, we will highlight the advancement of our understanding in lung regeneration and development of stem cell mediated therapeutic strategies in combating incurable lung diseases.

Erythroid cells in vitro: from developmental biology to blood transfusion products.

PubMed

Migliaccio, Anna Rita; Whitsett, Carolyn; Migliaccio, Giovanni

2009-07-01

Red blood cells (RBCs) transfusion plays a critical role in numerous therapies. Disruption of blood collection by political unrest, natural disasters and emerging infections and implementation of restrictions on the use of erythropoiesis-stimulating agents in cancer may impact blood availability in the near future. These considerations highlight the importance of developing alternative blood products. Knowledge about the processes that control RBC production has been applied to the establishment of culture conditions allowing ex-vivo generation of RBCs in numbers close to those (2.5 x 10 cells/ml) present in a transfusion, from cord blood, donated blood units or embryonic stem cells. In addition, experimental studies demonstrate that such cells protect mice from lethal bleeding. Therefore, erythroid cells generated ex vivo may be suitable for transfusion provided they can be produced safely in adequate numbers. However, much remains to be done to translate a theoretical production of approximately 2.5 x 10 RBCs in the laboratory into a 'clinical grade production process'. This review summarizes the state-of-the-art in establishing ex-vivo culture conditions for erythroid cells and discusses the most compelling issues to be addressed to translate this progress into a clinical grade transfusion product.

The desmoplakin–intermediate filament linkage regulates cell mechanics

PubMed Central

Broussard, Joshua A.; Yang, Ruiguo; Huang, Changjin; Nathamgari, S. Shiva P.; Beese, Allison M.; Godsel, Lisa M.; Hegazy, Marihan H.; Lee, Sherry; Zhou, Fan; Sniadecki, Nathan J.; Green, Kathleen J.; Espinosa, Horacio D.

2017-01-01

The translation of mechanical forces into biochemical signals plays a central role in guiding normal physiological processes during tissue development and homeostasis. Interfering with this process contributes to cardiovascular disease, cancer progression, and inherited disorders. The actin-based cytoskeleton and its associated adherens junctions are well-established contributors to mechanosensing and transduction machinery; however, the role of the desmosome–intermediate filament (DSM–IF) network is poorly understood in this context. Because a force balance among different cytoskeletal systems is important to maintain normal tissue function, knowing the relative contributions of these structurally integrated systems to cell mechanics is critical. Here we modulated the interaction between DSMs and IFs using mutant forms of desmoplakin, the protein bridging these structures. Using micropillar arrays and atomic force microscopy, we demonstrate that strengthening the DSM–IF interaction increases cell–substrate and cell–cell forces and cell stiffness both in cell pairs and sheets of cells. In contrast, disrupting the interaction leads to a decrease in these forces. These alterations in cell mechanics are abrogated when the actin cytoskeleton is dismantled. These data suggest that the tissue-specific variability in DSM–IF network composition provides an opportunity to differentially regulate tissue mechanics by balancing and tuning forces among cytoskeletal systems. PMID:28495795

Hydroxychloroquine protects the annexin A5 anticoagulant shield from disruption by antiphospholipid antibodies: evidence for a novel effect for an old antimalarial drug.

PubMed

Rand, Jacob H; Wu, Xiao-Xuan; Quinn, Anthony S; Ashton, Anthony W; Chen, Pojen P; Hathcock, James J; Andree, Harry A M; Taatjes, Douglas J

2010-03-18

Annexin A5 (AnxA5) is a potent anticoagulant protein that crystallizes over phospholipid bilayers (PLBs), blocking their availability for coagulation reactions. Antiphospholipid antibodies disrupt AnxA5 binding, thereby accelerating coagulation reactions. This disruption may contribute to thrombosis and miscarriages in the antiphospholipid syndrome (APS). We investigated whether the antimalarial drug, hydroxychloroquine (HCQ), might affect this prothrombotic mechanism. Binding of AnxA5 to PLBs was measured with labeled AnxA5 and also imaged with atomic force microscopy. Immunoglobulin G levels, AnxA5, and plasma coagulation times were measured on cultured human umbilical vein endothelial cells and a syncytialized trophoblast cell line. AnxA5 anticoagulant activities of APS patient plasmas were also determined. HCQ reversed the effect of antiphospholipid antibodies on AnxA5 and restored AnxA5 binding to PLBs, an effect corroborated by atomic force microscopy. Similar reversals of antiphospholipid-induced abnormalities were measured on the surfaces of human umbilical vein endothelial cells and syncytialized trophoblast cell lines, wherein HCQ reduced the binding of antiphospholipid antibodies, increased cell-surface AnxA5 concentrations, and prolonged plasma coagulation to control levels. In addition, HCQ increased the AnxA5 anticoagulant activities of APS patient plasmas. In conclusion, HCQ reversed antiphospholipid-mediated disruptions of AnxA5 on PLBs and cultured cells, and in APS patient plasmas. These results support the concept of novel therapeutic approaches that address specific APS disease mechanisms.

Ionizing radiation induces heritable disruption of epithelial cell interactions

NASA Technical Reports Server (NTRS)

Park, Catherine C.; Henshall-Powell, Rhonda L.; Erickson, Anna C.; Talhouk, Rabih; Parvin, Bahram; Bissell, Mina J.; Barcellos-Hoff, Mary Helen; Chatterjee, A. (Principal Investigator)

2003-01-01

Ionizing radiation (IR) is a known human breast carcinogen. Although the mutagenic capacity of IR is widely acknowledged as the basis for its action as a carcinogen, we and others have shown that IR can also induce growth factors and extracellular matrix remodeling. As a consequence, we have proposed that an additional factor contributing to IR carcinogenesis is the potential disruption of critical constraints that are imposed by normal cell interactions. To test this hypothesis, we asked whether IR affected the ability of nonmalignant human mammary epithelial cells (HMEC) to undergo tissue-specific morphogenesis in culture by using confocal microscopy and imaging bioinformatics. We found that irradiated single HMEC gave rise to colonies exhibiting decreased localization of E-cadherin, beta-catenin, and connexin-43, proteins necessary for the establishment of polarity and communication. Severely compromised acinar organization was manifested by the majority of irradiated HMEC progeny as quantified by image analysis. Disrupted cell-cell communication, aberrant cell-extracellular matrix interactions, and loss of tissue-specific architecture observed in the daughters of irradiated HMEC are characteristic of neoplastic progression. These data point to a heritable, nonmutational mechanism whereby IR compromises cell polarity and multicellular organization.

The cis decoy against the estrogen response element suppresses breast cancer cells via target disrupting c-fos not mitogen-activated protein kinase activity.

PubMed

Wang, Li Hua; Yang, Xiao Yi; Zhang, Xiaohu; Mihalic, Kelly; Xiao, Weihua; Farrar, William L

2003-05-01

Breast cancer, the most common malignancy in women, has been demonstrated to be associated with the steroid hormone estrogen and its receptor (ER), a ligand-activated transcription factor. Therefore, we developed a phosphorothiolate cis-element decoy against the estrogen response element (ERE decoy) to target disruption of ER DNA binding and transcriptional activity. Here, we showed that the ERE decoy potently ablated the 17beta-estrogen-inducible cell proliferation and induced apoptosis of human breast carcinoma cells by functionally affecting expression of c-fos gene and AP-1 luciferase gene reporter activity. Specificity of the decoy was demonstrated by its ability to directly block ER binding to a cis-element probe and transactivation. Moreover, the decoy failed to inhibit ER-mediated mitogen-activated protein kinase signaling pathways and cell growth of ER-negative breast cancer cells. Taken together, these data suggest that estrogen-mediated cell growth of breast cancer cells can be preferentially restricted via targeted disruption of ER at the level of DNA binding by a novel and specific decoy strategy applied to steroid nuclear receptors.

