WorldWideScience

Sample records for explants

  1. Explant cultures of human colon

    DEFF Research Database (Denmark)

    Autrup, Herman; Barrett, L.A.; Jackson, F.E.

    1978-01-01

    . The ability to maintain colonic mucosa in culture was subject to both intra- and interindividual variation. Cultured human colonic mucosa also activated a chemical procarcinogen, benzo[a]pyrene, into metabolites which bound to cellular DNA. A 100-fold interindividual variation in this binding was observed.......Human colonic epithelium has been cultured as explants in a chemically defined medium for periods of 1 to 20 days. The viability of the explants was shown by the preservation of the ultrastructural features of the colonic epithelial cells and by active incorporation of radioactive precursors...... into cellular DNA and protein. A progressive decrease in the number of goblet cells, decrease in the depth of the crypts, and a change from a columnar to a cuboidal epithelium were observed. After 20 days in culture the colonic mucosa consisted of a single layer of cuboidal epithelial cells and a few glands...

  2. Causes of IOL explantation in Spain.

    Science.gov (United States)

    Fernández-Buenaga, Roberto; Alio, Jorge L; Muñoz-Negrete, Francisco J; Barraquer Compte, Rafael I; Alio-Del Barrio, Jorge L

    2012-01-01

    To study the reasons and the demography of pseudophakic intraocular lens (IOL) explantation in Spain. In this observational multicenter retrospective study, the cases studied correspond to the 15 centers that constitute the Nodo Calidad Visual y Cirugia Refractiva of the Red Tematica de Investigacion Cooperativa (RETICS) sponsored by the Spanish Ministry of Health. Clinical data from all the patients who underwent explantation were assessed. The different reasons that caused the decision of explantation were analyzed. A total of 257 explanted pseudophakic IOLs have been studied. Patients' mean age when explantation occurred was 67.5 years (SD 13.5 [22-99]) and 135 were female (52.5%). The main causes for explantation were dislocation/decentration in 145 cases (56.3%), incorrect lens power in 33 cases (12.8%), IOL opacification in 29 eyes (11.3%), neuroadaptation failure in 16 cases (6.2%), pseudophakic bullous keratopathy in 6 eyes (2.3%), endophthalmitis in 5 cases (1.9%), and "other causes" in 23 eyes (8.9%). Treatment after explantation was posterior chamber IOL implantation in 149 eyes (58%), anterior chamber IOL implantation in 49 eyes (19.1%), aphakia in 39 eyes (15.2%), and missing information in 20 cases (7.8%). Mean time from implantation to explantation was 3.97 (SD 4.68 [0.005-21.1]) years. Dislocation/decentration (most of the time with lens in the bag) is the main cause for explantation in Spain, followed by incorrect lens power (which decreased greatly over past years) and IOL opacification. Posterior chamber IOL implantation is the most elected treatment after explantation.

  3. Lichen explants and natural occurrence of lichens

    Energy Technology Data Exchange (ETDEWEB)

    Kirschbaum, A.; Klee, R.

    1971-01-01

    Studies with lichen explants and with naturally occurring lichens, conducted in the Lower Main region in West Germany within the framework of an air hydgienic and meteorologic model study of that region, are described. Parmelia physodes explants from oak trees growing in nonpolluted areas were exposed in polluted areas, such as in an industrial area, an airport, a petroleum refinery, and near a large chemical plant. The degree of air pollution in the exposure site was evaluated by the degree of the lichen damage in seven grades. The large-scale average distribution of air pollution in the survey area was studied by surveying the natural occurrence of lichen species on 10 apple trees in area units of 6.25 sq km each. The lichen explant and lichen survey methods compared by the study of naturally occurring lichens were near the exposure site of lichen explants.

  4. Stabilization of gene expression and cell morphology after explant recycling during fin explant culture in goldfish

    Energy Technology Data Exchange (ETDEWEB)

    Chenais, Nathalie; Lareyre, Jean-Jacques; Le Bail, Pierre-Yves; Labbe, Catherine, E-mail: catherine.labbe@rennes.inra.fr

    2015-07-01

    The development of fin primary cell cultures for in vitro cellular and physiological studies is hampered by slow cell outgrowth, low proliferation rate, poor viability, and sparse cell characterization. Here, we investigated whether the recycling of fresh explants after a first conventional culture could improve physiological stability and sustainability of the culture. The recycled explants were able to give a supplementary cell culture showing faster outgrowth, cleaner cell layers and higher net cell production. The cells exhibited a highly stabilized profile for marker gene expression including a low cytokeratin 49 (epithelial marker) and a high collagen 1a1 (mesenchymal marker) expression. Added to the cell spindle-shaped morphology, motility behavior, and actin organization, this suggests that the cells bore stable mesenchymal characteristics. This contrast with the time-evolving expression pattern observed in the control fresh explants during the first 2 weeks of culture: a sharp decrease in cytokeratin 49 expression was concomitant with a gradual increase in col1a1. We surmise that such loss of epithelial features for the benefit of mesenchymal ones was triggered by an epithelial to mesenchymal transition (EMT) process or by way of a progressive population replacement process. Overall, our findings provide a comprehensive characterization of this new primary culture model bearing mesenchymal features and whose stability over culture time makes those cells good candidates for cell reprogramming prior to nuclear transfer, in a context of fish genome preservation. - Highlights: • Recycled fin explants outgrow cells bearing stable mesenchymal traits. • Cell production and quality is enhanced in the recycled explant culture system. • Fresh fin primary culture is highly variable and loose epithelial traits over time.

  5. In vitro shoot regeneration from preconditioned explants of chickpea ...

    African Journals Online (AJOL)

    The present study reports the successful shoot regeneration of preconditioned mature embryo and embryonic axis explants of chickpea cv. Gokce. Explants were preconditioned with 10 mgl benzylaminopurine (BA) for 7 days followed by culture on Murashige and Skoog (MS) medium containing 0.25, 0.50, 1.00 and 2.00 ...

  6. In vitro plant regeneration from different explants of Cardiospermum ...

    African Journals Online (AJOL)

    A rapid and efficient protocol was developed for inducing indirect organogenesis using stem and leaf explants of Cardiospermum halicacabum L. Explants were cultured on MS medium supplemented with different concentrations of IAA, NAA and 2, 4-D (1.0 to 2.0 mg/l) combined with 0.5 mg/l BAP for callus induction.

  7. Effect of explant age, hormones on somatic embryogenesis and ...

    African Journals Online (AJOL)

    STORAGESEVER

    2009-02-18

    Feb 18, 2009 ... from Solanum melongena L. (eggplant) cotyledon explants is associated with hormone-modulated enhancement of polyamine biosynthesis and conjugation. Protoplasma. 211:51-63. Sharma P, Rajam MV (1995). GenoType, explant and position effects on organogenesis and somatic embryogenesis in ...

  8. Cytokine manipulation of explanted Dupuytren's affected human palmar fascia.

    Science.gov (United States)

    Kuhn, M A; Payne, W G; Kierney, P C; Pu, L L; Smith, P D; Siegler, K; Ko, F; Wang, X; Robson, M C

    2001-01-01

    Dupuytren's disease plagues human hands and digits producing fibrotic nodules and fascial cords with resultant debilitating flexion contracture deformities. Interest in this condition is great but because the disease is specific to humans and study has been hampered by the lack of an in vivo model. By utilizing an in vivo "nude" rat model it is possible to maintain and study explanted Dupuytren's contracted palmar fascia for prolonged periods of time. Human specimens were divided into four, one for in vitro analysis, and three for model explantation. The explanted tissue was perfused with either transforming growth factor beta-2 (TGFbeta2), its antibody, or a control vehicle. Explant biopsies were obtained at 30 and 60 days and compared to tissue prior to explantation. Immunohistochemistry of collagen I and III, DNA synthesis, protein production, and fibroblast kinetics were serially determined. Perfusion of explanted Dupuytren's tissue by TGFbeta2 upregulated collagen I and III from biopsies obtained from the explants at 30 days when compared to vehicle control (P < 0.001). Perfusion with antibody prevented this upregulation when compared to vehicle control (P < 0.001). Cell cultures derived from fibroblasts obtained from biopsies of the explants perfused with TGFbeta2 increased DNA synthesis, protein production and fibroblast kinetics. These findings paralleled those from other fibroproliferative disorders suggesting a role for TGFbeta2 in the pathogenesis of Dupuytren's contracture as well as possible novel treatment approaches.

  9. Adventitious shoot regeneration from leaf explants of the valuable ...

    African Journals Online (AJOL)

    The objective of this study was to develop an efficient protocol for adventitious shoot regeneration for Plectranthus barbatus Andrews using leaf explants. The explants were cultured on MS (Murashige and Skoog, 1962) medium containing various concentration of kinetin (KN), 6-benzylaminopurine (BAP) and thidiazuron ...

  10. Effect of season, explants, growth regulators and sugar level on ...

    African Journals Online (AJOL)

    The effects of antioxidant treatments, 2,4-D concentrations and sucrose level were examined in order to optimize the induction and long term maintenance of callus cultures of Ficus religiosa L. from different explants including nodal segments, inter-nodal segments and shoot apices. The explants subjected to incubation for ...

  11. Efficient regeneration of plants from shoot tip explants of ...

    African Journals Online (AJOL)

    Dendrobium densiflorum Lindl. is one of the horticulturally important orchids of Nepal due to its beautiful yellowish flower and medicinal properties. The present study was carried out for plant regeneration from shoot tip explants of D. densiflorum by tissue culture technique. The shoot tip explants of this species, obtained ...

  12. Strategies for the selection of uncontaminated Eucalyptus explants ...

    African Journals Online (AJOL)

    The potential high yields of Eucalyptus shoot multiplication achieved with a temporary immersion culture system such as RITA® are compromised by losses caused by microbial contamination particularly bacteria, characteristic of the explants used to initiate the cultures. Disinfection of the explants through antibiotic ...

  13. Optimization of explants surface sterilization condition for field grown ...

    African Journals Online (AJOL)

    The aim of this work was to sterilize nodal explants, so as to mitigate microbial contamination in peach micropropagation. The nodal explants were treated with three concentrations levels (0.15, 0.25 and 0.5% (w/v) active ingredient of chlorine) of locally produced bleach, sodium hypochlorite (NaOCl) for varying exposure ...

  14. Auxin Transport in Explants of Coleus 1

    Science.gov (United States)

    Gorter, Chr. J.; Veen, H.

    1966-01-01

    α-Naphthaleneacetic acid-C14, labeled in the carboxyl group, was applied in blocks of agar to the distal and to the proximal (either apical or basal) ends of explants of Coleus. The radioactivity in receiver blocks at the opposite ends was measured. Acropetal transport was slight, only 4% of the basipetal transport. Translocation of NAA-C14 was polar in basipetal direction. Only 1.4% of the radioactivity lost from donor blocks at the apical position reached the receiver blocks; the greatest part remained in the tissue and was immobilized there. All activity found in receiver blocks at the basal end appeared to be still in the form of NAA. There were no differences between petiole tissue and stem tissue, so far as the transport of NAA is concerned. PMID:16656237

  15. Stabilization of gene expression and cell morphology after explant recycling during fin explant culture in goldfish.

    Science.gov (United States)

    Chenais, Nathalie; Lareyre, Jean-Jacques; Le Bail, Pierre-Yves; Labbe, Catherine

    2015-07-01

    The development of fin primary cell cultures for in vitro cellular and physiological studies is hampered by slow cell outgrowth, low proliferation rate, poor viability, and sparse cell characterization. Here, we investigated whether the recycling of fresh explants after a first conventional culture could improve physiological stability and sustainability of the culture. The recycled explants were able to give a supplementary cell culture showing faster outgrowth, cleaner cell layers and higher net cell production. The cells exhibited a highly stabilized profile for marker gene expression including a low cytokeratin 49 (epithelial marker) and a high collagen 1a1 (mesenchymal marker) expression. Added to the cell spindle-shaped morphology, motility behavior, and actin organization, this suggests that the cells bore stable mesenchymal characteristics. This contrast with the time-evolving expression pattern observed in the control fresh explants during the first 2 weeks of culture: a sharp decrease in cytokeratin 49 expression was concomitant with a gradual increase in col1a1. We surmise that such loss of epithelial features for the benefit of mesenchymal ones was triggered by an epithelial to mesenchymal transition (EMT) process or by way of a progressive population replacement process. Overall, our findings provide a comprehensive characterization of this new primary culture model bearing mesenchymal features and whose stability over culture time makes those cells good candidates for cell reprogramming prior to nuclear transfer, in a context of fish genome preservation. Copyright © 2015 Elsevier Inc. All rights reserved.

  16. Explantation of the novel Ahmed glaucoma valve M4 implant.

    Science.gov (United States)

    Hu, Wanda D; Pro, Michael J; Fudemberg, Scott J; Moster, Marlene R

    2015-02-01

    To report a series of cases involving Ahmed Glaucoma Valve M4 (AGV) explantation and to discuss the surgical technique to remove the drainage device. Four cases were identified that presented with AGV M4 postoperative complications necessitating tube shunt removal. Three patients presented with conjunctival erosion and 1 patient with persistent diplopia. AGV M4 implants were removed successfully between 1.5 and 9 months after implantation. Successful explantation of the AGV M4 novel implant was achieved in all cases without intraoperative or postoperative complications. If necessary, AGV M4 explantation can be successfully performed in the early postoperative period.

  17. Making post-mortem implantable cardioverter defibrillator explantation safe

    DEFF Research Database (Denmark)

    Räder, Sune B E W; Zeijlemaker, Volkert; Pehrson, Steen

    2009-01-01

    that the resting voltage over the operating person would not exceed 50 V. CONCLUSION: The use of intact medical gloves made of latex, neoprene, or plastic eliminates the potential electrical risk during explantation of an ICD. Two gloves on each hand offer sufficient protection. We will recommend the use......AIMS: The aim of this study is to investigate whether protection with rubber or plastic gloves during post-mortem explantation of an implantable cardioverter defibrillator (ICD) offers enough protection for the explanting operator during a worst-case scenario (i.e. ICD shock). METHODS AND RESULTS......: We investigated the insulating properties of rubber and plastic gloves (double layer) within the first 60 min exposure (mimicking the maximum time of an explantation procedure) to saline (simulating the effects of body fluids on the gloves). For latex gloves, we measured an increase in voltage up...

  18. An ovine tracheal explant culture model for allergic airway inflammation

    Directory of Open Access Journals (Sweden)

    Abeynaike Latasha

    2010-08-01

    Full Text Available Abstract Background The airway epithelium is thought to play an important role in the pathogenesis of asthmatic disease. However, much of our understanding of airway epithelial cell function in asthma has been derived from in vitro studies that may not accurately reflect the interactive cellular and molecular pathways active between different tissue constituents in vivo. Methods Using a sheep model of allergic asthma, tracheal explants from normal sheep and allergic sheep exposed to house dust mite (HDM allergen were established to investigate airway mucosal responses ex vivo. Explants were cultured for up to 48 h and tissues were stained to identify apoptotic cells, goblet cells, mast cells and eosinophils. The release of cytokines (IL-1α, IL-6 and TNF-α by cultured tracheal explants, was assessed by ELISA. Results The general morphology and epithelial structure of the tracheal explants was well maintained in culture although evidence of advanced apoptosis within the mucosal layer was noted after culture for 48 h. The number of alcian blue/PAS positive mucus-secreting cells within the epithelial layer was reduced in all cultured explants compared with pre-cultured (0 h explants, but the loss of staining was most evident in allergic tissues. Mast cell and eosinophil numbers were elevated in the allergic tracheal tissues compared to naïve controls, and in the allergic tissues there was a significant decline in mast cells after 24 h culture in the presence or absence of HDM allergen. IL-6 was released by allergic tracheal explants in culture but was undetected in cultured control explants. Conclusions Sheep tracheal explants maintain characteristics of the airway mucosa that may not be replicated when studying isolated cell populations in vitro. There were key differences identified in explants from allergic compared to control airways and in their responses in culture for 24 h. Importantly, this study establishes the potential for the

  19. Citrus tissue culture employing vegetative explants.

    Science.gov (United States)

    Chaturvedi, H C; Singh, S K; Sharma, A K; Agnihotri, S

    2001-11-01

    Citrus being a number one fruit of the world due to its high nutritional value, huge production of fruits and fruit products, the citrus industry may be considered a major fruit industry. Though citrus orchard area in India is comparable to USA, the produce is far less, while its export is nil. Biotechnology has played an outstanding role in boosting the citrus industry, e.g., in Spain, which is now the biggest exporter of citrus fruit with the application of micrografting. Amongst the fruit trees, perhaps the maximum tissue culture research has been done in citrus during the past four decades, however, the results of practical value are meagre. The shortfalls in citrus tissue culture research and some advancements made in this direction along with bright prospects are highlighted, restricting the review to vegetative explants only. Whilst utilization of nucellar embryogenesis is limited to rootstocks, the other aspects, like, regeneration and proliferation of shoot meristems measuring 200 microm in length--a global breakthrough--of two commercially important scion species, Citrus aurantifolia and C. sinensis and an important rootstock, C. limonia, improvement of micrografting technique, cloning of the same two scion species as well as some Indian rootstock species, employing nodal stem segments of mature trees, of immense practical value have been elaborated. A rare phenomenon of shift in the morphogenetic pattern of differentiation from shoot bud differentiation to embryoid formation occurred during the long-term culture of stem callus of C. grandis. Stem callus-regenerated plants of C. aurantifolia, C. sinensis and C. grandis showed variation in their ploidy levels and a somaclonal variant of C. sinensis, which produced seedless fruits was isolated. Tailoring of rooting in microshoots to a tap root-like system by changing the inorganic salt composition of the rooting medium, resulting in 100% transplant success, and germplasm preservation through normal growth

  20. Intermittent cyclic loading of cartilage explants modulates fibronectin metabolism.

    Science.gov (United States)

    Steinmeyer, J; Ackermann, B; Raiss, R X

    1997-09-01

    The aim of this study was to evaluate systematically the effect of tissue load, its amplitude, time of intermittence and duration of loading on the biosynthesis and release of fibronectin by intermittently loaded mature bovine articular cartilage explants. Cyclic compressive pressure was introduced using a sinusoidal waveform of 0.5 Hz-frequency with a peak stress of 0.1, 0.5 or 1.0 MPa for a period of 10 s followed by an unloaded period lasting 10, 100 or 1000 s. Fibronectin and total proteins were radiolabeled with 10 microCi/ml [3H]-phenylalanine during the final 18 h of the 1, 3 or 6 day experiments. The content of endogenous fibronectin was determined using enzyme-linked immunosorbant assay (ELISA), whereas the viability of explants was measured using sections of cartilage explants stained with fluorescein diacetate and propidium iodide. The deformation of loaded explants was determined using a load-displacement transducer system. The mechanical factor time of intermittence significantly altered the synthesis and release of fibronectin by cartilage explants, whereas the tested range of load magnitudes, as well as the duration of loading, seemed to be of subordinate importance. Loading affected the viability of the superficial zone in the cartilage, whereas the chondrocytes of the intermediate and deep zone remained viable. The compression of loaded explants was dependent on the magnitude of stress, as well as on the duration of unloading between each loading cycle. Synthesis of fibronectin, the retention of newly synthesized fibronectin within the extracellular matrix, and the portion of newly synthesized proteins that were fibronectin was significantly increased in cartilage explants which were cyclically compressed with 0.5 MPa for 10 s followed by a period of unloading lasting 100 s. Previous studies reporting that cartilage explants of human and animal osteoarthritic joints synthesize and retain elevated amounts of fibronectin imply that in our experiments

  1. Correlated response of in vitro regeneration capacity from different source of explants inCucumis melo.

    Science.gov (United States)

    Molina, R V; Nuez, F

    1995-01-01

    The variation among and within different populations of the regeneration ability from leaf, cotyledon and hypocotyl explants has been studied. A control population and two lines selected by their regeneration capacity from leaf explants were used. Significant differences among the plants of the control population,for the organogenic response, were detected. The regeneration capacity varies depending on the type of explant. Selection in order to improve the regeneration frequency from leaf explants also raises the organogenic response in the other explant types. This result suggests the presence of a partial common genetic system controlling the regeneration frequency of the diverse types of explants.

  2. Explant age, auxin concentrations and media type affect callus ...

    African Journals Online (AJOL)

    The effects of explant age of oil palm (Elaeis huineensis) embryo axes, 15 and 18 weeks after anthesis (WAA), media type (Eeuwens and Murashige and Skoog) supplemented with various concentrations of 2,4-D on callus production employing standard in vitro techniques were investigated. The results of the study showed ...

  3. Usual interstitial pneumonia: histologic study of biopsy and explant specimens.

    Science.gov (United States)

    Katzenstein, Anna-Luise A; Zisman, David A; Litzky, Leslie A; Nguyen, Binh T; Kotloff, Robert M

    2002-12-01

    The pathologic findings in biopsy and subsequent explant specimens from 20 patients with usual interstitial pneumonia (UIP) were reviewed to refine histologic criteria for diagnosis, to identify factors that may confound diagnosis, and to assess the relationship of UIP and nonspecific interstitial pneumonia (NSIP). One case of NSIP was also identified and included for comparison. Surgical biopsies from 15 of the 20 UIP cases were diagnosed as UIP, whereas 5 showed only nondiagnostic changes. An important new observation is that areas resembling nonspecific interstitial pneumonia (NSIP-like areas) are present in the majority of UIP cases in both biopsy and explant specimens, and they are extensive in some. Ten of the 15 UIP biopsies were considered straightforward, with typical patchy interstitial fibrosis, honeycomb change, and fibroblast foci. Five cases were considered difficult because of prominent NSIP-like areas in two, extensive honeycomb change in one, superimposed diffuse alveolar damage in one, and superimposed bronchiolitis obliterans-organizing pneumonia in one. The most helpful feature for diagnosing UIP in difficult cases was the presence of a distinct patchwork appearance to the characteristic uneven or variegated parenchymal involvement along with evidence of architectural derangement. No explant showing UIP was preceded by biopsy findings of NSIP, and the one NSIP case appeared similar at biopsy and explant. NSIP or NSIP-like areas and UIP may reflect different mechanisms of fibrosis related either to different severity of injury or to different injuries.

  4. High frequency plant regeneration from shoot tip explants of ...

    African Journals Online (AJOL)

    A high frequency and rapid regeneration protocol was developed from shoot tip explants of Citrullus colocynthis on Murashige and Skoog (MS) medium supplemented with N6-benzylamino-purine (BAP, 0.5 mg/l) and α-naphthalene acetic acid (NAA, 0.5 mg/l). Highest number of shoots (23.0 ± 0.567) was obtained on MS ...

  5. Adventitious shoot regeneration from in vitro stem explants of ...

    African Journals Online (AJOL)

    An efficient in vitro plant regeneration system from stem explants was established in Phellodendron amurense. Factors influencing shoot regeneration from stems including culture medium type, combinations of plant growth regulators and carbon source in the medium were investigated. Adventitious shoot regeneration was ...

  6. Plant regeneration via somatic embryogenesis from root explants of ...

    African Journals Online (AJOL)

    A system for induction of callus and plant regeneration via somatic embryogenesis from root explants of Hevea brasiliensis Muell. Arg. clone Reyan 87-6-62 was evaluated. The influence of plant growth regulators (PGRs) including 2,4-dichlorophenoxyacetic acid (2,4-D), 6-benzylaminopurine (6-BA) and kinetin (KT) on ...

  7. Somatic embryogenesis from leaf explants of hermaphrodite Carica ...

    African Journals Online (AJOL)

    In culture medium supplemented with 2,4-dichlorophenoxyacetic, FEC overgrew into a yellowish friable mass that fully covered the leaf explants. The somatic embryogenesis process occurred asynchronously, with new globular embryos continuously forming from the FEC. Torpedo and early cotyledonary somatic embryos ...

  8. Direct and indirect plant regeneration from various explants of ...

    African Journals Online (AJOL)

    user

    2011-04-18

    Apr 18, 2011 ... 1700s from North America. In Turkey, it is an exotic species of poplar and was introduced in the late 1960s from the USA. ..... Balkan H, Tanrıyar H, Calikoglu M, Ogras T, Ozden O, Tulukcu M,. Tank T (1998). Genotype differencies in direct plant regeneration from stem explants of Populus tremula in Turkey.

  9. Influence of explanting season on in vitro multiplication of the ...

    African Journals Online (AJOL)

    STORAGESEVER

    2009-07-20

    Jul 20, 2009 ... and multiple shoot formation was induced between July and September on MS medium supplemented with BAP ... Table 1. Response of apical bud and nodal explants of T. procumbens cultured on MS medium supplemented with different ... through TLC on silica gel-G plates using the method of Holloway.

  10. Effects of genotype, explant type and nutrient medium components ...

    African Journals Online (AJOL)

    The objective of the study was to develop an efficient method for shoot regeneration of canola (Brassica napus L.) and to compare the regeneration capacity of different explants on MS medium with several combinations of plant growth regulators. The experiments showed that the morphogenetical potential of canola ...

  11. Explants, hormones and sucrose influence in vitro shoot ...

    African Journals Online (AJOL)

    Zantedeschia is an important and rapidly expanding cut flower in Kenya today. A protocol for in vitro shoot regeneration of Zantedeschia, using tuber, leaf and shoot primordium explants on Murashige and Skoog (MS) (1962) basal salts, supplemented with 6-benzyalamino purine (BAP) or Kinetin is described. Of the four ...

  12. Effect of plant growth regulators, explants type and efficient plantlet ...

    African Journals Online (AJOL)

    use

    2011-12-05

    Dec 5, 2011 ... nodal segment and shoot tip) and different concentrations of plant growth regulators. Callus formation and shoot differentiation ... Key words: Naringi crenulata, callus, regeneration, leaf explants, peroxidase, total soluble protein. INTRODUCTION ... primary health care (Mousumi et al., 2007). The success.

  13. The effect of plant growth regulators, explants and cultivars on ...

    African Journals Online (AJOL)

    Spinach (Spinacia oleracea L.) is an important vegetable crop of which dioecy in nature has made cultivar improvement difficult using traditional breeding methods; therefore, production of high amount of disease free spinach is critical. To achieve the best explants and media for spinach tissue culture, the effects of two ...

  14. Simple, effective and economical explant-surface sterilization ...

    African Journals Online (AJOL)

    STORAGESEVER

    2009-10-19

    Oct 19, 2009 ... Three different surface sterilization methods were evaluated using seeds and excised embryos of cowpea, rice and sorghum as explants: Method 1: Ethanol alone in concentrations of 95, 90, 85 and 70% at different time intervals and observed at different days. Method 2: Locally produced bleaching.

  15. AAV Serotype Testing on Cultured Human Donor Retinal Explants

    NARCIS (Netherlands)

    Buck, Thilo M; Pellissier, Lucie P; Vos, Rogier M; van Dijk, Elon H C; Boon, Camiel J F; Wijnholds, J.

    2018-01-01

    This protocol details on a screening method for infectivity and tropism of different serotypes of adeno-associated viruses (AAVs) on human retinal explants with cell-type specific or ubiquitous green fluorescent protein (GFP) expression vectors. Eyes from deceased adult human donors are enucleated

  16. Somatic embryogenesis and plant regeneration from leaf explants of ...

    African Journals Online (AJOL)

    An attempt was made to study the somatic embryogenesis and plant regeneration from the in vitro leaf explants of Rumex vesicarius L. a renowned medicinal plant, which belongs to polygonaceae family. Effective in vitro regeneration of R. vesicarius was achieved via young leaf derived somatic embryo cultures.

  17. Efficient plant regeneration from leaf explants of Solanum americanum

    African Journals Online (AJOL)

    A very efficient system for direct plant regeneration from in vitro–derived leaf explants of Solanum americanum was developed. S. americanum is a tropical plant with important medical properties. The in vitro procedure that was established consists of (i) induction of shoots from leaf tissue, (ii) elongation of shoots, and (iii) ...

  18. Regeneration potential of seedling explants of chilli ( Capsicum ...

    African Journals Online (AJOL)

    A study was conducted with hypocotyl, cotyledon and shoot tip of chilli as explants for regeneration on MS medium supplemented with different concentrations and combinations of auxins and cytokinins. Regeneration potential was determined by two ways. One is regeneration of shoot via callus formation from hypocotyls ...

  19. High-frequency shoot regeneration of nodal explants from ...

    African Journals Online (AJOL)

    This paper describes the shoot regeneration of nodal segments from a medicinal plant, Tetrastigma hemsleyanum Diels et Gilg (Vitaceae). The highest number of shoots (7.27 shoots per explant) was observed in MS medium supplemented with 4 mg/l BA after six weeks of inoculation. 2 mg/l BA in combination with 0.1 mg/l ...

  20. callus induction from epicotyl and hypocotyl explants of

    African Journals Online (AJOL)

    Department of Botany, Obafemi Awolowo University, Ile-Ife. Nigeria. (Submitted: 31 May 2004; Accepted: 31 October 2004). Abstract. Epicotyl and hypocotyl explants of Parkia biglobosa (Locust bean) were cultured in vitro to investigate their callogenic capacity. Established cultures were obtained and maintained on MS ...

  1. High-frequency shoot regeneration of nodal explants from ...

    African Journals Online (AJOL)

    Jane

    2011-06-29

    Jun 29, 2011 ... develop a rapid and efficient in vitro multiplication and regeneration system using nodal explants. MATERIALS AND METHODS. Plant material and initiation of in vitro shoot cultures. Young in vivo shoots with six to eight nodes of T. hemsleyanum were collected from wild population in Zhejiang Province, ...

  2. Regeneration of plantlets from nodal and shoot tip explants of ...

    African Journals Online (AJOL)

    Anoectochilus elatus Lindley is an endangered terrestrial orchid. A procedure for the regeneration of complete plantlets of A. elatus Lindley through node and shoot tip explants resulted directly in shoots when cultured on a full strength Murashige and Skoog (1962) medium supplemented with cytokinins at different ...

  3. In vitro propagation of Alstroemeria using rhizome explants derived ...

    African Journals Online (AJOL)

    SERVER

    2007-09-19

    Sep 19, 2007 ... For surface sterilization of rhizome, those obtained from pot plants, (3 cm in length) were excised and washed thoroughly under running tap water for 10 min. Thereafter, the rhizome explants sterilized by immersion for 35 min in 40% (v/v) commercial bleach (containing 5.54% sodium hypochlorite) and.

  4. In vitro shoot regeneration from preconditioned explants of chickpea ...

    African Journals Online (AJOL)

    user

    2011-03-14

    Mar 14, 2011 ... cultivated edible legumes used as vegetable. Chickpeas are grown in the .... the culture medium had negative effect on shoot length and resulted in smaller shoots on both ... Effect of pre-conditioning on number of shoots and shoot length of mature embryo and embryonic axis explant of chickpea cv. Gokce.

  5. Adventitious shoot regeneration from cultured leaf explants of Petunia

    African Journals Online (AJOL)

    DIRECTOR

    2012-06-26

    Jun 26, 2012 ... the first bedding plant; in addition Petunia is the most ... leaf explants of Petunia using MS medium containing. 2 mgL .... suggested that differences in BA uptake and metabolism ... cation might have an inhibitory effect on shoot.

  6. The Removal of Hydrogel Explants: An Analysis of 467 Consecutive Cases

    NARCIS (Netherlands)

    Crama, N.; Klevering, B.J.

    2016-01-01

    PURPOSE: To describe the complications associated with hydrogel explants and to describe the indications, surgical technique, and risks involved in the removal of a hydrogel explant. DESIGN: Single-center, retrospective interventional case series. PARTICIPANTS: Patients who underwent surgical

  7. Morphologic differences observed by scanning electron microscopy according to the reason for pseudophakic IOL explantation

    DEFF Research Database (Denmark)

    Fernandez-Buenaga, Roberto; Alio, Jorge L.; Ramirez, Jose M.

    2015-01-01

    Purpose To compare variations in surface morphology, as studied by scanning electron microscopy (SEM), of explanted intraocular lenses (IOLs) concerning the cause leading to the explantation surgery. Methods In this prospective multicenter study, explanted IOLs were analyzed by SEM and energy-dis...

  8. Effect of storage media and time on fin explants culture in the ...

    African Journals Online (AJOL)

    On the outgrowth of cells, fin explants stored for seven days before culturing showed significantly higher growth (P<0.05) as observed on the fin explants stored in Dulbecco's modified Eagles medium (DMEM, 84.44%), phosphate buffered saline (PBS, 62.42%) and in control/fresh fin explants (100%), compared with the ...

  9. Absorção de macronutrientes por explantes de bananeira in vitro Macronutrient absorption by banana explants in vitro

    Directory of Open Access Journals (Sweden)

    Josefa Diva Nogueira Diniz

    1999-07-01

    Full Text Available Com o objetivo de estudar a absorção de macronutrientes (N, P, K, Ca, Mg e S em explantes de bananeira cv. Prata Anã, foram utilizados explantes de plantas estabelecidas in vitro, inoculados em meio básico de Murashige & Skoog (1962 contendo sacarose (30 g/L, e BAP (3,5 mg/L com sete tratamentos, representados pelos períodos de 0, 10, 20, 30, 40, 50 e 60 dias de cultivo e três repetições. As quantidades de macronutrientes totais absorvidas pelos explantes seguiram a ordem: K > N > Ca > ou = P > Mg @ S. O P foi o nutriente absorvido mais rapidamente pelos explantes, com 75% extraído do meio de cultivo nos primeiros 30 dias, cessando sua absorção aos 50 dias, restando ainda 9% no meio de cultivo. A absorção do S cessou também aos 50 dias, quando 66% deste nutriente ainda permanecia no meio de cultivo. Este resultado sugere haver uma relação, quanto à absorção, entre esses dois nutrientes. As maiores taxas de absorção de todos os nutrientes foram verificadas nos primeiros 20 dias. O rizoma, o pseudocaule e as folhas, se diferenciaram quanto à concentração e extração ou acúmulo de nutrientes.The absorption of the nutrients (N, P, K, Ca, Mg and S by banana (Musa sp. cv. Prata Anã explants on the basic medium of Murashige & Skoog (1962 supplemented with sucrose (30 g/L and BAP (3.5 mg/L were evaluated at 0, 10, 20, 30, 40, 50 and 60 days after inoculation. The seven treatments were arranged on a completely randomized design with three replicates. The sequence of nutrient absorption by the explants was K > N > Ca > or = P > Mg @ S. The P was the nutrient with the fastest absorption rate and at the 30th day the explants had already absorbed 75% of the P from the medium. The P absorption stopped by the 50th day. The S absorption stopped at the 50th day with 66% of it remaining in the medium. The results suggested a close relationship between these two nutrients. The highest rates of nutrient absorption were observed during the

  10. Laboratory analyses of two explanted hydrophobic acrylic intraocular lenses

    Directory of Open Access Journals (Sweden)

    Yunhai Dai

    2014-01-01

    Full Text Available Two three-piece hydrophobic acrylic intraocular lenses (IOLs were explanted from two patients at 7 and 9 years, respectively, after implantation, because of poor fundus visualisation and/or a clinically significant decrease in visual acuity related to their opacified IOLs. In addition to light microscopy, scanning electron microscopy and energy dispersive X-ray spectroscopy, confocal laser scanning microscopy was used for the first time to observe the explanted IOLs. The clinical aspect seemed to correspond to the phenomenon of surface light scattering, while laboratory analyses showed dense glistenings in the central layer of the IOL optic, which had no change next to the surface. Further studies on these phenomena are needed.

  11. Callus induction from epicotyl and hypocotyl explants of Parkia ...

    African Journals Online (AJOL)

    Established cultures were obtained and maintained on MS medium supplemented with either 2,4-D or NAA, each of concentration range of 0.4 – 1.0 mg/L. In general, while higher concentrations of 0.8 and 1.0 mg/L NAA appeared to have favoured callus production from these explants, the same concentrations of 2, 4-D ...

  12. Diverse response of tomato fruit explants to high temperature

    Directory of Open Access Journals (Sweden)

    Zofia Starck

    2014-01-01

    Full Text Available Tomato explants (fruit with a pedicel and a piece of peduncle, with fruit growth stimulated by treating the flowers with NOA + GA3 (NG-series were used as a model system for studying the effect of high temperature on C-sucrose uptake, its distribution and Ca retranslocation. Two cultivars with contrasting responses to high temperature were compared. In sensitive cv. Roma heat stress during 22h (40oC for 10h and 30oC for 12h, drastically depressed the uptake of 14C-sucrose coinciding with diminished fruit 14C-supply. It also decreased the specific activity of soluble acid invertase and the calcium content. All these strong negative responses to high temperature were markedly reduced in the NG-treated series involving remobilization of Ca to the fruits and a higher stability of the invertase activity. This indicates the indirect role of flower treatment with NG in addaptation to heat stress. In tolerant cv. Robin even higher temperatures (42oC for 10h and 34oC for 12h were not stressful. They did not affect the 14C-sucrose uptake and stimulated 14C-supply to the fruit. Increased specific activity of acid invertase and a higher calcium content were also recorded but only in the control explants. In contrast to cv. Roma elevated temperature was slightly stressful for cv. Robin explants of NG-series. The differences in response of both cultivar explants to elevated temperature, based on unequal fruit supply with 14C-sucrose, seem to be causaly connected with two factors: the invertase activity being more or less sensitive to the heat stress, the ability to translocate Ca to the heated fruits.

  13. Regeneration potential of seedling explants of chilli (Capsicum ...

    African Journals Online (AJOL)

    STORAGESEVER

    2009-02-18

    Feb 18, 2009 ... gation techniques for mass multiplication have become imperative. Conventional ..... the shooting media. For direct shoot regeneration, shoot tip explants were cultured onto MS medium supplemented with 5.0 mg L-1. BAP + 3.0 mg L-1 AgN03, 5.0 mg L-1 BAP + 0.1 mg L-1. NAA. It might be concluded from ...

  14. Shoot Regeneration from Leaf Explants of Withania somnifera (L. Dunal

    Directory of Open Access Journals (Sweden)

    Aruna Girish JOSHI

    2010-03-01

    Full Text Available Regeneration from leaf explants of Withania somnifera (L. for mass propagation was studied on Murashige and Skoog�s medium supplemented with Kinetin (Kn and 6-benzylaminopurine (BAP alone or in combination. Shoot buds were induced from the midrib on the abaxial side in presence of Kn and BAP (4 �M. These shoot buds developed into shoots on the same medium. Rooting of these shoots was achieved in 0.5 �M of IBA.

  15. Shoot Regeneration from Leaf Explants of Withania somnifera (L.) Dunal

    OpenAIRE

    Aruna Girish JOSHI; Mainavati A. PADHYA

    2010-01-01

    Regeneration from leaf explants of Withania somnifera (L.) for mass propagation was studied on Murashige and Skoog�s medium supplemented with Kinetin (Kn) and 6-benzylaminopurine (BAP) alone or in combination. Shoot buds were induced from the midrib on the abaxial side in presence of Kn and BAP (4 �M). These shoot buds developed into shoots on the same medium. Rooting of these shoots was achieved in 0.5 �M of IBA.

  16. Shoot Regeneration from Leaf Explants of Withania somnifera (L. Dunal

    Directory of Open Access Journals (Sweden)

    Aruna Girish JOSHI

    2010-03-01

    Full Text Available Regeneration from leaf explants of Withania somnifera (L. for mass propagation was studied on Murashige and Skoogs medium supplemented with Kinetin (Kn and 6-benzylaminopurine (BAP alone or in combination. Shoot buds were induced from the midrib on the abaxial side in presence of Kn and BAP (4 M. These shoot buds developed into shoots on the same medium. Rooting of these shoots was achieved in 0.5 M of IBA.

  17. callus induction and proliferation from cotyledon explants in ...

    African Journals Online (AJOL)

    ACSS

    2013-07-19

    amélioration de la qualité des graines est fortement souhaitable. Toutefois, cela exige au préalable l'existence de systèmes efficaces de régénération de plantes. L'induction de cals à partir des explants cotylédon a été étudiée chez ...

  18. An efficient plant regeneration protocol from petiole explants of ...

    African Journals Online (AJOL)

    The highest percentage of shoot buds induction (64.0%) was observed on MS medium supplemented with 0.52 mgL-1 TDZ with organic additives; adenine sulphate (50 mgL-1) + glutamine (100 mgL-1) + L-arginine (25 mgL-1) + citric acid (0.0025%) + ascorbic acid (0.005%). A maximum of six shoots per explant were ...

  19. In vitro regeneration from internodal explants of bitter melon ...

    African Journals Online (AJOL)

    Adventitious shoots were produced from organogenic callus when it was transferred to MS medium supplemented with 4.0 μM TDZ, 1.5 μM 2,4-D and 0.07 mM L-glutamine with shoot induction frequency of 96.5% and regeneration of adventitious shoots from callus (48 shoots per explant). Shoot proliferation occurred when ...

  20. Tropic responses of potato single-node explant cultures

    OpenAIRE

    Vinterhalter D.; Vinterhalter Branka

    2012-01-01

    A special in-vitro protocol was elaborated enabling the production of potato single-node explant plantlets that can be used as objects for tropic studies. In light-grown plantlets, achievement of a full (90°) phototropic (PT) curvature required 75 to 120 min of continuous unilateral blue light irradiation or 120-135 min of gravitropic stimulation (GT). Time-lapse photography revealed that the curves describing PT and GT bending have a sigmoid shape. Continuous BL irradiation was necessa...

  1. Materials characterization of explanted polypropylene hernia mesh: Patient factor correlation.

    Science.gov (United States)

    Smith, Sarah E; Cozad, Matthew J; Grant, David A; Ramshaw, Bruce J; Grant, Sheila A

    2016-02-01

    This study quantitatively assessed polypropylene (PP) hernia mesh degradation and its correlation with patient factors including body mass index, tobacco use, and diabetes status with the goal of improving hernia repair outcomes through patient-matched mesh. Thirty PP hernia mesh explants were subjected to a tissue removal process followed by assessment of their in vivo degradation using Fourier transform infrared, differential scanning calorimetry, and thermogravimetric analysis analyses. Results were then analyzed with respect to patient factors (body mass index, tobacco use, and diabetes status) to determine their influence on in vivo hernia mesh oxidation and degradation. Twenty of the explants show significant surface oxidation. Tobacco use exhibits a positive correlation with modulated differential scanning calorimetry melt temperature and exhibits significantly lower TGA decomposition temperatures than non-/past users. Chemical and thermal characterization of the explanted meshes indicate measurable degradation while in vivo regardless of the patient population; however, tobacco use is correlated with less oxidation and degradation of the polymeric mesh possibly due to a reduced inflammatory response. © The Author(s) 2015.

  2. Enhanced micropropagation and tiller formation in sugarcane through pretreatment of explants with thidiazuron (TDZ).

    Science.gov (United States)

    Kumari, Kavita; Lal, Madan; Saxena, Sangeeta

    2017-10-01

    An efficient, simple and commercially applicable protocol for rapid micropropagation of sugarcane has been designed using variety Co 05011. Pretreatment of shoot tip explants with thidiazuron (TDZ) induced high frequency regeneration of shoot cultures with improved multiplication ratio. The highest frequency (80%) of shoot initiation in explants pretreated with 10 mg/l of TDZ was obtained during the study. Maximum 65% shoot cultures could be established from the explants pretreated with TDZ as compared to minimum 40% establishment in explants without pretreatment. The explants pretreated with 10 mg/l of TDZ required minimum 40 days for the establishment of shoot cultures as compared to untreated explants which required 60 days. The highest average number of shoots per culture (19.1) could be obtained from the explants pretreated with 10 mg/l of TDZ, indicating the highest multiplication ratio (1:6). Highest rooting (over 94%) was obtained in shoots regenerated from pretreated explants on ½ strength MS medium containing 5.0 mg/l of NAA and 50 g/l of sucrose within 15 days. Higher number of tillers/clump (15.3) could be counted in plants regenerated from pretreated explants than untreated ones (10.9 tillers/clump) in field condition, three months after transplantation. Molecular analysis using RAPD and DAMD markers suggested that the pretreatment of explants with TDZ did not adversely affect the genetic stability of regenerated plants and maintained high clonal purity.

  3. Optimization of Brassica napus (canola) explant regeneration for genetic transformation.

    Science.gov (United States)

    Maheshwari, Priti; Selvaraj, Gopalan; Kovalchuk, Igor

    2011-12-15

    Brassica napus (canola) is the second largest oilseed crop in the world. It is among the first crops to be genetically transformed, and genetically modified cultivars are in commercial production at very significant levels. Despite the early lead with respect to transgenesis, there remain cultivars that are recalcitrant to transformation. To address this, we have conducted an elaborate investigation of the conditions for regenerating shoots from hypocotyl explants from four genetic lines: Invigor 5020, Westar and Topas as well as a microspore culture derived line of Topas (Line 4079). We analyzed the effect of hormonal combinations in regeneration medium, donor plant age and explant type on the regeneration capacity of these plants. The analysis showed that hypocotyls of eight-day-old seedlings grown on media supplemented with 1mg/L dinitrophenylhydrazine (2,4-D) produced the most shoots. Globular somatic embryos emerged following two weeks of 2,4-D treatment. When transferred to the medium containing 5mg/L benzyladenine (BA), approximately 82% of embryos produced shoots within six weeks. Invigor plants were shown to regenerate more efficiently than Topas; the number of plantlets regenerated from Invigor was approximately 40-50% more as compared to Topas or Line 4079. When hypocotyl explants were co-cultivated with the Agrobacterium strain GV3101 harboring a binary vector carrying a firefly luciferase reporter gene (LUC), significant numbers of plantlets were LUC-positive in a luciferase assay. Frequency of such plants were: Invigor 5020 (54.2 ± 2.5%), Westar (53.7 ± 5.3), Topas (16.0 ± 0.24) and Line 4079 (13.4 ± 4). Copyright © 2011 Elsevier B.V. All rights reserved.

  4. Absorção de micronutrientes por explantes de bananeira in vitro Micronutrient absorption by banana explants in vitro

    Directory of Open Access Journals (Sweden)

    Josefa Diva Nogueira Diniz

    1999-08-01

    Full Text Available Foi estudada a absorção dos micronutrientes B, Cu, Fe, Mn e Zn em explantes de bananeira (Musa sp., cultivar Prata Anã em meio básico de Murashige & Skoog suplementado com 30g/L de sacarose e 3,5mg/L de BAP. O experimento foi realizado em delineamento completamente casualizado, com três repetições. Na matéria seca dos propágulos inteiros, rizomas, pseudocaules e folhas foram avaliadas a concentração e extração de nutrientes, e, no meio de cultivo, a quantidade remanescente aos 0, 10, 20, 30, 40, 50 e 60 dias. A maior quantidade de micronutrientes extraída pelos propágulos foi observada nos primeiros 20 dias, exceto no tocante ao Mn, que foi aproximadamente constante durante todo o período. O Fe e o Cu foram os micronutrientes absorvidos em maior e menor quantidade, respectivamente. As concentrações de B, Zn, Mn, e Cu remanescentes no meio de cultivo aos 60 dias foram de 52, 61, 77 e 78%, respectivamente, o que sugere que podem ser reduzidas no meio básico MS para o cultivo de explantes de bananeira.The absorption of the micronutrients B, Cu, Fe, Mn and Zn by banana (Musa sp. cultivar "Prata Anã" explants on the basic medium of Murashige & Skoog supplemented with BAP (3.5 mg/L and sucrose (30 g/L were evaluated at 0, 10, 20, 30, 40, 50 and 60 days after the inoculation. The experiment was arranged on a completely randomized design with three replications. Concentration of the micronutrients in the medium and in dry matter of the whole propagule and in the rhizome, pseudostem and leaves was also evaluated. Absorption of Mn was approximately constant during all the period of the experiment, while the other micronutrients had their higher absorption observed on the first 20 days. At the end of the experiment concentrations of B, Zn, Mn and Cu in the medium were 52, 61, 77 and 78%, respectively. These results point out that it is possible to reduce the concentration of these micronutrients on the basic medium MS for banana

  5. Microscopic and spectroscopic investigation of an explanted opacified intraocular lens

    Energy Technology Data Exchange (ETDEWEB)

    Simon, V., E-mail: viosimon@phys.ubbcluj.ro [Babeş-Bolyai University, Faculty of Physics and Interdisciplinary Research Institute on Bio-Nano-Sciences, 400084 Cluj-Napoca (Romania); Radu, T.; Vulpoi, A. [Babeş-Bolyai University, Faculty of Physics and Interdisciplinary Research Institute on Bio-Nano-Sciences, 400084 Cluj-Napoca (Romania); Rosca, C. [Optilens Clinic of Ophthalmology, 400604 Cluj-Napoca (Romania); Eniu, D. [Iuliu Haţieganu University of Medicine and Pharmacy, Department of Molecular Sciences, 400349 Cluj-Napoca (Romania)

    2015-01-15

    Highlights: • Changes on intraocular lens (IOL) surface after implantation. • Partial opacification of IOL central area. • Elemental composition on IOL surface prior to and after implantation. • First XPS depth profiling examination of the opacifying deposits. • Cell-mediated hydroxyapatite structuring. - Abstract: The investigated polymethylmethacrylate intraocular lens explanted an year after implantation presented a fine granularity consisting of ring-like grains of about 15 μm in diameter. In order to evidence the changes occurred on intraocular lens relative to morphology, elemental composition and atomic environments, microscopic and spectroscopic analyses were carried out using scanning electron microscopy (SEM), Fourier transform infrared (FTIR), energy-dispersive X-ray (EDS), and X-ray photoelectron (XPS) spectroscopies. The results revealed that the grains contain hydroxyapatite mineral phase. A protein layer covers the lens both in opacified and transparent zones. The amide II band is like in basal epithelial cells. The shape and size of the grains, and the XPS depth profiling results indicate the possibility of a cell-mediated process involving lens epithelial cells which fagocitated apoptotic epithelial cells, and in which the debris derived from cell necrosis were calcified. To the best of our knowledge, this is the first investigation on explanted intraocular lenses using XPS depth profiling in order to examine the inside of the opacifying deposits.

  6. Bovine explant model of degeneration of the intervertebral disc

    Directory of Open Access Journals (Sweden)

    Sivan Sarit

    2008-02-01

    Full Text Available Abstract Background Many new treatments for degeneration of the intervertebral disc are being developed which can be delivered through a needle. These require testing in model systems before being used in human patients. Unfortunately, because of differences in anatomy, there are no ideal animal models of disc degeneration. Bovine explant model systems have many advantages but it is not possible to inject any significant volume into an intact disc. Therefore we have attempted to mimic disc degeneration in an explant bovine model via enzymatic digestion. Methods Bovine coccygeal discs were incubated with different concentrations of the proteolytic enzymes, trypsin and papain, and maintained in culture for up to 3 weeks. A radio-opaque solution was injected to visualise cavities generated. Degenerative features were monitored histologically and biochemically (water and glycosaminoglycan content, via dimethylmethylene blue. Results and Conclusion The central region of both papain and trypsin treated discs was macro- and microscopically fragmented, with severe loss of metachromasia. The integrity of the surrounding tissue was mostly in tact with cells in the outer annulus appearing viable. Biochemical analysis demonstrated greatly reduced glycosaminoglycan content in these compared to untreated discs. We have shown that bovine coccygeal discs, treated with proteolytic enzymes can provide a useful in vitro model system for developing and testing potential new treatments of disc degeneration, such as injectable implants or biological therapies.

  7. Micropropagation of Cyrtopodium paludicolum (Orchidaceae from root tip explants

    Directory of Open Access Journals (Sweden)

    Dayana Rotili Nunes Picolotto

    2017-06-01

    Full Text Available An efficient protocol for in vitro plant propagation of Cyrtopodium paludicolum has been developed using root tips dissected from well-developed seedlings. Root tips were cultured on Knudson medium supplemented with α-naphthaleneacetic acid (NAA, and/or thidiazuron (TDZ. TDZ did not induce protocorm-like bodies (PLBs in the NAA absence, indicating phytoregulators synergistic effect. Medium supplemented with 1.34 μM NAA and 2.27 μM TDZ resulted in better response on PBLs, and subsequent shoot differentiation (55.25 shoots per explant, and in better rooting number and root length responses, favoring acclimatization with 90% of survived plants. However, the medium supplemented with only NAA (1.34 μM resulted in 33.50 shoots per explant. Histological sections confirmed that only one PLB was induced per responsive root tip, and it showed numerous dispersed and extended meristemoids, or division centers that originated new PBLs. Additionally, this protocol could be an excellent model to study molecular aspects of root to shoot conversion.

  8. Effect of media composition and explant type on the regeneration of ...

    African Journals Online (AJOL)

    Two as well as three way interactions of three eggplant genotypes, media compositions and explants (hypocotyl, cotyledon and leaf) showed significant differences for plant regeneration. Among three explants, hypocotyl induced highest percent callusing, but cotyledon showed best results for somatic embryogenesis on all ...

  9. Effect of 2,4-D, explants type and cultivar on the callogenesis ...

    African Journals Online (AJOL)

    Although, statistically insignificant, the 8 mg/l 2,4-D concentration was visually the best in callus formation from explants both within and across the cassava cultivars. This study had shown that different cassava explants respond differently to tissue culture conditions established for callus culture formation. Key words: Auxin ...

  10. Influence of genotype and age of explant source on the capacity for ...

    African Journals Online (AJOL)

    ONOS

    2010-04-12

    Apr 12, 2010 ... The embryogenic capacities of flower explants from one- and two-week-old male inflorescence buds from Musa acuminata Cavendish, AAA, genotypes 'Williams' and 'Grand Naine' were investigated. Explants of hands with immature flowers were excised and induced for embryogenesis. Highly significant ...

  11. In vitro morphogenic events in culture of Lotus corniculatus L. seedling root explants

    Directory of Open Access Journals (Sweden)

    Jan J. Rybczyński

    2011-01-01

    Full Text Available The experiments were carried out on Lotus corniculatus (L. seedling root explants of the cultivar varieties Skrzeszowicka, Caroll A10 and strain 175. Callus formation and shoot regeneration were the major explant response depended mainly on of the studied genotype and used plant growth regulators (PGRs. Primary cortex of proximal and distal end of explant was the most active tissue for callus proliferation. For shoot primordia differentiation deeper zones of cortex took a part. The process of meristematic centre initiation was not uniform and various level of shoot differentiation events were observed not earlier than 3 weeks of culture. Usually, the shoot primordia regeneration began on proximal rather than distal end of the explant. BAP rather than urea derivatives stimulated shoot proliferation in extended cultures. Increasing of BAP and TDZ concentrations brought about the explant polarity and expansion of the meristematic zones. The explant position in root did not have significant influence on the number of regenerated shoots. The cultures only had better bud formation by TDZ when compared to BAP. BAP stimulated bud formation and development of the shoots from them. Short term of TDZ treatment of explants stimulated meristem formation which developed into buds and shoots. CPPU stimulated callus proliferation and bud formation when explants pretreatment was prolonged from 12 to 36 hrs.

  12. EXPLANTATION OF MESANGIAL CELL HILLOCKS - A METHOD FOR OBTAINING HUMAN MESANGIAL CELLS IN CULTURE

    NARCIS (Netherlands)

    MULLER, EW; KIM, Y; MICHAEL, AF; VERNIER, RL; VANDERHEM, GK; VANDERWOUDE, FJ

    A simple method is presented for selective cell culture of human mesangial cells using explanatation of mesangial cell hillocks. Glomeruli which had been incubated with collagenase were explanted on plastic tissue culture flasks. Three to 6 weeks after explantation, a rapidly growing multilayer of

  13. A simple technique of intraocular lenses explantation for single-piece foldable lenses

    Directory of Open Access Journals (Sweden)

    Arup Bhaumik

    2017-01-01

    Full Text Available Foldable intraocular lenses (IOLs are most commonly used in modern-day cataract surgery. Explantation of these IOLs is not frequently encountered, but sometimes extreme situations may demand the same. Commonly explantation is achieved by bisecting the IOL inside the anterior chamber with a cutter and delivering the pieces out one by one. This may require corneal wound extension with associated damage and endothelial loss leading to visual deterioration. We devised a simple, innovative IOL explantation technique utilizing a modified Alcon A cartridge and snare. This can successfully refold the IOL to be explanted inside the eye and deliver it out through the same wound. The device has limitations with very thick optic lenses, multipiece, and silicon IOLs. In conclusion, we describe a simple, innovative, and reproducible technique to explant almost any single piece IOL without compromising the original surgery and yielding very satisfactory outcomes.

  14. Hypoxia inhibits hypertrophic differentiation and endochondral ossification in explanted tibiae.

    Directory of Open Access Journals (Sweden)

    Jeroen C H Leijten

    Full Text Available Hypertrophic differentiation of growth plate chondrocytes induces angiogenesis which alleviates hypoxia normally present in cartilage. In the current study, we aim to determine whether alleviation of hypoxia is merely a downstream effect of hypertrophic differentiation as previously described or whether alleviation of hypoxia and consequent changes in oxygen tension mediated signaling events also plays an active role in regulating the hypertrophic differentiation process itself.Fetal mouse tibiae (E17.5 explants were cultured up to 21 days under normoxic or hypoxic conditions (21% and 2.5% oxygen respectively. Tibiae were analyzed on growth kinetics, histology, gene expression and protein secretion.The oxygen level had a strong influence on the development of explanted fetal tibiae. Compared to hypoxia, normoxia increased the length of the tibiae, length of the hypertrophic zone, calcification of the cartilage and mRNA levels of hypertrophic differentiation-related genes e.g. MMP9, MMP13, RUNX2, COL10A1 and ALPL. Compared to normoxia, hypoxia increased the size of the cartilaginous epiphysis, length of the resting zone, calcification of the bone and mRNA levels of hyaline cartilage-related genes e.g. ACAN, COL2A1 and SOX9. Additionally, hypoxia enhanced the mRNA and protein expression of the secreted articular cartilage markers GREM1, FRZB and DKK1, which are able to inhibit hypertrophic differentiation.Collectively our data suggests that oxygen levels play an active role in the regulation of hypertrophic differentiation of hyaline chondrocytes. Normoxia stimulates hypertrophic differentiation evidenced by the expression of hypertrophic differentiation related genes. In contrast, hypoxia suppresses hypertrophic differentiation of chondrocytes, which might be at least partially explained by the induction of GREM1, FRZB and DKK1 expression.

  15. Histologic Evaluation of Explanted Tissue-Engineered Bovine Pericardium (CardioCel).

    Science.gov (United States)

    Prabhu, Sudesh; Armes, Jane E; Bell, Douglas; Justo, Robert; Venugopal, Prem; Karl, Tom; Alphonso, Nelson

    2017-01-01

    CardioCel is a bovine pericardium that is subjected to a novel anticalcification tissue-engineering process. We present the histopathologic findings of human explants of CardioCel that were used in operations for congenital heart disease in children. Six explants were identified from 140 patients undergoing CardioCel implants from October 2012 to March 2015. CardioCel explants were evaluated histologically using hematoxylin and eosin, Masson trichrome, and immunohistochemical staining. A variable inflammatory response was seen in the surrounding native tissue, but not within the CardioCel graft in any of the explants. A neointimal layer of varying thickness developed on the visceral surface of 5 CardioCel explants with endothelialization of the longest duration explant. A granulation tissue layer developed on the parietal surface of the graft (consistently thicker than the neointima). Maintained collagen fiber architecture (laminated) and variable fibroblastic invasion (which increased with the age of the implant) were identified in all 6 cases. Scattered capillary vessels were noted in the majority of the explants with new collagen fibers in one, suggesting early remodeling. Calcium was seen in 1 explant at the interface of the graft and inflammatory response on its parietal surface. Evidence of graft remodeling was noted in the majority of the explants without inflammatory cells or calcification within the explanted graft material. A noticeable feature was the differential thickness of the host reaction to the parietal compared with the visceral surface of the graft. We will continue to evaluate CardioCel as a cardiovascular substitute for extracardiac and intracardiac reconstructions. Copyright © 2017 Elsevier Inc. All rights reserved.

  16. Correlation of Pre-Explant Lactate Dehydrogenase Concentrations and Findings During Post-Explant Pump Analysis of the HeartMate II Left Ventricular Assist Device.

    Science.gov (United States)

    Sood, Vikram; Alam, Osman; Tchantchaleishvili, Vakhtang; Pagani, Francis D; Aaronson, Keith D; Haft, Jonathan; Joyce, David L; Joyce, Lyle D; Daly, Richard C; Maltais, Simon; Stulak, John M

    2017-04-01

    Analyses of the HeartMate II left ventricular assist device are routinely performed after explant if returned to the manufacturer. Findings from manufacturer-reported pump analyses have not been correlated with pre-explant serum lactate dehydrogenase (LDH) values. Between May 2004 and December 2014, 502 patients underwent primary HeartMate II implantation. Seventy pumps were explanted in 58 patients and returned to the manufacturer for pump analysis: 51 (73%) for suspected pump thrombosis, 12 (17%) for device-related infection, and 7 (13%) for percutaneous lead fracture. Median time from implant to explant was 12.4 months (range, 1 to 57 months). Pump thrombus was confirmed in 53 of 70 pumps (76%). Stratified by major clinical indications for explant of suspected pump thrombus or infection and lead fracture, the presence of pump thrombus was identified in 47 of 51 (92%) and 6 of 19 (32%), respectively. The median of 1) all LDH values, 2) maximum LDH values, and 3) interval change in LDH between 6 and 2 months before device explant were 1,061, 1,940, and -27 IU/L with thrombus present and 533, 504, and 13 IU/L in the absence of thrombus. Density estimation of LDH concentrations identified that a LDH value of 1,155 IU/L in the 6 months preceding pump thrombus had a 78% sensitivity and 90% specificity for findings of pump thrombus at the time of pump analysis. Pre-explant LDH concentrations significantly correlated with findings on manufacture-performed pump analysis. These data validate LDH surveillance as an important clinical tool for identification of pump thrombus. Copyright © 2017 The Society of Thoracic Surgeons. Published by Elsevier Inc. All rights reserved.

  17. Azithromycin is able to control Toxoplasma gondii infection in human villous explants

    Science.gov (United States)

    2014-01-01

    Background Although Toxoplasma gondii infection is normally asymptomatic, severe cases of toxoplasmosis may occur in immunosuppressed patients or congenitally infected newborns. When a fetal infection is established, the recommended treatment is a combination of pyrimethamine, sulfadiazine and folinic acid (PSA). The aim of the present study was to evaluate the efficacy of azithromycin to control T. gondii infection in human villous explants. Methods Cultures of third trimester human villous explants were infected with T. gondii and simultaneously treated with either PSA or azithromycin. Proliferation of T. gondii, as well as production of cytokines and hormones by chorionic villous explants, was analyzed. Results Treatment with either azithromycin or PSA was able to control T. gondii infection in villous explants. After azithromycin or PSA treatment, TNF-α, IL-17A or TGF-β1 levels secreted by infected villous explants did not present significant differences. However, PSA-treated villous explants had decreased levels of IL-10 and increased IL-12 levels, while treatment with azithromycin increased production of IL-6. Additionally, T. gondii-infected villous explants increased secretion of estradiol, progesterone and HCG + β, while treatments with azithromycin or PSA reduced secretion of these hormones concurrently with decrease of parasite load. Conclusions In conclusion, these results suggest that azithromycin may be defined as an effective alternative drug to control T. gondii infection at the fetal-maternal interface. PMID:24885122

  18. The metabolic dynamics of cartilage explants over a long-term culture period

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    E.K Moo

    2011-01-01

    Full Text Available INTRODUCTION: Although previous studies have been performed on cartilage explant cultures, the generalized dynamics of cartilage metabolism after extraction from the host are still poorly understood due to differences in the experimental setups across studies, which in turn prevent building a complete picture. METHODS: In this study, we investigated the response of cartilage to the trauma sustained during extraction and determined the time needed for the cartilage to stabilize. Explants were extracted aseptically from bovine metacarpal-phalangeal joints and cultured for up to 17 days. RESULTS: The cell viability, cell number, proteoglycan content, and collagen content of the harvested explants were analyzed at 0, 2, 10, and 17 days after explantation. A high percentage of the cartilage explants were found to be viable. The cell density initially increased significantly but stabilized after two days. The proteoglycan content decreased gradually over time, but it did not decrease to a significant level due to leakage through the distorted peripheral collagen network and into the bathing medium. The collagen content remained stable for most of the culture period until it dropped abruptly on day 17. CONCLUSION: Overall, the tested cartilage explants were sustainable over long-term culture. They were most stable from day 2 to day 10. The degradation of the collagen on day 17 did not reach diseased levels, but it indicated the potential of the cultures to develop into degenerated cartilage. These findings have implications for the application of cartilage explants in pathophysiological fields.

  19. INDIRECT ORGANOGENESIS FROM LEAF EXPLANTS AND IN VITRO SHOOTS MULTIPLICATION OF Eucalyptus benthamii X Eucalyptus dunnii

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    Yohana de Oliveira-Cauduro

    2014-06-01

    Full Text Available http://dx.doi.org/10.5902/1980509814572The aims of this research were to evaluate different culture media for indirect organogenesis and shoot multiplication of Eucalyptus benthamii x Eucalyptus dunnii. For organogenesis, leaf explants were used to test the following treatments: two culture media (MS N/2 and JADS supplemented with 0.1 μM 1-naphthaleneacetic acid (NAA and thidiazuron (TDZ (0.1 or 0.5 μM, with or without PVP- 40 (250 mg L-1. The percentage of oxidized explants, callus forming explants, explants with anthocyanin, buds, shoots and the shoot number per explant were evaluated. In the multiplication experiment, isolated shoots were cultivated in MS, JADS and WPM media, all supplemented with 1.11 μM BAP. Four subcultures were carried out every 28 days. In every subculture the explant oxidation, partial or total leaf chlorosis, fresh mass and mean number of shoot per explant were evaluated. The MS N/2 medium supplemented with 0.1 μM NAA and 0.5 μM TDZ promoted the highest rate of organogenesis (8.3% and the culture media MS supplemented with 1.11 μM BAP the multiplication rate was higher than in the other media, in the first and the second subcultures (9.28 and 9.24, respectively, without differences between the three media in the following subcultures. 

  20. An ex vivo porcine nasal mucosa explants model to study MRSA colonization.

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    Pawel Tulinski

    Full Text Available Staphylococcus aureus is an opportunistic pathogen able to colonize the upper respiratory tract and skin surfaces in mammals. Methicillin-resistant S. aureus ST398 is prevalent in pigs in Europe and North America. However, the mechanism of successful pig colonization by MRSA ST398 is poorly understood. To study MRSA colonization in pigs, an ex vivo model consisting of porcine nasal mucosa explants cultured at an air-liquid interface was evaluated. In cultured mucosa explants from the surfaces of the ventral turbinates and septum of the pig nose no changes in cell morphology and viability were observed up to 72 h. MRSA colonization on the explants was evaluated followed for three MRSA ST398 isolates for 180 minutes. The explants were incubated with 3×10(8 CFU/ml in PBS for 2 h to allow bacteria to adhere to the explants surface. Next the explants were washed and in the first 30 minutes post adhering time, a decline in the number of CFU was observed for all MRSA. Subsequently, the isolates showed either: bacterial growth, no growth, or a further reduction in bacterial numbers. The MRSA were either localized as clusters between the cilia or as single bacteria on the cilia surface. No morphological changes in the epithelium layer were observed during the incubation with MRSA. We conclude that porcine nasal mucosa explants are a valuable ex vivo model to unravel the interaction of MRSA with nasal tissue.

  1. Genome-wide transcriptome and expression profile analysis of Phalaenopsis during explant browning.

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    Chuanjun Xu

    Full Text Available Explant browning presents a major problem for in vitro culture, and can lead to the death of the explant and failure of regeneration. Considerable work has examined the physiological mechanisms underlying Phalaenopsis leaf explant browning, but the molecular mechanisms of browning remain elusive. In this study, we used whole genome RNA sequencing to examine Phalaenopsis leaf explant browning at genome-wide level.We first used Illumina high-throughput technology to sequence the transcriptome of Phalaenopsis and then performed de novo transcriptome assembly. We assembled 79,434,350 clean reads into 31,708 isogenes and generated 26,565 annotated unigenes. We assigned Gene Ontology (GO terms, Kyoto Encyclopedia of Genes and Genomes (KEGG annotations, and potential Pfam domains to each transcript. Using the transcriptome data as a reference, we next analyzed the differential gene expression of explants cultured for 0, 3, and 6 d, respectively. We then identified differentially expressed genes (DEGs before and after Phalaenopsis explant browning. We also performed GO, KEGG functional enrichment and Pfam analysis of all DEGs. Finally, we selected 11 genes for quantitative real-time PCR (qPCR analysis to confirm the expression profile analysis.Here, we report the first comprehensive analysis of transcriptome and expression profiles during Phalaenopsis explant browning. Our results suggest that Phalaenopsis explant browning may be due in part to gene expression changes that affect the secondary metabolism, such as: phenylpropanoid pathway and flavonoid biosynthesis. Genes involved in photosynthesis and ATPase activity have been found to be changed at transcription level; these changes may perturb energy metabolism and thus lead to the decay of plant cells and tissues. This study provides comprehensive gene expression data for Phalaenopsis browning. Our data constitute an important resource for further functional studies to prevent explant browning.

  2. Reproducible simulation of respiratory motion in porcine lung explants

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    Biederer, J. [Dept. of Diagnostic Radiology, Univ. Hospital Schleswig-Holstein, Campus Kiel (Germany); Dept. of Radiology, German Cancer Research Center, Heidelberg (Germany); Plathow, C. [Dept. of Diagnostic Radiology, Eberhard-Karls-Univ. Tuebingen (Germany); Dept. of Radiology, German Cancer Research Center, Heidelberg (Germany); Schoebinger, M.; Meinzer, H.P. [Dept. of Medical and Biological Informatics, German Cancer Research Center, Heidelberg (Germany); Tetzlaff, R.; Puderbach, M.; Zaporozhan, J.; Kauczor, H.U. [Dept. of Radiology, German Cancer Research Center, Heidelberg (Germany); Bolte, H.; Heller, M. [Dept. of Diagnostic Radiology, Univ. Hospital Schleswig-Holstein, Campus Kiel (Germany)

    2006-11-15

    Purpose: To develop a model for exactly reproducible respiration motion simulations of animal lung explants inside an MR-compatible chest phantom. Materials and Methods: The materials included a piston pump and a flexible silicone reconstruction of a porcine diaphragm and were used in combination with an established MR-compatible chest phantom for porcine heart-lung preparations. The rhythmic inflation and deflation of the diaphragm at the bottom of the artificial thorax with water (1-1.5 L) induced lung tissue displacement resembling diaphragmatic breathing. This system was tested on five porcine heart-lung preparations using 1.5T MRI with transverse and coronal 3D-GRE (TR/TE=3.63/1.58, 256 x 256 matrix, 350 mm FOV, 4 mm slices) and half Fourier T2-FSE (TR/TE=545/29, 256 x 192, 350 mm, 6 mm) as well as multiple row detector CT (16 x 1 mm collimation, pitch 1.5, FOV 400 mm, 120 mAs) acquired at five fixed inspiration levels. Dynamic CT scans and coronal MRI with dynamic 2D-GRE and 2D-SS-GRE sequences (image frequencies of 10/sec and 3/sec, respectively) were acquired during continuous 'breathing' (7/minute). The position of the piston pump was visually correlated with the respiratory motion visible through the transparent wall of the phantom and with dynamic displays of CT and MR images. An elastic body splines analysis of the respiratory motion was performed using CT data. Results: Visual evaluation of MRI and CT showed three-dimensional movement of the lung tissue throughout the respiration cycle. Local tissue displacement inside the lung explants was documented with motion maps calculated from CT. The maximum displacement at the top of the diaphragm (mean 26.26 [SD 1.9] mm on CT and 27.16 [SD 1.5] mm on MRI, respectively [p=0.25; Wilcoxon test]) was in the range of tidal breathing in human patients. Conclusion: The chest phantom with a diaphragmatic pump is a promising platform for multi-modality imaging studies of the effects of respiratory lung

  3. Morphogenetic response of cotyledon and leaf explants of melon (Cucumis melo L. cv. Amarillo Oro

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    Fernanda Vidigal Duarte Souza

    2006-01-01

    Full Text Available Callus cultures from cotyledon and leaf explants of a Spanish cultivar of melon (Amarillo Oro were tested for growth and morphogenic capacity on several culture media with different concentrations of IAA (indole-3-acetic acid in combination with 1.0 mg.L-1 BA (6-benzylaminopurine or 6.0 mg.L-1 KIN (kinetin. The best results were achieved with cotyledon explants. The leaf explants presented low bud formation capacity. Variability of organogenic response on cotyledons of different age (7, 5, 3 and 1-day-old was evaluated. The age of explant had a significant influence on bud induction. Cotyledon explants from 7-day-old seedlings showed higher organogenic index and development of shoots when cultured onto MS medium supplemented with 1.5 mg.L-1 of IAA and 1.0 mg.L-1 of BA. The effect of cut type of cotyledonary explants on organogenic response was also investigated. Explants cut transversally showed the best results. The addition of copper sulfate in the culture medium promoted a qualitative improvement of the regenerated shoots.Explantes de cotilédones e folhas do cultivar de melão Amarillo Oro foram cultivados para avaliação do potencial morfogenético em diferentes meios de cultura com diversas concentrações de ácido indolacético (AIA, em combinação com 1,0 mg.L-1 benziladenina (BA e 6,0 mg.L-1 de cinetina (CIN. Os melhores resultados foram obtidos com explantes de cotilédones, sendo que explantes de folhas mostraram uma capacidade baixa na indução de gemas. A variação na resposta organogênica em cotilédones de diferentes idades fisiológicas (1, 3, 5 e 7 dias após a germinação mostraram que os melhores resultados foram obtidos com cotilédones de sete dias cultivados no meio MS suplementado com 1,5 mg.L-1 de AIA e 1,0 mg.L-1 de BA. O efeito do tipo de corte na resposta organogênica de explantes cotiledonares foi também avaliado. Os melhores resultados foram obtidos com explantes cortados transversalmente. Adi

  4. Micropropagation Of Merremia Quinquefolia (L. Hallier F. From Nodal Explants

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    Kher Mafatlal M.

    2015-06-01

    Full Text Available Merremia quinquefolia, is an important medicinal plant of the family Convolvulaceae known for its vasoconstrictor, uterotonic, neurohormonic, sympathicolytic and sedative effects. In the present investigation effect of cytokinins 6-benzylaminopurine (BAP, kinetin (Kn and thidiazuron (TDZ, at concentrations 1.0, 2.0, 3.0, 4.0 and 5.0 mg·dm−3 on in vitro shoot multiplication from nodal explants of M. quinquefolia was evaluated. Bud breaking and emergence of shoots started within 10-15 days of inoculation in all media containing cytokinin. Murashige and Skoog (MS medium supplemented with 4.0 mg·dm−3 BAP resulted in maximum number of shoots from single node within 45 days. In vitro raised shoots were successfully rooted on ½ mineral salts of MS medium with 3% sucrose supplemented with 2.0 mg·dm−3 indole-3-butyric acid (IBA. This is the first report on in vitro propagation of Merremia quinquefolia. This study can be useful for development of micropropagation protocols for related taxa.

  5. Shoot meristem: an ideal explant for Zea mays L. transformation.

    Science.gov (United States)

    Sairam, R V; Parani, M; Franklin, G; Lifeng, Z; Smith, B; MacDougall, J; Wilber, C; Sheikhi, H; Kashikar, N; Meeker, K; Al-Abed, D; Berry, K; Vierling, R; Goldman, S L

    2003-04-01

    We report on a rapid high-frequency somatic embryogenesis and plant regeneration protocol for Zea mays. Maize plants were regenerated from complete shoot meristem (3-4 mm) explants via organogenesis and somatic embryogenesis. In organogenesis, the shoot meristems were directly cultured on a high-cytokinin medium comprising 5-10 mg x L(-1) 6-benzylaminopurine (BAP). The number of multiple shoots produced per meristem varied from six to eight Plantlet regeneration through organogenesis resulted in just four weeks. Callus was induced in five days of incubation on an auxin-modified Murashige and Skoog (MS) medium. Prolific callus, with numerous somatic embryos, developed within 3-4 weeks when cultured on an auxin medium containing 5 mg 2,4-dichlorophenoxyacetic acid x L(-1). The number of multiple shoots varied from three to six per callus. Using R23 (Pioneer, Hi-Bred, Johnston, Iowa), the frequency of callus induction was consistently in excess of 80% and plant regeneration ranged between 47 and 64%. All regenerated plantlets survived in the greenhouse and produced normal plants. Each transgenic plant produced leaves, glumes, and anthers that uniformly expressed green fluorescent protein (GFP). The GFP gene segregated in the pollen. Based on this data it is concluded that the transgenics arose from single-cell somatic embryos. The rate of transfer DNA (T-DNA) transfer to complete shoot meristems of Zea mays was high on the auxin medium and was independent of using super-virulent strains of Agrobacterium.

  6. Tropic responses of potato single-node explant cultures

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    Vinterhalter D.

    2012-01-01

    Full Text Available A special in-vitro protocol was elaborated enabling the production of potato single-node explant plantlets that can be used as objects for tropic studies. In light-grown plantlets, achievement of a full (90° phototropic (PT curvature required 75 to 120 min of continuous unilateral blue light irradiation or 120-135 min of gravitropic stimulation (GT. Time-lapse photography revealed that the curves describing PT and GT bending have a sigmoid shape. Continuous BL irradiation was necessary for the induction of continuous PT bending. If the BL was turned off after 30-50 min of PT stimulation, the bending gradually decreased and stopped in darkness after 25.0 ± 2.0 min. Within this period, curvature increased by 15.5 ± 1.5°. When the BL was turned off upon completion of PT bending (when the plantlets reached an angle of 90°, the plantlets entered the phase of fast straightening. The 90° PT curvature was significantly exaggerated in darkness by turning the jars from a vertical to horizontal position providing 120.74 ± 2.5° as the final curvature angle after two more hours in darkness.

