Glutathione transferase-M2-2 secreted from glioblastoma cell protects SH-SY5Y cells from aminochrome neurotoxicity.

Science.gov (United States)

Cuevas, Carlos; Huenchuguala, Sandro; Muñoz, Patricia; Villa, Monica; Paris, Irmgard; Mannervik, Bengt; Segura-Aguilar, Juan

2015-04-01

U373MG cells are able to take up aminochrome that induces glutathione transferase M2-2 (GSTM2) expression in a concentration-dependent manner where 100 µM aminochrome increases GSTM2 expression by 2.1-fold (P protects SH-SY5Y cells incubated with 10 µM aminochrome. The significant protection provided by U373MG-conditioned medium in SH-SY5Y cells incubated with aminochrome was dependent on GSTM2 internalization into SH-SY5Y cells as evidenced by (i) uptake of (14)C-GSTM2 released from U373MG cells into SH-SY5Y cells, a process inhibited by anti-GSTM2 antiserum; (ii) lack of protection of U373MG-conditioned medium in the presence of anti-GSTM2 antiserum on SH-SY5Y cells treated with aminochrome; and (iii) lack of protection of conditioned medium from U373MGsiGST6 that expresses an siRNA directed against GSTM2 on SH-SY5Y cells treated with aminochrome. In conclusion, our results demonstrated that U373MG cells protect SH-SY5Y cells against aminochrome neurotoxicity by releasing GSTM2 into the conditioned medium and subsequent internalization of GSTM2 into SH-SY5Y cells. These results suggest a new mechanism of protection of dopaminergic neurons mediated by astrocytes by releasing GSTM2 into the intersynaptic space and subsequent internalization into dopaminergic neuron in order to protect these cells against aminochrome neurotoxicity.

Defective recoveryfrom potentially lethal damage in some human fibroblast cell strains

International Nuclear Information System (INIS)

Arlett, C.F.; Priestley, A.

1983-01-01

The repair of potentially lethal damage following treatment by gamma radiation was investigated in human fibroblasts held in a non-cycling state by maintenance in a medium containing 0.5 per cent foetal calf serum. Variation in their capacity to repair PLD was noted between three normal cell strains. A failure to repair PLD in ataxia-telangiectasia cells (AT5BI) was confirmed. In three cell strains which were intermediate between normals and A-T cells in their sensitivity, XP3BR, 46BR and GB1142, a limited capacity for the repair of PLD was observed. Two other cell strains, 47BR and 67BR, which showed little if any hypersensitivity could be clearly distinguished from normals after a 24 hour period for the repair of PLD. Thus the technique might permit better discrimination between cell strains. One other cell strain, H15617, could be distinguished from normals by proving hypersensitive under all conditions. Here, however, the repair of PLD appeared to be normal. (author)

Abnormal sensitivity of some Cockayne's syndrome cell strains to UV- and γ-rays

International Nuclear Information System (INIS)

Deschavanne, P.J.; Chavaudra, N.; Fertil, B.; Malaise, E.P.

1984-01-01

Cockayne's syndrome (CS) is a rare autosomal recessive genetic disease. Using a colony assay in vitro, we studied the sensitivity of 5 CS cell strains and two normal ones to UV- and γ-irradiation. The 5 CS strains appear to be UV-hypersensitive but the sensitivity varies widely from one strain to another. Hypersensitivity to γ-rays has been reported for 4 out of the 5 CS cell strains investigated. Repair of potentially lethal damage (PLD) after UV- and γ-irradiation was investigated by using unfed plateau cell cultures. Under these conditions, control cells show a great capacity to repair PLD. The two CS strains, which are hypersensitive to both UV- and γ-irradiation, exhibit no or only little PLD repair after treatment. The response of CS cell strains after γ-irradiation suggests a genetic heterogeneity. Three complementation groups are described in CS cells when dealing with UV radiosensitivity. However, variations in γ-ray sensitivity are reported for cells within the same UV complementation group. (Auth.)

Adhesion of Two Lactobacillus gasseri Probiotic Strains on Caco-2 Cells

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Mojca Narat

2003-01-01

Full Text Available Previous in vitro and in vivo studies showed that two human isolates of Lactobacillus gasseri, LF221 and K7 are able to survive the passage through the gastrointestinal tract and to colonise intestines of pigs at least temporarily. The aim of this study was to examine the adhesion ability of LF221 and K7 strains to Caco-2 cells. Adhesion of lactobacilli from early stationary growth phase was examined at two pH values of DMEM buffer (4.5 and 7. Lactobacillus rhamnosus GG, a widely used strain with clinical evidences of its efficiency, served as a positive control. The number of lactobacilli added to each well was found to be crucial in the adhesion assay. When added, lactobacilli were in range of 2.5 · 106 to 2.5 · 108 cfu/well, the linear correlation between the number of adhered cells (log cfu and the number of added cells (log cfu was found for all three strains (R2 > 0.99 at both pH values (4.5 and 7. At the highest concentration of added K7 and GG cells tested (app. 109 cfu/well, the efficiency of adhesion was reduced. pH value of the medium strongly affected the adhesion, which was promoted in acidic conditions (pH=4.5. The adhesion of K7 strain was slightly weaker compared to GG strain at both pH values, while at pH=4.5 the adhesion of LF221 strain was even better than GG adhesion, at least at lower concentration of lactobacilli. The direct comparison of these strains was possible by regression analysis. At lower concentration of lactobacilli (2.5 · 106, the best efficiency of adhesion (% of adhered bacteria was observed for the strain LF221, reaching the values of 7.8 and 1.9 % at pH=4.5 and 7, respectively, while at higher lactobacilli concentration the ration of adhesion was higher for GG strain (3.3 % at pH=4.5. In conclusion, strains LF221 and K7 were demonstrated to be adhesive, especially in acidic conditions. The level of adhesion of K7 and GG strains positively correlates with the number of added lactobacilli only up to the

Reduction of Mo(VI) by the bacterium Serratia sp. strain DRY5.

Science.gov (United States)

Rahman, M F A; Shukor, M Y; Suhaili, Z; Mustafa, S; Shamaan, N A; Syed, M A

2009-01-01

The need to isolate efficient heavy metal reducers for cost effective bioremediation strategy have resulted in the isolation of a potent molybdenum-reducing bacterium. The isolate was tentatively identified as Serratia sp. strain DRY5 based on the Biolog GN carbon utilization profiles and partial 16S rDNA molecular phylogeny. Strain DRY5 produced 2.3 times the amount of Mo-blue than S. marcescens strain Dr.Y6, 23 times more than E. coli K12 and 7 times more than E. cloacae strain 48. Strain DRY5 required 37 degrees C and pH 7.0 for optimum molybdenum reduction. Carbon sources such as sucrose, maltose, glucose and glycerol, supported cellular growth and molybdate reduction after 24 hr of static incubation. The most optimum carbon source that supported reduction was sucrose at 1.0% (w/v). Ammonium sulphate, ammonium chloride, glutamic acid, cysteine, and valine supported growth and molybdate reduction with ammonium sulphate as the optimum nitrogen source at 0. 2% (w/v). Molybdate reduction was optimally supported by 30 mM molybdate. The optimum concentration of phosphate for molybdate reduction was 5 mM when molybdate concentration was fixed at 30 mM and molybdate reduction was totally inhibited at 100 mM phosphate. Mo-blue produced by this strain shows a unique characteristic absorption profile with a maximum peak at 865 nm and a shoulder at 700 nm, Dialysis tubing experiment showed that 95.42% of Mo-blue was found in the dialysis tubing suggesting that the molybdate reduction seen in this bacterium was catalyzed by enzyme(s). The characteristics of isolate DRY5 suggest that it would be useful in the bioremediation ofmolybdenum-containing waste.

Is TrpM5 a reliable marker for chemosensory cells? Multiple types of microvillous cells in the main olfactory epithelium of mice

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Finger Thomas E

2008-12-01

Full Text Available Abstract Background In the past, ciliated receptor neurons, basal cells, and supporting cells were considered the principal components of the main olfactory epithelium. Several studies reported the presence of microvillous cells but their function is unknown. A recent report showed cells in the main olfactory epithelium that express the transient receptor potential channel TrpM5 claiming that these cells are chemosensory and that TrpM5 is an intrinsic signaling component of mammalian chemosensory organs. We asked whether the TrpM5-positive cells in the olfactory epithelium are microvillous and whether they belong to a chemosensory system, i.e. are olfactory neurons or trigeminally-innervated solitary chemosensory cells. Results We investigated the main olfactory epithelium of mice at the light and electron microscopic level and describe several subpopulations of microvillous cells. The ultrastructure of the microvillous cells reveals at least three morphologically different types two of which express the TrpM5 channel. None of these cells have an axon that projects to the olfactory bulb. Tests with a large panel of cell markers indicate that the TrpM5-positive cells are not sensory since they express neither neuronal markers nor are contacted by trigeminal nerve fibers. Conclusion We conclude that TrpM5 is not a reliable marker for chemosensory cells. The TrpM5-positive cells of the olfactory epithelium are microvillous and may be chemoresponsive albeit not part of the sensory apparatus. Activity of these microvillous cells may however influence functionality of local elements of the olfactory system.

The Epstein-Barr Virus BART miRNA Cluster of the M81 Strain Modulates Multiple Functions in Primary B Cells

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Lin, Xiaochen; Tsai, Ming-Han; Shumilov, Anatoliy; Poirey, Remy; Bannert, Helmut; Middeldorp, Jaap M.; Feederle, Regina; Delecluse, Henri-Jacques

2015-01-01

The Epstein-Barr virus (EBV) is a B lymphotropic virus that infects the majority of the human population. All EBV strains transform B lymphocytes, but some strains, such as M81, also induce spontaneous virus replication. EBV encodes 22 microRNAs (miRNAs) that form a cluster within the BART region of the virus and have been previously been found to stimulate tumor cell growth. Here we describe their functions in B cells infected by M81. We found that the BART miRNAs are downregulated in replicating cells, and that exposure of B cells in vitro or in vivo in humanized mice to a BART miRNA knockout virus resulted in an increased proportion of spontaneously replicating cells, relative to wild type virus. The BART miRNAs subcluster 1, and to a lesser extent subcluster 2, prevented expression of BZLF1, the key protein for initiation of lytic replication. Thus, multiple BART miRNAs cooperate to repress lytic replication. The BART miRNAs also downregulated pro- and anti-apoptotic mediators such as caspase 3 and LMP1, and their deletion did not sensitize B-cells to apoptosis. To the contrary, the majority of humanized mice infected with the BART miRNA knockout mutant developed tumors more rapidly, probably due to enhanced LMP1 expression, although deletion of the BART miRNAs did not modify the virus transforming abilities in vitro. This ability to slow cell growth could be confirmed in non-humanized immunocompromized mice. Injection of resting B cells exposed to a virus that lacks the BART miRNAs resulted in accelerated tumor growth, relative to wild type controls. Therefore, we found that the M81 BART miRNAs do not enhance B-cell tumorigenesis but rather repress it. The repressive effects of the BART miRNAs on potentially pathogenic viral functions in infected B cells are likely to facilitate long-term persistence of the virus in the infected host. PMID:26694854

Cyclic stress at mHz frequencies aligns fibroblasts in direction of zero strain.

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Uta Faust

Full Text Available Recognition of external mechanical signals is vital for mammalian cells. Cyclic stretch, e.g. around blood vessels, is one such signal that induces cell reorientation from parallel to almost perpendicular to the direction of stretch. Here, we present quantitative analyses of both, cell and cytoskeletal reorientation of umbilical cord fibroblasts. Cyclic strain of preset amplitudes was applied at mHz frequencies. Elastomeric chambers were specifically designed and characterized to distinguish between zero strain and minimal stress directions and to allow accurate theoretical modeling. Reorientation was only induced when the applied stretch exceeded a specific amplitude, suggesting a non-linear response. However, on very soft substrates no mechanoresponse occurs even for high strain. For all stretch amplitudes, the angular distributions of reoriented cells are in very good agreement with a theory modeling stretched cells as active force dipoles. Cyclic stretch increases the number of stress fibers and the coupling to adhesions. We show that changes in cell shape follow cytoskeletal reorientation with a significant temporal delay. Our data identify the importance of environmental stiffness for cell reorientation, here in direction of zero strain. These in vitro experiments on cultured cells argue for the necessity of rather stiff environmental conditions to induce cellular reorientation in mammalian tissues.

Do mechanical strain and TNF-α interact to amplify pro-inflammatory cytokine production in human annulus fibrosus cells?

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Likhitpanichkul, Morakot; Torre, Olivia M; Gruen, Jadry; Walter, Benjamin A; Hecht, Andrew C; Iatridis, James C

2016-05-03

During intervertebral disc (IVD) injury and degeneration, annulus fibrosus (AF) cells experience large mechanical strains in a pro-inflammatory milieu. We hypothesized that TNF-α, an initiator of IVD inflammation, modifies AF cell mechanobiology via cytoskeletal changes, and interacts with mechanical strain to enhance pro-inflammatory cytokine production. Human AF cells (N=5, Thompson grades 2-4) were stretched uniaxially on collagen-I coated chambers to 0%, 5% (physiological) or 15% (pathologic) strains at 0.5Hz for 24h under hypoxic conditions with or without TNF-α (10ng/mL). AF cells were treated with anti-TNF-α and anti-IL-6. ELISA assessed IL-1β, IL-6, and IL-8 production and immunocytochemistry measured F-actin, vinculin and α-tubulin in AF cells. TNF-α significantly increased AF cell pro-inflammatory cytokine production compared to basal conditions (IL-1β:2.0±1.4-84.0±77.3, IL-6:10.6±9.9-280.9±214.1, IL-8:23.9±26.0-5125.1±4170.8pg/ml for basal and TNF-α treatment, respectively) as expected, but mechanical strain did not. Pathologic strain in combination with TNF-α increased IL-1β, and IL-8 but not IL-6 production of AF cells. TNF-α treatment altered F-actin and α-tubulin in AF cells, suggestive of altered cytoskeletal stiffness. Anti-TNF-α (infliximab) significantly inhibited pro-inflammatory cytokine production while anti-IL-6 (atlizumab) did not. In conclusion, TNF-α altered AF cell mechanobiology with cytoskeletal remodeling that potentially sensitized AF cells to mechanical strain and increased TNF-α-induced pro-inflammatory cytokine production. Results suggest an interaction between TNF-α and mechanical strain and future mechanistic studies are required to validate these observations. Copyright © 2016 Elsevier Ltd. All rights reserved.

The structural proteins of epidemic and historical strains of Zika virus differ in their ability to initiate viral infection in human host cells.

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Bos, Sandra; Viranaicken, Wildriss; Turpin, Jonathan; El-Kalamouni, Chaker; Roche, Marjolaine; Krejbich-Trotot, Pascale; Desprès, Philippe; Gadea, Gilles

2018-03-01

Mosquito-borne Zika virus (ZIKV) recently emerged in South Pacific islands and Americas where large epidemics were documented. In the present study, we investigated the contribution of the structural proteins C, prM and E in the permissiveness of human host cells to epidemic strains of ZIKV. To this end, we evaluated the capacity of the epidemic strain BeH819015 to infect epithelial A549 and neuronal SH-SY5Y cells in comparison to the African historical MR766 strain. For that purpose, we generated a molecular clone of BeH819015 and a chimeric clone of MR766 which contains the BeH819015 structural protein region. We showed that ZIKV containing BeH819015 structural proteins was much less efficient in cell-attachment leading to a reduced susceptibility of A549 and SH-SY5Y cells to viral infection. Our data illustrate a previously underrated role for C, prM, and E in ZIKV epidemic strain ability to initiate viral infection in human host cells. Copyright © 2018 The Authors. Published by Elsevier Inc. All rights reserved.

Biodegradation of cypermethrin by immobilized cells of Micrococcus sp. strain CPN 1.

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Tallur, Preeti N; Mulla, Sikandar I; Megadi, Veena B; Talwar, Manjunatha P; Ninnekar, Harichandra Z

2015-01-01

Pyrethroid pesticide cypermethrin is a environmental pollutant because of its widespread use, toxicity and persistence. Biodegradation of such chemicals by microorganisms may provide an cost-effective method for their detoxification. We have investigated the degradation of cypermethrin by immobilized cells of Micrococcus sp. strain CPN 1 in various matrices such as, polyurethane foam (PUF), polyacrylamide, sodium alginate and agar. The optimum temperature and pH for the degradation of cypermethrin by immobilized cells of Micrococcus sp. were found to be 30 °C and 7.0, respectively. The rate of degradation of 10 and 20 mM of cypermethrin by freely suspended cells were compared with that of immobilized cells in batches and semi-continuous with shaken cultures. PUF-immobilized cells showed higher degradation of cypermethrin (10 mM and 20 mM) than freely suspended cells and cells immobilized in other matrices. The PUF-immobilized cells of Micrococcus sp. strain CPN 1 were retain their degradation capacity. Thus, they can be reused for more than 32 cycles, without losing their degradation capacity. Hence, the PUF-immobilized cells of Micrococcus sp. could potentially be used in the bioremediation of cypermethrin contaminated water.

Biodegradation of cypermethrin by immobilized cells of Micrococcus sp. strain CPN 1

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Preeti N. Tallur

2015-09-01

Full Text Available Pyrethroid pesticide cypermethrin is a environmental pollutant because of its widespread use, toxicity and persistence. Biodegradation of such chemicals by microorganisms may provide an cost-effective method for their detoxification. We have investigated the degradation of cypermethrin by immobilized cells of Micrococcus sp. strain CPN 1 in various matrices such as, polyurethane foam (PUF, polyacrylamide, sodium alginate and agar. The optimum temperature and pH for the degradation of cypermethrin by immobilized cells of Micrococcus sp. were found to be 30 °C and 7.0, respectively. The rate of degradation of 10 and 20 mM of cypermethrin by freely suspended cells were compared with that of immobilized cells in batches and semi-continuous with shaken cultures. PUF-immobilized cells showed higher degradation of cypermethrin (10 mM and 20 mM than freely suspended cells and cells immobilized in other matrices. The PUF-immobilized cells of Micrococcus sp. strain CPN 1 were retain their degradation capacity. Thus, they can be reused for more than 32 cycles, without losing their degradation capacity. Hence, the PUF-immobilized cells of Micrococcus sp. could potentially be used in the bioremediation of cypermethrin contaminated water.

) m /SrVO3 ( m = 5, 6) Superlattices

KAUST Repository

Dai, Qingqing

2018-05-04

The (LaV3+O3)m/SrV4+O3 (m = 5, 6) superlattices are investigated by first principles calculations. While bulk LaVO3 is a C‐type antiferromagnetic semiconductor and bulk SrVO3 is a paramagnetic metal, semiconducting A‐type antiferromagnetic states for both superlattices are found due to epitaxial strain. At the interfaces, however, the V spins couple antiferromagnetically for m = 5 and ferromagnetically for m = 6 (m‐dependence of the magnetization). Electronic reconstruction in form of charge ordering is predicted to occur with V3+ and V4+ states arranged in a checkerboard pattern on both sides of the SrO layer. As compared to bulk LaVO3, the presence of V4+ ions introduces in‐gap states that strongly reduce the bandgap and influence the orbital occupation and ordering.

Structure and expression of MHC class Ib genes of the central M region in rat and mouse: M4, M5, and M6.

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Lambracht-Washington, Doris; Moore, Yuki F; Wonigeit, Kurt; Lindahl, Kirsten Fischer

2008-04-01

The M region at the telomeric end of the murine major histocompatibility complex (MHC) contains class I genes that are highly conserved in rat and mouse. We have sequenced a cosmid clone of the LEW rat strain (RT1 haplotype) containing three class I genes, RT1.M6-1, RT1.M4, and RT1.M5. The sequences of allelic genes of the BN strain (RT1n haplotype) were obtained either from cDNAs or genomic clones. For the coding parts of the genes few differences were found between the two RT1 haplotypes. In LEW, however, only RT1.M5 and RT1.M6 have open reading frames; whereas in BN all three genes were intact. In line with the findings in BN, transcription was found for all three rat genes in several tissues from strain Sprague Dawley. Protein expression in transfectants could be demonstrated for RT1.M6-1 using the monoclonal antibody OX18. By sequencing of transcripts obtained by RT-PCR, a second, transcribed M6 gene, RT1.M6-2, was discovered, which maps next to RT1.M6-1 outside of the region covered by the cosmid. In addition, alternatively spliced forms for RT1.M5 and RT1.M6 were detected. Of the orthologous mouse genes, H2-M4, H2-M5, and H2-M6, only H2-M5 has an open reading frame. Other important differences between the corresponding parts of the M region of the two species are insertion of long LINE repeats, duplication of RT1.M6, and the inversion of RT1.M5 in the rat. This demonstrates substantial evolutionary dynamics in this region despite conservation of the class I gene sequences themselves.

Cells as strain-cued automata

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Cox, Brian N.; Snead, Malcolm L.

2016-02-01

We argue in favor of representing living cells as automata and review demonstrations that autonomous cells can form patterns by responding to local variations in the strain fields that arise from their individual or collective motions. An autonomous cell's response to strain stimuli is assumed to be effected by internally-generated, internally-powered forces, which generally move the cell in directions other than those implied by external energy gradients. Evidence of cells acting as strain-cued automata have been inferred from patterns observed in nature and from experiments conducted in vitro. Simulations that mimic particular cases of pattern forming share the idealization that cells are assumed to pass information among themselves solely via mechanical boundary conditions, i.e., the tractions and displacements present at their membranes. This assumption opens three mechanisms for pattern formation in large cell populations: wavelike behavior, kinematic feedback in cell motility that can lead to sliding and rotational patterns, and directed migration during invasions. Wavelike behavior among ameloblast cells during amelogenesis (the formation of dental enamel) has been inferred from enamel microstructure, while strain waves in populations of epithelial cells have been observed in vitro. One hypothesized kinematic feedback mechanism, "enhanced shear motility", accounts successfully for the spontaneous formation of layered patterns during amelogenesis in the mouse incisor. Directed migration is exemplified by a theory of invader cells that sense and respond to the strains they themselves create in the host population as they invade it: analysis shows that the strain fields contain positional information that could aid the formation of cell network structures, stabilizing the slender geometry of branches and helping govern the frequency of branch bifurcation and branch coalescence (the formation of closed networks). In simulations of pattern formation in

Actin and microtubule networks contribute differently to cell response for small and large strains

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Kubitschke, H.; Schnauss, J.; Nnetu, K. D.; Warmt, E.; Stange, R.; Kaes, J.

2017-09-01

Cytoskeletal filaments provide cells with mechanical stability and organization. The main key players are actin filaments and microtubules governing a cell’s response to mechanical stimuli. We investigated the specific influences of these crucial components by deforming MCF-7 epithelial cells at small (≤5% deformation) and large strains (>5% deformation). To understand specific contributions of actin filaments and microtubules, we systematically studied cellular responses after treatment with cytoskeleton influencing drugs. Quantification with the microfluidic optical stretcher allowed capturing the relative deformation and relaxation of cells under different conditions. We separated distinctive deformational and relaxational contributions to cell mechanics for actin and microtubule networks for two orders of magnitude of drug dosages. Disrupting actin filaments via latrunculin A, for instance, revealed a strain-independent softening. Stabilizing these filaments by treatment with jasplakinolide yielded cell softening for small strains but showed no significant change at large strains. In contrast, cells treated with nocodazole to disrupt microtubules displayed a softening at large strains but remained unchanged at small strains. Stabilizing microtubules within the cells via paclitaxel revealed no significant changes for deformations at small strains, but concentration-dependent impact at large strains. This suggests that for suspended cells, the actin cortex is probed at small strains, while at larger strains; the whole cell is probed with a significant contribution from the microtubules.

Transcription pattern of UL131A-128 mRNA in clinical strains of ...

Indian Academy of Sciences (India)

Human cytomegalovirus (HCMV) mRNA was obtained from human embryonic lung fibroblast cells infected by HCMV clinical strains from urine samples of infants at different kinetic periods. The cDNA of UL131A-128 mRNAs was amplified using reverse transcription-polymerase chain reaction (RT-PCR) and analysed by ...

Radioresistant cell strain of human fibrosarcoma cells obtained after long-term exposure to X-rays

International Nuclear Information System (INIS)

Wei, K.; Kodym, R.

1998-01-01

A radioresistant cell strain from human fibrosarcoma HT1080 has been obtained after prolonged exposure to x-rays for 7 months (2 Gy per day, 5 days per week). This new strain, HT1080R, differs from HT1080 in a significantly increased ability of clonogenical survival, with coefficient α decreasing from 0.161 to 0.123 Gy -1 and coefficient β decreasing from 0.0950 to 0.0565 Gy -2 . Furthermore, the radioresistance of HT1080R proved to be stable in long-term passaged cultures as well as in frozen samples. Differences between the two cell lines are also observed in the G-banded karyotype; the new cell line shows monosomy of chromosome 17 and loss of 5p + and 11q + present in the parental cells. These data suggest that the radioresistance may have been caused by radiation-induced cell mutation and that the resistant cells may have been selected by repeated irradiations. In order to characterize this new strain, the ability of the cells to rejoin DNA double-strand breaks, the cell cycle distribution and the amount of apoptosis after irradiation have been estimated; however, no differences are observed between these two cell strains. Although the mechanism of the elevated radioresistance remains unknown, this pair of cell strains can provide a new model system for further investigations with regard to the mechanisms of cellular radioresistance. The results also show that any type of irradiation similar to the schedules used in radiotherapy can lead to the formation and selection of more radioresistant cell clones in vitro, a phenomenon with possible implications for radiotherapy. (orig.)

Cloning and Characterization of a Unique Cytotoxic Protein Parasporin-5 Produced by Bacillus thuringiensis A1100 Strain

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Keisuke Ekino

2014-06-01

Full Text Available Parasporin is the cytocidal protein present in the parasporal inclusion of the non-insecticidal Bacillus thuringiensis strains, which has no hemolytic activity but has cytocidal activities, preferentially killing cancer cells. In this study, we characterized a cytocidal protein that belongs to this category, which was designated parasporin-5 (PS5. PS5 was purified from B. thuringiensis serovar tohokuensis strain A1100 based on its cytocidal activity against human leukemic T cells (MOLT-4. The 50% effective concentration (EC50 of PS5 to MOLT-4 cells was approximately 0.075 μg/mL. PS5 was expressed as a 33.8-kDa inactive precursor protein and exhibited cytocidal activity only when degraded by protease at the C-terminal into smaller molecules of 29.8 kDa. Although PS5 showed no significant homology with other known parasporins, a Position Specific Iterative-Basic Local Alignment Search Tool (PSI-BLAST search revealed that the protein showed slight homology to, not only some B. thuringiensis Cry toxins, but also to aerolysin-type β-pore-forming toxins (β-PFTs. The recombinant PS5 protein could be obtained as an active protein only when it was expressed in a precursor followed by processing with proteinase K. The cytotoxic activities of the protein against various mammalian cell lines were evaluated. PS5 showed strong cytocidal activity to seven of 18 mammalian cell lines tested, and low to no cytotoxicity to the others.

Aging enhances the vulnerability of mesenchymal stromal cells to uniaxial tensile strain-induced apoptosis.

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McKayed, Katey; Prendergast, Patrick J; Campbell, Veronica A

2016-02-08

Mechanical priming can be employed in tissue engineering strategies to control the fate and differentiation pattern of mesenchymal stromal cells. This is relevant to regenerative medicine whereby mechanical cues can promote the regeneration of a specific tissue type from mesenchymal precursors. The ability of cells to respond to mechanical forces is dependent upon mechanotransduction pathways that involve membrane-associated proteins, such as integrins. During the aging process changes in the mechanotransduction machinery may influence how cells from aged individuals respond to mechanical priming. In this study mesenchymal stromal cells were prepared from young adult and aged rats and exposed to uniaxial tensile strain at 5% and 10% for 3 days, or 2.5% for 7 days. Application of 5% tensile strain had no impact on cell viability. In contrast, application of 10% tensile strain evoked apoptosis and the strain-induced apoptosis was significantly higher in the mesenchymal stromal cells prepared from the aged rats. In parallel to the age-related difference in cellular responsiveness to strain, an age-related decrease in expression of α2 integrin and actin, and enhanced lipid peroxidation was observed. This study demonstrates that mesenchymal stem cells from aged animals have an altered membrane environment, are more vulnerable to the pro-apoptotic effects of 10% tensile strain and less responsive to the pro-osteogenic effects of 2.5% tensile strain. Thus, it is essential to consider how aged cells respond to mechanical stimuli in order to identify optimal mechanical priming strategies that minimise cell loss, particularly if this approach is to be applied to an aged population. Copyright © 2015 Elsevier Ltd. All rights reserved.

Strain preservation of experimental animals: vitrification of two-cell stage embryos for multiple mouse strains.

Science.gov (United States)

Eto, Tomoo; Takahashi, Riichi; Kamisako, Tsutomu

2015-04-01

Strain preservation of experimental animals is crucial for experimental reproducibility. Maintaining complete animal strains, however, is costly and there is a risk for genetic mutations as well as complete loss due to disasters or illness. Therefore, the development of effective vitrification techniques for cryopreservation of multiple experimental animal strains is important. We examined whether a vitrification method using cryoprotectant solutions, P10 and PEPeS, is suitable for preservation of multiple inbred and outbred mouse strains. First, we investigated whether our vitrification method using cryoprotectant solutions was suitable for two-cell stage mouse embryos. In vitro development of embryos exposed to the cryoprotectant solutions was similar to that of fresh controls. Further, the survival rate of the vitrified embryos was extremely high (98.1%). Next, we collected and vitrified two-cell stage embryos of 14 mouse strains. The average number of embryos obtained from one female was 7.3-33.3. The survival rate of vitrified embryos ranged from 92.8% to 99.1%, with no significant differences among mouse strains. In vivo development did not differ significantly between fresh controls and vitrified embryos of each strain. For strain preservation using cryopreserved embryos, two offspring for inbred lines and one offspring for outbred lines must be produced from two-cell stage embryos collected from one female. The expected number of surviving fetuses obtained from embryos collected from one female of either the inbred or outbred strains ranged from 2.9 to 19.5. The findings of the present study indicated that this vitrification method is suitable for strain preservation of multiple mouse strains. Copyright © 2015 The Authors. Published by Elsevier Inc. All rights reserved.

Strain elastography in the characterization of renal cell carcinoma and angiomyolipoma

Science.gov (United States)

Keskin, Suat; Güven, Selçuk; Keskin, Zeynep; Özbiner, Hüseyin; Kerimoğlu, Ülkü; Yeşildağ, Ahmet

2015-01-01

Introduction: We evaluate the diagnostic performance of strain elastography to differentiate renal cell carcinoma (RCC) from angiomyolipoma (AML). Methods: Strain elastography was performed in 65 patients (mean age 55.5 years; range: 32–81) who had renal lesions (24 AMLs and 41 RCCs) prospectively. Lesions were classified according to lesion size and histological subtypes. The strain ratios of the RCCs and AMLs were evaluated by a radiologist. The area under the curve and the cut-off point were used to assess diagnostic performance. Sensitivity, specificity, and positive and negative predictive values were obtained. Results: In assessing the mean strain ratio, we divided the groups in 3 according to size: (1) 40-mm lesions; the respective mean strain ratios were: 1.5 ± 0.5 (range: 0.06–5.92), 2.8 ± 0.4 (range: 0.17–9.92), 2.7 ± 0.3 (range: 0.08–6.15). When RCCs and AMLs were compared, there was a statistically significant difference in the strain ratio among the 3 groups divided per lesion size (p < 0.01). For the strain ratio, the mean ± standard deviation was 1.1 ± 0.1 for AMLs and 3.4 ± 0.3 for RCCs (p < 0.01). When lesion subtypes were compared, there was a statistically significant difference in the strain ratio between the AML and clear cell RCC (p < 0.01). Conclusions: For assessing renal lesions, strain elastography and strain ratio values may be useful in differentiating RCCs from AMLs. PMID:25737764

5-(2-Carboxyethenyl) isatin derivative induces G2/M cell cycle arrest and apoptosis in human leukemia K562 cells

International Nuclear Information System (INIS)

Zhou, Yao; Zhao, Hong-Ye; Han, Kai-Lin; Yang, Yao; Song, Bin-Bin; Guo, Qian-Nan; Fan, Zhen-Chuan; Zhang, Yong-Min; Teng, Yu-Ou; Yu, Peng

2014-01-01

Highlights: • 5-(2-Carboxyethenyl) isatin derivative (HKL 2H) inhibited K562’s proliferation. • HKL 2H caused the morphology change of G 2 /M phase arrest and typical apoptosis. • HKL 2H induced G2/M cell cycle phase arrest in K562 cells. • HKL 2H induced apoptosis in K562 cells through the mitochondrial pathway. - Abstract: Our previous study successfully identified that the novel isatin derivative (E)-methyl 3-(1-(4-methoxybenzyl)-2,3-dioxoindolin-5-yl) acrylate (HKL 2H) acts as an anticancer agent at an inhibitory concentration (IC 50 ) level of 3 nM. In this study, the molecular mechanism how HKL 2H induces cytotoxic activity in the human chronic myelogenous leukemia K562 cells was investigated. Flow cytometric analysis showed that the cells were arrested in the G 2 /M phase and accumulated subsequently in the sub-G 1 phase in the presence of HKL 2H. HKL 2H treatment down-regulated the expressions of CDK1 and cyclin B but up-regulated the level of phosphorylated CDK1. Annexin-V staining and the classic DNA ladder studies showed that HKL 2H induced the apoptosis of K562 cells. Our study further showed that HKL 2H treatment caused the dissipation of mitochondrial membrane potential, activated caspase-3 and lowered the Bcl-2/Bax ratio in K562 cells, suggesting that the HKL 2H-causing programmed cell death of K562 cells was caused via the mitochondrial apoptotic pathway. Taken together, our data demonstrated that HKL 2H, a 5-(2-carboxyethenyl) isatin derivative, notably induces G 2 /M cell cycle arrest and mitochondrial-mediated apoptosis in K562 cells, indicating that this compound could be a promising anticancer candidate for further investigation

Heterologous expression of Ralp3 in Streptococcus pyogenes M2 and M6 strains affects the virulence characteristics.

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Siemens, Nikolai; Kreikemeyer, Bernd

2013-01-01

Ralp3 is a transcriptional regulator present in a serotype specific fashion on the chromosome of the human pathogen Streptococcus pyogenes (group A streptococci, GAS). In serotypes harbouring the ralp3 gene either positive or negative effects on important metabolic and virulence genes involved in colonization and immune evasion in the human host were observed. A previous study revealed that deletion of ralp3 in a GAS M49 serotype significantly attenuated many virulence traits and caused metabolic disadvantages. This leads to two questions: (i) which kind of consequences could Ralp3 expression have in GAS serotypes naturally lacking this gene, and (ii) is Ralp3 actively lost during evolution in these serotypes. We investigated the role of Ralp3 in GAS M2 and M6 pathogenesis. Both serotypes lack ralp3 on their chromosome. The heterologous expression of ralp3 in both serotypes resulted in reduced attachment to and internalization into the majority of tested epithelial cells. Both ralp3 expression strains showed a decreased ability to survive in human blood and exclusively M2::ralp3 showed decreased survival in human serum. Both mutants secreted more active SpeB in the supernatant, resulting in a higher activity compared to wild type strains. The respective M2 and M6 wild type strains outcompeted the ralp3 expression strains in direct metabolic competition assays. The phenotypic changes observed in the M2:ralp3 and M6:ralp3 were verified on the transcriptional level. Consistent with the virulence data, tested genes showed transcript level changes in the same direction. Together these data suggest that Ralp3 can take over transcriptional control of virulence genes in serotypes lacking the ralp3 gene. Those serotypes most likely lost Ralp3 during evolution since obviously expression of this gene is disadvantageous for metabolism and pathogenesis.

A Benzothiazole Derivative (5g) Induces DNA Damage And Potent G2/M Arrest In Cancer Cells.

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Hegde, Mahesh; Vartak, Supriya V; Kavitha, Chandagirikoppal V; Ananda, Hanumappa; Prasanna, Doddakunche S; Gopalakrishnan, Vidya; Choudhary, Bibha; Rangappa, Kanchugarakoppal S; Raghavan, Sathees C

2017-05-31

Chemically synthesized small molecules play important role in anticancer therapy. Several chemical compounds have been reported to damage the DNA, either directly or indirectly slowing down the cancer cell progression by causing a cell cycle arrest. Direct or indirect reactive oxygen species formation causes DNA damage leading to cell cycle arrest and subsequent cell death. Therefore, identification of chemically synthesized compounds with anticancer potential is important. Here we investigate the effect of benzothiazole derivative (5g) for its ability to inhibit cell proliferation in different cancer models. Interestingly, 5g interfered with cell proliferation in both, cell lines and tumor cells leading to significant G2/M arrest. 5g treatment resulted in elevated levels of ROS and subsequently, DNA double-strand breaks (DSBs) explaining observed G2/M arrest. Consistently, we observed deregulation of many cell cycle associated proteins such as CDK1, BCL2 and their phosphorylated form, CyclinB1, CDC25c etc. Besides, 5g treatment led to decreased levels of mitochondrial membrane potential and activation of apoptosis. Interestingly, 5g administration inhibited tumor growth in mice without significant side effects. Thus, our study identifies 5g as a potent biochemical inhibitor to induce G2/M phase arrest of the cell cycle, and demonstrates its anticancer properties both ex vivo and in vivo.

Effects of Saponins against Clinical E. coli Strains and Eukaryotic Cell Line

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Arabski, Michał; Węgierek-Ciuk, Aneta; Czerwonka, Grzegorz; Lankoff, Anna; Kaca, Wiesław

2012-01-01

Saponins are detergent-like substances showing antibacterial as well as anticancer potential. In this study, the effects of saponins from Quillaja saponaria were analyzed against prokaryotic and eukaryotic cells. Multidrug-resistant clinical E. coli strains were isolated from human urine. As eukaryotic cells, the CHO-K1 cell lines were applied. Antibacterial effect of ampicillin, streptomycin, and ciprofloxacin in the presence of saponins was measured by cultivation methods. Properties of saponins against CHO-K1 cells were measured by the MTT test, hemolysis assay and flow cytometry. Saponin from Quillaja saponaria has a cytotoxic effect at concentrations higher than 25 μg/mL and in the range of 12–50 μg/mL significantly increases the level of early apoptotic cells. Saponin at dose of 12 μg/mL enhances the six E. coli strains growth. We postulate that saponins increase the influx of nutrients from the medium into E. coli cells. Saponins do not have synergetic effects on antibacterial action of tested antibiotics. In contrary, in the presence of saponins and antibiotics, more CFU/mL E. coli cells were observed. This effect was similar to saponins action alone towards E. coli cells. In conclusion, saponins was cytotoxic against CHO-K1 cells, whereas against E. coli cells this effect was not observed. PMID:22500084

Effects of Saponins against Clinical E. coli Strains and Eukaryotic Cell Line

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Michał Arabski

2012-01-01

Full Text Available Saponins are detergent-like substances showing antibacterial as well as anticancer potential. In this study, the effects of saponins from Quillaja saponaria were analyzed against prokaryotic and eukaryotic cells. Multidrug-resistant clinical E. coli strains were isolated from human urine. As eukaryotic cells, the CHO-K1 cell lines were applied. Antibacterial effect of ampicillin, streptomycin, and ciprofloxacin in the presence of saponins was measured by cultivation methods. Properties of saponins against CHO-K1 cells were measured by the MTT test, hemolysis assay and flow cytometry. Saponin from Quillaja saponaria has a cytotoxic effect at concentrations higher than 25 μg/mL and in the range of 12–50 μg/mL significantly increases the level of early apoptotic cells. Saponin at dose of 12 μg/mL enhances the six E. coli strains growth. We postulate that saponins increase the influx of nutrients from the medium into E. coli cells. Saponins do not have synergetic effects on antibacterial action of tested antibiotics. In contrary, in the presence of saponins and antibiotics, more CFU/mL E. coli cells were observed. This effect was similar to saponins action alone towards E. coli cells. In conclusion, saponins was cytotoxic against CHO-K1 cells, whereas against E. coli cells this effect was not observed.

A rapid minor groove binder PCR method for distinguishing the vaccine strain Brucella abortus 104M.

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Nan, Wenlong; Qin, Lide; Wang, Yong; Zhang, Yueyong; Tan, Pengfei; Chen, Yuqi; Mao, Kairong; Chen, Yiping

2018-01-24

Brucellosis is a widespread zoonotic disease caused by Gram-negative Brucella bacteria. Immunisation with attenuated vaccine is an effective method of prevention, but it can interfere with diagnosis. Live, attenuated Brucella abortus strain 104M has been used for the prevention of human brucellosis in China since 1965. However, at present, no fast and reliable method exists that can distinguish this strain from field strains. Single nucleotide polymorphism (SNP)-based assays offer a new approach for such discrimination. SNP-based minor groove binder (MGB) and Cycleave assays have been used for rapid identification of four Brucella vaccine strains (B. abortus strains S19, A19 and RB51, and B. melitensis Rev1). The main objective of this study was to develop a PCR assay for rapid and specific detection of strain 104M. We developed a SNP-based MGB PCR assay that could successfully distinguish strain 104M from 18 representative strains of Brucella (B. abortus biovars 1, 2, 3, 4, 5, 6, 7 and 9, B. melitensis biovars 1, 2 and 3, B. suis biovars 1, 2, 3 and 4, B. canis, B. neotomae, and B. ovis), four Brucella vaccine strains (A19, S19, S2, M5), and 55 Brucella clinical field strains. The assay gave a negative reaction with four non-Brucella species (Escherichia coli, Pasteurella multocida, Streptococcus suis and Pseudomonas aeruginosa). The minimum sensitivity of the assay, evaluated using 10-fold dilutions of chromosomal DNA, was 220 fg for the 104M strain and 76 fg for the single non-104M Brucella strain tested (B. abortus A19). The assay was also reproducible (intra- and inter-assay coefficients of variation = 0.006-0.022 and 0.012-0.044, respectively). A SNP-based MGB PCR assay was developed that could straightforwardly and unambiguously distinguish B. abortus vaccine strain 104M from non-104M Brucella strains. Compared to the classical isolation and identification approaches of bacteriology, this real-time PCR assay has substantial advantages in terms of

Biodegradation of cyanide by acetonitrile-induced cells of Rhodococcus sp. UKMP-5M.

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Nallapan Maniyam, Maegala; Sjahrir, Fridelina; Ibrahim, Abdul Latif; Cass, Anthony E G

2013-01-01

A Rhodococcus sp. UKMP-5M isolate was shown to detoxify cyanide successfully, suggesting the presence of an intrinsic property in the bacterium which required no prior cyanide exposure for induction of this property. However, in order to promote growth, Rhodococcus sp. UKMP-5M was fully acclimatized to cyanide after 7 successive subcultures in 0.1 mM KCN for 30 days. To further shorten the lag phase and simultaneously increase the tolerance towards higher cyanide concentrations, the bacterium was induced with various nitrile compounds sharing a similar degradatory pathway to cyanide. Acetonitrile emerged as the most favored inducer and the induced cells were able to degrade 0.1 mM KCN almost completely within 18 h. With the addition of subsequent aliquots of 0.1 mM KCN a shorter period for complete removal of cyanide was required, which proved to be advantageous economically. Both resting cells and crude enzyme of Rhodococcus sp. UKMP-5M were able to biodegrade cyanide to ammonia and formate without the formation of formamide, implying the identification of a simple hydrolytic cyanide degradation pathway involving the enzyme cyanidase. Further verification with SDS-PAGE revealed that the molecular weight of the active enzyme was estimated to be 38 kDa, which is consistent with previously reported cyanidases. Since the recent advancement in the application of biological methods in treating cyanide-bearing wastewater has been promising, the discovery of this new bacterium will add value by diversifying the existing microbial populations capable of cyanide detoxification.

Synergistic Action of D-Glucose and Acetosyringone on Agrobacterium Strains for Efficient Dunaliella Transformation.

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Ramachandran Srinivasan

Full Text Available An effective transformation protocol for Dunaliella, a β-carotene producer, was developed using the synergistic mechanism of D-glucose and Acetosyringone on three different Agrobacterium strains (EHA105, GV3101 and LBA4404. In the present study, we investigated the pre-induction of Agrobacterium strains harboring pMDC45 binary vector in TAP media at varying concentrations of D-glucose (5 mM, 10 mM, and 15mM and 100 μM of Acetosyringone for co-cultivation. Induction of Agrobacterium strains with 10 mM D-glucose and 100 μM Acetosyringone showed higher rates of efficiency compared to other treatments. The presence of GFP and HPT transgenes as a measure of transformation efficiency from the transgenic lines were determined using fluorescent microscopy, PCR, and southern blot analyzes. Highest transformation rate was obtained with the Agrobacterium strain LBA4404 (181 ± 3.78 cfu per 106 cells followed by GV3101 (128 ± 5.29 cfu per 106 cells and EHA105 (61 ± 5.03 cfu per 106 cells. However, the Agrobacterium strain GV3101 exhibited more efficient single copy transgene (HPT transfer into the genome of D. salina than LBA4404. Therefore, future studies dealing with genetic modifications in D. salina can utilize GV3101 as an optimal Agrobacterium strain for gene transfer.

Synergistic Action of D-Glucose and Acetosyringone on Agrobacterium Strains for Efficient Dunaliella Transformation.

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Srinivasan, Ramachandran; Gothandam, Kodiveri Muthukalianan

2016-01-01

An effective transformation protocol for Dunaliella, a β-carotene producer, was developed using the synergistic mechanism of D-glucose and Acetosyringone on three different Agrobacterium strains (EHA105, GV3101 and LBA4404). In the present study, we investigated the pre-induction of Agrobacterium strains harboring pMDC45 binary vector in TAP media at varying concentrations of D-glucose (5 mM, 10 mM, and 15mM) and 100 μM of Acetosyringone for co-cultivation. Induction of Agrobacterium strains with 10 mM D-glucose and 100 μM Acetosyringone showed higher rates of efficiency compared to other treatments. The presence of GFP and HPT transgenes as a measure of transformation efficiency from the transgenic lines were determined using fluorescent microscopy, PCR, and southern blot analyzes. Highest transformation rate was obtained with the Agrobacterium strain LBA4404 (181 ± 3.78 cfu per 106 cells) followed by GV3101 (128 ± 5.29 cfu per 106 cells) and EHA105 (61 ± 5.03 cfu per 106 cells). However, the Agrobacterium strain GV3101 exhibited more efficient single copy transgene (HPT) transfer into the genome of D. salina than LBA4404. Therefore, future studies dealing with genetic modifications in D. salina can utilize GV3101 as an optimal Agrobacterium strain for gene transfer.

Extracellular proteases of Halobacillus blutaparonensis strain M9, a new moderately halophilic bacterium.

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Santos, Anderson F; Valle, Roberta S; Pacheco, Clarissa A; Alvarez, Vanessa M; Seldin, Lucy; Santos, André L S

2013-12-01

Halophilic microorganisms are source of potential hydrolytic enzymes to be used in industrial and/or biotechnological processes. In the present study, we have investigated the ability of the moderately halophilic bacterium Halobacillus blutaparonensis (strain M9), a novel species described by our group, to release proteolytic enzymes. This bacterial strain abundantly proliferated in Luria-Bertani broth supplemented with 2.5% NaCl as well as secreted proteases to the extracellular environment. The production of proteases occurred in bacterial cells grown under different concentration of salt, ranging from 0.5% to 10% NaCl, in a similar way. The proteases secreted by H. blutaparonensis presented the following properties: (i) molecular masses ranging from 30 to 80 kDa, (ii) better hydrolytic activities under neutral-alkaline pH range, (iii) expression modulated according to the culture age, (iv) susceptibility to phenylmethylsulphonyl fluoride, classifying them as serine-type proteases, (v) specific cleavage over the chymotrypsin substrate, and (vi) enzymatic stability in the presence of salt (up to 20% NaCl) and organic solvents (e.g., ether, isooctane and cyclohexane). The proteases described herein are promising for industrial practices due to its haloalkaline properties.

Cell culture isolation and sequence analysis of genetically diverse US porcine epidemic diarrhea virus strains including a novel strain with a large deletion in the spike gene.

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Oka, Tomoichiro; Saif, Linda J; Marthaler, Douglas; Esseili, Malak A; Meulia, Tea; Lin, Chun-Ming; Vlasova, Anastasia N; Jung, Kwonil; Zhang, Yan; Wang, Qiuhong

2014-10-10

The highly contagious and deadly porcine epidemic diarrhea virus (PEDV) first appeared in the US in April 2013. Since then the virus has spread rapidly nationwide and to Canada and Mexico causing high mortality among nursing piglets and significant economic losses. Currently there are no efficacious preventive measures or therapeutic tools to control PEDV in the US. The isolation of PEDV in cell culture is the first step toward the development of an attenuated vaccine, to study the biology of PEDV and to develop in vitro PEDV immunoassays, inactivation assays and screen for PEDV antivirals. In this study, nine of 88 US PEDV strains were isolated successfully on Vero cells with supplemental trypsin and subjected to genomic sequence analysis. They differed genetically mainly in the N-terminal S protein region as follows: (1) strains (n=7) similar to the highly virulent US PEDV strains; (2) one similar to the reportedly US S INDEL PEDV strain; and (3) one novel strain most closely related to highly virulent US PEDV strains, but with a large (197aa) deletion in the S protein. Representative strains of these three genetic groups were passaged serially and grew to titers of ∼5-6log10 plaque forming units/mL. To our knowledge, this is the first report of the isolation in cell culture of an S INDEL PEDV strain and a PEDV strain with a large (197aa) deletion in the S protein. We also designed primer sets to detect these genetically diverse US PEDV strains. Copyright © 2014 Elsevier B.V. All rights reserved.

Autologous Hematopoietic Stem Cells transplantation and genetic modification of CCR5 m303/m303 mutant patient for HIV/AIDS.

