Isolation and purification of a granulosis virus from infected larvae of the Indian meal moth, Plodia interpunctella.

PubMed

Tweeten, K A; Bulla, L A; Consigli, R A

1977-09-01

A procedure was developed for purification of a granulosis virus inclusion body produced in vivo in the Indian meal moth, Plodia interpunctella (Hübner). Purification was accomplished by differential centrifugation, treatment with sodium deoxycholate, and velocity sedimentation in sucrose gradients. The adequacy of the procedure was confirmed by mixing experiments in which uninfected, radioactively labeled larvae were mixed with infected, unlabeled larvae. After purification, the virus was shown to be free of host tissue, to retain its physical integrity, and to be highly infectious per os. Preparations of purified virus consisted of homogeneous populations of intact inclusion bodies (210 by 380 nm) whose buoyant density was 1.271 g/cm3 when centrifuged to equilibrium in sucrose gradients. Electron microscopy of thin-sectioned virus or of virus sequentially disrupted on electron microscope grids demonstrated three components: protein matrix, envelope, and nucleocapsid.

Isolation and purification of a granulosis virus from infected larvae of the Indian meal moth, Plodia interpunctella.

PubMed Central

Tweeten, K A; Bulla, L A; Consigli, R A

1977-01-01

A procedure was developed for purification of a granulosis virus inclusion body produced in vivo in the Indian meal moth, Plodia interpunctella (Hübner). Purification was accomplished by differential centrifugation, treatment with sodium deoxycholate, and velocity sedimentation in sucrose gradients. The adequacy of the procedure was confirmed by mixing experiments in which uninfected, radioactively labeled larvae were mixed with infected, unlabeled larvae. After purification, the virus was shown to be free of host tissue, to retain its physical integrity, and to be highly infectious per os. Preparations of purified virus consisted of homogeneous populations of intact inclusion bodies (210 by 380 nm) whose buoyant density was 1.271 g/cm3 when centrifuged to equilibrium in sucrose gradients. Electron microscopy of thin-sectioned virus or of virus sequentially disrupted on electron microscope grids demonstrated three components: protein matrix, envelope, and nucleocapsid. Images PMID:334076

Separation of Bacteria, Protozoa and Carbon Nanotubes by Density Gradient Centrifugation

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

Mortimer, Monika; Petersen, Elijah; Buchholz, Bruce

Sustainable production and use of carbon nanotube (CNT)-enabled materials require efficient assessment of CNT environmental hazards, including the potential for CNT bioaccumulation and biomagnification in environmental receptors. Microbes, as abundant organisms responsible for nutrient cycling in soil and water, are important ecological receptors for studying the effects of CNTs. Quantification of CNT association with microbial cells requires efficient separation of CNT-associated cells from individually dispersed CNTs and CNT agglomerates. Here in this paper, we designed, optimized, and demonstrated procedures for separating bacteria (Pseudomonas aeruginosa) from unbound multiwall carbon nanotubes (MWCNTs) and MWCNT agglomerates using sucrose density gradient centrifugation. We demonstratemore » separation of protozoa (Tetrahymena thermophila) from MWCNTs, bacterial agglomerates, and protozoan fecal pellets by centrifugation in an iodixanol solution. The presence of MWCNTs in the density gradients after centrifugation was determined by quantification of 14C-labeled MWCNTs; the recovery of microbes from the density gradient media was confirmed by optical microscopy. Protozoan intracellular contents of MWCNTs and of bacteria were also unaffected by the designed separation process. Lastly, the optimized methods contribute to improved efficiency and accuracy in quantifying MWCNT association with bacteria and MWCNT accumulation in protozoan cells, thus supporting improved assessment of CNT bioaccumulation.« less

Separation of Bacteria, Protozoa and Carbon Nanotubes by Density Gradient Centrifugation

DOE PAGES

Mortimer, Monika; Petersen, Elijah; Buchholz, Bruce; ...

2016-10-12

Sustainable production and use of carbon nanotube (CNT)-enabled materials require efficient assessment of CNT environmental hazards, including the potential for CNT bioaccumulation and biomagnification in environmental receptors. Microbes, as abundant organisms responsible for nutrient cycling in soil and water, are important ecological receptors for studying the effects of CNTs. Quantification of CNT association with microbial cells requires efficient separation of CNT-associated cells from individually dispersed CNTs and CNT agglomerates. Here in this paper, we designed, optimized, and demonstrated procedures for separating bacteria (Pseudomonas aeruginosa) from unbound multiwall carbon nanotubes (MWCNTs) and MWCNT agglomerates using sucrose density gradient centrifugation. We demonstratemore » separation of protozoa (Tetrahymena thermophila) from MWCNTs, bacterial agglomerates, and protozoan fecal pellets by centrifugation in an iodixanol solution. The presence of MWCNTs in the density gradients after centrifugation was determined by quantification of 14C-labeled MWCNTs; the recovery of microbes from the density gradient media was confirmed by optical microscopy. Protozoan intracellular contents of MWCNTs and of bacteria were also unaffected by the designed separation process. Lastly, the optimized methods contribute to improved efficiency and accuracy in quantifying MWCNT association with bacteria and MWCNT accumulation in protozoan cells, thus supporting improved assessment of CNT bioaccumulation.« less

Separation of Bacteria, Protozoa and Carbon Nanotubes by Density Gradient Centrifugation

PubMed Central

Mortimer, Monika; Petersen, Elijah J.; Buchholz, Bruce A.; Holden, Patricia A.

2016-01-01

Sustainable production and use of carbon nanotube (CNT)-enabled materials require efficient assessment of CNT environmental hazards, including the potential for CNT bioaccumulation and biomagnification in environmental receptors. Microbes, as abundant organisms responsible for nutrient cycling in soil and water, are important ecological receptors for studying the effects of CNTs. Quantification of CNT association with microbial cells requires efficient separation of CNT-associated cells from individually dispersed CNTs and CNT agglomerates. Here, we designed, optimized, and demonstrated procedures for separating bacteria (Pseudomonas aeruginosa) from unbound multiwall carbon nanotubes (MWCNTs) and MWCNT agglomerates using sucrose density gradient centrifugation. We demonstrate separation of protozoa (Tetrahymena thermophila) from MWCNTs, bacterial agglomerates, and protozoan fecal pellets by centrifugation in an iodixanol solution. The presence of MWCNTs in the density gradients after centrifugation was determined by quantification of 14C-labeled MWCNTs; the recovery of microbes from the density gradient media was confirmed by optical microscopy. Protozoan intracellular contents of MWCNTs and of bacteria were also unaffected by the designed separation process. The optimized methods contribute to improved efficiency and accuracy in quantifying MWCNT association with bacteria and MWCNT accumulation in protozoan cells, thus supporting improved assessment of CNT bioaccumulation. PMID:27917301

Gating connexin 43 channels reconstituted in lipid vesicles by mitogen-activated protein kinase phosphorylation.

PubMed

Kim, D Y; Kam, Y; Koo, S K; Joe, C O

1999-02-26

The regulation of gap junctional permeability by phosphorylation was examined in a model system in which connexin 43 (Cx43) gap junction hemichannels were reconstituted in lipid vesicles. Cx43 was immunoaffinity-purified from rat brain, and Cx43 channels were reconstituted into unilamellar phospholipid liposomes. The activities of the reconstituted channels were measured by monitoring liposome permeability. Liposomes containing the Cx43 protein were fractionated on the basis of permeability to sucrose using sedimentation in an iso-osmolar density gradient. The gradient allowed separation of the sucrose-permeable and -impermeable liposomes. Liposomes that were permeable to sucrose were also permeable to the communicating dye molecule lucifer yellow. Permeability, and therefore activity of the reconstituted Cx43 channels, were directly dependent on the state of Cx43 phosphorylation. The permeability of liposomes containing Cx43 channels was increased by treatment of liposomes with calf intestinal phosphatase. Moreover, liposomes formed with Cx43 that had been dephosphorylated by calf intestinal phosphatase treatment showed increased permeability to sucrose. The role of phosphorylation in the gating mechanism of Cx43 channels was supported further by the observation that phosphorylation of Cx43 by mitogen-activated protein kinase reversibly reduced the permeability of liposomes containing dephosphorylated Cx43. Our results show a direct correlation between gap junctional permeability and the phosphorylation state of Cx43.

Differential compartmentation of sucrose and gentianose in the cytosol and vacuoles of storage root protoplasts from Gentiana Lutea L.

PubMed

Keller, F; Wiemken, A

1982-12-01

The storage roots of perennial Gentiana lutea L.plants contain several sugars. The predominant carbohydrate reserve is gentianose (β-D-glucopyranosyl-(1 → 6)-α-D-glucopyranosyl-(1 ↔ 2)-β-D-fructofuranoside). Vacuoles were isolated from root protoplasts and purified through a betaine density gradient. The yield was about 75%. Gentianose and gentiobiose were localized to 100% in the vacuoles, fructose and glucose to about 80%, and sucrose to only about 50%. Taking the volumes of the vacuolar and extravacuolar (cytosolic) compartments into account it is inferred that gentianose is located exclusively in the vacuoles, whilst sucrose is much more concentrated in the cytosol where it may play a role as a cryoprotectant. The concentration of fructose and glucose appeared to be similar on both sides of the tonoplast.

Respiratory Syncytial Virus Isolation by Combined Continuous Flow-Isopycnic Banding Centrifugation

PubMed Central

Cline, G. B.; Coates, Helen; Anderson, N. G.; Chanock, R. M.; Harris, W. W.

1967-01-01

A new zonal centrifuge rotor (B-IX) which combines continuous sample flow centrifugation with isopycnic banding has been used to isolate and concentrate respiratory syncytial virus from liter volumes of culture fluid. This isolation technique utilizes a sucrose density gradient to trap and isopycnically band the virus particles, and permits recovery of the particles from the rotor in an unaggregated condition. PMID:5621468

Purification of Giardia muris cysts by velocity sedimentation.

PubMed Central

Sauch, J F

1984-01-01

Giardia muris cysts were separated from fecal contaminants in primary isolates by unit gravity velocity sedimentation. Crude isolates obtained by centrifugation over 1.0 M sucrose were overlaid onto a Percoll density gradient, 1.01 to 1.03 g/ml. G. muris cysts were well separated from faster-sedimenting fecal debris and slower-sedimenting Spironucleus muris and bacteria in 1.5 h. PMID:6486790

Subcellular distribution of gluconeogenetic enzymes in germinating castor bean endosperm

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

Nishimura, M.; Beevers, H.

1979-07-01

The intracellular distribution of enzymes capable of catalyzing the reactions from oxaloacetate to sucrose in germinating castor bean endosperm has been studied by sucrose density gradient centrifugation. One set of glycolytic enzyme activities was detected in the plastids and another in the cytosol. The percentages of their activities in the plastids were less than 10% of total activities except for aldolase and fructose diphosphatase. The activities of several of the enzymes present in the plastids seem to be too low to account for the in vivo rate of gluconeogenesis whereas those in the cytosol are quite adequate. Furthermore, phosphoenolypyruvate carboxykinase,more » sucrose phosphate synthetase, and sucrose synthetase, which catalyze the first and final steps in the conversion of oxaloacetate to sucrose, were found only in the cytosol. It is deduced that in germinating castor bean endosperm the complete conversion of oxaloacetate to sucrose and CO/sub 2/ occurs in the cytosol. The plastids contain some enzymes of the pentose phosphate pathway, pyruvate dehydrogenase and fatty acid synthetase in addition to the set of glycolytic enzymes. This suggests that the role of the plastid in the endosperm of germinating castor bean is the production of fatty acids from sugar phosphates, as it is known to be in the endosperm during seed development.« less

Phloem-localized, proton-coupled sucrose carrier ZmSUT1 mediates sucrose efflux under the control of the sucrose gradient and the proton motive force.

PubMed

Carpaneto, Armando; Geiger, Dietmar; Bamberg, Ernst; Sauer, Norbert; Fromm, Jörg; Hedrich, Rainer

2005-06-03

The phloem network is as essential for plants as the vascular system is for humans. This network, assembled by nucleus- and vacuole-free interconnected living cells, represents a long distance transport pathway for nutrients and information. According to the Münch hypothesis, osmolytes such as sucrose generate the hydrostatic pressure that drives nutrient and water flow between the source and the sink phloem (Münch, E. (1930) Die Stoffbewegungen in der Pflanze, Gustav Fischer, Jena, Germany). Although proton-coupled sucrose carriers have been localized to the sieve tube and the companion cell plasma membrane of both source and sink tissues, knowledge of the molecular representatives and the mechanism of the sucrose phloem efflux is still scant. We expressed ZmSUT1, a maize sucrose/proton symporter, in Xenopus oocytes and studied the transport characteristics of the carrier by electrophysiological methods. Using the patch clamp techniques in the giant inside-out patch mode, we altered the chemical and electrochemical gradient across the sucrose carrier and analyzed the currents generated by the proton flux. Thereby we could show that ZmSUT1 is capable of mediating both the sucrose uptake into the phloem in mature leaves (source) as well as the desorption of sugar from the phloem vessels into heterotrophic tissues (sink). As predicted from a perfect molecular machine, the ZmSUT1-mediated sucrose-coupled proton current was reversible and depended on the direction of the sucrose and pH gradient as well as the membrane potential across the transporter.

Lipase activities in castor bean endosperm during germinaion. [Ricinus communis; glyoxysomes

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

Muto, S.; Beevers, H.

1974-01-01

Two lipases were found in extracts from castor bean (Ricinus communis L.) endosperm. One, with optimal activity at pH 5.0 (acid lipase), was present in dry seeds and displayed high activity during the first 2 days of germination. The second, with an alkaline pH optimum (alkaline lipase), was particularly active during days 3 to 5. When total homogenates of endosperm were fractionated into fat layer, supernatant, and particulate fractions, the acid lipase was recovered in the fat layer, and the alkaline lipase was located primarily in the particulate fraction. Sucrose density gradient centrifugation showed that the alkaline lipase was locatedmore » mainly in glyoxysomes, with some 30 percent of the activity in the endoplasmic reticulum. When glyoxysomes were broken by osmotic shock and exposed to KCl, which solubilizes most of the enzymes, the alkaline lipase remained particulate and was recovered with the glyoxysomal ''ghosts'' at equilibrium density 1.21 g/cm/sup 3/ on the sucrose gradient. Association of the lipase with the glyoxysomal membrane was supported by the responses to detergents and to butanol. The alkaline lipase hydrolyzed only monosubstituted glycerols. The roles of the two lipases in lipid utilization during germination of castor bean are discussed.« less

Reverse Transcriptase-Containing Particles Induced in Rous Sarcoma Virus-Transformed Rat Cells by Arginine Deprivation

PubMed Central

Kotler, Moshe; Weinberg, Eynat; Haspel, Osnat; Becker, Yechiel

1972-01-01

Incubation of rat cells transformed by Rous sarcoma virus (RSV) in an arginine-deficient medium resulted in accumulation of particles in the culture medium. Such particles did not appear when the transformed rat cells were incubated in a complete medium nor in the medium of primary rat cells which were incubated either in arginine-deficient or complete media. The particles which were released from the arginine-deprived transformed rat cells resemble C-type particles in their properties. These particles band in sucrose gradients at a density of 1.16 g/ml and contain 35S ribonucleic acid (RNA) molecules and a reverse transcriptase activity. Analysis of the cytoplasm of transformed and primary rat cells, deprived and undeprived of arginine, revealed the presence of reverse transcriptase-containing particles which banded in sucrose gradients at a density of 1.14 g/ml. These particles differed from the particles released into the medium by the arginine-deprived RSV-transformed rat cells. The deoxyribonucleic acid (DNA) molecules synthesized in vitro by the reverse transcriptase present in the particles isolated from the medium of arginine-deprived cells hybridized to RSV RNA, whereas the DNA synthesized by the cell-bound enzyme had no homology to RSV RNA. PMID:4116137

Different subcellular localization of neurotensin-receptor and neurotensin-acceptor sites in the rat brain dopaminergic system.

PubMed

Schotte, A; Rostène, W; Laduron, P M

1988-04-01

The subcellular localization of neurotensin-receptor sites (NT2 sites) and neurotensin-acceptor sites (NT1 sites) was studied in rat caudate-putamen by isopycnic centrifugation in sucrose density gradients. [3H]Neurotensin binding to NT2 sites occurred as a major peak at higher sucrose densities, colocalized with [3H]dopamine uptake, and as a small peak at a lower density; whereas binding to NT1 sites occurred as a single large peak at an intermediate density. 6-Hydroxydopamine lesions of the median forebrain bundle resulted in a total loss of NT2 sites in the caudate-putamen but did not affect NT2 sites in the nucleus accumbens and the olfactory tubercle. NT1 sites were not affected. Kainic acid injections into the rat caudate-putamen led to a partial decrease of NT1 sites in this region 5 days later. After a few weeks they returned to normal. Therefore NT2 sites are probably associated with presynaptic nigrostriatal dopaminergic terminals in the caudate-putamen but not in the nucleus accumbens and the olfactory tubercle. A possible association of NT1 sites with glial cells is suggested.

Intracellular Virus-Specific Structures and RNAs in Oncornavirus-Producing Human Cells

PubMed Central

Bukrinskaya, A. G.; Miller, G. G.; Lebedeva, E. N.; Zhdanov, V. M.

1974-01-01

Two kinds of virus-specific structures were isolated from the cytoplasm of Detroit-6 and human amnion cells producing oncornavirus-like particles. These structures represented A particles with the diameter of 70 to 80 nm and aggregated strands of nucleocapsids with the diameter of 3 and 6 nm. The structures were separated from cellular contaminants by isopycnic banding in linear sucrose gradients and subsequently further purified by sedimentation in velocity sucrose gradients. Their sedimentation coefficient was 250 and 150S, respectively. Both structures contain 60, 45, and 35S RNA species, and 150S structures also contained 20S RNA. The 35 and 20S RNA from the 150S structure formed hybrids with DNA enzymatically synthesized on extracellular virions. The structures displayed endogeneous polymerase activity, DNA product of the reaction being predominantly associated with 60S RNA. No 70S RNA was found in the cell structures of various densities. Also, the virions purified from tissue culture fluid contained 70S RNA. These findings are consistent with those on extracellular maturation of oncornavirus RNA. Images PMID:4810779

The Isolation and Partial Characterization of a Membrane Fraction Containing Phytochrome 12

PubMed Central

Marmé, Dieter; Mackenzie, John M.; Boisard, Jean; Briggs, Winslow R.

1974-01-01

If 4-day-old dark-grown zucchini squash seedlings (Cucurbita pepo L. cv. Black Beauty) are exposed briefly to red light, subsequent cell fractionation yields about 40% of the total extractable phytochrome in the far red-absorbing form bound to a particulate fraction. The amount of far red-absorbing phytochrome in the pellet is strongly dependent on the Mg concentration in the extraction medium. The apparent density of the Pfr-containing particles following sedimentation on sucrose gradients corresponds to 15% (w/w) sucrose with 0.1 mm Mg and 40% sucrose with 10 mm Mg. This particulate fraction could be readily separated from mitochondria and other particulate material by taking advantage of these apparent density changes with changes in Mg concentration. Electron microscopy of negatively stained preparations shows that with 1 mm Mg only minute particles are present. These were too small to reveal structural detail with this technique. With 3 mm Mg, separate membranous vesicles between 400 and 600 Ångstroms in diameter appear. At higher Mg concentrations, the vesicles aggregate, causing obvious turbity. The effect of Mg on vesicle formation and aggregation is completely reversible. Above 10 mm Mg, vesicle aggregation persists, but the percentage of bound Pfr decreases. Images PMID:16658871

Western blot (immunoblot) assay of small, round-structured virus associated with an acute gastroenteritis outbreak in Tokyo.

PubMed

Hayashi, Y; Ando, T; Utagawa, E; Sekine, S; Okada, S; Yabuuchi, K; Miki, T; Ohashi, M

1989-08-01

Small, round-structured virus (SRSV) was detected in a stool specimen of a patient during an acute gastroenteritis outbreak in Tokyo and was tentatively named SRSV-9. SRSV-9 was purified by sucrose velocity gradient centrifugation after CsCl density gradient centrifugation. The buoyant density of SRSV-9 appeared to be 1.36 g/ml in CsCl. A Western blot (immunoblot) assay using the biotin-avidin system revealed that SRSV-9 was antigenically related to the Hawaii agent but distinct from the Norwalk agent and contained a single major structural protein with a molecular size of 63.0 +/- 0.6 kilodaltons. The prevalence of SRSV-9 infection in Tokyo was surveyed by the Western blot antibody assay by using a crude virus preparation as the antigen. Seroconversion was observed in 56.5% of the patients involved in the outbreaks from which SRSV was detected by electron microscopy.

In vitro degradation of ribosomes.

PubMed

Mora, G; Rivas, A

1976-12-01

The cytoplasmic ribosomes from Euglena gracilis var. bacillaris are found to be of two types taking into consideration their stability "in vitro". In the group of unstable ribosomes the large subunit is degraded. The other group apparently does not suffer any degradation under the conditions described. However the RNAs extracted from both types of ribosomes are degraded during sucrose density gradients. The degradation of the largest RNA species has been reported previously, but no comment has been made about the stability of the ribosome itself.

Isolation and characterization of a mucosal triacylglycerol pool undergoing hydrolysis

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

Tipton AD IV; Frase, S.; Mansbach, C.M. II

1989-12-01

Absorbed and processed mucosal neutral lipid has been shown to be composed of at least two pools of triacylglycerol. One is likely to subserve chylomicron formation, and the other appears to be transported from the intestine via a nonlymphatic route. In the present study, 50 +/- 5% of the mucosal lipid pellets was centrifuged at 75,000 g.min (low-speed pellet (LSP)). Discontinuous sucrose density gradient centrifugation of LSP showed that 61 +/- 7% of the lipid banded at the 0.25-0.86 M sucrose interface. Neutral lipid analysis showed that this subfraction was only 58% triacylglycerol, suggesting it was undergoing hydrolysis. Active lipolyticmore » activity in vitro was found on incubation. The lipase had an alkaline pH optimum (pH 8.5) and persisted despite pancreatic ductular diversion. Lipolysis in vivo in a LSP fraction was shown by infusing (14C)glyceryltrioleate for 3.5 h followed by (3H)glyceryltrioleate for 30 min. Discontinuous sucrose density centrifugation of the LSP followed by an analysis of the lipids at the 0.25-0.86 M sucrose interface showed that 14C-neutral lipids were only 70 +/- 6% triacylglycerol, whereas 3H-neutral lipids were 88 +/- 2% triacylglycerol. 3H entered LSP slowly compared with the floating lipid in the same centrifuge tube. These studies suggest both in vivo and in vitro mucosal lipolysis by a specific, alkaline-active lipase. The turnover rate of LSP is likely to be slow by comparison with neutral lipid floating to the top of the centrifuge tube.« less

Selective production of sealed plasma membrane vesicles from red beet (Beta vulgaris L.) storage tissue.

PubMed

Giannini, J L; Gildensoph, L H; Briskin, D P

1987-05-01

Modification of our previous procedure for the isolation of microsomal membrane vesicles from red beet (Beta vulgaris L.) storage tissue allowed the recovery of sealed membrane vesicles displaying proton transport activity sensitive to both nitrate and orthovanadate. In the absence of a high salt concentration in the homogenization medium, contributions of nitrate-sensitive (tonoplast) and vanadate-sensitive (plasma membrane) proton transport were roughly equal. The addition of 0.25 M KCl to the homogenization medium increased the relative amount of nitrate-inhibited proton transport activity while the addition of 0.25 M KI resulted in proton pumping vesicles displaying inhibition by vanadate but stimulation by nitrate. These effects appeared to result from selective sealing of either plasma membrane or tonoplast membrane vesicles during homogenization in the presence of the two salts. Following centrifugation on linear sucrose gradients it was shown that the nitrate-sensitive, proton-transporting vesicles banded at low density and comigrated with nitrate-sensitive ATPase activity while the vanadate-sensitive, proton-transporting vesicles banded at a much higher density and comigrated with vanadate-sensitive ATPase. The properties of the vanadate-sensitive proton pumping vesicles were further characterized in microsomal membrane fractions produced by homogenization in the presence of 0.25 M KI and centrifugation on discontinuous sucrose density gradients. Proton transport was substrate specific for ATP, displayed a sharp pH optimum at 6.5, and was insensitive to azide but inhibited by N'-N-dicyclohexylcarbodiimide, diethylstilbestrol, and fluoride. The Km of proton transport for Mg:ATP was 0.67 mM and the K0.5 for vanadate inhibition was at about 50 microM. These properties are identical to those displayed by the plasma membrane ATPase and confirm a plasma membrane origin for the vesicles.

In vitro growth of Curcuma longa L. in response to five mineral elements and plant density in fed-batch culture systems.

PubMed

El-Hawaz, Rabia F; Bridges, William C; Adelberg, Jeffrey W

2015-01-01

Plant density was varied with P, Ca, Mg, and KNO3 in a multifactor experiment to improve Curcuma longa L. micropropagation, biomass and microrhizome development in fed-batch liquid culture. The experiment had two paired D-optimal designs, testing sucrose fed-batch and nutrient sucrose fed-batch techniques. When sucrose became depleted, volume was restored to 5% m/v sucrose in 200 ml of modified liquid MS medium by adding sucrose solutions. Similarly, nutrient sucrose fed-batch was restored to set points with double concentration of treatments' macronutrient and MS micronutrient solutions, along with sucrose solutions. Changes in the amounts of water and sucrose supplementations were driven by the interaction of P and KNO3 concentrations. Increasing P from 1.25 to 6.25 mM increased both multiplication and biomass. The multiplication ratio was greatest in the nutrient sucrose fed-batch technique with the highest level of P, 6 buds/vessel, and the lowest level of Ca and KNO3. The highest density (18 buds/vessel) produced the highest fresh biomass at the highest concentrations of KNO3 and P with nutrient sucrose fed-batch, and moderate Ca and Mg concentrations. However, maximal rhizome dry biomass required highest P, sucrose fed-batch, and a moderate plant density. Different media formulations and fed-batch techniques were identified to maximize the propagation and storage organ responses. A single experimental design was used to optimize these dual purposes.

In Vitro Growth of Curcuma longa L. in Response to Five Mineral Elements and Plant Density in Fed-Batch Culture Systems

PubMed Central

El-Hawaz, Rabia F.; Bridges, William C.; Adelberg, Jeffrey W.

2015-01-01

Plant density was varied with P, Ca, Mg, and KNO3 in a multifactor experiment to improve Curcuma longa L. micropropagation, biomass and microrhizome development in fed-batch liquid culture. The experiment had two paired D-optimal designs, testing sucrose fed-batch and nutrient sucrose fed-batch techniques. When sucrose became depleted, volume was restored to 5% m/v sucrose in 200 ml of modified liquid MS medium by adding sucrose solutions. Similarly, nutrient sucrose fed-batch was restored to set points with double concentration of treatments’ macronutrient and MS micronutrient solutions, along with sucrose solutions. Changes in the amounts of water and sucrose supplementations were driven by the interaction of P and KNO3 concentrations. Increasing P from 1.25 to 6.25 mM increased both multiplication and biomass. The multiplication ratio was greatest in the nutrient sucrose fed-batch technique with the highest level of P, 6 buds/vessel, and the lowest level of Ca and KNO3. The highest density (18 buds/vessel) produced the highest fresh biomass at the highest concentrations of KNO3 and P with nutrient sucrose fed-batch, and moderate Ca and Mg concentrations. However, maximal rhizome dry biomass required highest P, sucrose fed-batch, and a moderate plant density. Different media formulations and fed-batch techniques were identified to maximize the propagation and storage organ responses. A single experimental design was used to optimize these dual purposes. PMID:25830292

Precursor binding to an 880-kDa Toc complex as an early step during active import of protein into chloroplasts.

PubMed

Chen, Kuan-Yu; Li, Hsou-min

2007-01-01

The import of protein into chloroplasts is mediated by translocon components located in the chloroplast outer (the Toc proteins) and inner (the Tic proteins) envelope membranes. To identify intermediate steps during active import, we used sucrose density gradient centrifugation and blue-native polyacrylamide gel electrophoresis (BN-PAGE) to identify complexes of translocon components associated with precursor proteins under active import conditions instead of arrested binding conditions. Importing precursor proteins in solubilized chloroplast membranes formed a two-peak distribution in the sucrose density gradient. The heavier peak was in a similar position as the previously reported Tic/Toc supercomplex and was too large to be analyzed by BN-PAGE. The BN-PAGE analyses of the lighter peak revealed that precursors accumulated in at least two complexes. The first complex migrated at a position close to the ferritin dimer (approximately 880 kDa) and contained only the Toc components. Kinetic analyses suggested that this Toc complex represented an earlier step in the import process than the Tic/Toc supercomplex. The second complex in the lighter peak migrated at the position of the ferritin trimer (approximately 1320 kDa). It contained, in addition to the Toc components, Tic110, Hsp93, and an hsp70 homolog, but not Tic40. Two different precursor proteins were shown to associate with the same complexes. Processed mature proteins first appeared in the membranes at the same fractions as the Tic/Toc supercomplex, suggesting that processing of transit peptides occurs while precursors are still associated with the supercomplex.

Precursor binding to an 880-kDa Toc complex as an early step during active import of protein into chloroplasts

PubMed Central

Chen, Kuan-Yu; Li, Hsou-min

2007-01-01

The import of protein into chloroplasts is mediated by translocon components located in the chloroplast outer (the Toc proteins) and inner (the Tic proteins) envelope membranes. To identify intermediate steps during active import, we used sucrose density gradient centrifugation and blue-native polyacrylamide gel electrophoresis (BN-PAGE) to identify complexes of translocon components associated with precursor proteins under active import conditions instead of arrested binding conditions. Importing precursor proteins in solubilized chloroplast membranes formed a two-peak distribution in the sucrose density gradient. The heavier peak was in a similar position as the previously reported Tic/Toc supercomplex and was too large to be analyzed by BN-PAGE. The BN-PAGE analyses of the lighter peak revealed that precursors accumulated in at least two complexes. The first complex migrated at a position close to the ferritin dimer (approximately 880 kDa) and contained only the Toc components. Kinetic analyses suggested that this Toc complex represented an earlier step in the import process than the Tic/Toc supercomplex. The second complex in the lighter peak migrated at the position of the ferritin trimer (approximately 1320 kDa). It contained, in addition to the Toc components, Tic110, Hsp93, and an hsp70 homolog, but not Tic40. Two different precursor proteins were shown to associate with the same complexes. Processed mature proteins first appeared in the membranes at the same fractions as the Tic/Toc supercomplex, suggesting that processing of transit peptides occurs while precursors are still associated with the supercomplex. PMID:17144891

Receptor-mediated endocytosis of asialoglycoproteins by rat liver hepatocytes: biochemical characterization of the endosomal compartments

PubMed Central

1985-01-01

The endocytic compartments of the asialoglycoprotein (ASGP) pathway in rat hepatocytes were studied using a combined morphological and biochemical approach in the isolated perfused liver. Use of electron microscopic tracers and a temperature-shift protocol to synchronize ligand entry confirmed the route of ASGP internalization observed in our previous in vivo studies (1) and established conditions under which we could label the contents of successive compartments in the pathway for subcellular fractionation studies. Three endosomal compartments were demonstrated in which ASGPs appear after they enter the cell via coated pits and vesicles but before they reach their site of degradation in lysosomes. These three compartments could be distinguished by their location within the hepatocyte, by their morphological appearance in situ, and by their density in sucrose gradients. The distributions of ASGP receptors, both accessible and latent (revealed by detergent permeabilization), were also examined and compared with that of ligand during subcellular fractionation. Most accessible ASGP receptors co-distributed with conventional plasma membrane markers. However, hepatocytes contain a substantial intracellular pool of latent ASGP binding sites that exceeds the number of cell surface receptors and whose presence is not dependent on ASGP exposure. The distribution of these latent ASGP receptors on sucrose gradients (detected either immunologically or by binding assays) was coincident with that of ligand sequestered within the early endosome compartments. In addition, both early endosomes and the membrane vesicles containing latent ASGP receptors had high cholesterol content, because both shifted markedly in density upon exposure to digitonin. PMID:2866191

A model for melanosome biogenesis based on the purification and analysis of early melanosomes

PubMed Central

Kushimoto, Tsuneto; Basrur, Venkatesha; Valencia, Julio; Matsunaga, Jun; Vieira, Wilfred D.; Ferrans, Victor J.; Muller, Jacqueline; Appella, Ettore; Hearing, Vincent J.

2001-01-01

Melanosome biogenesis and function were studied after purification of early stage melanosomes and characterization of specific proteins sorted to that organelle. Melanosomes were isolated from highly pigmented human MNT1 melanoma cells after disruption and initial separation by sucrose density gradient centrifugation. Low-density sucrose fractions were found by electron microscopy to be enriched in stage I and stage II melanosomes, and these fractions were further separated and purified by free flow electrophoresis. Tyrosinase and dopachrome tautomerase (DCT) activities were found exclusively in stage II melanosomes, even though DCT (and to some extent tyrosinase) proteins were sorted to stage I melanosomes. Western immunoblotting revealed that these catalytic proteins, as well as TYRP1, MART1, and GP100, were cleaved and inactivated in stage I melanosomes. Proteolytic cleavage was critical for the refolding of GP100 within the melanosomal milieu, and subsequent reorganization of amorphous stage I melanosomes into fibrillar, ovoid, and highly organized stage II melanosomes appears to stabilize the catalytic functions of melanosomal enzymes and allows melanin biosynthesis to begin. These results provide a better understanding of the structural features seen during melanosome biogenesis, and they yield further clues as to the physiological regulation of pigmentation. PMID:11526213

Application of diffusion ordered-1H-nuclear magnetic resonance spectroscopy to quantify sucrose in beverages.

PubMed

Cao, Ruge; Nonaka, Airi; Komura, Fusae; Matsui, Toshiro

2015-03-15

This work focuses on a quantitative analysis of sucrose using diffusion ordered-quantitative (1)H-nuclear magnetic resonance spectroscopy (DOSY-qNMR), where an analyte can be isolated from interference based on its characteristic diffusion coefficient (D) in gradient magnetic fields. The D value of sucrose in deuterium oxide at 30°C was 4.9 × 10(-10)m(2)/s at field gradient pulse from 5.0 × 10(-2) to 3.0 × 10(-1)T/m, separated from other carbohydrates (glucose and fructose). Good linearity (r(2)=0.9999) was obtained between sucrose (0.5-20.0 g/L) and the resonance area of target glucopyranosyl-α-C1 proton normalised to that of cellobiose C1 proton (100.0 g/L, as an internal standard) in 1D sliced DOSY spectrum. The DOSY-qNMR method was successfully applied to quantify sucrose in orange juice (36.1 ± 0.5 g/L), pineapple juice (53.5 ± 1.1g/L) and a sports drink (24.7 ± 0.6g/L), in good agreement with the results obtained by an F-kit method. Copyright © 2014 Elsevier Ltd. All rights reserved.

Morphologic and proteomic characterization of exosomes released by cultured extravillous trophoblast cells

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

Atay, Safinur; Gercel-Taylor, Cicek; Kesimer, Mehmet

Exosomes represent an important intercellular communication vehicle, mediating events essential for the decidual microenvironment. While we have demonstrated exosome induction of pro-inflammatory cytokines, to date, no extensive characterization of trophoblast-derived exosomes has been provided. Our objective was to provide a morphologic and proteomic characterization of these exosomes. Exosomes were isolated from the conditioned media of Swan71 human trophoblast cells by ultrafiltration and ultracentrifugation. These were analyzed for density (sucrose density gradient centrifugation), morphology (electron microscopy), size (dynamic light scattering) and protein composition (Ion Trap mass spectrometry and western immunoblotting). Based on density gradient centrifugation, microvesicles from Sw71 cells exhibit amore » density between 1.134 and 1.173 g/ml. Electron microscopy demonstrated that microvesicles from Sw71 cells exhibit the characteristic cup-shaped morphology of exosomes. Dynamic light scattering showed a bell-shaped curve, indicating a homogeneous population with a mean size of 165 nm {+-} 0.5 nm. Ion Trap mass spectrometry demonstrated the presence of exosome marker proteins (including CD81, Alix, cytoskeleton related proteins, and Rab family). The MS results were confirmed by western immunoblotting. Based on morphology, density, size and protein composition, we defined the release of exosomes from extravillous trophoblast cells and provide their first extensive characterization. This characterization is essential in furthering our understanding of 'normal' early pregnancy.« less

Interpretation of sucrose gradient sedimentation pattern of deoxyribonucleic acid fragments resulting from random breaks.

PubMed

Litwin, S; Shahn, E; Kozinski, A W

1969-07-01

Mass distribution in a sucrose gradient of deoxyribonucleic acid (DNA) fragments arising as a result of random breaks is predicted by analytical means from which computer evaluations are plotted. The analytical results are compared with the results of verifying experiments: (i) a Monte Carlo computer experiment in which simulated molecules of DNA were individuals of unit length subjected to random "breaks" applied by a random number generator, and (ii) an in vitro experiment in which molecules of T4 DNA, highly labeled with (32)P, were stored in liquid nitrogen for variable periods of time during which a precisely known number of (32)P atoms decayed, causing single-stranded breaks. The distribution of sizes of the resulting fragments was measured in an alkaline sucrose gradient. The profiles obtained in this fashion were compared with the mathematical predictions. Both experiments agree with the analytical approach and thus permit the use of the graphs obtained from the latter as a means of determining the average number of random breaks in DNA from distributions obtained experimentally in a sucrose gradient. An example of the application of this procedure to a previously unresolved problem is provided in the case of DNA from ultraviolet-irradiated phage which undergoes a dose-dependent intracellular breakdown. The relationship between the number of lethal hits and the number of single-stranded breaks was not previously established. A comparison of the calculated number of nicks per strand of DNA with the known dose in phage-lethal hits reveals a relationship closely approximating one lethal hit to one single-stranded break.

Characterization and role in morphogenesis of a new subviral particle (55S) isolated from poliovirus-infected cells.

PubMed Central

Corrias, M V; Flore, O; Broi, E; Marongiu, M E; Pani, A; Torelli, S; La Colla, P

1987-01-01

A previously undetected subviral particle, designated the 55S particle because of its position in sucrose density gradients, has been found in cytoplasmic extracts of poliovirus-infected cells. It contains no RNA, is composed of equimolar amounts of the structural polypeptides P1AB, P1C, and P1D, and is stable in vitro under a variety of conditions: presence or absence of EDTA, dilution in low- or high-ionic-strength buffers, suspension in buffers up to pH 10, incubation at 37 degrees C, and centrifugation to equilibrium in CsCl gradients (where it bands at a density of 1.285 g/cm3). Conventional pulse-chase experiments show that 55S particles are the products of the assembly of 14S subunits and the precursors of virions. These data led to the formulation of a model of poliovirus morphogenesis in which the conversion of capsomers into 73S empty capsids does not occur directly, but through the formation of an intermediate structure, the 55S particle. PMID:3027383

The Purification and Concentration of Hog Cholera Virus*

PubMed Central

Cunliffe, H. R.; Rebers, P. A.

1968-01-01

Partial purification of hog cholera virus (HCV) using a simple batch-type chromatographic procedure with magnetic ferric oxide (MFO) is described. Infectious HCV was adsorbed from isotonic solutions to MFO and was eluted under conditions of low ionic strength and high pH. Aqueous solutions of 0.01 M sodium cyanide or 0.0003 M ammonium hydroxide effectively dissociated MFO-HCV complexes. The data indicate that 50 to 100% of the original HCV infectivity was recovered concomitant with a 90 to 95% reduction of extraneous organic nitrogen. MFO-purified HCV was concentrated by density gradient type centrifugations in buffered solutions of cesium chloride and sucrose. Prolonged isodensity centrifugations of concentrated MFO-purified HCV indicated a buoyant density of 1.14 to 1.15 gm/ml for the strain of virus used. PMID:15846899

Surface-Active Agents for Isolation of the Core Component of Avian Myeloblastosis Virus 1

PubMed Central

Stromberg, Kurt

1972-01-01

Sixty-one surface-active agents were evaluated in a procedure designed to assess their ability to remove the envelope from the core component of avian myeloblastosis virus (AMV). The procedure consisted of centrifugation of intact AMV through a series of sucrose gradients each containing an upper layer of agent at one of eight concentrations between 0.01 and 10%. The effectiveness of an agent in producing AMV cores was indicated by (i) the appearance of light-scattering bands in the region of core buoyant density in gradient tubes; (ii) the range of surfactant concentration over which these bands appeared; and (iii) an electron microscopy assessment by the negative-staining technique of the relative proportion of core to non-core material in each of these bands. Six nonionic surfactants were selected by this screening method for comparison in regard to recovery of core protein and endogenous ribonucleic acid (RNA)-dependent deoxyribonucleic acid (DNA) polymerase activity, as well as further morphologic evaluation by electron microscopy. The nonionic surfactants of the polyoxyethylene alcohol class (particularly, Sterox SL) were most effective. Nonionic surfactants of the polyoxyethylene alkylphenol class (particularly, Nonidet P-40) were also effective. Sterox SL and Nonidet P-40 each gave a more than fivefold increase in specific activity of endogenous RNA-dependent DNA polymerase, and each gave a low recovery of core protein. Sterox SL did not interfere to the extent that Nonidet P-40 did in procedures which involved spectrophotometric assay at 260 nm. The use of Sterox SL resulted in the least envelope contamination of core preparations by electron microscopy examination, the most recovery of protein and endogenous RNA-dependent DNA polymerase activity, and a core buoyant density in sucrose of 1.27 g/ml. Images PMID:4112071

Accumulation of Amyloid β-Protein in the Low-Density Membrane Domain Accurately Reflects the Extent of β-Amyloid Deposition in the Brain

PubMed Central

Oshima, Noriko; Morishima-Kawashima, Maho; Yamaguchi, Haruyasu; Yoshimura, Masahiro; Sugihara, Shiro; Khan, Karen; Games, Dora; Schenk, Dale; Ihara, Yasuo

2001-01-01

To learn more about the process of amyloid β-protein (Aβ) deposition in the brain, human prefrontal cortices were fractionated by sucrose density gradient centrifugation, and the Aβ content in each fraction was quantified by a two-site enzyme-linked immunosorbent assay. The fractionation protocol revealed two pools of insoluble Aβ. One corresponded to a low-density membrane domain; the other was primarily composed of extracellular Aβ deposits in those cases in which Aβ accumulated to significant levels. Aβ42 levels in the low-density membrane domain were proportional to the extent of total Aβ42 accumulation, which is known to correlate well with overall amyloid burden. In PDAPP mice that form senile plaques and accumulate Aβ in a similar manner to aging humans, Aβ42 accumulation in the low-density membrane domain also increased as Aβ deposition progressed with aging. These observations indicate that the Aβ42 associated with low-density membrane domains is tightly coupled with the process of extracellular Aβ deposition. PMID:11395399

Glucokinase is an integral component of the insulin granules in glucose-responsive insulin secretory cells and does not translocate during glucose stimulation.

PubMed

Arden, Catherine; Harbottle, Andrew; Baltrusch, Simone; Tiedge, Markus; Agius, Loranne

2004-09-01

The association of glucokinase with insulin secretory granules has been shown by cell microscopy techniques. We used MIN6 insulin-secretory cells and organelle fractionation to determine the effects of glucose on the subcellular distribution of glucokinase. After permeabilization with digitonin, 50% of total glucokinase remained bound intracellularly, while 30% was associated with the 13,000g particulate fraction. After density gradient fractionation of the organelles, immunoreactive glucokinase was distributed approximately equally between dense insulin granules and low-density organelles that cofractionate with mitochondria. Although MIN6 cells show glucose-responsive insulin secretion, glucokinase association with the granules and low-density organelles was not affected by glucose. Subfractionation of the insulin granule components by hypotonic lysis followed by sucrose gradient centrifugation showed that glucokinase colocalized with the granule membrane marker phogrin and not with insulin. PFK2 (6-phosphofructo-2-kinase-2/fructose-2,6-bisphosphatase)/FDPase-2, a glucokinase-binding protein, and glyceraldehyde phosphate dehydrogenase, which has been implicated in granule fusion, also colocalized with glucokinase after hypotonic lysis or detergent extaction of the granules. The results suggest that glucokinase is an integral component of the granule and does not translocate during glucose stimulation.

Characterization of dynamics in complex lyophilized formulations: II. Analysis of density variations in terms of glass dynamics and comparisons with global mobility, fast dynamics, and Positron Annihilation Lifetime Spectroscopy (PALS)

PubMed Central

Chieng, Norman; Cicerone, Marcus T.; Zhong, Qin; Liu, Ming; Pikal, Michael J.

2013-01-01

Amorphous HES/disaccharide (trehalose or sucrose) formulations, with and without added polyols (glycerol and sorbitol) and disaccharide formulations of human growth hormone (hGH), were prepared by freeze drying and characterized with particular interest in methodology for using high precision density measurements to evaluate free volume changes and a focus on comparisons between “free volume” changes obtained from analysis of density data, fast dynamics (local mobility), and PALS characterization of “free volume” hole size. Density measurements were performed using a helium gas pycnometer, and fast dynamics was characterized using incoherent neutron scattering spectrometer. Addition of sucrose and trehalose to hGH decreases free volume in the system with sucrose marginally more effective than trehalose, consistent with superior pharmaceutical stability of sucrose hGH formulations well below Tg relative to trehalose. We find that density data may be analyzed in terms of free volume changes by evaluation of volume changes on mixing and calculation of apparent specific volumes from the densities. Addition of sucrose to HES decreases free volume, but the effect of trehalose is not detectable above experimental error. Addition of sorbitol or glycerol to HES/trehalose base formulations appears to significantly decrease free volume, consistent with the positive impact of such additions on pharmaceutical stability (i.e., degradation) in the glassy state. Free volume changes, evaluated from density data, fast dynamics amplitude of local motion, and PALS hole size data generally are in qualitative agreement for the HES/disaccharide systems studied. All predict decreasing molecular mobility as disaccharides are added to HES. Global mobility as measured by enthalpy relaxation times, increases as disaccharides, particularly sucrose, are added to HES. PMID:23623797

( sup 14 C)-Sucrose uptake by guard cell protoplasts of pisum sativum, argenteum mutant

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

Rohrig, K.; Raschke, K.

1991-05-01

Guard cells rely on import for their supply with reduced carbon. The authors tested by silicone oil centrifugation the ability of guard cell protoplasts to accumulated ({sup 14}C)-sucrose. Uptake rates were corrected after measurement of {sup 14}C-sorbitol and {sup 3}H{sub 2}O spaces. Sucrose uptake followed biphasic kinetics, with a high-affinity component below 1 mM external sucrose (apparent K{sub m} 0.8 mM at 25C) and a low-affinity nonsaturable component above. Uptake depended on pH (optimum at pH 5.0). Variations in the concentrations of external KCl, CCCP, and valinomycin indicated that about one-half of the sucrose uptake rate could be related tomore » an electrochemical gradient across the plasmalemma. Total uptake rates measured at 5 mM external sucrose seem to be sufficient to replenish emptied plastids with starch within a few hours.« less

Involvement of the Golgi apparatus in the synthesis and secretion of hydroxyproline-rich cell wall glycoproteins. [carrot

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

Gardiner, M.; Chrispeels, M.J.

1975-01-01

Pulse labeling of carrot root phloem parenchyma (Daucus carota L. ev. Nantes) tissue with /sup 14/C-proline followed by fractionation of the cytoplasmic organelles on sucrose gradients was used to determine the identiy of the membranous organelles involved in the secretion of the hydroxyproline-rich glycoproteins of the cell wall. Identification of the organelles was done through electron-microscopical observations and through the localization of marker enzymes on the sucrose gradients. Enrichment of the organelles involved in secretion was determined by measuring the percentage of the incorporated radioactivity present as /sup 14/C-hydroxyproline. The Golgi apparatus (dictyosome) was found to be a major sitemore » of glycoprotein transport. This identification was based on the observed enrichment of dictyosomes paralleling the purification of newly synthesized cell-wall glycoproteins. A marker enzyme for the Golgi apparatus, inosinediphosphatase, banded with the newly synthesized cell wall glycoproteins on sequential isopycnic and rate zonal sucrose gradients. Marker enzymes for the endoplasmic reticulum and the plasma memebrane were clearly separated from the dictyosome-rich fraction. UDP-arabinose arabinosyl transferase, an enzyme involved in the glycosylation of the peptide moiety of this glycoprotein, also banded with the dictyosomes on both kinds of gradients. The results suggest an important role of the Golgi apparatus in the biosynthesis and the secretion of the cell wall glycoproteins of higher plants. (auth)« less

Development anmd testing of electrophoresis solutions. Task I.1: Development of optimal buffer system

NASA Technical Reports Server (NTRS)

1985-01-01

Two buffers were explored for testing: low ionic strength electrophoresis buffer with and without density gradient material. It was found that the electrophoresis routine was better tolerated when Ficoll was present. The results of a viability study of primary human fetal kidney (HFK-1) cells at the first passage are shown. Cell strain HFK-1 was used in several experiments at the first and second passage. The HFK consisted mainly of fibroblasts, and HFK-1 has a high epithelioid cell content. The chromosomes of HFK were examined and found to be euploid. The stock medium for cell electrophoresis is described. In this solution density gradient solutes such as sucrose and Ficoll are dissolved to bring the osmolarity to 0.30. Its ionic strength is less than 0.01M, and its conductivity is usually 0.0011 mho/cm. Methods for viability determination included direct microscopic counting of the percent cells attached and spread within 24 hr of plating test cultures or electrophoretically separated fractions. The Cytograf viability assay concept was tested, and shown that blue stained cells scatter less light into the 0.8 to 3.3 deg angular interval than do unstained cells.

Effects of vial packing density on drying rate during freeze-drying of carbohydrates or a model protein measured using a vial-weighing technique.

PubMed

Gieseler, Henning; Lee, Geoffrey

2008-02-01

To determine the effects of vial packing density in a laboratory freeze dryer on drying rate profiles of crystalline and amorphous formulations. The Christ freeze-drying balance measured cumulative water loss, m(t), and instantaneous drying rate, m(t), of water, mannitol, sucrose and sucrose/BSA formulations in commercial vials. Crystalline mannitol shows drying rate behaviour indicative of a largely homogeneous dried-product layer. The drying rate behaviour of amorphous sucrose indicates structural heterogeneity, postulated to come from shrinkage or microcollapse. Trehalose dries more slowly than sucrose. Addition of BSA to either disaccharide decreases primary drying time. Higher vial packing density greatly reduces drying rate because of effects of radiation heat transfer from chamber walls to test vial. Plots of m(t) versus radical t and m(t) versus layer thickness (either ice or dried-product) allow interpretation of changes in internal cake morphology during drying. Vial packing density greatly influences these profiles.

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

Lukeman, S.; Fanestil, D.

Although the PBS has been identified in many organs, its function and cellular location are speculative. Using rapid filtration, binding of (/sup 3/H)RO 5-4864 (*RO) (.75 nM) was assessed in four subcellular fractions (.3 mg/ml) derived from depapillated rat kidney by differential centrifugation: N (450g x 2 min), O (13,000 x 10), P (105,000 x 30), and S. The binding distribution was: N-18%, O-74%, P-6%, and S-2%. Marker enzyme analysis revealed that O was enriched in mitochondria (M), lysosomes (L), peroxisomes (P), and endoplasmic reticulum (ER), but not plasma membrane, and that N contained small amounts (10-15%) of markers formore » the above. Repeated washing of O removed ER enzymes but preserved *RO binding. O was further fractionated with centrifugation (57,000g x 4 hr) on a linear sucrose gradient (18-65%); *RO binding then comigrated with M but not P and L markers. Centrifugation of isolated M (5500 x 10 min) on another linear sucrose gradient (37-65%) gave low and high density bands, which contained 65% and 35% of *RO binding activity, resp. *RO binding in O was specific, saturable, reversible, and inhibited by diuretics. Inhibitors with the highest potency were indacrinone (K/sub d/ = 35 ..mu..M), hydrochlorothiazide (100 ..mu..M), and ethacrynic acid (325 ..mu..M). Low potency inhibitors (K/sub d/ greater than or equal to 1 mM) included amiloride, triamterene, furosemide, bumetanide, and ozolinone.« less

Downregulation of surface sodium pumps by endocytosis during meiotic maturation of Xenopus laevis oocytes

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

Schmalzing, G.; Eckard, P.; Kroener, S.P.

1990-01-01

During meiotic maturation, plasma membranes of Xenopus laevis oocytes completely lose the capacity to transport Na and K and to bind ouabain. To explore whether the downregulation might be due to an internalization of the sodium pump molecules, the intracellular binding of ouabain was determined. Selective permeabilization of the plasma membrane of mature oocytes (eggs) by digitonin almost failed to disclose ouabain binding sites. However, when the eggs were additionally treated with 0.02% sodium dodecyl sulfate (SDS) to permeabilize inner membranes, all sodium pumps present before maturation were recovered. Phosphorylation by (gamma-32P)ATP combined with SDS-polyacrylamide gel electrophoresis (PAGE) and autoradiographymore » showed that sodium pumps were greatly reduced in isolated plasma membranes of eggs. According to sucrose gradient fractionation, maturation induced a shift of sodium pumps from the plasma membrane fraction to membranes of lower buoyant density with a protein composition different from that of the plasma membrane. Endocytosed sodium pumps identified on the sucrose gradient from (3H)ouabain bound to the cell surface before maturation could be phosphorylated with inorganic (32P)phosphate. The findings suggest that downregulation of sodium pumps during maturation is brought about by translocation of surface sodium pumps to an intracellular compartment, presumably endosomes. This contrasts the mechanism of downregulation of Na-dependent cotransport systems, the activities of which are reduced as a consequence of a maturation-induced depolarization of the membrane without a removal of the corresponding transporter from the plasma membrane.« less

Detergent-resistant membrane subfractions containing proteins of plasma membrane, mitochondrial, and internal membrane origins.

PubMed

Mellgren, Ronald L

2008-04-24

HEK293 cell detergent-resistant membranes (DRMs) isolated by the standard homogenization protocol employing a Teflon pestle homogenizer yielded a prominent opaque band at approximately 16% sucrose upon density gradient ultracentrifugation. In contrast, cell disruption using a ground glass tissue homogenizer generated three distinct DRM populations migrating at approximately 10%, 14%, and 20% sucrose, named DRM subfractions A, B, and C, respectively. Separation of the DRM subfractions by mechanical disruption suggested that they are physically associated within the cellular environment, but can be dissociated by shear forces generated during vigorous homogenization. All three DRM subfractions possessed cholesterol and ganglioside GM1, but differed in protein composition. Subfraction A was enriched in flotillin-1 and contained little caveolin-1. In contrast, subfractions B and C were enriched in caveolin-1. Subfraction C contained several mitochondrial membrane proteins, including mitofilin and porins. Only subfraction B appeared to contain significant amounts of plasma membrane-associated proteins, as revealed by cell surface labeling studies. A similar distribution of DRM subfractions, as assessed by separation of flotillin-1 and caveolin-1 immunoreactivities, was observed in CHO cells, in 3T3-L1 adipocytes, and in HEK293 cells lysed in detergent-free carbonate. Teflon pestle homogenization of HEK293 cells in the presence of the actin-disrupting agent latrunculin B generated DRM subfractions A-C. The microtubule-disrupting agent vinblastine did not facilitate DRM subfraction separation, and DRMs prepared from fibroblasts of vimentin-null mice were present as a single major band on sucrose gradients, unless pre-treated with latrunculin B. These results suggest that the DRM subfractions are interconnected by the actin cytoskeleton, and not by microtubes or vimentin intermediate filaments. The subfractions described may prove useful in studying discrete protein populations associated with detergent-resistant membranes, and their potential interactions in cell signaling.

A dual mechanism of cellulose deficiency in shv3svl1

PubMed Central

Yeats, Trevor H.; Somerville, Chris R.

2016-01-01

ABSTRACT SHAVEN3 (SHV3) and its homolog SHAVEN3-like 1 (SVL1) encode glycosylphosphatidylinositol (GPI)-anchored proteins (GAPs) that are involved in cellulose biosynthesis and hypocotyl elongation in Arabidopsis thaliana. In a recent report, we showed that the cellulose and hypocotyl elongation defects of the shv3svl1 double mutant are greatly enhanced by exogenous sucrose in the growth medium. Further investigation of this phenomenon showed that shv3svl1 exhibits a hyperpolarized plasma membrane (PM) proton gradient that is coupled with enhanced accumulation of sucrose via the PM sucrose/proton symporter SUC1. The resulting high intracellular sucrose concentration appears to favor starch synthesis at the expense of cellulose synthesis. Here, we describe our interpretation of these results in terms of 2 potential regulators of cellulose synthesis: intracellular sucrose concentration and a putative signaling pathway that involves SHV3-like proteins. PMID:27494413

Apoplastic and symplastic phloem loading in Quercus robur and Fraxinus excelsior

PubMed Central

Lohaus, Gertrud

2014-01-01

Whereas most of the research on phloem loading is performed on herbaceous plants, less is known about phloem loading strategies in trees. In this study, the phloem loading mechanisms of Quercus robur and Fraxinus excelsior were analysed. The following features were examined: the minor vein structure, the sugar concentrations in phloem sap by the laser–aphid–stylet technique, the distribution of photoassimilates in the mesophyll cells by non-aqueous fractionation, gradients of sugar concentrations and osmotic pressure, and the expression of sucrose transporters. The minor vein configurations of Q. robur and F. excelsior belong to the open type. Quercus robur contained companion cells in the minor veins whereas F. excelsior showed intermediary cells in addition to ordinary companion cells. The main carbon transport form in Q. robur was sucrose (~1M). In F. excelsior high amounts of raffinose and stachyose were also transported. However, in both tree species, the osmolality of phloem sap was higher than the osmolality of the mesophyll cells. The concentration gradients between phloem sap and the cytoplasm of mesophyll cells for sucrose were 16-fold and 14-fold for Q. robur and F. excelsior, respectively. Independent of the type of translocated sugars, sucrose transporter cDNAs were cloned from both species. The results indicate that phloem loading of sucrose and other metabolites must involve active loading steps in both tree species. Quercus robur seems to be an apoplastic phloem loader while F. excelsior shows indications of being a symplastic or mixed symplastic–apoplastic phloem loader. PMID:24591056

Proteomic Analysis of Detergent-resistant Membrane Microdomains in Trophozoite Blood Stage of the Human Malaria Parasite Plasmodium falciparum*

PubMed Central

Yam, Xue Yan; Birago, Cecilia; Fratini, Federica; Di Girolamo, Francesco; Raggi, Carla; Sargiacomo, Massimo; Bachi, Angela; Berry, Laurence; Fall, Gamou; Currà, Chiara; Pizzi, Elisabetta; Breton, Catherine Braun; Ponzi, Marta

2013-01-01

Intracellular pathogens contribute to a significant proportion of infectious diseases worldwide. The successful strategy of evading the immune system by hiding inside host cells is common to all the microorganism classes, which exploit membrane microdomains, enriched in cholesterol and sphingolipids, to invade and colonize the host cell. These assemblies, with distinct biochemical properties, can be isolated by means of flotation in sucrose density gradient centrifugation because they are insoluble in nonionic detergents at low temperature. We analyzed the protein and lipid contents of detergent-resistant membranes from erythrocytes infected by Plasmodium falciparum, the most deadly human malaria parasite. Proteins associated with membrane microdomains of trophic parasite blood stages (trophozoites) include an abundance of chaperones, molecules involved in vesicular trafficking, and enzymes implicated in host hemoglobin degradation. About 60% of the identified proteins contain a predicted localization signal suggesting a role of membrane microdomains in protein sorting/trafficking. To validate our proteomic data, we raised antibodies against six Plasmodium proteins not characterized previously. All the selected candidates were recovered in floating low-density fractions after density gradient centrifugation. The analyzed proteins localized either to internal organelles, such as the mitochondrion and the endoplasmic reticulum, or to exported membrane structures, the parasitophorous vacuole membrane and Maurer's clefts, implicated in targeting parasite proteins to the host erythrocyte cytosol or surface. The relative abundance of cholesterol and phospholipid species varies in gradient fractions containing detergent-resistant membranes, suggesting heterogeneity in the lipid composition of the isolated microdomain population. This study is the first report showing the presence of cholesterol-rich microdomains with distinct properties and subcellular localization in trophic stages of Plasmodium falciparum. PMID:24045696

Influence of Long-Term Fertilization on Spore Density and Colonization of Arbuscular Mycorrhizal Fungi in a Brown Soil

NASA Astrophysics Data System (ADS)

Li, Dongdong; Luo, Peiyu; Yang, Jinfeng

2017-12-01

This study aims to explore changes of long-term fertilization on spore density and colonization of AMF (Arbuscular mycorrhizal fungi) under a 38-y long-term fertilization in a brown soil. Soil samples (0-20 cm,20-40cm,40-60cm)were taken from the six treatments of the long-term fertilization trial in October 2016:no fertilizer (CK), N1(mineral nitrogen fertilizer), N1P (mineral nitrogen and phosphate fertilizer), N1PK (mineral nitrogen, phosphate and potassic fertilizer), pig manure (M2), M2N1P (pig manure, mineral nitrogen andphosphate fertilizer).Spores were isolated from soils by wet sieving and sucrose density gradient centrifugation; mycorrhizal colonization levels were determined by the gridline intersect. The spore density was highest in the topsoils (0-20 cm), and was decreased with increasing of soil depth in each treatment. The spores density of M2N1P treatment was significantly higher than that of other treatments in each soil layer. Application of inorganic fertilizer (especially inorganic with organic fertilizer) can greatly improve the level of colonization. Our results suggested that long-term fertilization significantly affects spore density and colonization of AMF, however, spore density is not related to colonization rate.

von Willebrand's disease antigen II. A new plasma and platelet antigen deficient in severe von Willebrand's disease.

PubMed Central

Montgomery, R R; Zimmerman, T S

1978-01-01

Factor VIII-related antigen (VIIIag) is deficient in plasma and platelets of patients with severe von Willebrand's disease. This study reports a second von Willebrand's disease antigen (vWagII), distinct from VIIIag, that is also deficient in the platelets and plasma of patients with severe von Willebrand's disease. VIIIag and vWagII are separable by molecular exclusion chromatography, sucrose density gradient ultracentrifugation, and crossed immunoelectrophoresis. They show reactions of immunologic nonidentity with each other, and thus, do not share a precursor-product relationship. vWagII is released from normal platelets during blood clotting, accounting for a fourfold higher concentration of vWagII in serum over plasma. Images PMID:307007

Expression and self-assembly of cowpea chlorotic mottle virus-like particles in Pseudomonas fluorescens.

PubMed

Phelps, Jamie P; Dao, Philip; Jin, Hongfan; Rasochova, Lada

2007-02-01

Coat protein of the cowpea chlorotic mottle virus (CCMV), a plant bromovirus, has been expressed in a soluble form in a prokaryote, Pseudomonas fluorescens, and assembled into virus-like particles (VLPs) in vivo that were structurally similar to the native CCMV particles derived from plants. The CCMV VLPs were purified by PEG precipitation followed by separation on a sucrose density gradient and analyzed by size exclusion chromatography, UV spectrometry, and transmission electron microscopy. DNA microarray experiments revealed that the VLPs encapsulated very large numbers of different host RNAs in a non-specific manner. The development of a P. fluorescens expression system now enables production of CCMV VLPs by bacterial fermentation for use in pharmaceutical or nanotechnology applications.

Isolation of Chromoplasts and Suborganellar Compartments from Tomato and Bell Pepper Fruit.

PubMed

Barsan, Cristina; Kuntz, Marcel; Pech, Jean-Claude

2017-01-01

Tomato is a model for fruit development and ripening. The isolation of intact plastids from this organism is therefore important for metabolic and proteomic analyses. Pepper, a species from the same family, is also of interest since it allows isolation of intact chromoplasts in large amounts. Here, we provide a detailed protocol for the isolation of tomato plastids at three fruit developmental stages, namely, nascent chromoplasts from the mature green stage, chromoplasts from an intermediate stage, and fully differentiated red chromoplasts. The method relies on sucrose density gradient centrifugations. It yields high purity organelles suitable for proteome analyses. Enzymatic and microscopy assays are summarized to assess purity and intactness. A method is also described for subfractionation of pepper chromoplast lipoprotein structures.

Effect of fluorodeoxyuridine on the sedimentation of nucleoids from HeLa cells in sucrose gradients.

PubMed

Synzynys, B I; Brozmanová, J; Saenko, A S

1987-01-01

Sedimentation properties of nucleoids from HeLa cells cultured for 6 or 24 h with 10(-6) M fluorodeoxyuridine (FdUrd) were studied in neutral sucrose gradients. Independently on the presence and concentrations of ethidium bromide in the gradient, nucleoids from FdUrd treated cells sedimented farther than those from untreated cells. However, the maximum relaxation of supercoiled DNA, observed at the concentration of 5 micrograms/ml of ethidium bromide, was significantly lower in cells treated with FdUrd, which indicated that prior incubation with FdUrd did not increase the degree of DNA supercoiling but altered by some way the conformation of DNA in nucleus. Previously we have found, that treatment of HeLa cells with FdUrd resulted in the stimulation of DNA synthesis, which proved to be resistant to ultraviolet and gamma-irradiation. From the present results it is possible to suggest, that alterations of chromatine structure should be included in facilitating of DNA synthesis on DNA template damaged by ultraviolet or gamma irradiation.

[Effect of exogenous sucrose on growth and active ingredient content of licorice seedlings under salt stress conditions].

PubMed

Liu, Fu-zhi; Yang, Jun

2015-11-01

Licorice seedlings were taken as experimental materials, an experiment was conducted to study the effects of exogenous sucrose on growth and active ingredient content of licorice seedlings under NaCl stress conditions. The results of this study showed that under salt stress conditions, after adding a certain concentration of exogenous sucrose, the licorice seedlings day of relative growth rate was increasing, and this stress can be a significant weakening effect, indicating that exogenous sucrose salt stress-relieving effect. The total flavonoids and phenylalanine ammonia lyase (PAL) activity were significantly increased, the exogenous sucrose can mitigated the seedling roots under salt stress, the licorice flavonoid content in the enhanced growth was largely due to the activity of PAL an increased, when the concentration of exogenous sucrose wae 10 mmol x L(-1), PAL activity reaching a maximum, when the concentration of exogenous sucrose was 15 mmol x L(-1), PAL activity turned into a downward trend, the results indicating that this mitigation has concentration effect. After applying different concentrations of exogenous sugar, the contents of liquiritin changes with the change of flavonoids content was similar. After applying different concentrations of exogenous sucrose, the content of licorice acid under salt stress was higher than the levels were not reached during salt stress, the impact of exogenous sucrose concentration gradient of licorice acid accumulation was not obvious.

Maximizing Exosome Colloidal Stability Following Electroporation

PubMed Central

Hood, Joshua L.; Scott, Michael J.; Wickline, Samuel A.

2014-01-01

Development of exosome based semi-synthetic nanovesicles for diagnostic and therapeutic purposes requires novel approaches to load exosomes with cargo. Electroporation has previously been used to load exosomes with RNA. However, investigations into exosome colloidal stability following electroporation have not been considered. Herein, we report the development of a unique trehalose pulse media (TPM) that minimizes exosome aggregation following electroporation. Dynamic light scattering (DLS) and RNA absorbance were employed to determine the extent of exosome aggregation and electroextraction post electroporation in TPM compared to common PBS pulse media or sucrose pulse media (SPM). Use of TPM to disaggregate melanoma exosomes post electroporation was dependent on both exosome concentration and electric field strength. TPM maximized exosome dispersal post electroporation for both homogenous B16 melanoma and heterogeneous human serum derived populations of exosomes. Moreover, TPM enabled heavy cargo loading of melanoma exosomes with 5 nm superparamagnetic iron oxide nanoparticles (SPION5) while maintaining original exosome size and minimizing exosome aggregation as evidenced by transmission electron microscopy. Loading exosomes with SPION5 increased exosome density on sucrose gradients. This provides a simple, label free means to enrich exogenously modified exosomes and introduces the potential for MRI driven theranostic exosome investigations in vivo. PMID:24333249

Maximizing exosome colloidal stability following electroporation.

PubMed

Hood, Joshua L; Scott, Michael J; Wickline, Samuel A

2014-03-01

Development of exosome-based semisynthetic nanovesicles for diagnostic and therapeutic purposes requires novel approaches to load exosomes with cargo. Electroporation has previously been used to load exosomes with RNA. However, investigations into exosome colloidal stability following electroporation have not been considered. Herein, we report the development of a unique trehalose pulse media (TPM) that minimizes exosome aggregation following electroporation. Dynamic light scattering (DLS) and RNA absorbance were employed to determine the extent of exosome aggregation and electroextraction post electroporation in TPM compared to common PBS pulse media or sucrose pulse media (SPM). Use of TPM to disaggregate melanoma exosomes post electroporation was dependent on both exosome concentration and electric field strength. TPM maximized exosome dispersal post electroporation for both homogenous B16 melanoma and heterogeneous human serum-derived populations of exosomes. Moreover, TPM enabled heavy cargo loading of melanoma exosomes with 5nm superparamagnetic iron oxide nanoparticles (SPION5) while maintaining original exosome size and minimizing exosome aggregation as evidenced by transmission electron microscopy. Loading exosomes with SPION5 increased exosome density on sucrose gradients. This provides a simple, label-free means of enriching exogenously modified exosomes and introduces the potential for MRI-driven theranostic exosome investigations in vivo. Copyright © 2013 Elsevier Inc. All rights reserved.

STUDIES ON ISOLATED NUCLEI. I. ISOLATION AND CHEMICAL CHARACTERIZATION OF A NUCLEAR FRACTION FROM GUINEA PIG LIVER.

PubMed

MAGGIO, R; SIEKEVITZ, P; PALADE, G E

1963-08-01

This article describes a method for the isolation of nuclei from guinea pig liver. It involves the homogenization of the tissue in 0.88 M sucrose-1.5 mM CaCl(2) followed by centrifugation in a discontinuous density gradient in which the upper phase is the homogenate and the lower phase is 2.2 M sucrose-0.5 mM CaCl(2). Based on DNA recovery, the isolated fraction contains 25 to 30 per cent of the nuclei of the original homogenate. Electron microscopical observations showed that approximately 88 per cent of the isolated nuclei come from liver cells (the rest from von Kupffer cells and leucocytes) and that approximately 90 per cent of the nuclei appear intact, with well preserved nucleoli, nucleoplasm, nuclear envelope, and pores. Cytoplasmic contamination is minimal and consists primarily of the nuclear envelope and its attached ribosomes. The nuclear fraction consists of approximately 22.3 per cent DNA, approximately 4.7 per cent RNA, and approximately 73 per cent protein, the DNA/RNA ratio being 4.7. Data on RNA extractibility by phosphate and salt and on the base composition of total nuclear RNA are included.

Characterization of outer membranes isolated from Treponema pallidum, the syphilis spirochete.

PubMed

Radolf, J D; Robinson, E J; Bourell, K W; Akins, D R; Porcella, S F; Weigel, L M; Jones, J D; Norgard, M V

1995-11-01

Previous freeze-fracture electron microscopy (EM) studies have shown that the outer membrane (OM) of Treponema pallidum contains sparse transmembrane proteins. One strategy for molecular characterization of these rare OM proteins involves isolation of T. pallidum OMs. Here we describe a simple and extremely gentle method for OM isolation based upon isopycnic sucrose density gradient ultracentrifugation of treponemes following plasmolysis in 20% sucrose. Evidence that T. pallidum OMs were isolated included (i) the extremely low protein/lipid ratio of the putative OM fraction, (ii) a paucity of antigenic and/or biochemical markers for periplasmic, cytoplasmic membrane, and cytosolic compartments, and (iii) freeze-fracture EM demonstrating that the putative OMs contained intramembranous particles highly similar in size and density to those in native T. pallidum OMs. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis revealed that the OMs contained a relatively small number of treponemal proteins, including several which did not appear to correspond to previously characterized T. pallidum antigens. Interestingly, these candidate rare OM proteins reacted poorly with syphilitic sera as determined by both conventional immunoblotting and enhanced chemiluminescence. Compared with whole cells, T. pallidum OMs were deficient in cardiolipin, the major lipoidal antigen reactive with antibodies in syphilitic sera. Also noteworthy was that other lipoidal constituents of OMs, including the recently discovered glycolipids, did not react with human syphilitic sera. These latter observations suggest that the poor antigenicity of virulent T. pallidum is a function of both the lipid composition and the low protein content of its OM.

GP3 is a structural component of the PRRSV type II (US) virion

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

Lima, M. de; Departamento de Microbiologia e Parasitologia, Universidade Federal Fluminense, Niteroi, RJ; Ansari, I.H.

2009-07-20

Glycoprotein 3 (GP3) is a highly glycosylated PRRSV envelope protein which has been reported as being present in the virions of PRRSV type I, while missing in the type II PRRSV (US) virions. We herein present evidence that GP3 is indeed incorporated in the virus particles of a North American strain of PRRSV (FL12), at a density that is consistent with the minor structural role assigned to GP3 in members of the Arterivirus genus. Two 15aa peptides corresponding to two different immunodominant linear epitopes of GP3 derived from the North American strain of PRRSV (FL12) were used as antigen tomore » generate a rabbit monospecific antiserum to this protein. The specificity of this anti-GP3 antiserum was confirmed by radioimmunoprecipitation (RIP) assay using BHK-21 cells transfected with GP3 expressing plasmid, MARC-145 cells infected with FL12 PRRSV, as well as by confocal microscopy on PRRSV-infected MARC-145 cells. To test if GP3 is a structural component of the virion, {sup 35}S-labelled PRRSV virions were pelleted through a 30% sucrose cushion, followed by a second round of purification on a sucrose gradient (20-60%). Virions were detected in specific gradient fractions by radioactive counts and further confirmed by viral infectivity assay in MARC 145 cells. The GP3 was detected in gradient fractions containing purified virions by RIP using anti-GP3 antiserum. Predictably, the GP3 was less abundant in purified virions than other major structural envelope proteins such as GP5 and M. Further evidence of the presence of GP3 at the level of PRRSV FL12 envelope was obtained by immunogold staining of purified virions from the supernatant of infected cells with anti-GP3 antiserum. Taken together, these results indicate that GP3 is a minor structural component of the PRRSV type II (FL12 strain) virion, as had been previously described for PRRSV type I.« less

An Easy Method for Plant Polysome Profiling.

PubMed

Lecampion, Cécile; Floris, Maïna; Fantino, Jean Raphaël; Robaglia, Christophe; Laloi, Christophe

2016-08-28

Translation of mRNA to protein is a fundamental and highly regulated biological process. Polysome profiling is considered as a gold standard for the analysis of translational regulation. The method described here is an easy and economical way for fractionating polysomes from various plant tissues. A sucrose gradient is made without the need for a gradient maker by sequentially freezing each layer. Cytosolic extracts are then prepared in a buffer containing cycloheximide and chloramphenicol to immobilize the cytosolic and chloroplastic ribosomes to mRNA and are loaded onto the sucrose gradient. After centrifugation, six fractions are directly collected from the bottom to the top of the gradient, without piercing the ultracentrifugation tube. During collection, the absorbance at 260 nm is read continuously to generate a polysome profile that gives a snapshot of global translational activity. Fractions are then pooled to prepare three different mRNA populations: the polysomes, mRNAs bound to several ribosomes; the monosomes, mRNAs bound to one ribosome; and mRNAs that are not bound to ribosomes. mRNAs are then extracted. This protocol has been validated for different plants and tissues including Arabidopsis thaliana seedlings and adult plants, Nicotiana benthamiana, Solanum lycopersicum, and Oryza sativa leaves.

The Ozobranchus leech is a candidate mechanical vector for the fibropapilloma-associated turtle herpesvirus found latently infecting skin tumors on Hawaiian green turtles (Chelonia mydas)

USGS Publications Warehouse

Greenblatt, R.J.; Work, Thierry M.; Balazs, G.; Sutton, C.A.; Casey, R.N.; Casey, J.W.

2004-01-01

Fibropapillomatosis (FP) of marine turtles is a neoplastic disease of ecological concern. A fibropapilloma-associated turtle herpesvirus (FPTHV) is consistently present, usually at loads exceeding one virus copy per tumor cell. DNA from an array of parasites of green turtles (Chelonia mydas) was examined with quantitative PCR (qPCR) to determine whether any carried viral loads are sufficient to implicate them as vectors for FPTHV. Marine leeches (Ozobranchus spp.) were found to carry high viral DNA loads; some samples approached 10 million copies per leech. Isopycnic sucrose density gradient/qPCR analysis confirmed that some of these copies were associated with particles of the density of enveloped viruses. The data implicate the marine leech Ozobranchus as a mechanical vector for FPTHV. Quantitative RT-PCR analysis of FPTHV gene expression indicated that most of the FPTHV copies in a fibropapilloma have restricted DNA polymerase expression, suggestive of latent infection.

Characterization, localization, and seasonal changes of the sucrose transporter FeSUT1 in the phloem of Fraxinus excelsior

PubMed Central

Öner-Sieben, Soner; Rappl, Christine; Sauer, Norbert; Stadler, Ruth; Lohaus, Gertrud

2015-01-01

Trees are generally assumed to be symplastic phloem loaders. A typical feature for most wooden species is an open minor vein structure with symplastic connections between mesophyll cells and phloem cells, which allow sucrose to move cell-to-cell through the plasmodesmata into the phloem. Fraxinus excelsior (Oleaceae) also translocates raffinose family oligosaccharides in addition to sucrose. Sucrose concentration was recently shown to be higher in the phloem sap than in the mesophyll cells. This suggests the involvement of apoplastic steps and the activity of sucrose transporters in addition to symplastic phloem-loading processes. In this study, the sucrose transporter FeSUT1 from F. excelsior was analysed. Heterologous expression in baker’s yeast showed that FeSUT1 mediates the uptake of sucrose. Immunohistochemical analyses revealed that FeSUT1 was exclusively located in phloem cells of minor veins and in the transport phloem of F. excelsior. Further characterization identified these cells as sieve elements and possibly ordinary companion cells but not as intermediary cells. The localization and expression pattern point towards functions of FeSUT1 in phloem loading of sucrose as well as in sucrose retrieval. FeSUT1 is most likely responsible for the observed sucrose gradient between mesophyll and phloem. The elevated expression level of FeSUT1 indicated an increased apoplastic carbon export activity from the leaves during spring and late autumn. It is hypothesized that the importance of apoplastic loading is high under low-sucrose conditions and that the availability of two different phloem-loading mechanisms confers advantages for temperate woody species like F. excelsior. PMID:26022258

Identification and characterization of Vibrio cholerae surface proteins by radioiodination.

PubMed Central

Richardson, K; Parker, C D

1985-01-01

Whole cells and isolated outer membrane from Vibrio cholerae (Classical, Inaba) were radiolabeled with Iodogen or Iodo-beads as catalyst. Radiolabeling of whole cells was shown to be surface specific by sodium dodecyl sulfate-urea polyacrylamide gel electrophoresis of whole cells and cell fractions. Surface-labeled whole cells regularly showed 16 distinguishable protein species, of which nine were found in radiolabeled outer membrane preparations obtained by a lithium chloride-lithium acetate procedure. Eight of these proteins were found in outer membranes prepared by sucrose density gradient centrifugation and Triton X-100 extraction of radiolabeled whole cells. The mobility of several proteins was shown to be affected by temperature, and the major protein species exposed on the cell surface was shown to consist of at least two different peptides. Images PMID:3980099

Subcellular fractionation on Percoll gradient of mossy fiber synaptosomes: evoked release of glutamate, GABA, aspartate and glutamate decarboxylase activity in control and degranulated rat hippocampus.

PubMed

Taupin, P; Ben-Ari, Y; Roisin, M P

1994-05-02

Using discontinuous density gradient centrifugation in isotonic Percoll sucrose, we have characterized two subcellular fractions (PII and PIII) enriched in mossy fiber synaptosomes and two others (SII and SIII) enriched in small synaptosomes. These synaptosomal fractions were compared with those obtained from adult hippocampus irradiated at neonatal stage to destroy granule cells and their mossy fibers. Synaptosomes were viable as judged by their ability to release aspartate, glutamate and GABA upon K+ depolarization. After irradiation, compared to the control values, the release of glutamate and GABA was decreased by 57 and 74% in the PIII fraction, but not in the other fractions and the content of glutamate, aspartate and GABA was also decreased in PIII fraction by 62, 44 and 52% respectively. These results suggest that mossy fiber (MF) synaptosomes contain and release glutamate and GABA. Measurement of the GABA synthesizing enzyme, glutamate decarboxylase, exhibited no significant difference after irradiation, suggesting that GABA is not synthesized by this enzyme in mossy fibers.

Insulin stimulation of glucose transport in isolated rat adipocytes. Functional evidence for insulin activation of intrinsic transporter activity within the plasma membrane.