Estrogen-related receptor gamma disruption of source water and drinking water treatment processes extracts.

PubMed

Li, Na; Jiang, Weiwei; Rao, Kaifeng; Ma, Mei; Wang, Zijian; Kumaran, Satyanarayanan Senthik

2011-01-01

Environmental chemicals in drinking water can impact human health through nuclear receptors. Additionally, estrogen-related receptors (ERRs) are vulnerable to endocrine-disrupting effects. To date, however, ERR disruption of drinking water potency has not been reported. We used ERRgamma two-hybrid yeast assay to screen ERRgamma disrupting activities in a drinking water treatment plant (DWTP) located in north China and in source water from a reservoir, focusing on agonistic, antagonistic, and inverse agonistic activity to 4-hydroxytamoxifen (4-OHT). Water treatment processes in the DWTP consisted of pre-chlorination, coagulation, coal and sand filtration, activated carbon filtration, and secondary chlorination processes. Samples were extracted by solid phase extraction. Results showed that ERRgamma antagonistic activities were found in all sample extracts, but agonistic and inverse agonistic activity to 4-OHT was not found. When calibrated with the toxic equivalent of 4-OHT, antagonistic effluent effects ranged from 3.4 to 33.1 microg/L. In the treatment processes, secondary chlorination was effective in removing ERRgamma antagonists, but the coagulation process led to significantly increased ERRgamma antagonistic activity. The drinking water treatment processes removed 73.5% of ERRgamma antagonists. To our knowledge, the occurrence of ERRgamma disruption activities on source and drinking water in vitro had not been reported previously. It is vital, therefore, to increase our understanding of ERRy disrupting activities in drinking water.

Di-(2-ethylhexyl) phthalate could disrupt the insulin signaling pathway in liver of SD rats and L02 cells via PPARγ

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

Zhang, Wang; Shen, Xin-Yue; Zhang, Wen-wen

Di-(2-ethylhexyl)-phthalate (DEHP), a ubiquitous industrial pollutant in our daily life, has been reported to cause adverse effects on glucose homeostasis and insulin sensitivity in epidemiological studies previously. Recently, it has been reported to be an endocrine disrupter and ligand to peroxisome proliferator activated receptor, which could influence the homeostasis of liver metabolic systems and contribute to the development of type-2 diabetes. However, the potential mechanisms are not known yet. This study was designed to solve these problems with male SD rats and normal human hepatocyte line, L02 cells, exposed to DEHP for toxicological experiments. Adult male SD rats were dividedmore » into four groups, normal group fed with regular diets and three DEHP-treated groups (dissolved in olive oil at doses of 0.05, 5 and 500 mg/kg body weight, respectively, once daily through gastric intubations for 15 weeks). L02 cells were divided into 6 groups, normal group with 5, 10, 25, 50, and 100 μmol/l DEHP groups. DEHP-exposed rats exhibited significant liver damage, glucose tolerance, and insulin tolerance along with reduced expression of insulin receptor and GLUT4 proteins in the liver tissues. The results of in vitro experiments could determine that the DEHP-induced activation of peroxisome proliferator activated receptor γ (PPARγ) played a key role in the production of oxidative stress and down-regulated expression of insulin receptor and GLUT4 proteins in L02 cells. This conclusion could be supported by the results of in vitro experiments, in which the cells were exposed to DEHP with GW9662 (PPARγ inhibitor). In general, these results highlight the key role of PPARγ in the process of insulin resistance induced by DEHP. - Highlights: • DEHP exacerbates insulin resistance both in liver tissues and cells. • Expression of insulin receptor and GLUT4 were altered with PPARγ. • DEHP can induce oxidative stress to disrupt the metabolic homeostasis. • The dose of exposed DEHP is closed to daily exposure by human. • Determine the key role of PPARγ to insulin resistance.« less

Dexamethasone disrupts cytoskeleton organization and migration of T47D Human breast cancer cells by modulating the AKT/mTOR/RhoA pathway.

PubMed

Meng, Xian-Guo; Yue, Shou-Wei

2014-01-01

Glucocorticoids are commonly co-administered with chemotherapy to prevent drug-induced allergic reactions, nausea, and vomiting, and have anti-tumor functions clinically; however, the distinct effects of GC on subtypes of tumor cells, especially in breast cancer cells, are still not well understood. In this study, we aimed to clarify the effect of GC on subtypes of T47D breast cancer cells by focusing on apoptosis, cell organization and migration, and underluing molecular mechanisms. The cell scratch test was performed to observe the cell migration rate in T47D cells treated with dexamethasone (Dex). Hoechst and MTT assays were conducted to detect cell survival and rhodamine-labeled phalloidin staining to observe cytoskeleton dynamics. Related factors in the AKT/mTOR pathway were determined by Western blotting. Dex treatment could effectively inhibit T47D breast cancer cell migration with disruption of the cytoskeletal dynamic organization. Moreover, the effect of Dex on cell migration and cytoskeleton may be mediated by AKT/ mTOR/RhoA pathway. Although Dex inhibited T47D cell migration, it alone may not induce cell apoptosis in T47D cells. Dex in T47D human breast cancer cells could effectively inhibit cell migration by disrupting the cytoskeletal dynamic organization, which may be mediated by the AKT/mTOR/RhoA pathway. Our work suggests that glucocorticoid/Dex clinical use may prove helpful for the treatment of breast cancer metastasis.

Combretastatin A-4 derived 5-(1-methyl-4-phenyl-imidazol-5-yl)indoles with superior cytotoxic and anti-vascular effects on chemoresistant cancer cells and tumors.

PubMed

Mahal, Katharina; Biersack, Bernhard; Schruefer, Sebastian; Resch, Marcus; Ficner, Ralf; Schobert, Rainer; Mueller, Thomas

2016-08-08

5-(1-Methyl-4-phenyl-imidazol-5-yl)indoles 5 were prepared and tested as analogs of the natural vascular-disrupting agent combretastatin A-4 (CA-4). The 3-bromo-4,5-dimethoxyphenyl derivative 5c was far more active than CA-4 with low nanomolar IC50 concentrations against multidrug-resistant KB-V1/Vbl cervix and MCF-7/Topo mamma carcinoma cells, and also against CA-4-resistant HT-29 colon carcinoma cells. While not interfering markedly with the polymerization of tubulin in vitro, indole 5c completely disrupted the microtubule cytoskeleton of cancer cells at low concentrations. It also destroyed real blood vessels, both in the chorioallantoic membrane (CAM) of fertilized chicken eggs and within tumor xenografts in mice, without harming embryo or mouse, respectively. Indole 5c was less toxic than CA-4 to endothelial cells, fibroblasts, and cardiomyocytes. In highly vascularized xenograft tumors 5c induced distinct discolorations and histological features typical of vascular-disrupting agents, such as disrupted vessel structures, hemorrhages, and extensive necrosis. In a first preliminary therapy trial, indole 5c retarded the growth of resistant xenograft tumors in mice. © 2016 Elsevier Science Ltd. All rights reserved. Copyright © 2016 Elsevier Masson SAS. All rights reserved.