  7. Cultivar, explant type and culture medium influencing embryogenesis and organogenegenesis in Anthurium spp.

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    Yaowaphan Sontikun

    2006-07-01

    Full Text Available The effect of cultivars, culture media, explant type and wounding on culture response of anthurium were studied. Valantino gave the highest callus formation (83.73% significantly different from Sonat (78.73% and Plew Thien Phuket (45.66%. Valantino and Plew Thien Phuket gave meristematic nodular callus (MNC whereas Sonat produced embryogenic-like callus (ELC. Modified Murashige and Skoog (MMS medium gave the highest callus formation from both leaf (86.6% and node (100%. Callus obtained in Nitsch and Nitsch (NN and MMS was MNC while woody plant (WPM medium provided ELC. For explant types, internode gave the highest callus formation (72.63%. Nodal and internodal explant gave ELC while the leaf explant yielded MNC. Wounding leaf blades tended to promote more MNC.

  8. Proteomic Profile of Brucella abortus-Infected Bovine Chorioallantoic Membrane Explants

    Science.gov (United States)

    Mol, Juliana P. S.; Pires, Simone F.; Chapeaurouge, Alexander D.; Perales, Jonas; Santos, Renato L.; Andrade, Hélida M.; Lage, Andrey P.

    2016-01-01

    Brucella abortus is the etiological agent of bovine brucellosis, a zoonotic disease that causes significant economic losses worldwide. The differential proteomic profile of bovine chorioallantoic membrane (CAM) explants at early stages of infection with B. abortus (0.5, 2, 4, and 8 h) was determined. Analysis of CAM explants at 0.5 and 4 h showed the highest differences between uninfected and infected CAM explants, and therefore were used for the Differential Gel Electrophoresis (DIGE). A total of 103 spots were present in only one experimental group and were selected for identification by mass spectrometry (MALDI/ToF-ToF). Proteins only identified in extracts of CAM explants infected with B. abortus were related to recognition of PAMPs by TLR, production of reactive oxygen species, intracellular trafficking, and inflammation. PMID:27104343

  9. Organ explant culture of neonatal rat ventricles: a new model to study gene and cell therapy.

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    A Dénise den Haan

    Full Text Available Testing cardiac gene and cell therapies in vitro requires a tissue substrate that survives for several days in culture while maintaining its physiological properties. The purpose of this study was to test whether culture of intact cardiac tissue of neonatal rat ventricles (organ explant culture may be used as a model to study gene and cell therapy. We compared (immuno histology and electrophysiology of organ explant cultures to both freshly isolated neonatal rat ventricular tissue and monolayers. (Immuno histologic studies showed that organ explant cultures retained their fiber orientation, and that expression patterns of α-actinin, connexin-43, and α-smooth muscle actin did not change during culture. Intracellular voltage recordings showed that spontaneous beating was rare in organ explant cultures (20% and freshly isolated tissue (17%, but common (82% in monolayers. Accordingly, resting membrane potential was -83.9±4.4 mV in organ explant cultures, -80.5±3.5 mV in freshly isolated tissue, and -60.9±4.3 mV in monolayers. Conduction velocity, measured by optical mapping, was 18.2±1.0 cm/s in organ explant cultures, 18.0±1.2 cm/s in freshly isolated tissue, and 24.3±0.7 cm/s in monolayers. We found no differences in action potential duration (APD between organ explant cultures and freshly isolated tissue, while APD of monolayers was prolonged (APD at 70% repolarization 88.8±7.8, 79.1±2.9, and 134.0±4.5 ms, respectively. Organ explant cultures and freshly isolated tissue could be paced up to frequencies within the normal range for neonatal rat (CL 150 ms, while monolayers could not. Successful lentiviral (LV transduction was shown via Egfp gene transfer. Co-culture of organ explant cultures with spontaneously beating cardiomyocytes increased the occurrence of spontaneous beating activity of organ explant cultures to 86%. We conclude that organ explant cultures of neonatal rat ventricle are structurally and electrophysiologically similar

  10. Effects of sodium hyaluronate and methylprednisolone acetate on proteoglycan synthesis in equine articular cartilage explants.

    Science.gov (United States)

    Doyle, Aimie J; Stewart, Allison A; Constable, Peter D; Eurell, Jo Ann C; Freeman, David E; Griffon, Dominique J

    2005-01-01

    To determine effects of sodium hyaluronate (HA) on corticosteroid-induced cartilage matrix catabolism in equine articular cartilage explants. 30 articular cartilage explants from fetlock joints of 5 adult horses without joint disease. Articular cartilage explants were treated with control medium or medium containing methylprednisolone acetate (MPA; 0.05, 0.5, or 5.0 mg/mL), HA (0.1, 1.0, or 1.5 mg/mL), or both. Proteoglycan (PG) synthesis was measured by incorporation of sulfur 35-labeled sodium sulphate into PGs, and PG degradation was measured by release of radiolabeled PGs into the medium. Total glycosaminoglycan (GAG) content in media and explants and total explant DNA were determined. Methylprednisolone acetate caused a decrease in PG synthesis, whereas HA had no effect. Only the combination of MPA at a concentration of 0.05 mg/mL and HA at a concentration of 1.0 mg/mL increased PG synthesis, compared with control explants. Methylprednisolone acetate increased degradation of newly synthesized PGs into the medium, compared with control explants, and HA alone had no effect. Hyaluronate had no effect on MPA-induced PG degradation and release into media. Neither MPA alone nor HA alone had an effect on total cartilage GAG content. Methylprednisolone acetate caused an increase in release of GAG into the medium at 48 and 72 hours after treatment. In combination, HA had no protective effect on MPA-induced GAG release into the medium. Total cartilage DNA content was not affected by treatments. Our results indicate that HA addition has little effect on corticosteroid-induced cartilage matrix PG catabolism in articular cartilage explants.

  11. Facilitation of granulocyte migration into bovine pulmonary artery intimal explants by intact viable endothelium.

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    Niedermeyer, M E; Meyrick, B.; Parl, F.F.; Brigham, K L

    1984-01-01

    To characterize the role of normal endothelium in granulocyte chemotaxis, the authors measured granulocyte adherence to and migration into bovine pulmonary artery intimal explants. Explants were placed, endothelium uppermost, in chemotaxis chambers with zymosan-activated plasma in the lower well and 5 X 10(6)/ml 51Cr-labeled granulocytes in the upper well. After 15, 30, 60, 120, 180, or 240 minutes incubation at 37 C, granulocyte adherence was measured by removal of adherent granulocytes from...

  12. Impact of mold infections in explanted lungs on outcomes of lung transplantation.

    Science.gov (United States)

    Vadnerkar, Aniket; Clancy, Cornelius J; Celik, Umit; Yousem, Samuel A; Mitsani, Dimitra; Toyoda, Yoshiya; Nguyen, Minh-Ly; Kwak, Eun J; Pilewski, Joseph; Silveira, Fernanda P; Crespo, Maria; Nguyen, M Hong

    2010-01-27

    Little is known about the incidence or significance of mold infections in the explanted lungs of lung transplant recipients. We reviewed the histopathology of the explanted lungs from 304 patients who underwent lung transplantation at our institution from 2005 to 2007 and received alemtuzumab induction therapy and posttransplant voriconazole prophylaxis. Invasive mold infections were present in the explanted lungs of 5% (14 of 304) of patients, including chronic necrotizing pneumonias (n=7), mycetomas (n=4), and invasive fungal pneumonias (n=3). Only 21% (3 of 14) received immunosuppressive therapy within 1 year before lung transplantation, suggesting that lung damage itself predisposed patients to mold infections. The risk of mold infection was higher in patients with cystic fibrosis (11%, 4 of 35) than other underlying lung diseases (4%, 10 of 269). Pulmonary mold infections were not diagnosed or suspected in 57% (8 of 14) of patients. Despite secondary voriconazole prophylaxis, fungal infections developed in 43% (6 of 14) of patients with mold infections of the explanted lungs compared with 14% (42 of 290) of patients without mold infections (P=0.01). Three patients developed invasive fungal infections while on voriconazole prophylaxis and three developed fungal infections more than 8 months after the discontinuation of voriconazole. The mortality attributable to invasive fungal infections among patients with mold infections of the explanted lungs was 29% (4 of 14). Invasive mold infections in the explanted lungs are often not recognized before lung transplantation and are associated with poor outcomes.

  13. Indução de calos embriogênicos em explantes de cupuaçuzeiro Induction of embryogenics calli in cupuassu explants

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    Maria das Graças Rodrigues Ferreira

    2004-08-01

    Full Text Available Objetivou-se a indução de calos embriogênicos em cupuaçuzeiro, em função do tipo de explante e meio de cultura. Foram testados como explantes, segmentos cotiledonares e eixos embrionários divididos em três partes: região da plúmula, radícula e hipocótilo. Os explantes foram cultivados em 2 diferentes meios de cultura: 1 MS suplementado com 2,4-D (1 mg L-1 e Cinetina (0,25 mg L-1; 2 MS acrescido de ANA (5 mg L-1 e Cinetina (0,25 mg L-1. Constatou-se que a região do hipocótilo foi a parte mais responsiva do eixo embrionário, formando calos com aspecto branco e friável. As auxinas testadas nos meios não estimularam o processo embriogênico em calos de cupuaçuzeiro.It was studied the induction of embryogenics calli in cupuassu, in function of kind of explant and culture medium. Cotyledons segments and embryonic axes were tested and divided in three parts: region of plumule, radicule and hypocotile. The explants were cultivated in two different culture medium: 1 MS supplemented with 2,4-D (1 mg L-1 and Kinetin (0,25 mg L-1; 2 MS supplemented with NAA (5 mg L-1 and Kinetin (0,25 mg L-1. The hypocotile region demonstrated to be more responsive segment of the embryonic axe, forming callus with white and friable aspect. No somatic embryogenesis was evidenced in callus of cupuassu with auxines testeds in the medium.

  14. Primary cilia expression in bone marrow in response to mechanical stimulation in explant bioreactor culture.

    Science.gov (United States)

    Coughlin, T R; Schiavi, J; Alyssa Varsanik, M; Voisin, M; Birmingham, E; Haugh, M G; McNamara, L M; Niebur, G L

    2016-07-19

    Bone marrow contains a multitude of mechanically sensitive cells that may participate in mechanotransduction. Primary cilia are sensory organelles expressed on mesenchymal stem cells (MSCs), osteoblasts, osteocytes, and other cell types that sense fluid flow in monolayer culture. In marrow, cilia could similarly facilitate the sensation of relative motion between adjacent cells or interstitial fluid. The goal of this study was to determine the response of cilia to mechanical stimulation of the marrow. Bioreactors were used to supply trabecular bone explants with low magnitude mechanical stimulation (LMMS) of 0.3 ×g at 30 Hz for 1 h/d, 5 d/week, inducing shear stresses in the marrow. Four groups were studied: unstimulated (UNSTIM), stimulated (LMMS), and with and without chloral hydrate (UNSTIM+CH and LMMS+CH, respectively), which was used to disrupt cilia. After 19 days of culture, immunohistochemistry for acetylated α-tubulin revealed that more cells expressed cilia in culture compared to in vivo controls. Stimulation decreased the number of cells expressing cilia in untreated explants, but not in CH-treated explants. MSCs represented a greater fraction of marrow cells in the untreated explants than CH-treated explants. MSCs harvested from the stimulated groups were more proliferative than in the unstimulated explants, but this effect was absent from CH treated explants. In contrast to the marrow, neither LMMS nor CH treatment affected bone formation as measured by mineralising surface. Computational models indicated that LMMS does not induce bone strain, and the reported effects were thus attributed to shear stress in the marrow. From a clinical perspective, genetic or pharmaceutical alterations of cilia expression may affect marrow health and function.

  15. Efficient Regeneration of �Caralis� Alstroemeria Cultivar from Rhizome Explants

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    Amir Ghaffar SHAHRIARI

    2012-05-01

    Full Text Available In this paper, the effects of a number of growth regulators as well as supplements to the Murashige and Skoog (MS basal medium were evaluated on the regeneration of Alstroemeria rhizome explants. In the first experiment the effects of three cytokinins (BA, TDZ and 2IP each at 0.5, 1 and 2 mg/l in combination with NAA (0.2 mg/l, followed by another PGR combination of 2IP (at 0.5, 1 and 2 mg/l with NAA (0 and 0.2 mg/l, on regeneration of rhizome-derived explants, was investigated. Through the second experiment, the effects of a number of supplements, including glucose (30 g/l as the alternative for sucrose, casein hydrolysate (1 g/l, asparagine and glutamine, (each at 30 mg/l added to MS medium, containing 1 mg/l BA and 0.2 mg/l NAA, was examined on rhizome explants regeneration. Among the tested cytokinins, BA induced better regeneration of rhizome explants, resulting in a higher number of shoots compared to the other cytokinins. A medium supplemented with 1 mg/l BA and 0.2 mg/l NAA proved to be the most effective, with an average of 4.16 regenerated shoots per explant. In the second PGR combination, addition of NAA at 0.2 mg/l improved regeneration, compared to NAA-free treatments. In the second experiment, glucose substitution for sucrose improved regeneration with an average of 5.10 regenerated shoots per explant, compared to 4.16 shoots in sucrose-containing medium; whereas glutamine and asparagine (with 2.66 shoots and casein hydrolysate (with 3.80 shoots showed a negative influence on rhizome explants regeneration.

  16. Efficient culture protocol for plant regeneration from cotyledonary petiole explants of Jatropha curcas L.

    Directory of Open Access Journals (Sweden)

    Ying Liu

    2016-09-01

    Full Text Available A high-frequency and reproducible protocol for induction of adventitious shoot buds and plant regeneration from cotyledonary petiole explants of Jatropha curcas L. has been developed. The cotyledonary petiole explants of J. curcas cultured directly on medium supplemented with thidiazuron (TDZ induce regeneration of poor quality shoot buds that have a low regeneration frequency. However, treating the explants with high concentrations (10–60 mg/L of TDZ solution for certain time periods (5–80 min significantly increased the regeneration frequency and improved the quality of the regenerated shoot buds. The best shoot buds induction (88.42% and number of shoot buds (12.67 per explant were observed when in vitro explants were treated with 20 mg/L TDZ solution for 20 min before being transferred on hormone-free medium after 30 days. Regeneration was also influenced by the orientation (horizontal or vertical of the explants on the medium, and by the origin of the cotyledonary petioles (in vitro or in vivo used for the preparation of explants. We performed subsequent experiments for elongation and rooting of the regenerated shoot buds. Addition of L-arginine to the medium was conducive to the elongation of the shoot buds. A concentration of 7.5 mg/L L-arginine yielded the best results. The elongated shoots could initiate roots to become intact plantlets in half-strength Murashige and Skoog medium containing 0.1 mg/L indole-3-butyric acid. After acclimatization, these plantlets could be transplanted to the soil and the growth was normal. Therefore, application of the methods described here helped to increase plant regeneration efficiency.

  17. Plant regeneration from cotyledonary explants of Eucalyptus camaldulensis Regeneração de plantas de Eucalyptus camaldulensis a partir das explantes cotiledonares

    Directory of Open Access Journals (Sweden)

    Roberson Dibax

    2005-08-01

    Full Text Available Breeding methods based on genetic transformation techniques need to be implemented for Eucalyptus camaldulensis to shorten the long breeding cycles and avoid manipulation of adult trees; that requires the development of plant regeneration protocols enabling development of plants from transformed tissues. The present work aimed to optimise the regeneration process already established for the species. Cotyledonary leaves of E. camaldulensis were cultured in MS medium supplemented with naphthaleneacetic acid (NAA and 6-benzylaminopurine (BAP combinations. The most efficient treatment for bud indirect regeneration (2.7 µmol L-1 NAA and 4.44 µmol L-1 BAP was used for further experiments. When explants were kept in the dark during the first 30 days, the percentage of explants forming calluses increased and explant necrosis was reduced in comparison with light-cultured explants. Mineral medium modifications were compared and half-strength MS mineral medium turned out to be as efficient as full-strength medium, producing 54% and 47% of explants with buds, respectively. For shoot elongation, MS medium with half-strength nitrate and ammonium salts, and 0.2% activated charcoal yielded rooted shoots 1 to 8 cm high after one month. The procedure is an efficient protocol for E. camadulensis plant regeneration, reducing the stages necessary for the obtention of complete plants.A implementação, para espécies florestais, de técnicas de melhoramento baseadas em métodos de transformação genética, permitirá reduzir os longos ciclos de melhoramento e evitar a manipulação de árvores adultas. Isto implica dispor de um protocolo de regeneração que permita o desenvolvimento de plantas a partir de tecidos transformados. Este trabalho teve como objetivo otimizar este protocolo de regeneração para Eucalyptus camaldulensis. Folhas cotiledonares foram cultivadas em meio de cultura MS suplementado com combinações de ácido naftalenoacético (ANA e 6

  18. Changes in glycemic control and body weight after explantation of the duodenal-jejunal bypass liner.

    Science.gov (United States)

    Betzel, Bark; Koehestanie, Parviez; Homan, Jens; Aarts, Edo O; Janssen, Ignace M C; de Boer, Hans; Wahab, Peter J; Groenen, Marcel J M; Berends, Frits J

    2017-02-01

    The duodenal-jejunal bypass liner (DJBL) is an endoscopic device that induces weight loss and improves glycemic control in patients with type 2 diabetes mellitus (T2DM). The aim of the current study was to assess the effects of DJBL explantation on glycemic control and body weight. This prospective, observational study included only patients with T2DM who had the DJBL implanted for at least 6 months and had a follow-up of at least 12 months after explantation. The primary endpoints were changes in glycosylated hemoglobin A1c (HbA1c) and body weight during the 12 months after explantation. Secondary endpoints were changes in fasting plasma glucose, blood pressure, and plasma lipid levels. In total, 59 patients completed the 12-month follow-up after explantation. During this period body weight increased by 5.6 (standard deviation, 6.4) kg (P body weight remained 8.0 (SD 8.6) kg (P body weight loss of 7.4% (SD 7.6) (P weight gain and worsening of glycemic control, although some beneficial effects remained detectable 12 months after explantation. A change in strategy is needed to preserve the beneficial effects of DJBL treatment. (Clinical trial registration number: 746∖100111.). Copyright © 2017 American Society for Gastrointestinal Endoscopy. Published by Elsevier Inc. All rights reserved.

  19. Equine oviduct explant culture: a basic model to decipher embryo-maternal communication.

    Science.gov (United States)

    Nelis, Hilde; D'Herde, Katharina; Goossens, Karen; Vandenberghe, Lynn; Leemans, Bart; Forier, Katrien; Smits, Katrien; Braeckmans, Kevin; Peelman, Luc; Van Soom, Ann

    2014-08-01

    Equine embryos remain for 6 days in the oviduct and thus there is a need for an in vitro model to study embryo-oviductal interactions in the horse, since this subtle way of communication is very difficult to analyse in vivo. Until now, no equine oviduct explant culture model has been characterised both morphologically and functionally. Therefore, we established a culture system for equine oviduct explants that maintained epithelial morphology during 6 days of culture, as revealed by light microscopy and transmission electron microscopy. We demonstrated the presence of highly differentiated, tall columnar, pseudostratified epithelium with basal nuclei, numerous nucleoli, secretory granules and apical cilia, which is very similar to the in vivo situation. Both epithelium and stromal cells originating from the lamina propria are represented in the explants. Moreover, at least 98% of the cells remained membrane intact and fewer than 2% of the cells were apoptotic after 6 days of culture. Although dark-cell degeneration, which is a hypoxia-related type of cell death, was observed in the centre of the explants, quantitative real-time PCR failed to detect upregulation of the hypoxia-related marker genes HIF1A, VEGFA, uPA, GLUT1 and PAI1. Since the explants remained morphologically and functionally intact and since the system is easy to set up, it appears to be an excellent tool for proteome, transcriptome and miRNome analysis in order to unravel embryo-maternal interactions in the horse.

  20. Optical and atomic force microscopy of an explanted AcrySof intraocular lens with glistenings.

    Science.gov (United States)

    Dogru, M; Tetsumoto, K; Tagami, Y; Kato, K; Nakamae, K

    2000-04-01

    To assess the surface morphology and cause of glistenings in an explanted AcrySof intraocular lens (IOL). Shakai Hoken Kobe Central Hospital, Kobe, Japan. A 63-year-old Japanese man had implantation of an AcrySof IOL in the capsular bag. One month postoperatively, he had a neodymium:YAG laser capsulotomy for posterior capsule opacification, which changed the IOL's position in the capsular bag. A few months later, the patient developed disabling night glare from intralenticular glistenings and progressive hyperopic refractive error. The IOL was explanted and then analyzed by optical microscopy and atomic force microscopy (AFM). Laboratory analysis of control AcrySof IOLs kept in a balanced salt solution at steady room and body temperature for 2 months was also performed to evaluate the cause of the glistenings observed clinically. Optical microscopy showed that the explanted AcrySof IOL had several microvacuoles; no abnormalities were observed in the control AcrySof IOLs before or after folding at the room and body temperatures. The AFM analysis showed a significant change in the surface morphology of the explanted IOL, including vacuolar formations in the posterior surface as well as numerous anterior surface irregularities. No microvacuoles or surface morphology alterations were observed in the control AcrySof IOLs by AFM analysis. The glistenings in the explanted AcrySof IOL were likely caused by temperature changes and not mechanical stress from folding.

  1. Ex Vivo Produced Oral Mucosa Equivalent by Using the Direct Explant Cell Culture Technique

    Directory of Open Access Journals (Sweden)

    Kamile Öztürk

    2012-09-01

    Full Text Available Objective: The aim of this study is the histological and immunohistochemical evaluation of ex vivo produced oral mucosal equivalents using keratinocytes cultured by direct explant technique.Material and Methods: Oral mucosa tissue samples were obtained from the keratinized gingival tissues of 14 healthy human subjects. Human oral mucosa keratinocytes from an oral mucosa biopsy specimen were dissociated by the explant technique. Once a sufficient population of keratinocytes was reached, they were seeded onto the type IV collagen coated “AlloDerm” and taken for histological and immunohistochemical examinations at 11 days postseeding of the keratinocytes on the cadaveric human dermal matrix.Results: Histopathologically and immunohistochemically, 12 out of 14 successful ex vivo produced oral mucosa equivalents (EVPOME that consisted of a stratified epidermis on a dermal matrix have been developed with keratinocytes cultured by the explant technique.Conclusion: The technical handling involved in the direct explant method at the beginning of the process has fewer steps than the enzymatic method and use of the direct explant technique protocol for culturing of human oral mucosa keratinocyte may be more adequate for EVPOME production.

  2. Organogênese de explante foliar de clones de Eucalyptus grandis x E. urophylla Organogenesis of the leaf explant of Eucalyptus grandis x E. urophylla clones

    Directory of Open Access Journals (Sweden)

    Elisa Cristina Soares de Carvalho Alves

    2004-05-01

    Full Text Available O objetivo deste trabalho foi avaliar os efeitos dos reguladores de crescimento TDZ [1-fenil-3-(1,2,3-tia-diazol-5-iluréia], BAP (6-benzilaminopurina e ANA (ácido naftalenoacético no desempenho da propagação in vitro por organogênese de explante foliar de três clones híbridos de Eucalyptus grandis x Eucalyptus urophylla. Houve resposta diferenciada dos clones quanto a intensidade, textura e coloração dos calos, em razão dos tratamentos com os reguladores de crescimento. Os melhores resultados de calejamento dos três genótipos foram observados nos tratamentos com a combinação dos reguladores de crescimento TDZ (0,5 mg L-1 e ANA (0,1 mg L-1, obtendo-se 100% de calejamento no explante foliar. Os piores resultados de calejamento foram observados nos tratamentos com a combinação dos reguladores de crescimento BAP (0,1 mg L-1 e ANA (0,1 mg L-1. Em relação à regeneração, a melhor resposta foi obtida com 1,0 mg L-1 BAP em que 8% dos calos formados a partir de explantes foliares regeneraram gemas, com número médio destas formadas por calo igual a 4,2.The aim of this work was to evaluate the effects of growth regulators TDZ [1-phenil-3-(1,2,3-thiadiazol-5-yl urea], BAP (6-benzilaminopurine e NAA (Naphthalene acetic acid on the in vitro propagation by organogenesis from foliar explants of Eucalyptus grandis x E. urophylla. Depending on the clone used, there were singular responses to growth regulators treatment regarding callusing intensity, texture and color. The best results of the three genotypes used were observed with the TDZ (0.5 mg L-1 and NAA (0.1 mg L-1 treatment, where 100% of the foliar explants presented callus. The worst results were observed with the BAP (0.1 mg L-1 and NAA (0.1 mg L-1 treatment. Subsequently, considering the regeneration process, the best response was achieved with 1.0 mg L-1 BAP, in which 8% of the calli regenerated buds, with an average of 4.2 buds per explant.

  3. Explant culture of human peripheral lung. I. Metabolism of benzo[alpha]pyrene

    DEFF Research Database (Denmark)

    Stoner, G.D.; Harris, C.C.; Autrup, Herman

    1978-01-01

    hydroxylase activity and could metabolize BP into forms that were bound to cellular DNA and protein. Peripheral lung had significantly lower aryl hydrocarbon hydroxylase activity than cultured bronchus but both tissues had similar binding levels of BP to DNA. Radioautographic studies indicated that all cell......Human lung explants have been maintained in vitro for a period of 25 days. Autoradiographic studies indicated that the broncholar epithelial cells, type 2 alveolar epithelial cells, and stromal fibroblasts incorporated 3H-thymidine during the culture. After 7 to 10 days, type 2 cells were...... the predominant alveolar epithelial cell type. Lamellar inclusion bodies were released from the type 2 cells and accumulated in the alveolar spaces. The metabolism of benzo[alpha]pyrene (BP) in human lung explants cultured for up to 7 days was investigated. Human lung explants had measurable aryl hydrocarbon...

  4. Resolving browning during the establishment of explant cultures in Vicia faba L. for genetic transformation

    Directory of Open Access Journals (Sweden)

    Helena Klenotičová

    2013-01-01

    Full Text Available Optimisation of in vitro regeneration systems of two explant types for low-tannine cultivars of faba bean based on culturing of shoot apices and cotyledonary nodes were provided by usage of various antioxidants - ascorbic acid, citric acid, glutathione and activated charcoal. In subsequent testing, the combined effects of antioxidants with transformation co-cultivation compounds acetosyringone and L-cysteine was studied. The application of antioxidants lead to decreased callogenesis, citric acids treatments (50 mg.l−1 dramatically decreased necrotic response of explants. However, citric acid, used together with ascorbic acid completely inhibited shoot growth in shoot apex cultures. Glutathion evoked hyperhydricity of explants. Activated charcoal induced rooting on media which are commonly used for shoot proliferation. Combination of acetosyringone with antioxidants influenced shoot proliferation, except of variant with ascorbic acid. Citric acid was the best and universal antioxidant in faba bean in vitro cultures and its use is recommended for faba bean genetic transformation experiments.

  5. Comparative effects of plant growth regulators on leaf and stem explants of Labisia pumila var. alata.

    Science.gov (United States)

    Ling, Anna Pick Kiong; Tan, Kinn Poay; Hussein, Sobri

    2013-07-01

    Labisia pumila var. alata, commonly known as 'Kacip Fatimah' or 'Selusuh Fatimah' in Southeast Asia, is traditionally used by members of the Malay community because of its post-partum medicinal properties. Its various pharmaceutical applications cause an excessive harvesting and lead to serious shortage in natural habitat. Thus, this in vitro propagation study investigated the effects of different plant growth regulators (PGRs) on in vitro leaf and stem explants of L. pumila. The capabilities of callus, shoot, and root formation were evaluated by culturing both explants on Murashige and Skoog (MS) medium supplemented with various PGRs at the concentrations of 0, 1, 3, 5, and 7 mg/L. Medium supplemented with 3 mg/L indole-3-butyric acid (IBA) showed the optimal callogenesis from both leaf and stem explants with (72.34 ± 19.55)% and (70.40 ± 14.14)% efficacy, respectively. IBA was also found to be the most efficient PGR for root induction. A total of (50.00 ± 7.07)% and (77.78 ± 16.47)% of root formation were obtained from the in vitro stem and leaf explants after being cultured for (26.5 ± 5.0) and (30.0 ± 8.5) d in the medium supplemented with 1 and 3 mg/L of IBA, respectively. Shoot formation was only observed in stem explant, with the maximum percentage of formation ((100.00 ± 0.00)%) that was obtained in 1 mg/L zeatin after (11.0 ± 2.8) d of culture. Callus, roots, and shoots can be induced from in vitro leaf and stem explants of L. pumila through the manipulation of types and concentrations of PGRs.

  6. Prolactin expression and secretion by human breast glandular and adipose tissue explants.

    Science.gov (United States)

    Zinger, Michael; McFarland, Molly; Ben-Jonathan, Nira

    2003-02-01

    Prolactin (PRL) is a 23-kDa hormone produced by the pituitary and extrapituitary sites. The main target of PRL is the breast, where it affects cellular growth, differentiation, and milk production. Recent evidence suggests that locally produced PRL plays a role in breast tumorigenesis. Our objective was to examine PRL synthesis/release in different tissues of the human breast and determine the effect of ovarian steroids. Breast tissue, obtained from women undergoing mastectomy or breast reduction, was separated into glandular (nonmalignant) and adipose explants and incubated for 10 d. Conditioned media were analyzed for PRL by a bioassay. PRL release from glandular explants decreased by 60% from d 1-3, followed by a 4-fold increase on d 10. PRL release from adipose explants was unchanged from d 1-3 and increased more than 10-fold by d 10. PRL gene expression, determined by RT-PCR, was low on d 0 and markedly increased on d 10 in both types of explants. De novo synthesis of PRL was confirmed by metabolic labeling. Progesterone suppressed PRL release from glandular explants without affecting adipose explants. Estradiol did not alter PRL release from either tissue. In conclusion, the human breast produces and releases bioactive PRL, with a higher release rate by adipose than glandular tissue. The time-dependent rise in PRL release suggests removal from inhibitory control. Progesterone may be one of the factors that suppresses PRL production in the glandular compartment, whereas the factor(s) that regulate adipose PRL are unknown. These data suggest an autocrine/paracrine role for PRL in human glandular and adipose breast tissue.

  7. Analysis of Explanted Magnetically Controlled Growing Rods From Seven UK Spinal Centers.

    Science.gov (United States)

    Joyce, Thomas J; Smith, Simon L; Rushton, Paul R P; Bowey, Andrew J; Gibson, Michael J

    2018-01-01

    Analysis of explanted MAGnetic Expansion Control (MAGEC) growing rods. To analyze explanted MAGEC rods used in management of early onset scoliosis and identify the mode of failure in such cases. Magnetically controlled growing rods are increasingly used as the option of choice for early onset scoliosis. However, being more complex than conventional growing rods they are perhaps more likely to succumb to multifarious failure modes. In addition, metallosis has been reported around failed MAGEC rods. Explanted MAGEC rods from seven UK spinal centers were obtained for independent analysis. Thirty-four MAGEC rods, from 18 children, explanted for reasons including failure of rod lengthening and maximum rod distraction reached, were cut open to allow internal components to be evaluated and assessed. Externally, all MAGEC rods showed localized marks, which were termed "growth marks" as they indicated growth of the rod in vivo, on the extending bar component. After cutting open, titanium wear debris was found inside all 34 (100%) MAGEC rods. Ninety-one percent (31/34) of MAGEC rods showed measurable wear of the extending bar, towards the magnet end. Substantial damage to the radial bearing was seen inside 74% (25/34) of MAGEC rods while O-ring seal failure was seen in 53% (18/34) of cases. In 44% (15/34) of MAGEC rods the drive pin was fractured but this was felt to be an effect of rod failure, not a cause. The combination of high volumes of titanium wear debris alongside O-ring seal damage likely accounts for the metallosis reported clinically around some MAGEC rods. Based on this explant data, a failure mechanism in MAGEC rods due to the natural off axis loading in the spine was proposed. This is the largest data set reporting a complete analysis of explanted MAGEC rods to date. 4.

  8. The effect of glucocorticoids on tendon cell viability in human tendon explants

    Science.gov (United States)

    Lui, Wai Ting; Chuen Fu, Sai; Man Lee, Kwong

    2009-01-01

    Background and purpose Previous studies on the culture of human tenocytes have shown that dexamethasone and triamcino-lone reduce cell viability, suppress cell proliferation, and reduce collagen synthesis. However, such cell cultures lack the extracellular matrix and three-dimensional structure of normal tendons, which affects their response to stimuli. We established a human tendon explant culture system and tested the effects of dexamethasone and triamcinolone on cell viability. Methods Primary human tendon explant cultures were prepared from healthy hamstring tendons. Tendon strips were harvested from hamstring tendons and cultured in 24-well plates in Dulbecco’s modification of Eagle’s Medium (DMEM) supplemented with 2% fetal calf serum. The tendon explants were treated with 0 μM (control), 10 μM, or 100 μM dexamethasone sodium phosphate or 0 μM (control), 10 μM, or 100 μM triamcinolone acetonide in DMEM for 96 h. Cell viability was measured by Alamar blue assay before and after glucocorticoid treatment. Results Incubation with 10 μM and 100 μM dexamethasone reduced cell viability in human tendon explants by 35% and 45%, respectively, as compared to a 6% increase in the controls (p = 0.01, mixed-effects ANOVA). Triamcinolone at 10 μM and 100 μM reduced cell viability by 33% and 36%, respectively, as compared to a 9% increase in the controls (p = 0.07, mixed-effects ANOVA). Interpretation Human tendon explant cultures can be used to study the effects of glucocorticoids on human tendon. Dexamethasone and triamcinolone suppress the cell viability of human tendon in its natural 3-dimensional environment with matrix anchorage. Human tendon explant cultures provide a species-specific model for further investigation of the effects of glucocorticoids on the metabolism of the extracellular matrix of human tendon, and on its mechanical properties. PMID:19421908

  9. Effect of explant type, medium and soil mixture content on Dianthus deltoides L. rooting and acclimatization

    Directory of Open Access Journals (Sweden)

    Marković Marija

    2012-01-01

    Full Text Available The possibility of developing roots and shoots on Dianthus deltoides nodal and apical cuttings on rooting media was investigated in vitro conditions. The explants developed roots and shoots successfully, and the best results were achieved on MS medium without plant hormones. Number of roots per shoot, length of roots and ramification of roots were not significantly dependent on the medium (NAA-naphthaleneacetic acid concentration, or on the type of explant used (nodal or apical cuttings. The acclimatization rate of microplants was influenced by root numbers and ramification. The transfer to soil was successful, maximum survival rates (96% were achieved on peat - sand (4:1 mixture.

  10. Adherence of Candida albicans to bladder mucosa: development and application of a tissue explant assay.

    Science.gov (United States)

    Lyman, C A; Navarro, E; Garrett, K F; Roberts, D D; Pizzo, P A; Walsh, T J

    1999-01-01

    In order to study the interactions between Candida species and uroepithelial tissue, a tissue explant assay was developed using bladder mucosa harvested from New Zealand white rabbits. Blastoconidia of Candida albicans, Candida tropicalis and Candida glabrata attached to the uroepithelial tissue in similar quantities. However, there was significantly more adherence to the uroepithelium by pre-germinated C. albicans compared with C. albicans blastoconidia. Furthermore, the amount of uroepithelial tissue injury was directly related to the length of exposure of the tissue to Candida. Thus, this tissue explant assay may provide a useful method for investigating properties related to fungal adherence to transitional uroepithelium and organism-mediated tissue injury.

  11. Induction of bulb organogenesis inin vitrocultures of tarda tulip (Tulipa tardaStapf.) from seed-derived explants.

    Science.gov (United States)

    Maślanka, Małgorzata; Bach, Anna

    2014-01-01

    A protocol for obtaining bulbs via in vitro organogenesis was developed for tarda tulip ( Tulipa tarda Stapf). Scale explants were obtained from bulbs formed at the base of seedlings or from adventitious bulbs that developed from callus tissue forming on stolons or on germinating seeds. Some explants were subjected to chilling at 5°C for 12 wk. The culture media contained 3 or 6% sucrose and was supplemented with either no growth regulators, either 0.5 μM 6-benzyl-aminopurine (BAP) or 18.9 or 94.6 μM abscisic acid (ABA). Cultures were maintained in the dark at 20°C. Callus tissue developed mainly on media without growth regulators or with BAP. Callus was formed from up to 96% of explants derived from non-chilled adventitious bulbs that were treated with 3% sucrose and 0.5 μM BAP. Less callus was formed from chilled explants compared with non-chilled explants. Newly formed adventitious bulbs appeared on the explants via direct and indirect organogenesis. The media with BAP promoted the formation of adventitious bulbs at a rate of 56-92% from non-chilled explants, whereas a maximum rate of 36% was observed from chilled explants. ABA inhibited the induction of adventitious bulbs and callus. The adventitious bulbs obtained in these experiments contained a meristem, which was evidence that they had developed properly.

  12. An in vitro model for detecting skin irritants: methyl green-pyronine staining of human skin explant cultures

    NARCIS (Netherlands)

    Jacobs, J. J. L.; Lehé, C.; Cammans, K. D. A.; Das, P. K.; Elliott, G. R.

    2002-01-01

    We evaluated the potential of human organotypic skin explant cultures (hOSECs) for screening skin irritants. Test chemicals were applied to the epidermis of the skin explants which were incubated for 4, 24 or 48 h in tissue culture medium. A decrease in epidermal RNA staining, visualised in frozen

  13. Low-intensity pulsed ultrasound (LIPUS) and pulsed electromagnetic field (PEMF) treatments affect degeneration of cultured articular cartilage explants

    NARCIS (Netherlands)

    Tan, Lijun; Tan, Lijun; Ren, Yijin; van Kooten, Theo G.; Grijpma, Dirk W.; Kuijer, Roel

    2015-01-01

    Purpose: Articular cartilage has some capacity for self-repair. Clinically used low-intensity pulsed ultrasound (LIPUS) and pulsed electromagnetic field (PEMF) treatments were compared in their potency to prevent degeneration using an explant model of porcine cartilage. Methods: Explants of porcine

  14. Low-intensity pulsed ultrasound (LIPUS) and pulsed electromagnetic field (PEMF) treatments affect degeneration of cultured articular cartilage explants

    NARCIS (Netherlands)

    Tan, Lijun; Ren, Yijin; van Kooten, Theo G.; Grijpma, Dirk W.; Kuijer, Roelof

    PURPOSE: Articular cartilage has some capacity for self-repair. Clinically used low-intensity pulsed ultrasound (LIPUS) and pulsed electromagnetic field (PEMF) treatments were compared in their potency to prevent degeneration using an explant model of porcine cartilage. METHODS: Explants of porcine

  15. Callus induction and plant regeneration from different explant types of Miscanthus x ogiformis Honda 'Giganteus'

    DEFF Research Database (Denmark)

    Holme, Inger Bæksted; Petersen, Karen Koefoed

    1996-01-01

    Different explants of Miscanthus x ogiformis Honda 'Giganteus' were tested in order to develop an efficient tissue culture system. Shoot apices, leaf and root sections from in vitro-propagated plants, and leaf and immature inflorescence sections from 6-month-old greenhouse-grown plants were used....

  16. Effects of electromagnetic fields on proteoglycan metabolism of bovine articular cartilage explants

    NARCIS (Netherlands)

    De Mattei, M; Pasello, M; Pellati, A; Stabellini, G; Massari, L; Gemmati, D; Caruso, A

    2003-01-01

    Electromagnetic field (EMF) exposure has been proposed for the treatment of osteoarthritis. In this study, we investigated the effects of EMF (75 Hz, 2,3 mT) on proteoglycan (PG) metabolism of bovine articular cartilage explants cultured in vitro, both under basal conditions and in the presence of

  17. Hypoxia preferentially destroys GABAergic neurons in developing rat neocortex explants in culture

    NARCIS (Netherlands)

    Romijn, H. J.; Ruijter, J. M.; Wolters, P. S.

    1988-01-01

    The hypothesis that hypoxic ischemia before or during the human birth process preferentially destroys GABAergic nerve cells, particularly in the neocortex, was tested in a tissue culture model system. To that end, rat neocortex explants dissected from 6-day-old rat pups and cultured to a

  18. Influence of genotype and age of explant source on the capacity for ...

    African Journals Online (AJOL)

    Influence of genotype and age of explant source on the capacity for somatic embryogenesis of two Cavendish banana cultivars (Musa acuminata Colla, AAA). M Youssef, A James, A Mayo-Mosqueda, JR Ku-Cauich, R Grijalva-Arango, RM Escobedo-GM ...

  19. Effect of growth regulators and explant types on callus induction in ...

    African Journals Online (AJOL)

    Different concentrations of growth regulators and three types of explants were investigated for their efficiency on callus induction in Telfairia occidentalis with a view of providing baseline information for the development of a callus initiation protocol. Three concentrations of kinetin (KN) (0.1, 3.3 and 5.0 mg/L) in combination ...

  20. A RIFAMPICINA NA DESCONTAMINAÇÃO BACTERIANA DE EXPLANTES DE MAMOEIRO PROVENIENTES DO CAMPO

    Directory of Open Access Journals (Sweden)

    GIOVANNI RODRIGUES VIANNA

    1997-01-01

    Full Text Available Observou-se alta contaminação bacteriana nos explantes de mamoeiro introduzidos in vitro, a partir de plantas matrizes desenvolvidas no campo, independentemente da época do ano em que se realizaram as coletas. O uso de desinfestantes superficiais, como álcool e hipoclorito de sódio, garantiram níveis aceitáveis de controle apenas para fungos, não para bactérias. A rifampicina, por tratamento de imersão ou introdução em meio de cultura, controlou satisfatoriamente as contaminações de caráter endofítico, obtendo-se 70% de explantes sadios, sem sinais de fitotoxicidade.High contamination by bacteria was observed in papaya tissue cuttings introduced in vitro from plants grown in the field, independent of the period of the year that samples were collected. The use of alcohol and sodium hypoclorite did not guarantee good bacteria control. Rifampicin, added as an immersion solution treatment or in the culture media, controlled the internal contamination of explants, without damaging the cuttings. Up to 70% of healthy tissue explants were obtained by the use of rifampicin.

  1. How to Perform a Late Surgical Explantation of a CoreValve Aortic Bioprothesis.

    Science.gov (United States)

    Hernandez-Vaquero, Daniel; Pascual, Isaac; Diaz, Rocío; Álvarez-Cabo, Rubén; Moris, César; Silva, Jacobo

    2017-06-01

    As transcatheter techniques expand to younger patients, cardiac surgeons need to know a safe surgical technique to extract these kinds of prostheses. We describe here an adequate surgical strategy for the explantation of a CoreValve prosthesis that was implanted more than 5 years previously. Copyright © 2017 The Society of Thoracic Surgeons. Published by Elsevier Inc. All rights reserved.

  2. Microbiology of Explanted Suture Segments from Infected and Noninfected Surgical Patients

    Science.gov (United States)

    Krepel, Candace J.; Marks, Richard M.; Rossi, Peter J.; Sanger, James; Goldblatt, Matthew; Graham, Mary Beth; Rothenburger, Stephen; Collier, John; Seabrook, Gary R.

    2013-01-01

    Sutures under selective host/environmental factors can potentiate postoperative surgical site infection (SSI). The present investigation characterized microbial recovery and biofilm formation from explanted absorbable (AB) and nonabsorbable (NAB) sutures from infected and noninfected sites. AB and NAB sutures were harvested from noninfected (70.9%) and infected (29.1%) sites in 158 patients. At explantation, devices were sonicated and processed for qualitative/quantitative bacteriology; selective sutures were processed for scanning electron microscopy (SEM). Bacteria were recovered from 85 (53.8%) explanted sites; 39 sites were noninfected, and 46 were infected. Suture recovery ranged from 11.1 to 574.6 days postinsertion. A significant difference in mean microbial recovery between noninfected (1.2 isolates) and infected (2.7 isolates) devices (P sutures was noted. Biofilm was present in 100% and 66.6% of infected and noninfected devices, respectively (P sutures provide a hospitable surface for microbial adherence: (i) a significant difference in microbial recovery from infected and noninfected sutures was noted, (ii) infected sutures harbored a mixed flora, including multidrug-resistant health care-associated pathogens, and (iii) a significant difference in the presence or absence of a biofilm in infected versus noninfected explanted devices was noted. Further studies to document the benefit of focused risk reduction strategies to minimize suture contamination and biofilm formation postimplantation are warranted. PMID:23175247

  3. Embryogenic cells in Dactylis glomerata L. (Poaceae) explants identified by cell tracking and by SERK expression

    NARCIS (Netherlands)

    Somleva, M.N.; Schmidt, E.D.L.; Vries, de S.C.

    2000-01-01

    Single mesophyll cells in leaf explants of Dactylis glomerata L. (Dactylis) that were competent to form somatic embryos directly or through callus were identified by semi-automatic cell tracking. These competent cells were a subpopulation of small, isodiametric, cytoplasm-rich cells located close to

  4. Re-use of explanted DDD pacemakers as VDD- clinical utility and cost effectiveness.

    Science.gov (United States)

    Namboodiri, K K N; Sharma, Y P; Bali, H K; Grover, A

    2004-01-01

    Re-use of DDD pulse generators explanted from patients died of unrelated causes is associated with an additional cost of two transvenous leads if implanted as DDD itself, and high rate of infection according to some studies. We studied the clinical and economical aspects of reutilization of explanted DDD pacemakers programmed to VDD mode. Out of 28 patients who received VDD pacemaker during the period, October 2000- September 2001 in the Department of Cardiology, PGIMER, Chandigarh, 5 poor patients were implanted with explanted DDD pulse generators programmed to VDD mode. Each implantation was planned and carried out according to a standard protocol. The age ranged from 45 to 75 (mean-61) years. The indications for pacing were complete heart block (4) and second degree AV block (1). The clinical profile, costs and complications, if any were noted and followed up at regular intervals. The results were compared with patients who received new DDD pulse generators during this period. The additional cost for the atrial lead was not required in these patients. None of these patients had any local site infection. Compared to the two-lead system, the single lead system provided more rapid implantation and minimized complications associated with placement of an atrial lead. The explanted DDD pacemaker can be safely reused as VDD mode with same efficacy in selected patient population. This is associated with lower cost and complications compared to reimplantation as DDD itself.

  5. Production of immunoglobulins in gingival tissue explant cultures from juvenile periodontitis patients

    Energy Technology Data Exchange (ETDEWEB)

    Hall, E.R.; Falkler, W.A. Jr.; Suzuki, J.B. (Univ. of Maryland Dental School, Baltimore (USA))

    1990-10-01

    B lymphocytes and plasma cells are histologically observed in granulomatous periodontal tissues of juvenile periodontitis (JP) patients. Local immune processes may participate in protective or immunopathologic roles in the pathogenesis of this disease. An in vitro explant culture system was utilized to demonstrate the production of immunoglobulins by diseased JP tissues. Immunodiffusion studies using goat anti-human gamma, alpha, or mu chain serum revealed IgG to be the major immunoglobulin present in 92% of the day 1 supernatant fluids (SF) of the 47 JP gingival tissue explant cultures. IgA was present in 15% of the SF; however, no IgM was detected. Staph Protein A isolated 14C-labeled IgG from the SF, when allowed to react with goat anti-human gamma chain serum, formed lines of precipitation. Positive autoradiographs confirmed the biosynthesis of IgG by the explant cultures. The in vitro gingival tissue explant culture system described provides a useful model for the study of localized immunoglobulins produced by diseased tissues of JP patients.

  6. Staphylococcus aureus induces hypoxia and cellular damage in porcine dermal explants

    Science.gov (United States)

    Methicillin-resistant Staphylococcus aureus (MRSA) can infect wounds and produce difficult-to- treat biofilms. To determine the extent that MRSA biofilms can deplete oxygen, change pH and damage host tissue, we developed a porcine dermal explant model on which we cultured GFP-labeled MRSA biofilms. ...

  7. CALLUS INDUCTION AND PLANT REGENERATION IN PUNICA GRANATUM L. ?NANA' FROM LEAF EXPLANTS

    Directory of Open Access Journals (Sweden)

    Alireza Bonyanpour

    2013-09-01

    Full Text Available ABSTRACT In this investigation, leaf explants of a local cultivar of dwarf pomegranate were placed on Murashige and Skoog (1962 (MS medium supplemented with various concentrations of 6-benzyl adenin (BA and naphthalene acetic acid (NAA for callus induction. After 40 days, maximum callus induction was observed on a media containing 1 mg L-1 BA and 0.2 to 0.4 mg L-1 NAA. However, the highest callus growth was obtained on a medium containing 1 mg L-1 BA and 1 mg L-1 NAA. The highest number of shoots (7 shoots per explants was obtained by transferring the calli to the media containing 5 mg L-1 BA with 0.1 mg L-1 NAA. Maximum shoot proliferation was observed when shoots were cultured on woody plant medium (WPM supplemented with 5 mg L-1 kinetin (Kin. In this treatment, after 4 subcultures, 36 shoots were produced from one original explant. Among treatments used in rooting experiments, shoots cultured on WPM medium containing 0.2 mg L-1 indol butyric acid (IBA had the maximum root percentage (100% and good root growth (2.06 cm mean length and 2 roots in each explants. Rooted plantlets were cultured in a soil mixture containing vermiculite (60%, perlite (30% and coco peat (10% v/v. After 2 months, 80% of plants survived and transferred to the greenhouse.

  8. Investigating the Skoog-Miller Model for Organogenesis Using Sweet Potato Root Explants.

    Science.gov (United States)

    Delany, William; And Others

    1994-01-01

    Describes an experiment in which groups of students in a plant tissue culture course worked together to test application of the Skoog-Miller model (developed by Skoog and Miller in regeneration of tobacco experiments to demonstrate organogenesis) to sweet potato root explants. (ZWH)

  9. Growth Response of Explants of Irvingia Gabonensis (O'rorke, Baill ...

    African Journals Online (AJOL)

    Growth response of explants of Irvingia gabonensis to in vitro treatment was investigated using full, half and one quarter strength mineral components based on Murashige and Skoog medium. Plant growth regulator (kinetin-Kin) with concentration levels of 0, 1, 2, 3, 4 and 5mg/l were used for shoots initiation, while axillary ...

  10. EXPRESSION OF AHR AND ARNT MRNA IN CULTURED HUMAN ENDOMETRIAL EXPLANTS EXPOSED TO TCDD

    Science.gov (United States)

    Expression of AhR and ARNT mRNA in cultured human endometrial explants exposed to TCDD.Pitt JA, Feng L, Abbott BD, Schmid J, Batt RE, Costich TG, Koury ST, Bofinger DP.Curriculum in Toxicology, University of North Carolina, Chapel Hill, NC 27599, USA.Endom...

  11. Adventitious shoot regeneration from leaf explants of southern highbush blueberry cultivars

    Science.gov (United States)

    Protocols were developed to optimize adventitious shoot regeneration from four southern highbush blueberry cultivars. Leaf explants from six-week-old shoots of the four cultivars were excised and cultured on ten WPM (woody plant medium)-based regeneration media each containing thidiazuron (TDZ) (4.5...

  12. High frequency organogenesis in hypocotyl, cotyledon, leaf and petiole explants of broccoli (Brassica oleracea L. var. italica), an important vegetable crop

    National Research Council Canada - National Science Library

    Kumar, Pankaj; Srivastava, D K

    2015-01-01

    ... organogenesis from hypocotyl, cotyledon, leaf and petiole explants of broccoli (Brassica oleracea L. var. italica cv. Solan green head) has been developed. Hypocotyl and cotyledon explants were used from 10 to 12...

  13. Efficient Regeneration of �Caralis� Alstroemeria Cultivar from Rhizome Explants

    Directory of Open Access Journals (Sweden)

    Amir Ghaffar SHAHRIARI

    2012-05-01

    Full Text Available In this paper, the effects of a number of growth regulators as well as supplements to the Murashige and Skoog (MS basal medium were evaluated on the regeneration of Alstroemeria rhizome explants. In the first experiment the effects of three cytokinins (BA, TDZ and 2IP each at 0.5, 1 and 2 mg/l in combination with NAA (0.2 mg/l, followed by another PGR combination of 2IP (at 0.5, 1 and 2 mg/l with NAA (0 and 0.2 mg/l, on regeneration of rhizome-derived explants, was investigated. Through the second experiment, the effects of a number of supplements, including glucose (30 g/l as the alternative for sucrose, casein hydrolysate (1 g/l, asparagine and glutamine, (each at 30 mg/l added to MS medium, containing 1 mg/l BA and 0.2 mg/l NAA, was examined on rhizome explants� regeneration. Among the tested cytokinins, BA induced better regeneration of rhizome explants, resulting in a higher number of shoots compared to the other cytokinins. A medium supplemented with 1 mg/l BA and 0.2 mg/l NAA proved to be the most effective, with an average of 4.16 regenerated shoots per explant. In the second PGR combination, addition of NAA at 0.2 mg/l improved regeneration, compared to NAA-free treatments. In the second experiment, glucose substitution for sucrose improved regeneration with an average of 5.10 regenerated shoots per explant, compared to 4.16 shoots in sucrose-containing medium; whereas glutamine and asparagine (with 2.66 shoots and casein hydrolysate (with 3.80 shoots showed a negative influence on rhizome explants� regeneration.

  14. Milk Thistle Extract and Silymarin Inhibit Lipopolysaccharide Induced Lamellar Separation of Hoof Explants in Vitro

    Directory of Open Access Journals (Sweden)

    Nicole Reisinger

    2014-10-01

    Full Text Available The pathogenesis of laminitis is not completely identified and the role of endotoxins (lipopolysaccharides, LPS in this process remains unclear. Phytogenic substances, like milk thistle (MT and silymarin, are known for their anti-inflammatory and antioxidant properties and might therefore have the potential to counteract endotoxin induced effects on the hoof lamellar tissue. The aim of our study was to investigate the influence of endotoxins on lamellar tissue integrity and to test if MT and silymarin are capable of inhibiting LPS-induced effects in an in vitro/ex vivo model. In preliminary tests, LPS neutralization efficiency of these phytogenics was determined in an in vitro neutralization assay. Furthermore, tissue explants gained from hooves of slaughter horses were tested for lamellar separation after incubation with different concentrations of LPS. By combined incubation of explants with LPS and either Polymyxin B (PMB; positive control, MT or silymarin, the influence of these substances on LPS-induced effects was assessed. In the in vitro neutralization assay, MT and silymarin reduced LPS concentrations by 64% and 75%, respectively, in comparison PMB reduced 98% of the LPS concentration. In hoof explants, LPS led to a concentration dependent separation. Accordantly, separation force was significantly decreased by 10 µg/mL LPS. PMB, MT and silymarin could significantly improve tissue integrity of explants incubated with 10 µg/mL LPS. This study showed that LPS had a negative influence on the structure of hoof explants in vitro. MT and silymarin reduced endotoxin activity and inhibited LPS-induced effects on the lamellar tissue. Hence, MT and silymarin might be used to support the prevention of laminitis and should be further evaluated for this application.

  15. Influencing micropropagation in Clitoria ternatea L. through the manipulation of TDZ levels and use of different explant types.

    Science.gov (United States)

    Mukhtar, Seemab; Ahmad, Naseem; Khan, Md Imran; Anis, Mohammad; Aref, Ibrahim M

    2012-10-01

    A comparative performance of two explants types (CN and Nodal) for their efficiency to induce multiple shoot regeneration in Clitoria ternatea has been carried out. Thidiazuron (TDZ) in different concentrations (0.05-2.5 μM) was used as a supplement to the Murashige and Skoog's (MS) basal media. Explant type apart, two factors viz. concentration and exposure duration to TDZ played an important role in affecting multiple shoot regeneration. Cotyledonary node explants produced the best results at 0.1 μM TDZ, while in nodal explants the highest rate of shoot formation was achieved on MS medium supplemented with 1.0 μM TDZ. In both the explants, shoot multiplication increased when the regenerated shoots were subcultured on hormone free MS medium after 4 weeks of exposure to TDZ. Among the two, cotyledonary node explants produced considerably higher number of shoots at a comparatively lower concentration of TDZ than nodal explants. The regenerated shoots rooted best on MS medium containing 1.0 μM indole-3-butyric acid (IBA) and were successfully established in pots containing garden soil with 88 % survival rate. All the regenerated plants showed normal morphology and growth characteristics.

  16. Inhibition of TGFβ cell signaling for limbal explant culture in serumless, defined xeno-free conditions.

    Science.gov (United States)

    Zamudio, Aldo; Wang, Zheng; Chung, So-Hyang; Wolosin, J Mario

    2016-04-01

    Outgrowths of limbal epithelium by explant culture are used to treat limbal stem cell deficiency (LSCD). The explant culture medium is always complemented with serum, a complex solution which includes TGFβ. Since TGFβ is a cytostatic effector for epithelial proliferation we examined its effect on these cultures. Limbal biopsies were set on explant culture in DMEM/F12 with 5 ng/ml EGF and cholera toxin (ChT), ITS, and 5% FBS, henceforth SHEM or a) SHEMSB=SHEM plus SB431542 an inhibitor of TGFβ signaling; b) sfSHEM = SHEM with FBS replaced by 0.05% Albumax II; and c) sfSHEMSB and sfSHEMA83 = sfSHEM plus, respectively, SB431542 or A-83-01, another TGFβ inhibitor. After the initial outgrowths reached 3 cm in diameter, the limbal biopsies were serially transferred up to six times onto new inserts. Biopsy explant outgrowths were trypsinized and cell yield, morphology and stem-cell related JC-1 exclusion (IOVS, 52:4330) were determined by flow cytometry. Cells we plated at low density seeding to compare relative clonal proliferative activity. The expression of three proteins whose levels are associated with growth and differentiation states, Krt3, connexin 43 and p63 were determined by immunohistology and/or Western blot. Cell yield in rabbit, relative to SHEM (in %) were, SHEMSB, 104 ± 13 (p > 0.95); sfSHEM: 5 ± 3; and sfSHEMSB, 94 ± 18 (p > 0.95). Cell size and morphology, JC1 dye exclusion, Krt3, p63 and connexin 43 content, proliferation efficiency and the preservation of extended proliferative potential of the serially cultured biopsies were similar for SHEM, SHEMSB and sfSHEMSB. The only differences observed where reduced expression of Krt3 and increased preservation of p63 in the FBS-free medium. Removal of EGF from sfSHEMSB reduced yield by 92 ± 6% (p free medium caused a small, non-statistical decrease in growth rates. Equivalent results were observed in a preliminary experiment in human. These results suggest that in the absence serum

  17. A Novel 3D Skin Explant Model to Study Anaerobic Bacterial Infection

    Directory of Open Access Journals (Sweden)

    Grazieli Maboni

    2017-09-01

    Full Text Available Skin infection studies are often limited by financial and ethical constraints, and alternatives, such as monolayer cell culture, do not reflect many cellular processes limiting their application. For a more functional replacement, 3D skin culture models offer many advantages such as the maintenance of the tissue structure and the cell types present in the host environment. A 3D skin culture model can be set up using tissues acquired from surgical procedures or post slaughter, making it a cost effective and attractive alternative to animal experimentation. The majority of 3D culture models have been established for aerobic pathogens, but currently there are no models for anaerobic skin infections. Footrot is an anaerobic bacterial infection which affects the ovine interdigital skin causing a substantial animal welfare and financial impact worldwide. Dichelobacter nodosus is a Gram-negative anaerobic bacterium and the causative agent of footrot. The mechanism of infection and host immune response to D. nodosus is poorly understood. Here we present a novel 3D skin ex vivo model to study anaerobic bacterial infections using ovine skin explants infected with D. nodosus. Our results demonstrate that D. nodosus can invade the skin explant, and that altered expression of key inflammatory markers could be quantified in the culture media. The viability of explants was assessed by tissue integrity (histopathological features and cell death (DNA fragmentation over 76 h showing the model was stable for 28 h. D. nodosus was quantified in all infected skin explants by qPCR and the bacterium was visualized invading the epidermis by Fluorescent in situ Hybridization. Measurement of pro-inflammatory cytokines/chemokines in the culture media revealed that the explants released IL1β in response to bacteria. In contrast, levels of CXCL8 production were no different to mock-infected explants. The 3D skin model realistically simulates the interdigital skin and has

  18. One-stage explant-implant procedure of exposed porous orbital implants

    DEFF Research Database (Denmark)

    Toft, Peter B; Rasmussen, Marie L Roed; Prause, Jan Ulrik

    2011-01-01

    Purpose:  To investigate the risks of implant exposure after a combined explant-implant procedure in patients with an exposed porous orbital implant. Methods:  Twenty-four consecutive patients who had a combined explant-implant procedure of an exposed hydroxyapatite (21) or porous polyethylene (3......) orbital implant from January 2000 to February 2009 were included. The patient records were reviewed; patients were interviewed by telephone and invited for a clinical examination. Histopathological examination was carried out on the removed implants. Main outcome measures were: presence of exposure...... of the new implant or not, patient graded satisfaction with the cosmetic result, and presence of poor motility. Results:  None of the new implants became exposed or infected in the follow-up period of 25 [3-94] months (median [range]). The patients scored their satisfaction with the cosmetic result...

  19. Incubation under fluid dynamic conditions markedly improves the structural preservation in vitro of explanted skeletal muscles.

    Science.gov (United States)

    Carton, Flavia; Calderan, Laura; Malatesta, Manuela

    2017-11-28

    Explanted organs and tissues represent suitable experimental systems mimicking the functional and structural complexity of the living organism, with positive ethical and economic impact on research activities. However, their preservation in culture is generally limited, thus hindering their application as experimental models for biomedical research. In the present study, we investigated the potential of an innovative fluid dynamic culture system to improve the structural preservation in vitro of explanted mouse skeletal muscles (soleus). We used light and transmission electron microscopy to compare the morphological features of muscles maintained either in multiwell plates under conventional conditions or in a bioreactor mimicking the flow of physiological fluids. Our results demonstrate that fluid dynamic conditions markedly slowed the progressive structural deterioration of the muscle tissue occurring during the permanence in the culture medium, prolonging the preservation of some organelles such as mitochondria up to 48 h.

  20. Effect of explant origin and different growth regulators on micropropagation of Pistacia atlantica ssp. mutica

    Directory of Open Access Journals (Sweden)

    Ali-Ashraf Mehrabi

    2015-06-01

    Full Text Available Propagation of wild pistachio as a multipurpose woody species is a hard and tedious task. In this research, an effective in vitro protocol was developed for rapid proliferation of wild pistachio (Pistacia atlantica ssp. mutica in MS medium supplemented with B5 vitamins and different growth regulators. Rooting of plantlets was tested by two treatments containing Rhizopon and IBA in ex vitro. With respect to the results, the nodal segments explants, produced the highest shoot frequency, leaf frequency and the tallest shoots. On the other hand, the tallest shoots were generated from shoot tip explant and medium containing of TDZ plus IAA. Both treatments (Rhizopon and IBA led to a remarkable increase in the number of roots, root length and rooting percentage compared to the control. These results may be applied for rapid proliferation to spread the pistachio trees and shrubs that are difficult and time consuming.

  1. Acetylcholine causes rooting in leaf explants of in vitro raised tomato (Lycopersicon esculentum Miller) seedlings.

    Science.gov (United States)

    Bamel, Kiran; Gupta, Shrish Chandra; Gupta, Rajendra

    2007-05-30

    The animal neurotransmitter acetylcholine (ACh) induces rooting and promotes secondary root formation in leaf explants of tomato (Lycopersicon esculentum Miller var. Pusa Ruby), cultured in vitro on Murashige and Skoog's medium. The roots originate from the midrib of leaf explants and resemble taproot. ACh at 10(-5) M was found to be the optimum over a wide range of effective concentrations between 10(-7) and 10(-3) M. The breakdown products, choline and acetate were ineffective even at 10(-3) M concentration. ACh appears to have a natural role in tomato rhizogenesis because exogenous application of neostigmine, an inhibitor of ACh hydrolysis, could mimic the effect of ACh. Neostigmine, if applied in combination with ACh, potentiated the ACh effect.

  2. Effects of glucosamine on proteoglycan loss by tendon, ligament and joint capsule explant cultures.

    Science.gov (United States)

    Ilic, M Z; Martinac, B; Samiric, T; Handley, C J

    2008-12-01

    To investigate the effect of glucosamine on the loss of newly synthesized radiolabeled large and small proteoglycans by bovine tendon, ligament and joint capsule. The kinetics of loss of (35)S-labeled large and small proteoglycans from explant cultures of tendon, ligament and joint capsule treated with 10mM glucosamine was investigated over a 10-day culture period. The kinetics of loss of (35)S-labeled small proteoglycans and the formation of free [(35)S]sulfate were determined for the last 10 days of a 15-day culture period. The proteoglycan core proteins were analyzed by gel electrophoresis followed by fluorography. The metabolism of tendon, ligament and joint capsule explants exposed to 10mM glucosamine was evaluated by incorporation of [(3)H]serine and [(35)S]sulfate into protein and glycosaminoglycans, respectively. Glucosamine at 10mM stimulated the loss of small proteoglycans from ligament explant cultures. This was due to the increased loss of both macromolecular and free [(35)S]sulfate to the medium indicating that glucosamine affected the release of small proteoglycans as well as their intracellular degradation. The degradation pattern of small proteoglycans in ligament was not affected by glucosamine. In contrast, glucosamine did not have an effect on the loss of large or small proteoglycans from tendon and joint capsule or large proteoglycans from ligament explant cultures. The metabolism of cells in tendon, ligament and joint capsule was not impaired by the presence of 10mM glucosamine. Glucosamine stimulated the loss of small proteoglycans from ligament but did not have an effect on small proteoglycan catabolism in joint capsule and tendon or large proteoglycan catabolism in ligament, tendon or synovial capsule. The consequences of glucosamine therapy at clinically relevant concentrations on proteoglycan catabolism in joint fibrous connective tissues need to be further assessed in an animal model.

  3. Ultrastructural analysis of initial stages of dedifferentiation of root explants of Gentiana cruciata seedlings

    Directory of Open Access Journals (Sweden)

    Anna Mikuła

    2014-01-01

    Full Text Available The studies were carried out on isolated roots of 10-day old seedlings of Gentiana cruciata, which were placed and cultured on induction medium of Murashige and Skoog (1962 supplemented with 1.0 mg/dm3 dicamba + 0.l mg/dm3 NAA + 2.00 mg/dm3 BAP + 80.0 mg/dm3 adenine sulphate. Changes in explants from the 3rd to the l lth day of culture with the help of light and electron microscope were observed. Observations showed gradual dedifferentiation of root tissues, which was seen earliest in cortex at the proximal end of the explant and shifted gradually inwards the root and towards distal parts of its elongation zone. The most intensive callus formation appeared at cut surface of explant, where proliferation of cells in both cortex and axial cylinder was recognised. In the distal part of the elongation zone, cell divisions occurred only in endoderm and in axial cylinder. The meristematic part of the root was inactive. Finally, the following areas were distinguished in the explant: (I an area of intensive cell divisions, i.e., the elongation zone; (II an area of cell dispersion; and (III the inactive meristem. The ultrastructure brought evidences of cell reorganisation as the meaning of cell readiness to the division. Observations showed an increased activity of mitochondria and Golgi structures, thickening of walls and disappearance of plasmodesmal connections. Amyloplasts and lipid bodies in tissues in which they had been scarce or had not appeared before founding. Intensively dividing cells showed features of meristematic cells. They had dense cytoplasm with numerous organelles, large centrally located nuclei, and "nucleolar vacuoles" inside nucleoli. Cortex-derived callus formed aggregates. Both pericycle and endoderm produced callus of characteristic dense structure and regular type of divisions.

  4. Effect of explant density and medium culture volumes on cassava micropropagation in Temporal Immersion System

    OpenAIRE

    Milagros Basail; Victor Medero; Marilyn Martínez; José de la C. Ventura; Jorge López; Magaly García; Manuel Cabrera; Arletys Santos; Aymé Rayas; Carmen Pons; Maricel Bauta; Miguel Álvarez; Jesús García

    2003-01-01

    Due to the need of producing high quality planting material available to cassava growers, it has been necessary to look for alternatives in order to increase the efficiancy of in vitro propagation methods and their automation, such as the use of the Temporal Immersion Systems (RITA®). This work was carried out to increase the multiplication coefficient for cassava mass propagation through out Temporal Immersion Systems. The clone ‘CMC-40’ was used. Different medium volumes per explant, and ma...

  5. The Impact of Carbon Source, Explants and Growth Regulators on Callogenesis and Organogenesis of Artemisia annua

    Directory of Open Access Journals (Sweden)

    Bita GHASSEMI

    2015-12-01

    Full Text Available Artemisinin, a sesquiterpene lactone isolated from Artemisia annua L. plant is known for its antimalarial activity. The low content of artemisinin has stimulated researchers to enhance its production through biotechnological approaches such as tissue culture. The present study was initiated to study the effect of some important factors alone and in combination, on the callogenesis and organogenesis of Artemisia annua. The type of carbon source had a significant effect on NAA efficiency to callogenesis of A. annua, whereas the best callogenesis of A. absinthium was observed at 2 mg/L BAP + 30 g/L sucrose and in the absence of NAA, with root explants. Presence of BAP also had an important effect on callogenesis, especially in high concentrations. A suitable suspension culture was obtained in the MS basal medium containing 0.5 mg/L NAA and BAP, with 30 g/L glucose. Artemisinin was naturally production was at least 0.03 mg/g (w.dt at the first day and peaked on the 16th day with 0.31 mg/g (w.dt in the cell culture of A. annua. The maximum number of shoots (2.167 ± 1.484 was induced at 0.5 mg/L BAP + 0.1 mg/L NAA + glucose (30 g/L with leaf explants. However, treatments containing glucose did not show a good shoot induction. Longer shoots were induced in the medium containing either 0.5 mg/l NAA + 0.5 mg/l BAP + sucrose with leaf explants (1.493 cm ± 0.342 or 0.5 mg/l NAA + sucrose with stem explants (0.697 cm ± 0.930. Medium containing 0.5 or 2 mg/l NAA and sucrose (without BAP induced more roots though.

  6. Adventitious shoot regeneration from leaf explants of eastern cottonwood (Populus deltoides) cultured under photoautotrophic conditions.

    Science.gov (United States)

    Mingozzi, Marco; Montello, Paul; Merkle, Scott

    2009-03-01

    Effects of photoautotrophic and photomixotrophic growth conditions on adventitious shoot regeneration from leaf explants of eastern cottonwood (Populus deltoides Bartr. ex Marsh.) were investigated. Rooting and proliferating shoot cultures (Stage I) were grown in either an elevated (1500 ppm) CO(2) concentration ([CO(2)]) at high photosynthetic photon flux (PPF; ~ 150 micromol m(-2) s(-1)) (photoautotrophic condition) with 0, 10 or 30 g l(-1) sucrose or under standard conditions (ambient (360 ppm) [CO(2)] at low PPF (~ 60 micromol m(-2) s(-1)) with 30 g l(-1) sucrose). Leaves harvested from these cultures were analyzed for soluble sugars and were used as explants for adventitious shoot regeneration (Stage II), which was also carried out under photoautotrophic and standard conditions. Photoautotrophic conditions during Stage I promoted growth of rooting shoots but inhibited axillary shoot proliferation. Photoautotrophic conditions during Stage II suppressed callus and adventitious bud production from leaf explants compared with standard conditions. The regeneration environment appeared to be more important in controlling bud formation than the conditions under which the donor shoots were grown. Regardless of Stage I treatment, bud production was up to 100-fold higher for leaves cultured under standard conditions than under photoautotrophic conditions. Once adventitious buds were differentiated from the leaf tissues, however, their elongation was faster under photoautotrophic conditions than that under standard conditions, with some shoots reaching 10 mm in length on leaf explants cultured under photoautotrophic conditions. Because total leaf soluble sugar concentration was always lowest in shoots under standard conditions, which also yielded the highest bud production, the results suggest that endogenous starvation enhanced shoot production.

  7. Characteristic of c-Kit+ progenitor cells in explanted human hearts

    OpenAIRE

    Matuszczak, Sybilla; Czapla, Justyna; Jarosz-Biej, Magdalena; Wiśniewska, Ewa; Cichoń, Tomasz; Smolarczyk, Ryszard; Kobusińska, Magdalena; Gajda, Karolina; Wilczek, Piotr; Śliwka, Joanna; Zembala, Michał; Zembala, Marian; Szala, Stanisław

    2014-01-01

    According to literature data, self-renewing, multipotent, and clonogenic cardiac c-Kit+ progenitor cells occur within human myocardium. The aim of this study was to isolate and characterize c-Kit+ progenitor cells from explanted human hearts. Experimental material was obtained from 19 adult and 7 pediatric patients. Successful isolation and culture was achieved for 95 samples (84.1 %) derived from five different regions of the heart: right and left ventricles, atrium, intraventricular septum,...

  8. Explant culture: An advantageous method for isolation of mesenchymal stem cells from human tissues.

    Science.gov (United States)

    Hendijani, Fatemeh

    2017-04-01

    Mesenchymal stem cell (MSC) research progressively moves towards clinical phases. Accordingly, a wide range of different procedures were presented in the literature for MSC isolation from human tissues; however, there is not yet any close focus on the details to offer precise information for best method selection. Choosing a proper isolation method is a critical step in obtaining cells with optimal quality and yield in companion with clinical and economical considerations. In this concern, current review widely discusses advantages of omitting proteolysis step in isolation process and presence of tissue pieces in primary culture of MSCs, including removal of lytic stress on cells, reduction of in vivo to in vitro transition stress for migrated/isolated cells, reduction of price, processing time and labour, removal of viral contamination risk, and addition of supporting functions of extracellular matrix and released growth factors from tissue explant. In next sections, it provides an overall report of technical highlights and molecular events of explant culture method for isolation of MSCs from human tissues including adipose tissue, bone marrow, dental pulp, hair follicle, cornea, umbilical cord and placenta. Focusing on informative collection of molecular and methodological data about explant methods can make it easy for researchers to choose an optimal method for their experiments/clinical studies and also stimulate them to investigate and optimize more efficient procedures according to clinical and economical benefits. © 2017 John Wiley & Sons Ltd.

  9. Bioassay chamber for angiogenesis with perfused explanted arteries and electrospun scaffolding.

    Science.gov (United States)

    Rubenstein, David; Han, Dong; Goldgraben, Sara; El-Gendi, Hebah; Gouma, Pelagia-Irene; Frame, Mary D

    2007-01-01

    The purpose of this study was to test the hypothesis that explanted perfused arteries can serve as the initial endothelial cell culture source to evaluate the onset of angiogenesis in a cellulose acetate electrospun scaffold. Electrospun scaffolds with fiber diameters roughly controlled in three broad ranges: 0.01 to 0.2, 0.2 to 1, and 1 to 5 microm (Nanomed Nanotechnol Biol Med 2:37-41, 2006), were used in cell culture to determine which provides the best culture topology. This scaffold was then tested in a bioassay chamber whose cellular source was an explanted abdominal aorta from donated euthanized mice. Scaffolds were draped over a cannulated vessel perfused for 24 h. Cell viability, density, and morphology were quantified. The largest fiber diameter group provided the best culture topology for human umbilical vein endothelial cells, showing high cell viability and density, and enhanced elongated cell morphology. Addition of single-walled carbon nanotubes decreased cell density significantly but chitosan heightened cell density and promoted spontaneous capillary tube like structure. Viability of endothelial cells increased with higher flow in the bioassay chamber. Endothelial cells showed a growth preference towards larger diameter fibers. Addition of chitosan improved culture conditions. Thus, this study provides a proof of principle for the possibility of co-culturing tissue engineered vascular networks from a perfused explant.

  10. Characterization of Myelomonocytoid Progenitor Cells with Mesenchymal Differentiation Potential Obtained by Outgrowth from Pancreas Explants

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    Marc-Estienne Roehrich

    2012-01-01

    Full Text Available Progenitor cells can be obtained by outgrowth from tissue explants during primary ex vivo tissue culture. We have isolated and characterized cells outgrown from neonatal mouse pancreatic explants. A relatively uniform population of cells showing a distinctive morphology emerged over time in culture. This population expressed monocyte/macrophage and hematopoietic markers (CD11b+ and CD45+, and some stromal-related markers (CD44+ and CD29+, but not mesenchymal stem cell (MSC-defining markers (CD90− and CD105− nor endothelial (CD31− or stem cell-associated markers (CD133− and stem cell antigen-1; Sca-1−. Cells could be maintained in culture as a plastic-adherent monolayer in culture medium (MesenCult MSC for more than 1 year. Cells spontaneously formed sphere clusters “pancreatospheres” which, however, were nonclonal. When cultured in appropriate media, cells differentiated into multiple mesenchymal lineages (fat, cartilage, and bone. Positive dithizone staining suggested that a subset of cells differentiated into insulin-producing cells. However, further studies are needed to characterize the endocrine potential of these cells. These findings indicate that a myelomonocytoid population from pancreatic explant outgrowths has mesenchymal differentiation potential. These results are in line with recent data onmonocyte-derivedmesenchymal progenitors (MOMPs.

