WorldWideScience

Sample records for isolated intact mouse

  1. Intact calcium signaling in adrenergic-deficient embryonic mouse hearts.

    Science.gov (United States)

    Peoples, Jessica N; Taylor, David G; Katchman, Alexander N; Ebert, Steven N

    2018-01-22

    Mouse embryos that lack the ability to produce the adrenergic hormones, norepinephrine (NE) and epinephrine (EPI), due to disruption of the dopamine beta-hydroxylase (Dbh -/- ) gene inevitably perish from heart failure during mid-gestation. Since adrenergic stimulation is well-known to enhance calcium signaling in developing as well as adult myocardium, and impairments in calcium signaling are typically associated with heart failure, we hypothesized that adrenergic-deficient embryonic hearts would display deficiencies in cardiac calcium signaling relative to adrenergic-competent controls at a developmental stage immediately preceding the onset of heart failure, which first appears beginning or shortly after mouse embryonic day 10.5 (E10.5). To test this hypothesis, we used ratiometric fluorescent calcium imaging techniques to measure cytosolic calcium transients, [Ca 2+ ] i in isolated E10.5 mouse hearts. Our results show that spontaneous [Ca 2+ ] i oscillations were intact and robustly responded to a variety of stimuli including extracellular calcium (5 mM), caffeine (5 mM), and NE (100 nM) in a manner that was indistinguishable from controls. Further, we show similar patterns of distribution (via immunofluorescent histochemical staining) and activity (via patch-clamp recording techniques) for the major voltage-gated plasma membrane calcium channel responsible for the L-type calcium current, I Ca,L , in adrenergic-deficient and control embryonic cardiac cells. These results demonstrate that despite the absence of vital adrenergic hormones that consistently leads to embryonic lethality in vivo, intracellular and extracellular calcium signaling remain essentially intact and functional in embryonic mouse hearts through E10.5. These findings suggest that adrenergic stimulation is not required for the development of intracellular calcium oscillations or extracellular calcium signaling through I Ca,L and that aberrant calcium signaling does not likely contribute

  2. Dihydrotestosterone activates the MAPK pathway and modulates maximum isometric force through the EGF receptor in isolated intact mouse skeletal muscle fibres.

    Science.gov (United States)

    Hamdi, M M; Mutungi, G

    2010-02-01

    It is generally believed that steroid hormones have both genomic and non-genomic (rapid) actions. Although the latter form an important component of the physiological response of these hormones, little is known about the cellular signalling pathway(s) mediating these effects and their physiological functions in adult mammalian skeletal muscle fibres. Therefore, the primary aim of this study was to investigate the non-genomic actions of dihydrotestosterone (DHT) and their physiological role in isolated intact mammalian skeletal muscle fibre bundles. Our results show that treating the fibre bundles with physiological concentrations of DHT increases both twitch and tetanic contractions in fast twitch fibres. However, it decreases them in slow twitch fibres. These changes in force are accompanied by an increase in the phosphorylation of MAPK/ERK1/2 in both fibre types and that of regulatory myosin light chains in fast twitch fibres. Both effects were insensitive to inhibitors of Src kinase, androgen receptor, insulin-like growth factor 1 receptor and platelet-derived growth factor receptor. However, they were abolished by the MAPK/ERK1/2 kinase inhibitor PD98059 and the epidermal growth factor (EGF) receptor inhibitor tyrphostin AG 1478. In contrast, testosterone had no effect on force and increased the phosphorylation of ERK1/2 in slow twitch fibres only. From these results we conclude that sex steroids have non-genomic actions in isolated intact mammalian skeletal muscle fibres. These are mediated through the EGF receptor and one of their main physiological functions is the enhancement of force production in fast twitch skeletal muscle fibres.

  3. Isolation of intact elastin fibers devoid of microfibrils.

    NARCIS (Netherlands)

    Daamen, W.F.; Hafmans, T.G.M.; Veerkamp, J.H.; Kuppevelt, A.H.M.S.M. van

    2005-01-01

    Purification protocols for elastin generally result in greatly damaged elastin fibers and this likely influences the biological response. We here describe a novel protocol for the isolation of elastin whereby the fibers stay intact, and introduce the term "elastin fiber" for intact elastic fibers

  4. Radiation-induced adaptive response in the intact mouse

    International Nuclear Information System (INIS)

    Yonezawa, Morio

    2009-01-01

    The author and coworkers have revealed that radiation adaptive response (AR) is seen also in the bone marrow of the intact mouse, of which details are described here. First, SPF ICR mice were pre-irradiated (PI) with 0-0.1 Gy of X-ray and after 2 months, subsequently irradiated (SI) with 7.75 Gy. Survival rates at 30 days after SI were about 14% in mice with PI 0-0.025 Gy whereas 40% or more in animals with PI 0.05-0.1 Gy: bone marrow death was found significantly suppressed in this effective PI dose range. The death 2 weeks after SI was found also inhibited at PI 0.3-0.5 Gy. Second, PI doses and interval between PI and SI for acquiring the radio-resistance (RR) were studied and third, the PI 0.3-0.5 Gy with SI 8.0 Gy at 9-17 days later revealed that regional PI of the head (central nervous system) was found unnecessary for RR and of abdomen (systems of hemopoiesis, immunity and digestion), essential. Fourth, strain difference of RR was shown by the fact that RR was observed only in C57BL mouse as well, but neither in BALB/c nor C3H strain. Next, at 12 days after SI 4.25-6.75 Gy (PI 0.5 Gy at 14 days before), mouse spleen cells were subjected to colony formation analysis by counting the endogenous hemopoietic stem cells, which revealed that those cells were increased to about 5 times by PI. Suppression of SI-induced hemorrhage was found in mice with PI by the decreased fecal hemoglobin content. Finally, AR was similarly studied in p53 +/+ and its knockout C57BL mice and was not found in the latter animal, indicating the participation of p53 in AR of the intact mouse. Elucidation of AR mechanisms in the intact animal seems to require somewhat different aspect from that in cells. The results were controvertible to the general concept that radiation risk is proportional to cumulative dose, suggesting that low dose radiation differs from high dose one in biological effect. (K.T.)

  5. Isolation and Properties of Intact Chromoplasts from Tomato Fruits

    OpenAIRE

    Norio, Iwatsuki; Ryuichi, Moriyama; Tadashi, Asahi; Laboratory of Biochemistry, Faculty of Agriculture, Nagoya University; Laboratory of Biochemistry, Faculty of Agriculture, Nagoya University; Laboratory of Biochemistry, Faculty of Agriculture, Nagoya University

    1984-01-01

    Intact chromoplasts were isolated from tomato fruits at different ripening stages by Percoll density gradient centrifugation. The isolated chromoplast fractions were contaminated very little by other organelles, although the fraction from fully ripened fruits contained some mitochondria and microbodies. As the transformation of chloroplasts to chromoplasts proceeded, the density of the plastids decreased from 1.096 to 1.075g・cm^ and the decrease was related to a decrease in chlorophyll and an...

  6. Isolation of intact sub-dermal secretory cavities from Eucalyptus

    Directory of Open Access Journals (Sweden)

    Goodger Jason QD

    2010-09-01

    Full Text Available Abstract Background The biosynthesis of plant natural products in sub-dermal secretory cavities is poorly understood at the molecular level, largely due to the difficulty of physically isolating these structures for study. Our aim was to develop a protocol for isolating live and intact sub-dermal secretory cavities, and to do this, we used leaves from three species of Eucalyptus with cavities that are relatively large and rich in essential oils. Results Leaves were digested using a variety of commercially available enzymes. A pectinase from Aspergillus niger was found to allow isolation of intact cavities after a relatively short incubation (12 h, with no visible artifacts from digestion and no loss of cellular integrity or cavity contents. Several measurements indicated the potential of the isolated cavities for further functional studies. First, the cavities were found to consume oxygen at a rate that is comparable to that estimated from leaf respiratory rates. Second, mRNA was extracted from cavities, and it was used to amplify a cDNA fragment with high similarity to that of a monoterpene synthase. Third, the contents of the cavity lumen were extracted, showing an unexpectedly low abundance of volatile essential oils and a sizeable amount of non-volatile material, which is contrary to the widely accepted role of secretory cavities as predominantly essential oil repositories. Conclusions The protocol described herein is likely to be adaptable to a range of Eucalyptus species with sub-dermal secretory cavities, and should find wide application in studies of the developmental and functional biology of these structures, and the biosynthesis of the plant natural products they contain.

  7. Fluorescent-protein stabilization and high-resolution imaging of cleared, intact mouse brains.

    Directory of Open Access Journals (Sweden)

    Martin K Schwarz

    Full Text Available In order to observe and quantify long-range neuronal connections in intact mouse brain by light microscopy, it is first necessary to clear the brain, thus suppressing refractive-index variations. Here we describe a method that clears the brain and preserves the signal from proteinaceous fluorophores using a pH-adjusted non-aqueous index-matching medium. Successful clearing is enabled through the use of either 1-propanol or tert-butanol during dehydration whilst maintaining a basic pH. We show that high-resolution fluorescence imaging of entire, structurally intact juvenile and adult mouse brains is possible at subcellular resolution, even following many months in clearing solution. We also show that axonal long-range projections that are EGFP-labelled by modified Rabies virus can be imaged throughout the brain using a purpose-built light-sheet fluorescence microscope. To demonstrate the viability of the technique, we determined a detailed map of the monosynaptic projections onto a target cell population in the lateral entorhinal cortex. This example demonstrates that our method permits the quantification of whole-brain connectivity patterns at the subcellular level in the uncut brain.

  8. Isolation, culture and adenoviral transduction of parietal cells from mouse gastric mucosa

    International Nuclear Information System (INIS)

    Gliddon, Briony L; Nguyen, Nhung V; Gunn, Priscilla A; Gleeson, Paul A; Driel, Ian R van

    2008-01-01

    Here we describe a method for the isolation of intact gastric glands from mice and primary culture and transfection of mouse gastric epithelial cells. Collagenase digestion of PBS-perfused mouse stomachs released large intact gastric glands that were plated on a basement membrane matrix. The heterogeneous gland cell cultures typically contain ∼60% parietal cells. Isolated mouse parietal cells remain viable in culture for up to 5 days and react strongly with an antibody specific to the gastric H + /K + ATPase. Isolated intact mouse gastric glands and primary cultures of mouse parietal cells respond to the secretagogue, histamine. Typical morphological changes from a resting to an acid-secreting active parietal cell were observed. In resting cultures of mouse parietal cells, the H + /K + ATPase displayed a cytoplasmic punctate staining pattern consistent with tubulovesicle element structures. Following histamine stimulation, an expansion of internal apical vacuole structures was observed together with a pronounced redistribution of the H + /K + ATPase from the cytoplasm to the apical vacuoles. A reproducible procedure to express genes of interest exogenously in these cultures of mouse parietal cells was also established. This method combines recombinant adenoviral transduction with magnetic field-assisted transfection resulting in ∼30% transduced parietal cells. Adenoviral-transduced parietal cells maintain their ability to undergo agonist-induced activation. This protocol will be useful for the isolation, culture and expression of genes in parietal cells from genetically modified mice and as such will be an invaluable tool for studying the complex exocytic and endocytic trafficking events of the H + /K + ATPase which underpin the regulation of acid secretion

  9. Intact fetal ovarian cord formation promotes mouse oocyte survival and development

    Directory of Open Access Journals (Sweden)

    Pera Renee

    2010-01-01

    Full Text Available Abstract Background Female reproductive potential, or the ability to propagate life, is limited in mammals with the majority of oocytes lost before birth. In mice, surviving perinatal oocytes are enclosed in ovarian follicles for subsequent oocyte development and function in the adult. Before birth, fetal germ cells of both sexes develop in clusters, or germline cysts, in the undifferentiated gonad. Upon sex determination of the fetal gonad, germ cell cysts become organized into testicular or ovarian cord-like structures and begin to interact with gonadal somatic cells. Although germline cysts and testicular cords are required for spermatogenesis, the role of cyst and ovarian cord formation in mammalian oocyte development and female fertility has not been determined. Results Here, we examine whether intact fetal ovarian germ and somatic cell cord structures are required for oocyte development using mouse gonad re-aggregation and transplantation to disrupt gonadal organization. We observed that germ cells from disrupted female gonad prior to embryonic day e13.5 completed prophase I of meiosis but did not survive following transplantation. Furthermore, re-aggregated ovaries from e13.5 to e15.5 developed with a reduced number of oocytes. Oocyte loss occurred before follicle formation and was associated with an absence of ovarian cord structure and ovary disorganization. However, disrupted ovaries from e16.5 or later were resistant to the re-aggregation impairment and supported robust oocyte survival and development in follicles. Conclusions Thus, we demonstrate a critical window of oocyte development from e13.5 to e16.5 in the intact fetal mouse ovary, corresponding to the establishment of ovarian cord structure, which promotes oocyte interaction with neighboring ovarian somatic granulosa cells before birth and imparts oocytes with competence to survive and develop in follicles. Because germline cyst and ovarian cord structures are conserved in the

  10. Beta Adrenergic Regulation of Intrapulmonary Arteriovenous Anastomoses in Intact Rat and Isolated Rat Lungs

    Directory of Open Access Journals (Sweden)

    Melissa L. Bates

    2017-04-01

    Full Text Available Intrapulmonary arteriovenous anastomoses (IPAVA allow large diameter particles of venous origin to bypass the pulmonary capillary bed and embolize the systemic arterial circulation. IPAVA have been routinely observed in healthy humans with exercise, hypoxia, and catecholamine infusion, but the mechanism by which they are recruited is not well-defined. We hypothesized that beta-adrenergic receptor stimulation recruits IPAVA and that receptor blockade would limit hypoxia-induced IPAVA recruitment. To test our hypothesis, we evaluated the transpulmonary passage of microspheres in intact rats and isolated rats lung infused with the beta-adrenergic receptor agonist isoproterenol. We also evaluated IPAVA recruitment in intact rats with hypoxia and the beta-adrenergic receptor blocker propranolol. We found that IPAVA are recruited in the intact rat by isoproterenol and their recruitment by hypoxia can be minimized by propranolol, suggesting a role for the adrenergic system in the recruitment of IPAVA by hypoxia. IPAVA recruitment is completely abolished by ventilation with 100% oxygen. Isoproterenol also recruits IPAVA in isolated rat lungs. The fact that isoproterenol can recruit IPAVA in isolated lungs, without increased pulmonary flow, suggests that elevated cardiac output is not required for IPAVA recruitment.

  11. Effect of temperature on crossbridge force changes during fatigue and recovery in intact mouse muscle fibers.

    Directory of Open Access Journals (Sweden)

    Marta Nocella

    Full Text Available Repetitive or prolonged muscle contractions induce muscular fatigue, defined as the inability of the muscle to maintain the initial tension or power output. In the present experiments, made on intact fiber bundles from FDB mouse, fatigue and recovery from fatigue were investigated at 24°C and 35°C. Force and stiffness were measured during tetani elicited every 90 s during the pre-fatigue control phase and recovery and every 1.5 s during the fatiguing phase made of 105 consecutive tetani. The results showed that force decline could be split in an initial phase followed by a later one. Loss of force during the first phase was smaller and slower at 35°C than at 24°C, whereas force decline during the later phase was greater at 35°C so that total force depression at the end of fatigue was the same at both temperatures. The initial force decline occurred without great reduction of fiber stiffness and was attributed to a decrease of the average force per attached crossbridge. Force decline during the later phase was accompanied by a proportional stiffness decrease and was attributed to a decrease of the number of attached crossbridge. Similarly to fatigue, at both 24 and 35°C, force recovery occurred in two phases: the first associated with the recovery of the average force per attached crossbridge and the second due to the recovery of the pre-fatigue attached crossbridge number. These changes, symmetrical to those occurring during fatigue, are consistent with the idea that, i initial phase is due to the direct fast inhibitory effect of [Pi]i increase during fatigue on crossbridge force; ii the second phase is due to the delayed reduction of Ca(2+ release and /or reduction of the Ca(2+ sensitivity of the myofibrils due to high [Pi]i.

  12. Isolation of Functionally Intact Rhodoplasts from Griffithsia monilis (Ceramiaceae, Rhodophyta) 1

    Science.gov (United States)

    Lilley, Ross McC.; Larkum, Anthony W. D.

    1981-01-01

    A procedure is described for isolating photosynthetically active rhodoplasts (“red algal chloroplasts”) from the marine alga Griffithsia monilis. The rhodoplasts exhibited rates of CO2 fixation and CO2-dependent O2 evolution in the order of 200 micromoles per milligram chlorophyll a per hour when illuminated with red or green light and were approximately 80% intact. The response of the rate of photosynthesis to the inorganic phosphate and pyrophosphate concentrations in the medium was qualitatively similar to that previously reported for spinach chloroplasts. Osmotically shocked rhodoplasts evolved O2 from ferricyanide in red, but not in green, light and were completely uncoupled. Rhodoplast envelope rupture appeared to be accompanied by phycobilisome loss from the thylakoids. Images PMID:16661632

  13. Isolation of tissues and preservation of RNA from intact, germinated barley grain.

    Science.gov (United States)

    Betts, Natalie S; Berkowitz, Oliver; Liu, Ruijie; Collins, Helen M; Skadhauge, Birgitte; Dockter, Christoph; Burton, Rachel A; Whelan, James; Fincher, Geoffrey B

    2017-08-01

    Isolated barley (Hordeum vulgare L.) aleurone layers have been widely used as a model system for studying gene expression and hormonal regulation in germinating cereal grains. A serious technological limitation of this approach has been the inability to confidently extrapolate conclusions obtained from isolated tissues back to the whole grain, where the co-location of several living and non-living tissues results in complex tissue-tissue interactions and regulatory pathways coordinated across the multiple tissues. Here we have developed methods for isolating fragments of aleurone, starchy endosperm, embryo, scutellum, pericarp-testa, husk and crushed cell layers from germinated grain. An important step in the procedure involves the rapid fixation of the intact grain to freeze the transcriptional activity of individual tissues while dissection is effected for subsequent transcriptomic analyses. The developmental profiles of 19 611 gene transcripts were precisely defined in the purified tissues and in whole grain during the first 24 h of germination by RNA sequencing. Spatial and temporal patterns of transcription were validated against well-defined data on enzyme activities in both whole grain and isolated tissues. Transcript profiles of genes involved in mitochondrial assembly and function were used to validate the very early stages of germination, while the profiles of genes involved in starch and cell wall mobilisation matched existing data on activities of corresponding enzymes. The data will be broadly applicable for the interrogation of co-expression and differential expression patterns and for the identification of transcription factors that are important in the early stages of grain and seed germination. © 2017 The Authors The Plant Journal © 2017 John Wiley & Sons Ltd.

  14. A cytotoxic serine proteinase isolated from mouse submandibular gland.

    Science.gov (United States)

    Shimamura, T; Nagumo, N; Ikigai, H; Murakami, K; Okubo, S; Toda, M; Ohnishi, R; Tomita, M

    1989-08-01

    We have isolated a novel cytotoxic factor from the submandibular glands of male BALB/c mice by Sephadex G-50 gel filtration chromatography and reverse-phase HPLC. The cytotoxic factor is a serine proteinase, which belongs to the mouse glandular kallikrein (mGK) family, with an Mr of approximately 27,000. The purified serine proteinase showed cytotoxic activity against mouse thymocytes in a dose-dependent manner, and a serine proteinase inhibitor, diisopropyl fluorophosphate, blocked its cytotoxic activity.

  15. Subcellular distribution of glycogen and decreased tetanic Ca2+ in fatigued single intact mouse muscle fibres

    DEFF Research Database (Denmark)

    Nielsen, Joachim; Cheng, Arthur J; Ørtenblad, Niels

    2014-01-01

    In skeletal muscle fibres, glycogen has been shown to be stored at different subcellular locations: (i) between the myofibrils (intermyofibrillar); (ii) within the myofibrils (intramyofibrillar); and (iii) subsarcolemmal. Of these, intramyofibrillar glycogen has been implied as a critical regulator...... of sarcoplasmic reticulum Ca(2+) release. The aim of the present study was to test directly how the decrease in cytoplasmic free Ca(2+) ([Ca(2+)]i) during repeated tetanic contractions relates to the subcellular glycogen distribution. Single fibres of mouse flexor digitorum brevis muscles were fatigued with 70 Hz...... in tetanic [Ca(2+)]i, and hence force, is accompanied by major reductions in inter- and intramyofibrillar glycogen. The stronger correlation between decreased tetanic [Ca(2+)]i and reduced intramyofibrillar glycogen implies that sarcoplasmic reticulum Ca(2+) release critically depends on energy supply from...

  16. Isolation of intact RNA from murine CD4+ T cells after intracellular cytokine staining and fluorescence-activated cell sorting.

    Science.gov (United States)

    Kunnath-Velayudhan, Shajo; Porcelli, Steven A

    2018-05-01

    Intracellular cytokine staining (ICS) is a powerful method for identifying functionally distinct lymphocyte subsets, and for isolating these by fluorescence activated cell sorting (FACS). Although transcriptomic analysis of cells sorted on the basis of ICS has many potential applications, this is rarely performed because of the difficulty in isolating intact RNA from cells processed using standard fixation and permeabilization buffers for ICS. To address this issue, we compared three buffers shown previously to preserve RNA in nonhematopoietic cells subjected to intracellular staining for their effects on RNA isolated from T lymphocytes processed for ICS. Our results showed that buffers containing the recombinant ribonuclease inhibitor RNasin or high molar concentrations of salt yielded intact RNA from fixed and permeabilized T cells. As proof of principle, we successfully used the buffer containing RNasin to isolate intact RNA from CD4 + T cells that were sorted by FACS on the basis of specific cytokine production, thus demonstrating the potential of this approach for coupling ICS with transcriptomic analysis. Copyright © 2018 Elsevier B.V. All rights reserved.

  17. The cag PAI is intact and functional but HP0521 varies significantly in Helicobacter pylori isolates from Malaysia and Singapore.

    Science.gov (United States)

    Schmidt, H-M A; Andres, S; Nilsson, C; Kovach, Z; Kaakoush, N O; Engstrand, L; Goh, K-L; Fock, K M; Forman, D; Mitchell, H

    2010-04-01

    Helicobacter pylori-related disease is at least partially attributable to the genotype of the infecting strain, particularly the presence of specific virulence factors. We investigated the prevalence of a novel combination of H. pylori virulence factors, including the cag pathogenicity island (PAI), and their association with severe disease in isolates from the three major ethnicities in Malaysia and Singapore, and evaluated whether the cag PAI was intact and functional in vitro. Polymerase chain reaction (PCR) was used to detect dupA, cagA, cagE, cagT, cagL and babA, and to type vacA, the EPIYA motifs, HP0521 alleles and oipA ON status in 159 H. pylori clinical isolates. Twenty-two strains were investigated for IL-8 induction and CagA translocation in vitro. The prevalence of cagA, cagE, cagL, cagT, babA, oipA ON and vacA s1 and i1 was >85%, irrespective of the disease state or ethnicity. The prevalence of dupA and the predominant HP0521 allele and EPIYA motif varied significantly with ethnicity (p < 0.05). A high prevalence of an intact cag PAI was found in all ethnic groups; however, no association was observed between any virulence factor and disease state. The novel association between the HP0521 alleles, EPIYA motifs and host ethnicity indicates that further studies to determine the function of this gene are important.

  18. Role of Aquaporin-4 in Airspace-to-Capillary Water Permeability in Intact Mouse Lung Measured by a Novel Gravimetric Method

    Science.gov (United States)

    Song, Yuanlin; Ma, Tonghui; Matthay, Michael A.; Verkman, A.S.

    2000-01-01

    The mammalian peripheral lung contains at least three aquaporin (AQP) water channels: AQP1 in microvascular endothelia, AQP4 in airway epithelia, and AQP5 in alveolar epithelia. In this study, we determined the role of AQP4 in airspace-to-capillary water transport by comparing water permeability in wild-type mice and transgenic null mice lacking AQP1, AQP4, or AQP1/AQP4 together. An apparatus was constructed to measure lung weight continuously during pulmonary artery perfusion of isolated mouse lungs. Osmotically induced water flux (Jv) between the airspace and capillary compartments was measured from the kinetics of lung weight change in saline-filled lungs in response to changes in perfusate osmolality. Jv in wild-type mice varied linearly with osmotic gradient size (4.4 × 10−5 cm3 s−1 mOsm−1) and was symmetric, independent of perfusate osmolyte size, weakly temperature dependent, and decreased 11-fold by AQP1 deletion. Transcapillary osmotic water permeability was greatly reduced by AQP1 deletion, as measured by the same method except that the airspace saline was replaced by an inert perfluorocarbon. Hydrostatically induced lung edema was characterized by lung weight changes in response to changes in pulmonary arterial inflow or pulmonary venous outflow pressure. At 5 cm H2O outflow pressure, the filtration coefficient was 4.7 cm3 s−1 mOsm−1 and reduced 1.4-fold by AQP1 deletion. To study the role of AQP4 in lung water transport, AQP1/AQP4 double knockout mice were generated by crossbreeding of AQP1 and AQP4 null mice. Jv were (cm3 s−1 mOsm−1 × 10−5, SEM, n = 7–12 mice): 3.8 ± 0.4 (wild type), 0.35 ± 0.02 (AQP1 null), 3.7 ± 0.4 (AQP4 null), and 0.25 ± 0.01 (AQP1/AQP4 null). The significant reduction in P f in AQP1 vs. AQP1/AQP4 null mice was confirmed by an independent pleural surface fluorescence method showing a 1.6 ± 0.2-fold (SEM, five mice) reduced P f in the AQP1/AQP4 double knockout mice vs. AQP1 null mice. These results establish

  19. Isolation of intact and pure chloroplasts from leaves of Arabidopsis thaliana plants acclimated to low irradiance for studies on Rubisco regulation

    Directory of Open Access Journals (Sweden)

    Magda Grabsztunowicz

    2012-11-01

    Full Text Available A protocol is presented for low-cost and fast isolation of intact and pure chloroplasts from leaves of plants acclimated to low irradiance. The protocol is based on a differential centrifugation of cleared leaf homogenate and omits a centrifugation on Percoll gradient step. The intactness and purity of the chloroplasts isolated from leaves of low irradiance-acclimated plants by using this protocol (confirmed by phase contrast microscopy as well as enzymatic and immunological approaches allows plausible studies on low irradiance-dependent Rubisco regulation.

  20. Colocalization properties of elementary Ca(2+) release signals with structures specific to the contractile filaments and the tubular system of intact mouse skeletal muscle fibers.

    Science.gov (United States)

    Georgiev, Tihomir; Zapiec, Bolek; Förderer, Moritz; Fink, Rainer H A; Vogel, Martin

    2015-12-01

    Ca(2+) regulates several important intracellular processes. We combined second harmonic generation (SHG) and two photon excited fluorescence microscopy (2PFM) to simultaneously record the SHG signal of the myosin filaments and localized elementary Ca(2+) release signals (LCSs). We found LCSs associated with Y-shaped structures of the myosin filament pattern (YMs), so called verniers, in intact mouse skeletal muscle fibers under hypertonic treatment. Ion channels crucial for the Ca(2+) regulation are located in the tubular system, a system that is important for Ca(2+) regulation and excitation-contraction coupling. We investigated the tubular system of intact, living mouse skeletal muscle fibers using 2PFM and the fluorescent Ca(2+) indicator Fluo-4 dissolved in the external solution or the membrane dye di-8-ANEPPS. We simultaneously measured the SHG signal from the myosin filaments of the skeletal muscle fibers. We found that at least a subset of the YMs observed in SHG images are closely juxtaposed with Y-shaped structures of the transverse tubules (YTs). The distances of corresponding YMs and YTs yield values between 1.3 μm and 4.1 μm including pixel uncertainty with a mean distance of 2.52±0.10 μm (S.E.M., n=41). Additionally, we observed that some of the linear-shaped areas in the tubular system are colocalized with linear-shaped areas in the SHG images. Copyright © 2015 Elsevier Inc. All rights reserved.

  1. [Isolation, purification and primary culture of adult mouse cardiac fibroblasts].

    Science.gov (United States)

    Li, Rujun; Gong, Kaizheng; Zhang, Zhengang

    2017-01-01

    Objective To establish a method for primary culture of adult mouse cardiac fibroblasts. Methods Myocardial tissues from adult mice were digested with 1 g/L trypsin and 0.8 g/L collagenase IV by oscillating water bath for a short time repeatedly. Cardiac fibroblasts and myocardial cells were isolated with differential adhesion method. Immunofluorescence staining was used to assess the purity of cardiac fibroblasts. The cell morphology was observed under an inverted phase contrast microscope. The proliferation of cardiac fibroblasts was analyzed by growth curve and CCK-8 assay. The Smad2/3 phosphorylation induced by TGF-β1 was detected by Western blotting. Results After 90 minutes of differential adhesion, adherent fibroblasts formed spherical cell mass and after 3 days, cells were spindle-shaped and proliferated rapidly. Cells were confluent after 5 days and the growth curve presented nearly "S" shape. The positive expression rate of vimentin was 95%. CCK-8 assay showed that the optimal cell proliferating activity was found from day 3 to day 5. The level of phosphorylated Smad2/3 obviously increased at the second passage induced by TGF-β1. Conclusion This method is economical and stable to isolate cardiac fibroblasts with high activity and high purity from adult mice.

  2. An optimized method for mouse liver sinusoidal endothelial cell isolation

    Energy Technology Data Exchange (ETDEWEB)

    Meyer, Jeremy, E-mail: jeremy.meyer@hcuge.ch [Division of Digestive and Transplantation Surgery, University Hospitals of Geneva, Rue Gabrielle-Perret-Gentil 4, 1211 Genève 14 (Switzerland); Unit of Surgical Research, University of Geneva, Rue Michel-Servet 1, 1206 Genève (Switzerland); Lacotte, Stéphanie, E-mail: stephanie.lacotte@unige.ch [Unit of Surgical Research, University of Geneva, Rue Michel-Servet 1, 1206 Genève (Switzerland); Morel, Philippe, E-mail: philippe.morel@hcuge.ch [Division of Digestive and Transplantation Surgery, University Hospitals of Geneva, Rue Gabrielle-Perret-Gentil 4, 1211 Genève 14 (Switzerland); Unit of Surgical Research, University of Geneva, Rue Michel-Servet 1, 1206 Genève (Switzerland); Gonelle-Gispert, Carmen, E-mail: carmen.gonelle@unige.ch [Unit of Surgical Research, University of Geneva, Rue Michel-Servet 1, 1206 Genève (Switzerland); Bühler, Léo, E-mail: leo.buhler@hcuge.ch [Division of Digestive and Transplantation Surgery, University Hospitals of Geneva, Rue Gabrielle-Perret-Gentil 4, 1211 Genève 14 (Switzerland); Unit of Surgical Research, University of Geneva, Rue Michel-Servet 1, 1206 Genève (Switzerland)

    2016-12-10

    The objective of the present study was to develop an accurate and reproducible method for liver sinusoidal endothelial cell (LSEC) isolation in mice. Non-parenchymal cells were isolated using a modified two-step collagenase digestion combined with Optiprep density gradient centrifugation. LSEC were further purified using two prevalent methods, short-term selective adherence and CD146+ magnetic-activated cell sorting (MACS), and compared in terms of cell yield, viability and purity to our purification technique using CD11b cell depletion combined with long-term selective adherence. LSEC purification using our technique allowed to obtain 7.07±3.80 million LSEC per liver, while CD146+ MACS and short-term selective adherence yielded 2.94±1.28 and 0.99±0.66 million LSEC, respectively. Purity of the final cell preparation reached 95.10±2.58% when using our method. In contrast, CD146+ MACS and short-term selective adherence gave purities of 86.75±3.26% and 47.95±9.82%, respectively. Similarly, contamination by non-LSEC was the lowest when purification was performed using our technique, with a proportion of contaminating macrophages of only 1.87±0.77%. Further, isolated cells analysed by scanning electron microscopy presented typical LSEC fenestrations organized in sieve plates, demonstrating that the technique allowed to isolate bona fide LSEC. In conclusion, we described a reliable and reproducible technique for the isolation of high yields of pure LSEC in mice. This protocol provides an efficient method to prepare LSEC for studying their biological functions. - Highlights: • This protocol provides an efficient method to prepare primary mouse LSEC for studying their biological functions. • The liver cell dispersion step was improved by performing a retrograde cannulation of the liver. • The cell yield and the purity obtained were higher than comparative techniques in mice. • Contaminating macrophages were removed by introducing a CD11b- magnetic

  3. An optimized method for mouse liver sinusoidal endothelial cell isolation

    International Nuclear Information System (INIS)

    Meyer, Jeremy; Lacotte, Stéphanie; Morel, Philippe; Gonelle-Gispert, Carmen; Bühler, Léo

    2016-01-01

    The objective of the present study was to develop an accurate and reproducible method for liver sinusoidal endothelial cell (LSEC) isolation in mice. Non-parenchymal cells were isolated using a modified two-step collagenase digestion combined with Optiprep density gradient centrifugation. LSEC were further purified using two prevalent methods, short-term selective adherence and CD146+ magnetic-activated cell sorting (MACS), and compared in terms of cell yield, viability and purity to our purification technique using CD11b cell depletion combined with long-term selective adherence. LSEC purification using our technique allowed to obtain 7.07±3.80 million LSEC per liver, while CD146+ MACS and short-term selective adherence yielded 2.94±1.28 and 0.99±0.66 million LSEC, respectively. Purity of the final cell preparation reached 95.10±2.58% when using our method. In contrast, CD146+ MACS and short-term selective adherence gave purities of 86.75±3.26% and 47.95±9.82%, respectively. Similarly, contamination by non-LSEC was the lowest when purification was performed using our technique, with a proportion of contaminating macrophages of only 1.87±0.77%. Further, isolated cells analysed by scanning electron microscopy presented typical LSEC fenestrations organized in sieve plates, demonstrating that the technique allowed to isolate bona fide LSEC. In conclusion, we described a reliable and reproducible technique for the isolation of high yields of pure LSEC in mice. This protocol provides an efficient method to prepare LSEC for studying their biological functions. - Highlights: • This protocol provides an efficient method to prepare primary mouse LSEC for studying their biological functions. • The liver cell dispersion step was improved by performing a retrograde cannulation of the liver. • The cell yield and the purity obtained were higher than comparative techniques in mice. • Contaminating macrophages were removed by introducing a CD11b- magnetic

  4. RNA transcription in isolated chloroplasts during senescence and rejuvenation of intact cotyledons of CUCURBITA PEPO L. (ZUCCHINI)

    International Nuclear Information System (INIS)

    Mishev, K.; Ananiev, E.; Denev, L.; Radeva, G.

    2006-01-01

    RNA transcription was studied in intact chloroplasts isolated from cotyledons of Cucurbita pepoL. (zucchini) during their growth and development including natural senescence and rejuvenation. Rejuvenation of cotyledons was studied after decapitation of the epicotyl above the senescing yellow cotyledons. Maximal incorporation of [32P] UTP into overall chloroplast RNA was measured two days after exposure of seedlings to light (day 6 th after the onset of germination), followed by a gradual decrease reaching minimal values at the age of 25-28 days when cotyledons began to yellow and eventually die. Rejuvenation of cotyledons completely restored chloroplast RNA synthesis and fifteen days after decapitation (at the age of 40 days), the values of chloroplast transcription even exceeded that of the maximal transcriptional activity in young cotyledons. Inhibitory analysis with tagetitoxin (a specific inhibitor of plastid encoded chloroplast RNA polymerase (PEP)) showed that in young and rejuvenated cotyledons about 85% of chloroplast RNA polymerase activity was due to PEP and only 15% corresponded to the nuclear encoded plastid RNA polymerase (NEP). Definite regions of two chloroplast encoded genes were amplified by means of PCR technique using specific DNA primers for Rubisco large subunit gene (rbcL) and the housekeeping gene for chloroplast 16S rRNA as well as chloroplast DNA as a template. The appropriate lengths of the amplified DNA fragments were checked by restriction analysis

  5. Presence of orally administered rice bran oil γ-oryzanol in its intact form in mouse plasma.

    Science.gov (United States)

    Kobayashi, Eri; Ito, Junya; Kato, Shunji; Sawada, Kazue; Matsuki, Midori; Hashimoto, Hiroyuki; Miyazawa, Teruo; Nakagawa, Kiyotaka

    2016-12-07

    Although the beneficial effects (e.g., lipid-lowering activity) of γ-oryzanol (OZ), a mixture of ferulic acid esters of plant sterols and triterpene alcohols, have been extensively investigated, few studies have evaluated the absorption and metabolism of OZ. Moreover, it is unclear whether OZ, once ingested, is directly absorbed by the intestine into the bloodstream at a sufficient level to exhibit activity. Here, we prepared OZ concentrate from purified rice bran oil (Rice Oil OZ), determined the concentration of OZ in the preparation (cycloartenyl ferulate equivalent concentration; 52.2%), and then carried out chromatography-mass spectrometry analysis of plasma samples from mice after oral administration of Rice Oil OZ. The OZ concentrations of plasma from the control (vehicle-treated) mice were low (trace levels); however, at 5 h after a single oral administration of the Rice Oil OZ (600 mg per kg body weight), the levels significantly increased, reaching 17.6 ng mL -1 for cycloartenyl ferulate, 28.2 ng mL -1 for 24-methylenecycloartanyl ferulate isomers, 15.6 ng mL -1 for campesteryl ferulate, and 5.1 ng mL -1 for β-sitosteryl ferulate, respectively, expressed in equivalence of cycloartenyl ferulate in plasma. These results provided the first mass spectrometric evidence suggesting that a portion of orally administered OZ is directly absorbed by the intestine and is present in the intact form in plasma. The presence of a significant amount of OZ in its intact form in plasma may explain the beneficial effects of OZ in vivo.

  6. The Majority of Genotypes of the Virulence Gene inlA Are Intact among Natural Watershed Isolates of Listeria monocytogenes from the Central California Coast.

    Directory of Open Access Journals (Sweden)

    Lisa Gorski

    Full Text Available Internalin A is an essential virulence gene involved in the uptake of the foodborne pathogen Listeria monocytogenes into host cells. It is intact in clinical strains and often truncated due to Premature Stop Codons (PMSCs in isolates from processed foods and processing facilities. Less information is known about environmental isolates. We sequenced the inlA alleles and did Multi Locus Variable Number Tandem Repeat Analysis (MLVA on 112 L. monocytogenes isolates from a 3-year period from naturally contaminated watersheds near a leafy green growing area in Central California. The collection contained 14 serotype 1/2a, 12 serotype 1/2b, and 86 serotype 4b strains. Twenty-seven different inlA alleles were found. Twenty-three of the alleles are predicted to encode intact copies of InlA, while three contain PMSCs. Another allele has a 9-nucleotide deletion, previously described for a clinical strain, indicating that it is still functional. Intact inlA genes were found in 101 isolates, and 8 isolates contained the allele predicted to contain the 3-amino acid deletion. Both allele types were found throughout the 3-year sampling period. Three strains contained inlA alleles with PMSCs, and these were found only during the first 3 months of the study. SNP analysis of the intact alleles indicated clustering of alleles based on serotype and lineage with serotypes 1/2b and 4b (lineage I strains clustering together, and serotype 1/2a (lineage II strains clustering separately. The combination of serotype, MLVA types, and inlA allele types indicate that the 112 isolates reflect at least 49 different strains of L. monocytogenes. The finding that 90% of environmental L. monocytogenes isolates contain intact inlA alleles varies significantly from isolates found in processing plants. This information is important to public health labs and growers as to the varieties of L. monocytogenes that could potentially contaminate fresh produce in the field by various means.

  7. Isolation of Kupffer Cells and Hepatocytes from a Single Mouse Liver

    DEFF Research Database (Denmark)

    Aparicio-Vergara, Marcela; Tencerova, Michaela; Morgantini, Cecilia

    2017-01-01

    Liver perfusion is a common technique used to isolate parenchymal and non-parenchymal liver cells for in vitro experiments. This method allows hepatic cells to be separated based on their size and weight, by centrifugation using a density gradient. To date, other methods allow the isolation of only...... one viable hepatic cellular fraction from a single mouse; either parenchymal (hepatocytes) or non-parenchymal cells (i.e., Kupffer cells or hepatic stellate cells). Here, we describe a method to isolate both hepatocytes and Kupffer cells from a single mouse liver, thereby providing the unique...... advantage of studying different liver cell types that have been isolated from the same organism....

  8. Connective tissue growth factor/CCN2-null mouse embryonic fibroblasts retain intact transforming growth factor-β responsiveness

    International Nuclear Information System (INIS)

    Mori, Yasuji; Hinchcliff, Monique; Wu, Minghua; Warner-Blankenship, Matthew; Lyons, Karen M.; Varga, John

    2008-01-01

    Background: The matricellular protein connective tissue growth factor (CCN2) has been implicated in pathological fibrosis, but its physiologic role remains elusive. In vitro, transforming growth factor-β (TGF-β) induces CCN2 expression in mesenchymal cells. Because CCN2 can enhance profibrotic responses elicited by TGF-β, it has been proposed that CCN2 functions as an essential downstream signaling mediator for TGF-β. To explore this notion, we characterized TGF-β-induced activation of fibroblasts from CCN2-null (CCN2 -/- ) mouse embryos. Methods: The regulation of CCN2 expression was examined in vivo in a model of fibrosis induced by bleomycin. Cellular TGF-β signal transduction and regulation of collagen gene expression were examined in CCN2 -/- MEFs by immunohistochemistry, Northern, Western and RT-PCR analysis, immunocytochemistry and transient transfection assays. Results: Bleomycin-induced skin fibrosis in the mouse was associated with substantial CCN2 up-regulation in lesional fibroblasts. Whereas in vitro proliferation rate of CCN2 -/- MEFs was markedly reduced compared to wild type MEFs, TGF-β-induced activation of the Smad pathways, including Smad2 phosphorylation, Smad2/3 and Smad4 nuclear accumulation and Smad-dependent transcriptional responses, were unaffected by loss of CCN2. The stimulation of COL1A2 and fibronectin mRNA expression and promoter activity, and of corresponding protein levels, showed comparable time and dose-response in wild type and CCN2 -/- MEFs, whereas stimulation of alpha smooth muscle actin and myofibroblast transdifferentiation showed subtle impairment in MEFs lacking CCN2. Conclusion: Whereas endogenous CCN2 plays a role in regulation of proliferation and TGF-β-induced myofibroblast transdifferentiation, it appears to be dispensable for Smad-dependent stimulation of collagen and extracellular matrix synthesis in murine embryonic fibroblasts

  9. A cytocidal tissue kallikrein isolated from mouse submandibular glands.

    Science.gov (United States)

    Murakami, K; Ikigai, H; Nagumo, N; Tomita, M; Shimamura, T

    1989-11-06

    A cytocidal factor against mouse thymocytes was purified from the submandibular glands of female BALB/c mice using Sephadex G-50 gel filtration chromatography and reverse-phase HPLC. SDS-PAGE and amino acid sequence analysis revealed that the cytocidal factor was mouse glandular kallikrein (mGK)-6. mGK-6 showed an optimal enzyme activity at pH 10 and a cytocidal activity against thymocytes in a dose-dependent manner.

  10. Differential effect of r-56865 on ouabain binding to isolated sarcolemma and intact atrial tissue of guinea-pig

    NARCIS (Netherlands)

    HEERS, C; SCHEUFLER, E; WILHELM, D; WERMELSKIRCHEN, D; WILFFERT, B; Peters, Thies

    1 R 56865 (N-[1-[4-(4-fluorophenoxy)-butyl]-4-piperidinyl]-N-methyl-2-benzolamine) is a compound known to antagonize cardiac glycoside intoxication. Therefore, the effect of the compound on ouabain binding to intact cardiac tissue as well as cardiac membrane preparations was investigated. 2 The

  11. Variations in Local Calcium Signaling in Adjacent Cardiac Myocytes of the Intact Mouse Heart Detected with Two-Dimensional Confocal Microscopy

    Directory of Open Access Journals (Sweden)

    Karin P Hammer

    2015-01-01

    Full Text Available Dyssynchronous local Ca release within individual cardiac myocytes has been linked to cellular contractile dysfunction. Differences in Ca kinetics in adjacent cells may also provide a substrate for inefficient contraction and arrhythmias. In a new approach we quantify variation in local Ca transients between adjacent myocytes in the whole heart.Langendorff-perfused mouse hearts were loaded with Fluo-8 AM to detect Ca and Di-4-ANEPPS to visualize cell membranes. A spinning disc confocal microscope with a fast camera allowed us to record Ca signals within an area of 465 µm by 315 µm with an acquisition speed of 55 fps. Images from multiple transients recorded at steady state were registered to their time point in the cardiac cycle to restore averaged local Ca transients with a higher temporal resolution. Local Ca transients within and between adjacent myocytes were compared with regard to amplitude, time to peak and decay at steady state stimulation (250 ms cycle length.Image registration from multiple sequential Ca transients allowed reconstruction of high temporal resolution (2.4 ±1.3ms local CaT in 2D image sets (N= 4 hearts, n= 8 regions. During steady state stimulation, spatial Ca gradients were homogeneous within cells in both directions and independent of distance between measured points. Variation in CaT amplitudes was similar across the short and the long side of neighboring cells. Variations in TAU and TTP were similar in both directions. Isoproterenol enhanced the CaT but not the overall pattern of spatial heterogeneities.Here we detected and analyzed local Ca signals in intact mouse hearts with high temporal and spatial resolution, taking into account 2D arrangement of the cells. We observed significant differences in the variation of CaT amplitude along the long and short axis of cardiac myocytes. Variations of Ca signals between neighboring cells may contribute to the substrate of cardiac remodeling.

  12. Variations in DNA synthesis and mitotic indices in hepatocytes and sinusoid litoral cells of adult intact male mouse along a circadian time span.

    Science.gov (United States)

    Surur, J M; Moreno, F R; Badrán, A F; Llanos, J M

    1985-01-01

    Variations of DNA synthesis (DNAS) and mitotic indices along a circadian time span are described in the hepatocyte and sinusoid litoral cell populations of adult intact male mouse liver. Standardized (light from 0600 to 1800) mice were killed in groups of six to nine animals, every 2-4 hr along a circadian time span. Hepatocytes show significant peaks in the synthesis of DNA and the mitotic activity at 0200 and 1400, respectively. These results correspond to those previously described by us in young immature liver, regenerating liver and hepatomas. The phase differences between these peaks and the differences between their absolute values are discussed. Also considered are the practical consequences of our findings for experimental design. The curve of DNA synthesis of sinusoid litoral cells show a peak at 0200. The mitotic index show a bimodal waveform with peaks at 0800 and 2000. The existence of four different cell populations composing the so called sinusoid litoral cells and also the migration into and out of the liver of some macrophages considered as litoral (Kupffer) cells in our counts, makes interpretation of the curves somewhat complicated and deserves further analysis.

  13. Kinetic identification of an intracellular calcium compartment sensitive to phosphate and dinitrophenol in intact isolated rabbit aorta

    International Nuclear Information System (INIS)

    Hai, C.M.; Phair, R.D.

    1986-01-01

    Previous work from this laboratory revealed the presence of at least three distinct intracellular calcium compartments in intact segments of rabbit aorta. In this study one of these intracellular compartments is shown to be sensitive to dinitrophenol and to increased extracellular phosphate. Intact aortic segments were loaded with 45 Ca in bicarbonate-buffered physiologic salt solution for 1 hour, and then transferred to a flow-through chamber perfused with physiologic salt solution. Effluent from the chamber was collected for 8 hours, and 45 Ca efflux curves were analyzed using compartmental analysis. When aortic segments were loaded and washed out in dinitrophenol, the slowest component of the efflux curve was less prominent; in high phosphate it was more prominent. The rate constant changes required to account for these data were primarily in the exchange between the cytosolic and slowest intracellular calcium compartment, suggesting that the slowest calcium compartment resolved during the 8-hour washout was mitochondrial. This compartment contained 5.4 +/- 3.2 nmol calcium/g wet wt. tissue. The calcium flux across its membranes was 0.32 +/- 0.04 nmol min-1g-1. Because this flux is much smaller than the plasma-membrane calcium flux, we suggest that, in normal physiological circumstances, plasma-membrane extrusion is more important for the removal of Ca from the smooth muscle cytosol than is uptake into this slow intracellular compartment

  14. Temperature-Sensitive Mutants of Mouse Hepatitis Virus Strain A59: Isolation, Characterization and Neuropathogenic Properties.

    NARCIS (Netherlands)

    M.J.M. Koolen (Marck); A.D.M.E. Osterhaus (Albert); G. van Steenis (Bert); M.C. Horzinek; B.A.M. van der Zeijst (Ben)

    1983-01-01

    textabstractTwenty 5-fluorouracil-induced temperature-sensitive (ts) mutants of mouse hepatitis virus strain A59 were isolated from 1284 virus clones. Mutants were preselected on the basis of their inability to induce syncytia in infected cells at the restrictive temperature (40 degrees) vs the

  15. RNA isolation for transcriptomics of human and mouse small skin biopsies

    Directory of Open Access Journals (Sweden)

    Breit Timo M

    2011-10-01

    Full Text Available Abstract Background Isolation of RNA from skin biopsies presents a challenge, due to the tough nature of skin tissue and a high presence of RNases. As we lacked the dedicated equipment, i.e. homogenizer or bead-beater, needed for the available RNA from skin isolation methods, we adapted and tested our zebrafish single-embryo RNA-isolation protocol for RNA isolation from skin punch biopsies. Findings We tested our new RNA-isolation protocol in two experiments: a large-scale study with 97 human skin samples, and a small study with 16 mouse skin samples. Human skin was sampled with 4.0 mm biopsy punches and for the mouse skin different punch diameter sizes were tested; 1.0, 1.5, 2.0, and 2.5 mm. The average RNA yield in human samples was 1.5 μg with an average RNA quality RIN value of 8.1. For the mouse biopsies, the average RNA yield was 2.4 μg with an average RIN value of 7.5. For 96% of the human biopsies and 100% of the mouse biopsies we obtained enough high-quality RNA. The RNA samples were successfully tested in a transcriptomics analysis using the Affymetrix and Roche NimbleGen platforms. Conclusions Using our new RNA-isolation protocol, we were able to consistently isolate high-quality RNA, which is apt for further transcriptomics analysis. Furthermore, this method is already useable on biopsy material obtained with a punch diameter as small as 1.5 mm.

  16. Isolation and culture of adult mouse vestibular nucleus neurons

    Science.gov (United States)

    Him, Aydın; Altuntaş, Serap; Öztürk, Gürkan; Erdoğan, Ender; Cengiz, Nureddin

    2017-12-19

    Background/aim: Isolated cell cultures are widely used to study neuronal properties due to their advantages. Although embryonic animals are preferred for culturing, their morphological or electrophysiological properties may not reflect adult neurons, which may be important in neurodegenerative diseases. This paper aims to develop a method for preparing isolated cell cultures of medial vestibular nucleus (MVN) from adult mice and describe its morphological and electrophysiological properties.Materials and methods: Vestibular nucleus neurons were mechanically and enzymatically isolated and cultured using a defined medium with known growth factors. Cell survival was measured with propidium iodide, and electrophysiological properties were investigated with current-clamp recording.Results: Vestibular neurons grew neurites in cultures, gaining adult-like morphological properties, and stayed viable for 3 days in culture. Adding bovine calf serum, nerve growth factor, or insulin-like growth factor into the culture medium enhanced neuronal viability. Current-clamp recording of the cultured neurons revealed tonic and phasic-type neurons with similar input resistance, resting membrane potential, action potential amplitude, and duration. Conclusion: Vestibular neurons from adult mice can be cultured, and regenerate axons in a medium containing appropriate growth factors. Culturing adult vestibular neurons provides a new method to study age-related pathologies of the vestibular system.

  17. Experimental mouse lethality of Escherichia coli strains isolated from free ranging Tibetan yaks.

    Science.gov (United States)

    Rehman, Mujeeb Ur; Zhang, Hui; Wang, Yajing; Mehmood, Khalid; Huang, Shucheng; Iqbal, Muhammad Kashif; Li, Jiakui

    2017-08-01

    The present study has examined the virulence potential of Escherichia coli isolates harboring at least one virulence gene (associated with ExPEC or InPEC pathotype and belonging to different phylogenetic groups: A, B1, B2 or D), isolated from free ranging Tibetan yak feces. The E. coli isolates (n = 87) were characterized for different serogroups and a mouse model of subcutaneous-infection was used to envisage the virulence within these E. coli strains. Of the 87 E. coli isolates examined, 23% of the E. coli isolates caused lethal infections in a mouse model of subcutaneous infection and were classified as killer. Moreover, the majority of the killer strains belonged to phylogroup A (65%) and serogroup O 60 or O 101 (35%). Phylogroup B1, serogroups O 60 and O 101 were statistically associated with the killer status (P 1) were observed between the killer status isolates and all other bacterial virulence traits. This study comprises the first report on the virulence potential of E. coli strains isolated from free-ranging Tibetan yaks feces. Our findings suggest that pathogenic E. coli of free ranging yaks is highly worrisome, as these feces are used as manures by farmers and therewith pose a health risk to humans upon exposure. Copyright © 2017 Elsevier Ltd. All rights reserved.

  18. The mechanism of anthracene interaction with photosynthetic apparatus: A study using intact cells, thylakoid membranes and PS II complexes isolated from Chlamydomonas reinhardtii

    International Nuclear Information System (INIS)

    Aksmann, Anna; Shutova, Tatiana; Samuelsson, Goeran; Tukaj, Zbigniew

    2011-01-01

    Intact cells of Chlamydomonas reinhardtii as well as isolated thylakoid membranes and photosystem II complexes were used to examine a possible mechanism of anthracene (ANT) interaction with the photosynthetic apparatus. Since ANT concentrations above 1 mM were required to significantly inhibit the rate of oxygen evolution in PS II membrane fragments it may indicate that the toxicant did not directly interact with this photosystem. On the other hand, stimulation of oxygen uptake by ANT-treated thylakoids suggested that ANT could either act as an artificial electron acceptor in the photosynthetic electron transport chain or function as an uncoupler. Electron transfer from excited chlorophyll to ANT is impossible due to the very low reduction potential of ANT and therefore we propose that toxic concentrations of ANT increase the thylakoid membrane permeability and thereby function as an uncoupler, enhancing electron transport in vitro. Hence, its unspecific interference with photosynthetic membranes in vitro suggests that the inhibitory effect observed on intact cell photosynthesis is caused by uncoupling of phosphorylation.

  19. Acrolein relaxes mouse isolated tracheal smooth muscle via a TRPA1-dependent mechanism.

    Science.gov (United States)

    Cheah, Esther Y; Burcham, Philip C; Mann, Tracy S; Henry, Peter J

    2014-05-01

    Airway sensory C-fibres express TRPA1 channels which have recently been identified as a key chemosensory receptor for acrolein, a toxic and highly prevalent component of smoke. TRPA1 likely plays an intermediary role in eliciting a range of effects induced by acrolein including cough and neurogenic inflammation. Currently, it is not known whether acrolein-induced activation of TRPA1 produces other airway effects including relaxation of mouse airway smooth muscle. The aims of this study were to examine the effects of acrolein on airway smooth muscle tone in mouse isolated trachea, and to characterise the cellular and molecular mechanisms underpinning the effects of acrolein. Isometric tension recording studies were conducted on mouse isolated tracheal segments to characterise acrolein-induced relaxation responses. Release of the relaxant PGE₂ was measured by EIA to examine its role in the response. Use of selective antagonists/inhibitors permitted pharmacological characterisation of the molecular and cellular mechanisms underlying this relaxation response. Acrolein induced dose-dependent relaxation responses in mouse isolated tracheal segments. Importantly, these relaxation responses were significantly inhibited by the TRPA1 antagonists AP-18 and HC-030031, an NK₁ receptor antagonist RP-67580, and the EP₂ receptor antagonist PF-04418948, whilst completely abolished by the non-selective COX inhibitor indomethacin. Acrolein also caused rapid PGE₂ release which was suppressed by HC-030031. In summary, acrolein induced a novel bronchodilator response in mouse airways. Pharmacologic studies indicate that acrolein-induced relaxation likely involves interplay between TRPA1-expressing airway sensory C-fibres, NK₁ receptor-expressing epithelial cells, and EP₂-receptor expressing airway smooth muscle cells. Copyright © 2014 Elsevier Inc. All rights reserved.

  20. Isolation of Single-Domain Antibody Fragments That Preferentially Detect Intact (146S Particles of Foot-and-Mouth Disease Virus for Use in Vaccine Quality Control

    Directory of Open Access Journals (Sweden)

    Michiel M. Harmsen

    2017-08-01

    Full Text Available Intact (146S foot-and-mouth disease virus (FMDVs can dissociate into specific (12S viral capsid degradation products. FMD vaccines normally consist of inactivated virions. Vaccine quality is dependent on 146S virus particles rather than 12S particles. We earlier isolated two llama single-domain antibody fragments (VHHs that specifically recognize 146S particles of FMDV strain O1 Manisa and shown their potential use in quality control of FMD vaccines during manufacturing. These 146S-specific VHHs were specific for particular O serotype strains and did not bind strains from other FMDV serotypes. Here, we describe the isolation of 146S-specific VHHs against FMDV SAT2 and Asia 1 strains by phage display selection from llama immune libraries. VHHs that bind both 12S and 146S particles were readily isolated but VHHs that bind specifically to 146S particles could only be isolated by phage display selection using prior depletion for 12S particles. We obtained one 146S-specific VHH—M332F—that binds to strain Asia 1 Shamir and several VHHs that preferentially bind 146S particles of SAT2 strain SAU/2/00, from which we selected VHH M379F for further characterization. Both M332F and M379F did not bind FMDV strains from other serotypes. In a sandwich enzyme-linked immunosorbent assay (ELISA employing unlabeled and biotinylated versions of the same VHH M332F showed high specificity for 146S particles but M379F showed lower 146S-specificity with some cross-reaction with 12S particles. These ELISAs could detect 146S particle concentrations as low as 2.3–4.6 µg/l. They can be used for FMD vaccine quality control and research and development, for example, to identify virion stabilizing excipients.

  1. Effects of cartap on isolated mouse phrenic nerve diaphragm and its related mechanism.

    Science.gov (United States)

    Liao, J W; Kang, J J; Liu, S H; Jeng, C R; Cheng, Y W; Hu, C M; Tsai, S F; Wang, S C; Pang, V F

    2000-06-01

    Cartap, a nereistoxin analogue pesticide, is reported to have no irritation to eyes in rabbits. However, we have demonstrated recently that cartap could actually cause acute death in rabbits via ocular exposure. Our preliminary study with isolated mouse phrenic nerve diaphragms has shown that instead of neuromuscular blockade, cartap caused muscular contracture. The objective of the study was to examine the effect of cartap on the neuromuscular junction in more detail and to investigate its possible underlying mechanism with isolated mouse phrenic nerve diaphragms and sarcoplasmic reticulum (SR) vesicles. Cartap or nereistoxin at various concentrations was added in the organ bath with isolated mouse phrenic nerve diaphragm and both nerve- and muscle-evoked twitches were recorded. Instead of blocking the neuromuscular transmission as nereistoxin did, cartap caused contracture in stimulated or quiescent isolated mouse phrenic nerve diaphragm. Both the cartap-induced muscular contracture force and the time interval to initiate the contracture were dose-dependent. The contracture induced by cartap was not affected by the pretreatment of the diaphragm with the acetylcholine receptor blocker alpha-bungarotoxin; the Na(+) channel blocker tetrodotoxin; or various Ca(2+) channel blockers, NiCl(2), verapamil, and nifedipine. On the contrary, the contracture was significantly inhibited when the diaphragm was pretreated with ryanodine or EGTA containing Ca(2+)-free Krebs solution or in combination. This suggested that both internal and extracellular Ca(2+) might participate in cartap-induced skeletal muscle contracture. Moreover, cartap inhibited the [(3)H]-ryanodine binding to the Ca(2+) release channel of SR in a dose-dependent manner. Additionally, cartap could induce a significant reduction in Ca(2+)-ATPase activity of SR vesicles at a relatively high dose. The results suggested that cartap might cause the influx of extracellular Ca(2+) and the release of internal Ca(2

  2. Pharmacological and physiological assessment of serotonin formation and degradation in isolated preparations from mouse and human hearts.

    Science.gov (United States)

    Gergs, Ulrich; Jung, Franziska; Buchwalow, Igor B; Hofmann, Britt; Simm, Andreas; Treede, Hendrik; Neumann, Joachim

    2017-12-01

    Using transgenic (TG) mice that overexpress the human serotonin (5-HT) 4a receptor specifically in cardiomyocytes, we wanted to know whether 5-HT can be formed and degraded in the mammalian heart and whether this can likewise lead to inotropic and chronotropic effects in this TG model. We noted that the 5-HT precursor 5-hydroxy-tryptophan (5-HTP) can exert inotropic and chronotropic effects in cardiac preparations from TG mice but not from wild-type (WT) mice; similar results were found in human atrial preparations as well as in intact TG animals using echocardiography. Moreover, by immunohistochemistry we could detect 5-HT metabolizing enzymes and 5-HT transporters in mouse hearts as well as in human atria. Hence, in the presence of an inhibitor of aromatic l-amino acid decarboxylase, the positive inotropic effects of 5-HTP were absent in TG and isolated human atrial preparations, and, moreover, inhibitors of enzymes involved in 5-HT degradation enhanced the efficacy of 5-HT in TG atria. A releaser of neurotransmitters increased inotropy in the isolated TG atrium, and this effect could be blocked by a 5-HT 4a receptor antagonist. Fluoxetine, an inhibitor of 5-HT uptake, elevated the potency of 5-HT to increase contractility in the TG atrium. In addition, inhibitors of organic cation and monoamine transporters apparently reduced the positive inotropic potency of 5-HT in the TG atrium. Hence, we tentatively conclude that a local production and degradation of 5-HT in the mammalian heart and more specifically in mammalian myocytes probably occurs. Conceivably, this formation of 5-HT and possibly impaired degradation may be clinically relevant in cases of unexplained tachycardia and other arrhythmias. NEW & NOTEWORTHY The present work suggests that inotropically active serotonin (5-HT) can be formed in the mouse and human heart and probably by cardiomyocytes themselves. Moreover, active degradation of 5-HT seems to occur in the mammalian heart. These findings may again

  3. Gender differences in methionine accumulation and metabolism in freshly isolated mouse hepatocytes: Potential roles in toxicity

    International Nuclear Information System (INIS)

    Dever, Joseph T.; Elfarra, Adnan A.

    2009-01-01

    L-Methionine (Met) is hepatotoxic at high concentrations. Because Met toxicity in freshly isolated mouse hepatocytes is gender-dependent, the goal of this study was to assess the roles of Met accumulation and metabolism in the increased sensitivity of male hepatocytes to Met toxicity compared with female hepatocytes. Male hepatocytes incubated with Met (30 mM) at 37 o C exhibited higher levels of intracellular Met at 0.5, 1.0, and 1.5 h, respectively, compared to female hepatocytes. Conversely, female hepatocytes had higher levels of S-adenosyl-L-methionine compared to male hepatocytes. Female hepatocytes also exhibited higher L-methionine-L-sulfoxide levels relative to control hepatocytes, whereas the increases in L-methionine-D-sulfoxide (Met-D-O) levels were similar in hepatocytes of both genders. Addition of aminooxyacetic acid (AOAA), an inhibitor of Met transamination, significantly increased Met levels at 1.5 h and increased Met-D-O levels at 1.0 and 1.5 h only in Met-exposed male hepatocytes. No gender differences in cytosolic Met transamination activity by glutamine transaminase K were detected. However, female mouse liver cytosol exhibited higher methionine-DL-sulfoxide (MetO) reductase activity than male mouse liver cytosol at low (0.25 and 0.5 mM) MetO concentrations. Collectively, these results suggest that increased cellular Met accumulation, decreased Met transmethylation, and increased Met and MetO transamination in male mouse hepatocytes may be contributing to the higher sensitivity of the male mouse hepatocytes to Met toxicity in comparison with female mouse hepatocytes.

  4. Group and Individual Variability in Mouse Pup Isolation Calls Recorded on the Same Day Show Stability

    Directory of Open Access Journals (Sweden)

    Terra D. Barnes

    2017-12-01

    Full Text Available Mice produce ultrasonic vocalizations (USVs in a variety of social situations, and USVs have been leveraged to study many neurological diseases including verbal dyspraxia, depression, autism and stuttering. Pups produce isolation calls, a common USV, spontaneously when they are isolated from their mother during the first 2 weeks of life. Several genetic manipulations affect (and often reduce pup isolation calls in mice. To facilitate the use of this assay as a means of testing whether significant functional differences in genotypes exist instead of contextual differences, we test the variability inherent in many commons measures of mouse vocalizations. Here we use biological consistency as a way of determining which are reproducible in mouse pup vocalizations. We present a comprehensive analysis of the normal variability of these vocalizations in groups of mice, individual mice and different strains of mice. To control for maturation effects, we recorded pup isolation calls in the same group of C57BL/6J 5 days old mice twice, with 1 h of rest in between recordings. In almost all cases, the group averages between the first and second recordings were the same. We also found that there were high correlations in some parameters in individual mice across recording while others were not well correlated. These findings could be replicated for the majority of features in a separate group of C57BL/6J mice and a group of 129/SvEvBrd-C57BL/6J mice. The averages of these mouse USV features are highly consistent and represent a robust assay to test the effects of genetic and other interventions in the experimental setting.

  5. Cardiac endothelial cells isolated from mouse heart - a novel model for radiobiology

    International Nuclear Information System (INIS)

    Jelonek, K.; Walaszczyk, A.; Gabrys, D.; Pietrowska, M.; Widlak, P.; Kanthou, Ch.

    2011-01-01

    Cardiovascular disease is recognized as an important clinical problem in radiotherapy and radiation protection. However, only few radiobiological models relevant for assessment of cardiotoxic effects of ionizing radiation are available. Here we describe the isolation of mouse primary cardiac endothelial cells, a possible target for cardiotoxic effects of radiation. Cells isolated from hearts of juvenile mice were cultured and irradiated in vitro. In addition, cells isolated from hearts of locally irradiated adult animals (up to 6 days after irradiation) were tested. A dose-dependent formation of histone γH 2 A.X foci was observed after in vitro irradiation of cultured cells. However, such cells were resistant to radiation-induced apoptosis. Increased levels of actin stress fibres were observed in the cytoplasm of cardiac endothelial cells irradiated in vitro or isolated from irradiated animals. A high dose of 16 Gy did not increase permeability to Dextran in monolayers formed by endothelial cells. Up-regulated expression of Vcam1, Sele and Hsp70i genes was detected after irradiation in vitro and in cells isolated few days after irradiation in vivo. The increased level of actin stress fibres and enhanced expression of stress-response genes in irradiated endothelial cells are potentially involved in cardiotoxic effects of ionizing radiation. (authors)

  6. Lactobacillus apodemi sp. nov., a tannase-producing species isolated from wild mouse faeces.

    Science.gov (United States)

    Osawa, Ro; Fujisawa, Tomohiko; Pukall, Rüdiger

    2006-07-01

    A Gram-positive, rod-shaped, non-endospore-forming bacterium, strain ASB1(T), able to degrade tannin, was isolated from faeces of the Japanese large wood mouse, Apodemus speciosus. Comparative analysis of the 16S rRNA gene sequence revealed that the strain could be assigned as a member of the genus Lactobacillus. The nearest phylogenetic neighbours were determined as Lactobacillus animalis DSM 20602(T) (98.9 % 16S rRNA gene sequence similarity) and Lactobacillus murinus ASF 361 (98.9 %). Subsequent polyphasic analysis, including automated ribotyping and DNA-DNA hybridization experiments, confirmed that the isolate represents a novel species, for which the name Lactobacillus apodemi sp. nov. is proposed. The DNA G+C content of the novel strain is 38.5 mol%. The cell-wall peptidoglycan is of type A4alpha L-lys-D-asp. The type strain is ASB1(T) (=DSM 16634(T)=CIP 108913(T)).

  7. Assessing Mitochondrial Bioenergetics in Isolated Mitochondria from Various Mouse Tissues Using Seahorse XF96 Analyzer.

    Science.gov (United States)

    Iuso, Arcangela; Repp, Birgit; Biagosch, Caroline; Terrile, Caterina; Prokisch, Holger

    2017-01-01

    Working with isolated mitochondria is the gold standard approach to investigate the function of the electron transport chain in tissues, free from the influence of other cellular factors. In this chapter, we outline a detailed protocol to measure the rate of oxygen consumption (OCR) with the high-throughput analyzer Seahorse XF96. More importantly, this protocol wants to provide practical tips for handling many different samples at once, and take a real advantage of using a high-throughput system. As a proof of concept, we have isolated mitochondria from brain, heart, liver, muscle, kidney, and lung of a wild-type mouse, and measured basal respiration (State II), ADP-stimulated respiration (State III), non-ADP-stimulated respiration (State IV o ), and FCCP-stimulated respiration (State III u ) using respiratory substrates specific to the respiratory chain complex I (RCCI) and complex II (RCCII). Mitochondrial purification and Seahorse runs were performed in less than eight working hours.

  8. Isolation of an 18,000-dalton hypusine-containing protein from cultured mouse neuroblastoma cells

    International Nuclear Information System (INIS)

    Dou, Q.P.; Chen, K.Y.

    1987-01-01

    An 18,000-dalton protein can be metabolically labeled by [ 3 H]putrescine or spermidine in mammalian cells. The labeling is due to a post-translational conversion of a lysine residue to hypusine residue. Previous studies indicated that the labeling is growth-dependent and is greatly diminished in mouse neuroblastoma cells after differentiation. To further study the physiological functions of this protein in the differentiation of mouse neuroblastoma cells, they have developed a simple procedure to purify this protein from cultured NB-15 mouse neuroblastoma cells. The 4-steps procedure included a Cibacron-Blue column, an omega-diaminooctyl-agarose column, a Sephadex G-50 column, and a Mono Q column. The procedure resulted in a 500-fold purification and the preparation appeared to be homogenous as judged by SDS-PAGE. Peptide map analysis using V-8 protease digestion method indicated that the 18,000-dalton hypusine-containing protein from NB-15 cells was identical to eukaryotic initiation factor 4D isolated from rabbit reticulocytes. This purification scheme also enabled them to detect a very faintly labeled protein in NB-15 cells. This weakly labeled protein had an apparent molecular weight of 22,000-dalton and pI of 5.0

  9. Inherently variable responses to glucocorticoid stress among endogenous retroviruses isolated from 23 mouse strains.

    Science.gov (United States)

    Hsu, Karen; Lee, Young-Kwan; Chew, Alex; Chiu, Sophia; Lim, Debora; Greenhalgh, David G; Cho, Kiho

    2017-10-01

    Active participation of endogenous retroviruses (ERVs) in disease processes has been exemplified by the finding that the HERV (human ERV)-W envelope protein is involved in the pathogenesis of multiple sclerosis, an autoimmune disease. We also demonstrated that injury-elicited stressors alter the expression of murine ERVs (MuERVs), both murine leukemia virus-type and mouse mammary tumor virus (MMTV)-type (MMTV-MuERV). In this study, to evaluate MMTV-MuERVs' responses to stress (e.g., injury, infection)-elicited systemic glucocorticoid (GC) levels, we examined the GC-stress response of 64 MMTV-MuERV promoters isolated from the genomes of 23 mouse strains. All 64 promoters responded to treatment with a synthetic GC, dexamethasone (DEX), at a wide range from a 0.6- to 85.7-fold increase in reporter activity compared to no treatment. An analysis of the 10 lowest and 10 highest DEX responders revealed specific promoter elements exclusively present in either the three lowest or the two highest responders. Each promoter had a unique profile of transcription regulatory elements and the glucocorticoid response element (GRE) was identified in all promoters with the number of GREs ranging from 2 to 7. The three lowest DEX responders were the only promoters with two GREs. The findings from this study suggest that certain MMTV-MuERVs are more responsive to stress-elicited systemic GC elevation compared to the others. The mouse strain-specific genomic MMTV-MuERV profiles and individual MMTV-MuERVs' differential responses to GC-stress might explain, at least in part, the variable inflammatory responses to injury and/or infection, often observed among different mouse strains. This article is part of a Special Issue entitled: Immune and Metabolic Alterations in Trauma and Sepsis edited by Dr. Raghavan Raju. Copyright © 2016 Elsevier B.V. All rights reserved.

  10. Isolation and characterization of neural stem cells from dystrophic mdx mouse

    International Nuclear Information System (INIS)

    Annese, Tiziana; Corsi, Patrizia; Ruggieri, Simona; Tamma, Roberto; Marinaccio, Christian; Picocci, Sabrina; Errede, Mariella; Specchia, Giorgina; De Luca, Annamaria; Frassanito, Maria Antonia; Desantis, Vanessa; Vacca, Angelo; Ribatti, Domenico; Nico, Beatrice

    2016-01-01

    The blood-brain barrier (BBB) is altered in mdx mouse, an animal model to study Duchenne muscular dystrophy (DMD). Our previous work demonstrated that perivascular glial endfeet control the selective exchanges between blood and neuropil as well as the BBB development and integrity; the alterations of dystrophin and dystrophin-associated protein complex (DAPs) in the glial cells of mdx mouse, parallel damages of the BBB and increase in vascular permeability. The aim of this study was to improve our knowledge about brain cellular components in the mdx mouse through the isolation, for the first time, of the adult neural stem cells (ANSCs). We characterized them by FACS, electron microscopy, confocal immunofluorescence microscopy, Real Time-PCR and western blotting, and we studied the expression of the DAPs aquaporin-4 (AQP4), potassium channel Kir4.1, α- and β-dystroglycan (αDG, βDG), α-syntrophin (αSyn), and short dystrophin isoform Dp71 proteins. The results showed that the mdx ANSCs expressed CD133 and Nestin receptor as the control ones, but showed a reduction in Notch receptor and altered cell proliferation with an increment in the apoptotic nuclei. Ultrastructurally, they appeared 50% size reduced compared to control ones, with a few cytoplasmic organelles. Moreover, the mdx ANSCs are devoid in full length dystrophin 427, and they expressed post-transcriptional reduction in the Dp71 in parallel with the ubiquitin proteasome activation, and decrement of DAPs proteins which appeared diffused in the cytoplasm and not polarized on the stem cells plasmamembrane, as prevalently observed in the controls. Overall, these results indicate that structural and molecular alterations affect the neural stem cells in the dystrophic brain, whose increased apoptosis and reduced Dp71 and DAPs proteins expression, together with loss in Dp427 dystrophin, could be responsible of the altered mdx glial maintenance and differentiation and consequent failure in the vessels barrier

  11. Isolation and characterization of neural stem cells from dystrophic mdx mouse

    Energy Technology Data Exchange (ETDEWEB)

    Annese, Tiziana [Department of Basic Medical Sciences, Neurosciences and Sensory Organs, Section of Human Anatomy and Histology, University of Bari Medical School, Bari (Italy); Corsi, Patrizia [Department of Basic Medical Sciences, Neurosciences and Sensory Organs, Section of Physiology, University of Bari Medical School, Bari (Italy); Ruggieri, Simona; Tamma, Roberto; Marinaccio, Christian [Department of Basic Medical Sciences, Neurosciences and Sensory Organs, Section of Human Anatomy and Histology, University of Bari Medical School, Bari (Italy); Picocci, Sabrina [Department of Basic Medical Sciences, Neurosciences and Sensory Organs, Section of Physiology, University of Bari Medical School, Bari (Italy); Errede, Mariella [Department of Basic Medical Sciences, Neurosciences and Sensory Organs, Section of Human Anatomy and Histology, University of Bari Medical School, Bari (Italy); Specchia, Giorgina [Department of Emergency and Transplantation, Section of Hematology, University of Bari Medical School, Bari (Italy); De Luca, Annamaria [Department of Bioscience, Biotechnology and Pharmacological Sciences, Section of Pharmacology, University of Bari (Italy); Frassanito, Maria Antonia; Desantis, Vanessa; Vacca, Angelo [Department of Internal Medicine and Oncology, University of Bari Medical School, Bari (Italy); Ribatti, Domenico, E-mail: domenico.ribatti@uniba.it [Department of Basic Medical Sciences, Neurosciences and Sensory Organs, Section of Human Anatomy and Histology, University of Bari Medical School, Bari (Italy); National Cancer Institute “Giovanni Paolo II”, Bari (Italy); Nico, Beatrice, E-mail: beatrice.nico@uniba.it [Department of Basic Medical Sciences, Neurosciences and Sensory Organs, Section of Human Anatomy and Histology, University of Bari Medical School, Bari (Italy)

    2016-05-01

    The blood-brain barrier (BBB) is altered in mdx mouse, an animal model to study Duchenne muscular dystrophy (DMD). Our previous work demonstrated that perivascular glial endfeet control the selective exchanges between blood and neuropil as well as the BBB development and integrity; the alterations of dystrophin and dystrophin-associated protein complex (DAPs) in the glial cells of mdx mouse, parallel damages of the BBB and increase in vascular permeability. The aim of this study was to improve our knowledge about brain cellular components in the mdx mouse through the isolation, for the first time, of the adult neural stem cells (ANSCs). We characterized them by FACS, electron microscopy, confocal immunofluorescence microscopy, Real Time-PCR and western blotting, and we studied the expression of the DAPs aquaporin-4 (AQP4), potassium channel Kir4.1, α- and β-dystroglycan (αDG, βDG), α-syntrophin (αSyn), and short dystrophin isoform Dp71 proteins. The results showed that the mdx ANSCs expressed CD133 and Nestin receptor as the control ones, but showed a reduction in Notch receptor and altered cell proliferation with an increment in the apoptotic nuclei. Ultrastructurally, they appeared 50% size reduced compared to control ones, with a few cytoplasmic organelles. Moreover, the mdx ANSCs are devoid in full length dystrophin 427, and they expressed post-transcriptional reduction in the Dp71 in parallel with the ubiquitin proteasome activation, and decrement of DAPs proteins which appeared diffused in the cytoplasm and not polarized on the stem cells plasmamembrane, as prevalently observed in the controls. Overall, these results indicate that structural and molecular alterations affect the neural stem cells in the dystrophic brain, whose increased apoptosis and reduced Dp71 and DAPs proteins expression, together with loss in Dp427 dystrophin, could be responsible of the altered mdx glial maintenance and differentiation and consequent failure in the vessels barrier

  12. Isolation of three novel rat and mouse papillomaviruses and their genomic characterization.

    Directory of Open Access Journals (Sweden)

    Eric Schulz

    Full Text Available Despite a growing knowledge about the biological diversity of papillomaviruses (PV, only little is known about non-human PV in general and about PV mice models in particular. We cloned and sequenced the complete genomes of two novel PV types from the Norway rat (Rattus norvegicus; RnPV2 and the wood mouse (Apodemus sylvaticus; AsPV1 as well as a novel variant of the recently described MmuPV1 (originally designated as MusPV from a house mouse (Mus musculus; MmuPV1 variant. In addition, we conducted phylogenetic analyses using a systematically representative set of 79 PV types, including the novel sequences. As inferred from concatenated amino acid sequences of six proteins, MmuPV1 variant and AsPV1 nested within the Beta+Xi-PV super taxon as members of the Pi-PV. RnPV2 is a member of the Iota-PV that has a distant phylogenetic position from Pi-PV. The phylogenetic results support a complex scenario of PV diversification driven by different evolutionary forces including co-divergence with hosts and adaptive radiations to new environments. PV types particularly isolated from mice and rats are the basis for new animal models, which are valuable to study PV induced tumors and new treatment options.