Mechanism of action of coumarin and silver(I)-coumarin complexes against the pathogenic yeast Candida albicans.

PubMed

Thati, Bhumika; Noble, Andy; Rowan, Raymond; Creaven, Bernadette S; Walsh, Maureen; McCann, Malachy; Egan, Denise; Kavanagh, Kevin

2007-08-01

The anti-fungal activity and mode of action of a range of silver(I)-coumarin complexes was examined. The most potent silver(I)-coumarin complexes, namely 7-hydroxycoumarin-3-carboxylatosilver(I), 6-hydroxycoumarin-3-carboxylatosilver(I) and 4-oxy-3-nitrocoumarinbis(1,10-phenanthroline)silver(I), had MIC80 values of between 69.1 and 4.6 microM against the pathogenic yeast Candida albicans. These compounds also reduced respiration, lowered the ergosterol content of cells and increased the trans-membrane leakage of amino acids. A number of the complexes disrupted cytochrome synthesis in the cell and induced the appearance of morphological features consistent with cell death by apoptosis. These compounds appear to act by disrupting the synthesis of cytochromes which directly affects the cell's ability to respire. A reduction in respiration leads to a depletion in ergosterol biosynthesis and a consequent disruption of the integrity of the cell membrane. Disruption of cytochrome biosynthesis may induce the onset of apoptosis which has been shown previously to be triggered by alteration in the location of cytochrome c. Silver(I)-coumarin complexes demonstrate good anti-fungal activity and manifest a mode of action distinct to that of the conventional azole and polyene drugs thus raising the possibility of their use when resistance to conventional drug has emerged or in combination with such drugs.

The antifungal activity and membrane-disruptive action of dioscin extracted from Dioscorea nipponica.

PubMed

Cho, Jaeyong; Choi, Hyemin; Lee, Juneyoung; Kim, Mi-Sun; Sohn, Ho-Yong; Lee, Dong Gun

2013-03-01

Dioscin is a kind of steroidal saponin isolated from the root bark of wild yam Dioscorea nipponica. We investigated the antifungal effect of dioscin against different fungal strains and its antifungal mechanism(s) in Candida albicans cells. Using the propidium iodide assay and calcein-leakage measurement, we confirmed that dioscin caused fungal membrane damage. Furthermore, we evaluated the ability of dioscin to disrupt the plasma membrane potential, using 3,3'-dipropylthiadicarbocyanine iodide [DiSC(3)(5)] and bis-(1,3-dibarbituric acid)-trimethine oxanol [DiBAC(4)(3)]. Cells stained with the dyes had a significant increase in fluorescent intensity after exposure to dioscin, indicating that dioscin has an effect on the membrane potential. To visualize the effect of dioscin on the cell membrane, we synthesized rhodamine-labeled giant unilamellar vesicles (GUVs) mimicking the outer leaflet of the plasma membrane of C. albicans. As seen in the result, the membrane disruptive action of dioscin caused morphological change and rhodamine leakage of the GUVs. In three-dimensional contour-plot analysis using flow cytometry, we observed a decrease in cell size, which is in agreement with our result from the GUV assay. These results suggest that dioscin exerts a considerable antifungal activity by disrupting the structure in membrane after invading into the fungal membrane, resulting in fungal cell death. Copyright © 2012 Elsevier B.V. All rights reserved.

Assessing the carcinogenic potential of low-dose exposures to chemical mixtures in the environment: focus on the cancer hallmark of tumor angiogenesis.

PubMed

Hu, Zhiwei; Brooks, Samira A; Dormoy, Valérian; Hsu, Chia-Wen; Hsu, Hsue-Yin; Lin, Liang-Tzung; Massfelder, Thierry; Rathmell, W Kimryn; Xia, Menghang; Al-Mulla, Fahd; Al-Temaimi, Rabeah; Amedei, Amedeo; Brown, Dustin G; Prudhomme, Kalan R; Colacci, Annamaria; Hamid, Roslida A; Mondello, Chiara; Raju, Jayadev; Ryan, Elizabeth P; Woodrick, Jordan; Scovassi, A Ivana; Singh, Neetu; Vaccari, Monica; Roy, Rabindra; Forte, Stefano; Memeo, Lorenzo; Salem, Hosni K; Lowe, Leroy; Jensen, Lasse; Bisson, William H; Kleinstreuer, Nicole

2015-06-01

One of the important 'hallmarks' of cancer is angiogenesis, which is the process of formation of new blood vessels that are necessary for tumor expansion, invasion and metastasis. Under normal physiological conditions, angiogenesis is well balanced and controlled by endogenous proangiogenic factors and antiangiogenic factors. However, factors produced by cancer cells, cancer stem cells and other cell types in the tumor stroma can disrupt the balance so that the tumor microenvironment favors tumor angiogenesis. These factors include vascular endothelial growth factor, endothelial tissue factor and other membrane bound receptors that mediate multiple intracellular signaling pathways that contribute to tumor angiogenesis. Though environmental exposures to certain chemicals have been found to initiate and promote tumor development, the role of these exposures (particularly to low doses of multiple substances), is largely unknown in relation to tumor angiogenesis. This review summarizes the evidence of the role of environmental chemical bioactivity and exposure in tumor angiogenesis and carcinogenesis. We identify a number of ubiquitous (prototypical) chemicals with disruptive potential that may warrant further investigation given their selectivity for high-throughput screening assay targets associated with proangiogenic pathways. We also consider the cross-hallmark relationships of a number of important angiogenic pathway targets with other cancer hallmarks and we make recommendations for future research. Understanding of the role of low-dose exposure of chemicals with disruptive potential could help us refine our approach to cancer risk assessment, and may ultimately aid in preventing cancer by reducing or eliminating exposures to synergistic mixtures of chemicals with carcinogenic potential. © The Author 2015. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com.

Vascularization, High-Volume Solution Flow, and Localized Roles for Enzymes of Sucrose Metabolism during Tumorigenesis by Agrobacterium tumefaciens1

PubMed Central

Wächter, Rebecca; Langhans, Markus; Aloni, Roni; Götz, Simone; Weilmünster, Anke; Koops, Ariane; Temguia, Leopoldine; Mistrik, Igor; Pavlovkin, Jan; Rascher, Uwe; Schwalm, Katja; Koch, Karen E.; Ullrich, Cornelia I.