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Esmaeilzadeh, Abdolreza; Farshbaf, Alieh; Erfanmanesh, Maryam

2015-03-01

HIV and AIDS is one of the biggest challenges all over the world. There are an approximately 34 million people living with the virus, and a large number of them become infected each year. Although there are some antiviral drugs for HIV viral load reduction, they are not sufficient. There is no cure for AIDS. Nowadays natural resistance or immunity has absorbed attentions. Because in some HIV positive patients progression trend is slow or even they indicate resistance to AIDS. One of the most interesting approaches in this category is CCR5 gene. CCR5 is a main cc-chemokine co-receptor that facilitates HIV-1 entry to macrophage and CD4(+) T cells. To now, many polymorphisms have been known by CCR5 gene that produces a truncated protein with no function. So, HIV-1 could not entry to immune-cells and the body resistant to HIV/AIDS. Δ32/Δ32 and m303/m303 homozygotes are example of mutations that could create this resistance mechanism. There is a new treatment, such as Hematopoietic Stem Cell transplantation (HSCT) in Berlin and Boston patients for Δ32/Δ32 mutation. It could eliminate co-receptor antagonist and highly-active-anti retroviral therapy (HAART) drugs problems such as toxicity, low safety and side-effects. Now there, the aim of this hypothesis will be evaluation of a new mutation CCR5 m303/m303 as autologous HSCT. This novel hypothesis indicates that autologous HSCT for m303/m303 could be effective treatment for anyone HIV/AIDS affected patient worldwide. Copyright © 2015 Elsevier Ltd. All rights reserved.

The C5a/C5aR1 axis controls the development of experimental allergic asthma independent of LysM-expressing pulmonary immune cells.

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Anna V Wiese

Full Text Available C5a regulates the development of maladaptive immune responses in allergic asthma mainly through the activation of C5a receptor 1 (C5aR1. Yet, the cell types and the mechanisms underlying this regulation are ill-defined. Recently, we described increased C5aR1 expression in lung tissue eosinophils but decreased expression in airway and pulmonary macrophages as well as in pulmonary CD11b+ conventional dendritic cells (cDCs and monocyte-derived DCs (moDCs during the allergic effector phase using a floxed green fluorescent protein (GFP-C5aR1 knock-in mouse. Here, we determined the role of C5aR1 signaling in neutrophils, moDCs and macrophages for the pulmonary recruitment of such cells and the importance of C5aR1-mediated activation of LysM-expressing cells for the development of allergic asthma. We used LysM-C5aR1 KO mice with a specific deletion of C5aR1 in LysMCre-expressing cells and confirmed the specific deletion of C5aR1 in neutrophils, macrophages and moDCs in the airways and/or the lung tissue. We found that alveolar macrophage numbers were significantly increased in LysM-C5aR1 KO mice. Induction of ovalbumin (OVA-driven experimental allergic asthma in GFP-C5aR1fl/fl and LysM-C5aR1 KO mice resulted in strong but similar airway resistance, mucus production and Th2/Th17 cytokine production. In contrast, the number of airway but not of pulmonary neutrophils was lower in LysM-C5aR1 KO as compared with GFP-C5aR1fl/fl mice. The recruitment of macrophages, cDCs, moDCs, T cells and type 2 innate lymphoid cells was not altered in LysM-C5aR1 KO mice. Our findings demonstrate that C5aR1 is critical for steady state control of alveolar macrophage numbers and the transition of neutrophils from the lung into the airways in OVA-driven allergic asthma. However, C5aR1 activation of LysM-expressing cells plays a surprisingly minor role in the recruitment and activation of such cells and the development of the allergic phenotype in OVA-driven experimental

Human prostate supports more efficient replication of HIV-1 R5 than X4 strains ex vivo

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Denis Hélène

2008-12-01

Full Text Available Abstract Background In order to determine whether human prostate can be productively infected by HIV-1 strains with different tropism, and thus represent a potential source of HIV in semen, an organotypic culture of prostate from men undergoing prostatic adenomectomy for benign prostate hypertrophy (BPH was developed. The presence of potential HIV target cells in prostate tissues was investigated using immunohistochemistry. The infection of prostate explants following exposures with HIV-1 R5, R5X4 and X4 strains was analyzed through the measure of RT activity in culture supernatants, the quantification of HIV DNA in the explants and the detection of HIV RNA+ cells in situ. Results The overall prostate characteristics were retained for 21/2 weeks in culture. Numerous potential HIV-1 target cells were detected in the prostate stroma. Whilst HIV-1 R5SF162 strain consistently productively infected prostatic T lymphocytes and macrophages, the prototypic X4IIIB strain and a primary R5X4 strain showed less efficient replication in this organ. Conclusion The BPH prostate is a site of HIV-1 R5 replication that could contribute virus to semen. A limited spreading of HIV-1 X4 and R5X4 in this organ could participate to the preferential sexual transmission of HIV-1 R5 strains.

An RNA-binding protein, Qki5, regulates embryonic neural stem cells through pre-mRNA processing in cell adhesion signaling.

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Hayakawa-Yano, Yoshika; Suyama, Satoshi; Nogami, Masahiro; Yugami, Masato; Koya, Ikuko; Furukawa, Takako; Zhou, Li; Abe, Manabu; Sakimura, Kenji; Takebayashi, Hirohide; Nakanishi, Atsushi; Okano, Hideyuki; Yano, Masato

2017-09-15

Cell type-specific transcriptomes are enabled by the action of multiple regulators, which are frequently expressed within restricted tissue regions. In the present study, we identify one such regulator, Quaking 5 (Qki5), as an RNA-binding protein (RNABP) that is expressed in early embryonic neural stem cells and subsequently down-regulated during neurogenesis. mRNA sequencing analysis in neural stem cell culture indicates that Qki proteins play supporting roles in the neural stem cell transcriptome and various forms of mRNA processing that may result from regionally restricted expression and subcellular localization. Also, our in utero electroporation gain-of-function study suggests that the nuclear-type Qki isoform Qki5 supports the neural stem cell state. We next performed in vivo transcriptome-wide protein-RNA interaction mapping to search for direct targets of Qki5 and elucidate how Qki5 regulates neural stem cell function. Combined with our transcriptome analysis, this mapping analysis yielded a bona fide map of Qki5-RNA interaction at single-nucleotide resolution, the identification of 892 Qki5 direct target genes, and an accurate Qki5-dependent alternative splicing rule in the developing brain. Last, our target gene list provides the first compelling evidence that Qki5 is associated with specific biological events; namely, cell-cell adhesion. This prediction was confirmed by histological analysis of mice in which Qki proteins were genetically ablated, which revealed disruption of the apical surface of the lateral wall in the developing brain. These data collectively indicate that Qki5 regulates communication between neural stem cells by mediating numerous RNA processing events and suggest new links between splicing regulation and neural stem cell states. © 2017 Hayakawa-Yano et al.; Published by Cold Spring Harbor Laboratory Press.

Characterization of Selected Lactobacillus Strains for Use as Probiotics.

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Song, Minyu; Yun, Bohyun; Moon, Jae-Hak; Park, Dong-June; Lim, Kwangsei; Oh, Sejong

2015-01-01

The aim of this study was to evaluate the functional properties of lactic acid bacteria from various sources and to identify strains for use as probiotics. Ten Lactobacillus strains were selected and their properties such as bile tolerance, acid resistance, cholesterol assimilation activity, and adherence to HT-29 cells were assessed to determine their potential as probiotics. Lactobacillus sp. JNU 8829, L. casei MB3, L. sakei MA9, L. sakei CH8, and L. acidophilus M23 were found to show full tolerance to the 0.3% bile acid. All strains without L. acidophilus M23 were the most acid-tolerant strains. After incubating the strains at pH 2.5 for 2 h, their viability decreased by 3 Log cells. Some strains survived at pH 2.5 in the presence of pepsin and 0.3% bile acid. Lactobacillus sp. JNU 8829, L. acidophilus KU41, L. acidophilus M23, L. fermentum NS2, L. plantarum M13, and L. plantarum NS3 were found to reduce cholesterol levels by >50% in vitro. In the adhesion assay, Lactobacillus sp. JNU 8829, L. casei MB3, L. sakei MA9, and L. sakei CH8 showed higher adhesion activities after 2 h of co-incubation with the intestinal cells. The results of this comprehensive analysis shows that this new probiotic strain named, Lactobacillus sp. JNU 8829 could be a promising candidate for dairy products.

Enhanced degradation of 2-nitrotoluene by immobilized cells of Micrococcus sp. strain SMN-1.

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Mulla, Sikandar I; Talwar, Manjunatha P; Bagewadi, Zabin K; Hoskeri, Robertcyril S; Ninnekar, Harichandra Z

2013-02-01

Nitrotoluenes are the toxic pollutants of the environment because of their large scale use in the production of explosives. Biodegradation of such chemicals by microorganisms may provide an effective method for their detoxification. We have studied the degradation of 2-nitrotoluene by cells of Micrococcus sp. strain SMN-1 immobilized in various matrices such as polyurethane foam (PUF), sodium alginate (SA), sodium alginate-polyvinyl alcohol (SA-PVA), agar and polyacrylamide. The rate of degradation of 15 and 30 mM 2-nitrotoluene by freely suspended cells and immobilized cells in batches and fed-batch with shaken cultures were compared. The PUF-immobilized cells achieved higher degradation of 15 and 30 mM 2-nitrotoluene than freely suspended cells and the cells immobilized in SA-PVA, polyacrylamide, SA and agar. The PUF-immobilized cells could be reused more than 24 cycles without loosing their degradation capacity and showed more tolerance to pH and temperature changes than freely suspended cells. These results revealed the enhanced rate of degradation of 2-nitrotoluene by PUF-immobilized cells of Micrococcus sp. strain SMN-1. Copyright © 2012 Elsevier Ltd. All rights reserved.

5-(2-Carboxyethenyl) isatin derivative induces G{sub 2}/M cell cycle arrest and apoptosis in human leukemia K562 cells

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Zhou, Yao; Zhao, Hong-Ye; Han, Kai-Lin; Yang, Yao; Song, Bin-Bin; Guo, Qian-Nan [Key Laboratory of Industrial Microbiology, Ministry of Education, College of Biotechnology, Tianjin University of Science and Technology, Tianjin 300457 (China); Tianjin Key Laboratory of Industry Microbiology, College of Biotechnology, Tianjin University of Science and Technology, Tianjin 300457 (China); Fan, Zhen-Chuan [Key Laboratory of Food Nutrition and Safety (Tianjin University of Science and Technology), Ministry of Education, Tianjin 300457 (China); Obesita and Algaegen LLC, College Station, TX 77845 (United States); Zhang, Yong-Min [Université Pierre et Marie Curie-Paris 6, Institut Parisien de Chimie Moléculaire UMR CNRS 8232, 4 Place Jussieu, 75005 Paris (France); Teng, Yu-Ou, E-mail: tyo201485@tust.edu.cn [Key Laboratory of Industrial Microbiology, Ministry of Education, College of Biotechnology, Tianjin University of Science and Technology, Tianjin 300457 (China); Tianjin Key Laboratory of Industry Microbiology, College of Biotechnology, Tianjin University of Science and Technology, Tianjin 300457 (China); Yu, Peng, E-mail: yupeng@tust.edu.cn [Key Laboratory of Industrial Microbiology, Ministry of Education, College of Biotechnology, Tianjin University of Science and Technology, Tianjin 300457 (China); Tianjin Key Laboratory of Industry Microbiology, College of Biotechnology, Tianjin University of Science and Technology, Tianjin 300457 (China)

2014-08-08

Highlights: • 5-(2-Carboxyethenyl) isatin derivative (HKL 2H) inhibited K562’s proliferation. • HKL 2H caused the morphology change of G{sub 2}/M phase arrest and typical apoptosis. • HKL 2H induced G2/M cell cycle phase arrest in K562 cells. • HKL 2H induced apoptosis in K562 cells through the mitochondrial pathway. - Abstract: Our previous study successfully identified that the novel isatin derivative (E)-methyl 3-(1-(4-methoxybenzyl)-2,3-dioxoindolin-5-yl) acrylate (HKL 2H) acts as an anticancer agent at an inhibitory concentration (IC{sub 50}) level of 3 nM. In this study, the molecular mechanism how HKL 2H induces cytotoxic activity in the human chronic myelogenous leukemia K562 cells was investigated. Flow cytometric analysis showed that the cells were arrested in the G{sub 2}/M phase and accumulated subsequently in the sub-G{sub 1} phase in the presence of HKL 2H. HKL 2H treatment down-regulated the expressions of CDK1 and cyclin B but up-regulated the level of phosphorylated CDK1. Annexin-V staining and the classic DNA ladder studies showed that HKL 2H induced the apoptosis of K562 cells. Our study further showed that HKL 2H treatment caused the dissipation of mitochondrial membrane potential, activated caspase-3 and lowered the Bcl-2/Bax ratio in K562 cells, suggesting that the HKL 2H-causing programmed cell death of K562 cells was caused via the mitochondrial apoptotic pathway. Taken together, our data demonstrated that HKL 2H, a 5-(2-carboxyethenyl) isatin derivative, notably induces G{sub 2}/M cell cycle arrest and mitochondrial-mediated apoptosis in K562 cells, indicating that this compound could be a promising anticancer candidate for further investigation.

Cyclic strain-induced endothelial MMP-2: role in vascular smooth muscle cell migration

International Nuclear Information System (INIS)

Sweeney, Nicholas von Offenberg; Cummins, Philip M.; Birney, Yvonne A.; Redmond, Eileen M.; Cahill, Paul A.

2004-01-01

Matrix metalloproteinases (MMPs) play a vital role in vasculature response to hemodynamic stimuli via the degradation of extracellular matrix substrates. In this study, we investigated the putative role of cyclic strain-induced endothelial MMP-2 (and MMP-9) expression and release in modulating bovine aortic smooth muscle cell (BASMC) migration in vitro. Equibiaxial cyclic strain of bovine aortic endothelial cells (BAECs) leads to elevation in cellular MMP-2 (and MMP-9) expression, activity, and secretion into conditioned media, events which were time- and force-dependent. Subsequent incubation of BASMCs with conditioned media from chronically strained BAECs (5%, 24 h) significantly reduces BASMC migration (38 ± 6%), an inhibitory effect which could be completely reversed by targeted siRNA 'knock-down' of MMP-2 (but not MMP-9) expression and activity in BAECs. Moreover, inhibition of strain-mediated MMP-2 expression in BAECs by protein tyrosine kinase (PTK) blockade with genistein (50 μM) was also found to completely reverse this inhibitory effect on BASMC migration. Finally, direct supplementation of recombinant MMP-2 into the BASMC migration assay was found to have no significant effect on migration. However, the effect on BASMC migration of MMP-2 siRNA transfection in BAECs could be reversed by supplementation of recombinant MMP-2 into BAEC media prior to (and for the duration of) strain. These findings reveal a potentially novel role for strain-induced endothelial MMP-2 in regulating vascular SMC migration

The GAS5/miR-222 Axis Regulates Proliferation of Gastric Cancer Cells Through the PTEN/Akt/mTOR Pathway.

Science.gov (United States)

Li, Yanhua; Gu, Junjiao; Lu, Hong

2017-12-01

Several lines of evidence have indicated that growth arrest-specific transcript 5 (GAS5) functions as a tumor suppressor and is aberrantly expressed in multiple cancers. GAS5 was found to be downregulated in gastric cancer (GC) tissues, and ectopic expression of GAS5 inhibited GC cell proliferation. The present study aimed to explore the underlying mechanisms of GAS5 involved in GC cell proliferation. GAS5 and miR-222 expressions in GC cell lines were estimated by quantitative real-time polymerase chain reaction. The effects of GAS5 and miR-222 on GC cell proliferation were assessed by MTT assay and 5-bromo-2-deoxyuridine (BrdU) incorporation assays. The interaction between GAS5 and miR-222 was confirmed by luciferase reporter assay and RNA immunoprecipitation assay. The protein levels of the phosphatase and tensin homolog (PTEN), phosphorylated protein kinase B (Akt) (p-Akt), Akt, phosphorylated mammalian target of rapamycin (mTOR) (p-mTOR), and mTOR were determined by western blot. GAS5 was downregulated and miR-222 was upregulated in GC cells. GAS5 directly targeted and suppressed miR-222 expression. GAS5 overexpression and miR-222 inhibition suppressed cell proliferation, increased PTEN protein level and decreased p-Akt and p-mTOR protein levels in GC cells while GAS5 knockdown and miR-222 overexpression exhibited the opposite effects. Moreover, mechanistic analyses revealed that GAS5 regulated GC cell proliferation through the PTEN/Akt/mTOR pathway by negatively regulating miR-222. GAS5/miR-222 axis regulated proliferation of GC cells through the PTEN/Akt/mTOR pathway, which facilitated the development of lncRNA-directed therapy against this deadly disease.

Solitary waves in morphogenesis: Determination fronts as strain-cued strain transformations among automatous cells

Science.gov (United States)

Cox, Brian N.; Landis, Chad M.

2018-02-01

We present a simple theory of a strain pulse propagating as a solitary wave through a continuous two-dimensional population of cells. A critical strain is assumed to trigger a strain transformation, while, simultaneously, cells move as automata to tend to restore a preferred cell density. We consider systems in which the strain transformation is a shape change, a burst of proliferation, or the commencement of growth (which changes the shape of the population sheet), and demonstrate isomorphism among these cases. Numerical and analytical solutions describe a strain pulse whose height does not depend on how the strain disturbance was first launched, or the rate at which the strain transformation is achieved, or the rate constant in the rule for the restorative cell motion. The strain pulse is therefore very stable, surviving the imposition of strong perturbations: it would serve well as a timing signal in development. The automatous wave formulation is simple, with few model parameters. A strong case exists for the presence of a strain pulse during amelogenesis. Quantitative analysis reveals a simple relationship between the velocity of the leading edge of the pulse in amelogenesis and the known speed of migration of ameloblast cells. This result and energy arguments support the depiction of wave motion as an automatous cell response to strain, rather than as a response to an elastic energy gradient. The theory may also contribute to understanding the determination front in somitogenesis, moving fronts of convergent-extension transformation, and mitotic wavefronts in the syncytial drosophila embryo.

Heterologous expression of Ralp3 in Streptococcus pyogenes M2 and M6 strains affects the virulence characteristics.

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Nikolai Siemens

Full Text Available BACKGROUND: Ralp3 is a transcriptional regulator present in a serotype specific fashion on the chromosome of the human pathogen Streptococcus pyogenes (group A streptococci, GAS. In serotypes harbouring the ralp3 gene either positive or negative effects on important metabolic and virulence genes involved in colonization and immune evasion in the human host were observed. A previous study revealed that deletion of ralp3 in a GAS M49 serotype significantly attenuated many virulence traits and caused metabolic disadvantages. This leads to two questions: (i which kind of consequences could Ralp3 expression have in GAS serotypes naturally lacking this gene, and (ii is Ralp3 actively lost during evolution in these serotypes. METHODOLOGY/PRINCIPAL FINDINGS: We investigated the role of Ralp3 in GAS M2 and M6 pathogenesis. Both serotypes lack ralp3 on their chromosome. The heterologous expression of ralp3 in both serotypes resulted in reduced attachment to and internalization into the majority of tested epithelial cells. Both ralp3 expression strains showed a decreased ability to survive in human blood and exclusively M2::ralp3 showed decreased survival in human serum. Both mutants secreted more active SpeB in the supernatant, resulting in a higher activity compared to wild type strains. The respective M2 and M6 wild type strains outcompeted the ralp3 expression strains in direct metabolic competition assays. The phenotypic changes observed in the M2:ralp3 and M6:ralp3 were verified on the transcriptional level. Consistent with the virulence data, tested genes showed transcript level changes in the same direction. CONCLUSIONS/SIGNIFICANCE: Together these data suggest that Ralp3 can take over transcriptional control of virulence genes in serotypes lacking the ralp3 gene. Those serotypes most likely lost Ralp3 during evolution since obviously expression of this gene is disadvantageous for metabolism and pathogenesis.

A new human male diploid cell strain, TIG-7: its age-related changes and comparison with a matched female TIG-1 cell strain.

Science.gov (United States)

Yamamoto, K; Kaji, K; Kondo, H; Matsuo, M; Shibata, Y; Tasaki, Y; Utakoji, T; Ooka, H

1991-01-01

A new human diploid cell strain, TIG-7, which has the male karyotype, was established and characterized. Isozyme and histocompatibility typing of the cell strain was performed. The average in vitro life span of the cells is 73 population doublings. Changes in cell volume, doubling time, saturation density, the efficiency of cell attachment, plating efficiency, and relative DNA content were examined during in vitro cellular aging. Hydrocortisone slightly prolongs the life span of the cell strain when the hormone is administered to the cultures during middle passages. The age-related changes in the parameters of TIG-7 are not appreciably different from those of the previously established TIG-1 cell strain. These results show that this cell strain is useful for research on cellular aging; further profit is anticipated from research using a combination of these two sexually different cell strains.

Carbon-flux distribution within Streptomyces coelicolor metabolism: a comparison between the actinorhodin-producing strain M145 and its non-producing derivative M1146.

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Fabien Coze

Full Text Available Metabolic Flux Analysis is now viewed as essential to elucidate the metabolic pattern of cells and to design appropriate genetic engineering strategies to improve strain performance and production processes. Here, we investigated carbon flux distribution in two Streptomyces coelicolor A3 (2 strains: the wild type M145 and its derivative mutant M1146, in which gene clusters encoding the four main antibiotic biosynthetic pathways were deleted. Metabolic Flux Analysis and (13C-labeling allowed us to reconstruct a flux map under steady-state conditions for both strains. The mutant strain M1146 showed a higher growth rate, a higher flux through the pentose phosphate pathway and a higher flux through the anaplerotic phosphoenolpyruvate carboxylase. In that strain, glucose uptake and the flux through the Krebs cycle were lower than in M145. The enhanced flux through the pentose phosphate pathway in M1146 is thought to generate NADPH enough to face higher needs for biomass biosynthesis and other processes. In both strains, the production of NADPH was higher than NADPH needs, suggesting a key role for nicotinamide nucleotide transhydrogenase for redox homeostasis. ATP production is also likely to exceed metabolic ATP needs, indicating that ATP consumption for maintenance is substantial.Our results further suggest a possible competition between actinorhodin and triacylglycerol biosynthetic pathways for their common precursor, acetyl-CoA. These findings may be instrumental in developing new strategies exploiting S. coelicolor as a platform for the production of bio-based products of industrial interest.

Genome Sequence of Selenium-Solubilizing Bacterium Caulobacter vibrioides T5M6

DEFF Research Database (Denmark)

Wang, Yihua; Qin, Yanan; Kot, Witold

2016-01-01

Caulobacter vibrioides T5M6 is a Gram-negative strain that strongly solubilizes selenium (Se) mineral into Se(IV) and was isolated from a selenium mining area in Enshi, southwest China. This strain produces the phytohormone IAA and promotes plant growth. Here we present the genome of this strain...

Susceptibilidad "in vitro" de cepas de Cryptococcus a 5 drogas antifungicas "In vitro" susceptibility of Cryptococcus strains to 5 antifungal drugs

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A. J. Bava

1989-10-01

Full Text Available Se estudió la susceptibilidad "in vitro" de 24 cepas de 3 especies del género Cryptococcus a 5 drogas antifúngicas (anfotericina B, 5 fluorocitosina, ketoconazol, itraconazol y miconazol. Las mismas se agruparon según su especie, variedad y origen de aislamiento. Para determinar la concentración inhibitoria mínima (C.I.M. de cada droga se empleó el método de dilución en agar con el medio básico nitrogenado para levaduras, adicionado de glucosa. Se obtuvo además la media geométrica de estos valores para cada grupo y se comparó cada uno de ellos. Los resultados obtenidos fueron homogéneos con la sola excepción de las cepas de Cryptococcus sp (no neoformans, en las cuales se detectaron elevados valores de C.I.M. para la 5 fluorocitosina.A comparative study of the "in vitro" susceptibility of 24 Cryptococcus strains to 5 antifungal drugs (amphotericin B, 5 fluorocytosine, miconazole, itraconazole and ketoconazole, was carried out. These strains were grouped according to species, varieties and isolation's origins. The minimum inhibitory concentration (M.I.C. was determinated by the agar dilution technique in yeast nitrogen base agar with dextrose. The mean geometrical of the M.I.C. values of each group was compared with the others. The results obtained were homogeneous with the only exception of the "non neoformans" strains, in which, higher M.I.C. to 5 fluorocytosine values were detected.

A cell clone strain from Mythimna separata (Lepidoptera: Noctuidae) highly susceptible to Autographa californica multiple nucleopolyhedrovirus (AcMNPV) and M. separata NPV (MsNPV).

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Meng, Xiang-Qian; Zheng, Gui-Ling; Zhao, Chuan-De; Wan, Fang-Hao; Li, Chang-You

2017-08-01

In this study, we describe a cell line, Ms-10C, cloned from the line QAU-Ms-E-10 (simplified Ms-10), an embryonic line from Mythimna separata. The cloned cell line was significantly more sensitive to nucleopolyhedrovirus (NPV). Ms-10C cells were mainly spherical with a diameter of 14.42 ± 2.23 μm. DNA amplification fingerprinting (DAF) confirmed the profile of PCR-amplified bands of the cloned cell line was consistent with those of the parental cell line, Ms-10. The sequencing result of the mitochondrial cytochrome c oxidase I (mtCO I) fragment confirmed that the amplified 636-bps mtCOI fragment was 100% identical to that of M. separata. Its chromosomes exhibited the typical characters of lepidopteran cell lines. Its population doubling time was 42.2 h at 27°C. Ms-10C was more sensitive than Ms-10 to both Autographa californica multiple nucleopolyhedrovirus (AcMNPV) and M. separata nucleopolyhedrovirus (MsNPV). At 4 d post infection, the infection rates of two viruses reached 94.2 and 92.3%, respectively. The availability of this cell clone strain will provide a useful tool for the basic research on nucleopolyhedrovirus and for potential application in expression of recombinant proteins with baculovirus expression vector system.

Angiogenesis is induced by airway smooth muscle strain.

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Hasaneen, Nadia A; Zucker, Stanley; Lin, Richard Z; Vaday, Gayle G; Panettieri, Reynold A; Foda, Hussein D

2007-10-01

Angiogenesis is an important feature of airway remodeling in both chronic asthma and chronic obstructive pulmonary disease (COPD). Airways in those conditions are exposed to excessive mechanical strain during periods of acute exacerbations. We recently reported that mechanical strain of human airway smooth muscle (HASM) led to an increase in their proliferation and migration. Sustained growth in airway smooth muscle in vivo requires an increase in the nutritional supply to these muscles, hence angiogenesis. In this study, we examined the hypothesis that cyclic mechanical strain of HASM produces factors promoting angiogenic events in the surrounding vascular endothelial cells. Our results show: 1) a significant increase in human lung microvascular endothelial cell (HMVEC-L) proliferation, migration, and tube formation following incubation in conditioned media (CM) from HASM cells exposed to mechanical strain; 2) mechanical strain of HASM cells induced VEGF expression and release; 3) VEGF neutralizing antibodies inhibited the proliferation, migration, and tube formations of HMVEC-L induced by the strained airway smooth muscle CM; 4) mechanical strain of HASM induced a significant increase in hypoxia-inducible factor-1alpha (HIF-1alpha) mRNA and protein, a transcription factor required for VEGF gene transcription; and 5) mechanical strain of HASM induced HIF-1alpha/VEGF through dual phosphatidylinositol 3-kinase (PI3K)/Akt/mammalian target of rapamycin (mTOR) and ERK pathways. In conclusion, exposing HASM cells to mechanical strain induces signal transduction pathway through PI3K/Akt/mTOR and ERK pathways that lead to an increase in HIF-1alpha, a transcription factor required for VEGF expression. VEGF release by mechanical strain of HASM may contribute to the angiogenesis seen with repeated exacerbation of asthma and COPD.

Serotyping of Actinobacillus pleuropneumoniae serotype 5 strains using a monoclonal-based polystyrene agglutination test

DEFF Research Database (Denmark)

Dubreuil, J.D.; Letellier, A.; Stenbæk, Eva

1996-01-01

A polystyrene agglutination test has been developed for serotyping Actinobacillus pleuropneumoniae serotype 5a and 5b strains. Protein A-coated polystyrene microparticles were sensitized with a murine monoclonal antibody recognizing an epitope on serotype 5 LPS-O chain as shown by SDS-PAGE and We......A polystyrene agglutination test has been developed for serotyping Actinobacillus pleuropneumoniae serotype 5a and 5b strains. Protein A-coated polystyrene microparticles were sensitized with a murine monoclonal antibody recognizing an epitope on serotype 5 LPS-O chain as shown by SDS...... suspension of bacterial cells grown for 18 h. All A, pleuropneumoniae strains had been previously serotyped using standard procedures, The polystyrene agglutination test was rapid (less than 3 min) and easy to perform. Overall a very good correlation (97.3%) with the standard techniques was found...

Differences in 5-HT2A and mGlu2 Receptor Expression Levels and Repressive Epigenetic Modifications at the 5-HT2A Promoter Region in the Roman Low- (RLA-I) and High- (RHA-I) Avoidance Rat Strains

DEFF Research Database (Denmark)

Fomsgaard, Luna; Moreno, Jose L; de la Fuente Revenga, Mario

2018-01-01

The serotonin 2A (5-HT2A) and metabotropic glutamate 2 (mGlu2) receptors regulate each other and are associated with schizophrenia. The Roman high- (RHA-I) and the Roman low- (RLA-I) avoidance rat strains present well-differentiated behavioral profiles, with the RHA-I strain emerging as a putativ...

Analysis of the Catecholaminergic Phenotype in Human SH-SY5Y and BE(2-M17 Neuroblastoma Cell Lines upon Differentiation.

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Roberta Filograna

Full Text Available Human cell lines are often used to investigate cellular pathways relevant for physiological or pathological processes or to evaluate cell toxicity or protection induced by different compounds, including potential drugs. In this study, we analyzed and compared the differentiating activities of three agents (retinoic acid, staurosporine and 12-O-tetradecanoylphorbol-13-acetate on the human neuroblastoma SH-SY5Y and BE(2-M17 cell lines; the first cell line is largely used in the field of neuroscience, while the second is still poorly characterized. After evaluating their effects in terms of cell proliferation and morphology, we investigated their catecholaminergic properties by assessing the expression profiles of the major genes involved in catecholamine synthesis and storage and the cellular concentrations of the neurotransmitters dopamine and noradrenaline. Our results demonstrate that the two cell lines possess similar abilities to differentiate and acquire a neuron-like morphology. The most evident effects in SH-SY5Y cells were observed in the presence of staurosporine, while in BE(2-M17 cells, retinoic acid induced the strongest effects. Undifferentiated SH-SY5Y and BE(2-M17 cells are characterized by the production of both NA and DA, but their levels are considerably higher in BE(2-M17 cells. Moreover, the NAergic phenotype appears to be more pronounced in SH-SY5Y cells, while BE(2-M17 cells have a more prominent DAergic phenotype. Finally, the catecholamine concentration strongly increases upon differentiation induced by staurosporine in both cell lines. In conclusion, in this work the catecholaminergic phenotype of the human BE(2-M17 cell line upon differentiation was characterized for the first time. Our data suggest that SH-SY5Y and BE(2-M17 represent two alternative cell models for the neuroscience field.

Analysis of the Catecholaminergic Phenotype in Human SH-SY5Y and BE(2)-M17 Neuroblastoma Cell Lines upon Differentiation.

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Filograna, Roberta; Civiero, Laura; Ferrari, Vanni; Codolo, Gaia; Greggio, Elisa; Bubacco, Luigi; Beltramini, Mariano; Bisaglia, Marco

2015-01-01

Human cell lines are often used to investigate cellular pathways relevant for physiological or pathological processes or to evaluate cell toxicity or protection induced by different compounds, including potential drugs. In this study, we analyzed and compared the differentiating activities of three agents (retinoic acid, staurosporine and 12-O-tetradecanoylphorbol-13-acetate) on the human neuroblastoma SH-SY5Y and BE(2)-M17 cell lines; the first cell line is largely used in the field of neuroscience, while the second is still poorly characterized. After evaluating their effects in terms of cell proliferation and morphology, we investigated their catecholaminergic properties by assessing the expression profiles of the major genes involved in catecholamine synthesis and storage and the cellular concentrations of the neurotransmitters dopamine and noradrenaline. Our results demonstrate that the two cell lines possess similar abilities to differentiate and acquire a neuron-like morphology. The most evident effects in SH-SY5Y cells were observed in the presence of staurosporine, while in BE(2)-M17 cells, retinoic acid induced the strongest effects. Undifferentiated SH-SY5Y and BE(2)-M17 cells are characterized by the production of both NA and DA, but their levels are considerably higher in BE(2)-M17 cells. Moreover, the NAergic phenotype appears to be more pronounced in SH-SY5Y cells, while BE(2)-M17 cells have a more prominent DAergic phenotype. Finally, the catecholamine concentration strongly increases upon differentiation induced by staurosporine in both cell lines. In conclusion, in this work the catecholaminergic phenotype of the human BE(2)-M17 cell line upon differentiation was characterized for the first time. Our data suggest that SH-SY5Y and BE(2)-M17 represent two alternative cell models for the neuroscience field.

Development of a serum-free co-culture of human intestinal epithelium cell-lines (Caco-2/HT29-5M21)

Science.gov (United States)

Nollevaux, Géraldine; Devillé, Christelle; El Moualij, Benaïssa; Zorzi, Willy; Deloyer, Patricia; Schneider, Yves-Jacques; Peulen, Olivier; Dandrifosse, Guy

2006-01-01

Background The absorptive and goblet cells are the main cellular types encountered in the intestine epithelium. The cell lineage Caco-2 is a model commonly used to reproduce the features of the bowel epithelium. However, there is a strong debate regarding the value of Caco-2 cell culture to mimick in vivo situation. Indeed, some authors report in Caco-2 a low paracellular permeability and an ease of access of highly diffusible small molecules to the microvilli, due to an almost complete lack of mucus. The HT29-5M21 intestinal cell lineage is a mucin-secreting cellular population. A co-culture system carried out in a serum-free medium and comprising both Caco-2 and HT29-5M21 cells was developed. The systematic use of a co-culture system requires the characterization of the monolayer under a given experimental procedure. Results In this study, we investigated the activity and localization of the alkaline phosphatase and the expression of IAP and MUC5AC genes to determine a correlation between these markers and the cellular composition of a differentiated monolayer obtained from a mixture of Caco-2 and HT29-5M21 cells. We observed that the culture conditions used (serum-free medium) did not change the phenotype of each cell type, and produced a reproducible model. The alkaline phosphatase expression characterizing Caco-2 cells was influenced by the presence of HT29-5M21 cells. Conclusion The culture formed by 75% Caco-2 and 25% HT29-5M21 produce a monolayer containing the two main cell types of human intestinal epithelium and characterized by a reduced permeability to macromolecules. PMID:16670004

Trichoderma reesei xylanase 5 is defective in the reference strain QM6a but functional alleles are present in other wild-type strains.

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Ramoni, Jonas; Marchetti-Deschmann, Martina; Seidl-Seiboth, Verena; Seiboth, Bernhard

2017-05-01

Trichoderma reesei is a paradigm for the regulation and industrial production of plant cell wall-degrading enzymes. Among these, five xylanases, including the glycoside hydrolase (GH) family 11 XYN1 and XYN2, the GH10 XYN3, and the GH30 XYN4 and XYN6, were described. By genome mining and transcriptome analysis, a further putative xylanase, encoded by xyn5, was identified. Analysis of xyn5 from the genome-sequenced reference strain T. reesei QM6a shows that it encodes a non-functional, truncated form of XYN5. However, non-truncated orthologues are present in other genome sequenced Trichoderma spp., and sequencing of xyn5 in other T. reesei wild-type isolates shows that they harbor a putative functional xyn5 allele. In silico analysis and 3D modeling revealed that the encoded XYN5 has significant structural similarities to xylanases of the GH11 family, including a GH-typical substrate binding groove and a carboxylate pair in the active site. The xyn5 of wild-type strain TUCIM1282 was recombinantly expressed in a T. reesei strain with a (hemi)cellulase-free background and the corresponding protein purified to apparent homogeneity. The pH and temperature optima and the kinetic parameters of the purified XYN5 were pH 4, 50 °C, and V max  = 2646 nkat/mg with a K m of 9.68 mg/ml. This functional xyn5 allele was used to replace the mutated version which led to an overall increase of the xylanolytic activity. These findings are of particular importance as GH11 xylanases are of high biotechnological relevance, and T. reesei is one of the main industrial producers of such lignocellulose-degrading enzymes.

UBL5 is essential for pre-mRNA splicing and sister chromatid cohesion in human cells

DEFF Research Database (Denmark)

Oka, Yasuyoshi; Varmark, Hanne; Vitting-Seerup, Kristoffer

2014-01-01

UBL5 is an atypical ubiquitin-like protein, whose function in metazoans remains largely unexplored. We show that UBL5 is required for sister chromatid cohesion maintenance in human cells. UBL5 primarily associates with spliceosomal proteins, and UBL5 depletion decreases pre-mRNA splicing efficien...

Development of a serum-free co-culture of human intestinal epithelium cell-lines (Caco-2/HT29-5M21

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Schneider Yves-Jacques

2006-05-01

Full Text Available Abstract Background The absorptive and goblet cells are the main cellular types encountered in the intestine epithelium. The cell lineage Caco-2 is a model commonly used to reproduce the features of the bowel epithelium. However, there is a strong debate regarding the value of Caco-2 cell culture to mimick in vivo situation. Indeed, some authors report in Caco-2 a low paracellular permeability and an ease of access of highly diffusible small molecules to the microvilli, due to an almost complete lack of mucus. The HT29-5M21 intestinal cell lineage is a mucin-secreting cellular population. A co-culture system carried out in a serum-free medium and comprising both Caco-2 and HT29-5M21 cells was developed. The systematic use of a co-culture system requires the characterization of the monolayer under a given experimental procedure. Results In this study, we investigated the activity and localization of the alkaline phosphatase and the expression of IAP and MUC5AC genes to determine a correlation between these markers and the cellular composition of a differentiated monolayer obtained from a mixture of Caco-2 and HT29-5M21 cells. We observed that the culture conditions used (serum-free medium did not change the phenotype of each cell type, and produced a reproducible model. The alkaline phosphatase expression characterizing Caco-2 cells was influenced by the presence of HT29-5M21 cells. Conclusion The culture formed by 75% Caco-2 and 25% HT29-5M21 produce a monolayer containing the two main cell types of human intestinal epithelium and characterized by a reduced permeability to macromolecules.

Fixation of potentially lethal radiation damage by post-irradiation exposure of Chinese hamster cells to 0.5 M or 1.5 M NaCl solutions

International Nuclear Information System (INIS)

Raaphorst, G.P.; Dewey, W.C.

1979-01-01

The effect of 0.05 M and 1.5 M NaCl treatments on CHO cells during and after irradiation has been examined. Treatment with either hypotonic or hypertonic salt solutions during and after irradiation resulted in the fixation of radiation damage which would otherwise not be expressed. The half time for fixation was 4 to 5 min, and the increased expression of the potentially lethal damage by anisotonic solutions was mainly characterized by large decreases in the shoulder of the survival curve, as well as by decreases in Dsub(o). Fixation of radiation damage at 37 0 C occurred to a much greater extent for the hypertonic treatment than for the hypotonic treatment and was greater at 37 0 C than at 20 0 C. Although both the hypotonic and hypertonic treatments during and after irradiation reduced or eliminated the repair of sublethal and potentially lethal damage, treatment during irradiation only, radiosensitized the cells when the treatment was hypotonic, and radioprotected the cells when the treatment was hypertonic. These observations are discussed in relation to salt treatments and different temperatures altering competition between repair and fixation of potentially lethal lesions, the number of which depends on the particular salt treatment at the time of irradiation. (author)

MiR-137 inhibited cell proliferation and migration of vascular smooth muscle cells via targeting IGFBP-5 and modulating the mTOR/STAT3 signaling.

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Jin Pan

Full Text Available Abnormal proliferation of vascular smooth muscle cells (VSMCs contributes to the development of cardiovascular diseases. Studies have shown the great impact of microRNAs (miRNAs on the cell proliferation of VSMCs. This study examined the effects of miR-137 on the cell proliferation and migration of VSMCs and also explored the underlying molecular mechanisms. The mRNA and protein expression levels were determined by qRT-PCR and western blot assays, respectively. The CCK-8 assay, wound healing assay and transwell migration assay were performed to measure cell proliferation and migration of VSMCs. The miR-137-targeted 3'untranslated region of insulin-like growth factor-binding protein-5 (IGFBP-5 was confirmed by luciferase reporter assay. Platelet-derived growth factor-bb (PDGF-bb treatment enhanced cell proliferation and suppressed the expression of miR-137 in VSMCs. The gain-of-function and loss-of-function assays showed that overexpression of miR-137 suppressed the cell proliferation and migration, and also inhibited the expression of matrix genes of VSMCs; down-regulation of miR-137 had the opposite effects on VSMCs. Bioinformatics analysis and luciferase report assay results showed that IGFBP-5 was a direct target of miR-137, and miR-137 overexpression suppressed the IGFBP-5 expression and down-regulation of miR-137 increased the IGFBP-5 expression in VSMCs. PDGF-bb treatment also increased the IGFBP-5 mRNA expression. In addition, enforced expression of IGFBP-5 reversed the inhibitory effects of miR-137 on cell proliferation and migration of VSMCs. More importantly, overexpression of miR-137 also suppressed the activity of mTOR/STAT3 signaling in VSMCs. Taken together, the results suggest that miR-137 may suppress cell proliferation and migration of VSMCs via targeting IGFBP-5 and modulating mTOR/STAT3 signaling pathway.

Identification of nucleotides in the 5'UTR and amino acids substitutions that are essential for the infectivity of 5'UTR-NS5A recombinant of hepatitis C virus genotype 1b (strain Con1).

Science.gov (United States)

Li, Jinqian; Feng, Shengjun; Liu, Xi; Guo, Mingzhe; Chen, Mingxiao; Chen, Yiyi; Rong, Liang; Xia, Jinyu; Zhou, Yuanping; Zhong, Jin; Li, Yi-Ping

2018-05-01

Genotype 1b strain Con1 represents an important reference in the study of hepatitis C virus (HCV). Here, we aimed to develop an advanced infectious Con1 recombinant. We found that previously identified mutations A1226G/F1464L/A1672S/Q1773H permitted culture adaption of Con1 Core-NS5A (C-5A) recombinant containing 5'UTR and NS5B-3'UTR from JFH1 (genotype 2a), thus acquired additional mutations L725H/F886L/D2415G. C-5A containing all seven mutations (C-5A_7m) replicated efficiently in Huh7.5 and Huh7.5.1 cells and had an increased infectivity in SEC14L2-expressing Huh7.5.1 cells. Incorporation of Con1 NS5B was deleterious to C-5A_7m, however Con1 5'UTR was permissive but attenuated the virus. Nucleotides G1, A4, and G35 primarily accounted for the viral attenuation without affecting RNA translation. C-5A_7m was inhibited dose-dependently by simeprevir and daclatasvir, and substitutions at A4, A29, A34, and G35 conferred resistance to miR-122 antagonism. The novel Con1 5'UTR-NS5A recombinant, adaptive mutations, and critical nucleotides described here will facilitate future studies of HCV culture systems and virus-host interaction. Copyright © 2018 Elsevier Inc. All rights reserved.

Development of the radiation-resistant strain of Moraxella osloensis and effect of penicillin G on its growth

International Nuclear Information System (INIS)

Lim, Sangyong; Yun, Hyejeong; Joe, Minho; Kim, Dongho

2009-01-01

A series of repeated exposures to γ-radiation with intervening outgrowth of survivors was used to develop radioresistant cultures of Moraxella osloensis that have been recognized as potential pathogenic microorganism. The D 10 value of the radiation-resistant strain, 5.903±0.006 kGy, was increased by four-fold compared to the parent wild-type strain, 1.637±0.004 kGy. Since most strains of M. osloensis are sensitive to penicillin, we have surveyed the sensitivity of radiation-resistant strain to this antibiotic. When the optical density was monitored after the addition of penicillin G, the radioresistant strain appeared to be more resistant to only a low concentration of penicillin G (0.5 U/ml) than the parent strain. Interestingly, however, there was no apparent difference in the number of viable cells between both strains. Scanning electron microscope data showed that the resistance cells were generally larger than the parent cells, suggesting that this increase in size may cause a higher optical density of radioresistant cells. In conclusion, radiation mutation does not affect the penicillin resistance of M. osloensis.

Development of the radiation-resistant strain of Moraxella osloensis and effect of penicillin G on its growth

Science.gov (United States)

Lim, Sangyong; Yun, Hyejeong; Joe, Minho; Kim, Dongho

2009-07-01

A series of repeated exposures to γ-radiation with intervening outgrowth of survivors was used to develop radioresistant cultures of Moraxella osloensis that have been recognized as potential pathogenic microorganism. The D10 value of the radiation-resistant strain, 5.903±0.006 kGy, was increased by four-fold compared to the parent wild-type strain, 1.637±0.004 kGy. Since most strains of M. osloensis are sensitive to penicillin, we have surveyed the sensitivity of radiation-resistant strain to this antibiotic. When the optical density was monitored after the addition of penicillin G, the radioresistant strain appeared to be more resistant to only a low concentration of penicillin G (0.5 U/ml) than the parent strain. Interestingly, however, there was no apparent difference in the number of viable cells between both strains. Scanning electron microscope data showed that the resistance cells were generally larger than the parent cells, suggesting that this increase in size may cause a higher optical density of radioresistant cells. In conclusion, radiation mutation does not affect the penicillin resistance of M. osloensis.

Development of the radiation-resistant strain of Moraxella osloensis and effect of penicillin G on its growth

Energy Technology Data Exchange (ETDEWEB)

Lim, Sangyong; Yun, Hyejeong; Joe, Minho [Radiation Research Division for Biotechnology, Korea Atomic Energy Research Institute, Jeongeup 580-185 (Korea, Republic of); Kim, Dongho [Radiation Research Division for Biotechnology, Korea Atomic Energy Research Institute, Jeongeup 580-185 (Korea, Republic of)], E-mail: fungikim@kaeri.re.kr

2009-07-15

A series of repeated exposures to {gamma}-radiation with intervening outgrowth of survivors was used to develop radioresistant cultures of Moraxella osloensis that have been recognized as potential pathogenic microorganism. The D{sub 10} value of the radiation-resistant strain, 5.903{+-}0.006 kGy, was increased by four-fold compared to the parent wild-type strain, 1.637{+-}0.004 kGy. Since most strains of M. osloensis are sensitive to penicillin, we have surveyed the sensitivity of radiation-resistant strain to this antibiotic. When the optical density was monitored after the addition of penicillin G, the radioresistant strain appeared to be more resistant to only a low concentration of penicillin G (0.5 U/ml) than the parent strain. Interestingly, however, there was no apparent difference in the number of viable cells between both strains. Scanning electron microscope data showed that the resistance cells were generally larger than the parent cells, suggesting that this increase in size may cause a higher optical density of radioresistant cells. In conclusion, radiation mutation does not affect the penicillin resistance of M. osloensis.

Work strain in decontamination of hot cells, 2

International Nuclear Information System (INIS)

Kinouchi, Nobuyuki; Ikezawa, Yoshio

1991-01-01

In decontamination of hot cells, the workers should wear suitable protective clothing to protect them from internal exposure and skin contamination. But such protective clothing causes some work strain, especially heat-stress. As a simple method to evaluate quantitative work strain, we used sweat rates of the wearers. In the previous paper, sweat rates for workers with two types of protective clothing were reported. In the present paper, sweat rates under severer working conditions are measured for three types: (1) pressure ventilated blouse; (2) full-face mask and polyethylene coverall; (3) full-face mask and vinyl anorak. The measured values for 65 subjects widely scatter from 0.2 to 2.5 l/h for all the protective clothing. Based on these values, the effects of protective clothing and working conditions (ambient temperature and humidity) on work strain are discussed. (author)

Diversification of the vacAs1m1 and vacAs2m2 strains of Helicobacter pylori in Meriones unguiculatus

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Sandra Mendoza Elizalde

2016-11-01

Full Text Available The bacterium Helicobacter pylori exhibits great genetic diversity, and the pathogenic roles of its virulence factors have been widely studied. However, the evolutionary dynamics of H. pylori strains during stomach colonization are not well characterized. Here, we analyzed the microevolutionary dynamics of the toxigenic strain vacAs1m1, the non-toxigenic strain vacAs2m2, and a combination of both strains in an animal model over time. Meriones unguiculatus were inoculated with the following bacteria: group 1–toxigenic strain vacAs1m1/cagA+/cagE+/babA2+; ST181, group 2–non-toxigenic strain vacAs2m2/ cagA+/ cagE+/ babA2+; ST2901, and group 3–both strains. The gerbils were euthanized at different time points (3, 6, 12 and 18 months. In group 1, genetic alterations were observed at 6 and 12 months. With the combination of both strains, group 3 also exhibited genetic alterations at 3 and 18 months; moreover, a chimera, vacA m1-m2, was detected. Additionally, four new sequence types (STs were reported in the PubMLST database for H. pylori. Synonymous and non-synonymous mutations were analyzed and associated with alterations in amino acids. Microevolutionary analysis of the STs (PHYLOViZ identified in each group revealed many mutational changes in the toxigenic (vacAs1m1 and non-toxigenic (vacAs2m2 strains. Phylogenetic assessments (eBURST did not reveal clonal complexes. Our findings indicate that the toxigenic strain, vacAs1m1, and a combination of toxigenic and non-toxigenic strains acquired genetic material by recombination. The allelic combination, vacAs2m1, displayed the best adaptation in the animal model over time, and a chimera, m1-m2, was also identified, which confirmed previous reports.

Complete Genome Sequence of Escherichia coli Strain WG5

DEFF Research Database (Denmark)

Imamovic, Lejla; Misiakou, Maria-Anna; van der Helm, Eric

2018-01-01

Escherichia coli strain WG5 is a widely used host for phage detection, including somatic coliphages employed as standard ISO method 10705-1 (2000). Here, we present the complete genome sequence of a commercial E. coli WG5 strain.......Escherichia coli strain WG5 is a widely used host for phage detection, including somatic coliphages employed as standard ISO method 10705-1 (2000). Here, we present the complete genome sequence of a commercial E. coli WG5 strain....

Genome analysis of Mycoplasma synoviae strain MS-H, the most common M. synoviae strain with a worldwide distribution.