  13. Isolation and characterization of node/notochord-like cells from mouse embryonic stem cells

    DEFF Research Database (Denmark)

    Winzi, Maria K.; Hyttel, Poul; Dale, Jacqueline Kim

    2011-01-01

    The homeobox gene Noto is expressed in the node and its derivative the notochord. Here we use a targeted Noto-GFP reporter to isolate and characterize node/notochord-like cells derived from mouse embryonic stem cells. We find very few Noto-expressing cells after spontaneous differentiation. However......, the number of Noto-expressing cells was increased when using Activin A to induce a Foxa2- and Brachyury-expressing progenitor population, whose further differentiation into Noto-expressing cells was improved by simultaneous inhibition of BMP, Wnt, and retinoic acid signaling. Noto-GFP(+) cells expressed...... the node/notochord markers Noto, Foxa2, Shh, Noggin, Chordin, Foxj1, and Brachyury; showed a vacuolarization characteristic of notochord cells; and can integrate into midline structures when grafted into Hensen's node of gastrulating chicken embryos. The ability to generate node/notochord-like cells...

  14. Isolation and characterization of node/notochord-like cells from mouse embryonic stem cells.

    Science.gov (United States)

    Winzi, Maria K; Hyttel, Poul; Dale, Jacqueline Kim; Serup, Palle

    2011-11-01

    The homeobox gene Noto is expressed in the node and its derivative the notochord. Here we use a targeted Noto-GFP reporter to isolate and characterize node/notochord-like cells derived from mouse embryonic stem cells. We find very few Noto-expressing cells after spontaneous differentiation. However, the number of Noto-expressing cells was increased when using Activin A to induce a Foxa2- and Brachyury-expressing progenitor population, whose further differentiation into Noto-expressing cells was improved by simultaneous inhibition of BMP, Wnt, and retinoic acid signaling. Noto-GFP(+) cells expressed the node/notochord markers Noto, Foxa2, Shh, Noggin, Chordin, Foxj1, and Brachyury; showed a vacuolarization characteristic of notochord cells; and can integrate into midline structures when grafted into Hensen's node of gastrulating chicken embryos. The ability to generate node/notochord-like cells in vitro will aid the biochemical characterization of these developmentally important structures.

  15. Isolation of sphere-forming stem cells from the mouse inner ear.

    Science.gov (United States)

    Oshima, Kazuo; Senn, Pascal; Heller, Stefan

    2009-01-01

    The mammalian inner ear has very limited ability to regenerate lost sensory hair cells. This deficiency becomes apparent when hair cell loss leads to hearing loss as a result of either ototoxic insult or the aging process. Coincidently, with this inability to regenerate lost hair cells, the adult cochlea does not appear to harbor cells with a proliferative capacity that could serve as progenitor cells for lost cells. In contrast, adult mammalian vestibular sensory epithelia display a limited ability for hair cell regeneration, and sphere-forming cells with stem cell features can be isolated from the adult murine vestibular system. The neonatal inner ear, however, does harbor sphere-forming stem cells residing in cochlear and vestibular tissues. Here, we provide protocols to isolate sphere-forming stem cells from neonatal vestibular and cochlear sensory epithelia as well as from the spiral ganglion. We further describe procedures for sphere propagation, cell differentiation, and characterization of inner ear cell types derived from spheres. Sphere-forming stem cells from the mouse inner ear are an important tool for the development of cellular replacement strategies of damaged inner ears and are a bona fide progenitor cell source for transplantation studies.

  16. Diverse pathogenicity of equine herpesvirus 1 (EHV-1) isolates in CBA mouse model.

    Science.gov (United States)

    Yu, Mi Htay Htay; Kasem, Samy Gomaa Ahmed; Tsujimura, Koji; Matsumura, Tomio; Yanai, Tokuma; Yamaguchi, Tsuyoshi; Ohya, Kenji; Fukushi, Hideto

    2010-03-01

    The pathogenicity of equine herpesvirus 1 (EHV-1) isolates of Japan were evaluated by using the CBA mouse model. CBA mice were inoculated with eight Japanese EHV-1 strains (89c1, 90c16, 90c18, 97c11, 98c12, 00c19, 01c1 and HH-1) and one British strain (Ab4p). 89c1 caused slight body weight loss and nervous signs in mice at 8 days post infection (dpi). Severe weight loss and nervous signs were observed in mice inoculated with Ab4p at 6 dpi. The other strains did not cause apparent clinical signs. Infectious viruses were recovered from the lungs of all groups at 2 dpi. Histopathological analysis revealed interstitial pneumonia in the lungs of all mice inoculated with EHV-1. Encephalitis or meningoencephalitis was observed in the brains of mice inoculated with 89c1, 90c18, 97c11, 98c12, 01c1 and Ab4p. Japanese EHV-1 strains showed low pathogenicity in CBA mice, whereas the sequential affects of infection are similar to those of the highly pathogenic strain Ab4p. These results suggest that field isolates of EHV-1 have varying degrees of pathogenicity in CBA mice.

  17. Automated classification of mouse pup isolation syllables: from cluster analysis to an Excel based ‘mouse pup syllable classification calculator’

    Directory of Open Access Journals (Sweden)

    Jasmine eGrimsley

    2013-01-01

    Full Text Available Mouse pups vocalize at high rates when they are cold or isolated from the nest. The proportions of each syllable type produced carry information about disease state and are being used as behavioral markers for the internal state of animals. Manual classifications of these vocalizations identified ten syllable types based on their spectro-temporal features. However, manual classification of mouse syllables is time consuming and vulnerable to experimenter bias. This study uses an automated cluster analysis to identify acoustically distinct syllable types produced by CBA/CaJ mouse pups, and then compares the results to prior manual classification methods. The cluster analysis identified two syllable types, based on their frequency bands, that have continuous frequency-time structure, and two syllable types featuring abrupt frequency transitions. Although cluster analysis computed fewer syllable types than manual classification, the clusters represented well the probability distributions of the acoustic features within syllables. These probability distributions indicate that some of the manually classified syllable types are not statistically distinct. The characteristics of the four classified clusters were used to generate a Microsoft Excel-based mouse syllable classifier that rapidly categorizes syllables, with over a 90% match, into the syllable types determined by cluster analysis.

  18. Isolation and characterization of proteins of the mouse mammary tumour virus

    International Nuclear Information System (INIS)

    Westenbrink, F.

    1980-01-01

    A vaccination procedure was developed to mouse mammary tumor virus (MuMTV) induced mouse mammary tumorigenesis. The structural proteins of MuMTV were purified so that their immunogenic qualities were retained. Radioimmunoassays were developed for the proteins. (Auth.)

  19. Intramacrophage survival of uropathogenic Escherichia coli: Differences between diverse clinical isolates and between mouse and human macrophages

    DEFF Research Database (Denmark)

    Bokil, Nilesh J.; Totsika, Makrina; Carey, Alison J.

    2011-01-01

    assays, CFT073 was able to survive within primary mouse bone marrow-derived macrophages (BMM) up to 24h post-infection. Three additional well-characterized clinical UPEC isolates associated with distinct UTI symptomatologies displayed variable long-term survival within BMM. UPEC strains UTI89 and VR50...... or initial uptake of bacteria. E. coli UTI89 localized to a Lamp1+ vesicular compartment within BMM. In contrast to survival within mouse BMM, intracellular bacterial loads of VR50 were low in both human monocyte-derived macrophages (HMDM) and in human T24 bladder epithelial cells. Collectively, these data...

  20. Isolation and characterization of conditionally immortalized mouse glomerular endothelial cell lines.

    Science.gov (United States)

    Rops, Angelique L; van der Vlag, Johan; Jacobs, Cor W; Dijkman, Henry B; Lensen, Joost F; Wijnhoven, Tessa J; van den Heuvel, Lambert P; van Kuppevelt, Toin H; Berden, Jo H

    2004-12-01

    The culture and establishment of glomerular cell lines has proven to be an important tool for the understanding of glomerular cell functions in glomerular physiology and pathology. Especially, the recent establishment of a conditionally immortalized visceral epithelial cell line has greatly boosted the research on podocyte biology. Glomeruli were isolated from H-2Kb-tsA58 transgenic mice that contain a gene encoding a temperature-sensitive variant of the SV40 large tumor antigen, facilitating proliferative growth at 33 degrees C and differentiation at 37 degrees C. Glomerular endothelial cells were isolated from glomerular outgrowth by magnetic beads loaded with CD31, CD105, GSL I-B4, and ULEX. Clonal cell lines were characterized by immunofluorescence staining with antibodies/lectins specific for markers of endothelial cells, podocytes, and mesangial cells. Putative glomerular endothelial cell lines were analyzed for (1) cytokine-induced expression of adhesion molecules; (2) tube formation on Matrigel coating; and (3) the presence of fenestrae. As judged by immunostaining for Wilms tumor-1, smooth muscle actin (SMA), podocalyxin, and von Willebrand factor (vWF), we obtained putative endothelial, podocyte and mesangial cell lines. The mouse glomerular endothelial cell clone #1 (mGEnC-1) was positive for vWF, podocalyxin, CD31, CD105, VE-cadherin, GSL I-B4, and ULEX, internalized acetylated-low-density lipoprotein (LDL), and showed increased expression of adhesion molecules after activation with proinflammatory cytokines. Furthermore, mGEnC-1 formed tubes and contained nondiaphragmed fenestrae. The mGEnC-1 represents a conditionally immortalized cell line with various characteristics of differentiated glomerular endothelial cells when cultured at 37 degrees C. Most important, mGEnC-1 contains nondiaphragmed fenestrae, which is a unique feature of glomerular endothelial cells.

  1. Radiation-induced apoptosis in sensitive and resistant cells isolated from a mouse lymphoma

    International Nuclear Information System (INIS)

    Story, M.D.; Voehringer, D.W.; Malone, C.G.; Hobbs, M.L.; Meyn, R.E.

    1994-01-01

    Cells were isolated from a mouse lymphoma (LY-TH) and grown in vitro. They were susceptible to radiation-induced apoptosis after low doses with the appearance of endonucleolytically fragmented DNA 1 h after irradiation. Four hours after receiving 5 Gy, 80% of the DNA was endonucleolytically cleaved. Apoptosis induction by DNA double-strand break (dsb) formation was more effective compared with induction by single-strand break (ssb) formation. After long-term culturing, LY-TH cultures became refractory to apoptosis. Apoptosis-permissive cells (LY-as, cloned from LY-TH cells) were three times more radiosensitive than clonally expanded apoptosis-refractory cells (LY-ar). Low dose-rate irradiation and maintenance at 25 o C for 5 h postirradiation was sparing in LY-ar but not LY-as cells, suggesting a repair deficiency in LY-as cells. Analysis of dsb rejoining kinetics revealed no difference in the initial phase of dsb rejoining. After 1 h, however, relative dsbs in the LY-as variant increased as endonucleolytic cleavage was initiated. Signalling for radiation-induced apoptosis in LY-as cells was independent of the DNA dsb repair pathway and appeared determined by initial events, whereas in LY-ar cells, because of an inhibition in the apoptotic pathway, survival was enhanced and modifiable by repair processes. (author)

  2. Effect of social odor context on the emission of isolation-induced ultrasonic vocalizations in the BTBR T+tf/J mouse model for autism

    Science.gov (United States)

    Wöhr, Markus

    2015-01-01

    An important diagnostic criterion for social communication deficits in autism spectrum disorders (ASD) are difficulties in adjusting behavior to suit different social contexts. While the BTBR T+tf/J (BTBR) inbred strain of mice is one of the most commonly used mouse models for ASD, little is known about whether BTBR mice display deficits in detecting changes in social context and their ability to adjust to them. Here, it was tested therefore whether the emission of isolation-induced ultrasonic vocalizations (USV) in BTBR mouse pups is affected by the social odor context, in comparison to the standard control strain with high sociability, C57BL/6J (B6). It is known that the presence of odors from mothers and littermates leads to a calming of the isolated mouse pup, and hence to a reduction in isolation-induced USV emission. In accordance with their behavioral phenotypes with relevance to all diagnostic core symptoms of ASD, it was predicted that BTBR mouse pups would not display a calming response when tested under soiled bedding conditions with home cage bedding material containing maternal odors, and that similar isolation-induced USV emission rates would be seen in BTBR mice tested under clean and soiled bedding conditions. Unexpectedly, however, the present findings show that BTBR mouse pups display such a calming response and emit fewer isolation-induced USV when tested under soiled as compared to clean bedding conditions, similar to B6 mouse pups. Yet, in contrast to B6 mouse pups, which emitted isolation-induced USV with shorter call durations and lower levels of frequency modulation under soiled bedding conditions, social odor context had no effect on acoustic call features in BTBR mouse pups. This indicates that the BTBR mouse model for ASD does not display deficits in detecting changes in social context, but has a limited ability and/or reduced motivation to adjust to them. PMID:25852455

  3. Intramacrophage survival of uropathogenic Escherichia coli: Differences between diverse clinical isolates and between mouse and human macrophages

    KAUST Repository

    Bokil, Nilesh J.

    2011-11-01

    Uropathogenic E. coli (UPEC) are the primary cause of urinary tract infections. Recent studies have demonstrated that UPEC can invade and replicate within epithelial cells, suggesting that this bacterial pathogen may occupy an intracellular niche within the host. Given that many intracellular pathogens target macrophages, we assessed the interactions between UPEC and macrophages. Colonization of the mouse bladder by UPEC strain CFT073 resulted in increased expression of myeloid-restricted genes, consistent with the recruitment of inflammatory macrophages to the site of infection. In in vitro assays, CFT073 was able to survive within primary mouse bone marrow-derived macrophages (BMM) up to 24h post-infection. Three additional well-characterized clinical UPEC isolates associated with distinct UTI symptomatologies displayed variable long-term survival within BMM. UPEC strains UTI89 and VR50, originally isolated from patients with cystitis and asymptomatic bacteriuria respectively, showed elevated bacterial loads in BMM at 24h post-infection as compared to CFT073 and the asymptomatic bacteriuria strain 83972. These differences did not correlate with differential effects on macrophage survival or initial uptake of bacteria. E. coli UTI89 localized to a Lamp1 + vesicular compartment within BMM. In contrast to survival within mouse BMM, intracellular bacterial loads of VR50 were low in both human monocyte-derived macrophages (HMDM) and in human T24 bladder epithelial cells. Collectively, these data suggest that some UPEC isolates may subvert macrophage anti-microbial pathways, and that host species differences may impact on intracellular UPEC survival. © 2011 Elsevier GmbH.

  4. Defined Conditions for the Isolation and Expansion of Basal Prostate Progenitor Cells of Mouse and Human Origin

    Directory of Open Access Journals (Sweden)

    Thomas Höfner

    2015-03-01

    Full Text Available Methods to isolate and culture primary prostate epithelial stem/progenitor cells (PESCs have proven difficult and ineffective. Here, we present a method to grow and expand both murine and human basal PESCs long term in serum- and feeder-free conditions. The method enriches for adherent mouse basal PESCs with a Lin−SCA-1+CD49f+TROP2high phenotype. Progesterone and sodium selenite are additionally required for the growth of human Lin−CD49f+TROP2high PESCs. The gene-expression profiles of expanded basal PESCs show similarities to ESCs, and NF-kB function is critical for epithelial differentiation of sphere-cultured PESCs. When transplanted in combination with urogenital sinus mesenchyme, expanded mouse and human PESCs generate ectopic prostatic tubules, demonstrating their stem cell activity in vivo. This novel method will facilitate the molecular, genomic, and functional characterization of normal and pathologic prostate glands of mouse and human origin.

  5. Isoprenoid, lipid, and protein contents in intact plastids isolated from mesocarp cells of traditional and high-pigment tomato cultivars at different ripening stages.

    Science.gov (United States)

    Lenucci, Marcello S; Serrone, Lucia; De Caroli, Monica; Fraser, Paul D; Bramley, Peter M; Piro, Gabriella; Dalessandro, Giuseppe

    2012-02-22

    This study reports quali-quantitative analyses on isoprenoids, phospholipids, neutral lipids, phytosterols, and proteins in purified plastids isolated from fresh fruits of traditional (Donald and Incas) and high-pigment (Kalvert and HLY-18) tomato cultivars at four ripening stages. In all of the investigated cultivars, lycopene, β-catotene, lutein, and total carotenoids varied significantly during ripening. Chromoplasts of red-ripe tomato fruits of high-pigment cultivars accumulated twice as much as lycopene (307.6 and 319.2 μg/mg of plastid proteins in Kalvert and HLY-18, respectively) than ordinary cultivars (178.6 and 151.7 μg/mg of plastid proteins in Donald and Incas, respectively); differences in chlorophyll and α-tocopherol contents were also evidenced. Phospholipids and phytosterols increased during ripening, whereas triglycerides showed a general decrease. Regardless of the stage of ripening, palmitic acid was the major fatty acid in all cultivars (ranging from 35 to 52% of the total fatty acids), followed by stearic, oleic, linoleic, linolenic, and myristic acids, but their relative percentage was affected by ripening. Most of the bands detected on the SDS-PAGEs of plastid proteins were constantly present during chloroplast-to-chromoplast conversion, some others disappeared, and only one, with a molecular weight of ~41.6 kDa, was found to increase in intensity.

  6. Chloroplast to chromoplast transition in tomato fruit: spectral confocal microscopy analyses of carotenoids and chlorophylls in isolated plastids and time-lapse recording on intact live tissue.

    Science.gov (United States)

    Egea, Isabel; Bian, Wanping; Barsan, Cristina; Jauneau, Alain; Pech, Jean-Claude; Latché, Alain; Li, Zhengguo; Chervin, Christian

    2011-08-01

    There are several studies suggesting that tomato (Solanum lycopersicum) chromoplasts arise from chloroplasts, but there is still no report showing the fluorescence of both chlorophylls and carotenoids in an intermediate plastid, and no video showing this transition phase. Pigment fluorescence within individual plastids, isolated from tomato fruit using sucrose gradients, was observed at different ripening stages, and an in situ real-time recording of pigment fluorescence was performed on live tomato fruit slices. At the mature green and red stages, homogenous fractions of chloroplasts and chromoplasts were obtained, respectively. At the breaker stage, spectral confocal microscopy showed that intermediate plastids contained both chlorophylls and carotenoids. Furthermore, an in situ real-time recording (a) showed that the chloroplast to chromoplast transition was synchronous for all plastids of a single cell; and (b) confirmed that all chromoplasts derived from pre-existing chloroplasts. These results give details of the early steps of tomato chromoplast biogenesis from chloroplasts, with the formation of intermediate plastids containing both carotenoids and chlorophylls. They provide information at the sub-cellular level on the synchronism of plastid transition and pigment changes.

  7. Social isolation induces autophagy in the mouse mammary gland: link to increased mammary cancer risk.

    Science.gov (United States)

    Sumis, Allison; Cook, Katherine L; Andrade, Fabia O; Hu, Rong; Kidney, Emma; Zhang, Xiyuan; Kim, Dominic; Carney, Elissa; Nguyen, Nguyen; Yu, Wei; Bouker, Kerrie B; Cruz, Idalia; Clarke, Robert; Hilakivi-Clarke, Leena

    2016-10-01

    Social isolation is a strong predictor of early all-cause mortality and consistently increases breast cancer risk in both women and animal models. Because social isolation increases body weight, we compared its effects to those caused by a consumption of obesity-inducing diet (OID) in C57BL/6 mice. Social isolation and OID impaired insulin and glucose sensitivity. In socially isolated, OID-fed mice (I-OID), insulin resistance was linked to reduced Pparg expression and increased neuropeptide Y levels, but in group-housed OID fed mice (G-OID), it was linked to increased leptin and reduced adiponectin levels, indicating that the pathways leading to insulin resistance are different. Carcinogen-induced mammary tumorigenesis was significantly higher in I-OID mice than in the other groups, but cancer risk was also increased in socially isolated, control diet-fed mice (I-C) and G-OID mice compared with that in controls. Unfolded protein response (UPR) signaling (GRP78; IRE1) was upregulated in the mammary glands of OID-fed mice, but not in control diet-fed, socially isolated I-C mice. In contrast, expression of BECLIN1, ATG7 and LC3II were increased, and p62 was downregulated by social isolation, indicating increased autophagy. In the mammary glands of socially isolated mice, but not in G-OID mice, mRNA expressions of p53 and the p53-regulated autophagy inducer Dram1 were upregulated, and nuclear p53 staining was strong. Our findings further indicated that autophagy and tumorigenesis were not increased in Atg7(+/-) mice kept in social isolation and fed OID. Thus, social isolation may increase breast cancer risk by inducing autophagy, independent of changes in body weight. © 2016 Society for Endocrinology.

  8. Mouse-virulent Toxoplasma gondii isolated from feral cats on Mona Island, Puerto Rico.

    Science.gov (United States)

    Dubey, J P; López-Torres, H Y; Sundar, N; Velmurugan, G V; Ajzenberg, D; Kwok, O C H; Hill, R; Dardé, M L; Su, C

    2007-12-01

    Cats are essential in the life cycle of Toxoplasma gondii because they are the only hosts that can excrete the environmentally resistant oocysts. Samples of serum, feces, and tissues from cats from Mona, a remote island off the coast of Puerto Rico, were examined for T. gondii infection. Antibodies to T. gondii were assayed by the modified agglutination test and found in 16 of 19 (84.2%) of cats, with titers of 1:10 in 2, 1:80 in 1, 1:160 in 4, 1:320 in 3, and 1:1,280 or higher in 6. Tissues of 19 of the 20 cats were bioassayed in mice for T. gondii infection. Toxoplasma gondii was isolated from tissues of 12 cats: from the hearts of 9, skeletal muscle of 10, and brain of 1 cat. All infected mice from 10 of 12 isolates died of acute toxoplasmosis during primary infection. Genotyping of these 12 T. gondii isolates (designated (TgCatPr 1-12) by 10 multilocus PCR-RFLP markers, i.e., SAG1, SAG2, SAG3, BTUB, GRA6, c22-8, c29-2, L358, PK1, and an apicoplast marker Apico, and the 6 multilocus microsatellite markers TUB2, W35, TgM-A, B18, B17, and M33, revealed 7 genotypes; 5 isolates had Type I alleles at all loci except at 1 microsatellite locus, and the remainder were atypical. The latter isolates of T. gondii were different biologically and phenotypically from the feline isolates from the rest of the Americas. One isolate (TgCatPr 12) was a mixed infection with 2 genotypes.

  9. Role of adenosine 5'-monophosphate-activated protein kinase in interleukin-6 release from isolated mouse skeletal muscle

    DEFF Research Database (Denmark)

    Glund, Stephan; Treebak, Jonas Thue; Long, Yun Chau

    2009-01-01

    IL-6 is released from skeletal muscle during exercise and has consequently been implicated to mediate beneficial effects on whole-body metabolism. Using 5-aminoimidazole-4-carboxamide-1-beta-4-ribofuranoside (AICAR), a pharmacological activator of 5'-AMP-activated protein kinase (AMPK), we tested......-type mice was also incubated with the AMPK activator A-769662. Incubation of mouse glycolytic extensor digitorum longus and oxidative soleus muscle for 2 h was associated with profound IL-6 mRNA production and protein release, which was suppressed by AICAR (P ... the hypothesis that AMPK modulates IL-6 release from isolated muscle. Skeletal muscle from AMPKalpha2 kinase-dead transgenic, AMPKalpha1 knockout (KO) and AMPKgamma3 KO mice and respective wild-type littermates was incubated in vitro, in the absence or presence of 2 mmol/liter AICAR. Skeletal muscle from wild...

  10. Ion selectivity of the cation transport system of isolated intact cattle rod outer segments: evidence for a direct communication between the rod plasma membrane and the rod disk membranes.

    Science.gov (United States)

    Schnetkamp, P P

    1980-05-08

    The ion selectivity of cation transport through the plasma membrane of isolated intact cattle rod outer segments (rods) is investigated by means of 45Ca-exchange experiments and light-scattering experiments. These techniques appear to provide complementary information: the 45Ca experiments (45Ca fluxes in rods) describe electroneutral antiport, whereas the light-scattering experiments (shrinkage and swelling of rods upon hypertonic shocks with various electrolytes) reveal electrogenic uniport. Electroneutral symport of ions (salt transport) does not take place without addition of external ionophores and application of salts of weak acids. 1. Intact rods recover from a hypertonic shock in the presence of FCCP when lithium, sodium and potassium acetate are applied, but not when ammonium chloride, calcium and magnesium acetate are used. This indicates that the plasma membrane of isolated intact cattle rods is relatively permeable to net transport of Na+, Li+ and K+, and relatively impermeable to net transport of Cl-, Mg2+ and Ca2+ under conditions that do not give rise to diffusion potentials. 2. Rapid (t1/2 exchange diffusion of internal 45Ca with external Na+, Ca2+, Sr2+ and Ba2+, respectively. 3. All tested cations lower the rate of 45Ca uptake. The latter can be described by a single rate constant indicating a homogeneous rod preparation and a homogeneous endogenous Ca2+ pool. However, only those cations which stimulate 45Ca efflux from preloaded rods lower the final equilibrium of 45Ca uptake. Except for the effects of K+, Rb+ and Cs+ the reduction of the rate of 45Ca uptake by external cations appears to arise from competition for a common site on the plasms membrane. The observed affinities for this site do not correlate with actual transport (as indicated by the ability to stimulate 45Ca efflux). 4. K+ increases the affinity of the exchange diffusion system to Ca2+ from 1 microM to 0.15 microM and changes the relative affinities with respect to Ca2+ for the

  11. Early-Life Social Isolation Influences Mouse Ultrasonic Vocalizations during Male-Male Social Encounters.

    Science.gov (United States)

    Keesom, Sarah M; Finton, Caitlyn J; Sell, Gabrielle L; Hurley, Laura M

    2017-01-01

    Early-life social isolation has profound effects on adult social competence. This is often expressed as increased aggression or inappropriate displays of courtship-related behaviors. The social incompetence exhibited by isolated animals could be in part due to an altered ability to participate in communicatory exchanges. House mice (Mus musculus) present an excellent model for exploring this idea, because social isolation has a well-established influence on their social behavior, and mice engage in communication via multiple sensory modalities. Here, we tested the prediction that social isolation during early life would influence ultrasonic vocalizations (USVs) emitted by adult male mice during same-sex social encounters. Starting at three weeks of age, male mice were housed individually or in social groups of four males for five weeks, after which they were placed in one of three types of paired social encounters. Pair types consisted of: two individually housed males, two socially housed males, or an individually housed and a socially housed male ("mixed" pairs). Vocal behavior (USVs) and non-vocal behaviors were recorded from these 15-minute social interactions. Pairs of mice consisting of at least one individually housed male emitted more and longer USVs, with a greater proportional use of USVs containing frequency jumps and 50-kHz components. Individually housed males in the mixed social pairs exhibited increased levels of mounting behavior towards the socially housed males. Mounting in these pairs was positively correlated with increased number and duration of USVs as well as increased proportional use of spectrally more complex USVs. These findings demonstrate that USVs are part of the suite of social behaviors influenced by early-life social isolation, and suggest that altered vocal communication following isolation reflects reduced social competence.

  12. Isolation and Molecular Profiling of Primary Mouse Retinal Ganglion Cells: Comparison of Phenotypes from Healthy and Glaucomatous Retinas.

    Science.gov (United States)

    Chintalapudi, Sumana R; Djenderedjian, Levon; Stiemke, Andrew B; Steinle, Jena J; Jablonski, Monica M; Morales-Tirado, Vanessa M

    2016-01-01

    Loss of functional retinal ganglion cells (RGC) is an element of retinal degeneration that is poorly understood. This is in part due to the lack of a reliable and validated protocol for the isolation of primary RGCs. Here we optimize a feasible, reproducible, standardized flow cytometry-based protocol for the isolation and enrichment of homogeneous RGC with the Thy1.2(hi)CD48(neg)CD15(neg)CD57(neg) surface phenotype. A three-step validation process was performed by: (1) genomic profiling of 25-genes associated with retinal cells; (2) intracellular labeling of homogeneous sorted cells for the intracellular RGC-markers SNCG, brain-specific homeobox/POU domain protein 3A (BRN3A), TUJ1, and RNA-binding protein with multiple splicing (RBPMS); and (3) by applying the methodology on RGC from a mouse model with elevated intraocular pressure (IOP) and optic nerve damage. Use of primary RGC cultures will allow for future careful assessment of important cell specific pathways in RGC to provide mechanistic insights into the declining of visual acuity in aged populations and those suffering from retinal neurodegenerative diseases.

  13. Isolation and Molecular Profiling of Primary Mouse Retinal Ganglion Cells: Comparison of Phenotypes from Healthy and Glaucomatous Retinas

    Science.gov (United States)

    Chintalapudi, Sumana R.; Djenderedjian, Levon; Stiemke, Andrew B.; Steinle, Jena J.; Jablonski, Monica M.; Morales-Tirado, Vanessa M.

    2016-01-01

    Loss of functional retinal ganglion cells (RGC) is an element of retinal degeneration that is poorly understood. This is in part due to the lack of a reliable and validated protocol for the isolation of primary RGCs. Here we optimize a feasible, reproducible, standardized flow cytometry-based protocol for the isolation and enrichment of homogeneous RGC with the Thy1.2hiCD48negCD15negCD57neg surface phenotype. A three-step validation process was performed by: (1) genomic profiling of 25-genes associated with retinal cells; (2) intracellular labeling of homogeneous sorted cells for the intracellular RGC-markers SNCG, brain-specific homeobox/POU domain protein 3A (BRN3A), TUJ1, and RNA-binding protein with multiple splicing (RBPMS); and (3) by applying the methodology on RGC from a mouse model with elevated intraocular pressure (IOP) and optic nerve damage. Use of primary RGC cultures will allow for future careful assessment of important cell specific pathways in RGC to provide mechanistic insights into the declining of visual acuity in aged populations and those suffering from retinal neurodegenerative diseases. PMID:27242509

  14. Isolation of UV-sensitive mutants of mouse L5178Y cells by a cell suspension spotting method

    International Nuclear Information System (INIS)

    Shiomi, T.; Hieda-Shiomi, N.; Sato, K.

    1982-01-01

    We have isolated 56 UV-sensitive mutant clones from a mouse L51 T/t line of L5178Y cells by a cell suspension spotting method. Five mutants have also been isolated from L51 T/t and L5178Y cells by the method reported by Thompson and coworkers. We divided the mutants into two groups, highly sensitive and moderately sensitive mutants, according to their sensitivity to UV irradiation. Fifty-eight mutants were highly sensitive and three were moderately sensitive to UV. The reconstruction experiments indicate that more than 90% of highly sensitive mutants were recovered by the cell suspension spotting method. Frequencies of recovered mutants highly sensitive to UV increased with increasing dose of mutagens. Recovered mutant frequency reached 10(-2) after treatment with 1.5 micrograms/ml of N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) (survival 0.2%). Eight UV-sensitive mutants were divided into four complementation groups. These mutants were 2-6 times more sensitive to UV than parental L51 T/t cells in terms of D37 (dose required to reduce survival to 37%). Four representative UV-sensitive mutants which are classified into different complementation groups were examined for their sensitivity to killing by UV, 4-nitroquinoline-1-oxide (4NQO), mitomycin C (MMC), X-rays, and MNNG. All four classes of mutants were found to be cross-sensitive to UV, 4NQO, and MMC, but not sensitive to X-rays and MNNG

  15. Contribution of α-adrenoceptor stimulation by phenylephrine to basal nitric oxide production in the isolated mouse aorta.

    Science.gov (United States)

    van Langen, Johanna T H; Van Hove, Cor E; Schrijvers, Dorien M; Martinet, Wim; De Meyer, Guido R Y; Fransen, Paul; Bult, Hidde

    2013-04-01

    In the mouse aorta, contractions evoked by the α(1)-adrenoceptor agonist phenylephrine are strongly suppressed by the continuous production of nitric oxide (NO). We investigated whether phenylephrine itself stimulated NO production by activating endothelial α(2)-adrenoceptors. On a prostaglandin F(2α) contraction, the α(2)-adrenoceptor agonist 5-bromo-N-(4,5-dihydro-1H-imidazol-2-yl)-6-quinoxalinamine (UK14304) induced 29.3 ± 7.4% relaxation, which was inhibited by 0.1 μM 2-[(4,5-Dihydro-1H-imidazol-2-yl)methyl]-2,3-dihydro-1-methyl-1H-isoindole (BRL44408) with a pKB' corresponding to α(2)-antagonism. In the presence of NO synthase blockers, UK14304 elicited small contractions above 1 μM that were inhibited by 0.1 μM prazosin, but not influenced by 0.1 μM rauwolscine. At 3 μM or higher concentrations, phenylephrine caused only modest relaxation (up to 7.4 ± 2.3%) of segments constricted with prostaglandin F(2α) in the presence of prazosin, which was abolished with 0.1 μM BRL44408. Furthermore, BRL44408 did not increase contractions induced with 1 μM phenylephrine. These results confirm that α(1)- but not α(2)-adrenoceptors are expressed on aortic smooth muscle cells, whereas endothelial cells only express α(2)-adrenoceptors. Moreover, phenylephrine exerted a very modest relaxing effect through nonspecific stimulation of α(2)-adrenoceptors, but only at concentrations higher than 1 μM. It is concluded that the high basal output of NO in the isolated mouse aorta is not due to stimulation of α-adrenoceptors.

  16. Lipid composition of membrane rafts, isolated with and without detergent, from the spleen of a mouse model of Gaucher disease.

    Science.gov (United States)

    Hattersley, Kathryn J; Hein, Leanne K; Fuller, Maria

    2013-12-06

    Biological membranes are composed of functionally relevant liquid-ordered and liquid-disordered domains that coexist. Within the liquid-ordered domains are low-density microdomains known as rafts with a unique lipid composition that is crucial for their structure and function. Lipid raft composition is altered in sphingolipid storage disorders, and here we determined the lipid composition using a detergent and detergent-free method in spleen tissue, the primary site of pathology, in a mouse model of the sphingolipid storage disorder, Gaucher disease. The accumulating lipid, glucosylceramide, was 30- and 50-fold elevated in the rafts with the detergent and detergent-free method, respectively. Secondary accumulation of di- and trihexosylceramide resided primarily in the rafts with both methods. The phospholipids distributed differently with more than half residing in the rafts with the detergent-free method and less than 10% with the detergent method, with the exception of the fully saturated species that were primarily in the rafts. Individual isoforms of sphingomyelin correlated with detergent-free extraction and more than half resided in the raft fractions. However, this correlation was not seen with the detergent extraction method as sphingomyelin species were spread across both the raft and non-raft domains. Therefore caution must be exercised when interpreting phospholipid distribution in raft domains as it differs considerably depending on the method of isolation. Importantly, both methods revealed the same lipid alterations in the raft domains in the spleen of the Gaucher disease mouse model highlighting that either method is appropriate to determine membrane lipid changes in the diseased state. Copyright © 2013 Elsevier Inc. All rights reserved.

  17. Differences in pathogenicity of three animal isolates of Mycobacterium species in a mouse model.

    Directory of Open Access Journals (Sweden)

    Haodi Dong

    Full Text Available Animal mycobacterioses are among the most important zoonoses worldwide. These are generally caused by either Mycobacterium tuberculosis (MTB, M. bovis (MBO or M. avium (MAV. To test the hypothesis that different species of pathogenic mycobacteria isolated from varied anatomic locations or animal species differ in virulence and pathogenicity, we performed experiments with three mycobacteria strains (NTSE-3(MTB, NTSE-4(MBO and NTSE-5 (MAV obtained from animal species. Spoligotyping analysis was used to confirm both MTB and MBO strains while the MAV strain was confirmed by 16s rDNA sequencing. BALB/c mice were intranasally infected with the three strains at low and high CFU doses to evaluate variations in pathogenicity. Clinical and pathological parameters were assessed. Infected mice were euthanized at 80 days post-inoculation (dpi. Measures of lung and body weights indicated that the MBO infected group had higher mortality, more weight loss, higher bacterial burden and more severe lesions in lungs than the other two groups. Cytokine profiles showed higher levels of TNF-α for MBO versus MTB, while MAV had the highest amounts of IFN-β in vitro and in vivo. In vitro levels of other cytokines such as IL-1β, IL-10, IL-12, IL-17, and IFN-β showed that Th1 cells had the strongest response in MBO infected mice and that Th2 cells were inhibited. We found that the level of virulence among the three isolates decreased in the following order MBO>MTB>MAV.

  18. (Photosynthesis in intact plants)

    Energy Technology Data Exchange (ETDEWEB)

    1990-01-01

    Progress in the two years since the last renewal application has been excellent. We have made substantial contributions on both main fronts of the projects, and are particularly happy with the progress of our research on intact plants. The approach of basing our field work on a sound foundation of laboratory studies has enabled is to use methods which provide unambiguous assays of well characterized reactions. We have also made excellent progress in several laboratory studies which will have direct applications in future field work, and have introduced to the laboratory a range of molecular genetics techniques which will allow us to explore new options in the attempt to understand function at the level of molecular structure.

  19. Non-intact zona improves development of murine preimplantation ...

    African Journals Online (AJOL)

    ajl5

    2012-09-25

    Sep 25, 2012 ... 2College of Animal Science and Technology, Northwest A & F University, Yangling, ... Key words: Mouse, non-intact zona embryos, adenovirus vector with green fluorescent protein (pAd-GFP), .... Based on microscopic examination, the ZP of some ..... permeable structure of ZP that allowed penetration of.

  20. Patterns of cross-sensitivity in the responses of clonal subpopulations isolated from the RIF-1 mouse sarcoma to selected nitrosoureas and nitrogen mustards.

    OpenAIRE

    Reeve, J. G.; Wright, K. A.; Workman, P.

    1984-01-01

    The response of clonal subpopulations isolated from the RIF-1 mouse sarcoma to melphalan treatment is independent of cell ploidy, whereas a clear relationship exists between ploidy and cell sensitivity to CCNU treatment. In the present study RIF-1 clones have been exposed to nitrogen mustard, aniline mustard and chlorambucil, and to nitrosoureas BCNU, MeCCNU and chlorozotocin, in order to evaluate whether or not the different physiochemical and biological activities of these agents would affe...

  1. A brain-specific gene cluster isolated from the region of the mouse obesity locus is expressed in the adult hypothalamus and during mouse development

    Energy Technology Data Exchange (ETDEWEB)

    Laig-Webster, M.; Lim, M.E.; Chehab, F.F. [Univ. of California, San Francisco, CA (United States)

    1994-09-01

    The molecular defect underlying an autosomal recessive form of genetic obesity in a classical mouse model C57 BL/6J-ob/ob has not yet been elucidated. Whereas metabolic and physiological disturbances such as diabetes and hypertension are associated with obesity, the site of expression and the nature of the primary lesion responsible for this cascade of events remains elusive. Our efforts aimed at the positional cloning of the ob gene by YAC contig mapping and gene identification have resulted in the cloning of a brain-specific gene cluster from the ob critical region. The expression of this gene cluster is remarkably complex owing to the multitude of brain-specific mRNA transcripts detected on Northern blots. cDNA cloning of these transcripts suggests that they are expressed from different genes as well as by alternate splicing mechanisms. Furthermore, the genomic organization of the cluster appears to consist of at least two identical promoters displaying CpG islands characteristic of housekeeping genes, yet clearly involving tissue-specific expression. Sense and anti-sense synthetic RNA probes were derived from a common DNA sequence on 3 cDNA clones and hybridized to 8-16 days mouse embryonic stages and mouse adult brain sections. Expression in development was noticeable as of the 11th day of gestation and confined to the central nervous system mainly in the telencephalon and spinal cord. Coronal and sagittal sections of the adult mouse brain showed expression only in 3 different regions of the brain stem. In situ hybridization to mouse hypothalamus sections revealed the presence of a localized and specialized group of cells expressing high levels of mRNA, suggesting that this gene cluster may also be involved in the regulation of hypothalamic activities. The hypothalamus has long been hypothesized as a primary candidate tissue for the expression of the obesity gene mainly because of its well-established role in the regulation of energy metabolism and food intake.

  2. Expression, purification, crystallization and preliminary crystallographic study of isolated modules of the mouse coactivator-associated arginine methyltransferase 1

    Energy Technology Data Exchange (ETDEWEB)

    Troffer-Charlier, Nathalie; Cura, Vincent; Hassenboehler, Pierre; Moras, Dino; Cavarelli, Jean, E-mail: cava@igbmc.u-strasbg.fr [IGBMC (Institut de Génétique et de Biologie Moléculaire et Cellulaire), Département de Biologie et Génomique Structurales, 1 Rue Laurent Fries, Illkirch, F-67404 (France); INSERM, U596, Illkirch, F-67400 (France); CNRS, UMR7104, Illkirch, F-67400 (France); Université Louis Pasteur, Faculté des Sciences de la Vie, Strasbourg, F-67000 (France)

    2007-04-01

    Isolated modules of mouse coactivator-associated arginine methyltransferase 1 encompassing the protein arginine N-methyltransferase catalytic domain have been overexpressed, purified and crystallized. X-ray diffraction data have been collected and have enabled determination of the structures by multiple isomorphous replacement using anomalous scattering. Coactivator-associated arginine methyltransferase 1 (CARM1) plays a crucial role in gene expression as a coactivator of several nuclear hormone receptors and also of non-nuclear receptor systems. Its recruitment by the transcriptional machinery induces protein methylation, leading to chromatin remodelling and gene activation. CARM1{sub 28–507} and two structural states of CARM1{sub 140–480} were expressed, purified and crystallized. Crystals of CARM1{sub 28–507} belong to space group P6{sub 2}22, with unit-cell parameters a = b = 136.0, c = 125.3 Å; they diffract to beyond 2.5 Å resolution using synchrotron radiation and contain one monomer in the asymmetric unit. The structure of CARM1{sub 28–507} was solved by multiple isomorphous replacement and anomalous scattering methods. Crystals of apo CARM1{sub 140–480} belong to space group I222, with unit-cell parameters a = 74.6, b = 99.0, c = 207.4 Å; they diffract to beyond 2.7 Å resolution and contain two monomers in the asymmetric unit. Crystals of CARM1{sub 140–480} in complex with S-adenosyl-l-homocysteine belong to space P2{sub 1}2{sub 1}2, with unit-cell parameters a = 74.6, b = 98.65, c = 206.08 Å; they diffract to beyond 2.6 Å resolution and contain four monomers in the asymmetric unit. The structures of apo and holo CARM1{sub 140–480} were solved by molecular-replacement techniques from the structure of CARM1{sub 28–507}.

  3. Effects of salicylic acid on post-ischaemic ventricular function and purine efflux in isolated mouse hearts.

    Science.gov (United States)

    Farthing, Don; Gehr, Lynne; Karnes, H Thomas; Sica, Domenic; Gehr, Todd; Larus, Terri; Farthing, Christine; Xi, Lei

    2007-01-01

    Acetyl salicylic acid (aspirin) is one of the most widely used drugs in the world. Various plasma concentrations of aspirin and its predominant metabolite, salicylic acid, are required for its antiarthritic (1.5-2.5 mM), anti-inflammatory (0.5-5.0 mM) or antiplatelet (0.18-0.36 mM) actions. A recent study demonstrated the inhibitory effects of both aspirin and salicylic acid on oxidative phosphorylation and ATP synthesis in isolated rat cardiac mitochondria in a dose-dependent manner (0-10 mM concentration range). In this context, the present study was conducted to determine the effects of salicylic acid on inosine efflux (a potential biomarker of acute cardiac ischaemia) as well as cardiac contractile function in the isolated mouse heart following 20 min of zero-flow global ischaemia. Inosine efflux was found at significantly higher concentrations in ischaemic hearts perfused with Krebs buffer fortified with 1.0 mM salicylic acid compared with those without salicylic acid (12575+/-3319 vs. 1437+/-348 ng ml(-1) min(-1), mean+/-SEM, n=6 per group, psalicylic acid potentiates 8.8-fold ATP nucleotide purine catabolism into its metabolites (e.g. inosine, hypoxanthine). Salicylic acid (0.1 or 1.0 mM) did not appreciably inhibit purine nucleoside phosphorylase (the enzyme converts inosine to hypoxanthine) suggesting the augmented inosine efflux was due to the salicylic acid effect on upstream elements of cellular respiration. Whereas post-ischaemic cardiac function was further depressed by 1.0 mM salicylic acid, perfusion with 0.1 mM salicylic acid led to a remarkable functional improvement despite moderately increased inosine efflux (2.7-fold). We conclude that inosine is a sensitive biomarker for detecting cardiac ischaemia and salicylic acid-induced effects on cellular respiration. However, the inosine efflux level appears to be a poor predictor of the individual post-ischaemic cardiac functional recovery in this ex vivo model.

  4. Social isolation stress-induced oxidative damage in mouse brain and its modulation by majonoside-R2, a Vietnamese ginseng saponin.

    Science.gov (United States)

    Huong, Nguyen Thi Thu; Murakami, Yukihisa; Tohda, Michihisa; Watanabe, Hiroshi; Matsumoto, Kinzo

    2005-08-01

    Stressors with a physical factor such as immobilization, electric foot shock, cold swim, etc., have been shown to produce oxidative damage to membrane lipids in the brain. In this study, we investigated the effect of protracted social isolation stress on lipid peroxidation activity in the mouse brain and elucidated the protective effect of majonoside-R2, a major saponin component of Vietnamese ginseng, in mice exposed to social isolation stress. Thiobarbituric acid reactive substance levels, one of the end products of lipid peroxidation reaction, were increased in the brains of mice subjected to 6-8 weeks of social isolation stress. Measurements of nitric oxide (NO) metabolites (NO(x)(-)) also revealed a significant increase of NO production in the brains of socially isolated mice. Moreover, the depletion of brain glutathione content, an endogenous antioxidant, in socially isolated animals occurred in association with the rise in lipid peroxidation. The intraperitoneal administration of majonoside-R2 (10-50 mg/kg) had no effect on thiobarbituric acid reactive substances (TBARS), NO, or glutathione levels in the brains of group-housed control mice but it significantly suppressed the increase in TBARS and NO levels and the decrease in glutathione levels caused by social isolation stress. These results suggest that mice subjected to 6-8 weeks of social isolation stress produces oxidative damage in the brain partly via enhancement of NO production, and that majonoside-R2 exerts a protective effect by modulating NO and glutathione systems in the brain.

  5. Social isolation stress and chronic glutathione deficiency have a common effect on the glutamine-to-glutamate ratio and myo-inositol concentration in the mouse frontal cortex.

    Science.gov (United States)

    Corcoba, Alberto; Gruetter, Rolf; Do, Kim Q; Duarte, João M N

    2017-09-01

    Environmental stress can interact with genetic predisposition to increase the risk of developing psychopathology. In this work, we tested the hypothesis that social isolation stress interacts with impaired glutathione synthesis and have cumulative effects on the neurochemical profile of the frontal cortex. A mouse model with chronic glutathione deficit induced by knockout (-/-) of the glutamate-cysteine ligase modulatory subunit (Gclm) was exposed to social isolation stress from weaning to post-natal day 65. Using magnetic resonance methods at high-field (14.1 T), we analysed the neurochemical profile in the frontal cortex, brain size and ventricular volume of adult animals. Glutathione deficit was accompanied by elevated concentrations of N-acetylaspartate, alanine, and glutamine, as well as the ratio of glutamine-to-glutamate (Gln/Glu), and by a reduction in levels of myo-inositol and choline-containing compounds in the frontal cortex of -/- animals with respect to wild-type littermates. Although there was no significant interaction between social isolation stress and glutathione deficiency, mice reared in isolation displayed lower myo-inositol concentration (-8.4%, p social isolation had no effect on these parameters. We conclude that social isolation caused neurochemical alterations that may add to those associated to impaired glutathione synthesis. © 2017 International Society for Neurochemistry.

  6. Efficient isolation of pure and functional mitochondria from mouse tissues using automated tissue disruption and enrichment with anti-TOM22 magnetic beads.

    Directory of Open Access Journals (Sweden)

    Andras Franko

    Full Text Available To better understand molecular mechanisms regulating changes in metabolism, as observed e.g. in diabetes or neuronal disorders, the function of mitochondria needs to be precisely determined. The usual isolation methods such as differential centrifugation result in isolates of highly variable quality and quantity. To fulfill the need of a reproducible isolation method from solid tissues, which is suitable to handle parallel samples simultaneously, we developed a protocol based on anti-TOM22 (translocase of outer mitochondrial membrane 22 homolog antibody-coupled magnetic beads. To measure oxygen consumption rate in isolated mitochondria from various mouse tissues, a traditional Clark electrode and the high-throughput XF Extracellular Flux Analyzer were used. Furthermore, Western blots, transmission electron microscopic and proteomic studies were performed to analyze the purity and integrity of the mitochondrial preparations. Mitochondrial fractions isolated from liver, brain and skeletal muscle by anti-TOM22 magnetic beads showed oxygen consumption capacities comparable to previously reported values and little contamination with other organelles. The purity and quality of isolated mitochondria using anti-TOM22 magnetic beads was compared to traditional differential centrifugation protocol in liver and the results indicated an obvious advantage of the magnetic beads method compared to the traditional differential centrifugation technique.

  7. Intramacrophage survival of uropathogenic Escherichia coli: Differences between diverse clinical isolates and between mouse and human macrophages

    KAUST Repository

    Bokil, Nilesh J.; Totsika, Makrina; Carey, Alison J.; Stacey, Katryn J.; Hancock, Viktoria; Saunders, Bernadette M.; Ravasi, Timothy; Ulett, Glen C.; Schembri, Mark A.; Sweet, Matthew J.

    2011-01-01

    within the host. Given that many intracellular pathogens target macrophages, we assessed the interactions between UPEC and macrophages. Colonization of the mouse bladder by UPEC strain CFT073 resulted in increased expression of myeloid-restricted genes

  8. LH-21 and abnormal cannabidiol improve β-cell function in isolated human and mouse islets through GPR55-dependent and -independent signalling.

    Science.gov (United States)

    Ruz-Maldonado, Inmaculada; Pingitore, Attilio; Liu, Bo; Atanes, Patricio; Huang, Guo Cai; Baker, David; Alonso, Francisco José; Bermúdez-Silva, Francisco Javier; Persaud, Shanta J

    2018-04-01

    To examine the effects of Abn-CBD (GPR55 agonist) and LH-21 (CB1 antagonist) on human and mouse islet function, and to determine signalling via GPR55 using islets from GPR55 -/- mice. Islets isolated from human organ donors and mice were incubated in the absence or presence of Abn-CBD or LH-21, and insulin secretion, [Ca 2+ ] i, cAMP , apoptosis, β-cell proliferation and CREB and AKT phosphorylation were examined using standard techniques. Abn-CBD potentiated glucose-stimulated insulin secretion and elevated [Ca 2+ ] i in human islets and islets from both GPR55 +/+ and GPR55 -/- mice. LH-21 also increased insulin secretion and [Ca 2+ ] i in human islets and GPR55 +/+ mouse islets, but concentrations of LH-21 up to 0.1 μM were ineffective in islets from GPR55 -/- mice. Neither ligand affected basal insulin secretion or islet cAMP levels. Abn-CBD and LH-21 reduced cytokine-induced apoptosis in human islets and GPR55 +/+ mouse islets, and these effects were suppressed after GPR55 deletion. They also increased β-cell proliferation: the effects of Abn-CBD were preserved in islets from GPR55 -/- mice, while those of LH-21 were abolished. Abn-CBD and LH-21 increased AKT phosphorylation in mouse and human islets. This study showed that Abn-CBD and LH-21 improve human and mouse islet β-cell function and viability. Use of islets from GPR55 -/- mice suggests that designation of Abn-CBD and LH-21 as a GPR55 agonist and a CB1 antagonist, should be revised. © 2017 John Wiley & Sons Ltd.

  9. Effects of Lactobacillus kefiranofaciens M1 isolated from kefir grains on enterohemorrhagic Escherichia coli infection using mouse and intestinal cell models.

    Science.gov (United States)

    Chen, Y P; Lee, T Y; Hong, W S; Hsieh, H H; Chen, M J

    2013-01-01

    A potential probiotic strain, Lactobacillus kefiranofaciens M1, was previously isolated from kefir grains, which are used to manufacture the traditional fermented drink kefir. The aim of this study was to investigate the effects of Lb. kefiranofaciens M1 on enterohemorrhagic Escherichia coli (EHEC) infection, using mice and intestinal cell models. BALB/c mice were daily administrated with either phosphate buffered saline or Lb. kefiranofaciens M1 at 2×10(8) cfu/mouse per day intragastrically for 7 d. Intragastric challenges with EHEC (2×10(9) cfu/mouse) were conducted on d 0, 4, and 7 after treatment. Administration of Lb. kefiranofaciens M1 was able to prevent EHEC infection-induced symptoms, intestinal damage, renal damage, bacterial translocation, and Shiga toxin penetration. Furthermore, the mucosal EHEC-specific IgA responses were increased after Lb. kefiranofaciens M1 administration in the EHEC-infected mouse system. Additionally, in vitro, Lb. kefiranofaciens M1 was shown to have a protective effect on Caco-2 intestinal epithelial cells and Caco-2 intestinal epithelial cell monolayers; the bacteria limited EHEC-induced cell death and reduced the loss of epithelial integrity. These findings support the potential of Lb. kefiranofaciens M1 treatment as an approach to preventing EHEC infection and its effects. Copyright © 2013 American Dairy Science Association. Published by Elsevier Inc. All rights reserved.

  10. Reversed-phase high-performance liquid chromatographic analyses of insulin biosynthesis in isolated rat and mouse islets

    DEFF Research Database (Denmark)

    Linde, S; Hansen, Bruno A.; Welinder, B S

    1989-01-01

    deletion compared to rat C-peptide I. A marked species difference in the ratio between insulin I and II was observed, i.e., 2:1 in the rat and 1:2 in the mouse. Pulse-chase experiments in rat islets have demonstrated that the ratio between insulin I and II in newly synthesized insulin is higher than...

  11. Electron microscopy and in vitro deneddylation reveal similar architectures and biochemistry of isolated human and Flag-mouse COP9 signalosome complexes

    International Nuclear Information System (INIS)

    Rockel, Beate; Schmaler, Tilo; Huang, Xiaohua; Dubiel, Wolfgang

    2014-01-01

    Highlights: • Deneddylation rates of human erythrocyte and mouse fibroblast CSN are very similar. • 3D models of native human and mouse CSN reveal common architectures. • The cryo-structure of native mammalian CSN shows a horseshoe subunit arrangement. - Abstract: The COP9 signalosome (CSN) is a regulator of the ubiquitin (Ub) proteasome system (UPS). In the UPS, proteins are Ub-labeled for degradation by Ub ligases conferring substrate specificity. The CSN controls a large family of Ub ligases called cullin-RING ligases (CRLs), which ubiquitinate cell cycle regulators, transcription factors and DNA damage response proteins. The CSN possesses structural similarities with the 26S proteasome Lid complex and the translation initiation complex 3 (eIF3) indicating similar ancestry and function. Initial structures were obtained 14 years ago by 2D electron microscopy (EM). Recently, first 3D molecular models of the CSN were created on the basis of negative-stain EM and single-particle analysis, mostly with recombinant complexes. Here, we compare deneddylating activity and structural features of CSN complexes purified in an elaborate procedure from human erythrocytes and efficiently pulled down from mouse Flag-CSN2 B8 fibroblasts. In an in vitro deneddylation assay both the human and the mouse CSN complexes deneddylated Nedd8-Cul1 with comparable rates. 3D structural models of the erythrocyte CSN as well as of the mouse Flag-CSN were generated by negative stain EM and by cryo-EM. Both complexes show a central U-shaped segment from which several arms emanate. This structure, called the horseshoe, is formed by the PCI domain subunits. CSN5 and CSN6 point away from the horseshoe. Compared to 3D models of negatively stained CSN complexes, densities assigned to CSN2 and CSN4 are better defined in the cryo-map. Because biochemical and structural results obtained with CSN complexes isolated from human erythrocytes and purified by Flag-CSN pulldown from mouse B8 fibroblasts

  12. Electron microscopy and in vitro deneddylation reveal similar architectures and biochemistry of isolated human and Flag-mouse COP9 signalosome complexes

    Energy Technology Data Exchange (ETDEWEB)

    Rockel, Beate [Department of Molecular Structural Biology, Max-Planck-Institute of Biochemistry, Am Klopferspitz 18, 82152 Martinsried (Germany); Schmaler, Tilo; Huang, Xiaohua [Division of Molecular Biology, Department of General, Visceral, Vascular and Thoracic Surgery, Charité – Universitätsmedizin Berlin, Charitéplatz 1, 10117 Berlin (Germany); Dubiel, Wolfgang, E-mail: Wolfgang.dubiel@charite.de [Division of Molecular Biology, Department of General, Visceral, Vascular and Thoracic Surgery, Charité – Universitätsmedizin Berlin, Charitéplatz 1, 10117 Berlin (Germany)

    2014-07-25

    Highlights: • Deneddylation rates of human erythrocyte and mouse fibroblast CSN are very similar. • 3D models of native human and mouse CSN reveal common architectures. • The cryo-structure of native mammalian CSN shows a horseshoe subunit arrangement. - Abstract: The COP9 signalosome (CSN) is a regulator of the ubiquitin (Ub) proteasome system (UPS). In the UPS, proteins are Ub-labeled for degradation by Ub ligases conferring substrate specificity. The CSN controls a large family of Ub ligases called cullin-RING ligases (CRLs), which ubiquitinate cell cycle regulators, transcription factors and DNA damage response proteins. The CSN possesses structural similarities with the 26S proteasome Lid complex and the translation initiation complex 3 (eIF3) indicating similar ancestry and function. Initial structures were obtained 14 years ago by 2D electron microscopy (EM). Recently, first 3D molecular models of the CSN were created on the basis of negative-stain EM and single-particle analysis, mostly with recombinant complexes. Here, we compare deneddylating activity and structural features of CSN complexes purified in an elaborate procedure from human erythrocytes and efficiently pulled down from mouse Flag-CSN2 B8 fibroblasts. In an in vitro deneddylation assay both the human and the mouse CSN complexes deneddylated Nedd8-Cul1 with comparable rates. 3D structural models of the erythrocyte CSN as well as of the mouse Flag-CSN were generated by negative stain EM and by cryo-EM. Both complexes show a central U-shaped segment from which several arms emanate. This structure, called the horseshoe, is formed by the PCI domain subunits. CSN5 and CSN6 point away from the horseshoe. Compared to 3D models of negatively stained CSN complexes, densities assigned to CSN2 and CSN4 are better defined in the cryo-map. Because biochemical and structural results obtained with CSN complexes isolated from human erythrocytes and purified by Flag-CSN pulldown from mouse B8 fibroblasts

  13. Isolation of mouse mesenchymal stem cells with normal ploidy from bone marrows by reducing oxidative stress in combination with extracellular matrix

    Directory of Open Access Journals (Sweden)

    Wang Fang

    2011-07-01

    Full Text Available Abstract Background Isolation of mouse MSCs (mMSCs with normal ploidy from bone marrow remains challenging. mMSCs isolated under 20% O2 are frequently contaminated by overgrown hematopoietic cells, and could also be especially vulnerable to oxidative damage, resulting in chromosomal instability. Culture under low oxygen or extracellular matrix (ECM improves proliferation of MSCs in several species. We tested the hypothesis that culture under low oxygen in combination with ECM prepared from mouse embryonic fibroblast (MEF-ECM could be used to purify proliferative mMSCs, and to reduce oxidative damage and maintain their chromosomal stability. Results Optimization of culture conditions under 20% O2 resulted in immortalization of mMSCs, showing extensive chromosome abnormalities, consistent with previous studies. In contrast, culture under low oxygen (2% O2 improved proliferation of mMSCs and reduced oxidative damage, such that mMSCs were purified simply by plating at low density under 2% O2. MEF-ECM reduced oxidative damage and enhanced proliferation of mMSCs. However, these isolated mMSCs still exhibited high frequency of chromosome abnormalities, suggesting that low oxygen or in combination with MEF-ECM was insufficient to fully protect mMSCs from oxidative damage. Notably, antioxidants (alpha -phenyl-t-butyl nitrone (PBN and N-acetylcysteine (NAC further reduced DNA damage and chromosomal abnormalities, and increased proliferation of mMSCs. mMSCs isolated by the combination method were successfully used to generate induced pluripotent stem (iPS cells by ectopic expression of Oct4, Sox2, Klf4 and c-Myc. Conclusions We have developed a technique that allows to reduce the number of karyotypic abnormalities for isolation of primary mMSCs and for limited culture period by combination of low oxygen, MEF-ECM, antioxidants and low density plating strategy. The effectiveness of the new combination method is demonstrated by successful generation of i

  14. Phosphorylation of intact erythrocytes in human muscular dystrophy

    International Nuclear Information System (INIS)

    Johnson, R.M.; Nigro, M.

    1986-01-01

    The uptake of exogenous 32 Pi into the membrane proteins of intact erythrocytes was measured in 8 patients with Duchenne muscular dystrophy. No abnormalities were noted after autoradiographic analysis. This contrasts with earlier results obtained when isolated membranes were phosphorylated with gamma-[ 32 P]ATP, and suggests a possible reinterpretation of those experiments

  15. Purple Corn (Zea mays L.) Phenolic Compounds Profile and Its Assessment as an Agent Against Oxidative Stress in Isolated Mouse Organs

    Science.gov (United States)

    Ramos-Escudero, Fernando; Muñoz, Ana María; Alvarado-Ortíz, Carlos; Alvarado, Ángel

    2012-01-01

    Abstract This study was designed to determine the contents of total polyphenols, flavonoids, flavonols, flavanols, and anthocyanins of purple corn (Zea mays L.) extracts obtained with different methanol:water concentrations, acidified with 1% HCl (1 N). Another objective was to determine the antioxidant activity by 1,1-diphenyl-2-picrylhydrazyl (DPPH), 2,2′-azinobis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS), ferric reducing antioxidant power (FRAP), and deoxyribose assay, individual phenolic compounds by high-performance liquid chromatography (HPLC), and endogenous antioxidant enzyme (superoxide dismutase [SOD], catalase [CAT], and total peroxidase [TPX]) activity and lipid peroxidation activity (thiobarbituric acid–reactive substances [TBARS] assay) in isolated mouse organs. Overall, the highest total content of polyphenols, anthocyanins, flavonoids, flavonols, and flavanols was obtained with the 80:20 methanol:water extract, acidified with 1% HCl (1 N). The 50% inhibitory concentration values obtained by the DPPH and ABTS assays with this extract were 66.3 μg/mL and 250 μg/mL, respectively. The antioxidant activity by the FRAP assay was 26.1 μM Trolox equivalents/g, whereas the deoxyribose assay presented 93.6% inhibition. Because of these results, the 80:20 methanol:water extract, acidified with 1% HCl (1 N), was used for the remaining tests. Eight phenolic compounds were identified by HPLC: chlorogenic acid, caffeic acid, rutin, ferulic acid, morin, quercetin, naringenin, and kaempferol. Furthermore, it was observed that the purple corn extract was capable of significantly reducing lipid peroxidation (lower malondialdehyde [MDA] concentrations by the TBARS assay) and at the same time increasing endogenous antioxidant enzyme (CAT, TPX, and SOD) activities in isolated mouse kidney, liver, and brain. On the basis of the results, it was concluded that the purple corn extract contained various bioactive phenolic compounds that exhibited

  16. Purple corn (Zea mays L.) phenolic compounds profile and its assessment as an agent against oxidative stress in isolated mouse organs.

    Science.gov (United States)

    Ramos-Escudero, Fernando; Muñoz, Ana María; Alvarado-Ortíz, Carlos; Alvarado, Ángel; Yáñez, Jaime A

    2012-02-01

    This study was designed to determine the contents of total polyphenols, flavonoids, flavonols, flavanols, and anthocyanins of purple corn (Zea mays L.) extracts obtained with different methanol:water concentrations, acidified with 1% HCl (1 N). Another objective was to determine the antioxidant activity by 1,1-diphenyl-2-picrylhydrazyl (DPPH), 2,2'-azinobis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS), ferric reducing antioxidant power (FRAP), and deoxyribose assay, individual phenolic compounds by high-performance liquid chromatography (HPLC), and endogenous antioxidant enzyme (superoxide dismutase [SOD], catalase [CAT], and total peroxidase [TPX]) activity and lipid peroxidation activity (thiobarbituric acid-reactive substances [TBARS] assay) in isolated mouse organs. Overall, the highest total content of polyphenols, anthocyanins, flavonoids, flavonols, and flavanols was obtained with the 80:20 methanol:water extract, acidified with 1% HCl (1 N). The 50% inhibitory concentration values obtained by the DPPH and ABTS assays with this extract were 66.3 μg/mL and 250 μg/mL, respectively. The antioxidant activity by the FRAP assay was 26.1 μM Trolox equivalents/g, whereas the deoxyribose assay presented 93.6% inhibition. Because of these results, the 80:20 methanol:water extract, acidified with 1% HCl (1 N), was used for the remaining tests. Eight phenolic compounds were identified by HPLC: chlorogenic acid, caffeic acid, rutin, ferulic acid, morin, quercetin, naringenin, and kaempferol. Furthermore, it was observed that the purple corn extract was capable of significantly reducing lipid peroxidation (lower malondialdehyde [MDA] concentrations by the TBARS assay) and at the same time increasing endogenous antioxidant enzyme (CAT, TPX, and SOD) activities in isolated mouse kidney, liver, and brain. On the basis of the results, it was concluded that the purple corn extract contained various bioactive phenolic compounds that exhibited considerable in vitro

  17. Isolation, Culture, Functional Assays, and Immunofluorescence of Myofiber-Associated Satellite Cells.

    Science.gov (United States)

    Vogler, Thomas O; Gadek, Katherine E; Cadwallader, Adam B; Elston, Tiffany L; Olwin, Bradley B

    2016-01-01

    Adult skeletal muscle stem cells, termed satellite cells, regenerate and repair the functional contractile cells in adult skeletal muscle called myofibers. Satellite cells reside in a niche between the basal lamina and sarcolemma of myofibers. Isolating single myofibers and their associated satellite cells provides a culture system that partially mimics the in vivo environment. We describe methods for isolating and culturing intact individual myofibers and their associated satellite cells from the mouse extensor digitorum longus muscle. Following dissection and isolation of individual myofibers we provide protocols for myofiber transplantation, satellite cell transfection, immune detection of satellite cell antigens, and assays to examine satellite cell self-renewal and proliferation.

  18. Social isolation mediated anxiety like behavior is associated with enhanced expression and regulation of BDNF in the female mouse brain.

    Science.gov (United States)

    Kumari, Anita; Singh, Padmanabh; Baghel, Meghraj Singh; Thakur, M K

    2016-05-01

    Adverse early life experience is prominent risk factors for numerous psychiatric illnesses, including mood and anxiety disorders. It imposes serious long-term costs on the individual as well as health and social systems. Hence, developing therapies that prevent the long-term consequences of early life stress is of utmost importance, and necessitates a better understanding of the mechanisms by which early life stress triggers long-lasting alterations in gene expression and behavior. Post-weaning isolation rearing of rodents models the behavioral consequences of adverse early life experiences in humans and it is reported to cause anxiety like behavior which is more common in case of females. Therefore, in the present study, we have studied the impact of social isolation of young female mice for 8weeks on the anxiety like behavior and the underlying molecular mechanism. Elevated plus maze and open field test revealed that social isolation caused anxiety like behavior. BDNF, a well-known molecule implicated in the anxiety like behavior, was up-regulated both at the message and protein level in cerebral cortex by social isolation. CREB-1 and CBP, which play a crucial role in BDNF transcription, were up-regulated at mRNA level in cerebral cortex by social isolation. HDAC-2, which negatively regulates BDNF expression, was down-regulated at mRNA and protein level in cerebral cortex by social isolation. Furthermore, BDNF acts in concert with Limk-1, miRNA-132 and miRNA-134 for the regulation of structural and morphological plasticity. Social isolation resulted in up-regulation of Limk-1 mRNA and miRNA-132 expression in the cerebral cortex. MiRNA-134, which inhibits the translation of Limk-1, was decreased in cerebral cortex by social isolation. Taken together, our study suggests that social isolation mediated anxiety like behavior is associated with up-regulation of BDNF expression and concomitant increase in the expression of CBP, CREB-1, Limk-1 and miRNA-132, and decrease

  19. Neuronal activity in the isolated mouse spinal cord during spontaneous deletions in fictive locomotion: insights into locomotor central pattern generator organization

    Science.gov (United States)

    Zhong, Guisheng; Shevtsova, Natalia A; Rybak, Ilya A; Harris-Warrick, Ronald M

    2012-01-01

    We explored the organization of the spinal central pattern generator (CPG) for locomotion by analysing the activity of spinal interneurons and motoneurons during spontaneous deletions occurring during fictive locomotion in the isolated neonatal mouse spinal cord, following earlier work on locomotor deletions in the cat. In the isolated mouse spinal cord, most spontaneous deletions were non-resetting, with rhythmic activity resuming after an integer number of cycles. Flexor and extensor deletions showed marked asymmetry: flexor deletions were accompanied by sustained ipsilateral extensor activity, whereas rhythmic flexor bursting was not perturbed during extensor deletions. Rhythmic activity on one side of the cord was not perturbed during non-resetting spontaneous deletions on the other side, and these deletions could occur with no input from the other side of the cord. These results suggest that the locomotor CPG has a two-level organization with rhythm-generating (RG) and pattern-forming (PF) networks, in which only the flexor RG network is intrinsically rhythmic. To further explore the neuronal organization of the CPG, we monitored activity of motoneurons and selected identified interneurons during spontaneous non-resetting deletions. Motoneurons lost rhythmic synaptic drive during ipsilateral deletions. Flexor-related commissural interneurons continued to fire rhythmically during non-resetting ipsilateral flexor deletions. Deletion analysis revealed two classes of rhythmic V2a interneurons. Type I V2a interneurons retained rhythmic synaptic drive and firing during ipsilateral motor deletions, while type II V2a interneurons lost rhythmic synaptic input and fell silent during deletions. This suggests that the type I neurons are components of the RG, whereas the type II neurons are components of the PF network. We propose a computational model of the spinal locomotor CPG that reproduces our experimental results. The results may provide novel insights into the

  20. Isolation and Molecular Profiling of Primary Mouse Retinal Ganglion Cells: Comparison of Phenotypes from Healthy and Glaucomatous Retinas

    OpenAIRE

    Chintalapudi, Sumana R.; Djenderedjian, Levon; Stiemke, Andrew B.; Steinle, Jena J.; Jablonski, Monica M.; Morales-Tirado, Vanessa M.

    2016-01-01

    Loss of functional retinal ganglion cells (RGC) is an element of retinal degeneration that is poorly understood. This is in part due to the lack of a reliable and validated protocol for the isolation of primary RGCs. Here we optimize a feasible, reproducible, standardized flow cytometry-based protocol for the isolation and enrichment of homogeneous RGC with the Thy1.2hiCD48negCD15negCD57neg surface phenotype. A three-step validation process was performed by: (1) genomic profiling of 25-genes ...

  1. A rapid, efficient, and economic device and method for the isolation and purification of mouse islet cells.

    Directory of Open Access Journals (Sweden)

    Yin Zongyi

    Full Text Available Rapid, efficient, and economic method for the isolation and purification of islets has been pursued by numerous islet-related researchers. In this study, we compared the advantages and disadvantages of our developed patented method with those of commonly used conventional methods (Ficoll-400, 1077, and handpicking methods. Cell viability was assayed using Trypan blue, cell purity and yield were assayed using diphenylthiocarbazone, and islet function was assayed using acridine orange/ethidium bromide staining and enzyme-linked immunosorbent assay-glucose stimulation testing 4 days after cultivation. The results showed that our islet isolation and purification method required 12 ± 3 min, which was significantly shorter than the time required in Ficoll-400, 1077, and HPU groups (34 ± 3, 41 ± 4, and 30 ± 4 min, respectively; P 1000 islets. In summary, the MCT method is a rapid, efficient, and economic method for isolating and purifying murine islet cell clumps. This method overcomes some of the shortcomings of conventional methods, showing a relatively higher quality and yield of islets within a shorter duration at a lower cost. Therefore, the current method provides researchers with an alternative option for islet isolation and should be widely generalized.

  2. Glucose metabolite patterns as markers of functional differentiation in freshly isolated and cultured mouse mammary epithelial cells

    International Nuclear Information System (INIS)

    Emerman, J.T.; Bartley, J.C.; Bissel, M.J.

    1981-01-01

    In the mammary gland of non-ruminant animals, glucose is utilized in a characteristic and unique way during lacation. By measuring the incorporation of glucose carbon from [U- 14 C]glucose into intermediary metabolitees and metabolic products in mammary epithelia cells from virgin, pregnant, and lacating mice, we domonstrate that glucose metabolite patterns can be used to recognize stages of differentiated function. For these cells, the rates of synthesis of glycogen and lactose, the ratio of lactate to alanine, and the ratio of citrate to malate are important parameters in identifying the degree of expression of differentiation. We further show that these patterns can be used as markers to determine the differentiated state of cultured mammary epithelial cells. Cells maintained on plastic substrates lose their distinctive glucose metabolite patterns while those on floating collagen gels do not. Cells isolated from pregnant mice and cultured on collagen gels have a pattern similar to that of their freshly isolated counter-parts. When isolated from lacating mice, the metabolite patterns of cells cultured on collagen gels are different from that of the cells of origin, and resembles that of freshly isolated cells from pregnant mice. Our findings suggest that the floating collagen gels under the culture conditions used in these experiments provide an environment for the functional expression of the pregnant state, while additional factors are needed for the expression of the lactating state

  3. The uncharacterized gene 1700093K21Rik and flanking regions are correlated with reproductive isolation in the house mouse, Mus musculus.

    Science.gov (United States)

    Kass, David H; Janoušek, Václav; Wang, Liuyang; Tucker, Priscilla K

    2014-06-01

    Reproductive barriers exist between the house mouse subspecies, Mus musculus musculus and M. m. domesticus, members of the Mus musculus species complex, primarily as a result of hybrid male infertility, and a hybrid zone exists where their ranges intersect in Europe. Using single nucleotide polymorphisms (SNPs) diagnostic for the two taxa, the extent of introgression across the genome was previously compared in these hybrid populations. Sixty-nine of 1316 autosomal SNPs exhibited reduced introgression in two hybrid zone transects suggesting maladaptive interactions among certain loci. One of these markers is within a region on chromosome 11 that, in other studies, has been associated with hybrid male sterility of these subspecies. We assessed sequence variation in a 20 Mb region on chromosome 11 flanking this marker, and observed its inclusion within a roughly 150 kb stretch of DNA showing elevated sequence differentiation between the two subspecies. Four genes are associated with this genomic subregion, with two entirely encompassed. One of the two genes, the uncharacterized 1700093K21Rik gene, displays distinguishing features consistent with a potential role in reproductive isolation between these subspecies. Along with its expression specifically within spermatogenic cells, we present various sequence analyses that demonstrate a high rate of molecular evolution of this gene, as well as identify a subspecies amino acid variant resulting in a structural difference. Taken together, the data suggest a role for this gene in reproductive isolation.

  4. Chaotic electrical activity of living β-cells in the mouse pancreatic islet

    Science.gov (United States)

    Kanno, Takahiro; Miyano, Takaya; Tokuda, Isao; Galvanovskis, Juris; Wakui, Makoto

    2007-02-01

    To test for chaotic dynamics of the insulin producing β-cell and explore its biological role, we observed the action potentials with the perforated patch clamp technique, for isolated cells as well as for intact cells of the mouse pancreatic islet. The time series obtained were analyzed using nonlinear diagnostic algorithms associated with the surrogate method. The isolated cells exhibited short-term predictability and visible determinism, in the steady state response to 10 mM glucose, while the intact cells did not. In the latter case, determinism became visible after the application of a gap junction inhibitor. This tendency was enhanced by the stimulation with tolbutamide. Our observations suggest that, thanks to the integration of individual chaotic dynamics via gap junction coupling, the β-cells will lose memory of fluctuations occurring at any instant in their electrical activity more rapidly with time. This is likely to contribute to the functional stability of the islet against uncertain perturbations.

  5. Immune-modulating effects in mouse dendritic cells of lactobacilli and bifidobacteria isolated from individuals following omnivorous, vegetarian and vegan diets.

    Science.gov (United States)

    Luongo, Diomira; Treppiccione, Lucia; Sorrentino, Alida; Ferrocino, Ilario; Turroni, Silvia; Gatti, Monica; Di Cagno, Raffaella; Sanz, Yolanda; Rossi, Mauro

    2017-09-01

    Lactobacilli and bifidobacteria play a primary role in modulation of gut immunity. By considering that microbiota composition depends on various factors, including diet, we asked whether functional differences could characterize faecal populations of lactobacilli and bifidobacteria isolated from individuals with different dietary habits. 155 healthy volunteers who followed omnivorous, ovo-lacto-vegetarian or vegan diets were recruited at four Italian centres (Turin, Parma, Bologna and Bari). Faecal samples were collected; lactobacilli and bifidobacteria were isolated on selective media and their immunomodulatory activity was tested in mouse dendritic cells (DCs). Pre-incubation with lactobacilli increased LPS-induced expression of the maturation markers CD80 and CD86, whereas pre-incubation with bifidobacteria decreased such expression. Analysis of the cytokine profile indicated that strains of both genera induced down-regulation of IL-12 and up-regulation of IL-10, whereas expression of TNF-α was not modulated. Notably, analysis of anti-inflammatory potential (IL-10/IL-12 ratio) showed that lactobacilli evoked a greater anti-inflammatory effect than did bifidobacteria in the omnivorous group (P<0.05). We also found significantly reduced anti-inflammatory potential in the bacterial strains isolated from Bari's volunteers in comparison with those from the cognate groups from the other centres. In conclusion, lactobacilli and bifidobacteria showed a genus-specific ability of modulating in vitro innate immunity associated with a specific dietary habit. Furthermore, the geographical area had a significant impact on the anti-inflammatory potential of some components of faecal microbiota. Copyright © 2017 Elsevier Ltd. All rights reserved.