  11. Pistacia lentiscus fruit oil reduces oxidative stress in human skin explants caused by hydrogen peroxide.

    Science.gov (United States)

    Ben Khedir, S; Moalla, D; Jardak, N; Mzid, M; Sahnoun, Z; Rebai, T

    2016-10-01

    We investigated the efficacy of Pistacia lentiscus fruit oil (PLFO) for protecting human skin from damage due to oxidative stress. PLFO contains natural antioxidants including polyphenols, sterols and tocopherols. We compared the antioxidant potential of PLFO with extra virgin olive oil (EVOO). Explants of healthy adult human skin were grown in culture with either PLFO or EVOO before adding hydrogen peroxide (H2O2). We also used cultured skin explants to investigate the effects of PLFO on lipid oxidation and depletion of endogenous antioxidant defense enzymes including glutathione peroxidase (GPx), superoxide dismutase (SOD) and catalase (CAT) one day after 2 h exposure to H2O2. We found that PLFO scavenged radicals and protected skin against oxidative injury. PLFO exhibited greater antioxidant and free radical scavenging activity than EVOO. Skin explants treated with PLFO inhibited H2O2 induced MDA formation by inhibition of lipid oxidation. In addition, the oil inhibited H2O2 induced depletion of antioxidant defense enzymes including GPx, SOD and CAT. We found that treatment with PLFO repaired skin damage owing to its antioxidant properties.

  12. In vitro regeneration from petiole explants of non-toxic Jatropha curcas

    KAUST Repository

    Kumar, Nitish

    2011-01-01

    Jatropha curcas, a multipurpose shrub has acquired significant economic potential as biodiesel plant. The seeds or pressed cake is toxic due to the presence of toxic substances and is not useful as food/fodder despite having the best protein composition. A simple, efficient, and reproducible method for plant regeneration through direct organogenesis from petiole explants of non-toxic J. curcas was developed using Murashige and Skoog (MS) medium supplemented with different concentrations of thidiazuron (TDZ). The best induction of shoot buds (57.61%), and number of shoot buds (4.98) per explant were obtained when in vitro petiole explants were placed horizontally on MS medium supplemented with 2.27 mu M TDZ. The Induced shoot buds were transferred to MS medium containing 10 mu M kinetin (Kn), 4.5 mu M 6-benzyl aminopurine (BA), and 5.5 mu M alpha-naphthaleneacetic acid (NAA) for shoot proliferation and subsequent elongation was achieved on MS medium supplemented with 2.25 mu M BA and 8.5 mu M IAA. The elongated shoots could be rooted on half-strength MS medium with 15 mu M IBA, 11.4 mu M IAA and 5.5 mu M NAA with more than 90% survival rate. (C) 2010 Elsevier B.V. All rights reserved.

  13. Pericardium Plug in the Repair of the Corneoscleral Fistula After Ahmed Glaucoma Valve Explantation

    Science.gov (United States)

    Yoo, Chungkwon; Kwon, Sung Wook

    2008-01-01

    We report four cases in which a pericardium (Tutoplast®) plug was used to repair a corneoscleral fistula after Ahmed Glaucoma Valve (AGV) explantation. In four cases in which the AGV tube had been exposed, AGV explantation was performed using a pericardium (Tutoplast®) plug to seal the defect previously occupied by the tube. After debridement of the fistula, a piece of processed pericardium (Tutoplast®), measured 1 mm in width, was plugged into the fistula and secured with two interrupted 10-0 nylon sutures. To control intraocular pressure, a new AGV was implanted elsewhere in case 1, phaco-trabeculectomy was performed concurrently in case 2, cyclophotocoagulation was performed postoperatively in case 3 and anti-glaucomatous medication was added in case 4. No complication related to the fistula developed at the latest follow-up (range: 12~26 months). The pericardium (Tutoplast®) plug seems to be an effective method in the repair of corneoscleral fistulas resulting from explantation of glaucoma drainage implants. PMID:19096247

  14. Endogenous cytokinins in Cocos nucifera L. in vitro cultures obtained from plumular explants.

    Science.gov (United States)

    Sáenz, L; Azpeitia, A; Oropeza, C; Jones, L H; Fuchsova, K; Spichal, L; Strnad, M

    2010-11-01

    Auxin induces in vitro somatic embryogenesis in coconut plumular explants through callus formation. Embryogenic calli and non-embryogenic calli can be formed from the initial calli. Analysis of endogenous cytokinins showed the occurrence of cytokinins with aromatic and aliphatic side chains. Fourteen aliphatic cytokinins and four aromatic cytokinins were analysed in the three types of calli and all the cytokinins were found in each type, although some in larger proportions than others. The most abundant cytokinins in each type of callus were isopentenyladenine-9-glucoside, zeatin-9-glucoside, zeatin riboside, isopentenyladenine riboside, dihydrozeatin and dihydrozeatin riboside in decreasing order. Total cytokinin content was compared between the three types of calli, and it was found to be lower in embryogenic calli compared to non-embryogenic calli or initial calli. The same pattern was observed for individual cytokinins. When explants were cultured in media containing exogenously added cytokinins, the formation of embryogenic calli in the explants was reduced. When 8-azaadenine (an anticytokinin) was added the formation of embryogenic calli and somatic embryos was increased. These results suggest that the difference in somatic embryo formation capacity observed between embryogenic calli and non-embryogenic calli is related to their endogenous cytokinin contents.

  15. Somatic embryogenesis from bud and leaf explants of date palm (Phoenix dactylifera L.) cv. Najda.

    Science.gov (United States)

    Mazri, Mouaad Amine; Belkoura, Ilham; Meziani, Reda; Mokhless, Boutaïna; Nour, Souad

    2017-05-01

    An efficient regeneration system through somatic embryogenesis was developed for date palm cv. Najda. Adventitious bud and proximal leaf segments cultured on Murashige and Skoog (MS) medium supplemented with various combinations of auxins and cytokinins induced embryogenesis after at least 6 months of culture. Somatic embryogenesis induction seemed correlated with the type of the explant, the induction period and the auxin used. The highest rate of somatic embryogenesis (86.0%) was obtained on bud explants cultured on MS medium supplemented with 45.0 µM 2,4-dichlorophenoxyacetic acid (2,4-D), and 4.5 µM kinetin or 4.5 µM 6-(dimethylallylamino) purine (2iP). Whereas, low levels of embryogenesis were obtained on media supplemented with 1-naphthalene acetic acid (NAA) or 2-naphthoxyacetic acid (NOA). Proximal leaf segments showed somatic embryogenesis only when cultured on media supplemented with 2,4-D or picloram. Statistical analysis revealed significant effects of explant type and plant growth regulators (PGRs) combination on somatic embryogenesis. Somatic embryos were germinated successfully on PGR-free MS medium with or without activated charcoal (50.0-60.0 and 26.6-36.6%, respectively), and 80.0% of plantlets survived after transferring to a glasshouse for 6 months. Our results will be useful for large-scale propagation of date palm cv. Najda, characterized by high fruit quality and bayoud disease resistance.

  16. Callogenesis in root explants of four species of the family Solanaceae after inducing by Agrobacterium rhizogenes

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    Zahra Shakeran

    2015-09-01

    Full Text Available Studying explants affected by Agrobacterium rhizogenes shows that in addition to possible formation of hairy roots, it is likely that callogenesis can be induced in these tissues. The T-DNA region of A. rhizogenes codes enzymes that participate in biosynthesis of plants growth hormones. These hormones also affect callogenesis, hence, the formation of various calluses with different morphological properties are possible. It is very likely that the level of biosynthetic growth hormone, the plasmid carried by each bacteria strain, the position of T-DNA, and the level of gene expression contribute to this morphologic variation. In this study, the root explants of four species of the family Solanaceae namely Atropa belladonna, Datura metel, D. stramonium and Hyoscyamus niger were induced by using different strains of A. rhizogenes (A4, A7, AR15834, AR318, AR9402 and AR9543. Some of these explants entered callus phase and formed various calluses with different colors and shapes. Moreover, in some callus samples hairy roots were also appeared. These variations were probably caused by variations in the levels and ratios of auxin and cytokinine hormons after the induction. As shown in previous studies, the amount of secondary metabolites is reduced due to undifferentiated tissue produced in the callogenesis process.

  17. Bystander-induced differentiation: A major response to targeted irradiation of a urothelial explant model

    Energy Technology Data Exchange (ETDEWEB)

    Belyakov, Oleg V. [Gray Cancer Institute, P.O. Box 100, Mount Vernon Hospital, Northwood, Middlesex HA6 2JR (United Kingdom) and Radiation and Environmental Science Centre, Dublin Institute of Technology, Focas Institute, Kevin St., Dublin 9 (Ireland)]. E-mail: oleg.belyakov@stuk.fi; Folkard, Melvyn [Gray Cancer Institute, P.O. Box 100, Mount Vernon Hospital, Northwood, Middlesex HA6 2JR (United Kingdom); Mothersill, Carmel [Radiation and Environmental Science Centre, Dublin Institute of Technology, Focas Institute, Kevin St., Dublin 9 (Ireland); Prise, Kevin M. [Gray Cancer Institute, P.O. Box 100, Mount Vernon Hospital, Northwood, Middlesex HA6 2JR (United Kingdom); Michael, Barry D. [Gray Cancer Institute, P.O. Box 100, Mount Vernon Hospital, Northwood, Middlesex HA6 2JR (United Kingdom)

    2006-05-11

    A ureter primary explant technique, using porcine tissue sections was developed to study bystander effects under in vivo like conditions where dividing and differentiated cells are present. Targeted irradiations of ureter tissue fragments were performed with the Gray Cancer Institute charged particle microbeam at a single location (2 {mu}m precision) with 10 {sup 3}He{sup 2+} particles (5 MeV; LET 70 keV/{mu}m). After irradiation the ureter tissue section was incubated for 7 days allowing explant outgrowth to be formed. Differentiation was estimated using antibodies to Uroplakin III, a specific marker of terminal urothelial differentiation. Even although only a single region of the tissue section was targeted, thousands of additional cells were found to undergo bystander-induced differentiation in the explant outgrowth. This resulted in an overall increase in the fraction of differentiated cells from 63.5 {+-} 5.4% to 76.6 {+-} 5.6%. These changes are much greater than that observed for the induction of damage in this model. One interpretation of these results is that in the tissue environment, differentiation is a much more significant response to targeted irradiation and potentially a protective mechanism.

  18. Cell-mediated contraction of vitreous explants from chicken embryo: Possibility of screening for therapeutic agents against proliferative vitreoretinal diseases

    Science.gov (United States)

    Oki, Keitaro; Shimada, Arata; Nagase, Terumasa; Katsura, Yoshiya; Kosano, Hiroshi

    2013-01-01

    Purpose We aimed to establish a novel screening system for identifying potential therapeutic agents for treating proliferative vitreoretinal diseases (PVDs). In this study, we focused on vitreous explants from chicken embryos and evaluated the usefulness of quantitatively analyzing the effects of potential candidates on cell-mediated vitreous contraction, which leads to blindness in PVDs. Methods Vitreous explants were extracted from 19-day-old embryonic chickens and then incubated with retinal Müller cells or endothelial cells to permit cell adhesion. After cell adhesion occurred, we examined the effect of the attached cells on the wet weight of vitreous explants as an index of vitreous contraction. We also performed hematoxylin and eosin staining to characterize the cell morphology on the vitreous surface. Results Contraction of the vitreous explants was observed after cell adhesion of not only retinal Müller cells but also endothelial cells. We confirmed the adhesion of these cells on vitreous explants and estimated the number of adherent cells with 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) analysis. The cells on the vitreous surface presented an elongated fibroblast-like phenotype. Integrin was found to be a receptor involved in cell adhesion on the vitreous surface. Discussion Our results suggest that vitreous explants from chicken embryos may be novel useful tools for screening antiadhesion therapeutic agents in PVDs. This preliminary study must be validated with human vitreous and human retinal pigment epithelial cells. PMID:24319331

  19. A rooting procedure for lentil (Lens culinaris Medik.) and other hypogeous legumes (pea, chickpea and Lathyrus) based on explant polarity.

    Science.gov (United States)

    Fratini, R; Ruiz, M L

    2003-04-01

    The present study assessed the rooting response of lentil nodal segments in relation to explant polarity, hormone, salt and carbohydrate concentrations of the medium. Nodal segments of lentil with an axillary bud cultured in an inverted orientation (apical end in medium) showed higher rooting frequencies than explants cultured in a normal orientation (basal end in medium). The highest rooting percentage (95.35%) and average number of shoots regenerated per explant (2.4) were obtained from explants placed in an inverted orientation on Murashige and Skoog (MS) medium salts with 3% sucrose, supplemented with 5 microM indole acetic acid (IAA) and 1 microM kinetin (KN). Reducing or increasing phytohormone concentration did not alter significantly root regeneration of inverted explants. Sucrose at 3% allowed higher root regeneration frequencies compared to 1.5% sucrose. MS full concentration permitted regeneration of longer shoots with more nodes per regenerated shoot, compared to MS half-strength, which regenerated more shoots of shorter length and with less nodes. Inverted nodal segments of other hypogeous legumes (pea, chickpea and Lathyrus) also exhibited higher rooting frequencies than explants cultured in a normal orientation on MS medium with 3% sucrose and supplemented with 5 microM IAA and 1 microM KN. The most novel application of this study is the culture of nodal segments of hypogeous legumes in an inverted orientation. This procedure is a considerable improvement over other published procedures concerning in vitro rooting of lentil, pea, chickpea and Lathyrus.

  20. Sealing and explant types on the mangaba micropropagation Tipo de vedação e explantes na micropropagação de mangabeira

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    Aline de Jesus Sá

    2012-08-01

    Full Text Available In micropropagation, especially for mangaba tree botanical variety of Northeastern Brazil, limiting aspects such as ethylene accumulation in the cultivation flask and loss of vigor in subcultures have been observed. This study was aimed at assessing the technical and scientific knowledge of the in vitro propagation of botanical mangaba tree variety and at improving the micropropagation protocol, establishing the in vitro cultivation time, the best type of flask sealing and explant at different micropropagation stages. For the establishment phase and for the first and second subcultures, the MS medium with 3% sucrose and 0.6% agar, supplemented with 1 mg L-1 IAA and 1 mg L-1 BA was used. Evaluations were performed at 30, 50 and 65 days of in vitro cultivation. The best types of flask sealing for the establishment phase were the PVC film and Para-film® and for the first subculture the Para-film® seal. In the second subculture the PVC film and Para-film® seals promoted the best growth. The median and basal nodal segments presented the best performance in the first subculture. No significant effect of explant type was observed in the second subculture. The ideal subculture interval in the establishment phase and the first and second subcultures is 50 days.Na micropropagação, especialmente para mangaba, variedade botânica da árvore do Nordeste do Brasil, aspectos limitantes, como acúmulo de etileno no recipiente de cultivo e perda de vigor em subculturas têm sido observados. Neste estudo, objetivou-se avaliar o conhecimento técnico e científico da propagação in vitro de mangabeira, variedade botânica do Nordeste do Brasil, e melhorar o protocolo de micropropagação, o melhor tipo de vedação frasco e explante em diferentes etapas. Para a fase de estabelecimento e para as subculturas primeiro e segundo, foi utlizado o meio MS com 3% de sacarose e agar 0,6%, suplementado com 1 mg L-1 de AIA e 1 mg L-1 de BAP. As avaliações foram

  1. Studies on Callus Induction and Regeneration of Medicinal Plant Chicory (Cichorium intybus L. from Leaf and Petiole Explants

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    H. Hadizadeh

    2016-07-01

    Full Text Available Introduction: Chicory (Cichorium intybus L. belongs to Asteraceae family is commonly known as witloof chicory. The leaves and the roots of this medicinal plant are edible and commonly used as salad. Some varieties are also cultivated as coffee substitute after roasting the roots. All parts of the plant contain these volatile oils, with the majority of the toxic components concentrated in the plant's root. In folk medicine, the plant is used for the treatment of diarrhea, spleen enlargement, fever, and vomiting. Antihepatotoxic activity on damaged rat’s liver sections and anti-bacterial activity of this crop has been recently reported. In vitro regeneration from leaf explants with various hormonal combinations has been reported previously. Moreover, in vitro regeneration of Chicory from cotyledon explants using different combinations of plant growth regulators has been studied. Also, a protocol for the regeneration of plantlets from leaf and petiole explants of witloof chicory has been developed. The aim of the present investigation was optimization of callus induction and shoot regeneration from leaf and petiole tissues of Chicory (Esfahan genotype. Materials and Methods: In this investigation, Esfahan genotype was used for callus induction and direct shoot regeneration. Seeds were first washed with running tap water for 30 min then seeds were surface sterilized by dipping in 70% ethanol for 90 s and rinsed with sterile distilled water, followed by immersing in 5% sodium hypochlorite solution for 25 min and thereafter rinsed for 30 min with sterile distilled water. The basal medium used in this investigation was MS. For shoot regeneration, leaf and petiole explants (5 mm segments were excised from 4-week-old sterile seedlings and cultured on MS medium containing different combinations of NAA / BA and KIN / BA in two separate experiments. Experiments were performed factorial based on completely randomized design. Cultures were incubated at 25

  2. Effects of thidiazuron and paclobutrazol on regeneration potential of tulip flower stalk explants in vitro and subsequent shoot multiplication

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    Małgorzata Podwyszyńska

    2011-01-01

    Full Text Available The effects of TDZ and paclobutrazol on the primary regeneration on tulip flower stalk explants of six cultivars and subsequent shoot multiplication were examined. Explants, flower stalk slices, were excised from cooled and subsequently forced bulbs. The explants were incubated for two months in darkness on medium containing NAA and cytokinins, 2iP and BAP, as control, or TDZ (0.5-4 mg l-1 and paclobutrazol (0.05-0.4 mg l-1. Then, the regenerating explants were subcultured on medium with TDZ and NAA applied at low concentrations. Different regeneration capabilities were found depending on cultivar and growth regulators. The percentage of explants forming leaf-like structures ranged, on the control medium, from 80% in 'Blue Parrot' and 'Prominence' to below 30% in 'Apeldoorn' and 'Mirjoran'. TDZ, applied at optimum for each cultivar concentration, greatly increased the regeneration potential up to 70-100%. Paclobutrazol, added to the TDZ-containing medium, significantly enhanced the response of explants, resulting in high numbers of leaf-like structures formed per explant (13.7-22.8. The structures developed gradually into characteristic forms: the growing up cotyledonary leaf, the probable root primordium formed at its base, the growing downwards stolon and the shoot meristem developed finely on its tip. It is suggested that such primary regeneration may have a nature of somatic embryogenesis. Then, the adventitious shoots developed and formed clusters, which were divided into 2-3 smaller ones every two months. The growth regulators, used at initial stage, markedly influenced subsequent shoot multiplication. Thus, the most intensive shoot formation was noted with TDZ at concentrations of 0.5-2 mg l-1 and paclobutrazol of 0.05-0.1 mg l-1.

  3. Simulação da soja geneticamente modificada tolerante ao glyphosate por meio do cultivo de explantes Simulation of the transgenic soybean tolerant to glyphosate through explant cultivation

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    Sérgio C. Siqueira

    1999-04-01

    Full Text Available O objetivo do experimento consistiu na simulação in vitro da soja transgênica tolerante ao glyphosate, através do cultivo de explantes em meios de cultura contendo aminoácidos aromáticos. As avaliações basearam-se nos efeitos do glyphosate sobre sementes oriundas de explantes de soja (Glycine max (L. Merr. cv. UFV-16. Para tanto, explantes de soja foram cultivados em meios de cultura líquidos com pH em torno de 5,0. Cada explante constou de um legume completamente expandido contendo duas sementes de aproximadamente 100 mg, conectada a um segmento de caule de 45 mm de comprimento. Os tratamentos testados foram: A = glutamina (Gln; B = Gln + fenilalanina (Phe + tirosina (Tyr + triptofano (Trp; C = Gln + glyphosate; D = Gln + Phe + Tyr + Trp + glyphosate. O experimento foi conduzido sob irradiância de 80 mmol-2s-1 a 25oC por 204 horas. Nos tratamentos que receberam aminoácidos aromáticos e glutamina, o herbicida não afetou as massas fresca e seca das sementes, como também, não afetou seus constituintes bioquímicos (proteínas, óleo, ácidos graxos, carboidratos e clorofilas. Portanto, a suplementação exógena de aminoácidos aromáticos suprime os efeitos fitotóxicos do glyphosate sobre explantes de soja, permitindo estudos sobre o seu modo de ação e metabolismo nas sementes, uma vez que os explantes se comportaram analogamente à soja transgênica não suscetível ao herbicida.The objective of this experiment consisted in simulation in vitro of the transgenic soybean tolerant to glyphosate through explant cultivation in culture medium containing aromatic amino acids. The effects of glyphosate on soybean (Glycine max (L. Merrill were evaluated in seeds harvested from explants of cv UFV-16. The soybean explants were cultivated in liquid medium culture with pH about 5.0. Each explant consisted of one fruit completely expanded, containing two seeds of 100 mg approximately, and connected to a stem segment of 45 mm length. The

  4. Influência do cloreto de cálcio no crescimento de explantes de Gypsophila paniculata L. (Caryophyllaceae, cultivados in vitro Influence of calcium chloride on the growth of Gypsophila paniculata L. (Caryophyllaceae explants, cultivated in vitro

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    R. Jun Takane

    1994-08-01

    Full Text Available Este trabalho teve por finalidade verificar a influência do Cloreto de Cálcio (CaCl12 no crescimento de explantes de Gypsophila paniculata L., cultivados em meio de cultura a fim de fornecer subsídios para a micropropagação desta cultura. Foram utilizados como explantes somente as gemas apicais das plantas em fase de crescimento vegetativo. Os explantes foram inoculados em meio de cultura MS (Murashigue & Skoog modificado com diferentes concentrações de CaCl2: a 440; b 880; c 1.760; d 2.640 e e 3.520 mg/1. Os explantes foram deixados em câmara de crescimento sob uma temperatura constante de 25°C ± 2°C sob fotoperíodo de 16 horas. Os explantes do meio de cultura MS com 1.760 mg/1 de CaCl2 foram os de melhor resultado, com crescimento vigoroso e presença de coloração verde intensa nas folhas. Os explantes do meio de cultura MS com 440 mg/1 de CaCl2 não apresentaram um crescimento satisfatório, com estiolamento e coloração verde pouco intensa nas folhas; os explantes do meio de cultura MS com 3.520 mg/1 de CaCl2, também não apresentaram um crescimento satisfatório, pois com 10 dias de inoculação já se percebia uma menor indução no crescimento, podendo tal efeito ser conseqüência de dois fatores: excesso de cálcio no meio de cultura ou a toxidez ocasionada pelo cloro.Shoot tips from plants still in the vegetative growth phase were used as explants. They were sterilised with tetraciclin 1% for four minutes, sodium hypochloridre (commercial sanitary water with 5% of active cloride 20% (v/v for 15 minutes and alcohol 70 GL for 2 minutes. The explants were inoculated in a growth medium MS (Murashigue & Skoog, with different concentrations of CaCl2: a 440; b 880; c 1,760; d 2,640 and e 3,520 mg/1. Explants were kept in a growth chamber at constant temperature (25°C ± 2°C with a photo period of 16 hours. The explants in the medium modified with 1,760 mg/1 of CaCl2, presented the best results, with a vigorous growth and

  5. Adventitious Shoot Regeneration from Leaf Explant of Dwarf Hygro (Hygrophila polysperma (Roxb. T. Anderson

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    Mehmet Karataş

    2013-01-01

    Full Text Available Dwarf hygro (Hygrophila polysperma is an ornamental aquatic plant that changes its leaf colours to pinkish in high light. It is listed as a medicinal plant in medicinal plant lists of Indian states of West Bengal and Karnataka. It is also used as a screening tool for toxicities and a bioindicator to detect and control algae. The study reported in vitro adventitious shoot regeneration from leaf explants cultured on MS medium containing 0.10–1.60 mg/L Kin/TDZ with or without 0.10 mg/L IBA and 500 mg/L Amoklavin to eradicate endogenic bacterial contamination. Direct adventitious shoot regeneration started within one week from both culture mediums followed by late callus induction which was more prominent on TDZ containing media compared to Kin containing media. Addition of 0.10 mg/L IBA with both Kin and TDZ increased shoot regeneration frequency, mean number of shoots per explant, and mean shoot length. Maximum number of 16.33 and 20.55 shoots per explant was obtained on MS medium containing 0.80+0.10 mg/L Kin-IBA and 0.10+0.10 mg/L TDZ-IBA, respectively. Regenerated shoots were rooted on MS medium containing 0.20–1.00 mg/L IBA followed by successfull acclimatization in aquariums. Regenerated plantlets were also tested in jars containing distilled water that showed the pH 6–9 for the best plant growth and development.

  6. Glucocorticoids affect 24 h clock genes expression in human adipose tissue explant cultures.

    Directory of Open Access Journals (Sweden)

    Purificación Gómez-Abellán

    Full Text Available to examine firstly whether CLOCK exhibits a circadian expression in human visceral (V and subcutaneous (S adipose tissue (AT in vitro as compared with BMAL1 and PER2, and secondly to investigate the possible effect of the glucocorticoid analogue dexamethasone (DEX on positive and negative clock genes expression.VAT and SAT biopsies were obtained from morbid obese women (body mass index ≥ 40 kg/m(2 (n = 6. In order to investigate rhythmic expression pattern of clock genes and the effect of DEX on CLOCK, PER2 and BMAL1 expression, control AT (without DEX and AT explants treated with DEX (2 hours were cultured during 24 h and gene expression was analyzed at the following times: 10:00 h, 14:00 h, 18:00 h, 22:00 h, 02:00 h and 06:00 h, using qRT-PCR.CLOCK, BMAL1 and PER2 expression exhibited circadian patterns in both VAT and SAT explants that were adjusted to a typical 24 h sinusoidal curve. PER2 expression (negative element was in antiphase with respect to CLOCK and in phase with BMAL1 expression (both positive elements in the SAT (situation not present in VAT. A marked effect of DEX exposure on both positive and negative clock genes expression patterns was observed. Indeed, DEX treatment modified the rhythmicity pattern towards altered patterns with a period lower than 24 hours in all genes and in both tissues.24 h patterns in CLOCK and BMAL1 (positive clock elements and PER2 (negative element mRNA levels were observed in human adipose explants. These patterns were altered by dexamethasone exposure.

  7. Propagation of goldenrod (Solidago canadensis L. from leaf and nodal explants

    Directory of Open Access Journals (Sweden)

    Jun Li

    2012-02-01

    Full Text Available Goldenrod (Solidago canadensis L. is an invasive plant species in many countries except North America but a cut-flower species worldwide. There is a need to generate and propagate goldenrod clones efficiently for research and commercial purposes. A callus induction and plantlet regeneration system was developed by studying the influence of explant type and different concentrations of plant growth regulators. The highest callus production from leaf segments was obtained on Murashige and Skoog’s medium (MS medium supplemented with 1.0 mg/L naphthalene acetic acid (NAA and 1.0 mg/L 6-benzylaminopurine (BA. Adventitious shoots could be regenerated directly from leaf explants without an intermediate callus phase with the highest shoot induction percentage of 87.2%. The largest number of adventitious shoots per leaf explant (3.2 was obtained on MS medium supplemented with 0.4 mg/L NAA and 2.0 mg/L BA. MS medium supplemented with 0.1 mg/L NAA and 1.0 mg/L BA was the best medium for axillary shoot regeneration from nodal segments. The highest root number and longest roots occurred on half-strength MS without the addition of any growth regulator. Rooted plantlets were then transferred to a soil-based growth medium, placed in a greenhouse, and acclimatized with 100% success. All surviving plants grew normally without showing any morphological varia­tion when compared to those grow from seed. This regeneration protocol may be used to produce certain biotypes of goldenrod suitable for genetic transformation rapid propagation of goldenrod for commercial purposes or for screening fungi and toxins as potential biocontrol agents against this weed.

  8. Agrobacterium-mediated transformation of oat (Avena sativa L.) cultivars via immature embryo and leaf explants.

    Science.gov (United States)

    Gasparis, Sebastian; Bregier, Cezary; Orczyk, Waclaw; Nadolska-Orczyk, Anna

    2008-11-01

    This paper reports on the successful Agrobacterium-mediated transformation of oat, and on some factors influencing this process. In the first step of the experiments, three cultivars, two types of explant, and three combinations of strain/vectors, which were successfully used for transformation of other cereals were tested. Transgenic plants were obtained from the immature embryos of cvs. Bajka, Slawko and Akt and from leaf base explants of cv. Bajka after transformation with A. thumefaciens strain LBA4404(pTOK233). The highest transformation rate (12.3%) was obtained for immature embryos of cv. Bajka. About 79% of the selected plants proved to be transgenic; however, only 14.3% of the T(0) plants and 27.5% of the T(1) showed GUS expression. Cell competence of both types of explant differed in terms of their transformation ability and transgene expression. The next step of the study was to test the suitability for oat transformation of the pGreen binary vector combined with different selection cassettes: nptII or bar under the nos or 35S promoter. Transgenic plants were selected in combinations transformed with nos::nptII, 35S::nptII and nos::bar. The highest transformation efficiency (5.3%) was obtained for cv. Akt transformed with nos::nptII. A detailed analysis of the T(0) plants selected from a given callus line and their progeny revealed that they were the mixture of transgenic, chimeric-transgenic and non-transgenic individuals. Southern blot analysis of T(0) and T(1) showed simple integration pattern with the low copy number of the introduced transgenes.

  9. Stable genetic transformation of Jatropha curcas via Agrobacterium tumefaciens-mediated gene transfer using leaf explants

    KAUST Repository

    Kumar, Nitish

    2010-07-01

    Jatropha curcas is an oil bearing species with multiple uses and considerable economic potential as a biofuel crop. A simple and reproducible protocol was developed for Agrobacterium tumefaciens-mediated stable genetic transformation of J. curcas using leaf explains. Agrobacterium strain LBA 4404 harbouring the binary vector pCAMBIA 1304 having sense-dehydration responsive element binding (S-DREB2A), beta-glucuronidase (gus), and hygromycin-phosphotransferase (hpt) genes were used for gene transfer. A number of parameters such as preculture of explains, wounding of leaf explants, Agrobacterium growth phase (OD), infection duration, co-cultivation period, co-cultivation medium pH, and acetosyringone, were studied to optimized transformation efficiency. The highest transformation efficiency was achieved using 4-day precultured, non-wounded leaf explants infected with Agrobacterium culture corresponding to OD(600)=0.6 for 20 min, followed by co-cultivation for 4 days in a co-cultivation medium containing 100 mu M acetosyringone, pH 5.7. Co-cultivated leaf explants were initially cultured on Murashige and Skoog (MS) medium supplemented with 2.27 mu M thidiazuron (TDZ) for regeneration of shoot buds, followed by selection on same medium with 5 mu g ml(-1) hygromycin. Selected shoot buds were transferred to MS medium containing 10 mu M kinetin (Kn), 4.5 mu M 6-benzyl aminopurine (BA), and 5.5 mu M alpha-naphthaleneacetic acid (NAA) for proliferation. The proliferated shoots were elongated on MS medium supplemented with 2.25 mu M BA and 8.5 mu M indole-3-acetic acid (IAA). The elongated shoots were rooted on half strength MS medium supplemented with 15 mu M indole-3-butyric acid (IBA), 5.7 mu M IAA, 5.5 mu M NAA, and 0.25 mg l(-1) activated charcoal. GUS histochemical analysis of the transgenic tissues further confirmed the transformation event. PCR and DNA gel blot hybridization were performed to confirm the presence of transgene. A transformation efficiency of 29% was

  10. Cultivar-Dependent Direct Organogenesis of Date Palm from Shoot Tip Explants.

    Science.gov (United States)

    Abahmane, Larbi

    2017-01-01

    A number of public and private laboratories are working on date palm micropropagation to meet the increasing worldwide demand for date palm planting material. A standardized direct organogenesis protocol exists for the production of date palm plantlets to maintain the genetic fidelity of regenerated plants. Organogenesis has the advantage of using low concentrations of plant growth regulators and avoiding the callus phase. In addition, direct regeneration of vegetative buds minimizes the risk of somaclonal variation among plant regenerants. However, in vitro multiplication cycles should be limited in duration by frequent renewal of plant material. This chapter describes a simple and routine organogenesis protocol for date palm multiplication using shoot tip explants.

  11. Explant culture of rat colon: A model system for studying metabolism of chemical carcinogens

    DEFF Research Database (Denmark)

    Autrup, Herman; Stoner, G.D.; Jackson, F.

    1978-01-01

    . The explants were incubated at 30 degrees C. The viability of the tissue was measured both by incorporation of specific precursors into cellular macromolecules and by monitoring of tissue morphology with light and electron microscopy. Cultured rat colon was able to metabolize benzo[alpha]pyrene, 7.......5% bovine albumin or 5% fetal bovine serum. The dishes were placed in a controlled-atmosphere chamber which was gassed with 95% O2 and 5% CO2. The chamber then was placed on a rocker platform which rocked at 10 cycles per min causing the medium to flow intermittently over the epithelial surface...

  12. Mercury-induced ethylene formation and abscission in Citrus and Coleus explants

    Energy Technology Data Exchange (ETDEWEB)

    Goren, R.; Siegel, S.M.

    1976-04-01

    Mercury vapor induces ethylene formation and abscission in Citrus and Coleus explants. Both responses are markedly greater in the absence of CO/sub 2/. The stimulation of these metabolically complex processes indicates that the action of mercury vapor is not consistent with the more popular conception of mercury toxicity. This was manifested in its complete failure to disturb respiratory gas exchange, and in the total absence of any necrosis. Accordingly, the effect of mercury appears to be highly specific. The overall significance of these findings is discussed with respect to physiological, environmental, and methodological aspects.

  13. Influence de la taille des vitroplants et du type d'explant sur la ...

    African Journals Online (AJOL)

    L'aptitude à la callogenèse chez le cotonnier a été estimée par le taux d'induction et le poids sec des cals en fonction de la source de l'explant et de la taille des plantules. Le taux d'induction des cals augmente avec la taille des plantules alors que le poids sec des cals ; résultat de la croissance des cals, évolue en sens ...

  14. Organotypic Culture of Breast Tumor Explants as a Multicellular System for the Screening of Natural Compounds with Antineoplastic Potential

    Directory of Open Access Journals (Sweden)

    Irma Edith Carranza-Torres

    2015-01-01

    Full Text Available Breast cancer is the leading cause of death in women worldwide. The search for novel compounds with antitumor activity, with less adverse effects and higher efficacy, and the development of methods to evaluate their toxicity is an area of ​​intense research. In this study we implemented the preparation and culture of breast tumor explants, which were obtained from precision-cut breast tumor slices. In order to validate the model we are proposing to screen antineoplastic effect of natural compounds, we selected caffeic acid, ursolic acid, and rosmarinic acid. Using the Krumdieck tissue slicer, precision-cut tissue slices were prepared from breast cancer samples; from these slices, 4 mm explants were obtained and incubated with the selected compounds. Viability was assessed by Alamar Blue assay, LDH release, and histopathological criteria. Results showed that the viability of the explants cultured in the presence of paclitaxel (positive control decreased significantly (P<0.05; however, tumor samples responded differently to each compound. When the explants were coincubated with paclitaxel and compounds, a synergic effect was observed. This study shows that ex vivo culture of breast cancer explants offers a suitable alternative model for evaluating natural or synthetic compounds with antitumor properties within the complex microenvironment of the tumor.

  15. Personalization of the medical treatment of solid tumours using patient-derived tumour explants (Review).

    Science.gov (United States)

    Louandre, Christophe; Donnadieu, Jérome; Lachaier, Emma; Page, Cyril; Chauffert, Bruno; Galmiche, Antoine

    2016-03-01

    Improving the pre-clinical characterization of therapeutic approaches and developing new biological assays that will enable treatment personalization for individual patients are promising developments in oncology. Here we describe a new approach consisting of culturing human tumour explants. This approach involves the preparation of slices from freshly-obtained, surgically-resected material that can be maintained ex vivo for several days. Recent studies have provided proof of principle that this approach can be easily implemented in order to explore the mode of action of various anticancer drugs and the responses of 'real' tumours at the individual patient level. We present the practical aspects and highlight the versatility of this approach, which allows for the analysis of the susceptibility of any individual tumour to multiple anticancer drugs in parallel. We discuss its potential as a companion assay in the design of optimal clinical trials and as a guide for the prescription of medical treatment. We discuss which future clinical and biological studies are needed to validate the information gathered from cultured tumour explants, and to integrate this information with that gathered from other assays in order to optimize the medical treatment of cancer.

  16. FluoroGold-Labeled Organotypic Retinal Explant Culture for Neurotoxicity Screening Studies

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    Adrian Smedowski

    2018-01-01

    Full Text Available Preclinical toxicity screening of the new retinal compounds is an absolute requirement in the pathway of further drug development. Since retinal neuron cultivation and in vivo studies are relatively expensive and time consuming, we aimed to create a fast and reproducible ex vivo system for retinal toxicity screening. For this purpose, we used rat retinal explant culture that was retrogradely labeled with the FluoroGold before the isolation. Explants were exposed to a toxic concentration of gentamicin and ciliary neurotrophic factor (CNTF, a known neuroprotective agent. The measured outcomes showed the cell density in retinal ganglion cell layer (GCL and the activity of lactate dehydrogenase (LDH in the culture medium. Gentamicin-induced oxidative stress resulted in retinal cell damage and rapid LDH release to the culture medium (p0.05. As well as this, the LDH activity was directly correlated with the cell count in GCL (R=−0.84, p<0.00001, making a sensitive marker of retinal neuron damage. The FLOREC protocol could be considered as a fast, reproducible, and sensitive method to detect neurotoxicity in the screening studies of the retinal drugs.

  17. Effect of explant density and medium culture volumes on cassava micropropagation in Temporal Immersion System

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    Milagros Basail

    2003-04-01

    Full Text Available Due to the need of producing high quality planting material available to cassava growers, it has been necessary to look for alternatives in order to increase the efficiancy of in vitro propagation methods and their automation, such as the use of the Temporal Immersion Systems (RITA®. This work was carried out to increase the multiplication coefficient for cassava mass propagation through out Temporal Immersion Systems. The clone ‘CMC-40’ was used. Different medium volumes per explant, and material density per unit at a given Immersion frequency were tested. The highest results were obtained in the 2.8 multiplication coefficient with 20 ml culture medium volume and 3.2 using a density of 40 explants/flask. When the Temporal Immersion System is used with these results, a more efficient method for cassava micropropagation is established and also higher quality vitroplants for the rooting stage and further acclimatization in field conditions are produced. Key Words: Tissue Culture, liquid culture medium, Manihot esculenta Crantz

  18. Propagation of Indian Rhubarh (Rheum emodi Wall.) using shoot-tip and leaf explant culture.

    Science.gov (United States)

    Lal, N; Ahuja, P S

    1989-12-01

    Shoot-tip explants of Rheum emodi Wall. (Polygonaceae) gave rise to multiple shoots when cultured on a Murashige and Skoog (1962) medium (MS) with 2.0 mg/l 6-benzylaminopurine (BAP) and 1.0 mg/l indole-3-butyric acid (IBA). Also, shoot buds developed from leaf explants using MS medium with 2.0 mg/l BAP and 0.25 to 1.0 mg/l indole-3-acetic acid (IAA) or IBA. Roots were induced when the resulting shoots were placed on MS medium with 1.0 mg/l IBA. Both regeneration procedures gave rise to healthy plantlets that were established in soil under glasshouse conditions at 80% frequency after hardening phase of two weeks. Regenerated plants showed a constant chromosome number of 2n=2x=22, same as the parent plant. The use of liquid shake cultures minimized the time and culture medium requirements for propagation. This procedure can be applied for the conservation and utilization of elite clones of R. emodi.

  19. Two-Stage Explantation of a Magnetic Lower Esophageal Sphincter Augmentation Device Due to Esophageal Erosion.

    Science.gov (United States)

    Parmar, Abhishek D; Tessler, Robert A; Chang, Howard Y; Svahn, Jonathan D

    2017-08-01

    Implanting a magnetic lower esophageal sphincter augmentation device (LINX, Torax Medical) has become an increasingly common option in the surgical management of gastroesophageal reflux disease. As the enthusiasm for placing this device increases, experience in the management of device-related complications-including erosion-is necessary. We report a staged approach to LINX removal in a 64-year-old female with symptoms of odynophagia secondary to partial erosion of a LINX device into the esophagus. The patient had a 12-bead LINX device placed in 2011 at an outside, international facility. In late 2013, she began experiencing symptoms of odynophagia. An esophagogastroduodenoscopy at our institution in October 2015 demonstrated two metallic beads eroding through the distal esophageal lumen. An elective endoscopic removal of the two visible beads was performed. A postoperative esophagram confirmed that there was no resulting esophageal perforation. The patient noted mild improvement in her symptoms. After a 12-week period to allow for complete healing, the remaining 10 beads of the LINX device were explanted laparoscopically without complication. No further procedures were undertaken. At 2 months' follow-up, the patient noted complete resolution of her symptoms. Transmural erosion of the LINX device into the esophageal lumen is a rare occurrence, with only five such complications reported in the published literature. We present the first account of LINX explantation for esophageal erosion in the United States. We demonstrated that a staged laparoendoscopic approach to LINX removal in these cases is feasible with minimal morbidity.

  20. Increased proliferation of explanted vascular smooth muscle cells: a marker presaging atherogenesis.

    Science.gov (United States)

    Absher, P M; Schneider, D J; Baldor, L C; Russell, J C; Sobel, B E

    1997-06-01

    The JCR:LA-cp homozygous cp/cp corpulent rat is genetically predisposed to develop atherosclerosis evident after 9 and 18 months of age in males and females and to manifest metabolic derangements resembling those seen in type II diabetes in humans (hyperinsulinemia, insulin resistance, hyperglycemia and hypertriglyceridemia). The present study was undertaken to determine whether vascular smooth muscle cells (SMCs) explanted from vessels destined to become atherosclerotic later in life exhibit intrinsic properties ex vivo that presage atherogenesis to provide a means for evaluating promptly intervention designed to modify it. SMCs were cultured from aortic explants of JCR:LA-cp corpulent (cp/cp) and lean control (+/+) rats of 4, 5, 6, and 9 months of age. Compared with SMCs from controls, SMCs from cp/cp rats exhibited increased proliferation, higher saturation density, increased augmentation of proliferation in response to selected mitogens and greater adherence to extracellular matrix proteins. The increased proliferative activity ex vivo anteceded by several months the development of atherosclerotic lesions in vivo. Thus, it is a promising marker in assessments of the efficacy of interventions designed to retard or prevent atherosclerosis.

  1. Induction of Tetraploids from Petiole Explants through Colchicine Treatments in Echinacea purpurea L.

    Directory of Open Access Journals (Sweden)

    Dahanayake Nilanthi

    2009-01-01

    Full Text Available Petiole explants were obtained from in vitro grown diploid (2x=22 Echinacea purpurea plantlets. Shoots were regenerated by culturing the explants on MS basal medium containing 0.3 mg/L benzyladenine (BA, 0.01 mg/L naphthaleneacetic acid (NAA and four concentrations (30, 60, 120, and 240 mg/L of colchicine for 30 days, or 120 mg/L of colchicine for various durations (7, 14, 21, and 28 days. The regenerated shoots were induced to root on MS basal medium with 0.01 mg/L NAA, and then the root-tips of the regenerated shoots were sampled for count of chromosome number. It was found that a treatment duration of >7 days was necessary for induction of tetraploid (4x=44 shoots, and treatment with 120 mg/L colchicine for 28 days was the most efficient for induction of tetraploids, yielding 23.5% of tetraploids among all the regenerated shoots. Chimeras were observed in almost all the treatments. However, the ratio of tetraploid to diploid cells in a chimeric plant was usually low. In comparison with diploid plants, tetraploid plants in vitro had larger stomata and thicker roots with more root branches, and had prominently shorter inflorescence stalk when mature.

  2. Utilization of Aseptic Seedling Explants for In vitro Propagation of Indian Red Wood

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    Kishore Kumar CHIRUVELLA

    2013-12-01

    Full Text Available Micropropagation has been advocated as one of the most viable biotechnological tool for ex situ conservation of rare, endangered endemic medicinal plants germplasm. Rapid clonal micropropagation protocol for large-scale multiplication of an endemic medicinal plant Soymida febrifuga (Meliaceae was established from 15-day aseptic seedling cotyledonary node and shoot tip explants. High frequency of sprouting and shoot differentiation was observed from cotyledonary node explants compared to shoot tip, on Murashige and Skoog (MS medium fortified with BA, KN, 2-iP and CM. Of the cytokinins used, BA (3.0 mgl-1 supported highest average number and maximum multiple shoot differentiation (16.6. In vitro proliferated shoots were multiplied rapidly by culturing nodal segments as microcuttings, further subcultured on the same media for elongation. Elongated shoots upon transfer to MS medium fortified with IBA showed rooting within two weeks of culture. Rooted plantlets were successfully hardened and 75% of rooted shoots successfully survived on establishment to the soil. Plants looked healthy with no visually detectable phenotypic variations. This protocol provides a successful and rapid technique that can be used for ex situ conservation minimizing the pressure on wild populations and contributes to the conservation of this endemic medicinally potent flora.

  3. Ultrastructural Localization of Polygalacturonase in Ethylene-Stimulated Abscission of Tomato Pedicel Explants

    Directory of Open Access Journals (Sweden)

    Ming-Fang Qi

    2014-01-01

    Full Text Available Polygalacturonase (PG is crucial in plant organ abscission process. This paper investigated the cellular and subcellular localization of PG in ethylene-stimulated abscission of tomato pedicel explants. Confocal laser scanning microscopy of abscission zone sections with the fluorescent probe Cy3 revealed that PG was initially accumulated in parenchyma cells in cortical and vascular tissues after 8 h of ethylene treatment and then extended throughout the abscission zone when the abscission zone separated at 24 h after ethylene treatment. At the subcellular level, transmission electron microscopy with immunogold staining showed that PG showed abundant accumulation in the cortical and vascular tissues at 8 h after ethylene treatment, and the distribution area extended to the central parenchyma cells at 16 h after ethylene treatment. In addition, PGs were observed in the distal and proximal parts of the tomato pedicel explants throughout the abscission process. The results provided a visualized distribution of PG in the pedicel abscission zone and proved that PG was closely related to abscission.

  4. In vitro direct shoot regeneration from proximal, middle and distal segment of Coleus forskohlii leaf explants.

    Science.gov (United States)

    Krishna, Gaurav; Sairam Reddy, P; Anoop Nair, N; Ramteke, P W; Bhattacharya, P S

    2010-04-01

    Coleus forskohlii is an endangered multipurpose medicinal plant that has widespread applications. In spite of this, there have been few attempts to propagate its cultivation in India. The present communication presents an in vitro rapid regeneration method using leaf explants of Coleus forskohlii through direct organogenesis. Leaf explants that were excised into three different segments i.e. proximal (P), middle (M) and distal (D) were cultured on Murashige and Skoog (MS) basal medium supplemented with cytokinins. MS Media containing 5.0 mg L(-1) BAP (6-Benzylaminopurine) promoted regeneration of multiple shoots through direct organogenesis from the leaf, which were further elongated on MS media augmented with 0.1 mg L-1 BAP and 0.1 mg L(-1) IAA (Indole-3-acetic acid), cytokinin and auxin combination. Regenerated and elongated shoots, when transferred to ose resulted in profuse rooting plants that were transferred to soil after acclimatization and maintained in a green house. The current protocol offers a direct, mass propagation method bypassing the callus phase of C. forskohlii and is suitable for conservation, large-scale commercial cultivation, and genetic transformation with agronomically desirable traits.

  5. Neisseria gonorrhoeae Challenge Increases Matrix Metalloproteinase-8 Expression in Fallopian Tube Explants

    Directory of Open Access Journals (Sweden)

    Natalia E. Juica

    2017-09-01

    Full Text Available Background:Neisseria gonorrhoeae (Ngo is the etiological agent of gonorrhea, a sexually transmitted infection that initially infects the female lower genital tract. In untreated women, the bacteria can ascend to the upper genital reproductive tract and infect the fallopian tube (FTs, which is associated with salpingitis and can lead to impaired FT function and infertility. The extracellular matrix (ECM plays an important role in cell migration and differentiation in the female genital tract, and some pathogens modify the ECM to establish successful infections. The ECM is regulated by matrix metalloproteinases (MMPs and tissue inhibitors of metalloproteinases (TIMPs, their endogenous inhibitors; MMP deregulation causes pathological conditions in a variety of tissues.Results: The aim of this work was to analyze the expression and localization of MMP-3, MMP-8, MMP-9, and TIMP-1 in FT explants during Ngo infection using real-time PCR, immunohistochemistry, zymography and ELISA. No significant variations in MMP-3, MMP-9, and TIMP-1 transcript levels were observed. In contrast, a significant increase (p < 0.05 was observed for MMP-8 expression and was accompanied by stromal immunoreactivity in infected explants. ELISA results supported these findings and showed that MMP-8 release increased upon gonococcal infection.Conclusions: Our results indicate that gonococcal infection induces increased MMP-8 expression, which might contribute to FT damage during infection.

  6. Effect of Genotype, Explant and Hormonal Concentration on in Vitro Response of Eggplant

    Directory of Open Access Journals (Sweden)

    Dhavala V. N. CHAKRAVARTHI

    2010-09-01

    Full Text Available Three wild relatives Solanum: S. trilobatum L. (P1, S. indicum L. (P2, S. surattense Burm F. (P3 and four cultivars of S. melongena L.: �Pusa Kranthi� (P4, �Pusa purple long� (P5, �Pusa purple cluster� (P6 and �Green round� (P7 were evaluated for their callus induction frequency, total callus quantity, embryogenic callus quantity and plant regeneration ability. The present experiment revealed that in vitro response was significantly affected by the genotype, hormonal concentration and age of the explants. Cotyledon at walking stick stage of explants responded more than the opened leaves stage in all genotypes except in P1. Out of seven genotypes, P3 exhibited the highest callus induction, E-callus and regeneration response followed by P5 while highest total callus quantity obtained in P4. MS medium supplemented with 2 mg/l NAA+2.5 mg/l BAP was optimum for callus initiation, total callus quantity and embryogenic callus formation, and MS medium supplemented with 2.5 mg/l each BAP and KN were optimum for regeneration. Rooting of shoots occurred on half strength MS medium supplemented with 1 and 1.5 mg/l IBA respectively. The rooted plant lets were well accomplished with a survival frequency of 85.5�5%. Moreover, there were no phenotypic differences observed between the in vitro regenerated and in vivo plantlets.

  7. Effect of antibiotics selection on survival rate of nodal explant and gene transformation in Anthurium andraeanum cv. Sonate

    Directory of Open Access Journals (Sweden)

    Te-chato, S.

    2007-05-01

    Full Text Available The effects of various concentrations of hygromycin antibiotic supplemented in modified Murashige and Skoog (MMS on the survival rate of nodal explant of Anthurium andraeanum cv. Sonate were determined.The use of hygromycin at 50 mg/l caused absolute death of nodal tissue after 4 weeks of culture. Dipping nodal explant with agrobacterium, EHA 105 containing pCAMBIA1301 for 15 min followed by co-cultureon filter paper laid on MMS with 0.5 mg/l benzyladenine (BA, 0.5 mg/l thidiazuron (TDZ and 200 mg/l cefotaxime for 2 days then transferring the explant to culture on MMS supplemented with the abovephytohormones and 50 mg/l hygromycin resulted in the highest survival rate at 26.6% with 4 shoots/callus. Histochemical analysis of gus activity was found in callus after 6 weeks of nodal culture and in leaf fromshoot derived from the callus.

  8. Differentiation of presumptive primordial germ cell (pPGC)-like cells in explants into PGCs in experimental tadpoles

    Energy Technology Data Exchange (ETDEWEB)

    Ikenishi, K.; Okuda, T.; Nakazato, S.

    1984-05-01

    A single blastomere containing the ''germ plasm'' of 32-cell stage Xenopus embryos was cultured with (/sup 3/H)thymidine until the control embryos developed to the neurula stage. The explants, showing a spherical mass in which the nuclei of all cells were labeled, were implanted into the prospective place of presumptive primordial germ cells (pPGCs) in the endodermal cell mass of unlabeled host embryos of the neurula stage. Labeled PGCs as well as unlabeled, host PGCs were found in the genital ridges of experimental tadpoles. This indicates that the precursor of germ cells, corresponding to pPGCs in normal embryos of the neurula stage, in the explants migrated to genital ridges just at the right moment to become PGCs, and suggests that the developmental process progressed normally, even in the explants, as far as the differentiation of pPGCs is concerned.

  9. Antiviral activity of tiazofurin and mycophenolic acid against Grapevine leafroll-associated virus 3 in Vitis vinifera explants.

    Science.gov (United States)

    Panattoni, A; D'Anna, F; Triolo, E

    2007-03-01

    The ability to control plant viral diseases with chemicals has great potential value for agriculture, but few chemicals are available to date due to the difficulty in obtaining effective drugs. IMP dehydrogenase is an enzyme which catalyzes the conversion of inosine 5'-monophosphate to xanthosine 5'-monophosphate in the de novo purine nucleotide synthetic pathway, and is considered a sensitive target for antiviral drugs. Two IMPDH inhibitors, tiazofurin (TR) and mycophenolic acid (MPA), were tested for their inhibitory effect on Grapevine leafroll-associated virus 3 (GLRaV-3) in in vitro grapevine explants. TR administration produced plantlets characterized by negative ELISA readings. No PCR products were obtained from these samples. This was confirmed by the absence of viral particles. MPA was essentially ineffective against GLRaV-3 replication in Sangiovese explants. This is the first report of GLRaV-3 eradication in grapevine explants following TR administration.

  10. Opacification of hydrophilic MemoryLens U940A intraocular lenses: analysis of 2 explanted lenses.

    Science.gov (United States)

    Mattová, Jana; Bohácová, Eulália; Murgasová, Zuzana; Kadlec, Róbert; Forgác, Frantisek; Klobusická, Erika; Durcanský, Dusan

    2004-09-01

    To determine the rate of opacification of hydrophilic MemoryLens U940A intraocular lenses (IOLs) (Mentor Ophthalmics, Inc.) in the given cohort and perform a histopathological and spectrophotometer analysis of 2 explanted opacified IOLs. Ophthalmology Department, Faculty Hospital, Nitra, Slovakia. This retrospective study comprised 182 patients (205 eyes) who had implantation of a MemoryLens U940A IOL from June 1997 to June 2000. The patients were examined using a slitlamp to detect the presence of IOL opacification. In 4 cases, the lenses were explanted because of significant opacification and patient-reported problems; 2 lenses were provided for further analysis. One unused reference MemoryLens U940A IOL was also evaluated. All IOL were stained with von Kossa to determine the presence of calcium in the opacification. To confirm the components presence of an ultraviolet (UV) absorber, the IOLs were examined with an Avatar 330 Fourier transfer infrared (IR) spectroscope and a UV visible spectrophotometer (Philips). The IR spectrums for the IOL were identified using an IR spectrum atlas. The opacified IOLs, reference IOL, and the IOL packaging were further examined to determine the presence of silicone. Various amounts of opacification were found on the MemoryLens U940A IOL in 30 eyes (30 patients) (14.63%). Two explanted IOLs were positive for von Kossa staining, proving the presence of calcium deposits; the reference lens staining was negative. Spectrophotometry showed that the reference IOL and opacified IOLs were of the same polymer. The presence of the UV absorber on the benzophenone base was seen in the reference lens but not the opacified IOLs. In contrast, an increased concentration of low-molecular-weight components generated during the degradation of the polymer was present in the opacified lenses. The white cover pf the IOL is of polydimethyl siloxane, a silicone rubber. However, no silicone rubber was present in any examined lens, perhaps because the

  11. Identification of a benzyladenine disaccharide conjugate produced during shoot organogenesis in Petunia leaf explants

    Energy Technology Data Exchange (ETDEWEB)

    Auer, C.A. (Univ. of Connecticute, Storrs, CT (United States)); Cohen, J.D. (Beltsville Agricultural Research Ctr., MD (United States))

    1993-06-01

    Prior studies of benzyladenine (BA) metabolism in Petunia hybrida Vilm. leaf explants during shoot organogenesis revealed the presence of an abundant unidentified BA conjugate. The BA conjugate, compound C, made up to 39% of the total pool of BA conjugates in two Petunia lines and was associated with an increased shoot organogenic response when compared with a third Petunia line that did not produce any compound C. Structural analysis of compound C utilizing fast atom bombardment mass spectrometry, two methods of carbohydrate analysis, ultraviolet absorption spectra, and Fourier transform infrared spectra identified it as a new cytokinin conjugate, 6-benzylamino-9-[O-glucopyranosyl-(1[yields]3)-ribofuranosyl]-purine. Based on our prior biological studies and the similarity of this compound to related cytokinin conjugates, this disaccharide cytokinin conjugate may be part of the interconvertible pool of cytokinins active in Petunia shoot organogenesis. 17 refs., 2 figs., 2 tabs.

  12. Chemical and Structural Characterization of Several Mid-Term Explanted Breast Prostheses

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    Angela Amoresano

    2016-08-01

    Full Text Available The recent scandal of poly implant prostheses (PIP, which were found in some cases to be made of non-medical grade silicone (as reported by the European Scientific Committee on Emerging and Newly Identified Health Risks, had a great social impact. Thousands of patients asked for implant removal with significant costs for public health care systems. We analysed, by a multidisciplinary approach, sixteen different breast implants after explantation by using several analytical and structural techniques, such as Fourier Transform infrared spectroscopy (FT-IR, mass spectrometry equipped by ion coupled plasma (ICP-MS, gas-chromatography (GC-MS, and tensile testing. Traces of organic (fatty acid and inorganic (Fe, Cr, Pt, Na, and other metals substances were found in all samples, and, even if these values are under danger threshold levels, our study results highlight the possibility of bioaccumulation and tissue contamination, implying the need for continuous medical surveillance and monitoring of material aging.

  13. Freezing preservation of the mammalian cardiac explant. V. Cryoprotection by ethanol.

    Science.gov (United States)

    Wang, T; Banker, M C; Claydon, M; Hicks, G L; Layne, J R

    1992-08-01

    We studied the colligative cryoprotective effect of ethanol (EtOH) in preserving the isolated rat heart frozen at -3.4 degrees C or unfrozen at -1.4 degrees C. Addition of 4.7% (v/v) EtOH to a cardioplegic solution, CP-14, raised the osmolality from 280 to 1100 mOsm/kg H2O and lowered the melting point from -0.52 to -2.1 degrees C. Freezing of the cardiac explant at -3.4 degrees C for 6 h resulted in 34.3 +/- 1.9% of the tissue water as ice; recovery of cardiac output (CO) was 50%. Polyethylene glycol, which at 5% (w/v) has been shown to cryoprotect the hearts during freezing at -1.4 degrees C, did not improve the protective effect of 4.7% EtOH. CP-14 + 4.7% EtOH did not freeze at -1.4 degrees C. After 6 h storage, CO in hearts flushed with CP-14 + 4.7% EtOH oxygenated with 95% O2/5%CO2 returned to almost control level and was much higher than that in hearts flushed with 100% O2 saturated-CP-14 + 4.7% EtOH. Storage of 8 and 12 h reduced CO to 87 +/- 9 and 60 +/- 5% of control. By employing EtOH as a colligative cryoprotectant, we preserved the adult mammalian heart frozen at -3.4 degrees C or unfrozen at -1.4 degrees C, suggesting that this small molecular weight, penetrating substance may be a suitable cryoprotectant for long-term storage of the cardiac explant at high subzero temperatures.

  14. Callus induction and biomass accumulation in vitro in explants from chokeberry (Aronia melanocarpa (Michx. Elliot fruit

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    Tatiana I. Calalb

    2014-09-01

    Full Text Available In this study, the following features were determined: biological (the optimal histogen as explant and the optimal age of donor fruit, biotechnological (type, dosage and combination of growth regulators supplements in culture medium Murashige and Skoog as well as sucrose dosage, and physical (light regime, to induce callusing and biomass accumulation in vitro from the succulent chokeberry (Aronia melanocarpa (Michx. Elliot fruit. It turned out that it was much easier to induce callus from explants composed of the epicarp and hypoderm cut from fruits at 50–60 days after flowering. The role of light regime and varied supplementation of the basic MS medium with different doses of growth regulators was established; they resulted in four pigmented carpomass: violet, cream-pink, cream-white and green. The best combinations for the proliferation of fruit callus were culture media with 0.2–2.5 mg × dm-3 2,4-D+0.5 mg × dm-3 KIN +60 g × dm-3sucrose, while for fruit biomass accumulation enriched with phenolic substances – 2.5–3.5 mg × dm-3 NAA+0.5 mg × dm-3 KIN+60 g × dm-3sucrose. The chemical study of phenolic compounds by HPLC coupled with the mass spectrometry method identified chlorogenic acid, hiperozide, quercetrin, isoquercitrin and rutozide quantitatively and qualitatively in all pigmented carpomass and fruits; an exception is p-coumaric present only qualitatively in green carpomass and absent in fruit and quercetol absent in green carpomass.

  15. Effects of High Hydrostatic Pressure on Bacterial Growth on Human Ossicles Explanted from Cholesteatoma Patients

    Science.gov (United States)

    Ostwald, Jürgen; Lindner, Tobias; Zautner, Andreas Erich; Arndt, Kathleen; Pau, Hans Wilhelm; Podbielski, Andreas

    2012-01-01

    Background High hydrostatic pressure (HHP) treatment can eliminate cholesteatoma cells from explanted human ossicles prior to re-insertion. We analyzed the effects of HHP treatment on the microbial flora on ossicles and on the planktonic and biofilm states of selected isolates. Methodology Twenty-six ossicles were explanted from cholesteatoma patients. Five ossicles were directly analyzed for microbial growth without further treatment. Fifteen ossicles were cut into two pieces. One piece was exposed to HHP of 350 MPa for 10 minutes. Both the treated and untreated (control) pieces were then assessed semi-quantitatively. Three ossicles were cut into two pieces and exposed to identical pressure conditions with or without the addition of one of two different combinations of antibiotics to the medium. Differential effects of 10-minute in vitro exposure of planktonic and biofilm bacteria to pressures of 100 MPa, 250 MPa, 400 MPa and 540 MPa in isotonic and hypotonic media were analyzed using two patient isolates of Staphylococcus epidermidis and Neisseria subflava. Bacterial cell inactivation and biofilm destruction were assessed by colony counting and electron microscopy. Principal Findings A variety of microorganisms were isolated from the ossicles. Irrespective of the medium, HHP treatment at 350 MPa for 10 minutes led to satisfying but incomplete inactivation especially of Gram-negative bacteria. The addition of antibiotics increased the efficacy of elimination. A comparison of HHP treatment of planktonic and biofilm cells showed that the effects of HPP were reduced by about one decadic logarithmic unit when HPP was applied to biofilms. High hydrostatic pressure conditions that are suitable to inactivate cholesteatoma cells fail to completely sterilize ossicles even if antibiotics are added. As a result of the reduced microbial load and the viability loss of surviving bacteria, however, there is a lower risk of re-infection after re-insertion. PMID:22291908

  16. Effects of high hydrostatic pressure on bacterial growth on human ossicles explanted from cholesteatoma patients.

    Directory of Open Access Journals (Sweden)

    Steffen Dommerich

    Full Text Available BACKGROUND: High hydrostatic pressure (HHP treatment can eliminate cholesteatoma cells from explanted human ossicles prior to re-insertion. We analyzed the effects of HHP treatment on the microbial flora on ossicles and on the planktonic and biofilm states of selected isolates. METHODOLOGY: Twenty-six ossicles were explanted from cholesteatoma patients. Five ossicles were directly analyzed for microbial growth without further treatment. Fifteen ossicles were cut into two pieces. One piece was exposed to HHP of 350 MPa for 10 minutes. Both the treated and untreated (control pieces were then assessed semi-quantitatively. Three ossicles were cut into two pieces and exposed to identical pressure conditions with or without the addition of one of two different combinations of antibiotics to the medium. Differential effects of 10-minute in vitro exposure of planktonic and biofilm bacteria to pressures of 100 MPa, 250 MPa, 400 MPa and 540 MPa in isotonic and hypotonic media were analyzed using two patient isolates of Staphylococcus epidermidis and Neisseria subflava. Bacterial cell inactivation and biofilm destruction were assessed by colony counting and electron microscopy. PRINCIPAL FINDINGS: A variety of microorganisms were isolated from the ossicles. Irrespective of the medium, HHP treatment at 350 MPa for 10 minutes led to satisfying but incomplete inactivation especially of gram-negative bacteria. The addition of antibiotics increased the efficacy of elimination. A comparison of HHP treatment of planktonic and biofilm cells showed that the effects of HPP were reduced by about one decadic logarithmic unit when HPP was applied to biofilms. High hydrostatic pressure conditions that are suitable to inactivate cholesteatoma cells fail to completely sterilize ossicles even if antibiotics are added. As a result of the reduced microbial load and the viability loss of surviving bacteria, however, there is a lower risk of re-infection after re-insertion.

  17. Characteristic of c-Kit+ progenitor cells in explanted human hearts.

    Science.gov (United States)

    Matuszczak, Sybilla; Czapla, Justyna; Jarosz-Biej, Magdalena; Wiśniewska, Ewa; Cichoń, Tomasz; Smolarczyk, Ryszard; Kobusińska, Magdalena; Gajda, Karolina; Wilczek, Piotr; Sliwka, Joanna; Zembala, Michał; Zembala, Marian; Szala, Stanisław

    2014-09-01

    According to literature data, self-renewing, multipotent, and clonogenic cardiac c-Kit(+) progenitor cells occur within human myocardium. The aim of this study was to isolate and characterize c-Kit(+) progenitor cells from explanted human hearts. Experimental material was obtained from 19 adult and 7 pediatric patients. Successful isolation and culture was achieved for 95 samples (84.1%) derived from five different regions of the heart: right and left ventricles, atrium, intraventricular septum, and apex. The average percentage of c-Kit(+) cells, as assessed by FACS, ranged between 0.7 and 0.9%. In contrast to published data we do not observed statistically significant differences in the number of c-Kit(+) cells between disease-specific groups, parts of the heart or sexes. Nevertheless, c-Kit(+) cells were present in significant numbers (11-24%) in samples derived from three explanted pediatric hearts. c-Kit(+) cells were also positive for CD105 and a majority of them was positive for CD31 and CD34 (83.7 ± 8.6 and 75.7 ± 11.4%, respectively). Immunohistochemical analysis of the heart tissue revealed that most cells possessing the c-Kit antigen were also positive for tryptase, a specific mast cell marker. However, flow cytometry analysis has shown cultured c-Kit(+) cells to be negative for hematopoietic marker CD45 and mast cell marker CD33. Isolated c-Kit(+) cells display mesenchymal stem cell features and are thought to differentiate into endothelial cells.

  18. Interference in adhesion of bacteria and yeasts isolated from explanted voice prostheses to silicone rubber by rhamnolipid biosurfactants

    NARCIS (Netherlands)

    Rodrigues, LR; Banat, IM; van der Mei, HC; Teixeira, JA; Oliveira, R

    Aims: The effects and extent of adhesion of four different bacterial and two yeast strains isolated from explanted voice prostheses to silicone rubber with and without an adsorbed rhamnolipid biosurfactant layer obtained from Pseudomonasaeruginosa DS10-129 was studied. Methods and Results: The

  19. Explant-dependent receptivity to isolation and a cell-wall resynthesis in protoplast culture of recalcitrant yellow lupin

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    Alina WISZNIEWSKA

    2013-03-01

    Full Text Available Cell-wall resynthesis was studied in protoplast culture of yellow lupin (Lupinus luteus L.. We optimized protoplast isolation and found that explants excised from young seedling were more suitable sources of protoplasts, in contrast to callus tissue. Incubation in 2% cellulase R-10, 1% pectinase and 0.5% macerozyme solution for 3h effectively released protoplasts from majority of tested explants. Furthermore, we determined the optimal developmental age of explants which was 4, 21, 25 and 35 days for hypocotyls, cotyledons, in-vitro leaf mesophyll and ex-vitro leaf mesophyll, respectively. Explant type, culture medium and genotype influenced both a rate and a pattern of the cell wall regeneration. After 10 days of culture, the number of regenerated cells reached 44%-59% in hypocotyl, 84%-91% in cotyledonary, and 31%-42% in mesophyll protoplasts. Our results show that the earlier wall regeneration begins, the wall surface will be more incomplete. We suggest that unbalanced and inefficient cell-wall resynthesis likely contributes to recalcitrance of yellow lupin to manipulations in protoplast technology.

  20. Participation of intracellular cysteine proteinases, in particular cathepsin B, in degradation of collagen in periosteal tissue explants

    NARCIS (Netherlands)

    Creemers, L. B.; Hoeben, K. A.; Jansen, D. C.; Buttle, D. J.; Beertsen, W.; Everts, V.

    1998-01-01

    The involvement of cysteine proteinases in the degradation of soft connective tissue collagen was studied in cultured periosteal explants. Using cysteine proteinase inhibitors that were active intracellularly or extracellularly (Ep453 and Ep475, respectively), it was shown that over-all collagen

  1. Chitinase, beta-1,3-glucanase, osmotin, and extensin are expressed in tobacco explants during flower formation

    DEFF Research Database (Denmark)

    Neale, A D; Wahleithner, J A; Lund, Marianne

    1990-01-01

    Sequence analysis of five gene families that were isolated from tobacco thin cell layer explants initiating floral development [Meeks-Wagner et al. (1989). Plant Cell 1, 25-35] showed that two encode the pathogenesis-related proteins basic chitinase and basic beta-1,3-glucanase, while a third...

  2. Development of efficient plant regeneration and transformation system for impatiens using Agrobacterium tumefaciens and multiple bud cultures as explants.

    Science.gov (United States)

    Dan, Yinghui; Baxter, Aaron; Zhang, Song; Pantazis, Christopher J; Veilleux, Richard E

    2010-08-09

    Impatiens (Impatiens walleriana) is a top selling floriculture crop. The potential for genetic transformation of Impatiens to introduce novel flower colors or virus resistance has been limited by its general recalcitrance to tissue culture and transformation manipulations. We have established a regeneration and transformation system for Impatiens that provides new alternatives to genetic improvement of this crop. In a first step towards the development of transgenic INSV-resistant Impatiens, we developed an efficient plant regeneration system using hypocotyl segments containing cotyledonary nodes as explants. With this regeneration system, 80% of explants produced an average of 32.3 elongated shoots per initial explant plated, with up to 167 elongated shoots produced per explant. Rooting efficiency was high, and 100% of shoots produced roots within 12 days under optimal conditions, allowing plant regeneration within approximately 8 weeks. Using this regeneration system, we developed an efficient Agrobacterium-mediated Impatiens transformation method using in vitro multiple bud cultures as explants and a binary plasmid (pHB2892) bearing gfp and nptII genes. Transgenic Impatiens plants, with a frequency up to 58.9%, were obtained within 12 to 16 weeks from inoculation to transfer of transgenic plants to soil. Transgenic plants were confirmed by Southern blot, phenotypic assays and T1 segregation analysis. Transgene expression was observed in leaves, stems, roots, flowers, and fruit. The transgenic plants were fertile and phenotypically normal. We report the development of a simple and efficient Agrobacterium-mediated transformation system for Impatiens. To the best of our knowledge, there have been no reports of Agrobacterium-mediated transformation of Impatiens with experimental evidence of stable integration of T-DNA and of Agrobacterium-mediated transformation method for plants using in vitro maintained multiple bud cultures as explants. This transformation system

  3. Calogênese e rizogênese em explantes de mogno (Swietenia macrophylla King cultivados in vitro.

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    Silvana Cruz da Rocha

    2010-08-01

    Full Text Available A exploração de árvores tropicais realizada de forma indiscriminada, buscando espécies de alto valor econômico, tem levado várias espécies, como o mogno (Swietenia macrophylla King, ao perigo de extinção. O desenvolvimento de uma metodologia de regeneração de gemas, direta ou indireta, poderia auxiliar na obtenção de um grande número de mudas e constituir uma perspectiva à propagação sexuada. Essa última é limitada pelo fato das sementes perderem rapidamente a capacidade germinativa. No presente trabalho, foram utilizados dois tipos de explantes: fragmentos foliares e de raízes de plantas cultivadas in vitro. Após desinfestação, os explantes foram colocados em meio de cultura de Murashige e Skoog (1962 contendo três quartos da concentração de sais, vitaminas do mesmo meio, 30g.L-1 de sacarose, auxina (ácido naftaleno-acético, ANA, 0,11 µM e 0,54 µM, citocinina (cinetina, CIN, 1,2 µM, 2,3 µM, 4,7 µM e 9,3 µM; 6-benziladenina, BA, 2,2 µM, 4,4 µM e 8,8 µM ou 2-isopenteniladenina, 2-iP, 2,5 µM e 7g.L-1 de ágar. As variáveis testadas foram a concentração e o tipo de regulador de crescimento e a origem dos explantes. A cada 30 dias, os explantes foram avaliados pela contagem do número de explantes formando calos ou raízes e a consistência dos calos. Foram obtidos calos a com base nos dois tipos de explantes. Nos explantes foliares, 90% deles formaram calos em meios de cultura contendo BA 4,4 µM com ANA 0,54 µM e BA 8,9 µM com ANA 0,11 ou 0,54 µM. Nos explantes de raízes, a maior percentagem de explantes com calos foi de 55%, no meio de cultura com BA 2,2 µM e ANA 0,54 µM. Raízes adventícias foram obtidas partindo de calos e do limbo dos explantes foliares, em meios de cultura com CIN e ANA. Não foi observada a formação de gemas adventícias.

  4. Blockade of Toll-like receptor 2 prevents spontaneous cytokine release from rheumatoid arthritis ex vivo synovial explant cultures

    LENUS (Irish Health Repository)

    Nic An Ultaigh, Sinead

    2011-02-23

    Abstract Introduction The aim of this study was to examine the effect of blocking Toll-like receptor 2 (TLR2) in rheumatoid arthritis (RA) synovial cells. Methods RA synovial tissue biopsies, obtained under direct visualization at arthroscopy, were established as synovial explant cultures ex vivo or snap frozen for immunohistology. Mononuclear cell cultures were isolated from peripheral blood and synovial fluid of RA patients. Cultures were incubated with the TLR1\\/2 ligand, Pam3CSK4 (200 ng, 1 and 10 μg\\/ml), an anti-TLR2 antibody (OPN301, 1 μg\\/ml) or an immunoglobulin G (IgG) (1 μg\\/ml) matched control. The comparative effect of OPN301 and adalimumab (anti-tumour necrosis factor alpha) on spontaneous release of proinflammatory cytokines from RA synovial explants was determined using quantitative cytokine MSD multiplex assays or ELISA. OPN301 penetration into RA synovial tissue explants cultures was assessed by immunohistology. Results Pam3CSK4 significantly upregulated interleukin (IL)-6 and IL-8 in RA peripheral blood mononuclear cells (PBMCs), RA synovial fluid mononuclear cells (SFMCs) and RA synovial explant cultures (P < 0.05). OPN301 significantly decreased Pam3CSK4-induced cytokine production of tumour necrosis factor alpha (TNF-α), IL-1β, IL-6, interferon (IFN)-γ and IL-8 compared to IgG control in RA PBMCs and SFMCs cultures (all P < 0.05). OPN301 penetration of RA synovial tissue cultures was detected in the lining layer and perivascular regions. OPN301 significantly decreased spontaneous cytokine production of TNF-α, IL-1β, IFN-γ and IL-8 from RA synovial tissue explant cultures (all P < 0.05). Importantly, the inhibitory effect of OPN on spontaneous cytokine secretion was comparable to inhibition by anti-TNFα monoclonal antibody adalimumab. Conclusions These findings further support targeting TLR2 as a potential therapeutic agent for the treatment of RA.

  5. ORGANOGÊNESE INDIRETA A PARTIR DE EXPLANTES FOLIARES E MULTIPLICAÇÃO IN VITRO DE BROTAÇÕES DE Eucalyptus benthamii X Eucalyptus dunnii

    Directory of Open Access Journals (Sweden)

    Yohana de Oliveira-Cauduro

    2014-01-01

    Full Text Available The aims of this research were to evaluate different culture media for indirect organogenesis and shoot multiplication of Eucalyptus benthamii x Eucalyptus dunnii . For organogenesis, leaf explants were used to test the following treatments: two culture media (MS N/2 and JADS supplemented with 0.1 μM 1-naphthaleneacetic acid (NAA and thidiazuron (TDZ (0.1 or 0.5 μ M, with or without PVP- 40 (250 mg L -1 . The percentage of oxidized explants, callus forming explants, explants with anthocyanin,buds, shoots and the shoot number per explant were evaluated. In the multiplication experiment, isolated shoots were cultivated in MS, JADS and WPM media, all supplemented with 1.11 μ M BAP. Four subcultures were carried out every 28 days. In every subculture the explant oxidation, partial or total leaf chlorosis, fresh mass and mean number of shoot per explant were evaluated. The MS N/2 medium supplemented with 0.1 μM NAA and 0.5 μM TDZ promoted the highest rate of organogenesis (8.3% and the culture media MS supplemented with 1.11 μ M BAP the multiplication rate was higher than in the other media, in the first and the second subcultures (9.28 and 9.24, respectively, without differences between the three media in the following subcultures.

  6. Effect of environmental and cultural conditions on medium pH and explant growth performance of Douglas-fir (Pseudotsuga menziesii shoot cultures [version 2; referees: 2 approved

    Directory of Open Access Journals (Sweden)

    Chien-Chih Chen

    2015-05-01

    Full Text Available The medium pH level of plant tissue cultures has been shown to be essential to many aspects of explant development and growth. Sensitivity or tolerance of medium pH change in vitro varies according to specific requirements of individual species. The objectives of this study are to 1 determine medium pH change over time in storage conditions and with presence of explants, 2 evaluate the effects of medium pH change on explant growth performance and 3 assess the effects of adding a pH stabilizer, 2-(N-morpholinoethanesulfonic acid (MES that is commonly used in Douglas-fir micropropagation medium. Vegetative buds were collected in the spring before breaking dormancy from juvenile and mature donor trees for conducting these evaluations. Medium, with or without MES, was pre-adjusted to five pH levels before adding MES, agar and autoclaving. Medium pH changes and explant growth parameters were measured at eight different incubation times. Overall, MES provided a more stable medium pH, relative to starting pH values, under both light and dark storage conditions as well as with presence of explants. A general trend of decreasing medium pH over time was found comparing explants from juvenile and mature donor genotypes. Explant height and weight growth increased over time, but differ among explants from juvenile and mature donor genotypes. Our findings suggest that a 21-day subculture practice may best sustain medium freshness, medium pH level and desirable explant growth.