Science.gov (United States)

Zhu, Ling; Shahid, Muhammad A; Markham, John; Browning, Glenn F; Noormohammadi, Amir H; Marenda, Marc S

2018-02-02

The bacterial pathogen Mycoplasma synoviae can cause subclinical respiratory disease, synovitis, airsacculitis and reproductive tract disease in poultry and is a major cause of economic loss worldwide. The M. synoviae strain MS-H was developed by chemical mutagenesis of an Australian isolate and has been used as a live attenuated vaccine in many countries over the past two decades. As a result it may now be the most prevalent strain of M. synoviae globally. Differentiation of the MS-H vaccine from local field strains is important for epidemiological investigations and is often required for registration of the vaccine. The complete genomic sequence of the MS-H strain was determined using a combination of Illumina and Nanopore methods and compared to WVU-1853, the M. synoviae type strain isolated in the USA 30 years before the parent strain of MS-H, and MS53, a more recent isolate from Brazil. The vaccine strain genome had a slightly larger number of pseudogenes than the two other strains and contained a unique 55 kb chromosomal inversion partially affecting a putative genomic island. Variations in gene content were also noted, including a deoxyribose-phosphate aldolase (deoC) fragment and an ATP-dependent DNA helicase gene found only in MS-H. Some of these sequences may have been acquired horizontally from other avian mycoplasma species. MS-H was somewhat more similar to WVU-1853 than to MS53. The genome sequence of MS-H will enable identification of vaccine-specific genetic markers for use as diagnostic and epidemiological tools to better control M. synoviae.

Regulation of nicotinic receptor subtypes following chronic nicotinic agonist exposure in M10 and SH-SY5Y neuroblastoma cells

DEFF Research Database (Denmark)

Warpman, U; Friberg, L; Gillespie, A

1998-01-01

investigated in human neuroblastoma SH-SY5Y cells (expressing alpha3, alpha5, beta2, and beta4 nAChR subunits). Nicotine exhibited a 14 times lower affinity for the nAChRs in SH-SY5Y cells as compared with M10 cells, whereas epibatidine showed similar affinities for the nAChRs expressed in the two cell lines...

[Vitamin B12-independent strains of Methylophaga marina isolated from Red Sea algae].

Science.gov (United States)

Li, Ts D; Doronina, N V; Ivanova, E G; Trotsenko, Iu A

2007-01-01

Two strains (KM3 and KM5) of halophilic methylobacteria isolated from Red Sea algae do not require vitamin B12 for growth and can use methanol, methylamine, dimethylamine, trimethylamine, dimethyl sulfide, and fructose as sources of carbon and energy. The cells of these strains are gram-negative motile monotrichous (strain KM3) or peritrichous (strain KM5) rods. The strains are strictly aerobic and require Na+ ions but not growth factors for growth. They are oxidase- and catalase-positive and reduce nitrates to nitrites. Both strains can grow in a temperature range of 4 to 37 degrees C (with optimal growth at 29-34 degrees C), at pH between 5.5 and 8.5 (with optimal growth at pH 7.5-8.0), and in a range of salt concentrations between 0.5 and 15% NaCl (with optimal growth at 5-9% NaCl). The phospholipids of these strains are dominated by phosphatidylethanolamine and phosphatidylglycerol and also include phosphatidylcholine, phosphatidylserine, and cardiolipin. The dominant fatty acids are C(16:1omega7c) and C(16:0). The major ubiquinone is Q8. The cells accumulate ectoin, glutamate, and sucrose as intracellular osmoprotectants. The strains implement the 2-keto-3-deoxy-6-phosphogluconate-dependent variant of the ribulose monophosphate pathway. The G+C content of the DNA is 44.4-44.7 mol %. Analysis of the 16S rRNA genes showed that both strains belong to Gammaproteobacteria and have a high degree of homology (99.4%) to Methylophaga marina ATCC 35842T . Based on the data of polyphasic taxonomy, strains KM3 and KM5 are identified as new strains M. marina KM3 (VKM B-2386) and M. marina KM5 (VKM B-2387). The ability of these strains to produce auxins (indole-3-acetic acid) suggests their metabolic association with marine algae.

Cloning and expression analysis of genes encoding lytic endopeptidases L1 and L5 from Lysobacter sp. strain XL1.

Science.gov (United States)

Lapteva, Y S; Zolova, O E; Shlyapnikov, M G; Tsfasman, I M; Muranova, T A; Stepnaya, O A; Kulaev, I S; Granovsky, I E

2012-10-01

Lytic enzymes are the group of hydrolases that break down structural polymers of the cell walls of various microorganisms. In this work, we determined the nucleotide sequences of the Lysobacter sp. strain XL1 alpA and alpB genes, which code for, respectively, secreted lytic endopeptidases L1 (AlpA) and L5 (AlpB). In silico analysis of their amino acid sequences showed these endopeptidases to be homologous proteins synthesized as precursors similar in structural organization: the mature enzyme sequence is preceded by an N-terminal signal peptide and a pro region. On the basis of phylogenetic analysis, endopeptidases AlpA and AlpB were assigned to the S1E family [clan PA(S)] of serine peptidases. Expression of the alpA and alpB open reading frames (ORFs) in Escherichia coli confirmed that they code for functionally active lytic enzymes. Each ORF was predicted to have the Shine-Dalgarno sequence located at a canonical distance from the start codon and a potential Rho-independent transcription terminator immediately after the stop codon. The alpA and alpB mRNAs were experimentally found to be monocistronic; transcription start points were determined for both mRNAs. The synthesis of the alpA and alpB mRNAs was shown to occur predominantly in the late logarithmic growth phase. The amount of alpA mRNA in cells of Lysobacter sp. strain XL1 was much higher, which correlates with greater production of endopeptidase L1 than of L5.

Comparative analysis of conventional natural killer cell responses to acute infection with Toxoplasma gondii strains of different virulence

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Daria L Ivanova

2016-09-01

Full Text Available Conventional Natural Killer Cells (cNK, members of group 1 innate lymphoid cells, are a diverse cell subpopulation based on surface receptor expression, maturation and functional potential. cNK cells are critical for early immunity to T. gondii via IFNγ production. Acute cNK cell responses to infection with different strains of T. gondii have not yet been characterized in detail. Here we comprehensively performed this analysis with Type I virulent RH, and Type II avirulent ME49 and fully attenuated Type I cps1-1 strains. In response to these three parasite strains, murine cNK cells produce IFNγ, become cytotoxic and polyfunctional (IFNγ+CD107a+ at the site of infection. In contrast to virulent RH and avirulent ME49 T. gondii strains, attenuated cps1-1 induced only local cNK cell responses. Infections with RH and ME49 parasites significantly decreased cNK cell frequency and numbers in spleen 5 days post infection compared to cps1-1 parasites. cNK cell subsets expressing activating receptors Ly49H, Ly49D, NKG2D and inhibitory receptors Ly49I and NKG2A/CD94 were similar when compared between the strains and at 5 days post infection. cNK cells were not proliferating (Ki67- 5 days post infection with any of the strains. cNK cell maturation as measured by CD27, CD11b and KLRG1 was affected after infection with different parasite strains. RH and ME49 infection significantly reduced mature cNK cell frequency and increased immature cNK cell populations compared to cps1-1 infection. Interestingly, KLRG1 was highly expressed on immature cNK cells after RH infection. After RH and ME49 infections, CD69+ cNK cells were present at higher numbers than after cps1-1 infection, which may correlate with loss of the mature cNK cell population. Cytokine multiplex analysis indicated cNK cell responses correlated with peritoneal exudate cell (PEC, spleen and serum proinflammatory cytokine levels including IL-12. qPCR analysis of parasite-specific B1 gene revealed

Uranium uptake by immobilized cells of Pseudomonas strain EPS 5028

International Nuclear Information System (INIS)

Pons, M.P.; Fuste, M.C.

1993-01-01

Polyacrylamide-gel-immobilized cells of Pseudomonas strain EPS 5028 were effective in the removal of uranium (U) from synthetic effluents. Metal accumulation was performed in an open system in columns filled with immobilized cells that were challenged with continuous flows containing U. Possible variable of the system were studied. Uranium uptake by the immobilized cells of this microorganism was affected by pH but not by temperature or flow rate. In addition, U binding could be interpreted in terms of the Freundlich adsorption isotherm indicating single-layer adsorption. The feasibility of reusing the immobilized cells was suggested after the recovery of U with a solution of 0.1 M sodium carbonate. (orig.)

Bioactivity characterization of Lactobacillus strains isolated from dairy products

Science.gov (United States)

Haghshenas, Babak; Nami, Yousef; Haghshenas, Minoo; Abdullah, Norhafizah; Rosli, Rozita; Radiah, Dayang; Yari Khosroushahi, Ahmad

2015-01-01

This study aimed to find candidate strains of Lactobacillus isolated from sheep dairy products (yogurt and ewe colostrum) with probiotic and anticancer activity. A total of 100 samples were randomly collected from yogurt and colostrum and 125 lactic acid bacteria were isolated. Of these, 17 Lactobacillus strains belonging to five species (L. delbrueckii, L. plantarum, L. rhamnosus, L. paracasei, and L. casei) were identified. L. plantarum 17C and 13C, which isolated from colostrums, demonstrated remarkable results such as resistant to low pH and high concentrations of bile salts, susceptible to some antibiotics and good antimicrobial activity that candidate them as potential probiotics. Seven strains (1C, 5C, 12C, 13C, 17C, 7M, and 40M), the most resistant to simulated digestion, were further investigated to evaluate their capability to adhere to human intestinal Caco-2 cells. L. plantarum 17C was the most adherent strain. The bioactivity assessment of L. plantarum 17C showed anticancer effects via the induction of apoptosis on HT-29 human cancer cells and negligible side effects on one human epithelial normal cell line (FHs 74). The metabolites produced by this strain can be used as alternative pharmaceutical compounds with promising therapeutic indices because they are not cytotoxic to normal mammalian cells. PMID:26219634

Survey of radiosensitivity in a variety of human cell strains

Energy Technology Data Exchange (ETDEWEB)

Arlett, C.F.; Harcourt, S.A.

1980-03-01

Gamma-ray sensitivity for cell killing was assayed in 54 human cell strains, including some derived from individuals suffering from certain hereditary diseases. The overall range of Do values in this study was 38 to 180 rads, indicating a considerable range of variability in humans. The normal sensitivity was described by a range of Do values of 97 to 180 rads. All ten ataxia telangiectasia cell strains tested proved radiosensitive and gave a mean Do value of 57 +- 15 (S.E.) rads, and these represent the most radiosensitive human skin fibroblasts currently available. Representative cell strains from familial retinoblastoma, Fanconi's anemia, and Hutchinson-Gilford progeria occupied positions of intermediate sensitivity, as did one of two ataxia telangiectasia heterozygotes. Six xeroderma pigmentosum cell strains together with two Cockayne's syndrome cell strains (all known to be sensitive to ultraviolet light) fell into the normal range, indicating an absence of cross-sensitivity between ultraviolet light and gamma-irradiation.

Trichoderma strains- Silybum marianum hairy root cultures interactions

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T. Hasanloo

2015-03-01

Full Text Available Background and objectives: Silymarin is a unique flavonoid complex with documented hepatoprotective properties. Silybum  marianum hairy root culture as a source for producing silymarin has been an important strategy for study the cell signaling pathway. In the present investigation Trichoderma strains- Silybum marianum hairy root cultures interactions have been studied. Methods: The effects of two Trichoderma Strains (KHB and G46-7 (0, 0.5, 1, 2 and 4 mg/ 50 mL culture in 6 different exposure times (0, 24, 48, 72, 96 and 120 h have been investigated on flavonolignans production. The flavonolignans were analyzed by High Performance Liquid Chromatography method. Cell signaling pathway was evaluated by determination of H2O2 content, peroxidase and ascorbate peroxidase activities. Results:The elicitation effects of two Trichoderma Strains (KHB and G46-7 were examined on flavonolignans accumulation and the activation of cell defense system in S. marianum hairy root cultures. The results indicated that the highest silymarin accumulation (0.45 and 0.33 mg/g DW was obtained in media elicited with 0.5 mg/50 mL cultures of T. harzianum Strains (KHB and G46-3, respectively after 120 h. Feeding time experiments indicated that a significant higher content of silymarin production was achieved after 120 and 72 h in media treated with 0.5 mg/50 mL cultures of KHB and G46-3, respectively. Our results showed that S. marianum treated by KHB strain, increased taxifolin, silychristin, isosilybin and silydianin productions significantly. The H2O2 content in the control hairy root cultures remained lower than the treated cultures. There was significant enhancement in both peroxidase and ascorbate peroxidase activities in treated hairy roots reaching a peak after 72 h. Conclusion: These findings suggested that some Trichoderma strains are positive elicitors for promoting silymarin accumulation in S. marianum hairy root cultures. The results also suggested the presence

Mechanical strain modulates age-related changes in the proliferation and differentiation of mouse adipose-derived stromal cells

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Chiang Wen-Sheng

2010-03-01

Full Text Available Abstract Background Previous studies on the effects of aging in human and mouse mesenchymal stem cells suggest that a decline in the number and differentiation potential of stem cells may contribute to aging and aging-related diseases. In this report, we used stromal cells isolated from adipose tissue (ADSCs of young (8-10 weeks, adult (5 months, and old (21 months mice to test the hypothesis that mechanical loading modifies aging-related changes in the self-renewal and osteogenic and adipogenic differentiation potential of these cells. Results We show that aging significantly reduced the proliferation and increased the adipogenesis of ADSCs, while the osteogenic potential is not significantly reduced by aging. Mechanical loading (10% cyclic stretching, 0.5 Hz, 48 h increased the subsequent proliferation of ADSCs from mice of all ages. Although the number of osteogenic colonies with calcium deposition was increased in ADSCs subjected to pre-strain, it resulted from an increase in colony number rather than from an increase in osteogenic potential after strain. Pre-strain significantly reduced the number of oil droplets and the expression of adipogenic marker genes in adult and old ADSCs. Simultaneously subjecting ADSCs to mechanical loading and adipogenic induction resulted in a stronger inhibition of adipogenesis than that caused by pre-strain. The reduction of adipogenesis by mechanical strain was loading-magnitude dependent: loading with 2% strain only resulted in a partial inhibition, and loading with 0.5% strain could not inhibit adipogenesis in ADSCs. Conclusions We demonstrate that mechanical stretching counteracts the loss of self-renewal in aging ADSCs by enhancing their proliferation and, at the same time, reduces the heightened adipogenesis of old cells. These findings are important for the further study of stem cell control and treatment for a variety of aging related diseases.

RibM from Streptomyces davawensis is a riboflavin/roseoflavin transporter and may be useful for the optimization of riboflavin production strains

Science.gov (United States)

2011-01-01

Background The bacterium Bacillus subtilis, which is not a natural riboflavin overproducer, has been converted into an excellent production strain by classical mutagenesis and metabolic engineering. To our knowledge, the enhancement of riboflavin excretion from the cytoplasm of overproducing cells has not yet been considered as a target for (further) strain improvement. Here we evaluate the flavin transporter RibM from Streptomyces davawensis with respect to improvement of a riboflavin production strain. Results The gene ribM from S. davawensis, coding for a putative facilitator of riboflavin uptake, was codon optimized (ribMopt) for expression in B. subtilis. The gene ribMopt was functionally introduced into B. subtilis using the isopropyl-β-thiogalactopyranoside (IPTG)-inducible expression plasmid pHT01: Northern-blot analysis of total RNA from IPTG treated recombinant B. subtilis cells revealed a ribMopt specific transcript. Western blot analysis showed that the his6-tagged heterologous gene product RibM was present in the cytoplasmic membrane. Expression of ribM in Escherichia coli increased [14C]riboflavin uptake, which was not affected by the protonophore carbonyl cyanide m-chlorophenylhydrazone (CCCP). Expression of ribMopt supported growth of a B. subtilis ΔribB::Ermr ΔribU::Kanr double mutant deficient in riboflavin synthesis (ΔribB) and also deficient with respect to riboflavin uptake (ΔribU). Expression of ribMopt increased roseoflavin (a toxic riboflavin analog produced by S. davawensis) sensitivity of a B. subtilis ΔribU::Kanr strain. Riboflavin synthesis by a model riboflavin B. subtilis production strain overproducing RibM was increased significantly depending on the amount of the inducer IPTG. Conclusions The energy independent flavin facilitator RibM could in principle catalyze riboflavin export and thus may be useful to increase the riboflavin yield in a riboflavin production process using a recombinant RibM overproducing B. subtilis strain

Regulation of thrombomodulin expression and release in human aortic endothelial cells by cyclic strain.

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Fiona A Martin

Full Text Available Thrombomodulin (TM, an integral membrane glycoprotein expressed on the lumenal surface of vascular endothelial cells, promotes anti-coagulant and anti-inflammatory properties. Release of functional TM from the endothelium surface into plasma has also been reported. Much is still unknown however about how endothelial TM is regulated by physiologic hemodynamic forces (and particularly cyclic strain intrinsic to endothelial-mediated vascular homeostasis.This study employed human aortic endothelial cells (HAECs to investigate the effects of equibiaxial cyclic strain (7.5%, 60 cycles/min, 24 hrs, and to a lesser extent, laminar shear stress (10 dynes/cm2, 24 hrs, on TM expression and release. Time-, dose- and frequency-dependency studies were performed.Our initial studies demonstrated that cyclic strain strongly downregulated TM expression in a p38- and receptor tyrosine kinase-dependent manner. This was in contrast to the upregulatory effect of shear stress. Moreover, both forces significantly upregulated TM release over a 48 hr period. With continuing focus on the cyclic strain-induced TM release, we noted both dose (0-7.5% and frequency (0.5-2.0 Hz dependency, with no attenuation of strain-induced TM release observed following inhibition of MAP kinases (p38, ERK-1/2, receptor tyrosine kinase, or eNOS. The concerted impact of cyclic strain and inflammatory mediators on TM release from HAECs was also investigated. In this respect, both TNFα (100 ng/ml and ox-LDL (10-50 µg/ml appeared to potentiate strain-induced TM release. Finally, inhibition of neither MMPs (GM6001 nor rhomboids (3,4-dichloroisocoumarin had any effect on strain-induced TM release. However, significantly elevated levels (2.1 fold of TM were observed in isolated microparticle fractions following 7.5% strain for 24 hrs.A preliminary in vitro investigation into the effects of cyclic strain on TM in HAECs is presented. Physiologic cyclic strain was observed to downregulate TM

Activation of mitochondrial promoter PH-binding protein in a radio-resistant Chinese hamster cell strain associated with Bcl-2

International Nuclear Information System (INIS)

Roychoudhury, Paromita; Ghosh, Utpal; Bhattacharyya, Nitai P.; Chaudhuri, Keya

2006-01-01

The cellular response to ionizing radiation is mediated by a complex interaction of number of proteins involving different pathways. Previously, we have shown that up regulation of mitochondrial genes ND1, ND4, and COX1 transcribed from the heavy strand promoter (P H ) has been increased in a radio-resistant cell strain designated as M5 in comparison with the parental Chinese hamster V79 cells. These genes are also up regulated in Chinese hamster V79 cells VB13 that express exogenous human Bcl2. In the present study, the expression of the gene ND6 that is expressed from the light strand promoter (P L ) was found to be similar in both the cell lines, as determined by RT-PCR. To test the possibility that this differential expression of mitochondrial genes under these two promoters was mediated by differences in proteins' affinity to interact with these promoters, we have carried out electrophoretic mobility shift assay (EMSA) using mitochondrial cell extracts from these two cell lines. Our result of these experiments revealed that two different proteins formed complex with the synthetic promoters and higher amount of protein from M5 cell extracts interacted with the P H promoter in comparison to that observed with cell extracts from Chinese hamster V79 cells. The promoter-specific differential binding of proteins was also observed in VB13. These results showed that differential mitochondrial gene expression observed earlier in the radio-resistant M5 cells was due to enhanced interaction proteins with the promoters P H and mediated by the expression of Bcl2

Strain and Vibration in Mesenchymal Stem Cells

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Brooke McClarren

2018-01-01

Full Text Available Mesenchymal stem cells (MSCs are multipotent cells capable of differentiating into any mesenchymal tissue, including bone, cartilage, muscle, and fat. MSC differentiation can be influenced by a variety of stimuli, including environmental and mechanical stimulation, scaffold physical properties, or applied loads. Numerous studies have evaluated the effects of vibration or cyclic tensile strain on MSCs towards developing a mechanically based method of differentiation, but there is no consensus between studies and each investigation uses different culture conditions, which also influence MSC fate. Here we present an overview of the response of MSCs to vibration and cyclic tension, focusing on the effect of various culture conditions and strain or vibration parameters. Our review reveals that scaffold type (e.g., natural versus synthetic; 2D versus 3D can influence cell response to vibration and strain to the same degree as loading parameters. Hence, in the efforts to use mechanical loading as a reliable method to differentiate cells, scaffold selection is as important as method of loading.

CYP3A5 mRNA degradation by nonsense-mediated mRNA decay.

Science.gov (United States)

Busi, Florent; Cresteil, Thierry

2005-09-01

The total CYP3A5 mRNA level is significantly greater in carriers of the CYP3A5*1 allele than in CYP3A5*3 homozygotes. Most of the CYP3A5*3 mRNA includes an intronic sequence (exon 3B) containing premature termination codons (PTCs) between exons 3 and 4. Two models were used to investigate the degradation of CYP3A5 mRNA: a CYP3A5 minigene consisting of CYP3A5 exons and introns 3 to 6 transfected into MCF7 cells, and the endogenous CYP3A5 gene expressed in HepG2 cells. The 3'-untranslated region g.31611C>T mutation has no effect on CYP3A5 mRNA decay. Splice variants containing exon 3B were more unstable than wild-type (wt) CYP3A5 mRNA. Cycloheximide prevents the recognition of PTCs by ribosomes: in transfected MCF7 and HepG2 cells, cycloheximide slowed down the degradation of exon 3B-containing splice variants, suggesting the participation of nonsense-mediated decay (NMD). When PTCs were removed from pseudoexon 3B or when UPF1 small interfering RNA was used to impair the NMD mechanism, the decay of the splice variant was reduced, confirming the involvement of NMD in the degradation of CYP3A5 splice variants. Induction could represent a source of variability for CYP3A5 expression and could modify the proportion of splice variants. The extent of CYP3A5 induction was investigated after exposure to barbiturates or steroids: CYP3A4 was markedly induced in a pediatric population compared with untreated neonates. However, no effect could be detected in either the total CYP3A5 RNA, the proportion of splice variant RNA, or the protein level. Therefore, in these carriers, induction is unlikely to switch on the phenotypic CYP3A5 expression in carriers of CYP3A5*3/*3.

5-hydroxytryptamine1C receptor density and mRNA levels in choroid plexus epithelial cells after treatment with mianserin and (-)-1-(4-bromo-2,5-dimethoxyphenyl)-2-aminopropane.

Science.gov (United States)

Barker, E L; Sanders-Bush, E

1993-10-01

5-Hydroxytryptamine (5HT)1C and 5HT2 receptors display paradoxical down-regulation when exposed to receptor antagonists in vivo, a property that is unique to these two subtypes of serotonin (5HT) receptors. Because of the absence of cell culture model systems, the mechanisms involved in this paradoxical down-regulation have been difficult to explore. The present study focuses on the regulation of 5HT1C receptors in primary cultures of rat choroid plexus epithelial cells. Exposure of the epithelial cell cultures to 100 nM mianserin, a receptor antagonist, or (-)-1-(4-bromo-2,5-dimethoxyphenyl)-2-aminopropane, an agonist, for 72 hr caused a loss of 5HT1C receptor binding sites, as determined by [3H]mesulergine binding to crude membrane preparations. No significant changes in Kd values were observed. Neither the agonist nor antagonist caused a significant change in binding sites after 24 hr. A solution hybridization assay was used to determine whether the down-regulation by mianserin or (-)-1-(4-bromo-2,5-dimethoxyphenyl)-2-aminopropane was accompanied by a decrease in the steady state level of 5HT1C receptor mRNA. These studies showed that neither treatment caused an alteration in the levels of 5HT1C receptor mRNA. Thus, it is possible to reproduce the in vivo regulatory effects of drugs on 5HT1C receptors in choroid plexus epithelial cells in culture, including the atypical down-regulation by receptor antagonists. Using this cell culture model system, indirect transynaptic effects and decreases in receptor mRNA levels have been ruled out as mechanisms accounting for the down-regulation.

Modifying the 5'-Cap for Click Reactions of Eukaryotic mRNA and To Tune Translation Efficiency in Living Cells.

Science.gov (United States)

Holstein, Josephin M; Anhäuser, Lea; Rentmeister, Andrea

2016-08-26

The 5'-cap is a hallmark of eukaryotic mRNAs and plays fundamental roles in RNA metabolism, ranging from quality control to export and translation. Modifying the 5'-cap may thus enable modulation of the underlying processes and investigation or tuning of several biological functions. A straightforward approach is presented for the efficient production of a range of N7-modified caps based on the highly promiscuous methyltransferase Ecm1. We show that these, as well as N(2) -modified 5'-caps, can be used to tune translation of the respective mRNAs both in vitro and in cells. Appropriate modifications allow subsequent bioorthogonal chemistry, as demonstrated by intracellular live-cell labeling of a target mRNA. The efficient and versatile N7 manipulation of the mRNA cap makes mRNAs amenable to both modulation of their biological function and intracellular labeling, and represents a valuable addition to the chemical biology toolbox. © 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Thermal behavior of Ni (99.967% and 99.5% purity) deformed to an ultra-high strain by high pressure torsion

DEFF Research Database (Denmark)

Zhang, H.W.; Huang, Xiaoxu; Pippan, R.

2010-01-01

Polycrystalline Ni of two purities (99.967% (4N) and 99.5% (2N)) was deformed to an ultra-high strain of εvM = 100 (εvM, von Mises strain) by high pressure torsion at room temperature. The 4N and 2N samples at this strain are nanostructured with an average boundary spacing of 100 nm, a high density...

Phage-resistance linked to cell heterogeneity in the commercial strain Lactobacillus delbrueckii subsp. lactis Ab1.

Science.gov (United States)

Suárez, Viviana B; Maciel, Natalia; Guglielmotti, Daniela; Zago, Miriam; Giraffa, Giorgio; Reinheimer, Jorge

2008-12-10

The aim of this work was to study the relationship between the cell morphological heterogeneity and the phage-resistance in the commercial strain Lactobacillus delbrueckii subsp. lactis Ab1. Two morphological variants (named C and T) were isolated from this strain. Phage-resistant derivatives were isolated from them and the percentage of occurrence of confirmed phage-resistant cells was 0.001% of the total cellular population. Within these phage-resistant cell derivatives there were T (3 out of 4 total isolates) and C (1 out of 4 total isolates) variants. The study of some technological properties (e.g. proteolytic and acidifying activities) demonstrated that most of phage-resistant derivatives were not as good as the parental strain. However, for one derivative (a T variant), the technological properties were better than those of the parental strain. On the other hand, it was possible to determinate that the system of phage-resistance in the T variants was interference in adsorption step, with adsorption rates M.

Methane and Trichloroethylene Oxidation by an Estuarine Methanotroph, Methylobacter sp. Strain BB5.1

OpenAIRE

Smith, Kelly S.; Costello, Andria M.; Lidstrom, Mary E.

1998-01-01

An estuarine methanotroph was isolated from sediment enrichments and designated Methylobacter sp. strain BB5.1. In cells grown on medium with added copper, oxidation of methane and trichloroethylene occurred with similar Ks values, but the Vmax for trichloroethylene oxidation was only 0.1% of the methane oxidation Vmax. Cells grown on low-copper medium did not oxidize trichloroethylene and showed a variable rate of methane oxidation.

Cell surface groups of two picocyanobacteria strains studied by zeta potential investigations, potentiometric titration, and infrared spectroscopy.

Science.gov (United States)

Dittrich, Maria; Sibler, Sabine

2005-06-15

In order to clarify the role of picocyanobacteria in aquatic biogeochemical processes (e.g., calcite precipitation), cell surface properties need to be investigated. An experimental study of the cell surface characteristics of two Synechococcus-type unicellular autotrophic picocyanobacterial strains was carried out. One strain was isolated from Lake Plon and contained phycocyanin, the other strain came from Lago Maggiore and was rich in phycoerythrin. Potentiometric titrations were conducted to determine the different types of sites present on the bacteria cell walls. Infrared spectroscopy allowed characterization of the various functional groups (RNH(2), RCOOH, ROH, RPO(2)) and investigations of zeta potential provided insight into the isoelectrical points of the strains. Titrations reveal three distinct sites on the bacterial surfaces of phycocyanin- and phycoerythrin-rich strains with pK values of 4.8+/-0.3/5.0+/-0.2, 6.6+/-0.2/6.7+/-0.4, and 8.8+/-0.1/8.7+/-0.2, corresponding to carboxyl, phosphate, and amine groups with surface densities of 2.6+/-0.4/7.4+/-1.6 x 10(-4), 1.9+/-0.5/4.4+/-0.8 x 10(-4), and 2.5+/-0.4/4.8+/-0.7 x 10(-4) mol/g of dry bacteria. The deprotonation constants are similar to those of bacterial strains and site densities are also within an order of magnitude of other strains. The phycoerythrin-rich strain had a higher number of binding sites than the phycocyanin-rich strain. The results showed that picocyanobacteria may adsorb either calcium cations or carbonate anions and therefore strongly influence the biogeochemical cycling of calcite in pelagic systems.

Temperature-stress phase diagram of strain glass Ti48.5Ni51.5

International Nuclear Information System (INIS)

Wang, Y.; Ren, X.; Otsuka, K.; Saxena, A.

2008-01-01

The temperature and stress dependence of the properties of a recently discovered strain glass Ti 48.5 Ni 51.5 , which is a glass of frozen local lattice strains, was investigated systematically. It was found that the ideal freezing temperature (T 0 ) of the strain glass decreases with increasing stress. When the stress exceeds a critical value σ c (T), the pseudo-B2 strain glass transforms into B19' martensite. However, the stress-strain behavior associated with such a stress-induced transition showed a crossover at a crossover temperature T CR , which is ∼20 K below T 0 . Above T CR , the sample showed superelastic behavior; however, below T CR , the sample demonstrated plastic behavior. More interestingly, the σ c vs. temperature relation for unfrozen strain glass obeys the Clausius-Clapyeron relationship, whereas that for frozen strain glass disobeys this universal thermodynamic law. A phenomenological explanation is provided for all the phenomena observed, and it is shown that all the anomalous effects come from the broken ergodicity of the glass system and a temperature-dependent relative stability of the martensitic phase. Based on experimental observations, a temperature-stress phase diagram is constructed for this strain glass, which may serve as a guide map for understanding and predicting the properties of strain glass

Novel Acetone Metabolism in a Propane-Utilizing Bacterium, Gordonia sp. Strain TY-5▿

Science.gov (United States)

Kotani, Tetsuya; Yurimoto, Hiroya; Kato, Nobuo; Sakai, Yasuyoshi

2007-01-01

In the propane-utilizing bacterium Gordonia sp. strain TY-5, propane was shown to be oxidized to 2-propanol and then further oxidized to acetone. In this study, the subsequent metabolism of acetone was studied. Acetone-induced proteins were found in extracts of cells induced by acetone, and a gene cluster designated acmAB was cloned on the basis of the N-terminal amino acid sequences of acetone-induced proteins. The acmA and acmB genes encode a Baeyer-Villiger monooxygenase (BVMO) and esterase, respectively. The BVMO encoded by acmA was purified from acetone-induced cells of Gordonia sp. strain TY-5 and characterized. The BVMO exhibited NADPH-dependent oxidation activity for linear ketones (C3 to C10) and cyclic ketones (C4 to C8). Escherichia coli expressing the acmA gene oxidized acetone to methyl acetate, and E. coli expressing the acmB gene hydrolyzed methyl acetate. Northern blot analyses revealed that polycistronic transcription of the acmAB gene cluster was induced by propane, 2-propanol, and acetone. These results indicate that the acmAB gene products play an important role in the metabolism of acetone derived from propane oxidation and clarify the propane metabolism pathway of strain TY-5 (propane → 2-propanol → acetone → methyl acetate → acetic acid + methanol). This paper provides the first evidence for BVMO-dependent acetone metabolism. PMID:17071761

Radioprotective effect of cysteamine in glutathione synthetase-deficient cells

International Nuclear Information System (INIS)

Deschavanne, P.J.; Debieu, D.; Malaise, E.P.; Midander, J.; Revesz, L.

1986-01-01

The radioprotective role of endogenous and exogenous thiols was investigated, with survival as the end-point, after radiation exposure of cells under oxic and hypoxic conditions. Human cell strains originating from a 5-oxoprolinuria patient and from a related control were used. Due to a genetic deficiency in glutathione synthetase, the level of free SH groups, and in particular that of glutathione, is decreased in 5-oxoprolinuria cells. The glutathione synthetase deficient cells have a reduced oxygen enhancement ratio (1.5) compared to control cells (2.7). The radiosensitivity was assessed for both cell strains in the presence of different concentrations of an exogenous radioprotector:cysteamine. At concentrations varying between 0.1 and 20 mM, cysteamine protected the two cell strains to the same extent when irradiated under oxic and hypoxic conditions. The protective effect of cysteamine was lower under hypoxia than under oxic conditions for both cell strains. Consequently, the oxygen enhancement ratio decreased for both cell strains when cysteamine concentration increased. These results suggest that cysteamine cannot replace endogenous thiols as far as they are implicated in the radiobiological oxygen effect. (author)

The correlation between the ultrasonic elastic strain ratio of thyroid cancer and the malignant biological characteristics of cancer cells in the lesions

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Li Ma

2017-04-01

Full Text Available Objective: To study the correlation between the ultrasonic elastic strain ratio of thyroid cancer and the malignant biological characteristics of cancer cells in the lesions. Methods: A total of 90 patients with thyroid cancer who accepted surgical treatment in our hospital between March 2015 and September 2016 were selected as the observation group, and 50 patients who received surgery in our hospital during the same period and were with clear pathological diagnosis of thyroid adenoma were selected as the control group. According to the median of ultrasonic elastic strain ratio, the observing group were further divided into high elastic strain ratio group and low elastic strain ratio group (n=45. RT-PCR was used to determine the mRNA expression of proliferation genes, invasion genes and autophagy genes in lesions of all groups of patients. Results: The mean elastic strain ratio of observation group was significantly lower than that of control group. Proliferation gene FOXA1 mRNA expression in lesions of low elastic strain ratio group and high elastic strain ratio group were higher than those of control group while PP4R1 and Smac mRNA expression were lower than those of control group, and as the elastic strain ratio decreased, the FOXA1 mRNA expression increased while PP4R1 and Smac mRNA expression decreased; invasion genes BCORL1, SATB1, Twist1 and CXCR4 mRNA expression in lesions of low elastic strain ratio group and high elastic strain ratio group were higher than those of control group, and as the elastic strain ratio decreased, the BCORL1, SATB1, Twist1 and CXCR4 mRNA expression increased; autophagy genes ARHI, Beclin1, LC3 and PTEN mRNA expression in lesions of low elastic strain ratio group and high elastic strain ratio group were lower than those of control group, and as the elastic strain ratio decreased, the ARHI, Beclin1, LC3 and PTEN mRNA expression decreased. Conclusion: The ultrasonic elastic strain ratio of thyroid cancer is

Revealing strategies of quorum sensing in Azospirillum brasilense strains Ab-V5 and Ab-V6.

Science.gov (United States)

Fukami, Josiane; Abrantes, Julia Laura Fernandes; Del Cerro, Pablo; Nogueira, Marco Antonio; Ollero, Francisco Javier; Megías, Manuel; Hungria, Mariangela

2018-01-01

Azospirillum brasilense is an important plant-growth promoting bacterium (PGPB) that requires several critical steps for root colonization, including biofilm and exopolysaccharide (EPS) synthesis and cell motility. In several bacteria these mechanisms are mediated by quorum sensing (QS) systems that regulate the expression of specific genes mediated by the autoinducers N-acyl-homoserine lactones (AHLs). We investigated QS mechanisms in strains Ab-V5 and Ab-V6 of A. brasilense, which are broadly used in commercial inoculants in Brazil. Neither of these strains carries a luxI gene, but there are several luxR solos that might perceive AHL molecules. By adding external AHLs we verified that biofilm and EPS production and cell motility (swimming and swarming) were regulated via QS in Ab-V5, but not in Ab-V6. Differences were observed not only between strains, but also in the specificity of LuxR-type receptors to AHL molecules. However, Ab-V6 was outstanding in indole acetic acid (IAA) synthesis and this molecule might mimic AHL signals. We also applied the quorum quenching (QQ) strategy, obtaining transconjugants of Ab-V5 and Ab-V6 carrying a plasmid with acyl-homoserine lactonase. When maize (Zea mays L.) was inoculated with the wild-type and transconjugant strains, plant growth was decreased with the transconjugant of Ab-V5-confirming the importance of an AHL-mediated QS system-but did not affect plant growth promotion by Ab-V6.

Pravastatin and simvastatin inhibit the adhesion, replication and proliferation of Toxoplasma gondii (RH strain) in HeLa cells.

Science.gov (United States)

Sanfelice, Raquel Arruda; da Silva, Suelen Santos; Bosqui, Larissa Rodrigues; Miranda-Sapla, Milena Menegazzo; Barbosa, Bellisa Freitas; Silva, Rafaela José; Ferro, Eloísa A Vieira; Panagio, Luciano Aparecido; Navarro, Italmar Teodorico; Bordignon, Juliano; Conchon-Costa, Ivete; Pavanelli, Wander Rogerio; Almeida, Ricardo Sergio; Costa, Idessania Nazareth

2017-03-01

The conventional treatment for toxoplasmosis with pyrimethamine and sulfadiazine shows toxic effects to the host, and it is therefore necessary to search for new drugs. Some studies suggest the use of statins, which inhibit cholesterol synthesis in humans and also the initial processes of isoprenoid biosynthesis in the parasite. Thus, the objective of this study was to evaluate the activity of the statins pravastatin and simvastatin in HeLa cells infected in vitro with the RH strain of T. gondii. HeLa cells (1×10 5 ) were infected with T. gondii tachyzoites (5×10 5 ) following two different treatment protocols. In the first protocol, T. gondii tachyzoites were pretreated with pravastatin (50 and 100μg/mL) and simvastatin (1.56 and 3.125μg/mL) for 30min prior to infection. In the second, HeLa cells were first infected (5×10 5 ) with tachyzoites and subsequently treated with pravastatin and simvastatin for 24h at the concentrations noted above. Initially, we evaluated the cytotoxicity of drugs by the MTT assay, number of tachyzoites adhered to cells, number of infected cells, and viability of tachyzoites by trypan blue exclusion. The supernatant of the cell cultures was collected post-treatment for determination of the pattern of Th1/Th2/Th17 cytokines by cytometric bead array. There was no cytotoxicity to HeLa cells with 50 and 100μg/mL pravastatin and 1.56 and 3.125μg/mL simvastatin. There was no change in the viability of tachyzoites that received pretreatment. Regarding the pre- and post-treatment of the cells with pravastatin and simvastatin alone, there was a reduction in adhesion, invasion and proliferation of cells to T. gondii. As for the production of cytokines, we found that IL-6 and IL-17 were significantly reduced in cells infected with T. gondii and treated with pravastatin and simvastatin, when compared to control. Based on these results, we can infer that pravastatin and simvastatin alone possess antiproliferative effects on tachyzoites forms

Effect of growth at low pH on the cell surface properties of a typical strain of Lactobacillus casei group.

Science.gov (United States)

Hossein Nezhad, M; Stenzel, Dj; Britz, Ml

2010-09-01

Although members of the Lactobacillus casei group are known to survive under acidic conditions, the underlying mechanisms of growth at acidic condition and the impact of low pH on the relative level of protein expression at the cell surface remain poorly studied. After confirming the taxonomy of L. casei strain GCRL 12 which was originally isolated from cheese and confirmed by 16S rRNA sequence analysis, the impact of acidic pH on growth rate was determined. Late log-phase cells cultured at pH 4.0 showed obvious changes in Gram staining properties while transmission electron microscopy analysis revealed evidence of structural distortions of the cell surface relative to the controls cultured at pH 6.5. When comparing cytosolic or whole cell preparations on SDS-PAGE, few changes in protein profiles were observed under the two growth conditions. However, analysis of surface protein extracted by 5M LiCl demonstrated changes in the proportions of proteins present in the molecular weight range of 10 to 80 kDa, with some proteins more dominant at pH 6.5 and other at pH 4. These data suggest that surface proteins of this strain are associated with growth and survival at low pH. The function of these proteins is subject to further investigation.

The non-structural protein 5 and matrix protein are antigenic targets of T cell immunity to genotype 1 porcine reproductive and respiratory syndrome viruses

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Helen eMokhtar

2016-02-01

Full Text Available The porcine reproductive and respiratory syndrome virus (PRRSV is the cause of one of the most economically important diseases affecting swine worldwide. Efforts to develop a next-generation vaccine have largely focussed on envelope glycoproteins to target virus-neutralising antibody responses. However, these approaches have failed to demonstrate the necessary efficacy to progress towards market. T cells are crucial to the control of many viruses through cytolysis and cytokine secretion. Since control of PRRSV infection is not dependent on the development of neutralising antibodies, it has been proposed that T cell mediated immunity plays a key role. We therefore hypothesised that conserved T cell antigens represent prime candidates for the development a novel PRRS vaccine. Antigens were identified by screening a proteome-wide synthetic peptide library with T cells from cohorts of pigs rendered immune by experimental infections with a closely-related (subtype 1 or divergent (subtype 3 PRRSV-1 strain. Dominant T cell IFN-γ responses were directed against the non-structural protein 5 (NSP5, and to a lesser extent, the matrix (M protein. The majority of NSP5-specific CD8 T cells and M-specific CD4 T cells expressed a putative effector memory phenotype and were polyfunctional as assessed by co-expression of TNF-α and mobilisation of the cytotoxic degranulation marker CD107a. Both antigens were generally well conserved amongst strains of both PRRSV genotypes. Thus M and NSP5 represent attractive vaccine candidate T cell antigens which should be evaluated further in the context of PRRSV vaccine development.

GHGKHKNK Octapeptide (P-5m Inhibits Metastasis of HCCLM3 Cell Lines via Regulation of MMP-2 Expression in in Vitro and in Vivo Studies

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Xun Zhu

2012-02-01

Full Text Available P-5m, an octapeptide derived from domain 5 of HKa, was initially found to inhibit the invasion and migration of melanoma cells. The high metastatic potential of melanoma cells was prevented by the HGK motif in the P-5m peptide in vitro and in an experimental lung metastasis model, suggesting that P-5m may play an important role in the regulation of tumor metastasis. The aim of this study was to measure the effect of P-5m on tumor metastasis of human hepatocarcinoma cell line (HCCLM3 in vitro and in vivo in a nude mouse model of hepatocellular carcinoma (HCC, and detect the mechanisms involved in P-5m-induced anti-metastasis. By gelatin zymography, matrix metallo-proteinases 2 (MMP-2 activity in HCCLM3 was dramatically diminished by P-5m peptide. In addition, the migration and metastasis of HCCLM3 cells was also inhibited by the peptide in vitro. In an orthotopic model of HCC in nude mice, P-5m treatment effectively reduced the lung metastasis as well as the expression of MMP-2 in the tumor tissues. Overall, these observations indicate an important role for P-5m peptide in HCC invasion and metastasis, at least partially through modulation MMP-2 expression. These data suggests that P-5m may have therapeutic potential in metastatic human hepatocarcinoma.

Incorrect strain information for mouse cell lines: sequential influence of misidentification on sublines.

Science.gov (United States)

Uchio-Yamada, Kozue; Kasai, Fumio; Ozawa, Midori; Kohara, Arihiro

2017-03-01

Misidentification or cross-contamination of cell lines can cause serious issues. Human cell lines have been authenticated by short tandem repeat profiling; however, mouse cell lines have not been adequately assessed. In this study, mouse cell lines registered with the JCRB cell bank were examined by simple sequence length polymorphism (SSLP) analysis to identify their strains. Based on comparisons with 7 major inbred strains, our results revealed their strains in 80 of 90 cell lines. However, 12 of the 80 cell lines (15%) were found to differ from registered information. Of them, 4 cell lines originated from the same mouse, which had been generated through mating between two different inbred strains. The genotype of the mouse sample had not been examined after the backcross, leading to strain misidentification in those cell lines. Although 8 other cell lines had been established as sublines of a BALB/c cell line, their SSLP profiles are similar to a Swiss cell line. This affects differences in genotypes between inbred and outbred strains. Because the use of inbred samples and interbreeding between strains are not involved in human materials, our results suggest that the cause and influence of misidentification in mouse cell lines are different from those in human.

The prophylactic effect of probiotic Enterococcus lactis IW5 against different human cancer cells

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YOUSEF eNAMI

2015-11-01

Full Text Available Enterococcus lactis IW5 was obtained from human gut and the potential probiotic characteristics of this organism were then evaluated. Results showed that this strain was highly resistant to low pH and high bile salt and adhered strongly to Caco-2 human epithelial colorectal cell lines. The supernatant of E. lactis IW5 strongly inhibited the growth of several pathogenic bacteria and decreased the viability of different cancer cells, such as HeLa, AGS, HT-29, and MCF-7. Conversely, E. lactis IW5 did not inhibit the viability of normal FHs-74 cells. This strain did not generate toxic enzymes, including β-glucosidase, β-glucuronidase, and N-acetyl-β-glucosaminidase and was highly susceptible to ampicillin, gentamycin, penicillin, vancomycin, clindamycin, sulfamethoxazol, and chloramphenicol but resistant to erythromycin and tetracyclin. This study provided evidence for the effect of E. lactis IW5 on cancer cells. Therefore, E. lactis IW5, as a bioactive therapeutics, should be subjected to other relevant tests to verify the therapeutic suitability of this strain for clinical applications.

Abnormal G1 arrest in the cell lines from LEC strain rats after X-irradiation

International Nuclear Information System (INIS)

Hayashi, M.; Uehara, K.; Kirisawa, R.; Endoh, D.; Arai, S.; Okui, T.

1997-01-01

The effect of X-irradiation of cell lines from LEC and WKAH strain rats on a progression o cell cycle was investigated. When WKAH rat ells were exposed to 5 Gy of X-rays and their cell cycle distribution was determined by a flow cytometer, the proportion of S-phase cells decrease and that of G2/M-phase cells in creased at 8 hr post-irradiation. At 18 and 24 hr post-irradiation, approximately 80% of the cells appeared in the G1 phase. On the contrary, the proportion of S-phase cells increased and that of G1-phase cells decreased in LEC rats during 8-24 hr post-irradiation, compared with that at 0 hr post-irradiation. Thus, radiation-induced delay in the progression from the G1 phase to S phase (G1 arrest) was observed inWKAH rat cells but not in LEC rat cells. In the case of WKAH rat cells, the intensities of the bands of p53 protein increased at 1 and 2 hr after X-irradiation at 5 Gy, compared with those of un-irradiated cells and at 0 hr post-irradiation. In contrast, the intensities of the bands were faint and did not significantly increase in LEC rat ells during 0-6 hr incubation after X-irradiation. Present results suggested that the radioresistant DNA synthesis in LEC rat cells is thought to be due to the abnormal G1 arrest following X-irradiation

Sequences within the 5' untranslated region regulate the levels of a kinetoplast DNA topoisomerase mRNA during the cell cycle.

Science.gov (United States)

Pasion, S G; Hines, J C; Ou, X; Mahmood, R; Ray, D S

1996-12-01

Gene expression in trypanosomatids appears to be regulated largely at the posttranscriptional level and involves maturation of mRNA precursors by trans splicing of a 39-nucleotide miniexon sequence to the 5' end of the mRNA and cleavage and polyadenylation at the 3' end of the mRNA. To initiate the identification of sequences involved in the periodic expression of DNA replication genes in trypanosomatids, we have mapped splice acceptor sites in the 5' flanking region of the TOP2 gene, which encodes the kinetoplast DNA topoisomerase, and have carried out deletion analysis of this region on a plasmid-encoded TOP2 gene. Block deletions within the 5' untranslated region (UTR) identified two regions (-608 to -388 and -387 to -186) responsible for periodic accumulation of the mRNA. Deletion of one or the other of these sequences had no effect on periodic expression of the mRNA, while deletion of both regions resulted in constitutive expression of the mRNA throughout the cell cycle. Subcloning of these sequences into the 5' UTR of a construct lacking both regions of the TOP2 5' UTR has shown that an octamer consensus sequence present in the 5' UTR of the TOP2, RPA1, and DHFR-TS mRNAs is required for normal cycling of the TOP2 mRNA. Mutation of the consensus octamer sequence in the TOP2 5' UTR in a plasmid construct containing only a single consensus octamer and that shows normal cycling of the plasmid-encoded TOP2 mRNA resulted in substantial reduction of the cycling of the mRNA level. These results imply a negative regulation of TOP2 mRNA during the cell cycle by a mechanism involving redundant elements containing one or more copies of a conserved octamer sequence within the 5' UTR of TOP2 mRNA.

Studies on Post-Irradiation DNA Degradation in Micrococcus Radiodurans, Strain RII51

DEFF Research Database (Denmark)

Auda, H.; Emborg, C.

1973-01-01

The influence of irradiation condition on post-irradiation DNA degradation was studied in a radiation resistant mutant of M. radiodurans, strain ${\\rm R}_{{\\rm II}}5$. After irradiation with 1 Mrad or higher more DNA is degraded in cells irradiated in wet condition than in cells irradiated with t...

Cell Factory Stability and Genetic Circuits for Improved Strain Development

DEFF Research Database (Denmark)

Rugbjerg, Peter

. However, all synthetic gene systems -­ including the target metabolic pathways themselves -­ represent a possible fitness burden to the cell and thus constitute a threat to strain stability. In this thesis, several studies served to develop genetic systems for optimizing cell factory development...... systems can challenge the stability of strain designs. A metabolite-­producing Escherichia coli strain was long-­term cultured to study production stability and the dynamic effects of mutations within the cell population. A genetic error landscape of pathway disruptions was identified including particular......Development of new chemical-­‐producing microbial cell factories is an iterative trial-­and-­error process, and to screen candidate cells at high throughput, genetic biosensor systems are appealing. Each biosensor has distinct biological parameters, making modular tuning networks attractive...