PubMed Central

Hyslop, P A; Kuhn, C E; Sauerheber, R D

1985-01-01

We examined the effects of the membrane-impermeant amino-group-modifying agent fluorescein isothiocyanate (FITC) on the basal and insulin-stimulated hexose-transport activity of isolated rat adipocytes. Pre-treatment of cells with FITC causes irreversible inhibition of transport measured in subsequently washed cells. Transport activity was inhibited by approx. 50% with 2 mM-FITC in 8 min. The cells respond to insulin, after FITC treatment and removal, and the fold increase in transport above the basal value caused by maximal concentrations of insulin was independent of the concentration of FITC used for pre-treatment over the range 0-2 mM, where basal activity was progressively inhibited. The ability of FITC to modify selectively hexose transporters accessible only to the external milieu was evaluated by two methods. (1) Free intracellular FITC, and the distribution of FITC bound to cellular components, were assessed after dialysis of the homogenate and subcellular fractionation on sucrose gradients by direct spectroscopic measurement of fluorescein. Most (98%) of the FITC was associated with the non-diffusible fractions. Equilibrium sucrose-density-gradient centrifugation of the homogenate demonstrated that the subcellular distribution of the bound FITC correlated with the density distribution of a plasma-membrane marker, but not markers for Golgi, endoplasmic reticulum, mitochondria or protein. Exposing the cellular homogenate, rather than the intact cell preparation, to 2 mM-FITC resulted in a 4-5-fold increase in total bound FITC, and the density-distribution profile more closely resembled the distribution of total protein. (2) Incubation of hexokinase preparations with FITC rapidly and irreversibly inactivates this protein. However, both intracellular hexokinase total activity and its apparent Michaelis constant for glucose were unaffected in FITC-treated intact cells. Further control experiments demonstrated that FITC pre-treatment of cells had no effect on the intracellular ATP concentration or the dose-response curve of insulin stimulation of hexose transport. Since the fold increase of hexose transport induced by insulin is constant over the range of inhibition of surface-labelled hexose transporters, we suggest that insulin-induced insertion of additional transporters into the plasma membrane may not be the major locus of acceleration of hexose transport by the hormone. PMID:3910027

The long-term effects of feeding honey compared with sucrose and a sugar-free diet on weight gain, lipid profiles, and DEXA measurements in rats.

PubMed

Chepulis, L; Starkey, N

2008-01-01

To determine whether honey and sucrose would have differential effects on weight gain during long-term feeding, 45 2-mo-old Sprague Dawley rats were fed a powdered diet that was either sugar-free or contained 7.9% sucrose or 10% honey ad libitum for 52 wk (honey is 21% water). Weight gain was assessed every 1 to 2 wk and food intake was measured every 2 mo. At the completion of the study blood samples were removed for measurement of blood sugar (HbA1c) and a fasting lipid profile. DEXA analyses were then performed to determine body composition and bone mineral densities. Overall weight gain and body fat levels were significantly higher in sucrose-fed rats and similar for those fed honey or a sugar-free diet. HbA1c levels were significantly reduced, and HDL-cholesterol significantly increased, in honey-fed compared with rats fed sucrose or a sugar free diet, but no other differences in lipid profiles were found. No differences in bone mineral density were observed between honey- and sucrose-fed rats, although it was significantly increased in honey-fed rats compared with those fed the sugar-free diet.

Reconstitution of a physical complex between the N-formyl chemotactic peptide receptor and G protein. Inhibition by pertussis toxin-catalyzed ADP ribosylation.

PubMed

Bommakanti, R K; Bokoch, G M; Tolley, J O; Schreiber, R E; Siemsen, D W; Klotz, K N; Jesaitis, A J

1992-04-15

Photoaffinity-labeled N-formyl chemotactic peptide receptors from human neutrophils solubilized in octyl glucoside exhibit two forms upon sucrose density gradient sedimentation, with apparent sedimentation coefficients of approximately 4 and 7 S. The 7 S form can be converted to the 4 S form by guanosine 5'-O-(3-thiotriphosphate) (GTP gamma S) with an EC50 of approximately 20 nM, suggesting that the 7 S form may represent a physical complex of the receptor with endogenous G protein (Jesaitis, A. J., Tolley, J. O., Bokoch, G. M., and Allen, R. A. (1989) J. Cell Biol. 109, 2783-2790). To probe the nature of the 7 S form, we reconstituted the 7 S form from the 4 S form by adding purified G protein. The 4 S form, obtained by solubilizing GTP gamma S-treated neutrophil plasma membranes, was incubated with purified (greater than 95%) Gi protein from bovine brain (containing both Gi alpha 1 and Gi alpha 2) or with neutrophil G protein (Gn), and formation of the 7 S complex was analyzed on sucrose density gradients. The EC50 of 7 S complex formation induced by the two G proteins was 70 +/- 25 and 170 +/- 40 nM for Gn and Gi, respectively. No complexation was measurable when bovine transducin (Gt) was used up to 30 times the EC50 for Gn. The EC50 for Gi was the same for receptors, obtained from formyl peptide-stimulated or unstimulated cells. The addition of 10 microM GTP gamma S to the reconstituted 7 S complex caused a complete revision of the receptor to the 4 S form, and anti-Gi peptide antisera immunosedimented the 7 S form. ADP-ribosylation of Gi prevented formation of the 7 S form even at 20 times the concentration of unribosylated Gi normally used to attain 50% conversion to the 7 S form. These observations suggest that the 7 S species is a physical complex containing N-formyl chemotactic peptide receptor and G protein.

Density of ocular components of the bovine eye.

PubMed

Su, Xiao; Vesco, Christina; Fleming, Jacquelyn; Choh, Vivian

2009-10-01

Density is essential for acoustic characterization of tissues and provides a basic input for ultrasound backscatter and absorption models. Despite the existence of extensive compilations of acoustic properties, neither unified data on ocular density nor comparisons of the densities between all ocular components can be found. This study was undertaken to determine the mass density of all the ocular components of the bovine eye. Liquid components were measured through mass/volume ratio, whereas solid tissues were measured with two different densitometry techniques based on Archimedes Principle. The first method determines the density by measuring dry and wet weight of the tissues. The second method consists of immersing the tissues in sucrose solutions of varying densities and observing their buoyancy. Although the mean densities for all tissues were found to be within 0.02 g/cm by both methods, only the sucrose solution method offered a consistent relative order for all measured ocular components, as well as a considerably smaller standard deviation (a maximum standard deviation of 0.004 g/cm for cornea). The lens was found to be the densest component, followed by the sclera, cornea, choroid, retina, aqueous, and vitreous humors. The consistent results of the sucrose solution tests suggest that the ocular mass density is a physical property that is more dependent on the compositional and structural characteristics of the tissue and than on population variability.

Interaction of dietary sucrose and fiber on serum lipids in healthy young men fed high carbohydrate diets.

PubMed

Albrink, M J; Ullrich, I H

1986-03-01

High sucrose diets may cause increased serum triglycerides and decreased high density lipoprotein concentration. To determine whether dietary fiber protects against these effects, four groups of six healthy young men were assigned to one of four very high carbohydrate diets providing 0, 18, 36, or 52% of calories as sucrose. Each diet was fed in both low (less than 14 g) and high (greater than 34 g) levels of dietary fiber for 10 days each. Triglycerides increased during the 36 and 52% sucrose diets compared to 0 and 18% sucrose diets, and fiber protected partially against this rise. Serum cholesterol and LDL cholesterol were lower during the 0 and 18% sucrose diets than the 36 or 52% sucrose diets but fiber had no effect. HDL cholesterol decreased during all low fat diets, with a trend toward a greater decrease during the high sucrose diets. The results suggest that fiber protects against carbohydrate-induced lipemia but has no effect on cholesterol during very high carbohydrate diets.

Preparation of membrane rafts.

PubMed

Waugh, Mark G; Hsuan, J Justin

2009-01-01

The concept that biological membranes contain microdomains of specialized lipid and protein composition has attracted great attention in recent years. Initially, the focus in the field was very much on the characterization of cholesterol-and sphingolipid-rich plasma membrane microdomains that were resistant to solubilization in the cold non-ionic detergent Triton X-100. Such detergent-insoluble membrane domains were of low buoyant density and could be readily purified on sucrose equilibrium density gradients. The intrinsic buoyancy of the detergent-insoluble domains gave rise to the term "lipid rafts." Cholesterol- and sphingolipid-rich rafts at the plasma membrane have been implicated in a wide range of cellular processes, including pathogen invasion, receptor signaling, and endocytosis. However, work with other non-ionic detergents such as Lubrol WX and Brij-98 has revealed the existence of various raft subtypes with differing lipid compositions and proposed functions. More recently, there has been some focus on isolating lipid rafts from intracellular organelles, in particular membranes from the Golgi-endosomal pathway, where raft lipids have been proposed to function in processes such as the sorting of vesicular cargo and the processing of amyloid precursor protein. While there remains a large degree of controversy surrounding the purity, the physiological importance, and even the existence of different types of lipid rafts in intact cells, the ability to routinely purify such domains has led to significant progress in understanding the functional architecture of biological membranes. We describe a number of widely used methods to prepare rafts, based on early preparations of caveolae by density gradient ultracentrifugation and immunoaffinity precipitation.

The mechanism of phloem loading in rice (Oryza sativa).

PubMed

Eom, Joon-Seob; Choi, Sang-Bong; Ward, John M; Jeon, Jong-Seong

2012-05-01

Carbohydrates, mainly sucrose, that are synthesized in source organs are transported to sink organs to support growth and development. Phloem loading of sucrose is a crucial step that drives long-distance transport by elevating hydrostatic pressure in the phloem. Three phloem loading strategies have been identified, two active mechanisms, apoplastic loading via sucrose transporters and symplastic polymer trapping, and one passive mechanism. The first two active loading mechanisms require metabolic energy, carbohydrate is loaded into the phloem against a concentration gradient. The passive process, diffusion, involves equilibration of sucrose and other metabolites between cells through plasmodesmata. Many higher plant species including Arabidopsis utilize the active loading mechanisms to increase carbohydrate in the phloem to higher concentrations than that in mesophyll cells. In contrast, recent data revealed that a large number of plants, especially woody species, load sucrose passively by maintaining a high concentration in mesophyll cells. However, it still remains to be determined how the worldwide important cereal crop, rice, loads sucrose into the phloem in source organs. Based on the literature and our results, we propose a potential strategy of phloem loading in rice. Elucidation of the phloem loading mechanism should improve our understanding of rice development and facilitate its manipulation towards the increase of crop productivity.

Sucrose affects the developmental transition of rhizomes in Oryza longistaminata.

PubMed

Bessho-Uehara, Kanako; Nugroho, Jovano Erris; Kondo, Hirono; Angeles-Shim, Rosalyn B; Ashikari, Motoyuki

2018-05-08

Oryza longistaminata, the African wild rice, can propagate vegetatively through rhizomes. Rhizomes elongate horizontally underground as sink organs, however, they undergo a developmental transition that shifts their growth to the surface of the ground to become aerial stems. This particular stage is essential for the establishment of new ramets. While several determinants such as abiotic stimuli and plant hormones have been reported as key factors effecting developmental transition in aerial stem, the cause of this phenomenon in rhizome remains elusive. This study shows that depletion of nutrients, particularly sucrose, is the key stimulus that induces the developmental transition in rhizomes, as indicated by the gradient of sugars from the base to the tip of the rhizome. Sugar treatments revealed that sucrose specifically represses the developmental transition from rhizome to aerial stem by inhibiting the expression of sugar metabolism and hormone synthesis genes at the bending point. Sucrose depletion affected several factors contributing to the developmental transition of rhizome including signal transduction, transcriptional regulation and plant hormone balance.

Isolation of the Lateral Border Recycling Compartment using a diaminobenzidine-induced density shift

PubMed Central

Sullivan, David P.; Rüffer, Claas; Muller, William A.

2014-01-01

The migration of leukocytes across the endothelium and into tissue is critical to mounting an inflammatory response. The Lateral Border Recycling Compartment (LBRC), a complex vesicular-tubule invagination of the plasma membrane found at endothelial cell borders, plays an important role in the this process. Although a few proteins have been shown to be present in the LBRC, no unique marker is known. Here we detail methods that can be used to characterize a subcellular compartment that lacks an identifying marker. Initial characterization of the LBRC was performed using standard subcellular fractionation with sucrose gradients and took advantage of the observation that the compartment migrated at a lower density than other membrane compartments. To isolate larger quantities of the compartment, we modified a classic technique known as a diaminobenzidine (DAB)-induced density shift. The DAB-induced density shift allowed for specific isolation of membranes labeled with HRP conjugated antibody. Because the LBRC could be differentially labeled at 4°C and 37°C, we were able to identify proteins that are enriched in the compartment, despite lacking a unique marker. These methods serve as a model to others studying poorly characterized compartments and organelles and are applicable to a wide variety of biological systems. PMID:24915828

Intestinal secretion of immunoglobulins and antibodies to Escherichia coli in the pig

PubMed Central

Porter, P.; Noakes, D. E.; Allen, W. D.

1970-01-01

Immunoglobulins and antibodies against Escherichia coli 0141 have been studied in porcine intestinal secretions obtained from Thiry Vella loops prepared in the mid jejunum of 4 animals. The molecular size of the secreted immunoglobulins were investigated by gel filtration and sucrose density gradient ultra-centrifugation. Intestinal IgM was found to have 7S characteristics and intestinal IgA mainly 11S characteristics similar to secretory IgA isolated from porcine milk. Immune inhibition studies with rabbit anti-IgA-globulin serum produced complete elimination of E. coli 0141 antibodies detected by direct haemagglutination. In one animal incomplete antibody assayed by antiglobulin haemagglutination was identified in fractions associated with IgM and IgG. Immunofluorescent studies were made to correlate immunoglobulins in the small intestinal tissue with weaning. ImagesFIG. 1FIG. 2FIG. 7 PMID:4193669

Intracellular Distribution of Enzymes of the Cytidine Diphosphate Choline Pathway in Castor Bean Endosperm

PubMed Central

Lord, J. M.; Kagawa, T.; Beevers, Harry

1972-01-01

The occurrence and subcellular distribution of enzymes of the cytidine diphosphate choline pathway of lecithin synthesis have been examined. Choline kinase (EC 2.7.1.32) was completely soluble, while phosphorylcholine-cytidyl transferase (EC 2.7.7.15) and phosphorylcholine-glyceride transferase (EC 2.7.8.2) were associated with particulate fractions. Although components sedimenting at 10,000 to 100,000 × g contained both enzymes, phosphorylcholine-cytidyl transferase and particularly phosphorylcholine-glyceride transferase were present in the 10,000 × g pellet, which contained the major organelles, mitochondria, and glyoxysomes. When the crude homogenate was centrifuged on a sucrose density gradient, four major bands of particulate protein were recovered. A band at density 1.24 g/cm3 contained the glyoxysomes and was devoid of phosphorylcholine-cytidyl transferase and phosphorylcholine-glyceride transferase activity. Enzyme activity was barely detectable in the mitochondria, at density 1.18 g/cm2. Phosphorylcholine-glyceride transferase was found almost exclusively in a sharp band at density 1.12 g/cm3, and phosphorylcholinecytidyl transferase was found in the uppermost band at density 1.08 g/cm3. Thus, for the synthesis of lecithin in their membranes, the glyoxysomes and mitochondria depend on enzymes elsewhere in the cell; the final two steps in lecithin formation occur, apparently exclusively, in separate particulate cell components. Images PMID:4506764

Retrovirus purification: method that conserves envelope glycoprotein and maximizes infectivity.

PubMed Central

McGrath, M; Witte, O; Pincus, T; Weissman, I L

1978-01-01

A Sepharose 4B chromatographic method for purification of retroviruses is described which was less time consuming, increased purified virus yields, conserved viral glycoprotein, and increased recovery of biological infectivity in comparison with conventional sucrose gradient ultracentrifugation techniques. Images PMID:205680

ENHANCED CONCENTRATION AND ISOLATION OF CYCLOSPORA CAYETANENSIS OOCYSTS FROM HUMAN FECAL SAMPLES

EPA Science Inventory

Cyclospora cayetanensis is the causative agent of cyclosporiasis, an emerging infections disease. A new method for the purification of Cycloposra cayetanensis oocysts from fecal matter has been developed, using a modified detachment solution and a Renocal-sucrose gradient sedimen...

Activation of muscarinic receptors in rat parotid acinar cells induces AQP5 trafficking to nuclei and apical plasma membrane.

PubMed

Cho, Gota; Bragiel, Aneta M; Wang, Di; Pieczonka, Tomasz D; Skowronski, Mariusz T; Shono, Masayuki; Nielsen, Søren; Ishikawa, Yasuko

2015-04-01

The subcellular distribution of aquaporin-5 (AQP5) in rat parotid acinar cells in response to muscarinic acetylcholine receptor (mAChR) activation remains unclear. Immunoconfocal and immunoelectron microscopy were used to visualize the distribution of AQP5 in parotid acinar cells. Western blotting was used to analyze AQP5 levels in membranes. To clarify the characteristics of membrane domains associated with AQP5, detergent solubility and sucrose-density flotation experiments were performed. Under control conditions, AQP5 was diffusely distributed on the apical plasma membrane (APM) and apical plasmalemmal region and throughout the cytoplasm. Upon mAChR activation, AQP5 was predominantly located in the nucleus, APM and lateral plasma membrane (LPM). Subsequently, localization of AQP5 in the nucleus, APM and LPM was decreased. Prolonged atropine treatment inhibited mAChR agonist-induced translocation of AQP5 to the nucleus, APM and LPM. AQP5 levels were enhanced in isolated nuclei and nuclear membranes prepared from parotid tissues incubated with mAChR agonist. mAChR agonist induced AQP5 levels in both soluble and insoluble nuclear fractions solubilized with Triton X-100 or Lubrol WX. Small amounts of AQP5 in nuclei were detected using low-density sucrose gradient. When AQP5 was present in the nuclear membrane, nuclear size decreased. The activation of mAChR induced AQP5 translocation to the nucleus, APM and LPM, and AQP5 may trigger water transport across the nuclear membrane and plasma membrane in rat parotid acinar cells. AQP5 translocates to the nuclear membrane and may trigger the movement of water, inducing shrinkage of the nucleus and the start of nuclear functions. Copyright © 2015 Elsevier B.V. All rights reserved.

Differences in the protein composition of bovine retinal rod outer segment disk and plasma membranes isolated by a ricin-gold-dextran density perturbation method

PubMed Central

1987-01-01

The plasma membrane and disk membranes of bovine retinal rod outer segments (ROS) have been purified by a novel density-gradient perturbation method for analysis of their protein compositions. Purified ROS were treated with neuraminidase to expose galactose residues on plasma membrane-specific glycoproteins and labeled with ricin-gold-dextran particles. After the ROS were lysed in hypotonic buffer, the plasma membrane was dissociated from the disks by either mild trypsin digestion or prolonged exposure to low ionic strength buffer. The dense ricin-gold-dextran-labeled plasma membrane was separated from disks by sucrose gradient centrifugation. Electron microscopy was used to follow this fractionation procedure. The dense red pellet primarily consisted of inverted plasma membrane vesicles containing gold particles; the membrane fraction of density 1.13 g/cc consisted of unlabeled intact disks and vesicles. Ricin-binding studies indicated that the plasma membrane from trypsin-treated ROS was purified between 10-15-fold. The protein composition of plasma membranes and disks was significantly different as analyzed by SDS gels and Western blots labeled with lectins and monoclonal antibodies. ROS plasma membrane exhibited three major proteins of 36 (rhodopsin), 38, and 52 kD, three ricin-binding glycoproteins of 230, 160, and 110 kD, and numerous minor proteins in the range of 14-270 kD. In disk membranes rhodopsin appeared as the only major protein. A 220-kD concanavalin A-binding glycoprotein and peripherin, a rim-specific protein, were also present along with minor proteins of 43 and 57-63 kD. Radioimmune assays indicated that the ROS plasma membrane contained about half as much rhodopsin as disk membranes. PMID:2447095

Enhanced extracellular production of trans-resveratrol in Vitis vinifera suspension cultured cells by using cyclodextrins and methyljasmonate.

PubMed

Belchí-Navarro, Sarai; Almagro, Lorena; Lijavetzky, Diego; Bru, Roque; Pedreño, María A

2012-01-01

In this work, the effect of different inducing factors on trans-resveratrol extracellular production in Monastrell grapevine suspension cultured cells is evaluated. A detailed analysis provides the optimal concentrations of cyclodextrins, methyljasmonate and UV irradiation dosage, optimal cell density, elicitation time and sucrose content in the culture media. The results indicate that trans-resveratrol production decreases as the initial cell density increases for a constant elicitor concentration in Monastrell suspension cultured cells treated with cyclodextrins individually or in combination with methyljasmonate; the decrease observed in cell cultures elicited with cyclodextrins alone is far more drastic than those observed in the combined treatment. trans-Resveratrol extracellular production observed by the joint use of cyclodextrins and methyljasmonate (1,447.8 ± 60.4 μmol trans-resveratrol g(-1) dry weight) is lower when these chemical compounds are combined with UV light short exposure (669.9 ± 45.2 μmol trans-resveratrol g(-1) dry weight). Likewise, trans-resveratrol production is dependent on levels of sucrose in the elicitation medium with the maximal levels observed with 20 g l(-1) sucrose and the joint action of cyclodextrins and 100 μM methyljasmonate. The sucrose concentration did not seem to limit the process although it affects significantly the specific productivity since the lowest sucrose concentration is 10 g l(-1), the highest productivity is reached (100.7 ± 5.8 μmol trans-resveratrol g(-1) dry weight g(-1) sucrose) using cyclodextrins and 25 μM methyljasmonate.

Rapid screening for plasmid DNA.

PubMed

Hughes, C; Meynell, G G

1977-03-07

A procedure is described for demonstrating plasmid DNA and its molecular weight, based on rate zonal centrifugation of unlabelled DNA in neutral sucrose gradients containing a low concentration of ethidium bromide. Each DNA species is then visualized as a discrete fluorescent band when the centrifuge tube is illuminated with ultra-violet light. Plasmids exist as closed circular and as relaxed circular molecules, which sediment separately, but during preparation of lysates, closed circular molecules are nicked so that each plasmid forms only a single band of relaxed circles within the gradient.

The composition of the incubation medium influences the sensitivity of mitochondrial permeability transition to cyclosporin A.

PubMed

Chávez, Edmundo; García, Noemi; Zazueta, Cecilia; Correa, Francisco; Avilés, César; García, Gerardo; Balam, Eros O

2003-04-01

The aim of this work was to study permeability transition, and the influence of the composition of the incubation medium, on the inhibitory action of cyclosporin A. It was found that cyclosporin inhibited the opening of a nonspecific pore, as induced by the uncoupler carbonyl cyanide m-chlorophenylhydrazone, provided K+ was present in the incubation medium, but failed to do so if mitochondria are incubated in sucrose or Na+-based medium. It was also found that the sensitivity of mitochondria to the uncoupler depended on the incubation mixture, being more sensitive when sucrose was the osmotic support. Matrix Ca2+ release, large amplitude swelling, and drop in transmembrane electric gradient revealed permeability transition. The titration of membrane thiol groups shows them to be increased in mitochondria incubated in sucrose medium, in comparison with the values found in mitochondria incubated in KCl or NaCl medium. Our proposal is that the incubation in sucrose medium propitiated a conformational change of membrane proteins in such a way that cyclosporin was unable to bind to its target site.

Glyoxysomes in Megagamethophyte of Germinating Ponderosa Pine Seeds 12

PubMed Central

Ching, Te May

1970-01-01

Decoated ponderosa pine (Pinus ponderosa Laws) seeds contained 40% lipids, which were mainly stored in megagametophytic tissue and were utilized or converted to sugars via the glyoxylate cycle during germination. Mitochondria and glyoxysomes were isolated from the tissue by sucrose density gradient centrifugation at different stages of germination. It was found that isocitrate lyase, malate synthase, and catalase were mainly bound in glyoxysomes. Aconitase and fumarase were chiefly localized in mitochondria, whereas citrate synthase was common for both. Both organelles increased in quantity and specific activity of their respective marker enzymes with the advancement of germination. When the megagametophyte was exhausted at the end of germination, the quantity of these organelles and the activity of their marker enzymes decreased abruptly. At the stage of highest lipolysis, the isolated mitochondria and glyoxysomes were able to synthesize protein from labeled amino acids. Both organellar fractions contained RNA and DNA. Some degree of autonomy in glyoxysomes is indicated. Images PMID:16657489

Alteration in lipid composition of plasma membranes of sensitive and resistant Guerin carcinoma cells due to the action of free and liposomal form of cisplatin.

PubMed

Naleskina, L A; Todor, I N; Nosko, M M; Lukianova, N Y; Pivnyuk, V M; Chekhun, V F

2013-09-01

To study in vivo changes of lipid composition of plasma membranes of sensitive and resistant to cisplatin Guerin carcinoma cells under influence of free and liposomal cisplatin forms. The isolation of plasma membranes from parental (sensitive) and resistant to cisplatin Guerin carcinoma cells was by differential ultracentrifugation in sucrose density gradient. Lipids were detected by method of thin-layer chromatography. It was determined that more effective action of cisplatin liposomal form on resistant cells is associated with essential abnormalities of conformation of plasma membrane due to change of lipid components and architectonics of rafts. It results in the increase of membrane fluidity. Reconstructions in lipid composition of plasma membranes of cisplatin-resistant Guerin carcinoma cells provide more intensive delivery of drug into the cells, increase of its concentration and more effective interaction with cellular structural elements.

Lidocaine effect on flotillin-2 distribution in detergent-resistant membranes of equine jejunal smooth muscle in vitro.

PubMed

Tappenbeck, Karen; Schmidt, Sonja; Feige, Karsten; Naim, Hassan Y; Huber, Korinna

2014-05-01

Lidocaine is the most commonly chosen prokinetic for treating postoperative ileus in horses, a motility disorder associated with ischaemia-reperfusion injury of intestinal tissues. Despite the frequent use of lidocaine, the mechanism underlying its prokinetic effects is still unclear. Previous studies suggested that lidocaine altered cell membrane characteristics of smooth muscle cells. Therefore, the present study aimed to elucidate effects of lidocaine administration on characteristics of detergent-resistant membranes in equine jejunal smooth muscle. Lidocaine administration caused significant redistribution of flotillin-2, a protein marker of detergent-resistant membranes, in fractions of sucrose-density-gradients obtained from ischaemia-reperfusion injured smooth muscle solubilised with Triton X-100. It was concluded that lidocaine induced disruption of detergent-resistant membranes which might affect ion channel activity and therefore enhance smooth muscle contractility. Copyright © 2014 Elsevier Ltd. All rights reserved.

Phosphatidylglycerol synthesis in castor bean endosperm. [Ricinus communis

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

Moore, T.S. Jr.

1974-01-01

The synthesis of phosphatidylglycerol in castor bean (Ricinus communis var. Hale) endosperm tissue was found to be located in both the endoplasmic reticulum and mitochondrial fractions separated on sucrose density gradients. The enzyme of both fractions attained maximum activity at 5 mM Mn/sup 2 +/, 0.075 percent Triton X-100, and pH 7.3. The addition of dithiothreitol produced little effect, but sulfhydryl inhibitors reduced activity in both systems. Cytidine diphosphate-diglyceride exhibited an apparent Michaelis constant for the endoplasmic reticulum enzyme of 2.8 ..mu..M and for the mitochondrial enzyme of 2.0 ..mu..M; the maximum reaction rate was achieved at about 20 ..mu..M.more » For the second substrate, glycerol-phosphate, the apparent Michaelis constant for both fractions was about 50 ..mu..M and maximum velocity was reached at 400 ..mu..M. The specific activity of the mitochondrial enzyme was generally twice that of the endoplasmic reticulum.« less

[Isolation and purification of nonspecific nuclease of cyanobacterium Plectonema boryanum CALU 465].

PubMed

Tsymbal, N V; Samoĭlenko, V A; Syrchin, S A; Mendzhul, M I

2004-01-01

Nonspecific nuclease has been isolated from the cells of cyanobacterium Plectonema boryanum and purified to homogenic state. It has been established that the method of centrifugation of cell-free culture extract in the sucrose density gradient is efficient for the separation of pigment proteins and enzyme concentration. Under the successive use of two ion-exchangers the nuclease activity was determined in the concentration range of NaCl 0.065-0.085 M after separation of the cell-free cyanobacterium extract on the column with phosphocellulose in the range of 0.2-0.25 M, on the column with DEAE--Toyopearl respectively. The molecular mass of nuclease which is 40 kDa, has been determined by electrophoresis in polyacrylamide gel under denaturating conditions and gel-filtration on Sephadex G-100. It has been also established that the given enzyme is monosubunitary as to its structure.

Multiple sedimenting species of properdin in human serum and interaction of purified properdin with the third component of complement

PubMed Central

1976-01-01

Normal human serum subjected to sucrose density gradient analysis exhibited multiple sedimenting species of properdin antigen. Properdin antigen distribution was dependent on serum concentration, ionic strength, temperature, and the presence of C3, and was not dependent on the presence of divalent metal cations or blood coagulation. In mixtures of purified components, properdin sedimented heavier in the presence of C3, C3b, or C3c. Addition of factor B to mixtures containing C3 and properdin was without effect. These data provide insights into earlier discrepancies concerning the sedimentation behavior of partially purified properdin, indicate a propensity of some constituents of the alternative pathway to form protein-protein complexes, and suggest caution in interpretation of immunopathological studies in which properdin deposits are found in the presence of C3. PMID:2647

Relationship between Photoconvertible and Nonphotoconvertible Protochlorophyllides.

PubMed

Murray, A E; Klein, A O

1971-10-01

Two forms of protochlorophyllide are found in dark-grown bean (Phaseolus vulgaris, var. Black Velentine) leaves, one (protochlorophyllide(650)) which is directly photoconvertible to chlorophyllide and another (protochlorophyllide(632)) which is not. Dark-grown leaves placed in solutions of delta-aminolevulinic acid accumulate protochlorophyllide(632). Protochlorophyllide(650) and protochlorophyllide(632) can be partially separated on sucrose density gradients. A nitrogen atmosphere blocks chlorophyll synthesis in light or the regeneration of protochlorophyllide(650) in the dark, even in the presence of excess delta-aminolevulinic acid, except when a stockpile of protochlorophyllide(632) is present in the leaf. Under the latter conditions chlorophyll synthesis or protochlorophyllide(650) regeneration is accompanied by a decrease in protochlorophyllide(632). These experiments suggest that protochlorophyllide(632) may be converted to protochlorophyllide(650).Cycloheximide inhibited greening only after an "action-dependent" delay, requiring a predictable minimal period of illumination. This inhibition could be relieved for a time by feeding delta-aminolevulinic acid.

Combining Ultracentrifugation and Peptide Termini Group-specific Immunoprecipitation for Multiplex Plasma Protein Analysis

PubMed Central

Volk, Sonja; Schreiber, Thomas D.; Eisen, David; Wiese, Calvin; Planatscher, Hannes; Pynn, Christopher J.; Stoll, Dieter; Templin, Markus F.; Joos, Thomas O.; Pötz, Oliver

2012-01-01

Blood plasma is a valuable source of potential biomarkers. However, its complexity and the huge dynamic concentration range of its constituents complicate its analysis. To tackle this problem, an immunoprecipitation strategy was employed using antibodies directed against short terminal epitope tags (triple X proteomics antibodies), which allow the enrichment of groups of signature peptides derived from trypsin-digested plasma. Isolated signature peptides are subsequently detected using MALDI-TOF/TOF mass spectrometry. Sensitivity of the immunoaffinity approach was, however, compromised by the presence of contaminant peaks derived from the peptides of nontargeted high abundant proteins. A closer analysis of the enrichment strategy revealed nonspecific peptide binding to the solid phase affinity matrix as the major source of the contaminating peptides. We therefore implemented a sucrose density gradient ultracentrifugation separation step into the procedure. This yielded a 99% depletion of contaminating peptides from a sucrose fraction containing 70% of the peptide-antibody complexes and enabled the detection of the previously undetected low abundance protein filamin-A. Assessment of this novel approach using 15 different triple X proteomics antibodies demonstrated a more consistent detection of a greater number of targeted peptides and a significant reduction in the intensity of nonspecific peptides. Ultracentrifugation coupled with immunoaffinity MS approaches presents a powerful tool for multiplexed plasma protein analysis without the requirement for demanding liquid chromatography separation techniques. PMID:22527512

Accurate, Streamlined Analysis of mRNA Translation by Sucrose Gradient Fractionation

PubMed Central

Aboulhouda, Soufiane; Di Santo, Rachael; Therizols, Gabriel; Weinberg, David

2017-01-01

The efficiency with which proteins are produced from mRNA molecules can vary widely across transcripts, cell types, and cellular states. Methods that accurately assay the translational efficiency of mRNAs are critical to gaining a mechanistic understanding of post-transcriptional gene regulation. One way to measure translational efficiency is to determine the number of ribosomes associated with an mRNA molecule, normalized to the length of the coding sequence. The primary method for this analysis of individual mRNAs is sucrose gradient fractionation, which physically separates mRNAs based on the number of bound ribosomes. Here, we describe a streamlined protocol for accurate analysis of mRNA association with ribosomes. Compared to previous protocols, our method incorporates internal controls and improved buffer conditions that together reduce artifacts caused by non-specific mRNA–ribosome interactions. Moreover, our direct-from-fraction qRT-PCR protocol eliminates the need for RNA purification from gradient fractions, which greatly reduces the amount of hands-on time required and facilitates parallel analysis of multiple conditions or gene targets. Additionally, no phenol waste is generated during the procedure. We initially developed the protocol to investigate the translationally repressed state of the HAC1 mRNA in S. cerevisiae, but we also detail adapted procedures for mammalian cell lines and tissues. PMID:29170751

Production of the short peptide surfactant DAMP4 from glucose or sucrose in high cell density cultures of Escherichia coli BL21(DE3).

PubMed

Bruschi, Michele; Krömer, Jens O; Steen, Jennifer A; Nielsen, Lars K

2014-08-19

Peptides are increasingly used in industry as highly functional materials. Bacterial production of recombinant peptides has the potential to provide large amounts of renewable and low cost peptides, however, achieving high product titers from Chemically Defined Media (CDM) supplemented with simple sugars remains challenging. In this work, the short peptide surfactant, DAMP4, was used as a model peptide to investigate production in Escherichia coli BL21(DE3), a classical strain used for protein production. Under the same fermentation conditions, switching production of DAMP4 from rich complex media to CDM resulted in a reduction in yield that could be attributed to the reduction in final cell density more so than a significant reduction in specific productivity. To maximize product titer, cell density at induction was maximized using a fed-batch approach. In fed-batch DAMP4 product titer increased 9-fold compared to batch, while maintaining 60% specific productivity. Under the fed-batch conditions, the final product titer of DAMP4 reached more than 7 g/L which is the highest titer of DAMP4 reported to date. To investigate production from sucrose, sucrose metabolism was engineered into BL21(DE3) using a simple plasmid approach. Using this strain, growth and DAMP4 production characteristics obtained from CDM supplemented with sucrose were similar to those obtained when culturing the parent strain on CDM supplemented with glucose. Production of a model peptide was increased to several grams per liter using a CDM medium with either glucose or sucrose feedstock. It is hoped that this work will contribute cost reduction for production of designer peptide surfactants to facilitate their commercial application.

Metabolic Labeling and Membrane Fractionation for Comparative Proteomic Analysis of Arabidopsis thaliana Suspension Cell Cultures

PubMed Central

Szymanski, Witold G.; Kierszniowska, Sylwia; Schulze, Waltraud X.

2013-01-01

Plasma membrane microdomains are features based on the physical properties of the lipid and sterol environment and have particular roles in signaling processes. Extracting sterol-enriched membrane microdomains from plant cells for proteomic analysis is a difficult task mainly due to multiple preparation steps and sources for contaminations from other cellular compartments. The plasma membrane constitutes only about 5-20% of all the membranes in a plant cell, and therefore isolation of highly purified plasma membrane fraction is challenging. A frequently used method involves aqueous two-phase partitioning in polyethylene glycol and dextran, which yields plasma membrane vesicles with a purity of 95% 1. Sterol-rich membrane microdomains within the plasma membrane are insoluble upon treatment with cold nonionic detergents at alkaline pH. This detergent-resistant membrane fraction can be separated from the bulk plasma membrane by ultracentrifugation in a sucrose gradient 2. Subsequently, proteins can be extracted from the low density band of the sucrose gradient by methanol/chloroform precipitation. Extracted protein will then be trypsin digested, desalted and finally analyzed by LC-MS/MS. Our extraction protocol for sterol-rich microdomains is optimized for the preparation of clean detergent-resistant membrane fractions from Arabidopsis thaliana cell cultures. We use full metabolic labeling of Arabidopsis thaliana suspension cell cultures with K15NO3 as the only nitrogen source for quantitative comparative proteomic studies following biological treatment of interest 3. By mixing equal ratios of labeled and unlabeled cell cultures for joint protein extraction the influence of preparation steps on final quantitative result is kept at a minimum. Also loss of material during extraction will affect both control and treatment samples in the same way, and therefore the ratio of light and heave peptide will remain constant. In the proposed method either labeled or unlabeled cell culture undergoes a biological treatment, while the other serves as control 4. PMID:24121251

Alteration of Proteins and Pigments Influence the Function of Photosystem I under Iron Deficiency from Chlamydomonas reinhardtii

PubMed Central

Yadavalli, Venkateswarlu; Jolley, Craig C.; Malleda, Chandramouli; Thangaraj, Balakumar; Fromme, Petra; Subramanyam, Rajagopal

2012-01-01

Background Iron is an essential micronutrient for all organisms because it is a component of enzyme cofactors that catalyze redox reactions in fundamental metabolic processes. Even though iron is abundant on earth, it is often present in the insoluble ferric [Fe (III)] state, leaving many surface environments Fe-limited. The haploid green alga Chlamydomonas reinhardtii is used as a model organism for studying eukaryotic photosynthesis. This study explores structural and functional changes in PSI-LHCI supercomplexes under Fe deficiency as the eukaryotic photosynthetic apparatus adapts to Fe deficiency. Results 77K emission spectra and sucrose density gradient data show that PSI and LHCI subunits are affected under iron deficiency conditions. The visible circular dichroism (CD) spectra associated with strongly-coupled chlorophyll dimers increases in intensity. The change in CD signals of pigments originates from the modification of interactions between pigment molecules. Evidence from sucrose gradients and non-denaturing (green) gels indicates that PSI-LHCI levels were reduced after cells were grown for 72 h in Fe-deficient medium. Ultrafast fluorescence spectroscopy suggests that red-shifted pigments in the PSI-LHCI antenna were lost during Fe stress. Further, denaturing gel electrophoresis and immunoblot analysis reveals that levels of the PSI subunits PsaC and PsaD decreased, while PsaE was completely absent after Fe stress. The light harvesting complexes were also susceptible to iron deficiency, with Lhca1 and Lhca9 showing the most dramatic decreases. These changes in the number and composition of PSI-LHCI supercomplexes may be caused by reactive oxygen species, which increase under Fe deficiency conditions. Conclusions Fe deficiency induces rapid reduction of the levels of photosynthetic pigments due to a decrease in chlorophyll synthesis. Chlorophyll is important not only as a light-harvesting pigment, but also has a structural role, particularly in the pigment-rich LHCI subunits. The reduced level of chlorophyll molecules inhibits the formation of large PSI-LHCI supercomplexes, further decreasing the photosynthetic efficiency. PMID:22514709

Failure of Lactoperoxidase to Iodinate Specifically the Plasma Membrane of Cucurbita Tissue Segments

PubMed Central

Quail, Peter H.; Browning, Alan

1977-01-01

An attempt has been made to use lactoperoxidase-catalyzed iodination of excised Cucurbita hypocotyl hooks to monitor the distribution of plasma membrane fragments relative to that of phytochrome in particulate fractions from this tissue. Upon fractionation, the iodinated tissue yields a 20,000g pellet which contains 58% of the trichloroacetic acid-precipitable 125I at a specific radioactivity 12 times that of the proteins in the supernatant. On sucrose gradients, the labeled fraction has a mean isopycnic density of 1.15 g · cm−3. The distribution profile is distinct from that of the total particulate protein and does not coincide with either mitochondrial or endoplasmic reticulum markers. These observations satisfy operational criteria commonly accepted in other systems as indices of selective labeling of the cell surface. The sucrose gradient profiles of the phytochrome and 125I in the 20,000g pellets are noncoincident. In the absence of more direct evidence, this is readily interpreted to indicate a lack of association of the pigment with the plasma membrane. Autoradiographic analysis indicates, however, that the 125I is almost exclusively associated with an amorphous film (possibly phloem-exudate protein) overlying the cut cells at the point of prelabeling excision and along the outer physical surface of the hypocotyl cuticle. No evidence of plasma membrane labeling is apparent. The observed membrane-like behavior of the iodinated material upon cell fractionation is attributed to the preferential posthomogenization association of this material with a particular membrane fraction(s). These data indicate that in addition to the well recognized potential for spurious labeling of the internal cytoplasmic proteins of leaky cells, a further source of ambiguity in surface-labeling experiments should be considered. That is, the potential for labeling extracellular proteins of nonplasma membrane origin but with a capacity to become associated with membranes upon homogenization. Images PMID:16659933

Effects of xylitol as a sugar substitute on diabetes-related parameters in nondiabetic rats.

PubMed

Islam, Md Shahidul

2011-05-01

Abstract The present study was examined the effects of xylitol feeding on diabetes-associated parameters in nondiabetic rats. Seven-week-old male Sprague-Dawley rats were randomly divided into three groups: control (five rats), sucrose (six rats), and xylitol (six rats). Animal had free access to a commercial rat pellet diet, and ad libitum water, 10% sucrose solution, and 10% xylitol solution were supplied to the control, sucrose, and xylitol groups, respectively. After 3 weeks of feeding of experimental diets, food intakes were significantly (P<.05) lower in the sucrose and xylitol groups compared with the control group. Drink intake was significantly higher in the sucrose group but significantly lower in the xylitol group compared with the control group. Body weight gain was significantly lower in the xylitol group compared with the sucrose group. Weekly nonfasting blood glucose was significantly increased, but fasting blood glucose was significantly decreased, in the sucrose group compared with the control and xylitol groups. Significantly better glucose tolerance was observed in the xylitol group compared with the control and sucrose groups. Serum insulin and fructosamine concentrations were not significantly influenced by the feeding of xylitol or sucrose. Relative liver weight and liver glycogen were significantly increased in the xylitol group compared with the sucrose group, whereas no difference was observed between the xylitol and control groups. Serum total cholesterol and low-density lipoprotein-cholesterol were significantly decreased in the sucrose and xylitol groups, and serum triglyceride of the xylitol group, but not the sucrose group, was significantly increased compared with the control group. Data of this study suggest that xylitol can be a better sweetener than sucrose to maintain diabetes-related parameters at a physiologically safer and stable condition.

Physiological Aspects of Sugar Exchange between the Gametophyte and the Sporophyte of Polytrichum formosum

PubMed Central

Renault, Sylvie; Bonnemain, Jean Louis; Faye, Loïc; Gaudillere, Jean Pierre

1992-01-01

The sporophyte of bryophytes is dependent on the gametophyte for its carbon nutrition. This is especially true of the sporophytes of Polytrichum species, and it was generally thought that sucrose was the main form of sugar for long distance transport in the leptom. In Polytrichum formosum, sucrose was the main soluble sugar of the sporophyte and gametophyte tissues, and the highest concentration (about 230 mm) was found in the haustorium. In contrast, sugars collected from the vaginula apoplast were mainly hexoses, with traces of sucrose and trehalose. p-Chloromercuribenzene sulfonate, a nonpermeant inhibitor of the cell wall invertase, strongly reduced the hexose to sucrose ratio. The highest cell wall invertase activity (pH 4.5) was located in the vaginula, whereas the highest activity of a soluble invertase (pH 7.0) was found in both the vaginula and the haustorium. Glucose uptake was carrier-mediated but only weakly dependent on the external pH and the transmembrane electrical gradient, in contrast to amino acid uptake (S. Renault, C. Despeghel-Caussin, J.L. Bonnemain, S. Delrot [1989] Plant Physiol 90: 913-920). Furthermore, addition of 5 or 50 mm glucose to the incubation medium induced a marginal depolarization of the transmembrane potential difference of the transfer cells and had no effect on the pH of this medium. Glucose was converted to sucrose after its absorption into the haustorium. These results demonstrate the noncontinuity of sucrose at the gametophyte/sporophyte interface. They suggest that its conversion to glucose and fructose at this interface, and the subsequent reconversion to sucrose after hexose absorption by haustorium cells, mainly governs sugar accumulation in this latter organ. PMID:16653202

Honey promotes lower weight gain, adiposity, and triglycerides than sucrose in rats.

PubMed

Nemoseck, Tricia M; Carmody, Erin G; Furchner-Evanson, Allison; Gleason, Marsa; Li, Amy; Potter, Hayley; Rezende, Lauren M; Lane, Kelly J; Kern, Mark

2011-01-01

Various dietary carbohydrates have been linked to obesity and altered adipose metabolism; however, the influences of honey vs common sweeteners have not been fully explored. We hypothesized that in comparison with sucrose, a honey-based diet would promote lower weight gain, adiposity, and related biomarkers (leptin, insulin, and adiponectin) as well as a better blood lipid profile. Thirty-six male Sprague-Dawley rats (228.1 ± 12.5 g) were equally divided by weight into 2 groups (n = 18) and provided free access to 1 of 2 diets of equal energy densities differing only in a portion of the carbohydrate. Diets contained 20% carbohydrate (by weight of total diet) from either clover honey or sucrose. After 33 days, epididymal fat pads were excised and weighed, and blood was collected for analyses of serum concentrations of lipids, glucose, and markers of adiposity and inflammation. Body weight gain was 14.7% lower (P ≤ .05) for rats fed honey, corresponding to a 13.3% lower (P ≤ .05) consumption of food/energy, whereas food efficiency ratios were nearly identical. Epididymal fat weight was 20.1% lower (P ≤ .05) for rats fed honey. Serum concentrations of triglycerides and leptin were lower (P ≤ .05) by 29.6% and 21.6%, respectively, and non-high-density lipoprotein cholesterol was higher (P ≤ .05) by 16.8% for honey-fed rats. No significant differences in serum total cholesterol, high-density lipoprotein cholesterol, adiponectin, C-reactive protein, monocyte chemoattractant protein-1, glucose, or insulin were detected. These results suggest that in comparison with sucrose, honey may reduce weight gain and adiposity, presumably due to lower food intake, and promote lower serum triglycerides but higher non-high-density lipoprotein cholesterol concentrations. Copyright © 2011 Elsevier Inc. All rights reserved.

Composition, quaternary structure, and catalytic properties of D-ribulose-1, 5-bisphosphate carboxylase from Euglena gracilis.

PubMed

McFadden, B A; Lord, J M; Rowe, A; Dilks, S

1975-05-01

D-Ribulose-1,5-bisphosphate carboxylase has been purified in one step by sedimenting extracts of autotrophically-grown Euglena gracilis into a linear 0.2-0.8 M sucrose density gradient. The resultant product was pure by the criteria of disc electrophoresis in gels polymerized from 5 or 7.5% acrylamide and sedimentation. The molecular weight of the enzyme estimated by density gradient centrifugation and electrophoresis in gels polymerized from various concentrations of acrylamide was 5.25 X 10(5). The S20,W was 16.4 S. Dissociation and polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulphate established that the enzyme was composed of two types of subunits (mr 50,000 and 15,000). The oligomeric structure was visualized through negative staining and transmission electron microscopy leading to a model for the quaternary structure. Although the enzyme was moderately unstable, the estimated maximal specific activity was 1.6 mumol CO2 fixed min-1 mg protien-1 at 30 degrees C and pH 8.0 Km values were 2.2 m M, 15. 1 MUM and 0.63 mM for Mg2+, ribulose 1,5-bisphosphate, and CO2, respectively, when measured under air. 6-Phospho-D-gluconate was a noncompetitive inhibitor with respect to ribulose 1,5-bisphosphate (Ki = 0.04 mM). Oxygen was a competitive inhibitor with respect to CO2 suggesting that the enzyme was also an oxygenase. The latter was confirmed by experiments showing a molar equivalence between ribulose-1,5-bisphosphate-dependent oxygen consumption and phosphoglycerate production.

A membrane-anchored E-type endo-1,4-beta-glucanase is localized on Golgi and plasma membranes of higher plants.

PubMed

Brummell, D A; Catala, C; Lashbrook, C C; Bennett, A B

1997-04-29

Endo-1,4-beta-D-glucanases (EGases, EC 3.2.1.4) are enzymes produced in bacteria, fungi, and plants that hydrolyze polysaccharides possessing a 1,4-beta-D-glucan backbone. All previously identified plant EGases are E-type endoglucanases that possess signal sequences for endoplasmic reticulum entry and are secreted to the cell wall. Here we report the characterization of a novel E-type plant EGase (tomato Cel3) with a hydrophobic transmembrane domain and structure typical of type II integral membrane proteins. The predicted protein is composed of 617 amino acids and possesses seven potential sites for N-glycosylation. Cel3 mRNA accumulates in young vegetative tissues with highest abundance during periods of rapid cell expansion, but is not hormonally regulated. Antibodies raised to a recombinant Cel3 protein specifically recognized three proteins, with apparent molecular masses of 93, 88, and 53 kDa, in tomato root microsomal membranes separated by sucrose density centrifugation. The 53-kDa protein comigrated in the gradient with plasma membrane markers, the 88-kDa protein with Golgi membrane markers, and the 93-kDa protein with markers for both Golgi and plasma membranes. EGase enzyme activity was also found in regions of the density gradient corresponding to both Golgi and plasma membranes, suggesting that Cel3 EGase resides in both membrane systems, the sites of cell wall polymer biosynthesis. The in vivo function of Cel3 is not known, but the only other known membrane-anchored EGase is present in Agrobacterium tumefaciens where it is required for cellulose biosynthesis.

Lysis of Bacillus subtilis Cells by Glycerol and Sucrose Esters of Fatty Acids

PubMed Central

Tsuchido, Tetsuaki; Ahn, Yung-Hoon; Takano, Mitsuo

1987-01-01

The lytic action of glycerol and sucrose esters of fatty acids with different carbon chain lengths on the exponentially growing cells of Bacillus subtilis 168 was investigated. Of each series of esters, glycerol dodecanoate and sucrose hexadecanoate were the most active. Lysis at 1 h after the addition of 0.1 mM glycerol dodecanoate or 20 μg of sucrose hexadecanoate per ml was 81 or 79%, respectively, as evaluated by the reduction in optical density. During this treatment a great loss of viability occurred that preceded lysis. The results that were obtained suggest that autolysis is induced by these esters. The esters caused morphological changes in the cells, but a seeming adaptation of the cells to esters was seen. Images PMID:16347300

CLC-Nt1, a putative chloride channel protein of tobacco, co-localizes with mitochondrial membrane markers.

PubMed Central

Lurin, C; Güclü, J; Cheniclet, C; Carde, J P; Barbier-Brygoo, H; Maurel, C

2000-01-01

The voltage-dependent chloride channel (CLC) family of membrane proteins has cognates in animals, yeast, bacteria and plants, and chloride-channel activity has been assigned to most of the animal homologues. Lack of evidence of CLC functions in plants prompted us to characterize the cellular localization of the tobacco CLC-Nt1 protein. Specific polyclonal antibodies were raised against an N-terminal polypeptide of CLC-Nt1. These antibodies were used to probe membrane proteins prepared by various cell-fractionation methods. These included aqueous two-phase partitioning (for plasma membranes), free-flow electrophoresis (for vacuolar and plasma membranes), intact vacuole isolation, Percoll-gradient centrifugation (for plastids and mitochondria) and stepped, linear, sucrose-density-gradient centrifugation (for mitochondria). Each purified membrane fraction was characterized with specific marker enzyme activities or antibodies. Our studies ruled out the possibility that the major cell localization of CLC-Nt1 was the vacuolar or plasma membranes, the endoplasmic reticulum, the Golgi apparatus or the plastids. In contrast, we showed that the tobacco CLC-Nt1 specifically co-localized with the markers of the mitochondrial inner membrane, cytochrome c oxidase and NAD9 protein. CLC-Nt1 may correspond to the inner membrane anion channel ('IMAC') described previously in animal and plant mitochondria. PMID:10816421

CLC-Nt1, a putative chloride channel protein of tobacco, co-localizes with mitochondrial membrane markers.

PubMed

Lurin, C; Güclü, J; Cheniclet, C; Carde, J P; Barbier-Brygoo, H; Maurel, C

2000-06-01

The voltage-dependent chloride channel (CLC) family of membrane proteins has cognates in animals, yeast, bacteria and plants, and chloride-channel activity has been assigned to most of the animal homologues. Lack of evidence of CLC functions in plants prompted us to characterize the cellular localization of the tobacco CLC-Nt1 protein. Specific polyclonal antibodies were raised against an N-terminal polypeptide of CLC-Nt1. These antibodies were used to probe membrane proteins prepared by various cell-fractionation methods. These included aqueous two-phase partitioning (for plasma membranes), free-flow electrophoresis (for vacuolar and plasma membranes), intact vacuole isolation, Percoll-gradient centrifugation (for plastids and mitochondria) and stepped, linear, sucrose-density-gradient centrifugation (for mitochondria). Each purified membrane fraction was characterized with specific marker enzyme activities or antibodies. Our studies ruled out the possibility that the major cell localization of CLC-Nt1 was the vacuolar or plasma membranes, the endoplasmic reticulum, the Golgi apparatus or the plastids. In contrast, we showed that the tobacco CLC-Nt1 specifically co-localized with the markers of the mitochondrial inner membrane, cytochrome c oxidase and NAD9 protein. CLC-Nt1 may correspond to the inner membrane anion channel ('IMAC') described previously in animal and plant mitochondria.

Subnuclear systems for synthesis of simian virus 40 DNA in vitro.

PubMed Central

Edenberg, H J; Waqar, M A; Huberman, J A

1976-01-01

We have developed two subnuclear systems for synthesis of DNA of simian virus 40 in vitro. We prepare chromatin from infected cells by the method of Hancock [(1974) J. Mol. Biol. 86, 649-663]; these "chromatin bodies" can be disrupted and large debris can be pelleted, leaving a supernatant ("soluble system"). Both chromatin bodies and the soluble system incorporate deoxyribonucleoside triphosphates into nucleoprotein complexes that contain simian virus 40 DNA. The DNA labeled in short pulses sediments in neutral sucrose gradients slightly faster than mature simian virus 40 DNA, as expected for replicating intermediate. When rebanded in alkaline sucrose gradients, about half of the radioactivity is found in short strands (200-300 nucleotides) and half in longer strands (up to full viral size). When these systems are supplemented with a cytoplasmic preparation from HeLa cells, synthesis is stimulated about 5-fold, and the short strands are converted into strands of up to full viral length as well as into covalently closed circles. These subnuclear DNA-replicating systems should be useful for biochemical fractionation and characterization of some of the proteins required for DNA replication. PMID:188037

Cytolytic pore-forming protein associated with the surface membrane of Naegleria fowleri

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

Lowrey, D.M.

1985-01-01

Whole cell homogenates of Naegleria fowleri were examined by hemolytic and /sup 51/Cr-release assays for the presence of cytolytic molecules which may participate in the cytopathogenic action of this amoeba. Two distinct cytolytic activities were found. A surface membrane cytolysin was identified which was found to be avidly associated with membranes possessing an equilibrium density of 1.135 g/cm/sup 3/ in isopycnic sucrose gradients. The activity of the surface membrane cytolysin was not affected by heating at 75/sup 0/C for 30 min. The second cytolytic activity was found in putative lysosomes possessing an equilibrium density of 1.162 g/cm/sup 3/ and wasmore » completely inactivated by heating at 75/sup 0/C for 30 min. Cytolysis produced in the presence of both cytolysins was consistently synergistic with respect to the activity of either cytolysin alone. The lesions produced on erythrocytes by this cooperative process were characterized by electron microscopy as transmembrane pores resembling a number of other cytolytic effector molecules including the ninth component of complement, perforins of cytolytic T lymphocytes, and the alphatoxin of Staphylococcus aureus.« less

Extracellular vesicles mediate signaling between the aqueous humor producing and draining cells in the ocular system

PubMed Central

Lerner, Natalie; Avissar, Sofia

2017-01-01

Purpose Canonical Wnt signaling is associated with glaucoma pathogenesis and intraocular pressure (IOP) regulation. Our goal was to gain insight into the influence of non-pigmented ciliary epithelium (NPCE)-derived exosomes on Wnt signaling by trabecular meshwork (TM) cells. The potential impact of exosomes on Wnt signaling in the ocular drainage system remains poorly understood. Methods Exosomes isolated from media collected from cultured NPCE cells by differential ultracentrifugation were characterized by dynamic light scattering (DLS), tunable resistive pulse sensing (TRPS), and nanoparticle tracking analysis (NTA), sucrose density gradient migration and transmission electron microscopy (TEM). The cellular target specificity of the NPCE-derived exosomes was investigated by confocal microscopy-based monitoring of the uptake of DiD-labeled exosomes over time, as compared to uptake by various cell lines. Changes in Wnt protein levels in TM cells induced by NPCE exosomes were evaluated by Western blot. Results Exosomes derived from NPCE cells were purified and detected as small rounded 50–140 nm membrane vesicles, as defined by DLS, NTA, TRPS and TEM. Western blot analysis indicated that the nanovesicles were positive for classic exosome markers, including Tsg101 and Alix. Isolated nanoparticles were found in sucrose density fractions typical of exosomes (1.118–1.188 g/mL sucrose). Using confocal microscopy, we demonstrated time-dependent specific accumulation of the NPCE-derived exosomes in NTM cells. Other cell lines investigated hardly revealed any exosome uptake. We further showed that exosomes induced changes in Wnt signaling protein expression in the TM cells. Western blot analysis further revealed decreased phosphorylation of GKS3β and reduced β-catenin levels. Finally, we found that treatment of NTM cells with exosomes resulted in a greater than 2-fold decrease in the level of β-catenin in the cytosolic fraction. In contrast, no remarkable difference in the amount of β-catenin in the nuclear fraction was noted, relative to the control. Conclusions The data suggest that NPCE cells release exosome-like vesicles and that these nanoparticles affect canonical Wnt signaling in TM cells. These findings may have therapeutic relevance since canonical Wnt pathway is involved in intra-ocular pressure regulation. Further understanding of NPCE-derived exosome-responsive signaling pathways may reveal new targets for pharmacological intervention within the drainage system as a target for glaucoma therapy. PMID:28241021

Extracellular vesicles mediate signaling between the aqueous humor producing and draining cells in the ocular system.

PubMed

Lerner, Natalie; Avissar, Sofia; Beit-Yannai, Elie

2017-01-01

Canonical Wnt signaling is associated with glaucoma pathogenesis and intraocular pressure (IOP) regulation. Our goal was to gain insight into the influence of non-pigmented ciliary epithelium (NPCE)-derived exosomes on Wnt signaling by trabecular meshwork (TM) cells. The potential impact of exosomes on Wnt signaling in the ocular drainage system remains poorly understood. Exosomes isolated from media collected from cultured NPCE cells by differential ultracentrifugation were characterized by dynamic light scattering (DLS), tunable resistive pulse sensing (TRPS), and nanoparticle tracking analysis (NTA), sucrose density gradient migration and transmission electron microscopy (TEM). The cellular target specificity of the NPCE-derived exosomes was investigated by confocal microscopy-based monitoring of the uptake of DiD-labeled exosomes over time, as compared to uptake by various cell lines. Changes in Wnt protein levels in TM cells induced by NPCE exosomes were evaluated by Western blot. Exosomes derived from NPCE cells were purified and detected as small rounded 50-140 nm membrane vesicles, as defined by DLS, NTA, TRPS and TEM. Western blot analysis indicated that the nanovesicles were positive for classic exosome markers, including Tsg101 and Alix. Isolated nanoparticles were found in sucrose density fractions typical of exosomes (1.118-1.188 g/mL sucrose). Using confocal microscopy, we demonstrated time-dependent specific accumulation of the NPCE-derived exosomes in NTM cells. Other cell lines investigated hardly revealed any exosome uptake. We further showed that exosomes induced changes in Wnt signaling protein expression in the TM cells. Western blot analysis further revealed decreased phosphorylation of GKS3β and reduced β-catenin levels. Finally, we found that treatment of NTM cells with exosomes resulted in a greater than 2-fold decrease in the level of β-catenin in the cytosolic fraction. In contrast, no remarkable difference in the amount of β-catenin in the nuclear fraction was noted, relative to the control. The data suggest that NPCE cells release exosome-like vesicles and that these nanoparticles affect canonical Wnt signaling in TM cells. These findings may have therapeutic relevance since canonical Wnt pathway is involved in intra-ocular pressure regulation. Further understanding of NPCE-derived exosome-responsive signaling pathways may reveal new targets for pharmacological intervention within the drainage system as a target for glaucoma therapy.

Measuring the Density of a Sugar Solution: A General Chemistry Experiment Using a Student-Prepared Unknown

ERIC Educational Resources Information Center

Peterson, Karen I.

2008-01-01

The experiment developed in this article addresses the concept of equipment calibration for reducing systematic error. It also suggests simple student-prepared sucrose solutions for which accurate densities are known, but not readily available to students. Densities are measured with simple glassware that has been calibrated using the density of…

Combining Step Gradients and Linear Gradients in Density.

PubMed

Kumar, Ashok A; Walz, Jenna A; Gonidec, Mathieu; Mace, Charles R; Whitesides, George M

2015-06-16

Combining aqueous multiphase systems (AMPS) and magnetic levitation (MagLev) provides a method to produce hybrid gradients in apparent density. AMPS—solutions of different polymers, salts, or surfactants that spontaneously separate into immiscible but predominantly aqueous phases—offer thermodynamically stable steps in density that can be tuned by the concentration of solutes. MagLev—the levitation of diamagnetic objects in a paramagnetic fluid within a magnetic field gradient—can be arranged to provide a near-linear gradient in effective density where the height of a levitating object above the surface of the magnet corresponds to its density; the strength of the gradient in effective density can be tuned by the choice of paramagnetic salt and its concentrations and by the strength and gradient in the magnetic field. Including paramagnetic salts (e.g., MnSO4 or MnCl2) in AMPS, and placing them in a magnetic field gradient, enables their use as media for MagLev. The potential to create large steps in density with AMPS allows separations of objects across a range of densities. The gradients produced by MagLev provide resolution over a continuous range of densities. By combining these approaches, mixtures of objects with large differences in density can be separated and analyzed simultaneously. Using MagLev to add an effective gradient in density also enables tuning the range of densities captured at an interface of an AMPS by simply changing the position of the container in the magnetic field. Further, by creating AMPS in which phases have different concentrations of paramagnetic ions, the phases can provide different resolutions in density. These results suggest that combining steps in density with gradients in density can enable new classes of separations based on density.

Switching the mode of sucrose utilization by Saccharomyces cerevisiae

PubMed Central

Badotti, Fernanda; Dário, Marcelo G; Alves, Sergio L; Cordioli, Maria Luiza A; Miletti, Luiz C; de Araujo, Pedro S; Stambuk, Boris U

2008-01-01

Background Overflow metabolism is an undesirable characteristic of aerobic cultures of Saccharomyces cerevisiae during biomass-directed processes. It results from elevated sugar consumption rates that cause a high substrate conversion to ethanol and other bi-products, severely affecting cell physiology, bioprocess performance, and biomass yields. Fed-batch culture, where sucrose consumption rates are controlled by the external addition of sugar aiming at its low concentrations in the fermentor, is the classical bioprocessing alternative to prevent sugar fermentation by yeasts. However, fed-batch fermentations present drawbacks that could be overcome by simpler batch cultures at relatively high (e.g. 20 g/L) initial sugar concentrations. In this study, a S. cerevisiae strain lacking invertase activity was engineered to transport sucrose into the cells through a low-affinity and low-capacity sucrose-H+ symport activity, and the growth kinetics and biomass yields on sucrose analyzed using simple batch cultures. Results We have deleted from the genome of a S. cerevisiae strain lacking invertase the high-affinity sucrose-H+ symporter encoded by the AGT1 gene. This strain could still grow efficiently on sucrose due to a low-affinity and low-capacity sucrose-H+ symport activity mediated by the MALx1 maltose permeases, and its further intracellular hydrolysis by cytoplasmic maltases. Although sucrose consumption by this engineered yeast strain was slower than with the parental yeast strain, the cells grew efficiently on sucrose due to an increased respiration of the carbon source. Consequently, this engineered yeast strain produced less ethanol and 1.5 to 2 times more biomass when cultivated in simple batch mode using 20 g/L sucrose as the carbon source. Conclusion Higher cell densities during batch cultures on 20 g/L sucrose were achieved by using a S. cerevisiae strain engineered in the sucrose uptake system. Such result was accomplished by effectively reducing sucrose uptake by the yeast cells, avoiding overflow metabolism, with the concomitant reduction in ethanol production. The use of this modified yeast strain in simpler batch culture mode can be a viable option to more complicated traditional sucrose-limited fed-batch cultures for biomass-directed processes of S. cerevisiae. PMID:18304329

Switching the mode of sucrose utilization by Saccharomyces cerevisiae.

PubMed

Badotti, Fernanda; Dário, Marcelo G; Alves, Sergio L; Cordioli, Maria Luiza A; Miletti, Luiz C; de Araujo, Pedro S; Stambuk, Boris U

2008-02-27

Overflow metabolism is an undesirable characteristic of aerobic cultures of Saccharomyces cerevisiae during biomass-directed processes. It results from elevated sugar consumption rates that cause a high substrate conversion to ethanol and other bi-products, severely affecting cell physiology, bioprocess performance, and biomass yields. Fed-batch culture, where sucrose consumption rates are controlled by the external addition of sugar aiming at its low concentrations in the fermentor, is the classical bioprocessing alternative to prevent sugar fermentation by yeasts. However, fed-batch fermentations present drawbacks that could be overcome by simpler batch cultures at relatively high (e.g. 20 g/L) initial sugar concentrations. In this study, a S. cerevisiae strain lacking invertase activity was engineered to transport sucrose into the cells through a low-affinity and low-capacity sucrose-H+ symport activity, and the growth kinetics and biomass yields on sucrose analyzed using simple batch cultures. We have deleted from the genome of a S. cerevisiae strain lacking invertase the high-affinity sucrose-H+ symporter encoded by the AGT1 gene. This strain could still grow efficiently on sucrose due to a low-affinity and low-capacity sucrose-H+ symport activity mediated by the MALx1 maltose permeases, and its further intracellular hydrolysis by cytoplasmic maltases. Although sucrose consumption by this engineered yeast strain was slower than with the parental yeast strain, the cells grew efficiently on sucrose due to an increased respiration of the carbon source. Consequently, this engineered yeast strain produced less ethanol and 1.5 to 2 times more biomass when cultivated in simple batch mode using 20 g/L sucrose as the carbon source. Higher cell densities during batch cultures on 20 g/L sucrose were achieved by using a S. cerevisiae strain engineered in the sucrose uptake system. Such result was accomplished by effectively reducing sucrose uptake by the yeast cells, avoiding overflow metabolism, with the concomitant reduction in ethanol production. The use of this modified yeast strain in simpler batch culture mode can be a viable option to more complicated traditional sucrose-limited fed-batch cultures for biomass-directed processes of S. cerevisiae.

Electrophysiological approach to determine kinetic parameters of sucrose uptake by single sieve elements or phloem parenchyma cells in intact Vicia faba plants.

PubMed

Hafke, Jens B; Höll, Sabina-Roxana; Kühn, Christina; van Bel, Aart J E

2013-01-01

Apart from cut aphid stylets in combination with electrophysiology, no attempts have been made thus far to measure in vivo sucrose-uptake properties of sieve elements. We investigated the kinetics of sucrose uptake by single sieve elements and phloem parenchyma cells in Vicia faba plants. To this end, microelectrodes were inserted into free-lying phloem cells in the main vein of the youngest fully-expanded leaf, half-way along the stem, in the transition zone between the autotrophic and heterotrophic part of the stem, and in the root axis. A top-to-bottom membrane potential gradient of sieve elements was observed along the stem (-130 mV to -110 mV), while the membrane potential of the phloem parenchyma cells was stable (approx. -100 mV). In roots, the membrane potential of sieve elements dropped abruptly to -55 mV. Bathing solutions having various sucrose concentrations were administered and sucrose/H(+)-induced depolarizations were recorded. Data analysis by non-linear least-square data fittings as well as by linear Eadie-Hofstee (EH) -transformations pointed at biphasic Michaelis-Menten kinetics (2 MM, EH: K m1 1.2-1.8 mM, K m2 6.6-9.0 mM) of sucrose uptake by sieve elements. However, Akaike's Information Criterion (AIC) favored single MM kinetics. Using single MM as the best-fitting model, K m values for sucrose uptake by sieve elements decreased along the plant axis from 1 to 7 mM. For phloem parenchyma cells, higher K m values (EH: K m1 10 mM, K m2 70 mM) as compared to sieve elements were found. In preliminary patch-clamp experiments with sieve-element protoplasts, small sucrose-coupled proton currents (-0.1 to -0.3 pA/pF) were detected in the whole-cell mode. In conclusion (a) K m values for sucrose uptake measured by electrophysiology are similar to those obtained with heterologous systems, (b) electrophysiology provides a useful tool for in situ determination of K m values, (c) As yet, it remains unclear if one or two uptake systems are involved in sucrose uptake by sieve elements, (d) Affinity for sucrose uptake by sieve elements exceeds by far that by phloem parenchyma cells, (e) Patch-clamp studies provide a feasible basis for quantification of sucrose uptake by single cells. The consequences of the findings for whole-plant carbohydrate partitioning are discussed.

Electrophysiological approach to determine kinetic parameters of sucrose uptake by single sieve elements or phloem parenchyma cells in intact Vicia faba plants

PubMed Central

Hafke, Jens B.; Höll, Sabina-Roxana; Kühn, Christina; van Bel, Aart J. E.

2013-01-01

Apart from cut aphid stylets in combination with electrophysiology, no attempts have been made thus far to measure in vivo sucrose-uptake properties of sieve elements. We investigated the kinetics of sucrose uptake by single sieve elements and phloem parenchyma cells in Vicia faba plants. To this end, microelectrodes were inserted into free-lying phloem cells in the main vein of the youngest fully-expanded leaf, half-way along the stem, in the transition zone between the autotrophic and heterotrophic part of the stem, and in the root axis. A top-to-bottom membrane potential gradient of sieve elements was observed along the stem (−130 mV to −110 mV), while the membrane potential of the phloem parenchyma cells was stable (approx. −100 mV). In roots, the membrane potential of sieve elements dropped abruptly to −55 mV. Bathing solutions having various sucrose concentrations were administered and sucrose/H+-induced depolarizations were recorded. Data analysis by non-linear least-square data fittings as well as by linear Eadie–Hofstee (EH) -transformations pointed at biphasic Michaelis–Menten kinetics (2 MM, EH: Km1 1.2–1.8 mM, Km2 6.6–9.0 mM) of sucrose uptake by sieve elements. However, Akaike's Information Criterion (AIC) favored single MM kinetics. Using single MM as the best-fitting model, Km values for sucrose uptake by sieve elements decreased along the plant axis from 1 to 7 mM. For phloem parenchyma cells, higher Km values (EH: Km1 10 mM, Km2 70 mM) as compared to sieve elements were found. In preliminary patch-clamp experiments with sieve-element protoplasts, small sucrose-coupled proton currents (−0.1 to −0.3 pA/pF) were detected in the whole-cell mode. In conclusion (a) Km values for sucrose uptake measured by electrophysiology are similar to those obtained with heterologous systems, (b) electrophysiology provides a useful tool for in situ determination of Km values, (c) As yet, it remains unclear if one or two uptake systems are involved in sucrose uptake by sieve elements, (d) Affinity for sucrose uptake by sieve elements exceeds by far that by phloem parenchyma cells, (e) Patch-clamp studies provide a feasible basis for quantification of sucrose uptake by single cells. The consequences of the findings for whole-plant carbohydrate partitioning are discussed. PMID:23914194

Avocado oil supplementation modifies cardiovascular risk profile markers in a rat model of sucrose-induced metabolic changes.

PubMed

Carvajal-Zarrabal, Octavio; Nolasco-Hipolito, Cirilo; Aguilar-Uscanga, M Guadalupe; Melo-Santiesteban, Guadalupe; Hayward-Jones, Patricia M; Barradas-Dermitz, Dulce M

2014-01-01

The purpose of this study was to evaluate the effects of avocado oil administration on biochemical markers of cardiovascular risk profile in rats with metabolic changes induced by sucrose ingestion. Twenty-five rats were divided into five groups: a control group (CG; basic diet), a sick group (MC; basic diet plus 30% sucrose solution), and three other groups (MCao, MCac, and MCas; basic diet plus 30% sucrose solution plus olive oil and avocado oil extracted by centrifugation or using solvent, resp.). Glucose, total cholesterol, triglycerides, phospholipids, low- and high-density lipoproteins (LDL, HDL), very low-density lipoprotein (VLDL), lactic dehydrogenase, creatine kinase, and high sensitivity C-reactive protein concentration were analyzed. Avocado oil reduces TG, VLDL, and LDL levels, in the LDL case significantly so, without affecting HDL levels. An effect was exhibited by avocado oil similar to olive oil, with no significant difference between avocado oil extracted either by centrifugation or solvent in myocardial injury biochemical indicators. Avocado oil decreased hs-CRP levels, indicating that inflammatory processes were partially reversed. These findings suggested that avocado oil supplementation has a positive health outcome because it reduces inflammatory events and produces positive changes in the biochemical indicators studied, related to the development of metabolic syndrome.

Analysis of soybean leaf metabolism and seed coat transcriptome reveal sink strength is maintained under abiotic stress conditions

USDA-ARS?s Scientific Manuscript database

The seed coat is a vital tissue for directing the flow of photosynthate from source leaves to the embryo and cotyledons during seed development. By forming a sucrose gradient, the seed coat promotes transport of sugars from source leaves to seeds, thereby establishing sink strength. Understanding th...

Cariogenicity features of Streptococcus mutans in presence of rubusoside.

PubMed

Chu, Jinpu; Zhang, Tieting; He, Kexin

2016-05-11

One promising way of reducing caries is by using sucrose substitutes in food. rubusoside is a prototype sweet substance isolated from the leaves of the plant Rubrus suavissimus S. Lee. (Rosaceae), and is rated sweeter than sucrose. The purpose of this study was to investigate the effects of rubusoside on Streptococcus mutans growth, acidogenicity, and adherence to glass in vitro. The effects of rubusoside on the growth and glass surface adhering of Streptococcus mutans were investigated by measuring the optical density of the culture at 540 nm with a spectrophotometer. Rubusoside influence on Streptococcus mutans acidogenicity was determined by measuring the pH of the culture. Sucrose, glucose, maltose, fructose and xylitol were designed to compare with rubusoside. S. mutans growth in the rubusoside-treated group was significantly lower than that in the sucrose, glucose, maltose and fructose groups (p < 0.05) except for xylitol group (p > 0.05). Sucrose-treated S. mutans exhibited the highest adherence to glass, and rubusoside-treated S. mutans exhibited the lowest. S. mutans adherence to a glass surface and acidogenicity with sucrose were significantly reduced by rubusoside. Rubusoside may have some potential as a non-cariogenic, non-caloric sweetener.

Hyperphagia induced by sucrose: relation to circulating and CSF glucose and corticosterone and orexigenic peptides in the arcuate nucleus.

PubMed

Gaysinskaya, V A; Karatayev, O; Shuluk, J; Leibowitz, S F

2011-01-01

Sucrose-rich diets compared to starch-rich diets are known to stimulate overeating under chronic conditions. The present study in normal-weight rats established an acute "preload-to-test meal" paradigm for demonstrating sucrose-induced hyperphagia and investigating possible mechanisms that mediate this behavioral phenomenon. In this acute paradigm, the rats were first given a small (15 kcal) sucrose preload (30% sucrose) for 30 min compared to an equicaloric, starch preload (25% starch with 5% sucrose) and then allowed to freely consume a subsequent test meal of lab chow. The sucrose preload, when compared to a starch preload equal in energy density and palatability, consistently increased food intake in the subsequent test meal occurring between 60 and 120 min after the end of the preload. Measurements of hormones, metabolites and hypothalamic peptides immediately preceding this hyperphagia revealed marked differences between the sucrose vs starch groups that could contribute to the increase in food intake. Whereas the sucrose group compared to the starch group immediately after the preload (at 10 min) had elevated levels of glucose in serum and cerebrospinal fluid (CSF) along with reduced expressions of neuropeptide Y (NPY) and agouti-related protein (AgRP) in the arcuate nucleus (ARC), the subsequent effects (at 30-60 min) just preceding the test meal hyperphagia were the reverse. Along with lower levels of glucose, they included markedly elevated serum and CSF levels of corticosterone and mRNA levels of NPY and AgRP in the ARC. In addition to establishing an animal model for sucrose-induced hyperphagia, these results demonstrate peripheral and central mechanisms that may mediate this behavioral phenomenon. Copyright © 2010 Elsevier Inc. All rights reserved.

HYPERPHAGIA INDUCED BY SUCROSE: RELATION TO CIRCULATING AND CSF GLUCOSE AND CORTICOSTERONE AND OREXIGENIC PEPTIDES IN THE ARCUATE NUCLEUS

PubMed Central

Gaysinskaya, V. A.; Karatayev, O.; Shuluk, J.; Leibowitz, S. F.

2010-01-01

Sucrose-rich diets compared to starch-rich diets are known to stimulate overeating under chronic conditions. The present study in normal-weight rats established an acute “preload-to-test meal” paradigm for demonstrating sucrose-induced hyperphagia and investigating possible mechanisms that mediate this behavioral phenomenon. In this acute paradigm, the rats were first given a small (15 kcals) sucrose preload (30% sucrose) for 30 min compared to an equicaloric, starch preload (25% starch with 5% sucrose) and then allowed to freely consume a subsequent test meal of lab chow. The sucrose preload, when compared to a starch preload equal in energy density and palatability, consistently increased food intake in the subsequent test meal occurring between 60–120 min after the end of the preload. Measurements of hormones, metabolites and hypothalamic peptides immediately preceding this hyperphagia revealed marked differences between the sucrose vs starch groups that could contribute to the increase in food intake. Whereas the sucrose group compared to starch group immediately after the preload (at 10 min) had elevated levels of glucose in serum and cerebrospinal fluid (CSF) along with reduced expression of neuropeptide Y (NPY) and agouti-related protein (AgRP) in the arcuate nucleus (ARC), the subsequent effects (at 30–60 min) just preceding the test meal hyperphagia were the reverse. Along with lower levels of glucose, they included markedly elevated serum and CSF levels of corticosterone and mRNA levels of NPY and AgRP in the ARC. In addition to establishing an animal model for sucrose-induced hyperphagia, these results demonstrate peripheral and central mechanisms that may mediate this behavioral phenomenon. PMID:21036188

Incomplete antibodies and immunoglobulin characterization in adult urodeles, Pleurodeles waltlii Michah. and Triturus alpestris Laur.

PubMed Central

Tournefier, A

1975-01-01

Humoral immunoglobulin synthesis has been studied in two adult urodeles, Pleurodeles waltlii Michah. and Triturus alpestris Laur. following SRBC immunization. The specific antibody response is detected after a long period of immunization and is due exclusively to 'incomplete' antibodies which are unable to induce agglutination. The antibody titre is essentially dependent on the number of stimulations rather than on the dose or nature of the antigen (papainized or normal erythrocytes). Antibodies are detected in only 50 per cent of the immunized animals, 50 per cent never respond. This suggests that the latter group does not possess the genetic equipment (Ir genes) to recognize the antigenic determinants and to synthesize the corresponding antibodies. The sedimentation coefficient of the synthesized immunoglobulins was investigated by sucrose density gradient centrifugation and their characterization was carried out by starch and polyacrylamide gel electrophoresis. With this peculiar antigen even after a booster injection, only one class of immunoglobulin, an 18-2S IgM could be detected. PMID:49296

d-Alanine Oxidase from Escherichia coli: Localization and Induction by l-Alanine

PubMed Central

Raunio, R. P.; Jenkins, W. T.

1973-01-01

Dialyzed membranes of Escherichia coli prepared by an ethylenediaminetetraacetic acid-lysozyme method catalyze the oxidation of both l-alanine and d-alanine. The specific activities for the oxidations of both d-alanine and l-alanine are increased fivefold when the cells are grown in the presence of either l-alanine or dl-alanine, but are increased only slightly when grown in the presence of d-alanine. In the dl-alanine-induced system, the specific activities for the oxidations of some other d-amino acids are also raised. dl-alanine also induces two other alanine catabolizing enzymes, alanine dehydrogenase and alanine-glutamate aminotransferase which are found in the “soluble” fraction of lysozyme-treated cells. The oxidations of both l-alanine and d-alanine were associated with the membranes of induced cells. After the membranes were disintegrated by sonic treatment, both l-alanine and d-alanine oxidation catalysts sedimented in a sucrose density gradient together with d-lactate and l-lactate dehydrogenases, apparently as a single multienzyme complex. PMID:4146872

Giardia and Cryptosporidium spp. dissemination during wastewater treatment and comparative detection via immunofluorescence assay (IFA), nested polymerase chain reaction (nested PCR) and loop mediated isothermal amplification (LAMP).