  7. In vitro plant regeneration of Albizia lebbeck (L. from seed explants

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    S. Perveen

    2013-07-01

    Full Text Available Objectives: An efficient and reproducible regeneration protocol for rapid multiplication of Albizia lebbeck (L. was developed by using intact seed explants.Methods: Murashige and Skoog's (MS medium supplemented with different hormones (BA, Kn, GA3 and TDZ was used for the induction of multiple shoots from the seed explants. Ex-vitro rooting was performed by using pulse treatment method in auxins (IBA and NAA and the complete plantlets were transferred to the field.Results: High frequency direct shoot induction was found in aseptic seed cultures of A. lebbeck on Murashige and Skoog medium supplemented with 5.0 µM TDZ (Thiadiazuron. Seeds were germinated after 7 days of culture and induced maximum 8 shoots from the region adjacent to the apex of the primary shoot of the seedling upto 25 days of incubation. Proliferating shoot cultures with increased shoot length was established by sub-culture of excised sprouting epicotyls on MS medium supplied with reduced concentrations of TDZ. Maximum shoot regeneration frequency (76 % with  highest number of shoots (21 and shoot length (5.1 cm per sprouting epicotyl was observed in the MS medium supplemented with 0.5 µM TDZ after 8 weeks of culture. Different concentrations of Indole-3-butyric acid (IBA and α-naphthalene acetic acid (NAA were tested to determine the optimal conditions for ex-vitro rooting of the microshoots. The best treatment for maximum ex-vitro root induction frequency (81 % was accomplished with IBA (250 µM pulse treatment given to the basal end of the microshoots for 30 min followed by their transfer in plastic cups containing soilrite and eventually established in normal garden soil + soilrite (1:1 with 78 % survival rate. In addition, histological study was undertaken to gain a better understanding of the regenerated shoots from the epicotyl region.Conclusion: The findings will be fruitful in getting a time saving and cost effective protocol for the in vitro propagation of Albizia

  8. Effects of dosage titration of methylprednisolone acetate and triamcinolone acetonide on interleukin-1-conditioned equine articular cartilage explants in vitro.

    Science.gov (United States)

    Dechant, J E; Baxter, G M; Frisbie, D D; Trotter, G W; McIlwraith, C W

    2003-07-01

    Osteoarthritis is a frequent sequela of joint disease, especially with severe injuries or if attempts at therapy are unsuccessful. Negative and positive effects of corticosteroid treatment of articular cartilage have been demonstrated by in vitro and in vivo studies. To assess the metabolic effects of varying dosages of methylprednisolone acetate (MPA) and triamcinolone acetonide (TA) on interleukin-1alpha (IL-1) conditioned equine cartilage explants. Our hypothesis was that lower dosages of corticosteroids would be less detrimental to cartilage metabolism than higher dosages. TA would be less detrimental to cartilage metabolism than MPA. Treatment groups included articular cartilage explants with no IL-1 (control), IL-1 alone, and IL-1 plus 10, 5, 1 and 0.5 mg/ml MPA or 1.2, 0.6, 0.12 and 0.06 mg/ml TA. Explants were labelled with 35SO4 prior to the beginning and end of the experiment to assess glycosaminoglycan (GAG) degradation and synthesis, respectively. Total GAG content in media and explants and total cartilage DNA were also analysed. MPA and TA reduced GAG synthesis compared to control and IL-1 alone. The highest dosage of MPA (10 mg/ml) reduced GAG synthesis less than lower dosages of MPA and all dosages of TA. Compared to IL-1 alone, all dosages of TA and lower dosages of MPA increased GAG degradation. MPA at 10 mg/ml reduced GAG degradation. Both MPA and TA increased media GAG content compared to control and IL-1 explants. Total cartilage GAGs were unchanged with MPA, but reduced with TA, compared with IL-1 alone. Total cartilage DNA was decreased with MPA and increased with TA compared to IL-1 and control explants. MPA and TA did not counteract the negative effects of IL-1 and did not maintain cartilage metabolism at control levels. Lower dosages of MPA and TA were not less detrimental to cartilage metabolism than higher dosages. TA did not appear to be less harmful than MPA on cartilage metabolism. The results of this study differ from the findings of

  9. Effects of carbon ion beam irradiation on the shoot regeneration from in vitro axillary bud explants of the Impatiens hawkeri

    Science.gov (United States)

    Zhou, Libin; Zhou, Libin; Li, Wenjian; Li, Ping; Dong, Xicun; Qu, Ying; Ma, Shuang; Li, Qiang

    Accelerated ion beams is an excellent mutagen in plant breeding which can induce higher mutation frequencies and wider mutation spectrum than those of low linear energy transfer (LET) irradiations, such as X-rays (Okamura et al. 2003, Yamaguchi et al. 2003). Mutation breeding operation of two Saintpaulia ionahta cultivars using the method combining plant tissue culture technique and carbon ion beam irradiations were set out at Institute of Modern Physics from 2005 (Zhou et al. 2006). The effects of 960 MeV carbon ion beam and 8 MeV X-ray irradiations on regenerated shoots of Impatiens hawkeri from another kind of explants named in vitro axillary buds explants were studied recently. The biology endpoints in this study included relative number of roots (RNR), relative length of roots (RLR), relative height of shoots (RHS), relative number of nodes (RNN), survival fraction (SF) and morphology changes in the regenerated shoots. The experimental results showed that carbon ion beams inhibited the root and stem developments of axillary bud explants more severely than X-rays did. And the 50% lethal dose (LD50 ) is about 23.3 Gy for the carbon ion beam and 49.1 Gy for the X-rays, respectively. Relative biological effectiveness (RBE) of Impatiens hawkeri with respect to X-rays according to 50% SF was about two. Secondly, the percentage of shoots regenerated with malformed shoots including curliness, carnification, nicks in all Impatiens hawkeri axillary bud explants irradiated with carbon ion beam at 20 Gy accounted for 55.6%, while the highest number for the 40 Gy X-ray irradiation was 40%. Last, many regenerated shoots whose vascular bundle fused together were obtained only from explants irradiated with carbon ion beams. Based on the results above, it can be concluded that the effect of mutation induction by carbon ion beam irradiation on the axillary explants of Impatiens hawkeri is better than that by X-ray irradiation; and the optimal mutagenic dose varies from 20 Gy

  10. Indução in vitro de raízes adventícias em explantes de salix (Salix humboldtiana Willdenow(Salix humboldtiana Willdenow In vitro induction of adventicious roots in salix (salix humboldtiana willdenow explants

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    Vespasiano Borges de Paiva Neto

    1998-06-01

    Full Text Available With the objective to investigate the in vitro behavior of salix (Salix humboldtiana Willdenow, leaf and nodal segments were inoculated in WPM medium supplemented with NAA and IBA. In leaf explants, the presence of these growth regulators, isolated or in combination, induced the formation of adventicious roots. Root hairs were only observed when NAA was present. In nodal explants, while the combination of 2,68 µM ANA + 2,46 µM IBA induced bud growth, the other treatments (4,92 µM IBA; 2,68µM ANA + 4,92 µM IBA; 5,37 µM ANA + 4,92 µM IBA; 5,37µM ANA induced the formation of adventicious roots.

  11. Plant regeneration of Rhabdadenia Ragonesei (Apocynaceae by in vitro culturing of leaf explants REGENERACIÓN DE PLANTAS DE RHABDADENIA RAGONESEI (APOCYNACEAE POR CULTIVO IN VITRO DE EXPLANTES FOLIARES

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    Eduardo Flachsland

    2010-08-01

    Full Text Available Plants of Rhabdadenia Ragonesei Woodson (Apocynaceae were regenerated in vitro from leaves explants. The procedure employed includes: 1 Surface sterilization of leaves by immersion in 70% ethanol (10 s followed by 1,1%NaOCl (15 min and three wash with sterile distilled water. 2 Callus and buds induction by culture on Murashige and Skoog medium (MS + 3 mg/L benzyladenine (BAP. 3 Subculture of callus and buds on MS + 1 mg/L BAP, and 4 Rooting on MS + 0.5 mg/L naftalenacetic acid Se regeneraron plantas de Rhabdadenia Ragonesei Woodson (Apocynaceae mediante el cultivo in vitro de explantes foliares en condiciones ambientales controladas. El procedimiento consistió en: 1 Desinfección de las hojas por inmersión en etanol al 70% (10 s seguida de Inmersión en NaOCl al 1,1% (15 min y lavado tres veces con agua destilada estéril. 2 Inducción de callos y yemas mediante el cultivo de explantes foliares en el medio de Murashige y Skoog (MS + 3 mg/L de benciladenina (BAP. 3 Subcultivo de callos y yemas en MS + 1 mg/L de BAP y 4 Enraizamiento de los vastagos obtenidos en MS + 0,5 mg/L de ácido naftalenacético

  12. The slice culture method for following development of tooth germs in explant culture.

    Science.gov (United States)

    Alfaqeeh, Sarah A; Tucker, Abigail S

    2013-11-13

    Explant culture allows manipulation of developing organs at specific time points and is therefore an important method for the developmental biologist. For many organs it is difficult to access developing tissue to allow monitoring during ex vivo culture. The slice culture method allows access to tissue so that morphogenetic movements can be followed and specific cell populations can be targeted for manipulation or lineage tracing. In this paper we describe a method of slice culture that has been very successful for culture of tooth germs in a range of species. The method provides excellent access to the tooth germs, which develop at a similar rate to that observed in vivo, surrounded by the other jaw tissues. This allows tissue interactions between the tooth and surrounding tissue to be monitored. Although this paper concentrates on tooth germs, the same protocol can be applied to follow development of a number of other organs, such as salivary glands, Meckel's cartilage, nasal glands, tongue, and ear.

  13. Characterising the effects of in vitro mechanical stimulation on morphogenesis of developing limb explants.

    Science.gov (United States)

    Chandaria, Vikesh V; McGinty, James; Nowlan, Niamh C

    2016-11-07

    Mechanical forces due to fetal movements play an important role in joint shape morphogenesis, and abnormalities of the joints relating to abnormal fetal movements can have long-term health implications. While mechanical stimulation during development has been shown to be important for joint shape, the relationship between the quantity of mechanical stimulation and the growth and shape change of developing cartilage has not been quantified. In this study, we culture embryonic chick limb explants in vitro in order to reveal how the magnitude of applied movement affects key aspects of the developing joint shape. We hypothesise that joint shape is affected by movement magnitude in a dose-dependent manner, and that a movement regime most representative of physiological fetal movements will promote characteristics of normal shape development. Chick hindlimbs harvested at seven days of incubation were cultured for six days, under either static conditions or one of three different dynamic movement regimes, then assessed for joint shape, cell survival and proliferation. We demonstrate that a physiological magnitude of movement in vitro promotes the most normal progression of joint morphogenesis, and that either under-stimulation or over-stimulation has detrimental effects. Providing insight into the optimal level of mechanical stimulation for cartilage growth and morphogenesis is pertinent to gaining a greater understanding of the etiology of conditions such as developmental dysplasia of the hip, and is also valuable for cartilage tissue engineering. Copyright © 2016 The Authors. Published by Elsevier Ltd.. All rights reserved.

  14. Efficient soybean regeneration and Agrobacterium-mediated transformation using a whole cotyledonary node as an explant.

    Science.gov (United States)

    Zhang, Fuli; Chen, Can; Ge, Honglian; Liu, Jinmei; Luo, Yunling; Liu, Kun; Chen, Long; Xu, Kedong; Zhang, Yi; Tan, Guangxuan; Li, Chengwei

    2014-01-01

    An optimized regeneration and Agrobacterium-mediated transformation protocol based on whole cotyledonary node explants was developed in soybean (Glycine max) cultivar Zhong Huang 13. Adding 6-benzylaminopurine (BAP) in a germinating medium could significantly increase regeneration efficiency; the optimal BAP concentration for shoot formation was 0.5 mg/L. The concentrations of plant growth regulators in a shoot induction medium were optimized by the orthogonal test [L9 (3(3))]. The best combination for shoot regeneration was a medium of Murashige & Skoog salts with B5 vitamins (MSB) supplemented with 3.5 mg/L BAP, 0.2 mg/L indole-3-butyric acid (IBA), and 0.2 mg/L kinetin (KT). Under this favorable condition, one node could regenerate 28-30 shoots. Soybean whole cotyledonary nodes were transformed by inoculation with A. tumefaciens strain EHA105 harboring a vector pBI121 containing a β-glucuronidase gene (gus). GUS assay, polymerase chain reaction, and Southern blot analysis indicated that the gus gene was transformed into soybean plants with 23.1% transformation efficiency. Transgenic plants could be obtained within 5-6 weeks, which was about 4 weeks less than that of a traditional single cotyledonary node method. © 2014 International Union of Biochemistry and Molecular Biology, Inc.

  15. Insulin, a key regulator of hormone responsive milk protein synthesis during lactogenesis in murine mammary explants.

    Science.gov (United States)

    Menzies, Karensa K; Lee, Heather J; Lefèvre, Christophe; Ormandy, Christopher J; Macmillan, Keith L; Nicholas, Kevin R

    2010-03-01

    Murine milk protein gene expression requires insulin, hydrocortisone, and prolactin; however, the role of insulin is not well understood. This study, therefore, examined the requirement of insulin for milk protein synthesis. Mammary explants were cultured in various combinations of the lactogenic hormones and global changes in gene expression analysed using Affymetrix microarray. The expression of 164 genes was responsive to insulin, and 18 were involved in protein synthesis at the level of transcription and posttranscription, as well as amino acid uptake and metabolism. The folate receptor gene was increased by fivefold, highlighting a potentially important role for the hormone in folate metabolism, a process that is emerging to be central for protein synthesis. Interestingly, gene expression of two milk protein transcription factors, Stat5a and Elf5, previously identified as key components of prolactin signalling, both showed an essential requirement for insulin. Subsequent experiments in HCll cells confirmed that Stat5a and Elf5 gene expression could be induced in the absence of prolactin but in the presence of insulin. Whereas prolactin plays an essential role in phosphorylating and activating Stat5a, gene expression is only induced when insulin is present. This indicates insulin plays a crucial role in the transcription of the milk protein genes.

  16. In Vitro Shoot Bud Differentiation from Hypocotyl Explants of Chili Peppers (Capsicum annuum L.

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    Owk ANIEL KUMAR

    2010-03-01

    Full Text Available Chili pepper (Capsicum annuum L. is an economically important spice crop in tropical and subtropical countries. In vitro plant regeneration was obtained from 15th day old hypocotyl explants of three chili pepper cultivars (Capsicum annuum L., var. �X-235�, var. �PC-1� and var. �Pusa Jwala�. Among the genotypes of Capsicum L. var. �X-235� responded better than the var. �PC-1� and var. �Pusa Jwala�. MS medium containing BAP (4.0 mg/l and IAA (0.5 mg/l was found to be the best medium for the production of maximum number of shoot buds in all the genotypes of chili pepper i.e., 6.80�0.16 (var. �X-235�, 5.00�0.19 (var. �PC-1� and 4.80�0.12 (var. �Pusa Jwala�. The shoots were rooted on MS medium fortified with IBA (0.5 mg/l. Rooted plants were hardened and transplanted to the soil. The plants showed 80-90% survival during transplantation.

  17. In Vitro Regeneration of Shoots From Nodal Explants of Dendrobium Chrysotoxum Lindl

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    Kaur Saranjeet

    2017-06-01

    Full Text Available Transverse sections (2 mm thickness of stem-nodes from in vitro raised seedlings had morphogenic potential on semisolid and liquid Murashige and Skoog medium supplemented with cytokinins N6-benzyladenine (BA 4.44 μM, furfurylaminopurine (KIN 4.65 μM and auxin α-naphthalene acetic acid (NAA 5.37 μM individually and in combinations. The regeneration response was influenced by both the type of growth regulator and physical state of the medium. The explants produced either shoot buds on cytokinincontaining media or protocorm-like bodies (PLBs on NAA containing media both solid and liquid. More neo-formations were produced on liquid media, especially those containing only NAA. They were formed at nodal and inter-nodal regions. The secondary buds were produced on the surface of primary PLBs. The plantlets were developed on MS medium containing banana homogenate 50 g·dm-3. The current study is the first ever report on successful regeneration of Dendrobium chrysotoxum from stem-node segments.

  18. Micropropagation of Origanum acutidens (HAND.-MAZZ.) IETSWAART Using Stem Node Explants

    Science.gov (United States)

    Yildirim, Mehmet Ugur

    2013-01-01

    Origanum acutidens (HAND.-MAZZ.) IETSWAART is a promising ornamental plant that can be widely used in landscape management. It is endemic to Eastern Anatolian region of Turkey. Tissue culture has not been used to micropropagate it. The study reports stem node explants from one-week-old seedlings of the plant for successful micropropagation. The stem nodes were cultured on MS medium containing 0.6, 1.2, 1.8, and 2.4 mg/L BAP with 0.2 mg/L NAA. Visible effects of culture media on shoot proliferation were recorded. Shoot regeneration rate was maximum on MS medium containing 1.80 mg/L BAP-0.2 mg/L NAA. The micropropagated shoots were rooted on MS medium containing 0.2 mg/L NAA. All microrooted plantlets survived during acclimatisation on peat moss. It was concluded that O. acutidens can be successfully micropropagated under in vitro conditions. PMID:23983625

  19. Proliferation, maturation and germination of Castanea sativa Mill. Somatic embryos originated from leaf explants.

    Science.gov (United States)

    Corredoira, E; Ballester, A; Vieitez, A M

    2003-07-01

    Experiments were performed to determine the influence of proliferation medium on the maintenance of embryogenic competence and on repetitive embryogenesis in Castanea sativa Mill. somatic embryos derived from leaf explants. Somatic embryo proliferation was carried out by both direct secondary embryogenesis and by the culture of nodular callus tissue originated from cotyledons of somatic embryos. Both systems led to the production of cotyledonary somatic embryos on Murashige and Skoog proliferation medium supplemented with 0.1 mg l-1 benzyladenine and 0.1 mg l-1 naphthaleneacetic acid. Carbon source and concentration had a marked influence on maturation and subsequent germination ability of chestnut somatic embryos. Plantlet conversion was achieved in embryos matured on media with 6 % sucrose, and on 3 or 6 % maltose, whereas mean shoot length, root length and leaf number of produced plants were not significantly affected by these maturation media. Overall, the best results were obtained with 3 % maltose-matured somatic embryos, giving rise to 6 % plant recovery in addition to 33 % of embryos exhibiting only shoot development. The application of a 2-month cold treatment at 4 degrees C to somatic embryos matured on medium with 3 % maltose was necessary for achieving plant conversion, while partial desiccation did not appear to influence this response. A total of 39 % of embryos eventually produced plants either through conversion to plantlets or indirectly through rooting of shoots. Shoots formed by somatic embryos could be excised, multiplied and rooted following the micropropagation procedures previously developed for chestnut.

  20. In vitro neurotoxic effects of 1 GeV/n iron particles assessed in retinal explants.

    Science.gov (United States)

    Vazquez, M E; Kirk, E

    2000-01-01

    The heavy ion component of the cosmic radiation remains problematic to the assessment of risk in manned space flight. The biological effectiveness of HZE particles has yet to be established, particularly with regard to nervous tissue. Using heavy ions accelerated at the AGS of Brookhaven National Laboratory, we study the neurotoxic effects of iron particles. We exposed retinal explants, taken from chick embryos, to determine the dose response relationships for neurite outgrowth. Morphometric techniques were used to evaluate the in vitro effects of 1 GeV/a iron particles (LET 148 keV/micrometer). Iron particles produced a dose-dependent reduction of neurite outgrowth with a maximal effect achieved with a dose of 100 cGy. Doses as low as 10-50 cGy were able to induce reductions of the neurite outgrowth as compared to the control group. Neurite generation is a more sensitive parameter than neurite elongation, suggesting different mechanism of radiation damage in our model. These results showed that low doses/fluences of iron particles could impair the retinal ganglion cells' capacity to generate neurites indicating the highly neurotoxic capability of this heavy charged particle.

  1. Analysis of explanted ePTFE cardiovascular grafts (modified BT shunt)

    Energy Technology Data Exchange (ETDEWEB)

    Doble, Mukesh [Department of Biotechnology, Indian Institute of Technology, Chennai 600 036 (India); Makadia, Nilesh; Pavithran, Sreeja; Kumar, R Suresh [Department of Pediatric Cardiology, Institute of Cardio-Vascular Diseases, Madras Medical Mission, Chennai, 600 037 (India)], E-mail: mukeshd@iitm.ac.in

    2008-09-01

    Structural, chemical, mechanical and surface changes were studied in expanded polytetrafluroethylene vascular grafts explanted from children undergoing planned surgical management of congenital heart disease. These grafts were implanted when recipients were aged 7 days to 8 years (median-48 weeks) and they had been in circulation for a period of 10-52 months (median-74 weeks). While no chemical changes were observed in the shunt, on average the tensile strength had decreased by 50%, total elongation by 61% and crystallinity by 3%. No salt deposits were observed on the surface of the graft. Soluble and insoluble proteins were bound to the polymer surface, which had made the surface hydrophilic. The external surface roughness had increased by 254.5 and the internal surface roughness by 2.6 times the initial value. The fine polymer structure had become fused and clumped. The fusing of strands on the polymer surface became more pronounced with longer duration of implantation. In one instance of previously documented graft stenosis, the heat capacity was found to be more than that of the unimplanted sample, indicating an increase in crystallinity. A longer period of study with a larger sample size would likely shed more light on the relation between physico-chemical changes and graft stenosis.

  2. Morphogenic responses of three explants of Lupinus montanus (H.B.K. cultured in vitro

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    Gabriel Ramírez-González

    2015-01-01

    Full Text Available Resumen El necrosamiento y la respuesta morfogénica de explantes de cotiledón, epicótilo e hipocótilo de plántulas de Lupinus montanus (H.B.K. germinadas in vitro se evaluaron para establecer las condiciones básicas de micropropagación. El necrosamiento se evaluó en medio MS con 0.40 mg·litro-1 de tiamina, 100 mg·litro-1 de mioinositol, 3 % de sacarosa y 7 g·litro-1 de agar-agar y distintas dosis de carbón activado (CA y ácido cítrico. Los tratamientos con CA (50 mg·litro-1 y 100 mg·litro-1 fueron los mejores en el control del necrosamiento (P = 0.001; el cotiledón tuvo mayor nivel de necrosamiento ( 70 % a diferencia del cotiledón, cuya respuesta fue primordialmente callogénica (50 %. El epicótilo cultivado en medio MS con 3.0 μM de AIA y 1.0 μM de BA tuvo el mayor número de brotes (10 y altura (11.4 ± 2.6 cm.

  3. Antimicrobial dressing efficacy against mature Pseudomonas aeruginosa biofilm on porcine skin explants.

    Science.gov (United States)

    Phillips, Priscilla L; Yang, Qingping; Davis, Stephen; Sampson, Edith M; Azeke, John I; Hamad, Afifa; Schultz, Gregory S

    2015-08-01

    An ex vivo porcine skin explant biofilm model that preserves key properties of biofilm attached to skin at different levels of maturity (0-3 days) was used to assess the efficacy of commercially available antimicrobial dressings and topical treatments. Assays were also performed on the subpopulation of antibiotic tolerant biofilm generated by 24 hours of pre-treatment with gentamicin (120× minimal inhibitory concentration) prior to agent exposure. Five types of antimicrobial agents (iodine, silver, polyhexamethylene biguanide, honey and ethanol) and four types of moisture dressings (cotton gauze, sodium carboxymethylcellulose fibre, calcium alginate fibre and cadexomer beads) were assessed. Time-release silver gel and cadexomer iodine dressings were the most effective in reducing mature biofilm [between 5 and 7 logarithmic (log) of 7-log total], whereas all other dressing formulations reduced biofilm between 0·3 and 2 log in 24 or 72 hours with a single exposure. Similar results were found after 24-hour exposure to silver release dressings using an in vivo pig burn wound model, demonstrating correlation between the ex vivo and in vivo models. Results of this study indicate that commonly used microbicidal wound dressings vary widely in their ability to kill mature biofilm and the efficacy is influenced by time of exposure, number of applications, moisture level and agent formulation (sustained release). © 2013 The Authors. International Wound Journal published by Medicalhelplines.com Inc and John Wiley & Sons Ltd.

  4. Flask sealing on in vitro seed germination and morphogenesis of two types of ornamental pepper explants

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    Diego Silva Batista

    Full Text Available ABSTRACT: The influence of flask sealing and explant source on the in vitro morphogenesis of pepper, were evaluated in Capsicum annuum. Seeds were sterilized and inoculated in Murashige and Skoog media supplemented with vitamins, myo-inositol, sucrose, and agar. Gas exchange was evaluated in the germination stage by comparing 3 flask-sealing systems: rigid polypropylene lids (PLs without vents, PLs with 1 vent, and PLs with 2 vents covered with membranes. In the regeneration stage, cotyledon and hypocotyl segments were transferred to the organogenesis-inducing media, being the different sealing types also tested in a factorial scheme. Photosynthetic pigments, morphological and hystological analyses were conducted for each treatment. Plants maintained in glass flasks capped with vented lids showed more vigorous growth and differentiated anatomical structures. These treatments resulted in taller plants, higher numbers and more expanded leaves, higher fresh and dry weights, and an increase in photosynthetic pigments. Cultivation of C. annuum in flasks with reduced gas exchange was more effective on callus induction. During the regeneration stage, hypocotyls were more effective than cotyledons. Sealing type influenced the morphogenic responses of pepper, demonstrating that an increase in gas exchange has a positive effect on biomass production and acclimatization of the plantlets.

  5. In vitro plant regeneration of two cucumber (Cucumis sativum L. genotypes: Effects of explant types and culture medium

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    Grozeva Stanislava

    2014-01-01

    Full Text Available The effect of different phytohormone concentrations on callusogenesis and organogenesis in two cucumber genotypes were studied. It was established that the rate of plant regeneration depends on genotype, explant type and culture medium. Hypocotyls were found to be more responsive than cotyledons in morphogenesis. In vitro planlet-regenerants have been obtained in hypocotyls explants on culture medium with 1.0 and 2.0 mgL-1 BA for cultivar Gergana and in 1.0 and 3.0 mgL-1K-line 15B. Induction of regeneration in cotyledons were established only in cultivar Gergana on culture medium supplemented with 3.0 mgL-1 BA and in combination of 0.5 mgL-1IAA.

  6. Callus induction and plant regeneration from explants of commercial cultivars of leek (Allium ampeloprasum var. porrum L.).

    Science.gov (United States)

    Buiteveld, J; van der Valk, P; Jansen, J; Creemers-Molenaar, J; Colijn-Hooymans, C M

    1993-05-01

    Plant regeneration capacity was studied for 8 cultivars and 4 accessions of leek (A. ampeloprasum var. porrum L.). Compact callus was induced on embryo and leaf explants on three different media. The highest frequency of compact callus formation (up to 90%) was obtained when mature, zygotic embryos were cultured on MS medium, containing 30 g/l sucrose and 1 mg/l 2,4-D. Regeneration occurred through somatic embryogenesis on MS medium, supplemented with 1 mg/l kinetin. Plants could be regenerated from all cultivars and accessions tested. These cultivars and accessions could be classified into three groups with respect to shoot formation frequency. The results suggest a distinct influence of the genotype on the morphogenic response of leek embryo explants in vitro.

  7. Microvesicle-mediated release of soluble LH/hCG receptor (LHCGR from transfected cells and placenta explants

    Directory of Open Access Journals (Sweden)

    Randeva Harpal

    2011-05-01

    Full Text Available Abstract Placental hCG and pitutary LH transduce signals in target tissues through a common receptor (LHCGR. We demonstrate that recombinant LHCGR proteins which include the hormone-binding domain are secreted from transfected cells and that natural LHCGR is also secreted from human placental explants. LHCGR recombinant proteins representing varying lengths of the N-terminal extracellular domain were expressed in Chinese Hamster Ovary cells in suspension culture. Secretion was minimal up to 72h but by 96h 24-37% of the LHCGR had been released into the culture medium. The secreted proteins were folded and sensitive to glycosidases suggesting N-linked glycosylation. Secretion was independent of recombinant size and was mediated via structurally defined membrane vesicles (50-150nm. Similarly cultured human early pregnancy placental explants also released LHCGR via microvesicles. These studies provide the first experimental evidence of the possible mechanistic basis of the secretion of LHCGR.

  8. The effect of protease inhibitors on the induction of osteoarthritis-related biomarkers in bovine full-depth cartilage explants

    DEFF Research Database (Denmark)

    He, Yi; Zheng, Qinlong; Jiang, Mengmeng

    2015-01-01

    Objective The specific degradation of type II collagen and aggrecan by matrix metalloproteinase (MMP)-9, -13 and ADAMTS-4 and -5 (aggrecanase-1 and -2) in the cartilage matrix is a critical step in pathology of osteoarthritis (OA). The aims of this study were: i) To investigate the relative...... contribution of ADAMTS-4 and ADAMTS-5 to cartilage degradation upon catabolic stimulation; ii) To investigate the effect of regulating the activities of key enzymes by mean of broad-spectrum inhibitors. Methods Bovine full-depth cartilage explants stimulated with tumor necrosis factor alpha (TNF...... protease for the generation of 374ARGS aggrecan fragment in the TNF-α/OSM stimulated bovine cartilage explants. This study addresses the need to determine the roles of ADAMTS-4 and ADAMTS-5 in human articular degradation in OA and hence identify the attractive target for slowing down human cartilage...

  9. The Impact of Carbon Source, Explants and Growth Regulators on Callogenesis and Organogenesis of Artemisia annua

    Directory of Open Access Journals (Sweden)

    Bita GHASSEMI

    2015-12-01

    Full Text Available Artemisinin, a sesquiterpene lactone isolated from Artemisia annua L. plant is known for its antimalarial activity. The low content of artemisinin has stimulated researchers to enhance its production through biotechnological approaches such as tissue culture. The present study was initiated to study the effect of some important factors alone and in combination, on the callogenesis and organogenesis of Artemisia annua. The type of carbon source had a significant effect on NAA efficiency to callogenesis of A. annua, whereas the best callogenesis of A. absinthium was observed at 2 mg/L BAP + 30 g/L sucrose and in the absence of NAA, with root explants. Presence of BAP also had an important effect on callogenesis, especially in high concentrations. A suitable suspension culture was obtained in the MS basal medium containing 0.5 mg/L NAA and BAP, with 30 g/L glucose. Artemisinin was naturally production was at least 0.03 mg/g (w.dt at the first day and peaked on the 16th day with 0.31 mg/g (w.dt in the cell culture of A. annua. The maximum number of shoots (2.167 ± 1.484 was induced at 0.5 mg/L BAP + 0.1 mg/L NAA + glucose (30 g/L with leaf explants. However, treatments containing glucose did not show a good shoot induction. Longer shoots were induced in the medium containing either 0.5 mg/l NAA + 0.5 mg/l BAP + sucrose with leaf explants (1.493 cm ± 0.342 or 0.5 mg/l NAA + sucrose with stem explants (0.697 cm ± 0.930. Medium containing 0.5 or 2 mg/l NAA and sucrose (without BAP induced more roots though.

  10. Evaluation of the efficacy of dasatinib, a Src/Abl inhibitor, in colorectal cancer cell lines and explant mouse model.

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    Aaron J Scott

    Full Text Available Dysregulation of the Src pathway has been shown to be important at various stages of cancer. Dasatinib is a potent Src/Abl inhibitor and has demonstrated to have anti-proliferative and anti-invasive activity in many preclinical models. The objective of this study was to determine the anti-tumor activity of dasatinib using in vitro and in vivo preclinical colorectal (CRC models.CRC cell lines and patient-derived tumor explant (PDX models were used to investigate the efficacy of dasatinib. We treated 50 CRC cell lines with dasatinib for 72 hours and proliferation was assayed by a sulforhodamine B (SRB assay; an IC50 ≤ 0.08 μmol/L was considered sensitive. We treated 17 patient-derived CRC explants with dasatinib (50 mg/kg/day, administered once-daily for 28 days to determine in vivo efficacy. Tumor growth inhibition (TGI ≥ 50% was considered sensitive.We found that 8 out of 50 CRC cell lines reached an IC50 ≤ 0.08 μmol/L with dasatinib treatment. In addition, of 17 CRC explants grown in the xenograft mouse model, 2 showed sensitivity to dasatinib. The anti-tumor effects observed in this study were a result of G1 cell cycle arrest as the dasatinib sensitive CRC cell lines exhibited G1 inhibition. Moreover, those CRC cell lines that were responsive (0.08 μmol/L to treatment demonstrated a significant baseline increase in Src and FAK gene expression.Dasatinib demonstrated significant anti-proliferative activity in a subset of CRC cell lines in vitro, especially in those with increased Src expression at baseline, but only showed modest efficacy in CRC explants. Dasatinib is currently being studied in combination with chemotherapy in patients with advanced CRC, as its use as a single agent appears limited.

  11. Plant regeneration of non-toxic Jatropha curcas—impacts of plant growth regulators, source and type of explants

    KAUST Repository

    Kumar, Nitish

    2011-01-28

    Jatropha curcas is an oil bearing species with multiple uses and considerable economic potential as a biofuel plant, however, oil and deoiled cake are toxic. A non-toxic variety of J. curcas is reported from Mexico. The present investigation explores the effects of different plant growth regulators (PGRs) viz. 6-benzyl aminopurine (BAP) or thidiazuron (TDZ) individually and in combination with indole-3-butyric acid (IBA), on regeneration from in vitro and field-grown mature leaf explants, in vitro and glasshouse-grown seedlings cotyledonary leaf explants of non-toxic J. curcas. In all the tested parameters maximum regeneration efficiency (81.07%) and the number of shoot buds per explants (20.17) was observed on 9.08 μM TDZ containing Murashige and Skoog’s (MS) medium from in vitro cotyledonary leaf explants. The regenerated shoot buds were transferred to MS medium containing 10 μM kinetin (Kn), 4.5 μM BAP and 5.5 μM α-naphthaleneacetic acid (NAA) for shoot proliferation. The proliferated shoots could be elongated on MS medium supplemented with 2.25 μM BAP and 8.5 μM IAA. Rooting was achieved when the basal cut end of elongated shoots were dipped in half strength MS liquid medium containing different concentrations and combinations of IBA, IAA and NAA for four days followed by transfer to growth regulators free half strength MS medium supplemented 0.25 mg/l activated charcoal. The rooted plants could be established in soil with more than 90% survival rate.

  12. Effects of ion beam irradiation on adventitious shoot regeneration from in vitro leaf explants of Septennial ionahta

    Energy Technology Data Exchange (ETDEWEB)

    Zhou, L.B. [Radiobiology Department, Institute of Modern Physics, Chinese Academy of Sciences, Nanchang Road 509, Lanzhou 730000 (China)]. E-mail: libinzhou@impcas.ac.cn; Li, W.J. [Radiobiology Department, Institute of Modern Physics, Chinese Academy of Sciences, Nanchang Road 509, Lanzhou 730000 (China); Ma, S. [Radiobiology Department, Institute of Modern Physics, Chinese Academy of Sciences, Nanchang Road 509, Lanzhou 730000 (China); Dong, X.C. [Radiobiology Department, Institute of Modern Physics, Chinese Academy of Sciences, Nanchang Road 509, Lanzhou 730000 (China); Yu, L.X. [Radiobiology Department, Institute of Modern Physics, Chinese Academy of Sciences, Nanchang Road 509, Lanzhou 730000 (China); Li, Q. [Radiobiology Department, Institute of Modern Physics, Chinese Academy of Sciences, Nanchang Road 509, Lanzhou 730000 (China); Zhou, G.M. [Radiobiology Department, Institute of Modern Physics, Chinese Academy of Sciences, Nanchang Road 509, Lanzhou 730000 (China); Gao, Q.X. [Institute of Cell Biology, School of Life Science, Lanzhou University, Southern Tianshui Road 222, Lanzhou 730000 (China)

    2006-03-15

    The effects of 960 MeV carbon ion beam and 8 MeV X-ray irradiation on adventitious shoots from in vitro leaf explants of two different Saintpaulia ionahta (Mauve and Indikon) cultivars were studied with regard to tissue increase, shoots differentiation and morphology changes in the shoots. The experimental results showed that the survival fraction of shoot formation for the Mauve and Indikon irradiated with the carbon ion beam at 20 Gy were 0.715 and 0.600, respectively, while those for both the cultivars exposed to the X-ray irradiation at the same dose were 1.000. Relative biological effectiveness (RBE) of Mauve with respect to X-ray was about two. Secondly, the percentage of regenerating explants with malformed shoots in all Mauve regenerating explants irradiated with carbon ion beam at 20 Gy accounted for 49.6%, while that irradiated with the same dose of X-ray irradiation was only 4.7%; as for Saintpaulia ionahta Indikon irradiated with 20 Gy carbon ion beam, the percentage was 43.3%, which was higher than that of X-ray irradiation. Last, many chlorophyll deficient and other varieties of mutants were obtained in this study. Based on the results above, it can be concluded that the effect of mutation induction by carbon ion beam irradiation on the leaf explants of Saintpaulia ionahta is better than that by X-ray irradiation; and the optimal mutagenic dose varies from 20 Gy to 25 Gy for carbon ion beam irradiation.

  13. Effects of ion beam irradiation on adventitious shoot regeneration from in vitro leaf explants of Saintpaulia ionahta

    Science.gov (United States)

    Zhou, L. B.; Li, W. J.; Ma, S.; Dong, X. C.; Yu, L. X.; Li, Q.; Zhou, G. M.; Gao, Q. X.

    2006-03-01

    The effects of 960 MeV carbon ion beam and 8 MeV X-ray irradiation on adventitious shoots from in vitro leaf explants of two different Saintpaulia ionahta (Mauve and Indikon) cultivars were studied with regard to tissue increase, shoots differentiation and morphology changes in the shoots. The experimental results showed that the survival fraction of shoot formation for the Mauve and Indikon irradiated with the carbon ion beam at 20 Gy were 0.715 and 0.600, respectively, while those for both the cultivars exposed to the X-ray irradiation at the same dose were 1.000. Relative biological effectiveness (RBE) of Mauve with respect to X-ray was about two. Secondly, the percentage of regenerating explants with malformed shoots in all Mauve regenerating explants irradiated with carbon ion beam at 20 Gy accounted for 49.6%, while that irradiated with the same dose of X-ray irradiation was only 4.7%; as for Saintpaulia ionahta Indikon irradiated with 20 Gy carbon ion beam, the percentage was 43.3%, which was higher than that of X-ray irradiation. Last, many chlorophyll deficient and other varieties of mutants were obtained in this study. Based on the results above, it can be concluded that the effect of mutation induction by carbon ion beam irradiation on the leaf explants of Saintpaulia ionahta is better than that by X-ray irradiation; and the optimal mutagenic dose varies from 20 Gy to 25 Gy for carbon ion beam irradiation.

  14. Direct regeneration of Periwinkle (Catharanthus roseus) via node explants culture and different combinations of plant growth regulators

    OpenAIRE

    M. Talebi; F. Etesam; B.E. Sayed-Tabatabaei; Gh. Khaksar

    2012-01-01

    Periwinkle (Catharanthus roseus L., Apocynaceae) contains more than 130 different terpenoid indole alkaloids (TIAs), of which two dimeric alkaloids, Vinblastine and Vincristine, have antineoplastic activity and are useful in treatment of various cancers. Specific production of some alkaloids in differentiated tissues such as leaf and stem led to use direct regeneration of explants in order to increase the production of these important alkaloids in the plant. In this research, 30 combinations ...

  15. The role of the sewing ring in tissue healing: a pathologic study of explanted mechanical heart valves.

    Science.gov (United States)

    Kurian, Mathew V; Sabareeswaran, Arumugam; Kanchanamala, Muniraj; Balachandran, Seetharaman; Mogileswari, Dhananjayan; Subban, Vijayakumar

    2012-03-01

    Serious complications may occur after heart valve replacement, and many such patients will require reoperation. The study aim was to identify the pattern of tissue response around the sewing ring of those valves that have been explanted as a result of various valve-associated complications. A total of 51 mechanical heart valves (MHVs) was explanted from 45 patients who had undergone reoperation for valve-related complications. The examination of the valves included an analysis of the operative findings, macroscopic findings, histopathology, and dissection of the sewing ring. The extent of tissue hyperplasia was variable around the sewing rings of valves explanted for various pathologies. In pannus, the hyperplastic tissue extended into the valve orifice and produced an obstruction to flow, whereas in thrombosed valves the thrombus was attached to the tissue at the annulus. In non-infective pathologies, the histology revealed cellular infiltration that was limited to the peripheral fabric layers of the sewing ring, though the extent of infiltration was not increased with the duration of implantation. In prosthetic valve endocarditis (PVE), the surrounding hyperplastic tissue was granulomatous, but cellular infiltration into the sewing ring was absent. The dissection of various models of explanted valves revealed that different types of filler and fixing mechanisms had increased the bulk of the sewing ring. Tissue hyperplasia of varying extent occurs around the sewing ring after MHV implantation. The cellular elements grow into the peripheral layers, but not the deeper layers, of the sewing ring. In PVE, there was an absence of cellular infiltration into the sewing ring.

  16. Effect of ZnO nanoparticles on Brassica nigra seedlings and stem explants: growth dynamics and antioxidative response

    Directory of Open Access Journals (Sweden)

    Hira eZafar

    2016-04-01

    Full Text Available Nanoparticles (NPs have diverse properties in comparison to respective chemicals due to structure, surface area ratio, morphology, and reactivity. Toxicological effects of metallic NPs to organisms including plants have been reported. However, to the authors’ knowledge there is no report on the effect of NPs on in vitro culture of plant explants. In this study, ZnO NPs at 500-1500 mg/L badly affected Brassica nigra seed germination and seedling growth and raised antioxidative activities and antioxidants concentrations. On the other hand, culturing the stem explants of B. nigra on Murashige and Skoog (MS medium in presence of low concentration of ZnO NPs (1-20 mg/L produced white thin roots with thick root hairs. At 10 mg/L ZnO NPs shoots emergence was also observed. The developed calli/roots showed 79% DPPH (2,2-diphenyl-1-picryl hydrazyl radical scavenging activity at 10 mg/L. While total antioxidant and reducing power potential were also significantly different in presence of ZnO NPs. Non enzymatic antioxidative molecules, phenolics (up to 0.15 µg GAE/mg FW and flavonoids (up to 0.22 µg QE/mg FW, also raised and found NPs concentration dependent. We state that ZnO NPs may induce roots from explants cultured on appropriate medium and can be cultured for production of valuable secondary metabolites.

  17. Thidiazuron: A potent cytokinin for efficient plant regeneration in Himalayan poplar (Populus ciliata Wall. using leaf explants

    Directory of Open Access Journals (Sweden)

    Gaurav Aggarwal

    2012-11-01

    Full Text Available Populus species are important resource for certain branches of industry and have special roles for scientific study on biological and agricultural systems. The present investigation was undertaken with an objective of enhancing the frequency of plant regeneration in Himalayan poplar (Populus ciliata Wall.. The effect of Thiadizuron (TDZ alone and in combination with adenine and α-Naphthalene acetic acid (NAA were studied on the regeneration potential of leaf explants. A high efficiency of shoot regeneration was observed in leaf (80.00% explants on MS basal medium supplemented with 0.024 mg/l TDZ and 79.7 mg/l adenine. Elongation and multiplication of shoots were obtained on Murashige and Skoog (MS basal medium, containing 0.5 mg/l 6. Benzyl aminopurine (BAP + 0.2mg/l Indole 3-acetic acid (IAA + 0.3 mg/l Gibberellic acid (GA3. High frequency root regeneration from in vitro developed shoots was observed on MS basal medium supplemented with 0.10 mg/l Indole 3-butyric acid(IBA. Maximum of the in vitro rooted plantlets were well accomplished to the mixture of sand: soil (1:1 and exhibited similar morphology with the field plants. A high efficiency plant regeneration protocol has been developedfrom leaf explants in Himalayan poplar (Populus ciliata Wall..

  18. Antibodies produced by clonally expanded plasma cells in multiple sclerosis cerebrospinal fluid cause demyelination of spinal cord explants.

    Science.gov (United States)

    Blauth, Kevin; Soltys, John; Matschulat, Adeline; Reiter, Cory R; Ritchie, Alanna; Baird, Nicholas L; Bennett, Jeffrey L; Owens, Gregory P

    2015-12-01

    B cells are implicated in the etiology of multiple sclerosis (MS). Intrathecal IgG synthesis, cerebrospinal fluid (CSF) oligoclonal bands and lesional IgG deposition suggest a role for antibody-mediated pathology. We examined the binding of IgG1 monoclonal recombinant antibodies (rAbs) derived from MS patient CSF expanded B cell clones to central nervous system (CNS) tissue. MS rAbs displaying CNS binding to mouse and human CNS tissue were further tested for their ability to induce complement-mediated tissue injury in ex vivo spinal cord explant cultures. The staining of CNS tissue, primary human astrocytes and human neurons revealed a measurable bias in MS rAb binding to antigens preferentially expressed on astrocytes and neurons. MS rAbs that recognize myelin-enriched antigens were rarely detected. Both myelin-specific and some astrocyte/neuronal-specific MS rAbs caused significant myelin loss and astrocyte activation when applied to spinal cord explant cultures in the presence of complement. Overall, the intrathecal B cell response in multiple sclerosis binds to both glial and neuronal targets and produces demyelination in spinal cord explant cultures implicating intrathecal IgG in MS pathogenesis.

  19. Thidiazuron: A potent cytokinin for efficient plant regeneration in Himalayan poplar (Populus ciliata Wall. using leaf explants

    Directory of Open Access Journals (Sweden)

    Gaurav Aggarwal

    2012-12-01

    Full Text Available Populus species are important resource for certain branches of industry and have special roles for scientific study on biological and agricultural systems. The present investigation was undertaken with an objective of enhancing the frequency of plant regeneration in Himalayan poplar (Populus ciliataWall.. The effect of Thiadizuron (TDZ alone and in combination with adenine and alpha-Naphthalene acetic acid (NAA were studied on the regeneration potential of leaf explants. A high efficiency of shoot regeneration was observed in leaf (80.00% explants on MS basal medium supplemented with 0.024 mg/l TDZ and 79.7 mg/l adenine. Elongation and multiplication of shoots were obtained on Murashige and Skoog (MS basal medium, containing 0.5 mg/l 6. Benzyl aminopurine (BAP + 0.2mg/l Indole 3-acetic acid (IAA + 0.3 mg/l Gibberellic acid (GA3. High frequency root regeneration from in vitro developed shoots was observed on MS basal medium supplemented with 0.10 mg/l Indole 3-butyric acid (IBA. Maximum of the in vitro rooted plantlets were well accomplished to the mixture of sand: soil (1:1 and exhibited similar morphology with the field plants. A high efficiency plant regeneration protocol has been developed from leaf explants in Himalayan poplar (Populus ciliata Wall.. 

  20. Study on Effect of Type of Explant and Hormone on Callus Induction and Regeneration in Saffron (Crocus sativus L.

    Directory of Open Access Journals (Sweden)

    Mohsen Sajjadi

    2015-10-01

    Full Text Available Saffron (Crocus sativus L. is one of the medicinal plants that contain active components and medicinal materials. Tissue culture of saffron can improve the quality and quantity of the saffron product, increase its export and the farmers’ income. In this study, 36 different types of hormone combinations in the dark and 9 different treatments of hormone combinations in cold (4°C, using different saffron explants (bulb, leaf, scales around leaf and distal parts of the leaf were studied in tissue culture. To investigate the growth of corms, the callus formation and the regeneration rate, three replications for each treatment were used and the length of shoot (cm, the callus formation percentage and the regeneration percentage were measured and statistical analysis was performed. Among the types of explants, only explants from bulbs produced the callus on MS medium containing 2 mg.l-1 BAP and 1 mg.l-1 IBA in both the dark and cold conditions. The highest percentage of regeneration was obtained in MS medium with hormonal composition of 0.3 mg.l-1 TDZ, 1 mg.l-1 BAP, 2 mg.l-1 IBA and 0.01 mg.l-1 GA3 in the cold conditions.

  1. Indirect organogenesis from various explants of Hildegardia populifolia (Roxb. Schott & Endl. – A threatened tree species from Eastern Ghats of Tamil Nadu, India

    Directory of Open Access Journals (Sweden)

    A.R. Lavanya

    2014-12-01

    Full Text Available Hildegardia species are an important resource for fiber industry. This investigation was conducted to develop a plant regeneration protocol for Hildegardia populifolia (Roxb. Schott & Endl. via indirect organogenesis Callus was obtained from leaf, internode and petiole explants, among these explants internode explant gave best result on MS medium supplemented with different concentrations of 2,4-Dichlorophenoxy acetic acid (2,4-D. The highest percentage (100% of regeneration was obtained with benzyladenine (BA (2.0 mg/l + indole-3-acetic acid (IAA (0.1 mg/l + glutamine (25 mg/l + thidiazuron (TDZ (0.5 mg/l from internode explants. Shootlets were highly rooted on MS medium supplemented with 3.0 mg/l indole-3-butyric acid (IBA. In vitro rooted seedlings were successfully acclimatized. This in vitro regeneration system will facilitate further development of reliable procedures for this genus.

  2. Evaluación de diferentes combinaciones fitohormonales en la regeneración de Solanum tuberosum (Solanaceae) Var. Pastusa Suprema a partir de explantes internodales

    National Research Council Canada - National Science Library

    Jenny Paola Jiménez Barreto; Alejandro Chaparro Giraldo; Jennifer Blanco

    2009-01-01

    .... The effect of zeatin riboside (ZR), gibberellic acid (GA3) and naphthalene acetic acid (NAA) phytohormones used in specific combinations was evaluated regarding callus induction and bud regeneration and number per explant...

  3. High frequency organogenesis in hypocotyl, cotyledon, leaf and petiole explants of broccoli (Brassica oleracea L. var. italica), an important vegetable crop.

    Science.gov (United States)

    Kumar, Pankaj; Srivastava, D K

    2015-04-01

    Broccoli (Brassica oleracea L. var. italica) is an important, nutritionally rich vegetable crop, but severely affected by environmental stresses, pests and diseases which cause massive yield and quality losses. Genetic manipulation is becoming an important method for broccoli improvement. In the present study, a reproducible and highly efficient protocol for obtaining organogenesis from hypocotyl, cotyledon, leaf and petiole explants of broccoli (Brassica oleracea L. var. italica cv. Solan green head) has been developed. Hypocotyl and cotyledon explants were used from 10 to 12 days old aseptically grown seedlings whereas leaf and petiole explants were excised from 18 to 20 days old green house grown seedlings and surface sterilized. These explants were cultured on shoot induction medium containing different concentration and combination of BAP and NAA. High efficiency shoot regeneration has been achieved in hypocotyl (83.33 %), cotyledon (90.11 %), leaf (62.96 %) and petiole (91.10 %) explants on MS medium supplemented with 3.5 mg/l BAP + 0.019 mg/l NAA 2.5 mg/l BAP + 0.5 mg/l NAA, 4.0 mg/l BAP + 0.5 mg/l NAA and 4.5 mg/l BAP + 0.019 mg/l NAA respectively. Petiole explants showed maximum shoot regeneration response as compared to other explants. MS medium supplemented with 0.10 mg/l NAA was found best for root regeneration (100 %) from in vitro developed shoots. The regenerated complete plantlets were transferred to the pots containing cocopeat and successfully acclimatized. This optimized regeneration protocol can be efficiently used for genetic transformation in broccoli. This is the first comparative report on multiple shoot induction using four different types of explants viz. hypocotyl, cotyledon, leaf and petiole.

  4. Effect Of Explant Source And Different Medium Culture On Friable Embryogenic Callus Induction Of Four Cultivars Of Cassava Manihot Esculenta Crantz

    Directory of Open Access Journals (Sweden)

    Simplice Prosper Yandia

    2015-08-01

    Full Text Available In order to obtain Friable Embryogenic Callus FEC for protoplast isolation we have evaluated in this research the competance for Friable Embryogenic Callus FEC of four cassava cultivars M61033 Rendre Yalipe and Six-mois in media containing MS supplemented with 8mgl 24-D MS supplemented with 10 mgl BAP and GD supplemented with 12mgl picloram using apical bud AB and immature leaves lobes ILL as explants. In general in the medium GD12mgl picloram the highest efficiencies of FEC ranged from 58 to 87 and the highest score of FEC ranged from 4.2 to 5.4 with explants AB however we have observed with explants ILL the efficiencies of somatic embryos ranged form 41 to 75 and the score ranged from 4.1 to 4.4. The mediums MS28 mgl 24-D have induced with explants AB the efficiencies of FEC ranged from 43 to 57 and the score ranged from 3.1 to 3.8 however with ILL explants the efficiencies of FEC ranged from 39 to 49 and the score ranged from 2.9 to 3.7. The least FEC were observed in the medium MS210 mgl BAP with BA explants however the efficiencies ranged from 6 to 11 and the score ranged from 1.1 to 1.8. Whereas the efficiencies of FEC with ILL explants ranged from 4 to 7 and the score ranged from 0.5 to 0.8. All of four cultivars showed capability of producing FEC although their efficiency varied according to gonotype donors explants and medium taking into acount. Abbreviations GD Gressoff and Doy MS Murashige and Skoog 24-dichloro phenoxyacetic acid BAP Benzylamino-purin-Acid AB Apical Bud ILL Immature Leaves lobes

  5. Sacha Inchi Oil (Plukenetia volubilis L.), effect on adherence of Staphylococus aureus to human skin explant and keratinocytes in vitro.

    Science.gov (United States)

    Gonzalez-Aspajo, German; Belkhelfa, Haouaria; Haddioui-Hbabi, Laïla; Bourdy, Geneviève; Deharo, Eric

    2015-08-02

    Plukenetia volubilis L. (Euphorbiaceae) is a domesticated vine distributed from the high-altitude Andean rain forest to the lowlands of the Peruvian Amazon. Oil from the cold-pressed seeds, sold under the commercial name of Sacha Inchi Oil (SIO) is actually much in favour because it contains a high percentage of omega 3 and omega 6, and is hence used as a dietary supplement. SIO is also used traditionally for skin care, in order to maintain skin softness, and for the treatment of wounds, insect bites and skin infections, in a tropical context where the skin is frequently damaged. This study was designed in order to verify whether the traditional use of SIO for skin care would have any impact on Staphylococcus aureus growth and skin adherence, as S. aureus is involved in many skin pathologies (impetigo, folliculitis, furuncles and subcutaneous abscesses) being one if the main pathogens that can be found on the skin. Therefore, our objective was to assess SIO bactericidal activity and interference with adherence to human skin explants and the keratinocyte cell line. Cytotoxicity on that cells was also determined. The activity of SIO was compared to coconut oil (CocO), which is widely used for skin care but has different unsaturated fatty acids contents. Laboratory testing with certified oil, determined antibacterial activity against radio labelled S. aureus. Cytotoxic effects were measured with XTT on keratinocyte cells and with neutral red on human skin explants; phenol was used as cytotoxic control. Adherence assays were carried out by mixing H3-labelled S. aureus bacteria with keratinocyte cells and human skin explants, incubated with oils 2h before (to determine the inhibition of adherence, assimilated to a preventive effect) or 2h after the contact of the biological material with S. aureus (to assess the detachment of the bacteria, assimilated to a curative effect). Residual radioactivity measured after washings made it possible to determine the adherence

  6. Growth of vegetative explant Moringa oleifera on different composition of auxin and cytokinin and its synthetic seed germination

    Science.gov (United States)

    Muslihatin, Wirdhatul; Jadid, Nurul; Puspitasari, Ika D.; Safitri, Chusnul E.

    2017-06-01

    The spread of Moringa oleifera is also rare for seed germination and viability or survival are low, and the lack of vegetative propagation method. The purpose of this study are to determine the effect of auxin and cytokinin on growth vegetative explants Moringa oleifera and its synthetic seed germination. The explants grown on MS medium with sucrose content of 30% and a range of additional hormone. Addition concentration and different types of hormone made in order to know the sensitivity and response explant growth on a variety of media to get a good callus and embryosomatic. The composition of the hormone given is MS + 2.4 D 3 ppm; MS + 2,4D 2 ppm + BAP 2 ppm; MS + NAA + 0.5 ppm kinetin 1 ppm; MS + NAA 1 ppm + kinetin 1 ppm; MS + NAA 1 ppm + 0.5 ppm kinetin. The explants were incubated at a temperature of 18-20 ° C with a photoperiod 16/8. Explants and MS medium is incubated to form embryonic callus. Seeds synthetic made from embryonic callus growing on medium 1 ppm kinetin + NAA 1 ppm with encapsulation method with sodium alginate 2%. Seed synthetic germinated in some kind of medium that medium ms0 solid (M1), ms0 liquid (M2), MS0 semi-solid (M3), MS solid NAA 1ppm + Kinetin 1 ppm (M4), MS liquid NAA 1 ppm + kinetin (M5), and semi-solid MS + NAA 1 ppm kinetin 1 ppm (M6). Synthetic seed viability was observed with the parameters of the fresh weight of synthetic seed, germination percentage and seedling. Chlorophyll content was measured by spectrophotometric method with solvent asseton. Best callus generated in this study are embryonic callus that grew on media NAA 1 ppm + kinetin 1 ppm. Embryonic callus on M6 + NAA 1 ppm kinetin 1 ppm capable of germination with an average weight of callus and sprouts of 40.38 mg. Of the entire amount of a synthetic seed on M6, just 5 seed germinate, so the percentage of germination of seeds is equal to 41.67%. with an average length of sprouts 1 cm with an average total chlorophyll content of 8.66 mg / g.

  7. Replication of avian, human and swine influenza viruses in porcine respiratory explants and association with sialic acid distribution

    Directory of Open Access Journals (Sweden)

    Nauwynck Hans J

    2010-02-01

    Full Text Available Abstract Background Throughout the history of human influenza pandemics, pigs have been considered the most likely "mixing vessel" for reassortment between human and avian influenza viruses (AIVs. However, the replication efficiencies of influenza viruses from various hosts, as well as the expression of sialic acid (Sia receptor variants in the entire porcine respiratory tract have never been studied in detail. Therefore, we established porcine nasal, tracheal, bronchial and lung explants, which cover the entire porcine respiratory tract with maximal similarity to the in vivo situation. Subsequently, we assessed virus yields of three porcine, two human and six AIVs in these explants. Since our results on virus replication were in disagreement with the previously reported presence of putative avian virus receptors in the trachea, we additionally studied the distribution of sialic acid receptors by means of lectin histochemistry. Human (Siaα2-6Gal and avian virus receptors (Siaα2-3Gal were identified with Sambucus Nigra and Maackia amurensis lectins respectively. Results Compared to swine and human influenza viruses, replication of the AIVs was limited in all cultures but most strikingly in nasal and tracheal explants. Results of virus titrations were confirmed by quantification of infected cells using immunohistochemistry. By lectin histochemistry we found moderate to abundant expression of the human-like virus receptors in all explant systems but minimal binding of the lectins that identify avian-like receptors, especially in the nasal, tracheal and bronchial epithelium. Conclusions The species barrier that restricts the transmission of influenza viruses from one host to another remains preserved in our porcine respiratory explants. Therefore this system offers a valuable alternative to study virus and/or host properties required for adaptation or reassortment of influenza viruses. Our results indicate that, based on the expression of Sia

  8. Efficient plant regeneration protocol through callus for Saussurea obvallata (DC.) Edgew. (Asteraceae): effect of explant type, age and plant growth regulators.

    Science.gov (United States)

    Dhar, Uppeandra; Joshi, Mitali

    2005-06-01

    A callus induction and in vitro plantlet regeneration system for the endangered state flower of Uttaranchal (Saussurea obvallata) was optimized by studying the influence of explant type (root, hypocotyl, cotyledon and leaf), age and different concentrations of plant growth regulators. Explants from 10 to 15-day-old seedlings showed maximum callus induction. Callus formation and shoot differentiation was initiated on Murashige-Skoog (MS) medium containing 6-benzyladenine (BA) and alpha-naphthalene acetic acid (NAA) in all explant types. The best results were obtained using leaf explants: 100% callusing was achieved in MS medium supplemented with 2.5 microM BA and 1.0 microM NAA, and 100% differentiation along with a multiplication rate of 12 shoots per explant with a combination of 5.0 microM BA and 1.0 microM NAA. However, the results reflected the existence of high inter-explant variability in response to growth regulators. In vitro rooting of shoots was achieved at an efficiency of 100% in one-half strength MS medium supplemented with 2.5 microM indole-3-butyric acid. Application of this protocol has potential for mass multiplication of the target species in a limited time period.

  9. Early somatic embryogenesis in Heliconia chartacea Lane ex Barreiros cv. Sexy Pink ovary section explants

    Directory of Open Access Journals (Sweden)

    Cláudia Ulisses

    2010-02-01

    Full Text Available The present work evaluated the development of embryogenic callus from transversal ovary sections. The experiments were carried out under two experimental regimes using combinations of IAA (0; 5.71; 8.56; 11.42; 14.27μM and 2,4-D (0; 13.57; 18.10; 22.62μM or combinations of 2,4-D with BA (0; 4.43; 6.65; 8.87; 11.09μM. Assessments were made of anatomical aspects of the callus and for the presence of embryogenic structures using cytochemical and histological analyses and stereomicroscopic and scanning electronic microscopic observations. Treatments with 2,4-D and IAA produced friable calluses demonstrating cellular acquisition of morphogenetic competence as well as the formation of pro-embryogenic sectors. The expression of embryogenic program could be observed, with proembryogenic cell clusters developing into globular embryos. These results offer the possibility of using new types of explants for culturing helicons that avoid the growth of endophytic bacteria.Este trabalho teve como objetivo avaliar a resposta de secções transversais de ovários e o desenvolvimento de calos embriogênicos. O experimento constou de dois ensaios. No primeiro avaliou-se combinações entre AIA (0; 5.71; 8.56; 11.42; 14.27μM e 2,4-D (0; 13.57; 18.10; 22.62μM e no segundo avaliou-se as concentrações de 2,4-D supracitadas, combinadas com concentrações de BA (0; 4.43; 6.65; 8.87; 11.09μM. Os calos formados foram avaliados quanto à presença de estruturas embriogênicas utilizando-se estereomicroscópio, microscópio eletrônico de varredura, além de análises citoquímicas e histológicas. Combinações entre 2,4-D e AIA induziram a formação de calos friáveis com setores pró-embriogênicos, refletindo a aquisição de competência morfogenética. Posteriormente foi observada a expressão do programa embriogênico quando massas pró-embriogências desenvolveram-se formando embriões somáticos. Esses resultados apresentam uma alternativa para a utiliza

  10. Shoot regeneration from cotyledonary leaf explants of jatropha curcas: A biodiesel plant

    KAUST Repository

    Kumar, Nitish Chandramohana

    2010-03-07

    A simple, high frequency, and reproducible method for plant regeneration through direct organogenesis from cotyledonary leaf explants of Jatropha curcas was developed using Murashige and Skoog (MS) medium supplemented with different concentrations of thidiazuron (TDZ) or 6-benzyl aminopurine (BAP). Medium containing TDZ has greater influence on regeneration as compared to BAP. The induced shoot buds were transferred to MS medium containing 10 lM kinetin (Kn), 4.5 lM BAP, and 5.5 lM a-naphthaleneacetic acid (NAA) for shoot proliferation. The proliferated shoots could be elongated on MS medium supplemented with different concentrations and combinations of BAP, indole-3-acetic acid (IAA), NAA, and indole-3-butyric acid (IBA). MS medium with 2.25 lM BAP and 8.5 lM IAA was found to be the best combination for shoot elongation. However, significant differences in plant regeneration and shoot elongation were observed among the genotypes studied. Rooting was achieved when the basal cut end of elongated shoots were dipped in half strength MS liquid medium containing dif- ferent concentrations and combinations of IBA, IAA, and NAA for 4 days, followed by transfer to growth regulators free half strength MS medium supplemented 0.25 mg l-1 activated charcoal. Elongated shoot treated with 15 lM IBA, 5.7 lM IAA, and 11 lM NAA resulted in highest percent rooting. The rooted plants could be established in soil with more than 90% survival rate. The method developed may be useful in improvement of J. curcas through genetic modification. © Franciszek Górski Institute of Plant Physiology, Polish Academy of Sciences, Kraków 2010.

  11. Indirect organogenesis in milkweed ( Calotropis procera from mature zygotic embryo explants

    Directory of Open Access Journals (Sweden)

    Hojatollah Abbasi

    2018-01-01

    Full Text Available Milkweed ( Calotropis procera is a valuable medicinal plant which grows in many regions of Iran. Its significant medicinal properties have made it an important crop which is cultivated commercially. This plant is propagated from seeds as well as root and shoot cuttings. Due to problems in the usage of these reproduction methods, new propagation methods such as tissue culturing should be developed. This study was aimed at obtaining appropriate concentrations of plant hormones for indirect organogenesis of milkweed. The experiment was arranged in a completely randomized design (CRD with 3 replications. The effects of various concentrations of (2,4-dichlorophenoxyacetic acid 2,4-D (0.1, 0.5, 1, 2 and 3 mg/l were studied in terms of callus induction and shoot regeneration on an MS based medium supplemented with BA (benzyl amino purine and NAA (naphthalene acetic acid at the same concentration. Mature embryos were used as explants and morphological traits such as embryo size, callus size, number and size of shoots and roots were recorded. The results showed that 2,4-D significantly increased the size of cultured embryos (P < 0.05. The largest embryo volume was observed in cultures treated with 3 mg/l 2,4-D. The highest callusing was recorded in 2 mg/l 2,4-D. The effects of BA and NAA concentrations on shoot regeneration were significant and the highest values were observed for a combination of 1 mg/l BA and 2 mg/l NAA. 1 mg/l IBA (Indole 3-butyric acid was able to induce the highest number of better quality roots and shoots.

  12. Exploring the potentials of nurture: 2(nd) and 3(rd) generation explant human skin equivalents.

    Science.gov (United States)

    Danso, Mogbekeloluwa O; van Drongelen, Vincent; Mulder, Aat; Gooris, Gert; van Smeden, Jeroen; El Ghalbzouri, Abdoelwaheb; Bouwstra, Joke A

    2015-02-01

    Explant human skin equivalents (Ex-HSEs) can be generated by placing a 4mm skin biopsy onto a dermal equivalent. The keratinocytes migrate from the biopsy onto the dermal equivalent, differentiate and form the epidermis of 1(st) generation Ex-HSEs. This is especially suitable for the expansion of skin material from which only small fragments of skin can be harvested e.g. diseased skin. We evaluated whether 2(nd) and 3(rd) generation Ex-HSEs can also be generated from a single skin biopsy whilst maintaining the epidermal properties of 1(st) generation Ex-HSEs and native human skin. 2(nd) generation Ex-HSEs were produced by placing a biopsy from the 1(st) generation Ex-HSE onto a new dermal equivalent. Likewise, the 3(rd) generation Ex-HSEs were generated from a 2(nd) generation Ex-HSE biopsy. We show for the first time that Ex-HSEs can be passaged to the 2(nd) and 3(rd) generation and display similar epidermal morphology and expression of differentiation markers as in native human skin and 1(st) generation Ex-HSEs except for involucrin. The 2(nd) and 3(rd) generation Ex-HSEs also show many similarities with 1(st) generation Ex-HSEs in lipid properties e.g. presence of all lipid classes, similar fatty acid chain length distribution and lamellar lipid organization. However, some differences arise in increased level of hexagonal lateral packing and a change in ceramide profiling. The changes in specific lipid classes were also accompanied by changes in the expression of the enzymes responsible for their synthesis. The expansion of skin biopsies to the 2(nd) and 3(rd) generation Ex-HSEs could be a promising method to expand valuable epidermal tissue to analyze morphological and differentiation parameters in the native epidermis. Copyright © 2015 Japanese Society for Investigative Dermatology. Published by Elsevier Ireland Ltd. All rights reserved.

  13. Effects of compression on the loss of newly synthesized proteoglycans and proteins from cartilage explants

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    Sah, R.L.; Doong, J.Y.; Grodzinsky, A.J.; Plaas, A.H.; Sandy, J.D. (Department of Electrical Engineering and Computer Science, Massachusetts Institute of Technology Harvard-M.I.T., Cambridge (United States))

    1991-04-01

    The effects of mechanical compression of calf cartilage explants on the catabolism and loss into the medium of proteoglycans and proteins radiolabeled with (35S)sulfate and (3H)proline were examined. A single 2- or 12-h compression of 3-mm diameter cartilage disks from a thickness of 1.25 to 0.50 mm, or slow cyclic compression (2 h on/2 h off) from 1.25 mm to 1.00, 0.75, or 0.50 mm for 24 h led to transient alterations and/or sustained increases in loss of radiolabeled macromolecules. The effects of imposing or removing loads were consistent with several compression-induced physical mediators including fluid flow, diffusion, and matrix disruption. Cyclic compression induced convective fluid flow and enhanced the loss of 35S- and 3H-labeled macromolecules from tissue into medium. In contrast, prolonged static compression induced matrix consolidation and appeared to hinder the diffusional transport and loss of 35S- and 3H-labeled macromolecules. Since high amplitude cyclic compression led to a sustained increase in the rate of loss of 3H- and 35S-labeled macromolecules that was accompanied by an increase in the rate of loss of (3H)hydroxyproline residues and an increase in tissue hydration, such compression may have caused disruption of the collagen meshwork. The 35S-labeled proteoglycans lost during such cyclic compression were of smaller average size than those from controls, but contained a similarly low proportion (approximately 15%) that could form aggregates with excess hyaluronate and link protein. The size distribution and aggregability of the remaining tissue proteoglycans and 35S-labeled proteoglycans were not markedly affected. The loss of tissue proteoglycan paralleled the loss of 35S-labeled macromolecules.

  14. Regeneración de Solanum tuberosum L. variedad pastusa suprema a partir de explantes internodales

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    Jenny Paola Jiménez Barreto

    2006-01-01

    adecuado (26% +/- 16. Los tratamientos en los que se utilizó una concentración baja de ANA (0,02 mg/L, fueron eficientes tanto en la formación de callo, como en la regeneración (86,0 +/- 13 y 70 +/- 22%. El nivel de zeatina ribósido y ácido giberélico utilizados en el medio, demostraron también ser estadísticamente significativos en el comportamiento callogénico y regenerativo. Los ensayos realizados demuestran la importancia de la combinación de los tres tipos hormonales en la inducción de callo, y subsecuente regeneración; probablemente, su interacción a nivel fisiológico crea las condiciones necesarias para que el tejido se desdiferencie y exprese su totipotencialidad. Comparando los resultados obtenidos en los 12 tratamientos para cada una de las variables estudiadas se puede concluir que la adición de 3 mg/L de zeatina ribósido, 0,02 mg/L de ácido naftalénacetico y 1,0 mg/L de ácido gibérelico en el medio de cultivo, constituye una formulación hormonal adecuada para inducir el proceso de organogénesis indirecta sobre la variedad de papa Pastusa Suprema, obteniendo porcentajes de regeneración de 92,9 % +/- 4,9 y un número medio de regenerantes por explante de 6,37 +/- 1,03 en un tiempo total de ocho semanas.

  15. Mechanical spectroscopy of retina explants at the protein level employing nanostructured scaffolds.

    Science.gov (United States)

    Mayazur Rahman, S; Reichenbach, Andreas; Zink, Mareike; Mayr, Stefan G

    2016-04-14

    Development of neuronal tissue, such as folding of the brain, and formation of the fovea centralis in the human retina are intimately connected with the mechanical properties of the underlying cells and the extracellular matrix. In particular for neuronal tissue as complex as the vertebrate retina, mechanical properties are still a matter of debate due to their relation to numerous diseases as well as surgery, where the tension of the retina can result in tissue detachment during cutting. However, measuring the elasticity of adult retina wholemounts is difficult and until now only the mechanical properties at the surface have been characterized with micrometer resolution. Many processes, however, such as pathological changes prone to cause tissue rupture and detachment, respectively, are reflected in variations of retina elasticity at smaller length scales at the protein level. In the present work we demonstrate that freely oscillating cantilevers composed of nanostructured TiO2 scaffolds can be employed to study the frequency-dependent mechanical response of adult mammalian retina explants at the nanoscale. Constituting highly versatile scaffolds with strong tissue attachment for long-term organotypic culture atop, these scaffolds perform damped vibrations as fingerprints of the mechanical tissue properties that are derived using finite element calculations. Since the tissue adheres to the nanostructures via constitutive proteins on the photoreceptor side of the retina, the latter are stretched and compressed during vibration of the underlying scaffold. Probing mechanical response of individual proteins within the tissue, the proposed mechanical spectroscopy approach opens the way for studying tissue mechanics, diseases and the effect of drugs at the protein level.

