The Allosterically Unregulated Isoform of ADP-Glucose Pyrophosphorylase from Barley Endosperm Is the Most Likely Source of ADP-Glucose Incorporated into Endosperm Starch1

Science.gov (United States)

Doan, Danny N.P.; Rudi, Heidi; Olsen, Odd-Arne

1999-01-01

We present the results of studies of an unmodified version of the recombinant major barley (Hordeum vulgare) endosperm ADP-glucose pyrophoshorylase (AGPase) expressed in insect cells, which corroborate previous data that this isoform of the enzyme acts independently of the allosteric regulators 3-phosphoglycerate and inorganic phosphate. We also present a characterization of the individual subunits expressed separately in insect cells, showing that the SS AGPase is active in the presence of 3-phosphoglycerate and is inhibited by inorganic phosphate. As a step toward the elucidation of the role of the two AGPase isoforms in barley, the temporal and spatial expression profile of the four barley AGPase transcripts encoding these isoforms were studied. The results show that the steady-state level of beps and bepl, the transcripts encoding the major endosperm isoform, correlated positively with the rate of endosperm starch accumulation. In contrast, blps and blpl, the transcripts encoding the major leaf isoform, were constitutively expressed at a very low steady-state level throughout the barley plant. The implications of these findings for the evolution of plant AGPases are discussed. PMID:10557246

Comparison of starch granule development and physicochemical properties of starches in wheat pericarp and endosperm.

Science.gov (United States)

Yu, Xurun; Zhou, Liang; Zhang, Jing; Yu, Heng; Xiong, Fei; Wang, Zhong

2015-01-01

The objectives of this study were: (i) to characterize structural development of starch granule in pericarp and endosperm during wheat caryopsis growth; (ii) to compare physicochemical properties of starches in pericarp and endosperm; (iii) to further discover the relationships between pericarp starches and endosperm starches. Wheat pericarp and endosperm at different development stages were observed by light microscopy and scanning electron microscopy, respectively. Structural properties of starches were determined using X-ray power diffraction and (13) C solid nuclear magnetic resonance. Pericarp starch granules (PSG) accumulated in amyloplasts and chloroplasts, and showed a typical accumulation peak at 5 days after fertilization (DAF), and then gradually decomposed during 5-22 DAF. PSG in the abdominal region showed a higher rate of decomposition compared to the dorsal region of pericarp. Endosperm starch granules (ESG) accumulated in amyloplasts, and occurred in endosperm cells at 5 DAF, then rapidly enriched the endosperm cells until 22 DAF. Compared with ESG, PSG were compound granules of irregular shape and small size distribution. The results also suggested lower amylose content and V-type single-helix content and higher proportions of double helices for PSG compared to ESG. Based on the structural development of PSG and ESG, we speculated that the saccharides resulting from decomposition of PSG, on one hand, enabled the pericarp to survive before maturity of wheat caryopsis and, on the other hand, provided extra nutrition for the growth of ESG. © 2014 Society of Chemical Industry.

21 CFR 73.315 - Corn endosperm oil.

Science.gov (United States)

2010-04-01

... 21 Food and Drugs 1 2010-04-01 2010-04-01 false Corn endosperm oil. 73.315 Section 73.315 Food and... ADDITIVES EXEMPT FROM CERTIFICATION Foods § 73.315 Corn endosperm oil. (a) Identity. (1) The color additive corn endosperm oil is a reddish-brown liquid composed chiefly of glycerides, fatty acids, sitosterols...

Mannanase production by the lettuce endosperm : Control by the embryo.

Science.gov (United States)

Halmer, P; Bewley, J D

1979-01-01

Endo-β-mannanase (EC 3.2.1.78) is produced and secreted by the cells of the endosperm of lettuce (lactuca sativa L.) "seeds" (achenes). In imbibed intact seeds, production is prevented by inhibitors. If the endosperm is incubated alone, these inhibitors can be removed by leaching, allowing mannanase production. Abscisic acid, a component of lettuce seeds, inhibits the production of mannanase in the isolated endosperm, and may be involved in regulation of mannanase production in intact seeds. During germination the inhibition is removed, beginning 4-8 h after red-light irradiation, which was given 4 h from sowing. The cotyledons participate in this process, and are controlled by events occuring in the axis within 4 h from red-light irradiation. This control by the axis apparently depends on the exchange of diffusible substances. Both benzyladenine and gibberellic acid can replace the influence of the axis if the latter is removed, and may therefore be involved in the control by the axis of the rest of the seed.

Degradation of the endosperm cell walls of Lactuca sativa L., cv. grand rapids in relation to the mobilisation of proteins and the production of hydrolytic enzymes in the axis, cotyledons and endosperm.

Science.gov (United States)

Leung, D W; Reid, J S; Bewley, J D

1979-01-01

The timing of changes in total nitrogen and soluble amino nitrogen content, and in the activities of proteinase (pH 7.0), isocitrate lyase, catalase, phytase, phosphatase (pH 5.0), α-galactosidase and β-mannosidase were studied in extracts from the cotyledons, axis and endosperms of germinating and germinated light-promoted lettuce seeds. The largest amount of total nitrogen (2.7% seed dry weight) occurs within the cotyledons, as storage protein. As this decreases the total nitrogen content of the axis increases and the soluble amino nitrogen in the cotyledons and axis increases. Proteinase activity in the cotyledons increases coincidentally with the depletion of total nitrogen therein. Enzymes for phytate mobilisation and for gluconeogenesis of hydrolysed lipids increase in activity in the cotyledons as the appropriate stored reserves decline. Beta-mannosidase, an enzyme involved in the hydrolysis of oligo-mannans released by the action of endo-β-mannase on mannan reserves in the endosperm, arises within the cotyledons. This indicates that complete hydrolysis of mannans to the monomer does not occur within the endosperm. Mobilisation of all cotyledon reserves occurs after the endosperm has been degraded, providing further evidence that the endosperm is an early source of food reserves for the growing embryo.

Effect of high temperature on cell structure and gluten protein accumulation in the endosperm of the developing wheat (Triticum aestivum L.) grain

Science.gov (United States)

High temperature during grain fill is one of the more significant environmental factors that alters wheat yield and flour quality. To identify endosperm responses to high temperature, cell structure and gluten protein composition were investigated in developing wheat (Triticum aestivum L. cv. Butte ...

Brassica napus seed endosperm - metabolism and signaling in a dead end tissue.

Science.gov (United States)

Lorenz, Christin; Rolletschek, Hardy; Sunderhaus, Stephanie; Braun, Hans-Peter

2014-08-28

Oilseeds are an important element of human nutrition and of increasing significance for the production of industrial materials. The development of the seeds is based on a coordinated interplay of the embryo and its surrounding tissue, the endosperm. This study aims to give insights into the physiological role of endosperm for seed development in the oilseed crop Brassica napus. Using protein separation by two-dimensional (2D) isoelectric focusing (IEF)/SDS polyacrylamide gel electrophoresis (PAGE) and protein identification by mass spectrometry three proteome projects were carried out: (i) establishment of an endosperm proteome reference map, (ii) proteomic characterization of endosperm development and (iii) comparison of endosperm and embryo proteomes. The endosperm proteome reference map comprises 930 distinct proteins, including enzymes involved in genetic information processing, carbohydrate metabolism, environmental information processing, energy metabolism, cellular processes and amino acid metabolism. To investigate dynamic changes in protein abundance during seed development, total soluble proteins were extracted from embryo and endosperm fractions at defined time points. Proteins involved in sugar converting and recycling processes, ascorbate metabolism, amino acid biosynthesis and redox balancing were found to be of special importance for seed development in B. napus. Implications for the seed filling process and the function of the endosperm for seed development are discussed. The endosperm is of key importance for embryo development during seed formation in plants. We present a broad study for characterizing endosperm proteins in the oilseed plant B. napus. Furthermore, a project on the biochemical interplay between the embryo and the endosperm during seed development is presented. We provide evidence that the endosperm includes a complete set of enzymes necessary for plant primary metabolism. Combination of our results with metabolome data will further

Diurnal oscillation of SBE expression in sorghum endosperm

Energy Technology Data Exchange (ETDEWEB)

Sun, Chuanxin; Mutisya, J.; Rosenquist, S.; Baguma, Y.; Jansson, C.

2009-01-15

Spatial and temporal expression patterns of the sorghum SBEI, SBEIIA and SBEIIB genes, encoding, respectively, starch branching enzyme (SBE) I, IIA and IIB, in the developing endosperm of sorghum (Sorghum bicolor) were studied. Full-length genomic and cDNA clones for sorghum was cloned and the SBEIIA cDNA was used together with gene-specific probes for sorghum SBEIIB and SBEI. In contrast to sorghum SBEIIB, which was expressed primarily in endosperm and embryo, SBEIIA was expressed also in vegetative tissues. All three genes shared a similar temporal expression profile during endosperm development, with a maximum activity at 15-24 days after pollination. This is different from barley and maize where SBEI gene activity showed a significantly later onset compared to that of SBEIIA and SBEIIB. Expression of the three SBE genes in the sorghum endosperm exhibited a diurnal rhythm during a 24-h cycle.

The effect of high temperature on cell structure and gluten protein accumulation in the endosperm of the developing wheat (Triticum aestivum L.) grain

Science.gov (United States)

High temperature during grain fill is one of the more significant environmental factors that alters wheat yield and flour quality. To identify endosperm responses to high temperature, cell structure and gluten protein composition were investigated in developing wheat (Triticum aestivum L. cv. Butte ...

EFFECT OF ENDOSPERM HARDNESS ON AN ETHANOL PROCESS USING A GRANULAR STARCH HYDROLYZING ENZYME

Energy Technology Data Exchange (ETDEWEB)

Wang, P; W Liu, D B; Johnston, K D; Rausch, S J; Schmidt, M E; Tumbleson, V Singh

2010-01-01

Granular starch hydrolyzing enzymes (GSHE) can hydrolyze starch at low temperature (32°C). The dry grind process using GSHE (GSH process) has fewer unit operations and no changes in process conditions (pH 4.0 and 32°C) compared to the conventional process because it dispenses with the cooking and liquefaction step. In this study, the effects of endosperm hardness, protease, urea, and GSHE levels on GSH process were evaluated. Ground corn, soft endosperm, and hard endosperm were processed using two GSHE levels (0.1 and 0.4 mL per 100 g ground material) and four treatments of protease and urea addition. Soft and hard endosperm materials were obtained by grinding and sifting flaking grits from a dry milling pilot plant; classifications were confirmed using scanning electron microscopy. During 72 h of simultaneous granular starch hydrolysis and fermentation (GSHF), ethanol and glucose profiles were determined using HPLC. Soft endosperm resulted in higher final ethanol concentrations compared to ground corn or hard endosperm. Addition of urea increased final ethanol concentrations for soft and hard endosperm. Protease addition increased ethanol concentrations and fermentation rates for soft endosperm, hard endosperm, and ground corn. The effect of protease addition on ethanol concentrations and fermentation rates was most predominant for soft endosperm, less for hard endosperm, and least for ground corn. Samples (soft endosperm, hard endosperm, or corn) with protease resulted in higher (1.0% to 10.5% v/v) ethanol concentration compared to samples with urea. The GSH process with protease requires little or no urea addition. For fermentation of soft endosperm, GSHE dose can be reduced. Due to nutrients (lipids, minerals, and soluble proteins) present in corn that enhance yeast growth, ground corn fermented faster at the beginning than hard and soft endosperm.

FRAKSINASI ENZIM LIPASE DARI ENDOSPERM KELAPA DENGAN METODE SALTING OUT (Lipase fractionation of Coconut Endosperm by Salting out Method

Directory of Open Access Journals (Sweden)

Moh. Su'i

2014-02-01

Full Text Available This research learns about fractionation of lipases activity from coconut endosperm by using ammonium sulphate of 0–15%; 15-30 %, 30–45 %, 45–60 %, 60–75 % and 75–90 %. The results showed that the fractions of 0–15% ; 30–45 %, 45–60 % and 60–75 % have lipase activity. Meanwhile, the highest activity was fractions of 60-75%. fractions of 15-30% and 75-90%  have no lipase enzym activity. Molecule weigh of lipase enzyme was 72 kDa. Keywords: Lipases, endosperm, coconut, fractionation, ammonium sulphate   ABSTRAK Penelitian ini mempelajari fraksinasi enzim lipase dari endosperm kelapa menggunakan ammonium sulfat. fraksinasi dilakukan dengan variasi konsentrasi ammonium sulfat 0–15% ; 15-30%; 30–45 %, 45–60 %, 60–75 % dan 75–90 %. Hasil penelitian menunjukkan bahwa enzim lipase terdapat pada fraksi 0–15% ; 30–45 %, 45–60 % dan fraksi 60–75 % dengan aktivitas enzim tertinggi pada fraksi 60-75%. Sedangkan fraksi 15-30% dan 75-90% tidak ada enzim lipase. Berat molekul enzim lipase pada semua fraksi 72 kDa. Kata kunci: Lipase, endosperm, fraksinasi, ammonium sulfat

Rhinanthus serotinus (Schönheit) Oborny (Scrophulariaceae): immunohistochemical and ultrastructural studies of endosperm chalazal haustorium development.

Science.gov (United States)

Świerczyńska, Joanna; Kozieradzka-Kiszkurno, Małgorzata; Bohdanowicz, Jerzy

2013-12-01

Chalazal endosperm haustorium in Rhinanthus serotinus consists of a single large binucleate cell. It originates from the primary endosperm cell dividing transversely into two unequal cells: a smaller micropylar cell and a larger chalazal cell. The chalazal cell undergoes a single mitotic division, then lengthens significantly during development and functions as a chalazal endosperm haustorium. In this paper, immunofluorescent techniques, rhodamine phalloidin assay, and electron microscopy were used to examine the actin and tubulin cytoskeleton during the development of the chalazal haustorium. During the differentiation stage, numerous longitudinally oriented bundles of microfilaments ran along the axis of transvacuolar strands in haustorium. Microtubules formed intensely fluorescent areas near the nuclear envelope and also formed radial perinuclear microtubule arrays. In the fully differentiated haustorium cell, the actin cytoskeleton formed dense clusters of microfilaments on the chalazal and micropylar poles of the haustorium. Numerous microfilament bundles occurred near wall ingrowths on the chalazal wall. There were numerous clusters of microfilaments and microtubules around the huge lobed polytenic haustorial nuclei. The microfilaments were oriented longitudinally to the long axis of the haustorium cell and surrounded both nuclei. The microtubules formed radial perinuclear systems which were appeared to radiate from the surface of the nuclear envelope. The early stage of degeneration of the chalazal haustorium was accompanied by the degradation of microtubules and disruption of the parallel orientation of microtubules in the chalazal area of the cell. The degree of vacuolization increased, autophagous vacuoles appeared and the number of vesicles decreased.

Transcriptome Dynamics during Maize Endosperm Development.

Directory of Open Access Journals (Sweden)

Jianzhou Qu

Full Text Available The endosperm is a major organ of the seed that plays vital roles in determining seed weight and quality. However, genome-wide transcriptome patterns throughout maize endosperm development have not been comprehensively investigated to date. Accordingly, we performed a high-throughput RNA sequencing (RNA-seq analysis of the maize endosperm transcriptome at 5, 10, 15 and 20 days after pollination (DAP. We found that more than 11,000 protein-coding genes underwent alternative splicing (AS events during the four developmental stages studied. These genes were mainly involved in intracellular protein transport, signal transmission, cellular carbohydrate metabolism, cellular lipid metabolism, lipid biosynthesis, protein modification, histone modification, cellular amino acid metabolism, and DNA repair. Additionally, 7,633 genes, including 473 transcription factors (TFs, were differentially expressed among the four developmental stages. The differentially expressed TFs were from 50 families, including the bZIP, WRKY, GeBP and ARF families. Further analysis of the stage-specific TFs showed that binding, nucleus and ligand-dependent nuclear receptor activities might be important at 5 DAP, that immune responses, signalling, binding and lumen development are involved at 10 DAP, that protein metabolic processes and the cytoplasm might be important at 15 DAP, and that the responses to various stimuli are different at 20 DAP compared with the other developmental stages. This RNA-seq analysis provides novel, comprehensive insights into the transcriptome dynamics during early endosperm development in maize.

Mutagenic effects of endosperm of triticum aestivum implanted by heavy ion beams

International Nuclear Information System (INIS)

Xie Hongmei; Li Xinglin; Wei Zengquan; Xie Zhongkui

2004-01-01

75 MeV/u 16 O 8+ ions (degraded to 36 MeV/u) were used to implant into endosperm about 2.4 mm on top of the seeds. Germination started after a 'grafting' technique was employed. Chromosomal aberration frequency and micronucleus frequency of the root-tip cells in M 0 were measured. The results indicate that the frequencies were proportional to implanted dose. Antioxidant enzyme activity, MDA content and protein content of present generation M 0 were assayed. Farm culture was carried out in many generations. Short-stem and various variation of ear-type were obtained and the variation possess heredity. It showed that the endosperm implanted by the ions not only affected biological repair system, but also induced the mutation of offspring

Replication of DNA during barley endosperm development

DEFF Research Database (Denmark)

Giese, H.

1992-01-01

The incorporation of [6-H-3]-thymidine into DNA of developing barley end sperm was examined by autoradiography of cross sections of seeds and DNA analysis. The majority of nuclear divisions took place in the very young endosperm, but as late as 25 days after anthesis there was evidence for DNA...... replication. The DNA content of the endosperm increases during development and in response to nitrogen application in parallel to the storage protein synthesis profile. The hordein genes were hypersensitive to DNase I treatment throughout development....

Sugar uptake and starch biosynthesis by slices of developing maize endosperm

International Nuclear Information System (INIS)

Felker, F.C.; Liu, Kangchien; Shannon, J.C.

1990-01-01

14 C-Sugar uptake and incorporation into starch by slices of developing maize (Zea mays L.) endosperm were examined and compared with sugar uptake by maize endosperm-derived suspension cultures. Rates of sucrose, fructose, and D- and L-glucose uptake by slices were similar, whereas uptake rates for these sugars differed greatly in suspension cultures. Concentration dependence of sucrose, fructose, and D-glucose uptake was biphasic (consisting of linear plus saturable components) with suspension cultures but linear with slices. These and other differences suggest that endosperm slices are freely permeable to sugars. After diffusion into the slices, sugars were metabolized and incorporated into starch. Starch synthesis, but not sugar accumulation, was greatly reduced by 2.5 millimolar p-chloromercuribenzenesulfonic acid and 0.1 millimolar carbonyl cyanide m-chlorophenylhydrazone. Starch synthesis was dependent on kernel age and incubation temperature, but not on external pH (5 through 8). Competing sugars generally did not affect the distribution of 14 C among the soluble sugars extracted from endosperm slices incubated in 14 C-sugars. Competing hexoses reduced the incorporation of 14 C into starch, but competing sucrose did not, suggesting that sucrose is not a necessary intermediate in starch biosynthesis. The bidirectional permeability of endosperm slices to sugars makes the characterization of sugar transport into endosperm slices impossible, however the model system is useful for experiments dealing with starch biosynthesis which occurs in the metabolically active tissue

DNA endoreplication level in endosperm during seed development in three monocotyledonous species

Directory of Open Access Journals (Sweden)

Kazimierz Marciniak

2014-01-01

Full Text Available The DNA content after the Feulgen reaction in the endosperm of three monocotyledonous plant species (Asparagus officinalis, Muscari comosom, Haemanthus kurharinae differing in their 2C DNA content, was cytophotometrically measured. During endosperm development 1-6 endoreplication cycles take place, depending on the species. Differences in nuclear DNA endoreplication dynamics in the tested species are similar to those occurring in root parenchyma, but the endoreplication level in the endosperm is higher.

Circadian oscillation of starch branching enzyme gene expression in the sorghum endosperm

Energy Technology Data Exchange (ETDEWEB)

Mutisya, J.; Sun, C.; Jansson, C.

2009-08-31

Expression of the three SBE genes, encoding starch branching enzymes, in the sorghum endosperm exhibited a diurnal rhythm during a 24-h cycle. Remarkably, the oscillation in SBE expression was maintained in cultured spikes after a 48-h dark treatment, also when fed a continuous solution of sucrose or abscisic acid. Our findings suggest that the rhythmicity in SBE expression in the endosperm is independent of cues from the photosynthetic source and that the oscillator resides within the endosperm itself.

Effect of nitrogen fertilizer on distribution of starch granules in different regions of wheat endosperm

Directory of Open Access Journals (Sweden)

Fei Xiong

2014-02-01

Full Text Available This study provided visual evidence of a nitrogen effect on starch granules (SGs in wheat endosperm. Winter wheat (Titicum aestivum L. cultivar Xumai 30 was cultured under no nitrogen (control and 240 kg ha− 1 of nitrogen applied at the booting stage. The number, morphology, and size of A- and B-type SGs in subaleurone of dorsal endosperm (SDE, center of dorsal endosperm (CDE, modified aleurone (MA, subaleurone of ventral endosperm (SVE, and center of ventral endosperm (CVE were observed under light and electron microscopes. (1 The distribution of SGs in SDE was similar to that in SVE, the distributions of SGs in CDE and CVE were similar, but the distribution of SGs in MA was different from those in the other four endosperm regions. The number of SGs in the five endosperm regions was in the order SDE > CDE > SVE > CVE > MA. (2 Nitrogen increased the number of A- and B-type SGs in SDE and SVE. Nitrogen also increased the number of B-type SGs but decreased the number of A-type SGs in CDE and CVE. Nitrogen decreased the numbers of A-type and B-type SGs in MA. The results suggest that increased N fertilizer application mainly increased the numbers of small SGs and decreased the numbers of large SGs, but that the results varied in different regions of the wheat endosperm.

Endosperm imprinting: a child custody battle?

Science.gov (United States)

Becraft, Philip W

2012-02-07

Endosperm gene imprinting has long been speculated to control nutrient allocation to seeds. For the first time, an imprinted gene directly involved in this process has been identified. Copyright © 2012 Elsevier Ltd. All rights reserved.

Effect of endosperm mutants on maize seed germination

Directory of Open Access Journals (Sweden)

Pajić Zorica

2004-01-01

Full Text Available The expression of genetic potential of yielding and quality of a certain genotype depends among other factors on seed quality. Seed is very important not only for the reproduction of the particular plant species, but also, for the contemporary plant production. Each part of maize seed (pericarp endosperm and germ has a specific function in the complex process of germination and emergence. The following three genotypes of different endosperm types were observed: ZPSC 42A (standard grain quality dent hybrid ZPSC 504 su (sweet maize hybrid with a sugary gene and ZPSyn.II sh2 (synthetic population with a shranken2 gene. Seed viability of the stated genotypes was determined by the accepted ISTA methods: standard method accelerating age and cold test. Obtained results point out to differences in the germination capacity of the observed genotypes. The greatest reduction of the germination capacity and the emergence rate was expressed by the application of the accelerating ageing method. Appeared differences are probably a result of the endosperm texture (type, grain weight, sugar content and pericarp thickens and composition.

Effect of salicylhydroxamic acid on endosperm strength and embryo growth of Lactuca sativa L. cv Waldmann's Green seeds

Science.gov (United States)

Brooks, C. A.; Mitchell, C. A.

1988-01-01

Salicylhydroxamic acid (SHAM) stimulated germination of photosensitive lettuce (Lactuca sativa L. cv Waldmann's Green) seeds in darkness. To determine whether SHAM acts on the embryo or the endosperm, we investigated separately effects of SHAM on growth potential of isolated embryos as well as on endosperm strength. Embryo growth potential was quantified by incubating decoated embryos in various concentrations of osmoticum and measuring subsequent radicle elongation. Growth potential of embryos isolated from seeds pretreated with 4 millimolar SHAM was equal to that of untreated controls. Rupture strength of endosperm tissue excised from seeds pretreated with SHAM was 33% less than that of controls in the micropylar region. To determine if the embryo must be in contact with the endosperm of SHAM to weaken the endosperm, some endosperms were incubated with SHAM only after dissection from seeds. Rupture strength of SHAM-treated, isolated endosperms in the micropylar region was 25% less than that of untreated controls. There was no difference in rupture strength in the cotyledonary region of endosperm isolated from seeds treated with SHAM in buffer or buffer alone. SHAM therefore stimulates germination not by enhancing embryo growth potential, but by weakening the micropylar region of the endosperm enclosing the embryo.

Comparative metabolome analysis of wheat embryo and endosperm reveals the dynamic changes of metabolites during seed germination.

Science.gov (United States)

Han, Caixia; Zhen, Shoumin; Zhu, Gengrui; Bian, Yanwei; Yan, Yueming

2017-06-01

In this study, we performed the first comparative metabolomic analysis of the wheat embryo and endosperm during seed germination using GC-MS/MS. In total, 82 metabolites were identified in the embryo and endosperm. Principal component analysis (PCA), metabolite-metabolite correlation and hierarchical cluster analysis (HCA) revealed distinct dynamic changes in metabolites between the embryo and endosperm during seed germination. Generally, the metabolite changes in the embryo were much greater than those in the endosperm, suggesting that the embryo is more active than the endosperm during seed germination. Most amino acids were upregulated in both embryo and endosperm, while polysaccharides and organic acids associated with sugars were mainly downregulated in the embryo. Most of the sugars showed an upregulated trend in the endosperm, but significant changes in lipids occurred only in the embryo. Our results suggest that the embryo mobilises mainly protein and lipid metabolism, while the endosperm mobilises storage starch and minor protein metabolism during seed germination. The primary energy was generated mainly in the embryo by glycolysis during seed imbibition. The embryo containing most of the genetic information showed increased nucleotides during seed germination process, indicating more active transcription and translation metabolisms. Copyright © 2017 Elsevier Masson SAS. All rights reserved.

Abscisic acid and ethephon regulation of cellulase in the endosperm cap and radicle during lettuce seed germination.

Science.gov (United States)

Chen, Bingxian; Ma, Jun; Xu, Zhenjiang; Wang, Xiaofeng

2016-10-01

The purpose of this study was to investigate the role of cellulase in endosperm cap weakening and radicle elongation during lettuce (Lactuca sativa L.) seed germination. The application of abscisic acid (ABA) or ethephon inhibits or promotes germination, respectively, by affecting endosperm cap weakening and radicle elongation. Cellulase activities, and related protein and transcript abundances of two lettuce cellulase genes, LsCEL1 and LsCEL2, increase in the endosperm cap and radicle prior to radicle protrusion following imbibition in water. ABA or ethephon reduce or elevate, respectively, cellulase activity, and related protein and transcript abundances in the endosperm cap. Taken together, these observations suggest that cellulase plays a role in endosperm cap weakening and radicle elongation during lettuce seed germination, and that the regulation of cellulase in the endosperm cap by ABA and ethephon play a role in endosperm cap weakening. However, the influence of ABA and ethephon on radicle elongation may not be through their effects on cellulase. © 2016 Institute of Botany, Chinese Academy of Sciences.

Genetic analysis of vitreous endosperms derived from homozygotic plants for opaque-2 gene in maize (Zea mays L.)

International Nuclear Information System (INIS)

Prioli, A.J.; Barbosa, H.M.; Sant'Anna, R.

1980-01-01

From experiments in which opaque-2 maize seeds were treated with gamma rays and ethil methanesulfonate, and their respective untreated controls, seeds with hard, vitreous endosperms were obtained. Some of these were completely vitreous, with no evidence of opaque endosperm tissue. Others had very small and few (one to three) areas of opaque tissue. Plants derived from completely vitreous endosperm seeds were self pollinated and crossed to an opaque-2 inbred. The segregation of vitreous to opaque seeds indicated that the normal allele at the opaque-2 locus was responsible for the vitreousity of the endosperm. Lysine content of the vitreous endosperm was comparable to that of normal endosperms. Plants derived from vitreous seeds with few and tiny spots of opaque tissue produced, upon selfing or crossing to the opaque-2 inbred, only opaque-2 seeds. It is concluded that: (a) induced mutation may not be an effective tool to obtain vitreous opaque-2 endosperm with high lysine content; and, (b) there are unknown genetic systems which severely modify the expression of the opaque-2 gene. (Author) [pt

Disruption of endosperm development: an inbreeding effect in almond (Prunus dulcis).

Science.gov (United States)

Ortega, Encarnación; Martínez-García, Pedro J; Dicenta, Federico; Egea, José

2010-06-01

A homozygous self-compatible almond, originated from self-fertilization of a self-compatible genotype and producing a reasonable yield following open pollination, exhibited a very high fruit drop rate when self-pollinated. To investigate whether fruit dropping in this individual is related to an abnormal development of the embryo sac following self-fertilization, histological sections of ovaries from self and cross-pollinated flowers were observed by light microscopy. Additionally, the presence of pollen tubes in the ovary and fruit set were determined for both types of pollination. Despite pollen tubes reached the ovary after both pollinations, differences in embryo sac and endosperm development after fertilization were found. Thus, while for cross-fertilized ovules a pro-embryo and an endosperm with abundant nuclei were generally observed, most self-fertilized ovules remained in a previous developmental stage in which the embryo sac was not elongated and endosperm nuclei were absent. Although 30 days after pollination fruit set was similar for both pollination types, at 60 days it was significantly reduced for self-pollination. These results provide evidence that the high fruit drop in this genotype is the consequence of a disrupted development of the endosperm, what could be an expression of its high level of inbreeding.

Involvement of reactive oxygen species in endosperm cap weakening and embryo elongation growth during lettuce seed germination

Science.gov (United States)

Zhang, Yu; Chen, Bingxian; Xu, Zhenjiang; Shi, Zhaowan; Chen, Shanli; Huang, Xi; Chen, Jianxun; Wang, Xiaofeng

2014-01-01

Endosperm cap (CAP) weakening and embryo elongation growth are prerequisites for the completion of lettuce seed germination. Although it has been proposed that the cell wall loosening underlying these processes results from an enzymatic mechanism, it is still unclear which enzymes are involved. Here it is shown that reactive oxygen species (ROS), which are non-enzymatic factors, may be involved in the two processes. In Guasihong lettuce seeds imbibed in water, O2·– and H2O2 accumulated and peroxidase activity increased in the CAP, whereas its puncture force decreased. In addition, in the radicle, the increase in embryo growth potential was accompanied by accumulation of O2·– and an increase in peroxidase activity. Imbibing seeds in 0.3% sodium dichloroisocyanurate (SDIC) reduced endosperm viability and the levels of O2·–, H2O2, and peroxidase activity in the CAP, whereas the decrease in its puncture force was inhibited. However, in the embryo, SDIC did not affect the accumulation of O2·–, peroxidase activity, and the embryo growth potential. As a result, SDIC caused atypical germination, in which the endosperm ruptured at the boundary between the CAP and lateral endosperm. ROS scavengers and ROS generation inhibitors inhibited the CAP weakening and also decreased the embryo growth potential, thus decreasing the percentage of seed germination. Exogenous ROS and ROS generation inducers increased the percentage of CAP rupture to some extent, and the addition of H2O2 to 0.3% SDIC enabled some seeds to undergo typical germination. PMID:24744430

An Endosperm-Associated Cuticle Is Required for Arabidopsis Seed Viability, Dormancy and Early Control of Germination.

Directory of Open Access Journals (Sweden)

Julien De Giorgi

2015-12-01

Full Text Available Cuticular layers and seeds are prominent plant adaptations to terrestrial life that appeared early and late during plant evolution, respectively. The cuticle is a waterproof film covering plant aerial organs preventing excessive water loss and protecting against biotic and abiotic stresses. Cutin, consisting of crosslinked fatty acid monomers, is the most abundant and studied cuticular component. Seeds are dry, metabolically inert structures promoting plant dispersal by keeping the plant embryo in an arrested protected state. In Arabidopsis thaliana seeds, the embryo is surrounded by a single cell endosperm layer itself surrounded by a seed coat layer, the testa. Whole genome analyses lead us to identify cutin biosynthesis genes as regulatory targets of the phytohormones gibberellins (GA and abscisic acid (ABA signaling pathways that control seed germination. Cutin-containing layers are present in seed coats of numerous species, including Arabidopsis, where they regulate permeability to outer compounds. However, the role of cutin in mature seed physiology and germination remains poorly understood. Here we identify in mature seeds a thick cuticular film covering the entire outer surface of the endosperm. This seed cuticle is defective in cutin-deficient bodyguard1 seeds, which is associated with alterations in endospermic permeability. Furthermore, mutants affected in cutin biosynthesis display low seed dormancy and viability levels, which correlates with higher levels of seed lipid oxidative stress. Upon seed imbibition cutin biosynthesis genes are essential to prevent endosperm cellular expansion and testa rupture in response to low GA synthesis. Taken together, our findings suggest that in the course of land plant evolution cuticular structures were co-opted to achieve key physiological seed properties.

[Starch synthesis in the maize endosperm as affected by starch-synthesizing mutants]. [Annual report, March 1994--June 30, 1995

Energy Technology Data Exchange (ETDEWEB)

Nelson, O.

1995-07-01

Progress is reported in several areas relevant to maize endosperm development. These areas are (1) The tentative identification of the enzymatic deficiency in a previously unknown endosperm mutant, sugary3-1 (su3-1). The evidence leading to this conclusion will be presented below. (2) The recognition that the endosperm mutant that produces an interesting starch resembling some starches that have been chemically modified is actually an unusual, hypomorphic allele (8132) at the brittle2 (bt2) locus; (3) The orange endosperm color present in some progenies derived from a cross between the original bt2-8132 and W22N apparently results from an interaction between two genes, one of which behaves as though linked to the bt2 locus. In the orange endosperm derivative, our limited evidence suggests that the quantity of all the carotinoids present in the yellow endosperm stocks appear to be increased proportionally.

Purification and characterization of a serine protease (CESP) from mature coconut endosperm

Science.gov (United States)

Panicker, Leelamma M; Usha, Rajamma; Roy, Samir; Mandal, Chhabinath

2009-01-01

Background In plants, proteases execute an important role in the overall process of protein turnover during seed development, germination and senescence. The limited knowledge on the proteolytic machinery that operates during seed development in coconut (Cocos nucifera L.) prompted us to search for proteases in the coconut endosperm. Findings We have identified and purified a coconut endosperm protease (CESP) to apparent homogeneity. CESP is a single polypeptide enzyme of approximate molecular mass of 68 kDa and possesses pH optimum of 8.5 for the hydrolysis of BAPNA. Studies relating to substrate specificity and pattern of inhibition by various protease inhibitors indicated that CESP is a serine protease with cleavage specificity to peptide bonds after arginine. Purified CESP was often autolysed to two polypeptides of 41.6 kDa (CESP1) and 26.7 kDa (CESP2) and is confirmed by immunochemistry. We have shown the expression of CESP in all varieties of coconut and in all stages of coconut endosperm development with maximum amount in fully matured coconut. Conclusion Since the involvement of proteases in the processing of pre-proteins and maintenance of intracellular protein levels in seeds are well known, we suspect this CESP might play an important role in the coconut endosperm development. However this need to be confirmed using further studies. PMID:19426537

Changes in Nuclear Structure During Wheat Endosperm Development

NARCIS (Netherlands)

Wegel, E.

2005-01-01

This thesis is an investigation into the structure of wheat endosperm nuclei starting with nuclear divisions and migration during syncytium formation followed by the development of nuclear shape and positioning of chromosome territories and ending with changes in subchromosomal structure during the

Transport and metabolism of a sucrose analog (1'-fluorosucrose) into Zea mays L. Endosperm without invertase hydrolysis

International Nuclear Information System (INIS)

Schmalstig, J.G.; Hitz, W.D.

1987-01-01

1'-fluorosucrose (FS), a sucrose analog resistant to hydrolysis by invertase, was transported from husk leaves into maize (Zea mays L.) kernels with the same magnitude and kinetics as sucrose. 14 C-Label from [ 14 C]FS and [ 14 C]sucrose in separate experiments was distributed similarly between the pedicel, endosperm, and embryo with time. FS passed through maternal tissue and was adsorbed intact into the endosperm where it was metabolized and used in synthesis of sucrose and methanol-chloroform-water insolubles. Accumulation of [ 14 C]sucrose from supplied [ 14 C]glucosyl-FS indicated that the glucose moiety from the breakdown of sucrose (here FS), which normally occurs in the process of starch synthesis in maize endosperm, was available to the pool of substrates for resynthesis of sucrose. Uptake of FS into maize endosperm without hydrolysis suggest that despite the presence of invertase in maternal tissues and the hydrolysis of a large percentage of sucrose unloaded form the phloem, hexoses are not specifically needed for uptake into maize endosperm

Map-Based Cloning of Seed Dormancy1-2 Identified a Gibberellin Synthesis Gene Regulating the Development of Endosperm-Imposed Dormancy in Rice.

Science.gov (United States)

Ye, Heng; Feng, Jiuhuan; Zhang, Lihua; Zhang, Jinfeng; Mispan, Muhamad S; Cao, Zhuanqin; Beighley, Donn H; Yang, Jianchang; Gu, Xing-You

2015-11-01

Natural variation in seed dormancy is controlled by multiple genes mapped as quantitative trait loci in major crop or model plants. This research aimed to clone and characterize the Seed Dormancy1-2 (qSD1-2) locus associated with endosperm-imposed dormancy and plant height in rice (Oryza sativa). qSD1-2 was delimited to a 20-kb region, which contains OsGA20ox2 and had an additive effect on germination. Naturally occurring or induced loss-of-function mutations of the gibberellin (GA) synthesis gene enhanced seed dormancy and also reduced plant height. Expression of this gene in seeds (including endospermic cells) during early development increased GA accumulation to promote tissue morphogenesis and maturation programs. The mutant allele prevalent in semidwarf cultivars reduced the seed GA content by up to 2-fold at the early stage, which decelerated tissue morphogenesis including endosperm cell differentiation, delayed abscisic acid accumulation by a shift in the temporal distribution pattern, and postponed dehydration, physiological maturity, and germinability development. As the endosperm of developing seeds dominates the moisture equilibrium and desiccation status of the embryo in cereal crops, qSD1-2 is proposed to control primary dormancy by a GA-regulated dehydration mechanism. Allelic distribution of OsGA20ox2, the rice Green Revolution gene, was associated with the indica and japonica subspeciation. However, this research provided no evidence that the primitive indica- and common japonica-specific alleles at the presumably domestication-related locus functionally differentiate in plant height and seed dormancy. Thus, the evolutionary mechanism of this agriculturally important gene remains open for discussion. © 2015 American Society of Plant Biologists. All Rights Reserved.

The effects of calcium regulation of endosperm reserve protein ...

African Journals Online (AJOL)

Administrator

2011-06-15

Jun 15, 2011 ... on barley endosperm protein mobilization during malting. Although, the site and ... fractionating head of the digesting vigreux column. The digest was ... growth and enormous reductions in malting loss (Ezeogu and Okolo ...

Disruption of endosperm development is a major cause of hybrid seed inviability between Mimulus guttatus and Mimulus nudatus.

Science.gov (United States)

Oneal, Elen; Willis, John H; Franks, Robert G

2016-05-01

Divergence of developmental mechanisms within populations could lead to hybrid developmental failure, and might be a factor driving speciation in angiosperms. We investigate patterns of endosperm and embryo development in Mimulus guttatus and the closely related, serpentine endemic Mimulus nudatus, and compare them to those of reciprocal hybrid seed. We address whether disruption in hybrid seed development is the primary source of reproductive isolation between these sympatric taxa. M. guttatus and M. nudatus differ in the pattern and timing of endosperm and embryo development. Some hybrid seeds exhibit early disruption of endosperm development and are completely inviable, while others develop relatively normally at first, but later exhibit impaired endosperm proliferation and low germination success. These developmental patterns are reflected in mature hybrid seeds, which are either small and flat (indicating little to no endosperm) or shriveled (indicating reduced endosperm volume). Hybrid seed inviability forms a potent reproductive barrier between M. guttatus and M. nudatus. We shed light on the extent of developmental variation between closely related species within the M. guttatus species complex, an important ecological model system, and provide a partial mechanism for the hybrid barrier between M. guttatus and M. nudatus. © 2016 The Authors. New Phytologist © 2016 New Phytologist Trust.

Map-Based Cloning of Seed Dormancy1-2 Identified a Gibberellin Synthesis Gene Regulating the Development of Endosperm-Imposed Dormancy in Rice1

Science.gov (United States)

Ye, Heng; Feng, Jiuhuan; Zhang, Lihua; Zhang, Jinfeng; Mispan, Muhamad S.; Cao, Zhuanqin; Beighley, Donn H.; Yang, Jianchang; Gu, Xing-You

2015-01-01

Natural variation in seed dormancy is controlled by multiple genes mapped as quantitative trait loci in major crop or model plants. This research aimed to clone and characterize the Seed Dormancy1-2 (qSD1-2) locus associated with endosperm-imposed dormancy and plant height in rice (Oryza sativa). qSD1-2 was delimited to a 20-kb region, which contains OsGA20ox2 and had an additive effect on germination. Naturally occurring or induced loss-of-function mutations of the gibberellin (GA) synthesis gene enhanced seed dormancy and also reduced plant height. Expression of this gene in seeds (including endospermic cells) during early development increased GA accumulation to promote tissue morphogenesis and maturation programs. The mutant allele prevalent in semidwarf cultivars reduced the seed GA content by up to 2-fold at the early stage, which decelerated tissue morphogenesis including endosperm cell differentiation, delayed abscisic acid accumulation by a shift in the temporal distribution pattern, and postponed dehydration, physiological maturity, and germinability development. As the endosperm of developing seeds dominates the moisture equilibrium and desiccation status of the embryo in cereal crops, qSD1-2 is proposed to control primary dormancy by a GA-regulated dehydration mechanism. Allelic distribution of OsGA20ox2, the rice Green Revolution gene, was associated with the indica and japonica subspeciation. However, this research provided no evidence that the primitive indica- and common japonica-specific alleles at the presumably domestication-related locus functionally differentiate in plant height and seed dormancy. Thus, the evolutionary mechanism of this agriculturally important gene remains open for discussion. PMID:26373662

Investigations on embryo and endosperm development in gamma-irradiated Capsicum annuum L. and Capsicum pendulum Willd. seeds

Energy Technology Data Exchange (ETDEWEB)

Ilieva, I; Molkhova, E [Akademiya na Selskostopanskite Nauki, Sofia (Bulgaria). Inst. po Genetika

1976-01-01

Investigations were carried out concerning the effect of ionizing rays on pepper embryo development and on the radiosensitivity of single phases of embryogenesis. A single gamma-irradiation was effected with doses 1000, 1500, 2000 and 2500 rad, 7 days after flower pollination, when the preembryo had two cells. As a result of irradiation a shortening of the suspensor was established as well as delayed development or even totally blocked growth and degeneration of the embryo. Blocked cell division and degeneration of endospermal cells were observed. These disturbances lead to histologic changes in the seeds and to their non-viability.

Investigations on embryo and endosperm development in gamma-irradiated Capsicum annuum L. and Capsicum pendulum Willd. seeds

International Nuclear Information System (INIS)

Ilieva, I.; Molkhova, E.

1976-01-01

Investigations were carried out concerning the effect of ionizing rays on pepper embryo development and on the radiosensitivity of single phases of embryogenesis. A single gamma-irradiation was effected with doses 1000, 1500, 2000 and 2500 rad, 7 days after flower pollination, when the preembryo had two cells. As a result of irradiation a shortening of the suspensor was established as well as delayed development or even totally blocked growth and degeneration of the embryo. Blocked cell division and degeneration of endospermal cells were observed. These disturbances lead to histologic changes in the seeds and to their non-viability. (author)

Microwave fixation enhances gluten fibril formation in wheat endosperm

Science.gov (United States)

The wheat storage proteins, primarily glutenin and gliadin, contribute unique functional properties in food products and play a critical role in determining the end-use quality of wheat. In the wheat endosperm these proteins form a proteinaceous matrix deposited among starch granules only to be brou...

The effects of calcium regulation of endosperm reserve protein ...

African Journals Online (AJOL)

The effects of steep liquor calcium ion on sorghum endosperm reserve protein mobilization were evaluated using two improved Nigeria sorghum cultivars (ICSV 400 and KSV 8). The key protein modification factors evaluated were free amino nitrogen (FAN), total non protein nitrogen (TNPN) and soluble protein of cold water ...

Induction and multiplication of callus from endosperm of Cycas ...

African Journals Online (AJOL)

The usage of medicinal plants in traditional medication has gained the attraction from global and local markets, mainly to cure diseases or simply for health maintenance. Callus cultures were initiated from the endosperm of the medicinal plant Cycas revoluta, cultured on half-strength Murashige and Skoog (MS) medium ...

Modeling of the endosperm crush response profile of hard red spring wheat using a single kernel characterization system

Science.gov (United States)

When a wheat endosperm is crushed the force profile shows viscoelastic response and the modulus of elasticity is an important parameter that might have substantial influence on wheat milling. An experiment was performed to model endosperm crush response profile (ECRP) and to determine the modulus o...

Engineering the provitamin A (beta-carotene) biosynthetic pathway into (carotenoid-free) rice endosperm.

Science.gov (United States)

Ye, X; Al-Babili, S; Klöti, A; Zhang, J; Lucca, P; Beyer, P; Potrykus, I

2000-01-14

Rice (Oryza sativa), a major staple food, is usually milled to remove the oil-rich aleurone layer that turns rancid upon storage, especially in tropical areas. The remaining edible part of rice grains, the endosperm, lacks several essential nutrients, such as provitamin A. Thus, predominant rice consumption promotes vitamin A deficiency, a serious public health problem in at least 26 countries, including highly populated areas of Asia, Africa, and Latin America. Recombinant DNA technology was used to improve its nutritional value in this respect. A combination of transgenes enabled biosynthesis of provitamin A in the endosperm.

Characterization of the imprinting and expression patterns of ZAG2 in maize endosperm and embryo

Directory of Open Access Journals (Sweden)

Chaoxian Liu

2015-02-01

Full Text Available ZAG2 has been identified as a maternally expressed imprinted gene in maize endosperm. Our study revealed that paternally inherited ZAG2 alleles were imprinted in maize endosperm and embryo at 14 days after pollination (DAP, and consistently imprinted in endosperm at 10, 12, 16, 18, 20, 22, 24, 26, and 28 DAP in reciprocal crosses between B73 and Mo17. ZAG2 alleles were also imprinted in reciprocal crosses between Zheng 58 and Chang 7-2 and between Huang C and 178. ZAG2 alleles exhibited differential imprinting in hybrids of 178 × Huang C and B73 × Mo17, while in other hybrids ZAG2 alleles exhibited binary imprinting. The tissue-specific expression pattern of ZAG2 showed that ZAG2 was expressed at a high level in immature ears, suggesting that ZAG2 plays important roles in not only kernel but ear development.

Normal and hetero-yellow endosperm grain sorghum as substitute ...

African Journals Online (AJOL)

housed in flat deck-type cages, 1,6 x 1 m, fitted with a self- feeder and an automatic water nipple. Temperatures in the ... adiabatic bomb calorimeter. Amino acid analyses, following acid hydrolysis in a .... the hetero-yellow endosperm type sorghum had the highest avarage daily gains (ADGs), whereas pigs fed the maize-.

Rapid Elimination of the Persistent Synergid through a Cell Fusion Mechanism

KAUST Repository

Maruyama, Daisuke; Volz, Ronny; Takeuchi, Hidenori; Mori, Toshiyuki; Igawa, Tomoko; Kurihara, Daisuke; Kawashima, Tomokazu; Ueda, Minako; Ito, Masaki; Umeda, Masaaki; Nishikawa, Shuhichi; Groß -Hardt, Rita; Higashiyama, Tetsuya

2015-01-01

the endosperm proliferation, preventing attractions of excess number of pollen tubes (polytubey). The synergid-endosperm fusion is induced by fertilization of the central cell, while the egg cell fertilization predominantly activates ethylene signaling

EFFECT OF PHYSIOLOGICAL AGE AND GROWTH REGULATORS ON CALLUS BROWNING OF COCONUT ENDOSPERM CULTURE IN VITRO

Directory of Open Access Journals (Sweden)

LAZARUS AGUS SUKAMTO

2011-01-01

Full Text Available The possibility of physiological age and growth regulators affecting callus browning ofcoconut endosperm was investigated. Solid endosperm explants of four coconut fruits fromsame brunches of two coconut cultivars “Samoan Dwarf ” were grown on modified Murashigeand Skoog (MS formula with addition of 10 mg l putresine, 2.50 g l activated charcoal (AC,1.70 g l phytagel, 0, 10 , 10 , 10 , 10 M 2,4-dichlorophenoxyacetic acid (2,4-D or 4-amino-3,5,6-trichloropicolinic acid (Picloram combined with 10 M 6-benzylaminopurine (BA.Callogenesis occurred on 98.83% of explants. Callus browning between different physiologicalages (antipodal and micropylar tissues of coconut endosperm at 9, 26 and 31 weeks of culture(WOC was significantly different, but not at 16 and 21 WOC. Auxins of 2,4-D and Picloramdid not affect significantly callus browning of endosperm cultures. Auxin doses at 10 , 10 , and10 M decreased significantly callus browning at 9 and 16 WOC, respectively, but at 10 Mbrowning was less significant compared to other doses at 21 WOC. Auxin dose at 10 M causedless significant browning compared to other doses at 31 WOC. The addition of BA decreasedsignificantly callus browning at 9 WOC, but did not affect callus browning thereafter.

Rapid Elimination of the Persistent Synergid through a Cell Fusion Mechanism

KAUST Repository

Maruyama, Daisuke

2015-05-01

In flowering plants, fertilization-dependent degeneration of the persistent synergid cell ensures one-on-one pairings of male and female gametes. Here, we report that the fusion of the persistent synergid cell and the endosperm selectively inactivates the persistent synergid cell in Arabidopsis thaliana. The synergid-endosperm fusion causes rapid dilution of pre-secreted pollen tube attractant in the persistent synergid cell and selective disorganization of the synergid nucleus during the endosperm proliferation, preventing attractions of excess number of pollen tubes (polytubey). The synergid-endosperm fusion is induced by fertilization of the central cell, while the egg cell fertilization predominantly activates ethylene signaling, an inducer of the synergid nuclear disorganization. Therefore, two female gametes (the egg and the central cell) control independent pathways yet coordinately accomplish the elimination of the persistent synergid cell by double fertilization. Two female gametes (the egg cell and the central cell) in flowering plants coordinately prevent attractions of excess number of pollen tubes via two mechanisms to inactivate persistent synergid cell. © 2015 Elsevier Inc.

An Integrated “Multi-Omics” Comparison of Embryo and Endosperm Tissue-Specific Features and Their Impact on Rice Seed Quality

Directory of Open Access Journals (Sweden)

Marc Galland

2017-11-01

Full Text Available Although rice is a key crop species, few studies have addressed both rice seed physiological and nutritional quality, especially at the tissue level. In this study, an exhaustive “multi-omics” dataset on the mature rice seed was obtained by combining transcriptomics, label-free shotgun proteomics and metabolomics from embryo and endosperm, independently. These high-throughput analyses provide a new insight on the tissue-specificity related to rice seed quality. Foremost, we pinpointed that extensive post-transcriptional regulations occur at the end of rice seed development such that the embryo proteome becomes much more diversified than the endosperm proteome. Secondly, we observed that survival in the dry state in each seed compartment depends on contrasted metabolic and enzymatic apparatus in the embryo and the endosperm, respectively. Thirdly, it was remarkable to identify two different sets of starch biosynthesis enzymes as well as seed storage proteins (glutelins in both embryo and endosperm consistently with the supernumerary embryo hypothesis origin of the endosperm. The presence of a putative new glutelin with a possible embryonic favored abundance is described here for the first time. Finally, we quantified the rate of mRNA translation into proteins. Consistently, the embryonic panel of protein translation initiation factors is much more diverse than that of the endosperm. This work emphasizes the value of tissue-specificity-centered “multi-omics” study in the seed to highlight new features even from well-characterized pathways. It paves the way for future studies of critical genetic determinants of rice seed physiological and nutritional quality.

The molecular biology and biochemistry of rice endosperm α-globulin

International Nuclear Information System (INIS)

Shorrosh, B.S.

1989-01-01

The author's first objective was to isolate a cDNA clone that encodes the rice endosperm α-globulin. Purified antibodies against a rice storage protein, α-globulin, were used to screen a λgt11 cDNA expression library constructed from immature rice seed endosperm. The cDNA insert of clone 4A1 (identified by antibody screening) was used as a probe to identify long cDNA inserts in the library. The deduced amino acid sequence of clone A3-12 cDNA insert (identified by cDNA screening) contained the amino acid sequences of three cyanogen bromide peptides fragment of α-globulin. The calculated molecular weight and amino acid composition of the deduced amino acid sequence were similar to the α-globulin protein. Northern blot analysis indicated that mRNA of one size, approximately 1.0 kb, is expressed. Southern genomic blot analysis revealed one band with EcoRI or Hind III digestion. Cell-free translation and immunoprecipitation showed that the initial translation product is approximately 2,000 daltons larger than the mature protein. The amino acid sequence of α-globulin revealed limited regions of similarities with wheat storage proteins. The author concludes that the cDNA insert in clone A3-12 contained the entire coding region of α-globulin protein and that α-globulin is encoded by a single gene. My second objective was to inhibit the degradation of α-globulin in the salt extract of rice flour. The salt extract of rice flour contained an acid protease whose optimal pH was 3 for 3 H-casein hydrolysis. A polypeptide with molecular weight of 20,000 was immunologically reactive with α-globulin antibodies and is produced by limited proteolysis in the extract. Pepstatin inhibited the proteolysis of 3H-casein and slowed the proteolysis of α-globulin

OsbZIP58, a basic leucine zipper transcription factor, regulates starch biosynthesis in rice endosperm.

Science.gov (United States)

Wang, Jie-Chen; Xu, Heng; Zhu, Ying; Liu, Qiao-Quan; Cai, Xiu-Ling

2013-08-01

Starch composition and the amount in endosperm, both of which contribute dramatically to seed yield, cooking quality, and taste in cereals, are determined by a series of complex biochemical reactions. However, the mechanism regulating starch biosynthesis in cereal seeds is not well understood. This study showed that OsbZIP58, a bZIP transcription factor, is a key transcriptional regulator controlling starch synthesis in rice endosperm. OsbZIP58 was expressed mainly in endosperm during active starch synthesis. osbzip58 null mutants displayed abnormal seed morphology with altered starch accumulation in the white belly region and decreased amounts of total starch and amylose. Moreover, osbzip58 had a higher proportion of short chains and a lower proportion of intermediate chains of amylopectin. Furthermore, OsbZIP58 was shown to bind directly to the promoters of six starch-synthesizing genes, OsAGPL3, Wx, OsSSIIa, SBE1, OsBEIIb, and ISA2, and to regulate their expression. These findings indicate that OsbZIP58 functions as a key regulator of starch synthesis in rice seeds and provide new insights into seed quality control.

Dissection of Molecular Mechanisms Regulating Protein Body Formation in Maize Endosperm - DE-FG03-95-ER20183

International Nuclear Information System (INIS)

Larkins, Brian A.

2003-01-01

Dissection of Molecular Mechanisms Regulating Protein Body Formation in Maize Endosperm - DE-FG03-95-ER20183 Final Technical Report and Patent Summary Dr. Brian A. Larkins, Department of Plant Sciences, University of Arizona, Tucson, AZ 85721 Endosperm texture is an important quality trait in maize, as it influences the shipping characteristics of the grain, its susceptibility to insects, the yield of grits from dry milling, energy costs during wet milling, and the baking and digestibility properties of the flour. There appears to be a causal relationship between kernel hardness and the formation of zein-containing protein bodies, as mutations affecting protein body number and structure are associated with a soft, starchy kernel. In this project we used a variety of approaches to better understand this relationship and investigate the molecular and biochemical changes associated with starchy endosperm mutants. We characterized the distribution of zein mRNAs on endosperm rough endoplasmic reticulum (RER) membranes and the interactions between zein proteins, as each of these could influence the structure of protein bodies. Based on in situ hybridization, mRNAs encoding the 22-kD alpha- and 27-kD gamma-zeins are randomly distributed on RER; hence, mRNA targeting does not appear to influence the formation of protein bodies. Investigation of the interactions between zein proteins (alpha, beta, gamma, delta) with the yeast two-hybrid system showed that interactions between the 19- and 22-alpha-zeins are relatively weak, although each of them interacted strongly with the 10-kD delta-zein. Strong interactions were detected between the alpha- and delta-zeins and the 16-kD gamma- and 15-kD beta-zeins; however, the 50-kD and 27-kD gamma-zeins did not interact detectably with the alpha- and delta-zein proteins. The NH2- and COOH-terminal domains of the 22-kD alpha-zein were found to interact most strongly with the 15-kD beta- and 16-kD gamma-zeins, suggesting the 16-kD and 15

Differential Synthesis in Vitro of Barley Aleurone and Starchy Endosperm Proteins

DEFF Research Database (Denmark)

Mundy, John; Hejgaard, Jørn; Hansen, Annette

1986-01-01

RNAs from isolated endosperm and aleurone tissues (developing and mature grain) and from cultured (germinating) aleurone layers treated with abscisic acid (ABA) and GA(3). B and C hordein polypeptides and the salt-soluble proteins beta-amylase, protein Z, protein C, the chymotrypsin inhibitors (CI-1 and 2...

High Temperature-Induced Expression of Rice α-Amylases in Developing Endosperm Produces Chalky Grains

Directory of Open Access Journals (Sweden)

Masaru Nakata

2017-12-01

Full Text Available Global warming impairs grain filling in rice and reduces starch accumulation in the endosperm, leading to chalky-appearing grains, which damages their market value. We found previously that high temperature-induced expression of starch-lytic α-amylases during ripening is crucial for grain chalkiness. Because the rice genome carries at least eight functional α-amylase genes, identification of the α-amylase(s that contribute most strongly to the production of chalky grains could accelerate efficient breeding. To identify α-amylase genes responsible for the production of chalky grains, we characterized the histological expression pattern of eight α-amylase genes and the influences of their overexpression on grain appearance and carbohydrate components through a series of experiments with transgenic rice plants. The promoter activity of most α-amylase genes was elevated to various extents at high temperature. Among them, the expression of Amy1A and Amy3C was induced in the internal, especially basal to dorsal, region of developing endosperm, whereas that of Amy3D was confined near the ventral aleurone. These regions coincided with the site of occurrence of chalkiness, which was in clear contrast to conventionally known expression patterns of the enzyme in the scutellum and aleurone during seed germination. Furthermore, overexpression of α-amylase genes, except for Amy3E, in developing endosperm produced various degrees of chalky grains without heat exposure, whereas that of Amy3E yielded normal translucent grains, as was the case in the vector control, even though Amy3E-overexpressing grains contained enhanced α-amylase activities. The weight of the chalky grains was decreased due to reduced amounts of starch, and microscopic observation of the chalky part of these grains revealed that their endosperm consisted of loosely packed round starch granules that had numerous pits on their surface, confirming the hydrolysis of the starch reserve by

The α-Amylase Induction in Endosperm during Rice Seed Germination Is Caused by Gibberellin Synthesized in Epithelium1

Science.gov (United States)

Kaneko, Miyuki; Itoh, Hironori; Ueguchi-Tanaka, Miyako; Ashikari, Motoyuki; Matsuoka, Makoto

2002-01-01

We recently isolated two genes (OsGA3ox1 and OsGA3ox2) from rice (Oryza sativa) encoding 3β-hydroxylase, which catalyzes the final step of active gibberellin (GA) biosynthesis (H. Itoh, M. Ueguchi-Tanaka, N. Sentoku, H. Kitano, M. Matsuoka, M. Kobayashi [2001] Proc Natl Acad Sci USA 98: 8909–8914). Using these cloned cDNAs, we analyzed the temporal and spatial expression patterns of the 3β-hydroxylase genes and also an α-amylase gene (RAmy1A) during rice seed germination to investigate the relationship between GA biosynthesis and α-amylase expression. Northern-blot analyses revealed that RAmy1A expression in the embryo occurs before the induction of 3β-hydroxylase expression, whereas in the endosperm, a high level of RAmy1A expression occurs 1 to 2 d after the peak of OsGA3ox2 expression and only in the absence of uniconazol. Based on the analysis of an OsGA3ox2 null mutant (d18-Akibare dwarf), we determined that 3β-hydroxylase produced by OsGA3ox2 is important for the induction of RAmy1A expression and that the OsGA3ox1 product is not essential for α-amylase induction. The expression of OsGA3ox2 was localized to the shoot region and epithelium of the embryo, strongly suggesting that active GA biosynthesis occurs in these two regions. The synthesis of active GA in the epithelium is important for α-amylase expression in the endosperm, because an embryonic mutant defective in shoot formation, but which developed epithelium cells, induced α-amylase expression in the endosperm, whereas a mutant defective in epithelium development did not. PMID:11950975

Correlation-maximizing surrogate gene space for visual mining of gene expression patterns in developing barley endosperm tissue

Directory of Open Access Journals (Sweden)

Usadel Björn

2007-05-01

Full Text Available Abstract Background Micro- and macroarray technologies help acquire thousands of gene expression patterns covering important biological processes during plant ontogeny. Particularly, faithful visualization methods are beneficial for revealing interesting gene expression patterns and functional relationships of coexpressed genes. Such screening helps to gain deeper insights into regulatory behavior and cellular responses, as will be discussed for expression data of developing barley endosperm tissue. For that purpose, high-throughput multidimensional scaling (HiT-MDS, a recent method for similarity-preserving data embedding, is substantially refined and used for (a assessing the quality and reliability of centroid gene expression patterns, and for (b derivation of functional relationships of coexpressed genes of endosperm tissue during barley grain development (0–26 days after flowering. Results Temporal expression profiles of 4824 genes at 14 time points are faithfully embedded into two-dimensional displays. Thereby, similar shapes of coexpressed genes get closely grouped by a correlation-based similarity measure. As a main result, by using power transformation of correlation terms, a characteristic cloud of points with bipolar sandglass shape is obtained that is inherently connected to expression patterns of pre-storage, intermediate and storage phase of endosperm development. Conclusion The new HiT-MDS-2 method helps to create global views of expression patterns and to validate centroids obtained from clustering programs. Furthermore, functional gene annotation for developing endosperm barley tissue is successfully mapped to the visualization, making easy localization of major centroids of enriched functional categories possible.