Entrance and Survival of Brucella pinnipedialis Hooded Seal Strain in Human Macrophages and Epithelial Cells

Science.gov (United States)

Briquemont, Benjamin; Sørensen, Karen K.; Godfroid, Jacques

2013-01-01

Marine mammal Brucella spp. have been isolated from pinnipeds (B. pinnipedialis) and cetaceans (B. ceti) from around the world. Although the zoonotic potential of marine mammal brucellae is largely unknown, reports of human disease exist. There are few studies of the mechanisms of bacterial intracellular invasion and multiplication involving the marine mammal Brucella spp. We examined the infective capacity of two genetically different B. pinnipedialis strains (reference strain; NTCT 12890 and a hooded seal isolate; B17) by measuring the ability of the bacteria to enter and replicate in cultured phagocytes and epithelial cells. Human macrophage-like cells (THP-1), two murine macrophage cell lines (RAW264.7 and J774A.1), and a human malignant epithelial cell line (HeLa S3) were challenged with bacteria in a gentamicin protection assay. Our results show that B. pinnipedialis is internalized, but is then gradually eliminated during the next 72 – 96 hours. Confocal microscopy revealed that intracellular B. pinnipedialis hooded seal strain colocalized with lysosomal compartments at 1.5 and 24 hours after infection. Intracellular presence of B. pinnipedialis hooded seal strain was verified by transmission electron microscopy. By using a cholesterol-scavenging lipid inhibitor, entrance of B. pinnipedialis hooded seal strain in human macrophages was significantly reduced by 65.8 % (± 17.3), suggesting involvement of lipid-rafts in intracellular entry. Murine macrophages invaded by B. pinnipedialis do not release nitric oxide (NO) and intracellular bacterial presence does not induce cell death. In summary, B. pinnipedialis hooded seal strain can enter human and murine macrophages, as well as human epithelial cells. Intracellular entry of B. pinnipedialis hooded seal strain involves, but seems not to be limited to, lipid-rafts in human macrophages. Brucella pinnipedialis does not multiply or survive for prolonged periods intracellulary. PMID:24376851

ISOLATION AND CHARACTERIZATION OF A MOLYBDENUM-REDUCING AND GLYPHOSATE-DEGRADING Klebsiella oxytoca STRAIN SAW-5 IN SOILS FROM SARAWAK

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M.K. Sabullah

2016-02-01

Full Text Available Bioremediation of pollutants including heavy metals and xenobiotics is an economic and environmentally friendly process. A novel molybdenum-reducing bacterium with the ability to utilize the pesticide glyphosate as a carbon source is reported. The characterization works were carried out utilizing bacterial resting cells in a microplate format. The bacterium reduces molybdate to Mo-blue optimally between pH 6.3 and 6.8 and at 34oC. Glucose was the best electron donor for supporting molybdate reduction followed by lactose, maltose, melibiose, raffinose, d-mannitol, d-xylose, l-rhamnose, l-arabinose, dulcitol, myo-inositol and glycerol in descending order. Other requirements include a phosphate concentration at 5.0 mM and a molybdate concentration between 20 and 30 mM. The molybdenum blue exhibited an absorption spectrum resembling a reduced phosphomolybdate. Molybdenum reduction was inhibited by mercury, silver, cadmium and copper at 2 ppm by 45.5, 26.0, 18.5 and 16.3%, respectively. Biochemical analysis identified the bacterium as Klebsiella oxytoca strain Saw-5. To conclude, the capacity of this bacterium to reduce molybdenum into a less toxic form and to grow on glyphosate is novel and makes the bacterium an important instrument for bioremediation of these pollutants.

A novel podoplanin-GFPCre mouse strain for gene deletion in lymphatic endothelial cells.

Science.gov (United States)

Gil, Hyea Jin; Ma, Wanshu; Oliver, Guillermo

2018-04-01

The lymphatic vascular system is a one-direction network of thin-walled capillaries and larger vessels covered by a continuous layer of endothelial cells responsible for maintaining fluid homeostasis. Some of the main functions of the lymphatic vasculature are to drain fluid from the extracellular spaces and return it back to the blood circulation, lipid absorption from the intestinal tract, and transport of immune cells to lymphoid organs. A number of genes controlling the development of the mammalian lymphatic vasculature have been identified in the last few years, and their functional roles started to be characterized using gene inactivation approaches in mice. Unfortunately, only few mouse Cre strains relatively specific for lymphatic endothelial cells (LECs) are currently available. In this article, we report the generation of a novel Podoplanin (Pdpn) GFPCre transgenic mouse strain using its 5' regulatory region. Pdpn encodes a transmembrane mucin-type O-glycoprotein that is expressed on the surface of embryonic and postnatal LECs, in addition to few other cell types. Our detailed characterization of this novel strain indicates that it will be a valuable additional genetic tool for the analysis of gene function in LECs. © 2018 Wiley Periodicals, Inc.

Efficient infectious cell culture systems of the hepatitis C virus (HCV) prototype strains HCV-1 and H77

DEFF Research Database (Denmark)

Li, Yi-Ping; Ramirez, Santseharay; Mikkelsen, Lotte

2015-01-01

UNLABELLED: The first discovered and sequenced hepatitis C virus (HCV) genome and the first in vivo infectious HCV clones originated from the HCV prototype strains HCV-1 and H77, respectively, both widely used in research of this important human pathogen. In the present study, we developed...... efficiently after transfection and subsequent infection of naive Huh7.5 cells, reaching titers of 10(3.5) and 10(4.4) FFU/ml, respectively. IMPORTANCE: Hepatitis C virus (HCV) was discovered in 1989 with the cloning of the prototype strain HCV-1 genome. In 1997, two molecular clones of H77, the other HCV...... prototype strain, were shown to be infectious in chimpanzees, but not in vitro. HCV research was hampered by a lack of infectious cell culture systems, which became available only in 2005 with the discovery of JFH1 (genotype 2a), a genome that could establish infection in Huh7.5 cells. Recently, we...

BCG strain S4-Jena: An early BCG strain is capable to reduce the proliferation of bladder cancer cells by induction of apoptosis

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Hermann Inge-Marie

2010-06-01

Full Text Available Abstract Background Intravesical immunotherapy with Mycobacterium bovis bacillus Calmette-Guérin has been established as the most effective adjuvant treatment for high risk non-muscle-invasive bladder cancer (NMIBC. We investigated the differences between the S4-Jena BCG strain and commercially available BCG strains. We tested the genotypic varieties between S4-Jena and other BCG strains and analysed the effect of the BCG strains TICE and S4-Jena on two bladder cancer cell lines. Results In contrast to commercially available BCG strains the S4-Jena strain shows genotypic differences. Spoligotyping verifies the S4-Jena strain as a BCG strain. Infection with viable S4-Jena or TICE decreased proliferation in the T24 cell line. Additionally, hallmarks of apoptosis were detectable. In contrast, Cal29 cells showed only a slightly decreased proliferation with TICE. Cal29 cells infected with S4-Jena, though, showed a significantly decreased proliferation in contrast to TICE. Concordantly with these results, infection with TICE had no effect on the morphology and hallmarks of apoptosis of Cal29 cells. However, S4-Jena strain led to clearly visible morphological changes and caspases 3/7 activation and PS flip. Conclusions S4-Jena strain has a direct influence on bladder cancer cell lines as shown by inhibition of cell proliferation and induction of apoptosis. The data implicate that the T24 cells are responder for S4-Jena and TICE BCG. However, the Cal29 cells are only responder for S4-Jena and they are non-responder for TICE BCG. S4-Jena strain may represent an effective therapeutic agent for NMIBC.

Optimisation of 1.3 μm strained-layer semiconductor lasers

International Nuclear Information System (INIS)

Pacey, C.

1999-03-01

The objectives of the research undertaken have been to investigate the properties of semiconductor lasers operating at around 1.3 μm. The aim of the investigation is to suggest modifications which give rise to improved operating characteristics especially in the high temperature (approaching 85 deg. C) range. The investigation can be divided into 2 sections: a theoretical approach and an experimental section. The theoretical study examined the performance of compressively strained InGaAsP/InP multiple quantum-well lasers emitting at 1.3 μm. in order to investigate the important factors and trends in the threshold current density and differential gain with strain, well width and well number. Structures with a fixed compressive strain of 1% but variable well width, and also with a fixed well width but variable strain from 0% to 1.4% have been considered. It has been found that there is little benefit to having compressive strains greater than 1%. For structures with a fixed 1% compressive strain and unstrained barriers, an optimum structure for lowest threshold current density and a high differential gain has been found to consist of six 35 A quantum-wells. In addition, compensated strain (CS) structures with compressive wells and tensile barriers have been examined. It is shown that the conduction band offset can be significantly increased and the valence band offset reduced in such structures, to give band-offset ratios comparable with aluminium based 1.3 μm devices. The gain calculations performed suggest that there is little degradation in the threshold carrier density or differential gain due to these alterations in the band offsets; and hence a better laser performance is expected due to a reduction in thermal leakage currents due to the improved electron confinement. The experimental study concentrates on looking at certain key design parameters to investigate their effect on the laser performance. These design parameters range from the number of quantum

From the Macro to the Micro: Gel Mapping to Differentiate between Sporozoites of Two Immunologically Distinct Strains of Eimeria maxima (Strains M6 and Guelph).

Science.gov (United States)

El-Ashram, Saeed; Yin, Qing; Liu, Hongbin; Al Nasr, Ibrahim; Liu, Xianyong; Suo, Xun; Barta, John

2015-01-01

Two immunologically distinct strains of E. maxima were examined in this study: the M6 strain and the Guelph strain. The differential expression between the sporozoites of the two strains of E. maxima was determined by image analysis of 100 μg of protein from each strain separated by standard one- and conventional two-dimensional polyacrylamide gel electrophoresis. In addition to differences in both molecular weight and the electrophoretic mobility, differences in the intensity of polypeptide bands for example, GS 136.4 and M6 169 were explored. Pooled gels were prepared from each strain. A representative 2D-PAGE gel spanning a non-linear pH range of 3-10 of E. maxima strain M6 consisted of approximately 694 polypeptide spots with about 67 (9.6%) of the polypeptide spots being unique relative to the other strain. E. maxima strain GS had about 696 discernable polypeptide spots with 69 spots (9.9%) that differed from those of the M6 strain. In-depth characterization of the variable polypeptide spots; unique polypeptide spots (absence or presence) and shared polypeptide spots with modifications may lead to novel vaccine target in the form of multi-component, multi-stage, multi-immunovariant strains, multi-species subunit vaccine, and diagnostic probe for E. maxima.

Activation of the adenylyl cyclase/cyclic AMP/protein kinase A pathway in endothelial cells exposed to cyclic strain

Science.gov (United States)

Cohen, C. R.; Mills, I.; Du, W.; Kamal, K.; Sumpio, B. E.

1997-01-01

The aim of this study was to assess the involvement of the adenylyl cyclase/cyclic AMP/protein kinase A pathway (AC) in endothelial cells (EC) exposed to different levels of mechanical strain. Bovine aortic EC were seeded to confluence on flexible membrane-bottom wells. The membranes were deformed with either 150 mm Hg (average 10% strain) or 37.5 mm Hg (average 6% strain) vacuum at 60 cycles per minute (0.5 s strain; 0.5 s relaxation) for 0-60 min. The results demonstrate that at 10% average strain (but not 6% average strain) there was a 1.5- to 2.2-fold increase in AC, cAMP, and PKA activity by 15 min when compared to unstretched controls. Further studies revealed an increase in cAMP response element binding protein in EC subjected to the 10% average strain (but not 6% average strain). These data support the hypothesis that cyclic strain activates the AC/cAMP/PKA signal transduction pathway in EC which may occur by exceeding a strain threshold and suggest that cyclic strain may stimulate the expression of genes containing cAMP-responsive promoter elements.

Incorrect strain information for mouse cell lines: sequential influence of misidentification on sublines

OpenAIRE

Uchio-Yamada, Kozue; Kasai, Fumio; Ozawa, Midori; Kohara, Arihiro

2016-01-01

Misidentification or cross-contamination of cell lines can cause serious issues. Human cell lines have been authenticated by short tandem repeat profiling; however, mouse cell lines have not been adequately assessed. In this study, mouse cell lines registered with the JCRB cell bank were examined by simple sequence length polymorphism (SSLP) analysis to identify their strains. Based on comparisons with 7 major inbred strains, our results revealed their strains in 80 of 90 cell lines. However,...

Finite-Element Modeling of Viscoelastic Cells During High-Frequency Cyclic Strain

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David W. Holdsworth

2012-03-01

Full Text Available Mechanotransduction refers to the mechanisms by which cells sense and respond to local loads and forces. The process of mechanotransduction plays an important role both in maintaining tissue viability and in remodeling to repair damage; moreover, it may be involved in the initiation and progression of diseases such as osteoarthritis and osteoporosis. An understanding of the mechanisms by which cells respond to surrounding tissue matrices or artificial biomaterials is crucial in regenerative medicine and in influencing cellular differentiation. Recent studies have shown that some cells may be most sensitive to low-amplitude, high-frequency (i.e., 1–100 Hz mechanical stimulation. Advances in finite-element modeling have made it possible to simulate high-frequency mechanical loading of cells. We have developed a viscoelastic finite-element model of an osteoblastic cell (including cytoskeletal actin stress fibers, attached to an elastomeric membrane undergoing cyclic isotropic radial strain with a peak value of 1,000 µstrain. The results indicate that cells experience significant stress and strain amplification when undergoing high-frequency strain, with peak values of cytoplasmic strain five times higher at 45 Hz than at 1 Hz, and peak Von Mises stress in the nucleus increased by a factor of two. Focal stress and strain amplification in cells undergoing high-frequency mechanical stimulation may play an important role in mechanotransduction.

Establishment of an immortalized mouse dermal papilla cell strain with optimized culture strategy

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Haiying Guo

2018-01-01

Full Text Available Dermal papilla (DP plays important roles in hair follicle regeneration. Long-term culture of mouse DP cells can provide enough cells for research and application of DP cells. We optimized the culture strategy for DP cells from three dimensions: stepwise dissection, collagen I coating, and optimized culture medium. Based on the optimized culture strategy, we immortalized primary DP cells with SV40 large T antigen, and established several immortalized DP cell strains. By comparing molecular expression and morphologic characteristics with primary DP cells, we found one cell strain named iDP6 was similar with primary DP cells. Further identifications illustrate that iDP6 expresses FGF7 and α-SMA, and has activity of alkaline phosphatase. During the process of characterization of immortalized DP cell strains, we also found that cells in DP were heterogeneous. We successfully optimized culture strategy for DP cells, and established an immortalized DP cell strain suitable for research and application of DP cells.

Reduction of molybdate to molybdenum blue by Klebsiella sp. strain hkeem.

Science.gov (United States)

Lim, H K; Syed, M A; Shukor, M Y

2012-06-01

A novel molybdate-reducing bacterium, tentatively identified as Klebsiella sp. strain hkeem and based on partial 16s rDNA gene sequencing and phylogenetic analysis, has been isolated. Strain hkeem produced 3 times more molybdenum blue than Serratia sp. strain Dr.Y8; the most potent Mo-reducing bacterium isolated to date. Molybdate was optimally reduced to molybdenum blue using 4.5 mM phosphate, 80 mM molybdate and using 1% (w/v) fructose as a carbon source. Molybdate reduction was optimum at 30 °C and at pH 7.3. The molybdenum blue produced from cellular reduction exhibited absorption spectrum with a maximum peak at 865 nm and a shoulder at 700 nm. Inhibitors of electron transport system such as antimycin A, rotenone, sodium azide, and potassium cyanide did not inhibit the molybdenum-reducing enzyme. Mercury, silver, and copper at 1 ppm inhibited molybdenum blue formation in whole cells of strain hkeem. Copyright © 2012 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Human cyclin T1 expression ameliorates a T-cell-specific transcriptional limitation for HIV in transgenic rats, but is not sufficient for a spreading infection of prototypic R5 HIV-1 strains ex vivo

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Littman Dan R

2009-01-01

Full Text Available Abstract Background Cells derived from native rodents have limits at distinct steps of HIV replication. Rat primary CD4 T-cells, but not macrophages, display a profound transcriptional deficit that is ameliorated by transient trans-complementation with the human Tat-interacting protein Cyclin T1 (hCycT1. Results Here, we generated transgenic rats that selectively express hCycT1 in CD4 T-cells and macrophages. hCycT1 expression in rat T-cells boosted early HIV gene expression to levels approaching those in infected primary human T-cells. hCycT1 expression was necessary, but not sufficient, to enhance HIV transcription in T-cells from individual transgenic animals, indicating that endogenous cellular factors are critical co-regulators of HIV gene expression in rats. T-cells from hCD4/hCCR5/hCycT1-transgenic rats did not support productive infection of prototypic wild-type R5 HIV-1 strains ex vivo, suggesting one or more significant limitation in the late phase of the replication cycle in this primary rodent cell type. Remarkably, we identify a replication-competent HIV-1 GFP reporter strain (R7/3 YU-2 Env that displays characteristics of a spreading, primarily cell-to-cell-mediated infection in primary T-cells from hCD4/hCCR5-transgenic rats. Moreover, the replication of this recombinant HIV-1 strain was significantly enhanced by hCycT1 transgenesis. The viral determinants of this so far unique replicative ability are currently unknown. Conclusion Thus, hCycT1 expression is beneficial to de novo HIV infection in a transgenic rat model, but additional genetic manipulations of the host or virus are required to achieve full permissivity.

Newly identified CpG ODNs, M5-30 and M6-395, stimulate mouse immune cells to secrete TNF-alpha and enhance Th1-mediated immunity.

Science.gov (United States)

Choi, Sun-Shim; Chung, Eunkyung; Jung, Yu-Jin

2010-08-01

Bacterial CpG motifs are known to induce both innate and adaptive immunity in infected hosts via toll-like receptor 9 (TLR9). Because small oligonucleotides (ODNs) mimicking bacterial CpG motifs are easily synthesized, they have found use as immunomodulatory agents in a number of disease models. We have developed a novel bioinformatics approach to identify effective CpG ODN sequences and evaluate their function as TLR9 ligands in a murine system. Among the CpG ODNs we identified, M5-30 and M6-395 showed significant ability to stimulate TNF-alpha and IFN-gamma production in a mouse macrophage cell line and mouse splenocytes, respectively. We also found that these CpG ODNs activated cells through the canonical NF-kappa B signaling pathway. Moreover, both CpG ODNs were able to induce Th1-mediated immunity in Mycobacterium tuberculosis (Mtb)-infected mice. Our results demonstrate that M5-30 and M6-395 function as TLR9-specific ligands, making them useful in the study of TLR9 functionality and signaling in mice.

Progress toward characterization of the group A Streptococcus metagenome: complete genome sequence of a macrolide-resistant serotype M6 strain.

Science.gov (United States)

Banks, David J; Porcella, Stephen F; Barbian, Kent D; Beres, Stephen B; Philips, Lauren E; Voyich, Jovanka M; DeLeo, Frank R; Martin, Judith M; Somerville, Greg A; Musser, James M

2004-08-15

We describe the genome sequence of a macrolide-resistant strain (MGAS10394) of serotype M6 group A Streptococcus (GAS). The genome is 1,900,156 bp in length, and 8 prophage-like elements or remnants compose 12.4% of the chromosome. A 8.3-kb prophage remnant encodes the SpeA4 variant of streptococcal pyrogenic exotoxin A. The genome of strain MGAS10394 contains a chimeric genetic element composed of prophage genes and a transposon encoding the mefA gene conferring macrolide resistance. This chimeric element also has a gene encoding a novel surface-exposed protein (designated "R6 protein"), with an LPKTG cell-anchor motif located at the carboxyterminus. Surface expression of this protein was confirmed by flow cytometry. Humans with GAS pharyngitis caused by serotype M6 strains had antibody against the R6 protein present in convalescent, but not acute, serum samples. Our studies add to the theme that GAS prophage-encoded extracellular proteins contribute to host-pathogen interactions in a strain-specific fashion.

Influence of a reducing agent used to prepare radiopharmaceuticals labeled with technetium-99m on the adhesive properties of Corynebacterium diphtheriae strains

International Nuclear Information System (INIS)

Souza, S.M.; Bernardo-Filho, M.; Hirata, R. Jr.; Moreira, L.O.; Mattos-Guaraldi, A.L.

2002-01-01

Aim: In this work we investigated the influence of stannous chloride (SnCl 2 ) used in nuclear medicine as a reducing agent to prepare radiopharmaceuticals labeled with technetium-99m, on the survival and adhesive properties of two toxigenic C. diphtheriae of the sucrose fermenting (241strain) and non fermenting (CDC-E8392 strain) biotypes. Materials and Methods: Bacterial strains were submitted to survival and filamentation induction assays using different concentrations of SnCl 2 . The influence of SnCl 2 on the adhesive properties of C.diphtheriae were evaluated by bacterial autoaggregation, haemagglutination, adherence to glass surface and lectin-binding assays. Results: Differences in survival fractions suggested differences in susceptibility of microorganisms to bactericidal effect of stannous chloride. A percentage of 0.4% bacterial cells of no.241 strain and 0.04% of CDC-E8392 strain survived after 220 μl ml -1 SnCl 2 treatment. Results of both polystyrene and spontaneous autoaggregation tests showed an increase in the hydrophobic surface properties of C. diphtheriae strains. SnCl 2 induced spontaneous bacterial autoaggregation of sucrose fermenting 241 strain. SnCl 2 enhanced adherence to glass and totally inhibited the haemagglutinating activity of the non-sucrose fermenting strain CDC-E8392 strain (original titer=32). Decrease in haemagglutinatination was dependent on the concentration of SnCl 2 used. Lectin-binding assays demonstrated increase in the expression of cell surface receptors to lectin with affinity for molecules containing mannose residues after treatment with SnCl 2 . The presence of SnCl 2 induced differences in the expression of bacterial surface carbohydrates possibly related with differences in degrees of haemagglutination and adherence to glass of diphtheria bacilli. Conclusion: The presence of SnCl 2 may influence on the adhesive properties of bacterial pathogens. The occurrence of cell filamentation suggests a potential genotoxicity

Distinct CCR2(+) Gr1(+) cells control growth of the Yersinia pestis ΔyopM mutant in liver and spleen during systemic plague.

Science.gov (United States)

Ye, Zhan; Uittenbogaard, Annette M; Cohen, Donald A; Kaplan, Alan M; Ambati, Jayakrishna; Straley, Susan C

2011-02-01

We are using a systemic plague model to identify the cells and pathways that are undermined by the virulence protein YopM of the plague bacterium Yersinia pestis. In this study, we pursued previous findings that Gr1(+) cells are required to selectively limit growth of ΔyopM Y. pestis and that CD11b(+) cells other than polymorphonuclear leukocytes (PMNs) are selectively lost in spleens infected with parent Y. pestis. When PMNs were ablated from mice, ΔyopM Y. pestis grew as well as the parent strain in liver but not in spleen, showing that these cells are critical for controlling growth of the mutant in liver but not spleen. In mice lacking expression of the chemokine receptor CCR2, wild-type growth was restored to ΔyopM Y. pestis in both organs. In spleen, the Gr1(+) cells differentially recruited by parent and ΔyopM Y. pestis infections were CCR2(+) Gr1(+) CD11b(+) CD11c(Lo-Int) MAC3(+) iNOS(+) (inducible nitric oxide synthase-positive) inflammatory dendritic cells (iDCs), and their recruitment to spleen from blood was blocked when YopM was present in the infecting strain. Consistent with influx of iDCs being affected by YopM in spleen, the growth defect of the ΔyopM mutant was relieved by the parent Y. pestis strain in a coinfection assay in which the parent strain could affect the fate of the mutant in trans. In a mouse model of bubonic plague, CCR2 also was shown to be required for ΔyopM Y. pestis to show wild-type growth in skin. The data imply that YopM's pathogenic effect indirectly undermines signaling through CCR2. We propose a model for how YopM exerts its different effects in liver and spleen.

Cell cycle analysis of cultured mammalian cells after exposure to 4,5',8-trimethylpsoralen and long-wave ultraviolet light

International Nuclear Information System (INIS)

Cohen, S.R.; Burkholder, D.E.; Varga, J.M.; Carter, D.M.; Bartholomew, J.C.

1981-01-01

Cell cycle analysis was used to study the the effect of 4,5'8-trimethylpsoralen (TMP) and long-wave ultraviolet light (UV-A) on cultured mammalian cells. DNA distribution patterns were measured for murine melanoma cells (a cloned line of Cloudman S91) and a strain of diploid human skin fibroblasts (CRL 1295) using both a microfluorimetry procedure and flow cytometry. The untreated cells and those receiving TMP along and UV-A alone had identical DNA content as assessed at several posttreatment intervals (0-72 hr). The majority of cells in control groups contained a G1 DNA content, whereas exposure to TMP (2 x 10(-7) M) plus UV-A (1 Joule/cm2) led to the accumulation of cells in the G2 phase. These observations were similar for each cell type and both analytical techniques were in excellent agreement. The finding that psoralen plus UV-A induces a phase-specific G2 blockade in cultured cells has important implications for understanding the mechanisms which account for enhanced pigmentation and suppression of cellular proliferation following exposure to these agents in vivo

Characterization of the psychrotolerant acetogen strain SyrA5 and the emended description of the species Acetobacterium carbinolicum.

Science.gov (United States)

Paarup, Maiken; Friedrich, Michael W; Tindall, Brian J; Finster, Kai

2006-01-01

A psychrotolerant, obligate anaerobic, acetogenic bacterium designated strain SyrA5 was isolated from black anoxic sediment of a brackish fjord. Cells were Gram-positive, non-sporeforming rods. The isolate utilized H(2)/CO(2), CO, fructose, glucose, ethanol, ethylene glycol, glycerol, pyruvate, lactate, betaine and the methyl-groups of several methoxylated benzoic derivatives such as syringate, trimethoxybenzoate and vallinate. The optimum temperature for growth was 29 degrees C, whilst slow growth occurred at 2 degrees C. The strain grew optimally with NaCl concentrations below 2.7% (w/v), but growth occurred up to 4.3% (w/v) NaCl. Growth was observed in the range from pH 5.9 to 8.5, optimum at pH 8. The G+C content was 44.1 mol%. Based upon 16S rRNA gene sequence analysis and DNA-DNA reassociation studies, the organism was classified in the genus Acetobacterium. Strain SyrA5 shared a 16S rRNA sequence similarity with A. carbinolicum of 100%, a fthfs gene (which codes for the N5,N10 tetrahydrofolate synthetase) sequence identity of 98.5-98.7% (amino acid sequence similarities were 99.4-100%) and a RNA-DNA hybridization homology of 64-68%. Despite a number of phenotypic differences between strain SyrA5 and A. carbinolicum we propose including strain SyrA5 as a subspecies of A. carbinolicum for which we propose the name Acetobacterium carbinolicum subspecies kysingense. The type strain is SyrA5 (=DSM 16427(T), ATCC BAA-990).

Invasion of Porphyromonas gingivalis strains into vascular cells and tissue

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Ingar Olsen

2015-08-01

Full Text Available Porphyromonas gingivalis is considered a major pathogen in adult periodontitis and is also associated with multiple systemic diseases, for example, cardiovascular diseases. One of its most important virulence factors is invasion of host cells. The invasion process includes attachment, entry/internalization, trafficking, persistence, and exit. The present review discusses these processes related to P. gingivalis in cardiovascular cells and tissue. Although most P. gingivalis strains invade, the invasion capacity of strains and the mechanisms of invasion including intracellular trafficking among them differ. This is consistent with the fact that there are significant differences in the pathogenicity of P. gingivalis strains. P. gingivalis invasion mechanisms are also dependent on types of host cells. Although much is known about the invasion process of P. gingivalis, we still have little knowledge of its exit mechanisms. Nevertheless, it is intriguing that P. gingivalis can remain viable in human cardiovascular cells and atherosclerotic plaque and later exit and re-enter previously uninfected host cells.

Gold nanoparticle-based beacon to detect STAT5b mRNA expression in living cells: a case optimized by bioinformatics screen.

Science.gov (United States)

Deng, Dawei; Li, Yang; Xue, Jianpeng; Wang, Jie; Ai, Guanhua; Li, Xin; Gu, Yueqing

2015-01-01

Messenger RNA (mRNA), a single-strand ribonucleic acid with functional gene information is usually abnormally expressed in cancer cells and has become a promising biomarker for the study of tumor progress. Hairpin DNA-coated gold nanoparticle (hDAuNP) beacon containing a bare gold nanoparticle (AuNP) as fluorescence quencher and thiol-terminated fluorescently labeled stem-loop-stem oligonucleotide sequences attached by Au-S bond is currently a new nanoscale biodiagnostic platform capable of mRNA detection, in which the design of the loop region sequence is crucial for hybridizing with the target mRNA. Hence, in this study, to improve the sensitivity and selectivity of hDAuNP beacon simultaneously, the loop region of hairpin DNA was screened by bioinformatics strategy. Here, signal transducer and activator of transcription 5b (STAT5b) mRNA was selected and used as a practical example. The results from the combined characterizations using optical techniques, flow cytometry assay, and cell microscopic imaging showed that after optimization, the as-prepared hDAuNP beacon had higher selectivity and sensitivity for the detection of STAT5b mRNA in living cells, as compared with our previous beacon. Thus, the bioinformatics method may be a promising new strategy for assisting in the designing of the hDAuNP beacon, extending its application in the detection of mRNA expression and the resultant mRNA-based biological processes and disease pathogenesis.

Avian metapneumovirus M2:2 protein inhibits replication in Vero cells: modification facilitates live vaccine development.

Science.gov (United States)

Clubbe, Jayne; Naylor, Clive J

2011-11-28

Throughout the world, avian metapneumovirus (AMPV) infection of subtype A is principally controlled by two live vaccines both derived from UK field strain #8544. Improvements of those vaccines by use of reverse genetics technology was found to be hampered by the inability of #8544 to replicate in the commonly exploited Vero cell based reverse genetics system. A systematic reverse genetics based genome modification of a DNA copy of #8544, employing sequence data from a Vero grown, #8544 derived, live vaccine; was used to determine mutations required to facilitate virus recovery and replication in Vero cells. This identified a single coding substitution in the M2:2 reading frame as responsible. Furthermore, ablation of M2:2 was found to elicit the same outcome. M2:2 sequence analysis of seven AMPVs found Vero cell adaption to be associated with non similar amino acid changes in M2:2. The study shows that M2:2 modification of field virus #8544 will enable research leading to improved vaccines. This may have more general application to other AMPV field strains. Copyright © 2011 Elsevier Ltd. All rights reserved.

The 5'-poly(A leader of poxvirus mRNA confers a translational advantage that can be achieved in cells with impaired cap-dependent translation.

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Pragyesh Dhungel

2017-08-01

Full Text Available The poly(A leader at the 5'-untranslated region (5'-UTR is an unusually striking feature of all poxvirus mRNAs transcribed after viral DNA replication (post-replicative mRNAs. These poly(A leaders are non-templated and of heterogeneous lengths; and their function during poxvirus infection remains a long-standing question. Here, we discovered that a 5'-poly(A leader conferred a selective translational advantage to mRNA in poxvirus-infected cells. A constitutive and uninterrupted 5'-poly(A leader with 12 residues was optimal. Because the most frequent lengths of the 5'-poly(A leaders are 8-12 residues, the result suggests that the poly(A leader has been evolutionarily optimized to boost poxvirus protein production. A 5'-poly(A leader also could increase protein production in the bacteriophage T7 promoter-based expression system of vaccinia virus, the prototypic member of poxviruses. Interestingly, although vaccinia virus post-replicative mRNAs do have 5'- methylated guanosine caps and can use cap-dependent translation, in vaccinia virus-infected cells, mRNA with a 5'-poly(A leader could also be efficiently translated in cells with impaired cap-dependent translation. However, the translation was not mediated through an internal ribosome entry site (IRES. These results point to a fundamental mechanism poxvirus uses to efficiently translate its post-replicative mRNAs.

mGluR5 stimulating Homer–PIKE formation initiates icariin induced cardiomyogenesis of mouse embryonic stem cells by activating reactive oxygen species

Energy Technology Data Exchange (ETDEWEB)

Zhou, Limin; Huang, Yujie; Zhang, Yingying [Institute of Pharmacology, Toxicology and Biochemical Pharmaceutics, College of Pharmaceutical Sciences, Zhejiang University, No. 866, Yu Hang Tang Road, Hangzhou 310058 (China); Zhao, Qingwei [The First Affiliated Hospital, College of Medicine, Zhejiang University, No. 79, Qing Chun Road, Hangzhou 310003 (China); Zheng, Bei; Lou, Yijia [Institute of Pharmacology, Toxicology and Biochemical Pharmaceutics, College of Pharmaceutical Sciences, Zhejiang University, No. 866, Yu Hang Tang Road, Hangzhou 310058 (China); Zhu, Danyan, E-mail: zdyzxb@zju.edu.cn [Institute of Pharmacology, Toxicology and Biochemical Pharmaceutics, College of Pharmaceutical Sciences, Zhejiang University, No. 866, Yu Hang Tang Road, Hangzhou 310058 (China)

2013-06-10

Icariin (ICA) has been reported to facilitate cardiac differentiation of mouse embryonic stem (ES) cells; however, the mechanism by which ICA induced cardiomyogenesis has not been fully elucidated yet. Here, an underlying signaling network including metabotropic glutamate receptor 5 (mGluR5), Homer, phosphatidylinositol 3-Kinase Enhancer (PIKE), phosphatidylinositol 3-Kinase (PI3K), reactive oxygen species (ROS) and nuclear factor-kappaB (NF-κB) was investigated in ICA induced cardiomyogenesis. Our results showed that the co-expression of mGluR5 together with α-actinin or Troponin T in embryoid bodies (EBs) treated with ICA was elevated to 10.86% and 9.62%, compared with the case in the control (4.04% and 3.45%, respectively). Exposure of EBs to ICA for 2 h remarkably increased the dimeric form of mGluR5, which was inhibited by small interfering RNA targeting mGluR5 (si-mGluR5). Moreover, the extracellular glutamate concentration in ICA treatment medium was elevated to 28.9±3.5 μM. Furthermore, the activation of mGluR5 by ICA triggered the formation of Homer–PIKE complex and activated PI3K, stimulating ROS generation and NF-κB nuclear translocation. Knockdown of mGluR5 or inhibition of PI3K by LY294002 blocked ICA induced cardiomyogenesis via repressing mGluR5 pathway, reducing ROS and NF-κB activation. These results revealed that the inducible mechanisms of ICA were related to activate mGluR5 pathway. -- Highlights: • ICA increased mGluR5 expression in cardiac differentiation of ES cells. • ICA enhanced the glutamate level and the receptor mGluR5 dimerization, stimulating the formation of Homer–PIKE complex. • Knockdown of mGluR5 or inhibition of PI3K by LY294002 inhibited ICA induced ROS generation and NF-κB nuclear translocation.

Adhesion of some probiotic and dairy Lactobacillus strains to Caco-2 cell cultures.

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Tuomola, E M; Salminen, S J

1998-05-05

The adhesion of 12 different Lactobacillus strains was studied using Caco-2 cell line as an in vitro model for intestinal epithelium. Some of the strains tested have been used as probiotics, and most of them are used in the dairy and food industry. Human and bovine enterotoxigenic Escherichia coli strains were used as positive and negative control, respectively. Bacterial adhesion to Caco-2 cell cultures was quantitated using radiolabelled bacteria. The adherence of bacteria was also observed microscopically after Gram staining. Viability of bacteria prior to adhesion was verified using flow cytometry. Among the tested strains, L. casei (Fyos) was the most adhesive strain and L. casei var. rhamnosus (Lactophilus) was the least adhesive strain, approximately 14 and 3% of the added bacteria adhered to Caco-2 cell cultures, respectively. The corresponding values for positive and negative control E. coli strains were 14 and 4%, respectively. The Lactobacillus strains tested could not be divided into distinctly adhesive or non-adhesive strains, since there was a continuation of adhesion rates. The four most adhesive strains were L. casei (Fyos), L. acidophilus 1 (LC1), L. rhamnosus LC-705 and Lactobacillus GG (ATCC 53103). No significant differences in the percentage adhesion were observed between these strains. Adhesion of all the strains was dependent on the number of bacteria used, since an approximately constant number of Caco-2 cells was used, indicating that the Caco-2 cell binding sites were not saturated. Viability of bacteria was high since approximately 90% of the bacteria were viable with the exception of L. acidophilus 1 which was 74% viable. Microscopic evaluations agreed with the radiolabelled binding as evidenced by observing more bacteria in Gram-stained preparations of good adhering strains compared to poorly adhering strains.

Gold nanoparticle-based beacon to detect STAT5b mRNA expression in living cells: a case optimized by bioinformatics screen

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Deng D

2015-04-01

Full Text Available Dawei Deng,* Yang Li,* Jianpeng Xue, Jie Wang, Guanhua Ai, Xin Li, Yueqing GuDepartment of Biomedical Engineering, China Pharmaceutical University, Nanjing, People’s Republic of China*These authors contributed equally to this workAbstract: Messenger RNA (mRNA, a single-strand ribonucleic acid with functional gene information is usually abnormally expressed in cancer cells and has become a promising biomarker for the study of tumor progress. Hairpin DNA-coated gold nanoparticle (hDAuNP beacon containing a bare gold nanoparticle (AuNP as fluorescence quencher and thiol-terminated fluorescently labeled stem–loop–stem oligonucleotide sequences attached by Au–S bond is currently a new nanoscale biodiagnostic platform capable of mRNA detection, in which the design of the loop region sequence is crucial for hybridizing with the target mRNA. Hence, in this study, to improve the sensitivity and selectivity of hDAuNP beacon simultaneously, the loop region of hairpin DNA was screened by bioinformatics strategy. Here, signal transducer and activator of transcription 5b (STAT5b mRNA was selected and used as a practical example. The results from the combined characterizations using optical techniques, flow cytometry assay, and cell microscopic imaging showed that after optimization, the as-prepared hDAuNP beacon had higher selectivity and sensitivity for the detection of STAT5b mRNA in living cells, as compared with our previous beacon. Thus, the bioinformatics method may be a promising new strategy for assisting in the designing of the hDAuNP beacon, extending its application in the detection of mRNA expression and the resultant mRNA-based biological processes and disease pathogenesis.Keywords: molecular beacon, bioinformatics, gold nanoparticle, STAT5b mRNA, visual detection

Sensitivity to fuel diesel oil and cell wall structure of some Scenedesmus (Chlorococcales strains

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Zbigniew Tukaj

2014-01-01

Full Text Available Sensitivity of three Scenedesmus strains exposed to aqueous fuel-oil extract (AFOE is strongly strain-dependent S. quadricauda is the most resistant, S. armatus moderately tolerant whereas the most sensitive appears to be S. microspina. The sensitivity of tested species increases parallel with decreasing of cell size and cell number in coenobium. The values of the cell surface/cell volumes ratios only partly explain the above relationships. Electron microscope investigations reveal that the sensitivity may depend on cell wall structure of the strains. Cell wall of all here investigated strains is built of two layers: the inner so-called cellulosic layer and the outer one showing a three-laminar structure (TLS. The latter contains an acetolysis-resistant biopolymer (ARB. These two layers are similar in thickness in the three strains tested, but the surface of Scenedesmus is covered with various epistructures that are characteristic of strains. Some of them as the tightly fitting warty layer of S. armatus and especially the loosely fitting reticulate layer of S. quadricauda may contribute to lower permeability of cell wall. The structure of the rosettes also appears to be correlated with the sensitivity of strains. Presence of invaginations of plasmalemma in areas under rosettes indicates their role in transport processes inside/outside the cells.

Modified nucleotides m2G966/m5C967 of Escherichia coli 16S rRNA are required for attenuation of tryptophan operon

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Prokhorova, Irina V.; Osterman, Ilya A.; Burakovsky, Dmitry E.; Serebryakova, Marina V.; Galyamina, Maria A.; Pobeguts, Olga V.; Altukhov, Ilya; Kovalchuk, Sergey; Alexeev, Dmitry G.; Govorun, Vadim M.; Bogdanov, Alexey A.; Sergiev, Petr V.; Dontsova, Olga A.

2013-11-01

Ribosomes contain a number of modifications in rRNA, the function of which is unclear. Here we show - using proteomic analysis and dual fluorescence reporter in vivo assays - that m2G966 and m5C967 in 16S rRNA of Escherichia coli ribosomes are necessary for correct attenuation of tryptophan (trp) operon. Expression of trp operon is upregulated in the strain where RsmD and RsmB methyltransferases were deleted, which results in the lack of m2G966 and m5C967 modifications. The upregulation requires the trpL attenuator, but is independent of the promotor of trp operon, ribosome binding site of the trpE gene, which follows trp attenuator and even Trp codons in the trpL sequence. Suboptimal translation initiation efficiency in the rsmB/rsmD knockout strain is likely to cause a delay in translation relative to transcription which causes misregulation of attenuation control of trp operon.

Macrophage activation and histopathological findings in Calomys callosus and Swiss mice infected with several strains of Trypanosoma cruzi

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Monamaris Marques Borges

1992-12-01

Full Text Available Peritoneal macrophage activation as measured by H2O2 release and histopathology was compared between Swiss mice and Calomys callosus, a wild rodent, reservoir of Trypanosoma cruzi, during the course of infection with four strains of this parasite. In mice F and Y strain infections result in high parasitemia and mortality while with silvatic strains Costalimai and M226 parasitemia is sub-patent, with very low mortality. H2O2 release peaked at 33,6 and 59 nM/2 x 10(elevado a sexta potência cells for strains Y and F, respectively, 48 and 50 nM/2 x 10 (elevado a sexta potência for strains Costalimai and M226, at different days after infection. Histopathological findings of myositis, myocarditis, necrotizing artheritis and abscence of macrophage parasitism were foud for strains F and Costalimai. Y strain infection presented moderate myocarditis and myositis, with parasites multiplying within macrophages. In C. callosus all four strains resulted in patent parasitemia wich was eventually overcome, with scarce mortality. H2O2 release for strains Y or F was comparable to that of mice-peaks of 27 and 53 nM/2 x 10 (elevado a sexta potência cells, with lower values for strains Costalimai and M226 - 16.5 and 4.6 nM/2 x 10(elevado a sexta potênciacells, respectively. Histopathological lesions with Y and F strain injected animals were comparable to those of mice at the onset of infections; they subsided completely at the later stages with Y strain and partially with F strain infected C. callosus. In Costalimai infected C. callosus practically no histopathological alterations were observed.

Antagonistic activity exerted in vitro and in vivo by Lactobacillus casei (strain GG) against Salmonella typhimurium C5 infection.

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Hudault, S; Liévin, V; Bernet-Camard, M F; Servin, A L

1997-02-01

The aim of this study was to compare the antagonistic properties of Lactobacillus casei GG exerted in vitro against Salmonella typhimurium C5 in a cellular model, cultured enterocyte-like Caco-2 cells, to those exerted in vivo in an animal model, C3H/He/Oujco mice. Our results show that a 1-h contact between the invading strain C5 and either the culture or the supernatant of L. casei GG impeded the invasion by the Salmonella strain in Caco-2 cells, without modifying the viability of the strain. After neutralization at pH 7, no inhibition of the invasion by C5 was observed. The antagonistic activity of L. casei GG was examined in C3H/He/Oujco mice orally infected with C5 as follows: (i) L. casei GG was given daily to conventional animals as a probiotic, and (ii) it was given once to germ-free animals in order to study the effect of the population of L. casei GG established in the different segments of the gut. In vivo experiments show that after a single challenge with C5, this strain survives and persists at a higher level in the feces of the untreated conventional mice than in those of the treated group. In L. casei GG germ-free mice, establishment of L. casei GG in the gut significantly delayed the occurrence of 100% mortality of the animals (15 days after C5 challenge versus 9 days in germ-free mice [P L. casei GG population level in the gut dramatically decreased in these animals.

Lactococcus lactis subsp. cremoris strain JFR1 attenuates Salmonella adhesion to human intestinal cells in vitro.

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Zhang, Justina Su; Guri, Anilda; Corredig, Milena; Morales-Rayas, Rocio; Hassan, Ashraf; Griffiths, Mansel; LaPointe, Gisèle

2016-12-01

Lactococcus lactis subsp. cremoris JFR1 has been studied in reduced fat cheese due to its ability to produce exopolysaccharides (EPS) in situ, contributing to improved textural and organoleptic properties. In this study, the effect of strain JFR1 on virulence gene expression and attachment of Salmonella to HT-29 human colon carcinoma cells was investigated. Overnight cultures of L. lactis subsp. cremoris JFR1 containing EPS, grown in M17 media with 0.5% glucose supplementation, decreased attachment as well as down regulated virulence gene expression in Salmonella enterica subsp. enterica when tested on HT-29 cells. However, EPS isolated from milk fermented with L. lactis subsp. cremoris JFR1 did not affect Salmonella virulence gene expression or attachment to HT-29 cells. These results suggest that EPS does not contribute to the attachment of Salmonella to human intestinal cells. However, the possibility that the isolation process may have affected the structural features of EPS cannot be ruled out. Copyright © 2016 Elsevier Ltd. All rights reserved.

Clostridium botulinum strains producing BoNT/F4 or BoNT/F5.

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Raphael, Brian H; Bradshaw, Marite; Kalb, Suzanne R; Joseph, Lavin A; Lúquez, Carolina; Barr, John R; Johnson, Eric A; Maslanka, Susan E

2014-05-01

Botulinum neurotoxin type F (BoNT/F) may be produced by Clostridium botulinum alone or in combination with another toxin type such as BoNT/A or BoNT/B. Type F neurotoxin gene sequences have been further classified into seven toxin subtypes. Recently, the genome sequence of one strain of C. botulinum (Af84) was shown to contain three neurotoxin genes (bont/F4, bont/F5, and bont/A2). In this study, eight strains containing bont/F4 and seven strains containing bont/F5 were examined. Culture supernatants produced by these strains were incubated with BoNT/F-specific peptide substrates. Cleavage products of these peptides were subjected to mass spectral analysis, allowing detection of the BoNT/F subtypes present in the culture supernatants. PCR analysis demonstrated that a plasmid-specific marker (PL-6) was observed only among strains containing bont/F5. Among these strains, Southern hybridization revealed the presence of an approximately 242-kb plasmid harboring bont/F5. Genome sequencing of four of these strains revealed that the genomic backgrounds of strains harboring either bont/F4 or bont/F5 are diverse. None of the strains analyzed in this study were shown to produce BoNT/F4 and BoNT/F5 simultaneously, suggesting that strain Af84 is unusual. Finally, these data support a role for the mobility of a bont/F5-carrying plasmid among strains of diverse genomic backgrounds.

The Effect of Tensile Strain on Optical Anisotropy and Exciton of m-Plane ZnO

KAUST Repository

Wang, H. H.

2015-03-20

The near band edge emission of the tensile-strained m-plane ZnO film grown on (112)LaAlO3 substrates shows abnormal low polarization degree (ρ = 0.1). The temperature dependency of polarization degree clarifies the origins of different emission peaks. In tensile-strained m-plane ZnO, the [0001] polarized state is upper shifted and is overlapping with the [112̅0] polarized state. This phenomenon causes the abnormal low polarization degree and reveals the effect of strain on the emission anisotropy of m-plane ZnO.

The cochaperone BAG3 coordinates protein synthesis and autophagy under mechanical strain through spatial regulation of mTORC1.

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Kathage, Barbara; Gehlert, Sebastian; Ulbricht, Anna; Lüdecke, Laura; Tapia, Victor E; Orfanos, Zacharias; Wenzel, Daniela; Bloch, Wilhelm; Volkmer, Rudolf; Fleischmann, Bernd K; Fürst, Dieter O; Höhfeld, Jörg

2017-01-01

The cochaperone BAG3 is a central protein homeostasis factor in mechanically strained mammalian cells. It mediates the degradation of unfolded and damaged forms of the actin-crosslinker filamin through chaperone-assisted selective autophagy (CASA). In addition, BAG3 stimulates filamin transcription in order to compensate autophagic disposal and to maintain the actin cytoskeleton under strain. Here we demonstrate that BAG3 coordinates protein synthesis and autophagy through spatial regulation of the mammalian target of rapamycin complex 1 (mTORC1). The cochaperone utilizes its WW domain to contact a proline-rich motif in the tuberous sclerosis protein TSC1 that functions as an mTORC1 inhibitor in association with TSC2. Interaction with BAG3 results in a recruitment of TSC complexes to actin stress fibers, where the complexes act on a subpopulation of mTOR-positive vesicles associated with the cytoskeleton. Local inhibition of mTORC1 is essential to initiate autophagy at sites of filamin unfolding and damage. At the same time, BAG3-mediated sequestration of TSC1/TSC2 relieves mTORC1 inhibition in the remaining cytoplasm, which stimulates protein translation. In human muscle, an exercise-induced association of TSC1 with the cytoskeleton coincides with mTORC1 activation in the cytoplasm. The spatial regulation of mTORC1 exerted by BAG3 apparently provides the basis for a simultaneous induction of autophagy and protein synthesis to maintain the proteome under mechanical strain. Copyright © 2016 Elsevier B.V. All rights reserved.

A novel and simple cell-based electrochemical biosensor for evaluating the antioxidant capacity of Lactobacillus plantarum strains isolated from Chinese dry-cured ham.