  16. In vitro cell cultures obtained from different explants of Corylus avellana produce Taxol and taxanes

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    Cavalli Francesca

    2006-12-01

    Full Text Available Abstract Background Taxol is an effective antineoplastic agent, originally extracted from the bark of Taxus brevifolia with a low yield. Many attempts have been made to produce Taxol by chemical synthesis, semi-synthesis and plant tissue cultures. However, to date, the availability of this compound is not sufficient to satisfy the commercial requirements. The aim of the present work was to produce suspension cell cultures from plants not belonging to Taxus genus and to verify whether they produced Taxol and taxanes. For this purpose different explants of hazel (Corylus avellana species were used to optimize the protocol for inducing in vitro callus, an undifferentiated tissue from which suspension cell cultures were established. Results Calli were successfully induced from stems, leaves and seeds grown in various hormone concentrations and combinations. The most suitable callus to establish suspension cell cultures was obtained from seeds. Media recovered from suspension cell cultures contained taxanes, and showed antiproliferative activity on human tumour cells. Taxol, 10-deacetyltaxol and 10-deacetylbaccatin III were the main taxanes identified. The level of Taxol recovered from the media of hazel cultures was similar to that found in yew cultures. Moreover, the production of taxanes in hazel cell cultures increased when elicitors were used. Conclusion Here we show that hazel cell cultures produce Taxol and taxanes under controlled conditions. This result suggests that hazel possesses the enzymes for Taxol production, which until now was considered to be a pathway particular to Taxus genus. The main benefit of producing taxanes through hazel cell cultures is that hazel is widely available, grows at a much faster rate in vivo, and is easier to cultivate in vitro than yew. In addition, the production of callus directly from hazel seeds shortens the culture time and minimizes the probability of contamination. Therefore, hazel could become a

  17. Regeneración de Solanum tuberosum L. variedad pastusa suprema a partir de explantes internodales

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    Chaparro Giraldo Alejandro

    2006-06-01

    Full Text Available Una de las vías posibles para aumentar la eficacia de los métodos de transformación genética mediante Agrobacterium tumefaciens es la optimización de la capacidad de regeneración de las plantas de interés. En la Universidad Nacional de Colombia, Sede Bogotá, el grupo de Ingeniería Genética de Plantas (IGP, ha venido trabajando en la obtención de plantas transgénicas a partir de la variedad de papa Diacol Capiro. Es de interés llevar a cabo este tipo de procedimientos en otras variedades de papa colombiana. La variedad Pastusa Suprema
    es un cultivar obtenido mediante mejoramiento genético convencional, que presenta cualidades superiores a otros cultivares. Es importante desarrollar un sistema eficiente de regeneración en esta variedad, como soporte para ulteriores estudios de transferencia de genes que permitan optimizar aún más su rendimiento. En este trabajo se evaluó el efecto de diferentes combinaciones hormonales adicionadas a un medio básico compuesto de sales Murashige y Skogg (1962, sobre el comportamiento callogénico y regenerativo de la variedad
    de Pastusa Suprema. Se realizó un diseño experimental factorial, en arreglo completamente al azar. Los factores corresponden a las hormonas ácido naftalénacetico (ANA, ácido giberélico (AG3 y ZR (Zeatina Ribósido, y los niveles son las concentraciones por factor ZR: 2,0 mg/L y 3,0 mg/L; ANA 0,0 mg/L, 0,02 mg/L, y 0,2 mg/L; y AG3: 0,02 mg/L, 1,0 mg/L. Los tratamientos probados corresponden a todas las combinaciones posibles entre los niveles de hormona evaluados, dando lugar a un total de doce tratamientos. La eficiencia del medio de cultivo se evaluó con base en las variables: porcentaje de inducción de callos, porcentaje de regeneración, y número de regenerantes por explante. El análisis de los datos se realizó con el software estadístico
    SAS versión 8.0. La presencia de ANA demostró ser esencial en la respuesta callogénica y regenerativa de

  18. Effect of 6-BA on nodal explant bud sproutings of Coffea arabica cv. Mundo Novo Efeito de 6-BA na brotação de gemas de explantes nodais de Coffea arabica cv. Mundo Novo

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    Luis Carlos da Silva Ramos

    2005-01-01

    Full Text Available Coffee plants can be micropropagated by nodal bud sprouting using the 6-benzylaminopurine (6-BA hormone. However, literature reports the use of a wide range of 6-BA, from 0.5 to 88.8 µM L-1. So, this study was performed to narrow that range. Nodal explants of Coffea arabica cv Mundo Novo obtained from in vitro plantlets were inoculated on gelled-MS medium supplemented with different concentrations of 6-BA. Two assays were carried out: in the first one, 6-BA was used at concentrations of 0, 5, 25, 50, and 100 µM L-1, being evaluated at 43 and 123 days. In the second experiment, dosis of 10, 20 and 30 µM L-1, have evaluated at 65 and 100 days. Treatments with 6-BA induced multiple sprouting from the nodal explants, which were best characterized around 100 days after inoculation. The nodal explants grew taller and showed multiple shoots, whereas the effect of 6-BA at 5 to 25 µM L-1 was similar to that with higher concentrations (50 and 100 µM L-1. Nodal explants yielded from 2.9 to 6.0 buds per node, achieving height of 1.3 to 1.5 cm at 5 to 25 µM L-1 of 6-BA, whereas they yielded from 4.3 to 4.9 buds per node but the sprouting grew about 0.8 cm at 50 and 100 µM L-1 of 6-BA. This study indicated that multiple sprouting of lateral buds can be induced by lower concentrations of 6-BA, for example, from 10 to 30 µM L-1, diminishing possible risks of somaclonal variation due to high levels of hormone concentration.O cafeeiro pode ser micropropagado via brotação de gemas laterais, aplicando o regulador de crescimento 6-benzilaminopurina (6-BA. Entretanto, a literatura apresenta ampla variação da dose empregada, desde 0.5 a 88.8 µM L-1. Assim, este estudo visou otimizar doses para explantes nodais do cafeeiro C. arabica cv Mundo Novo. Explantes nodais, obtidos de plântulas cultivadas in vitro, foram inoculados em meio MS geleificado, com adição de diferentes concentrações de 6-BA. Foram feitos dois experimentos: no primeiro, 6-BA foi

  19. Indução de calos friáveis em explantes foliares de Salix (Salyx humboldtiana Willd Induction of friable callus in leaf explants of Salix (Salyx humboldtiana Willd

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    Breno Régis Santos

    2005-06-01

    Full Text Available O salix é uma árvore ornamental adaptada a terrenos úmidos, sendo considerada uma espécie importante para a recomposição de áreas ciliares degradadas. A madeira pode ser empregada na indústria em geral. Apesar de produzir grande quantidade de sementes, estas não possuem alta percentagem de germinação. O presente trabalho teve como objetivo realizar o estabelecimento in vitro de salix através da indução e formação de calos friáveis em explantes foliares, visando a uma posterior regeneração de plantas para a propagação massal desta espécie. Explantes foliares foram inoculados em meio de cultura MS (MURASHIGE & SKOOG, 1962, acrescido de diferentes concentrações de ácido 2,4-diclorofenoxiacético (0; 1,0; 2,0; 4,0; 6,0; 8,0; 10,0 e 12,0mg L-1 e combinações entre ácido naftalenoacético e benzilaminopurina, ambos em concentrações de 0,0; 1,0; 2,0; 4,0; 6,0 e 8,0mg L-1. Os resultados demonstram que explantes inoculados na ausência de reguladores de crescimento não apresentam a formação de calos friáveis. Significativa produção de calos friáveis (90% é obtida utilizando-se 6,0mg L-1 de ácido 2,4-diclorofenoxiacético. A utilização de concentração individual de ácido naftalenoacético ou benzilaminopurina, além de induzir a calogênese também foi capaz de promover rizogênese.Salix is an ornamental tree adapted to humid soils being considered an important species used in depleted areas. Its wood may be used in all kinds of industries. Although the production of seeds is large, the germination is reduced. The objective of this work was to establish salix in vitro through the induction and formation of friable callus of leaf explants to provide future regeneration of plants for mass propagation of the species. Leaf explants were inoculated in MS medium (MURASHIGE & SKOOG, 1962 supplemented with different concentrations of 2-4-diclorofenoxiacetic acid (0; 1.0; 2.0; 4.0; 6.0; 8.0 and 12.0mg L-1 and combinations

  20. Indução de brotações em explantes de segmentos de folhas de plântulas de urucueiro em diferentes citocininas Induction of shoot buds in leaf explants of annatto seedlings in differents cytokinins

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    Jacqueline Leite Almeida

    1996-04-01

    Full Text Available Segmentos de folhas de plântulas de urucum (Bixa orellana L, foram cultivados in vitro, em um meio básico idealizado por Murashige & Skoog (1962, suplementado com 0,5mg/l dos reguladores do crescimento, benzilaminopurina (BAP, cinetina (KN e isopentenil-adenina (2iP, isolados ou combinados entre si, com o objetivo de avaliar qual a melhor citocinina ou combinação destas para indução de brotações de boa qualidade visando à micropropagação. Após 60 dias de cultivo, as gemas obtidas foram transferidas para um meio MS modificado, contendo a metade da formulação de sais minerais, sem a suplementação de reguladores do crescimento. Gemas de melhor qualidade foram obtidas nos tratamentos onde a KN e o BAP estavam em combinação. A KN sozinha não foi eficiente na indução de brotações. O maior número de gemas por explante foi obtido com KN, BAP e 2iP e no tratamento com apenas BAP.Excised leaf explants of annatto (Bixa orellana L. seedlings were cultivated on a Murashige & Skoog basal medium containing benzylaminopurine (BAP, kinetin (KN and isopentenyl-adenine (2iP individually or in combination, each at concentration of 0.5mg/l, with the purpose of evaluated the best cytokinins or cytokinins combination for shoot bud formation. After 60 days, regenerated shoots were transferred to half strenght MS medium without growth regulators. Best shoots were observed where KN and BAP were added in combination. Isolated KN was ineffective for shoot bud formation. The maximum average number of shoot buds per leaf explant was obtained on medium with BAP, KN and 2iP in combination and on medium supplied of isolated BAP.

  1. Meio de cultura e tipo de explante no estabelecimento in vitro de espécies de maracujazeiro Culture medium and type of explant in the in vitro establishment of passion fruit species

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    Gláucia Amorim Faria

    2007-01-01

    Full Text Available O Brasil é um dos principais centros de dispersão da variabilidade genética do gênero Passiflora. Sua auto-incompatibilidade aliada à incidência de doenças do sistema radicular e da parte aérea, desmatamentos e monocultivos promovem perda de material genético. Tendo-se em vista o risco de erosão genética, torna-se necessário a conservação da variabilidade em bancos de germoplasma, de grande interesse no melhoramento de plantas. Estudos em relação ao tipo de explante e concentração dos meios de cultivo são necessários para se determinar protocolo de estabelecimento e conservação in vitro de germoplasma de maracujá. O objetivo deste trabalho foi avaliar a influência da concentração dos sais e nutrientes do meio MS e tipos de explantes no estabelecimento e crescimento das espécies de maracujazeiro Passiflora giberti N. E. Brown, P. edulis Sims e P. laurifolia L. Em cada espécie de Passiflora havia características próprias quanto ao desenvolvimento in vitro. O meio de cultura MS completo e os segmentos nodais que continham a segunda gema axilar tiveram melhores resultados em relação às demais.Brazil is one of the main centers of genetic variability dispersion of the Passiflora genera. Its self incompatibility as well as disease incidence in its leaves and root system and, deforestation and monocultivation, promote loss of genetic material. Considering the risk of genetic erosion, the conservation of the variability in germplasm banks, which is of great interest in plant breeding, is necessary. Studies regarding the type of explant and concentration of the culture media are necessary in order to determine protocols of establishment and in vitro conservation of passion-fruit germplasm. The objective of the present work was to evaluate the influence of the salt and nutrient concentration in the MS culture medium and types of explants in the establishment and growth of the Passion fruit species: Passiflora giberti N. E

  2. The Effect of Plant Growth Regulators and Different Explants on the Response of Tissue Culture and Cell Suspension Cultures of German Chamomile (Matricaria chamomilla L.

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    L. Koohi,

    2014-07-01

    Full Text Available German chamomile (Matricaria chamomilla L. is one of the most important medicinal plants that its essential oils used in different medicinal industries. In this study which was carried out in 2013 growing season at the Faculty of Agricultural Sciences of the University of Mohaghegh Ardabili, the in vitro response of leaf and hypocotyl explants of German Chamomile in B5 medium supplemented with different levels of plant growth regulators including 2,4-D, naphthalene acetic acid (NAA, kinetin and 6-benzylaminopurine (BAP were investigated in a factorial experiment based on completely randomized design (CRD.In addition, cell suspension cultures were established and characterized. Hypocotyl and leaf explants exhibited cell proliferation and produced callus within 1-2 weeks. The highest fresh weight of the callus (264.1 mg was produced by leaf explants in the medium supplemented with 0.5 mg/l 2,4-D and 1 mg/l BAP. However, the leaf explants cultured on medium containing 1.5 mg/l 2,4-D showed the lowest cell proliferation and callus yield (40.42 mg. The highest percentage of root induction from leaf explants (58.73% was observed on the medium containing 4 mg/l 2,4-D and 1 mg/l Kin, and from hypocotyl explants (48.61% was observed on medium supplemented with 1.5 mg/l NAA. The 42.22% of calli derived from hypocotyl explants on B5 medium supplemented with 4 mg/l NAA and 3 mg/l BAP, were friable. Cell suspension cultures of German chamomile were established by transferring of hypocotyl-derived friable calli into the MS medium supplemented with 1.5 mg/l 2,4-D and 1 mg/l kinetin. The growth curve of cell proliferations started 4 days after culture and continued to grow until day 13th, where the cells entered stationary phase.

  3. Synovial Cytokines and the MSIS Criteria Are Not Useful for Determining Infection Resolution After Periprosthetic Joint Infection Explantation.

    Science.gov (United States)

    Frangiamore, Salvatore J; Siqueira, Marcelo B P; Saleh, Anas; Daly, Thomas; Higuera, Carlos A; Barsoum, Wael K

    2016-07-01

    Diagnosing periprosthetic joint infection (PJI) requires a combination of clinical and laboratory parameters, which may be expensive and difficult to interpret. Synovial fluid cytokines have been shown to accurately differentiate septic from aseptic failed total knee (TKA) and hip (THA) arthroplasties. However, after first-stage explantation, there is still no reliable test to rule out PJI before a second-stage reimplantation procedure. (1) Which synovial fluid cytokines have the highest diagnostic accuracy for PJI? (2) Which cytokine shows the greatest decrease associated with the resolution of infection in the same patient between explantation and subsequent reimplantation of an infected arthroplasty? (3) What is the accuracy of synovial fluid cytokines and the Musculoskeletal Infection Society (MSIS) criteria to rule out PJI after first-stage explantation? (4) What are the most studied synovial fluid cytokines for diagnosing PJI as reported in the literature and what are their cumulative diagnostic accuracy? Between May 2013 and March 2014, 104 patients with painful THA and TKA evaluated for possible PJI were included in our study. Of these, 90 (87%) had cytokine levels measured from synovial fluid samples collected as part of this prospective study (n = 33 hips, n = 57 knees). A second group of 35 patients (n = 36 samples) who presented during the same time period with an antibiotic spacer also had synovial cytokines measured before second-stage reimplantation. For the first group of 90 patients, the MSIS definition classified each joint at the time of surgery as infected (n = 31) or not infected (n = 59) and was used as the standard to test the accuracy in diagnosing PJI. Of the 35 patients with synovial marker data before second-stage surgery, 15 patients had cytokine measurements both at explantation and reimplantation and were used to quantify the change between stages. The reimplantation group had a minimum 1-year followup (with four [11%] patients lost to

  4. In Vitro propagation of enterolobium cyclocarpum (guanacaste from nodal explants of axenic seedlings

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    Araceli Rodríguez Sahagún

    2007-01-01

    Full Text Available Enterolobium cyclocarpum (Jacq. Griseb. es un árbol leguminoso de uso múltiple, el cual es considerado una especie amenazada, resultado de la sobreexplotación y las bajas tasas de propagación natural debidas a las características intrínsecas del árbol. Una alternativa para superar este problema es el establecimiento de sistemas para su propagación masiva en tiempos cortos. En este trabajo, se investigó un protocolo para la propagación in vitro de E. cyclocarpum utilizando los segmentos nodales axénicos obtenidos de plántulas germinadas in vitro. Las semillas colectadas en dos comunidades mexicanas fueron germinadas tanto ex vitro como in vitro, y se evaluó el efecto de un pre-tratamiento de escarificación térmica. Para los experimentos de propagación se seleccionaron semillas provenientes de sólo una de las comunidades, debido a que presentaban una menor variabilidad genética de acuerdo con marcadores RAPD y a que existía una gran variación en las respuestas observadas en lotes de semillas mezclados. Esta variación fisiológica presente en semillas mezcladas, probablemente refleja un efecto del genotipo.Los segmentos nodales obtenidos de las plántulas fueron cultivados en medio basal MS suplementado con 30 g/L de sacarosa en presencia de distintas concentraciones de ácido 1-naftalenacético (ANA en combinación con benziladenina (BA o kinetina (KIN. La mayor tasa de multiplicación (de 4.75 brotes por explante en promedio se obtuvo cuando el medio MS fue suplementado con 2.2 µM BA y 10.7 µM ANA. Los brotes obtenidos fueron enraizados en medio MS con la mitad de concentración de sales y sin reguladores de crecimiento. Las plántulas micropropagadas fueron aclimatadas y transferidas exitosamente a suelo con una tasa de sobrevivencia del 90%. Estas plantas eran morfológicamente similares a la planta madre y no se detectó variación entre ellas por el uso de marcadores RAPD, lo cual hace posible el uso de este

  5. Plant regeneration from cotyledonary explants of Eucalyptus camaldulensis dehn and histological study of organogenesis in vitro

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    Roberson Dibax

    2010-04-01

    Full Text Available The present work aimed at regenerating plants of Eucalyptus camaldulensis from the cotyledonary explants and describing the anatomy of the tissues during callogenesis and organogenesis processes, in order to determine the origin of the buds. The cotyledonary leaves of E. camaldulensis were cultured in Murashige and Skoog (MS, WPM and JADS media supplemented with 2.7 µM NAA and 4.44 µM BAP. The best results for bud regeneration were obtained on MS and WPM media (57.5 and 55% of calluses formed buds, respectively. Shoot elongation and rooting (80% were obtained on MS/2 medium (with half-strength salt concentration with 0.2% activated charcoal. Acclimatization was performed in the growth chamber for 48 h and then the plants were transferred to a soil:vermiculite mixture and cultured in a greenhouse. Histological studies revealed that the callogenesis initiated in palisade parenchyma cells and that the adventitious buds were formed from the calluses, indicating indirect organogenesis.Este trabalho teve como objetivo a obtenção de plantas de Eucalyptus camaldulensis a partir de folhas cotiledonares e o estudo da anatomia dos tecidos durante a calogênese e organogênese para determinar a origem das gemas. Folhas cotiledonares foram cultivadas em meios de cultura MS, WPM e JADS suplementados com 2,7 µM de ANA e 4,44 µM de BAP. Os melhores resultados para a regeneração de gemas foram obtidos com os meios MS e WPM. Para o alongamento e enraizamento, o meio de cultura MS/2 contendo 0,2% de carvão ativado apresentou-se eficiente para ambas as etapas. A aclimatização foi realizada mediante a abertura dos frascos na sala de crescimento por 48 horas, seguido da transferência para casa-de-vegetação com nebulização intermitente. Estudos histológicos foram conduzidos e revelaram que a calogênese teve início nas células do parênquima paliçádico e que as gemas adventícias formaram-se a partir dos calos, indicando a organogênese indireta.

  6. Development of efficient catharanthus roseus regeneration and transformation system using agrobacterium tumefaciens and hypocotyls as explants

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    Wang Quan

    2012-06-01

    Full Text Available Abstract Background As a valuable medicinal plant, Madagascar periwinkle (Catharanthus roseus produces many terpenoid indole alkaloids (TIAs, such as vindoline, ajamlicine, serpentine, catharanthine, vinblastine and vincristine et al. Some of them are important components of drugs treating cancer and hypertension. However, the yields of these TIAs are low in wild-type plants, and the total chemical synthesis is impractical in large scale due to high-cost and their complicated structures. The recent development of metabolic engineering strategy offers a promising solution. In order to improve the production of TIAs in C. roseus, the establishment of an efficient genetic transformation method is required. Results To develop a genetic transformation method for C. roseus, Agrobacterium tumefaciens strain EHA105 was employed which harbors a binary vector pCAMBIA2301 containing a report β-glucuronidase (GUS gene and a selectable marker neomycin phosphotransferase II gene (NTPII. The influential factors were investigated systematically and the optimal transformation condition was achieved using hypocotyls as explants, including the sonication treatment of 10 min with 80 W, A. tumefaciens infection of 30 min and co-cultivation of 2 d in 1/2 MS medium containing 100 μM acetosyringone. With a series of selection in callus, shoot and root inducing kanamycin-containing resistance media, we successfully obtained stable transgenic regeneration plants. The expression of GUS gene was confirmed by histochemistry, polymerase chain reaction, and genomic southern blot analysis. To prove the efficiency of the established genetic transformation system, the rate-limiting gene in TIAs biosynthetic pathway, DAT, which encodes deacetylvindoline-4-O-acetyltransferase, was transferred into C. roseus using this established system and 9 independent transgenic plants were obtained. The results of metabolite analysis using high performance liquid chromatography (HPLC

  7. Development of efficient Catharanthus roseus regeneration and transformation system using agrobacterium tumefaciens and hypocotyls as explants.

    Science.gov (United States)

    Wang, Quan; Xing, Shihai; Pan, Qifang; Yuan, Fang; Zhao, Jingya; Tian, Yuesheng; Chen, Yu; Wang, Guofeng; Tang, Kexuan

    2012-06-29

    As a valuable medicinal plant, Madagascar periwinkle (Catharanthus roseus) produces many terpenoid indole alkaloids (TIAs), such as vindoline, ajamlicine, serpentine, catharanthine, vinblastine and vincristine et al. Some of them are important components of drugs treating cancer and hypertension. However, the yields of these TIAs are low in wild-type plants, and the total chemical synthesis is impractical in large scale due to high-cost and their complicated structures. The recent development of metabolic engineering strategy offers a promising solution. In order to improve the production of TIAs in C. roseus, the establishment of an efficient genetic transformation method is required. To develop a genetic transformation method for C. roseus, Agrobacterium tumefaciens strain EHA105 was employed which harbors a binary vector pCAMBIA2301 containing a report β-glucuronidase (GUS) gene and a selectable marker neomycin phosphotransferase II gene (NTPII). The influential factors were investigated systematically and the optimal transformation condition was achieved using hypocotyls as explants, including the sonication treatment of 10 min with 80 W, A. tumefaciens infection of 30 min and co-cultivation of 2 d in 1/2 MS medium containing 100 μM acetosyringone. With a series of selection in callus, shoot and root inducing kanamycin-containing resistance media, we successfully obtained stable transgenic regeneration plants. The expression of GUS gene was confirmed by histochemistry, polymerase chain reaction, and genomic southern blot analysis. To prove the efficiency of the established genetic transformation system, the rate-limiting gene in TIAs biosynthetic pathway, DAT, which encodes deacetylvindoline-4-O-acetyltransferase, was transferred into C. roseus using this established system and 9 independent transgenic plants were obtained. The results of metabolite analysis using high performance liquid chromatography (HPLC) showed that overexpression of DAT

  8. High throughput Agrobacterium tumefaciens-mediated germline transformation of mechanically isolated meristem explants of cotton (Gossypium hirsutum L.).

    Science.gov (United States)

    Chen, Yurong; Rivlin, Anatoly; Lange, Andrea; Ye, Xudong; Vaghchhipawala, Zarir; Eisinger, Elizabeth; Dersch, Erik; Paris, Miriam; Martinell, Brian; Wan, Yuechun

    2014-01-01

    Agrobacterium tumefaciens mediates high frequency of germline transformation of cotton meristem explants. The meristem transformation system we developed is rapid, high throughput and genotype-flexible. We have developed a high throughput cotton transformation system based on direct Agrobacterium inoculation of mechanically isolated meristem explants of cotton (Gossypium hirsutum L.). The explants were inoculated with a disarmed A. tumefaciens strain, AB33 harboring a 2 T-DNA binary vector pMON114908. This vector contained a gene of interest, an intron-disrupted β-glucuronidase gene in one T-DNA, and a selectable marker gene, aadA in the other T-DNA. Critical factors, such as method of co-culture, culture temperature during selection, composition of selection medium, and selection scheme were found to influence transformation frequency. The cycle time from initial inoculation to the transplanting of transgenic plants to soil was 7-8 weeks. Stable integration of transgenes and their transmission to progeny were confirmed by molecular and genetic analyses. Transgenes segregated in the expected Mendelian fashion in the T1 generation for most of the transgenic events. It was possible to recover marker-free events in the T1 generation when utilizing a binary vector that contained the selectable marker and gene of interest expression cassettes on independent T-DNAs. The procedure presented here has been used to regenerate thousands of independent transgenic events from multiple varieties with numerous constructs, and we believe it represents a major step forward in cotton transformation technology.

  9. Effect of Explant Source and Growth Regulators on in vitro Callus Growth of Taxus baccata L. Washingtonii

    Directory of Open Access Journals (Sweden)

    Ivana Bjedov

    2002-01-01

    Full Text Available The effects of explant source, medium composition and growth regulators were examined in order to optimize the induction and selection of fast growing callus lines of European yew (Taxus baccata L. Washingtonii. Callus cultures were induced from isolated mature zygotic embryos or from segments of juvenile branches. Following two months of growth on induction medium (MS + 3.0 mg/L NAA, 0.5 mg/L kinetin, 100 mg/L arginine, 2.5 % sucrose and 0.8 % agar, callus proliferation was induced in 86.4 % of embryo explants and 100 % of branch-cutting explants. The growth potential of established callus lines was found to vary in response to genetic potential and culture medium composition. The growth rate of stem-derived callus obtained on induction medium was superior to that obtained using all other tested media modifications (duplication time 9.6 days. However, the growth of embryo-derived callus lines was enhanced by increasing the iron content from 27.8 to 55.6 mg/L FeSO4 ∙7H2O in the maintaining MS medium (duplication time for line E2 was 8.5 days. In two out of three embryo-derived lines, tissue growth was further improved by transferring onto modified B5 medium (duplication time for lines E2 end E5 was 4 and 5.7 days, respectively. HPLC analysis confirmed the presence of the anticancer agent cephalomannine in calli grown on B5 medium and a taxane-like substance in calli grown on MS medium.

  10. High-frequency in vitro plantlet regeneration from apical bud as a novel explant of Carum copticum L.

    Science.gov (United States)

    Salehi, Mansoureh; Hosseini, Bahman; Jabbarzadeh, Zohreh

    2014-05-01

    To develop an in vitro regeneration system to increase the recovery of Carum copticum L. plantlets as a part of developing a metabolic engineering program. The efficacy of different concentrations and combinations of 6-benzyladenine, indole-3-acetic acid and indole butyric acid on direct shoot regeneration and rooting of ajowan from apical bud explants were assessed. All explants were cultured on Murashige and Skoog (MS) medium supplemented with different combinations of 6-benzyl amino purine (BAP) (0, 2.2, 4.4, 8.8 µmol/L) and indole-3-acetic acid (IAA) (0, 0.5, 1.1, 2.2 µmol/L). The maximum shoot regeneration frequency (97.5%) and the highest number of shoots produced from apical buds (34 shoots per explant) were obtained on MS medium fortified with BAP (4.4 µmol/L) and IAA (0.5 µmol/L). Low shoot regeneration frequency was observed in BAP free treatments. The effects of different strengths of MS medium and various concentrations of IAA and indole-3- butyric acid on rooting rate, length and average number of roots were also investigated. Application of indole-3- butyric acid (6 µmol/L) in full-strength MS medium, was more effective than IAA and resulted in highest shoot regeneration frequency with the rooting rate of 100% and highest mean number of roots per shoot (41.8). The rooted plantlets were acclimatized successfully in greenhouse conditions with a survival rate of 90%. In this study, a simple and reliable regeneration and acclimatization protocol for Carum copticum has been presented. This protocol can be found very advantageous for a variety of purposes, including mass multiplication of Carum species, medicinal plant breeding studies and transgenic plant production.

  11. Influence de l'âge des explants primaires sur la régénération des ...

    African Journals Online (AJOL)

    Méthodologie et résultats: Les explants primaires prélevés sur les tiges âgées de 14, 21, 35 et 60 jours ont été désinfectés et mis en culture sur deux milieux de culture ... Conclusions et application des résultats: La conservation de toutes les variétés d'igname de la Côte d'Ivoire sous forme de vitroplants par la technique de ...

  12. Plant regeneration from in vitro-derived leaf and petiole explants of Viola cornuta L. 'Lutea Splendens'

    OpenAIRE

    Antonić, Dragana; Trajković, Milena; Cingel, Aleksandar; Subotić, Angelina; Jevremović, Slađana

    2017-01-01

    In vitro-derived petiole and leaf explants of Viola cornuta L. 'Lutea Splendens' were cultured on half-strength Murashige and Skoog (MS) medium with several concentrations of 6-benzylaminopurin (BAP), 6-furfurylaminopurine (KIN), N-phenyl-N'(1,2,3-thidiazol-5-yl) urea (TDZ) or N-(2-chloro-4-pyridyl)-N'phenylurea (CPPU) alone or supplemented with indole-3-acetic acid (IAA) or a-naphthaleneacetic acid (NAA). The most efficient direct adventitious shoot induction (21%) without callus formation w...

  13. Use of explant models as a tool to assess the anti HIV activity of microbicides in preclinical studies

    OpenAIRE

    Villegas, Guillermo

    2016-01-01

    Villegas, G. (2016). Use of explant models as a tool to assess the anti HIV activity of microbicides in preclinical studies. (Tesis de posgrado). Bernal, Argentina: Universidad Nacional de Quilmes. Actualmente existe una necesidad, no alcanzada todavía de proteger a las mujeres contra las enfermedades de transmisión sexual y el embarazo no deseado. En 2013 se produjeron 2,1 millones de nuevas infecciones por VIH y 1,5 millones de muertes debidas al VIH/SIDA en todo el mundo. Los casos repo...

  14. Linear energy transfer dependence of the effects of carbon ion beams on adventitious shoot regeneration from in vitro leaf explants of Saintpaulia ionahta.

    Science.gov (United States)

    Zhou, Libin; Li, Wenjian; Yu, Lixia; Li, Ping; Li, Qiang; Ma, Shuang; Dong, Xicun; Zhou, Guangming; Leloup, Corinne

    2006-07-01

    To determine the effects of carbon ion beams with five different linear energy transfer (LET) values on adventitious shoots from in vitro leaf explants of Saintpaulia ionahta Mauve cultivar with regard to tissue increase, shoots differentiation and morphology changes in the shoots. In vitro leaf explant samples were irradiated with carbon ion beams with LET values in the range of 31 approximately 151 keV/microm or 8 MeV of X-rays (LET = 0.2 keV/microm) at different doses. Fresh weight increase, surviving fraction and percentage of the explants with regenerated malformed shoots in all the irradiated leaf explants were statistically analysed. The fresh weight increase (FWI) and surviving fraction (SF) decreased dramatically with increasing LET at the same doses. In addition, malformed shoots, including curliness, carnification, nicks and chlorophyll deficiency, occurred in both carbon ion beam and X-ray irradiations. The induction frequency with the former, however, was far more than that with the X-rays. This work demonstrated the LET dependence of the relative biological effectiveness (RBE) of tissue culture of Saintpaulia ionahta according to 50% FWI and 50% SF. After irradiating leaf explants with 5 Gy of a 221 MeV carbon ion beam having a LET value of 96 keV/microm throughout the sample, a chlorophyll-deficient (CD) mutant, which could transmit the character of chlorophyll deficiency to its progeny through three continuous tissue culture cycles, and plantlets with other malformations were obtained.

  15. Investigations on the growth and metabolism of cultured explants of Daucus carota : III. The range of responses induced in carrot explants by exogenous growth factors and by trace elements.

    Science.gov (United States)

    Steward, F C; Rao, K V

    1970-06-01

    The responses of carrot explants to various growth-promoting agents and to certain trace elements with which they interact have been investigated. A great range in the metabolic behavior of the tissue may be brought about in this way. The responses to the exogenously applied substances are described in terms of the growth of the carrot explants in fresh weight and number of cells and also in terms of their metabolism, as shown by the final content and composition of the non-protein N compounds, by the relations between protein and non-protein (alcohol-soluble) N and by the content of nucleic acid in the cultured tissue.The growth-promoting agents employed consisted of (1) the balanced complex of factors found in coconut milk, (2) an active isolate from Aesculus (AFaesc), which is one of a class of growth factors (AF1) that interact with inositol (AF1+inositol) and which in this sense comprise growth-promoting System I, (3) the substance zeatin (Zeat) which is typical of a class of active factors (AF2) that interact with indoleacetic acid (AF2+IAA) and which, therefore, function as a growth promoting complex termed System II in the culture of carrot tissue.The carrot explants stimulated by coconut milk grew better than those stimulated by the other combinations of growth factors and they converted their soluble N more effectively to protein. The growth, whether it was induced by coconut milk or by System I or II, and other specific effects attributable to the growth factors employed were markedly affected also by the elements iron and molybdenum.The carrot explants that had responded to coconut milk emphasized alanine in their soluble, non-protein, nitrogenous pool, whereas those subjected to the active components of System I or of System II as clearly emphasized glutamine as the prominent non-protein, nitrogen-rich compound.The partial effects due to the component parts of System I (AFaesc or inositol) and to the component parts of System II (Zeat. or IAA), as

  16. THE PHENOLS ACCUMULATION IN TRANSFORMED ROOT CULTURES OF DIFFERENT EXPLANTS SOURCES OF COMMON BUCKWHEAT (Fagopyrum esculentum Moench

    Directory of Open Access Journals (Sweden)

    O. V. Sytar

    2013-06-01

    Full Text Available The growth parameters of transformed root cultures, total phenolic content and phenolic acids composition has been studied in root cultures, which were obtained from various explants of buckwheat by Agrobacterium rhizogenes strains A4. The methods of obtaining of the transformed root cultures, total phenol estimation, gas-liquid chromatography and polymerase chain reaction has been used. Elevated levels of total phenols in transformed roots of buckwheat from different sources of explants have been found. The high content of chlorogenic, p-hydroxybenzoic, p-anisic and caffeic acids has been discovered in the root cultures, which can be used for their industrial production. Maximal root growth was equal 21.2 g/l of dry weight in the roots as source for root culture, 17.7 g/l with leaves and 14.6 g/l with stems at 3 week after placement. Molecular analysis by polymerase chain reaction amplification was confirmed that the rol B gene (652 bp which transferred info hairy roots from Ri-plasmid in Agrobacterium rhizogenes is responsible for induction of root from plant species.

  17. The Effects of Gamma Irradiation on the Growth and Propagation of In-Vitro Chrysanthemum Shoot Explants (cv. Yellow Puma

    Directory of Open Access Journals (Sweden)

    I. Dwimahyani

    2010-08-01

    Full Text Available The study on the effect of gamma irradiation on in-vitro shoot growth of chrysanthemum cv. Yellow Puma has been carried out. The aim of the study was to observe genetic variability of shoot growth caused by gamma irradiation. Shoot explants with four leaves were irradiated by gamma with dose of 10, 15 and 20 Gy with 3 replications at each of dose. The irradiated shoot explants were then transferred into fresh MS solid medium and placed in a growth room. Observation was performed on number of leaves and branches on M1V0 generation, while plantlets height and number of branches were observed a M1V1 generation. Number of survival plantlets and multiplication rate on three subsequent subcultures were observed as well. Results showed that gamma rays with dose of 20 Gy inhibited growth of leaves as much as 50% compared to control (shoots without irradiation, and branches 73.7% in three weeks. Observation on multiplication rate at M1V1 generation showed that gamma irradiation with dose of 10 Gy promoted multiplication rate as much as 10% higher than control. It can be concluded that in vitro mutagenesis using gamma iradiation with dose of 10 to 15 Gy can be used for inducing genetic variability of chrysanthemum cv. Yellow Puma.

  18. Influence of genotype and explant source on indirect organogenesis by in vitro culture of leaves of Melia azedarach L.

    Science.gov (United States)

    Vila, S K; Rey, H Y; Mroginski, L A

    2004-04-01

    In vitro regeneration of shoots from leaf explants of the Paradise tree (Melia azedarach L.) was studied. Three different portions (proximal portion, distal portion and rachis of the leaflets) of three developmental stages (folded, young still expanding and completely expanded) of leaves of 10-15 year old plants of seven genotypes were cultured on Murashige and Skoog (1962) medium (MS) supplemented with 1 mg x l(-1) benzylaminopurine (BAP) + 0.1 mg x l(-1) kinetin (KIN) + 3 mg x l(-1) adenine sulphate (ADS). The rachis of the leaflets of the completely expanded leaves was found to be the most responsive tissue, in most of the genotypes employed. Shoot regeneration occurred in leaf explants of all the genotypes tested. The best genotype for shoot regeneration was clone 4. Rooting was induced on MS medium supplemented with 2.5 mg x l(-1) 3-indolebutyric acid, IBA, (4 days) followed by subculture on MS lacking growth regulators (26 days). Complete plants were transferred to soil.

  19. Plant Explants Grown on Medium Supplemented with Fe3O4 Nanoparticles Have a Significant Increase in Embryogenesis

    Directory of Open Access Journals (Sweden)

    Inese Kokina

    2017-01-01

    Full Text Available Development of nanotechnology leads to the increasing release of nanoparticles in the environment that results in accumulation of different NPs in living organisms including plants. This can lead to serious changes in plant cultures which leads to genotoxicity. The aims of the present study were to detect if iron oxide NPs pass through the flax cell wall, to compare callus morphology, and to estimate the genotoxicity in Linum usitatissimum L. callus cultures induced by different concentrations of Fe3O4 nanoparticles. Two parallel experiments were performed: experiment A, where flax explants were grown on medium supplemented with 0.5 mg/l, 1 mg/l, and 1.5 mg/l Fe3O4 NPs for callus culture obtaining, and experiment B, where calluses obtained from basal MS medium were transported into medium supplemented with concentrations of NPs identical to experiment A. Obtained results demonstrate similarly in both experiments that 25 nm Fe3O4 NPs pass into callus cells and induce low toxicity level in the callus cultures. Nevertheless, calluses from experiment A showed 100% embryogenesis in comparison with experiment B where 100% rhizogenesis was noticed. It could be associated with different stress levels and adaptation time for explants and calluses that were transported into medium with Fe3O4 NPs supplementation.

  20. A dose titration of triamcinolone acetonide on insulin-like growth factor-1 and interleukin-1-conditioned equine cartilage explants.

    Science.gov (United States)

    Sandler, E A; Frisbie, D D; McIlwraith, C W

    2004-01-01

    Previous in vitro pilot studies have defined a potentially beneficial effect of insulin-like growth factor-1 (IGF-1) and triamcinolone acetonide (TA) on interleukin-1 (IL-1)-conditioned equine cartilage. Furthermore, an optimal dose for IGF-1 treatment alone has been documented previously using the same test system as in the current project. To perform a dose titration of TA on IL-1-conditioned equine articular cartilage explants in the presence of an optimised IGF-1 dose, in order to optimise a triamcinolone concentration for use in combination with IGF-1 for future investigations. Cartilage explants were harvested from the distal femur of a normal horse. The effect of a clinically relevant TA dose range was evaluated in the presence of IL-1 and IGF-1 through measurement of proteoglycan (PG) matrix metabolism (synthesis and degradation). TA and IGF-1 in combination inhibited the IL-1-induced release of PG matrix components (glycosaminoglycan or GAG) from the articular cartilage, as well as producing a significant increase in GAG synthesis. This experiment provided proof of principle that a combination treatment appears to be able to combat the IL-1-induced matrix depletion, while enhancing anabolic metabolism within the articular cartilage. The use of IGF-1 in conjunction with TA in vivo has the potential to provide beneficial anabolic effects not seen with TA alone.

  1. MRI screening for silicone breast implant rupture: accuracy, inter- and intraobserver variability using explantation results as reference standard

    Energy Technology Data Exchange (ETDEWEB)

    Maijers, M.C.; Ritt, M.J.P.F. [VU University Medical Centre, Department of Plastic, Reconstructive and Hand Surgery, De Boelelaan 1117, PO Box 7057, Amsterdam (Netherlands); Niessen, F.B. [VU University Medical Centre, Department of Plastic, Reconstructive and Hand Surgery, De Boelelaan 1117, PO Box 7057, Amsterdam (Netherlands); Jan van Goyen Clinic, Department of Plastic Surgery, Amsterdam (Netherlands); Veldhuizen, J.F.H. [MRI Centre, Amsterdam (Netherlands); Manoliu, R.A. [MRI Centre, Amsterdam (Netherlands); VU University Medical Centre, Department of Radiology, Amsterdam (Netherlands)

    2014-06-15

    The recall of Poly Implant Prothese (PIP) silicone breast implants in 2010 resulted in large numbers of asymptomatic women with implants who underwent magnetic resonance imaging (MRI) screening. This study's aim was to assess the accuracy and interobserver variability of MRI screening in the detection of rupture and extracapsular silicone leakage. A prospective study included 107 women with 214 PIP implants who underwent explantation preceded by MRI. In 2013, two radiologists blinded for previous MRI findings or outcome at surgery, independently re-evaluated all MRI examinations. A structured protocol described the MRI findings. The ex vivo findings served as reference standard. In 208 of the 214 explanted prostheses, radiologists agreed independently about the condition of the implants. In five of the six cases they disagreed (2.6 %), but subsequently reached consensus. A sensitivity of 93 %, specificity of 93 %, positive predictive value of 77 % and negative predictive value of 98 % was found. The interobserver agreement was excellent (kappa value of 0.92). MRI has a high accuracy in diagnosing rupture in silicone breast implants. Considering the high kappa value of interobserver agreement, MRI appears to be a consistent diagnostic test. A simple, uniform classification, may improve communication between radiologist and plastic surgeon. (orig.)

  2. Toxic and therapeutic effects of Nifurtimox and Benznidazol on Trypanosoma cruzi ex vivo infection of human placental chorionic villi explants.

    Science.gov (United States)

    Rojo, Gemma; Castillo, Christian; Duaso, Juan; Liempi, Ana; Droguett, Daniel; Galanti, Norbel; Maya, Juan Diego; López-Muñoz, Rodrigo; Kemmerling, Ulrike

    2014-04-01

    Nifurtimox (Nfx) and Benznidazole (Bnz) are the only available drugs in use for the treatment of Chagas disease. These drugs are recommended but not fully validated in evidence-based medicine and reports about the differential toxicity of both drugs are controversial. Here, we evaluated the toxic and therapeutic effects of Nfx and Bnz on human placental chorionic villi explants (HPCVE) during ex vivo infection of Trypanosoma cruzi, performing histopathological, histochemical, immunohistochemical as well as immunofluorescence analysis of the tissue. Additionally, we determined the effect of both drugs on parasite load by real time PCR. Bnz prevents the parasite induced tissue damage in ex vivo infected HPCVE compared to Nfx, which is toxic per se. The presence of T. cruzi antigens and DNA in infected explants suggests that these drugs do not impair parasite invasion into the HPCVE. Additionally, our results confirm reports suggesting that Bnz is less toxic than Nfx and support the need for the development of more effective and better-tolerated drugs. Copyright © 2014 Elsevier B.V. All rights reserved.

  3. Pre-culturing of nodal explants in thidiazuron supplemented liquid medium improves in vitro shoot multiplication of Cassia angustifolia.

    Science.gov (United States)

    Siddique, I; Abdullwahab Bukhari, N; Perveen, K; Siddiqui, I; Anis, M

    2013-09-01

    An in vitro propagation system for Cassia angustifolia Vahl. has been developed. Due to the presence of sennosides, the demand of this plant has increased manyfold in global market. Multiple shoots were induced by culturing nodal explants excised from mature plants on a liquid Murashige and Skoog [8] medium supplemented with 5-100 μM of thidiazuron (TDZ) for different treatment duration (4, 8, 12 and 16 d). The optimal level of TDZ supplemented to the culture medium was 75 μM for 12 d induction period followed by subculturing in MS medium devoid of TDZ as it produced maximum regeneration frequency (87%), mean number of shoots (9.6 ± 0.33) and shoot length (4.4 ± 0.46 cm) per explant. A culture period longer than 12 d with TDZ resulted in the formation of fasciated or distorted shoots. Ex vitro rooting was achieved when the basal cut end of regenerated shoots was dipped in 200 μM indole-3-butyric acid (IBA) for half an hour followed by their transplantation in plastic pots filled with sterile soilrite where 85% plantlets grew well and all exhibited normal development. The present findings describe an efficient and rapid plant regeneration protocol that can further be used for genetic transformation studies.

  4. Suppression of MMP activity in bovine cartilage explants cultures has little if any effect on the release of aggrecanase-derived aggrecan fragments

    DEFF Research Database (Denmark)

    Wang, Bijue; Chen, Pingping; Jensen, Anne-Christine Bay

    2009-01-01

    -epitope specific immunoassays; (1) sandwich (342)FFGVG-G2 ELISA, (2) competition NITEGE(373)ELISA (3) sandwich G1-NITEGE(373 )ELISA (4) competition (374)ARGSV ELISA, and (5) sandwich (374)ARGSV-G2 ELISA all detecting aggrecan fragments, and (6) sandwich CTX-II ELISA, detecting C-telopeptides of type II collagen......- and aggrecanase-derived fragments of aggrecan and type II collagen into the supernatant of bovine cartilage explants cultures using neo-epitope specific immunoassays, and to associate the release of these fragments with the activity of proteolytic enzymes using inhibitors. FINDINGS: Bovine cartilage explants were....... We found that (1) aggrecanase-derived aggrecan fragments are released in the early (day 2-7) and mid phase (day 9-14) into the supernatant from bovine explants cultures stimulated with catabolic cytokines, (2) the release of NITEGE(373 )neo-epitopes are delayed compared to the corresponding (374...

  5. Agrobacterium-mediated genetic transformation of Pogostemon cablin (Blanco) Benth. Using leaf explants: bactericidal effect of leaf extracts and counteracting strategies.

    Science.gov (United States)

    Paul, Anamika; Bakshi, Souvika; Sahoo, Debee Prasad; Kalita, Mohan Chandra; Sahoo, Lingaraj

    2012-04-01

    An optimized protocol for Agrobacterium tumefaciens-mediated transformation of patchouli using leaf disk explants is reported. In vitro antibacterial activity of leaf extracts of the plants revealed Agrobacterium sensitivity to the extracts. Fluorometric assay of bacterial cell viability indicated dose-dependent cytotoxic activity of callus extract against Agrobacterium cells. Addition of 0.1% Tween 20 and 2 g/l L-glutamine to Agrobacterium infection medium counteracted the bactericidal effect and significantly increased the T-DNA delivery to explants. A short preculture of explants for 2 days followed by infection with Agrobacterium in medium containing 150 μM of acetosyringone were found essential for efficient T-DNA delivery. Cocultivation for 3 days at 22 °C in conjunction with other optimized factors resulted in maximum T-DNA delivery. The Agrobacterium-mediated transformation of leaf disk explants were found significantly related to physiological age of the explants, age and origin of the of the donor plant. Leaf explants from second node of the 3-month-old in vivo plants showed highest transformation efficiency (94.3%) revealed by transient GUS expression assay. Plants selected on medium containing 20 mg/l kanamycin showed stable GUS expression in leaves and stem. The elongated shoots readily developed roots on kanamycin-free rooting medium and on transfer to soil, plants were successfully established. Polymerase chain reaction (PCR) and reverse-transcriptase PCR analysis in putative plants confirmed their transgenic nature. The established transformation method should provide new opportunities for the genetic improvement of patchouli for desirable trait.

  6. Inferior dermoglandular flap for autologous breast remodeling following explantation of breast implants in ptotic breasts: a case report and literature search

    Directory of Open Access Journals (Sweden)

    Umar Daraz Khan

    2015-04-01

    Full Text Available Explantation following aesthetic mammoplasty without implant replacement is quite uncommon and often leaves the patient worse off than prior to mammoplasty. A case is presented here in which patient's own tissue was used as an inferior dermoglandular flap for autologous breast remodeling. Inferior dermal flap has been described for breast reconstruction and simultaneous augmentation mammoplasty with mastopexy for prosthesis cover in the lower pole of the breast, but its use following explantation without implant replacement has not been described for breast remodeling and volume conservation.

  7. Screening for in vitro shoot-forming capacity of seedling explants in bell pepper (Capsicum annuum L.) genotypes and efficient plant regeneration using thidiazuron.

    Science.gov (United States)

    Szász, A; Nervo, G; Fári, M

    1995-07-01

    In vitro shoot regeneration ability of 17 (7 Italian and 10 Hungarian) bell pepper genotypes was investigated using excised cotyledons and rooted hypocotyls as explants. Most of the Italian genotypes and two of the Hungarian genotypes responded well, producing shoots from rooted hypocotyls. Only two genotypes (one Italian and one Hungarian) gave a weak response using cotyledons. For direct shoot induction in these explants, in addition to the methods cited in the relevant papers, a new method was applied using thidiazuron as a cytokinin. Shoots were successfully regenerated from cotyledons of two Italian and two Hungarian genotypes using thidiazuron which were considered to be non responsive to the usual methods.

  8. Tumours with elevated levels of the Notch and Wnt pathways exhibit efficacy to PF-03084014, a γ-secretase inhibitor, in a preclinical colorectal explant model

    OpenAIRE

    Arcaroli, J J; Quackenbush, K S; Purkey, A; Powell, R W; Pitts, T.M.; Bagby, S.; Tan, A C; Cross, B; McPhillips, K; Song, E-K; Tai, W. M.; Winn, R A; Bikkavilli, K; VanScoyk, M; Eckhardt, S G

    2013-01-01

    Background: Dysregulation of the Notch pathway has been identified to play an important role in the development and progression of colorectal cancer (CRC). In this study, we used a patient-derived CRC explant model to investigate the efficacy of the clinical γ-secretase inhibitor (GSI) PF-03084014. Methods: A total of 16 CRC explants were treated with PF-03084014. Knockdown of RBPj κ gene was used to determine the specificity of PF-03084014. Evaluation of the Notch and Wnt pathways in CRC exp...

  9. Sour orange bud regeneration and in vitro plant development related to culture medium composition and explant type Regeneração de gemas de laranja-azeda e desenvolvimento in vitro de plantas em função da composição do meio de cultura e tipo de explante

    Directory of Open Access Journals (Sweden)

    Rosely Pereira da Silva

    2010-03-01

    Full Text Available In order to evaluate the formation of adventitious buds and in vitro regeneration of sour orange plants (Citrus aurantium L. two organogenesis-inducing experiments were conducted. In the first experiment, the induction and in vitro regeneration of adventitious buds were tested on epicotyl and internodal segments under the influence of BAP or KIN associated with NAA. The second experiment evaluated the in vitro regeneration of sour orange plants related to different explant types (epicotyls segments, internodal segments of in vitro germinated plantlets and internodal segments of greenhouse cultivated plants. Data collected on both experiments included the percentage of responsive explants (explants that formed buds, and the number of buds per explant. The addition of BAP showed the best organogenic response. In vitro germinated epicotyl segments and internodal segments are recommended as explants for sour orange in vitro organogenesis. Rooting of regenerated shoots was achieved without the need of auxin in the medium.Com o objetivo de avaliar a formação de gemas adventícias e regeneração in vitro de plantas de laranja-azeda (Citrus aurantium L., foram realizados dois experimentos de indução à organogênese. No primeiro experimento, a indução e a regeneração in vitro de gemas adventícias foram investigadas a partir de segmentos internodais e segmentos de epicótilo sob o efeito de BAP ou CIN associados com ANA. O segundo experimento avaliou a regeneração in vitro de plantas de laranja-azeda em função do tipo de explante (segmentos de epicótilo, segmentos internodais de plantas germinadas in vitro e segmentos internodais de plantas cultivadas em casa de vegetação. Os dados coletados em ambos os experimentos incluíram a porcentagem de explantes responsivos (explantes que formaram gemas e número de gemas por explante. A adição de BAP revelou a melhor resposta organogenética. Segmentos de epicótilo e segmentos internodais s

  10. Micropropagação das bananeiras 'Prata-Anã' e 'FHIA 01' a partir de explantes de plantas tratadas com paclobutrazol Micropropagation of banana trees 'Prata Anã' and 'FHIA 01' explants from plants treated with paclobutrazol

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    Djalmary de Souza e Souza

    2010-06-01

    Full Text Available Com o objetivo de avaliar o desenvolvimento in vitro de explantes das bananeiras 'Prata-Anã' e 'FHIA 01' provenientes de plantas tratadas com paclobutrazol (PBZ, conduziu-se este experimento no Laboratório de Cultura de Células e Tecidos Vegetais, Setor de Fruticultura, DFT/UFV. Utilizou-se esquema fatorial 2 x 5, correspondendo às duas cultivares ('Prata-Anã' e 'FHIA 01' e cinco doses de PBZ (0,0; 0,5; 1,0; 1,5 e 2,0 g i.a. planta-1, em delineamento experimental inteiramente ao acaso, com número variável de repetições. Avaliaram-se, na parte aérea: taxa de brotação, altura e diâmetro, número de folhas, massa fresca e seca e intensidade da cor verde; e no sistema radicular: porcentagem de enraizamento, número de raízes, comprimento da maior raiz, massa fresca e seca, e a relação massa da raiz:massa da parte aérea. Foi observado que a altura da parte aérea foi reduzida a partir de 1,13 g i.a. planta-1, em ambas as cultivares. O diâmetro não foi alterado com o aumento das doses, mas nos explantes da 'Prata Anã' os diâmetros foram maiores que 'FHIA 01'. Para o número de folhas, as massas fresca e seca não apresentaram diferenças entre as doses e entre as cultivares. A intensidade da cor verde aumentou linearmente com o aumento das doses, em ambas as cultivares. A partir de 1,07 g i.a. planta-1, a taxa de brotação da 'Prata-Anã' foi inibida, enquanto a da 'FHIA 01' foi estimulada a partir de 0,85 g i.a. planta-1. Houve interação significativa para a porcentagem de enraizamento, onde se observaram valores mais baixos para a cultivar 'Prata-Anã' (64,71%, em relação à 'FHIA 01' (95,83%, na maior dose. Não foi observado efeito das doses de PBZ sobre a porcentagem de enraizamento de explantes da 'Prata-Anã', enquanto para 'FHIA 01' houve aumento linear do enraizamento com o aumento das doses, sendo que o maior valor encontrado foi de 95,18%. As demais características aumentaram linearmente com o aumento das doses de

  11. Nicotine promotes rooting in leaf explants of in vitro raised seedlings of tomato, Lycopersicon esculentum Miller var. Pusa Ruby.

    Science.gov (United States)

    Bamel, Kiran; Gupta, Rajendra; Gupta, Shrish C

    2015-11-01

    Nicotine promotes rooting in leaf explants of tomato (Lycopersicon esculentum Miller var. Pusa Ruby). Nicotine at 10(-9) to 10(-3) M concentrations was added to the MS basal medium. The optimum response (three-fold increase in rooting) was obtained at 10(-7) M nicotine-enriched MS medium. At the same level i.e. 10(-7) M Nicotine induced dramatic increase (11-fold) in the number of secondary roots per root. We have shown earlier that exogenous acetylcholine induces a similar response in tomato leaves. Since nicotine is an agonist of one of the two acetylcholine receptors in animals, its ability to simulate ACh action in a plant system suggests the presence of the same molecular mechanism operative in both, animal and plant cells. Copyright © 2015 Elsevier B.V. All rights reserved.

  12. Pulmonary arterial lesions in explanted lungs after transplantation correlate with severity of pulmonary hypertension in chronic obstructive pulmonary disease

    DEFF Research Database (Denmark)

    Carlsen, Jørn; Hasseriis Andersen, Kasper; Boesgaard, Søren

    2013-01-01

    BACKGROUND: Pulmonary vascular findings are largely unreported in end-stage chronic obstructive pulmonary disease (COPD). METHODS: Pulmonary vascular lesions in explanted lungs from 70 patients with COPD/emphysema or α-1-antitrypsin deficiency were analyzed retrospectively. Patients were stratified...... by the presence and severity of pulmonary hypertension (PH) assessed by right-heart catheterization in 3 hemodynamically distinct groups: (1) non-PH (mean pulmonary arterial pressure [mPAP]50 mm Hg; median HE Grade 4 (range 3-6), with generalized arterial dilatation and plexiform lesions. CONCLUSIONS: The extent...... of pulmonary vascular lesions in COPD correlate with the severity of PH. Morphologic lesions similar to those characteristic of IPAH can be observed as PH in COPD progresses to levels characteristic of IPAH....

  13. The gallium complex KP46 exerts strong activity against primary explanted melanoma cells and induces apoptosis in melanoma cell lines

    Science.gov (United States)

    Valiahdi, Seied Mojtaba; Heffeter, Petra; Jakupec, Michael A.; Marculescu, Rodrig; Berger, Walter; Rappersberger, Klemens; Keppler, Bernhard K.

    2012-01-01

    The antineoplastic properties of gallium are well documented. Owing to their robust accumulation of gallium, melanoma cells should be amenable to gallium-based anticancer drugs. With the aim of improving the disappointingly low activity of inorganic gallium salts, we have developed the orally bioavailable gallium complex KP46 [tris(8-quinolinolato)gallium(III)] that was already successfully studied in a phase I clinical trial. To assess its therapeutic potential in malignant melanoma, its antiproliferative effects were investigated in series of human cell lines and primary explanted melanoma samples by means of the MTT [3-(4,5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide] assay and the Human Tumor Cloning Assay, respectively. When compared with other cell lines, the majority of melanoma cells rank among the KP46-sensitive cell lines (50% inhibitory concentration values: 0.8–3.7 μmol/l). Clinically achievable concentrations of KP46 proved to be highly effective in melanoma cells from primary explants of cutaneous and lymph node metastases. Colony growth was inhibited in 10 of 10 specimens by 5 lmol/l KP46 (corresponding to the steady-state plasma concentration measured earlier in a study patient) and in four of 10 specimens by 0.5 μmol/l KP46. In-vitro potency of KP46 is higher than that of dacarbazine or fotemustine and comparable with that of cisplatin. The effects induced by KP46 in melanoma cell lines involve cell cycle perturbations (S-phase arrest) and apoptosis (activation of caspase-9, PARP [poly(ADP-ribose) polymerase] cleavage, formation of apoptotic bodies). No effects on DNA secondary structure could be observed in an electrophoretic mobility shift assay using double-stranded plasmid DNA. Thus, further studies on the therapeutic applicability of KP46 in malignant melanoma are warranted. PMID:19584767

  14. A valued Indian medicinal plant – Begonia malabarica Lam. : Successful plant regeneration through various explants and field performance

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    Sevanan Rajeshkumar

    2009-05-01

    Full Text Available A cost-effective and efficient protocol has been described in the present work for large-scale and rapid in vitro propagation of a valuable medicinal herb Begonia malabarica Lam. (Begoniaceae by shoot auxillary-bud proliferation and organogenesis on MS medium supplemented with 6-benzylaminopurine (BA; 0.0-8.8 mg/l and indole-3-acetic acid (IAA; 0.0-2.88 mg/l at different concentrations, either alone or in combinations. Initiation of callus formation from the base of the leaf lamina was observed on MS supplemented with BA, IAA and adenine sulphate. Root induction on shoots was achieved on full strength MS with IAA/ indole-3-butyric acid (IBA at different concentrations. MS medium with 4.4 mg/l BA and 1.4 mg/l IAA elicited the maximum number of shoots (10 multiple shoots from nodal explants. Leaf-based callus differentiated into more than 28 shoots on MS with 150 mg/l adenine sulphate. The regenerated shoots were rooted on MS with 1.2 mg/l IBA within ten days. Almost 95% of the rooted shoots survived hardening when transferred to the field. The regenerated plants did not show any morphological change and variation in levels of secondary metabolites when compared with the mother stock. Thus, a reproduction of B. malabarica was established through nodal and leaf explants. This protocol can be exploited for conservation and commercial propagation of this medical plant in the Indian subcontinent and might be useful for genetic improvement programs.

  15. Monocyte imaging after myocardial infarction with 19F MRI at 3 T: a pilot study in explanted porcine hearts.

    Science.gov (United States)

    Bönner, F; Merx, M W; Klingel, K; Begovatz, P; Flögel, U; Sager, M; Temme, S; Jacoby, C; Salehi Ravesh, M; Grapentin, C; Schubert, R; Bunke, J; Roden, M; Kelm, M; Schrader, J

    2015-06-01

    Inflammation is a hallmark of cardiac healing after myocardial infarction and it determines subsequent cardiovascular morbidity and mortality. The aim of the present study was to explore whether inflammation imaging with two perfluorocarbon (PFC) nanoemulsions and fluorine magnetic resonance imaging ((19)F MRI) is feasible at 3.0 T with sufficient signal-to-noise ratio (SNR) using explanted hearts, an (19)F surface coil and dedicated MR sequences. Acute myocardial infarction (AMI) was induced by balloon angioplasty (50 min) of the distal left anterior descending artery in 12 pigs. One day thereafter, PFCs were injected intravenously to label circulating monocytes. Either emulsified perfluoro-15-crown-5 ether or already clinically applied perfluorooctyl bromide (PFOB) was applied. Four days after AMI and immediately after gadolinium administration, hearts were explanted and imaged with a 3.0 T Achieva MRI scanner. (19)F MRI could be acquired with an SNR of >15 using an in-plane resolution of 2 × 2 mm(2) within <20 min for both agents. Combined late gadolinium enhancement (LGE) and (19)F MRI revealed that (19)F signal was inhomogenously distributed across LGE myocardium reflecting patchy macrophage infiltration as confirmed by histology. In whole hearts, we found an apico-basal (19)F gradient within LGE-positive myocardium. The (19)F-positive volume was always smaller than LGE volume. Ex vivo experiments on isolated monocytes revealed that pig and human cells phagocytize PFCs even more avidly than mouse monocytes. This pilot study demonstrates that (19)F MRI at 3.0 T with clinically applicable PFOB is feasible, thus highlighting the potential of (19)F MRI to monitor the inflammatory response after AMI. Published on behalf of the European Society of Cardiology. All rights reserved. © The Author 2015. For permissions please email: journals.permissions@oup.com.

  16. Maturation and germination of oak somatic embryos originated from leaf and stem explants: RAPD markers for genetic analysis of regenerants.

    Science.gov (United States)

    Sánchez, M Concepción; Martínez, M Teresa; Valladares, Silvia; Ferro, Enrique; Viéitez, Ana M

    2003-06-01

    Experiments were performed to determine the influence of maturation medium carbohydrate content on the rates of germination and plantlet conversion (root and shoot growth) of somatic embryos from four embryogenic lines derived from leaf or internode explants of Quercus robur L. seedlings. The conversion rate was favoured by high carbohydrate content as long as the maturation medium contained at least 2% sucrose, which was necessary for healthy embryo development. Given this, sorbitol and mannitol favoured the conversion rate more efficiently than sucrose, the highest rate, 32%, being achieved by medium with 6% sorbitol and 3% sucrose. Maturation treatment did not affect the root or shoot lengths of converted embryos. In supplementary experiments, 2 weeks of gibberellic acid treatment between maturation and germination treatments did not improve germination rates, but did reduce root length and the number of leaves per regenerated plantlet. In the four embryogenic lines tested, plant recovery rate was enhanced by inclusion of benzyladenine into the germination medium following culture of the embryos on maturation medium with 6% sorbitol and 2-3% sucrose. In embryogenic systems it is important to assess the uniformity of the regenerants. Random amplified polymorphic DNA (RAPD) analysis using 32 arbitrary oligonucleotide primers was performed to study variability in DNA sequences within and between four embryogenic lines. No intraclonal nor interclonal polymorphism was detected between embryogenic lines originating from different types of explant from the same seedling, but every one of the primers detected enough polymorphism among clones originating from different plants to allow these three origins to be distinguished. No differences in DNA sequences between regenerated plantlets and their somatic embryos of origin were detected, but a nodular callus line that had lost its embryogenic capacity was found to be mutant with respect to three other clones originating

  17. Transforming growth factor-β1 regulated phosphorylated AKT and interferon gamma expressions are associated with epithelial cell survival in rhesus macaque colon explants.

    Science.gov (United States)

    Pahar, Bapi; Pan, Diganta; Lala, Wendy; Kenway-Lynch, Carys S; Das, Arpita

    2015-05-01

    Transforming growth factor-β1 (TGF-β1) is an important immunoregulatory cytokine that plays an obligate role in regulating T-cell functions. Here, we demonstrated the role of TGF-β1 in regulating the survival of intestinal epithelial cells (ECs) in rhesus colon explant cultures using either anti-TGF-β1 antibody or recombinant TGF-β1 proteins. Neutralization of endogenous TGF-β1 using anti-TGF-β1 antibodies induced apoptosis of both intestinal ECs and lamina propria (LP) cells. Additionally, endogenous TGF-β1 blocking significantly increased expression of IFNγ, TNFα, CD107a and Perforin in LP cells compared to media and isotype controls. A significant decrease in pAKT expression was detected in anti-TGF-β1 MAbs treated explants compared to isotype and rTGF-β1 protein treated explants. Our results demonstrated TGF-β1 regulated pAKT and IFNγ expressions were associated with epithelial cell survival in rhesus macaque colon explants and suggest a potential role of mucosal TGF-β1 in regulating intestinal homeostasis and EC integrity. Copyright © 2015 Elsevier Inc. All rights reserved.

  18. Protocols for Callus and Somatic Embryo Initiation for Hibiscus sabdariffa L. (Malvaceae): Influence of Explant Type, Sugar, and Plant Growth Regulators

    Science.gov (United States)

    A significant work on callus induction and somatic embryogenesis was realized for Hibiscus sabdariffa. Two genotypes (Hibiscus sabdariffa and Hibiscus sabdariffa var. altissima) two sugars (sucrose and glucose) and three concentrations (1 %, 2%, 3%) of each sugar, 3 explant types (root, hypocotyl, c...

  19. Immature tassels as alternative explants in somatic embryogenesis and plant regeneration in south Brazilian maize genotypes - doi: 10.4025/actasciagron.v35i1.15545

    Directory of Open Access Journals (Sweden)

    Magali Ferrari Grando

    2012-08-01

    Full Text Available Somatic embryogenesis and in vitro plant regeneration are fundamental processes in the obtainment of transgenic maize plants. Explant, genotype and culture medium are determining factors in these processes. Immature embryo explants and the American Hi-II genotype have been widely employed to acquire genetically modified plants in this species. However, the use of more readily available explants is desired as well as the development of genetic transformation protocols for productive genotypes adapted to local conditions. This study provides an evaluation of immature tassel explants in relation to embryogenic callus production and plant regeneration in South Brazilian maize genotypes for their use in genetic transformation experiments. Immature tassels from 5 hybrids were cultivated in different callus-induction media. The frequency and the fresh mass of embryogenic calli were evaluated. The frequency was influenced by genotype, and the fresh mass was influenced by genotype and culture medium. In plant regeneration, shoots, complete seedlings and acclimatized and fertile plants were quantified. Treatments producing long term embryogenic calli from immature tassels of South Brazilian genotypes with the capacity to regenerate were identified.

  20. Season and genotype influence in morphogenic and biochemical response of foliar explants of Coffea canephora P. var. Robusta used for callus formation

    Directory of Open Access Journals (Sweden)

    María Esther González

    2005-04-01

    Full Text Available The seasonal change is one from the factors to consider in the embryogenic answer of diverse crops, the variations of temperature, humidity and precipitations cause fluctuations in the plants that influence in the behavior in vitro of the explants. This study was carried out with the objective of evaluating the influence of the time of the year in which the explants were taken on the answer in vitro of Robusta genotypes. Samples were carried out during every month of the year, the explants were cultivated in a culture medium with the mineral salts MS, and 0.5 and 2.0 mg.l-1 of 2,4-D and kinetina, respectively. It was observed that the time of the year in that the samples were selected exercised a marked effect in the answer of the explants. For the studied conditions was favorable to take the leaves samples in the periods May - June, January - February and November - December, due to the few index of peroxidases activity, phenolic oxidation and fungous contamination, as well as a high percentage of callus formation and bigger content of proteins in the same ones. It was determined that the enzymatic peroxidases activity could constitute an important marker for the selection of samples in coffee. Key words: fungous contamination, peroxidases activity,phenolic oxidation

  1. The release of bystander factor(s) from tissue explant cultures of rainbow trout (Onchorhynchus mykiss) after exposure to gamma radiation.

    Science.gov (United States)

    O'Dowd, Colm; Mothersill, Carmel E; Cairns, Michael T; Austin, Brian; McClean, Brendan; Lyng, Fiona M; Murphy, James E J

    2006-10-01

    The bystander response has been documented in cell lines and cell cultures derived from aquatic species over the past several years. However, little work has been undertaken to identify a similar bystander response in tissue explant cultures from fish. In this study, indirect effects of ionizing gamma radiation on tissue explant cultures of fish were investigated. Tissue explants in culture were exposed to 0.5 Gy and 5 Gy gamma radiation from a 60Co teletherapy unit. A bystander response in Epithelioma papulosum cyprini (EPC) cells exposed to gamma-irradiated tissue conditioned medium from rainbow trout explants was investigated, and the effects on cell survival were quantified by the clonogenic survival assay. Dichlorofluorescein and rhodamine 123 fluorescent dyes were used to identify alterations in reactive oxygen species (ROS) and mitochondrial membrane potential (MMP), respectively. Results indicate a different response for the three tissue types investigated. Clonogenic assay results vary from a decrease in cell survival (gill) to no effect (skin) to a stimulatory effect (spleen). Results from fluorescence assays of ROS and MMP show similarities to clonogenic assay results. This study identifies a useful model for further studies relating to the bystander effect in aquatic organisms in vivo and ex vivo.

  2. Chondrogenic Priming at Reduced Cell Density Enhances Cartilage Adhesion of Equine Allogeneic MSCs - a Loading Sensitive Phenomenon in an Organ Culture Study with 180 Explants.

    Science.gov (United States)

    Spaas, Jan H; Broeckx, Sarah Y; Chiers, Koen; Ferguson, Stephen J; Casarosa, Marco; Van Bruaene, Nathalie; Forsyth, Ramses; Duchateau, Luc; Franco-Obregón, Alfredo; Wuertz-Kozak, Karin

    2015-01-01

    Clinical results of regenerative treatments for osteoarthritis are becoming increasingly significant. However, several questions remain UNANSWERED concerning mesenchymal stem cell (MSC) adhesion and incorporation into cartilage. To this end, peripheral blood (PB) MSCs were chondrogenically induced and/or stimulated with pulsed electromagnetic fields (PEMFs) for a brief period of time just sufficient to prime differentiation. In an organ culture study, PKH26 labelled MSCs were added at two different cell densities (0.5 x106 vs 1.0 x106). In total, 180 explants of six horses (30 per horse) were divided into five groups: no lesion (i), lesion alone (ii), lesion with naïve MSCs (iii), lesion with chondrogenically-induced MSCs (iv) and lesion with chondrogenically-induced and PEMF-stimulated MSCs (v). Half of the explants were mechanically loaded and compared with the unloaded equivalents. Within each circumstance, six explants were histologically evaluated at different time points (day 1, 5 and 14). COMP expression was selectively increased by chondrogenic induction (p = 0.0488). PEMF stimulation (1mT for 10 minutes) further augmented COL II expression over induced values (p = 0.0405). On the other hand, MSC markers remained constant over time after induction, indicating a largely predifferentiated state. In the unloaded group, MSCs adhered to the surface in 92.6% of the explants and penetrated into 40.7% of the lesions. On the other hand, physiological loading significantly reduced surface adherence (1.9%) and lesion filling (3.7%) in all the different conditions (p < 0.0001). Remarkably, homogenous cell distribution was characteristic for chondrogenic induced MSCs (+/- PEMFs), whereas clump formation occurred in 39% of uninduced MSC treated cartilage explants. Finally, unloaded explants seeded with a moderately low density of MSCs exhibited greater lesion filling (p = 0.0022) and surface adherence (p = 0.0161) than explants seeded with higher densities of MSCs. In

  3. Flower-bud formation in explants of photoperiodic and day-neutral Nicotiana biotypes and its bearing on the regulation of flower formation

    Energy Technology Data Exchange (ETDEWEB)

    Rajeevan, M.S.; Lang, A. (Michigan State Univ., East Lansing (United States))

    1993-05-15

    The capacity to form flower buds in thin-layer explants was studied in Nicotiana of several species, cultivars, and lines of differing in their response to photoperiod. This capacity was found in all biotypes examined and could extend into sepals and corolla. It varied depending on genotype, source tissue and its developmental state, and composition of the culture medium, particularly the levels of glucose, auxin, and cytokinin. It was greatest in the two day-neutral plants examined, Samsun tobacco and Nicotiana rustica, where it extended from the inflorescence region down the vegetative stem, in a basipetally decreasing gradient; it was least in the two qualitative photoperiodic plants studied, the long-day plant Nicotiana silvestris and the short-day plant Maryland Mammoth tobacco, the quantitative long-day plant Nicotiana alata and the quantitative short-day plant Nicotiana otophora line 38-G-81, where it was limited to the pedicels (and, in some cases, the sepals). Regardless of the photoperiodic response of the source plants, the response was the same in explants cultured under long and short days. The capacity to form flow buds in explants is present in all Nicotiana biotypes studied supports the idea that it is regulated by the same mechanism(s), regardless of the plant's photoperiodic character. However, flower formation in the explants is not identical with de novo flower formation in a hitherto vegetative plant: it is rather the expression of a floral state already established in the plant, although it can vary widely in extent and spatial distribution. Culture conditions that permit flower-bud formation in an explant are conditions that maintain the floral state and encourage its expression; conditions under which no flower buds are formed reduce this state and/or prevent its expression. 14 refs., 5 figs., 3 tabs.

  4. Desinfestação e estabelecimento in vitro de explantes de bananeira 'Grande Naine' em diferentes concentrações de hipoclorito de sódio

    Directory of Open Access Journals (Sweden)

    Gustavo Alves Pereira

    2011-10-01

    Full Text Available A maioria dos plantios de bananeira ainda é realizada utilizando mudas tradicionais, mas outros métodos de propagação, como a micropropagação in vitro, vêm sendo desenvolvidos e aperfeiçoados, para elevar a taxa de multiplicação em curto espaço de tempo e melhorar a qualidade da produção de mudas. Contudo, a contaminação é um dos maiores problemas desta técnica. Este trabalho teve por objetivo avaliar a eficiência da descontaminação de explantes de bananeira com o uso de diferentes concentrações de cloro ativo durante a assepsia do explante. O delineamento experimental utilizado foi inteiramente casualizado e constituído de cinco tratamentos e cinco repetições, sendo cada repetição representada por 5 explantes em diferentes concentrações de cloro ativo, sendo: T1 (testemunha, sem cloro ativo; T2 (0,5%; T3 (1,0%; T4 (1,5%, e T5 (2%. Os dados obtidos foram submetidos à análise de variância, e as médias, comparadas pelo teste de Tukey, a 5% de probabilidade. Os resultados permitiram concluir que a maior eficiência dentre os tratamentos testados foi a imersão dos explantes em hipoclorito de sódio com 2% de cloro ativo, sendo as doses testadas não tóxicas aos explantes, permitindo o desenvolvimento normal dos mesmos, concluindo assim que essa concentração possa ser utilizada para o controle de contaminações para micropropagação de bananeira cv. Grande Naine.

  5. Effect of explant density and volume of cultivation medium on in-vitro multiplication of blueberry (Vaccinium corymbosum L. varieties "Brigitta" and "Legacy"

    Directory of Open Access Journals (Sweden)

    Mario Rodríguez Beraud

    2015-03-01

    Full Text Available The objective of the investigation was to evaluate the in-vitro multiplication of two varieties of blueberry (Vaccinium corymbosum L., “Brigitta” and “Legacy” in response to five explants densities (5, 10, 15, 20 and 25 and four flask volumes (10, 20, 30 and 40 mL for cultivation. For both varieties the cultivation medium WPM (Woody Plant Medium was used. The experiment was completely randomized with 20 treatments and 12 repetitions per treatment. After 45 days of cultivation we evaluated the height of shoots, number of shoots/explant, number of nodes/shoot and number of shoots/flask. Variety “Brigitta” had highest shoots at higher densities and flask volumes, while variety “Legacy” had the highest average shoot height with intermediate densities and high volumes. Regarding the number of shoots/explant, the volume of the medium had no influence on “Brigitta”, however, higher plant densities affected this parameter. With variety “Legacy” the maximum number of shoots was achieved with lower plant densities and intermediate culture volumes per flask. In relation to the number of nodes per explant "Brigitta had lower numbers as compared to “Legacy”, but with both varieties the number of nodes decresed with smaller volumes of medium in the flasks. For the number of shoots per flask, “Brigitta” responsed best at higher densities exceeding 40 shoots per flask. In contrast, “Legacy” produced maximum results at density of 25 explants in 30 mL of medium. It is concluded that for the optimum multiplication of both varieties the correct selection of both, the planting density and the volume of multiplication medium are important.

  6. The effect of auxin 2,4-D and cytokinin 2-ip on direct somatic embryogenesis formation of Coffea arabica L. leaf explant

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    Rina Arimarsetiowati

    2011-08-01

    Full Text Available One of the propagation technique for coffee plant production is tissue culture. Tissue culture technique for Coffea arabica L. faces some problems, mainly in the planlet formation regenerated from explants. The objective of this experiment was to examine the effect 2,4-D and 2-ip combination on the formation of direct somatic embryogenesis of Coffea arabica L. in leaves explant. Auxin (2,4-D and cytokinin (2-ip concentrations of, respectively, 1; 5 µM and 5; 10; 15; 20 were used as treatments. This research was conducted using completely randomized design with 10 replications. Observation to induce somatic embryos was done by quantitatively on number of callus from explant and number of embryogenic callus. Beside that, observation by qualitative descriptive was also done on deve lopment of embryogenesis. The results showed that Arabica coffee leaves explant of AS 2K clones could be induced in all medium combination except 5µM 2,4-D and 20µM 2-ip combination. Arabica coffee leaves explant of S 795, Sigararutang and AS 1 varieties could be induced in all medium combination. The highest frequency of callus formation was found in AS 2K, Sigararutang and AS 1 varieties on medium containing 1µM 2,4-D in combination with 10µM 2-ip, whereas for the S 795 variety on medium containing 5µM 2,4-D in combination with 10µM 2-ip. The highest frequency of embriogenic callus in all Arabica coffee variety could be reached on medium containing 5µM 2,4-D in combination with 15µM 2-ip. Key words : Coffea arabica L., somatic embryogenesis, 2,4-D, 2-ip, tissue culture, leaves, callus embryogenic.

  7. UV-independent induction of beta defensin 3 in neonatal human skin explants [v2; ref status: indexed, http://f1000r.es/53b

    Directory of Open Access Journals (Sweden)

    Erin Wolf Horrell

    2015-02-01

    Full Text Available In order to determine the effect of UV radiation on β-defensin 3 (BD3 expression in human skin, freshly-isolated UV-naïve skin was obtained from newborn male infants undergoing planned circumcision.  Skin explants sustained ex vivo dermis side down on RPMI media were exposed to 0.5 kJ/m2 UVB, and biopsies were taken from the explant through 72 hours after radiation.  mRNA expression was measured by qRTPCR and normalized to TATA-binding protein.  BD3 expression at each time point was compared with an untreated control taken at time 0 within each skin sample.  Extensive variability in both the timing and magnitude of BD3 induction across individuals was noted and was not predicted by skin pigment phenotype, suggesting that BD3 induction was not influenced by epidermal melanization.  However, a mock-irradiated time course demonstrated UV-independent BD3 mRNA increases across multiple donors which was not further augmented by treatment with UV radiation, suggesting that factors other than UV damage promoted increased BD3 expression in the skin explants.  We conclude that BD3 expression is induced in a UV-independent manner in human skin explants processed and maintained in standard culture conditions, and that neonatal skin explants are an inappropriate model with which to study the effects of UV on BD3 induction in whole human skin.

  8. UV-independent induction of beta defensin 3 in neonatal human skin explants [v1; ref status: indexed, http://f1000r.es/4s2

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    Erin Wolf Horrell

    2014-11-01

    Full Text Available In order to determine the effect of UV radiation on β-defensin 3 (BD3 expression in human skin, freshly-isolated UV-naïve skin was obtained from newborn male infants undergoing planned circumcision.  Skin explants sustained ex vivo dermis side down on RPMI media were exposed to 0.5 kJ/m2 UVB, and biopsies were taken from the explant through 72 hours after radiation.  mRNA expression was measured by qRTPCR and normalized to TATA-binding protein.  BD3 expression at each time point was compared with an untreated control taken at time 0 within each skin sample.  Extensive variability in both the timing and magnitude of BD3 induction across individuals was noted and was not predicted by skin pigment phenotype, suggesting that BD3 induction was not influenced by epidermal melanization.  However, a mock-irradiated time course demonstrated UV-independent BD3 mRNA increases across multiple donors which was not further augmented by treatment with UV radiation, suggesting that factors other than UV damage promoted increased BD3 expression in the skin explants.  We conclude that BD3 expression is induced in a UV-independent manner in human skin explants processed and maintained in standard culture conditions, and that neonatal skin explants are an inappropriate model with which to study the effects of UV on BD3 induction in whole human skin.

  9. Changes in the metabolic footprint of placental explant-conditioned medium cultured in different oxygen tensions from placentas of small for gestational age and normal pregnancies.