  6. Incubating Isolated Mouse EDL Muscles with Creatine Improves Force Production and Twitch Kinetics in Fatigue Due to Reduction in Ionic Strength

    Science.gov (United States)

    Head, Stewart I.; Greenaway, Bronwen; Chan, Stephen

    2011-01-01

    Background Creatine supplementation can improve performance during high intensity exercise in humans and improve muscle strength in certain myopathies. In this present study, we investigated the direct effects of acute creatine incubation on isolated mouse fast-twitch EDL muscles, and examined how these effects change with fatigue. Methods and Results The extensor digitorum longus muscle from mice aged 12–14 weeks was isolated and stimulated with field electrodes to measure force characteristics in 3 different states: (i) before fatigue; (ii) immediately after a fatigue protocol; and (iii) after recovery. These served as the control measurements for the muscle. The muscle was then incubated in a creatine solution and washed. The measurement of force characteristics in the 3 different states was then repeated. In un-fatigued muscle, creatine incubation increased the maximal tetanic force. In fatigued muscle, creatine treatment increased the force produced at all frequencies of stimulation. Incubation also increased the rate of twitch relaxation and twitch contraction in fatigued muscle. During repetitive fatiguing stimulation, creatine-treated muscles took 55.1±9.5% longer than control muscles to lose half of their original force. Measurement of weight changes showed that creatine incubation increased EDL muscle mass by 7%. Conclusion Acute creatine application improves force production in isolated fast-twitch EDL muscle, and these improvements are particularly apparent when the muscle is fatigued. One likely mechanism for this improvement is an increase in Ca2+ sensitivity of contractile proteins as a result of ionic strength decreases following creatine incubation. PMID:21850234

  7. Incubating isolated mouse EDL muscles with creatine improves force production and twitch kinetics in fatigue due to reduction in ionic strength.

    Directory of Open Access Journals (Sweden)

    Stewart I Head

    Full Text Available BACKGROUND: Creatine supplementation can improve performance during high intensity exercise in humans and improve muscle strength in certain myopathies. In this present study, we investigated the direct effects of acute creatine incubation on isolated mouse fast-twitch EDL muscles, and examined how these effects change with fatigue. METHODS AND RESULTS: The extensor digitorum longus muscle from mice aged 12-14 weeks was isolated and stimulated with field electrodes to measure force characteristics in 3 different states: (i before fatigue; (ii immediately after a fatigue protocol; and (iii after recovery. These served as the control measurements for the muscle. The muscle was then incubated in a creatine solution and washed. The measurement of force characteristics in the 3 different states was then repeated. In un-fatigued muscle, creatine incubation increased the maximal tetanic force. In fatigued muscle, creatine treatment increased the force produced at all frequencies of stimulation. Incubation also increased the rate of twitch relaxation and twitch contraction in fatigued muscle. During repetitive fatiguing stimulation, creatine-treated muscles took 55.1±9.5% longer than control muscles to lose half of their original force. Measurement of weight changes showed that creatine incubation increased EDL muscle mass by 7%. CONCLUSION: Acute creatine application improves force production in isolated fast-twitch EDL muscle, and these improvements are particularly apparent when the muscle is fatigued. One likely mechanism for this improvement is an increase in Ca(2+ sensitivity of contractile proteins as a result of ionic strength decreases following creatine incubation.

  8. Isolation and characterization of a new cell line from spontaneous mouse mammary tumour, MBL-6, for in vivo cancer studies

    Directory of Open Access Journals (Sweden)

    Ladan Langroudi

    2017-12-01

    Full Text Available In search for treatments against breast cancer, cell lines are one of the basic resources, particularly as in vitro models. Additionally, animal models of cancer are used as the successive step in therapeutics research. In this regard, human breast cancer cell lines provide fundamental models in vitro. However, in vivo studies require immunodeficient mice, which lack the influence of other in vivo factors such as the native microenvironment and the immune system. There are few standard models to study the pathogenic mechanism at molecular level and cell signaling pathway of breast cancer. In this study, a new mouse breast cancer cell line, MBL-6, was successfully established and characterized from tissues of a spontaneous mammary tumor. The cell line had epithelial morphology, formed adherent monolayer, maintained continuously in vitro and was able to form new tumors when injected subcutaneously in syngeneic mice. The growth pattern and metastasis evaluations revealed a considerable in situ duration before invading distant organs. Real time polymerase chain reaction (PCR analysis showed the expression of ER-, PR- and Her-2 receptors. The chromosome analysis showed numerous chromosomal abnormalities. Aggressive tumorigenecity in tumorigenesis test and the IC50 to cyclophosphamide (CTX, celecoxib (CLX and cisplatin (CPN was also evaluated. The numerous tests performed on the new MBL-6 cell line suggest that it is in good quality and may be used in animal models of breast cancer studies.

  9. Dehydrodiconiferyl Alcohol Isolated from Cucurbita moschata Shows Anti-adipogenic and Anti-lipogenic Effects in 3T3-L1 Cells and Primary Mouse Embryonic Fibroblasts*

    Science.gov (United States)

    Lee, Junghun; Kim, Donghyun; Choi, Jonghyun; Choi, Hyounjeong; Ryu, Jae-Ha; Jeong, Jinhyun; Park, Eun-Jin; Kim, Seon-Hee; Kim, Sunyoung

    2012-01-01

    A water-soluble extract from the stems of Cucurbita moschata, code named PG105, was previously found to contain strong anti-obesity activities in a high fat diet-induced obesity mouse model. One of its biological characteristics is that it inhibits 3T3-L1 adipocyte differentiation. To isolate the biologically active compound(s), conventional solvent fractionation was performed, and the various fractions were tested for anti-adipogenic activity using Oil Red O staining method. A single spot on thin layer chromatography of the chloroform fraction showed a potent anti-adipogenic activity. When purified, the structure of its major component was resolved as dehydrodiconiferyl alcohol (DHCA), a lignan, by NMR and mass spectrometry analysis. In 3T3-L1 cells, synthesized DHCA significantly reduced the expression of several adipocyte marker genes, including peroxisome proliferator-activated receptor γ (Pparg), CCAAT/enhancer-binding protein α (Cebpa), fatty acid-binding protein 4 (Fabp4), sterol response element-binding protein-1c (Srebp1c), and stearoyl-coenzyme A desaturase-1 (Scd), and decreased lipid accumulation without affecting cell viability. DHCA also suppressed the mitotic clonal expansion of preadipocytes (an early event of adipogenesis), probably by suppressing the DNA binding activity of C/EBPβ, and lowered the production level of cyclinA and cyclin-dependent kinase 2 (Cdk2), coinciding with the decrease in DNA synthesis and cell division. In addition, DHCA directly inhibited the expression of SREBP-1c and SCD-1. Similar observations were made, using primary mouse embryonic fibroblasts. Taken together, our data indicate that DHCA may contain dual activities, affecting both adipogenesis and lipogenesis. PMID:22262865

  10. Electrophysiological properties of myocytes isolated from the mouse atrioventricular node: L-type ICa, IKr, If, and Na-Ca exchange.

    Science.gov (United States)

    Choisy, Stéphanie C; Cheng, Hongwei; Orchard, Clive H; James, Andrew F; Hancox, Jules C

    2015-11-01

    The atrioventricular node (AVN) is a key component of the cardiac pacemaker-conduction system. This study investigated the electrophysiology of cells isolated from the AVN region of adult mouse hearts, and compared murine ionic current magnitude with that of cells from the more extensively studied rabbit AVN. Whole-cell patch-clamp recordings of ionic currents, and perforated-patch recordings of action potentials (APs), were made at 35-37°C. Hyperpolarizing voltage commands from -40 mV elicited a Ba(2+)-sensitive inward rectifier current that was small at diastolic potentials. Some cells (Type 1; 33.4 ± 2.2 pF; n = 19) lacked the pacemaker current, If, whilst others (Type 2; 34.2 ± 1.5 pF; n = 21) exhibited a clear If, which was larger than in rabbit AVN cells. On depolarization from -40 mV L-type Ca(2+) current, IC a,L, was elicited with a half maximal activation voltage (V0.5) of -7.6 ± 1.2 mV (n = 24). IC a,L density was smaller than in rabbit AVN cells. Rapid delayed rectifier (IK r) tail currents sensitive to E-4031 (5 μmol/L) were observed on repolarization to -40 mV, with an activation V0.5 of -10.7 ± 4.7 mV (n = 8). The IK r magnitude was similar in mouse and rabbit AVN. Under Na-Ca exchange selective conditions, mouse AVN cells exhibited 5 mmol/L Ni-sensitive exchange current that was inwardly directed negative to the holding potential (-40 mV). Spontaneous APs (5.2 ± 0.5 sec(-1); n = 6) exhibited an upstroke velocity of 37.7 ± 16.2 V/s and ceased following inhibition of sarcoplasmic reticulum Ca(2+) release by 1 μmol/L ryanodine, implicating intracellular Ca(2+) cycling in murine AVN cell electrogenesis. © 2015 The Authors. Physiological Reports published by Wiley Periodicals, Inc. on behalf of the American Physiological Society and The Physiological Society.

  11. Comparison of the virulence of three H3N2 canine influenza virus isolates from Korea and China in mouse and Guinea pig models.

    Science.gov (United States)

    Xie, Xing; Na, Woonsung; Kang, Aram; Yeom, Minjoo; Yuk, Heejun; Moon, Hyoungjoon; Kim, Sung-Jae; Kim, Hyun-Woo; Kim, Jeong-Ki; Pang, Maoda; Wang, Yongshan; Liu, Yongjie; Song, Daesub

    2018-05-02

    Avian-origin H3N2 canine influenza virus (CIV) has been the most common subtype in Korea and China since 2007. Here, we compared the pathogenicity and transmissibility of three H3N2 CIV strains [Chinese CIV (JS/10), Korean CIV (KR/07), and Korean recombinant CIV between the classic H3N2 CIV and the pandemic H1N1 virus (MV/12)] in BALB/c mouse and guinea pig models. The pandemic H1N1 (CA/09) strain served as the control. BALB/c mice infected with H1N1 had high mortality and obvious body weight loss, whereas no overt disease symptoms were observed in mice inoculated with H3N2 CIV strains. The viral titers were higher in the group MV/12 than those in groups JS/10 and KR/07, while the mice infected with JS/10 showed higher viral titers in all tissues (except for the lung) than the mice infected with KR/07. The data obtained in guinea pigs also demonstrated that group MV/12 presented the highest loads in most of the tissues, followed by group JS/10 and KR/07. Also, direct contact transmissions of all the three CIV strains could be observed in guinea pigs, and for the inoculated and the contact groups, the viral titer of group MV/12 and KR/07 was higher than that of group JS/10 in nasal swabs. These findings indicated that the matrix (M) gene obtained from the pandemic H1N1 may enhance viral replication of classic H3N2 CIV; JS/10 has stronger viral replication ability in tissues as compared to KR/07, whereas KR/07 infected guinea pigs have more viral shedding than JS/10 infected guinea pigs. There exists a discrepancy in pathobiology among CIV isolates. Reverse genetics regarding the genomes of CIV isolates will be helpful to further explain the virus characteristics.

  12. Comparative costs of the Mouse Inoculation Test (MIT) and Virus Isolation in Cell Culture (VICC) for use in rabies diagnosis in Brazil.

    Science.gov (United States)

    Bones, Vanessa C; Gameiro, Augusto H; Castilho, Juliana G; Molento, Carla F M

    2015-05-01

    The decision to use laboratory animals rather than in vitro methods is frequently based on the financial costs involved, so the objective of our study was to compare the costs of performing the Mouse Inoculation Test (MIT) and Virus Isolation in Cell Culture (VICC) for use in rabies diagnosis in Brazil. Based on observations of laboratory routines at the Pasteur Institute, São Paulo, we listed the fixed cost (FC) and variable cost (VC) items necessary to perform both tests. Considering that 200 MITs are equivalent to 350 VICC assays, in terms of facilities and staff-hours needed per month, we calculated, for both tests, the average total cost per sample, the costs of the implementation of the laboratory structure, and the costs of routine use. With regard to absolute values, the total cost was mainly influenced by FC items, as they represented 60% of the cost for the MIT and 86% of the cost for VICC. A sample analysed by the MIT costs around 205% more than one analysed by using VICC. The MIT costs 74% and 406% more than VICC, when implementation costs and routine use per month, respectively, are taken into account. Our results can assist in the resolution of costing disputes that could hinder the replacement of animals for rabies diagnosis in Brazil. The method demonstrated here might also be useful for cost comparisons in other situations where animal use still continues when validated alternatives exist. 2015 FRAME.

  13. Patterns of cross-sensitivity in the responses of clonal subpopulations isolated from the RIF-1 mouse sarcoma to selected nitrosoureas and nitrogen mustards.

    Science.gov (United States)

    Reeve, J. G.; Wright, K. A.; Workman, P.

    1984-01-01

    The response of clonal subpopulations isolated from the RIF-1 mouse sarcoma to melphalan treatment is independent of cell ploidy, whereas a clear relationship exists between ploidy and cell sensitivity to CCNU treatment. In the present study RIF-1 clones have been exposed to nitrogen mustard, aniline mustard and chlorambucil, and to nitrosoureas BCNU, MeCCNU and chlorozotocin, in order to evaluate whether or not the different physiochemical and biological activities of these agents would affect the patterns of drug sensitivity obtained for melphalan and CCNU. Irrespective of the different lipophilicities, transport properties and chemical reactivities of the nitrogen mustards, RIF-1 clones showed the same pattern of sensitivity as previously observed for melphalan. Similarly, RIF-1 clones when exposed to nitrosoureas BCNU, MeCCNU and chlorozotocin, showed the same pattern of sensitivity as that obtained for CCNU exposure. These data suggest (a) that the variation in the sensitivity of RIF-1 clones to treatment by the nitrogen mustards is unlikely to reflect differences in either membrane permeability or in drug transport and (b) that the ploidy dependent nitrosourea responses shown by RIF-1 clones similarly do not reflect differences in drug uptake. PMID:6466534

  14. Diffusion of [2-14C]diazepam across hairless mouse skin and human skin

    International Nuclear Information System (INIS)

    Koch, R.L.; Palicharla, P.; Groves, M.J.

    1987-01-01

    The objectives of this study were to investigate the absorption of diazepam applied topically to the hairless mouse in vivo and to determine the diffusion of diazepam across isolated hairless mouse skin and human skin. [ 14 C]Diazepam was readily absorbed after topical administration to the intact hairless mouse, a total of 75.8% of the 14 C-label applied being recovered in urine and feces. Diazepam was found to diffuse across human and hairless mouse skin unchanged in experiments with twin-chambered diffusion cells. The variation in diffusion rate or the flux for both human and mouse tissues was greater among specimens than between duplicate or triplicate trials for a single specimen. Fluxes for mouse skin (stratum corneum, epidermis, and dermis) were greater than for human skin (stratum corneum and epidermis): 0.35-0.61 microgram/cm2/h for mouse skin vs 0.24-0.42 microgram/cm2/h for human skin. The permeability coefficients for mouse skin ranged from 1.4-2.4 X 10(-2)cm/h compared with 0.8-1.4 X 10(-2)cm/h for human skin. Although human stratum corneum is almost twice the thickness of that of the hairless mouse, the diffusion coefficients for human skin were 3-12 times greater (0.76-3.31 X 10(-6) cm2/h for human skin vs 0.12-0.27 X 10(-6) cm2/h for hairless mouse) because of a shorter lag time for diffusion across human skin. These differences between the diffusion coefficients and diffusion rates (or permeability coefficients) suggest that the presence of the dermis may present some barrier properties. In vitro the dermis may require complete saturation before the diazepam can be detected in the receiving chamber

  15. Isolation of a novel chronic lymphocytic leukemic (CLL) cell line and development of an in vivo mouse model of CLL.

    Science.gov (United States)

    Kellner, Joshua; Wierda, William; Shpall, Elizabeth; Keating, Michael; McNiece, Ian

    2016-01-01

    Leukemic cell lines have become important tools for studies of disease providing a monoclonal cell population that can be extensively expanded in vitro while preserving leukemic cellular characteristics. However, studies of chronic lymphocytic leukemia (CLL) have been impeded in part by the lack of continuous human cell lines. CLL cells have a high spontaneous apoptosis rate in vitro and exhibit minimal proliferation in xenograft models. Therefore, there is a need for development of primary CLL cell lines and we describe the isolation of such a line from the bone marrow of a CLL patient (17p deletion and TP53 mutation) which has been in long term culture for more than 12 months with continuous proliferation. The CLL cell line (termed MDA-BM5) which was generated in vitro with continuous co-culture on autologous stromal cells is CD19+CD5+ and shows an identical pattern of somatic hypermutation as determined in the patient's bone marrow (BM), confirming the origin of the cells from the original CLL clone. MDA-BM5 cells were readily transplantable in NOD/SCID gamma null mice (NSG) with disease developing in the BM, liver and spleen. BM cells from quaternary serial transplantation in NSG mice demonstrated the presence of CD19+CD5+ cells with Ig restricted to lambda which is consistent with the original patient cells. These studies describe a new CLL cell line from a patient with del(17p) that provides a unique model for in vitro and in vivo studies. Copyright © 2015 Elsevier Ltd. All rights reserved.

  16. [Isolation and purification of BMScs of GFP transgenic mouse using the method of adhering to cuture plastic in different time].

    Science.gov (United States)

    Li, Fu-Qiang; Zhou, Hong-Ying; Yang, Hui-Lun; Xiang, Tao; Mei, Yan; Hu, Huo-Zhen; Wang, Ting-Hua

    2006-03-01

    To adopt the method of adhering to culture plastic in different time for cultivating and purifying BMSCs of green fluorescent protein (GFP) transgenic mice. Bone marrow cells isolated from GFP transgenic mice are directly planted in culture flask and an exchange of the total volume medium is made at different time. Then the cells adhering to culture plastic are differently counted according to the cell types and are examined by immunohistochemistry using the antibodies of CD44, CD45 and CD54 in three days. Moreover, the cells after the exchange of the total volume medium in 4 hours, 8 hours and 24 hours are selected and successively subcultured down to the fifth passage. Then the result of amplification is calculated and the cells are examined by immunohistochemistry using the antibodies of CD44, CD45 and CD54. With the extending of the time for the first exchange of medium, the density of cells adhering to culture plastic increased accordingly, but the BMSCs proportion decreased. The cells after first exchange of medium in 4 hours had high BMSCs proportion but low BMSCs density, and the cells in 24 hours had high BMSCs density and low BMSCs proportion. However, the cells in 8-10 hours had high BMSCs density and also high BMSCs proportion. The subcultured BMSCs could stably express GFP. The method of adhering to culture plastic in different time for cultivating and purifying BMSCs of GFP transgenic mice is effective. It is suitable to make the first exchange of total volume medium in 8-10 hours. The subcultured cell has the capacity for amplification and will probably be a seed cell for the research of tissue engineering and gene therapy.

  17. Excitons in intact cells of photosynthetic bacteria.

    Science.gov (United States)

    Freiberg, Arvi; Pajusalu, Mihkel; Rätsep, Margus

    2013-09-26

    Live cells and regular crystals seem fundamentally incompatible. Still, effects characteristic to ideal crystals, such as coherent sharing of excitation, have been recently used in many studies to explain the behavior of several photosynthetic complexes, especially the inner workings of the light-harvesting apparatus of the oldest known photosynthetic organisms, the purple bacteria. To this date, there has been no concrete evidence that the same effects are instrumental in real living cells, leaving a possibility that this is an artifact of unnatural study conditions, not a real effect relevant to the biological operation of bacteria. Hereby, we demonstrate survival of collective coherent excitations (excitons) in intact cells of photosynthetic purple bacteria. This is done by using excitation anisotropy spectroscopy for tracking the temperature-dependent evolution of exciton bands in light-harvesting systems of increasing structural complexity. The temperature was gradually raised from 4.5 K to ambient temperature, and the complexity of the systems ranged from detergent-isolated complexes to complete bacterial cells. The results provide conclusive evidence that excitons are indeed one of the key elements contributing to the energetic and dynamic properties of photosynthetic organisms.

  18. Dihydrotestosterone treatment rescues the decline in protein synthesis as a result of sarcopenia in isolated mouse skeletal muscle fibres.

    Science.gov (United States)

    Wendowski, Oskar; Redshaw, Zoe; Mutungi, Gabriel

    2017-02-01

    Sarcopenia, the progressive decline in skeletal muscle mass and function with age, is a debilitating condition. It leads to inactivity, falls, and loss of independence. Despite this, its cause(s) and the underlying mechanism(s) are still poorly understood. In this study, small skeletal muscle fibre bundles isolated from the extensor digitorum longus (a fast-twitch muscle) and the soleus (a slow-twitch muscle) of adult mice of different ages (range 100-900 days old) were used to investigate the effects of ageing and dihydrotestosterone (DHT) treatment on protein synthesis as well as the expression and function of two amino acid transporters; the sodium-coupled neutral amino acid transporter (SNAT) 2, and the sodium-independent L-type amino-acid transporter (LAT) 2. At all ages investigated, protein synthesis was always higher in the slow-twitch than in the fast-twitch muscle fibres and decreased with age in both fibre types. However, the decline was greater in the fast-twitch than in the slow-twitch fibres and was accompanied by a reduction in the expression of SNAT2 and LAT2 at the protein level. Again, the decrease in the expression of the amino acid transporters was greater in the fast-twitch than in the slow-twitch fibres. In contrast, ageing had no effect on SNAT2 and LAT2 expressions at the mRNA level. Treating the muscle fibre bundles with physiological concentrations (~2 nM) of DHT for 1 h completely reversed the effects of ageing on protein synthesis and the expression of SNAT2 and LAT2 protein in both fibre types. From the observations that ageing is accompanied by a reduction in protein synthesis and transporter expression and that these effects are reversed by DHT treatment, we conclude that sarcopenia arises from an age-dependent reduction in protein synthesis caused, in part, by the lack of or by the low bioavailability of the male sex steroid, DHT.

  19. Strategies for Analyzing Data from Intact Groups.

    Science.gov (United States)

    Cross, Lawrence H.; Lane, Carolyn E.

    Action research often necessitates the use of intact groups for the comparison of educational treatments or programs. This paper considers several analytical methods that might be used for such situations when pretest scores indicate that these intact groups differ significantly initially. The methods considered include gain score analysis of…

  20. Exploratory investigations of hypervelocity intact capture spectroscopy

    Science.gov (United States)

    Tsou, P.; Griffiths, D. J.

    1993-01-01

    The ability to capture hypervelocity projectiles intact opens a new technique available for hypervelocity research. A determination of the reactions taking place between the projectile and the capture medium during the process of intact capture is extremely important to an understanding of the intact capture phenomenon, to improving the capture technique, and to developing a theory describing the phenomenon. The intact capture of hypervelocity projectiles by underdense media generates spectra, characteristic of the material species of projectile and capture medium involved. Initial exploratory results into real-time characterization of hypervelocity intact capture techniques by spectroscopy include ultra-violet and visible spectra obtained by use of reflecting gratings, transmitting gratings, and prisms, and recorded by photographic and electronic means. Spectrometry proved to be a valuable real-time diagnostic tool for hypervelocity intact capture events, offering understanding of the interactions of the projectile and the capture medium during the initial period and providing information not obtainable by other characterizations. Preliminary results and analyses of spectra produced by the intact capture of hypervelocity aluminum spheres in polyethylene (PE), polystyrene (PS), and polyurethane (PU) foams are presented. Included are tentative emission species identifications, as well as gray body temperatures produced in the intact capture process.

  1. Intact glycopeptide characterization using mass spectrometry

    OpenAIRE

    Cao, Li; Qu, Yi; Zhang, Zhaorui; Wang, Zhe; Prykova, Iya; Wu, Si

    2016-01-01

    Glycosylation is one of the most prominent and extensively studied protein post-translational modifications. However, traditional proteomic studies at the peptide level (bottom-up) rarely characterize intact glycopeptides (glycosylated peptides without removing glycans), so no glycoprotein heterogeneity information is retained. Intact glycopeptide characterization, on the other hand, provides opportunities to simultaneously elucidate the glycan structure and the glycosylation site needed to r...

  2. Does human leukocyte elastase degrade intact skin elastin?

    DEFF Research Database (Denmark)

    Schmelzer, Christian E H; Jung, Michael C; Wohlrab, Johannes

    2012-01-01

    This study aimed to investigate the susceptibility of intact fibrillar human elastin to human leukocyte elastase and cathepsin G. Elastin is a vital protein of the extracellular matrix of vertebrates, and provides exceptional properties including elasticity and tensile strength to many tissues...... and organs, including the aorta, lung, cartilage, elastic ligaments and skin, and is thus critical for their long-term function. Mature elastin is an insoluble and extremely durable protein that undergoes very little turnover, but sustained exposure to proteases may lead to irreversible and severe damage......, and thus to functional loss of the elastic fiber network. Hence, it is a key issue to understand which enzymes actually initiate elastolysis under certain pathological conditions or during intrinsic aging. In this paper, we provide a complete workflow for isolation of pure and intact elastin from very...

  3. Potentiation by substance P of contractions of the isolated vas deferens of the mouse elicited by electric field stimulation and by drugs

    International Nuclear Information System (INIS)

    Blackwell, M.; James, T.A.; Starr, M.S.

    1978-01-01

    Isolated vasa deferentia from the mouse were opened longitudinally and suspended in Krebs solution at 37 0 C in an organ bath. Contractions of the muscle were elicited by electric field stimulation, noradrenaline (10 -6 M) and acetylcholine (10 -6 M). Continued transmural stimulation evoked a biphasic response comprising a rapid twitch followed by about 10 s later by a smaller, sustained rise in muscle tone. The amplitudes of nerve-mediated and drug-induced responses were considerably potentiated by substances P (SP) in the dose range 10 -12 to 10 -7 M. Higher concentrations of SP were directly spasmogenic. The sensitizing property of SP was dose-dependent and was usually well maintained, but always disappeared quickly on washing the preparation. In some experiments SP facilitated the twitch, but not the subsequent phase of the electrically-induced contraction or the response to externally applied noradrenaline. Phentolamine (10 -6 M) failed to block this effect of SP, but itself potentiated the nerve-mediated twitch, and completely abolished the sustained secondary contraction. Desmethylimipramine (10 -6 M) enhanced the delayed contraction but not the immediate contraction. The uptake of tritiated noradrenaline (3 x 10 -7 M) by vasa was inhibited by desmethylimipramine (10 -6 M) and increased by nialamide (3 x 10 -5 M), but was not modified by SP (10 -6 M). Nerve-mediated release of accumulated radioactivity was accelerated by phentolamine, but not by SP or desmethylimipramine. These findings suggest that SP sensitizes the muscle cells to depolarizing stimuli but that it has no facilitatory effect on sympathetic neural elements. (author)

  4. Marrow-isolated adult multilineage inducible cells embedded within a biologically-inspired construct promote recovery in a mouse model of peripheral vascular disease.

    Science.gov (United States)

    Grau-Monge, Cristina; Delcroix, Gaëtan J-R; Bonnin-Marquez, Andrea; Valdes, Mike; Awadallah, Ead Lewis Mazen; Quevedo, Daniel F; Armour, Maxime R; Montero, Ramon B; Schiller, Paul C; Andreopoulos, Fotios M; D'Ippolito, Gianluca

    2017-02-17

    Peripheral vascular disease is one of the major vascular complications in individuals suffering from diabetes and in the elderly that is associated with significant burden in terms of morbidity and mortality. Stem cell therapy is being tested as an attractive alternative to traditional surgery to prevent and treat this disorder. The goal of this study was to enhance the protective and reparative potential of marrow-isolated adult multilineage inducible (MIAMI) cells by incorporating them within a bio-inspired construct (BIC) made of two layers of gelatin B electrospun nanofibers. We hypothesized that the BIC would enhance MIAMI cell survival and engraftment, ultimately leading to a better functional recovery of the injured limb in our mouse model of critical limb ischemia compared to MIAMI cells used alone. Our study demonstrated that MIAMI cell-seeded BIC resulted in a wide range of positive outcomes with an almost full recovery of blood flow in the injured limb, thereby limiting the extent of ischemia and necrosis. Functional recovery was also the greatest when MIAMI cells were combined with BICs, compared to MIAMI cells alone or BICs in the absence of cells. Histology was performed 28 days after grafting the animals to explore the mechanisms at the source of these positive outcomes. We observed that our critical limb ischemia model induces an extensive loss of muscular fibers that are replaced by intermuscular adipose tissue (IMAT), together with a highly disorganized vascular structure. The use of MIAMI cells-seeded BIC prevented IMAT infiltration with some clear evidence of muscular fibers regeneration.

  5. Intact glycopeptide characterization using mass spectrometry.

    Science.gov (United States)

    Cao, Li; Qu, Yi; Zhang, Zhaorui; Wang, Zhe; Prytkova, Iya; Wu, Si

    2016-05-01

    Glycosylation is one of the most prominent and extensively studied protein post-translational modifications. However, traditional proteomic studies at the peptide level (bottom-up) rarely characterize intact glycopeptides (glycosylated peptides without removing glycans), so no glycoprotein heterogeneity information is retained. Intact glycopeptide characterization, on the other hand, provides opportunities to simultaneously elucidate the glycan structure and the glycosylation site needed to reveal the actual biological function of protein glycosylation. Recently, significant improvements have been made in the characterization of intact glycopeptides, ranging from enrichment and separation, mass spectroscopy (MS) detection, to bioinformatics analysis. In this review, we recapitulated currently available intact glycopeptide characterization methods with respect to their advantages and limitations as well as their potential applications.

  6. Mouse fetal antigen 1 (mFA1), the circulating gene product of mdlk, pref-1 and SCP-1: isolation, characterization and biology

    DEFF Research Database (Denmark)

    Bachmann, E; Krogh, T N; Højrup, P

    1996-01-01

    The mouse homologue to human fetal antigen 1 (hFA1) was purified from mouse amniotic fluid by cation exchange chromatography and immunospecific affinity chromatography. Mouse FA1 (mFA1) is a single chain glycoprotein with an M(r) of 42-50 kDa (SDS-PAGE). The N-terminal amino acid sequence (39...... residues) revealed 74% identity to hFA1 and 100% identity to the translated cDNAs referred to as mouse dlk, pref-1 and SCP-1. mFA1 is the secreted processed molecule encoded by the mRNA defined by these identical mouse cDNAs. Monospecific rabbit anti-mFA1 antibodies, purified by ammonium sulfate...... precipitation and immunospecific affinity chromatography, were used for immunohistochemical and quantitative ELISA techniques. The indirect immunoperoxidase technique demonstrated mFA1 within the endocrine structures of adult mouse pancreas, whereas the exocrine tissue remained unstained. FA1-positive staining...

  7. Investigation of Mitochondrial Dysfunction by Sequential Microplate-Based Respiration Measurements from Intact and Permeabilized Neurons

    Science.gov (United States)

    Clerc, Pascaline; Polster, Brian M.

    2012-01-01

    Mitochondrial dysfunction is a component of many neurodegenerative conditions. Measurement of oxygen consumption from intact neurons enables evaluation of mitochondrial bioenergetics under conditions that are more physiologically realistic compared to isolated mitochondria. However, mechanistic analysis of mitochondrial function in cells is complicated by changing energy demands and lack of substrate control. Here we describe a technique for sequentially measuring respiration from intact and saponin-permeabilized cortical neurons on single microplates. This technique allows control of substrates to individual electron transport chain complexes following permeabilization, as well as side-by-side comparisons to intact cells. To illustrate the utility of the technique, we demonstrate that inhibition of respiration by the drug KB-R7943 in intact neurons is relieved by delivery of the complex II substrate succinate, but not by complex I substrates, via acute saponin permeabilization. In contrast, methyl succinate, a putative cell permeable complex II substrate, failed to rescue respiration in intact neurons and was a poor complex II substrate in permeabilized cells. Sequential measurements of intact and permeabilized cell respiration should be particularly useful for evaluating indirect mitochondrial toxicity due to drugs or cellular signaling events which cannot be readily studied using isolated mitochondria. PMID:22496810

  8. Differentiation of mouse embryonic stem cells into cardiomyocytes via the hanging-drop and mass culture methods.

    Science.gov (United States)

    Fuegemann, Christopher J; Samraj, Ajoy K; Walsh, Stuart; Fleischmann, Bernd K; Jovinge, Stefan; Breitbach, Martin

    2010-12-01

    Herein, we describe two protocols for the in vitro differentiation of mouse embryonic stem cells (mESCs) into cardiomyocytes. mESCs are pluripotent and can be differentiated into cells of all three germ layers, including cardiomyocytes. The methods described here facilitate the differentiation of mESCs into the different cardiac subtypes (atrial-, ventricular-, nodal-like cells). The duration of cell culture determines whether preferentially early- or late-developmental stage cardiomyocytes can be obtained preferentially. This approach allows the investigation of cardiomyocyte development and differentiation in vitro, and also allows for the enrichment and isolation of physiologically intact cardiomyocytes for transplantation purposes. © 2010 by John Wiley & Sons, Inc.

  9. The uptake of radioactive iodine in rat intact Graafian follicles

    International Nuclear Information System (INIS)

    Lieberman, L.M.; Lieberman, G.L.; Lieberman, M.E.

    1984-01-01

    The concentration of iodine-131 in the ovaries of mammals has important implications in the use of I-131 for the diagnosis and treatment of thyroid disease in women. The authors studied the I-131 uptake in whole ovaries and in isolated Graafian follicles of sexually mature rats. Adult female Sprague-Dawley rats, in groups of 5-6 animals, were injected IP with 10-50 μCi of I-131, at 3, 12, and 24 hrs prior to the day of proestrus and killed on the day of proestrus. The thyroid gland and ovaries were removed intact and these organs, as well as eight other tissue specimens, were weighed. The large preovulatory follicles (6-9/ovary) were then isolated under a dissecting microscope and the remaining ovary weighed. All samples were counted in a gamma well counter and the % dose/g estimated. The thyroid gland showed 23.7% dose/organ at 24 hrs. Blood decreased from 1.6% dose/g at 3 hrs to 0.5% dose/g at 24 hrs with the uterus showing 1.1% dose/g and 0.4% dose/g at the same times. Ovarian tissue was 0.5, 0.1, and 0.1% dose/g at 3,12, and 24 hrs respectively, while the intact Graafian follicles had from one-tenth to one-third the concentration of the ovary at the same times. (0.05, 0.03, and 0.03% dose/g). The authors found that the intact Graafian follicle concentrates approximately one-thirtieth to one-sixteenth of the I-131 in the blood and one-tenth to one-third of the I-131 in the ovary. This suggests that there is no active uptake of I-131 in the follicle or follicular fluid

  10. Accurate assessment of in situ isometric contractile properties of hindlimb plantar and dorsal flexor muscle complex of intact mice

    NARCIS (Netherlands)

    Gorselink, M.; Drost, M.R.; Louw, de J.; Willems, P.J.B.; Rosielle, P.C.J.N.; Janssen, J.D.; Vusse, van der G.J.

    2000-01-01

    An isometric torque sensor for measuring in situ contractions of plantar or dorsal flexors of intact mouse hindlimb has been developed and evaluated. With this device, muscle torque can be accurately measured within the range of -14 mN·m to +14 mN·m. Special attention was paid to fixation of the

  11. A Transgenic Tri-Modality Reporter Mouse

    OpenAIRE

    Yan, Xinrui; Ray, Pritha; Paulmurugan, Ramasamy; Tong, Ricky; Gong, Yongquan; Sathirachinda, Ataya; Wu, Joseph C.; Gambhir, Sanjiv S.

    2013-01-01

    Transgenic mouse with a stably integrated reporter gene(s) can be a valuable resource for obtaining uniformly labeled stem cells, tissues, and organs for various applications. We have generated a transgenic mouse model that ubiquitously expresses a tri-fusion reporter gene (fluc2-tdTomato-ttk) driven by a constitutive chicken β-actin promoter. This "Tri-Modality Reporter Mouse" system allows one to isolate most cells from this donor mouse and image them for bioluminescent (fluc2), fluorescent...

  12. Spontaneous and light-induced photon emission from intact brains of chick embryos

    Institute of Scientific and Technical Information of China (English)

    张锦珠; 于文斗; 孙彤

    1997-01-01

    Photon emission (PE) and light-induced photon emission(LPE) of intact brains isolated from chick embryos have been measured by using the single photon counting device. Experimental results showed that the intensi-ty level of photon emission was detected to be higher from intact brain than from the medium in which the brain was immerged during measuring, and the emission intensity was related to the developmental stages, the healthy situation of the measured embryos, and the freshness of isolated brains as well. After white light illumination, a short-life de-layed emission from intact brains was observed, and its relaxation behavior followed a hyperbolic rather than an expo-nential law. According to the hypothesis of biophoton emission originating from a delocalized coherent electromagnetic field and Frohlich’s idea of coherent long-range interactions in biological systems, discussions were made on the signifi-cance of photon emission in studying cell communication, biological regulation, living system’

  13. Single guard cell recordings in intact plants : light-induced hyperpolarization of the plasma membrane

    NARCIS (Netherlands)

    Roelfsema, MRG; Steinmeyer, R; Staal, M; Hedrich, R

    Guard cells are electrically isolated from other plant cells and therefore offer the unique possibility to conduct current- and voltage-clamp recordings on single cells in an intact plant. Guard cells in their natural environment were impaled with double-barreled electrodes and found to exhibit

  14. Intacting Integrity in coping with health issues

    DEFF Research Database (Denmark)

    Jepsen, Stine Leegaard; Bastrup Jørgensen, Lene; Fridlund, Bengt

    2017-01-01

    The aim of this study was to develop a formal substantive theory (FST) on the multidimensional behavioral process of coping with health issues. Intacting integrity while coping with health issues emerged as the core category of this FST. People facing health issues strive to safeguard and keep...

  15. Epispadias in boys with an intact prepuce

    NARCIS (Netherlands)

    Bos, E. M. E.; Kuijper, C. F.; Chrzan, R. J.; Dik, P.; Klijn, A. J.; de Jong, T. P. V. M.

    2014-01-01

    To present an overview of the clinical presentation and pathological anatomy, and the results of surgical correction of 7 cases of epispadias with intact prepuce; a rare condition that has only occasionally been reported in literature. A retrospective search was performed in the surgical and

  16. HYDROCARBON VAPOR DIFFUSION IN INTACT CORE SLEEVES

    Science.gov (United States)

    The diffusion of 2,2,4-trimethylpentane (TMP) and 2,2,5-trimethylhexane (TMH) vapors put of residually contaminated sandy soil from the U.S. Environmental Protection Agency (EPA) field research site at Traverse City, Michigan, was measured and modeled. The headspace of an intact ...

  17. Small Particles Intact Capture Experiment (SPICE)

    Science.gov (United States)

    Nishioka, Ken-Ji; Carle, G. C.; Bunch, T. E.; Mendez, David J.; Ryder, J. T.

    1994-01-01

    The Small Particles Intact Capture Experiment (SPICE) will develop technologies and engineering techniques necessary to capture nearly intact, uncontaminated cosmic and interplanetary dust particles (IDP's). Successful capture of such particles will benefit the exobiology and planetary science communities by providing particulate samples that may have survived unaltered since the formation of the solar system. Characterization of these particles may contribute fundamental data to our knowledge of how these particles could have formed into our planet Earth and, perhaps, contributed to the beginnings of life. The term 'uncontaminated' means that captured cosmic and IDP particles are free of organic contamination from the capture process and the term 'nearly intact capture' means that their chemical and elemental components are not materially altered during capture. The key to capturing cosmic and IDP particles that are organic-contamination free and nearly intact is the capture medium. Initial screening of capture media included organic foams, multiple thin foil layers, and aerogel (a silica gel); but, with the exception of aerogel, the requirements of no contamination or nearly intact capture were not met. To ensure no contamination of particles in the capture process, high-purity aerogel was chosen. High-purity aerogel results in high clarity (visual clearness), a useful quality in detection and recovery of embedded captured particles from the aerogel. P. Tsou at the Jet Propulsion Laboratory (JPL) originally described the use of aerogel for this purpose and reported laboratory test results. He has flown aerogel as a 'GAS-can Lid' payload on STS-47 and is evaluating the results. The Timeband Capture Cell Experiment (TICCE), a Eureca 1 experiment, is also flying aerogel and is scheduled for recovery in late April.

  18. Mouse pancreas tissue slice culture facilitates long-term studies of exocrine and endocrine cell physiology in situ.

    Science.gov (United States)

    Marciniak, Anja; Selck, Claudia; Friedrich, Betty; Speier, Stephan

    2013-01-01

    Studies on pancreatic cell physiology rely on the investigation of exocrine and endocrine cells in vitro. Particularly, in the case of the exocrine tissue these studies have suffered from a reduced functional viability of acinar cells in culture. As a result not only investigations on dispersed acinar cells and isolated acini were limited in their potential, but also prolonged studies on pancreatic exocrine and endocrine cells in an intact pancreatic tissue environment were unfeasible. To overcome these limitations, we aimed to establish a pancreas tissue slice culture platform to allow long-term studies on exocrine and endocrine cells in the intact pancreatic environment. Mouse pancreas tissue slice morphology was assessed to determine optimal long-term culture settings for intact pancreatic tissue. Utilizing optimized culture conditions, cell specificity and function of exocrine acinar cells and endocrine beta cells were characterized over a culture period of 7 days. We found pancreas tissue slices cultured under optimized conditions to have intact tissue specific morphology for the entire culture period. Amylase positive intact acini were present at all time points of culture and acinar cells displayed a typical strong cell polarity. Amylase release from pancreas tissue slices decreased during culture, but maintained the characteristic bell-shaped dose-response curve to increasing caerulein concentrations and a ca. 4-fold maximal over basal release. Additionally, endocrine beta cell viability and function was well preserved until the end of the observation period. Our results show that the tissue slice culture platform provides unprecedented maintenance of pancreatic tissue specific morphology and function over a culture period for at least 4 days and in part even up to 1 week. This analytical advancement now allows mid -to long-term studies on the cell biology of pancreatic disorder pathogenesis and therapy in an intact surrounding in situ.

  19. Mouse pancreas tissue slice culture facilitates long-term studies of exocrine and endocrine cell physiology in situ.

    Directory of Open Access Journals (Sweden)

    Anja Marciniak

    Full Text Available Studies on pancreatic cell physiology rely on the investigation of exocrine and endocrine cells in vitro. Particularly, in the case of the exocrine tissue these studies have suffered from a reduced functional viability of acinar cells in culture. As a result not only investigations on dispersed acinar cells and isolated acini were limited in their potential, but also prolonged studies on pancreatic exocrine and endocrine cells in an intact pancreatic tissue environment were unfeasible. To overcome these limitations, we aimed to establish a pancreas tissue slice culture platform to allow long-term studies on exocrine and endocrine cells in the intact pancreatic environment. Mouse pancreas tissue slice morphology was assessed to determine optimal long-term culture settings for intact pancreatic tissue. Utilizing optimized culture conditions, cell specificity and function of exocrine acinar cells and endocrine beta cells were characterized over a culture period of 7 days. We found pancreas tissue slices cultured under optimized conditions to have intact tissue specific morphology for the entire culture period. Amylase positive intact acini were present at all time points of culture and acinar cells displayed a typical strong cell polarity. Amylase release from pancreas tissue slices decreased during culture, but maintained the characteristic bell-shaped dose-response curve to increasing caerulein concentrations and a ca. 4-fold maximal over basal release. Additionally, endocrine beta cell viability and function was well preserved until the end of the observation period. Our results show that the tissue slice culture platform provides unprecedented maintenance of pancreatic tissue specific morphology and function over a culture period for at least 4 days and in part even up to 1 week. This analytical advancement now allows mid -to long-term studies on the cell biology of pancreatic disorder pathogenesis and therapy in an intact surrounding in situ.

  20. A Method to Identify and Isolate Pluripotent Human Stem Cells and Mouse Epiblast Stem Cells Using Lipid Body-Associated Retinyl Ester Fluorescence

    OpenAIRE

    Thangaselvam Muthusamy; Odity Mukherjee; Radhika Menon; Megha Prakash Bangalore; Mitradas M. Panicker

    2014-01-01

    Summary We describe the use of a characteristic blue fluorescence to identify and isolate pluripotent human embryonic stem cells and human-induced pluripotent stem cells. The blue fluorescence emission (450–500 nm) is readily observed by fluorescence microscopy and correlates with the expression of pluripotency markers (OCT4, SOX2, and NANOG). It allows easy identification and isolation of undifferentiated human pluripotent stem cells, high-throughput fluorescence sorting and subsequent propa...

  1. Measurement of diffusive properties of intact rock

    Energy Technology Data Exchange (ETDEWEB)

    Harvey, K B

    1996-12-01

    In the Postclosure Assessment of a Reference System for the Disposal of Canada`s Nuclear Fuel Waste (Goodwin et al. 1994) the disposal vault is assumed to be surrounded by a zone of intact rock, referred to as the `exclusion zone.` A sensitivity analysis of the relative effectiveness of the several engineered and natural barriers that contribute to the safety of the reference disposal system has shown that this zone of intact rock is the most effective of these barriers to the movement of radionuclides through the reference system. Peer review of the geosphere model used in the case study for the EIS (Environmental Impact Statement) of the Canadian Nuclear Fuel Waste Management Program has identified the need to quantify the properties of the intact rock surrounding the disposal vault that would control the transport of radionuclides by diffusion. The Postclosure Assessment also identified the need for appropriate values of the free water diffusion coefficient (D{sub o}) for {sup 129}1 and {sup 14}C. The measurement of rock resistivity allows the calculation of the Formation Factor for a rock This review describes the Formation Factor, diffusivity, permeability, and porosity, and how these properties might be measured or inferred for insitu rock under the conditions that apply to the intact rock surrounding a potential disposal vault. The importance of measuring the intrinsic diffusion coefficient (D{sup i}) of diffusing species under solution salinities simulating those of groundwaters is emphasised, and a method of measurement is described that is independent of the diffusing medium, and which would be appropriate for measurements made in chemically complex media such as groundwaters. (author). 95 refs., 4 tabs., 39 figs.

  2. Measurement of diffusive properties of intact rock

    International Nuclear Information System (INIS)

    Harvey, K.B.

    1996-12-01

    In the Postclosure Assessment of a Reference System for the Disposal of Canada's Nuclear Fuel Waste (Goodwin et al. 1994) the disposal vault is assumed to be surrounded by a zone of intact rock, referred to as the 'exclusion zone.' A sensitivity analysis of the relative effectiveness of the several engineered and natural barriers that contribute to the safety of the reference disposal system has shown that this zone of intact rock is the most effective of these barriers to the movement of radionuclides through the reference system. Peer review of the geosphere model used in the case study for the EIS (Environmental Impact Statement) of the Canadian Nuclear Fuel Waste Management Program has identified the need to quantify the properties of the intact rock surrounding the disposal vault that would control the transport of radionuclides by diffusion. The Postclosure Assessment also identified the need for appropriate values of the free water diffusion coefficient (D o ) for 129 1 and 14 C. The measurement of rock resistivity allows the calculation of the Formation Factor for a rock This review describes the Formation Factor, diffusivity, permeability, and porosity, and how these properties might be measured or inferred for insitu rock under the conditions that apply to the intact rock surrounding a potential disposal vault. The importance of measuring the intrinsic diffusion coefficient (D i ) of diffusing species under solution salinities simulating those of groundwaters is emphasised, and a method of measurement is described that is independent of the diffusing medium, and which would be appropriate for measurements made in chemically complex media such as groundwaters. (author). 95 refs., 4 tabs., 39 figs

  3. Autism Spectrum Disorder and intact executive functioning.

    Science.gov (United States)

    Ferrara, R; Ansermet, F; Massoni, F; Petrone, L; Onofri, E; Ricci, P; Archer, T; Ricci, S

    2016-01-01

    Earliest notions concerning autism (Autism Spectrum Disorders, ASD) describe the disturbance in executive functioning. Despite altered definition, executive functioning, expressed as higher cognitive skills required complex behaviors linked to the prefrontal cortex, are defective in autism. Specific difficulties in children presenting autism or verbal disabilities at executive functioning levels have been identified. Nevertheless, the developmental deficit of executive functioning in autism is highly diversified with huge individual variation and may even be absent. The aim of the present study to examine the current standing of intact executive functioning intact in ASD. Analysis of ASD populations, whether high-functioning, Asperger's or autism Broad Phenotype, studied over a range of executive functions including response inhibition, planning, cognitive flexibility, cognitive inhibition, and alerting networks indicates an absence of damage/impairment compared to the typically-developed normal control subjects. These findings of intact executive functioning in ASD subjects provide a strong foundation on which to construct applications for growth environments and the rehabilitation of autistic subjects.

  4. Social Isolation During the Critical Period Reduces Synaptic and Intrinsic Excitability of a Subtype of Pyramidal Cell in Mouse Prefrontal Cortex.

    Science.gov (United States)

    Yamamuro, Kazuhiko; Yoshino, Hiroki; Ogawa, Yoichi; Makinodan, Manabu; Toritsuka, Michihiro; Yamashita, Masayuki; Corfas, Gabriel; Kishimoto, Toshifumi

    2018-03-01

    Juvenile social experience is crucial for the functional development of forebrain regions, especially the prefrontal cortex (PFC). We previously reported that social isolation for 2 weeks after weaning induces prefrontal cortex dysfunction and hypomyelination. However, the effect of social isolation on physiological properties of PFC neuronal circuit remained unknown. Since hypomyelination due to isolation is prominent in deep-layer of medial PFC (mPFC), we focused on 2 types of Layer-5 pyramidal cells in the mPFC: prominent h-current (PH) cells and nonprominent h-current (non-PH) cells. We found that a 2-week social isolation after weaning leads to a specific deterioration in action potential properties and reduction in excitatory synaptic inputs in PH cells. The effects of social isolation on PH cells, which involve reduction in functional glutamatergic synapses and α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid/N-methyl-d-aspartate charge ratio, are specific to the 2 weeks after weaning and to the mPFC. We conclude that juvenile social experience plays crucial roles in the functional development in a subtype of Layer-5 pyramidal cells in the mPFC. Since these neurons project to subcortical structures, a deficit in social experience during the critical period may result in immature neural circuitry between mPFC and subcortical targets. © The Author 2017. Published by Oxford University Press. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com.

  5. Comparison of allergenicity and immunogenicity of an intact allergen vaccine and commercially available allergoid products for birch pollen immunotherapy.

    Science.gov (United States)

    Lund, L; Henmar, H; Würtzen, P A; Lund, G; Hjortskov, N; Larsen, J N

    2007-04-01

    Specific immunotherapy with intact allergen vaccine is a well-documented treatment for allergic diseases. Different vaccine formulations are currently commercially available, the active ingredient either being intact allergens or chemically modified allergoids. The rationale behind allergoids is to decrease allergenicity while maintaining immunogenicity. However, data from the German health authorities based on reporting of adverse events over a 10-year period did not indicate increased safety of allergoids over intact allergens. The objective of this study was to investigate the effect of chemical modification on allergenicity and immunogenicity comparing four commercial allergoid products for birch pollen immunotherapy with an intact allergen vaccine. Solid-phase IgE inhibition and histamine release assays were selected as model systems for allergenicity, and a combination of human T cell proliferation and IgG titres following mouse immunizations were used to address the immunogenicity of the intact allergen vaccine and the four allergoids. In all assays, the products were normalized with respect to the manufacturer's recommended maintenance dose. IgE inhibition experiments showed a change in epitope composition comparing intact allergen vaccine with allergoid. One allergoid product induced enhanced histamine release compared to the intact allergens, while the other three allergoids showed reduced release. Standard T cell stimulation assays using lines from allergic patients showed a reduced response for all allergoids compared with the intact allergen vaccine regardless of the cell type used for antigen presentation. All allergoids showed reduced capacity to induce allergen-specific IgG responses in mice. While some allergoids were associated with reduced allergenicity, a clear reduction in immunogenicity was observed for all allergoid products compared with the intact allergen vaccine, and the commercial allergoids tested therefore do not fulfil the allergoid

  6. Performance of intact and partially degraded concrete barriers in limiting fluid flow

    International Nuclear Information System (INIS)

    Walton, J.C.; Seitz, R.R.

    1991-07-01

    Concrete barriers will play a critical role in the long-term isolation of low-level radioactive wastes. Over time the barriers will degrade, and in many cases, the fundamental processes controlling performance of the barriers will be different for intact and degraded conditions. This document examines factors controlling fluid flow through intact and degraded concrete disposal facilities. Simplified models are presented fro predicting build up of fluid above a vault; fluid flow through and around intact vaults, through flaws in coatings/liners applied to a vault, and through cracks in a concrete vault; and the influence of different backfill materials around the outside of the vault. Example calculations are presented to illustrate the parameters and processes that influence fluid flow. 46 refs., 49 figs., 2 tabs

  7. Isolation of Mal d 1 and Api g 1 - specific recombinant antibodies from mouse IgG Fab fragment libraries - Mal d 1-specific antibody exhibits cross-reactivity against Bet v 1.

    Science.gov (United States)

    Haka, Jaana; Niemi, Merja H; Iljin, Kristiina; Reddy, Vanga Siva; Takkinen, Kristiina; Laukkanen, Marja-Leena

    2015-05-27

    Around 3-5% of the population suffer from IgE-mediated food allergies in Western countries and the number of food-allergenic people is increasing. Individuals with certain pollen allergies may also suffer from a sensitisation to proteins in the food products. As an example a person sensitised to the major birch pollen allergen, Bet v 1, is often sensitised to its homologues, such as the major allergens of apple, Mal d 1, and celery, Api g 1, as well. Development of tools for the reliable, sensitive and quick detection of allergens present in various food products is essential for allergic persons to prevent the consumption of substances causing mild and even life-threatening immune responses. The use of monoclonal antibodies would ensure the specific detection of the harmful food content for a sensitised person. Mouse IgG antibody libraries were constructed from immunised mice and specific recombinant antibodies for Mal d 1 and Api g 1 were isolated from the libraries by phage display. More detailed characterisation of the resulting antibodies was carried out using ELISA, SPR experiments and immunoprecipitation assays. The allergen-specific Fab fragments exhibited high affinity towards the target recombinant allergens. Furthermore, the Fab fragments also recognised native allergens from natural sources. Interestingly, isolated Mal d 1-specific antibody bound also to Bet v 1, the main allergen eliciting the cross-reactivity syndrome between the birch pollen and apple. Despite the similarities in Api g 1 and Bet v 1 tertiary structures, the isolated Api g 1-specific antibodies showed no cross-reactivity to Bet v 1. Here, high-affinity allergen-specific recombinant antibodies were isolated with interesting binding properties. With further development, these antibodies can be utilised as tools for the specific and reliable detection of allergens from different consumable products. This study gives new preliminary insights to elucidate the mechanism behind the pollen

  8. Cysteine-rich whey protein isolate (Immunocal®) ameliorates deficits in the GFAP.HMOX1 mouse model of schizophrenia.

    Science.gov (United States)

    Song, Wei; Tavitian, Ayda; Cressatti, Marisa; Galindez, Carmela; Liberman, Adrienne; Schipper, Hyman M

    2017-09-01

    Schizophrenia is a neuropsychiatric disorder that features neural oxidative stress and glutathione (GSH) deficits. Oxidative stress is augmented in brain tissue of GFAP.HMOX1 transgenic mice which exhibit schizophrenia-relevant characteristics. The whey protein isolate, Immunocal® serves as a GSH precursor upon oral administration. In this study, we treated GFAP.HMOX1 transgenic mice daily with either Immunocal (33mg/ml drinking water) or equivalent concentrations of casein (control) between the ages of 5 and 6.5 months. Immunocal attenuated many of the behavioral, neurochemical and redox abnormalities observed in GFAP.HMOX1 mice. In addition to restoring GSH homeostasis in the CNS of the transgenic mice, the whey protein isolate augmented GSH reserves in the brains of wild-type animals. These results demonstrate that consumption of whey protein isolate augments GSH stores and antioxidant defenses in the healthy and diseased mammalian brain. Whey protein isolate supplementation (Immunocal) may constitute a safe and effective modality for the management of schizophrenia, an unmet clinical imperative. Copyright © 2017 Elsevier Inc. All rights reserved.

  9. How can we conserve intact tropical peatlands?

    Science.gov (United States)

    Lawson, Ian; Roucoux, Katherine

    2017-04-01

    The scientific community has, for more than three decades, been expressing increasing alarm about the fate of peatlands in parts of Indonesia and Malaysia, where extensive land-use conversion and drainage for rice and oil palm have greatly compromised peatland hydrology, ecology, biological richness, and carbon storage. The discourse in the literature on these peatlands is now moving on from attempts to preserve the last remaining fragments of peat-swamp forest, towards discussion of how best to restore damaged ecosystems, and whether it is possible to manage plantations more 'sustainably'. It is becoming increasingly clear, however, that peatlands occur quite widely in other parts of the lowland tropics, including parts of Amazonia and the Congo Basin, and many of these peatlands can reasonably be described as 'intact': although few if any parts of the tropics are totally unaffected by human actions, the hydrology and functional ecology of these systems appear to be close to a 'natural' state. The question then arises as to what should be done with the knowledge of their existence. Here we analyse the arguments in favour of protecting intact peatlands, and the potential conflicts with other priorities such as economic development and social justice. We evaluate alternative mechanisms for protecting intact peatlands, focusing on the particular issues raised by peatlands as opposed to other kinds of tropical ecosystem. We identify ways in which natural science agendas can help to inform these arguments, using our own contributions in palaeoecology and carbon mapping as examples. Finally, we argue for a radical reconsideration of research agendas in tropical peatlands, highlighting the potential contribution of methodologies borrowed from the social sciences and humanities.

  10. Sorption of cesium in intact rock

    International Nuclear Information System (INIS)

    Puukko, E.

    2014-04-01

    The mass distribution coefficient K d is used in performance assessment (PA) to describe sorption of a radionuclide on rock. The R d is determined using crushed rock which causes uncertainty in converting the R d values to K d values for intact rock. This work describes a method to determine the equilibrium of sorption on intact rock. The rock types of the planned Olkiluoto waste disposal site were T-series mica gneiss (T-MGN), T-series tonalite granodiorite granite gneiss (T-TGG), P-series tonalite granodiorite granite gneiss (P-TGG) and pegmatitic granite (PGR). These rocks contain different amount of biotite which is the main sorbing mineral. The sorption of cesium on intact rock slices was studied by applying an electrical field to speed up migration of cesium into the rock. Cesium is in the solution as a noncomplex cation Cs + and it is sorbed by ion exchange. The tracer used in the experiments was 134 Cs. The experimental sorption on the intact rock is compared with values calculated using the in house cation exchange sorption model (HYRL model) in PHREEQC program. The observed sorption on T-MGN and T-TGG rocks was close to the calculated values. Two PGR samples were from a depth of 70 m and three samples were from a depth of 150 m. Cesium sorbed more than predicted on the two 70 m PGR samples. The sorption of Cs on the three 150 m PGR samples was small which was consistent with the calculations. The pegmatitic granite PGR has the smallest content of biotite of the four rock types. In the case of P-TGG rock the observed values of sorption were only half of the calculated values. Two kind of slices were cut from P-TGG drill core. The slices were against and to the direction of the foliation of the biotite rims. The sorption of cesium on P-TGG rock was same in both cases. The results indicated that there was no effect of the directions of the electric field and the foliation of biotite in the P-TGG rock. (orig.)

  11. Quantitative proteomic analysis of intact plastids.

    Science.gov (United States)

    Shiraya, Takeshi; Kaneko, Kentaro; Mitsui, Toshiaki

    2014-01-01

    Plastids are specialized cell organelles in plant cells that are differentiated into various forms including chloroplasts, chromoplasts, and amyloplasts, and fulfill important functions in maintaining the overall cell metabolism and sensing environmental factors such as sunlight. It is therefore important to grasp the mechanisms of differentiation and functional changes of plastids in order to enhance the understanding of vegetality. In this chapter, details of a method for the extraction of intact plastids that makes analysis possible while maintaining the plastid functions are provided; in addition, a quantitative shotgun method for analyzing the composition and changes in the content of proteins in plastids as a result of environmental impacts is described.

  12. Oxidation of molecular tritium by intact soils

    International Nuclear Information System (INIS)

    Sweet, C.W.; Murphy, C.E. Jr.

    1980-01-01

    The effects of environmental factors on the rate of oxidation of molecular tritium (T 2 ) to tritiated water (HTO) were determined for intact soils during field exposures. Maximum deposition velocities of approximately 0.03 cm/sec were measured for T 2 at low wind speeds for a variety of soils over a wide range of conditions. Deposition velocities were slightly inhibited in wet soils and at 0 0 C. In dry soils, oxidation of T 2 to HTO occurred deeper in the soil profile, but deposition velocities were unaffected

  13. A method to detect transfected chloramphenicol acetyltransferase gene expression in intact animals

    International Nuclear Information System (INIS)

    Narayanan, R.; Jastreboff, M.M.; Chiu, Chang Fang; Ito, Etsuro; Bertino, J.R.

    1988-01-01

    A rapid procedure is described for assaying chloramphenicol acetyltransferase enzyme activity in intact animals following transfection of the RSV CAT plasmid into mouse bone marrow cells by electroporation. The reconstituted mice were injected with [ 14 C]chloramphenicol and ethyl acetate extracts of 24-h urine samples were analyzed by TLC autoradiography for the excretion of 14 C-labeled metabolites. CAT expression in vivo can be detected by the presence of acetylated 14 C-labeled metabolites in the urine within 1 week after bone marrow transplantation and, under the conditions described, these metabolites can be detected for at least 3 months. CAT expression in intact mice as monitored by the urine assay correlates with the CAT expression in the hematopoietic tissues assayed in vitro. This method offers a quick mode of screening for introduced CAT gene expression in vivo without sacrificing the mice

  14. Mammary Gland Pathology Subsequent to Acute Infection with Strong versus Weak Biofilm Forming Staphylococcus aureus Bovine Mastitis Isolates: A Pilot Study Using Non-Invasive Mouse Mastitis Model.

    Directory of Open Access Journals (Sweden)

    Jully Gogoi-Tiwari

    Full Text Available Biofilm formation by Staphylococcus aureus is an important virulence attribute because of its potential to induce persistent antibiotic resistance, retard phagocytosis and either attenuate or promote inflammation, depending upon the disease syndrome, in vivo. This study was undertaken to evaluate the potential significance of strength of biofilm formation by clinical bovine mastitis-associated S. aureus in mammary tissue damage by using a mouse mastitis model.Two S. aureus strains of the same capsular phenotype with different biofilm forming strengths were used to non-invasively infect mammary glands of lactating mice. Biofilm forming potential of these strains were determined by tissue culture plate method, ica typing and virulence gene profile per detection by PCR. Delivery of the infectious dose of S. aureus was directly through the teat lactiferous duct without invasive scraping of the teat surface. Both bacteriological and histological methods were used for analysis of mammary gland pathology of mice post-infection.Histopathological analysis of the infected mammary glands revealed that mice inoculated with the strong biofilm forming S. aureus strain produced marked acute mastitic lesions, showing profuse infiltration predominantly with neutrophils, with evidence of necrosis in the affected mammary glands. In contrast, the damage was significantly less severe in mammary glands of mice infected with the weak biofilm-forming S. aureus strain. Although both IL-1β and TNF-α inflammatory biomarkers were produced in infected mice, level of TNF-α produced was significantly higher (p<0.05 in mice inoculated with strong biofilm forming S. aureus than the weak biofilm forming strain.This finding suggests an important role of TNF-α in mammary gland pathology post-infection with strong biofilm-forming S. aureus in the acute mouse mastitis model, and offers an opportunity for the development of novel strategies for reduction of mammary tissue damage

  15. NIH mouse study finds gut microorganisms may determine cancer treatment outcome

    Science.gov (United States)

    An intact gut commensal microbiota, which is a population of microorganisms living in the intestine, is required for optimal response to cancer therapy, according to a mouse study by scientists at the National Cancer Institute (NCI)

  16. Mouse adhalin

    DEFF Research Database (Denmark)

    Liu, L; Vachon, P H; Kuang, W

    1997-01-01

    . To analyze the biological roles of adhalin, we cloned the mouse adhalin cDNA, raised peptide-specific antibodies to its cytoplasmic domain, and examined its expression and localization in vivo and in vitro. The mouse adhalin sequence was 80% identical to that of human, rabbit, and hamster. Adhalin...... was specifically expressed in striated muscle cells and their immediate precursors, and absent in many other cell types. Adhalin expression in embryonic mouse muscle was coincident with primary myogenesis. Its expression was found to be up-regulated at mRNA and protein levels during myogenic differentiation...

  17. Effects of hydrolysed casein, intact casein and intact whey protein on energy expenditure and appetite regulation

    DEFF Research Database (Denmark)

    Bendtsen, Line Quist; Lorenzen, Janne Kunchel; Gomes, Sisse

    2014-01-01

    Casein and whey differ in amino acid composition and in the rate of absorption; however, the absorption rate of casein can be increased to mimic that of whey by exogenous hydrolysis. The objective of the present study was to compare the effects of hydrolysed casein (HC), intact casein (IC......) and intact whey (IW) on energy expenditure (EE) and appetite regulation, and thereby to investigate the influence of amino acid composition and the rate of absorption. In the present randomised cross-over study, twenty-four overweight and moderately obese young men and women consumed three isoenergetic...

  18. Xenopus egg cytoplasm with intact actin.

    Science.gov (United States)

    Field, Christine M; Nguyen, Phuong A; Ishihara, Keisuke; Groen, Aaron C; Mitchison, Timothy J

    2014-01-01

    We report optimized methods for preparing Xenopus egg extracts without cytochalasin D, that we term "actin-intact egg extract." These are undiluted egg cytoplasm that contains abundant organelles, and glycogen which supplies energy, and represents the least perturbed cell-free cytoplasm preparation we know of. We used this system to probe cell cycle regulation of actin and myosin-II dynamics (Field et al., 2011), and to reconstitute the large, interphase asters that organize early Xenopus embryos (Mitchison et al., 2012; Wühr, Tan, Parker, Detrich, & Mitchison, 2010). Actin-intact Xenopus egg extracts are useful for analysis of actin dynamics, and interaction of actin with other cytoplasmic systems, in a cell-free system that closely mimics egg physiology, and more generally for probing the biochemistry and biophysics of the egg, zygote, and early embryo. Detailed protocols are provided along with assays used to check cell cycle state and tips for handling and storing undiluted egg extracts. © 2014 Elsevier Inc. All rights reserved.

  19. Intactness of cell wall structure controls the in vitro digestion of starch in legumes.

    Science.gov (United States)

    Dhital, Sushil; Bhattarai, Rewati R; Gorham, John; Gidley, Michael J

    2016-03-01

    Increasing the level of starch that is not digested by the end of the small intestine and therefore enters the colon ('resistant starch') is a major opportunity for improving the nutritional profile of foods. One mechanism that has been shown to be successful is entrapment of starch within an intact plant tissue structure. However, the level of tissue intactness required for resistance to amylase digestion has not been defined. In this study, intact cells were isolated from a range of legumes after thermal treatment at 60 °C (starch not gelatinised) or 95 °C (starch gelatinised) followed by hydrolysis using pancreatic alpha amylase. It was found that intact cells, isolated at either temperature, were impervious to amylase. However, application of mechanical force damaged the cell wall and made starch accessible to digestive enzymes. This shows that the access of enzymes to the entrapped swollen starch is the rate limiting step controlling hydrolysis of starch in cooked legumes. The results suggest that a single cell wall could be sufficient to provide an effective delivery of starch to the large intestine with consequent nutritional benefits, provided that mechanical damage during digestion is avoided.

  20. In vitro culture and characterization of human lung cancer circulating tumor cells isolated by size exclusion from an orthotopic nude-mouse model expressing fluorescent protein.

    Science.gov (United States)

    Kolostova, Katarina; Zhang, Yong; Hoffman, Robert M; Bobek, Vladimir

    2014-09-01

    In the present study, we demonstrate an animal model and recently introduced size-based exclusion method for circulating tumor cells (CTCs) isolation. The methodology enables subsequent in vitro CTC-culture and characterization. Human lung cancer cell line H460, expressing red fluorescent protein (H460-RFP), was orthotopically implanted in nude mice. CTCs were isolated by a size-based filtration method and successfully cultured in vitro on the separating membrane (MetaCell®), analyzed by means of time-lapse imaging. The cultured CTCs were heterogeneous in size and morphology even though they originated from a single tumor. The outer CTC-membranes were blebbing in general. Abnormal mitosis resulting in three daughter cells was frequently observed. The expression of RFP ensured that the CTCs originated from lung tumor. These readily isolatable, identifiable and cultivable CTCs can be used to characterize individual patient cancers and for screening of more effective treatment.

  1. Characterization of the anion sensitive ATPase in intact vacuoles of Kalanchoe diagremontiana

    Energy Technology Data Exchange (ETDEWEB)

    Kobza, J.; Uribe, E.G.

    1986-04-01

    A method for the isolation of intact vacuoles from K. daigremontiana was developed which produced high yields of relatively pure vacuoles as determined by marker enzyme contamination. Upon isolation, the vacuoles were stabilized by the inclusion of 5% (w/v) ficoll. Enzyme activity was insensitive to vanadate and azide but was strongly inhibited by DCCD. Enzyme activity was strictly dependent on the inclusion of Mg/sup 2 +/ and was stimulated by anions as depicted by the series, NO/sub 3//sup -/ < Br/sup -/ < SO/sub 4//sup -/ < HCO/sub 3//sup -/ < Cl/sup -/. It was found that in intact vacuoles the ATPase activity was stimulated by phosphate to a level equivalent to that found with the chloride. The enzyme exhibited Michaelis-Menten kinetics with a Km for Mg-ATP complex of 0.51 mM.

  2. Proghrelin-derived peptides influence the secretion of insulin, glucagon, pancreatic polypeptide and somatostatin: a study on isolated islets from mouse and rat pancreas

    DEFF Research Database (Denmark)

    Qader, S.S.; Hakanson, R.; Lundquist, I.

    2008-01-01

    ghrelin, and to the 23-amino acid peptide obestatin, claimed to be a physiological opponent of acyl ghrelin. This study examines the effects of the proghrelin products, alone and in combinations, on the secretion of insulin, glucagon, pancreatic polypeptide (PP) and somatostatin from isolated islets...... times higher concentration than acyl ghrelin (corresponding to the ratio of the two peptides in circulation), desacyl ghrelin abolished the effects of acyl ghrelin but not those of obestatin. Acyl ghrelin and obestatin affected the secretion of glucagon, PP and somatostatin at physiologically relevant...

  3. Protein methylation reactions in intact pea chloroplasts

    International Nuclear Information System (INIS)

    Niemi, K.J.

    1989-01-01

    Post-translational protein methylation was investigated in Pisum sativum chloroplasts. Intact pea chloroplasts were incubated with ( 3 H-methyl)-S-adenosylmethionine under various conditions. The chloroplasts were then separated into stromal and thylakoid fractions and analyzed for radioactivity transferred to protein. Light enhanced the magnitude of labeling in both fractions. One thylakoid polypeptide with an apparent molecular mass of 43 kDa was labeled only in the light. Several other thylakoid and stromal proteins were labeled in both light and dark-labeling conditions. Both base-labile methylation, carboxy-methylesters and base-stable groups, N-methylations were found. Further characterization of the methyl-transfer reactions will be presented

  4. A new infusion pathway intactness monitoring system.

    Science.gov (United States)

    Ogawa, Hidekuni; Yonezawa, Yoshiharu; Maki, Hiromichi; Ninomiya, Ishio; Sata, Koji; Hamada, Shingo; Caldwell, W Morton

    2006-01-01

    A new infusion pathway monitoring system has been developed for hospital and home use. The system consists of linear integrated circuits and a low-power 8-bit single chip microcomputer which constantly monitors the infusion pathway intactness. An AC (alternating current) voltage is induced on the patient's body by electrostatic coupling from the normal 100 volt, 60 Hz AC power line wiring field in the patient's room. The induced AC voltage can be recorded by a main electrode wrapped around the infusion polyvinyl chloride tube. A reference electrode is wrapped on the electrode to monitor the AC voltage around the main electrode. If the injection needle or infusion tube becomes detached, then the system detects changes in the induced AC voltages and alerts the nursing station, via the nurse call system or PHS (personal handy phone system).

  5. Reconciling certification and intact forest landscape conservation.

    Science.gov (United States)

    Kleinschroth, Fritz; Garcia, Claude; Ghazoul, Jaboury

    2018-05-29

    In 2014, the Forest Stewardship Council (FSC) added a new criterion to its principles that requires protection of intact forest landscapes (IFLs). An IFL is an extensive area of forest that lacks roads and other signs of human activity as detected through remote sensing. In the Congo basin, our analysis of road networks in formally approved concessionary logging areas revealed greater loss of IFL in certified than in noncertified concessions. In areas of informal (i.e., nonregulated) extraction, road networks are known to be less detectable by remote sensing. Under the current definition of IFL, companies certified under FSC standards are likely to be penalized relative to the noncertified as well as the informal logging sector on account of their planned road networks, despite an otherwise better standard of forest management. This could ultimately undermine certification and its wider adoption, with implications for the future of sustainable forest management.

  6. Mammary Gland Pathology Subsequent to Acute Infection with Strong versus Weak Biofilm Forming Staphylococcus aureus Bovine Mastitis Isolates: A Pilot Study Using Non-Invasive Mouse Mastitis Model.

    Science.gov (United States)

    Gogoi-Tiwari, Jully; Williams, Vincent; Waryah, Charlene Babra; Costantino, Paul; Al-Salami, Hani; Mathavan, Sangeetha; Wells, Kelsi; Tiwari, Harish Kumar; Hegde, Nagendra; Isloor, Shrikrishna; Al-Sallami, Hesham; Mukkur, Trilochan

    2017-01-01

    Biofilm formation by Staphylococcus aureus is an important virulence attribute because of its potential to induce persistent antibiotic resistance, retard phagocytosis and either attenuate or promote inflammation, depending upon the disease syndrome, in vivo. This study was undertaken to evaluate the potential significance of strength of biofilm formation by clinical bovine mastitis-associated S. aureus in mammary tissue damage by using a mouse mastitis model. Two S. aureus strains of the same capsular phenotype with different biofilm forming strengths were used to non-invasively infect mammary glands of lactating mice. Biofilm forming potential of these strains were determined by tissue culture plate method, ica typing and virulence gene profile per detection by PCR. Delivery of the infectious dose of S. aureus was directly through the teat lactiferous duct without invasive scraping of the teat surface. Both bacteriological and histological methods were used for analysis of mammary gland pathology of mice post-infection. Histopathological analysis of the infected mammary glands revealed that mice inoculated with the strong biofilm forming S. aureus strain produced marked acute mastitic lesions, showing profuse infiltration predominantly with neutrophils, with evidence of necrosis in the affected mammary glands. In contrast, the damage was significantly less severe in mammary glands of mice infected with the weak biofilm-forming S. aureus strain. Although both IL-1β and TNF-α inflammatory biomarkers were produced in infected mice, level of TNF-α produced was significantly higher (pmastitis model, and offers an opportunity for the development of novel strategies for reduction of mammary tissue damage, with or without use of antimicrobials and/or anti-inflammatory compounds for the treatment of bovine mastitis.

  7. Sequence and chromosomal localization of the mouse brevican gene

    DEFF Research Database (Denmark)

    Rauch, U; Meyer, H; Brakebusch, C

    1997-01-01

    Brevican is a brain-specific proteoglycan belonging to the aggrecan family. Phage clones containing the complete mouse brevican open reading frame of 2649 bp and the complete 3'-untranslated region of 341 bp were isolated from a mouse brain cDNA library, and cosmid clones containing the mouse...

  8. Triterpene alcohol isolation from oil shale.

    Science.gov (United States)

    Albrecht, P; Ourisson, G

    1969-03-14

    Isoarborinol, an intact pentacyclic unsaturated alcohol, was isolated from the Messel oil shale (about 50 x 106 years old). Complex organic substances, even those very sensitive to oxidation, reduction, or acidic conditions, can thus survive without alteration for long periods.

  9. Antioxidant status and gut microbiota change in an aging mouse model as influenced by exopolysaccharide produced by Lactobacillus plantarum YW11 isolated from Tibetan kefir.

    Science.gov (United States)

    Zhang, Jian; Zhao, Xiao; Jiang, Yunyun; Zhao, Wen; Guo, Ting; Cao, Yongqiang; Teng, Junwei; Hao, Xiaona; Zhao, Juan; Yang, Zhennai

    2017-08-01

    This study investigated the effect of exopolysaccharide (EPS) produced by Lactobacillus plantarum YW11 on the oxidative status and gut microbiota in an aging mouse model induced with d-galactose. The in vitro assay of the antioxidant activity of the EPS showed concentration-dependent (0.25-3.0 mg/mL) activities. At 3.0 mg/mL, the EPS reached the highest scavenging activities with half maximal inhibitory concentration values against hydroxyl radicals at 75.10% and 1.22 mg/mL, superoxide anion at 62.71% and 1.54 mg/mL, 2, 2-diphenyl-1-picrylhydrazyl at 35.11% and 0.63 mg/mL, and the maximal chelating rate on ferrous ion and the half-maximal chelating concentration of the EPS at 41.09% and 1.07 mg/mL, respectively. High doses of EPS (50 mg/kg per day) effectively relieved the oxidative stress in the aging mice with increased levels of glutathione peroxidase, superoxide dismutase, catalase, and total antioxidant capacity in mice serum by 21.55, 33.14, 61.09, and 38.18%, respectively, and decreased malondialdehyde level from 11.69 to 5.89 mmol/mL compared with those in the untreated aging mice model. The analysis of pyrosequencing sequence data from the gut microbiota revealed that the EPS could recover the microbiota diversity and phylotypes decreased or eliminated by the d-galactose treatment. The EPS could selectively decrease the abundance of Flexispira (37.5 fold), and increase the abundance of Blautia (36.5 fold) and Butyricicoccus (9.5 fold), which correspondingly decreased the content of nitrogen oxides to 9.87% and increased the content of short-chain fatty acids by 2.23 fold, thereby improving the oxidative and health conditions of the host intestinal tract. Further correlation analysis of core-microbiota variation induced by different treatments showed a strong correlation with oxidative phenotypes [catalase, goodness of prediction (Q 2 ) = 0.49; total antioxidant capacity, Q 2 = 0.45; nitrogen oxides, Q 2 = 0.67; short-chain fatty acids, Q 2 = 0.55]. The

  10. Comparison of Intact PTH and Bio-Intact PTH Assays Among Non-Dialysis Dependent Chronic Kidney Disease Patients.

    Science.gov (United States)

    Einbinder, Yael; Benchetrit, Sydney; Golan, Eliezer; Zitman-Gal, Tali

    2017-09-01

    The third-generation bio-intact parathyroid hormone (PTH) (1-84) assay was designed to overcome problems associated with the detection of C-terminal fragments by the second-generation intact PTH assay. The two assays have been compared primarily among dialysis populations. The present study evaluated the correlations and differences between these two PTH assays among patients with chronic kidney disease (CKD) stages 3 to 5 not yet on dialysis. Blood samples were collected from 98 patients with CKD stages 3 to 5. PTH concentrations were measured simultaneously by using the second-generation - PTH intact-STAT and third-generation bio-intact 1-84 PTH assays. Other serum biomarkers of bone mineral disorders were also assessed. CKD stage was calculated by using the CKD-Epidemiology Collaboration (EPI) formula. Serum bio-intact PTH concentrations were strongly correlated but significantly lower than the intact PTH concentrations (r=0.963, Pbio-intact PTH) positively correlated with urea (r=0.523, r=0.504; P=0.002, respectively), phosphorus (r=0.532, r=0.521; Pbio-intact PTH assay detected significantly lower PTH concentrations compared with intact PTH assay. Additional studies that correlate the diagnosis and management of CKD mineral and bone disorders with bone histomorphometric findings are needed to determine whether bio-intact PTH assay results are better surrogate markers in these early stages of CKD. © The Korean Society for Laboratory Medicine

  11. Effect of Kuwanon G isolated from the root bark of Morus alba on ovalbumin-induced allergic response in a mouse model of asthma.