Science.gov (United States)

Ge, Qingfeng; Ge, Panwei; Jiang, Donglei; Du, Nan; Chen, Jiahui; Yuan, Limin; Yu, Hai; Xu, Xin; Wu, Mangang; Zhang, Wangang; Zhou, Guanghong

2018-01-15

The analysis of antioxidants in foodstuffs has become an active area of research, leading to the recent development of numerous methods for assessing antioxidant capacity. Here we described the fabrication and validation of a novel and simple cell-based electrochemical biosensor for this purpose. The biosensor is used to assess the antioxidant capacity of cell-free extracts from Lactobacillus plantarum strains isolated from Chinese dry-cured ham. The biosensor relies on the determination of cellular reactive oxygen species (ROS) (the flux of H 2 O 2 released from RAW 264.7 macrophage cells) to indirectly assess changes in intracellular oxidative stress level as influenced by L. plantarum strains. A one-step acidified manganese dioxide (a-MnO 2 ) modified gold electrode (GE) was used to immobilize RAW 264.7 macrophage cells, which were then encapsulated in a 3D cell culture system consisting of alginate/ graphene oxide (NaAlg/GO). The biosensor exhibited a rapid and sensitive response for the detection of H 2 O 2 released from RAW264.7 cells. The detection limit was 0.02μM with a linear response from 0.05μM to 0.85μM and the biosensor was shown to have good stability and outstanding repeatability. This technique was then used for evaluating the antioxidant ability of extracts from L. plantarum NJAU-01. According to the electrochemical investigations and assays of SEM, TEM, and ROS, these cell-free extracts effectively reduced the oxidative stress levels in RAW264.7 cells under external stimulation. Extracts from L. plantarum strains at a dose of 10 10 CFU/mL showed the highest antioxidant activities with a relative antioxidant capacity (RAC) rate of 88.94%. Hence, this work provides a simple and efficient electrochemical biosensing platform based on RAW264.7 cells for fast, sensitive and quantitative assessment of antioxidant capacity of L. plantarum strains. The method demonstrates its potential for rapid screening for evaluating antioxidant properties of

Novel thermostable clostridial strains through protoplast fusion for enhanced biobutanol production at higher temperature—preliminary study

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Muhammad Ferhan

2016-01-01

Full Text Available The objective of this study is to improve the thermal stability of clostridium strains for enhanced biobutanol production. Thermostable clostridia species were developed through protoplast fusion between mesophilic clostridial species (i.e., Clostridium beijerinckii and Clostridium acetobutylicum and thermophilic clostridial species (i.e., Clostridium thermocellum. Production of biobutanol was examined in the present preliminary study using the clostridium strains and their protoplast fusants using sugar mixture with composition identical to that of wheat straw acid hydrolysate. Maximum biobutanol production of 9.4 g/L was achieved by a fused strain at 45 °C with total sugar consumption of 66% compared to that at 35 °C (i.e., 8.4 g/L production and 64% total sugar consumption. Glucose and xylose uptake rates were generally higher compared to all other individual sugars in the feedstock. In general, average cell concentrations were in close proximity for all parenting and fused strains at 35 °C; i.e., in the range of 5.12 × 107 to 5.49 × 107 cells/mL. Average cell concentration of fusants between the mesophilic clostridial species and the thermophilic clostridial species slightly increased to ~ 5.62 × 107 cells/mL at a higher temperature of 45 °C. These results, in addition to the ones obtained for the butanol production, demonstrate enhanced thermal stability of both fusants at a higher temperature (45 °C.

Prymnesium parvum revisited: Relationship between allelopathy, ichthyotoxicity, and chemical profiles in 5 strains

Energy Technology Data Exchange (ETDEWEB)

Blossom, Hannah E., E-mail: hblossom@bio.ku.dk [Marine Biological Section, University of Copenhagen, Strandpromenaden 5, 3000 Helsingør (Denmark); Rasmussen, Silas A. [Department of Systems Biology, Technical University of Denmark, Søltofts Plads, Building 221, 2800 Kgs Lyngby (Denmark); Andersen, Nikolaj G. [Marine Biological Section, University of Copenhagen, Strandpromenaden 5, 3000 Helsingør (Denmark); Larsen, Thomas O.; Nielsen, Kristian F. [Department of Systems Biology, Technical University of Denmark, Søltofts Plads, Building 221, 2800 Kgs Lyngby (Denmark); Hansen, Per J. [Marine Biological Section, University of Copenhagen, Strandpromenaden 5, 3000 Helsingør (Denmark)

2014-12-15

Highlights: • Five strains of P. parvum were tested for toxicity towards rainbow trout and microalgae. • Toxicity towards microalgae was not correlated to toxicity towards fish. • A microalgal bioassay cannot be used as a reliable proxy for ichthyotoxicity. • Concentrations of GATs were low and not correlated to effects on fish or on algae. • P. parvum does not produce oleamide based on {sup 13}C labeling and extraction in glass. - Abstract: Bioassay-guided discovery of ichthyotoxic algal compounds using in vivo fish assays is labor intensive, costly, and highly regulated. Since the mode of action of most known algal-mediated fish-killing toxins is damage to the cell membranes in the gills, various types of cell-based bioassays are often used for bioassay guided purification of new ichthyotoxins. Here we tested the hypothesis that allelopathy is related to ichthyotoxicity and thus that a microalgal bioassay can be used as a proxy for ichthyotoxicity by comparing the toxicity of five strains of Prymnesium parvum toward rainbow trout (Oncorhynchus mykiss, 10 g) and the microalga Teleaulax acuta. No relationship between median effective concentrations (EC{sub 50}s) on fish and median lethal concentrations (LC{sub 50}s) on algae was observed in the 5 strains showing that a microalgal bioassay cannot be used as a proxy for ichthyotoxicity. Fish were more sensitive to P. parvum with EC{sub 50}s ranging from 6 × 10{sup 3} to 40 × 10{sup 3} cells ml{sup −1}, compared to the test alga where LC{sub 50}s ranged from 30 × 10{sup 3} to nearly non-toxic at 500 × 10{sup 3} cells ml{sup −1}. In addition, the cellular concentrations of two recently suggested ichthyotoxins produced by P. parvum, the “golden algae toxins”, GAT 512 and a novel GAT 510, did not show any relationship to either ichthyotoxicity or allelopathy, and are not the biologically relevant toxins, but are simply lipids found in algal chloroplasts. Finally, we demonstrate that the recently

Kushenin induces the apoptosis of HCV-infected cells by blocking the PI3K-Akt-mTOR pathway via inhibiting NS5A

Energy Technology Data Exchange (ETDEWEB)

Zhou, Yi; Chen, Na; Liu, Xiaojing; Lin, Shumei [Department of Infectious Diseases, The First Affiliated Hospital of Xi’an Jiaotong University, Xi’an, Shaanxi 710061 (China); Luo, Wenjuan, E-mail: wenjuanluoxa@163.com [School of Pharmacy, Xi’an Jiaotong University, Xi’an, Shaanxi 710061 (China); Liu, Min, E-mail: minliusx@163.com [Department of Infectious Diseases, The First Affiliated Hospital of Xi’an Jiaotong University, Xi’an, Shaanxi 710061 (China)

2016-07-01

With the increased burden induced by HCV, there is an urgent need to develop better-tolerated agents with good safety. In this study, we evaluated the anti-HCV capability of kushenin, as well as the possible mechanism to Huh7.5-HCV cells. The results demonstrated that kushenin significantly inhibited the HCV-RNA level. Similarly, the expression of HCV-specific protein NS5A was also decreased. Molecular docking results displayed that kushenin bonded well to the active pockets of HCV NS5A, further confirming the effects of kushenin on HCV replication. Coimmunoprecipitation assay determined that kushenin suppressed the interaction between PI3K and NS5A in HCV-replicon cells. Furthermore, kushenin exerted an obviously induced function on HCV-replicon cells apoptosis by inhibiting PI3K-Akt-mTOR pathway, which could be ameliorated by the specific activator IGF-1 addition. Taken together, kushenin possesses the ability to inhibit HCV replication, and contributes to the increased apoptosis of HCV-infected cells by blocking the PI3K-Akt-mTOR pathway via inhibiting NS5A. Our results provide important evidence for a better understanding of the pathogenesis of HCV infection, and suggest that kushenin has the potential to treat HCV disease. - Highlights: • Kushenin inhibits HCV replication. • Kushenin bonds directly to NS5A protein. • Kushenin induces the apoptosis of HCV-infected cells. • kushenin suppresses the interaction between PI3K and NS5A. • Kushenin inhibits PI3K-Akt-mTOR pathway.

Kushenin induces the apoptosis of HCV-infected cells by blocking the PI3K-Akt-mTOR pathway via inhibiting NS5A

International Nuclear Information System (INIS)

Zhou, Yi; Chen, Na; Liu, Xiaojing; Lin, Shumei; Luo, Wenjuan; Liu, Min

2016-01-01

With the increased burden induced by HCV, there is an urgent need to develop better-tolerated agents with good safety. In this study, we evaluated the anti-HCV capability of kushenin, as well as the possible mechanism to Huh7.5-HCV cells. The results demonstrated that kushenin significantly inhibited the HCV-RNA level. Similarly, the expression of HCV-specific protein NS5A was also decreased. Molecular docking results displayed that kushenin bonded well to the active pockets of HCV NS5A, further confirming the effects of kushenin on HCV replication. Coimmunoprecipitation assay determined that kushenin suppressed the interaction between PI3K and NS5A in HCV-replicon cells. Furthermore, kushenin exerted an obviously induced function on HCV-replicon cells apoptosis by inhibiting PI3K-Akt-mTOR pathway, which could be ameliorated by the specific activator IGF-1 addition. Taken together, kushenin possesses the ability to inhibit HCV replication, and contributes to the increased apoptosis of HCV-infected cells by blocking the PI3K-Akt-mTOR pathway via inhibiting NS5A. Our results provide important evidence for a better understanding of the pathogenesis of HCV infection, and suggest that kushenin has the potential to treat HCV disease. - Highlights: • Kushenin inhibits HCV replication. • Kushenin bonds directly to NS5A protein. • Kushenin induces the apoptosis of HCV-infected cells. • kushenin suppresses the interaction between PI3K and NS5A. • Kushenin inhibits PI3K-Akt-mTOR pathway.

Efficient infectious cell culture systems of the hepatitis C virus (HCV) prototype strains HCV-1 and H77.

Science.gov (United States)

Li, Yi-Ping; Ramirez, Santseharay; Mikkelsen, Lotte; Bukh, Jens

2015-01-01

The first discovered and sequenced hepatitis C virus (HCV) genome and the first in vivo infectious HCV clones originated from the HCV prototype strains HCV-1 and H77, respectively, both widely used in research of this important human pathogen. In the present study, we developed efficient infectious cell culture systems for these genotype 1a strains by using the HCV-1/SF9_A and H77C in vivo infectious clones. We initially adapted a genome with the HCV-1 5'UTR-NS5A (where UTR stands for untranslated region) and the JFH1 NS5B-3'UTR (5-5A recombinant), including the genotype 2a-derived mutations F1464L/A1672S/D2979G (LSG), to grow efficiently in Huh7.5 cells, thus identifying the E2 mutation S399F. The combination of LSG/S399F and reported TNcc(1a)-adaptive mutations A1226G/Q1773H/N1927T/Y2981F/F2994S promoted adaptation of the full-length HCV-1 clone. An HCV-1 recombinant with 17 mutations (HCV1cc) replicated efficiently in Huh7.5 cells and produced supernatant infectivity titers of 10(4.0) focus-forming units (FFU)/ml. Eight of these mutations were identified from passaged HCV-1 viruses, and the A970T/I1312V/C2419R/A2919T mutations were essential for infectious particle production. Using CD81-deficient Huh7 cells, we further demonstrated the importance of A970T/I1312V/A2919T or A970T/C2419R/A2919T for virus assembly and that the I1312V/C2419R combination played a major role in virus release. Using a similar approach, we found that NS5B mutation F2994R, identified here from culture-adapted full-length TN viruses and a common NS3 helicase mutation (S1368P) derived from viable H77C and HCV-1 5-5A recombinants, initiated replication and culture adaptation of H77C containing LSG and TNcc(1a)-adaptive mutations. An H77C recombinant harboring 19 mutations (H77Ccc) replicated and spread efficiently after transfection and subsequent infection of naive Huh7.5 cells, reaching titers of 10(3.5) and 10(4.4) FFU/ml, respectively. Hepatitis C virus (HCV) was discovered in 1989 with

Genomic, Transcriptomic and Metabolomic Studies of Two Well-Characterized, Laboratory-Derived Vancomycin-Intermediate Staphylococcus aureus Strains Derived from the Same Parent Strain

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Dipti S. Hattangady

2015-02-01

Full Text Available Complete genome comparisons, transcriptomic and metabolomic studies were performed on two laboratory-selected, well-characterized vancomycin-intermediate Staphylococcus aureus (VISA derived from the same parent MRSA that have changes in cell wall composition and decreased autolysis. A variety of mutations were found in the VISA, with more in strain 13136p−m+V20 (vancomycin MIC = 16 µg/mL than strain 13136p−m+V5 (MIC = 8 µg/mL. Most of the mutations have not previously been associated with the VISA phenotype; some were associated with cell wall metabolism and many with stress responses, notably relating to DNA damage. The genomes and transcriptomes of the two VISA support the importance of gene expression regulation to the VISA phenotype. Similarities in overall transcriptomic and metabolomic data indicated that the VISA physiologic state includes elements of the stringent response, such as downregulation of protein and nucleotide synthesis, the pentose phosphate pathway and nutrient transport systems. Gene expression for secreted virulence determinants was generally downregulated, but was more variable for surface-associated virulence determinants, although capsule formation was clearly inhibited. The importance of activated stress response elements could be seen across all three analyses, as in the accumulation of osmoprotectant metabolites such as proline and glutamate. Concentrations of potential cell wall precursor amino acids and glucosamine were increased in the VISA strains. Polyamines were decreased in the VISA, which may facilitate the accrual of mutations. Overall, the studies confirm the wide variability in mutations and gene expression patterns that can lead to the VISA phenotype.

Characterization of the Metabolically Modified Heavy Metal-Resistant Cupriavidus metallidurans Strain MSR33 Generated for Mercury Bioremediation

Science.gov (United States)

Rojas, Luis A.; Yáñez, Carolina; González, Myriam; Lobos, Soledad; Smalla, Kornelia; Seeger, Michael

2011-01-01

Background Mercury-polluted environments are often contaminated with other heavy metals. Therefore, bacteria with resistance to several heavy metals may be useful for bioremediation. Cupriavidus metallidurans CH34 is a model heavy metal-resistant bacterium, but possesses a low resistance to mercury compounds. Methodology/Principal Findings To improve inorganic and organic mercury resistance of strain CH34, the IncP-1β plasmid pTP6 that provides novel merB, merG genes and additional other mer genes was introduced into the bacterium by biparental mating. The transconjugant Cupriavidus metallidurans strain MSR33 was genetically and biochemically characterized. Strain MSR33 maintained stably the plasmid pTP6 over 70 generations under non-selective conditions. The organomercurial lyase protein MerB and the mercuric reductase MerA of strain MSR33 were synthesized in presence of Hg2+. The minimum inhibitory concentrations (mM) for strain MSR33 were: Hg2+, 0.12 and CH3Hg+, 0.08. The addition of Hg2+ (0.04 mM) at exponential phase had not an effect on the growth rate of strain MSR33. In contrast, after Hg2+ addition at exponential phase the parental strain CH34 showed an immediate cessation of cell growth. During exposure to Hg2+ no effects in the morphology of MSR33 cells were observed, whereas CH34 cells exposed to Hg2+ showed a fuzzy outer membrane. Bioremediation with strain MSR33 of two mercury-contaminated aqueous solutions was evaluated. Hg2+ (0.10 and 0.15 mM) was completely volatilized by strain MSR33 from the polluted waters in presence of thioglycolate (5 mM) after 2 h. Conclusions/Significance A broad-spectrum mercury-resistant strain MSR33 was generated by incorporation of plasmid pTP6 that was directly isolated from the environment into C. metallidurans CH34. Strain MSR33 is capable to remove mercury from polluted waters. This is the first study to use an IncP-1β plasmid directly isolated from the environment, to generate a novel and stable bacterial strain

Characterization of the metabolically modified heavy metal-resistant Cupriavidus metallidurans strain MSR33 generated for mercury bioremediation.

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Luis A Rojas

Full Text Available BACKGROUND: Mercury-polluted environments are often contaminated with other heavy metals. Therefore, bacteria with resistance to several heavy metals may be useful for bioremediation. Cupriavidus metallidurans CH34 is a model heavy metal-resistant bacterium, but possesses a low resistance to mercury compounds. METHODOLOGY/PRINCIPAL FINDINGS: To improve inorganic and organic mercury resistance of strain CH34, the IncP-1β plasmid pTP6 that provides novel merB, merG genes and additional other mer genes was introduced into the bacterium by biparental mating. The transconjugant Cupriavidus metallidurans strain MSR33 was genetically and biochemically characterized. Strain MSR33 maintained stably the plasmid pTP6 over 70 generations under non-selective conditions. The organomercurial lyase protein MerB and the mercuric reductase MerA of strain MSR33 were synthesized in presence of Hg(2+. The minimum inhibitory concentrations (mM for strain MSR33 were: Hg(2+, 0.12 and CH(3Hg(+, 0.08. The addition of Hg(2+ (0.04 mM at exponential phase had not an effect on the growth rate of strain MSR33. In contrast, after Hg(2+ addition at exponential phase the parental strain CH34 showed an immediate cessation of cell growth. During exposure to Hg(2+ no effects in the morphology of MSR33 cells were observed, whereas CH34 cells exposed to Hg(2+ showed a fuzzy outer membrane. Bioremediation with strain MSR33 of two mercury-contaminated aqueous solutions was evaluated. Hg(2+ (0.10 and 0.15 mM was completely volatilized by strain MSR33 from the polluted waters in presence of thioglycolate (5 mM after 2 h. CONCLUSIONS/SIGNIFICANCE: A broad-spectrum mercury-resistant strain MSR33 was generated by incorporation of plasmid pTP6 that was directly isolated from the environment into C. metallidurans CH34. Strain MSR33 is capable to remove mercury from polluted waters. This is the first study to use an IncP-1β plasmid directly isolated from the environment, to generate a novel

Production of high titer attenuated poliovirus strains on the serum-free PER.C6(®) cell culture platform for the generation of safe and affordable next generation IPV.

Science.gov (United States)

Sanders, Barbara P; Oakes, Isabel de los Rios; van Hoek, Vladimir; Liu, Ying; Marissen, Wilfred; Minor, Philip D; Wimmer, Eckard; Schuitemaker, Hanneke; Custers, Jerome H H V; Macadam, Andrew; Cello, Jeronimo; Edo-Matas, Diana

2015-11-27

As poliovirus eradication draws closer, alternative Inactivated Poliovirus Vaccines (IPV) are needed to overcome the risks associated with continued use of the Oral Poliovirus Vaccine and of neurovirulent strains used during manufacture of conventional (c) IPV. We have previously demonstrated the susceptibility of the PER.C6(®) cell line to cIPV strains; here we investigated the suspension cell culture platform for growth of attenuated poliovirus strains. We examined attenuated Sabin strain productivity on the PER.C6(®) cell platform compared to the conventional Vero cell platform. The suitability of the suspension cell platform for propagation of rationally-attenuated poliovirus strains (stabilized Sabin type 3 S19 derivatives and genetically attenuated and stabilized MonoCre(X) strains), was also assessed. Yields were quantified by infectious titer determination and D-antigen ELISA using either serotype-specific polyclonal rabbit sera for Sabin strains or monoclonal cIPV-strain-specific antibodies for cIPV, S19 and MonoCre(X) strains. PER.C6(®) cells supported the replication of Sabin strains to yields of infectious titers that were in the range of cIPV strains at 32.5°C. Sabin strains achieved 30-fold higher yields (pSabin strain productivity on the PER.C6(®) cell platform was maintained at 10l scale. Yields of infectious titers of S19 and MonoCre(X) strains were 0.5-1 log10 lower than seen for cIPV strains, whereas D-antigen yield and productivities in doses/ml using rationally-attenuated strains were in line with yields reported for cIPV strains. Sabin and rationally-attenuated polioviruses can be grown to high infectious titers and D-antigen yields. Sabin strain infection shows increased productivity on the PER.C6(®) cell platform as compared to the conventional Vero cell platform. Novel cell platforms with the potential for higher yields could contribute to increased affordability of a next generation of IPV vaccines needed for achieving and

Accumulation of Tc-99m-MIBI and Tc-99m-tetrofosmin in tumor cells. Uptake and washout studies

International Nuclear Information System (INIS)

Rodrigues, M.; Aghajanian, A.A.; Sinzinger, H.; Kalinowska, W.; Zielinski, C.

2002-01-01

Aim: To investigate in-vitro the uptake and washout of Tc-99m-MIBI and Tc-99m-tetrofosmin in human breast adenocarcinoma and soft tissue sarcoma cell lines. Methods: The uptake of Tc-99m-MIBI and Tc-99m-tetrofosmin (at 37 0 C, 10, 30 and 60 minutes after incubation with 7.4x10E5 Bq each tracer) was investigated in breast adenocarcinoma MCF-7 and SK-BR-3 cells, synovial sarcoma SW 982 cells and chondrosarcoma SW 1353 cells (concentration of 1x10E6 cells/ml incubation medium). Tracer uptake in cells incubated with ouabain (Na/K-ATPase pump inhibitor; 100 μM and 1mM; 15 and 30 minutes), nigericin (increases mitochondrial potential and disrupts cell membrane potential; 5 and 50 μg/ml; 15 minutes) and carbonyl cyanide m-chlorophenylhydrazone (CCCP) (depolarizes mitochondrial membrane; 10 and 100 μM; 30 minutes) was compared to that in cells without incubation with chemical agents (control cells). The washout (at 37 0 C, 10-60 minutes, 30 and 60 minutes after tracer incubation) of Tc-99m-MIBI and Tc-99m-tetrofosmin was studied in MCF-7 cells, SK-BR-3 cells , SW 1353 cells and fibrosarcoma SW 684 cells. Results: Cellular tracer uptake decreased with ouabain (decrease of Tc-99m-tetrofosmin > Tc-99m-MIBI in SK-BR-3 cells and SW 982 cells) and increased with nigericin (increase of Tc-99m-MIBI > Tc-99m-tetrofosmin in all cells) as compared to the uptake in control cells. With CCCP, decrease of Tc-99m-MIBI uptake in cells preincubated with nigericin was higher than that in cells under basal conditions, whereas a similar decrease of Tc-99m-tetrofosmin uptake in these two group of cells was found. Washout of Tc-99m-MIBI from all cells was lower than that of Tc-99m-tetrofosmin. No significant difference in cell-associated activity of both tracers was found between washout after 30 minutes and that after 60 minutes of incubation, in all cells. Washout of Tc-99m-MIBI from SK-BR-3 cells Tc-99m-tetrofosmin) and Na/K-ATPase pump (Tc-99m-tetrofosmin > Tc-99m-MIBI). Tc-99m-MIBI and

Targeted Delivery of GP5 Antigen of PRRSV to M Cells Enhances the Antigen-Specific Systemic and Mucosal Immune Responses

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Luping Du

2018-01-01

Full Text Available Efficient delivery of antigens through oral immunization is a first and critical step for successful induction of mucosal immunity, which can provide protection against pathogens invading the mucosa. Membranous/microfold cells (M cells within the mucosa can transcytose internalized antigen without degradation and thus play an important role in initiating antigen-specific mucosal immune responses through inducing secretory IgA production. In this research, we modified poly (D, L-lactide-co-glycolide (PLGA nanoparticles (NPs with Ulex europaeus agglutinin 1 (UEA-1 and successfully prepared an oral vaccine delivery system, UEA-1/PLGA NPs. PLGA NPs were prepared using a standard double emulsion solvent evaporation technique, which can protect the entrapped PRRSV DNA vaccine [pcDNA3.1-SynORF5 (synthetic ORF5] or subunit vaccine ORF5-encoded glycoprotein (GP5 from exposure to the gastrointestinal (GI tract and release the plasmids in a controlled manner. With UEA-1 modification, the UEA-1/PLGA NPs can be effectively transported by M-cells. We investigated immune response induced by UEA-1/PLGA-SynORF5 or UEA-1/PLGA-GP5 following inoculation in mice and piglets. Compared with PLGA-SynORF5 or PLGA-GP5 NPs, UEA-1/PLGA-SynORF5, or UEA-1/PLGA-GP5 NPs stimulated significantly increased serum IgG levels and augmented intestinal IgA levels in mice and piglets (P < 0.05. Our findings indicate UEA-1/PLGA NPs can be applied as a promising and universally robust oral vaccine delivery system.

Targeted Delivery of GP5 Antigen of PRRSV to M Cells Enhances the Antigen-Specific Systemic and Mucosal Immune Responses

Science.gov (United States)

Du, Luping; Yu, Zhengyu; Pang, Fengjiao; Xu, Xiangwei; Mao, Aihua; Yuan, Wanzhe; He, Kongwang; Li, Bin

2018-01-01

Efficient delivery of antigens through oral immunization is a first and critical step for successful induction of mucosal immunity, which can provide protection against pathogens invading the mucosa. Membranous/microfold cells (M cells) within the mucosa can transcytose internalized antigen without degradation and thus play an important role in initiating antigen-specific mucosal immune responses through inducing secretory IgA production. In this research, we modified poly (D, L-lactide-co-glycolide) (PLGA) nanoparticles (NPs) with Ulex europaeus agglutinin 1 (UEA-1) and successfully prepared an oral vaccine delivery system, UEA-1/PLGA NPs. PLGA NPs were prepared using a standard double emulsion solvent evaporation technique, which can protect the entrapped PRRSV DNA vaccine [pcDNA3.1-SynORF5 (synthetic ORF5)] or subunit vaccine ORF5-encoded glycoprotein (GP5) from exposure to the gastrointestinal (GI) tract and release the plasmids in a controlled manner. With UEA-1 modification, the UEA-1/PLGA NPs can be effectively transported by M-cells. We investigated immune response induced by UEA-1/PLGA-SynORF5 or UEA-1/PLGA-GP5 following inoculation in mice and piglets. Compared with PLGA-SynORF5 or PLGA-GP5 NPs, UEA-1/PLGA-SynORF5, or UEA-1/PLGA-GP5 NPs stimulated significantly increased serum IgG levels and augmented intestinal IgA levels in mice and piglets (P < 0.05). Our findings indicate UEA-1/PLGA NPs can be applied as a promising and universally robust oral vaccine delivery system. PMID:29423381

Identification of mutations in the M RNA of a candidate vaccine strain of Rift Valley fever virus.

Science.gov (United States)

Takehara, K; Min, M K; Battles, J K; Sugiyama, K; Emery, V C; Dalrymple, J M; Bishop, D H

1989-04-01

The M RNA species of a candidate vaccine strain of Rift Valley fever virus (RVFV ZH-548M12), derived by consecutive high level mutagenesis using 5-fluorouracil (H. Caplen, C. J. Peters, and D. H. L. Bishop, J. Gen. Virol., 66, 2271-2277, 1985), has been cloned and the cDNA sequenced. The data have been compared to those obtained for the parent virus strain RVFV ZH-548 as well as the previously published data for RVFV ZH-501 (M. S. Collett, A. F. Purchio, K. Keegan, S. Frazier, W. Hays, D. K. Anderson, M. D. Parker, C. Schmaljohn, J. Schmidt, and J. M. Dalrymple, Virology, 144, 228-245, 1985). Some eight nucleotide and three amino acid differences were identified between the M RNAs of ZH-501 and ZH-548. Between the M RNAs of ZH-548 and that of the M12 mutant there were 12 nucleotide and 7 amino acid changes. Unique to the mutant virus is a new AUG codon upstream of that which initiates the open reading frame of the RVFV M gene product (the viral glycoprotein precursor). The significance of this and other differences in the mutant RNA with regard to the derivation and potential attenuation of the candidate vaccine is discussed.

Residual strain, scale effects, and time-dependent behaviour at the 240-m level of the underground research laboratory

International Nuclear Information System (INIS)

Read, R.S.

1990-01-01

Two subhorizontal, orthogonal boreholes were monitored continuously during concentric overcoring at the 240-m level of the Underground Research Laboratory (URL). The magnitude and orientation of principal residual strain components in the near-field stress regime were determined assuming linear elastic behaviour of the rock mass and isotropic conditions. In terms of magnitude, results compared favourably with those from previous tests at the 240-m level. However, orientation results were inconclusive. The effects of scale and borehole orientation relative to the principal stress direction on the results from a modified CSIR triaxial cell overcore test were also investigated; no scale effects were apparent in the experiment, but borehole orientation did affect results. Finally, time-dependent behaviour was detected in the Lac du Bonnet granite, and was monitored between successive overcore tests in one of the boreholes. Results on residual strain, scale effects, and time-dependent behaviour are presented, along with limitations and possible modifications to the testing procedure

Biotransformation of Tributyltin chloride by Pseudomonas stutzeri strain DN2

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Dnyanada S. Khanolkar

2014-12-01

Full Text Available A bacterial isolate capable of utilizing tributyltin chloride (TBTCl as sole carbon source was isolated from estuarine sediments of west coast of India and identified as Pseudomonas stutzeri based on biochemical tests and Fatty acid methyl ester (FAME analysis. This isolate was designated as strain DN2. Although this bacterial isolate could resist up to 3 mM TBTCl level, it showed maximum growth at 2 mM TBTCl in mineral salt medium (MSM. Pseudomonas stutzeri DN2 exposed to 2 mM TBTCl revealed significant alteration in cell morphology as elongation and shrinkage in cell size along with roughness of cell surface. FTIR and NMR analysis of TBTCl degradation product extracted using chloroform and purified using column chromatography clearly revealed biotransformation of TBTCl into Dibutyltin dichloride (DBTCl2 through debutylation process. Therefore, Pseudomonas stutzeri strain DN2 may be used as a potential bacterial strain for bioremediation of TBTCl contaminated aquatic environmental sites.

Biotransformation of Tributyltin chloride by Pseudomonas stutzeri strain DN2

Science.gov (United States)

Khanolkar, Dnyanada S.; Naik, Milind Mohan; Dubey, Santosh Kumar

2014-01-01

A bacterial isolate capable of utilizing tributyltin chloride (TBTCl) as sole carbon source was isolated from estuarine sediments of west coast of India and identified as Pseudomonas stutzeri based on biochemical tests and Fatty acid methyl ester (FAME) analysis. This isolate was designated as strain DN2. Although this bacterial isolate could resist up to 3 mM TBTCl level, it showed maximum growth at 2 mM TBTCl in mineral salt medium (MSM). Pseudomonas stutzeri DN2 exposed to 2 mM TBTCl revealed significant alteration in cell morphology as elongation and shrinkage in cell size along with roughness of cell surface. FTIR and NMR analysis of TBTCl degradation product extracted using chloroform and purified using column chromatography clearly revealed biotransformation of TBTCl into Dibutyltin dichloride (DBTCl2) through debutylation process. Therefore, Pseudomonas stutzeri strain DN2 may be used as a potential bacterial strain for bioremediation of TBTCl contaminated aquatic environmental sites. PMID:25763027

Bioaccumulation of uranium from waste water using different strains of Saccharomyces cerevisiae

International Nuclear Information System (INIS)

Tykva, R.; Novak, J.; Podracka, E.; Popa, K.

2009-01-01

Five different strains of Saccharomyces cerevisiae were tested for their abilities to accumulate uranium from waste water containing competitive ions. Samples of water passing out from a previous uranium mill were used. The strains tested possess different abilities to accumulate uranium. The kinetics of bioaccumulation, the leaching degree, the influence of cell density and their origin were investigated. Under the applied experimental conditions, more than a half of the total activity (uranium and the decay products) could be accumulated after 60 min contact time of 1 mL (S. cerevisiae) suspension and 5 mL of water. The other cations present in solution effectively competed for the uranium accumulation. 226 Ra and its decay products were completely retained using all tested strains. (authors)

Antifungal activity of plant essential oils and selected Pseudomonas strains against Phomopsis theicola

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Starović Mira

2017-01-01

Full Text Available Development of natural plant protection products as an alternative to synthetic fungicides is of significant importance regarding the environment. This study was carried out with an objective to investigate in vitro antifungal activities of several essential oils extracted from oregano, basil, myrtle and Turkish pickling herb, and the plant growth-promoting rhizobacteria in the genus Pseudomonas, against the phytopathogenic fungus Phomopsis theicola. Microdilution methods were used to determine the minimum inhibitory concentrations (MIC of selected antimicrobial essential oils (EOs. All EOs exhibited significant levels of antifungal activity against the tested fungal isolates. The oregano EO was found the most potent one (MIC - 5.5 µg/mL, followed by basil (MIC - 75.0µg/mL, myrtle (MIC - 775 µg/mL and Turkish pickling herb (MIC - 7750 µg/mL. Inhibition of Ph. theicola mycelial growth was observed for all tested Pseudomonas spp. strains. K113 and L1 strains were highly effective and achieved more than 60% of fungal growth inhibition using the overnight culture and more than 57% inhibition by applying cell-free supernatants of both strains. A future field trial with K113 and L1 cultures and cell-free supernatants, containing extracellular metabolites toward Ph. theicola, will estimate their effectiveness and applicability as an alternative to chemical protection of apple trees.

Characterisation of the simultaneous molybdenum reduction and glyphosate degradation by Burkholderia vietnamiensis AQ5-12 and Burkholderia sp. AQ5-13.

Science.gov (United States)

Manogaran, Motharasan; Ahmad, Siti Aqlima; Yasid, Nur Adeela; Yakasai, Hafeez Muhammad; Shukor, Mohd Yunus

2018-02-01

In this novel study, we report on the use of two molybdenum-reducing bacteria with the ability to utilise the herbicide glyphosate as the phosphorus source. The bacteria reduced sodium molybdate to molybdenum blue (Mo-blue), a colloidal and insoluble product, which is less toxic. The characterisation of the molybdenum-reducing bacteria was carried out using resting cells immersed in low-phosphate molybdenum media. Two glyphosate-degrading bacteria, namely Burkholderia vietnamiensis AQ5-12 and Burkholderia sp. AQ5-13, were able to use glyphosate as a phosphorous source to support molybdenum reduction to Mo-blue. The bacteria optimally reduced molybdenum between the pHs of 6.25 and 8. The optimum concentrations of molybdate for strain Burkholderia vietnamiensis strain AQ5-12 was observed to be between 40 and 60 mM, while for Burkholderia sp. AQ5-13, the optimum molybdate concentration occurred between 40 and 50 mM. Furthermore, 5 mM of phosphate was seen as the optimum concentration supporting molybdenum reduction for both bacteria. The optimum temperature aiding Mo-blue formation ranged from 30 to 40 °C for Burkholderia vietnamiensis strain AQ5-12, whereas for Burkholderia sp. AQ5-13, the range was from 35 to 40 °C. Glucose was the best electron donor for supporting molybdate reduction, followed by sucrose, fructose and galactose for both strains. Ammonium sulphate was the best nitrogen source in supporting molybdenum reduction. Interestingly, increasing the glyphosate concentrations beyond 100 and 300 ppm for Burkholderia vietnamiensis strain AQ5-12 and Burkholderia sp. AQ5-13, respectively, significantly inhibited molybdenum reduction. The ability of these bacteria to reduce molybdenum while degrading glyphosate is a useful process for the bioremediation of both toxicants.

Monitoring the ethanol stress response of a sigM deletion strain of B. cereus ATCC 14579.

NARCIS (Netherlands)

Voort, van der M.

2008-01-01

Here, the role of σM and its regulon in stress response and survival of B. cereus ATCC 14579 was assessed by comparative transciptome and phenotypic analysis of this strain and its sigM deletion strain. Exposure of B. cereus ATCC 14579 to a wide range of stresses revealed expression of sigM,

Molecular-beam epitaxy growth and characterization of 5-μm quantum cascade laser

International Nuclear Information System (INIS)

Mamutin, V V; Ustinov, V M; Ilyinskaya, N D; Baydakova, M V; Ber, B Ya; Kasantsev, D Yu

2011-01-01

Molecular-beam epitaxy growth of 5 μm emitting strain-compensated quantum semiconductor laser (QCL) is reported. The QCL structure is characterized by complementary techniques: high-resolution X-ray diffraction and dynamical secondary-ion mass-spectrometry, that reveal the high quality of QCL structure and in-depth distribution of chemical composition, respectively.

Acid resistance and response to pH-induced stress in two Lactobacillus plantarum strains with probiotic potential.

Science.gov (United States)

Šeme, H; Gjuračić, K; Kos, B; Fujs, Š; Štempelj, M; Petković, H; Šušković, J; Bogovič Matijašić, B; Kosec, G

2015-01-01

Two new Lactobacillus plantarum strains, KR6-DSM 28780 and M5 isolated from sour turnip and traditional dried fresh cheese, respectively, were evaluated for species identity, antibiotic susceptibility, resistance to gastrointestinal conditions and adaptive response to low pH. Resistance mechanisms involved in the adaptation to acid-induced stress in these two strains were investigated by quantitative PCR of the atpA, cfa1, mleS and hisD genes. In addition to absence of antibiotic resistance, the two L. plantarum strains showed excellent survival rates at pH values as low as 2.4. Adaptive response to low pH was clearly observed in both strains; strain KR6 was superior to M5, as demonstrated by its ability to survive during 3 h incubation at pH 2.0 upon adaptation to moderately acidic conditions. In contrast, acid adaptation did not significantly affect the survival rate during simulated passage through the gastrointestinal tract. In both strains, induction of histidine biosynthesis (hisD) was upregulated during the acid adaptation response. In addition, significant upregulation of the cfa1 gene, involved in modulation of membrane fatty acid composition, was observed during the adaptation phase in strain KR6 but not in strain M5. Cells adapted to moderately acidic conditions also showed a significantly increased viability after the lyophilisation procedure, a cross-protection phenomenon providing additional advantage in probiotic application.

Three-dimensional development of tensile pre-strained annulus fibrosus cells for tissue regeneration: An in-vitro study

Energy Technology Data Exchange (ETDEWEB)

Chuah, Yon Jin [School of Chemical and Biomedical Engineering, Nanyang Technological University, 62 Nanyang Drive, Singapore 637459 (Singapore); Lee, Wu Chean [University Hospital Conventry & Warwickshire NHS Trust, Clifford Bridge Road, West Midlands CV2, 2DX (United Kingdom); Wong, Hee Kit [Department of Orthopedic Surgery, National University Health System, NUHS Tower Block Level 11, 1E Kent Ridge Road, Singapore 119228 (Singapore); Kang, Yuejun, E-mail: yuejun.kang@ntu.edu.sg [School of Chemical and Biomedical Engineering, Nanyang Technological University, 62 Nanyang Drive, Singapore 637459 (Singapore); Hee, Hwan Tak, E-mail: HTHee@ntu.edu.sg [School of Chemical and Biomedical Engineering, Nanyang Technological University, 62 Nanyang Drive, Singapore 637459 (Singapore); Pinnacle Spine & Scoliosis Centre, 3 Mount Elizabeth, Mount Elizabeth Medical Centre, #04-07, Singapore 228510 (Singapore); School of Physical and Mathematical Sciences, Nanyang Technological University, 21 Nanyang Link, Singapore 637459 (Singapore)

2015-02-01

Prior research has investigated the immediate response after application of tensile strain on annulus fibrosus (AF) cells for the past decade. Although mechanical strain can produce either catabolic or anabolic consequences to the cell monolayer, little is known on how to translate these findings into further tissue engineering applications. Till to date, the application and effect of tensile pre-strained cells to construct a three-dimensional (3D) AF tissue remains unknown. This study aims to investigate the effect of tensile pre-strained exposure of 1 to 24 h on the development of AF pellet culture for 3 weeks. Equibiaxial cyclic tensile strain was applied on AF monolayer cells over a period of 24 h, which was subsequently developed into a cell pellet. Investigation on cellular proliferation, phenotypic gene expression, and histological changes revealed that tensile pre-strain for 24 h had significant and lasting effect on the AF tissue development, with enhanced cell proliferation, and up-regulation of collagen type I, II, and aggrecan expression. Our results demonstrated the regenerative ability of AF cell pellets subjected to 24 h tensile pre-straining. Knowledge on the effects of tensile pre-strain exposure is necessary to optimize AF development for tissue reconstruction. Moreover, the tensile pre-strained cells may further be utilized in either cell therapy to treat mild disc degeneration disease, or the development of a disc construct for total disc replacement. - Highlights: • Establishment of tensile pre-strained cell line population for annulus development. • Tensile strain limits collagen gene expression declination in monolayer culture. • Tensile pre-strained cells up-regulate their matrix protein in 3D pellet culture.

Synthesis, Antifungal and Antitumor Activity of Novel (Z-5-Hetarylmethylidene-1,3-thiazol-4-ones and (Z-5-Ethylidene-1,3-thiazol-4-ones

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Susana A. Zacchino

2013-05-01

Full Text Available New hetaryl- and alkylidenerhodanine derivatives 3a–d, 3e, and 4a–d were prepared from heterocyclic aldehydes 1a–d or acetaldehyde 1e. The treatment of several rhodanine derivatives 3a–d and 3e with piperidine or morpholine in THF under reflux, afforded (Z-5-(hetarylmethylidene-2-(piperidin-1-ylthiazol-4(5H-ones and 2-morpholinothiazol-4(5H-ones 5a–d, 6a–d, and (Z-5-ethylidene-2-morpholinothiazol-4(5H-one (5e, respectively, in good yields. Structures of all compounds were determined by IR, 1D and 2D NMR and mass spectrometry. Several of these compounds were screened by the U.S. National Cancer Institute (NCI to assess their antitumor activity against 60 different human tumor cell lines. Compound 3c showed high activity against HOP-92 (Non-Small Cell Lung Cancer, which was the most sensitive cell line, with GI50 = 0.62 μM and LC50 > 100 μM from the in vitro assays. In vitro antifungal activity of these compounds was also determined against 10 fungal strains. Compound 3e showed activity against all fungal strains tested, but showed high activity against Saccharomyces cerevisiae (MIC 3.9 μg/mL.

Test results from Fermilab 1.5 m model SSC collider dipole magnets

International Nuclear Information System (INIS)

Koska, W.; Bossert, R.; Carson, J.; Coulter, K.J.; Delchamps, S.; Gourlay, S.; Jaffery, T.S.; Kinney, W.; Lamm, M.J.; Ozelis, J.P.; Strait, J.; Wake, M.

1991-09-01

We will present results from tests of 1.5 m model SSC collider dipole magnets. These R ampersand D magnets are identical to the 15 m full length dipoles currently being assembled at Fermilab in all important aspects except length. Because of their small size they can be built faster and tested more extensively than the long magnets. The model magnets are used to optimize design parameters for, and to indicate the performance which can be expected from, the 15 m magnets. The are instrumented with voltage taps over the first two current blocks for quench localization and with several arrays of strain gauge transducers for the study of mechanical behavior. The stress at the poles of the inner and outer coils is monitored during construction and, along with end force and shell strain, during excitation. Magnetic measurements are made several times during each magnet's lifetime, including at operating temperature and field. We will report on studies of the quench performance, mechanical behavior and magnetic field of these magnets

Immune responses of mature chicken bone-marrow-derived dendritic cells infected with Newcastle disease virus strains with differing pathogenicity.

Science.gov (United States)

Xiang, Bin; Zhu, Wenxian; Li, Yaling; Gao, Pei; Liang, Jianpeng; Liu, Di; Ding, Chan; Liao, Ming; Kang, Yinfeng; Ren, Tao

2018-06-01

Infection of chickens with virulent Newcastle disease virus (NDV) is associated with severe pathology and increased morbidity and mortality. The innate immune response contributes to the pathogenicity of NDV. As professional antigen-presenting cells, dendritic cells (DCs) play a unique role in innate immunity. However, the contribution of DCs to NDV infection has not been investigated in chickens. In this study, we selected two representative NDV strains, i.e., the velogenic NDV strain Chicken/Guangdong/GM/2014 (GM) and the lentogenic NDV strain La Sota, to investigate whether NDVs could infect LPS-activated chicken bone-derived marrow DCs (mature chicken BM-DCs). We compared the viral titres and innate immune responses in mature chicken BM-DCs following infection with those strains. Both NDV strains could infect mature chicken BM-DC, but the GM strain showed stronger replication capacity than the La Sota strain in mature chicken BM-DCs. Gene expression profiling showed that MDA5, LGP2, TLR3, TLR7, IFN-α, IFN-β, IFN-γ, IL-1β, IL-6, IL-18, IL-8, CCL5, IL-10, IL-12, MHC-I, and MHC-II levels were altered in mature DCs after infection with NDVs at all evaluated times postinfection. Notably, the GM strain triggered stronger innate immune responses than the La Sota strain in chicken BM-DCs. However, both strains were able to suppress the expression of some cytokines, such as IL-6 and IFN-α, in mature chicken DCs at 24 hpi. These data provide a foundation for further investigation of the role of chicken DCs in NDV infection.

Strain diversity of CTX-M-producing Enterobacteriaceae in individual pigs

DEFF Research Database (Denmark)

Hansen, Katrine Hartung; Bortolaia, Valeria; Damborg, Peter Panduro

2014-01-01

of CTX-M-2/97-producing Proteus mirabilis or Providencia rettgeri was observed in two sows and two offspring. At least two genetically unrelated CTX-M-producing E. coli strains were isolated from approximately one fourth of the samples with remarkable differences between isolates obtained by enrichment...

PRAS40 and PRR5-like protein are new mTOR interactors that regulate apoptosis.

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Kathrin Thedieck

Full Text Available TOR (Target of Rapamycin is a highly conserved protein kinase and a central controller of cell growth. TOR is found in two functionally and structurally distinct multiprotein complexes termed TOR complex 1 (TORC1 and TOR complex 2 (TORC2. In the present study, we developed a two-dimensional liquid chromatography tandem mass spectrometry (2D LC-MS/MS based proteomic strategy to identify new mammalian TOR (mTOR binding proteins. We report the identification of Proline-rich Akt substrate (PRAS40 and the hypothetical protein Q6MZQ0/FLJ14213/CAE45978 as new mTOR binding proteins. PRAS40 binds mTORC1 via Raptor, and is an mTOR phosphorylation substrate. PRAS40 inhibits mTORC1 autophosphorylation and mTORC1 kinase activity toward eIF-4E binding protein (4E-BP and PRAS40 itself. HeLa cells in which PRAS40 was knocked down were protected against induction of apoptosis by TNFalpha and cycloheximide. Rapamycin failed to mimic the pro-apoptotic effect of PRAS40, suggesting that PRAS40 mediates apoptosis independently of its inhibitory effect on mTORC1. Q6MZQ0 is structurally similar to proline rich protein 5 (PRR5 and was therefore named PRR5-Like (PRR5L. PRR5L binds specifically to mTORC2, via Rictor and/or SIN1. Unlike other mTORC2 members, PRR5L is not required for mTORC2 integrity or kinase activity, but dissociates from mTORC2 upon knock down of tuberous sclerosis complex 1 (TSC1 and TSC2. Hyperactivation of mTOR by TSC1/2 knock down enhanced apoptosis whereas PRR5L knock down reduced apoptosis. PRR5L knock down reduced apoptosis also in mTORC2 deficient cells. The above suggests that mTORC2-dissociated PRR5L may promote apoptosis when mTOR is hyperactive. Thus, PRAS40 and PRR5L are novel mTOR-associated proteins that control the balance between cell growth and cell death.

Effects of different magnitudes of mechanical strain on Osteoblasts in vitro

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Tang Lin; Lin Zhu; Li Yongming

2006-01-01

In addition to systemic and local factors, mechanical strain plays a crucial role in bone remodeling during growth, development, and fracture healing, and especially in orthodontic tooth movement. Although many papers have been published on the effects of mechanical stress on osteoblasts or osteoblastic cells, little is known about the effects of different magnitudes of mechanical strain on such cells. In the present study, we investigated how different magnitudes of cyclic tensile strain affected osteoblasts. MC3T3-E1 osteoblastic cells were subjected to 0%, 6%, 12% or 18% elongation for 24 h using a Flexercell Strain Unit, and then the mRNA and protein expressions of osteoprotegerin (OPG) and receptor activator of nuclear factor-κB ligand (RANKL) were examined. The results showed that cyclic tensile strain induced a magnitude-dependent increase (0%, 6%, 12%, and 18%) in OPG synthesis and a concomitant decrease in RANKL mRNA expression and sRANKL release from the osteoblasts. Furthermore, the induction of OPG mRNA expression by stretching was inhibited by indomethacin or genistein, and the stretch-induced reduction of RANKL mRNA was inhibited by PD098059. These results indicate that different magnitudes of cyclic tensile strain influence the biological behavior of osteoblasts, which profoundly affects bone remodeling

Study of probiotic potential of four wild Lactobacillus rhamnosus strains.

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Tuo, Yanfeng; Zhang, Weiqin; Zhang, Lanwei; Ai, Lianzhong; Zhang, Yingchun; Han, Xue; Yi, Huaxi

2013-06-01

The four wild Lactobacillus rhamnosus strains were examined in vitro for resistance to simulated gastro and intestinal juices, adhesion to HT-29 cells, antagonistic activity against enteric pathogens and immunomodulating activity. The strains L. rhamnosus SB5L, J5L and IN1L were able to survive in simulated gastro juice while the strain L. rhamnosus SB31L lost viability exposed to simulated gastro juice for 3 h. The four strains had high viability in simulated small intestinal juice with little loss (<1.0 cycle reduction). The strains SB5L, J5L and IN1L antagonized against Escherichia coli ATCC 25922, Salmonella enterica serovar Typhimurium ATCC 14028, Shigella sonnei ATCC 25931. The strain L. rhamnosus IN1L had the highest adhesive capability to HT-29 cells in vitro (251 bacteria cells per 100 HT-29 cells) compared to the other three L. rhamnosus strains. The live bacteria, cell wall and DNA of the four L. rhamnosus induced the secretion of pro-inflammatory cytokines IL-12 (p70), IFN-γ and TNF-α by human peripheral blood mononuclear cells (PBMCs). The levels of IL-12 (p70), IFN-γ and TNF-α produced by stimulated PBMCs were significantly higher (P < 0.05) than those of the control. Those data indicated that the four L. rhamnosus strains have the potential as the probiotic for human being use, although further studies are still needed. Crown Copyright © 2013. Published by Elsevier Ltd. All rights reserved.

Comparison of hydrogen-production capability of four different Enterobacteriaceae strains under growing and non-growing conditions

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Seol, Eunhee; Kim, Seohyoung; Raj, S. Mohan; Park, Sunghoon [Department of Chemical and Biochemical Engineering, Pusan National University, Busan 609-735 (Korea)

2008-10-15

Non-growing cells can function as whole-cell biocatalysts for hydrogen (H{sub 2}) production, a process that has recently drawn much attention. In order to evaluate their potential as whole-cell biocatalysts, we compared the H{sub 2}-production capability of four Enterobacteriaceae strains (Citrobacter amalonaticus Y19, Escherichia coli K-12 MG1655, Escherichia coli DJT135, and Enterobacter aerogenes) under growing and non-growing conditions. We evaluated their H{sub 2}-production activity at varying temperatures (25-45 C) and pH conditions (6.0-8.0) using glucose or formate as the carbon source. Under growing conditions with 10 mM glucose as a substrate, E. aerogenes exhibited the highest H{sub 2}-production activity (17.0 {+-} 0.2 {mu}mol H{sub 2} mg cell{sup -1} h{sup -1}) among the four strains, but the final H{sub 2} yield was similar (1.7-1.8 mol H{sub 2} mol{sup -1} glucose) in all four strains. H{sub 2} production in the four strains proceeded through a formate-dependent pathway that involved the formate hydrogen lyase (FHL) complex. Under non-growing conditions with 20 mM formate as a substrate, we obtained high H{sub 2}-production activities, in the range of 95.5-195.2 {mu}mol H{sub 2} mg cell{sup -1} h{sup -1}, with E. coli DJT135 exhibiting the highest activity (195.2 {mu}mol H{sub 2} mg{sup -1} h{sup -1}) at pH 6.0 and 45 C. In contrast, using glucose as the carbon substrate in non-growing cell experiments greatly reduced the H{sub 2}-production activity to 6.1-7.7 {mu}mol H{sub 2} mg cell{sup -1} h{sup -1}. This study indicated that formate is a better substrate than glucose for H{sub 2} production by non-growing cells, and that the H{sub 2}-production performance among the strains did not vary significantly, with the exception of E. coli K-12 MG1655. (author)

Effects of single- and multi-strain probiotics on biofilm formation and in vitro adhesion to bladder cells by urinary tract pathogens.