Non-reciprocal Interspecies Hybridization Barriers in the Capsella Genus Are Established in the Endosperm.

Directory of Open Access Journals (Sweden)

Carolin A Rebernig

2015-06-01

Full Text Available The transition to selfing in Capsella rubella accompanies its recent divergence from the ancestral outcrossing C. grandiflora species about 100,000 years ago. Whether the change in mating system was accompanied by the evolution of additional reproductive barriers that enforced species divergence remained unknown. Here, we show that C. rubella and C. grandiflora are reproductively separated by an endosperm-based, non-reciprocal postzygotic hybridization barrier. While hybridizations of C. rubella maternal plants with C. grandiflora pollen donors resulted in complete seed abortion caused by endosperm cellularization failure, the reciprocal hybridization resulted in the formation of small seeds with precociously cellularized endosperm. Strikingly, the transcriptomic response of both hybridizations mimicked respectively the response of paternal and maternal excess hybridizations in Arabidopsis thaliana, suggesting unbalanced genome strength causes hybridization failure in both species. These results provide strong support for the theory that crosses between plants of different mating systems will be unbalanced, with the outcrosser behaving like a plant of increased ploidy, evoking a response that resembles an interploidy-type seed failure. Seed incompatilibity of C. rubella pollinated by C. grandiflora followed the Bateson-Dobzhansky-Muller model, involving negative genetic interaction of multiple paternal C. grandiflora loci with at least one maternal C. rubella locus. Given that both species only recently diverged, our data suggest that a fast evolving mechanism underlies the post-zygotic hybridization barrier(s separating both species.

Dissection of Molecular Mechanisms Regulating Protein Body Formation in Maize Endosperm - DE-FG03-95-ER20183 B139

Energy Technology Data Exchange (ETDEWEB)

Brian A. Larkins

2003-03-21

Dissection of Molecular Mechanisms Regulating Protein Body Formation in Maize Endosperm - DE-FG03-95-ER20183 Final Technical Report and Patent Summary Dr. Brian A. Larkins, Department of Plant Sciences, University of Arizona, Tucson, AZ 85721 Endosperm texture is an important quality trait in maize, as it influences the shipping characteristics of the grain, its susceptibility to insects, the yield of grits from dry milling, energy costs during wet milling, and the baking and digestibility properties of the flour. There appears to be a causal relationship between kernel hardness and the formation of zein-containing protein bodies, as mutations affecting protein body number and structure are associated with a soft, starchy kernel. In this project we used a variety of approaches to better understand this relationship and investigate the molecular and biochemical changes associated with starchy endosperm mutants. We characterized the distribution of zein mRNAs on endosperm rough endoplasmic reticulum (RER) membranes and the interactions between zein proteins, as each of these could influence the structure of protein bodies. Based on in situ hybridization, mRNAs encoding the 22-kD alpha- and 27-kD gamma-zeins are randomly distributed on RER; hence, mRNA targeting does not appear to influence the formation of protein bodies. Investigation of the interactions between zein proteins (alpha, beta, gamma, delta) with the yeast two-hybrid system showed that interactions between the 19- and 22-alpha-zeins are relatively weak, although each of them interacted strongly with the 10-kD delta-zein. Strong interactions were detected between the alpha- and delta-zeins and the 16-kD gamma- and 15-kD beta-zeins; however, the 50-kD and 27-kD gamma-zeins did not interact detectably with the alpha- and delta-zein proteins. The NH2- and COOH-terminal domains of the 22-kD alpha-zein were found to interact most strongly with the 15-kD beta- and 16-kD gamma-zeins, suggesting the 16-kD and 15

Myrigalone A Inhibits Lepidium sativum Seed Germination by Interference with Gibberellin Metabolism and Apoplastic Superoxide Production Required for Embryo Extension Growth and Endosperm Rupture

Czech Academy of Sciences Publication Activity Database

Oracz, K.; Voegele, A.; Tarkowská, Danuše; Jacquemoud, D.; Turečková, Veronika; Urbanová, Terezie; Strnad, Miroslav; Sliwinska, E.; Leubner-Metzger, G.

2012-01-01

Roč. 53, č. 1 (2012), s. 81-95 ISSN 0032-0781 R&D Projects: GA AV ČR KAN200380801; GA MŠk ED0007/01/01; GA ČR GD522/08/H003 Keywords : Embryo cell extension growth * Endoreduplication * Endosperm rupture * Gibberellin metabolism * Lepidium sativum * Myrica gale * Phytotoxicity * Reactive oxygen species Subject RIV: EF - Botanics Impact factor: 4.134, year: 2012

Improved evidence-based genome-scale metabolic models for maize leaf, embryo, and endosperm

Energy Technology Data Exchange (ETDEWEB)

Seaver, Samuel M. D.; Bradbury, Louis M. T.; Frelin, Océane; Zarecki, Raphy; Ruppin, Eytan; Hanson, Andrew D.; Henry, Christopher S.

2015-03-10

There is a growing demand for genome-scale metabolic reconstructions for plants, fueled by the need to understand the metabolic basis of crop yield and by progress in genome and transcriptome sequencing. Methods are also required to enable the interpretation of plant transcriptome data to study how cellular metabolic activity varies under different growth conditions or even within different organs, tissues, and developmental stages. Such methods depend extensively on the accuracy with which genes have been mapped to the biochemical reactions in the plant metabolic pathways. Errors in these mappings lead to metabolic reconstructions with an inflated number of reactions and possible generation of unreliable metabolic phenotype predictions. Here we introduce a new evidence-based genome-scale metabolic reconstruction of maize, with significant improvements in the quality of the gene-reaction associations included within our model. We also present a new approach for applying our model to predict active metabolic genes based on transcriptome data. This method includes a minimal set of reactions associated with low expression genes to enable activity of a maximum number of reactions associated with high expression genes. We apply this method to construct an organ-specific model for the maize leaf, and tissue specific models for maize embryo and endosperm cells. We validate our models using fluxomics data for the endosperm and embryo, demonstrating an improved capacity of our models to fit the available fluxomics data. All models are publicly available via the DOE Systems Biology Knowledgebase and PlantSEED, and our new method is generally applicable for analysis transcript profiles from any plant, paving the way for further in silico studies with a wide variety of plant genomes.

[Calcium distribution in the central cell of lettuce (Lactuca sativa L.) before and after pollination].

Science.gov (United States)

Qiu, Yi Lan; Liu, Ru Shi; Ye, Lv; Tian, Hui

2008-02-01

Potassium antimonite precipitation was used to locate calcium in the central cell of lettuce (Lactuca sativa L.) before and after pollination. At 3d before anthesis, two polar nuclei of central cell separately located at two polarity of the cell, and few calcium precipitates (ppts) appeared in the polar nuclei and cytoplasm, but some ppts in its small vacuoles. At 2d before anthesis, two polar nuclei moved toward the middle of the cell and fused to form a secondary nucleus, and the ppts evidently increased in the nucleus and cytoplasm. At 1d before anthesis, secondary nucleus again moved toward micropylar end and located near the egg to prepare for fertilization. Calcium precipitates were mainly accumulated in the secondary nucleus. After pollination and before fertilization, the distribution of calcium ppts was similar to that before pollination. At 4h after pollination, the central cell was fertilized, and calcium ppts evidently increased in the cell and numerous were accumulated in its nucleus and cytoplasm. At 6h after pollination, the primary endosperm nucleus completed its first division and formed two dissociate endosperm nuclei, and still many calcium precipitates appeared in the nucleus and cytoplasm. With endosperm development, calcium ppts decreased in the endosperm cell. At 1d after emasculated and without pollination, the secondary nucleus of the cell still bordered on the egg and some calcium ppts appeared in the secondary nucleus. The results indicated that the temporal and spatial changes of calcium in the central cell may play an important physiological role during the development of the central cell and endosperm.

Structure and Composition of Protein Bodies from Wild-Type and High-Lysine Barley Endosperm

DEFF Research Database (Denmark)

Ingversen, J.

1975-01-01

Protein bodies were isolated from 13 and 28 day old endosperms of barley mutant 1508 and its wild type, Bomi barley. The fine structure of the isolated protein bodies was determined by electron microscopy, and the proteins present in the preparations characterized by amino-acid analysis and SDS......-polyacrylamidegel electrophoresis. Sections through pellets of isolated protein bodies from both the mutant and the wild type revealed protein body structures corresponding with those observed in sections through the intact starchy endosperms. The majority of the wild-type protein bodies was homogeneous spheres accompanied...... that the wild-type protein bodies contained large amounts of prolamines (the storage protein group which is soluble in 55 % isopropanol) and some glutelins (the storage proteins soluble in dilute alkali), whereas the mutant protein bodies have glutelin as the major component and little prolamines...

Phosphorylation of glyoxysomal malate synthase from castor oil seed endosperm and cucumber cotyledon

International Nuclear Information System (INIS)

Yang, Y.P; Randall, D.D.

1989-01-01

Glyoxysomal malate synthase (MS) was purified to apparent homogeneity from 3-d germinating castor oil seed endosperm by a relatively simple procedure including two sucrose density gradient centrifugations. Antibodies raised to the caster oil seed MS crossreacted with MS from cucumber cotyledon. MS was phosphorylated in both tissues in an MgATP dependent reaction. The phosphorylation pattern was similar for both enzymes and both enzymes were inhibited by NaF, NaMo, (NH 4 )SO 4 , glyoxylate and high concentration of MgCl 2 (60 mM), but was not inhibited by NaCl and malate. Further characterization of the phosphorylation of MS from castor oil seed endosperms showed that the 5S form of MS is the form which is labelled by 32 P. The addition of exogenous alkaline phosphatase to MS not only decreased enzyme activity, but could also dephosphorylate phospho-MS. The relationship between dephosphorylation of MS and the decrease of MS activity is currently under investigation

The MADS Box Genes ABS, SHP1, and SHP2 Are Essential for the Coordination of Cell Divisions in Ovule and Seed Coat Development and for Endosperm Formation in Arabidopsis thaliana.

Science.gov (United States)

Ehlers, Katrin; Bhide, Amey S; Tekleyohans, Dawit G; Wittkop, Benjamin; Snowdon, Rod J; Becker, Annette

2016-01-01

Seed formation is a pivotal process in plant reproduction and dispersal. It begins with megagametophyte development in the ovule, followed by fertilization and subsequently coordinated development of embryo, endosperm, and maternal seed coat. Two closely related MADS-box genes, SHATTERPROOF 1 and 2 (SHP1 and SHP2) are involved in specifying ovule integument identity in Arabidopsis thaliana. The MADS box gene ARABIDOPSIS BSISTER (ABS or TT16) is required, together with SEEDSTICK (STK) for the formation of endothelium, part of the seed coat and innermost tissue layer formed by the maternal plant. Little is known about the genetic interaction of SHP1 and SHP2 with ABS and the coordination of endosperm and seed coat development. In this work, mutant and expression analysis shed light on this aspect of concerted development. Triple tt16 shp1 shp2 mutants produce malformed seedlings, seed coat formation defects, fewer seeds, and mucilage reduction. While shp1 shp2 mutants fail to coordinate the timely development of ovules, tt16 mutants show less peripheral endosperm after fertilization. Failure in coordinated division of the innermost integument layer in early ovule stages leads to inner seed coat defects in tt16 and tt16 shp1 shp2 triple mutant seeds. An antagonistic action of ABS and SHP1/SHP2 is observed in inner seed coat layer formation. Expression analysis also indicates that ABS represses SHP1, SHP2, and FRUITFUL expression. Our work shows that the evolutionary conserved Bsister genes are required not only for endothelium but also for endosperm development and genetically interact with SHP1 and SHP2 in a partially antagonistic manner.

The MADS Box Genes ABS, SHP1, and SHP2 Are Essential for the Coordination of Cell Divisions in Ovule and Seed Coat Development and for Endosperm Formation in Arabidopsis thaliana.

Directory of Open Access Journals (Sweden)

Katrin Ehlers

Full Text Available Seed formation is a pivotal process in plant reproduction and dispersal. It begins with megagametophyte development in the ovule, followed by fertilization and subsequently coordinated development of embryo, endosperm, and maternal seed coat. Two closely related MADS-box genes, SHATTERPROOF 1 and 2 (SHP1 and SHP2 are involved in specifying ovule integument identity in Arabidopsis thaliana. The MADS box gene ARABIDOPSIS BSISTER (ABS or TT16 is required, together with SEEDSTICK (STK for the formation of endothelium, part of the seed coat and innermost tissue layer formed by the maternal plant. Little is known about the genetic interaction of SHP1 and SHP2 with ABS and the coordination of endosperm and seed coat development. In this work, mutant and expression analysis shed light on this aspect of concerted development. Triple tt16 shp1 shp2 mutants produce malformed seedlings, seed coat formation defects, fewer seeds, and mucilage reduction. While shp1 shp2 mutants fail to coordinate the timely development of ovules, tt16 mutants show less peripheral endosperm after fertilization. Failure in coordinated division of the innermost integument layer in early ovule stages leads to inner seed coat defects in tt16 and tt16 shp1 shp2 triple mutant seeds. An antagonistic action of ABS and SHP1/SHP2 is observed in inner seed coat layer formation. Expression analysis also indicates that ABS represses SHP1, SHP2, and FRUITFUL expression. Our work shows that the evolutionary conserved Bsister genes are required not only for endothelium but also for endosperm development and genetically interact with SHP1 and SHP2 in a partially antagonistic manner.

Development of marker-free transgenic Jatropha curcas producing curcin-deficient seeds through endosperm-specific RNAi-mediated gene silencing.

Science.gov (United States)

Gu, Keyu; Tian, Dongsheng; Mao, Huizhu; Wu, Lifang; Yin, Zhongchao

2015-10-08

Jatropha curcas L. is a potential biofuel plant and its seed oil is suitable for biodiesel production. Despite this promising application, jatropha seeds contain two major toxic components, namely phorbol esters and curcins. These compounds would reduce commercial value of seed cake and raise safety and environment concerns on jatropha plantation and processing. Curcins are Type I ribosome inactivating proteins. Several curcin genes have been identified in the jatropha genome. Among which, the Curcin 1 (C1) gene is identified to be specifically expressed in endosperm, whereas the Curcin 2A (C2A) is mainly expressed in young leaves. A marker-free RNAi construct carrying a β-estradiol-regulated Cre/loxP system and a C1 promoter-driven RNAi cassette for C1 gene was made and used to generate marker-free transgenic RNAi plants to specifically silence the C1 gene in the endosperm of J. curcas. Plants of transgenic line L1, derived from T0-1, carry two copies of marker-free RNAi cassette, whereas plants of L35, derived from T0-35, harbored one copy of marker-free RNAi cassette and three copies of closely linked and yet truncated Hpt genes. The C1 protein content in endosperm of L1 and L35 seeds was greatly reduced or undetectable, while the C2A proteins in young leaves of T0-1 and T0-35 plants were unaffected. In addition, the C1 mRNA transcripts were undetectable in the endosperm of T3 seeds of L1 and L35. The results demonstrated that the expression of the C1 gene was specifically down-regulated or silenced by the double-stranded RNA-mediated RNA interference generated from the RNAi cassette. The C1 promoter-driven RNAi cassette for the C1 gene in transgenic plants was functional and heritable. Both C1 transcripts and C1 proteins were greatly down-regulated or silenced in the endosperm of transgenic J. curcas. The marker-free transgenic plants and curcin-deficient seeds developed in this study provided a solution for the toxicity of curcins in jatropha seeds and

Myosin-Va-dependent cell-to-cell transfer of RNA from Schwann cells to axons.

Directory of Open Access Journals (Sweden)

José R Sotelo

Full Text Available To better understand the role of protein synthesis in axons, we have identified the source of a portion of axonal RNA. We show that proximal segments of transected sciatic nerves accumulate newly-synthesized RNA in axons. This RNA is synthesized in Schwann cells because the RNA was labeled in the complete absence of neuronal cell bodies both in vitro and in vivo. We also demonstrate that the transfer is prevented by disruption of actin and that it fails to occur in the absence of myosin-Va. Our results demonstrate cell-to-cell transfer of RNA and identify part of the mechanism required for transfer. The induction of cell-to-cell RNA transfer by injury suggests that interventions following injury or degeneration, particularly gene therapy, may be accomplished by applying them to nearby glial cells (or implanted stem cells at the site of injury to promote regeneration.

Myosin-Va-dependent cell-to-cell transfer of RNA from Schwann cells to axons.

Science.gov (United States)

Sotelo, José R; Canclini, Lucía; Kun, Alejandra; Sotelo-Silveira, José R; Xu, Lei; Wallrabe, Horst; Calliari, Aldo; Rosso, Gonzalo; Cal, Karina; Mercer, John A

2013-01-01

To better understand the role of protein synthesis in axons, we have identified the source of a portion of axonal RNA. We show that proximal segments of transected sciatic nerves accumulate newly-synthesized RNA in axons. This RNA is synthesized in Schwann cells because the RNA was labeled in the complete absence of neuronal cell bodies both in vitro and in vivo. We also demonstrate that the transfer is prevented by disruption of actin and that it fails to occur in the absence of myosin-Va. Our results demonstrate cell-to-cell transfer of RNA and identify part of the mechanism required for transfer. The induction of cell-to-cell RNA transfer by injury suggests that interventions following injury or degeneration, particularly gene therapy, may be accomplished by applying them to nearby glial cells (or implanted stem cells) at the site of injury to promote regeneration.

High-Throughput Sequencing of Small RNA Transcriptomes in Maize Kernel Identifies miRNAs Involved in Embryo and Endosperm Development.

Science.gov (United States)

Xing, Lijuan; Zhu, Ming; Zhang, Min; Li, Wenzong; Jiang, Haiyang; Zou, Junjie; Wang, Lei; Xu, Miaoyun

2017-12-14

Maize kernel development is a complex biological process that involves the temporal and spatial expression of many genes and fine gene regulation at a transcriptional and post-transcriptional level, and microRNAs (miRNAs) play vital roles during this process. To gain insight into miRNA-mediated regulation of maize kernel development, a deep-sequencing technique was used to investigate the dynamic expression of miRNAs in the embryo and endosperm at three developmental stages in B73. By miRNA transcriptomic analysis, we characterized 132 known miRNAs and six novel miRNAs in developing maize kernel, among which, 15 and 14 miRNAs were commonly differentially expressed between the embryo and endosperm at 9 days after pollination (DAP), 15 DAP and 20 DAP respectively. Conserved miRNA families such as miR159, miR160, miR166, miR390, miR319, miR528 and miR529 were highly expressed in developing embryos; miR164, miR171, miR393 and miR2118 were highly expressed in developing endosperm. Genes targeted by those highly expressed miRNAs were found to be largely related to a regulation category, including the transcription, macromolecule biosynthetic and metabolic process in the embryo as well as the vitamin biosynthetic and metabolic process in the endosperm. Quantitative reverse transcription-PCR (qRT-PCR) analysis showed that these miRNAs displayed a negative correlation with the levels of their corresponding target genes. Importantly, our findings revealed that members of the miR169 family were highly and dynamically expressed in the developing kernel, which will help to exploit new players functioning in maize kernel development.

Proteomic Analysis of the Endosperm Ontogeny of Jatropha curcas L. Seeds.

Science.gov (United States)

Shah, Mohibullah; Soares, Emanoella L; Carvalho, Paulo C; Soares, Arlete A; Domont, Gilberto B; Nogueira, Fábio C S; Campos, Francisco A P

2015-06-05

Seeds of Jatropha curcas L. represent a potential source of raw material for the production of biodiesel. However, this use is hampered by the lack of basic information on the biosynthetic pathways associated with synthesis of toxic diterpenes, fatty acids, and triacylglycerols, as well as the pattern of deposition of storage proteins during seed development. In this study, we performed an in-depth proteome analysis of the endosperm isolated from five developmental stages which resulted in the identification of 1517, 1256, 1033, 752, and 307 proteins, respectively, summing up 1760 different proteins. Proteins with similar label free quantitation expression pattern were grouped into five clusters. The biological significance of these identifications is discussed with special focus on the analysis of seed storage proteins, proteins involved in the metabolism of fatty acids, carbohydrates, toxic components and proteolytic processing. Although several enzymes belonging to the biosynthesis of diterpenoid precursors were identified, we were unable to find any terpene synthase/cyclase, indicating that the synthesis of phorbol esters, the main toxic diterpenes, does not occur in seeds. The strategy used enabled us to provide a first in depth proteome analysis of the developing endosperm of this biodiesel plant, providing an important glimpse into the enzymatic machinery devoted to the production of C and N sources to sustain seed development.

Effect of heat stress on the pattern of protein synthesis in wheat endosperm

International Nuclear Information System (INIS)

Inwood, W.; Bernardin, J.

1990-01-01

The exposure of detached wheat heads (T. aestivum L. cv Cheyenne) to elevated temperatures resulted not only in the induction of a typical set of high and low molecular weight heat shock proteins (hsps), but also in a differential effect on the synthesis of wheat storage proteins in endosperm tissue when monitored by SDS PAGE of 35 S-labeled polypeptides. The synthesis of hsps in the endosperm had a rapid onset, reached a maximum rate within the first 2 hours at 40 degree C, and then steadily decreased during the next four hours. When heads were returned to 25 degree C after 3 hours at 40 degree C, hsp synthesis did not cease abruptly, but gradually declined over the next several hours. High molecular weight glutenin protein synthesis was drastically reduced with the same time course as heat shock protein synthesis was induced at 40 degree C. Conversely, the synthesis of gliadin proteins remained at a high level at 40 degree C. The synthesis rates for glutenin and gliadin proteins remained at low and high levels, respectively, for as long as the elevated temperature was maintained up to 7 hours

AGL61 interacts with AGL80 and is required for central cell development in Arabidopsis.

Science.gov (United States)

Steffen, Joshua G; Kang, Il-Ho; Portereiko, Michael F; Lloyd, Alan; Drews, Gary N

2008-09-01

The central cell of the female gametophyte plays a role in pollen tube guidance and in regulating the initiation of endosperm development. Following fertilization, the central cell gives rise to the seed's endosperm, which nourishes the developing embryo within the seed. The molecular mechanisms controlling specification and differentiation of the central cell are poorly understood. We identified AGL61 in a screen for transcription factor genes expressed in the female gametophyte. AGL61 encodes a Type I MADS domain protein, which likely functions as a transcription factor. Consistent with this, an AGL61-green fluorescent protein fusion protein is localized to the nucleus. In the context of the ovule and seed, AGL61 is expressed exclusively in the central cell and early endosperm. agl61 female gametophytes are affected in the central cell specifically. The morphological defects include an overall reduction in size of the central cell and a reduced or absent central cell vacuole. When fertilized with wild-type pollen, agl61 central cells fail to give rise to endosperm. In addition, synergid- and antipodal-expressed genes are ectopically expressed in agl61 central cells. The expression pattern and mutant phenotype of AGL61 are similar to those of AGL80, suggesting that AGL61 may function as a heterodimer with AGL80 within the central cell; consistent with this, AGL61 and AGL80 interact in yeast two-hybrid assays. Together, these data suggest that AGL61 functions as a transcription factor and controls the expression of downstream genes during central cell development.

Bovine somatic cell nuclear transfer.

Science.gov (United States)

Ross, Pablo J; Cibelli, Jose B

2010-01-01

Somatic cell nuclear transfer (SCNT) is a technique by which the nucleus of a differentiated cell is introduced into an oocyte from which its genetic material has been removed by a process called enucleation. In mammals, the reconstructed embryo is artificially induced to initiate embryonic development (activation). The oocyte turns the somatic cell nucleus into an embryonic nucleus. This process is called nuclear reprogramming and involves an important change of cell fate, by which the somatic cell nucleus becomes capable of generating all the cell types required for the formation of a new individual, including extraembryonic tissues. Therefore, after transfer of a cloned embryo to a surrogate mother, an offspring genetically identical to the animal from which the somatic cells where isolated, is born. Cloning by nuclear transfer has potential applications in agriculture and biomedicine, but is limited by low efficiency. Cattle were the second mammalian species to be cloned after Dolly the sheep, and it is probably the most widely used species for SCNT experiments. This is, in part due to the high availability of bovine oocytes and the relatively higher efficiency levels usually obtained in cattle. Given the wide utilization of this species for cloning, several alternatives to this basic protocol can be found in the literature. Here we describe a basic protocol for bovine SCNT currently being used in our laboratory, which is amenable for the use of the nuclear transplantation technique for research or commercial purposes.

Generation of transgenic wheat (Triticum aestivum L.) accumulating heterologous endo-xylanase or ferulic acid esterase in the endosperm

DEFF Research Database (Denmark)

Harholt, Jesper; Bach, Inga Christensen; Lind Bouquin, Solveig

2010-01-01

Endo-xylanase (from Bacillus subtilis) or ferulic acid esterase (from Aspergillus niger) were expressed in wheat under the control of the endosperm-specific 1DX5 glutenin promoter. Constructs both with and without the endoplasmic reticulum retention signal (Lys-Asp-Glu-Leu) KDEL were used....... Extensive analysis of the cell walls showed a 10%-15% increase in arabinose to xylose ratio, a 50% increase in the proportion of water-extractable arabinoxylan, and a shift in the MW of the water-extractable arabinoxylan from being mainly larger than 85 kD to being between 2 and 85 kD. Ferulic acid esterase......-expressing grains were also shrivelled, and the seed weight was decreased by 20%-50%. No ferulic acid esterase activity could be detected in wild-type grains whereas ferulic acid esterase activity was detected in transgenic lines. The grain cell walls had 15%-40% increase in water-unextractable arabinoxylan...

Hormonal responses during early embryogenesis in maize.

Science.gov (United States)

Chen, Junyi; Lausser, Andreas; Dresselhaus, Thomas

2014-04-01

Plant hormones have been shown to regulate key processes during embryogenesis in the model plant Arabidopsis thaliana, but the mechanisms that determine the peculiar embryo pattern formation of monocots are largely unknown. Using the auxin and cytokinin response markers DR5 and TCSv2 (two-component system, cytokinin-responsive promoter version #2), as well as the auxin efflux carrier protein PIN1a (PINFORMED1a), we have studied the hormonal response during early embryogenesis (zygote towards transition stage) in the model and crop plant maize. Compared with the hormonal response in Arabidopsis, we found that detectable hormone activities inside the developing maize embryo appeared much later. Our observations indicate further an important role of auxin, PIN1a and cytokinin in endosperm formation shortly after fertilization. Apparent auxin signals within adaxial endosperm cells and cytokinin responses in the basal endosperm transfer layer as well as chalazal endosperm are characteristic for early seed development in maize. Moreover, auxin signalling in endosperm cells is likely to be involved in exogenous embryo patterning as auxin responses in the endosperm located around the embryo proper correlate with adaxial embryo differentiation and outgrowth. Overall, the comparison between Arabidopsis and maize hormone response and flux suggests intriguing mechanisms in monocots that are used to direct their embryo patterning, which is significantly different from that of eudicots.

Functional dissection of a napin gene promoter: identification of promoter elements required for embryo and endosperm-specific transcription.

Science.gov (United States)

Ellerström, M; Stålberg, K; Ezcurra, I; Rask, L

1996-12-01

The promoter region (-309 to +44) of the Brassica napus storage protein gene napA was studied in transgenic tobacco by successive 5' as well as internal deletions fused to the reporter gene GUS (beta-glucuronidase). The expression in the two main tissues of the seed, the endosperm and the embryo, was shown to be differentially regulated. This tissue-specific regulation within the seed was found to affect the developmental expression during seed development. The region between -309 to -152, which has a large effect on quantitative expression, was shown to harbour four elements regulating embryo and one regulating endosperm expression. This region also displayed enhancer activity. Deletion of eight bp from position -152 to position -144 totally abolished the activity of the napA promoter. This deletion disrupted a cis element with similarity to an ABA-responsive element (ABRE) overlapping with an E-box, demonstrating its crucial importance for quantitative expression. An internal deletion of the region -133 to -120, resulted in increased activity in both leaves and endosperm and a decreased activity in the embryo. Within this region, a cis element similar to the (CA)n element, found in other storage protein promoters, was identified. This suggest that the (CA)n element is important for conferring seed specificity by serving both as an activator and a repressor element.

Regenerative abilities of mesenchymal stem cells through mitochondrial transfer.

Science.gov (United States)

Paliwal, Swati; Chaudhuri, Rituparna; Agrawal, Anurag; Mohanty, Sujata

2018-03-30

The past decade has witnessed an upsurge in studies demonstrating mitochondrial transfer as one of the emerging mechanisms through which mesenchymal stem cells (MSCs) can regenerate and repair damaged cells or tissues. It has been found to play a critical role in healing several diseases related to brain injury, cardiac myopathies, muscle sepsis, lung disorders and acute respiratory disorders. Several studies have shown that various mechanisms are involved in mitochondrial transfer that includes tunnel tube formation, micro vesicle formation, gap junctions, cell fusion and others modes of transfer. Few studies have investigated the mechanisms that contribute to mitochondrial transfer, primarily comprising of signaling pathways involved in tunnel tube formation that facilitates tunnel tube formation for movement of mitochondria from one cell to another. Various stress signals such as release of damaged mitochondria, mtDNA and mitochondrial products along with elevated reactive oxygen species levels trigger the transfer of mitochondria from MSCs to recipient cells. However, extensive cell signaling pathways that lead to mitochondrial transfer from healthy cells are still under investigation and the changes that contribute to restoration of mitochondrial bioenergetics in recipient cells remain largely elusive. In this review, we have discussed the phenomenon of mitochondrial transfer from MSCs to neighboring stressed cells, and how this aids in cellular repair and regeneration of different organs such as lung, heart, eye, brain and kidney. The potential scope of mitochondrial transfer in providing novel therapeutic strategies for treatment of various pathophysiological conditions has also been discussed.

Influence of instrument rigidity and specimen geometry on calculations of compressive strength properties of wheat endosperm

Science.gov (United States)

Endosperm texture is one of the most important quality features in wheat that defines milling energy requirements and the suitability of flour or semolina for the various food products such as pan breads, crackers, cakes, and pastas. Rooted in low molecular weight proteins known as puroindolines a a...

Shielded cells transfer automation

International Nuclear Information System (INIS)

Fisher, J.J.

1984-01-01

Nuclear waste from shielded cells is removed, packaged, and transferred manually in many nuclear facilities. Radiation exposure is absorbed by operators during these operations and limited only through procedural controls. Technological advances in automation using robotics have allowed a production waste removal operation to be automated to reduce radiation exposure. The robotic system bags waste containers out of glove box and transfers them to a shielded container. Operators control the system outside the system work area via television cameras. 9 figures

PEMANFAATAN FRAKSI KAYA ASAM LAURAT HASIL HIDROLISIS DARI ENDOSPERM KELAPA MENGGUNAKAN LIPASE ENDOGENEUS SEBAGAI PENGAWET SUSU KEDELAI KEMASAN (Utilization of High Lauric Fraction that Produced from Coconut Endosperm Using Lipase Endogenous as Preservation of Soybean Milk Packaging

Directory of Open Access Journals (Sweden)

Moh. Su'i

2016-10-01

Full Text Available Results of previous studies show that the high lauric fraction isolated from coconut endosperm is able to inhibit pathogenic and non-pathogenic bacteria. This research aims to study the addition of high lauric fraction that hydrolysed of coconut endosperm of the storability of soy milk packaging. High lauric fraction isolated from coconut milk, then the fraction analized of the fatty acid composition with gas chromatography (GC and then used as a preservative soy milk. The fraction is added to the soy milk with concentrations of 0, 10, 15 and 20%, then stored for 3 days. Every day is observed until soy milk damaged. The results showed that the fraction isolated from coconut milk contains 50.45% lauric acid, 17.52% myristic acid, 7.02% palmitic acid, 6.46% capric acid, 5.52% caprylic acid, 5.12% linoleic acid, 1.89% oleic acid, and 0.11% caproic acid. The addition of lauric acid-rich fraction of 20% were able to preserve soy milk for 2 days with a total microbe 1.00 x 104 cfu/ml, free fatty acids 0.12 m mol/ml, pH 5.05 and a balanced aroma 4 (nice. Keywords: Coconut, lauric acid, soy milk, storage ABSTRAK Hasil penelitian sebelumnya menunjukkan bahwa fraksi kaya asam laurat hasil isolasi dari endosperm kelapa mampu menghambat bakteri patogen dan non patogen. Penelitian ini bertujuan mempelajari penambahan fraksi kaya asam laurat hasil hidrolisis dari endosperm kelapa terhadap daya simpan susu kedelai kemasan. Fraksi yang kaya asam laurat diisolasi dari santan kelapa kemudian fraksi tersebut diuji komposisi asam lemaknya menggunakan chromatografi gas (GC dan selanjutnya digunakan sebagai bahan pengawet susu kedelai. Fraksi kaya asam laurat ditambahkan ke dalam susu kedelai dengan konsentrasi 0, 10, 15 dan 20%, kemudian disimpan selama 3 hari. Setiap hari dilakukan pengamatan hingga susu mengalami kerusakan. Hasil penelitian menunjukkan bahwa fraksi hasil isolasi dari santan kelapa mengandung asam laurat 50,45%, asam miristat 17,52%, asam palmitat

Alpha-synuclein cell-to-cell transfer and seeding in grafted dopaminergic neurons in vivo.

Directory of Open Access Journals (Sweden)

Elodie Angot

Full Text Available Several people with Parkinson's disease have been treated with intrastriatal grafts of fetal dopaminergic neurons. Following autopsy, 10-22 years after surgery, some of the grafted neurons contained Lewy bodies similar to those observed in the host brain. Numerous studies have attempted to explain these findings in cell and animal models. In cell culture, α-synuclein has been found to transfer from one cell to another, via mechanisms that include exosomal transport and endocytosis, and in certain cases seed aggregation in the recipient cell. In animal models, transfer of α-synuclein from host brain cells to grafted neurons has been shown, but the reported frequency of the event has been relatively low and little is known about the underlying mechanisms as well as the fate of the transferred α-synuclein. We now demonstrate frequent transfer of α-synuclein from a rat brain engineered to overexpress human α-synuclein to grafted dopaminergic neurons. Further, we show that this model can be used to explore mechanisms underlying cell-to-cell transfer of α-synuclein. Thus, we present evidence both for the involvement of endocytosis in α-synuclein uptake in vivo, and for seeding of aggregation of endogenous α-synuclein in the recipient neuron by the transferred α-synuclein. Finally, we show that, at least in a subset of the studied cells, the transmitted α-synuclein is sensitive to proteinase K. Our new model system could be used to test compounds that inhibit cell-to-cell transfer of α-synuclein and therefore might retard progression of Parkinson neuropathology.

Cloning and functional expression of a cDNA encoding stearoyl-ACP Δ9-desaturase from the endosperm of coconut (Cocos nucifera L.).

Science.gov (United States)

Gao, Lingchao; Sun, Ruhao; Liang, Yuanxue; Zhang, Mengdan; Zheng, Yusheng; Li, Dongdong

2014-10-01

Coconut (Cocos nucifera L.) is an economically tropical fruit tree with special fatty acid compositions. The stearoyl-acyl carrier protein (ACP) desaturase (SAD) plays a key role in the properties of the majority of cellular glycerolipids. In this paper, a full-length cDNA of a stearoyl-acyl carrier protein desaturase, designated CocoFAD, was isolated from cDNA library prepared from the endosperm of coconut (C. nucifera L.). An 1176 bp cDNA from overlapped PCR products containing ORF encoding a 391-amino acid (aa) protein was obtained. The coded protein was virtually identical and shared the homology to other Δ9-desaturase plant sequences (greater than 80% as similarity to that of Elaeis guineensis Jacq). The real-time fluorescent quantitative PCR result indicated that the yield of CocoFAD was the highest in the endosperm of 8-month-old coconut and leaf, and the yield was reduced to 50% of the highest level in the endosperm of 15-month-old coconut. The coding region showed heterologous expression in strain INVSc1 of yeast (Saccharomyces cerevisiae). GC-MS analysis showed that the levels of palmitoleic acid (16:1) and oleic acid (18:1) were improved significantly; meanwhile stearic acid (18:0) was reduced. These results indicated that the plastidial Δ9 desaturase from the endosperm of coconut was involved in the biosynthesis of hexadecenoic acid and octadecenoic acid, which was similar with other plants. These results may be valuable for understanding the mechanism of fatty acid metabolism and the genetic improvement of CocoFAD gene in palm plants in the future. Copyright © 2014 Elsevier B.V. All rights reserved.

Effect of high temperature on grain filling period, yield, amylose content and activity of starch biosynthesis enzymes in endosperm of basmati rice.

Science.gov (United States)

Ahmed, Nisar; Tetlow, Ian J; Nawaz, Sehar; Iqbal, Ahsan; Mubin, Muhammad; Nawaz ul Rehman, Muhammad Shah; Butt, Aisha; Lightfoot, David A; Maekawa, Masahiko

2015-08-30

High temperature during grain filling affects yield, starch amylose content and activity of starch biosynthesis enzymes in basmati rice. To investigate the physiological mechanisms underpinning the effects of high temperature on rice grain, basmati rice was grown under two temperature conditions - 32 and 22 °C - during grain filling. High temperature decreased the grain filling period from 32 to 26 days, reducing yield by 6%, and caused a reduction in total starch (3.1%) and amylose content (22%). Measurable activities of key enzymes involved in sucrose to starch conversion, sucrose synthase, ADP-glucose pyrophosphorylase, starch phosphorylase and soluble starch synthase in endosperms developed at 32 °C were lower than those at 22 °C compared with similar ripening stage on an endosperm basis. In particular, granule-bound starch synthase (GBSS) activity was significantly lower than corresponding activity in endosperms developing at 22 °C during all developmental stages analyzed. Results suggest changes in amylose/amylopectin ratio observed in plants grown at 32 °C was attributable to a reduction in activity of GBSS, the sole enzyme responsible for amylose biosynthesis. © 2014 Society of Chemical Industry.

Do rice suspension-cultured cells treated with abscisic acid mimic developing seeds?

Science.gov (United States)

Matsuno, Koya; Fujimura, Tatsuhito

2015-08-01

Starch synthesis is activated in the endosperm during seed development and also in rice suspension cells cultured with abscisic acid. In the anticipation that the mechanisms of starch synthesis are similar between the endosperm and the suspension cells cultured with abscisic acid, expression of genes involved in starch synthesis was evaluated in the suspension cells after abscisic acid treatment. However, it was found that the regulatory mechanism of starch synthesis in the suspension cells cultured with abscisic acid was different from that in developing seeds. Expression analyses of genes involved in oil bodies, which accumulate in the embryo and aleurone layer, and seed storage proteins, which accumulate mainly in the endosperm, showed that the former were activated in the suspension cells cultured with abscisic acid, but the latter were not. Master regulators for embryogenesis, OsVP1 (homologue of AtABI3) and OsLFL1 (homologue of AtFUS3 or AtLFL2), were expressed in the suspension cells at levels comparable to those in the embryo. From these results, it is suggested that interactions between regulators and abscisic acid control the synthesis of phytic acid and oil bodies in the cultured cells and embryo. We suggest that the system of suspension cells cultured with abscisic acid helps to reveal the mechanisms of phytic acid and oil body synthesis in embryo.

Gene Transfer in Eukaryotic Cells Using Activated Dendrimers

Science.gov (United States)

Dennig, Jörg

Gene transfer into eukaryotic cells plays an important role in cell biology. Over the last 30 years a number of transfection methods have been developed to mediate gene transfer into eukaryotic cells. Classical methods include co-precipitation of DNA with calcium phosphate, charge-dependent precipitation of DNA with DEAE-dextran, electroporation of nucleic acids, and formation of transfection complexes between DNA and cationic liposomes. Gene transfer technologies based on activated PAMAM-dendrimers provide another class of transfection reagents. PAMAM-dendrimers are highly branched, spherical molecules. Activation of newly synthesized dendrimers involves hydrolytic removal of some of the branches, and results in a molecule with a higher degree of flexibility. Activated dendrimers assemble DNA into compact structures via charge interactions. Activated dendrimer - DNA complexes bind to the cell membrane of eukaryotic cells, and are transported into the cell by non-specific endocytosis. A structural model of the activated dendrimer - DNA complex and a potential mechanism for its uptake into cells will be discussed.

Regulation of FA and TAG biosynthesis pathway genes in endosperms and embryos of high and low oil content genotypes of Jatropha curcas L.

Science.gov (United States)

Sood, Archit; Chauhan, Rajinder Singh

2015-09-01

The rising demand for biofuels has raised concerns about selecting alternate and promising renewable energy crops which do not compete with food supply. Jatropha (Jatropha curcas L.), a non-edible energy crop of the family euphorbiaceae, has the potential of providing biodiesel feedstock due to the presence of high proportion of unsaturated fatty acids (75%) in seed oil which is mainly accumulated in endosperm and embryo. The molecular basis of seed oil biosynthesis machinery has been studied in J. curcas, however, what genetic differences contribute to differential oil biosynthesis and accumulation in genotypes varying for oil content is poorly understood. We investigated expression profile of 18 FA and TAG biosynthetic pathway genes in different developmental stages of embryo and endosperm from high (42%) and low (30%) oil content genotypes grown at two geographical locations. Most of the genes showed relatively higher expression in endosperms of high oil content genotype, whereas no significant difference was observed in endosperms versus embryos of low oil content genotype. The promoter regions of key genes from FA and TAG biosynthetic pathways as well as other genes implicated in oil accumulation were analyzed for regulatory elements and transcription factors specific to oil or lipid accumulation in plants such as Dof, CBF (LEC1), SORLIP, GATA and Skn-1_motif etc. Identification of key genes from oil biosynthesis and regulatory elements specific to oil deposition will be useful not only in dissecting the molecular basis of high oil content but also improving seed oil content through transgenic or molecular breeding approaches. Copyright © 2015 Elsevier Masson SAS. All rights reserved.

Blast cells transfer experimental hypersensitivity pneumonitis in guinea pigs

International Nuclear Information System (INIS)

Schuyler, M.; Cook, C.; Listrom, M.; Fengolio-Preiser, C.

1988-01-01

We previously demonstrated that experimental hypersensitivity pneumonitis (HP) can be transferred by lymph node cells (LNC) cultured in vitro with antigen. The purpose of this study was to identify the cells responsible for transfer and to determine if pulmonary cells can transfer HP. We cultured LNC from sensitized Strain 2 guinea pigs with a soluble extract of Micropolyspora faeni for 72 h, separated lymphoblasts from small lymphocytes, and transferred both subpopulations intravenously to syngeneic recipients. We also transferred irradiated lymphoblasts (1,500 rads), macrophage-depleted, lymphoblast-enriched populations, and pulmonary cells either without culture or after culture with M. faeni. Control animals received an equal volume of medium. All recipient animals were challenged intratracheally (i.t.) with M. faeni 48 h after the cell transfer, and they were killed 4 days after i.t. challenge. Randomly selected microscopic fields of the lung (250/animal) were judged to be normal or abnormal without knowledge of treatment. This measurement was reproducible (r = 0.95 for duplicate measurements, n = 55). All guinea pigs were maintained in HEPA-filtered air. There was a low level of pulmonary response to an i.t. challenge of M. faeni in animals that received medium. Animals that received pulmonary cells, either cultured or noncultured, did not differ from those in the control group. There was a substantial increase (p less than 0.01) in the extent of pulmonary abnormalities in the recipients of the lymphoblast population, with significant correlation (r = 0.87, p less than 0.01) between the number of lymphoblasts transferred and the extent of pulmonary abnormalities

Method of transferring regular shaped vessel into cell

International Nuclear Information System (INIS)

Murai, Tsunehiko.

1997-01-01

The present invention concerns a method of transferring regular shaped vessels from a non-contaminated area to a contaminated cell. A passage hole for allowing the regular shaped vessels to pass in the longitudinal direction is formed to a partitioning wall at the bottom of the contaminated cell. A plurality of regular shaped vessel are stacked in multiple stages in a vertical direction from the non-contaminated area present below the passage hole, allowed to pass while being urged and transferred successively into the contaminated cell. As a result, since they are transferred while substantially closing the passage hole by the regular shaped vessels, radiation rays or contaminated materials are prevented from discharging from the contaminated cell to the non-contaminated area. Since there is no requirement to open/close an isolation door frequently, the workability upon transfer can be improved remarkably. In addition, the sealing member for sealing the gap between the regular shaped vessel passing through the passage hole and the partitioning wall of the bottom is disposed to the passage hole, the contaminated materials in the contaminated cells can be prevented from discharging from the gap to the non-contaminated area. (N.H.)

Transfer of immunity by transfer of bone marrow cells: T-cell dependency

International Nuclear Information System (INIS)

Marusic, M.

1978-01-01

Thymectomized, lethally irradiated mice reconstituted with normal bone marrow cells succumbed when challenged ip with rat Yoshida ascites sarcoma (YAS) cells 40 days after irradiation and reconstitution. In contrast, thymectomized irradiated mice reconstituted with bone marrow cells from YAS-immune donors rejected the subsequent tumor challenge. Pretreatment of the bone marrow cells from immune donors with anti-Thy 1.2 antiserum and complement completely abolished the transfer of anti-YAS resistance. Bone marrow cells from donors thymectomized 2 months before immunization enabled almost all recipients to reject YAS, but bone marrow cells from donors thymectomized 8 months before immunization protected only 50 percent of the recipients. Further analysis showed that mice thymectomized 8 months before immunization failed to generate anti-YAS antibody response, whereas the antibody response of mice thymectomized 2 months before immunization did not differ from that of non-thymectomized age-matched control mice. The data suggest that the immune reaction of mice against xenogeneic YAS requires long-lived T 2 lymphocytes

Cell-printing and transfer technology applications for bone defects in mice.

Science.gov (United States)

Tsugawa, Junichi; Komaki, Motohiro; Yoshida, Tomoko; Nakahama, Ken-ichi; Amagasa, Teruo; Morita, Ikuo

2011-10-01

Bone regeneration therapy based on the delivery of osteogenic factors and/or cells has received a lot of attention in recent years since the discovery of pluripotent stem cells. We reported previously that the implantation of capillary networks engineered ex vivo by the use of cell-printing technology could improve blood perfusion. Here, we developed a new substrate prepared by coating glass with polyethylene glycol (PEG) to create a non-adhesive surface and subsequent photo-lithography to finely tune the adhesive property for efficient cell transfer. We examined the cell-transfer efficiency onto amniotic membrane and bone regenerative efficiency in murine calvarial bone defect. Cell transfer of KUSA-A1 cells (murine osteoblasts) to amniotic membrane was performed for 1 h using the substrates. Cell transfer using the substrate facilitated cell engraftment onto the amniotic membrane compared to that by direct cell inoculation. KUSA-A1 cells transferred onto the amniotic membrane were applied to critical-sized calvarial bone defects in mice. Micro-computed tomography (micro-CT) analysis showed rapid and effective bone formation by the cell-equipped amniotic membrane. These results indicate that the cell-printing and transfer technology used to create the cell-equipped amniotic membrane was beneficial for the cell delivery system. Our findings support the development of a biologically stable and effective bone regeneration therapy. Copyright © 2011 John Wiley & Sons, Ltd.

delta 6 Hexadecenoic acid is synthesized by the activity of a soluble delta 6 palmitoyl-acyl carrier protein desaturase in Thunbergia alata endosperm.

Science.gov (United States)

Cahoon, E B; Cranmer, A M; Shanklin, J; Ohlrogge, J B

1994-11-04

delta 6 Hexadecenoic acid (16:1 delta 6) composes more than 80% of the seed oil of Thunbergia alata. Studies were conducted to determine the biosynthetic origin of the double bond of this unusual fatty acid. Assays of fractions of developing T. alata seed endosperm with [1-14C]palmitoyl (16:0)-acyl carrier protein (ACP) revealed the presence of a soluble delta 6 desaturase activity. This activity was greatest when 16:0-ACP was provided as a substrate, whereas no desaturation of the coenzyme A ester of this fatty acid was detected. In addition, delta 6 16:0-ACP desaturase activity in T. alata endosperm extracts was dependent on the presence of ferredoxin and molecular oxygen and was stimulated by catalase. To further characterize this enzyme, a cDNA encoding a diverged acyl-ACP desaturase was isolated from a T. alata endosperm cDNA library using polymerase chain reaction with degenerate oligonucleotides corresponding to conserved amino acid sequences in delta 9 stearoyl (18:0)- and delta 4 16:0-ACP desaturases. The primary structure of the mature peptide encoded by this cDNA shares 66% identity with the mature castor delta 9 18:0-ACP desaturase and 57% identity with the mature coriander delta 4 16:0-ACP desaturase. Extracts of Escherichia coli that express the T. alata cDNA catalyzed the delta 6 desaturation of 16:0-ACP. These results demonstrate that 16:1 delta 6 in T. alata endosperm is formed by the activity of a soluble delta 6 16:0-ACP desaturase that is structurally related to the delta 9 18:0- and delta 4 16:0-ACP desaturases. Implications of this work to an understanding of active site structures of acyl-ACP desaturases are discussed.

Entwicklung transgener Gerste (Hordeum vulgare L.) mit dem Ziel der Lysin- und Threoninanreicherung im Endosperm

OpenAIRE

Ibrahim, Ahmed Shawky Ahmed

2006-01-01

An efficient Agrobacterium-mediated barley transformation system was established with a transformation rate of 13.4 % on average. Towards improving the nutritional value of barley, a set of novel transformation vectors was developed including the dapA and lysC genes encoding the feed-back-inhibition insensitive form of the dihydrodipicolinate synthase (DHDPS) and aspartate kinase (AK) respectively. Both genes under the control of the endosperm-specific D-hordein promoter or the constitutive u...

Evidence for Transfer of Membranes from Mesenchymal Stem Cells to HL-1 Cardiac Cells.

Science.gov (United States)

Boomsma, Robert A; Geenen, David L

2014-01-01

This study examined the interaction of mouse bone marrow mesenchymal stem cells (MSC) with cardiac HL-1 cells during coculture by fluorescent dye labeling and then flow cytometry. MSC were layered onto confluent HL-1 cell cultures in a 1 : 4 ratio. MSC gained gap junction permeant calcein from HL-1 cells after 4 hours which was partially reduced by oleamide. After 20 hours, 99% MSC gained calcein, unaffected by oleamide. Double-labeling HL-1 cells with calcein and the membrane dye DiO resulted in transfer of both calcein and DiO to MSC. When HL-1 cells were labeled with calcein and MSC with DiO, MSC gained calcein while HL-1 cells gained DiO. Very little fusion was observed since more than 90% Sca-1 positive MSC gained DiO from HL-1 cells while less than 9% gained gap junction impermeant CMFDA after 20 hours with no Sca-1 transfer to HL-1 cells. Time dependent transfer of membrane DiD was observed from HL-1 cells to MSC (100%) and vice versa (50%) after 20 hours with more limited transfer of CMFDA. These results demonstrate that MSC and HL-1 cells exchange membrane components which may account for some of the beneficial effect of MSC in the heart after myocardial infarction.

Human somatic cell nuclear transfer and cloning.

Science.gov (United States)

2012-10-01

This document presents arguments that conclude that it is unethical to use somatic cell nuclear transfer (SCNT) for infertility treatment due to concerns about safety; the unknown impact of SCNT on children, families, and society; and the availability of other ethically acceptable means of assisted reproduction. This document replaces the ASRM Ethics Committee report titled, "Human somatic cell nuclear transfer (cloning)," last published in Fertil Steril 2000;74:873-6. Copyright © 2012 American Society for Reproductive Medicine. Published by Elsevier Inc. All rights reserved.

Stem cell collection and gene transfer in Fanconi anemia.

Science.gov (United States)

Kelly, Patrick F; Radtke, Susan; von Kalle, Christof; Balcik, Brenden; Bohn, Kimberley; Mueller, Robin; Schuesler, Todd; Haren, Moira; Reeves, Lilith; Cancelas, Jose A; Leemhuis, Thomas; Harris, Richard; Auerbach, Arleen D; Smith, Franklin O; Davies, Stella M; Williams, David A

2007-01-01

Fanconi anemia (FA) is a rare genetic syndrome characterized by progressive bone marrow failure (BMF), congenital anomalies, and a predisposition to malignancy. Successful gene transfer into hematopoietic stem cells (HSCs) could reverse BMF in this disease. We developed clinical trials to determine whether a sufficient number of CD34(+) stem cells could be collected for gene modification and to evaluate the safety and efficacy of HSC-corrective gene transfer in FA genotype A (FANCA) patients. Here, we report that FA patients have significant depletion of their BM CD34(+) cell compartment even before severe pancytopenia is present. However, oncoretroviral-mediated ex vivo gene transfer was efficient in clinical scale in FA-A cells, leading to reversal of the cellular phenotype in a significant percentage of CD34(+) cells. Re-infusion of gene-corrected products in two patients was safe and well tolerated and accompanied by transient improvements in hemoglobin and platelet counts. Gene correction was transient, likely owing to the low dose of gene-corrected cells infused. Our early experience shows that stem cell collection is well tolerated in FA patients and suggests that collection be considered as early as possible in patients who are potential candidates for future gene transfer trials.

Analysis of the arabinoxylan arabinofuranohydrolase gene family in barley does not support their involvement in the remodelling of endosperm cell walls during development.

Science.gov (United States)

Laidlaw, Hunter K C; Lahnstein, Jelle; Burton, Rachel A; Fincher, Geoffrey B; Jobling, Stephen A

2012-05-01

Arabinoxylan arabinofuranohydrolases (AXAHs) are family GH51 enzymes that have been implicated in the removal of arabinofuranosyl residues from the (1,4)-β-xylan backbone of heteroxylans. Five genes encoding barley AXAHs range in size from 4.6 kb to 7.1 kb and each contains 16 introns. The barley HvAXAH genes map to chromosomes 2H, 4H, and 5H. A small cluster of three HvAXAH genes is located on chromosome 4H and there is evidence for gene duplication and the presence of pseudogenes in barley. The cDNAs corresponding to barley and wheat AXAH genes were cloned, and transcript levels of the genes were profiled across a range of tissues at different developmental stages. Two HvAXAH cDNAs that were successfully expressed in Nicotiana benthamiana leaves exhibited similar activities against 4-nitrophenyl α-L-arabinofuranoside, but HvAXAH2 activity was significantly higher against wheat flour arabinoxylan, compared with HvAXAH1. HvAXAH2 also displayed activity against (1,5)-α-L-arabinopentaose and debranched arabinan. Western blotting with an anti-HvAXAH antibody was used to define further the locations of the AXAH enzymes in developing barley grain, where high levels were detected in the outer layers of the grain but little or no protein was detected in the endosperm. The chromosomal locations of the genes do not correspond to any previously identified genomic regions shown to influence heteroxylan structure. The data are therefore consistent with a role for AXAH in depolymerizing arabinoxylans in maternal tissues during grain development, but do not provide compelling evidence for a role in remodelling arabinoxylans during endosperm or coleoptile development in barley as previously proposed.

NREL Solar Cell Wins Federal Technology Transfer Prize | News | NREL

Science.gov (United States)

Solar Cell Wins Federal Technology Transfer Prize News Release: NREL Solar Cell Wins Federal Technology Transfer Prize May 7, 2009 A new class of ultra-light, high-efficiency solar cells developed by the U.S. Department of Energy's National Renewable Energy Laboratory has been awarded a national prize

Endosperm and whole grain rye breads are characterized by low post-prandial insulin response and a beneficial blood glucose profile

Directory of Open Access Journals (Sweden)

Östman Elin M

2009-09-01

Full Text Available Abstract Background Rye products have previously been shown to induce comparatively low post-prandial insulin responses; irrespectively of their glycaemic indices (GI. However, the mechanism behind this lowered insulin demand remains unknown. An improved insulin economy might contribute to the benefits seen in epidemiological studies with whole grain diets on metabolic risk factors and weight regulation. The objective of this study was to explore the mechanism for a reduced post-prandial insulin demand with rye products. Methods 12 healthy subjects were given flour based rye products made from endosperm, whole grain or bran, produced with different methods (baking, simulated sour-dough baking and boiling as breakfasts in random order in a cross-over design. White wheat bread (WWB was used as a reference. Blood glucose, serum insulin, plasma ghrelin and subjective satiety were measured during 180 minutes. To evaluate the course of post-meal glycaemia, a measure of the glycaemic profile (GP was introduced defined as the duration for the incremental post-prandial blood glucose response divided with the blood glucose incremental peak (min/mM. Results The study shows that whole grain rye breads and endosperm rye products induced significantly (p Conclusion Our study shows that endosperm and wholegrain rye products induce low acute insulinaemic responses and improved glycaemic profiles. The results also suggest that the rye products possess beneficial appetite regulating properties. Further studies are needed to identify the unknown property or bioactive component(s responsible for these beneficial metabolic features of rye.

Improving zinc accumulation in cereal endosperm using HvMTP1, a transition metal transporter

DEFF Research Database (Denmark)

Menguer, Paloma K; Vincent, Thomas; Miller, Anthony J

2018-01-01

Zinc (Zn) is essential for all life forms, including humans. It is estimated that around two billion people are deficient in their Zn intake. Human dietary Zn intake relies heavily on plants, which in many developing countries consists mainly of cereals. The inner part of cereal grain......) vacuolar Zn transporter HvMTP1 was expressed under the control of the endosperm-specific D-hordein promoter. Transformed plants exhibited no significant change in growth but had higher total grain Zn concentration, as measured by ICP-OES, compared to parental controls. Compared with Zn, transformants had...

In vitro biochemical characterization of all barley endosperm starch synthases

DEFF Research Database (Denmark)

Cuesta-Seijo, Jose A.; Nielsen, Morten M.; Ruzanski, Christian

2016-01-01

Starch is the main storage polysaccharide in cereals and the major source of calories in the human diet. It is synthesized by a panel of enzymes including five classes of starch synthases (SSs). While the overall starch synthase (SS) reaction is known, the functional differences between the five SS....... Here we provide a detailed biochemical study of the activity of all five classes of SSs in barley endosperm. Each enzyme was produced recombinantly in E. coli and the properties and modes of action in vitro were studied in isolation from other SSs and other substrate modifying activities. Our results...... define the mode of action of each SS class in unprecedented detail; we analyze their substrate selection, temperature dependence and stability, substrate affinity and temporal abundance during barley development. Our results are at variance with some generally accepted ideas about starch biosynthesis...

Transfer of experimental autoimmune thyroiditis with T cell clones

International Nuclear Information System (INIS)

Romball, C.G.; Weigle, W.O.