PubMed

Gallas-Lindemann, Carmen; Sotiriadou, Isaia; Plutzer, Judit; Noack, Michael J; Mahmoudi, Mohammad Reza; Karanis, Panagiotis

2016-06-01

Environmental water samples from the Lower Rhine area in Germany were investigated via immunofluorescence assays (IFAs), nested polymerase chain reaction (nested PCR) and loop-mediated isothermal amplification (LAMP) to detect the presence of Giardia spp. (n=185) and Cryptosporidium spp. (n=227). The samples were concentrated through filtration or flocculation, and oocysts were purified via centrifugation through a sucrose density gradient. For all samples, IFA was performed first, followed by DNA extraction for the nested PCR and LAMP assays. Giardia cysts were detected in 105 samples (56.8%) by IFA, 62 samples (33.5%) by nested PCR and 79 samples (42.7%) by LAMP. Cryptosporidium spp. were detected in 69 samples (30.4%) by IFA, 95 samples (41.9%) by nested PCR and 99 samples (43.6%) by LAMP. According to these results, the three detection methods are complementary for monitoring Giardia and Cryptosporidium in environmental waters. Copyright © 2016 Elsevier B.V. All rights reserved.

Adrenocorticotropin receptors: Functional expression from rat adrenal mRNA in Xenopus laevis oocytes

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

Mertz, L.M.; Catt, K.J.

1991-10-01

The adrenocorticotropin (ACTH) receptor, which binds corticotropin and stimulates adenylate cyclase and steroidogenesis in adrenocortical cells, was expressed in Xenopus laevis oocytes microinjected with rat adrenal poly(A){sup +} RNA. Expression of the ACTH receptor in individual stage 5 and 6 oocytes was monitored by radioimmunoassay of ligand-stimulated cAMP production. Injection of 5-40 ng of adrenal mRNA caused dose-dependent increases in ACTH-responsive cAMP production. Size fractionation of rat adrenal poly(A){sup +}RNA by sucrose density-gradient centrifugation revealed that mRNA encoding the ACTH receptor was present in the 1.1-to 2.0-kilobase fraction. These data indicate that ACTH receptors can be expressed from adrenal mRNAmore » in Xenopus oocytes and are fully functional in terms of ligand specificity and signal generation. The extracellular cAMP response to ACTH is a sensitive and convenient index of receptor expression. This system should permit more complete characterization and expression cloning of the ACTH receptor.« less

Enzymatic dehalogenation of pentachlorophenol by extracts from Arthrobacter sp. strain ATCC 33790.

PubMed Central

Schenk, T; Müller, R; Mörsberger, F; Otto, M K; Lingens, F

1989-01-01

Arthrobacter sp. strain ATCC 33790 was grown with pentachlorophenol (PCP) as the sole source of carbon and energy. Crude extracts, which were prepared by disruption of the bacteria with a French pressure cell, showed no dehalogenating activity with PCP as the substrate. After sucrose density ultracentrifugation of the crude extract at 145,000 x g, various layers were found in the gradient. One yellow layer showed enzymatic conversion of PCP. One chloride ion was released per molecule of PCP. The product of the enzymatic conversion was tetrachlorohydroquinone. NADPH and oxygen were essential for this reaction. EDTA stimulated the enzymatic activity by 67%. The optimum pH for the enzyme activity was 7.5, and the temperature optimum was 25 degrees C. Enzymatic activity was also detected with 2,4,5-trichlorophenol, 2,3,4-trichlorophenol, 2,4,6-trichlorophenol, and 2,3,4,5-tetrachlorophenol as substrates, whereas 3,4,5-trichlorophenol, 2,4-dichlorophenol, 3,4-dichlorophenol, and 4-chlorophenol did not serve as substrates. PMID:2793827

Investigations on gel forming media use in low gravity bioseparations research

NASA Technical Reports Server (NTRS)

Todd, Paul; Szlag, David C.; Plank, Lindsay D.; Delcourt, Scott G.; Kunze, M. Elaine

1989-01-01

Research on gelling media and conditions suitable for the preservation of the spatial configuration of cell suspensions and macromolecular solutions after separation in free fluid during low gravity experiments is presented. The examples studied included free electrophoresis of cells in a cylindrical column and two-phase aqueous polymer separation. Microgravity electrophoresis experiments were simulated by separating model cell types (animal or human) in a vertical density gradient containing low-conductivity buffer, 1.7-6.5 percent Ficoll, 6.8-5.0 percent sucrose, and 1 percent SeaPrep low-melting temperature agarose. Upon cooling, a gel formed in the column and cells could be captured at the forming locations. Two-phase extraction experiments were simulated using two-polymer solutions in which phase separation occurs in normal saline at temperatures compatible with cell viability and in which one or both phases form a gel upon cooling. Suitable polymers included commercial agaroses (1-2 percent), maltodextrin (5-7 percent), and gelatin (5-20 percent).

Identification and partial characterization of a latent ATP, Mg-dependent protein phosphatase in rabbit skeletal muscle cytosol.

PubMed

Vandenheede, J R; Staquet, S; Merlevede, W

1989-05-04

Fractionation of rabbit skeletal muscle cytosol on Aminohexyl-Sepharose has resulted in the identification of a latent ATP, Mg-dependent protein phosphatase whose catalytic subunit is in the active conformation, but is inhibited by the presence of more than one modulator unit. The partially purified enzyme is converted to an inactive, kinase FA-dependent form upon incubation at 30 degrees C unless modulator-specific polyclonal antibodies are added to the preparation. The immunoglobulins also relieve the inhibition which is responsible for the low basal phosphatase activity of the enzyme, and they counteract all of the heat-stable inhibitor activity present in the preparation. Addition of free catalytic subunit abolishes the inhibition of the latent enzyme in a dose-dependent way, but cannot prevent the inactivation process. The inactivated phosphatase and the original latent enzyme exhibit the same apparent Mr in sucrose density-gradient centrifugation (70,000) and in gel filtration (110,000).

Characterization of antioxidant enzymes and peroxisomes of olive (Olea europaea L.) fruits.

PubMed

Lopez-Huertas, Eduardo; del Río, Luis A

2014-10-15

The presence of peroxisomes in olive (Olea europaea L.) fruits and different antioxidant enzymes occurring in this plant tissue is reported for the first time. Ultrastructural analysis showed that olive cells were characterized by the presence of large vacuoles and lipid drops. Plastids, mitochondria and peroxisomes were placed near the cell wall, showing some type of association with it. Olive fruit peroxisomes were purified by sucrose density-gradient centrifugation, and catalase, glutathione reductase and ascorbate peroxidase were found in peroxisomes. In olive fruit tissue the presence of a battery of antioxidant enzymes was demonstrated, including catalase, four superoxide dismutase isozymes (mainly an Fe-SOD plus 2 Cu,Zn-SOD and a Mn-SOD), all the enzymes of the ascorbate-glutathione cycle, reduced and oxidized glutathione, ascorbate, and four NADPH-recycling dehydrogenases. The knowledge of the full composition of antioxidants (enzymatic and non-enzymatic) in olive fruits is crucial to be able to understand the processes regulating the antioxidant composition of olive oil. Copyright © 2014 Elsevier GmbH. All rights reserved.

High-Throughput Analysis of Sucrose Fatty Acid Esters by Supercritical Fluid Chromatography/Tandem Mass Spectrometry

PubMed Central

Hori, Katsuhito; Tsumura, Kazunobu; Fukusaki, Eiichiro; Bamba, Takeshi

2014-01-01

Supercritical fluid chromatography (SFC) coupled with triple quadrupole mass spectrometry was applied to the profiling of sucrose fatty acid esters (SEs). The SFC conditions (column and modifier gradient) were optimized for the effective separation of SEs. In the column test, a silica gel reversed-phase column was selected. Then, the method was used for the detailed characterization of commercial SEs and the successful analysis of SEs containing different fatty acids. The present method allowed for fast and high-resolution separation of monoesters to tetra-esters within a shorter time (15 min) as compared to the conventional high-performance liquid chromatography. The applicability of our method for the analysis of SEs was thus demonstrated. PMID:26819875

Oxygen isotope fractionations across individual leaf carbohydrates in grass and tree species.

PubMed

Lehmann, Marco M; Gamarra, Bruno; Kahmen, Ansgar; Siegwolf, Rolf T W; Saurer, Matthias

2017-08-01

Almost no δ 18 O data are available for leaf carbohydrates, leaving a gap in the understanding of the δ 18 O relationship between leaf water and cellulose. We measured δ 18 O values of bulk leaf water (δ 18 O LW ) and individual leaf carbohydrates (e.g. fructose, glucose and sucrose) in grass and tree species and δ 18 O of leaf cellulose in grasses. The grasses were grown under two relative humidity (rH) conditions. Sucrose was generally 18 O-enriched compared with hexoses across all species with an apparent biosynthetic fractionation factor (ε bio ) of more than 27‰ relative to δ 18 O LW , which might be explained by isotopic leaf water and sucrose synthesis gradients. δ 18 O LW and δ 18 O values of carbohydrates and cellulose in grasses were strongly related, indicating that the leaf water signal in carbohydrates was transferred to cellulose (ε bio  = 25.1‰). Interestingly, damping factor p ex p x , which reflects oxygen isotope exchange with less enriched water during cellulose synthesis, responded to rH conditions if modelled from δ 18 O LW but not if modelled directly from δ 18 O of individual carbohydrates. We conclude that δ 18 O LW is not always a good substitute for δ 18 O of synthesis water due to isotopic leaf water gradients. Thus, compound-specific δ 18 O analyses of individual carbohydrates are helpful to better constrain (post-)photosynthetic isotope fractionation processes in plants. © 2017 John Wiley & Sons Ltd.

Phosphoinositide 3-kinase regulates maturation of lysosomes in rat hepatocytes.

PubMed Central

Mousavi, Seyed Ali; Brech, Andreas; Berg, Trond; Kjeken, Rune

2003-01-01

To obtain information about the role of phosphoinositide 3-kinase (PI3K) in the endocytic pathway in hepatocytes, the uptake and intracellular transport of asialo-orosomucoid (ASOR) was followed in cells treated with wortmannin or LY294002. The two inhibitors, at concentrations known to inhibit the enzyme, did not affect internalization or the number of surface asialoglycoprotein receptors, but they caused a paradoxical increase (approx. 50% above control values) in the degradation of ASOR labelled with [(125)I]tyramine cellobiose ([(125)I]TC). Wortmannin or LY204002 inhibited the autophagic sequestration of lactate dehydrogenase very effectively, and the enhanced degradation of [(125)I]TC-ASOR could be an indirect effect of reduced autophagy, as an amino acid mixture known to inhibit autophagy also caused increased degradation of [(125)I]TC-ASOR, and its effect was not additive to that of wortmannin or LY294002. Wortmannin or LY294002 had pronounced effects on the late parts of the endocytic pathway in the hepatocytes: first, dense lysosomes disappeared and were replaced by swollen vesicles; secondly, degradation of [(125)I]TC-ASOR took place in an organelle of lower buoyant density (in a sucrose gradient) than the bulk of lysosomes (identified in the gradient by lysosomal marker enzymes). With increasing length of incubation with wortmannin or LY294002, the density distributions of the lysosomal markers also shifted to lower density and gradually approached that of the labelled degradation products. The labelled degradation products formed from [(125)I]TC-labelled proteins were trapped at the site of formation, because they did not penetrate the vesicle membranes. The results obtained indicate that internalization and intracellular transport of ASOR to lysomes may take place in the absence of PI3K activity in rat hepatocytes. On the other hand, fusion of late endosomes with lysosomes seems to produce 'hybrid organelles' (active lysosomes) that are unable to mature into dense lysosomes. PMID:12646047

Baclofen-induced reductions in optional food intake depend upon food composition.

PubMed

Wojnicki, F H E; Charny, G; Corwin, R L W

2013-05-01

Baclofen reduces intake of some foods but stimulates intake or has no effect on others. The reasons for these differences are not known. The present study examined effects of baclofen when composition, energy density, preference, presentation and intake of optional foods varied. Semi-solid fat emulsions and sucrose products were presented for brief periods to non-food-deprived rats. In Experiment 1, fat and sucrose composition were varied while controlling energy density. In Experiment 2A, schedule of access and the number of optional foods were varied. In Experiment 2B, the biopolymer (thickener) was examined. Baclofen reduced intake of fat and/or sugar options with different energy densities (1.28-9kcal/g), when presented daily or intermittently, and when intakes were relatively high or low. However, the efficacy of baclofen was affected by the biopolymer used to thicken the options: baclofen had no effect when options were thickened with one biopolymer (3173), but reduced intake when options were thickened with another biopolymer (515). Baclofen failed to reduce intake of a concentrated sugar option (64% sucrose), regardless of biopolymer. Based upon these results, caution is urged when interpreting results obtained with products using different thickening agents. Systematic research is needed when designing products used in rat models of food intake. Copyright © 2013 Elsevier Ltd. All rights reserved.

BACLOFEN-INDUCED REDUCTIONS IN OPTIONAL FOOD INTAKE DEPEND UPON FOOD COMPOSITION

PubMed Central

Wojnicki, F.H.E.; Charny, G.; Corwin, R.L.W

2013-01-01

Baclofen reduces intake of some foods but stimulates intake or has no effect on others. The reasons for these differences are not known. The present study examined effects of baclofen when composition, energy density, preference, presentation and intake of optional foods varied. Semi-solid fat emulsions and sucrose products were presented for brief periods to non-food-deprived rats. In Experiment 1, fat and sucrose composition were varied while controlling energy density. In Experiment 2A, schedule of access and the number of optional foods were varied. In Experiment 2B, the biopolymer (thickener) was examined. Baclofen reduced intake of fat and/or sugar options with different energy densities (1.28-9 kcal/g), when presented daily or intermittently, and when intakes were relatively high or low. However, the efficacy of baclofen was affected by the biopolymer used to thicken the options: baclofen had no effect when options were thickened with one biopolymer (3173), but reduced intake when options were thickened with another biopolymer (515). Baclofen failed to reduce intake of a concentrated sugar option (64% sucrose), regardless of biopolymer. Based upon these results, caution is urged when interpreting results obtained with products using different thickening agents. Systematic research is needed when designing products used in rat models of food intake. PMID:23321345

Association of gamma-secretase with lipid rafts in post-Golgi and endosome membranes.

PubMed

Vetrivel, Kulandaivelu S; Cheng, Haipeng; Lin, William; Sakurai, Takashi; Li, Tong; Nukina, Nobuyuki; Wong, Philip C; Xu, Huaxi; Thinakaran, Gopal

2004-10-22

Alzheimer's disease-associated beta-amyloid peptides (Abeta) are generated by the sequential proteolytic processing of amyloid precursor protein (APP) by beta- and gamma-secretases. There is growing evidence that cholesterol- and sphingolipid-rich membrane microdomains are involved in regulating trafficking and processing of APP. BACE1, the major beta-secretase in neurons is a palmitoylated transmembrane protein that resides in lipid rafts. A subset of APP is subject to amyloidogenic processing by BACE1 in lipid rafts, and this process depends on the integrity of lipid rafts. Here we describe the association of all four components of the gamma-secretase complex, namely presenilin 1 (PS1)-derived fragments, mature nicastrin, APH-1, and PEN-2, with cholesterol-rich detergent insoluble membrane (DIM) domains of non-neuronal cells and neurons that fulfill the criteria of lipid rafts. In PS1(-/-)/PS2(-/-) and NCT(-/-) fibroblasts, gamma-secretase components that still remain fail to become detergent-resistant, suggesting that raft association requires gamma-secretase complex assembly. Biochemical evidence shows that subunits of the gamma-secretase complex and three TGN/endosome-resident SNAREs cofractionate in sucrose density gradients, and show similar solubility or insolubility characteristics in distinct non-ionic and zwitterionic detergents, indicative of their co-residence in membrane microdomains with similar protein-lipid composition. This notion is confirmed using magnetic immunoisolation of PS1- or syntaxin 6-positive membrane patches from a mixture of membranes with similar buoyant densities following Lubrol WX extraction or sonication, and gradient centrifugation. These findings are consistent with the localization of gamma-secretase in lipid raft microdomains of post-Golgi and endosomes, organelles previously implicated in amyloidogenic processing of APP.

Association of γ-Secretase with Lipid Rafts in Post-Golgi and Endosome Membranes*

PubMed Central

Vetrivel, Kulandaivelu S.; Cheng, Haipeng; Lin, William; Sakurai, Takashi; Li, Tong; Nukina, Nobuyuki; Wong, Philip C.; Xu, Huaxi; Thinakaran, Gopal

2005-01-01

Alzheimer’s disease-associated β-amyloid peptides (Aβ) are generated by the sequential proteolytic processing of amyloid precursor protein (APP) by β- and γ-secretases. There is growing evidence that cholesterol- and sphingolipid-rich membrane microdomains are involved in regulating trafficking and processing of APP. BACE1, the major γ-secretase in neurons is a palmi-toylated transmembrane protein that resides in lipid rafts. A subset of APP is subject to amyloidogenic processing by BACE1 in lipid rafts, and this process depends on the integrity of lipid rafts. Here we describe the association of all four components of the γ-secretase complex, namely presenilin 1 (PS1)-derived fragments, mature nicastrin, APH-1, and PEN-2, with cholesterol-rich detergent insoluble membrane (DIM) domains of non-neuronal cells and neurons that fulfill the criteria of lipid rafts. In PS1−/−/PS2−/− and NCT−/− fibroblasts, γ-secretase components that still remain fail to become detergent-resistant, suggesting that raft association requires γ-secretase complex assembly. Biochemical evidence shows that subunits of the γ-secretase complex and three TGN/endosome-resident SNAREs cofractionate in sucrose density gradients, and show similar solubility or insolubility characteristics in distinct non-ionic and zwitterionic detergents, indicative of their co-residence in membrane microdomains with similar protein-lipid composition. This notion is confirmed using magnetic immunoisolation of PS1- or syntaxin 6-positive membrane patches from a mixture of membranes with similar buoyant densities following Lubrol WX extraction or sonication, and gradient centrifugation. These findings are consistent with the localization of γ-secretase in lipid raft microdomains of post-Golgi and endosomes, organelles previously implicated in amyloidogenic processing of APP. PMID:15322084

Heat Induction of Prophage φ105 in Bacillus subtilis: Replication of the Bacterial and Bacteriophage Genomes

PubMed Central

Armentrout, Richard W.; Rutberg, Lars

1971-01-01

A temperature-inducible mutant of temperate Bacillus bacteriophage φ105 was isolated and used to lysogenize a thymine-requiring strain of Bacillus subtilis 168. Synthesis of phage and bacterial deoxyribonucleic acid (DNA) was studied by sucrose gradient centrifugation and density equilibrium centrifugation of DNA extracted from induced bacteria. The distribution of DNA in the gradients was measured by differential isotope and density labeling of DNA before and after induction and by measuring the biological activity of the DNA in genetic transformation, in rescue of phage markers, and in infectivity assays. At early times after induction, but after at least one round of replication, phage DNA remains associated with high-molecular-weight DNA, whereas, later in the infection, phage DNA is associated with material of decreasing molecular weight. Genetic linkage between phage and bacterial markers can be demonstrated in replicated DNA from induced cells. Prophage induction is shown to affect replication of the bacterial chromosome. The overall rate of replication of prelabeled bacterial DNA is identical in temperature-induced lysogenics and in “mock-induced” wild-type φ105 lysogenics. The rate of replication of the bacterial marker phe-1 (and also of nia-38), located close to the prophage in direction of the terminus of the bacterial chromosome, is increased in induced cells, however, relative to other bacterial markers tested. In temperature-inducible lysogenics, where the prophage also carries a ts mutation which blocks phage DNA synthesis, replication of both phage and bacterial DNA stops after about 50% of the phage DNA has replicated once. The results of these experiments suggest that the prophage is not initially excised in induced cells, but rather it is specifically replicated in situ together with adjacent parts of the bacterial chromosome. PMID:5002012

DNA of a Human Hepatitis B Virus Candidate

PubMed Central

Robinson, William S.; Clayton, David A.; Greenman, Richard L.

1974-01-01

Particles containing DNA polymerase (Dane particles) were purified from the plasma of chronic carriers of hepatitis B antigen. After a DNA polymerase reaction with purified Dane particle preparations treated with Nonidet P-40 detergent, Dane particle core structures containing radioactive DNA product were isolated by sedimentation in a sucrose density gradient. The radioactive DNA was extracted with sodium dodecyl sulfate and isolated by band sedimentation in a preformed CsCl gradient. Examination of the radioactive DNA band by electron microscopy revealed exclusively circular double-stranded DNA molecules approximately 0.78 μm in length. Identical circular molecules were observed when DNA was isolated by a similar procedure from particles that had not undergone a DNA polymerase reaction. The molecules were completely degraded by DNase 1. When Dane particle core structures were treated with DNase 1 before DNA extraction, only 0.78-μm circular DNA molecules were detected. Without DNase treatment of core structures, linear molecules with lengths between 0.5 and 12 μm, in addition to the 0.78-μm circles were found. These results suggest that the 0.78-μm circular molecules were in a protected position within Dane particle cores and the linear molecules were not within core structures. Length measurements on 225 circular molecules revealed a mean length of 0.78 ± 0.09 μm which would correspond to a molecular weight of around 1.6 × 106. The circular molecules probably serve as primer-template for the DNA polymerase reaction carried out by Dane particle cores. Thermal denaturation and buoyant density measurements on the Dane particle DNA polymerase reaction product revealed a guanosine plus cytosine content of 48 to 49%. Images PMID:4847328

Postphloem, Nonvascular Transfer in Citrus

PubMed Central

Koch, Karen E.; Avigne, Wayne T.

1990-01-01

Postphloem, nonvascular assimilate transport occurs over an unusually long area in citrus fruit and thus facilitates investigation of this process relative to sugar entry into many sink structures. Labeled photosynthates moving into juice tissues of grapefruit (Citrus paradisi Macf.) slowed dramatically after entering the postphloem transport path (parenchyma cells, narrow portions of segment epidermis, and hair-like, parenchymatous stalks of juice sacs). Kinetic, metabolic, and compositional data indicated that transfer through the nonvascular area was delayed many hours by temporary storage and/or equilibration with sugars in compartments along the postphloem path. Labeled assimilates were generally recovered as sucrose throughout the path, and extent of hexose formation enroute bore no apparent relationship to the assimilate transfer process. Even after 24 hours, radiolabel was restricted to discrete, highly localized areas directly between vascular bundles and juice sacs. Postphloem transfer occurred against an ascending sucrose concentration gradient in young fruit, whereas a descending gradient (favoring diffusion/cytoplasmic streaming) developed only later in maturation. Involvement of a postphloem bulk flow is complicated in the present instance by the extremely limited water loss from juice sacs either via transpiration or fluid backflow. Nonetheless, tissue expansion can account for a collective water inflow of at least 1.0 milliliter per day throughout the majority of juice sac development, thus providing a modest, but potentially important means of nonvascular solution flow. Overall, data indicate postphloem transfer (a) can follow highly localized paths through sizable nonvascular areas (up to 3.0 centimeters total), (b) appears to involve temporary storage and/or equilibration with compartmentalized sugars enroute, (c) can occur either against an overall up-hill sugar gradient (young tissues) or along a descending gradient (near full expansion), and (d) appears to involve at least some contribution by nonvascular mass flow accommodated by tissue expansion. Images Figure 1 Figure 4 PMID:16667632

Stabilization of electron-scale turbulence by electron density gradient in national spherical torus experiment

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

Ruiz Ruiz, J.; White, A. E.; Ren, Y.

2015-12-15

Theory and experiments have shown that electron temperature gradient (ETG) turbulence on the electron gyro-scale, k{sub ⊥}ρ{sub e} ≲ 1, can be responsible for anomalous electron thermal transport in NSTX. Electron scale (high-k) turbulence is diagnosed in NSTX with a high-k microwave scattering system [D. R. Smith et al., Rev. Sci. Instrum. 79, 123501 (2008)]. Here we report on stabilization effects of the electron density gradient on electron-scale density fluctuations in a set of neutral beam injection heated H-mode plasmas. We found that the absence of high-k density fluctuations from measurements is correlated with large equilibrium density gradient, which ismore » shown to be consistent with linear stabilization of ETG modes due to the density gradient using the analytical ETG linear threshold in F. Jenko et al. [Phys. Plasmas 8, 4096 (2001)] and linear gyrokinetic simulations with GS2 [M. Kotschenreuther et al., Comput. Phys. Commun. 88, 128 (1995)]. We also found that the observed power of electron-scale turbulence (when it exists) is anti-correlated with the equilibrium density gradient, suggesting density gradient as a nonlinear stabilizing mechanism. Higher density gradients give rise to lower values of the plasma frame frequency, calculated based on the Doppler shift of the measured density fluctuations. Linear gyrokinetic simulations show that higher values of the electron density gradient reduce the value of the real frequency, in agreement with experimental observation. Nonlinear electron-scale gyrokinetic simulations show that high electron density gradient reduces electron heat flux and stiffness, and increases the ETG nonlinear threshold, consistent with experimental observations.« less

Effect of Environmental Density and Buoyancy on Growth and Gravitropic Response in Maize Roots

NASA Astrophysics Data System (ADS)

Robbins, J. L.; Mulkey, T. J.

2008-06-01

The mechanism by which plants sense gravity is not fully understood. The hydrostatic model was proposed as an alternative to the statolith model. These experiments are designed to provide further understanding about the underlying mechanism of the gravitropic sensing. Primary roots of maize with a length of about 1 cm were used. The roots were placed in environments of various density and buoyancy using air, water, sucrose, sucrose/polyethylene glycol 4000 (PEG), PEG 8000, and Ficoll PM 400. The rates of growth and gravitropic curvature were monitored using time-lapse video and digital recordings. Comparison of roots in air to roots in oxygenated water indicate that there is no significant difference in growth rates but the higher density of water and the other test solutions significantly slows the gravitropic response. Altering the environmental density and buoyancy of the solution surrounding the root does not appear to alter sedimentation of statoliths within the root tip.

Preparation of Rocky Mountain spotted fever vaccine suitable for human immunization.

PubMed Central

Kenyon, R H; Pedersen, C E

1975-01-01

Rocky Mountain spotted fever vaccine was produced from rickettsiae grown in chicken embryo cells in roller bottle cultures. The rickettsiae were concentrated and purified by passage through a sucrose gradient and inactivated with formalin. This vaccine satisfactorily passed preinactivation and final container testing and is believed to be superior to the presently available yolk sac vaccine. PMID:809483

A Method for Isolation of Extracellular Vesicles and Characterization of Exosomes from Brain Extracellular Space.

PubMed

Pérez-González, Rocío; Gauthier, Sebastien A; Kumar, Asok; Saito, Mitsuo; Saito, Mariko; Levy, Efrat

2017-01-01

Extracellular vesicles (EV), including exosomes, secreted vesicles of endocytic origin, and microvesicles derived from the plasma membrane, have been widely isolated and characterized from conditioned culture media and bodily fluids. The difficulty in isolating EV from tissues, however, has hindered their study in vivo. Here, we describe a novel method designed to isolate EV and characterize exosomes from the extracellular space of brain tissues. The purification of EV is achieved by gentle dissociation of the tissue to free the brain extracellular space, followed by sequential low-speed centrifugations, filtration, and ultracentrifugations. To further purify EV from other extracellular components, they are separated on a sucrose step gradient. Characterization of the sucrose step gradient fractions by electron microscopy demonstrates that this method yields pure EV preparations free of large vesicles, subcellular organelles, or debris. The level of EV secretion and content are determined by assays for acetylcholinesterase activity and total protein estimation, and exosomal identification and protein content are analyzed by Western blot and immuno-electron microscopy. Additionally, we present here a method to delipidate EV in order to improve the resolution of downstream electrophoretic analysis of EV proteins.

Density Gradients in Chemistry Teaching

ERIC Educational Resources Information Center

Miller, P. J.

1972-01-01

Outlines experiments in which a density gradient might be used to advantage. A density gradient consists of a column of liquid, the composition and density of which varies along its length. The procedure can be used in analysis of solutions and mixtures and in density measures of solids. (Author/TS)

Evidence for retrovirus infections in green turtles Chelonia mydas from the Hawaiian islands

USGS Publications Warehouse

Casey, R.N.; Quackenbush, S.L.; Work, Thierry M.; Balazs, G.H.; Bowser, P.R.; Casey, J.W.

1997-01-01

Apparently normal Hawaiian green turtles Chelonia mydas and those displaying fibropapillomas were analyzed for infection by retroviruses. Strikingly, all samples were positive for polymerase enhanced reverse transcriptase (PERT) with levels high enough to quantitate by the conventional reverse transcriptase (RT) assay. However, samples of skin, even from asymptomatic turtles, were RT positive, although the levels of enzyme activity in healthy turtles hatched and raised in captivity were much lower than those observed in asymptomatic free-ranging turtles. Turtles with fibropapillomas displayed a broad range of reverse transcriptase activity. Skin and eye fibropapillomas and a heart tumor were further analyzed and shown to have reverse transcriptase activity that banded in a sucrose gradient at 1.17 g ml-1. The reverse transcriptase activity purified from the heart tumor displayed a temperature optimum of 37??C and showed a preference for Mn2+ over Mg2+. Sucrose gradient fractions of this sample displaying elevated reverse transcriptase activity contained primarily retrovitalsized particles with prominent envelope spikes, when negatively stained and examined by electron microscopy. Sodium dodecylsulfate-polyacrylamide gel electrophoresis (SDS-PAGE) analysis of gradient-purified virions revealed a conserved profile among 4 independent tumors and showed 7 prominent proteins having molecular weights of 116, 83, 51, 43, 40, 20 and 14 kDa. The data suggest that retroviral infections are widespread in Hawaiian green turtles and a comprehensive investigation is warranted to address the possibility that these agents cause green turtle fibropapillomatosis (GTFP).

Pig Brain Mitochondria as a Biological Model for Study of Mitochondrial Respiration.

PubMed

Fišar, Z; Hroudová, J

2016-01-01

Oxidative phosphorylation is a key process of intracellular energy transfer by which mitochondria produce ATP. Isolated mitochondria serve as a biological model for understanding the mitochondrial respiration control, effects of various biologically active substances, and pathophysiology of mitochondrial diseases. The aim of our study was to evaluate pig brain mitochondria as a proper biological model for investigation of activity of the mitochondrial electron transport chain. Oxygen consumption rates of isolated pig brain mitochondria were measured using high-resolution respirometry. Mitochondrial respiration of crude mitochondrial fraction, mitochondria purified in sucrose gradient, and mitochondria purified in Percoll gradient were assayed as a function of storage time. Oxygen flux and various mitochondrial respiratory control ratios were not changed within two days of mitochondria storage on ice. Leak respiration was found higher and Complex I-linked respiration lower in purified mitochondria compared to the crude mitochondrial fraction. Damage to both outer and inner mitochondrial membrane caused by the isolation procedure was the greatest after purification in a sucrose gradient. We confirmed that pig brain mitochondria can serve as a biological model for investigation of mitochondrial respiration. The advantage of this biological model is the stability of respiratory parameters for more than 48 h and the possibility to isolate large amounts of mitochondria from specific brain areas without the need to kill laboratory animals. We suggest the use of high-resolution respirometry of pig brain mitochondria for research of the neuroprotective effects and/or mitochondrial toxicity of new medical drugs.

Episodic sucrose intake during food restriction increases synaptic abundance of AMPA receptors in nucleus accumbens and augments intake of sucrose following restoration of ad libitum feeding.

PubMed

Peng, X-X; Lister, A; Rabinowitsch, A; Kolaric, R; Cabeza de Vaca, S; Ziff, E B; Carr, K D

2015-06-04

Weight-loss dieting often leads to loss of control, rebound weight gain, and is a risk factor for binge pathology. Based on findings that food restriction (FR) upregulates sucrose-induced trafficking of glutamatergic AMPA receptors to the nucleus accumbens (NAc) postsynaptic density (PSD), this study was an initial test of the hypothesis that episodic "breakthrough" intake of forbidden food during dieting interacts with upregulated mechanisms of synaptic plasticity to increase reward-driven feeding. Ad libitum (AL) fed and FR subjects consumed a limited amount of 10% sucrose, or had access to water, every other day for 10 occasions. Beginning three weeks after return of FR rats to AL feeding, when 24-h chow intake and rate of body weight gain had normalized, subjects with a history of sucrose intake during FR consumed more sucrose during a four week intermittent access protocol than the two AL groups and the group that had access to water during FR. In an experiment that substituted noncontingent administration of d-amphetamine for sucrose, FR subjects displayed an enhanced locomotor response during active FR but a blunted response, relative to AL subjects, during recovery from FR. This result suggests that the enduring increase in sucrose consumption is unlikely to be explained by residual enhancing effects of FR on dopamine signaling. In a biochemical experiment which paralleled the sucrose behavioral experiment, rats with a history of sucrose intake during FR displayed increased abundance of pSer845-GluA1, GluA2, and GluA3 in the NAc PSD relative to rats with a history of FR without sucrose access and rats that had been AL throughout, whether they had a history of episodic sucrose intake or not. A history of FR, with or without a history of sucrose intake, was associated with increased abundance of GluA1. A terminal 15-min bout of sucrose intake produced a further increase in pSer845-GluA1 and GluA2 in subjects with a history of sucrose intake during FR. Generally, neither a history of sucrose intake nor a terminal bout of sucrose intake affected AMPA receptor abundance in the NAc PSD of AL subjects. Together, these results are consistent with the hypothesis, but the functional contribution of increased synaptic incorporation of AMPA receptors remains to be established. Copyright © 2015 IBRO. Published by Elsevier Ltd. All rights reserved.

A novel technique for real-time estimation of edge pedestal density gradients via reflectometer time delay data

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

Zeng, L., E-mail: zeng@fusion.gat.com; Doyle, E. J.; Rhodes, T. L.

2016-11-15

A new model-based technique for fast estimation of the pedestal electron density gradient has been developed. The technique uses ordinary mode polarization profile reflectometer time delay data and does not require direct profile inversion. Because of its simple data processing, the technique can be readily implemented via a Field-Programmable Gate Array, so as to provide a real-time density gradient estimate, suitable for use in plasma control systems such as envisioned for ITER, and possibly for DIII-D and Experimental Advanced Superconducting Tokamak. The method is based on a simple edge plasma model with a linear pedestal density gradient and low scrape-off-layermore » density. By measuring reflectometer time delays for three adjacent frequencies, the pedestal density gradient can be estimated analytically via the new approach. Using existing DIII-D profile reflectometer data, the estimated density gradients obtained from the new technique are found to be in good agreement with the actual density gradients for a number of dynamic DIII-D plasma conditions.« less

Reduction of spiked porcine circovirus during the manufacture of a Vero cell-derived vaccine.

PubMed

Lackner, Cornelia; Leydold, Sandra M; Modrof, Jens; Farcet, Maria R; Grillberger, Leopold; Schäfer, Birgit; Anderle, Heinz; Kreil, Thomas R

2014-04-11

Porcine circovirus-1 (PCV1) was recently identified as a contaminant in live Rotavirus vaccines, which was likely caused by contaminated porcine trypsin. The event triggered the development of new regulatory guidance on the use of porcine trypsin which shall ensure that cell lines and porcine trypsin in use are free from PCV1. In addition, manufacturing processes of biologicals other than live vaccines include virus clearance steps that may prevent and mitigate any potential virus contamination of product. In this work, artificial spiking of down-scaled models for the manufacturing process of an inactivated pandemic influenza virus vaccine were used to investigate inactivation of PCV1 and the physico-chemically related porcine parvovirus (PPV) by formalin and ultraviolet-C (UV-C) treatment as well as removal by the purification step sucrose gradient ultracentrifugation. A PCV1 infectivity assay, using a real-time PCR infectivity readout was established. The formalin treatment (0.05% for 48h) showed substantial inactivation for both PCV1 and PPV with reduction factors of 3.0log10 and 6.8log10, respectively, whereas UV-C treatment resulted in complete PPV (≥5.9log10) inactivation already at a dose of 13mJ/cm but merely 1.7log10 at 24mJ/cm(2) for PCV1. The UV-C inactivation results with PPV were confirmed using minute virus of mice (MVM), indicating that parvoviruses are far more sensitive to UV-C than PCV1. The sucrose density gradient ultracentrifugation also contributed to PCV1 clearance with a reduction factor of 2log10. The low pH treatment during the production of procine trypsin was investigated and showed effective inactivation for both PCV1 (4.5log10) and PPV (6.4log10). In conclusion, PCV1 in general appears to be more resistant to virus inactivation than PPV. Still, the inactivated pandemic influenza vaccine manufacturing process provides for robust virus reduction, in addition to the already implemented testing for PCV1 to avoid any contaminations. Copyright © 2014 Elsevier Ltd. All rights reserved.

Membrane-association of mRNA decapping factors is independent of stress in budding yeast

PubMed Central

Huch, Susanne; Gommlich, Jessie; Muppavarapu, Mridula; Beckham, Carla; Nissan, Tracy

2016-01-01

Recent evidence has suggested that the degradation of mRNA occurs on translating ribosomes or alternatively within RNA granules called P bodies, which are aggregates whose core constituents are mRNA decay proteins and RNA. In this study, we examined the mRNA decapping proteins, Dcp1, Dcp2, and Dhh1, using subcellular fractionation. We found that decapping factors co-sediment in the polysome fraction of a sucrose gradient and do not alter their behaviour with stress, inhibition of translation or inhibition of the P body formation. Importantly, their localisation to the polysome fraction is independent of the RNA, suggesting that these factors may be constitutively localised to the polysome. Conversely, polysomal and post-polysomal sedimentation of the decapping proteins was abolished with the addition of a detergent, which shifts the factors to the non-translating RNP fraction and is consistent with membrane association. Using a membrane flotation assay, we observed the mRNA decapping factors in the lower density fractions at the buoyant density of membrane-associated proteins. These observations provide further evidence that mRNA decapping factors interact with subcellular membranes, and we suggest a model in which the mRNA decapping factors interact with membranes to facilitate regulation of mRNA degradation. PMID:27146487

Membrane-association of mRNA decapping factors is independent of stress in budding yeast.

PubMed

Huch, Susanne; Gommlich, Jessie; Muppavarapu, Mridula; Beckham, Carla; Nissan, Tracy

2016-05-05

Recent evidence has suggested that the degradation of mRNA occurs on translating ribosomes or alternatively within RNA granules called P bodies, which are aggregates whose core constituents are mRNA decay proteins and RNA. In this study, we examined the mRNA decapping proteins, Dcp1, Dcp2, and Dhh1, using subcellular fractionation. We found that decapping factors co-sediment in the polysome fraction of a sucrose gradient and do not alter their behaviour with stress, inhibition of translation or inhibition of the P body formation. Importantly, their localisation to the polysome fraction is independent of the RNA, suggesting that these factors may be constitutively localised to the polysome. Conversely, polysomal and post-polysomal sedimentation of the decapping proteins was abolished with the addition of a detergent, which shifts the factors to the non-translating RNP fraction and is consistent with membrane association. Using a membrane flotation assay, we observed the mRNA decapping factors in the lower density fractions at the buoyant density of membrane-associated proteins. These observations provide further evidence that mRNA decapping factors interact with subcellular membranes, and we suggest a model in which the mRNA decapping factors interact with membranes to facilitate regulation of mRNA degradation.

ISOLATION AND CHARACTERIZATION OF LAMELLAR BODIES AND TUBULAR MYELIN FROM RAT LUNG HOMOGENATES

PubMed Central

Gil, Joan; Reiss, Oscar K.

1973-01-01

Three surface-active fractions which differ in their morphology have been isolated from rat lung homogenates by ultracentrifugation in a discontinuous sucrose density gradient. In order of increasing density, the fractions consisted, as shown by electron microscopy, primarily of common myelin figures, lamellar bodies, and tubular myelin figures. The lipid of all three fractions contained approximately 94% polar lipids and 2% cholesterol. In the case of the common myelin figures and the lamellar bodies, the polar lipids consisted of 73% phosphatidylcholines, 9% phosphatidylserines and inositols, and 8% phosphatidylethanolamines. In the case of the tubular myelin figures, the respective percentages were 58, 19, and 5. Over 90% of the fatty acids of the lecithins of all three fractions were saturated. Electrophoresis of the proteins of the fractions in sodium dodecyl sulfate or Triton X-100 revealed that the lamellar bodies and the tubular myelin figures differed in the mobilities of their proteins. The common myelin figures, however, contained proteins from both of the other fractions. These data indicate that, whereas the lipids of the extracellular, alveolar surfactant(s) originate in the lamellar bodies, the proteins arise from another source. It is further postulated that the tubular myelin figures represent a liquid crystalline state of the alveolar surface-active lipoproteins. PMID:4726305

Liquid density analysis of sucrose and alcoholic beverages using polyimide guided Love-mode acoustic wave sensors

NASA Astrophysics Data System (ADS)

Turton, Andrew; Bhattacharyya, Debabrata; Wood, David

2006-02-01

A liquid density sensor using Love-mode acoustic waves has been developed which is suitable for use in the food and drinks industries. The sensor has an open flat surface allowing immersion into a sample and simple cleaning. A polyimide waveguide layer allows cheap and simple fabrication combined with a robust chemically resistant surface. The low shear modulus of polyimide allows thin guiding layers giving a high sensitivity. A dual structure with a smooth reference device exhibiting viscous coupling with the wave, and a patterned sense area to trap the liquid causing mass loading, allows discrimination of the liquid density from the square root of the density-viscosity product (ρη)0.5. Frequency shift and insertion loss change were proportional to (ρη)0.5 with a non-linear response due to the non-Newtonian nature of viscous liquids at high frequencies. Measurements were made with sucrose solutions up to 50% and different alcoholic drinks. A maximum sensitivity of 0.13 µg cm-3 Hz-1 was achieved, with a linear frequency response to density. This is the highest liquid density sensitivity obtained for acoustic mode sensors to the best of our knowledge.

Hormonal interactions during cluster-root development in phosphate-deficient white lupin (Lupinus albus L.).

PubMed

Wang, Zhengrui; Rahman, A B M Moshiur; Wang, Guoying; Ludewig, Uwe; Shen, Jianbo; Neumann, Günter

2015-04-01

This study addresses hormonal interactions involved in cluster-root (CR) development of phosphate (Pi)-deficient white lupin (Lupinus albus), which represents the most efficient plant strategy for root-induced mobilisation of sparingly soluble soil phosphorus (P) sources. Shoot-to-root translocation of auxin was unaffected by P-limitation, while strong stimulatory effects of external sucrose on CR formation, even in P-sufficient plants, suggest sucrose, rather than auxins, acts as a shoot-borne signal, triggering the induction of CR primordia. Ethylene may act as mediator of the sucrose signal, as indicated by moderately increased expression of genes involved in ethylene biosynthesis in pre-emergent clusters and by strong inhibitory effects of the ethylene antagonist CoCl2 on CR formation induced by sucrose amendments or P-limitation. As reported in other plants, moderately increased production of brassinosteroids (BRs) and cytokinin, in pre-emergent clusters, may be required for the formation of auxin gradients necessary for induction of CR primordia via interference with auxin biosynthesis and transport. The well-documented inhibition of root elongation by high doses of ethylene may be involved in the growth inhibition of lateral rootlets during CR maturation, indicated by a massive increased expression of gene involved in ethylene production, associated with a declined expression of transcripts with stimulatory effects (BR and auxin-related genes). Copyright © 2014 Elsevier GmbH. All rights reserved.

Connections between density, wall-normal velocity, and coherent structure in a heated turbulent boundary layer

NASA Astrophysics Data System (ADS)

Saxton-Fox, Theresa; Gordeyev, Stanislav; Smith, Adam; McKeon, Beverley

2015-11-01

Strong density gradients associated with turbulent structure were measured in a mildly heated turbulent boundary layer using an optical sensor (Malley probe). The Malley probe measured index of refraction gradients integrated along the wall-normal direction, which, due to the proportionality of index of refraction and density in air, was equivalently an integral measure of density gradients. The integral output was observed to be dominated by strong, localized density gradients. Conditional averaging and Pearson correlations identified connections between the streamwise gradient of density and the streamwise gradient of wall-normal velocity. The trends were suggestive of a process of pick-up and transport of heat away from the wall. Additionally, by considering the density field as a passive marker of structure, the role of the wall-normal velocity in shaping turbulent structure in a sheared flow was examined. Connections were developed between sharp gradients in the density and flow fields and strong vertical velocity fluctuations. This research is made possible by the Department of Defense through the National Defense & Engineering Graduate Fellowship (NDSEG) Program and by the Air Force Office of Scientific Research Grant # FA9550-12-1-0060.

Single fiber lignin distributions based on the density gradient column method

Treesearch

Brian Boyer; Alan W. Rudie

2007-01-01

The density gradient column method was used to determine the effects of uniform and non-uniform pulping processes on variation in individual fiber lignin concentrations of the resulting pulps. A density gradient column uses solvents of different densities and a mixing process to produce a column of liquid with a smooth transition from higher density at the bottom to...

Peroxisomal Ascorbate Peroxidase Resides within a Subdomain of Rough Endoplasmic Reticulum in Wild-Type Arabidopsis Cells1

PubMed Central

Lisenbee, Cayle S.; Heinze, Michael; Trelease, Richard N.

2003-01-01

Previously we reported (R.T. Mullen, C.S. Lisenbee, J.A. Miernyk, R.N. Trelease [1999] Plant Cell 11: 2167–2185) that overexpressed ascorbate peroxidase (APX), a peroxisomal membrane protein, sorted indirectly to Bright Yellow-2 cell peroxisomes via a subdomain of the endoplasmic reticulum (ER; peroxisomal endoplasmic reticulum [pER]). More recently, a pER-like compartment also was identified in pumpkin (Cucurbita pepo) and transformed Arabidopsis cells (K. Nito, K. Yamaguchi, M. Kondo, M. Hayashi, M. Nishimura [2001] Plant Cell Physiol 42: 20–27). Here, we characterize more extensively the localization of endogenous Arabidopsis peroxisomal APX (AtAPX) in cultured wild-type Arabidopsis cells (Arabidopsis var. Landsberg erecta). AtAPX was detected in peroxisomes, but not in an ER subcompartment, using immunofluorescence microscopy. However, AtAPX was detected readily with immunoblots in both peroxisomal and ER fractions recovered from sucrose (Suc) density gradients. Most AtAPX in microsomes (200,000g, 1 h pellet) applied to gradients exhibited a Mg2+-induced shift from a distribution throughout gradients (approximately 18%–40% [w/w] Suc) to ≥42% (w/w) Suc regions of gradients, including pellets, indicative of localization in rough ER vesicles. Immunogold electron microscopy of the latter fractions verified these findings. Further analyses of peroxisomal and rough ER vesicle fractions revealed that AtAPX in both fractions was similarly associated with and located mostly on the cytosolic face of the membranes. Thus, at the steady state, endogenous peroxisomal AtAPX resides at different levels in rough ER and peroxisomes. Collectively, these findings show that rather than being a transiently induced sorting compartment formed in response to overexpressed peroxisomal APX, portions of rough ER (pER) in wild-type cells serve as a constitutive sorting compartment likely involved in posttranslational routing of constitutively synthesized peroxisomal APX. PMID:12805617

Effects of carbon amendment on in situ atrazine degradation and total microbial biomass.

PubMed

Ngigi, Anastasiah N; Getenga, Zachary M; Dörfler, Ulrike; Boga, Hamadi I; Kuria, Benson; Ndalut, Paul; Schroll, Reiner

2013-01-01

This study elucidates the effects of carbon amendment on metabolic degradation of atrazine (6-chloro-N(2)-ethyl-N(4)-isopropyl-1,3,5-triazine-2,4-diamine) and total microbial biomass in soil. Degradation of (14)C-ring-labelled atrazine was monitored in laboratory incubations of soils supplemented with 0, 10, 100 and 1000 μg g(-1) sucrose concentrations. An experiment to determine the effect of carbon amendment on total microbial biomass and soil respiration was carried out with different concentrations of sucrose and non-labelled atrazine. The soils were incubated at a constant temperature and constant soil moisture at water potential of -15 kPa and a soil density of 1.3 g cm(-3). Mineralization of (14)C-ring-labelled atrazine was monitored continuously over a period of 59 d in the first experiment. The CO(2) production was monitored for 62 d in the second experiment and microbial biomass determined at the end of the incubation period. The addition of 1000 μg g(-1) sucrose reduced atrazine mineralization to 43.5% compared to 51.7% of the applied amount for the treatment without sucrose. The addition of 1000 μg g(-1) sucrose modified the transformation products to 1.08 μg g(-1) deisopropylatrazine (DIA), 0.32 μg g(-1) desethylatrazine (DEA) and 0.18 μg g(-1) deisopropyl-2-hydroxyatrazine (OH-DIA). Treatment without sucrose resulted in formation of 0.64 μg g(-1) hydroxyatrazine (HA), 0.28 μg g(-1) DIA and 0.20 μg g(-1) OH-DIA. Atrazine dealkylation was enhanced in treatments with 100 and 1000 μg g(-1) of sucrose added. HA metabolite was formed in the control (no sucrose) and in the presence of 10 μg g(-1) of sucrose, whereas DEA was only detected in treatment with 1000 μg g(-1) sucrose. Results indicate that total microbial biomass increased significantly (P < 0.001) with the addition of 1000 μg g(-1) sucrose.

Consumption of sucrose from infancy increases the visceral fat accumulation, concentration of triglycerides, insulin and leptin, and generates abnormalities in the adrenal gland.

PubMed

Díaz-Aguila, Yadira; Castelán, Francisco; Cuevas, Estela; Zambrano, Elena; Martínez-Gómez, Margarita; Muñoz, Alvaro; Rodríguez-Antolín, Jorge; Nicolás-Toledo, Leticia

2016-03-01

Consumption of sugar-sweetened beverages promotes the development of metabolic syndrome (MetS) and type 2 diabetes mellitus in humans. One factor related to the appearance of MetS components is the dysfunction of the adrenal gland. In fact, the experimental generation of hyperglycemia has been associated with morphological and microvascular changes in the adrenal glands of rats. We hypothesized that high sucrose consumption from infancy promotes histological disruption of the adrenal glands associated with the appearance of metabolic syndrome indicators. Male Wistar rats were separated at weaning (21 days old) into two groups: free access to tap water (control group, C) or 30 % sucrose diluted in water (sugar-fed group). After 12 weeks, high sucrose consumption promoted an increase in visceral fat accumulation, adipose cell number, and insulin resistance. Also, a rise in the concentration of triglycerides, very low-density lipoprotein, insulin and leptin was observed. In control rats, a histomorphometric asymmetry between the right and left adrenal glands was found. In the sugar-fed group, sucrose consumption produced a major change in adrenal gland asymmetry. No changes in corticosterone serum level were observed in either group. Our results suggest that a high sucrose liquid-diet from early life alters the morphology of adrenocortical zones, leading to MetS indicators.

Protein-based microhydraulic transport for controllable actuation

NASA Astrophysics Data System (ADS)

Sundaresan, Vishnu Baba; Leo, Donald J.

2006-03-01

Plants have the ability to develop large mechanical force from chemical energy available with bio-fuels. The energy released by the cleavage of a terminal phosphate ion during the hydrolysis of a bio- fuel assists the transport of ions and fluids in cellular homeostasis. Materials that develop pressure and hence strain similar to the response of plants to an external stimuli are classified as nastic materials. Calculations for controlled actuation of an active material inspired by biological transport mechanism demonstrated the feasibility of developing such a material with actuation energy densities on the order of 100 kJ/m 3. The mathematical model for a simplified proof of concept actuator referred to as micro hydraulic actuator uses ion transporters extracted from plants reconstituted on a synthetic bilayer lipid membrane (BLM). Thermodynamic model of the concept actuator predicted the ability to develop 5 percent normalized deformation in thickness of the micro- hydraulic actuator. Controlled fluid transport through AtSUT4 (Proton-sucrose co-transporter from Arabidopsis thaliana) reconstituted on a 1-Palmitoyl-2-Oleoyl-sn-Glycero-3-[Phospho-L- Serine] (Sodium Salt) (POPS), 1-Palmitoyl-2-Oleoyl-sn-Glycero-3- Phosphoethanolamine (POPE) BLM on a porous lead silicate glass plate (50μm with 61μm pitch) was driven by proton gradient. Bulk fluid flux of 1.2 μl/min was observed for each microliter of AtSUT4 transporter suspension (16.6 mg/ml in pH7.0 medium) reconstituted on the BLM. The flux rate is observed to be dependent on the concentration of sucrose present in pH4 buffer. Flux rate of 10 μl/min is observed for 5 mM sucrose in the first 10 minutes. The observed flux scales linearly with BLM area and the amount of proteins reconstituted on the lipid membrane. This article details the next step in the development of the micro hydraulic actuator - fluid transport driven by exergonic Adenosine triphosphate (ATP) hydrolysis reaction in the presence of ATP-phosphohydrolase (red beet ATP-ase) enzyme in the reconstituted bilayer.

Water stress, shoot growth and storage of non-structural carbohydrates along a tree height gradient in a tall conifer

Treesearch

David R. Woodruff; Frederick C. Meinzer

2011-01-01

We analyzed concentrations of starch, sucrose, glucose and fructose in upper branch wood, foliage and trunk sapwood of Douglas-fir trees in height classes ranging from ~2 to ~57 m. Mean concentrations of non-structural carbohydrates (NSC) for all tissues were highest in the tallest height class and lowest in the lowest height class, and height-related trends in NSC...

Maturation-Associated Destabilization of Hepatitis B Virus Nucleocapsid

PubMed Central

Cui, Xiuji; Ludgate, Laurie; Ning, Xiaojun

2013-01-01

The mature nucleocapsid (NC) of hepatitis B virus containing the relaxed circular (RC) DNA genome can be secreted extracellularly as virions after envelopment with the viral surface proteins or, alternatively, can be disassembled to release RC DNA (i.e., uncoating) into the host cell nucleus to form the covalently closed circular (CCC) DNA, which sustains viral replication and persistence. In contrast, immature NCs containing the viral single-stranded DNA or the pregenomic RNA are incompetent for either envelopment or uncoating. Little is currently known about how mature NCs, and not the immature ones, are specifically selected for these processes. Here, we have carried out a biochemical analysis of the different NC populations upon their separation through sucrose gradient centrifugation. We have found that the maturation of NCs is associated with their destabilization, manifested as increased protease and nuclease sensitivity, altered sedimentation during sucrose gradient centrifugation, and retarded mobility during native agarose gel electrophoresis. Also, three distinct populations of intracellular mature NCs could be differentiated based on these characteristics. Furthermore, mature NCs generated in vitro under cell-free conditions acquired similar properties. These results have thus revealed significant structural changes associated with NC maturation that likely play a role in the selective uncoating of the mature NC for CCC DNA formation and/or its preferential envelopment for virion secretion. PMID:23966388

Uptake and intracellular fate of [14C]sucrose-insulin in perfused rat livers.

PubMed

Surmacz, C A; Wert, J J; Ward, W F; Mortimore, G E

1988-07-01

Insulin was covalently linked to [14C]sucrose by means of cyanuric chloride to provide a label that would remain entrapped within the vacuolar system. The uptake of the conjugate by the perfused rat liver was rapid (half-life = 2.9 min), competitively inhibited by native insulin, and abolished by alkali denaturation. As assessed by its distribution on self-generating gradients of colloidal silica-povidone, label in lysosome-enriched samples of liver taken at different times after the addition of the conjugate moved progressively during 15 min from the plasma membrane into an intermediate peak and then to dense lysosomal fractions. After 30-60 min, the label had equilibrated throughout the lysosomal-vacuolar system. The initial movement from the plasma membrane to the intermediate peak occurred between 2 and 5 min. Because label in the peak could be physically separated from the lysosomal marker, beta-acetylglucosaminidase, by dispersing the sample through the gradient mixture before centrifugation rather than layering it, we concluded that the intermediate particles in question were not lysosomal in nature. On gel-filtration chromatography, label extracted from the intermediate peak did not move with insulin but rather as a broad band of lower molecular weight products, suggesting that insulin is subject to early proteolytic attack within a nonlysosomal compartment.

Proteins of Vasicular Stomatitis Virus

PubMed Central

Kang, C. Y.; Prevec, L.

1969-01-01

Infection of L cells with vesicular stomatitis virus results in the release, into the cell-free fluid, of four antigenic components separable by rate zonal centrifugation on sucrose gradients. The largest antigens are the infectious (B) particle and a shorter noninfectious, autointerfering (T) particle. The two small antigens are characterized by sedimentation coefficients of approximately 20S and 6S. Treatment of purified B or T particles with sodium deoxycholate results in the release from the particle of a nucleoprotein core which can be purified on sucrose gradient and which has a sedimentation coefficient characteristic of the virus from which it arose. Utilizing purified antigens labeled with 14C-amino acids during growth, we examined the protein constituents of each antigen by acrylamide-gel electrophoresis. The proteins of B and T particles are identical, each containing one minor (virus protein 1) and three major (virus proteins 2, 3, and 4) proteins, numbered in order of increasing mobility. Virus protein 3 originates from the nucleoprotein core, whereas proteins 2 and 4 come from the coat. The origin of virus protein 1 is not known. The 20S antigen contains a single protein equivalent to virus protein 3, whereas the 6S antigen shows a single protein which is similar to, but probably distinct from, virus protein 2. PMID:4306195

A straightforward method for measuring the range of apparent density of microplastics.

PubMed

Li, Lingyun; Li, Mengmeng; Deng, Hua; Cai, Li; Cai, Huiwen; Yan, Beizhan; Hu, Jun; Shi, Huahong

2018-10-15

Density of microplastics has been regarded as the primary property that affect the distribution and bioavailability of microplastics in the water column. For measuring the density of microplastis, we developed a simple and rapid method based on density gradient solutions. In this study, we tested four solvents to make the density gradient solutions, i.e., ethanol (0.8 g/cm 3 ), ultrapure water (1.0 g/cm 3 ), saturated NaI (1.8 g/cm 3 ) and ZnCl 2 (1.8 g/cm 3 ). Density of microplastics was measured via observing the float or sink status in the density gradient solutions. We found that density gradient solutions made from ZnCl 2 had a larger uncertainty in measuring density than that from NaI, most likely due to a higher surface tension of ZnCl 2 solution. Solutions made from ethanol, ultrapure water, and NaI showed consistent density results with listed densities of commercial products, indicating that these density gradient solutions were suitable for measuring microplastics with a density range of 0.8-1.8 g/cm 3 . Copyright © 2018 Elsevier B.V. All rights reserved.

Fast simultaneous analysis of caffeine, trigonelline, nicotinic acid and sucrose in coffee by liquid chromatography-mass spectrometry.

PubMed

Perrone, Daniel; Donangelo, Carmen Marino; Farah, Adriana

2008-10-15

A rapid liquid chromatography-mass spectrometry method for the simultaneous quantification of caffeine, trigonelline, nicotinic acid and sucrose in coffee was developed and validated. The method involved extraction with hot water, clarification with basic lead acetate and membrane filtration, followed by chromatographic separation using a Spherisorb(®) S5 ODS2, 5μm chromatographic column and gradient elution with 0.3% aqueous formic acid/methanol at a flow rate of 0.2mL/min. The electrospray ionization source was operated in the negative mode to generate sucrose ions and in the positive mode to generate caffeine, trigonelline and nicotinic acid ions. Ionization suppression of all analytes was found due to matrix effect. Calibrations curves prepared in green and roasted coffee extracts were linear with r(2)>0.999. Roasted coffee was spiked and recoveries ranged from 93.0% to 105.1% for caffeine, from 85.2% to 116.2% for trigonelline, from 89.6% to 113.5% for nicotinic acid and from 94.1% to 109.7% for sucrose. Good repeatibilities (RSD<5%) were found for all analytes in the matrix. The limit of detection (LOD), calculated on the basis of signal-to-noise ratios of 3:1, was 11.9, 36.4, 18.5 and 5.0ng/mL for caffeine, trigonelline, nicotinic acid and sucrose, respectively. Analysis of 11 coffee samples (regular or decaffeinated green, ground roasted and instant) gave results in agreement with the literature. The method showed to be suitable for different types of coffee available in the market thus appearing as a fast and reliable alternative method to be used for routine coffee analysis. Copyright © 2008 Elsevier Ltd. All rights reserved.

The tonoplast-localized sucrose transporter in Populus (PtaSUT4) regulates whole-plant water relations, responses to water stress, and photosynthesis.

PubMed

Frost, Christopher J; Nyamdari, Batbayar; Tsai, Chung-Jui; Harding, Scott A

2012-01-01

The Populus sucrose (Suc) transporter 4 (PtaSUT4), like its orthologs in other plant taxa, is tonoplast localized and thought to mediate Suc export from the vacuole into the cytosol. In source leaves of Populus, SUT4 is the predominantly expressed gene family member, with transcript levels several times higher than those of plasma membrane SUTs. A hypothesis is advanced that SUT4-mediated tonoplast sucrose fluxes contribute to the regulation of osmotic gradients between cellular compartments, with the potential to mediate both sink provisioning and drought tolerance in Populus. Here, we describe the effects of PtaSUT4-RNA interference (RNAi) on sucrose levels and raffinose family oligosaccharides (RFO) induction, photosynthesis, and water uptake, retention and loss during acute and chronic drought stresses. Under normal water-replete growing conditions, SUT4-RNAi plants had generally higher shoot water contents than wild-type plants. In response to soil drying during a short-term, acute drought, RNAi plants exhibited reduced rates of water uptake and delayed wilting relative to wild-type plants. SUT4-RNAi plants had larger leaf areas and lower photosynthesis rates than wild-type plants under well-watered, but not under chronic water-limiting conditions. Moreover, the magnitude of shoot water content, height growth, and photosynthesis responses to contrasting soil moisture regimes was greater in RNAi than wild-type plants. The concentrations of stress-responsive RFOs increased in wild-type plants but were unaffected in SUT4-RNAi plants under chronically dry conditions. We discuss a model in which the subcellular compartmentalization of sucrose mediated by PtaSUT4 is regulated in response to both sink demand and plant water status in Populus.

Fluoroquinolone resistance of Serratia marcescens: sucrose, salicylate, temperature, and pH induction of phenotypic resistance.

PubMed

Begic, Sanela; Worobec, Elizabeth A

2007-11-01

Serratia marcescens is a nosocomial agent with a natural resistance to a broad spectrum of antibiotics, making the treatment of its infections very challenging. This study examines the influence of salicylate, sucrose, temperature, and pH variability on membrane permeability and susceptibility of S. marcescens to norfloxacin (hydrophilic fluoroquinolone) and nalidixic acid (hydrophobic quinolone). Resistance of wild-type S. marcescens UOC-67 (ATCC 13880) to norfloxacin and nalidixic acid was assessed by minimal inhibitory concentration (MIC) assays after growth in the presence of various concentrations of sucrose and salicylate and different temperatures and pH values. Norfloxacin and nalidixic acid accumulation was determined in the absence and presence of (i) carbonyl cyanide m-chlorophenylhydrazone (CCCP), a proton-motive-force collapser, and (ii) Phe-Arg beta-naphthylamide (PAbetaN), an efflux pump inhibitor. Accumulation of norfloxacin decreased when S. marcescens was grown in high concentrations of salicylate (8 mmol/L) and sucrose (10% m/v), at high temperature (42 degrees C), and at pH 6, and it was restored in the presence of CCCP because of the collapse of proton-gradient-dependent efflux in S. marcescens. Although nalidixic acid accumulation was observed, it was not affected by salicylate, sucrose, pH, or temperature changes. In the absence of PAbetaN, and either in the presence or absence of CCCP, a plateau was reached in the nalidixic acid accumulation for all environmental conditions. With the addition of 20 mg/L PAbetaN nalidixic acid accumulation is restored for all environmental conditions, suggesting that this quinolone is recognized by a yet to be identified S. marcescens pump that does not use proton motive force as its energy source.

Studies on sex-organ development. Changes in nuclear and chromatin composition and genomic activity during spermatogenesis in the maturing rooster testis.

PubMed Central

Mezquita, C; Teng, C S

1977-01-01

We developed a technique to separate nuclei of rooster testis by centrifugation through a discontinuous sucrose density gradient and by sedimentation at unit gravity. Four different major fractions obtained from testicular nuclei and one from the vas deferens were characterized according to their velocity of sedimentation, morphology and DNA content. The ratios (w/w) of basic proteins, non-histone proteins and RNA to DNA decreased during spermiogenesis both in nuclei and chromatin. Changes in the electrophoretic patterns of histones and non-histone proteins were detected especially in the elongated spermatids. The lack of uptake of [3H]uridine in elongating and elongated spermatids and in spermatozoa was demonstrated by radioautography and by the detection of labelled RNA extracted from different fractions of nuclei. Template activity for RNA synthesis and the binding of actinomycin D by testicular nuclei reached a peak in the elongated spermatid stage, when the histones are replaced by the protamine. Images PLATE 1 PLATE 2 PLATE 3 PLATE 4 PLATE 5 PMID:560187

Subunit stoichiometry of human muscle chloride channels.

PubMed

Fahlke, C; Knittle, T; Gurnett, C A; Campbell, K P; George, A L

1997-01-01

Voltage-gated Cl- channels belonging to the ClC family appear to function as homomultimers, but the number of subunits needed to form a functional channel is controversial. To determine subunit stoichiometry, we constructed dimeric human skeletal muscle Cl- channels in which one subunit was tagged by a mutation (D136G) that causes profound changes in voltage-dependent gating. Sucrose-density gradient centrifugation experiments indicate that both monomeric and dimeric hClC-1 channels in their native configurations exhibit similar sedimentation properties consistent with a multimeric complex having a molecular mass of a dimer. Expression of the heterodimeric channel in a mammalian cell line results in a homogenous population of Cl- channels exhibiting novel gating properties that are best explained by the formation of heteromultimeric channels with an even number of subunits. Heteromultimeric channels were not evident in cells cotransfected with homodimeric WT-WT and D136G-D136G constructs excluding the possibility that functional hClC-1 channels are assembled from more than two subunits. These results demonstrate that the functional hClC-1 unit consists of two subunits.

Expression of hepatitis B surface antigen in transgenic plants.

PubMed Central

Mason, H S; Lam, D M; Arntzen, C J

1992-01-01

Tobacco plants were genetically transformed with the gene encoding hepatitis B surface antigen (HBsAg) linked to a nominally constitutive promoter. Enzyme-linked immunoassays using a monoclonal antibody directed against human serum-derived HBsAg revealed the presence of HBsAg in extracts of transformed leaves at levels that correlated with mRNA abundance. This suggests that there were no major inherent limitations of transcription or translation of this foreign gene in plants. Recombinant HBsAg was purified from transgenic plants by immunoaffinity chromatography and examined by electron microscopy. Spherical particles with an average diameter of 22 nm were observed in negatively stained preparations. Sedimentation of transgenic plant extracts in sucrose and cesium chloride density gradients showed that the recombinant HBsAg and human serum-derived HBsAg had similar physical properties. Because the HBsAg produced in transgenic plants is antigenically and physically similar to the HBsAg particles derived from human serum and recombinant yeast, which are used as vaccines, we conclude that transgenic plants hold promise as low-cost vaccine production systems. Images PMID:1465391

Production of infectious ferret hepatitis E virus in a human hepatocarcinoma cell line PLC/PRF/5.

PubMed

Li, Tian-Cheng; Yoshizaki, Sayaka; Yang, Tingting; Kataoka, Michiyo; Nakamura, Tomofumi; Ami, Yasushi; Yuriko, Suzaki; Takeda, Naokazu; Wakita, Takaji

2016-02-02

A strain of ferret hepatitis E virus (HEV), sF4370, isolated from an imported ferret was used to inoculate a human hepatocarcinoma cell line, PLC/PRF/5. The virus genome and capsid protein were detected in the cell culture supernatant. Immunofluorescence microscopy indicated that the capsid protein was located in the cytoplasm. The virus particles were purified from the culture supernatant by sucrose gradient ultracentrifugation. The capsid protein with molecular mass of ∼72 kDa was detected in fractions with density of 1.150-1.162 g/cm(3), and particles of ferret HEV was associated with cell membrane. The virus recovered from the supernatant was serially passaged with PLC/PRF/5 cells and had the ability to infect ferrets by oral inoculation, indicating that the ferret HEV grown in PLC/PRF/5 was infectious. The establishment of ferret HEV cell culture system might be useful to understand the life cycle, mechanism of infection and replication of ferret HEV. Copyright © 2015 Elsevier B.V. All rights reserved.

Calmodulin stimulation of calcium transport in carrot microsomal vesicles. [Daucus carota

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

Pierce, W.S.; Sze, H.

1987-04-01

ATP-dependent /sup 45/Ca/sup 2 +/ uptake into microsomal vesicles isolated from cultured carrot cells (Daucus carota Danvers) was stimulated 2-3 fold by 5 ug/ml calmodulin (CaM). Microsomal vesicles separated with a linear sucrose gradient showed two peaks with CaM-stimulated Ca/sup 2 +/ uptake activities. One peak (at 1.12 g/cc) comigrated with the activity of the antimycin A-insensitive NADH-dependent cytochrome c reductase. This transport activity was enhanced 10-20 fold by 10 mM oxalate and appeared to be associates with vesicles derived primarily from the ER. The other peak of CaM-stimulated Ca/sup 2 +/ uptake (at 1.17 g/cc) was not affected bymore » oxalate. These vesicles are probably derived from the plasma membrane. Preliminary experiments with the low-density vesicles (ER) vesicles, indicate that inositol-1,4,5-trisphosphate caused a transient reduction in intravesicular Ca/sup 2 +/. These results are consistent with the ER being an important site of intracellular Ca/sup 2 +/ regulation.« less

Isolation and characterization of a novel mycovirus from Penicillium digitatum.

PubMed

Niu, Yuhui; Zhang, Tingfu; Zhu, Ying; Yuan, Yongze; Wang, Shengqiang; Liu, Jing; Liu, Deli

2016-07-01

A novel double-stranded RNA virus designated Penicillium digitatum virus 1 (PdV1) was isolated from the citrus fruit rot pathogen P. digitatum (HS-RH1). The full-length cDNA sequence of the dsRNA/PdV1 (5211bp) possesses two partially overlapping open reading frames, which encode a coat protein (CP) and a putative RNA-dependent RNA polymerase (RdRp), respectively. Phylogenetic analysis based on multiple alignments of the amino acid sequences of the RdRp and CP indicated that PdV1 tentatively belongs to the genus Victorivirus in the Totiviridae family. Electron micrographs of negatively stained viral particles purified from the peak fraction of sucrose density gradient centrifugation showed spherical particles ~35nm in diameter. Transfection experiments with purified virions indicated that PdV1 could reduce the vegetative growth and virulence of P. digitatum strain HS-F6. In summary, we report the first isolation and characterization of a mycovirus from P. digitatum that contributes to the hypovirulence phenotypes of the host strain. Copyright © 2016 Elsevier Inc. All rights reserved.

Isolation of plasma membrane fractions from the intestinal epithelial model T84.

PubMed

Kaoutzani, P; Parkos, C A; Delp-Archer, C; Madara, J L

1993-05-01

The human intestinal epithelial cell line T84 is widely used as a model for studies of Cl- secretion and crypt cell biology. We report a fractionation approach that permits separation of purified apical and basolateral T84 plasma membrane domains. T84 cellular membranes were isolated by nitrogen cavitation and differential centrifugation from monolayers grown on permeable supports. Membranes were then fractionated by isopycnic sucrose density gradient sedimentation, and fractions were assessed, using enzymatic and Western blot techniques, for apical (alkaline phosphatase) and basolateral (Na(+)-K(+)-ATPase) plasma membrane markers and for cytosolic, lysosomal, Golgi, and mitochondrial markers. Buffer conditions were defined that permitted separation of enriched apical and basolateral markers. The validity of the selected markers for the apical and basolateral domains was verified by selective apical and basolateral surface labeling studies using trace iodinated wheat germ agglutinin or biotinylation. This approach allows for separation of apical and basolateral plasma membranes of T84 cells for biochemical analyses and should thus be of broad utility in studies of this model polarized and transporting epithelium.

Above and below ground carbohydrate allocation differs between ash (Fraxinus excelsior L.) and beech (Fagus sylvatica L.)

PubMed Central

Thoms, Ronny; Köhler, Michael; Gessler, Arthur

2017-01-01

We investigated soluble carbohydrate transport in trees that differed in their phloem loading strategies in order to better understand the transport of photosynthetic products into the roots and the rhizosphere as this knowledge is needed to better understand the respiratory processes in the rhizosphere. We compared beech, which is suggested to use mainly passive loading of transport sugars along a concentration gradient into the phloem, with ash that uses active loading and polymer trapping of raffinose family oligosaccharides (RFOs). We pulse-labeled 20 four-year old European beech and 20 four-year old ash trees with 13CO2 and tracked the fate of the label within different plant compartments. We extracted soluble carbohydrates from leaves, bark of stems and branches, and fine roots, measured their amount and isotopic content and calculated their turnover times. In beech one part of the sucrose was rapidly transported into sink tissues without major exchange with storage pools whereas another part of sucrose was strongly exchanged with unlabeled possibly stored sucrose. In contrast the storage and allocation patterns in ash depended on the identity of the transported sugars. RFO were the most important transport sugars that had highest turnover in all shoot compartments. However, the turnover of RFOs in the roots was uncoupled from the shoot. The only significant relation between sugars in the stem base and in the roots of ash was found for the amount (r2 = 0.50; p = 0.001) and isotopic content (r2 = 0.47; p = 0.01) of sucrose. The negative relation of the amounts suggested an active transport of sucrose into the roots of ash. Sucrose concentration in the root also best explained the concentration of RFOs in the roots suggesting that RFO in the roots of ash may be resynthesized from sucrose. Our results interestingly suggest that in both tree species only sucrose directly entered the fine root system and that in ash RFOs are transported indirectly into the fine roots only. The direct transport of sucrose might be passive in beech but active in ash (sustained active up- and unloading to co-cells), which would correspond to the phloem loading strategies. Our results give first hints that the transport of carbohydrates between shoot and root is not necessarily continuous and involves passive (beech) and active (ash) transport processes, which may be controlled by the phloem unloading. PMID:28934229

Characterization of human glucocorticoid receptor complexes formed with DNA fragments containing or lacking glucocorticoid response elements

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

Tully, D.B.; Cidlowski, J.A.

1989-03-07

Sucrose density gradient shift assays were used to study the interactions of human glucocorticoid receptors (GR) with small DNA fragments either containing or lacking glucocorticoid response element (GRE) DNA consensus sequences. When crude cytoplasmic extracts containing ({sup 3}H)triamcinolone acetonide (({sup 3}H)TA) labeled GR were incubated with unlabeled DNA under conditions of DNA excess, a GRE-containing DNA fragment obtained from the 5' long terminal repeat of mouse mammary tumor virus (MMTV LTR) formed a stable 12-16S complex with activated, but not nonactivated, ({sup 3}H)TA receptor. By contrast, if the cytosols were treated with calf thymus DNA-cellulose to deplete non-GR-DNA-binding proteins priormore » to heat activation, a smaller 7-10S complex was formed with the MMTV LTR DNA fragment. Activated ({sup 3}H)TA receptor from DNA-cellulose pretreated cytosols also interacted with two similarly sized fragments from pBR322 DNA. Stability of the complexes formed between GR and these three DNA fragments was strongly affected by even moderate alterations in either the salt concentration or the pH of the gradient buffer. Under all conditions tested, the complex formed with the MMTV LTR DNA fragment was more stable than the complexes formed with either of the pBR322 DNA fragments. Together these observations indicate that the formation of stable complexes between activated GR and isolated DNA fragments requires the presence of GRE consensus sequences in the DNA.« less

A modified gradient approach for the growth of low-density InAs quantum dot molecules by molecular beam epitaxy

NASA Astrophysics Data System (ADS)

Sharma, Nandlal; Reuter, Dirk

2017-11-01

Two vertically stacked quantum dots that are electronically coupled, so called quantum dot molecules, are of great interest for the realization of solid state building blocks for quantum communication networks. We present a modified gradient approach to realize InAs quantum dot molecules with a low areal density so that single quantum dot molecules can be optically addressed. The individual quantum dot layers were prepared by solid source molecular beam epitaxy depositing InAs on GaAs(100). The bottom quantum dot layer has been grown without substrate rotation resulting in an In-gradient across the surface, which translated into a density gradient with low quantum dot density in a certain region of the wafer. For the top quantum dot layer, separated from the bottom quantum dot layer by a 6 nm thick GaAs barrier, various InAs amounts were deposited without an In-gradient. In spite of the absence of an In-gradient, a pronounced density gradient is observed for the top quantum dots. Even for an In-amount slightly below the critical thickness for a single dot layer, a density gradient in the top quantum dot layer, which seems to reproduce the density gradient in the bottom layer, is observed. For more or less In, respectively, deviations from this behavior occur. We suggest that the obvious influence of the bottom quantum dot layer on the growth of the top quantum dots is due to the strain field induced by the buried dots.

Endogenous New World primate type C viruses isolated from owl monkey (Aotus trivirgatus) kidney cell line.

PubMed Central

Todaro, G J; Sherr, C J; Sen, A; King, N; Daniel, M D; Fleckenstein, B

1978-01-01

A type C virus (OMC-1) detected in a culture of owl monkey kidney cells resembled typical type C viruses morphologically, but was slightly larger than previously characterized mammalian type C viruses. OMC-1 can be transmitted to bat lung cells and cat embryo fibroblasts. The virions band at a density of 1.16 g/ml in isopycnic sucrose density gradients and contain reverse transcriptase and a 60-65S RNA genome composed of approximately 32S subunits. The reverse transcriptase is immunologically and biochemically distinct from the polymerases of othe retroviruses. Radioimmunoassays directed to the interspecies antigenic determinants of the major structure proteins of other type C viruses do not detect a related antigen in OMC-1. Nucleic acid hybridization experiments using labeled viral genomic RNA or proviral cDNA transcripts to normal cellular DNA of different species show that OMC-1 is an endogenous virus with multiple virogene copies (20-50 per haploid genome) present in normal owl monkey cells and is distinct from previously isolated type C and D viruses. Sequences related to the OMC-1 genome can be detected in other New World monkeys. Thus, similar to the Old World primates (e.g., baboons as a prototype), the New World monkeys contain endogenous type C viral genes that appear to have been transmitted in the primate germ line. Images PMID:76312

Heterotrophic cultivation of Chlorella pyrenoidosa using sucrose as the sole carbon source by co-culture with Rhodotorula glutinis.

PubMed

Wang, Shikai; Wu, Yong; Wang, Xu

2016-11-01

Heterotrophic cultivation of microalgae is a feasible alternative strategy to avoid the light limitation of photoautotrophic culture, but the heterotrophic utilization of disaccharides is difficult for microalgae. Aimed at this problem, a co-culture system was developed by mix culture of C. pyrenoidosa and R. glutinis using sucrose as the sole carbon source. In this system, C. pyrenoidosa could utilize glucose and fructose which were hydrolyzed from sucrose by R. glutinis. The highest specific growth rate and final cell number proportion of algae was 1.02day(-1) and 45%, respectively, when cultured at the initial algal cell number proportion of 95.24% and the final algal cell density was 111.48×10(6)cells/mL. In addition, the lipid content was also promoted due to the synergistic effects in mix culture. This study provides a novel approach using sucrose-riched wastes for the heterotrophic culture of microalgae and may effectively decrease the cost of carbon source. Copyright © 2016 Elsevier Ltd. All rights reserved.

Engineering zonal cartilage through bioprinting collagen type II hydrogel constructs with biomimetic chondrocyte density gradient.

PubMed

Ren, Xiang; Wang, Fuyou; Chen, Cheng; Gong, Xiaoyuan; Yin, Li; Yang, Liu

2016-07-20

Cartilage tissue engineering is a promising approach for repairing and regenerating cartilage tissue. To date, attempts have been made to construct zonal cartilage that mimics the cartilaginous matrix in different zones. However, little attention has been paid to the chondrocyte density gradient within the articular cartilage. We hypothesized that the chondrocyte density gradient plays an important role in forming the zonal distribution of extracellular matrix (ECM). In this study, collagen type II hydrogel/chondrocyte constructs were fabricated using a bioprinter. Three groups were created according to the total cell seeding density in collagen type II pre-gel: Group A, 2 × 10(7) cells/mL; Group B, 1 × 10(7) cells/mL; and Group C, 0.5 × 10(7) cells/mL. Each group included two types of construct: one with a biomimetic chondrocyte density gradient and the other with a single cell density. The constructs were cultured in vitro and harvested at 0, 1, 2, and 3 weeks for cell viability testing, reverse-transcription quantitative PCR (RT-qPCR), biochemical assays, and histological analysis. We found that total ECM production was positively correlated with the total cell density in the early culture stage, that the cell density gradient distribution resulted in a gradient distribution of ECM, and that the chondrocytes' biosynthetic ability was affected by both the total cell density and the cell distribution pattern. Our results suggested that zonal engineered cartilage could be fabricated by bioprinting collagen type II hydrogel constructs with a biomimetic cell density gradient. Both the total cell density and the cell distribution pattern should be optimized to achieve synergistic biological effects.

A High-Saturated-Fat, High-Sucrose Diet Aggravates Bone Loss in Ovariectomized Female Rats.