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Chapman, C M C; Gibson, G R; Rowland, I

2014-06-01

There is increasing evidence that probiotic bacteria can inhibit and/or prevent urinary tract infections. Possible mechanisms include prevention of adhesion of pathogens to the bladder epithelium and inhibition of biofilm formation. Currently there is interest in the comparative efficacy of single probiotics vs. strain mixtures. We have therefore tested the inhibitory activity of four single probiotics and four probiotic mixtures towards the urinary tract pathogens Escherichia coli NCTC 9001 and Enterococcus faecalis NCTC 00775. Inhibition of biofilm formation by cell-free supernatants was tested using the Crystal Violet assay, while prevention of pathogen adhesion to host cells was tested by using bladder cancer cells as a model for the human urinary tract. Under pH-controlled conditions, there was no significant inhibition of biofilm formation by any treatment. Without pH control, 5/8 treatments significantly inhibited biofilm production by E. coli, while 5/8 treatments inhibited production by E. faecalis. Using data from all Crystal Violet assays, there was no significant difference in the ability of single- and multi-strain probiotics to inhibit biofilm formation. In the cell culture assays, all treatments were able to significantly reduce numbers of pathogenic cells adhering to host cells by 2.5-3.5 logs. No significant difference was observed between the displacement caused by single strains and mixtures for either pathogen. Inhibition of biofilm seems to be a major mechanism of urinary tract pathogen exclusion, related to, and possibly dependent upon, the probiotic ability to reduce environmental pH. Exclusion via competition of binding sites is a possible in vivo mechanism for these probiotics. If an additive or synergistic effect exists between strains within a mixture, it does not manifest itself in a greater effect through these two inhibitory mechanisms. Copyright © 2014 Elsevier Ltd. All rights reserved.

Development of Functional Microfold (M Cells from Intestinal Stem Cells in Primary Human Enteroids.

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Joshua D Rouch

Full Text Available Intestinal microfold (M cells are specialized epithelial cells that act as gatekeepers of luminal antigens in the intestinal tract. They play a critical role in the intestinal mucosal immune response through transport of viruses, bacteria and other particles and antigens across the epithelium to immune cells within Peyer's patch regions and other mucosal sites. Recent studies in mice have demonstrated that M cells are generated from Lgr5+ intestinal stem cells (ISCs, and that infection with Salmonella enterica serovar Typhimurium increases M cell formation. However, it is not known whether and how these findings apply to primary human small intestinal epithelium propagated in an in vitro setting.Human intestinal crypts were grown as monolayers with growth factors and treated with recombinant RANKL, and assessed for mRNA transcripts, immunofluorescence and uptake of microparticles and S. Typhimurium.Functional M cells were generated by short-term culture of freshly isolated human intestinal crypts in a dose- and time-dependent fashion. RANKL stimulation of the monolayer cultures caused dramatic induction of the M cell-specific markers, SPIB, and Glycoprotein-2 (GP2 in a process primed by canonical WNT signaling. Confocal microscopy demonstrated a pseudopod phenotype of GP2-positive M cells that preferentially take up microparticles. Furthermore, infection of the M cell-enriched cultures with the M cell-tropic enteric pathogen, S. Typhimurium, led to preferential association of the bacteria with M cells, particularly at lower inoculum sizes. Larger inocula caused rapid induction of M cells.Human intestinal crypts containing ISCs can be cultured and differentiate into an epithelial layer with functional M cells with characteristic morphological and functional properties. This study is the first to demonstrate that M cells can be induced to form from primary human intestinal epithelium, and that S. Typhimurium preferentially infect these cells in an

Design and Validation of a Cyclic Strain Bioreactor to Condition Spatially-Selective Scaffolds in Dual Strain Regimes

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J. Matthew Goodhart

2014-03-01

Full Text Available The objective of this study was to design and validate a unique bioreactor design for applying spatially selective, linear, cyclic strain to degradable and non-degradable polymeric fabric scaffolds. This system uses a novel three-clamp design to apply cyclic strain via a computer controlled linear actuator to a specified zone of a scaffold while isolating the remainder of the scaffold from strain. Image analysis of polyethylene terephthalate (PET woven scaffolds subjected to a 3% mechanical stretch demonstrated that the stretched portion of the scaffold experienced 2.97% ± 0.13% strain (mean ± standard deviation while the unstretched portion experienced 0.02% ± 0.18% strain. NIH-3T3 fibroblast cells were cultured on the PET scaffolds and half of each scaffold was stretched 5% at 0.5 Hz for one hour per day for 14 days in the bioreactor. Cells were checked for viability and proliferation at the end of the 14 day period and levels of glycosaminoglycan (GAG and collagen (hydroxyproline were measured as indicators of extracellular matrix production. Scaffolds in the bioreactor showed a seven-fold increase in cell number over scaffolds cultured statically in tissue culture plastic petri dishes (control. Bioreactor scaffolds showed a lower concentration of GAG deposition per cell as compared to the control scaffolds largely due to the great increase in cell number. A 75% increase in hydroxyproline concentration per cell was seen in the bioreactor stretched scaffolds as compared to the control scaffolds. Surprisingly, little differences were experienced between the stretched and unstretched portions of the scaffolds for this study. This was largely attributed to the conditioned and shared media effect. Results indicate that the bioreactor system is capable of applying spatially-selective, linear, cyclic strain to cells growing on polymeric fabric scaffolds and evaluating the cellular and matrix responses to the applied strains.

Cloning and expression of pab gene of M. tuberculosis isolated from pulmonary TB patient in E.coli DH5α

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Tri Y. M. Raras

2011-11-01

Full Text Available Background: Mycobacterium tuberculosis antigen38 is a potent serodiagnostic agent containing two M. tuberculosisspecific B-cell epitopes. The high price of imported diagnostic agents hinders realization of fast clinical TB diagnosis in developing countries. Therefore, we produced recombinant antigen38 (recAg38M from M. tuberculosis local strain, which might be used to produce economical tuberculosis serodiagnostic kit.Methods: Pab gene that was isolated from pulmonary TB patient in Malang was cloned into a plasmid vector (pGEMTeasy to construct pMB38. The E.coli DH5α clone carrying pMb38 was selected on X-gal medium. The expression of pab was mediated using pPRoExHTc under the control of Trc promoter and E.coli DH5α as host.Results: Alignment of the pab sequence from the white E.coli DH5α clones with that of M. tuberculosis H37Rv showed 98% homology. The recombinant protein in which the signal peptide has been deleted to prevent the protein being secreted into medium was found in the cytoplasm.Conclusion: pab gene of M. tuberculosis isolated from a TB patient could be expressed in heterologous system in E.coliDH5α. (Med J Indones 2011; 20:247-54Keywords: Mycobacterium tuberculosis, Pab gene expression, recombinant antigen38

Appearance and evolution of the specific chromosomal rearrangements associated with malignant transformation of mouse m5S cells

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Kodama, S.; Okumura, Y.; Komatsu, K.; Sasaki, M.S.

1991-01-01

Chromosomal alterations were studied during the acquisition of malignant phenotypes in two karyotypically distinct cells isolated from transformed foci induced by x-irradiation in mouse m5S cells. Because the transformants, despite foci origin, showed low ability to grow in agar, they were cultured in vitro with serial transfer schedules to allow further cell generations and assayed for anchorage independence (AI) at each passage level. The AI frequency increased with the cell doubling numbers. Chromosome analysis showed that a focus was one cell origin, but the transformants showed karyotypic instability during cell proliferation, giving rise to the rearrangements clustered in the distal region of the specific chromosomes. These rearrangements appeared to be directed toward the acquisition of malignant phenotypes. Analysis of the types and sites of rearrangements indicated that a mechanism exists that induces frequent rearrangements of the specific region of a chromosome during the process of transformation into the malignant state

Antagonistic Activity of Lactobacillus reuteri Strains on the Adhesion Characteristics of Selected Pathogens.

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Singh, Tejinder P; Kaur, Gurpreet; Kapila, Suman; Malik, Ravinder K

2017-01-01

Adhesion ability of probiotics is the key factor that decides their colonization in the gastrointestinal tract and potential to inhibit pathogens. Therefore, adhesion ability can be considered as a key determinant for probiotic efficacy. Presents study documents the antagonistic activity of viable/untreated, Lithium chloride (LiCl) treated or heat-killed forms of eight probiotic Lactobacillus reuteri strains on the adhesion characteristics of selected pathogens. All strains investigated were able to adhere to Caco-2 cells. L. reuteri strains tested were able to inhibit and displace ( P strain L. reuteri LR6 showed the strongest adhesion and pathogen inhibition ability among the eight L. reuteri strains tested. In addition, the abilities to inhibit and to displace adhered pathogens depended on both the probiotic and the pathogen strains tested suggesting the involvement of various mechanisms. The adhesion and antagonistic potential of the probiotic strains were significantly decreased upon exposure to 5 M LiCl, showing that surface molecules, proteinaceous in nature, are involved. The heat-killed forms of the probiotic L. reuteri strains also inhibited the attachment of selected pathogens to Caco-2 cells. In conclusion, in vitro assays showed that L. reuteri strains, as viable or heat-killed forms, are adherent to Caco-2 cells and are highly antagonistic to pathogens tested in which surface associated proteins play an important role.

Effectiveness of 5-Pyrrolidone-2-carboxylic Acid and Copper Sulfate Pentahydrate Association against Drug Resistant Staphylococcus Strains.

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Governa, Paolo; Miraldi, Elisabetta; De Fina, Gianna; Biagi, Marco

2016-04-01

Bacterial resistance is an ongoing challenge for pharmacotherapy and pharmaceutical chemistry. Staphylococcus aureus is the bacterial species which makes it most difficult to treat skin and soft tissue infections and it is seen in thousands of hospitalization cases each year. Severe but often underrated infectious diseases, such as complicated nasal infections, are primarily caused by MRSA and S. epidermidis too. With the aim of studying new drugs with antimicrobial activity and effectiveness on drug resistant Staphylococcus strains, our attention in this study was drawn on the activity of a new association between two natural products: 5-pyrrolidone-2-carboxylic acid (PCA), naturally produced by certain Lactobacillus species, and copper sulfate pentahydrate (CS). The antimicrobial susceptibility test was conducted taking into account 12 different Staphylococcus strains, comprising 6 clinical isolates and 6 resistant strains. PCA 4%, w/w, and CS 0.002%, w/w, association in distilled water solution was found to have bactericidal activity against all tested strains. Antimicrobial kinetics highlighted that PCA 4%, w/w, and CS 0.002% association could reduce by 5 log10 viable bacterial counts of MRSA and oxacillin resistant S. epidennidis in less than 5 and 3 minutes respectively. Microscopic investigations suggest a cell wall targeting mechanism of action. Being very safe and highly tolerated, the natural product PCA and CS association proved to be a promising antimicrobial agent to treat Staphylococcus related infections.

Comparison analysis of microRNAs in response to dengue virus type 2 infection between the Vero cell-adapted strain and its source, the clinical C6/36 isolated strain.

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Yang, Jiajia; Lin, Yao; Jiang, Liming; Xi, Juemin; Wang, Xiaodan; Guan, Jiaoqiong; Chen, Junying; Pan, Yue; Luo, Jia; Ye, Chao; Sun, Qiangming

2018-05-02

To elucidate the differences in microRNAs during dengue virus infection between Vero cell-adapted strain (DENV-2-Vero) and its source, the clinical C6/36 isolated strain (DENV-2-C6/36), a comparison analysis was performed in Vero cells by high throughput sequencing. The results showed that the expression of 16 known and 3 novel miRNAs exhibited marked differences. 5 known miRNAs were up-regulated in DENV-2-C6/36 group, while 11 known microRNAs were down-regulated in DENV-2-Vero group. The GO enrichment and KEGG pathway analysis showed that there was a distinct difference in regulating viral replication between two strains. In DENV-2-Vero infection group, significantly enriched GO terms included virion attachment to host cells, viral structural protein/genome processing and packaging. Meanwhile, the regulation of cell death and apoptosis between two groups were different in the early stage of infection. KEGG enrichment analysis showed that DENV-2-C6/36 infection induced more intense regulation of immune-related pathways, including Fc gamma R-mediated phagocytosis, etc. DENV-2-Vero infection could partially alleviate the immune defense of Vero cells compared with DENV-2-C6/36. The results indicated that the distinct microRNA changes induced by two DENV-2 strains may be partly related to their infective abilities. Our data provide useful insights that help elucidate the host-pathogen interactions following DENV infection. Copyright © 2018 Elsevier B.V. All rights reserved.

Characterisation of iunH gene knockout strain from Mycobacterium tuberculosis

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Anne Drumond Villela

Full Text Available BACKGROUND Tuberculosis (TB is an infectious disease caused mainly by the bacillus Mycobacterium tuberculosis. The better understanding of important metabolic pathways from M. tuberculosis can contribute to the development of novel therapeutic and prophylactic strategies to combat TB. Nucleoside hydrolase (MtIAGU-NH, encoded by iunH gene (Rv3393, is an enzyme from purine salvage pathway in M. tuberculosis. MtIAGU-NH accepts inosine, adenosine, guanosine, and uridine as substrates, which may point to a pivotal metabolic role. OBJECTIVES Our aim was to construct a M. tuberculosis knockout strain for iunH gene, to evaluate in vitro growth and the effect of iunH deletion in M. tuberculosis in non-activated and activated macrophages models of infection. METHODS A M. tuberculosis knockout strain for iunH gene was obtained by allelic replacement, using pPR27xylE plasmid. The complemented strain was constructed by the transformation of the knockout strain with pNIP40::iunH. MtIAGU-NH expression was analysed by Western blot and LC-MS/MS. In vitro growth was evaluated in Sauton’s medium. Bacterial load of non-activated and interferon-γ activated RAW 264.7 cells infected with knockout strain was compared with wild-type and complemented strains. FINDINGS Western blot and LC-MS/MS validated iunH deletion at protein level. The iunH knockout led to a delay in M. tuberculosis growth kinetics in Sauton’s medium during log phase, but did not affect bases and nucleosides pool in vitro. No significant difference in bacterial load of knockout strain was observed when compared with both wild-type and complemented strains after infection of non-activated and interferon-γ activated RAW 264.7 cells. MAIN CONCLUSION The disruption of iunH gene does not influence M. tuberculosis growth in both non-activated and activated RAW 264.7 cells, which show that iunH gene is not important for macrophage invasion and virulence. Our results indicated that MtIAGU-NH is not a

Evaluation of Synergistic Interactions Between Cell-Free Supernatant of Lactobacillus Strains and Amikacin and Genetamicin Against Pseudomonas aeruginosa.

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Aminnezhad, Sargol; Kermanshahi, Rouha Kasra; Ranjbar, Reza

2015-04-01

The indiscriminate use of antibiotics in the treatment of infectious diseases can increase the development of antibiotic resistance. Therefore, there is a big demand for new sources of antimicrobial agents and alternative treatments for reduction of antibiotic dosage required to decrease the associated side effects. In this study, the synergistic action of aminoglycoside antibiotics and cell-free supernatant (CFS) of probiotic (Lactobacillus rahmnosus and L. casei) against Pseudomonas aeruginosa PTCC 1430 was evaluated. A growth medium for culturing of probiotic bacteria was separated by centrifugation. The antimicrobial effects of CFS of probiotic bacteria were evaluated using the agar well diffusion assay. The minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) were evaluated using the micro dilution method. Finally, an interaction between CFS and amikacin or gentamicin against P. aeruginosa PTCC 1430 was examined through the checkerboard method and fractional inhibitory concentration (FIC). Furthermore, CFSs from Lactobacillus strains were analyzed by reversed phase HPLC (RP-HPLC) for antimicrobial compounds. The results showed a significant effect of CFS on the growth of P. aeruginosa. The MIC and MBC of CFS from L. casei were 62.5 µL⁄mL while the MIC and MBC of CFS from L. rhamnosus were 62.5 μL⁄mL and 125 μL⁄mL, respectively. Using the FIC indices, synergistic interactions were observed in combination of CFS and antibiotics. Fractional Inhibitory Concentration indices of CFS from L. casei and aminoglycoside antibiotics were 0.124 and 0.312 while FIC indices of CFS from L. rhamnosus and aminoglycoside antibiotics were 0.124 and 0.56, respectively showing a synergism effect. The results of RP-HPLC showed that CFS of Lactobacillus strains contained acetic acid, lactic acid and hydrogen peroxide (H2O2). Our findings indicate that probiotic bacterial strains of Lactobacillus have a significant inhibitory effect on the

Characterization of exoelectrogenic bacteria enterobacter strains isolated from a microbial fuel cell exposed to copper shock load.

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Cuijie Feng

Full Text Available Microorganisms capable of generating electricity in microbial fuel cells (MFCs have gained increasing interest. Here fourteen exoelectrogenic bacterial strains were isolated from the anodic biofilm in an MFC before and after copper (Cu shock load by Hungate roll-tube technique with solid ferric (III oxide as an electron acceptor and acetate as an electron donor. Phylogenetic analysis of the 16S rRNA gene sequences revealed that they were all closely related to Enterobacter ludwigii DSM 16688T within the Enterobacteriaceae family, although these isolated bacteria showed slightly different morphology before and after Cu shock load. Two representative strains R2B1 (before Cu shock load and B4B2 (after Cu shock load were chosen for further analysis. B4B2 is resistant to 200 mg L-1 of Cu(II while R2B1 is not, which indicated the potential selection of the Cu shock load. Raman analysis revealed that both R2B1 and B4B2 contained c-type cytochromes. Cyclic voltammetry measurements revealed that strain R2B1 had the capacity to transfer electrons to electrodes. The experimental results demonstrated that strain R2B1 was capable of utilizing a wide range of substrates, including Luria-Bertani (LB broth, cellulose, acetate, citrate, glucose, sucrose, glycerol and lactose to generate electricity, with the highest current density of 440 mA·m-2 generated from LB-fed MFC. Further experiments indicated that the bacterial cell density had potential correlation with the current density.

Effects of Pre-Strains on Failure Assessment Analysis to API 5L X65 Pipeline

International Nuclear Information System (INIS)

Baek, Jong Hyun; Kim, Young Pyo; Kim, Woo Sik; Seok, Chang Sung

2009-01-01

This paper prescribed the structural integrity of the API 5 L X6 5 pipeline subjected to tensile pre-strain. The effects of pre-strain on the mechanical properties of API 5 L X6 5 pipe were substantially investigated through a variety of the experimental procedures. Axial tensile pre-strain of 1.5, 5 and 10% was applied to plate-type tensile specimens cut from the pipe body prior to mechanical testing. Tensile test revealed that yield strength and tensile strength were increased with increasing tensile pre-strain. The increasing rate of the yield strength owing to the pre-strain is greater than that of the tensile strength. However, the pre-strain up to 5% had a little effect on the decreasing of the fracture toughness. The structural integrity of the API 5 L X6 5 pipeline subjected to large plastic deformation was evaluated through the fitness-for service code.

DNA-specific labelling by deoxyribonucleoside 5'-monophosphates in Saccharomyces cerevisiae

International Nuclear Information System (INIS)

Brendel, M.; Faeth, W.W.; Toper, R.

1975-01-01

Growth of 5'-dTMP low-requiring strains is inhibited by exogenous 5'-dGMP and 5'-GMP at concentrations higher than 5 x 10 -4 M. Synthesis of nucleic acids ceases and cells remain fixed in their respective place in the cell cycle. At concentrations lower than 10 -5 M deoxyribonucleoside 5'-monophosphates may be employed for radioactive labelling, the label being preferentially used for DNA synthesis. Affinity to DNA of the 5'-dNMPs is in the order of 5'-dAMPS > 5'-dGMP > 5'-dCMP > 5'-dUMP. DNA-specific label is achieved with 5'-dAMP when the medium is supplemented with adenine and deoxyadenosine. (orig.) [de

Improvement of Fish Sauce Quality by Strain CMC5-3-1: A Novel Species of Staphylococcus sp.

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Udomsil, Natteewan; Rodtong, Sureelak; Tanasupawat, Somboon; Yongsawatdigul, Jirawat

2015-09-01

Staphylococcus sp. CMC5-3-1 and CMS5-7-5 isolated from fermented fish sauce at 3 to 7 mo, respectively, showed different characteristics on protein hydrolysis and volatile formation. These Gram-positive cocci were able to grow in up to 15% NaCl with the optimum at 0.5% to 5% NaCl in tryptic soy broth. Based on ribosomal 16S rRNA gene sequences, Staphylococcus sp. CMC5-3-1 and CMS5-7-5 showed 99.0% similarity to that of Staphylococcus piscifermentans JCM 6057(T) , but DNA-DNA relatedness was sauce inoculated with Staphylococcus sp. CMC5-3-1 was 740.5 mM, which was higher than that inoculated by the strain CMS5-7-5 (662.14 mM, P sauce inoculated with Staphylococcus sp. CMC5-3-1 showed the highest content of total glutamic acid (P sauce inoculated with Staphylococcus sp. CMC5-3-1 was 2-methypropanal, contributing to the desirable dark chocolate note. Staphylococcus sp. CMC5-3-1 could be applied as a starter culture to improve the umami and aroma of fish sauce. © 2015 Institute of Food Technologists®

X-ray microbeam measurements of individual dislocation cell elastic strains in deformed single-crystal copper

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Levine, Lyle E. [National Institute of Standards and Technology (NIST); Larson, Ben C [ORNL; Yang, Wenge [Carnegie Institution of Washington; Kassner, Michael E. [University of Southern California; Tischler, Jonathan Zachary [ORNL; Delos-Reyes, Michael A. [University of Southern California; Fields, Richard J. [National Institute of Standards and Technology (NIST); Liu, Wenjun [Argonne National Laboratory (ANL)

2006-01-01

The distribution of elastic strains (and thus stresses) at the sub-micrometer length scale within deformed metal single crystals has surprisingly broad implications for our understanding of important physical phenomena. These include the evolution of the complex dislocation structures that govern mechanical behavior within individual grains [1-4], the transport of dislocations through such structures [5-7], changes in mechanical properties that occur during reverse loading [8-10] (e.g. sheet metal forming), and the analyses of diffraction line profiles for microstructural studies of these phenomena [11-17]. We present the first direct, spatially-resolved measurements of the elastic strains within individual dislocation cells in copper single crystals deformed in tension and compression along <100> axes. Broad distributions of elastic strains are found, with profound implications for theories of dislocation structure evolution [4,18], dislocation transport [5-7], and the extraction of dislocation parameters from X-ray line profiles [11-17,19].

Generation of Elf5-Cre knockin mouse strain for trophoblast-specific gene manipulation.

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Kong, Shuangbo; Liang, Guixian; Tu, Zhaowei; Chen, Dunjin; Wang, Haibin; Lu, Jinhua

2018-04-01

Placental development is a complex and highly controlled process during which trophoblast stem cells differentiate to various trophoblast subtypes. The early embryonic death of systemic gene knockout models hampers the investigation of these genes that might play important roles during placentation. A trophoblast specific Cre mouse model would be of great help for dissecting out the potential roles of these genes during placental development. For this purpose, we generate a transgenic mouse with the Cre recombinase inserted into the endogenous locus of Elf5 gene that is expressed specifically in placental trophoblast cells. To analyze the specificity and efficiency of Cre recombinase activity in Elf5-Cre mice, we mated Elf5-Cre mice with Rosa26 mT/mG reporter mice, and found that Elf5-Cre transgene is expressed specifically in the trophoectoderm as early as embryonic day 4.5 (E4.5). By E12.5, the activity of Elf5-Cre transgene was detected exclusively in all derivatives of trophoblast lineages, including spongiotrophoblast, giant cells, and labyrinth trophoblasts. In addition, Elf5-Cre transgene was also active during spermatogenesis, from spermatids to mature sperms, which is consistent with the endogenous Elf5 expression in testis. Collectively, our results provide a unique tool to delete specific genes selectively and efficiently in trophoblast lineage during placentation. © 2018 Wiley Periodicals, Inc.

Effect of Enterococcus faecium M74 strain on egg yolk fat and cholesterol

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Mária Angelovičová

2013-05-01

Full Text Available Abstract The aim was to evaluate the functional efficiency of a probiotic strain Enterococcus faecium M74 in the feed on egg yolk weight, egg yolk fat and cholesterol contents of Shaver Starcross 288 hens.  Feed in the experimental group was enriched with a probiotic additive containing of 5*109 viable Enterococcus faecium per g. Egg samples a total 30 pcs per group were collected during the first egg-laying period at week 28 and 38 of hens´ age. Non-significantly lower of egg yolk weight was observed in the experimental group at all sampling times compared with their respective controls. Non-significantly lower of egg yolk weight was observed in the experimental group at all sampling times compared with their respective controls. Significantly lower concentrations of egg yolk cholesterol were found in the experimental group at week 28, and week 38 in compare with controls. In conclusion, the addition of probiotic strains Enterococcus faecium M74 to the feed of Shaver Starcross 288 hens reduced cholesterol in egg yolk at all sampling times. Even though the hypocholesterolemic mechanism of probiotics has not yet been fully understood, it is an established fact that cholesterol and bile salt metabolism are closely linked. However, the hypocholesterolemic mechanism of probiotics based on the bile salt hydrolase activity hypothesis has not yet been sufficiently elucidated.

Specificity and Strain-Typing Capabilities of Nanorod Array-Surface Enhanced Raman Spectroscopy for Mycoplasma pneumoniae Detection.

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Kelley C Henderson

Full Text Available Mycoplasma pneumoniae is a cell wall-less bacterial pathogen of the human respiratory tract that accounts for > 20% of all community-acquired pneumonia (CAP. At present the most effective means for detection and strain-typing is quantitative polymerase chain reaction (qPCR, which can exhibit excellent sensitivity and specificity but requires separate tests for detection and genotyping, lacks standardization between available tests and between labs, and has limited practicality for widespread, point-of-care use. We have developed and previously described a silver nanorod array-surface enhanced Raman Spectroscopy (NA-SERS biosensing platform capable of detecting M. pneumoniae with statistically significant specificity and sensitivity in simulated and true clinical throat swab samples, and the ability to distinguish between reference strains of the two main genotypes of M. pneumoniae. Furthermore, we have established a qualitative lower endpoint of detection for NA-SERS of < 1 genome equivalent (cell/μl and a quantitative multivariate detection limit of 5.3 ± 1 cells/μl. Here we demonstrate using partial least squares- discriminatory analysis (PLS-DA of sample spectra that NA-SERS correctly identified M. pneumoniae clinical isolates from globally diverse origins and distinguished these from a panel of 12 other human commensal and pathogenic mycoplasma species with 100% cross-validated statistical accuracy. Furthermore, PLS-DA correctly classified by strain type all 30 clinical isolates with 96% cross-validated accuracy for type 1 strains, 98% cross-validated accuracy for type 2 strains, and 90% cross-validated accuracy for type 2V strains.

Comparative genomic analyses of Mycoplasma hyopneumoniae pathogenic 168 strain and its high-passaged attenuated strain

Science.gov (United States)

2013-01-01

Background Mycoplasma hyopneumoniae is the causative agent of porcine enzootic pneumonia (EP), a mild, chronic pneumonia of swine. Despite presenting with low direct mortality, EP is responsible for major economic losses in the pig industry. To identify the virulence-associated determinants of M. hyopneumoniae, we determined the whole genome sequence of M. hyopneumoniae strain 168 and its attenuated high-passage strain 168-L and carried out comparative genomic analyses. Results We performed the first comprehensive analysis of M. hyopneumoniae strain 168 and its attenuated strain and made a preliminary survey of coding sequences (CDSs) that may be related to virulence. The 168-L genome has a highly similar gene content and order to that of 168, but is 4,483 bp smaller because there are 60 insertions and 43 deletions in 168-L. Besides these indels, 227 single nucleotide variations (SNVs) were identified. We further investigated the variants that affected CDSs, and compared them to reported virulence determinants. Notably, almost all of the reported virulence determinants are included in these variants affected CDSs. In addition to variations previously described in mycoplasma adhesins (P97, P102, P146, P159, P216, and LppT), cell envelope proteins (P95), cell surface antigens (P36), secreted proteins and chaperone protein (DnaK), mutations in genes related to metabolism and growth may also contribute to the attenuated virulence in 168-L. Furthermore, many mutations were located in the previously described repeat motif, which may be of primary importance for virulence. Conclusions We studied the virulence attenuation mechanism of M. hyopneumoniae by comparative genomic analysis of virulent strain 168 and its attenuated high-passage strain 168-L. Our findings provide a preliminary survey of CDSs that may be related to virulence. While these include reported virulence-related genes, other novel virulence determinants were also detected. This new information will form

Genomic Comparison among Lethal Invasive Strains of Streptococcus pyogenes Serotype M1

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Gabriel R. Fernandes

2017-10-01

Full Text Available Streptococcus pyogenes, also known as group A Streptococcus (GAS, is a human pathogen that causes diverse human diseases including streptococcal toxic shock syndrome (STSS. A GAS outbreak occurred in Brasilia, Brazil, during the second half of the year 2011, causing 26 deaths. Whole genome sequencing was performed using Illumina platform. The sequences were assembled and genes were predicted for comparative analysis with emm type 1 strains: MGAS5005 and M1 GAS. Genomics comparison revealed one of the invasive strains that differ from others isolates and from emm 1 reference genomes. Also, the new invasive strain showed differences in the content of virulence factors compared to other isolated in the same outbreak. The evolution of contemporary GAS strains is strongly associated with horizontal gene transfer. This is the first genomic study of a Streptococcal emm 1 outbreak in Brazil, and revealed the rapid bacterial evolution leading to new clones. The emergence of new invasive strains can be a consequence of the injudicious use of antibiotics in Brazil during the past decades.

Genome sequencing and analysis of BCG vaccine strains.

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Wen Zhang

Full Text Available BACKGROUND: Although the Bacillus Calmette-Guérin (BCG vaccine against tuberculosis (TB has been available for more than 75 years, one third of the world's population is still infected with Mycobacterium tuberculosis and approximately 2 million people die of TB every year. To reduce this immense TB burden, a clearer understanding of the functional genes underlying the action of BCG and the development of new vaccines are urgently needed. METHODS AND FINDINGS: Comparative genomic analysis of 19 M. tuberculosis complex strains showed that BCG strains underwent repeated human manipulation, had higher region of deletion rates than those of natural M. tuberculosis strains, and lost several essential components such as T-cell epitopes. A total of 188 BCG strain T-cell epitopes were lost to various degrees. The non-virulent BCG Tokyo strain, which has the largest number of T-cell epitopes (359, lost 124. Here we propose that BCG strain protection variability results from different epitopes. This study is the first to present BCG as a model organism for genetics research. BCG strains have a very well-documented history and now detailed genome information. Genome comparison revealed the selection process of BCG strains under human manipulation (1908-1966. CONCLUSIONS: Our results revealed the cause of BCG vaccine strain protection variability at the genome level and supported the hypothesis that the restoration of lost BCG Tokyo epitopes is a useful future vaccine development strategy. Furthermore, these detailed BCG vaccine genome investigation results will be useful in microbial genetics, microbial engineering and other research fields.

Effect of plastic straining on the F and M centres kinetics in γ-irradiated NaCl

International Nuclear Information System (INIS)

Agullo Lopez, F.

1966-01-01

The effect of plastic straining on the room-temperature F and H growth curves in a γ-radiation field has been analyzed. Cristal are strained after F-saturation is reached and then irradiation is continued. The new F growth curve consists of an initial fast growing stage due to additional vacancies created by deformation being turned into F centre, followed by a linear stage. Its slope is higher than that prior to straining. Also the role of straining on M centre thermal decay as well as on the F→M reaction under F light, has been investigated. This reaction has been shown to preferentially occur where intense gliding has developed. (Author) 44 refs

Characterization of a novel chitinase from a moderately halophilic bacterium, Virgibacillus marismortui strain M3-23

OpenAIRE

Essghaier, Badiaa; Hedi, Abdeljabbar; Bajji, Mohammed; Jijakli, Haissam; Boudabous, Abdellatif; Sadfi-Zouaoui, Najla

2012-01-01

A new chitinase produced by the moderately halophilic bacterium Virgibacillus marismortui strain M3- 23 was identified and characterized. Distinguishable characteristics of high activity and stability at different pH, temperatures and salinity of M3-23 chitinase are reported. Analysis of the catalytic domain sequence from the enzyme highlighted its relationship to glycosyl hydrolase family 18. Comparison of the deduced chitinase sequence from strain M3-23 to known chitinases from Bacillus spe...

Extracellular matrix metabolism disorder induced by mechanical strain on human parametrial ligament fibroblasts.

Science.gov (United States)

Min, Jie; Li, Bingshu; Liu, Cheng; Guo, Wenjun; Hong, Shasha; Tang, Jianming; Hong, Li

2017-05-01

Pelvic organ prolapse (POP) is a global health problem that may seriously impact the quality of life of the sufferer. The present study aimed to investigate the potential mechanisms underlying alterations in extracellular matrix (ECM) metabolism in the pathogenesis of POP, by investigating the expression of ECM components in human parametrial ligament fibroblasts (hPLFs) subject to various mechanical strain loads. Fibroblasts derived from parametrial ligaments were cultured from patients with POP and without malignant tumors, who underwent vaginal hysterectomy surgery. Fibroblasts at generations 3‑6 of exponential phase cells were selected, and a four‑point bending device was used for 0, 1,333 or 5,333 µ mechanical loading of cells at 0.5 Hz for 4 h. mRNA and protein expression levels of collagen type I α 1 chain (COL1A1), collagen type III α 1 chain (COL3A1), elastin, matrix metalloproteinase (MMP) ‑2 and ‑9, and transforming growth factor (TGF)‑β1 were detected by reverse transcription‑quantitative polymerase chain reaction and western blotting, respectively. Under increased mechanical strain (5,333 µ), mRNA and protein expression levels of COL1A1, COL3A1 elastin and TGF‑β1 decreased, particularly COL1A1; however, mRNA and protein expression levels of MMP‑2 and ‑9 were significantly increased, compared with the control group (0 µ strain). Following 1,333 µ mechanical strain, mRNA and protein expression levels of COL1A1, COL3A1 elastin and MMP‑2 increased, and MMP‑9 decreased, whereas no significant differences were observed in TGF‑β1 mRNA and protein expression levels. In conclusion, ECM alterations may be involved in pathogenesis of POP, with decreased synthesis and increased degradation of collagen and elastin. Furthermore, the TGF‑β1 signaling pathway may serve an important role in this process and thus may supply a new target and strategy for understanding the etiology and therapy of POP.

PAX5О± and PAX5ОІ mRNA expression in breast Cancer: Relation ...

African Journals Online (AJOL)

Manal Basyouni Ahmed

mRNA expression of PAX5a and PAX5b in breast cancer and assessing their underlying pathological roles through ... the molecular alterations that contribute to disease initiation and ... ring growth and survival of cancer cells [3]. PAX5 is ..... and CA15-3 are prognostic parameters for different molecular subtypes of · breast ...

The M1 muscarinic receptor and its second messenger coupling in human neuroblastoma cells and transfected murine fibroblast cells

International Nuclear Information System (INIS)

Mei, Lin.

1989-01-01

The data of this study indicate that pirenzepine (PZ)-high affinity muscarinic receptors (mAChRs) are coupled to the hydrolysis of inositol lipids and not to the adenylate cyclase system in human neuroblastoma SH-SY5Y cells. The maximal carbachol(CCh)-stimulated [ 3 H]IP 1 accumulation in the SH-SY5Y cells was decreased in the presence of 1μg/ml pertussis toxin, suggesting that a pertussis toxin sensitive G-protein may be involved in the coupling. Several cell clones which express only M 1 mAChR were generated by transfecting the murine fibroblast B82 cells with the cloned rat genomic m 1 gene. The transfected B82 cells (cTB10) showed specific [ 3 H](-)QNB binding activity. The mAChRs in these cells are of the M 1 type defined by their high affinity for PZ and low affinity for AF-DX 116 and coupled to hydrolysis of inositol lipids, possibly via a pertussis toxin sensitive G protein. The relationship between the M 1 mAChR density and the receptor-mediated hydrolysis of inositol lipids was studied in 7 clones. The M 1 mAChR densities in these cells characterized by [ 3 H](-)MQNB binding ranged from 12 fmol/10 6 cells in LK3-1 cells to 260 fmol/10 6 cells in the LK3-8 cells

Strain-specific helper T cell profile in the gut-associated lymphoid tissue.

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Stanisavljević, Suzana; Đedović, Neda; Vujičić, Milica; Saksida, Tamara; Jevtić, Bojan; Milovanović, Boško; Momčilović, Miljana; Miljković, Đorđe; Stojanović, Ivana

2017-10-01

C57BL/6, BALB/c and NOD mice are among the most frequently used strains in autoimmunity research. NOD mice spontaneously develop type 1 diabetes (T1D) and they are prone to induction of experimental autoimmune encephalomyelitis (EAE). Both diseases can be routinely induced in C57BL/6 mice, but not in BALB/c mice. Also, C57BL/6 mice are generally considered T helper (Th)1-biased and BALB/c Th2-biased mice. Having in mind increasingly appreciated role of gut associated lymphoid tissue (GALT) cells in autoimmunity, especially in relation to gut Th17 and regulatory T (Treg) cells, our aim was to determine if there are differences in proportion of CD4 + T cell populations in mesenteric lymph nodes and Peyer's patches of these mouse strains. Lower proportion of Treg was observed in NOD PP, Th2 cells dominated in BALB/c mice in mesenteric lymph nodes (MLN) and Peyer's patches (PP), while Th1 cells prevailed in C57BL/6 MLN. Intradermal immunization of mice with complete Freund's adjuvant resulted in significant difference in Th cell distribution in GALT of NOD mice. Differences were less pronounced in C57BL/6 mice, while GALT of BALB/c mice was almost unresponsive to the immunization. The observed strain- and tissue-dependent changes in Treg proportion after the immunization was probably a consequence of different CCR2 or CCR6-related migration patterns and/or in situ Treg proliferation. In conclusion, NOD, a highly autoimmunity-prone mouse strain, exhibits more profound GALT-related immune response upon immunization compared to the strains that are less prone to autoimmunity. Copyright © 2017 European Federation of Immunological Societies. Published by Elsevier B.V. All rights reserved.

[Production of monoclonal antibodies against a wild strain of rabies virus].

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Akacem, O; Benmansour, A; Coulon, P; Brahimi, M; Benhassine, M

1992-01-01

Production of monoclonal antibodies against a wild strain of rabies virus. Cell fusion of SP 2/O, a murine myeloma against a wild strain of rabies virus has originated five monoclonal antibodies (M.A.) specific for virus nucleocapsid , one M.A. specific for virus glycoprotein and one M.A. specific for a viral membrane protein.

Zika Virus Strains Potentially Display Different Infectious Profiles in Human Neural Cells

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Yannick Simonin

2016-10-01

Full Text Available The recent Zika virus (ZIKV epidemic has highlighted the poor knowledge on its physiopathology. Recent studies showed that ZIKV of the Asian lineage, responsible for this international outbreak, causes neuropathology in vitro and in vivo. However, two African lineages exist and the virus is currently found circulating in Africa. The original African strain was also suggested to be neurovirulent but its laboratory usage has been criticized due to its multiple passages. In this study, we compared the French Polynesian (Asian ZIKV strain to an African strain isolated in Central African Republic and show a difference in infectivity and cellular response between both strains in human neural stem cells and astrocytes. Consistently, this African strain led to a higher infection rate and viral production, as well as stronger cell death and anti-viral response. Our results highlight the need to better characterize the physiopathology and predict neurological impairment associated with African ZIKV.

Growth characteristics and enzyme production optimization of lipase Producing Strain

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Zheng, Chaocheng

2018-01-01

55 samples from different regions were selected and screened by Rhodamine B flat transparent circle method to observe lipase producing effect, among which, LHY-1, identified as Serratia sp. has the characteristics of fast growth, high enzyme production and stable ability. The colony of this strain is white, the edge is smooth and tidy, the surface is moist, the cell is straight, rod-shaped, gram negative, 0.1-0.2 μm in diameter and, length 0.3-0.5 μm in length.

Deletion of ALS5, ALS6 or ALS7 increases adhesion of Candida albicans to human vascular endothelial and buccal epithelial cells

OpenAIRE

ZHAO, XIAOMIN; OH, SOON-HWAN; HOYER, LOIS L.

2007-01-01

C. albicans yeast forms deleted for ALS5, ALS6 or ALS7 are more adherent than a relevant control strain to human vascular endothelial cell monolayers and buccal epithelial cells. In the buccal and vaginal reconstituted human epithelium (RHE) disease models, however, mutant and control strains caused a similar degree of tissue destruction. Deletion of ALS5 or ALS6 significantly slowed growth of the mutant strain; this phenotype was not affected by addition of excess uridine to the culture medi...

Strain improvement and metabolic flux analysis in the wild-type and a mutant Lactobacillus lactis strain for L(+)-lactic acid production.

Science.gov (United States)

Bai, Dong-Mei; Zhao, Xue-Ming; Li, Xin-Gang; Xu, Shi-Min

2004-12-20

The effects of initial glucose concentration and calcium lactate concentration on the lactic acid production by the parent strain, Lactobacillus lactis BME5-18, were studied. The results of the experiments indicated that glucose and lactate repressed the cell growth and the lactic acid production by Lactobacillus lactis BME5-18. A L(+)-lactic acid overproducing strain, Lactobacillus lactis BME5-18M, was screened by mutagenizing the parent strain with ultraviolet (UV) light irradiation and selecting the high glucose and lactate calcium concentration repression resistant mutant. Starting with a concentration of 100g L(-1) glucose, the mutant produced 98.6 g L(-1) lactic acid after 60 h in flasks, 73.9% higher than that of the parent strain. The L(+)-lactic acid purity was 98.1% by weight based on the amount of total lactic acid. The culture of the parent strain could not be analyzed well by conventional metabolic flux analysis techniques, since some pyruvate were accumulated intracellularly. Therefore, a revised flux analysis method was proposed by introducing intracellular pyruvate pool. Further studies demonstrate that there is a high level of NADH oxidase activity (12.11 mmol mg(-1) min(-1)) in the parent strain. The molecular mechanisms of the strain improvement were proposed, i.e., the high level of NADH oxidase activity was eliminated and the uptake rate of glucose was increased from 82.1 C-mmol (g DW h)(-1) to 98.9 C-mmol (g DW h)(-1) by mutagenizing the parent strain with UV, and therefore the mutant strain converts mostly pyruvate to lactic acid with a higher productivity (1.76 g L(-1) h(-1)) than the parent strain (0.95 g L(-1) h(-1)).

Novel image analysis methods for quantification of in situ 3-D tendon cell and matrix strain.

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Fung, Ashley K; Paredes, J J; Andarawis-Puri, Nelly

2018-01-23

Macroscopic tendon loads modulate the cellular microenvironment leading to biological outcomes such as degeneration or repair. Previous studies have shown that damage accumulation and the phases of tendon healing are marked by significant changes in the extracellular matrix, but it remains unknown how mechanical forces of the extracellular matrix are translated to mechanotransduction pathways that ultimately drive the biological response. Our overarching hypothesis is that the unique relationship between extracellular matrix strain and cell deformation will dictate biological outcomes, prompting the need for quantitative methods to characterize the local strain environment. While 2-D methods have successfully calculated matrix strain and cell deformation, 3-D methods are necessary to capture the increased complexity that can arise due to high levels of anisotropy and out-of-plane motion, particularly in the disorganized, highly cellular, injured state. In this study, we validated the use of digital volume correlation methods to quantify 3-D matrix strain using images of naïve tendon cells, the collagen fiber matrix, and injured tendon cells. Additionally, naïve tendon cell images were used to develop novel methods for 3-D cell deformation and 3-D cell-matrix strain, which is defined as a quantitative measure of the relationship between matrix strain and cell deformation. The results support that these methods can be used to detect strains with high accuracy and can be further extended to an in vivo setting for observing temporal changes in cell and matrix mechanics during degeneration and healing. Copyright © 2017. Published by Elsevier Ltd.

Simvastatin and metformin inhibit cell growth in hepatitis C virus infected cells via mTOR increasing PTEN and autophagy.

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José A Del Campo

Full Text Available Hepatitis C virus (HCV infection has been related to increased risk of development of hepatocellular carcinoma (HCC while metformin (M and statins treatment seemed to protect against HCC development. In this work, we aim to identify the mechanisms by which metformin and simvastatin (S could protect from liver cancer. Huh7.5 cells were infected with HCV particles and treated with M+S. Human primary hepatocytes were treated with M+S. Treatment with both drugs inhibited Huh7.5 cell growth and HCV infection. In non-infected cells S increased translational controlled tumor protein (TCTP and phosphatase and tensin homolog (PTEN proteins while M inhibited mammalian target of rapamycin (mTOR and TCTP. Simvastatin and metformin co-administered down-regulated mTOR and TCTP, while PTEN was increased. In cells infected by HCV, mTOR, TCTP, p62 and light chain 3B II (LC3BII were increased and PTEN was decreased. S+M treatment increased PTEN, p62 and LC3BII in Huh7.5 cells. In human primary hepatocytes, metformin treatment inhibited mTOR and PTEN, but up-regulated p62, LC3BII and Caspase 3. In conclusion, simvastatin and metformin inhibited cell growth and HCV infection in vitro. In human hepatocytes, metformin increased cell-death markers. These findings suggest that M+S treatment could be useful in therapeutic prevention of HCV-related hepatocellular carcinoma.

IER5 gene's mRNA expression after irradiation

International Nuclear Information System (INIS)

Ding Kuke; Shen Jingjing; Xu Lili; Li Yanling; Zhou Ping; Ma Binrong; Zhao Zengqiang; Sui Jianli; Zhou Pingkun

2008-01-01

Objective: To explore the effect of irradiation on IER5 gene expression. Methods: Two kinds of cells (AHH-1 and HeLa) and the BALB/c-nu mice inoculated with tumor cells were exposed to 60 Co γ- rays and analyzed by real-time PCR. The above-mentioned irradiated objects were firstly divided into groups by different doses and post-radiation time, then mRNA were extracted and reverse-transcripted to DNA before real-time PCR test. Results: Under the same condition, AHH-1 was more sensitive to radiation than HeLa. The dose level corresponding to the expression peak of AHH-1 was less than that of HeLa. For AHH-1 cells, the response to 2 Gy irradiation was earlier than that to 10 Gy. But there was not remarkable difference for HeLa response between 2 and 10 Gy, and the top transcriptional levels for both cells nearly simultaneously appeared at 2 h after irradiation. In addition, the IER5 gene of human liver tumor was more sensitive than that of lung cancer and brain tumor. Conclusions: IER5 might be a candidate biomarker of radiation injury, and had the potential value in radiation-therapy for liver tumor. (authors)

Sulfur Metabolism of Hydrogenovibrio thermophilus Strain S5 and Its Adaptations to Deep-Sea Hydrothermal Vent Environment

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Lijing Jiang

2017-12-01

Full Text Available Hydrogenovibrio bacteria are ubiquitous in global deep-sea hydrothermal vents. However, their adaptations enabling survival in these harsh environments are not well understood. In this study, we characterized the physiology and metabolic mechanisms of Hydrogenovibrio thermophilus strain S5, which was first isolated from an active hydrothermal vent chimney on the Southwest Indian Ridge. Physiological characterizations showed that it is a microaerobic chemolithomixotroph that can utilize sulfide, thiosulfate, elemental sulfur, tetrathionate, thiocyanate or hydrogen as energy sources and molecular oxygen as the sole electron acceptor. During thiosulfate oxidation, the strain produced extracellular sulfur globules 0.7–6.0 μm in diameter that were mainly composed of elemental sulfur and carbon. Some organic substrates including amino acids, tryptone, yeast extract, casamino acids, casein, acetate, formate, citrate, propionate, tartrate, succinate, glucose and fructose can also serve as carbon sources, but growth is weaker than under CO2 conditions, indicating that strain S5 prefers to be chemolithoautotrophic. None of the tested organic carbons could function as energy sources. Growth tests under various conditions confirmed its adaption to a mesophilic mixing zone of hydrothermal vents in which vent fluid was mixed with cold seawater, preferring moderate temperatures (optimal 37°C, alkaline pH (optimal pH 8.0, microaerobic conditions (optimal 4% O2, and reduced sulfur compounds (e.g., sulfide, optimal 100 μM. Comparative genomics showed that strain S5 possesses more complex sulfur metabolism systems than other members of genus Hydrogenovibrio. The genes encoding the intracellular sulfur oxidation protein (DsrEF and assimilatory sulfate reduction were first reported in the genus Hydrogenovibrio. In summary, the versatility in energy and carbon sources, and unique physiological properties of this bacterium have facilitated its adaptation to deep

The plane strain tests in the PROMETRA program

International Nuclear Information System (INIS)

Cazalis, B.; Desquines, J.; Carassou, S.; Le Jolu, T.; Bernaudat, C.

2016-01-01

A fuel cladding mechanical test, performed under conditions of plane strain deformation in the transverse direction of tube axis, was originally developed at Pennsylvania State University. It was decided to implement this original test within the PROMETRA program using the same experimental procedure and its optimization for a ring mechanical testing on plane strain conditions (PST tests) in hot cells laboratory. This paper presents a detailed description and an interpretation of the Plane Strain Tensile (PST) tests performed in the framework of the PROMETRA program on fresh and irradiated claddings. At first, the context of the PST tests is situated and the specificities of these tests implemented at CEA are justified. Indeed, a significant adjustment of the original experimental procedure is carried out in order to test the irradiated fuel cladding in the best possible conditions. Then, the tests results on fresh Zircaloy-4 and on irradiated Zircaloy-4, M5™ and ZIRLO ® specimens are gathered. The main analyses in support of these tests, such as metallographies, fractographic examinations and finite element simulations are detailed. Finally, a synthesis of the interpretation of the tests is proposed. The PST test seems only representative of plane strain fracture conditions when the test material is very ductile (fresh or high temperature or low hydride material like M5TM). However, it provides a relevant representation of the RIA rupture initiation which is observed in irradiated cladding resulting from hydride rim damage due to the strong irradiation of a fuel rod. - Highlights: • A plane strain mechanical test performed on fuel rod claddings is described. • The tests are performed in the framework of the French PROMETRA program. • Fresh Zircaloy-4 and irradiated Zircaloy-4, M5 and ZIRLO specimens are tested. • The main analyses in support of these tests are detailed. • A synthesis of the interpretation of the PST tests is proposed.

