Thermoinactivation analysis of vacuolar H(+)-pyrophosphatase.

PubMed

Yang, Su J; Jiang, Shih S; Hsiao, Yi Y; Van, Ru C; Pan, Yih J; Pan, Rong L

2004-06-07

Vacuolar H(+)-translocating pyrophosphatase (H(+)-PPase; EC 3.6.1.1) catalyzes both the hydrolysis of PP(i) and the electrogenic translocation of proton from the cytosol to the lumen of the vacuole. Vacuolar H(+)-PPase, purified from etiolated hypocotyls of mung bean (Vigna radiata L.), is a homodimer with a molecular mass of 145 kDa. To investigate the relationship between structure and function of this H(+)-translocating enzyme, thermoinactivation analysis was employed. Thermoinactivation studies suggested that vacuolar H(+)-PPase consists of two distinct states upon heat treatment and exhibited different transition temperatures in the presence and absence of ligands (substrate and inhibitors). Substrate protection of H(+)-PPase stabilizes enzyme structure by increasing activation energy from 54.9 to 70.2 kJ/mol. We believe that the conformation of this enzyme was altered in the presence of substrate to protect against the thermoinactivation. In contrast, the modification of H(+)-PPase by inhibitor (fluorescein 5'-isothiocyanate; FITC) augmented the inactivation by heat treatment. The native, substrate-bound, and FITC-labeled vacuolar H(+)-PPases possess probably distinct conformation and show different modes of susceptibility to thermoinactivation. Our results also indicate that the structure of one subunit of this homodimer exerts long distance effect on the other, suggesting a specific subunit-subunit interaction in vacuolar H(+)-PPase. A working model was proposed to interpret the relationship of the structure and function of vacuolar H(+)-PPase.

Differential expression of vacuolar and defective cell wall invertase genes in roots and seeds of metalliferous and non-metalliferous populations of Rumex dentatus under copper stress.

PubMed

Xu, Zhong-Rui; Cai, Shen-Wen; Huang, Wu-Xing; Liu, Rong-Xiang; Xiong, Zhi-Ting

2018-01-01

Acid invertase activities in roots and young seeds of a metalliferous population (MP) of Rumex dentatus were previously observed to be significantly higher than those of a non-metalliferous population (NMP) under Cu stress. To date, no acid invertase gene has been cloned from R. dentatus. Here, we isolated four full-length cDNAs from the two populations of R. dentatus, presumably encoding cell wall (RdnCIN1 and RdmCIN1 from the NMP and MP, respectively) and vacuolar invertases (RdnVIN1 and RdmVIN1 from the NMP and MP, respectively). Unexpectedly, RdnCIN1 and RdmCIN1 most likely encode special defective invertases with highly attenuated sucrose-hydrolyzing capacity. The transcript levels of RdmCIN1 were significantly higher than those of RdnCIN1 in roots and young seeds under Cu stress, whereas under control conditions, the former was initially lower than the latter. Unexpected high correlations were observed between the transcript levels of RdnCIN1 and RdmCIN1 and the activity of cell wall invertase, even though RdnCIN1 and RdmCIN1 do not encode catalytically active invertases. Similarly, the transcript levels of RdmVIN1 in roots and young seeds were increased under Cu stress, whereas those of RdnVIN1 were decreased. The high correlations between the transcript levels of RdnVIN1 and RdmVIN1 and the activity of vacuolar invertase indicate that RdnVIN1 and RdmVIN1 might control distinct vacuolar invertase activities in the two populations. Moreover, a possible indirect role for acid invertases in Cu tolerance, mediated by generating a range of sugars used as nutrients and signaling molecules, is discussed. Copyright © 2017 Elsevier Inc. All rights reserved.

The plant homolog to the human sodium/dicarboxylic cotransporter is the vacuolar malate carrier

PubMed Central

Emmerlich, Vera; Linka, Nicole; Reinhold, Thomas; Hurth, Marco A.; Traub, Michaela; Martinoia, Enrico; Neuhaus, H. Ekkehard

2003-01-01

Malate plays a central role in plant metabolism. It is an intermediate in the Krebs and glyoxylate cycles, it is the store for CO2 in C4 and crassulacean acid metabolism plants, it protects plants from aluminum toxicity, it is essential for maintaining the osmotic pressure and charge balance, and it is therefore involved in regulation of stomatal aperture. To fulfil many of these roles, malate has to be accumulated within the large central vacuole. Many unsuccessful efforts have been made in the past to identify the vacuolar malate transporter; here, we describe the identification of the vacuolar malate transporter [A. thaliana tonoplast dicarboxylate transporter (AttDT)]. This transporter exhibits highest sequence similarity to the human sodium/dicarboxylate cotransporter. Independent T-DNA [portion of the Ti (tumor-inducing) plasmid that is transferred to plant cells] Arabidopsis mutants exhibit substantially reduced levels of leaf malate, but respire exogenously applied [14C]malate faster than the WT. An AttDT-GFP fusion protein was localized to vacuole. Vacuoles isolated from Arabidopsis WT leaves exhibited carbonylcyanide m-chlorophenylhydrazone and citrate inhibitable malate transport, which was not stimulated by sodium. Vacuoles isolated from mutant plants import [14C]-malate at strongly reduced rates, confirming that this protein is the vacuolar malate transporter. PMID:12947042

The plant homolog to the human sodium/dicarboxylic cotransporter is the vacuolar malate carrier.

PubMed

Emmerlich, Vera; Linka, Nicole; Reinhold, Thomas; Hurth, Marco A; Traub, Michaela; Martinoia, Enrico; Neuhaus, H Ekkehard

2003-09-16

Malate plays a central role in plant metabolism. It is an intermediate in the Krebs and glyoxylate cycles, it is the store for CO2 in C4 and crassulacean acid metabolism plants, it protects plants from aluminum toxicity, it is essential for maintaining the osmotic pressure and charge balance, and it is therefore involved in regulation of stomatal aperture. To fulfil many of these roles, malate has to be accumulated within the large central vacuole. Many unsuccessful efforts have been made in the past to identify the vacuolar malate transporter; here, we describe the identification of the vacuolar malate transporter [A. thaliana tonoplast dicarboxylate transporter (AttDT)]. This transporter exhibits highest sequence similarity to the human sodium/dicarboxylate cotransporter. Independent T-DNA [portion of the Ti (tumor-inducing) plasmid that is transferred to plant cells] Arabidopsis mutants exhibit substantially reduced levels of leaf malate, but respire exogenously applied [14C]malate faster than the WT. An AttDT-GFP fusion protein was localized to vacuole. Vacuoles isolated from Arabidopsis WT leaves exhibited carbonylcyanide m-chlorophenylhydrazone and citrate inhibitable malate transport, which was not stimulated by sodium. Vacuoles isolated from mutant plants import [14C]-malate at strongly reduced rates, confirming that this protein is the vacuolar malate transporter.