PubMed

Dong, Xiao-Li; Li, Chun-Mei; Cao, Si-Si; Zhou, Li-Ping; Wong, Man-Sau

2016-06-01

Estrogen deficiency in women and high-saturated fat, high-sucrose (HFS) diets have both been recognized as risk factors for metabolic syndrome. Studies on the combined actions of these 2 detrimental factors on the bone in females are limited. We sought to determine the interactive actions of estrogen deficiency and an HFS diet on bone properties and to investigate the underlying mechanisms. Six-month-old Sprague Dawley sham or ovariectomized (OVX) rats were pair fed the same amount of either a low-saturated-fat, low-sucrose (LFS) diet (13% fat calories; 15% sucrose calories) or an HFS diet (42% fat calories; 30% sucrose calories) for 12 wk. Blood, liver, and bone were collected for correspondent parameters measurement. Ovariectomy decreased bone mineral density in the tibia head (TH) by 62% and the femoral end (FE) by 49% (P < 0.0001). The HFS diet aggravated bone loss in OVX rats by an additional 41% in the TH and 37% in the FE (P < 0.05). Bone loss in the HFS-OVX rats was accompanied by increased urinary deoxypyridinoline concentrations by 28% (P < 0.05). The HFS diet induced cathepsin K by 145% but reduced osteoprotegerin mRNA expression at the FE of the HFS-sham rats by 71% (P < 0.05). Ovariectomy significantly increased peroxisome proliferator-activated receptor γ mRNA expression by 136% and 170% at the FE of the LFS- and HFS-OVX rats, respectively (P < 0.05). The HFS diet aggravated ovariectomy-induced lipid deposition and oxidative stress (OS) in rat livers (P < 0.05). Trabecular bone mineral density at the FE was negatively correlated with rat liver malondialdehyde concentrations (R(2) = 0.39; P < 0.01). The detrimental actions of the HFS diet and ovariectomy on bone properties in rats occurred mainly in cancellous bones and were characterized by a high degree of bone resorption and alterations in OS. © 2016 American Society for Nutrition.

In vitro cell-mediated immunity after thermal injury is not impaired. Density gradient purification of mononuclear cells is associated with spurious (artifactual) immunosuppression.

PubMed Central

Xu, D Z; Deitch, E A; Sittig, K; Qi, L; McDonald, J C

1988-01-01

Mononuclear cells isolated by density gradient centrifugation from the peripheral blood of burn patients, but not healthy volunteers, are contaminated with large numbers of nonmononuclear cells. These contaminating leukocytes could cause artifactual alterations in standard in vitro tests of lymphocyte function. Thus, we compared the in vitro blastogenic response of density gradient purified leukocytes and T-cell purified lymphocytes from 13 burn patients to mitogenic (PHA) and antigenic stimuli. The mitogenic and antigenic response of the patients' density gradient purified leukocytes were impaired compared to healthy volunteers (p less than 0.01). However, when the contaminating nonlymphocytes were removed, the patients' cells responded normally to both stimuli. Thus, density gradient purified mononuclear cells from burn patients are contaminated by leukocytes that are not phenotypically or functionally lymphocytes. Since the lymphocytes from burn patients respond normally to PHA and alloantigens after the contaminating nonlymphocyte cell population has been removed, it appears that in vitro assays of lymphocyte function using density gradient purified leukocytes may give spurious results. PMID:2973771

Schizosaccharomyces pombe Polysome Profile Analysis and RNA Purification.

PubMed

Wolf, Dieter A; Bähler, Jürg; Wise, Jo Ann

2017-04-03

Polysome profile analysis is widely used by investigators studying the mechanism and regulation of translation. The method described here uses high-velocity centrifugation of whole cell extracts on linear sucrose gradients to separate 40S and 60S ribosomal subunits from 80S monosomes and polysomes. Cycloheximide is included in the lysis buffer to "freeze" polysomes by blocking translation. After centrifugation, the gradient is fractionated and RNA (and/or protein) is prepared from each fraction for subsequent analysis of individual species using northern or western blots. The entire RNA population in each fraction can be analyzed by hybridization to microarrays or by high-throughput RNA sequencing, and the proteins present can be identified by mass spectrometry analysis. © 2017 Cold Spring Harbor Laboratory Press.

Virus purification by CsCl density gradient using general centrifugation.

PubMed

Nasukawa, Tadahiro; Uchiyama, Jumpei; Taharaguchi, Satoshi; Ota, Sumire; Ujihara, Takako; Matsuzaki, Shigenobu; Murakami, Hironobu; Mizukami, Keijirou; Sakaguchi, Masahiro

2017-11-01

Virus purification by cesium chloride (CsCl) density gradient, which generally requires an expensive ultracentrifuge, is an essential technique in virology. Here, we optimized virus purification by CsCl density gradient using general centrifugation (40,000 × g, 2 h, 4 °C), which showed almost the same purification ability as conventional CsCl density gradient ultracentrifugation (100,000 × g, 1 h, 4 °C) using phages S13' and φEF24C. Moreover, adenovirus strain JM1/1 was also successfully purified by this method. We suggest that general centrifugation can become a less costly alternative to ultracentrifugation for virus purification by CsCl densiy gradient and will thus encourage research in virology.

Use of Total Electron Content data to analyze ionosphere electron density gradients

NASA Astrophysics Data System (ADS)

Nava, B.; Radicella, S. M.; Leitinger, R.; Coisson, P.

In presence of electron density gradients the thin shell approximation for the ionosphere used together with a simple mapping function to convert slant Total Electron Content TEC to vertical TEC could lead to TEC conversion errors Therefore these mapping function errors can be used to identify the effects of the electron density gradients in the ionosphere In the present work high precision GPS derived slant TEC data have been used to investigate the effects of the electron density gradients in the middle and low latitude ionosphere under geomagnetic quiet and disturbed conditions In particular the data corresponding to the geographic area of the American sector for the days 5-7 April 2000 have been used to perform a complete analysis of mapping function errors based on the coinciding pierce point technique The results clearly illustrate the electron density gradient effects according to the locations considered and to the actual levels of disturbance of the ionosphere

First-row diatomics: Calculation of the geometry and energetics using self-consistent gradient-functional approximations

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

Kutzler, F.W.; Painter, G.S.

1992-02-15

A fully self-consistent series of nonlocal (gradient) density-functional calculations has been carried out using the augmented-Gaussian-orbital method to determine the magnitude of gradient corrections to the potential-energy curves of the first-row diatomics, Li{sub 2} through F{sub 2}. Both the Langreth-Mehl-Hu and the Perdew-Wang gradient-density functionals were used in calculations of the binding energy, bond length, and vibrational frequency for each dimer. Comparison with results obtained in the local-spin-density approximation (LSDA) using the Vosko-Wilk-Nusair functional, and with experiment, reveals that bond lengths and vibrational frequencies are rather insensitive to details of the gradient functionals, including self-consistency effects, but the gradient correctionsmore » reduce the overbinding commonly observed in the LSDA calculations of first-row diatomics (with the exception of Li{sub 2}, the gradient-functional binding-energy error is only 50--12 % of the LSDA error). The improved binding energies result from a large differential energy lowering, which occurs in open-shell atoms relative to the diatomics. The stabilization of the atom arises from the use of nonspherical charge and spin densities in the gradient-functional calculations. This stabilization is negligibly small in LSDA calculations performed with nonspherical densities.« less

Larger aggregates of mutant seipin in Celia's Encephalopathy, a new protein misfolding neurodegenerative disease.

PubMed

Ruiz-Riquelme, Alejandro; Sánchez-Iglesias, Sofía; Rábano, Alberto; Guillén-Navarro, Encarna; Domingo-Jiménez, Rosario; Ramos, Adriana; Rosa, Isaac; Senra, Ana; Nilsson, Peter; García, Ángel; Araújo-Vilar, David; Requena, Jesús R

2015-11-01

Celia's Encephalopathy (MIM #615924) is a recently discovered fatal neurodegenerative syndrome associated with a new BSCL2 mutation (c.985C>T) that results in an aberrant isoform of seipin (Celia seipin). This mutation is lethal in both homozygosity and compounded heterozygosity with a lipodystrophic BSCL2 mutation, resulting in a progressive encephalopathy with fatal outcomes at ages 6-8. Strikingly, heterozygous carriers are asymptomatic, conflicting with the gain of toxic function attributed to this mutation. Here we report new key insights about the molecular pathogenic mechanism of this new syndrome. Intranuclear inclusions containing mutant seipin were found in brain tissue from a homozygous patient suggesting a pathogenic mechanism similar to other neurodegenerative diseases featuring brain accumulation of aggregated, misfolded proteins. Sucrose gradient distribution showed that mutant seipin forms much larger aggregates as compared with wild type (wt) seipin, indicating an impaired oligomerization. On the other hand, the interaction between wt and Celia seipin confirmed by coimmunoprecipitation (CoIP) assays, together with the identification of mixed oligomers in sucrose gradient fractionation experiments can explain the lack of symptoms in heterozygous carriers. We propose that the increased aggregation and subsequent impaired oligomerization of Celia seipin leads to cell death. In heterozygous carriers, wt seipin might prevent the damage caused by mutant seipin through its sequestration into harmless mixed oligomers. Copyright © 2015 Elsevier Inc. All rights reserved.

Sonication-based isolation and enrichment of Chlorella protothecoides chloroplasts for illumina genome sequencing

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

Angelova, Angelina; Park, Sang-Hycuk; Kyndt, John

2013-09-01

With the increasing world demand for biofuel, a number of oleaginous algal species are being considered as renewable sources of oil. Chlorella protothecoides Krüger synthesizes triacylglycerols (TAGs) as storage compounds that can be converted into renewable fuel utilizing an anabolic pathway that is poorly understood. The paucity of algal chloroplast genome sequences has been an important constraint to chloroplast transformation and for studying gene expression in TAGs pathways. In this study, the intact chloroplasts were released from algal cells using sonication followed by sucrose gradient centrifugation, resulting in a 2.36-fold enrichment of chloroplasts from C. protothecoides, based on qPCR analysis.more » The C. protothecoides chloroplast genome (cpDNA) was determined using the Illumina HiSeq 2000 sequencing platform and found to be 84,576 Kb in size (8.57 Kb) in size, with a GC content of 30.8 %. This is the first report of an optimized protocol that uses a sonication step, followed by sucrose gradient centrifugation, to release and enrich intact chloroplasts from a microalga (C. prototheocoides) of sufficient quality to permit chloroplast genome sequencing with high coverage, while minimizing nuclear genome contamination. The approach is expected to guide chloroplast isolation from other oleaginous algal species for a variety of uses that benefit from enrichment of chloroplasts, ranging from biochemical analysis to genomics studies.« less

Use of Trypanosoma equiperdum infected rabbits as a source of splenic mRNA; construction of cDNA clones and identification of a rabbit mu heavy chain clone.

PubMed

Bernstein, K E; Pavirani, A; Alexander, C; Jacobsen, F; Fitzmaurice, L; Mage, R

1983-01-01

Rabbits were infected by Trypanosoma equiperdum and the splenic mRNA was isolated. In vitro translation of this RNA and immunoprecipitation with anti-light chain, anti-heavy chain, anti-mu and anti-VH antibodies demonstrated that T. equiperdum infection elicits large quantities of splenic mRNA encoding mu and kappa chains. The mu and gamma heavy chains and the kappa light chains synthesized in the cell-free translation system were specifically immunoprecipitated by antisera to heavy chain VHa and light chain kappa b allotypes. In vitro labeling of spleen cells from trypanosome-infected animals demonstrated that the biosynthetically labeled IgM has a mu chain of higher molecular weight than the mu chain synthesized by in vitro translation, a difference that is largely abolished when cellular glycosylation is blocked with the antibiotic tunicamycin. Enrichment for heavy chain or light chain mRNA was achieved by fractionating mRNA from trypanosome-infected animals on a sucrose gradient. cDNA clones carrying mu heavy chain sequences were produced using a 'one tube' protocol and identified by cross species hybridization and hybridization selection. Infection of rabbits with T. equiperdum followed by sucrose gradient enrichment of splenic mRNA has provided sufficient quantities of mRNA encoding mu heavy chain suitable for cDNA cloning.

The size of adenylate cyclase and guanylate cyclase from the rat renal medulla.

PubMed

Neer, E J

1976-01-01

The size distribution of adenylate cyclase from the rat renal medulla solubilized with the nonionic detergents Triton X-100 and Lubrol PX was determined by gel filtration and by centrifugation in sucrose density gradients made up in H2O or D2O. The physical parameters of the predominant form in Triton X-100 are s20,w, 5.9S; Strokes radius, 62 A; partial specific volume (v), 0.74 ml/g; mass, 159,000 daltons; f/f0, 1.6; axial ratio (prolate ellipsoid), 11. For the minor form the values are: s20w, 3.0; Stokes radius, 28 A; mass, 38,000 daltons; f/f0, 1.2. The corresponding values determined in Lubrol PX are similar. The value for V for the enzyme indicates that it binds less than 0.2 mg detergent/mg protein. Since interactions with detergents probably substitute for interactions with lipids and hydrophobic amino acid side chains, these findings suggest that no more than 5% of the surface of adenylate cyclase is involved in hydrophobic interactions with other membrane components. Thus, most of the mass of the enzyme is not deeply embedded in the lipid bilayer of the plasma membrane. Similar studies have been performed on the soluble guanylate cyclase of the rat renal medulla. In the absence of detergent, the molecular properties of this enzyme are: s20w, 6.3S; Stokes radius, 54 A, V, 0.75 ml/g; mass, 154,000 daltons f/f0, 1.4; Axial ratio, 7. The addition of 0.1% Lubrol PX to this soluble enzyme increases it activity two- to fourfold and changes the physical properties to: s20,w, 5.5S; Stokes radius, 62 A; V, 0.74 ml/g; mass, 148,000 daltons, f/f0, 1.6; axial ratio, 11. These results show that Lubrol PX activates the enzyme by causing a conformational change with unfolding on the polypeptide chain. Guanylate cyclase from the particulate cell fraction can be solubilized with Lubrol PX but has properties quite different from those of the enzyme in the soluble cell fraction. It is a heterogeneous aggregate with s20,w, 10S; Stokes radius, 65 A; mass about 300,000 daltons. The conditions which solubilize guanylate cyclase also solubilize adenylate cyclase and the two activities can be separated on the same sucrose gradient.

Particle simulation of electromagnetic emissions from electrostatic instability driven by an electron ring beam on the density gradient

NASA Astrophysics Data System (ADS)

Horký, Miroslav; Omura, Yoshiharu; Santolík, Ondřej

2018-04-01

This paper presents the wave mode conversion between electrostatic and electromagnetic waves on the plasma density gradient. We use 2-D electromagnetic code KEMPO2 implemented with the generation of density gradient to simulate such a conversion process. In the dense region, we use ring beam instability to generate electron Bernstein waves and we study the temporal evolution of wave spectra, velocity distributions, Poynting flux, and electric and magnetic energies to observe the wave mode conversion. Such a conversion process can be a source of electromagnetic emissions which are routinely measured by spacecraft on the plasmapause density gradient.

Density-Gradient-Driven trapped-electron-modes in improved-confinement RFP plasmas

NASA Astrophysics Data System (ADS)

Duff, James; Sarff, John; Ding, Weixing; Brower, David; Parke, Eli; Chapman, Brett; Terry, Paul; Pueschel, M. J.; Williams, Zach

2017-10-01

Short wavelength density fluctuations in improved-confinement MST plasmas exhibit multiple features characteristic of the trapped-electron-mode (TEM). Core transport in the RFP is normally governed by magnetic stochasticity stemming from long wavelength tearing modes that arise from current profile peaking, which are suppressed via inductive control for this work. The improved confinement is associated with an increase in the pressure gradient that can destabilize drift waves. The measured density fluctuations have f 50 kHz, kϕρs < 0.14 , and propagate in the electron drift direction. Their spectral emergence coincides with a sharp decrease in global tearing mode associated fluctuations, their amplitude increases with local density gradient, and they exhibit a density-gradient threshold at R /Ln 15 . The GENE code, modified for the RFP, predicts the onset of density-gradient-driven TEM for these strong-gradient plasma conditions. While nonlinear analysis shows a large Dimits shift associated with predicted strong zonal flows, the inclusion of residual magnetic fluctuations, comparable to experimental magnetic fluctuations, causes a collapse of the zonal flows and an increase in the predicted transport to a level close to the experimentally measured heat flux. Work supported by US DOE.

Drought response transcriptomes are altered in poplar with reduced tonoplast sucrose transporter expression

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

Xue, Liang-Jiao; Frost, Christopher J.; Tsai, Chung-Jui

Transgenic Populus tremula x alba (717-1B4) plants with reduced expression of a tonoplast sucrose efflux transporter, PtaSUT4, exhibit reduced shoot growth compared to wild type (WT) under sustained mild drought. The present study was undertaken to determine whether SUT4-RNAi directly or indirectly altered poplar predisposition and/or response to changes in soil water availability. While sucrose and hexose levels were constitutively elevated in shoot organs, expression responses to drought were most altered in the root tips of SUT4-RNAi plants. Prior to any drought treatment, constitutively elevated transcript levels of abscisic acid biosynthetic genes and bark/vegetative storage proteins suggested altered metabolism inmore » root tips of RNAi plants. Stronger drought-stimulation of stress-inducible genes encoding late-embryogenesis-abundant proteins in transgenic roots was consistent with increased vulnerability to soil drying. Transcript evidence suggested an RNAi effect on intercellular water trafficking by aquaporins in stem xylem during soil drying and recovery. Co-expression network analysis predicted altered integration of abscisic acid sensing/signaling with ethylene and jasmonate sensing/signaling in RNAi compared to WT roots. The overall conclusion is that steepened shoot-root sugar gradient in RNAi plants increased sensitivity of root tips to decreasing soil water availability.« less

Drought response transcriptomes are altered in poplar with reduced tonoplast sucrose transporter expression

DOE PAGES

Xue, Liang-Jiao; Frost, Christopher J.; Tsai, Chung-Jui; ...

2016-09-19

Transgenic Populus tremula x alba (717-1B4) plants with reduced expression of a tonoplast sucrose efflux transporter, PtaSUT4, exhibit reduced shoot growth compared to wild type (WT) under sustained mild drought. The present study was undertaken to determine whether SUT4-RNAi directly or indirectly altered poplar predisposition and/or response to changes in soil water availability. While sucrose and hexose levels were constitutively elevated in shoot organs, expression responses to drought were most altered in the root tips of SUT4-RNAi plants. Prior to any drought treatment, constitutively elevated transcript levels of abscisic acid biosynthetic genes and bark/vegetative storage proteins suggested altered metabolism inmore » root tips of RNAi plants. Stronger drought-stimulation of stress-inducible genes encoding late-embryogenesis-abundant proteins in transgenic roots was consistent with increased vulnerability to soil drying. Transcript evidence suggested an RNAi effect on intercellular water trafficking by aquaporins in stem xylem during soil drying and recovery. Co-expression network analysis predicted altered integration of abscisic acid sensing/signaling with ethylene and jasmonate sensing/signaling in RNAi compared to WT roots. The overall conclusion is that steepened shoot-root sugar gradient in RNAi plants increased sensitivity of root tips to decreasing soil water availability.« less

The effect of density gradients on hydrometers

NASA Astrophysics Data System (ADS)

Heinonen, Martti; Sillanpää, Sampo

2003-05-01

Hydrometers are simple but effective instruments for measuring the density of liquids. In this work, we studied the effect of non-uniform density of liquid on a hydrometer reading. The effect induced by vertical temperature gradients was investigated theoretically and experimentally. A method for compensating for the effect mathematically was developed and tested with experimental data obtained with the MIKES hydrometer calibration system. In the tests, the method was found reliable. However, the reliability depends on the available information on the hydrometer dimensions and density gradients.

Density-Gradient-Driven trapped-electron-modes in improved-confinement RFP plasmas

NASA Astrophysics Data System (ADS)

Duff, James

2016-10-01

Short wavelength density fluctuations in improved-confinement MST plasmas exhibit multiple features characteristic of the trapped-electron-mode (TEM), strong evidence that drift wave turbulence emerges in RFP plasmas when transport associated with MHD tearing is reduced. Core transport in the RFP is normally governed by magnetic stochasticity stemming from long wavelength tearing modes that arise from current profile peaking. Using inductive control, the tearing modes are reduced and global confinement is increased to values expected for a comparable tokamak plasma. The improved confinement is associated with a large increase in the pressure gradient that can destabilize drift waves. The measured density fluctuations have frequencies >50 kHz, wavenumbers k_phi*rho_s<0.14, and propagate in the electron drift direction. Their spectral emergence coincides with a sharp decrease in fluctuations associated with global tearing modes. Their amplitude increases with the local density gradient, and they exhibit a density-gradient threshold at R/L_n 15, higher than in tokamak plasmas by R/a. the GENE code, modified for RFP equilibria, predicts the onset of microinstability for these strong-gradient plasma conditions. The density-gradient-driven TEM is the dominant instability in the region where the measured density fluctuations are largest, and the experimental threshold-gradient is close to the predicted critical gradient for linear stability. While nonlinear analysis shows a large Dimits shift associated with predicted strong zonal flows, the inclusion of residual magnetic fluctuations causes a collapse of the zonal flows and an increase in the predicted transport to a level close to the experimentally measured heat flux. Similar circumstances could occur in the edge region of tokamak plasmas when resonant magnetic perturbations are applied for the control of ELMs. Work supported by US DOE.

Horizontal density-gradient effects on simulation of flow and transport in the Potomac Estuary

USGS Publications Warehouse

Schaffranek, Raymond W.; Baltzer, Robert A.; ,

1990-01-01

A two-dimensional, depth-integrated, hydrodynamic/transport model of the Potomac Estuary between Indian Head and Morgantown, Md., has been extended to include treatment of baroclinic forcing due to horizontal density gradients. The finite-difference model numerically integrates equations of mass and momentum conservation in conjunction with a transport equation for heat, salt, and constituent fluxes. Lateral and longitudinal density gradients are determined from salinity distributions computed from the convection-diffusion equation and an equation of state that expresses density as a function of temperature and salinity; thus, the hydrodynamic and transport computations are directly coupled. Horizontal density variations are shown to contribute significantly to momentum fluxes determined in the hydrodynamic computation. These fluxes lead to enchanced tidal pumping, and consequently greater dispersion, as is evidenced by numerical simulations. Density gradient effects on tidal propagation and transport behavior are discussed and demonstrated.

Inhibition of vaccinia virus maturation by zinc chloride.

PubMed Central

Katz, E; Margalith, E

1981-01-01

Zinc chloride (0.1 mM) inhibited by 96.4% the growth of vaccinia virus in HeLa cells. Approximately 50% inhibition in formation of particles that sedimented in sucrose gradients similarly to vaccinia virions occurred in the presence of zinc ions. Whereas the synthesis of the viral deoxyribonucleic acid was not affected by zinc chloride, a decrease in the overall synthesis of viral polypeptides and inhibition of the cleavage of precursors to the core polypeptides were observed. Images PMID:7347557

[A preparative method for isolating the synaptonemal complexes from mammalian spermatocytes].

PubMed

Dadashev, S Ia; Bogdanov, Iu F; Gorach, G G; Kolomiets, O L; Karpova, O I

1993-01-01

A method of isolation of synaptonemal complexes (SC) from mouse, rat and Syrian hamster spermatocytes is described. A fraction of pachytene spermatocyte nuclei was obtained by centrifugation of the testis homogenate in stepwise sucrose gradient and then lysed. The resulting chromatine was hydrolysed with DNAse II, and a fraction of isolated SCs was obtained by ultracentrifugation of the hydrolysate. The method can be applied for obtaining the SC fraction from spermatocytes sufficient for cytological, biochemical and molecular biology studies.

Use of total electron content data to analyze ionosphere electron density gradients

NASA Astrophysics Data System (ADS)

Nava, B.; Radicella, S. M.; Leitinger, R.; Coïsson, P.

In the presence of electron density gradients the thin shell approximation for the ionosphere, used together with a simple mapping function to convert slant total electron content (TEC) to vertical TEC, could lead to TEC conversion errors. These "mapping function errors" can therefore be used to detect the electron density gradients in the ionosphere. In the present work GPS derived slant TEC data have been used to investigate the effects of the electron density gradients in the middle and low latitude ionosphere under geomagnetic quiet and disturbed conditions. In particular the data corresponding to the geographic area of the American Sector for the days 5-7 April 2000 have been used to perform a complete analysis of mapping function errors based on the "coinciding pierce point technique". The results clearly illustrate the electron density gradient effects according to the locations considered and to the actual levels of disturbance of the ionosphere. In addition, the possibility to assess an ionospheric shell height able to minimize the mapping function errors has been verified.

The effect of shear flow and the density gradient on the Weibel instability growth rate in the dense plasma

NASA Astrophysics Data System (ADS)

Amininasab, S.; Sadighi-Bonabi, R.; Khodadadi Azadboni, F.

2018-02-01

Shear stress effect has been often neglected in calculation of the Weibel instability growth rate in laser-plasma interactions. In the present work, the role of the shear stress in the Weibel instability growth rate in the dense plasma with density gradient is explored. By increasing the density gradient, the shear stress threshold is increasing and the range of the propagation angles of growing modes is limited. Therefore, by increasing steps of the density gradient plasma near the relativistic electron beam-emitting region, the Weibel instability occurs at a higher stress flow. Calculations show that the minimum value of the stress rate threshold for linear polarization is greater than that of circular polarization. The Wiebel instability growth rate for linear polarization is 18.3 times circular polarization. One sees that for increasing stress and density gradient effects, there are smaller maximal growth rates for the range of the propagation angles of growing modes /π 2 < θ m i n < π and /3 π 2 < θ m i n < 2 π in circular polarized plasma and for /k c ω p < 4 in linear polarized plasma. Therefore, the shear stress and density gradient tend to stabilize the Weibel instability for /k c ω p < 4 in linear polarized plasma. Also, the shear stress and density gradient tend to stabilize the Weibel instability for the range of the propagation angles of growing modes /π 2 < θ m i n < π and /3 π 2 < θ m i n < 2 π in circular polarized plasma.

Comparison of two sperm processing techniques for low complexity assisted fertilization: sperm washing followed by swim-up and discontinuous density gradient centrifugation.

PubMed

Fácio, Cássio L; Previato, Lígia F; Machado-Paula, Ligiane A; Matheus, Paulo Cs; Araújo, Edilberto

2016-12-01

This study aimed to assess and compare sperm motility, concentration, and morphology recovery rates, before and after processing through sperm washing followed by swim-up or discontinuous density gradient centrifugation in normospermic individuals. Fifty-eight semen samples were used in double intrauterine insemination procedures; 17 samples (group 1) were prepared with sperm washing followed by swim-up, and 41 (group 2) by discontinuous density gradient centrifugation. This prospective non-randomized study assessed seminal parameters before and after semen processing. A dependent t-test was used for the same technique to analyze seminal parameters before and after semen processing; an independent t-test was used to compare the results before and after processing for both techniques. The two techniques produced decreases in sample concentration (sperm washing followed by swim-up: P<0.000006; discontinuous density gradient centrifugation: P=0.008457) and increases in motility and normal morphology sperm rates after processing. The difference in sperm motility between the two techniques was not statistically significant. Sperm washing followed by swim-up had better morphology recovery rates than discontinuous density gradient centrifugation (P=0.0095); and the density gradient group had better concentration recovery rates than the swim-up group (P=0.0027). The two methods successfully recovered the minimum sperm values needed to perform intrauterine insemination. Sperm washing followed by swim-up is indicated for semen with high sperm concentration and better morphology recovery rates. Discontinuous density gradient centrifugation produced improved concentration recovery rates.

Density Gradient Columns for Chemical Displays.

ERIC Educational Resources Information Center

Guenther, William B.

1986-01-01

Procedures for preparing density gradient columns for chemical displays are presented. They include displays illustrating acid-base reactions, metal ion equilibria, and liquid density. The lifetime of these metastable displays is surprising, some lasting for months in display cabinets. (JN)

Effects of low central fuelling on density and ion temperature profiles in reversed shear plasmas on JT-60U

NASA Astrophysics Data System (ADS)

Takenaga, H.; Ide, S.; Sakamoto, Y.; Fujita, T.; JT-60 Team

2008-07-01

Effects of low central fuelling on density and ion temperature profiles have been investigated using negative ion based neutral beam injection and electron cyclotron heating (ECH) in reversed shear plasmas on JT-60U. Strong internal transport barrier (ITB) was maintained in density and ion temperature profiles, when central fuelling was decreased by switching positive ion based neutral beam injection to ECH after the strong ITB formation. Similar density and ion temperature ITBs were formed for the low and high central fuelling cases during the plasma current ramp-up phase. Strong correlation between the density gradient and the ion temperature gradient was observed, indicating that particle transport and ion thermal transport are strongly coupled or the density gradient assists the ion temperature ITB formation through suppression of drift wave instabilities such as ion temperature gradient mode. These results support that the density and ion temperature ITBs can be formed under reactor relevant conditions.

Is Urografin density gradient centrifugation suitable to separate nonculturable cells from Escherichia coli populations?

PubMed

Arana, Inés; Orruño, Maite; Seco, Carolina; Muela, Alicia; Barcina, Isabel

2008-03-01

The ability of Urografin or Percoll density gradient centrifugations to separate nonculturable subpopulations from heterogeneous Escherichia coli populations was analysed. Bacterial counts (total, active and culturable cells) and flow cytometric analyses were carried out in all recovered bands. After Urografin centrifugation, and despite the different origin of E. coli populations, a common pattern was obtained. High-density bands were formed mainly by nonculturable cells. However, the increase in cell density would not be common to all nonculturable cells, since part of this subpopulations banded in low-density zones, mixed with culturable cells. Bands obtained after Percoll centrifugation were heterogeneous and culturable and nonculturable cells were recovered along the gradient. Thus, fractionation in Urografin cannot be only attributed to changes in buoyant densities during the transition from culturable to nonculturable state. Urografin density gradients allow us to obtain enriched fractions in nonculturable subpopulations from a heterogeneous population, but working conditions should be carefully chosen to avoid Urografin toxicity.

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

Haas, M.; Forbush, B. III

(Na + K + Cl) cotransport is the major mechanism of salt transport across the apical membrane of the epithelial cells of the thick ascending limb of Henle's loop of mammalian kidney and the site of action of loop diuretics such as furosemide and bumetanide. We have identified a 150-kDa protein in membranes from dog kidney cortex that is photolabeled by a radiolabeled, benzophenone analogue of bumetanide, (/sup 3/H)4-benzoyl-5-sulfamoyl-3-(3-thenyloxy)benzoic acid ((/sup 3/H)BSTBA). Several pieces of evidence strongly suggest that this 150-kDa protein is at least part of the (Na + K + Cl) cotransport system. 1) Photoincorporation of (/sup 3/H)BSTBAmore » into this protein is completely blocked by inclusion of 10 microM unlabeled bumetanide in the photolysis medium. 2) Photoincorporation of (/sup 3/H)BSTBA into this protein shows a saturable dependence on (/sup 3/H)BSTBA concentration, with a K 1/2 (approximately 0.1 microM) very similar to that for reversible (/sup 3/H)BSTBA binding to kidney membranes. 3) Photolabeling of this protein by (/sup 3/H)BSTBA requires the simultaneous presence of Na, K, and Cl in the photolysis medium. 4) When crude membranes from dog kidney cortex are centrifuged on sucrose density gradients, saturable (/sup 3/H)bumetanide binding and photoincorporation of (/sup 3/H)BSTBA in the 150-kDa region show a very similar distribution among the 15 gradient fractions collected. (/sup 3/H)BSTBA is also photoincorporated into at least two lower molecular mass proteins, the largest of which is approximately 50 kDa.« less

Effect of density gradient centrifugation on reactive oxygen species in human semen.

PubMed

Takeshima, Teppei; Yumura, Yasushi; Kuroda, Shinnosuke; Kawahara, Takashi; Uemura, Hiroji; Iwasaki, Akira

2017-06-01

Density gradient centrifugation can separate motile sperm from immotile sperm and other cells for assisted reproduction, but may also remove antioxidants from seminal plasma, resulting in oxidative stress. Therefore, we investigated reactive oxygen species (ROS) concentrations and distribution in semen before and after density gradient centrifugation. We assessed semen volume, sperm concentration, sperm motility, and ROS levels before and after density gradient centrifugation (300 x g for 20 minutes) in 143 semen samples from 118 patients. The ROS removal rate was evaluated in ROS-positive samples and ROS formation rate in ROS-negative samples. Thirty-eight of 143 untreated samples (26.6%) were ROS-positive; sperm motility was significantly lower in these samples than in ROS-negative samples (p < 0.05). After density gradient centrifugation, only seven of the 38 ROS-positive samples (18.42%) exhibited a ROS-positive lower layer (containing motile sperm) with a ROS removal rate of 81.58%, whereas the upper layer was ROS-positive in 24 samples (63.16%). In the ROS-negative group (n = 105), ROS was detected in 19 samples after centrifugation (18.10%, ROS generation rate), of which 18 were ROS-positive only in the upper layer or interface and the other was ROS-positive in both layers. Density gradient centrifugation can separate motile sperm from immotile sperm as well as remove ROS (including newly generated ROS). This data supports the view that density gradient centrifugation can select motile spermatozoa without enhancing oxidative stress. ROS: reactive oxygen species; SOD: superoxide dismutase; GPx: glutathione peroxidase; DNA: deoxyribonucleic acid; DGC: density gradient centrifugation; IUI: intrauterine insemination; IVF: in vitro fertilization; HEPES: 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid; EDTA: ethylenediaminetetraacetic acid; HTF: HEPES-buffered human tubal fluid; IMSI: intracytoplasmic morphologically selected sperm injection; SMAS: sperm motility analyzing system; CASA: computer-assisted semen analyzer; WHO: World Health Organization.

Renewable Hydrogen Carrier - Carbohydrate: Constructing the Carbon-Neutral Carbohydrate Economy

DTIC Science & Technology

2011-01-31

temperature. High fructose corn syrup , low-cost sucrose replacement, is made by stabilized glucose isomerase, which can work at ~60 °C for even about two...sustainable production, high -density storage, costly infrastructure, to eliminating safety concern. The use of renewable carbohydrate as a high -density...100% selectivity of enzymes, modest reaction conditions, and high -purity of generated hydrogen, carbohydrate is a promising hydrogen carrier for end

On the identity of the last known stable radical in X-irradiated sucrose

NASA Astrophysics Data System (ADS)

Kusakovskij, Jevgenij; De Cooman, Hendrik; Sagstuen, Einar; Callens, Freddy; Vrielinck, Henk

2017-04-01

Identification of radiation-induced radicals in relatively simple molecules is a prerequisite for the understanding of reaction pathways of the radiation chemistry of complex systems. Sucrose presents an additional practical interest as a versatile radiation dosimetric system. In this work, we present a periodic density functional theory study aimed to identify the fourth stable radical species in this carbohydrate. The proposed model is a fragment suspended in the lattice by hydrogen bonds with an unpaired electron at the original C5' carbon of the fructose unit. It requires a double scission of the ring accompanied by substantial chemical and geometric reorganization.

Growth Kinetics, Carbohydrate, and Leaf Phosphate Content of Clover (Trifolium subterraneum L.) after Transfer to a High CO2 Atmosphere or to High Light and Ambient Air 1

PubMed Central

Morin, Francoise; André, Marcel; Betsche, Thomas

1992-01-01

Intact air-grown (photosynthetic photon flux density, 400 microeinsteins per square meter per second) clover plants (Trifolium subterraneum L.) were transfered to high CO2 (4000 microliters CO2 per liter; photosynthetic photon flux density, 400 microeinsteins per square meter per second) or to high light (340 microliters CO2 per liter; photosynthetic photon flux density, 800 microeinsteins per square meter per second) to similarly stimulate photosynthetic net CO2 uptake. The daily increment of net CO2 uptake declined transiently in high CO2, but not in high light, below the values in air/standard light. After about 3 days in high CO2, the daily increment of net CO2 uptake increased but did not reach the high light values. Nightly CO2 release increased immediately in high light, whereas there was a 3-day lag phase in high CO2. During this time, starch accumulated to a high level, and leaf deterioration was observed only in high CO2. After 12 days, starch was two- to threefold higher in high CO2 than in high light, whereas sucrose was similar. Leaf carbohydrates were determined during the first and fourth day in high CO2. Starch increased rapidly throughout the day. Early in the day, sucrose was low and similar in high CO2 and ambient air (same light). Later, sucrose increased considerably in high CO2. The findings that (a) much more photosynthetic carbon was partitioned into the leaf starch pool in high CO2 than in high light, although net CO2 uptake was similar, and that (b) rapid starch formation occurred in high CO2 even when leaf sucrose was only slightly elevated suggest that low sink capacity was not the main constraint in high CO2. It is proposed that carbon partitioning between starch (chloroplast) and sucrose (cytosol) was perturbed by high CO2 because of the lack of photorespiration. Total phosphate pools were determined in leaves. Concentrations based on fresh weight of orthophosphate, soluble esterified phosphate, and total phosphate markedly declined during 13 days of exposure of the plants to high CO2 but changed little in high light/ambient air. During this time, the ratio of orthophosphate to soluble esterified phosphate decreased considerably in high CO2 and increased slightly in high light/ambient air. It appears that phosphate uptake and growth were similarly stimulated by high light, whereas the coordination was weak in high CO2. PMID:16668889

Nucleoprotein Complexes Containing Replicating Simian Virus 40 DNA: Comparison with Polyoma Nucleoprotein Complexes

PubMed Central

Hall, Mark R.; Meinke, William; Goldstein, David A.

1973-01-01

Procedures for isolating nucleoprotein complexes containing replicating polyoma DNA from infected mouse cells were used to prepare short-lived nucleoprotein complexes (r-SV40 complexes) containing replicating simian virus 40 (SV40) DNA from infected monkey cells. Like the polyoma complexes, r-SV40 complexes were only partially released from nuclei by cell lysis but could be extracted from nuclei by prolonged treatment with solutions containing Triton X-100. r-SV40 complexes sedimented faster than complexes containing SV40 supercoiled DNA (SV40 complex) in sucrose gradients, and both types of SV40 nucleoprotein complexes sedimented ahead of polyoma complexes containing supercoiled polyoma DNA (py complex). The sedimentation rates of py complex and SV40 complex were 56 and 61S, respectively, based on the sedimentation rate of the mouse large ribosomal subunit as a marker. r-SV40 complexes sedimented as multiple peaks between 56 and 75S. Sedimentation and buoyant density measurements indicated that protein is bound to all forms of SV40 DNA at about the same ratio of protein to DNA (1-2/1) as was reported for polyoma nucleoproteins. PMID:4359958

Isolation and separation of physicochemically distinct fimbrial types expressed on a single culture of Escherichia coli O7:K1:H6.

PubMed Central

Karch, H; Leying, H; Büscher, K H; Kroll, H P; Opferkuch, W

1985-01-01

The fimbrial (pili) profile of a single strain of Escherichia coli O7:K1:H6 (WF96) was evaluated. Fimbriae were isolated by sucrose density gradient ultracentrifugation, purified from flagellae by the use of 0.4% sodium dodecyl sulfate (SDS), and separated into distinct fimbrial types. Analysis of the purified WF96 fimbriae by SDS-polyacrylamide gel electrophoresis revealed two polypeptide bands with molecular weights of 16,000 and 21,000. Treatment of the fimbrial mixture with saturated guanidine hydrochloride resulted in the appearance of a third band with a molecular weight of 19,500. The relative susceptibilities of the WF96 fimbrial types to disrupting chemicals (octyl-glucoside, urea, SDS, and guanidine hydrochloride) were assessed by exposure of the fimbrial mixture to each agent, separation of the depolymerized fimbriae from intact fimbriae by gel filtration on Sepharose CL-4B, and identification of the disaggregated fimbrial types by SDS-polyacrylamide gel electrophoresis of column fractions. The physicochemical heterogeneity of the three fimbrial types coexpressed on WF96 was exploited to develop a method for separation of individual fimbriae. Images PMID:2857155

Preparation of protein samples for mass spectrometry and N-terminal sequencing.

PubMed

Glenn, Gary

2014-01-01

The preparation of protein samples for mass spectrometry and N-terminal sequencing is a key step in successfully identifying proteins. Mass spectrometry is a very sensitive technique, and as such, samples must be prepared carefully since they can be subject to contamination of the sample (e.g., due to incomplete subcellular fractionation or purification of a multiprotein complex), overwhelming of the sample by highly abundant proteins, and contamination from skin or hair (keratin can be a very common hit). One goal of sample preparation for mass spec is to reduce the complexity of the sample - in the example presented here, mitochondria are purified, solubilized, and fractionated by sucrose density gradient sedimentation prior to preparative 1D SDS-PAGE. It is important to verify the purity and integrity of the sample so that you can have confidence in the hits obtained. More protein is needed for N-terminal sequencing and ideally it should be purified to a single band when run on an SDS-polyacrylamide gel. The example presented here involves stably expressing a tagged protein in HEK293 cells and then isolating the protein by affinity purification and SDS-PAGE. © 2014 Elsevier Inc. All rights reserved.

LH-RH binding to purified pituitary plasma membranes: absence of adenylate cyclase activation.

PubMed

Clayton, R N; Shakespear, R A; Marshall, J C

1978-06-01

Purified bovine pituitary plasma membranes possess two specific LH-RH binding sites. The high affinity site (2.5 X 10(9) l/mol) has low capacity (9 X 10(-15) mol/mg membrane protein) while the low affinity site 6.1 X 10(5) l/mol) has a much higher capacity (1.1 X 10(-10) mol/mg). Specific LH-RH binding to plasma membranes is increased 8.5-fold during purification from homogenate whilst adenylate cyclase activity is enriched 7--8-fold. Distribution of specific LH-RH binding to sucrose density gradient interface fractions parallels that of adenylate cyclase activity. Mg2+ and Ca2+ inhibit specific [125I]LH-RH binding at micromolar concentrations. Synthetic LH-RH, up to 250 microgram/ml, failed to stimulate adenylase cyclase activity of the purified bovine membranes. Using a crude 10,800 g rat pituitary membrane preparation, LH-RH similarly failed to activate adenylate cyclase even in the presence of guanyl nucleotides. These data confirm the presence of LH-RH receptor sites on pituitary plasma membranes and suggest that LH-RH-induced gonadotrophin release may be mediated by mechanisms other than activation of adenylate cyclase.

A polygalacturonase localized in the Golgi apparatus in Pisum sativum.

PubMed

Ohashi, Takao; Jinno, Jun; Inoue, Yoshiyuki; Ito, Shoko; Fujiyama, Kazuhito; Ishimizu, Takeshi

2017-09-01

Pectin is a plant cell wall constituent that is mainly composed of polygalacturonic acid (PGA), a linear α1,4-d-galacturonic acid (GalUA) backbone. Polygalacturonase (PG) hydrolyzes the α1,4-linkages in PGA. Nearly all plant PGs identified thus far are secreted as soluble proteins. Here we describe the microsomal PG activity in pea (Pisum sativum) epicotyls and present biochemical evidence that it was localized to the Golgi apparatus, where pectins are biosynthesized. The microsomal PG was purified, and it was enzymatically characterized. The purified enzyme showed maximum activity towards pyridylaminated oligogalacturonic acids with six degrees of polymerization (PA-GalUA6), with a Km value of 11 μM for PA-GalUA6. The substrate preference of the enzyme was complementary to that of PGA synthase. The main PG activity in microsomes was detected in the Golgi fraction by sucrose density gradient ultracentrifugation. The activity of the microsomal PG was lower in rapidly growing epicotyls, in contrast to the high expression of PGA synthase. The role of this PG in the regulation of pectin biosynthesis or plant growth is discussed. © The Authors 2017. Published by Oxford University Press on behalf of the Japanese Biochemical Society. All rights reserved.

Association of the HNK-1 epitope with the detergent-soluble G4 isoform of acetylcholinesterase from human neuroblastoma cells.

PubMed

Johnson, G; Moore, S W

2001-07-01

The HNK-1 carbohydrate epitope is expressed in neural and natural killer cells and is a mediator of cell adhesion. It is well documented that acetylcholinesterase has a secondary function in cell adhesion and differentiation. The presence of HNK-1 on isoforms of Torpedo and Electrophorus acetylcholinesterase, as well as isoforms from the bovine central nervous system has been described. In this paper, we have investigated the association of the epitope with acetylcholinesterase from human neuroblastoma cells. Acetylcholinesterase was extracted, with or without detergent, purified on immunoaffinity columns and the isoforms separated by sucrose density gradient sedimentation. Secreted acetylcholinesterase, from spent serum-free culture medium, was similarly treated. The presence of the HNK-1 epitope was determined by ELISA using the anti-HNK-1 and Elec 39 monoclonal antibodies. The epitope was found to be associated with the detergent-soluble G4 isoform, but not with the hydrophilic G1 nor the secreted hydrophilic G4 isoforms. Likewise, no HNK-1 was observed associated with human erythrocyte acetylcholinesterase. These results indicate that acetylcholinesterase-G4, anchored in the extracellular membrane, is capable of mediating cell-substrate adhesion through HNK-1.

Molecular size of the gamma-aminobutyric acidA receptor purified from mammalian cerebral cortex.

PubMed

Mamalaki, C; Barnard, E A; Stephenson, F A

1989-01-01

The hydrodynamic behaviour of both the soluble and purified gamma-aminobutyric acidA (GABAA) receptor of bovine or rat cerebral cortex has been investigated in solution in Triton X-100 or in 3-[(3-cholamidopropyl)-dimethylammonio]-1-propanesulphonate (CHAPS). In all the hydrodynamic separations made, it was found that the binding activities for GABA, benzodiazepine, and (where detectable) t-butylbicyclophosphorothionate comigrated. Conditions were established for gel exclusion chromatography and for sucrose density gradient velocity sedimentation that maintain the GABAA receptor in a nonaggregated form. Using these conditions, the molecular weight of the bovine GABAA receptor in the above-mentioned detergents was calculated using the H2O/2H2O method. A value of Mr 230,000-240,000 was calculated for the bovine pure GABAA receptor purified in sodium deoxycholate/Triton X-100 media. A value of Mr 284,000-290,000 was calculated for the nonaggregated bovine or rat cortex receptor in CHAPS, but the Stokes radius is smaller in the latter than in the former medium and the detergent binding in CHAPS is underestimated. Thus the deduced Mr, 240,000, is the best estimate by this method.

Salmonella Enterica Serovar Typhimurium BipA Exhibits Two Distinct Ribosome Binding Modes

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

deLivron, M.; Robinson, V

BipA is a highly conserved prokaryotic GTPase that functions to influence numerous cellular processes in bacteria. In Escherichia coli and Salmonella enterica serovar Typhimurium, BipA has been implicated in controlling bacterial motility, modulating attachment and effacement processes, and upregulating the expression of virulence genes and is also responsible for avoidance of host defense mechanisms. In addition, BipA is thought to be involved in bacterial stress responses, such as those associated with virulence, temperature, and symbiosis. Thus, BipA is necessary for securing bacterial survival and successful invasion of the host. Steady-state kinetic analysis and pelleting assays were used to assess themore » GTPase and ribosome-binding properties of S. enterica BipA. Under normal bacterial growth, BipA associates with the ribosome in the GTP-bound state. However, using sucrose density gradients, we demonstrate that the association of BipA and the ribosome is altered under stress conditions in bacteria similar to those experienced during virulence. The data show that this differential binding is brought about by the presence of ppGpp, an alarmone that signals the onset of stress-related events in bacteria.« less

Environmentally Benign Battlefield Effects Black Smoke Simulator

DTIC Science & Technology

2006-11-01

tested and results Fuel Oxidizer Color of Smoke Density of Smoke Sugar (Sucrose) KNO3 Grey Medium Dextrin KNO3 Grey Thin Microcrystalline...design. 3.5 Initial Prototype Scale Fiberboard Testing Several quality black smoke formulations were identified in the small pellet testing to

Effects of agave nectar versus sucrose on weight gain, adiposity, blood glucose, insulin, and lipid responses in mice.

PubMed

Hooshmand, Shirin; Holloway, Brittany; Nemoseck, Tricia; Cole, Sarah; Petrisko, Yumi; Hong, Mee Young; Kern, Mark

2014-09-01

Agave nectar is a fructose-rich liquid sweetener derived from a plant, and is often promoted as a low glycemic alternative to refined sugar. However, little scientific research has been conducted in animals or humans to determine its metabolic and/or health effects. The aim of this study was to explore the influence of agave nectar versus sucrose on weight gain, adiposity, fasting plasma blood glucose, insulin, and lipid levels. Eighteen (n=18) male ICR mice (33.8±1.6 g) were divided into two groups (n=6 for agave nectar and n=12 for sucrose) and provided free access to one of two diets of equal energy densities differing only in a portion of the carbohydrate provided. Diets contained 20% carbohydrate (by weight of total diet) from either raw agave nectar or sucrose. Epididymal fat pads were excised, and blood was collected after 34 days. Weight gain (4.3±2.2 vs. 8.4±3.4 g), fat pad weights (0.95±0.54 vs. 1.75±0.66 g), plasma glucose (77.8±12.2 vs. 111.0±27.9 mg/dL), and insulin (0.61±0.29 vs. 1.46±0.81 ng/mL) were significantly lower (P≤.05) for agave nectar-fed mice compared to sucrose-fed mice respectively. No statistically significant differences in total cholesterol or triglycerides were detected. These results suggest that in comparison to sucrose, agave nectar may have a positive influence on weight gain and glucose control. However, more research with a larger sample of animals and/or with human subjects is warranted.

Analytic Expressions for the Gravity Gradient Tensor of 3D Prisms with Depth-Dependent Density

NASA Astrophysics Data System (ADS)

Jiang, Li; Liu, Jie; Zhang, Jianzhong; Feng, Zhibing

2017-12-01

Variable-density sources have been paid more attention in gravity modeling. We conduct the computation of gravity gradient tensor of given mass sources with variable density in this paper. 3D rectangular prisms, as simple building blocks, can be used to approximate well 3D irregular-shaped sources. A polynomial function of depth can represent flexibly the complicated density variations in each prism. Hence, we derive the analytic expressions in closed form for computing all components of the gravity gradient tensor due to a 3D right rectangular prism with an arbitrary-order polynomial density function of depth. The singularity of the expressions is analyzed. The singular points distribute at the corners of the prism or on some of the lines through the edges of the prism in the lower semi-space containing the prism. The expressions are validated, and their numerical stability is also evaluated through numerical tests. The numerical examples with variable-density prism and basin models show that the expressions within their range of numerical stability are superior in computational accuracy and efficiency to the common solution that sums up the effects of a collection of uniform subprisms, and provide an effective method for computing gravity gradient tensor of 3D irregular-shaped sources with complicated density variation. In addition, the tensor computed with variable density is different in magnitude from that with constant density. It demonstrates the importance of the gravity gradient tensor modeling with variable density.

Mobilization of Ca2+ by Cyclic ADP-Ribose from the Endoplasmic Reticulum of Cauliflower Florets1

PubMed Central

Navazio, Lorella; Mariani, Paola; Sanders, Dale

2001-01-01

The NAD+ metabolite cADP-Rib (cADPR) elevates cytosolic free Ca2+ in plants and thereby plays a central role in signal transduction pathways evoked by the drought and stress hormone abscisic acid. cADPR is known to mobilize Ca2+ from the large vacuole of mature cells. To determine whether additional sites for cADPR-gated Ca2+ release reside in plant cells, microsomes from cauliflower (Brassica oleracea) inflorescences were subfractionated on sucrose density gradients, and the distribution of cADPR-elicited Ca2+ release was monitored. cADPR-gated Ca2+ release was detected in the heavy-density fractions associated with rough endoplasmic reticulum (ER). cADPR-dependent Ca2+ release co-migrated with two ER markers, calnexin and antimycin A-insensitive NADH-cytochrome c reductase activity. To investigate the possibility that contaminating plasma membrane in the ER-rich fractions was responsible for the observed release, plasma membrane vesicles were purified by aqueous two-phase partitioning, everted with Brij-58, and loaded with Ca2+: These vesicles failed to respond to cADPR. Ca2+ release evoked by cADPR at the ER was fully inhibited by ruthenium red and 8-NH2-cADPR, a specific antagonist of cADPR-gated Ca2+ release in animal cells. The presence of a Ca2+ release pathway activated by cADPR at higher plant ER reinforces the notion that, alongside the vacuole, the ER participates in Ca2+ signaling. PMID:11299392

Lack of detection of a putative retrovirus associated with haemic neoplasia in the soft shell clam Mya arenaria.

PubMed

AboElkhair, M; Iwamoto, T; Clark, K F; McKenna, P; Siah, A; Greenwood, S J; Berthe, F C J; Casey, J W; Cepica, A

2012-01-01

Haemic neoplasia (HN) is a leukemia-like disease that affects at least 20 species of marine bivalves including soft shell clam, Mya arenaria. Since the disease was discovered in 1969, the etiology remains unknown. A retroviral etiology has been suggested based on the detection of reverse transcriptase activity and electron microscopic observation of retroviral-like particles using negative staining. To date, however no virus isolate and no retroviral sequence from HN has been obtained. Moreover, transmission of the disease by cell-free filtrate from affected clams has not been reproduced. In the current study, we reinvestigated the association of HN with a putative retrovirus. Sucrose gradient centrifugation followed by assessment of reverse transcriptase activity, electrophoretic analysis of protein and RNA, and electron microscopic examinations of fractions corresponding to retroviral density were employed. Detection of retroviral pol sequences using degenerate RT-PCR approaches was also attempted. Our results showed visible bands at the expected density of retrovirus in HN-positive and HN-negative clam tissues and both with reverse transcriptase activity. Electron microscopy, RNA analysis, protein analysis, and PCR systems targeting the pol gene of retroviruses did not however provide clear evidence supporting presence of a retrovirus. We point out that the retrovirus etiology of HN of Mya arenaria proposed some 25 years ago should be reconsidered in the absence of a virus isolate or virus sequences. Copyright © 2011 Elsevier Inc. All rights reserved.

Overnutrition in spiny mice (Acomys cahirinus): beta-cell expansion leading to rupture and overt diabetes on fat-rich diet and protective energy-wasting elevation in thyroid hormone on sucrose-rich diet.

PubMed

Shafrir, E

2000-01-01

The investigation of diabetes propensity in spiny mice, performed in Geneva and Jerusalem colonies, is reviewed. Spiny mice live in semi-desert regions of the eastern Mediterranean countries. Those transferred to Geneva in the 1950s were maintained on a rodent diet supplemented by fat-rich seeds. They became obese, exhibited pancreatic islet hyperplasia and hypertrophy. Low insulin secretion response was characteristic of this species, despite ample pancreatic content of insulin. After a few months, diabetes with ketosis occurred, often suddenly, in association with islet cell disintegration. In Jerusalem the spiny mice were collected from their native habitat and placed on diets containing 50% sucrose or fat-rich seed diets. On a sucrose-rich diet, spiny mice developed hepatomegaly, lipogenic enzyme hyperactivity, and elevation in very low density lipoproteins as a result of metabolism of the fructose component mainly in the liver. No overt diabetes or pancreatic islet disintegration were observed, although insulin content and beta-cell hypertrophy and hyperplasia were apparent. On a fat-rich diet, spiny mice exhibited marked weight gain, adipose tissue growth and low hepatic lipogenesis. The obesity was accompanied by mild hyperglycemia and hyperinsulinemia with glucose intolerance leading to an occasional glucosuria after several months on the diet. The sucrose diet induced an extrathyroidal elevation of triiodothyronine (T(3)). Serum T(3) level and hepatic T(4)-T(3) conversion were increased, while serum T(4) levels tended to decrease. The activity of the T(3)-inducible hepatic mitochondrial FAD-glycerophosphate oxidase and K(+)/Na(+)-ATPase, as well as body temperature were increased, indicating that the sucrose diet was associated with enhanced thermogenesis and energy-wasting metabolic cycling. The sucrose-rich diet might exert an adaptive thermogenesis-mediated defense mechanism, protecting against excessive weight gain and disruptive pancreatic islet lesion. After 18 months maintenance on sucrose-rich versus fat-rich diets the number of animals surviving was significantly higher on the sucrose diet whereas on the fat diet a significant number of animals succumbed to expansive islet cell disruption and diabetes.

On the biogeography of salt limitation: A study of ant communities

PubMed Central

Kaspari, Michael; Yanoviak, Stephen P.; Dudley, Robert

2008-01-01

Sodium is an essential nutrient whose deposition in rainfall decreases with distance inland. The herbivores and microbial decomposers that feed on sodium-poor vegetation should be particularly constrained along gradients of decreasing sodium. We studied the use of sucrose and NaCl baits in 17 New World ant communities located 4–2757 km inland. Sodium use was higher in genera and subfamilies characterized as omnivores/herbivores compared with those classified as carnivores and was lower in communities embedded in forest litter than in those embedded in abundant vegetation. Sodium use was increased in ant communities further inland, as was preference for the baits with the highest sodium concentration. Sucrose use, a measure of ant activity, peaked in communities 10–100 km inland. We suggest that the geography of ant activity is shaped by sodium toxicity near the shore and by sodium deficit farther inland. Given the importance of ants in terrestrial ecosystems, changing patterns of rainfall with global change may ramify through inland food webs. PMID:19004798

Development and validation of a critical gradient energetic particle driven Alfven eigenmode transport model for DIII-D tilted neutral beam experiments

DOE PAGES

Waltz, Ronald E.; Bass, Eric M.; Heidbrink, William W.; ...

2015-10-30

Recent experiments with the DIII-D tilted neutral beam injection (NBI) varying the beam energetic particle (EP) source profiles have provided strong evidence that unstable Alfven eigenmodes (AE) drive stiff EP transport at a critical EP density gradient. Here the critical gradient is identified by the local AE growth rate being equal to the local ITG/TEM growth rate at the same low toroidal mode number. The growth rates are taken from the gyrokinetic code GYRO. Simulation show that the slowing down beam-like EP distribution has a slightly lower critical gradient than the Maxwellian. The ALPHA EP density transport code, used tomore » validate the model, combines the low-n stiff EP critical density gradient AE mid-core transport with the energy independent high-n ITG/TEM density transport model controling the central core EP density profile. For the on-axis NBI heated DIII-D shot 146102, while the net loss to the edge is small, about half the birth fast ions are transported from the central core r/a < 0.5 and the central density is about half the slowing down density. Lastly, these results are in good agreement with experimental fast ion pressure profiles inferred from MSE constrained EFIT equilibria.« less

Plasma membrane cholesterol level and agonist-induced internalization of δ-opioid receptors; colocalization study with intracellular membrane markers of Rab family.

PubMed

Brejchova, Jana; Vosahlikova, Miroslava; Roubalova, Lenka; Parenti, Marco; Mauri, Mario; Chernyavskiy, Oleksandr; Svoboda, Petr

2016-08-01

Decrease of cholesterol level in plasma membrane of living HEK293 cells transiently expressing FLAG-δ-OR by β-cyclodextrin (β-CDX) resulted in a slight internalization of δ-OR. Massive internalization of δ-OR induced by specific agonist DADLE was diminished in cholesterol-depleted cells. These results suggest that agonist-induced internalization of δ-OR, which has been traditionally attributed exclusively to clathrin-mediated pathway, proceeds at least partially via membrane domains. Identification of internalized pools of FLAG-δ-OR by colocalization studies with proteins of Rab family indicated the decreased presence of receptors in early endosomes (Rab5), late endosomes and lysosomes (Rab7) and fast recycling vesicles (Rab4). Slow type of recycling (Rab11) was unchanged by cholesterol depletion. As expected, agonist-induced internalization of oxytocin receptors was totally suppressed in β-CDX-treated cells. Determination of average fluorescence lifetime of TMA-DPH, the polar derivative of hydrophobic membrane probe diphenylhexatriene, in live cells by FLIM indicated a significant alteration of the overall PM structure which may be interpreted as an increased "water-accessible space" within PM area. Data obtained by studies of HEK293 cells transiently expressing FLAG-δ-OR by "antibody feeding" method were extended by analysis of the effect of cholesterol depletion on distribution of FLAG-δ-OR in sucrose density gradients prepared from HEK293 cells stably expressing FLAG-δ-OR. Major part of FLAG-δ-OR was co-localized with plasma membrane marker Na,K-ATPase and β-CDX treatment resulted in shift of PM fragments containing both FLAG-δ-OR and Na,K-ATPase to higher density. Thus, the decrease in content of the major lipid constituent of PM resulted in increased density of resulting PM fragments.

Plasma membrane isolation using immobilized concanavalin A magnetic beads.

PubMed

Lee, Yu-Chen; Srajer Gajdosik, Martina; Josic, Djuro; Lin, Sue-Hwa

2012-01-01

Isolation of highly purified plasma membranes is the key step in constructing the plasma membrane proteome. Traditional plasma membrane isolation method takes advantage of the differential density of organelles. While differential centrifugation methods are sufficient to enrich for plasma membranes, the procedure is lengthy and results in low recovery of the membrane fraction. Importantly, there is significant contamination of the plasma membranes with other organelles. The traditional agarose affinity matrix is suitable for isolating proteins but has limitation in separating organelles due to the density of agarose. Immobilization of affinity ligands to magnetic beads allows separation of affinity matrix from organelles through magnets and could be developed for the isolation of organelles. We have developed a simple method for isolating plasma membranes using lectin concanavalin A (ConA) magnetic beads. ConA is immobilized onto magnetic beads by binding biotinylated ConA to streptavidin magnetic beads. The ConA magnetic beads are used to bind glycosylated proteins present in the membranes. The bound membranes are solubilized from the magnetic beads with a detergent containing the competing sugar alpha methyl mannoside. In this study, we describe the procedure of isolating rat liver plasma membranes using sucrose density gradient centrifugation as described by Neville. We then further purify the membrane fraction by using ConA magnetic beads. After this purification step, main liver plasma membrane proteins, especially the highly glycosylated ones and proteins containing transmembrane domains could be identified by LC-ESI-MS/MS. While not described here, the magnetic bead method can also be used to isolate plasma membranes from cell lysates. This membrane purification method should expedite the cataloging of plasma membrane proteome.

Biosynthesis and Intracellular Transport of 11S Globulin in Developing Pumpkin Cotyledons 1

PubMed Central

Hara-Nishimura, Ikuko; Nishimura, Mikio; Akazawa, Takashi

1985-01-01

In vitro studies to explore the biosynthesis of 11S globulin developing cotyledons of pumpkin (Cucurbita sp.) demonstrated that 11S globulin is synthesized on membrane-bound polysomes. Mr of the translation products (preproglobulin) synthesized by the poly(A)+-RNA isolated from developing cotyledons were determined to be 64,000 and 59,000, which are larger than those of the mature globulin subunit (62,000 and 57,000). Preproglobulin is then cotranslationally processed by cleavage of the signal peptide to produce proglobulin. In vivo pulse-chase experiments showed the sequential transformation of the single-chain proglobulin to mature globulin subunit (disulfide-linked doublet polypeptides) indicating posttranslational modification of the proglobulin. Subcellular fractionation of the pulse-chased intact cotyledons showed that the [35S]methionine label is detectable in proglobulin in rough endoplasmic reticulum shortly after the pulse label. With time, the labeled proteins move into other cellular fractions: proglobulin in the density = 1.24 grams per cubic centimeter fractions after 30 minutes and mature globulin subunit associated with protein bodies after 1 to 2 hours. The distribution of proglobulin in sucrose density gradients did not correspond with those of catalase (microbody marker) or fumarase (mitochondria marker). An accumulation of proglobulin occurred in the density = 1.24 grams per cubic centimeter fractions, whereas the mature globulin was scarcely detectable in this fraction. In contrast, proglobulin was not detected by immunochemical blotting analysis in the protein bodies prepared under the mild conditions from cotyledon protoplasts. The results suggest that the d = 1.24 grams per cubic centimeter fractions are engaged in the translocation of proglobulin into the protein bodies. Images Fig. 1 Fig. 2 Fig. 3 Fig. 4 Fig. 5 PMID:16664128

Resolution of G(s)alpha and G(q)alpha/G(11)alpha proteins in membrane domains by two-dimensional electrophoresis: the effect of long-term agonist stimulation.

PubMed

Matousek, P; Novotný, J; Svoboda, P

2004-01-01

Low-density membrane-domain fractions were prepared from S49 lymphoma cells and clone e2m11 of HEK293 cells expressing a large number of thyrotropin-releasing hormone receptor (TRH-R) and G(11)alpha by flotation on sucrose density gradients. The intact cell structure was broken by detergent-extraction, alkaline-treatment or drastic homogenization. Three types of low-density membranes were resolved by two-dimensional electrophoresis and analyzed for G(s)alpha (S49) or G(q)alpha/G11) (e2m11) content. Four individual immunoblot signals of Gsalpha protein were identified in S49 lymphoma cells indicating complete resolution of the long G(s)alpha L+/-ser and short G(s)alpha S+/-ser variants of G(s)alpha. All these were diminished by prolonged agonist (isoprenaline) stimulation. In e2m11-HEK cells, five different immunoblot signals were detected indicating post-translational modification of G proteins of G(q)alpha/G(11)alpha family. The two major spots corresponding to exogenously (over)expressed G(11)alpha and endogenous G(q)alpha were reduced; the minor spots diminished by hormonal stimulation. Parallel analysis by silver staining of the total protein content indicated that no major changes in protein composition occurred under these conditions. Our data thus indicate that agonist-stimulation of target cells results in down-regulation of all different members of G(s) and G(q)/G(11) families. This agonist-specific effect may be demonstrated in crude membrane as well as domain/raft preparations and it is not accompanied by changes in overall protein composition.

Assembly of the epithelial Na+ channel evaluated using sucrose gradient sedimentation analysis.

PubMed

Cheng, C; Prince, L S; Snyder, P M; Welsh, M J

1998-08-28

Three subunits, alpha, beta, and gamma, contribute to the formation of the epithelial Na+ channel. To investigate the oligomeric assembly of the channel complex, we used sucrose gradient sedimentation analysis to determine the sedimentation properties of individual subunits and heteromultimers comprised of multiple subunits. When the alpha subunit was expressed alone, it first formed an oligomeric complex with a sedimentation coefficient of 11 S, and then generated a higher order multimer of 25 S. In contrast, individual beta and gamma subunits predominately assembled into 11 S complexes. We obtained similar results with expression in cells and in vitro. When we co-expressed beta with alpha or with alpha plus gamma, the beta subunit assembled into a 25 S complex. Glycosylation of the alpha subunit was not required for assembly into a 25 S complex. We found that the alpha subunit formed intra-chain disulfide bonds. Although such bonds were not required to generate an oligomeric complex, under nonreducing conditions the alpha subunit formed a complex that migrated more homogeneously at 25 S. This suggests that intra-chain disulfide bonds may stabilize the complex. These data suggest that the epithelial Na+ channel subunits form high order oligomeric complexes and that the alpha subunit contains the information that facilitates such formation. Interestingly, the ability of the alpha, but not the beta or gamma, subunit to assemble into a 25 S homomeric complex correlates with the ability of these subunits to generate functional channels when expressed alone.

X-ray tomography characterization of density gradient aerogel in laser targets

NASA Astrophysics Data System (ADS)

Borisenko, L.; Orekhov, A.; Musgrave, C.; Nazarov, W.; Merkuliev, Yu; Borisenko, N.

2016-04-01

The low-density solid laser target characterization studies begun with the SkyScan 1074 computer microtomograph (CMT) [1, 2] are now continued with higher resolution of SkyScan 1174. The research is particularly focused on the possibility to obtain, control and measure precisely the gradient density polymers for laser target production. Repeatability of the samples and possibility to obtain stable gradients are analysed. The measurements were performed on the mm-scale divinyl benzene (DVB) rods.

Interconfigurational energies in transition-metal atoms using gradient-corrected density-functional theory

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

Kutzler, F.W.; Painter, G.S.

1991-03-15

The rapid variation of charge and spin densities in atoms and molecules provides a severe test for local-density-functional theory and for the use of gradient corrections. In the study reported in this paper, we use the Langreth, Mehl, and Hu (LMH) functional and the generalized gradient approximation (GGA) of Perdew and Yue to calculate {ital s}-{ital d} transition energies, 4{ital s} ionization energies, and 3{ital d} ionization energies for the 3{ital d} transition-metal atoms. These calculations are compared with results from the local-density functional of Vosko, Wilk, and Nusair. By comparison with experimental energies, we find that the gradient functionalsmore » are only marginally more successful than the local-density approximation in calculating energy differences between states in transition-metal atoms. The GGA approximation is somewhat better than the LMH functional for most of the atoms studied, although there are several exceptions.« less

Semi-local machine-learned kinetic energy density functional with third-order gradients of electron density

NASA Astrophysics Data System (ADS)

Seino, Junji; Kageyama, Ryo; Fujinami, Mikito; Ikabata, Yasuhiro; Nakai, Hiromi

2018-06-01

A semi-local kinetic energy density functional (KEDF) was constructed based on machine learning (ML). The present scheme adopts electron densities and their gradients up to third-order as the explanatory variables for ML and the Kohn-Sham (KS) kinetic energy density as the response variable in atoms and molecules. Numerical assessments of the present scheme were performed in atomic and molecular systems, including first- and second-period elements. The results of 37 conventional KEDFs with explicit formulae were also compared with those of the ML KEDF with an implicit formula. The inclusion of the higher order gradients reduces the deviation of the total kinetic energies from the KS calculations in a stepwise manner. Furthermore, our scheme with the third-order gradient resulted in the closest kinetic energies to the KS calculations out of the presented functionals.

Properties of ion temperature gradient and trapped electron modes in tokamak plasmas with inverted density profiles

NASA Astrophysics Data System (ADS)

Du, Huarong; Jhang, Hogun; Hahm, T. S.; Dong, J. Q.; Wang, Z. X.

2017-12-01

We perform a numerical study of linear stability of the ion temperature gradient (ITG) mode and the trapped electron mode (TEM) in tokamak plasmas with inverted density profiles. A local gyrokinetic integral equation is applied for this study. From comprehensive parametric scans, we obtain stability diagrams for ITG modes and TEMs in terms of density and temperature gradient scale lengths. The results show that, for the inverted density profile, there exists a normalized threshold temperature gradient above which the ITG mode and the TEM are either separately or simultaneously unstable. The instability threshold of the TEM for the inverted density profile is substantially different from that for normal and flat density profiles. In addition, deviations are found on the ITG threshold from an early analytic theory in sheared slab geometry with the adiabatic electron response [T. S. Hahm and W. M. Tang, Phys. Fluids B 1, 1185 (1989)]. A possible implication of this work on particle transport in pellet fueled tokamak plasmas is discussed.

Density gradients at hydrogel interfaces for enhanced cell penetration.

PubMed

Simona, B R; Hirt, L; Demkó, L; Zambelli, T; Vörös, J; Ehrbar, M; Milleret, V

2015-04-01

We report that stiffness gradients facilitate infiltration of cells through otherwise cell-impermeable hydrogel interfaces. By enabling the separation of hydrogel manufacturing and cell seeding, and by improving cell colonization of additively manufactured hydrogel elements, interfacial density gradients present a promising strategy to progress in the creation of 3D tissue models.

Merging Features and Optical-Near Infrared Color Gradients of Early-type Galaxies

NASA Astrophysics Data System (ADS)

Kim, Duho; Im, M.

2012-01-01

It has been suggested that merging plays an important role in the formation and the evolution of early-type galaxies (ETGs). Optical-NIR color gradients of ETGs in high density environments are found to be less steep than those of ETGs in low density environments, hinting frequent merger activities in ETGs in high density environments. In order to examine if the flat color gradients are the result of dry mergers, we studied the relations between merging features, color gradient, and environments of 198 low redshift ETGs selected from Sloan Digital Sky Survey (SDSS) Stripe82. Near Infrared (NIR) images are taken from UKIRT Infrared Deep Sky Survey (UKIDSS) Large Area Survey (LAS). Color(r-K) gradients of ETGs with tidal features are a little flatter than relaxed ETGs, but not significant. We found that massive (>1011.3 M⊙) relaxed ETGs have 2.5 times less scattered color gradients than less massive ETGs. The less scattered color gradients of massive ETGs could be evidence of dry merger processes in the evolution of massive ETGs. We found no relation between color gradients of ETGs and their environments.

Method and means for a spatial and temporal probe for laser-generated plumes based on density gradients

DOEpatents

Yeung, Edward S.; Chen, Guoying

1990-05-01

A method and means for a spatial and temporal probe for laser generated plumes based on density gradients includes generation of a plume of vaporized material from a surface by an energy source. The probe laser beam is positioned so that the plume passes through the probe laser beam. Movement of the probe laser beam caused by refraction from the density gradient of the plume is monitored. Spatial and temporal information, correlated to one another, is then derived.

Insecticidal and repellent activities of insecticide-sucrose solutions to Culex pipiens molestus (Diptera: Culicidae) under laboratory and field conditions.

PubMed

Shin, Ehyun; Park, Chan; Ahn, Young-Joon; Lee, Dong-Kyu; Chang, Kyu-Sik

2011-06-01

Culex pipiens molestus Forskal has been reported as a dominant species in underground structures of urban areas in the Republic of Korea (ROK) during all seasons and becomes bothersome to humans in late autumn and winter. Most Cx. pipiens molestus in septic tanks are controlled in the ROK using larvicides such as Bt and IGR. However, there are a number of problems associated with larvicides, such as high cost and requirement for frequent use. In the present work, a new control method for Cx. pipiens molestus in septic tanks by using mixtures of sucrose solution with insecticides was investigated. The insecticidal and repellent activities of ten insecticides were evaluated for best control of Cx. pipiens molestus in septic tanks. Firstly, differences in susceptibilities to insecticides were evaluated in topical assays by forced direct contact bioassay and in a screened wire cage by free direct contact bioassay. The difference in insecticide susceptibility in the mosquitoes was the result of repellency by the insecticides. In three septic tanks, the density of Culex mosquitoes was sharply reduced by a deltamethrin-sucrose solution kit. The results demonstrated the potential for mosquito control by deltamethrin-sucrose solution, and the study offers basic information related to mosquito control in septic tanks. Copyright © 2011 Society of Chemical Industry.

[In vitro utilization of fructooligosaccharide by streptococci mutans].

PubMed

Linardi, M M; Rosa, O P; Buzalaf, M A; Torres, S A

2001-01-01

Neosugar is the trade name of a fructooligosaccharide (FOS) whose utilization by oral bacteria is not well known yet. The aim of the present study was to evaluate in vitro the effect of this product on the growth, fermentation and production of plaque by mutans streptococci: S. mutans, serotypes c, e and f, S. sobrinus, serotype d, S. downei, serotype h, S. cricetus, serotype a and S. rattus, serotype b. The evaluation of growth was carried out in Brain Heart Infusion (BHI) broths containing or not sucrose and FOS and in buffered broths having glucose or FOS as carbon sources, through optical density reading in spectrophotometer after 24 hours of incubation at 37 degrees C. Thereafter the reading of pH was made in the same media. The plaque produced on glass sticks in BHI broths containing 5% sucrose or FOS was weighed and carbohydrates and proteins were assayed. The possible cariogenicity of Neosugar was confirmed, since it sustained the same growth and intensity of fermentation of sucrose in BHI broth for all streptococci and permitted in vitro production of plaque by some of them. The amount of plaque as well as its content of proteins and carbohydrates were smaller than those produced with sucrose, although the difference was statistically significant only for carbohydrates.

New biobased high functionality polyols and their use in polyurethane coatings.

PubMed

Pan, Xiao; Webster, Dean C

2012-02-13

High-functionality polyols for application in polyurethanes (PUs) were prepared by epoxide ring-opening reactions from epoxidized sucrose esters of soybean oil-epoxidized sucrose soyates-in which secondary hydroxyl groups were generated from epoxides on fatty acid chains. Ester polyols were prepared by using a base-catalyzed acid-epoxy reaction with carboxylic acids (e.g., acetic acid); ether polyols were prepared by using an acid-catalyzed alcohol-epoxy reaction with monoalcohols (e.g., methanol). The polyols were characterized by using gel permeation chromatography, FTIR spectroscopy, (1)H NMR spectroscopy, differential scanning calorimetry (DSC), and viscosity measurements. PU thermosets were prepared by using aliphatic polyisocyanates based on isophorone diisocyanate and hexamethylene diisocyanate. The properties of the PUs were studied by performing tensile testing, dynamic mechanical analysis, DSC, and thermogravimetric analysis. The properties of PU coatings on steel substrates were evaluated by using ASTM methods to determine coating hardness, adhesion, solvent resistance, and ductility. Compared to a soy triglyceride polyol, sucrose soyate polyols provide greater hardness and range of cross-link density to PU thermosets because of the unique structure of these macromolecules: well-defined compact structures with a rigid sucrose core coupled with high hydroxyl group functionality. Copyright © 2012 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Product suitability of wood...determined by density gradients across growth rings

Treesearch

Robert M. Echols

1972-01-01

The suitability of wood for various uses can be determined by synthesizing single growth-ring density curves from accumulated means of wood density classes. Wood density gradients across growth rings were measured in large increment cores from 46-year-old ponderosa pines (Pinus ponderosa Laws.) by using X-rays. Of the 48 trees analyzed, 36 had been...

Numerical Study on Density Gradient Carbon-Carbon Composite for Vertical Launching System

NASA Astrophysics Data System (ADS)

Yoon, Jin-Young; Kim, Chun-Gon; Lim, Juhwan

2018-04-01

This study presents new carbon-carbon (C/C) composite that has a density gradient within single material, and estimates its heat conduction performance by a numerical method. To address the high heat conduction of a high-density C/C, which can cause adhesion separation in the steel structures of vertical launching systems, density gradient carbon-carbon (DGCC) composite is proposed due to its exhibiting low thermal conductivity as well as excellent ablative resistance. DGCC is manufactured by hybridizing two different carbonization processes into a single carbon preform. One part exhibits a low density using phenolic resin carbonization to reduce heat conduction, and the other exhibits a high density using thermal gradient-chemical vapor infiltration for excellent ablative resistance. Numerical analysis for DGCC is performed with a heat conduction problem, and internal temperature distributions are estimated by the forward finite difference method. Material properties of the transition density layer, which is inevitably formed during DGCC manufacturing, are assumed to a combination of two density layers for numerical analysis. By comparing numerical results with experimental data, we validate that DGCC exhibits a low thermal conductivity, and it can serve as highly effective ablative material for vertical launching systems.

A refined model of sedimentary rock cover in the southeastern part of the Congo basin from GOCE gravity and vertical gravity gradient observations

NASA Astrophysics Data System (ADS)

Martinec, Zdeněk; Fullea, Javier

2015-03-01

We aim to interpret the vertical gravity and vertical gravity gradient of the GOCE-GRACE combined gravity model over the southeastern part of the Congo basin to refine the published model of sedimentary rock cover. We use the GOCO03S gravity model and evaluate its spherical harmonic representation at or near the Earth's surface. In this case, the gradiometry signals are enhanced as compared to the original measured GOCE gradients at satellite height and better emphasize the spatial pattern of sedimentary geology. To avoid aliasing, the omission error of the modelled gravity induced by the sedimentary rocks is adjusted to that of the GOCO03S gravity model. The mass-density Green's functions derived for the a priori structure of the sediments show a slightly greater sensitivity to the GOCO03S vertical gravity gradient than to the vertical gravity. Hence, the refinement of the sedimentary model is carried out for the vertical gravity gradient over the basin, such that a few anomalous values of the GOCO03S-derived vertical gravity gradient are adjusted by refining the model. We apply the 5-parameter Helmert's transformation, defined by 2 translations, 1 rotation and 2 scale parameters that are searched for by the steepest descent method. The refined sedimentary model is only slightly changed with respect to the original map, but it significantly improves the fit of the vertical gravity and vertical gravity gradient over the basin. However, there are still spatial features in the gravity and gradiometric data that remain unfitted by the refined model. These may be due to lateral density variation that is not contained in the model, a density contrast at the Moho discontinuity, lithospheric density stratifications or mantle convection. In a second step, the refined sedimentary model is used to find the vertical density stratification of sedimentary rocks. Although the gravity data can be interpreted by a constant sedimentary density, such a model does not correspond to the gravitational compaction of sedimentary rocks. Therefore, the density model is extended by including a linear increase in density with depth. Subsequent L2 and L∞ norm minimization procedures are applied to find the density parameters by adjusting both the vertical gravity and the vertical gravity gradient. We found that including the vertical gravity gradient in the interpretation of the GOCO03S-derived data reduces the non-uniqueness of the inverse gradiometric problem for density determination. The density structure of the sedimentary formations that provide the optimum predictions of the GOCO03S-derived gravity and vertical gradient of gravity consists of a surface density contrast with respect to surrounding rocks of 0.24-0.28 g/cm3 and its decrease with depth of 0.05-0.25 g/cm3 per 10 km. Moreover, the case where the sedimentary rocks are gravitationally completely compacted in the deepest parts of the basin is supported by L∞ norm minimization. However, this minimization also allows a remaining density contrast at the deepest parts of the sedimentary basin of about 0.1 g/cm3.

Density functional theory for molecular and periodic systems using density fitting and continuous fast multipole method: Analytical gradients.