    Science.gov (United States)

    Jung, Hyo Won; Kang, Seok Yong; Kang, Jong Seong; Kim, A Ryun; Woo, Eun-Rhan; Park, Yong-Ki

    2014-11-01

    The root bark of Morus alba L. (Mori Cortex Radicis; MCR) is traditionally used in Korean medicine for upper respiratory diseases. In this study, we investigated the antiasthmatic effect of kuwanon G isolated from MCR on ovalbumin (OVA)-induced allergic asthma in mice. Kuwanon G (1 and 10 mg/kg) was administered orally in mice once a day for 7 days during OVA airway challenge. We measured the levels of OVA-specific IgE and Th2 cytokines (IL-4, IL-5, and IL-13) in the sera or bronchoalveolar lavage (BAL) fluids and also counted the immune cells in BAL fluids. Histopathological changes in the lung tissues were analyzed. Kuwanon G significantly decreased the levels of OVA-specific IgE and IL-4, IL-5, and IL-13 in the sera and BAL fluids of asthma mice. Kuwanon G reduced the numbers of inflammatory cells in the BAL fluids of asthma mice. Furthermore, the pathological feature of lungs including infiltration of inflammatory cells, thickened epithelium of bronchioles, mucus, and collagen accumulation was inhibited by kuwanon G. These results indicate that kuwanon G prevents the pathological progression of allergic asthma through the inhibition of lung destruction by inflammation and immune stimulation. Copyright © 2014 John Wiley & Sons, Ltd.

  12. Evidence for an intact polysaccharide capsule in Bordetella pertussis.

    Science.gov (United States)

    Neo, YiLin; Li, Rui; Howe, Josephine; Hoo, Regina; Pant, Aakanksha; Ho, SiYing; Alonso, Sylvie

    2010-03-01

    Polysaccharide capsules contribute to the pathogenesis of many bacteria species by providing resistance against various defense mechanisms. The production of a capsule in Bordetella pertussis, the etiologic agent of whooping cough, has remained controversial; earlier studies reported this pathogen as a capsulated microorganism whereas the recent B. pertussis genome analysis revealed the presence of a truncated capsule locus. In this work, using transmission electron microscopy and immunostaining approaches, we provide a formal evidence for the presence of an intact microcapsule produced at the surface of both laboratory strain and clinical isolates of B. pertussis. In agreement with previous studies, we found that the capsule is optimally produced in avirulent phase. Unexpectedly, the presence of the capsule was also detected at the surface of virulent B. pertussis bacteria. Consistently, a substantial transcriptional activity of the capsule operon was detected in virulent phase, suggesting that the capsular polysaccharide may play a role during pertussis pathogenesis. In vitro assays indicated that the presence of the capsule does not affect B. pertussis adherence to mammalian cells and does not further protect the bacterium from phagocytosis, complement-mediated killing or antimicrobial peptide attack. Copyright 2009. Published by Elsevier SAS.

  13. Pathophysiology of preterm labor with intact membranes.

    Science.gov (United States)

    Talati, Asha N; Hackney, David N; Mesiano, Sam

    2017-11-01

    Preterm labor with intact membranes is a major cause of spontaneous preterm birth (sPTB). To prevent sPTB a clear understanding is needed of the hormonal interactions that initiate labor. The steroid hormone progesterone acting via its nuclear progesterone receptors (PRs) in uterine cells is essential for the establishment and maintenance of pregnancy and disruption of PR signaling (i.e., functional progesterone/PR withdrawal) is key trigger for labor. The process of parturition is also associated with inflammation within the uterine tissues and it is now generally accepted that inflammatory stimuli from multiple extrinsic and intrinsic sources induce labor. Recent studies suggest inflammatory stimuli induce labor by affecting PR transcriptional activity in uterine cells to cause functional progesterone/PR withdrawal. Advances in understanding the functional interaction of inflammatory load on the pregnancy uterus and progesterone/PR signaling is opening novel areas of research and may lead to rational therapeutic strategies to effectively prevent sPTB. Copyright © 2017 Elsevier Inc. All rights reserved.

  14. Positive Youth Development, Life Satisfaction and Problem Behaviors of Adolescents in Intact and Non-Intact Families in Hong Kong

    Directory of Open Access Journals (Sweden)

    Daniel Tan Lei Shek

    2013-08-01

    Full Text Available This study investigated whether Chinese adolescents living in intact and non-intact families differed in their positive development, life satisfaction, and risk behavior. A total of 3,328 Secondary 1 students responded to measures of positive youth development (such as resilience and psychosocial competencies, life satisfaction, and risk behavior (substance abuse, delinquency, Internet addiction, consumption of pornographic materials, self-harm, and behavioral intention to engage in problem behavior. Findings revealed that adolescents growing up in intact families reported higher levels of positive developmental outcomes and life satisfaction as compared with adolescents from non-intact families. Adolescents in non-intact families also reported higher levels of risk behaviors than those growing up in intact families.

  15. Increase of extracellular glutamate concentration increases its oxidation and diminishes glucose oxidation in isolated mouse hippocampus: reversible by TFB-TBOA.

    Science.gov (United States)

    Torres, Felipe Vasconcelos; Hansen, Fernanda; Locks-Coelho, Lucas Doridio

    2013-08-01

    Glutamate concentration at the synaptic level must be kept low in order to prevent excitotoxicity. Astrocytes play a key role in brain energetics, and also astrocytic glutamate transporters are responsible for the vast majority of glutamate uptake in CNS. Experiments with primary astrocytic cultures suggest that increased influx of glutamate cotransported with sodium at astrocytes favors its flux to the tricarboxylic acid cycle instead of the glutamate-glutamine cycle. Although metabolic coupling can be considered an emergent field of research with important recent discoveries, some basic aspects of glutamate metabolism still have not been characterized in brain tissue. Therefore, the aim of this study was to investigate whether the presence of extracellular glutamate is able to modulate the use of glutamate and glucose as energetic substrates. For this purpose, isolated hippocampi of mice were incubated with radiolabeled substrates, and CO2 radioactivity and extracellular lactate were measured. Our results point to a diminished oxidation of glucose with increasing extracellular glutamate concentration, glutamate presumably being the fuel, and might suggest that oxidation of glutamate could buffer excitotoxic conditions by high glutamate concentrations. In addition, these findings were reversed when glutamate uptake by astrocytes was impaired by the presence of (3S)-3-[[3-[[4-(trifluoromethyl)benzoyl]amino]phenyl]methoxy]-L-aspartic acid (TFB-TBOA). Taken together, our findings argue against the lactate shuttle theory, because glutamate did not cause any detectable increase in extracellular lactate content (or, presumably, in glycolysis), because the glutamate is being used as fuel instead of going to glutamine and back to neurons. Copyright © 2013 Wiley Periodicals, Inc.

  16. Calculation of ion currents across the inner membrane of functionally intact mitochondria

    Science.gov (United States)

    Kane, Daniel A; Pavlov, Evgeny V

    2013-01-01

    Mitochondrial ion transport systems play a central role in cell physiology. Rates of Ca2+ and K+ transport across the inner mitochondrial membrane have been derived from the measurement of ion accumulation over time within functional isolated mitochondria or mitochondria of cultured cells. Alternatively, the electrical currents generated by ionic flux have been directly measured in purified and swollen mitochondrial samples (mitoplasts) or reconstituted channels, and typically range from 1 pA to several 100s pA. However, the direct electrophysiological approach necessarily requires extensive processing of the mitochondria prior to measurement, which can only be performed on isolated mitoplasts. To compare rates of mitochondrial ion transport measured in electrophysiological experiments to those measured in intact mitochondria and cells, we converted published rates of mitochondrial ion uptake into units of ionic current. We estimate that for monovalent ions, uptake by intact mitochondria at the rate of 1 nmol ∙ mg−1 protein ∙ min−1 is equivalent to 0.2 fA of current per whole single mitochondrion (0.4 fA for divalent ions). In intact mitochondria, estimated rates of electrogenic cation uptake are limited to 1–100 fA of integral current per single mitochondrion. These estimates are orders of magnitude lower than the currents through mitochondrial channels directly measured via patch-clamp or artificial lipid bilayer approaches. PMID:24037064

  17. 50 CFR 622.38 - Landing fish intact.

    Science.gov (United States)

    2010-10-01

    ... that is operating under the respective trip limits. Such cut-off fish also may be sold. A maximum of... 50 Wildlife and Fisheries 8 2010-10-01 2010-10-01 false Landing fish intact. 622.38 Section 622.38... Landing fish intact. The operator of a vessel that fishes in the EEZ is responsible for ensuring that fish...

  18. Intact Four-atom Organic Tetracation Stabilized by Charge Localization in the Gas Phase.

    Science.gov (United States)

    Yatsuhashi, Tomoyuki; Toyota, Kazuo; Mitsubayashi, Naoya; Kozaki, Masatoshi; Okada, Keiji; Nakashima, Nobuaki

    2016-10-05

    Several features distinguish intact multiply charged molecular cations (MMCs) from other species such as monocations and polycations: high potential energy, high electron affinity, a high density of electronic states with various spin multiplicities, and charge-dependent reactions. However, repulsive Coulombic interactions make MMCs quite unstable, and hence small organic MMCs are currently not readily available. Herein, we report that the isolated four-atom molecule diiodoacetylene survives after the removal of four electrons via tunneling. We show that the tetracation remains metastable towards dissociation because of the localization (91-95 %) of the positive charges on the terminal iodine atoms, ensuring minimum Coulomb repulsion between adjacent atoms as well as maximum charge-induced attractive dipole interactions between iodine and carbon. Our approach making use of iodines as the positively charged sites enables small organic MMCs to remain intact. © 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  19. Genistein Stimulates Jejunum Chloride Secretion via an Akt-Mediated Pathway in Intact Female Mice

    Directory of Open Access Journals (Sweden)

    Lana Leung

    2015-02-01

    Full Text Available Background/Aims: We have previously shown that daily subcutaneous injections with the naturally occurring phytoestrogen genistein (600 mg genistein/kg body weight/day, 600G results in a significantly increased basal intestinal chloride, Cl-, secretion (Isc, a measure of transepithelial secretion in intact C57BL/6J female mice after 1-week of treatment, compared to controls (DMSO vehicle injected. Removal of endogenous estrogen via ovariectomy (OVX had no effect on the 600G-mediated increase in basal Isc. Methods: Given the estrogen-like characteristics of genistein, we compared the effects of daily estradiol (E2 injections (10 mg E2/kg body weight/day, 10E2 on basal Isc in intact and OVX mice. In intact mice, 10E2 was without effect on basal Isc, however, in OVX mice, 10E2 significantly increased basal Isc (mimicked 600G. The goal of the current study was to characterize the intracellular signaling pathways responsible for mediating 600G- or 10E2-stimulated increases in basal Isc in intact female or OVX mice. Results: We measured total protein expression in isolated segments of jejunum using western blot from the following six groups of mice; intact or OVX with; 600G, 10E2 or control. The proteins of interest were: Akt, p-Akt, p-PDK1, p-PTEN, p-c-Raf, p-GSK-3β, rap-1 and ERK1/2. All blots were normalized to GAPDH levels (n = 6-18/group. Conclusion: These data suggest that the presence of the endogenous sex steroid, estrogen, modifies the intracellular signaling pathway required to mediate Cl- secretion when the intestine is exposed to exogenous 600G or E2. These studies may have relevance for designing pharmacological tools for women with intestinal chloride secretory dysfunctions.

  20. Analysis of 16S libraries of mouse gastrointestinal microflora reveals a large new group of mouse intestinal bacteria

    NARCIS (Netherlands)

    Salzman, NH; de Jong, H; Paterson, Y; Harmsen, HJM; Welling, GW; Bos, NA

    2002-01-01

    Total genomic DNA from samples of intact mouse small intestine, large intestine, caecum and faeces was used as template for PCR amplification of 16S rRNA gene sequences with conserved bacterial primers. Phylogenetic analysis of the amplification products revealed 40 unique 16S rDNA sequences. Of

  1. Serum steroid levels in intact and endocrine ablated BALB/c nude mice and their intact littermates

    DEFF Research Database (Denmark)

    Brünner, N; Svenstrup, B; Spang-Thomsen, M

    1986-01-01

    An investigation was made of the serum steroid levels found in intact and endocrine ablated nude mice of both sexes and in their intact homozygous littermates. The results showed that nude mice have a normal steroidogenesis, but with decreased levels of circulating steroids compared to those...

  2. Human cord blood-derived platelet lysate enhances the therapeutic activity of adipose-derived mesenchymal stromal cells isolated from Crohn's disease patients in a mouse model of colitis.

    Science.gov (United States)

    Forte, Dorian; Ciciarello, Marilena; Valerii, Maria Chiara; De Fazio, Luigia; Cavazza, Elena; Giordano, Rosaria; Parazzi, Valentina; Lazzari, Lorenza; Laureti, Silvio; Rizzello, Fernando; Cavo, Michele; Curti, Antonio; Lemoli, Roberto M; Spisni, Enzo; Catani, Lucia

    2015-09-09

    Due to their immunomodulatory properties, mesenchymal stromal cells (MSCs) have been used for auto-immune disease treatment. Crohn disease (CD) and ulcerative colitis are two major inflammatory bowel diseases (IBDs), resulting from pathological immune responses to environmental or microbial antigens. Preclinical and clinical studies have suggested that MSC-based cellular therapy hold promising potential for IBD treatment. However, open issues include the selection of the proper cell dose, the source and the optimal route of administration of MSCs for more effective results. Platelet lysate has gained clinical interest due to its efficacy in accelerating wound healing. Thus, we propose to combine the administration of MSCs with a human umbilical cord blood-derived platelet lysate (hCBPL) as a novel strategy to improve MSC-based therapy for IBD resolution. Colitis was induced in 8-week-old C57BL/6J mice by daily oral administration of dextran sulphate sodium (DSS) (1.5 % w/v in tap water) for 9 days. MSCs were isolated from adipose tissue of CD patients (adCD-MSCs), expanded in proliferation medium, resuspended in hCBPL or PBS and administrated via enema for three times (1 × 10(6) cells/mouse/time) every other day starting on day +7 from DSS induction. The colitis evolution was evaluated by daily monitoring of body weight, stool consistency and bleeding. Histopathological analysis was performed. Inflammatory cytokine plasma levels were determined. adCD-MSCs stained with lipophilic membrane dye Nile Red, were injected in DSS mice as described above. Colon section of mice sacrificed 24 hours after last cell administration, were analyzed by confocal microscopy. We found that adCD-MSCs could be easily isolated and expanded from CD patients. Upon injection, adCD-MSCs exerted a therapeutic effect on DSS-induced colitis. Moreover, hCBPL increased adCD-MSCs efficacy by significantly reducing colitis scores, extension of the colon inflamed area and plasma levels of

  3. Action of DTPA on hepatic plutonium. II. DTPA-induced removal of monomeric plutonium from mouse liver parenchymal cells

    International Nuclear Information System (INIS)

    Bhattacharyya, M.H.; Peterson, D.P.; Lindenbaum, A.

    1978-01-01

    Liver parenchymal cells were isolated 6 and 24 hr following the administration of diethylenetriaminepentaacetic acid (DTPA, 0.25 mmole/kg) to mice previously injected with 239 Pu-citrate (4.4 μCi/kg). Isolated parenchymal cells contained 440 dpm Pu/10 6 cells at 24 hr after Pu injection, just prior to DTPA administration. The PU content decreased to 330 dpm/10 6 cells at 6 hr and 140 dpm/10 6 cells at 24 hr after DTPA administration. Thus DTPA induced a striking decrease in the Pu content of isolated liver parenchymal cells. Parenchymal cells isolated from control mice not treated with DTPA changed little in Pu content from 24 to 48 hr after Pu injection. By 24 hr after DTPA treatment, the decrease in the Pu content of isolated liver parenchymal cells could account for the DTPA-induced release of Pu from the intact liver. Thus in the liver DTPA appears to act preferentially on the Pu associated with parenchymal cells. Liver parenchymal cells isolated 6 hr after DTPA administration and containing 330 dpm Pu/10 6 cells were incubated in vitro in the absence of added DTPA. After 18 hr of incubation the cells contained 130 dpm Pu/10 6 cells. This level corresponds to the level observed in cells isolated 24 hr after DTPA administration. Cells isolated from untreated mice lost only 15% of their Pu content during a similar in vitro incubation. Thus, by 6 hr after DTPA administration to the mouse, isolated liver parenchymal cells appeared to retain their ability to release Pu in vitro with no need for additional exposure to DTPA. The physiological significance of this finding is discussed

  4. Allergenicity, immunogenicity and dose-relationship of three intact allergen vaccines and four allergoid vaccines for subcutaneous grass pollen immunotherapy.

    Science.gov (United States)

    Henmar, H; Lund, G; Lund, L; Petersen, A; Würtzen, P A

    2008-09-01

    Different vaccines containing intact allergens or chemically modified allergoids as active ingredients are commercially available for specific immunotherapy. Allergoids are claimed to have decreased allergenicity without loss of immunogenicity and this is stated to allow administration of high allergoid doses. We compared the allergenicity and immunogenicity of four commercially available chemically modified grass pollen allergoid products with three commercially available intact grass pollen allergen vaccines. The allergenicity was investigated with immunoglobulin (Ig)E-inhibition and basophil activation assays. Human T cell proliferation and specific IgG-titres following mouse immunizations were used to address immunogenicity. Furthermore, intact allergen vaccines with different contents of active ingredients were selected to study the influence of the allergen dose. In general, a lower allergenicity for allergen vaccines was clearly linked to a reduced immunogenicity. Compared with the vaccine with the highest amount of intact allergen, the allergoids caused reduced basophil activation as well as diminished immunogenicity demonstrated by reduced T cell activation and/or reduced induction of murine grass-specific IgG antibodies. Interestingly, intact allergen vaccines with lower content of active ingredient exhibited similarly reduced allergenicity, while immunogenicity was still higher or equal to that of allergoids. The low allergenicity observed for some allergoids was inherently linked to a significantly lower immunogenic response questioning the rationale behind the chemical modification into allergoids. In addition, the linkage between allergenicity, immunogenicity and dose found for intact allergen vaccines and the immunogen as well as allergenic immune responses observed for allergoids suggest that the modified allergen vaccines do not contain high doses of immunologically active ingredients.

  5. Glucose metabolism, islet architecture, and genetic homogeneity in imprinting of [Ca2+](i and insulin rhythms in mouse islets.

    Directory of Open Access Journals (Sweden)

    Craig S Nunemaker

    2009-12-01

    Full Text Available We reported previously that islets isolated from individual, outbred Swiss-Webster mice displayed oscillations in intracellular calcium ([Ca2+](i that varied little between islets of a single mouse but considerably between mice, a phenomenon we termed "islet imprinting." We have now confirmed and extended these findings in several respects. First, imprinting occurs in both inbred (C57BL/6J as well as outbred mouse strains (Swiss-Webster; CD1. Second, imprinting was observed in NAD(PH oscillations, indicating a metabolic component. Further, short-term exposure to a glucose-free solution, which transiently silenced [Ca2+](i oscillations, reset the oscillatory patterns to a higher frequency. This suggests a key role for glucose metabolism in maintaining imprinting, as transiently suppressing the oscillations with diazoxide, a K(ATP-channel opener that blocks [Ca2+](i influx downstream of glucose metabolism, did not change the imprinted patterns. Third, imprinting was not as readily observed at the level of single beta cells, as the [Ca2+](i oscillations of single cells isolated from imprinted islets exhibited highly variable, and typically slower [Ca2+](i oscillations. Lastly, to test whether the imprinted [Ca2+](i patterns were of functional significance, a novel microchip platform was used to monitor insulin release from multiple islets in real time. Insulin release patterns correlated closely with [Ca2+](i oscillations and showed significant mouse-to-mouse differences, indicating imprinting. These results indicate that islet imprinting is a general feature of islets and is likely to be of physiological significance. While islet imprinting did not depend on the genetic background of the mice, glucose metabolism and intact islet architecture may be important for the imprinting phenomenon.

  6. Intact skin and not stripped skin is crucial for the safety and efficacy of peanut epicutaneous immunotherapy (EPIT in mice

    Directory of Open Access Journals (Sweden)

    Mondoulet Lucie

    2012-11-01

    Full Text Available Abstract Background Epicutaneous immunotherapy (EPIT on intact skin with an epicutaneous delivery system has already been used in preclinical and clinical studies. In epicutaneous vaccination and immunotherapy, the stripping of skin before application of the allergen is suggested to facilitate the passage of allergen through immune cells. Objectives The aim of this study was to compare the immunological response induced by EPIT performed on intact and stripped skin in a mouse model of peanut allergy. Methods After oral sensitization with peanut and cholera toxin, BALB/c mice were epicutaneously treated using an epicutaneous delivery system (Viaskin® (DBV Technologies, Paris applied either on intact skin or on stripped skin. Following EPIT, mice received an exclusive oral peanut regimen, aimed at triggering esophageal and jejunal lesions. We assessed eosinophil infiltration by histology, mRNA expression in the esophagus, antibody levels and peripheral T-cell response. Results EPIT on intact skin significantly reduced Th2 immunological response (IgE response and splenocyte secretion of Th2 cytokines as well as esophageal eosinophilia (2.7 ± 0.9, compared to Sham 19.9 ± 1.5, p  Conclusions Epicutaneous allergen-specific immunotherapy needs the integrity of superficial layers of the stratum corneum to warranty safety of treatment and to induce a tolerogenic profile of the immune response.

  7. Genistein exposure inhibits growth and alters steroidogenesis in adult mouse antral follicles

    Energy Technology Data Exchange (ETDEWEB)

    Patel, Shreya, E-mail: Shreya.patel214@gmail.com [Department of Comparative Biosciences, University of Illinois, 2001 S. Lincoln Ave, Urbana, IL 61802 (United States); Peretz, Jackye, E-mail: Jackye.peretz@gmail.com [Department of Comparative Biosciences, University of Illinois, 2001 S. Lincoln Ave, Urbana, IL 61802 (United States); Pan, Yuan-Xiang, E-mail: yxpan@illinois.edu [Department of Food Science and Human Nutrition, University of Illinois, 905 S. Goodwin, Urbana, IL 61801 (United States); Helferich, William G., E-mail: helferic@illinois.edu [Department of Food Science and Human Nutrition, University of Illinois, 905 S. Goodwin, Urbana, IL 61801 (United States); Flaws, Jodi A., E-mail: jflaws@illinois.edu [Department of Comparative Biosciences, University of Illinois, 2001 S. Lincoln Ave, Urbana, IL 61802 (United States)

    2016-02-15

    Genistein is a naturally occurring isoflavone phytoestrogen commonly found in plant products such as soybeans, lentils, and chickpeas. Genistein, like other phytoestrogens, has the potential to mimic, enhance, or impair the estradiol biosynthesis pathway, thereby potentially altering ovarian follicle growth. Previous studies have inconsistently indicated that genistein exposure may alter granulosa cell proliferation and hormone production, but no studies have examined the effects of genistein on intact antral follicles. Thus, this study was designed to test the hypothesis that genistein exposure inhibits follicle growth and steroidogenesis in intact antral follicles. To test this hypothesis, antral follicles isolated from CD-1 mice were cultured with vehicle (dimethyl sulfoxide; DMSO) or genistein (6.0 and 36 μM) for 18–96 h. Every 24 h, follicle diameters were measured to assess growth. At the end of each culture period, the media were pooled to measure hormone levels, and the cultured follicles were collected to measure expression of cell cycle regulators and steroidogenic enzymes. The results indicate that genistein (36 μM) inhibits growth of mouse antral follicles. Additionally, genistein (6.0 and 36 μM) increases progesterone, testosterone, and dehydroepiandrosterone (DHEA) levels, but decreases estrone and estradiol levels. The results also indicate that genistein alters the expression of steroidogenic enzymes at 24, 72 and 96 h, and the expression of cell cycle regulators at 18 h. These data indicate that genistein exposure inhibits antral follicle growth by inhibiting the cell cycle, alters sex steroid hormone levels, and dysregulates steroidogenic enzymes in cultured mouse antral follicles. - Highlights: • Genistein exposure inhibits antral follicle growth. • Genistein exposure alters expression of cell cycle regulators. • Genistein exposure alters sex steroid hormones. • Genistein exposure alters expression of steroidogenic enzymes.

  8. Genistein exposure inhibits growth and alters steroidogenesis in adult mouse antral follicles

    International Nuclear Information System (INIS)

    Patel, Shreya; Peretz, Jackye; Pan, Yuan-Xiang; Helferich, William G.; Flaws, Jodi A.

    2016-01-01

    Genistein is a naturally occurring isoflavone phytoestrogen commonly found in plant products such as soybeans, lentils, and chickpeas. Genistein, like other phytoestrogens, has the potential to mimic, enhance, or impair the estradiol biosynthesis pathway, thereby potentially altering ovarian follicle growth. Previous studies have inconsistently indicated that genistein exposure may alter granulosa cell proliferation and hormone production, but no studies have examined the effects of genistein on intact antral follicles. Thus, this study was designed to test the hypothesis that genistein exposure inhibits follicle growth and steroidogenesis in intact antral follicles. To test this hypothesis, antral follicles isolated from CD-1 mice were cultured with vehicle (dimethyl sulfoxide; DMSO) or genistein (6.0 and 36 μM) for 18–96 h. Every 24 h, follicle diameters were measured to assess growth. At the end of each culture period, the media were pooled to measure hormone levels, and the cultured follicles were collected to measure expression of cell cycle regulators and steroidogenic enzymes. The results indicate that genistein (36 μM) inhibits growth of mouse antral follicles. Additionally, genistein (6.0 and 36 μM) increases progesterone, testosterone, and dehydroepiandrosterone (DHEA) levels, but decreases estrone and estradiol levels. The results also indicate that genistein alters the expression of steroidogenic enzymes at 24, 72 and 96 h, and the expression of cell cycle regulators at 18 h. These data indicate that genistein exposure inhibits antral follicle growth by inhibiting the cell cycle, alters sex steroid hormone levels, and dysregulates steroidogenic enzymes in cultured mouse antral follicles. - Highlights: • Genistein exposure inhibits antral follicle growth. • Genistein exposure alters expression of cell cycle regulators. • Genistein exposure alters sex steroid hormones. • Genistein exposure alters expression of steroidogenic enzymes.

  9. In vivo robotics: the automation of neuroscience and other intact-system biological fields.

    Science.gov (United States)

    Kodandaramaiah, Suhasa B; Boyden, Edward S; Forest, Craig R

    2013-12-01

    Robotic and automation technologies have played a huge role in in vitro biological science, having proved critical for scientific endeavors such as genome sequencing and high-throughput screening. Robotic and automation strategies are beginning to play a greater role in in vivo and in situ sciences, especially when it comes to the difficult in vivo experiments required for understanding the neural mechanisms of behavior and disease. In this perspective, we discuss the prospects for robotics and automation to influence neuroscientific and intact-system biology fields. We discuss how robotic innovations might be created to open up new frontiers in basic and applied neuroscience and present a concrete example with our recent automation of in vivo whole-cell patch clamp electrophysiology of neurons in the living mouse brain. © 2013 New York Academy of Sciences.

  10. Sympathetic Nervous Regulation of Calcium and Action Potential Alternans in the Intact Heart.

    Science.gov (United States)

    Winter, James; Bishop, Martin J; Wilder, Catherine D E; O'Shea, Christopher; Pavlovic, Davor; Shattock, Michael J

    2018-01-01

    Rationale: Arrhythmogenic cardiac alternans are thought to be an important determinant for the initiation of ventricular fibrillation. There is limited information on the effects of sympathetic nerve stimulation (SNS) on alternans in the intact heart and the conclusions of existing studies, focused on investigating electrical alternans, are conflicted. Meanwhile, several lines of evidence implicate instabilities in Ca handling, not electrical restitution, as the primary mechanism underpinning alternans. Despite this, there have been no studies on Ca alternans and SNS in the intact heart. The present study sought to address this, by application of voltage and Ca optical mapping for the simultaneous study of APD and Ca alternans in the intact guinea pig heart during direct SNS. Objective : To determine the effects of SNS on APD and Ca alternans in the intact guinea pig heart and to examine the mechanism(s) by which the effects of SNS are mediated. Methods and Results : Studies utilized simultaneous voltage and Ca optical mapping in isolated guinea pig hearts with intact innervation. Alternans were induced using a rapid dynamic pacing protocol. SNS was associated with rate-independent shortening of action potential duration (APD) and the suppression of APD and Ca alternans, as indicated by a shift in the alternans threshold to faster pacing rates. Qualitatively similar results were observed with exogenous noradrenaline perfusion. In contrast with previous reports, both SNS and noradrenaline acted to flatten the slope of the electrical restitution curve. Pharmacological block of the slow delayed rectifying potassium current (I Ks ), sufficient to abolish I Ks -mediated APD-adaptation, partially reversed the effects of SNS on pacing-induced alternans. Treatment with cyclopiazonic acid, an inhibitor of the sarco(endo)plasmic reticulum ATPase, had opposite effects to that of SNS, acting to increase susceptibility to alternans, and suggesting that accelerated Ca reuptake

  11. Intact skull chronic windows for mesoscopic wide-field imaging in awake mice

    Science.gov (United States)

    Silasi, Gergely; Xiao, Dongsheng; Vanni, Matthieu P.; Chen, Andrew C. N.; Murphy, Timothy H.

    2016-01-01

    Background Craniotomy-based window implants are commonly used for microscopic imaging, in head-fixed rodents, however their field of view is typically small and incompatible with mesoscopic functional mapping of cortex. New Method We describe a reproducible and simple procedure for chronic through-bone wide-field imaging in awake head-fixed mice providing stable optical access for chronic imaging over large areas of the cortex for months. Results The preparation is produced by applying clear-drying dental cement to the intact mouse skull, followed by a glass coverslip to create a partially transparent imaging surface. Surgery time takes about 30 minutes. A single set-screw provides a stable means of attachment for mesoscale assessment without obscuring the cortical field of view. Comparison with Existing Methods We demonstrate the utility of this method by showing seed-pixel functional connectivity maps generated from spontaneous cortical activity of GCAMP6 signals in both awake and anesthetized mice. Conclusions We propose that the intact skull preparation described here may be used for most longitudinal studies that do not require micron scale resolution and where cortical neural or vascular signals are recorded with intrinsic sensors. PMID:27102043

  12. Genome-wide identification of estrogen receptor alpha-binding sites in mouse liver

    DEFF Research Database (Denmark)

    Gao, Hui; Fält, Susann; Sandelin, Albin

    2007-01-01

    We report the genome-wide identification of estrogen receptor alpha (ERalpha)-binding regions in mouse liver using a combination of chromatin immunoprecipitation and tiled microarrays that cover all nonrepetitive sequences in the mouse genome. This analysis identified 5568 ERalpha-binding regions...... genes. The majority of ERalpha-binding regions lie in regions that are evolutionarily conserved between human and mouse. Motif-finding algorithms identified the estrogen response element, and variants thereof, together with binding sites for activator protein 1, basic-helix-loop-helix proteins, ETS...... signaling in mouse liver, by characterizing the first step in this signaling cascade, the binding of ERalpha to DNA in intact chromatin....

  13. Uptake and clearance of plutonium-238 from intact liver and liver cells transplanted into fat pads of F344/N rats

    International Nuclear Information System (INIS)

    Brooks, A.L.; Guilmette, R.A.; Hahn, F.F.; Jirtle, R.L.

    1985-01-01

    An understanding of the role of liver cells and the intact liver in plutonium biokinetics is needed. Liver cells were isolated from rats, injected into fat pads of recipient rats, and allowed 21 days to form cell colonies. Rats then received a single intraperitoneal injection of 1 μCi 238 Pu-citrate and were serially sacrificed. Uptake, retention, and distribution of Pu in intact liver and in liver cells growing in fat pads were determined. Intact liver cells took up about twice as much 238 Pu as liver cells transplanted into fat pads. However, the retention kinetics of Pu were similar for both the liver cells in the fat pads and the intact liver cells when the retention was expressed as activity per cell. 4 references, 1 figure, 1 table

  14. Assessment of geomechanical properties of intact Opalinus Clay - Expert report

    International Nuclear Information System (INIS)

    Amann, F.; Vogelhuber, M.

    2015-11-01

    This comprehensive report published by the Swiss Federal Nuclear Safety Inspectorate ENSI presents an expert report published on the assessment of the geomechanical properties of intact Opalinus Clay. This review report addresses the conceptual constitutive framework for repositories in Opalinus Clay. The author addresses the geomechanical fundamentals that are necessary in order to adequately judge experiments on intact Opalinus Clay and the interpretation of the results. The report assesses in detail the various test series on intact Opalinus Clay carried out along with the interpretations made by experts and NAGRA. Further assessments are quoted including those on sample geometries tested, effective strength properties, undrained shear strength properties and elastic properties. The results of work done by other experts are also presented and discussed. The report is completed with a list of relevant literature

  15. Assessment of geomechanical properties of intact Opalinus Clay - Expert report

    Energy Technology Data Exchange (ETDEWEB)

    Amann, F. [Eidgenössische Technische Hochschule ETHZ, Zürich (Switzerland); Vogelhuber, M. [Dr. von Moos AG, Geotechnisches Büro, Zürich (Switzerland)

    2015-11-15

    This comprehensive report published by the Swiss Federal Nuclear Safety Inspectorate ENSI presents an expert report published on the assessment of the geomechanical properties of intact Opalinus Clay. This review report addresses the conceptual constitutive framework for repositories in Opalinus Clay. The author addresses the geomechanical fundamentals that are necessary in order to adequately judge experiments on intact Opalinus Clay and the interpretation of the results. The report assesses in detail the various test series on intact Opalinus Clay carried out along with the interpretations made by experts and NAGRA. Further assessments are quoted including those on sample geometries tested, effective strength properties, undrained shear strength properties and elastic properties. The results of work done by other experts are also presented and discussed. The report is completed with a list of relevant literature.

  16. pGlyco 2.0 enables precision N-glycoproteomics with comprehensive quality control and one-step mass spectrometry for intact glycopeptide identification.

    Science.gov (United States)

    Liu, Ming-Qi; Zeng, Wen-Feng; Fang, Pan; Cao, Wei-Qian; Liu, Chao; Yan, Guo-Quan; Zhang, Yang; Peng, Chao; Wu, Jian-Qiang; Zhang, Xiao-Jin; Tu, Hui-Jun; Chi, Hao; Sun, Rui-Xiang; Cao, Yong; Dong, Meng-Qiu; Jiang, Bi-Yun; Huang, Jiang-Ming; Shen, Hua-Li; Wong, Catherine C L; He, Si-Min; Yang, Peng-Yuan

    2017-09-05

    The precise and large-scale identification of intact glycopeptides is a critical step in glycoproteomics. Owing to the complexity of glycosylation, the current overall throughput, data quality and accessibility of intact glycopeptide identification lack behind those in routine proteomic analyses. Here, we propose a workflow for the precise high-throughput identification of intact N-glycopeptides at the proteome scale using stepped-energy fragmentation and a dedicated search engine. pGlyco 2.0 conducts comprehensive quality control including false discovery rate evaluation at all three levels of matches to glycans, peptides and glycopeptides, improving the current level of accuracy of intact glycopeptide identification. The N-glycoproteome of samples metabolically labeled with 15 N/ 13 C were analyzed quantitatively and utilized to validate the glycopeptide identification, which could be used as a novel benchmark pipeline to compare different search engines. Finally, we report a large-scale glycoproteome dataset consisting of 10,009 distinct site-specific N-glycans on 1988 glycosylation sites from 955 glycoproteins in five mouse tissues.Protein glycosylation is a heterogeneous post-translational modification that generates greater proteomic diversity that is difficult to analyze. Here the authors describe pGlyco 2.0, a workflow for the precise one step identification of intact N-glycopeptides at the proteome scale.

  17. Centralized mouse repositories.

    Science.gov (United States)

    Donahue, Leah Rae; Hrabe de Angelis, Martin; Hagn, Michael; Franklin, Craig; Lloyd, K C Kent; Magnuson, Terry; McKerlie, Colin; Nakagata, Naomi; Obata, Yuichi; Read, Stuart; Wurst, Wolfgang; Hörlein, Andreas; Davisson, Muriel T

    2012-10-01

    Because the mouse is used so widely for biomedical research and the number of mouse models being generated is increasing rapidly, centralized repositories are essential if the valuable mouse strains and models that have been developed are to be securely preserved and fully exploited. Ensuring the ongoing availability of these mouse strains preserves the investment made in creating and characterizing them and creates a global resource of enormous value. The establishment of centralized mouse repositories around the world for distributing and archiving these resources has provided critical access to and preservation of these strains. This article describes the common and specialized activities provided by major mouse repositories around the world.

  18. Bioavailability and in vivo metabolism of intact glucosinolates

    DEFF Research Database (Denmark)

    Sørensen, Jens Christian; Frandsen, Heidi Blok; Jensen, Søren Krogh

    2016-01-01

    Health benefits associated with consumption of cruciferous vegetables have received considerable attention with a hitherto focus on the role and bioactivity of glucosinolate degradation products. We investigated the in vivo metabolism of intact glucosinolates by following their fate in digesta an...

  19. Capillary zone electrophoresis-mass spectromet of intact proteins

    NARCIS (Netherlands)

    Domínguez-Vega, Elena; Haselberg, Rob; Somsen, Govert W.

    2016-01-01

    Capillary electrophoresis (CE) coupled with mass spectrometry (MS) has proven to be a powerful analytical tool for the characterization of intact proteins. It combines the high separation efficiency, short analysis time, and versatility of CE with the mass selectivity and sensitivity offered by MS

  20. Intact collagen and atelocollagen sponges: Characterization and ESEM observation

    International Nuclear Information System (INIS)

    Ruozi, Barbara; Tosi, Giovanni; Leo, Eliana; Parma, Bruna; Vismara, Susanna; Forni, Flavio; Vandelli, Maria Angela

    2007-01-01

    In this study we have investigated the chemical-physical and morphological properties of intact and atelocollagen sponges used for tissue engineering. The porous sponges were prepared by lyophilization and their physico-chemical characteristics (water binding capacity, denaturing temperature, amino group content) were investigated. Considering the importance of the 'in vivo' interactions between these sponges and the tissue, our attention was addressed (a) to clarify the relationships between the morphology and the amount of water absorbed and (b) to evaluate the influence of pepsin-alkaline treatment on the reorganization of the atelocollagen fibres. Conventional scanning electron microscopy (SEM) and environmental scanning electron microscopy (ESEM) were employed to study the morphology and wetting behaviour of the intact and atelocollagen sponges. The observations by SEM indicated remarkable differences both in the structure and dimension of the pores between intact and atelocollagen sponges. At the data are related to a different water binding capacity. However, the ESEM observations, achieved by changing the relative humidity in the operative chamber, demonstrated that the water adsorbed can be removed with major difficulty from atelocollagen sponges than from intact ones

  1. Behavior Management Style of Single Parents and Intact Families.

    Science.gov (United States)

    Smith, Douglas K.; And Others

    Studies examining the behavior management styles of parents as a function of family intactness and parent employment status are lacking. To assess parental style of behavior management, the Parental Management Questionnaire (PMQ) was completed by 1,957 parents of elementary school children (50% response rate). The PMQ is based on Aronfreed's…

  2. Clonal expansion of genome-intact HIV-1 in functionally polarized Th1 CD4+ T cells.

    Science.gov (United States)

    Lee, Guinevere Q; Orlova-Fink, Nina; Einkauf, Kevin; Chowdhury, Fatema Z; Sun, Xiaoming; Harrington, Sean; Kuo, Hsiao-Hsuan; Hua, Stephane; Chen, Hsiao-Rong; Ouyang, Zhengyu; Reddy, Kavidha; Dong, Krista; Ndung'u, Thumbi; Walker, Bruce D; Rosenberg, Eric S; Yu, Xu G; Lichterfeld, Mathias

    2017-06-30

    HIV-1 causes a chronic, incurable disease due to its persistence in CD4+ T cells that contain replication-competent provirus, but exhibit little or no active viral gene expression and effectively resist combination antiretroviral therapy (cART). These latently infected T cells represent an extremely small proportion of all circulating CD4+ T cells but possess a remarkable long-term stability and typically persist throughout life, for reasons that are not fully understood. Here we performed massive single-genome, near-full-length next-generation sequencing of HIV-1 DNA derived from unfractionated peripheral blood mononuclear cells, ex vivo-isolated CD4+ T cells, and subsets of functionally polarized memory CD4+ T cells. This approach identified multiple sets of independent, near-full-length proviral sequences from cART-treated individuals that were completely identical, consistent with clonal expansion of CD4+ T cells harboring intact HIV-1. Intact, near-full-genome HIV-1 DNA sequences that were derived from such clonally expanded CD4+ T cells constituted 62% of all analyzed genome-intact sequences in memory CD4 T cells, were preferentially observed in Th1-polarized cells, were longitudinally detected over a duration of up to 5 years, and were fully replication- and infection-competent. Together, these data suggest that clonal proliferation of Th1-polarized CD4+ T cells encoding for intact HIV-1 represents a driving force for stabilizing the pool of latently infected CD4+ T cells.

  3. Pharmacokinetics of erythropoietin in intact and anephric dogs

    International Nuclear Information System (INIS)

    Fu, J.S.; Lertora, J.J.; Brookins, J.; Rice, J.C.; Fisher, J.W.

    1988-01-01

    The present studies were performed to determine the pharmacokinetic parameters of erythropoietin in intact and anephric dogs by use of unlabeled crude native erythropoietin (nEp) and iodine 125-labeled purified recombinant erythropoietin (rEp) given by intravenous infusion for 15 minutes. Sephadex G-75 gel filtration was used to confirm that the 125I-rEp molecule remained iodinated in dog plasma during the 24-hour period of these studies. The plasma disappearance of erythropoietin conformed to a biexponential equation for both nEp and 125I-rEp, with the central compartment being larger than the peripheral compartment. The mean distribution half-life of 75.3 +/- 21.2 minutes for nEp was significantly (p less than 0.05) longer than that of 125I-rEp (23.7 +/- 5.0 minutes) in intact dogs. The intercompartmental clearance (CIic) for nEp (0.018 +/- 0.006 L/kg/hr) was significantly smaller than that of 125I-rEp (0.068 +/- 0.018 L/kg/hr) in intact dogs (p less than 0.05). There were no significant differences in apparent volume of distribution, elimination half-life, and elimination clearance (CIe) for nEp and rEp in intact dogs. The mean elimination half-life for 125I-rEp in intact dogs (9.0 +/- 0.6 hours) and anephric dogs (13.8 +/- 1.4 hours) was significantly different (p less than 0.05). The CIe for 125I-rEp in anephric dogs (0.008 +/- 0.001 L/kg/hr) was significantly (p less than 0.05) smaller than that of 125I-rEp in intact dogs (0.011 +/- 0.001 L/kg/hr). There were no significant differences in apparent volume of distribution, distribution half-life, and CIic for 125I-rEp in intact and anephric dogs

  4. Threats to intact tropical peatlands and opportunities for their conservation.

    Science.gov (United States)

    Roucoux, K H; Lawson, I T; Baker, T R; Del Castillo Torres, D; Draper, F C; Lähteenoja, O; Gilmore, M P; Honorio Coronado, E N; Kelly, T J; Mitchard, E T A; Vriesendorp, C F

    2017-12-01

    Large, intact areas of tropical peatland are highly threatened at a global scale by the expansion of commercial agriculture and other forms of economic development. Conserving peatlands on a landscape scale, with their hydrology intact, is of international conservation importance to preserve their distinctive biodiversity and ecosystem services and maintain their resilience to future environmental change. We explored threats to and opportunities for conserving remaining intact tropical peatlands; thus, we excluded peatlands of Indonesia and Malaysia, where extensive deforestation, drainage, and conversion to plantations means conservation in this region can protect only small fragments of the original ecosystem. We focused on a case study, the Pastaza-Marañón Foreland Basin (PMFB) in Peru, which is among the largest known intact tropical peatland landscapes in the world and is representative of peatland vulnerability. Maintenance of the hydrological conditions critical for carbon storage and ecosystem function of peatlands is, in the PMFB, primarily threatened by expansion of commercial agriculture linked to new transport infrastructure that is facilitating access to remote areas. There remain opportunities in the PMFB and elsewhere to develop alternative, more sustainable land-use practices. Although some of the peatlands in the PMFB fall within existing legally protected areas, this protection does not include the most carbon-dense (domed pole forest) areas. New carbon-based conservation instruments (e.g., REDD+, Green Climate Fund), developing markets for sustainable peatland products, transferring land title to local communities, and expanding protected areas offer pathways to increased protection for intact tropical peatlands in Amazonia and elsewhere, such as those in New Guinea and Central Africa which remain, for the moment, broadly beyond the frontier of commercial development. © 2017 The Authors. Conservation Biology published by Wiley Periodicals, Inc

  5. Enzymatic methylation of band 3 anion transporter in intact human erythrocytes

    International Nuclear Information System (INIS)

    Lou, L.L.; Clarke, S.

    1987-01-01

    Band 3, the anion transport protein of erythrocyte membranes, is a major methyl-accepting substrate of the intracellular erythrocyte protein carboxyl methyltransferase (S-adenosyl-L-methionine: protein-D-aspartate O-methyltransferase; EC 2.1.1.77). The localization of methylation sites in intact cells by analysis of proteolytic fragments indicated that sites were present in the cytoplasmic N-terminal domain as well as the membranous C-terminal portion of the polypeptide. The amino acid residues that serve as carboxyl methylation sites of the erythrocyte anion transporter were also investigated. 3 H-Methylated band 3 was purified from intact erythrocytes incubated with L-[methyl- 3 H]methionine and from trypsinized and lysed erythrocytes incubated with S-adenosyl-L-[methyl- 3 H]methionine. After proteolytic digestion with carboxypeptidase Y, D-aspartic acid beta-[ 3 H]methyl ester was isolated in low yields (9% and 1%, respectively) from each preparation. The bulk of the radioactivity was recovered as [ 3 H]methanol, and the amino acid residue(s) originally associated with these methyl groups could not be determined. No L-aspartic acid beta-[ 3 H]methyl ester or glutamyl gamma-[ 3 H]methyl ester was detected. The formation of D-aspartic acid beta-[ 3 H]methyl esters in this protein in intact cells resulted from protein carboxyl methyltransferase activity since it was inhibited by adenosine and homocysteine thiolactone, which increases the intracellular concentration of the potent product inhibitor S-adenosylhomocysteine, and cycloleucine, which prevents the formation of the substrate S-adenosyl-L-[methyl- 3 H]methionine

  6. The Multilevel Mixed Intact Group Analysis: A Mixed Method to Seek, Detect, Describe, and Explain Differences Among Intact Groups

    Science.gov (United States)

    Schoonenboom, Judith

    2016-01-01

    Educational innovations often involve intact subgroups, such as school classes or university departments. In small-scale educational evaluation research, typically involving 1 to 20 subgroups, differences among these subgroups are often neglected. This article presents a mixed method from a qualitative perspective, in which differences among…

  7. The Multilevel Mixed Intact Group Analysis: A Mixed Method to Seek, Detect, Describe and Explain Differences Between Intact Groups

    NARCIS (Netherlands)

    Schoonenboom, J.I.

    2014-01-01

    Educational innovations often involve intact subgroups, such as school classes or university departments. In small-scale educational evaluation research, typically involving 1 to 20 subgroups, differences among these subgroups are often neglected. This article presents a mixed method from a

  8. Intestinimonas butyriciproducens gen. nov., sp. nov., a novel butyrate-producing bacterium from the mouse intestine

    NARCIS (Netherlands)

    Kläring, K.; Hanske, L.; Bui, T.P.N.; Charrier, C.; Blaut, M.; Haller, D.; Plugge, C.M.; Clavel, T.

    2013-01-01

    Whilst creating a bacterial collection of strains from the mouse intestine, we isolated a Gram-negative, spore-forming, non-motile and strictly anaerobic rod-shaped bacterium from the caecal content of a TNFdeltaARE mouse. The isolate, referred to as strain SRB-521-5-IT, was originally cultured on a

  9. Less Daily Computer Use is Related to Smaller Hippocampal Volumes in Cognitively Intact Elderly.

    Science.gov (United States)

    Silbert, Lisa C; Dodge, Hiroko H; Lahna, David; Promjunyakul, Nutta-On; Austin, Daniel; Mattek, Nora; Erten-Lyons, Deniz; Kaye, Jeffrey A

    2016-01-01

    Computer use is becoming a common activity in the daily life of older individuals and declines over time in those with mild cognitive impairment (MCI). The relationship between daily computer use (DCU) and imaging markers of neurodegeneration is unknown. The objective of this study was to examine the relationship between average DCU and volumetric markers of neurodegeneration on brain MRI. Cognitively intact volunteers enrolled in the Intelligent Systems for Assessing Aging Change study underwent MRI. Total in-home computer use per day was calculated using mouse movement detection and averaged over a one-month period surrounding the MRI. Spearman's rank order correlation (univariate analysis) and linear regression models (multivariate analysis) examined hippocampal, gray matter (GM), white matter hyperintensity (WMH), and ventricular cerebral spinal fluid (vCSF) volumes in relation to DCU. A voxel-based morphometry analysis identified relationships between regional GM density and DCU. Twenty-seven cognitively intact participants used their computer for 51.3 minutes per day on average. Less DCU was associated with smaller hippocampal volumes (r = 0.48, p = 0.01), but not total GM, WMH, or vCSF volumes. After adjusting for age, education, and gender, less DCU remained associated with smaller hippocampal volume (p = 0.01). Voxel-wise analysis demonstrated that less daily computer use was associated with decreased GM density in the bilateral hippocampi and temporal lobes. Less daily computer use is associated with smaller brain volume in regions that are integral to memory function and known to be involved early with Alzheimer's pathology and conversion to dementia. Continuous monitoring of daily computer use may detect signs of preclinical neurodegeneration in older individuals at risk for dementia.

  10. Imaging with 11B of intact tissues using magnetic resonance gradient echoes

    International Nuclear Information System (INIS)

    Richards, T.L.; Bradshaw, K.M.; Freeman, D.M.; Sotak, C.H.; Gavin, P.R.

    1988-01-01

    Boron neutron capture therapy (BNCT) is a proposed method of treating Glioblastoma Multiforme. BNCT is based on 10 B intake by the tumor and in-situ activation by neutron beam. It is estimated that to have successful BNCT, a 10 B delivery mechanism must deposit 20 ppM or more of 10 B within the tumor. To study and understand this delivery mechanism, 11 B can be used instead of 10 B. The pharmacokinetics of any compound using 11 B will be the same as 10 B. The advantage of using 11 B over 10 B is its greater nuclear magnetic resonance sensitivity for both spectroscopy and imaging. The use of 11 B imaging to detect and quantitate boron uptake non-invasively in animal tumor modes will facilitate continued work with 10 B. Preliminary work has shown that 11 B nuclear magnetic resonance (NMR) spectroscopy (nonlocalized) can detect 11 B in intact mouse tissues and the area under the boron peak correlates with the total boron content (correlation coefficient of 0.997). Once the ability to non-invasively measure the boron compound is established using magnetic resonance imaging (MRI) combined with spectroscopy, we will be able to address the following questions: (1) what is the optimum method of boron administration for maximum tumor selective uptake, (2) at what time is peak tumor boron concentration after infusion, and (3) what is the dose distribution in the head (based on neutron radiation and boron concentration)? The purpose of this study was to test the feasibility of imaging 11 B in intact tissues using magnetic resonance

  11. [Management of threatening preterm labor with intact membranes: indications for antibiotics].

    Science.gov (United States)

    Winer, N

    2002-11-01

    To analyse the benefits and disadvantages of systematic antibiotic therapy in patient presenting a preterm labor with intact membranes. We reviewed French and English reports on Medline using to the following key words: "antibiotic therapy and preterm labor, preterm labor, streptococcus B, vaginose, mycoplasma, antenatal infection". The systematic prescription of antibiotics is not recommended for patients presenting preterm labor who have intact membranes and no symptoms of infection. The benefit of antibiotics is small and shows a tendency to prolong the pregnancy and the reduction of maternal infection. No benefit has been shown for neonatal results. When early-onset neonatal sepsis develops in a case in which antepartum chemoprophylaxis was used, the isolated bacteria will present an increasing risk of bacterial drug resistance. Local treatment (cream or pessary) do not belong in the treatment of threatening preterm labor and are not recommended for the prevention of prematurity or materno fetal infection. Risk groups of patients who present a positive vaginal colonization are subject to discussion. Studies do not allow us to ascertain that antibiotics have a beneficial effect on prematurity in these groups. Antibiotics are recommended for the treatment of asymptomatic bacteriuria. This treatment reduces prematurity and maternal infections. Despite poor consensus criteria, if threatening preterm labor is associated with a bacteriuria, experts usually recommend treatment.

  12. Screening of intact yeasts and cell extracts to reduce Scrapie prions during biotransformation of food waste.

    Science.gov (United States)

    Huyben, David; Boqvist, Sofia; Passoth, Volkmar; Renström, Lena; Allard Bengtsson, Ulrika; Andréoletti, Olivier; Kiessling, Anders; Lundh, Torbjörn; Vågsholm, Ivar

    2018-02-08

    Yeasts can be used to convert organic food wastes to protein-rich animal feed in order to recapture nutrients. However, the reuse of animal-derived waste poses a risk for the transmission of infectious prions that can cause neurodegeneration and fatality in humans and animals. The aim of this study was to investigate the ability of yeasts to reduce prion activity during the biotransformation of waste substrates-thereby becoming a biosafety hurdle in such a circular food system. During pre-screening, 30 yeast isolates were spiked with Classical Scrapie prions and incubated for 72 h in casein substrate, as a waste substitute. Based on reduced Scrapie seeding activity, waste biotransformation and protease activities, intact cells and cell extracts of 10 yeasts were further tested. Prion analysis showed that five yeast species reduced Scrapie seeding activity by approximately 1 log10 or 90%. Cryptococcus laurentii showed the most potential to reduce prion activity since both intact and extracted cells reduced Scrapie by 1 log10 and achieved the highest protease activity. These results show that select forms of yeast can act as a prion hurdle during the biotransformation of waste. However, the limited ability of yeasts to reduce prion activity warrants caution as a sole barrier to transmission as higher log reductions are needed before using waste-cultured yeast in circular food systems.

  13. Differential effects of androgens on coronary blood flow regulation and arteriolar diameter in intact and castrated swine

    Directory of Open Access Journals (Sweden)

    O’Connor Erin K

    2012-05-01

    Full Text Available Abstract Background Low endogenous testosterone levels have been shown to be a risk factor for the development of cardiovascular disease and cardiovascular benefits associated with testosterone replacement therapy are being advocated; however, the effects of endogenous testosterone levels on acute coronary vasomotor responses to androgen administration are not clear. The objective of this study was to compare the effects of acute androgen administration on in vivo coronary conductance and in vitro coronary microvascular diameter in intact and castrated male swine. Methods Pigs received intracoronary infusions of physiologic levels (1–100 nM of testosterone, the metabolite 5α-dihydrotestosterone, and the epimer epitestosterone while left anterior descending coronary blood flow and mean arterial pressure were continuously monitored. Following sacrifice, coronary arterioles were isolated, cannulated, and exposed to physiologic concentrations (1–100 nM of testosterone, 5α-dihydrotestosterone, and epitestosterone. To evaluate effects of the androgen receptor on acute androgen dilation responses, real-time PCR and immunohistochemistry for androgen receptor were performed on conduit and resistance coronary vessels. Results In vivo, testosterone and 5α-dihydrotestosterone produced greater increases in coronary conductance in the intact compared to the castrated males. In vitro, percent maximal dilation of microvessels was similar between intact and castrated males for testosterone and 5α-dihydrotestosterone. In both studies epitestosterone produced significant increases in conductance and microvessel diameter from baseline in the intact males. Androgen receptor mRNA expression and immunohistochemical staining were similar in intact and castrated males. Conclusions Acute coronary vascular responses to exogenous androgen administration are increased by endogenous testosterone, an effect unrelated to changes in androgen receptor expression.

  14. Structural determination of intact proteins using mass spectrometry

    Science.gov (United States)

    Kruppa, Gary [San Francisco, CA; Schoeniger, Joseph S [Oakland, CA; Young, Malin M [Livermore, CA

    2008-05-06

    The present invention relates to novel methods of determining the sequence and structure of proteins. Specifically, the present invention allows for the analysis of intact proteins within a mass spectrometer. Therefore, preparatory separations need not be performed prior to introducing a protein sample into the mass spectrometer. Also disclosed herein are new instrumental developments for enhancing the signal from the desired modified proteins, methods for producing controlled protein fragments in the mass spectrometer, eliminating complex microseparations, and protein preparatory chemical steps necessary for cross-linking based protein structure determination.Additionally, the preferred method of the present invention involves the determination of protein structures utilizing a top-down analysis of protein structures to search for covalent modifications. In the preferred method, intact proteins are ionized and fragmented within the mass spectrometer.

  15. SOLITARY CHEMORECEPTOR CELL SURVIVAL IS INDEPENDENT OF INTACT TRIGEMINAL INNERVATION

    Science.gov (United States)

    Gulbransen, Brian; Silver, Wayne; Finger, Tom

    2008-01-01

    Nasal solitary chemoreceptor cells (SCCs) are a population of specialized chemosensory epithelial cells presumed to broaden trigeminal chemoreceptivity in mammals (Finger et al., 2003). SCCs are innervated by peptidergic trigeminal nerve fibers (Finger et al., 2003) but it is currently unknown if intact innervation is necessary for SCC development or survival. We tested the dependence of SCCs on innervation by eliminating trigeminal nerve fibers during development with neurogenin-1 knockout mice, during early postnatal development with capsaicin desensitization, and during adulthood with trigeminal lesioning. Our results demonstrate that elimination of innervation at any of these times does not result in decreased SCC numbers. In conclusion, neither SCC development nor mature cell maintenance is dependent on intact trigeminal innervation. PMID:18300260

  16. Direct detection of radicals in intact soybean nodules

    DEFF Research Database (Denmark)

    Mathieu, C; Moreau, S; Frendo, P

    1998-01-01

    Electron paramagnetic resonance spectroscopy has been employed to examine the nature of the metal ions and radicals present in intact root nodules of soybean plants grown in the absence of nitrate. The spectra obtained from nodules of different ages using this non-invasive technique show dramatic...... differences, suggesting that there are both qualitative and quantitative changes in the metal ion and radical species present. A major component of the spectra obtained from young nodules is assigned to a complex (Lb-NO) of nitric oxide (NO.) with the heme protein leghemoglobin (Lb). This Lb-NO species, which...... has not been previously detected in intact root nodules of plants grown in the absence of nitrate, is thought to be formed by reaction of nitric oxide with iron(II) leghemoglobin. The nitric oxide may be generated from arginine via a nitric oxide synthase-like activity present in the nodules...

  17. Global forest loss disproportionately erodes biodiversity in intact landscapes.

    Science.gov (United States)

    Betts, Matthew G; Wolf, Christopher; Ripple, William J; Phalan, Ben; Millers, Kimberley A; Duarte, Adam; Butchart, Stuart H M; Levi, Taal

    2017-07-27

    Global biodiversity loss is a critical environmental crisis, yet the lack of spatial data on biodiversity threats has hindered conservation strategies. Theory predicts that abrupt biodiversity declines are most likely to occur when habitat availability is reduced to very low levels in the landscape (10-30%). Alternatively, recent evidence indicates that biodiversity is best conserved by minimizing human intrusion into intact and relatively unfragmented landscapes. Here we use recently available forest loss data to test deforestation effects on International Union for Conservation of Nature Red List categories of extinction risk for 19,432 vertebrate species worldwide. As expected, deforestation substantially increased the odds of a species being listed as threatened, undergoing recent upgrading to a higher threat category and exhibiting declining populations. More importantly, we show that these risks were disproportionately high in relatively intact landscapes; even minimal deforestation has had severe consequences for vertebrate biodiversity. We found little support for the alternative hypothesis that forest loss is most detrimental in already fragmented landscapes. Spatial analysis revealed high-risk hot spots in Borneo, the central Amazon and the Congo Basin. In these regions, our model predicts that 121-219 species will become threatened under current rates of forest loss over the next 30 years. Given that only 17.9% of these high-risk areas are formally protected and only 8.9% have strict protection, new large-scale conservation efforts to protect intact forests are necessary to slow deforestation rates and to avert a new wave of global extinctions.

  18. Antimicrobial activity of a new intact skin antisepsis formulation.

    Science.gov (United States)

    Russo, Antonello; Viotti, Pier Luigi; Vitali, Matteo; Clementi, Massimo

    2003-04-01

    Different antiseptic formulations have shown limitations when applied to disinfecting intact skin, notably short-term tolerability and/or efficacy. The purpose of this study was optimizing a new antiseptic formulation specifically targeted at intact skin disinfection and evaluating its in vitro microbicidal activity and in vivo efficacy. The biocidal properties of the antiseptic solution containing 0.5% chloramine-T diluted in 50% isopropyl alcohol (Cloral; Eurospital SpA Trieste, Italy) were measured in vitro versus gram-positive-, gram-negative-, and acid-alcohol-resistant germs and fungi with standard suspension tests in the presence of fetal bovine serum. Virus-inhibiting activity was evaluated in vitro against human cytomegalovirus, herpes simplex virus, poliovirus, hepatitis B virus, and hepatitis C virus. Tests used different methods for the different biologic and in vitro replication capacity of these human viruses. Lastly, Cloral tolerability and skin colonization retardation efficacy after disinfection were studied in vivo. The antiseptic under review showed fast and sustained antimicrobial activity. The efficacy of Cloral against clinically important bacterial and viral pathogens and fungi was highlighted under the experimental conditions described in this article. Finally, microbial regrowth lag and no side effects were documented in vivo after disinfection of 11 volunteers. A stable chloramine-T solution in isopropyl alcohol may be suggested for intact skin antisepsis.

  19. AVP-stimulated nucleotide secretion in perfused mouse medullary thick ascending limb and cortical collecting duct

    DEFF Research Database (Denmark)

    Odgaard, Elvin V. P.; Prætorius, Helle; Leipziger, Jens Georg

    2009-01-01

    is stimulated remain elusive. Here, we investigate the phenomenon of nucleotide secretion in intact, perfused mouse medullary thick ascending limb (mTAL) and cortical collecting duct (CCD). The nucleotide secretion was monitored by a biosensor adapted to register nucleotides in the tubular outflow...

  20. High Throughput Microplate Respiratory Measurements Using Minimal Quantities Of Isolated Mitochondria

    Science.gov (United States)

    Rogers, George W.; Brand, Martin D.; Petrosyan, Susanna; Ashok, Deepthi; Elorza, Alvaro A.; Ferrick, David A.; Murphy, Anne N.

    2011-01-01

    Recently developed technologies have enabled multi-well measurement of O2 consumption, facilitating the rate of mitochondrial research, particularly regarding the mechanism of action of drugs and proteins that modulate metabolism. Among these technologies, the Seahorse XF24 Analyzer was designed for use with intact cells attached in a monolayer to a multi-well tissue culture plate. In order to have a high throughput assay system in which both energy demand and substrate availability can be tightly controlled, we have developed a protocol to expand the application of the XF24 Analyzer to include isolated mitochondria. Acquisition of optimal rates requires assay conditions that are unexpectedly distinct from those of conventional polarography. The optimized conditions, derived from experiments with isolated mouse liver mitochondria, allow multi-well assessment of rates of respiration and proton production by mitochondria attached to the bottom of the XF assay plate, and require extremely small quantities of material (1–10 µg of mitochondrial protein per well). Sequential measurement of basal, State 3, State 4, and uncoupler-stimulated respiration can be made in each well through additions of reagents from the injection ports. We describe optimization and validation of this technique using isolated mouse liver and rat heart mitochondria, and apply the approach to discover that inclusion of phosphatase inhibitors in the preparation of the heart mitochondria results in a specific decrease in rates of Complex I-dependent respiration. We believe this new technique will be particularly useful for drug screening and for generating previously unobtainable respiratory data on small mitochondrial samples. PMID:21799747

  1. High throughput microplate respiratory measurements using minimal quantities of isolated mitochondria.

    Directory of Open Access Journals (Sweden)

    George W Rogers

    Full Text Available Recently developed technologies have enabled multi-well measurement of O(2 consumption, facilitating the rate of mitochondrial research, particularly regarding the mechanism of action of drugs and proteins that modulate metabolism. Among these technologies, the Seahorse XF24 Analyzer was designed for use with intact cells attached in a monolayer to a multi-well tissue culture plate. In order to have a high throughput assay system in which both energy demand and substrate availability can be tightly controlled, we have developed a protocol to expand the application of the XF24 Analyzer to include isolated mitochondria. Acquisition of optimal rates requires assay conditions that are unexpectedly distinct from those of conventional polarography. The optimized conditions, derived from experiments with isolated mouse liver mitochondria, allow multi-well assessment of rates of respiration and proton production by mitochondria attached to the bottom of the XF assay plate, and require extremely small quantities of material (1-10 µg of mitochondrial protein per well. Sequential measurement of basal, State 3, State 4, and uncoupler-stimulated respiration can be made in each well through additions of reagents from the injection ports. We describe optimization and validation of this technique using isolated mouse liver and rat heart mitochondria, and apply the approach to discover that inclusion of phosphatase inhibitors in the preparation of the heart mitochondria results in a specific decrease in rates of Complex I-dependent respiration. We believe this new technique will be particularly useful for drug screening and for generating previously unobtainable respiratory data on small mitochondrial samples.

  2. Altered ingestive behavior, weight changes, and intact olfactory sense in an APP overexpression model.

    Science.gov (United States)

    Vloeberghs, Ellen; Van Dam, Debby; Franck, Frieda; Serroyen, Jan; Geert, Molenberghs; Staufenbiel, Matthias; De Deyn, Peter Paul

    2008-06-01

    Transgenic APP23 mice were generated to model Alzheimer's disease. The APP23 model develops pathological features, learning deficits, and memory deficits analogous to dementing patients. In this report, transgenic mice exhibited several behavioral disturbances indicating the presence of neuropsychiatric symptoms of dementia. Aiming to verify whether the model also develops other behavioral problems, the authors investigated ingestive behavior in APP23 males of 3, 6 and 12 months. In addition, body weights of a naive male group were longitudinally monitored starting at weaning. Olfactory acuity was evaluated in mice of different age groups. Although olfactory functioning of APP23 mice appeared intact, they drank more and took more food pellets compared with wild-type littermates during a 1-week registration period. From the age of 4.5 weeks onward, APP23 males weighed significantly less than their control littermates, whereas this difference became more prominent with increasing age. Our results suggest the presence of a hypermetabolic state in this model. This is the first report, evidencing the presence of changes in eating and drinking behavior in a single transgenic Alzheimer mouse model. (Copyright) 2008 APA, all rights reserved.

  3. Genomic Relatedness of Chlamydia Isolates Determined by Amplified Fragment Length Polymorphism Analysis

    OpenAIRE

    Meijer, Adam; Morré, Servaas A.; Van Den Brule, Adriaan J. C.; Savelkoul, Paul H. M.; Ossewaarde, Jacobus M.

    1999-01-01

    The genomic relatedness of 19 Chlamydia pneumoniae isolates (17 from respiratory origin and 2 from atherosclerotic origin), 21 Chlamydia trachomatis isolates (all serovars from the human biovar, an isolate from the mouse biovar, and a porcine isolate), 6 Chlamydia psittaci isolates (5 avian isolates and 1 feline isolate), and 1 Chlamydia pecorum isolate was studied by analyzing genomic amplified fragment length polymorphism (AFLP) fingerprints. The AFLP procedure was adapted from a previously...

  4. Gene expression changes in the colon epithelium are similar to those of intact colon during late inflammation in interleukin-10 gene deficient mice.

    Directory of Open Access Journals (Sweden)

    Anna E Russ

    Full Text Available In addition to their role in absorption and secretion, epithelial cells play an important role in the protection of the colon mucosa from the resident microbiota and are important for the maintenance of homeostasis. Microarray analysis of intact colon samples is widely used to gain an overview of the cellular pathways and processes that are active in the colon during inflammation. Laser microdissection of colon epithelial cells allows a more targeted analysis of molecular pathways in the mucosa, preceding and during inflammation, with potentially increased sensitivity to changes in specific cell populations. The aim of this study was to investigate the molecular changes that occur in early and late inflammation stages in colon epithelium of a mouse model of inflammatory bowel diseases. Microarray analysis of intact colon samples and microdissected colon epithelial cell samples from interleukin-10 gene deficient and control mice at 6 and 12 weeks of age was undertaken. Results of gene set enrichment analysis showed that more immune-related pathways were identified between interleukin-10 gene deficient and control mice at 6 weeks of age in epithelial cells than intact colon. This suggests that targeting epithelial cells could increase sensitivity for detecting immune changes that occur early in the inflammatory process. However, in the later stages of inflammation, microarray analyses of intact colon and epithelium both provide a similar overview of gene expression changes in the colon mucosa at the pathway level.

  5. Feasible pickup from intact ossicular chain with floating piezoelectric microphone.

    Science.gov (United States)

    Kang, Hou-Yong; Na, Gao; Chi, Fang-Lu; Jin, Kai; Pan, Tie-Zheng; Gao, Zhen

    2012-02-22

    Many microphones have been developed to meet with the implantable requirement of totally implantable cochlear implant (TICI). However, a biocompatible one without destroying the intactness of the ossicular chain still remains under investigation. Such an implantable floating piezoelectric microphone (FPM) has been manufactured and shows an efficient electroacoustic performance in vitro test at our lab. We examined whether it pick up sensitively from the intact ossicular chain and postulated whether it be an optimal implantable one. Animal controlled experiment: five adult cats (eight ears) were sacrificed as the model to test the electroacoustic performance of the FPM. Three groups were studied: (1) the experiment group (on malleus): the FPM glued onto the handle of the malleus of the intact ossicular chains; (2) negative control group (in vivo): the FPM only hung into the tympanic cavity; (3) positive control group (Hy-M30): a HiFi commercial microphone placed close to the site of the experiment ear. The testing speaker played pure tones orderly ranged from 0.25 to 8.0 kHz. The FPM inside the ear and the HiFi microphone simultaneously picked up acoustic vibration which recorded as .wav files to analyze. The FPM transformed acoustic vibration sensitively and flatly as did the in vitro test across the frequencies above 2.0 kHz, whereas inefficiently below 1.0 kHz for its overloading mass. Although the HiFi microphone presented more efficiently than the FPM did, there was no significant difference at 3.0 kHz and 8.0 kHz. It is feasible to develop such an implantable FPM for future TICIs and TIHAs system on condition that the improvement of Micro Electromechanical System and piezoelectric ceramic material technology would be applied to reduce its weight and minimize its size.

  6. Feasible pickup from intact ossicular chain with floating piezoelectric microphone

    Directory of Open Access Journals (Sweden)

    Kang Hou-Yong

    2012-02-01

    Full Text Available Abstract Objectives Many microphones have been developed to meet with the implantable requirement of totally implantable cochlear implant (TICI. However, a biocompatible one without destroying the intactness of the ossicular chain still remains under investigation. Such an implantable floating piezoelectric microphone (FPM has been manufactured and shows an efficient electroacoustic performance in vitro test at our lab. We examined whether it pick up sensitively from the intact ossicular chain and postulated whether it be an optimal implantable one. Methods Animal controlled experiment: five adult cats (eight ears were sacrificed as the model to test the electroacoustic performance of the FPM. Three groups were studied: (1 the experiment group (on malleus: the FPM glued onto the handle of the malleus of the intact ossicular chains; (2 negative control group (in vivo: the FPM only hung into the tympanic cavity; (3 positive control group (Hy-M30: a HiFi commercial microphone placed close to the site of the experiment ear. The testing speaker played pure tones orderly ranged from 0.25 to 8.0 kHz. The FPM inside the ear and the HiFi microphone simultaneously picked up acoustic vibration which recorded as .wav files to analyze. Results The FPM transformed acoustic vibration sensitively and flatly as did the in vitro test across the frequencies above 2.0 kHz, whereas inefficiently below 1.0 kHz for its overloading mass. Although the HiFi microphone presented more efficiently than the FPM did, there was no significant difference at 3.0 kHz and 8.0 kHz. Conclusions It is feasible to develop such an implantable FPM for future TICIs and TIHAs system on condition that the improvement of Micro Electromechanical System and piezoelectric ceramic material technology would be applied to reduce its weight and minimize its size.

  7. Comparative characteristic of transmembrane currents and caffeine-induced responses of intact and irradiated small intestine smooth muscle cells

    International Nuclear Information System (INIS)

    Stepanov, Yu.V.; Gordienko, D.V.; Preobrazhenskaya, T.D.; Stepanova, L.I.; Vojtsitskij, V.M.

    1994-01-01

    A comparative investigation of transmembrane ion currents and caffeine-induced responses of single smooth muscle cells isolated from the circular layer of rat small intestine was curried out by the method of 'patch-clamp'. No reliable difference in potential-dependent and amplitude-kinetic characteristics of transmembrane ion currents in cells of intact and irradiated with dose of 3 Gy rats was revealed. In cells of irradiated animals external application of caffeine (4 mM) was not accompanied by strong quick-inactivated transient Ca 2+ -dependent potassium current as in control

  8. Radionuclide sorption on crushed and intact granitic rock

    International Nuclear Information System (INIS)

    Eriksen, Tryggve E.; Locklund, Birgitta

    1989-05-01

    The specific surface areas and distribution ratios for sorption of 85 Sr, 137 Cs and 152 Eu were measured for crushed and intact granite rock. The experimental data can be accommodated by a sorption model encompassing sorption on outer and inner surface. It is clearly demonstrated that the time required to obtain reliable Kd-values for the sorption of strongly sorbing radionuclides like 152 Eu is very long due to solution depletion and slow diffusion into the rock. A combination of surface area measurements and batch sorption with small particles may therefore be preferable when studying strongly sorbing nuclides. (authors) (17 figs., 6 tabs.)

  9. Radioiodination of an outer membrane protein in intact Rickettsia prowazekii

    International Nuclear Information System (INIS)

    Smith, D.K.; Winkler, H.H.

    1980-01-01

    Intact Rickettsia prowazekii was radiolabeled with the glucose oxidase-lactoperoxidase method of iodination. Separation of the rickettsial extract into cytoplasmic, outer and inner membrane fractions demonstrated that the outer membrane was preferentially labeled. Analysis of the polypeptides of these fractions on high-resolution slab polyacrylamide gels showed that most of the 125 I was in polypeptide T49, an outer membrane constituent. Additional outer membrane polypeptides were iodinated in broken envelope preparations, demonstrating that T49 is uniquely accessible to the external environment and the asymmetric polypeptide organization of the outer membrane

  10. Bone marrow dosimetry in rats using direct tissue counting after injection of radio-iodinated intact monoclonal antibodies or F(ab')2 fragments

    International Nuclear Information System (INIS)

    Buchegger, F.; Chalandon, Y.; Pelegrin, A.; Hardman, N.; Mach, J.P.

    1991-01-01

    Normal rats were injected intravenously with 131I- and 125I-labeled intact murine and chimeric mouse-human monoclonal antibodies directed against carcinoembryonic antigen or with the corresponding F(ab')2 fragments. At different times after injection, individual animals were killed and radioactivity of blood and major organs, including bones and bone marrow, was determined. Ratios comparing radioactivity concentration in different tissues with that of bone marrow were calculated and found to remain stable during several effective half-lives of the antibodies. Mean bone marrow radioactivity was 35% (range, 29%-40%) of that of blood and 126% (range, 108%-147%) of that of liver after injection of intact Mabs or F(ab')2 fragments. In nude rats bearing human colon carcinoma xenografts producing carcinoembryonic antigen, relative bone marrow radioactivity was slightly lower than that in normal rats

  11. Equol inhibits growth, induces atresia, and inhibits steroidogenesis of mouse antral follicles in vitro

    International Nuclear Information System (INIS)

    Mahalingam, Sharada; Gao, Liying; Gonnering, Marni; Helferich, William; Flaws, Jodi A.