2003-01-01

Vascular differentiation and epidermal disruption are associated with establishment of tumors induced by Agrobacterium tumefaciens. Here, we address the relationship of these processes to the redirection of nutrient-bearing water flow and carbohydrate delivery for tumor growth within the castor bean (Ricinus communis) host. Treatment with aminoethoxyvinyl-glycine showed that vascular differentiation and epidermal disruption were central to ethylene-dependent tumor establishment. CO2 release paralleled tumor growth, but water flow increased dramatically during the first 3 weeks. However, tumor water loss contributed little to water flow to host shoots. Tumor water loss was followed by accumulation of the osmoprotectants, sucrose (Suc) and proline, in the tumor periphery, shifting hexose-to-Suc balance in favor of sugar signals for maturation and desiccation tolerance. Concurrent activities and sites of action for enzymes of Suc metabolism changed: Vacuolar invertase predominated during initial import of Suc into the symplastic continuum, corresponding to hexose concentrations in expanding tumors. Later, Suc synthase (SuSy) and cell wall invertase rose in the tumor periphery to modulate both Suc accumulation and descending turgor for import by metabolization. Sites of abscisic acid immunolocalization correlated with both central vacuolar invertase and peripheral cell wall invertase. Vascular roles were indicated by SuSy immunolocalization in xylem parenchyma for inorganic nutrient uptake and in phloem, where resolution allowed SuSy identification in sieve elements and companion cells, which has widespread implications for SuSy function in transport. Together, data indicate key roles for ethylene-dependent vascularization and cuticular disruption in the redirection of water flow and carbohydrate transport for successful tumor establishment. PMID:14526106

cAMP-specific PDE4 Phosphodiesterases and AIP in the Pathogenesis of Pituitary Tumors

PubMed Central

Bolger, Graeme B.; Bizzi, Mariana Ferreira; Brant Pinheiro, Sergio Veloso; Trivellin, Giampaolo; Smoot, Lisa; Accavitti, Mary-Ann; Korbonits, Márta; Ribeiro-Oliveira, Antonio

2016-01-01

PDE4 cyclic nucleotide phosphodiesterases regulate cAMP abundance in cells and thereby regulate numerous processes, including cell growth and differentiation. The rat PDE4A5 isoform (human homologue PDE4A4) interacts with the AIP protein (also called XAP2 or ARA-9). Germline mutations in AIP occur in approximately 20% of patients with Familial Isolated Pituitary Adenoma (FIPA) and 20% of childhood-onset simplex somatotroph adenomas. We therefore examined the protein expression of PDE4A4 and the closely-related isoform PDE4A8 in normal human pituitary tissue and in pituitary adenomas. PDE4A4 had low expression in normal pituitary, but was significantly over-expressed in somatotroph, lactotroph, corticotroph and clinically non-functioning gonadotroph adenomas (P<0.0001 for all subtypes). Likewise, PDE4A8 was expressed in normal pituitary and was also significantly over-expressed in the adenoma subtypes (P<0.0001 for all). Among the different adenoma subtypes, corticotroph and lactotroph adenomas were the highest and lowest expressed for PDE4A4, respectively, whereas the opposite was observed for PDE4A8. Naturally occurring oncogenic variants in AIP were shown by a two-hybrid assay to disrupt the ability of AIP to interact with PDE4A5. A reverse-two-hybrid screen identified numerous additional variants in the TPR region of AIP that also disrupted its ability to interact with PDE4A5. The expression of PDE4A4 and PDE4A8 in normal pituitary, their increased expression in adenomatous pituitary cells where AIP is meant to participate, and the disruption of the PDE4A4-AIP interaction by AIP mutants may play a role in pituitary tumorigenesis. PMID:27267386

Anti-angiogenic and vascular disrupting effects of C9, a new microtubule-depolymerizing agent

PubMed Central

Ren, Xuan; Dai, Mei; Lin, Li-Ping; Li, Pui-Kai; Ding, Jian

2009-01-01

Background and purpose: The critical role of blood supply in the growth of solid tumours makes blood vessels an ideal target for anti-tumour drug discovery. The anti-angiogenic and vascular disrupting activities of C9, a newly synthesized microtubule-depolymerizing agent, were investigated with several in vitro and in vivo models. Possible mechanisms involved in its activity were also assessed. Experimental approach: Microtubule-depolymerizing actions were assessed by surface plasmon resonance binding, competitive inhibition and cytoskeleton immunofluorescence. Anti-angiogenic and vascular disrupting activities were tested on proliferation, migration, tube formation with human umbilical vein endothelial cells, and in rat aortic ring, chick chorioallantoic membrane and Matrigel plug assays. Western blots and Rho activation assays were employed to examine the role of Raf-MEK-ERK (mitogen-activated ERK kinase, extracellular signal-regulated kinase) and Rho/Rho kinase signalling. Key results: C9 inhibited proliferation, migration and tube formation of endothelial cells and inhibited angiogenesis in aortic ring and chick chorioallantoic membrane assays. C9 induced disassembly of microtubules in endothelial cells and down-regulated Raf-MEK-ERK signalling activated by pro-angiogenic factors. In addition, C9 disrupted capillary-like networks and newly formed vessels in vitro and rapidly decreased perfusion of neovasculature in vivo. Endothelial cell contraction and membrane blebbing induced by C9 in neovasculature was dependent on the Rho/Rho kinase pathway. Conclusions and implications: Anti-angiogenic and vascular disruption by C9 was associated with changes in morphology and function of endothelial cells, involving the Raf-MEK-ERK and Rho/Rho kinase signalling pathways. These findings strongly suggest that C9 is a new microtubule-binding agent that could effectively target tumour vasculature. PMID:19302593

KML001 displays vascular disrupting properties and irinotecan combined antitumor activities in a murine tumor model.

PubMed

Moon, Chang Hoon; Lee, Seung Ju; Lee, Ho Yong; Lee, Jong Cheol; Cha, Heejeong; Cho, Wha Ja; Park, Jeong Woo; Park, Hyun Jin; Seo, Jin; Lee, Young Han; Song, Ho-Taek; Min, Young Joo

2013-01-01

KML001 is sodium metaarsenite, and has shown cytotoxic activity in human tumor cell lines. The anti-cancer mechanism of KML001 involves cancer cell destruction due to DNA damage at the telomeres of cancer cell chromosomes. In this study, we assessed the vascular disrupting properties of KML001 and investigated whether KML001 as VDA is able to increase anti-tumor activity in irinotecan combined treatment. We used a murine model of the CT26 colon carcinoma cell line. CT26 isograft mice treated intraperitoneally with 10 mg/kg KML001 displayed extensive central necrosis of tumor by 24 h. The vascular disrupting effects of KML001 were assessed by dynamic contrast enhanced magnetic resonance imaging. Gadopentetic acid-diethylene triaminepentaacetic acid contrast enhancement was markedly decreased in KML001-treated mice one day after treatment, whereas persistently high signal enhancement was observed in mice injected with saline. Rate constant K(ep) value representing capillary permeability was significantly decreased (p<0.05) in mice treated with KML001. Cytoskeletal changes of human umbilical vein endothelial cells (HUVECs) treated with 10 uM KML001 were assessed by immune blotting and confocal imaging. KML001 degraded tubulin protein in HUVECs, which may be related to vascular disrupting properties of KML001. Finally, in the mouse CT26 isograft model, KML001 combined with irinotecan significantly delayed tumor growth as compared to control and irinotecan alone. These results suggest that KML001 is a novel vascular disrupting agent, which exhibits significant vascular shut-down activity and enhances anti-tumor activity in combination with chemotherapy. These data further suggest an avenue for effective combination therapy in treating solid tumors.