PubMed

Łazarski, Roman; Burow, Asbjörn Manfred; Grajciar, Lukáš; Sierka, Marek

2016-10-30

A full implementation of analytical energy gradients for molecular and periodic systems is reported in the TURBOMOLE program package within the framework of Kohn-Sham density functional theory using Gaussian-type orbitals as basis functions. Its key component is a combination of density fitting (DF) approximation and continuous fast multipole method (CFMM) that allows for an efficient calculation of the Coulomb energy gradient. For exchange-correlation part the hierarchical numerical integration scheme (Burow and Sierka, Journal of Chemical Theory and Computation 2011, 7, 3097) is extended to energy gradients. Computational efficiency and asymptotic O(N) scaling behavior of the implementation is demonstrated for various molecular and periodic model systems, with the largest unit cell of hematite containing 640 atoms and 19,072 basis functions. The overall computational effort of energy gradient is comparable to that of the Kohn-Sham matrix formation. © 2016 Wiley Periodicals, Inc. © 2016 Wiley Periodicals, Inc.

Tripolar vortex formation in dense quantum plasma with ion-temperature-gradients

NASA Astrophysics Data System (ADS)

Qamar, Anisa; Ata-ur-Rahman, Mirza, Arshad M.

2012-05-01

We have derived system of nonlinear equations governing the dynamics of low-frequency electrostatic toroidal ion-temperature-gradient mode for dense quantum magnetoplasma. For some specific profiles of the equilibrium density, temperature, and ion velocity gradients, the nonlinear equations admit a stationary solution in the form of a tripolar vortex. These results are relevant to understand nonlinear structure formation in dense quantum plasmas in the presence of equilibrium ion-temperature and density gradients.

High resolution of heterogeneity among human neutrophil granules: physical, biochemical, and ultrastructural properties of isolated fractions.

PubMed

Rice, W G; Kinkade, J M; Parmley, R T

1986-08-01

Previous studies on the fractionation of human neutrophil granules have identified two major populations: myeloperoxidase (MPO)-containing azurophil, or primary, granules and MPO-deficient specific, or secondary, granules. Peripheral blood neutrophils from individual donors were lysed in sucrose-free media by either hypotonic shock or nitrogen cavitation. Using a novel two-gradient Percoll density centrifugation system, the granule-rich postnuclear supernatant was rapidly (ten minutes) and reproducibly resolved into 13 granule fractions (L1 through L8 and H1 through H5). Granule flotation and recentrifugation experiments on both continuous, self-generated and multiple-step gradients using individual and mixed isolated fractions demonstrated that the banding patterns were isopycnic and nonartifactual. Isolated granules were intact based on the findings that biochemical latency of several granule enzymes was greater than 95%, and thin-sectioned electron micrographs demonstrated intact granule profiles. Biochemical analyses of the granule marker proteins MPO, beta-glucuronidase, lysozyme, and lactoferrin indicated that a number of the fractions were related to the major azurophil and specific granule populations. Lactoferrin was found in ten of 13 fractions (L1 through L8, H1 to H2), whereas MPO was found in every fraction. Consistent with these biochemical data, all fractions exhibited varying degrees of heterogeneity based on ultrastructural morphology and cytochemistry, including diaminobenzidine (DAB) reactivity for peroxidase and periodate-thiocarbohydrazide-silver proteinate (PA-TCH-SP) staining for complex glycoconjugates. A variable but significant percentage (23% to 70%) of the granules in fractions L1 through L8 and H1 and H2 showed DAB reactivity, while about 90% of the granules in fractions H3 through H5 were peroxidase positive. These results demonstrated that DAB-reactive granules spanned the entire range of granule size and density. Ultrastructural PA-TCH-SP staining of isolated granule fractions revealed patterns similar to those of granules in intact neutrophils at different stages of development. Granules from human acute promyelocytic leukemia cells (HL-60) exhibited a surprisingly low density compared with typical azurophil granules from normal, mature neutrophils. The data suggest that both functional and maturational differences contribute to granule heterogeneity, and provide a new practical and conceptual framework for further defining the phenomenon of neutrophil granule heterogeneity.

Generalization of the van der Pauw Method: Analyzing Longitudinal Magnetoresistance Asymmetry to Quantify Doping Gradients

NASA Astrophysics Data System (ADS)

Grayson, M.; Zhou, Wang; Yoo, Heun-Mo; Prabhu-Gaunkar, S.; Tiemann, L.; Reichl, C.; Wegscheider, W.

A longitudinal magnetoresistance asymmetry (LMA) between a positive and negative magnetic field is known to occur in both the extreme quantum limit and the classical Drude limit in samples with a nonuniform doping density. By analyzing the current stream function in van der Pauw measurement geometry, it is shown that the electron density gradient can be quantitatively deduced from this LMA in the Drude regime. Results agree with gradients interpolated from local densities calibrated across an entire wafer, establishing a generalization of the van der Pauw method to quantify density gradients. Results will be shown of various semoconductor systems where this method is applied, from bulk doped semiconductors, to exfoliated 2D materials. McCormick Catalyst Award from Northwestern University, EECS Bridge Funding, and AFOSR FA9550-15-1-0247.

Postreplication Repair of Ultraviolet Damage in Haemophilus influenzae

PubMed Central

Leclerc, J. Eugene; Setlow, Jane K.

1972-01-01

The deoxyribonucleic acid (DNA) synthesized following ultraviolet (UV) irradiation of wild-type (Rd) and recombination-defective strains of Haemophilus influenzae has been analyzed by alkaline sucrose gradient sedimentation. Strain Rd and a UV-resistant, recombination-defective strain Rd(DB117) rec− are able to carry out postreplication repair, i.e., close the single-strand gaps in the newly synthesized DNA; in the UV-sensitive, recombination-defective strain DB117, the gaps remain open. The lack of postreplication repair in this strain may be the result of degradation of the newly synthesized DNA. PMID:4537422

Inactivation of Encephalomyocarditis Virus in Aerosols: Fate of Virus Protein and Ribonucleic Acid

PubMed Central

De Jong, J. C.; Harmsen, M.; Trouwborst, T.; Winkler, K. C.

1974-01-01

After aerosolization at relative humidities of 50% or lower, encephalomyocarditis virus is rapidly inactivated. In this process the protein coat of the virion is damaged. This appears as a loss of hemagglutination activity and loss of affinity for hemagglutination inhibiting antibodies. The ribonucleic acid of the virus retains its infectivity but it becomes susceptible to ribonuclease. It sediments in sucrose gradients when centrifuged at high speed with the same velocity as free infectious ribonucleic acid extracted with phenol from intact encephalomyocarditis virus. PMID:4358862

Identification and Characterization of Distinct Apoptotic Pathways in Cancer Cells Activated in Response to Treatment with Different Anti-Cancer Agents

DTIC Science & Technology

2000-08-01

caspases precursors in different cell type induce apoptosis (Miura et al. 1993; Fernandes- 3 Alnemri et al. 1994; Kumar et al. 1994; Wang et al. 1994; Boldin ...association of ribosomes with mRNA. The lysates were sedimented through a linear sucrose gradient and fractions were collected. RNA was extracted from...precursors in different cell type induces apoptosis (Miura et al. 1993; Fernandes- Alnemri et al. 1994; Kumar et al. 1994; Wang et al. 1994; Boldin et al

Temperature Gradients on the Cell Wall in the Critical Viscosity Experiment

NASA Technical Reports Server (NTRS)

Berg, Robert F.; Moldover, Michael R.

1993-01-01

Because of the diverging susceptibility delta rho/delta Tau near the liquid-vapor critical point, temperature gradients must be kept small to maintain adequate sample homogeneity. In our Science Requirements Document we paid particular attention to radial density gradients caused by equilibration of the xenon sample. Axial density gradients were addressed through the requirement that the cell's copper wall have a gradient less than 22 microK/m. This report re-examines the cell wall's temperature distribution in more detail by estimating all known significant contributions to temperature differences on the cell's wall.

Binding in pair potentials of liquid simple metals from nonlocality in electronic kinetic energy

NASA Technical Reports Server (NTRS)

Perrot, F.; March, N. H.

1990-01-01

The paper presents an explicit expression for the pair potential in liquid simple metals from low-order density-gradient theory when the superposition of single-center displaced charges is employed. Numerical results are presented for the gradient expansion pair interaction in liquid Na and Be. The low-order density-gradient equation for the pair potential is presented.

MEMS cantilever based magnetic field gradient sensor

NASA Astrophysics Data System (ADS)

Dabsch, Alexander; Rosenberg, Christoph; Stifter, Michael; Keplinger, Franz

2017-05-01

This paper describes major contributions to a MEMS magnetic field gradient sensor. An H-shaped structure supported by four arms with two circuit paths on the surface is designed for measuring two components of the magnetic flux density and one component of the gradient. The structure is produced from silicon wafers by a dry etching process. The gold leads on the surface carry the alternating current which interacts with the magnetic field component perpendicular to the direction of the current. If the excitation frequency is near to a mechanical resonance, vibrations with an amplitude within the range of 1-103 nm are expected. Both theoretical (simulations and analytic calculations) and experimental analysis have been carried out to optimize the structures for different strength of the magnetic gradient. In the same way the impact of the coupling structure on the resonance frequency and of different operating modes to simultaneously measure two components of the flux density were tested. For measuring the local gradient of the flux density the structure was operated at the first symmetrical and the first anti-symmetrical mode. Depending on the design, flux densities of approximately 2.5 µT and gradients starting from 1 µT mm-1 can be measured.

Acetylcholinesterase from Apis mellifera head. Evidence for amphiphilic and hydrophilic forms characterized by Triton X-114 phase separation.

PubMed Central

Belzunces, L P; Toutant, J P; Bounias, M

1988-01-01

The polymorphism of bee acetylcholinesterase was studied by sucrose-gradient-sedimentation analysis and non-denaturing electrophoretic analysis of fresh extracts. Lubrol-containing extracts exhibited only one form, which sedimented at 5 S when analysed on high-salt Lubrol-containing gradients and 6 S when analysed on low-salt Lubrol-containing gradients. The 5 S/6 S form aggregated upon removal of the detergent when sedimented on detergent-free gradients and was recovered in the detergent phase after Triton X-114 phase separation. Thus the 5 S/6 S enzyme corresponds to an amphiphilic acetylcholinesterase form. In detergent-free extracts three forms, whose apparent sedimentation coefficients are 14 S, 11 S and 7 S, were observed when sedimentations were performed on detergent-free gradients. Sedimentation analyses on detergent-containing gradients showed only a 5 S peak in high-salt detergent-free extracts and a 6 S peak, with a shoulder at about 7 S, in low-salt detergent-free extracts. Electrophoretic analysis in the presence of detergent demonstrated that the 14 S and 11 S peaks corresponded to aggregates of the 5 S/6 S form, whereas the 7 S peak corresponded to a hydrophilic acetylcholinesterase form which was recovered in the aqueous phase following Triton X-114 phase separation. The 5 S/6 S amphiphilic form could be converted into a 7.1 S hydrophilic form by phosphatidylinositol-specific phospholipase C digestion. Images Fig. 3. Fig. 6. PMID:2849414

Method and means for a spatial and temporal probe for laser-generated plumes based on density gradients

DOEpatents

Yeung, E.S.; Chen, G.

1990-05-01

A method and means are disclosed for a spatial and temporal probe for laser generated plumes based on density gradients includes generation of a plume of vaporized material from a surface by an energy source. The probe laser beam is positioned so that the plume passes through the probe laser beam. Movement of the probe laser beam caused by refraction from the density gradient of the plume is monitored. Spatial and temporal information, correlated to one another, is then derived. 15 figs.

Evaluation of effects of geometrical parameters on density distribution in compaction of PM gears

NASA Astrophysics Data System (ADS)

Khodaee, Alireza; Melander, Arne

2017-10-01

The usage of powder metallurgy (PM) for manufacturing of transmission components in automotive industries has been studied by many researchers. PM components have become of interest in recent years due to advancements in post processing possibilities such as hot isostatic pressing (HIP). Still in many of the forming process routes for making components from PM materials, the compaction of the powder into green component is the first step. Compaction is required to put the powder into the near net shape of the desired component and it causes a density gradient in the body of the green component. Basically the friction between powder particles and between the powder particles and die walls are the well-known roots for such density gradients in the compacted component. Looking at forming of PM gears, the gradient in density is one of the most important roots of problems in the processing of PM gears as well. That is because making a gear with full density and no pores will be very costly if large density gradients exist in the green component. The purpose of this study is to find the possible relations between the gear geometry and the density gradients in the green component after compaction in addition to the friction effects. For this purpose several gears should be tested. To reduce the research costs, the finite element (FE) method is used. First a FE model of the compaction process is developed and verified. To investigate the relations between the density gradients and the gear parameters such as addendum diameter (da) and the face width (b) several gear geometries have been studied. The compaction of selected gears is simulated using the FE model. The simulations results which are the distribution of density in the green component are evaluated and discussed and conclusion are made based on them.

Correlations between quasi-coherent fluctuations and the pedestal evolution during the inter-edge localized modes phase on DIII-D

DOE PAGES

Diallo, A.; Groebner, R. J.; Rhodes, T. L.; ...

2015-05-15

Direct measurements of the pedestal recovery during an edge-localized mode cycle provide evidence that quasi-coherent fluctuations (QCFs) play a role in the inter-ELM pedestal dynamics. When using fast Thomson scattering measurements, we found that the pedestal density and temperature evolutions are probed on sub-millisecond time scales to show a fast recovery of the density gradient compared to the temperature gradient. The temperature gradient appears to provide a drive for the onset of quasi-coherent fluctuations (as measured with the magnetic probe and the density diagnostics) localized in the pedestal. The amplitude evolution of these QCFs tracks the temperature gradient evolution includingmore » its saturation. Such correlation suggests that these QCFs play a key role in limiting the pedestal temperature gradient. Moreover, the saturation of the QCFs coincides with the pressure gradient reaching the kinetic-ballooning mode (KBM) critical gradient as predicted by EPED1. Furthermore, linear microinstability analysis using GS2 indicates that the steep gradient is near the KBM threshold. Finally, the modeling and the observations together suggest that QCFs are consistent with dominant KBMs, although microtearing cannot be excluded as subdominant.« less

Relationship between renal pathology and the size of circulating immune complexes in patients with systemic lupus erythematosus

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

Wener, M.H.; Mannik, M.; Schwartz, M.M.

1987-03-01

Sera from 35 patients with biopsy-proven diffuse proliferative (WHO class IV) or membranous (WHO class V) lupus nephritis were analyzed for the presence and size of circulating immune complexes. Elevations of the C1q solid-phase assay (C1qSP) for immune complexes were found in sera from all patients with diffuse proliferative nephritis, with a mean +/- 1 SEM of 166.8 +/- 42.0 micrograms/AHG-equivalents/ml serum, and in 71.4% of the patients with membranous nephritis (83.1 +/- 26.7, p = 0.06). Using the WHO criteria for subclasses of membranous lupus nephritis, we also designated renal biopsies as nonproliferative (WHO classes Va and Vb) ormore » proliferative (WHO classes IV and Vc). Employing the latter groupings, we observed significant differences between C1qSP results of patients with nonproliferative (30.3 +/- 8.8) and proliferative (172.8 +/- 36.8, p less than 0.001) lupus nephritis. These data suggest that the presence of C1q-binding material in serum is pathophysiologically related to proliferative glomerular lesions, and that levels of C1qSP binding reflect renal lesions in SLE patients. Sucrose density gradient ultracentrifugation was performed on each serum, and gradient fractions analyzed for C1qSP-binding and total IgG, using techniques to minimize losses of immune complexes. The predominant peak of C1qSP activity sedimented with the 6.6S monomeric IgG. The 6.6S C1q-binding IgG was increased only in 1 of 10 patients with membranous lupus nephritis without proliferative changes, and was elevated in 16 of 25 patients with proliferative lesions (WHO classes IV and Vc).« less

Ultrastructural and biochemical localization of N-RAP at the interface between myofibrils and intercalated disks in the mouse heart.

PubMed

Zhang, J Q; Elzey, B; Williams, G; Lu, S; Law, D J; Horowits, R

2001-12-11

N-RAP is a recently discovered muscle-specific protein found at cardiac intercalated disks. Double immunogold labeling of mouse cardiac muscle reveals that vinculin is located immediately adjacent to the fascia adherens region of the intercalated disk membrane, while N-RAP extends approximately 100 nm further toward the interior of the cell. We partially purified cardiac intercalated disks using low- and high-salt extractions followed by density gradient centrifugation. Immunoblots show that this preparation is highly enriched in desmin and junctional proteins, including N-RAP, talin, vinculin, beta1-integrin, N-cadherin, and connexin 43. Electron microscopy and immunolabeling demonstrate that N-RAP and vinculin are associated with the large fragments of intercalated disks that are present in this preparation, which also contains numerous membrane vesicles. Detergent treatment of the partially purified intercalated disks removed the membrane vesicles and extracted vinculin and beta1-integrin. Further separation on a sucrose gradient removed residual actin and myosin and yielded a fraction morphologically similar to fasciae adherentes that was highly enriched in N-RAP, N-cadherin, connexin 43, talin, desmin, and alpha-actinin. The finding that N-RAP copurifies with detergent-extracted intercalated disk fragments even though beta-integrin and vinculin have been completely removed suggests that N-RAP association with the adherens junction region is mediated by the cadherin system. Consistent with this hypothesis, we found that recombinant N-RAP fragments bind alpha-actinin in a gel overlay assay. In addition, immunofluorescence shows that N-RAP remains bound at the ends of isolated, detergent-treated cardiac myofibrils. These results demonstrate that N-RAP remains tightly bound to myofibrils and fasciae adherentes during biochemical purification and may be a key constituent in the mechanical link between these two structures.

Metabolic effects of aspartame in adulthood: A systematic review and meta-analysis of randomized clinical trials.

PubMed

Santos, Natalia Cardoso; de Araujo, Laiza Magalhaes; De Luca Canto, Graziela; Guerra, Eliete Neves Silva; Coelho, Michella Soares; Borin, Maria de Fatima

2017-04-10

Data about harms or benefits associated with the consumption of aspartame, a nonnutritive sweetener worldwide consumed, are still controversial. This systematic review and meta-analysis of randomized controlled clinical trials aimed to assess the effect of aspartame consumption on metabolic parameters related to diabetes and obesity. The search was performed on Cochrane, LILACS, PubMed, SCOPUS, Web of Science databases, and on a gray literature using Open Grey, Google Scholar, and ProQuest Dissertations & Theses Global. Searches across all databases were conducted from the earliest available date up to April 13, 2016, without date and language restrictions. Pooled mean differences were calculated using a random or fixed-effects model for heterogeneous and homogenous studies, respectively. Twenty-nine articles were included in qualitative synthesis and twelve, presenting numeric results, were used in meta-analysis. Fasting blood glucose (mmol/L), insulin levels (μU/mL), total cholesterol (mmol/L), triglycerides concentrations (mmol/L), high-density lipoprotein cholesterol (mmol/L), body weight (kg), and energy intake (MJ) were considered as the main outcomes in subjects that consumed aspartame, and results were presented as mean difference; % confidence interval, range. Aspartame consumption was not associated with alterations on blood glucose levels compared to control (-0.03 mmol/L; 95% CI, -0.21 to 0.14) or to sucrose (0.31 mmol/L; 95% CI, -0.05 to 0.67) and on insulin levels compared to control (0.13 μU/mL; 95% CI, -0.69 to 0.95) or to sucrose (2.54 μU/mL; 95% CI, -6.29 to 11.37). Total cholesterol was not affected by aspartame consumption compared to control (-0.02 mmol/L; 95% CI, -0.31 to 0.27) or to sucrose (-0.24 mmol/L; 95% CI, -0.89 to 0.42). Triglycerides concentrations were not affected by aspartame consumption compared to control (0.00 mmol/L; 95% CI, -0.04 to 0.05) or to sucrose (0.00 mmol/L; 95% CI, -0.09 to 0.09). High-density lipoprotein cholesterol serum levels were higher on aspartame compared to control (-0.03 mmol/L; 95% CI, -0.06 to -0.01) and lower on aspartame compared to sucrose (0.05 mmol/L; 95% CI, 0.02 to 0.09). Body weight did not change after aspartame consumption compared to control (5.00 kg; 95% CI, -1.56 to 11.56) or to sucrose (3.78 kg; 95% CI, -2.18 to 9.74). Energy intake was not altered by aspartame consumption compared to control (-0.49 MJ; 95% CI, -1.21 to 0.22) or to sucrose (-0.17 MJ; 95% CI, -2.03 to 1.69). Data concerning effects of aspartame on main metabolic variables associated to diabetes and obesity do not support a beneficial related to its consumption.

Development and validation of a critical gradient energetic particle driven Alfven eigenmode transport model for DIII-D tilted neutral beam experiments

NASA Astrophysics Data System (ADS)

Waltz, R. E.; Bass, E. M.; Heidbrink, W. W.; VanZeeland, M. A.

2015-11-01

Recent experiments with the DIII-D tilted neutral beam injection (NBI) varying the beam energetic particle (EP) source profiles have provided strong evidence that unstable Alfven eigenmodes (AE) drive stiff EP transport at a critical EP density gradient [Heidbrink et al 2013 Nucl. Fusion 53 093006]. Here the critical gradient is identified by the local AE growth rate being equal to the local ITG/TEM growth rate at the same low toroidal mode number. The growth rates are taken from the gyrokinetic code GYRO. Simulation show that the slowing down beam-like EP distribution has a slightly lower critical gradient than the Maxwellian. The ALPHA EP density transport code [Waltz and Bass 2014 Nucl. Fusion 54 104006], used to validate the model, combines the low-n stiff EP critical density gradient AE mid-core transport with the Angioni et al (2009 Nucl. Fusion 49 055013) energy independent high-n ITG/TEM density transport model controling the central core EP density profile. For the on-axis NBI heated DIII-D shot 146102, while the net loss to the edge is small, about half the birth fast ions are transported from the central core r/a  <  0.5 and the central density is about half the slowing down density. These results are in good agreement with experimental fast ion pressure profiles inferred from MSE constrained EFIT equilibria.

Steep, Transient Density Gradients in the Martian Ionosphere Similar to the Ionopause at Venus

NASA Astrophysics Data System (ADS)

Duru, Firdevs; Gurnett, Donald; Frahm, Rudy; Winningham, D. L.; Morgan, David; Howes, Gregory

Using Mars Advanced Radar for Subsurface and Ionospheric Sounding (MARSIS) on the Mars Express (MEX) spacecraft, the electron density can be measured by two methods: from the excitation of local plasma oscillations and from remote sounding. A study of the local electron density versus time for 1664 orbits revealed that in 132 orbits very sharp gradients in the electron density occurred that are similar to the ionopause boundary commonly observed at Venus. In 40 of these cases, remote sounding data have also confirmed identical locations of steep ionopause-like density gradients. Measurements from the Analyzer of Space Plasma and Energetic Atoms (ASPERA-3) Electron Spectrometer (ELS) and Ion Mass Analyzer (IMA) instruments (also on Mars Express) verify that these sharp decreases in the electron density occur somewhere between the end of the region where ionospheric photoelectrons are dominant and the magnetosheath. Combined studies of the two experiments reveal that the steep density gradients define a boundary where the magnetic fields change from open to closed. This study shows that, although the individual cases are from a wide range of altitudes, the average altitude of the boundary as a function of solar zenith angle is almost constant. The average altitude is approximately 500 km up to solar zenith angles of 60o, after which it shows a slight increase. The average thickness of the boundary is about 22 km according to remote sounding measurements. The altitude of the steep gradients shows an increase at locations with strong crustal magnetic fields.

Flow convergence caused by a salinity minimum in a tidal channel

USGS Publications Warehouse

Warner, John C.; Schoellhamer, David H.; Burau, Jon R.; Schladow, S. Geoffrey

2006-01-01

Residence times of dissolved substances and sedimentation rates in tidal channels are affected by residual (tidally averaged) circulation patterns. One influence on these circulation patterns is the longitudinal density gradient. In most estuaries the longitudinal density gradient typically maintains a constant direction. However, a junction of tidal channels can create a local reversal (change in sign) of the density gradient. This can occur due to a difference in the phase of tidal currents in each channel. In San Francisco Bay, the phasing of the currents at the junction of Mare Island Strait and Carquinez Strait produces a local salinity minimum in Mare Island Strait. At the location of a local salinity minimum the longitudinal density gradient reverses direction. This paper presents four numerical models that were used to investigate the circulation caused by the salinity minimum: (1) A simple one-dimensional (1D) finite difference model demonstrates that a local salinity minimum is advected into Mare Island Strait from the junction with Carquinez Strait during flood tide. (2) A three-dimensional (3D) hydrodynamic finite element model is used to compute the tidally averaged circulation in a channel that contains a salinity minimum (a change in the sign of the longitudinal density gradient) and compares that to a channel that contains a longitudinal density gradient in a constant direction. The tidally averaged circulation produced by the salinity minimum is characterized by converging flow at the bed and diverging flow at the surface, whereas the circulation produced by the constant direction gradient is characterized by converging flow at the bed and downstream surface currents. These velocity fields are used to drive both a particle tracking and a sediment transport model. (3) A particle tracking model demonstrates a 30 percent increase in the residence time of neutrally buoyant particles transported through the salinity minimum, as compared to transport through a constant direction density gradient. (4) A sediment transport model demonstrates increased deposition at the near-bed null point of the salinity minimum, as compared to the constant direction gradient null point. These results are corroborated by historically noted large sedimentation rates and a local maximum of selenium accumulation in clams at the null point in Mare Island Strait.

A density gradient of VAPG peptides on a cell-resisting surface achieves selective adhesion and directional migration of smooth muscle cells over fibroblasts.

PubMed

Yu, Shan; Zuo, Xingang; Shen, Tao; Duan, Yiyuan; Mao, Zhengwei; Gao, Changyou

2018-05-01

Selective adhesion and migration of smooth muscle cells (SMCs) over fibroblasts (FIBs) is required to prevent adventitia fibrosis in vascular regeneration. In this study, a uniform cell-resisting layer of poly(ethylene glycol) (PEG) with a density gradient of azide groups was generated on a substrate by immobilizing two kinds of PEG molecules in a gradient manner. A density gradient of alkynyl-functionalized Val-Ala-Pro-Gly (VAPG) peptides was then prepared on the PEG layer via click chemistry. The VAPG density gradient was characterized by fluorescence imaging, revealing the gradual enhancement of the fluorescent intensity along the substrate direction. The adhesion and mobility of SMCs were selectively enhanced on the VAPG density gradient, leading to directional migration toward the higher peptide density (up to 84%). In contrast, the adhesion and mobility of FIBs were significantly weakened. The net displacement of SMCs also significantly increased compared with that on tissue culture polystyrene (TCPS) and that of FIBs on the gradient. The mitogen-activated protein kinase (MAPK) signaling pathways related to cell migration were studied, showing higher expressions of functional proteins from SMCs on the VAPG-modified surface in a density-dependent manner. For the first time the selective adhesion and directional migration of SMCs over FIBs was achieved by an elaborative design of a gradient surface, leading to a new insight in design of novel vascular regenerative materials. Selective cell adhesion and migration guided by regenerative biomaterials are extremely important for the regeneration of targeted tissues, which can avoid the drawbacks of incorrect and uncontrolled responses of tissue cells to implants. For example, selectivity of smooth muscle cells (SMCs) over fibroblasts (FIBs) is required to prevent adventitia fibrosis in vascular regeneration. Herein we prepare a uniform cell-repelling layer, on which SMCs-selective Val-Ala-Pro-Gly (VAPG) peptides are immobilized in a continuous manner. Selective adhesion and enhanced and directional migration of SMCs over FIBs are achieved by the interplay of cell-repelling layer and gradient SMCs-selective VAPG peptides, paving a new way for the design of novel vascular grafts with enhanced biological performance. Copyright © 2018 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.

Generation of multiple toroidal dust vortices by a non-monotonic density gradient in a direct current glow discharge plasma

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

Kaur, Manjit, E-mail: manjit@ipr.res.in; Bose, Sayak; Chattopadhyay, P. K.

2015-09-15

Observation of two well-separated dust vortices in an unmagnetized parallel plate DC glow discharge plasma is reported in this paper. A non-monotonic radial density profile, achieved by an especially designed cathode structure using a concentric metallic disk and ring of different radii, is observed to produce double dust tori between cathode and anode. PIV analysis of the still images of the double tori shows oppositely rotating dust structures between the central disk and the ring. Langmuir probe measurements of background plasma shows a non-uniform plasma density profile between the disk and the ring. Location and sense of rotation of themore » dust vortices coincides with the location and direction of the radial gradient in the ion drag force caused by the radial density gradient. The experimentally observed dust vorticity matches well with the calculated one using hydrodynamic formulations with shear in ion drag dominating over the dust charge gradient. These results corroborate that a radial gradient in the ion drag force directed towards cathode is the principal cause of dust rotation.« less

Enviromental Effects on Internal Color Gradients of Early-Type Galaxies

NASA Astrophysics Data System (ADS)

La Barbera, F.; de Carvalho, R. R.; Gal, R. R.; Busarello, G.; Haines, C. P.; Mercurio, A.; Merluzzi, P.; Capaccioli, M.; Djorgovski, S. G.

2007-05-01

One of the most debated issues of observational and theoretical cosmology is that of how the environment affects the formation and evolution of galaxies. To gain new insight into this subject, we have derived surface photometry for a sample of 3,000 early-type galaxies belonging to 163 clusters with different richness, spanning a redshift range of 0.05 to 0.25. This large data-set is used to analyze how the color distribution inside galaxies depends on several parameters, such as cluster richness, local galaxy density, galaxy luminosity and redshift. We find that the internal color profile of galaxies strongly depends on the environment where galaxies reside. Galaxies in poor and rich clusters are found to follow two distinct trends in the color gradient vs. redshift diagram, with color gradients beeing less steep in rich rather than in poor clusters. No dependence of color gradients on galaxy luminosity is detected both for poor and rich clusters. We find that color gradients strongly depend on local galaxy density, with more shallow gradients in high density regions. Interestingly, this result holds only for low richness clusters, with color gradients of galaxies in rich clusters showing no dependence on local galaxy density. Our results support a reasonable picture whereby young early-type galaxies form in a dissipative collapse process, and then undergo increased (either major or minor) merging activity in richer rather than in poor clusters.

Biochemical characterization of detergent-resistant membranes: a systematic approach

PubMed Central

Babiychuk, Eduard B.; Draeger, Annette

2006-01-01

Lateral segregation of cholesterol- and sphingomyelin-rich rafts and glycerophospholipid-containing non-raft microdomains has been proposed to play a role in a variety of biological processes. The most compelling evidence for membrane segregation is based on the observation that extraction with non-ionic detergents leads to solubilization of a subset of membrane components only. However, one decade later, a large body of inconsistent detergent-extraction data is threatening the very concept of membrane segregation. We have assessed the validity of the existing paradigms and we show the following. (i) The localization of a membrane component within a particular fraction of a sucrose gradient cannot be taken as a yardstick for its solubility: a variable localization of the DRMs (detergent-resistant membranes) in sucrose gradients is the result of complex associations between the membrane skeleton and the lipid bilayer. (ii) DRMs of variable composition can be generated by using a single detergent, the increasing concentration of which gradually extracts one protein/lipid after another. Therefore any extraction pattern obtained by a single concentration experiment is bound to be ‘investigator-specific’. It follows that comparison of DRMs obtained by different detergents in a single concentration experiment is prone to misinterpretations. (iii) Depletion of cholesterol has a graded effect on membrane solubility. (iv) Differences in detergent solubility of the members of the annexin protein family arise from their association with chemically different membrane compartments; however, these cannot be attributed to the ‘brick-like’ raft-building blocks of fixed size and chemical composition. Our findings demonstrate a need for critical re-evaluation of the accumulated detergent-extraction data. PMID:16608442

Biochemical characterization of detergent-resistant membranes: a systematic approach.

PubMed

Babiychuk, Eduard B; Draeger, Annette

2006-08-01

Lateral segregation of cholesterol- and sphingomyelin-rich rafts and glycerophospholipid-containing non-raft microdomains has been proposed to play a role in a variety of biological processes. The most compelling evidence for membrane segregation is based on the observation that extraction with non-ionic detergents leads to solubilization of a subset of membrane components only. However, one decade later, a large body of inconsistent detergent-extraction data is threatening the very concept of membrane segregation. We have assessed the validity of the existing paradigms and we show the following. (i) The localization of a membrane component within a particular fraction of a sucrose gradient cannot be taken as a yardstick for its solubility: a variable localization of the DRMs (detergent-resistant membranes) in sucrose gradients is the result of complex associations between the membrane skeleton and the lipid bilayer. (ii) DRMs of variable composition can be generated by using a single detergent, the increasing concentration of which gradually extracts one protein/lipid after another. Therefore any extraction pattern obtained by a single concentration experiment is bound to be 'investigator-specific'. It follows that comparison of DRMs obtained by different detergents in a single concentration experiment is prone to misinterpretations. (iii) Depletion of cholesterol has a graded effect on membrane solubility. (iv) Differences in detergent solubility of the members of the annexin protein family arise from their association with chemically different membrane compartments; however, these cannot be attributed to the 'brick-like' raft-building blocks of fixed size and chemical composition. Our findings demonstrate a need for critical re-evaluation of the accumulated detergent-extraction data.

Degradation of surfactant-associated protein B (SP-B) during in vitro conversion of large to small surfactant aggregates.

PubMed Central

Veldhuizen, R A; Inchley, K; Hearn, S A; Lewis, J F; Possmayer, F

1993-01-01

Pulmonary surfactant obtained from lung lavages can be separated by differential centrifugation into two distinct subfractions known as large surfactant aggregates and small surfactant aggregates. The large-aggregate fraction is the precursor of the small-aggregate fraction. The ratio of the small non-surface-active to large surface-active surfactant aggregates increases after birth and in several types of lung injury. We have utilized an in vitro system, surface area cycling, to study the conversion of large into small aggregates. Small aggregates generated by surface area cycling were separated from large aggregates by centrifugation at 40,000 g for 15 min rather than by the normal sucrose gradient centrifugation. This new separation method was validated by morphological studies. Surface-tension-reducing activity of total surfactant extracts, as measured with a pulsating-bubble surfactometer, was impaired after surface area cycling. This impairment was related to the generation of small aggregates. Immunoblot analysis of large and small aggregates separated by sucrose gradient centrifugation revealed the presence of detectable amounts of surfactant-associated protein B (SP-B) in large aggregates but not in small aggregates. SP-A was detectable in both large and small aggregates. PAGE of cycled and non-cycled surfactant showed a reduction in SP-B after surface area cycling. We conclude that SP-B is degraded during the formation of small aggregates in vitro and that a change in surface area appears to be necessary for exposing SP-B to protease activity. Images Figure 2 Figure 5 Figure 6 Figure 7 PMID:8216208

Role of density gradient driven trapped electron mode turbulence in the H-mode inner core with electron heating

DOE PAGES

Ernst, D. R.; Burrell, K. H.; Guttenfelder, W.; ...

2016-05-10

In a series of DIII-D [J. L. Luxon, Nucl. Fusion 42 614 (2002)] low torque quiescent H-mode experiments show that density gradient driven TEM (DGTEM) turbulence dominates the inner core of H-Mode plasmas during strong electron cyclotron heating (ECH). By adding 3.4 MW ECH doubles T e/T i from 0.5 to 1.0, which halves the linear DGTEM critical density gradient, locally reducing density peaking, while transport in all channels displays extreme stiffness in the density gradient. This then suggests fusion -heating may degrade inner core confinement in H-Mode plasmas with moderate density peaking and low collisionality, with equal electron andmore » ion temperatures, key conditions expected in burning plasmas. Gyrokinetic simulations using GYRO [J. Candy and R. E. Waltz, J. Comp. Phys. 186 545 (2003)] (and GENE [F. Jenko et al., Phys. Plasmas 7, 1904 (2000)]) closely match not only particle, energy, and momentum fluxes, but also density fluctuation spectra from Doppler Backscattering (DBS), with and without ECH. Inner core DBS density fluctuations display discrete frequencies with adjacent toroidal mode numbers, which we identify as DGTEMs. GS2 [W. Dorland et al., Phys. Rev. Lett. 85 5579 (2000)] predictions show the DGTEM can be suppressed, to avoid degradation with electron heating, by broadening the current density profile to attain q 0 > q min > 1.« less

Enhanced recovery of valuable metals from spent lithium-ion batteries through optimization of organic acids produced by Aspergillus niger.

PubMed

Bahaloo-Horeh, Nazanin; Mousavi, Seyyed Mohammad

2017-02-01

In the present study, spent medium bioleaching method was performed using organic acids produced by Aspergillus niger to dissolve Ni, Co, Mn, Li, Cu and Al from spent lithium-ion batteries (LIBs). Response surface methodology was used to investigate the effects and interactions between the effective factors of sucrose concentration, initial pH, and inoculum size to optimize organic acid production. Maximum citric acid, malic acid, and gluconic acid concentrations of 26,478, 1832.53 and 8433.76ppm, respectively, and a minimum oxalic acid concentration of 305.558ppm were obtained under optimal conditions of 116.90 (gl -1 ) sucrose concentration, 3.45% (vv -1 ) inoculum size, and a pH value of 5.44. Biogenically-produced organic acids are used for leaching of spent LIBs at different pulp densities. The highest metal recovery of 100% Cu, 100% Li, 77% Mn, and 75% Al occurred at 2% (wv -1 ) pulp density; 64% Co and 54% Ni recovery occurred at 1% (wv -1 ) pulp density. The bioleaching of metals from spent LIBs can decrease the environmental impact of this waste. The results of this study suggest that the process can be used for large scale industrial purposes. Copyright © 2016 Elsevier Ltd. All rights reserved.

The isolation and subfractionation of plasma membrane from the cellular slime mould Dictyostelium discoideum

PubMed Central

Green, Anita A.; Newell, Peter C.

1974-01-01

A procedure for the isolation and separation of three different subfractions of plasma membrane from the cellular slime mould Dictyostelium discoideum is described. The cells were disrupted by freeze-thawing in liquid N2 and plasma membranes were purified by equilibrium centrifugation in a sucrose gradient. The cell surface was labelled with radioactive iodide by using the lactoperoxidase iodination method. Alkaline phosphatase was identified as a plasma-membrane marker by its co-distribution with [125I]iodide. 5′-Nucleotidase, which has been widely described as a plasma-membrane marker enzyme in mammalian tissues, was not localized to any marked extent in D. discoideum plasma membrane. The isolated plasma membranes showed a 24-fold enrichment of alkaline phosphatase specific activity relative to the homogenate and a yield of 50% of the total plasma membranes. Determination of succinate dehydrogenase and NADPH–cytochrome c reductase activities indicated that the preparation contained 2% of the total mitochondria and 3% of the endoplasmic reticulum. When the plasma-membrane preparation was further disrupted in a tight-fitting homogenizer, three plasma-membrane subfractions of different densities were obtained by isopycnic centrifugation. The enrichment of alkaline phosphatase was greatest in the subfraction with the lowest density. This fraction was enriched 36-fold relative to the homogenate and contained 19% of the total alkaline phosphatase activity but only 0.08% of the succinate dehydrogenase activity and 0.34% of the NADPH–cytochrome c reductase activity. Electron microscopy of this fraction showed it to consist of smooth membrane vesicles with no recognizable contaminants. ImagesPLATE 1 PMID:4156170

Fusicoccin-Binding Proteins in Arabidopsis thaliana (L.) Heynh. 1

PubMed Central

Meyer, Christiane; Feyerabend, Martin; Weiler, Elmar W.

1989-01-01

Using the novel radioligand, [3H]-9′-nor-fusicoccin-8′-alcohol, high affinity binding sites for fusicoccin were characterized in preparations from leaves of Arabidopsis thaliana (L.) Heynh. The binding site copartitioned with the plasmalemma marker, vanadate-sensitive K+, Mg2+-ATPase, when microsomal fractions were further purified by aqueous two-phase partitioning in polyethylene glycol-dextran phase systems and sedimented at an equilibrium density of 1.17 grams per cubic centimeter in continuous sucrose density gradients, as did the ATPase marker. The binding of [3H]-9′-nor-fusicoccin-8′-alcohol was saturable and Scatchard analysis revealed a biphasic plot with two apparent dissociation constants (KD), KD1 = 1.5 nanomolar and KD2 = 42 nanomolar, for the radioligand. Binding was optimal at pH 6, thermolabile, and was reduced by 70% when the membrane vesicles were pretreated with trypsin. The data are consistent with the presence of one or several binding proteins for fusicoccin at the plasma membrane of A. thaliana. Binding of the radioligand was unaffected by pretreatment of the sites with various alkylating and reducing agents, but was reduced by 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide, diethylpyrocarbonate, chloramine T, and periodate. A number of detergents were tested to find optimum conditions for solubilization. Nonanoyl-N-methylglucamide (50 millimolar) solubilized 70% of the radioligand-binding protein complex in undissociated form. Photoaffinity labeling of membrane preparations with a tritiated azido analog of fusicoccin resulted in the labeling of a 34 ± 1 kilodalton polypeptide. Labeling of this polypeptide, presumably the fusicoccin-binding protein, was severely reduced in the presence of unlabeled fusicoccin. PMID:16666603

Plant regeneration from leaf protoplasts of Solanum torvum.

PubMed

Guri, A; Volokita, M; Sink, K C

1987-07-01

A protocol to obtain regenerated plants from protoplasts of Solanum torvum Sw a wild species of eggplant resistant to Verticillium wilt is reported. Leaf protoplasts were enzymatically isolated from six-week old seedlings grown in a controlled environment chamber. Protoplasts were plated on modified KM medium (0.4 M glucose)+(mg/l): 1.0 p-chlorophenoxyacetic acid (CPA)+1.0 naphthaleneacetic acid (NAA)+0.5 6-benzylaminopurine (BAP) and 0.02 abscisic acid (ABA). The protoplast density was 5×10(4) per ml with 5 ml placed in each of two quadrants in X-dishes (100×15 mm). The reservoir medium was modified KM+(mg/l): 0.1 NAA+0.5 BAP+0.1 M sucrose+0.1 M mannitol+0.6% washed agar+1% activated charcoal. Dishes were initially placed in the dark at 27°C. Protoplast division was initiated in 1-2 weeks and 4 weeks later p-calli were 1-3 mm. Plating efficiency was 11% when measured at 3 weeks. Six-week old p-calli were transferred individually onto Whatman No. 1 filter paper layered on modified KM (0.15 M sucrose)+mg/l: 2.0 indoleacetic acid (IAA)+2.0 zeatin+0.5% washed agar for 2 weeks. Subsequently, shoots occurred within 4 weeks at 70% efficiency on MS+30 g/l sucrose+2 mg/l zeatin. Shoots were rooted on half strength MS+10 g/l sucrose.

The effect of normally consumed amounts of sucrose or high fructose corn syrup on lipid profiles, body composition and related parameters in overweight/obese subjects.

PubMed

Lowndes, Joshua; Sinnett, Stephanie; Pardo, Sabrina; Nguyen, Von T; Melanson, Kathleen J; Yu, Zhiping; Lowther, Britte E; Rippe, James M

2014-03-17

The American Heart Association (AHA) has advocated that women and men not consume more than 100 and 150 kcal/day, respectively, from added sugars. These levels are currently exceeded by over 90% of the adult population in the United States. Few data exist on longer-term metabolic effects when sucrose and High Fructose Corn Syrup (HFCS), the principal sources of added dietary sugars, are consumed at levels typical of the general population. Sixty five overweight and obese individuals were placed on a eucaloric (weight stable) diet for 10-weeks, which incorporated sucrose- or HFCS-sweetened, low-fat milk at 10% or 20% of calories in a randomized, double-blinded study. All groups responded similarly (interaction p > 0.05). There was no change in body weight in any of the groups over the 10-week study, or in systolic or diastolic blood pressure. Likewise, there were no changes in total cholesterol, triglycerides, low-density lipoprotein (LDL), or apolipoprotein B (Apo B). We conclude that (1) when consumed as part of a eucaloric diet fructose--when given with glucose (as normally consumed) does not promote weight gain or an atherogenic lipid profile even when consumed at two to four times the level recently recommended by the AHA. (2) There were no differences between HFCS and sucrose on these parameters.

The Effect of Normally Consumed Amounts of Sucrose or High Fructose Corn Syrup on Lipid Profiles, Body Composition and Related Parameters in Overweight/Obese Subjects

PubMed Central

Lowndes, Joshua; Sinnett, Stephanie; Pardo, Sabrina; Nguyen, Von T.; Melanson, Kathleen J.; Yu, Zhiping; Lowther, Britte E.; Rippe, James M.

2014-01-01

The American Heart Association (AHA) has advocated that women and men not consume more than 100 and 150 kcal/day, respectively, from added sugars. These levels are currently exceeded by over 90% of the adult population in the United States. Few data exist on longer-term metabolic effects when sucrose and High Fructose Corn Syrup (HFCS), the principal sources of added dietary sugars, are consumed at levels typical of the general population. Sixty five overweight and obese individuals were placed on a eucaloric (weight stable) diet for 10-weeks, which incorporated sucrose- or HFCS-sweetened, low-fat milk at 10% or 20% of calories in a randomized, double-blinded study. All groups responded similarly (interaction p > 0.05). There was no change in body weight in any of the groups over the 10-week study, or in systolic or diastolic blood pressure. Likewise, there were no changes in total cholesterol, triglycerides, low-density lipoprotein (LDL), or apolipoprotein B (Apo B). We conclude that (1) when consumed as part of a eucaloric diet fructose—when given with glucose (as normally consumed) does not promote weight gain or an atherogenic lipid profile even when consumed at two to four times the level recently recommended by the AHA. (2) There were no differences between HFCS and sucrose on these parameters. PMID:24642950

Persistence of sucrose fermenting and nonfermenting vibrios in tissues of Manila clam species, Ruditapes philippinarum, depurated in seawater at two different temperatures.

PubMed

Lopez-Joven, C; Ruiz-Zarzuela, I; de Blas, I; Furones, M D; Roque, A

2011-08-01

Effective post-harvest treatment to eliminate toxigenic vibrios is an important measure to reduce risk associated with seafood consumption. In the present study, we examined whether natural populations of sucrose nonfermenting (SNFV) and sucrose fermenting vibrios (SFV) persisted in Ruditapes philippinarum, after depuration at two different temperatures. Two experiments (one in Spring and one in Summer) were performed. Clams were depurated in duplicate tanks in an open-circuit seawater system using filtration and ultraviolet treatment. Clams were sampled daily (9 days), individually processed and inoculated on thiosulphate-citrate-bile salts-sucrose (TCBS) agar plates to estimate the density of SNFV and SFV. Results showed that depuration was less effective in Summer than in Spring, for SNFV and SFV. Despite an initial lower presence in the Summer experiment, clearance of SNFV was less efficient than in Spring, with 22.2% of clams presenting SNFV on day 8 and without significant changes on day 9. When compared, daily proportion of presence of SNFV and SFV in Spring, it was observed that SFV were more frequent than SNFV on days 8 and 9. In Summer these differences were significant on days 7-9. Present study demonstrated that SNFV (involved in food poisoning) are purged, at any temperature, faster than SFV. Copyright © 2011 Elsevier Ltd. All rights reserved.

The Relationships Between ELM Suppression, Pedestal Profiles, and Lithium Wall Coatings in NSTX

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

D.P. Boyle, R. Maingi, P.B. Snyder, J. Manickam, T.H. Osborne, R.E. Bell, B.P. LeBlanc, and the NSTX Team

2012-08-17

Recently in the National Spherical Torus Experiment (NSTX), increasing lithium wall coatings suppressed edge localized modes (ELMs), gradually but not quite monotonically. This work details profile and stability analysis as ELMs disappeared throughout the lithium scan. While the quantity of lithium deposited between discharges did not uniquely determine the presence of ELMs, profile analysis demonstrated that lithium was correlated to wider density and pressure pedestals with peak gradients farther from the separatrix. Moreover, the ELMy and ELM-free discharges were cleanly separated by their density and pedestal widths and peak gradient locations. Ultimately, ELMs were only suppressed when lithium caused themore » density pedestal to widen and shift inward. These changes in the density gradient were directly reflected in the pressure gradient and calculated bootstrap current. This supports the theory that ELMs in NSTX are caused by peeling and/or ballooning modes, as kink/peeling modes are stabilized when the edge current and pressure gradient shift away from the separatrix. Edge stability analysis using ELITE corroborated this picture, as reconstructed equilibria from ELM-free discharges were generally farther from their kink/peeling stability boundaries than ELMy discharges. We conclude that density profile control provided by lithium is the key first step to ELM suppression in NSTX« less

The relationships between edge localized modes suppression, pedestal profiles and lithium wall coatings in NSTX

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

Boyle, D. P.; Maingi, R.; Snyder, P. B.

2011-01-01

Recently in the National Spherical Torus Experiment (NSTX), increasing lithium wall coatings suppressed edge localized modes (ELMs), gradually but not quite monotonically. This work details profile and stability analysis as ELMs disappeared throughout the lithium scan. While the quantity of lithium deposited between discharges did not uniquely determine the presence of ELMs, profile analysis demonstrated that lithium was correlated with wider density and pressure pedestals with peak gradients farther from the separatrix. Moreover, the ELMy and ELM-free discharges were cleanly separated by their density and pedestal widths and peak gradient locations. Ultimately, ELMs were only suppressed when lithium caused themore » density pedestal to widen and shift inward. These changes in the density gradient were directly reflected in the pressure gradient and calculated bootstrap current. This supports the theory that ELMs in NSTX are caused by peeling and/or ballooning modes, as kink/peeling modes are stabilized when the edge current and pressure gradient shift away from the separatrix. Edge stability analysis using ELITE corroborated this picture, as reconstructed equilibria from ELM-free discharges were generally farther from their kink/peeling stability boundaries than ELMy discharges. We conclude that density profile control provided by lithium is the key first step to ELM suppression in NSTX.« less

Acclimation of Hydrilla verticillata to sediment anoxia in vegetation restoration in eutrophic waters.

PubMed

Wu, Juan; Dai, Yanran; Rui, Shengyang; Cui, Naxin; Zhong, Fei; Cheng, Shuiping

2015-12-01

Sediment anoxia generally results from intense organic enrichment and is a limiting factor in the restoration of vegetation in eutrophic waters. To investigate the effect of sediment anoxia on a typical pollution-tolerant submerged macrophyte species, Hydrilla verticillata, and acclimation mechanisms in the plant, a gradient of sediment anoxia was simulated with additions of sucrose to the sediment, which can stimulate increased concentrations of total nitrogen, NH4(+) and Fe in pore water. H. verticillata growth was significantly affected by highly anoxic conditions, as indicated by reduced total biomass in the 0.5 and 1% sucrose treatments. However, slight anoxia (0.1% sucrose addition) promoted growth, and the shoot biomass was 22.64% higher than in the control. In addition to morphologic alterations, H. verticillata showed physiological acclimations to anoxia, including increased anaerobic respiration and changes in carbon and nitrogen metabolism in roots. The soluble protein and soluble carbohydrate contents in roots of the 1% treatment were both significantly higher compared with those in the control. The increase in alcohol dehydrogenase activity and pyruvate content in the roots suggested that H. verticillata has a well-developed capacity for anaerobic fermentation. This study suggests that highly anoxic sediments inhibit the growth of H. verticillata and the species has a degree of tolerance to anoxic conditions. Further in situ investigations should be conducted on the interactions between sediment conditions and macrophytes to comprehensively evaluate the roles of sediment in the restoration of vegetation in eutrophic waters.

ISOLATION OF SMOOTH VESICLES AND FREE RIBOSOMES FROM RAT LIVER MICROSOMES

PubMed Central

Chauveau, J.; Moulé, Y.; Rouiller, C.; Schneebeli, J.

1962-01-01

Microsomes, isolated from rat liver homogenate in 0.88 M sucrose, have been fractionated by differential centrifugation. The 2nd microsomal fraction, sedimented between 60 minutes at 105,000 g and 3 hours at 145,000 g, consists mainly of smooth vesicles, free ribosomes, and ferritin. By utilizing the differences in density existing between the membranes and the granular elements it has been possible to separate the smooth membranes from the free ribosomes and ferritin. The procedure is to resuspend the 2nd microsomal fraction in a sucrose solution of 1.21 or 1.25 density and centrifuge it at 145,000 g for 20 or 40 hours. A centripetal migration of membranes and a centrifugal sedimentation of granular elements are obtained. Phospholipids, as well as the enzymatic activities DPNH-cytochrome c reductase, glucose-6-phosphatase and esterase are localized in the membranes. The free ribosomes have been purified by washing. A concentration of 200 µg RNA per mg nitrogen has been reached. RNA is also present in the membranes. These results are discussed in relation to current views on microsomal structure and chemistry. PMID:13878497

[Simultaneous determination of sugar alcohols and sugars in functional foods by precolumn ultraviolet derivatization--high performance liquid chromatography].

PubMed

Liu, Ya-Pan; Ran, Xue-Qin; Chen, Lu-Ying; Zhang, Jing; Ruan, Jia; Li, Yong-Xin; Sun, Chengjun

2014-09-01

To establish a method using precolumn ultraviolet derivatization coupled with high performance liquid chromatography (HPLC) for simultaneous determination of erythritol, xylitol, galactitol, sorbitol, mannitol, maltitol, glucose and sucrose in functional foods. Target sugar alcohols and sugars in food samples were extracted in water by ultrasonic method and then reacted with benzoyl chloride to form violet-absorbing products, which were separated on a C18 column with gradient elution using methanol and water as mobile phase. The experiment was performed using a flow rate of 1.00 mL/min, column temperature at 30 degrees C and detected wavelength at 232 nm. The linear correlation coefficients of all the derivatives were more than 0. 999. The detection limits of the method were as low as 2. 2 microg/mL. The average recoveries were 89.6%-117.0%, with intraday relative standard derivations lower than 5%. This method is simple, inexpensive and easy to operate and it is suitable for the determination of sugar alcohols and glucose and sucrose in functional foods.

Complex mountain terrain and disturbance history drive variation in forest aboveground live carbon density in the western Oregon Cascades, USA

Treesearch

Harold S.J. Zald; Thomas A. Spies; Rupert Seidl; Robert J. Pabst; Keith A. Olsen; Ashley Steel

2016-01-01

Forest carbon (C) density varies tremendously across space due to the inherent heterogeneity of forest ecosystems. Variation of forest C density is especially pronounced in mountainous terrain, where environmental gradients are compressed and vary at multiple spatial scales. Additionally, the influence of environmental gradients may vary with forest age and...

Controls on stand transpiration and soil water utilization along a tree density gradient in a Neotropical savanna

Treesearch

Sandra J. Bucci; Fabian G. Scholz; Guillermo Goldstein; William A. Hoffmann; Frederick C. Meinzer; Augusto C. Franco; Thomas Giambelluca; Fernando Miralles-Wilhelm

2008-01-01

Environmental controls of stand-level tree transpiration (E) and seasonal patterns of soil water utilization were studied in five central Brazilian savanna (Cerrado) sites differing in tree density. Tree density of Cerrado vegetation in the study area consistently changes along topographic gradients from ~1,000 trees ha-1 in open savannas (campo...

[Studies of progestin specific binding protein in the human prostate. [III]; Sodium molybdate effect on SDG analysis].

PubMed

Imai, K; Kumasaka, F; Kobayashi, M; Takahashi, Y; Takahashi, E; Yamanaka, H

1985-08-20

The effect of sodium molybdate on the specific binding protein (SBP) of synthetic progestin 17 alpha-methyl-[3H]-promegestone (R5020) in the cytosol of the human prostate was studied. In a sucrose density gradient analysis, two R5020 SBP components at 4S and 7-8S were observed. It was apparent that the 4S component was reduced and the 7-8S component increased with the addition of 10mM sodium molybdate into the cytosol. Therefore, the molybdate enhancement degree on total SBP amount (4S plus 7-8S) was decided by the relationship between the decreasing rate at 4S and the increasing one at 7-8S. It was shown that the molybdate effect was time-dependent and was not related to the SBP state, whether it was bounded with steroid or not. Moreover, it was estimated that the molybdate effect was not related to phosphatase inhibition since R5020 SBP in SDG was not enhanced by the addition of sodium fluoride which was a phosphatase inhibitor. In this report, the possibility of the existence of the 7-8S forming factor in the human prostate and the relationship between it and sodium molybdate was also discussed through an experiment on a Sephadex G-25.

Evidence for the identity and some comparative properties of alpha-ketoglutarate and 2-keto-4-hydroxyglutarate dehydrogenase activity.

PubMed

Gupta, S C; Dekker, E E

1980-02-10

Enzyme preparations of pig heart and Escherichia coli are shown to catalyze a NAD+- and CoASH-dependent oxidation of 2-keto-4-hydroxyglutarate. Several independent lines of evidence support the conclusion that this hydroxyketo acid is a substrate for the well known alpha-ketoglutarate dehydrogenase complex of the citric acid cycle. The evidence includes (a) a constant ratio of specific activity values for the two substrates through several steps of purification, (b) identical elution profiles from a calcium phosphate gel-cellulose column and a constant ratio of specific activity toward the two substrates throughout the activity peak, (c) identical inactivation curves in controlled heat denaturation studies, (d) the same pH activity curves, (e) no effect on the oxidation of either keto acid by repeated freezing and thawing of dehydrogenase preparations, and (f) the same activity pattern when the E. coli complex is distributed into several fractions by sucrose density gradient centrifugation. Additionally, the same cofactors are required for maximal activity and glyoxylate inhibits the oxidation of either substrate noncompetitively. Ferricyanide-linked oxidation of 2-keto-4-hydroxyglutarate yields malate as the product and a 1:2:1 stoichiometric relationship is obtained between the amount of hydroxyketo acid oxidized, ferricyanide reduced, and malate formed.

Preliminary X-ray data analysis of crystalline hibiscus chlorotic ringspot virus

PubMed Central

Cheng, Ao; Speir, Jeffrey A.; Yuan, Y. Adam; Johnson, John E.; Wong, Sek-Man

2009-01-01

Hibiscus chlorotic ringspot virus (HCRSV) is a positive-sense monopartite single-stranded RNA virus that belongs to the Carmovirus genus of the Tombusviridae family, which includes carnation mottle virus (CarMV). The HCRSV virion has a 30 nm diameter icosahedral capsid with T = 3 quasi-symmetry containing 180 copies of a 38 kDa coat protein (CP) and encapsidates a full-length 3.9 kb genomic RNA. Authentic virus was harvested from infected host kenaf leaves and was purified by saturated ammonium sulfate precipitation, sucrose density-gradient centrifugation and anion-exchange chromatography. Virus crystals were grown in multiple conditions; one of the crystals diffracted to 3.2 Å resolution and allowed the collection of a partial data set. The crystal belonged to space group R32, with unit-cell parameters a = b = 336.4, c = 798.5 Å. Packing considerations and rotation-function analysis determined that there were three particles per unit cell, all of which have the same orientation and fixed positions, and resulted in tenfold noncrystallography symmetry for real-space averaging. The crystals used for the structure determination of southern bean mosaic virus (SBMV) have nearly identical characteristics. Together, these findings will greatly aid the high-resolution structure determination of HCRSV. PMID:19478438

Detection of Toxoplasma gondii oocysts in water: proposition of a strategy and evaluation in Champagne-Ardenne Region, France.

PubMed

Aubert, D; Villena, I

2009-03-01

Water is a vehicle for disseminating human and veterinary toxoplasmosis due to oocyst contamination. Several outbreaks of toxoplasmosis throughout the world have been related to contaminated drinking water. We have developed a method for the detection of Toxoplasma gondii oocysts in water and we propose a strategy for the detection of multiple waterborne parasites, including Cryptosporidium spp. and Giardia. Water samples were filtered to recover Toxoplasma oocysts and, after the detection of Cryptosporidium oocysts and Giardia cysts by immunofluorescence, as recommended by French norm procedure NF T 90-455, the samples were purified on a sucrose density gradient. Detection of Toxoplasma was based on PCR amplification and mouse inoculation to determine the presence and infectivity of recovered oocysts. After experimental seeding assays, we determined that the PCR assay was more sensitive than the bioassay. This strategy was then applied to 482 environmental water samples collected since 2001. We detected Toxoplasma DNA in 37 environmental samples (7.7%), including public drinking water; however, none of them were positive by bioassay. This strategy efficiently detects Toxoplasma oocysts in water and may be suitable as a public health sentinel method. Alternative methods can be used in conjunction with this one to determine the infectivity of parasites that were detected by molecular methods.

Preliminary X-ray Data Analysis of Crystalline Hibiscus Chlorotic Ringspot Virus

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

Cheng, A.; Speir, J; Yuan, Y

Hibiscus chlorotic ringspot virus (HCRSV) is a positive-sense monopartite single-stranded RNA virus that belongs to the Carmovirus genus of the Tombusviridae family, which includes carnation mottle virus (CarMV). The HCRSV virion has a 30 nm diameter icosahedral capsid with T = 3 quasi-symmetry containing 180 copies of a 38 kDa coat protein (CP) and encapsidates a full-length 3.9 kb genomic RNA. Authentic virus was harvested from infected host kenaf leaves and was purified by saturated ammonium sulfate precipitation, sucrose density-gradient centrifugation and anion-exchange chromatography. Virus crystals were grown in multiple conditions; one of the crystals diffracted to 3Synchrotron .2 Amore » resolution and allowed the collection of a partial data set. The crystal belonged to space group R32, with unit-cell parameters a = b = 336.4, c = 798.5 . Packing considerations and rotation-function analysis determined that there were three particles per unit cell, all of which have the same orientation and fixed positions, and resulted in tenfold noncrystallography symmetry for real-space averaging. The crystals used for the structure determination of southern bean mosaic virus (SBMV) have nearly identical characteristics. Together, these findings will greatly aid the high-resolution structure determination of HCRSV.« less

Gadd45a Is an RNA Binding Protein and Is Localized in Nuclear Speckles

PubMed Central

Sytnikova, Yuliya A.; Kubarenko, Andriy V.; Schäfer, Andrea; Weber, Alexander N. R.; Niehrs, Christof

2011-01-01

Background The Gadd45 proteins play important roles in growth control, maintenance of genomic stability, DNA repair, and apoptosis. Recently, Gadd45 proteins have also been implicated in epigenetic gene regulation by promoting active DNA demethylation. Gadd45 proteins have sequence homology with the L7Ae/L30e/S12e RNA binding superfamily of ribosomal proteins, which raises the question if they may interact directly with nucleic acids. Principal Findings Here we show that Gadd45a binds RNA but not single- or double stranded DNA or methylated DNA in vitro. Sucrose density gradient centrifugation experiments demonstrate that Gadd45a is present in high molecular weight particles, which are RNase sensitive. Gadd45a displays RNase-sensitive colocalization in nuclear speckles with the RNA helicase p68 and the RNA binding protein SC35. A K45A point mutation defective in RNA binding was still active in DNA demethylation. This suggests that RNA binding is not absolutely essential for demethylation of an artificial substrate. A point mutation at G39 impared RNA binding, nuclear speckle localization and DNA demethylation, emphasizing its relevance for Gadd45a function. Significance The results implicate RNA in Gadd45a function and suggest that Gadd45a is associated with a ribonucleoprotein particle. PMID:21249130

Membranous appendices of spherosomes (oleosomes) : Possible role in fat utilization in germinating oil seeds.

PubMed

Wanner, G; Theimer, R R

1978-01-01

Spherosomes (oleosomes) of cotyledons of rape (Brassica napus L.), sunflower (Helianthus annuus L.), and watermelon (Citrullus vulgaris, Schrad.) seedlings are delimited by a "half unit membrane" that appears to be continuous with each of the osmiophilic layers of a tripartite unit membrane forming a handlelike appendix of the spherosomes. Prior to any noticeable utilization of the spherosomal storage fat, ribosomes were found to be attached to these "handles". At later stages appendices of the spherosomes are smooth, showing a diameter of about 22 nm that greatly exceeds the thickness of any other unit membrane profiles identical in structure and diameter osomes appears to be continuous with the thick lipid layer of the handles. In intermediate stages of fat depletion the spherosomal bodies become invaginated with cytoplasmic material. Finally vesicles with cytoplasmic contents surrounded by a membrane with a typically thick lipid layer are left in the cells. Membrane profiles indentical in structure and diameter to the spherosomal appendices were also present in electron micrographs of the lipolytic membrane fraction recovered from sucrose density gradients after centrifugation of a microsomal cell fraction. The ultrastructural observations are taken for evidence that the spherosomal appendices represent the lipase-carrying membranes isolated previously (Theimer and Rosnitschek, 1978). A novel hypothesis for development and utilization of fat-storing spherosomes is also proposed.

In Vitro Product of a Ribonucleic Acid Polymerase Induced by Influenza Virus

PubMed Central

Mahy, B. W. J.; Bromley, P. A.

1970-01-01

The ribonucleic acid (RNA)-dependent RNA polymerase induced in the microsomal fraction of cells infected with influenza virus synthesized a mixture of single-and double-stranded RNA in vitro. The single-stranded RNA sedimented mainly in the 8S region on sucrose density gradients, with a smaller proportion of the RNA sedimenting at 18S. This sedimentation pattern corresponds closely to that of incomplete influenza virus RNA. The double-stranded RNA formed in vitro sedimented at 11S, but molecules which may be replicative intermediate, sedimenting at 14 to 20S, were also detected in the in vitro reaction product. Similar species of RNA were detected in vivo by pulse-labeling infected cells at the time of polymerase harvest, but the proportion of each RNA species was different, most of the RNA being single-stranded and sedimenting in the 18S region. An 11S double-stranded RNA was also synthesized in vivo. Pulse chase analysis of the double-stranded RNA synthesized in vitro showed that most is stable, and only a small proportion turns over during the reaction. A proportion of the RNA formed in vitro could be annealed to RNA formed in infected cells and to RNA extracted from purified virus. PMID:5480408

Sinking velocities of phytoplankton measured on a stable density gradient by laser scanning

PubMed Central

Walsby, Anthony E; Holland, Daryl P

2005-01-01

Two particular difficulties in measuring the sinking velocities of phytoplankton cells are preventing convection within the sedimenting medium and determining the changing depth of the cells. These problems are overcome by using a density-stabilized sedimentation column scanned by a laser. For freshwater species, a suspension of phytoplankton is layered over a vertical density gradient of Percoll solution; as the cells sink down the column their relative concentration is measured by the forward scattering of light from a laser beam that repeatedly scans up and down the column. The Percoll gradient stabilizes the column, preventing vertical mixing by convection, radiation or perturbation of density by the descending cells. Measurements were made on suspensions of 15 μm polystyrene microspheres with a density of 1050 kg m−3; the mean velocity was 6.28 μm s−1, within 1.5% of that calculated by the Stokes equation, 6.36 μm s−1. Measurements made on the filamentous cyanobacterium Planktothrix rubescens gave mean velocities within the theoretical range of values based on the range of size, shape, orientation and density of the particles in a modified Stokes equation. Measurements on marine phytoplankton may require density gradients prepared with other substances. PMID:16849271

Sorting process of nanoparticles and applications of same

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

Tyler, Timothy P.; Henry, Anne-Isabelle; Van Duyne, Richard P.

In one aspect of the present invention, a method for sorting nanoparticles includes preparing a high-viscosity density gradient medium filled in a container, dispersing nanoparticles into an aqueous solution to form a suspension of the nanoparticles, each nanoparticle having one or more cores and a shell encapsulating the one or more cores, layering the suspension of the nanoparticles on the top of the high-viscosity density gradient medium in the container, and centrifugating the layered suspension of the nanoparticles on the top of the high-viscosity density gradient medium in the container at a predetermined speed for a predetermined period of timemore » to form a gradient of fractions of the nanoparticles along the container, where each fraction comprises nanoparticles in a respective one of aggregation states of the nanoparticles.« less

Food restriction-induced hyperactivity: addiction or adaptation to famine?

PubMed

Duclos, Martine; Ouerdani, Amel; Mormède, Pierre; Konsman, Jan Pieter

2013-06-01

Increased physical activity is present in 30-80% of anorexia nervosa patients. To explain the paradox of low food intake and excessive exercise in humans and other animals, it has been proposed that increased physical activity along with food restriction activates brain reward circuits and is addictive. Alternatively, the fleeing-famine hypothesis postulates that refusal of known scarce energy-low food sources and hyperactivity facilitate migration towards new habitats that potentially contain new energy-rich foodstuffs. The use of rewarding compounds that differ in energy density, such as the energy-free sweetener saccharin and the energy rich sucrose makes it possible to critically test the reward-addiction and fleeing-famine hypotheses. The aims of the present work were to study if sucrose and/or saccharin could attenuate food restriction-induced hyperactivity, weight loss, increased plasma corticosterone, and activation of brain structures involved in neuroendocrine control, energy balance, physical activity, and reward signaling in rats. Its major findings are that access to sucrose, but not to saccharin, attenuated food restriction-induced running wheel activity, weight loss, rises in plasma corticosterone, and expression of the cellular activation marker c-Fos in the paraventricular and arcuate hypothalamus and in the nucleus accumbens. These findings suggest that the energy-richness and easy availability of sucrose interrupted a fleeing-famine-like hyperactivity response. Since corticosterone mediates food restriction-induced wheel running (Duclos et al., 2009), we propose that the attenuating effect of sucrose consumption on plasma corticosterone plays a role in reduced wheel running and weight loss by lowering activation of the nucleus accumbens and arcuate hypothalamus in these animals. Copyright © 2012 Elsevier Ltd. All rights reserved.

Rimonabant’s Reductive Effects on High Densities of Food Reinforcement, but not Palatability, in Lean and Obese Zucker Rats

PubMed Central

Buckley, Jessica Lynn; Rasmussen, Erin B.

2014-01-01

Rationale Cannabinoid antagonists purportedly have greater effects in reducing the intake of highly palatable food compared to less palatable food. However, this assertion is based on free-feeding studies in which the amount of palatable food eaten under baseline conditions is often confounded with other variables, such as unequal access to both food options and differences in qualitative features of the foods. Objective We attempted to reduce these confounds by using a model of choice that programmed the delivery rates of sucrose and carrot-flavored pellets. Methods Lever-pressing of ten lean (Fa/Fa or Fa/fa) and ten obese (fa/fa) Zucker rats was placed under three conditions in which programmed ratios for food pellets on two levers were 5:1, 1:1, and 1:5. In Phase 1, responses on the two levers produced one type of pellet (sucrose or carrot); in Phase 2, responses on one lever produced sucrose pellets and on the other lever produced carrot pellets. After responses stabilized under each food ratio, acute doses of rimonabant (0, 3, and 10 mg/kg) were administered before experimental sessions. The number of reinforcers and responses earned per session under each ratio and from each lever was compared. Results and Conclusions Rimonabant reduced reinforcers in 1:5 and 5:1 food ratios in Phase 1, and across all ratios in Phase 2. Rimonabant reduced sucrose and carrot-flavored pellet consumption similarly; rimonabant did not affect bias toward sucrose, but increased sensitivity to amount differences in lean rats. This suggests that relative amount of food, not palatability, may be an important behavioral mechanism in the effects of rimonabant. PMID:24398820

Rimonabant's reductive effects on high densities of food reinforcement, but not palatability, in lean and obese Zucker rats.

PubMed

Buckley, Jessica L; Rasmussen, Erin B

2014-05-01

Cannabinoid antagonists purportedly have greater effects in reducing the intake of highly palatable food compared to less palatable food. However, this assertion is based on free-feeding studies in which the amount of palatable food eaten under baseline conditions is often confounded with other variables, such as unequal access to both food options and differences in qualitative features of the foods. We attempted to reduce these confounds by using a model of choice that programmed the delivery rates of sucrose and carrot-flavored pellets. Lever pressing of ten lean (Fa/Fa or Fa/fa) and ten obese (fa/fa) Zucker rats was placed under three conditions in which programmed ratios for food pellets on two levers were 5:1, 1:1, and 1:5. In phase 1, responses on the two levers produced one type of pellet (sucrose or carrot); in phase 2, responses on one lever produced sucrose pellets and on the other lever produced carrot pellets. After responses stabilized under each food ratio, acute doses of rimonabant (0, 3, and 10 mg/kg) were administered before experimental sessions. The number of reinforcers and responses earned per session under each ratio and from each lever was compared. Rimonabant reduced reinforcers in 1:5 and 5:1 food ratios in phase 1, and across all ratios in phase 2. Rimonabant reduced sucrose and carrot-flavored pellet consumption similarly; rimonabant did not affect bias toward sucrose, but increased sensitivity to amount differences in lean rats. This suggests that relative amount of food, not palatability, may be an important behavioral mechanism in the effects of rimonabant.

The in planta transcriptome of Ralstonia solanacearum: conserved physiological and virulence strategies during bacterial wilt of tomato.

PubMed

Jacobs, Jonathan M; Babujee, Lavanya; Meng, Fanhong; Milling, Annett; Allen, Caitilyn

2012-01-01

Plant xylem fluid is considered a nutrient-poor environment, but the bacterial wilt pathogen Ralstonia solanacearum is well adapted to it, growing to 10(8) to 10(9) CFU/g tomato stem. To better understand how R. solanacearum succeeds in this habitat, we analyzed the transcriptomes of two phylogenetically distinct R. solanacearum strains that both wilt tomato, strains UW551 (phylotype II) and GMI1000 (phylotype I). We profiled bacterial gene expression at ~6 × 10(8) CFU/ml in culture or in plant xylem during early tomato bacterial wilt pathogenesis. Despite phylogenetic differences, these two strains expressed their 3,477 common orthologous genes in generally similar patterns, with about 12% of their transcriptomes significantly altered in planta versus in rich medium. Several primary metabolic pathways were highly expressed during pathogenesis. These pathways included sucrose uptake and catabolism, and components of these pathways were encoded by genes in the scrABY cluster. A UW551 scrA mutant was significantly reduced in virulence on resistant and susceptible tomato as well as on potato and the epidemiologically important weed host Solanum dulcamara. Functional scrA contributed to pathogen competitive fitness during colonization of tomato xylem, which contained ~300 µM sucrose. scrA expression was induced by sucrose, but to a much greater degree by growth in planta. Unexpectedly, 45% of the genes directly regulated by HrpB, the transcriptional activator of the type 3 secretion system (T3SS), were upregulated in planta at high cell densities. This result modifies a regulatory model based on bacterial behavior in culture, where this key virulence factor is repressed at high cell densities. The active transcription of these genes in wilting plants suggests that T3SS has a biological role throughout the disease cycle. IMPORTANCE Ralstonia solanacearum is a widespread plant pathogen that causes bacterial wilt disease. It inflicts serious crop losses on tropical farmers, with major economic and human consequences. It is also a model for the many destructive microbes that colonize the water-conducting plant xylem tissue, which is low in nutrients and oxygen. We extracted bacteria from infected tomato plants and globally identified the biological functions that R. solanacearum expresses during plant pathogenesis. This revealed the unexpected presence of sucrose in tomato xylem fluid and the pathogen's dependence on host sucrose for virulence on tomato, potato, and the common weed bittersweet nightshade. Further, R. solanacearum was highly responsive to the plant environment, expressing several metabolic and virulence functions quite differently in the plant than in pure culture. These results reinforce the utility of studying pathogens in interaction with hosts and suggest that selecting for reduced sucrose levels could generate wilt-resistant crops.

Natural honey lowers plasma glucose, C-reactive protein, homocysteine, and blood lipids in healthy, diabetic, and hyperlipidemic subjects: comparison with dextrose and sucrose.

PubMed

Al-Waili, Noori S

2004-01-01

This study included the following experiments: (1) effects of dextrose solution (250 mL of water containing 75 g of dextrose) or honey solution (250 mL of water containing 75 g of natural honey) on plasma glucose level (PGL), plasma insulin, and plasma C-peptide (eight subjects); (2) effects of dextrose, honey, or artificial honey (250 mL of water containing 35 g of dextrose and 40 g of fructose) on cholesterol and triglycerides (TG) (nine subjects); (3) effects of honey solution, administered for 15 days, on PGL, blood lipids, C-reactive protein (CRP), and homocysteine (eight subjects); (4) effects of honey or artificial honey on cholesterol and TG in six patients with hypercholesterolemia and five patients with hypertriglyceridemia; (5) effects of honey for 15 days on blood lipid and CRP in five patients with elevated cholesterol and CRP; (6) effects of 70 g of dextrose or 90 g of honey on PGL in seven patients with type 2 diabetes mellitus; and (7) effects of 30 g of sucrose or 30 g of honey on PGL, plasma insulin, and plasma C-peptide in five diabetic patients. In healthy subjects, dextrose elevated PGL at 1 (53%) and 2 (3%) hours, and decreased PGL after 3 hours (20%). Honey elevated PGL after 1 hour (14%) and decreased it after 3 hours (10%). Elevation of insulin and C-peptide was significantly higher after dextrose than after honey. Dextrose slightly reduced cholesterol and low-density lipoprotein-cholesterol (LDL-C) after 1 hour and significantly after 2 hours, and increased TG after 1, 2, and 3 hours. Artificial honey slightly decreased cholesterol and LDL-C and elevated TG. Honey reduced cholesterol, LDL-C, and TG and slightly elevated high-density lipoprotein-cholesterol (HDL-C). Honey consumed for 15 days decreased cholesterol (7%), LDL-C (1%), TG (2%), CRP (7%), homocysteine (6%), and PGL (6%), and increased HDL-C (2%). In patients with hypertriglyceridemia, artificial honey increased TG, while honey decreased TG. In patients with hyperlipidemia, artificial honey increased LDL-C, while honey decreased LDL-C. Honey decreased cholesterol (8%), LDL-C (11%), and CRP (75%) after 15 days. In diabetic patients, honey compared with dextrose caused a significantly lower rise of PGL. Elevation of PGL was greater after honey than after sucrose at 30 minutes, and was lower after honey than it was after sucrose at 60, 120, and 180 minutes. Honey caused greater elevation of insulin than sucrose did after 30, 120, and 180 minutes. Honey reduces blood lipids, homocysteine, and CRP in normal and hyperlipidemic subjects. Honey compared with dextrose and sucrose caused lower elevation of PGL in diabetics.

A cross-sectional analysis of the relationship between tobacco and alcohol outlet density and neighbourhood deprivation.

PubMed

Shortt, Niamh K; Tisch, Catherine; Pearce, Jamie; Mitchell, Richard; Richardson, Elizabeth A; Hill, Sarah; Collin, Jeff

2015-10-05

There is a strong socio-economic gradient in both tobacco-and alcohol-related harm. One possible factor contributing to this social gradient may be greater availability of tobacco and alcohol in more socially-deprived areas. A higher density of tobacco and alcohol outlets is not only likely to increase supply but also to raise awareness of tobacco/alcohol brands, create a competitive local market that reduces product costs, and influence local social norms relating to tobacco and alcohol consumption. This paper examines the association between the density of alcohol and tobacco outlets and neighbourhood-level income deprivation. Using a national tobacco retailer register and alcohol licensing data this paper calculates the density of alcohol and tobacco retail outlets per 10,000 population for small neighbourhoods across the whole of Scotland. Average outlet density was calculated for neighbourhoods grouped by their level of income deprivation. Associations between outlet density and deprivation were analysed using one way analysis of variance. There was a positive linear relationship between neighbourhood deprivation and outlets for both tobacco (p <0.001) and off-sales alcohol (p <0.001); the most deprived quintile of neighbourhoods had the highest densities of both. In contrast, the least deprived quintile had the lowest density of tobacco and both off-sales and on-sales alcohol outlets. The social gradient evident in alcohol and tobacco supply may be a contributing factor to the social gradient in alcohol- and tobacco-related disease. Policymakers should consider such gradients when creating tobacco and alcohol control policies. The potential contribution to public health, and health inequalities, of reducing the physical availability of both alcohol and tobacco products should be examined in developing broader supply-side interventions.

Analytic energy gradients for the coupled-cluster singles and doubles method with the density-fitting approximation

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

Bozkaya, Uğur, E-mail: ugur.bozkaya@hacettepe.edu.tr; Department of Chemistry, Atatürk University, Erzurum 25240; Sherrill, C. David

2016-05-07

An efficient implementation is presented for analytic gradients of the coupled-cluster singles and doubles (CCSD) method with the density-fitting approximation, denoted DF-CCSD. Frozen core terms are also included. When applied to a set of alkanes, the DF-CCSD analytic gradients are significantly accelerated compared to conventional CCSD for larger molecules. The efficiency of our DF-CCSD algorithm arises from the acceleration of several different terms, which are designated as the “gradient terms”: computation of particle density matrices (PDMs), generalized Fock-matrix (GFM), solution of the Z-vector equation, formation of the relaxed PDMs and GFM, back-transformation of PDMs and GFM to the atomic orbitalmore » (AO) basis, and evaluation of gradients in the AO basis. For the largest member of the alkane set (C{sub 10}H{sub 22}), the computational times for the gradient terms (with the cc-pVTZ basis set) are 2582.6 (CCSD) and 310.7 (DF-CCSD) min, respectively, a speed up of more than 8-folds. For gradient related terms, the DF approach avoids the usage of four-index electron repulsion integrals. Based on our previous study [U. Bozkaya, J. Chem. Phys. 141, 124108 (2014)], our formalism completely avoids construction or storage of the 4-index two-particle density matrix (TPDM), using instead 2- and 3-index TPDMs. The DF approach introduces negligible errors for equilibrium bond lengths and harmonic vibrational frequencies.« less

[A DNA study of rat liver oligonucleosomes enriched by transcriptionally active genes during induction due to the administration of an amino acid mixture].