1987-01-01

We have investigated three T lymphocyte clones isolated from CBA/CaJ mice primed with mouse thyroid extract (MTE) in adjuvant. All three clones are L3T4+, Ig-, and Lyt2- and proliferate to MTE, mouse thyroglobulin (MTG) and rat thyroid extract. Clones A7 and B7 transfer thyroiditis to irradiated (475 rad) syngeneic mice, but not to normal recipients. The thyroid lesion induced by the B7 clone is characterized by the infiltration of both mononuclear and polymorphonuclear cells. The thyroiditis is transient in that lesions are apparent 7 and 14 days after transfer, but thyroids return to normal by day 21. Clone B7 showed helper activity for trinitrophenyl-keyhole limpet hemocyanin-primed B cells in vitro when stimulated with trinitrophenyl-MTG and also stimulated the production of anti-MTG antibody in recipient mice. Clone A7 induced thyroid lesions characterized by infiltration of the thyroid with mononuclear cells, with virtually no polymorphonuclear cell infiltration. This clone has shown no helper activity following stimulation with trinitrophenyl-MTG. The third clone (D2) proliferates to and shows helper activity to MTG, but fails to transfer thyroiditis to syngeneic, irradiated mice. On continuous culture, clone B7 lost its surface Thy. The loss of Thy appears unrelated to the ability to transfer thyroiditis since subclones of B7 with markedly different percentages of Thy+ cells transferred disease equally well

T-cell receptor transfer into human T cells with ecotropic retroviral vectors.

Science.gov (United States)

Koste, L; Beissert, T; Hoff, H; Pretsch, L; Türeci, Ö; Sahin, U

2014-05-01

Adoptive T-cell transfer for cancer immunotherapy requires genetic modification of T cells with recombinant T-cell receptors (TCRs). Amphotropic retroviral vectors (RVs) used for TCR transduction for this purpose are considered safe in principle. Despite this, TCR-coding and packaging vectors could theoretically recombine to produce replication competent vectors (RCVs), and transduced T-cell preparations must be proven free of RCV. To eliminate the need for RCV testing, we transduced human T cells with ecotropic RVs so potential RCV would be non-infectious for human cells. We show that transfection of synthetic messenger RNA encoding murine cationic amino-acid transporter 1 (mCAT-1), the receptor for murine retroviruses, enables efficient transient ecotropic transduction of human T cells. mCAT-1-dependent transduction was more efficient than amphotropic transduction performed in parallel, and preferentially targeted naive T cells. Moreover, we demonstrate that ecotropic TCR transduction results in antigen-specific restimulation of primary human T cells. Thus, ecotropic RVs represent a versatile, safe and potent tool to prepare T cells for the adoptive transfer.

Molecular analysis of endo-β-mannanase genes upon seed imbibition suggest a cross-talk between radicle and micropylar endosperm during germination of Arabidopsis thaliana

Science.gov (United States)

Iglesias-Fernández, Raquel; del Carmen Rodríguez-Gacio, María; Barrero-Sicilia, Cristina; Carbonero, Pilar

2011-01-01

The endo-β-mannanase (MAN) family is represented in the Arabidopsis genome by eight members, all with canonical signal peptides and only half of them being expressed in germinating seeds. The transcripts of these genes were localized in the radicle and micropylar endosperm (ME) before radicle protrusion and this expression disappears as soon as the endosperm is broken by the emerging radicle tip. However, only three of these MAN genes, AtMAN5, AtMAN7 and especially AtMAN6 influence the germination time (t50) as assessed by the analysis of the corresponding knock-out lines. The data suggest a possible interaction between embryo and ME regarding the role of MAN during the Arabidopsis germination process. PMID:21301215

Mechanisms of ion-bombardment-induced DNA transfer into bacterial E. coli cells

Energy Technology Data Exchange (ETDEWEB)

Yu, L.D., E-mail: yuld@thep-center.org [Thailand Center of Excellence in Physics, Commission on Higher Education, 328 Si Ayutthaya Road, Bangkok 10400 (Thailand); Plasma and Beam Physics Research Facility, Department of Physics and Materials Science, Faculty of Science, Chiang Mai University, Chiang Mai 50200 (Thailand); Sangwijit, K. [Molecular Biology Laboratory, Department of Biology, Faculty of Science, Chiang Mai University, Chiang Mai 50200 (Thailand); Prakrajang, K. [Plasma and Beam Physics Research Facility, Department of Physics and Materials Science, Faculty of Science, Chiang Mai University, Chiang Mai 50200 (Thailand); Faculty of Science, Maejo University, Chiang Mai 50290 (Thailand); Phanchaisri, B. [Institute of Science and Technology Research, Chiang Mai University, Chiang Mai 50200 (Thailand); Thongkumkoon, P. [Thailand Center of Excellence in Physics, Commission on Higher Education, 328 Si Ayutthaya Road, Bangkok 10400 (Thailand); Plasma and Beam Physics Research Facility, Department of Physics and Materials Science, Faculty of Science, Chiang Mai University, Chiang Mai 50200 (Thailand); Thopan, P. [Plasma and Beam Physics Research Facility, Department of Physics and Materials Science, Faculty of Science, Chiang Mai University, Chiang Mai 50200 (Thailand); Singkarat, S. [Thailand Center of Excellence in Physics, Commission on Higher Education, 328 Si Ayutthaya Road, Bangkok 10400 (Thailand); Plasma and Beam Physics Research Facility, Department of Physics and Materials Science, Faculty of Science, Chiang Mai University, Chiang Mai 50200 (Thailand); Anuntalabhochai, S. [Molecular Biology Laboratory, Department of Biology, Faculty of Science, Chiang Mai University, Chiang Mai 50200 (Thailand)

2014-05-01

Highlights: • Ion bombardment could induce DNA transfer into E. coli cells. • The DNA transfer induction depended on ion energy and fluence. • The mechanism was associated with the bacterial cell envelope structure. • A mechanism phase diagram was proposed to summarize the mechanism. - Abstract: As a useful ion beam biotechnology, ion-bombardment-induced DNA transfer into bacterial Escherichia coli (E. coli) cells has been successfully operated using argon ions. In the process ion bombardment of the bacterial cells modifies the cell envelope materials to favor the exogenous DNA molecules to pass through the envelope to enter the cell. The occurrence of the DNA transfer induction was found ion energy and fluence dependent in a complex manner. At ion energy of a few keV and a few tens of keV to moderate fluences the DNA transfer could be induced by ion bombardment of the bacterial cells, while at the same ion energy but to high fluences DNA transfer could not be induced. On the other hand, when the ion energy was medium, about 10–20 keV, the DNA transfer could not be induced by ion bombardment of the cells. The complexity of the experimental results indicated a complex mechanism which should be related to the complex structure of the bacterial E. coli cell envelope. A phase diagram was proposed to interpret different mechanisms involved as functions of the ion energy and fluence.

Mechanisms of ion-bombardment-induced DNA transfer into bacterial E. coli cells

International Nuclear Information System (INIS)

Yu, L.D.; Sangwijit, K.; Prakrajang, K.; Phanchaisri, B.; Thongkumkoon, P.; Thopan, P.; Singkarat, S.; Anuntalabhochai, S.

2014-01-01

Highlights: • Ion bombardment could induce DNA transfer into E. coli cells. • The DNA transfer induction depended on ion energy and fluence. • The mechanism was associated with the bacterial cell envelope structure. • A mechanism phase diagram was proposed to summarize the mechanism. - Abstract: As a useful ion beam biotechnology, ion-bombardment-induced DNA transfer into bacterial Escherichia coli (E. coli) cells has been successfully operated using argon ions. In the process ion bombardment of the bacterial cells modifies the cell envelope materials to favor the exogenous DNA molecules to pass through the envelope to enter the cell. The occurrence of the DNA transfer induction was found ion energy and fluence dependent in a complex manner. At ion energy of a few keV and a few tens of keV to moderate fluences the DNA transfer could be induced by ion bombardment of the bacterial cells, while at the same ion energy but to high fluences DNA transfer could not be induced. On the other hand, when the ion energy was medium, about 10–20 keV, the DNA transfer could not be induced by ion bombardment of the cells. The complexity of the experimental results indicated a complex mechanism which should be related to the complex structure of the bacterial E. coli cell envelope. A phase diagram was proposed to interpret different mechanisms involved as functions of the ion energy and fluence

Sugar transport by maize endosperm suspension cultures

International Nuclear Information System (INIS)

Felker, F.C.; Goodwin, J.C.

1987-01-01

To determine the mechanism of sugar uptake by suspension cultures derived from developing maize (Zea mays L.) endosperm, incorporation of radioactivity from 14 C-sugars by the tissue in the mid-log phase of growth was examined. Among the sugars tested was l'-deoxy-l'-fluorosucrose (FS), a derivative not hydrolyzed by invertase but recognized by sucrose carriers in other systems. At 40 mM, uptake of label from FS was 23% of that from sucrose, while uptake of label from L-glucose (used as a control for medium carry-over and adsorption) was 16% of that from sucrose. Uptake of label from sucrose did not increase at concentrations above 50 mM, possibly due to a rate-limiting requirement for extracellular hydrolysis. Kinetic analysis revealed both saturable and linear components of uptake for glucose and fructose. The rate of fructose uptake exceeded that of glucose at all concentrations. Fructose uptake at 20 mM was inhibited by NaN 3 , HgCl 2 , dinitrophenol, carbonyl cyanide m-chlorophenylhydrazone, and p-chloromercuribenzenesulfonic acid. Results suggest that sucrose is hydrolyzed prior to uptake, and that fructose is transported preferentially by a carrier sensitive to an external sulfhydryl group inhibitor. Metabolic activity is required for sugar uptake. The specificity of the hexose transporter is currently being investigated

Quantitative control of mitochondria transfer between live single cells using a microfluidic device

Directory of Open Access Journals (Sweden)

Ken-Ichi Wada

2017-12-01

Full Text Available Quantitative control of mitochondria transfer between live cells is a promising approach for genetic manipulation of mitochondrial DNA (mtDNA because single mitochondrion transfer to a mtDNA-less (ρ0 cell potentially leads to homoplasmy of mtDNA. In this paper, we describe a method for quantitative control of mitochondria transfer between live single cells. For this purpose, we fabricated novel microfluidic devices having cell paring structures with a 4.1, 5.6 or 10.0 μm-length microtunnel. When cells were fused through a microtunnel using the Sendai virus envelope-based method, a strictured cytoplasmic connection was achieved with a length corresponding to that of the microtunnel. Elongation of the cytoplasmic connection led to a decrease in mitochondria transfer to the fusion partner. Moreover, some cell pairs that fused through a 10.0 μm-length microtunnel showed single mitochondrion transfer. Fused cells were spontaneously disconnected from each other when they were recovered in a normal culture medium. These results suggest that our cell fusion method can perform quantitative control of mitochondria transfer that includes a single mitochondrion transfer.

Cloning mice and ES cells by nuclear transfer from somatic stem cells and fully differentiated cells.

Science.gov (United States)

Wang, Zhongde

2011-01-01

Cloning animals by nuclear transfer (NT) has been successful in several mammalian species. In addition to cloning live animals (reproductive cloning), this technique has also been used in several species to establish cloned embryonic stem (ntES) cell lines from somatic cells. It is the latter application of this technique that has been heralded as being the potential means to produce isogenic embryonic stem cells from patients for cell therapy (therapeutic cloning). These two types of cloning differ only in the steps after cloned embryos are produced: for reproductive cloning the cloned embryos are transferred to surrogate mothers to allow them to develop to full term and for therapeutic cloning the cloned embryos are used to derive ntES cells. In this chapter, a detailed NT protocol in mouse by using somatic stem cells (neuron and skin stem cells) and fully differentiated somatic cells (cumulus cells and fibroblast cells) as nuclear donors is described.

Sacrificial-layer free transfer of mammalian cells using near infrared femtosecond laser pulses

Science.gov (United States)

Zhang, Jun; Hartmann, Bastian; Siegel, Julian; Marchi, Gabriele; Clausen-Schaumann, Hauke; Sudhop, Stefanie; Huber, Heinz P.

2018-01-01

Laser-induced cell transfer has been developed in recent years for the flexible and gentle printing of cells. Because of the high transfer rates and the superior cell survival rates, this technique has great potential for tissue engineering applications. However, the fact that material from an inorganic sacrificial layer, which is required for laser energy absorption, is usually transferred to the printed target structure, constitutes a major drawback of laser based cell printing. Therefore alternative approaches using deep UV laser sources and protein based acceptor films for energy absorption, have been introduced. Nevertheless, deep UV radiation can introduce DNA double strand breaks, thereby imposing the risk of carcinogenesis. Here we present a method for the laser-induced transfer of hydrogels and mammalian cells, which neither requires any sacrificial material for energy absorption, nor the use of UV lasers. Instead, we focus a near infrared femtosecond (fs) laser pulse (λ = 1030 nm, 450 fs) directly underneath a thin cell layer, suspended on top of a hydrogel reservoir, to induce a rapidly expanding cavitation bubble in the gel, which generates a jet of material, transferring cells and hydrogel from the gel/cell reservoir to an acceptor stage. By controlling laser pulse energy, well-defined cell-laden droplets can be transferred with high spatial resolution. The transferred human (SCP1) and murine (B16F1) cells show high survival rates, and good cell viability. Time laps microscopy reveals unaffected cell behavior including normal cell proliferation. PMID:29718923

Focus on Adoptive T Cell Transfer Trials in Melanoma

Directory of Open Access Journals (Sweden)

Liat Hershkovitz

2010-01-01

Full Text Available Adoptive Cell Transfer (ACT of Tumor-Infiltrating Lymphocytes (TIL in combination with lymphodepletion has proven to be an effective treatment for metastatic melanoma patients, with an objective response rate in 50%–70% of the patients. It is based on the ex vivo expansion and activation of tumor-specific T lymphocytes extracted from the tumor and their administration back to the patient. Various TIL-ACT trials, which differ in their TIL generation procedures and patient preconditioning, have been reported. In the latest clinical studies, genetically engineered peripheral T cells were utilized instead of TIL. Further improvement of adoptive T cell transfer depends on new investigations which seek higher TIL quality, increased durable response rates, and aim to treat more patients. Simplifying this therapy may encourage cancer centers worldwide to adopt this promising technology. This paper focuses on the latest progress regarding adoptive T cell transfer, comparing the currently available protocols and discussing their advantages, disadvantages, and implication in the future.

Cellular transfer of magnetic nanoparticles via cell microvesicles: impact on cell tracking by magnetic resonance imaging.

Science.gov (United States)

Silva, Amanda K Andriola; Wilhelm, Claire; Kolosnjaj-Tabi, Jelena; Luciani, Nathalie; Gazeau, Florence

2012-05-01

Cell labeling with magnetic nanoparticles can be used to monitor the fate of transplanted cells in vivo by magnetic resonance imaging. However, nanoparticles initially internalized in administered cells might end up in other cells of the host organism. We investigated a mechanism of intercellular cross-transfer of magnetic nanoparticles to different types of recipient cells via cell microvesicles released under cellular stress. Three cell types (mesenchymal stem cells, endothelial cells and macrophages) were labeled with 8-nm iron oxide nanoparticles. Then cells underwent starvation stress, during which they produced microvesicles that were subsequently transferred to unlabeled recipient cells. The analysis of the magnetophoretic mobility of donor cells indicated that magnetic load was partially lost under cell stress. Microvesicles shed by stressed cells participated in the release of magnetic label. Moreover, such microvesicles were uptaken by naïve cells, resulting in cellular redistribution of nanoparticles. Iron load of recipient cells allowed their detection by MRI. Cell microvesicles released under stress may be disseminated throughout the organism, where they can be uptaken by host cells. The transferred cargo may be sufficient to allow MRI detection of these secondarily labeled cells, leading to misinterpretations of the effectiveness of transplanted cells.

Review of somatic cell nuclear transfer in pig | Muenthaisong ...

African Journals Online (AJOL)

It is now more than 8 years, since the first cloned pig from nuclear transfer was reported. Success of somatic cell nuclear transfer (SCNT) in pig is still low compared to that in bovine. Embryonic and neonatal abnormalities of cloned piglets are probably a result of incorrect or incomplete reprogramming of the transferred ...

Mortalin antibody-conjugated quantum dot transfer from human mesenchymal stromal cells to breast cancer cells requires cell–cell interaction

Energy Technology Data Exchange (ETDEWEB)

Pietilä, Mika [National Institute of Advanced industrial Sciences and Technology, Tsukuba, Ibaraki 305 8562 (Japan); Lehenkari, Petri [Institute of Biomedicine, Department of Anatomy and Cell Biology, University of Oulu, Aapistie 7, P.O. Box 5000, FIN-90014 (Finland); Institute of Clinical Medicine, Division of Surgery, University of Oulu and Clinical Research Centre, Department of Surgery and Intensive Care, Oulu University Hospital, Aapistie 5a, P.O. Box 5000, FIN-90014 (Finland); Kuvaja, Paula [Institute of Biomedicine, Department of Anatomy and Cell Biology, University of Oulu, Aapistie 7, P.O. Box 5000, FIN-90014 (Finland); Department of Pathology, Oulu University Hospital, P.O. Box 50, FIN-90029 OYS, Oulu (Finland); Kaakinen, Mika [Biocenter Oulu, University of Oulu, P.O. Box 5000, FI-90014 (Finland); Kaul, Sunil C.; Wadhwa, Renu [National Institute of Advanced industrial Sciences and Technology, Tsukuba, Ibaraki 305 8562 (Japan); Uemura, Toshimasa, E-mail: t.uemura@aist.go.jp [National Institute of Advanced industrial Sciences and Technology, Tsukuba, Ibaraki 305 8562 (Japan)

2013-11-01

The role of tumor stroma in regulation of breast cancer growth has been widely studied. However, the details on the type of heterocellular cross-talk between stromal and breast cancer cells (BCCs) are still poorly known. In the present study, in order to investigate the intercellular communication between human mesenchymal stromal cells (hMSCs) and breast cancer cells (BCCs, MDA-MB-231), we recruited cell-internalizing quantum dots (i-QD) generated by conjugation of cell-internalizing anti-mortalin antibody and quantum dots (QD). Co-culture of illuminated and color-coded hMSCs (QD655) and BCCs (QD585) revealed the intercellular transfer of QD655 signal from hMSCs to BCCs. The amount of QD double positive BCCs increased gradually within 48 h of co-culture. We found prominent intercellular transfer of QD655 in hanging drop co-culture system and it was non-existent when hMSCs and BBCs cells were co-cultured in trans-well system lacking imminent cell–cell contact. Fluorescent and electron microscope analyses also supported that the direct cell-to-cell interactions may be required for the intercellular transfer of QD655 from hMSCs to BCCs. To the best of our knowledge, the study provides a first demonstration of transcellular crosstalk between stromal cells and BCCs that involve direct contact and may also include a transfer of mortalin, an anti-apoptotic and growth-promoting factor enriched in cancer cells.

Role of donor lymphoid cells in the transfer of allograft tolerance

International Nuclear Information System (INIS)

Pierce, G.E.; Watts, L.M.

1985-01-01

Tolerance to murine skin allografts across a MHC disparity was induced by conditioning primary hosts with sublethal fractionated total-body irradiation (FTBI) and transfusion of allogeneic bone marrow (BM). Tolerance could be adoptively transferred to secondary hosts conditioned by FTBI with infusion of spleen cells from hosts bearing intact skin allografts greater than 60 days. Tolerance could not be transferred by tolerant host spleen (THS) preparations from which cells of the donor genotype had been deleted by cytotoxic alloantisera. Deletion of host genotype cells, however, did not diminish the capability of THS to transfer tolerance. All of the tolerizing activity of THS appeared to reside within cells of the donor genotype. Small numbers of normal donor spleen cells could induce tolerance in FTBI hosts but only at the expense of very high mortality, in contrast to the low mortality observed with tolerizing injections of allogeneic donor cells from THS or injections of normal semiallogeneic F1 hybrid spleen cells. If an active immune response is responsible for tolerance induction/transfer in this model, allogeneic donor lymphoid cells derived from BM, in contrast to donor spleen cells, must be capable of mounting this response without concomitant severe GVHD. In future experiments, cells of donor genotype can be isolated from THS and purified in sufficient numbers to compare their tolerizing efficiency vs. that of normal donor cells, detect possible suppression of normal host cell alloreactivity in vitro and identify the donor cell phenotypes involved

Effect of estrone on somatic and female gametophyte cell division and differentiation in Arabidospis thaliana cultured in vitro

Directory of Open Access Journals (Sweden)

Piotr Żabicki

2014-04-01

Full Text Available The aim of the study was to determine the effect of the mammalian female sex hormone estrone on differentiation of somatic tissues and on induction of autonomous endosperm in culture of female gametophyte cells of Arabidopsis thaliana ecotype Columbia (Col-0. In culture, estrone-stimulated development of autonomous endosperm (AE occurred in 14.7% of unpollinated pistils. The AE represented development stages similar to those of young endosperm after fertilization and AE of fis mutants in vivo. In the majority of ovules the AE was in a few-nucleate young stage. Some ovules showed more advanced stages of AE development, with nuclei and cytoplasm forming characteristic nuclear cytoplasmic domains (NCDs. Sporadically, AE was divided into regions characteristic for Arabidopsis endosperm formed after fertilization. Direct organogenesis (caulogenesis, rhizogenesis, callus proliferation and formation of trichome-like structures were observed during in vitro culture of hypocotyls and cotyledons of 3-day-old seedlings cultured on medium supplemented with estrone for 28 days. Histological analysis showed adventitious root formation and changes in explant anatomy caused by estrone.

From discovery of high lysine barley endosperm mutants in the 1960-70 ties to new holistic spectral models of the phenome and of pleiotropy in 2008

International Nuclear Information System (INIS)

Munck, L.; Moeller Jespersen, B.

2008-01-01

As documented by eight IAEA/FAO symposia 1968-82 on nutritionally improved seeds, a wide range of high lysine endosperm mutants were isolated in maize, sorghum and barley. These mutants observed by new spectroscopic screening methods can now be exploited to advance basic biological research and theory. Since 1982 effective methods to overview the physiochemical composition of seeds by Near Infrared Spectroscopy evaluated by chemometric data analysis have developed. Spectroscopic analyses by calibration have now substituted for the wet analyses in industry. In genetics there has traditionally been a differentiation between major genes for qualitative and minor 'polygenes' for quantitative traits. This view has been coupled to an incomplete understanding of pleiotropy. It is shown that seed spectra from isogenic barley endosperm mutants represent a coarse-grained physiochemical overview of the phenome that can be classified by chemometrics. Pleiotropy expressed by a gene is quantified as a whole pattern by the gene specific mutant spectrum subtracted by the spectrum of the parent variety. Selection for an improved plumpness (starch) in a breeding material with the lys3.a mutant visualises in spectra the effect of enriching 'minor polygenes' for an increased content of starch in a mutant gene background. Morphological, spectroscopic and chemical analyses suggest that mutant genes have both qualitative and quantitative expressions. They produce qualitative pleiotropic phenomenological patterns that can be observed as more or less severe changes in macro and microstructures of the plant and seed phenotype. Behind are quantitative chemical changes that by spectroscopy and chemometrics can be transferred to qualitative patterns. In fact one major gene for a qualitative trait can act as several apparent minor polygenes for quantitative variables. This explains the reduced need for the previously expected several hundred thousands of genes and gene modifiers down to the

Adoptive Transfer of Dying Cells Causes Bystander-Induced Apoptosis

Science.gov (United States)

Schwulst, Steven J.; Davis, Christopher G.; Coopersmith, Craig M.; Hotchkiss, Richard S.

2009-01-01

The anti-apoptotic Bcl-2 protein has the remarkable ability to prevent cell death from several noxious stimuli. Intriguingly, Bcl-2 overexpression in one cell type has been reported to protect against cell death in neighboring non-Bcl-2 overexpressing cell types. The mechanism of this “trans” protection has been speculated to be secondary to the release of a cytoprotective factor by Bcl-2 overexpressing cells. We employed a series of adoptive transfer experiments in which lymphocytes that overexpress Bcl-2 were administered to either wild type mice or mice lacking mature T and B cells (Rag-1-/-) to detect the presence or absence of the putative protective factor. We were unable to demonstrate “trans” protection. However, adoptive transfer of apoptotic or necrotic cells exacerbated the degree of apoptotic death in neighboring non-Bcl-2 overexpressing cells (p≤0.05). Therefore, this data suggests that dying cells emit signals triggering cell death in neighboring non-Bcl-2 overexpressing cells, i.e. a “trans” destructive effect. PMID:17194455

Gene transfer to primary corneal epithelial cells with an integrating lentiviral vector

Directory of Open Access Journals (Sweden)

Lauro Augusto de Oliveira

2010-10-01

Full Text Available PURPOSE: To evaluate the transfer of heterologous genes carrying a Green Fluorescent Protein (GFP reporter cassette to primary corneal epithelial cells ex vivo. METHODS: Freshly enucleated rabbit corneoscleral tissue was used to obtain corneal epithelial cell suspension via enzymatic digestion. Cells were plated at a density of 5×10³ cells/cm² and allowed to grow for 5 days (to 70-80% confluency prior to transduction. Gene transfer was monitored using fluorescence microscopy and fluorescence activated cell sorter (FACS. We evaluated the transduction efficiency (TE over time and the dose-response effect of different lentiviral particles. One set of cells were dual sorted by fluorescence activated cell sorter for green fluorescent protein expression as well as Hoechst dye exclusion to evaluate the transduction of potentially corneal epithelial stem cells (side-population phenotypic cells. RESULTS: Green fluorescent protein expressing lentiviral vectors were able to effectively transduce rabbit primary epithelial cells cultured ex vivo. Live cell imaging post-transduction demonstrated GFP-positive cells with normal epithelial cell morphology and growth. The transduction efficiency over time was higher at the 5th post-transduction day (14.1% and tended to stabilize after the 8th day. The number of transduced cells was dose-dependent, and at the highest lentivirus concentrations approached 7%. When double sorted by fluorescence activated cell sorter to isolate both green fluorescent protein positive and side population cells, transduced side population cells were identified. CONCLUSIONS: Lentiviral vectors can effectively transfer heterologous genes to primary corneal epithelial cells expanded ex vivo. Genes were stably expressed over time, transferred in a dose-dependence fashion, and could be transferred to mature corneal cells as well as presumable putative stem cells.

Proliferation-linked apoptosis of adoptively transferred T cells after IL-15 administration in macaques.

Directory of Open Access Journals (Sweden)

Carolina Berger

Full Text Available The adoptive transfer of antigen-specific effector T cells is being used to treat human infections and malignancy. T cell persistence is a prerequisite for therapeutic efficacy, but reliably establishing a high-level and durable T cell response by transferring cultured CD8(+ T cells remains challenging. Thus, strategies that promote a transferred high-level T cell response may improve the efficacy of T cell therapy. Lymphodepletion enhances persistence of transferred T cells in mice in part by reducing competition for IL-15, a common γ-chain cytokine that promotes T cell memory, but lymphodepleting regimens have toxicity. IL-15 can be safely administered and has minimal effects on CD4(+ regulatory T cells at low doses, making it an attractive adjunct in adoptive T cell therapy. Here, we show in lymphoreplete macaca nemestrina, that proliferation of adoptively transferred central memory-derived CD8(+ effector T (T(CM/E cells is enhanced in vivo by administering IL-15. T(CM/E cells migrated to memory niches, persisted, and acquired both central memory and effector memory phenotypes regardless of the cytokine treatment. Unexpectedly, despite maintaining T cell proliferation, IL-15 did not augment the magnitude of the transferred T cell response in blood, bone marrow, or lymph nodes. T cells induced to proliferate by IL-15 displayed increased apoptosis demonstrating that enhanced cycling was balanced by cell death. These results suggest that homeostatic mechanisms that regulate T cell numbers may interfere with strategies to augment a high-level T cell response by adoptive transfer of CD8(+ T(CM/E cells in lymphoreplete hosts.

Lymphoma immunotherapy: vaccines, adoptive cell transfer and immunotransplant

Science.gov (United States)

Brody, Joshua; Levy, Ronald

2017-01-01

Therapy for non-Hodgkin lymphoma has benefited greatly from basic science and clinical research such that chemotherapy and monoclonal antibody therapy have changed some lymphoma subtypes from uniformly lethal to curable, but the majority of lymphoma patients remain incurable. Novel therapies with less toxicity and more specific targeting of tumor cells are needed and immunotherapy is among the most promising of these. Recently completed randomized trials of idiotype vaccines and earlier-phase trials of other vaccine types have shown the ability to induce antitumor T cells and some clinical responses. More recently, trials of adoptive transfer of antitumor T cells have demonstrated techniques to increase the persistence and antitumor effect of these cells. Herein, we discuss lymphoma immunotherapy clinical trial results and what lessons can be taken to improve their effect, including the combination of vaccination and adoptive transfer in an approach we have dubbed ‘immunotransplant’. PMID:20636025

Transfer plate radioassay using cell monolayers to detect anti-cell surface antibodies synthesized by lymphocyte hybridomas

International Nuclear Information System (INIS)

Schneider, M.D.; Eisenbarth, G.S.

1979-01-01

A solid phase [ 125 I] Protein A radioassay for anti-cell surface antibodies is described, which employs target cell monolayers cultured on fenestrated polyvinyl chloride 96-well plates ('transfer plates'). The calibrated aperture in the bottom of each well is small enough to retain fluid contents by surface tension during monolayer growth, but also permits fluid to enter the wells when transfer plate are lowered into receptacles containing washing buffer on test sera. To assay for antibodies directed against target cell surface antigens, transfer plates bearing monolayers are inserted into microculture plates with corresponding 96-well geometry, thereby simultaneously sampling 96 wells. This assay allows rapid screening of hundreds of hybrid cell colonies for production of antibodies with desired tissue specificity. (Auth.)

Propagation of senescent mice using nuclear transfer embryonic stem cell lines.

Science.gov (United States)

Mizutani, Eiji; Ono, Tetsuo; Li, Chong; Maki-Suetsugu, Rinako; Wakayama, Teruhiko

2008-09-01

Senescent mice are often infertile, and the cloning success rate decreases with age, making it almost impossible to produce cloned progeny directly from such animals. In this study, we tried to produce offspring from such "unclonable" senescent mice using nuclear transfer techniques. Donor fibroblasts were obtained from the tail tips of mice aged up to 2 years and 9 months. Although most attempts failed to produce cloned mice by direct somatic cell nuclear transfer, we managed to establish nuclear transfer embryonic stem (ntES) cell lines from all aged mice with an establishment rate of 10-25%, irrespective of sex or strain. Finally, cloned mice were obtained from these ntES cells by a second round of nuclear transfer. In addition, healthy offspring was obtained from all aged donors via germline transmission of ntES cells in chimeric mice. This technique is thus applicable to the propagation of a variety of animals, irrespective of age or fertile potential.

Determination and correlation of mass transfer coefficients in a stirred cell

International Nuclear Information System (INIS)

Herranz, J.; Bloxom, S.R.; Keeler, J.B.; Roth, S.R.

1975-01-01

In the proposed Molten Salt Breeder Reactor flowsheet, a fraction of the rare earth fission products is removed from the fuel salt in mass transfer cells. To obtain design parameters for this extraction, the effect of cell size, blade diameter, phase volume, and agitation rate on the mass transfer for a high density ratio system (mercury/water) in nondispersing square cross section contactors was determined. Aqueous side mass transfer coefficients were measured by polarography over a wide range of operating conditions. Correlations for the experimental mass transfer coefficients as functions of the operating parameters are presented. Several techniques for measuring mercury-side mass transfer coefficients were evaluated and a new one is recommended

Isolation of the endosperm-specific LPAAT gene promoter from coconut (Cocos nucifera L.) and its functional analysis in transgenic rice plants.

Science.gov (United States)

Xu, Li; Ye, Rongjian; Zheng, Yusheng; Wang, Zhekui; Zhou, Peng; Lin, Yongjun; Li, Dongdong

2010-09-01

As one of the key tropical crops, coconut (Cocos nucifera L.) is a member of the monocotyledonous family Aracaceae (Palmaceae). In this study, we amplified the upstream region of an endosperm-specific expression gene, Lysophosphatidyl acyltransferase (LPAAT), from the coconut genomic DNA by chromosome walking. In this sequence, we found several types of promoter-related elements including TATA-box, CAAT-box and Skn1-motif. In order to further examine its function, three different 5'-deletion fragments were inserted into pBI101.3, a plant expression vector harboring the LPAAT upstream sequence, leading to pBI101.3-L1, pBI101.3-L2 and pBI101.3-L3, respectively. We obtained transgenic plants of rice by Agrobacterium-mediated callus transformation and plant regeneration and detected the expression of gus gene by histochemical staining and fluorometric determination. We found that gus gene driven by the three deletion fragments was specifically expressed in the endosperm of rice seeds, but not in the empty vector of pBI101.3 and other tissues. The highest expression level of GUS was at 15 DAF in pBI101.3-L3 and pBI101.3-L2 transgenic lines, while the same level was detected at 10 DAF in pBI101.3-L1. The expression driven by the whole fragment was up to 1.76- and 2.8-fold higher than those driven by the -817 bp and -453 bp upstream fragments, and 10.7-fold higher than that driven by the vector without the promoter. Taken together, our results strongly suggest that these promoter fragments from coconut have a significant potential in genetically improving endosperm in main crops.

Preferential transfer of certain plasma membrane proteins onto T and B cells by trogocytosis.

Directory of Open Access Journals (Sweden)

Sandrine Daubeuf

2010-01-01

Full Text Available T and B cells capture antigens via membrane fragments of antigen presenting cells (APC in a process termed trogocytosis. Whether (and how a preferential transfer of some APC components occurs during trogocytosis is still largely unknown. We analyzed the transfer onto murine T and B cells of a large panel of fluorescent proteins with different intra-cellular localizations in the APC or various types of anchors in the plasma membrane (PM. Only the latter were transferred by trogocytosis, albeit with different efficiencies. Unexpectedly, proteins anchored to the PM's cytoplasmic face, or recruited to it via interaction with phosphinositides, were more efficiently transferred than those facing the outside of the cell. For proteins spanning the PM's whole width, transfer efficiency was found to vary quite substantially, with tetraspanins, CD4 and FcRgamma found among the most efficiently transferred proteins. We exploited our findings to set immunodiagnostic assays based on the capture of preferentially transferred components onto T or B cells. The preferential transfer documented here should prove useful in deciphering the cellular structures involved in trogocytosis.

An improved out-cell to in-cell rapid transfer system at the HFEF-south

International Nuclear Information System (INIS)

Bacca, J.P.; Sherman, E.K.

1990-01-01

The Argonne National Laboratory (ANL) Hot Fuel Examination Facility-South (HFEF-S), located at the ANL-West site of the Idaho National Engineering Laboratory, is currently undergoing extensive refurbishment and modifications in preparation for its use, beginning in 1991, in demonstrating remote recycling of fast reactor, metal-alloy fuel as part of the US Department of Energy liquid-metal reactor, Integral Fast Reactor (IFR) program. Included in these improvements to HFEF-S is a new, small-item, rapid transfer system (RTS). When installed, this system will enable the rapid transfer of small items from the hot-cell exterior into the argon cell (argon-gas atmosphere) of the facility without necessitating the use of time-consuming and laborious procedures. The new RTS will also provide another important function associated with HFEF-S hot-cell operation in the IFR Fuel Recycle Program; namely, the rapid insertion of clean, radioactive contamination-measuring smear paper specimens into the hot cells for area surveys, and the expedited removal of these contaminated (including alpha as well as beta/gamma contamination) smears from the argon cell for transfer to an adjacent health physics field laboratory in the facility for nuclear contamination/radiation counting

Accumulation and conversion of sugars by developing wheat grains. VII. Effect of changes in sieve tube and endosperm cavity sap concentrations on the grain filling rate

International Nuclear Information System (INIS)

Fisher, D.B.; Gifford, R.M.

1987-01-01

The extent to which wheat grain growth is dependent on transport pool solute concentration was investigated by the use of illumination and partial grain removal to vary solute concentrations in the sieve tube and endosperm cavity saps of the wheat ear (Triticum aestivum L.). Short-term grain growth rates were estimated indirectly from the product of phloem area, sieve tube sap concentration, and 32 P translocation velocity. On a per grain basis, calculated rates of mass transport through the peduncle were fairly constant over a substantial range in other transport parameters (i.e. velocity, concentration, phloem area, and grain number). The rates were about 40% higher than expected; this probably reflects some unavoidable bias on faster-moving tracer in the velocity estimates. Sieve tube sap concentration increased in all experiments (by 20 to 64%), with a concomitant decline in velocity (to as low as 8% of the initial value). Endosperm cavity sucrose concentration also increased in all experiments, but cavity sap osmolality and total amino acid concentration remained nearly constant. No evidence was found for an increase in the rate of mass transport per grain through the peduncle in response to the treatments. This apparent unresponsiveness of grain growth rate to increased cavity sap sucrose concentration conflicts with earlier in vitro endosperm studies showing that sucrose uptake increased with increasing external sucrose concentration up to 150 to 200 millimolar

An improved out-cell to in-cell rapid transfer system at the HFEF/South

International Nuclear Information System (INIS)

Bacca, J.P.; Sherman, E.K.

1991-01-01

This paper reports on Argonne National Laboratory's Fuel Cycle Facility (FCF) (formerly named Hot Fuel Examination Facility-South) (HFEF/South) which is currently being refurbished and upgraded in preparation for demonstrating remote, fast reactor metal-fuel reprocessing and refabrication, as part of the Integral Fast Reactor (IFR) Program. Among the FCF hot-cell system upgrades being provided is a newly fabricated, direct, out-of-cell to in-cell, small-item transfer system for the FCF argon cell. This system will enable the rapid transfer of selected small items from the hot cell exterior into the argon cell (argon-gas atmosphere) of the facility, without necessitating the use of formerly employed, very time-consuming, and quite laborious procedures. The new system will be especially valuable for the rapid insertion of IFR fuel processing makeup materials and small tools into the argon cell, and for use in argon cell and overall FCF radioactive contamination-control activities

Organic solar cells: understanding the role of Förster resonance energy transfer.

Science.gov (United States)

Feron, Krishna; Belcher, Warwick J; Fell, Christopher J; Dastoor, Paul C

2012-12-12

Organic solar cells have the potential to become a low-cost sustainable energy source. Understanding the photoconversion mechanism is key to the design of efficient organic solar cells. In this review, we discuss the processes involved in the photo-electron conversion mechanism, which may be subdivided into exciton harvesting, exciton transport, exciton dissociation, charge transport and extraction stages. In particular, we focus on the role of energy transfer as described by F¨orster resonance energy transfer (FRET) theory in the photoconversion mechanism. FRET plays a major role in exciton transport, harvesting and dissociation. The spectral absorption range of organic solar cells may be extended using sensitizers that efficiently transfer absorbed energy to the photoactive materials. The limitations of F¨orster theory to accurately calculate energy transfer rates are discussed. Energy transfer is the first step of an efficient two-step exciton dissociation process and may also be used to preferentially transport excitons to the heterointerface, where efficient exciton dissociation may occur. However, FRET also competes with charge transfer at the heterointerface turning it in a potential loss mechanism. An energy cascade comprising both energy transfer and charge transfer may aid in separating charges and is briefly discussed. Considering the extent to which the photo-electron conversion efficiency is governed by energy transfer, optimisation of this process offers the prospect of improved organic photovoltaic performance and thus aids in realising the potential of organic solar cells.

Organic Solar Cells: Understanding the Role of Förster Resonance Energy Transfer

Directory of Open Access Journals (Sweden)

Paul C. Dastoor

2012-12-01

Full Text Available Organic solar cells have the potential to become a low-cost sustainable energy source. Understanding the photoconversion mechanism is key to the design of efficient organic solar cells. In this review, we discuss the processes involved in the photo-electron conversion mechanism, which may be subdivided into exciton harvesting, exciton transport, exciton dissociation, charge transport and extraction stages. In particular, we focus on the role of energy transfer as described by F¨orster resonance energy transfer (FRET theory in the photoconversion mechanism. FRET plays a major role in exciton transport, harvesting and dissociation. The spectral absorption range of organic solar cells may be extended using sensitizers that efficiently transfer absorbed energy to the photoactive materials. The limitations of F¨orster theory to accurately calculate energy transfer rates are discussed. Energy transfer is the first step of an efficient two-step exciton dissociation process and may also be used to preferentially transport excitons to the heterointerface, where efficient exciton dissociation may occur. However, FRET also competes with charge transfer at the heterointerface turning it in a potential loss mechanism. An energy cascade comprising both energy transfer and charge transfer may aid in separating charges and is briefly discussed. Considering the extent to which the photo-electron conversion efficiency is governed by energy transfer, optimisation of this process offers the prospect of improved organic photovoltaic performance and thus aids in realising the potential of organic solar cells.

Cloning Endangered Felids by Interspecies Somatic Cell Nuclear Transfer.

Science.gov (United States)

Gómez, Martha C; Pope, C Earle

2015-01-01

In 2003, the first wild felid was produced by interspecies somatic cell nuclear transfer. Since then other wild felid clone offspring have been produced by using the same technique with minor modifications. This chapter describes detailed protocols used in our laboratory for (1) the isolation, culture, and preparation of fibroblast cells as donor nucleus, and (2) embryo reconstruction with domestic cat enucleated oocytes to produce cloned embryos that develop to the blastocyst stage in vitro and, after transfer into synchronized recipients, establish successful pregnancies.

Determination of Endosperm Protein Secondary Structure in Hard Wheat Breeding Lines using Synchrotron Infrared Microspectroscopy

International Nuclear Information System (INIS)

Bonwell, E.; Fisher, T.; Fritz, A.; Wetzel, D.

2008-01-01

One molecular aspect of mature hard wheat protein quality for breadmaking is the relative amount of endosperm protein in the a-helix form compared with that in other secondary structure forms including β-sheet. Modeling of a-helix and β-sheet absorption bands that contribute to the amide I band at 1650 cm-1 was applied to more than 1500 spectra in this study. The microscopic view of wheat endosperm is dominated by many large starch granules with protein in between. The spectrum produced from in situ microspectroscopy of this mixture is dominated by carbohydrate bands from the large starch granules that fill up the field. The high spatial resolution achievable with synchrotron infrared microspectroscopy enables revealing good in situ spectra of the protein located interstitially. Synchrotron infrared microspectroscopic mapping of 4 μm thick frozen sections of endosperm in the subaleurone region provides spectra from a large number of pixels. Pixels with protein-dominated spectra are sorted out from among adjacent pixels to minimize the starch absorption and scattering contributions. Subsequent data treatment to extract information from the amide I band requires a high signal to noise ratio. Although spectral interference of the carbohydrate band on the amide band is not a problem, the scattering produced by the large starch granules diminishes the signal to noise ratio throughout the spectrum. High density mapping was done on beamlines U2B and U10B at the National Synchrotron Light Source at Brookhaven National Laboratory, Upton, NY. Mapping with a single masked spot size of 5.5 μm diameter or confocal 5 μm x 5 μm spot size, respectively, on the two beamlines used produced spectra for new breeding lines under current consideration. Appropriate data treatment allows calculation of a numerical estimate of the a-helix population relative to other secondary protein structures from the position and shape of the amide I absorption band. Current breeding lines show a

Efficient gene transfer into lymphoma cells using adenoviral vectors combined with lipofection.

Science.gov (United States)

Buttgereit, P; Weineck, S; Röpke, G; Märten, A; Brand, K; Heinicke, T; Caselmann, W H; Huhn, D; Schmidt-Wolf, I G

2000-08-01

Tumor cells, such as lymphoma cells, are possible targets for gene therapy. In general, gene therapeutic approaches require efficient gene transfer to host cells and sufficient transgene expression. However, lymphoma cells previously have been demonstrated to be resistant to most of the currently available gene transfer methods. The aim of this study was to analyze various methods for transfection of lymphoma cells and to improve the efficiency of gene delivery. In accordance with previously published reports, lymphoma cells were demonstrated to be resistant to lipofection and electroporation. In contrast, we present an improved adenoviral protocol leading to highly efficient gene transfer to lymphoma cell lines derived from B cells as well as primary lymphoma cells being achieved with an adenoviral vector system encoding the beta-galactosidase protein. At a multiplicity of infection of 200, up to 100% of Daudi cells and Raji cells and 70% of OCI-Ly8-LAM53 cells could be transfected. Even at high adenoviral concentrations, no marked toxicity was observed, and the growth characteristics of the lymphoma cell lines were not impaired. The transfection rates in primary cells derived from six patients with non-Hodgkin's lymphoma were 30-65%, respectively. Transfection efficiency could be further increased by addition of cationic liposomes to adenoviral gene transfer. Furthermore, we examined the expression of the Coxsackie-adenoviral receptor (CAR) and the integrin receptors on the lymphoma cell surface. Flow cytometric analysis showed that 88% of Daudi cells, 69% of Raji cells, and 6% of OCI-Ly8-LAM53 cells expressed CAR on the cell surface. According to our data, adenoviral infection of lymphoma cells seems to be mediated by CAR. In contrast, integrin receptors are unlikely to play a major role, because lymphoma cells were negative for alphavbeta3-integrins and negative for alphavbeta5-integrins. In conclusion, this study demonstrates that B-lymphoma cell lines and

Transfer cell wall ingrowths and vein loading characteristics in pea leaf discs

International Nuclear Information System (INIS)

Wimmers, L.E.; Turgeon, R.

1987-01-01

Transfer cell wall ingrowths are thought to increase transport capacity by increasing plasmalemma surface area. Leaf minor vein phloem transfer cells presumably enhance phloem loading. In Pisum sativum cv. Little marvel grown under different light regimes (150 to 1000 μmol photons m -2 sec -1 ) there is a positive correlation between light intensity and wall ingrowth area in phloem transfer cells. The extent of ingrowth and correlation to light intensity is greatest in minor veins, decreasing as vein size increases. Vein loading was assayed by floating abraded leaf discs on 14 C-sucrose (10 mM). There is a positive correlation between uptake and transfer cell wall area, although the latter increased more than the former. The difference in uptake is stable throughout the photoperiod, and is also stable in mature leaves for at least four days after plants are transfered to a different light intensity. Sucrose uptake is biphasic. The saturable component of uptake is sensitive to light intensity, the Km for sucrose is negatively correlated to light intensity, while V/sub max/remains unchanged

Recombinant human proinsulin from transgenic corn endosperm: solvent screening and extraction studies

Directory of Open Access Journals (Sweden)

C. S. Farinas

2007-09-01

Full Text Available Recombinant pharmaceutical proteins are being produced in different systems such as bacteria and mammalian cell cultures. The use of transgenic plants as bioreactors has recently arisen as an alternative system offering many practical and economic advantages. However, finding an optimum strategy for the downstream processing (DSP of recombinant proteins from plants still remains a challenge. In this work, we studied the extraction of recombinant human proinsulin (rhProinsulin produced in the endosperm of transgenic corn seeds. An efficient extraction solvent was selected and the effects of temperature, solvent-to-solid ratio, time, and impeller rotational speed on the extraction were evaluated using an experimental design. After an extraction kinetics study, temperature was further evaluated to maximize rhProinsulin concentration in the extracts and to minimize the native corn components carbohydrates, phenolic compounds, and proteins. A high efficiency condition for extracting rhProinsulin with the selected solvent - 50 mM sodium bicarbonate buffer pH 10.0 and 5 mM DTT - was an extraction time of 2 h at a solvent-to-solid ratio of 10:1 and 25º C. The maximum rhProinsulin concentration in the extracts at that condition was 18.87 mg l-1 or 0.42% of the total soluble protein. These values are within the range in which the production of pharmaceutical proteins in plants can be competitive with other expression systems. The results presented provide information for the development of an additional production platform for the hormone insulin.

Light transfer in agar immobilized microalgae cell cultures

Science.gov (United States)

Kandilian, Razmig; Jesus, Bruno; Legrand, Jack; Pilon, Laurent; Pruvost, Jérémy

2017-09-01

This paper experimentally and theoretically investigates light transfer in agar-immobilized cell cultures. Certain biotechnological applications such as production of metabolites secreted by photosynthetic microorganisms require cells to be immobilized in biopolymers to minimize contamination and to facilitate metabolite recovery. In such applications, light absorption by cells is one of the most important parameters affecting cell growth or metabolite productivity. Modeling light transfer therein can aid design and optimize immobilized-cell reactors. In this study, Parachlorella kessleri cells with areal biomass concentrations ranging from 0.36 to 16.9 g/m2 were immobilized in 2.6 mm thick agar gels. The average absorption and scattering cross-sections as well as the scattering phase function of P. kessleri cells were measured. Then, the absorption and transport scattering coefficients of the agar gel were determined using an inverse method based on the modified two-flux approximation. The forward model was used to predict the normal-hemispherical transmittance and reflectance of the immobilized-cell films accounting for absorption and scattering by both microalgae and the agar gel. Good agreement was found between the measured and predicted normal-hemispherical transmittance and reflectance provided absorption and scattering by agar were taken into account. Moreover, good agreement was found between experimentally measured and predicted mean rate of photon absorption. Finally, optimal areal biomass concentration was determined to achieve complete absorption of the incident radiation.

Soya beans and maize : the effect of chemical and physical structure of cell wall polysaccharides on fermentation kinetics

NARCIS (Netherlands)

Laar, van H.

2000-01-01

The analysis of the relationship between cell wall composition and fermentation of endosperm cell walls of soya beans and maize was approached from three different angles. Firstly, the fermentation (rate and extent of fermentation, the sugar degradation pattern, and volatile fatty acid

Sensing lymphoma cells based on a cell-penetrating/apoptosis-inducing/electron-transfer peptide probe

International Nuclear Information System (INIS)

Sugawara, Kazuharu; Shinohara, Hiroki; Kadoya, Toshihiko; Kuramitz, Hideki

2016-01-01

To electrochemically sense lymphoma cells (U937), we fabricated a multifunctional peptide probe that consists of cell-penetrating/apoptosis-inducing/electron-transfer peptides. Electron-transfer peptides derive from cysteine residue combined with the C-terminals of four tyrosine residues (Y_4). A peptide whereby Y_4C is bound to the C-terminals of protegrin 1 (RGGRLCYCRRRFCVCVGR-NH_2) is known to be an apoptosis-inducing agent against U937 cells, and is referred to as a peptide-1 probe. An oxidation response of the peptide-1 probe has been observed due to a phenolic hydroxyl group, and this response is decreased by the uptake of the peptide probe into the cells. To improve the cell membrane permeability against U937 cells, the RGGR at the N-terminals of the peptide-1 probe was replaced by RRRR (peptide-2 probe). In contrast, RNRCKGTDVQAWY_4C (peptide-3 probe), which recognizes ovalbumin, was constructed as a control. Compared with the other probes, the change in the peak current of the peptide-2 probe was the greatest at low concentrations and occurred in a short amount of time. Therefore, the cell membrane permeability of the peptide-2 probe was increased based on the arginine residues and the apoptosis-inducing peptides. The peak current was linear and ranged from 100 to 1000 cells/ml. The relative standard deviation of 600 cells/ml was 5.0% (n = 5). Furthermore, the membrane permeability of the peptide probes was confirmed using fluorescent dye. - Highlights: • We constructed a multifunctional peptide probe for the electrochemical sensing of lymphoma cells. • The peptide probe consists of cell-penetrating/apoptosis-inducing/electron-transfer peptides. • The electrode response of the peptide probe changes due to selective uptake into the cells.

Sensing lymphoma cells based on a cell-penetrating/apoptosis-inducing/electron-transfer peptide probe

Energy Technology Data Exchange (ETDEWEB)

Sugawara, Kazuharu, E-mail: kzsuga@maebashi-it.ac.jp [Maebashi Institute of Technology, Gunma 371-0816 (Japan); Shinohara, Hiroki; Kadoya, Toshihiko [Maebashi Institute of Technology, Gunma 371-0816 (Japan); Kuramitz, Hideki [Department of Environmental Biology and Chemistry, Graduate School of Science and Engineering for Research, University of Toyama, Toyama 930-8555 (Japan)

2016-06-14

To electrochemically sense lymphoma cells (U937), we fabricated a multifunctional peptide probe that consists of cell-penetrating/apoptosis-inducing/electron-transfer peptides. Electron-transfer peptides derive from cysteine residue combined with the C-terminals of four tyrosine residues (Y{sub 4}). A peptide whereby Y{sub 4}C is bound to the C-terminals of protegrin 1 (RGGRLCYCRRRFCVCVGR-NH{sub 2}) is known to be an apoptosis-inducing agent against U937 cells, and is referred to as a peptide-1 probe. An oxidation response of the peptide-1 probe has been observed due to a phenolic hydroxyl group, and this response is decreased by the uptake of the peptide probe into the cells. To improve the cell membrane permeability against U937 cells, the RGGR at the N-terminals of the peptide-1 probe was replaced by RRRR (peptide-2 probe). In contrast, RNRCKGTDVQAWY{sub 4}C (peptide-3 probe), which recognizes ovalbumin, was constructed as a control. Compared with the other probes, the change in the peak current of the peptide-2 probe was the greatest at low concentrations and occurred in a short amount of time. Therefore, the cell membrane permeability of the peptide-2 probe was increased based on the arginine residues and the apoptosis-inducing peptides. The peak current was linear and ranged from 100 to 1000 cells/ml. The relative standard deviation of 600 cells/ml was 5.0% (n = 5). Furthermore, the membrane permeability of the peptide probes was confirmed using fluorescent dye. - Highlights: • We constructed a multifunctional peptide probe for the electrochemical sensing of lymphoma cells. • The peptide probe consists of cell-penetrating/apoptosis-inducing/electron-transfer peptides. • The electrode response of the peptide probe changes due to selective uptake into the cells.

Culture promotes transfer of thyroid epithelial cell hyperplasia and proliferation by reducing regulatory T cell numbers.

Science.gov (United States)

Kayes, Timothy D; Braley-Mullen, Helen

2013-01-01

IFN-γ(-/-) NOD.H-2h4 mice develop a spontaneous autoimmune thyroid disease, thyroid epithelial cell hyperplasia and proliferation (TEC H/P) when given NaI in their water for 7+ mo. TEC H/P can be transferred to IFN-γ(-/-) SCID mice by splenocytes from mice with severe (4-5+) disease, and transfer of TEC H/P is improved when splenocytes are cultured prior to transfer. Older (9+ mo) IFN-γ(-/-) NOD.H-2h4 mice have elevated numbers of FoxP3(+) T reg cells, up to 2-fold greater than younger (2 mo) mice. During culture, the number of T reg decreases and this allows the improved transfer of TEC H/P. Co-culture with IL-2 prior to transfer prevents the decrease of T reg and improves their in vitro suppressive ability resulting in reduced TEC H/P in recipient mice. Therefore, culturing splenocytes improves transfer of TEC H/P by reducing the number of T reg and IL-2 inhibits transfer by preserving T reg number and function. Copyright © 2013 Elsevier Inc. All rights reserved.

Exosomes Promote Ovarian Cancer Cell Invasion through Transfer of CD44 to Peritoneal Mesothelial Cells.

Science.gov (United States)

Nakamura, Koji; Sawada, Kenjiro; Kinose, Yasuto; Yoshimura, Akihiko; Toda, Aska; Nakatsuka, Erika; Hashimoto, Kae; Mabuchi, Seiji; Morishige, Ken-Ichirou; Kurachi, Hirohisa; Lengyel, Ernst; Kimura, Tadashi

2017-01-01

Epithelial ovarian cancer (EOC) cells metastasize within the peritoneal cavity and directly encounter human peritoneal mesothelial cells (HPMC) as the initial step of metastasis. The contact between ovarian cancer cells and the single layer of mesothelial cells involves direct communications that modulate cancer progression but the mechanisms are unclear. One candidate mediating cell-cell communications is exosomes, 30-100 nm membrane vesicles of endocytic origin, through the cell-cell transfer of proteins, mRNAs, or microRNAs. Therefore, the goal was to mechanistically characterize how EOC-derived exosomes modulate metastasis. Exosomes from ovarian cancer cells were fluorescently labeled and cocultured with HPMCs which internalized the exosomes. Upon exosome uptake, HPMCs underwent a change in cellular morphology to a mesenchymal, spindle phenotype. CD44, a cell surface glycoprotein, was found to be enriched in the cancer cell-derived exosomes, transferred, and internalized to HPMCs, leading to high levels of CD44 in HPMCs. This increased CD44 expression in HPMCs promoted cancer invasion by inducing the HPMCs to secrete MMP9 and by cleaning the mesothelial barrier for improved cancer cell invasion. When CD44 expression was knocked down in cancer cells, exosomes had fewer effects on HPMCs. The inhibition of exosome release from cancer cells blocked CD44 internalization in HPMCs and suppressed ovarian cancer invasion. In ovarian cancer omental metastasis, positive CD44 expression was observed in those mesothelial cells that directly interacted with cancer cells, whereas CD44 expression was negative in the mesothelial cells remote from the invading edge. This study indicates that ovarian cancer-derived exosomes transfer CD44 to HPMCs, facilitating cancer invasion. Mechanistic insight from the current study suggests that therapeutic targeting of exosomes may be beneficial in treating ovarian cancer. Mol Cancer Res; 15(1); 78-92. ©2016 AACR. ©2016 American

Nano-ranged low-energy ion-beam-induced DNA transfer in biological cells

Energy Technology Data Exchange (ETDEWEB)

Yu, L.D., E-mail: yuld@fnrf.science.cmu.ac.th [Thailand Center of Excellence in Physics, Commission on Higher Education, 328 Si Ayutthaya Road, Bangkok 10400 (Thailand); Plasma and Beam Physics Research Facility, Department of Physics and Materials Science, Faculty of Science, Chiang Mai University, Chiang Mai 50200 (Thailand); Wongkham, W. [Department of Biology, Faculty of Science, Chiang Mai University, Chiang Mai 50200 (Thailand); Prakrajang, K. [Plasma and Beam Physics Research Facility, Department of Physics and Materials Science, Faculty of Science, Chiang Mai University, Chiang Mai 50200 (Thailand); Sangwijit, K.; Inthanon, K. [Department of Biology, Faculty of Science, Chiang Mai University, Chiang Mai 50200 (Thailand); Thongkumkoon, P. [Thailand Center of Excellence in Physics, Commission on Higher Education, 328 Si Ayutthaya Road, Bangkok 10400 (Thailand); Plasma and Beam Physics Research Facility, Department of Physics and Materials Science, Faculty of Science, Chiang Mai University, Chiang Mai 50200 (Thailand); Wanichapichart, P. [Thailand Center of Excellence in Physics, Commission on Higher Education, 328 Si Ayutthaya Road, Bangkok 10400 (Thailand); Membrane Science and Technology Research Center, Department of Physics, Faculty of Science, Prince of Songkla University, Hat Yai, Songkla 90112 (Thailand); Anuntalabhochai, S. [Department of Biology, Faculty of Science, Chiang Mai University, Chiang Mai 50200 (Thailand)

2013-06-15

Low-energy ion beams at a few tens of keV were demonstrated to be able to induce exogenous macromolecules to transfer into plant and bacterial cells. In the process, the ion beam with well controlled energy and fluence bombarded living cells to cause certain degree damage in the cell envelope in nanoscales to facilitate the macromolecules such as DNA to pass through the cell envelope and enter the cell. Consequently, the technique was applied for manipulating positive improvements in the biological species. This physical DNA transfer method was highly efficient and had less risk of side-effects compared with chemical and biological methods. For better understanding of mechanisms involved in the process, a systematic study on the mechanisms was carried out. Applications of the technique were also expanded from DNA transfer in plant and bacterial cells to DNA transfection in human cancer cells potentially for the stem cell therapy purpose. Low-energy nitrogen and argon ion beams that were applied in our experiments had ranges of 100 nm or less in the cell envelope membrane which was majorly composed of polymeric cellulose. The ion beam bombardment caused chain-scission dominant damage in the polymer and electrical property changes such as increase in the impedance in the envelope membrane. These nano-modifications of the cell envelope eventually enhanced the permeability of the envelope membrane to favor the DNA transfer. The paper reports details of our research in this direction.

Nano-ranged low-energy ion-beam-induced DNA transfer in biological cells

International Nuclear Information System (INIS)

Yu, L.D.; Wongkham, W.; Prakrajang, K.; Sangwijit, K.; Inthanon, K.; Thongkumkoon, P.; Wanichapichart, P.; Anuntalabhochai, S.