KML001 Displays Vascular Disrupting Properties and Irinotecan Combined Antitumor Activities in a Murine Tumor Model

PubMed Central

Moon, Chang Hoon; Lee, Seung Ju; Lee, Ho Yong; Lee, Jong Cheol; Cha, HeeJeong; Cho, Wha Ja; Park, Jeong Woo; Park, Hyun Jin; Seo, Jin; Lee, Young Han; Song, Ho-Taek; Min, Young Joo

2013-01-01

KML001 is sodium metaarsenite, and has shown cytotoxic activity in human tumor cell lines. The anti-cancer mechanism of KML001 involves cancer cell destruction due to DNA damage at the telomeres of cancer cell chromosomes. In this study, we assessed the vascular disrupting properties of KML001 and investigated whether KML001 as VDA is able to increase anti-tumor activity in irinotecan combined treatment. We used a murine model of the CT26 colon carcinoma cell line. CT26 isograft mice treated intraperitoneally with 10 mg/kg KML001 displayed extensive central necrosis of tumor by 24 h. The vascular disrupting effects of KML001 were assessed by dynamic contrast enhanced magnetic resonance imaging. Gadopentetic acid-diethylene triaminepentaacetic acid contrast enhancement was markedly decreased in KML001-treated mice one day after treatment, whereas persistently high signal enhancement was observed in mice injected with saline. Rate constant Kep value representing capillary permeability was significantly decreased (p<0.05) in mice treated with KML001. Cytoskeletal changes of human umbilical vein endothelial cells (HUVECs) treated with 10 uM KML001 were assessed by immune blotting and confocal imaging. KML001 degraded tubulin protein in HUVECs, which may be related to vascular disrupting properties of KML001. Finally, in the mouse CT26 isograft model, KML001 combined with irinotecan significantly delayed tumor growth as compared to control and irinotecan alone. These results suggest that KML001 is a novel vascular disrupting agent, which exhibits significant vascular shut-down activity and enhances anti-tumor activity in combination with chemotherapy. These data further suggest an avenue for effective combination therapy in treating solid tumors. PMID:23326531

Circadian Disruption and Diet-Induced Obesity Synergize to Promote Development of β-Cell Failure and Diabetes in Male Rats

PubMed Central

Qian, Jingyi; Yeh, Bonnie; Rakshit, Kuntol; Colwell, Christopher S.

2015-01-01

There are clear epidemiological associations between circadian disruption, obesity, and pathogenesis of type 2 diabetes. The mechanisms driving these associations are unclear. In the current study, we hypothesized that continuous exposure to constant light (LL) compromises pancreatic β-cell functional and morphological adaption to diet-induced obesity leading to development of type 2 diabetes. To address this hypothesis, we studied wild type Sprague Dawley as well as Period-1 luciferase reporter transgenic rats (Per1-Luc) for 10 weeks under standard light-dark cycle (LD) or LL with concomitant ad libitum access to either standard chow or 60% high-fat diet (HFD). Exposure to HFD led to a comparable increase in food intake, body weight, and adiposity in both LD- and LL-treated rats. However, LL rats displayed profound loss of behavioral circadian rhythms as well as disrupted pancreatic islet clock function characterized by the impairment in the amplitude and the phase islet clock oscillations. Under LD cycle, HFD did not adversely alter diurnal glycemia, diurnal insulinemia, β-cell secretory function as well as β-cell survival, indicating successful adaptation to increased metabolic demand. In contrast, concomitant exposure to LL and HFD resulted in development of hyperglycemia characterized by loss of diurnal changes in insulin secretion, compromised β-cell function, and induction of β-cell apoptosis. This study suggests that circadian disruption and diet-induced obesity synergize to promote development of β-cell failure, likely mediated as a consequence of impaired islet clock function. PMID:26348474

Lipid recovery from wet oleaginous microbial biomass for biofuel production: A critical review

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

Dong, Tao; Knoshaug, Eric P.; Pienkos, Philip T.

Biological lipids derived from oleaginous microorganisms are promising precursors for renewable biofuel productions. Direct lipid extraction from wet cell-biomass is favored because it eliminates the need for costly dehydration. However, the development of a practical and scalable process for extracting lipids from wet cell-biomass is far from ready to be commercialized, instead, requiring intensive research and development to understand the lipid accessibility, mechanisms in mass transfer and establish robust lipid extraction approaches that are practical for industrial applications. Furthermore, this paper aims to present a critical review on lipid recovery in the context of biofuel productions with special attention tomore » cell disruption and lipid mass transfer to support extraction from wet biomass.« less

Lipid recovery from wet oleaginous microbial biomass for biofuel production: A critical review

DOE PAGES

Dong, Tao; Knoshaug, Eric P.; Pienkos, Philip T.; ...

2016-06-15

Biological lipids derived from oleaginous microorganisms are promising precursors for renewable biofuel productions. Direct lipid extraction from wet cell-biomass is favored because it eliminates the need for costly dehydration. However, the development of a practical and scalable process for extracting lipids from wet cell-biomass is far from ready to be commercialized, instead, requiring intensive research and development to understand the lipid accessibility, mechanisms in mass transfer and establish robust lipid extraction approaches that are practical for industrial applications. Furthermore, this paper aims to present a critical review on lipid recovery in the context of biofuel productions with special attention tomore » cell disruption and lipid mass transfer to support extraction from wet biomass.« less

Yersinia vs. host Immunity: how a pathogen evades or triggers a protective response

PubMed Central

Chung, Lawton K.; Bliska, James B.

2015-01-01

The human pathogenic Yersinia species cause diseases that represent a significant source of morbidity and mortality. Despite this, specific mechanisms underlying Yersinia pathogenesis and protective host responses remain poorly understood. Recent studies have shown that Yersinia disrupt cell death pathways, perturb inflammatory processes and exploit immune cells to promote disease. The ensuing host responses following Yersinia infection include coordination of innate and adaptive immune responses in an attempt to control bacterial replication. Here, we highlight current advances in our understanding of the interactions between the pathogenic yersiniae and host cells, as well as the protective host responses mobilized to counteract these pathogens. Together, these studies enhance our understanding of Yersinia pathogenesis and highlight the ongoing battle between host and microbe. PMID:26638030

Microtubule Binding and Disruption and Induction of Premature Senescence by Disorazole C1S⃞

PubMed Central

Tierno, Marni Brisson; Kitchens, Carolyn A.; Petrik, Bethany; Graham, Thomas H.; Wipf, Peter; Xu, Fengfeng L.; Saunders, William S.; Raccor, Brianne S.; Balachandran, Raghavan; Day, Billy W.; Stout, Jane R.; Walczak, Claire E.; Ducruet, Alexander P.; Reese, Celeste E.; Lazo, John S.