The plane strain tests in the PROMETRA program

Energy Technology Data Exchange (ETDEWEB)

Cazalis, B., E-mail: bernard.cazalis@irsn.fr [Institut de Radioprotection et de Sûreté Nucléaire, IRSN/PSN-RES, F-13115 Saint-Paul Lez Durance BP3 (France); Desquines, J. [Institut de Radioprotection et de Sûreté Nucléaire, IRSN/PSN-RES, F-13115 Saint-Paul Lez Durance BP3 (France); Carassou, S.; Le Jolu, T. [Commissariat à l' Energie Atomique, CEA/DEN/DMN, F- 91191 Gif-sur-Yvette (France); Bernaudat, C. [Electricité de France, EDF/SEPTEN, F-69628 Villeurbanne (France)

2016-04-15

A fuel cladding mechanical test, performed under conditions of plane strain deformation in the transverse direction of tube axis, was originally developed at Pennsylvania State University. It was decided to implement this original test within the PROMETRA program using the same experimental procedure and its optimization for a ring mechanical testing on plane strain conditions (PST tests) in hot cells laboratory. This paper presents a detailed description and an interpretation of the Plane Strain Tensile (PST) tests performed in the framework of the PROMETRA program on fresh and irradiated claddings. At first, the context of the PST tests is situated and the specificities of these tests implemented at CEA are justified. Indeed, a significant adjustment of the original experimental procedure is carried out in order to test the irradiated fuel cladding in the best possible conditions. Then, the tests results on fresh Zircaloy-4 and on irradiated Zircaloy-4, M5™ and ZIRLO{sup ®} specimens are gathered. The main analyses in support of these tests, such as metallographies, fractographic examinations and finite element simulations are detailed. Finally, a synthesis of the interpretation of the tests is proposed. The PST test seems only representative of plane strain fracture conditions when the test material is very ductile (fresh or high temperature or low hydride material like M5TM). However, it provides a relevant representation of the RIA rupture initiation which is observed in irradiated cladding resulting from hydride rim damage due to the strong irradiation of a fuel rod. - Highlights: • A plane strain mechanical test performed on fuel rod claddings is described. • The tests are performed in the framework of the French PROMETRA program. • Fresh Zircaloy-4 and irradiated Zircaloy-4, M5 and ZIRLO specimens are tested. • The main analyses in support of these tests are detailed. • A synthesis of the interpretation of the PST tests is proposed.

Probiotic attributes of Lactobacillus strains isolated from food and of human origin.

Science.gov (United States)

Gaudana, Sandeep B; Dhanani, Akhilesh S; Bagchi, Tamishraha

2010-06-01

Lactobacilli isolated from various sources were identified on the basis of 16S-23S rRNA gene intergenic region amplification and subsequent sequencing of the smaller intergenic region. An in vitro analysis of probiotic properties including binding, ability to tolerate different concentrations of bile, survival in acidic buffer and antimicrobial activity of four different isolates and two standard strains (Lactobacillus plantarum American Type Culture Collection (ATCC) 8014 and L. rhamnosus GG (LGG)) was carried out. The ability of each isolate to stimulate Caco-2 cells, human peripheral blood mononuclear cells (PBMC) and THP-1 cells resulting in immunomodulation of these cells was analysed. Isolates L. rhamnosus CS25 and L. delbrueckii M and standard strain ATCC 8014 showed broad antimicrobial activity, and isolates CS25 (percentage of survival 6.9 % at pH 2.5, 5.1 % at pH 2.0) and L. plantarum CS23 (5.7 % at pH 2.5, 4.9 % at pH 2.0) have shown good tolerance to acidic pH. Isolate CS23 showed a good survival (14 %) after 2 h incubation in de Man, Rogosa and Sharpe (MRS) medium containing 3 % bile salts. Isolates CS23, CS25 and L. fermentum ASt1 could stimulate Caco-2 cells, human PBMC and THP-1 cells for a strong and varied immunomodulatory response in these cells. Though LGG showed poor antimicrobial activity as well as bile and acid tolerance, it was found to be the best binding strain tested. Child faecal isolate CS23 from the present study showed high binding ability (seventeen bacteria/Caco-2), high tolerance to acidic pH and bile salts and significant immunomodulation; therefore it is a good potential probiotic candidate.

Cyclic mechanical strain maintains Nanog expression through PI3K/Akt signaling in mouse embryonic stem cells

International Nuclear Information System (INIS)

Horiuchi, Rie; Akimoto, Takayuki; Hong, Zhang; Ushida, Takashi

2012-01-01

Mechanical strain has been reported to affect the proliferation/differentiation of many cell types; however, the effects of mechanotransduction on self-renewal as well as pluripotency of embryonic stem (ES) cells remains unknown. To investigate the effects of mechanical strain on mouse ES cell fate, we examined the expression of Nanog, which is an essential regulator of self-renewal and pluripotency as well as Nanog-associated intracellular signaling during uniaxial cyclic mechanical strain. The mouse ES cell line, CCE was plated onto elastic membranes, and we applied 10% strain at 0.17 Hz. The expression of Nanog was reduced during ES cell differentiation in response to the withdrawal of leukemia inhibitory factor (LIF); however, two days of cyclic mechanical strain attenuated this reduction of Nanog expression. On the other hand, the cyclic mechanical strain promoted PI3K-Akt signaling, which is reported as an upstream of Nanog transcription. The cyclic mechanical strain-induced Akt phosphorylation was blunted by the PI3K inhibitor wortmannin. Furthermore, cytochalasin D, an inhibitor of actin polymerization, also inhibited the mechanical strain-induced increase in phospho-Akt. These findings imply that mechanical force plays a role in regulating Nanog expression in ES cells through the actin cytoskeleton-PI3K-Akt signaling. -- Highlights: ► The expression of Nanog, which is an essential regulator of “stemness” was reduced during embryonic stem (ES) cell differentiation. ► Cyclic mechanical strain attenuated the reduction of Nanog expression. ► Cyclic mechanical strain promoted PI3K-Akt signaling and mechanical strain-induced Akt phosphorylation was blunted by the PI3K inhibitor and an inhibitor of actin polymerization.

CCL5 and CCR5 interaction promotes cell motility in human osteosarcoma.

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Shih-Wei Wang

Full Text Available BACKGROUND: Osteosarcoma is characterized by a high malignant and metastatic potential. CCL5 (previously called RANTES was originally recognized as a product of activated T cells, and plays a crucial role in the migration and metastasis of human cancer cells. It has been reported that the effect of CCL5 is mediated via CCR receptors. However, the effect of CCL5 on migration activity and integrin expression in human osteosarcoma cells is mostly unknown. METHODOLOGY/PRINCIPAL FINDINGS: Here we found that CCL5 increased the migration and expression of αvβ3 integrin in human osteosarcoma cells. Stimulation of cells with CCL5 increased CCR5 but not CCR1 and CCR3 expression. CCR5 mAb, inhibitor, and siRNA reduced the CCL5-enhanced the migration and integrin up-regulation of osteosarcoma cells. Activations of MEK, ERK, and NF-κB pathways after CCL5 treatment were demonstrated, and CCL5-induced expression of integrin and migration activity was inhibited by the specific inhibitor and mutant of MEK, ERK, and NF-κB cascades. In addition, over-expression of CCL5 shRNA inhibited the migratory ability and integrin expression in osteosarcoma cells. CONCLUSIONS/SIGNIFICANCE: CCL5 and CCR5 interaction acts through MEK, ERK, which in turn activates NF-κB, resulting in the activations of αvβ3 integrin and contributing the migration of human osteosarcoma cells.

Copper tolerance in Frankia sp. strain EuI1c involves surface binding and copper transport.

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Rehan, Medhat; Furnholm, Teal; Finethy, Ryan H; Chu, Feixia; El-Fadly, Gomaah; Tisa, Louis S

2014-09-01

Several Frankia strains have been shown to be copper-tolerant. The mechanism of their copper tolerance was investigated for Frankia sp. strain EuI1c. Copper binding was shown by binding studies. Unusual globular structures were observed on the surface of the bacterium. These globular structures were composed of aggregates containing many relatively smaller "leaf-like" structures. Scanning electron microscopy with energy-dispersive X-ray (SEM-EDAX) analysis of these structures indicated elevated copper and phosphate levels compared to the control cells. Fourier transform infrared spectroscopy (FTIR) analysis indicated an increase in extracellular phosphate on the cell surface of copper-stressed cells. Bioinformatics' analysis of the Frankia sp. strain EuI1c genome revealed five potential cop genes: copA, copZ, copC, copCD, and copD. Experiments with Frankia sp. strain EuI1c using qRT-PCR indicated an increase in messenger RNA (mRNA) levels of the five cop genes upon Cu(2+) stress. After 5 days of Cu(2+) stress, the copA, copZ, copC, copCD, and copD mRNA levels increased 25-, 8-, 18-, 18-, and 25-fold, respectively. The protein profile of Cu(2+)-stressed Frankia sp. strain EuI1c cells revealed the upregulation of a 36.7 kDa protein that was identified as FraEuI1c_1092 (sulfate-binding periplasmic transport protein). Homologues of this gene were only present in the genomes of the Cu(2+)-resistant Frankia strains (EuI1c, DC12, and CN3). These data indicate that copper tolerance by Frankia sp. strain EuI1c involved the binding of copper to the cell surface and transport proteins.

Layered perovskite LaBaCuMO{sub 5+x} (M = Fe, Co) cathodes for intermediate-temperature protonic ceramic membrane fuel cells

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Ling Yihan; Lin Bin; Zhao Ling; Zhang Xiaozhen; Yu Jia; Peng Ranran; Meng Guangyao [CAS Key Laboratory of Materials for Energy Conversion, Department of Materials Science and Engineering, University of Science and Technology of China (USTC), Hefei, Anhui 230026 (China); Liu Xingqin, E-mail: lyhyy@mail.ustc.edu.c [CAS Key Laboratory of Materials for Energy Conversion, Department of Materials Science and Engineering, University of Science and Technology of China (USTC), Hefei, Anhui 230026 (China)

2010-03-18

Layered perovskite LaBaCuFeO{sub 5+x} (LBCF) and LaBaCuCoO{sub 5+x} (LBCC) oxides are synthesized by a modified Pechini method and examined as potential cathode materials for intermediate-temperature protonic ceramic membrane fuel cells (IT-PCMFCs). Thin proton-conducting BaZr{sub 0.1}Ce{sub 0.7}Y{sub 0.2}O{sub 3-{delta}} (BZCY) electrolyte and NiO-BaZr{sub 0.1}Ce{sub 0.7}Y{sub 0.2}O{sub 3-{delta}} (NiO-BZCY) anode functional layer are prepared over porous anode substrates composed of NiO-BaZr{sub 0.1}Ce{sub 0.7}Y{sub 0.2}O{sub 3-{delta}} by a one-step dry-pressing/co-firing process. Laboratory-sized quad-layer cells of NiO-BYCZ/NiO-BYCZ/BYCZ/LBCF (LBCC) are operated from 550 to 700 {sup o}C with humidified hydrogen ({approx}3% H{sub 2}O) as fuel and the static air as oxidant. The single cell with LBCF cathode shows peak power densities of only 327 mW cm{sup -2} at 700 {sup o}C and 105 mW cm{sup -2} for 550 {sup o}C, while the single cell with LBCC cathode shows peak power densities of 432 and 171 mW cm{sup -2} at 700 and 550 {sup o}C, respectively. The dramatic improvement of cell performance is attributed to higher cobaltites catalytic activity than that of ferrites for IT-PCMFCs, which is in good agreement with the results of impedance measurement.

Strain differences in the response of mouse testicular stem cells to fractionated radiation

International Nuclear Information System (INIS)

Meistrich, M.L.; Finch, M.; Lu, C.C.; de Ruiter-Bootsma, A.L.; de Rooij, D.G.; Davids, J.A.G.

1984-01-01

The survival of spermatogonial stem cells in CBA and C3H mice after single and split-dose (24-hr interval) irradiation with fission neutrons and gamma rays was compared. The first doses of the fractionated regimes were either 150 rad (neutrons) or 600 rad (gamma). For both strains the neutron survival curves were exponential. The D 0 value of stem cells in CBA decreased from 83 to 25 rad upon fractionation; that of C3H stem cells decreased only from 54 to 36 rad. The survival curves for gamma irradiation, which all showed shoulders, indicated that C3H stem cells had larger repair capacities than CBA stem cells. However, the most striking difference between the two strains in response to gamma radiation was in the slopes of the second-dose curves. Whereas C3H stem cells showed a small increase of the D 0 upon fractionation (from 196 to 218 rad), CBA stem cells showed a marked decrease (from 243 to 148 rad). The decreases in D 0 upon fractionation, observed in both strains with neutron irradiation and also with gamma irradiation in CBA, are most likely the result of recruitment or progression of radioresistant survivors to a more sensitive state of proliferation or cell cycle phase. It may be that the survivng stem cells in C3H mice are recruited less rapidly and synchronously into active cycle than in CBA mice. Thus, it appears that the strain differences may be quantitative, rather than qualitative

Thyrotropin Receptor and Membrane Interactions in FRTL-5 Thyroid Cell Strain in Microgravity

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Albi, E.; Ambesi-Impiombato, F. S.; Peverini, M.; Damaskopoulou, E.; Fontanini, E.; Lazzarini, R.; Curcio, F.; Perrella, G.

2011-01-01

The aim of this work was to analyze the possible alteration of thyrotropin (TSH) receptors in microgravity, which could explain the absence of thyroid cell proliferation in the space environment. Several forms of the TSH receptor are localized on the plasma membrane associated with caveolae and lipid rafts. The TSH regulates the fluidity of the cell membrane and the presence of its receptors in microdomains that are rich in sphingomyelin and cholesterol. TSH also stimulates cyclic adenosine monophosphate (cAMP) accumulation and cell proliferation. Reported here are the results of an experiment in which the FRTL-5 thyroid cell line was exposed to microgravity during the Texus-44 mission (launched February 7, 2008, from Kiruna, Sweden). When the parabolic flight brought the sounding rocket to an altitude of 264km, the culture media were injected with or without TSH in the different samples, and weightlessness prevailed on board for 6 minutes and 19 seconds. Control experiments were performed, in parallel, in an onboard 1g centrifuge and on the ground in Kiruna laboratory. Cell morphology and function were analyzed. Results show that in microgravity conditions the cells do not respond to TSH treatment and present an irregular shape with condensed chromatin, a modification of the cell membrane with shedding of the TSH receptor in the culture medium, and an increase of sphingomyelin-synthase and Bax proteins. It is possible that real microgravity induces a rearrangement of specific sections of the cell membrane, which act as platforms for molecular receptors, thus influencing thyroid cell function in astronauts during space missions.

Lgr5-EGFP marks taste bud stem/progenitor cells in posterior tongue.

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Yee, Karen K; Li, Yan; Redding, Kevin M; Iwatsuki, Ken; Margolskee, Robert F; Jiang, Peihua

2013-05-01

Until recently, reliable markers for adult stem cells have been lacking for many regenerative mammalian tissues. Lgr5 (leucine-rich repeat-containing G-protein-coupled receptor 5) has been identified as a marker for adult stem cells in intestine, stomach, and hair follicle; Lgr5-expressing cells give rise to all types of cells in these tissues. Taste epithelium also regenerates constantly, yet the identity of adult taste stem cells remains elusive. In this study, we found that Lgr5 is strongly expressed in cells at the bottom of trench areas at the base of circumvallate (CV) and foliate taste papillae and weakly expressed in the basal area of taste buds and that Lgr5-expressing cells in posterior tongue are a subset of K14-positive epithelial cells. Lineage-tracing experiments using an inducible Cre knockin allele in combination with Rosa26-LacZ and Rosa26-tdTomato reporter strains showed that Lgr5-expressing cells gave rise to taste cells, perigemmal cells, along with self-renewing cells at the bottom of trench areas at the base of CV and foliate papillae. Moreover, using subtype-specific taste markers, we found that Lgr5-expressing cell progeny include all three major types of adult taste cells. Our results indicate that Lgr5 may mark adult taste stem or progenitor cells in the posterior portion of the tongue. Copyright © 2013 AlphaMed Press.

Improved production of 2,5-furandicarboxylic acid by overexpression of 5-hydroxymethylfurfural oxidase and 5-hydroxymethylfurfural/furfural oxidoreductase in Raoultella ornithinolytica BF60.

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Yuan, Haibo; Li, Jianghua; Shin, Hyun-Dong; Du, Guocheng; Chen, Jian; Shi, Zhongping; Liu, Long

2018-01-01

2,5-Furandicarboxylic acid (FDCA) is a promising bio-based building block and can be produced by biotransformation of 5-hydroxymethylfurfural (HMF). To improve the FDCA production, two genes-one encoding HMF oxidase (HMFO; from Methylovorus sp. strain MP688) and another encoding for HMF/Furfural oxidoreductase (HmfH; from Cupriavidus basilensis HMF14)-were introduced into Raoultella ornithinolytica BF60. The FDCA production in the engineered whole-cell biocatalyst increased from 51.0 to 93.6mM, and the molar conversion ratio of HMF to FDCA increased from 51.0 to 93.6%. Copyright © 2017 Elsevier Ltd. All rights reserved.

Purification of foot-and-mouth disease virus by heparin as ligand for certain strains.

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Du, Ping; Sun, Shiqi; Dong, Jinjie; Zhi, Xiaoying; Chang, Yanyan; Teng, Zhidong; Guo, Huichen; Liu, Zaixin

2017-04-01

The goal of this project was to develop an easily operable and scalable process for the recovery and purification of foot-and-mouth disease virus (FMDV) from cell culture. Heparin resins HipTrap Heparin HP and AF-Heparin HC-650 were utilized to purify FMDV O/HN/CHA/93. Results showed that the purity of AF-Heparin HC-650 was ideal. Then, the O/HN/CHA/93, O/Tibet/CHA/99, Asia I/HN/06, and A/CHA/HB/2009 strains were purified by AF-Heparin HC-650. Their affinity/virus recoveries were approximately 51.2%/45.8%, 71.5%/70.9%, 96.4%/73.5, and 59.5%/42.1%, respectively. During a stepwise elution strategy, the viral particles were mainly eluted at 300mM ionic strength peaks. The heparin affinity chromatography process removed more than 94% of cellular and medium proteins. Anion exchange resin Capto Q captured four FMD virus particles; 40% of binding proteins and 80%-90% of viral particles were eluted at 450mM NaCl. Moreover, ionic strength varied from 30 to 450mM had no effect on the immunity to FMDV. The results revealed that heparin sulfate may be the main receptor for CHA/99 strain attachment-susceptible cells. Heparin affinity chromatography can reach perfect results, especially when used as a ligand of the virus. Anion exchange is useful only as previous step for further purification. Copyright © 2016. Published by Elsevier B.V.

[Genetic diversity of extraintestinal Escherichia coli strains producers of beta-lactamases TEM, SHV and CTX-M associated with healthcare].

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Varela, Yasmin; Millán, Beatriz; Araque, María

2017-06-01

There are few reports from Venezuela describing the genetic basis that sustains the pathogenic potential and phylogenetics of Escherichia coli extraintestinal strains isolated in health care units. To establish the genetic diversity of extraintestinal E. coli strains producers of betalactamases TEM, SHV and CTX-M associated with healthcare. We studied a collection of 12 strains of extraintestinal E. coli with diminished sensitivity to broad-spectrum cephalosporins. Antimicrobial susceptibility was determined by minimum inhibitory concentration. We determined the phylogenetic groups, virulence factors and genes encoding antimicrobial resistance using PCR, and clonal characterization by repetitive element palindromic-PCR rep-PCR. All strains showed resistance to cephalosporins and joint resistance to quinolones and aminoglycosides. The phylogenetic distribution showed that the A and B1 groups were the most frequent, followed by D and B2. We found all the virulence factors analyzed in the B2 group, and fimH gene was the most frequent among them. We found blaCTX-M in all strains,with a higher prevalence of blaCTX-M-8; two of these strains showed coproduction of blaCTX-M-9 and were genetically identified as blaCTXM-65 and blaCTX-M-147 by sequencing. The strains under study showed genetic diversity, hosting a variety of virulence genes, as well as antimicrobial resistance with no particular phylogroup prevalence. This is the first report of blaCTX-M alleles in Venezuela and in the world associated to non-genetically related strains isolated in health care units, a situation that deserves attention, as well as the rationalization of antimicrobials use.

Whole transcriptome analysis of Acinetobacter baumannii assessed by RNA-sequencing reveals different mRNA expression profiles in biofilm compared to planktonic cells.

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Soraya Rumbo-Feal

Full Text Available Acinetobacterbaumannii has emerged as a dangerous opportunistic pathogen, with many strains able to form biofilms and thus cause persistent infections. The aim of the present study was to use high-throughput sequencing techniques to establish complete transcriptome profiles of planktonic (free-living and sessile (biofilm forms of A. baumannii ATCC 17978 and thereby identify differences in their gene expression patterns. Collections of mRNA from planktonic (both exponential and stationary phase cultures and sessile (biofilm cells were sequenced. Six mRNA libraries were prepared following the mRNA-Seq protocols from Illumina. Reads were obtained in a HiScanSQ platform and mapped against the complete genome to describe the complete mRNA transcriptomes of planktonic and sessile cells. The results showed that the gene expression pattern of A. baumannii biofilm cells was distinct from that of planktonic cells, including 1621 genes over-expressed in biofilms relative to stationary phase cells and 55 genes expressed only in biofilms. These differences suggested important changes in amino acid and fatty acid metabolism, motility, active transport, DNA-methylation, iron acquisition, transcriptional regulation, and quorum sensing, among other processes. Disruption or deletion of five of these genes caused a significant decrease in biofilm formation ability in the corresponding mutant strains. Among the genes over-expressed in biofilm cells were those in an operon involved in quorum sensing. One of them, encoding an acyl carrier protein, was shown to be involved in biofilm formation as demonstrated by the significant decrease in biofilm formation by the corresponding knockout strain. The present work serves as a basis for future studies examining the complex network systems that regulate bacterial biofilm formation and maintenance.

mTOR inhibition sensitizes ONC201-induced anti-colorectal cancer cell activity.

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Jin, Zhe-Zhu; Wang, Wei; Fang, Di-Long; Jin, Yong-Jun

2016-09-30

We here tested the anti-colorectal cancer (CRC) activity by a first-in-class small molecule TRAIL inducer ONC201. The potential effect of mTOR on ONC201's actions was also examined. ONC201 induced moderate cytotoxicity against CRC cell lines (HT-29, HCT-116 and DLD-1) and primary human CRC cells. Significantly, AZD-8055, a mTOR kinase inhibitor, sensitized ONC201-induced cytotoxicity in CRC cells. Meanwhile, ONC201-induced TRAIL/death receptor-5 (DR-5) expression, caspase-8 activation and CRC cell apoptosis were also potentiated with AZD-8055 co-treatment. Reversely, TRAIL sequestering antibody RIK-2 or the caspase-8 specific inhibitor z-IETD-fmk attenuated AZD-8055 plus ONC201-induced CRC cell death. Further, mTOR kinase-dead mutation (Asp-2338-Ala) or shRNA knockdown significantly sensitized ONC201's activity in CRC cells, leading to profound cell death and apoptosis. On the other hand, expression of a constitutively-active S6K1 (T389E) attenuated ONC201-induced CRC cell apoptosis. For the mechanism study, we showed that ONC201 blocked Akt, but only slightly inhibited mTOR in CRC cells. Co-treatment with AZD-8055 also concurrently blocked mTOR activation. These results suggest that mTOR could be a primary resistance factor of ONC201 in CRC cells. Copyright © 2016 Elsevier Inc. All rights reserved.

A New Heterotrophic Strain for Bioleaching of Low Grade Complex Copper Ore

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Kaijian Hu

2016-02-01

Full Text Available A new heterotrophic strain, named Providencia sp. JAT-1, was isolated and used in bioleaching of low-grade complex copper ore. The strain uses sodium citrate as a carbon source and urea as a nitrogen source to produce ammonia. The optimal growth condition of the strain is 30 C, initial pH 8, sodium citrate 10 g/L and urea 20 g/L, under which the cell density and ammonia concentration in the medium reached a maximum of 4.83 × 108 cells/mL and 14 g/L, respectively. Ammonia produced by the strain is used as the main lixiviant in bioleaching. Bioleaching results revealed that higher strain growth led to a higher copper recovery, while higher pulp density will cause a greater inhibitory effect on strain growth and ammonia production. The copper extraction reached the highest value of 54.5% at the pulp density of 1%. Malachite, chrysocolla and chalcocite are easy to leach out in this bioleaching system while chalcopyrite is difficult. Results of comparative leaching experiments show that bioleaching using JAT-1 is superior to ammonia leaching at the same condition. The metabolites produced by the strain other than ammonia are also involved in bioleaching.

Cyclic mechanical strain maintains Nanog expression through PI3K/Akt signaling in mouse embryonic stem cells

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Horiuchi, Rie [Division of Regenerative Medical Engineering, Center for Disease Biology and Integrative Medicine, Graduate School of Medicine, The University of Tokyo, 7-3-1 Hongo, Bunkyo, Tokyo 113-0033 (Japan); Akimoto, Takayuki, E-mail: akimoto@m.u-tokyo.ac.jp [Division of Regenerative Medical Engineering, Center for Disease Biology and Integrative Medicine, Graduate School of Medicine, The University of Tokyo, 7-3-1 Hongo, Bunkyo, Tokyo 113-0033 (Japan); Institute for Biomedical Engineering, Consolidated Research Institute for Advanced Science and Medical Care, Waseda University, 513 Waseda-tsurumaki, Shinjuku, Tokyo 162-0041 (Japan); Hong, Zhang [Institute for Biomedical Engineering, Consolidated Research Institute for Advanced Science and Medical Care, Waseda University, 513 Waseda-tsurumaki, Shinjuku, Tokyo 162-0041 (Japan); Ushida, Takashi [Division of Regenerative Medical Engineering, Center for Disease Biology and Integrative Medicine, Graduate School of Medicine, The University of Tokyo, 7-3-1 Hongo, Bunkyo, Tokyo 113-0033 (Japan)

2012-08-15

Mechanical strain has been reported to affect the proliferation/differentiation of many cell types; however, the effects of mechanotransduction on self-renewal as well as pluripotency of embryonic stem (ES) cells remains unknown. To investigate the effects of mechanical strain on mouse ES cell fate, we examined the expression of Nanog, which is an essential regulator of self-renewal and pluripotency as well as Nanog-associated intracellular signaling during uniaxial cyclic mechanical strain. The mouse ES cell line, CCE was plated onto elastic membranes, and we applied 10% strain at 0.17 Hz. The expression of Nanog was reduced during ES cell differentiation in response to the withdrawal of leukemia inhibitory factor (LIF); however, two days of cyclic mechanical strain attenuated this reduction of Nanog expression. On the other hand, the cyclic mechanical strain promoted PI3K-Akt signaling, which is reported as an upstream of Nanog transcription. The cyclic mechanical strain-induced Akt phosphorylation was blunted by the PI3K inhibitor wortmannin. Furthermore, cytochalasin D, an inhibitor of actin polymerization, also inhibited the mechanical strain-induced increase in phospho-Akt. These findings imply that mechanical force plays a role in regulating Nanog expression in ES cells through the actin cytoskeleton-PI3K-Akt signaling. -- Highlights: Black-Right-Pointing-Pointer The expression of Nanog, which is an essential regulator of 'stemness' was reduced during embryonic stem (ES) cell differentiation. Black-Right-Pointing-Pointer Cyclic mechanical strain attenuated the reduction of Nanog expression. Black-Right-Pointing-Pointer Cyclic mechanical strain promoted PI3K-Akt signaling and mechanical strain-induced Akt phosphorylation was blunted by the PI3K inhibitor and an inhibitor of actin polymerization.

Mitofusin 1 is degraded at G2/M phase through ubiquitylation by MARCH5

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Park Yong-Yea

2012-12-01

Full Text Available Abstract Background Mitochondria exhibit a dynamic morphology in cells and their biogenesis and function are integrated with the nuclear cell cycle. In mitotic cells, the filamentous network structure of mitochondria takes on a fragmented form. To date, however, whether mitochondrial fusion activity is regulated in mitosis has yet to be elucidated. Findings Here, we report that mitochondria were found to be fragmented in G2 phase prior to mitotic entry. Mitofusin 1 (Mfn1, a mitochondrial fusion protein, interacted with cyclin B1, and their interactions became stronger in G2/M phase. In addition, MARCH5, a mitochondrial E3 ubiquitin ligase, reduced Mfn1 levels and the MARCH5-mediated Mfn1 ubiquitylation were enhanced in G2/M phase. Conclusions Mfn1 is degraded through the MARCH5-mediated ubiquitylation in G2/M phase and the cell cycle-dependent degradation of Mfn1 could be facilitated by interaction with cyclin B1/Cdk1 complexes.

A viral microRNA down-regulates multiple cell cycle genes through mRNA 5'UTRs.

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Finn Grey

2010-06-01

Full Text Available Global gene expression data combined with bioinformatic analysis provides strong evidence that mammalian miRNAs mediate repression of gene expression primarily through binding sites within the 3' untranslated region (UTR. Using RNA induced silencing complex immunoprecipitation (RISC-IP techniques we have identified multiple cellular targets for a human cytomegalovirus (HCMV miRNA, miR-US25-1. Strikingly, this miRNA binds target sites primarily within 5'UTRs, mediating significant reduction in gene expression. Intriguingly, many of the genes targeted by miR-US25-1 are associated with cell cycle control, including cyclin E2, BRCC3, EID1, MAPRE2, and CD147, suggesting that miR-US25-1 is targeting genes within a related pathway. Deletion of miR-US25-1 from HCMV results in over expression of cyclin E2 in the context of viral infection. Our studies demonstrate that a viral miRNA mediates translational repression of multiple cellular genes by targeting mRNA 5'UTRs.

Raspberry wine fermentation with suspended and immobilized yeast cells of two strains of Saccharomyces cerevisiae.

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Djordjević, Radovan; Gibson, Brian; Sandell, Mari; de Billerbeck, Gustavo M; Bugarski, Branko; Leskošek-Čukalović, Ida; Vunduk, Jovana; Nikićević, Ninoslav; Nedović, Viktor

2015-01-01

The objectives of this study were to assess the differences in fermentative behaviour of two different strains of Saccharomyces cerevisiae (EC1118 and RC212) and to determine the differences in composition and sensory properties of raspberry wines fermented with immobilized and suspended yeast cells of both strains at 15 °C. Analyses of aroma compounds, glycerol, acetic acid and ethanol, as well as the kinetics of fermentation and a sensory evaluation of the wines, were performed. All fermentations with immobilized yeast cells had a shorter lag phase and faster utilization of sugars and ethanol production than those fermented with suspended cells. Slower fermentation kinetics were observed in all the samples that were fermented with strain RC212 (suspended and immobilized) than in samples fermented with strain EC1118. Significantly higher amounts of acetic acid were detected in all samples fermented with strain RC212 than in those fermented with strain EC1118 (0.282 and 0.602 g/l, respectively). Slightly higher amounts of glycerol were observed in samples fermented with strain EC1118 than in those fermented with strain RC212. Copyright © 2014 John Wiley & Sons, Ltd.

Comprehensive genomic analysis of a plant growth-promoting rhizobacterium Pantoea agglomerans strain P5.

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Shariati J, Vahid; Malboobi, Mohammad Ali; Tabrizi, Zeinab; Tavakol, Elahe; Owilia, Parviz; Safari, Maryam

2017-11-15

In this study, we provide a comparative genomic analysis of Pantoea agglomerans strain P5 and 10 closely related strains based on phylogenetic analyses. A next-generation shotgun strategy was implemented using the Illumina HiSeq 2500 technology followed by core- and pan-genome analysis. The genome of P. agglomerans strain P5 contains an assembly size of 5082485 bp with 55.4% G + C content. P. agglomerans consists of 2981 core and 3159 accessory genes for Coding DNA Sequences (CDSs) based on the pan-genome analysis. Strain P5 can be grouped closely with strains PG734 and 299 R using pan and core genes, respectively. All the predicted and annotated gene sequences were allocated to KEGG pathways. Accordingly,  genes involved in plant growth-promoting (PGP) ability, including phosphate solubilization, IAA and siderophore production, acetoin and 2,3-butanediol synthesis and bacterial secretion, were assigned. This study provides an in-depth view of the PGP characteristics of strain P5, highlighting its potential use in agriculture as a biofertilizer.

Biological assay of attenuated strain NADL-2 and virulent strain NADL-8 of porcine parvovirus.

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Mengeling, W L; Pejsak, Z; Paul, P S

1984-11-01

Attenuated strain NADL-2 and virulent strain NADL-8 of porcine parvovirus (PPV) were titrated in vivo and in vitro under similar conditions to provide a better understanding of some of the factors involved in virulence of PPV in causing maternal reproductive failure of swine. Both strains cause fetal death when they are injected directly into fetal fluids, but only strain NADL-8 does so when administered to pregnant swine. The strains were tested for their hemagglutinating activity (HA), median cell culture infective dose (CCID50), median fetal infective dose (FID50), and median fetal lethal dose (FLD50). The FID50 and FLD50 were determined by injecting virus directly into the amniotic fluid of fetuses in utero at 44 +/- 2 days of gestation and collecting the fetuses 15 +/- 1 days later. Both strains had an HA titer of 64, suggesting that there is a similar number of virions in stock preparations. However, other measurements differed markedly. The CCID50, FID50, and FLD50 were 10(5.5), 10(3.5), and 10(0.5), respectively, for strain NADL-2, and 10(4.5), 10(7.7), and 10(6.3), respectively, for strain NADL-8. Collectively, the values indicate that more than 10,000 times as much strain NADL-2 would need to reach the conceptus transplacentally to establish infection. These observations may help to explain the different consequences of oronasal exposure of pregnant swine to these strains of PPV.

Recombinant ArtinM activates mast cells.

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Barbosa-Lorenzi, Valéria Cintra; Cecilio, Nerry Tatiana; de Almeida Buranello, Patricia Andressa; Pranchevicius, Maria Cristina; Goldman, Maria Helena S; Pereira-da-Silva, Gabriela; Roque-Barreira, Maria Cristina; Jamur, Maria Célia; Oliver, Constance

2016-07-04

Mast cells are hematopoietically derived cells that play a role in inflammatory processes such as allergy, as well as in the immune response against pathogens by the selective and rapid release of preformed and lipid mediators, and the delayed release of cytokines. The native homotetrameric lectin ArtinM, a D-mannose binding lectin purified from Artocarpus heterophyllus seeds, is one of several lectins that are able to activate mast cells. Besides activating mast cells, ArtinM has been shown to affect several biological responses, including immunomodulation and acceleration of wound healing. Because of the potential pharmacological application of ArtinM, a recombinant ArtinM (rArtinM) was produced in Escherichia coli. The current study evaluated the ability of rArtinM to induce mast cell degranulation and activation. The glycan binding specificity of rArtinM was similar to that of jArtinM. rArtinM, via its CRD, was able to degranulate, releasing β-hexosaminidase and TNF-α, and to promote morphological changes on the mast cell surface. Moreover, rArtinM induced the release of the newly-synthesized mediator, IL-4. rArtinM does not have a co-stimulatory effect on the FcεRI degranulation via. The IgE-dependent mast cell activation triggered by rArtinM seems to be dependent on NFkB activation. The lectin rArtinM has the ability to activate and degranulate mast cells via their CRDs. The present study indicates that rArtinM is a suitable substitute for the native form, jArtinM, and that rArtinM may serve as an important and reliable pharmacological agent.

Growth Phase-Dependent Proteomes of the Malaysian Isolated Lactococcus lactis Dairy Strain M4 Using Label-Free Qualitative Shotgun Proteomics Analysis

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Theresa Wan Chen Yap

2014-01-01

Full Text Available Lactococcus lactis is the most studied mesophilic fermentative lactic acid bacterium. It is used extensively in the food industry and plays a pivotal role as a cell factory and also as vaccine delivery platforms. The proteome of the Malaysian isolated L. lactis M4 dairy strain, obtained from the milk of locally bred cows, was studied to elucidate the physiological changes occurring between the growth phases of this bacterium. In this study, ultraperformance liquid chromatography nanoflow electrospray ionization tandem mass spectrometry (UPLC- nano-ESI-MSE approach was used for qualitative proteomic analysis. A total of 100 and 121 proteins were identified from the midexponential and early stationary growth phases, respectively, of the L. lactis strain M4. During the exponential phase, the most important reaction was the generation of sufficient energy, whereas, in the early stationary phase, the metabolic energy pathways decreased and the biosynthesis of proteins became more important. Thus, the metabolism of the cells shifted from energy production in the exponential phase to the synthesis of macromolecules in the stationary phase. The resultant proteomes are essential in providing an improved view of the cellular machinery of L. lactis during the transition of growth phases and hence provide insight into various biotechnological applications.

Growth phase-dependent proteomes of the Malaysian isolated Lactococcus lactis dairy strain M4 using label-free qualitative shotgun proteomics analysis.

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Yap, Theresa Wan Chen; Rabu, Amir; Abu Bakar, Farah Diba; Rahim, Raha Abdul; Mahadi, Nor Muhammad; Illias, Rosli Md; Murad, Abdul Munir Abdul

2014-01-01

Lactococcus lactis is the most studied mesophilic fermentative lactic acid bacterium. It is used extensively in the food industry and plays a pivotal role as a cell factory and also as vaccine delivery platforms. The proteome of the Malaysian isolated L. lactis M4 dairy strain, obtained from the milk of locally bred cows, was studied to elucidate the physiological changes occurring between the growth phases of this bacterium. In this study, ultraperformance liquid chromatography nanoflow electrospray ionization tandem mass spectrometry (UPLC- nano-ESI-MS(E)) approach was used for qualitative proteomic analysis. A total of 100 and 121 proteins were identified from the midexponential and early stationary growth phases, respectively, of the L. lactis strain M4. During the exponential phase, the most important reaction was the generation of sufficient energy, whereas, in the early stationary phase, the metabolic energy pathways decreased and the biosynthesis of proteins became more important. Thus, the metabolism of the cells shifted from energy production in the exponential phase to the synthesis of macromolecules in the stationary phase. The resultant proteomes are essential in providing an improved view of the cellular machinery of L. lactis during the transition of growth phases and hence provide insight into various biotechnological applications.

Growth Phase-Dependent Proteomes of the Malaysian Isolated Lactococcus lactis Dairy Strain M4 Using Label-Free Qualitative Shotgun Proteomics Analysis

Science.gov (United States)

Yap, Theresa Wan Chen; Rabu, Amir; Abu Bakar, Farah Diba; Abdul Rahim, Raha; Mahadi, Nor Muhammad; Illias, Rosli Md.

2014-01-01

Lactococcus lactis is the most studied mesophilic fermentative lactic acid bacterium. It is used extensively in the food industry and plays a pivotal role as a cell factory and also as vaccine delivery platforms. The proteome of the Malaysian isolated L. lactis M4 dairy strain, obtained from the milk of locally bred cows, was studied to elucidate the physiological changes occurring between the growth phases of this bacterium. In this study, ultraperformance liquid chromatography nanoflow electrospray ionization tandem mass spectrometry (UPLC- nano-ESI-MSE) approach was used for qualitative proteomic analysis. A total of 100 and 121 proteins were identified from the midexponential and early stationary growth phases, respectively, of the L. lactis strain M4. During the exponential phase, the most important reaction was the generation of sufficient energy, whereas, in the early stationary phase, the metabolic energy pathways decreased and the biosynthesis of proteins became more important. Thus, the metabolism of the cells shifted from energy production in the exponential phase to the synthesis of macromolecules in the stationary phase. The resultant proteomes are essential in providing an improved view of the cellular machinery of L. lactis during the transition of growth phases and hence provide insight into various biotechnological applications. PMID:24982972

Comparative genomic analysis of Brucella abortus vaccine strain 104M reveals a set of candidate genes associated with its virulence attenuation.

Science.gov (United States)

Yu, Dong; Hui, Yiming; Zai, Xiaodong; Xu, Junjie; Liang, Long; Wang, Bingxiang; Yue, Junjie; Li, Shanhu

2015-01-01

The Brucella abortus strain 104M, a spontaneously attenuated strain, has been used as a vaccine strain in humans against brucellosis for 6 decades in China. Despite many studies, the molecular mechanisms that cause the attenuation are still unclear. Here, we determined the whole-genome sequence of 104M and conducted a comprehensive comparative analysis against the whole genome sequences of the virulent strain, A13334, and other reference strains. This analysis revealed a highly similar genome structure between 104M and A13334. The further comparative genomic analysis between 104M and A13334 revealed a set of genes missing in 104M. Some of these genes were identified to be directly or indirectly associated with virulence. Similarly, a set of mutations in the virulence-related genes was also identified, which may be related to virulence alteration. This study provides a set of candidate genes associated with virulence attenuation in B.abortus vaccine strain 104M.

Microbial production of squalene by a nicotinic acid-resistant mutant derived from Fusarium sp. No.5-128B

International Nuclear Information System (INIS)

Ogawa, T.; Kojima, I.; Takeda, N.; Fukuda, H.

1994-01-01

A nicotinic acid-resistant mutant, designated NA201, was obtained from Fusarium sp. no.5-128B by treatment with ultraviolet light. This mutant strain could grow in the presence of up to 500mM nicotinic acid in the culture medium, although the parent strain could not grow at concentrations of nicotinic acid above 200 mM. The Na201 strain exhibited morphological mutations, neither forming aerial hyphae nor secreting a red-brown pigment. However, it retained the resistance to kabicidin at 25 mg-l(-1) of the parent strain. The mutant NA201 cells contained high levels of squalene and low levels of ergosterol, about 53 times higher and five to six times lower, respectively, than those of the parent strain under standard culture conditions. The volumetric oxygen transfer coefficient (Kd) affected the level of squalene in the mutant cells. The Kd for the maximum production of squalene by the mutant was 24 mmol O2 l(-1)h(-1)atm(-1) and the level of squalene in the mutant cells was 26 mg (g cell)(-1) on a dry weight basis. The greatest accumulation of squalene by the Na201 strain, corresponding to 323 mg per liter of culture medium and 35 mg (g cell)(-1) on a dry weight basis, was achieved in a culture in which the Kd was changed from a high to a low value on the third day, with the simultaneous addition of 3% glucose (w/v)

Mechanisms of tolerance and high degradation capacity of the herbicide mesotrione by Escherichia coli strain DH5-α.

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Luiz R Olchanheski

Full Text Available The intensive use of agrochemicals has played an important role in increasing agricultural production. One of the impacts of agrochemical use has been changes in population structure of soil microbiota. The aim of this work was to analyze the adaptive strategies that bacteria use to overcome oxidative stress caused by mesotrione, which inhibits 4-hydroxyphenylpyruvate dioxygenase. We also examined antioxidative stress systems, saturation changes of lipid membranes, and the capacity of bacteria to degrade mesotrione. Escherichia coli DH5-á was chosen as a non-environmental strain, which is already a model bacterium for studying metabolism and adaptation. The results showed that this bacterium was able to tolerate high doses of the herbicide (10× field rate, and completely degraded mesotrione after 3 h of exposure, as determined by a High Performance Liquid Chromatography. Growth rates in the presence of mesotrione were lower than in the control, prior to the period of degradation, showing toxic effects of this herbicide on bacterial cells. Changes in the saturation of the membrane lipids reduced the damage caused by reactive oxygen species and possibly hindered the entry of xenobiotics in the cell, while activating glutathione-S-transferase enzyme in the antioxidant system and in the metabolizing process of the herbicide. Considering that E. coli DH5-α is a non-environmental strain and it had no previous contact with mesotrione, the defense system found in this strain could be considered non-specific. This bacterium system response may be a general adaptation mechanism by which bacterial strains resist to damage from the presence of herbicides in agricultural soils.

Mycobacterium tuberculosis infection causes different levels of apoptosis and necrosis in human macrophages and alveolar epithelial cells.

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Danelishvili, Lia; McGarvey, Jeffery; Li, Yong-Jun; Bermudez, Luiz E

2003-09-01

Mycobacterium tuberculosis interacts with macrophages and epithelial cells in the alveolar space of the lung, where it is able to invade and replicate in both cell types. M. tuberculosis-associated cytotoxicity to these cells has been well documented, but the mechanisms of host cell death are not well understood. We examined the induction of apoptosis and necrosis of human macrophages (U937) and type II alveolar epithelial cells (A549) by virulent (H37Rv) and attenuated (H37Ra) M. tuberculosis strains. Apoptosis was determined by both enzyme-linked immunosorbent assay (ELISA) and TdT-mediated dUTP nick end labelling (TUNEL) assay, whereas necrosis was evaluated by the release of lactate dehydrogenase (LDH). Both virulent and attenuated M. tuberculosis induced apoptosis in macrophages; however, the attenuated strain resulted in significantly more apoptosis than the virulent strain after 5 days of infection. In contrast, cytotoxicity of alveolar cells was the result of necrosis, but not apoptosis. Although infection with M. tuberculosis strains resulted in apoptosis of 14% of the cells on the monolayer, cell death associated with necrosis was observed in 59% of alveolar epithelial cells after 5 days of infection. Infection with M. tuberculosis suppressed apoptosis of alveolar epithelial cells induced by the kinase inhibitor, staurosporine. Because our findings suggest that M. tuberculosis can modulate the apoptotic response of macrophages and epithelial cells, we carried out an apoptosis pathway-specific cDNA microarray analysis of human macrophages and alveolar epithelial cells. Whereas the inhibitors of apoptosis, bcl-2 and Rb, were upregulated over 2.5-fold in infected (48 h) alveolar epithelial cells, the proapoptotic genes, bad and bax, were downregulated. The opposite was observed when U937 macrophages were infected with M. tuberculosis. Upon infection of alveolar epithelial cells with M. tuberculosis, the generation of apoptosis, as determined by the

Effect of growth conditions on production of rhamnose-containing cell wall and capsular polysaccharides by strains of Lactobacillus casei subsp. rhamnosus.

Science.gov (United States)

Wicken, A J; Ayres, A; Campbell, L K; Knox, K W

1983-01-01

Strains of Lactobacillus casei subsp. rhamnosus possessing two cell wall polysaccharides, a hexosamine-containing H-polysaccharide and a rhamnose-containing R-polysaccharide, were examined for the effect of growth conditions on the production of these two components. In strain NCTC 6375, R- and H-polysaccharides accounted for an estimated 44 and 20%, respectively, of the cell wall for organisms grown in batch culture with glucose as the carbohydrate source. Growth on fructose-containing media reduced the amount of R-polysaccharide by approximately 50% without affecting the amount of H-polysaccharide. Subculture of fructose-grown organisms in glucose restored the original proportions of the two polysaccharides. Galactose- and sucrose-grown cells behaved similarly to glucose-grown cells with respect to polysaccharide production, whereas growth in rhamnose or ribose showed values close to those for fructose-grown cells. Continuous culture of strain NCTC 6375 for more than 100 generations showed a gradual and irreversible reduction of the R-polysaccharide to less than 5% of the cell wall and an increase of the H-polysaccharide to 40% of the cell wall. Other type culture strains of L. casei subsp. rhamnosus, NCIB 7473 and ATCC 7469, behaved similarly in batch and continuous culture. In contrast, strains of L. casei subsp. rhamnosus isolated at the Institute of Dental Research showed phenotypic stability with respect to the relative proportions of R- and H-polysaccharides in both batch and continuous culture. Changes in polysaccharide composition of type culture strains were also mirrored in changes in the immunogenicity of the two components and resistance to the rate of enzymic lysis of whole organisms. For L. casei subsp. rhamnosus strain NCTC 10302 the R-polysaccharide is present entirely as capsular material. The amount of R-polysaccharide produced was also markedly dependent on the carbohydrate component of the medium in batch culture and both dilution rate and

Comparative cytotoxicity study of nicotine and cotinine on MRC-5 cell line

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Ana-Maria Vlasceanu

2018-04-01

Full Text Available Nicotine has several health hazards regarding carcinogenic potential. It also imparts increased risk for respiratory, cardiovascular, and gastrointestinal disorders. Several mechanisms have been proposed for the carcinogenic potential, including effects on cell proliferation, inducing oxidative stress, DNA mutation, or inhibition of apoptosis. The cotinine metabolite is generally thought to have effects similar to nicotine in some experimental systems. The purpose of this study was to assess the nicotine and cotinine cytotoxicity on MRC-5 lung fibroblasts. The pulmonary fibroblasts were treated with various concentrations of nicotine or cotinine (in the range 1 µM – 2 mM for 24 or 48 h and analyzed for cell viability by MTT test. The results indicated that high nicotine concentrations (2 mM induced marked cell death (about 50% in MRC-5 cell line. Cotinine showed lower toxicity than nicotine on the MRC-5 cells. In contrast to nicotine treatment, cells treated with cotinine continued to proliferate after the 48h incubation period.

Transport of EDTA into cells of the EDTA-degrading bacterial strain DSM 9103.

Science.gov (United States)

Witschel, M; Egli, T; Zehnder, A J; Wehrli, E; Spycher, M

1999-04-01

In the bacterial strain DSM 9103, which is able to grow with the complexing agent EDTA as the sole source of carbon, nitrogen and energy, the transport of EDTA into whole cells was investigated. EDTA uptake was found to be dependent on speciation: free EDTA and metal-EDTA complexes with low stability constants were readily taken up, whereas those with stability constants higher than 1016 were not transported. In EDTA-grown cells, initial transport rates of CaEDTA showed substrate-saturation kinetics with a high apparent affinity for CaEDTA (affinity constant Kt= 0.39 microM). Several uncouplers had an inhibitory effect on CaEDTA transport. CaEDTA uptake was also significantly reduced in the presence of an inhibitor of ATPase and the ionophore nigericin, which dissipates the proton gradient. Valinomycin, however, which affects the electrical potential, had little effect on uptake, indicating that EDTA transport is probably driven by the proton gradient. Of various structurally related compounds tested only Ca2+-complexed diethylenetriaminepentaacetate (CaDTPA) competitively inhibited CaEDTA transport. Uptake in fumarate-grown cells was low compared to that measured in EDTA-grown bacteria. These results strongly suggest that the first step in EDTA degradation by strain DSM 9103 consists of transport by an inducible energy-dependent carrier. Uptake experiments with 45Ca2+ in the presence and absence of EDTA indicated that Ca2+ is transported together with EDTA into the cells. In addition, these transport studies and electron-dispersive X-ray analysis of electron-dense intracellular bodies present in EDTA-grown cells suggest that two mechanisms acting simultaneously allow the cells to cope with the large amounts of metal ions taken up together with EDTA. In one mechanism the metal ions are excreted, in the other they are inactivated intracellularly in polyphosphate granules.