PubMed

Vardevanian, P O; Davtian, A M; Tiratsuian, S G; Vardevanian, A O

1990-01-01

A highly active fraction of rat liver oligonucleosome DNA has been isolated and studied by means of thermal denaturation after induction by amino acid mixture or hydrocortisone. A considerable redistribution of DNA content has been shown in sucrose gradient fractions during these forms of induction. The changes are revealed in melting temperature, differential melting profile of DNA, isolated from actively transcribed chromatine fractions. Analysis of melting profiles shows changes of GC content of oligonucleosome DNA, suggesting that there are differences in activation during two studied forms of induction.

Fully kinetic Biermann battery and associated generation of pressure anisotropy

NASA Astrophysics Data System (ADS)

Schoeffler, K. M.; Loureiro, N. F.; Silva, L. O.

2018-03-01

The dynamical evolution of a fully kinetic, collisionless system with imposed background density and temperature gradients is investigated analytically. The temperature gradient leads to the generation of temperature anisotropy, with the temperature along the gradient becoming larger than that in the direction perpendicular to it. This causes the system to become unstable to pressure anisotropy driven instabilities, dominantly to the electron Weibel instability. When both density and temperature gradients are present and nonparallel to each other, we obtain a Biermann-like linear-in-time magnetic field growth. Accompanying particle-in-cell numerical simulations are shown to confirm our analytical results.

Generation of a wakefield undulator in plasma with transverse density gradient

DOE PAGES

Stupakov, Gennady V.

2017-11-30

Here, we show that a short relativistic electron beam propagating in a plasma with a density gradient perpendicular to the direction of motion generates a wakefield in which a witness bunch experiences a transverse force. A density gradient oscillating along the beam path would create a periodically varying force$-$an undulator, with an estimated strength of the equivalent magnetic field more than ten Tesla. This opens an avenue for creation of a high-strength, short-period undulators, which eventually may lead to all-plasma, free electron lasers where a plasma wakefield acceleration is naturally combined with a plasma undulator in a unifying, compact setup.

Generation of a wakefield undulator in plasma with transverse density gradient

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

Stupakov, Gennady V.

Here, we show that a short relativistic electron beam propagating in a plasma with a density gradient perpendicular to the direction of motion generates a wakefield in which a witness bunch experiences a transverse force. A density gradient oscillating along the beam path would create a periodically varying force$-$an undulator, with an estimated strength of the equivalent magnetic field more than ten Tesla. This opens an avenue for creation of a high-strength, short-period undulators, which eventually may lead to all-plasma, free electron lasers where a plasma wakefield acceleration is naturally combined with a plasma undulator in a unifying, compact setup.

Response of bird species densities to habitat structure and fire history along a Midwestern open-forest gradient

USGS Publications Warehouse

Grundel, R.; Pavlovic, N.B.

2007-01-01

Oak savannas were historically common but are currently rare in the Midwestern United States. We assessed possible associations of bird species with savannas and other threatened habitats in the region by relating fire frequency and vegetation characteristics to seasonal densities of 72 bird species distributed across an open-forest gradient in northwestern Indiana. About one-third of the species did not exhibit statistically significant relationships with any combination of seven vegetation characteristics that included vegetation cover in five vertical strata, dead tree density, and tree height. For 40% of the remaining species, models best predicting species density incorporated tree density. Therefore, management based solely on manipulating tree density may not be an adequate strategy for managing bird populations along this open-forest gradient. Few species exhibited sharp peaks in predicted density under habitat conditions expected in restored savannas, suggesting that few savanna specialists occur among Midwestern bird species. When fire frequency, measured over fifteen years, was added to vegetation characteristics as a predictor of species density, it was incorporated into models for about one-quarter of species, suggesting that fire may modify habitat characteristics in ways that are important for birds but not captured by the structural habitat variables measured. Among those species, similar numbers had peaks in predicted density at low, intermediate, or high fire frequency. For species suggested by previous studies to have a preference for oak savannas along the open-forest gradient, estimated density was maximized at an average fire return interval of about one fire every three years. ?? The Cooper Ornithological Society 2007.

Is the bulk mode conversion important in high density helicon plasma?

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

Isayama, Shogo; Hada, Tohru; Shinohara, Shunjiro

2016-06-15

In a high-density helicon plasma production process, a contribution of Trivelpiece-Gould (TG) wave for surface power deposition is widely accepted. The TG wave can be excited either due to an abrupt density gradient near the plasma edge (surface conversion) or due to linear mode conversion from the helicon wave in a density gradient in the bulk region (bulk mode conversion). By numerically solving the boundary value problem of linear coupling between the helicon and the TG waves in a background with density gradient, we show that the efficiency of the bulk mode conversion strongly depends on the dissipation included inmore » the plasma, and the bulk mode conversion is important when the dissipation is small. Also, by performing FDTD simulation, we show the time evolution of energy flux associated with the helicon and the TG waves.« less

Hydrodynamic Model for Density Gradients Instability in Hall Plasmas Thrusters

NASA Astrophysics Data System (ADS)

Singh, Sukhmander

2017-10-01

There is an increasing interest for a correct understanding of purely growing electromagnetic and electrostatic instabilities driven by a plasma gradient in a Hall thruster devices. In Hall thrusters, which are typically operated with xenon, the thrust is provided by the acceleration of ions in the plasma generated in a discharge chamber. The goal of this paper is to study the instabilities due to gradients of plasma density and conditions for the growth rate and real part of the frequency for Hall thruster plasmas. Inhomogeneous plasmas prone a wide class of eigen modes induced by inhomogeneities of plasma density and called drift waves and instabilities. The growth rate of the instability has a dependences on the magnetic field, plasma density, ion temperature and wave numbers and initial drift velocities of the plasma species.

Gravity Gradient Tensor of Arbitrary 3D Polyhedral Bodies with up to Third-Order Polynomial Horizontal and Vertical Mass Contrasts

NASA Astrophysics Data System (ADS)

Ren, Zhengyong; Zhong, Yiyuan; Chen, Chaojian; Tang, Jingtian; Kalscheuer, Thomas; Maurer, Hansruedi; Li, Yang

2018-03-01

During the last 20 years, geophysicists have developed great interest in using gravity gradient tensor signals to study bodies of anomalous density in the Earth. Deriving exact solutions of the gravity gradient tensor signals has become a dominating task in exploration geophysics or geodetic fields. In this study, we developed a compact and simple framework to derive exact solutions of gravity gradient tensor measurements for polyhedral bodies, in which the density contrast is represented by a general polynomial function. The polynomial mass contrast can continuously vary in both horizontal and vertical directions. In our framework, the original three-dimensional volume integral of gravity gradient tensor signals is transformed into a set of one-dimensional line integrals along edges of the polyhedral body by sequentially invoking the volume and surface gradient (divergence) theorems. In terms of an orthogonal local coordinate system defined on these edges, exact solutions are derived for these line integrals. We successfully derived a set of unified exact solutions of gravity gradient tensors for constant, linear, quadratic and cubic polynomial orders. The exact solutions for constant and linear cases cover all previously published vertex-type exact solutions of the gravity gradient tensor for a polygonal body, though the associated algorithms may differ in numerical stability. In addition, to our best knowledge, it is the first time that exact solutions of gravity gradient tensor signals are derived for a polyhedral body with a polynomial mass contrast of order higher than one (that is quadratic and cubic orders). Three synthetic models (a prismatic body with depth-dependent density contrasts, an irregular polyhedron with linear density contrast and a tetrahedral body with horizontally and vertically varying density contrasts) are used to verify the correctness and the efficiency of our newly developed closed-form solutions. Excellent agreements are obtained between our solutions and other published exact solutions. In addition, stability tests are performed to demonstrate that our exact solutions can safely be used to detect shallow subsurface targets.

Density-Gradient Theory: A Macroscopic Approach to Quantum Confinement and Tunneling in Semiconductor Devices

DTIC Science & Technology

2011-01-01

that are attractive as luminescent biolabels, and possibly also for optoelectronic devices and solar cells . The equilibrium nature of such situations...The boundary layers as- sociated with the diffusion and Debye lengths are familiar, while that of LQ defines the layer in which the quantum in...circuits, transmission lines Diffusion -drift, density-gradient Semi-classical electron dynamics, Boltzmann transport Schrödinger, density- matrix, Wigner

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

Constantin, Lucian A.; Fabiano, Eduardo; Della Sala, Fabio

We introduce a novel non-local ingredient for the construction of exchange density functionals: the reduced Hartree parameter, which is invariant under the uniform scaling of the density and represents the exact exchange enhancement factor for one- and two-electron systems. The reduced Hartree parameter is used together with the conventional meta-generalized gradient approximation (meta-GGA) semilocal ingredients (i.e., the electron density, its gradient, and the kinetic energy density) to construct a new generation exchange functional, termed u-meta-GGA. This u-meta-GGA functional is exact for the exchange of any one- and two-electron systems, is size-consistent and non-empirical, satisfies the uniform density scaling relation, andmore » recovers the modified gradient expansion derived from the semiclassical atom theory. For atoms, ions, jellium spheres, and molecules, it shows a good accuracy, being often better than meta-GGA exchange functionals. Our construction validates the use of the reduced Hartree ingredient in exchange-correlation functional development, opening the way to an additional rung in the Jacob’s ladder classification of non-empirical density functionals.« less

Isoelectric focusing of red blood cells in a density gradient stabilized column

NASA Technical Reports Server (NTRS)

Smolka, A. J. K.; Miller, T. Y.

1980-01-01

The effects of Ficoll and cell application pH on red blood cell electrophoretic mobility and focusing pH were investigated by focusing cells in a density gradient stabilized column. Sample loading, cell dispersion, column conductivity, resolution of separation, and the effect of Ampholines were examined.

Expression of cardiac sarcolemmal Na sup + -Ca sup 2+ exchange activity in Xenopus laevis oocytes

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

Longoni, S.; Coady, M.J.; Ikeda, T.

1988-12-01

Injection of Xenopus laevis oocytes with rabbit heart poly(A){sup +}RNA results in expression of Na{sup +} inside (Na{sub i}{sup +})-dependent Ca{sup 2+} uptake activity. The activity was measured by first loading the oocytes with Na{sup +} using nystatin and then incubating the oocytes in K{sup +} or Na{sup +} medium containing {sup 45}Ca. The expressed Na{sup +} gradient-dependent Ca{sup 2+} uptake was five to eight times that observed with water-injected oocytes or with poly(A){sup +}RNA-injected oocytes for which the Na{sup +} load step had been omitted. Induced activity was related to the amount of RNA injected and was insensitive tomore » nifedipine. Fractionation of the poly(A){sup +}RNA on a sucrose gradient determined that the active message had a size range between 3 and 8 kb. The properties of the Na{sup +} gradient-dependent Ca{sup 2+} uptake indicated that Na{sup +}-Ca{sup 2+} exchange activity had been expressed in X. laevis oocytes. The result may be useful for cloning and identifying the molecular component responsible for Na{sup +}-Ca{sup 2+} exchange.« less

Nonlinear Upshift of Trapped Electron Mode Critical Density Gradient: Simulation and Experiment

NASA Astrophysics Data System (ADS)

Ernst, D. R.

2012-10-01

A new nonlinear critical density gradient for pure trapped electron mode (TEM) turbulence increases strongly with collisionality, saturating at several times the linear threshold. The nonlinear TEM threshold appears to limit the density gradient in new experiments subjecting Alcator C-Mod internal transport barriers to modulated radio-frequency heating. Gyrokinetic simulations show the nonlinear upshift of the TEM critical density gradient is associated with long-lived zonal flow dominated states [1]. This introduces a strong temperature dependence that allows external RF heating to control TEM turbulent transport. During pulsed on-axis heating of ITB discharges, core electron temperature modulations of 50% were produced. Bursts of line-integrated density fluctuations, observed on phase contrast imaging, closely follow modulations of core electron temperature inside the ITB foot. Multiple edge fluctuation measurements show the edge response to modulated heating is out of phase with the core response. A new limit cycle stability diagram shows the density gradient appears to be clamped during on-axis heating by the nonlinear TEM critical density gradient, rather than by the much lower linear threshold. Fluctuation wavelength spectra will be quantitatively compared with nonlinear TRINITY/GS2 gyrokinetic transport simulations, using an improved synthetic diagnostic. In related work, we are implementing the first gyrokinetic exact linearized Fokker Planck collision operator [2]. Initial results show short wavelength TEMs are fully stabilized by finite-gyroradius collisional effects for realistic collisionalities. The nonlinear TEM threshold and its collisionality dependence may impact predictions of density peaking based on quasilinear theory, which excludes zonal flows.[4pt] In collaboration with M. Churchill, A. Dominguez, C. L. Fiore, Y. Podpaly, M. L. Reinke, J. Rice, J. L. Terry, N. Tsujii, M. A. Barnes, I. Bespamyatnov, R. Granetz, M. Greenwald, A. Hubbard, J. W. Hughes, M. Landreman, B. Li, Y. Ma, P. Phillips, M. Porkolab, W. Rowan, S. Wolfe, and S. Wukitch.[4pt] [1] D. R. Ernst et al., Proc. 21st IAEA Fusion Energy Conference, Chengdu, China, paper IAEA-CN-149/TH/1-3 (2006). http://www-pub.iaea.org/MTCD/Meetings/FEC200/th1-3.pdf[0pt] [2] B. Li and D.R. Ernst, Phys. Rev. Lett. 106, 195002 (2011).

Functionalization of quantum rods with oligonucleotides for programmable assembly with DNA origami

NASA Astrophysics Data System (ADS)

Doane, Tennyson L.; Alam, Rabeka; Maye, Mathew M.

2015-02-01

The DNA-mediated self-assembly of CdSe/CdS quantum rods (QRs) onto DNA origami is described. Two QR types with unique optical emission and high polarization were synthesized, and then functionalized with oligonucleotides (ssDNA) using a novel protection-deprotection approach, which harnessed ssDNA's tailorable rigidity and denaturation temperature to increase DNA coverage by reducing non-specific coordination and wrapping. The QR assembly was programmable, and occurred at two different assembly zones that had capture strands in parallel alignment. QRs with different optical properties were assembled, opening up future studies on orientation dependent QR FRET. The QR-origami conjugates could be purified via gel electrophoresis and sucrose gradient ultracentrifugation. Assembly yields, QR stoichiometry and orientation, as well as energy transfer implications were studied in light of QR distances, origami flexibility, and conditions.The DNA-mediated self-assembly of CdSe/CdS quantum rods (QRs) onto DNA origami is described. Two QR types with unique optical emission and high polarization were synthesized, and then functionalized with oligonucleotides (ssDNA) using a novel protection-deprotection approach, which harnessed ssDNA's tailorable rigidity and denaturation temperature to increase DNA coverage by reducing non-specific coordination and wrapping. The QR assembly was programmable, and occurred at two different assembly zones that had capture strands in parallel alignment. QRs with different optical properties were assembled, opening up future studies on orientation dependent QR FRET. The QR-origami conjugates could be purified via gel electrophoresis and sucrose gradient ultracentrifugation. Assembly yields, QR stoichiometry and orientation, as well as energy transfer implications were studied in light of QR distances, origami flexibility, and conditions. Electronic supplementary information (ESI) available: Experimental conditions, DNA origami blueprint and sequences, FRET calculations. Additional Fig. S1-S13. See DOI: 10.1039/c4nr07662a

Direct isolation of a functional violaxanthin cycle domain from thylakoid membranes of higher plants.

PubMed

Goss, Reimund; Greifenhagen, Anne; Bergner, Juliane; Volke, Daniela; Hoffmann, Ralf; Wilhelm, Christian; Schaller-Laudel, Susann

2017-04-01

A special domain of the thylakoid membrane of higher plants has been isolated which carries out the de-epoxidation of the xanthophyll cycle pigment violaxanthin to zeaxanthin. Recent models indicate that in the chloroplast of higher plants, the violaxanthin (V) cycle takes place within specialized domains in the thylakoid membrane. Here, we describe a new procedure to directly isolate such a domain in functional state. The procedure consists of a thylakoid membrane isolation at a pH value of 5.2 which realizes the binding of the enzyme V de-epoxidase (VDE) to the membrane throughout the preparation process. Isolated thylakoid membranes are then solubilized with the very mild detergent n-dodecyl α-D-maltoside and the pigment-protein complexes are separated by sucrose gradient ultracentrifugation. The upper main fraction of the sucrose gradient represents a V cycle domain which consists of the major light-harvesting complex of photosystem II (LHCII), a special lipid composition with an enrichment of the galactolipid monogalactosyldiacylglycerol (MGDG) and the VDE. The domain is isolated in functional state as evidenced by the ability to convert the LHCII-associated V to zeaxanthin. The direct isolation of a V cycle domain proves the most important hypotheses concerning the de-epoxidation reaction in intact thylakoid membranes. It shows that the VDE binds to the thylakoid membrane at low pH values of the thylakoid lumen, that it binds to membrane regions enriched in LHCII, and that the domain contains high amounts of MGDG. The last point is in line with the importance of the galactolipid for V solubilisation and, by providing inverted hexagonal lipid structures, for VDE activity.

Local relative density modulates failure and strength in vertically aligned carbon nanotubes.

PubMed

Pathak, Siddhartha; Mohan, Nisha; Decolvenaere, Elizabeth; Needleman, Alan; Bedewy, Mostafa; Hart, A John; Greer, Julia R

2013-10-22

Micromechanical experiments, image analysis, and theoretical modeling revealed that local failure events and compressive stresses of vertically aligned carbon nanotubes (VACNTs) were uniquely linked to relative density gradients. Edge detection analysis of systematically obtained scanning electron micrographs was used to quantify a microstructural figure-of-merit related to relative local density along VACNT heights. Sequential bottom-to-top buckling and hardening in stress-strain response were observed in samples with smaller relative density at the bottom. When density gradient was insubstantial or reversed, bottom regions always buckled last, and a flat stress plateau was obtained. These findings were consistent with predictions of a 2D material model based on a viscoplastic solid with plastic non-normality and a hardening-softening-hardening plastic flow relation. The hardening slope in compression generated by the model was directly related to the stiffness gradient along the sample height, and hence to the local relative density. These results demonstrate that a microstructural figure-of-merit, the effective relative density, can be used to quantify and predict the mechanical response.

3D Imaging of Density Gradients Using Plenoptic BOS

NASA Astrophysics Data System (ADS)

Klemkowsky, Jenna; Clifford, Chris; Fahringer, Timothy; Thurow, Brian

2016-11-01

The combination of background oriented schlieren (BOS) and a plenoptic camera, termed Plenoptic BOS, is explored through two proof-of-concept experiments. The motivation of this work is to provide a 3D technique capable of observing density disturbances. BOS uses the relationship between density and refractive index gradients to observe an apparent shift in a patterned background through image comparison. Conventional BOS systems acquire a single line-of-sight measurement, and require complex configurations to obtain 3D measurements, which are not always conducive to experimental facilities. Plenoptic BOS exploits the plenoptic camera's ability to generate multiple perspective views and refocused images from a single raw plenoptic image during post processing. Using such capabilities, with regards to BOS, provides multiple line-of-sight measurements of density disturbances, which can be collectively used to generate refocused BOS images. Such refocused images allow the position of density disturbances to be qualitatively and quantitatively determined. The image that provides the sharpest density gradient signature corresponds to a specific depth. These results offer motivation to advance Plenoptic BOS with an ultimate goal of reconstructing a 3D density field.

Automated Processing of Plasma Samples for Lipoprotein Separation by Rate-Zonal Ultracentrifugation.

PubMed

Peters, Carl N; Evans, Iain E J

2016-12-01

Plasma lipoproteins are the primary means of lipid transport among tissues. Defining alterations in lipid metabolism is critical to our understanding of disease processes. However, lipoprotein measurement is limited to specialized centers. Preparation for ultracentrifugation involves the formation of complex density gradients that is both laborious and subject to handling errors. We created a fully automated device capable of forming the required gradient. The design has been made freely available for download by the authors. It is inexpensive relative to commercial density gradient formers, which generally create linear gradients unsuitable for rate-zonal ultracentrifugation. The design can easily be modified to suit user requirements and any potential future improvements. Evaluation of the device showed reliable peristaltic pump accuracy and precision for fluid delivery. We also demonstrate accurate fluid layering with reduced mixing at the gradient layers when compared to usual practice by experienced laboratory personnel. Reduction in layer mixing is of critical importance, as it is crucial for reliable lipoprotein separation. The automated device significantly reduces laboratory staff input and reduces the likelihood of error. Overall, this device creates a simple and effective solution to formation of complex density gradients. © 2015 Society for Laboratory Automation and Screening.

DNA stable-isotope probing (DNA-SIP).

PubMed

Dunford, Eric A; Neufeld, Josh D

2010-08-02

DNA stable-isotope probing (DNA-SIP) is a powerful technique for identifying active microorganisms that assimilate particular carbon substrates and nutrients into cellular biomass. As such, this cultivation-independent technique has been an important methodology for assigning metabolic function to the diverse communities inhabiting a wide range of terrestrial and aquatic environments. Following the incubation of an environmental sample with stable-isotope labelled compounds, extracted nucleic acid is subjected to density gradient ultracentrifugation and subsequent gradient fractionation to separate nucleic acids of differing densities. Purification of DNA from cesium chloride retrieves labelled and unlabelled DNA for subsequent molecular characterization (e.g. fingerprinting, microarrays, clone libraries, metagenomics). This JoVE video protocol provides visual step-by-step explanations of the protocol for density gradient ultracentrifugation, gradient fractionation and recovery of labelled DNA. The protocol also includes sample SIP data and highlights important tips and cautions that must be considered to ensure a successful DNA-SIP analysis.

Prediction of solubility parameters and miscibility of pharmaceutical compounds by molecular dynamics simulations.

PubMed

Gupta, Jasmine; Nunes, Cletus; Vyas, Shyam; Jonnalagadda, Sriramakamal

2011-03-10

The objectives of this study were (i) to develop a computational model based on molecular dynamics technique to predict the miscibility of indomethacin in carriers (polyethylene oxide, glucose, and sucrose) and (ii) to experimentally verify the in silico predictions by characterizing the drug-carrier mixtures using thermoanalytical techniques. Molecular dynamics (MD) simulations were performed using the COMPASS force field, and the cohesive energy density and the solubility parameters were determined for the model compounds. The magnitude of difference in the solubility parameters of drug and carrier is indicative of their miscibility. The MD simulations predicted indomethacin to be miscible with polyethylene oxide and to be borderline miscible with sucrose and immiscible with glucose. The solubility parameter values obtained using the MD simulations values were in reasonable agreement with those calculated using group contribution methods. Differential scanning calorimetry showed melting point depression of polyethylene oxide with increasing levels of indomethacin accompanied by peak broadening, confirming miscibility. In contrast, thermal analysis of blends of indomethacin with sucrose and glucose verified general immiscibility. The findings demonstrate that molecular modeling is a powerful technique for determining the solubility parameters and predicting miscibility of pharmaceutical compounds. © 2011 American Chemical Society

Three-Dimensional Printed Graphene Foams.

PubMed

Sha, Junwei; Li, Yilun; Villegas Salvatierra, Rodrigo; Wang, Tuo; Dong, Pei; Ji, Yongsung; Lee, Seoung-Ki; Zhang, Chenhao; Zhang, Jibo; Smith, Robert H; Ajayan, Pulickel M; Lou, Jun; Zhao, Naiqin; Tour, James M

2017-07-25

An automated metal powder three-dimensional (3D) printing method for in situ synthesis of free-standing 3D graphene foams (GFs) was successfully modeled by manually placing a mixture of Ni and sucrose onto a platform and then using a commercial CO 2 laser to convert the Ni/sucrose mixture into 3D GFs. The sucrose acted as the solid carbon source for graphene, and the sintered Ni metal acted as the catalyst and template for graphene growth. This simple and efficient method combines powder metallurgy templating with 3D printing techniques and enables direct in situ 3D printing of GFs with no high-temperature furnace or lengthy growth process required. The 3D printed GFs show high-porosity (∼99.3%), low-density (∼0.015g cm -3 ), high-quality, and multilayered graphene features. The GFs have an electrical conductivity of ∼8.7 S cm -1 , a remarkable storage modulus of ∼11 kPa, and a high damping capacity of ∼0.06. These excellent physical properties of 3D printed GFs indicate potential applications in fields requiring rapid design and manufacturing of 3D carbon materials, for example, energy storage devices, damping materials, and sound absorption.

Plasma Irregularities on the Leading and Trailing Edges of Polar Cap Patches

NASA Astrophysics Data System (ADS)

Lamarche, L. J.; Varney, R. H.; Gillies, R.; Chartier, A.; Mitchell, C. N.

2017-12-01

Plasma irregularities in the polar cap have often been attributed to the gradient drift instability (GDI). Traditional fluid theories of GDI predicts irregularity growth only on the trailing edge of polar patches, where the plasma density gradient is parallel to the plasma drift velocity, however many observations show irregularities also form on the leading edge of patches. We consider decameter-scale irregularities detected by polar-latitude SuperDARN (Super Dual Auroral Radar Network) radars with any relationship between the background density gradients and drift velocity. Global electron density from the Multi-Instrument Data Analysis System (MIDAS), a GPS tomography routine, is used to provide context for where irregularities are observed relative to polar patches and finer-scale background density gradients are found from 3D imaging from both the North and Canada faces of the Resolute Bay Incoherent Scatter Radars (RISR-N and RISR-C) jointly. Shear-based instabilities are considered as mechanisms by which plasma irregularities could form on the leading edge of patches. Theoretical predictions of instability growth from both GDI and shear instabilities are compared with irregularity observations for the October 13, 2016 storm.

Mimicking lichens: incorporation of yeast strains together with sucrose-secreting cyanobacteria improves survival, growth, ROS removal, and lipid production in a stable mutualistic co-culture production platform

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

Li, Tingting; Li, Chien-Ting; Butler, Kirk

The feasibility of heterotrophic-phototrophic symbioses was tested via pairing of yeast strains Cryptococcus curvatus, Rhodotorula glutinis, or Saccharomyces cerevisiae with a sucrose-secreting cyanobacterium Synechococcus elongatus. The phototroph S. elongatus showed no growth in standard BG-11 medium with yeast extract, but grew well in BG-11 medium alone or supplemented with yeast nitrogen base without amino acids (YNB w/o aa). Among three yeast species, C. curvatus and R. glutinis adapted well to the BG-11 medium supplemented with YNB w/o aa, sucrose, and various concentrations of NaCl needed to maintain sucrose secretion from S. elongatus, while growth of S. cerevisiae was highly dependentmore » on sucrose levels. R. glutinis and C. curvatus grew efficiently and utilized sucrose produced by the partner in co-culture. Co-cultures of S. elongatus and R. glutinis were sustained over 1 month in both batch and in semi-continuous culture, with the final biomass and overall lipid yields in the batch co-culture 40 to 60% higher compared to batch mono-cultures of S. elongatus. The co-cultures showed enhanced levels of palmitoleic and linoleic acids. Furthermore, cyanobacterial growth in co-culture with R. glutinis was significantly superior to axenic growth, as S. elongatus was unable to grow in the absence of the yeast partner when cultivated at lower densities in liquid medium. Accumulated reactive oxygen species was observed to severely inhibit axenic growth of cyanobacteria, which was efficiently alleviated through catalase supply and even more effectively with co-cultures of R. glutinis. In conclusion, the pairing of a cyanobacterium and eukaryotic heterotroph in the artificial lichen of this study demonstrates the importance of mutual interactions between phototrophs and heterotrophs, e.g., phototrophs provide a carbon source to heterotrophs, and heterotrophs assist phototrophic growth and survival by removing/eliminating oxidative stress. Our results establish a potential stable production platform that combines the metabolic capability of photoautotrophs to capture inorganic carbon with the channeling of the resulting organic carbon directly to a robust heterotroph partner for producing biofuel and other chemical precursors.« less

Mimicking lichens: incorporation of yeast strains together with sucrose-secreting cyanobacteria improves survival, growth, ROS removal, and lipid production in a stable mutualistic co-culture production platform

DOE PAGES

Li, Tingting; Li, Chien-Ting; Butler, Kirk; ...

2017-03-21

The feasibility of heterotrophic-phototrophic symbioses was tested via pairing of yeast strains Cryptococcus curvatus, Rhodotorula glutinis, or Saccharomyces cerevisiae with a sucrose-secreting cyanobacterium Synechococcus elongatus. The phototroph S. elongatus showed no growth in standard BG-11 medium with yeast extract, but grew well in BG-11 medium alone or supplemented with yeast nitrogen base without amino acids (YNB w/o aa). Among three yeast species, C. curvatus and R. glutinis adapted well to the BG-11 medium supplemented with YNB w/o aa, sucrose, and various concentrations of NaCl needed to maintain sucrose secretion from S. elongatus, while growth of S. cerevisiae was highly dependentmore » on sucrose levels. R. glutinis and C. curvatus grew efficiently and utilized sucrose produced by the partner in co-culture. Co-cultures of S. elongatus and R. glutinis were sustained over 1 month in both batch and in semi-continuous culture, with the final biomass and overall lipid yields in the batch co-culture 40 to 60% higher compared to batch mono-cultures of S. elongatus. The co-cultures showed enhanced levels of palmitoleic and linoleic acids. Furthermore, cyanobacterial growth in co-culture with R. glutinis was significantly superior to axenic growth, as S. elongatus was unable to grow in the absence of the yeast partner when cultivated at lower densities in liquid medium. Accumulated reactive oxygen species was observed to severely inhibit axenic growth of cyanobacteria, which was efficiently alleviated through catalase supply and even more effectively with co-cultures of R. glutinis. In conclusion, the pairing of a cyanobacterium and eukaryotic heterotroph in the artificial lichen of this study demonstrates the importance of mutual interactions between phototrophs and heterotrophs, e.g., phototrophs provide a carbon source to heterotrophs, and heterotrophs assist phototrophic growth and survival by removing/eliminating oxidative stress. Our results establish a potential stable production platform that combines the metabolic capability of photoautotrophs to capture inorganic carbon with the channeling of the resulting organic carbon directly to a robust heterotroph partner for producing biofuel and other chemical precursors.« less

Density gradient in SiO 2 films on silicon as revealed by positron annihilation spectroscopy

NASA Astrophysics Data System (ADS)

Revesz, A. G.; Anwand, W.; Brauer, G.; Hughes, H. L.; Skorupa, W.

2002-06-01

Positron annihilation spectroscopy of thermally grown and deposited SiO 2 films on silicon shows in a non-destructive manner that these films have a gradient in their density. The gradient is most pronounced for the oxide grown in dry oxygen. Oxidation in water-containing ambient results in an oxide with reduced gradient, similarly to the gradient in the deposited oxide. These observations are in accordance with earlier optical and other studies using stepwise etching or a set of samples of varying thickness. The effective oxygen charge, which is very likely one of the reasons for the difference in the W parameters of silica glass and quartz crystal, could be even higher at some localized configurations in the SiO 2 films resulting in increased positron trapping.

Evaluation of the qualitative and quantitative effectiveness of three media of centrifugation (Maxifreeze, Cushion Fluid Equine, and PureSperm 100) in preparation of fresh or frozen-thawed brown bear spermatozoa.

PubMed

Nicolas, M; Alvarez, M; Borragán, S; Martinez-Pastor, F; Chamorro, C A; Alvarez-Rodriguez, M; de Paz, P; Anel, L

2012-04-01

Centrifugation is a crucial procedure in sperm cryopreservation protocols of brown bear (Ursus arctos), because the semen must be processed to increase sperm concentration and/or clean urine-contaminated samples. The efficacy of three media for centrifugation (Maxifreeze [IMV technologies, L'Aigle, France], Cushion Fluid Equine (Minitübe, Tiefenbach, Germany), and PureSperm [Nidacon, Gothenburg, Sweden]) on the quality of bear spermatozoa was evaluated. In experiment one, two cushioned media used for protecting against mechanical stress during centrifugation were analyzed. In experiment two, a density gradient based on PureSperm was assessed in relation to the maximum retrieval and the quality of fresh spermatozoa, and the freezability of the spermatozoa selected in this density gradient was studied in experiment three. Finally, the selection of frozen-thawed sperm using PureSperm was analyzed in experiment four. Our results indicate that the use of dense isotonic cushion solutions (Maxifreeze, Cushion Fluid Equine) in centrifugation did not improve the quality of recovered spermatozoa compared with standard centrifugation. However, a density gradient prepared with PureSperm improved the quality of spermatozoa in fresh semen and frozen-thawed semen, but the spermatozoa selected from the fresh sample with this density gradient did not show a better resistance to freezing with this density gradient in comparison with the control sample. Copyright © 2012 Elsevier Inc. All rights reserved.

Effects of semen storage and separation techniques on sperm DNA fragmentation.

PubMed

Jackson, Robert E; Bormann, Charles L; Hassun, Pericles A; Rocha, André M; Motta, Eduardo L A; Serafini, Paulo C; Smith, Gary D

2010-12-01

To determine the effect of semen storage and separation techniques on sperm DNA fragmentation. Controlled clinical study. An assisted reproductive technology laboratory. Thirty normoozospermic semen samples obtained from patients undergoing infertility evaluation. One aliquot from each sample was immediately prepared (control) for the sperm chromatin dispersion assay (SCD). Aliquots used to assess storage techniques were treated in the following ways: snap frozen by liquid nitrogen immersion, slow frozen with Tris-yolk buffer and glycerol, kept on ice for 24 hours or maintained at room temperature for 4 and 24 hours. Aliquots used to assess separation techniques were processed by the following methods: washed and centrifuged in media, swim-up from washed sperm pellet, density gradient separation, density gradient followed by swim-up. DNA integrity was then measured by SCD. DNA fragmentation as measured by SCD. There was no significant difference in fragmentation among the snap frozen, slow frozen, and wet-ice groups. Compared to other storage methods short-term storage at room temperature did not impact DNA fragmentation yet 24 hours storage significantly increased fragmentation. Swim-up, density gradient and density gradient/swim-up had significantly reduced DNA fragmentation levels compared with washed semen. Postincubation, density gradient/swim-up showed the lowest fragmentation levels. The effect of sperm processing methods on DNA fragmentation should be considered when selecting storage or separation techniques for clinical use. Copyright © 2010 American Society for Reproductive Medicine. Published by Elsevier Inc. All rights reserved.

Brain regions mediating α3β4 nicotinic antagonist effects of 18-MC on methamphetamine and sucrose self-administration

PubMed Central

Glick, Stanley D.; Sell, Elizabeth M.; Maisonneuve, Isabelle M.

2008-01-01

The novel iboga alkaloid congener 18-methoxycoronaridine (18-MC) is a putative anti-addictive agent that has been shown, in rats, to decrease the self-administration of several drugs of abuse. Previous work has established that 18-MC is a potent antagonist at α3β4 nicotinic receptors. Because high densities of α3β4 nicotinic receptors occur in the medial habenula and the interpeduncular nucleus and moderate densities occur in the dorsolateral tegmentum, ventral tegmental area, and basolateral amygdala, the present study was conducted to determine if 18-MC could act in these brain areas to modulate methamphetamine self-administration in rats. Local administration of 18-MC into either the medial habenula, the interpeduncular area or the basolateral amygdala decreased methamphetamine self-administration. Similar results were produced by local administration into the same brain areas of two other α3β4 nicotinic antagonists, mecamylamine and α-conotoxin AuIB. Local administration of 18-MC, or the other antagonists, into the dorsolateral tegmentum or the ventral tegmental area had no effect on methamphetamine self-administration. In contrast, local administration of 18-MC and the other antagonists decreased sucrose self-administration when administered into the dorsolateral tegmentum or basolateral amygdala but had no effect when infused into the medial habenula, interpeduncular nucleus, or ventral tegmental area. These data are consistent with the hypothesis that 18-MC decreases methamphetamine self-administration by indirectly modulating the dopaminergic mesolimbic pathway via blockade of α3β4 nicotinic receptors in the habenulo-interpeduncular pathway and the basolateral amygdala. The data also suggest that the basolateral amygdala along with a different pathway involving α3β4 receptors in the dorsolateral tegmentum mediate the effect of 18-MC on sucrose self-administration. PMID:18930043

Evidence of toroidally localized turbulence with applied 3D fields in the DIII-D tokamak

DOE PAGES

Wilcox, R. S.; Shafer, M. W.; Ferraro, N. M.; ...

2016-09-21

New evidence indicates that there is significant 3D variation in density fluctuations near the boundary of weakly 3D tokamak plasmas when resonant magnetic perturbations are applied to suppress transient edge instabilities. The increase in fluctuations is concomitant with an increase in the measured density gradient, suggesting that this toroidally localized gradient increase could be a mechanism for turbulence destabilization in localized flux tubes. Two-fluid magnetohydrodynamic simulations find that, although changes to the magnetic field topology are small, there is a significant 3D variation of the density gradient within the flux surfaces that is extended along field lines. This modeling agreesmore » qualitatively with the measurements. The observed gradient and fluctuation asymmetries are proposed as a mechanism by which global profile gradients in the pedestal could be relaxed due to a local change in the 3D equilibrium. In conclusion, these processes may play an important role in pedestal and scrape-off layer transport in ITER and other future tokamak devices with small applied 3D fields.« less

Method and apparatus for ceramic analysis

DOEpatents

Jankowiak, Ryszard J.; Schilling, Chris; Small, Gerald J.; Tomasik, Piotr

2003-04-01

The present invention relates to a method and apparatus for ceramic analysis, in particular, a method for analyzing density, density gradients and/or microcracks, including an apparatus with optical instrumentation for analysis of density, density gradients and/or microcracks in ceramics. The method provides analyzing density of a ceramic comprising exciting a component on a surface/subsurface of the ceramic by exposing the material to excitation energy. The method may further include the step of obtaining a measurement of an emitted energy from the component. The method may additionally include comparing the measurement of the emitted energy from the component with a predetermined reference measurement so as to obtain a density for said ceramic.

A Theory of Density Layering in Stratified Turbulence using Statistical State Dynamics

NASA Astrophysics Data System (ADS)

Fitzgerald, J.; Farrell, B.

2016-12-01

Stably stratified turbulent fluids commonly develop density structures that are layered in the vertical direction (e.g., Manucharyan et al., 2015). Within layers, density is approximately constant and stratification is weak. Between layers, density varies rapidly and stratification is strong. A common explanation for the existence of layers invokes the negative diffusion mechanism of Phillips (1972) & Posmentier (1977). The physical principle underlying this mechanism is that the flux-gradient relationship connecting the turbulent fluxes of buoyancy to the background stratification must have the special property of weakening fluxes with strengthening gradient. Under these conditions, the evolution of the stratification is governed by a negative diffusion problem which gives rise to spontaneous layer formation. In previous work on stratified layering, this flux-gradient property is often assumed (e.g, Posmentier, 1977) or drawn from phenomenological models of turbulence (e.g., Balmforth et al., 1998).In this work we develop the theoretical underpinnings of layer formation by applying stochastic turbulence modeling and statistical state dynamics (SSD) to predict the flux-gradient relation and analyze layer formation directly from the equations of motion. We show that for stochastically-forced homogeneous 2D Boussinesq turbulence, the flux-gradient relation can be obtained analytically and indicates that the fluxes always strengthen with stratification. The Phillips mechanism thus does not operate in this maximally simplified scenario. However, when the problem is augmented to include a large scale background shear, we show that the flux-gradient relationship is modified so that the fluxes weaken with stratification. Sheared and stratified 2D Boussinesq turbulence thus spontaneously forms density layers through the Phillips mechanism. Using SSD (Farrell & Ioannou 2003), we obtain a closed, deterministic dynamics for the stratification and the statistical turbulent state. We show that density layers form as a linear instability of the sheared turbulence, associated with a supercritical bifurcation. We further show that SSD predicts the nonlinear equilibration and maintenance of the layers, and captures the phenomena of layer growth and mergers (Radko, 2007).

Plasma Irregularity Production in the Polar Cap F-Region Ionosphere

NASA Astrophysics Data System (ADS)

Lamarche, Leslie

Plasma in the Earth's ionosphere is highly irregular on scales ranging between a few centimeters and hundreds of kilometers. Small-scale irregularities or plasma waves can scatter radio waves resulting in a loss of signal for navigation and communication networks. The polar region is particularly susceptible to strong disturbances due to its direct connection with the Sun's magnetic field and energetic particles. In this thesis, factors that contribute to the production of decameter-scale plasma irregularities in the polar F region ionosphere are investigated. Both global and local control of irregularity production are studied, i.e. we consider global solar control through solar illumination and solar wind as well as much more local control by plasma density gradients and convection electric field. In the first experimental study, solar control of irregularity production is investigated using the Super Dual Auroral Radar Network (SuperDARN) radar at McMurdo, Antarctica. The occurrence trends for irregularities are analyzed statistically and a model is developed that describes the location of radar echoes within the radar's field-of-view. The trends are explained through variations in background plasma density with solar illumination affecting radar beam propagation. However, it is found that the irregularity occurrence during the night is higher than expected from ray tracing simulations based on a standard ionospheric density model. The high occurrence at night implies an additional source of plasma density and it is proposed that large-scale density enhancements called polar patches may be the source of this density. Additionally, occurrence maximizes around the terminator due to different competing irregularity production processes that favor a more or less sunlit ionosphere. The second study is concerned with modeling irregularity characteristics near a large-scale density gradient reversal, such as those expected near polar patches, with a particular focus on the asymmetry of the irregularity growth rate across the gradient reversal. Directional dependencies on the plasma density gradient, plasma drift, and wavevector are analyzed in the context of the recently developed general fluid theory of the gradient-drift instability. In the ionospheric F region, the strongest asymmetry is found when an elongated structure is oriented along the radar's boresight and moving perpendicular to its direction of elongation. These results have important implications for finding optimal configurations for oblique-scanning ionospheric radars such as SuperDARN to observe gradient reversals. To test the predictions of the developed model and the general theory of the gradient-drift instability, an experimental investigation is presented focusing on decameter-scale irregularities near a polar patch and the previously uninvestigated directional dependence of irregularity characteristics. Backscatter power and occurrence of irregularities are analyzed using measurements from the SuperDARN radar at Rankin Inlet, Canada, while background density gradients and convection electric fields are found from the north face of the Resolute Bay Incoherent Scatter Radar. It is shown that irregularity occurrence tends to follow the expected trends better than irregularity power, suggesting that while the gradient-drift instability may be a dominant process in generating small-scale irregularities, other mechanisms such as a shear-driven instability or nonlinear process may exert greater control over their intensity. It is concluded from this body of work that the production of small-scale plasma irregularities in the polar F-region ionosphere is controlled both by global factors such as solar illumination as well as local plasma density gradients and electric fields. In general, linear gradient-drift instability theory describes small-scale irregularity production well, particularly for low-amplitude perturbations. The production of irregularities is complex, and while ground-based radars are invaluable tools to study the ionosphere, care must be taken to interpret results correctly.

Double-Diffusive Finger Convection: Flow Field Evolution in a Hele-Shaw Cell

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

COOPER,CLAY A.; GLASS JR.,ROBERT J.; TYLER,SCOTT W.

Double-diffusive finger convection is a hydrodynamic instability that can occur when two components with different diffusivities are oppositely stratified with respect to the fluid density gradient as a critical condition is exceeded. Laboratory experiments were designed using sodium chloride and sucrose solutions in a Hele-Shaw cell. A high resolution, full field, light transmission technique was used to study the development of the instability. The initial buoyancy ratio (R{sub p}), which is a ratio of fluid density contributions by the two solutes, was varied systematically in the experiments so that the range of parameter space spanned conditions that were nearly stablemore » (R{sub p} = 2.8) to those that were moderately unstable (R{sub p} = 1.4). In systems of low R{sub p}, fingers develop within several minutes, merge with adjacent fingers, form conduits, and stall before newer-generated fingers travel through the conduits and continue the process. Solute fluxes in low R{sub p} systems quickly reach steady state and are on the order of 10{sup {minus}6} m{sup 2} sec{sup {minus}1}. In the higher R{sub p} experiments, fingers are slower to evolve and do not interact as dynamically as in the lower R{sub p} systems. Our experiment with initial R{sub p} = 2.8 exhibited flux on the order of that expected for a similar diffusive system (i.e., 10{sup {minus}7} m{sup 2} sec{sup {minus}1}), although the structures were very different than the pattern of transport expected in a diffusing system. Mass flux decayed as t{sup 1/2} in two experiments each with initial R{sub p} = 2.4 and 2.8.« less

Citrulline diet supplementation improves specific age-related raft changes in wild-type rodent hippocampus.

PubMed

Marquet-de Rougé, Perrine; Clamagirand, Christine; Facchinetti, Patricia; Rose, Christiane; Sargueil, Françoise; Guihenneuc-Jouyaux, Chantal; Cynober, Luc; Moinard, Christophe; Allinquant, Bernadette

2013-10-01

The levels of molecules crucial for signal transduction processing change in the brain with aging. Lipid rafts are membrane microdomains involved in cell signaling. We describe here substantial biophysical and biochemical changes occurring within the rafts in hippocampus neurons from aging wild-type rats and mice. Using continuous sucrose density gradients, we observed light-, medium-, and heavy raft subpopulations in young adult rodent hippocampus neurons containing very low levels of amyloid precursor protein (APP) and almost no caveolin-1 (CAV-1). By contrast, old rodents had a homogeneous age-specific high-density caveolar raft subpopulation containing significantly more cholesterol (CHOL), CAV-1, and APP. C99-APP-Cter fragment detection demonstrates that the first step of amyloidogenic APP processing takes place in this caveolar structure during physiological aging of the rat brain. In this age-specific caveolar raft subpopulation, levels of the C99-APP-Cter fragment are exponentially correlated with those of APP, suggesting that high APP concentrations may be associated with a risk of large increases in beta-amyloid peptide levels. Citrulline (an intermediate amino acid of the urea cycle) supplementation in the diet of aged rats for 3 months reduced these age-related hippocampus raft changes, resulting in raft patterns tightly close to those in young animals: CHOL, CAV-1, and APP concentrations were significantly lower and the C99-APP-Cter fragment was less abundant in the heavy raft subpopulation than in controls. Thus, we report substantial changes in raft structures during the aging of rodent hippocampus and describe new and promising areas of investigation concerning the possible protective effect of citrulline on brain function during aging.

Role of turbulence regime on determining the local density gradient

DOE PAGES

Wang, X.; Mordijck, Saskia; Doyle, E. J.; ...

2017-11-16

In this study we show that the local density gradient in the plasma core depends on the calculated mode-frequency of the most unstable linear mode and reaches a maximum when this frequency is close to zero. Previous theoretical and experimental work on AUG has shown that the ratio of electron to ion temperature, and as such the frequency of the dominant linear gyrokinetic mode, affects the local density gradient close to ρ = 0.3 [1, 2]. On DIII-D we find that by adding Electron Cyclotron Heating (ECH), we modify the dominant unstable linear gyro kinetic mode from an Ion Temperaturemore » Gradient (ITG) mode to a Trapped Electron Mode (TEM), which means that the frequency of the dominant mode changes sign (from the ion to the electron direction). Local density peaking around mid-radius increases by 50% right around the cross-over between the ITG and TEM regimes. By comparing how the particle flux changes, through the derivative of the electron density, n e, with respect to time, ∂n e/∂t, we find that the particle flux also exhibits the same trend versus mode frequency. As a result, we find that the changes in local particle transport are inversely proportional to the changes in electron density, indicating that the changes are driven by a change in thermo-diffusive pinch.« less

Double-hybrid density-functional theory with meta-generalized-gradient approximations

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

Souvi, Sidi M. O., E-mail: sidi.souvi@irsn.fr; Sharkas, Kamal; Toulouse, Julien, E-mail: julien.toulouse@upmc.fr

2014-02-28

We extend the previously proposed one-parameter double-hybrid density-functional theory [K. Sharkas, J. Toulouse, and A. Savin, J. Chem. Phys. 134, 064113 (2011)] to meta-generalized-gradient-approximation (meta-GGA) exchange-correlation density functionals. We construct several variants of one-parameter double-hybrid approximations using the Tao-Perdew-Staroverov-Scuseria (TPSS) meta-GGA functional and test them on test sets of atomization energies and reaction barrier heights. The most accurate variant uses the uniform coordinate scaling of the density and of the kinetic energy density in the correlation functional, and improves over both standard Kohn-Sham TPSS and second-order Møller-Plesset calculations.

Assembly of multiple cell gradients directed by three-dimensional microfluidic channels.

PubMed

Li, Yiwei; Feng, Xiaojun; Wang, Yachao; Du, Wei; Chen, Peng; Liu, Chao; Liu, Bi-Feng

2015-08-07

Active control over the cell gradient is essential for understanding biological systems and the reconstitution of the functionality of many types of tissues, particularly for organ-on-a-chip. Here, we propose a three-dimensional (3D) microfluidic strategy for generating controllable cell gradients. In this approach, a homogeneous cell suspension is loaded into a 3D stair-shaped PDMS microchannel to generate a cell gradient within 10 min by sedimentation. We demonstrate that cell gradients of various profiles (exponential and piecewise linear) can be achieved by precisely controlling the height of each layer during the fabrication. With sequential seeding, we further demonstrate the generation of two overlapping cell gradients on the same glass substrate with pre-defined designs. The cell gradient-based QD cytotoxicity assay also demonstrated that cell behaviors and resistances were regulated by the changes in cell density. These results reveal that the proposed 3D microfluidic strategy provides a simple and versatile means for establishing controllable gradients in cell density, opening up a new avenue for reconstructing functional tissues.

Friend Leukemogenic Virus-neutralizing Antibody from Mouse Ascitic Fluid

PubMed Central

March, R. W.; Chirigos, M. A.; Hook, W. A.; Burka, B. L.

1967-01-01

Ascitic fluid antibody produced in C57/B1 mice immunized with Friend leukemogenic virus exhibited potent neutralizing activity. In vitro neutralization tests revealed that a mean neutralization index of 3.0 was achieved, and it was shown by sucrose gradient ultracentrifugation that this antibody resembled the 7S type. A mean yield of 6.7 ml of fluid per mouse per weekly paracentesis was obtained over an 8-week period. The ascitic fluid antibody to Friend virus was also active in vivo. Mice given antibody 3, 5, 7, and 9 days after infection with Friend virus did not develop the splenomegaly characteristic of Friend disease. PMID:16349755

Analysis of the structure of fish lymphocystis disease virions from skin tumours of pleuronectes.

PubMed

Samalecos, C P

1986-01-01

Virions of fish lymphocystis disease (FLDV) from tumour-carrying-fishes (flounder, dab, plaice and gurnard) collected in the North Sea were isolated directly from the tumours and purified by sucrose and subsequent caesium chloride gradient centrifugation. They were studied by electron microscopy using embedding methods, negative staining and using metal shadowing methods. Tumours of dermal connective fish tissue showed particles with hexagonal outlines. Occasionally, an "empty structure" was observed. Subunits of the virus membrane were identified after 3 per cent phosphotungstic acid (PTA) or 0.2 per cent Ruthenium red (RR) staining. FLDV measures 199-227 nm in diameter.

Molecular assembly and subcellular distribution of ATP-sensitive potassium channel proteins in rat hearts.

PubMed

Kuniyasu, Akihiko; Kaneko, Kazuyoshi; Kawahara, Kohichi; Nakayama, Hitoshi

2003-09-25

Cardiac ATP-sensitive K(+) (K(ATP)) channels are proposed to contribute to cardio-protection and ischemic preconditioning. Although mRNAs for all subunits of K(ATP) channels (Kir6.0 and sulfonylurea receptors SURs) were detected in hearts, subcellular localization of their proteins and the subunit combination are not well elucidated. We address these questions in rat hearts, using anti-peptide antibodies raised against each subunit. By immunoblot analysis, all of the subunits were detected in microsomal fractions including sarcolemmal membranes, while they were not detected in mitochondrial fractions at all. Immunoprecipitation and sucrose gradient sedimentation of the digitonin-solubilized microsomes indicated that Kir6.2 exclusively assembled with SUR2A. The molecular mass of the Kir6.2-SUR2A complex estimated by sucrose sedimentation was 1150 kDa, significantly larger than the calculated value for (Kir6.2)(4)-(SUR2A)(4), suggesting a potential formation of micellar complex with digitonin but no indication of hybrid channel formation under the conditions. These findings provide additional information on the structural and functional relationships of cardiac K(ATP) channel proteins involving subcellular localization and roles for cardioprotection and ischemic preconditioning.

Cell wall invertase in tobacco crown gall cells : enzyme properties and regulation by auxin.

PubMed

Weil, M; Rausch, T

1990-12-01

The cell wall invertase from an Agrobacterium tumefaciens-transformed Nicotiana tabacum cell line (SR1-C58) was purified. The heterogeneously glycosylated enzyme has the following properties: M(r) 63,000, pH optimum at 4.7, K(m sucrose) 0.6 millimolar (at pH 4.7), pl 9.5. Enzyme activity is inhibited by micromolar concentrations of HgCl(2) but is insensitive to H(2)O(2), N-ethylmaleimide and dithiothreitol. Upon transfer of transformed cells from the stationary phase to fresh medium, a cycloheximide- and tunicamycin-sensitive de novo formation of cell wall invertase is demonstrated in the absence or presence of sucrose. While in an auxin mutant (lacking gene 1;SR1-3845) 1 micromolar 1-naphthaleneacetic acid led to a further increased activity, the wild-type transformed cell line (SR1-C58) responded with a decreased activity compared to the control. An analysis of cell wall invertase in and around tumors initiated with Agrobacterium tumefaciens (strain C58) on Nicotiana tabacum stem and Kalanchoë daigremontiana leaves revealed gradients of activity. The results indicate that the auxin-stimulated cell wall invertase is essential for the establishment of the tumor sink.

Radiation effects on bovine taste bud membranes

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

Shatzman, A.R.; Mossman, K.L.

1982-11-01

In order to investigate the mechanisms of radiation-induced taste loss, the effects of radiation on preparations of enriched bovine taste bud membranes were studied. Taste buds containing circumvallate papilae, and surrounding control epithelial tissues devoid of taste buds, were obtained from steers and given radiation doses of 0-7000 cGy (rad). Tissue fractions were isolated into membrane-enriched and heterogeneous components using differential and sucrose gradient centrifugation of tissue homogenates. The yield of membranes, as measured by protein content in the buoyant membrane-enriched fractions, was reduced in quantity with increasing radiation dose. The relation between radiation dose and membrane quantity in membrane-enrichedmore » fractions could be fit by a simple exponential model with taste bud-derived membranes twice as radiosensitive as membranes from control epithelial tissue. Binding of sucrose, sodium, and acetate and fluoride stimulation of adenylate cyclase were nearly identical in both irradiated and nonirradiated intact membranes. Radiation had no effect on fractions of heterogeneous components. While it is not clear what changes are occurring in enriched taste cell membranes, damage to membranes may play an important role in the taste loss observed in patients following radiotherapy.« less

Single-strand breakage of DNA in UV-irradiated uvrA, uvrB, and uvrC mutants of Escherichia coli.

PubMed Central

Tang, M S; Ross, L

1985-01-01

We transduced the uvrA6, uvrB5, uvrC34, and uvrC56 markers from the original mutagenized strains into an HF4714 background. Although in the original mutagenized strains uvrA6 cells are more UV sensitive than uvrB5 and uvrC34 cells, in the new background no significant difference in UV sensitivity is observed among uvrA6, uvrB5, and uvrC34 cells. No DNA single-strand breaks are detected in UV-irradiated uvrA6 or uvrB5 cells, whereas in contrast a significant number of single-strand breaks are detected in both UV-irradiated uvrC34 and uvrC56 cells. The number of single-strand breaks in these cells reaches a plateau at 20-J/m2 irradiation. Since these single-strand breaks can be detected by both alkaline sucrose and neutral formamide-sucrose gradient sedimentation, we concluded that the single-strand breaks observed in UV-irradiated uvrC cells are due to phosphodiester bond interruptions in DNA and are not due to apurinic/apyrimidinic sites. PMID:3882671

Density and fluence dependence of lithium cell damage and recovery characteristics

NASA Technical Reports Server (NTRS)

Faith, T. J.

1971-01-01

Experimental results on lithium-containing solar cells point toward the lithium donor density gradient dN sub L/dw as being the crucial parameter in the prediction of cell behavior after irradiation by electrons. Recovery measurements on a large number of oxygen-rich and oxygen-lean lithium cells have confirmed that cell recovery speed is directly proportional to the value of the lithium gradient for electron fluences. Gradient measurements have also been correlated with lithium diffusion schedules. Results have shown that long diffusion times (25 h) with a paint-on source result in large cell-to-cell variations in gradient, probably due to a loss of the lithium source with time.

The effect of a longitudinal density gradient on electron plasma wake field acceleration

NASA Astrophysics Data System (ADS)

Tsiklauri, David

2016-12-01

Three-dimensional, particle-in-cell, fully electromagnetic simulations of electron plasma wake field acceleration in the blow-out regime are presented. Earlier results are extended by (i) studying the effect of a longitudinal density gradient, (ii) avoiding the use of a co-moving simulation box, (iii) inclusion of ion motion, and (iv) studying fully electromagnetic plasma wake fields. It is established that injecting driving and trailing electron bunches into a positive density gradient of 10-fold increasing density over 10 cm long lithium vapour plasma results in spatially more compact and three times larger, compared with the uniform density case, electric fields (-6.4×1010 V m-1), leading to acceleration of the trailing bunch up to 24.4 GeV (starting from an initial 20.4 GeV), with energy transfer efficiencies from the leading to trailing bunch of 75%. In the uniform density case, a -2.5×1010 V m-1 wake is created leading to acceleration of the trailing bunch up to 22.4 GeV, with energy transfer efficiencies of 65%. It is also established that injecting the electron bunches into a negative density gradient of 10-fold decreasing density over 10 cm long plasma results in spatially more spread and two and a half smaller electric fields (-1.0×1010 V m-1), leading to a weaker acceleration of the trailing bunch up to 21.4 GeV, with energy transfer efficiencies of 45%. Taking ion motions into consideration shows that in the plasma wake ion number density can increase over a few times the background value. It is also shown that transverse electromagnetic fields in a plasma wake are of the same order as the longitudinal (electrostatic) ones.

Visualisation and orbital-free parametrisation of the large-Z scaling of the kinetic energy density of atoms

NASA Astrophysics Data System (ADS)

Cancio, Antonio C.; Redd, Jeremy J.

2017-03-01

The scaling of neutral atoms to large Z, combining periodicity with a gradual trend to homogeneity, is a fundamental probe of density functional theory, one that has driven recent advances in understanding both the kinetic and exchange-correlation energies. Although research focus is normally upon the scaling of integrated energies, insights can also be gained from energy densities. We visualise the scaling of the positive-definite kinetic energy density (KED) in closed-shell atoms, in comparison to invariant quantities based upon the gradient and Laplacian of the density. We notice a striking fit of the KED within the core of any atom to a gradient expansion using both the gradient and the Laplacian, appearing as an asymptotic limit around which the KED oscillates. The gradient expansion is qualitatively different from that derived from first principles for a slowly varying electron gas and is correlated with a nonzero Pauli contribution to the KED near the nucleus. We propose and explore orbital-free meta-GGA models for the kinetic energy to describe these features, with some success, but the effects of quantum oscillations in the inner shells of atoms make a complete parametrisation difficult. We discuss implications for improved orbital-free description of molecular properties.

Study of adhesion and friction properties on a nanoparticle gradient surface: transition from JKR to DMT contact mechanics.

PubMed

Ramakrishna, Shivaprakash N; Nalam, Prathima C; Clasohm, Lucy Y; Spencer, Nicholas D

2013-01-08

We have previously investigated the dependence of adhesion on nanometer-scale surface roughness by employing a roughness gradient. In this study, we correlate the obtained adhesion forces on nanometer-scale rough surfaces to their frictional properties. A roughness gradient with varying silica particle (diameter ≈ 12 nm) density was prepared, and adhesion and frictional forces were measured across the gradient surface in perfluorodecalin by means of atomic force microscopy with a polyethylene colloidal probe. Similarly to the pull-off measurements, the frictional forces initially showed a reduction with decreasing particle density and later an abrupt increase as the colloidal sphere began to touch the flat substrate beneath, at very low particle densities. The friction-load relation is found to depend on the real contact area (A(real)) between the colloid probe and the underlying particles. At high particle density, the colloidal sphere undergoes large deformations over several nanoparticles, and the contact adhesion (JKR type) dominates the frictional response. However, at low particle density (before the colloidal probe is in contact with the underlying surface), the colloidal sphere is suspended by a few particles only, resulting in local deformations of the colloid sphere, with the frictional response to the applied load being dominated by long-range, noncontact (DMT-type) interactions with the substrate beneath.

Material and Optical Densities

ERIC Educational Resources Information Center

Gluck, Paul

2007-01-01

The bending of a laser beam in a medium with a density and refractive index gradient in the same direction has been described previously. When a transparent container is half filled with a salt or sugar solution and an equal amount of water is floated on top of it, then diffusion will create a concentration gradient from top to bottom. A laser…

Particle Size Effects on CL-20 Initiation and Detonation

NASA Astrophysics Data System (ADS)

Valancius, Cole; Bainbridge, Joe; Love, Cody; Richardson, Duane

2017-06-01

Particle size or specific surface area effects on explosives has been of interest to the explosives community for both application and modeling of initiation and detonation. Different particles sizes of CL-20 were used in detonator experiments to determine the effects of particle size on initiation, run-up to steady state detonation, and steady state detonation. Historical tests have demonstrated a direct relationship between particle size and initiation. However, historical tests inadvertently employed density gradients, making it difficult to discern the effects of particle size from the effects of density. Density gradients were removed from these tests using a larger diameter, shorter charge column, allowing for similar loading across different particle sizes. Without the density gradient, the effects of particle size on initiation and detonation are easier to determine. The results of which contrast with historical results, showing particle size does not directly affect initiation threshold.

Possible influence of the Kuramoto length in a photo-catalytic water splitting reaction revealed by Poisson-Nernst-Planck equations involving ionization in a weak electrolyte

NASA Astrophysics Data System (ADS)

Suzuki, Yohichi; Seki, Kazuhiko

2018-03-01

We studied ion concentration profiles and the charge density gradient caused by electrode reactions in weak electrolytes by using the Poisson-Nernst-Planck equations without assuming charge neutrality. In weak electrolytes, only a small fraction of molecules is ionized in bulk. Ion concentration profiles depend on not only ion transport but also the ionization of molecules. We considered the ionization of molecules and ion association in weak electrolytes and obtained analytical expressions for ion densities, electrostatic potential profiles, and ion currents. We found the case that the total ion density gradient was given by the Kuramoto length which characterized the distance over which an ion diffuses before association. The charge density gradient is characterized by the Debye length for 1:1 weak electrolytes. We discuss the role of these length scales for efficient water splitting reactions using photo-electrocatalytic electrodes.

High Efficacy but Low Potency of δ-Opioid Receptor-G Protein Coupling in Brij-58-Treated, Low-Density Plasma Membrane Fragments.

PubMed

Roubalova, Lenka; Vosahlikova, Miroslava; Brejchova, Jana; Sykora, Jan; Rudajev, Vladimir; Svoboda, Petr

2015-01-01

HEK293 cells stably expressing PTX-insensitive δ-opioid receptor-Gi1α (C351I) fusion protein were homogenized, treated with low concentrations of non-ionic detergent Brij-58 at 0°C and fractionated by flotation in sucrose density gradient. In optimum range of detergent concentrations (0.025-0.05% w/v), Brij-58-treated, low-density membranes exhibited 2-3-fold higher efficacy of DADLE-stimulated, high-affinity [32P]GTPase and [35S]GTPγS binding than membranes of the same density prepared in the absence of detergent. The potency of agonist DADLE response was significantly decreased. At high detergent concentrations (>0.1%), the functional coupling between δ-opioid receptors and G proteins was completely diminished. The same detergent effects were measured in plasma membranes isolated from PTX-treated cells. Therefore, the effect of Brij-58 on δ-opioid receptor-G protein coupling was not restricted to the covalently bound Gi1α within δ-opioid receptor-Gi1α fusion protein, but it was also valid for PTX-sensitive G proteins of Gi/Go family endogenously expressed in HEK293 cells. Characterization of the direct effect of Brij-58 on the hydrophobic interior of isolated plasma membranes by steady-state anisotropy of diphenylhexatriene (DPH) fluorescence indicated a marked increase of membrane fluidity. The time-resolved analysis of decay of DPH fluorescence by the "wobble in cone" model of DPH motion in the membrane indicated that the exposure to the increasing concentrations of Brij-58 led to a decreased order and higher motional freedom of the dye. Limited perturbation of plasma membrane integrity by low concentrations of non-ionic detergent Brij-58 results in alteration of δ-OR-G protein coupling. Maximum G protein-response to agonist stimulation (efficacy) is increased; affinity of response (potency) is decreased. The total degradation plasma membrane structure at high detergent concentrations results in diminution of functional coupling between δ-opioid receptors and G proteins.

Sucrose-mediated transcriptional regulation of sucrose symporter activity in the phloem.

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

Matt Vaughn Greg Harrington Daniel R Bush

2002-08-06

This project was based on our discovery that sucrose acts as a signaling molecule that regulates the activity of a proton-sucrose symporter in sugar beet leaf tissue. A major objective here was determining how sucrose transporter activity is being regulated. When sucrose accumulates in the phloem sucrose transport activity drops dramatically. Western blots of plasma membrane proteins isolated from sucrose treated leaves showed that the loss of sucrose transport activity was proportional to a decline in symporter abundance, demonstrating that sucrose transport is regulated by changes in the amount of BvSUT1 protein. BvSUT1 transcript levels decreased in parallel with themore » loss of sucrose transport activity. Nuclear run-on experiments demonstrated that BvSUT1 gene transcription was repressed significantly in nuclei from leaves fed 100 mM exogenous sucrose, showing that sucrose-dependent modulation of BvSUT1 mRNA levels is mediated by changes in transcription. To identify which secondary messenger systems might be involved in regulating symporter activity, we used a variety of pharmacological agents to probe for a role of calcium or protein phosphorylation in sucrose signaling. In a detailed analysis, only okadaic acid altered sucrose transport activity. These results suggest a protein phosphatase is involved. We hypothesized that protein kinase inhibitors would have a neutral affect or increase symporter transcription. Transpirational feeding of the protein kinase inhibitor staurosporine had no impact on sucrose transport while calphostin C, an inhibitor of protein kinase C, caused a 60% increase. These data provided good evidence that protein phosphorylation plays a central role in regulating sucrose symporter expression and sucrose transport activity. To determine whether protein phosphorylation is involved in sucrose regulation of proton-sucrose symporter activity, we pre-fed leaves with staurosporine for 4 h and then fed the treated leaves water or 100 mM sucrose for an additional 20 h. Sucrose transport activity was higher than the water control in both staurosporine/water- and staurosporine/sucrose-fed leaves. In contrast, sucrose transport activity was only 40% of the water control in sucrose-fed leaves. Taken together, these results showed that a phosphorylation-dependent signal transduction pathway is involved in sucrose-mediated regulation of BvSUT1 gene expression, sucrose transport activity, and ultimately phloem loading. Publications originating from this work: Vaughn MW, GN. Harrington, and DR Bush 2002. Sucrose-mediated transcriptional regulation of sucrose symporter activity in the phloem. Proc. Natl. Acad. Sci. USA 99:10876-10880 Ransom-Hodgkins W, MW Vaughn, and DR Bush 2003. Protein phosphorylation mediates a key step in sucrose-regulation of the expression and transport activity of a beet proton-sucrose symporter. Planta 217:483-489 Harrington GN and Bush DR 2003. The bifunctional role of hexokinase in metabolism and glucose signaling. Plant Cell 15: 2493-2496« less

Investigation of Photographic Image Quality Estimators

DTIC Science & Technology

1980-04-01

WORDS (Conltnu* wi r« y «f •• »iä* ll n«c»*aarr «M läm’lly by ftloc* nuwWo Resolving Power Acutance SENTINEL SIGMA Math Model Modulation Transfer...Bibeman (1973) describes acutance as being "expressed in terms of the mean square of the gradient of . . . density (in a photographic image) with...the density difference AD. for each interval from the (smoothed) microdensitometer trace (calibrated in density units). 4. Compute the gradient -77

Characterization of the endomannosidase pathway for the processing of N-linked oligosaccharides in glucosidase II-deficient and parent mouse lymphoma cells.

PubMed

Moore, S E; Spiro, R G

1992-04-25

Studies on N-linked oligosaccharide processing in the mouse lymphoma glucosidase II-deficient mutant cell line (PHAR2.7) as well as the parent BW5147 cells indicated that the former maintain their capacity to synthesize complex carbohydrate units through the use of the deglucosylation mechanism provided by endomannosidase. The in vivo activity of this enzyme was evident in the mutant cells from their production of substantial amounts of glucosylated mannose saccharides, predominantly Glc2Man; moreover, in the presence of 1-deoxymannojirimycin or kifunensine to prevent processing by mannosidase I, N-linked Man8GlcNAc2 was observed entirely in the form of the characteristic isomer in which the terminal mannose of the alpha 1,3-linked branch is missing (isomer A). In contrast, parent lymphoma cells, as well as HepG2 cells in the presence of 1-deoxymannojirimycin accumulated Man9GlcNAc2 as the primary deglucosylated N-linked oligosaccharide and contained only about 16% of their Man8GlcNAc2 as isomer A. In the presence of the glucosidase inhibitor castanospermine the mutant released Glc3Man instead of Glc2Man, and the parent cells converted their deglucosylation machinery to the endomannosidase route. Despite the mutant's capacity to accommodate a large traffic through this pathway no increase in the in vitro determined endomannosidase activity was evident. The exclusive utilization of endomannosidase by the mutant for the deglucosylation of its predominant N-linked Glc2Man9GlcNAc2 permitted an exploration of the in vivo site of this enzyme's action. Pulse-chase studies utilizing sucrose-D2O density gradient centrifugation indicated that the Glc2Man9GlcNAc2 to Man8GlcNAc2 conversion is a relatively late event that is temporally separated from the endoplasmic reticulum-situated processing of Glc3Man9GlcNAc2 to Glc2Man9GlcNAc2 and in contrast to the latter takes place in the Golgi compartment.

Astacin Family Metallopeptidases and Serine Peptidase Inhibitors in Spider Digestive Fluid

PubMed Central

Foradori, Matthew J.; Tillinghast, Edward K.; Smith, J. Stephen; Townley, Mark A.; Mooney, Robert E.

2006-01-01

Digestive fluid of the araneid spider Argiope aurantia is known to contain zinc metallopeptidases. Using anion-exchange chromatography, size-exclusion chromatography, sucrose density gradient centrifugation, and gel electrophoresis, we isolated two lower-molecular-mass peptidases, designated p16 and p18. The N-terminal amino acid sequences of p16 (37 residues) and p18 (20 residues) are 85% identical over the first 20 residues and are most similar to the N-terminal sequences of the fully active form of meprin (β subunits) from several vertebrates (47–52% and 50–60% identical, respectively). Meprin is a peptidase in the astacin (M12A) subfamily of the astacin (M12) family. Additionally, a 66-residue internal sequence obtained from p16 aligns with the conserved astacin subfamily domain. Thus, at least some spider digestive peptidases appear related to astacin of decapod crustaceans. However, important differences between spider and crustacean metallopeptidases with regard to isoelectric point and their susceptibility to hemolymph-borne inhibitors are demonstrated. Anomalous behavior of the lower-molecular-mass Argiope peptidases during certain fractionation procedures indicates that these peptidases may take part in reversible associations with each other or with other proteins. A. aurantia digestive fluid also contains inhibitory activity effective against insect digestive peptidases. Here we present evidence for at least thirteen, heat-stable serine peptidase inhibitors ranging in molecular mass from about 15 to 32 kDa. PMID:16458560

Detection of Toxoplasma gondii oocysts in different water resources by Loop Mediated Isothermal Amplification (LAMP).

PubMed

Gallas-Lindemann, Carmen; Sotiriadou, Isaia; Mahmoodi, Mohammad Reza; Karanis, Panagiotis

2013-02-01

Human toxoplasmosis is potentially contracted due to consumption of contaminated drinking water and represents an increasing public health risk worldwide. Toxoplasma gondii oocysts can be resistant to standard disinfection processes, including UV radiation. Increased awareness of the risk of waterborne toxoplasmosis outbreaks has led to an increase in research interest in the detection of oocysts in environmental water systems. Ninety-five environmental water samples from the Lower Rhine area in Germany have been included in the study and examined for the presence of Toxoplasma. Water samples were filtered or flocculated by aluminum sulfate and purified by sucrose density gradient. DNA was then extracted, and the DNA samples were then examined by LAMP analysis. T. gondii DNA was detected in eight out of 83 (9.6%) influent and effluent samples obtained from wastewater treatment plants. All samples (n=12) from the surface, ground, raw and tap waters tested negative. The purpose of this work was to investigate the occurrence and distribution of Toxoplasma oocysts on the Lower Rhine in Germany. Our study provides evidence that the assay is a sensitive, specific, rapid and cost effective method for the detection of T. gondii and is useful for both the investigations of cases of waterborne outbreaks and for identifying the source of contamination. Copyright © 2012 Elsevier B.V. All rights reserved.

DEAD-Box RNA Helicase Dbp4 Is Required for Small-Subunit Processome Formation and Function

PubMed Central

Soltanieh, Sahar; Osheim, Yvonne N.; Spasov, Krasimir; Trahan, Christian; Beyer, Ann L.

2014-01-01

DEAD-box RNA helicase Dbp4 is required for 18S rRNA synthesis: cellular depletion of Dbp4 impairs the early cleavage reactions of the pre-rRNA and causes U14 small nucleolar RNA (snoRNA) to remain associated with pre-rRNA. Immunoprecipitation experiments (IPs) carried out with whole-cell extracts (WCEs) revealed that hemagglutinin (HA)-tagged Dbp4 is associated with U3 snoRNA but not with U14 snoRNA. IPs with WCEs also showed association with the U3-specific protein Mpp10, which suggests that Dbp4 interacts with the functionally active U3 RNP; this particle, called the small-subunit (SSU) processome, can be observed at the 5′ end of nascent pre-rRNA. Electron microscopy analyses indicated that depletion of Dbp4 compromised SSU processome formation and cotranscriptional cleavage of the pre-rRNA. Sucrose density gradient analyses revealed that depletion of U3 snoRNA or the Mpp10 protein inhibited the release of U14 snoRNA from pre-rRNA, just as was seen with Dbp4-depleted cells, indicating that alteration of SSU processome components has significant consequences for U14 snoRNA dynamics. We also found that the C-terminal extension flanking the catalytic core of Dbp4 plays an important role in the release of U14 snoRNA from pre-rRNA. PMID:25535329

Functional Characterization of Lpt3 and Lpt6, the Inner-Core Lipooligosaccharide Phosphoethanolamine Transferases from Neisseria meningitidis▿

PubMed Central

Wenzel, Cory Q.; St. Michael, Frank; Stupak, Jacek; Li, Jianjun; Cox, Andrew D.; Richards, James C.

2010-01-01

The lipooligosaccharide (LOS) of Neisseria meningitidis contains heptose (Hep) residues that are modified with phosphoethanolamine (PEtn) at the 3 (3-PEtn) and/or 6 (6-PEtn) position. The lpt3 (NMB2010) and lpt6 (NMA0408) genes of N. meningitidis, which are proposed to encode the required HepII 3- and 6-PEtn transferases, respectively, were cloned and overexpressed as C-terminally polyhistidine-tagged fusion proteins in Escherichia coli and found to localize to the inner membrane, based on sucrose density gradient centrifugation. Lpt3-His6 and Lpt6-His6 were purified from Triton X-100-solubilized membranes by nickel chelation chromatography, and dot blot analysis of enzymatic reactions with 3-PEtn- and 6-PEtn-specific monoclonal antibodies demonstrated conclusively that Lpt3 and Lpt6 are phosphatidylethanolamine-dependent LOS HepII 3- and 6-PEtn transferases, respectively, and that both enzymes are capable of transferring PEtn to both fully acylated LOS and de-O-acylated (de-O-Ac) LOS. Further enzymatic studies using capillary electrophoresis-mass spectrometry (MS) demonstrated that both Lpt3 and Lpt6 are capable of transferring PEtn to de-O-Ac LOS molecules already containing PEtn at the 6 and 3 positions of HepII, respectively, demonstrating that there is no obligate order of PEtn addition in the generation of 3,6-di-PEtn LOS moieties in vitro. PMID:19854897

Studies on proinsulin and proglucagon biosynthesis and conversion at the subcellular level: I. Fractionation procedure and characterization of the subcellular fractions

PubMed Central

Noe, BD; Baste, CA; Bauer, GE

1977-01-01

Anglerfish islets were homogenized in 0.25 M sucrose and separated into seven separate subcellular fractions by differential and discontinuous density gradient centrifugation. The objective was to isolate microsomes and secretory granules in a highly purified state. The fractions were characterized by electron microscopy and chemical analyses. Each fraction was assayed for its content of protein, RNA, DNA, immunoreactive insulin (IRI), and immunoreactive glucagon (IRG). Ultrastructural examination showed that two of the seven subcellular fractions contain primarily mitochondria, and that two others consist almost exclusively of secretory granules. A fifth fraction contains rough and smooth microsomal vesicles. The remaining two fractions are the cell supernate and the nuclei and cell debris. The content of DNA and RNA in all fractions is consistent with the observed ultrastructure. More than 82 percent of the total cellular IRI and 89(percent) of the total cellular IRG are found in the fractions of secretory granules. The combined fractions of secretory granules and microsomes consistently yield >93 percent of the total IRG. These results indicate that the fractionation procedure employed yields fractions of microsomes and secretory granules that contain nearly all the immunoassayable insulin and glucagons found in whole islet tissue. These fractions are thus considered suitable for study of proinsulin and proglucagon biosynthesis and their metabolic conversion at the subcellular level. PMID:328517

Subcellular fractionation by zonal centrifugation of glucose-repressed anaerobically grown Saccharomyces carlsbergensis

PubMed Central

Cartledge, T. G.; Lloyd, D.

1972-01-01

1. Homogenates were prepared from sphaeroplasts of anaerobically grown, glucoserepressed Saccharomyces carlsbergensis, and the distributions of marker enzymes investigated after zonal centrifugation on sucrose gradients containing 2mm-MgCl2. 2. These homogenates contained no detectable cytochrome c oxidase, succinate–cytochrome c oxidoreductase, succinate–ferricyanide oxidoreductase, l(+)-lactate–cytochrome c oxidoreductase or catalase. Cytochromes a+a3 and c were not detected. 3. Zonal centrifugation of whole homogenates indicated complex density distributions of the sedimentable portions of NADH– and NADPH–cytochrome c oxidoreductases, adenosine triphosphatases (ATPases), adenosine pyrophosphatase (ADPase), pyrophosphatase and acid p-nitrophenyl phosphatase. Several different ATPases were distinguished on the basis of their sensitivities to oligomycin and ouabain. 4. Differential centrifugation of whole homogenates at 105g-min left 80–90% of the protein, dithionite-reducible cytochrome b, acid hydrolases and pyrophosphatase in a supernatant (S1) together with 65 and 56% of the NADH– and NADPH–cytochrome c oxidoreductases respectively, 25% of the ATPases and 71% of the adenosine monophosphatase. 5. Further analysis of supernatant S1 revealed the presence of a class of small particles containing NADPH–cytochrome c oxidoreductases and ATPases. 6. At least four different populations of large particles were distinguished. 7. Electron microscopy indicated that one of these corresponded to `promitochondria' as described by other workers. ImagesPLATE 1PLATE 2PLATE 3 PMID:4405573

Quantitative Detection of the Foot-And-Mouth Disease Virus Serotype O 146S Antigen for Vaccine Production Using a Double-Antibody Sandwich ELISA and Nonlinear Standard Curves.

PubMed

Feng, Xia; Ma, Jun-Wu; Sun, Shi-Qi; Guo, Hui-Chen; Yang, Ya-Min; Jin, Ye; Zhou, Guang-Qing; He, Ji-Jun; Guo, Jian-Hong; Qi, Shu-yun; Lin, Mi; Cai, Hu; Liu, Xiang-Tao

2016-01-01

The efficacy of an inactivated foot-and-mouth disease (FMD) vaccine is mainly dependent on the integrity of the foot-and-mouth disease virus (FMDV) particles. At present, the standard method to quantify the active component, the 146S antigen, of FMD vaccines is sucrose density gradient (SDG) analysis. However, this method is highly operator dependent and difficult to automate. In contrast, the enzyme-linked immunosorbent assay (ELISA) is a time-saving technique that provides greater simplicity and sensitivity. To establish a valid method to detect and quantify the 146S antigen of a serotype O FMD vaccine, a double-antibody sandwich (DAS) ELISA was compared with an SDG analysis. The DAS ELISA was highly correlated with the SDG method (R2 = 0.9215, P<0.01). In contrast to the SDG method, the DAS ELISA was rapid, robust, repeatable and highly sensitive, with a minimum quantification limit of 0.06 μg/mL. This method can be used to determine the effective antigen yields in inactivated vaccines and thus represents an alternative for assessing the potency of FMD vaccines in vitro. But it still needs to be prospectively validated by analyzing a new vaccine preparation and determining the proper protective dose followed by an in vivo vaccination-challenge study to confirm the ELISA findings.

Subcellular localization and cytoplasmic complex status of endogenous Keap1.