LegC3, an Effector Protein from Legionella pneumophila, Inhibits Homotypic Yeast Vacuole Fusion In Vivo and In Vitro

PubMed Central

Bennett, Terry L.; Kraft, Shannon M.; Reaves, Barbara J.; Mima, Joji; O’Brien, Kevin M.; Starai, Vincent J.

2013-01-01

During infection, the intracellular pathogenic bacterium Legionella pneumophila causes an extensive remodeling of host membrane trafficking pathways, both in the construction of a replication-competent vacuole comprised of ER-derived vesicles and plasma membrane components, and in the inhibition of normal phagosome:endosome/lysosome fusion pathways. Here, we identify the LegC3 secreted effector protein from L. pneumophila as able to inhibit a SNARE- and Rab GTPase-dependent membrane fusion pathway in vitro, the homotypic fusion of yeast vacuoles (lysosomes). This vacuole fusion inhibition appeared to be specific, as similar secreted coiled-coiled domain containing proteins from L. pneumophila, LegC7/YlfA and LegC2/YlfB, did not inhibit vacuole fusion. The LegC3-mediated fusion inhibition was reversible by a yeast cytosolic extract, as well as by a purified soluble SNARE, Vam7p. LegC3 blocked the formation of trans-SNARE complexes during vacuole fusion, although we did not detect a direct interaction of LegC3 with the vacuolar SNARE protein complexes required for fusion. Additionally, LegC3 was incapable of inhibiting a defined synthetic model of vacuolar SNARE-driven membrane fusion, further suggesting that LegC3 does not directly inhibit the activity of vacuolar SNAREs, HOPS complex, or Sec17p/18p during membrane fusion. LegC3 is likely utilized by Legionella to modulate eukaryotic membrane fusion events during pathogenesis. PMID:23437241

Simulation of defects in fusion plasma first wall materials

NASA Astrophysics Data System (ADS)

T, Troev; N, Nankov; T, Yoshiie

2014-06-01

Numerical calculations of radiation damages in beryllium, alpha-iron and tungsten irradiated by fusion neutrons were performed using molecular dynamics (MD) simulations. The displacement cascades efficiency has been calculated using the Norgett-Robinson-Torrens (NRT) formula, the universal pair-potential of Ziegler-Biersack-Littmark (ZBL) and the EAM inter-atomic potential. The pair potential overestimates the defects production by a factor of 2. The ZBL pair potential results and the EAM are comparable at higher primary knock-on atom (PKA) energies (E > 100 keV). We found that the most common types of defects are single vacancies, di-vacancies, interstitials and small number of interstitial clusters. On the bases of calculated results, the behavior of vacancies, empty nano-voids and nano-voids with hydrogen and helium were discussed.

Relation between lung perfusion defects and intravascular clots in acute pulmonary thromboembolism: assessment with breath-hold SPECT-CT pulmonary angiography fusion images.

PubMed

Suga, Kazuyoshi; Yasuhiko, Kawakami; Iwanaga, Hideyuki; Tokuda, Osamu; Matsunaga, Naofumi

2008-09-01

The relation between lung perfusion defects and intravascular clots in acute pulmonary thromboembolism (PTE) was comprehensively assessed on deep-inspiratory breath-hold (DIBrH) perfusion SPECT-computed tomographic pulmonary angiography (CTPA) fusion images. Subjects were 34 acute PTE patients, who had successfully performed DIBrH perfusion SPECT using a dual-headed SPECT and a respiratory tracking system. Automated DIBrH SPECT-CTPA fusion images were used to assess the relation between lung perfusion defects and intravascular clots detected by CTPA. DIBrH SPECT visualized 175 lobar/segmental or subsegmental defects in 34 patients, and CTPA visualized 61 intravascular clots at variable locations in 30 (88%) patients, but no clots in four (12%) patients. In 30 patients with clots, the fusion images confirmed that 69 (41%) perfusion defects (20 segmental, 45 subsegmental and 4 lobar defects) of total 166 defects were located in lung territories without clots, although the remaining 97 (58%) defects were located in lung territories with clots. Perfusion defect was absent in lung territories with clots (one lobar branch and three segmental branches) in four (12%) of these patients. In four patients without clots, nine perfusion defects including four segmental ones were present. Because of unexpected dissociation between intravascular clots and lung perfusion defects, the present fusion images will be a useful adjunct to CTPA in the diagnosis of acute PTE.

The plant vacuolar Na+/H+ antiport.

PubMed

Barkla, B J; Apse, M P; Manolson, M F; Blumwald, E

1994-01-01

Salt stress imposes severe limitations on plant growth, however, the extent of growth reduction depends upon the soil salinity level and the plant species. One of the mechanisms employed by salt tolerant plants is the effective vacuolar compartmentalization of sodium. The sequestration of sodium into the vacuole occurs by the operation of a Na+/H+ antiport located at the tonoplast. Evidence for a plant vacuolar Na+/H+ antiport has been demonstrated in tissues, intact vacuoles and isolated tonoplast vesicles. In sugar beet cell suspensions, the activity of the vacuolar Na+/H+ antiport increased with increasing NaCl concentrations in the growth medium. This increased activity was correlated with the increased synthesis of a 170 kDa tonoplast polypeptide. In vivo labelling of tonoplast proteins showed the enhanced synthesis of the 170 kDa polypeptide not only upon exposure of the cells to salt, but also when the cells were grown in the presence of amiloride. Exposure of the cells to amiloride also resulted in increased vacuolar Na+/H+ antiport activity. Polyclonal antibodies raised against the 170 kDa polypeptide almost completely inhibited the antiport activity, suggesting the association of this protein with the plant vacuolar Na+/H+ antiport. Antibodies against the Na+/H+ antiport-associated polypeptide were used to screen a Beta lambda ZAP expression library. A partial clone of 1.65 kb was sequenced and found to encode a polypeptide with a putative transmembrane domain and a large hydrophilic C terminus. This clone showed no homology to any previously cloned gene at either the nucleic acid or the amino acid level.