    2016-01-01

    Equol is a non-steroidal estrogen metabolite produced by microbial conversion of daidzein, a major soy isoflavone, in the gut of some humans and many animal species. Isoflavones and their metabolites can affect endogenous estradiol production, action, and metabolism, potentially influencing ovarian follicle function. However, no studies have examined the effects of equol on intact ovarian antral follicles, which are responsible for sex steroid synthesis and further development into ovulatory follicles. Thus, the present study tested the hypothesis that equol inhibits antral follicle growth, increases follicle atresia, and inhibits steroidogenesis in the adult mouse ovary. To test this hypothesis, antral follicles isolated from adult CD-1 mice were cultured with vehicle control (dimethyl sulfoxide; DMSO) or equol (600 nM, 6 μM, 36 μM, and 100 μM) for 48 and 96 h. Every 24 h, follicle diameters were measured to monitor growth. At 48 and 96 h, the culture medium was subjected to measurement of hormone levels, and the cultured follicles were subjected to gene expression analysis. Additionally, follicles were histologically evaluated for signs of atresia after 96 h of culture. The results indicate that equol (100 μM) inhibited follicle growth, altered the mRNA levels of bcl2-associated X protein and B cell leukemia/lymphoma 2, and induced follicle atresia. Further, equol decreased the levels of estradiol, testosterone, androstenedione, and progesterone, and it decreased mRNA levels of cholesterol side-chain cleavage, steroid 17-α-hydroxalase, and aromatase. Collectively, these data indicate that equol inhibits growth, increases atresia, and inhibits steroidogenesis of cultured mouse antral follicles. - Highlights: • Equol exposure inhibits antral follicle growth. • Equol exposure increases follicle atresia. • Equol exposure inhibits sex steroid hormone levels. • Equol exposure inhibits mRNA levels of certain steroidogenic enzymes.

  12. Equol inhibits growth, induces atresia, and inhibits steroidogenesis of mouse antral follicles in vitro

    Energy Technology Data Exchange (ETDEWEB)

    Mahalingam, Sharada, E-mail: mahalin2@illinois.edu [Department of Comparative Biosciences, College of Veterinary Medicine, University of Illinois, 2001 S. Lincoln Ave, Urbana, IL 61802 (United States); Gao, Liying, E-mail: lgao@uiuc.edu [Department of Comparative Biosciences, College of Veterinary Medicine, University of Illinois, 2001 S. Lincoln Ave, Urbana, IL 61802 (United States); Gonnering, Marni, E-mail: mgonne2@illinois.edu [Department of Comparative Biosciences, College of Veterinary Medicine, University of Illinois, 2001 S. Lincoln Ave, Urbana, IL 61802 (United States); Helferich, William, E-mail: helferic@illinois.edu [Department of Food Science and Human Nutrition, University of Illinois, 905 S. Goodwin, Urbana, IL 61801 (United States); Flaws, Jodi A., E-mail: jflaws@illinois.edu [Department of Comparative Biosciences, College of Veterinary Medicine, University of Illinois, 2001 S. Lincoln Ave, Urbana, IL 61802 (United States)

    2016-03-15

    Equol is a non-steroidal estrogen metabolite produced by microbial conversion of daidzein, a major soy isoflavone, in the gut of some humans and many animal species. Isoflavones and their metabolites can affect endogenous estradiol production, action, and metabolism, potentially influencing ovarian follicle function. However, no studies have examined the effects of equol on intact ovarian antral follicles, which are responsible for sex steroid synthesis and further development into ovulatory follicles. Thus, the present study tested the hypothesis that equol inhibits antral follicle growth, increases follicle atresia, and inhibits steroidogenesis in the adult mouse ovary. To test this hypothesis, antral follicles isolated from adult CD-1 mice were cultured with vehicle control (dimethyl sulfoxide; DMSO) or equol (600 nM, 6 μM, 36 μM, and 100 μM) for 48 and 96 h. Every 24 h, follicle diameters were measured to monitor growth. At 48 and 96 h, the culture medium was subjected to measurement of hormone levels, and the cultured follicles were subjected to gene expression analysis. Additionally, follicles were histologically evaluated for signs of atresia after 96 h of culture. The results indicate that equol (100 μM) inhibited follicle growth, altered the mRNA levels of bcl2-associated X protein and B cell leukemia/lymphoma 2, and induced follicle atresia. Further, equol decreased the levels of estradiol, testosterone, androstenedione, and progesterone, and it decreased mRNA levels of cholesterol side-chain cleavage, steroid 17-α-hydroxalase, and aromatase. Collectively, these data indicate that equol inhibits growth, increases atresia, and inhibits steroidogenesis of cultured mouse antral follicles. - Highlights: • Equol exposure inhibits antral follicle growth. • Equol exposure increases follicle atresia. • Equol exposure inhibits sex steroid hormone levels. • Equol exposure inhibits mRNA levels of certain steroidogenic enzymes.

  13. Metabolism of inhaled ethane and pentane by the intact rat

    International Nuclear Information System (INIS)

    Daugherty, M.S.; Luddent, T.M.; Burk, R.F.

    1986-01-01

    Measurement of exhaled ethane or pentane is a noninvasive technique for studying in vivo lipid peroxidation. Many past studies have assumed that pentane and ethane are not metabolized. Radiolabeled ( 14 C) ethane and pentane were used to study the disposition of these compounds in intact rats. Rats were placed for 8 h in a closed plexiglass chamber fitted with a system for replenishing chamber atmospheric O 2 . Evolved CO 2 was trapped by recirculating chamber air through 3 N NaOH contained in a vessel external to the chamber. Radiolabeled ethane or pentane was injected into the chamber at the start of each experiment. The percent of 14 C-activity added to the chamber recovered in the CO 2 trap, urine, and chamber air at the end of the experiment (8 h) in the [ 14 C]-ethane (n=5) and [ 14 C]-pentane (n=4) studies are presented. Results indicate that both ethane and pentane are metabolized to CO 2 in the intact rat. Possible changes in ethane and pentane metabolism must be considered if the exhalation rates of these hydrocarbons are to be used as indices of in vivo lipid peroxidation

  14. Surface plasmon resonance sensing: from purified biomolecules to intact cells.

    Science.gov (United States)

    Su, Yu-Wen; Wang, Wei

    2018-04-12

    Surface plasmon resonance (SPR) has become a well-recognized label-free technique for measuring the binding kinetics between biomolecules since the invention of the first SPR-based immunosensor in 1980s. The most popular and traditional format for SPR analysis is to monitor the real-time optical signals when a solution containing ligand molecules is flowing over a sensor substrate functionalized with purified receptor molecules. In recent years, rapid development of several kinds of SPR imaging techniques have allowed for mapping the dynamic distribution of local mass density within single living cells with high spatial and temporal resolutions and reliable sensitivity. Such capability immediately enabled one to investigate the interaction between important biomolecules and intact cells in a label-free, quantitative, and single cell manner, leading to an exciting new trend of cell-based SPR bioanalysis. In this Trend Article, we first describe the principle and technical features of two types of SPR imaging techniques based on prism and objective, respectively. Then we survey the intact cell-based applications in both fundamental cell biology and drug discovery. We conclude the article with comments and perspectives on the future developments. Graphical abstract Recent developments in surface plasmon resonance (SPR) imaging techniques allow for label-free mapping the mass-distribution within single living cells, leading to great expansions in biomolecular interactions studies from homogeneous substrates functionalized with purified biomolecules to heterogeneous substrates containing individual living cells.

  15. Metabolic impact of adult-onset, isolated, growth hormone deficiency (AOiGHD due to destruction of pituitary somatotropes.

    Directory of Open Access Journals (Sweden)

    Raul M Luque

    2011-01-01

    Full Text Available Growth hormone (GH inhibits fat accumulation and promotes protein accretion, therefore the fall in GH observed with weight gain and normal aging may contribute to metabolic dysfunction. To directly test this hypothesis a novel mouse model of adult onset-isolated GH deficiency (AOiGHD was generated by cross breeding rat GH promoter-driven Cre recombinase mice (Cre with inducible diphtheria toxin receptor mice (iDTR and treating adult Cre(+/-,iDTR(+/- offspring with DT to selectively destroy the somatotrope population of the anterior pituitary gland, leading to a reduction in circulating GH and IGF-I levels. DT-treated Cre(-/-,iDTR(+/- mice were used as GH-intact controls. AOiGHD improved whole body insulin sensitivity in both low-fat and high-fat fed mice. Consistent with improved insulin sensitivity, indirect calorimetry revealed AOiGHD mice preferentially utilized carbohydrates for energy metabolism, as compared to GH-intact controls. In high-fat, but not low-fat fed AOiGHD mice, fat mass increased, hepatic lipids decreased and glucose clearance and insulin output were impaired. These results suggest the age-related decline in GH helps to preserve systemic insulin sensitivity, and in the context of moderate caloric intake, prevents the deterioration in metabolic function. However, in the context of excess caloric intake, low GH leads to impaired insulin output, and thereby could contribute to the development of diabetes.

  16. Maximizing Selective Cleavages at Aspartic Acid and Proline Residues for the Identification of Intact Proteins

    Science.gov (United States)

    Foreman, David J.; Dziekonski, Eric T.; McLuckey, Scott A.

    2018-04-01

    A new approach for the identification of intact proteins has been developed that relies on the generation of relatively few abundant products from specific cleavage sites. This strategy is intended to complement standard approaches that seek to generate many fragments relatively non-selectively. Specifically, this strategy seeks to maximize selective cleavage at aspartic acid and proline residues via collisional activation of precursor ions formed via electrospray ionization (ESI) under denaturing conditions. A statistical analysis of the SWISS-PROT database was used to predict the number of arginine residues for a given intact protein mass and predict a m/z range where the protein carries a similar charge to the number of arginine residues thereby enhancing cleavage at aspartic acid residues by limiting proton mobility. Cleavage at aspartic acid residues is predicted to be most favorable in the m/z range of 1500-2500, a range higher than that normally generated by ESI at low pH. Gas-phase proton transfer ion/ion reactions are therefore used for precursor ion concentration from relatively high charge states followed by ion isolation and subsequent generation of precursor ions within the optimal m/z range via a second proton transfer reaction step. It is shown that the majority of product ion abundance is concentrated into cleavages C-terminal to aspartic acid residues and N-terminal to proline residues for ions generated by this process. Implementation of a scoring system that weights both ion fragment type and ion fragment area demonstrated identification of standard proteins, ranging in mass from 8.5 to 29.0 kDa. [Figure not available: see fulltext.

  17. Gaze beats mouse

    DEFF Research Database (Denmark)

    Mateo, Julio C.; San Agustin, Javier; Hansen, John Paulin

    2008-01-01

    Facial EMG for selection is fast, easy and, combined with gaze pointing, it can provide completely hands-free interaction. In this pilot study, 5 participants performed a simple point-and-select task using mouse or gaze for pointing and a mouse button or a facial-EMG switch for selection. Gaze...

  18. Cotton leaf curl Burewala virus with intact or mutant transcriptional activator proteins: complexity of cotton leaf curl disease.

    Science.gov (United States)

    Kumar, Jitendra; Gunapati, Samatha; Alok, Anshu; Lalit, Adarsh; Gadre, Rekha; Sharma, Naresh C; Roy, Joy K; Singh, Sudhir P

    2015-05-01

    Cotton leaf curl disease (CLCuD) is a serious disease of cotton on the Indian subcontinent. In the present study, three cotton leaf curl viruses, cotton leaf curl Burewala virus (CLCuBuV), cotton leaf curl Kokhran virus (CLCuKoV) and cotton leaf curl Multan virus (CLCuMV), and their associated satellites, cotton leaf curl Multan betasatellite (CLCuMB) and cotton leaf curl Multan alphasatellite (CLCuMA), were detected. CLCuBuV with either intact (CLCuBuV-1) or mutant (CLCuBuV-2) transcriptional activator protein (TrAP) were detected in different plants. Agroinoculation with CLCuBuV-1 or CLCuBuV-2 together with CLCuMB and CLCuMA, resulted in typical leaf curling and stunting of tobacco plants. Inoculation with CLCuKoV or an isolate of CLCuMV (CLCuMV-2), together with CLCuMB and CLCuMA, induced severe leaf curling, while the other isolate of CLCuMV (CLCuMV-1), which was recombinant in origin, showed mild leaf curling in tobacco. To investigate the effect of intact or mutant TrAP and also the recombination events, CLCuBuV-1, CLCuBuV-2, CLCuMV-1 or CLCuMV-2 together with the satellites (CLCuMA and CLCuMB) were transferred to cotton via whitefly-mediated transmission. Cotton plants containing CLCuBuV-1, CLCuBuV-2 or CLCuMV-2 together with satellites showed curling and stunting, whereas the plants having CLCuMV-1 and the satellites showed only mild and indistinguishable symptoms. CLCuBuV-1 (intact TrAP) showed severe symptoms in comparison to CLCuBuV-2 (mutant TrAP). The present study reveals that two types of CLCuBuV, one with an intact TrAP and the other with a mutant TrAP, exist in natural infection of cotton in India. Additionally, CLCuMuV-1, which has a recombinant origin, induces mild symptoms in comparison to the other CLCuMV isolates.

  19. Transport of indoleacetic acid in intact corn coleoptiles

    International Nuclear Information System (INIS)

    Parker, K.E.; Briggs, W.R.

    1990-01-01

    We have characterized the transport of [ 3 H]indoleacetic acid (IAA) in intact corn (Zea mays L.) coleoptiles. We have used a wide range of concentrations of added IAA (28 femtomoles to 100 picomoles taken up over 60 minutes). The shape of the transport curve varies with the concentration of added IAA, although the rate of movement of the observed front of tracer is invariant with concentration. At the lowest concentration of tracer used, the labeled IAA in the transport stream is not detectably metabolized or immobilized, curvature does not develop as a result of tracer application, and normal phototropic and gravitropic responsiveness are not affected. Therefore we believe we are observing the transport of true tracer quantities of labeled auxin at this lowest concentration

  20. Simple Genome Editing of Rodent Intact Embryos by Electroporation.

    Directory of Open Access Journals (Sweden)

    Takehito Kaneko

    Full Text Available The clustered regularly interspaced short palindromic repeat (CRISPR/CRISPR-associated (Cas system is a powerful tool for genome editing in animals. Recently, new technology has been developed to genetically modify animals without using highly skilled techniques, such as pronuclear microinjection of endonucleases. Technique for animal knockout system by electroporation (TAKE method is a simple and effective technology that produces knockout rats by introducing endonuclease mRNAs into intact embryos using electroporation. Using TAKE method and CRISPR/Cas system, the present study successfully produced knockout and knock-in mice and rats. The mice and rats derived from embryos electroporated with Cas9 mRNA, gRNA and single-stranded oligodeoxynucleotide (ssODN comprised the edited targeted gene as a knockout (67% of mice and 88% of rats or knock-in (both 33%. The TAKE method could be widely used as a powerful tool to produce genetically modified animals by genome editing.

  1. Selective dansylation of M protein within intact influenza virions

    Energy Technology Data Exchange (ETDEWEB)

    Robertson, B.H.; Bennett, J.C.; Compans, R.W.

    1982-12-01

    Exposure of purified influenza virions to (/sup 14/C)dansyl chloride resulted in the covalent attachment of the dansyl chromophore to the virion. Gel electrophoresis revealed that the dansyl chromophore was specifically coupled to the internal membrane (M) protein. Purification of the M protein by gel filtration followed by cyanogen bromide cleavage and peptide fractionation revealed that four of six peptide peaks contained dansyl label. Acid hydrolysis of the separated peptide peaks followed by thin-layer chromatography revealed that dansyl label was coupled to lysine residues present in these peptides. The results of these investigations have demonstrated that the M protein molecule is the major viral polypeptide labeled when intact virions are exposed to dansyl chloride.

  2. Selective dansylation of M protein within intact influenza virions

    International Nuclear Information System (INIS)

    Robertson, B.H.; Bennett, J.C.; Compans, R.W.

    1982-01-01

    Exposure of purified influenza virions to [ 14 C]dansyl chloride resulted in the covalent attachment of the dansyl chromophore to the virion. Gel electrophoresis revealed that the dansyl chromophore was specifically coupled to the internal membrane (M) protein. Purification of the M protein by gel filtration followed by cyanogen bromide cleavage and peptide fractionation revealed that four of six peptide peaks contained dansyl label. Acid hydrolysis of the separated peptide peaks followed by thin-layer chromatography revealed that dansyl label was coupled to lysine residues present in these peptides. The results of these investigations have demonstrated that the M protein molecule is the major viral polypeptide labeled when intact virions are exposed to dansyl chloride

  3. Optimized Planning Target Volume for Intact Cervical Cancer

    International Nuclear Information System (INIS)

    Khan, Alvin; Jensen, Lindsay G.; Sun Shuai; Song, William Y.; Yashar, Catheryn M.; Mundt, Arno J.; Zhang Fuquan; Jiang, Steve B.; Mell, Loren K.

    2012-01-01

    Purpose: To model interfraction clinical target volume (CTV) variation in patients with intact cervical cancer and design a planning target volume (PTV) that minimizes normal tissue dose while maximizing CTV coverage. Methods and Materials: We analyzed 50 patients undergoing external-beam radiotherapy for intact cervical cancer using daily online cone-beam computed tomography (CBCT). The CBCTs (n = 972) for each patient were rigidly registered to the planning CT. The CTV was delineated on the planning CT (CTV 0 ) and the set of CBCTs ({CTV 1 –CTV 25 }). Manual (n = 98) and automated (n = 668) landmarks were placed over the surface of CTV 0 with reference to defined anatomic structures. Normal vectors were extended from each landmark, and the minimum length required for a given probability of encompassing CTV 1 –CTV 25 was computed. The resulting expansions were used to generate an optimized PTV. Results: The mean (SD; range) normal vector length to ensure 95% coverage was 4.3 mm (2.7 mm; 1–16 mm). The uniform expansion required to ensure 95% probability of CTV coverage was 13 mm. An anisotropic margin of 20 mm anteriorly and posteriorly and 10 mm superiorly, inferiorly, and laterally also would have ensured a 95% probability of CTV coverage. The volume of the 95% optimized PTV (1470 cm 3 ) was significantly lower than both the anisotropic PTV (2220 cm 3 ) and the uniformly expanded PTV (2110 cm 3 ) (p 0 , 5–10 mm along the interfaces of CTV 0 with the bladder and rectum, and 10–14 mm along the anterior surface of CTV 0 at the level of the uterus. Conclusion: Optimizing PTV definition according to surface landmarking resulted in a high probability of CTV coverage with reduced PTV volumes. Our results provide data justifying planning margins to use in practice and clinical trials.

  4. Isolated galaxies

    International Nuclear Information System (INIS)

    Einasto, Maret

    1990-01-01

    To test for the possible presence of really isolated galaxies, which form a randomly distributed population in voids, we compare the distribution of most isolated galaxies in an observed sample with distributions of the same number of random points using the nearest neighbour test. The results show that the random population of really isolated galaxies does not exist - even the most isolated galaxies are connected with systems of galaxies, forming their outlying parts. (author)

  5. NIH Mouse Metabolic Phenotyping Centers: the power of centralized phenotyping.

    Science.gov (United States)

    Laughlin, Maren R; Lloyd, K C Kent; Cline, Gary W; Wasserman, David H

    2012-10-01

    The Mouse Metabolic Phenotyping Centers (MMPCs) were founded in 2001 by the National Institutes of Health (NIH) to advance biomedical research by providing the scientific community with standardized, high-quality phenotyping services for mouse models of diabetes, obesity, and their complications. The intent is to allow researchers to take optimum advantage of the many new mouse models produced in labs and in high-throughput public efforts. The six MMPCs are located at universities around the country and perform complex metabolic tests in intact mice and hormone and analyte assays in tissues on a fee-for-service basis. Testing is subsidized by the NIH in order to reduce the barriers for mouse researchers. Although data derived from these tests belong to the researcher submitting mice or tissues, these data are archived after publication in a public database run by the MMPC Coordinating and Bioinformatics Unit. It is hoped that data from experiments performed in many mouse models of metabolic diseases, using standard protocols, will be useful in understanding the nature of these complex disorders. The current areas of expertise include energy balance and body composition, insulin action and secretion, whole-body and tissue carbohydrate and lipid metabolism, cardiovascular and renal function, and metabolic pathway kinetics. In addition to providing services, the MMPC staff provides expertise and advice to researchers, and works to develop and refine test protocols to best meet the community's needs in light of current scientific developments. Test technology is disseminated by publications and through annual courses.

  6. Mouse Genome Informatics (MGI)

    Data.gov (United States)

    U.S. Department of Health & Human Services — MGI is the international database resource for the laboratory mouse, providing integrated genetic, genomic, and biological data to facilitate the study of human...

  7. Mouse Phenome Database (MPD)

    Data.gov (United States)

    U.S. Department of Health & Human Services — The Mouse Phenome Database (MPD) has characterizations of hundreds of strains of laboratory mice to facilitate translational discoveries and to assist in selection...

  8. TL transgenic mouse strains

    International Nuclear Information System (INIS)

    Obata, Y.; Matsudaira, Y.; Hasegawa, H.; Tamaki, H.; Takahashi, T.; Morita, A.; Kasai, K.

    1993-01-01

    As a result of abnormal development of the thymus of these mice, TCR αβ lineage of the T cell differentiation is disturbed and cells belonging to the TCR γδ CD4 - CD8 - double negative (DN) lineage become preponderant. The γδ DN cells migrate into peripheral lymphoid organs and constitute nearly 50% of peripheral T cells. Immune function of the transgenic mice is severely impaired, indicating that the γδ cells are incapable of participating in these reactions. Molecular and serological analyses of T-cell lymphomas reveal that they belong to the γδ lineage. Tg.Tla a -3-1 mice should be useful in defining the role of TL in normal and abnormal T cell differentiation as well as in the development of T-cell lymphomas, and further they should facilitate studies on the differentiation and function of γδ T cells. We isolated T3 b -TL gene from B6 mice and constructed a chimeric gene in which T3 b -TL is driven by the promoter of H-2K b . With the chimeric gene, two transgenic mouse strains, Tg. Con.3-1 and -2 have been derived in C3H background. Both strains express TL antigen in various tissues including skin. The skin graft of transgenic mice on C3H and (B6 X C3H)F 1 mice were rejected. In the mice which rejected the grafts, CD8 + TCRαβ cytotoxic T cells (CTL) against TL antigens were recognized. The recognition of TL by CTL did not require the antigen presentation by H-2 molecules. The results indicated that TL antigen in the skin becomes a transplantation antigen and behaves like a typical allogeneic MHC class I antigen. The facts that (B6 X C3H)F 1 mice rejected the skin expressing T3 b -TL antigen and induced CTL that killed TL + lymphomas of B6 origin revealed that TL antigen encoded by T3 b -TL is recognized as non-self in B6 mice. Experiments are now extended to analyze immune responses to TL antigen expressed on autochthonous T cell lymphomas. (J.P.N.)

  9. Mouse models of long QT syndrome

    Science.gov (United States)

    Salama, Guy; London, Barry

    2007-01-01

    Congenital long QT syndrome is a rare inherited condition characterized by prolongation of action potential duration (APD) in cardiac myocytes, prolongation of the QT interval on the surface electrocardiogram (ECG), and an increased risk of syncope and sudden death due to ventricular tachyarrhythmias. Mutations of cardiac ion channel genes that affect repolarization cause the majority of the congenital cases. Despite detailed characterizations of the mutated ion channels at the molecular level, a complete understanding of the mechanisms by which individual mutations may lead to arrhythmias and sudden death requires study of the intact heart and its modulation by the autonomic nervous system. Here, we will review studies of molecularly engineered mice with mutations in the genes (a) known to cause long QT syndrome in humans and (b) specific to cardiac repolarization in the mouse. Our goal is to provide the reader with a comprehensive overview of mouse models with long QT syndrome and to emphasize the advantages and limitations of these models. PMID:17038432

  10. Hyperoxia decreases glycolytic capacity, glycolytic reserve and oxidative phosphorylation in MLE-12 cells and inhibits complex I and II function, but not complex IV in isolated mouse lung mitochondria.

    Directory of Open Access Journals (Sweden)

    Kumuda C Das

    Full Text Available High levels of oxygen (hyperoxia are frequently used in critical care units and in conditions of respiratory insufficiencies in adults, as well as in infants. However, hyperoxia has been implicated in a number of pulmonary disorders including bronchopulmonary dysplasia (BPD and adult respiratory distress syndrome (ARDS. Hyperoxia increases the generation of reactive oxygen species (ROS in the mitochondria that could impair the function of the mitochondrial electron transport chain. We analyzed lung mitochondrial function in hyperoxia using the XF24 analyzer (extracellular flux and optimized the assay for lung epithelial cells and mitochondria isolated from lungs of mice. Our data show that hyperoxia decreases basal oxygen consumption rate (OCR, spare respiratory capacity, maximal respiration and ATP turnover in MLE-12 cells. There was significant decrease in glycolytic capacity and glycolytic reserve in MLE-12 cells exposed to hyperoxia. Using mitochondria isolated from lungs of mice exposed to hyperoxia or normoxia we have shown that hyperoxia decreased the basal, state 3 and state3 μ (respiration in an uncoupled state respirations. Further, using substrate or inhibitor of a specific complex we show that the OCR via complex I and II, but not complex IV was decreased, demonstrating that complexes I and II are specific targets of hyperoxia. Further, the activities of complex I (NADH dehydrogenase, NADH-DH and complex II (succinate dehydrogenase, SDH were decreased in hyperoxia, but the activity of complex IV (cytochrome oxidase, COX remains unchanged. Taken together, our study show that hyperoxia impairs glycolytic and mitochondrial energy metabolism in in tact cells, as well as in lungs of mice by selectively inactivating components of electron transport system.

  11. Digested BLG can induce tolerance when co-administered with intact BLG in Brown Norway rats

    DEFF Research Database (Denmark)

    Bøgh, Katrine Lindholm; Barkholt, Vibeke; Madsen, Charlotte Bernhard

    the human gastro-duodenal digestion process. Four different fractions of BLG-digest was made, based on sizes of peptides or aggregates hereof. Intact BLG and the four fractions of BLG-digesta were characterized by protein chemical analyses. Brown Norway (BN) rats were immunised i.p. three times without......Background: Milk is a major constituent of small children’s diet. Milk allergy is also one of the most common allergies in small children. Prevention, treatment and general understanding of this allergy are therefore important. Methods: Intact BLG was digested in an in vitro model simulating...... the use of adjuvant with either PBS (control), 200 µg of intact BLG, 30 µg of intact BLG, 200 µg of digested BLG (with 30 µg of intact BLG), 200 µg of digested BLG, 200 µg of a fraction of large complexes or 200 µg of a fraction of small complexes (all three without intact BLG). Sera from BN rats were...

  12. Molecular characteristics and virulence potential of Listeria monocytogenes isolates from Chinese food systems.

    Science.gov (United States)

    Chen, Jianshun; Luo, Xiaokai; Jiang, Lingli; Jin, Peijie; Wei, Wei; Liu, Dongyou; Fang, Weihuan

    2009-02-01

    In this study, we examined Listeria monocytogenes isolates from Chinese food sources in an attempt to gain further insights on the molecular characteristics and virulence potential of this important foodborne pathogen. Of the 88 L. monocytogenes food isolates recovered, 42 (47.7%) were of serovars 1/2a or 3a; 23 (26.1%) of serovars 1/2b or 3b; 15 (17.0%) of 1/2c or 3c; 6 (6.8%) of serovars 4b, 4d or 4e; and 2 (2.2%) of serovars 4a or 4c. In contrast to inlAB locus conserved in all serovars, internalin cluster between ascB and dapE varies with different serovars, with inlC2DE, inlGC2DE and inlGHE predominantly in serovars 1/2b or 4b, serovar 1/2a and serovar 1/2c. While inlF existed in all the inlGHE- and inlGC2DE-containing isolates but 17.4% of those having inlC2DE, lmo2026 existed in all the inlGHE-containing isolates but 20.0% of those bearing inlGC2DE, suggesting that inlF might have co-evolved with inlGC2DE and inlGHE while lmo2026 with inlGHE only. With the exception of serovar 4a isolate, most serovar isolates demonstrated remarkable ability to form plaques on L929 cells and produced significant mouse mortality irrespective of the internalin gene organization and whether an intact actA gene is present or not. These results indicate that majority of these food isolates may have the potential to cause human diseases if ingested via contaminated foods. Given that serovar 4b accounts for nearly half of human clinical listeriosis cases documented, the relative low proportion of serovar 4b food isolates suggests that this serovar is probably more tolerant of the adverse conditions in the host's stomach and/or more efficient in entering host cells than serovars 1/2a, 1/2b and 1/2c.

  13. Enhanced Automatic Action Imitation and Intact Imitation-Inhibition in Schizophrenia.

    Science.gov (United States)

    Simonsen, Arndis; Fusaroli, Riccardo; Skewes, Joshua Charles; Roepstorff, Andreas; Campbell-Meiklejohn, Daniel; Mors, Ole; Bliksted, Vibeke

    2018-02-21

    Imitation plays a key role in social learning and in facilitating social interactions and likely constitutes a basic building block of social cognition that supports higher-level social abilities. Recent findings suggest that patients with schizophrenia have imitation impairments that could contribute to the social impairments associated with the disorder. However, extant studies have specifically assessed voluntary imitation or automatic imitation of emotional stimuli without controlling for potential confounders. The imitation impairments seen might therefore be secondary to other cognitive, motoric, or emotional deficits associated with the disorder. To overcome this issue, we used an automatic imitation paradigm with nonemotional stimuli to assess automatic imitation and the top-down modulation of imitation where participants were required to lift one of 2 fingers according to a number shown on the screen while observing the same or the other finger movement. In addition, we used a control task with a visual cue in place of a moving finger, to isolate the effect of observing finger movement from other visual cueing effects. Data from 33 patients (31 medicated) and 40 matched healthy controls were analyzed. Patients displayed enhanced imitation and intact top-down modulation of imitation. The enhanced imitation seen in patients may have been medication induced as larger effects were seen in patients receiving higher antipsychotic doses. In sum, we did not find an imitation impairment in schizophrenia. The results suggest that previous findings of impaired imitation in schizophrenia might have been due to other cognitive, motoric, and/or emotional deficits.

  14. Characterization of methanotrophic bacteria on the basis of intact phospholipid profiles.

    Science.gov (United States)

    Fang, J; Barcelona, M J; Semrau, J D

    2000-08-01

    The intact phospholipid profiles (IPPs) of seven species of methanotrophs from all three physiological groups, type I, II and X, were determined using liquid chromatography/electrospray ionization/mass spectrometry. In these methanotrophs, two major classes of phospholipids were found, phosphatidylglycerol (PG) and phosphatidylethanolamine (PE) as well as its derivatives phosphatidylmethylethanolamine (PME) and phosphatidyldimethylethanolamine (PDME). Specifically, the type I methanotrophs, Methylomonas methanica, Methylomonas rubra and Methylomicrobium album BG8 were characterized by PE and PG phospholipids with predominantly C16:1 fatty acids. The type II methanotrophs, Methylosinus trichosporium OB3b and CSC1 were characterized by phospholipids of PG, PME and PDME with predominantly C18:1 fatty acids. Methylococcus capsulatus Bath, a representative of type X methanotrophs, contained mostly PE (89% of the total phospholipids). Finally, the IPPs of a recently isolated acidophilic methanotroph, Methylocella palustris, showed it had a preponderance of PME phospholipids with 18:1 fatty acids (94% of total). Principal component analysis showed these methanotrophs could be clearly distinguished based on phospholipid profiles. Results from this study suggest that IPP can be very useful in bacterial chemotaxonomy.

  15. Babesia divergens: protection of intact calves against heterologous challenge by the injection of irradiated piroplasms

    Energy Technology Data Exchange (ETDEWEB)

    Lewis, D; Purnell, R E; Brocklesby, D W [Agricultural Research Council, Compton (UK). Inst. for Research on Animal Diseases

    1980-03-01

    Blood from a splenectomized calf infected with Babesia divergens was divided into 20 ml aliquots which were ..gamma..-irradiated at doses of 0, 24, 28, 32, 36 and 40 krad and then inoculated into groups of three intact calves. Animals receiving non-irradiated blood had typical mild B. divergens reactions, but those receiving irradiated blood had either very mild reactions or no overt reaction. When the calves were challenged with a similar number (7.5 X 10/sup 9/) of heterologous parasites of a recently-isolated field strain, those which had received blood irradiated at 0, 24, 28 and 32 krad were all immune whereas four of the five surviving animals which had received blood irradiated at 36 or 40 krad were susceptible. The immune status of individual cattle was reflected accurately in the results of the micro-ELISA test, which detected a significant rise in serum antibody titre of the four susceptible animals by day 21 after challenge.

  16. Intact Automatic Imitation and Typical Spatial Compatibility in Autism Spectrum Disorder: Challenging the Broken Mirror Theory.

    Science.gov (United States)

    Sowden, Sophie; Koehne, Svenja; Catmur, Caroline; Dziobek, Isabel; Bird, Geoffrey

    2016-02-01

    A lack of imitative behavior is frequently described as a core feature of Autism Spectrum Disorder (ASD), and is consistent with claims of mirror neuron system dysfunction in these individuals. Previous research has questioned this characterization of ASD however, arguing that when tests of automatic imitation are used--which do not require higher-level cognitive processing--imitative behavior is intact or even enhanced in individuals with ASD. In Experiment 1, 60 adult individuals with ASD and a matched Control group completed an automatic imitation task in which they were required to perform an index or a middle finger lift while observing a hand making either the same, or the alternate, finger movement. Both groups demonstrated a significant imitation effect whereby actions were executed faster when preceded by observation of the same action, than when preceded by the alternate action. The magnitude of this "imitation effect" was statistically indistinguishable in the ASD and Control groups. Experiment 2 utilized an improved automatic imitation paradigm to demonstrate that, when automatic imitation effects are isolated from those due to spatial compatibility, increasing autism symptom severity is associated with an increased tendency to imitate. Notably, there was no association between autism symptom severity and spatial compatibility, demonstrating the specificity of the link between ASD symptoms and increased imitation. These results provide evidence against claims of a lack of imitative behavior in ASD, and challenge the "Broken Mirror Theory of Autism." © 2015 International Society for Autism Research, Wiley Periodicals, Inc.

  17. Direct comparison of phosphate uptake by adnate and loosely attached microalgae within an intact biofilm matrix

    International Nuclear Information System (INIS)

    Burkholder, J.M.; Wetzel, R.G.; Klomparens, K.L.

    1990-01-01

    We report a direct comparison of phosphate uptake by adnate and loosely attached microalgae in an intact biofilm matrix, with resolution at the level of individual cells. Track scanning electron microscope autoradiography enabled assay of [ 33 P]phosphate uptake from the overlying water by adnate algae left undisturbed on mature leaves of the macrophyte Potamogeton illinoensis or on artificial plant mimics. The epiphyte communities developed in either phosphate-poor or moderately phosphate-enriched water, and they were assayed on both natural and artificial plants. All adnate taxa examined from both natural and artificial plants in both habitats took up significantly less radiolabel when assayed beneath the overlying matrix than when they were exposed to the water upon removal of the overstory material. Track scanning electron microscope autoradiography and track light microscope autoradiography were intercalibrated to enable comparison of [ 33 P]phosphate uptake by adnate and loosely attached components of the epiphyte matrix. Loosley attached cells on substrata from both habitats took up significantly more radiolabel than did underlying adnate cells, indicating that access to phosphate supplies from the water depended on the position of microbial cells in the matrix. In this short-term assay, the adnate microalgae were relatively isolated from the water column nutrient source

  18. Babesia divergens: protection of intact calves against heterologous challenge by the injection of irradiated piroplasms

    International Nuclear Information System (INIS)

    Lewis, D.; Purnell, R.E.; Brocklesby, D.W.

    1980-01-01

    Blood from a splenectomized calf infected with Babesia divergens was divided into 20 ml aliquots which were γ-irradiated at doses of 0, 24, 28, 32, 36 and 40 krad and then inoculated into groups of three intact calves. Animals receiving non-irradiated blood had typical mild B. divergens reactions, but those receiving irradiated blood had either very mild reactions or no overt reaction. When the calves were challenged with a similar number (7.5 X 10 9 ) of heterologous parasites of a recently-isolated field strain, those which had received blood irradiated at 0, 24, 28 and 32 krad were all immune whereas four of the five surviving animals which had received blood irradiated at 36 or 40 krad were susceptible. The immune status of individual cattle was reflected accurately in the results of the micro-ELISA test, which detected a significant rise in serum antibody titre of the four susceptible animals by day 21 after challenge. (Auth.)

  19. The role of inorganic phosphate in intact human erythrocytes

    International Nuclear Information System (INIS)

    Nishiguchi, Eiko; Umeda, Masahiro.

    1988-01-01

    The role of inorganic phosphate in intact human erythrocytes was investigated by phosphorus-31 nuclear magnetic resonance ( 31 P NMR). When erythrocytes stored for 5 weeks were incubated at 37 deg C, pH 7.4, in medium containing 2 mM adenine and 10 mM inosine, with or without 5 mM glucose, a substance of around 4 ppm, as assessed by 31 P NMR chemical shift, was detected in the mixture. However, this substance disappeared by the addition of inorganic phosphate. When erythrocytes stored for 4 weeks in acid citrate dextrose (ACD) solution were incubated with 2 mM adenine, 10 mM inosine, 5 mM glucose, 50 mM inorganic phosphate and 10 mM pyruvate at 37 deg C, pH 7.4, the 2,3-DPG level increased gradually, whereas the ATP level initially increased and then decreased. Intracellular inorganic phosphate appeared to be used for the synthesis of ATP and 2,3-DPG during the first 30 min. of the reaction. These results suggests that the inorganic phosphate accelerates glycolysis by increasing the activity of glycolytic enzymes rather than its direct involvement in synthesizing organic phosphorus compounds in stored erythrocytes. The results also suggests that the reserve energy from ATP synthesis is not sufficient for the synthesis of 2,3-DPG. (author)

  20. Basic timing abilities stay intact in patients with musician's dystonia.

    Directory of Open Access Journals (Sweden)

    M C van der Steen

    Full Text Available Task-specific focal dystonia is a movement disorder that is characterized by the loss of voluntary motor control in extensively trained movements. Musician's dystonia is a type of task-specific dystonia that is elicited in professional musicians during instrumental playing. The disorder has been associated with deficits in timing. In order to test the hypothesis that basic timing abilities are affected by musician's dystonia, we investigated a group of patients (N = 15 and a matched control group (N = 15 on a battery of sensory and sensorimotor synchronization tasks. Results did not show any deficits in auditory-motor processing for patients relative to controls. Both groups benefited from a pacing sequence that adapted to their timing (in a sensorimotor synchronization task at a stable tempo. In a purely perceptual task, both groups were able to detect a misaligned metronome when it was late rather than early relative to a musical beat. Overall, the results suggest that basic timing abilities stay intact in patients with musician's dystonia. This supports the idea that musician's dystonia is a highly task-specific movement disorder in which patients are mostly impaired in tasks closely related to the demands of actually playing their instrument.

  1. Basic Timing Abilities Stay Intact in Patients with Musician's Dystonia

    Science.gov (United States)

    van der Steen, M. C.; van Vugt, Floris T.; Keller, Peter E.; Altenmüller, Eckart

    2014-01-01

    Task-specific focal dystonia is a movement disorder that is characterized by the loss of voluntary motor control in extensively trained movements. Musician's dystonia is a type of task-specific dystonia that is elicited in professional musicians during instrumental playing. The disorder has been associated with deficits in timing. In order to test the hypothesis that basic timing abilities are affected by musician's dystonia, we investigated a group of patients (N = 15) and a matched control group (N = 15) on a battery of sensory and sensorimotor synchronization tasks. Results did not show any deficits in auditory-motor processing for patients relative to controls. Both groups benefited from a pacing sequence that adapted to their timing (in a sensorimotor synchronization task at a stable tempo). In a purely perceptual task, both groups were able to detect a misaligned metronome when it was late rather than early relative to a musical beat. Overall, the results suggest that basic timing abilities stay intact in patients with musician's dystonia. This supports the idea that musician's dystonia is a highly task-specific movement disorder in which patients are mostly impaired in tasks closely related to the demands of actually playing their instrument. PMID:24667273

  2. Intact information sampling in mesial temporal lobe epilepsy.

    Science.gov (United States)

    Zamarian, Laura; Trinka, Eugen; Kuchukhidze, Giorgi; Bodner, Thomas; Unterberger, Iris; Luef, Gerhard; Delazer, Margarete

    2015-11-01

    Previous studies have reported deficits in decision making under ambiguity for patients with mesial temporal lobe epilepsy (mTLE). It is unknown whether mTLE is also associated with alterations at a predecisional stage. This study aimed to gain insight into predecisional processing of patients with mTLE. We compared performance of patients with mTLE (n = 25) with that of healthy controls (n = 75) on the information sampling task (IST), a task assessing reflection-impulsivity and predecisional information sampling. Patients and healthy controls showed a similar performance pattern in both conditions of the IST as indicated by the amount of information gathered, the degree of uncertainty tolerated, and the number of decision errors made. They both also demonstrated a significant sensitivity to the different reward characteristics of the task. For the patient group, we found no significant effects on performance on the IST of epilepsy lateralization, abnormality side, structural abnormality (hippocampus vs. amygdala), and medication (monotherapy vs. polytherapy). Reflection processes and predecisional information sampling as tested by the IST are intact in mTLE. Patients collect as much information as healthy individuals and adapt their behavior according to the changing reward conditions. Our findings indicate that in well-defined risk situations, where memory demands are sufficiently minimized, patients with mTLE should be able to gather sufficient information, weight risks and benefits, and make advantageous decisions. (c) 2015 APA, all rights reserved).

  3. Assessment of mechanical strain in the intact plantar fascia.

    Science.gov (United States)

    Clark, Ross A; Franklyn-Miller, Andrew; Falvey, Eanna; Bryant, Adam L; Bartold, Simon; McCrory, Paul

    2009-09-01

    A method of measuring tri-axial plantar fascia strain that is minimally affected by external compressive force has not previously been reported. The purpose of this study was to assess the use of micro-strain gauges to examine strain in the different axes of the plantar fascia. Two intact limbs from a thawed, fresh-frozen cadaver were dissected, and a combination of five linear and one three-way rosette gauges were attached to the fascia of the foot and ankle. Strain was assessed during two trials, both consisting of an identical controlled, loaded dorsiflexion. An ICC analysis of the results revealed that the majority of gauge placement sites produced reliable measures (ICC>0.75). Strain mapping of the plantar fascia indicates that the majority of the strain is centrally longitudinal, which provides supportive evidence for finite element model analysis. Although micro-strain gauges do possess the limitation of calibration difficulty, they provide a repeatable measure of fascial strain and may provide benefits in situations that require tri-axial assessment or external compression.

  4. Temporal Ventriloquism Reveals Intact Audiovisual Temporal Integration in Amblyopia.

    Science.gov (United States)

    Richards, Michael D; Goltz, Herbert C; Wong, Agnes M F

    2018-02-01

    We have shown previously that amblyopia involves impaired detection of asynchrony between auditory and visual events. To distinguish whether this impairment represents a defect in temporal integration or nonintegrative multisensory processing (e.g., cross-modal matching), we used the temporal ventriloquism effect in which visual temporal order judgment (TOJ) is normally enhanced by a lagging auditory click. Participants with amblyopia (n = 9) and normally sighted controls (n = 9) performed a visual TOJ task. Pairs of clicks accompanied the two lights such that the first click preceded the first light, or second click lagged the second light by 100, 200, or 450 ms. Baseline audiovisual synchrony and visual-only conditions also were tested. Within both groups, just noticeable differences for the visual TOJ task were significantly reduced compared with baseline in the 100- and 200-ms click lag conditions. Within the amblyopia group, poorer stereo acuity and poorer visual acuity in the amblyopic eye were significantly associated with greater enhancement in visual TOJ performance in the 200-ms click lag condition. Audiovisual temporal integration is intact in amblyopia, as indicated by perceptual enhancement in the temporal ventriloquism effect. Furthermore, poorer stereo acuity and poorer visual acuity in the amblyopic eye are associated with a widened temporal binding window for the effect. These findings suggest that previously reported abnormalities in audiovisual multisensory processing may result from impaired cross-modal matching rather than a diminished capacity for temporal audiovisual integration.

  5. Intact unconscious processing of eye contact in schizophrenia

    Directory of Open Access Journals (Sweden)

    Kiley Seymour

    2016-03-01

    Full Text Available The perception of eye gaze is crucial for social interaction, providing essential information about another person’s goals, intentions, and focus of attention. People with schizophrenia suffer a wide range of social cognitive deficits, including abnormalities in eye gaze perception. For instance, patients have shown an increased bias to misjudge averted gaze as being directed toward them. In this study we probed early unconscious mechanisms of gaze processing in schizophrenia using a technique known as continuous flash suppression. Previous research using this technique to render faces with direct and averted gaze initially invisible reveals that direct eye contact gains privileged access to conscious awareness in healthy adults. We found that patients, as with healthy control subjects, showed the same effect: faces with direct eye gaze became visible significantly faster than faces with averted gaze. This suggests that early unconscious processing of eye gaze is intact in schizophrenia and implies that any misjudgments of gaze direction must manifest at a later conscious stage of gaze processing where deficits and/or biases in attributing mental states to gaze and/or beliefs about being watched may play a role.

  6. Stress dependence of permeability of intact and fractured shale cores.

    Science.gov (United States)

    van Noort, Reinier; Yarushina, Viktoriya

    2016-04-01

    Whether a shale acts as a caprock, source rock, or reservoir, understanding fluid flow through shale is of major importance for understanding fluid flow in geological systems. Because of the low permeability of shale, flow is thought to be largely confined to fractures and similar features. In fracking operations, fractures are induced specifically to allow for hydrocarbon exploration. We have constructed an experimental setup to measure core permeabilities, using constant flow or a transient pulse. In this setup, we have measured the permeability of intact and fractured shale core samples, using either water or supercritical CO2 as the transporting fluid. Our measurements show decreasing permeability with increasing confining pressure, mainly due to time-dependent creep. Furthermore, our measurements show that for a simple splitting fracture, time-dependent creep will also eliminate any significant effect of this fracture on permeability. This effect of confinement on fracture permeability can have important implications regarding the effects of fracturing on shale permeability, and hence for operations depending on that.

  7. Research on intact marine ecosystems: a lost era.

    Science.gov (United States)

    Stachowitsch, Michael

    2003-07-01

    It is proposed that a new, fifth era should be added to the four historical phases of marine research identified by Rupert Riedl, specifically an era devoted to studying and ameliorating disturbed marine ecosystems. In an age of global environmental deterioration, many marine ecosystems and organisms are high on the list of threatened entities. This poor status prompts research that would otherwise have been unnecessary and hinders research that would normally have been conducted. I argue that research into intact marine ecosystems is becoming increasingly difficult, and that most of our future insights into marine habitats will stem from knowledge gained by examining various disfunctions of those systems rather than their functions. The new era will therefore differ from past research in its underlying aim, the range of topics studied, the selection and funding of those topics, the validity of its conclusions, and in its urgency. Sea turtles and cetaceans are cited as case studies at the organismic level, shallow-water benthic communities, including coral reefs, at the ecosystem level.

  8. Measurement of tritiated norepinephrine metabolism in intact rat brain

    International Nuclear Information System (INIS)

    Levitt, M.; Kowalik, S.; Barkai, A.I.

    1983-01-01

    A procedure for the study of NE metabolism in the intact rat brain is described. The method involves ventriculocisternal perfusion of the adult male rat with artificial CSF containing [ 3 H]NE. Radioactivity in the perfusate associated with NE and its metabolites 3,4-dihydroxymandelic acid (DOMA), 3,4-dihydroxphenylethyleneglycol (DHPG), 3-methoxy-4-hydroxymandelic acid (VMA), 3-methoxy-4-hydroxyphenylethyleneglycol (MHPG), and normetanephrine (NMN) is separated using high-performance liquid chromatography (HPLC). After 80 min the radioactivity in the perfusate reaches an apparent steady-state. Analysis of the steady-state samples shows higher activity in the fractions corresponding to DHPG and MHPG than in those corresponding to DOMA and VMA, confirming glycol formation as the major pathway of NE metabolism in rat brain. Pretreatment with an MAO inhibitor (tranylcypromine) results in a marked decrease in the deaminated metabolites DHPG and MHPG and a concurrent increase in NMN. The results indicate this to be a sensitive procedure for the in vivo determination of changes in NE metabolism. (Auth.)

  9. Structure of the intact ATM/Tel1 kinase

    Science.gov (United States)

    Wang, Xuejuan; Chu, Huanyu; Lv, Mengjuan; Zhang, Zhihui; Qiu, Shuwan; Liu, Haiyan; Shen, Xuetong; Wang, Weiwu; Cai, Gang

    2016-05-01

    The ataxia-telangiectasia mutated (ATM) protein is an apical kinase that orchestrates the multifaceted DNA-damage response. Normally, ATM kinase is in an inactive, homodimer form and is transformed into monomers upon activation. Besides a conserved kinase domain at the C terminus, ATM contains three other structural modules, referred to as FAT, FATC and N-terminal helical solenoid. Here we report the first cryo-EM structure of ATM kinase, which is an intact homodimeric ATM/Tel1 from Schizosaccharomyces pombe. We show that two monomers directly contact head-to-head through the FAT and kinase domains. The tandem N-terminal helical solenoid tightly packs against the FAT and kinase domains. The structure suggests that ATM/Tel1 dimer interface and the consecutive HEAT repeats inhibit the binding of kinase substrates and regulators by steric hindrance. Our study provides a structural framework for understanding the mechanisms of ATM/Tel1 regulation as well as the development of new therapeutic agents.

  10. Genetic disruptions of Drosophila Pavlovian learning leave extinction learning intact.

    Science.gov (United States)

    Qin, H; Dubnau, J

    2010-03-01

    Individuals who experience traumatic events may develop persistent posttraumatic stress disorder. Patients with this disorder are commonly treated with exposure therapy, which has had limited long-term success. In experimental neurobiology, fear extinction is a model for exposure therapy. In this behavioral paradigm, animals are repeatedly exposed in a safe environment to the fearful stimulus, which leads to greatly reduced fear. Studying animal models of extinction already has lead to better therapeutic strategies and development of new candidate drugs. Lack of a powerful genetic model of extinction, however, has limited progress in identifying underlying molecular and genetic factors. In this study, we established a robust behavioral paradigm to study the short-term effect (acquisition) of extinction in Drosophila melanogaster. We focused on the extinction of olfactory aversive 1-day memory with a task that has been the main workhorse for genetics of memory in flies. Using this paradigm, we show that extinction can inhibit each of two genetically distinct forms of consolidated memory. We then used a series of single-gene mutants with known impact on associative learning to examine the effects on extinction. We find that extinction is intact in each of these mutants, suggesting that extinction learning relies on different molecular mechanisms than does Pavlovian learning.

  11. Proteasomes remain intact, but show early focal alteration in their composition in a mouse model of amyotrophic lateral sclerosis.

    Science.gov (United States)

    Kabashi, Edor; Agar, Jeffrey N; Hong, Yu; Taylor, David M; Minotti, Sandra; Figlewicz, Denise A; Durham, Heather D

    2008-06-01

    In amyotrophic lateral sclerosis caused by mutations in Cu/Zn-superoxide dismutase (SOD1), altered solubility and aggregation of the mutant protein implicates failure of pathways for detecting and catabolizing misfolded proteins. Our previous studies demonstrated early reduction of proteasome-mediated proteolytic activity in lumbar spinal cord of SOD1(G93A) transgenic mice, tissue particularly vulnerable to disease. The purpose of this study was to identify any underlying abnormalities in proteasomal structure. In lumbar spinal cord of pre-symptomatic mice [postnatal day 45 (P45) and P75], normal levels of structural 20S alpha subunits were incorporated into 20S/26S proteasomes; however, proteasomal complexes separated by native gel electrophoresis showed decreased immunoreactivity with antibodies to beta3, a structural subunit of the 20S proteasome core, and beta5, the subunit with chymotrypsin-like activity. This occurred prior to increase in beta5i immunoproteasomal subunit. mRNA levels were maintained and no association of mutant SOD1 with proteasomes was identified, implicating post-transcriptional mechanisms. mRNAs also were maintained in laser captured motor neurons at a later stage of disease (P100) in which multiple 20S proteins are reduced relative to the surrounding neuropil. Increase in detergent-insoluble, ubiquitinated proteins at P75 provided further evidence of stress on mechanisms of protein quality control in multiple cell types prior to significant motor neuron death.

  12. A Novel Immune-Intact Mouse Model of Prostate Cancer Bone Metastasis: Mechanisms of Chemotaxis and Bone Colonization

    Science.gov (United States)

    2017-10-01

    Colonization PRINCIPAL INVESTIGATOR: Srinivas Nandana CONTRACTING ORGANIZATION: Cedars-Sinai Medical Center Los Angeles, CA, 90048 REPORT DATE...8. PERFORMING ORGANIZATION REPORT NUMBER Cedars-Sinai Medical Center, 8700 Beverly Blvd, Los Angeles, CA, 90048 9. SPONSORING / MONITORING...Revised Statement of Work (SOW): PROPOSED START DATE Sep 30, 2016 ACTUAL START DATE – Sep 18, 2017 Site: Uro -Oncology, Dept of Medicine

  13. Cytosolic Phosphoenolpyruvate Carboxykinase Does Not Solely Control the Rate of Hepatic Gluconeogenesis in the Intact Mouse Liver

    OpenAIRE

    Burgess, Shawn C.; He, TianTeng; Yan, Zheng; Lindner, Jill; Sherry, A. Dean; Malloy, Craig R.; Browning, Jeffrey D.; Magnuson, Mark A.

    2007-01-01

    When dietary carbohydrate is unavailable, glucose required to support metabolism in vital tissues is generated via gluconeogenesis in the liver. Expression of phosphoenolpyruvate carboxykinase (PEPCK), commonly considered the control point for liver gluconeogenesis, is normally regulated by circulating hormones to match systemic glucose demand. However, this regulation fails in diabetes. Because other molecular and metabolic factors can also influence gluconeogenesis, the explicit role of PEP...

  14. Perturbations of NAD+ salvage systems impact mitochondrial function and energy homeostasis in mouse myoblasts and intact skeletal muscle

    DEFF Research Database (Denmark)

    Andersen, Marianne Agerholm; Dall, Morten; Jensen, Benjamin Anderschou Holbech

    2018-01-01

    Nicotinamide adenine dinucleotide (NAD+) can be synthesized by nicotinamide phosphoribosyltransferase (NAMPT). We aimed to determine the role of NAMPT for maintaining NAD+ levels, mitochondrial function, and metabolic homeostasis in skeletal muscle cells. We generated stable Nampt knockdown (sh......Nampt KD) C2C12 cells using a shRNA lentiviral approach. Moreover, we applied gene electrotransfer to express cre recombinase in tibialis anterior muscle of floxed Nampt mice. In shNampt KD C2C12 myoblasts, Nampt and NAD+ levels were reduced by 70% and 50%, respectively, and maximal respiratory capacity...... was reduced by 25%. Moreover, anaerobic glycolytic flux increased by 55% and 2-deoxyglucose uptake increased by 25% in shNampt KD cells. Treatment with the NAD+ precursor nicotinamide riboside restored NAD+ levels in shNampt cells and increased maximal respiratory capacity by 18% and 32% in control and sh...

  15. Development of the leptospirosis by experimental infection in hamsters (Mesocricetus auratus with Leptospira interrogans serovar Canicola, strain LO4, by intact and scratched skin exposures

    Directory of Open Access Journals (Sweden)

    Carolina de Sousa Américo Batista

    2010-10-01

    Full Text Available The establishment and evolution of leptospirosis in hamster (Mesocricetus auratus by experimental infection with Leptospira interrogans serovar Canicola, LO4 strain, by intact and scratched skin exposures, having as control the intraperitoneal route, were evaluated. Hundred-twenty female hamsters distributed in two groups according to inoculation route (intact and scratched skin were used. Infectious inoculum was constituted by a pure culture of L. interrogans serovar Canicola (strain LO4, isolated from liver from a slaughtered swine in Londrina, Paraná state and typified by agglutinins adsortion technique with monoclonal antibody kit at the Royal Tropical Institute, Amsterdam, the Netherlands. The animals were observed twice a day during 21 days. Animals that died were necropsied and kidneys, liver, genital tract (uterus and ovaries and brain were aseptically collected. On the 21st post-inoculation day, surviving animals were euthanized. In these animals, serum samples were also collected by cardiac puncture to antileptospires agglutinins research using microscopic agglutination test (MAT. Fresh direct microscopy and microbiological culture were used for the detection of leptospires. Scratched skin route induced larger lethality when compared to intact skin route, with establishment and evolution of leptospirosis. On the other hand, intact skin route induced renal and/or genital carrier state more frequently. LO4 strain presented low immunogenic power, characterized by soroconversion at the MAT in only one inoculated animal.

  16. Novel insights on the relationship between T-tubular defects and contractile dysfunction in a mouse model of hypertrophic cardiomyopathy.

    Science.gov (United States)

    Crocini, C; Ferrantini, C; Scardigli, M; Coppini, R; Mazzoni, L; Lazzeri, E; Pioner, J M; Scellini, B; Guo, A; Song, L S; Yan, P; Loew, L M; Tardiff, J; Tesi, C; Vanzi, F; Cerbai, E; Pavone, F S; Sacconi, L; Poggesi, C

    2016-02-01

    Abnormalities of cardiomyocyte Ca(2+) homeostasis and excitation-contraction (E-C) coupling are early events in the pathogenesis of hypertrophic cardiomyopathy (HCM) and concomitant determinants of the diastolic dysfunction and arrhythmias typical of the disease. T-tubule remodelling has been reported to occur in HCM but little is known about its role in the E-C coupling alterations of HCM. Here, the role of T-tubule remodelling in the electro-mechanical dysfunction associated to HCM is investigated in the Δ160E cTnT mouse model that expresses a clinically-relevant HCM mutation. Contractile function of intact ventricular trabeculae is assessed in Δ160E mice and wild-type siblings. As compared with wild-type, Δ160E trabeculae show prolonged kinetics of force development and relaxation, blunted force-frequency response with reduced active tension at high stimulation frequency, and increased occurrence of spontaneous contractions. Consistently, prolonged Ca(2+) transient in terms of rise and duration are also observed in Δ160E trabeculae and isolated cardiomyocytes. Confocal imaging in cells isolated from Δ160E mice reveals significant, though modest, remodelling of T-tubular architecture. A two-photon random access microscope is employed to dissect the spatio-temporal relationship between T-tubular electrical activity and local Ca(2+) release in isolated cardiomyocytes. In Δ160E cardiomyocytes, a significant number of T-tubules (>20%) fails to propagate action potentials, with consequent delay of local Ca(2+) release. At variance with wild-type, we also observe significantly increased variability of local Ca(2+) transient rise as well as higher Ca(2+)-spark frequency. Although T-tubule structural remodelling in Δ160E myocytes is modest, T-tubule functional defects determine non-homogeneous Ca(2+) release and delayed myofilament activation that significantly contribute to mechanical dysfunction. Copyright © 2015 The Authors. Published by Elsevier Ltd.. All

  17. Intact long-type dupA as a marker for gastroduodenal diseases in Okinawan subpopulation, Japan.

    Science.gov (United States)

    Takahashi, Ayaka; Shiota, Seiji; Matsunari, Osamu; Watada, Masahide; Suzuki, Rumiko; Nakachi, Saori; Kinjo, Nagisa; Kinjo, Fukunori; Yamaoka, Yoshio

    2013-02-01

    Helicobacter pylori dupA can be divided into two types according to the presence or absence of the mutation. In addition, full-sequenced data revealed that dupA has two types with different lengths depend on the presence of approximately 600 bp in the putative 5' region (presence; long-type and absence; short-type), which has not been taken into account in previous studies. A total of 319 strains isolated from Okinawa, the south islands of Japan, were included. The status of dupA and cagA was determined by polymerase chain reaction. The presence of mutations in long-type dupA was determined by DNA sequencing. The prevalence of long-type dupA was 26.3% (84/319). Sequence analysis showed that there were only six cases (7.1%) with point mutations lead to stop codon among 84 long-type dupA strains studied. Interestingly, intact long-type dupA without frameshift mutation, but not short-type dupA, was significantly associated with gastric ulcer and gastric cancer than gastritis (p = .001 and p = .019, respectively). After adjustment by age, gender, and cagA, the presence of intact long-type dupA was significantly associated with gastric ulcer and gastric cancer compared with gastritis (odds ratio [OR] = 3.35, 95% confidence interval [CI] = 1.55-7.24 and OR = 4.14, 95% CI = 1.23-13.94, respectively). Intact long-type dupA is a real virulence marker for severe outcomes in Okinawa, Japan. The previous information gained from PCR-based methods without taking long-type dupA into account must be interpreted with caution. © 2012 Blackwell Publishing Ltd.

  18. The intact capture of hypervelocity dust particles using underdense foams

    Science.gov (United States)

    Maag, Carl R.; Borg, J.; Tanner, William G.; Stevenson, T. J.; Bibring, J.-P.

    1994-01-01

    probability of survival for the impacting particle. The primary objectives of the experiment are to (1) Examine the morphology of primary and secondary hypervelocity impact craters. Primary attention will be paid to craters caused by ejecta during hypervelocity impacts of different substrates. (2) Determine the size distribution of ejecta by means of witness plates and collect fragments of ejecta from craters by means of momentum-sensitive mcropore foam. (3) Assess the directionality of the flux by means of penetration-hole alignment of thin films placed above the cells. (4) Capture intact the particles that perforated the thin film and entered the cell. Capture media consisted of both previously flight-tested micropore foams and aerogel. The foams had different latent heats of fusion and, accordingly, will capture particles over a range of momenta. Aerogel was incorporated into the cells to determine the minimum diameter than can be captured intact.

  19. Intensity modulated tangential beam irradiation of the intact breast

    International Nuclear Information System (INIS)

    Hong, L.; Hunt, M.; Chui, C.; Forster, K.; Lee, H.; Lutz, W.; Yahalom, J.; Kutcher, G.J.; McCormick, B.

    1997-01-01

    Purpose/Objective: The purpose of this study was to evaluate the potential benefits of intensity modulated tangential beams in the irradiation of the intact breast. The primary goal was to develop an intensity modulated treatment which would substantially decrease the dose to coronary arteries, lung and contralateral breast while still using a standard tangential beam arrangement. Improved target dose homogeneity, within the limits imposed by opposed fields, was also desired. Since a major goal of the study was the development of a technique which was practical for use on a large population of patients, the design of 'standard' intensity profiles analogous in function to conventional wedges was also investigated. Materials and Methods: Three dimensional treatment planning was performed using both conventional and intensity modulated tangential beams. Plans were developed for both the right and left breast for a range of patient sizes and shapes. For each patient, PTV, lung, heart, origin and peripheral branches of the coronary artery, and contralateral breast were contoured. Optimum tangential beam direction and shape were designed using Beams-Eye-View display and then used for both the conventional and intensity modulated plans. For the conventional plan, the optimum wedge combination and beam weighting were chosen based on the dose distribution in a single transverse plane through the field center. Intensity modulated plans were designed using an algorithm which allows the user to specify the prescribed, maximum and minimum acceptable doses and dose volume constraints for each organ of interest. Plans were compared using multiple dose distributions and DVHs. Results: Significant improvements in the doses to critical structures were achieved using the intensity modulated plan. Coronary artery dose decreased substantially for patients treated to the left breast. Ipsilateral lung and contralateral breast doses decreased for all patients. For one patient treated to

  20. ZNF 197L is dispensable in mouse development

    African Journals Online (AJOL)

    Jane

    2011-07-27

    protein interactions (Kim et al., 1996; Friedman et .... A fragment of pU17 vector was used as a probe to detect the trapping ... RNA was isolated from adult mouse brain, heart, lung, .... Zinc finger peptides for the regulation of gene.

  1. Intact suppression of increased false recognition in schizophrenia.

    Science.gov (United States)

    Weiss, Anthony P; Dodson, Chad S; Goff, Donald C; Schacter, Daniel L; Heckers, Stephan

    2002-09-01

    Recognition memory is impaired in patients with schizophrenia, as they rely largely on item familiarity, rather than conscious recollection, to make mnemonic decisions. False recognition of novel items (foils) is increased in schizophrenia and may relate to this deficit in conscious recollection. By studying pictures of the target word during encoding, healthy adults can suppress false recognition. This study examined the effect of pictorial encoding on subsequent recognition of repeated foils in patients with schizophrenia. The study included 40 patients with schizophrenia and 32 healthy comparison subjects. After incidental encoding of 60 words or pictures, subjects were tested for recognition of target items intermixed with 60 new foils. These new foils were subsequently repeated following either a two- or 24-word delay. Subjects were instructed to label these repeated foils as new and not to mistake them for old target words. Schizophrenic patients showed greater overall false recognition of repeated foils. The rate of false recognition of repeated foils was lower after picture encoding than after word encoding. Despite higher levels of false recognition of repeated new items, patients and comparison subjects demonstrated a similar degree of false recognition suppression after picture, as compared to word, encoding. Patients with schizophrenia displayed greater false recognition of repeated foils than comparison subjects, suggesting both a decrement of item- (or source-) specific recollection and a consequent reliance on familiarity in schizophrenia. Despite these deficits, presenting pictorial information at encoding allowed schizophrenic subjects to suppress false recognition to a similar degree as the comparison group, implying the intact use of a high-level cognitive strategy in this population.

  2. Mapping the World's Intact Forest Landscapes by Remote Sensing

    Directory of Open Access Journals (Sweden)

    Peter Potapov

    2008-12-01

    Full Text Available Protection of large natural forest landscapes is a highly important task to help fulfill different international strategic initiatives to protect forest biodiversity, to reduce carbon emissions from deforestation and forest degradation, and to stimulate sustainable forest management practices. This paper introduces a new approach for mapping large intact forest landscapes (IFL, defined as an unbroken expanse of natural ecosystems within areas of current forest extent, without signs of significant human activity, and having an area of at least 500 km2. We have created a global IFL map using existing fine-scale maps and a global coverage of high spatial resolution satellite imagery. We estimate the global area of IFL within the current extent of forest ecosystems (forest zone to be 13.1 million km2 or 23.5% of the forest zone. The vast majority of IFL are found in two biomes: Dense Tropical and Subtropical Forests (45.3% and Boreal Forests (43.8%. The lowest proportion of IFL is found in Temperate Broadleaf and Mixed Forests. The IFL exist in 66 of the 149 countries that together make up the forest zone. Three of them - Canada, Russia, and Brazil - contain 63.8% of the total IFL area. Of the world's IFL area, 18.9% has some form of protection, but only 9.7% is strictly protected, i.e., belongs to IUCN protected areas categories I-III. The world IFL map presented here is intended to underpin the development of a general strategy for nature conservation at the global and regional scales. It also defines a baseline for monitoring deforestation and forest degradation that is well suited for use with operational and cost-effective satellite data. All project results and IFL maps are available on a dedicated web site (http://www.intactforests.org.

  3. Preferential flow through intact soil cores: Effects of matrix head

    Energy Technology Data Exchange (ETDEWEB)

    Langner, H.W.; Gaber, H.M.; Wraith, J.M.; Huwe, B.; Inskeep, W.P.

    1999-12-01

    Continuous soil pores may act as pathways for preferential flow depending on their size and water status (filled or drained), the latter being largely controlled by the soil matrix head (h). The literature contains a wide range of proposed minimal pore sizes that may contribute to preferential flow. The objective of this study was to examine the relationship between h (and corresponding pore sizes) and preferential solute transport in a naturally structured soil. Tracer ({sup 3}H{sub 2}O and pentafluorobenzoic acid, [PFBA]) miscible displacement experiments were performed at several h values in intact soil cores (15-cm diameter, 30-cm length) using an apparatus especially suited to maintain constant h while collecting large effluent volumes. To test for the occurrence of preferential flow, observed breakthrough curves (BTCs) were evaluated for physical nonequilibrium (PNE) using a comparison between fitted local equilibrium (PNE) and PNE models. Fitting results of the observed BTCs indicated absence of PNE in all solute transport experiments at h {le} {minus}10 cm. Experiments at h {ge} {minus}5 cm consistently exhibited PNE conditions, indicating the presence of preferential flow. These results suggest that soil pores with effective radii of 150 {micro}m and smaller (water-filled at h = {minus}10 cm) do not contribute to preferential flow. Observed pore water velocities were not indicative of the presence or absence of preferential flow conditions. Continuous measurements of soil water content ({theta}) using time domain reflectometry (TDR) revealed that at h = {minus}10 cm, <2% of the soil volume had drained.

  4. Caloric restriction mimetic 2-deoxyglucose maintains cytoarchitecture and reduces tau phosphorylation in primary culture of mouse hippocampal pyramidal neurons.

    Science.gov (United States)

    Bele, M S; Gajare, K A; Deshmukh, A A

    2015-06-01

    Typical form of neurons is crucially important for their functions. This is maintained by microtubules and associated proteins like tau. Hyperphosphorylation of tau is a major concern in neurodegenerative diseases. Glycogen synthase kinase3β (GSK3β) and cyclin-dependent protein kinase 5 (Cdk5) are the enzymes that govern tau phosphorylation. Currently, efforts are being made to target GSK3β and Cdk5 as possible therapeutic avenues to control tau phosphorylation and treat neurodegenerative diseases related to taupathies. In a number of studies, caloric restriction mimetic 2-deoxyglucose (C6H12O5) was found to be beneficial in improving the brain functions. However, no reports are available on the effect of 2-deoxyglucose 2-DG on tau phosphorylation. In the present study, hippocampal pyramidal neurons from E17 mouse embryos were isolated and cultured on poly-L-lysine-coated coverslips. Neurons from the experimental group were treated with 10 mM 2-deoxyglucose. The treatment of 2-DG resulted in healthier neuronal morphology in terms of significantly lower number of cytoplasmic vacuoles, little or no membrane blebbings, maintained axon hillock and intact neurites. There were decreased immunofluorescence signals for GSK3β, pTau at Ser262, Cdk5 and pTau at Ser235 suggesting decreased tau phosphorylation, which was further confirmed by Western blotting. The results indicate the beneficial effects of 2-DG in controlling the tau phosphorylation and maintaining the healthy neuronal cytoarchitecture.

  5. Limitations of the colloidal silica method in mapping the endothelial plasma membrane proteome of the mouse heart.

    Science.gov (United States)

    Arjunan, Selvam; Reinartz, Michael; Emde, Barbara; Zanger, Klaus; Schrader, Jürgen

    2009-01-01

    The endothelial cell (EC) membrane is an important interface, which plays a crucial role in signal transduction. Our aim was to selectively purify luminal EC membrane proteins from the coronary vasculature of the isolated perfused mouse heart and analyze its composition with mass spectrometry (MS). To specifically label coronary ECs in the intact heart, the colloidal silica method was applied, which is based on the binding of positively charged colloidal silica to the surface of EC membranes. Transmission electron microscopy revealed the specific labeling of ECs of macro and microvessels. Two different methods of tissue homogenization (Teflon pestle and ultra blade) together with density centrifugation were used for membrane protein enrichment. Enrichment and purity was controlled by Western blot analysis using the EC-specific protein caveolin 1 and various intracellular marker proteins. The ultra blade method resulted in a tenfold enrichment of caveolin 1, while there was negligible contamination as judged by Western blot. However, protein yield was low and required pooling of ten hearts for MS. When enriched endothelial membrane proteins were digested with trypsin and analyzed by LC-MS, a total of 56 proteins could be identified, of which only 12 were membrane proteins. We conclude that coronary endothelial membranes can be conveniently labeled with colloidal silica. However, due to the ionic nature of interaction of colloidal silica with the EC membrane the shear rate required for cardiac homogenization resulted in a substantial loss of specificity.

  6. Immunostimulatory mouse granuloma protein.

    Science.gov (United States)

    Fontan, E; Fauve, R M; Hevin, B; Jusforgues, H

    1983-10-01

    Earlier studies have shown that from subcutaneous talc-induced granuloma in mice, a fraction could be extracted that fully protected mice against Listeria monocytogenes. Using standard biochemical procedures--i.e., ammonium sulfate fractionation, preparative electrophoresis, gel filtration chromatography, isoelectric focusing, and preparative polyacrylamide gel electrophoresis--we have now purified an active factor to homogeneity. A single band was obtained in NaDodSO4/polyacrylamide gel with an apparent Mr of 55,000. It migrated with alpha 1-globulins and the isoelectric point was 5 +/- 0.1. The biological activity was destroyed with Pronase but not with trypsin and a monospecific polyclonal rabbit antiserum was obtained. The intravenous injection of 5 micrograms of this "mouse granuloma protein" fully protects mice against a lethal inoculum of L. monocytogenes. Moreover, after their incubation with 10 nM mouse granuloma protein, mouse peritoneal cells became cytostatic against Lewis carcinoma cells.

  7. Burn mouse models

    DEFF Research Database (Denmark)

    Calum, Henrik; Høiby, Niels; Moser, Claus

    2014-01-01

    Severe thermal injury induces immunosuppression, involving all parts of the immune system, especially when large fractions of the total body surface area are affected. An animal model was established to characterize the burn-induced immunosuppression. In our novel mouse model a 6 % third-degree b......Severe thermal injury induces immunosuppression, involving all parts of the immune system, especially when large fractions of the total body surface area are affected. An animal model was established to characterize the burn-induced immunosuppression. In our novel mouse model a 6 % third...... with infected burn wound compared with the burn wound only group. The burn mouse model resembles the clinical situation and provides an opportunity to examine or develop new strategies like new antibiotics and immune therapy, in handling burn wound victims much....

  8. Differences in School Behavior and Achievement between Children from Intact, Reconstituted, and Single-Parent Families.

    Science.gov (United States)

    Featherstone, Darin R.; And Others

    1992-01-01

    Analyzed differences in school behavior and achievement among students (n=530) in grades six through nine from intact, reconstituted, and single-parent families. Students from intact, two-parent families had fewer absences and tardies, higher grade point averages, and fewer negative and more positive teacher behavioral ratings than did those from…

  9. Visual Speech Fills in Both Discrimination and Identification of Non-Intact Auditory Speech in Children

    Science.gov (United States)

    Jerger, Susan; Damian, Markus F.; McAlpine, Rachel P.; Abdi, Herve

    2018-01-01

    To communicate, children must discriminate and identify speech sounds. Because visual speech plays an important role in this process, we explored how visual speech influences phoneme discrimination and identification by children. Critical items had intact visual speech (e.g. baez) coupled to non-intact (excised onsets) auditory speech (signified…

  10. Number and location of mouse mammary tumor virus proviral DNA in mouse DNA of normal tissue and of mammary tumors.

    Science.gov (United States)

    Groner, B; Hynes, N E

    1980-01-01

    The Southern DNA filter transfer technique was used to characterize the genomic location of the mouse mammary tumor proviral DNA in different inbred strains of mice. Two of the strains (C3H and CBA) arose from a cross of a Bagg albino (BALB/c) mouse and a DBA mouse. The mouse mammary tumor virus-containing restriction enzyme DNA fragments of these strains had similar patterns, suggesting that the proviruses of these mice are in similar genomic locations. Conversely, the pattern arising from the DNA of the GR mouse, a strain genetically unrelated to the others, appeared different, suggesting that its mouse mammary tumor proviruses are located in different genomic sites. The structure of another gene, that coding for beta-globin, was also compared. The mice strains which we studied can be categorized into two classes, expressing either one or two beta-globin proteins. The macroenvironment of the beta-globin gene appeared similar among the mice strains belonging to one genetic class. Female mice of the C3H strain exogenously transmit mouse mammary tumor virus via the milk, and their offspring have a high incidence of mammary tumor occurrence. DNA isolated from individual mammary tumors taken from C3H mice or from BALB/c mice foster nursed on C3H mothers was analyzed by the DNA filter transfer technique. Additional mouse mammary tumor virus-containing fragments were found in the DNA isolated from each mammary tumor. These proviral sequences were integrated into different genomic sites in each tumor. Images PMID:6245257

  11. Peri/nuclear localization of intact insulin-like growth factor binding protein-2 and a distinct carboxyl-terminal IGFBP-2 fragment in vivo

    International Nuclear Information System (INIS)

    Hoeflich, A.; Reisinger, R.; Schuett, B.S.; Elmlinger, M.W.; Russo, V.C.; Vargas, G.A.; Jehle, P.M.; Lahm, H.; Renner-Mueller, I.; Wolf, E.

    2004-01-01

    Insulin-like growth factor binding protein-2 (IGFBP-2) as one of the most important IGFBPs has never been assessed in the intracellular compartment in vivo. Since there is evidence for novel intracellular functions of distinct IGFBPs, we investigated the presence of IGFBP-2 inside the cell. In peri/nuclear fractions of various tissues isolated from IGFBP-2 transgenic and non-transgenic mice we were able to show the presence of intact IGFBP-2. In addition, we demonstrate the presence of a highly conserved carboxyl-terminal IGFBP-2 fragment in the peri/nuclear fraction by using different peptide-induced antibodies. In pancreatic sections, confocal microscopy revealed the presence of IGFBP-2 on the nuclear surface but not within the nucleus. Our findings suggest novel functions of intact IGFBP-2 and IGFBP-2 fragments within the cell

  12. Liver polyribosome distribution in intact and adrenalectomized rats exposed to. gamma. radiation

    Energy Technology Data Exchange (ETDEWEB)

    Yatvin, M B; Abdel-Halim, M N [Wisconsin Univ., Madison (USA). Dept. of Radiology; Wisconsin Univ., Madison (USA). Dept. of Human Oncology)

    1978-06-01

    The mechanism(s) by which gamma radiation influences liver polyribosome distribution was studied in groups of intact and adrenalectomized male rats. A shift from light to heavy aggregates occurred in the polyribosomes of both intact and adrenalectomized rats after they were exposed to gamma rays. In irradiated adrenalectomized rats, however, the shift to heavier aggregates was not as great as that which occurred in irradiated adrenal-intact animals. Subcutaneous injection of cortisone acetate (10 mg/100 g body weight) also altered the liver polyribosome patterns of both intact and adrenalectomized rats within 8 hours of its administration. The shift which occurred following cortisone administration, however, was less striking than that seen after irradiation only. Thus, although adrenal glucocorticoids contribute to the radiation-indu ied shift in liver polyribosomes in adrenal-intact rats, other factors appear to be involved, since the shift is also obtained in adrenalectomized animals.

  13. Study of the metabolism of 13C labeled substrates by 13C NMR spectroscopy of intact cells, tissues, and organs

    International Nuclear Information System (INIS)

    Matwiyoff, N.A.; London, R.E.; Hutson, J.Y.

    1982-01-01

    Carbon-13 nuclear magnetic resonance spectroscopy, in conjunction with carbon-13 labeling, has become an important analytical technique for the study of biological systems and biologically important molecules. The growing list of its well established applications to isolated molecules in solution includes the investigation of: metabolic pathways; the microenvironments of ligands bound to proteins; the architecture and dynamics of macromolecules; the structures of coenzymes and other natural products; and the mechanisms of reactions. Recently interest has been reawakened in the use of the technique for the study of metabolic pathways and structural components in intact organelles, cells, and tissues. The promise and problems in the use of 13 C labeling in such investigations can be illustrated by the results on suspensions of the yeast, Candida utilis

  14. Intensity-modulated tangential beam irradiation of the intact breast

    International Nuclear Information System (INIS)

    Hong, L.; Hunt, M.; Chui, C.; Spirou, S.; Forster, K.; Lee, H.; Yahalom, J.; Kutcher, G.J.; McCormick, B.