2013-01-01

Low-energy ion beams at a few tens of keV were demonstrated to be able to induce exogenous macromolecules to transfer into plant and bacterial cells. In the process, the ion beam with well controlled energy and fluence bombarded living cells to cause certain degree damage in the cell envelope in nanoscales to facilitate the macromolecules such as DNA to pass through the cell envelope and enter the cell. Consequently, the technique was applied for manipulating positive improvements in the biological species. This physical DNA transfer method was highly efficient and had less risk of side-effects compared with chemical and biological methods. For better understanding of mechanisms involved in the process, a systematic study on the mechanisms was carried out. Applications of the technique were also expanded from DNA transfer in plant and bacterial cells to DNA transfection in human cancer cells potentially for the stem cell therapy purpose. Low-energy nitrogen and argon ion beams that were applied in our experiments had ranges of 100 nm or less in the cell envelope membrane which was majorly composed of polymeric cellulose. The ion beam bombardment caused chain-scission dominant damage in the polymer and electrical property changes such as increase in the impedance in the envelope membrane. These nano-modifications of the cell envelope eventually enhanced the permeability of the envelope membrane to favor the DNA transfer. The paper reports details of our research in this direction.

Rabbit embryonic stem cell lines derived from fertilized, parthenogenetic or somatic cell nuclear transfer embryos

International Nuclear Information System (INIS)

Fang, Zhen F.; Gai, Hui; Huang, You Z.; Li, Shan G.; Chen, Xue J.; Shi, Jian J.; Wu, Li; Liu, Ailian; Xu, Ping; Sheng, Hui Z.

2006-01-01

Embryonic stem cells were isolated from rabbit blastocysts derived from fertilization (conventional rbES cells), parthenogenesis (pES cells) and nuclear transfer (ntES cells), and propagated in a serum-free culture system. Rabbit ES (rbES) cells proliferated for a prolonged time in an undifferentiated state and maintained a normal karyotype. These cells grew in a monolayer with a high nuclear/cytoplasm ratio and contained a high level of alkaline phosphate activity. In addition, rbES cells expressed the pluripotent marker Oct-4, as well as EBAF2, FGF4, TDGF1, but not antigens recognized by antibodies against SSEA-1, SSEA-3, SSEA-4, TRA-1-10 and TRA-1-81. All 3 types of ES cells formed embryoid bodies and generated teratoma that contained tissue types of all three germ layers. rbES cells exhibited a high cloning efficiency, were genetically modified readily and were used as nuclear donors to generate a viable rabbit through somatic cell nuclear transfer. In combination with genetic engineering, the ES cell technology should facilitate the creation of new rabbit lines

Heavy ion induced DNA transfer in biological cells

International Nuclear Information System (INIS)

Vilaithong, T.; Yu, L.D.; Apavatjrut, P.; Phanchaisri, B.; Sangyuenyongpipat, S.; Anuntalabhochai, S.; Brown, I.G.

2004-01-01

Low-energy ion beam bombardment of biological materials for genetic modification purposes has experienced rapid growth in the last decade, particularly for the direct DNA transfer into living organisms including both plants and bacteria. Attempts have been made to understand the mechanisms involved in ion-bombardment-induced direct gene transfer into biological cells. Here we summarize the present status of the application of low-energy ions for genetic modification of living sample materials

Nitrogen and phosphorus compounds in the aleurone grains of Iris pseudoacorus endosperm and Pisum sativum cotyledons

Directory of Open Access Journals (Sweden)

Ligia Konopska

2015-01-01

Full Text Available Aleurone grains from Iris pseudoacorus endosperm and Pisum sativum cotyledons were isolated partly according to Tombs's method (1967. Nitrogen compounds content was determined in them by Kjeldahl's micromethod, and in the particular fractions after Thiman and Laloraya (1960. Mainly protein N was detected in the aleurone grains, constituting 14.8 and 15.2 per cent of the dry mass of pea and Iris seeds, respectively. Moreover, phosphorus compounds were fractionated according to Holden and Pirie (1955. Analyses demonstrated the presence in aleurone grains of inorganic P, acid-soluble organophosphorus compounds, phospholipids and RNA.

Biosynthesis of 12α-and 13-hydroxylated gibberellins in a cell-free system from Cucurbita maxima endosperm and the identification of new endogenous gibberellins.

Science.gov (United States)

Lange, T; Hedden, P; Graebe, J E

1993-03-01

Gibberellin (GA) biosynthesis in cell-free systems from Cucurbita maxima L. endosperm was reinvestigated using incubation conditions different from those employed in previous work. The metabolism of GA12 yielded GA13, GA43 and 12α-hydroxyGA43 as major products, GA4, GA37, GA39, GA46 and four unidentified compounds as minor products. The intermediates GA15, GA24 and GA25 accumulated at low protein concentrations. The structure of the previously uncharacterised 12α-hydroxyGA43 was inferred from its mass spectrum and by its formation from both GA39 and GA43. Gibberellin A39 and 12α-hydroxyGA43 were formed by a soluble 12α-hydroxylase that had not been detected before. Gibberellin A12-aldehyde was metabolised to essentially the same products as GA12 but with less efficiency. A new 13-hydroxylation pathway was found. Gibberellin A53, formed from GA12 by a microsomal oxidase, was converted by soluble 2-oxoglutarate-dependent oxidases to GA1 GA23, GA28, GA44, and putative 2β-hydroxyGA28. Minor products were GA19, GA20, GA38 and three unidentified GAs. Microsomal 13-hydroxylation (the formation of GA53) was suppressed by the cofactors for 2-oxoglutarate-dependent enzymes. Reinvestigation of the endogenous GAs confirmed the significance of the new metabolic products. In addition to the endogenous GAs reported by Blechschmidt et al. (1984, Phytochemistry 23, 553-558), GA1, GA8, GA25, GA28, GA36, GA48 and 12α-hydroxyGA43 were identified by full-scan capillary gas chromatography-mass spectrometry and Kovats retention indices. Thus both the 12α-hydroxylation and the 13-hydroxylation pathways found in the cell-free system operate also in vivo, giving rise to 12α-hydroxyGA43 and GA1 (or GA8), respectively, as their end products. Evidence for endogenous GA20 and GA24 was also obtained but it was less conclusive due to interference.

Morphological classification of plant cell deaths

DEFF Research Database (Denmark)

van Doorn, W.G.; Beers, E.P.; Dangl, J.L.

2011-01-01

, which can express features of both necrosis and vacuolar cell death, PCD in starchy cereal endosperm and during self-incompatibility. The present classification is not static, but will be subject to further revision, especially when specific biochemical pathways are better defined....... the classification of PCD in plants. Here we suggest a classification based on morphological criteria. According to this classification, the use of the term 'apoptosis' is not justified in plants, but at least two classes of PCD can be distinguished: vacuolar cell death and necrosis. During vacuolar cell death...

Adoptive T cell transfer for cancer immunotherapy in the era of synthetic biology.

Science.gov (United States)

Kalos, Michael; June, Carl H

2013-07-25

Adoptive T cell transfer for cancer and chronic infection is an emerging field that shows promise in recent trials. Synthetic-biology-based engineering of T lymphocytes to express high-affinity antigen receptors can overcome immune tolerance, which has been a major limitation of immunotherapy-based strategies. Advances in cell engineering and culture approaches to enable efficient gene transfer and ex vivo cell expansion have facilitated broader evaluation of this technology, moving adoptive transfer from a "boutique" application to the cusp of a mainstream technology. The major challenge currently facing the field is to increase the specificity of engineered T cells for tumors, because targeting shared antigens has the potential to lead to on-target off-tumor toxicities, as observed in recent trials. As the field of adoptive transfer technology matures, the major engineering challenge is the development of automated cell culture systems, so that the approach can extend beyond specialized academic centers and become widely available. Copyright © 2013 Elsevier Inc. All rights reserved.

The biomechanics of seed germination.

Science.gov (United States)

Steinbrecher, Tina; Leubner-Metzger, Gerhard

2017-02-01

From a biomechanical perspective, the completion of seed (and fruit) germination depends on the balance of two opposing forces: the growth potential of the embryonic axis (radicle-hypocotyl growth zone) and the restraint of the seed-covering layers (endosperm, testa, and pericarp). The diverse seed tissues are composite materials which differ in their dynamic properties based on their distinct cell wall composition and water uptake capacities. The biomechanics of embryo cell growth during seed germination depend on irreversible cell wall loosening followed by water uptake due to the decreasing turgor, and this leads to embryo elongation and eventually radicle emergence. Endosperm weakening as a prerequisite for radicle emergence is a widespread phenomenon among angiosperms. Research into the biochemistry and biomechanics of endosperm weakening has demonstrated that the reduction in puncture force of a seed's micropylar endosperm is environmentally and hormonally regulated and involves tissue-specific expression of cell wall remodelling proteins such as expansins, diverse hydrolases, and the production of directly acting apoplastic reactive oxygen. The endosperm-weakening biomechanics and its underlying cell wall biochemistry differ between the micropylar (ME) and chalazal (CE) endosperm domains. In the ME, they involve cell wall loosening, cell separation, and programmed cell death to provide decreased and localized ME tissue resistance, autolysis, and finally the formation of an ME hole required for radicle emergence. Future work will further unravel the molecular mechanisms, environmental regulation, and evolution of the diverse biomechanical cell wall changes underpinning the control of germination by endosperm weakening. © The Author 2016. Published by Oxford University Press on behalf of the Society for Experimental Biology. All rights reserved. For permissions, please email: journals.permissions@oup.com.

Influences of somatic donor cell sex on and embryo development following somatic cell nuclear transfer in pigs

Directory of Open Access Journals (Sweden)

Jae-Gyu Yoo

2017-04-01

Full Text Available Objective The present study investigates pre- and post-implantation developmental competence of nuclear-transferred porcine embryos derived from male and female fetal fibroblasts. Methods Male and female fetal fibroblasts were transferred to in vitro-matured enucleated oocytes and in vitro and in vivo developmental competence of reconstructed embryos was investigated. And, a total of 6,789 female fibroblast nuclear-transferred embryos were surgically transferred into 41 surrogate gilts and 4,746 male fibroblast nuclear-transferred embryos were surgically transferred into 25 surrogate gilts. Results The competence to develop into blastocysts was not significantly different between the sexes. The mean cell number of female and male cloned blastocysts obtained by in vivo culture (143.8±10.5 to 159.2±14.8 was higher than that of in vitro culture of somatic cell nuclear transfer (SCNT groups (31.4±8.3 to 33.4±11.1. After embryo transfer, 5 pregnant gilts from each treatment delivered 15 female and 22 male piglets. The average birth weight of the cloned piglets, gestation length, and the postnatal survival rates were not significantly different (p<0.05 between sexes. Conclusion The present study found that the sex difference of the nuclear donor does not affect the developmental rate of porcine SCNT embryos. Furthermore, postnatal survivability of the cloned piglets was not affected by the sex of the donor cell.

Role of zein proteins in structure and assembly of protein bodies and endosperm texture. Progress report and appendix 1 - preliminary data

Energy Technology Data Exchange (ETDEWEB)

Larkins, B.

1997-05-01

Although funding for this project was initiated less than two years ago, we have made significant progress with our research objectives. We have cloned the gene responsible for the fl2 mutation. In fl2, the mutant phenotype appears to result from a defective signal peptide in an alpha-zein protein. As a consequence, the signal peptide remains attached when the protein accumulates in the protein body. A mutation like fl2 could explain other semidominant and dominant opaque mutants on the basis of abnormal zein polypeptides. A manuscript describing the research that led to the cloning of fl2 is in press, and a second manuscript on the characterization of this gene has been prepared for publication. We found that increased amounts of the 27-kD gamma-zein protein enlarge the proportion of vitreous endosperm and increases the hardness of o2 mutants. This protein also enhances these properties in wild type seeds. The mechanism by which the gamma-zein protein brings about these changes is unclear, and is under investigation. We have found and characterized several mutants that reduce gamma-zein synthesis. The mutations do not significantly affect synthesis of any other type of zein protein. They appear to create an opaque phenotype by reducing the number rather than the size of protein bodies. Interestingly, the mutant seeds fail to germinate. A manuscript describing one of these mutants, o15, has been prepared for publication. We have created a number of transgenic tobacco plants that can produce alpha-, beta-, gamma(27-kD)-, or delta-zeins, as well as combinations of these proteins. Analysis of seeds from these plants and crosses of these plants has shown that tobacco endosperm can serve as a heterologous system to study zein interactions. We have obtained evidence that interactions between alpha- and gamma-zein proteins are required for stable accumulation of alpha-zeins in the endosperm. These and other preliminary results are illustrated in Appendix 1.

Biochip-based study of unidirectional mitochondrial transfer from stem cells to myocytes via tunneling nanotubes

International Nuclear Information System (INIS)

Yang, Huaxiao; Gao, Bruce Z; Borg, Thomas K; Markwald, Roger; Ma, Zhen; Xu, Meifeng; Wetzel, George; Saraf, Laxmikant V; Runyan, Raymond B

2016-01-01

Tunneling nanotubes (TNTs) are small membranous tubes of 50–1000 nm diameter observed to connect cells in culture. Transfer of subcellular organelles through TNTs was observed in vitro and in vivo, but the formation and significance of these structures is not well understood. A polydimethylsiloxane biochip-based coculture model was devised to constrain TNT orientation and explore both TNT-formation and TNT-mediated mitochondrial transfer. Two parallel microfluidic channels connected by an array of smaller microchannels enabled localization of stem cell and cardiomyocyte populations while allowing connections to form between them. Stem cells and cardiomyocytes were deposited in their respective microfluidic channels, and stem cell-cardiomyocyte pairs were formed via the microchannels. Formation of TNTs and transfer of stained mitochondria through TNTs was observed by 24 h real-time video recording. The data show that stem cells are 7.7 times more likely to initiate contact by initial extension of filopodia. By 24 h, 67% of nanotube connections through the microchannels are composed of cardiomyocyte membrane. Filopodial extension and retraction by stem cells draws an extension of TNTs from cardiomyocytes. MitoTracker staining shows that unidirectional transfer of mitochondria between stem cell-cardiomyocyte pairs invariably originates from stem cells. Control experiments with cardiac fibroblasts and cardiomyocytes show little nanotube formation between homotypic or mixed cell pairs and no mitochondrial transfer. These data identify a novel biological process, unidirectional mitochondrial transfer, mediated by heterotypic TNT connections. This suggests that the enhancement of cardiomyocyte function seen after stem-cell injection may be due to a bioenergetic stimulus provided by mitochondrial transfer. (paper)

Towards a Biohybrid Lung: Endothelial Cells Promote Oxygen Transfer through Gas Permeable Membranes.

Science.gov (United States)

Menzel, Sarah; Finocchiaro, Nicole; Donay, Christine; Thiebes, Anja Lena; Hesselmann, Felix; Arens, Jutta; Djeljadini, Suzana; Wessling, Matthias; Schmitz-Rode, Thomas; Jockenhoevel, Stefan; Cornelissen, Christian Gabriel

2017-01-01

In patients with respiratory failure, extracorporeal lung support can ensure the vital gas exchange via gas permeable membranes but its application is restricted by limited long-term stability and hemocompatibility of the gas permeable membranes, which are in contact with the blood. Endothelial cells lining these membranes promise physiological hemocompatibility and should enable prolonged application. However, the endothelial cells increase the diffusion barrier of the blood-gas interface and thus affect gas transfer. In this study, we evaluated how the endothelial cells affect the gas exchange to optimize performance while maintaining an integral cell layer. Human umbilical vein endothelial cells were seeded on gas permeable cell culture membranes and cultivated in a custom-made bioreactor. Oxygen transfer rates of blank and endothelialized membranes in endothelial culture medium were determined. Cell morphology was assessed by microscopy and immunohistochemistry. Both setups provided oxygenation of the test fluid featuring small standard deviations of the measurements. Throughout the measuring range, the endothelial cells seem to promote gas transfer to a certain extent exceeding the blank membranes gas transfer performance by up to 120%. Although the underlying principles hereof still need to be clarified, the results represent a significant step towards the development of a biohybrid lung.

Uptake and utilization of nutrients by developing kernels of Zea mays L

International Nuclear Information System (INIS)

Lyznik, L.A.

1987-01-01

The mechanisms involved in amino acid and sugar uptake by developing maize kernels were investigated. In the pedicel region of maize kernel, the site of nutrient unloading from phloem terminals, amino acids are accumulated in considerable amounts and undergo significant interconversion. A wide spectrum of enzymatic activities involved in the metabolism of amino acids is observed in these tissues. Subsequently, amino acids are taken up by the endosperm tissue in processes which require energy and the presence of carrier proteins. Conversely, no evidence was found that energy and carriers are involved in sugar uptake. This process of sugar uptake is not inhibited by metabolic inhibitors and shows nonsaturable kinetics, but the uptake is pH-dependent. L-glucose is taken up at a significantly reduced rate in comparison to D-glucose uptake. Based on analysis of radioactivity distribution among sugar fractions after incubations of kernels with radiolabeled D-glucose, it seems that sucrose is not efficiently resynthesized from D-glucose in the endosperm tissue. Thus, the proposed mechanism of sucrose transport involving sucrose hydrolysis in the pedicel region and subsequent resynthesis in endosperm cells may not be the main pathway. The evidence that transfer cells play an active role in D-glucose transport is presented

Cloning animals by somatic cell nuclear transfer – biological factors

Science.gov (United States)

Tian, X Cindy; Kubota, Chikara; Enright, Brian; Yang, Xiangzhong

2003-01-01

Cloning by nuclear transfer using mammalian somatic cells has enormous potential application. However, somatic cloning has been inefficient in all species in which live clones have been produced. High abortion and fetal mortality rates are commonly observed. These developmental defects have been attributed to incomplete reprogramming of the somatic nuclei by the cloning process. Various strategies have been used to improve the efficiency of nuclear transfer, however, significant breakthroughs are yet to happen. In this review we will discuss studies conducted, in our laboratories and those of others, to gain a better understanding of nuclear reprogramming. Because cattle are a species widely used for nuclear transfer studies, and more laboratories have succeeded in cloning cattle than any other specie, this review will be focused on somatic cell cloning of cattle. PMID:14614770

Excimer laser forward transfer of mammalian cells using a novel triazene absorbing layer

International Nuclear Information System (INIS)

Doraiswamy, A.; Narayan, R.J.; Lippert, T.; Urech, L.; Wokaun, A.; Nagel, M.; Hopp, B.; Dinescu, M.; Modi, R.; Auyeung, R.C.Y.; Chrisey, D.B.

2006-01-01

We present a novel laser-based approach for developing tissue engineered constructs and other cell-based assembly's. We have deposited mesoscopic patterns of viable B35 neuroblasts using a soft direct approach of the matrix assisted pulsed laser evaporation direct write (MAPLE DW) process. As a development of the conventional direct write process, an intermediate layer of absorbing triazene polymer is used to provide gentler and efficient transfers. Transferred cells were examined for viability and proliferation and compared with that of as-seeded cells to determine the efficacy of the process. Results suggest that successful transfers can be achieved at lower fluences than usual by the incorporation of the intermediate absorbing layer thus avoiding any damage to cells and other delicate materials. MAPLE DW offers rapid computer-controlled deposition of mesoscopic voxels at high spatial resolutions, with extreme versatility in depositing combinations of natural/synthetic, living/non-living, organic/inorganic and hard/soft materials. Our approach offers a gentle and efficient transfer of viable cells which when combined with a variety of matrix materials allows development of constructs and bioactive systems in bioengineering

Role of T cells in sex differences in syngeneic bone marrow transfers

International Nuclear Information System (INIS)

Raveche, E.S.; Santoro, T.; Brecher, G.; Tjio, J.H.

1985-01-01

Transferred marrow cells will proliferate in normal mice not exposed to irradiation or any other type of stem cell depletion when five consecutive transfers of 40 million cells are given. Approximately 25% of the mitotic cells are of male donor origin observed cytogenetically in all of the female recipient spleens and marrow analyzed from two weeks to one and one-half years after transfusions. Male donor stem cells are accepted and form a stable component of the self-renewing stem cell pool. In contrast, only 5% female cells are found in male recipients. This sex difference in engraftment is not hormonal since castration of recipients does not alter the percentage of donor cells. Rigorous T depletion of female donor bone marrow, however, increases the percentage of donor engraftment to the level observed when male marrow, either whole or T depleted, is transferred to female recipients. The success of T-depleted female stem cells to seed male recipients is observed in both C57BL/6 and CBA/J. In addition, recipient nude BALB/c males, which lack a thymus, fail to accept whole bone marrow from BALB/c females. However, male bone marrow cells seed BALB/c nude females. These studies demonstrate that the poor engraftment of female cells in transfused male recipients is abrogated by the removal of T cells from the donor female marrow

Membrane microparticles mediate transfer of P-glycoprotein to drug sensitive cancer cells.

Science.gov (United States)

Bebawy, M; Combes, V; Lee, E; Jaiswal, R; Gong, J; Bonhoure, A; Grau, G E R

2009-09-01

Multidrug resistance (MDR), a significant impediment to the successful treatment of cancer clinically, has been attributed to the overexpression of P-glycoprotein (P-gp), a plasma membrane multidrug efflux transporter. P-gp maintains sublethal intracellular drug concentrations by virtue of its drug efflux capacity. The cellular regulation of P-gp expression is currently known to occur at either pre- or post-transcriptional levels. In this study, we identify a 'non-genetic' mechanism whereby microparticles (MPs) serve as vectors in the acquisition and spread of MDR. MPs isolated from drug-resistant cancer cells (VLB(100)) were co-cultured with drug sensitive cells (CCRF-CEM) over a 4 h period to allow for MP binding and P-gp transfer. Presence of P-gp on MPs was established using flow cytometry (FCM) and western blotting. Whole-cell drug accumulation assays using rhodamine 123 and daunorubicin (DNR) were carried out to validate the transfer of functional P-gp after co-culture. We establish that MPs shed in vitro from drug-resistant cancer cells incorporate cell surface P-gp from their donor cells, effectively bind to drug-sensitive recipient cells and transfer functional P-gp to the latter. These findings serve to substantially advance our understanding of the molecular basis for the emergence of MDR in cancer clinically and lead to new treatment strategies which target and inhibit MP mediated transfer of P-gp during the course of treatment.

Generation of transgenic wheat (Triticum aestivum L.) accumulating heterologous endo-xylanase or ferulic acid esterase in the endosperm.

Science.gov (United States)

Harholt, Jesper; Bach, Inga C; Lind-Bouquin, Solveig; Nunan, Kylie J; Madrid, Susan M; Brinch-Pedersen, Henrik; Holm, Preben B; Scheller, Henrik V

2010-04-01

Endo-xylanase (from Bacillus subtilis) or ferulic acid esterase (from Aspergillus niger) were expressed in wheat under the control of the endosperm-specific 1DX5 glutenin promoter. Constructs both with and without the endoplasmic reticulum retention signal (Lys-Asp-Glu-Leu) KDEL were used. Transgenic plants were recovered in all four cases but no qualitative differences could be observed whether KDEL was added or not. Endo-xylanase activity in transgenic grains was increased between two and threefold relative to wild type. The grains were shrivelled and had a 25%-33% decrease in mass. Extensive analysis of the cell walls showed a 10%-15% increase in arabinose to xylose ratio, a 50% increase in the proportion of water-extractable arabinoxylan, and a shift in the MW of the water-extractable arabinoxylan from being mainly larger than 85 kD to being between 2 and 85 kD. Ferulic acid esterase-expressing grains were also shrivelled, and the seed weight was decreased by 20%-50%. No ferulic acid esterase activity could be detected in wild-type grains whereas ferulic acid esterase activity was detected in transgenic lines. The grain cell walls had 15%-40% increase in water-unextractable arabinoxylan and a decrease in monomeric ferulic acid between 13% and 34%. In all the plants, the observed changes are consistent with a plant response that serves to minimize the effect of the heterologously expressed enzymes by increasing arabinoxylan biosynthesis and cross-linking.

Generation of transgenic wheat (Triticum aestivum L.) accumulating heterologous endo-xylanase or ferulic acid esterase in the endosperm

Energy Technology Data Exchange (ETDEWEB)

Harholt, Jesper; Bach, Inga C; Lind-Bouquin, Solveig; Nunan, Kylie J.; Madrid, Susan M.; Brinch-Pedersen, Henrik; Holm, Preben B.; Scheller, Henrik V.

2009-12-08

Endo-xylanase (from Bacillus subtilis) or ferulic acid esterase (from Aspergillus niger) were expressed in wheat under the control of the endosperm specific 1DX5 glutenin promoter. Constructs both with and without the endoplasmic reticulum retention signal KDEL were used. Transgenic plants were recovered in all four cases but no qualitative differences could be observed whether KDEL was added or not. Endo-xylanase activity in transgenic grains was increased between two and three fold relative to wild type. The grains were shriveled and had a 25-33% decrease in mass. Extensive analysis of the cell walls showed a 10-15% increase in arabinose to xylose ratio, a 50% increase in the proportion of water extractable arabinoxylan, and a shift in the MW of the water extractable arabinoxylan from being mainly larger than 85 kD to being between 2 kD and 85 kD. Ferulic acid esterase expressing grains were also shriveled and the seed weight was decreased by 20-50%. No ferulic acid esterase activity could be detected in wild type grains whereas ferulic acid esterase activity was detected in transgenic lines. The grain cell walls had 15-40% increase in water unextractable arabinoxylan and a decrease in monomeric ferulic acid between 13 and 34%. In all the plants the observed changes are consistent with a plant response that serves to minimize the effect of the heterologously expressed enzymes by increasing arabinoxylan biosynthesis and cross-linking.

On the track of transfer cell formation by specialized plant-parasitic nematodes.

Science.gov (United States)

Rodiuc, Natalia; Vieira, Paulo; Banora, Mohamed Youssef; de Almeida Engler, Janice

2014-01-01

Transfer cells are ubiquitous plant cells that play an important role in plant development as well as in responses to biotic and abiotic stresses. They are highly specialized and differentiated cells playing a central role in the acquisition, distribution and exchange of nutrients. Their unique structural traits are characterized by augmented ingrowths of invaginated secondary wall material, unsheathed by an amplified area of plasma membrane enriched in a suite of solute transporters. Similar morphological features can be perceived in vascular root feeding cells induced by sedentary plant-parasitic nematodes, such as root-knot and cyst nematodes, in a wide range of plant hosts. Despite their close phylogenetic relationship, these obligatory biotrophic plant pathogens engage different approaches when reprogramming root cells into giant cells or syncytia, respectively. Both nematode feeding-cells types will serve as the main source of nutrients until the end of the nematode life cycle. In both cases, these nematodes are able to remarkably maneuver and reprogram plant host cells. In this review we will discuss the structure, function and formation of these specialized multinucleate cells that act as nutrient transfer cells accumulating and synthesizing components needed for survival and successful offspring of plant-parasitic nematodes. Plant cells with transfer-like functions are also a renowned subject of interest involving still poorly understood molecular and cellular transport processes.

On the track of transfer cells formation by specialized plant-parasitic nematodes

Directory of Open Access Journals (Sweden)

Natalia eRodiuc

2014-05-01

Full Text Available Transfer cells are ubiquitous plant cells that play an important role in plant development as well as in responses to biotic and abiotic stresses. They are highly specialized and differentiated cells playing a central role in the acquisition, distribution and exchange of nutrients. Their unique structural traits are characterized by augmented ingrowths of invaginated secondary wall material, unsheathed by an amplified area of plasma membrane enriched in a suite of solute transporters. Similar morphological features can be perceived in vascular root feeding cells induced by sedentary plant-parasitic nematodes, such as root-knot and cyst nematodes, in a wide range of plant hosts. Despite their close phylogenetic relationship, these obligatory biotrophic plant pathogens engage different approaches when reprogramming root cells into giant cells or syncytia, respectively. Both nematode feeding-cells types will serve as the main source of nutrients until the end of the nematode life cycle. In both cases, these nematodes are able to remarkably maneuver and reprogram plant host cells. In this review we will discuss the structural, functional and morphogenetic characteristics function and formation of these specialized multinucleate cells that act as nutrient transfer cells to accumulate and synthesize components needed for survival and successful offspring of plant-parasitic nematodes. Plant cells with transfer-like functions are also a renowned subject of interest involving still poorly understood molecular and cellular transport processes.

In vitro study for laser gene transfer in BHK-21 fibroblast cell line

Science.gov (United States)

Abdel Aziz, M.; Salem, D. S.; Salama, M. S.; Badr, Y.

2009-02-01

Modifications to our previously introduced system for laser microbeam cell surgery were carried out in the present work to match animal cells. These modifications included: 1- Using other laser system that used before, Excimer laser with 193 and 308 nm wavelengths. The used laser here, is He-Cd with low power and 441.5 nm wavelength in the visible region. 2- Instead of using pulsed laser, we used here CW He-Cd chopped by electrical chopper, which is synchronized with the mechanical motion of the mobile stage with step 40 microns, according to cell dimensions to avoid puncturing the same cell twice. The advantages of the modified here laser setup for gene transfer is: it is less damaging to the sensitive animal cell which has thin cell membrane. The present work aimed to: 1- Design a modified laser microbeam cell surgery, applicable to animal cells, such as fibroblast cells 2- To examine the efficiency of such system. 3- To assure gene transfer and its expression in the used cells. 4- To evaluate the ultra damages produced from using the laser beam as a modality for gene transfer. On the other wards, to introduce: safe, efficient and less damaging modality for gene transfer in animal cells. To achieve these goals, we applied the introduced here home-made laser setup with its synchronized parameters to introduce pBK-CMV phagemid, containing LacZ and neomycin resistance (neor )genes into BHK-21 fibroblast cell line. The results of the present work showed that: 1- Our modified laser microbeam cell surgery setup proved to be useful and efficient tool for gene transfer into fibroblast cells. 2- The presence and expression of LacZ gene was achieved using histochemical LacZ assay. 3- Selection of G418 antibiotic sensitivity assay confirmed the presence and expression towards stability of neor gene with time. 4- Presence of LacZ and neor genes in the genomic DNA of transfected fibroblast cells was indicated using PCR analysis. 5- Transmission electron microscopy indicated

Inducible cell death in plant immunity

DEFF Research Database (Denmark)

Hofius, Daniel; Tsitsigiannis, Dimitrios I; Jones, Jonathan D G

2006-01-01

Programmed cell death (PCD) occurs during vegetative and reproductive plant growth, as typified by autumnal leaf senescence and the terminal differentiation of the endosperm of cereals which provide our major source of food. PCD also occurs in response to environmental stress and pathogen attack......, and these inducible PCD forms are intensively studied due their experimental tractability. In general, evidence exists for plant cell death pathways which have similarities to the apoptotic, autophagic and necrotic forms described in yeast and metazoans. Recent research aiming to understand these pathways...

Functional and structural characterization of plastidic starch phosphorylase during barley endosperm development

DEFF Research Database (Denmark)

Cuesta-Seijo, Jose A.; Ruzanski, Christian; Krucewicz, Katarzyna

2017-01-01

The production of starch is essential for human nutrition and represents a major metabolic flux in the biosphere. The biosynthesis of starch in storage organs like barley endosperm operates via two main pathways using different substrates: starch synthases use ADP-glucose to produce amylose......,4-glucans using HvPho1 from G1P as the sole substrate. The structural properties of HvPho1 provide insights into the low affinity of HvPho1 for large polysaccharides like starch or amylopectin. Our results suggest that HvPho1 may play a role during the initiation of starch biosynthesis in barley....... and amylopectin, the two major components of starch, whereas starch phosphorylase (Pho1) uses glucose-1-phosphate (G1P), a precursor for ADP-glucose production, to produce α-1,4 glucans. The significance of the Pho1 pathway in starch biosynthesis has remained unclear. To elucidate the importance of barley Pho1...

Telomere Elongation and Naive Pluripotent Stem Cells Achieved from Telomerase Haplo-Insufficient Cells by Somatic Cell Nuclear Transfer

Directory of Open Access Journals (Sweden)

Li-Ying Sung

2014-12-01

Full Text Available Summary: Haplo-insufficiency of telomerase genes in humans leads to telomere syndromes such as dyskeratosis congenital and idiopathic pulmonary fibrosis. Generation of pluripotent stem cells from telomerase haplo-insufficient donor cells would provide unique opportunities toward the realization of patient-specific stem cell therapies. Recently, pluripotent human embryonic stem cells (ntESCs have been efficiently achieved by somatic cell nuclear transfer (SCNT. We tested the hypothesis that SCNT could effectively elongate shortening telomeres of telomerase haplo-insufficient cells in the ntESCs with relevant mouse models. Indeed, telomeres of telomerase haplo-insufficient (Terc+/− mouse cells are elongated in ntESCs. Moreover, ntESCs derived from Terc+/− cells exhibit naive pluripotency as evidenced by generation of Terc+/− ntESC clone pups by tetraploid embryo complementation, the most stringent test of naive pluripotency. These data suggest that SCNT could offer a powerful tool to reprogram telomeres and to discover the factors for robust restoration of telomeres and pluripotency of telomerase haplo-insufficient somatic cells. : Sung et al. demonstrate in a mouse model that telomeres of telomerase haplo-insufficient cells can be elongated by somatic cell nuclear transfer. Moreover, ntESCs derived from Terc+/− cells exhibit pluripotency evidenced by generation of Terc+/−ntESC clone pups by tetraploid embryo complementation, the most stringent test of naive pluripotency.

EXOSOMES AND TRANSFER OF (EPIGENETIC INFORMATION BY TUMOR CELLS

Directory of Open Access Journals (Sweden)

E. M. Tchevkina

2015-01-01

Full Text Available In this review, we will introduce the current knowledge about exosomes – vesicles that are generated in the cells and released into the extracellular space. Exosomes are forming in the cell plasma membrane and represent the spherical shapes restricted by their membrane and contained the various biomolecules including nucleic acids, proteins, lipids etc. The intent interest to exosomes is based on their ability to horizontal transfer between the cells, to permeate into vascular system reaching the different tissues and to incorporate into the recipient cells. It was shown that exosome incorporation into the cells lead to remarkable changes in the recipient cells both in genomic level (via the integration of exosomal DNA into the host DNA and in epigenomic level (via the modulation of the content and/or activity of the signaling proteins, microRNA etc.. Undoubtedly, one of the most interesting and perspective achievements in the exosome study is the demonstration of exosome ability to provide the horizontal transfer of the genetic information from cell to cell – the fact supported in the different studies with the various cell models. Here, we will discuss the recent data regarding the main characteristics and properties of exosomes, the role of exosomes in the tumorigenesis including neoplastic transformation, metastasis, multi-drug resistance. The final part of the review involves the most growing area in the exosome study – the possible usage of exosomes in the cancer treatment, in particular – as the specific drug delivery system.

Pollination with heavily irradiated pollen in Nicotiana: induced parthenogenesis and embryological study

International Nuclear Information System (INIS)

Musial, K.; Przywara, L.

1999-01-01

Nicotiana crosses were pollinated in situ and in vitro with heavily irradiated pollen (500, 700, 1000 Gy) to induce parthenogenesis and to study the development of embryo and endosperm. Haploids were obtained after in situ pollination only; however, parthenogenetic proembryos occurred also after in vitro pollination. It was demonstrated that ovule culture following pollination offers a better chance to produce haploids than undisturbed pollination does. Pollination with irradiated pollen (PwIP) stron gly decreased the number of endosperm cells and the size of embryo sacs, and it affected the development of embryos; no significant differences between applied irradiation doses were found. Ovules with endosperm only, embryo only, and with both embryo and endosperm were observed. The most frequent were the ovules with endosperm only, the rarest with embryo only. A small amount of storage products occurred in the endosperm cells. The diploid chromosome number counted in the endosperm produced after PwIP points to their origin without fertilization. An interesting phenomenon observed after PwIP was vigorous growth of endothelium. (author)

Viruses transfer the antiviral second messenger cGAMP between cells.

Science.gov (United States)

Bridgeman, A; Maelfait, J; Davenne, T; Partridge, T; Peng, Y; Mayer, A; Dong, T; Kaever, V; Borrow, P; Rehwinkel, J

2015-09-11

Cyclic GMP-AMP synthase (cGAS) detects cytosolic DNA during virus infection and induces an antiviral state. cGAS signals by synthesis of a second messenger, cyclic GMP-AMP (cGAMP), which activates stimulator of interferon genes (STING). We show that cGAMP is incorporated into viral particles, including lentivirus and herpesvirus virions, when these are produced in cGAS-expressing cells. Virions transferred cGAMP to newly infected cells and triggered a STING-dependent antiviral program. These effects were independent of exosomes and viral nucleic acids. Our results reveal a way by which a signal for innate immunity is transferred between cells, potentially accelerating and broadening antiviral responses. Moreover, infection of dendritic cells with cGAMP-loaded lentiviruses enhanced their activation. Loading viral vectors with cGAMP therefore holds promise for vaccine development. Copyright © 2015, American Association for the Advancement of Science.

Efficient gene transfer into nondividing cells by adeno-associated virus-based vectors.

Science.gov (United States)

Podsakoff, G; Wong, K K; Chatterjee, S

1994-09-01

Gene transfer vectors based on adeno-associated virus (AAV) are emerging as highly promising for use in human gene therapy by virtue of their characteristics of wide host range, high transduction efficiencies, and lack of cytopathogenicity. To better define the biology of AAV-mediated gene transfer, we tested the ability of an AAV vector to efficiently introduce transgenes into nonproliferating cell populations. Cells were induced into a nonproliferative state by treatment with the DNA synthesis inhibitors fluorodeoxyuridine and aphidicolin or by contact inhibition induced by confluence and serum starvation. Cells in logarithmic growth or DNA synthesis arrest were transduced with vCWR:beta gal, an AAV-based vector encoding beta-galactosidase under Rous sarcoma virus long terminal repeat promoter control. Under each condition tested, vCWR:beta Gal expression in nondividing cells was at least equivalent to that in actively proliferating cells, suggesting that mechanisms for virus attachment, nuclear transport, virion uncoating, and perhaps some limited second-strand synthesis of AAV vectors were present in nondividing cells. Southern hybridization analysis of vector sequences from cells transduced while in DNA synthetic arrest and expanded after release of the block confirmed ultimate integration of the vector genome into cellular chromosomal DNA. These findings may provide the basis for the use of AAV-based vectors for gene transfer into quiescent cell populations such as totipotent hematopoietic stem cells.

A novel mechanism of bacterial toxin transfer within host blood cell-derived microvesicles.

Directory of Open Access Journals (Sweden)

Anne-lie Ståhl

2015-02-01

Full Text Available Shiga toxin (Stx is the main virulence factor of enterohemorrhagic Escherichia coli, which are non-invasive strains that can lead to hemolytic uremic syndrome (HUS, associated with renal failure and death. Although bacteremia does not occur, bacterial virulence factors gain access to the circulation and are thereafter presumed to cause target organ damage. Stx was previously shown to circulate bound to blood cells but the mechanism by which it would potentially transfer to target organ cells has not been elucidated. Here we show that blood cell-derived microvesicles, shed during HUS, contain Stx and are found within patient renal cortical cells. The finding was reproduced in mice infected with Stx-producing Escherichia coli exhibiting Stx-containing blood cell-derived microvesicles in the circulation that reached the kidney where they were transferred into glomerular and peritubular capillary endothelial cells and further through their basement membranes followed by podocytes and tubular epithelial cells, respectively. In vitro studies demonstrated that blood cell-derived microvesicles containing Stx undergo endocytosis in glomerular endothelial cells leading to cell death secondary to inhibited protein synthesis. This study demonstrates a novel virulence mechanism whereby bacterial toxin is transferred within host blood cell-derived microvesicles in which it may evade the host immune system.

Highly Efficient Transfer of Chromosomes to a Broad Range of Target Cells Using Chinese Hamster Ovary Cells Expressing Murine Leukemia Virus-Derived Envelope Proteins.

Directory of Open Access Journals (Sweden)

Teruhiko Suzuki

Full Text Available Microcell-mediated chromosome transfer (MMCT is an essential step for introducing chromosomes from donor cells to recipient cells. MMCT allows not only for genetic/epigenetic analysis of specific chromosomes, but also for utilization of human and mouse artificial chromosomes (HACs/MACs as gene delivery vectors. Although the scientific demand for genome scale analyses is increasing, the poor transfer efficiency of the current method has hampered the application of chromosome engineering technology. Here, we developed a highly efficient chromosome transfer method, called retro-MMCT, which is based on Chinese hamster ovary cells expressing envelope proteins derived from ecotropic or amphotropic murine leukemia viruses. Using this method, we transferred MACs to NIH3T3 cells with 26.5 times greater efficiency than that obtained using the conventional MMCT method. Retro-MMCT was applicable to a variety of recipient cells, including embryonic stem cells. Moreover, retro-MMCT enabled efficient transfer of MAC to recipient cells derived from humans, monkeys, mice, rats, and rabbits. These results demonstrate the utility of retro-MMCT for the efficient transfer of chromosomes to various types of target cell.

Membrane Transfer from Mononuclear Cells to Polymorphonuclear Neutrophils Transduces Cell Survival and Activation Signals in the Recipient Cells via Anti-Extrinsic Apoptotic and MAP Kinase Signaling Pathways.

Science.gov (United States)

Li, Ko-Jen; Wu, Cheng-Han; Shen, Chieh-Yu; Kuo, Yu-Min; Yu, Chia-Li; Hsieh, Song-Chou

2016-01-01

The biological significance of membrane transfer (trogocytosis) between polymorphonuclear neutrophils (PMNs) and mononuclear cells (MNCs) remains unclear. We investigated the biological/immunological effects and molecular basis of trogocytosis among various immune cells in healthy individuals and patients with active systemic lupus erythematosus (SLE). By flow cytometry, we determined that molecules in the immunological synapse, including HLA class-I and-II, CD11b and LFA-1, along with CXCR1, are exchanged among autologous PMNs, CD4+ T cells, and U937 cells (monocytes) after cell-cell contact. Small interfering RNA knockdown of the integrin adhesion molecule CD11a in U937 unexpectedly enhanced the level of total membrane transfer from U937 to PMN cells. Functionally, phagocytosis and IL-8 production by PMNs were enhanced after co-culture with T cells. Total membrane transfer from CD4+ T to PMNs delayed PMN apoptosis by suppressing the extrinsic apoptotic molecules, BAX, MYC and caspase 8. This enhancement of activities of PMNs by T cells was found to be mediated via p38- and P44/42-Akt-MAP kinase pathways and inhibited by the actin-polymerization inhibitor, latrunculin B, the clathrin inhibitor, Pitstop-2, and human immunoglobulin G, but not by the caveolin inhibitor, methyl-β-cyclodextrin. In addition, membrane transfer from PMNs enhanced IL-2 production by recipient anti-CD3/anti-CD28 activated MNCs, and this was suppressed by inhibitors of mitogen-activated protein kinase (PD98059) and protein kinase C (Rottlerin). Of clinical significance, decreased total membrane transfer from PMNs to MNCs in patients with active SLE suppressed mononuclear IL-2 production. In conclusion, membrane transfer from MNCs to PMNs, mainly at the immunological synapse, transduces survival and activation signals to enhance PMN functions and is dependent on actin polymerization, clathrin activation, and Fcγ receptors, while membrane transfer from PMNs to MNCs depends on MAP kinase and

Membrane Transfer from Mononuclear Cells to Polymorphonuclear Neutrophils Transduces Cell Survival and Activation Signals in the Recipient Cells via Anti-Extrinsic Apoptotic and MAP Kinase Signaling Pathways.

Directory of Open Access Journals (Sweden)

Ko-Jen Li

Full Text Available The biological significance of membrane transfer (trogocytosis between polymorphonuclear neutrophils (PMNs and mononuclear cells (MNCs remains unclear. We investigated the biological/immunological effects and molecular basis of trogocytosis among various immune cells in healthy individuals and patients with active systemic lupus erythematosus (SLE. By flow cytometry, we determined that molecules in the immunological synapse, including HLA class-I and-II, CD11b and LFA-1, along with CXCR1, are exchanged among autologous PMNs, CD4+ T cells, and U937 cells (monocytes after cell-cell contact. Small interfering RNA knockdown of the integrin adhesion molecule CD11a in U937 unexpectedly enhanced the level of total membrane transfer from U937 to PMN cells. Functionally, phagocytosis and IL-8 production by PMNs were enhanced after co-culture with T cells. Total membrane transfer from CD4+ T to PMNs delayed PMN apoptosis by suppressing the extrinsic apoptotic molecules, BAX, MYC and caspase 8. This enhancement of activities of PMNs by T cells was found to be mediated via p38- and P44/42-Akt-MAP kinase pathways and inhibited by the actin-polymerization inhibitor, latrunculin B, the clathrin inhibitor, Pitstop-2, and human immunoglobulin G, but not by the caveolin inhibitor, methyl-β-cyclodextrin. In addition, membrane transfer from PMNs enhanced IL-2 production by recipient anti-CD3/anti-CD28 activated MNCs, and this was suppressed by inhibitors of mitogen-activated protein kinase (PD98059 and protein kinase C (Rottlerin. Of clinical significance, decreased total membrane transfer from PMNs to MNCs in patients with active SLE suppressed mononuclear IL-2 production. In conclusion, membrane transfer from MNCs to PMNs, mainly at the immunological synapse, transduces survival and activation signals to enhance PMN functions and is dependent on actin polymerization, clathrin activation, and Fcγ receptors, while membrane transfer from PMNs to MNCs depends on

Efficient gene transfer into nondividing cells by adeno-associated virus-based vectors.

OpenAIRE

Podsakoff, G; Wong, K K; Chatterjee, S

1994-01-01

Gene transfer vectors based on adeno-associated virus (AAV) are emerging as highly promising for use in human gene therapy by virtue of their characteristics of wide host range, high transduction efficiencies, and lack of cytopathogenicity. To better define the biology of AAV-mediated gene transfer, we tested the ability of an AAV vector to efficiently introduce transgenes into nonproliferating cell populations. Cells were induced into a nonproliferative state by treatment with the DNA synthe...

Enzymes in biogenesis of plant cell wall polysaccharides. Enzyme characterization using tracer techniques

International Nuclear Information System (INIS)

Dickinson, D.B.

1975-01-01

Enzymes and metabolic pathways, by which starch and cell wall polysaccharides are formed, were investigated in order to learn how these processes are regulated and to identify the enzymatic regulatory mechanisms involved. Germinating lily pollen was used for studies of cell wall formation, and pollen and maize endosperm for studies of starch biosynthesis. Hexokinase being the first step in conversion of hexoses to starch, wall polysaccharides and respiratory substrates, maize endosperm enzyme was assayed by its conversion of 14 C-hexose to 14 C-hexose-6-P, and rapid separation of the two labelled compounds on anion-exchange paper. This enzyme did not appear to be under tight regulation by feed-back inhibition or activation, nor to be severely inhibited by glucose-6-P or activated by citrate. ADP-glucose pyrophosphorylase and other pyrophosphorylases were assayed radiochemically with 14 C-glucose-1-P (forward direction) or 32-PPsub(i) (reverse direction). They showed that the maize endosperm enzyme was activated by the glycolytic intermediates fructose-6-P and 3-phosphoglycerate, and that low levels of the enzyme were present in the high sucrose-low starch mutant named shrunken-2. Under optimal in-vitro assay conditions, the pollen enzyme reacted four times faster than the observed in-vivo rate of starch accumulation. Biogenesis of plant cell wall polysaccharides requires the conversion of hexose phosphates to various sugar nucleotides and utilization of the latter by the appropriate polysaccharide synthetases. Lily pollen possesses a β-1,3-glucan synthetase which is activated up to six-fold by β-linked oligosaccharides. Hence, the in-vivo activity of this enzyme may be modulated by such effector molecules

T-cell involvement in adoptive transfer of line 10 tumor immunity in strain 2 guinea pigs

International Nuclear Information System (INIS)

de Jong, W.H.; Steerenberg, P.A.; van de Plas, M.M.; Kruizinga, W.; Ruitenberg, J.

1985-01-01

Several aspects of adoptive transfer of tumor immunity were studied in the line 10 hepatocarcinoma in the syngeneic Sewall-Wright strain 2 guinea pig. In particular, the need for cooperation between donor and recipient T-cells was investigated. Donor immune spleen cells remained immunologically capable of inducing tumor rejection for at least 160 days after adoptive transfer. Irradiated (1,000 rad) or mitomycin-treated immune spleen cells lacked tumor-rejection activity, which is indicative of the necessity for in vivo proliferation after adoptive transfer of immunity. Furthermore, adoptive transfer of tumor immunity was abrogated after treatment of the line 10 immune spleen cells with rabbit anti-guinea pig-thymocyte serum (ATS) plus complement. The role of recipient T-cells was investigated in strain 2 guinea pigs which were T-cell depleted by thymectomy, irradiation, and bone marrow reconstitution (T-XBM animals). Severe suppression of T-cell activity was present at 2 and 6 weeks after irradiation and bone marrow reconstitution. At 10 weeks nonspecific T-cell activity was partially restored. The induction of antigen-specific responses, measured by delayed-type hypersensitivity skin testing in vivo and antigenic stimulation in vitro, was suppressed at 2 weeks after irradiation and bone marrow reconstitution. Additional in vivo treatment of T-XBM animals with a rabbit ATS improved the T-cell depletion only moderately. Tumor growth and tumor rejection after adoptive transfer of immunity were equal in normal and T-cell-deprived recipient animals, thus indicating that recipient T-cells are not needed for tumor rejection after adoptive transfer of line 10 tumor immunity

Storage sites in seeds of Caesalpinia echinata and C. ferrea (Leguminosae with considerations on nutrients flow

Directory of Open Access Journals (Sweden)

Simone de Pádua Teixeira

2008-02-01

Full Text Available The seeds of Caesalpinia echinata and C. ferrea behaved as typical endospermic seeds, despite their different morphological classification (exendospermic seeds were described for C. echinata and endospermic seeds for C. ferrea. Then, the aim of this work was to compare, under ultrastructural and histochemical terms, the nature of the storage substances and their accumulation sites, as well as the nutrient flow in seeds of these species. Cotyledons in C. echinata accumulate carbohydrates, lipids and proteins, which are mobilized from the outer to the inner parts as revealed by the position of plasmodesmata. Endosperm in C. ferrea accumulates carbohydrates and in C. echinata accumulates substances during the initial embryogenic phases. Such tissue develops a chalazal haustorium that is responsible for the transport of substances into the endosperm itself and from it into the embryo, confirmed by the presence of transference cells.As sementes de Caesalpinia echinata e C. ferrea comportam-se como endospérmicas, apesar de descritas na literatura como exendospérmicas e endospérmicas, respectivamente. Desta forma, o objetivo deste trabalho foi comparar, em termos ultra-estrutural e histoquímico, a natureza das substâncias de reserva e seus tecidos acumuladores, bem como o fluxo de nutrientes nas sementes destas espécies. Os cotilédones em C. echinata acumulam carboidratos, lipídios e proteínas, mobilizados da periferia para o centro, como visto pelo posicionamento dos plasmodesmas. O endosperma em C. ferrea acumula carboidratos e lipídios, e em C. echinata, acumula substâncias nos estádios iniciais da embriogênese. Este tecido desenvolve um haustório calazal agressivo, que transporta substâncias para o endosperma propriamente dito e deste para o embrião, fato confirmado pela presença de células de transferência no endosperma.

Light harvesting via energy transfer in the dye solar cell

Energy Technology Data Exchange (ETDEWEB)

Siegers, Conrad

2007-11-09

The PhD-thesis ''Light Harvesting via Energy Transfer in the Dye Solar Cell'' (University of Freiburg, July 2007) describes the conceptual design, synthesis and testing of energy donor acceptor sensitizers for the dye solar cell (DSC). Under monochromatic illumination solar cells sensitized with the novel donor acceptor systems revealed a higher power conversion efficiency than cells containing exclusively the acceptor component. The following approach led to this conclusion: (i) the choice of suitable chromophores as energy donor and acceptor moieties according to the Foerster-theory, (ii) the synthesis of different donor acceptor systems, (iii) the development of a methodology allowing the quantification of energy transfer within dye solar cells, and (iv) the evaluation of characteristics of DSCs that were sensitized with the different donor acceptor systems. The acceptor chromophores used in this work were derived from [Ru(dcbpy)2acac]Cl (dcbpy = 4,4'-dicarboxy-2,2'-bipyridin, acac = acetylacetonato). This complex offered the opportunity to introduce substituents at the acac-ligand's terminal CH3 groups without significantly affecting its excellent photoelectrochemical properties. Alkylated 4-amino-1,8-naphthalimides (termed Fluorols in the following) were used as energy donor chromophores. This class of compounds fulfils the requirements for efficient energy transfer to [Ru(dcbpy)2acac]Cl. Covalently linking donor and acceptor chromophores to one another was achieved by two different concepts. A dyad comprising one donor and one acceptor chromophore was synthesized by subsequent hydrosilylation steps of an olefin-bearing donor and an acceptor precursor to the dihydrosilane HSiMe2-CH2CH2-SiMe2H. A series of polymers comprising multiple donor and acceptor units was made by the addition of alkyne-bearing chromophores to hyperbranched polyglycerol azide (''Click-chemistry''). In this series the donor acceptor

Transfer plate radioassay using adsorbed anti-insulin antibody to detect insulin secreted by islet cell cultures

International Nuclear Information System (INIS)

Scearce, R.M.; Oie, H.K.; Gazdar, A.F.; Chick, W.L.; Eisenbarth, G.S.

1981-01-01

A solid-phase radioimmunoassay for detection of insulin synthesized by islet cell clones is described. This assay employs anti-insulin antibody adsorbed onto fenestrated polyvinyl chloride 96-well plates ('transfer plates'). The calibrated aperture in the bottom of each transfer plate well permits fluid to enter the wells when transfer plates are lowered into microculture wells containing insulin. With this assay it is possible to rapidly screen hundreds of islet cell cultures for insulin production. The authors have used this assay to facilitate cloning of the RIN rat insulinoma cell line. The assay readily detects insulin synthesis by RIN cells and [ 125 I]insulin is not displaced by culture medium from cells which do not produce insulin. The transfer plate format should be applicable to semiautomate other radioimmunoassays. (Auth.)

A vertically integrated dynamic RAM-cell: Buried bit line memory cell with floating transfer layer

NARCIS (Netherlands)

Mouthaan, A.J.; Vertregt, Maarten

1986-01-01

A charge injection device has been realized in which charge can be injected on to an MOS-capacitor from a buried layer via an isolated transfer layer. The cell is positioned vertically between word and bit line. LOCOS (local oxidation) is used to isolate the cells and (deep) ion implantation to

Calcineurin Orchestrates Lateral Transfer of Aspergillus fumigatus during Macrophage Cell Death.

Science.gov (United States)

Shah, Anand; Kannambath, Shichina; Herbst, Susanne; Rogers, Andrew; Soresi, Simona; Carby, Martin; Reed, Anna; Mostowy, Serge; Fisher, Matthew C; Shaunak, Sunil; Armstrong-James, Darius P

2016-11-01

Pulmonary aspergillosis is a lethal mold infection in the immunocompromised host. Understanding initial control of infection and how this is altered in the immunocompromised host are key goals for comprehension of the pathogenesis of pulmonary aspergillosis. To characterize the outcome of human macrophage infection with Aspergillus fumigatus and how this is altered in transplant recipients on calcineurin inhibitor immunosuppressants. We defined the outcome of human macrophage infection with A. fumigatus, as well as the impact of calcineurin inhibitors, through a combination of single-cell fluorescence imaging, transcriptomics, proteomics, and in vivo studies. Macrophage phagocytosis of A. fumigatus enabled control of 90% of fungal germination. However, fungal germination in the late phagosome led to macrophage necrosis. During programmed necroptosis, we observed frequent cell-cell transfer of A. fumigatus between macrophages, which assists subsequent control of germination in recipient macrophages. Lateral transfer occurred through actin-dependent exocytosis of the late endosome in a vasodilator-stimulated phosphoprotein envelope. Its relevance to the control of fungal germination was also shown by direct visualization in our zebrafish aspergillosis model in vivo. The calcineurin inhibitor FK506 (tacrolimus) reduced cell death and lateral transfer in vitro by 50%. This resulted in uncontrolled fungal germination in macrophages and also resulted in hyphal escape. These observations identify programmed, necrosis-dependent lateral transfer of A. fumigatus between macrophages as an important host strategy for controlling fungal germination. This process is critically dependent on calcineurin. Our studies provide fundamental insights into the pathogenesis of pulmonary aspergillosis in the immunocompromised host.

Laser-induced transfer of gel microdroplets for cell printing

Science.gov (United States)

Yusupov, V. I.; Zhigar'kov, V. S.; Churbanova, E. S.; Chutko, E. A.; Evlashin, S. A.; Gorlenko, M. V.; Cheptsov, V. S.; Minaev, N. V.; Bagratashvili, V. N.

2017-12-01

We study thermal and transport processes involved in the transfer of gel microdroplets under the conditions of laser cell microprinting. The specific features of the interaction of pulsed laser radiation ( λ = 1.064 µm, pulse duration 4 - 200 ns, energy 2 µJ - 1 mJ) with the absorbing gold film deposited on the glass donor substrate are determined. The investigation of the dynamics of transport processes by means of fast optical video recording and optoacoustic methods makes it possible to determine the characteristics of the produced gel jets as functions of the laser operation regimes. The hydrodynamic process of interaction between the laser radiation and the gold coating with the hydrogel layer on it is considered and the temperature in the region of the laser pulse action is estimated. It is shown that in the mechanism of laser-induced transfer a significant role is played by the processes of explosive boiling of water (in gel) and gold. The amount of gold nanoparticles arriving at the acceptor plate in the process of the laser transfer is determined. For the laser pulse duration 8 ns and small energies (less than 10 µJ), the fraction of gold nanoparticles in the gel microdroplets is negligibly small, and their quantity linearly grows with increasing pulse energy. The performed studies offer a base for optimising the processes of laser transfer of gel microdroplets in the rapidly developing technologies of cell microprinting.

Development of a high-titer retrovirus producer cell line capable of gene transfer into rhesus monkey hematopoietic stem cells

International Nuclear Information System (INIS)

Bodine, D.M.; McDonagh, K.T.; Brandt, S.J.; Ney, P.A.; Agricola, B.; Byrne, E.; Nienhuis, A.W.

1990-01-01

Retroviral-mediated gene transfer into primitive hematopoietic cells has been difficult to achieve in large-animal models. The authors have developed an amphotropic producer clone that generates >10 10 recombinant retroviral particles (colony-forming units) per ml of culture medium. Autologous rhesus monkey bone marrow cells were cocultured with either high or low titer producer clones for 4-6 days and reinfused into sublethally irradiated animals. The proviral genome was detected in blood and bone-marrow cells from all three animals reconstituted with cells cocultured with the high-titer producer cells. In contrast, three animals reconstituted with bone marrow cocultured with the low-titer producer clone exhibited no evidence of gene transfer

Differentiated cells are more efficient than adult stem cells for cloning by somatic cell nuclear transfer.

Science.gov (United States)

Sung, Li-Ying; Gao, Shaorong; Shen, Hongmei; Yu, Hui; Song, Yifang; Smith, Sadie L; Chang, Ching-Chien; Inoue, Kimiko; Kuo, Lynn; Lian, Jin; Li, Ao; Tian, X Cindy; Tuck, David P; Weissman, Sherman M; Yang, Xiangzhong; Cheng, Tao

2006-11-01

Since the creation of Dolly via somatic cell nuclear transfer (SCNT), more than a dozen species of mammals have been cloned using this technology. One hypothesis for the limited success of cloning via SCNT (1%-5%) is that the clones are likely to be derived from adult stem cells. Support for this hypothesis comes from the findings that the reproductive cloning efficiency for embryonic stem cells is five to ten times higher than that for somatic cells as donors and that cloned pups cannot be produced directly from cloned embryos derived from differentiated B and T cells or neuronal cells. The question remains as to whether SCNT-derived animal clones can be derived from truly differentiated somatic cells. We tested this hypothesis with mouse hematopoietic cells at different differentiation stages: hematopoietic stem cells, progenitor cells and granulocytes. We found that cloning efficiency increases over the differentiation hierarchy, and terminally differentiated postmitotic granulocytes yield cloned pups with the greatest cloning efficiency.