PubMed

Watai, Yoriko; Kobayashi, Akira; Nagase, Hiroko; Mizukami, Mio; McEvoy, Justina; Singer, Jeffrey D; Itoh, Ken; Yamamoto, Masayuki

2007-10-01

Keap1 acts as a sensor for oxidative/electrophilic stress, an adaptor for Cullin-3-based ubiquitin ligase, and a regulator of Nrf2 activity through the interaction with Nrf2 Neh2 domain. However, the mechanism(s) of Nrf2 migration into the nucleus in response to stress remains largely unknown due to the lack of a reliable antibody for the detection of endogenous Keap1 molecule. Here, we report the generation of a new monoclonal antibody for the detection of endogenous Keap1 molecules. Immunocytochemical analysis of mouse embryonic fibroblasts with the antibody revealed that under normal, unstressed condition, Keap1 is localized primarily in the cytoplasm with minimal amount in the nucleus and endoplasmic reticulum. This subcellular localization profile of Keap1 appears unchanged after treatment of cells with diethyl maleate, an electrophile, and/or Leptomycin B, a nuclear export inhibitor. Subcellular fractionation analysis of mouse liver cells showed similar results. No substantial change in the subcellular distribution profile could be observed in cells isolated from butylated hydroxyanisole-treated mice. Analyses of sucrose density gradient centrifugation of mouse liver cells indicated that Keap1 appears to form multiprotein complexes in the cytoplasm. These results demonstrate that endogenous Keap1 remains mostly in the cytoplasm, and electrophiles promote nuclear accumulation of Nrf2 without altering the subcellular localization of Keap1.

DNA Packaging Mutant: Repression of the Vaccinia Virus A32 Gene Results in Noninfectious, DNA-Deficient, Spherical, Enveloped Particles

PubMed Central

Cassetti, Maria Cristina; Merchlinsky, Michael; Wolffe, Elizabeth J.; Weisberg, Andrea S.; Moss, Bernard

1998-01-01

The vaccinia virus A32 open reading frame was predicted to encode a protein with a nucleoside triphosphate-binding motif and a mass of 34 kDa. To investigate the role of this protein, we constructed a mutant in which the original A32 gene was replaced by an inducible copy. The recombinant virus, vA32i, has a conditional lethal phenotype: infectious virus formation was dependent on isopropyl-β-d-thiogalactopyranoside (IPTG). Under nonpermissive conditions, the mutant synthesized early- and late-stage viral proteins, as well as viral DNA that was processed into unit-length genomes. Electron microscopy of cells infected in the absence of IPTG revealed normal-appearing crescents and immature virus particles but very few with nucleoids. Instead of brick-shaped mature particles with defined core structures, there were numerous electron-dense, spherical particles. Some of these spherical particles were wrapped with cisternal membranes, analogous to intracellular and extracellular enveloped virions. Mutant viral particles, purified by sucrose density gradient centrifugation, had low infectivity and transcriptional activity, and the majority were spherical and lacked DNA. Nevertheless, the particle preparation contained representative membrane proteins, cleaved and uncleaved core proteins, the viral RNA polymerase, the early transcription factor and several enzymes, suggesting that incorporation of these components is not strictly coupled to DNA packaging. PMID:9621036

Quantitative Detection of the Foot-And-Mouth Disease Virus Serotype O 146S Antigen for Vaccine Production Using a Double-Antibody Sandwich ELISA and Nonlinear Standard Curves

PubMed Central

Feng, Xia; Ma, Jun-Wu; Sun, Shi-Qi; Guo, Hui-Chen; Yang, Ya-Min; Jin, Ye; Zhou, Guang-Qing; He, Ji-Jun; Guo, Jian-Hong; Qi, Shu-yun; Lin, Mi; Cai, Hu; Liu, Xiang-Tao

2016-01-01

The efficacy of an inactivated foot-and-mouth disease (FMD) vaccine is mainly dependent on the integrity of the foot-and-mouth disease virus (FMDV) particles. At present, the standard method to quantify the active component, the 146S antigen, of FMD vaccines is sucrose density gradient (SDG) analysis. However, this method is highly operator dependent and difficult to automate. In contrast, the enzyme-linked immunosorbent assay (ELISA) is a time-saving technique that provides greater simplicity and sensitivity. To establish a valid method to detect and quantify the 146S antigen of a serotype O FMD vaccine, a double-antibody sandwich (DAS) ELISA was compared with an SDG analysis. The DAS ELISA was highly correlated with the SDG method (R2 = 0.9215, P<0.01). In contrast to the SDG method, the DAS ELISA was rapid, robust, repeatable and highly sensitive, with a minimum quantification limit of 0.06 μg/mL. This method can be used to determine the effective antigen yields in inactivated vaccines and thus represents an alternative for assessing the potency of FMD vaccines in vitro. But it still needs to be prospectively validated by analyzing a new vaccine preparation and determining the proper protective dose followed by an in vivo vaccination-challenge study to confirm the ELISA findings. PMID:26930597

Isolation of circulating immune complexes using Raji cells. Separation of antigens from immune complexes and production of antiserum.

PubMed Central

Theofilopoulos, A N; Eisenberg, R A; Dixon, F J

1978-01-01

Raji cells were used for the isolation of complement-fixing antigen-antibody complexes from serum. Immune complexes bound to these cells were radiolabeled at the cell surface with lactoperoxidase. The complexes were then eluted from the cells with isotonic citrate buffer pH 3.2 or recovered by immunoprecipitation of cell lysates. The antigen and antibody moieties of the complexes were isolated by dissociating sucrose density gradient centrifugation or by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. A variety of preformed immune complexes were successfully isolated from serum with this approach. In addition, these techniques were used to isolate and identify the antigens in immune complexes in the serum of rabbits with chronic serum sickness and rats with Moloney virus-induced sarcomas. Methods were also developed for the production of antisera against the antigenic moiety of immune complexes isolated from serum. Repeated challenge of rabbits with whole Raji cells with bound complexes or eluates from such cells resulted in antibody production against the antigens of the immune complexes, although reactivity against cellular and serum components was also elicited. Monospecific antisera against the antigens in immune complexes were produced by immunizing rabbits with the alum-precipitated antigen isolated on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. These techniques may be useful in isolating antigens in immune complex-associated diseases of unknown etiology. Images PMID:659616

Isolation and characterization of a Treponema pallidum major 60-kilodalton protein resembling the groEL protein of Escherichia coli.

PubMed Central

Houston, L S; Cook, R G; Norris, S J

1990-01-01

A native structure containing the major 60-kilodalton common antigen polypeptide (designated TpN60) was isolated from Treponema pallidum subsp. pallidum (Nichols strain) through a combination of differential centrifugation and sucrose density gradient sedimentation. Gel filtration chromatography indicated that this structure is a high-molecular-weight homo-oligomer of TpN60. Antisera to TpN60 reacted with the groEL polypeptide of Escherichia coli, as determined by immunoperoxidase staining of two-dimensional electroblots. Electron microscopy of the isolated complex revealed a ringlike structure with a diameter of approximately 16 nm which was very similar in appearance to the groEL protein. Comparison of the N-terminal amino acid sequence of TpN60 with the deduced sequences of the E. coli groEL protein, related chaperonin proteins from mycobacteria and Coxiella burnetti, the hsp60 protein of Saccharomyces cerevisiae, the wheat ribulose bisphosphate carboxylase-oxygenase-subunit-binding protein (alpha subunit), and the human P1 mitochondrial protein indicated sequence identity at 8 of 22 to 10 of 22 residues (36 to 45% identity). We conclude that the oligomer of TpN60 is homologous to the groEL protein and related chaperonins found in a wide variety of procaryotes and eucaryotes and thus may represent a heat shock protein involved in protein folding and assembly. Images PMID:1971618

Characterization of Leachianone G 2″ -Dimethylallyltransferase, a Novel Prenyl Side-Chain Elongation Enzyme for the Formation of the Lavandulyl Group of Sophoraflavanone G in Sophora flavescens Ait. Cell Suspension Cultures

PubMed Central

Zhao, Ping; Inoue, Kenichiro; Kouno, Isao; Yamamoto, Hirobumi

2003-01-01

Leachianone G (LG) 2′′-dimethylallyltransferase, a novel prenyl side-chain elongation enzyme, was identified in Sophora flavescens Ait. cultured cells. The enzyme transfers a dimethylallyl group to the 2′′ position of another dimethylallyl group attached at position 8 of LG to form sophoraflavanone G, a branched monoterpenoid-conjugated flavanone characteristic to this plant. This membrane-bound dimethylallyltransferase required Mg2+ (optimum concentration was 10 mm) for the reaction and had an optimum pH of 8.8. It utilized dimethylallyl diphosphate as the sole prenyl donor, and the 2′-hydroxy function in LG was indispensable to the activity. The apparent Km values for dimethylallyl diphosphate and LG were 59 and 2.3 μm, respectively. Subcellular localization of three enzymes that participated in the formation of the lavandulyl group was also investigated by sucrose density gradient centrifugation. Two prenyltransferases, naringenin 8-dimethylallyltransferase and LG 2′′-dimethylallyltransferase, were localized in the plastids, whereas 8-dimethylallylnaringenin 2′-hydroxylase, which catalyzes the crucial step in the lavandulyl-group formation, was associated with the endoplasmic reticulum. These results suggest the close cooperation between the plastids and the endoplasmic reticulum in the formation of lavandulyl groups. PMID:14551337

Dimerization and actin-bundling properties of villin and its role in the assembly of epithelial cell brush borders.

PubMed

George, Sudeep P; Wang, Yaohong; Mathew, Sijo; Srinivasan, Kamalakkannan; Khurana, Seema

2007-09-07

Villin is a major actin-bundling protein in the brush border of epithelial cells. In this study we demonstrate for the first time that villin can bundle actin filaments using a single F-actin binding site, because it has the ability to self-associate. Using fluorescence resonance energy transfer, we demonstrate villin self-association in living cells in microvilli and in growth factor-stimulated cells in membrane ruffles and lamellipodia. Using sucrose density gradient, size-exclusion chromatography, and matrix-assisted laser desorption ionization time-of-flight, the majority of villin was identified as a monomer or dimer. Villin dimers were also identified in Caco-2 cells, which endogenously express villin and Madin-Darby canine kidney cells that ectopically express villin. Using truncation mutants of villin, site-directed mutagenesis, and fluorescence resonance energy transfer, an amino-terminal dimerization site was identified that regulated villin self-association in parallel conformation as well as actin bundling by villin. This detailed analysis describes for the first time microvillus assembly by villin, redefines the actin-bundling function of villin, and provides a molecular mechanism for actin bundling by villin, which could have wider implications for other actin cross-linking proteins that share a villin-like headpiece domain. Our study also provides a molecular basis to separate the morphologically distinct actin-severing and actin-bundling properties of villin.

Three Members of the LC8/DYNLL Family Are Required for Outer Arm Dynein Motor Function

PubMed Central

Tanner, Christopher A.; Rompolas, Panteleimon; Patel-King, Ramila S.; Gorbatyuk, Oksana; Wakabayashi, Ken-ichi; Pazour, Gregory J.

2008-01-01

The highly conserved LC8/DYNLL family proteins were originally identified in axonemal dyneins and subsequently found to function in multiple enzyme systems. Genomic analysis uncovered a third member (LC10) of this protein class in Chlamydomonas. The LC10 protein is extracted from flagellar axonemes with 0.6 M NaCl and cofractionates with the outer dynein arm in sucrose density gradients. Furthermore, LC10 is specifically missing only from axonemes of those strains that fail to assemble outer dynein arms. Previously, the oda12-1 insertional allele was shown to lack the Tctex2-related dynein light chain LC2. The LC10 gene is located ∼2 kb from that of LC2 and is also completely missing from this mutant but not from oda12-2, which lacks only the 3′ end of the LC2 gene. Although oda12-1 cells assemble outer arms that lack only LC2 and LC10, this strain exhibits a flagellar beat frequency that is consistently less than that observed for strains that fail to assemble the entire outer arm and docking complex (e.g., oda1). These results support a key regulatory role for the intermediate chain/light chain complex that is an integral and highly conserved feature of all oligomeric dynein motors. PMID:18579685

Coinfection with recombinant vaccinia viruses expressing poliovirus P1 and P3 proteins results in polyprotein processing and formation of empty capsid structures.

PubMed

Ansardi, D C; Porter, D C; Morrow, C D

1991-04-01

The assembly process of poliovirus occurs via an ordered proteolytic processing of the capsid precursor protein, P1, by the virus-encoded proteinase 3CD. To further delineate this process, we have isolated a recombinant vaccinia virus which expresses, upon infection, the poliovirus P1 capsid precursor polyprotein with an authentic carboxy terminus. Coinfection of HeLa cells with the P1-expressing vaccinia virus and with a second recombinant vaccinia virus which expresses the poliovirus proteinase 3CD resulted in the correct processing of P1 to yield the three individual capsid proteins VP0, VP3, and VP1. When extracts from coinfected cells were fractionated on sucrose density gradients, the VP0, VP3, and VP1 capsid proteins were immunoprecipitated with type 1 poliovirus antisera from fractions corresponding to a sedimentation consistent for poliovirus 75S procapsids. Examination of these fractions by electron microscopy revealed structures which lacked electron-dense cores and which corresponded in size and shape to those expected for poliovirus empty capsids. We conclude that the expression of the two poliovirus proteins P1 and 3CD in coinfected cells is sufficient for the correct processing of the capsid precursor to VP0, VP3, and VP1 as well as for the assembly of poliovirus empty capsid-like structures.

Compositional changes in lipid microdomains of air-blood barrier plasma membranes in pulmonary interstitial edema.

PubMed

Palestini, Paola; Calvi, Chiara; Conforti, Elena; Daffara, Rossella; Botto, Laura; Miserocchi, Giuseppe

2003-10-01

We evaluated in anesthetized rabbits the compositional changes of plasmalemmal lipid microdomains from lung tissue samples after inducing pulmonary interstitial edema (0.5 ml/kg for 3 h, leading to approximately 5% increase in extravascular water). Lipid microdomains (lipid rafts and caveolae) were present in the detergent-resistant fraction (DRF) obtained after discontinuous sucrose density gradient. DRF was enriched in caveolin-1, flotillin, aquaporin-1, GM1, cholesterol, sphingomyelin, and phosphatidylserine, and their contents significantly increased in interstitial edema. The higher DRF content in caveolin, flotillin, and aquaporin-1 and of the ganglioside GM1 suggests an increase both in caveolar domains and in lipid rafts, respectively. Compositional changes could be ascribed to endothelial and epithelial cells that provide most of plasma membrane surface area in the air-blood barrier. Alterations in lipid components in the plasma membrane may reflect rearrangement of floating lipid platforms within the membrane and/or lipid translocation from intracellular stores. Lipid traffic could be stimulated by the marked increase in hydraulic interstitial pressure after initial water accumulation, from approximately -10 to 5 cmH2O, due to the low compliance of the pulmonary tissue, in particular in the basement membranes and in the interfibrillar substance. Compositional changes in lipid microdomains represent a sign of cellular activation and suggest the potential role of mechanotransduction in response to developing interstitial edema.

Postnatal ocular expression of tyrosinase and related proteins: disruption by the pink-eyed unstable (p(un)) mutation.

PubMed

Chiu, E; Lamoreux, M L; Orlow, S J

1993-09-01

Ocular pigmentation in the mouse occurs primarily postnatally as a result of the melanization of neural crest-derived melanocytes. Using immunologic and biochemical techniques, we demonstrate that in normal mice the expression of tyrosinase and the related proteins TRP-1 and TRP-2, rises during the first week of life, remains elevated for a week, and then steadily declines to low levels by adulthood. Sucrose gradient density centrifugation demonstrates that tyrosinase, TRP-1 and TRP-2 are present in high molecular weight forms in the eyes of wild-type mice. The normal time course is disrupted in mice carrying the pink-eyed unstable (p(un)) mutation at the P-locus, a model for tyrosinase-positive albinism in man. Tyrosinase and TRP-2 are present at wild-type levels in the eyes of p(un)/p(un) mice at birth, but, rather than rising, their levels rapidly decline over the first week of life. TRP-1 is almost undetectable, even at birth. High molecular weight complexes could not be detected in eyes of p(un)/p(un) mice. Our results suggest that postnatal ocular melanogenesis in the mouse presents an attractive model for the study of the orderly expression and action of the proteins involved in eumelanin synthesis, and that the p(un) mutation disrupts this temporally controlled process.

Antibody class capture assay (ACCA) for rubella-specific IgM antibody.

PubMed

Isaac, M; Payne, R A

1982-01-01

Enzyme-linked immunosorbent assays for IgM antirubella were carried out on 1,546 sera, using an IgM capture method with a F (ab')2 conjugate (ACCA). Under the conditions described, sera containing IgM antirubella bound up to 15 times as much enzyme activity as negative specimens. Paired serum specimens from 27 patients, serial serum specimens from 6 patients, and single serum specimens from 15 patients who had had recent rubella were examined by the haemagglutination inhibition test (HAI) in the presence and absence of 2-mercaptoethanol following sucrose density gradient centrifugation (SDGC). ACCA confirmed all the results found with HAI following SDGC. Specimens were examined from ten patients with congenital rubella; ACCA confirmed the results found with both immunofluorescence following SDGC and radioimmunoassay. Pre- and post-vaccination specimens from 123 patients who had been vaccinated against rubella were examined. An IgM response could only be demonstrated in the 57 cases when IgG was absent in the first specimen. The specificity of the assay was confirmed by testing 31 serum specimens from rubella immune patients that also contained rheumatoid factor, 163 serum specimens from patients with acute infections other than rubella, and 12 serum specimens from infants with miscellaneous neonatal abnormalities other than congenital rubella. The ACCA proved a simple, sensitive, and specific test for IgM antirubella and the results compared favourably with those obtained by the SDGC technique.

On the derivation of empirical limits on the helium abundance in coronal holes below 1.5 solar radius

NASA Technical Reports Server (NTRS)

Habbal, Shadia Rifai; Esser, Ruth

1994-01-01

We present a simple technique describing how limits on the helium abundance, alpha, defined as the ratio of helium to proton number density, can be inferred from measurements of the electron density and temperature below 1.5 solar radius. As an illustration, we apply this technique to two different data sets: emission-line intensities in the extreme ultraviolet (EUV) and white-light observations, both measured in polar coronal holes. For the EUV data, the temperature gradient is derived from line intensity ratios, and the density gradient is replaced by the gradient of the line intensity. The lower limit on alpha derived from these data is 0.2-0.3 at 1 solar radius and drops very sharply to interplanetary values of a few percent below 1.06 solar radius. The white-light observations yield density gradients in the inner corona beyond 1.25 solar radius but do not have corresponding temperature gradients. In this case we consider an isothermal atmosphere, and derive an upper limit of 0.2 for alpha. These examples are used to illustrate how this technique could be applicable to the more extensive data to be obtained with the upcoming SOHO mission. Although only ranges on alpha can be derived, the application of the technique to data currently available merely points to the fact that alpha can be significantly large in the inner corona.

Purification of Piscirickettsia salmonis and partial characterisation of antigens

USGS Publications Warehouse

Barnes, M.N.; Landolt, M.L.; Powell, D.B.; Winton, J.R.

1998-01-01

Piscirickettsia salmonis is the etiological agent of salmonid rickettsial septicemia, an economically significant disease affecting the salmon aquaculture industry. As with other rickettsial pathogens, antigenic analysis of P. salmonis has been limited by the inherent difficulties of purifying an intracellular organism away from host cell material. In this report, we describe the use of diatrizoate meglumine and diatrizoate sodium (DMDS) density gradient centrifugation to purify P. salmonis grown in chinook salmon embryo (CHSE-214) cells. Plaque assay titers and total protein assays confirmed that viable P. salmonis was consistently concentrated in a visible band within the DMDS density gradient at a density of 1.15 to 1.16 g ml-1. Recovery of purified, viable organisms from DMDS density gradients varied from 0.6 to 3%. Preparations of uninfected CHSE-214 cells, CHSE-214 cells infected with P. salmonis, and gradient-purified P. salmonis were compared using sodium dodecyl sulfate polyacrylamide gel electrophoresis to assess the degree of purification and to identify P. salmonis-specific proteins. Although gradient-purified P. salmonis preparations were not completely free of host cell material, 8 bacterial proteins were identified. Polyclonal rabbit antiserum was used in an immunoblot of proteins from purified P. salmonis to identify 3 major and 5 minor antigens. The major antigens of 56, 30 and 20 kDa were potential candidates for experimental vaccines and development of novel diagnostic assays.

The influence of double-diffusive processes on the melting of ice in the Arctic Ocean: laboratory analogue experiments and their interpretation

NASA Astrophysics Data System (ADS)

Turner, J. S.; Veronis, G.

2004-03-01

This study has been motivated by two oceanographic observations: an increased rate of melting of sea ice in the Arctic Ocean, and the advance of an anomalously warm tongue of Atlantic water across the Arctic below the halocline over the last few decades. A series of laboratory experiments has been carried out in order to explore the physical principles underlying these phenomena, and the possibility that the extra heating at depth is responsible for the enhanced melting rate. A tank was filled with salt solution having various constant vertical density gradients. A block of ice one third of the length of the tank was floated on the surface at one end, and the rest of the surface and the walls of the tank were insulated. When no extra heat was supplied the melting rate (loss of weight of the ice in 1 h) systematically decreased as the stratification was changed from homogeneous fluid to increasingly large density gradients, while keeping the salinity of the solution in contact with the ice constant. An analogue of the intruding Atlantic water was produced by heating the lower portion of the vertical end wall at the end of the tank opposite to the ice end, keeping its temperature constant, and using the same range of salinity gradients as in the unheated experiments. Again the melting rate decreased as the density gradient was increased, but for low gradients it was larger than that in the unheated experiments. Above a certain intermediate gradient there was no significant difference in melting rate between the unheated and heated runs. The melting data were supplemented by photographs and vertical temperature and salinity profiles. The upward transfer of heat from the body of the fluid to melt the ice was clearly double-diffusive: overturning layers, separated by 'diffusive' interfaces, were visible on shadowgraphs, and the thickness of the layers decreased as the density gradient increased. The mean thickness of the layers through the depth of the tank also systematically decreased as the density gradient increased. With weak gradients an extra heat flux to the ice came from the intruding heated layer, but at large gradients this tongue of warm water at depth did not add to the flux near the surface. Though they were obtained in a simple, arbitrary and fixed geometry, we believe that the results of these experiments can be used as the basis for a better physical understanding of the melting rates of ice in the Arctic under various conditions.

Avifaunal responses to fire in southwestern montane forests along a burn severity gradient

USGS Publications Warehouse

Kotliar, N.B.; Kennedy, P.L.; Ferree, K.

2007-01-01

The effects of burn severity on avian communities are poorly understood, yet this information is crucial to fire management programs. To quantify avian response patterns along a burn severity gradient, we sampled 49 random plots (2001-2002) at the 17 351-ha Cerro Grande Fire (2000) in New Mexico, USA. Additionally, pre-fire avian surveys (1986-1988, 1990) created a unique opportunity to quantify avifaunal changes in 13 pre-fire transects (resampled in 2002) and to compare two designs for analyzing the effects of unplanned disturbances: after-only analysis and before-after comparisons. Distance analysis was used to calculate densities. We analyzed after-only densities for 21 species using gradient analysis, which detected a broad range of responses to increasing burn severity: (I) large significant declines, (II) weak, but significant declines, (III) no significant density changes, (IV) peak densities in low- or moderate-severity patches, (V) weak, but significant increases, and (VI) large significant increases. Overall, 71% of the species included in the after-only gradient analysis exhibited either positive or neutral density responses to fire effects across all or portions of the severity gradient (responses III-VI). We used pre/post pairs analysis to quantify density changes for 15 species using before-after comparisons; spatiotemporal variation in densities was large and confounded fire effects for most species. Only four species demonstrated significant effects of burn severity, and their densities were all higher in burned compared to unburned forests. Pre- and post-fire community similarity was high except in high-severity areas. Species richness was similar pre- and post-fire across all burn severities. Thus, ecosystem restoration programs based on the assumption that recent severe fires in Southwestern ponderosa pine forests have overriding negative ecological effects are not supported by our study of post-fire avian communities. This study illustrates the importance of quantifying burn severity and controlling confounding sources of spatiotemporal variation in studies of fire effects. After-only gradient analysis can be an efficient tool for quantifying fire effects. This analysis can also augment historical data sets that have small samples sizes coupled with high non-process variation, which limits the power of before-after comparisons. ?? 2007 by the Ecological Society of America.

The effects of density gradients on the convective amplification of upper hybrid waves in the magnetosphere

NASA Technical Reports Server (NTRS)

Engel, J.; Kennel, C.

1985-01-01

Intense (at least 10 mV/m) electrostatic plasma waves have been detected near the upper hybrid frequency between + or -50 deg MLAT during recent GEOS-1 crossings. Wave growth rate and convective amplification calculations were carried out in order to explain the occurrence of intense upper hybrid (IUH) events over such a wide range of latitudes. The effects of wave refractions were taken into account in the convective amplification calculations. Specific results are presented for the upper hybrid wave growth of an IUH event occurring at 10 deg MLAT. It is shown that a density gradient may be necessary to explain the observed amplification at 10 deg MLAT. At the equator, however, the long scale length of the magnetic field gradient enables large amplitudes to be attained without a density gradient. The results of a UH ray tracing analysis are discussed within the framework of current theories concerning magnetospheric continuum radiation.

Kuroshio Graduate Student Support

DTIC Science & Technology

2018-06-06

875 North Randolph Street Arlington, VA 22203-1995 11. SPONSOR/MONITOR’S REPORT NUMBER(S) 12. DISTRIBUTION / AVAILABILITY ...strong horizontal density gradients (e.g., midlatitude subtropical gyres), with a small fraction occurring in regions of deep mixed layers (e.g., high ...Society homogenous in the presence of sharp horizontal density contrasts (e.g., Fig. 1a). These sharp gradients provide a source of available

Pulsed field gradients in simulations of one- and two-dimensional NMR spectra.

PubMed

Meresi, G H; Cuperlovic, M; Palke, W E; Gerig, J T

1999-03-01

A method for the inclusion of the effects of z-axis pulsed field gradients in computer simulations of an arbitrary pulsed NMR experiment with spin (1/2) nuclei is described. Recognizing that the phase acquired by a coherence following the application of a z-axis pulsed field gradient bears a fixed relation to its order and the spatial position of the spins in the sample tube, the sample is regarded as a collection of volume elements, each phase-encoded by a characteristic, spatially dependent precession frequency. The evolution of the sample's density matrix is thus obtained by computing the evolution of the density matrix for each volume element. Following the last gradient pulse, these density matrices are combined to form a composite density matrix which evolves through the rest of the experiment to yield the observable signal. This approach is implemented in a program which includes capabilities for rigorous inclusion of spin relaxation by dipole-dipole, chemical shift anisotropy, and random field mechanisms, plus the effects of arbitrary RF fields. Mathematical procedures for accelerating these calculations are described. The approach is illustrated by simulations of representative one- and two-dimensional NMR experiments. Copyright 1999 Academic Press.

SDSS-IV MaNGA: environmental dependence of stellar age and metallicity gradients in nearby galaxies

NASA Astrophysics Data System (ADS)

Zheng, Zheng; Wang, Huiyuan; Ge, Junqiang; Mao, Shude; Li, Cheng; Li, Ran; Mo, Houjun; Goddard, Daniel; Bundy, Kevin; Li, Hongyu; Nair, Preethi; Lin, Lihwai; Long, R. J.; Riffel, Rogério; Thomas, Daniel; Masters, Karen; Bizyaev, Dmitry; Brownstein, Joel R.; Zhang, Kai; Law, David R.; Drory, Niv; Roman Lopes, Alexandre; Malanushenko, Olena

2017-03-01

We present a study on the stellar age and metallicity distributions for 1105 galaxies using the STARLIGHT software on MaNGA (Mapping Nearby Galaxies at APO) integral field spectra. We derive age and metallicity gradients by fitting straight lines to the radial profiles, and explore their correlations with total stellar mass M*, NUV - r colour and environments, as identified by both the large-scale structure (LSS) type and the local density. We find that the mean age and metallicity gradients are close to zero but slightly negative, which is consistent with the inside-out formation scenario. Within our sample, we find that both the age and metallicity gradients show weak or no correlation with either the LSS type or local density environment. In addition, we also study the environmental dependence of age and metallicity values at the effective radii. The age and metallicity values are highly correlated with M* and NUV - r and are also dependent on LSS type as well as local density. Low-mass galaxies tend to be younger and have lower metallicity in low-density environments while high-mass galaxies are less affected by environment.

Protamine ratio and the level of histone retention in sperm selected from a density gradient preparation.

PubMed

Hammoud, S; Liu, L; Carrell, D T

2009-04-01

Fertile males express two forms of sperm nuclear proteins, protamine 1 (P1) and protamine 2 (P2), in roughly equal quantities, whereas some infertile men have been shown to have a reduction in protamine content and an increase in the level of histones retained in mature sperm. In this study, we assessed histone and protamine levels in spermatozoa isolated from different layers of a density gradient centrifugation column to evaluate the nuclear protein content of the sperm population selected. Protamine levels were measured using acid gel electrophoresis and immunofluorescence, and the percentage of cells retaining histones was evaluated using aniline staining and immunofluorescence. Our data suggests that there is an inverse correlation between P1/P2 ratio and the level of histone expression in the different layers of the density gradient. Paradoxically, the 90% layer had a lower P1/P2 ratio, which corresponded with an increase in histone expression. It is concluded that although the sperm population selected in the 90% layer of the density gradient columns had a lower P1/P2 ratio, it was yet similar to the P1/P2 ratio observed in previously screened fertile donors.

Elimination of sucrose transport and hydrolysis in Saccharomyces cerevisiae: a platform strain for engineering sucrose metabolism

PubMed Central

Marques, Wesley Leoricy; Mans, Robert; Marella, Eko Roy; Cordeiro, Rosa Lorizolla; van den Broek, Marcel; Daran, Jean-Marc G.; Pronk, Jack T.; Gombert, Andreas K.; van Maris, Antonius J.A.

2017-01-01

Abstract Many relevant options to improve efficacy and kinetics of sucrose metabolism in Saccharomyces cerevisiae and, thereby, the economics of sucrose-based processes remain to be investigated. An essential first step is to identify all native sucrose-hydrolysing enzymes and sucrose transporters in this yeast, including those that can be activated by suppressor mutations in sucrose-negative strains. A strain in which all known sucrose-transporter genes (MAL11, MAL21, MAL31, MPH2, MPH3) were deleted did not grow on sucrose after 2 months of incubation. In contrast, a strain with deletions in genes encoding sucrose-hydrolysing enzymes (SUC2, MAL12, MAL22, MAL32) still grew on sucrose. Its specific growth rate increased from 0.08 to 0.25 h−1 after sequential batch cultivation. This increase was accompanied by a 3-fold increase of in vitro sucrose-hydrolysis and isomaltase activities, as well as by a 3- to 5-fold upregulation of the isomaltase-encoding genes IMA1 and IMA5. One-step Cas9-mediated deletion of all isomaltase-encoding genes (IMA1-5) completely abolished sucrose hydrolysis. Even after 2 months of incubation, the resulting strain did not grow on sucrose. This sucrose-negative strain can be used as a platform to test metabolic engineering strategies and for fundamental studies into sucrose hydrolysis or transport. PMID:28087672

Diffusion of Conserved Charges in Relativistic Heavy Ion Collisions

NASA Astrophysics Data System (ADS)

Greif, Moritz; Fotakis, Jan. A.; Denicol, Gabriel S.; Greiner, Carsten

2018-06-01

We demonstrate that the diffusion currents do not depend only on gradients of their corresponding charge density, but that the different diffusion charge currents are coupled. This happens in such a way that it is possible for density gradients of a given charge to generate dissipative currents of another charge. Within this scheme, the charge diffusion coefficient is best viewed as a matrix, in which the diagonal terms correspond to the usual charge diffusion coefficients, while the off-diagonal terms describe the coupling between the different currents. In this Letter, we calculate for the first time the complete diffusion matrix for hot and dense nuclear matter, including baryon, electric, and strangeness charges. We find that the baryon diffusion current is strongly affected by baryon charge gradients but also by its coupling to gradients in strangeness. The electric charge diffusion current is found to be strongly affected by electric and strangeness gradients, whereas strangeness currents depend mostly on strange and baryon gradients.

Preparation of an Arg-Glu-Asp-Val Peptide Density Gradient on Hyaluronic Acid-Coated Poly(ε-caprolactone) Film and Its Influence on the Selective Adhesion and Directional Migration of Endothelial Cells.

PubMed

Yu, Shan; Gao, Ying; Mei, Xu; Ren, Tanchen; Liang, Su; Mao, Zhengwei; Gao, Changyou

2016-11-02

Selective adhesion and migration of endothelial cells (ECs) over smooth muscle cells (SMCs) is very important in the rapid endothelialization of blood-contacting implants to prevent vascular restenosis. In this study, a uniform cell-resistant layer of methacrylate-functionalized hyaluronic acid (HA) was first immobilized on a poly(ε-caprolactone) (PCL) film via polydopamine coupling. Then, a density gradient of thiol-functionalized Arg-Glu-Asp-Val (REDV) peptide was prepared on the HA layer via thiol-ene click chemistry and the continuous injection method. The REDV gradient selectively enhanced EC adhesion and preferential directional migration toward the region of higher REDV density, reaching 86% directionality in the middle of the gradient. The migration rate of ECs was also significantly enhanced twofold compared with that on tissue culture polystyrene (TCPS). In contrast, the gradient significantly weakened the adhesion of SMCs to 25% of that on TCPS but had no obvious impact on the migration rate and directionality. Successful modulation of the selective adhesion and directional migration of ECs over SMCs on biodegradable polymers serves as an important step toward practical applications for guided tissue regeneration.

Integrating Reverse-Electrodialysis Stacks with Flow Batteries for Improved Energy Recovery from Salinity Gradients and Energy Storage.

PubMed

Zhu, Xiuping; Kim, Taeyoung; Rahimi, Mohammad; Gorski, Christopher A; Logan, Bruce E

2017-02-22

Salinity gradient energy can be directly converted into electrical power by using reverse electrodialysis (RED) and other technologies, but reported power densities have been too low for practical applications. Herein, the RED stack performance was improved by using 2,6-dihydroxyanthraquinone and ferrocyanide as redox couples. These electrolytes were then used in a flow battery to produce an integrated RED stack and flow battery (RED-FB) system capable of capturing, storing, and discharging salinity gradient energy. Energy captured from the RED stack was discharged in the flow battery at a maximum power density of 3.0 kW m -2 -anode, which was similar to the flow batteries charged by electrical power and could be used for practical applications. Salinity gradient energy captured from the RED stack was recovered from the electrolytes as electricity with 30 % efficiency, and the maximum energy density of the system was 2.4 kWh m -3 -anolyte. The combined RED-FB system overcomes many limitations of previous approaches to capture, store, and use salinity gradient energy from natural or engineered sources. © 2017 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.

Implementation and application of a gradient enhanced crystal plasticity model

NASA Astrophysics Data System (ADS)

Soyarslan, C.; Perdahcıoǧlu, E. S.; Aşık, E. E.; van den Boogaard, A. H.; Bargmann, S.

2017-10-01

A rate-independent crystal plasticity model is implemented in which description of the hardening of the material is given as a function of the total dislocation density. The evolution of statistically stored dislocations (SSDs) is described using a saturating type evolution law. The evolution of geometrically necessary dislocations (GNDs) on the other hand is described using the gradient of the plastic strain tensor in a non-local manner. The gradient of the incremental plastic strain tensor is computed explicitly during an implicit FE simulation after each converged step. Using the plastic strain tensor stored as state variables at each integration point and an efficient numerical algorithm to find the gradients, the GND density is obtained. This results in a weak coupling of the equilibrium solution and the gradient enhancement. The algorithm is applied to an academic test problem which considers growth of a cylindrical void in a single crystal matrix.

Growth and Survival of Some Probiotic Strains in Simulated Ice Cream Conditions

NASA Astrophysics Data System (ADS)

Homayouni, A.; Ehsani, M. R.; Azizi, A.; Razavi, S. H.; Yarmand, M. S.

A Completely Randomized Design (CRD) experiment was applied in triplicates to evaluate the survival of four probiotic strains in simulated ice cream conditions. The growth and survival rate of these probiotic strains (Lactobacillus acidophilus, Lactobacillus casei, Bifidobacterium bifidum and Bifidobacterium longum) in varying amount of sucrose (10, 15, 20 and 25%), oxygen scavenging components (0.05% L-cysteine and 0.05% L-ascorbate) and temperatures (4 and -20°C) during different periods of time (1, 2 and 3 months) were evaluated in MRS-broth medium. Optical density at 580 nm was used to measure growth. Lactobacilli strains proved to be highly resistant in comparison with Biffidobacteria strains. The viable cell number of Lactobacillus casei in different sucrose concentrations, different oxidoreduction potentials and refrigeration temperature was 1x1010, 2x108 and 5x107 cfu mL-1, respectively. Growth and survival rate of Lactobacillus casei showed to be the highest.

Proliferation and glucosinolates accumulation of broccoli adventitious roots in liquid medium

NASA Astrophysics Data System (ADS)

Nhut, Nguyen Minh; Tien, Le Thi Thuy

2017-09-01

Cotyledons from 7-day-old in vitro broccoli seedling were used as explant source in adventitious root induction on MS medium supplemented with 30 g/l sucrose, 1.6 mg/l IBA and 7 g/l agar. Adventitious roots from cotyledons were transferred to liquid medium containing the same components as rooting medium for two weeks, then subcultured to MS medium with diferent sugar, macrominerals and casein hydrolysate concentrations. The best adventitious root growth was observed in half-strength MS medium supplemented with 40 g/l sucrose, 600 mg/l casein hydrolysate and 1.6 mg/l IBA (growth index of 4.00 in about 14 culture days with inoculum density of 1.0 g fresh weight / 30 ml of culture medium). The culturing process can be stopped on the 28th day for root biomass and on the 35th day for glucosinolates.

Cellulose Deficiency Is Enhanced on Hyper Accumulation of Sucrose by a H+-Coupled Sucrose Symporter1[OPEN

PubMed Central

Yeats, Trevor H.; Sorek, Hagit

2016-01-01

In order to understand factors controlling the synthesis and deposition of cellulose, we have studied the Arabidopsis (Arabidopsis thaliana) double mutant shaven3 shaven3-like1 (shv3svl1), which was shown previously to exhibit a marked cellulose deficiency. We discovered that exogenous sucrose (Suc) in growth medium greatly enhances the reduction in hypocotyl elongation and cellulose content of shv3svl1. This effect was specific to Suc and was not observed with other sugars or osmoticum. Live-cell imaging of fluorescently labeled cellulose synthase complexes revealed a slowing of cellulose synthase complexes in shv3svl1 compared with the wild type that is enhanced in a Suc-conditional manner. Solid-state nuclear magnetic resonance confirmed a cellulose deficiency of shv3svl1 but indicated that cellulose crystallinity was unaffected in the mutant. A genetic suppressor screen identified mutants of the plasma membrane Suc/H+ symporter SUC1, indicating that the accumulation of Suc underlies the Suc-dependent enhancement of shv3svl1 phenotypes. While other cellulose-deficient mutants were not specifically sensitive to exogenous Suc, the feronia (fer) receptor kinase mutant partially phenocopied shv3svl1 and exhibited a similar Suc-conditional cellulose defect. We demonstrate that shv3svl1, like fer, exhibits a hyperpolarized plasma membrane H+ gradient that likely underlies the enhanced accumulation of Suc via Suc/H+ symporters. Enhanced intracellular Suc abundance appears to favor the partitioning of carbon to starch rather than cellulose in both mutants. We conclude that SHV3-like proteins may be involved in signaling during cell expansion that coordinates proton pumping and cellulose synthesis. PMID:27013021

Light and nitrogen nutrition regulate apical control in Rosa hybrida L.

PubMed

Furet, Pierre-Maxime; Lothier, Jérémy; Demotes-Mainard, Sabine; Travier, Sandrine; Henry, Clémence; Guérin, Vincent; Vian, Alain

2014-03-01

Apical control is defined as the inhibition of basal axillary bud outgrowth by an upper actively growing axillary axis, whose regulation is poorly understood yet differs markedly from the better-known apical dominance. We studied the regulation of apical control by environmental factors in decapitated Rosa hybrida in order to remove the apical hormonal influence and nutrient sink. In this plant model, all the buds along the main axis have a similar morphology and are able to burst in vitro. We concentrated on the involvement of light intensity and nitrate nutrition on bud break and axillary bud elongation in the primary axis pruned above the fifth leaf of each rose bush. We observed that apical control took place in low light (92 μmol m(-2)s(-1)), where only the 2-apical buds grew out, both in low (0.25 mM) and high (12.25 mM) nitrate. In contrast, in high light (453 μmol m(-2)s(-1)), the apical control only operates in low nitrate while all the buds along the stem grew out when the plant was supplied with a high level of nitrate. We found a decreasing photosynthetic activity from the top to the base of the plant concomitant with a light gradient along the stem. The quantity of sucrose, fructose, glucose and starch are higher in high light conditions in leaves and stem. The expression of the sucrose transporter RhSUC2 was higher in internodes and buds in this lighting condition, suggesting an increased capacity for sucrose transport. We propose that light intensity and nitrogen availability both contribute to the establishment of apical control. Copyright © 2013 Elsevier GmbH. All rights reserved.

Profile of the intestinal mucosal corticosteroid receptors in the domestic duck

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

DiBattista, J.A.; Mehdi, A.Z.; Sandor, T.

The corticosteroid receptor profile of the intestinal tract of the domestic duck (maintained on either a low-sodium (LS) or a high-sodium (HS) diet) was investigated. Using tritiated triamcinolone acetonide (TA), corticosterone, or aldosterone as ligands, cytoplasmic mineralocorticoid receptors (MR, type I) and glucocorticoid receptors (GR, type II) were found in the mucosal cytosol of the jejunum and colon. The diet little influenced the GR binding parameters, while the MR (aldosterone) binding parameters showed a down-regulation following LS diets. The competition hierarchy of radioinert steroids on the formation of the (TH)corticosterone-receptor complex was corticosterone = cortisol = 11-deoxycorticosterone greater than aldosteronemore » = TA = dexamethasone much greater than 11-deoxycortisol; with (TH)aldosterone, the competition was corticosterone = progesterone = 11-deoxycorticosterone greater than aldosterone = cortisol = TA = dexamethasone greater than 11-deoxycortisol greater than 11-dehydrocorticosterone. On linear sucrose gradients, receptor-ligand complexes sedimented with a single peak at 8.5 S (hypotonic gradient) and 4.0-4.5 S (hypertonic gradient), respectively. Heat-activated (TH)TA- and (TH)aldosterone-receptor complexes bound avidly to DNA-cellulose and, upon ion-exchange chromatography on DEAE-Sephacel, the presence of the negatively charged unactivated and the more positively charged activated complexes could be shown.« less

Plant species invasions along the latitudinal gradient in the United States

USGS Publications Warehouse

Stohlgren, T.J.; Barnett, D.; Flather, C.; Kartesz, J.; Peterjohn, B.

2005-01-01

It has been long established that the richness of vascular plant species and many animal taxa decreases with increasing latitude, a pattern that very generally follows declines in actual and potential evapotranspiration, solar radiation, temperature, and thus, total productivity. Using county-level data on vascular plants from the United States (3000 counties in the conterminous 48 states), we used the Akaike Information Criterion (AIC) to evaluate competing models predicting native and nonnative plant species density (number of species per square kilometer in a county) from various combinations of biotic variables (e.g., native bird species density, vegetation carbon, normalized difference vegetation index), environmental/topographic variables (elevation, variation in elevation, the number of land cover classes in the county; radiation, mean precipitation, actual evapotranspiration, and potential evapotranspiration), and human variables (human population density, crop-land, and percentage of disturbed lands in a county). We found no evidence of a latitudinal gradient for the density of native plant species and a significant, slightly positive latitudinal gradient for the density of nonnative plant species. We found stronger evidence of a significant, positive productivity gradient (vegetation carbon) for the density of native plant species and nonnative plant species. We found much stronger significant relationships when biotic, environmental/topographic, and human variables were used to predict native plant species density and nonnative plant species density. Biotic variables generally had far greater influence in multivariate models than human or environmental/topographic variables. Later, we found that the best, single, positive predictor of the density of nonnative plant species in a county was the density of native plant species in a county. While further study is needed, it may be that, while humans facilitate the initial establishment invasions of nonnative plant species, the spread and subsequent distributions of nonnative species are controlled largely by biotic and environmental factors.

Nitric oxide inhibits the production of soluble endothelin converting enzyme-1.

PubMed

Kuruppu, Sanjaya; Rajapakse, Niwanthi W; Dunstan, Rhys A; Smith, A Ian

2014-11-01

This study examined the effect of nitric oxide on the production of soluble ECE-1. Activity of ECE-1 in media was measured using a quenched fluorescent substrate assay, and expressed as a percentage of control. Endothelial cells were incubated with the nitric oxide donor Diethylenetriamine NONOate (DETA; 250-800 µM), NOS substrate L-Arg (200-1,000 µM), a L-Arg transport inhibitor (L-Lys; 10 µM) and NOS inhibitors (L-Gln and N5-[imino(nitroamino)methyl]-L-ornithine, methyl ester, monohydrochloride (L-NAME); 10-100 µM). The effect of L-Arg (1,000 µM) was also tested in the presence of L-Lys (10 µM), L-Gln (100 µM) and L-NAME (10-100 µM). Ultracentrifugation (100,000×g, 4 °C, 1 h) completely removed ECE-1 activity from the supernatant. In addition, fractionation of concentrated media on a sucrose density gradient indicated that ECE-1 activity was localised to the mid portion of the gradient, thus suggesting the possible role of exosomes in ECE-1 release. Production of soluble ECE-1 by Ea.hy926 cells was inhibited significantly (P < 0.05, unpaired t test, n = 4) in the presence of DETA (75.31 ± 3.59; 800 µM) and L-Arg (60.97 ± 9.22; 1,000 µM). L-Arg-mediated reduction in the release of soluble ECE-1 was blocked by the inhibition of NOS using L-NAME (100 µM; 99.19 ± 0.58) and L-Gln (100 µM; 104.41 ± 0.65). In addition, the presence of L-Lys (10 µM) significantly blocked the L-Arg (1,000 µM)-induced reduction in soluble ECE-1 levels (122.38 ± 13.16). These treatments had no effect on the expression of ECE-1 on the cell surface. Our data provide evidence that NO can inhibit the production of soluble ECE-1 by endothelial cells.

Incubation of sucrose craving: effects of reduced training and sucrose pre-loading

PubMed Central

Grimm, Jeffrey W.; Fyall, Amber M.; Osincup, Dan P.

2010-01-01

Time-dependent increases in cue-induced reward seeking after forced abstinence were described in rats with a history of cocaine or sucrose self-administration, suggesting reward craving incubates over time. In the present study, we examined the effects of reduced training experience, or sucrose pre-loading just prior to testing, on the incubation of sucrose craving. Sucrose seeking (responding in extinction and then for a sucrose-paired cue) increased over time in groups of rats that self-administered sucrose 6 h/day for 10 days and were tested at 1, 7, or 30 days of forced abstinence. We found that groups of rats that had self-administered 2 instead of 6 h/day showed a similar profile of responding. Incubation of sucrose craving was attenuated by free access to sucrose in home cages for 17 h immediately prior to testing assessed as extinction responding on days 1 and 30. However, this sucrose pre-loading had no effect on the time-dependent increase in responding for the sucrose-paired cue. In summary, reducing the training experience had no effect on the incubation of sucrose craving and free access to sucrose had only a limited effect–attenuating extinction responding. These results illustrate the strength of the incubation of craving and further suggest long-term changes in brain motivational circuitry following sucrose self-administration. PMID:15642609

Thermospheric neutral density estimates from heater-induced ion up-flow at EISCAT

NASA Astrophysics Data System (ADS)

Kosch, Michael; Ogawa, Yasunobu; Yamazaki, Yosuke; Vickers, Hannah; Blagoveshchenskaya, Nataly

We exploit a recently-developed technique to estimate the upper thermospheric neutral density using measurements of ionospheric plasma parameters made by the EISCAT UHF radar during ionospheric modification experiments. Heating the electrons changes the balance between upward plasma pressure gradient and downward gravity, resulting in ion up-flow up to ~200 m/s. This field-aligned flow is retarded by collisions, which is directly related to the neutral density. Whilst the ion up-flow is consistent with the plasma pressure gradient, the estimated thermospheric neutral density depends on the assumed composition, which varies with altitude. Results in the topside ionosphere are presented.

Derivation of general analytic gradient expressions for density-fitted post-Hartree-Fock methods: An efficient implementation for the density-fitted second-order Møller–Plesset perturbation theory

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

Bozkaya, Uğur, E-mail: ugur.bozkaya@atauni.edu.tr

General analytic gradient expressions (with the frozen-core approximation) are presented for density-fitted post-HF methods. An efficient implementation of frozen-core analytic gradients for the second-order Møller–Plesset perturbation theory (MP2) with the density-fitting (DF) approximation (applying to both reference and correlation energies), which is denoted as DF-MP2, is reported. The DF-MP2 method is applied to a set of alkanes, conjugated dienes, and noncovalent interaction complexes to compare the computational cost of single point analytic gradients with MP2 with the resolution of the identity approach (RI-MP2) [F. Weigend and M. Häser, Theor. Chem. Acc. 97, 331 (1997); R. A. Distasio, R. P. Steele,more » Y. M. Rhee, Y. Shao, and M. Head-Gordon, J. Comput. Chem. 28, 839 (2007)]. In the RI-MP2 method, the DF approach is used only for the correlation energy. Our results demonstrate that the DF-MP2 method substantially accelerate the RI-MP2 method for analytic gradient computations due to the reduced input/output (I/O) time. Because in the DF-MP2 method the DF approach is used for both reference and correlation energies, the storage of 4-index electron repulsion integrals (ERIs) are avoided, 3-index ERI tensors are employed instead. Further, as in case of integrals, our gradient equation is completely avoid construction or storage of the 4-index two-particle density matrix (TPDM), instead we use 2- and 3-index TPDMs. Hence, the I/O bottleneck of a gradient computation is significantly overcome. Therefore, the cost of the generalized-Fock matrix (GFM), TPDM, solution of Z-vector equations, the back transformation of TPDM, and integral derivatives are substantially reduced when the DF approach is used for the entire energy expression. Further application results show that the DF approach introduce negligible errors for closed-shell reaction energies and equilibrium bond lengths.« less

Copper-induced alteration in sucrose partitioning and its relationship to the root growth of two Elsholtzia haichowensis Sun populations.

PubMed

Li, Min-Jing; Xiong, Zhi-Ting; Liu, Hui; Kuo, Yi-Ming; Tong, Lei

2016-10-02

Hydroponic culture was used to comparatively investigate the copper (Cu)-induced alteration to sucrose metabolism and biomass allocation in two Elsholtzia haichowensis Sun populations with one from a Cu-contaminated site (CS) and the other from a non-contaminated site (NCS). Experimental results revealed that biomass allocation preferred roots over shoots in CS population, and shoots over roots in NCS population under Cu exposure. The difference in biomass allocation was correlated with the difference in sucrose partitioning between the two populations. Cu treatment (45 μM) significantly decreased leaf sucrose content and increased root sucrose content in CS population as a result of the increased activities of leaf sucrose synthesis enzymes (sucrose phosphate synthetase and sucrose synthase) and root sucrose cleavage enzyme (vacuolar invertase), which led to increased sucrose transport from leaves to roots. In contrast, higher Cu treatment increased sucrose content in leaves and decreased sucrose content in roots in NCS population as a result of the decreased activities of root sucrose cleavage enzymes (vacuolar and cell wall invertases) that led to less sucrose transport from leaves to roots. These results provide important insights into carbon resource partitioning and biomass allocation strategies in metallophytes and are beneficial for the implementation of phytoremediation techniques.

Reinforcement value and substitutability of sucrose and wheel running: implications for activity anorexia.

PubMed

Belke, Terry W; Pierce, W David; Duncan, Ian D

2006-09-01

Choice between sucrose and wheel-running reinforcement was assessed in two experiments. In the first experiment, ten male Wistar rats were exposed to concurrent VI 30 s VI 30 s schedules of wheel-running and sucrose reinforcement. Sucrose concentration varied across concentrations of 2.5, 7.5, and 12.5%. As concentration increased, more behavior was allocated to sucrose and more reinforcements were obtained from that alternative. Allocation of behavior to wheel running decreased, but obtained wheel-running reinforcement did not change. Overall, the results suggested that food-deprived rats were sensitive to qualitative changes in food supply (sucrose concentration) while continuing to defend a level of physical activity (wheel running). In the second study, 15 female Long Evans rats were exposed to concurrent variable ratio schedules of sucrose and wheel-running, wheel-running and wheel-running, and sucrose and sucrose reinforcement. For each pair of reinforcers, substitutability was assessed by the effect of income-compensated price changes on consumption of the two reinforcers. Results showed that, as expected, sucrose substituted for sucrose and wheel running substituted for wheel running. Wheel running, however, did not substitute for sucrose; but sucrose partially substituted for wheel running. We address the implications of the interrelationships of sucrose and wheel running for an understanding of activity anorexia.

Reinforcement Value and Substitutability of Sucrose and Wheel Running: Implications for Activity Anorexia

PubMed Central

Belke, Terry W; Duncan, Ian D; David Pierce, W

2006-01-01

Choice between sucrose and wheel-running reinforcement was assessed in two experiments. In the first experiment, ten male Wistar rats were exposed to concurrent VI 30 s VI 30 s schedules of wheel-running and sucrose reinforcement. Sucrose concentration varied across concentrations of 2.5, 7.5, and 12.5%. As concentration increased, more behavior was allocated to sucrose and more reinforcements were obtained from that alternative. Allocation of behavior to wheel running decreased, but obtained wheel-running reinforcement did not change. Overall, the results suggested that food-deprived rats were sensitive to qualitative changes in food supply (sucrose concentration) while continuing to defend a level of physical activity (wheel running). In the second study, 15 female Long Evans rats were exposed to concurrent variable ratio schedules of sucrose and wheel-running, wheel-running and wheel-running, and sucrose and sucrose reinforcement. For each pair of reinforcers, substitutability was assessed by the effect of income-compensated price changes on consumption of the two reinforcers. Results showed that, as expected, sucrose substituted for sucrose and wheel running substituted for wheel running. Wheel running, however, did not substitute for sucrose; but sucrose partially substituted for wheel running. We address the implications of the interrelationships of sucrose and wheel running for an understanding of activity anorexia. PMID:17002224

Cs2SO4 Gradients Containing Both Hg2+ and Ag+ Effect the Complete Separation of Satellite Deoxyribonucleic Acids Having Identical Densities in Neutral CsCl Gradients

PubMed Central

Skinner, Dorothy M.; Beattie, Wanda G.

1973-01-01

A combination of both Ag+ and Hg2+ in Cs2SO4 effects the complete separation of two DNAs having identical densities in CsCl. Satellite DNAs of hermit crab, Pagurus pollicaris, and lobster, Homarus americanus, have been isolated by this means. PMID:4522292

High frequency drift instabilities in a dusty plasma

NASA Technical Reports Server (NTRS)

Rosenberg, M.; Krall, N. A.

1994-01-01

High frequency drift instabilities with omega(sub ce) much greater than omega which is greater than omega(sub ci) are investigated in a dusty magnetized plasma in which locally there is an electron density gradient which is opposite in sign to a dust density gradient. Two different equilibria are considered, characterized by rho(sub d) greater than L(sub d) and less than L(sub d), where rho(sub d) is the dust gyroradius and L(sub nd) is the dust density scale length. Possible application to Saturn's F-ring is discussed.

Recent developments in LIBXC - A comprehensive library of functionals for density functional theory

NASA Astrophysics Data System (ADS)

Lehtola, Susi; Steigemann, Conrad; Oliveira, Micael J. T.; Marques, Miguel A. L.

2018-01-01

LIBXC is a library of exchange-correlation functionals for density-functional theory. We are concerned with semi-local functionals (or the semi-local part of hybrid functionals), namely local-density approximations, generalized-gradient approximations, and meta-generalized-gradient approximations. Currently we include around 400 functionals for the exchange, correlation, and the kinetic energy, spanning more than 50 years of research. Moreover, LIBXC is by now used by more than 20 codes, not only from the atomic, molecular, and solid-state physics, but also from the quantum chemistry communities.

3D joint inversion of gravity-gradient and borehole gravity data

NASA Astrophysics Data System (ADS)

Geng, Meixia; Yang, Qingjie; Huang, Danian

2017-12-01

Borehole gravity is increasingly used in mineral exploration due to the advent of slim-hole gravimeters. Given the full-tensor gradiometry data available nowadays, joint inversion of surface and borehole data is a logical next step. Here, we base our inversions on cokriging, which is a geostatistical method of estimation where the error variance is minimised by applying cross-correlation between several variables. In this study, the density estimates are derived using gravity-gradient data, borehole gravity and known densities along the borehole as a secondary variable and the density as the primary variable. Cokriging is non-iterative and therefore is computationally efficient. In addition, cokriging inversion provides estimates of the error variance for each model, which allows direct assessment of the inverse model. Examples are shown involving data from a single borehole, from multiple boreholes, and combinations of borehole gravity and gravity-gradient data. The results clearly show that the depth resolution of gravity-gradient inversion can be improved significantly by including borehole data in addition to gravity-gradient data. However, the resolution of borehole data falls off rapidly as the distance between the borehole and the feature of interest increases. In the case where the borehole is far away from the target of interest, the inverted result can be improved by incorporating gravity-gradient data, especially all five independent components for inversion.

Characterization of Sugar Contents and Sucrose Metabolizing Enzymes in Developing Leaves of Hevea brasiliensis

PubMed Central

Zhu, Jinheng; Qi, Jiyan; Fang, Yongjun; Xiao, Xiaohu; Li, Jiuhui; Lan, Jixian; Tang, Chaorong

2018-01-01

Sucrose-metabolizing enzymes in plant leaves have hitherto been investigated mainly in temperate plants, and rarely conducted in tandem with gene expression and sugar analysis. Here, we investigated the sugar content, gene expression, and the activity of sucrose-metabolizing enzymes in the leaves of Hevea brasiliensis, a tropical tree widely cultivated for natural rubber. Sucrose, fructose and glucose were the major sugars detected in Hevea leaves at four developmental stages (I to IV), with starch and quebrachitol as minor saccharides. Fructose and glucose contents increased until stage III, but decreased strongly at stage IV (mature leaves). On the other hand, sucrose increased continuously throughout leaf development. Activities of all sucrose-cleaving enzymes decreased markedly at maturation, consistent with transcript decline for most of their encoding genes. Activity of sucrose phosphate synthase (SPS) was low in spite of its high transcript levels at maturation. Hence, the high sucrose content in mature leaves was not due to increased sucrose-synthesizing activity, but more to the decline in sucrose cleavage. Gene expression and activities of sucrose-metabolizing enzymes in Hevea leaves showed striking differences compared with other plants. Unlike in most other species where vacuolar invertase predominates in sucrose cleavage in developing leaves, cytoplasmic invertase and sucrose synthase (cleavage direction) also featured prominently in Hevea. Whereas SPS is normally responsible for sucrose synthesis in plant leaves, sucrose synthase (synthesis direction) was comparable or higher than that of SPS in Hevea leaves. Mature Hevea leaves had an unusually high sucrose:starch ratio of about 11, the highest reported to date in plants. PMID:29449852

Sucrose and Related Oligosaccharides

NASA Astrophysics Data System (ADS)

Eggleston, Gillian

Sucrose (α-D-glucopyranosyl-(1↔2)-β-D-fructofuranoside) is the most common low-molecular-weight sugar found in the plant kingdom. It is ubiquitously known as common table sugar and primarily produced industrially from sugarcane (Saccharum officinarum) and sugar beet (Beta vulgaris); the basics of the industrial manufacture of sucrose are outlined in this chapter. Commercial sucrose has a very high purity (> 99.9%) making it one of the purest organic substances produced on an industrial scale. Value-addition to sucrose via chemical and biotechnological reactions is becoming more important for the diversification of the sugar industry to maintain the industries' competitiveness in a world increasingly turning to a bio-based economy. The basis for the chemical reactivity of sucrose is the eight hydroxyl groups present on the molecule, although, sucrose chemical reactivity is regarded as difficult. Increasing use of enzymatic biotechnological techniques to derivatize sucrose is expected, to add special functionalities to sucrose products like biodegradability, biocompatibility, and non-toxicity. Analysis of sucrose by colorimetric, enzymatic, oxidation-reduction and chromatography methods are discussed. Oligosaccharides related to sucrose are outlined in detail and include sucrose-based plant, honey and in vitro oligosaccharides.

Work-function calculations for a symmetrical total-charge-density profile at the metallic surface

NASA Astrophysics Data System (ADS)

Wojciechowski, K. F.; Sobańska-Nowotnik, M.

1983-07-01

It is shown that, if the total-charge-density profile nT(x) at the surface of jellium satisfies the Budd-Vannimenus constraint and also is a symmetrical function of x, relative to the ordinate axis, then the work-function variation versus the Wigner-Seitz radius rs does not depend on the form of nT(x). Also the simple linear-density profile is used to calculate the work function by application of the variational principle for the energy, including the first and second density-gradient corrections to the kinetic energy and the first gradient correction to the exchange and correlation energy. The results for the work function are in good agreement with the polycrystalline values for low-density metals.

Great Lakes Region Morphology and Impacts of March 17, 2015 SED Geomagnetic Storm

NASA Astrophysics Data System (ADS)

Heine, T.; Moldwin, M.; Zou, S.

2015-12-01

Under quiet geomagnetic conditions, the mid-latitude ionosphere is relatively uniform with little spatial variation in electron density. However, during intense geomagnetic storms, density gradients associated with Storm Enhanced Density (SED) plumes and Sub-auroral Polarization Streams (SAPS) can move across the dayside mid-latitude ionosphere producing small spatial scale density structure that may be connected to ionospheric scintillation. The evolution of the SED plume during the March 17, 2015 "St. Patrick's Day Storm" is investigated using aggregated data from high resolution GPS receivers at the University of Michigan and throughout the Great Lakes region. Structural density features in the SED gradient can be observed and compared to GPS scintillation measurements—providing insight into the physical mechanisms behind ionospheric scintillation.

Spatial patterns of distribution, abundance, and species diversity of small odontocetes estimated using density surface modeling with line transect sampling

NASA Astrophysics Data System (ADS)

Kanaji, Yu; Okazaki, Makoto; Miyashita, Tomio

2017-06-01

Spatial patterns of distribution, abundance, and species diversity of small odontocetes including species in the Delphinidae and Phocoenidae families were investigated using long-term dedicated sighting survey data collected between 1983 and 2006 in the North Pacific. Species diversity indices were calculated from abundance estimated using density surface modeling of line-transect data. The estimated abundance ranged from 19,521 individuals in killer whale to 1,886,022 in pantropical spotted dolphin. The predicted density maps showed that the habitats of small odontocetes corresponded well with distinct oceanic domains. Species richness was estimated to be highest between 30 and 40°N where warm- and cold-water currents converge. Simpson's Diversity Index showed latitudinal diversity gradients of decreasing species numbers toward the poles. Higher diversity was also estimated in the coastal areas and the zonal areas around 35-42°N. Coastal-offshore gradients and latitudinal gradients are known for many taxa. The zonal areas around 35°N and 40°N coincide with the Kuroshio Current and its extension and the subarctic boundary, respectively. These results suggest that the species diversity of small odontocetes primarily follows general patterns of latitudinal and longitudinal gradients, while the confluence of faunas originating in distinct water masses increases species diversify in frontal waters around 30-40°N. Population densities tended to be higher for the species inhabiting higher latitudes, but were highest for intermediate latitudes at approximately 35-40°N. According to latitudinal gradients in water temperature and biological productivity, the costs for thermoregulation will decrease in warmer low latitudes, while feeding efficiency will increase in colder high latitudes. These trade-offs could optimize population density in intermediate latitudes.

Impact of a large density gradient on linear and nonlinear edge-localized mode simulations

DOE PAGES

Xi, P. W.; Xu, X. Q.; Xia, T. Y.; ...

2013-09-27

Here, the impact of a large density gradient on edge-localized modes (ELMs) is studied linearly and nonlinearly by employing both two-fluid and gyro-fluid simulations. In two-fluid simulations, the ion diamagnetic stabilization on high-n modes disappears when the large density gradient is taken into account. But gyro-fluid simulations show that the finite Larmor radius (FLR) effect can effectively stabilize high-n modes, so the ion diamagnetic effect alone is not sufficient to represent the FLR stabilizing effect. We further demonstrate that additional gyroviscous terms must be kept in the two-fluid model to recover the linear results from the gyro-fluid model. Nonlinear simulations show that the density variation significantly weakens the E × B shearing at the top of the pedestal and thus leads to more energy loss during ELMs. The turbulence spectrum after an ELM crash is measured and has the relation ofmore » $$P(k_{z})\\propto k_{z}^{-3.3}$$ .« less

A representation for the turbulent mass flux contribution to Reynolds-stress and two-equation closures for compressible turbulence

NASA Technical Reports Server (NTRS)

Ristorcelli, J. R.

1993-01-01

The turbulent mass flux, or equivalently the fluctuating Favre velocity mean, appears in the first and second moment equations of compressible kappa-epsilon and Reynolds stress closures. Mathematically it is the difference between the unweighted and density-weighted averages of the velocity field and is therefore a measure of the effects of compressibility through variations in density. It appears to be fundamental to an inhomogeneous compressible turbulence, in which it characterizes the effects of the mean density gradients, in the same way the anisotropy tensor characterizes the effects of the mean velocity gradients. An evolution equation for the turbulent mass flux is derived. A truncation of this equation produces an algebraic expression for the mass flux. The mass flux is found to be proportional to the mean density gradients with a tensor eddy-viscosity that depends on both the mean deformation and the Reynolds stresses. The model is tested in a wall bounded DNS at Mach 4.5 with notable results.

Hierarchical porous carbon materials prepared using nano-ZnO as a template and activation agent for ultrahigh power supercapacitors.

PubMed

Wang, Haoran; Yu, Shukai; Xu, Bin

2016-09-20

Hierarchical porous carbon materials with high surface areas and a localized graphitic structure were simply prepared from sucrose using nano-ZnO as a hard template, activation agent and graphitization catalyst simultaneously, which exhibit an outstanding high-rate performance and can endure an ultrafast scan rate of 20 V s -1 and ultrahigh current density of 1000 A g -1 .

Oxygen exposure promotes fuel diversity for Shewanella oneidensis microbial fuel cells.

PubMed

Biffinger, Justin C; Byrd, Jacqueline N; Dudley, Breanna L; Ringeisen, Bradley R

2008-01-18

Miniature microbial fuel cells (mini-MFCs) were used to monitor the current generated by Shewanella oneidensis DSP10 under both anaerobic and aerobic conditions when exposed to glucose as a potential electron donor. In addition to glucose, other carbon fuels including fructose, sucrose, acetate, and ascorbic acid were also tested. When the anolyte containing S. oneidensis was grown in the presence of oxygen, power densities of 270+/-10, 350+/-20, and 120+/-10 W/m(3) were recorded from the mini-MFC for glucose, fructose, and ascorbic acid electron donors, respectively, while sucrose and acetate produced no response. The power produced from glucose decreased considerably (

The equivalent magnetizing method applied to the design of gradient coils for MRI.

PubMed

Lopez, Hector Sanchez; Liu, Feng; Crozier, Stuart

2008-01-01

This paper presents a new method for the design of gradient coils for Magnetic Resonance Imaging systems. The method is based on the equivalence between a magnetized volume surrounded by a conducting surface and its equivalent representation in surface current/charge density. We demonstrate that the curl of the vertical magnetization induces a surface current density whose stream line defines the coil current pattern. This method can be applied for coils wounds on arbitrary surface shapes. A single layer unshielded transverse gradient coil is designed and compared, with the designs obtained using two conventional methods. Through the presented example we demonstrate that the generated unconventional current patterns obtained using the magnetizing current method produces a superior gradient coil performance than coils designed by applying conventional methods.

Breaks induced in the deoxyribonucleic acid of aerosolized Escherichia coli by ozonized cyclohexene.

PubMed Central

De Mik, G; De Groot, I

1978-01-01

The inactivation of aerosolized Escherichia coli by ozone, cyclohexene, and ozonized cyclohexene was studied. The parameters for damage were loss of reproduction and introduction of breaks in the deoxyribonucleic acid (DNA). Aerosolization of E. coli in clean air at 80 percent relative humidity or in air containing either ozone or cyclohexene hardly affected survival; however, some breaks per DNA molecule were induced, as shown by sucrose gradient sedimentation of the DNA. Aerosolization of E. coli in air containing ozonized cyclohexene at 80 percent relative humidity decreased the survival by a factor of 10(3) or more after 1 h of exposure and induced many breaks in the DNA. PMID:341811

Isolation of Autolysosomes from Tobacco BY-2 Cells.

PubMed

Takatsuka, Chihiro; Inoue-Aono, Yuko; Moriyasu, Yuji

2017-01-01

Autolysosomes are organelles that sequester and degrade a portion of the cytoplasm during autophagy. Although autophagosomes are short lived compared to other organelles such as mitochondria, plastids, and peroxisomes, many autolysosomes accumulate in tobacco BY-2 cells cultured under sucrose starvation conditions in the presence of a cysteine protease inhibitor. We here describe our methodology for isolating autolysosomes from BY-2 cells by conventional cell fractionation using a Percoll gradient. The autolysosome fraction separates clearly from fractions containing mitochondria and peroxisomes. It contains acid phosphatase, vacuolar H + -ATPase, and protease activity. Electron micrographs show that the fraction contains partially degraded cytoplasm seen in autolysosomes before isolation although an autolysosome structure is only partially preserved.

Optimization of magnetic flux density measurement using multiple RF receiver coils and multi-echo in MREIT.

PubMed

Jeong, Woo Chul; Chauhan, Munish; Sajib, Saurav Z K; Kim, Hyung Joong; Serša, Igor; Kwon, Oh In; Woo, Eung Je

2014-09-07

Magnetic Resonance Electrical Impedance Tomography (MREIT) is an MRI method that enables mapping of internal conductivity and/or current density via measurements of magnetic flux density signals. The MREIT measures only the z-component of the induced magnetic flux density B = (Bx, By, Bz) by external current injection. The measured noise of Bz complicates recovery of magnetic flux density maps, resulting in lower quality conductivity and current-density maps. We present a new method for more accurate measurement of the spatial gradient of the magnetic flux density gradient (∇ Bz). The method relies on the use of multiple radio-frequency receiver coils and an interleaved multi-echo pulse sequence that acquires multiple sampling points within each repetition time. The noise level of the measured magnetic flux density Bz depends on the decay rate of the signal magnitude, the injection current duration, and the coil sensitivity map. The proposed method uses three key steps. The first step is to determine a representative magnetic flux density gradient from multiple receiver coils by using a weighted combination and by denoising the measured noisy data. The second step is to optimize the magnetic flux density gradient by using multi-echo magnetic flux densities at each pixel in order to reduce the noise level of ∇ Bz and the third step is to remove a random noise component from the recovered ∇ Bz by solving an elliptic partial differential equation in a region of interest. Numerical simulation experiments using a cylindrical phantom model with included regions of low MRI signal to noise ('defects') verified the proposed method. Experimental results using a real phantom experiment, that included three different kinds of anomalies, demonstrated that the proposed method reduced the noise level of the measured magnetic flux density. The quality of the recovered conductivity maps using denoised ∇ Bz data showed that the proposed method reduced the conductivity noise level up to 3-4 times at each anomaly region in comparison to the conventional method.

A transferable sucrose utilization approach for non-sucrose-utilizing Escherichia coli strains.

PubMed

Bruschi, Michele; Boyes, Simon J; Sugiarto, Haryadi; Nielsen, Lars K; Vickers, Claudia E

2012-01-01

Sucrose has economic and environmental advantages over glucose as a feedstock for bioprocesses. E. coli is widely used in industry, but the majority of current industrial E. coli strains cannot utilize sucrose. Previous attempts to transfer sucrose catabolic capabilities into non-sucrose-utilizing strains have met with limited success due to low growth rates on sucrose and phenotypic instability of the engineered strains. To address these problems, we developed a transferrable sucrose utilization cassette which confers efficient sucrose catabolism when integrated onto the E. coli chromosome. The cassette was based on the csc genes from E. coli W, a strain which grows very quickly on sucrose. Both plasmid-borne expression and chromosomal integration of a repressor-less sucrose utilizing cassette were investigated in E. coli strains K-12, B and C. In contrast to previous studies, strains harboring chromosomal cassettes could grow at the same rate as they do on glucose. Interestingly, we also discovered that spontaneous chromosomal integration of the csc genes was required to allow efficient growth from plasmid-transformed strains. The ability to engineer industrial strains for efficient sucrose utilization will allow substitution of sucrose for glucose in industrial fermentations. This will encourage the use of sucrose as a carbon source and assist in transition of our petrochemical-based economy to a bio-based economy. Crown Copyright © 2011. Published by Elsevier Inc. All rights reserved.

Joint Analysis of GOCE Gravity Gradients Data with Seismological and Geodynamic Observations to Infer Mantle Properties

NASA Astrophysics Data System (ADS)

Metivier, L.; Greff-Lefftz, M.; Panet, I.; Pajot-Métivier, G.; Caron, L.

2014-12-01

Joint inversion of the observed geoid and seismic velocities has been commonly used to constrain the viscosity profile within the mantle as well as the lateral density variations. Recent satellite measurements of the second-order derivatives of the Earth's gravity potential give new possibilities to understand these mantle properties. We use lateral density variations in the Earth's mantle based on slab history or deduced from seismic tomography. The main uncertainties are the relationship between seismic velocity and density -the so-called density/velocity scaling factor- and the variation with depth of the density contrast between the cold slabs and the surrounding mantle, introduced here as a scaling factor with respect to a constant value. The geoid, gravity and gravity gradients at the altitude of the GOCE satellite (about 255 km) are derived using geoid kernels for given viscosity depth profiles. We assume a layered mantle model with viscosity and conversion factor constant in each layer, and we fix the viscosity of the lithosphere. We perform a Monte Carlo search for the viscosity and the density/velocity scaling factor profiles within the mantle which allow to fit the observed geoid, gravity and gradients of gravity. We test a 2-layer, a 3-layer and 4-layer mantle. For each model, we compute the posterior probability distribution of the unknown parameters, and we discuss the respective contributions of the geoid, gravity and gravity gradients in the inversion. Finally, for the best fit, we present the viscosity and scaling factor profiles obtained for the lateral density variations derived from seismic velocities and for slabs sinking into the mantle.

Time-dependent decreases in nucleus accumbens AMPA/NMDA ratio and incubation of sucrose craving in adolescent and adult rats.

PubMed

Counotte, Danielle S; Schiefer, Christopher; Shaham, Yavin; O'Donnell, Patricio

2014-04-01

There is evidence that cue-induced sucrose seeking progressively increases after cessation of oral sucrose self-administration (incubation of sucrose craving) in both adolescent and adult rats. The synaptic plasticity changes associated with this incubation at different age groups are unknown. We assessed whether incubation of sucrose craving in rats trained to self-administer sucrose as young adolescents, adolescents, or adults is associated with changes in 2-amino-3-(3-hydroxy-5-methyl-isoxazol-4-yl)propanoic acid (AMPA)/N-methyl-D-aspartate (NMDA) ratio (a measure of postsynaptic changes in synaptic strength) in nucleus accumbens. Three age groups initiated oral sucrose self-administration training (10 days) on postnatal day (P) 35 (young adolescents), P42 (adolescents), or P70 (adults). They were then tested for cue-induced sucrose seeking (assessed in an extinction test) on abstinence days 1 and 21. Separate groups of rats were trained to self-administer sucrose or water (a control condition), and assessed for AMPA/NMDA ratio in nucleus accumbens on abstinence days 1-3 and 21. Adult rats earned more sucrose rewards, but sucrose intake per body weight was higher in young adolescent rats. Time-dependent increases in cue-induced sucrose seeking (incubation of sucrose craving) were more pronounced in adult rats, less pronounced in adolescents, and not detected in young adolescents. On abstinence day 21, but not days 1-3, AMPA/NMDA ratio in nucleus accumbens were decreased in rats that self-administered sucrose as adults and adolescents, but not young adolescents. Our data demonstrate age-dependent changes in magnitude of incubation of sucrose craving and nucleus accumbens synaptic plasticity after cessation of sucrose self-administration.

Method for providing a low density high strength polyurethane foam

DOEpatents

Whinnery, Jr., Leroy L.; Goods, Steven H.; Skala, Dawn M.; Henderson, Craig C.; Keifer, Patrick N.

2013-06-18

Disclosed is a method for making a polyurethane closed-cell foam material exhibiting a bulk density below 4 lbs/ft.sup.3 and high strength. The present embodiment uses the reaction product of a modified MDI and a sucrose/glycerine based polyether polyol resin wherein a small measured quantity of the polyol resin is "pre-reacted" with a larger quantity of the isocyanate in a defined ratio such that when the necessary remaining quantity of the polyol resin is added to the "pre-reacted" resin together with a tertiary amine catalyst and water as a blowing agent, the polymerization proceeds slowly enough to provide a stable foam body.

A Geophysical Flow Experiment in a Compressible Critical Fluid

NASA Technical Reports Server (NTRS)

Hegseth, John; Garcia, Laudelino

1996-01-01

The first objective of this experiment is to build an experimental system in which, in analogy to a geophysical system, a compressible fluid in a spherical annulus becomes radially stratified in density through an A.C. electric field. When this density gradient is demonstrated, the system will be augmented so that the fluid can be driven by heating and rotation and tested in preparation for a microgravity experiment. This apparatus consists of a spherical capacitor filled with critical fluid in a temperature controlled environment. To make the fluid critical, the apparatus will be operated near the critical pressure, critical density, and critical temperature of the fluid. This will result in a highly compressible fluid because of the properties of the fluid near its critical point. A high voltage A.C. source applied across the capacitor will create a spherically symmetric central force because of the dielectric properties of the fluid in an electric field gradient. This central force will induce a spherically symmetric density gradient that is analogous to a geophysical fluid system. To generate such a density gradient the system must be small (approx. 1 inch diameter). This small cell will also be capable of driving the critical fluid by heating and rotation. Since a spherically symmetric density gradient can only be made in microgravity, another small cell, of the same geometry, will be built that uses incompressible fluid. The driving of the fluid by rotation and heating in these small cells will be developed. The resulting instabilities from the driving in these two systems will then be studied. The second objective is to study the pattern forming instabilities (bifurcations) resulting from the well controlled experimental conditions in the critical fluid cell. This experiment will come close to producing conditions that are geophysically similar and will be studied as the driving parameters are changed.

C library for topological study of the electronic charge density.

PubMed

Vega, David; Aray, Yosslen; Rodríguez, Jesús

2012-12-05

The topological study of the electronic charge density is useful to obtain information about the kinds of bonds (ionic or covalent) and the atom charges on a molecule or crystal. For this study, it is necessary to calculate, at every space point, the electronic density and its electronic density derivatives values up to second order. In this work, a grid-based method for these calculations is described. The library, implemented for three dimensions, is based on a multidimensional Lagrange interpolation in a regular grid; by differentiating the resulting polynomial, the gradient vector, the Hessian matrix and the Laplacian formulas were obtained for every space point. More complex functions such as the Newton-Raphson method (to find the critical points, where the gradient is null) and the Cash-Karp Runge-Kutta method (used to make the gradient paths) were programmed. As in some crystals, the unit cell has angles different from 90°, the described library includes linear transformations to correct the gradient and Hessian when the grid is distorted (inclined). Functions were also developed to handle grid containing files (grd from DMol® program, CUBE from Gaussian® program and CHGCAR from VASP® program). Each one of these files contains the data for a molecular or crystal electronic property (such as charge density, spin density, electrostatic potential, and others) in a three-dimensional (3D) grid. The library can be adapted to make the topological study in any regular 3D grid by modifying the code of these functions. Copyright © 2012 Wiley Periodicals, Inc.

Upper hybrid wave excitation due to O-mode interaction with density gradient in the ionosphere

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

Antani, S.N.; Kaup, D.J.; Rao, N.N.

1995-12-31

It has been well recognized that upper hybrid (UH) waves play a key role in various wave processes occurring in the upper hybrid resonance (UHR) region of the ionosphere leading to the observed stimulated electromagnetic emissions (SEE) during artificial heating by ordinary mode (O-mode) electromagnetic waves. Hence it is important to investigate how the UH waves get excited from the incident O-mode. It has been generally suggested that the UH waves are excited by O-mode interaction with nonuniform ionospheric plasma. For instance, direct conversion of the O-mode into UH waves due to pre-existing short scale irregularities was reported earlier. Heremore » the authors consider the role of large-scale, smooth density gradient in exciting the UH waves from the O-mode. The model used is that of a driven harmonic oscillator in which the source term arises from the O-mode interaction with local density gradient. For a slab model with density gradient in the x-direction, and the geomagnetic field in the z-direction, they obtain an inhomogeneous fourth order ordinary differential equation governing the UH wave excitation. This equation has been analyzed in the vicinity of the UHR. The pertinent solutions will be presented and discussed for the typical parameters of heating experiments.« less

THE EFFECT OF DIETARY WHEAT BRAN ON SUCROSE-INDUCED CHANGES OF SERUM GLUCOSE AND LIPIDS IN RATS.