    1999-01-01

    Purpose: To evaluate the potential benefits of intensity modulated tangential beams in the irradiation of the intact breast. Methods and Materials: Three-dimensional treatment planning was performed on five left and five right breasts using standard wedged and intensity modulated (IM) tangential beams. Optimal beam parameters were chosen using beams-eye-view display. For the standard plans, the optimal wedge angles were chosen based on dose distributions in the central plane calculated without inhomogeneity corrections, according to our standard protocol. Intensity-modulated plans were generated using an inverse planning algorithm and a standard set of target and critical structure optimization criteria. Plans were compared using multiple dose distributions and dose volume histograms for the planning target volume (PTV), ipsilateral lung, coronary arteries, and contralateral breast. Results: Significant improvements in the doses to critical structures were achieved using intensity modulation. Compared with a standard-wedged plan prescribed to 46 Gy, the dose from the IM plan encompassing 20% of the coronary artery region decreased by 25% (from 36 to 27 Gy) for patients treated to the left breast; the mean dose to the contralateral breast decreased by 42% (from 1.2 to 0.7 Gy); the ipsilateral lung volume receiving more than 46 Gy decreased by 30% (from 10% to 7%); the volume of surrounding soft tissue receiving more than 46 Gy decreased by 31% (from 48% to 33%). Dose homogeneity within the target volume improved greatest in the superior and inferior regions of the breast (approximately 8%), although some decrease in the medial and lateral high-dose regions (approximately 4%) was also observed. Conclusion: Intensity modulation with a standard tangential beam arrangement significantly reduces the dose to the coronary arteries, ipsilateral lung, contralateral breast, and surrounding soft tissues. Improvements in dose homogeneity throughout the target volume can also be

  15. Isolation of Dobrava Virus from Apodemus flavicollis in Greece

    Science.gov (United States)

    Papa, Anna; Nemirov, Kirill; Henttonen, Heikki; Niemimaa, Jukka; Antoniadis, Antonis; Vaheri, Antti; Plyusnin, Alexander; Vapalahti, Olli

    2001-01-01

    Dobrava virus (DOBV) carried by Apodemus flavicollis is the causative agent of severe hemorrhagic fever with renal syndrome (HFRS). DOBV was isolated from an A. flavicollis mouse trapped in northeastern Greece. This is the third DOBV cell culture isolate in the world, clustering together with other Greek DOBV sequences from HFRS patients and rodents. PMID:11376073

  16. Gene expression profile data for mouse facial development

    Directory of Open Access Journals (Sweden)

    Sonia M. Leach

    2017-08-01

    Full Text Available This article contains data related to the research articles "Spatial and Temporal Analysis of Gene Expression during Growth and Fusion of the Mouse Facial Prominences" (Feng et al., 2009 [1] and “Systems Biology of facial development: contributions of ectoderm and mesenchyme” (Hooper et al., 2017 In press [2]. Embryonic mammalian craniofacial development is a complex process involving the growth, morphogenesis, and fusion of distinct facial prominences into a functional whole. Aberrant gene regulation during this process can lead to severe craniofacial birth defects, including orofacial clefting. As a means to understand the genes involved in facial development, we had previously dissected the embryonic mouse face into distinct prominences: the mandibular, maxillary or nasal between E10.5 and E12.5. The prominences were then processed intact, or separated into ectoderm and mesenchyme layers, prior analysis of RNA expression using microarrays (Feng et al., 2009, Hooper et al., 2017 in press [1,2]. Here, individual gene expression profiles have been built from these datasets that illustrate the timing of gene expression in whole prominences or in the separated tissue layers. The data profiles are presented as an indexed and clickable list of the genes each linked to a graphical image of that gene׳s expression profile in the ectoderm, mesenchyme, or intact prominence. These data files will enable investigators to obtain a rapid assessment of the relative expression level of any gene on the array with respect to time, tissue, prominence, and expression trajectory.

  17. methoxyethanol by a new bacterium isolate Pseudomonas sp. Strain

    African Journals Online (AJOL)

    Michael Horsfall

    A 2-methoxyethanol degrading bacterium was isolated from anaerobic sludge of a municipal sewage from ... Stoichiometrically, the strain utilized one mole of oxygen per one mole of 2-methoxyethanol instead of ... physiological and biochemical characterization of the .... observed with acetate and the intact resting cells.

  18. Impact of physiological variables and genetic background on myocardial frequency-resistivity relations in the intact beating murine heart.

    Science.gov (United States)

    Reyes, Maricela; Steinhelper, Mark E; Alvarez, Jorge A; Escobedo, Daniel; Pearce, John; Valvano, Jonathan W; Pollock, Brad H; Wei, Chia-Ling; Kottam, Anil; Altman, David; Bailey, Steven; Thomsen, Sharon; Lee, Shuko; Colston, James T; Oh, Jung Hwan; Freeman, Gregory L; Feldman, Marc D

    2006-10-01

    Conductance measurements for generation of an instantaneous left ventricular (LV) volume signal in the mouse are limited, because the volume signal is a combination of blood and LV muscle, and only the blood signal is desired. We have developed a conductance system that operates at two simultaneous frequencies to identify and remove the myocardial contribution to the instantaneous volume signal. This system is based on the observation that myocardial resistivity varies with frequency, whereas blood resistivity does not. For calculation of LV blood volume with the dual-frequency conductance system in mice, in vivo murine myocardial resistivity was measured and combined with an analytic approach. The goals of the present study were to identify and minimize the sources of error in the measurement of myocardial resistivity to enhance the accuracy of the dual-frequency conductance system. We extended these findings to a gene-altered mouse model to determine the impact of measured myocardial resistivity on the calculation of LV pressure-volume relations. We examined the impact of temperature, timing of the measurement during the cardiac cycle, breeding strain, anisotropy, and intrameasurement and interanimal variability on the measurement of intact murine myocardial resistivity. Applying this knowledge to diabetic and nondiabetic 11- and 20- to 24-wk-old mice, we demonstrated differences in myocardial resistivity at low frequencies, enhancement of LV systolic function at 11 wk and LV dilation at 20-24 wk, and histological and electron-microscopic studies demonstrating greater glycogen deposition in the diabetic mice. This study demonstrated the accurate technique of measuring myocardial resistivity and its impact on the determination of LV pressure-volume relations in gene-altered mice.

  19. Colonization, mouse-style

    Directory of Open Access Journals (Sweden)

    Searle Jeremy B

    2010-10-01

    Full Text Available Abstract Several recent papers, including one in BMC Evolutionary Biology, examine the colonization history of house mice. As well as background for the analysis of mouse adaptation, such studies offer a perspective on the history of movements of the humans that accidentally transported the mice. See research article: http://www.biomedcentral.com/1471-2148/10/325

  20. Nonstructural Protein L* Species Specificity Supports a Mouse Origin for Vilyuisk Human Encephalitis Virus.

    Science.gov (United States)

    Drappier, Melissa; Opperdoes, Fred R; Michiels, Thomas

    2017-07-15

    Vilyuisk human encephalitis virus (VHEV) is a picornavirus related to Theiler's murine encephalomyelitis virus (TMEV). VHEV was isolated from human material passaged in mice. Whether this VHEV is of human or mouse origin is therefore unclear. We took advantage of the species-specific activity of the nonstructural L* protein of theiloviruses to track the origin of TMEV isolates. TMEV L* inhibits RNase L, the effector enzyme of the interferon pathway. By using coimmunoprecipitation and functional RNase L assays, the species specificity of RNase L antagonism was tested for L* from mouse (DA) and rat (RTV-1) TMEV strains as well as for VHEV. Coimmunoprecipitation and functional assay data confirmed the species specificity of L* activity and showed that L* from rat strain RTV-1 inhibited rat but not mouse or human RNase L. Next, we showed that the VHEV L* protein was phylogenetically related to L* of mouse viruses and that it failed to inhibit human RNase L but readily antagonized mouse RNase L, unambiguously showing the mouse origin of VHEV. IMPORTANCE Defining the natural host of a virus can be a thorny issue, especially when the virus was isolated only once or when the isolation story is complex. The species Theilovirus includes Theiler's murine encephalomyelitis virus (TMEV), infecting mice and rats, and Saffold virus (SAFV), infecting humans. One TMEV strain, Vilyuisk human encephalitis virus (VHEV), however, was isolated from mice that were inoculated with cerebrospinal fluid of a patient presenting with chronic encephalitis. It is therefore unclear whether VHEV was derived from the human sample or from the inoculated mouse. The L* protein encoded by TMEV inhibits RNase L, a cellular enzyme involved in innate immunity, in a species-specific manner. Using binding and functional assays, we show that this species specificity even allows discrimination between TMEV strains of mouse and of rat origins. The VHEV L* protein clearly inhibited mouse but not human RNase L

  1. Proteolysis breaks tolerance toward intact α345(IV) collagen, eliciting novel anti-GBM autoantibodies specific for α345NC1 hexamers

    Science.gov (United States)

    Olaru, Florina; Wang, Xu-Ping; Luo, Wentian; Ge, Linna; Miner, Jeffrey H; Kleinau, Sandra; Geiger, Xochiquetzal J.; Wasiluk, Andrew; Heidet, Laurence; Kitching, A. Richard; Borza, Dorin-Bogdan

    2012-01-01

    Goodpasture disease is an autoimmune kidney disease mediated by autoAbs against NC1 monomers of α3(IV) collagen that bind to the glomerular basement membrane (GBM), usually causing rapidly progressive glomerulonephritis. We identified a novel type of human IgG4-restricted anti-GBM autoAbs associated with mild non-progressive glomerulonephritis, which specifically targeted α345NC1 hexamers but not α3NC1 monomers. The mechanisms eliciting these anti-GBM autoAbs were investigated in mouse models recapitulating this phenotype. Wild type and FcγRIIB−/− mice immunized with autologous murine GBM NC1 hexamers produced mouse IgG1-restricted autoAbs specific for α345NC1 hexamers, which bound to the GBM in vivo but did not cause glomerulonephritis. In these mice, intact collagen IV from murine GBM was not immunogenic. However, in Col4a3−/− Alport mice, both intact collagen IV and NC1 hexamers from murine GBM elicited IgG antibodies specific for α3α4α5NC1 hexamers, which were not subclass restricted. As heterologous antigen in COL4A3-humanized mice, murine GBM NC1 hexamers elicited mouse IgG1, IgG2a and IgG2b autoAbs specific for α345NC1 hexamers and induced anti-GBM Ab glomerulonephritis. These findings indicate that tolerance toward autologous intact α3α4α5(IV) collagen is established in hosts expressing this antigen, even though autoreactive B cells specific for α345NC1 hexamers are not purged from their repertoire. Proteolysis selectively breaches this tolerance by generating autoimmunogenic α3α4α5NC1 hexamers. This provides a mechanism eliciting autoAbs specific for α345NC1 hexamers, which are restricted to non-inflammatory IgG subclasses and non-nephritogenic. In Alport syndrome, lack of tolerance toward α3α4α5(IV) collagen promotes production of alloantibodies to α345NC1 hexamers, including pro-inflammatory IgG subclasses which mediate post-transplant anti-GBM nephritis. PMID:23303673

  2. Blunt liver injury with intact ribs under impacts on the abdomen: a biomechanical investigation.

    Directory of Open Access Journals (Sweden)

    Yu Shao

    Full Text Available Abdominal trauma accounts for nearly 20% of all severe traffic injuries and can often result from intentional physical violence, from which blunt liver injury is regarded as the most common result and is associated with a high mortality rate. Liver injury may be caused by a direct impact with a certain velocity and energy on the abdomen, which may result in a lacerated liver by penetration of fractured ribs. However, liver ruptures without rib cage fractures were found in autopsies in a series of cases. All the victims sustained punches on the abdomen by fist. Many studies have been dedicated to determining the mechanism underlying hepatic injury following abdominal trauma, but most have been empirical. The actual process and biomechanism of liver injury induced by blunt impact on the abdomen, especially with intact ribs remained, are still inexhaustive. In order to investigate this, finite element methods and numerical simulation technology were used. A finite element human torso model was developed from high resolution CT data. The model consists of geometrically-detailed liver and rib cage models and simplified models of soft tissues, thoracic and abdominal organs. Then, the torso model was used in simulations in which the right hypochondrium was punched by a fist from the frontal, lateral, and rear directions, and in each direction with several impact velocities. Overall, the results showed that liver rupture was primarily caused by a direct strike of the ribs induced by blunt impact to the abdomen. Among three impact directions, a lateral impact was most likely to cause liver injury with a minimum punch speed of 5 m/s (the momentum was about 2.447 kg.m/s. Liver injuries could occur in isolation and were not accompanied by rib fractures due to different material characteristics and injury tolerance.

  3. Staurosporine Inhibits Frequency-Dependent Myofilament Desensitization in Intact Rabbit Cardiac Trabeculae

    Directory of Open Access Journals (Sweden)

    Kenneth D. Varian

    2012-01-01

    Full Text Available Myofilament calcium sensitivity decreases with frequency in intact healthy rabbit trabeculae and associates with Troponin I and Myosin light chain-2 phosphorylation. We here tested whether serine-threonine kinase activity is primarily responsible for this frequency-dependent modulations of myofilament calcium sensitivity. Right ventricular trabeculae were isolated from New Zealand White rabbit hearts and iontophoretically loaded with bis-fura-2. Twitch force-calcium relationships and steady state force-calcium relationships were measured at frequencies of 1 and 4 Hz at 37 °C. Staurosporine (100 nM, a nonspecific serine-threonine kinase inhibitor, or vehicle (DMSO was included in the superfusion solution before and during the contractures. Staurosporine had no frequency-dependent effect on force development, kinetics, calcium transient amplitude, or rate of calcium transient decline. The shift in the pCa50 of the force-calcium relationship was significant from 6.05±0.04 at 1 Hz versus 5.88±0.06 at 4 Hz under control conditions (vehicle, P<0.001 but not in presence of staurosporine (5.89±0.08 at 1 Hz versus 5.94±0.07 at 4 Hz, P=NS. Phosphoprotein analysis (Pro-Q Diamond stain confirmed that staurosporine significantly blunted the frequency-dependent phosphorylation at Troponin I and Myosin light chain-2. We conclude that frequency-dependent modulation of calcium sensitivity is mediated through a kinase-specific effect involving phosphorylation of myofilament proteins.

  4. Intact reflexive but deficient voluntary social orienting in autism spectrum disorder

    Directory of Open Access Journals (Sweden)

    Megan Anne Kirchgessner

    2015-12-01

    Full Text Available Impairment in social interactions is a primary characteristic of people diagnosed with autism spectrum disorder (ASD. Although these individuals tend to orient less to naturalistic social cues than do typically developing (TD individuals, laboratory experiments testing social orienting in ASD have been inconclusive, possibly because of a failure to fully isolate reflexive (stimulus-driven and voluntary (goal-directed social orienting processes. The purpose of the present study was to separately examine potential reflexive and/or voluntary social orienting differences in individuals with ASD relative to TD controls. Subjects (ages 7-14 with high-functioning ASD and a matched control group completed three gaze cueing tasks on an iPad in which individuals briefly saw a face with averted gaze followed by a target after a variable delay. Two tasks were 100% predictive with either all congruent (target appears in gaze direction or all incongruent (target appears opposite from gaze direction trials, respectively. Another task was non-predictive with these same trials (half congruent and half incongruent intermixed randomly. Response times (RTs to the target were used to calculate reflexive (incongruent condition RT – congruent condition RT and voluntary (non-predictive condition RT – predictive condition RT gaze cueing effects. Subjects also completed two additional non-social orienting tasks (ProPoint and AntiPoint. Subjects with ASD demonstrate intact reflexive but deficient voluntary gaze following. Similar results were found in a separate test of non-social orienting. This suggests problems with using social cues, but only in a goal-directed fashion, in our sample of high-functioning individuals with ASD. Such findings may not only explain inconclusive previous findings but more importantly be critical for understanding social dysfunctions in ASD and for developing future interventions.

  5. DNA synthesis in periportal and perivenous hepatocytes of intact and hepatectomized young mice.

    Science.gov (United States)

    Fernández-Blanco, A; Inda, A M; Errecalde, A L

    2015-01-01

    DNA synthesis of hepatocytes in two areas of Intact and Hepatectomized young mice liver along a circadian period was studied. DNA synthesis was significantly different at all analyzed time points in Intact and Hepatectomized animals. Differences between periportal and perivenous hepatocytes were found in hepatectomized animals at 04/42 and 08/46 hr of day/hour post-hepatectomy. DNAs peak in periportal hepatocytes regenerating liver occurs 4 hr earlier than in perivenous hepatocytes, probably reflecting their shorter G1 phase. Besides, daily mean values of regenerating livers were higher than those observed in Intact animals, as a consequence of surgical removal.

  6. Astonishing advances in mouse genetic tools for biomedical research.

    Science.gov (United States)

    Kaczmarczyk, Lech; Jackson, Walker S

    2015-01-01

    The humble house mouse has long been a workhorse model system in biomedical research. The technology for introducing site-specific genome modifications led to Nobel Prizes for its pioneers and opened a new era of mouse genetics. However, this technology was very time-consuming and technically demanding. As a result, many investigators continued to employ easier genome manipulation methods, though resulting models can suffer from overlooked or underestimated consequences. Another breakthrough, invaluable for the molecular dissection of disease mechanisms, was the invention of high-throughput methods to measure the expression of a plethora of genes in parallel. However, the use of samples containing material from multiple cell types could obfuscate data, and thus interpretations. In this review we highlight some important issues in experimental approaches using mouse models for biomedical research. We then discuss recent technological advances in mouse genetics that are revolutionising human disease research. Mouse genomes are now easily manipulated at precise locations thanks to guided endonucleases, such as transcription activator-like effector nucleases (TALENs) or the CRISPR/Cas9 system, both also having the potential to turn the dream of human gene therapy into reality. Newly developed methods of cell type-specific isolation of transcriptomes from crude tissue homogenates, followed by detection with next generation sequencing (NGS), are vastly improving gene regulation studies. Taken together, these amazing tools simplify the creation of much more accurate mouse models of human disease, and enable the extraction of hitherto unobtainable data.

  7. In vitro culture of pre-implanted mouse embryos. A model system for studying combined effects

    International Nuclear Information System (INIS)

    Streffer, C.; Beuningen, D. van; Molls, M.; Pon, A.; Schulz, S.; Zamboglou, N.

    1978-01-01

    Studies on combined effects, e.g. interaction between chemical toxicants and ionizing radiation, are difficult to perform, as they are dependent on many factors (substance concentration, radiation dose, sequence of treatments, etc.). In order to obtain data from such studies it is necessary to establish a comparatively simple experimental model system. We have established such a model system by studying combined effects on pre-implanted mouse embryos cultured in vitro. This system has the following advantages: (1) The embryos can be cultivated for several days in vitro; (2) Their physiological intactness can be tested; and (3) Cell proliferation, cell killing and chromosomal damage can be investigated comparatively easily. The embryos are isolated at the 2-cell stage and incubated in a culture medium in vitro. The development of the embryos is followed under the microscope until the development of blastocysts or the hatching of blastocysts is observed. These blastocysts can be transplanted to fostered mice and the development of normal animals determined. The proliferation kinetics can be studied easily, and the methods are described. A method has also been developed to measure the DNA content of individual cells by microscope fluorometry. After treatment of the embryos with ionizing radiation or drugs the release of micronuclei has been observed from the cell nuclei, which is an expression for chromosomal damage. Substances or radionuclides can be added to the culture medium or external irradiation can be performed during the culture period. Also the combined effects of radiation and heating can be studied. The effects of X-rays and tritiated compounds have also been investigated. The combined effects of radiation with antibiotics such as actinomycin D, and environmental toxicants such as lead, have been determined. The system described has been useful to evaluate cytological, teratogenic and cytogenetic effects

  8. The Mouse That Soared

    Science.gov (United States)

    2004-09-01

    Astronomers have used an X-ray image to make the first detailed study of the behavior of high-energy particles around a fast moving pulsar. The image, from NASA's Chandra X-ray Observatory, shows the shock wave created as a pulsar plows supersonically through interstellar space. These results will provide insight into theories for the production of powerful winds of matter and antimatter by pulsars. Chandra's image of the glowing cloud, known as the Mouse, shows a stubby bright column of high-energy particles, about four light years in length, swept back by the pulsar's interaction with interstellar gas. The intense source at the head of the X-ray column is the pulsar, estimated to be moving through space at about 1.3 million miles per hour. VLA Radio Image of the Mouse, Full Field VLA Radio Image of the Mouse, Full Field A cone-shaped cloud of radio-wave-emitting particles envelopes the X-ray column. The Mouse, a.k.a. G359.23-0.82, was discovered in 1987 by radio astronomers using the National Science Foundation's Very Large Array in New Mexico. It gets its name from its appearance in radio images that show a compact snout, a bulbous body, and a remarkable long, narrow, tail that extends for about 55 light years. "A few dozen pulsar wind nebulae are known, including the spectacular Crab Nebula, but none have the Mouse's combination of relatively young age and incredibly rapid motion through interstellar space," said Bryan Gaensler of the Harvard-Smithsonian Center for Astrophysics and lead author of a paper on the Mouse that will appear in an upcoming issue of The Astrophysical Journal. "We effectively are seeing a supersonic cosmic wind tunnel, in which we can study the effects of a pulsar's motion on its pulsar wind nebula, and test current theories." Illustration of the Mouse System Illustration of the Mouse System Pulsars are known to be rapidly spinning, highly magnetized neutron stars -- objects so dense that a mass equal to that of the Sun is packed into a

  9. 31P NMR study of phosphate metabolites in intact developing seeds of wheat, soybean and mustard

    International Nuclear Information System (INIS)

    Gambhir, P.N.; Pande, P.C.; Ratcliffe, R.G.

    1994-01-01

    The study of 31 P NMR spectra of intact developing seeds of wheat, soybean and mustard and its possible use for assessing the relative degree of hypoxia under in vivo conditions are reported. 7 refs., 2 figs

  10. Toward best practices in data processing and analysis for intact biotherapeutics by MS in quantitative bioanalysis.

    Science.gov (United States)

    Kellie, John F; Kehler, Jonathan R; Karlinsey, Molly Z; Summerfield, Scott G

    2017-12-01

    Typically, quantitation of biotherapeutics from biological matrices by LC-MS is based on a surrogate peptide approach to determine molecule concentration. Recent efforts have focused on quantitation of the intact protein molecules or larger mass subunits of monoclonal antibodies. To date, there has been limited guidance for large or intact protein mass quantitation for quantitative bioanalysis. Intact- and subunit-level analyses of biotherapeutics from biological matrices are performed at 12-25 kDa mass range with quantitation data presented. Linearity, bias and other metrics are presented along with recommendations made on the viability of existing quantitation approaches. This communication is intended to start a discussion around intact protein data analysis and processing, recognizing that other published contributions will be required.

  11. Near-infrared reflectance spectroscopy for predicting amino acids content in intact processed animal proteins.

    Science.gov (United States)

    De la Haba, Maria José; Garrido-Varo, Ana; Guerrero-Ginel, José Emilio; Pérez-Marín, Dolores C

    2006-10-04

    Near-infrared calibrations were developed for the instantaneous prediction of amino acids composition of processed animal proteins (PAPs). Two sample presentation modes were compared (ground vs intact) for demonstrating the viability of the analysis in the intact form, avoiding the need for milling. Modified partial least-squares (MPLS) equations for the prediction of amino acids in PAPs were developed using the same set of samples (N = 92 PAPs) analyzed in ground and intact form and in three cups differing in the optical window size. The standard error for cross validation (SECV) and the coefficient of determination (1-VR) values yielded with the calibrations developed using the samples analyzed in the intact form showed similar or even better accuracy than those obtained with finely ground samples. The excellent predictive ability (1-VR > 0.90; CV marketing of these important protein feed ingredients, alleviating the costs and time associated with the routine quality controls.

  12. Cholesteatoma behind an intact tympanic membrane: histopathologic evidence for a tympanic membrane origin.

    Science.gov (United States)

    Sudhoff, H; Linthicum, F H

    2001-07-01

    Several theories have been proposed with respect to the origin and pathogenesis of cholesteatoma behind an intact tympanic membrane. The authors describe a case of cholesteatoma behind an intact tympanic membrane in a 71-year-old man with a history of tympanic membrane retraction fixed to the incus without evidence of a perforation. The membrane eventually became detached, and remnants of keratinizing squamous epithelium were found on the incus. Mechanisms such as metaplasia, ectopic epidermis rests, or ingrowth of meatal epidermis have been proposed to explain the pathogenesis of cholesteatoma behind an intact tympanic membrane. These findings, based on temporal bone histopathology, support the role of an acquired epidermal rest. This case report provides evidence that cholesteatoma behind an intact tympanic membrane can be established from a resolved retraction of the pars tensa of the tympanic membrane.

  13. Superolateral Dislocation of Intact Mandibular Condyle: A Case Report and Review of Literature

    OpenAIRE

    Saikrishna, Degala; Shyam Sundar, S.; Mamata, K. S.

    2015-01-01

    Anteromedial fracture dislocation of the mandibular condyle is common but a superolateral dislocation of an intact condyle is quite rare. This type of dislocation is often misdiagnosed or completely overlooked and hence inadequately addressed. We report a case of a 41-year-old male patient who experienced superolateral dislocation of the intact condyle with symphysis fracture and panfacial fracture following a road-traffic accident, and review of literature of superolateral dislocations from ...

  14. Impulse radar scanning of intact salt at the Avery Island Mine

    International Nuclear Information System (INIS)

    Cook, C.W.

    1980-05-01

    A series of experiments was run in the Avery Island Mine to evaluate the capability of an impulse radar to locate anomalies and simulated waste targets in intact dome salt. Voids in salt were difficult to detect. On the positive side, metal targets and simulated waste (glass) were easily located in intact salt. Radar scanning at ranges of greater than 25 meters and short-range resolution of target positions to within a few centimeters were achieved

  15. Prediction of valid acidity in intact apples with Fourier transform near infrared spectroscopy*

    OpenAIRE

    Liu, Yan-de; Ying, Yi-bin; Fu, Xia-ping

    2005-01-01

    To develop nondestructive acidity prediction for intact Fuji apples, the potential of Fourier transform near infrared (FT-NIR) method with fiber optics in interactance mode was investigated. Interactance in the 800 nm to 2619 nm region was measured for intact apples, harvested from early to late maturity stages. Spectral data were analyzed by two multivariate calibration techniques including partial least squares (PLS) and principal component regression (PCR) methods. A total of 120 Fuji appl...

  16. Occurence of translocations between irradiated and intact chromosomes of Drosophila melanogaster

    International Nuclear Information System (INIS)

    Myasnyankina, E.N.; Abeleva, Eh.A.; Generalova, M.V.

    1980-01-01

    Two translocations between irradiated father and intact mother autosomes are obtained in Drosophila melanogaster. Five out of 283 regular translocations (between the second and the third chromosomes of an irradiated male) are accompanied by a recombination over the second or the third chromosomes. Nine flies out of twenty considered to be recombinants, could originate due to mutations. The data obtained prove that intact female autosomes can take part in the exchange with homologic (recombinations) and heterologic (translocations) irradiated male autosomes

  17. Risks for Conduct Disorder Symptoms Associated with Parental Alcoholism in Stepfather Families versus Intact Families from a Community Sample

    Science.gov (United States)

    Foley, Debra L.; Pickles, Andrew; Rutter, Michael; Gardner, Charles O.; Maes, Hermine H.; Silberg, Judy L.; Eaves, Lindon J.

    2004-01-01

    Background: It is not known if the prevalence of parental psychiatric disorders is higher in stepfather than intact families, or if parental alcoholism is differentially associated with risk for conduct disorder (CD) symptoms in stepfather families versus intact families. Method: The sample comprised 839 girls and 741 boys from 792 intact families…

  18. Hypophysectomy eliminates and growth hormone (GH) maintains the midpregnancy elevation in GH receptor and serum binding protein in the mouse

    International Nuclear Information System (INIS)

    Sanchez-Jimenez, F.; Fielder, P.J.; Martinez, R.R.; Smith, W.C.; Talamantes, F.

    1990-01-01

    [ 125 I]Iodomouse GH [( 125 I]iodo-mGH) binding to samples of serum and hepatic microsomal membranes was measured in hypophysectomized pregnant, sham-operated pregnant, intact pregnant, and intact adult virgin mice. Surgeries were carried out on day 11 of pregnancy, and the animals were killed on day 14. The binding of mGH to both serum and hepatic microsomal membranes of intact virgin mice was much lower than to those of intact pregnant mice. In hypophysectomized mice, the mGH-binding capacity of both serum and hepatic microsomes decreased to values similar to those of nonpregnant mice. No significant differences were observed between intact and sham-operated pregnant animals in the maternal serum mGH concentration, the serum GH-binding protein concentration, or the hepatic GH receptor concentration. GH receptor and binding protein-encoding mRNAs were also higher in intact and sham-operated pregnant mice than in virgin and hypophysectomized mice. Hypophysectomized mice were treated with 200 micrograms/day bovine GH, administered by osmotic minipump; after 3 days of treatment, a significant elevation of hepatic GH receptor and serum GH-binding protein levels was observed. These results demonstrate an up-regulation of hepatic GH receptors and serum GH-binding protein by GH during pregnancy in the mouse

  19. Isolation and characterization of mouse innate lymphoid cells.

    Science.gov (United States)

    Halim, Timotheus Y F; Takei, Fumio

    2014-08-01

    Innate lymphoid cells (ILCs) are rare populations of cytokine-producing lymphocytes and are divided into three groups, namely ILC1, ILC2, and ILC3, based on the cytokines that they produce. They comprise less than 1% of lymphocytes in mucosal tissues and express no unique cell surface markers. Therefore, they can only be identified by combinations of multiple cell surface markers and further characterized by cytokine production in vitro. Thus, multicolor flow cytometry is the only reliable method to purify and characterize ILCs. Here we describe the methods for cell preparation, flow cytometric analysis, and purification of murine ILC2 and ILC3. Copyright © 2014 John Wiley & Sons, Inc.

  20. CYP1A1 and CYP1A2 expression: Comparing 'humanized' mouse lines and wild-type mice; comparing human and mouse hepatoma-derived cell lines

    International Nuclear Information System (INIS)

    Uno, Shigeyuki; Endo, Kaori; Ishida, Yuji; Tateno, Chise; Makishima, Makoto; Yoshizato, Katsutoshi; Nebert, Daniel W.

    2009-01-01

    Human and rodent cytochrome P450 (CYP) enzymes sometimes exhibit striking species-specific differences in substrate preference and rate of metabolism. Human risk assessment of CYP substrates might therefore best be evaluated in the intact mouse by replacing mouse Cyp genes with human CYP orthologs; however, how 'human-like' can human gene expression be expected in mouse tissues? Previously a bacterial-artificial-chromosome-transgenic mouse, carrying the human CYP1A1 C YP1A2 locus and lacking the mouse Cyp1a1 and Cyp1a2 orthologs, was shown to express robustly human dioxin-inducible CYP1A1 and basal versus inducible CYP1A2 (mRNAs, proteins, enzyme activities) in each of nine mouse tissues examined. Chimeric mice carrying humanized liver have also been generated, by transplanting human hepatocytes into a urokinase-type plasminogen activator(+/+) s evere-combined-immunodeficiency (uPA/SCID) line with most of its mouse hepatocytes ablated. Herein we compare basal and dioxin-induced CYP1A mRNA copy numbers, protein levels, and four enzymes (benzo[a]pyrene hydroxylase, ethoxyresorufin O-deethylase, acetanilide 4-hydroxylase, methoxyresorufin O-demethylase) in liver of these two humanized mouse lines versus wild-type mice; we also compare these same parameters in mouse Hepa-1c1c7 and human HepG2 hepatoma-derived established cell lines. Most strikingly, mouse liver CYP1A1-specific enzyme activities are between 38- and 170-fold higher than human CYP1A1-specific enzyme activities (per unit of mRNA), whereas mouse versus human CYP1A2 enzyme activities (per unit of mRNA) are within 2.5-fold of one another. Moreover, both the mouse and human hepatoma cell lines exhibit striking differences in CYP1A mRNA levels and enzyme activities. These findings are relevant to risk assessment involving human CYP1A1 and CYP1A2 substrates, when administered to mice as environmental toxicants or drugs.

  1. Nonspecific airway reactivity in a mouse model of asthma

    Energy Technology Data Exchange (ETDEWEB)

    Collie, D.D.; Wilder, J.A.; Bice, D.E.

    1995-12-01

    Animal models are indispensable for studies requiring an intact immune system, especially for studying the pathogenic mechanisms in atopic diseases, regulation of IgE production, and related biologic effects. Mice are particularly suitable and have been used extensively for such studies because their immune system is well characterized. Further, large numbers of mutants or inbred strains of mice are available that express deficiencies of individual immunologic processes, inflammatory cells, or mediator systems. By comparing reactions in such mice with appropriate control animals, the unique roles of individual cells or mediators may be characterized more precisely in the pathogenesis of atopic respiratory diseases including asthma. However, given that asthma in humans is characterized by the presence of airway hyperresponsiveness to specific and nonspecific stimuli, it is important that animal models of this disease exhibit similar physiologic abnormalities. In the past, the size of the mouse has limited its versatility in this regard. However, recent studies indicate the feasibility of measuring pulmonary responses in living mice, thus facilitating the physiologic evaluation of putative mouse models of human asthma that have been well charcterized at the immunologic and patholigic level. Future work will provide details of the morphometry of the methacholine-induced bronchoconstriction and will further seek to determine the relationship between cigarette smoke exposure and the development of NS-AHR in the transgenic mouse model.

  2. Analysis of 16S libraries of mouse gastrointestinal microflora reveals a large new group of mouse intestinal bacteria.

    Science.gov (United States)

    Salzman, Nita H; de Jong, Hendrik; Paterson, Yvonne; Harmsen, Hermie J M; Welling, Gjalt W; Bos, Nicolaas A

    2002-11-01

    Total genomic DNA from samples of intact mouse small intestine, large intestine, caecum and faeces was used as template for PCR amplification of 16S rRNA gene sequences with conserved bacterial primers. Phylogenetic analysis of the amplification products revealed 40 unique 16S rDNA sequences. Of these sequences, 25% (10/40) corresponded to described intestinal organisms of the mouse, including Lactobacillus spp., Helicobacter spp., segmented filamentous bacteria and members of the altered Schaedler flora (ASF360, ASF361, ASF502 and ASF519); 75% (30/40) represented novel sequences. A large number (11/40) of the novel sequences revealed a new operational taxonomic unit (OTU) belonging to the Cytophaga-Flavobacter-Bacteroides phylum, which the authors named 'mouse intestinal bacteria'. 16S rRNA probes were developed for this new OTU. Upon analysis of the novel sequences, eight were found to cluster within the Eubacterium rectale-Clostridium coccoides group and three clustered within the Bacteroides group. One of the novel sequences was distantly related to Verrucomicrobium spinosum and one was distantly related to Bacillus mycoides. Oligonucleotide probes specific for the 16S rRNA of these novel clones were generated. Using a combination of four previously described and four newly designed probes, approximately 80% of bacteria recovered from the murine large intestine and 71% of bacteria recovered from the murine caecum could be identified by fluorescence in situ hybridization (FISH).

  3. Symbiodinium isolation by NaOH treatment.

    Science.gov (United States)

    Zamoum, Thamilla; Furla, Paola

    2012-11-15

    The presence of photosynthetic zooxanthellae (dinoflagellates) in the tissue of many cnidarians is the main reason for their ecological success (i.e. coral reefs). It could also be the main cause of their demise, as the worldwide bleaching of reef-building coral is nothing less than the breakdown of this symbiotic association. The stability of this relationship is the principal marker for the biomonitoring of cnidarian health. We have therefore developed a new, simple method to isolate zooxanthellae in a few steps using NaOH solution. The protocol was validated in three symbiotic cnidarian species: a sea anemone, a gorgonian and a coral. Our method allows the isolation of intact and viable zooxanthellae with better yields than classic methods, especially for species with a calcareous skeleton. Moreover, the isolated zooxanthellae were free of host nucleic contaminants, facilitating subsequent specific molecular analyses.

  4. Isolation World

    OpenAIRE

    Núñez Martín, Eugeni

    2012-01-01

    El trabajo de fin de grado tiene como nombre “Isolation World”, que en su traducción literal significa “Aislamiento del mundo”, un videojuego diseñado y creado desde cero en su totalidad, utilizando herramientas y conocimiento de lógica en programación que se han ido aprendiendo y desarrollando a lo largo de la carrera.

  5. Isolation and expression of a Pax-6 gene in the regenerating and intact Planarian Dugesia(G)tigrina

    Science.gov (United States)

    Callaerts, P.; Munoz-Marmol, A. M.; Glardon, S.; Castillo, E.; Sun, H.; Li, W.-H.; Gehring, W. J.; Salo, E.

    1999-01-01

    The Pax-6 gene encodes a transcription factor containing both a paired and a homeodomain and is highly conserved among Metazoa. In both vertebrates and invertebrates, Pax-6 is required for eye morphogenesis, development of parts of the central nervous system, and, in some phyla, for the development of olfactory sense organs. Ectopic expression of Pax-6 from insects, mammals, cephalopods, and ascidians induces ectopic eyes in Drosophila, suggesting that Pax-6 may be a universal master control gene for eye morphogenesis. Platyhelminthes are an ancient phylum, originating from the base of spiralian protostomes, that bear primitive eyes, consisting of a group of rhabdomeric photoreceptor cells enclosed in a cup of pigment cells. The analysis of Pax-6 and its expression pattern should provide insights into the ancestral function of Pax-6 in eye morphogenesis. We have identified the Pax-6 gene of the planarian Dugesia(G)tigrina (Platyhelminthes; Turbellaria; Tricladida). This gene shares significant sequence identity and conserved genomic organization with Pax-6 proteins from other phyla. Phylogenetic analysis indicates that it clusters with the other Pax-6 genes, but in the most basal position. DtPax-6 is expressed as a single transcript in both regenerating and fully grown eyes, and electron microscopy studies show strong expression in the perykarion of both photoreceptor and pigment cells. Very low levels of expression also are detectable in other body regions. Because a bona fide Pax-6 homolog so far has not been detected in diploblastic animals, we speculate that Pax-6 may be typical for triploblasts and that the appearance of additional Pax genes may have coincided with increasingly complex body plans. PMID:9892672

  6. Effect of taurine on the insuline secretion isolated by the pancreatic tissue of intact and irradiated rats

    International Nuclear Information System (INIS)

    Dokshina, G.A.; Silaeva, T.Yu.

    1976-01-01

    The whole-body irradiation of rats (700 rads) inhibits the secretory activity of insular pancreatic tissue. Administration of taurine (200 mg/kg), on the fifth day after irradiation, five times every second day normalizes the secretory function of pancreatic islands. In the experiments in vitro, taurine (1.5 and 3.0 mg/ml) stimulated hormone secretion. The stimulating action of the amino acid manifests itself when β-receptors are blocked by obsidane (0.5 μg/ml). It is suggested that insuline secretion by β-cells of pancreas is restored and enhanced by taurine not merely through the adenylatecyclase system; other ways are also possible

  7. Effect of taurine on the insuline secretion isolated by the pancreatic tissue of intact and irradiated rats

    Energy Technology Data Exchange (ETDEWEB)

    Dokshina, G A; Silaeva, T Yu [Tomskij Gosudarstvennyj Univ. (USSR). Nauchno-Issledovatel' skij Inst. Biologii i Biofiziki

    1976-05-01

    The whole-body irradiation of rats (700 rads) inhibits the secretory activity of insular pancreatic tissue. Administration of taurine (200 mg/kg), on the fifth day after irradiation, five times every second day normalizes the secretory function of pancreatic islands. In the experiments in vitro, taurine (1.5 and 3.0 mg/ml) stimulated hormone secretion. The stimulating action of the amino acid manifests itself when ..beta..-receptors are blocked by obsidane (0.5 ..mu..g/ml). It is suggested that insuline secretion by ..beta..-cells of pancreas is restored and enhanced by taurine not merely through the adenylatecyclase system; other ways are also possible.

  8. Performance of intact and castrated beef cattle in an intensive croppasture rotation system

    Directory of Open Access Journals (Sweden)

    Tercilio Turini

    2015-07-01

    Full Text Available This research had as objective to evaluate the performance of intact or castrated beef cattle in a croppasture rotation system. The experiment was conducted during 2004 and 2005, and carried out at the Cooperativa Agropecuária Mourãoense (COAMO Experimental Farm, in Campo Mourão city, Paraná state. It was used a completely randomized design, with two treatments, intact or castrated. Forty ½Angus+½Nelore crossbred animals, with average age of nine months, were used. Half of the animals were castrated at weaning, and the other half was kept intact. Pasture was composed of two areas. The winter field, established after soybean crop, was composed by a mixture of black oat (Avena strigosa and Italian ryegrass (Lolium multiforum. The summer field was composed by stargrass (Cynodon nlemfuensis and Mombaça grass (Panicum maximum. During the winter time it was used a continues grazing system, with regulator animals (put and take, and during the summer an intensive rotational system, with regulator animals and fixed grazing period. Intact animals presented higher average daily weight gain (0.907 vs 0.698 kg, slaughter weight (490.9 vs 442.2 kg, and hot carcass weight (250.2 vs 232.6 kg. Slaughter age was influenced by sexual condition, being lesser in the intact animals. Carcass dressing percentage was similar for the groups. Castrated animals showed better finishing fat cover and backfat thickness (3.45 vs 2.70 mm compared to intact ones. Therefore, it can be concluded that intact animals presents better performance than castrated ones when finished in an intensive crop-pasture rotation system, however, they may not present the minimum required fat cover, when slaughter at young ages.

  9. The Sycp1 loci of the mouse genome: successive retropositions of a meiotic gene during the recent evolution of the genus.

    Science.gov (United States)

    Sage, J; Yuan, L; Martin, L; Mattei, M G; Guénet, J L; Liu, J G; Hoög, C; Rassoulzadegan, M; Cuzin, F

    1997-08-15

    The murine Sycp1 gene is expressed at the early stages of meiosis. We show that it is composed of a number of small exons and localized on mouse chromosome 3. In the laboratory strains, two retrogenes were also identified. The first one (Sycp1-ps1), on chromosome 7, has accumulated point mutations and deletions and is not transcribed. A second retrogene (Sycp1-ps2), on chromosome 8, is inserted within the continuity of a moderately repeated element, in an intron of another gene (Cad11). The two retroposition events can be dated to distinct periods in the evolution of the Muridae. Sycp1-ps2 has kept features indicative of a relatively recent origin, namely a nearly intact coding region, a poly(A) tail, and 14-bp terminal repeats. Its recent origin was confirmed by the fact that it is found in all the laboratory strains of mice, but neither in a recent isolate from Mus musculus domesticus wild stocks nor in the closely related subspecies M. musculus musculus, M. m. molossinus, M. m. castaneus, and M. m. bactrianus. Appearance of the more ancient Sycp1-ps1 retrogene is concomitant with the radiation of the genus. It is present in various Mus species (M. spretus, M. spicilegus, M. macedonicus, and M. cookii), but neither in the rat nor in the more closely related Pyromis genus. Transposition of retrotranscripts during meiosis and their hereditary establishment thus appear to occur relatively frequently. They may, therefore, play a significant role in the evolutionary process.

  10. Cyanine 5.5 conjugated nanobubbles as a tumor selective contrast agent for dual ultrasound-fluorescence imaging in a mouse model.

    Directory of Open Access Journals (Sweden)

    Liyi Mai

    Full Text Available Nanobubbles and microbubbles are non-invasive ultrasound imaging contrast agents that may potentially enhance diagnosis of tumors. However, to date, both nanobubbles and microbubbles display poor in vivo tumor-selectivity over non-targeted organs such as liver. We report here cyanine 5.5 conjugated nanobubbles (cy5.5-nanobubbles of a biocompatible chitosan-vitamin C lipid system as a dual ultrasound-fluorescence contrast agent that achieved tumor-selective imaging in a mouse tumor model. Cy5.5-nanobubble suspension contained single bubble spheres and clusters of bubble spheres with the size ranging between 400-800 nm. In the in vivo mouse study, enhancement of ultrasound signals at tumor site was found to persist over 2 h while tumor-selective fluorescence emission was persistently observed over 24 h with intravenous injection of cy5.5-nanobubbles. In vitro cell study indicated that cy5.5-flurescence dye was able to accumulate in cancer cells due to the unique conjugated nanobubble structure. Further in vivo fluorescence study suggested that cy5.5-nanobubbles were mainly located at tumor site and in the bladder of mice. Subsequent analysis confirmed that accumulation of high fluorescence was present at the intact subcutaneous tumor site and in isolated tumor tissue but not in liver tissue post intravenous injection of cy5.5-nanobubbles. All these results led to the conclusion that cy5.5-nanobubbles with unique crosslinked chitosan-vitamin C lipid system have achieved tumor-selective imaging in vivo.

  11. Isolation of Chromoplasts and Suborganellar Compartments from Tomato and Bell Pepper Fruit.

    Science.gov (United States)

    Barsan, Cristina; Kuntz, Marcel; Pech, Jean-Claude

    2017-01-01

    Tomato is a model for fruit development and ripening. The isolation of intact plastids from this organism is therefore important for metabolic and proteomic analyses. Pepper, a species from the same family, is also of interest since it allows isolation of intact chromoplasts in large amounts. Here, we provide a detailed protocol for the isolation of tomato plastids at three fruit developmental stages, namely, nascent chromoplasts from the mature green stage, chromoplasts from an intermediate stage, and fully differentiated red chromoplasts. The method relies on sucrose density gradient centrifugations. It yields high purity organelles suitable for proteome analyses. Enzymatic and microscopy assays are summarized to assess purity and intactness. A method is also described for subfractionation of pepper chromoplast lipoprotein structures.

  12. Isolation of Toxoplasma gondii in a pig from a local abattoir in ...

    African Journals Online (AJOL)

    Inoculation of pepsin digested muscle (mixed heart and diaphragm) from a slaughter pig into six Balb C mice resulted in the isolation of Toxoplasma gondii which proved to be pathogenic to mice. These infected mice were retarded in growth when compared to the two uninoculated control animals. Mouse No.3 and Mouse ...

  13. Evaluation of an in vitro toxicogenetic mouse model for hepatotoxicity

    International Nuclear Information System (INIS)

    Martinez, Stephanie M.; Bradford, Blair U.; Soldatow, Valerie Y.; Kosyk, Oksana; Sandot, Amelia; Witek, Rafal; Kaiser, Robert; Stewart, Todd; Amaral, Kirsten; Freeman, Kimberly; Black, Chris; LeCluyse, Edward L.; Ferguson, Stephen S.; Rusyn, Ivan

    2010-01-01

    Numerous studies support the fact that a genetically diverse mouse population may be useful as an animal model to understand and predict toxicity in humans. We hypothesized that cultures of hepatocytes obtained from a large panel of inbred mouse strains can produce data indicative of inter-individual differences in in vivo responses to hepato-toxicants. In order to test this hypothesis and establish whether in vitro studies using cultured hepatocytes from genetically distinct mouse strains are feasible, we aimed to determine whether viable cells may be isolated from different mouse inbred strains, evaluate the reproducibility of cell yield, viability and functionality over subsequent isolations, and assess the utility of the model for toxicity screening. Hepatocytes were isolated from 15 strains of mice (A/J, B6C3F1, BALB/cJ, C3H/HeJ, C57BL/6J, CAST/EiJ, DBA/2J, FVB/NJ, BALB/cByJ, AKR/J, MRL/MpJ, NOD/LtJ, NZW/LacJ, PWD/PhJ and WSB/EiJ males) and cultured for up to 7 days in traditional 2-dimensional culture. Cells from B6C3F1, C57BL/6J, and NOD/LtJ strains were treated with acetaminophen, WY-14,643 or rifampin and concentration-response effects on viability and function were established. Our data suggest that high yield and viability can be achieved across a panel of strains. Cell function and expression of key liver-specific genes of hepatocytes isolated from different strains and cultured under standardized conditions are comparable. Strain-specific responses to toxicant exposure have been observed in cultured hepatocytes and these experiments open new opportunities for further developments of in vitro models of hepatotoxicity in a genetically diverse population.

  14. Properties of internalization factors contributing to the uptake of extracellular DNA into tumor-initiating stem cells of mouse Krebs-2 cell line.

    Science.gov (United States)

    Dolgova, Evgeniya V; Potter, Ekaterina A; Proskurina, Anastasiya S; Minkevich, Alexandra M; Chernych, Elena R; Ostanin, Alexandr A; Efremov, Yaroslav R; Bayborodin, Sergey I; Nikolin, Valeriy P; Popova, Nelly A; Kolchanov, Nikolay A; Bogachev, Sergey S

    2016-05-25

    Previously, we demonstrated that poorly differentiated cells of various origins, including tumor-initiating stem cells present in the ascites form of mouse cancer cell line Krebs-2, are capable of naturally internalizing both linear double-stranded DNA and circular plasmid DNA. The method of co-incubating Krebs-2 cells with extracellular plasmid DNA (pUC19) or TAMRA-5'-dUTP-labeled polymerase chain reaction (PCR) product was used. It was found that internalized plasmid DNA isolated from Krebs-2 can be transformed into competent Escherichia coli cells. Thus, the internalization processes taking place in the Krebs-2 cell subpopulation have been analyzed and compared, as assayed by E. coli colony formation assay (plasmid DNA) and cytofluorescence (TAMRA-DNA). We showed that extracellular DNA both in the form of plasmid DNA and a PCR product is internalized by the same subpopulation of Krebs-2 cells. We found that the saturation threshold for Krebs-2 ascites cells is 0.5 μg DNA/10(6) cells. Supercoiled plasmid DNA, human high-molecular weight DNA, and 500 bp PCR fragments are internalized into the Krebs-2 tumor-initiating stem cells via distinct, non-competing internalization pathways. Under our experimental conditions, each cell may harbor 340-2600 copies of intact plasmid material, or up to 3.097 ± 0.044×10(6) plasmid copies (intact or not), as detected by quantitative PCR. The internalization dynamics of extracellular DNA, copy number of the plasmids taken up by the cells, and competition between different types of double-stranded DNA upon internalization into tumor-initiating stem cells of mouse ascites Krebs-2 have been comprehensively analyzed. Investigation of the extracellular DNA internalization into tumor-initiating stem cells is an important part of understanding their properties and possible destruction mechanisms. For example, a TAMRA-labeled DNA probe may serve as an instrument to develop a target for the therapy of cancer, aiming at elimination of

  15. Notochord manipulation does not impact oesophageal and tracheal formation from isolated foregut in 3D explant culture.

    Science.gov (United States)

    Mc Laughlin, Danielle; Murphy, Paula; Puri, Prem

    2016-01-01

    Tracheo-oesophageal malformations result from disturbed foregut separation during early development. The notochord, a specialised embryonic structure, forms immediately adjacent to the dividing foregut. In the Adriamycin mouse model of oesophageal atresia, foregut and notochord abnormalities co-exist, and the site and severity of foregut malformations closely correlate to the position and extent of the notochord defects. Notochord and foregut abnormalities also co-exist in the Noggin Knockout mouse as well in a small number of human cases. The notochord is a source of powerful molecular signals during early embryogenesis, being particularly important for neural crest development. The influence of notochord signaling on the adjacent foregut is not known. The purpose of this study was to examine the impact of notochord manipulation on foregut separation using a robust 3D explant method for culturing isolated foregut which permits oeosphageal and tracheal formation in vitro. Foregut was micro-dissected from embryonic day 9 mice (License B100/4447 Irish Medicines Board), embedded in collagen and cultured for 48 h with native notochord intact (n = 6), notochord removed (n = 10) or additional notochord transplanted from stage matched controls (n = 8). Specimens were analysed for foregut morphology and molecular patterning using immunohistochemistry for Hnf3b (an endoderm marker) and Sox2 (a notochord and oesophageal marker) on cryosections. Foregut separation into distinct oesophagus and trachea was observed in isolated foregut specimens with or without their native notochord. In specimens with additional notochord transplants, foregut morphology and molecular patterning were comparable to controls whether or not the native notochord was maintained. In particular foregut separation was not disrupted by the transplantation of additional notochord at the dorsal foregut endoderm. The relationship between the embryonic foregut and notochord is complex and ill

  16. Self-etching adhesive on intact enamel, with and without pre-etching.

    Science.gov (United States)

    Devarasa, G M; Subba Reddy, V V; Chaitra, N L; Swarna, Y M

    2012-05-01

    Bond strengths of composite resin to enamel using self-etch adhesive (SEA) Clearfil SE bond system on intact enamel and enamel pre-etched with phosphoric acid were compared. The objective was to determine if the pre-etching would increase the bond strengths of the SEA systems to intact enamel and to evaluate the effect of pre-etching on bond formation of self-etch adhesives on intact enamel. Labial surfaces of 40 caries free permanent upper central and lateral incisors were cleaned, sectioned of their roots. All specimens were mounted on acrylic block and divided randomly into four groups. In two groups the application of self-etch adhesive, Clearfil SE bond was carried as per manufacturer's instructions, composite cylinders were built, whereas in the other two groups, 37% phosphoric acid etching was done before the application of self-etching adhesives. Then the resin tags were analyzed using scanning electron microscope and shear bond strength was measured using Instron universal testing machine. When phosphoric acid was used, there was significant increase in the depth of penetration of resin tags and in the Shear Bond Strength of composite to enamel. The results indicate that out of both treatment groups, pre-etching the intact enamel with 37% phosphoric acid resulted in formation of longer resin tags and higher depth of penetration of resin tags of the Clearfil SE bond, and attaining higher bond strength of the Clearfil SE bond to intact enamel. Copyright © 2011 Wiley Periodicals, Inc.

  17. Axenic isolation of viable Giardia muris trophozoites.

    Science.gov (United States)

    Tillotson, K D; Buret, A; Olson, M E

    1991-06-01

    Large numbers of viable Giardia muris trophozoites were isolated from the duodenum of experimentally infected mice 6 days after inoculation with 1,000 G. muris cysts. A series of shaking, incubation, and washing steps in the presence of the broad-spectrum antibiotic piperacillin readily provided 4.9 +/- 1.5 x 10(5) G. muris trophozoites per mouse, free of detectable contaminant organisms. Anaerobic and microaerophilic culturing and scanning electron microscopy demonstrated axenic status and high purity of the isolates. The viability of trophozoites was 98 +/- 2%. Application of this technique should permit novel immunological and epidemiological analyses of G. muris infection and biochemical investigations of this protozoan parasite.

  18. Formation of DNA adducts in mouse tissues after 1-nitropyrene administration

    International Nuclear Information System (INIS)

    Mitchell, C.E.

    1986-01-01

    DNA adducts were isolated and characterized in mouse lung, liver and kidney after intratracheal instillation of [ 3 H]-1-nitropyrene (1-NP). HPLC analysis of the enzymatically digested DNA indicated the presence of multiple DNA adducts in mouse lung, liver and kidney. These results indicate that DNA adducts of 1-NP are formed in mouse lung, liver and kidney after intratracheal instillation of 1-NP; the HPLC profiles of the multiple adducts suggests that adducts may be formed via metabolic pathways that involve both nitroreduction and ring-oxidation. 6 references, 1 figure

  19. Quantitative Analysis of Human Salivary Gland-Derived Intact Proteome Using Top-Down Mass Spectrometry

    Energy Technology Data Exchange (ETDEWEB)

    Wu, Si; Brown, Joseph N.; Tolic, Nikola; Meng, Da; Liu, Xiaowen; Zhang, Haizhen; Zhao, Rui; Moore, Ronald J.; Pevzner, Pavel A.; Smith, Richard D.; Pasa-Tolic, Ljiljana

    2014-05-31

    There are several notable challenges inherent to fully characterizing the entirety of the human saliva proteome using bottom-up approaches, including polymorphic isoforms, post-translational modifications, unique splice variants, deletions, and truncations. To address these challenges, we have developed a top-down based liquid chromatography-mass spectrometry (LC-MS) approach, which cataloged 20 major human salivary proteins with a total of 83 proteoforms, containing a broad range of post-translational modifications. Among these proteins, several previously reported disease biomarker proteins were identified at the intact protein level, such as beta-2 microglobulin (B2M). In addition, intact glycosylated proteoforms of several saliva proteins were also characterized, including intact N-glycosylated protein prolactin inducible protein (PIP) and O-glycosylated acidic protein rich protein (aPRP). These characterized proteoforms constitute an intact saliva proteoform database, which was used for quantitative comparison of intact salivary proteoforms among six healthy individuals. Human parotid (PS) and submandibular/sublingual gland (SMSL) secretion samples (2 μg of protein each) from six healthy individuals were compared using RPLC coupled with the 12T FTICR mass spectrometer. Significantly different protein and PTM patterns were resolved with high reproducibility between PS and SMSL glands. The results from this study provide further insight into the potential mechanisms of PTM pathways in oral glandular secretion, expanding our knowledge of this complex yet easily accessible fluid. Intact protein LC-MS approach presented herein can potentially be applied for rapid and accurate identification of biomarkers from only a few microliters of human glandular saliva.

  20. Neural activation differences in amputees during imitation of intact versus amputee movements

    Directory of Open Access Journals (Sweden)

    William F Cusack

    2012-06-01

    Full Text Available The mirror neuron system has been attributed with increased activation in motor-related cortical areas upon viewing of another’s actions. Recent work suggests that limb movements that are similar and dissimilar in appearance to that of the viewer equivalently activate the mirror neuron system. It is unclear if this result can be observed in the action encoding areas in amputees who use prosthetic devices. Intact subjects and upper extremity amputee prosthesis users were recruited to view video demonstrations of tools being used by an intact actor and a prosthetic device user. All subjects were asked to pantomime the movements seen in the video while recording electroencephalography. Intact subjects showed equivalent left parietofrontal activity during imitation after watching the intact or prosthetic arm. Likewise, when prosthesis users imitated prosthesis demonstrations, typical left parietofrontal activation was observed during planning. When prosthesis users imitated intact actors, a new pattern was revealed which showed greater bilateral parietal and occipital activity during movement planning (p<0.001. This change may be required for prosthesis users to imitate movements in which the limb states between the observed and the observer do not match. The finding that prosthesis users imitating other prosthesis users showed typical left parietofrontal activation suggests that these subjects engage normal planning related activity when they are able to imitate a limb matching their own. This result has significant implications on rehabilitation, as standard therapy involves training with an intact occupational therapist, which could necessitate atypical planning mechanisms in amputees when learning to use their prosthesis.

  1. Losing a jewel—Rapid declines in Myanmar’s intact forests from 2002-2014

    Science.gov (United States)

    Horning, Ned; Khaing, Thiri; Thein, Zaw Min; Aung, Kyaw Moe; Aung, Kyaw Htet; Phyo, Paing; Tun, Ye Lin; Oo, Aung Htat; Neil, Anthony; Thu, Win Myo; Songer, Melissa; Huang, Qiongyu; Connette, Grant; Leimgruber, Peter

    2017-01-01

    New and rapid political and economic changes in Myanmar are increasing the pressures on the country’s forests. Yet, little is known about the past and current condition of these forests and how fast they are declining. We mapped forest cover in Myanmar through a consortium of international organizations and environmental non-governmental groups, using freely-available public domain data and open source software tools. We used Landsat satellite imagery to assess the condition and spatial distribution of Myanmar’s intact and degraded forests with special focus on changes in intact forest between 2002 and 2014. We found that forests cover 42,365,729 ha or 63% of Myanmar, making it one of the most forested countries in the region. However, severe logging, expanding plantations, and degradation pose increasing threats. Only 38% of the country’s forests can be considered intact with canopy cover >80%. Between 2002 and 2014, intact forests declined at a rate of 0.94% annually, totaling more than 2 million ha forest loss. Losses can be extremely high locally and we identified 9 townships as forest conversion hotspots. We also delineated 13 large (>100,000 ha) and contiguous intact forest landscapes, which are dispersed across Myanmar. The Northern Forest Complex supports four of these landscapes, totaling over 6.1 million ha of intact forest, followed by the Southern Forest Complex with three landscapes, comprising 1.5 million ha. These remaining contiguous forest landscape should have high priority for protection. Our project demonstrates how open source data and software can be used to develop and share critical information on forests when such data are not readily available elsewhere. We provide all data, code, and outputs freely via the internet at (for scripts: https://bitbucket.org/rsbiodiv/; for the data: http://geonode.themimu.info/layers/geonode%3Amyan_lvl2_smoothed_dec2015_resamp) PMID:28520726

  2. Isotope inequilibrium of glucose metabolites in intact cells and particlefree supernatants of Ehrlich ascites tumor

    International Nuclear Information System (INIS)

    Daehnfeldt, J.L.; Winge, P.

    1975-01-01

    With an enzyme degradative technique, isotope inequilibrium of glucose metabolites was demonstrated in intact cells and particle-free supernatants of Ehrlich ascites tumor using I- 14 C-glucose as tracer. Inequilibrium was found between glucose and glucose-6-phosphate, glucose and fructose-6-phosphate, glucose and 6-phosphogluconate, while glucose-6-phosphate and fructose-6-phosphate were found to be in near equilibrium within the incubation time investigated. Glucose and lactate were found to be in near equilibrium after 8 min in intact cells. Calculations based on the equilibrium levels found, showed that these inequilibria could not be explained by the effects of the pentose cycle. (U.S.)

  3. Boron-11 MRI and MRS of intact animals infused with a boron neutron capture agent

    International Nuclear Information System (INIS)

    Kabalka, G.W.; Davis, M.; Bendel, P.

    1988-01-01

    Boron neutron capture therapy (BNCT) depends on the delivery of boron-containing drugs to a targeted lesion. Currently, the verification and quantification of in vivo boron content is a difficult problem. Boron-11 spectroscopy was utilized to confirm the presence of a dimeric sulfhydryl dodecaborane BNCT agent contained in an intact animal. Spectroscopy experiments revealed that the decay time of transverse magnetization of the boron-11 spins was less than 1 ms which precluded the use of a 2DFT imaging protocol. A back-projection protocol was developed and utilized to generate the first boron-11 image of a BNCT agent in the liver of an intact Fisher 344 rat

  4. Clonal outbreaks of [ Pasteurella] pneumotropica biovar Heyl in two mouse colonies

    DEFF Research Database (Denmark)

    Adhikary, Sadhana; Bisgaard, Magne; Dagnæs-Hansen, Frederik

    2017-01-01

    The aim of this study was to document the pathogenic role of biovar Heyl of [Pasteurella] pneumotropica in mouse colonies. Fifty-three isolates associated with mastitis and orbital, cutaneous and vaginal abscesses as well as isolates from the nose and vagina of healthy mice were investigated...... strains with the same rpoB sequence type, as shown by the PFGE profiles. The investigation documented that members of biovar Heyl of [P.] pneumotropica caused disease outbreaks in mouse colonies since the clonality indicated a primary role of [P.] pneumotropica biovar Heyl in the infections observed....

  5. The impact of family intactness on family functioning, parental control and parent-child relational qualities in a Chinese context

    Directory of Open Access Journals (Sweden)

    Daniel Tan Lei Shek

    2015-01-01

    Full Text Available The current study investigated the differences between intact and non-intact families in family processes, including systematic family functioning, parental behavioral control, parental psychological control, and parent-child relational qualities. The participants were 3,328 Secondary One students, with a mean age of 12.59 years, recruited from 28 secondary schools in Hong Kong. Four validated scales were used to assess family processes. Results showed that adolescents in non-intact families perceived relatively poorer family functioning, lower level of paternal and maternal behavioral control, lower level of paternal psychological control and poorer parent-child relational qualities than did adolescents in intact families. This generally indicated that family processes were poorer in non-intact families, compared with those in intact families. The theoretical and practical implications of the findings were discussed.

  6. Isolation of plasmodesmata from Arabidopsis suspension culture cells.

    Science.gov (United States)

    Grison, Magali S; Fernandez-Calvino, Lourdes; Mongrand, Sébastien; Bayer, Emmanuelle M F

    2015-01-01

    Due to their position firmly anchored within the plant cell wall, plasmodesmata (PD) are notoriously difficult to isolate from plant tissue. Yet, getting access to isolated PD represents the most straightforward strategy for the identification of their molecular components. Proteomic and lipidomic analyses of such PD fractions have provided and will continue to provide critical information on the functional and structural elements that define these membranous nano-pores. Here, we describe a two-step simple purification procedure that allows isolation of pure PD-derived membranes from Arabidopsis suspension cells. The first step of this procedure consists in isolating cell wall fragments containing intact PD while free of contamination from other cellular compartments. The second step relies on an enzymatic degradation of the wall matrix and the subsequent release of "free" PD. Isolated PD membranes provide a suitable starting material for the analysis of PD-associated proteins and lipids.

  7. Chronic pretrigeminal and cerveau isolé cats.

    Science.gov (United States)

    Slósarska, M; Zernicki, B

    1973-01-01

    Ten pretrigeminal and ten cerveau isole cats were observed chronically. During 24-36 h sessions EEG activity was continuously recorded and the EEG and ocular responses to visual and olfactory stimuli were studied. In the pretrigeminal cat acute and chronic stages were distinguished, and in the cerveau isole, acute, "early chronic" and "late chronic" stages. During the acute stage, the pretrigeminal cat is continuously awake, whereas the cerveau isole is comatose. During the "early chronic stage", which lasted at least about 3 weeks, the cerveau isole is semicomatose. During the chronic stage in the pretrigeminal cat and the "late chronic stage" in the cerveau isole, the sleep-waking cycle is present. In both preparations alert wakefulness, drowsiness, light .synchronized sleep and deep synchronized sleep occupy, respectively, about 30 percent, 45 percent, 15 percent and 10 percent of the time. Thus, synchronized sleep is strikingly reduced in comparison with an intact cat, while desynchronized sleep is absent.

  8. Genetic analysis of radiation-induced mouse thymic lymphomas

    International Nuclear Information System (INIS)

    Kominami, R.; Wakabayashi, Y.; Niwa, O.

    2003-01-01

    Mouse thymic lymphomas are one of the classic models of radiation-induced malignancies, and the model has been used for the study of genes involved in carcinogenesis. ras oncogenes are the first isolate which undergoes mutations in 10 to 30 % of lymphomas, and p16INK4a and p19ARF in the INK4a-ARF locus are also frequently inactivated. In our previous study, the inactivation of Ikaros, a key regurator of lymphoid system, was found in those lymphomas, and it was suggested that there are other responsible genes yet to be discovered. On the other hand, genetic predisposition to radiation-induced lymphoma often differs in different strains, and this reflects the presence of low penetrance genes that can modify the impact of a given mutation. Little study of such modifiers or susceptibility genes has been performed, either. Recent availability of databases on mouse genome information and the power of mouse genetic system underline usefulness of the lymphoma model in search for novel genes involved, which may provide clues to molecular mechanisms of development of the radiogenic lymphoma and also genes involved in human lymphomas and other malignancies. Accordingly, we have carried out positional cloning for the two different types of tumor-related genes. In this symposium, our current progress is presented that includes genetic mapping of susceptibility/ resistance loci on mouse chromosomes 4, 5 and 19, and also functional analysis of a novel tumor suppressor gene, Rit1/Bcl11b, that has been isolated from allelic loss (LOH) mapping and sequence analysis for γ -ray induced mouse thymic lymphomas

  9. Isolation of nucleoli from Ehrlich ascites tumor cells and dynamics of nascent RNA within isolated nucleoli.

    Science.gov (United States)

    Thiry, Marc; Ploton, Dominique

    2008-01-01

    Here we describe a new, rapid method for isolating nucleoli from Ehrlich tumor cells that preserves their morphological integrity and high transcriptional activity. Until now, methods for isolation of nucleoli were generally assumed to empty one of their three main compartments, the fibrillar center, of its contents. This new method consists of sonicating cells in an isotonic medium containing MgSO(4), spermidine, and spermine, followed by separation of nucleoli through a Percoll density gradient. Using the nonisotopic approach of labelling with BrUTP, we have further investigated the dynamics of nascent ribosomal RNAs (rRNAs) within morphologically intact isolated nucleoli at the electron microscope level. We show that ribosomal transcripts are elongated in the cortex of the fibrillar center and then enter the surrounding dense fibrillar component.

  10. Individual tree detection in intact forest and degraded forest areas in the north region of Mato Grosso State, Brazilian Amazon

    Science.gov (United States)

    Santos, E. G.; Jorge, A.; Shimabukuro, Y. E.; Gasparini, K.

    2017-12-01

    The State of Mato Grosso - MT has the second largest area with degraded forest among the states of the Brazilian Legal Amazon. Land use and land cover change processes that occur in this region cause the loss of forest biomass, releasing greenhouse gases that contribute to the increase of temperature on earth. These degraded forest areas lose biomass according to the intensity and magnitude of the degradation type. The estimate of forest biomass, commonly performed by forest inventory through sample plots, shows high variance in degraded forest areas. Due to this variance and complexity of tropical forests, the aim of this work was to estimate forest biomass using LiDAR point clouds in three distinct forest areas: one degraded by fire, another by selective logging and one area of intact forest. The approach applied in these areas was the Individual Tree Detection (ITD). To isolate the trees, we generated Canopy Height Models (CHM) images, which are obtained by subtracting the Digital Elevation Model (MDE) and the Digital Terrain Model (MDT), created by the cloud of LiDAR points. The trees in the CHM images are isolated by an algorithm provided by the Quantitative Ecology research group at the School of Forestry at Northern Arizona University (SILVA, 2015). With these points, metrics were calculated for some areas, which were used in the model of biomass estimation. The methodology used in this work was expected to reduce the error in biomass estimate in the study area. The cloud points of the most representative trees were analyzed, and thus field data was correlated with the individual trees found by the proposed algorithm. In a pilot study, the proposed methodology was applied generating the individual tree metrics: total height and area of the crown. When correlating 339 isolated trees, an unsatisfactory R² was obtained, as heights found by the algorithm were lower than those obtained in the field, with an average difference of 2.43 m. This shows that the

  11. Metabolic Characterization of Intact Cells Reveals Intracellular Amyloid Beta but Not Its Precursor Protein to Reduce Mitochondrial Respiration

    Science.gov (United States)

    Schaefer, Patrick M.; von Einem, Bjoern; Walther, Paul; Calzia, Enrico; von Arnim, Christine A. F.

    2016-01-01

    One hallmark of Alzheimer´s disease are senile plaques consisting of amyloid beta (Aβ), which derives from the processing of the amyloid precursor protein (APP). Mitochondrial dysfunction has been linked to the pathogenesis of Alzheimer´s disease and both Aβ and APP have been reported to affect mitochondrial function in isolated systems. However, in intact cells, considering a physiological localization of APP and Aβ, it is pending what triggers the mitochondrial defect. Thus, the aim of this study was to dissect the impact of APP versus Aβ in inducing mitochondrial alterations with respect to their subcellular localization. We performed an overexpression of APP or beta-site amyloid precursor protein cleaving enzyme 1 (BACE1), increasing APP and Aβ levels or Aβ alone, respectively. Conducting a comprehensive metabolic characterization we demonstrate that only APP overexpression reduced mitochondrial respiration, despite lower extracellular Aβ levels compared to BACE overexpression. Surprisingly, this could be rescued by a gamma secretase inhibitor, oppositionally indicating an Aβ-mediated mitochondrial toxicity. Analyzing Aβ localization revealed that intracellular levels of Aβ and an increased spatial association of APP/Aβ with mitochondria are associated with reduced mitochondrial respiration. Thus, our data provide marked evidence for a prominent role of intracellular Aβ accumulation in Alzheimer´s disease associated mitochondrial dysfunction. Thereby it highlights the importance of the localization of APP processing and intracellular transport as a decisive factor for mitochondrial function, linking two prominent hallmarks of neurodegenerative diseases. PMID:28005987

  12. A non mouse-adapted dengue virus strain as a new model of severe dengue infection in AG129 mice.

    Directory of Open Access Journals (Sweden)

    Grace K Tan

    Full Text Available The spread of dengue (DEN worldwide combined with an increased severity of the DEN-associated clinical outcomes have made this mosquito-borne virus of great global public health importance. Progress in understanding DEN pathogenesis and in developing effective treatments has been hampered by the lack of a suitable small animal model. Most of the DEN clinical isolates and cell culture-passaged DEN virus strains reported so far require either host adaptation, inoculation with a high dose and/or intravenous administration to elicit a virulent phenotype in mice which results, at best, in a productive infection with no, few, or irrelevant disease manifestations, and with mice dying within few days at the peak of viremia. Here we describe a non-mouse-adapted DEN2 virus strain (D2Y98P that is highly infectious in AG129 mice (lacking interferon-alpha/beta and -gamma receptors upon intraperitoneal administration. Infection with a high dose of D2Y98P induced cytokine storm, massive organ damage, and severe vascular leakage, leading to haemorrhage and rapid death of the animals at the peak of viremia. In contrast, very interestingly and uniquely, infection with a low dose of D2Y98P led to asymptomatic viral dissemination and replication in relevant organs, followed by non-paralytic death of the animals few days after virus clearance, similar to the disease kinetic in humans. Spleen damage, liver dysfunction and increased vascular permeability, but no haemorrhage, were observed in moribund animals, suggesting intact vascular integrity, a cardinal feature in DEN shock syndrome. Infection with D2Y98P thus offers the opportunity to further decipher some of the aspects of dengue pathogenesis and provides a new platform for drug and vaccine testing.

  13. Fertility, pregnancy, and delivery after biventricular repair for pulmonary atresia with an intact ventricular septum

    NARCIS (Netherlands)

    Drenthen, Willem; Pieper, Petronella G.; Roos-Hesselink, Jollen W.; Zoon, Nicole; Voors, Adrlaan A.; Mulder, Barbara J. M.; van Dijk, Arie P. J.; Vliegen, Hubert W.; Sollie, Krystyna M.; Ebels, Tjark; van Veldhuisen, Dirk J.

    2006-01-01

    The objective of the present study to investigate fertility, pregnancy, and delivery in women with biventricular repair for pulmonary atresia with an intact ventricular septum (PAIVS). Using a nationwide registry (CONCOR), 37 patients with pulmonary atresia were identified, 6 of whom (aged 21 to 34

  14. Fertility, pregnancy, and delivery after biventricular repair for pulmonary atresia with an intact ventricular septum.

    NARCIS (Netherlands)

    Drenthen, W.; Pieper, P.G.; Roos-Hesselink, J.W.; Zoon, N.; Voors, A.A.; Mulder, B.J.M.; Dijk, A.P.J. van; Vliegen, H.W.; Sollie, K.M.; Ebels, T.; Veldhuisen, D.J. van

    2006-01-01

    The objective of the present study to investigate fertility, pregnancy, and delivery in women with biventricular repair for pulmonary atresia with an intact ventricular septum (PAIVS). Using a nationwide registry (CONCOR), 37 patients with pulmonary atresia were identified, 6 of whom (aged 21 to 34

  15. Family Conflict and Children's Self-Concepts: A Comparison of Intact and Single-Parent Families.

    Science.gov (United States)

    Raschke, Helen J.; Raschke, Vernon J.

    1979-01-01

    Using the Piers-Harris Children's Self-Concept Scale to measure self-concept, and self-reports for family structure and family conflict, no significant differences in self-concept scores of children from intact, single-parent, reconstituted, or other types of families were found. Self-concept scores were significantly lower for children reporting…

  16. Family Relationships and the Psychosocial Adjustment of School-Aged Children in Intact Families

    Science.gov (United States)

    Hakvoort, Esther M.; Bos, Henny M. W.; Van Balen, Frank; Hermanns, Jo M. A.

    2010-01-01

    The authors investigated whether the quality of three family relationships (i.e., marital, parent-child, sibling) in intact families are associated with each other and with children's psychosocial adjustment. Data were collected by means of maternal and child reports (N = 88) using standardized instruments (i.e., Marital Satisfaction Scale,…

  17. 46 CFR 173.020 - Intact stability standards: Counterballasted and non-counterballasted vessels.

    Science.gov (United States)

    2010-10-01

    ...) SUBDIVISION AND STABILITY SPECIAL RULES PERTAINING TO VESSEL USE Lifting § 173.020 Intact stability standards... vessel that is not equipped to counter-ballast while lifting must be shown by design calculations to... and crane radius. (b) Each vessel must have a righting arm curve with the following characteristics...

  18. 46 CFR 173.025 - Additional intact stability standards: Counterballasted vessels.

    Science.gov (United States)

    2010-10-01

    ...) SUBDIVISION AND STABILITY SPECIAL RULES PERTAINING TO VESSEL USE Lifting § 173.025 Additional intact stability standards: Counterballasted vessels. (a) Each vessel equipped to counterballast while lifting must be shown... loading and operation and at each combination of hook load and crane radius. (b) When doing the...

  19. Detection of intact megadalton protein assemblies of vanillyl-alcohol oxidase by mass spectrometry

    NARCIS (Netherlands)

    Berkel, van W.J.H.; Heuvel, van den R.H.H.; Versluis, C.; Heck, A.

    2000-01-01

    Well-resolved ion signals of intact large protein assemblies, with molecular masses extending above one million Dalton, have been detected and mass analyzed using electrospray ionization mass spectrometry, with an uncertainty in mass of <0.2&Eth;The mass spectral data seem to reflect known

  20. Prevalence of depressive symptoms in older nursing home residents with intact cognitive function in Taiwan.

    Science.gov (United States)

    Hu, Sophia H; Chuang, Yeu-Hui; Ting, Yeh-Feng; Lin, Kuan-Yu; Hsieh, Chia-Jung

    2018-03-25

    The investigators aimed to explore the prevalence of depressive symptoms and associated factors among older residents with intact cognitive function in nursing homes in Taiwan. A cross-sectional descriptive and correlational research design was used. A convenience sample of 178 older residents without cognitive impairment was recruited from 36 nursing homes in Southern Taiwan. The questionnaires included demographic data; the Barthel Index, which assesses the ability to perform activities of daily living; and the Geriatric Depression Scale Short Form. Among older residents in nursing homes with intact cognitive function, 39.3% had depressive symptoms. Age, religion, previous living status, previous working status, being totally dependent in physical function, and being severely dependent in physical function were significant predictors of depressive symptoms among cognitively intact older residents. The findings highlight the critical mental healthcare issues among older residents with intact cognitive function in nursing homes. Practical strategies for preventing the occurrence of depressive symptoms and caring for those who have depressive symptoms should be developed, especially for younger or dependent older residents or residents who have never been employed, have no religious beliefs, or have lived alone before they moved into an institution. © 2018 Wiley Periodicals, Inc.