2009-01-01

Disorazoles comprise a family of 29 macrocyclic polyketides isolated from the fermentation broth of the myxobacterium Sorangium cellulosum. The major fermentation product, disorazole A1, was found previously to irreversibly bind to tubulin and to have potent cytotoxic activity against tumor cells, possibly because of its highly electrophilic epoxide moiety. To test this hypothesis, we synthesized the epoxide-free disorazole C1 and found it retained potent antiproliferative activity against tumor cells, causing prominent G2/M phase arrest and inhibition of in vitro tubulin polymerization. Furthermore, disorazole C1 produced disorganized microtubules at interphase, misaligned chromosomes during mitosis, apoptosis, and premature senescence in the surviving cell populations. Using a tubulin polymerization assay, we found disorazole C1 inhibited purified bovine tubulin polymerization, with an IC50 of 11.8 ± 0.4 μM, and inhibited [3H]vinblastine binding noncompetitively, with a Ki of 4.5 ± 0.6 μM. We also found noncompetitive inhibition of [3H]dolastatin 10 binding by disorazole C1, with a Ki of 10.6 ± 1.5 μM, indicating that disorazole C1 bound tubulin uniquely among known antimitotic agents. Disorazole C1 could be a valuable chemical probe for studying the process of mitotic spindle disruption and its relationship to premature senescence. PMID:19066338

Defined tetra-allelic gene disruption of the 4-coumarate:coenzyme A ligase 1 (Pv4CL1) gene by CRISPR/Cas9 in switchgrass results in lignin reduction and improved sugar release

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

Park, Jong -Jin; Yoo, Chang Geun; Flanagan, Amy

The development of genome editing technologies offers new prospects in improving bioenergy crops like switchgrass (Panicum virgatum). Switchgrass is an outcrossing species with an allotetraploid genome (2n = 4x = 36), a complexity which forms an impediment to generating homozygous knock-out plants. Lignin, a major component of the plant cell wall and a contributor to cellulosic feedstock’s recalcitrance to decomposition, stands as a barrier to efficient biofuel production by limiting enzyme access to cell wall polymers during the fermentation process.

Defined tetra-allelic gene disruption of the 4-coumarate:coenzyme A ligase 1 (Pv4CL1) gene by CRISPR/Cas9 in switchgrass results in lignin reduction and improved sugar release

DOE PAGES

Park, Jong -Jin; Yoo, Chang Geun; Flanagan, Amy; ...

2017-11-30

The development of genome editing technologies offers new prospects in improving bioenergy crops like switchgrass (Panicum virgatum). Switchgrass is an outcrossing species with an allotetraploid genome (2n = 4x = 36), a complexity which forms an impediment to generating homozygous knock-out plants. Lignin, a major component of the plant cell wall and a contributor to cellulosic feedstock’s recalcitrance to decomposition, stands as a barrier to efficient biofuel production by limiting enzyme access to cell wall polymers during the fermentation process.

Camouflage and Misdirection: The Full-On Assault of Ebola Virus Disease

PubMed Central

Misasi, John; Sullivan, Nancy J.

2014-01-01

Ebolaviruses cause a severe hemorrhagic fever syndrome that is rapidly fatal to humans and non-human primates. Ebola protein interactions with host cellular proteins disrupt Type I and Type II interferon responses, RNAi anti-viral responses, antigen presentation, T-cell mediated antibody responses, humoral antibodies and cell mediated immunity. This multifaceted approach to evasion and suppression of innate and adaptive immune responses in their target hosts leads to the severe immune dysregulation and “cytokine storm” that is characteristic of fatal ebolavirus infection. Here we highlight some of the processes by which Ebola interacts with its mammalian hosts to evade anti-viral defenses. PMID:25417101

Electrophysiologic and morphologic effects of ophthalmic preparations on rabbit cornea epithelium.

PubMed

Burstein, N L; Klyce, S D

1977-10-01

The effects of several components of ophthalmic preparations on isolated rabbit cornea were studied by continuous electrophysiologic monitoring followed by fixation for scanning electron microscopy (SEM). Benzalkonium chloride (0.001 percent), thimerosal (0.0004 percent), and amphotericin B (0.0025 percent) all briefly increased ion transport, then greatly decreased epithelial resistance. Severe disruption of surface cell layers occurred simultaneously with resistance decrease. Silver nitrate (0.00017 percent) stimulated transport with less accompanying morphologic damage. Tetracaine (0.05 percent) disrupted epithelial function and caused exfoliation of several cell layers. Chlorobutanol (0.1 percent) produced a nearly complete loss of the squamous cell layer. Chloramphenicol, epinephrine, and pilocarpine produced minor changes in structure and electrophysiology at full clinical concentration. It was concluded that low concentrations of preservatives in ophthalmic preparations disrupt the barrier and transport properties of the corneal epithelium.

Endoplasmic Reticulum Stress and Type 2 Diabetes

PubMed Central

Back, Sung Hoon; Kaufman, Randal J.

2013-01-01

Given the functional importance of the endoplasmic reticulum (ER), an organelle that performs folding, modification, and trafficking of secretory and membrane proteins to the Golgi compartment, the maintenance of ER homeostasis in insulin-secreting β-cells is very important. When ER homeostasis is disrupted, the ER generates adaptive signaling pathways, called the unfolded protein response (UPR), to maintain homeostasis of this organelle. However, if homeostasis fails to be restored, the ER initiates death signaling pathways. New observations suggest that both chronic hyperglycemia and hyperlipidemia, known as important causative factors of type 2 diabetes (T2D), disrupt ER homeostasis to induce unresolvable UPR activation and β-cell death. This review examines how the UPR pathways, induced by high glucose and free fatty acids (FFAs), interact to disrupt ER function and cause β-cell dysfunction and death. PMID:22443930

Cell migration and antigen capture are antagonistic processes coupled by myosin II in dendritic cells

PubMed Central

Chabaud, Mélanie; Heuzé, Mélina L.; Bretou, Marine; Vargas, Pablo; Maiuri, Paolo; Solanes, Paola; Maurin, Mathieu; Terriac, Emmanuel; Le Berre, Maël; Lankar, Danielle; Piolot, Tristan; Adelstein, Robert S.; Zhang, Yingfan; Sixt, Michael; Jacobelli, Jordan; Bénichou, Olivier; Voituriez, Raphaël; Piel, Matthieu; Lennon-Duménil, Ana-Maria

2015-01-01

The immune response relies on the migration of leukocytes and on their ability to stop in precise anatomical locations to fulfil their task. How leukocyte migration and function are coordinated is unknown. Here we show that in immature dendritic cells, which patrol their environment by engulfing extracellular material, cell migration and antigen capture are antagonistic. This antagonism results from transient enrichment of myosin IIA at the cell front, which disrupts the back-to-front gradient of the motor protein, slowing down locomotion but promoting antigen capture. We further highlight that myosin IIA enrichment at the cell front requires the MHC class II-associated invariant chain (Ii). Thus, by controlling myosin IIA localization, Ii imposes on dendritic cells an intermittent antigen capture behaviour that might facilitate environment patrolling. We propose that the requirement for myosin II in both cell migration and specific cell functions may provide a general mechanism for their coordination in time and space. PMID:26109323

Altered Dynamics of a Lipid Raft Associated Protein in a Kidney Model of Fabry Disease

PubMed Central

Labilloy, Anatália; Youker, Robert T.; Bruns, Jennifer R.; Kukic, Ira; Kiselyov, Kirill; Halfter, Willi; Finegold, David; do Monte, Semiramis Jamil Hadad; Weisz, Ora A.