Dual effects of adenovirus-mediated thrombopoietin gene transfer on hepatic oval cell proliferation and platelet counts

International Nuclear Information System (INIS)

Ichiba, Miho; Shimomura, Takashi; Murai, Rie; Hashiguchi, Koichi; Saeki, Toshiya; Yoshida, Yoko; Kanbe, Takamasa; Tanabe, Naotada; Tsuchiya, Hiroyuki; Miura, Norimasa; Tajima, Fumihito; Kurimasa, Akihiro; Hamada, Hirofumi; Shiota, Goshi

2005-01-01

Thrombopoietin (TPO) is the growth factor for megakaryocytes and platelets, however, it also acts as a potent regulator of stem cell proliferation. To examine the significance of TPO expression in proliferation of hepatic oval cells, the effect of adenovirus-mediated TPO gene transfer into livers of the Solt-Farber model, which mimics the condition where liver regeneration is impaired, was examined. Hepatic TPO mRNA peaked its expression at 2 days after gene transduction and then gradually decreased. The peripheral platelet number began to increase at 4 days (P < 0.05) and reached its plateau at 9 days (P < 0.01). Oval cells expressed c-Mpl, a receptor for TPO as well as immature hematopoietic and hepatocytic surface markers such as CD34 and AFP. The proliferating cell nuclear antigen-positive oval cells in rats into which adenovirus-TPO gene was transferred at 7 and 9 days were significantly greater than those in adenovirus-LacZ gene transferred (P < 0.05, each), and the total numbers of oval cells in the adenovirus-TPO gene transferred at 9 and 13 days were also significantly greater than those in adenovirus-LacZ gene transferred (P < 0.05, each). Expression of SCF protein was increased at 4, 7, and 9 days by TPO gene administration and that of c-Kit was increased at 4 and 7 days. These data suggest that adenovirus-mediated TPO gene transfer stimulated oval cell proliferation in liver as well as increasing peripheral platelet counts, emphasizing the significance of the TPO/c-Mpl system in proliferation of hepatic oval cells

Human somatic cell nuclear transfer and reproductive cloning: an Ethics Committee opinion.

Science.gov (United States)

2016-04-01

This document presents arguments that conclude that it is unethical to use somatic cell nuclear transfer (SCNT) for infertility treatment due to concerns about safety; the unknown impact of SCNT on children, families, and society; and the availability of other ethically acceptable means of assisted reproduction. This document replaces the ASRM Ethics Committee report titled, "Human somatic cell nuclear transfer and cloning," last published in Fertil Steril 2012;98:804-7. Copyright © 2016 American Society for Reproductive Medicine. Published by Elsevier Inc. All rights reserved.

Study of charge transfer reactions in a microbial fuel cell

Energy Technology Data Exchange (ETDEWEB)

Martin, E.; Savadogo, O. [Ecole Polytechnique, Montreal, PQ (Canada). Dept. de Genie Chimique; National Research Council of Canada, Montreal, PQ (Canada). Biotechnology Research Inst.; Tartakovsky, B. [National Research Council of Canada, Montreal, PQ (Canada). Biotechnology Research Inst.

2008-07-01

Electron transfer reactions in a microbial fuel cell (MFC) were evaluated. The MFC was inoculated with anaerobic mesophilic sludge and operated with carbon felt, carbon cloth, and platinum (Pt) coated carbon cloth. The MFC was then fed with either acetate or glucose as a source of fuel and operated at a temperature of 25 degrees C and a pH of 7. Scanning electron microscopy (SEM) micrographs demonstrated that the micro-organisms colonized the anodes. Cyclic voltammetry and polarization tests were conducted using different fractions of the anodophilic biofilm in order to determine charge transfer routes. The study characterized the electron transfer mechanisms used by the exoelectrogenic micro-organisms to produce electricity. It was concluded that further research is needed to characterize reaction transfer routes. 2 refs., 1 fig.

Production of human CD59-transgenic pigs by embryonic germ cell nuclear transfer

International Nuclear Information System (INIS)

Ahn, Kwang Sung; Won, Ji Young; Park, Jin-Ki; Sorrell, Alice M.; Heo, Soon Young; Kang, Jee Hyun; Woo, Jae-Seok; Choi, Bong-Hwan; Chang, Won-Kyong; Shim, Hosup

2010-01-01

Research highlights: → Human CD59 (hCD59) gene was introduced into porcine embryonic germ (EG) cells. → hCD59-transgenic EG cells were resistant to hyperacute rejection in cytolytic assay. → hCD59-transgenic pigs were produced by EG cell nuclear transfer. -- Abstract: This study was performed to produce transgenic pigs expressing the human complement regulatory protein CD59 (hCD59) using the nuclear transfer (NT) of embryonic germ (EG) cells, which are undifferentiated stem cells derived from primordial germ cells. Because EG cells can be cultured indefinitely in an undifferentiated state, they may provide an inexhaustible source of nuclear donor cells for NT to produce transgenic pigs. A total of 1980 NT embryos derived from hCD59-transgenic EG cells were transferred to ten recipients, resulting in the birth of fifteen piglets from three pregnancies. Among these offspring, ten were alive without overt health problems. Based on PCR analysis, all fifteen piglets were confirmed as hCD59 transgenic. The expression of the hCD59 transgene in the ten living piglets was verified by RT-PCR. Western analysis showed the expression of the hCD59 protein in four of the ten RT-PCR-positive piglets. These results demonstrate that hCD59-transgenic pigs could effectively be produced by EG cell NT and that such transgenic pigs may be used as organ donors in pig-to-human xenotransplantation.

Production of human CD59-transgenic pigs by embryonic germ cell nuclear transfer

Energy Technology Data Exchange (ETDEWEB)

Ahn, Kwang Sung; Won, Ji Young [Department of Physiology, Dankook University School of Medicine, Cheonan (Korea, Republic of); Park, Jin-Ki [Animal Biotechnology Division, National Institute of Animal Science, Suwon (Korea, Republic of); Sorrell, Alice M. [Department of Physiology, Dankook University School of Medicine, Cheonan (Korea, Republic of); Heo, Soon Young; Kang, Jee Hyun [Department of Nanobiomedical Science, Dankook University, Cheonan (Korea, Republic of); Woo, Jae-Seok [Animal Biotechnology Division, National Institute of Animal Science, Suwon (Korea, Republic of); Choi, Bong-Hwan [Genomics and Bioinformatics Division, National Institute of Animal Science, Suwon (Korea, Republic of); Chang, Won-Kyong [Animal Biotechnology Division, National Institute of Animal Science, Suwon (Korea, Republic of); Shim, Hosup, E-mail: shim@dku.edu [Department of Nanobiomedical Science, Dankook University, Cheonan (Korea, Republic of); Institute of Tissue Regeneration Engineering, Dankook University, Cheonan (Korea, Republic of)

2010-10-01

Research highlights: {yields} Human CD59 (hCD59) gene was introduced into porcine embryonic germ (EG) cells. {yields} hCD59-transgenic EG cells were resistant to hyperacute rejection in cytolytic assay. {yields} hCD59-transgenic pigs were produced by EG cell nuclear transfer. -- Abstract: This study was performed to produce transgenic pigs expressing the human complement regulatory protein CD59 (hCD59) using the nuclear transfer (NT) of embryonic germ (EG) cells, which are undifferentiated stem cells derived from primordial germ cells. Because EG cells can be cultured indefinitely in an undifferentiated state, they may provide an inexhaustible source of nuclear donor cells for NT to produce transgenic pigs. A total of 1980 NT embryos derived from hCD59-transgenic EG cells were transferred to ten recipients, resulting in the birth of fifteen piglets from three pregnancies. Among these offspring, ten were alive without overt health problems. Based on PCR analysis, all fifteen piglets were confirmed as hCD59 transgenic. The expression of the hCD59 transgene in the ten living piglets was verified by RT-PCR. Western analysis showed the expression of the hCD59 protein in four of the ten RT-PCR-positive piglets. These results demonstrate that hCD59-transgenic pigs could effectively be produced by EG cell NT and that such transgenic pigs may be used as organ donors in pig-to-human xenotransplantation.

Cloning of ES cells and mice by nuclear transfer.

Science.gov (United States)

Wakayama, Sayaka; Kishigami, Satoshi; Wakayama, Teruhiko

2009-01-01

We have been able to develop a stable nuclear transfer (NT) method in the mouse, in which donor nuclei are directly injected into the oocyte using a piezo-actuated micromanipulator. Although the piezo unit is a complex tool, once mastered it is of great help not only in NT experiments, but also in almost all other forms of micromanipulation. Using this technique, embryonic stem (ntES) cell lines established from somatic cell nuclei can be generated relatively easily from a variety of mouse genotypes and cell types. Such ntES cells can be used not only for experimental models of human therapeutic cloning but also as a means of preserving mouse genomes instead of preserving germ cells. Here, we describe our most recent protocols for mouse cloning.

Potential of primary kidney cells for somatic cell nuclear transfer mediated transgenesis in pig

Directory of Open Access Journals (Sweden)

Richter Anne

2012-11-01

Full Text Available Abstract Background Somatic cell nuclear transfer (SCNT is currently the most efficient and precise method to generate genetically tailored pig models for biomedical research. However, the efficiency of this approach is crucially dependent on the source of nuclear donor cells. In this study, we evaluate the potential of primary porcine kidney cells (PKCs as cell source for SCNT, including their proliferation capacity, transfection efficiency, and capacity to support full term development of SCNT embryos after additive gene transfer or homologous recombination. Results PKCs could be maintained in culture with stable karyotype for up to 71 passages, whereas porcine fetal fibroblasts (PFFs and porcine ear fibroblasts (PEFs could be hardly passaged more than 20 times. Compared with PFFs and PEFs, PKCs exhibited a higher proliferation rate and resulted in a 2-fold higher blastocyst rate after SCNT and in vitro cultivation. Among the four transfection methods tested with a GFP expression plasmid, best results were obtained with the NucleofectorTM technology, resulting in transfection efficiencies of 70% to 89% with high fluorescence intensity, low cytotoxicity, good cell proliferation, and almost no morphological signs of cell stress. Usage of genetically modified PKCs in SCNT resulted in approximately 150 piglets carrying at least one of 18 different transgenes. Several of those pigs originated from PKCs that underwent homologous recombination and antibiotic selection before SCNT. Conclusion The high proliferation capacity of PKCs facilitates the introduction of precise and complex genetic modifications in vitro. PKCs are thus a valuable cell source for the generation of porcine biomedical models by SCNT.

DNA-mediated gene transfer into ataxia-telangiectasia cells

International Nuclear Information System (INIS)

Crescenzi, M.; Pulciani, S.; Carbonari, M.; Tedesco, L.; Russo, G.; Gaetano, C.; Fiorilli, M.

1986-01-01

The complete description of the genetic lesion(s) underlying the AT mutation might, therefore, highlight not only a DNA-repair pathwa, but also an important aspect of the physiology of lymphocytes. DNA-mediated gene transfer into eukaryotic cells has proved a powerful tool for the molecular cloning of certain mammalian genes. The possibility to clone a given gene using this technology depends, basically, on the availability of a selectable marker associated with the expression of the transfected gene in the recipient cell. Recently, a human DNA repair gene has been cloned in CHO mutant cells by taking advantage of the increased resistance to ultraviolet radiation of the transformants. As a preliminary step toward the molecular cloning of the AT gene(s), the authors have attempted to confer radioresistance to AT cells by transfection with normal human DNA

Development of buffalo (Bubalus bubalis embryonic stem cell lines from somatic cell nuclear transferred blastocysts

Directory of Open Access Journals (Sweden)

Syed Mohmad Shah

2015-11-01

Full Text Available We developed buffalo embryonic stem cell lines from somatic cell nuclear transfer derived blastocysts, produced by hand-guided cloning technique. The inner cell mass of the blastocyst was cut mechanically using a Microblade and cultured onto feeder cells in buffalo embryonic stem (ES cell culture medium at 38 °C in a 5% CO2 incubator. The stem cell colonies were characterized for alkaline phosphatase activity, karyotype, pluripotency and self-renewal markers like OCT4, NANOG, SOX2, c-Myc, FOXD3, SSEA-1, SSEA-4, TRA-1-60, TRA-1-81 and CD90. The cell lines also possessed the capability to differentiate across all the three germ layers under spontaneous differentiation conditions.

Complementation of a DNA repair defect in xeroderma pigmentosum cells by transfer of human chromosome 9

International Nuclear Information System (INIS)

Kaur, G.P.; Athwal, R.S.

1989-01-01

Complementation of the repair defect in xeroderma pigmentosum cells of complementation group A was achieved by the transfer of human chromosome 9. A set of mouse-human hybrid cell lines, each containing a single Ecogpt-marked human chromosome, was used as a source of donor chromosomes. Chromosome transfer to XPTG-1 cells, a hypoxanthine/guanine phosphoribosyltransferase-deficient mutant of simian virus 40-transformed complementation group A cells, was achieved by microcell fusion and selection for Ecogpt. Chromosome-transfer clones of XPTG-1 cells, each containing a different human donor chromosome, were analyzed for complementation of sensitivity to UV irradiation. Among all the clones, increased levels of resistance to UV was observed only in clones containing chromosome 9. Since our recipient cell line XPTG-1 is hypoxanthine/guanine phosphoribosyltransferase deficient, cultivation of Ecogpt+ clones in medium containing 6-thioguanine permits selection of cells for loss of the marker and, by inference, transferred chromosome 9. Clones isolated for growth in 6-thioguanine, which have lost the Ecogpt-marked chromosome, exhibited a UV-sensitive phenotype, confirming the presence of the repair gene(s) for complementation group A on chromosome 9

Recent advancements in cloning by somatic cell nuclear transfer.

Science.gov (United States)

Ogura, Atsuo; Inoue, Kimiko; Wakayama, Teruhiko

2013-01-05

Somatic cell nuclear transfer (SCNT) cloning is the sole reproductive engineering technology that endows the somatic cell genome with totipotency. Since the first report on the birth of a cloned sheep from adult somatic cells in 1997, many technical improvements in SCNT have been made by using different epigenetic approaches, including enhancement of the levels of histone acetylation in the chromatin of the reconstructed embryos. Although it will take a considerable time before we fully understand the nature of genomic programming and totipotency, we may expect that somatic cell cloning technology will soon become broadly applicable to practical purposes, including medicine, pharmaceutical manufacturing and agriculture. Here we review recent progress in somatic cell cloning, with a special emphasis on epigenetic studies using the laboratory mouse as a model.

Recent advancements in cloning by somatic cell nuclear transfer

Science.gov (United States)

Ogura, Atsuo; Inoue, Kimiko; Wakayama, Teruhiko

2013-01-01

Somatic cell nuclear transfer (SCNT) cloning is the sole reproductive engineering technology that endows the somatic cell genome with totipotency. Since the first report on the birth of a cloned sheep from adult somatic cells in 1997, many technical improvements in SCNT have been made by using different epigenetic approaches, including enhancement of the levels of histone acetylation in the chromatin of the reconstructed embryos. Although it will take a considerable time before we fully understand the nature of genomic programming and totipotency, we may expect that somatic cell cloning technology will soon become broadly applicable to practical purposes, including medicine, pharmaceutical manufacturing and agriculture. Here we review recent progress in somatic cell cloning, with a special emphasis on epigenetic studies using the laboratory mouse as a model. PMID:23166393

Analysis of bone marrow stromal cell transferred bacterial {beta}-galactosidase gene by PIXE

Energy Technology Data Exchange (ETDEWEB)

Kumakawa, Toshiro [Tokyo Metropolitan Geriatric Hospital, Tokyo (Japan). Dept. of Blood Transfusion and Hematology; Hibino, Hitoshi; Tani, Kenzaburo; Asano, Shigetaka; Futatugawa, Shouji; Sera, Kouichiro

1997-12-31

PIXE, Particle Induced X-ray Emission, is a powerful, multi-elemental analysis method which has many distinguishing features and has been used in varies research fields. Recently the method of applying baby cyclotrons for nuclear medicine to PIXE has been developed. This enables us to study biomedical phenomena from the physical point of view. Mouse bone marrow stromal cells were transferred bacterial {beta}-galactosidase gene (LacZ gene) by murine retroviral vectors. Analysis of the bone marrow stromal cells with the LacZ gene by PIXE revealed remarkable changes of intracellular trace elements compared with the normal control cells. These results indicate that gene transfer by retroviral vectors may bring about a dynamic change of intracellular circumstances of the target cell. (author)

Baking quality parameters of wheat in relation to endosperm storage proteins

Directory of Open Access Journals (Sweden)

Daniela Horvat

2012-01-01

Full Text Available Wheat storage proteins of twelve winter wheat cultivars grown at the experimental field of the Agricultural Institute Osijek in 2009 were studied for their contribution to the baking quality. Composition of high molecular weight glutenin subunits (HMW-GS was analyzed by SDS-PAGE method, while the proportions of endosperm storage proteins were determined by RP-HPLC method. Regarding the proportion of storage proteins, results of the linear correlation (p<0.05 showed that protein (P and wet gluten (WG content were highly negatively correlated with albumins and globulins (AG and positively with α- gliadins (GLI. A strong negative correlation between AG and water absorption (WA capacity of flour was found, while α- GLI had positive influence on this property. Dough development time (DDT was positively significantly correlated with HMW-GS and negatively with AG. Degree of dough softening (DS was strongly positively affected by γ- GLI and gliadins to glutenins ratio (GLI/GLU and negatively by total GLU and HMW-GS. Dough energy (E and maximum resistance (RMAX were significantly positively affected by Glu-1 score and negatively by GLI/GLU ratio. Resistance to extensibility ratio (R/EXT was significantly negatively correlated with total GLI. Bread volume was significantly negatively influenced by AG.

Isolation and characterization of the messenger RNA and the gene coding for a proline-rich zein from corn endosperm

International Nuclear Information System (INIS)

Wang, S.Z.

1985-01-01

Gamma-zein, a proline-rich protein from corn endosperm, was investigated at the molecular level. Immunological and electrophoretic data indicated that gamma-zein was deposited into protein bodies in corn endosperm. Both isolated polysomes and poly(A) + mRNA were found to direct into vitro synthesis of gamma-zein in a wheat germ system. In vitro synthesized gamma-zein was immunoprecipitated from the total in vitro translation products. A cDNA expression library was constructed by reverse transcription of total poly(A) + mRNA using pUC8 plasmid as vector and E. coli strain DH1 as host. The library was screened for the expression of gamma-zein and alpha-zein by specific antibodies. The library was also screened with 32 P-labeled gamma-zein and alpha-zein cDNA probes. The results indicated that gamma-zein and its fragments were readily expressed in E. coli while alpha-zein was not. Seven independently selected clones, six of which were selected by antibody and one by a cDNA probe, were sequenced. A comparison of sequence information from seven clones revealed that their overlapping regions were identical. This suggests that gamma-zein is encoded by a single gene. This finding is in conflict with what was expected on the basis of extensive charge heterogeneity of gamma-zein in isoelectric focusing. Individual bands cut from an IEF gel were rerun and shown to give several bands suggesting that the charge heterogeneity of gamma-zein may be an artifact. Sequence information of gamma-zein indicated that the gene encodes a mature protein whose primary structure includes 204 amino acids and has a molecular weight of 21,824 daltons

Cell intrinsic immunity spreads to bystander cells via the intercellular transfer of cGAMP.

Science.gov (United States)

Ablasser, Andrea; Schmid-Burgk, Jonathan L; Hemmerling, Inga; Horvath, Gabor L; Schmidt, Tobias; Latz, Eicke; Hornung, Veit

2013-11-28

The innate immune defence of multicellular organisms against microbial pathogens requires cellular collaboration. Information exchange allowing immune cells to collaborate is generally attributed to soluble protein factors secreted by pathogen-sensing cells. Cytokines, such as type I interferons (IFNs), serve to alert non-infected cells to the possibility of pathogen challenge. Moreover, in conjunction with chemokines they can instruct specialized immune cells to contain and eradicate microbial infection. Several receptors and signalling pathways exist that couple pathogen sensing to the induction of cytokines, whereas cytosolic recognition of nucleic acids seems to be exquisitely important for the activation of type I IFNs, master regulators of antiviral immunity. Cytosolic DNA is sensed by the receptor cyclic GMP-AMP (cGAMP) synthase (cGAS), which catalyses the synthesis of the second messenger cGAMP(2'-5'). This molecule in turn activates the endoplasmic reticulum (ER)-resident receptor STING, thereby inducing an antiviral state and the secretion of type I IFNs. Here we find in murine and human cells that cGAS-synthesized cGAMP(2'-5') is transferred from producing cells to neighbouring cells through gap junctions, where it promotes STING activation and thus antiviral immunity independently of type I IFN signalling. In line with the limited cargo specificity of connexins, the proteins that assemble gap junction channels, most connexins tested were able to confer this bystander immunity, thus indicating a broad physiological relevance of this local immune collaboration. Collectively, these observations identify cGAS-triggered cGAMP(2'-5') transfer as a novel host strategy that serves to rapidly convey antiviral immunity in a transcription-independent, horizontal manner.

Transfer of mRNA Encoding Invariant NKT Cell Receptors Imparts Glycolipid Specific Responses to T Cells and γδT Cells.

Science.gov (United States)

Shimizu, Kanako; Shinga, Jun; Yamasaki, Satoru; Kawamura, Masami; Dörrie, Jan; Schaft, Niels; Sato, Yusuke; Iyoda, Tomonori; Fujii, Shin-Ichiro

2015-01-01

Cell-based therapies using genetically engineered lymphocytes expressing antigen-specific T cell receptors (TCRs) hold promise for the treatment of several types of cancers. Almost all studies using this modality have focused on transfer of TCR from CD8 cytotoxic T lymphocytes (CTLs). The transfer of TCR from innate lymphocytes to other lymphocytes has not been studied. In the current study, innate and adaptive lymphocytes were transfected with the human NKT cell-derived TCRα and β chain mRNA (the Vα24 and Vβ11 TCR chains). When primary T cells transfected with NKT cell-derived TCR were subsequently stimulated with the NKT ligand, α-galactosylceramide (α-GalCer), they secreted IFN-γ in a ligand-specific manner. Furthermore when γδT cells were transfected with NKT cell-derived TCR mRNA, they demonstrated enhanced proliferation, IFN-γ production and antitumor effects after α-GalCer stimulation as compared to parental γδT cells. Importantly, NKT cell TCR-transfected γδT cells responded to both NKT cell and γδT cell ligands, rendering them bi-potential innate lymphocytes. Because NKT cell receptors are unique and universal invariant receptors in humans, the TCR chains do not yield mispaired receptors with endogenous TCR α and β chains after the transfection. The transfection of NKT cell TCR has the potential to be a new approach to tumor immunotherapy in patients with various types of cancer.

DOT1L inhibitor improves early development of porcine somatic cell nuclear transfer embryos

DEFF Research Database (Denmark)

Tao, Jia; Zhang, Yu; Zuo, Xiaoyuan

2017-01-01

Incomplete epigenetic reprogramming of the genome of donor cells causes poor early and full-term developmental efficiency of somatic cell nuclear transfer (SCNT) embryos. Previous research indicate that inhibition of the histone H3 K79 methyltransferase DOT1L, using a selective pharmacological...... inhibitor EPZ004777 (EPZ), significantly improved reprogramming efficiency during the generation of mouse induced pluripotent stem cells. However, the roles of DOT1L in porcine nuclear transfer-mediated cellular reprogramming are not yet known. Here we showed that DOT1L inhibition via 0.5 nM EPZ treatment...

Transfer of experimental allergic encephalomyelitis to bone marrow chimeras. Endothelial cells are not a restricting element

International Nuclear Information System (INIS)

Hinrichs, D.J.; Wegmann, K.W.; Dietsch, G.N.

1987-01-01

The adoptive transfer of clinical and histopathologic signs of experimental allergic encephalomyelitis (EAE) requires MHC compatibility between cell donor and cell recipient. The results of adoptive transfer studies using F1 to parent bone marrow chimeras as recipients of parental-derived BP-sensitive spleen cells indicate that this restriction is not expressed at the level of the endothelial cell but is confined to the cells of bone marrow derivation. Furthermore, these results indicate that the development of EAE is not dependent on the activity of MHC-restricted cytotoxic cells

Transfer of Chinese hamster DNA repair gene(s) into repair-deficient human cells (Xeroderma pigmentosum)

International Nuclear Information System (INIS)

Karentz, D.; Cleaver, J.E.

1985-01-01

Transfer of repair genes by DNA transfection into repair-deficient Xeroderma pigmentosum (XP) cells has thus far been unsuccessful, presenting an obstacle to cloning XP genes. The authors chose an indirect route to transfer repair genes in chromosome fragments. DNA repair-competent (UV resistant) hybrid cell lines were established by PEG-mediated fusions of DNA repair-deficient (UV sensitive) human fibroblasts (XP12RO) with wild type Chinese hamster (CHO) cells (AA8). CHO cells were exposed to 5 Krad X-rays prior to fusions, predisposing hybrid cells to lose CHO chromosome fragments preferentially. Repair-competent hybrids were selected by periodic exposures to UV light. Secondary and tertiary hybrid cell lines were developed by fusion of X-irradiated hybrids to XP12RO. The hybrid cell lines exhibit resistance to UV that is comparable to that of CHO cells and they are proficient at repair replication after UV exposure. Whole cell DNA-DNA hybridizations indicate that the hybrids have greater homology to CHO DNA than is evident between XP12RO and CHO. These observations indicate that CHO DNA sequences which can function in repair of UV-damaged DNA in human cells have been transferred into the genome of the repair-deficient XP12RO cells

Outward electron transfer by Saccharomyces cerevisiae monitored with a bi-cathodic microbial fuel cell-type activity sensor.

Science.gov (United States)

Ducommun, Raphaël; Favre, Marie-France; Carrard, Delphine; Fischer, Fabian

2010-03-01

A Janus head-like bi-cathodic microbial fuel cell was constructed to monitor the electron transfer from Saccharomyces cerevisiae to a woven carbon anode. The experiments were conducted during an ethanol cultivation of 170 g/l glucose in the presence and absence of yeast-peptone medium. First, using a basic fuel-cell type activity sensor, it was shown that yeast-peptone medium contains electroactive compounds. For this purpose, 1% solutions of soy peptone and yeast extract were subjected to oxidative conditions, using a microbial fuel cell set-up corresponding to a typical galvanic cell, consisting of culture medium in the anodic half-cell and 0.5 M K(3)Fe(CN)(6) in the cathodic half-cell. Second, using a bi-cathodic microbial fuel cell, it was shown that electrons were transferred from yeast cells to the carbon anode. The participation of electroactive compounds in the electron transport was separated as background current. This result was verified by applying medium-free conditions, where only glucose was fed, confirming that electrons are transferred from yeast cells to the woven carbon anode. Knowledge about the electron transfer through the cell membrane is of importance in amperometric online monitoring of yeast fermentations and for electricity production with microbial fuel cells. Copyright (c) 2009 John Wiley & Sons, Ltd.

Electroluminescence from charge transfer states in Donor/Acceptor solar cells

DEFF Research Database (Denmark)

Sherafatipour, Golenaz; Madsen, Morten

Charge photocurrent generation is a key process in solar energy conversion systems. Effective dissociation of the photo-generated electron-hole pairs (excitons) has a strong influence on the efficiency of the organic solar cells. Charge dissociation takes place at the donor/acceptor interface via...... which the maximum open-circuit voltage can be estimated, and further can be used in the modeling and optimization of the OPV devices. [1] C. Deibe, T. Strobe, and V. Dyakonov, “Role of the charge transfer state in organic donor-acceptor solar cells,” Adv. Mater., vol. 22, pp. 4097–4111, 2010. [2] K...... charge transfer (CT) excitons, which is Coulombically bound interfacial electron- hole pairs residing at the donor/acceptor heterojunctions. The CT state represents an intermediate state between the exciton dissociation and recombination back to the ground state. Since the recombination of photo...

Pollination with gamma-irradiated pollen and seed development in kiwifruit (Actinidia deliciosa var. deliciosa)

International Nuclear Information System (INIS)

Musial, K.

1997-01-01

Full text. The effects of pollen irradiation at 70 and 90 kr on seed set were studied in Actinidia deliciosa var. deliciosa. Pollination with irradiated pollen affected seed development and contents. Rising irradiation doses increased the percentages of empty seeds and decreased the percentages of seeds containing embryos with endosperm. Moreover, pollination with heavily irradiated pollen led to the formation of seeds containing the endosperm only. Embryo and endosperm size was also strongly influenced by irradiated pollen. The length of endosperms was reduced at all levels of pollen irradiation compared to the non-irradiated controls; the embryo development was conspicuously retarded. Cells in endosperm resulting from the treatments differed in the presence and number of starch grains. (author)

A cytochemical and immunocytochemical analysis of the wall labyrinth apparatus in leaf transfer cells in Elodea canadensis.

Science.gov (United States)

Ligrone, Roberto; Vaughn, Kevin C; Rascio, Nicoletta

2011-04-01

Transfer cells are plant cells specialized in apoplast/symplast transport and characterized by a distinctive wall labyrinth apparatus. The molecular architecture and biochemistry of the labyrinth apparatus are poorly known. The leaf lamina in the aquatic angiosperm Elodea canadensis consists of only two cell layers, with the abaxial cells developing as transfer cells. The present study investigated biochemical properties of wall ingrowths and associated plasmalemma in these cells. Leaves of Elodea were examined by light and electron microscopy and ATPase activity was localized cytochemically. Immunogold electron microscopy was employed to localize carbohydrate epitopes associated with major cell wall polysaccharides and glycoproteins. The plasmalemma associated with the wall labyrinth is strongly enriched in light-dependent ATPase activity. The wall ingrowths and an underlying wall layer share an LM11 epitope probably associated with glucuronoarabinoxylan and a CCRC-M7 epitope typically associated with rhamnogalacturonan I. No labelling was observed with LM10, an antibody that recognizes low-substituted and unsubstituted xylan, a polysaccharide consistently associated with secondary cell walls. The JIM5 and JIM7 epitopes, associated with homogalacturonan with different degrees of methylation, appear to be absent in the wall labyrinth but present in the rest of cell walls. The wall labyrinth apparatus of leaf transfer cells in Elodea is a specialized structure with distinctive biochemical properties. The high level of light-dependent ATPase activity in the plasmalemma lining the wall labyrinth is consistent with a formerly suggested role of leaf transfer cells in enhancing inorganic carbon inflow. The wall labyrinth is a part of the primary cell wall. The discovery that the wall ingrowths in Elodea have an antibody-binding pattern divergent, in part, from that of the rest of cell wall suggests that their carbohydrate composition is modulated in relation to transfer

Transcriptional reprogramming of gene expression in bovine somatic cell chromatin transfer embryos

Directory of Open Access Journals (Sweden)

Page Grier P

2009-04-01

Full Text Available Abstract Background Successful reprogramming of a somatic genome to produce a healthy clone by somatic cells nuclear transfer (SCNT is a rare event and the mechanisms involved in this process are poorly defined. When serial or successive rounds of cloning are performed, blastocyst and full term development rates decline even further with the increasing rounds of cloning. Identifying the "cumulative errors" could reveal the epigenetic reprogramming blocks in animal cloning. Results Bovine clones from up to four generations of successive cloning were produced by chromatin transfer (CT. Using Affymetrix bovine microarrays we determined that the transcriptomes of blastocysts derived from the first and the fourth rounds of cloning (CT1 and CT4 respectively have undergone an extensive reprogramming and were more similar to blastocysts derived from in vitro fertilization (IVF than to the donor cells used for the first and the fourth rounds of chromatin transfer (DC1 and DC4 respectively. However a set of transcripts in the cloned embryos showed a misregulated pattern when compared to IVF embryos. Among the genes consistently upregulated in both CT groups compared to the IVF embryos were genes involved in regulation of cytoskeleton and cell shape. Among the genes consistently upregulated in IVF embryos compared to both CT groups were genes involved in chromatin remodelling and stress coping. Conclusion The present study provides a data set that could contribute in our understanding of epigenetic errors in somatic cell chromatin transfer. Identifying "cumulative errors" after serial cloning could reveal some of the epigenetic reprogramming blocks shedding light on the reprogramming process, important for both basic and applied research.

Alterations in radioresistance of eucaryotic cells after the transfer of genomic wildtype DNA and metallothionein genes

International Nuclear Information System (INIS)

Lohrer, H.

1987-01-01

The presented paper describes experiments concerning the alteration of radiosensitivity of eucaryotic cells after gene transfer. Ionizing radiation (γ- or X-ray) induces DNA single- or double strand breaks, which are religated by an unknown repair system. Repair deficient cells are highly sensitive to ionizing radiation. In the experiments described, cells from a patient with the heritable disease Ataxia telangiectasia were used as well as two X-ray sensitive CHO mutant cell lines. After gene transfer of an intact human DNA repair gene or a metallothionein gene the cells should regain radioresistance. (orig.) [de

Observações Citológicas em coffea: VI — Desenvolvimento do embrião e do endosferma em Coffea Arabica l.

Directory of Open Access Journals (Sweden)

A. J. T. Mendes

1942-04-01

Full Text Available The ovule of C. arabica L. consists õf a single integument and a small nucellús which disappears as the ovule matures. Three of the four macrospores resulting from the'division of the macrosporocyte, degenerate. The remaining chalazal cell gives rise to a "normal'' embryo sac, which is ready for fertilization at the time of the flower opening. Double fertilization occurs, as a rule, the day the flower opens. The embryo sac then increases in volume and compresses the inner integument cells. The outer cells of the integument, however, multiply actively, giving rise to the "perisperm". After degeneration of the synergids and antipodals, the zygote stays near the micro-pyle in a resting stage, while the primary endosperm nucleus divides. This first division of the endosperm occurs from 21 to 27 days after flower opening. The cytoplasm condenses around the newly formed nuclei, permitting the adjacent tissues to sink into the embryo sac. Since the separating walls were not seen at the binueleate stage and were present at the four-nucleate stage, it seems that the endosperm belongs to the' "nuclear type". As the number of endosperm cells increases, the "perisperm" cells are again compressed and give more and more room to the new tissue. The first division in the zygote occurs from sixty to seventy days after flower opening, when the endosperm is already multinucleate. A differentiated embryo develops, with a hypocotyl and two small cotyledons in the ripe seed. In the ripe seed the "perisperm" disappears almost completely: its remains form the thin "silver skin" which envelops the endosperm. The parchment layer which envelops the seed is the endocarp.

Role of ooplasm in nuclear and nucleolar remodeling of intergeneric somatic cell nuclear transfer embryos during the first cell cycle

DEFF Research Database (Denmark)

Østrup, Olga; Strejcek, Frantisek; Petrovicova, Ida

2011-01-01

Initially, development of the zygote is under control of the oocyte ooplasm. However, it is presently unknown if and to what extent is the ooplasm able to interact with a transferred somatic cell from another species in the context of interspecies somatic cell nuclear transfer (SCNT). Here, one-c...... in sequence-specific interactions between the ooplasm and chromatin of another genus. In conclusion, the results demonstrate a possible reason why the intergeneric SCNT embryos never reached the full term....

Theoretical study of electronic transfer current rate at dye-sensitized solar cells

Science.gov (United States)

AL-Agealy, Hadi J. M.; AlMaadhede, Taif Saad; Hassooni, Mohsin A.; Sadoon, Abbas K.; Ashweik, Ahmed M.; Mahdi, Hind Abdlmajeed; Ghadhban, Rawnaq Qays

2018-05-01

In this research, we present a theoretical study of electronic transfer kinetics rate in N719/TiO2 and N719/ZnO dye-sensitized solar cells (DSSC) systems using a simple model depending on the postulate of quantum mechanics theory. The evaluation of the electronic transition current rate in DSSC systems are function of many parameters such that; the reorientation transition energies ΛSe m D y e , the transition coupling parameter ℂT(0), potential exponential effect e-(E/C-EF ) kBT , unit cell volume VSem, and temperature T. Furthermore, the analysis of electronic transfer current rate in N719/TiO2 and N719/ZnO systems show that the rate upon dye-sensitization solar cell increases with increases of transition coupling parameter, decreasing potential that building at interface a results of different material in this devices and increasing with reorientation transition energy. On the other hand, we can find the electronic transfer behavior is dependent of the dye absorption spectrum and mainly depending on the reorientation of transition energy. The replacement of the solvents in both DSSC system caused increasing of current rates dramatically depending on polarity of solvent in subset devices. This change in current rate of electron transfer were attributed to much more available of recombination sites introduced by the solvents medium. The electronic transfer current dynamics are shown to occurs in N719/TiO2 system faster many time compare to ocuures at N719/ZnO system, this indicate that TiO2 a is a good and active material compare with ZnO to using in dye sensitized solar cell devices. In contrast, the large current rate in N719/TiO2 comparing to ZnO of N719/ZnO systems indicate that using TiO2 with N719 dye lead to increasing the efficiency of DSSC.

Ability of spleen cells from tumor bearing mice to transfer immunologic memory

Energy Technology Data Exchange (ETDEWEB)

Plavsic, B.; Jurin, M. (Zagreb Univ. (Yugoslavia)); Ugarkovic, B. (Institut Rudjer Boskovic, Zagreb (Yugoslavia))

1983-01-01

The ability of splenocytes from tumorous mice to transfer immunologic memory was tested. Three syngeneic experimental tumors from highly inbred strains were used; fibrosarcoma, lymphoma and Lewis lung carcinoma. Splenocytes from tumorous mice were collected after rejection of allogeneic skin which had been grafted at different stages of the tumor disease, and injected into lethally irradiated syngeneic recipients. These secondary hosts were grafted with the same allogeneic skin graft as their donors and the ability of cells transplanted from tumorous donors to transfer memory to allograft was tested. Tumorous mice seemed to have more memory cells (T lymphocytes) in their spleens than the controls.

Fluorescence resonance energy transfer imaging of CFP/YFP labeled NDH in cyanobacterium cell

International Nuclear Information System (INIS)

Ji Dongmei; Lv Wei; Huang Zhengxi; Xia Andong; Xu Min; Ma Weimin; Mi Hualing; Ogawa Teruo

2007-01-01

The laser confocal scanning microscopy combined with time-correlated single photon counting imaging technique to obtain fluorescence intensity and fluorescence lifetime images for fluorescence resonance energy transfer measurement is reported. Both the fluorescence lifetime imaging microscopy (FLIM) and intensity images show inhomogeneous cyan fluorescent protein and yellow fluorescent protein (CFP /YFP) expression or inhomogeneous energy transfer between CFP and YFP over whole cell. The results presented in this work show that FLIM could be a potential method to reveal the structure-function behavior of NAD(P)H dehydrogenase complexes in living cell

Inside Job: Viruses Transfer cGAMP between Cells.

Science.gov (United States)

Gulen, Muhammet F; Ablasser, Andrea

2015-09-09

The DNA sensor, cyclic GMP-AMP synthase (cGAS), is essential for the detection of viral infection. In a recent issue of Science, two studies, Bridgeman et al. (2015) and Gentili et al. (2015), report a novel mechanism for propagating an antiviral signal between cells, based on the transfer of the cGAS enzymatic product, cyclic GMP-AMP (cGAMP), in viral particles. Copyright © 2015 Elsevier Inc. All rights reserved.

Gene transfer preferentially selects MHC class I positive tumour cells and enhances tumour immunogenicity.

Science.gov (United States)

Hacker, Ulrich T; Schildhauer, Ines; Barroso, Margarita Céspedes; Kofler, David M; Gerner, Franz M; Mysliwietz, Josef; Buening, Hildegard; Hallek, Michael; King, Susan B S

2006-05-01

The modulated expression of MHC class I on tumour tissue is well documented. Although the effect of MHC class I expression on the tumorigenicity and immunogenicity of MHC class I negative tumour cell lines has been rigorously studied, less is known about the validity of gene transfer and selection in cell lines with a mixed MHC class I phenotype. To address this issue we identified a C26 cell subline that consists of distinct populations of MHC class I (H-2D/K) positive and negative cells. Transient transfection experiments using liposome-based transfer showed a lower transgene expression in MHC class I negative cells. In addition, MHC class I negative cells were more sensitive to antibiotic selection. This led to the generation of fully MHC class I positive cell lines. In contrast to C26 cells, all transfectants were rejected in vivo and induced protection against the parental tumour cells in rechallenge experiments. Tumour cell specificity of the immune response was demonstrated in in vitro cytokine secretion and cytotoxicity assays. Transfectants expressing CD40 ligand and hygromycin phosphotransferase were not more immunogenic than cells expressing hygromycin resistance alone. We suggest that the MHC class I positive phenotype of the C26 transfectants had a bearing on their immunogenicity, because selected MHC class I positive cells were more immunogenic than parental C26 cells and could induce specific anti-tumour immune responses. These data demonstrate that the generation of tumour cell transfectants can lead to the selection of subpopulations that show an altered phenotype compared to the parental cell line and display altered immunogenicity independent of selection marker genes or other immune modulatory genes. Our results show the importance of monitoring gene transfer in the whole tumour cell population, especially for the evaluation of in vivo therapies targeted to heterogeneous tumour cell populations.

Area-Specific Cell Stimulation via Surface-Mediated Gene Transfer Using Apatite-Based Composite Layers

Directory of Open Access Journals (Sweden)

Yushin Yazaki

2015-04-01

Full Text Available Surface-mediated gene transfer systems using biocompatible calcium phosphate (CaP-based composite layers have attracted attention as a tool for controlling cell behaviors. In the present study we aimed to demonstrate the potential of CaP-based composite layers to mediate area-specific dual gene transfer and to stimulate cells on an area-by-area basis in the same well. For this purpose we prepared two pairs of DNA–fibronectin–apatite composite (DF-Ap layers using a pair of reporter genes and pair of differentiation factor genes. The results of the area-specific dual gene transfer successfully demonstrated that the cells cultured on a pair of DF-Ap layers that were adjacently placed in the same well showed specific gene expression patterns depending on the gene that was immobilized in theunderlying layer. Moreover, preliminary real-time PCR results indicated that multipotential C3H10T1/2 cells may have a potential to change into different types of cells depending on the differentiation factor gene that was immobilized in the underlying layer, even in the same well. Because DF-Ap layers have a potential to mediate area-specific cell stimulation on their surfaces, they could be useful in tissue engineering applications.

Exosome-mediated transfer of miR-10b promotes cell invasion in breast cancer.

Science.gov (United States)

Singh, Ramesh; Pochampally, Radhika; Watabe, Kounosuke; Lu, Zhaohui; Mo, Yin-Yuan

2014-11-26

Exosomes are 30-100 nm membrane vesicles of endocytic origin, mediating diverse biological functions including tumor cell invasion, cell-cell communication and antigen presentation through transfer of proteins, mRNAs and microRNAs. Recent evidence suggests that microRNAs can be released through ceramide-dependent secretory machinery regulated by neutral sphingomyelinase 2 (nSMase2) enzyme encoded by the smpd3 gene that triggers exosome secretion. However, whether exosome-mediated microRNA transfer plays any role in cell invasion remains poorly understood. Thus, the aim of this study was to identify the exosomal microRNAs involved in breast cancer invasion. The expression level of endogenous and exosomal miRNAs were examined by real time PCR and the expression level of target proteins were detected by western blot. Scanning electron and confocal microscopy were used to characterize exosomes and to study its uptake and transfer. Luciferase reporter plasmids and its mutant were used to confirm direct targeting. Furthermore, the functional significance of exosomal miR-10b was estimated by invasion assay. In this study, we demonstrate that microRNA carrying exosomes can be transferred among different cell lines through direct uptake. miR-10b is highly expressed in metastatic breast cancer MDA-MB-231 cells as compared to non-metastatic breast cancer cells or non-malignant breast cells; it is actively secreted into medium via exosomes. In particular, nSMase2 or ceramide promotes the exosome-mediated miR-10b secretion whereas ceramide inhibitor suppresses this secretion. Moreover, upon uptake, miR-10b can suppress the protein level of its target genes such as HOXD10 and KLF4, indicating its functional significance. Finally, treatment with exosomes derived from MDA-MB-231 cells could induce the invasion ability of non-malignant HMLE cells. Together, our results suggest that a set of specific microRNAs may play an important role in modulating tumor microenvironment through

Studies on the transfer of protective immunity with lymphoid cells from mice immune to malaria sporozoites

International Nuclear Information System (INIS)

Verhave, J.P.; Strickland, G.T.; Jaffe, H.A.; Ahmed, A.

1978-01-01

In an effort to understand the mechanisms involved in the protective immunity to malarial sporozoites, an A/J mouse/Plasmodium berghei model was studied. Protective immunity could consistently be adoptively transferred only by using sublethal irradiation of recipients (500 R); a spleen equivalent (100 x 10 6 ) of donor cells from immune syngeneic mice; and a small booster immunization (1 x 10 4 ) of recipients with irradiation-attenuated sporozoites. Recipient animals treated in this manner were protected from lethal challenge with 1 x 10 4 nonattenuated sporozoites. Immune and nonimmune serum and spleen cells from nonimmune animals did not protect recipient mice. Fewer immune spleen cells (50 x 10 6 ) protected some recipients. In vitro treatment of immune spleen cells with anti-theta sera and complement abolished their ability to transfer protection. This preliminary study suggests that protective sporozoite immunity can be transferred with cells, and that it is T cell dependent

Microfluidic monitoring of programmed cell death in living plant seed tissue

DEFF Research Database (Denmark)

Mark, Christina; Heiskanen, Arto; Zor, Kinga

, et al., (2006), BioEssays, 28, p. 1091). Microfluidic cell culture enables in vitro experiments to approach in vivo conditions. Combining microfluidics with the Lab-On-a-Chip concept allows implementing a wide range of assays for real-time monitoring of effects in a biological system of factors...... such as concentration of selected compounds, external pH, oxygen consumption, redox state and cell viability. The aleurone layer of the barley seed is a 2-3 single cell type thick tissue that can be dissected from the embryo and starchy endosperm. During incubation in vitro this mechanically very robust maintains...

An analysis of expressed sequence tags of developing castor endosperm using a full-length cDNA library

Directory of Open Access Journals (Sweden)

Wallis James G

2007-07-01

Full Text Available Abstract Background Castor seeds are a major source for ricinoleate, an important industrial raw material. Genomics studies of castor plant will provide critical information for understanding seed metabolism, for effectively engineering ricinoleate production in transgenic oilseeds, or for genetically improving castor plants by eliminating toxic and allergic proteins in seeds. Results Full-length cDNAs are useful resources in annotating genes and in providing functional analysis of genes and their products. We constructed a full-length cDNA library from developing castor endosperm, and obtained 4,720 ESTs from 5'-ends of the cDNA clones representing 1,908 unique sequences. The most abundant transcripts are genes encoding storage proteins, ricin, agglutinin and oleosins. Several other sequences are also very numerous, including two acidic triacylglycerol lipases, and the oleate hydroxylase (FAH12 gene that is responsible for ricinoleate biosynthesis. The role(s of the lipases in developing castor seeds are not clear, and co-expressing of a lipase and the FAH12 did not result in significant changes in hydroxy fatty acid accumulation in transgenic Arabidopsis seeds. Only one oleate desaturase (FAD2 gene was identified in our cDNA sequences. Sequence and functional analyses of the castor FAD2 were carried out since it had not been characterized previously. Overexpression of castor FAD2 in a FAH12-expressing Arabidopsis line resulted in decreased accumulation of hydroxy fatty acids in transgenic seeds. Conclusion Our results suggest that transcriptional regulation of FAD2 and FAH12 genes maybe one of the mechanisms that contribute to a high level of ricinoleate accumulation in castor endosperm. The full-length cDNA library will be used to search for additional genes that affect ricinoleate accumulation in seed oils. Our EST sequences will also be useful to annotate the castor genome, which whole sequence is being generated by shotgun sequencing at

Simplification of Bovine Somatic Cell Nuclear Transfer by Application of a Zona-Free Manipulation Technique

DEFF Research Database (Denmark)

Booth, Paul J; Tan, Shijian; Reipurth, Rikke

2001-01-01

Contemporary nuclear transfer techniques often require the involvement of skilled personnel and extended periods of micromanipulation. Here, we present details of the development of a nuclear transfer technique for somatic cells that is both simpler and faster than traditional methods. The techni......Contemporary nuclear transfer techniques often require the involvement of skilled personnel and extended periods of micromanipulation. Here, we present details of the development of a nuclear transfer technique for somatic cells that is both simpler and faster than traditional methods....... The technique comprises the bisection of zona-free oocytes and the reconstruction of embryos comprising two half cytoplasts and a somatic cell by adherence using phytohaemagglutinin-P (PHA) followed by an electropulse and subsequent culture in microwells (termed WOWs--well of the well). The development......-intact zygotes were not different in either blastocyst yield (44.6 +/- 2.4% versus 51.8 +/- 13.5% [mean +/- SEM]) or quality (126.3 +/- 48.4 versus 119.9 +/- 32.6 total cells), and exposure of zygotes to PHA-P did not reduce blastocyst yields compared to vehicle control (40.8 +/- 11.6% versus 47.1 +/- 20...

Application of Adoptive T-Cell Therapy Using Tumor Antigen-Specific T-Cell Receptor Gene Transfer for the Treatment of Human Leukemia

Directory of Open Access Journals (Sweden)

Toshiki Ochi

2010-01-01

Full Text Available The last decade has seen great strides in the field of cancer immunotherapy, especially the treatment of melanoma. Beginning with the identification of cancer antigens, followed by the clinical application of anti-cancer peptide vaccination, it has now been proven that adoptive T-cell therapy (ACT using cancer antigen-specific T cells is the most effective option. Despite the apparent clinical efficacy of ACT, the timely preparation of a sufficient number of cancer antigen-specific T cells for each patient has been recognized as its biggest limitation. Currently, therefore, attention is being focused on ACT with engineered T cells produced using cancer antigen-specific T-cell receptor (TCR gene transfer. With regard to human leukemia, ACT using engineered T cells bearing the leukemia antigen-specific TCR gene still remains in its infancy. However, several reports have provided preclinical data on TCR gene transfer using Wilms' tumor gene product 1 (WT1, and also preclinical and clinical data on TCR gene transfer involving minor histocompatibility antigen, both of which have been suggested to provide additional clinical benefit. In this review, we examine the current status of anti-leukemia ACT with engineered T cells carrying the leukemia antigen-specific TCR gene, and discuss the existing barriers to progress in this area.

Transfer of stem cells carrying engineered chromosomes with XY clone laser system.

Science.gov (United States)

Sinko, Ildiko; Katona, Robert L

2011-01-01

Current transgenic technologies for gene transfer into the germline of mammals cause a random integration of exogenous naked DNA into the host genome that can generate undesirable position effects as well as insertional mutations. The vectors used to generate transgenic animals are limited by the amount of foreign DNA they can carry. Mammalian artificial chromosomes have large DNA-carrying capacity and ability to replicate in parallel with, but without integration into, the host genome. Hence they are attractive vectors for transgenesis, cellular protein production, and gene therapy applications as well. ES cells mediated chromosome transfer by conventional blastocyst injection has a limitation in unpredictable germline transmission. The demonstrated protocol of laser-assisted microinjection of artificial chromosome containing ES cells into eight-cell mouse embryos protocol described here can solve the problem for faster production of germline transchromosomic mice.

Mass and Heat Transfer in Ion-Exchange Membranes Applicable to Solid Polymer Fuel Cells

Energy Technology Data Exchange (ETDEWEB)

Otteroey, M

1996-04-01

In this doctoral thesis, an improved emf method for determination of transference numbers of two counter ions in ion-exchange membranes is presented. Transference numbers were obtained as a continuous function of the composition. The method avoids problems with diffusion by using a stack of membranes. Water transference coefficients in ion-exchange membranes is discussed and reversible and irreversible water transfer is studied by emf methods. Efforts were made to get data relevant to the solid polymer fuel cell. The results support the findings of other researchers that the reversible water transfer is lower than earlier predicted. A chapter on the conductivity of ion-exchange membranes establishes a method to separate the very thin liquid layers surrounding the membranes in a stack. Using the method it was found that the conductivity is obtained with high accuracy and that the liquid layer in a membrane stack can contribute significantly to the total measured resistance. A four point impedance method was tested to measure the conductivity of membranes under fuel cell conditions. Finally, there is a discussion of reversible heat effects and heat transfer in ion-exchange membranes. 155 refs., 45 figs., 13 tabs.

Adoptively transferred immune T cells eradicate established tumors in spite of cancer-induced immune suppression

Science.gov (United States)

Arina, Ainhoa; Schreiber, Karin; Binder, David C.; Karrison, Theodore; Liu, Rebecca B.; Schreiber, Hans

2014-01-01

Myeloid-derived CD11b+Gr1+ suppressor cells (MDSC) and tumor-associated macrophages (TAM) are considered a major obstacle for effective adoptive T cell therapy. Myeloid cells suppress naive T cell proliferation ex vivo and can prevent the generation of T cell responses in vivo. We find, however, that immune T cells adoptively transferred eradicate well-established tumors in the presence of MDSC and TAM which are strongly immunosuppressive ex vivo. These MDSC and TAM were comparable in levels and immunosuppression among different tumor models. Longitudinal microscopy of tumors in vivo revealed that after T cell transfer tumor vasculature and cancer cells disappeared simultaneously. During T-cell mediated tumor destruction, the tumor stroma contained abundant myeloid cells (mainly TAM) that retained their suppressive properties. Preimmunized but not naive mice resisted immune suppression caused by an unrelated tumor-burden supporting the idea that in vivo, myeloid immunosuppressive cells can suppress naive but not memory T cell responses. PMID:24367029

Islet-specific T cell clones transfer diabetes to nonobese diabetic (NOD) F1 mice.

Science.gov (United States)

Peterson, J D; Pike, B; McDuffie, M; Haskins, K

1994-09-15

To investigate diabetes resistance to T cell-mediated disease transfer, we administered islet-specific T cell clones to the F1 progeny of nonobese diabetic (NOD) mice that were crossed with various nondiabetes-prone inbred mouse strains. We investigated four diabetogenic CD4+ T cell clones and all induced insulitis and full development of diabetes in (SWR x NOD)F1, (SJL x NOD)F1, and (C57BL/6 x NOD)F1 mice. In contrast, (BALB/c x NOD)F1 and (CBA x NOD)F1 mice were susceptible to disease transfer by some T cell clones but not others, and (C57/L x NOD)F1 mice seemed to be resistant to both insulitis and disease transfer by all of the clones tested. Disease induced by the T cell clones in susceptible F1 strains was age dependent and could only be observed in recipients younger than 13 days old. Full or partial disease resistance did not correlate with the presence or absence of I-E, different levels of Ag expression in islet cells, or differences in APC function. The results from this study suggest that there may be multiple factors contributing to susceptibility of F1 mice to T cell clone-mediated induction of diabetes, including non-MHC-related genetic background, the immunologic maturity of the recipient, and individual characteristics of the T cell clones.

Differential antibody production by adherent and nonadherent spleen cells transferred to irradiated and cyclophosphamide-treated recipient mice

International Nuclear Information System (INIS)

Albright, J.F.; Deitchman, J.W.; Hassell, S.A.; Ozato, K.

1975-01-01

Mouse spleen cells were separated into adherent (Ad) and nonadherent (Nad) populations by incubation in plastic petri dishes. Adherent, Nad and unfractionated cell preparations (UCP) were transferred into syngeneic recipient mice that had been either irradiated or cyclophosphamide (CY) treated and the adoptive humoral Ab responses were studied by assessment of hemolytic Ab-forming cells (PFC) or humoral serum Ab production. Adherent cells failed to produce PFC in irradiated recipients, but functioned vigorously in CY-treated recipients. Nonadherent cells generated PFC in either type of host, as did UCP. Studies of comparative responses in CY-treated recipients revealed that: (a) Ad-cells generated 2 / 3 the number of PFC given by equivalent numbers of transferred Nad cells and UCP; (b) per equivalent numbers of transferred cells the Ad fraction generated 5 times more and 16 times more Ab than did the Nad cells and UCP, respectively. Spleen cells taken from mice 6 hr after CY treatment failed to respond to the mitogens phytohemagglutinin and bacterial lipopolysaccharide, showing that all cells were temporarily incapable of proliferation. Transfer of spleen cells from donor mice 16 hr after CY treatment, into thymectomized, irradiated, bone marrow-reconstituted recipients revealed substantial T-helper cell activity. We conclude that: (a) Ad preparations lacked T cells that were supplied by CY-treated recipients although T cell proliferation was temporarily inhibited in the latter; (b) B cells present in the Ad fraction were removed from some type of inhibitor of Ab synthesis and/or secretion, the production of which may be associated with T cells present in Nad preparations and UCP; (c) T-helper cells were only transiently affected by CY

A flexible thermoresponsive cell culture substrate for direct transfer of keratinocyte cell sheets.

Science.gov (United States)

Praveen, Wulligundam; Madathil, Bernadette K; Sajin Raj, R S; Kumary, T V; Anil Kumar, P R

2017-10-25

Most cell sheet engineering systems require a support or carrier to handle the harvested cell sheets. In this study, polyethylene terephthalate-based overhead projection transparency sheets (OHPS) were subjected to surface hydrolysis by alkali treatment to increase pliability and hydrophilicity and enable poly(N-isopropylacrylamide-co-glycidylmethacrylate) copolymer (NGMA) coating to impart thermoresponsiveness. NGMA was applied on the modified OHPS by the technique of spin coating using an indigenously designed spin coater. The spin coating had the advantage of using low volumes of the polymer and a reduced coating time. The surface chemistry and thermoresponsive coating was analyzed by Fourier transform infrared spectroscopy and water contact angle. Human keratinocyte cells were cultured on the spin coated surface and scaffold-free cell sheets were successfully harvested by simple variation of temperature. These cell sheets were found to be viable, exhibited epithelial characteristic and cell-cell contact as confirmed by positive immunostaining for ZO-1. The integrity and morphology of the cell sheet was confirmed by stereomicroscopy and E-SEM. These results highlight the potential of the NGMA spin coated modified OHPS to serve as a thermoresponsive culture surface-cum-flexible transfer tool.

A Structurally Specialized Uniform Wall Layer is Essential for Constructing Wall Ingrowth Papillae in Transfer Cells

Science.gov (United States)

Xia, Xue; Zhang, Hui-Ming; Offler, Christina E.; Patrick, John W.

2017-01-01

Transfer cells are characterized by wall labyrinths with either a flange or reticulate architecture. A literature survey established that reticulate wall ingrowth papillae ubiquitously arise from a modified component of their wall labyrinth, termed the uniform wall layer; a structure absent from flange transfer cells. This finding sparked an investigation of the deposition characteristics and role of the uniform wall layer using a Vicia faba cotyledon culture system. On transfer of cotyledons to culture, their adaxial epidermal cells spontaneously trans-differentiate to a reticulate architecture comparable to their abaxial epidermal transfer cell counterparts formed in planta. Uniform wall layer construction commenced once adaxial epidermal cell expansion had ceased to overlay the original outer periclinal wall on its inner surface. In contrast to the dense ring-like lattice of cellulose microfibrils in the original primary wall, the uniform wall layer was characterized by a sparsely dispersed array of linear cellulose microfibrils. A re-modeled cortical microtubule array exerted no influence on uniform wall layer formation or on its cellulose microfibril organization. Surprisingly, formation of the uniform wall layer was not dependent upon depositing a cellulose scaffold. In contrast, uniform wall cellulose microfibrils were essential precursors for constructing wall ingrowth papillae. On converging to form wall ingrowth papillae, the cellulose microfibril diameters increased 3-fold. This event correlated with up-regulated differential, and transfer-cell specific, expression of VfCesA3B while transcript levels of other cellulose biosynthetic-related genes linked with primary wall construction were substantially down-regulated. PMID:29259611

Energy Transfer Kinetics in Photosynthesis as an Inspiration for Improving Organic Solar Cells.

Science.gov (United States)

Nganou, Collins; Lackner, Gerhard; Teschome, Bezu; Deen, M Jamal; Adir, Noam; Pouhe, David; Lupascu, Doru C; Mkandawire, Martin

2017-06-07

Clues to designing highly efficient organic solar cells may lie in understanding the architecture of light-harvesting systems and exciton energy transfer (EET) processes in very efficient photosynthetic organisms. Here, we compare the kinetics of excitation energy tunnelling from the intact phycobilisome (PBS) light-harvesting antenna system to the reaction center in photosystem II in intact cells of the cyanobacterium Acaryochloris marina with the charge transfer after conversion of photons into photocurrent in vertically aligned carbon nanotube (va-CNT) organic solar cells with poly(3-hexyl)thiophene (P3HT) as the pigment. We find that the kinetics in electron hole creation following excitation at 600 nm in both PBS and va-CNT solar cells to be 450 and 500 fs, respectively. The EET process has a 3 and 14 ps pathway in the PBS, while in va-CNT solar cell devices, the charge trapping in the CNT takes 11 and 258 ps. We show that the main hindrance to efficiency of va-CNT organic solar cells is the slow migration of the charges after exciton formation.