  1. Proceedings of the international workshop on mechanistic understanding of radionuclide migration in compacted/intact systems

    International Nuclear Information System (INIS)

    Tachi, Yukio; Yui, Mikazu

    2010-03-01

    The international workshop on mechanistic understanding of radionuclide migration in compacted / intact systems was held at ENTRY, JAEA, Tokai on 21st - 23rd January, 2009. This workshop was hosted by Japan Atomic Energy Agency (JAEA) as part of the project on the mechanistic model/database development for radionuclide sorption and diffusion behavior in compacted / intact systems. The overall goal of the project is to develop the mechanistic model / database for a consistent understanding and prediction of migration parameters and its uncertainties for performance assessment of geological disposal of radioactive waste. The objective of the workshop is to integrate the state-of-the-art of mechanistic sorption and diffusion model in compacted / intact systems, especially in bentonite / clay systems, and discuss the JAEA's mechanistic approaches and future challenges, especially the following discussions points; 1) What's the status and difficulties for mechanistic model/database development? 2) What's the status and difficulties for applicability of mechanistic model to the compacted/intact system? 3) What's the status and difficulties for obtaining evidences for mechanistic model? 4) What's the status and difficulties for standardization of experimental methodology for batch sorption and diffusion? 5) What's the uncertainties of transport parameters in radionuclides migration analysis due to a lack of understanding/experimental methodologies, and how do we derive them? This report includes workshop program, overview and materials of each presentation, summary of discussions. (author)

  2. The action of cobra venom phospholipase A2 isoenzymes towards intact human erythrocytes

    NARCIS (Netherlands)

    Roelofsen, B.; Sibenius Trip, M.; Verheij, H.M.; Zevenbergen, J.L.

    1980-01-01

    1. 1. Cobra venom phospholipase A2 from three different sources has been fractionated into different isoenzymes by DEAE ion-exchange chromatography. 2. 2. Treatment of intact human erythrocytes with the various isoenzymes revealed significant differences in the degree of phosphatidylcholine

  3. Collagen fibril size and crimp morphology in ruptured and intact Achilles tendons

    DEFF Research Database (Denmark)

    Magnusson, S P; Qvortrup, K; Larsen, Jytte Overgaard

    2002-01-01

    tendons. Crimp angle did not display any region-specific differences, or any difference between the rupture and intact tendons. In conclusion, these data suggest that although crimp morphology is unchanged there appears to be a site-specific loss of larger fibrils in the core and periphery of the Achilles...

  4. Mechanisms of blood pressure changes following renal irradiation of intact, adrenalectomized and adrenal regenerating rats

    International Nuclear Information System (INIS)

    Rosenblum, M.

    1976-01-01

    Results are reported from studies on the differences in changes in systolic arterial blood pressure following renal x-irradiation (1100 R) in adrenal-intact, adrenalectomized, and adrenal-regenerating rats and the roles of the kidneys and of the adrenal glands in the blood pressure changes

  5. Spectrophotometric Evaluation of the Pulpal Peroxide Levels in Intact and Restored Teeth - An Invitro Study.

    Science.gov (United States)

    Patri, Gaurav; Acharya, Gourismita; Agrawal, Pratik; Panda, Vijeta

    2016-08-01

    Hydrogen peroxide (30%) is a commonly used "in office" bleaching agent. Deleterious effects of hydrogen peroxide on the pulp have been observed. The present study was conducted with the aim to evaluate the penetration of 30% hydrogen peroxide into the pulp chamber through intact teeth and through the surface of teeth, restored with either hybrid composite or Resin Modified Glass Ionomer Cement (RMGIC). Sixty extracted human maxillary central incisors were selected and divided into six groups. Two groups were restored with hybrid composite resin and two with RMGIC, while two groups were left intact. The teeth with acetate buffer solution in their pulp cavity were then immersed in either 30% hydrogen peroxide or distilled water depending upon the group, for 60 minutes at 37°C. Then horseradish peroxidase and leucocrystal violet were added to the acetate buffer solution present in the pulp chamber after it was transferred to a test tube and the optical density of the resultant blue solution obtained was measured spectrophotometrically. The data obtained were analyzed using one way ANOVA and Student's t-test. The data obtained established that hydrogen peroxide penetrated into the pulp from the bleaching agent used. Hydrogen peroxide (30%) showed the highest pulpal peroxide level in teeth restored with RMGIC followed by teeth restored with hybrid composite resin and the least amount of penetration was observed in intact teeth. The amount of peroxide penetration into the tooth is more through restored tooth than intact tooth and is also dependant on the type of restorative materials used.

  6. Decubitus grade IV (deep pressure sore) with intact skin in a patient with spinal cord injury

    NARCIS (Netherlands)

    Theunissen, C.C.W.; Zeilstra, J.T.; van Voorst Vader, P.C.; Kardaun, S.H.; Leeman, F.W.J.

    2006-01-01

    Even with intact skin the possibility of pressure sores should not be dismissed. Early recognition of a pressure sore is important for adequate treatment and prevention of progression. Multidisciplinary intervention is essential. A wheelchair patient with spinal cord injury is described, who

  7. Family relationships and the psychosocial adjustment of school-aged children in intact families

    NARCIS (Netherlands)

    Hakvoort, E.M.; Bos, H.M.W.; van Balen, F.; Hermanns, J.M.A.

    2010-01-01

    The authors investigated whether the quality of three family relationships (i.e., marital, parent-child, sibling) in intact families are associated with each other and with children's psychosocial adjustment. Data were collected by means of maternal and child reports (N = 88) using standardized

  8. Effects of radiation quality on the opening of stomata in intact Phaseolus vulgaris leaves

    International Nuclear Information System (INIS)

    Sikorska, K.; Kozłowska, B.; Ciereszko, I.; Maleszewski, S.

    1997-01-01

    In intact French bean (Phaseolus vulgaris L.) leaves blue radiation enhanced opening of stomata both when it was used individually and when it was used as preirradiation before ''white light'' irradiation. Effects of red radiation were just the contrary

  9. Predictors of Attachment Security in Preschool Children from Intact and Divorced Families

    Science.gov (United States)

    Nair, Hira; Murray, Ann D.

    2005-01-01

    The authors selected 58 mother-child dyads from divorced and intact families to participate in a study on the impact of divorce on preschoolers' attachment security. The authors explored pathways that lead to security of attachment. They found that mothers from divorced families were younger, had lower income levels, and had lower levels of…

  10. Aboveground biomass variability across intact and degraded forests in the Brazilian Amazon

    Science.gov (United States)

    Marcos Longo; Michael Keller; Maiza N. dos-Santos; Veronika Leitold; Ekena R. Pinagé; Alessandro Baccini; Sassan Saatchi; Euler M. Nogueira; Mateus Batistella; Douglas C. Morton

    2016-01-01

    Deforestation rates have declined in the Brazilian Amazon since 2005, yet degradation from logging, fire, and fragmentation has continued in frontier forests. In this study we quantified the aboveground carbon density (ACD) in intact and degraded forests using the largest data set of integrated forest inventory plots (n = 359) and airborne lidar data (18,000 ha)...

  11. Forest loss in protected areas and intact forest landscapes : A global analysis

    NARCIS (Netherlands)

    Heino, Matias; Kummu, Matti; Makkonen, Marika; Mulligan, Mark; Verburg, Peter H.; Jalava, Mika; Räsänen, Timo A.

    2015-01-01

    In spite of the high importance of forests, global forest loss has remained alarmingly high during the last decades. Forest loss at a global scale has been unveiled with increasingly finer spatial resolution, but the forest extent and loss in protected areas (PAs) and in large intact forest

  12. Using Spores for Fusarium spp. Classification by MALDI-Based Intact Cell/Spore Mass Spectrometry

    Directory of Open Access Journals (Sweden)

    Wolfgang Winkler

    2012-01-01

    Full Text Available Fusarium is a widespread genus of filamentous fungi and a member of the soil microbial community. Certain subspecies are health threatening because of their mycotoxin production that affects the human and animal food chain. Thus, for early and effective pest control, species identification is of particular interest; however, differentiation on the subspecies level is challenging and time-consuming for this fungus. In the present study, we show the possibilities of intact cell mass spectrometry for spore analysis of 22 different Fusarium strains belonging to six Fusarium subspecies. We found that species differentiation is possible if mass spectrometric analyses are performed under well-defined conditions with fixed parameters. A critical point for analysis is a proper sample preparation of spores, which increases the quality of mass spectra with respect to signal intensity and m/z value variations. It was concluded that data acquistion has to be performed automatically; otherwise, user-specific variations are introduced generating data which cannot fit the existing datasets. Data that show clearly that matrix-assisted laser desorption ionization-based intact cell/intact spore mass spectrometry (IC/ISMS can be applied to differentiate closely related Fusarium spp. are presented. Results show a potential to build a database on Fusarium species for accurate species identification, for fast response in the case of infections in the cornfield. We furthermore demonstrate the high precision of our approach in classification of intact Fusarium species according to the location of their collection.

  13. Tolerance of a standard intact protein formula versus a partially hydrolyzed formula in healthy, term infants

    Directory of Open Access Journals (Sweden)

    Marunycz John D

    2009-06-01

    Full Text Available Abstract Background Parents who perceive common infant behaviors as formula intolerance-related often switch formulas without consulting a health professional. Up to one-half of formula-fed infants experience a formula change during the first six months of life. Methods The objective of this study was to assess discontinuance due to study physician-assessed formula intolerance in healthy, term infants. Infants (335 were randomized to receive either a standard intact cow milk protein formula (INTACT or a partially hydrolyzed cow milk protein formula (PH in a 60 day non-inferiority trial. Discontinuance due to study physician-assessed formula intolerance was the primary outcome. Secondary outcomes included number of infants who discontinued for any reason, including parent-assessed. Results Formula intolerance between groups (INTACT, 12.3% vs. PH, 13.7% was similar for infants who completed the study or discontinued due to study physician-assessed formula intolerance. Overall study discontinuance based on parent- vs. study physician-assessed intolerance for all infants (14.4 vs.11.1% was significantly different (P = 0.001. Conclusion This study demonstrated no difference in infant tolerance of intact vs. partially hydrolyzed cow milk protein formulas for healthy, term infants over a 60-day feeding trial, suggesting nonstandard partially hydrolyzed formulas are not necessary as a first-choice for healthy infants. Parents frequently perceived infant behavior as formula intolerance, paralleling previous reports of unnecessary formula changes. Trial Registration clinicaltrials.gov: NCT00666120

  14. Operative findings of conductive hearing loss with intact tympanic membrane and normal temporal bone computed tomography.

    Science.gov (United States)

    Kim, Se-Hyung; Cho, Yang-Sun; Kim, Hye Jeong; Kim, Hyung-Jin

    2014-06-01

    Despite recent technological advances in diagnostic methods including imaging technology, it is often difficult to establish a preoperative diagnosis of conductive hearing loss (CHL) in patients with an intact tympanic membrane (TM). Especially, in patients with a normal temporal bone computed tomography (TBCT), preoperative diagnosis is more difficult. We investigated middle ear disorders encountered in patients with CHL involving an intact TM and normal TBCT. We also analyzed the surgical results with special reference to the pathology. We reviewed the medical records of 365 patients with intact TM, who underwent exploratory tympanotomy for CHL. Fifty nine patients (67 ears, eight bilateral surgeries) had a normal preoperative TBCT findings reported by neuro-radiologists. Demographic data, otologic history, TM findings, preoperative imaging findings, intraoperative findings, and pre- and postoperative audiologic data were obtained and analyzed. Exploration was performed most frequently in the second and fifth decades. The most common postoperative diagnosis was stapedial fixation with non-progressive hearing loss. The most commonly performed hearing-restoring procedure was stapedotomy with piston wire prosthesis insertion. Various types of hearing-restoring procedures during exploration resulted in effective hearing improvement, especially with better outcome in the ossicular chain fixation group. In patients with CHL who have intact TM and normal TBCT, we should consider an exploratory tympanotomy for exact diagnosis and hearing improvement. Information of the common operative findings from this study may help in preoperative counseling.

  15. Raman spectroscopy of normal oral buccal mucosa tissues: study on intact and incised biopsies

    Science.gov (United States)

    Deshmukh, Atul; Singh, S. P.; Chaturvedi, Pankaj; Krishna, C. Murali

    2011-12-01

    Oral squamous cell carcinoma is one of among the top 10 malignancies. Optical spectroscopy, including Raman, is being actively pursued as alternative/adjunct for cancer diagnosis. Earlier studies have demonstrated the feasibility of classifying normal, premalignant, and malignant oral ex vivo tissues. Spectral features showed predominance of lipids and proteins in normal and cancer conditions, respectively, which were attributed to membrane lipids and surface proteins. In view of recent developments in deep tissue Raman spectroscopy, we have recorded Raman spectra from superior and inferior surfaces of 10 normal oral tissues on intact, as well as incised, biopsies after separation of epithelium from connective tissue. Spectral variations and similarities among different groups were explored by unsupervised (principal component analysis) and supervised (linear discriminant analysis, factorial discriminant analysis) methodologies. Clusters of spectra from superior and inferior surfaces of intact tissues show a high overlap; whereas spectra from separated epithelium and connective tissue sections yielded clear clusters, though they also overlap on clusters of intact tissues. Spectra of all four groups of normal tissues gave exclusive clusters when tested against malignant spectra. Thus, this study demonstrates that spectra recorded from the superior surface of an intact tissue may have contributions from deeper layers but has no bearing from the classification of a malignant tissues point of view.

  16. Impact of location of CaCO3 precipitation on the development of intact anaerobic sludge

    NARCIS (Netherlands)

    Langerak, van E.P.A.; Ramaekers, H.; Wiechers, J.; Veeken, A.H.M.; Hamelers, H.V.M.; Lettinga, G.

    2000-01-01

    The development of intact anaerobic granular sludge was studied in UASB reactors under varying conditions of CaCO3 precipitation and biomass yield. Varying precipitating quantities were obtained using different calcium concentrations in the influent and different biomass yields were obtained by

  17. Biotransformation of the citrus flavone tangeretin in rats. Identification of metabolites with intact flavane nucleus

    DEFF Research Database (Denmark)

    Nielsen, S.E.; Breinholt, V.; Cornett, C.

    2000-01-01

    were separated and identified by HPLC and the structures elucidated by LC/MS and H-1 NMR. Ten new, major metabolites with intact flavonoid structure were identified. The metabolites identified were either demethylated or hydroxylated derivatives of the parent compound and metabolic changes were found...

  18. Automated setup for characterization of intact histone tails in Suz12-/- stem cells

    DEFF Research Database (Denmark)

    Sidoli, Simone; Schwämmle, Veit; Hansen, Thomas Aarup

    Epigenetics is defined as the study of heritable changes that occur without modifying the DNA sequence. Histone proteins are crucial components of epigenetic mechanisms and regulation, since they are fundamental for chromatin structure. Mass spectrometry-based proteomics is already an integrated...... developed a high-resolving and automated LC-MS/MS setup to characterize intact histone tails (middle-down strategy)...

  19. Assessment of characteristic failure envelopes for intact rock using results from triaxial tests

    OpenAIRE

    Muralha, J.; Lamas, L.

    2014-01-01

    The paper presents contributions to the statistical study of the parameters of the Mohr-Coulomb and Hoek-Brown strength criteria, in order to assess the characteristic failure envelopes for intact rock, based on the results of several sets of triaxial tests performed by LNEC. 10p DBB/NMMR

  20. Predicting water-holding capacity of intact chicken broiler breast fillets with Vis/NIR spectroscopy

    Science.gov (United States)

    The ability of using visible and near-infrared (Vis/NIR) spectroscopy to predict water-holding capacity (WHC) of intact chicken broiler breast fillets (pectoralis major) was assessed in this study. Boneless and skinless chicken fillets (214 in total) were procured from a commercial processing plant ...

  1. Secretion of intact proteins and peptide fragments by lysosomal pathways of protein degradation

    International Nuclear Information System (INIS)

    Isenman, L.D.; Dice, J.F.

    1989-01-01

    We report that degradation of proteins microinjected into human fibroblasts is accompanied by release into the culture medium of peptide fragments and intact proteins as well as single amino acids. For the nine proteins and polypeptides microinjected, acid-precipitable radioactivity, i.e. peptide fragments and/or intact proteins, ranged from 10 to 67% of the total released radioactivity. Peptide fragments and/or intact protein accounted for 60% of the radioactivity released into the medium by cells microinjected with ribonuclease A. Two major radiolabeled peptide fragments were found, and one was of an appropriate size to function as an antigen in antigen-presenting cells. The peptides released from microinjected ribonuclease A were derived from lysosomal pathways of proteolysis based on several lines of evidence. Previous studies have shown that microinjected ribonuclease A is degraded to single amino acids entirely within lysosomes. We show that release of free amino acids and peptide fragments and/or intact protein was equivalently stimulated by serum deprivation and equivalently inhibited by NH4Cl. We also show that lysosomal degradation of endocytosed [3H]ribonuclease A was accompanied by the release of two peptide fragments similar in size and charge to those from microinjected [ 3 H]ribonuclease A. These findings demonstrate that degradation within lysosomes occurs in a manner that spares specific peptides; they also suggest a previously unsuspected pathway by which cells can secrete cytosol-derived polypeptides

  2. Conservation experiments applying radiation-curable impregnating agents to intact and artifically decayed wood samples

    International Nuclear Information System (INIS)

    Schaudy, R.; Slais, E.

    1983-02-01

    Conservation experiments have been performed applying 10 selected impregnating agents to intact and chemically as well as biologically decayed wood samples. The quality of the radiation-curable impregnating agents could be valued by determination of the monomer uptake, the alteration of dimensions and volume and the deformation of the samples. The results are to be discussed. (Author) [de

  3. Transbilayer distribution and mobility of phosphatidylcholine in intact erythrocyte membranes. A study with phosphatidylcholine exchange protein

    NARCIS (Netherlands)

    van Meer, G.; Poorthuis, B. J.; Wirtz, K. W.; Op den Kamp, J. A.; van Deenen, L. L.

    1980-01-01

    1. The exchange of phosphatidylcholine between intact human or rat erythrocytes and rat liver microsomes was greatly stimulated by phosphatidylcholine-specific exchange proteins from rat liver and beef liver. It was found, however, that compared to the exchange reaction between phospholipid vesicles

  4. Transbilayer distribution and mobility of phosphatidylcholine in intact erythrocyte membranes. A study with phosphatidylcholine exchange protein

    NARCIS (Netherlands)

    van Meer, G.|info:eu-repo/dai/nl/068570368; Poorthuis, B.J.H.M.; Wirtz, K.W.A.|info:eu-repo/dai/nl/068427956; op den Kamp, J.A.F.; van Deenen, L.L.M.

    1980-01-01

    The exchange of phosphatidylcholine between intact human or rat erythrocytes and rat liver microsomes was greatly stimulated by phosphatidylcholine-specific exchange proteins from rat liver and beef liver. It was found, however, that compared to the exchange reaction between phospholipid vesicles

  5. The Fate of Dissolved Creosote Compounds in an Intact Fratured Clay Column

    DEFF Research Database (Denmark)

    Broholm, Kim; Arvin, Erik; Hansen, Asger

    1995-01-01

    The fate of 16 different organics typical for creosote was studied under aerobic conditions in a large intact fractured clay column experiment. Some of the organics (benzene, toluene, o-xylene, phenol, and o-cresol) were transported at the same rate as bromide through the fractured clay, whereas ...

  6. Isolation of Mycobacterium paratuberculosis from Milk by Immunomagnetic Separation

    OpenAIRE

    Grant, Irene R.; Ball, Hywel J.; Rowe, Michael T.

    1998-01-01

    An immunomagnetic separation (IMS) technique was developed to facilitate selective isolation of Mycobacterium paratuberculosis cells from milk. Rabbit polyclonal antibodies against radiation-killed intact M. paratuberculosis cells were produced and used to coat sheep anti-rabbit immunoglobulin G (IgG) type M-280 Dynabeads. The rabbit anti-M. paratuberculosis IgG-coated beads (IMB) reacted strongly with laboratory strains of M. paratuberculosis as determined by slide agglutination, and microsc...

  7. Aboveground Biomass Variability Across Intact and Degraded Forests in the Brazilian Amazon

    Science.gov (United States)

    Longo, Marcos; Keller, Michael; Dos-Santos, Maiza N.; Leitold, Veronika; Pinage, Ekena R.; Baccini, Alessandro; Saatchi, Sassan; Nogueira, Euler M.; Batistella, Mateus; Morton, Douglas C.

    2016-01-01

    Deforestation rates have declined in the Brazilian Amazon since 2005, yet degradation from logging, re, and fragmentation has continued in frontier forests. In this study we quantified the aboveground carbon density (ACD) in intact and degraded forests using the largest data set of integrated forest inventory plots (n 359) and airborne lidar data (18,000 ha) assembled to date for the Brazilian Amazon. We developed statistical models relating inventory ACD estimates to lidar metrics that explained70 of the variance across forest types. Airborne lidar-ACD estimates for intact forests ranged between 5.0 +/- 2.5 and 31.9 +/- 10.8 kg C m(exp -2). Degradation carbon losses were large and persistent. Sites that burned multiple times within a decade lost up to 15.0 +/- 0.7 kg C m(-2)(94%) of ACD. Forests that burned nearly15 years ago had between 4.1 +/- 0.5 and 6.8 +/- 0.3 kg C m(exp -2) (22-40%) less ACD than intact forests. Even for low-impact logging disturbances, ACD was between 0.7 +/- 0.3 and 4.4 +/- 0.4 kg C m(exp -2)(4-21%) lower than unlogged forests. Comparing biomass estimates from airborne lidar to existing biomass maps, we found that regional and pan-tropical products consistently overestimated ACD in degraded forests, under-estimated ACD in intact forests, and showed little sensitivity to res and logging. Fine-scale heterogeneity in ACD across intact and degraded forests highlights the benefits of airborne lidar for carbon mapping. Differences between airborne lidar and regional biomass maps underscore the need to improve and update biomass estimates for dynamic land use frontiers, to better characterize deforestation and degradation carbon emissions for regional carbon budgets and Reduce Emissions from Deforestation and forest Degradation(REDD+).

  8. Visual speech alters the discrimination and identification of non-intact auditory speech in children with hearing loss.

    Science.gov (United States)

    Jerger, Susan; Damian, Markus F; McAlpine, Rachel P; Abdi, Hervé

    2017-03-01

    Understanding spoken language is an audiovisual event that depends critically on the ability to discriminate and identify phonemes yet we have little evidence about the role of early auditory experience and visual speech on the development of these fundamental perceptual skills. Objectives of this research were to determine 1) how visual speech influences phoneme discrimination and identification; 2) whether visual speech influences these two processes in a like manner, such that discrimination predicts identification; and 3) how the degree of hearing loss affects this relationship. Such evidence is crucial for developing effective intervention strategies to mitigate the effects of hearing loss on language development. Participants were 58 children with early-onset sensorineural hearing loss (CHL, 53% girls, M = 9;4 yrs) and 58 children with normal hearing (CNH, 53% girls, M = 9;4 yrs). Test items were consonant-vowel (CV) syllables and nonwords with intact visual speech coupled to non-intact auditory speech (excised onsets) as, for example, an intact consonant/rhyme in the visual track (Baa or Baz) coupled to non-intact onset/rhyme in the auditory track (/-B/aa or/-B/az). The items started with an easy-to-speechread/B/or difficult-to-speechread/G/onset and were presented in the auditory (static face) vs. audiovisual (dynamic face) modes. We assessed discrimination for intact vs. non-intact different pairs (e.g., Baa:/-B/aa). We predicted that visual speech would cause the non-intact onset to be perceived as intact and would therefore generate more same-as opposed to different-responses in the audiovisual than auditory mode. We assessed identification by repetition of nonwords with non-intact onsets (e.g.,/-B/az). We predicted that visual speech would cause the non-intact onset to be perceived as intact and would therefore generate more Baz-as opposed to az- responses in the audiovisual than auditory mode. Performance in the audiovisual mode showed more same

  9. Visual Speech Alters the Discrimination and Identification of Non-Intact Auditory Speech in Children with Hearing Loss

    Science.gov (United States)

    Jerger, Susan; Damian, Markus F.; McAlpine, Rachel P.; Abdi, Hervé

    2017-01-01

    Objectives Understanding spoken language is an audiovisual event that depends critically on the ability to discriminate and identify phonemes yet we have little evidence about the role of early auditory experience and visual speech on the development of these fundamental perceptual skills. Objectives of this research were to determine 1) how visual speech influences phoneme discrimination and identification; 2) whether visual speech influences these two processes in a like manner, such that discrimination predicts identification; and 3) how the degree of hearing loss affects this relationship. Such evidence is crucial for developing effective intervention strategies to mitigate the effects of hearing loss on language development. Methods Participants were 58 children with early-onset sensorineural hearing loss (CHL, 53% girls, M = 9;4 yrs) and 58 children with normal hearing (CNH, 53% girls, M = 9;4 yrs). Test items were consonant-vowel (CV) syllables and nonwords with intact visual speech coupled to non-intact auditory speech (excised onsets) as, for example, an intact consonant/rhyme in the visual track (Baa or Baz) coupled to non-intact onset/rhyme in the auditory track (/–B/aa or /–B/az). The items started with an easy-to-speechread /B/ or difficult-to-speechread /G/ onset and were presented in the auditory (static face) vs. audiovisual (dynamic face) modes. We assessed discrimination for intact vs. non-intact different pairs (e.g., Baa:/–B/aa). We predicted that visual speech would cause the non-intact onset to be perceived as intact and would therefore generate more same—as opposed to different—responses in the audiovisual than auditory mode. We assessed identification by repetition of nonwords with non-intact onsets (e.g., /–B/az). We predicted that visual speech would cause the non-intact onset to be perceived as intact and would therefore generate more Baz—as opposed to az— responses in the audiovisual than auditory mode. Results

  10. Isolation and propagation of colon cancer stem cells

    NARCIS (Netherlands)

    Prasetyanti, Pramudita R.; Zimberlin, Cheryl; de Sousa E Melo, Felipe; Medema, Jan Paul

    2013-01-01

    The design of tissue culture conditions that faithfully reproduce the characteristics of cells in their native environment remains one of the main challenges of cancer stem cell (CSC) biology. Here we describe a detailed methodology for the isolation and expansion of both human colon CSCs and mouse

  11. Cutaneous leishmaniosis in naturally infected dogs in Paraná, Brazil, and the epidemiological implications of Leishmania (Viannia) braziliensis detection in internal organs and intact skin.

    Science.gov (United States)

    Marquez, Ellen de Souza; de Castro, Edilene Alcântara; Nabut, Luciene Biazono; da Costa-Ribeiro, Magda Clara Vieira; Dela Coletta Troiano Araújo, Ludmilla; Poubel, Saloe Bispo; Gonçalves, André Luiz; Cruz, Mariza Fordellone Rosa; Dos Santos Trad, Ana Paula Millet Evangelista; Dias, Rafael Andre Ferreira; Navarro, Italmar Teodorico; Thomaz-Soccol, Vanete

    2017-08-30

    Environmental changes have occurred over the years, altering the eco-epidemiological pattern of leishmaniosis in the State of Paraná, Brazil, involving the pillars of the cycle (parasite, vectors, reservoir, and environment) and their interaction. Much has been discussed about the dog's role as a reservoir of the Leishmania (Viannia) braziliensis Vianna, 1911 transmission cycle. However, this question remains unanswered. The purpose of this study was to investigate, using parasitological and molecular methods, different samples in eight naturally infected dogs from an endemic rural locality where only L. (V.) braziliensis is present, and where human cases have been previously notified. Blood and biopsied organ samples from naturally infected dogs were analyzed by culture media, PCR, random amplified polymorphic DNA and sequencing methodologies. Only skin lesions from all dogs yielded positive cultures and when PCR was performed, L. (V.) braziliensis DNA was amplified from intact skin, peripheral blood, bone marrow, spleen, liver and lymph nodes. RAPD was also applied to isolates from the skin lesions, exhibiting the genetic variability of the parasite identified. To confirm which species of Leishmania was amplified in PCR, the sequencing method was performed, verifying 100% similarity with the Viannia subgenus. This study showed that L. (V.) braziliensis can spread to other sites besides the ulcerous lesions, such as intact skin, peripheral blood and internal organs, making it possibility for dogs to serve as active sources of parasite transmission. For definitive proof, xenodiagnostic test on intact skin of infected dogs, should be done. Copyright © 2017 Elsevier B.V. All rights reserved.

  12. The Mouse Solitary Odorant Receptor Gene Promoters as Models for the Study of Odorant Receptor Gene Choice.

    Directory of Open Access Journals (Sweden)

    Andrea Degl'Innocenti

    Full Text Available In vertebrates, several anatomical regions located within the nasal cavity mediate olfaction. Among these, the main olfactory epithelium detects most conventional odorants. Olfactory sensory neurons, provided with cilia exposed to the air, detect volatile chemicals via an extremely large family of seven-transmembrane chemoreceptors named odorant receptors. Their genes are expressed in a monogenic and monoallelic fashion: a single allele of a single odorant receptor gene is transcribed in a given mature neuron, through a still uncharacterized molecular mechanism known as odorant receptor gene choice.Odorant receptor genes are typically arranged in genomic clusters, but a few are isolated (we call them solitary from the others within a region broader than 1 Mb upstream and downstream with respect to their transcript's coordinates. The study of clustered genes is problematic, because of redundancy and ambiguities in their regulatory elements: we propose to use the solitary genes as simplified models to understand odorant receptor gene choice.Here we define number and identity of the solitary genes in the mouse genome (C57BL/6J, and assess the conservation of the solitary status in some mammalian orthologs. Furthermore, we locate their putative promoters, predict their homeodomain binding sites (commonly present in the promoters of odorant receptor genes and compare candidate promoter sequences with those of wild-caught mice. We also provide expression data from histological sections.In the mouse genome there are eight intact solitary genes: Olfr19 (M12, Olfr49, Olfr266, Olfr267, Olfr370, Olfr371, Olfr466, Olfr1402; five are conserved as solitary in rat. These genes are all expressed in the main olfactory epithelium of three-day-old mice. The C57BL/6J candidate promoter of Olfr370 has considerably varied compared to its wild-type counterpart. Within the putative promoter for Olfr266 a homeodomain binding site is predicted. As a whole, our findings

  13. Multi-Axis Prosthetic Knee Resembles Alpine Skiing Movements of an Intact Leg

    Science.gov (United States)

    Demšar, Ivan; Duhovnik, Jože; Lešnik, Blaž; Supej, Matej

    2015-01-01

    The purpose of the study was to analyse the flexion angles of the ski boot, ankle and knee joints of an above-knee prosthesis and to compare them with an intact leg and a control group of skiers. One subject with an above-knee amputation of the right leg and eight healthy subjects simulated the movement of a skiing turn by performing two-leg squats in laboratory conditions. By adding additional loads in proportion to body weight (BW; +1/3 BW, +2/3 BW, +3/3 BW), various skiing regimes were simulated. Change of Flexion Angle (CoFA) and Range of Motion (RoM) in the ski boot, ankle and knee joints were calculated and compared. An average RoM in the skiing boot on the side of prosthesis (4.4 ± 1.1°) was significantly lower compared to an intact leg (5.9 ± 1.8°) and the control group (6.5 ± 2.3°). In the ankle joint, the average RoM was determined to be 13.2±2.9° in the prosthesis, 12.7 ± 2.8° in an intact leg and 14.8±3.6 in the control group. However, the RoM of the knee joint in the prosthesis (42.2 ± 4.2°) was significantly larger than that of the intact leg (34.7 ± 4.4°). The average RoM of the knee joint in the control group was 47.8 ± 5.4°. The influences of additional loads on the kinematics of the lower extremities were different on the side of the prosthesis and on the intact leg. In contrast, additional loads did not produce any significant differences in the control group. Although different CoFAs in the ski boot, ankle and knee joints were used, an above-knee prosthesis with a built-in multi-axis prosthetic knee enables comparable leg kinematics in simulated alpine skiing. Key points The RoM in the ski boot on the side of the prosthetic leg was smaller than the RoM of the intact leg and the control group of healthy subjects. The RoM in the ankle joint of prosthetic leg was comparable to that of the intact leg and the control group of healthy subjects. The RoM in the prosthetic knee joint was greater than the RoM in the knee joint of the

  14. Mouse endometrial stromal cells produce basement-membrane components

    DEFF Research Database (Denmark)

    Wewer, U M; Damjanov, A; Weiss, J

    1986-01-01

    During mouse pregnancy, uterine stromal cells transform into morphologically distinct decidual cells under the influence of the implanting embryo and a proper hormonal environment. Mechanical stimulation of hormonally primed uterine stromal cells leads to the same morphologic alterations. The dec......During mouse pregnancy, uterine stromal cells transform into morphologically distinct decidual cells under the influence of the implanting embryo and a proper hormonal environment. Mechanical stimulation of hormonally primed uterine stromal cells leads to the same morphologic alterations....... Mouse decidual cells isolated from 6- to 7-day pregnant uteri explanted in vitro continue to synthesize basement-membrane-like extracellular matrix. Using immunohistochemistry and metabolic labeling followed by immunoprecipitation, SDS-PAGE, and fluorography, it was shown that the decidual cells...... to undergo pseudodecidualization. We thus showed that stromal cells from pregnant and nonpregnant mouse uteri synthesize significant amounts of basement-membrane components in vitro, and hence could serve as a good model for the study of normal basement-membrane components....

  15. Secretion of neurotensin from isolated perfused porcine ileum

    DEFF Research Database (Denmark)

    Holst Pedersen, J; Knuthsen, S; Bernabei, M

    1988-01-01

    The secretion and molecular nature of immunoreactive neurotensin (NT) was studied following stimulation of an isolated perfused porcine ileal segment with glucose, triglyceride and intra-arterial infusion of gastrin-releasing peptide (GRP). Secreted peptides were separated using gel chromatography...... in doses from 10(-10) to 10(-8) M stimulated NT release in a dose-related manner. Following gel chromatography only the intact peptide and no smaller or larger molecular size immunoreactive components were observed. The study showed that both luminal and humoral stimuli release NT from the isolated pig...

  16. 40 CFR 261.40 - Conditional Exclusion for Used, Intact Cathode Ray Tubes (CRTs) Exported for Recycling.

    Science.gov (United States)

    2010-07-01

    ... 40 Protection of Environment 25 2010-07-01 2010-07-01 false Conditional Exclusion for Used, Intact Cathode Ray Tubes (CRTs) Exported for Recycling. 261.40 Section 261.40 Protection of Environment...) Exported for Recycling. Used, intact CRTs exported for recycling are not solid wastes if they meet the...

  17. Characterization of Leishmania isolates from Nepalese patients with visceral leishmaniasis.

    Science.gov (United States)

    Pandey, Kishor; Yanagi, Testuo; Pandey, Basu Dev; Mallik, Arun Kumar; Sherchand, Jeevan Bahadur; Kanbara, Hiroji

    2007-05-01

    In Nepal, visceral leishmaniasis (VL) is endemic in 13 districts of the central and eastern regions. A total of 166 bone-marrow aspirates were obtained from patients with suspected VL. Ninety-seven were identified as positive by microscopy, and 29 of those were successfully isolated and cultured. We characterized these isolates by molecular analysis and by their ability to infect mice. PCR-restriction fragment length polymorphism analysis of the mini-exon and the cysteine proteinase b gene showed that all isolates were Leishmania donovani, and the restriction pattern of the Nepalese isolates corresponded to the standard Indian strain of L. donovani but differed from that of the Kenyan strain. The single-strand conformation polymorphism analysis of ribosomal internal transcribed spacer showed no genetic heterogeneity within Nepalese isolates. Intraperitoneal inoculation with the promastigotes of all isolates resulted in amastigote proliferation in the spleen of 20 nude mice, of which ten isolates were highly infective, and ten were moderately infective, including one BALB/c mouse. Of the 20 amastigotes isolated from the spleen of nude mice, only the ten highly infective isolates infected BALB/c mice, of which, two isolates were considered to have low infectivity, three isolates were considered to be moderately infective, and five isolates were considered to be highly infective.

  18. Supravital dithizone staining in the isolation of human and rat pancreatic islets

    DEFF Research Database (Denmark)

    Hansen, W A; Christie, M R; Kahn, R

    1989-01-01

    Dithizone, a zinc chelating agent, is known to selectively stain the islets of Langerhans in the pancreas. In the present study, we have used this stain to aid the identification of islets in material obtained by collagenase digestion of human pancreas. Islets were shown to rapidly and reversibly...... techniques for the large scale isolation of functionally intact human islets....

  19. Isolation and Characterization of Erythrocyte and Parasite Membranes from Rhesus Red Cells Infected with P. knowlesi.

    Science.gov (United States)

    1981-06-01

    initiated experiments to separate and isolate the vacuolar membrane and the parasite plasma menbrane . For this, the surfaces of intact schizonts...controlled nitrogen decompression (1) is surrounded by two membranes, its own plasma membrane and the membrane of the parasitophorous vacuole. We have

  20. Isolated total RNA and protein are preserved after thawing for more than twenty-four hours

    Science.gov (United States)

    de Oliveira, Ivone Braga; Ramos, Débora Rothstein; Lopes, Karen Lucasechi; de Souza, Regiane Machado; Heimann, Joel Claudio; Furukawa, Luzia Naôko Shinohara

    2012-01-01

    OBJECTIVE: The preservation of biological samples at a low temperature is important for later biochemical and/or histological analyses. However, the molecular viability of thawed samples has not been studied sufficiently in depth. The present study was undertaken to evaluate the viability of intact tissues, tissue homogenates, and isolated total RNA after defrosting for more than twenty-four hours. METHODS: The molecular viability of the thawed samples (n = 82) was assessed using the A260/A280 ratio, the RNA concentration, the RNA integrity, the level of intact mRNA determined by reverse transcriptase polymerase chain reaction, the protein level determined by Western blotting, and an examination of the histological structure. RESULTS: The integrity of the total RNA was not preserved in the thawed intact tissue, but the RNA integrity and level of mRNA were perfectly preserved in isolated defrosted samples of total RNA. Additionally, the level of β-actin protein was preserved in both thawed intact tissue and homogenates. CONCLUSION: Isolated total RNA does not undergo degradation due to thawing for at least 24 hours, and it is recommended to isolate the total RNA as soon as possible after tissue collection. Moreover, the protein level is preserved in defrosted tissues. PMID:22473407

  1. Isolation and amino acid sequence of corticotropin-releasing factor from pig hypothalami.

    OpenAIRE

    Patthy, M; Horvath, J; Mason-Garcia, M; Szoke, B; Schlesinger, D H; Schally, A V

    1985-01-01

    A polypeptide was isolated from acid extracts of porcine hypothalami on the basis of its high ability to stimulate the release of corticotropin from superfused rat pituitary cells. After an initial separation by gel filtration on Sephadex G-25, further purification was carried out by reversed-phase HPLC. The isolated material was homogeneous chromatographically and by N-terminal sequencing. Based on automated gas-phase sequencing of the intact and CNBr-cleaved peptide and on carboxypeptidase ...

  2. Isolation, characterization, virulence and immunogenicity testing of field isolates of Pasteurella multocida, Staphylococcus aureus, and Streptococcus agalactiae in laboratory settings.

    Science.gov (United States)

    Qudratullah; Muhammad, G; Saqib, M; Bilal, M Qamar

    2017-08-01

    The present study was designed to investigate isolation, characterization, virulence and immunogenicity testing of field isolates of Pasteurella multocida, Staphylococcus aureus, and Streptococcus agalactiae in rabbits and mice. Isolates of P. multocida, S. aureus and Str. agalactiae recovered from field cases of Hemorragic septicemia and mastitis were scrutinized for virulence/pathogenicity and immunogenicity. Mouse LD 50 of P. multocida showed that P. multocida isolate No.1 was more virulent than isolates No. 2 and 3. Virulence of isolate No.1S. aureus and Str. agalactiae revealed that 100, 80% rabbits died within 18h of inoculation. Seven-digit numerical profiles of these 4 isolates with API ® Staph test strips isolates, No.1 (6736153) showed good identification (S. aureus id=90.3%). Indirect ELISA-based serum antibody titers to P. multocida isolate No.1, S. aureus No.1, Str. agalactiae, isolate No.1 elicited high antibody titers 1.9, 1.23, 1.12 respectively. All the pathogens of Isolate No. 1 (P. multocida, S. aureus Str. agalactiae), were high antibody than others isolates. Copyright © 2017 Elsevier B.V. All rights reserved.

  3. Effects of ignoring baseline on modeling transitions from intact cognition to dementia.

    Science.gov (United States)

    Yu, Lei; Tyas, Suzanne L; Snowdon, David A; Kryscio, Richard J

    2009-07-01

    This paper evaluates the effect of ignoring baseline when modeling transitions from intact cognition to dementia with mild cognitive impairment (MCI) and global impairment (GI) as intervening cognitive states. Transitions among states are modeled by a discrete-time Markov chain having three transient (intact cognition, MCI, and GI) and two competing absorbing states (death and dementia). Transition probabilities depend on two covariates, age and the presence/absence of an apolipoprotein E-epsilon4 allele, through a multinomial logistic model with shared random effects. Results are illustrated with an application to the Nun Study, a cohort of 678 participants 75+ years of age at baseline and followed longitudinally with up to ten cognitive assessments per nun.

  4. Blood epididymal barrier to [3H]-inulin in intact and vasectomized hamsters

    International Nuclear Information System (INIS)

    Turner, T.T.; D'Addario, D.A.; Howards, S.S.

    1981-01-01

    The net transport of [ 3 H]-inulin into the fluids of the hamster seminiferous and caput, corpus, and cauda epididymal tubules was examined in both intact animals and those vasectomized 10 months previously. Mean isotope concentrations in reproductive tract tubule fluids did not exceeded 10 per cent of blood plasma isotope concentrations during the experiment. There were no significant differences in net transport of [ 3 H]-inulin into any of the tubule fluids sampled. Ten months after vasectomy, the seminiferous tubule, and all regions of the epididymal tubule retain the capacity to exclude [ 3 H]-insulin. Thus in the hamster 10 months after vasectomy, the blood testis and blood epididymal barriers to inulin are intact

  5. Determination of the intracellular pH of intact erythrocytes by 1H NMR spectroscopy

    International Nuclear Information System (INIS)

    Rabenstein, D.L.; Isab, A.A.

    1982-01-01

    A method is described for determining the intracellular pH of intact erythrocytes by 1 H NMR. The determination is based on the pH dependence of the chemical shifts of resonances for carbon-bounded protons of an indicator molecule (imidazole) in intact cells. The imidazole is introduced into the erythrocytes by incubation in an isotonic saline solution of the indicator. The pH dependence of the chemical shifts of the imidazole resonances is calibrated from 1 H NMR spectra of the imidazole-containing red cell lysates whose pH is varied by the addition of acid or base and measured directly with a pH electrode. To reduce in intensity or eliminate the much more intense envelope of resonances from the hemoglobin, the 1 H NMR measurements are made by either the spin-echo Fourier transform technique or by the transfer-or-saturation by cross-relaxation method

  6. Superresolution and Fluorescence Dynamics Evidence Reveal That Intact Liposomes Do Not Cross the Human Skin Barrier

    DEFF Research Database (Denmark)

    Dreier, Jes; Sørensen, Jens A; Brewer, Jonathan R

    2016-01-01

    In this study we use the combination of super resolution optical microscopy and raster image correlation spectroscopy (RICS) to study the mechanism of action of liposomes as transdermal drug delivery systems in human skin. Two different compositions of liposomes were applied to newly excised human...... skin, a POPC liposome and a more flexible liposome containing the surfactant sodium cholate. Stimulated emission depletion microscopy (STED) images of intact skin and cryo-sections of skin treated with labeled liposomes were recorded displaying an optical resolution low enough to resolve the 100 nm...... liposomes in the skin. The images revealed that virtually none of the liposomes remained intact beneath the skin surface. RICS two color cross correlation diffusion measurements of double labeled liposomes confirmed these observations. Our results suggest that the liposomes do not act as carriers...

  7. Intact and Degraded Component Criticality Calculations of N Reactor Spent Nuclear Fuel

    International Nuclear Information System (INIS)

    L. Angers

    2001-01-01

    The objective of this calculation is to perform intact and degraded mode criticality evaluations of the Department of Energy's (DOE) N Reactor Spent Nuclear Fuel codisposed in a 2-Defense High-Level Waste (2-DHLW)/2-Multi-Canister Overpack (MCO) Waste Package (WP) and emplaced in a monitored geologic repository (MGR) (see Attachment I). The scope of this calculation is limited to the determination of the effective neutron multiplication factor (k eff ) for both intact and degraded mode internal configurations of the codisposal waste package. This calculation will support the analysis that will be performed to demonstrate the technical viability for disposing of U-metal (N Reactor) spent nuclear fuel in the potential MGR

  8. Cutaneous mast cell maturation does not depend on an intact bone marrow microenvironment

    International Nuclear Information System (INIS)

    Charley, M.R.; Mikhael, A.; Sontheimer, R.D.; Gilliam, J.N.; Bennett, M.

    1984-01-01

    A study was made to determine whether the maturation of murine cutaneous mast cells from stem cells depends on an intact bone marrow microenvironment. Normal bone marrow cells (+/+) were infused into 2 groups of mast cell-deficient mice: WBB6F1-W/Wv mice and 89 Sr-pretreated W/Wv mice. 89 Sr is a long-lived bone-seeking radioisotope which provides continuous irradiation of the marrow and thereby ablates the marrow microenvironment. Skin biopsies revealed that the 89 Sr-pretreated mice and the controls had repopulated their skin with mast cells equally well. Natural killer cell function was significantly depressed in the 89 Sr-treated mice, confirming that the marrow microenvironment had been functionally altered. It appears that, although the precursors for cutaneous mast cells are marrow derived, they do not need an intact marrow microenvironment for maturation

  9. Mapping of the Mouse Actin Capping Protein Beta Subunit Gene

    Directory of Open Access Journals (Sweden)

    Cooper John A

    2000-07-01

    Full Text Available Abstract Background Capping protein (CP, a heterodimer of α and β subunits, is found in all eukaryotes. CP binds to the barbed ends of actin filaments in vitro and controls actin assembly and cell motility in vivo. Vertebrates have three isoforms of CPβ produced by alternatively splicing from one gene; lower organisms have one gene and one isoform. Results We isolated genomic clones corresponding to the β subunit of mouse CP and identified its chromosomal location by interspecies backcross mapping. Conclusions The CPβ gene (Cappb1 mapped to Chromosome 4 between Cdc42 and D4Mit312. Three mouse mutations, snubnose, curly tail, and cribriform degeneration, map in the vicinity of the β gene.

  10. Dengue virus infection induces broadly cross-reactive human IgM antibodies that recognize intact virions in humanized BLT-NSG mice.

    Science.gov (United States)

    Jaiswal, Smita; Smith, Kenneth; Ramirez, Alejandro; Woda, Marcia; Pazoles, Pamela; Shultz, Leonard D; Greiner, Dale L; Brehm, Michael A; Mathew, Anuja

    2015-01-01

    The development of small animal models that elicit human immune responses to dengue virus (DENV) is important since prior immunity is a major risk factor for developing severe dengue disease. This study evaluated anti-DENV human antibody (hAb) responses generated from immortalized B cells after DENV-2 infection in NOD-scid IL2rγ(null) mice that were co-transplanted with human fetal thymus and liver tissues (BLT-NSG mice). DENV-specific human antibodies predominantly of the IgM isotype were isolated during acute infection and in convalescence. We found that while a few hAbs recognized the envelope protein produced as a soluble recombinant, a number of hAbs only recognized epitopes on intact virions. The majority of the hAbs isolated during acute infection and in immune mice were serotype-cross-reactive and poorly neutralizing. Viral titers in immune BLT-NSG mice were significantly decreased after challenge with a clinical strain of dengue. DENV-specific hAbs generated in BLT-NSG mice share some of the characteristics of Abs isolated in humans with natural infection. Humanized BLT-NSG mice provide an attractive preclinical platform to assess the immunogenicity of candidate dengue vaccines. © 2014 by the Society for Experimental Biology and Medicine.

  11. Rapid differentiation of Listeria monocytogenes epidemic clones III and IV and their intact compared with heat-killed populations using Fourier transform infrared spectroscopy and chemometrics.

    Science.gov (United States)

    Nyarko, Esmond B; Puzey, Kenneth A; Donnelly, Catherine W

    2014-06-01

    The objectives of this study were to determine if Fourier transform infrared (FT-IR) spectroscopy and multivariate statistical analysis (chemometrics) could be used to rapidly differentiate epidemic clones (ECs) of Listeria monocytogenes, as well as their intact compared with heat-killed populations. FT-IR spectra were collected from dried thin smears on infrared slides prepared from aliquots of 10 μL of each L. monocytogenes ECs (ECIII: J1-101 and R2-499; ECIV: J1-129 and J1-220), and also from intact and heat-killed cell populations of each EC strain using 250 scans at a resolution of 4 cm(-1) in the mid-infrared region in a reflectance mode. Chemometric analysis of spectra involved the application of the multivariate discriminant method for canonical variate analysis (CVA) and linear discriminant analysis (LDA). CVA of the spectra in the wavelength region 4000 to 600 cm(-1) separated the EC strains while LDA resulted in a 100% accurate classification of all spectra in the data set. Further, CVA separated intact and heat-killed cells of each EC strain and there was 100% accuracy in the classification of all spectra when LDA was applied. FT-IR spectral wavenumbers 1650 to 1390 cm(-1) were used to separate heat-killed and intact populations of L. monocytogenes. The FT-IR spectroscopy method allowed discrimination between strains that belong to the same EC. FT-IR is a highly discriminatory and reproducible method that can be used for the rapid subtyping of L. monocytogenes, as well as for the detection of live compared with dead populations of the organism. Fourier transform infrared (FT-IR) spectroscopy and multivariate statistical analysis can be used for L. monocytogenes source tracking and for clinical case isolate comparison during epidemiological investigations since the method is capable of differentiating epidemic clones and it uses a library of well-characterized strains. The FT-IR method is potentially less expensive and more rapid compared to genetic

  12. Sediment carbon and nutrient fluxes from cleared and intact temperate mangrove ecosystems and adjacent sandflats.

    Science.gov (United States)

    Bulmer, Richard H; Schwendenmann, Luitgard; Lohrer, Andrew M; Lundquist, Carolyn J

    2017-12-01

    The loss of mangrove ecosystems is associated with numerous impacts on coastal and estuarine function, including sediment carbon and nutrient cycling. In this study we compared in situ fluxes of carbon dioxide (CO 2 ) from the sediment to the atmosphere, and fluxes of dissolved inorganic nutrients and oxygen across the sediment-water interface, in intact and cleared mangrove and sandflat ecosystems in a temperate estuary. Measurements were made 20 and 25months after mangrove clearance, in summer and winter, respectively. Sediment CO 2 efflux was over two-fold higher from cleared than intact mangrove ecosystems at 20 and 25months after mangrove clearance. The higher CO 2 efflux from the cleared site was explained by an increase in respiration of dead root material along with sediment disturbance following mangrove clearance. In contrast, sediment CO 2 efflux from the sandflat site was negligible (≤9.13±1.18mmolm -2 d -1 ), associated with lower sediment organic matter content. The fluxes of inorganic nutrients (NH 4 + , NO x and PO 4 3- ) from intact and cleared mangrove sediments were low (≤20.37±18.66μmolm -2 h - 1 ). The highest NH 4 + fluxes were measured at the sandflat site (69.21±13.49μmolm -2 h - 1 ). Lower inorganic nutrient fluxes within the cleared and intact mangrove sites compared to the sandflat site were associated with lower abundance of larger burrowing macrofauna. Further, a higher fraction of organic matter, silt and clay content in mangrove sediments may have limited nutrient exchange. Copyright © 2017 Elsevier B.V. All rights reserved.

  13. A structured approach to the study of metabolic control principles in intact and impaired mitochondria

    OpenAIRE

    Huber, Heinrich J.; Connolly, Niamh M. C.; Dussmann, Heiko; Prehn, Jochen H. M.

    2012-01-01

    We devised an approach to extract control principles of cellular bioenergetics for intact and impaired mitochondria from ODE-based models and applied it to a recently established bioenergetic model of cancer cells. The approach used two methods for varying ODE model parameters to determine those model components that, either alone or in combination with other components, most decisively regulated bioenergetic state variables. We found that, while polarisation of the mitochondrial membrane pot...

  14. Ingested soluble CD14 from milk is transferred intact into the blood of newborn rats.

    Science.gov (United States)

    Ward, Tonya L; Spencer, William J; Davis, Laura D R; Harrold, Joann; Mack, David R; Altosaar, Illimar

    2014-02-01

    Milk acts as an edible immune system that is transferred from mother to newborn. Soluble Cluster of Differentiation 14 (sCD14) is a protein found in significant quantities in human milk (~8-29 µg/ml). At a 10-fold lower concentration in the blood (~3 µg/ml), the most notable role of sCD14 is to sequester lipopolysaccharides of Gram-negative bacteria from immune cells. To explore the pharmacodynamics of this milk protein and its biological fate, the biodistribution of radiolabeled sCD14 ((14)C, (125)I) was monitored in 10-d-old rat pups. Up to 3.4 ± 2.2% of the radiolabeled sCD14 administered was observed, intact, in the pup blood for up to 8 h post-ingestion. Additionally, 30.3 ± 13.0% of the radiolabeled sCD14 administered was observed degraded in the stomach at 8 h post-ingestion. A reservoir of intact, administered sCD14 (3.2 ± 0.3%), however, remained in the stomach at 8 h post-ingestion. Intact sCD14 was observed in the small intestine at 5.5 ± 1.6% of the dose fed at 8 h post-ingestion. The presence of intact sCD14 in the blood and the gastrointestinal tract of newborns post-ingestion has implications in the development of allergies, obesity, and other inflammation-related pathogeneses later in life.

  15. Forest Loss in Protected Areas and Intact Forest Landscapes: A Global Analysis.

    Science.gov (United States)

    Heino, Matias; Kummu, Matti; Makkonen, Marika; Mulligan, Mark; Verburg, Peter H; Jalava, Mika; Räsänen, Timo A

    2015-01-01

    In spite of the high importance of forests, global forest loss has remained alarmingly high during the last decades. Forest loss at a global scale has been unveiled with increasingly finer spatial resolution, but the forest extent and loss in protected areas (PAs) and in large intact forest landscapes (IFLs) have not so far been systematically assessed. Moreover, the impact of protection on preserving the IFLs is not well understood. In this study we conducted a consistent assessment of the global forest loss in PAs and IFLs over the period 2000-2012. We used recently published global remote sensing based spatial forest cover change data, being a uniform and consistent dataset over space and time, together with global datasets on PAs' and IFLs' locations. Our analyses revealed that on a global scale 3% of the protected forest, 2.5% of the intact forest, and 1.5% of the protected intact forest were lost during the study period. These forest loss rates are relatively high compared to global total forest loss of 5% for the same time period. The variation in forest losses and in protection effect was large among geographical regions and countries. In some regions the loss in protected forests exceeded 5% (e.g. in Australia and Oceania, and North America) and the relative forest loss was higher inside protected areas than outside those areas (e.g. in Mongolia and parts of Africa, Central Asia, and Europe). At the same time, protection was found to prevent forest loss in several countries (e.g. in South America and Southeast Asia). Globally, high area-weighted forest loss rates of protected and intact forests were associated with high gross domestic product and in the case of protected forests also with high proportions of agricultural land. Our findings reinforce the need for improved understanding of the reasons for the high forest losses in PAs and IFLs and strategies to prevent further losses.

  16. Microseismic Analysis of Fracture of an Intact Rock Asperity Traversing a Sawcut Fault

    Science.gov (United States)

    Mclaskey, G.; Lockner, D. A.

    2017-12-01

    Microseismic events carry information related to stress state, fault geometry, and other subsurface properties, but their relationship to large and potentially damaging earthquakes is not well defined. We conducted laboratory rock mechanics experiments that highlight the interaction between a sawcut fault and an asperity composed of an intact rock "pin". The sample is a 76 mm diameter cylinder of Westerly granite with a 21 mm diameter cylinder (the pin) of intact Westerly granite that crosses the sawcut fault. Upon loading to 80 MPa in a triaxial machine, we first observed a slip event that ruptured the sawcut fault, slipped about 35 mm, but was halted by the rock pin. With continued loading, the rock pin failed in a swarm of thousands of M -7 seismic events similar to the localized microcracking that occurs during the final fracture nucleation phase in an intact rock sample. Once the pin was fractured to a critical point, it permitted complete rupture events on the sawcut fault (stick-slip instabilities). No seismicity was detected on the sawcut fault plane until the pin was sheared. Subsequent slip events were preceded by 10s of foreshocks, all located on the fault plane. We also identified an aseismic zone on the fault plane surrounding the fractured rock pin. A post-mortem analysis of the sample showed a thick gouge layer where the pin intersected the fault, suggesting that this gouge propped open the fault and prevented microseismic events in its vicinity. This experiment is an excellent case study in microseismicity since the events separate neatly into three categories: slip on the sawcut fault, fracture of the intact rock pin, and off-fault seismicity associated with pin-related rock joints. The distinct locations, timing, and focal mechanisms of the different categories of microseismic events allow us to study how their occurrence is related to the mechanics of the deforming rock.

  17. Dissolution of intact UO2 pellet in batch and rotary dissolver conditions

    International Nuclear Information System (INIS)

    Jayendra Kumar Gelatar; Bijendra Kumar; Sampath, M.; Shekhar Kumar; Kamachi Mudali, U.; Natarajan, R.

    2015-01-01

    Comparative dissolution of intact un-irradiated UO 2 pellet of PHWR fuel dimensions was performed in batch and dynamic rotary dissolver conditions in aqueous nitric acid solutions at elevated temperatures. The extent of dissolution was estimated by determining the uranium concentration of the resulting aqueous solution. It was observed that rate of dissolution was much faster in dynamic conditions as compared to static batch conditions. (author)

  18. DETERMINATION OF THE MINIMUM INTACT DIMENSIONS AVAILABLE IN PRACTICAL APPLICATIONS OF LASER CUTTING

    Directory of Open Access Journals (Sweden)

    Peter Gyorgy HORVATH

    2016-06-01

    Full Text Available The reuse of wastes is one of the main requirements of sustainable wood processing. Technologies available for waste management however are currently suited to the reuse of large quantities. This study investigates the utilisation of individual pieces of sawn wood and veneer wastes by laser cutting, with special emphasis on the search for the practical minimum intact dimension that can be produced by laser cutting

  19. Temperature dependence of 1H NMR relaxation time, T2, for intact and neoplastic plant tissues

    Science.gov (United States)

    Lewa, Czesław J.; Lewa, Maria

    Temperature dependences of the spin-spin proton relaxation time, T2, have been shown for normal and tumorous tissues collected from kalus culture Nicotiana tabacum and from the plant Kalanchoe daigremontiana. For neoplastic plant tissues, time T2 was increased compared to that for intact plants, a finding similar to that for animal and human tissues. The temperature dependences obtained were compared to analogous relations observed with animal tissues.

  20. Fourier transform infrared spectroscopic study of intact cells of the nitrogen-fixing bacterium Azospirillum brasilense

    Science.gov (United States)

    Kamnev, A. A.; Ristić, M.; Antonyuk, L. P.; Chernyshev, A. V.; Ignatov, V. V.

    1997-06-01

    The data of Fourier transform infrared (FTIR) spectroscopic measurements performed on intact cells of the soil nitrogen-fixing bacterium Azospirillum brasilense grown in a standard medium and under the conditions of an increased metal uptake are compared and discussed. The structural FTIR information obtained is considered together with atomic absorption spectrometry (AAS) data on the content of metal cations in the bacterial cells. Some methodological aspects concerning preparation of bacterial cell samples for FTIR measurements are also discussed.

  1. Discrimination between intact and decayed pulp regions in carious teeth by ADC mapping.

    Science.gov (United States)

    Cankar, Ksenija; Nemeth, Lidija; Bajd, Franci; Vidmar, Jernej; Serša, Igor

    2014-01-01

    The aim of this study was to evaluate an advanced magnetic resonance imaging (MRI) method, apparent diffusion coefficient (ADC) mapping, in the functional assessment of carious teeth. 38 extracted human teeth with scores of 0, 3 and 6 according to International Caries Detection and Assessment System (ICDAS) criteria were screened and subsequently analyzed by MRI at 2.35 T. Histology sectioning of teeth was used for the gold standard by analyzing two extreme cases (intact and severely decayed). ADC maps of the same teeth were calculated from corresponding diffusion-weighted images and used to obtain ADC distributions along dental pulp as functions of the relative pulp length measured from the occlusal pulp side. The measured distributions were analyzed for the best fit by a four-parameter three-segment linear regression model for ADC distribution along the pulp. MRI results were in good agreement with findings in histological sections of identical teeth. The best fit model parameters, relative decayed region depth, relative transition region width and ADC values of intact and decayed pulp tissue, showed statistically significant differences between the ADC values of intact and decayed pulp tissue (1.0 × 10(-9) m(2)/s vs. 0.74-0.89 × 10(-9) m(2)/s) and the relative decayed region depth progressing with ICDAS score (3 vs. 46% with ICDAS 3 vs. ICDAS 6). The results of this feasibility study confirmed relevance of ADC mapping for the discrimination and localization of intact and decayed regions in dental pulps of carious teeth. © 2014 S. Karger AG, Basel.

  2. Selective Osmotic Shock (SOS)-Based Islet Isolation for Microencapsulation.

    Science.gov (United States)

    Enck, Kevin; McQuilling, John Patrick; Orlando, Giuseppe; Tamburrini, Riccardo; Sivanandane, Sittadjody; Opara, Emmanuel C

    2017-01-01

    Islet transplantation (IT) has recently been shown to be a promising alternative to pancreas transplantation for reversing diabetes. IT requires the isolation of the islets from the pancreas, and these islets can be used to fabricate a bio-artificial pancreas. Enzymatic digestion is the current gold standard procedure for islet isolation but has lingering concerns. One such concern is that it has been shown to damage the islets due to nonselective tissue digestion. This chapter provides a detailed description of a nonenzymatic method that we are exploring in our lab as an alternative to current enzymatic digestion procedures for islet isolation from human and nonhuman pancreatic tissues. This method is based on selective destruction and protection of specific cell types and has been shown to leave the extracellular matrix (ECM) of islets intact, which may thus enhance islet viability and functionality. We also show that these SOS-isolated islets can be microencapsulated for transplantation.

  3. Characterizing embryonic gene expression patterns in the mouse using nonredundant sequence-based selection

    DEFF Research Database (Denmark)

    Sousa-Nunes, Rita; Rana, Amer Ahmed; Kettleborough, Ross

    2003-01-01

    This article investigates the expression patterns of 160 genes that are expressed during early mouse development. The cDNAs were isolated from 7.5 d postcoitum (dpc) endoderm, a region that comprises visceral endoderm (VE), definitive endoderm, and the node-tissues that are required for the initi...

  4. Genomic organization of the mouse peroxisome proliferator-activated receptor beta/delta gene

    DEFF Research Database (Denmark)

    Larsen, Leif K; Amri, Ez-Zoubir; Mandrup, Susanne

    2002-01-01

    Peroxisome proliferator-activated receptor (PPAR) beta/delta is ubiquitously expressed, but the level of expression differs markedly between different cell types. In order to determine the molecular mechanisms governing PPARbeta/delta gene expression, we have isolated and characterized the mouse...

  5. Mechanisms of blood pressure changes following renal irradiation of intact, adrenalectomized, and adrenal regenerating rats

    International Nuclear Information System (INIS)

    Rosenblum, M.

    1977-01-01

    This study was conducted to determine the differences in changes in systolic arterial blood pressure following renal x irradiation (1100 R) in adrenal-intact, adrenalectomized, and adrenal-regenerating rats and to elucidate the involvement or roles of the kidneys and of the adrenal glands in the blood pressure changes. The parameters studied included the following: systolic blood pressure; body weight; food and fluid consumption; urine output; plasma and urine electrolytes; sodium balance; plasma renin activity; plasma corticosterone; renal vascular volume; renal vascular permeability (using 125 I-polyvinylpyrrolidone extravasation rate as an indicator); renal blood flow (using 42 K extraction); kidney weight; hematocrit; and total vascular, plasma, and red cell volumes. Renal x irradiation of intact rats caused polydipsia, polyuria, and reduced urine concentrations of sodium and potassium without significantly affecting blood pressure during the period of study (80 days); plasma renin activity was significantly lowered and had a positive correlation with blood volume; an abnormal blood volume-plasma renin activity relationship is suggested. Adrenalectomy caused prolonged hypotension in saline-maintained rats even though their sodium balance was more positive than that in adrenal-intact or adrenal-regenerating rats with normal or elevated blood pressure. The blood pressure of renally irrradiated, adrenalectomized rats was greater than non-irradiated adrenalectomized rats, but with only borderline significance; it is concluded that the absence of the adrenal glands does not affect the degree or duration of the effects of renal irradiation on blood pressure

  6. The effect of self-transcendence on depression in cognitively intact nursing home patients.

    Science.gov (United States)

    Haugan, Gørill; Innstrand, Siw Tone

    2012-01-01

    Aims. This study's aim was to test the effects of self-transcendence on depression among cognitively intact nursing home patients. Background. Depression is considered the most frequent mental disorder among the elderly population. Specifically, the depression rate among nursing home patients is three to four times higher than that among community-dwelling elderly. Therefore, finding new and alternative ways to prevent and decrease depression is of great importance for nursing home patients' well-being. Self-transcendence is related to spiritual as well as nonspiritual factors, and it is described as a correlate and resource for well-being among vulnerable populations and at the end of life. Methods. A two-factor construct of the self-transcendence scale (interpersonal and intrapersonal) and the hospital anxiety and depression scale (HADS) was applied. A sample of 202 cognitively intact nursing home patients in central Norway was selected to respond to the questionnaires in 2008/2009. Results. A hypothesized SEM model demonstrated significant direct relationships and total effects of self-transcendence on depression. Conclusion and Implication for Practice. Facilitating patients' self-transcendence, both interpersonally and intrapersonally, might decrease depression among cognitively intact nursing home patients.

  7. Predictors of attachment security in preschool children from intact and divorced families.

    Science.gov (United States)

    Nair, Hira; Murray, Ann D

    2005-09-01

    The authors selected 58 mother-child dyads from divorced and intact families to participate in a study on the impact of divorce on preschoolers' attachment security. The authors explored pathways that lead to security of attachment. They found that mothers from divorced families were younger, had lower income levels, and had lower levels of education compared with their intact counterparts. Divorced mothers also reported significantly higher levels of stress, depression, need for social support, and conflict with their spouses. Mothers from intact families were more likely to use positive (authoritative) parenting styles compared with divorced mothers. Children in the divorced group had lower security scores on the Attachment Q-Set instrument (E. Waters, 1995). Regression analyses indicated that parenting style made a direct (independent) contribution to attachment security. In addition, temperament was related to attachment security, but temperament did not diminish the association of parenting style with attachment security. Furthermore, regression analyses indicated that the relationship of divorce to attachment security was mediated by parenting style.

  8. Estimating Carbon Dynamics in an Intact Lowland Mixed Dipterocarp Forest Using a Forest Carbon Model

    Directory of Open Access Journals (Sweden)

    Jongyeol Lee

    2017-04-01

    Full Text Available Intact dipterocarp forests in Asia act as crucial carbon (C reservoirs, and it is therefore important to investigate the C dynamics in these forests. We estimated C dynamics, together with net ecosystem production (NEP, in an intact tropical dipterocarp forest of Brunei Darussalam. Fifty-four simulation units (plots; 20 m × 20 m were established and initial C stocks were determined via direct field measurement. The C dynamics were annually simulated with a regression model and the Forest Biomass and Dead organic matter Carbon (FBDC model. The initial C stock (Mg C·ha−1 of biomass, litter, dead wood and mineral soil were 213.1 ± 104.8, 2.0 ± 0.8, 31.3 ± 38.8, and 80.7 ± 15.5, respectively. Their annual changes (Mg C·ha−1·year−1 were 3.2 ± 1.1, 0.2 ± 0.2, −3.7 ± 6.1, and −0.3 ± 1.1, respectively. NEP was −0.6 ± 6.1 Mg C·ha−1·year−1, showing large heterogeneity among the plots. The initial C stocks of biomass and dead wood, biomass turnover rates and dead wood decay rates were elucidated as dominant factors determining NEP in a sensitivity analysis. Accordingly, investigation on those input data can constrain an uncertainty in determining NEP in the intact tropical forests.

  9. Binding of radiolabelled luteinizing hormone to intact and ovariectomised rat uterus

    International Nuclear Information System (INIS)

    Sen, S.; Bhattacharya, S.

    1992-01-01

    Binding of ovine LH to uterine tissue preparation from intact and ovariectomised rat clearly indicates that uterus possesses specific binding sites for LH. Binding characteristics of LH to uterine tissue preparation from intact rat showed saturability with high affinity and low capacity. Scatchard plot analysis showed dissociation constant of the specific binding site to be 0.12 x 10 -9 mol/l and the number of binding sites was 2.31±0.05 fmol/mg protein. Ovariectomy did not change the binding affinity but effected a decrease in the number of binding sites (1.7 ± 0.08 f mol/mg protein). LH treatment of ovariectomized (ovx) rat had no effect on binding affinity but significantly increased the number of binding sites (3.23 ± 0.1 f mol/mg protein). Reduction of uterine weight due to ovariectomy and marked increase of ovx rat uterine weight by LH administration indicate a source of estrogen in ovx rat. An in vitro uterine tissue slice (from intact and ovx rat) incubation showed depletion of 17 β-estradiol (E 2 ) content in ovx rat which significantly elevated on LH addition. Data suggest the LH binding to rat uterine tissue has biological relevance. (author). 16 refs., 4 figs. 1 tab

  10. Conductive hearing loss with an intact tympanic membrane due to non-inflammatory causes.

    Science.gov (United States)

    Choi, Jin Hyuk; Lee, Min Young; Park, Ji Hye; Lee, Kyu-Yup; Lee, Sang Heun; Jang, Jeong Hun

    2016-04-01

    We analyzed audiologic and surgical findings in patients with conductive hearing loss (CHL) with an intact tympanic membrane (TM) that was of a non-inflammatory origin. We reviewed data from patients who underwent exploratory tympanotomy for CHL with intact TM from January 1995 to November 2012. Patients with diseases of non-inflammatory origin were enrolled (69 patients; 79 ears). Patients were categorized into two groups: non-trauma (50 ears) and trauma (29 ears). Demographic data, intraoperative findings, and audiologic results were obtained and analyzed. Overall, the second decade was the most common age of diagnosis in both the non-trauma and trauma groups. Operative findings showed that ossicular dislocation was more prevalent than ossicular fixation; all trauma group subjects had ossicular dislocation. Short columellization or partial ossicular replacement was the most frequently adopted surgical procedures in both groups. Overall, audiologically, air-conduction thresholds (ACs) and air-bone gaps were significantly improved over the short- and long-term period in both groups. However, the non-trauma group had significantly higher preoperative ACs than the trauma group, especially at low frequencies. This study provides clinicians with useful information regarding the clinical characteristics of CHL with intact TM of non-inflammatory origin. Copyright © 2015 Elsevier Ireland Ltd. All rights reserved.

  11. Differential transcriptional profiling of damaged and intact adjacent dorsal root ganglia neurons in neuropathic pain.

    Directory of Open Access Journals (Sweden)

    A K Reinhold

    Full Text Available Neuropathic pain, caused by a lesion in the somatosensory system, is a severely impairing mostly chronic disease. While its underlying molecular mechanisms are not thoroughly understood, neuroimmune interactions as well as changes in the pain pathway such as sensitization of nociceptors have been implicated. It has been shown that not only are different cell types involved in generation and maintenance of neuropathic pain, like neurons, immune and glial cells, but, also, intact adjacent neurons are relevant to the process. Here, we describe an experimental approach to discriminate damaged from intact adjacent neurons in the same dorsal root ganglion (DRG using differential fluorescent neuronal labelling and fluorescence-activated cell sorting (FACS. Two fluorescent tracers, Fluoroemerald (FE and 1-dioctadecyl-3,3,3,3-tetramethylindocarbocyanine perchlorate (DiI, were used, whose properties allow us to distinguish between damaged and intact neurons. Subsequent sorting permitted transcriptional analysis of both groups. Results and qPCR validation show a strong regulation in damaged neurons versus contralateral controls as well as a moderate regulation in adjacent neurons. Data for damaged neurons reveal an mRNA expression pattern consistent with established upregulated genes like galanin, which supports our approach. Moreover, novel genes were found strongly regulated such as corticotropin-releasing hormone (CRH, providing novel targets for further research. Differential fluorescent neuronal labelling and sorting allows for a clear distinction between primarily damaged neuropathic neurons and "bystanders," thereby facilitating a more detailed understanding of their respective roles in neuropathic processes in the DRG.

  12. Perineal hernia repair using an autologous tunica vaginalis communis in nine intact male dogs.

    Science.gov (United States)

    Pratummintra, Kittiya; Chuthatep, Suwicha; Banlunara, Wijit; Kalpravidh, Marissak

    2013-01-01

    Perineal hernia in dogs is very problematic and mostly requires surgical reconstruction of the weak pelvic diaphragm. Tissue or synthetic grafts have been used for the correction after failure of the conventional herniorrhaphy. Aim of this clinical trial was to assess the possible use of the autologous tunica vaginalis communis as a free graft for perineal hernia repair in intact male dogs. Seven unilateral and 2 bilateral perineal hernias in nine intact male dogs free from testicular and scrotal neoplasms were included in this study. The median surgical time for unilateral herniorrhaphy was 75 min. The median follow-up time was 13 months. The success of the autografting, based on no recurrence and comfort of the animals during urination and defecation, was found in ten of 11 hernias; giving a success rate of 90.91%. One hernia (9.09%) recurred 10 days after surgery. Histopathological examination of the apposing area between the graft and the adjacent tissue, taken during the repair of the recurred case at day 20, revealed neovascularization and connective tissue ingrowth. In conclusion, the tunica vaginalis autograft can be used for perineal herniorrhaphy in intact male dogs.

  13. Length of time domestic dogs (Canis familiaris) spend smelling urine of gonadectomised and intact conspecifics.

    Science.gov (United States)

    Riach, Anna C; Asquith, Rachel; Fallon, Melissa L D

    2017-09-01

    Domestic dogs (Canis familiaris) use urine to communicate among themselves, however, it is unknown whether the gonadectomy (neutering or spaying) of a dog affects this communication in anyway. Urine samples from 10 intact and 10 gonadectomised, unfamiliar dogs were presented to 12 tester dogs to sniff under controlled conditions in a pilot study. The amount of time the tester dogs spent sniffing each sample was recorded. Overall, tester dogs were recorded smelling the urine of gonadectomised individuals for a longer time. In addition to the type of urine sample, the result is likely to have been influenced by the sex and status (gonadectomised or intact) of the tester dogs. The observed increase in the length of time spent sniffing urine from gonadectomised individuals could be explained by the tester dogs experiencing more difficulty in gaining information from the urine or facing more confusion while analysing the urine compared to the intact urine they have evolved to smell. Copyright © 2017 Elsevier B.V. All rights reserved.

  14. EThcD Discrimination of Isomeric Leucine/Isoleucine Residues in Sequencing of the Intact Skin Frog Peptides with Intramolecular Disulfide Bond

    Science.gov (United States)

    Samgina, Tatiana Yu; Kovalev, Sergey V.; Tolpina, Miriam D.; Trebse, Polonca; Torkar, Gregor; Lebedev, Albert T.

    2018-05-01

    Our scientific interests involve de novo sequencing of non-tryptic natural amphibian skin peptides including those with intramolecular S-S bond by means of exclusively mass spectrometry. Reliable discrimination of the isomeric leucine/isoleucine residues during peptide sequencing by means of mass spectrometry represents a bottleneck in the workflow for complete automation of the primary structure elucidation of these compounds. MS3 is capable of solving the problem. Earlier we demonstrated the advanced efficiency of ETD-HCD method to discriminate Leu/Ile in individual peptides by consecutive application of ETD to the polyprotonated peptides followed by HCD applied to the manually selected primary z-ions with the targeted isomeric residues at their N-termini and registration of the characteristic w-ions. Later this approach was extended to deal with several (4-7) broad band mass ranges, without special isolation of the primary z-ions. The present paper demonstrates an advanced version of this method when EThcD is applied in the whole mass range to a complex mixture of natural non-tryptic peptides without their separation and intermediate isolation of the targeted z-ions. The proposed EThcD method showed over 81% efficiency for the large natural peptides with intact disulfide ring, while the interfering process of radical site migration is suppressed. Due to higher speed and sensitivity, the proposed EThcD approach facilitates the analytical procedure and allows for the automation of the entire experiment and data processing. Moreover, in some cases it gives a chance to establish the nature of the residues in the intact intramolecular disulfide loops. [Figure not available: see fulltext.

  15. Generation of a Novel T Cell Specific Interleukin-1 Receptor Type 1 Conditional Knock Out Mouse Reveals Intrinsic Defects in Survival, Expansion and Cytokine Production of CD4 T Cells.

    Directory of Open Access Journals (Sweden)

    Ilgiz A Mufazalov

    Full Text Available Interleukin-1 (IL-1 plays a crucial role in numerous inflammatory diseases via action on its only known signaling IL-1 receptor type 1 (IL-1R1. To investigate the role of IL-1 signaling in selected cell types, we generated a new mouse strain in which exon 5 of the Il1r1 gene is flanked by loxP sites. Crossing of these mice with CD4-Cre transgenic mice resulted in IL-1R1 loss of function specifically in T cells. These mice, termed IL-1R1ΔT, displayed normal development under steady state conditions. Importantly, isolated CD4 positive T cells retained their capacity to differentiate toward Th1 or Th17 cell lineages in vitro, and strongly proliferated in cultures supplemented with either anti-CD3/CD28 or Concanavalin A, but, as predicted, were completely unresponsive to IL-1β administration. Furthermore, IL-1R1ΔT mice were protected from gut inflammation in the anti-CD3 treatment model, due to dramatically reduced frequencies and absolute numbers of IL-17A and interferon (IFN-γ producing cells. Taken together, our data shows the necessity of intact IL-1 signaling for survival and expansion of CD4 T cells that were developed in an otherwise IL-1 sufficient environment.