Vacuolar respiration of nitrate coupled to energy conservation in filamentous Beggiatoaceae.

PubMed

Beutler, Martin; Milucka, Jana; Hinck, Susanne; Schreiber, Frank; Brock, Jörg; Mussmann, Marc; Schulz-Vogt, Heide N; de Beer, Dirk

2012-11-01

We show that the nitrate storing vacuole of the sulfide-oxidizing bacterium Candidatus Allobeggiatoa halophila has an electron transport chain (ETC), which generates a proton motive force (PMF) used for cellular energy conservation. Immunostaining by antibodies showed that cytochrome c oxidase, an ETC protein and a vacuolar ATPase are present in the vacuolar membrane and cytochrome c in the vacuolar lumen. The effect of different inhibitors on the vacuolar pH was studied by pH imaging. Inhibition of vacuolar ATPases and pyrophosphatases resulted in a pH decrease in the vacuole, showing that the proton gradient over the vacuolar membrane is used for ATP and pyrophosphate generation. Blockage of the ETC decreased the vacuolar PMF, indicating that the proton gradient is build up by an ETC. Furthermore, addition of nitrate resulted in an increase of the vacuolar PMF. Inhibition of nitrate reduction, led to a decreased PMF. Nitric oxide was detected in vacuoles of cells exposed to nitrate showing that nitrite, the product of nitrate reduction, is reduced inside the vacuole. These findings show consistently that nitrate respiration contributes to the high proton concentration within the vacuole and the PMF over the vacuolar membrane is actively used for energy conservation. © 2012 Society for Applied Microbiology and Blackwell Publishing Ltd.

Defects of Vps15 in skeletal muscles lead to autophagic vacuolar myopathy and lysosomal disease

PubMed Central

Nemazanyy, Ivan; Blaauw, Bert; Paolini, Cecilia; Caillaud, Catherine; Protasi, Feliciano; Mueller, Amelie; Proikas-Cezanne, Tassula; Russell, Ryan C; Guan, Kun-Liang; Nishino, Ichizo; Sandri, Marco; Pende, Mario; Panasyuk, Ganna

2013-01-01

The complex of Vacuolar Protein Sorting 34 and 15 (Vps34 and Vps15) has Class III phosphatidylinositol 3-kinase activity and putative roles in nutrient sensing, mammalian Target Of Rapamycin (mTOR) activation by amino acids, cell growth, vesicular trafficking and autophagy. Contrary to expectations, here we show that Vps15-deficient mouse tissues are competent for LC3-positive autophagosome formation and maintain mTOR activation. However, an impaired lysosomal function in mutant cells is traced by accumulation of adaptor protein p62, LC3 and Lamp2 positive vesicles, which can be reverted to normal levels after ectopic overexpression of Vps15. Mice lacking Vps15 in skeletal muscles, develop a severe myopathy. Distinct from the autophagy deficient Atg7−/− mutants, pathognomonic morphological hallmarks of autophagic vacuolar myopathy (AVM) are observed in Vps15−/− mutants, including elevated creatine kinase plasma levels, accumulation of autophagosomes, glycogen and sarcolemmal features within the fibres. Importantly, Vps34/Vps15 overexpression in myoblasts of Danon AVM disease patients alleviates the glycogen accumulation. Thus, the activity of the Vps34/Vps15 complex is critical in disease conditions such as AVMs, and possibly a variety of other lysosomal storage diseases. PMID:23630012

Decision-Level Fusion of Spatially Scattered Multi-Modal Data for Nondestructive Inspection of Surface Defects

PubMed Central

Heideklang, René; Shokouhi, Parisa

2016-01-01

This article focuses on the fusion of flaw indications from multi-sensor nondestructive materials testing. Because each testing method makes use of a different physical principle, a multi-method approach has the potential of effectively differentiating actual defect indications from the many false alarms, thus enhancing detection reliability. In this study, we propose a new technique for aggregating scattered two- or three-dimensional sensory data. Using a density-based approach, the proposed method explicitly addresses localization uncertainties such as registration errors. This feature marks one of the major of advantages of this approach over pixel-based image fusion techniques. We provide guidelines on how to set all the key parameters and demonstrate the technique’s robustness. Finally, we apply our fusion approach to experimental data and demonstrate its capability to locate small defects by substantially reducing false alarms under conditions where no single-sensor method is adequate. PMID:26784200

Vacuolar Transporters – Companions on a Longtime Journey[OPEN

PubMed Central

2018-01-01

Biochemical and electrophysiological studies on plant vacuolar transporters became feasible in the late 1970s and early 1980s, when methods to isolate large quantities of intact vacuoles and purified vacuolar membrane vesicles were established. However, with the exception of the H+-ATPase and H+-PPase, which could be followed due to their hydrolytic activities, attempts to purify tonoplast transporters were for a long time not successful. Heterologous complementation, T-DNA insertion mutants, and later proteomic studies allowed the next steps, starting from the 1990s. Nowadays, our knowledge about vacuolar transporters has increased greatly. Nevertheless, there are several transporters of central importance that have still to be identified at the molecular level or have even not been characterized biochemically. Furthermore, our knowledge about regulation of the vacuolar transporters is very limited, and much work is needed to get a holistic view about the interplay of the vacuolar transportome. The huge amount of information generated during the last 35 years cannot be summarized in such a review. Therefore, I decided to concentrate on some aspects where we were involved during my research on vacuolar transporters, for some our laboratories contributed more, while others contributed less. PMID:29295940

Spatial-time-state fusion algorithm for defect detection through eddy current pulsed thermography

NASA Astrophysics Data System (ADS)

Xiao, Xiang; Gao, Bin; Woo, Wai Lok; Tian, Gui Yun; Xiao, Xiao Ting

2018-05-01

Eddy Current Pulsed Thermography (ECPT) has received extensive attention due to its high sensitive of detectability on surface and subsurface cracks. However, it remains as a difficult challenge in unsupervised detection as to identify defects without knowing any prior knowledge. This paper presents a spatial-time-state features fusion algorithm to obtain fully profile of the defects by directional scanning. The proposed method is intended to conduct features extraction by using independent component analysis (ICA) and automatic features selection embedding genetic algorithm. Finally, the optimal feature of each step is fused to obtain defects reconstruction by applying common orthogonal basis extraction (COBE) method. Experiments have been conducted to validate the study and verify the efficacy of the proposed method on blind defect detection.