Exogenous ROS-induced cell sheet transfer based on hematoporphyrin-polyketone film via a one-step process.

Science.gov (United States)

Koo, Min-Ah; Lee, Mi Hee; Kwon, Byeong-Ju; Seon, Gyeung Mi; Kim, Min Sung; Kim, Dohyun; Nam, Ki Chang; Park, Jong-Chul

2018-04-01

To date, most of invasive cell sheet harvesting methods have used culture surface property variations, such as wettability, pH, electricity, and magnetism, to induce cell detachment. These methods that rely on surface property changes are effective when cell detachment prior to application is necessary, but of limited use when used for cell sheet transfer to target regions. The study reports a new reactive oxygen species (ROS)-induced strategy based on hematoporphyrin-incorporated polyketone film (Hp-PK film) to transfer cell sheets directly to target areas without an intermediate harvesting process. After green LED (510 nm) irradiation, production of exogenous ROS from the Hp-PK films induces cell sheet detachment and transfer. The study suggests that ROS-induced cell detachment property of the Hp-PK film is closely related to conformational changes of extracellular matrix (ECM) proteins. Also, this strategy with the Hp-PK film can be applied by regulating production rate of exogenous ROS in various types of cells, including fibroblasts, mesenchymal stem cells and keratinocytes. In conclusion, ROS-induced method using the Hp-PK film can be used for one-step cell sheet transplantation and has potential in biomedical applications. Copyright © 2018 Elsevier Ltd. All rights reserved.

Asymmetric transfer hydrogenation by synthetic catalysts in cancer cells

Science.gov (United States)

Coverdale, James P. C.; Romero-Canelón, Isolda; Sanchez-Cano, Carlos; Clarkson, Guy J.; Habtemariam, Abraha; Wills, Martin; Sadler, Peter J.

2018-03-01

Catalytic anticancer metallodrugs active at low doses could minimize side-effects, introduce novel mechanisms of action that combat resistance and widen the spectrum of anticancer-drug activity. Here we use highly stable chiral half-sandwich organometallic Os(II) arene sulfonyl diamine complexes, [Os(arene)(TsDPEN)] (TsDPEN, N-(p-toluenesulfonyl)-1,2-diphenylethylenediamine), to achieve a highly enantioselective reduction of pyruvate, a key intermediate in metabolic pathways. Reduction is shown both in aqueous model systems and in human cancer cells, with non-toxic concentrations of sodium formate used as a hydride source. The catalytic mechanism generates selectivity towards ovarian cancer cells versus non-cancerous fibroblasts (both ovarian and lung), which are commonly used as models of healthy proliferating cells. The formate precursor N-formylmethionine was explored as an alternative to formate in PC3 prostate cancer cells, which are known to overexpress a deformylase enzyme. Transfer-hydrogenation catalysts that generate reductive stress in cancer cells offer a new approach to cancer therapy.

Hordein gene dose effects in triploid endosperm of barley (Hordeum vulgare L.

Directory of Open Access Journals (Sweden)

Perović Dragan

2009-01-01

Full Text Available The presence of two maternal chromosome sets in triploid barley endosperm allows the distinction of maternal and paternal hordein bands in an electrophoregram: the maternal bands are stronger due to the higher gene dose. In the F1 generation there are differences between reciprocal crosses and in the F2 generation all 16 classes that are theoretically possible for a pair of polymorphic loci can be distinguished. This full classification is rarely possible in genetic studies, and allows more accurate estimates of recombination rates. Two hordein gene clusters (Hor1 and Hor2, corresponding to hordein C and hordein B respectively were analyzed in hybrids obtained by crossing two winter barley cultivars Partizan and HWV-247. Hordein separation was performed by acid-polyacrylamide gel electrophoresis at pH 3.2 (A-PAGE. A set of most informative bands of B and C hordeins was selected in each cross by two criteria: (1 presence or absence of bands in the parents and (2 signal strength to allow doses scoring. The average genetic distance between Hor1 and Hor2 loci was 11 cM. Distances in male and female maps were not significantly different, suggesting a similar recombination rate in male and female meiosis.

Control of DEMETER DNA demethylase gene transcription in male and female gamete companion cells in Arabidopsis thaliana.

Science.gov (United States)

Park, Jin-Sup; Frost, Jennifer M; Park, Kyunghyuk; Ohr, Hyonhwa; Park, Guen Tae; Kim, Seohyun; Eom, Hyunjoo; Lee, Ilha; Brooks, Janie S; Fischer, Robert L; Choi, Yeonhee

2017-02-21

The DEMETER (DME) DNA glycosylase initiates active DNA demethylation via the base-excision repair pathway and is vital for reproduction in Arabidopsis thaliana DME-mediated DNA demethylation is preferentially targeted to small, AT-rich, and nucleosome-depleted euchromatic transposable elements, influencing expression of adjacent genes and leading to imprinting in the endosperm. In the female gametophyte, DME expression and subsequent genome-wide DNA demethylation are confined to the companion cell of the egg, the central cell. Here, we show that, in the male gametophyte, DME expression is limited to the companion cell of sperm, the vegetative cell, and to a narrow window of time: immediately after separation of the companion cell lineage from the germline. We define transcriptional regulatory elements of DME using reporter genes, showing that a small region, which surprisingly lies within the DME gene, controls its expression in male and female companion cells. DME expression from this minimal promoter is sufficient to rescue seed abortion and the aberrant DNA methylome associated with the null dme-2 mutation. Within this minimal promoter, we found short, conserved enhancer sequences necessary for the transcriptional activities of DME and combined predicted binding motifs with published transcription factor binding coordinates to produce a list of candidate upstream pathway members in the genetic circuitry controlling DNA demethylation in gamete companion cells. These data show how DNA demethylation is regulated to facilitate endosperm gene imprinting and potential transgenerational epigenetic regulation, without subjecting the germline to potentially deleterious transposable element demethylation.

Grain Filling Characteristics and Their Relations with Endogenous Hormones in Large- and Small-Grain Mutants of Rice.

Science.gov (United States)

Zhang, Weiyang; Cao, Zhuanqin; Zhou, Qun; Chen, Jing; Xu, Gengwen; Gu, Junfei; Liu, Lijun; Wang, Zhiqin; Yang, Jianchang; Zhang, Hao

2016-01-01

This study determined if the variation in grain filling parameters between two different spikelet types of rice (Oryza sativa L.) is regulated by the hormonal levels in the grains. Two rice mutants, namely, a large-grain mutant (AZU-M) and a small-grain mutant (ZF802-M), and their respective wild types (AZU-WT and ZF802-WT) were grown in the field. The endosperm cell division rate, filling rate, and hormonal levels: zeatin + zeatin riboside (Z+ZR), indo-3-acetic acid (IAA), polyamines (PAs), and abscisic acid (ABA) were determined. The results showed that there was no significant difference between the filling and endosperm cell division rates. These rates were synchronous between the superior and inferior spikelets for both mutants. However, the abovementioned parameters were significantly different between the two spikelet types for the two wild types. The superior spikelets filled faster and their filling rate was higher compared to the inferior ones. Changes in the concentrations of plant hormones were consistent with the observed endosperm cell division rate and the filling rate for both types of spikelets of mutant and wild type plants. Regression analysis showed a significant positive correlation between cell division and filling rates with the concentrations of the investigated hormones. Exogenous chemical application verified the role of ABA, IAA, and PAs in grain filling. The results indicate that poor filling of inferior spikelets in rice occurs primarily due to the reduced hormone concentrations therein, leading to lower division rate of endosperm cells, fewer endosperm cells, slower filling rate, and smaller grain weight.

Grain Filling Characteristics and Their Relations with Endogenous Hormones in Large- and Small-Grain Mutants of Rice.

Directory of Open Access Journals (Sweden)

Weiyang Zhang

Full Text Available This study determined if the variation in grain filling parameters between two different spikelet types of rice (Oryza sativa L. is regulated by the hormonal levels in the grains. Two rice mutants, namely, a large-grain mutant (AZU-M and a small-grain mutant (ZF802-M, and their respective wild types (AZU-WT and ZF802-WT were grown in the field. The endosperm cell division rate, filling rate, and hormonal levels: zeatin + zeatin riboside (Z+ZR, indo-3-acetic acid (IAA, polyamines (PAs, and abscisic acid (ABA were determined. The results showed that there was no significant difference between the filling and endosperm cell division rates. These rates were synchronous between the superior and inferior spikelets for both mutants. However, the abovementioned parameters were significantly different between the two spikelet types for the two wild types. The superior spikelets filled faster and their filling rate was higher compared to the inferior ones. Changes in the concentrations of plant hormones were consistent with the observed endosperm cell division rate and the filling rate for both types of spikelets of mutant and wild type plants. Regression analysis showed a significant positive correlation between cell division and filling rates with the concentrations of the investigated hormones. Exogenous chemical application verified the role of ABA, IAA, and PAs in grain filling. The results indicate that poor filling of inferior spikelets in rice occurs primarily due to the reduced hormone concentrations therein, leading to lower division rate of endosperm cells, fewer endosperm cells, slower filling rate, and smaller grain weight.

Trial Watch: Adoptive cell transfer for oncological indications

Science.gov (United States)

Aranda, Fernando; Buqué, Aitziber; Bloy, Norma; Castoldi, Francesca; Eggermont, Alexander; Cremer, Isabelle; Fridman, Wolf Hervé; Fucikova, Jitka; Galon, Jérôme; Spisek, Radek; Tartour, Eric; Zitvogel, Laurence; Kroemer, Guido; Galluzzi, Lorenzo

2015-01-01

One particular paradigm of anticancer immunotherapy relies on the administration of (potentially) tumor-reactive immune effector cells. Generally, these cells are obtained from autologous peripheral blood lymphocytes (PBLs) ex vivo (in the context of appropriate expansion, activation and targeting protocols), and re-infused into lymphodepleted patients along with immunostimulatory agents. In spite of the consistent progress achieved throughout the past two decades in this field, no adoptive cell transfer (ACT)-based immunotherapeutic regimen is currently approved by regulatory agencies for use in cancer patients. Nonetheless, the interest of oncologists in ACT-based immunotherapy continues to increase. Accumulating clinical evidence indicates indeed that specific paradigms of ACT, such as the infusion of chimeric antigen receptor (CAR)-expressing autologous T cells, are associated with elevated rates of durable responses in patients affected by various neoplasms. In line with this notion, clinical trials investigating the safety and therapeutic activity of ACT in cancer patients are being initiated at an ever increasing pace. Here, we review recent preclinical and clinical advances in the development of ACT-based immunotherapy for oncological indications. PMID:26451319

Dataset on exogenous application of salicylic acid and methyljasmonate and the accumulation of caffeine in young leaf tissues and catabolically inactive endosperms

Directory of Open Access Journals (Sweden)

Avinash Kumar

2017-08-01

Full Text Available Exogenous exposure of coffee plants to 50 μM and 500 μM salicylic acid through liquid hydroponic medium or the exposure to volatile fumes of methyljasmonate was carried out to study the role of salicylic acid and methyljasmonate on the accumulation of caffeine and other methylxanthines like 7-methylxanthine, theobromine and theophylline. Transcript levels of the first, second and third N-methyltransferase involved in the core caffeine biosynthetic pathway namely, xanthosine methyltransferase (XMT, methylxanthine methyltransferase (MXMT and di-methylxanthine methyltransferase (DXMT was investigated by semi-quantitative RT-PCR for validating the reason behind the changes of caffeine biosynthetic potential under the influence of the two analogues of plant phytohormones. Maturing coffee fruits are known to be biologically inactive with respect to caffeine biosynthetic activity in the endosperms. To understand this, fruits were treated with different doses of salicylic acid in a time-course manner and the de-repression of tissue maturation-mediated knockdown of caffeine biosynthesis by exogenously applied salicylic acid was achieved. In our companion paper [1] it was shown that the repression of NMT genes during the dry weight accumulation phase of maturing endosperm could be relaxed by the exogenous application of salicylic acid and methyljasmonate. A probable model based on the work carried out therein and based on other literature [2–4] was proposed to describe that the crosstalk between salicylic acid or methyljasmonate and the ABA/ethylene pathway and might involve transcription factors downstream to the signaling cascade.

Microparticle-mediated transfer of the viral receptors CAR and CD46, and the CFTR channel in a CHO cell model confers new functions to target cells.

Directory of Open Access Journals (Sweden)

Gaëlle Gonzalez

Full Text Available Cell microparticles (MPs released in the extracellular milieu can embark plasma membrane and intracellular components which are specific of their cellular origin, and transfer them to target cells. The MP-mediated, cell-to-cell transfer of three human membrane glycoproteins of different degrees of complexity was investigated in the present study, using a CHO cell model system. We first tested the delivery of CAR and CD46, two monospanins which act as adenovirus receptors, to target CHO cells. CHO cells lack CAR and CD46, high affinity receptors for human adenovirus serotype 5 (HAdV5, and serotype 35 (HAdV35, respectively. We found that MPs derived from CHO cells (MP-donor cells constitutively expressing CAR (MP-CAR or CD46 (MP-CD46 were able to transfer CAR and CD46 to target CHO cells, and conferred selective permissiveness to HAdV5 and HAdV35. In addition, target CHO cells incubated with MP-CD46 acquired the CD46-associated function in complement regulation. We also explored the MP-mediated delivery of a dodecaspanin membrane glycoprotein, the CFTR to target CHO cells. CFTR functions as a chloride channel in human cells and is implicated in the genetic disease cystic fibrosis. Target CHO cells incubated with MPs produced by CHO cells constitutively expressing GFP-tagged CFTR (MP-GFP-CFTR were found to gain a new cellular function, the chloride channel activity associated to CFTR. Time-course analysis of the appearance of GFP-CFTR in target cells suggested that MPs could achieve the delivery of CFTR to target cells via two mechanisms: the transfer of mature, membrane-inserted CFTR glycoprotein, and the transfer of CFTR-encoding mRNA. These results confirmed that cell-derived MPs represent a new class of promising therapeutic vehicles for the delivery of bioactive macromolecules, proteins or mRNAs, the latter exerting the desired therapeutic effect in target cells via de novo synthesis of their encoded proteins.

In Vitro Biochemical Characterization of All Barley Endosperm Starch Synthases

Directory of Open Access Journals (Sweden)

Jose Antonio Cuesta-Seijo

2016-01-01

Full Text Available Starch is the main storage polysaccharide in cereals and the major source of calories in the human diet. It is synthesized by a panel of enzymes including five classes of starch synthases (SSs. While the overall starch synthase (SS reaction is known, the functional differences between the five SS classes are poorly understood. Much of our knowledge comes from analyzing mutant plants with altered SS activities, but the resulting data are often difficult to interpret as a result of pleitropic effects, competition between enzymes, overlaps in enzyme activity and disruption of multi-enzyme complexes. Here we provide a detailed biochemical study of the activity of all five classes of SSs in barley endosperm. Each enzyme was produced recombinantly in E. coli and the properties and modes of action in vitro were studied in isolation from other SSs and other substrate modifying activities. Our results define the mode of action of each SS class in unprecedented detail; we analyze their substrate selection, temperature dependence and stability, substrate affinity and temporal abundance during barley development. Our results are at variance with some generally accepted ideas about starch biosynthesis and might lead to the reinterpretation of results obtained in planta. In particular, they indicate that granule bound SS is capable of processive action even in the absence of a starch matrix, that SSI has no elongation limit, and that SSIV, believed to be critical for the initiation of starch granules, has maltoligosaccharides and not polysaccharides as its preferred substrates.

Molecular evolution of the endosperm starch synthesis pathway genes in rice (Oryza sativa L.) and its wild ancestor, O. rufipogon L.

Science.gov (United States)

Yu, Guoqin; Olsen, Kenneth M; Schaal, Barbara A

2011-01-01

The evolution of metabolic pathways is a fundamental but poorly understood aspect of evolutionary change. One approach for understanding the complexity of pathway evolution is to examine the molecular evolution of genes that together comprise an integrated metabolic pathway. The rice endosperm starch biosynthetic pathway is one of the most thoroughly characterized metabolic pathways in plants, and starch is a trait that has evolved in response to strong selection during rice domestication. In this study, we have examined six key genes (AGPL2, AGPS2b, SSIIa, SBEIIb, GBSSI, ISA1) in the rice endosperm starch biosynthesis pathway to investigate the evolution of these genes before and after rice domestication. Genome-wide sequence tagged sites data were used as a neutral reference to overcome the problems of detecting selection in species with complex demographic histories such as rice. Five variety groups of Oryza sativa (aus, indica, tropical japonica, temperate japonica, aromatic) and its wild ancestor (O. rufipogon) were sampled. Our results showed evidence of purifying selection at AGPL2 in O. rufipogon and strong evidence of positive selection at GBSSI in temperate japonica and tropical japonica varieties and at GBSSI and SBEIIb in aromatic varieties. All the other genes showed a pattern consistent with neutral evolution in both cultivated rice and its wild ancestor. These results indicate the important role of positive selection in the evolution of starch genes during rice domestication. We discuss the role of SBEIIb and GBSSI in the evolution of starch quality during rice domestication and the power and limitation of detecting selection using genome-wide data as a neutral reference.

Differential radiosensitivity on a tissue level in Delphinium ajacis

Energy Technology Data Exchange (ETDEWEB)

Mandal, S K; Basu, R K [Bose Research Inst., Calcutta (India). Cryogenetics Lab.

1980-09-01

Root, leaf, pollen mother cell and endosperm of D.ajacis showed differential sensitivity as measured by X-ray-induced chromosomal aberrations at mitotic anaphase and telophase stages of the first and second division cycles after irradiation. These tissues differed significantly in Interphase Chromosome Volume (ICV) values. In all the tissues the percentage of aberrant cells increased linearly with increase in X-ray dose. Though endosperm had the largest ICV value it was the most radioresistant tissue tested. The relative radiosensitivity of the other 3 tissues was positively correlated with ICV value. The radioresistance of endosperm is probably due to factors unique to this tissue which remained obscure.

Immunomodulatory Effects of Hemagglutinin- (HA- Modified A20 B-Cell Lymphoma Expanded as a Brain Tumor on Adoptively Transferred HA-Specific CD4+ T Cells

Directory of Open Access Journals (Sweden)

Valentin P. Shichkin

2014-01-01

Full Text Available Previously, the mouse A20 B-cell lymphoma engineered to express hemagglutinin (HA antigen (A20HA was used as a systemic tumor model. In this work, we used the A20HA cells as a brain tumor. HA-specific CD4+ T cells were transferred intravenously in a tail vein 5 days after A20HA intracranial inoculation and analyzed on days 2, 9, and 16 after the adoptive transfer by different methods. The transferred cells demonstrated state of activation as early as day 2 after the adoptive transfer and most the of viable HA-specific cells became anergic on day 16. Additionally, symptoms of systemic immunosuppression were observed in mice with massive brain tumors at a late stage of the brain tumor progression (days 20–24 after the A20HA inoculation. Despite that, a deal of HA-specific CD4+ T cells kept the functional activity even at the late stage of A20HA tumor growth. The activated HA-specific CD4+ T cells were found also in the brain of brain-tumor-bearing mice. These cells were still responding to reactivation with HA-peptide in vitro. Our data support an idea about sufficient role of both the tumor-specific and -nonspecific mechanisms inducing immunosuppression in cancer patients.

In vitro oocyte culture and somatic cell nuclear transfer used to produce a live-born cloned goat.

Science.gov (United States)

Ohkoshi, Katsuhiro; Takahashi, Seiya; Koyama, Shin-Ichiro; Akagi, Satoshi; Adachi, Noritaka; Furusawa, Tadashi; Fujimoto, Jun-Ichiro; Takeda, Kumiko; Kubo, Masanori; Izaike, Yoshiaki; Tokunaga, Tomoyuki

2003-01-01

The use of an in vitro culture system was examined for production of somatic cells suitable for nuclear transfer in the goat. Goat cumulus-oocyte complexes were incubated in tissue culture medium TCM-199 supplemented with 10% fetal bovine serum (FBS) for 20 h. In vitro matured (IVM) oocytes were enucleated and used as karyoplast recipients. Donor cells obtained from the anterior pituitary of an adult male were introduced into the perivitelline space of enucleated IVM oocytes and fused by an electrical pulse. Reconstituted oocytes were cultured in chemically defined medium for 9 days. Two hundred and twenty-eight oocytes (70%) were fused with donor cells. After in vitro culture, seven somatic cell nuclear transfer (SCNT) oocytes (3%) developed to the blastocyst stage. SCNT embryos were transferred to the oviducts of recipient females (four 8-cell embryos per female) or uterine horn (two blastocysts per female). One male clone (NT1) was produced at day 153 from an SCNT blastocyst and died 16 days after birth. This study demonstrates that nuclear transferred goat oocytes produced using an in vitro culture system could develop to term and that donor anterior pituitary cells have the developmental potential to produce term offspring. In this study, it suggested that the artificial control of endocrine system in domestic animal might become possible by the genetic modification to anterior pituitary cells.

Horizontal gene transfers with or without cell fusions in all categories of the living matter.

Science.gov (United States)

Sinkovics, Joseph G

2011-01-01

This article reviews the history of widespread exchanges of genetic segments initiated over 3 billion years ago, to be part of their life style, by sphero-protoplastic cells, the ancestors of archaea, prokaryota, and eukaryota. These primordial cells shared a hostile anaerobic and overheated environment and competed for survival. "Coexist with, or subdue and conquer, expropriate its most useful possessions, or symbiose with it, your competitor" remain cellular life's basic rules. This author emphasizes the role of viruses, both in mediating cell fusions, such as the formation of the first eukaryotic cell(s) from a united crenarchaeon and prokaryota, and the transfer of host cell genes integrated into viral (phages) genomes. After rising above the Darwinian threshold, rigid rules of speciation and vertical inheritance in the three domains of life were established, but horizontal gene transfers with or without cell fusions were never abolished. The author proves with extensive, yet highly selective documentation, that not only unicellular microorganisms, but the most complex multicellular entities of the highest ranks resort to, and practice, cell fusions, and donate and accept horizontally (laterally) transferred genes. Cell fusions and horizontally exchanged genetic materials remain the fundamental attributes and inherent characteristics of the living matter, whether occurring accidentally or sought after intentionally. These events occur to cells stagnating for some 3 milliard years at a lower yet amazingly sophisticated level of evolution, and to cells achieving the highest degree of differentiation, and thus functioning in dependence on the support of a most advanced multicellular host, like those of the human brain. No living cell is completely exempt from gene drains or gene insertions.

MicroRNA Transfer Between Bone Marrow Adipose and Multiple Myeloma Cells.

Science.gov (United States)

Soley, Luna; Falank, Carolyne; Reagan, Michaela R

2017-06-01

Multiple myeloma remains an incurable disease, largely due to the tumor-supportive role of the bone marrow microenvironment. Bone marrow adipose tissue (BMAT) is one component of the fertile microenvironment which is believed to contribute to myeloma progression and drug resistance, as well as participate in a vicious cycle of osteolysis and tumor growth. MicroRNAs (miRNAs) have recently emerged as instrumental regulators of cellular processes that enable the development and dissemination of cancer. This review highlights the intersection between two emerging research fields and pursues the scientific and clinical implications of miRNA transfer between BMAT and myeloma cells. This review provides a concise and provocative summary of the evidence to support exosome-mediated transfer of tumor-supportive miRNAs. The work may prompt researchers to better elucidate the mechanisms by which this novel means of genetic communication between tumor cells and their environment could someday yield targeted therapeutics.

Recombinant interleukin 2 stimulates in vivo proliferation of adoptively transferred lymphokine-activated killer (LAK) cells

International Nuclear Information System (INIS)

Ettinghausen, S.E.; Lipford, E.H. III; Mule, J.J.; Rosenberg, S.A.

1985-01-01

The authors previously reported that the adoptive transfer of lymphokine-activated killer (LAK) cells plus repetitive injections of recombinant interleukin 2 (IL 2) produced a marked reduction in established pulmonary metastases from a variety of murine sarcomas. The requirement for the exogenous administration of IL 2 prompted a subsequent examination of the role of IL 2 in the in vivo function of transferred LAK cells. The in vivo proliferation and migration patterns of lymphoid cells in C57BL/6 mice were examined after i.v. transfer of LAK cells alone, i.p. injection of IL 2 alone, or the combination of LAK cells and IL 2. A model for in vivo labeling of the DNA of dividing cells was used in which mice were injected with 5-[ 125 I]-iodo-2'-deoxyuridine ( 125 IUdR) and, 20 hr later, their tissues were removed and were counted in a gamma analyzer. A proliferation index (PI) was calculated by dividing the mean cpm of organs of experimentally treated mice by the mean cpm of organs of control mice. In animals given LAK cells alone, the lungs and liver demonstrated little if any uptake of 125 IUdR above saline-treated controls, whereas the same organs of mice receiving 6000 U of IL 2 alone displayed higher radiolabel incorporation. When mice were given LAK cells plus 6000 U of IL 2, their tissues showed an additional increase in 125 IUdR uptake

Parameter extraction of different fuel cell models with transferred adaptive differential evolution

International Nuclear Information System (INIS)

Gong, Wenyin; Yan, Xuesong; Liu, Xiaobo; Cai, Zhihua

2015-01-01

To improve the design and control of FC (fuel cell) models, it is important to extract their unknown parameters. Generally, the parameter extraction problems of FC models can be transformed as nonlinear and multi-variable optimization problems. To extract the parameters of different FC models exactly and fast, in this paper, we propose a transferred adaptive DE (differential evolution) framework, in which the successful parameters of the adaptive DE solving previous problems are properly transferred to solve new optimization problems in the similar problem-domains. Based on this framework, an improved adaptive DE method (TRADE, in short) is presented as an illustration. To verify the performance of our proposal, TRADE is used to extract the unknown parameters of two types of fuel cell models, i.e., PEMFC (proton exchange membrane fuel cell) and SOFC (solid oxide fuel cell). The results of TRADE are also compared with those of other state-of-the-art EAs (evolutionary algorithms). Even though the modification is very simple, the results indicate that TRADE can extract the parameters of both PEMFC and SOFC models exactly and fast. Moreover, the V–I characteristics obtained by TRADE agree well with the simulated and experimental data in all cases for both types of fuel cell models. Also, it improves the performance of the original adaptive DE significantly in terms of both the quality of final solutions and the convergence speed in all cases. Additionally, TRADE is able to provide better results compared with other EAs. - Highlights: • A framework of transferred adaptive differential evolution is proposed. • Based on the framework, an improved differential evolution (TRADE) is presented. • TRADE obtains very promising results to extract the parameters of PEMFC and SOFC models

A bipartite signal mediates the transfer of type IV secretion substrates of Bartonella henselae into human cells.

Science.gov (United States)

Schulein, Ralf; Guye, Patrick; Rhomberg, Thomas A; Schmid, Michael C; Schröder, Gunnar; Vergunst, Annette C; Carena, Ilaria; Dehio, Christoph

2005-01-18

Bacterial type IV secretion (T4S) systems mediate the transfer of macromolecular substrates into various target cells, e.g., the conjugative transfer of DNA into bacteria or the transfer of virulence proteins into eukaryotic host cells. The T4S apparatus VirB of the vascular tumor-inducing pathogen Bartonella henselae causes subversion of human endothelial cell (HEC) function. Here we report the identification of multiple protein substrates of VirB, which, upon translocation into HEC, mediate all known VirB-dependent cellular changes. These Bartonella-translocated effector proteins (Beps) A-G are encoded together with the VirB system and the T4S coupling protein VirD4 on a Bartonella-specific pathogenicity island. The Beps display a modular architecture, suggesting an evolution by extensive domain duplication and reshuffling. The C terminus of each Bep harbors at least one copy of the Bep-intracellular delivery domain and a short positively charged tail sequence. This biparte C terminus constitutes a transfer signal that is sufficient to mediate VirB/VirD4-dependent intracellular delivery of reporter protein fusions. The Bep-intracellular delivery domain is also present in conjugative relaxases of bacterial conjugation systems. We exemplarily show that the C terminus of such a conjugative relaxase mediates protein transfer through the Bartonella henselae VirB/VirD4 system into HEC. Conjugative relaxases may thus represent the evolutionary origin of the here defined T4S signal for protein transfer into human cells.

Somatic cell nuclear transfer: Infinite reproduction of a unique diploid genome

International Nuclear Information System (INIS)

Kishigami, Satoshi; Wakayama, Sayaka; Hosoi, Yoshihiko; Iritani, Akira; Wakayama, Teruhiko

2008-01-01

In mammals, a diploid genome of an individual following fertilization of an egg and a spermatozoon is unique and irreproducible. This implies that the generated unique diploid genome is doomed with the individual ending. Even as cultured cells from the individual, they cannot normally proliferate in perpetuity because of the 'Hayflick limit'. However, Dolly, the sheep cloned from an adult mammary gland cell, changes this scenario. Somatic cell nuclear transfer (SCNT) enables us to produce offspring without germ cells, that is, to 'passage' a unique diploid genome. Animal cloning has also proven to be a powerful research tool for reprogramming in many mammals, notably mouse and cow. The mechanism underlying reprogramming, however, remains largely unknown and, animal cloning has been inefficient as a result. More momentously, in addition to abortion and fetal mortality, some cloned animals display possible premature aging phenotypes including early death and short telomere lengths. Under these inauspicious conditions, is it really possible for SCNT to preserve a diploid genome? Delightfully, in mouse and recently in primate, using SCNT we can produce nuclear transfer ES cells (ntES) more efficiently, which can preserve the eternal lifespan for the 'passage' of a unique diploid genome. Further, new somatic cloning technique using histone-deacetylase inhibitors has been developed which can significantly increase the previous cloning rates two to six times. Here, we introduce SCNT and its value as a preservation tool for a diploid genome while reviewing aging of cloned animals on cellular and individual levels

Somatic cell nuclear transfer: infinite reproduction of a unique diploid genome.

Science.gov (United States)

Kishigami, Satoshi; Wakayama, Sayaka; Hosoi, Yoshihiko; Iritani, Akira; Wakayama, Teruhiko

2008-06-10

In mammals, a diploid genome of an individual following fertilization of an egg and a spermatozoon is unique and irreproducible. This implies that the generated unique diploid genome is doomed with the individual ending. Even as cultured cells from the individual, they cannot normally proliferate in perpetuity because of the "Hayflick limit". However, Dolly, the sheep cloned from an adult mammary gland cell, changes this scenario. Somatic cell nuclear transfer (SCNT) enables us to produce offspring without germ cells, that is, to "passage" a unique diploid genome. Animal cloning has also proven to be a powerful research tool for reprogramming in many mammals, notably mouse and cow. The mechanism underlying reprogramming, however, remains largely unknown and, animal cloning has been inefficient as a result. More momentously, in addition to abortion and fetal mortality, some cloned animals display possible premature aging phenotypes including early death and short telomere lengths. Under these inauspicious conditions, is it really possible for SCNT to preserve a diploid genome? Delightfully, in mouse and recently in primate, using SCNT we can produce nuclear transfer ES cells (ntES) more efficiently, which can preserve the eternal lifespan for the "passage" of a unique diploid genome. Further, new somatic cloning technique using histone-deacetylase inhibitors has been developed which can significantly increase the previous cloning rates two to six times. Here, we introduce SCNT and its value as a preservation tool for a diploid genome while reviewing aging of cloned animals on cellular and individual levels.

Intercellular transfer of P-glycoprotein from the drug resistant human bladder cancer cell line BIU-87 does not require cell-to-cell contact.

Science.gov (United States)

Zhou, Hui-liang; Zheng, Yong-jun; Cheng, Xiao-zhi; Lv, Yi-song; Gao, Rui; Mao, Hou-ping; Chen, Qin

2013-09-01

The efflux activity of transmembrane P-glycoprotein prevents various therapeutic drugs from reaching lethal concentrations in cancer cells, resulting in multidrug resistance. We investigated whether drug resistant bladder cancer cells could transfer functional P-glycoprotein to sensitive parental cells. Drug sensitive BIU-87 bladder cancer cells were co-cultured for 48 hours with BIU-87/ADM, a doxorubicin resistant derivative of the same cell line, in a Transwell® system that prevented cell-to-cell contact. The presence of P-glycoprotein in recipient cell membranes was established using fluorescein isothiocyanate, laser scanning confocal microscopy and Western blot. P-glycoprotein mRNA levels were compared between cell types. Rhodamine 123 efflux assay was done to confirm that P-glycoprotein was biologically active. The amount of P-glycoprotein protein in BIU-87 cells co-cultured with BIU-87/ADM was significantly higher than in BIU-87 cells (0.44 vs 0.25) and BIU-87/H33342 cells (0.44 vs 0.26, each p transfer. P-glycoprotein mRNA expression was significantly higher in BIU-87/ADM cells than in co-cultured BIU-87 cells (1.28 vs 0.30), BIU-87/H33342 (0.28) and BIU-87 cells (0.25, each p <0.001), ruling out a genetic mechanism. After 30 minutes of efflux, rhodamine 123 fluorescence intensity was significantly lower in BIU-87/ADM cells (5.55 vs 51.45, p = 0.004) and co-cultured BIU-87 cells than in BIU-87 cells (14.22 vs 51.45, p <0.001), indicating that P-glycoprotein was functional. Bladder cancer cells can acquire functional P-glycoprotein through a nongenetic mechanism that does not require direct cell contact. This mechanism is consistent with a microparticle mediated process. Copyright © 2013 American Urological Association Education and Research, Inc. Published by Elsevier Inc. All rights reserved.

Combinations of chromosome transfer and genome editing for the development of cell/animal models of human disease and humanized animal models.

Science.gov (United States)

Uno, Narumi; Abe, Satoshi; Oshimura, Mitsuo; Kazuki, Yasuhiro

2018-02-01

Chromosome transfer technology, including chromosome modification, enables the introduction of Mb-sized or multiple genes to desired cells or animals. This technology has allowed innovative developments to be made for models of human disease and humanized animals, including Down syndrome model mice and humanized transchromosomic (Tc) immunoglobulin mice. Genome editing techniques are developing rapidly, and permit modifications such as gene knockout and knockin to be performed in various cell lines and animals. This review summarizes chromosome transfer-related technologies and the combined technologies of chromosome transfer and genome editing mainly for the production of cell/animal models of human disease and humanized animal models. Specifically, these include: (1) chromosome modification with genome editing in Chinese hamster ovary cells and mouse A9 cells for efficient transfer to desired cell types; (2) single-nucleotide polymorphism modification in humanized Tc mice with genome editing; and (3) generation of a disease model of Down syndrome-associated hematopoiesis abnormalities by the transfer of human chromosome 21 to normal human embryonic stem cells and the induction of mutation(s) in the endogenous gene(s) with genome editing. These combinations of chromosome transfer and genome editing open up new avenues for drug development and therapy as well as for basic research.

β-Cell Replacement in Mice Using Human Type 1 Diabetes Nuclear Transfer Embryonic Stem Cells.

Science.gov (United States)

Sui, Lina; Danzl, Nichole; Campbell, Sean R; Viola, Ryan; Williams, Damian; Xing, Yuan; Wang, Yong; Phillips, Neil; Poffenberger, Greg; Johannesson, Bjarki; Oberholzer, Jose; Powers, Alvin C; Leibel, Rudolph L; Chen, Xiaojuan; Sykes, Megan; Egli, Dieter

2018-01-01

β-Cells derived from stem cells hold great promise for cell replacement therapy for diabetes. Here we examine the ability of nuclear transfer embryonic stem cells (NT-ESs) derived from a patient with type 1 diabetes to differentiate into β-cells and provide a source of autologous islets for cell replacement. NT-ESs differentiate in vitro with an average efficiency of 55% into C-peptide-positive cells, expressing markers of mature β-cells, including MAFA and NKX6.1. Upon transplantation in immunodeficient mice, grafted cells form vascularized islet-like structures containing MAFA/C-peptide-positive cells. These β-cells adapt insulin secretion to ambient metabolite status and show normal insulin processing. Importantly, NT-ES-β-cells maintain normal blood glucose levels after ablation of the mouse endogenous β-cells. Cystic structures, but no teratomas, were observed in NT-ES-β-cell grafts. Isogenic induced pluripotent stem cell lines showed greater variability in β-cell differentiation. Even though different methods of somatic cell reprogramming result in stem cell lines that are molecularly indistinguishable, full differentiation competence is more common in ES cell lines than in induced pluripotent stem cell lines. These results demonstrate the suitability of NT-ES-β-cells for cell replacement for type 1 diabetes and provide proof of principle for therapeutic cloning combined with cell therapy. © 2017 by the American Diabetes Association.

Direct electron transfer based enzymatic fuel cells

International Nuclear Information System (INIS)

Falk, Magnus; Blum, Zoltan; Shleev, Sergey

2012-01-01

In this mini-review we briefly describe some historical developments made in the field of enzymatic fuel cells (FCs), discussing important design considerations taken when constructing mediator-, cofactor-, and membrane-less biological FCs (BFCs). Since the topic is rather extensive, only BFCs utilizing direct electron transfer (DET) reactions on both the anodic and cathodic sides are considered. Moreover, the performance of mostly glucose/oxygen biodevices is analyzed and compared. We also present some unpublished results on mediator-, cofactor-, and membrane-less glucose/oxygen BFCs recently designed in our group and tested in different human physiological fluids, such as blood, plasma, saliva, and tears. Finally, further perspectives for BFC applications are highlighted.

Direct Electron Transfer of Dehydrogenases for Development of 3rd Generation Biosensors and Enzymatic Fuel Cells

Directory of Open Access Journals (Sweden)

Paolo Bollella

2018-04-01

Full Text Available Dehydrogenase based bioelectrocatalysis has been increasingly exploited in recent years in order to develop new bioelectrochemical devices, such as biosensors and biofuel cells, with improved performances. In some cases, dehydrogeases are able to directly exchange electrons with an appropriately designed electrode surface, without the need for an added redox mediator, allowing bioelectrocatalysis based on a direct electron transfer process. In this review we briefly describe the electron transfer mechanism of dehydrogenase enzymes and some of the characteristics required for bioelectrocatalysis reactions via a direct electron transfer mechanism. Special attention is given to cellobiose dehydrogenase and fructose dehydrogenase, which showed efficient direct electron transfer reactions. An overview of the most recent biosensors and biofuel cells based on the two dehydrogenases will be presented. The various strategies to prepare modified electrodes in order to improve the electron transfer properties of the device will be carefully investigated and all analytical parameters will be presented, discussed and compared.

Horizontal transfer of miR-106a/b from cisplatin resistant hepatocarcinoma cells can alter the sensitivity of cervical cancer cells to cisplatin.

Science.gov (United States)

Raji, Grace R; Sruthi, T V; Edatt, Lincy; Haritha, K; Sharath Shankar, S; Sameer Kumar, V B

2017-10-01

Recent studies indicate that horizontal transfer of genetic material can act as a communication tool between heterogenous populations of tumour cells, thus altering the chemosensitivity of tumour cells. The present study was designed to check whether the horizontal transfer of miRNAs released by cisplatin resistant (Cp-r) Hepatocarcinoma cells can alter the sensitivity of cervical cancer cells. For this exosomes secreted by cisplatin resistant and cisplatin sensitive HepG2 cells (EXres and EXsen) were isolated and characterised. Cytotoxicity analysis showed that EXres can make Hela cells resistant to cisplatin. Analysis of miR-106a/b levels in EXres and EXsen showed that their levels vary. Mechanistic studies showed that miR-106a/b play an important role in EXsen and EXres mediated change in chemosensitivity of Hela cells to cisplatin. Further SIRT1 was identified as a major target of miR-106a/b using in silico tools and this was proved by experimentation. Also the effect of miR-106a/b in chemosensitivity was seen to be dependent on regulation of SIRT1 by miR-106a/b. In brief, this study brings into light, the SIRT1 dependent mechanism of miR-106a/b mediated regulation of chemosensitivity upon the horizontal transfer from one cell type to another. Copyright © 2017 Elsevier Inc. All rights reserved.

The role of the thymus for maturation of transferred bursa cells into immuno-competent B cells in chickens treated with cyclophosphamide

International Nuclear Information System (INIS)

Hirota, Y.; Bito, Y.

1978-01-01

Chickens injected with cyclophosphamide and X-ray irradiated in the newly hatched period were immunized with a mixture of sheep red blood cells, Brucella abortus and Salmonella pullorum at 4, 5 and 6 weeks of age, and were examined for serum antibody titres, serum immunoglobulin concentration and bursal and splenic structures at 7 weeks of age. The neonatal treatments suppressed completely or almost completely antibody responses, immunoglobulin production and formation of bursal follicles and splenic germinal centres. The transplantation of bursa cells into the chickens immunologically impaired by the treatments restored these functions and structures. In contrast, the transfer of bursa cells into chickens thymectomized, cyclophosphamide-treated and X-ray irradiated did not result in efficient restoration of the bursa-dependent immune system; 10-day-old bursa cells hardly restore the system, although 4-week-old bursa cells did so slightly. The chickens thymectomized, cyclophosphamide-treated, X-ray irradiated and re-populated with 10-day-old bursa cells were examined for the existence of functional B cells with the use of a syngeneic cell transfer system. The experiments verified that immunocompetent B cells had not developed in the chickens thus treated. (author)

Effects of donor fibroblast cell type and transferred cloned embryo number on the efficiency of pig cloning.

Science.gov (United States)

Li, Zicong; Shi, Junsong; Liu, Dewu; Zhou, Rong; Zeng, Haiyu; Zhou, Xiu; Mai, Ranbiao; Zeng, Shaofen; Luo, Lvhua; Yu, Wanxian; Zhang, Shouquan; Wu, Zhenfang

2013-02-01

Currently, cloning efficiency in pigs is very low. Donor cell type and number of cloned embryos transferred to an individual surrogate are two major factors that affect the successful rate of somatic cell nuclear transfer (SCNT) in pigs. This study aimed to compare the influence of different donor fibroblast cell types and different transferred embryo numbers on recipients' pregnancy rate and delivery rate, the average number of total clones born, clones born alive and clones born healthy per litter, and the birth rate of healthy clones (=total number of healthy cloned piglets born /total number of transferred cloned embryos). Three types of donor fibroblasts were tested in large-scale production of cloned pigs, including fetal fibroblasts (FFBs) from four genetically similar Western swine breeds of Pietrain (P), Duroc (D), Landrace (L), and Yorkshire (Y), which are referred to as P,D,LY-FFBs, adult fibroblasts (AFBs) from the same four breeds, which are designated P,D,L,Y-AFBs, and AFBs from a Chinese pig breed of Laiwu (LW), which is referred to as LW-AFBs. Within each donor fibroblast cell type group, five transferred cloned embryo number groups were tested. In each embryo number group, 150-199, 200-249, 250-299, 300-349, or 350-450 cloned embryos were transferred to each individual recipient sow. For the entire experiment, 92,005 cloned embryos were generated from nearly 115,000 matured oocytes and transferred to 328 recipients; in total, 488 cloned piglets were produced. The results showed that the mean clones born healthy per litter resulted from transfer of embryos cloned from LW-AFBs (2.53 ± 0.34) was similar with that associated with P,D,L,Y-FFBs (2.72 ± 0.29), but was significantly higher than that resulted from P,D,L,Y-AFBs (1.47 ± 0.18). Use of LW-AFBs as donor cells for SCNT resulted in a significantly higher pregnancy rate (72.00% vs. 59.30% and 48.11%) and delivery rate (60.00% vs. 45.93% and 35.85%) for cloned embryo recipients, and a

Evaluation of porcine stem cells competence for somatic cell nuclear transfer and production of cloned animals

DEFF Research Database (Denmark)

Secher, Jan; Liu, Ying; Petkov, Stoyan

2017-01-01

Porcine somatic cell nuclear transfer (SCNT) has been used extensively to create genetically modified pigs, but the efficiency of the methodology is still low. It has been hypothesized that pluripotent or multipotent stem cells might result in increased SCNT efficacy as these cells are closer than...... somatic cells to the epigenetic state found in the blastomeres and therefore need less reprogramming. Our group has worked with porcine SCNT during the last 20 years and here we describe our experience with SCNT of 3 different stem cell lines. The porcine stem cells used were: Induced pluripotent stem...... cells (iPSCs) created by lentiviral doxycycline-dependent reprogramming and cultered with a GSK3β- and MEK-inhibitor (2i) and leukemia inhibitor factor (LIF) (2i LIF DOX-iPSCs), iPSCs created by a plasmid-based reprogramming and cultured with 2i and fibroblast growth factor (FGF) (2i FGF Pl...

The proteins of the grape (Vitis vinifera L.) seed endosperm: fractionation and identification of the major components.

Science.gov (United States)

Gazzola, Diana; Vincenzi, Simone; Gastaldon, Luca; Tolin, Serena; Pasini, Gabriella; Curioni, Andrea

2014-07-15

In the present study, grape (Vitis vinifera L.) seed endosperm proteins were characterized after sequential fractionation, according to a modified Osborne procedure. The salt-soluble fraction (albumins and globulins) comprised the majority (58.4%) of the total extracted protein. The protein fractions analysed by SDS-PAGE showed similar bands, indicating different solubility of the same protein components. SDS-PAGE in non-reducing and reducing conditions revealed the polypeptide composition of the protein bands. The main polypeptides, which were similar in all the grape varieties analysed, were identified by LC-MS/MS as homologous to the 11S globulin-like seed storage proteins of other plant species, while a monomeric 43 kDa protein presented high homology with the 7S globulins of legume seeds. The results provide new insights about the identity, structure and polypeptide composition of the grape seed storage proteins. Copyright © 2014 Elsevier Ltd. All rights reserved.

Equipment characterization to mitigate risks during transfers of cell culture manufacturing processes.

Science.gov (United States)

Sieblist, Christian; Jenzsch, Marco; Pohlscheidt, Michael

2016-08-01

The production of monoclonal antibodies by mammalian cell culture in bioreactors up to 25,000 L is state of the art technology in the biotech industry. During the lifecycle of a product, several scale up activities and technology transfers are typically executed to enable the supply chain strategy of a global pharmaceutical company. Given the sensitivity of mammalian cells to physicochemical culture conditions, process and equipment knowledge are critical to avoid impacts on timelines, product quantity and quality. Especially, the fluid dynamics of large scale bioreactors versus small scale models need to be described, and similarity demonstrated, in light of the Quality by Design approach promoted by the FDA. This approach comprises an associated design space which is established during process characterization and validation in bench scale bioreactors. Therefore the establishment of predictive models and simulation tools for major operating conditions of stirred vessels (mixing, mass transfer, and shear force.), based on fundamental engineering principles, have experienced a renaissance in the recent years. This work illustrates the systematic characterization of a large variety of bioreactor designs deployed in a global manufacturing network ranging from small bench scale equipment to large scale production equipment (25,000 L). Several traditional methods to determine power input, mixing, mass transfer and shear force have been used to create a data base and identify differences for various impeller types and configurations in operating ranges typically applied in cell culture processes at manufacturing scale. In addition, extrapolation of different empirical models, e.g. Cooke et al. (Paper presented at the proceedings of the 2nd international conference of bioreactor fluid dynamics, Cranfield, UK, 1988), have been assessed for their validity in these operational ranges. Results for selected designs are shown and serve as examples of structured

Developmental potential of human oocytes reconstructed by transferring somatic cell nuclei into polyspermic zygote cytoplasm

International Nuclear Information System (INIS)

Fan, Yong; Chen, Xinjie; Luo, Yumei; Chen, Xiaolin; Li, Shaoying; Huang, Yulin; Sun, Xiaofang

2009-01-01

The generation of patient-specific nuclear transfer embryonic stem cells holds huge promise in modern regenerative medicine and cell-based drug discovery. Since human in vivo matured oocytes are not readily available, human therapeutic cloning is developing slowly. Here, we investigated for the first time whether human polyspermic zygotes could support preimplantation development of cloned embryos. Our results showed that polyspermic zygotes could be used as recipients for human somatic cell nuclear transfer (SCNT). The preimplantation developmental potential of SCNT embryos from polyspermic zygotes was limited to the 8-cell stage. Since ES cell lines can be derived from single blastomeres, these results may have important significance for human ES cells derived by SCNT. In addition, confocal images demonstrated that all of the SCNT embryos that failed to cleave showed abnormal microtubule organization. The results of the present study suggest that polyspermic human zygotes could be used as a potential source of recipient cytoplasm for SCNT.

Developmental potential of human oocytes reconstructed by transferring somatic cell nuclei into polyspermic zygote cytoplasm

Energy Technology Data Exchange (ETDEWEB)

Fan, Yong; Chen, Xinjie; Luo, Yumei; Chen, Xiaolin; Li, Shaoying; Huang, Yulin [Institute of Gynecology and Obstetrics, The Third Affiliated Hospital of Guangzhou Medical College, Duobao Road 63, Guangzhou, Guangdong (China); Sun, Xiaofang, E-mail: xiaofangsun@hotmail.com [Institute of Gynecology and Obstetrics, The Third Affiliated Hospital of Guangzhou Medical College, Duobao Road 63, Guangzhou, Guangdong (China)

2009-04-24

The generation of patient-specific nuclear transfer embryonic stem cells holds huge promise in modern regenerative medicine and cell-based drug discovery. Since human in vivo matured oocytes are not readily available, human therapeutic cloning is developing slowly. Here, we investigated for the first time whether human polyspermic zygotes could support preimplantation development of cloned embryos. Our results showed that polyspermic zygotes could be used as recipients for human somatic cell nuclear transfer (SCNT). The preimplantation developmental potential of SCNT embryos from polyspermic zygotes was limited to the 8-cell stage. Since ES cell lines can be derived from single blastomeres, these results may have important significance for human ES cells derived by SCNT. In addition, confocal images demonstrated that all of the SCNT embryos that failed to cleave showed abnormal microtubule organization. The results of the present study suggest that polyspermic human zygotes could be used as a potential source of recipient cytoplasm for SCNT.

Method for somatic cell nuclear transfer in zebrafish.

Science.gov (United States)

Siripattarapravat, Kannika; Cibelli, Jose B

2011-01-01

Somatic cell nuclear transfer (SCNT) has been a well-known technique for decades and widely applied to generate identical animals, including ones with genetic alterations. The system has been demonstrated successfully in zebrafish. The elaborated requirements of SCNT, however, limit reproducibility of the established model to a few groups in zebrafish research community. In this chapter, we meticulously outline each step of the published protocol as well as preparations of equipments and reagents used in zebrafish SCNT. All describable detailed-tips are elaborated in texts and figures. Copyright © 2011 Elsevier Inc. All rights reserved.

Interferon-β gene transfer induces a strong cytotoxic bystander effect on melanoma cells.

Science.gov (United States)

Rossi, Úrsula A; Gil-Cardeza, María L; Villaverde, Marcela S; Finocchiaro, Liliana M E; Glikin, Gerardo C

2015-05-01

A local gene therapy scheme for the delivery of type I interferons could be an alternative for the treatment of melanoma. We evaluated the cytotoxic effects of interferon-β (IFNβ) gene lipofection on tumor cell lines derived from three human cutaneous and four canine mucosal melanomas. The cytotoxicity of human IFNβ gene lipofection resulted higher or equivalent to that of the corresponding addition of the recombinant protein (rhIFNβ) to human cells. IFNβ gene lipofection was not cytotoxic for only one canine melanoma cell line. When cultured as monolayers, three human and three canine IFNβ-lipofected melanoma cell lines displayed a remarkable bystander effect. As spheroids, the same six cell lines were sensitive to IFNβ gene transfer, two displaying a significant multicell resistance phenotype. The effects of conditioned IFNβ-lipofected canine melanoma cell culture media suggested the release of at least one soluble thermolabile cytotoxic factor that could not be detected in human melanoma cells. By using a secretion signal-free truncated human IFNβ, we showed that its intracellular expression was enough to induce cytotoxicity in two human melanoma cell lines. The lower cytoplasmatic levels of reactive oxygen species detected after intracellular IFNβ expression could be related to the resistance displayed by one human melanoma cell line. As IFNβ gene transfer was effective against most of the assayed melanomas in a way not limited by relatively low lipofection efficiencies, the clinical potential of this approach is strongly supported. Copyright © 2015 Elsevier Masson SAS. All rights reserved.

Proanthocyanidins in seed coat tegmen and endospermic cap inhibit seed germination in Sapium sebiferum.

Science.gov (United States)

Shah, Faheem Afzal; Ni, Jun; Chen, Jing; Wang, Qiaojian; Liu, Wenbo; Chen, Xue; Tang, Caiguo; Fu, Songling; Wu, Lifang

2018-01-01

Sapium sebiferum , an ornamental and bio-energetic plant, is propagated by seed. Its seed coat contains germination inhibitors and takes a long time to stratify for germination. In this study, we discovered that the S. sebiferum seed coat (especially the tegmen) and endospermic cap (ESC) contained high levels of proanthocyanidins (PAs). Seed coat and ESC removal induced seed germination, whereas exogenous application with seed coat extract (SCE) or PAs significantly inhibited this process, suggesting that PAs in the seed coat played a major role in regulating seed germination in S. sebiferum . We further investigated how SCE affected the expression of the seed-germination-related genes. The results showed that treatment with SCE upregulated the transcription level of the dormancy-related gene, gibberellins (GAs) suppressing genes, abscisic acid (ABA) biosynthesis and signalling genes. SCE decreased the transcript levels of ABA catabolic genes, GAs biosynthesis genes, reactive oxygen species genes and nitrates-signalling genes. Exogenous application of nordihydroguaiaretic acid, gibberellic acid, hydrogen peroxide and potassium nitrate recovered seed germination in seed-coat-extract supplemented medium. In this study, we highlighted the role of PAs, and their interactions with the other germination regulators, in the regulation of seed dormancy in S. sebiferum .

Plasma polymer-coated contact lenses for the culture and transfer of corneal epithelial cells in the treatment of limbal stem cell deficiency.

Science.gov (United States)

Brown, Karl David; Low, Suet; Mariappan, Indumathi; Abberton, Keren Maree; Short, Robert; Zhang, Hong; Maddileti, Savitri; Sangwan, Virender; Steele, David; Daniell, Mark

2014-02-01

Extensive damage to the limbal region of the cornea leads to a severe form of corneal blindness termed as limbal stem cell deficiency (LSCD). Whereas most cases of corneal opacity can be treated with full thickness corneal transplants, LSCD requires stem cell transplantation for successful ocular surface reconstruction. Current treatments for LSCD using limbal stem cell transplantation involve the use of murine NIH 3T3 cells and human amniotic membranes as culture substrates, which pose the threat of transmission of animal-derived pathogens and donor tissue-derived cryptic infections. In this study, we aimed to produce surface modified therapeutic contact lenses for the culture and delivery of corneal epithelial cells for the treatment of LSCD. This approach avoids the possibility of suture-related complications and is completely synthetic. We used plasma polymerization to deposit acid functional groups onto the lenses at various concentrations. Each surface was tested for its suitability to promote corneal epithelial cell adhesion, proliferation, retention of stem cells, and differentiation and found that acid-based chemistries promoted better cell adhesion and proliferation. We also found that the lenses coated with a higher percentage of acid functional groups resulted in a higher number of cells transferred onto the corneal wound bed in rabbit models of LSCD. Immunohistochemistry of the recipient cornea confirmed the presence of autologous, transplanted 5-bromo-2'-deoxyuridine (BrdU)-labeled cells. Hematoxylin staining has also revealed the presence of a stratified epithelium at 26 days post-transplantation. This study provides the first evidence for in vivo transfer and survival of cells transplanted from a contact lens to the wounded corneal surface. It also proposes the possibility of using plasma polymer-coated contact lenses with high acid functional groups as substrates for the culture and transfer of limbal cells in the treatment of LSCD.

Determination of Four Major Saponins in Skin and Endosperm of Seeds of Horse Chestnut (Aesculus Hippocastanum L.) Using High Performance Liquid Chromatography with Positive Confirmation by Thin Layer Chromatography

OpenAIRE

Abudayeh, Zead Helmi Mahmoud; Al Azzam, Khaldun Mohammad; Naddaf, Ahmad; Karpiuk, Uliana Vladimirovna; Kislichenko, Viktoria Sergeevna

2015-01-01

urpose: To separate and quantify four major saponins in the extracts of the skin and the endosperm of seeds of horse chestnut (Aesculus hippocastanum L.) using ultrasonic solvent extraction followed by a high performance liquid chromatography-diode array detector (HPLC-DAD) with positive confirmation by thin layer chromatography (TLC). Methods: The saponins: escin Ia, escin Ib, isoescin Ia and isoescin Ib were extracted using ultrasonic extraction method. The optimized ex...

Variability of barley aleurone layer induced by X-rays

Directory of Open Access Journals (Sweden)

Romuald Kosina

2015-05-01

Full Text Available A series of Hordeum vulgare cultivars was irradiated by X-rays to induce mutations in endosperm. Many structural defects of endosperm were revealed in plants irradiated 8 DAF. Change of a cell cycle was especially frequent and this was visible in the form of clones of small or large cells in the aleurone layer. X-irradiation appeared as a successful tool in the study of development.

Comparison of lentiviral and sleeping beauty mediated αβ T cell receptor gene transfer.

Directory of Open Access Journals (Sweden)

Anne-Christine Field

Full Text Available Transfer of tumour antigen-specific receptors to T cells requires efficient delivery and integration of transgenes, and currently most clinical studies are using gamma retroviral or lentiviral systems. Whilst important proof-of-principle data has been generated for both chimeric antigen receptors and αβ T cell receptors, the current platforms are costly, time-consuming and relatively inflexible. Alternative, more cost-effective, Sleeping Beauty transposon-based plasmid systems could offer a pathway to accelerated clinical testing of a more diverse repertoire of recombinant high affinity T cell receptors. Nucleofection of hyperactive SB100X transposase-mediated stable transposition of an optimised murine-human chimeric T cell receptor specific for Wilm's tumour antigen from a Sleeping Beauty transposon plasmid. Whilst transfer efficiency was lower than that mediated by lentiviral transduction, cells could be readily enriched and expanded, and mediated effective target cells lysis in vitro and in vivo. Integration sites of transposed TCR genes in primary T cells were almost randomly distributed, contrasting the predilection of lentiviral vectors for transcriptionally active sites. The results support exploitation of the Sleeping Beauty plasmid based system as a flexible and adaptable platform for accelerated, early-phase assessment of T cell receptor gene therapies.

Proteasen in pflanzlichen Organellen

OpenAIRE

Helm, Michael

2006-01-01

Der Programmierte Zelltod (PCD) ist essentiell für die Entwicklung der Pflanze, im Speziellen für den Abbau nicht mehr benötigter Gewebe zur Rückführung von Nährstoffen an die weiterlebenden Teile der Pflanze. Ricinus (R. communis) speichert Öl und Eiweiß in einem lebenden Endosperm, das die Kotyledonen umgibt. Die Speicherstoffe werden während der Keimung mobilisiert und den Kotyledonen zugeführt. Der PCD des Endosperm-Gewebes wird eingeleitet, sobald dieser Transfer abgeschlossen ist. Ein s...

Determination of the Fate and Function of Innate Lymphoid Cells Following Adoptive Transfer of Innate Lymphoid Cell Precursors.

Science.gov (United States)

O'Sullivan, Timothy E; Sun, Joseph C

2018-01-01

Innate lymphoid cells are a heterogeneous family of tissue-resident and circulating lymphocytes that play an important role in host immunity. Recent studies have profiled the developmental pathways of mature ILCs and have identified ILC progenitors in the bone marrow through the use of transcription factor reporter mice. Here we describe methodology to identify and isolate bone marrow CHILP and ILC2 progenitor (ILC2P) cells based on cell surface marker expression for adoptive transfer into lymphopenic mice to track the fate of developing ILCs.

Identification of new members of Fertilisation Independent Seed Polycomb Group pathway involved in the control of seed development in Arabidopsis thaliana.