  16. Development and Characterization of a Human and Mouse Intestinal Epithelial Cell Monolayer Platform

    Directory of Open Access Journals (Sweden)

    Kenji Kozuka

    2017-12-01

    Full Text Available Summary: We describe the development and characterization of a mouse and human epithelial cell monolayer platform of the small and large intestines, with a broad range of potential applications including the discovery and development of minimally systemic drug candidates. Culture conditions for each intestinal segment were optimized by correlating monolayer global gene expression with the corresponding tissue segment. The monolayers polarized, formed tight junctions, and contained a diversity of intestinal epithelial cell lineages. Ion transport phenotypes of monolayers from the proximal and distal colon and small intestine matched the known and unique physiology of these intestinal segments. The cultures secreted serotonin, GLP-1, and FGF19 and upregulated the epithelial sodium channel in response to known biologically active agents, suggesting intact secretory and absorptive functions. A screen of over 2,000 pharmacologically active compounds for inhibition of potassium ion transport in the mouse distal colon cultures led to the identification of a tool compound. : Siegel and colleagues describe their development of a human and mouse intestinal epithelial cell monolayer platform that maintains the cellular, molecular, and functional characteristics of tissue for each intestinal segment. They demonstrate the platform's application to drug discovery by screening a library of over 2,000 compounds to identify an inhibitor of potassium ion transport in the mouse distal colon. Keywords: intestinal epithelium, organoids, monolayer, colon, small intestine, phenotype screening assays, enteroid, colonoid

  17. 10. international mouse genome conference

    Energy Technology Data Exchange (ETDEWEB)

    Meisler, M.H.

    1996-12-31

    Ten years after hosting the First International Mammalian Genome Conference in Paris in 1986, Dr. Jean-Louis Guenet presided over the Tenth Conference at the Pasteur Institute, October 7--10, 1996. The 1986 conference was a satellite to the Human Gene Mapping Workshop and had approximately 50 attendees. The 1996 meeting was attended by 300 scientists from around the world. In the interim, the number of mapped loci in the mouse increased from 1,000 to over 20,000. This report contains a listing of the program and its participants, and two articles that review the meeting and the role of the laboratory mouse in the Human Genome project. More than 200 papers were presented at the conference covering the following topics: International mouse chromosome committee meetings; Mutant generation and identification; Physical and genetic maps; New technology and resources; Chromatin structure and gene regulation; Rate and hamster genetic maps; Informatics and databases; and Quantitative trait analysis.

  18. Teratology studies in the mouse.

    Science.gov (United States)

    Marsden, Edward; Leroy, Mariline

    2013-01-01

    The rat is the routine species of choice as the rodent model for regulatory safety testing of xenobiotics such as medicinal products, food additives, and other chemicals. However, the rat is not always suitable for pharmacological, toxicological, immunogenic, pharmacokinetic, or even practical reasons. Under such circumstances, the mouse offers an alternative for finding a suitable rodent model acceptable to the regulatory authorities. Since all essential routes of administration are possible, the short reproductive cycle and large litter size of the mouse make it a species well adapted for use in teratology studies. Given that good quality animals, including virgin mated females, can be acquired relatively easily and inexpensively, the mouse has been used in reproductive toxicity studies for decades and study protocols are well established.

  19. Mouse models of Fanconi anemia

    International Nuclear Information System (INIS)

    Parmar, Kalindi; D'Andrea, Alan; Niedernhofer, Laura J.

    2009-01-01

    Fanconi anemia is a rare inherited disease characterized by congenital anomalies, growth retardation, aplastic anemia and an increased risk of acute myeloid leukemia and squamous cell carcinomas. The disease is caused by mutation in genes encoding proteins required for the Fanconi anemia pathway, a response mechanism to replicative stress, including that caused by genotoxins that cause DNA interstrand crosslinks. Defects in the Fanconi anemia pathway lead to genomic instability and apoptosis of proliferating cells. To date, 13 complementation groups of Fanconi anemia were identified. Five of these genes have been deleted or mutated in the mouse, as well as a sixth key regulatory gene, to create mouse models of Fanconi anemia. This review summarizes the phenotype of each of the Fanconi anemia mouse models and highlights how genetic and interventional studies using the strains have yielded novel insight into therapeutic strategies for Fanconi anemia and into how the Fanconi anemia pathway protects against genomic instability.

  20. Mouse models of Fanconi anemia

    Energy Technology Data Exchange (ETDEWEB)

    Parmar, Kalindi; D' Andrea, Alan [Department of Radiation Oncology, Dana-Farber Cancer Institute, Harvard Medical School, 44 Binney Street, Boston, MA 02115 (United States); Niedernhofer, Laura J., E-mail: niedernhoferl@upmc.edu [Department of Microbiology and Molecular Genetics, University of Pittsburgh School of Medicine and Cancer Institute, 5117 Centre Avenue, Hillman Cancer Center, Research Pavilion 2.6, Pittsburgh, PA 15213-1863 (United States)

    2009-07-31

    Fanconi anemia is a rare inherited disease characterized by congenital anomalies, growth retardation, aplastic anemia and an increased risk of acute myeloid leukemia and squamous cell carcinomas. The disease is caused by mutation in genes encoding proteins required for the Fanconi anemia pathway, a response mechanism to replicative stress, including that caused by genotoxins that cause DNA interstrand crosslinks. Defects in the Fanconi anemia pathway lead to genomic instability and apoptosis of proliferating cells. To date, 13 complementation groups of Fanconi anemia were identified. Five of these genes have been deleted or mutated in the mouse, as well as a sixth key regulatory gene, to create mouse models of Fanconi anemia. This review summarizes the phenotype of each of the Fanconi anemia mouse models and highlights how genetic and interventional studies using the strains have yielded novel insight into therapeutic strategies for Fanconi anemia and into how the Fanconi anemia pathway protects against genomic instability.

  1. Regulation of Polysaccharide- and Protein- Specific Antibody Responses to Intact Extracellular Bacteria

    Science.gov (United States)

    2016-03-11

    OVA peptide (amino acids 323-339), presented by MHC-II I-Ad, were purchased from Taconic Farms (Hudson, NY). They were thereafter bred in our...overnight at 4°C and plates were then washed 3x with PBS + 0.1% Tween 20. Alkaline phosphatase-conjugated polyclonal goat anti-mouse IgG Abs 41 | P a g e...phosphatase-conjugated polyclonal goat anti-mouse IgG antibodies (200 ng/ml) in PBS + 1.0% BSA were then added, and plates were incubated at 37°C for

  2. Multi-Axis Prosthetic Knee Resembles Alpine Skiing Movements of an Intact Leg

    Directory of Open Access Journals (Sweden)

    Ivan Demšar, Jože Duhovnik, Blaž Lešnik, Matej Supej

    2015-12-01

    Full Text Available The purpose of the study was to analyse the flexion angles of the ski boot, ankle and knee joints of an above-knee prosthesis and to compare them with an intact leg and a control group of skiers. One subject with an above-knee amputation of the right leg and eight healthy subjects simulated the movement of a skiing turn by performing two-leg squats in laboratory conditions. By adding additional loads in proportion to body weight (BW; +1/3 BW, +2/3 BW, +3/3 BW, various skiing regimes were simulated. Change of Flexion Angle (CoFA and Range of Motion (RoM in the ski boot, ankle and knee joints were calculated and compared. An average RoM in the skiing boot on the side of prosthesis (4.4 ± 1.1° was significantly lower compared to an intact leg (5.9 ± 1.8° and the control group (6.5 ± 2.3°. In the ankle joint, the average RoM was determined to be 13.2±2.9° in the prosthesis, 12.7 ± 2.8° in an intact leg and 14.8±3.6 in the control group. However, the RoM of the knee joint in the prosthesis (42.2 ± 4.2° was significantly larger than that of the intact leg (34.7 ± 4.4°. The average RoM of the knee joint in the control group was 47.8 ± 5.4°. The influences of additional loads on the kinematics of the lower extremities were different on the side of the prosthesis and on the intact leg. In contrast, additional loads did not produce any significant differences in the control group. Although different CoFAs in the ski boot, ankle and knee joints were used, an above-knee prosthesis with a built-in multi-axis prosthetic knee enables comparable leg kinematics in simulated alpine skiing.

  3. The Ptch1DL mouse: a new model to study lambdoid craniosynostosis and basal cell nevus syndrome associated skeletal defects

    OpenAIRE

    Feng, Weiguo; Choi, Irene; Clouthier, David E.; Niswander, Lee; Williams, Trevor

    2013-01-01

    Mouse models provide valuable opportunities for probing the underlying pathology of human birth defects. Employing an ENU-based screen for recessive mutations affecting craniofacial anatomy we isolated a mouse strain, Dogface-like (DL), with abnormal skull and snout morphology. Examination of the skull indicated that these mice developed craniosynostosis of the lambdoid suture. Further analysis revealed skeletal defects related to the pathology of basal cell nevus syndrome (BCNS) including de...

  4. Induction and repair of strand breaks and 3'-hydroxy terminals in the DNA of mouse brain following gamma irradiation

    International Nuclear Information System (INIS)

    Yoshizawa, K.; Furuno, I.; Yada, T.; Matsudaira, H.

    1978-01-01

    DNA was isolated from mouse brain after in vivo γ-ray irradiation, treated with endonuclease S 1 from Aspergillus oryzae if necessary, and analysed further by alkaline and neutral sucrose gradient centrifugation. In parallel, its template activity was determined by DNA polymerase (EC 2.7.7.7, enzyme A of Klenow from Escherichia coli) assay as described previously. Similar experiments were performed with cultured mouse leukaemia cells (L5178Y) irradiated in vitro at 0 0 C. (Auth.)

  5. Mouse Resource Browser-a database of mouse databases

    NARCIS (Netherlands)

    Zouberakis, Michael; Chandras, Christina; Swertz, Morris; Smedley, Damian; Gruenberger, Michael; Bard, Jonathan; Schughart, Klaus; Rosenthal, Nadia; Hancock, John M.; Schofield, Paul N.; Kollias, George; Aidinis, Vassilis

    2010-01-01

    The laboratory mouse has become the organism of choice for discovering gene function and unravelling pathogenetic mechanisms of human diseases through the application of various functional genomic approaches. The resulting deluge of data has led to the deployment of numerous online resources and the

  6. Examining an underappreciated control on lignin decomposition in soils? Effects of reactive manganese species on intact plant cell walls

    Science.gov (United States)

    Keiluweit, M.; Bougoure, J.; Pett-Ridge, J.; Kleber, M.; Nico, P. S.

    2011-12-01

    Lignin comprises a dominant proportion of carbon fluxes into the soil (representing up to 50% of plant litter and roots). Two lines of evidence suggest that manganese (Mn) acts as a strong controlling factor on the residence time of lignin in soil ecosystems. First, Mn content is highly correlated with litter decomposition in temperate and boreal forest soil ecosystems and, second, microbial agents of lignin degradation have been reported to rely on reactive Mn(III)-complexes to specifically oxidize lignin. However, few attempts have been made to isolate the mechanisms responsible for the apparent Mn-dependence of lignin decomposition in soils. Here we tested the hypothesis that Mn(III)-oxalate complexes may act as a perforating 'pretreatment' for structurally intact plant cell walls. We propose that these diffusible oxidizers are small enough to penetrate and react with non-porous ligno-cellulose in cell walls. This process was investigated by reacting single Zinnia elegans tracheary elements with Mn(III)-oxalate complexes in a continuous flow-through microreactor. The uniformity of cultured tracheary elements allowed us to examine Mn(III)-induced changes in cell wall chemistry and ultrastructure on the micro-scale using fluorescence and electron microscopy as well as synchrotron-based infrared and X-ray spectromicroscopy. Our results show that Mn(III)-complexes substantially oxidize specific lignin components of the cell wall, solubilize decomposition products, severely undermine the cell wall integrity, and cause cell lysis. We conclude that Mn(III)-complexes induce oxidative damage in plant cell walls that renders ligno-cellulose substrates more accessible for microbial lignin- and cellulose-decomposing enzymes. Implications of our results for the rate limiting impact of soil Mn speciation and availability on litter decomposition in forest soils will be discussed.

  7. Intact nitric oxide production is obligatory for the sustained flow response during hypercapnic acidosis in guinea pig heart.

    Science.gov (United States)

    Heintz, Anke; Koch, Thea; Deussen, Andreas

    2005-04-01

    The mechanisms underlying hypercapnic coronary dilation remain unsettled. This study tests the hypothesis that flow dependent NO production is obligatory for the hypercapnic flow response. In isolated, constant pressure (CP) perfused guinea pig hearts a step change of arterial pCO(2) from 38.6 to 61.4 mm Hg induced a bi-phasic flow response with an early transient (maximum 60 s) and a consecutive persisting flow rise (121.6+/-6.6 (S.D.) % after 10 min). In contrast, when perfused with constant flow (CF), perfusion pressure only transiently (2 min) fell by 7.4+/-4.8 % following the step change of arterial pCO(2). In CP perfused hearts L-NAME (100 micromol/l) specifically abolished the delayed flow rise during hypercapnic acidosis (102.37+/-2.9% after 10 min), whereas the inhibitor had no effect on perfusion pressure response in CF perfused hearts. Under CP perfusion arterial hypercapnia resulted in a transient rise of coronary cGMP release (from 0.69+/-0.35 to 1.12+/-0.68 pmol/ml), which was abolished after L-NAME. Surprisingly, the K(+)ATP channel blocker glibenclamide did not have any significant effect on the hypercapnic flow response but largely blunted reactive hyperemia after a 20 s flow stop. The delayed steady state hypercapnic flow response in guinea pig heart requires intact NO production. The absence of a persisting decrease in coronary resistance under CF perfusion points to an important role of shear stress dependent NO production.

  8. Decomposition of intact chicken feathers by a thermophile in combination with an acidulocomposting garbage-treatment process.

    Science.gov (United States)

    Shigeri, Yasushi; Matsui, Tatsunobu; Watanabe, Kunihiko

    2009-11-01

    In order to develop a practical method for the decomposition of intact chicken feathers, a moderate thermophile strain, Meiothermus ruber H328, having strong keratinolytic activity, was used in a bio-type garbage-treatment machine working with an acidulocomposting process. The addition of strain H328 cells (15 g) combined with acidulocomposting in the garbage machine resulted in 70% degradation of intact chicken feathers (30 g) within 14 d. This degradation efficiency is comparable to a previous result employing the strain as a single bacterium in flask culture, and it indicates that strain H328 can promote intact feather degradation activity in a garbage machine currently on the market.

  9. Transfer Comparison Study Nitrogen on the Intact and Decapitated Legumes by Using the 15N Labeling Technique

    International Nuclear Information System (INIS)

    Widjayanto, Didik W.

    1998-01-01

    The experiment was done in order to evaluate the N transfer from the intact and decapitated legumes by using the 15 N labeling technique. Seven days after final labeling the above ground biomass from labeled legume species was removed and the remaining stalks capped to prevent regrowth. Twenty days after final labeling (fourteen days after capping) the all treatments were sample and analyzed. The decapitated legumes appeared to transfer more percentage N than the intact legumes. Although both decapitated and intact legumes transferred, the transfer of N did not incur a dry matter and N yield benefit

  10. A Functional Analysis on the Interspecies Interaction between Mouse LFA-1 and Human Intercellular Adhesion Molecule-1 at the Cell Level

    Directory of Open Access Journals (Sweden)

    David Núñez

    2017-12-01

    Full Text Available The interaction between intercellular adhesion molecules (ICAM and the integrin leukocyte function-associated antigen-1 (LFA-1 is crucial for the regulation of several physiological and pathophysiological processes like cell-mediated elimination of tumor or virus infected cells, cancer metastasis, or inflammatory and autoimmune processes. Using purified proteins it was reported a species restriction for the interaction of ICAM-1 and LFA-1, being mouse ICAM-1 able to interact with human LFA-1 but not human ICAM-1 with mouse LFA-1. However, in vivo results employing tumor cells transfected with human ICAM-1 suggest that functionally mouse LFA-1 can recognize human ICAM-1. In order to clarify the interspecies cross-reactivity of the ICAM-1/LFA-1 interaction, we have performed functional studies analyzing the ability of human soluble ICAM-1 and human/mouse LFA-1 derived peptides to inhibit cell aggregation and adhesion as well as cell-mediated cytotoxicity in both mouse and human systems. In parallel, the affinity of the interaction between mouse LFA-1-derived peptides and human ICAM-1 was determined by calorimetry assays. According to the results obtained, it seems that human ICAM-1 is able to interact with mouse LFA-1 on intact cells, which should be taking into account when using humanized mice and xenograft models for the study of immune-related processes.

  11. A Functional Analysis on the Interspecies Interaction between Mouse LFA-1 and Human Intercellular Adhesion Molecule-1 at the Cell Level.

    Science.gov (United States)

    Núñez, David; Comas, Laura; Lanuza, Pilar M; Sánchez-Martinez, Diego; Pérez-Hernández, Marta; Catalán, Elena; Domingo, María Pilar; Velázquez-Campoy, Adrián; Pardo, Julián; Gálvez, Eva M

    2017-01-01

    The interaction between intercellular adhesion molecules (ICAM) and the integrin leukocyte function-associated antigen-1 (LFA-1) is crucial for the regulation of several physiological and pathophysiological processes like cell-mediated elimination of tumor or virus infected cells, cancer metastasis, or inflammatory and autoimmune processes. Using purified proteins it was reported a species restriction for the interaction of ICAM-1 and LFA-1, being mouse ICAM-1 able to interact with human LFA-1 but not human ICAM-1 with mouse LFA-1. However, in vivo results employing tumor cells transfected with human ICAM-1 suggest that functionally mouse LFA-1 can recognize human ICAM-1. In order to clarify the interspecies cross-reactivity of the ICAM-1/LFA-1 interaction, we have performed functional studies analyzing the ability of human soluble ICAM-1 and human/mouse LFA-1 derived peptides to inhibit cell aggregation and adhesion as well as cell-mediated cytotoxicity in both mouse and human systems. In parallel, the affinity of the interaction between mouse LFA-1-derived peptides and human ICAM-1 was determined by calorimetry assays. According to the results obtained, it seems that human ICAM-1 is able to interact with mouse LFA-1 on intact cells, which should be taking into account when using humanized mice and xenograft models for the study of immune-related processes.

  12. Study and ICH validation of a reverse-phase liquid chromatographic method for the quantification of the intact monoclonal antibody cetuximab

    Directory of Open Access Journals (Sweden)

    Antonio Martínez-Ortega

    2016-04-01

    Full Text Available Cetuximab (CTX is a potent chimeric mouse/human monoclonal antibody (mAb approved worldwide for treatment of metastatic colorectal cancer. Among the various biological and physical analyses performed for full study on this biopharmaceutic, the determination of the concentration preparations throughout manufacturing and subsequent handling in hospital is particularly relevant. In the present work, the study and validation of a method for quantifying intact CTX by reverse-phase high-performance liquid chromatography with diode array detection ((RPHPLC/DAD is presented. With that end, we checked the performance of a chromatographic method for quantifying CTX and conducted a study to validate the method as stability-indicating in accordance with the International Conference on Harmonization guidelines (ICH for biotechnological drugs; therefore, we evaluated linearity, accuracy, precision, detection and quantification limits, robustness and system suitability. The specificity of the method and the robustness of the mAb formulation against external stress factors were estimated by comprehensive chromatographic analysis by subjecting CTX to several informative stress conditions. As demonstrated, the method is rapid, accurate, and reproducible for CTX quantification. It was also successfully used to quantify CTX in a long-term stability study performed under hospital conditions.

  13. Novel mouse model of chronic Pseudomonas aeruginosa lung infection mimicking cystic fibrosis

    DEFF Research Database (Denmark)

    Hoffmann, Nadine; Rasmussen, Thomas Bovbjerg; Jensen, Peter Østrup

    2005-01-01

    (NH57388C) from the mucoid isolate (NH57388A) and a nonmucoid isolate (NH57388B) deficient in AHL were almost cleared from the lungs of the mice. This model, in which P. aeruginosa is protected against the defense system of the lung by alginate, is similar to the clinical situation. Therefore...... pulmonary mouse model without artificial embedding. The model is based on a stable mucoid CF sputum isolate (NH57388A) with hyperproduction of alginate due to a deletion in mucA and functional N-acylhomoserine lactone (AHL)-based quorum-sensing systems. Chronic lung infection could be established in both CF...

  14. Excision of pyrimidine dimers from epidermal DNA and nonsemiconservative epidermal DNA synthesis following ultraviolet irradiation of mouse skin

    International Nuclear Information System (INIS)

    Bowden, G.T.; Trosko, J.E.; Shapas, B.G.; Boutwell, R.K.

    1975-01-01

    Pyrimidine dimer production and excision in epidermal DNA were studied at five different dose levels of ultraviolet light in the skin of intact mice. Dimer production increased with dose up to 50,400 ergs/sq mm. Approximately 30 percent of the thymine-containing dimers were excised by 24 hr after irradiation at three lower dose levels of ultraviolet light. Nonsemiconservative DNA replication in ultraviolet-irradiated mouse skin was shown to continue for at least 18 hr. The rate of nonsemiconservative replication decreased with time, but did so slowly. The initial rates of nonsemiconservative replication increased with ultraviolet light dose levels up to about 4200 ergs/sq mm, after which the initial rates were decreased. Semiconservative epidermal DNA synthesis was shown to be inhibited by hydroxyurea, but hydroxyurea had no effect on ultraviolet light-induced nonsemiconservative DNA replication. The observed pyrimidine dimer excision and nonsemiconservative DNA replication suggest that in the intact mouse the cells of the epidermis are capable of DNA excision repair after ultraviolet irradiation of mouse skin

  15. Characterization of mitomycin-C-sensitive mouse lymphoma L5178Y cell mutants

    International Nuclear Information System (INIS)

    Inaba, Hiroko; Shiomi, Naoko; Shiomi, Tadahiro; Sato, Koki; Yoshida, Michihiro.

    1985-01-01

    Twenty-six mutants showing high sensitivity to mytomicin-C (MMC) were isolated from mouse lymphoma L5178Y cells by a replica-plating technique. Twenty-five of the mutants were 5 - 10 times more sensitive to MMC than were parental cells, and showed normal sensitivity to U.V. light and x-rays. From a complementation analysis, 5 mutants (MC s ) isolated from independently mutagenized cell populations were classified into two groups. These mutants possessed recessive character for MMC-sensitivity and there were at least two genes involved in the MMC-sensitivity. As for DNA-damaging factors, such as photoadducts of 8-methoxypsoralen (8-MOP) and 3-carbethoxysoralen (3-CPs), MC s mutants showed higher sensitivity to photoadducts of 8-MOP than to (3-CPs). MC s mutants were also highly sensitive to a DNA cross-linking agent, cisplatin. Characterization of the sensitivity of mouse MC s mutants was analogous to that of Fanconi's anemia (FA)-derived cells. Low concentrations (10 ng/ml) of MMC induced chromosome aberration in a high incidence in mouse MC s cells, as well as in FA cells. The frequency of MMC-induced chromosome aberrations was normal in hybrid cells between normal human diploid somatic cells and mouse mutants and between FA cells and mouse wild cells, and hereditary deficiency became normal by hybrization. (Namekawa, K.)

  16. Prospective Validation of a High Dimensional Shape Model for Organ Motion in Intact Cervical Cancer

    Energy Technology Data Exchange (ETDEWEB)

    Williamson, Casey W.; Green, Garrett; Noticewala, Sonal S.; Li, Nan; Shen, Hanjie [Department of Radiation Medicine and Applied Sciences, University of California, San Diego, La Jolla, California (United States); Vaida, Florin [Division of Biostatistics and Bioinformatics, Department of Family Medicine and Public Health, University of California, San Diego, La Jolla, California (United States); Mell, Loren K., E-mail: lmell@ucsd.edu [Department of Radiation Medicine and Applied Sciences, University of California, San Diego, La Jolla, California (United States)

    2016-11-15

    Purpose: Validated models are needed to justify strategies to define planning target volumes (PTVs) for intact cervical cancer used in clinical practice. Our objective was to independently validate a previously published shape model, using data collected prospectively from clinical trials. Methods and Materials: We analyzed 42 patients with intact cervical cancer treated with daily fractionated pelvic intensity modulated radiation therapy and concurrent chemotherapy in one of 2 prospective clinical trials. We collected online cone beam computed tomography (CBCT) scans before each fraction. Clinical target volume (CTV) structures from the planning computed tomography scan were cast onto each CBCT scan after rigid registration and manually redrawn to account for organ motion and deformation. We applied the 95% isodose cloud from the planning computed tomography scan to each CBCT scan and computed any CTV outside the 95% isodose cloud. The primary aim was to determine the proportion of CTVs that were encompassed within the 95% isodose volume. A 1-sample t test was used to test the hypothesis that the probability of complete coverage was different from 95%. We used mixed-effects logistic regression to assess effects of time and patient variability. Results: The 95% isodose line completely encompassed 92.3% of all CTVs (95% confidence interval, 88.3%-96.4%), not significantly different from the 95% probability anticipated a priori (P=.19). The overall proportion of missed CTVs was small: the grand mean of covered CTVs was 99.9%, and 95.2% of misses were located in the anterior body of the uterus. Time did not affect coverage probability (P=.71). Conclusions: With the clinical implementation of a previously proposed PTV definition strategy based on a shape model for intact cervical cancer, the probability of CTV coverage was high and the volume of CTV missed was low. This PTV expansion strategy is acceptable for clinical trials and practice; however, we recommend daily

  17. Does intact pleura during internal mammary artery harvesting decreases pleuropulmonary complications

    International Nuclear Information System (INIS)

    Paracha, V.I.; Iqbal, M.A.; Rashid, A.

    2011-01-01

    Objective: To evaluate the effects on postoperative pulmonary function of pleural integrity versus opened pleura, in patients who receive a left internal mammary artery graft. Study Design: Randomized control trial. Place and Duration of study: Armed Forces Institute of Cardiology and National Institute of Heart Diseases, Rawalpindi from June 2009 to Feb 2010. Patients and Methods: The study was conducted on 62 patients who underwent elective coronary artery bypass grafting. The patients were divided into 2 groups: Group A consisted of patients who underwent internal mammary artery harvesting with opened pleura (n=31) while Group B consisted of patients having internal mammary artery harvested with intact pleura (n=31). To monitor pleuro-pulmonary complications, chest radiography was performed routinely 1 day before operation and on the second, fourth and sixth postoperative days. The preoperative, after extubation, and first postoperative day values of partial oxygen pressure (PaO/sub 2/), partial carbon dioxide pressure (PaCO/sub 2/), oxygen (O/sub 2/) saturation and haematocrit were recorded for comparison. The effect of intact pleura on IMA traction was also recorded by serial cardiac enzymes measurement and electrocardiography in the immediate postoperative period. Results: Pleural effusion occurred more often in patients of Group A (35.5% versus 12.9%) which was statistically significant (P 0.038). However, no significant differences were recorded in postoperative respiratory complications. Reoperation for postoperative bleeding and tamponade was done in 6.5% and 3.2% of cases in Group B respectively but none Group A; however it was not statistically significant (P 0.076). The mean postoperative hospital stay was 7.84 days for Group A and 7.58 days for Group B. Conclusion: Pleural integrity during IMA harvesting decreases pleural effusion. Although, no significant differences were recorded in postoperative lung complications, intact pleural technique of IMA

  18. Human skin penetration of silver nanoparticles through intact and damaged skin

    International Nuclear Information System (INIS)

    Larese, Francesca Filon; D'Agostin, Flavia; Crosera, Matteo; Adami, Gianpiero; Renzi, Nadia; Bovenzi, Massimo; Maina, Giovanni

    2009-01-01

    There is a growing interest on nanoparticle safety for topical use. The benefits of nanoparticles have been shown in several scientific fields, but little is known about their potential to penetrate the skin. This study aims at evaluating in vitro skin penetration of silver nanoparticles. Experiments were performed using the Franz diffusion cell method with intact and damaged human skin. Physiological solution was used as receiving phase and 70 μg/cm 2 of silver nanoparticles coated with polyvinylpirrolidone dispersed in synthetic sweat were applied as donor phase to the outer surface of the skin for 24 h. The receptor fluid measurements were performed by electro thermal atomic absorption spectroscopy (ETAAS). Human skin penetration was also determined by using transmission electron microscope (TEM) to verify the location of silver nanoparticles in exposed membranes. Median silver concentrations of 0.46 ng cm -2 (range -2 (range 0.43-11.6) were found in the receiving solutions of cells where the nanoparticles solution was applied on intact skin (eight cells) and on damaged skin (eight cells), respectively. Twenty-four hours silver flux permeation in damaged skin was 0.62 ± 0.2 ng cm -2 with a lag time <1 h. Our experimental data showed that silver nanoparticles absorption through intact and damaged skin was very low but detectable, and that in case of damaged skin it was possible an increasing permeation of silver applied as nanoparticles. Moreover, silver nanoparticles could be detected in the stratum corneum and the outermost surface of the epidermis by electron microscopy. We demonstrated for the first time that silver applied as nanoparticles coated with polyvinylpirrolidone is able to permeate the damaged skin in an in vitro diffusion cell system

  19. Bioengineered human IAS reconstructs with functional and molecular properties similar to intact IAS

    Science.gov (United States)

    Singh, Jagmohan

    2012-01-01

    Because of its critical importance in rectoanal incontinence, we determined the feasibility to reconstruct internal anal sphincter (IAS) from human IAS smooth muscle cells (SMCs) with functional and molecular attributes similar to the intact sphincter. The reconstructs were developed using SMCs from the circular smooth muscle layer of the human IAS, grown in smooth muscle differentiation media under sterile conditions in Sylgard-coated tissue culture plates with central Sylgard posts. The basal tone in the reconstructs and its changes were recorded following 0 Ca2+, KCl, bethanechol, isoproterenol, protein kinase C (PKC) activator phorbol 12,13-dibutyrate, and Rho kinase (ROCK) and PKC inhibitors Y-27632 and Gö-6850, respectively. Western blot (WB), immunofluorescence (IF), and immunocytochemical (IC) analyses were also performed. The reconstructs developed spontaneous tone (0.68 ± 0.26 mN). Bethanechol (a muscarinic agonist) and K+ depolarization produced contraction, whereas isoproterenol (β-adrenoceptor agonist) and Y-27632 produced a concentration-dependent decrease in the tone. Maximal decrease in basal tone with Y-27632 and Gö-6850 (each 10−5 M) was 80.45 ± 3.29 and 17.76 ± 3.50%, respectively. WB data with the IAS constructs′ SMCs revealed higher levels of RhoA/ROCK, protein kinase C-potentiated inhibitor or inhibitory phosphoprotein for myosin phosphatase (CPI-17), phospho-CPI-17, MYPT1, and 20-kDa myosin light chain vs. rectal smooth muscle. WB, IF, and IC studies of original SMCs and redispersed from the reconstructs for the relative distribution of different signal transduction proteins confirmed the feasibility of reconstruction of IAS with functional properties similar to intact IAS and demonstrated the development of myogenic tone with critical dependence on RhoA/ROCK. We conclude that it is feasible to bioengineer IAS constructs using human IAS SMCs that behave like intact IAS. PMID:22790596

  20. Investigations on the role of hemoglobin in sulfide metabolism by intact human red blood cells.

    Science.gov (United States)

    Bianco, Christopher L; Savitsky, Anton; Feelisch, Martin; Cortese-Krott, Miriam M

    2018-03-01

    In addition to their role as oxygen transporters, red blood cells (RBCs) contribute to cardiovascular homeostasis by regulating nitric oxide (NO) metabolism via interaction of hemoglobin (Hb) with nitrite and NO itself. RBCs were proposed to also participate in sulfide metabolism. Although Hb is known to react with sulfide, sulfide metabolism by intact RBCs has not been characterized so far. Therefore we explored the role of Hb in sulfide metabolism in intact human RBCs. We find that upon exposure of washed RBCs to sulfide, no changes in oxy/deoxyhemoglobin (oxy/deoxyHb) are observed by UV-vis and EPR spectroscopy. However, sulfide reacts with methemoglobin (metHb), forming a methemoglobin-sulfide (metHb-SH) complex. Moreover, while metHb-SH is stable in cell-free systems even in the presence of biologically relevant thiols, it gradually decomposes to produce oxyHb, inorganic polysulfides and thiosulfate in intact cells, as detected by EPR and mass spectrometry. Taken together, our results demonstrate that under physiological conditions RBCs are able to metabolize sulfide via intermediate formation of a metHb-SH complex, which subsequently decomposes to oxyHb. We speculate that decomposition of metHb-SH is preceded by an inner-sphere electron transfer, forming reduced Hb (which binds oxygen to form oxyHb) and thiyl radical (a process we here define as "reductive sulfhydration"), which upon release, gives rise to the oxidized products, thiosulfate and polysulfides. Thus, not only is metHb an efficient scavenger and regulator of sulfide in blood, intracellular sulfide itself may play a role in keeping Hb in the reduced oxygen-binding form and, therefore, be involved in RBC physiology and function. Copyright © 2018 The Authors. Published by Elsevier Inc. All rights reserved.

  1. Release of proteins from intact chloroplasts induced by reactive oxygen species during biotic and abiotic stress.

    Science.gov (United States)

    Kwon, Kwang-Chul; Verma, Dheeraj; Jin, Shuangxia; Singh, Nameirakpam D; Daniell, Henry

    2013-01-01

    Plastids sustain life on this planet by providing food, feed, essential biomolecules and oxygen. Such diverse metabolic and biosynthetic functions require efficient communication between plastids and the nucleus. However, specific factors, especially large molecules, released from plastids that regulate nuclear genes have not yet been fully elucidated. When tobacco and lettuce transplastomic plants expressing GFP within chloroplasts, were challenged with Erwinia carotovora (biotic stress) or paraquat (abiotic stress), GFP was released into the cytoplasm. During this process GFP moves gradually towards the envelope, creating a central red zone of chlorophyll fluorescence. GFP was then gradually released from intact chloroplasts into the cytoplasm with an intact vacuole and no other visible cellular damage. Different stages of GFP release were observed inside the same cell with a few chloroplasts completely releasing GFP with detection of only red chlorophyll fluorescence or with no reduction in GFP fluorescence or transitional steps between these two phases. Time lapse imaging by confocal microscopy clearly identified sequence of these events. Intactness of chloroplasts during this process was evident from chlorophyll fluorescence emanated from thylakoid membranes and in vivo Chla fluorescence measurements (maximum quantum yield of photosystem II) made before or after infection with pathogens to evaluate their photosynthetic competence. Hydrogen peroxide and superoxide anion serve as signal molecules for generation of reactive oxygen species and Tiron, scavenger of superoxide anion, blocked release of GFP from chloroplasts. Significant increase in ion leakage in the presence of paraquat and light suggests changes in the chloroplast envelope to facilitate protein release. Release of GFP-RC101 (an antimicrobial peptide), which was triggered by Erwinia infection, ceased after conferring protection, further confirming this export phenomenon. These results suggest a

  2. Release of proteins from intact chloroplasts induced by reactive oxygen species during biotic and abiotic stress.

    Directory of Open Access Journals (Sweden)

    Kwang-Chul Kwon

    Full Text Available Plastids sustain life on this planet by providing food, feed, essential biomolecules and oxygen. Such diverse metabolic and biosynthetic functions require efficient communication between plastids and the nucleus. However, specific factors, especially large molecules, released from plastids that regulate nuclear genes have not yet been fully elucidated. When tobacco and lettuce transplastomic plants expressing GFP within chloroplasts, were challenged with Erwinia carotovora (biotic stress or paraquat (abiotic stress, GFP was released into the cytoplasm. During this process GFP moves gradually towards the envelope, creating a central red zone of chlorophyll fluorescence. GFP was then gradually released from intact chloroplasts into the cytoplasm with an intact vacuole and no other visible cellular damage. Different stages of GFP release were observed inside the same cell with a few chloroplasts completely releasing GFP with detection of only red chlorophyll fluorescence or with no reduction in GFP fluorescence or transitional steps between these two phases. Time lapse imaging by confocal microscopy clearly identified sequence of these events. Intactness of chloroplasts during this process was evident from chlorophyll fluorescence emanated from thylakoid membranes and in vivo Chla fluorescence measurements (maximum quantum yield of photosystem II made before or after infection with pathogens to evaluate their photosynthetic competence. Hydrogen peroxide and superoxide anion serve as signal molecules for generation of reactive oxygen species and Tiron, scavenger of superoxide anion, blocked release of GFP from chloroplasts. Significant increase in ion leakage in the presence of paraquat and light suggests changes in the chloroplast envelope to facilitate protein release. Release of GFP-RC101 (an antimicrobial peptide, which was triggered by Erwinia infection, ceased after conferring protection, further confirming this export phenomenon. These

  3. Palladium nanoparticles exposure: Evaluation of permeation through damaged and intact human skin.

    Science.gov (United States)

    Larese Filon, Francesca; Crosera, Matteo; Mauro, Marcella; Baracchini, Elena; Bovenzi, Massimo; Montini, Tiziano; Fornasiero, Paolo; Adami, Gianpiero

    2016-07-01

    The intensified use of palladium nanoparticles (PdNPs) in many chemical reactions, jewellery, electronic devices, in car catalytic converters and in biomedical applications lead to a significant increase in palladium exposure. Pd can cause allergic contact dermatitis when in contact with the skin. However, there is still a lack of toxicological data related to nano-structured palladium and information on human cutaneous absorption. In fact, PdNPs, can be absorbed through the skin in higher amounts than bulk Pd because NPs can release more ions. In our study, we evaluated the absorption of PdNPs, with a size of 10.7 ± 2.8 nm, using intact and damaged human skin in Franz cells. 0.60 mg cm(-2) of PdNPs were applied on skin surface for 24 h. Pd concentrations in the receiving solutions at the end of experiments were 0.098 ± 0.067 μg cm(-2) and 1.06 ± 0.44 μg cm(-2) in intact skin and damaged skin, respectively. Pd flux permeation after 24 h was 0.005 ± 0.003 μg cm(-2) h(-1) and 0.057 ± 0.030 μg cm(-2) h(-1) and lag time 4.8 ± 1.7 and 4.2 ± 3.6 h, for intact and damaged skin respectively. This study indicates that Pd can penetrate human skin. Copyright © 2016 Elsevier Ltd. All rights reserved.

  4. Effects of reactive Mn(III)-oxalate complexes on structurally intact plant cell walls

    Science.gov (United States)

    Summering, J. A.; Keiluweit, M.; Goni, M. A.; Nico, P. S.; Kleber, M.

    2011-12-01

    Lignin components in the in plant litter are commonly assumed to have longer residence times in soil than many other compounds, which are supposedly, more easily degradable. The supposed resistance of lignin compounds to decomposition is generally attributed to the complex chain of biochemical steps required to create footholds in the non-porous structure of ligno-cellulose in cell walls. Interestingly, Mn(III) complexes have shown the ability to degrade ligno-cellulose. Mn(III) chelated by ligands such as oxalate are soluble oxidizers with a high affinity for lignin structures. Here we determined (i) the formation and decay kinetics of the Mn(III)-oxalate complexes in aqueous solution and (ii) the effects that these complexes have on intact ligno-cellulose. UV/vis spectroscopy and iodometric titrations confirmed the transient nature of Mn(III)-oxalate complexes with decay rates being in the order of hours. Zinnia elegans tracheary elements - a model ligno-cellulose substrate - were treated with Mn(III)-oxalate complexes in a newly developed flow-through reactor. Soluble decomposition products released during the treatment were analyzed by GC/MS and the degree of cell integrity was measured by cell counts, pre- and post-treatment counts indicate a decrease in intact Zinnia elegans as a result of Mn(III)-treatment. GC/MS results showed the release of a multitude of solubilized lignin breakdown products from plant cell walls. We conclude that Mn(III)-oxalate complexes have the ability to lyse intact plant cells and solubilize lignin. Lignin decomposition may thus be seen as resource dependent, with Mn(III) a powerful resource that should be abundant in terrestrial characterized by frequent redox fluctuations.

  5. Spatially Rearranged Object Parts Can Facilitate Perception of Intact Whole Objects

    Directory of Open Access Journals (Sweden)

    Laura eCacciamani

    2014-05-01

    Full Text Available The familiarity of an object depends on the spatial arrangement of its parts; when the parts are spatially rearranged, they form a novel, unrecognizable configuration. Yet the same collection of parts comprises both the familiar and novel configuration. Is it possible that the collection of familiar parts activates a representation of the intact familiar configuration even when they are spatially rearranged? We presented novel configurations as primes before test displays that assayed effects on figure-ground perception from memories of intact familiar objects. In our test displays, two equal-area regions shared a central border; one region depicted a portion of a familiar object. Previous research with such displays has shown that participants are more likely to perceive the region depicting a familiar object as the figure and the abutting region as its ground when the familiar object is depicted in its upright orientation rather than upside down. The novel primes comprised either the same or a different collection of parts as the familiar object in the test display (part-rearranged and control primes, respectively. We found that participants were more likely to perceive the familiar region as figure in upright vs. inverted displays following part-rearranged primes but not control primes. Thus, priming with a novel configuration comprising the same familiar parts as the upcoming figure-ground display facilitated orientation-dependent effects of object memories on figure assignment. Similar results were obtained when the spatially rearranged collection of parts was suggested on the groundside of the prime’s border, suggesting that familiar parts in novel configurations access the representation of their corresponding intact whole object before figure assignment. These data demonstrate that familiar parts access memories of familiar objects even when they are arranged in a novel configuration.

  6. Spatially rearranged object parts can facilitate perception of intact whole objects.

    Science.gov (United States)

    Cacciamani, Laura; Ayars, Alisabeth A; Peterson, Mary A

    2014-01-01

    The familiarity of an object depends on the spatial arrangement of its parts; when the parts are spatially rearranged, they form a novel, unrecognizable configuration. Yet the same collection of parts comprises both the familiar and novel configuration. Is it possible that the collection of familiar parts activates a representation of the intact familiar configuration even when they are spatially rearranged? We presented novel configurations as primes before test displays that assayed effects on figure-ground perception from memories of intact familiar objects. In our test displays, two equal-area regions shared a central border; one region depicted a portion of a familiar object. Previous research with such displays has shown that participants are more likely to perceive the region depicting a familiar object as the figure and the abutting region as its ground when the familiar object is depicted in its upright orientation rather than upside down. The novel primes comprised either the same or a different collection of parts as the familiar object in the test display (part-rearranged and control primes, respectively). We found that participants were more likely to perceive the familiar region as figure in upright vs. inverted displays following part-rearranged primes but not control primes. Thus, priming with a novel configuration comprising the same familiar parts as the upcoming figure-ground display facilitated orientation-dependent effects of object memories on figure assignment. Similar results were obtained when the spatially rearranged collection of parts was suggested on the groundside of the prime's border, suggesting that familiar parts in novel configurations access the representation of their corresponding intact whole object before figure assignment. These data demonstrate that familiar parts access memories of familiar objects even when they are arranged in a novel configuration.

  7. Prototypic and Arkypallidal Neurons in the Dopamine-Intact External Globus Pallidus

    Science.gov (United States)

    Abdi, Azzedine; Mallet, Nicolas; Mohamed, Foad Y.; Sharott, Andrew; Dodson, Paul D.; Nakamura, Kouichi C.; Suri, Sana; Avery, Sophie V.; Larvin, Joseph T.; Garas, Farid N.; Garas, Shady N.; Vinciati, Federica; Morin, Stéphanie; Bezard, Erwan

    2015-01-01

    Studies in dopamine-depleted rats indicate that the external globus pallidus (GPe) contains two main types of GABAergic projection cell; so-called “prototypic” and “arkypallidal” neurons. Here, we used correlative anatomical and electrophysiological approaches in rats to determine whether and how this dichotomous organization applies to the dopamine-intact GPe. Prototypic neurons coexpressed the transcription factors Nkx2-1 and Lhx6, comprised approximately two-thirds of all GPe neurons, and were the major GPe cell type innervating the subthalamic nucleus (STN). In contrast, arkypallidal neurons expressed the transcription factor FoxP2, constituted just over one-fourth of GPe neurons, and innervated the striatum but not STN. In anesthetized dopamine-intact rats, molecularly identified prototypic neurons fired at relatively high rates and with high regularity, regardless of brain state (slow-wave activity or spontaneous activation). On average, arkypallidal neurons fired at lower rates and regularities than prototypic neurons, and the two cell types could be further distinguished by the temporal coupling of their firing to ongoing cortical oscillations. Complementing the activity differences observed in vivo, the autonomous firing of identified arkypallidal neurons in vitro was slower and more variable than that of prototypic neurons, which tallied with arkypallidal neurons displaying lower amplitudes of a “persistent” sodium current important for such pacemaking. Arkypallidal neurons also exhibited weaker driven and rebound firing compared with prototypic neurons. In conclusion, our data support the concept that a dichotomous functional organization, as actioned by arkypallidal and prototypic neurons with specialized molecular, structural, and physiological properties, is fundamental to the operations of the dopamine-intact GPe. PMID:25926446

  8. Isolated traumatic aniridia after trabeculectomy in a pseudophakic eye

    Directory of Open Access Journals (Sweden)

    Subashini Kaliaperumal

    2014-01-01

    Full Text Available This is a single case report of an elderly patient who had blunt trauma in an eye that had phacoemulsification and two trabeculectomies. She had good vision with a well-functioning bleb before the trauma. She presented during her routine follow-up visit for glaucoma with isolated aniridia and an intact globe. The capsular bag, zonules, and the intraocular lens were intact. The cupping was 0.8, and the rest of the fundus and macula were normal. Pigments were seen over the sclera extending posteriorly upto the fornix. Gonioscopy revealed only faint pigments at the fistula. Following the trauma, the intraocular pressure had increased to 26 mm Hg. The mechanism and the management of the glaucoma are discussed.

  9. Clinical application of determination of plasma intact parathyroid hormone content in kidney disease

    International Nuclear Information System (INIS)

    Zhu Mei; Wang Zhaohui; Zhou Xiaoli; Ren Chunling; Chen Huaqian

    2011-01-01

    Objective: To observe intact parathyroid hormone in kidney disease with clinical application. Methods: Plasma i-PTH level was measured in 46 patients with chronic renal insufficiency lose compensation stage, 39 patients with chronic renal failure, 35 patients with uremia. Besides, control group (n=41) was established. Results: Result shown that plasma i-PTH levels were experiment group and control group were obvious difference (P<0.01), among experiment group plasma i-PTH level was obvious difference (P<0.01). Conclusion: Results suggested along with renal function were worsen that plasma i-PTH level increasing gradually during renal insufficiency. (authors)

  10. In vitro permeation of palladium powders through intact and damaged human skin.

    Science.gov (United States)

    Crosera, Matteo; Mauro, Marcella; Bovenzi, Massimo; Adami, Gianpiero; Baracchini, Elena; Maina, Giovanni; Larese Filon, Francesca

    2018-05-01

    The use of palladium (Pd) has grown in the last decades, commonly used in automotive catalytic converters, jewellery and dental restorations sectors. Both general and working population can be exposed to this metal, which may act as skin sensitizer. This study investigated in vitro palladium powders permeation through excised intact and damaged human skin using the Franz diffusion cell method and the effect of rapid skin decontamination using sodium laureth-sulphate. 1 mL of a 10 min sonicated suspension made of 2.5 g of Pd powder in 50 mL synthetic sweat at pH 4.5 and room temperature was applied to the outer surface of the skin membranes for 24 h. Pd permeation, assessed by ICP-MS, was higher when damaged skin was used (p = 0.03). Final flux permeation values and lag times were 0.02 ± 0.01 μg cm -2  h -1 and 6.00 ± 3.95 h for intact, and 0.10 ± 0.02 μg cm -2  h -1 and 2.05 ± 1.49 h for damaged skin samples, respectively. Damaged skin protocol enhances Pd skin penetration inside dermal layer (p = 0.04), thus making the metal available for systemic uptake. Pd penetration (p = 0.02) and permeation (p = 0.012) through intact skin decreased significantly when a cleaning procedure was applied. This study demonstrates that after skin exposure to Pd powders a small permeation of the metal happen both through intact and damaged skin and that an early decontamination with a common cleanser can significantly decrease the final amount of metal available forsystemic uptake. Copyright © 2018 Elsevier B.V. All rights reserved.

  11. Enzymatic changes in intact leaves of Phaseolus vulgaris following ozone fumigation

    Energy Technology Data Exchange (ETDEWEB)

    Dass, H C; Weaver, G M

    1972-01-01

    Enzymatic changes in the intact leaves of Phaseolus vulgaris cv. Seaway 65 were studied following ozone fumigation. It was found that peroxidase enzyme increased significantly with the ozone treatment in the first 48 h. Similarly, cellulase enzyme showed significant increase 48 h. following ozone treatment. Lactic dehydrogenase activity was not markedly affected by ozone treatment. Disc electrophoretic studies of peroxidase isoenzymes showed that ozone treatment induced a new band of peroxidase. The role of peroxidase, cellulase and lactic dehydrogenase enzymes is discussed in relation to ozone damage and the bronzing disorder in white beans. 22 references, 1 figure, 1 table.

  12. Assessment of renal function following irradiation of the intact spleen for hodgkin disease

    International Nuclear Information System (INIS)

    Birkhead, B.M.; Dobbs, C.E.; Beard, M.F.; Tyson, J.W.; Fuller, E.A.

    1979-01-01

    Twenty-three patients with clinical Stage IA to IIIB Hodgkin disease underwent extended-field radiotherapy, including the intact spleen. In 17 of those patients, there was little evidence of renal dysfunction resulting from partial irradiation of the left kidney, which is inherent in such treatment. While isotopic images revealed an anatomic defect in the upper pole of the left kidney in one-third of the patients, dynamic studies showed that this anatomic alteration was not accompanied by any demonstrable dysfunction. Consistently normal serum BUN and creatinine levels in all patients tended to affirm that conclusion

  13. Thermal deposition of intact tetrairon(III) single-molecule magnets in high-vacuum conditions.

    Science.gov (United States)

    Margheriti, Ludovica; Mannini, Matteo; Sorace, Lorenzo; Gorini, Lapo; Gatteschi, Dante; Caneschi, Andrea; Chiappe, Daniele; Moroni, Riccardo; de Mongeot, Francesco Buatier; Cornia, Andrea; Piras, Federica M; Magnani, Agnese; Sessoli, Roberta

    2009-06-01

    A tetrairon(III) single-molecule magnet is deposited using a thermal evaporation technique in high vacuum. The chemical integrity is demonstrated by time-of-flight secondary ion mass spectrometry on a film deposited on Al foil, while superconducting quantum interference device magnetometry and alternating current susceptometry of a film deposited on a kapton substrate show magnetic properties identical to the pristine powder. High-frequency electron paramagnetic resonance spectra confirm the characteristic behavior for a system with S = 5 and a large Ising-type magnetic anisotropy. All these results indicate that the molecules are not damaged during the deposition procedure keeping intact the single-molecule magnet behavior.

  14. Pulmonary atresia with intact ventricular septum and agenesis of the ductus arteriosus in a pup

    International Nuclear Information System (INIS)

    Brown, D.J.; Patterson, D.F.

    1989-01-01

    A 7-week-old Wire Fox Terrier was admitted with pulmonary atresia with intact ventricular septum. The right ventricle and tricuspid valve were hypoplastic, and venous return to the right atrium reached the left side through an atrial septal defect. Oxygenation was via hyperplastic bronchial arteries. There was no evidence of the ductus arteriosus. Physical examination, plain and contrast radiography, and electrocardiography were performed. Clinical findings for this combination of defects were similar to those of more common defects (tetralogy of Fallot, patent ductus arteriosus

  15. A Universal Protocol for Photochemical Covalent Immobilization of Intact Carbohydrates for the Preparation of Carbohydrate Microarrays

    Science.gov (United States)

    Wang, Huibin; Zhang, Yiming; Yuan, Xun; Chen, Yi; Yan, Mingdi

    2010-01-01

    A universal photochemical method has been established for the immobilization of intact carbohydrates and their analogues, and for the fabrication of carbohydrate microarrays. The method features the use of perfluorophenyl azide (PFPA)-modified substrates and the photochemical reaction of surface azido groups with printed carbohydrates. Various aldoses, ketoses, non-reducing sugars such as alditols and their derivatives can be directly arrayed on the PFPA-modified chips. The lectin-recognition ability of arrayed mannose, glucose and their oligo- and polysaccharides were confirmed using surface plasmon resonance imaging and laser-induced fluorescence imaging. PMID:21138274

  16. Superolateral dislocation of an intact mandibular condyle into the temporal fossa: case report and literature review.

    Science.gov (United States)

    Sharma, Divashree; Khasgiwala, Ankit; Maheshwari, Bharat; Singh, Charanpreet; Shakya, Neelam

    2017-02-01

    Temporomandibular joint dislocation refers to the dislodgement of mandibular condyle from the glenoid fossa. Anterior and anteromedial dislocations of the mandibular condyle are frequently reported in the literature, but superolateral dislocation is a rare presentation. This report outlines a case of superolateral dislocation of an intact mandibular condyle that occurred in conjunction with an ipsilateral mandibular parasymphysis fracture. A review of the clinical features of superolateral dislocation of the mandibular condyle and the possible techniques of its reduction ranging from the most conservative means to extensive surgical interventions is presented. © 2016 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

  17. Intact Pituitary Function is Decisive for the Catabolic Response to TNF-α

    DEFF Research Database (Denmark)

    Bach, Ermina; Møller, Andreas B; Jørgensen, Jens O L

    2015-01-01

    Context: TNF-α generates inflammatory responses and insulin resistance, lipolysis and protein breakdown. It is unclear whether these changes depend on intact hypothalamo-pituitary stress hormone responses triggering release of cortisol and growth hormone. Objective: To define differential effects......-α on lipase expression or regulation in fat. Conclusions: TNF-α increased both urea and amino acid fluxes and EGP significantly more in CTR compared to HP, suggesting that increases in endogenous cortisol and GH release are significant components of the metabolic response to TNF-α....

  18. Regulation of ion transport via apical purinergic receptors in intact rabbit airway epithelium

    DEFF Research Database (Denmark)

    Poulsen, Asser Nyander; Klausen, Thomas Levin; Pedersen, Peter Steen

    2005-01-01

    and unidirectional Cl- fluxes decreased significantly. The results suggest that nucleotides released to the airway surface liquid exert an autocrine regulation of epithelial NaCl absorption mainly by inhibiting the amiloride-sensitive epithelial Na+ channel (ENaC) and paracellular anion conductance via a P2Y......We investigated purinergic receptors involved in ion transport regulation in the intact rabbit nasal airway epithelium. Stimulation of apical membrane P2Y receptors with ATP or UTP (200 microM) induced transient increases in short-circuit current (Isc) of 13 and 6% followed by sustained inhibitions...

  19. The Megameatus, Intact Prepuce Variant of Hypospadias: Use of the Inframeatal Vascularized Flap for Surgical Correction

    OpenAIRE

    Marc Cendron

    2018-01-01

    Introduction: The megameatus intact prepuce (MIP) variant of hypospadias is a rare variant of hypospadias that is diagnosed either early at the time of circumcision or later as the foreskin is retracted. The true incidence of the anomaly is difficult to determine precisely as some patient never come to medical attention but is felt to under 5% of all cases of hypospadias. The purposes of this study are to review the embryology and clinical findings of MIP and then, in light of a personal expe...

  20. Use of electroporation to study the cytotoxic effects of fluorodeoxyuridylate in intact cells.

    Science.gov (United States)

    Jastreboff, M M; Sokoloski, J A; Bertino, J R; Narayanan, R

    1987-04-15

    The introduction of 2'-deoxyuridine 5'-monophosphate and its analog, 5-fluoro-2'-deoxyuridine 5'-monophosphate, into intact CCRF-CEM and NIH3T3 cells was achieved by electroporation. Following electroporation, cells were shown to be fully functional as monitored by the incorporation of deoxyuridylate, after conversion to thymidylate, into DNA. Pretreatment of cells with fluorodeoxyuridine completely abolished this effect. In contrast, introduction of the fluoro analog into cells by electroporation markedly inhibited both DNA synthesis and cell growth in a time-dependent manner. Thus, electroporation offers a powerful tool to permeabilize cells to a variety of cellular metabolites and antimetabolites.

  1. Pertussis toxin-catalyzed ADP-ribosylation of a G protein in mouse oocytes, eggs, and preimplantation embryos: Developmental changes and possible functional roles

    Energy Technology Data Exchange (ETDEWEB)

    Jones, J.; Schultz, R.M. (Univ. of Pennsylvania, Philadelphia (USA))

    1990-06-01

    G proteins, which in many somatic cells serve as mediators of signal transduction, were identified in preimplantation mouse embryos by their capacity to undergo pertussis toxin-catalyzed ADP-ribosylation. Two pertussis toxin (PT) substrates with Mr = 38,000 and 39,000 (alpha 38 and alpha 39) are present in approximately equal amounts. Relative to the amount in freshly isolated germinal vesicle (GV)-intact oocytes, the amount of PT-catalyzed ADP-ribosylation of alpha 38-39 falls during oocyte maturation, rises between the one- and two-cell stages, falls by the eight-cell and morula stages, and increases again by the blastocyst stage. The decrease in PT-catalyzed ADP-ribosylation of alpha 38-39 that occurs during oocyte maturation, however, does not require germinal vesicle breakdown (GVBD), since inhibiting GVBD with 3-isobutyl-1-methyl xanthine (IBMX) does not prevent the decrease in the extent of PT-catalyzed ADP-ribosylation. A biologically active phorbol diester (12-O-tetradecanoyl phorbol 13-acetate), but not an inactive one (4 alpha-phorbol 12,13-didecanoate, 4 alpha-PDD), totally inhibits the increase in PT-catalyzed ADP-ribosylation of alpha 38-39 that occurs between the one- and two-cell stage; TPA inhibits cleavage, but not transcriptional activation, which occurs in the two-cell embryo. In contrast, cytochalasin D, genistein, or aphidicolin, each of which inhibits cleavage of one-cell embryos, or alpha-amanitin or H8, each of which inhibits transcriptional activation but not cleavage of one-cell embryos, have little or inhibitory effects on the increase in PT-catalyzed ADP-ribosylation of alpha 38-39. Results of immunoblotting experiments using an antibody that is highly specific for alpha il-3 reveal the presence of a cross-reactive species of Mr = 38,000 (alpha 38) in the GV-intact oocyte, metaphase II-arrested egg, and one-, two-cell embryos.

  2. Ultralarge von Willebrand Factor Fibers Mediate Luminal Staphylococcus aureus Adhesion to an Intact Endothelial Cell Layer Under Shear Stress

    NARCIS (Netherlands)

    Pappelbaum, Karin I.; Gorzelanny, Christian; Graessle, Sandra; Suckau, Jan; Laschke, Matthias W.; Bischoff, Markus; Bauer, Corinne; Schorpp-Kistner, Marina; Weidenmaier, Christopher; Schneppenheim, Reinhard; Obser, Tobias; Sinha, Bhanu; Schneider, Stefan W.

    2013-01-01

    Background During pathogenesis of infective endocarditis, Staphylococcus aureus adherence often occurs without identifiable preexisting heart disease. However, molecular mechanisms mediating initial bacterial adhesion to morphologically intact endocardium are largely unknown. Methods and Results

  3. Novel molecular events associated with altered steroidogenesis induced by exposure to atrazine in the intact and castrate male rat

    Science.gov (United States)

    Toxicology is increasingly focused on molecular events comprising adverse outcome pathways. Atrazine activates the hypothalamic-pituitary adrenal axis, but relationships to gonadal alterations are unknown. We characterized hormone profiles and adrenal (intact and castrate) and te...

  4. Cinacalcet reduces plasma intact parathyroid hormone, serum phosphate and calcium levels in patients with secondary hyperparathyroidism irrespective of its severity.

    LENUS (Irish Health Repository)

    2011-09-01

    To evaluate the relationship between the severity of secondary hyperparathyroidism (SHPT) - defined in terms of baseline plasma intact parathyroid hormone (iPTH) level - and the magnitude of response to cinacalcet.

  5. Development of a Representative Mouse Model with Nonalcoholic Steatohepatitis.

    Science.gov (United States)

    Verbeek, Jef; Jacobs, Ans; Spincemaille, Pieter; Cassiman, David

    2016-06-01

    Non-alcoholic fatty liver disease (NAFLD) is the most prevalent liver disease in the Western world. It represents a disease spectrum ranging from isolated steatosis to non-alcoholic steatohepatitis (NASH). In particular, NASH can evolve to fibrosis, cirrhosis, hepatocellular carcinoma, and liver failure. The development of novel treatment strategies is hampered by the lack of representative NASH mouse models. Here, we describe a NASH mouse model, which is based on feeding non-genetically manipulated C57BL6/J mice a 'Western style' high-fat/high-sucrose diet (HF-HSD). HF-HSD leads to early obesity, insulin resistance, and hypercholesterolemia. After 12 weeks of HF-HSD, all mice exhibit the complete spectrum of features of NASH, including steatosis, hepatocyte ballooning, and lobular inflammation, together with fibrosis in the majority of mice. Hence, this model closely mimics the human disease. Implementation of this mouse model will lead to a standardized setup for the evaluation of (i) underlying mechanisms that contribute to the progression of NAFLD to NASH, and (ii) therapeutic interventions for NASH. © 2016 by John Wiley & Sons, Inc. Copyright © 2016 John Wiley & Sons, Inc.

  6. Separation of mouse testis cells on a Celsep (TM) apparatus and their usefulness as a source of high molecular weight DNA or RNA

    Science.gov (United States)

    Wolgemuth, D. J.; Gizang-Ginsberg, E.; Engelmyer, E.; Gavin, B. J.; Ponzetto, C.

    1985-01-01

    The use of a self-contained unit-gravity cell separation apparatus for separation of populations of mouse testicular cells is described. The apparatus, a Celsep (TM), maximizes the unit area over which sedimentation occurs, reduces the amount of separation medium employed, and is quite reproducible. Cells thus isolated have been good sources for isolation of DNA, and notably, high molecular weight RNA.

  7. Construction of disturbed and intact soil blocks to develop percolating soil based treatment systems for dirty water from dairy farms.

    Science.gov (United States)

    Brookman, S K E; Chadwick, D R; Headon, D M

    2002-03-01

    Intact soil blocks with a surface area of 1.8 x 1.6 m, 1.0 m deep, were excavated in a coarse sandy loam. The sides of the soil blocks were supported with plywood before using hydraulic rams to force a steel cutting plate beneath them. Disturbed soil blocks of the same depth as the intact blocks were also established. Experiments were conducted to determine purification efficiencies for biological oxygen demand (BOD), molybdate reactive phosphorus (MRP), nitrate and ammonium-N after the application of dirty water. A preliminary experiment is described where a low application of dirty water was applied to the soil blocks, 2 mm day(-1). In addition, a chloride tracer was conducted for the duration of the experiment. Disturbed soil had a purification efficiency for BOD of 99% compared to 96% from intact soil (Pammonium-N were 100 and 99%, respectively, for the intact and disturbed soils. Nitrate-N concentration increased in leachate from both treatments reaching maximum concentrations of 15 and 8 mg l(-1) from disturbed and intact soils, respectively. Chloride traces for each soil block followed similar patterns with 47 and 51% loss from disturbed and intact soils, respectively.

  8. The Mouse SAGE Site: database of public mouse SAGE libraries

    Czech Academy of Sciences Publication Activity Database

    Divina, Petr; Forejt, Jiří

    2004-01-01

    Roč. 32, - (2004), s. D482-D483 ISSN 0305-1048 R&D Projects: GA MŠk LN00A079; GA ČR GV204/98/K015 Grant - others:HHMI(US) 555000306 Institutional research plan: CEZ:AV0Z5052915 Keywords : mouse SAGE libraries * web -based database Subject RIV: EB - Genetics ; Molecular Biology Impact factor: 7.260, year: 2004

  9. Neutrophilia, gelatinase release and microvascular leakage induced by human mast cell tryptase in a mouse model: Lack of a role of protease-activated receptor 2 (PAR2).

    Science.gov (United States)

    Khedr, M E M S; Abdelmotelb, A M; Pender, S L F; Zhou, X; Walls, A F

    2018-05-01

    Tryptase, the most abundant protease of the human mast cell, has been implicated as a key mediator of allergic inflammation that acts through activation of PAR2. To investigate the contribution of PAR2 in the pro-inflammatory actions mediated by tryptase in a mice model. We have injected recombinant human βII-tryptase into the peritoneum of PAR2-deficient and wild-type C57BL/6 mice. After 6, 12 and 24 hours, mice were killed, peritoneal lavage performed and inflammatory changes investigated. Tryptase stimulated an increase in neutrophil numbers in the peritoneum, but responses did not differ between PAR2-deficient and wild-type mice. Heat inactivation of tryptase or pre-incubation with a selective tryptase inhibitor reduced neutrophilia, but neutrophil accumulation was not elicited with a peptide agonist of PAR2 (SLIGRL-NH 2 ). Zymography indicated that tryptase stimulated the release of matrix metalloproteinases (MMP) 2 and 9 in the peritoneum of both mouse strains. Studies involving immunomagnetic isolation of neutrophils suggested that neutrophils represent the major cellular source of tryptase-induced MMP2 and MMP9. At 24 hours after tryptase injection, there was increased microvascular leakage as indicated by high levels of albumin in peritoneal lavage fluid, and this appeared to be partially abolished by heat-inactivating tryptase or addition of a protease inhibitor. There was no corresponding increase in levels of histamine or total protein. The extent of tryptase-induced microvascular leakage or gelatinase release into the peritoneum did not differ between PAR2-deficient and wild-type mice. Our findings indicate that tryptase is a potent stimulus for neutrophil accumulation, MMP release and microvascular leakage. Although these actions required an intact catalytic site, the primary mechanism of tryptase in vivo would appear to involve processes independent of PAR2. © 2018 The Authors. Clinical & Experimental Allergy Published by John Wiley & Sons Ltd.

  10. Effect of low dose tritium on mouse lymphocyte DNA estimated by comet assay

    International Nuclear Information System (INIS)

    Ichimasa, Yusuke; Otsuka, Kensuke; Maruyama, Satoko; Tauchi, Hiroshi; Ichimasa, Michiko; Uda, Tatsuhiko

    2003-01-01

    This paper deals with low dose effect of HTO on mouse lymphocytes DNA (in vitro irradiation) estimated by the comet assay using ICR male mouse of 20 to 23 weeks old. Lymphocytes were isolated by centrifugation of whole blood sample on Ficoll-Paque solution and embedded in agarose gel just after mixed with HTO. After lymphocytes were exposed to 17-50 mGy of HTO, the agarose gel slides were washed to remove HTO and cell lysis treatment on the slides was conducted before electrophoresis. The individual comets on stained slides after electrophoresis were analyzed using imaging software. No significant DNA damages were observed. (author)

  11. Tumor penetration with intact MAb and fragments demonstrated in vitro on tumor spheroids and in vivo in the nude mouse model

    International Nuclear Information System (INIS)

    Buchegger, F.; Halpern, S.E.; Sutherland, R.M.; Schreyer, M.; Mach, J.P.

    1986-01-01

    Tumor spheroids grown in culture represent a good in vitro model for the study of tumor penetration phenomena of potential radiotherapeutics. Using this system, it was found that Fab-fragments penetrate tumors more quickly and deeply than complete antibodies. These results were confirmed in tumor bearing nephrectomized nude mice

  12. Characterization of a mouse-adapted Staphylococcus aureus strain.

    Directory of Open Access Journals (Sweden)

    Silva Holtfreter

    Full Text Available More effective antibiotics and a protective vaccine are desperately needed to combat the 'superbug' Staphylococcus aureus. While in vivo pathogenicity studies routinely involve infection of mice with human S. aureus isolates, recent genetic studies have demonstrated that S. aureus lineages are largely host-specific. The use of such animal-adapted S. aureus strains may therefore be a promising approach for developing more clinically relevant animal infection models. We have isolated a mouse-adapted S. aureus strain (JSNZ which caused a severe outbreak of preputial gland abscesses among male C57BL/6J mice. We aimed to extensively characterize this strain on a genomic level and determine its virulence potential in murine colonization and infection models. JSNZ belongs to the MLST type ST88, rare among human isolates, and lacks an hlb-converting phage encoding human-specific immune evasion factors. Naive mice were found to be more susceptible to nasal and gastrointestinal colonization with JSNZ than with the human-derived Newman strain. Furthermore, naïve mice required antibiotic pre-treatment to become colonized with Newman. In contrast, JSNZ was able to colonize mice in the absence of antibiotic treatment suggesting that this strain can compete with the natural flora for space and nutrients. In a renal abscess model, JSNZ caused more severe disease than Newman with greater weight loss and bacterial burden. In contrast to most other clinical isolates, JSNZ can also be readily genetically modified by phage transduction and electroporation. In conclusion, the mouse-adapted strain JSNZ may represent a valuable tool for studying aspects of mucosal colonization and for screening novel vaccines and therapies directed at preventing colonization.

  13. Isolation of Mitochondria from Potato Tubers

    DEFF Research Database (Denmark)

    Havelund, Jesper F.; Salvato, Fernanda; Chen, Mingjie

    2014-01-01

    One way to study the function of plant mitochondria is to extract them from plant tissues in an uncontaminated, intact and functional form. The reductionist assumption is that the components present in such a preparation and the in vitro measurable functions or activities reliably reflect...... the in vivo properties of the organelle inside the plant cell. Here, we describe a method to isolate mitochondria from a relatively homogeneous plant tissue, the dormant potato tuber. The homogenization is done using a juice extractor, which is a relatively gentle homogenization procedure where...... the mitochondria are only exposed to strong shearing forces once. After removal of starch and large tissue pieces by filtration, differential centrifugation is used to remove residual starch as well as larger organelles. The crude mitochondria are then first purified by using a step Percoll gradient...

  14. Mixed-mode reversed phase/positively charged repulsion chromatography for intact protein separation.

    Science.gov (United States)

    Ding, Ling; Guo, Zhimou; Hu, Zhuo; Liang, Xinmiao

    2017-05-10

    A mixed-mode reversed phase/positively charged repulsion stationary phase C8PN composed of octyl and amino group has been developed for separation of intact protein. Before the separation of proteins, a set of probe compounds were employed to evaluate the chromatographic properties of C8PN, demonstrating typical reversed phase/positively charged repulsion interaction on this stationary phase as estimated. Then the new C8PN stationary phase was used to separate a standard protein mixture on the reversed phase mode. Compared with a commercial C4 stationary phase, it showed different selectivity for some proteins. In order to better understand the properties of C8PN, the effect of acetonitrile content was investigated based on retention equation. Higher values of the equation parameters on C8PN demonstrated that the protein retentions were more sensitive to the change of acetonitrile content. Besides, the influences of buffer salt additives on the protein retentions were also studied. The retention factors of the proteins got larger with the increase of buffer salt concentration, which confirmed the positively charged repulsion interaction on the column. Finally, the C8PN was further applied to separate oxidized- and reduced- forms of Recombinant Human Growth Hormone. Our study indicated the advantages and application potential of mixed-mode reversed phase/positively charged repulsion stationary phase for intact protein separation. Copyright © 2017 Elsevier B.V. All rights reserved.

  15. Superresolution and Fluorescence Dynamics Evidence Reveal That Intact Liposomes Do Not Cross the Human Skin Barrier.

    Directory of Open Access Journals (Sweden)

    Jes Dreier

    Full Text Available In this study we use the combination of super resolution optical microscopy and raster image correlation spectroscopy (RICS to study the mechanism of action of liposomes as transdermal drug delivery systems in human skin. Two different compositions of liposomes were applied to newly excised human skin, a POPC liposome and a more flexible liposome containing the surfactant sodium cholate. Stimulated emission depletion microscopy (STED images of intact skin and cryo-sections of skin treated with labeled liposomes were recorded displaying an optical resolution low enough to resolve the 100 nm liposomes in the skin. The images revealed that virtually none of the liposomes remained intact beneath the skin surface. RICS two color cross correlation diffusion measurements of double labeled liposomes confirmed these observations. Our results suggest that the liposomes do not act as carriers that transport their cargo directly through the skin barrier, but mainly burst and fuse with the outer lipid layers of the stratum corneum. It was also found that the flexible liposomes showed a greater delivery of the fluorophore into the stratum corneum, indicating that they functioned as chemical permeability enhancers.

  16. Metabolic control over the oxygen consumption flux in intact skeletal muscle: in silico studies.

    Science.gov (United States)

    Liguzinski, Piotr; Korzeniewski, Bernard

    2006-12-01

    It has been postulated previously that a direct activation of all oxidative phosphorylation complexes in parallel with the activation of ATP usage and substrate dehydrogenation (the so-called each-step activation) is the main mechanism responsible for adjusting the rate of ATP production by mitochondria to the current energy demand during rest-to-work transition in intact skeletal muscle in vivo. The present in silico study, using a computer model of oxidative phosphorylation developed previously, analyzes the impact of the each-step-activation mechanism on the distribution of control (defined within Metabolic Control Analysis) over the oxygen consumption flux among the components of the bioenergetic system in intact oxidative skeletal muscle at different energy demands. It is demonstrated that in the absence of each-step activation, the oxidative phosphorylation complexes take over from ATP usage most of the control over the respiration rate and oxidative ATP production at higher (but still physiological) energy demands. This leads to a saturation of oxidative phosphorylation, impossibility of a further acceleration of oxidative ATP synthesis, and dramatic drop in the phosphorylation potential. On the other hand, the each-step-activation mechanism allows maintenance of a high degree of the control exerted by ATP usage over the ATP turnover and oxygen consumption flux even at high energy demands and thus enables a potentially very large increase in ATP turnover. It is also shown that low oxygen concentration shifts the metabolic control from ATP usage to cytochrome oxidase and thus limits the oxidative ATP production.

  17. Sequencing Larger Intact Proteins (30-70 kDa) with Activated Ion Electron Transfer Dissociation

    Science.gov (United States)

    Riley, Nicholas M.; Westphall, Michael S.; Coon, Joshua J.

    2018-01-01

    The analysis of intact proteins via mass spectrometry can offer several benefits to proteome characterization, although the majority of top-down experiments focus on proteoforms in a relatively low mass range (AI-ETD) to proteins in the 30-70 kDa range. AI-ETD leverages infrared photo-activation concurrent to ETD reactions to improve sequence-informative product ion generation. This method generates more product ions and greater sequence coverage than conventional ETD, higher-energy collisional dissociation (HCD), and ETD combined with supplemental HCD activation (EThcD). Importantly, AI-ETD provides the most thorough protein characterization for every precursor ion charge state investigated in this study, making it suitable as a universal fragmentation method in top-down experiments. Additionally, we highlight several acquisition strategies that can benefit characterization of larger proteins with AI-ETD, including combination of spectra from multiple ETD reaction times for a given precursor ion, multiple spectral acquisitions of the same precursor ion, and combination of spectra from two different dissociation methods (e.g., AI-ETD and HCD). In all, AI-ETD shows great promise as a method for dissociating larger intact protein ions as top-down proteomics continues to advance into larger mass ranges. [Figure not available: see fulltext.

  18. IgE epitopes of intact and digested Ara h 1

    DEFF Research Database (Denmark)

    Bøgh, Katrine Lindholm; Nielsen, H.; Madsen, Charlotte Bernhard

    2012-01-01

    epitopes have been suggested to be of great importance. ObjectiveThe aim of this study was to identify IgE specific epitopes of intact and digested Ara h 1, and to compare epitope patterns between humans and rats. MethodsSera from five peanut allergic patients and five Brown Norway rats were used...... to identify intact and digested Ara h 1-specific IgE epitopes by competitive immunoscreening of a phage-displayed random hepta-mer peptide library using polyclonal IgE from the individual sera. The resulting peptide sequences were mapped on the surface of a three-dimensional structure of the Ara h 1 molecule...... to mimic epitopes using a computer-based algorithm. ResultsPatients as well as rats were shown to have individual IgE epitope patterns. All epitope mimics were conformational and found to cluster into three different areas of the Ara h 1 molecule. Five epitope motifs were identified by patient IgE, which...

  19. Clean Transfer of Large Graphene Single Crystals for High-Intactness Suspended Membranes and Liquid Cells.

    Science.gov (United States)

    Zhang, Jincan; Lin, Li; Sun, Luzhao; Huang, Yucheng; Koh, Ai Leen; Dang, Wenhui; Yin, Jianbo; Wang, Mingzhan; Tan, Congwei; Li, Tianran; Tan, Zhenjun; Liu, Zhongfan; Peng, Hailin

    2017-07-01

    The atomically thin 2D nature of suspended graphene membranes holds promising in numerous technological applications. In particular, the outstanding transparency to electron beam endows graphene membranes great potential as a candidate for specimen support of transmission electron microscopy (TEM). However, major hurdles remain to be addressed to acquire an ultraclean, high-intactness, and defect-free suspended graphene membrane. Here, a polymer-free clean transfer of sub-centimeter-sized graphene single crystals onto TEM grids to fabricate large-area and high-quality suspended graphene membranes has been achieved. Through the control of interfacial force during the transfer, the intactness of large-area graphene membranes can be as high as 95%, prominently larger than reported values in previous works. Graphene liquid cells are readily prepared by π-π stacking two clean single-crystal graphene TEM grids, in which atomic-scale resolution imaging and temporal evolution of colloid Au nanoparticles are recorded. This facile and scalable production of clean and high-quality suspended graphene membrane is promising toward their wide applications for electron and optical microscopy. © 2017 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  20. On Plant Detection of Intact Tomato Fruits Using Image Analysis and Machine Learning Methods

    Directory of Open Access Journals (Sweden)

    Kyosuke Yamamoto

    2014-07-01

    Full Text Available Fully automated yield estimation of intact fruits prior to harvesting provides various benefits to farmers. Until now, several studies have been conducted to estimate fruit yield using image-processing technologies. However, most of these techniques require thresholds for features such as color, shape and size. In addition, their performance strongly depends on the thresholds used, although optimal thresholds tend to vary with images. Furthermore, most of these techniques have attempted to detect only mature and immature fruits, although the number of young fruits is more important for the prediction of long-term fluctuations in yield. In this study, we aimed to develop a method to accurately detect individual intact tomato fruits including mature, immature and young fruits on a plant using a conventional RGB digital camera in conjunction with machine learning approaches. The developed method did not require an adjustment of threshold values for fruit detection from each image because image segmentation was conducted based on classification models generated in accordance with the color, shape, texture and size of the images. The results of fruit detection in the test images showed that the developed method achieved a recall of 0.80, while the precision was 0.88. The recall values of mature, immature and young fruits were 1.00, 0.80 and 0.78, respectively.