Genes encoding the vacuolar Na+/H+ exchanger and flower coloration.

PubMed

Yamaguchi, T; Fukada-Tanaka, S; Inagaki, Y; Saito, N; Yonekura-Sakakibara, K; Tanaka, Y; Kusumi, T; Iida, S

2001-05-01

Vacuolar pH plays an important role in flower coloration: an increase in the vacuolar pH causes blueing of flower color. In the Japanese morning glory (Ipomoea nil or Pharbitis nil), a shift from reddish-purple buds to blue open flowers correlates with an increase in the vacuolar pH. We describe details of the characterization of a mutant that carries a recessive mutation in the Purple (Pr) gene encoding a vacuolar Na+/H+ exchanger termed InNHX1. The genome of I. nil carries one copy of the Pr (or InNHX1) gene and its pseudogene, and it showed functional complementation to the yeast nhx1 mutation. The mutant of I. nil, called purple (pr), showed a partial increase in the vacuolar pH during flower-opening and its reddish-purple buds change into purple open flowers. The vacuolar pH in the purple open flowers of the mutant was significantly lower than that in the blue open flowers. The InNHX1 gene is most abundantly expressed in the petals at around 12 h before flower-opening, accompanying the increase in the vacuolar pH for the blue flower coloration. No such massive expression was observed in the petunia flowers. Since the NHX1 genes that promote the transport of Na+ into the vacuoles have been regarded to be involved in salt tolerance by accumulating Na+ in the vacuoles, we can add a new biological role for blue flower coloration in the Japanese morning glory by the vacuolar alkalization.

The VPH1 gene encodes a 95-kDa integral membrane polypeptide required for in vivo assembly and activity of the yeast vacuolar H(+)-ATPase.

PubMed

Manolson, M F; Proteau, D; Preston, R A; Stenbit, A; Roberts, B T; Hoyt, M A; Preuss, D; Mulholland, J; Botstein, D; Jones, E W

1992-07-15

Yeast vacuolar acidification-defective (vph) mutants were identified using the pH-sensitive fluorescence of 6-carboxyfluorescein diacetate (Preston, R. A., Murphy, R. F., and Jones, E. W. (1989) Proc. Natl. Acad. Sci. U.S.A. 86, 7027-7031). Vacuoles purified from yeast bearing the vph1-1 mutation had no detectable bafilomycin-sensitive ATPase activity or ATP-dependent proton pumping. The peripherally bound nucleotide-binding subunits of the vacuolar H(+)-ATPase (60 and 69 kDa) were no longer associated with vacuolar membranes yet were present in wild type levels in yeast whole cell extracts. The VPH1 gene was cloned by complementation of the vph1-1 mutation and independently cloned by screening a lambda gt11 expression library with antibodies directed against a 95-kDa vacuolar integral membrane protein. Deletion disruption of the VPH1 gene revealed that the VPH1 gene is not essential for viability but is required for vacuolar H(+)-ATPase assembly and vacuolar acidification. VPH1 encodes a predicted polypeptide of 840 amino acid residues (molecular mass 95.6 kDa) and contains six putative membrane-spanning regions. Cell fractionation and immunodetection demonstrate that Vph1p is a vacuolar integral membrane protein that co-purifies with vacuolar H(+)-ATPase activity. Multiple sequence alignments show extensive homology over the entire lengths of the following four polypeptides: Vph1p, the 116-kDa polypeptide of the rat clathrin-coated vesicles/synaptic vesicle proton pump, the predicted polypeptide encoded by the yeast gene STV1 (Similar To VPH1, identified as an open reading frame next to the BUB2 gene), and the TJ6 mouse immune suppressor factor.

The Bimodal Lifestyle of Intracellular Salmonella in Epithelial Cells: Replication in the Cytosol Obscures Defects in Vacuolar Replication

PubMed Central

Steele-Mortimer, Olivia

2012-01-01

Salmonella enterica serovar Typhimurium invades and proliferates within epithelial cells. Intracellular bacteria replicate within a membrane bound vacuole known as the Salmonella containing vacuole. However, this bacterium can also replicate efficiently in the cytosol of epithelial cells and net intracellular growth is a product of both vacuolar and cytosolic replication. Here we have used semi-quantitative single-cell analyses to investigate the contribution of each of these replicative niches to intracellular proliferation in cultured epithelial cells. We show that cytosolic replication can account for the majority of net replication even though it occurs in less than 20% of infected cells. Consequently, assays for net growth in a population of infected cells, for example by recovery of colony forming units, are not good indicators of vacuolar proliferation. We also show that the Salmonella Type III Secretion System 2, which is required for SCV biogenesis, is not required for cytosolic replication. Altogether this study illustrates the value of single cell analyses when studying intracellular pathogens. PMID:22719929

Wheat Vacuolar Iron Transporter TaVIT2 Transports Fe and Mn and Is Effective for Biofortification.

PubMed

Connorton, James M; Jones, Eleanor R; Rodríguez-Ramiro, Ildefonso; Fairweather-Tait, Susan; Uauy, Cristobal; Balk, Janneke

2017-08-01

Increasing the intrinsic nutritional quality of crops, known as biofortification, is viewed as a sustainable approach to alleviate micronutrient deficiencies. In particular, iron deficiency anemia is a major global health issue, but the iron content of staple crops such as wheat ( Triticum aestivum ) is difficult to change because of genetic complexity and homeostasis mechanisms. To identify target genes for the biofortification of wheat, we functionally characterized homologs of the VACUOLAR IRON TRANSPORTER ( VIT ). The wheat genome contains two VIT paralogs, TaVIT1 and TaVIT2 , which have different expression patterns but are both low in the endosperm. TaVIT2, but not TaVIT1, was able to rescue the growth of a yeast ( Saccharomyces cerevisiae ) mutant defective in vacuolar iron transport. TaVIT2 also complemented a manganese transporter mutant but not a vacuolar zinc transporter mutant. By overexpressing TaVIT2 under the control of an endosperm-specific promoter, we achieved a greater than 2-fold increase in iron in white flour fractions, exceeding minimum legal fortification levels in countries such as the United Kingdom. The antinutrient phytate was not increased and the iron in the white flour fraction was bioavailable in vitro, suggesting that food products made from the biofortified flour could contribute to improved iron nutrition. The single-gene approach impacted minimally on plant growth and also was effective in barley ( Hordeum vulgare ). Our results show that by enhancing vacuolar iron transport in the endosperm, this essential micronutrient accumulated in this tissue, bypassing existing homeostatic mechanisms. © 2017 American Society of Plant Biologists. All Rights Reserved.