2013-01-01

Accumulation of globotriaosylceramide (Gb3) and other neutral glycosphingolipids with galactosyl residues is the hallmark of Fabry disease, a lysosomal storage disorder caused by deficiency of the enzyme alpha-galactosidase A (α-gal A). These lipids are incorporated into the plasma membrane and intracellular membranes, with a preference for lipid rafts. Disruption of raft mediated cell processes is implicated in the pathogenesis of several human diseases, but little is known about the effects of the accumulation of glycosphingolipids on raft dynamics in the context of Fabry disease. Using siRNA technology, we have generated a polarized renal epithelial cell model of Fabry disease in Madin-Darby canine kidney cells. These cells present increased levels of Gb3 and enlarged lysosomes, and progressively accumulate zebra bodies. The polarized delivery of both raft-associated and raft-independent proteins was unaffected by α-gal A knockdown, suggesting that accumulation of Gb3 does not disrupt biosynthetic trafficking pathways. To assess the effect of α-gal A silencing on lipid raft dynamics, we employed number and brightness (N&B) analysis to measure the oligomeric status and mobility of the model glycosylphosphatidylinositol (GPI)-anchored protein GFP-GPI. We observed a significant increase in the oligomeric size of antibody-induced clusters of GFP-GPI at the plasma membrane of α-gal A silenced cells compared with control cells. Our results suggest that the interaction of GFP-GPI with lipid rafts may be altered in the presence of accumulated Gb3. The implications of our results with respect to the pathogenesis of Fabry disease are discussed. PMID:24215843

Cell death mechanisms in GT1-7 GnRH cells exposed to polychlorinated biphenyls PCB74, PCB118, and PCB153

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

Dickerson, Sarah M.; Guevara, Esperanza; Woller, Michael J.

2009-06-01

Exposure to endocrine disrupting chemicals (EDCs) such as polychlorinated biphenyls (PCBs) causes functional deficits in neuroendocrine systems. We used an immortalized hypothalamic GT1-7 cell line, which synthesizes the neuroendocrine peptide gonadotropin-releasing hormone (GnRH), to examine the neurotoxic and endocrine disrupting effects of PCBs and their mechanisms of action. Cells were treated for 1, 4, 8, or 24 h with a range of doses of a representative PCB from each of three classes: coplanar (2,4,4',5-tetrachlorobiphenyl: PCB74), dioxin-like coplanar (2',3,4,4',5' pentachlorobiphenyl: PCB118), non-coplanar (2,2',4,4',5,5'-hexachlorobiphenyl: PCB153), or their combination. GnRH peptide concentrations, cell viability, apoptotic and necrotic cell death, and caspase activation weremore » quantified. In general, GnRH peptide levels were suppressed by high doses and longer durations of PCBs, and elevated at low doses and shorter timepoints. The suppression of GnRH peptide levels was partially reversed in cultures co-treated with the estrogen receptor antagonist ICI 182,780. All PCBs reduced viability and increased both apoptotic and necrotic cell death. Although the effects for the three classes of PCBs were often similar, subtle differences in responses, together with evidence that the combination of PCBs acted slightly different from individual PCBs, suggest that the three tested PCB compounds may act via slightly different or more than one mechanism. These results provide evidence that PCB congeners have endocrine disrupting and/or neurotoxic effects on the hypothalamic GnRH cell line, a finding that has implications for environmental endocrine disruption in animals.« less

Cell death mechanisms in GT1-7 GnRH cells exposed to polychlorinated biphenyls PCB74, PCB118, and PCB153

PubMed Central

Dickerson, Sarah M.; Guevara, Esperanza; Woller, Michael J.; Gore, Andrea C.

2009-01-01

Exposure to endocrine-disrupting chemicals (EDCs) such as polychlorinated biphenyls (PCBs) causes functional deficits in neuroendocrine systems. We used an immortalized hypothalamic GT1-7 cell line, which synthesizes the neuroendocrine peptide gonadotropin-releasing hormone (GnRH), to examine the neurotoxic and endocrine disrupting effects of PCBs and their mechanisms of action. Cells were treated for 1, 4, 8, or 24 h with a range of doses of a representative PCB from each of three classes: coplanar (2,4,4′,5-tetrachlorobiphenyl: PCB74), dioxin-like coplanar (2′,3,4,4′,5′ pentachlorobiphenyl: PCB118), non-coplanar (2,2′,4,4′,5,5′-hexachlorobiphenyl: PCB153), or their combination. GnRH peptide concentrations, cell viability, apoptotic and necrotic cell death, and caspase activation were quantified. In general, GnRH peptide levels were suppressed by high doses and longer durations of PCBs, and elevated at low doses and shorter time points. The suppression of GnRH peptide levels was partially reversed in cultures co-treated with the estrogen receptor antagonist ICI 182,780. All PCBs reduced viability and increased both apoptotic and necrotic cell death. Although the effects for the three classes of PCBs were often similar, subtle differences in responses, together with evidence that the combination of PCBs acted slightly differently from individual PCBs, suggest that the three tested PCB compounds may act via slightly different or more than one mechanism. These results provide evidence that PCB congeners have endocrine disrupting and/or neurotoxic effects on the hypothalamic GnRH cell line, a finding that has implications for environmental endocrine disruption in animals. PMID:19362103

InVivo Imaging of Myelination for Drug Discovery and Development in Multiple Sclerosis

DTIC Science & Technology

2012-10-01

oligodendrocyte precursor cells, which are subsequently activated and distributed to the damaged axons. However, the remyelination process is often disrupted in...emitting carbon -11, and used for PET imaging of myelination (Wu et al., 2008; Wu et al., 2010). We demonstrated that [11C]MeDAS can readily penetrate...conducted. The animals were killed by a transcardial perfusion of saline followed by 4% polyformaldhyde ( PFA ) under anesthesia. The spinal cord and brain

Endocrine Disruption and Human Prostate Cancer

DTIC Science & Technology

2008-03-01

decades in men . Briefly we use rodent stroma to create a niche in which human stem cells are directed to become human prostatic epithelia. Task 1 sought to...we used our model system to study human prostate development and maturation over 8-12 weeks, comparable to the process that takes decades in men ...was 100%. The early onset of prostatitis, is comparable to the onset of non-bacterial early-onset prostatitis in younger men , and implicates the EDC

Three-dimensional co-culture process

NASA Technical Reports Server (NTRS)

Wolf, David A. (Inventor); Goodwin, Thomas J. (Inventor)

1992-01-01

The present invention relates to a 3-dimensional co-culture process, more particularly to methods or co-culturing at least two types of cells in a culture environment, either in space or in unit gravity, with minimum shear stress, freedom for 3-dimensional spatial orientation of the suspended particles and localization of particles with differing or similar sedimentation properties in a similar spatial region to form 3-dimensional tissue-like structures. Several examples of multicellular 3-dimensional experiences are included. The protocol and procedure are also set forth. The process allows simultaneous culture of multiple cell types and supporting substrates in a manner which does not disrupt the 3-dimensional spatial orientation of these components. The co-cultured cells cause a mutual induction effect which mimics the natural hormonal signals and cell interactions found in the intact organism. This causes the tissues to differentiate and form higher 3-dimensional structures such as glands, junctional complexes polypoid geometries, and microvilli which represent the corresponding in-vitro structures to a greater degree than when the cell types are cultured individually or by conventional processes. This process was clearly demonstrated for the case of two epithelial derived colon cancer lines, each co-cultured with normal human fibroblasts and with microcarrier bead substrates. The results clearly demonstrate increased 3-dimensional tissue-like structure and biochemical evidence of an increased differentiation state. With the present invention a variety of cells may be co-cultured to produce tissue which has 3-dimensionality and has some of the characteristics of in-vitro tissue. The process provides enhanced 3-dimensional tissue which create a multicellular organoid differentiation model.