Four-point bending protocols to study the effects of dynamic strain in osteoblastic cells in vitro.

Science.gov (United States)

Galea, Gabriel L; Price, Joanna S

2015-01-01

Strain engendered within bone tissue by mechanical loading of the skeleton is a major influence on the processes of bone modeling and remodeling and so a critical determinant of bone mass and architecture. The cells best placed to respond to strain in bone tissue are the resident osteocytes and osteoblasts. To address the mechanisms of strain-related responses in osteoblast-like cells, our group uses both in vivo and in vitro approaches, including a system of four-point bending of the substrate on which cells are cultured. A range of cell lines can be studied using this system but we routinely compare their responses to those in primary cultures of osteoblast-like cells derived from explants of mouse long bones. These cells show a range of well-characterized responses to physiological levels of strain, including increased proliferation, which in vivo is a feature of the osteogenic response.

Anti-Inflammatory Lactobacillus rhamnosus CNCM I-3690 Strain Protects against Oxidative Stress and Increases Lifespan in Caenorhabditis elegans

Science.gov (United States)

Grompone, Gianfranco; Martorell, Patricia; Llopis, Silvia; González, Núria; Genovés, Salvador; Mulet, Ana Paula; Fernández-Calero, Tamara; Tiscornia, Inés; Bollati-Fogolín, Mariela; Chambaud, Isabelle; Foligné, Benoit; Montserrat, Agustín; Ramón, Daniel

2012-01-01

Numerous studies have shown that resistance to oxidative stress is crucial to stay healthy and to reduce the adverse effects of aging. Accordingly, nutritional interventions using antioxidant food-grade compounds or food products are currently an interesting option to help improve health and quality of life in the elderly. Live lactic acid bacteria (LAB) administered in food, such as probiotics, may be good antioxidant candidates. Nevertheless, information about LAB-induced oxidative stress protection is scarce. To identify and characterize new potential antioxidant probiotic strains, we have developed a new functional screening method using the nematode Caenorhabditis elegans as host. C. elegans were fed on different LAB strains (78 in total) and nematode viability was assessed after oxidative stress (3 mM and 5 mM H2O2). One strain, identified as Lactobacillus rhamnosus CNCM I-3690, protected worms by increasing their viability by 30% and, also, increased average worm lifespan by 20%. Moreover, transcriptomic analysis of C. elegans fed with this strain showed that increased lifespan is correlated with differential expression of the DAF-16/insulin-like pathway, which is highly conserved in humans. This strain also had a clear anti-inflammatory profile when co-cultured with HT-29 cells, stimulated by pro-inflammatory cytokines, and co-culture systems with HT-29 cells and DC in the presence of LPS. Finally, this Lactobacillus strain reduced inflammation in a murine model of colitis. This work suggests that C. elegans is a fast, predictive and convenient screening tool to identify new potential antioxidant probiotic strains for subsequent use in humans. PMID:23300685

Anti-inflammatory Lactobacillus rhamnosus CNCM I-3690 strain protects against oxidative stress and increases lifespan in Caenorhabditis elegans.

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Gianfranco Grompone

Full Text Available Numerous studies have shown that resistance to oxidative stress is crucial to stay healthy and to reduce the adverse effects of aging. Accordingly, nutritional interventions using antioxidant food-grade compounds or food products are currently an interesting option to help improve health and quality of life in the elderly. Live lactic acid bacteria (LAB administered in food, such as probiotics, may be good antioxidant candidates. Nevertheless, information about LAB-induced oxidative stress protection is scarce. To identify and characterize new potential antioxidant probiotic strains, we have developed a new functional screening method using the nematode Caenorhabditis elegans as host. C. elegans were fed on different LAB strains (78 in total and nematode viability was assessed after oxidative stress (3 mM and 5 mM H(2O(2. One strain, identified as Lactobacillus rhamnosus CNCM I-3690, protected worms by increasing their viability by 30% and, also, increased average worm lifespan by 20%. Moreover, transcriptomic analysis of C. elegans fed with this strain showed that increased lifespan is correlated with differential expression of the DAF-16/insulin-like pathway, which is highly conserved in humans. This strain also had a clear anti-inflammatory profile when co-cultured with HT-29 cells, stimulated by pro-inflammatory cytokines, and co-culture systems with HT-29 cells and DC in the presence of LPS. Finally, this Lactobacillus strain reduced inflammation in a murine model of colitis. This work suggests that C. elegans is a fast, predictive and convenient screening tool to identify new potential antioxidant probiotic strains for subsequent use in humans.

Stretching of red blood cells at high strain rates

Science.gov (United States)

Mancuso, J. E.; Ristenpart, W. D.

2017-10-01

Most work on the mechanical behavior of red blood cells (RBCs) in flow has focused on simple shear flows. Relatively little work has examined RBC deformations in the physiologically important extensional flow that occurs at the entrance to a constriction. In particular, previous work suggests that RBCs rapidly stretch out and then retract upon entering the constriction, but to date no model predicts this behavior for the extremely high strain rates typically experienced there. In this Rapid Communication, we use high speed video to perform systematic measurements of the dynamic stretching behavior of RBCs as they enter a microfluidic constriction. We demonstrate that both the Kelvin-Voigt and Skalak viscoelastic models capture the observed stretching dynamics, up to strain rates as high as 2000 s-1. The results indicate that the effective elastic modulus of the RBC membrane at these strain rates is an order of magnitude larger than moduli measured by micropipette aspiration or other low strain rate techniques.

Blood cell labeling with technetium-99m, (2)

International Nuclear Information System (INIS)

Uchida, Tatsumi; Yoshida, Hiroshi; Matsuda, Shin; Kimura, Hideo; Miura, Nobuo

1978-01-01

Using a labeling method with sup(99m)Tc-pertechnetate to red blood cells (RBC), circulating blood volume was measured in comparison with that from 51 Cr-labeled RBC method. The technique is easier than already published methods, because CIS kit for sup(99m)Tc-RBC labeling (TCK-11) became to be available recently. Two mls of ACD-anticoagulated blood were withdrawn and 0.5 ml of reducing reagent prepared just before use was added to blood, waiting 5 minutes and discarding the serum after centrifugation, then adding 100 μCi of sup(99m)Tc. After washing the labeled cells by isotonic saline, cells were re-suspended in 10 ml of saline and injected to the subject. Blood specimen was obtained 10, 30, 60 and 120 minutes after infusion and blood volume was calculated by the usual way. Circulating blood volume by sup(99m)Tc was well correlated with that by 51 Cr (=0.98, p 0.01), however, the value calculated from sup(99m)Tc were 4.8 percent higher than those by 51 Cr, which suggested the elution of sup(99m)Tc from labeled RBC. sup(99m)Tc method has the advantages that higher radioactivity can be obtained in small amount of blood, which is useful in the determination of blood volume in children or in small animals in the laboratory. The measurement of blood volume of the mouse was done by using sup(99m)Tc method. The results were 1.70 +- 0.06 ml (6.35 +- 0.18%/gm), which coincided with the values reported previously. Because of it's short half life and low radiation dosage to the patients, sup(99m)Tc method will be recommended in the field of pediatrics or in patients with polycythemia or congestive heart failure, who are requested the repeated measurement of blood volume. (auth.)

Antibodies induced by vaccination with purified chick embryo cell culture vaccine (PCECV) cross-neutralize non-classical bat lyssavirus strains.

Science.gov (United States)

Malerczyk, Claudius; Selhorst, Thomas; Tordo, Noël; Moore, Susan; Müller, Thomas

2009-08-27

Tissue-culture vaccines like purified chick embryo cell vaccine (PCECV) have been shown to provide protection against classical rabies virus (RABV) via pre-exposure or post-exposure prophylaxis. A cross-neutralization study was conducted using a panel of 100 human sera, to determine, to what extent after vaccination with PCECV protection exists against non-classical bat lyssavirus strains like European bat lyssavirus (EBLV) type 1 and 2 and Australian bat lyssavirus (ABLV). Virus neutralizing antibody (VNA) concentrations against the rabies virus variants CVS-11, ABLV, EBLV-1 and EBLV-2 were determined by using a modified rapid fluorescent focus inhibition test. For ABLV and EBLV-2, the comparison to CVS-11 revealed almost identical results (100% adequate VNA concentrations >or=0.5 IU/mL; correlation coefficient r(2)=0.69 and 0.77, respectively), while for EBLV-1 more scattering was observed (97% adequate VNA concentrations; r(2)=0.50). In conclusion, vaccination with PCECV produces adequate VNA concentrations against classical RABV as well as non-classical lyssavirus strains ABLV, EBLV-1, and EBLV-2.

Development of high temperature strain gage, (5)

International Nuclear Information System (INIS)

Yuuki, Hiroshi; Kobayashi, Yukio; Kanai, Kenji; Yamaura, Yoshio

1976-01-01

Development and improvement of resistance wire type strain gages usable for experimental measurement of thermal strains generated at high temperature in various structures and equipments that consist of a Fast Breeder Reactor have been carried out, and various characteristics of the strain gages have been investigated. Based on the results obtained up to now, development and research of this time mainly aim to improve strain and fatigue characteristics. As the results, characteristics of strain gages with sensing elements of nichrome V are improved, specifically mechanical hysteresis is decreased, strain limit is increased, etc. Also, improvement is recognized in thermal output, and it becomes clear that dummy gages work effectively. However, a filling method of MgO and an inserting method of active-dummy elements are selected as primary objects to improve strain characteristics, and many hours are taken for these objects, so confirmations of characteristics of platinum-tungsten strain gages, strain sensing elements of which are troublesome to produce, have not been completely done, though the performance of the gages has been improved in several points. As to nichrome V strain gages, there is a fair prospect of obtaining ones, specifications of which are quite close to the goal, though problems in manufacturing technics remain for future. As to platinum-tungsten strain gages, it is expected that similar strain gages to nichrome V are obtainable by improvement in manufacturing of sensing elements. (auth.)

Production of biosurfactant on crude date syrup under saline conditions by entrapped cells of Natrialba sp. strain E21, an extremely halophilic bacterium isolated from a solar saltern (Ain Salah, Algeria).

Science.gov (United States)

Kebbouche-Gana, Salima; Gana, Mohamed Lamine; Ferrioune, Imen; Khemili, Souad; Lenchi, Nesrine; Akmouci-Toumi, Sihem; Bouanane-Darenfed, Nabila Amel; Djelali, Nacer-Eddine

2013-11-01

A bacterial strain E21 was isolated from a sample of water collected in the salt lake located close to Ain Salah, Algeria. The analysis of 16S rRNA gene sequence had indicated that the strain had 93 % sequence similarity with the genus Natrialba sp. strain E21 (GenBank, FR750525.1) and was considered extremely halophilic. Production of biosurfactant by the strain E21 with free and entrapped cells was investigated using soluble starch in the saline conditions. Biosurfactant synthesis was followed by measuring the surface tension and emulsifying index 9 days under optimal conditions (40 °C, pH 7). Some diffusional limitations in alginate and agar beads affected the kinetics of biosurfactant production when compared to that obtained with free cells culture. The minimum values of surface tension were 27 and 30 mN m(-1) achieved after 9 days with free and immobilized cells, respectively, while the corresponding maximum E24 values were 65.3 and 62.3 %, respectively. The re-use of bacterial cells along with the limited cell losses provided by the immobilized system might lead to significant reduction of the biosurfactant production cost.

Optimization and validation of a neutralizing antibody assay for HIV-1 in A3R5 cells.

Science.gov (United States)

Sarzotti-Kelsoe, Marcella; Daniell, Xiaoju; Todd, Christopher A; Bilska, Miroslawa; Martelli, Amanda; LaBranche, Celia; Perez, Lautaro G; Ochsenbauer, Christina; Kappes, John C; Rountree, Wes; Denny, Thomas N; Montefiori, David C

2014-07-01

A3R5 is a human CD4(+) lymphoblastoid cell line that was engineered to express CCR5 and is useful for the detection of weak neutralizing antibody responses against tier 2 strains of HIV-1. Here we describe the optimization and validation of the HIV-1 neutralizing antibody assay that utilizes A3R5 cells, performed in compliance with Good Clinical Laboratory Practice (GCLP) guidelines. The assay utilizes Renilla luciferase-expressing replication competent infectious molecular clones (IMC) encoding heterologous env genes from different HIV-1 clades. Key assay validation parameters tested included specificity, accuracy, precision, limit of detection and quantitation, specificity, linearity and range, and robustness. Plasma samples demonstrated higher non-specific activity than serum samples in the A3R5 assay. This assay can tolerate a wide range of virus input but is more sensitive to cell concentration. The higher sensitivity of the A3R5 assay in neutralization responses to tier 2 strains of HIV-1 makes it complementary to, but not a substitute for the TZM-bl assay. The validated A3R5 assay is employed as an endpoint immunogenicity test for vaccine-elicited neutralizing antibodies against tier 2 strains of HIV-1, and to identify correlates of protection in HIV-1 vaccine trials conducted globally. Copyright © 2014 Elsevier B.V. All rights reserved.

Proteomic profile of culture filtrate from the Brazilian vaccine strain Mycobacterium bovis BCG Moreau compared to M. bovis BCG Pasteur

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Degrave Wim M

2011-04-01

Full Text Available Abstract Background Bacille Calmette-Guerin (BCG is currently the only available vaccine against tuberculosis (TB and comprises a heterogeneous family of sub-strains with genotypic and phenotypic differences. The World Health Organization (WHO affirms that the characterization of BCG sub-strains, both on genomic and proteomic levels, is crucial for a better comprehension of the vaccine. In addition, these studies can contribute in the development of a more efficient vaccine against TB. Here, we combine two-dimensional electrophoresis (2DE and mass spectrometry to analyse the proteomic profile of culture filtrate proteins (CFPs from M. bovis BCG Moreau, the Brazilian vaccine strain, comparing it to that of BCG Pasteur. CFPs are considered of great importance given their dominant immunogenicity and role in pathogenesis, being available for interaction with host cells since early infection. Results The 2DE proteomic map of M. bovis BCG Moreau CFPs in the pH range 3 - 8 allowed the identification of 158 spots corresponding to 101 different proteins, identified by MS/MS. Comparison to BCG Pasteur highlights the great similarity between these BCG strains. However, quantitative analysis shows a higher expression of immunogenic proteins such as Rv1860 (BCG1896, Apa, Rv1926c (BCG1965c, Mpb63 and Rv1886c (BCG1923c, Ag85B in BCG Moreau when compared to BCG Pasteur, while some heat shock proteins, such as Rv0440 (BCG0479, GroEL2 and Rv0350 (BCG0389, DnaK, show the opposite pattern. Conclusions Here we report the detailed 2DE profile of CFPs from M. bovis BCG Moreau and its comparison to BCG Pasteur, identifying differences that may provide relevant information on vaccine efficacy. These findings contribute to the detailed characterization of the Brazilian vaccine strain against TB, revealing aspects that may lead to a better understanding of the factors leading to BCG's variable protective efficacy against TB.

Strain induced magnetism in La0.5Ca0.5MnO3 systems

Energy Technology Data Exchange (ETDEWEB)

Tietze, Thomas; Nolle, Daniela; Schuetz, Gisela; Goering, Eberhard [Max-Planck-Institut fuer Metallforschung, Stuttgart (Germany); Aydogdu, Guelguen; Habermeier, Hanns-Ulrich [Max-Planck-Institut fuer Festkoerperforschung, Stuttgart (Germany)

2009-07-01

The La{sub 1-x}Ca{sub x}MnO{sub 3} exhibits, besides the double exchange relate magneto resistive effects, many interesting properties as a function of the doping level. Depending on the doping level x the system may exhibit ferromagnetism, antiferromagnetism, orbital ordering, and charge ordering. Epitaxial tensile and compressive strains are very important for the fine tuning of the lattice degree of freedom and therefore for the magnetic nearest neighbor coupling. By *adjusting* tensile and compressive strain with corresponding substrates one can switch between FM and AFM coupling between the FM ordered ab-planes. In order to investigate the influence of different substrates and relaxation effects element specific XMCD measurements were performed on La{sub 0.5}Ca{sub 0.5}MnO{sub 3} systems.

Mouse consomic strains: Exploiting genetic divergence between Mus m. musculus and Mus m. domesticus subspecies

Czech Academy of Sciences Publication Activity Database

Gregorová, Soňa; Divina, Petr; Storchová, Radka; Trachtulec, Zdeněk; Fotopulosová, Vladana; Svenson, K.L.; Donahue, K.L.; Paigen, B.; Forejt, Jiří

2008-01-01

Roč. 18, č. 3 (2008), s. 509-515 ISSN 1088-9051 R&D Projects: GA MŠk(CZ) 1M0520; GA ČR(CZ) GA301/07/1264 Grant - others:HHMI(US) HHMI55000306; NIH(US) 1R01HG00318; EC(XE) AnEUploidy 037627 Institutional research plan: CEZ:AV0Z50520514 Keywords : complex trait loci (QTLs) * chromosome substitution strains * PWD/Ph Subject RIV: EB - Genetics ; Molecular Biology Impact factor: 10.176, year: 2008

Control of RFM strain endogenous retrovirus in RFM mouse cells

International Nuclear Information System (INIS)

Tennant, R.W.; Otten, J.A.; Wang, T.W.; Liou, R.S.; Brown, A.; Yang, W.K.

1983-01-01

RFM/Un mice express an endogenous type C retrovirus throughout their life span in many tissues; primary or established embryo fibroblast cell cultures do not express a virus but can be induced by exposure to 5-iodo-2'-deoxyuridine. All of our sources yielded a single ecotropic virus (RFV) which appeared to be related more closely to the endogenous N-tropic virus (WN1802N) of BALB/c mice than to Gross leukemia virus on the basis of two-dimensional gel electropherograms of virion proteins. No xenotropic or recombinant viruses were isolated by cocultivation techniques. RFV is N-tropic, and RFM/Un cells possess the Fv-1/sup n/ allele, as indicated by restriction of B-tropic virus and susceptibility to Gross strain N-tropic virus. However, RFM cells are highly resistant to RFV and other endogenous N-tropic viruses. This resistance is expressed by two-hit titration kinetics and by inhibition of viral linear duplex DNA formation. This is similar to the effects of the Fv-1 locus, but preliminary work has shown no apparent genetic linkage between the two restrictions. The relative strength of the restriction, the presence of a single class of ecotropic virus, and the absence of recombinant viruses suggest that in RFM mice virus is expressed only in cells in which it is induced and not by cell-to-cell transmission

Bacterial degradation of m-nitrobenzoic acid.

OpenAIRE

Nadeau, L J; Spain, J C

1995-01-01

Pseudomonas sp. strain JS51 grows on m-nitrobenzoate (m-NBA) with stoichiometric release of nitrite. m-NBA-grown cells oxidized m-NBA and protocatechuate but not 3-hydroxybenzoate, 4-hydroxy-3-nitrobenzoate, 4-nitrocatechol, and 1,2,4-benzenetriol. Protocatechuate accumulated transiently when succinate-grown cells were transferred to media containing m-NBA. Respirometric experiments indicated that the conversion of m-NBA to protocatechuate required 1 mol of oxygen per mol of substrate. Conver...

Orthopoxvirus species and strain differences in cell entry

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Bengali, Zain; Satheshkumar, P.S. [Laboratory of Viral Diseases, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, MD 20892-3210 (United States); Moss, Bernard, E-mail: bmoss@nih.gov [Laboratory of Viral Diseases, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, MD 20892-3210 (United States)

2012-11-25

Vaccinia virus (VACV) enters cells by a low pH endosomal route or by direct fusion with the plasma membrane. We previously found differences in entry properties of several VACV strains: entry of WR was enhanced by low pH, reduced by bafilomycin A1 and relatively unaffected by heparin, whereas entry of IHD-J, Copenhagen and Elstree were oppositely affected. Since binding and entry modes may have been selected by specific conditions of in vitro propagation, we now examined the properties of three distinct, recently isolated cowpox viruses and a monkeypox virus as well as additional VACV and cowpox virus strains. The recent isolates were more similar to WR than to other VACV strains, underscoring the biological importance of endosomal entry by orthopoxviruses. Sequence comparisons, gene deletions and gene swapping experiments indicated that viral determinants, other than or in addition to the A26 and A25 'fusion-suppressor' proteins, impact entry properties.

Short-period strain (0.1-105 s): Near-source strain field for an earthquake (M L 3.2) near San Juan Bautista, California

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Johnston, M. J. S.; Borcherdt, R. D.; Linde, A. T.

1986-10-01

Measurements of dilational earth strain in the frequency band 25-10-5 Hz have been made on a deep borehole strainmeter installed near the San Andreas fault. These data are used to determine seismic radiation fields during nuclear explosions, teleseisms, local earthquakes, and ground noise during seismically quiet times. Strains of less than 10-10 on these instruments can be clearly resolved at short periods (< 10 s) and are recorded with wide dynamic range digital recorders. This permits measurement of the static and dynamic strain variations in the near field of local earthquakes. Noise spectra for earth strain referenced to 1 (strain)2/Hz show that strain resolution decreases at about 10 dB per decade of frequency from -150 dB at 10-4 Hz to -223 dB at 10 Hz. Exact expressions are derived to relate the volumetric strain and displacement field for a homogeneous P wave in a general viscoelastic solid as observed on colocated dilatometers and seismometers. A rare near-field recording of strain and seismic velocity was obtained on May 26, 1984, from an earthquake (ML 3.2) at a hypocentral distance of 3.2 km near the San Andreas fault at San Juan Bautista, California. While the data indicate no precursory strain release at the 5 × 10-11 strain level, a coseismic strain release of 1.86 nanostrain was observed. This change in strain is consistent with that calculated from a simple dislocation model of the event. Ground displacement spectra, determined from the downhole strain data and instrument-corrected surface seismic data, suggest that source parameters estimated from surface recordings may be contaminated by amplification effects in near-surface low-velocity materials.

Models to understand the population-level impact of mixed strain M. tuberculosis infections.

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Sergeev, Rinat; Colijn, Caroline; Cohen, Ted

2011-07-07

-resistant strains to persist, may provide opportunities for compensatory mutations to accumulate and for relatively fit, highly drug-resistant strains of M. tuberculosis to emerge. Published by Elsevier Ltd.

Capture of cell culture-derived influenza virus by lectins: strain independent, but host cell dependent.

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Opitz, Lars; Zimmermann, Anke; Lehmann, Sylvia; Genzel, Yvonne; Lübben, Holger; Reichl, Udo; Wolff, Michael W

2008-12-01

Strategies to control influenza outbreaks are focused mainly on prophylactic vaccination. Human influenza vaccines are trivalent blends of different virus subtypes. Therefore and due to frequent antigenic drifts, strain independent manufacturing processes are required for vaccine production. This study verifies the strain independency of a capture method based on Euonymus europaeus lectin-affinity chromatography (EEL-AC) for downstream processing of influenza viruses under various culture conditions propagated in MDCK cells. A comprehensive lectin binding screening was conducted for two influenza virus types from the season 2007/2008 (A/Wisconsin/67/2005, B/Malaysia/2506/2004) including a comparison of virus-lectin interaction by surface plasmon resonance technology. EEL-AC resulted in a reproducible high product recovery rate and a high degree of contaminant removal in the case of both MDCK cell-derived influenza virus types demonstrating clearly the general applicability of EEL-AC. In addition, host cell dependency of EEL-AC was studied with two industrial relevant cell lines: Vero and MDCK cells. However, the choice of the host cell lines is known to lead to different product glycosylation profiles. Hence, altered lectin specificities have been observed between the two cell lines, requiring process adaptations between different influenza vaccine production systems.

Mycobacterial secretion systems ESX-1 and ESX-5 play distinct roles in host cell death and inflammasome activation

KAUST Repository

Abdallah, Abdallah

2011-09-28

During infection of humans and animals, pathogenic mycobacteria manipulate the host cell causing severe diseases such as tuberculosis and leprosy. To understand the basis of mycobacterial pathogenicity, it is crucial to identify the molecular virulence mechanisms. In this study, we address the contribution of ESX-1 and ESX-5 - two homologous type VII secretion systems of mycobacteria that secrete distinct sets of immune modulators - during the macrophage infection cycle. Using wild-type, ESX-1- and ESX-5-deficient mycobacterial strains, we demonstrate that these secretion systems differentially affect subcellular localization and macrophage cell responses. We show that in contrast to ESX-1, the effector proteins secreted by ESX-5 are not required for the translocation of Mycobacterium tuberculosis or Mycobacterium marinum to the cytosol of host cells. However, the M. marinum ESX-5 mutant does not induce inflammasome activation and IL-1b activation. The ESX-5 system also induces a caspase-independent cell death after translocation has taken place. Importantly, by means of inhibitory agents and small interfering RNA experiments, we reveal that cathepsin B is involved in both the induction of cell death and inflammasome activation upon infection with wild-type mycobacteria. These results reveal distinct roles for two different type VII secretion systems during infection and shed light on how virulent mycobacteria manipulate the host cell in various ways to replicate and spread. Copyright © 2011 by The American Association of Immunologists, Inc.

The biological properties of different Epstein-Barr virus strains explain their association with various types of cancers.

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Tsai, Ming-Han; Lin, Xiaochen; Shumilov, Anatoliy; Bernhardt, Katharina; Feederle, Regina; Poirey, Remy; Kopp-Schneider, Annette; Pereira, Bruno; Almeida, Raquel; Delecluse, Henri-Jacques

2017-02-07

The Epstein-Barr virus (EBV) is etiologically associated with the development of multiple types of tumors, but it is unclear whether this diversity is due to infection with different EBV strains. We report a comparative characterization of SNU719, GP202, and YCCEL1, three EBV strains that were isolated from gastric carcinomas, M81, a virus isolated in a nasopharyngeal carcinoma and several well-characterized laboratory type A strains. We found that B95-8, Akata and GP202 induced cell growth more efficiently than YCCEL1, SNU719 and M81 and this correlated positively with the expression levels of the viral BHRF1 miRNAs. In infected B cells, all strains except Akata and B95-8 induced lytic replication, a risk factor for carcinoma development, although less efficiently than M81. The panel of viruses induced tumors in immunocompromised mice with variable speed and efficacy that did not strictly mirror their in vitro characteristics, suggesting that additional parameters play an important role. We found that YCCEL1 and M81 infected primary epithelial cells, gastric carcinoma cells and gastric spheroids more efficiently than Akata or B95-8. Reciprocally, Akata and B95-8 had a stronger tropism for B cells than YCCEL1 or M81. These data suggest that different EBV strains will induce the development of lymphoid tumors with variable efficacy in immunocompromised patients and that there is a parallel between the cell tropism of the viral strains and the lineage of the tumors they induce. Thus, EBV strains can be endowed with properties that will influence their transforming abilities and the type of tumor they induce.

(4-Methoxyphenyl)(3,4,5-trimethoxyphenyl)methanone inhibits tubulin polymerization, induces G2/M arrest, and triggers apoptosis in human leukemia HL-60 cells

International Nuclear Information System (INIS)

Magalhães, Hemerson I.F.; Wilke, Diego V.; Bezerra, Daniel P.; Cavalcanti, Bruno C.; Rotta, Rodrigo; Lima, Dênis P. de; Beatriz, Adilson; Moraes, Manoel O.; Diniz-Filho, Jairo; Pessoa, Claudia

2013-01-01

(4-Methoxyphenyl)(3,4,5-trimethoxyphenyl)methanone (PHT) is a known cytotoxic compound belonging to the phenstatin family. However, the exact mechanism of action of PHT-induced cell death remains to be determined. The aim of this study was to investigate the mechanisms underlying PHT-induced cytotoxicity. We found that PHT displayed potent cytotoxicity in different tumor cell lines, showing IC 50 values in the nanomolar range. Cell cycle arrest in G 2 /M phase along with the augmented metaphase cells was found. Cells treated with PHT also showed typical hallmarks of apoptosis such as cell shrinkage, chromatin condensation, phosphatidylserine exposure, increase of the caspase 3/7 and 8 activation, loss of mitochondrial membrane potential, and internucleosomal DNA fragmentation without affecting membrane integrity. Studies conducted with isolated tubulin and docking models confirmed that PHT binds to the colchicine site and interferes in the polymerization of microtubules. These results demonstrated that PHT inhibits tubulin polymerization, arrests cancer cells in G 2 /M phase of the cell cycle, and induces their apoptosis, exhibiting promising anticancer therapeutic potential. - Highlights: • PHT inhibits tubulin polymerization. • PHT arrests cancer cells in G 2 /M phase of the cell cycle. • PHT induces caspase-dependent apoptosis

Specific toxicity of 5-thio-D-glucose to hypoxic cells

International Nuclear Information System (INIS)

Schulz, R.J.; Bongiorni, P.

1984-01-01

The toxicity of 5-thio-D-glucose (5TG) to mammalian cells in culture has been studied with respect to oxygen tension, concentration, and temperature. At 37 0 C and at 5 mM concentration of the drug in normal growth medium, survival is 10 -3 for 4-hr exposure to 5 ppm O 2 ; this increases to 0.5 for 24-hr exposure to 200 ppm O 2 . The relationship between survival and oxygen tension is nonlinear with the greatest change occurring between 50 and 100 ppm. The drug is essentially nontoxic to aerated cells. Drug toxicity increases with concentration up to about 5 mM at which point a plateau is reached. The effect of elevated temperature is to reduce the time required to obtain a specific level of survival, but temperatures as high as 42 0 C had only a slight effect on drug toxicity for oxygen tensions higher than 100 ppm. The effect of D-glucose on the toxicity of 5TG was studied, and an inverse relationship was established. At D-glucose concentrations greater than 20 mM the toxicity of 5TG was nullified regardless of oxygen tension or 5TG concentration

Benefits of combinative application of probiotic, enterocin M-producing strain Enterococcus faecium AL41 and Eleutherococcus senticosus in rabbits.

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Lauková, Andrea; Simonová, Monika Pogány; Chrastinová, Ľubica; Plachá, Iveta; Čobanová, Klaudia; Formelová, Zuzana; Chrenková, Mária; Ondruška, Ľubomír; Strompfová, Viola

2016-03-01

This study presents the effects of the probiotic and enterocin M-producing strain Enterococcus faecium AL41 on microbiota, phagocytic activity (PA), oxidative stress, performance and biochemical parameters when applied individually or in combination with Eleutherococcus senticosus in rabbits. The novelty of the study lies in the use of our non-rabbit-derived strain (AL41 = CCM8558) which produces new enterocin M. Ninety-six post-weaned rabbits (Hyplus breed) aged 5 weeks were divided into three experimental groups, 24 in each: E. senticosus (ES, 30 g/100 kg) in feed, E. faecium AL41 (10(9) CFU/mL marked by rifampicin to differentiate it from other enterococci) in water, and ES + AL. AL41 colonized sufficiently in rabbits to reduce coliforms, staphylococci, pseudomonads and clostridia. Slight decrease in bacteria was also found in the caecum and appendix. Phagocytic activity was significantly increased in the experimental groups compared to the control group (CG) (p < 0.001; p < 0.05). Applications did not evoke oxidative stress. Biochemical parameters in blood and caecal organic acids were slightly influenced. Average daily weight gain was slightly higher in ES and AL + ES. Combinative application of E. faecium with E. senticosus can be beneficial in rabbits. AL41 strain alone and in combination with ES produced reduction in spoilage bacteria; the highest stimulation of PA was in the AL41 + ES group.

Polyvinylpyrrolidone-Capped Silver Nanoparticle Inhibits Infection of Carbapenem-Resistant Strain of Acinetobacter baumannii in the Human Pulmonary Epithelial Cell

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Vishvanath Tiwari

2017-08-01

Full Text Available Acinetobacter baumannii, an opportunistic ESKAPE pathogen, causes respiratory and urinary tract infections. Its prevalence increases gradually in the clinical setup. Pathogenicity of Acinetobacter is significantly influenced by its ability to infect and survive in human pulmonary cells. Therefore, it is important to study the infection of A. baumannii in human pulmonary host cell (A-549, monitoring surface interacting and internalized bacteria. It was found that during infection of A. baumannii, about 40% bacteria adhered to A-549, whereas 20% got internalized inside pulmonary cell and induces threefold increase in the reactive oxygen species production. We have synthesized polyvinylpyrrolidone (PVP-capped AgNPs using chemical methods and tested its efficacy against carbapenem-resistant strain of A. baumannii. PVP-capped silver nanoparticles (PVP-AgNPs (30 µM have shown antibacterial activity against carbapenem-resistant strain of A. baumannii and this concentration does not have any cytotoxic effect on the human pulmonary cell line (IC50 is 130 µM. Similarly, PVP-AgNPs treatment decreases 80% viability of intracellular bacteria, decreases adherence of A. baumannii to A-549 (40 to 2.2%, and decreases intracellular concentration (20 to 1.3% of A. baumannii. This concludes that PVP-AgNPs can be developed as a substitute for carbapenem to control the infection caused by carbapenem-resistant A. baumannii.

Holographic cosmology from a system of M2–M5 branes

International Nuclear Information System (INIS)

Sepehri, Alireza; Faizal, Mir; Setare, Mohammad Reza; Ali, Ahmed Farag

2016-01-01

In this paper, we analyze the holographic cosmology using a M2–M5 brane configuration. In this configuration, a M2-brane will be placed in between a M5-brane and an anti-M5-brane. The M2-brane will act as a channel for energy to flow from an anti-M5-brane to a M5-brane, and this will increase the degrees of freedom on the M5-brane causing inflation. The inflation will end when the M5-brane and anti-M5-brane get separated. However, at a later stage the distance between the M5-brane and the anti-M5-bran can reduce and this will cause the formation of tachyonic states. These tachyonic states will again open a bridge between the M5-branes and the anti-M5-branes, which will cause further acceleration of the universe.

Holographic cosmology from a system of M2–M5 branes

Energy Technology Data Exchange (ETDEWEB)

Sepehri, Alireza, E-mail: alireza.sepehri@uk.ac.ir [Faculty of Physics, Shahid Bahonar University, P.O. Box 76175, Kerman (Iran, Islamic Republic of); Research Institute for Astronomy and Astrophysics of Maragha (RIAAM), Maragha (Iran, Islamic Republic of); Faizal, Mir, E-mail: f2mir@uwaterloo.ca [Department of Physics and Astronomy, University of Waterloo, Waterloo, ON, N2L 3G1 (Canada); Setare, Mohammad Reza, E-mail: rezakord@ipm.ir [Department of Science, Campus of Bijar, University of Kurdistan, Bijar (Iran, Islamic Republic of); Ali, Ahmed Farag, E-mail: afali@fsu.edu [Department of Physics, Florida State University, Tallahassee, FL 32306 (United States); Department of Physics, Faculty of Science, Benha University, Benha 13518 (Egypt)

2016-05-15

In this paper, we analyze the holographic cosmology using a M2–M5 brane configuration. In this configuration, a M2-brane will be placed in between a M5-brane and an anti-M5-brane. The M2-brane will act as a channel for energy to flow from an anti-M5-brane to a M5-brane, and this will increase the degrees of freedom on the M5-brane causing inflation. The inflation will end when the M5-brane and anti-M5-brane get separated. However, at a later stage the distance between the M5-brane and the anti-M5-bran can reduce and this will cause the formation of tachyonic states. These tachyonic states will again open a bridge between the M5-branes and the anti-M5-branes, which will cause further acceleration of the universe.

Heat shock protein 70 modulates neural progenitor cells dynamics in human neuroblastoma SH-SY5Y cells exposed to high glucose content.

Science.gov (United States)

Salimi, Leila; Rahbarghazi, Reza; Jafarian, Vahab; Biray Avci, Çıgır; Goker Bagca, Bakiye; Pinar Ozates, Neslihan; Khaksar, Majid; Nourazarian, Alireza

2018-01-18

In the current experiment, detrimental effects of high glucose condition were investigated on human neuroblastoma cells. Human neuroblastoma cell line SH-SY5Y were exposed to 5, 40, and 70 mM glucose over a period of 72 h. Survival rate and the proliferation of cells were analyzed by MTT and BrdU incorporation assays. Apoptosis was studied by the assays of flow cytometry and PCR array. In order to investigate the trans-differentiation capacity of the cell into mature neurons, we used immunofluorescence imaging to follow NeuN protein level. The transcription level of HSP70 was shown by real-time PCR analysis. MMP-2 and -9 activities were shown by gelatin Zymography. According to data from MTT and BrdU incorporation assay, 70 mM glucose reduced cell viability and proliferation rate as compared to control (5 mM glucose) and cells treated with 40 mM glucose (P Cell exposure to 70 mM glucose had potential to induced apoptosis after 72 h (P SH-SY5Y cells to detrimental effects of high glucose condition during trans-differentiation into mature neuron-like cells. Real-time PCR analysis confirmed the expression of HSP70 in cells under high content glucose levels, demonstrating the possible cell compensatory response to an insulting condition (p control vs 70 mM group  cells being exposed to 70 mM glucose. High glucose condition could abrogate the dynamics of neural progenitor cells. The intracellular level of HSP70 was proportional to cell damage in high glucose condition. © 2018 Wiley Periodicals, Inc.

A microfabricated strain gauge array on polymer substrate for tactile neuroprostheses in rats

International Nuclear Information System (INIS)

Beygi, M; Mutlu, S; Güçlü, B

2016-01-01

In this study, we present the design, microfabrication and characterization of a tactile sensor system which can be used for sensory neuroprostheses in rats. The sensor system consists of an array of 2  ×  7 cells, each of which has a series combination of four strain gauges. Each group of four strain gauges is placed around a square membrane with a size of 2.5  ×  2.5 mm 2 . Unlike most common tactile sensors based on silicon substrates, we used 3D-printed polylactic acid as a substrate, because it is not brittle, and under local extremes, it would prevent the catastrophic failure of all cells. The strain gauges were fabricated by depositing and patterning a 50 nm thick aluminum (Al) film on a polyimide sheet with a thickness of 0.125 mm. Polydimethylsiloxane (PDMS) elastomer was bonded on the top surface of the PI membrane. The PDMS layer was prepared in two different thicknesses, 1.2 and 1.7 mm, to investigate its effect on the static response of the sensor. The sensitivity and the maximum allowable force, corresponding to the maximum deformation of 0.9 mm at the center of each cell, changed based on the thickness of the PDMS layer. Sensor cells operated linearly up to 3 N with an average sensitivity of 200 mΩ N −1 (0.7 Ω mm −1 ) for 1.2 mm thick PDMS. These values changed to 4 N and 70 mΩ N −1 (0.3 Ω mm −1 ), respectively, for 1.7 mm thick PDMS. The nonlinearity was less than 3%. The cells had low cross-talk (∼5 mΩ N −1 and 0.02 Ω mm −1 ) relative to the average sensitivity. Additionally, the dynamic response of the sensor was characterized at several frequencies by using a vibrotactile stimulation system previously designed for psychophysics experiments. The sensor was also tested inside the rat conditioning chamber to demonstrate the relevant signals in a tactile neuroprosthesis. (paper)

A microfabricated strain gauge array on polymer substrate for tactile neuroprostheses in rats

Science.gov (United States)

Beygi, M.; Mutlu, S.; Güçlü, B.

2016-08-01

In this study, we present the design, microfabrication and characterization of a tactile sensor system which can be used for sensory neuroprostheses in rats. The sensor system consists of an array of 2  ×  7 cells, each of which has a series combination of four strain gauges. Each group of four strain gauges is placed around a square membrane with a size of 2.5  ×  2.5 mm2. Unlike most common tactile sensors based on silicon substrates, we used 3D-printed polylactic acid as a substrate, because it is not brittle, and under local extremes, it would prevent the catastrophic failure of all cells. The strain gauges were fabricated by depositing and patterning a 50 nm thick aluminum (Al) film on a polyimide sheet with a thickness of 0.125 mm. Polydimethylsiloxane (PDMS) elastomer was bonded on the top surface of the PI membrane. The PDMS layer was prepared in two different thicknesses, 1.2 and 1.7 mm, to investigate its effect on the static response of the sensor. The sensitivity and the maximum allowable force, corresponding to the maximum deformation of 0.9 mm at the center of each cell, changed based on the thickness of the PDMS layer. Sensor cells operated linearly up to 3 N with an average sensitivity of 200 mΩ N-1 (0.7 Ω mm-1) for 1.2 mm thick PDMS. These values changed to 4 N and 70 mΩ N-1 (0.3 Ω mm-1), respectively, for 1.7 mm thick PDMS. The nonlinearity was less than 3%. The cells had low cross-talk (~5 mΩ N-1 and 0.02 Ω mm-1) relative to the average sensitivity. Additionally, the dynamic response of the sensor was characterized at several frequencies by using a vibrotactile stimulation system previously designed for psychophysics experiments. The sensor was also tested inside the rat conditioning chamber to demonstrate the relevant signals in a tactile neuroprosthesis.

Adaptation of high-growth influenza H5N1 vaccine virus in Vero cells: implications for pandemic preparedness.

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Yu-Fen Tseng

Full Text Available Current egg-based influenza vaccine production technology can't promptly meet the global demand during an influenza pandemic as shown in the 2009 H1N1 pandemic. Moreover, its manufacturing capacity would be vulnerable during pandemics caused by highly pathogenic avian influenza viruses. Therefore, vaccine production using mammalian cell technology is becoming attractive. Current influenza H5N1 vaccine strain (NIBRG-14, a reassortant virus between A/Vietnam/1194/2004 (H5N1 virus and egg-adapted high-growth A/PR/8/1934 virus, could grow efficiently in eggs and MDCK cells but not Vero cells which is the most popular cell line for manufacturing human vaccines. After serial passages and plaque purifications of the NIBRG-14 vaccine virus in Vero cells, one high-growth virus strain (Vero-15 was generated and can grow over 10(8 TCID(50/ml. In conclusion, one high-growth H5N1 vaccine virus was generated in Vero cells, which can be used to manufacture influenza H5N1 vaccines and prepare reassortant vaccine viruses for other influenza A subtypes.

Predicted strains in austenitic stainless steels at stresses above yield

International Nuclear Information System (INIS)

Hammond, J.P.; Sikka, V.K.

1977-01-01

Tensile results on austenitic stainless steels were analyzed to develop means for predicting strains at stresses above yield for reactor regulatory applications. Eight heats each of types 316 and 304 were tested at 24, 93, 204, and 316 0 C as mill-annealed and at 24 0 C after reannealing. The effects of heat-to-heat variations on total strain (to 5%) at discrete stress levels were portrayed by a rational polynomial incorporating three constants that relate to the basic features of the true-stress-true-strain diagram. Because these constants usually are interrelated, a single parameter, yield strength (YS), proved adequate to predict results. For predictions analytical expressions of yield strength, an average value (YSa), and a lower bound value [YSa - 1.65SEE (standard error of estimate)] were used. Using the rational polynomial with these parameters we determined (1) limits of total maximum strain and (2) ratios of strain of material of lower bound YS to that of average YS. These are recorded at regular increments of stress [34 MPa (5 ksi)] and at ASME Code-related stresses (S/sub y), S/sub m/, 1.2S/sub m/ and 1.5S/sub m/). At intermediate stresses, strain penalties for using material of lower bound strength were large, generally larger for type 316 than type 304. For mill-annealed type 316 at 24, 93, 204, and 316 0 C, the maximum ratios of strain were 8.8, 13.0, 14.1, and 14.9, respectively, whereas for type 304 they were 3.5, 3.4, 5.6, and 4.6. At 1.5S/sub m/ and 316 0 C, a maximum strain of 2.08% was predicted for type 316 and 1.66% for type 304, as contrasted to values of 0.14 and 0.39% for average strain

Type III methyltransferase M.NgoAX from Neisseria gonorrhoeae FA1090 regulates biofilm formation and human cell invasion

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Agnieszka eKwiatek

2015-12-01

Full Text Available Neisseria gonorrhoeae is the etiological factor of the sexually transmitted gonorrhea disease that may lead, under specific conditions, to systemic infections. The gonococcal genome encodes many Restriction Modification (RM systems, which main biological role is to defend the pathogen from potentially harmful foreign DNA. However, RM systems seem also to be involved in several other functions. In this study, we examined the effect of inactivation the N. gonorrhoeae FA1090 ngo0545 gene encoding M.NgoAX methyltransferase on the global gene expression, biofilm formation, interactions with human epithelial host cells and overall bacterial growth. Expression microarrays showed at least a two-fold deregulation of a total of 121 genes in the NgoAX knock-out mutant compared to the wt strain under standard grow conditions. As determined by the assay with crystal violet, the NgoAX knock-out strain formed a slightly larger biofilm biomass per cell than the wt strain (OD570/600 = 13.8  2.24 and 9.35  2.06, respectively. SCLM observations showed that the biofilm formed by the gonococcal ngo0545 gene mutant is more relaxed and dispersed than the one formed by the wt strain. Thickness of the biofilm formed by both strains was 48.3 (14.9 µm for the mutant and 28.6 (4.0 µm for the wt. This more relaxed feature of the biofilm in respect to adhesion and bacterial interactions seems advantageous for pathogenesis of the NgoAX-deficient gonococci at the stage of human epithelial cell invasion. Indeed, the overall adhesion of mutant bacterial cells to human cells was lower than adhesion of the wt gonococci (adhesion index = 0.672 ( 0.2 and 2.15 ( 1.53, respectively; yet, a higher number of mutant than wt bacteria were found inside the Hec-1-B epithelial cells (invasion index = 3.38 ( 0.93  105 for mutant and 4.67 ( 3.09  104 for the wt strain. These results indicate that NgoAX-deficient cells have lower ability to attach to human cells

RADIOAUTOGRAPHIC DEMONSTRATION OF 5-HYDROXYTRYPTAMINE-3H UPTAKE BY PULMONARY ENDOTHELIAL CELLS

Science.gov (United States)

Strum, Judy M.; Junod, Alain F.

1972-01-01

The lung is able to rapidly remove 5-hydroxytryptamme (5-HT) from the circulation by a Na+-dependent transport mechanism. In order to identify the sites of uptake, radioautographic studies were done on rat lungs which had been isolated and perfused with 5-HT-3H and 0 5 mM iproniazid, a monoamine oxidase inhibitor. In control experiments 10-4 M imipramine was added to the perfusate to inhibit the membrane transport of 5-HT At the light microscope level, silver grains were seen concentrated near capillaries and in the endothelium of large vessels From electron microscope radioautographs a semiquantitative grain count was made and 90% of the silver grains were observed over capillary endothelial cells. The grains were found over the nucleus and cytoplasm of the cell and shewed no preferential association with any particular cytoplasmic inclusion bodies, organelles, or vesicles Other cell types were unlabeled except for a few mast cells, certain vascular smooth muscle cells, and one nerve ending. This radioautographic demonstration of the cell type responsible for the rapid removal of 5-HT from the lung circulation clearly establishes the existence of a new metabolic role for pulmonary endothelial cells. PMID:5044755

Aggregative adherent strains of Corynebacterium pseudodiphtheriticum enter and survive within HEp-2 epithelial cells

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Monica Cristina de Souza

2012-06-01

Full Text Available Corynebacterium pseudodiphtheriticum is a well-known human pathogen that mainly causes respiratory disease and is associated with high mortality in compromised hosts. Little is known about the virulence factors and pathogenesis of C. pseudodiphtheriticum. In this study, cultured human epithelial (HEp-2 cells were used to analyse the adherence pattern, internalisation and intracellular survival of the ATCC 10700 type strain and two additional clinical isolates. These microorganisms exhibited an aggregative adherence-like pattern to HEp-2 cells characterised by clumps of bacteria with a "stacked-brick" appearance. The differences in the ability of these microorganisms to invade and survive within HEp-2 cells and replicate in the extracellular environment up to 24 h post infection were evaluated. The fluorescent actin staining test demonstrated that actin polymerisation is involved in the internalisation of the C. pseudodiphtheriticum strains. The depolymerisation of microfilaments by cytochalasin E significantly reduced the internalisation of C. pseudodiphtheriticum by HEp-2 cells. Bacterial internalisation and cytoskeletal rearrangement seemed to be partially triggered by the activation of tyrosine kinase activity. Although C. pseudodiphtheriticum strains did not demonstrate an ability to replicate intracellularly, HEp-2 cells were unable to fully clear the pathogen within 24 h. These characteristics may explain how some C. pseudodiphtheriticum strains cause severe infection in human patients.

Bifidobacterium breve M-16V as a Probiotic for Preterm Infants: A Strain-Specific Systematic Review.

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Athalye-Jape, Gayatri; Rao, Shripada; Simmer, Karen; Patole, Sanjay

2017-08-01

Bifidobacterium breve M-16V has been used as a probiotic in preterm infants. Probiotic strain-specific data are essential to guide clinical practice. To assess effects of B breve M-16V in preterm neonates. A systematic review of randomized controlled trials (RCTs) and non-RCTs of B breve M-16V in preterm infants was conducted. Multiple databases, proceedings of Pediatric Academy Society, and other relevant conferences were searched in September 2016 and on January 5, 2017. Five RCTs (n = 482) and 4 non-RCTs (n = 2496) were included. Of the 5 RCTs, 4 carried high/unclear risk of bias in many domains. Meta-analysis (fixed effects model) of RCTs showed no significant benefits on stage ≥2 necrotizing enterocolitis, late-onset sepsis, mortality, and postnatal age at full feeds. Meta-analysis of non-RCTs showed significant benefits on (1) late-onset sepsis-3 studies (n = 2452), odds ratio = 0.56 (95% CI, 0.45-0.71), P breve M-16V. On Grading of Recommendations, Assessment, Development, and Evaluation analysis, the overall quality of evidence was deemed very low. Current evidence is limited regarding the potential of B breve M-16V in preterm neonates. Adequately powered, preferably cluster RCTs are needed to confirm these findings.