Roles of histidine residues in plant vacuolar H(+)-pyrophosphatase.

PubMed

Hsiao, Yi Y; Van, Ru C; Hung, Shu H; Lin, Hsin H; Pan, Rong L

2004-02-15

Vacuolar proton pumping pyrophosphatase (H(+)-PPase; EC 3.6.1.1) plays a pivotal role in electrogenic translocation of protons from cytosol to the vacuolar lumen at the expense of PP(i) hydrolysis. Alignment analysis on amino acid sequence demonstrates that vacuolar H(+)-PPase of mung bean contains six highly conserved histidine residues. Previous evidence indicated possible involvement of histidine residue(s) in enzymatic activity and H(+)-translocation of vacuolar H(+)-PPase as determined by using histidine specific modifier, diethylpyrocarbonate [J. Protein Chem. 21 (2002) 51]. In this study, we further attempted to identify the roles of histidine residues in mung bean vacuolar H(+)-PPase by site-directed mutagenesis. A line of mutants with histidine residues singly replaced by alanine was constructed, over-expressed in Saccharomyces cerevisiae, and then used to determine their enzymatic activities and proton translocations. Among the mutants scrutinized, only the mutation of H716 significantly decreased the enzymatic activity, the proton transport, and the coupling ratio of vacuolar H(+)-PPase. The enzymatic activity of H716A is relatively resistant to inhibition by diethylpyrocarbonate as compared to wild-type and other mutants, indicating that H716 is probably the target residue for the attack by this modifier. The mutation at H716 of V-PPase shifted the optimum pH value but not the T(1/2) (pretreatment temperature at which half enzymatic activity is observed) for PP(i) hydrolytic activity. Mutation of histidine residues obviously induced conformational changes of vacuolar H(+)-PPase as determined by immunoblotting analysis after limited trypsin digestion. Furthermore, mutation of these histidine residues modified the inhibitory effects of F(-) and Na(+), but not that of Ca(2+). Single substitution of H704, H716 and H758 by alanine partially released the effect of K(+) stimulation, indicating possible location of K(+) binding in the vicinity of domains

Molecular dynamics simulation of the evolution of hydrophobic defects in one monolayer of a phosphatidylcholine bilayer: relevance for membrane fusion mechanisms.

PubMed Central

Tieleman, D Peter; Bentz, Joe

2002-01-01

The spontaneous formation of the phospholipid bilayer underlies the permeability barrier function of the biological membrane. Tears or defects that expose water to the acyl chains are spontaneously healed by lipid lateral diffusion. However, mechanical barriers, e.g., protein aggregates held in place, could sustain hydrophobic defects. Such defects have been postulated to occur in processes such as membrane fusion. This gives rise to a new question in bilayer structure: What do the lipids do in the absence of lipid lateral diffusion to minimize the free energy of a hydrophobic defect? As a first step to understand this rather fundamental question about bilayer structure, we performed molecular dynamic simulations of up to 10 ns of a planar bilayer from which lipids have been deleted randomly from one monolayer. In one set of simulations, approximately one-half of the lipids in the defect monolayer were restrained to form a mechanical barrier. In the second set, lipids were free to diffuse around. The question was simply whether the defects caused by removing a lipid would aggregate together, forming a large hydrophobic cavity, or whether the membrane would adjust in another way. When there are no mechanical barriers, the lipids in the defect monolayer simply spread out and thin with little effect on the other intact monolayer. In the presence of a mechanical barrier, the behavior of the lipids depends on the size of the defect. When 3 of 64 lipids are removed, the remaining lipids adjust the lower one-half of their chains, but the headgroup structure changes little and the intact monolayer is unaffected. When 6 to 12 lipids are removed, the defect monolayer thins, lipid disorder increases, and lipids from the intact monolayer move toward the defect monolayer. Whereas this is a highly simplified model of a fusion site, this engagement of the intact monolayer into the fusion defect is strikingly consistent with recent results for influenza hemagglutinin mediated

Diethylpyrocarbonate inhibition of vacuolar H+-pyrophosphatase possibly involves a histidine residue.

PubMed

Hsiao, Yi Yuong; Van, Ru Chuan; Hung, Hsiao Hui; Pan, Rong Long

2002-01-01

Vacuolar proton pumping pyrophosphatase (H+-PPase; EC 3.6.1.1) plays a pivotal role in electrogenic translocation of protons from cytosol to the vacuolar lumen at the expense of PPi hydrolysis. A histidine-specific modifier, diethylpyrocarbonate (DEPC), could substantially inhibit enzymic activity and H+-translocation of vacuolar H+-PPase in a concentration-dependent manner. Absorbance of vacuolar H+-PPase at 240 nm was increased upon incubation with DEPC, demonstrating that an N-carbethoxyhistidine moiety was probably formed. On the other hand, hydroxylamine, a reagent that can deacylate N-carbethoxyhistidine, could reverse the absorption change at 240 nm and partially restore PPi hydrolysis activity as well. The pKa of modified residues of the enzyme was determined to be 6.4, a value close to that of histidine. Thus, we speculate that inhibition of vacuolar H+-PPase by DEPC possibly could be attributed to the modification of histidyl residues on the enzyme. Furthermore, inhibition of vacuolar H+-PPase by DEPC follows pseudo-first-order rate kinetics. A reaction order of 0.85 was calculated from a double logarithmic plot of the apparent reaction constant against DEPC concentration, suggesting that the modification of one single histidine residue on the enzyme suffices to inhibit vacuolar H+-PPase. Inhibition of vacuolar H+-PPase by DEPC changes Vmax but not Km values. Moreover, DEPC inhibition of vacuolar H+-PPase could be substantially protected against by its physiological substrate, Mg2+-PPi. These results indicated that DEPC specifically competes with the substrate at the active site and the DEPC-labeled histidine residue might locate in or near the catalytic domain of the enzyme. Besides, pretreatment of the enzyme with N-ethylmaleimide decreased the degree of subsequent labeling of H+-PPase by DEPC. Taken together, we suggest that vacuolar H+-PPase likely contains a substrate-protectable histidine residue contributing to the inhibition of its activity by

A vacuolar iron transporter in tulip, TgVit1, is responsible for blue coloration in petal cells through iron accumulation.