    LENUS (Irish Health Repository)

    Horgan, R P

    2012-01-31

    Being born small for gestational age (SGA) confers significantly increased risks of perinatal morbidity and mortality. Accumulating evidence suggests that an SGA fetus results from a poorly perfused and abnormally developed placenta. Some of the placental features seen in SGA, such as abnormal cell turnover and impaired nutrient transport, can be reproduced by culture of placental explants in hypoxic conditions. Metabolic footprinting offers a hypothesis-generating strategy to investigate factors absorbed by and released from this tissue in vitro. Previously, metabolic footprinting of the conditioned culture media has identified differences in placental explants cultured under normoxic and hypoxic conditions and between normal pregnancies and those complicated by pre-eclampsia. In this study we aimed to examine the differences in the metabolic footprint of placental villous explants cultured at different oxygen (O(2)) tensions between women who deliver an SGA baby (n = 9) and those from normal controls (n = 8). Placental villous explants from cases and controls were cultured for 96 h in 1% (hypoxic), 6% (normoxic) and 20% (hyperoxic) O(2). Metabolic footprints were analysed by Ultra Performance Liquid Chromatography coupled to an electrospray hybrid LTQ-Orbitrap Mass Spectrometry (UPLC-MS). 574 metabolite features showed significant difference between SGA and normal at one or more of the oxygen tensions. SGA explant media cultured under hypoxic conditions was observed, on a univariate level, to exhibit the same metabolic signature as controls cultured under normoxic conditions in 49% of the metabolites of interest, suggesting that SGA tissue is acclimatised to hypoxic conditions in vivo. No such behaviour was observed under hyperoxic culture conditions. Glycerophospholipid and tryptophan metabolism were highlighted as areas of particular interest.

  10. Pattern of antioxidant enzyme activities and hydrogen peroxide content during developmental stages of rhizogenesis from hypocotyl explants of Mesembryanthemum crystallinum L.

    Science.gov (United States)

    Konieczny, Robert; Banaś, Agnieszka K; Surówka, Ewa; Michalec, Żaneta; Miszalski, Zbigniew; Libik-Konieczny, Marta

    2014-01-01

    H2O2 is necessary to elicit rhizogenic action of auxin. Activities of specific catalase and manganese superoxide dismutase forms mark roots development. Hypocotyl explants of Mesembryanthemum crystallinum regenerated roots on medium containing 2,4-dichlorophenoxyacetic acid. Explants became competent to respond to the rhizogenic action of auxin on day 3 of culture, when hydrogen peroxide content in cultured tissue was the highest. L-Ascorbic acid added to the medium at 5 μM lowered the H2O2 level, inhibited rhizogenesis and induced non-regenerative callus, suggesting that certain level of H2O2 is required to promote root initiation. Coincident with the onset of rhizogenic determination, meristemoids formed at the periphery of the hypocotyl stele and the activity of the manganese form of superoxide dismutase, MnSOD-2 was induced. Once induced, MnSOD-2 activity was maintained through the post-determination phase of rooting, involving root growth. MnSOD-2 activity was not found in non-rhizogenic explants maintained in the presence of AA. Analyses of the maximum photochemical efficiency of photosystem II and the oxygen uptake rate revealed that the explants were metabolically arrested during the predetermination stage of rhizogenesis. Respiratory and photosynthetic rates were high during root elongation and maturation. Changes in catalase and peroxidase activities correlated with fluctuations of endogenous H2O2 content throughout rhizogenic culture. Expression of a specific CAT-2 form accompanied the post-determination stage of rooting and a high rate of carbohydrate metabolism during root growth. On the other hand, the occurrence of MnSOD-2 activity did not depend on the metabolic status of explants. The expression of MnSOD-2 activity throughout root development seems to relate it specifically to root metabolism and indicates it as a molecular marker of rhizogenesis in M. crystallinum.

  11. Regeneração in vitro de urucum (Bixa orellana L. a partir de diferentes tipos de explantes In vitro regeneration of annatto (Bixa orellana L. from various explants

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    Jane Fiuza Rodrigues Portela de Carvalho

    2005-12-01

    Full Text Available Este trabalho teve como objetivo avaliar a regeneração in vitro de plantas de urucum (Bixa orellana L. a partir de diferentes tipos de explantes. Para definir o meio de cultura adequado para indução de brotações, diferentes concentrações e, ou, combinações da auxina AIA e das citocininas BAP e ZEA foram testadas. As melhores respostas de regeneração para segmentos de hipocótilo, nós cotiledonares e hipocótilos invertidos foram observadas em meios suplementados de ZEA (2,28 µM e AIA (0,30 µM, ZEA (4,56 µM e ZEA (4,56 µM, respectivamente. O meio de enraizamento mais eficaz foi o MS, com a metade de sua concentração salina e 5 µM de AIB. Análises citológicas, realizadas antes da aclimatação, confirmaram a estabilidade cromossômica das plantas cultivadas in vitro, não sendo detectado variação com relação ao número de cromossomos metafásicos (2n = 14.The present work aimed the establishment of a regeneration protocol for annatto (Bixa orellana L. from different juvenile explants. In order to promote shoot induction, different concentrations and/or combinations of IAA and the cytokinins BAP and ZEA were assessed. Better regeneration responses were achieved when segmented hypocotyl, cotiledonary nodes and inverted hypocotyl were cultured onto MS-based medium supplemented with ZEA (2.28 µM and IAA (0.30 µM, ZEA (4.56 µM or ZEA (4.56 µM, respectively. Rooting of elongated shoots displayed higher frequencies when half-strength MS medium with IBA (5 µM was used. No genetic variation was detected among regenerants as revealed by cytological analysis based on metaphasic chromosome countings (2n = 14.

  12. Rod and cone photoreceptor cells produce ROS in response to stress in a live retinal explant system.

    LENUS (Irish Health Repository)

    Bhatt, Lavinia

    2010-01-01

    PURPOSE: The production of reactive oxygen species (ROS) can lead to oxidative stress, which is a strong contributory factor to many ocular diseases. In this study, the removal of trophic factors is used as a model system to investigate the effects of stress in the retina. The aims were to determine if both rod and cone photoreceptor cells produce ROS when they are deprived of trophic factor support and to demonstrate if the nicotinamide adenine dinucleotide phosphate (NADPH) oxidase (Nox) enzymes are responsible for this ROS production. METHODS: Retinas were explanted from mice aged between postnatal days 8-10 and cultured overnight. The following morning, confocal microscopy combined with various fluorescent probes was used to detect the production of ROS. Each time peanut agglutinin (PNA), a cone photoreceptor marker, was used to facilitate orientation of the retina. Dihydroethidium and dihydrorhodamine 123 (DHR123) were used to determine which cells produce ROS. Subsequently, western blots of retinal serial sections were used to detect the presence of Noxs in the different retinal layers. The Nox inhibitor apocynin was then tested to determine if it altered the production of ROS within these cells. RESULTS: Live retinal explants, viewed at high magnifications using confocal microscopy, displayed an increase in the fluorescent products of dihydroethidium and DHR123 upon serum removal when compared to controls. DHR123 fluorescence, once oxidized, localized to mitochondria and was found in the same focal plane as the PNA staining. This showed that cones and rods produced ROS when stressed. Retinal serial sectioning established that the photoreceptor layer expressed Nox4, dual oxidase (Duox) 1, and Duox2 at varying levels. Finally, the Nox inhibitor apocynin decreased the burst stimulated by the stress of serum removal. CONCLUSIONS: Confocal microscopy and PNA staining allowed differentiation of cell types within the outermost layers of the retina, demonstrating

  13. Long-term gene therapy with Del1 fragment using nonviral vectors in mice with explanted tumors

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    Kitano H

    2016-01-01

    Full Text Available Hisataka Kitano,1 Atsushi Mamiya,1 Tomomi Ishikawa,2 Kayo Egoshi,3 Shinichiro Kokubun,2 Chiaki Hidai2 1Division of Dental Surgery, School of Medicine, Nihon University, Tokyo, Japan; 2Division of Physiology, Department of Biomedical Science, School of Medicine, Nihon University, Tokyo, Japan; 3School of Medicine, Nihon University, Tokyo, Japan Abstract: Cancer gene therapy using nonviral vectors is useful for long periods of treatment because such vectors are both safe and inexpensive, and thus can be used repeatedly. It has been reported that gene therapy with an E3C1 fragment of Del1 in a mouse explanted tumor model improved prognosis. The present study aimed to analyze the long-term effects of repeated nonviral gene transfer of E3C1. Mice with explanted tumors of SCCKN cells, a human squamous carcinoma, were treated with a plasmid encoding E3C1. Plasmids were injected locally every week using a transfection reagent. Control mice treated with mock DNA started to be euthanized on day 18, because the tumors had grown to over 15% of the body weight, and all of them had died by day 43. On the other hand, the tumors in two of ten mice treated with E3C1 had disappeared. The other eight mice started to be euthanized on day 46 and eight of ten mice had been euthanized by day 197. After 18 days of therapy, the tumor volume of control mice was 2,804±829 mm3 and that of the E3C1 mice was 197±159 mm3. Histochemical studies showed enhanced apoptosis in the E3C1-treated tumors, as compared with controls. Changes in cell morphology and decreased polymerized actin induced by E3C1 indicated disturbed cell adhesion to the matrix. In in vitro studies of SCCKN cells, prolonged administration of an E3C1 recombinant protein to cultured cells reduced adhesion-independent growth of cancer cells, as compared with control cells. These data suggest that E3C1 treatment induces anoikis. Keywords: cancer gene therapy, nonviral vector, apoptosis, anoikis, Del1

  14. Estabelecimento e desenvolvimento in vitro de Eugenia involucrata DC.: influência do tipo de explante e do meio nutritivo

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    Diego Pascoal Golle

    2012-03-01

    Full Text Available http://dx.doi.org/10.5902/198050985092Apresentando diversas características de interesse nos setores da silvicultura, fruticultura, meio ambiente e medicinal, Eugenia involucrata DC. (Myrtaceae é uma espécie florestal nativa de vários Estados brasileiros. Considerada a dificuldade da propagação por sementes, que são recalcitrantes, perdendo rapidamente sua viabilidade após a colheita, este trabalho objetivou avaliar a influência do uso de segmentos apicais e nodais cultivados em diferentes meios nutritivos no estabelecimento e no desenvolvimento in vitro desta espécie. Segmentos apicais e nodais coletados em plantas de três anos de idade, mantidas em casa de vegetação, foram inoculados nos meios nutritivos MS, ½ MS e WPM. Em um primeiro momento, adicionaram-se aos meios de cultura 1 μM de ANA e 5 μM de TDZ e, após, os explantes foram transferidos para frascos contendo os respectivos meios nutritivos frescos, na ausência de reguladores de crescimento, mas com o acréscimo de 1 g L-1 de carvão ativado, sendo cultivados por 30 dias adicionais. Os meios ½ MS e WPM são adequados para o estabelecimento e o desenvolvimento in vitro a partir do cultivo de segmentos nodais, enquanto que, com segmentos apicais, é apropriado o meio ½ MS. O meio ½ MS possibilitou os melhores índices de enraizamento nos explantes, juntamente com o meio WPM. O estabelecimento e o enraizamento in vitro foram promovidos pelo cultivo de segmentos apicais e nodais de Eugenia involucrata DC. Os meios nutritivos ½ MS e WPM são apropriados para o cultivo in vitro de segmentos nodais de E. involucrata, recomendando-se o meio ½ MS pela economia e praticidade. Para segmentos apicais, o uso do meio de cultura ½ MS é mais adequado que o dos meios MS e WPM.

  15. Upregulation of IL-17A/F from human lung tissue explants with cigarette smoke exposure: implications for COPD.

    Science.gov (United States)

    Chang, Ying; Al-Alwan, Laila; Alshakfa, Sama; Audusseau, Severine; Mogas, Andrea Karen; Chouiali, Fazila; Nair, Parameswaran; Baglole, Carolyn J; Hamid, Qutayba; Eidelman, David H

    2014-11-27

    Chronic obstructive pulmonary disease (COPD) is an inflammatory disorder marked by relative resistance to steroids. The IL-17 superfamily, which mediates cross-talk between the adaptive and innate immune systems, has been associated with diminished responses to steroids. Increasing evidence supports elevated IL-17 expression in the lung of COPD subjects. However, whether cells of the immune system (systemic) and/or local lung cells are contributing to the elevated IL-17 remains unclear. To address this issue, we utilized a human parenchymal lung tissue explant culture system with cigarette smoke exposure to investigate the expression of IL-17 and the mechanisms involved. Parenchymal lung tissue removed from 10 non-COPD and 8 COPD patients was sectioned and cultured with different concentrations of cigarette smoke extract (CSE) for 3 or 6 hours. Tissue viability was evaluated by LDH (lactate dehydrogenase) in culture supernatants. Western blot and real-time PCR were performed to evaluate IL-17A/F expression. To investigate the mechanisms, pharmacological inhibitors for MAPK p38, ERK1/2, NF-κB and PI3K pathways were added into the culture media. No tissue damage was observed after the cigarette smoke exposure for 3 h or 6 h compared with the control media. At the protein level, the expression of both IL-17A (2.4 ± 0.6 fold) and IL-17 F (3.7 ± 0.7 fold) in the tissue from non-COPD subjects was significantly increased by 5% of CSE at 3 h. For COPD subjects, IL-17A/F expression were significantly increased only at 6 h with 10% of CSE (IL-17A: 4.2 ± 0.8 fold; IL-17 F: 3.3 ± 0.8 fold). The increased expression of IL-17A/F is also regulated at the mRNA level. The inhibitors for NF-κB and PI3K pathways significantly inhibited CSE-induced IL-17A/F expression from lung tissue of non-COPD subjects. We found the evidence that the expression of both IL-17A and IL-17 F is increased by the cigarette smoke exposure in explants from both non-COPD and COPD subjects, supporting

  16. An Efficient Plant Regeneration and Transformation System of Ma Bamboo (Dendrocalamus latiflorus Munro) Started from Young Shoot as Explant

    Science.gov (United States)

    Ye, Shanwen; Cai, Changyang; Ren, Huibo; Wang, Wenjia; Xiang, Mengqi; Tang, Xiaoshan; Zhu, Caiping; Yin, Tengfei; Zhang, Li; Zhu, Qiang

    2017-01-01

    Genetic engineering technology has been successfully used in many plant species, but is limited in woody plants, especially in bamboos. Ma bamboo (Dendrocalamus latiflorus Munro) is one of the most important bamboo species in Asia, and its genetic improvement was largely restricted by the lack of an efficient regeneration and transformation method. Here we reported a plantlet regeneration and Agrobacterium-mediated transformation protocol by using Ma bamboo young shoots as explants. Under our optimized conditions, embryogenic calluses were successfully induced from the excised young shoots on callus induction medium and rapidly grew on callus multiplication medium. Shoots and roots were regenerated on shoot induction medium and root induction medium, respectively, with high efficiency. An Agrobacterium-mediated genetic transformation protocol of Ma bamboo was established, verified by PCR and GUS staining. Furthermore, the maize Lc gene under the control of the ubiquitin promoter was successfully introduced into Ma bamboo genome and generated an anthocyanin over-accumulation phenotype. Our methods established here will facilitate the basic research as well as genetic breeding of this important bamboo species. Key achievements: A stable and high efficiency regeneration and Agrobacterium-mediated transformation protocol for Ma bamboo from vegetative organ is established. PMID:28798758

  17. Proinflammatory and Anabolic Gene Expression Effects of Platelet-Rich Gel Supernatants on Equine Synovial Membrane Explants Challenged with Lipopolysaccharide.

    Science.gov (United States)

    Carmona, Jorge U; Ríos, Diana L; López, Catalina; Álvarez, María E; Pérez, Jorge E

    2017-01-01

    Platelet-rich plasma (PRP) preparations are used in horses with osteoarthritis (OA). However, some controversies remain regarding the ideal concentration of platelets and leukocytes to produce an adequate anti-inflammatory and anabolic response in the synovial membrane. The aims of this study were to study the influence of leukoconcentrated platelet-rich gel (Lc-PRG) and leukoreduced platelet-rich gel (Lr-PRG) supernatants on the quantitative expression of some proinflammatory and anabolic genes in equine synovial membrane explants (SMEs) challenged with lipopolysaccharide (LPS). SMEs from six horses were cultured over 96 h. Then, SMEs were harvested for RNA extraction and quantitative gene expression analysis by RT-qPCR for nuclear factor kappa B (NFκB), matrix metalloproteinase 13 (MMP-13), a disintegrin and metalloproteinase with thrombospondin motifs 4 (ADAMTS-4), collagen type I alpha 1 (COL1A1), collagen type II alpha 1 (COL2A1), and cartilage oligomeric matrix protein (COMP). The 25% and 50% Lc-PRG supernatants led to downregulation of NFκB, MMP-13, ADAMTS-4, COL1A1, COL2A1, and COMP in SMEs. Lr-PRG supernatants (particularly at the 50% concentration) induced downregulation of NFκB, MMP-13, ADAMTS-4, and COL1A1 and upregulation of COL2A1 and COMP. Lr-PRG supernatants should be used for the treatment of inflammatory arthropathies in horses because they have anti-inflammatory and anabolic effects in the synovial membrane.

  18. Effect of Growth Regulators on In Vitro Morphogenic Response of Boscia senegalensis (Pers.) Lam. Poir. Using Mature Zygotic Embryos Explants

    Science.gov (United States)

    Daffalla, Hussien H.; Abdellatef, Eltayb; Elhadi, Elsadig A.; Khalafalla, Mutasim M.

    2011-01-01

    The percent study describes the in vitro responses of mature zygotic embryos of Boscia senegalensis to different concentrations (0.0–5.0 mg/L) of 6-benzyladnine (BA), Thidiazuron (TDZ), α-Naphthalene acetic acid (NAA), and 2, 4-Dichlorophenoxyacetic acid (2, 4-D) supplemented on Murashige and Skoog medium (MS). The plant growth regulators (PGRs) were considerably affected the morphogenetic responses. BA produced adventitious shoots through two ways: direct organogenesis and auxiliary shoot formation. Both 2, 4-D and TDZ tend to produce callus, whereas NAA improve the development of embryos to seedlings. Maximum number of shoots/explant (14.8 ± 0.6) was obtained on MS medium supplemented with 3.0 mg/L BA. 67.0% of excised shoots were rooted either on 1/2 MS medium augmented with or without 0.25 mg/L IBA. The highest number of roots (1.2 ± 0.4) and root length (0.5 ± 0.2 cm) was produced on 0.25 mg/L IBA-containing medium. Regenerated plants were successfully acclimatized and transferred to the green house with 70% survival rate. All the plants appeared morphologically uniform with normal growth pattern. A rapid (30 days), efficient and without subculturing protocol for in vitro regeneration of B. senegalensis was developed. PMID:21687567

  19. Accuracy of hepatocellular carcinoma detection on multidetector CT in a transplant liver population with explant liver correlation

    Energy Technology Data Exchange (ETDEWEB)

    Addley, H.C., E-mail: helenclareaddley@hotmail.co.u [Department of Radiology, Addenbrooke' s Hospital, Cambridge University Hospitals NHS Foundation Trust, Cambridge (United Kingdom); Griffin, N.; Shaw, A.S.; Mannelli, L. [Department of Radiology, Addenbrooke' s Hospital, Cambridge University Hospitals NHS Foundation Trust, Cambridge (United Kingdom); Parker, R.A. [Department of Medical Statistics, Addenbrooke' s Hospital, Cambridge University Hospitals NHS Foundation Trust, Cambridge (United Kingdom); Aitken, S.; Wood, H. [Department of Radiology, Addenbrooke' s Hospital, Cambridge University Hospitals NHS Foundation Trust, Cambridge (United Kingdom); Davies, S. [Department of Histopathology, Addenbrooke' s Hospital, Cambridge University Hospitals NHS Foundation Trust, Cambridge (United Kingdom); Alexander, G.J. [Department of Hepatology, Addenbrooke' s Hospital, Cambridge University Hospitals NHS Foundation Trust, Cambridge (United Kingdom); Lomas, D.J. [Department of Radiology, Addenbrooke' s Hospital, Cambridge University Hospitals NHS Foundation Trust, Cambridge (United Kingdom)

    2011-04-15

    Aim: To evaluate the diagnostic accuracy of multidetector computed tomography (MDCT) for hepatocellular carcinoma (HCC) in cirrhotic patients undergoing liver transplantation. Secondary aims were to examine the effect of radiologist experience and lesion size on diagnostic accuracy. Materials and methods: Thirty-nine patients (72% male with a mean age of 56.5 years) underwent liver transplantation following preoperative triple-phase MDCT examination of the liver. MDCT examinations were retrospectively independently reviewed by three radiologists for the presence and location of suspected HCCs, with the diagnostic confidence recorded using a five-point confidence scale. MDCT examinations were compared with explant specimens for histopathological correlation. Results: Histopathological results demonstrated 46 HCCs in 29 of the 39 patients. Analysis demonstrated a sensitivity of 65-75% and specificity of 47-88% for detection of HCC lesions. The sensitivity dropped to 48-57% for lesions of size {<=}20 mm. As the diagnostic confidence increased, there was a further decrease in the sensitivity (4-26%). The radiologist with the greatest number of years experience was found to have a significantly higher accuracy of detection of HCC lesions compared with the least experienced radiologist. Conclusion: Larger lesion size of HCC and greater number of years experience of the radiologist resulted in significantly higher accuracy of HCC lesion detection. The overall sensitivity and specificity results for MDCT detection of HCC are comparable to previous helical CT imaging.

  20. Increased Agrobacterium-mediated transformation and rooting efficiencies in canola (Brassica napus L.) from hypocotyl segment explants

    Science.gov (United States)

    Cardoza, V.; Stewart, C. N.

    2003-01-01

    An efficient protocol for the production of transgenic Brassica napus cv. Westar plants was developed by optimizing two important parameters: preconditioning time and co-cultivation time. Agrobacterium tumefaciens-mediated transformation was performed using hypocotyls as explant tissue. Two variants of a green fluorescent protein (GFP)-encoding gene--mGFP5-ER and eGFP--both under the constitutive expression of the cauliflower mosaic virus 35S promoter, were used for the experiments. Optimizing the preconditioning time to 72 h and co-cultivation time with Agrobacterium to 48 h provided the increase in the transformation efficiency from a baseline of 4% to 25%. With mGFP5-ER, the transformation rate was 17% and with eGFP it was 25%. Transgenic shoots were selected on 200 mg/l kanamycin. Rooting efficiency was 100% on half-strength Murashige and Skoog medium with 10 g/l sucrose and 0.5 mg/l indole butyric acid in the presence of kanamycin.

  1. Thidiazuron induces shoot organogenesis at low concentrations and somatic embryogenesis at high concentrations on leaf and petiole explants of African violet (Saintpaulia ionantha Wendl).

    Science.gov (United States)

    Mithila, J; Hall, J C; Victor, J M R; Saxena, P K

    2003-01-01

    Regeneration via shoot organogenesis and somatic embryogenesis was observed from thidiazuron (TDZ)-treated leaf and petiole explants of greenhouse- and in vitro-grown African violet plants. The response of cultures to other growth regulators over a range of 0.5 microM to 10 microM was 50% less than that observed with TDZ. A comparative study among several cultivars of African violet indicated that "Benjamin" and "William" had the highest regeneration potential. In "Benjamin", higher frequencies of shoot organogenesis (twofold) and somatic embryogenesis (a 50% increase) were observed from in vitro- and greenhouse-grown plants, respectively. At concentrations lower than 2.5 microM, TDZ induced shoot organogenesis, whereas at higher doses (5-10 microM) somatic embryos were formed. These findings provide the first report of simultaneous shoot organogenesis and somatic embryogenesis of African violet explants in response to TDZ.

  2. Cochlear nucleus whole mount explants promote the differentiation of neuronal stem cells from the cochlear nucleus in co-culture experiments.

    Science.gov (United States)

    Rak, Kristen; Völker, Johannes; Jürgens, Lukas; Völker, Christine; Frenz, Silke; Scherzad, Agmal; Schendzielorz, Philipp; Jablonka, Sibylle; Mlynski, Robert; Radeloff, Andreas; Hagen, Rudolf

    2015-08-07

    The cochlear nucleus is the first brainstem nucleus to receive sensory input from the cochlea. Depriving this nucleus of auditory input leads to cellular and molecular disorganization which may potentially be counteracted by the activation or application of stem cells. Neuronal stem cells (NSCs) have recently been identified in the neonatal cochlear nucleus and a persistent neurogenic niche was demonstrated in this brainstem nucleus until adulthood. The present work investigates whether the neurogenic environment of the cochlear nucleus can promote the survival of engrafted NSCs and whether cochlear nucleus-derived NSCs can differentiate into neurons and glia in brain tissue. Therefore, cochlear nucleus whole-mount explants were co-cultured with NSCs extracted from either the cochlear nucleus or the hippocampus and compared to a second environment using whole-mount explants from the hippocampus. Factors that are known to induce neuronal differentiation were also investigated in these NSC-explant experiments. NSCs derived from the cochlear nucleus engrafted in the brain tissue and differentiated into all cells of the neuronal lineage. Hippocampal NSCs also immigrated in cochlear nucleus explants and differentiated into neurons, astrocytes and oligodendrocytes. Laminin expression was up-regulated in the cochlear nucleus whole-mounts and regulated the in vitro differentiation of NSCs from the cochlear nucleus. These experiments confirm a neurogenic environment in the cochlear nucleus and the capacity of cochlear nucleus-derived NSCs to differentiate into neurons and glia. Consequently, the presented results provide a first step for the possible application of stem cells to repair the disorganization of the cochlear nucleus, which occurs after hearing loss. Copyright © 2015 Elsevier B.V. All rights reserved.

  3. Improvement in Agrobacterium-mediated transformation of chickpea (Cicer arietinum L.) by the inhibition of polyphenolics released during wounding of cotyledonary node explants.

    Science.gov (United States)

    Yadav, Reena; Mehrotra, Meenakshi; Singh, Aditya K; Niranjan, Abhishek; Singh, Rani; Sanyal, Indraneel; Lehri, Alok; Pande, Veena; Amla, D V

    2017-01-01

    Agrobacterium-mediated transformation of chickpea (Cicer arietinum L.) has been performed using cotyledonary node explants (CNs), which release phenolics upon excision that are detrimental to the viability of Agrobacterium tumefaciens and result in low transformation frequency. Twelve low molecular weight phenolic compounds and salicylic acid were identified in the exudates released upon excision during the preparation of cotyledonary nodes by reverse phase high-performance liquid chromatography (RP-HPLC). Zone inhibition assays performed with the explant exudates released at periodic intervals after excision showed the inhibition of A. tumefaciens. Agroinoculation of freshly excised cotyledonary nodes of chickpea showed 98-99 % inhibition of colony forming units (cfu). Osmium tetraoxide fixation of excised tissues showed enhanced accumulation of phenolics in the sub-epidermal regions causing enzymatic browning, affecting the viability and performance of A. tumefaciens for T-DNA delivery. The periodic analysis of exudates released from excised CNs showed enhanced levels of gallic acid (0.2945 ± 0.014 μg/g), chlorogenic acid (0.0978 ± 0.0046 μg/g), and quercetin (0.0971 ± 0.0046 μg/g) fresh weight, which were detrimental to A. tumefaciens. Quantitative assays and the elution profile showed the maximum leaching of phenolics, flavonoids, and salicylic acid immediately after the excision of explants and continued till 4 to 8 h post-excision. Pre-treatment of excised explants with inhibitors of polyphenol oxidase like L-cysteine, DTT, and sodium thiosulfate before co-cultivation showed the recovery of A. tumefaciens cfu, decreased the accumulation of phenolics, and improved transformation frequency. Our results show the hypersensitive response of excision stress for the expression of defense response-related genes and synthesis of metabolites in grain legume chickpea against pathogen infestation including Agrobacterium.

  4. Post-explant visualization of thrombi in outflow grafts and their junction to a continuous-flow total artificial heart using a high-definition miniaturized camera.

    Science.gov (United States)

    Karimov, Jamshid H; Horvath, David; Sunagawa, Gengo; Byram, Nicole; Moazami, Nader; Golding, Leonard A R; Fukamachi, Kiyotaka

    2015-12-01

    Post-explant evaluation of the continuous-flow total artificial heart in preclinical studies can be extremely challenging because of the device's unique architecture. Determining the exact location of tissue regeneration, neointima formation, and thrombus is particularly important. In this report, we describe our first successful experience with visualizing the Cleveland Clinic continuous-flow total artificial heart using a custom-made high-definition miniature camera.

  5. CRESCIMENTO E OXIDAÇÃO DE EXPLANTES DE BANANEIRA-PRATA (Musa AAB IN VITRO: I. CONCENTRAÇÕES DE SAIS DE FERRO, COBRE E ZINCO

    Directory of Open Access Journals (Sweden)

    UTINO SERGIO

    2001-01-01

    Full Text Available Este experimento teve como objetivo avaliar a influência de diferentes concentrações de ferro, cobre e zinco do meio MS (MURASHIGE & SKOOG, 1962 no controle da oxidação de explantes iniciais de bananeira-Prata (Musa AAB. Foram utilizadas três concentrações (100, 50 e 0 miM de FeEDTA, duas concentrações (0,1 e 0miM de (CuSO4.5H2O e duas concentrações (30 e 0miM de (ZnSO4.7H2O, num delineamento inteiramente casualizado, arranjado em um fatorial completo 3 x 2 x 2, utilizando-se de 15 repetições. Ápices caulinares foram inoculados em meio MS modificado e, decorridos 28 dias após a inoculação, avaliaram-se a massa de matéria fresca, altura e grau de oxidação. Observou-se que esses micronutrientes são essenciais para o crescimento dos explantes e que a concentração de ferro influencia na oxidação de explantes, sendo que maiores graus de escurecimento foram observados nas concentrações mais elevadas. A redução ou retirada destes elementos do meio MS, isoladamente ou em combinações, não foi suficiente para eliminar a oxidação dos explantes.

  6. Prolactin-induced alpha-lactalbumin activity in mammary explants from pregnant rabbits. A role for epidermal growth factor and glucocorticoids.

    OpenAIRE

    Sankaran, L.; Topper, Y J

    1984-01-01

    Exogenous prolactin alone can induce alpha-lactalbumin activity in rabbit mammary explants. Under these conditions, exogenous corticosol has no effect. However, low levels of epidermal growth factor (EGF) can markedly inhibit the induction by prolactin, and this inhibitory effect, in turn, can be prevented by cortisol. The steroid can, in fact, convert EGF from a potent inhibitor into an agent which enhances the induction. None of the other growth factors tested inhibits induction of alpha-la...

  7. Chondrogenic Priming at Reduced Cell Density Enhances Cartilage Adhesion of Equine Allogeneic MSCs - a Loading Sensitive Phenomenon in an Organ Culture Study with 180 Explants

    Directory of Open Access Journals (Sweden)

    Jan H. Spaas

    2015-09-01

    Full Text Available Background: Clinical results of regenerative treatments for osteoarthritis are becoming increasingly significant. However, several questions remain unanswered concerning mesenchymal stem cell (MSC adhesion and incorporation into cartilage. Methods: To this end, peripheral blood (PB MSCs were chondrogenically induced and/or stimulated with pulsed electromagnetic fields (PEMFs for a brief period of time just sufficient to prime differentiation. In an organ culture study, PKH26 labelled MSCs were added at two different cell densities (0.5 x106 vs 1.0 x106. In total, 180 explants of six horses (30 per horse were divided into five groups: no lesion (i, lesion alone (ii, lesion with naïve MSCs (iii, lesion with chondrogenically-induced MSCs (iv and lesion with chondrogenically-induced and PEMF-stimulated MSCs (v. Half of the explants were mechanically loaded and compared with the unloaded equivalents. Within each circumstance, six explants were histologically evaluated at different time points (day 1, 5 and 14. Results: COMP expression was selectively increased by chondrogenic induction (p = 0.0488. PEMF stimulation (1mT for 10 minutes further augmented COL II expression over induced values (p = 0.0405. On the other hand, MSC markers remained constant over time after induction, indicating a largely predifferentiated state. In the unloaded group, MSCs adhered to the surface in 92.6% of the explants and penetrated into 40.7% of the lesions. On the other hand, physiological loading significantly reduced surface adherence (1.9% and lesion filling (3.7% in all the different conditions (p Conclusion: The present study demonstrates that primed chondrogenic induction of MSCs at a lower cell density without loading results in significantly enhanced and homogenous MSC adhesion and incorporation into equine cartilage.

  8. Effect of Hypoxia on the Calcium and Magnesium Content, Lipid Peroxidation Level, and Ca2+-ATPase Activity of Syncytiotrophoblast Plasma Membranes from Placental Explants

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    Delia I. Chiarello

    2014-01-01

    Full Text Available In the current study the possible relationship between the Ca2+/Mg2+ ratio of human syncytiotrophoblast plasma membranes and their lipid peroxidation and Ca2+-ATPase activity was determined. Syncytiotrophoblast plasma membranes of placental explants cultured under hypoxia increased their lipid peroxidation and Ca2+ content, diminished their Ca2+-ATPase activity, and kept their Mg2+ content unchanged. Membranes preincubated with different concentrations of Ca2+ increased their Ca2+ content without changes in their Mg2+ content. There is a direct relationship between Ca2+ content and lipid peroxidation of the membranes, as well as an inverse relationship between their Ca2+ content and Ca2+-ATPase activity. On the contrary, preincubation of membranes with different concentrations of Mg2+ showed a higher Mg2+ content without changing their lipid peroxidation and Ca2+-ATPase activity. Explants cultured under hypoxia in the presence of 4 mM MgSO4 showed similar values of lipid peroxidation and Ca2+-ATPase activity of their membranes compared to those of explants cultured under normoxia. Increased Ca2+ content of the membranes by interacting with negatively charged phospholipids could result in destabilizing effects of the membrane structure, exposing hydrocarbon chains of fatty acids to the action of free radicals. Mg2+ might exert a stabilizing effect of the membranes, avoiding their exposure to free radicals.

  9. Geldanamycin induces production of heat shock protein 70 and partially attenuates ototoxicity caused by gentamicin in the organ of Corti explants

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    Haupt Heidemarie

    2009-09-01

    Full Text Available Abstract Background Heat shock protein 70 (HSP70 protects inner ear cells from damage and death induced by e.g. heat or toxins. Benzoquinone ansamycin antibiotic geldanamycin (GA was demonstrated to induce the expression of HSP70 in various animal cell types. The aim of our study was to investigate whether GA induces HSP70 in the organ of Corti (OC, which contains the auditory sensory cells, and whether GA can protect these cells from toxicity caused by a common aminoglycoside antibiotic gentamicin. Methods To address these questions, we used the OC explants isolated from p3-p5 rats. As a read-out, we used RT-PCR, ELISA and immunofluorescence. Results We found that GA at the concentration of 2 μM efficiently induced HSP70 expression on mRNA and protein level in the OC explants. Confocal microscopy revealed that HSP70 induced by GA is expressed by hair cells and interdental cells of spiral limbus. Preincubation of explants with 2 μM GA prior to adding gentamicin (500 μM significantly reduced the loss of outer but not inner hair cells, suggesting different mechanisms of otoprotection needed for these two cell types. Conclusion GA induced HSP70 in the auditory sensory cells and partially protected them from toxicity of gentamicin. Understanding the molecular mechanisms of GA otoprotection may provide insights for preventative therapy of the hearing loss caused by aminoglycoside antibiotics.

  10. Release of Pro-Inflammatory Cytokines from Muscle and Bone Causes Tenocyte Death in a Novel Rotator Cuff In Vitro Explant Culture Model.

    Science.gov (United States)

    Connizzo, Brianne K; Grodzinsky, Alan J

    2018-02-15

    Tendinopathy is a significant clinical problem thought to be associated with altered mechanical loading. Explant culture models allow researchers to alter mechanical loading in a controlled in vitro environment while maintaining tenocytes in their native matrix. However, current models do not accurately represent commonly injured tendons, ignoring contributions of associated musculature and bone, as well as regional collagen structure. This study details the characterization of a mouse rotator cuff explant culture model, including bone, tendon, and muscle (BTM). Following harvest, BTM explants were maintained in stress-deprived culture for one week and tendon was then assessed for changes in cell viability, metabolism, matrix structure and content. Matrix turnover occurred throughout culture as manifested in both gene expression and biosynthesis, but this did not translate to net changes in total collagen or sulfated glycosaminoglycan content. Furthermore, tendon structure was not significantly altered throughout culture. However, we found significant cell death in BTM tendons after 3 days in culture, which we hypothesize is cytokine-induced. Using a targeted multiplex assay, we found high levels of pro-inflammatory cytokines released to the culture medium from muscle and bone, levels that did cause cell death in tendon-alone controls. Overall, this model presents an innovative approach to understanding rotator cuff injury and tenocyte mechanobiology in a clinically-relevant tendon structure. Our model can be a powerful tool to investigate how mechanical and biological stimuli can alter normal tendon health and lead to tendon degeneration, and may provide a testbed for therapeutics for tendon repair.

  11. Controlled release of C-type natriuretic peptide by microencapsulation dampens proinflammatory effects induced by IL-1β in cartilage explants.

    Science.gov (United States)

    Peake, Nick J; Pavlov, Anton M; D'Souza, Alveena; Pingguan-Murphy, Belinda; Sukhorukov, Gleb B; Hobbs, Adrian J; Chowdhury, Tina T

    2015-02-09

    C-type natriuretic peptide (CNP) exhibits potent anti-inflammatory effects in chondrocytes that have the potential to repair cartilage damage observed in osteoarthritis (OA). However, treatments for OA have been challenging due to poor targeting and delivery of therapeutics. The present study fabricated polyelectrolyte microcapsules loaded with CNP and examined whether the layer-by-layer (LbL) approach could have protective effects in cartilage explants treated with the pro-inflammatory cytokine, interleukin-1β (IL-1β). SEM showed uniform, 2 to 3 μm spherical microcapsules with morphological characteristic similar to templates loaded with or without CNP. The protein was localized around the external surface of the microcapsules with encapsulation efficiencies >82.9%. CNP release profiles were broadly similar following 9 days of culture. The presence of CNP microcapsules did not significantly affect cell viability (80%) with DNA values that remained stable throughout the culture conditions. Confocal imaging showed clustering of microcapsules in chondrocytes to natriuretic peptide receptor (Npr) 2 and 3. Treatment of cartilage explants with CNP microcapsules led to concentration-dependent inhibition of NO release in response to IL-1β and restoration of matrix synthesis. In summary, we demonstrate controlled delivery of CNP to dampen pro-inflammatory effects induced by IL-1β in cartilage explants. The LbL approach has the potential to promote cartilage repair in vivo.

  12. In vitro culture of Cucumis sativus L. VI. Histological analysis of leaf explants cultured on media with 2, 4-D or 2, 4, 5-T

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    Anna Nadolska-Orczyk

    2014-01-01

    Full Text Available The developmental sequence of callus initiation and somatic embryogenesis in leaf explants of Cucumis sativus cv. Borszczagowski was analysed and compared on media containing two different auxin phenoxy-derivatives (2,4-D and 2,4,5-T and cytokinin (BAP or 2iP. During the first 20 days of culture on media with 2,4,5-T proliferation of parenchymatic tissue occurred mainly and only small meristematic centers were observed. There was an intensive detachment of parenchymatic cells and dissociation of their cell walls near vessels and in the lower part of the explant adjacent to the medium. These cells were strongly plasmolysed. On the 2,4-D containing medium mostly meristematic tissue developed, proliferating around vascular bundles and forming meristematic centers or promeristem-like structures. After 35-50 days of culture, secondary callus was formed by separation of meristematic cells from the meristem surface in explants cultured on the 2,4-D containing medium. On medium supplemented with 2, 4, 5-T the detachment of parenchymatic and meristematic cells occurred, along with formation of a gel-like substance. The gel-like callus contained multi-cellular aggregates, proembryoids and embryoids. This type of callus tissue was initiated more intensively on medium with 2, 4, 5-T, but the frequency of somatic embryogenesis was much lower. The periferial cells of aggregates, proembryoids and embryoids showed the tendency to separate from the surface of the tissue. Many embryoids formed adventitious embryos.

  13. Agrobacterium-mediated genetic transformation of tea leaf explants: effects of counteracting bactericidity of leaf polyphenols without loss of bacterial virulence.

    Science.gov (United States)

    Sandal, Indra; Saini, Uksha; Lacroix, Benoît; Bhattacharya, Amita; Ahuja, Paramvir Singh; Citovsky, Vitaly

    2007-02-01

    Tea is one of the major crops in Asia and Africa, and its improvement by genetic modification is important for economy of many tea-producing regions. Although somatic embryos derived from cotyledon explants have been transformed with Agrobacterium, the leaves of several commercially important tea cultivars have remained recalcitrant to transformation, largely due to bactericidal effect of polyphenols that are exuded by tea leaves in vitro. Moreover, the commonly used polyphenol adsorbents and antioxidants cannot overcome this problem. Leaf explants, however, are more desirable than cotyledon-derived somatic embryos, especially when it is necessary to further improve a selected elite and also retain its superior traits. Thus, we developed a procedure for Agrobacterium-mediated genetic transformation of tea leaf explants which is based on the presence of L-glutamine in the co-cultivation medium. We then showed that the transformation process is facilitated via a protective action of L-glutamine against bactericidal effects of leaf polyphenols without affecting the bacterial virulence (vir) gene expression.

  14. Tumours with elevated levels of the Notch and Wnt pathways exhibit efficacy to PF-03084014, a γ-secretase inhibitor, in a preclinical colorectal explant model.

    Science.gov (United States)

    Arcaroli, J J; Quackenbush, K S; Purkey, A; Powell, R W; Pitts, T M; Bagby, S; Tan, A C; Cross, B; McPhillips, K; Song, E-K; Tai, W M; Winn, R A; Bikkavilli, K; Vanscoyk, M; Eckhardt, S G; Messersmith, W A

    2013-08-06

    Dysregulation of the Notch pathway has been identified to play an important role in the development and progression of colorectal cancer (CRC). In this study, we used a patient-derived CRC explant model to investigate the efficacy of the clinical γ-secretase inhibitor (GSI) PF-03084014. A total of 16 CRC explants were treated with PF-03084014. Knockdown of RBPjκ gene was used to determine the specificity of PF-03084014. Evaluation of the Notch and Wnt pathways in CRC explant tumours was performed by gene array and immunoblotting. We identified a subset of CRC tumours that exhibited elevations of the Notch and Wnt pathways sensitive to PF-03084014. Treatment with the GSI resulted in a significant reduction in cleaved Notch, Axin2 (Wnt-dependent gene) and active β-catenin. In addition, knockdown of the RBPjκ gene showed that PF-03084014 has specificity for the Notch pathway in an HCT116 cell line xenograft model. Finally, an increase in apoptosis was observed in CRC001- and CRC021-sensitive tumours. This study provides evidence that inhibition of γ-secretase may be beneficial in a subset of patients with elevated levels of the Wnt and Notch pathways.

  15. Differential Gene Expression in Explanted Human Retinal Pigment Epithelial Cells 24-Hours Post-Exposure to 532 nm, 3.0 ns Pulsed Laser Light and 1064 nm, 170 ps Pulsed Laser Light 12-Hours Post-Exposure: Results Compendium

    National Research Council Canada - National Science Library

    Obringer, John

    2004-01-01

    .... We assessed the sublethal insult to human retinal pigment epithelial cells using a cadaver organ donor explant system for genes differentially expressed 12 and 24 hours post- exposure using gene...

  16. Suppression of MMP activity in bovine cartilage explants cultures has little if any effect on the release of aggrecanase-derived aggrecan fragments

    Directory of Open Access Journals (Sweden)

    Sondergaard Bodil-Cecilie

    2009-12-01

    Full Text Available Abstract Background Progressive loss of articular cartilage is a central hallmark in many joint disease, however, the relative importance of individual proteolytic pathways leading to cartilage erosion is at present unknown. We therefore investigated the time-dependant release ex vivo of MMP- and aggrecanase-derived fragments of aggrecan and type II collagen into the supernatant of bovine cartilage explants cultures using neo-epitope specific immunoassays, and to associate the release of these fragments with the activity of proteolytic enzymes using inhibitors. Findings Bovine cartilage explants were cultured in the presence or absence of the catabolic cytokines oncostatin M (OSM and tumor necrosis factor alpha (TNFα. In parallel, explants were co-cultured with protease inhibitors such as GM6001, TIMP1, TIMP2 and TIMP3. Fragments released into the supernatant were determined using a range of neo-epitope specific immunoassays; (1 sandwich 342FFGVG-G2 ELISA, (2 competition NITEGE373ELISA (3 sandwich G1-NITEGE373 ELISA (4 competition 374ARGSV ELISA, and (5 sandwich 374ARGSV-G2 ELISA all detecting aggrecan fragments, and (6 sandwich CTX-II ELISA, detecting C-telopeptides of type II collagen. We found that (1 aggrecanase-derived aggrecan fragments are released in the early (day 2-7 and mid phase (day 9-14 into the supernatant from bovine explants cultures stimulated with catabolic cytokines, (2 the release of NITEGE373 neo-epitopes are delayed compared to the corresponding 374ARGSV fragments, (3 the MMP inhibitor GM6001 did not reduce the release of aggrecanase-derived fragment, but induced a further delay in the release of these fragments, and finally (4 the MMP-derived aggrecan and type II collagen fragments were released in the late phase (day 16-21 only. Conclusion Our data support the model, that aggrecanases and MMPs act independently in the processing of the aggrecan molecules, and furthermore that suppression of MMP-activity had little if

  17. Micropropagation and Microrhizome Induction in Costus pictus D. Don Using In Vitro and Ex Vitro Nodal Segments as Explant

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    Kshetrimayum PUNYARANI

    2012-05-01

    Full Text Available Costus pictus D. Don is a potent anti-diabetic plant used in folk, ayurvedic and homeopathic system of medicine. Gene and protein expression of key targets in insulin signaling pathway have revealed that methyl tetracosonoate, a bio-active molecule from Costus pictus extract has anti-diabetic activity. The axillary buds of Costus pictus are dormant. The dormancy of axillary buds were broken when cultured in Murashige and Skoog (MS medium supplemented with 3-4 ?M 6-benzylaminopurine (BAP in combination with 0.2-1 ?M naphthalene acetic acid (NAA. The highest bud-break percentage was achieved in those supplemented with 0.6 ?M NAA and 3 ?M BAP. The sprouted axillary buds were transferred onto medium supplemented with 0.6 ?M NAA and 6-10 ?M BAP for shoot multiplication. The maximum average number of shoot was achieved in medium supplemented with 0.6 ?M NAA and 8 ?M BAP. The shoots were successfully rooted when transferred onto media supplemented with 1-12 ?M NAA or indole-3-acetic acid (IAA and 3 ?M BAP. The maximum number of roots was found in 8 ?M NAA and 3 ?M BAP. The dormancy of in vitro axillary buds were also successfully broken in stems from which shoot apex were decapitated and cultured in MS medium with 0.6 ?M NAA, 7 ?M BAP and 5-13% sucrose. These sprouted in vitro axillary buds could be used as secondary explants for shoot multiplication. The maximum was in medium supplemented with 9% sucrose. Rhizomes were successfully induced when 4-month old plantlets were cultured on strength MS medium supplemented with 2.4 ?M NAA, 32 ?M BAP and 5-13% sucrose. Microrhizomes formed in 9% sucrose was largest in size with highest average fresh weight.

  18. Micropropagation and Microrhizome Induction in Costus pictus D. Don Using In Vitro and Ex Vitro Nodal Segments as Explant

    Directory of Open Access Journals (Sweden)

    Kshetrimayum PUNYARANI

    2012-05-01

    Full Text Available Costus pictus D. Don is a potent anti-diabetic plant used in folk, ayurvedic and homeopathic system of medicine. Gene and protein expression of key targets in insulin signaling pathway have revealed that methyl tetracosonoate, a bio-active molecule from Costus pictus extract has anti-diabetic activity. The axillary buds of Costus pictus are dormant. The dormancy of axillary buds were broken when cultured in Murashige and Skoog (MS medium supplemented with 3-4 ?M 6-benzylaminopurine (BAP in combination with 0.2-1 ?M naphthalene acetic acid (NAA. The highest bud-break percentage was achieved in those supplemented with 0.6 ?M NAA and 3 ?M BAP. The sprouted axillary buds were transferred onto medium supplemented with 0.6 ?M NAA and 6-10 ?M BAP for shoot multiplication. The maximum average number of shoot was achieved in medium supplemented with 0.6 ?M NAA and 8 ?M BAP. The shoots were successfully rooted when transferred onto media supplemented with 1-12 ?M NAA or indole-3-acetic acid (IAA and 3 ?M BAP. The maximum number of roots was found in 8 ?M NAA and 3 ?M BAP. The dormancy of in vitro axillary buds were also successfully broken in stems from which shoot apex were decapitated and cultured in MS medium with 0.6 ?M NAA, 7 ?M BAP and 5-13% sucrose. These sprouted in vitro axillary buds could be used as secondary explants for shoot multiplication. The maximum was in medium supplemented with 9% sucrose. Rhizomes were successfully induced when 4-month old plantlets were cultured on � strength MS medium supplemented with 2.4 ?M NAA, 32 ?M BAP and 5-13% sucrose. Microrhizomes formed in 9% sucrose was largest in size with highest average fresh weight.

  19. TGF-beta1, -beta2 and -beta3 cooperate to facilitate tubulogenesis in the explanted quail heart.

    Science.gov (United States)

    Holifield, Jennifer S; Arlen, Angela M; Runyan, Raymond B; Tomanek, Robert J

    2004-01-01

    Transforming growth factor-beta (TGF-beta) isoforms have been implicated as both pro- and anti-angiogenic modulators. In this study we addressed the roles of TGF-beta isoforms on coronary tubulogenesis. Embryonic (E6) quail ventricular specimens were explanted onto collagen gels allowing endothelial cells to migrate and form vascular tubes. Growth factors and/or neutralizing growth factor antibodies were added to the cultures. Endothelial cells were identified using a quail endothelial cell marker, QH1. Image analysis was used to quantify aggregate tube length. Addition of any isoform (TGF-beta(1), TGF-beta(2) or TGF-beta(3)) virtually prevented tubulogenesis (>95% inhibition), while stimulation of tubulogenesis occurred by adding neutralizing antibodies to TGF-beta(3), but not to TGF-beta(1) or -beta(2). When all three isoforms were added, tubulogenesis was enhanced, indicating the key role of TGF-beta(3). Documentation of the inhibitory effect of TGF-beta isoforms on tubulogenesis is further supported by our experiments in which the marked enhancement of tube formation by bFGF and VEGF was negated when exogenous TGF-beta(1), -beta(2), or -beta(3) were added to the cultures. (1) TGF-beta(1), -beta(2) and -beta(3) each inhibits angiogenesis; (2) cooperation between the three TGF-beta isoforms and other angiogenic factors is essential for the regulation of normal tubulogenesis and (3) the stimulatory effect of VEGF or bFGF on tubulogenesis is negated by exogenous TGB-betas. Copyright 2004 S. Karger AG, Basel.

  20. Establishment and Characterization of Primary Cultures from Iranian Oral Squamous Cell Carcinoma Patients by Enzymatic Method and Explant Culture

    Directory of Open Access Journals (Sweden)

    Meysam Ganjibakhsh

    2017-10-01

    Full Text Available Objectives: Oral Squamous Cell Carcinoma (OSCC is the most frequent oral cancer worldwide. It is known as the eighth most common cancer in men and as the fifth most common cancer in women. Cytogenetic and biochemical studies in recent decades have emphasized the necessity of providing an appropriate tool for such researches. Cancer cell culture is a useful tool for investigations on biochemical, genetic, molecular and immunological characteristics of different cancers, including oral cancer. Here, we explain the establishment process of five primary oral cancer cells derived from an Iranian population.Materials and Methods: The specimens were obtained from five oral cancer patients. Enzymatic, explant culture and magnetic-activated cell sorting (MACS methods were used for cell isolation. After quality control tests, characterization and authentication of primary oral cancer cells were performed by short tandem repeats (STR profiling, chromosome analysis, species identification, and monitoring the growth, morphology and the expression of CD326 and CD133 markers.Results: Five primary oral cancer cells were established from an Iranian population. The flow cytometry results showed that the isolated cells were positive for CD326 and CD133 markers. Furthermore, the cells were free from mycoplasma, bacterial and fungal contamination. No misidentified or cross-contaminated cells were detected by STR analysis.Conclusions: Human primary oral cancer cells provide an extremely useful platform for studying carcinogenesis pathways of oral cancer in Iranian population. They may be helpful in explaining the ethnic differences in cancer biology and the individuality in anticancer drug response in future studies.

  1. Tumorigenic risk of human induced pluripotent stem cell explants cultured on mouse SNL76/7 feeder cells

    Energy Technology Data Exchange (ETDEWEB)

    Kamada, Mizuna; Mitsui, Youji, E-mail: y-mitsui8310@hb.tp1.jp; Kumazaki, Tsutomu; Kawahara, Yuta; Matsuo, Taira; Takahashi, Tomoko, E-mail: t-takahashi@kph.bunri-u.ac.jp

    2014-10-24

    Highlights: • hiPS cell explants formed malignant tumors when SNL76/7 feeder cells were used. • Multi type tumors developed by interaction of SNL76/7 feeder cells with hiPS cells. • Tumorigenic risk occurs by co-culture of hiPS cells with SNL76/7 feeder cells. - Abstract: The potential for tumor formation from transplanted human induced pluripotent stem cell (hiPSC) derivatives represents a high risk in their application to regenerative medicine. We examined the genetic origin and characteristics of tumors, that were formed when 13 hiPSC lines, established by ourselves, and 201B7 hiPSC from Kyoto University were transplanted into severe combined immune-deficient (SCID) mice. Though teratomas formed in 58% of mice, five angiosarcomas, one malignant solitary fibrous tumor and one undifferentiated pleomorphic sarcoma formed in the remaining mice. Three malignant cell lines were established from the tumors, which were derived from mitomycin C (MMC)-treated SNL76/7 (MMC-SNL) feeder cells, as tumor development from fusion cells between MMC-SNL and hiPSCs was negative by genetic analysis. While parent SNL76/7 cells produced malignant tumors, neither MMC-SNL nor MMC-treated mouse embryo fibroblast (MEF) produced malignant tumors. When MMC-SNL feeder cells were co-cultured with hiPSCs, growing cell lines were generated, that expressed genes similar to the parent SNL76/7 cells. Thus, hiPSCs grown on MMC-SNL feeder cells have a high risk of generating feeder-derived malignant tumors. The possible mechanism(s) of growth restoration and the formation of multiple tumor types are discussed with respect of the interactions between MMC-SNL and hiPSC.

  2. Organogênese in vitro a partir de explante caulinar na regeneração de clones de Eucalyptus grandis W. Hill ex Maiden X E. urophylla S. T. Blake In vitro regeneration from stem explants of Eucalyptus grandis x E. urophylla clones through organogenesis

    Directory of Open Access Journals (Sweden)

    Elisa Cristina Soares de Carvalho Alves

    2004-10-01

    Full Text Available Com o objetivo de testar a regeneração in vitro por organogênese a partir de explante caulinar de três clones híbridos de Eucalyptus grandis x Eucalyptus urophylla, foram avaliados os efeitos dos reguladores de crescimento TDZ [1-fenil-3-(1,2,3-tiadiazol-5-iluréia], BAP (6-benzilaminopurina e ANA (ácido naftalenoacético. De modo geral, pôde-se observar resposta diferenciada dos clones quanto a intensidade, textura, coloração e grau de oxidação dos calos, em função dos tratamentos com os reguladores de crescimento. Os melhores resultados de calejamento foram dos tratamentos com a combinação dos reguladores de crescimento TDZ (0,5 mg L-1 e ANA (0,1 mg L-1, obtendo-se 100% de calejamento no explante caulinar. Houve a formação de estruturas nodulares compactas, principalmente na extremidade dos explantes caulinares, sendo essas regiões responsáveis pela regeneração de gemas adventícias. Em relação à regeneração, a melhor resposta foi obtida com 1,0 mg L-1 BAP.With the objective of testing in vitro regeneration through organogenesis from stem explants of three hybrid clones of Eucalyptus grandis x Eucalyptus urophylla, the effects of the growth regulators TDZ [1-phenil-3-(1, 2, 3-thiadiazol-5-ilurea], BAP (6-benzilaminopurine and NAA (Naphthalene acetic acid were evaluated. In general, a differentiated clone response in relation to intensity, texture, color and oxidation degree of calli was observed due to growth regulator treatments. The best results in callus formation were recorded for the treatments with the combination TDZ (0,5 mg L-1 and ANA (0,1 mg L-1, achieving 100% of callus formation from the stem explants. Compact node structures appeared mainly in the extremities of the stem explants, being those areas responsible for the regeneration of adventitious buds. The best regeneration response was obtained with 1,0 mg L-1 BAP.

  3. Germinação, avaliação do ácido giberélico e posição do explante no alongamento in vitro de Uncaria guianensis (AUBLET Gmelin Rubiaceae (UNHA-DE-GATO Germination, avaliation of giberelic acid and position of explants in vitro of Uncaria guianensis (Aublet Gmelin Rubiaceae (cat's claw

    Directory of Open Access Journals (Sweden)

    Rita de Cassia Alves Pereira

    2006-08-01

    Full Text Available Na Amazônia existe uma diversidade vegetal, onde se encontram muitas plantas com propriedades medicinais e, que durante milênios são utilizadas pelas comunidades nativas. Uma dessas plantas é a unha-de-gato (Uncaria guianensis, cujo valor medicinal se atribui a efeitos imuno-estimulantes, anti-inflamatório e inibidores de crescimento de células cancerígenas. Atualmente, a espécie vem sendo submetida a uma extração indiscriminada e intensiva, podendo levá-la a sua extinção. A micropropagação permite solucionar problemas dessa natureza. Objetivou-se com este trabalho identificar um protocolo de propagação in vitro desta espécie. O melhor meio para germinação dos embriões foi ¼ de MS independente da presença ou não de sacarose. A posição de inoculação do explante da espécie Uncaria guianensis demonstrou exercer influência no número médio de brotações/explante original, assim como no número de gemas iniciais. Presença ou ausência de ácido giberélico não exerceu efeito nesta característica, exceto quando explantes com uma única gema foram inoculados na horizontal.In the Amazon Rain Forest there are a great botanical diversity where can be found a lot of plants with medicinal proprieties, and for ages it has been used by the native people. One of those plants is Uncaria guianensis known as Cat's Claw to which is believed to have antinflammatories, immunostimulating and growth inhibitors effects on cancerigenic cells. Nowadays, those species have been under uncontrolled and long extraction that could take to extinction. The micropropagation can solve these problems. The purpose of this work was to identify in vitro propagation protocol from these specie. The best medium for embryo germination was ¼ MS independent from presence or absence of saccarose. The explant inoculation position from Uncaria guianensis specie influenciated the shoots average number/original explant, as well as, the initial buds numbers

  4. Successful Consecutive Expansion of Limbal Explants Using a Biosafe Culture Medium under Feeder Layer-Free Conditions.

    Science.gov (United States)

    López-Paniagua, Marina; Nieto-Miguel, Teresa; de la Mata, Ana; Galindo, Sara; Herreras, José M; Corrales, Rosa M; Calonge, Margarita

    2017-05-01

    Transplantation of in vitro cultured limbal epithelial stem cells (LESCs) is a treatment widely used for LESC deficiency. However, the number of limbal tissue donors is limited, and protocols for LESC cultivation often include compounds and/or feeder layers that can induce side effects and/or increase the cost of the culture procedure. We investigated the feasibility of obtaining more than one limbal primary culture (LPC) from the same biopsy using a culture medium in which several potentially harmful compounds were replaced at the same time by biosafe supplements, allowing the LESC cultivation without feeder layers. We established feeder layer-free LPCs with three culture media: (1) a modified supplemental hormonal epithelial medium, containing potential harmful components (cholera toxin, dimethylsulfoxide, and fetal bovine serum [FBS]), (2) IOBA-FBS, a medium with FBS but with no other harmful supplements, and (3) IOBA-HS, similar to IOBA-FBS but with human serum instead of FBS. Additionally, the same limbal explant was consecutively cultured with IOBA-HS producing three cultures. LPCs were characterized by real-time reverse transcription polymerase chain reaction and/or immunofluorescence. LPCs cultured with the three media under feeder layer-free conditions showed cuboidal cells and no significant differences in the percentage of positive cells for limbal (ABCG2, p63, and K14) and corneal (K3, K12) proteins. Except for ABCG2, the relative mRNA expression of the LESC markers was significantly higher when IOBA-FBS or IOBA-HS was used. LPC1 showed characteristics similar to LPC0, while LPC2 cell morphology became elongated and the expression of some LESC markers was diminished. IOBA-HS enables the culturing of up to two biosafe homologous LPCs from one limbal tissue under feeder layer-free conditions. The routine use of this culture medium could improve both the biosafety and the number of available LPCs for potential clinical transplantation, as well as decrease

  5. Somatic embryogenesis in anthurium (Anthurium andraeanum cv. Eidibel as affected by different explants - doi: 10.4025/actasciagron.v36i1.16557

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    Marcos Vinícius Marques Pinheiro

    2014-01-01

    Full Text Available This study establishes a protocol for the induction of somatic embryogenesis in Anthurium andraeanum cv. Eidibel. The experiment was arranged in a completely randomized 5 x 5 x 5 factorial design using five explant types (whole leaves, half leaves; petiole; nodal segments and root segments from in vitro plantlets; five auxins: indole-3-acetic acid (IAA, α-naphthaleneacetic acid (NAA, indole-3-butyric acid (IBA, 2,4-dichlorophenoxyacetic acid (2,4-D, and 4-amino-3,5,6-trichloropicolinic acid (Picloram; at five concentrations (0, 2.5, 5, 7.5 and 10 µM, with five replications using five Petri dishes. The cultures were maintained in a growth room at 25 ± 2ºC in the dark. The explant type was investigated for the induction of somatic embryogenesis in anthurium cv. Eidibel, and nodal segments were shown to be the most suitable explant for this process. After 60 days in culture, the highest number of embryogenic calli was recorded for the nodal segments cultured in NAA (5, 7.5 and 10 µM, 2,4-D (10 µM and Picloram (7.5 and 10 µM. The histological analysis confirmed the presence of embryos with established polarization, procambium, ground meristem and protoderm in the nodal segments cultured in Pierik medium containing 10 µM NAA. After the conversion of the somatic embryos into plantlets, these plantlets were acclimatized transferred to in vivo conditions and grown into normal plants.

  6. Role of TDZ in the quick regeneration of multiple shoots from nodal explant of Vitex trifolia L.--an important medicinal plant.

    Science.gov (United States)

    Ahmed, Md Rafique; Anis, Mohammad

    2012-11-01

    The effect of thidiazuron (TDZ) has been investigated in shoot multiplication for a simple, efficient, rapid, and commercially applicable regeneration protocol of an important medicinal plant, Vitex trifolia. Multiple shoots were induced in nodal explants obtained from a mature tree on Murashige and Skoog (MS) medium supplemented with TDZ in various concentrations (0.5, 1.0, 2.5, 5.0, 7.5, or 10.0 μM). Prolonged exposure of the culture to TDZ had an adverse affect. To avoid this, the cultures were transferred to TDZ-free MS medium or MS medium fortified with various concentrations of 6-benzyladenine (BA) alone or in combination with α-naphthalene acetic acid (NAA) to enhance multiplication, proliferation, and elongation of induced shoots. Optimum shoot multiplication and elongation was achieved when TDZ-exposed explants were repeatedly subcultured on MS media containing a combination of 1.0 μM BA and 0.5 μM NAA. The highest shoot regeneration frequency (90 %) and maximum number (22.3 ± 0.2) of shoots per explant with shoot length of (5.2 ± 0.2 cm) was recorded on MS medium fortified with 5.0 μM TDZ. In vitro rooting of isolated shoots was achieved best in half-strength MS medium containing 0.5 μM NAA. Properly rooted plantlets were successfully hardened off and acclimatized in thermocol cups containing sterile Soilrite. These plantlets were then transferred to pots containing different potting substrate; percentage survival of the plantlets was highest in vermiculite/garden soil mixture (1:1) and successfully transfer to greenhouse under sunlight.

  7. Comparison of the sensitivity of four native Canadian fish species to 17-α ethinylestradiol, using an in vitro liver explant assay.

    Science.gov (United States)

    Beitel, Shawn C; Doering, Jon A; Eisner, Bryanna K; Hecker, Markus

    2015-12-01

    Exposure to environmental estrogens and other endocrine-active chemicals can impact reproduction of freshwater fishes. While extensive data exists regarding the effect of estrogens on standard laboratory species, little is known about the sensitivity of freshwater fishes native to North America to these compounds. Current testing strategies for the toxicological assessment of contaminants still rely heavily on studies with live animals, which poses increasing concerns from an economical and ethical perspective. Therefore, the aim of the present study was to investigate the sensitivity of four native species, namely, northern pike (Esox lucius), walleye (Sander vitreus), white sucker (Catostomus commersoni), and juvenile white sturgeon (Acipenser transmontanus), to an environmental estrogen, 17α-ethinylestradiol (EE2), using an in vitro tissue explant approach. Transcript abundances of vitellogenin (VTG) as well as the estrogen receptors (ER) α and β were used as the measuring endpoints as they represent well established biomarkers previously used to assess exposure to estrogens. Transcript abundance of VTG was upregulated in a concentration-dependent manner in each species. Liver explants of male walleye were found to have the greatest sensitivity to EE2, with a lowest observable effect concentration of 300 ng/L (1.0 nM) for VTG transcript abundance, with juvenile white sturgeon having the greatest magnitude of VTG transcript upregulation in exposed tissue (15-fold relative to control). Exposure of liver explants to EE2 resulted in no alteration in transcript abundance of ERβ, whereas upregulation of ERα was observed in northern pike only. Based on in vitro expression of VTG, the species tested were among the species with greatest sensitivity to environmental estrogens tested to date.

  8. Indução de calos em sempre-viva (Syngonanthus mucugensis Giulietti, utilizando diferentes tipos de explantes e concentrações de BAP = Callus induction in sempre-viva (Syngonanthus mucugensis Giulietti using different types of explants and BAP concentrations

    Directory of Open Access Journals (Sweden)

    Janilza da Paixão Santos

    2008-04-01

    Full Text Available A sempre-viva-de-mucugê [Syngonanthus mucugensis – Eriocaulaceae] é uma planta com grande valor ornamental e caracteriza-se pela durabilidade de suas inflorescências que se mantém mesmo depois de coletadas e secas. A propagação sexuada dessa espécie resulta em plantas desuniformes por ser geneticamente segregante e, aliada aeste fato, existe a ameaça de extinção da espécie, sendo por isso restrita a coleta na sua região de origem. Desta forma, a cultura de tecidos torna-se uma alternativa viável para a formação de novas mudas e, por isso, objetivou-se, neste trabalho, avaliar a indução de calos in vitro de Syngonanthus mucugensis, utilizando diferentes concentrações de BAP (6-benzilaminapurina. O delineamento experimental foi inteiramente casualizado (DIC, sendo que cadatratamento continha quatro repetições e cada repetição era formada por quatro explantes. Os explantes utilizados foram plantas cultivadas in vitro e segmentos nodais delas. O meio de cultivo utilizado foi o MS (metade dos sais, suplementado com (0,0; 0,89; 1,78; 3,55; 7,10; 14,21 e 28,42 μM de BAP. Aos 60 dias, avaliaram-se a sobrevivência dos explantes e a porcentagem de formação de calos. Verificou-se que plantas inteiras e segmentos nodais deSyngonanthus mucugensis são explantes responsíveis à formação de calos friáveis, sendo que a produção significativa é obtida utilizando-se as concentrações de 1,78 e 3,55 μM de BAP.Sempre-viva-de-mucugê [Syngonanthus mucugensis – Eriocaulaceae] is a plant with great ornamental value characterized by the durability of its inflorescences, which remains even after collected and dried. Thesexual propagation of this species results in disuniform plants, as it is genetically segregating; in addition, this species is currently endangered, with collection therefore restricted to its region of origin. Thus, tissue culture becomes a viable alternative for theformation of new plants. This study aimed to

  9. Enrofloxacin and Toltrazuril Are Able to Reduce Toxoplasma gondii Growth in Human BeWo Trophoblastic Cells and Villous Explants from Human Third Trimester Pregnancy

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    Rafaela J. da Silva

    2017-07-01

    Full Text Available Classical treatment for congenital toxoplasmosis is based on combination of sulfadiazine and pyrimethamine plus folinic acid. Due to teratogenic effects and bone marrow suppression caused by pyrimethamine, the establishment of new therapeutic strategies is indispensable to minimize the side effects and improve the control of infection. Previous studies demonstrated that enrofloxacin and toltrazuril reduced the incidence of Neospora caninum and Toxoplasma gondii infection. The aim of the present study was to evaluate the efficacy of enrofloxacin and toltrazuril in the control of T. gondii infection in human trophoblast cells (BeWo line and in human villous explants from the third trimester. BeWo cells and villous were treated with several concentrations of enrofloxacin, toltrazuril, sulfadiazine, pyrimethamine, or combination of sulfadiazine+pyrimethamine, and the cellular or tissue viability was verified. Next, BeWo cells were infected by T. gondii (2F1 clone or the ME49 strain, whereas villous samples were only infected by the 2F1 clone. Then, infected cells and villous were treated with all antibiotics and the T. gondii intracellular proliferation as well as the cytokine production were analyzed. Finally, we evaluated the direct effect of enrofloxacin and toltrazuril in tachyzoites to verify possible changes in parasite structure. Enrofloxacin and toltrazuril did not decrease the viability of cells and villous in lower concentrations. Both drugs were able to significantly reduce the parasite intracellular proliferation in BeWo cells and villous explants when compared to untreated conditions. Regardless of the T. gondii strain, BeWo cells infected and treated with enrofloxacin or toltrazuril induced high levels of IL-6 and MIF. In villous explants, enrofloxacin induced high MIF production. Finally, the drugs increased the number of unviable parasites and triggered damage to tachyzoite structure. Taken together, it can be concluded that

  10. Enrofloxacin and Toltrazuril Are Able to Reduce Toxoplasma gondii Growth in Human BeWo Trophoblastic Cells and Villous Explants from Human Third Trimester Pregnancy.

    Science.gov (United States)

    da Silva, Rafaela J; Gomes, Angelica O; Franco, Priscila S; Pereira, Ariane S; Milian, Iliana C B; Ribeiro, Mayara; Fiorenzani, Paolo; Dos Santos, Maria C; Mineo, José R; da Silva, Neide M; Ferro, Eloisa A V; de Freitas Barbosa, Bellisa

    2017-01-01

    Classical treatment for congenital toxoplasmosis is based on combination of sulfadiazine and pyrimethamine plus folinic acid. Due to teratogenic effects and bone marrow suppression caused by pyrimethamine, the establishment of new therapeutic strategies is indispensable to minimize the side effects and improve the control of infection. Previous studies demonstrated that enrofloxacin and toltrazuril reduced the incidence of Neospora caninum and Toxoplasma gondii infection. The aim of the present study was to evaluate the efficacy of enrofloxacin and toltrazuril in the control of T. gondii infection in human trophoblast cells (BeWo line) and in human villous explants from the third trimester. BeWo cells and villous were treated with several concentrations of enrofloxacin, toltrazuril, sulfadiazine, pyrimethamine, or combination of sulfadiazine+pyrimethamine, and the cellular or tissue viability was verified. Next, BeWo cells were infected by T. gondii (2F1 clone or the ME49 strain), whereas villous samples were only infected by the 2F1 clone. Then, infected cells and villous were treated with all antibiotics and the T. gondii intracellular proliferation as well as the cytokine production were analyzed. Finally, we evaluated the direct effect of enrofloxacin and toltrazuril in tachyzoites to verify possible changes in parasite structure. Enrofloxacin and toltrazuril did not decrease the viability of cells and villous in lower concentrations. Both drugs were able to significantly reduce the parasite intracellular proliferation in BeWo cells and villous explants when compared to untreated conditions. Regardless of the T. gondii strain, BeWo cells infected and treated with enrofloxacin or toltrazuril induced high levels of IL-6 and MIF. In villous explants, enrofloxacin induced high MIF production. Finally, the drugs increased the number of unviable parasites and triggered damage to tachyzoite structure. Taken together, it can be concluded that enrofloxacin and

  11. Endothelial and smooth muscle cells derived from human cardiac explants demonstrate angiogenic potential and suitable for design of cell-containing vascular grafts.

    Science.gov (United States)

    Zakharova, I S; Zhiven', M K; Saaya, Sh B; Shevchenko, A I; Smirnova, A M; Strunov, A; Karpenko, A A; Pokushalov, E A; Ivanova, L N; Makarevich, P I; Parfyonova, Y V; Aboian, E; Zakian, S M

    2017-03-03

    Endothelial and smooth muscle cells are considered promising resources for regenerative medicine and cell replacement therapy. It has been shown that both types of cells are heterogeneous depending on the type of vessels and organs in which they are located. Therefore, isolation of endothelial and smooth muscle cells from tissues relevant to the area of research is necessary for the adequate study of specific pathologies. However, sources of specialized human endothelial and smooth muscle cells are limited, and the search for new sources is still relevant. The main goal of our study is to demonstrate that functional endothelial and smooth muscle cells can be obtained from an available source-post-surgically discarded cardiac tissue from the right atrial appendage and right ventricular myocardium. Heterogeneous primary cell cultures were enzymatically isolated from cardiac explants and then grown in specific endothelial and smooth muscle growth media on collagen IV-coated surfaces. The population of endothelial cells was further enriched by immunomagnetic sorting for CD31, and the culture thus obtained was characterized by immunocytochemistry, ultrastructural analysis and in vitro functional tests. The angiogenic potency of the cells was examined by injecting them, along with Matrigel, into immunodeficient mice. Cells were also seeded on characterized polycaprolactone/chitosan membranes with subsequent analysis of cell proliferation and function. Endothelial cells isolated from cardiac explants expressed CD31, VE-cadherin and VEGFR2 and showed typical properties, namely, cytoplasmic Weibel-Palade bodies, metabolism of acetylated low-density lipoproteins, formation of capillary-like structures in Matrigel, and production of extracellular matrix and angiogenic cytokines. Isolated smooth muscle cells expressed extracellular matrix components as well as α-actin and myosin heavy chain. Vascular cells derived from cardiac explants demonstrated the ability to stimulate

  12. Long-Term Cultures of Human Cornea Limbal Explants Form 3D Structures Ex Vivo - Implications for Tissue Engineering and Clinical Applications.

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    Dóra Júlia Szabó

    Full Text Available Long-term cultures of cornea limbal epithelial stem cells (LESCs were developed and characterized for future tissue engineering and clinical applications. The limbal tissue explants were cultivated and expanded for more than 3 months in medium containing serum as the only growth supplement and without use of scaffolds. Viable 3D cell outgrowth from the explants was observed within 4 weeks of cultivation. The outgrowing cells were examined by immunofluorescent staining for putative markers of stemness (ABCG2, CK15, CK19 and Vimentin, proliferation (p63α, Ki-67, limbal basal epithelial cells (CK8/18 and differentiated cornea epithelial cells (CK3 and CK12. Morphological and immunostaining analyses revealed that long-term culturing can form stratified 3D tissue layers with a clear extracellular matrix deposition and organization (collagen I, IV and V. The LESCs showed robust expression of p63α, ABCG2, and their surface marker fingerprint (CD117/c-kit, CXCR4, CD146/MCAM, CD166/ALCAM changed over time compared to short-term LESC cultures. Overall, we provide a model for generating stem cell-rich, long-standing 3D cultures from LESCs which can be used for further research purposes and clinical transplantation.

  13. Plant regeneration from pulse-treated longitudinally sliced half cotyledon node explants of Turkish ochrus chickling [Lathyrus ochrus (L. D.C.

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    Saglam S.

    2012-01-01

    Full Text Available The forage legume ochrus chickling [Lathyrus ochrus (L. D.C.] which is distributed in the Mediterranean region, is gaining importance in terms of economy and agriculture in Turkey. However, the full potential of the legume has yet to be realized due to the presence of neurotoxin, β-N-oxalyl-L-a, β-diaminopropionic acid (ODAP causing lathyrism. This study aimed to develop an efficient micropropagation system using longitudinally sliced cotyledon node explants for use in Agrobacterium-mediated genetic transformation in the future. The results show that the maximum number of shoots per explant was achieved on MS medium solidified with 8 g/l isubgol gelled medium containing 0.30 mg/l BA-0.2 mg/l NAA. Well-developed shoots were rooted by pulse treatment with 50 mg/l IBA and culturing on an 8 g/l isubgol gel solidified MS medium. The results showed 60% rooting in the treated shoots. The rooted plantlets were transferred to pots containing sand and organic matter and acclimatized.