Science.gov (United States)

Guitton, Anne-Elisabeth; Page, Damian R; Chambrier, Pierre; Lionnet, Claire; Faure, Jean-Emmanuel; Grossniklaus, Ueli; Berger, Frédéric

2004-06-01

In higher plants, double fertilisation initiates seed development. One sperm cell fuses with the egg cell and gives rise to the embryo, the second sperm cell fuses with the central cell and gives rise to the endosperm. The endosperm develops as a syncytium with the gradual organisation of domains along an anteroposterior axis defined by the position of the embryo at the anterior pole and by the attachment to the placenta at the posterior pole. We report that ontogenesis of the posterior pole in Arabidopsis thaliana involves oriented migration of nuclei in the syncytium. We show that this migration is impaired in mutants of the three founding members of the FERTILIZATION INDEPENDENT SEED (FIS) class, MEDEA (MEA), FIS2 and FERTILIZATION INDEPENDENT ENDOSPERM (FIE). A screen based on a green fluorescent protein (GFP) reporter line allowed us to identify two new loci in the FIS pathway, medicis and borgia. We have cloned the MEDICIS gene and show that it encodes the Arabidopsis homologue of the yeast WD40 domain protein MULTICOPY SUPRESSOR OF IRA (MSI1). The mutations at the new fis loci cause the same cellular defects in endosperm development as other fis mutations, including parthenogenetic development, absence of cellularisation, ectopic development of posterior structures and overexpression of the GFP marker.

Direct transfer of viral and cellular proteins from varicella-zoster virus-infected non-neuronal cells to human axons.

Science.gov (United States)

Grigoryan, Sergei; Yee, Michael B; Glick, Yair; Gerber, Doron; Kepten, Eldad; Garini, Yuval; Yang, In Hong; Kinchington, Paul R; Goldstein, Ronald S

2015-01-01

Varicella Zoster Virus (VZV), the alphaherpesvirus that causes varicella upon primary infection and Herpes zoster (shingles) following reactivation in latently infected neurons, is known to be fusogenic. It forms polynuclear syncytia in culture, in varicella skin lesions and in infected fetal human ganglia xenografted to mice. After axonal infection using VZV expressing green fluorescent protein (GFP) in compartmentalized microfluidic cultures there is diffuse filling of axons with GFP as well as punctate fluorescence corresponding to capsids. Use of viruses with fluorescent fusions to VZV proteins reveals that both proteins encoded by VZV genes and those of the infecting cell are transferred in bulk from infecting non-neuronal cells to axons. Similar transfer of protein to axons was observed following cell associated HSV1 infection. Fluorescence recovery after photobleaching (FRAP) experiments provide evidence that this transfer is by diffusion of proteins from the infecting cells into axons. Time-lapse movies and immunocytochemical experiments in co-cultures demonstrate that non-neuronal cells fuse with neuronal somata and proteins from both cell types are present in the syncytia formed. The fusogenic nature of VZV therefore may enable not only conventional entry of virions and capsids into axonal endings in the skin by classical entry mechanisms, but also by cytoplasmic fusion that permits viral protein transfer to neurons in bulk.

In vitro fermentation characteristics of novel fibers, coconut endosperm fiber and chicory pulp, using canine fecal inoculum.

Science.gov (United States)

de Godoy, M R C; Mitsuhashi, Y; Bauer, L L; Fahey, G C; Buff, P R; Swanson, K S

2015-01-01

The objective of this experiment was to determine the effects of in vitro fermentation of coconut endosperm fiber (CEF), chicory pulp (CHP), and selective blends of these substrates on SCFA production and changes in microbiota using canine fecal inocula. A total of 6 individual substrates, including short-chain fructooligosaccharide (scFOS; a well-established prebiotic source), pectin (PEC; used as a positive control), pelletized cellulose (PC; used as a negative control), beet pulp (BP; considered the gold standard fiber source in pet foods), CEF, and CHP, and 3 CEF:CHP blends (75:25% CEF:CHP [B1], 50:50% CEF:CHP [B2], and 25:75% CEF:CHP [B3]) were tested. Triplicate samples of each substrate were fermented for 0, 8, and 16 h after inoculation. A significant substrate × time interaction (P fiber substrates. Future research should investigate the effects of CEF, CHP, and their blends on gastrointestinal health and fecal quality in dogs.

Microvesicle and tunneling nanotube mediated intercellular transfer of g-protein coupled receptors in cell cultures

International Nuclear Information System (INIS)

Guescini, M.; Leo, G.; Genedani, S.; Carone, C.; Pederzoli, F.; Ciruela, F.; Guidolin, D.; Stocchi, V.; Mantuano, M.; Borroto-Escuela, D.O.; Fuxe, K.; Agnati, L.F.

2012-01-01

Recent evidence shows that cells exchange collections of signals via microvesicles (MVs) and tunneling nano-tubes (TNTs). In this paper we have investigated whether in cell cultures GPCRs can be transferred by means of MVs and TNTs from a source cell to target cells. Western blot, transmission electron microscopy and gene expression analyses demonstrate that A 2A and D 2 receptors are present in released MVs. In order to further demonstrate the involvement of MVs in cell-to-cell communication we created two populations of cells (HEK293T and COS-7) transiently transfected with D 2 R-CFP or A 2A R-YFP. These two types of cells were co-cultured, and FRET analysis demonstrated simultaneously positive cells to the D 2 R-CFP and A 2A R-YFP. Fluorescence microscopy analysis also showed that GPCRs can move from one cell to another also by means of TNTs. Finally, recipient cells pre-incubated for 24 h with A 2A R positive MVs were treated with the adenosine A 2A receptor agonist CGS-21680. The significant increase in cAMP accumulation clearly demonstrated that A 2A Rs were functionally competent in target cells. These findings demonstrate that A 2A receptors capable of recognizing and decoding extracellular signals can be safely transferred via MVs from source to target cells.

Conductive polymer layers to limit transfer of fuel reactants to catalysts of fuel cells to reduce reactant crossover

Science.gov (United States)

Stanis, Ronald J.; Lambert, Timothy N.

2016-12-06

An apparatus of an aspect includes a fuel cell catalyst layer. The fuel cell catalyst layer is operable to catalyze a reaction involving a fuel reactant. A fuel cell gas diffusion layer is coupled with the fuel cell catalyst layer. The fuel cell gas diffusion layer includes a porous electrically conductive material. The porous electrically conductive material is operable to allow the fuel reactant to transfer through the fuel cell gas diffusion layer to reach the fuel cell catalyst layer. The porous electrically conductive material is also operable to conduct electrons associated with the reaction through the fuel cell gas diffusion layer. An electrically conductive polymer material is coupled with the fuel cell gas diffusion layer. The electrically conductive polymer material is operable to limit transfer of the fuel reactant to the fuel cell catalyst layer.

Feedback control and heat transfer measurements in a Rayleigh-Bénard convection cell

Science.gov (United States)

Vial, M.; Hernández, R. H.

2017-07-01

We report experimental results on the heat transfer and instability onset of a Rayleigh-Bénard convection cell of aspect ratios 6:3:1 filled with a high Prandtl aqueous solution of glycerol under feedback control. We investigate the transient and stationary response of both local temperature readings and heat transfer fluxes on the Rayleigh Bénard cell in both conductive and convective states when we perform two independent feedback control actions on both hot and cold walls. We evaluate the performance of both controllers to maintain a temperature gradient independently if the system is below or above the convection threshold. As the convection cell can be rotated at 180° about the shorter axis of the cell, it was possible to perform transitions between thermal conduction and convection regimes and vice versa under a constant temperature difference maintained by both independent controllers. The experimental setup provided an accurate measurement of the critical Rayleigh number and the evolution of the Nusselt number as a function of the Rayleigh number in the moderately supercritical regime (R a cellular convection pattern formed by 6 transverse rolls throughout the range of Rayleigh numbers.

Extracellular ATP reduces HIV-1 transfer from immature dendritic cells to CD4+ T lymphocytes

Directory of Open Access Journals (Sweden)

Barat Corinne

2008-03-01

Full Text Available Abstract Background Dendritic cells (DCs are considered as key mediators of the early events in human immunodeficiency virus type 1 (HIV-1 infection at mucosal sites. Previous studies have shown that surface-bound virions and/or internalized viruses found in endocytic vacuoles of DCs are efficiently transferred to CD4+ T cells. Extracellular adenosine triphosphate (ATP either secreted or released from necrotic cells induces a distorted maturation of DCs, transiently increases their endocytic capacity and affects their migratory capacity. Knowing that high extracellular ATP concentrations are present in situations of tissue injury and inflammation, we investigated the effect of ATP on HIV-1 transmission from DCs to CD4+ T lymphocytes. Results In this study, we show that extracellular ATP reduces HIV-1 transfer from immature monocyte-derived DCs (iDCs to autologous CD4+ T cells. This observed decrease in viral replication was related to a lower proportion of infected CD4+ T cells following transfer, and was seen with both X4- and R5-tropic isolates of HIV-1. Extracellular ATP had no effect on direct CD4+ T cell infection as well as on productive HIV-1 infection of iDCs. These observations indicate that extracellular ATP affects HIV-1 infection of CD4+ T cells in trans with no effect on de novo virus production by iDCs. Additional experiments suggest that extracellular ATP might modulate the trafficking pathway of internalized virions within iDCs leading to an increased lysosomal degradation, which could be partly responsible for the decreased HIV-1 transmission. Conclusion These results suggest that extracellular ATP can act as a factor controlling HIV-1 propagation.

Optimizing viral and non-viral gene transfer methods for genetic modification of porcine mesenchymal stem cells

DEFF Research Database (Denmark)

Stiehler, Maik; Duch, Mogens; Mygind, Tina

2006-01-01

INTRODUCTION: Mesenchymal stem cells (MSCs) provide an excellent source of pluripotent progenitor cells for tissue-engineering applications due to their proliferation capacity and differentiation potential. Genetic modification of MSCs with genes encoding tissue-specific growth factors...... viral and non-viral ex vivo gene delivery systems with respect to gene transfer efficiency, maintenance of transgene expression, and safety issues using primary porcine MSCs as target cells. MATERIALS AND METHODS: MSCs were purified from bone marrow aspirates from the proximal tibiae of four 3-month......-old Danish landrace pigs by Ficoll step gradient separation and polystyrene adherence technique. Vectors expressing enhanced green fluorescent protein (eGFP) and human bone morphogenetic protein-2 (BMP-2) were transferred to the cells by different non-viral methods and by use of recombinant adeno...

Free tissue transfer in patients with sickle cell disease: Considerations for multi-disciplinary peri-operative management.

Science.gov (United States)

Cooper, Lilli; Seth, Rohit; Rhodes, Elizabeth; Alousi, Mohammed; Sivakumar, Bran

2017-01-01

Sickle cell disease (SCD) is an increasingly common condition in the UK. The safety of free tissue transfer in these patients is controversial, and no specific guidelines exist. The aim of this paper is to create recommendations for the plastic surgical multidisciplinary team for use in the assessment and management of SCD patients undergoing free tissue transfer and reconstruction. A literature review was performed in PubMed of 'sickle [TiAb] AND plast* adj3 surg*. Sickle cell disease is explained, as is the relative peri-operative risk in different genotypes of SCD. Acute and chronic manifestations of SCD are described by system, for consideration at pre-operative assessment and post-operative review. The evidence surrounding free tissue transfer and SCD is discussed and the outcomes in published cases summarised. An algorithm for peri-operative multi-disciplinary management is outlined and justified. Free tissue transfer theoretically carries a high risk of a crisis, due not only to long anaesthetic times, but the potential requirement for tourniquet use, and the relatively hypoxic state of the transferred tissue. This paper outlines a useful, practical algorithm to optimise the safety of free tissue transfer in patients with SCD. Copyright © 2016 British Association of Plastic, Reconstructive and Aesthetic Surgeons. Published by Elsevier Ltd. All rights reserved.

Viral-specific T-cell transfer from HSCT donor for the treatment of viral infections or diseases after HSCT.

Science.gov (United States)

Qian, C; Wang, Y; Reppel, L; D'aveni, M; Campidelli, A; Decot, V; Bensoussan, D

2018-02-01

Allogeneic hematopoietic stem cell transplantation (HSCT) is a curative option for treatment of some malignant and non-malignant hematological diseases. However, post-HSCT patients are severely immunocompromised and susceptible to viral infections, which are a major cause of morbidity and mortality. Although antiviral agents are now available for most types of viral infections, they are not devoid of side effects and their efficacy is limited when there is no concomitant antiviral immune reconstitution. In recent decades, adoptive transfer of viral-specific T cells (VSTs) became an alternative treatment for viral infection after HSCT. However, two major issues are concerned in VST transfer: the risk of GVHD and antiviral efficacy. We report an exhaustive review of the published studies that focus on prophylactic and/or curative therapy by donor VST transfer for post-HSCT common viral infections. A low incidence of GVHD and a good antiviral efficacy was observed after adoptive transfer of VSTs from HSCT donor. Viral-specific T-cell transfer is a promising approach for a broad clinical application. Nevertheless, a randomized controlled study in a large cohort of patients comparing antiviral treatment alone to antiviral treatment combined with VSTs is still needed to demonstrate efficacy and safety.

Horizontal transfer of mitochondria between mammalian cells: beyond co-culture approaches

Czech Academy of Sciences Publication Activity Database

Berridge, M.V.; McConnell, M. J.; Grasso, C.; Bajziková, Martina; Kovářová, Jaromíra; Neužil, Jiří

2016-01-01

Roč. 38, June (2016), s. 75-82 ISSN 0959-437X R&D Projects: GA ČR(CZ) GAP301/10/1937; GA MŠk(CZ) ED1.1.00/02.0109 Institutional support: RVO:86652036 Keywords : MESENCHYMAL STEM-CELLS * GENE-TRANSFER * SUPPORTING ASPARTATE BIOSYNTHESIS Subject RIV: EB - Genetics ; Molecular Biology Impact factor: 5.825, year: 2016

Retroviral-mediated transfer and expression of human β-globin genes in cultured murine and human erythroid cells

International Nuclear Information System (INIS)

Weber-Benarous, A.; Cone, R.D.; London, I.M.; Mulligan, R.C.

1988-01-01

The authors cloned human β-globin DNA sequences from a genomic library prepared from DNA isolated from the human leukemia cell line K562 and have used the retroviral vector pZip-NeoSV(X)1 to introduce a 3.0-kilobase segment encompassing the globin gene into mouse erythroleukemia cells. Whereas the endogenous K562 β-globin gene is repressed in K562 cells, when introduced into mouse erythroleukemia cells by retroviral-mediated gene transfer, the β-globin gene from K562 cells was transcribed and induced 5-20-fold after treatment of the cells with dimethyl sulfoxide. The transcripts were correctly initiated, and expression and regulation of the K562 gene were identical to the expression of a normal human β-globin gene transferred into mouse erythroleukemia cells in the same way. They have also introduced the normal human β-globin gene into K562 cells using the same retrovirus vector. SP6 analysis of the RNA isolated from the transduced cells showed that the normal β-globin gene was transcribed at a moderately high level, before or after treatment with hemin. Based on these data, they suggest that the lack of expression of the endogenous β-globin gene in K562 cells does not result from an alteration in the gene itself and may not result from a lack of factor(s) necessary for β-lobin gene transcription. Retroviral-mediated transfer of the human β-globin gene may, however, uniquely influence expression of the gene K562 cells

Concentrated Protein Body Product Derived from Rice Endosperm as an Oral Tolerogen for Allergen-Specific Immunotherapy—A New Mucosal Vaccine Formulation against Japanese Cedar Pollen Allergy

Science.gov (United States)

Wakasa, Yuhya; Takagi, Hidenori; Watanabe, Nobumasa; Kitamura, Noriko; Fujiwara, Yoshihiro; Ogo, Yuko; Hayashi, Shimpei; Yang, Lijun; Ohta, Masaru; Thet Tin, Wai Wai; Sekikawa, Kenji; Takano, Makoto; Ozawa, Kenjirou; Hiroi, Takachika; Takaiwa, Fumio

2015-01-01

The endoplasmic reticulum-derived type-I protein body (PB-I) from rice endosperm cells is an ideal candidate formulation for the oral delivery of bioencapsulated peptides as tolerogens for allergen-specific immunotherapy. In the present study, PBs containing the deconstructed Japanese cedar pollen allergens Cryptomeria japonica 1 (Cry j 1) and Cry j 2 were concentrated by treatment with thermostable α-amylase at 90°C to remove the starch from milled rice powder, which resulted in a 12.5-fold reduction of dry weight compared to the starting material. The modified Cry j 1 and Cry j 2 antigens in this concentrated PB product were more resistant to enzymatic digestion than those in the milled seed powder despite the absence of intact cell wall and starch, and remained stable for at least 10 months at room temperature without detectable loss or degradation. The high resistance of these allergens could be attributed to changes in protein physicochemical properties induced by the high temperature concentration process, as suggested by the decreased solubility of the antigens and seed proteins in PBs in step-wise-extraction experiments. Confocal microscopy showed that the morphology of antigen-containing PB-Is was preserved in the concentrated PB product. The concentrated PB product induced specific immune tolerance against Cry j 1 and Cry j 2 in mice when orally administered, supporting its potential use as a novel oral tolerogen formulation. PMID:25774686

Concentrated protein body product derived from rice endosperm as an oral tolerogen for allergen-specific immunotherapy--a new mucosal vaccine formulation against Japanese cedar pollen allergy.

Directory of Open Access Journals (Sweden)

Yuhya Wakasa

Full Text Available The endoplasmic reticulum-derived type-I protein body (PB-I from rice endosperm cells is an ideal candidate formulation for the oral delivery of bioencapsulated peptides as tolerogens for allergen-specific immunotherapy. In the present study, PBs containing the deconstructed Japanese cedar pollen allergens Cryptomeria japonica 1 (Cry j 1 and Cry j 2 were concentrated by treatment with thermostable α-amylase at 90°C to remove the starch from milled rice powder, which resulted in a 12.5-fold reduction of dry weight compared to the starting material. The modified Cry j 1 and Cry j 2 antigens in this concentrated PB product were more resistant to enzymatic digestion than those in the milled seed powder despite the absence of intact cell wall and starch, and remained stable for at least 10 months at room temperature without detectable loss or degradation. The high resistance of these allergens could be attributed to changes in protein physicochemical properties induced by the high temperature concentration process, as suggested by the decreased solubility of the antigens and seed proteins in PBs in step-wise-extraction experiments. Confocal microscopy showed that the morphology of antigen-containing PB-Is was preserved in the concentrated PB product. The concentrated PB product induced specific immune tolerance against Cry j 1 and Cry j 2 in mice when orally administered, supporting its potential use as a novel oral tolerogen formulation.

Charge transfer processes in hybrid solar cells composed of amorphous silicon and organic materials

Energy Technology Data Exchange (ETDEWEB)

Schaefer, Sebastian; Neher, Dieter [Universitaet Potsdam, Inst. Physik u. Astronomie, Karl-Liebknecht-Strasse 24/25, 14467 Potsdam-Golm (Germany); Schulze, Tim; Korte, Lars [Helmholtz Zentrum Berlin, Inst. fuer Silizium Photovoltaik, Kekulestrasse 5, 12489 Berlin (Germany)

2011-07-01

The efficiency of hybrid solar cells composed of organic materials and amorphous hydrogenated silicon (a-Si:H) strongly depends upon the efficiency of charge transfer processes at the inorganic-organic interface. We investigated the performance of devices comprising an ITO/a-Si:H(n-type)/a-Si:H(intrinsic)/organic/metal multilayer structure and using two different organic components: zinc phthalocyanine (ZnPc) and poly(3-hexylthiophene) (P3HT). The results show higher power conversion- and quantum efficiencies for the P3HT based cells, compared to ZnPc. This can be explained by larger energy-level offset at the interface between the organic layer and a-Si:H, which facilitates hole transfer from occupied states in the valence band tail to the HOMO of the organic material and additionally promotes exciton splitting. The performance of the a-Si:H/P3HT cells can be further improved by treatment of the amorphous silicon surface with hydrofluoric acid (HF) and p-type doping of P3HT with F4TCNQ. The improved cells reached maximum power conversion efficiencies of 1%.

Bortezomib Enhances the Antitumor Effects of Interferon-β Gene Transfer on Melanoma Cells.

Science.gov (United States)

Rossi, Ursula A; Finocchiaro, Liliana M E; Glikin, Gerardo C

2017-01-01

Malignant melanoma is a fast growing form of skin cancer with increasing global incidence. Clinically, canine malignant melanoma and human melanoma share comparable treatment-resistances, metastatic phenotypes and site selectivity. Both interferon-β (IFNβ) and bortezomib (BTZ) display inhibitory activities on melanoma cells. Here, we evaluated the cytotoxic effects of the combination of BTZ and IFNβ gene lipofection on cultured melanoma cell lines. Cell viability determined by the acid phosphatase method, cell migration mesasured by the wound healing assay, DNA fragmentation and cell cycle by flow cytometry after propidium iodide staining and reactive oxygen species (ROS) production by H2DCF-DA fluorescence. Four canine mucosal (Ak, Br, Bk and Ol) and two human dermal (A375 and SB2) melanoma cell lines were assayed. BTZ sub-pharmacological concentrations (5 nM) enhanced the cytotoxic effects of IFNβ transgene expression on melanoma cells monolayers and spheroids. The combination was also more effective than the single treatments when assayed for clonogenic survival and cell migration. The combined treatment produced a significant raise of apoptosis evidenced by DNA fragmentation as compared to either BTZ or IFNβ gene lipofection single treatments. Furthermore, BTZ significantly increased the intracellular ROS generation induced by IFNβ gene transfer in melanoma cells, an effect that was reversed by the addition of the ROS inhibitor N-acetyl-L-cystein. The present work encourages further studies about the potential of the combination of interferon gene transfer with proteasome inhibitors as a new combined therapy for malignant melanoma, both in veterinary and/or human clinical settings. Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.org.

Effect of adoptive transfer or depletion of regulatory T cells on triptolide-induced liver injury

Directory of Open Access Journals (Sweden)

Xinzhi eWang

2016-04-01

Full Text Available ObjectiveThe aim of this study is to clarify the role of regulatory T cell (Treg in triptolide (TP-induced hepatotoxicity. MethodsFemale C57BL/6 mice received either adoptive transfer of Tregs or depletion of Tregs, then underwent TP administration and were sacrificed 24 hours after TP administration. Liver injury was determined according to ALT and AST levels in serum and histopathological change in liver tissue. Hepatic frequencies of Treg cells and the mRNA expression levles of transcription factor FoxP3 and RORγt, IL-10, SOCS and Notch/Notch ligand were investigated.ResultsDuring TP-induced liver injury, hepatic Treg and IL-10 decreased, while Th17 cell transcription factor RORγt, SOCS signaling and Notch signaling increased, accompanied with liver inflammation. Adoptive transfer of Tregs ameliorated the severity of TP-induced liver injury, accompanied with increased levels of hepatic Treg and IL-10. Adoptive transfer of Tregs remarkably inhibited the expression of RORγt, SOCS3, Notch1 and Notch3. On the contrary, depletion of Treg cells in TP-administered mice resulted in a notable increase of RORγt, SOCS1, SOCS3 and Notch3, while the Treg and IL-10 of liver decreased. Consistent with the exacerbation of liver injury, higher serum levels of ALT and AST were detected in Treg-depleted mice. ConclusionsThese results showed that adoptive transfer or depletion of Tregs attenuated or aggravated TP-induced liver injury, suggesting that Tregs could play important roles in the progression of liver injury. SOCS proteins and Notch signaling affected Tregs, which may contribute to the pathogenesis of TP-induced hepatotoxicity.

Correction of Fanconi Anemia Group C Hematopoietic Stem Cells Following Intrafemoral Gene Transfer

Directory of Open Access Journals (Sweden)

Ouassila Habi

2010-01-01

Full Text Available The main cause of morbidity and mortality in Fanconi anemia patients is the development of bone marrow (BM failure; thus correction of hematopoietic stem cells (HSCs through gene transfer approaches would benefit FA patients. However, gene therapy trials for FA patients using ex vivo transduction protocols have failed to provide long-term correction. In addition, ex vivo cultures have been found to be hazardous for FA cells. To circumvent negative effects of ex vivo culture in FA stem cells, we tested the corrective ability of direct injection of recombinant lentiviral particles encoding FancC-EGFP into femurs of FancC−/− mice. Using this approach, we show that FancC−/− HSCs were efficiently corrected. Intrafemoral gene transfer of the FancC gene prevented the mitomycin C-induced BM failure. Moreover, we show that intrafemoral gene delivery into aplastic marrow restored the bone marrow cellularity and corrected the remaining HSCs. These results provide evidence that targeting FA-deficient HSCs directly in their environment enables efficient and long-term correction of BM defects in FA.

Electron Transfer Mediators for Photoelectrochemical Cells Based on Cu(I Metal Complexes

Directory of Open Access Journals (Sweden)

Michele Brugnati

2007-01-01

Full Text Available The preparation and the photoelectrochemical characterization of a series of bipyridine and pyridyl-quinoline Cu(I complexes, used as electron transfer mediators in regenerative photoelectrochemical cells, are reported. The best performing mediators produced maximum IPCEs of the order of 35–40%. The J-V curves recorded under monochromatic light showed that the selected Cu(I/(II couples generated higher Vocs and fill factors compared to an equivalent I-/I3- cell, due to a decreased dark current.

Adenovirus gene transfer to amelogenesis imperfecta ameloblast-like cells.

Directory of Open Access Journals (Sweden)

Anton V Borovjagin

Full Text Available To explore gene therapy strategies for amelogenesis imperfecta (AI, a human ameloblast-like cell population was established from third molars of an AI-affected patient. These cells were characterized by expression of cytokeratin 14, major enamel proteins and alkaline phosphatase staining. Suboptimal transduction of the ameloblast-like cells by an adenovirus type 5 (Ad5 vector was consistent with lower levels of the coxsackie-and-adenovirus receptor (CAR on those cells relative to CAR-positive A549 cells. To overcome CAR -deficiency, we evaluated capsid-modified Ad5 vectors with various genetic capsid modifications including "pK7" and/or "RGD" motif-containing short peptides incorporated in the capsid protein fiber as well as fiber chimera with the Ad serotype 3 (Ad3 fiber "knob" domain. All fiber modifications provided an augmented transduction of AI-ameloblasts, revealed following vector dose normalization in A549 cells with a superior effect (up to 404-fold of pK7/RGD double modification. This robust infectivity enhancement occurred through vector binding to both α(vβ3/α(vβ5 integrins and heparan sulfate proteoglycans (HSPGs highly expressed by AI-ameloblasts as revealed by gene transfer blocking experiments. This work thus not only pioneers establishment of human AI ameloblast-like cell population as a model for in vitro studies but also reveals an optimal infectivity-enhancement strategy for a potential Ad5 vector-mediated gene therapy for AI.

CpG Oligodeoxynucleotides Enhance the Efficacy of Adoptive Cell Transfer Using Tumor Infiltrating Lymphocytes by Modifying the Th1 Polarization and Local Infiltration of Th17 Cells

Directory of Open Access Journals (Sweden)

Lin Xu

2010-01-01

Full Text Available Adoptive cell transfer immunotherapy using tumor infiltrating lymphocytes (TILs was an important therapeutic strategy against tumors. But the efficacy remains limited and development of new strategies is urgent. Recent evidence suggested that CpG-ODNs might be a potent candidate for tumor immunotherapy. Here we firstly reported that CpG-ODNs could significantly enhance the antitumor efficacy of adoptively transferred TILs in vivo accompanied by enhanced activity capacity and proliferation of CD8+ T cells and CD8+ T cells, as well as a Th1 polarization immune response. Most importantly, we found that CpG-ODNs could significantly elevate the infiltration of Th17 cells in tumor mass, which contributed to anti-tumor efficacy of TILs in vivo. Our findings suggested that CpG ODNs could enhance the anti-tumor efficacy of adoptively transferred TILs through modifying Th1 polarization and local infiltration of Th17 cells, which might provide a clue for developing a new strategy for ACT based on TILs.

Transgenic expression of phytase in wheat endosperm increases bioavailability of iron and zinc in grains.

Science.gov (United States)

Abid, Nabeela; Khatoon, Asia; Maqbool, Asma; Irfan, Muhammad; Bashir, Aftab; Asif, Irsa; Shahid, Muhammad; Saeed, Asma; Brinch-Pedersen, Henrik; Malik, Kauser A

2017-02-01

Phytate is a major constituent of wheat seeds and chelates metal ions, thus reducing their bioavailability and so the nutritional value of grains. Transgenic plants expressing heterologous phytase are expected to enhance degradation of phytic acid stored in seeds and are proposed to increase the in vitro bioavailability of mineral nutrients. Wheat transgenic plants expressing Aspergillus japonicus phytase gene (phyA) in wheat endosperm were developed till T 3 generation. The transgenic lines exhibited 18-99 % increase in phytase activity and 12-76 % reduction of phytic acid content in seeds. The minimum phytic acid content was observed in chapatti (Asian bread) as compared to flour and dough. The transcript profiling of phyA mRNA indicated twofold to ninefold higher expression as compared to non transgenic controls. There was no significant difference in grain nutrient composition of transgenic and non-transgenic seeds. In vitro bioavailability assay for iron and zinc in dough and chapatti of transgenic lines revealed a significant increase in iron and zinc contents. The development of nutritionally enhanced cereals is a step forward to combat nutrition deficiency for iron and zinc in malnourished human population, especially women and children.

Junctional transfer in cultured vascular endothelium: II. Dye and nucleotide transfer

International Nuclear Information System (INIS)

Larson, D.M.; Sheridan, J.D.

1985-01-01

Vascular endothelial cultures, derived from large vessels, retain many of the characteristics of their in vivo counterparts. However, the observed reduction in size and complexity of intercellular gap and tight junctions in these cultured cells suggests that important functions, thought to be mediated by these structures, may be altered in vitro. In continuing studies on intercellular communication in vessel wall cells, the authors have quantitated the extent of junctional transfer of small molecular tracers (the fluorescent dye Lucifer Yellow CH and tritiated uridine nucleotides) in confluent cultures of calf aortic (BAEC) and umbilical vein (BVEC) endothelium. Both BAEC and BVEC show extensive (and quantitatively equivalent) dye and nucleotide transfer. As an analogue of intimal endothelium, the authors have also tested dye transfer in freshly isolated sheets of endothelium. Transfer in BAEC and BVEC sheets was more rapid, extensive and homogeneous than in the cultured cells, implying a reduction in molecular coupling as endothelium adapts to culture conditions. In addition, they have documented heterocellular nucleotide transfer between cultured endothelium and vascular smooth muscle cells, of particular interest considering the prevalence of ''myo-endothelial'' junctions in vivo. These data yield further information on junctional transfer in cultured vascular endothelium and have broad implications for the functional integration of the vessel wall in the physiology and pathophysiology of the vasculature

Microvesicle and tunneling nanotube mediated intercellular transfer of g-protein coupled receptors in cell cultures

Energy Technology Data Exchange (ETDEWEB)

Guescini, M. [Department of Biomolecular Sciences, University of Urbino ' Carlo Bo' , 61029 Urbino (Italy); Leo, G.; Genedani, S. [Department Biomedical Sciences, University of Modena and Reggio Emilia (Italy); Carone, C. [Department Biomedical Sciences, University of Modena and Reggio Emilia (Italy); IRCCS San Camillo Lido, Venezia (Italy); Pederzoli, F. [Department Biomedical Sciences, University of Modena and Reggio Emilia (Italy); Ciruela, F. [Departament Patologia i Terapeutica Experimental, Universitat de Barcelona (Spain); Guidolin, D. [Department of Human Anatomy and Physiology, University of Padua (Italy); Stocchi, V.; Mantuano, M. [Department of Biomolecular Sciences, University of Urbino ' Carlo Bo' , 61029 Urbino (Italy); Borroto-Escuela, D.O.; Fuxe, K. [Department of Neuroscience, Karolinska Institutet, Stockholm (Sweden); Agnati, L.F., E-mail: luigiagnati@tin.it [IRCCS San Camillo Lido, Venezia (Italy)

2012-03-10

Recent evidence shows that cells exchange collections of signals via microvesicles (MVs) and tunneling nano-tubes (TNTs). In this paper we have investigated whether in cell cultures GPCRs can be transferred by means of MVs and TNTs from a source cell to target cells. Western blot, transmission electron microscopy and gene expression analyses demonstrate that A{sub 2A} and D{sub 2} receptors are present in released MVs. In order to further demonstrate the involvement of MVs in cell-to-cell communication we created two populations of cells (HEK293T and COS-7) transiently transfected with D{sub 2}R-CFP or A{sub 2A}R-YFP. These two types of cells were co-cultured, and FRET analysis demonstrated simultaneously positive cells to the D{sub 2}R-CFP and A{sub 2A}R-YFP. Fluorescence microscopy analysis also showed that GPCRs can move from one cell to another also by means of TNTs. Finally, recipient cells pre-incubated for 24 h with A{sub 2A}R positive MVs were treated with the adenosine A{sub 2A} receptor agonist CGS-21680. The significant increase in cAMP accumulation clearly demonstrated that A{sub 2A}Rs were functionally competent in target cells. These findings demonstrate that A{sub 2A} receptors capable of recognizing and decoding extracellular signals can be safely transferred via MVs from source to target cells.

Antiangiogenic immunotherapy targeting Flk-1, DNA vaccine and adoptive T cell transfer, inhibits ocular neovascularization

Energy Technology Data Exchange (ETDEWEB)

Zhang, Han [Department of Ophthalmology, Graduate School of Medical Sciences, Kyushu University, 3-1-1 Maidashi, Higashi-Ku, Fukuoka 812-8582 (Japan); Sonoda, Koh-Hei, E-mail: sonodak@med.kyushu-u.ac.jp [Department of Ophthalmology, Graduate School of Medical Sciences, Kyushu University, 3-1-1 Maidashi, Higashi-Ku, Fukuoka 812-8582 (Japan); Hijioka, Kuniaki; Qiao, Hong; Oshima, Yuji; Ishibashi, Tatsuro [Department of Ophthalmology, Graduate School of Medical Sciences, Kyushu University, 3-1-1 Maidashi, Higashi-Ku, Fukuoka 812-8582 (Japan)

2009-04-17

Ocular neovascularization (NV) is the primary cause of blindness in a wide range of ocular diseases. The exact mechanism underlying the pathogenesis of ocular NV is not yet well understood, and so there is no satisfactory therapy for ocular NV. Here, we describe a strategy targeting Flk-1, a self-antigen overexpressed on proliferating endothelial cells in ocular NV, by antiangiogenic immunotherapy-DNA vaccine and adoptive T cell therapy. An oral DNA vaccine encoding Flk-1 carried by attenuated Salmonella typhimurium markedly suppressed development of laser-induced choroidal NV. We further demonstrated that adoptive transfer of vaccine-induced CD8{sup +} T cells reduced pathological preretinal NV, with a concomitant facilitation of physiological revascularization after oxygen-induced retinal vessel obliteration. However, physiological retinal vascular development was unaffected in neonatal mice transferred with vaccine-induced CD8{sup +} T cells. These findings suggested that antiangiogenic immunotherapy targeting Flk-1 such as vaccination and adoptive immunotherapy may contribute to future therapies for ocular NV.

Antiangiogenic immunotherapy targeting Flk-1, DNA vaccine and adoptive T cell transfer, inhibits ocular neovascularization

International Nuclear Information System (INIS)

Zhang, Han; Sonoda, Koh-Hei; Hijioka, Kuniaki; Qiao, Hong; Oshima, Yuji; Ishibashi, Tatsuro

2009-01-01

Ocular neovascularization (NV) is the primary cause of blindness in a wide range of ocular diseases. The exact mechanism underlying the pathogenesis of ocular NV is not yet well understood, and so there is no satisfactory therapy for ocular NV. Here, we describe a strategy targeting Flk-1, a self-antigen overexpressed on proliferating endothelial cells in ocular NV, by antiangiogenic immunotherapy-DNA vaccine and adoptive T cell therapy. An oral DNA vaccine encoding Flk-1 carried by attenuated Salmonella typhimurium markedly suppressed development of laser-induced choroidal NV. We further demonstrated that adoptive transfer of vaccine-induced CD8 + T cells reduced pathological preretinal NV, with a concomitant facilitation of physiological revascularization after oxygen-induced retinal vessel obliteration. However, physiological retinal vascular development was unaffected in neonatal mice transferred with vaccine-induced CD8 + T cells. These findings suggested that antiangiogenic immunotherapy targeting Flk-1 such as vaccination and adoptive immunotherapy may contribute to future therapies for ocular NV.

Proanthocyanidins in seed coat tegmen and endospermic cap inhibit seed germination in Sapium sebiferum

Directory of Open Access Journals (Sweden)

Faheem Afzal Shah

2018-04-01

Full Text Available Sapium sebiferum, an ornamental and bio-energetic plant, is propagated by seed. Its seed coat contains germination inhibitors and takes a long time to stratify for germination. In this study, we discovered that the S. sebiferum seed coat (especially the tegmen and endospermic cap (ESC contained high levels of proanthocyanidins (PAs. Seed coat and ESC removal induced seed germination, whereas exogenous application with seed coat extract (SCE or PAs significantly inhibited this process, suggesting that PAs in the seed coat played a major role in regulating seed germination in S. sebiferum. We further investigated how SCE affected the expression of the seed-germination-related genes. The results showed that treatment with SCE upregulated the transcription level of the dormancy-related gene, gibberellins (GAs suppressing genes, abscisic acid (ABA biosynthesis and signalling genes. SCE decreased the transcript levels of ABA catabolic genes, GAs biosynthesis genes, reactive oxygen species genes and nitrates-signalling genes. Exogenous application of nordihydroguaiaretic acid, gibberellic acid, hydrogen peroxide and potassium nitrate recovered seed germination in seed-coat-extract supplemented medium. In this study, we highlighted the role of PAs, and their interactions with the other germination regulators, in the regulation of seed dormancy in S. sebiferum.

Characterization of Tetraploid Somatic Cell Nuclear Transfer-Derived Human Embryonic Stem Cells.

Science.gov (United States)

Shin, Dong-Hyuk; Lee, Jeoung-Eun; Eum, Jin Hee; Chung, Young Gie; Lee, Hoon Taek; Lee, Dong Ryul

2017-12-01

Polyploidy is occurred by the process of endomitosis or cell fusion and usually represent terminally differentiated stage. Their effects on the developmental process were mainly investigated in the amphibian and fishes, and only observed in some rodents as mammalian model. Recently, we have established tetraploidy somatic cell nuclear transfer-derived human embryonic stem cells (SCNT-hESCs) and examined whether it could be available as a research model for the polyploidy cells existed in the human tissues. Two tetraploid hESC lines were artificially acquired by reintroduction of remained 1st polar body during the establishment of SCNT-hESC using MII oocytes obtained from female donors and dermal fibroblasts (DFB) from a 35-year-old adult male. These tetraploid SCNT-hESC lines (CHA-NT1 and CHA-NT3) were identified by the cytogenetic genotyping (91, XXXY,-6, t[2:6] / 92,XXXY,-12,+20) and have shown of indefinite proliferation, but slow speed when compared to euploid SCNT-hESCs. Using the eight Short Tendem Repeat (STR) markers, it was confirmed that both CHA-NT1 and CHA-NT3 lines contain both nuclear and oocyte donor genotypes. These hESCs expressed pluripotency markers and their embryoid bodies (EB) also expressed markers of the three embryonic germ layers and formed teratoma after transplantation into immune deficient mice. This study showed that tetraploidy does not affect the activities of proliferation and differentiation in SCNT-hESC. Therefore, tetraploid hESC lines established after SCNT procedure could be differentiated into various types of cells and could be an useful model for the study of the polyploidy cells in the tissues.

Innate immune cell-derived microparticles facilitate hepatocarcinoma metastasis by transferring integrin α(M)β₂ to tumor cells.

Science.gov (United States)

Ma, Jingwei; Cai, Wenqian; Zhang, Yi; Huang, Chunmei; Zhang, Huafeng; Liu, Jing; Tang, Ke; Xu, Pingwei; Katirai, Foad; Zhang, Jianmin; He, Wei; Ye, Duyun; Shen, Guan-Xin; Huang, Bo

2013-09-15

Mechanisms by which tumor cells metastasize to distant organs still remain enigmatic. Immune cells have been assumed to be the root of metastasis by their fusing with tumor cells. This fusion theory, although interpreting tumor metastasis analogically and intriguingly, is arguable to date. We show in this study an alternative explanation by immune cell-derived microparticles (MPs). Upon stimulation by PMA or tumor cell-derived supernatants, immune cells released membrane-based MPs, which were taken up by H22 tumor cells, leading to tumor cell migration in vitro and metastasis in vivo. The underlying molecular basis was involved in integrin α(M)β₂ (CD11b/CD18), which could be effectively relayed from stimulated innate immune cells to MPs, then to tumor cells. Blocking either CD11b or CD18 led to significant decreases in MP-mediated tumor cell metastasis. This MP-mediated transfer of immune phenotype to tumor cells might also occur in vivo. These findings suggest that tumor cells may usurp innate immune cell phenotypes via MP pathway for their metastasis, providing new insight into tumor metastatic mechanism.

Immune expulsion of Trichuris muris from resistant mice: suppression by irradiation and restoration by transfer of lymphoid cells

International Nuclear Information System (INIS)

Wakelin, D.; Selby, G.R.

1976-01-01

Lethal irradiation (850 rads of x rays) of mice made resistant to Trichuris muris markedly depressed their ability to expel a challenge infection. Expulsion was restored within 7 to 10 days when MLNC from uninfected mice were transferred on the day of infection, but no significant restoration was evident after transfer of immune serum. Transfer of Bm alone had no restorative effect within 10 days and no synergism was seen when both BM and MLNC were transferred. MLNC from uninfected donors did not restore challenge expulsion when transfer was delayed until day 7 and the mice were killed 3 days later, although MLNC from resistant donors were effective within this time. When irradiated mice were given BM and the challenge infection allowed to continue for 15 days expulsion was restored, as it was when challenge was delayed for 7 days after BM transfer in thymectomized mice. The results confirm that expulsion of T. muris involves both antibody-mediated and lymphoid cell-mediated phases and offer no evidence for the involvement of other cell types. (author)

Determination of charge transfer resistance and capacitance of microbial fuel cell through a transient response analysis of cell voltage.

Science.gov (United States)

Ha, Phuc Thi; Moon, Hyunsoo; Kim, Byung Hong; Ng, How Yong; Chang, In Seop

2010-03-15

An alternative method for determining the charge transfer resistance and double-layer capacitance of microbial fuel cells (MFCs), easily implemented without a potentiostat, was developed. A dynamic model with two parameters, the charge transfer resistance and double-layer capacitance of electrodes, was derived from a linear differential equation to depict the current generation with respect to activation overvoltage. This model was then used to fit the transient cell voltage response to the current step change during the continuous operation of a flat-plate type MFC fed with acetate. Variations of the charge transfer resistance and the capacitance value with respect to the MFC design conditions (biocatalyst existence and electrode area) and operating parameters (acetate concentration and buffer strength in the catholyte) were then determined to elucidate the validity of the proposed method. This model was able to describe the dynamic behavior of the MFC during current change in the activation loss region; having an R(2) value of over 0.99 in most tests. Variations of the charge transfer resistance value (thousands of Omega) according to the change of the design factors and operational factors were well-correlated with the corresponding MFC performances. However, though the capacitance values (approximately 0.02 F) reflected the expected trend according to the electrode area change and catalyst property, they did not show significant variation with changes in either the acetate concentration or buffer strength. (c) 2009 Elsevier B.V. All rights reserved.

Modeling the efficiency of Förster resonant energy transfer from energy relay dyes in dye-sensitized solar cells

KAUST Repository

Hoke, Eric T.; Hardin, Brian E.; McGehee, Michael D.

2010-01-01

Förster resonant energy transfer can improve the spectral breadth, absorption and energy conversion efficiency of dye sensitized solar cells. In this design, unattached relay dyes absorb the high energy photons and transfer the excitation

Characterization and Ectopic Expression of CoWRI1, an AP2/EREBP Domain-Containing Transcription Factor from Coconut (Cocos nucifera L.) Endosperm, Changes the Seeds Oil Content in Transgenic Arabidopsis thaliana and Rice (Oryza sativa L.).

Science.gov (United States)

Sun, RuHao; Ye, Rongjian; Gao, Lingchao; Zhang, Lin; Wang, Rui; Mao, Ting; Zheng, Yusheng; Li, Dongdong; Lin, Yongjun

2017-01-01

Coconut ( Cocos nucifera L.) is a key tropical crop and a member of the monocotyledonous family Arecaceae ( Palmaceae ). Few genes and related metabolic processes involved in coconut endosperm development have been investigated. In this study, a new member of the WRI1 gene family was isolated from coconut endosperm and was named CoWRI1 . Its transcriptional activities and interactions with the acetyl-CoA carboxylase ( BCCP2 ) promoter of CoWRI1 were confirmed by the yeast two-hybrid and yeast one-hybrid approaches, respectively. Functional characterization was carried out through seed-specific expression in Arabidopsis and endosperm-specific expression in rice. In transgenic Arabidopsis , high over-expressions of CoWRI1 in seven independent T2 lines were detected by quantitative real-time PCR. The relative mRNA accumulation of genes encoding enzymes involved in either fatty acid biosynthesis or triacylglycerols assembly (BCCP2, KASI, MAT, ENR, FATA, and GPDH) were also assayed in mature seeds. Furthermore, lipid and fatty acids C16:0 and C18:0 significantly increased. In two homozygous T2 transgenic rice lines (G5 and G2), different CoWRI1 expression levels were detected, but no CoWRI1 transcripts were detected in the wild type. Analyses of the seed oil content, starch content, and total protein content indicated that the two T2 transgenic lines showed a significant increase ( P oil content. The transgenic lines also showed a significant increase in starch content, whereas total protein content decreased significantly. Further analysis of the fatty acid composition revealed that palmitic acid (C16:0) and linolenic acid (C18:3) increased significantly in the seeds of the transgenic rice lines, but oleic acid (C18:1) levels significantly declined.

Function of donor cell centrosome in intraspecies and interspecies nuclear transfer embryos

International Nuclear Information System (INIS)

Zhong Zhisheng; Zhang Gang; Meng Xiaoqian; Zhang Yanling; Chen Dayuan; Schatten, Heide; Sun Qingyuan

2005-01-01

Centrosomes, the main microtubule-organizing centers (MTOCs) in most animal cells, are important for many cellular activities such as assembly of the mitotic spindle, establishment of cell polarity, and cell movement. In nuclear transfer (NT), MTOCs that are located at the poles of the meiotic spindle are removed from the recipient oocyte, while the centrosome of the donor cell is introduced. We used mouse MII oocytes as recipients, mouse fibroblasts, rat fibroblasts, or pig granulosa cells as donor cells to construct intraspecies and interspecies nuclear transfer embryos in order to observe centrosome dynamics and functions. Three antibodies against centrin, γ-tubulin, and NuMA, respectively, were used to stain the centrosome. Centrin was not detected either at the poles of transient spindles or at the poles of first mitotic spindles. γ-tubulin translocated into the two poles of the transient spindles, while no accumulated γ-tubulin aggregates were detected in the area adjacent to the two pseudo-pronuclei. At first mitotic metaphase, γ-tubulin was translocated to the spindle poles. The distribution of γ-tubulin was similar in mouse intraspecies and rat-mouse interspecies embryos. The NuMA antibody that we used can recognize porcine but not murine NuMA protein, so it was used to trace the NuMA protein of donor cell in reconstructed embryos. In the pig-mouse interspecies reconstructed embryos, NuMA concentrated between the disarrayed chromosomes soon after activation and translocated to the transient spindle poles. NuMA then immigrated into pseudo-pronuclei. After pseudo-pronuclear envelope breakdown, NuMA was located between the chromosomes and then translocated to the spindle poles of first mitotic metaphase. γ-tubulin antibody microinjection resulted in spindle disorganization and retardation of the first cell division. NuMA antibody microinjection also resulted in spindle disorganization. Our findings indicate that (1) the donor cell centrosome, defined as

Determination of Four Major Saponins in Skin and Endosperm of Seeds of Horse Chestnut (Aesculus Hippocastanum L.) Using High Performance Liquid Chromatography with Positive Confirmation by Thin Layer Chromatography.

Science.gov (United States)

Abudayeh, Zead Helmi Mahmoud; Al Azzam, Khaldun Mohammad; Naddaf, Ahmad; Karpiuk, Uliana Vladimirovna; Kislichenko, Viktoria Sergeevna

2015-11-01

To separate and quantify four major saponins in the extracts of the skin and the endosperm of seeds of horse chestnut (Aesculus hippocastanum L.) using ultrasonic solvent extraction followed by a high performance liquid chromatography-diode array detector (HPLC-DAD) with positive confirmation by thin layer chromatography (TLC). The saponins: escin Ia, escin Ib, isoescin Ia and isoescin Ib were extracted using ultrasonic extraction method. The optimized extraction conditions were: 70% methanol as extraction solvent, 80 °C as extraction temperature, and the extraction time was achieved in 4 hours. The HPLC conditions used: Zorbax SB-ODS-(150 mm × 2.1 mm, 3 μm) column, acetonitrile and 0.10% phosphoric acid solution (39:61 v/v) as mobile phase, flow rate was 0.5 mL min(-1) at 210 nm and 230 nm detection. The injection volume was 10 μL, and the separation was carried out isothermally at 30 °C in a heated chamber. The results indicated that the developed HPLC method is simple, sensitive and reliable. Moreover, the content of escins in seeds decreased by more than 30% in endosperm and by more than 40% in skin upon storage for two years. This assay can be readily utilized as a quality control method for horse chestnut and other related medicinal plants.

DNA Methylation in Peripheral Blood Cells of Pigs Cloned by Somatic Cell Nuclear Transfer

DEFF Research Database (Denmark)

Gao, Fei; Li, Shengting; Lin, Lin

2011-01-01

To date, the genome-wide DNA methylation status of cloned pigs has not been investigated. Due to the relatively low success rate of pig cloning by somatic cell nuclear transfer, a better understanding of the epigenetic reprogramming and the global methylation patterns associated with development...... in cloned pigs is required. In this study we applied methylation-specific digital karyotyping tag sequencing by Solexa technology and investigated the genome-wide DNA methylation profiles of peripheral blood cells in cloned pigs with normal phenotypes in comparison with their naturally bred controls....... In the result, we found that globally there was no significant difference of DNA methylation patterns between the two groups. Locus-specifically, some genes involved in embryonic development presented a generally increased level of methylation. Our findings suggest that in cloned pigs with normal phenotypes...

E+ subgroup PPR protein defective kernel 36 is required for multiple mitochondrial transcripts editing and seed development in maize and Arabidopsis.

Science.gov (United States)

Wang, Gang; Zhong, Mingyu; Shuai, Bilian; Song, Jiandong; Zhang, Jie; Han, Liang; Ling, Huiling; Tang, Yuanping; Wang, Guifeng; Song, Rentao

2017-06-01

Mitochondria are semi-autonomous organelles that are the powerhouse of the cells. Plant mitochondrial RNA editing guided by pentatricopeptide repeat (PPR) proteins is essential for energy production. We identify a maize defective kernel mutant dek36, which produces small and collapsed kernels, leading to embryos and/or seedlings lethality. Seed filling in dek36 is drastically impaired, in line with the defects observed in the organization of endosperm transfer tissue. Positional cloning reveals that DEK36, encoding a mitochondria-targeted E+ subgroup PPR protein, is required for mitochondrial RNA editing at atp4-59, nad7-383 and ccmF N -302, thus resulting in decreased activities of mitochondrial complex I, complex III and complex IV in dek36. Loss-of-function of its Arabidopsis ortholog At DEK36 causes arrested embryo and endosperm development, leading to embryo lethality. At_dek36 also has RNA editing defects in atp4, nad7, ccmF N 1 and ccmF N 2 , but at the nonconserved sites. Importantly, efficiency of all editing sites in ccmF N 1 , ccmF N 2 and rps12 is severely decreased in At_dek36, probably caused by the impairment of their RNA stabilization. These results suggest that the DEK36 orthologue pair are essential for embryo and endosperm development in both maize and Arabidopsis, but through divergent function in regulating RNA metabolism of their mitochondrial targets. © 2017 The Authors. New Phytologist © 2017 New Phytologist Trust.

Clinical responses to adoptive T-cell transfer can be modeled in an autologous immune-humanized mouse model

DEFF Research Database (Denmark)

Jespersen, Henrik; Lindberg, Mattias F; Donia, Marco

2017-01-01

Immune checkpoint inhibitors and adoptive cell transfer (ACT) of autologous tumor-infiltrating T cells have shown durable responses in patients with melanoma. To study ACT and immunotherapies in a humanized model, we have developed PDXv2.0 - a melanoma PDX model where tumor cells and tumor...

Single Molecule Spectroelectrochemistry of Interfacial Charge Transfer Dynamics In Hybrid Organic Solar Cell

Energy Technology Data Exchange (ETDEWEB)

Pan, Shanlin [Univ. of Alabama, Tuscaloosa, AL (United States)

2014-11-16

Our research under support of this DOE grant is focused on applied and fundamental aspects of model organic solar cell systems. Major accomplishments are: 1) we developed a spectroelectorchemistry technique of single molecule single nanoparticle method to study charge transfer between conjugated polymers and semiconductor at the single molecule level. The fluorescence of individual fluorescent polymers at semiconductor surfaces was shown to exhibit blinking behavior compared to molecules on glass substrates. Single molecule fluorescence excitation anisotropy measurements showed the conformation of the polymer molecules did not differ appreciably between glass and semiconductor substrates. The similarities in molecular conformation suggest that the observed differences in blinking activity are due to charge transfer between fluorescent polymer and semiconductor, which provides additional pathways between states of high and low fluorescence quantum efficiency. Similar spectroelectrochemistry work has been done for small organic dyes for understand their charge transfer dynamics on various substrates and electrochemical environments; 2) We developed a method of transferring semiconductor nanoparticles (NPs) and graphene oxide (GO) nanosheets into organic solvent for a potential electron acceptor in bulk heterojunction organic solar cells which employed polymer semiconductor as the electron donor. Electron transfer from the polymer semiconductor to semiconductor and GO in solutions and thin films was established through fluorescence spectroscopy and electroluminescence measurements. Solar cells containing these materials were constructed and evaluated using transient absorption spectroscopy and dynamic fluorescence techniques to understand the charge carrier generation and recombination events; 3) We invented a spectroelectorchemistry technique using light scattering and electroluminescence for rapid size determination and studying electrochemistry of single NPs in an

Efficiency of porcine somatic cell nuclear transfer – a retrospective study of factors related to embryo recipient and embryos transferred

Directory of Open Access Journals (Sweden)

Yongye Huang

2013-10-01

The successful generation of pigs via somatic cell nuclear transfer depends on reducing risk factors in several aspects. To provide an overview of some influencing factors related to embryo transfer, the follow-up data related to cloned pig production collected in our laboratory was examined. (i Spring showed a higher full-term pregnancy rate compared with winter (33.6% vs 18.6%, P = 0.006. Furthermore, a regression equation can be drawn between full-term pregnancy numbers and pregnancy numbers in different months (y = 0.692x−3.326. (ii There were no significant differences detected in the number of transferred embryos between surrogate sows exhibiting full-term development compared to those that did not. (iii Non-ovulating surrogate sows presented a higher percentage of full-term pregnancies compared with ovulating sows (32.0% vs 17.5%, P = 0.004; respectively. (iv Abortion was most likely to take place between Day 27 to Day 34. (v Based on Life Table Survival Analysis, delivery in normally fertilized and surrogate sows is expected to be completed before Day 117 or Day 125, respectively. Additionally, the length of pregnancy in surrogate sows was negatively correlated with the average litter size, which was not found for normally fertilized sows. In conclusion, performing embryo transfer in appropriate seasons, improving the quality of embryos transferred, optimizing the timing of embryo transfer, limiting the occurrence of abortion, combined with ameliorating the management of delivery, is expected to result in the harvest of a great number of surviving cloned piglets.

Heat and mass transfer effects in a direct methanol fuel cell: A 1D model

Energy Technology Data Exchange (ETDEWEB)

Oliveira, V.B.; Falcao, D.S.; Pinto, A.M.F.R. [Centro de Estudos de Fenomenos de Transporte, Departamento de Eng. Quimica, Faculdade de Engenharia da Universidade do Porto, Rua Dr. Roberto Frias, 4200-465 Porto (Portugal); Rangel, C.M. [INETI - Unidade de Electroquimica e Materiais, Paco do Lumiar, 22,1649-038 (Portugal)

2008-07-15

Models are a fundamental tool for the design process of fuel cells and fuel cell systems. In this work, a steady-state, one-dimensional model accounting for coupled heat and mass transfer, along with the electrochemical reactions occurring in the DMFC, is presented. The model output is the temperature profile through the cell and the water balance and methanol crossover between the anode and the cathode. The model predicts the correct trends for the influence of current density and methanol feed concentration on both methanol and water crossover. The model estimates the net water transfer coefficient through the membrane, {alpha}, a very important parameter to describe water management in the DMFC. Suitable operating ranges can be set up for different MEA structures maintaining the crossover of methanol and water within acceptable levels. The model is rapidly implemented and is therefore suitable for inclusion in real-time system level DMFC calculations. (author)

High in vitro development after somatic cell nuclear transfer and trichostatin A treatment of reconstructed porcine embryos

DEFF Research Database (Denmark)

Li, J.; Østrup, Olga; Villemoes, Klaus

2008-01-01

Abnormal epigenetic modification is supposed to be one of factors accounting for inefficient reprogramming of the donor cell nuclei in ooplasm after somatic cell nuclear transfer (SCNT). Trichostatin A (TSA) is an inhibitor of histone deacetylase, potentially enhancing cloning efficiency. The aim...... transferred to 2 recipients resulting in one pregnancy and birth of one live and five dead piglets. Our data demonstrate that TSA treatment after HMC in pigs may affect reprogramming of the somatic genome resulting in higher in vitro embryo development, and enable full-term in vivo development....

The cell agglutination agent, phytohemagglutinin-L, improves the efficiency of somatic nuclear transfer cloning in cattle (Bos taurus).

Science.gov (United States)

Du, Fuliang; Shen, Perng-Chih; Xu, Jie; Sung, Li-Ying; Jeong, B-Seon; Lucky Nedambale, Tshimangadzo; Riesen, John; Cindy Tian, X; Cheng, Winston T K; Lee, Shan-Nan; Yang, Xiangzhong

2006-02-01

One of the several factors that contribute to the low efficiency of mammalian somatic cloning is poor fusion between the small somatic donor cell and the large recipient oocyte. This study was designed to test phytohemagglutinin (PHA) agglutination activity on fusion rate, and subsequent developmental potential of cloned bovine embryos. The toxicity of PHA was established by examining its effects on the development of parthenogenetic bovine oocytes treated with different doses (Experiment 1), and for different durations (Experiment 2). The effective dose and duration of PHA treatment (150 microg/mL, 20 min incubation) was selected and used to compare membrane fusion efficiency and embryo development following somatic cell nuclear transfer (Experiment 3). Cloning with somatic donor fibroblasts versus cumulus cells was also compared, both with and without PHA treatment (150 microg/mL, 20 min). Fusion rate of nuclear donor fibroblasts, after phytohemagglutinin treatment, was increased from 33 to 61% (P cell nuclear donors. The nuclear transfer (NT) efficiency per oocyte used was improved following PHA treatment, for both fibroblast (13% versus 22%) as well as cumulus cells (17% versus 34%; P cloned embryos, both with and without PHA treatment, were subjected to vitrification and embryo transfer testing, and resulted in similar survival (approximately 90% hatching) and pregnancy rates (17-25%). Three calves were born following vitrification and embryo transfer of these embryos; two from the PHA-treated group, and one from non-PHA control group. We concluded that PHA treatment significantly improved the fusion efficiency of somatic NT in cattle, and therefore, increased the development of cloned blastocysts. Furthermore, within a determined range of dose and duration, PHA had no detrimental effect on embryo survival post-vitrification, nor on pregnancy or calving rates following embryo transfer.

The nutritional property of endosperm starch and its contribution to the health benefits of whole grain foods.