PubMed

Momonoi, Kazumi; Yoshida, Kumi; Mano, Shoji; Takahashi, Hideyuki; Nakamori, Chihiro; Shoji, Kazuaki; Nitta, Akira; Nishimura, Mikio

2009-08-01

Blue color in flowers is due mainly to anthocyanins, and a considerable part of blue coloration can be attributed to metal-complexed anthocyanins. However, the mechanism of metal ion transport into vacuoles and subsequent flower color development has yet to be fully explored. Previously, we studied the mechanism of blue color development specifically at the bottom of the inner perianth in purple tulip petals of Tulipa gesneriana cv. Murasakizuisho. We found that differences in iron content were associated with the development of blue- and purple-colored cells. Here, we identify a vacuolar iron transporter in T. gesneriana (TgVit1), and characterize the localization and function of this transporter protein in tulip petals. The amino acid sequence of TgVit1 is 85% similar that of the Arabidopsis thaliana vacuolar iron transporter AtVIT1, and also showed similarity to the AtVIT1 homolog in yeast, Ca(2+)-sensitive cross-complementer 1 (CCC1). The gene TgVit1 was expressed exclusively in blue-colored epidermal cells, and protein levels increased with increasing mRNA expression and blue coloration. Transient expression experiments revealed that TgVit1 localizes to the vacuolar membrane, and is responsible for the development of the blue color in purple cells. Expression of TgVit1 in yeast rescued the growth defect of ccc1 mutant cells in the presence of high concentrations of FeSO(4). Our results indicate that TgVit1 plays an essential role in blue coloration as a vacuolar iron transporter in tulip petals. These results suggest a new role for involvement of a vacuolar iron transporter in blue flower color development.

Anthocyanin Vacuolar Inclusions Form by a Microautophagy Mechanism

PubMed Central

Chanoca, Alexandra; Ueda, Takashi; Grotewold, Erich

2015-01-01

Anthocyanins are flavonoid pigments synthesized in the cytoplasm and stored inside vacuoles. Many plant species accumulate densely packed, 3- to 10-μm diameter anthocyanin deposits called anthocyanin vacuolar inclusions (AVIs). Despite their conspicuousness and importance in organ coloration, the origin and nature of AVIs have remained controversial for decades. We analyzed AVI formation in cotyledons of different Arabidopsis thaliana genotypes grown under anthocyanin inductive conditions and in purple petals of lisianthus (Eustoma grandiorum). We found that cytoplasmic anthocyanin aggregates in close contact with the vacuolar surface are directly engulfed by the vacuolar membrane in a process reminiscent of microautophagy. The engulfed anthocyanin aggregates are surrounded by a single membrane derived from the tonoplast and eventually become free in the vacuolar lumen like an autophagic body. Neither endosomal/prevacuolar trafficking nor the autophagy ATG5 protein is involved in the formation of AVIs. In Arabidopsis, formation of AVIs is promoted by both an increase in cyanidin 3-O-glucoside derivatives and by depletion of the glutathione S-transferase TT19. We hypothesize that this novel microautophagy mechanism also mediates the transport of other flavonoid aggregates into the vacuole. PMID:26342015

Wheat Vacuolar Iron Transporter TaVIT2 Transports Fe and Mn and Is Effective for Biofortification1[OPEN

PubMed Central

Jones, Eleanor R.; Rodríguez-Ramiro, Ildefonso

2017-01-01

Increasing the intrinsic nutritional quality of crops, known as biofortification, is viewed as a sustainable approach to alleviate micronutrient deficiencies. In particular, iron deficiency anemia is a major global health issue, but the iron content of staple crops such as wheat (Triticum aestivum) is difficult to change because of genetic complexity and homeostasis mechanisms. To identify target genes for the biofortification of wheat, we functionally characterized homologs of the VACUOLAR IRON TRANSPORTER (VIT). The wheat genome contains two VIT paralogs, TaVIT1 and TaVIT2, which have different expression patterns but are both low in the endosperm. TaVIT2, but not TaVIT1, was able to rescue the growth of a yeast (Saccharomyces cerevisiae) mutant defective in vacuolar iron transport. TaVIT2 also complemented a manganese transporter mutant but not a vacuolar zinc transporter mutant. By overexpressing TaVIT2 under the control of an endosperm-specific promoter, we achieved a greater than 2-fold increase in iron in white flour fractions, exceeding minimum legal fortification levels in countries such as the United Kingdom. The antinutrient phytate was not increased and the iron in the white flour fraction was bioavailable in vitro, suggesting that food products made from the biofortified flour could contribute to improved iron nutrition. The single-gene approach impacted minimally on plant growth and also was effective in barley (Hordeum vulgare). Our results show that by enhancing vacuolar iron transport in the endosperm, this essential micronutrient accumulated in this tissue, bypassing existing homeostatic mechanisms. PMID:28684433

ATP-dependent export of neutral amino acids by vacuolar membrane vesicles of Saccharomyces cerevisiae.

PubMed

Ishimoto, Masaya; Sugimoto, Naoko; Sekito, Takayuki; Kawano-Kawada, Miyuki; Kakinuma, Yoshimi

2012-01-01

Amino acid analysis of Saccharomyces cerevisiae cells indicated that neutral amino acids such as glycine and alanine were probably excluded from the vacuoles, and that vacuolar H(+)-ATPase (V-ATPase) was involved in the vacuolar compartmentalization of these amino acids. We found that vacuolar membrane vesicles export neutral amino acids in an ATP-dependent manner. This is important in identifying vacuolar transporters for neutral amino acids.

Deleting the DAG Kinase Dgk1 Augments Yeast Vacuole Fusion Through In-creased Ypt7 Activity and Altered Membrane Fluidity

PubMed Central

Miner, Gregory E.; Starr, Matthew L.; Hurst, Logan R.; Fratti, Rutilio A.