  14. Nivalenol Has a Greater Impact than Deoxynivalenol on Pig Jejunum Mucosa in Vitro on Explants and in Vivo on Intestinal Loops

    Science.gov (United States)

    Cheat, Sophal; Gerez, Juliana R.; Cognié, Juliette; Alassane-Kpembi, Imourana; Bracarense, Ana Paula F. L.; Raymond-Letron, Isabelle; Oswald, Isabelle P.; Kolf-Clauw, Martine

    2015-01-01

    The mycotoxins deoxynivalenol (DON) and nivalenol (NIV), worldwide cereal contaminants, raise concerns for animal and human gut health, following contaminated food or feed ingestion. The impact of DON and NIV on intestinal mucosa was investigated after acute exposure, in vitro and in vivo. The histological changes induced by DON and NIV were analyzed after four-hour exposure on pig jejunum explants and loops, two alternative models. On explants, dose-dependent increases in the histological changes were induced by DON and NIV, with a two-fold increase in lesion severity at 10 µM NIV. On loops, NIV had a greater impact on the mucosa than DON. The overall proliferative cells showed 30% and 13% decrease after NIV and DON exposure, respectively, and NIV increased the proliferative index of crypt enterocytes. NIV also increased apoptosis at the top of villi and reduced by almost half the proliferative/apoptotic cell ratio. Lamina propria cells (mainly immune cells) were more sensitive than enterocytes (epithelial cells) to apoptosis induced by NIV. Our results demonstrate a greater impact of NIV than DON on the intestinal mucosa, both in vitro and in vivo, and highlight the need of a specific hazard characterization for NIV risk assessment. PMID:26035490

  15. Combined effect of gamma irradiation methods and in vitro explant sources on mutation induction of flower color in Chrysanthemum morifoliun Ramat

    Energy Technology Data Exchange (ETDEWEB)

    Nagatomi, Shigeki [Inst. of Radiation Breeding, Omiya, Ibaraki (Japan); Miyahira, Eiken; Degi, Konosuke

    1997-11-01

    Effective radiation breeding method was searched by establishing an effective exposure method to induce a mutation involved in flower color of chrysanthemum and clarifying the effects of its combined use with cultured explants. A chrysanthemum `Taihei`, a variety suitable for cut-flower use was used as the subject, which was irradiated at a dose ranging from 0.25-1.5 Gy/day for 20 days. The floral petals, buds and leaves were used as the explants for callus induction culture. The flower color was evaluated using Japanese Standard Color chart for Horticultural Plants. The color spectrum of the adaxial surface of a petal was recorded by spectro-photometer TC-1800 MK-2. Thus, six mutants of flower color were registered as new varieties. Either of these mutants was derived from chronic irradiation. Three varieties from petal culture, two from bud one and one from cutting culture were obtained, showing that the combined method of chronic irradiation and organ culture is useful in practice for mutation breeding of flower species. Further, this method is applicable for production of non-chimeric mutants, enhancement of the mutation rate and widening the mutation spectra in vegetatively propagated plants. (M.N.)

  16. Agrobacterium-mediated genetic transformation and regeneration of transgenic plants using leaf midribs as explants in ramie [Boehmeria nivea (L.) Gaud].

    Science.gov (United States)

    An, Xia; Wang, Bo; Liu, Lijun; Jiang, Hui; Chen, Jie; Ye, Shengtuo; Chen, Leiyu; Guo, Pingan; Huang, Xing; Peng, Dingxiang

    2014-05-01

    In this study, leaf midribs, the elite explants, were used for the first time to develop an efficient regeneration and transformation protocol for ramie [Boehmeria nivea (L.) Gaud.] via Agrobacterium-mediated genetic transformation. Sensitivity of leaf midribs regeneration to kanamycin was evaluated, which showed that 40 mg l(-1) was the optimal concentration needed to create the necessary selection pressure. Factors affecting the ramie transformation efficiency were evaluated, including leaf age, Agrobacterium concentration, length of infection time for the Agrobacterium solution, acetosyringone concentration in the co-cultivation medium, and the co-cultivation period. The midrib explants from 40-day-old in vitro shoots, an Agrobacterium concentration at OD600 of 0.6, 10-min immersion in the bacteria solution, an acetosyringone concentration of 50 mg l(-1) in the co-cultivation medium and a 3-day co-cultivation period produced the highest efficiencies of regeneration and transformation. In this study, the average transformation rate was 23.25%. Polymerase chain reactions using GUS and NPTII gene-specific primers, Southern blot and histochemical GUS staining analyses further confirmed that the transgene was integrated into the ramie genome and expressed in the transgenic ramie. The establishment of this system of Agrobacterium-mediated genetic transformation and regeneration of transgenic plants will be used not only to introduce genes of interest into the ramie genome for the purpose of trait improvement, but also as a common means of testing gene function by enhancing or inhibiting the expression of target genes.

  17. Comparison of spiculogenesis in in vitro ADCP-primmorph and explants culture of marine sponge Hymeniacidon perleve with 3-TMOSPU supplementation.

    Science.gov (United States)

    Cao, Xupeng; Yu, Xingju; Zhang, Wei

    2007-01-01

    This study aims to test the feasibility of introducing functional chemical groups into biogenic silica spicules by examining the effect of supplementing a silican coupler [3-(trimethoxysilyl)propyl]urea (3-TMOSPU) as silica source in the cultures of archaeocytes-dominant-cell-population (ADCP) primmorphs and explants of the marine sponge Hymeniacidon perleve. Analysis by Fourier Transform Infrared Spectroscopy (FT-IR) confirmed that the organic group in 3-TMOSPU was introduced into silica spicules. By comparing ADCP-primmorph cultures when supplemented with Na2SiO3, 3-TMOSPU supplementation showed no notable effect on the primmorphs development and cell locomotion behaviors. A decline in silicatein expression quantified by real-time RT-PCR was, however, observed during spiculogenesis. The decline was slower for the 3-TMOSPU group whereas significantly fewer spicules were formed. When sponge papillae explants were cultured, 3-TMOSPU supplementation had no negative effect on sponge growth but inhibited the growth biofouling of the diatom Nitzschia closterium. By monitoring the detectable Si concentration, it seemed that 3-TMOSPU was converted by the sponge and its conversion was related to spiculogenesis. Analysis of spicule dimensional changes indicated that the inhibition of spiculogenesis by 3-TMOSPU supplementation was less in ADCP-primmorphs culture due to lower 3-TMOSPU/detectable Si ratio in the media.

  18. Nivalenol Has a Greater Impact than Deoxynivalenol on Pig Jejunum Mucosa in Vitro on Explants and in Vivo on Intestinal Loops

    Directory of Open Access Journals (Sweden)

    Sophal Cheat

    2015-05-01

    Full Text Available The mycotoxins deoxynivalenol (DON and nivalenol (NIV, worldwide cereal contaminants, raise concerns for animal and human gut health, following contaminated food or feed ingestion. The impact of DON and NIV on intestinal mucosa was investigated after acute exposure, in vitro and in vivo. The histological changes induced by DON and NIV were analyzed after four-hour exposure on pig jejunum explants and loops, two alternative models. On explants, dose-dependent increases in the histological changes were induced by DON and NIV, with a two-fold increase in lesion severity at 10 µM NIV. On loops, NIV had a greater impact on the mucosa than DON. The overall proliferative cells showed 30% and 13% decrease after NIV and DON exposure, respectively, and NIV increased the proliferative index of crypt enterocytes. NIV also increased apoptosis at the top of villi and reduced by almost half the proliferative/apoptotic cell ratio. Lamina propria cells (mainly immune cells were more sensitive than enterocytes (epithelial cells to apoptosis induced by NIV. Our results demonstrate a greater impact of NIV than DON on the intestinal mucosa, both in vitro and in vivo, and highlight the need of a specific hazard characterization for NIV risk assessment.

  19. Study of the effect of plant growth regulators, size, and cultivar of the grape inflorescence explant on production of phenolic compounds in an in vitro condition

    Directory of Open Access Journals (Sweden)

    Sedighi Azam

    2014-01-01

    Full Text Available Introduction: Phenolic compounds are a large number of secondary metabolites that have useful and desirable effects in the field of agriculture, medicine, and food. This research was aimed to achieve methods of in vitro propagation of grapevine in order to apply biotechnologies for correction, growth, and optimization of products and compounds of the cultivated plant in relation to phenol ratio. Methods: In this interventional study, the effects of cultivar and size of the inflorescence explant and the gibberellin hormone in two levels, benzylaminopurine, and auxin hormones in three levels with three replicates per treatment were evaluated in relation to phenol ratio, in order to evaluate the effect of plant growth regulators, the type and size of the grape inflorescence explant on the phenol production. Results: The type of plant growth regulators affected phenolic substances production. The production of phenolic substances decreased in a medium with the highest concentration of growth regulators, 4 and 2.5 μM concentration of benzylaminopurine, and 4.9 μM of auxin. Production of phenolic substances increased in the free-plant hormone medium. In smaller samples tendency to turn brown was more regarding high amount of the sugar. Conclusion: The plant sample and the cultivar as important factors in producing phenol environment are induced by environmental stimuli like sugar, light, temperature, stress, ozone, and wound and can be actually applied to increase phenol production.

  20. Effect of Basal Medium, Explants Size and Density on the In Vitro Proliferation and Growth of Date Palm (Phoenix dactylifera L. Cultivar ‘16-bis’

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    Mouaad Amine MAZRI

    2013-08-01

    Full Text Available The effect of basal medium, explant size and density on shoot multiplication, growth, rooting and acclimatization of date palm cv. ‘16-bis’ was evaluated. Bud clusters of different sizes (2, 3, 4 and 5 buds per cluster were cultured at density of 1, 2, 3 and 4 clusters on Murashige and Skoog medium (MS, woody plant medium (WPM and Nitsch medium (NM supplemented with 0.5 mg/L 2-naphthoxyacetic acid and 0.5 mg/L kinetin for three months (multiplication phase. Separated shoots of different sizes (<3 cm; 3 to 4.5 cm and 4.5 to 6 cm were cultured at density of 1, 2, 3 and 4 shoots on hormone free MS medium, WPM or NM for three months (Elongation-rooting phase. The proliferation and development of shoots were affected by the basal medium, explant size and density. The optimal shoot proliferation (18.1 was observed when 4 buds clusters were cultured at the density of 2 clusters per jar in MS medium. Separated shoots of 4.5 to 6 cm length exhibited the optimal in vitro development in terms of leaf length and greening, and root number and length when cultured on MS medium. In addition, these shoots reached the highest acclimation frequency with 80%. Our results would be utilized for an efficient propagation of plantlets of cv. ‘16-bis’, a selected date palm cultivar resistant to the bayoud disease.

  1. Influence of Explant Position on Growth of Talinum paniculatum Gaertn. Adventitious Root in Solid Medium and Enhance Production Biomass in Balloon Type Bubble Bioreactor

    Science.gov (United States)

    Solim, M. H.; Kristanti, A. N.; Manuhara, Y. S. W.

    2017-03-01

    Talinum paniculatum Gaertn. is one of traditional medicinal plant in Indonesia as an aphrodisiac. This plant has various compounds which is accumulated in roots. In vitro culture of this plant can enhance production of adventitious roots. The aim of this research was to know the influence of explants position on growth of T. paniculatum Gaertn. adventitious root in MS solid medium and enhance the production of biomass in balloon type bubble bioreactor. Explants from leaf were cultured at abaxial and adaxial positions in solid MS medium supplemented with IBA 2 mgL-1. Adventitious roots were cultured in bioreactor with various treatments (without IBA, supplemented with IBA 2 mgL-1 and supplemented with IBA 2 mgL-1 + buffer NaHCO3). Result showed that the main growth of abaxial root was higher than adaxial, however, the total of adaxial root branch was higher than abaxial. The highest biomass production of adventitious root cultured was achieved by MS medium supplemented with IBA 2 mgL-1 + buffer NaHCO3. This treatment has produced fresh biomass two fold of initial inoculum.

  2. An efficient in vitro shoot regeneration from leaf petiolar explants and ex vitro rooting of Bixa orellana L.- A dye yielding plant.

    Science.gov (United States)

    Mohammed, Arifullah; Chiruvella, Kishore K; Namsa, Nima D; Ghanta, Rama Gopal

    2015-07-01

    Bixa orellana L. (Bixaceae) is a multipurpose tree grown for the production of commercially important dyes. In the present study, an efficient, reproducible protocol was developed for direct plant regeneration from in vitro derived petiole explants of Bixa orellana L. Murashige and Skoog medium (MS) supplemented with 2-isopentenyl adenine (9.8 μM) and naphthalene acetic acid (10.7 μM) was found to be optimum for production of high frequency of shoot organogenesis. Subculturing of the shoots onto the fresh MS medium containing similar concentrations of 2-iP (9.8 μM) and NAA (10.7 μM) produced elongated shoots. Elongated shoots when placed onto MS medium supplemented with 1.7 μM indole-3-acetic acid and 14.7 μM 2-iP produced optimal rooting. Rooted plantlets were acclimatized and transplanted to the field successfully. Histological investigation revealed the origin of shoot primordia, from sub-epidermal cells of petiole explants. The regeneration protocol developed in this study can be useful for mass in vitro propagation and effective genetic transformation of commercially important edible dye yielding tree species.

  3. Adrenergic Activation of Melatonin Secretion in Ovine Pineal Explants in Short-Term Superfusion Culture Occurs via Protein Synthesis Independent and Dependent Phenomena

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    Bogdan Lewczuk

    2014-01-01

    Full Text Available The ovine pineal is generally considered as an interesting model for the study on adrenergic regulation of melatonin secretion due to some functional similarities with this gland in the human. The present investigations, performed in the superfusion culture of pineal explants, demonstrated that the norepinephrine-induced elevation of melatonin secretion in ovine pinealocytes comprised of two subsequent periods: a rapid increase phase and a slow increase phase. The first one included the quick rise in release of N-acetylserotonin and melatonin, occurring parallel to elevation of NE concentration in the medium surrounding explants. This rapid increase phase was not affected by inhibition of translation. The second, slow increase phase began after NE level had reached the maximum concentration in the culture medium and lasted about two hours. It was completely abolished by the treatment with translation inhibitors. The obtained results showed for the first time that the regulation of N-acetylserotonin synthesis in pinealocytes of some species like the sheep involves the on/off mechanism, which is completely independent of protein synthesis and works very fast. They provided strong evidence pointing to the need of revision of the current opinion that arylalkylamines N-acetyltransferase activity in pinealocytes is controlled exclusively by changes in enzyme abundance.

  4. A simple shoot multiplication procedure using internode explants, and its application for particle bombardment and Agrobacterium-mediated transformation in watercress.

    Science.gov (United States)

    Ogita, Shinjiro; Usui, Miki; Shibutani, Nanae; Kato, Yasuo

    2009-07-01

    A shoot multiplication system derived from internode explants was investigated with the aim of improving genetic characteristics of watercress (Nasturtium officinale R. Br.). Internodes of ca. 1 cm excised from in vitro stock shoot culture were placed on half-strength Murashige and Skoog (MS) medium supplemented with 3 muM 2,4-dichlorophenoxyacetic acid as a pre-treatment. Laser scanning microscopy indicated clearly that the first sign of meristematic cell division could be seen after 1-2 days of pre-culture, and meristematic tissues multiplied along the vascular cambium of the internode segment during 7 days of culture. Multiple shoots could be obtained from more than 90% of the pre-treated explants when they were subsequently transferred to MS medium supplemented with 1 muM thidiazuron for 3 weeks. These findings indicate that pre-treatment of the internodes for 7 days promoted their capacity for organogenesis. Using this pre-treatment, frequent generation of transgenic watercress plants was achieved by adapting particle bombardment and Agrobacterium-mediated transformation techniques with a construct expressing a synthetic green florescent protein gene.

  5. The effect of negative pressure wound therapy with periodic instillation using antimicrobial solutions on Pseudomonas aeruginosa biofilm on porcine skin explants.

    Science.gov (United States)

    Phillips, Priscilla L; Yang, Qingping; Schultz, Gregory S

    2013-12-01

    Negative pressure wound therapy with instillation (NPWTi) is increasingly used as an adjunct therapy for a wide variety of infected wounds. However, the effect of NPWTi on mature biofilm in wounds has not been determined. This study assessed the effects of NPWTi using saline or various antimicrobial solutions on mature Pseudomonas aeruginosa biofilm using an ex vivo porcine skin explant biofilm model. Treatment consisted of six cycles with 10-minute exposure to instillation solution followed by 4 hours of negative pressure at -125 mm Hg over a 24-hour period. NPWTi using saline reduced bacterial levels by 1-log (logarithmic) of 7-log total colony-forming units (CFUs). In contrast, instillation of 1% povidone iodine (2-log), L-solution (3-log), 0·05% chlorhexidine gluconate (3-log), 0·1% polyhexamethylene biguanide (4-log), 0·2% polydiallyldimethylammonium chloride (4-log) and 10% povidone iodine (5-log), all significantly reduced (P porcine skin explants, these ex vivo model data suggest that NPWTi with delivery of active antimicrobial agents enhances the reduction of CFUs by increasing destruction and removal of biofilm bacteria. These results must be confirmed in human studies. © 2013 The Authors. International Wound Journal © 2013 John Wiley & Sons Ltd and Medicalhelplines.com Inc.

  6. Efecto de la densidad de explantes y el volumen de medio de cultivo sobre la multiplicación in vitro de arándano (Vaccinium corymbosum L. variedades Brigitta y Legacy

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    Mario Rodríguez Beraud

    2015-01-01

    Full Text Available El objetivo fue evaluar la multiplicación in vitro de dos variedades de arándano (Vaccinium corymbosum L., “Brigitta” y “Legacy”, en respuesta a cinco densidades de explantes (5, 10, 15, 20 y 25 y cuatro volúmenes de medio (10, 20, 30 y 40 mL utilizando para ambas variedades el medio WPM (Woody Plant Medium en un diseño completamente al azar con 20 tratamientos y 12 repeticiones por tratamiento. Transcurrido 45 días se evaluó: altura de brote, número de brotes/explante, número de nudos/brote y número de brotes/frasco. Brigitta obtuvo la mayor altura del brote en tratamientos con densidades y volúmenes elevados, mientras que Legacy obtuvo el mayor promedio de altura del brote con densidades intermedias y volúmenes elevados. En cuanto al número de brotes/explante, el volumen de medio no influyó en la variedad Brigitta, en cambio, altas densidades la afectaron significativamente, mientras que en Legacy el máximo número de brotes se alcanzó con densidades bajas y volúmenes intermedios. En relación al número de nudos por explante Brigitta obtuvo los valores más bajos comparados con Legacy, pero en ambas variedades se produjo una disminución del número de nudos con menores volúmenes de medio. Para el número de brotes por frasco Brigitta obtuvo las mayores respuestas con densidades elevadas, superando los 40 brotes por frasco. En cambio, en Legacy el resultado máximo se obtuvo con la densidad 25 explantes en 30 mL de medio. Por lo tanto, se concluye que ambas variedades fueron influenciadas tanto por el volumen del medio como la densidad de explantes.

  7. Induction and flow cytometry identification of tetraploids from seed-derived explants through colchicine treatments in Catharanthus roseus (L.) G. Don.

    Science.gov (United States)

    Xing, Shi-Hai; Guo, Xin-Bo; Wang, Quan; Pan, Qi-Fang; Tian, Yue-Sheng; Liu, Pin; Zhao, Jing-Ya; Wang, Guo-Feng; Sun, Xiao-Fen; Tang, Ke-Xuan

    2011-01-01

    The tetraploid plants of Catharanthus roseus (L.) G. Don was obtained by colchicine induction from seeds explants, and the ploidy of the plants was identified by flow cytometry. The optimal treatment is 0.2% colchicine solution treated for 24 hours, and the induction rate reaches up to 30%. Comparing with morphological characteristics and growth habits between tetraploids and the control, we found that tetraploids of C. roseus had larger stoma and more branches and leaves. HPLC analysis showed tetraploidization could increase the contents of terpenoid indole alkaloids in C. roseus. Thus, tetraploidization could be used to produce higher alkaloids lines for commercial use. QRT-PCR results showed that the expression of enzymes involved in terpenoid indole alkaloids biosynthesis pathway had increased in the tetraploid plants. To our knowledge, this was the first paper to explore the secondary metabolism in autotetraploid C. roseus induced by colchicine.

  8. Effect of IAA on growth, organogenesis and RNA metabolism during the development of Cichorium intybus root explants cultured „in vitro"

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    E. Gwóźdź

    2015-01-01

    Full Text Available The effects of 3-indolylacetic acid (IAA on growth, organogenesis, RNA content, RNase activity and MAK elution profiles during the development of chicory root explants cultured in vitro were investigated. It was found that the intensive callus growth in the presence of IAA was accompanied by an increase in the RNA content, with simultaneous decrease of RNase activity. Fractionation of RNA by MAK column chromatography showed that the high content of RNA in the callus under the influence of IAA was due to an increased accumulation of the ribosomal fraction of RNA mainly. Experiments with actinomycin D demonstrated that this antibiotic abolished both the auxin-induced callus growth and the inhibitory effect of IAA on bud formation. No significant inhibition of spontaneous bud formation under the influence of actinomycin D was observed. The possible relationship between the IAA-affected morphogenetic processes and RNA metabolism is discussed.

  9. Organogenesis from in vitro-derived leaf and internode explants of Hoya wightii ssp. palniensis -a vulnerable species of Western Ghats

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    Subbaiah Revathi Lakshmi

    2013-06-01

    Full Text Available An efficient system was developed for indirect plant regeneration from in vitro-derived leaf and internode explants of Hoya wightii ssp. palniensis. Maximum percentage of the organogenic callus was obtained on MS medium supplemented with NAA (1.0 mg/l and 2,4-D (2.0 mg/l. The best shoot bud induction was observed on MS medium with BA (1.0 mg/l +IBA (0.5 mg/l. The coconut water (15% was better, resulting in a differentiation of the shoot initials in to well-developed shoots. The elongated shoots (› 3cm long were rooted on a full strength MS basal medium, supplemented with 0.2 mg/l of IBA. Finally, the rooted plants were transferred to the soil with 80% success rate. This protocol was utilized for the in vitro propagation of this endangered plant species.

  10. Induction and Flow Cytometry Identification of Tetraploids from Seed-Derived Explants through Colchicine Treatments in Catharanthus roseus (L. G. Don

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    Shi-Hai Xing

    2011-01-01

    Full Text Available The tetraploid plants of Catharanthus roseus (L. G. Don was obtained by colchicine induction from seeds explants, and the ploidy of the plants was identified by flow cytometry. The optimal treatment is 0.2% colchicine solution treated for 24 hours, and the induction rate reaches up to 30%. Comparing with morphological characteristics and growth habits between tetraploids and the control, we found that tetraploids of C. roseus had larger stoma and more branches and leaves. HPLC analysis showed tetraploidization could increase the contents of terpenoid indole alkaloids in C. roseus. Thus, tetraploidization could be used to produce higher alkaloids lines for commercial use. QRT-PCR results showed that the expression of enzymes involved in terpenoid indole alkaloids biosynthesis pathway had increased in the tetraploid plants. To our knowledge, this was the first paper to explore the secondary metabolism in autotetraploid C. roseus induced by colchicine.

  11. Induction and Flow Cytometry Identification of Tetraploids from Seed-Derived Explants through Colchicine Treatments in Catharanthus roseus (L.) G. Don

    Science.gov (United States)

    Xing, Shi-Hai; Guo, Xin-Bo; Wang, Quan; Pan, Qi-Fang; Tian, Yue-Sheng; Liu, Pin; Zhao, Jing-Ya; Wang, Guo-Feng; Sun, Xiao-Fen; Tang, Ke-Xuan

    2011-01-01

    The tetraploid plants of Catharanthus roseus (L.) G. Don was obtained by colchicine induction from seeds explants, and the ploidy of the plants was identified by flow cytometry. The optimal treatment is 0.2% colchicine solution treated for 24 hours, and the induction rate reaches up to 30%. Comparing with morphological characteristics and growth habits between tetraploids and the control, we found that tetraploids of C. roseus had larger stoma and more branches and leaves. HPLC analysis showed tetraploidization could increase the contents of terpenoid indole alkaloids in C. roseus. Thus, tetraploidization could be used to produce higher alkaloids lines for commercial use. QRT-PCR results showed that the expression of enzymes involved in terpenoid indole alkaloids biosynthesis pathway had increased in the tetraploid plants. To our knowledge, this was the first paper to explore the secondary metabolism in autotetraploid C. roseus induced by colchicine. PMID:21660143

  12. Effects of sainfoin (Onobrychis viciifolia) extract and monomers of condensed tannins on the association of abomasal nematode larvae with fundic explants.

    Science.gov (United States)

    Brunet, S; Jackson, F; Hoste, H

    2008-06-01

    Tannin-rich forages offer an alternative to anthelmintic chemicals to control gastrointestinal nematodes. However, the mode of action of such bioactive plants still needs to be assessed. Previous studies have shown that extracts of tannin-rich plants interfere with the first phase of host invasion, i.e., the exsheathment of infective larvae (L3s). In the current study, we examined the hypothesis that exposure to tannins could also affect the second phase of larval establishment, i.e., the tissue association/penetration of the exsheathed L3s into the digestive mucosae. An in vitro direct challenge technique using fundic explants was applied in this study. The main parasite model was Haemonchus contortus. The objectives were to verify: (i) whether a modification of the association/penetration of L3s with the mucosae occurred after contact with sainfoin extract; (ii) whether this is a dose-dependent phenomenon; (iii) whether tannins were responsible for these effects; (iv) whether these effects were dependent on the parasite species; and (v) how the biochemical structure of tannins might influence these effects. Following 3h contact with sainfoin extract at 1,200 microg/ml, the penetration of exsheathed L3s of H. contortus and Teladorsagia circumcincta into fundic explants was significantly reduced. Moreover, a dose-response relationship was found for H. contortus. For both nematodes, the changes were totally alleviated after addition of polyvinyl polypyrrolidone, an inhibitor of tannins, to the sainfoin extract, suggesting that tannins play a major role in the observed effects. Comparison of results obtained with different monomers of condensed tannins confirms a relationship between structure and activity, the prodelphinidin monomers and galloyl-derivatives being more effective than the procyanidin monomers. Combined with the delay or the inhibition of larval exsheathment previously shown, these effects could explain how tanniniferous plants reduce the establishment

  13. Effect of AgNO3 and BAP on Root as a Novel Explant in Date Palm (Phoenix dactylifera cv. Medjool) Somatic Embryogenesis.

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    Roshanfekrrad, Marjan; Zarghami, Reza; Hassani, Hassan; Zakizadeh, Hedayat; Salari, Ali

    2017-01-01

    Somatic embryogenesis techniques are used for cloning a wide range of varieties of date palms around the world. The aim of the present study was to develop an efficient method with the lowest cost and the greatest potential to obtain in vitro plantlets of date palm cv. Medjool. Also, produce embryogenic callus and somatic embryos without using 2,4-dichlorophenoxyacetic acid (2,4-D). In this study, produced plantlets through somatic embryogenesis were used in vitro roots as explant cultured on Murashige and Skoog (MS) media containing three level of Silver Nitrate (AgNO3) (0, 3 and 6 mg L-1) plus two level of 6-benzylaminopurine (BAP) (0 and 2 mg L-1) plus 0.1 mg L-1 1-naphthylacetic acid (NAA) for callus induction. After 12 weeks of culture, callus induction and after 16 weeks, production of embryogenic callus and embryos were occurred from root explants. According to the results, medium containing 2 mg L-1 BAP and 3 mg L-1 silver nitrate+0.1 mg L-1 NAA showed the highest amount of embryogenic callus fresh weight (1.38 g). This treatment also cause the highest number and length of embryos by production of 90.04 embryogenic callus with length of 11.18 mm. On the other hand, shoots were appeared from germinated embryos and white roots began to appear within 8 weeks. Medium contains 3 mg L-1 BAP and 0.1 mg L-1 NAA with average of 12.27 cm shoot length and 15.48 cm root length was the best. Control treatment had the lowest average shoot (3.71 cm) and root (5.03 cm) length. This study showed that certain concentration of silver nitrate and BAP has stimulating effect on growth of produced embryonic callus from root segments of Medjool cultivar of date palm.

  14. Agrobacterium-mediated transformation of Eucalyptus globulus using explants with shoot apex with introduction of bacterial choline oxidase gene to enhance salt tolerance.

    Science.gov (United States)

    Matsunaga, Etsuko; Nanto, Kazuya; Oishi, Masatoshi; Ebinuma, Hiroyasu; Morishita, Yoshihiko; Sakurai, Nozomu; Suzuki, Hideyuki; Shibata, Daisuke; Shimada, Teruhisa

    2012-01-01

    Eucalyptus globulus is one of the most economically important plantation hardwoods for paper making. However, its low transformation frequency has prevented genetic engineering of this species with useful genes. We found the hypocotyl section with a shoot apex has the highest regeneration ability among another hypocotyl sections, and have developed an efficient Agrobacterium-mediated transformation method using these materials. We then introduced a salt tolerance gene, namely a bacterial choline oxidase gene (codA) with a GUS reporter gene, into E. globulus. The highest frequency of transgenic shoot regeneration from hypocotyls with shoot apex was 7.4% and the average frequency in four experiments was 4.0%, 12-fold higher than that from hypocotyls without shoot apex. Using about 10,000 explants, over 250 regenerated buds were confirmed as transformants by GUS analysis. Southern blot analysis of 100 elongated shoots confirmed successful generation of stable transformants. Accumulation of glycinebetaine was investigated in 44 selected transgenic lines, which showed 1- to 12-fold higher glycinebetaine levels than non-transgenic controls. Rooting of 16 transgenic lines was successful using a photoautotrophic method under enrichment with 1,000 ppm CO(2). The transgenic whole plantlets were transplanted into potting soil and grown normally in a growth room. They showed salt tolerance to 300 mM NaCl. The points of our system are using explants with shoot apex as materials, inhibiting the elongation of the apex on the selection medium, and regenerating transgenic buds from the side opposite to the apex. This approach may also solve transformation problems in other important plants.

  15. Effect of Gliadin on Permeability of Intestinal Biopsy Explants from Celiac Disease Patients and Patients with Non-Celiac Gluten Sensitivity

    Science.gov (United States)

    Hollon, Justin; Leonard Puppa, Elaine; Greenwald, Bruce; Goldberg, Eric; Guerrerio, Anthony; Fasano, Alessio

    2015-01-01

    Background: Intestinal exposure to gliadin leads to zonulin upregulation and consequent disassembly of intercellular tight junctions and increased intestinal permeability. We aimed to study response to gliadin exposure, in terms of barrier function and cytokine secretion, using intestinal biopsies obtained from four groups: celiac patients with active disease (ACD), celiac patients in remission (RCD), non-celiac patients with gluten sensitivity (GS) and non-celiac controls (NC). Methods: Ex-vivo human duodenal biopsies were mounted in microsnapwells and luminally incubated with either gliadin or media alone. Changes in transepithelial electrical resistance were monitored over 120 min. Media was subsequently collected and cytokines quantified. Results: Intestinal explants from all groups (ACD (n = 6), RCD (n = 6), GS (n = 6), and NC (n = 5)) demonstrated a greater increase in permeability when exposed to gliadin vs. media alone. The increase in permeability in the ACD group was greater than in the RCD and NC groups. There was a greater increase in permeability in the GS group compared to the RCD group. There was no difference in permeability between the ACD and GS groups, between the RCD and NC groups, or between the NC and GS groups. IL-10 was significantly greater in the media of the NC group compared to the RCD and GS groups. Conclusions: Increased intestinal permeability after gliadin exposure occurs in all individuals. Following gliadin exposure, both patients with gluten sensitivity and those with active celiac disease demonstrate a greater increase in intestinal permeability than celiacs in disease remission. A higher concentration of IL-10 was measured in the media exposed to control explants compared to celiac disease in remission or gluten sensitivity. PMID:25734566

  16. Effect of Gliadin on Permeability of Intestinal Biopsy Explants from Celiac Disease Patients and Patients with Non-Celiac Gluten Sensitivity

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    Justin Hollon

    2015-02-01

    Full Text Available Background: Intestinal exposure to gliadin leads to zonulin upregulation and consequent disassembly of intercellular tight junctions and increased intestinal permeability. We aimed to study response to gliadin exposure, in terms of barrier function and cytokine secretion, using intestinal biopsies obtained from four groups: celiac patients with active disease (ACD, celiac patients in remission (RCD, non-celiac patients with gluten sensitivity (GS and non-celiac controls (NC. Methods: Ex-vivo human duodenal biopsies were mounted in microsnapwells and luminally incubated with either gliadin or media alone. Changes in transepithelial electrical resistance were monitored over 120 min. Media was subsequently collected and cytokines quantified. Results: Intestinal explants from all groups (ACD (n = 6, RCD (n = 6, GS (n = 6, and NC (n = 5 demonstrated a greater increase in permeability when exposed to gliadin vs. media alone. The increase in permeability in the ACD group was greater than in the RCD and NC groups. There was a greater increase in permeability in the GS group compared to the RCD group. There was no difference in permeability between the ACD and GS groups, between the RCD and NC groups, or between the NC and GS groups. IL-10 was significantly greater in the media of the NC group compared to the RCD and GS groups. Conclusions: Increased intestinal permeability after gliadin exposure occurs in all individuals. Following gliadin exposure, both patients with gluten sensitivity and those with active celiac disease demonstrate a greater increase in intestinal permeability than celiacs in disease remission. A higher concentration of IL-10 was measured in the media exposed to control explants compared to celiac disease in remission or gluten sensitivity.

  17. Decrease in neuronal nicotinic acetylcholine receptor subunit and PSD-93 transcript levels in the male mouse MPG after cavernous nerve injury or explant culture

    Science.gov (United States)

    Girard, Beatrice M.; Merriam, Laura A.; Tompkins, John D.; Vizzard, Margaret A.

    2013-01-01

    Quantitative real-time PCR was used to test whether cavernous nerve injury leads to a decrease in major pelvic ganglia (MPG) neuronal nicotinic ACh receptor (nAChR) subunit and postsynaptic density (PSD)-93 transcript levels. Subunits α3, β4, and α7, commonly expressed in the MPG, were selected for analysis. After 72 h in explant culture, MPG transcript levels for α3, β4, α7, and PSD-93 were significantly depressed. Three days after cavernous nerve axotomy or crush in vivo, transcript levels for α3, β4, and PSD-93, but not for α7, were significantly depressed. Three days after dissection of the cavernous nerve free of underlying tissue and application of a 5-mm lateral stretch (manipulation), transcript levels for α3 and PSD-93 were also significantly decreased. Seven days after all three surgical procedures, α3 transcript levels remained depressed, but PSD-93 transcript levels were still decreased only after axotomy or nerve crush. At 30 days postsurgery, transcript levels for the nAChR subunits and PSD-93 had recovered. ACh-induced currents were significantly smaller in MPG neurons dissociated from 3-day explant cultured ganglia than from those recorded in neurons dissociated from acutely isolated ganglia; this observation provides direct evidence showing that a decrease in nAChR function was coincident with a decrease in nAChR subunit transcript levels. We conclude that a downregulation of nAChR subunit and PSD-93 expression after cavernous nerve injury, or even manipulation, could interrupt synaptic transmission within the MPG and thus contribute to the loss of neural control of urogenital organs after pelvic surgeries. PMID:24049141

  18. Evaluación de medios de cultivo para propagación in vitro de semillas y explantes de especies silvestres de Solanum

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    Danita Andrade Díaz

    2013-01-01

    Full Text Available Se evaluaron los medios de cultivo Hussey-Stacey (A, Hendrix et al. (H, Atkinson et al. (AT y mitad de Murashyge y Skoog (½MS para la propagación in vitro de semillas y explantes de Solanum mammosum, S. marginatum, S. hirtum y S. umbellatum. En la fase de propagación sexual se evaluó el porcentaje de germinación, número de raíces, longitud de planta, días a formación de hojas y raíces, días a morfogénesis completa y materia seca. Se determinó el tipo de morfogénesis a través de callos, vástago y plantas totalmente formadas. Se estudiaron el número y longitud de brotes, número de raíces y hojas, producción de materia seca y días a morfogénesis completa. Para S. mammosum el mejor medio de germinación fue ½MS, para el desarrollo de plantas fue A y para propagación vegetativa, A-Nudos (Medio A con explantes tipo nudos. En el mismo orden, para S. marginatum fueron los medios A, H y ½MS y/o H con nudos, mientras que en S. hirtum fueron H,A y H-nudos. Para S. umbellatum no se encontraron diferencias en germinación entre H, ½MS y A. Para la formación de plantas el mejor medio fue ½ MS y para propagación vegetativa fue H-Nudo.

  19. Defining the inflammatory signature of human lung explant tissue in the presence and absence of glucocorticoid [version 1; referees: 2 approved

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    Tracy L Rimington

    2017-04-01

    Full Text Available Background: Airway inflammation is a feature of many respiratory diseases and there is a need for newer, more effective anti-inflammatory compounds. The aim of this study was to develop an ex vivo human lung explant model which can be used to help study the mechanisms underlying inflammatory responses and which can provide a tool to aid drug discovery for inflammatory respiratory diseases such as asthma and COPD. Method: Parenchymal lung tissue from 6 individual donors was dissected and cultured with two pro-inflammatory stimuli, lipopolysaccharide (LPS (1 µg/ml and interleukin-1 beta (IL-1β (10 ng/ml in the presence or absence of dexamethasone (1 µM.  Inflammatory responses were assessed using Luminex analysis of tissue culture supernatants to measure levels of 21 chemokines, growth factors and cytokines. Results: A robust and reproducible inflammatory signal was detected across all donors for 12 of the analytes measured following LPS stimulation with a modest fold increase (4-fold in CCL3, CCL4, GM-CSF, IL-10, TNF-α and IL-1β.  The inflammatory signal induced by IL-1β stimulation was less than that observed with LPS but resulted in elevated levels of 7 analytes (CXCL8, CCL3, CCL4, GM-CSF, IL-6, IL-10 and TNF-α.  The inflammatory responses induced by both stimulations was supressed by dexamethasone for the majority of analytes. Conclusions: These data provide proof of concept that this ex vivo human lung explant model is responsive to inflammatory signals and could be used to investigate the anti-inflammatory effects of existing and novel compounds.  In addition this model could be used to help define the mechanisms and pathways involved in development of inflammatory airway disease. Abbreviations: COPD: Chronic Obstructive Pulmonary Disease; ICS: inhaled corticosteroids; LPS: lipopolysaccharide; IL-1β: interleukin-1 beta; PSF: penicillin, streptomycin and fungizone

  20. Callus induction of leaf explant Piper betle L. Var Nigra with combination of plant growth regulators indole-3-acetic acid (IAA), benzyl amino purin (BAP) and kinetin

    Science.gov (United States)

    Junairiah, Zuraidassanaaz, Nabilah Istighfari; Izdihar, Fairuz Nabil; Manuhara, Yosephine Sri Wulan

    2017-09-01

    The purpose of this research was to determine the combination of plant growth regulators IAA, BAP and kinetin towards callus induction and growth of leaf explants Piper betle L. VarNigra. Explants from leaf of Piper betle L. VarNigra was cultured on MS medium with 24 treatment combinations of plant growth regulators IAA and BAP and 24 treatment combinations of plant growth regulators IAA and kinetin with 0.0;0.5;1.0;1.5;2.0 mg/L concentration respectively, the observed variable were the length of time the formation of callus, callus morphology, fresh and dry weight of callus. The results of this research showed that the combination of growth regulators IAA with BAP and kinetin had effects on leaf growth of Piper betle L. VarNigra. During 8 weeks observation, it indicated that the combination of concentration IAA 0.5 mg/L and BAP 2.0 mg/L showed fastest callus formation at 8.5 days. Combination of concentration IAA 1.0 mg/L and BAP 1.5 mg/L showed the highest of fresh weight at 0.6596 grams, and the highest dry weight was obtained from the combination of concentration IAA 0.5 mg/L and BAP 0.5 mg/L at 0.0727 grams. Combination of concentration IAA 1.0 mg/L and kinetin 1.5 mg/L had the highest of fresh weight at 0.2972 grams and the highest dry weight at 0.1660 grams. Callus of Piper betle L. VarNigra had two textures, that were compact and friable, and also showed various kind of colors, like white, greenish white, yellowish white, tanned white, brown and black. Based on this research, that concentration IAA 1.0 mg/L and 1.5 mg/L kinetin was the best combination for induction of callus from leaf of Piper betle L. Var Nigra.

  1. REGENERACION Y SENSIBILIDAD A MANOSA EN ENTRENUDOS DE PAPA (Solanum tuberosum spp. andígena Var Diacol Capiro. REGENERATION AND MANNOSE SENSIBILITY IN POTATO INTERNODAL EXPLANTS (Solanum tuberosum spp. andígena Var Diacol Capiro

    Directory of Open Access Journals (Sweden)

    MARY LUZ YAYA-LANCHEROS

    Full Text Available Se estableció un sistema de organogénesis indirecta para la obtención de brotes múltiples a partir de segmentos internodales de la variedad Diacol Capiro. La ubicación de explantes en medio Murashige y Skoog (MS suplementado con 2 mg/l de zeatina ribosido (ZR, 0,02 mg/l de ácido naftalenácetico (ANA y 0,02 mg/l de ácido giberélico (AG3, permite la obtención de plántulas entre la séptima y novena semana con una efectividad del 80-100%. Mediante ubicación de explantes previamente cocultivados con la cepa LBA4404 de Agrobacterium tumefaciens que contiene el plásmido recombinante pNOV022, se verificó la utilidad del medio para procesos de transformación, obteniéndose tasas hasta del 100% de regeneración. Finalmente, con el objetivo de determinar el uso potencial de la manosa como agente selectivo en procesos de transformación, se evaluó el efecto de diferentes concentraciones de manosa sobre la viabilidad y capacidad regenerativa de explantes.A system of indirect organogenesis for the multiple buds production from internode stem sections in Diacol Capiro variety was established. Explants on Murashige & Skoog (MS medium with zeatine riboside (ZR 2 mg/l, naftalenacetic acid (NAA 0.02 mg/l and giberelic acid (GA3 0.02 mg/l, produced plants ranging between 7 to 9 weeks with 80-100% effectiveness. In the same medium, explants infected with Agrobacterium LBA4404 strain which carries recombinant plasmid pNOV022, produced regeneration rates reached 100%, thus, the medium utility for trnsformation processes was verified. Finally, to determine the potential use of the mannose as selective agent in transformation processes, the effect of different mannose concentrations on explant viability and regenerative capacity was evaluated.

  2. A dark incubation period is important for Agrobacterium-mediated transformation of mature internode explants of sweet orange, grapefruit, citron, and a citrange rootstock.

    Science.gov (United States)

    Marutani-Hert, Mizuri; Bowman, Kim D; McCollum, Greg T; Mirkov, T Erik; Evens, Terence J; Niedz, Randall P

    2012-01-01

    Citrus has an extended juvenile phase and trees can take 2-20 years to transition to the adult reproductive phase and produce fruit. For citrus variety development this substantially prolongs the time before adult traits, such as fruit yield and quality, can be evaluated. Methods to transform tissue from mature citrus trees would shorten the evaluation period via the direct production of adult phase transgenic citrus trees. Factors important for promoting shoot regeneration from internode explants from adult phase citrus trees were identified and included a dark incubation period and the use of the cytokinin zeatin riboside. Transgenic trees were produced from four citrus types including sweet orange, citron, grapefruit, and a trifoliate hybrid using the identified factors and factor settings. The critical importance of a dark incubation period for shoot regeneration was established. These results confirm previous reports on the feasibility of transforming mature tissue from sweet orange and are the first to document the transformation of mature tissue from grapefruit, citron, and a trifoliate hybrid.

  3. A dark incubation period is important for Agrobacterium-mediated transformation of mature internode explants of sweet orange, grapefruit, citron, and a citrange rootstock.

    Directory of Open Access Journals (Sweden)

    Mizuri Marutani-Hert

    Full Text Available BACKGROUND: Citrus has an extended juvenile phase and trees can take 2-20 years to transition to the adult reproductive phase and produce fruit. For citrus variety development this substantially prolongs the time before adult traits, such as fruit yield and quality, can be evaluated. Methods to transform tissue from mature citrus trees would shorten the evaluation period via the direct production of adult phase transgenic citrus trees. METHODOLOGY/PRINCIPAL FINDINGS: Factors important for promoting shoot regeneration from internode explants from adult phase citrus trees were identified and included a dark incubation period and the use of the cytokinin zeatin riboside. Transgenic trees were produced from four citrus types including sweet orange, citron, grapefruit, and a trifoliate hybrid using the identified factors and factor settings. SIGNIFICANCE: The critical importance of a dark incubation period for shoot regeneration was established. These results confirm previous reports on the feasibility of transforming mature tissue from sweet orange and are the first to document the transformation of mature tissue from grapefruit, citron, and a trifoliate hybrid.

  4. High frequency direct shoot organogenesis of leaf explants and a comparative evaluation of phytochemicals, antioxidant potential of wild vs. in vitro plant extracts of Lysimachia laxa.

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    Gupta, Sanjoy; Seal, Tapan; Mao, A A; Sarma, Soneswar

    2017-08-01

    The present studies were attempted to develop direct shoot organogenesis from in vitro grown leaf explants of Lysimachia laxa and comparative evaluation of phytochemical and antioxidant potential of in vitro raised and wild plants extracts. The fresh leaves of this species are used for deworming gastrointestinal worm infection in traditional medicine. Overexploitation of this species and poor regeneration has led to rapid decline in wild population, therefore, present investigation was attempted to develop an efficient rapid mass propagation protocol for this species. Our result showed significantly (P rooting induction (100%) with average root number of 11.70 and length 7.35 cm. All rooted plants were successfully acclimatized in greenhouse and transferred to field condition with a survival rate of 97%. The contents of phenolic and flavonoid were higher in in vitro raised plant in compared to wild plant extracts. Antioxidants assay showed high radical scavenging activity of IC50 1.61 ± 0.07 mg dry material and reducing power of 49.79 ± 0.11 mg/g ascorbic acid equivalent by aqueous methanol extracts of in vitro raised 3-months-old plants in compare to the wild plants. The present protocol is a viable option for pharmaceutical or nutraceutical industries for sustainable utilization of L. laxa with enhanced of phytochemical and antioxidant potency which is not reported elsewhere.

  5. Villous explant culture using early gestation tissue from ongoing pregnancies with known normal outcomes: the effect of oxygen on trophoblast outgrowth and migration.

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    Seeho, S K M; Park, J H; Rowe, J; Morris, J M; Gallery, E D M

    2008-05-01

    Early placental and embryo development occur in a physiologically low oxygen environment, with a rise in oxygen tension within the placenta towards the end of the first trimester. Oxygen is implicated in the regulation of trophoblast differentiation and invasion. This study examined the effects of oxygen tension on extravillous trophoblast outgrowth and migration from normal pregnancies free of significant pathology. Early gestation villous tissue (11-14 weeks gestation), obtained by chorionic villus sampling, was cultured in 3 or 20% oxygen. Maternal and fetal outcomes were ascertained for all samples. The frequency and amount of trophoblast outgrowth and migration from villi were measured for up to 192 h. Significantly fewer explants produced outgrowths in 3% compared with 20% oxygen. The number of sites of trophoblast outgrowth and the extent of migration were also significantly less in 3% compared with 20% oxygen. In vitro hypoxia/reoxygenation further reduced trophoblast growth compared with 3% oxygen alone. HLA-G expression in extravillous trophoblasts was not affected by oxygen tension, with HLA-G positive extravillous trophoblasts being universally Ki67 negative. Human placental villi and extravillous trophoblasts in the late first trimester of pregnancy are sensitive to oxygen tension, with low oxygen inhibiting extravillous trophoblast outgrowth and migration.

  6. In vitro propagation and genetic fidelity study of plant regenerated from inverted hypocotyl explants of eggplant (Solanum melongena L.) cv. Arka Shirish.

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    Padma Mallaya, N; Ravishankar, G A

    2013-02-01

    Genetic variation due to somaclonal variation in micropropagated plants is a beneficial phenomenon for crop improvement. Genetic integrity of the plants derived through micropropagation becomes crucial if genetic transformation studies have to be carried out. Somaclonal variation in tissue culture is a common phenomenon which makes it mandatory to check for genetic stability of plants. Hypocotyl explants of Solanummelongena L. cv. Arka Shirish inoculated with inverted polarity in MS media supplemented with 0.5 mg L(-1) thidiazuron (TDZ) gave maximum number of shoot buds. Elongation of the shoot buds was achieved on MS medium supplemented with 0.5 mg L(-1) 2, 3, 5-triiodobenzoic acid (TIBA) and 0.1 mg L(-1) gibberellic acid (GA3). The elongated shoots were rooted in MS with 1 mg L(-1) indole-3-butyric acid (IBA), and the rooted plants were hardened in the greenhouse. Morphological characteristics were similar in both seed-propagated and micropropagated plants. Random amplified polymorphic DNA analysis carried out with 10 primers for genetic stability studies of the regenerated plants generated 96 scorable bands with a total of 1,056 bands for the primers. Comparison of the bands with the mother plant revealed the monomorphic nature and true-to-type clones. The above regeneration protocol will be useful for micropropagation and genetic transformation studies of S.melongena L. cv. Arka Shirish.

  7. Asymmetric motion distribution between components of a mobile-core lumbar disc prosthesis: an explanation of unequal wear distribution in explanted CHARITÉ polyethylene cores.

    Science.gov (United States)

    Patwardhan, Avinash G; Havey, Robert M; Wharton, Nicholas D; Tsitsopoulos, Parmenion P; Newman, Patrick; Carandang, Gerard; Voronov, Leonard I

    2012-05-02

    The biconvex mobile core of the CHARITÉ lumbar disc prosthesis forms two joints (spherical bearings) with the metal end plates. We quantified the intra-prosthesis motion to test the hypothesis that the total prosthesis motion would not be equally distributed between the two bearings of implanted CHARITÉ discs, which might explain the unequal wear distribution reported in explanted cores. The hypothesis was tested by studying the flexion-extension motion responses of (1) twenty-six monosegmental CHARITÉ III discs implanted in nineteen human cadaveric lumbar spines, and (2) twenty-one CHARITÉ III discs (fifteen monosegmental, six bisegmental) implanted in eighteen patients in other published clinical studies. Intra-prosthesis motions were quantified with use of a radiographic image analysis technique. Eighty-eight percent of the CHARITÉ discs implanted in cadaveric specimens exhibited larger motion at the superior bearing, with 54% demonstrating more than twice as much motion at the superior bearing as at the inferior bearing. The ratio of motion at the superior bearing to motion at the inferior bearing averaged 2.68 ± 1.84, which was significantly larger than 1.0 (p cases and 2.55 ± 2.66 for all cases; both ratios were significantly larger than 1.0 (p < 0.05). We found preferentially larger motion at the superior bearing of the CHARITÉ discs implanted in human cadaveric lumbar spines and in patients, regardless of the implanted level.

  8. A mouse ocular explant model that enables the study of living optic nerve head events after acute and chronic intraocular pressure elevation: Focusing on retinal ganglion cell axons and mitochondria.

    Science.gov (United States)

    Kimball, Elizabeth C; Pease, Mary E; Steinhart, Matthew R; Oglesby, Ericka N; Pitha, Ian; Nguyen, Cathy; Quigley, Harry A

    2017-07-01

    We developed an explant model of the mouse eye and optic nerve that facilitates the study of retinal ganglion cell axons and mitochondria in the living optic nerve head (ONH) in an ex vivo environment. Two transgenic mouse strains were used, one expressing yellow fluorescent protein in selected axons and a second strain expressing cyan fluorescent protein in all mitochondria. We viewed an explanted mouse eye and optic nerve by laser scanning microscopy at and behind the ONH, the site of glaucoma injury. Explants from previously untreated mice were studied with the intraocular pressure (IOP) set artificially at normal or elevated levels for several hours. Explants were also studied from eyes that had undergone chronic IOP elevation from 14 h to 6 weeks prior to ex vivo study. Image analysis in static images and video of individual mitochondria or axonal structure determined effects of acute and chronic IOP elevation. At normal IOP, fluorescent axonal structure was stable for up to 3 h under ex vivo conditions. After chronic IOP elevation, axonal integrity index values indicated fragmentation of axon structure in the ONH. In mice with fluorescent mitochondria, the normal density decreased with distance behind the ONH by 45% (p = 0.002, t-test). Density increased with prior chronic IOP elevation to 21,300 ± 4176 mitochondria/mm 2 compared to control 16,110 ± 3159 mitochondria/mm 2 (p = 0.025, t-test), but did not increase significantly after 4 h, acute IOP elevation (1.5% decrease in density, p = 0.83, t-test). Mean normal mitochondrial length of 2.3 ± 1.4 μm became 13% smaller after 4 h of IOP elevation ex vivo compared to baseline (p = 0.015, t-test, N-10). Normal mitochondrial speed of movement was significantly slower in the anterograde direction (towards the brain) than retrograde, but there were more mitochondria in motion and traveling longer lengths in anterograde direction. The percent of mitochondria in motion decreased by >50

  9. MiniCD4 microbicide prevents HIV infection of human mucosal explants and vaginal transmission of SHIV(162P3 in cynomolgus macaques.

    Directory of Open Access Journals (Sweden)

    Nathalie Dereuddre-Bosquet

    Full Text Available In complement to an effective vaccine, development of potent anti-HIV microbicides remains an important priority. We have previously shown that the miniCD4 M48U1, a functional mimetic of sCD4 presented on a 27 amino-acid stable scaffold, inhibits a broad range of HIV-1 isolates at sub-nanomolar concentrations in cellular models. Here, we report that M48U1 inhibits efficiently HIV-1(Ba-L in human mucosal explants of cervical and colorectal tissues. In vivo efficacy of M48U1 was evaluated in nonhuman primate (NHP model of mucosal challenge with SHIV(162P3 after assessing pharmacokinetics and pharmacodynamics of a miniCD4 gel formulation in sexually matured female cynomolgus macaques. Among 12 females, half were treated with hydroxyethylcellulose-based gel (control, the other half received the same gel containing 3 mg/g of M48U1, one hour before vaginal route challenge with 10 AID(50 of SHIV(162P3. All control animals were infected with a peak plasma viral load of 10(5-10(6 viral RNA (vRNA copies per mL. In animals treated with miniCD4, 5 out of 6 were fully protected from acquisition of infection, as assessed by qRT-PCR for vRNA detection in plasma, qPCR for viral DNA detection in PBMC and lymph node cells. The only infected animal in this group had a delayed peak of viremia of one week. These results demonstrate that M48U1 miniCD4 acts in vivo as a potent entry inhibitor, which may be considered in microbicide developments.

  10. Early transcriptional responses of bovine chorioallantoic membrane explants to wild type, ΔvirB2 or ΔbtpB Brucella abortus infection.

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    Juliana P S Mol

    Full Text Available The pathogenesis of the Brucella-induced inflammatory response in the bovine placenta is not completely understood. In this study we evaluated the role of the B. abortus Type IV secretion system and the anti-inflammatory factor BtpB in early interactions with bovine placental tissues. Transcription profiles of chorioallantoic membrane (CAM explants inoculated with wild type (strain 2308, ΔvirB2 or ΔbtpB Brucella abortus were compared by microarray analysis at 4 hours post infection. Transcripts with significant variation (>2 fold change; P<0.05 were functionally classified, and transcripts related to defense and inflammation were assessed by quantitative real time RT-PCR. Infection with wild type B. abortus resulted in slightly more genes with decreased than increased transcription levels. Conversely, infection of trophoblastic cells with the ΔvirB2 or the ΔbtpB mutant strains, that lack a functional T4SS or that has impaired inhibition of TLR signaling, respectively, induced more upregulated than downregulated genes. Wild type Brucella abortus impaired transcription of host genes related to immune response when compared to ΔvirB and ΔbtpB mutants. Our findings suggest that proinflammatory genes are negatively modulated in bovine trophoblastic cells at early stages of infection. The virB operon and btpB are directly or indirectly related to modulation of these host genes. These results shed light on the early interactions between B. abortus and placental tissue that ultimately culminate in inflammatory pathology and abortion.

  11. Preliminary experience with porcine intestinal submucosa (CorMatrix) for valve reconstruction in congenital heart disease: histologic evaluation of explanted valves.

    Science.gov (United States)

    Zaidi, Abbas Haider; Nathan, Meena; Emani, Sitaram; Baird, Christopher; del Nido, Pedro J; Gauvreau, Kimberlee; Harris, Marian; Sanders, Stephen P; Padera, Robert F

    2014-11-01

    We compared the histologic findings in explanted CorMatrix (9 patients) and autologous pericardium (9 patients) used for valvuloplasty of the aortic (7 patients) and/or mitral (11 patients) valve in patients with congenital heart defects. We used standard tissue stains and immunohistochemistry to identify the inflammatory cell type. CorMatrix was associated with an intense inflammatory response in the surrounding native tissue, extending into CorMatrix in 8 of 9 cases, continuing to the longest follow-up point (9 months). The typical response included macrophages and giant cells in contact with the material, surrounded by lymphocytes, macrophages, plasma cells, and eosinophils. The thickness of the residual CorMatrix material was 280 to 300 μm, similar to the nominal thickness at implantation and unrelated to the implantation duration. Only at the longest follow-up interval was any significant resorption of CorMatrix material evident. A neointima had formed on the surface of CorMatrix, increasing in thickness with the period in situ. Mild cellular infiltration of CorMatrix was noted in all cases; however, in no case, did it appear that CorMatrix was being remodeled into tissue resembling a 3-layered native valve. In contrast, a near absence of any inflammatory reaction was seen and no eosinophilia associated with autologous pericardium was present, irrespective of the duration in situ. Furthermore, we observed more tissue infiltration, remodeling, vascularization, and neointima formation with autologous pericardium. Although CorMatrix used for valve repair induced an intense inflammatory response, little or no remodeling to form tissue resembling a 3-layered native valve was seen at ≤9 months after implantation. Copyright © 2014 The American Association for Thoracic Surgery. Published by Elsevier Inc. All rights reserved.

  12. Oxidação fenólica, tipo de explante e meios de cultura no estabelecimento in vitro de canafístula (Peltophorum dubium (Spreng. Taub..

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    Josiana Scherer Bassan

    2010-08-01

    Full Text Available A canafístula, Peltophorum dubium (Spreng. Taub. é uma espécie florestal nativa e com ampla dispersão geográfica, desempenhando um papel pioneiro nas áreas abertas, em capoeiras e matas degradadas. Apresenta um rápido crescimento e se adapta facilmente, sendo muito recomendada para reflorestamento homogêneo. A madeira é utilizada em construções civis, indústria de móveis, construção naval e militar. A micropropagação é uma técnica utilizada com bastante sucesso, apresentando, entre outras vantagens, um rápido aumento no número de plantas geneticamente idênticas partindo de plantas selecionadas. Os objetivos do trabalho são avaliar a influência da luz no controle da oxidação fenólica dos explantes de canafístula (Peltophorum dubium (Spreng., determinar o meio nutritivo e o tipo de explante mais adequado para o estabelecimento in vitro de canafístula (Peltophorum dubium (Spreng.. Foram realizados dois experimentos. No primeiro, ápices caulinares foram cultivados em meio base MS a 25 ± 3°C, por 7 dias no escuro e, posteriormente, sob fotoperíodo de 16 horas de luz e intensidade luminosa de 20 μmol.m2.s-1, fornecida por lâmpada fluorescente branca durante 21 dias ou permaneceram na câmara de crescimento com exposição à luz durante todo o experimento. Foi empregado o delineamento inteiramente casualizado com cinco repetições contendo quatro unidades experimentais (UE. A oxidação fenólica foi observada após 21 dias de cultivo. Não ocorreu oxidação fenólica em nenhum dos tratamentos analisados. No segundo experimento, ápices caulinares e segmentos nodais foram cultivados em meio base MS e meio WPM. Os explantes foram mantidos em sala de crescimento com fotoperíodo de 16 horas de luz e intensidade luminosa de 20 μmol.m2.s-1, fornecidas por lâmpada fluorescente branca fria e temperatura de 25 ± 3°C. A UE foi composta por um frasco de vidro de 150 mL contendo 30 mL de meio nutritivo e um explante. O

  13. Analysis of mass spectrometry data from the secretome of an explant model of articular cartilage exposed to pro-inflammatory and anti-inflammatory stimuli using machine learning.

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    Swan, Anna L; Hillier, Kirsty L; Smith, Julia R; Allaway, David; Liddell, Susan; Bacardit, Jaume; Mobasheri, Ali

    2013-12-13

    Osteoarthritis (OA) is an inflammatory disease of synovial joints involving the loss and degeneration of articular cartilage. The gold standard for evaluating cartilage loss in OA is the measurement of joint space width on standard radiographs. However, in most cases the diagnosis is made well after the onset of the disease, when the symptoms are well established. Identification of early biomarkers of OA can facilitate earlier diagnosis, improve disease monitoring and predict responses to therapeutic interventions. This study describes the bioinformatic analysis of data generated from high throughput proteomics for identification of potential biomarkers of OA. The mass spectrometry data was generated using a canine explant model of articular cartilage treated with the pro-inflammatory cytokine interleukin 1 β (IL-1β). The bioinformatics analysis involved the application of machine learning and network analysis to the proteomic mass spectrometry data. A rule based machine learning technique, BioHEL, was used to create a model that classified the samples into their relevant treatment groups by identifying those proteins that separated samples into their respective groups. The proteins identified were considered to be potential biomarkers. Protein networks were also generated; from these networks, proteins pivotal to the classification were identified. BioHEL correctly classified eighteen out of twenty-three samples, giving a classification accuracy of 78.3% for the dataset. The dataset included the four classes of control, IL-1β, carprofen, and IL-1β and carprofen together. This exceeded the other machine learners that were used for a comparison, on the same dataset, with the exception of another rule-based method, JRip, which performed equally well. The proteins that were most frequently used in rules generated by BioHEL were found to include a number of relevant proteins including matrix metalloproteinase 3, interleukin 8 and matrix gla protein. Using this

  14. The liver in heart failure: a biopsy and explant series of the histopathologic and laboratory findings with a particular focus on pre-cardiac transplant evaluation.

    Science.gov (United States)

    Louie, Christine Y; Pham, Michael X; Daugherty, Tami J; Kambham, Neeraja; Higgins, John P T

    2015-07-01

    The pathologic liver changes in chronic heart failure have been characterized mostly based on autopsy series and include sinusoidal dilation and congestion progressing to pericellular fibrosis, bridging fibrosis, and ultimately to cardiac cirrhosis or sclerosis. Liver biopsies are commonly obtained as part of the work up before heart transplantation in patients with longstanding right heart failure, particularly if ascites, abnormal liver function tests or abnormal abdominal imaging are noted as part of the pre-transplant evaluation. In these cases, the liver biopsy findings may be used to further risk stratify patients for isolated heart or combined heart and liver transplantation. Thus, it is important to be able to correlate the histologic changes with post-transplant outcomes. We report the pathologic and clinical findings in liver explants from six patients who underwent combined heart-liver transplantation. We also report preoperative liver biopsy findings from 21 patients who underwent heart transplantation without simultaneous liver transplantation. We staged the changes related to chronic passive congestion as follows: stage 0-no fibrosis; stage I-pericellular fibrosis; stage II-bridging fibrosis; and stage III-regenerative nodules. Nineteen biopsies showed fibrosis with bridging fibrosis in 13 and regenerative nodules in 6. Fifteen patients were alive at 1 year post transplant. Only three patients had a post-operative course that was characterized by signs and symptoms of chronic liver disease. Pre-transplant liver biopsies from these patients all showed at least stage II fibrosis. These patients survived for 3, 6, and 10 months after cardiac transplant. The presence of bridging fibrosis was not significantly associated with post-operative survival (P=0.336) or post-operative liver failure (P=0.257). We conclude that patients with bridging fibrosis may still be considered viable candidates for isolated heart transplantation. Because the pattern of

  15. Melatonin promotes adventitious root regeneration in in vitro shoot tip explants of the commercial sweet cherry rootstocks CAB-6P (Prunus cerasus L.), Gisela 6 (P. cerasus × P. canescens), and MxM 60 (P. avium × P. mahaleb).

    Science.gov (United States)

    Sarropoulou, Virginia N; Therios, Ioannis N; Dimassi-Theriou, Kortessa N

    2012-01-01

    The objectives of this study were to test the effects of melatonin (N-acetyl-5-methoxytryptamine), a natural compound of edible plants on the rooting of certain commercial sweet cherry rootstocks. Shoot tip explants from previous in vitro cultures of the cherry rootstocks CAB-6P (Prunus cerasus L.), Gisela 6 (P. cerasus × P. canescens), and M × M 60 (P. avium × P. mahaleb) were included in the experiment. The effect of indole-3-acetic acid (IAA) and indole-3-butyric acid (IBA) alone or in combination with melatonin was tested concerning their rooting potential. Seven concentrations of melatonin (0, 0.05, 0.1, 0.5, 1, 5, and 10 μM) alone or in combination with 5.71 μM of IAA or 4.92 μM of IBA were tested. For each rootstock, 21 treatments were included. The explants were grown in glass tubes containing 10 mL of substrate. The parameters measured include rooting percentage, number of roots per rooted explant, root length, and callus formation. The data presented in this study show that melatonin has a rooting promoting effect at a low concentration but a growth inhibitory effect at high concentrations. In the absence of auxin, 1 μM melatonin had auxinic response concerning the number and length of roots, but 10 μM melatonin was inhibitory to rooting in all the tested rootstocks. The final conclusion of this experiment is that exogenously applied melatonin acted as a rooting promoter and its action was similar to that of IAA. © 2011 John Wiley & Sons A/S.

  16. Protocolo gerenciado de tratamento do potencial doador falecido reduz incidência de parada cardíaca antes do explante dos órgãos

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    Glauco Adrieno Westphal

    2012-12-01

    Full Text Available OBJETIVO: Avaliar o efeito da aplicação de um protocolo gerenciado de manutenção de potenciais doadores falecidos de múltiplos órgãos em duas unidades hospitalares. MÉTODOS: Estudo antes (Fase 1/depois (Fase 2 realizado em dois hospitais gerais que incluiu, consecutivamente, os potenciais doadores ingressados em duas unidades de terapia intensiva. Na Fase 1 (16 meses, os dados foram coletados retrospectivamente e as medidas de manutenção do potencial doador foram instituídas a critério do intensivista. Na Fase 2 (12 meses, a coleta de dados foi prospectiva e a manutenção foi guiada por um protocolo gerenciado. As duas fases foram comparadas entre si de acordo com variáveis demográficas, variáveis fisiológicas no diagnóstico da morte encefálica e ao final do processo, tempo necessário para realização do exame confirmatório de morte encefálica e final do processo, aderência aos conjuntos de medidas essenciais de manutenção (pacotes, perdas por parada cardíaca, perdas por negativa familiar, perdas por contraindicação e taxa de conversão de potenciais doadores em doadores reais. Foram aplicados os testes de t-Student e do qui-quadrado, e o valor de p<0,05 foi considerado significativo. RESULTADOS: Identificaram-se 42 potenciais doadores (18 na Fase 1 e 24 na Fase 2. Houve diminuição do tempo entre a primeira exploração clínica e o explante (Fase 1: 35,0±15,5 horas versus Fase 2: 24,6±6,2 horas; p=0,023. Houve aumento na aderência em 10 dos 19 itens essenciais de manutenção, e redução nas perdas por parada cardíaca (Fase 1: 27,8 versus 0% na Fase 2; p=0,006 com aumento de doadores reais (Fase 1: 44,4 versus 75% na Fase 2; p=0,044. Não houve mudança nas perdas por negativa familiar ou por contraindicação médica. CONCLUSÃO: A adoção de um protocolo gerenciado promove a aplicação de medidas essenciais no cuidado do potencial doador falecido e pode reduzir as perdas de potenciais doadores por parada

  17. [Activity of the corn Spm transposon system in transgenic plants Orychophragmus violaceus (L.) O.E. Schulz obtained by both direct transfer of DNA to protoplasts and agrobacterial transformation of root explants].

    Science.gov (United States)

    Sakhno, L A; Sytnik, E S; Cherep, N N; Komarnitskiĭ, I K; Kuchuk, N V; Klimiuk, V I

    2002-01-01

    Transposon mediated insertional mutagenesis is one of the approaches for the unique gene cloning. A wild species of Cruciferae family Orychophragmus violaceus (L.) O.E. Schulz, which is of interest for practical breeding as a donor of improved plant oil, was an object of the investigation. Plasmid construction used in the experiments included selective NPT II gene, reported GUS gene serving as an excision marker, structural BAR gene located within the dSpm element and Spm transposase. The GUS gene of this plasmid had not his own promoter and became functional only after Spm-transposition. Transformed Orychophragmus violaceus (L.) O.E. Schulz. plants were obtained by direct mesophyll protoplast transformation as well as Agrobacterium tumefaciens-mediated root explant transformation. Gene transfer and the transposition event were confirmed by the GUS activity and the PCR analysis. Relative transformation efficiency using protoplasts was 5.8%.

  18. Evaluación del efecto de las artemisininas provenientes del extracto etanólico de Artemisia cina sobre L3 de Haemonchus contortus en una técnica de explantes abomasales

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    Rosa Isabel Higuera-Piedrahita

    2016-01-01

    Full Text Available El nuevo enfoque de control integrado de parásitos obliga a buscar alternativas que consideren el medioambiente, es decir, sostenibles y sustentables. El objetivo de este trabajo fue fraccionar el extracto etanólico de la planta Artemisia cina, obtener artemisininas y conocer el efecto de estas sobre la capacidad de asociación de las L3 de Haemonchus contortus en explantes abomasales. El extracto etanólico se fraccionó por medio de la metodología establecida para Artemisia japónica, y se identificaron las artemisininas por medio de cromatografía en capa fina, teniendo como referencia artemisininas comerciales. Las artemisininas se utilizaron sobre L3 desenvainada, se realizó la técnica de explantes abomasales por triplicado y se compararon así: levamisol (7.5 mg/ml, artemisinina comercial (1 mg/ml, agua y seis diferentes fracciones de A. cina a dosis de 1 mg/ml: Ac3k, Ac3b, Ac3a, Ac3h, Ac3i, Ac4b. No se obtuvieron diferencias significativas entre las fracciones de A. cina y artemisinina comercial (p>0.05. Se concluye que no existió efecto de las artemisininas obtenidas del extracto etanólico de A. cina sobre la capacidad de asociación de las L3 de H. contortus al tejido abomasal. Es importante continuar con más estudios de la artemisinina para determinar sobre qué fase del parásito afecta su viabilidad.

  19. DESENVOLVIMENTO DE CALOS EM EXPLANTES DE CUPUAÇUZEIRO (Theobroma grandiflorum Schum. EM FUNÇÃO DA CONCENTRAÇÃO DE AUXINAS E DO MEIO LÍQUIDO

    Directory of Open Access Journals (Sweden)

    FERREIRA MARIA DAS GRAÇAS RODRIGUES

    2001-01-01

    Full Text Available Objetivou-se estudar o efeito da concentração de auxina e do meio líquido sobre o desenvolvimento de calos de cupuaçuzeiro. Segmentos de eixos embrionários e cotilédones, obtidos de frutos de cupuaçu dos tipos Mamorana e Redondo, foram cultivados em 4 meios de cultura diferentes: 1 meio MS (50%, suplementado com 2,4-D (1; 2; 4; 8 mg/L; 2 sais N6 (SIGMA (4 g/L, acrescidos de 2,4-D (0; 2; 4 mg/L e ANA (0; 3; 5 mg/L; 3 igual ao anterior, suplementado apenas com ANA (3 mg/L; e 4 meio MS, acrescido com ANA (1 mM. Calos com aspecto branco e brilhante foram observados em segmentos de eixos embrionários e cotilédones, cultivados nas menores concentrações de meio 1 (1 e 2 mg/L, enquanto nas maiores concentrações (4 e 8 mg/L se observou a formação de calos e massa calosa branco-opaca, em eixos embrionários e em segmentos cotiledonares, estas estruturas tornaram-se escuras dentro de oito semanas. Usando o meio 2, um grande número de raízes foram formadas, enquanto o mesmo meio suplementado apenas com ANA (3; 5 mg/L originou uma massa calosa. A combinação de ANA e 2,4-D, 3 e 2 mg/L, respectivamente, promoveu a formação de calos brancos e raízes. A transferência das culturas para meio líquido, sem regulador de crescimento, promoveu aumento de tamanho dos explantes e escurecimento dos mesmos. O cultivo desses explantes no meio 3 resultou no aparecimento de calos amarelos, com aspecto friável, que permaneceram com a mesma aparência no meio 4.

  20. Concurrent Liver Hodgkin Lymphoma and Nodular Regenerative Hyperplasia on an Explanted Liver with Clinical Diagnosis of Alcoholic Cirrhosis at University Hospital Fundación Santa Fe de Bogotá

    Directory of Open Access Journals (Sweden)

    R. López

    2014-01-01

    Full Text Available Liver involvement by Hodgkin lymphoma (HL is well documented. However, secondary liver failure to this neoplastic process is rare and usually presents late in the course of the disease. We present a case of a HL associated with nodular regenerative hyperplasia (NRH diagnosed on an explanted liver from a 53-year-old patient with clinical diagnosis of alcoholic cirrhosis. Hematoxylin and eosin stain (H&E showed abnormal liver architecture with hepatocytes nodules highlighted by reticulin stain with absent fibrosis on the trichrome stain. The portal spaces had diffuse infiltration by Reed-Sternberg cells positive for CD15, CD30, and latent membrane protein (LMP on immunohistochemical studies. The patient also had a concurrent hilar lymph node biopsy that also showed HL involvement. Liver failure as the initial presentation of Hodgkin’ lymphoma is rare. We believe that more research about the utility of performing liver biopsies in patients candidates for transplantation with noncirrhotic hepatic failure is needed in order to establish the etiology and the optimal treatment.

  1. No adaptation to digitalization as evaluated by digitalis receptor (Na,K-ATPase) quantification in explanted hearts from donors without heart disease and from digitalized recipients with end-stage heart failure.

    Science.gov (United States)

    Schmidt, T A; Allen, P D; Colucci, W S; Marsh, J D; Kjeldsen, K

    1993-01-01

    Speculations about development of tolerance to the inotropic effect of digitalis have been engendered since studies in various in vitro systems and tissues not representative of the heart have shown up-regulation of sodium potassium adenosine triphosphatase (Na,K-ATPase) when exposed to digitalis. Moreover the digitalis receptor (i.e., Na,K-ATPase) concentration in the normal, vital human left ventricle has not been previously determined. On this basis, digitalis receptor concentration was quantified in the left ventricle of explanted hearts from subjects without heart disease and from patients with end-stage heart failure who had received digitalis therapy. This was performed using vanadate-facilitated 3H-ouabain binding to intact tissue samples giving values of 728 +/- 58 (n = 5) and 467 +/- 55 pmol/g wet weight (n = 6) (mean +/- SEM) (p digitalization was associated with occupancy of digitalis receptors in the failing human heart of 24% (p < 0.02).(ABSTRACT TRUNCATED AT 250 WORDS)

  2. An efficient Agrobacterium-mediated transformation and regeneration system for leaf explants of two elite aspen hybrid clones Populus alba × P. berolinensis and Populus davidiana × P. bolleana.

    Science.gov (United States)

    Wang, Haihai; Wang, Cuiting; Liu, Hua; Tang, Renjie; Zhang, Hongxia

    2011-11-01

    Transgenic technology has been successfully used for gene function analyses and trait improvement in cereal plants. However, its usage is limited in woody plants, especially in the difficult-to-transform but commercially viable hybrid poplar. In this work, an efficient regeneration and transformation system was established for the production of two hybrid aspen clones: Populus alba × P. berolinensis and Populus davidiana × P. bolleana. A plant transformation vector designed to express the reporter gene uidA, encoding β-glucuronidase (GUS), driven by the cauliflower mosaic virus 35S promoter, was used to detect transformation event at early stages of plant regeneration, and to optimize the parameters that may affect poplar transformation efficiency. Bacterium strain and age of leaf explant are two major factors that affect transformation efficiency. Addition of thidiazuron (TDZ) improved both regeneration and transformation efficiency. The transformation efficiency is approximately 9.3% for P. alba × P. berolinensis and 16.4% for P. davidiana × P. bolleana. Using this system, transgenic plants were usually produced in less than 1 month after co-cultivation. The growth characteristics and morphology of transgenic plants were identical to the untransformed wild type plants, and the transgenes could be inherited by vegetative propagation, as confirmed by PCR, Southern blotting, RT-PCR and β-glucuronidase staining analyses. The establishment of this system will help to facilitate the studies of gene functions in tree growth and development at a genome level, and as well as the introduction of some valuable traits in aspen breeding.

  3. Efficiencies in alginate encapsulation of vegetative explants

    Science.gov (United States)

    The goal of this study was to improve a non-mechanized bulk encapsulation technique to standardize encapsulation procedures and reduce the labor time compared to encapsulating individual nodes. Four mm-long nodal segments from Stage II cultures of Hibiscus moscheutos L. ‘Lord Baltimore’ were encapsu...

  4. Enhanced regeneration in explants of tomato (Lycopersicon ...

    African Journals Online (AJOL)

    USER

    2010-06-14

    Jun 14, 2010 ... Significant differences for regeneration, time taken to regenerate and number of leaf primordial were observed for different ... caused by fungi, bacteria, viruses and various nematodes. Development of protocols for in vitro ... high quality tomato seedlings via tissue culture could help to reduce the price per ...

  5. Improvement of banana cv. Rasthali (Silk, AAB) against Fusarium oxysporum f.sp. cubense (VCG 0124/5) through induced mutagenesis: Determination of LD50 specific to mutagen, explants, toxins and in vitro and in vivo screening for Fusarium wilt resistance.

    Science.gov (United States)

    Saraswathi, M S; Kannan, G; Uma, S; Thangavelu, R; Backiyarani, S

    2016-05-01

    Shoot tips and in vitro grown proliferating buds of banana cv. Rasthali (Silk, AAB) were treated with various concentrations and durations of chemical mutagens viz., EMS, NaN3 and DES. LD50 for shoot tips based on 50% reduction in fresh weight was determined as 2% for 3 h, 0.02% for 5 h and 0.15% for 5 h, while for proliferating buds, they were 0.6% for 30 min, 0.01% for 2 h and 0.06% for 2 h for the mutagens EMS, NaN3 and DES, respectively. Subsequently, the mutated explants were screened in vitro against fusarium wilt using selection agents like fusaric acid and culture filtrate. LD50 for in vitro selection agents calculated based on 50% survival of explants was 0.050 mM and 7% for fusaric acid and culture filtrate, respectively and beyond which a rapid decline in growth was observed. This was followed by pot screening which led to the identification of three putative resistant mutants with an internal disease score of 1 (corm completely clean, no vascular discolouration). The putative mutants identified in the present study have also been mass multiplied in vitro.

  6. Efficient plant regeneration from cotyledonary node explants of ...

    African Journals Online (AJOL)

    An efficient regeneration system was developed using cotyledonary node of two inbred line of melon, Cucumis melo 'CM-15' and 'CM-23'. The induction of shoots was achieved on Murashige and Skoog (MS) solid medium supplemented with different combinations of 6-benzylaminopurine (BA) and indole- 3-acetic acid ...