Science.gov (United States)

Zhang, Genyi; Hamaker, Bruce R

2017-12-12

Purported health benefits of whole grain foods in lowering risk of obesity, type 2 diabetes, cardiovascular disease, and cancer are supported by epidemiological studies and scientific researches. Bioactive components including dietary fibers, phytochemicals, and various micronutrients present in the bran and germ are commonly considered as the basis for such benefits. Endosperm starch, as the major constituent of whole grains providing glucose to the body, has been less investigated regarding its nutritional property and contribution to the value of whole grain foods. Nutritional quality of starch is associated with its rate of digestion and glucose absorption. In whole grain foods, starch digestion and glucose delivery may vary depending on the form in which the food is delivered, some with starch being rapidly and others slowly digested. Furthermore, there are other inherent factors in whole grain products, such as phenolic compounds and dietary fibers, that may moderate glycemic profiles. A good understanding of the nutritional properties of whole grain starch is important to the development of food processing technologies to maximize their health benefits.

Improvement of mouse cloning using nuclear transfer-derived embryonic stem cells and/or histone deacetylase inhibitor.

Science.gov (United States)

Wakayama, Sayaka; Wakayama, Teruhiko

2010-01-01

Nuclear transfer-derived ES (ntES) cell lines can be established from somatic cell nuclei with a relatively high success rate. Although ntES cells have been shown to be equivalent to ES cells, there are ethical objections concerning human cells, such as the use of fresh oocyte donation from young healthy woman. In contrast, the use of induced pluripotent stem (iPS) cells for cloning poses few ethical problems and is a relatively easy technique compared with nuclear transfer. Therefore, although there are several reports proposing the use of ntES cells as a model of regenerative medicine, the use of these cells in preliminary medical research is waning. However, in theory, 5 to 10 donor cells can establish one ntES cell line and, once established, these cells will propagate indefinitely. These cells can be used to generate cloned animals from ntES cell lines using a second round of NT. Even in infertile and "unclonable" strains of mice, we can generate offspring from somatic cells by combining cloning with ntES technology. Moreover, cloned offspring can be generated potentially even from the nuclei of dead bodies or freeze-dried cells via ntES cells, such as from an extinct frozen animal. Currently, only the ntES technology is available for this purpose, because all other techniques, including iPS cell derivation, require significant numbers of living donor cells. This review describes how to improve the efficiency of cloning, the establishment of clone-derived embryonic stem cells and further applications.

Fluorescence lifetime microscopy for monitoring cell adhesion using metal induced energy transfer

Science.gov (United States)

Hwang, Wonsang; Seo, JinWon; Song, Jun ho; Kim, DongEun; Won, YoungJae; Choi, In-Hong; Yoo, Kyung-Hwa; Kim, Dug Young

2018-02-01

A precise control and a reliable monitoring tool for the adhesion properties of a cell are very important in atherosclerosis studies. If endothelial cells in contact with the intracellular membrane are not attached securely, low-density lipoprotein (LDL) particles can enter into the inner membrane. It is therefore necessary to measure conditions under which endothelial cell detachment occurs. When a cell is attached to a metal thin film, the lifetime of a fluorescence probe attached to the membrane of the cell is reduced by the metal-induced energy transfer (MIET). Fluorescence lifetime imaging microscopy (FLIM) is used to monitor the attachment condition of a cell to a metal surface using FRET. However, this requires high numerical aperture (NA) objective lens because axial confocal resolution must be smaller than the cell thickness. This requirement limits the field of view of the measurement specimen. In this study we provides a new method which can measure adhesion properties of endothelial cells even with a low NA objective lens by resolving two lifetime components in FLIM.

Study on Seed Morphogenesis of Orobanchaceae in Taiwan

Directory of Open Access Journals (Sweden)

Jao-Shien Chen

2011-11-01

Full Text Available Seed morphogenesis of Orobanchaceae was not completely investigated previously. Here, we observed seed development of Orobanchaceous species in Taiwan using light and scanning electron microscopies. Results indicated that seeds of Aeginetia indica, Boschniakia himalaica, and Orobanche caerulescens all consisted of embryo, endosperm and testa. Ontogeny of the embryo in A. indica was Solanad type, while in both B. himalaica and O. caerulescens was Onagrad type. The mature embryos of the three species lacked embryonic organs, and their endosperm development was the cellular type and, at maturity, appeared as several cell layers of storage tissue. Ontogeny of the testa was all non-multiplicative, with the residues of the outermost cell layer and reticulately-thickened secondary walls of its cells at maturity. Mature seeds of A. indica and O. caerulescens were ovate whereas those of B. himalaica were oblate. As for Christisonia hookeri, due to lack of samples, only the cellular-typed endosperm was determined. The comparative development of Orobanchaceous seeds was discussed.

Utilization of Cell-Transfer Technique for Molecular Testing on Hematoxylin-Eosin-Stained Sections: A Viable Option for Small Biopsies That Lack Tumor Tissues in Paraffin Block.

Science.gov (United States)

Wu, Howard H; Jovonovich, Stephen M; Randolph, Melissa; Post, Kristin M; Sen, Joyashree D; Curless, Kendra; Cheng, Liang

2016-12-01

- In some instances the standard method of doing molecular testing from formalin-fixed, paraffin-embedded block is not possible because of limited tissue. Tumor cell-enriched cell-transfer technique has been proven useful for performing immunocytochemistry and molecular testing on cytologic smears. - To establish the cell-transfer technique as a viable option for isolating tumor cells from hematoxylin-eosin (H&E)-stained slides. - Molecular testing was performed by using the cell-transfer technique on 97 archived H&E-stained slides from a variety of different tumors. Results were compared to the conventional method of molecular testing. - Polymerase chain reaction-based molecular testing via the cell-transfer technique was successfully performed on 82 of 97 samples (85%). This included 39 of 47 cases for EGFR, 10 of 11 cases for BRAF, and 33 of 39 cases for KRAS mutations. Eighty-one of 82 cell-transfer technique samples (99%) showed agreement with previous standard method results, including 4 mutations and 35 wild-type alleles for EGFR, 4 mutations and 6 wild-type alleles for BRAF, and 11 mutations and 21 wild-type alleles for KRAS. There was only 1 discrepancy: a cell-transfer technique with a false-negative >KRAS result (wild type versus G12C). - Molecular testing performed on H&E-stained sections via cell-transfer technique is useful when tissue from cell blocks and small surgical biopsy samples is exhausted and the only available material for testing is on H&E-stained slides.

Genomic stability of lyophilized sheep somatic cells before and after nuclear transfer.

Directory of Open Access Journals (Sweden)

Domenico Iuso

Full Text Available The unprecedented decline of biodiversity worldwide is urging scientists to collect and store biological material from seriously threatened animals, including large mammals. Lyophilization is being explored as a low-cost system for storage in bio-banks of cells that might be used to expand or restore endangered or extinct species through the procedure of Somatic Cell Nuclear Transfer (SCNT. Here we report that the genome is intact in about 60% of lyophylized sheep lymphocytes, whereas DNA damage occurs randomly in the remaining 40%. Remarkably, lyophilized nuclei injected into enucleated oocytes are repaired by a robust DNA repairing activity of the oocytes, and show normal developmental competence. Cloned embryos derived from lyophylized cells exhibited chromosome and cellular composition comparable to those of embryos derived from fresh donor cells. These findings support the feasibility of lyophylization as a storage procedure of mammalian cells to be used for SCNT.

Genomic stability of lyophilized sheep somatic cells before and after nuclear transfer.

Science.gov (United States)

Iuso, Domenico; Czernik, Marta; Di Egidio, Fiorella; Sampino, Silvestre; Zacchini, Federica; Bochenek, Michal; Smorag, Zdzislaw; Modlinski, Jacek A; Ptak, Grazyna; Loi, Pasqualino

2013-01-01

The unprecedented decline of biodiversity worldwide is urging scientists to collect and store biological material from seriously threatened animals, including large mammals. Lyophilization is being explored as a low-cost system for storage in bio-banks of cells that might be used to expand or restore endangered or extinct species through the procedure of Somatic Cell Nuclear Transfer (SCNT). Here we report that the genome is intact in about 60% of lyophylized sheep lymphocytes, whereas DNA damage occurs randomly in the remaining 40%. Remarkably, lyophilized nuclei injected into enucleated oocytes are repaired by a robust DNA repairing activity of the oocytes, and show normal developmental competence. Cloned embryos derived from lyophylized cells exhibited chromosome and cellular composition comparable to those of embryos derived from fresh donor cells. These findings support the feasibility of lyophylization as a storage procedure of mammalian cells to be used for SCNT.

Improving the Safety of Cell Therapy Products by Suicide Gene Transfer

Directory of Open Access Journals (Sweden)

Antonio eDi Stasi

2014-11-01

Full Text Available Adoptive T-cell therapy can involve donor lymphocyte infusion (DLI after allogeneic hematopoietic stem cell transplantation, the administration of tumor infiltrating lymphocyte (TILs expanded ex-vivo, or more recently the use of T cell receptor (TCR or chimeric antigen receptor (CAR redirected T cells. However cellular therapies can pose significant risks, including graft-versus-host-disease and other on and off-target effects, and therefore strategies need to be implemented to permanently reverse any sign of toxicity. A suicide gene is a genetically encoded molecule that allows selective destruction of adoptively transferred cells. Suicide gene addition to cellular therapeutic products can lead to selective ablation of gene-modified cells, preventing collateral damage to contiguous cells and/or tissues. The ‘ideal’ suicide gene would ensure the safety of gene modified cellular applications by granting irreversible elimination of ‘all’ and ‘only’ the cells responsible for the unwanted toxicity. This review presents the suicide gene safety systems reported to date, with a focus on the state-of-the-art and potential applications regarding two of the most extensively validated suicide genes, including the clinical setting: herpes-simplex-thymidine-kinase (HSV-TK and inducible-caspase-9 (iCasp9.

Successful cloning of coyotes through interspecies somatic cell nuclear transfer using domestic dog oocytes.

Science.gov (United States)

Hwang, Insung; Jeong, Yeon Woo; Kim, Joung Joo; Lee, Hyo Jeong; Kang, Mina; Park, Kang Bae; Park, Jung Hwan; Kim, Yeun Wook; Kim, Woo Tae; Shin, Taeyoung; Hyun, Sang Hwan; Jeung, Eui-Bae; Hwang, Woo Suk

2013-01-01

Interspecies somatic cell nuclear transfer (iSCNT) is an emerging assisted reproductive technology (ART) for preserving Nature's diversity. The scarcity of oocytes from some species makes utilisation of readily available oocytes inevitable. In the present study, we describe the successful cloning of coyotes (Canis latrans) through iSCNT using oocytes from domestic dogs (Canis lupus familiaris or dingo). Transfer of 320 interspecies-reconstructed embryos into 22 domestic dog recipients resulted in six pregnancies, from which eight viable offspring were delivered. Fusion rate and cloning efficiency during iSCNT cloning of coyotes were not significantly different from those observed during intraspecies cloning of domestic dogs. Using neonatal fibroblasts as donor cells significantly improved the cloning efficiency compared with cloning using adult fibroblast donor cells (Pcloning of coyotes in the present study holds promise for cloning other endangered species in the Canidae family using similar techniques. However, there are still limitations of the iSCNT technology, as demonstrated by births of morphologically abnormal coyotes and the clones' inheritance of maternal domestic dog mitochondrial DNA.

Control of Listeria innocua Biofilms on Food Contact Surfaces with Slightly Acidic Electrolyzed Water and the Risk of Biofilm Cells Transfer to Duck Meat.

Science.gov (United States)

Jeon, Hye Ri; Kwon, Mi Jin; Yoon, Ki Sun

2018-04-01

Biofilm formation on food contact surfaces is a potential hazard leading to cross-contamination during food processing. We investigated Listeria innocua biofilm formation on various food contact surfaces and compared the washing effect of slightly acidic electrolyzed water (SAEW) at 30, 50, 70, and 120 ppm with that of 200 ppm of sodium hypochlorite (NaClO) on biofilm cells. The risk of L. innocua biofilm transfer and growth on food at retail markets was also investigated. The viability of biofilms that formed on food contact surfaces and then transferred cells to duck meat was confirmed by fluorescence microscopy. L. innocua biofilm formation was greatest on rubber, followed by polypropylene, glass, and stainless steel. Regardless of sanitizer type, washing removed biofilms from polypropylene and stainless steel better than from rubber and glass. Among the various SAEW concentrations, washing with 70 ppm of SAEW for 5 min significantly reduced L. innocua biofilms on food contact surfaces during food processing. Efficiency of transfer of L. innocua biofilm cells was the highest on polypropylene and lowest on stainless steel. The transferred biofilm cells grew to the maximum population density, and the lag time of transferred biofilm cells was longer than that of planktonic cells. The biofilm cells that transferred to duck meat coexisted with live, injured, and dead cells, which indicates that effective washing is essential to remove biofilm on food contact surfaces during food processing to reduce the risk of foodborne disease outbreaks.

Distribution of gluten proteins in bread wheat (Triticum aestivum) grain.

Science.gov (United States)

Tosi, Paola; Gritsch, Cristina Sanchis; He, Jibin; Shewry, Peter R

2011-07-01

Gluten proteins are the major storage protein fraction in the mature wheat grain. They are restricted to the starchy endosperm, which forms white flour on milling, and interact during grain development to form large polymers which form a continuous proteinaceous network when flour is mixed with water to give dough. This network confers viscosity and elasticity to the dough, enabling the production of leavened products. The starchy endosperm is not a homogeneous tissue and quantitative and qualitative gradients exist for the major components: protein, starch and cell wall polysaccharides. Gradients in protein content and composition are the most evident and are of particular interest because of the major role played by the gluten proteins in determining grain processing quality. Protein gradients in the starchy endosperm were investigated using antibodies for specific gluten protein types for immunolocalization in developing grains and for western blot analysis of protein extracts from flour fractions obtained by sequential abrasion (pearling) to prepare tissue layers. Differential patterns of distribution were found for the high-molecular-weight subunits of glutenin (HMW-GS) and γ-gliadins when compared with the low-molecular-weight subunits of glutenin (LMW-GS), ω- and α-gliadins. The first two types of gluten protein are more abundant in the inner endosperm layers and the latter more abundant in the subaleurone. Immunolocalization also showed that segregation of gluten proteins occurs both between and within protein bodies during protein deposition and may still be retained in the mature grain. Quantitative and qualitative gradients in gluten protein composition are established during grain development. These gradients may be due to the origin of subaleurone cells, which unlike other starchy endosperm cells derive from the re-differentiation of aleurone cells, but could also result from the action of specific regulatory signals produced by the maternal tissue

[Nuclear transfer and therapeutic cloning].

Science.gov (United States)

Xu, Xiao-Ming; Lei, An-Min; Hua, Jin-Lian; Dou, Zhong-Ying

2005-03-01

Nuclear transfer and therapeutic cloning have widespread and attractive prospects in animal agriculture and biomedical applications. We reviewed that the quality of oocytes and nuclear reprogramming of somatic donor cells were the main reasons of the common abnormalities in cloned animals and the low efficiency of cloning and showed the problems and outlets in therapeutic cloning, such as some basic problems in nuclear transfer affected clinical applications of therapeutic cloning. Study on isolation and culture of nuclear transfer embryonic stem (ntES) cells and specific differentiation of ntES cells into important functional cells should be emphasized and could enhance the efficiency. Adult stem cells could help to cure some great diseases, but could not replace therapeutic cloning. Ethics also impeded the development of therapeutic cloning. It is necessary to improve many techniques and reinforce the research of some basic theories, then somatic nuclear transfer and therapeutic cloning may apply to agriculture reproduction and benefit to human life better.

Transfer of in vitro expanded T lymphocytes after activation with dendritomas prolonged survival of mice challenged with EL4 tumor cells.

Science.gov (United States)

Li, Jinhua; Theofanous, Leigh; Stickel, Sara; Bouton-Verville, Hilary; Burgin, Kelly E; Jakubchak, Susan; Wagner, Thomas E; Wei, Yanzhang

2007-07-01

Adoptive T cell transfer after in vitro expansion represents an attractive cancer immunotherapy. The majority of studies so far have been focusing on the expansion of tumor infiltrated lymphocytes (TIL) and some have shown very encouraging results. Recently, we have developed a unique tumor immune response activator, dendritomas, by fusion of dendritic cells and tumor cells. Animal studies and early clinical trials have shown that dendritomas are able to activate tumor specific immune responses. In this study, we hypothesized that naïve T cells can be primed with dendritomas and expanded in vitro to develop an adoptive transfer therapy for patients who do not have solid tumors, such as leukemia. T cells were isolated and purified from lymph nodes of mice. The cells were then incubated with dendritomas made from syngeneic DCs and tumor cells and expanded in vitro using Dynabeads mouse CD3/CD28 T cell expander for approximately three weeks. The in vitro primed and expanded T cells showed tumor cell specific CTL activity and increased secretion of IFN-gamma. Tumor bearing mice receiving the in vitro expanded T cells survived significantly longer than control mice. Furthermore, the depletion of regulator T cells enhanced the survival of the mice that received the adoptive transfer therapy.

Current status and applications of somatic cell nuclear transfer in dogs.

Science.gov (United States)

Jang, Goo; Kim, Min Kyu; Lee, Byeong Chun

2010-11-01

Although somatic cell nuclear transfer (SCNT) technology and applications are well developed in most domesticated and laboratory animals, their use in dogs has advanced only slowly. Many technical difficulties had to be overcome before preliminary experiments could be conducted. First, due to the very low efficiency of dog oocyte maturation in vitro, in vivo matured oocytes were generally used. The nucleus of an in vivo matured oocyte was removed and a donor cell (from fetal or adult fibroblasts) was injected into the oocyte. Secondly, fusion of the reconstructed oocytes was problematic, and it was found that a higher electrical voltage was necessary, in comparison to other mammalian species. By transferring the resulting fused oocytes into surrogate females, several cloned offspring were born. SCNT was also used for producing cloned wolves, validating reproductive technologies for aiding conservation of endangered or extinct breeds. Although examples of transgenesis in canine species are very sparse, SCNT studies are increasing, and together with the new field of gene targeting technology, they have been applied in many fields of veterinary or bio-medical science. This review summarizes the current status of SCNT in dogs and evaluates its potential future applications. Copyright © 2010 Elsevier Inc. All rights reserved.

Ultrastructure of central cell in female gametophyte of Castilleja wightii Elmer (Scrophulariaceae).

Science.gov (United States)

Ekici, Nuran; Dane, Feruzan; Olgun, Göksel

2013-09-01

Embryo sac cells are highly differentiated in plants. The central cell is one of the most important cells of the embryo sac. It forms endosperm by fusion with a sperm cell. Ultrastructure of the central cell in the mature embryo sac of Castilleja wightii was investigated in this study. Nucleolus which had a lot of vacuole in a large secondary nucleus and numerous dictyosomes, vesicles, mitochondria, amyloplasts in cytoplasm were seen in this cell. Also free ribosomes in the form of polysomes and large lipid bodies were detected in the cytoplasm. Numerous vacuoles of different size were observed and some of them had autophagic function. Both smooth and rough endoplasmic reticulums were seen. Although invaginations were seen in the plasmalemma of the central cell to the inside of the embryo sac, a thick cuticular layer was observed outer side on the cell wall. The aim of this study was to contribute studies about the ultrastructure of embryo sacs.

A highly efficient method for generation of therapeutic quality human pluripotent stem cells by using naive induced pluripotent stem cells nucleus for nuclear transfer

OpenAIRE

Sanal, Madhusudana Girija

2014-01-01

Even after several years since the discovery of human embryonic stem cells and induced pluripotent stem cells (iPSC), we are still unable to make any significant therapeutic benefits out of them such as cell therapy or generation of organs for transplantation. Recent success in somatic cell nuclear transfer (SCNT) made it possible to generate diploid embryonic stem cells, which opens up the way to make high-quality pluripotent stem cells. However, the process is highly inefficient and hence e...

Effects of PTEN transfer on cell cycle progression and expression of P27kipl followed by X-ray irradiation

International Nuclear Information System (INIS)

Tian Mei; Wu Congmei; Liu Linlin; Piao Chunji; Li Xiuyi

2007-01-01

Objective: To investigate the effect of pEgr-hPTEN stable transfer combined with irradiation on the cell cycle progression and the expression of cell cycle kinase inhibitor P27 kipl protein of SHG-44 human glioma cells. Methods: pEgr-hPTEN vector containing the exogenous wild type PTEN gene was transfected into SHG-44 cells under mediation of lipofectamine in vitro, the positive cell clones were selected and amplified by using G418. Western blotting was used to measure the expression of PTEN protein. Transmission electron microscope was adopted to detect the cell ultrastructural changes and flow cytometry was adopted to analysis the changes of cell cycle progression and the expression of P27 kipl in SHG-44-sPTEN cells followed by different doses of X-ray irradiation. Results: Egr-1 promoter could be induced and activated by irradiation and then enhanced the expression of downstream PTEN gene within 5 Gy. The ultrastructure of SHG-44-sPTEN cells had many degenerative changes and many early apoptotic changes including the chromosome condensate around the nuclear envelope. pEgr-hPTEN stable transfer combined with X-ray irradiation could significantly induce G 1 arrest. The expression of P27 kipl proteins increased in SHG-44-sPTEN stable transfected cells. Conclusion: PTEN stable transfer combined with irradiation can significantly induce G 1 arrest. The molecular basis may be correlated with the enhanced expression of PTEN induced by irradiation and increased expression of cell cycle kinase inhibitor P27 kipl . (authors)

Morphological characterization of pre- and peri-implantation in vitro cultured, somatic cell nuclear transfer and in vivo derived ovine embryos

DEFF Research Database (Denmark)

Tveden-Nyborg, Pernille Yde; Peura, T.T.; Hartwich, K.M.

2005-01-01

The processes of cellular differentiation were studied in somatic cell nuvlear transfer (SCNT), in vitro cultured (IVC) and in vivo developed (in vivo) ovine embryos on days 7, 9, 11, 13, 17 and 19. SCNT embryos were constructed from in vitro matured oocytes and granulosa cells, and IVC embryos...... were produced by in vitro culture of in vivo fertilized zygotes. Most SCNT and IVC embryos were transferred to recipients on day 6 while some remained in culture for day 7 processing. In vivo embryos were collected as zygotes, transferred to intermediate recipients and retransferred to final recipients...

The beginning of a seed: regulatory mechanisms of double fertilization.

Science.gov (United States)

Bleckmann, Andrea; Alter, Svenja; Dresselhaus, Thomas

2014-01-01

THE LAUNCH OF SEED DEVELOPMENT IN FLOWERING PLANTS (ANGIOSPERMS) IS INITIATED BY THE PROCESS OF DOUBLE FERTILIZATION: two male gametes (sperm cells) fuse with two female gametes (egg and central cell) to form the precursor cells of the two major seed components, the embryo and endosperm, respectively. The immobile sperm cells are delivered by the pollen tube toward the ovule harboring the female gametophyte by species-specific pollen tube guidance and attraction mechanisms. After pollen tube burst inside the female gametophyte, the two sperm cells fuse with the egg and central cell initiating seed development. The fertilized central cell forms the endosperm while the fertilized egg cell, the zygote, will form the actual embryo and suspensor. The latter structure connects the embryo with the sporophytic maternal tissues of the developing seed. The underlying mechanisms of double fertilization are tightly regulated to ensure delivery of functional sperm cells and the formation of both, a functional zygote and endosperm. In this review we will discuss the current state of knowledge about the processes of directed pollen tube growth and its communication with the synergid cells resulting in pollen tube burst, the interaction of the four gametes leading to cell fusion and finally discuss mechanisms how flowering plants prevent multiple sperm cell entry (polyspermy) to maximize their reproductive success.

Enhanced Performance of Dye-Sensitized Solar Cells with Nanostructure Graphene Electron Transfer Layer

Directory of Open Access Journals (Sweden)

Chih-Hung Hsu

2014-01-01

Full Text Available The utilization of nanostructure graphene thin films as electron transfer layer in dye-sensitized solar cells (DSSCs was demonstrated. The effect of a nanostructure graphene thin film in DSSC structure was examined. The nanostructure graphene thin films provides a great electron transfer channel for the photogenerated electrons from TiO2 to indium tin oxide (ITO glass. Obvious improvements in short-circuit current density of the DSSCs were observed by using the graphene electron transport layer modified photoelectrode. The graphene electron transport layer reduces effectively the back reaction in the interface between the ITO transparent conductive film and the electrolyte in the DSSC.

High linear-energy-transfer radiation can overcome radioresistance of glioma stem-like cells to low linear-energy-transfer radiation.

Science.gov (United States)

Hirota, Yuki; Masunaga, Shin-Ichiro; Kondo, Natsuko; Kawabata, Shinji; Hirakawa, Hirokazu; Yajima, Hirohiko; Fujimori, Akira; Ono, Koji; Kuroiwa, Toshihiko; Miyatake, Shin-Ichi

2014-01-01

Ionizing radiation is applied as the standard treatment for glioblastoma multiforme (GBM). However, radiotherapy remains merely palliative, not curative, because of the existence of glioma stem cells (GSCs), which are regarded as highly radioresistant to low linear-energy-transfer (LET) photons. Here we analyzed whether or not high-LET particles can overcome the radioresistance of GSCs. Glioma stem-like cells (GSLCs) were induced from the GBM cell line A172 in stem cell culture medium. The phenotypes of GSLCs and wild-type cells were confirmed using stem cell markers. These cells were irradiated with (60)Co gamma rays or reactor neutron beams. Under neutron-beam irradiation, high-LET proton particles can be produced through elastic scattering or nitrogen capture reaction. Radiosensitivity was assessed by a colony-forming assay, and the DNA double-strand breaks (DSBs) were assessed by a histone gamma-H2AX focus detection assay. In stem cell culture medium, GSLCs could form neurosphere-like cells and express neural stem cell markers (Sox2 and Musashi) abundantly in comparison with their parental cells. GSLCs were significantly more radioresistant to gamma rays than their parental cells, but neutron beams overcame this resistance. There were significantly fewer gamma-H2AX foci in the A172 GSLCs 24 h after irradiation with gamma rays than in their parental cultured cells, while there was no apparent difference following neutron-beam irradiation. High-LET radiation can overcome the radioresistance of GSLCs by producing unrepairable DNA DSBs. High-LET radiation therapy might have the potential to overcome GBM's resistance to X-rays in a clinical setting.

Supercapacitive Biosolar Cell Driven by Direct Electron Transfer between Photosynthetic Membranes and CNT Networks with Enhanced Performance

DEFF Research Database (Denmark)

Pankratov, Dmitry; Pankratova, Galina; Dyachkova, Tatiana P.

2017-01-01

enabled a 1.5-fold enhancement in photocurrent density. This system offers more advantages including a reduced charge-transfer resistance, a lower open-circuit potential, and an improved cell stability. More remarkably, the average power density of the optimized cells was 250 times higher than...

Using viral vectors as gene transfer tools (Cell Biology and Toxicology Special Issue: ETCS-UK 1 day meeting on genetic manipulation of cells).

Science.gov (United States)

Howarth, Joanna L; Lee, Youn Bok; Uney, James B

2010-02-01

In recent years, the development of powerful viral gene transfer techniques has greatly facilitated the study of gene function. This review summarises some of the viral delivery systems routinely used to mediate gene transfer into cell lines, primary cell cultures and in whole animal models. The systems described were originally discussed at a 1-day European Tissue Culture Society (ETCS-UK) workshop that was held at University College London on 1st April 2009. Recombinant-deficient viral vectors (viruses that are no longer able to replicate) are used to transduce dividing and post-mitotic cells, and they have been optimised to mediate regulatable, powerful, long-term and cell-specific expression. Hence, viral systems have become very widely used, especially in the field of neurobiology. This review introduces the main categories of viral vectors, focusing on their initial development and highlighting modifications and improvements made since their introduction. In particular, the use of specific promoters to restrict expression, translational enhancers and regulatory elements to boost expression from a single virion and the development of regulatable systems is described.

Impeding Xist expression from the active X chromosome improves mouse somatic cell nuclear transfer

NARCIS (Netherlands)

Inoue, K.; Kohda, T.; Sugimoto, M.; Sado, T.; Ogonuki, N.; Matoba, S.; Shiura, H.; Ikeda, R.; Mochida, K.; Fujii, T.; Sawai, K.; Otte, A.P.; Tian, X.C.; Yang, X.; Ishino, F.; Abe, K.; Ogura, A.

2010-01-01

Cloning mammals by means of somatic cell nuclear transfer (SCNT) is highly inefficient because of erroneous reprogramming of the donor genome. Reprogramming errors appear to arise randomly, but the nature of nonrandom, SCNT-specific errors remains elusive. We found that Xist, a noncoding RNA that

Nuclear and nuclear reprogramming during the first cell cycle in bovine nuclear transfer embryos

DEFF Research Database (Denmark)

Østrup, Olga; Petrovicova, Ida; Strejcek, Frantisek

2009-01-01

Abstract The immediate events of genomic reprogramming at somatic cell nuclear transfer (SCNT) are to high degree unknown. This study was designed to evaluate the nuclear and nucleolar changes during the first cell cycle. Bovine SCNT embryos were produced from starved bovine fibroblasts and fixed......, somatic cell nuclei introduced into enucleated oocytes displayed chromatin condensation, partial nuclear envelope breakdown, nucleolar desegregation and transcriptional quiescence already at 0.5 hpa. Somatic cell cytoplasm remained temporally attached to introduced nucleus and nucleolus was partially...... restored indicating somatic influence in the early SCNT phases. At 1-3 hpa, chromatin gradually decondensed toward the nucleus periphery and nuclear envelope reformed. From 4 hpa, the somatic cell nucleus gained a PN-like appearance and displayed NPBs suggesting ooplasmic control of development....

Molecular cloning of L-methylmalonyl-CoA mutase: Gene transfer and analysis of mut cell lines

International Nuclear Information System (INIS)

Ledley, F.D.; Lumetta, M.; Nguyen, P.N.; Kolhouse, J.F.; Allen, R.H.

1988-01-01

L-Methylmalonyl-CoA mutase (MCM, EC 5.4.99.2) is a mitochondrial adenosylcobalamin-requiring enzyme that catalyzes the isomerization of L-methylmalonyl-CoA to succinyl-CoA. This enzyme is deficient in methylmalonic acidemia, an often fatal disorder of organic acid metabolism. Antibody against human placental MCM was used to screen human placenta and liver cDNA expression libraries for MCM cDNA clones. One clone expressed epitopes that could affinity-purify antibodies against MCM. A cDNA corresponding in length to the mRNA was obtained and introduced into COS cells by DNA-mediated gene transfer. Cells transformed with this clone expressed increased levels of MCM enzymatic activity. RNA blot analysis of cells genetically deficient in MCM indicates that several deficient cell lines have a specific decrease in the amount of hybridizable mRNA. These data confirm the authenticity of the MCM cDNA clone, establish the feasibility of constituting MCM activity by gene transfer for biochemical analysis and gene therapy, and provide a preliminary picture of the genotypic spectrum underlying MCM deficiency

Immune mechanisms in the transfer of experimental autoimmune encephalomyelitis without adjuvant

International Nuclear Information System (INIS)

Silberg, D.G.

1985-01-01

Experimental autoimmune encephalomyelitis (EAE) can be induced in Lewis rats without the use of adjuvant. Spleen cells of naive rats were sensitized to myelin basic protein (MBP) in vitro. Transfer of these cells did not result in the development of EAE. However, spleen cells from primary recipients, taken 10 days post transfer, and cultured with MBP (secondary culture, transferred EAE to secondary recipients. EAE can be induced in primary recipients by the transfer of secondary cultured cells or cultured cells or challenge with MBP in complete Freund's adjuvant (CFA) or incomplete Freund's adjuvant (IFA) 10 days after injection of naive cultured cells. The finding that MBP-CFA challenged 1' recipients developed EAE, suggests that the rats have been primed to MBP through the naive cultured cell transfer. The cells from naive culture that sensitize the primary recipient were radioresistant (1500 R), probably macrophages. This is in contrast to the cells transferring EAE to the secondary recipient, which were radiosensitive. Unlike the spleen cells which transfer EAE from MBP-CFA sensitized rats, the cells in the secondary transfer could not be activated to transfer EAE when cultured with concanavalin A. Clinical EAE in the secondary recipient was more severe when these rats were irradiated (200 R) prior to transfer. There is evidence that low dose irradiation eliminates naturally occurring suppressor cells. EAE also developed in lethally irradiated (850 R) recipients of secondary cultured cells, suggesting that the transferred cells can induce EAE alone or by recruiting radioresistant cells in the secondary host

Microscopia eletrônica de varredura do endosperma de café (Coffea arabica L. durante o processo de secagem Scanning electron microscopy of the endosperm of coffee (Coffea arabica L. during the drying process

Directory of Open Access Journals (Sweden)

Reni Saath

2010-02-01

content to 11% (bu. During the drying process the coffee grains were randomly sampled and fragments of the endosperm were prepared for scanning electron microscopy and eletromicrographs were taken. Measurements of the cells were taken for evaluating changes in the plasma membrane of the endosperm cells in relation to the moisture content and drying period. The cell cytoplasm of the coffee grains with 11% moisture content was not affected when dried under sun light and at the temperature of 40°C. When dried at 60°C, changes in the cellular structures of the cytoplasm were observed for coffees with moisture content of 20%.

High linear-energy-transfer radiation can overcome radioresistance of glioma stem-like cells to low linear-energy-transfer radiation

International Nuclear Information System (INIS)

Hirota, Yuki; Kawabata, Shinji; Kuroiwa, Toshihiko; Miyatake, Shin-ichi; Masunaga, Shin-ichiro; Kondo, Natsuko; Ono, Koji; Hirakawa, Hirokazu; Yajima, Hirohiko; Fujimori, Akira

2014-01-01

Ionizing radiation is applied as the standard treatment for glioblastoma multiforme (GBM). However, radiotherapy remains merely palliative, not curative, because of the existence of glioma stem cells (GSCs), which are regarded as highly radioresistant to low linear-energy-transfer (LET) photons. Here we analyzed whether or not high-LET particles can overcome the radioresistance of GSCs. Glioma stem-like cells (GSLCs) were induced from the GBM cell line A172 in stem cell culture medium. The phenotypes of GSLCs and wild-type cells were confirmed using stem cell markers. These cells were irradiated with 60 Co gamma rays or reactor neutron beams. Under neutron-beam irradiation, high-LET proton particles can be produced through elastic scattering or nitrogen capture reaction. Radiosensitivity was assessed by a colony-forming assay, and the DNA double-strand breaks (DSBs) were assessed by a histone gamma-H2AX focus detection assay. In stem cell culture medium, GSLCs could form neurosphere-like cells and express neural stem cell markers (Sox2 and Musashi) abundantly in comparison with their parental cells. GSLCs were significantly more radioresistant to gamma rays than their parental cells, but neutron beams overcame this resistance. There were significantly fewer gamma-H2AX foci in the A172 GSLCs 24 h after irradiation with gamma rays than in their parental cultured cells, while there was no apparent difference following neutron-beam irradiation. High-LET radiation can overcome the radioresistance of GSLCs by producing unrepairable DNA DSBs. High-LET radiation therapy might have the potential to overcome GBM's resistance to X-rays in a clinical setting. (author)

Application of the comet assay in studies of programmed cell death (PCD in plants

Directory of Open Access Journals (Sweden)

Maria Charzyńska

2014-01-01

Full Text Available Programmed cell death (PCD in plants is an intensively investigated process. One of the main characteristics of PCD in both animal and plant organisms is the non-random, internucleosomal fragmentation of nuclear DNA, usually analysed using total DNA gel electrophoresis or TUNEL method. In this paper we present application of the "comet assay" (Single Cell Gel Electrophoresis for detection of nDNA degradation in studies of PCD during plant life cycle. We analyzed three types of tissue: anther tapetum, endosperm and mesophyll which were prepared in different ways to obtain a suspension of viable cells (without cell walls. The comet assay gives a possibility of examination of the nDNA degradation in individual cell. This method is significant for studies of the plant tissue differentiation and senescence especially in the cases when it is not possible to isolate large number of cells at the same developmental stage.

The Influence of Interspecies Somatic Cell Nuclear Transfer on Epigenetic Enzymes Transcription in Early Embryos

DEFF Research Database (Denmark)

Morovic, Martin; Murin, Matej; Strejcek, Frantisek

2016-01-01

in oocytes and early embryos of several species including bovine and porcine zygotes is species-dependent process and the incomplete DNA methylation correlates with the nuclear transfer failure rate in mammals. In this study the transcription of DNA methyltransferase 1 and 3a (DNMT1, DNMT3a) genes in early......One of the main reason for the incorrect development of embryos derived from somatic cell nuclear transfer is caused by insufficient demethylation of injected somatic chromatin to a state comparable with an early embryonic nucleus. It is already known that the epigenetic enzymes transcription....... In spite of the detection of ooplasmic DNA methyltransferases, the somatic genes for DNMT1 and DNMT3a enzymes were not expressed and the development of intergeneric embryos stopped at the 4-cell stage. Our results indicate that the epigenetic reprogramming during early mammalian development is strongly...

Hole transfer from CdSe nanoparticles to TQ1 polymer in hybrid solar cell device

Science.gov (United States)

Sohail, Muhammad; Shah, Zawar Hussain; Saeed, Shomaila; Bibi, Nasreen; Shahbaz, Sadia; Ahmed, Safeer; Shabbir, Saima; Siddiq, Muhammad; Iqbal, Azhar

2018-05-01

In view of realizing the economic viability, we fabricate a solar cell device containing low band gap and easily processable polymer 5-yl-8-(thiophene-2,5-diyl)-2,3-bis(3-(octyloxy)phenyl) quinoxaline (TQ1) and CdSe nanoparticles (NPs) and investigate its charge transport properties. When the TQ1 is combined with the CdSe NPs a strong photoluminescence quenching and shortening of photoluminescence lifetime of the TQ1 is observed indicating exciton transfer from TQ1 to the CdSe NPs. The time-resolved photoluminescence further reveals that the exciton transfer from the polymer to CdSe NPs is very efficient (68%) and it occurs in solar cell as compared to polymer only device. These observations suggest the importance of other II-VI semiconductor NPs to achieve higher efficiency for photovoltaic devices containing TQ1 polymer.

Anti-gp120 minibody gene transfer to female genital epithelial cells protects against HIV-1 virus challenge in vitro.

Directory of Open Access Journals (Sweden)

Ussama M Abdel-Motal

Full Text Available Although cervico-vaginal epithelial cells of the female lower genital tract provide the initial defense system against HIV-1 infection, the protection is sometimes incomplete. Thus, enhancing anti-HIV-1 humoral immunity at the mucosal cell surface by local expression of anti-HIV-1 broadly neutralizing antibodies (BnAb that block HIV-1 entry would provide an important new intervention that could slow the spread of HIV/AIDS.This study tested the hypothesis that adeno-associated virus (AAV-BnAb gene transfer to cervico-vaginal epithelial cells will lead to protection against HIV-1. Accordingly, a recombinant AAV vector that encodes human b12 anti-HIV gp120 BnAb as a single-chain variable fragment Fc fusion (scFvFc, or "minibody" was constructed. The secreted b12 minibody was shown to be biologically functional in binding to virus envelope protein, neutralizing HIV-1 and importantly, blocking transfer and infectivity of HIV-1(bal in an organotypic human vaginal epithelial cell (VEC model. Furthermore, cervico-vaginal epithelial stem cells were found to be efficiently transduced by the optimal AAV serotype mediated expression of GFP.This study provides the foundation for a novel microbicide strategy to protect against sexual transmission of HIV-1 by AAV transfer of broadly neutralizing antibody genes to cervico-vaginal epithelial stem cells that could replenish b12 BnAb secreting cells through multiple menstrual cycles.

Programmed cell death during quinoa perisperm development.

Science.gov (United States)

López-Fernández, María Paula; Maldonado, Sara

2013-08-01

At seed maturity, quinoa (Chenopodium quinoa Willd.) perisperm consists of uniform, non-living, thin-walled cells full of starch grains. The objective of the present study was to study quinoa perisperm development and describe the programme of cell death that affects the entire tissue. A number of parameters typically measured during programmed cell death (PCD), such as cellular morphological changes in nuclei and cytoplasm, endoreduplication, DNA fragmentation, and the participation of nucleases and caspase-like proteases in nucleus dismantling, were evaluated; morphological changes in cytoplasm included subcellular aspects related to starch accumulation. This study proved that, following fertilization, the perisperm of quinoa simultaneously accumulates storage reserves and degenerates, both processes mediated by a programme of developmentally controlled cell death. The novel findings regarding perisperm development provide a starting point for further research in the Amaranthaceae genera, such as comparing seeds with and without perisperm, and specifying phylogeny and evolution within this taxon. Wherever possible and appropriate, differences between quinoa perisperm and grass starchy endosperm--a morphologically and functionally similar, although genetically different tissue--were highlighted and discussed.

Proliferative lifespan is conserved after nuclear transfer.

Science.gov (United States)

Clark, A John; Ferrier, Patricia; Aslam, Samena; Burl, Sarah; Denning, Chris; Wylie, Diana; Ross, Arlene; de Sousa, Paul; Wilmut, Ian; Cui, Wei

2003-06-01

Cultured primary cells exhibit a finite proliferative lifespan, termed the Hayflick limit. Cloning by nuclear transfer can reverse this cellular ageing process and can be accomplished with cultured cells nearing senescence. Here we describe nuclear transfer experiments in which donor cell lines at different ages and with different proliferative capacities were used to clone foetuses and animals from which new primary cell lines were generated. The rederived lines had the same proliferative capacity and rate of telomere shortening as the donor cell lines, suggesting that these are innate, genetically determined, properties that are conserved by nuclear transfer.

DNA methylation in porcine preimplantation embryos developed in vivo and produced by in vitro fertilization, parthenogenetic activation and somatic cell nuclear transfer

DEFF Research Database (Denmark)

Deshmukh, Rahul Shahaji; Østrup, Olga; Østrup, Esben

2011-01-01

DNA demethylation and remethylation are crucial for reprogramming of the differentiated parental/somatic genome in the recipient ooplasm upon somatic cell nuclear transfer. Here, we analyzed the DNA methylation dynamics during porcine preimplantation development. Porcine in vivo developed (IV......), in vitro fertilized (IVF), somatic cell nuclear transfer (SCNT) and parthenogenetically activated (PA) embryos were evaluated for DNA methylation quantification at different developmental stages. Fertilized (IV and IVF) one-cell stages lacked a substantial active demethylation of the paternal genome...

DNA methylation in porcine preimplantation embryos developed in-vivo or produced by in-vitro fertilization, parthenogenetic activation and somatic cell nuclear transfer

DEFF Research Database (Denmark)

Deshmukh, Rahul Shahaji; Østrup, Olga; Østrup, Esben

2011-01-01

DNA demethylation and remethylation are crucial for reprogramming of the differentiated parental/somatic genome in the recipient ooplasm upon somatic cell nuclear transfer. Here, we analyzed the DNA methylation dynamics during porcine preimplantation development. Porcine in vivo developed (IV......), in vitro fertilized (IVF), somatic cell nuclear transfer (SCNT) and parthenogenetically activated (PA) embryos were evaluated for DNA methylation quantification at different developmental stages. Fertilized (IV and IVF) one-cell stages lacked a substantial active demethylation of the paternal genome...

Tolerogenic dendritic cells pulsed with enterobacterial extract suppress development of colitis in the severe combined immunodeficiency transfer model

DEFF Research Database (Denmark)

Pedersen, Anders Elm; Gad, M; Kristensen, N N

2007-01-01

Immunomodulatory dendritic cells (DCs) that induce antigen-specific T-cell tolerance upon in vivo adoptive transfer are promising candidates for immunotherapy of autoimmune diseases. The feasibility of such a strategy has recently proved its efficacy in animal models of allotransplantation and ex...

Polarized and persistent Ca²⁺ plumes define loci for formation of wall ingrowth papillae in transfer cells.

Science.gov (United States)

Zhang, Hui-Ming; Imtiaz, Mohammad S; Laver, Derek R; McCurdy, David W; Offler, Christina E; van Helden, Dirk F; Patrick, John W

2015-03-01

Transfer cell morphology is characterized by a polarized ingrowth wall comprising a uniform wall upon which wall ingrowth papillae develop at right angles into the cytoplasm. The hypothesis that positional information directing construction of wall ingrowth papillae is mediated by Ca(2+) signals generated by spatiotemporal alterations in cytosolic Ca(2+) ([Ca(2+)]cyt) of cells trans-differentiating to a transfer cell morphology was tested. This hypothesis was examined using Vicia faba cotyledons. On transferring cotyledons to culture, their adaxial epidermal cells synchronously trans-differentiate to epidermal transfer cells. A polarized and persistent Ca(2+) signal, generated during epidermal cell trans-differentiation, was found to co-localize with the site of ingrowth wall formation. Dampening Ca(2+) signal intensity, by withdrawing extracellular Ca(2+) or blocking Ca(2+) channel activity, inhibited formation of wall ingrowth papillae. Maintenance of Ca(2+) signal polarity and persistence depended upon a rapid turnover (minutes) of cytosolic Ca(2+) by co-operative functioning of plasma membrane Ca(2+)-permeable channels and Ca(2+)-ATPases. Viewed paradermally, and proximal to the cytosol-plasma membrane interface, the Ca(2+) signal was organized into discrete patches that aligned spatially with clusters of Ca(2+)-permeable channels. Mathematical modelling demonstrated that these patches of cytosolic Ca(2+) were consistent with inward-directed plumes of elevated [Ca(2+)]cyt. Plume formation depended upon an alternating distribution of Ca(2+)-permeable channels and Ca(2+)-ATPase clusters. On further inward diffusion, the Ca(2+) plumes coalesced into a uniform Ca(2+) signal. Blocking or dispersing the Ca(2+) plumes inhibited deposition of wall ingrowth papillae, while uniform wall formation remained unaltered. A working model envisages that cytosolic Ca(2+) plumes define the loci at which wall ingrowth papillae are deposited. © The Author 2014. Published by Oxford

Charge Transfer Dynamics of Highly Efficient Cyanidin-3-O- Glucoside Sensitizer for Dye-Sensitized Solar Cells

International Nuclear Information System (INIS)

Prima, E C; Yuliarto, B; Suyatman; Dipojono, H K

2016-01-01

This paper reports the novel efficiency achievement of black rice-based natural dye- sensitized solar cells. The higher dye concentration, the longer dye extraction as well as dye immersion onto a TiO 2 film, and the co-adsorption addition are key strategies for improved-cell performance compared to the highest previous achievement. The black rice dye containing 1.38 mM cyanidin-3-O-glucoside has been extracted without purification for 3 weeks at dark condition and room temperature. The anatase TiO 2 photoanode was dipped into dye solution within 4 days. Its electrode was firmly sealed to be a cell and was filled by I - /I 3 - electrolyte using vacuum technique. As a result, the overall solar-to-energy conversion efficiency was 1.49% at AM 1.5 illumination (100 mW.cm -2 ). The voltametric analysis has reported the interfacial electronic band edges of TiO 2 -Dye-Electrolyte. Furthermore, electrochemical impedance spectroscopy has shown the kinetic of interfacial electron transfer dynamics among TiO 2 -dye-electrolyte. The cell has the transfer resistance (Rt) of 12.5 ω, the recombination resistance (Rr) of 266.8 ω, effective electron diffusion coefficients (Dn) of 1.4 × 10 -3 cm 2 /s, Dye-TiO 2 effective electron transfer (τ d ) of 26.6 μs, effective diffusion length (L n )of 33.78 μm, chemical capacitance (C μ ) of 12.43 μF, and electron lifetime (τ n ) of 3.32 ms. (paper)

IR-FEL-induced green fluorescence protein (GFP) gene transfer into plant cell

CERN Document Server

Awazu, K; Tamiya, E

2002-01-01

A Free Electron Laser (FEL) holds potential for various biotechnological applications due to its characteristics such as flexible wavelength tunability, short pulse and high peak power. We could successfully introduce the Green Fluorescent Protein (GFP) gene into tobacco BY2 cells by IR-FEL laser irradiation. The irradiated area of the solution containing BY2 cells and plasmid was about 0.1 mm sup 2. FEL irradiation at a wavelength of 5.75 and 6.1 mu m, targeting absorption by the ester bond of the lipid and the amide I bond of the protein, respectively, was shown to cause the introduction of the fluorescent dye into the cell. On the other hand, transient expression of the GFP fluorescence was only observed after irradiation at 5.75 mu m. The maximum transfer efficiency was about 0.5%.

Models for mass transfer effects in semi-fuel cells and for a silver-zinc battery

Science.gov (United States)

Venkatraman, Murali Sankar

Semi-Fuel Cells (SFCs) and Silver-Zinc batteries have been recognized as batteries for high power applications. For channel flow between two parallel plates, featured in SFCs, obstacles may take the form of ordered asymmetrical porous nets. The net controls the spacing between the two electrode plates. The effect of the inert insulating net and its geometry on the heat and mass transfer characteristics in such a system is presented. The governing equations for momentum, continuity, and energy are solved in a three-dimensional domain using a commercial computational fluid dynamics software for fully developed flow with constant temperature boundary conditions. The local Nusselt number is calculated from the resulting temperature distribution. This net also affects the limiting current distribution in an SFC operating at limiting current because it disrupts the parabolic laminar flow velocity distribution. Hence, the current density distribution is obtained from the Nusselt number distribution through a heat and mass transfer analogy. The location, spacing, and number of the longitudinal and transverse ribs of the net are shown to affect the local and average current density distributions and Nusselt numbers on each of the two electrode plates. The results show that transverse ribs have a greater effect and that the enhancements of the average current density of 250% can be obtained for a spacing of 0.94 x 10-3 m with greater than 16 transverse ribs. A silver-zinc battery shows similar mass transfer limitations while discharged at moderate to high discharge rates. A one-dimensional mathematical model consisting of a negative (zinc) electrode, separator, and positive (silver) electrode, has been developed to study the performance and thermal behavior of the silver-zinc cell during discharge. The physical phenomena described here are reaction kinetics, mass transfer and heat generation. The analysis includes finite matrix conductivities (thermal and electrical

Cucumber (Cucumis sativus L.) embryo development in situ after pollination with irradiated pollen

International Nuclear Information System (INIS)

Faris, N.M.; Niemirowicz-Szczytt, K.

1999-01-01

Embryological studies were undertaken to compare the normal development of cucumber endosperm and embryo with that observed after pollination with gamma-irradiated pollen (0.1 and 0.3 kGy). Delayed penetration of the pollen tube occurred at both irradiation doses. Endosperm and embryo development was also delayed, but was initiated within 6 days after pollination in 100% of embryo sacs at 0.1 kGy and in 70-80% at 0.3 kGy. Various abnormalities in endosperm and embryo cell structure confirmed progressive degeneration, which occurred earlier with the higher dose of irradiation. Degeneration increased dramatically; only 30-40% of the embryos reached the globular stage 15 days after pollination. (author)

High Performing Ternary Solar Cells through Förster Resonance Energy Transfer between Nonfullerene Acceptors.

Science.gov (United States)

Yang, Lei; Gu, Wenxing; Hong, Ling; Mi, Yang; Liu, Feng; Liu, Ming; Yang, Yufei; Sharma, Bigyan; Liu, Xinfeng; Huang, Hui

2017-08-16

Nonradiative Förster resonance energy transfer (FRET) is an important mechanism of organic solar cells, which can improve the exciton migration over a long distance, resulting in improvement of efficiency of solar cells. However, the current observations of FRET are very limited, and the efficiencies are less than 9%. In this study, FRET effect was first observed between two nonfullerene acceptors in ternary solar cells, which improved both the absorption range and exciton harvesting, leading to the dramatic enhancement in the short circuit current and power conversion efficiency. Moreover, this strategy is proved to be a versatile platform for conjugated polymers with different bandgaps, resulting in a remarkable efficiency of 10.4%. These results demonstrated a novel method to enhance the efficiency of organic soar cells.

In utero hematopoietic stem cell transfer: current status and future strategies.

Science.gov (United States)

Surbek, D V; Gratwohl, A; Holzgreve, W

1999-07-01

Successful prenatal treatment of severe immunodeficiencies by allogeneic hematopoietic stem cell transplantation in utero has been reported. Though other diseases like hemoglobinopathies or storage diseases are potentially amenable to this novel therapeutic approach, no success has yet been achieved in recipients without severe immunodeficiency. Graft rejection by the developing fetus and/or lack of selective, competitive advantage of donor versus host stem cells preventing stable engraftment seem to be the major obstacles. Several strategies to overcome these hurdles are being explored in preclinical settings, including timing and repeated dosing of stem cell administration to the fetus, ex vivo modification of the transplant, using different fetal compartments as targets for early stem cell transfer, or inducing microchimerism for postnatal transplantation from the same donor. In addition, the exact definition of the basic concept of early fetal immunologic naivete and the understanding of the molecular basics of migration and homing in fetal hematopoiesis system seem mandatory for a successful approach. Gene therapy using ex vivo transduced autologous cord blood cells or direct gene targeting in utero are other potential means to correct hematopoietic and immunologic single gene disorders in utero, though this approach is still away from the stage of clinical trials.

Transfer of allogeneic CD4+ T cells rescues CD8+ T cells in anti-PD-L1–resistant tumors leading to tumor eradication

Science.gov (United States)

Arina, Ainhoa; Karrison, Theodore; Galka, Eva; Schreiber, Karin; Weichselbaum, Ralph R.; Schreiber, Hans

2017-01-01

Adoptively transferred CD8+ T cells can stabilize the size of solid tumors over long periods of time by exclusively recognizing antigen cross-presented on tumor stroma. However, these tumors eventually escape T cell–mediated growth control. The aim of this study was to eradicate such persistent cancers. In our model, the SIYRYYGL antigen is expressed by cancer cells that lack the MHC-I molecule Kb needed for direct presentation, but the antigen is picked up and cross-presented by tumor stroma. A single injection of antigen-specific 2C CD8+ T cells caused long-term inhibition of tumor growth, but without further intervention, tumors started to progress after approximately 3 months. Escape was associated with reduced numbers of circulating 2C cells. Tumor-infiltrating 2C cells produced significantly less TNFα and expressed more of the “exhaustion” markers PD-1 and Tim-3 than T cells from lymphoid organs. High-dose local ionizing radiation, depletion of myeloid-derived suppressor cells, infusions of additional 2C cells, and antibodies blocking PD-L1 did not prevent tumor escape. In contrast, adoptive transfer of allogeneic CD4+ T cells restored the numbers of circulating Ag-specific CD8+ T cells and their intratumoral function, resulting in tumor eradication. These CD4+ T cells had no antitumor effects in the absence of CD8+ T cells and recognized the alloantigen cross-presented on tumor stroma. CD4+ T cells might also be effective in cancer patients when PD1/PD-L1 blockade does not rescue intratumoral CD8+ T-cell function and tumors persist. PMID:28077434

Nanoimprint-Transfer-Patterned Solids Enhance Light Absorption in Colloidal Quantum Dot Solar Cells

KAUST Repository

Kim, Younghoon

2017-03-13

Colloidal quantum dot (CQD) materials are of interest in thin-film solar cells due to their size-tunable bandgap and low-cost solution-processing. However, CQD solar cells suffer from inefficient charge extraction over the film thicknesses required for complete absorption of solar light. Here we show a new strategy to enhance light absorption in CQD solar cells by nanostructuring the CQD film itself at the back interface. We use two-dimensional finite-difference time-domain (FDTD) simulations to study quantitatively the light absorption enhancement in nanostructured back interfaces in CQD solar cells. We implement this experimentally by demonstrating a nanoimprint-transfer-patterning (NTP) process for the fabrication of nanostructured CQD solids with highly ordered patterns. We show that this approach enables a boost in the power conversion efficiency in CQD solar cells primarily due to an increase in short-circuit current density as a result of enhanced absorption through light-trapping.

Dexamethasone/1alpha-25-dihydroxyvitamin D3-treated dendritic cells suppress colitis in the SCID T-cell transfer model

DEFF Research Database (Denmark)

Pedersen, Anders Elm; Schmidt, Esben Gjerløff Wedebye; Gad, Monika

2008-01-01

severe combined immunodeficient (SCID) mice adoptively transferred with CD4(+) CD25(-) T cells from the development of wasting disease and colitis. We therefore established an in vitro test that could predict the in vivo function of DCs and improve strategies for the preparation of immunomodulatory DCs...... in this model. Based on these in vitro findings, we here evaluate three methods for DC generation including short-term and long-term IL-10 exposure or DC exposure to dexamethasone in combination with vitamin D3 (Dex/D3). All DCs resulted in lower CD4(+) CD25(-) T-cell enteroantigen-specific responses in vitro...

Ex-Vivo Gene Therapy Using Lentiviral Mediated Gene Transfer Into Umbilical Cord Blood Derived Stem Cells

Directory of Open Access Journals (Sweden)

Hanieh Jalali

2016-02-01

Full Text Available Background Introduction of therapeutic genes into the injured site of nervous system can be achieved using transplantation of cellular vehicles containing desired gene. To transfer exogenous genes into the cellular vehicles, lentiviral vectors are one of interested vectors because of advantages such high transduction efficiency of dividing and non-dividing cells. Unrestricted somatic stem cells are subclasses of umbilical cord blood derived stem cells which are appreciate candidates to use as cellular vehicles for ex vivo gene therapy of nervous system. Objectives In current study we investigated the effect of lentiviral vector transduction on the neuronal related features of unrestricted somatic stem cells to indicate the probable and unwanted changes related to transduction procedure. Materials and Methods In this experimental study, lentiviral vector containing green fluorescent protein (GFP were transduced into unrestricted somatic stem cells and its effect was investigated with using MTT assay, qPCR and immunohistochemistry techniques. For statistical comparison of real time PCR results, REST software (2009, Qiagen was used. Results Obtained results showed lentiviral vector transduction did not have cytotoxic effects on unrestricted somatic stem cells and did not change neuronal differentiation capacity of them as well the expression of some neuronal related genes and preserved them in multilineage situation. Conclusions In conclusion, we suggested that lentiviral vectors could be proper vectors to transfer therapeutic gene into unrestricted somatic stem cells to provide a cellular vehicle for ex vivo gene therapy of nervous system disorders.

Production of rhesus monkey cloned embryos expressing monomeric red fluorescent protein by interspecies somatic cell nuclear transfer

International Nuclear Information System (INIS)

Zhu, Hai-Ying; Kang, Jin-Dan; Li, Suo; Jin, Jun-Xue; Hong, Yu; Jin, Long; Guo, Qing; Gao, Qing-Shan; Yan, Chang-Guo; Yin, Xi-Jun

2014-01-01

Highlights: • Rhesus monkey cells were electroporated with a plasmid containing mRFP1, and an mRFP1-expressing cell line was generated. • For the first time, mRFP1-expressing rhesus monkey cells were used as donor cells for iSCNT. • The effect of VPA on the development of embryos cloned using iSCNT was determined. - Abstract: Interspecies somatic cell nuclear transfer (iSCNT) is a promising method to clone endangered animals from which oocytes are difficult to obtain. Monomeric red fluorescent protein 1 (mRFP1) is an excellent selection marker for transgenically modified cloned embryos during somatic cell nuclear transfer (SCNT). In this study, mRFP-expressing rhesus monkey cells or porcine cells were transferred into enucleated porcine oocytes to generate iSCNT and SCNT embryos, respectively. The development of these embryos was studied in vitro. The percentage of embryos that underwent cleavage did not significantly differ between iSCNT and SCNT embryos (P > 0.05; 71.53% vs. 80.30%). However, significantly fewer iSCNT embryos than SCNT embryos reached the blastocyst stage (2.04% vs. 10.19%, P < 0.05). Valproic acid was used in an attempt to increase the percentage of iSCNT embryos that developed to the blastocyst stage. However, the percentages of embryos that underwent cleavage and reached the blastocyst stage were similar between untreated iSCNT embryos and iSCNT embryos treated with 2 mM valproic acid for 24 h (72.12% vs. 70.83% and 2.67% vs. 2.35%, respectively). These data suggest that porcine-rhesus monkey interspecies embryos can be generated that efficiently express mRFP1. However, a significantly lower proportion of iSCNT embryos than SCNT embryos reach the blastocyst stage. Valproic acid does not increase the percentage of porcine-rhesus monkey iSCNT embryos that reach the blastocyst stage. The mechanisms underling nuclear reprogramming and epigenetic modifications in iSCNT need to be investigated further