2017-01-01

Diacylglycerol (DAG) is a fusogenic lipid that can be produced through phospholipase C activity on phosphatidylinositol 4,5-bisphosphate [PI(4,5)P2], or through phosphatidic acid (PA) phosphatase activity. The fusion of Saccharomyces cerevisiae vacuoles requires DAG, PA and PI(4,5)P2, and the production of these lipids is thought to provide temporally specific stoichiometries that are critical for each stage of fusion. Furthermore, DAG and PA can be interconverted by the DAG kinase Dgk1 and the PA phosphatase Pah1. Previously we found that pah1Δ vacuoles were fragmented, blocked in SNARE priming and showed arrested endosomal maturation. In other pathways the effects of deleting PAH1 can be compensated for by additionally deleting DGK1, however deleting both genes did not rescue the pah1Δ vacuolar defects. Deleting DGK1 alone caused a marked increase in vacuole fusion that was attributed to elevated DAG levels. This was accompanied by a gain in resistance to the inhibitory effects of PA as well as inhibitors of Ypt7 activity. Together these data show that Dgk1 function can act as a negative regulator of vacuole fusion through the production of PA at the cost of depleting DAG and reducing Ypt7 activity. PMID:28276191

Pth1/Vam3p is the syntaxin homolog at the vacuolar membrane of Saccharomyces cerevisiae required for the delivery of vacuolar hydrolases.

PubMed Central

Srivastava, A; Jones, E W

1998-01-01

The PEP12 homolog Pth1p (Pep twelve homolog 1) is predicted to be similar in size to Pep12p, the endosomal syntaxin homolog that mediates docking of Golgi-derived transport vesicles and, like other members of the syntaxin family, is predicted to be a cytoplasmically oriented, integral membrane protein with a C-terminal transmembrane domain. Kinetic analyses indicate that deltapth1/vam3 mutants fail to process the soluble vacuolar hydrolase precursors and that PrA, PrB and most of CpY accumulate within the cell in their Golgi-modified P2 precursor forms. This is in contrast to a pep12 mutant in which P2CpY is secreted from the cell. Furthermore, pep12 is epistatic to pth1/vam3 with respect to the CpY secretion phenotype. Alkaline phosphatase, a vacuolar membrane hydrolase, accumulates in its precursor form in the deltapth1/vam3 mutant. Maturation of pro-aminopeptidase I, a hydrolase precursor delivered directly to the vacuole from the cytoplasm, is also blocked in the deltapth1/vam3 mutant. Subcellular fractionation localizes Pth1/Vam3p to vacuolar membranes. Based on these data, we propose that Pth1/Vam3p is the vacuolar syntaxin/t-SNARE homolog that participates in docking of transport vesicles at the vacuolar membrane and that the function of Pth1/Vam3p impinges on at least three routes of protein delivery to the yeast vacuole. PMID:9475723

Novel families of vacuolar amino acid transporters.

PubMed

Sekito, Takayuki; Fujiki, Yuki; Ohsumi, Yoshinori; Kakinuma, Yoshimi

2008-08-01

Amino acids are compartmentalized in the vacuoles of microorganisms and plants. In Saccharomyces cerevisiae, basic amino acids accumulate preferentially into vacuoles but acidic amino acids are almost excluded from them. This indicates that selective machineries operate at the vacuolar membrane. The members of the amino acid/auxin permease family and the major facilitator superfamily involved in the vacuolar compartmentalization of amino acids have been recently identified in studies using S. cerevisiae. Homologous genes for these transporters are also found in plant and mammalian genomes. The physiological significance in response to nitrogen starvation can now be discussed. (c) 2008 IUBMB

Hygromycin B hypersensitive (hhy) mutants implicate an intact trans-Golgi and late endosome interface in efficient Tor1 vacuolar localization and TORC1 function.

PubMed

Ejzykowicz, Daniele E; Locken, Kristopher M; Ruiz, Fiona J; Manandhar, Surya P; Olson, Daniel K; Gharakhanian, Editte

2017-06-01

Saccharomyces cerevisiae vacuoles are functionally analogous to mammalian lysosomes. Both also serve as physical platforms for Tor Complex 1 (TORC1) signal transduction, the master regulator of cellular growth and proliferation. Hygromycin B is a eukaryotic translation inhibitor. We recently reported on hygromycin B hypersensitive (hhy) mutants that fail to grow at subtranslation inhibitory concentrations of the drug and exhibit vacuolar defects (Banuelos et al. in Curr Genet 56:121-137, 2010). Here, we show that hhy phenotype is not due to increased sensitivity to translation inhibition and establish a super HHY (s-HHY) subgroup of genes comprised of ARF1, CHC1, DRS2, SAC1, VPS1, VPS34, VPS45, VPS52, and VPS54 that function exclusively or inclusively at trans-Golgi and late endosome interface. Live cell imaging of s-hhy mutants revealed that hygromycin B treatment disrupts vacuolar morphology and the localization of late endosome marker Pep12, but not that of late endosome-independent vacuolar SNARE Vam3. This, along with normal post-late endosome trafficking of the vital dye FM4-64, establishes that severe hypersensitivity to hygromycin B correlates specifically with compromised trans-Golgi and late endosome interface. We also show that Tor1p vacuolar localization and TORC1 anabolic functions, including growth promotion and phosphorylation of its direct substrate Sch9, are compromised in s-hhy mutants. Thus, an intact trans-Golgi and late endosome interface is a requisite for efficient Tor1 vacuolar localization and TORC1 function.

Characterization of the gag/fusion protein encoded by the defective Duplan retrovirus inducing murine acquired immunodeficiency syndrome.

PubMed Central

Huang, M; Jolicoeur, P

1990-01-01

Murine acquired immunodeficiency syndrome is induced by a defective retrovirus. Sequencing of this defective viral genome revealed a long open reading frame which encodes a putative gag/fusion protein, N-MA-p12-CA-NC-COOH, (D. C. Aziz, Z. Hanna, and P. Jolicoeur, Nature (London) 338:505-508, 1989). We raised a specific antibody to the unique p12 domain of this gag fusion precursor, Pr60gag. We found that Pr60gag was indeed encoded by the defective viral genome both in cell-free translation reticulocyte extracts and in infected mouse fibroblasts. Pr60gag was found to be myristylated, phosphorylated, and attached to the cell membrane, like other helper murine leukemia virus (MuLV) gag precursors. Pr60gag was not substantially cleaved within the nonproducer cells and was not released from these cells. However, in the presence of helper MuLV proteins, it formed phenotypically mixed particles. In these particles, Pr60gag was only partially cleaved. In helper MuLV-producing cells harboring the defective virus, a gag-related p40 intermediate was generated both intracellularly and extracellularly. In these cells, Pr60gag appeared to behave as a dominant negative mutant, interfering with proper cleavage of helper Pr65gag. Our data indicate that Pr60gag is a major (and possibly the only) gene product of the defective murine acquired immunodeficiency syndrome virus and is likely to harbor some determinants of pathogenicity of this virus. Images PMID:2243376