PubMed

Numan Ahmad, Mousa; Rabah Takruri, Hamid

2015-10-01

wheat bran has been known for many health benefits, but its glucose- and lipid-lowering activity still remains unresolved. to investigate effects of varying amounts of wheat bran and feeding period on serum glucose and lipids in sucrose-fed rats. eighty male Sprague-Dawley rats were assigned into 4 sucrose-based diets containing either 0, 5, 10 or 20% wheat bran (WB) and given ad libitum to rats for 4, 8, 12 or 16 weeks. Serum glucose, total cholesterol (TC), low- and high-density lipoprotein cholesterol (LDL-C and HDL-C), triglycerides (TG), phospholipids (PL) and total lipids (TL) were quantified at end of each feeding period and other biological parameters were assessed. in all feeding periods, food intake showed ascending linear trend (p < 0.05), whereas body weight did not respond to WB. Compared to 0%, 10 or 20% WB induced decrease (p < 0.05) in TC (16 weeks) and HDL-C (12 or 16 weeks), whereas 5, 10 or 20% WB induced similar decrease in PL (4 or 12 weeks), TL (all periods) and glucose (4 or 8 weeks). This glucose- and lipid- lowering effect was substantiated by descending linear responses (p < 0.05) to WB. PL and TL descending responses to WB were seen in all feeding periods. TG exhibited no change with WB, but linearly responded (4 or 8 weeks). Differences in glucose or lipid variables of rats fed WB diets for all feeding periods were less evident. results suggest that wheat bran reduces serum glucose and lipids mainly phospholipids in sucrose fed rats in an interaction that is likely to have clinical implications in cardiometabolic conditions. Copyright AULA MEDICA EDICIONES 2014. Published by AULA MEDICA. All rights reserved.

High sucrose consumption induces memory impairment in rats associated with electrophysiological modifications but not with metabolic changes in the hippocampus.

PubMed

Lemos, C; Rial, D; Gonçalves, F Q; Pires, J; Silva, H B; Matheus, F C; da Silva, A C; Marques, J M; Rodrigues, R J; Jarak, I; Prediger, R D; Reis, F; Carvalho, R A; Pereira, F C; Cunha, R A

2016-02-19

High sugar consumption is a risk factor for metabolic disturbances leading to memory impairment. Thus, rats subject to high sucrose intake (HSu) develop a metabolic syndrome and display memory deficits. We now investigated if these HSu-induced memory deficits were associated with metabolic and electrophysiological alterations in the hippocampus. Male Wistar rats were submitted for 9 weeks to a sucrose-rich diet (35% sucrose solution) and subsequently to a battery of behavioral tests; after sacrifice, their hippocampi were collected for ex vivo high-resolution magic angle spinning (HRMAS) metabolic characterization and electrophysiological extracellular recordings in slices. HSu rats displayed a decreased memory performance (object displacement and novel object recognition tasks) and helpless behavior (forced swimming test), without altered locomotion (open field). HRMAS analysis indicated a similar hippocampal metabolic profile of HSu and control rats. HSu rats also displayed no change of synaptic transmission and plasticity (long-term potentiation) in hippocampal Schaffer fibers-CA1 pyramid synapses, but had decreased amplitude of long-term depression in the temporoammonic (TA) pathway. Furthermore, HSu rats had an increased density of inhibitory adenosine A1 receptors (A1R), that translated into a greater potency of A1R in Schaffer fiber synapses, but not in the TA pathway, whereas the endogenous activation of A1R in HSu rats was preserved in the TA pathway but abolished in Schaffer fiber synapses. These results suggest that HSu triggers a hippocampal-dependent memory impairment that is not associated with altered hippocampal metabolism but is probably related to modified synaptic plasticity in hippocampal TA synapses. Copyright © 2015 IBRO. Published by Elsevier Ltd. All rights reserved.

The influence of temperature, sucrose and lactose on dilute solution properties of basil (Ocimumbasilicum) seed gum.

PubMed

Mirabolhassani, Seyed Elias; Rafe, Ali; Razavi, Seyed M A

2016-12-01

Hydrocolloid interactions with solvent/cosolutes play a vital role in the resolution of their functional properties. Basil seed gum (BSG) is a plant-derived hydrocolloid which has been found many applications in food formulations as stabilizer, emulsifier, thickener and gelling agents. Sucrose and lactose are the most effective sugars in textural and sensorial properties of bakery and dairy products which adding them to solutions containing hydrocolloids can be helpful to approach a proper formula. In this paper, the effect of temperature (25-65°C), sucrose (10, 20, 30 and 40%) and lactose (5, 10 and 15%) were investigated through some molecular parameters of BSG. Results revealed high flexible chain (665.35), intrinsic viscosity (11.38 dl/g) and hydrogel content (73%) of BSG, which may be attributed to some extent by its high molecular weight (1.73×10 6 Da). The density and intrinsic viscosity of BSG were diminished by growing temperature from 25 to 55°C. Among five models, which were applied to estimate intrinsic viscosity, Higiro-2 was the most suitable model at varying temperatures and cosolutes concentrations. The sugars showed a significant effect on the molecular parameters of BSG such as swollen specific volume, shape function, hydration parameter, and coil dimensions. The sugars showed more impact on the [η] of BSG and its molecular parameters than that of temperature. However, lactose had a more prominent effect on the BSG dimensions than that of sucrose, which can be related to its molecular conformation and spatial orientation. It is feasible to make a proper formula by BSG and explain some phenomena in its applications in food and pharmaceutical systems. Copyright © 2016 Elsevier B.V. All rights reserved.

Differential neural representation of oral ethanol by central taste-sensitive neurons in ethanol-preferring and genetically heterogeneous rats

PubMed Central

Wilson, David M.; Brasser, Susan M.

2011-01-01

In randomly bred rats, orally applied ethanol stimulates neural substrates for appetitive sweet taste. To study associations between ethanol's oral sensory characteristics and genetically mediated ethanol preference, we made electrophysiological recordings of oral responses (spike density) by taste-sensitive nucleus tractus solitarii neurons in anesthetized selectively bred ethanol-preferring (P) rats and their genetically heterogeneous Wistar (W) control strain. Stimuli (25 total) included ethanol [3%, 5%, 10%, 15%, 25%, and 40% (vol/vol)], a sucrose series (0.01, 0.03, 0.1, 0.3, 0.5, and 1 M), and other sweet, salt, acidic, and bitter stimuli; 50 P and 39 W neurons were sampled. k-means clustering applied to the sucrose response series identified cells showing high (S1) or relatively low (S0) sensitivity to sucrose. A three-way factorial analysis revealed that activity to ethanol was influenced by a neuron's sensitivity to sucrose, ethanol concentration, and rat line (P = 0.01). Ethanol produced concentration-dependent responses in S1 neurons that were larger than those in S0 cells. Although responses to ethanol by S1 cells did not differ between lines, neuronal firing rates to ethanol in S0 cells increased across concentration only in P rats. Correlation and multivariate analyses revealed that ethanol evoked responses in W neurons that were strongly and selectively associated with activity to sweet stimuli, whereas responses to ethanol by P neurons were not easily associated with activity to representative sweet, sodium salt, acidic, or bitter stimuli. These findings show differential central neural representation of oral ethanol between genetically heterogeneous rats and P rats genetically selected to prefer alcohol. PMID:21918002

Differential neural representation of oral ethanol by central taste-sensitive neurons in ethanol-preferring and genetically heterogeneous rats.

PubMed

Lemon, Christian H; Wilson, David M; Brasser, Susan M

2011-12-01

In randomly bred rats, orally applied ethanol stimulates neural substrates for appetitive sweet taste. To study associations between ethanol's oral sensory characteristics and genetically mediated ethanol preference, we made electrophysiological recordings of oral responses (spike density) by taste-sensitive nucleus tractus solitarii neurons in anesthetized selectively bred ethanol-preferring (P) rats and their genetically heterogeneous Wistar (W) control strain. Stimuli (25 total) included ethanol [3%, 5%, 10%, 15%, 25%, and 40% (vol/vol)], a sucrose series (0.01, 0.03, 0.1, 0.3, 0.5, and 1 M), and other sweet, salt, acidic, and bitter stimuli; 50 P and 39 W neurons were sampled. k-means clustering applied to the sucrose response series identified cells showing high (S(1)) or relatively low (S(0)) sensitivity to sucrose. A three-way factorial analysis revealed that activity to ethanol was influenced by a neuron's sensitivity to sucrose, ethanol concentration, and rat line (P = 0.01). Ethanol produced concentration-dependent responses in S(1) neurons that were larger than those in S(0) cells. Although responses to ethanol by S(1) cells did not differ between lines, neuronal firing rates to ethanol in S(0) cells increased across concentration only in P rats. Correlation and multivariate analyses revealed that ethanol evoked responses in W neurons that were strongly and selectively associated with activity to sweet stimuli, whereas responses to ethanol by P neurons were not easily associated with activity to representative sweet, sodium salt, acidic, or bitter stimuli. These findings show differential central neural representation of oral ethanol between genetically heterogeneous rats and P rats genetically selected to prefer alcohol.

Effect of centrifugal fractionation protocols on quality and recovery rate of equine sperm.

PubMed

Edmond, A J; Brinsko, S P; Love, C C; Blanchard, T L; Teague, S R; Varner, D D

2012-03-15

Centrifugal fractionation of semen is commonly done to improve quality of human semen in assisted-reproduction laboratories, allowing sperm separation based on their isopycnic points. Sperm with morphologic abnormalities are often more buoyant, promoting their retention above defined density media, with structurally normal sperm passing through the media following centrifugation. Three experiments were conducted to evaluate the effects of density-medium type, centrifuge-tube size, sperm number, and density-medium volume (column height) on stallion sperm quality and recovery rate in sperm pellets following centrifugation. In all three experiments, equine semen was initially centrifuged to increase sperm concentration. In Experiment 1, semen was layered over continuous or discontinuous gradients. For Experiment 2, semen was layered over three column heights of continuous gradients in 15- or 50-ml conical-bottom tubes. For Experiment 3, increasing sperm numbers were layered over continuous gradient in 15- or 50-ml conical-bottom tubes. Following centrifugation, sperm pellets were evaluated for sperm morphologic quality, motility, DNA integrity, and recovery rate. Centrifugal fractionation improved (P < 0.05) sperm morphology, motility, and DNA integrity, as compared to controls. The continuous gradient increased (P < 0.05) sperm recovery rate relative to the discontinuous gradient, whereas sperm processed in 15-ml tubes yielded higher velocity and higher recovery rates (P < 0.05 for each) than that processed in 50-ml tubes. Sperm recovery rate was not affected (P > 0.05) by column height of gradient. Increasing sperm number subjected to gradient centrifugation decreased (P < 0.05) sperm recovery rate when 15-ml tubes were used. Copyright © 2012 Elsevier Inc. All rights reserved.

[Sugar triggers our reward-system. Sweets release opiates which stimulates the appetite for sucrose--insulin can depress it].

PubMed

Erlanson-Albertsson, Charlotte

The consumption of sweet food has increased in Sweden, as in other Western countries. The type of food item has changed. The sweet is dominated by soft drinks. Appetite regulation for sucrose has been described in experimental animal models. It has been found that opioids stimulate appetite for sucrose. At the same time sucrose releases endogenous opioids so that a triggering of sucrose consumption occurs. Insulin has been shown to decrease sucrose intake by blocking the opioid response. Sucrose addiction has been described in rat model. With a concentrated sucrose solution to drink an opioid dependence developed with 1) increased consumption of sucrose 2) abstinence symptoms with no sucrose and 3) anxiety with an opiate blocker. Sucrose addiction in man has not been described in the scientific literature. There is an increased liking of sweets with alcoholic persons, which may be significant to support a strongly rewarding effect of sucrose, also in man. We should limit the access to sweet foods, in particular the sweet drinks. Insulin and insulin sensitivity may be an important factor to restrict the intake of sweet food.

Comment on ``Turbulent equipartition theory of toroidal momentum pinch'' [Phys. Plasmas 15, 055902 (2008)

NASA Astrophysics Data System (ADS)

Peeters, A. G.; Angioni, C.; Strintzi, D.

2009-03-01

The comment addresses questions raised on the derivation of the momentum pinch velocity due to the Coriolis drift effect [A. G. Peeters et al., Phys. Rev. Lett. 98, 265003 (2007)]. These concern the definition of the gradient, and the scaling with the density gradient length. It will be shown that the turbulent equipartition mechanism is included within the derivation using the Coriolis drift, with the density gradient scaling being the consequence of drift terms not considered in [T. S. Hahm et al., Phys. Plasmas 15, 055902 (2008)]. Finally the accuracy of the analytic models is assessed through a comparison with the full gyrokinetic solution.

The role of fluctuation-induced transport in a toroidal plasma with strong radial electric fields

NASA Technical Reports Server (NTRS)

Roth, J. R.; Krawczonek, W. M.; Powers, E. J.; Hong, J. Y.; Kim, Y. C.

1981-01-01

Previous work employing digitally implemented spectral analysis techniques is extended to demonstrate that radial fluctuation-induced transport is the dominant ion transport mechanism in an electric field dominated toroidal plasma. Such transport can be made to occur against a density gradient, and hence may have a very beneficial effect on confinement in toroidal plasmas of fusion interest. It is shown that Bohm or classical diffusion down a density gradient, the collisional Pedersen-current mechanism, and the collisionless electric field gradient mechanism described by Cole (1976) all played a minor role, if any, in the radial transport of this plasma.

Variation of arbuscular mycorrhizal fungal communities along an altitudinal gradient in rupestrian grasslands in Brazil.

PubMed

Coutinho, Etiene Silva; Fernandes, G Wilson; Berbara, Ricardo Luís Louro; Valério, Henrique Maia; Goto, Bruno Tomio

2015-11-01

Variation in arbuscular mycorrhizal fungi (AMF) communities is described for the first time in rupestrian grasslands in Brazil along an altitudinal gradient of 700 m (800 to 1400 m a.s.l.). Hypotheses tested were that soil properties influence the variation in AMF communities and that the frequency of the most common species of AMF is inversely influenced by the richness of other AMF. Field and laboratory data were collected on AMF community composition, richness, density, and frequency in the altitudinal gradient, and the relationships with several physical-chemical soil properties and altitude were evaluated. Fifty-one species of AMF were recorded, with 14 species being reported as possibly new to science and nine species representing new records for Brazil. This single elevation gradient alone contains 22% of the known world diversity of AMF. Soil properties and AMF community density and richness varied significantly along the elevation (p < 0.05). AMF density and richness were higher at the intermediate altitude, while AMF species composition differed statistically among the altitudes.

Plasma response to the injection of an electron beam

NASA Technical Reports Server (NTRS)

Singh, N.; Schunk, R. W.

1984-01-01

The results of Vlasov-Poisson-solver numerical simulations of the detailed temporal response of a Maxwellian plasma to the sudden injection of an electron beam are presented in graphs and maps and discussed. Phenomena characterized include ion bursts, electron shocks and holes, plasma heating and expulsion, density gradients; cavitons, deep-density-front and solitary-pulse propagation down the density gradient, and Bunemann-mode excitation leading to formation of a virtual cathode and double layers which are at first monotonic or have low-potential-side dips or high-potential-side bumps and become strong as the electron-current density decreases. The strength of the double layer is found to be roughly proportional to the beam energy.

Linear stability analysis of the Vlasov-Poisson equations in high density plasmas in the presence of crossed fields and density gradients

NASA Technical Reports Server (NTRS)

Kaup, D. J.; Hansen, P. J.; Choudhury, S. Roy; Thomas, Gary E.

1986-01-01

The equations for the single-particle orbits in a nonneutral high density plasma in the presence of inhomogeneous crossed fields are obtained. Using these orbits, the linearized Vlasov equation is solved as an expansion in the orbital radii in the presence of inhomogeneities and density gradients. A model distribution function is introduced whose cold-fluid limit is exactly the same as that used in many previous studies of the cold-fluid equations. This model function is used to reduce the linearized Vlasov-Poisson equations to a second-order ordinary differential equation for the linearized electrostatic potential whose eigenvalue is the perturbation frequency.

Non-Axisymmetric Line Driven Disc Winds II - Full Velocity Gradient

NASA Astrophysics Data System (ADS)

Dyda, Sergei; Proga, Daniel

2018-05-01

We study non-axisymetric features of 3D line driven winds in the Sobolev approximation, where the optical depth is calculated using the full velocity gradient. We find that non-axisymmetric density features, so called clumps, form primarily at the base of the wind on super-Sobolev length scales. The density of clumps differs by a factor of ˜3 from the azimuthal average, the magnitude of their velocity dispersion is comparable to the flow velocity and they produce ˜20% variations in the column density. Clumps may be observable because differences in density produce enhancements in emission and absorption profiles or through their velocity dispersion which enhances line broadening.

Effect of cell density on adipogenic differentiation of mesenchymal stem cells

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

Lu, Hongxu; Biomaterials Center, National Institute for Materials Science, 1-1 Namiki, Tsukuba, Ibaraki 305-0044; Guo, Likun

2009-04-10

The effect of cell density on the adipogenic differentiation of human bone marrow-derived mesenchymal stem cells (MSCs) was investigated by using a patterning technique to induce the formation of a cell density gradient on a micropatterned surface. The adipogenic differentiation of MSCs at a density gradient from 5 x 10{sup 3} to 3 x 10{sup 4} cells/cm{sup 2} was examined. Lipid vacuoles were observed at all cell densities after 1-3 weeks of culture in adipogenic differentiation medium although the lipid vacuoles were scarce at the low cell density and abundant at the high cell density. Real-time RT-PCR analysis showed thatmore » adipogenesis marker genes encoding peroxisome proliferator-activated receptor {gamma}2 (PPAR{gamma}2), lipoprotein lipase (LPL), and fatty acid binding protein-4 (FABP4) were detected in the MSCs cultured at all cell densities. The results suggest that there was no apparent effect of cell density on the adipogenic differentiation of human MSCs.« less

Gradients and anisotropies of high energy cosmic rays in the outer heliosphere

NASA Technical Reports Server (NTRS)

Fillius, W.; Roelof, E. C.; Smith, E. J.; Wood, D.; Ip, W. H.

1985-01-01

Two cosmic rays which pass through the same point going in opposite directions will, in the absence of scattering and inhomogeneities in the magnetic field, trace helices about adjacent flux tubes, whose centerlines are separated by one gyrodiameter. A directional anisotropy at the point suggests a difference in the number of cosmic rays loading the two flux tubes; that is, a density gradient over the baseline of a gyrodiameter. Previous studies at lower energies have shown that the cosmic ray density gradients vary in time and space. It is suggested that the radial gradient associated with solar cycle modulation is supported largely by narrow barriers which encircle the sun and propagate outward with the solar wind. If so, the anisotropy is a desirable way to detect spatial gradients, because it can be associated with the local solar wind and magnetic field conditions. Anisotropic measurements made by Cerenkov detectors on Pioneers 10 and 11 were studied. It was found that local anisotropy varies greatly, but that the long term average is consistent with the global radial gradient measured between two spacecraft over a baseline of many AU.

Behavioral and hormonal responses to stress in binge-like eating prone female rats.

PubMed

Calvez, Juliane; Timofeeva, Elena

2016-04-01

Binge eating episodes are frequently stimulated by stress. We developed a model of binge eating proneness based on individual sensitivity of young female Sprague Dawley rats to significantly increase sucrose consumption in response to stress. The rats were subjected to unpredictable intermittent 1-h access to 10% sucrose. After the stabilization of sucrose intake, rats were assessed for consistency of higher (for binge-like eating prone, BEP) or lower (for binge-like eating resistant, BER) sucrose intake in response to unpredictable episodes of foot-shock stress. The objectives of this study included demonstrating face validity of the BEP model and determining if some of the features of this model were pre-existing before exposure to intermittent access to sucrose and repeated stress. The BEP rats consumed a larger (20%>BER) amount of sucrose in a discrete (1-h) period of time compared to the BER phenotype in non-stressful conditions and significantly increased sucrose intake (50%>BER) under stress. Conversely, stress did not affect sucrose intake in BER rats. BEP rats showed higher sucrose intake compared to BER rats at the beginning of darkness as well as during the light period when they were sated and not physically hungry. Analyses of the sucrose licking microstructure revealed that BEP rats had a high motivational drive to consume sucrose in non-stressful condition and an increased hedonic value of sucrose when they were exposed to stressful conditions. BEP rats consumed sucrose much more rapidly under stressful conditions compared to BER rats. Finally, BEP rats demonstrated compulsive-like intake of sucrose (assessed in the light-dark box) and a blunted stress-induced increase in plasma corticosterone levels. Body weight and chow intake were not different between the phenotypes. Before exposure to intermittent access to sucrose and repeated stress, the BEP rats showed no clear evidence for compulsive sucrose intake. However, from the first 1-h access to sucrose, the BEP rats exhibited sucrose overeating; and from the first exposure to stress before intermittent access to sucrose, the BEP rats showed a blunted increase in corticosterone plasma levels. Innate sucrose hyperconsumption and altered reactivity of the hypothalamo-pituitary adrenal (HPA) axis to stress may be involved in the development of binge-like eating. Increased perceived hedonic value of palatable food and an increased motivation to consume this food despite aversive conditions as well as deregulated reactivity of the HPA axis may contribute to stress-induced bingeing on sucrose in BEP rats. Copyright © 2016 Elsevier Inc. All rights reserved.

Sucrose secreted by the engineered cyanobacterium and its fermentability

NASA Astrophysics Data System (ADS)

Duan, Yangkai; Luo, Quan; Liang, Feiyan; Lu, Xuefeng

2016-10-01

The unicellular cyanobacterium, Synechococcus elongatus PCC 7942 (Syn7942), synthesizes sucrose as the only compatible solute under salt stress. A series of engineered Syn7942 strains for sucrose production were constructed. The overexpression of the native sps (encoding a natively fused protein of sucrose phosphate synthase SPS and sucrose phosphate phosphatase SPP) in Syn7942 wild type caused a 93% improvement of sucrose productivity. The strain FL130 co-overexpressing sps and cscB (encoding a sucrose transporter) exhibited a 74% higher extracellular sucrose production than that overexpressing cscB only. Both results showed the significant improvement of sucrose productivity by the double functional protein SPS-SPP. Afterwards, FL130 was cultivated under a modified condition, and the cell-free culture medium containing 1.5 g L-1 sucrose was pre-treated with an acid hydrolysis technique. Cultivated with the neutralized hydrolysates as the starting media, two widely used microorganisms, Escherichia coli and Saccharomyces cerevisiae, showed a comparable growth with that in the control media supplemented with glucose. These results clearly demonstrated that the cell-free culture of sucrose-secreting cyanobacteria can be applied as starting media in microbial cultivation.

Multiconfiguration Pair-Density Functional Theory.

PubMed

Li Manni, Giovanni; Carlson, Rebecca K; Luo, Sijie; Ma, Dongxia; Olsen, Jeppe; Truhlar, Donald G; Gagliardi, Laura

2014-09-09

We present a new theoretical framework, called Multiconfiguration Pair-Density Functional Theory (MC-PDFT), which combines multiconfigurational wave functions with a generalization of density functional theory (DFT). A multiconfigurational self-consistent-field (MCSCF) wave function with correct spin and space symmetry is used to compute the total electronic density, its gradient, the on-top pair density, and the kinetic and Coulomb contributions to the total electronic energy. We then use a functional of the total density, its gradient, and the on-top pair density to calculate the remaining part of the energy, which we call the on-top-density-functional energy in contrast to the exchange-correlation energy of Kohn-Sham DFT. Because the on-top pair density is an element of the two-particle density matrix, this goes beyond the Hohenberg-Kohn theorem that refers only to the one-particle density. To illustrate the theory, we obtain first approximations to the required new type of density functionals by translating conventional density functionals of the spin densities using a simple prescription, and we perform post-SCF density functional calculations using the total density, density gradient, and on-top pair density from the MCSCF calculations. Double counting of dynamic correlation or exchange does not occur because the MCSCF energy is not used. The theory is illustrated by applications to the bond energies and potential energy curves of H2, N2, F2, CaO, Cr2, and NiCl and the electronic excitation energies of Be, C, N, N(+), O, O(+), Sc(+), Mn, Co, Mo, Ru, N2, HCHO, C4H6, c-C5H6, and pyrazine. The method presented has a computational cost and scaling similar to MCSCF, but a quantitative accuracy, even with the present first approximations to the new types of density functionals, that is comparable to much more expensive multireference perturbation theory methods.

Analysis of sucrose-induced small RNAs in Streptococcus mutans in the presence of different sucrose concentrations.

PubMed

Liu, Shan Shan; Zhu, Wen Hui; Zhi, Qing Hui; Liu, Jia; Wang, Yan; Lin, Huan Cai

2017-07-01

Streptococcus mutans (S. mutans) is the major pathogen contributing to dental caries. Sucrose is an important carbohydrate source for S. mutans and is crucial for dental caries. Small RNAs (sRNAs) are key post-transcriptional regulators of stress adaptation and virulence in bacteria. Here, for the first time, we created three replicate RNA libraries exposed to either 1 or 5% sucrose. The expression levels of sRNAs and target genes (gtfB, gtfC, and spaP) related to virulence were assessed. In addition, some phenotypic traits were evaluated. We obtained 2125 sRNA candidates with at least 100 average reads in 1% sucrose or 5% sucrose. Of these candidates, 2 were upregulated and 20 were downregulated in 1% sucrose. Six of these 22 differentially expressed sRNAs were validated by qRT-PCR. The expression level of target gene gtfB was higher in 1% sucrose. The adherence ratio of S. mutans was higher in 1% sucrose than in 5% sucrose. The synthesis of water-insoluble glucans (WIGs) was significantly higher in 5% sucrose than in 1% sucrose. These data suggest that a series of sRNAs can be induced in response to sucrose, and that some sRNAs might be involved in the regulation of phenotypes, providing new insight into the prevention of caries.

An analysis of the gradient-induced electric fields and current densities in human models when situated in a hybrid MRI-LINAC system

NASA Astrophysics Data System (ADS)

Liu, Limei; Trakic, Adnan; Sanchez-Lopez, Hector; Liu, Feng; Crozier, Stuart

2014-01-01

MRI-LINAC is a new image-guided radiotherapy treatment system that combines magnetic resonance imaging (MRI) with a linear accelerator (LINAC) in a single unit. One drawback is that the pulsing of the split gradient coils of the system induces an electric field and currents in the patient which need to be predicted and evaluated for patient safety. In this novel numerical study the in situ electric fields and associated current densities were evaluated inside tissue-accurate male and female human voxel models when a number of different split-geometry gradient coils were operated. The body models were located in the MRI-LINAC system along the axial and radial directions in three different body positions. Each model had a region of interest (ROI) suitable for image-guided radiotherapy. The simulation results show that the amplitudes and distributions of the field and current density induced by different split x-gradient coils were similar with one another in the ROI of the body model, but varied outside of the region. The fields and current densities induced by a split classic coil with the surface unconnected showed the largest deviation from those given by the conventional non-split coils. Another finding indicated that the distributions of the peak current densities varied when the body position, orientation or gender changed, while the peak electric fields mainly occurred in the skin and fat tissues.

fNL‑gNL mixing in the matter density field at higher orders

NASA Astrophysics Data System (ADS)

Gressel, Hedda A.; Bruni, Marco

2018-06-01

In this paper we examine how primordial non-Gaussianity contributes to nonlinear perturbative orders in the expansion of the density field at large scales in the matter dominated era. General Relativity is an intrinsically nonlinear theory, establishing a nonlinear relation between the metric and the density field. Representing the metric perturbations with the curvature perturbation ζ, it is known that nonlinearity produces effective non-Gaussian terms in the nonlinear perturbations of the matter density field δ, even if the primordial ζ is Gaussian. Here we generalise these results to the case of a non-Gaussian primordial ζ. Using a standard parametrization of primordial non-Gaussianity in ζ in terms of fNL, gNL, hNL\\ldots , we show how at higher order (from third and higher) nonlinearity also produces a mixing of these contributions to the density field at large scales, e.g. both fNL and gNL contribute to the third order in δ. This is the main result of this paper. Our analysis is based on the synergy between a gradient expansion (aka long-wavelength approximation) and standard perturbation theory at higher order. In essence, mathematically the equations for the gradient expansion are equivalent to those of first order perturbation theory, thus first-order results convert into gradient expansion results and, vice versa, the gradient expansion can be used to derive results in perturbation theory at higher order and large scales.

High power density yeast catalyzed microbial fuel cells

NASA Astrophysics Data System (ADS)

Ganguli, Rahul

Microbial fuel cells leverage whole cell biocatalysis to convert the energy stored in energy-rich renewable biomolecules such as sugar, directly to electrical energy at high efficiencies. Advantages of the process include ambient temperature operation, operation in natural streams such as wastewater without the need to clean electrodes, minimal balance-of-plant requirements compared to conventional fuel cells, and environmentally friendly operation. These make the technology very attractive as portable power sources and waste-to-energy converters. The principal problem facing the technology is the low power densities compared to other conventional portable power sources such as batteries and traditional fuel cells. In this work we examined the yeast catalyzed microbial fuel cell and developed methods to increase the power density from such fuel cells. A combination of cyclic voltammetry and optical absorption measurements were used to establish significant adsorption of electron mediators by the microbes. Mediator adsorption was demonstrated to be an important limitation in achieving high power densities in yeast-catalyzed microbial fuel cells. Specifically, the power densities are low for the length of time mediator adsorption continues to occur. Once the mediator adsorption stops, the power densities increase. Rotating disk chronoamperometry was used to extract reaction rate information, and a simple kinetic expression was developed for the current observed in the anodic half-cell. Since the rate expression showed that the current was directly related to microbe concentration close to the electrode, methods to increase cell mass attached to the anode was investigated. Electrically biased electrodes were demonstrated to develop biofilm-like layers of the Baker's yeast with a high concentration of cells directly connected to the electrode. The increased cell mass did increase the power density 2 times compared to a non biofilm fuel cell, but the power density increase was shown to quickly saturate with cell mass attached on the electrode. Based on recent modelling data that suggested that the electrode currents might be limited by the poor electrical conductivity of the anode, the power density versus electrical conductivity of a yeast-immobilized anode was investigated. Introduction of high aspect ratio carbon fiber filaments to the immobilization matrix increased the electrical conductivity of the anode. Although a higher electrical conductivity clearly led to an increase in power densities, it was shown that the principal limitation to power density increase was coming from proton transfer limitations in the immobilized anode. Partial overcoming of the gradients lead a power density of ca. 250 microW cm-2, which is the highest reported for yeast powered MFCs. A yeast-catalyzed microbial fuel cell was investigated as a power source for low power sensors using raw tree sap. It was shown that yeast can efficiently utilize the sucrose present in the raw tree sap to produce electricity when excess salt is added to the medium. Therefore the salinity of a potential energy source is an important consideration when MFCs are being considered for energy harvesting from natural sources.

Comparison of energy efficiency and power density in pressure retarded osmosis and reverse electrodialysis.

PubMed

Yip, Ngai Yin; Elimelech, Menachem

2014-09-16

Pressure retarded osmosis (PRO) and reverse electrodialysis (RED) are emerging membrane-based technologies that can convert chemical energy in salinity gradients to useful work. The two processes have intrinsically different working principles: controlled mixing in PRO is achieved by water permeation across salt-rejecting membranes, whereas RED is driven by ion flux across charged membranes. This study compares the energy efficiency and power density performance of PRO and RED with simulated technologically available membranes for natural, anthropogenic, and engineered salinity gradients (seawater-river water, desalination brine-wastewater, and synthetic hypersaline solutions, respectively). The analysis shows that PRO can achieve both greater efficiencies (54-56%) and higher power densities (2.4-38 W/m(2)) than RED (18-38% and 0.77-1.2 W/m(2)). The superior efficiency is attributed to the ability of PRO membranes to more effectively utilize the salinity difference to drive water permeation and better suppress the detrimental leakage of salts. On the other hand, the low conductivity of currently available ion exchange membranes impedes RED ion flux and, thus, constrains the power density. Both technologies exhibit a trade-off between efficiency and power density: employing more permeable but less selective membranes can enhance the power density, but undesired entropy production due to uncontrolled mixing increases and some efficiency is sacrificed. When the concentration difference is increased (i.e., natural → anthropogenic → engineered salinity gradients), PRO osmotic pressure difference rises proportionally but not so for RED Nernst potential, which has logarithmic dependence on the solution concentration. Because of this inherently different characteristic, RED is unable to take advantage of larger salinity gradients, whereas PRO power density is considerably enhanced. Additionally, high solution concentrations suppress the Donnan exclusion effect of the charged RED membranes, severely reducing the permselectivity and diminishing the energy conversion efficiency. This study indicates that PRO is more suitable to extract energy from a range of salinity gradients, while significant advancements in ion exchange membranes are likely necessary for RED to be competitive with PRO.

Comparison of Energy Efficiency and Power Density in Pressure Retarded Osmosis and Reverse Electrodialysis

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

Yip, NY; Elimelech, M

Pressure retarded osmosis (PRO) and reverse electrodialysis (RED) are emerging membrane-based technologies that can convert chemical energy in salinity gradients to useful work. The two processes have intrinsically different working principles: controlled mixing in PRO is achieved by water permeation across salt-rejecting membranes, whereas RED is driven by ion flux across charged membranes. This study compares the energy efficiency and power density performance of PRO and RED with simulated technologically available membranes for natural, anthropogenic, and engineered salinity gradients (seawater-river water, desalination brine-wastewater, and synthetic hypersaline solutions, respectively). The analysis shows that PRO can achieve both greater efficiencies (54-56%) andmore » higher power densities (2.4-38 W/m(2)) than RED (18-38% and 0.77-1.2 W/m(2)). The superior efficiency is attributed to the ability of PRO membranes to more effectively utilize the salinity difference to drive water permeation and better suppress the detrimental leakage of salts. On the other hand, the low conductivity of currently available ion exchange membranes impedes RED ion flux and, thus, constrains the power density. Both technologies exhibit a trade-off between efficiency and power density: employing more permeable but less selective membranes can enhance the power density, but undesired entropy production due to uncontrolled mixing increases and some efficiency is sacrificed. When the concentration difference is increased (i.e., natural -> anthropogenic -> engineered salinity gradients), PRO osmotic pressure difference rises proportionally but not so for RED Nernst potential, which has logarithmic dependence on the solution concentration. Because of this inherently different characteristic, RED is unable to take advantage of larger salinity gradients, whereas PRO power density is considerably enhanced. Additionally, high solution concentrations suppress the Donnan exclusion effect of the charged RED membranes, severely reducing the permselectivity and diminishing the energy conversion efficiency. This study indicates that PRO is more suitable to extract energy from a range of salinity gradients, while significant advancements in ion exchange membranes are likely necessary for RED to be competitive with PRO.« less

The effects of fruiting positions on cellulose synthesis and sucrose metabolism during cotton (Gossypium hirsutum L.) fiber development.

PubMed

Ma, Yina; Wang, Youhua; Liu, Jingran; Lv, Fengjuan; Chen, Ji; Zhou, Zhiguo

2014-01-01

Cotton (Gossypium hirsutum L.) boll positions on a fruiting branch vary in their contribution to yield and fiber quality. Fiber properties are dependent on deposition of cellulose in the fiber cell wall, but information about the enzymatic differences in sucrose metabolism between these fruiting positions is lacking. Therefore, two cotton cultivars with different sensitivities to low temperature were tested in 2010 and 2011 to quantify the effect of fruit positions (FPs) on fiber quality in relation to sucrose content, enzymatic activities and sucrose metabolism. The indices including sucrose content, sucrose transformation rate, cellulose content, and the activities of the key enzymes, sucrose phosphate synthase (SPS), acid invertase (AI) and sucrose synthase (SuSy) which inhibit cellulose synthesis and eventually affect fiber quality traits in cotton fiber, were determined. Results showed that as compared with those of FP1, cellulose content, sucrose content, and sucrose transformation rate of FP3 were all decreased, and the variations of cellulose content and sucrose transformation rate caused by FPs in Sumian 15 were larger than those in Kemian 1. Under FP effect, activities of SPS and AI in sucrose regulation were decreased, while SuSy activity in sucrose degradation was increased. The changes in activities of SuSy and SPS in response to FP effect displayed different and large change ranges between the two cultivars. These results indicate that restrained cellulose synthesis and sucrose metabolism in distal FPs are mainly attributed to the changes in the activities of these enzymes. The difference in fiber quality, cellulose synthesis and sucrose metabolism in response to FPs in fiber cells for the two cotton cultivars was mainly determined by the activities of both SuSy and SPS.

Starch Combined with Sucrose Provokes Greater Root Dentine Demineralization than Sucrose Alone.

PubMed

Souza, Samilly Evangelista; Sampaio, Aline Araújo; Del Bel Cury, Altair Antoninha; Cavalcanti, Yuri Wanderley; Ricomini Filho, Antônio Pedro; Cury, Jaime Aparecido

2018-02-14

Since there is no consensus about whether starch increases the cariogenic potential of sucrose, we used a validated 3-species biofilm model to evaluate if starch combined with sucrose provokes higher root dentine demineralization than sucrose alone. Biofilms (n = 18) composed by Streptococcus mutans (the most cariogenic bacteria), Actinomces naeslundii (which has amylolytic activity), and Streptococcus gordonii (which binds salivary amylase) were formed on root dentine slabs under exposure 8 ×/day to one of the following treatments: 0.9% NaCl, 1% starch, 10% sucrose, or a combination of 1% starch and 10% sucrose. Before each treatment, biofilms were pretreated with human whole saliva for 1 min. The pH of the culture medium was measured daily as an indicator of biofilm acidogenicity. After 96 h of growth, the biofilms were collected, and the biomass, bacteria viability, and polysaccharides were analyzed. Dentine demineralization was assessed by surface hardness loss (% SHL). Biofilm bioarchitecture was analyzed using confocal laser scanning microscopy. Treatment with a starch and sucrose combination provoked higher (p = 0.01) dentine demineralization than sucrose alone (% SHL = 53.2 ± 7.0 vs. 43.2 ± 8.7). This was supported by lower pH values (p = 0.007) of the culture medium after daily exposure to the starch and sucrose combination compared with sucrose (4.89 ± 0.29 vs. 5.19 ± 0.32). Microbiological and biochemical findings did not differ between biofilms treated with the combination of starch and sucrose and sucrose alone (p > 0.05). Our findings give support to the hypothesis that a starch and sucrose combination is more cariogenic for root dentine than sucrose alone. © 2018 S. Karger AG, Basel.

Calculated in situ rock density from gravity observations, UA-1 (Cannikin) emplacement hole, Amchitka Island, Alaska

USGS Publications Warehouse

Healey, D.L.

1971-01-01

Gravity observations were made on the ground surface and at a depth of 5,854 feet in drill hole UA-1. Two attempts to measure the free-air gradient utilizing the headframe over the drill hole were unsuccessful owing to mechanical vibrations in the structure. Because of the uncertainty in the measured free-air gradients these values were discarded and the average value (0.09406 mgal/ft) was used in the calculations. The calculated in situ bulk density is 2.36 g/cc. The weighted average bulk density determined from 47 core samples taken in the adjacent UAE-1 drill hole is also 2.36 g/cc. An analysis of selected portions of density logs provides an in situ bulk density of 2.37 g/cc.

Communication: A new class of non-empirical explicit density functionals on the third rung of Jacob's ladder

NASA Astrophysics Data System (ADS)

de Silva, Piotr; Corminboeuf, Clémence

2015-09-01

We construct an orbital-free non-empirical meta-generalized gradient approximation (GGA) functional, which depends explicitly on density through the density overlap regions indicator [P. de Silva and C. Corminboeuf, J. Chem. Theory Comput. 10, 3745 (2014)]. The functional does not depend on either the kinetic energy density or the density Laplacian; therefore, it opens a new class of meta-GGA functionals. By construction, our meta-GGA yields exact exchange and correlation energy for the hydrogen atom and recovers the second order gradient expansion for exchange in the slowly varying limit. We show that for molecular systems, overall performance is better than non-empirical GGAs. For atomization energies, performance is on par with revTPSS, without any dependence on Kohn-Sham orbitals.

Experiment study of mud to the moving process influent about viscous debris flow along slope

NASA Astrophysics Data System (ADS)

Jun, JiXian; Ying, Liang; Li, Pan Hua; Qiang, OuGuo

2018-01-01

Mud is the main component of viscous debris flow. The physical model experiments of viscous debris flow were carried out through the mixing mud with different density and fixed components of coarse particles. The width, longitudinal movement distance and motion velocity were recorded by video cameras during experiment. Through viscous debris flow physical model experiments, the influence of mud to transverse width, longitudinal movement distance and motion velocity was discussed. The physical model experiment results show that the motion forms change from inviscid particle flow to viscous debris flow and to the whole mass sliding with the increase of mud density; the width and the length along the slope decrease with mud density increasing; the movement process has classified phenomena about viscous debris flow composed by different mud densities: the velocity increases rapidly with time and the change gradient is steady when the density of mud is lower than 1.413g/cm3; the movement process can be divided into two stages when the density of mud is higher than 1.413g/cm3: the movement velocity is lower and the gradient change is small in the initial stage; but in the second stage, the movement velocity increases quickly, and the gradient is higher than the first stage, and with steady value.

Line length dependence of threshold current density and driving force in eutectic SnPb and SnAgCu solder electromigration

NASA Astrophysics Data System (ADS)

Yoon, Min-Seung; Ko, Min-Ku; Kim, Bit-Na; Kim, Byung-Joon; Park, Yong-Bae; Joo, Young-Chang

2008-04-01

The relationship between the threshold current density and the critical line length in eutectic SnPb and SnAgCu electromigrations were examined using solder lines with the various lengths ranging from 100to1000μm. When the electron wind-force was balanced by the back-stress gradient force, the net flux of electromigration is zero, at which the current density and line length are defined as the threshold current density and the critical length, respectively. It was found that in SnAgCu electromigration, the 1/L dependence on the threshold current density showed good agreement, whereas the threshold current densities of the eutectic SnPb deviated from the 1/L dependence. The balance between the electron wind-force and the back-stress gradient force was the main factor determining the threshold product of SnAgCu electromigration. On the other hand, in the case of eutectic SnPb, the chemical driving force is contributed as a back-flux force in addition to the back-stress gradient force. The existence of the chemical driving force was caused by the nonequilibrium Pb concentration inside the Pb-rich phases between the cathode and anode during the electromigration procedure.

Deconstructing the vanilla milkshake: the dominant effect of sucrose on self-administration of nutrient-flavor mixtures.

PubMed

Naleid, Amy M; Grimm, Jeffrey W; Kessler, David A; Sipols, Alfred J; Aliakbari, Sepideh; Bennett, Jennifer L; Wells, Jason; Figlewicz, Dianne P

2008-01-01

Rats and humans avidly consume flavored foods that contain sucrose and fat, presumably due to their rewarding qualities. In this study, we hypothesized that the complex mixture of corn oil, sucrose, and flavor is more reinforcing than any of these components alone. We observed a concentration-dependent increase in reinforcers of sucrose solutions received (0%, 3%, 6.25%, and 12.5%) in both fixed ratio and progressive ratio procedures, but with equicaloric corn oil solutions (0%, 1.4%, 2.8%, and 5.6%) this finding was replicated only in the fixed ratio procedure. Likewise, addition of 1.4% oil to 3% or 12.5% sucrose increased fixed ratio, but not progressive ratio, reinforcers received relative to those of sucrose alone. Finally, addition of 3% vanilla flavoring did not change self-administration of 3% sucrose or 3% sucrose+1.4% oil solutions. These data suggest that, calorie-for-calorie, sucrose is the dominant reinforcing component of novel foods that contain a mixture of fat, sucrose, and flavor.

The Structure of Sucrose Synthase-1 from Arabidopsis thaliana and Its Functional Implications

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

Zheng, Yi; Anderson, Spencer; Zhang, Yanfeng

2014-10-02

Sucrose transport is the central system for the allocation of carbon resources in vascular plants. During growth and development, plants control carbon distribution by coordinating sites of sucrose synthesis and cleavage in different plant organs and different cellular locations. Sucrose synthase, which reversibly catalyzes sucrose synthesis and cleavage, provides a direct and reversible means to regulate sucrose flux. Depending on the metabolic environment, sucrose synthase alters its cellular location to participate in cellulose, callose, and starch biosynthesis through its interactions with membranes, organelles, and cytoskeletal actin. The x-ray crystal structure of sucrose synthase isoform 1 from Arabidopsis thaliana (AtSus1) hasmore » been determined as a complex with UDP-glucose and as a complex with UDP and fructose, at 2.8- and 2.85-{angstrom} resolutions, respectively. The AtSus1 structure provides insights into sucrose catalysis and cleavage, as well as the regulation of sucrose synthase and its interactions with cellular targets.« less

Effects of resource availability and propagule supply on native species recruitment in sagebrush ecosystems invaded by Bromus tectorum

USGS Publications Warehouse

Mazzola, Monica B.; Chambers, Jeanne C.; Blank, Robert R.; Pyke, David A.; Schupp, Eugene W.; Allcock, Kimberly G.; Doescher, Paul S.; Nowak, Robert S.

2011-01-01

Resource availability and propagule supply are major factors influencing establishment and persistence of both native and invasive species. Increased soil nitrogen (N) availability and high propagule inputs contribute to the ability of annual invasive grasses to dominate disturbed ecosystems. Nitrogen reduction through carbon (C) additions can potentially immobilize soil N and reduce the competitiveness of annual invasive grasses. Native perennial species are more tolerant of resource limiting conditions and may benefit if N reduction decreases the competitive advantage of annual invaders and if sufficient propagules are available for their establishment. Bromus tectorum, an exotic annual grass in the sagebrush steppe of western North America, is rapidly displacing native plant species and causing widespread changes in ecosystem processes. We tested whether nitrogen reduction would negatively affect B. tectorum while creating an opportunity for establishment of native perennial species. A C source, sucrose, was added to the soil, and then plots were seeded with different densities of both B. tectorum (0, 150, 300, 600, and 1,200 viable seeds m-2) and native species (0, 150, 300, and 600 viable seeds m-2). Adding sucrose had short-term (1 year) negative effects on available nitrogen and B. tectorum density, biomass and seed numbers, but did not increase establishment of native species. Increasing propagule availability increased both B. tectorum and native species establishment. Effects of B. tectorum on native species were density dependent and native establishment increased as B. tectorum propagule availability decreased. Survival of native seedlings was low indicating that recruitment is governed by the seedling stage.

Molecular Diagnostics of the Internal Motions of Massive Cores

NASA Astrophysics Data System (ADS)

Pineda, Jorge; Velusamy, T.; Goldsmith, P.; Li, D.; Peng, R.; Langer, W.

2009-12-01

We present models of the internal kinematics of massive cores in the Orion molecular cloud. We use a sample of cores studied by Velusamy et al. (2008) that show red, blue, and no asymmetry in their HCO+ line profiles in equal proportion, and which therefore may represent a sample of cores in different kinematic states. We use the radiative transfer code RATRAN (Hogerheijde & van der Tak 2000) to model several transitions of HCO+ and H13CO+ as well as the dust continuum emission, of a spherical model cloud with radial density, temperature, and velocity gradients. We find that an excitation and velocity gradients are prerequisites to reproduce the observed line profiles. We use the dust continuum emission to constrain the density and temperature gradients. This allows us to narrow down the functional forms of the velocity gradient giving us the opportunity to test several theoretical predictions of velocity gradients produced by the effect of magnetic fields (e.g. Tassis et. al. 2007) and turbulence (e.g. Vasquez-Semanedi et al 2007).

Ion temperature gradient mode driven solitons and shocks

NASA Astrophysics Data System (ADS)

Zakir, U.; Adnan, Muhammad; Haque, Q.; Qamar, Anisa; Mirza, Arshad M.

2016-04-01

Ion temperature gradient (ITG) driven solitons and shocks are studied in a plasma having gradients in the equilibrium number density and equilibrium ion temperature. In the linear regime, it is found that the ion temperature and the ratio of the gradient scale lengths, ηi=Ln/LT , affect both the real frequency and the growth rate of the ITG driven wave instability. In the nonlinear regime, for the first time we derive a Korteweg de Vries-type equation for the ITG mode, which admits solitary wave solution. It is found that the ITG mode supports only compressive solitons. Further, it is noticed that the soliton amplitude and width are sensitive to the parameter ηi=Ln/LT . Second, in the presence of dissipation in the system, we obtain a Burger type equation, which admits the shock wave solution. This work may be useful to understand the low frequency electrostatic modes in inhomogeneous electron-ion plasma having density and ion temperature gradients. For illustration, the model has been applied to tokamak plasma.

Method of accelerating photons by a relativistic plasma wave

DOEpatents

Dawson, John M.; Wilks, Scott C.

1990-01-01

Photons of a laser pulse have their group velocity accelerated in a plasma as they are placed on a downward density gradient of a plasma wave of which the phase velocity nearly matches the group velocity of the photons. This acceleration results in a frequency upshift. If the unperturbed plasma has a slight density gradient in the direction of propagation, the photon frequencies can be continuously upshifted to significantly greater values.

Development of a vector-tensor system to measure the absolute magnetic flux density and its gradient in magnetically shielded rooms.

PubMed

Voigt, J; Knappe-Grüneberg, S; Gutkelch, D; Haueisen, J; Neuber, S; Schnabel, A; Burghoff, M

2015-05-01

Several experiments in fundamental physics demand an environment of very low, homogeneous, and stable magnetic fields. For the magnetic characterization of such environments, we present a portable SQUID system that measures the absolute magnetic flux density vector and the gradient tensor. This vector-tensor system contains 13 integrated low-critical temperature (LTc) superconducting quantum interference devices (SQUIDs) inside a small cylindrical liquid helium Dewar with a height of 31 cm and 37 cm in diameter. The achievable resolution depends on the flux density of the field under investigation and its temporal drift. Inside a seven-layer mu-metal shield, an accuracy better than ±23 pT for the components of the static magnetic field vector and ±2 pT/cm for each of the nine components of the gradient tensor is reached by using the shifting method.

Development of a vector-tensor system to measure the absolute magnetic flux density and its gradient in magnetically shielded rooms

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

Voigt, J.; Knappe-Grüneberg, S.; Gutkelch, D.

2015-05-15

Several experiments in fundamental physics demand an environment of very low, homogeneous, and stable magnetic fields. For the magnetic characterization of such environments, we present a portable SQUID system that measures the absolute magnetic flux density vector and the gradient tensor. This vector-tensor system contains 13 integrated low-critical temperature (LTc) superconducting quantum interference devices (SQUIDs) inside a small cylindrical liquid helium Dewar with a height of 31 cm and 37 cm in diameter. The achievable resolution depends on the flux density of the field under investigation and its temporal drift. Inside a seven-layer mu-metal shield, an accuracy better than ±23more » pT for the components of the static magnetic field vector and ±2 pT/cm for each of the nine components of the gradient tensor is reached by using the shifting method.« less

Analytic calculations of hyper-Raman spectra from density functional theory hyperpolarizability gradients

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

Ringholm, Magnus; Ruud, Kenneth; Bast, Radovan

We present the first analytic calculations of the geometrical gradients of the first hyperpolarizability tensors at the density-functional theory (DFT) level. We use the analytically calculated hyperpolarizability gradients to explore the importance of electron correlation effects, as described by DFT, on hyper-Raman spectra. In particular, we calculate the hyper-Raman spectra of the all-trans and 11-cis isomers of retinal at the Hartree-Fock (HF) and density-functional levels of theory, also allowing us to explore the sensitivity of the hyper-Raman spectra on the geometrical characteristics of these structurally related molecules. We show that the HF results, using B3LYP-calculated vibrational frequencies and force fields,more » reproduce the experimental data for all-trans-retinal well, and that electron correlation effects are of minor importance for the hyper-Raman intensities.« less

Improving Rydberg Excitations within Time-Dependent Density Functional Theory with Generalized Gradient Approximations: The Exchange-Enhancement-for-Large-Gradient Scheme.

PubMed

Li, Shaohong L; Truhlar, Donald G

2015-07-14

Time-dependent density functional theory (TDDFT) with conventional local and hybrid functionals such as the local and hybrid generalized gradient approximations (GGA) seriously underestimates the excitation energies of Rydberg states, which limits its usefulness for applications such as spectroscopy and photochemistry. We present here a scheme that modifies the exchange-enhancement factor to improve GGA functionals for Rydberg excitations within the TDDFT framework while retaining their accuracy for valence excitations and for the thermochemical energetics calculated by ground-state density functional theory. The scheme is applied to a popular hybrid GGA functional and tested on data sets of valence and Rydberg excitations and atomization energies, and the results are encouraging. The scheme is simple and flexible. It can be used to correct existing functionals, and it can also be used as a strategy for the development of new functionals.

Improving Rydberg Excitations within Time-Dependent Density Functional Theory with Generalized Gradient Approximations: The Exchange-Enhancement-for-Large-Gradient Scheme

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

Li, Shaohong L.; Truhlar, Donald G.

Time-dependent density functional theory (TDDFT) with conventional local and hybrid functionals such as the local and hybrid generalized gradient approximations (GGA) seriously underestimates the excitation energies of Rydberg states, which limits its usefulness for applications such as spectroscopy and photochemistry. We present here a scheme that modifies the exchange-enhancement factor to improve GGA functionals for Rydberg excitations within the TDDFT framework while retaining their accuracy for valence excitations and for the thermochemical energetics calculated by ground-state density functional theory. The scheme is applied to a popular hybrid GGA functional and tested on data sets of valence and Rydberg excitations andmore » atomization energies, and the results are encouraging. The scheme is simple and flexible. It can be used to correct existing functionals, and it can also be used as a strategy for the development of new functionals.« less

Improving Rydberg Excitations within Time-Dependent Density Functional Theory with Generalized Gradient Approximations: The Exchange-Enhancement-for-Large-Gradient Scheme

DOE PAGES

Li, Shaohong L.; Truhlar, Donald G.

2015-05-22

Time-dependent density functional theory (TDDFT) with conventional local and hybrid functionals such as the local and hybrid generalized gradient approximations (GGA) seriously underestimates the excitation energies of Rydberg states, which limits its usefulness for applications such as spectroscopy and photochemistry. We present here a scheme that modifies the exchange-enhancement factor to improve GGA functionals for Rydberg excitations within the TDDFT framework while retaining their accuracy for valence excitations and for the thermochemical energetics calculated by ground-state density functional theory. The scheme is applied to a popular hybrid GGA functional and tested on data sets of valence and Rydberg excitations andmore » atomization energies, and the results are encouraging. The scheme is simple and flexible. It can be used to correct existing functionals, and it can also be used as a strategy for the development of new functionals.« less

How mesoscopic staircases condense to macroscopic barriers in confined plasma turbulence

NASA Astrophysics Data System (ADS)

Ashourvan, Arash; Diamond, P. H.

2016-11-01

This Rapid Communication sets forth the mechanism by which mesoscale staircase structures condense to form macroscopic states of enhanced confinement. Density, vorticity, and turbulent potential enstrophy are the variables for this model. Formation of the staircase structures is due to inhomogeneous mixing of (generalized) potential vorticity (PV). Such mixing results in the local sharpening of density and vorticity gradients. When PV gradients steepen, the density staircase structure develops into a lattice of mesoscale "jumps" and "steps," which are, respectively, regions of local gradient steepening and flattening. The jumps then merge and migrate in radius, leading to the emergence of a new macroscale profile structure, so indicating that profile self-organization is a global process, which may be described by a local, but nonlinear model. This work predicts and demonstrates how mesoscale condensation of staircases leads to global states of enhanced confinement.

Pilot scale production of highly efficacious and stable enterovirus 71 vaccine candidates.

PubMed

Chou, Ai-Hsiang; Liu, Chia-Chyi; Chang, Cheng-Peng; Guo, Meng-Shin; Hsieh, Shih-Yang; Yang, Wen-Hsueh; Chao, Hsin-Ju; Wu, Chien-Long; Huang, Ju-Lan; Lee, Min-Shi; Hu, Alan Yung-Chi; Lin, Sue-Chen; Huang, Yu-Yun; Hu, Mei-Hua; Chow, Yen-Hung; Chiang, Jen-Ron; Chang, Jui-Yuan; Chong, Pele

2012-01-01

Enterovirus 71 (EV71) has caused several epidemics of hand, foot and mouth diseases (HFMD) in Asia and now is being recognized as an important neurotropic virus. Effective medications and prophylactic vaccine against EV71 infection are urgently needed. Based on the success of inactivated poliovirus vaccine, a prototype chemically inactivated EV71 vaccine candidate has been developed and currently in human phase 1 clinical trial. In this report, we present the development of a serum-free cell-based EV71 vaccine. The optimization at each step of the manufacturing process was investigated, characterized and quantified. In the up-stream process development, different commercially available cell culture media either containing serum or serum-free was screened for cell growth and virus yield using the roller-bottle technology. VP-SFM serum-free medium was selected based on the Vero cell growth profile and EV71 virus production. After the up-stream processes (virus harvest, diafiltration and concentration), a combination of gel-filtration liquid chromatography and/or sucrose-gradient ultracentrifugation down-stream purification processes were investigated at a pilot scale of 40 liters each. Although the combination of chromatography and sucrose-gradient ultracentrifugation produced extremely pure EV71 infectious virus particles, the overall yield of vaccine was 7-10% as determined by a VP2-based quantitative ELISA. Using chromatography as the downstream purification, the virus yield was 30-43%. To retain the integrity of virus neutralization epitopes and the stability of the vaccine product, the best virus inactivation was found to be 0.025% formalin-treatment at 37 °C for 3 to 6 days. Furthermore, the formalin-inactivated virion vaccine candidate was found to be stable for >18 months at 4 °C and a microgram of viral proteins formulated with alum adjuvant could induce strong virus-neutralizing antibody responses in mice, rats, rabbits, and non-human primates. These results provide valuable information supporting the current cell-based serum-free EV71 vaccine candidate going into human Phase I clinical trials.

Isolation of Cytoplasmic Pituitary Granules with Gonadotropic Activity

PubMed Central

Hartley, Marshall W.; McShan, W. H.; Ris, Hans

1960-01-01

A fraction isolated from the anterior pituitary glands of rats castrate for 8 weeks contained essentially a single cytoplasmic constituent with which the major portion of the gonadotropic hormone activity was associated. The glands were homogenized in an 0.25 M sucrose + 7.3 per cent polyvinylpyrrolidone (PVP) solution and fractionated by differential centrifugation to give a heterogeneous small granule fraction which contained almost all the gonadotropic hormone activity. The active supernatant containing this small granule fraction was separated into layers by isopycnic gradient centrifugation on a continuous 6 to 45 per cent sucrose + 17.5 per cent "diodrast" + 5 x 10-4 M "versene" gradient at 100,000 g for 2 hours. Three layers were obtained and the pellet from the active bottom layer was sectioned, examined with the electron microscope, and found to contain 200 mµ granules, mitochondria, ergastoplasm, and other cellular debris. This layer was fractionated further by isopycnic and differential centrifugation to obtain a pellet which contained the major portion of the gonadotropic hormone activity. Because of the heterogeneity of this fraction, due to the contamination of the 200 mµ granules with mitochondria and other cellular debris, the active layer and the resuspended active pellet, obtained by centrifuging this layer first at 17,000 g then diluting the supernatant and centrifuging at 30,000 g for 1 hour, were filtered through Millipore HA paper with a pore size of 0.45 µ. The cytoplasmic material containing the gonadotropic hormone activity passed through the filter paper and this activity was recovered in the pellets obtained by centrifuging at 100,000 g for 1 hour. These active pellets consisted almost entirely of 200 mµ granules with a minimum amount of contamination, and they contained the major portion of the gonadotropic hormone activity with practically none remaining in the supernatant fraction. These results are discussed in view of their importance to the cytology of the pituitary gland. PMID:14400127

The rate of uptake of cardiac glycosides into human cultured cells and the effects of chloroquine on it.

PubMed

Algharably, N; Owler, D; Lamb, J F

1986-10-15

HeLa cells grown on Petri dishes were either pulse labelled with various cardiac glycosides or grown in low concentrations of them for up to 2 days; either in the presence of chloroquine or not. The cells were then homogenised and the cell free homogenate layered on a continuous sucrose gradient; and the glycoside content and that of various markers measured. In another series of experiments HeLa cells were grown on plastic beads under the above conditions and then the content of glycosides and of some marker enzymes measured. The rate of internalisation of ouabain, digoxin and digitoxin from the plasma membrane preparation produced by the bead method is at 9% hr-1, similar to the rate of loss of digoxin and digitoxin from whole cells but much faster than that of ouabain. In the sucrose gradient experiments it was found that [3H]ouabain, digoxin and digitoxin all initially co-distribute with the plasma membrane marker, 5'-nucleotidase, and then leave this fraction of the homogenate at a fast rate when kept at 37 degrees, to co-distribute with the lysosomal marker, beta-hexosaminidase. At 2 degrees the ouabain remains co-distributed with the plasma membrane marker. The rate of transfer is estimated to be some 90% hr-1, much faster than previously thought. Chloroquine causes an increased retention of digoxin and digitoxin in the lysosomal fraction of the homogenate. These results are best explained by supposing that the sodium pump-glycoside complex rapidly enters a region of the peripheral cytoplasm, and that this region then controls the subsequent exit of digoxin and digitoxin from the cell. The main barrier for ouabain occurs at a stage later than this. The consequences of this model on other aspects of pump activity is discussed.

In vivo transport of three radioactive [18F]-fluorinated deoxysucrose analogs by the maize sucrose transporter ZmSUT1.

PubMed

Tran, Thu M; Hampton, Carissa S; Brossard, Tom W; Harmata, Michael; Robertson, J David; Jurisson, Silvia S; Braun, David M

2017-06-01

Sucrose transporter (SUT) proteins translocate sucrose across cell membranes; however, mechanistic aspects of sucrose binding by SUTs are not well resolved. Specific hydroxyl groups in sucrose participate in hydrogen bonding with SUT proteins. We previously reported that substituting a radioactive fluorine-18 [ 18 F] at the C-6' position within the fructosyl moiety of sucrose did not affect sucrose transport by the maize (Zea mays) ZmSUT1 protein. To determine how 18 F substitution of hydroxyl groups at two other positions within sucrose, the C-1' in the fructosyl moiety or the C-6 in the glucosyl moiety, impact sucrose transport, we synthesized 1'-[F 18 ]fluoro-1'-deoxysucrose and 6-[F 18 ]fluoro-6-deoxysucrose ([ 18 F]FDS) analogs. Each [ 18 F]FDS derivative was independently introduced into wild-type or sut1 mutant plants, which are defective in sucrose phloem loading. All three (1'-, 6'-, and 6-) [ 18 F]FDS derivatives were efficiently and equally translocated, similarly to carbon-14 [ 14 C]-labeled sucrose. Hence, individually replacing the hydroxyl groups at these positions within sucrose does not interfere with substrate recognition, binding, or membrane transport processes, and hydroxyl groups at these three positions are not essential for hydrogen bonding between sucrose and ZmSUT1. [ 18 F]FDS imaging afforded several advantages compared to [ 14 C]-sucrose detection. We calculated that 1'-[ 18 F]FDS was transported at approximately a rate of 0.90 ± 0.15 m.h-1 in wild-type leaves, and at 0.68 ± 0.25 m.h-1 in sut1 mutant leaves. Collectively, our data indicated that [ 18 F]FDS analogs are valuable tools to probe sucrose-SUT interactions and to monitor sucrose transport in plants. Copyright © 2017 Elsevier Masson SAS. All rights reserved.

Leaf dynamics in growth and reproduction of Xanthium canadense as influenced by stand density

PubMed Central

Ogawa, Takahiro; Oikawa, Shimpei; Hirose, Tadaki

2015-01-01

Background and Aims Leaf longevity is controlled by the light gradient in the canopy and also by the nitrogen (N) sink strength in the plant. Stand density may influence leaf dynamics through its effects on light gradient and on plant growth and reproduction. This study tests the hypothesis that the control by the light gradient is manifested more in the vegetative period, whereas the opposite is true when the plant becomes reproductive and develops a strong N sink. Methods Stands of Xanthium canadense were established at two densities. Emergence, growth and death of every leaf on the main stem and branches, and plant growth and N uptake were determined from germination to full senescence. Mean residence time and dry mass productivity were calculated per leaf number, leaf area, leaf mass and leaf N (collectively termed ‘leaf variables’) in order to analyse leaf dynamics and its effect on plant growth. Key Results Branching and reproductive activities were higher at low than at high density. Overall there was no significant difference in mean residence time of leaf variables between the two stands. However, early leaf cohorts on the main stem had a longer retention time at low density, whereas later cohorts had a longer retention time at high density. Branch leaves emerged earlier and tended to live longer at low than at high density. Leaf efficiencies, defined as carbon export per unit investment of leaf variables, were higher at low density in all leaf variables except for leaf number. Conclusions In the vegetative phase of plant growth, the light gradient strongly controls leaf longevity, whereas later the effects of branching and reproductive activities become stronger and over-rule the effect of light environment. As leaf N supports photosynthesis and also works as an N source for plant development, N use is pivotal in linking leaf dynamics with plant growth and reproduction. PMID:26248476

Sucrose metabolic pathways in sweetgum and pecan seedlings

Treesearch

S.S. Sung; P.P. Kormanik; D.P. Xu; C.C. Black

1989-01-01

Sucrose metabolism and glycolysis were studied in one- to two-year-old seedlings of sweetgum (Liquidambar styraciflua L.) and pecan (Carya illinoinensis (Wangenh.) C. Koch). The sucrose synthase pathway was identified as the dominant sucrose metabolic activity in sucrose sink tissues such as terminal buds and the root cambial...

Succinic acid production from sucrose by Actinobacillus succinogenes NJ113.

PubMed

Jiang, Min; Dai, Wenyu; Xi, Yonglan; Wu, Mingke; Kong, Xiangping; Ma, Jiangfeng; Zhang, Min; Chen, Kequan; Wei, Ping

2014-02-01

In this study, sucrose, a reproducible disaccharide extracted from plants, was used as the carbon source for the production of succinic acid by Actinobacillus succinogenes NJ113. During serum bottle fermentation, the succinic acid concentration reached 57.1g/L with a yield of 71.5%. Further analysis of the sucrose utilization pathways revealed that sucrose was transported and utilized via a sucrose phosphotransferase system, sucrose-6-phosphate hydrolase, and a fructose PTS. Compared to glucose utilization in single pathway, more pathways of A. succinogenes NJ113 are dependent on sucrose utilization. By changing the control strategy in a fed-batch culture to alleviate sucrose inhibition, 60.5g/L of succinic acid was accumulated with a yield of 82.9%, and the productivity increased by 35.2%, reaching 2.16g/L/h. Thus utilization of sucrose has considerable potential economics and environmental meaning. Copyright © 2014 Elsevier Ltd. All rights reserved.

Dissecting genomic hotspots underlying seed protein, oil, and sucrose content in an interspecific mapping population of soybean using high-density linkage mapping.

PubMed

Patil, Gunvant; Vuong, Tri D; Kale, Sandip; Valliyodan, Babu; Deshmukh, Rupesh; Zhu, Chengsong; Wu, Xiaolei; Bai, Yonghe; Yungbluth, Dennis; Lu, Fang; Kumpatla, Siva; Shannon, J Grover; Varshney, Rajeev K; Nguyen, Henry T

2018-04-04

The cultivated [Glycine max (L) Merr.] and wild [Glycine soja Siebold & Zucc.] soybean species comprise wide variation in seed composition traits. Compared to wild soybean, cultivated soybean contains low protein, high oil, and high sucrose. In this study, an interspecific population was derived from a cross between G. max (Williams 82) and G. soja (PI 483460B). This recombinant inbred line (RIL) population of 188 lines was sequenced at 0.3× depth. Based on 91 342 single nucleotide polymorphisms (SNPs), recombination events in RILs were defined, and a high-resolution bin map was developed (4070 bins). In addition to bin mapping, quantitative trait loci (QTL) analysis for protein, oil, and sucrose was performed using 3343 polymorphic SNPs (3K-SNP), derived from Illumina Infinium BeadChip sequencing platform. The QTL regions from both platforms were compared, and a significant concordance was observed between bin and 3K-SNP markers. Importantly, the bin map derived from next-generation sequencing technology enhanced mapping resolution (from 1325 to 50 Kb). A total of five, nine, and four QTLs were identified for protein, oil, and sucrose content, respectively, and some of the QTLs coincided with soybean domestication-related genomic loci. The major QTL for protein and oil were mapped on Chr. 20 (qPro_20) and suggested negative correlation between oil and protein. In terms of sucrose content, a novel and major QTL were identified on Chr. 8 (qSuc_08) and harbours putative genes involved in sugar transport. In addition, genome-wide association using 91 342 SNPs confirmed the genomic loci derived from QTL mapping. A QTL-based haplotype using whole-genome resequencing of 106 diverse soybean lines identified unique allelic variation in wild soybean that could be utilized to widen the genetic base in cultivated soybean. © 2018 The Authors. Plant Biotechnology Journal published by Society for Experimental Biology and The Association of Applied Biologists and John Wiley & Sons Ltd.

Study of homogeneous bubble nucleation in liquid carbon dioxide by a hybrid approach combining molecular dynamics simulation and density gradient theory

NASA Astrophysics Data System (ADS)

Langenbach, K.; Heilig, M.; Horsch, M.; Hasse, H.

2018-03-01

A new method for predicting homogeneous bubble nucleation rates of pure compounds from vapor-liquid equilibrium (VLE) data is presented. It combines molecular dynamics simulation on the one side with density gradient theory using an equation of state (EOS) on the other. The new method is applied here to predict bubble nucleation rates in metastable liquid carbon dioxide (CO2). The molecular model of CO2 is taken from previous work of our group. PC-SAFT is used as an EOS. The consistency between the molecular model and the EOS is achieved by adjusting the PC-SAFT parameters to VLE data obtained from the molecular model. The influence parameter of density gradient theory is fitted to the surface tension of the molecular model. Massively parallel molecular dynamics simulations are performed close to the spinodal to compute bubble nucleation rates. From these simulations, the kinetic prefactor of the hybrid nucleation theory is estimated, whereas the nucleation barrier is calculated from density gradient theory. This enables the extrapolation of molecular simulation data to the whole metastable range including technically relevant densities. The results are tested against available experimental data and found to be in good agreement. The new method does not suffer from typical deficiencies of classical nucleation theory concerning the thermodynamic barrier at the spinodal and the bubble size dependence of surface tension, which is typically neglected in classical nucleation theory. In addition, the density in the center of critical bubbles and their surface tension is determined as a function of their radius. The usual linear Tolman correction to the capillarity approximation is found to be invalid.

Study of homogeneous bubble nucleation in liquid carbon dioxide by a hybrid approach combining molecular dynamics simulation and density gradient theory.

PubMed

Langenbach, K; Heilig, M; Horsch, M; Hasse, H

2018-03-28

A new method for predicting homogeneous bubble nucleation rates of pure compounds from vapor-liquid equilibrium (VLE) data is presented. It combines molecular dynamics simulation on the one side with density gradient theory using an equation of state (EOS) on the other. The new method is applied here to predict bubble nucleation rates in metastable liquid carbon dioxide (CO 2 ). The molecular model of CO 2 is taken from previous work of our group. PC-SAFT is used as an EOS. The consistency between the molecular model and the EOS is achieved by adjusting the PC-SAFT parameters to VLE data obtained from the molecular model. The influence parameter of density gradient theory is fitted to the surface tension of the molecular model. Massively parallel molecular dynamics simulations are performed close to the spinodal to compute bubble nucleation rates. From these simulations, the kinetic prefactor of the hybrid nucleation theory is estimated, whereas the nucleation barrier is calculated from density gradient theory. This enables the extrapolation of molecular simulation data to the whole metastable range including technically relevant densities. The results are tested against available experimental data and found to be in good agreement. The new method does not suffer from typical deficiencies of classical nucleation theory concerning the thermodynamic barrier at the spinodal and the bubble size dependence of surface tension, which is typically neglected in classical nucleation theory. In addition, the density in the center of critical bubbles and their surface tension is determined as a function of their radius. The usual linear Tolman correction to the capillarity approximation is found to be invalid.

Conditional suicide system of Escherichia coli released into soil that uses the Bacillus subtilis sacB gene.

PubMed Central

Recorbet, G; Robert, C; Givaudan, A; Kudla, B; Normand, P; Faurie, G

1993-01-01

The sacB gene from Bacillus subtilis confers sucrose sensitivity upon gram-negative bacteria. The gene was investigated for use as a potential conditional suicide system for Escherichia coli released into soil. To ensure against the loss of the cell death function encoded under nonselective conditions, the nptI-sacR-B suicide cassette was inserted into the E. coli chromosome by using a circular nonreplicative integration vector. Stability studies yielded no loss of the suicide cassette in the integrated E. coli EL1026 strain. sacB induction in the absence of a selective pressure resulted in a lysis efficiency of up to 99.9%. The microcosm experiments confirmed the ability of the suicide cassette to limit the growth and reduce the survival of E. coli strains released into soil. Sucrose addition to sterile soil resulted in a 10(-3)-fold reduction of the final E. coli population density. sacB induction prevented the proliferation and triggered the rapid disappearance of E. coli from natural soil. Mutation to sucrose tolerance occurred at a frequency of 10(-5), making E. coli EL1026 a potential counterselectable donor strain for gene transfer studies. Specificity and potential adaptability to a wide range of gram-negative bacteria are additional conveniences of this conditional suicide system for the containment and counterselection of engineered microorganisms. PMID:8517732

Conditional suicide system of Escherichia coli released into soil that uses the Bacillus subtilis sacB gene.

PubMed

Recorbet, G; Robert, C; Givaudan, A; Kudla, B; Normand, P; Faurie, G

1993-05-01

The sacB gene from Bacillus subtilis confers sucrose sensitivity upon gram-negative bacteria. The gene was investigated for use as a potential conditional suicide system for Escherichia coli released into soil. To ensure against the loss of the cell death function encoded under nonselective conditions, the nptI-sacR-B suicide cassette was inserted into the E. coli chromosome by using a circular nonreplicative integration vector. Stability studies yielded no loss of the suicide cassette in the integrated E. coli EL1026 strain. sacB induction in the absence of a selective pressure resulted in a lysis efficiency of up to 99.9%. The microcosm experiments confirmed the ability of the suicide cassette to limit the growth and reduce the survival of E. coli strains released into soil. Sucrose addition to sterile soil resulted in a 10(-3)-fold reduction of the final E. coli population density. sacB induction prevented the proliferation and triggered the rapid disappearance of E. coli from natural soil. Mutation to sucrose tolerance occurred at a frequency of 10(-5), making E. coli EL1026 a potential counterselectable donor strain for gene transfer studies. Specificity and potential adaptability to a wide range of gram-negative bacteria are additional conveniences of this conditional suicide system for the containment and counterselection of engineered microorganisms.

Structural development of sucrose-sweetened and sucrose-free sponge cakes during baking.

PubMed

Baeva, Marianna Rousseva; Terzieva, Vesselina Velichkova; Panchev, Ivan Nedelchev

2003-06-01

The influence of sucrose, wheat starch and sorbitol upon the heat- and mass-exchanging processes forming the structure of sponge cake was studied. Under the influence of wheat starch and sorbitol the structure of the sucrose-free sponge cake was formed at more uniform total moisture release. This process was done at lower temperatures and smoother change of the sponge cake height with respect to the sucrose-sweetened sponge cake. The porous and steady structure of both cakes was finally formed at identical time--between 18th and 19th minute, at the applied conditions for baking of each batter (metal pan with diameter 15.4 cm and depth 6.2 cm containing 300 g of batter and placed in an electric oven "Rahovetz-02", Bulgaria for 30 min at 180 degrees C). The water-losses at the end of baking (10.30% and 10.40% for the sucrose-sweetened cake and sucrose-free cake, respectively) and the final temperatures reached in the crumb central layers (96.6 degrees C and 96.3 degrees C for the sucrose-sweetened cake and sucrose-free cake, respectively) during baking of both samples were not statistically different. The addition of wheat starch and sorbitol in sucrose-free sponge cake lead to the statistically different values for the porosity (76.15% and 72.98%) and the volume (1014.17 cm3 and 984.25 cm3) of the sucrose-sweetened and sucrose-free sponge cakes, respectively. As a result, the sucrose-free sponge cake formed during baking had a more homogeneous and finer microstructure with respect to that ofthe sucrose-sweetened one.

Biochemical characterization of native Usher protein complexes from a vesicular subfraction of tracheal epithelial cells.

PubMed

Zallocchi, Marisa; Sisson, Joseph H; Cosgrove, Dominic

2010-02-16

Usher syndrome is the major cause of deaf/blindness in the world. It is a genetic heterogeneous disorder, with nine genes already identified as causative for the disease. We noted expression of all known Usher proteins in bovine tracheal epithelial cells and exploited this system for large-scale biochemical analysis of Usher protein complexes. The dissected epithelia were homogenized in nondetergent buffer and sedimented on sucrose gradients. At least two complexes were evident after the first gradient: one formed by specific isoforms of CDH23, PCDH15, and VLGR-1 and a different one at the top of the gradient that included all of the Usher proteins and rab5, a transport vesicle marker. TEM analysis of these top fractions found them enriched in 100-200 nm vesicles, confirming a vesicular association of the Usher complex(es). Immunoisolation of these vesicles confirmed some of the associations already predicted and identified novel interactions. When the vesicles are lysed in the presence of phenylbutyrate, most of the Usher proteins cosediment into the gradient at a sedimentation coefficient of approximately 50 S, correlating with a predicted molecular mass of 2 x 10(6) Da. Although it is still unclear whether there is only one complex or several independent complexes that are trafficked within distinct vesicular pools, this work shows for the first time that native Usher protein complexes occur in vivo. This complex(es) is present primarily in transport vesicles at the apical pole of tracheal epithelial cells, predicting that Usher proteins may be directionally transported as complexes in hair cells and photoreceptors.

BIOCHEMICAL CHARACTERIZATION OF NATIVE USHER PROTEIN COMPLEXES FROM A VESICULAR SUBFRACTION OF TRACHEAL EPITHELIAL CELLS†

PubMed Central

Zallocchi, Marisa; Sisson, Joseph H.; Cosgrove, Dominic

2010-01-01

Usher syndrome is the major cause of deaf/blindness in the world. It is a genetic heterogeneous disorder, with nine genes already identified as causative for the disease. We noted expression of all known Usher proteins in bovine tracheal epithelial cells, and exploited this system for large-scale biochemical analysis of Usher protein complexes. The dissected epithelia were homogenized in non-detergent buffer, and sedimented on sucrose gradients. At least two complexes were evident after the first gradient: one formed by specific isoforms of CDH23, PCDH15 and VLGR-1, and a different one at the top of the gradient that included all the Usher proteins and rab5, a transport vesicle marker. TEM analysis of these top fractions found them enriched in 100–200 nm vesicles, confirming a vesicular association of the Usher complex(es). Immunoisolation of these vesicles confirmed some of the associations already predicted and identified novel interactions. When the vesicles are lysed in the presence of phenylbutyrate, most of the Usher proteins co-sediment into the gradient at a sedimentation coefficient of approximately 50S, correlating with a predicted molecular mass of 2 × 106 Daltons. Although it is still unclear whether there is only one complex or several independent complexes that are trafficked within distinct vesicular pools, this work shows for the first time that native Usher proteins complexes occur in vivo. This complex(es) is present primarily in transport vesicles at the apical pole of tracheal epithelial cells, predicting that Usher proteins may be directionally transported as complexes in hair cells and photoreceptors. PMID:20058854

Metabolic engineering to expand the substrate spectrum of Pseudomonas putida toward sucrose.

PubMed

Löwe, Hannes; Schmauder, Lukas; Hobmeier, Karina; Kremling, Andreas; Pflüger-Grau, Katharina

2017-08-01

Sucrose is an important disaccharide used as a substrate in many industrial applications. It is a major component of molasses, a cheap by-product of the sugar industry. Unfortunately, not all industrially relevant organisms, among them Pseudomonas putida, are capable of metabolizing sucrose. We chose a metabolic engineering approach to circumvent this blockage and equip P. putida with the activities necessary to consume sucrose. Therefore, we constructed a pair of broad-host range mini-transposons (pSST - sucrose splitting transposon), carrying either cscA, encoding an invertase able to split sucrose into glucose and fructose, or additionally cscB, encoding a sucrose permease. Introduction of cscA was sufficient to convey sucrose consumption and the additional presence of cscB had no further effect, though the sucrose permease was built and localized to the membrane. Sucrose was split extracellularly by the activity of the invertase CscA leaking out of the cell. The transposons were also used to confer sucrose consumption to Cupriavidus necator. Interestingly, in this strain, CscB acted as a glucose transporter, such that C. necator also gained the ability to grow on glucose. Thus, the pSST transposons are functional tools to extend the substrate spectrum of Gram-negative bacterial strains toward sucrose. © 2017 The Authors. MicrobiologyOpen published by John Wiley & Sons Ltd.