Evaluation of the impact of food matrix change on the in vitro bioaccessibility of carotenoids in pumpkin (Cucurbita moschata) slices during two drying processes.

PubMed

Zhang, Zhongyuan; Wang, Xiaoyan; Li, Yixiang; Wei, Qiuyu; Liu, Chunju; Nie, Meimei; Li, Dajing; Xiao, Yadong; Liu, Chunquan; Xu, Lang; Zhang, Min; Jiang, Ning

2017-12-13

The food matrix is a limiting factor in determining the bioaccessibility of carotenoids. The impact of food matrix change on the bioaccessibility of carotenoids during drying processes is still unknown. The effect of intermittent microwave vacuum-assisted drying (IMVD) and hot air drying (HAD) on the in vitro liberation and micellization of carotenoids in pumpkin slices was studied. This variable depended on the changes of the matrix driven by the drying process. Different changes in the cell morphology and carotenoid distribution of pumpkin slices during the two processing methods were observed. For IMVD, cell wall degradation and complete chromoplast organelle disruption contributed to the improvement in the liberation and micellization of carotenoids. In the HAD-dried sample, large pigment aggregates hindered the liberation of carotenoids. The carotenoid level in the micellar fraction appeared to be lower than that in the aqueous supernatant during the two processes, suggesting that the new obstacles formed during processing and/or digestion hindered the incorporation of carotenoids in mixed micelles.

Baculovirus infection induces disruption of the nuclear lamina.

PubMed

Zhang, Xiaomei; Xu, Kaiyan; Wei, Denghui; Wu, Wenbi; Yang, Kai; Yuan, Meijin

2017-08-10

Baculovirus nucleocapsids egress from the nucleus primarily via budding at the nuclear membrane. The nuclear lamina underlying the nuclear membrane represents a substantial barrier to nuclear egress. Whether the nuclear lamina undergoes disruption during baculovirus infection remains unknown. In this report, we generated a clonal cell line, Sf9-L, that stably expresses GFP-tagged Drosophila lamin B. GFP autofluorescence colocalized with immunofluorescent anti-lamin B at the nuclear rim of Sf9-L cells, indicating GFP-lamin B was incorporated into the nuclear lamina. Meanwhile, virus was able to replicate normally in Sf9-L cells. Next, we investigated alterations to the nuclear lamina during baculovirus infection in Sf9-L cells. A portion of GFP-lamin B localized diffusely at the nuclear rim, and some GFP-lamin B was redistributed within the nucleus during the late phase of infection, suggesting the nuclear lamina was partially disrupted. Immunoelectron microscopy revealed associations between GFP-lamin B and the edges of the electron-dense stromal mattes of the virogenic stroma, intranuclear microvesicles, and ODV envelopes and nucleocapsids within the nucleus, indicating the release of some GFP-lamin B from the nuclear lamina. Additionally, GFP-lamin B phosphorylation increased upon infection. Based on these data, baculovirus infection induced lamin B phosphorylation and disruption of the nuclear lamina.

Ventricular Zone Disruption in Human Neonates With Intraventricular Hemorrhage.

PubMed

McAllister, James P; Guerra, Maria Montserrat; Ruiz, Leandro Castaneyra; Jimenez, Antonio J; Dominguez-Pinos, Dolores; Sival, Deborah; den Dunnen, Wilfred; Morales, Diego M; Schmidt, Robert E; Rodriguez, Esteban M; Limbrick, David D

2017-05-01

To determine if ventricular zone (VZ) and subventricular zone (SVZ) alterations are associated with intraventricular hemorrhage (IVH) and posthemorrhagic hydrocephalus, we compared postmortem frontal and subcortical brain samples from 12 infants with IVH and 3 nonneurological disease controls without hemorrhages or ventriculomegaly. Birth and expiration estimated gestational ages were 23.0-39.1 and 23.7-44.1 weeks, respectively; survival ranges were 0-42 days (median, 2.0 days). Routine histology and immunohistochemistry for neural stem cells (NSCs), neural progenitors (NPs), multiciliated ependymal cells (ECs), astrocytes (AS), and cell adhesion molecules were performed. Controls exhibited monociliated NSCs and multiciliated ECs lining the ventricles, abundant NPs in the SVZ, and medial vs. lateral wall differences with a complex mosaic organization in the latter. In IVH cases, normal VZ/SVZ areas were mixed with foci of NSC and EC loss, eruption of cells into the ventricle, cytoplasmic transposition of N-cadherin, subependymal rosettes, and periventricular heterotopia. Mature AS populated areas believed to be sites of VZ disruption. The cytopathology and extension of the VZ disruption correlated with developmental age but not with brain hemorrhage grade or location. These results corroborate similar findings in congenital hydrocephalus in animals and humans and indicate that VZ disruption occurs consistently in premature neonates with IVH. © 2017 American Association of Neuropathologists, Inc. All rights reserved.

Lipopolysaccharide-induced blood-brain barrier disruption: roles of cyclooxygenase, oxidative stress, neuroinflammation, and elements of the neurovascular unit.

PubMed

Banks, William A; Gray, Alicia M; Erickson, Michelle A; Salameh, Therese S; Damodarasamy, Mamatha; Sheibani, Nader; Meabon, James S; Wing, Emily E; Morofuji, Yoichi; Cook, David G; Reed, May J

2015-11-25

Disruption of the blood-brain barrier (BBB) occurs in many diseases and is often mediated by inflammatory and neuroimmune mechanisms. Inflammation is well established as a cause of BBB disruption, but many mechanistic questions remain. We used lipopolysaccharide (LPS) to induce inflammation and BBB disruption in mice. BBB disruption was measured using (14)C-sucrose and radioactively labeled albumin. Brain cytokine responses were measured using multiplex technology and dependence on cyclooxygenase (COX) and oxidative stress determined by treatments with indomethacin and N-acetylcysteine. Astrocyte and microglia/macrophage responses were measured using brain immunohistochemistry. In vitro studies used Transwell cultures of primary brain endothelial cells co- or tri-cultured with astrocytes and pericytes to measure effects of LPS on transendothelial electrical resistance (TEER), cellular distribution of tight junction proteins, and permeability to (14)C-sucrose and radioactive albumin. In comparison to LPS-induced weight loss, the BBB was relatively resistant to LPS-induced disruption. Disruption occurred only with the highest dose of LPS and was most evident in the frontal cortex, thalamus, pons-medulla, and cerebellum with no disruption in the hypothalamus. The in vitro and in vivo patterns of LPS-induced disruption as measured with (14)C-sucrose, radioactive albumin, and TEER suggested involvement of both paracellular and transcytotic pathways. Disruption as measured with albumin and (14)C-sucrose, but not TEER, was blocked by indomethacin. N-acetylcysteine did not affect disruption. In vivo, the measures of neuroinflammation induced by LPS were mainly not reversed by indomethacin. In vitro, the effects on LPS and indomethacin were not altered when brain endothelial cells (BECs) were cultured with astrocytes or pericytes. The BBB is relatively resistant to LPS-induced disruption with some brain regions more vulnerable than others. LPS-induced disruption appears is to be dependent on COX but not on oxidative stress. Based on in vivo and in vitro measures of neuroinflammation, it appears that astrocytes, microglia/macrophages, and pericytes play little role in the LPS-mediated disruption of the BBB.