  7. An efficient plant regeneration protocol from petiole explants of ...

    African Journals Online (AJOL)

    saini

    2012-08-07

    Aug 7, 2012 ... medium supplemented with 1.0 mgL-1 indole-3-acetic acid (IAA) and 0.2 mgL-1 IBA. The rooted plantlets were established in soil ... number of options for the production of liquid fuel as an alternative source have been ..... Symposium on Jatropha, Managua, Nicaragua, Mexico, p. 15. Deore AC, Johnson TS ...

  8. Hypoxia Inhibits Hypertrophic Differentiation and Endochondral Ossification in Explanted Tibiae

    NARCIS (Netherlands)

    Leijten, Jeroen Christianus Hermanus; Moreira Teixeira, Liliana; Landman, Ellie; van Blitterswijk, Clemens; Karperien, Hermanus Bernardus Johannes

    2012-01-01

    Purpose: Hypertrophic differentiation of growth plate chondrocytes induces angiogenesis which alleviates hypoxia normally present in cartilage. In the current study, we aim to determine whether alleviation of hypoxia is merely a downstream effect of hypertrophic differentiation as previously

  9. 143 GROWTH RESPONSE OF EXPLANTS OF Irvingia gabonensis ...

    African Journals Online (AJOL)

    et al., 2006). Though micropropagation techniques have been developed for some woody species and have been successfully produced over the last few years, scanty reports exists on the micropropagation of. Irvingia gabonensis. Thus, the use of in vitro techniques in practical forestry with this plant is limited at present.

  10. In vitro performances of hypocotyl and cotyledon explants of tomato ...

    African Journals Online (AJOL)

    Jane

    2011-08-15

    Aug 15, 2011 ... lmos E, Hernendaze JA, Sevilla F, Hellin E (1994). Induction of several antioxidant enzymes in the selection of salt tolerant cell line of Pisum sativum. J. Plant Physiol. 144: 594-598. Potluri Sasikala DP, Devi Prasad PV (1994). Salinity on in vitro performance of some cultivars of potato. R. Bras. Fisiol. Veg.

  11. Efficient plant regeneration from leaf explants of Solanum americanum

    African Journals Online (AJOL)

    ONOS

    2010-09-06

    Sep 6, 2010 ... (Solanum dulcamara L.), a medicinal plant. Acta Soc. Bot. Pol. 77(4):. 275-280. Patiño-Torres C, Hoyos-Sánchez R, Afanador-Kafuri L (2007). Selección y regeneración in vitro de somaclones de tomate de árbol. (Solanum betacea cav. Sendt) utilizando filtrados de cultivo de. Colletotrichum acutatum con ...

  12. Direct and indirect plant regeneration from various explants of ...

    African Journals Online (AJOL)

    Populus species are important resource for certain branches of industry and have special roles for scientific study on biological and agricultural systems. Plant regeneration via direct and indirect organogenesis of four Populus deltoides Bartram ex Marsh. ssp. deltoides × Populus deltoides Bartram ex Marsh. ssp. deltoides ...

  13. In vitro REGENERATION OF PIGEON PEA USING LEAF EXPLANTS ...

    African Journals Online (AJOL)

    ACSS

    2016-05-19

    May 19, 2016 ... ranging from globular to torpedo-shaped (Fig. 1D). However, transfer of the somatic embryos to. ½ MS and MS with 0.5 mg l-1 BAP for 56 days only, increased their sizes but no germination was attained. Root induction. KAT 60/8 and ICEAP 00557 shoots displayed slow and scanty root development, when ...

  14. Vitrification of caudal fin explants from zebrafish adult specimens.

    Science.gov (United States)

    Cardona-Costa, J; Roig, J; Perez-Camps, M; García-Ximénez, F

    2006-01-01

    No data on vitrification of tissue samples are available in fishes. Three vitrification solutions were compared: V1: 20% ethylene glycol and 20% dimethyl sulphoxide; V2: 25% propylene glycol and 20% dimethyl sulphoxide, and; V3: 20% propylene glycol and 13% methanol, all three prepared in Hanks' buffered salt solution plus 20 percent FBS, following the same one step vitrification procedure developed in mammals. Caudal fin tissue pieces were vitrified into 0.25 ml plastic straws in 30s and stored in liquid nitrogen for 3 days minimum, warmed (10s in nitrogen vapour and 5s in a 25 degree C water bath) and cultured (L-15 plus 20% FBS at 28.5 degree C). At the third day of culture, both attachment and outgrowing rates were recorded. V3 led to the worst results (8% of attachment rate). V1 and V2 allow higher attachment rates (V1: 63% vs V2: 50%. P < 0.05) but not significantly different outgrowing rates (83% to 94%). Vitrification of caudal fin pieces is advantageous in fish biodiversity conservation, particularly in the wild, due to the simplicity of procedure and equipment.

  15. Adventitious shoot regeneration from leaf explants of miniature ...

    African Journals Online (AJOL)

    Seeds of miniature paprika (Capsicum annuum) 'Hivita Red' and 'Hivita Yellow' were decontaminated and placed in a petri dish containing a half-strength MS medium and then were incubated in the dark for 7 10 ... Morphology and fruit shape of regenerated plants were normal and plants set seeds as the same as to the ...

  16. Effect of season, explants, growth regulators and sugar level on ...

    African Journals Online (AJOL)

    Administrator

    2011-06-06

    Jun 6, 2011 ... petiolated, long tipped, leathery, heart-shaped leaves. It is very popular as a shade tree because of its good form and easy adaptability to various soil conditions and so very commonly planted as an avenue or roadside tree. The fruits and tender leaf buds are occasionally eaten in times of scarcity. The fruits ...

  17. In vitro propagation of Alstroemeria using rhizome explants derived ...

    African Journals Online (AJOL)

    Alstroemeria with beautiful and long shelf life of cut flower is one of the most important ornamental plants. This plant could propagate by splitting of the in vivo produced rhizomes but the propagation rate is rather low. In the present study, regeneration ability of plantlets was compared using in vitro and in vivo grown rhizome ...

  18. Plant regeneration via somatic embryogenesis from root explants of ...

    African Journals Online (AJOL)

    hope&shola

    2010-08-30

    benzylaminopurine (6-BA) and kinetin (KT) on callus induction of root ... The pH of the medium was adjusted to 5.8 and all media were .... embryos; g, successful soil transfer of regenerated plants. Scale bars represent 0.5 cm (b, e), 1 cm ...

  19. In vitro regeneration from internodal explants of bitter melon ...

    African Journals Online (AJOL)

    Thiru

    2012-04-24

    Apr 24, 2012 ... Organogenic callus induction and high frequency shoot regeneration were achieved from internodal ... (Momordica anti-Hiv protein), which suppresses HIV ..... Plant Cell Rep. 8: 116-119. Bourinbaiar AS, Lee-Huang S (1996). The activity of plant derived antiretroviral proteins MAP30 and GAP31 against ...

  20. Effect of explant plant source and acetosyringone concentration on ...

    African Journals Online (AJOL)

    Gene introduction into crop plants through genetic manipulation is a better alternative to conventional breeding for the improvement of stress tolerance. Agrobacterium-mediated transformation offers precise integration of genes into the genome with enhanced transgene stability. There are a number of factors which ...

  1. Simple, effective and economical explant-surface sterilization ...

    African Journals Online (AJOL)

    Reckitt and Benckiser (Nig) Ltd) containing 3.5% sodium hypochlorite was used instead of the pure sodium hypochlorite solution. Our results showed that Method 2 produced the highest reduction in bacterial and fungal contamination (0%) at ...

  2. What can we learn from explanted endovascular devices?

    Science.gov (United States)

    Riepe, G; Heintz, C; Kaiser, E; Chakfé, N; Morlock, M; Delling, M; Imig, H

    2002-08-01

    To examine the durability of the Stentor and Vanguard endovascular devices in human implants. The textile covering, the polypropylene ligatures and the stent metal of 34 devices (25 Stentor, 9 Vanguard) with a mean duration of implantation of 28.8 +/- 16 months was examined by means of stereomicroscopy and scanning electron microscopy. The polyester textile covering showed gaps along the sutured seam and isolated holes in the fabric. All of the examined polypropylene ligatures were worn, some ruptured. Four different types of stent corrosion were classified--pits (100%), bizarre craters (68%), large deficiencies (14%) and fractures (32%). Holes in the polyester fabric and frame dislocations are specific for the design of Stentor and Vanguard grafts. The early corrosion of the stent metal Nitinol in these devices is surprising. Until more experience is gained with other devices, we have to be reminded, that the "gold standard" for the long-term durability of artificial vascular grafts is still "today's" conventional graft.

  3. Dedifferentiation of leaf explants and antileukemia activity of an ...

    African Journals Online (AJOL)

    The crude extracts of the callus were evaluated in vitro for their activity against leukemia cells and hepatocarcinoma. Among the different concentrations, 2,4-D at 0.1 mg/l induced highest frequencies of callus growth index (7.8) when compared with other concentrations. Ethanolic extracts killed about 36% of abnormal cells ...

  4. High frequency plant regeneration from shoot tip explants of ...

    African Journals Online (AJOL)

    USER

    2010-08-02

    Aug 2, 2010 ... Sobhakumari VP, Lalithakumari D (2003). Direct plant regeneration from shoot tip cultures of Capsicum annuum L. cv. PLR-1. Phytomorphology, 53(3&4): 235-242. Suchitra B, Mehar Z, Susil K (1999). In vitro multiplication of Centella asiatica. Curr. Sci. 76: 147-158. Sunandakumari C, MaSurtin KP, Chithra ...

  5. Callus regeneration from stem explants of Pseudarthira viscida (L ...

    African Journals Online (AJOL)

    STORAGESEVER

    2009-09-01

    Sep 1, 2009 ... An in vitro propagation protocol has been developed for Pseudarthira viscida (L.) Wight and Arn. (Papilionaideae), a vulnerable medicinal plant from stem callus was used for axillary shoot multiplication. The plant was standardized by using MS (Murashige and Skoog) medium containing 3%. (w/v) sucrose ...

  6. Regeneration of plantlets from leaf and internode explants of ...

    African Journals Online (AJOL)

    STORAGESEVER

    2009-05-18

    May 18, 2009 ... potential restricts their multiplication. Micropropagation technique offers an alternative method for cloning these plants (Unander, 1991; Santos et al., 1994). This research describes the micro-propagation of P. amarus from leaf discs and internodes and successful establish- ment of plantlets in soil.

  7. Effect of plant growth regulators, explants type and efficient plantlet ...

    African Journals Online (AJOL)

    In vitro rooting of shoots was achieved on ½ strength MS medium supplemented with IBA. Best rooting was achieved on ½ strength MS medium supplemented with 1.0 mg/l IBA (indole-3-butyric acid). The highest total soluble protein contents and peroxidase activity was observed in the four weeks old callus cultures derived ...

  8. Adventitious shoot regeneration from cultured leaf explants of Petunia

    African Journals Online (AJOL)

    DIRECTOR

    2012-06-26

    Jun 26, 2012 ... trend of average number of shoots produced was observed. Key words: Cytokinins, petunia, regeneration, thidiazuron. INTRODUCTION. Petunia hybrida is an important floriculture ornamental crop of high commercial interest. It is considered to be the first bedding plant; in addition Petunia is the most.

  9. In vitro response from cotyledon and hypocotyls explants in tomato ...

    African Journals Online (AJOL)

    USER

    2010-07-26

    Jul 26, 2010 ... of variance (ANOVA) and LSD was used p = 0.05 for comparison between treatment means. RESULT AND DISCUSSION. The cytokinin BAP promotes cell division, shoot multipli- cation and auxiliary bud formation (Sutter, 1996). Com- bination of phytohormones (cytokines and auxins) has been reported to ...

  10. In vitro shoot multiplication and rooting from seedling explants of ...

    African Journals Online (AJOL)

    BAP at 5 mg/l gave higher shoot multiplication compared with 0 – 2 mg/l BAP. Although axillary buds opened on cuttings treated with 0.1 to 2 mg/l TDZ, no elongation occurred. Shoots placed on 0.5 – 5 mg/l 2-iP produced roots but no axillary shoots. Indole-3-butyric acid (IBA) and indole-3-acetic acid (IAA) were tested for ...

  11. 143 GROWTH RESPONSE OF EXPLANTS OF Irvingia gabonensis ...

    African Journals Online (AJOL)

    difference (LSD) at 0.05 level of probability. RESULTS. One quarter strength MS medium with 3mg/l of kinetin was optimum for shoot initiation of the species. There were significant (P<0.05) differences in shoot length among treatments with full (MS+Kin), half (1/2 MS+Kin) and one quarter (1/4 MS +Kin) strength media.

  12. The Influence of Explant Types and Orientation on in Vitro Culture

    Directory of Open Access Journals (Sweden)

    Kamnoon KANCHANAPOOM

    2011-08-01

    Full Text Available Inflorescence, apical and lateral buds of Musa balbisiana ‘Kluai Hin’ (BBB group were used to culture on MS medium supplemented with 22 μM BA and 15% (v/v coconut water. Comparison of bud orientation showed that the best response of in vitro shoot tip proliferation was obtained with abaxial surface of buds lying down i.e. one side touching the medium (tilt. Mass propagation of shoot tips was obtained when cultured buds on MS medium containing 44 μM BA. Rooting was achieved by transferring the isolated shoots to MS basal medium without growth regulators. Rooted plantlets were acclimatized and successfully established in soil.

  13. Tympanic membrane organ culture using cell culture well inserts engrafted with tympanic membrane tissue explants.

    Science.gov (United States)

    Liew, Lawrence J; Day, Richard M; Dilley, Rodney J

    2017-03-01

    Tissue engineering approaches using growth factors and various materials for repairing chronic perforations of the tympanic membrane are being developed, but there are surprisingly few relevant tissue culture models available to test new treatments. Here, we present a simple three-dimensional model system based on micro-dissecting the rat tympanic membrane umbo and grafting it into the membrane of a cell culture well insert. Cell outgrowth from the graft produced sufficient cells to populate a membrane of similar surface area to the human tympanic membrane within 2 weeks. Tissue grafts from the annulus region also showed cell outgrowth but were not as productive. The umbo organoid supported substantial cell proliferation and migration under the influence of keratinocyte growth medium. Cells from umbo grafts were enzymatically harvested from the polyethylene terephthalate (PET) membrane for expansion in routine culture and cells could be harvested consecutively from the same graft over multiple cycles. We used harvested cells to test cell migration properties and to engraft a porous silk scaffold material as proof-of-principle for tissue engineering applications. This model is simple enough to be widely adopted for tympanic membrane regeneration studies and has promise as a tissue-equivalent model alternative to animal testing.

  14. Prolific shoot regeneration from immature embryo explants of sainfoin (Onobrychis viciifolia Scop.).

    Science.gov (United States)

    Ozcan, S; Sevimay, C S; Yildiz, M; Sancak, C; Ozgen, M

    1996-12-01

    Immature cotyledons and embryo axes of sainfoin were cultured on Murashige and Skoog (MS) media supplemented with various concentrations of 6-benzylaminopurine (BAP) and a-naphthaleneacetic acid (NAA) to induce adventitious shoot regeneration. The highest frequency of shoot regeneration occurred following an initial callus growth on a MS medium containing 0.5 mg/l BAP and 2 mg/l NAA. Immature embryo axes showed higher regeneration capacity than immature cotyledons, however, shoot elongation was best achieved on immature cotyledons. Regenerated shoots were excised and rooted in half strength MS medium with 1 mg/l indole-butyric acid (IBA) or 1 mg/l NAA. The rooted plantlets were finally transferred to compost.

  15. Factors influencing plant regeneration from seedling explants of Hairy nightshade (Solanum sarrachoides)

    Science.gov (United States)

    A good model plant to investigate plant – pathogen interactions would be easy to grow, have a short life cycle, be a natural host of many pathogens, and be easy to manipulate genetically. Hairy nightshade (Solanum sarrachoides) is a ubiquitous, fast growing weed that produces copious amounts of see...

  16. Effects of plant growth regulators on in vitro cultured nodal explants ...

    African Journals Online (AJOL)

    SAM

    2014-07-09

    Jul 9, 2014 ... Smith et al. (1986) and Konan et al. (1997) were the first to report success in the production of an average of 5 to 6 shoots per bud from in vitro culture of ... also succeeded in in vitro nodal culture of cassava. (Konan et al., 2006; Medina et al., 2006; Escobar et al.,. 2009). Despite these reported successes, ...

  17. Intestinal surfactant permeation enhancers and their interaction with enterocyte cell membranes in a mucosal explant system

    DEFF Research Database (Denmark)

    Danielsen, E Michael; Hansen, Gert H

    2017-01-01

    Intestinal permeation enhancers (PEs) are agents aimed to improve oral delivery of therapeutic drugs with poor bioavailability. The main permeability barrier for oral delivery is the intestinal epithelium, and PEs act to increase the paracellular and/or transcellular passage of drugs. Transcellular...

  18. Characteristics of chrysanthemum mutants regenerated from in vitro explants irradiated with 12C5+ ion beam

    Energy Technology Data Exchange (ETDEWEB)

    Nagatomi, Shigeaki [Laboratory of Radiation Biotechnology, Institute of Radiation Breeding, NIAR, MAFF, Tsukuba, Ibaraki (Japan)

    1998-12-31

    As most of the mutagens used for mutant varieties registered in Japan are gamma ray and x ray, it is necessary to develop new and efficient mutagens for plant mutation breeding. In this report, radiobiological effects of 12C5+ ion beam on in vitro cultured materials and mutation induction of flower color of regenerated plants are demonstrated, the results being compared with those from gamma ray irradiation. (J.P.N.)

  19. [Intraocular lens explantation and secondary implantation of a light-adjustable lens. Video article].

    Science.gov (United States)

    Hengerer, F H

    2016-04-01

    The operation is carried out to exchange previously implanted intraocular lenses (IOL) because of deviation from the target refraction with aniseikonia and anisometropia, in cases of contact lens intolerance, dissatisfaction with the IOL due to side effects and particularly problems with multifocal lenses. The aim of this surgical approach is to retain the original implantation site of the exchange lens and capsular bag integrity with maximum sparing of the zonular fibers and secondary IOL implantation with stable positioning for fine adjustment of residual refractive errors. Using a non-astigmatic approach the primary IOL is first mobilized and removed. A light-adjustable IOL is then implanted in the capsular bag and residual refractive errors in the visual axis are adjusted using profiled UV light. The surgical technique is demonstrated in detail with the help of a video of the operation, which is available online. Residual postoperative deviations from the target refraction of sphere and cylinder up to 2 diopters can be precisely adjusted noninvasively. Individual alterations of asphericity can provide stable distance visual acuity while enhancing near and intermediate visual acuity.

  20. Patient-specific three-dimensional explant spheroids derived from human nasal airway epithelium

    DEFF Research Database (Denmark)

    Marthin, June Kehlet; Stevens, Elizabeth Munkebjerg; Larsen, Lars Allan

    2017-01-01

    surface facing the outside and accessible for analysis of ciliary function. METHODS: We performed a two-group comparison study of ciliary beat pattern and ciliary beat frequency in spheroids derived from nasal airway epithelium in patients with primary ciliary dyskinesia (PCD) and in healthy controls...

  1. Criteria and Planning Guidance for Ex-Plant Harvesting to Support Subsequent License Renewal

    Energy Technology Data Exchange (ETDEWEB)

    Ramuhalli, Pradeep [Pacific Northwest National Lab. (PNNL), Richland, WA (United States); Devanathan, Ram [Pacific Northwest National Lab. (PNNL), Richland, WA (United States); Meyer, Ryan M. [Pacific Northwest National Lab. (PNNL), Richland, WA (United States); Glass, Samuel W. [Pacific Northwest National Lab. (PNNL), Richland, WA (United States); Knobbs, Katherine J. [Pacific Northwest National Lab. (PNNL), Richland, WA (United States)

    2017-12-07

    As U.S. nuclear power plants look to subsequent license renewal (SLR) to operate for a 20-year period beyond 60 years, the U.S. Nuclear Regulatory Commission and the industry will be addressing technical issues around the capability of long-lived passive components to meet their functionality objectives. A key challenge will be to better understand likely materials degradation mechanisms in these components and their impacts on component functionality and margins to safety. Research addressing many of the remaining technical gaps in these areas for SLR may greatly benefit from materials sampled from plants (decommissioned or operating). Because of the cost and inefficiency of piecemeal sampling, there is a need for a strategic and systematic approach to sampling materials from structures, systems, and components (SSC) in both operating and decommissioned plants. This document describes a potential approach for sampling (harvesting) materials that focuses on prioritizing materials for sampling using a number of criteria. These criteria are based on an evaluation of technical gaps identified in the literature, research needs to address these technical gaps, and lessons learned from previous harvesting campaigns. The document also describes a process for planning future harvesting campaigns; such a plan would include an understanding of the harvesting priorities, available materials, and the planned use of the materials to address the technical gaps.

  2. Toxic Effects of Lipid-Mediated Gene Transfer in Ventral Mesencephalic Explant Cultures

    DEFF Research Database (Denmark)

    Bauer, Matthias; Kristensen, Bjarne Winther; Meyer, Morten

    2006-01-01

    of propidium iodide and release of cytosolic lactate dehydrogenase to the culture medium. Using a liposomal transfection reagent, cell membrane damage was already seen 24 hr after transfection. Nestin-positive target cells, which were used as morphological correlate, were severely diminished in some areas...

  3. Explantátové kultury vyšších rostlin 25

    OpenAIRE

    Vránová, Marie

    2006-01-01

    SOUHRN Získané poznatky z diplomové práce lze shrnout následovně: 1. Ze sterilních lístků Centella asiatica byly odvozeny kultury schopné dlouhodobé kultivace. 2. Byla prokázána schopnost kultury Centella asiatica glykosylovat hydrochinon na arbutin. 3. Zvýšením koncentrace prekurzoru hydrochinonu došlo ke zvýšení produkce arbutinu 4. V extraktech kalusů kultury Centella asiatica byla po přidání prekurzoru kyseliny 4- hydroxybenzoové opakovaně nalezena neznámá látka ve zvýšené koncentraci 5. ...

  4. The effect of hygromycin on regeneration in different Alstroemeria explant types after Agrobacterium-mediated transformation

    NARCIS (Netherlands)

    Kim, J.B.; Jeu, de M.J.; Raemakers, C.J.J.M.; Jacobsen, E.; Visser, R.G.F.

    2002-01-01

    This is the first successful report of Agrobacterium-mediated transformation in Alstroemeria by infection of FEC (Friable Embryogenic Callus) lines and leaves with axil tissues. Of the transformation methods, particle bombardment and Agrobacterium-mediated transformation have been widely used to

  5. Iron accumulates in the lavage and explanted lungs of cystic fibrosis patients.

    Science.gov (United States)

    Abstract Oxidative stress participates in the pathophysiology of cystic fibrosis (CF). An underlying disruption in iron homeostasis can frequently be demonstrated in injuries and diseases associated with an oxidative stress. We tested the hypothesis that iron accumulation and ...

  6. Phagocytosis of collagen fibrils by periosteal fibroblasts in long bone explants. Effect of concanavalin A

    NARCIS (Netherlands)

    Everts, V.; Beertsen, W.

    1992-01-01

    In an attempt to determine whether phagocytosis of collagen by fibroblasts involves binding of the fibril to the plasma membrane, the effect of the lectin concanavalin A (Con A) was studied in an in vitro model system. Metacarpal bone rudiments from 19-day-old mouse fetuses were incubated with

  7. Histologic, Molecular, and Clinical Evaluation of Explanted Breast Prostheses, Capsules, and Acellular Dermal Matrices for Bacteria

    Science.gov (United States)

    Poppler, Louis; Cohen, Justin; Dolen, Utku Can; Schriefer, Andrew E.; Tenenbaum, Marissa M.; Deeken, Corey; Chole, Richard A.; Myckatyn, Terence M.

    2015-01-01

    Background Subclinical infections, manifest as biofilms, are considered an important cause of capsular contracture. Acellular dermal matrices (ADMs) are frequently used in revision surgery to prevent recurrent capsular contractures. Objective We sought to identify an association between capsular contracture and biofilm formation on breast prostheses, capsules, and ADMs in a tissue expander/implant (TE/I) exchange clinical paradigm. Methods Biopsies of the prosthesis, capsule, and ADM from patients (N = 26) undergoing TE/I exchange for permanent breast implant were evaluated for subclinical infection. Capsular contracture was quantified with Baker Grade and intramammary pressure. Biofilm formation was evaluated with specialized cultures, rtPCR, bacterial taxonomy, live:dead staining, and scanning electron microscopy (SEM). Collagen distribution, capsular histology, and ADM remodeling were quantified following fluorescent and light microscopy. Results Prosthetic devices were implanted from 91 to 1115 days. Intramammary pressure increased with Baker Grade. Of 26 patients evaluated, one patient had a positive culture and one patient demonstrated convincing evidence of biofilm morphology on SEM. Following PCR amplification 5 samples randomly selected for 16S rRNA gene sequencing demonstrated an abundance of suborder Micrococcineae, consistent with contamination. Conclusions Our data suggest that bacterial biofilms likely contribute to a proportion, but not all diagnosed capsular contractures. Biofilm formation does not appear to differ significantly between ADMs or capsules. While capsular contracture remains an incompletely understood but common problem in breast implant surgery, advances in imaging, diagnostic, and molecular techniques can now provide more sophisticated insights into the pathophysiology of capsular contracture. Level of Evidence PMID:26229126

  8. Effects of plant growth regulators on in vitro cultured nodal explants ...

    African Journals Online (AJOL)

    Cassava (Manihot esculenta Crantz) is one of the major food security crops in Ethiopia. Recently, clean planting materials of improved cassava cultivars are in high demand. A limitation, however, is the low multiplication ratio (1:10) of the crop via conventional methods. Thus, a study was undertaken to develop an efficient in ...

  9. Plant regeneration from cotyledonary explants of Eucalyptus camaldulensis dehn and histological study of organogenesis in vitro

    National Research Council Canada - National Science Library

    Roberson Dibax; Regina Caetano Quisen; Cleusa Bona; Marguerite Quoirin

    2010-01-01

    ... a soil:vermiculite mixture and cultured in a greenhouse. Histological studies revealed that the callogenesis initiated in palisade parenchyma cells and that the adventitious buds were formed from the calluses, indicating indirect organogenesis...

  10. A Novel In Vitro Protocol for Inducing Direct Somatic Embryogenesis in Phalaenopsis aphrodite without Taking Explants

    Directory of Open Access Journals (Sweden)

    Jia-Hua Feng

    2014-01-01

    Full Text Available An alternative in vitro protocol for embryo induction directly from intact living seedlings of Phalaenopsis aphrodite subspecies formosana was established in this study. Without the supplementation of plant growth regulators (PGRs, no embryos were obtained from all the seedlings when cultured on the solid medium. In contrast, embryos formed from the seedlings on the 2-layer medium and the 2-step culture system without the use of PGRs. It was found that the age of the seedlings affected embryo induction. The 2-month-old seedlings typically had higher embryogenic responses when compared with the 4-month-old seedlings in the 2-layer medium or 2-step system. For the 2-month-old seedlings, 1 mg/L TDZ resulted in the highest number of embryos at the distal site of the shoot. However, on the leaves’ surface, 0.5 mg/L TDZ induced the highest number of embryos. When the 2-month-old seedlings were cultured using the 2-step method at 1 mg/L of TDZ, the highest embryogenic response was obtained, with an average of 44 embryos formed on each seedling. These adventitious embryos were able to convert into plantlets in a PGR-free 1/2 MS medium, and the plantlets had normal morphology and growth.

  11. Cultura Primária de Explant de Hiperplasia Prostática Benigna

    Directory of Open Access Journals (Sweden)

    Patrícia Borba Martiny

    2014-01-01

    Full Text Available A Hiperplasia Prostática Benigna (HPB é um crescimento patológico e não maligno da próstata causada principalmente pela proliferação exacerbada das células epiteliais e, principalmente, estromais. Essa neoplasia benigna acomete a maioria dos homens de idade avançada, sendo a condição crônica mais prevalente entre a população masculina. Como mostrado em estudos, o desenvolvimento prostático é intimamente dependente da interação entre as variantes celulares estromais e epiteliais presentes nesta glândula. Entretanto, a cocultura de ambos os tipos celulares é de difícil estabelecimento, uma vez que os dois tipos celulares têm diferentes características de adesão à placa de cultivo celular.

  12. Pulmonary artery rupture in a patient receiving an orthotopic heart transplant after total artificial heart explant.

    Science.gov (United States)

    Nomoto, Koichi; Weiner, Menachem M; Evans, Adam

    2014-02-01

    Our case illustrates a patient who suffered a pulmonary artery rupture despite previous total artificial heart implantation and replacement with orthotopic heart transplant. Pulmonary artery rupture during or following cardiac surgery has been reported to occur due to both pulmonary artery catheter use and surgical technique. Our case is the first to demonstrate the occurrence of this complication in the total artificial heart patient population.

  13. In vitro direct regeneration in mint from different explants on half ...

    African Journals Online (AJOL)

    STORAGESEVER

    2009-09-15

    Sep 15, 2009 ... 1Plant Cell and Tissue Culture Lab, Department of Biotechnology, Quaid-i-Azam University, Islamabad, Pakistan 45320. 2Agriculture Biotechnology ... clonal variants, somatic hybrids or transgenic plants. However a prerequisite to ..... Antifeeding, settling inhibitory and toxic activities of labiate essential oils ...

  14. A novel 3D skin explant model to study anaerobic bacterial infection

    DEFF Research Database (Denmark)

    Maboni, Grazieli; Davenport, Rebecca; Sessford, Kate

    2017-01-01

    Skin infection studies are often limited by financial and ethical constraints, and alternatives, such as monolayer cell culture, do not reflect many cellular processes limiting their application. For a more functional replacement, 3D skin culture models offer many advantages such as the maintenance...

  15. Synovial explant inflammatory mediator production corresponds to rheumatoid arthritis imaging hallmarks

    DEFF Research Database (Denmark)

    Andersen, Martin; Boesen, Mikael; Ellegaard, Karen

    2014-01-01

    INTRODUCTION: Despite the widespread use of magnetic resonance imaging (MRI) and Doppler ultrasound for the detection of rheumatoid arthritis (RA) disease activity, little is known regarding the association of imaging-detected activity and synovial pathology. The purpose of this study was to comp......INTRODUCTION: Despite the widespread use of magnetic resonance imaging (MRI) and Doppler ultrasound for the detection of rheumatoid arthritis (RA) disease activity, little is known regarding the association of imaging-detected activity and synovial pathology. The purpose of this study...... was to compare site-specific release of inflammatory mediators and evaluate the corresponding anatomical sites by examining colour Doppler ultrasound (CDUS) and MRI scans. METHODS: RA patients were evaluated on the basis of CDUS and 3-T MRI scans and subsequently underwent synovectomy using a needle arthroscopic...

  16. in tissue culture of lilium explants may become heavily contaminated by the standard initiation procedure

    NARCIS (Netherlands)

    Askari Rabori, N.; Wang, Y.G.; Klerk, de G.J.M.

    2014-01-01

    In tissue culture of Lilium, the standard initiation procedure brought about substantial contamination in two ways. (1) When scales were detached from the mother bulb, microorganisms could enter via the wound. This source of contamination was strongly enhanced by the negative hydrostatic pressure

  17. Mechanism of Trypanosoma cruzi Placenta Invasion and Infection: The Use of Human Chorionic Villi Explants

    Directory of Open Access Journals (Sweden)

    Ricardo E. Fretes

    2012-01-01

    Full Text Available Congenital Chagas disease, a neglected tropical disease, endemic in Latin America, is associated with premature labor and miscarriage. During vertical transmission the parasite Trypanosoma cruzi (T. cruzi crosses the placental barrier. However, the exact mechanism of the placental infection remains unclear. We review the congenital transmission of T. cruzi, particularly the role of possible local placental factors that contribute to the vertical transmission of the parasite. Additionally, we analyze the different methods available for studying the congenital transmission of the parasite. In that context, the ex vivo infection with T. cruzi trypomastigotes of human placental chorionic villi constitutes an excellent tool for studying parasite infection strategies as well as possible local antiparasitic mechanisms.

  18. Hepatocytes explanted in the spleen preferentially express carbamoylphosphate synthetase rather than glutamine synthetase

    NARCIS (Netherlands)

    Lamers, W. H.; Been, W.; Charles, R.; Moorman, A. F.

    1990-01-01

    Urea cycle enzymes and glutamine synthetase are essential for NH3 detoxification and systemic pH homeostasis in mammals. Carbamoylphosphate synthetase, the first and flux-determining enzyme of the cycle, is found only in a large periportal compartment, and glutamine synthetase is found only in a

  19. Explantation of infected aortic aneurysm and endograft with ascending aorta to mesenteric bypass for mesenteric ischemia.

    Science.gov (United States)

    Gates, Lindsay; Chin, Jason A; Bonde, Pramod N; Ochoa Chaar, Cassius I; Sumpio, Bauer E; Sarac, Timur P

    2017-01-01

    A 65-year-old man presented with an infected perivisceral aortic aneurysm after previous treatment of an abdominal aortic aneurysm with an endograft. On presentation, he was septic and had occlusion of the celiac, superior mesenteric, inferior mesenteric, and bilateral renal arteries. He underwent a three-stage procedure: first, axillobifemoral bypass; then resection of the thoracoabdominal aorta; and finally bypass from the ascending aorta to the celiac and superior mesenteric arteries with a rifampin-soaked Gelsoft graft (Vascutek, Renfrewshire, Scotland). The abdominal pain resolved, and the patient remains symptom free 10 months postoperatively. This rare surgical revascularization technique offered a nontraditional solution to a difficult surgical issue. Copyright © 2016 Society for Vascular Surgery. Published by Elsevier Inc. All rights reserved.

  20. The Stimulatory Effect of Notochordal Cell-Conditioned Medium in a Nucleus Pulposus Explant Culture

    NARCIS (Netherlands)

    de Vries, Stefan A H; van Doeselaar, Marina; Meij, Björn P; Tryfonidou, Marianna A; Ito, K|info:eu-repo/dai/nl/345809610

    2016-01-01

    Objectives: Notochordal cell-conditioned medium (NCCM) has previously shown to have a stimulatory effect on nucleus pulposus cells (NPCs) and bone marrow stromal cells (BMSCs) in alginate and pellet cultures. These culture methods provide a different environment than the nucleus pulposus (NP)

  1. The Stimulatory Effect of Notochordal-Cell Conditioned Medium in a Nucleus Pulposus Explant Culture

    NARCIS (Netherlands)

    de Vries, Stefan; Doeselaar, Marina van; Meij, Björn; Tryfonidou, M; Ito, Keita

    2015-01-01

    OBJECTIVES: Notochordal cell-conditioned medium (NCCM) has previously shown to have a stimulatory effect on nucleus pulposus cells (NPCs) and bone marrow stromal cells (BMSCs) in alginate and pellet cultures. These culture methods provide a different environment than the nucleus pulposus (NP)

  2. A REVIEW: MICROPROPAGATION OF PHALAENOPSIS sp FROM LEAF AND FLOWER STALK EXPLANTS

    Directory of Open Access Journals (Sweden)

    Meutia Zahara

    2017-09-01

    • Semiarti, E.; Indrianto, A.; Purwanto, A. Agrobacterium-Mediated transformation of Indonesian orchids for  micropropagation, genetic transformation, Prof. MarÃa Alvarez (Ed., ISBN: 978-953-307-364-4, InTech, 2011. Available from: http://www.intechopen.com/books/ genetic-transformation/agrobacterium-mediated-transformation-ofindonesian-orchids-for-micropropagation.

  3. Etiolation and flooding of donor plants enhance the capability of Arabidopsis explants to root

    NARCIS (Netherlands)

    Massoumi Bagherabadi, Mehdi; Krens, Frans A.; Visser, Richard G.F.; Klerk, de Geert Jan M.

    2017-01-01

    Rooting of cuttings depends not only on the rooting treatment and the genotype, but also on the condition of the cuttings at the time of excision. The physiological and developmental conditions of the donor plant may be decisive. We have examined in Arabidopsis the effect of two donor plant

  4. Alternative V kappa gene rearrangements in a murine B cell lymphoma. An explantation for idiotypic heterogeneity.

    Science.gov (United States)

    Carroll, W L; Starnes, C O; Levy, R; Levy, S

    1988-11-01

    Idiotype variants of 38C13, a murine B cell lymphoma, have been isolated by immunoselection with antiidiotype mAbs. The V region genes for the kappa light chains and mu heavy chains expressed by these tumor cells were sequenced and compared. There was no evidence for V region somatic point mutation in this tumor. However, while the heavy chain genes were all identical, the light chain genes were all different. The light chain genes of each variant were derived from the V kappa-Ox1 gene family and joined to J kappa 4, whereas the light chain gene of the parental tumor was derived from the V kappa 9 family and joined to J kappa 2. Two of the variants used the identical V kappa gene but differed by the inclusion of a variable number of additional nucleotides in the V/J joint. Thus, the idiotypic heterogeneity of this B cell lymphoma arises as a consequence of alternative light chain rearrangements rather than point mutation. This process repetitively uses members of the same V kappa gene family. Two of the variants use the identical V kappa and J kappa gene segments but differ by the presence of extra nucleotides at the V kappa/J kappa joint.

  5. Ethylene oxide gas sterilization: a simple technique for storing explanted skull bone. Technical note.

    Science.gov (United States)

    Jho, David H; Neckrysh, Sergey; Hardman, Julian; Charbel, Fady T; Amin-Hanjani, Sepideh

    2007-08-01

    The authors evaluated the effectiveness of a simple technique using ethylene oxide (EtO) gas sterilization and room temperature storage of autologous bone grafts for reconstructive cranioplasty following decompressive craniectomy. The authors retrospectively analyzed data in 103 consecutive patients who underwent cranioplasty following decompressive craniectomy for any cause at the University of Illinois at Chicago between 1999 and 2005. Patients with a pre-existing intracranial infection prior to craniectomy or lost to follow-up before reconstruction were excluded. Autologous bone grafts were cleansed of soft tissue, hermetically sealed in sterilization pouches for EtO gas sterilization, and stored at room temperature until reconstructive cranioplasty was performed. Cranioplasties were performed an average of 4 months after decompressive craniectomy, and the follow-up after reconstruction averaged 14 months. Excellent aesthetic and functional results after single-stage reconstruction were achieved in 95 patients (92.2%) as confirmed on computed tomography. An infection of the bone flap occurred in eight patients (7.8%), and the skull defects were eventually reconstructed using polymethylmethacrylate with satisfactory results. The mean preservation interval was 3.8 months in patients with uninfected flaps and 6.4 months in those with infected flaps (p = 0.02). A preservation time beyond 10 months was associated with a significantly increased risk of flap infection postcranioplasty (odds ratio [OR] 10.8, p = 0.02). Additionally, patients who had undergone multiple craniotomies demonstrated a trend toward increased infection rates (OR 3.0, p = 0.13). Data in this analysis support the effectiveness of this method, which can be performed at any institution that provides EtO gas sterilization services. The findings also suggest that bone flaps preserved beyond 10 months using this technique should be discarded or resterilized prior to reconstruction.

  6. Long-term culture of sponge explants: conditions enhancing survival and growth, and assessment of bioactivity

    NARCIS (Netherlands)

    Caralt, de S.; Agell, G.; Uriz, M.J.

    2003-01-01

    Sponges are an important source of secondary metabolites with pharmaceutical interest. This is the main reason for the increasing interest of sponge culture recent years. The optimal culture system depends on the species to be cultured: while some species easily produce sponge aggregates after

  7. Sprifermin (rhFGF18) modulates extracellular matrix turnover in cartilage explants ex vivo

    DEFF Research Database (Denmark)

    Reker, Ditte; Kjelgaard-Petersen, Cecilie Freja; Siebuhr, Anne Sofie

    2017-01-01

    or placebo at weekly intervals, similar to the dosing regimen used in clinical trials. Pre-culturing with oncostatin M and tumour necrosis factor-a, was also used to induce an inflammatory state before treatment. Metabolic activity was measured using AlamarBlue, and chondrocyte proliferation was visualized...

  8. The Tribology of Explanted Hip Resurfacings Following Early Fracture of the Femur.

    Science.gov (United States)

    Lord, James K; Langton, David J; Nargol, Antoni V F; Meek, R M Dominic; Joyce, Thomas J

    2015-10-15

    A recognized issue related to metal-on-metal hip resurfacings is early fracture of the femur. Most theories regarding the cause of fracture relate to clinical factors but an engineering analysis of failed hip resurfacings has not previously been reported. The objective of this work was to determine the wear volumes and surface roughness values of a cohort of retrieved hip resurfacings which were removed due to early femoral fracture, infection and avascular necrosis (AVN). Nine resurfacing femoral heads were obtained following early fracture of the femur, a further five were retrieved due to infection and AVN. All fourteen were measured for volumetric wear using a co-ordinate measuring machine. Wear rates were then calculated and regions of the articulating surface were divided into "worn" and "unworn". Roughness values in these regions were measured using a non-contacting profilometer. The mean time to fracture was 3.7 months compared with 44.4 months for retrieval due to infection and AVN. Average wear rates in the early fracture heads were 64 times greater than those in the infection and AVN retrievals. Given the high wear rates of the early fracture components, such wear may be linked to an increased risk of femoral neck fracture.

  9. Riata implantable cardioverter-defibrillator lead failure: analysis of explanted leads with a unique insulation defect.

    Science.gov (United States)

    Hauser, Robert G; McGriff, Deepa; Retel, Linda Kallinen

    2012-05-01

    The Riata family of implantable cardioverter-defibrillator leads (St Jude Medical, Sylmar, CA) appears prone to a unique failure mechanism whereby the conductor cables wear through the silicone insulation from inside-out and are seen outside the lead body (externalized conductors). To assess the extent of Riata lead damage associated with inside-out insulation defects and their clinical consequences. In September 2011, we searched the U.S. Food and Drug Administration's Manufacturers and User Defined Experience medical device database for reports describing Riata lead failures that had been analyzed by the manufacturer. The Manufacturers and User Defined Experience search identified 105 leads that had inside-out insulation defects. Eight-French single-coil Riata leads accounted for a higher-than-expected proportion (25.7%) of the leads with this defect. A total of 226 insulation defects were found in the 105 leads (2.2 defects per lead), including 143 inside-out defects (1.4 defects per lead). The most common location of insulation defects was distal to the proximal coil (n = 108). Twenty-eight leads (26.7%) had inside-out insulation defects underneath the shocking coils. Of 43 leads whose cables were assessed for the integrity of the ethylene-tetrafluoroethylene cable coating, 22 (51.2%) were found to be abraded, exposing the conductor surfaces. On X-ray radiography or fluoroscopy, 7 leads were found to have externalized cables; 2 of these leads had no electrical abnormalities, while 4 exhibited noise or increased impedance. Inappropriate shocks were experienced by 31 of the 105 patients (29.5%). Riata leads that have inside-out insulation defects often have multiple defects, including additional inside-out abrasions along the body of the lead and beneath the shocking coils. Eight-French single-coil Riata models may be more prone to externalized cables than are dual-coil and 7-F designs. Externalized cables are but one manifestation of interior insulation damage. Our findings question the durability of the ethylene-tetrafluoroethylene cable coating on exposed cables. Copyright © 2012 Heart Rhythm Society. Published by Elsevier Inc. All rights reserved.

  10. In Vitro propagation of enterolobium cyclocarpum (guanacaste) from nodal explants of axenic seedlings

    OpenAIRE

    Araceli Rodríguez Sahagún; Osvaldo A. Castellanos Hernández; Gustavo J. Acevedo Hernández

    2007-01-01

    Enterolobium cyclocarpum (Jacq.) Griseb. es un árbol leguminoso de uso múltiple, el cual es considerado una especie amenazada, resultado de la sobreexplotación y las bajas tasas de propagación natural debidas a las características intrínsecas del árbol. Una alternativa para superar este problema es el establecimiento de sistemas para su propagación masiva en tiempos cortos. En este trabajo, se investigó un protocolo para la propagación in vitro de E. cyclocarpum utilizando los segmen...

  11. Identificação e controle com antibióticos de bactérias endofíticas contaminantes em explantes de batata micropropagados Identification and antibiotic control of endophytic bacteria contaminants in micropropagated potato explants

    Directory of Open Access Journals (Sweden)

    Jonny Everson Scherwinski Pereira

    2003-07-01

    Full Text Available Este trabalho teve por objetivos isolar, caracterizar e identificar bactérias endofíticas contaminantes encontradas em tecidos de batata durante a micropropagação e selecionar antibióticos para o controle in vitro desses microrganismos por meio da determinação da concentração bactericida mínima inibitória. Brotações de batata apresentando contaminação bacteriana durante a etapa de multiplicação in vitro, foram superficialmente esterilizadas e os internódios transferidos para placas de Petri com ágar nutriente, onde permaneceram incubadas a 28°C por até cinco dias. Após purificação, as bactérias foram caracterizadas e identificadas por testes taxonômicos. Um total de oito estirpes bacterianas foram isoladas e identificadas como pertencentes às famílias Acetobacteriaceae (1 e Enterobacteriaceae (2 e aos gêneros Corynebacterium (3, Pseudomonas (1 e Xanthomonas (1. Os melhores resultados para a inibição do crescimento bacteriano foram obtidos com os antibióticos ampicilina, cloranfenicol, estreptomicina e tetraciclina em concentrações que variaram de 32 a 256 mg L-1.This work aimed to isolate, characterize and identify contaminant endophytic bacteria found in potato tissues during the micropropagation and to select antibiotics for in vitro control of these microorganisms by determining the inhibitory minimal bactericidal concentration. Potato shoots presenting bacterial contamination during the in vitro multiplication were superficially sterilized and the internodes transferred to Petri dishes with nutrient agar medium for up to five days at 28°C. After subcultures the grown bacteria were purified and identified through taxonomic tests. A total of eight bacterial endophytic strains were isolated and identified as belonging to Acetobacteriaceae (1 and Enterobacteriaceae (2 families and Corynebacterium (3, Pseudomonas (1 and Xanthomonas (1 genera. The best results for bacterial growth inhibition were obtained with ampicilin, chloramphenicol, streptomycin and tetracycline antibiotics in concentrations ranging from 32 to 256 mg L-1.

  12. Structures of benzo(a)pyrene-nucleic acid adducts formed in human and bovine bronchial explants

    DEFF Research Database (Denmark)

    Jeffrey, A.M.; Weinstein, I.B.; Jenette, K.W.

    1977-01-01

    PUBLICATION by Sims et al. of evidence that the 7,8-dihydrodiol-9,10-oxide of benzo(a)pyrene (BP) is a metabolic intermediate in the covalent binding of this ubiquitous polycyclic aromatic hydrocarbon to DNA in hamster embryo cells1 was followed by many related publications2. Grover et al. 3 also...

  13. Effect of triiodothyronine and insulin on glucose metabolism in tissue explants and isolated adipocytes from lean and obese Zucker rats

    Energy Technology Data Exchange (ETDEWEB)

    Bailey, J.W.

    1985-01-01

    Glucose metabolism in adipocytes from 6 week old lean and obese Zucker rats were sensitive to direct and chronic treatment with insulin and triidothyronine (T/sub 3/). Insulin had a large stimulatory effect on glucose metabolism in acutely isolated adipocytes. This effect was greater in the lean than in the obese. Fatty acid, CO/sub 2/, and glycerol-glyceride formation from radiolabeled glucose was elevated in the obese over the leans. Pretreatment of isolated adipocytes with pharmacological concentrations of T/sub 3/ for 30 minutes prior to the measurement of glucose metabolism had a greater effect on lean than obese adipocytes. The presence of insulin was required to observe the acute effects of T/sub 3/. A 2-hour exposure to physiological levels of T/sub 3/ in the presence of insulin in both lean and obese adipocytes decreased lipogenesis. In the absence of insulin, a 2 hour pretreatment with physiological levels of T/sub 3/ in tissue from a euthyroid animal produced increased lipogenesis.

  14. In Vitro Conservation of Date Palm Shoot-Tip Explants and Callus Cultures Under Minimal Growth Conditions.

    Science.gov (United States)

    El-Dawayati, Maiada M

    2017-01-01

    Date palm fruit production has great economic significance for many countries. There is a fundamental necessity to conserve valuable date palm germplasm, but there are various problems with in vivo and ex situ conservation. In vitro storage has several advantages over conventional germplasm conservation methods. The in vitro technique offers a developed method of slow-growth storage, which is considered as an alternate solution for short- and medium-term storage of date palm germplasm under controlled conditions. Minimal growth conditions for germplasm conservation are generally achieved by reducing growth rate through modification of environmental growing conditions and culture, by using low temperatures, and the addition of growth retardants and osmotic agents. This chapter describes a protocol for short-term in vitro conservation of date palm shoot-tip and callus cultures under slow-growth storage conditions, using sucrose as an osmotic agent and abscisic acid (ABA) as a growth retardant at 15 °C for 12 months.

  15. Subzero nonfreezing storage of the mammalian cardiac explant. I. Methanol, ethanol, ethylene glycol, and propylene glycol as colligative cryoprotectants.

    Science.gov (United States)

    Yang, X; Zhu, Q; Layne, J R; Claydon, M; Hicks, G L; Wang, T

    1993-08-01

    We employed hyperosmotic concentrations of penetrating cryoprotective agents (CPA) to store the isolated rat hearts unfrozen at subzero temperatures. The effect of acute exposure to CPA was assessed by flushing the hearts with CP-14, a cardioplegic solution, containing methanol (MeOH), ethanol (EtOH), ethylene glycol (EG), or propylene glycol (PG) for 2 min and reperfusing immediately with Krebs-Henseleit buffer in a working-heart model. The maximal doses that did not cause irreversible suppression of heart function were: MeOH, 1.78 M; EtOH, 1.27 M; EG, 0.84 M; and PG, 0.87 M. For nonfreezing storage, the hearts were flushed with CP-14 containing the highest tolerable concentrations of MeOH, EtOH, EG, or PG, stored for 6 h at -3.7, -2.8, and -1.4 degrees C, respectively, and then reperfused. Control cardiac output (CO) was 76.2 +/- 1.8 ml/min. Post-reperfusional recovery of CO was 86% in MeOH hearts, 82% in EtOH hearts, 76% in EG hearts, and 79% in PG hearts. Thus MeOH offered not only the least cardiac-suppressing effect but the lowest nonfreezing storage temperature. When storage time was extended, recovery and myocardial ATP level decreased with time in hearts flushed with CP-14 + 1.78 M MeOH and stored at -3.7 degrees C. The decay of function was faster than the decay of ATP level, suggesting energy was better preserved than function. The low return of function, however, may be related to CPA toxicity, osmotic stress, and ischemia/reperfusion injury. Nonfreezing storage at subzero temperatures using these CPAs may provide a novel approach to long-term cardiac preservation.

  16. Sacha Inchi Oil (Plukenetia volubilis L.), effect on adherence of Staphylococus aureus to human skin explant and keratinocytes in vitro

    National Research Council Canada - National Science Library

    Gonzalez-Aspajo, German; Belkhelfa, Haouaria; Haddioui-Hbabi, Laïla; Bourdy, Geneviève; Deharo, Eric

    2015-01-01

    .... Oil from the cold-pressed seeds, sold under the commercial name of Sacha Inchi Oil (SIO) is actually much in favour because it contains a high percentage of omega 3 and omega 6, and is hence used as a dietary supplement...

  17. Case presentation of florid cemento-osseous dysplasia with concomitant cemento-ossifying fibroma discovered during implant explantation.

    Science.gov (United States)

    Gerlach, Robert C; Dixon, Douglas R; Goksel, Tamer; Castle, James T; Henry, Walter A

    2013-03-01

    A 39-year-old African American woman presented for treatment of a symptomatic mandibular right first molar with a large, periapical radiolucency. After initial attempts at endodontic therapy, this tooth was ultimately extracted owing to unabated symptoms. The extraction site underwent ridge preservation grafting, implant placement, and restoration. After 26 months of implant function, the patient returned with clinical symptoms of pain, buccal swelling, and the sensation of a "loose" implant. This case report details a diagnosis of 2 distinct disease entities associated with the implant site, a cemento-ossifying fibroma and florid cemento-osseous dysplasia of the mandible. This diagnosis was determined from clinical, surgical, radiographic, and histopathologic evidence after biopsy and removal of the previously osseointegrated implant following postinsertion failure by fibrous encapsulation. Before implant therapy, it is essential to conduct a thorough radiographic evaluation of any dental arch with suspected bony lesions to prevent implant failure. Published by Mosby, Inc.

  18. Effects of Explant Source and Dark-preconditioning on Adventitious Bud Formation in Neofinetia falcata H. H. Hu in vitro

    National Research Council Canada - National Science Library

    箕作, 和彦; 森, 源治郎; 淨閑, 正史; 三柴, 啓一郎; 森川, 利信; 小田, 雅行

    2009-01-01

    フウラン(Neofinetia falcata H. H. Hu)の中でも観賞価値の高い品種は,形質が分離する種子繁殖ではなく,栄養繁殖が行われている.しかし,その増殖率は低い.そこで,幼植物の組織培養による増殖効率の向上について検討した.まず,無菌播種して形成したプロトコームを上下に 2...

  19. Regulation of EGF and Prostaglandin Expression during Neonatal Gastrointestinal Injury in a Non-Human Primate Explant Model

    Science.gov (United States)

    2017-05-05

    FHsIm7- d Prosta.gim:lin E.’q)IcSLoO During Neon tal Gastro!:n.r.e:;t!na.llDjury: Line M8ER: FOml 3D39 r 1 :2. DYE8 DA-E Ap ill? 2017 15. ACe\\" edo . N\\’l...efficiently replicates in vivo tissue behavior allowing for testing of confounding variables within the. same tissue and translation to an in vivo

  20. Reformation of tissue balls from tentacle explants of coral Goniopora lobata: self-organization process and response to environmental stresses.

    Science.gov (United States)

    Lu, Qiongxuan; Liu, Tao; Tang, Xianming; Dong, Bo; Guo, Huarong

    2017-02-01

    Coral has strong regeneration ability, which has been applied for coral production and biodiversity protection via tissue ball (TB) culture. However, the architecture, morphological processes, and effects of environmental factors on TB formation have not been well investigated. In this study, we first observed TB formation from the cutting tentacle of scleractinia coral Goniopora lobata and uncovered its inner organization and architecture by confocal microscopy. We then found that the cutting tentacle TB could self-organize and reform a solid TB (sTB) in the culture media. Using chemical drug treatment and dissection manipulation approaches, we demonstrated that the mechanical forces for bending and rounding of the cutting fragments came from the epithelial cells, and the cilia of epithelial cell played indispensable roles for the rounding process. Environmental stress experiments showed that high temperature, not CO2-induced acidification, affected TB and sTB formation. However, the combination of high temperature and acidification caused additional severe effects on sTB reformation. Our studies indicate that coral TB has strong regeneration ability and therefore could serve as a new model to further explore the molecular mechanism of TB formation and the effects of environmental stresses on coral survival and regeneration.

  1. Activation of Toll-like Receptor 2 in Human Synovium Explants Increase Tissue Turnover and Secretion of Interleukin-6

    DEFF Research Database (Denmark)

    Sharma, Neha; Kayed, Ashref; Kjelgaard-Petersen, Cecilie Freja

    2017-01-01

    Background/Purpose: The innate immune system is important for initiation and development of OA. Increased degradation of the cartilage release fragments into the synovial fluid, which can then bind to innate immune receptors in the synovium. The aim of this study was to investigate the effect of ...

  2. Establishing an efficient explant superficial sterilization protocol for in vitro micropropagation of bear’s garlic (Allium ursinum L.

    Directory of Open Access Journals (Sweden)

    Tomaszewska-Sowa Magdalena

    2015-12-01

    Full Text Available Introduction:Allium ursinum L. has a commercial value due to its high contents of bio-active compounds and mild, garlic-like taste. In vitro culture played an important role in obtaining Allium species with the desired characteristics and in the production of healthy reproductive material.

  3. The early effects of radiation on in vitro explants of mouse pancreas. A morphological and immunocytochemical study

    Energy Technology Data Exchange (ETDEWEB)

    Kosanlavit, R

    2001-07-01

    Prodromal radiation sickness involving the digestive system may occur less than an hour following whole-body or abdominal irradiation, and may be of such severity as to prevent cancer patients from completing their course of radiotherapy. The contribution of radiation-induced pancreatic damage to radiation sickness is poorly understood. This study seeks to demonstrate the early effects of X-rays (0.5-10 Gy) on mouse pancreas in vitro. The response of exocrine acinar cells, and endocrine cells from the islets of Langerhans was examined using immunocytochemistry, light and transmission electron microscopy, and morphometric analysis. There was an approximate 50% decrease in the mean number of zymogen granules in acinar cells following 10 Gy irradiation at 1 hour, which may have been due to the acceleration of enzyme secretion or the interruption of enzyme synthesis or a combination of both. The frequency distributions of zymogen granules diameter showed minor change. The gross structure of acinar cells appeared not to be affected by irradiation at the doses and times used. Following 5 and 10 Gy irradiation a few pancreatic endocrine cells within each islet lost their chromogranin A-immunoreactivity whereas other islet cells showed more intense immunostaining for chromogranin A. A dose of 10 Gy significantly decreased the volume density of glucagon-containing cells at 1 hour. Doses of 5 and 10 Gy slightly decreased the volume density of somatostatin-containing cells from 30 minutes to 3 hours. Such changes in the expression of endocrine products from these cells are likely to have profound physiological effects. Radiation induced no changes in the volume density of insulin and PP-containing cells. The results of the present study suggest that X-irradiation induce changes to exocrine and endocrine pancreatic cells, and that this may contribute to some of the symptoms of radiation sickness. (author)

  4. A Microperfusion and In-Bore Oxygenator System Designed for Magnetic Resonance Microscopy Studies on Living Tissue Explants

    Science.gov (United States)

    Flint, Jeremy J.; Menon, Kannan; Hansen, Brian; Forder, John; Blackband, Stephen J.

    2015-12-01

    Spectrometers now offer the field strengths necessary to visualize mammalian cells but were not designed to accommodate imaging of live tissues. As such, spectrometers pose significant challenges—the most evident of which are spatial limitations—to conducting experiments in living tissue. This limitation becomes problematic upon trying to employ commercial perfusion equipment which is bulky and—being designed almost exclusively for light microscopy or electrophysiology studies—seldom includes MR-compatibility as a design criterion. To overcome problems exclusive to ultra-high magnetic field environments with limited spatial access, we have designed microperfusion and in-bore oxygenation systems capable of interfacing with Bruker’s series of micro surface-coils. These devices are designed for supporting cellular resolution imaging in MR studies of excised, living tissue. The combined system allows for precise control of both dissolved gas and pH levels in the perfusate thus demonstrating applicability for a wide range of tissue types. Its compactness, linear architecture, and MR-compatible material content are key design features intended to provide a versatile hardware interface compatible with any NMR spectrometer. Such attributes will ensure the microperfusion rig’s continued utility as it may be used with a multitude of contemporary NMR systems in addition to those which are currently in development.

  5. A protocol for high frequency regeneration through nodal explant cultures and ex vitro rooting of Plumbago rosea L.

    Science.gov (United States)

    Jose, Binoy; Satheeshkumar, K; Seeni, S

    2007-01-15

    A rapid clonal multiplication scheme comprising direct multiple shoot initiation and downsizing of the node with buds proliferated upon during subculture was developed for Plumbago rosea. Sixty five per cent of the nodes (approximately 2.0 cm) dissected out of young shoots from field grown plants and cultured in MS agar medium containing 3% sucrose and 15.4 microM BAP remained contamination free and responded at 95% rate with callusing at basal cut end and axillary bud break in 5 days followed by the formation of 2.41 +/- 0.14 shoots of 0.87 +/- 0.14 cm length in 3 weeks. Though differences in frequency and number of buds formed between nodes of 1-5 positions from the young shoots was negligible, the shoots emanated from the youngest node were shorter (0.92 +/- 0.19 cm) than those (2.3 +/- 0.50 cm) of the mature 5th node. Synergistic influence of BAP and auxins on caulogenesis was absent. Bud emergence in shorter (approximately 0.5 cm) nodes was delayed up to 3 weeks and extensive callus proliferation from the cut basal end overlapped the 8.2 +/- 0.37 axillary shoots/buds formed after 7 weeks. Reduction in the size (downsized) of the 2.0 cm node with buds to 1.0 cm by dissecting out the basal internodal segment having the callus and subculture of them (approximately 1.0 cm) with buds in contact with the medium for 3 weeks contributed to maximum multiplication of 42.1 +/- 5.40 shoot buds. Division of the shoot cluster and transfer of 2-3 shoots (0.5-1.5 cm) in a clump to MS basal liquid medium induced elongation of the shoots to 4.1 +/- 0.18 cm in 2 weeks. Shoots of 3.0-4.2 cm length were rooted within 3 weeks at 100% efficiency in vitro or ex vitro without hardening. In vitro rhizogenesis in presence of 0.49 microM IBA is recommended for enhanced rooting and high yield of commercially important tuberous roots during cultivation in the field.

  6. High–frequency in vitro plantlet regeneration from apical bud as a novel explant of Carum copticum L.

    Directory of Open Access Journals (Sweden)

    Mansoureh Salehi

    2014-05-01

    Conclusion: In this study, a simple and reliable regeneration and acclimatization protocol for Carum copticum has been presented. This protocol can be found very advantageous for a variety of purposes, including mass multiplication of Carum species, medicinal plant breeding studies and transgenic plant production.

  7. 69. Explante de válvula aórtica transca téter corevalve

    Directory of Open Access Journals (Sweden)

    J.R. González Rodríguez

    2010-01-01

    Conclusiones: El implante de válvulas aórticas transcatéter no es una técnica exenta de complicaciones. Los diferentes modelos y accesos para implantarlas hace necesaria la colaboración de equipos multidisciplinarios en quirófanos híbridos.

  8. Knock-Out Serum Replacement and Melatonin Effects on Germ Cell Differentiation in Murine Testicular Explant Cultures

    OpenAIRE

    Reda, Ahmed; Albalushi, Halima; Montalvo, Sheyla Cisneros; Nurmio, Mirja; Sahin, Zeliha; Hou, Mi; Geijsen, Niels; Toppari, Jorma; S?der, Olle; Stukenborg, Jan-Bernd

    2017-01-01

    Finding robust culture conditions for in vitro maturation (IVM) of male germ cells is still a challenge. Recently, a testis organ culture method, using Knockout Serum Replacement (KSR), was suggested as a promising approach. However, the efficiency of that model is still not optimal. Hence, we have tried to establish the culture conditions in two laboratories, and to improve the reliability of the culture system to generate mature germ cells. Male mice at three days of age were sacrificed. Te...

  9. Knock-Out Serum Replacement and Melatonin Effects on Germ Cell Differentiation in Murine Testicular Explant Cultures.

    Science.gov (United States)

    Reda, Ahmed; Albalushi, Halima; Montalvo, Sheyla Cisneros; Nurmio, Mirja; Sahin, Zeliha; Hou, Mi; Geijsen, Niels; Toppari, Jorma; Söder, Olle; Stukenborg, Jan-Bernd

    2017-07-01

    Finding robust culture conditions for in vitro maturation (IVM) of male germ cells is still a challenge. Recently, a testis organ culture method, using Knockout Serum Replacement (KSR), was suggested as a promising approach. However, the efficiency of that model is still not optimal. Hence, we have tried to establish the culture conditions in two laboratories, and to improve the reliability of the culture system to generate mature germ cells. Male mice at three days of age were sacrificed. Testes were cut into small pieces which were cultured atop agarose stands, using Minimum Essential Medium alpha supplemented with different supplements; melatonin, Glutamax, and different concentrations of KSR. The results showed that the duration of culture beyond 18 days had an impact on the number of differentiated germ cells. Supplementation with melatonin and Glutamax revealed a positive influence on the efficiency of male germ cell differentiation in vitro. Furthermore, the results confirmed that KSR had a positive effect on germ cell maturation and testosterone production, with a concentration of at least 10%. In conclusion, this study emphasizes the beneficial role of at least 10% KSR in the IVM of germ cells.

  10. Knock-Out Serum Replacement and Melatonin Effects on Germ Cell Differentiation in Murine Testicular Explant Cultures

    NARCIS (Netherlands)

    Reda, Ahmed; Albalushi, Halima; Montalvo, Sheyla Cisneros; Nurmio, Mirja; Sahin, Zeliha; Hou, Mi; Geijsen, Niels; Toppari, Jorma; Söder, Olle; Stukenborg, Jan-Bernd

    Finding robust culture conditions for in vitro maturation (IVM) of male germ cells is still a challenge. Recently, a testis organ culture method, using Knockout Serum Replacement (KSR), was suggested as a promising approach. However, the efficiency of that model is still not optimal. Hence, we have

  11. Interactions of virulent and avirulent Yersinia ruckeri strains with isolated gill arches and intestinal explants of rainbow trout Oncorhynchus mykiss.

    Science.gov (United States)

    Tobback, E; Hermans, K; Decostere, A; Van den Broeck, W; Haesebrouck, F; Chiers, K

    2010-07-01

    Yersinia ruckeri is the causative agent of enteric redmouth disease leading to significant losses in salmonid aquaculture worldwide. Little information is available on the pathogenesis of this disease. Basic steps in the establishment of an infection include attachment to the epithelium followed by invasion at the portal of entry. In this study, the interactions of Y. ruckeri with the gills and the gut of rainbow trout Oncorhynchus mykiss (Walbaum, 1792) were studied using standardized perfusion models. Virulent and avirulent Y ruckeri isolates appeared to adhere to and invade both tissues without significant differences. For the first time, the gill and gut perfusion models are shown to be suitable to study bacterial invasiveness.

  12. A two-centre evaluation of the human organotypic skin explant culture model for screening contact allergens

    NARCIS (Netherlands)

    Lehé, Cynthia L.; Jacobs, John J. L.; Elliott, Graham R.; Das, Pranab K.

    2003-01-01

    Animal models are considered to be the "gold standard" for determining the potential contact allergenicity of low molecular weight chemicals. However, governmental regulations and ethical considerations limit the use of animals for such purposes. There is therefore a need for in vitro alternative

  13. A comparative surface topographical analysis of explanted total knee replacement prostheses: Oxidised zirconium vs cobalt chromium femoral components.

    Science.gov (United States)

    Kennard, Emma; Scholes, Susan C; Sidaginamale, Raghavendra; Gangadharan, Rajkumar; Weir, David J; Holland, James; Deehan, David; Joyce, Thomas J

    2017-12-01

    It has been proposed that an increased surface roughness of the femoral components of Total Knee Replacements (TKRs) may be a contributing factor to the accelerated wear of the polyethylene (PE) bearing and ultimately prosthesis failure. Oxidised Zirconium was introduced to the orthopaedic market in an attempt to reduce PE wear associated failures and increase the longevity of the prosthesis. In this study, non-contacting profilometry was used to measure the surface roughness of the femoral components of 6 retrieved TKRs (3 Oxidised Zirconium (OxZr) and 3 Cobalt Chromium alloy (CoCr) femoral components) and 2 as-manufactured femoral components (1 OxZr and 1 CoCr). A semi-quantitative method was used to analyse the damage on the retrieved PE components. The Sa values for the retrieved OxZr femoral components (Sa = 0.093 µm ± 0.014) and for the retrieved CoCr femoral components (Sa = 0.065 µm ± 0.005) were significantly greater (p < .05) than the roughness values for the as-manufactured femoral components (OxZr Sa = 0.061 µm ± 0.004 and CoCr Sa = 0.042 µm ± 0.003). No significant difference was seen between the surface roughness parameters of the retrieved OxZr and retrieved CoCr femoral components. There was no difference between the PE component damage scores for the retrieved OxZr TKRs compared to the retrieved CoCr TKRs. These results agree with other studies that both OxZr and CoCr femoral components roughen during time in vivo but the lack of difference between the surface roughness measurements of the two materials is in contrast to previous topographical reports. Further analysis of retrieved OxZr TKRs is recommended so that a fuller appreciation of their benefits and limitations be obtained. Copyright © 2017 IPEM. All rights reserved.

  14. The effect of explant cut and cytokinin type on micropropagation of fig (Ficus carica L.) ‘Brown Turkey’

    Science.gov (United States)

    This study was conducted to micropropagate ‘Brown Turkey’ fig, one of the most popular cultivars in California, USA for fresh and dried uses. Nodal segments cut lengthwise or not were cultured on Woody Plant Medium containing 0.3, 1.0 or 3.0 µM of N-6 benzyladenine (BA) or kinetin in a 3 (concentra...

  15. Quantitative assessment of magnetic resonance derived myocardial perfusion measurements using advanced techniques: microsphere validation in an explanted pig heart system.

    Science.gov (United States)

    Schuster, Andreas; Zarinabad, Niloufar; Ishida, Masaki; Sinclair, Matthew; van den Wijngaard, Jeroen Phm; Morton, Geraint; Hautvast, Gilion Ltf; Bigalke, Boris; van Horssen, Pepijn; Smith, Nicolas; Spaan, Jos Ae; Siebes, Maria; Chiribiri, Amedeo; Nagel, Eike

    2014-10-14

    Cardiovascular Magnetic Resonance (CMR) myocardial perfusion imaging has the potential to evolve into a method allowing full quantification of myocardial blood flow (MBF) in clinical routine. Multiple quantification pathways have been proposed. However at present it remains unclear which algorithm is the most accurate. An isolated perfused, magnetic resonance (MR) compatible pig heart model allows very accurate titration of MBF and in combination with high-resolution assessment of fluorescently-labeled microspheres represents a near optimal platform for validation. We sought to investigate which algorithm is most suited to quantify myocardial perfusion by CMR at 1.5 and 3 Tesla using state of the art CMR perfusion techniques and quantification algorithms. First-pass perfusion CMR was performed in an MR compatible blood perfused pig heart model. We acquired perfusion images at physiological flow ("rest"), reduced flow ("ischaemia") and during adenosine-induced hyperaemia ("hyperaemia") as well as during coronary occlusion. Perfusion CMR was performed at 1.5 Tesla (n = 4 animals) and at 3 Tesla (n = 4 animals). Fluorescently-labeled microspheres and externally controlled coronary blood flow served as reference standards for comparison of different quantification strategies, namely Fermi function deconvolution (Fermi), autoregressive moving average modelling (ARMA), exponential basis deconvolution (Exponential) and B-spline basis deconvolution (B-spline). All CMR derived MBF estimates significantly correlated with microsphere results. The best correlation was achieved with Fermi function deconvolution both at 1.5 Tesla (r = 0.93, p < 0.001) and at 3 Tesla (r = 0.9, p < 0.001). Fermi correlated significantly better with the microspheres than all other methods at 3 Tesla (p < 0.002). B-spline performed worse than Fermi and Exponential at 1.5 Tesla and showed the weakest correlation to microspheres (r = 0.74, p < 0.001). All other comparisons were not significant. At 3 Tesla exponential deconvolution performed worst (r = 0.49, p < 0.001). CMR derived quantitative blood flow estimates correlate with true myocardial blood flow in a controlled animal model. Amongst the different techniques, Fermi function deconvolution was the most accurate technique at both field strengths. Perfusion CMR based on Fermi function deconvolution may therefore emerge as a useful clinical tool providing accurate quantitative blood flow assessment.

  16. Production of Gymnemic Acid Depends on Medium, Explants, PGRs, Color Lights, Temperature, Photoperiod, and Sucrose Sources in Batch Culture of Gymnema sylvestre

    Directory of Open Access Journals (Sweden)

    A. Bakrudeen Ali Ahmed

    2012-01-01

    Full Text Available Gymnema sylvestre (R.Br. is an important diabetic medicinal plant which yields pharmaceutically active compounds called gymnemic acid (GA. The present study describes callus induction and the subsequent batch culture optimization and GA quantification determined by linearity, precision, accuracy, and recovery. Best callus induction of GA was noticed in MS medium combined with 2,4-D (1.5 mg/L and KN (0.5 mg/L. Evaluation and isolation of GA from the calluses derived from different plant parts, namely, leaf, stem and petioles have been done in the present case for the first time. Factors such as light, temperature, sucrose, and photoperiod were studied to observe their effect on GA production. Temperature conditions completely inhibited GA production. Out of the different sucrose concentrations tested, the highest yield (35.4 mg/g d.w was found at 5% sucrose followed by 12 h photoperiod (26.86 mg/g d.w. Maximum GA production (58.28 mg/g d.w was observed in blue light. The results showed that physical and chemical factors greatly influence the production of GA in callus cultures of G. sylvestre. The factors optimized for in vitro production of GA during the present study can successfully be employed for their large-scale production in bioreactors.

  17. High frequency organogenesis in hypocotyl, cotyledon, leaf and petiole explants of broccoli (Brassica oleracea L. var. italica), an important vegetable crop

    National Research Council Canada - National Science Library

    Kumar, Pankaj; Srivastava, D K

    2015-01-01

    Broccoli (Brassica oleracea L. var. italica) is an important, nutritionally rich vegetable crop, but severely affected by environmental stresses, pests and diseases which cause massive yield and quality losses...

  18. The Effect of Divergence in Feed Efficiency on the Intestinal Microbiota and the Intestinal Immune Response in Both Unchallenged and Lipopolysaccharide Challenged Ileal and Colonic Explants.

    Directory of Open Access Journals (Sweden)

    Stafford Vigors

    Full Text Available Feed efficiency is an important trait in pig production, with evidence to suggest that the efficiencies of a variety of biological systems contribute to variation in this trait. Little work has been conducted on the contribution of the intestinal innate immune response to divergence in feed efficiency. Hence, the objective of this study was to examine select bacterial populations and gene expression profiles of a range of targets relating to gut health and immunity in the intestine of pigs phenotypically divergent in feed efficiency in: a the basal state; and (b following an ex-vivo lipopolysaccharide (LPS challenge of ileal and colonic tissue. Male pigs (initial BW 22.4 kg (SD = 2.03 were fed a standard finishing diet for the final 43 days prior to slaughter to evaluate feed intake and growth for the purpose of calculating residual feed intake (RFI. On day 115, 16 animals (average weight 85 kg, SEM 2.8 kg, designated high RFI (HRFI and low RFI (LRFI were slaughtered. The LRFI pigs had increased lactobacillus spp. in the caecum compared to HRFI pigs (P 0.10. Interestingly, there was an interaction between RFI and LPS for the cytokines IL-8, IL-1, IL-6, TNF-α, Interferon-γ (IFN-γ and SOCS3, with the LRFI group having consistently lower gene expression in the colon following the LPS challenge, compared to the HRFI group. The lower gene expression of SOCS and cytokines following an ex vivo LPS challenge supports the theory that a possible energy saving mechanism exists in the intestinal innate immune response to an immune challenge in more feed efficient pigs.

  19. The Effect of Divergence in Feed Efficiency on the Intestinal Microbiota and the Intestinal Immune Response in Both Unchallenged and Lipopolysaccharide Challenged Ileal and Colonic Explants.

    Science.gov (United States)

    Vigors, Stafford; O'Doherty, John V; Kelly, Alan K; O'Shea, Cormac J; Sweeney, Torres

    2016-01-01

    Feed efficiency is an important trait in pig production, with evidence to suggest that the efficiencies of a variety of biological systems contribute to variation in this trait. Little work has been conducted on the contribution of the intestinal innate immune response to divergence in feed efficiency. Hence, the objective of this study was to examine select bacterial populations and gene expression profiles of a range of targets relating to gut health and immunity in the intestine of pigs phenotypically divergent in feed efficiency in: a) the basal state; and (b) following an ex-vivo lipopolysaccharide (LPS) challenge of ileal and colonic tissue. Male pigs (initial BW 22.4 kg (SD = 2.03)) were fed a standard finishing diet for the final 43 days prior to slaughter to evaluate feed intake and growth for the purpose of calculating residual feed intake (RFI). On day 115, 16 animals (average weight 85 kg, SEM 2.8 kg), designated high RFI (HRFI) and low RFI (LRFI) were slaughtered. The LRFI pigs had increased lactobacillus spp. in the caecum compared to HRFI pigs (P 0.10). Interestingly, there was an interaction between RFI and LPS for the cytokines IL-8, IL-1, IL-6, TNF-α, Interferon-γ (IFN-γ) and SOCS3, with the LRFI group having consistently lower gene expression in the colon following the LPS challenge, compared to the HRFI group. The lower gene expression of SOCS and cytokines following an ex vivo LPS challenge supports the theory that a possible energy saving mechanism exists in the intestinal innate immune response to an immune challenge in more feed efficient pigs.

  20. A Simple Method for Establishing Adherent Ex Vivo Explant Cultures from Human Eye Pathologies for Use in Subsequent Calcium Imaging and Inflammatory Studies

    Science.gov (United States)

    Veréb, Zoltán; Facskó, Andrea; Hawlina, Marko

    2014-01-01

    A novel, simple, and reproducible method for cultivating pathological tissues obtained from human eyes during surgery was developed using viscoelastic material as a tissue adherent to facilitate cell attachment and expansion and calcium imaging of cultured cells challenged by mechanical and acetylcholine (ACh) stimulation as well as inflammatory studies. Anterior lens capsule-lens epithelial cells (aLC-LECs) from cataract surgery and proliferative diabetic retinopathy (PDR) fibrovascular epiretinal membranes (fvERMs) from human eyes were used in the study. We hereby show calcium signaling in aLC-LECs by mechanical and acetylcholine (ACh) stimulation and indicate presence of ACh receptors in these cells. Furthermore, an ex vivo study model was established for measuring the inflammatory response in fvERMs and aLC-LECs upon TNFα treatment. PMID:25276840