Structural evolution dynamics in fusion of sumanenes and corannulenes: defects formation and self-healing mechanism

NASA Astrophysics Data System (ADS)

Sorkin, Anastassia; Su, Haibin

2018-06-01

The fusion processes of structures consisting of various combinations between sumanene and corannulene, leading to the formation of graphene nanoribbons (GNRs) under heating are simulated by density-functional-based tight-binding molecular dynamics. Distinct stages are unraveled in the course of GNR formation. Firstly, the carbon fragments coalescence into highly strained framework. Secondly, structural reconstruction invokes breaking most strained bonds to form a GNR structure containing numerous defects. Lastly, defects are remedied by the delicate ‘edge-facilitated self-healing’ process through two synergized edge-related effects: elevated mobility of defects and promoted structure reconstructions owing to the remarkable dynamics associated with edges. Importantly, detailed dynamics in the course of forming GNRs with defects and grain boundaries simulated in this work is valuable to provide better understanding at the atomistic scale of defect formation as well as self-healing in the context of the sp2 carbon network. In particular, edges play important roles in not only generating Stone–Wales (SW), 5-8-5 types of defects, 8-5-5-8 and pentagon–heptagon grain boundaries. In addition, our simulations predict the existence of one novel defect, coined as the Inverse SW defect, which is to be confirmed in future experimental studies. This study of dynamic structural evolution reveals that edges are prone to intrinsic and extrinsic modifications such as atomic-scale defects, structural distortions and inhomogeneity.

The Marine Natural Product Manzamine A Targets Vacuolar ATPases and Inhibits Autophagy in Pancreatic Cancer Cells

PubMed Central

Kallifatidis, Georgios; Hoepfner, Dominic; Jaeg, Tiphaine; Guzmán, Esther A.; Wright, Amy E.

2013-01-01

Manzamine A, a member of the manzamine alkaloids, was originally isolated from marine sponges of the genus Haliclona. It was recently shown to have activity against pancreatic cancer cells, but the precise mechanism of action remained unclear. To further our understanding of the mechanism of action of manzamine A, chemogenomic profiling in the yeast S. cerevisiae was performed, suggesting that manzamine A is an uncoupler of vacuolar ATPases. Fluorescence microscopy confirmed this effect on yeast vacuoles, where manzamine A produced a phenotype very similar to that of the established v-ATPase inhibitor bafilomycin A1. In pancreatic cancer cells, 10 µM manzamine A affected vacuolar ATPase activity and significantly increased the level of autophagosome marker LC3-II and p62/SQSTM1 as observed by western blot analysis. Treatment with manzamine A in combination with bafilomycin A1 (inhibitor of autophagosome-lysosome fusion) did not change the levels of LC3-II when compared to cells treated with bafilomycin A1 alone, suggesting that manzamine A is a potential inhibitor of autophagy by preventing autophagosome turnover. As autophagy is essential for pancreatic tumor growth, blocking this pathway with manzamine A suggests a promising strategy for the treatment of pancreatic cancer. PMID:24048269

Process defects and in situ monitoring methods in metal powder bed fusion: a review

NASA Astrophysics Data System (ADS)

Grasso, Marco; Colosimo, Bianca Maria

2017-04-01

Despite continuous technological enhancements of metal Additive Manufacturing (AM) systems, the lack of process repeatability and stability still represents a barrier for the industrial breakthrough. The most relevant metal AM applications currently involve industrial sectors (e.g. aerospace and bio-medical) where defects avoidance is fundamental. Because of this, there is the need to develop novel in situ monitoring tools able to keep under control the stability of the process on a layer-by-layer basis, and to detect the onset of defects as soon as possible. On the one hand, AM systems must be equipped with in situ sensing devices able to measure relevant quantities during the process, a.k.a. process signatures. On the other hand, in-process data analytics and statistical monitoring techniques are required to detect and localize the defects in an automated way. This paper reviews the literature and the commercial tools for in situ monitoring of powder bed fusion (PBF) processes. It explores the different categories of defects and their main causes, the most relevant process signatures and the in situ sensing approaches proposed so far. Particular attention is devoted to the development of automated defect detection rules and the study of process control strategies, which represent two critical fields for the development of future smart PBF systems.

Vacuolar protein sorting mechanisms in plants.

PubMed

Xiang, Li; Etxeberria, Ed; Van den Ende, Wim

2013-02-01

Plant vacuoles are unique, multifunctional organelles among eukaryotes. Considerable new insights in plant vacuolar protein sorting have been obtained recently. The basic machinery of protein export from the endoplasmic reticulum to the Golgi and the classical route to the lytic vacuole and the protein storage vacuole shows many similarities to vacuolar/lysosomal sorting in other eukaryotes. However, as a result of its unique functions in plant defence and as a storage compartment, some plant-specific entities and sorting determinants appear to exist. The alternative post-Golgi route, as found in animals and yeast, probably exists in plants as well. Likely, adaptor protein complex 3 fulfils a central role in this route. A Golgi-independent route involving plant-specific endoplasmic reticulum bodies appears to provide sedentary organisms such as plants with extra flexibility to cope with changing environmental conditions. © 2012 The Authors Journal compilation © 2012 FEBS.

Evidence for Avt6 as a vacuolar exporter of acidic amino acids in Saccharomyces cerevisiae cells.

PubMed

Chahomchuen, Thippayarat; Hondo, Kana; Ohsaki, Mariko; Sekito, Takayuki; Kakinuma, Yoshimi

2009-12-01

Here we examined the significance of Avt6, a vacuolar exporter of glutamate and aspartate suggested by the in vitro membrane vesicle experiment, in vacuolar compartmentalization of amino acids in Saccharomyces cerevisiae cells. Fluorescent microscopic observation of GFP-fused Avt6 revealed it to be exclusively localized to the vacuolar membrane, with the amount of Myc-tagged Avt6 significantly increased under nitrogen starvation. Glutamate uptake by cells was enhanced by deletion of the AVT6 gene, indicating indirect involvement of Avt6 in cellular glutamate accumulation. Differences in acidic amino acid content of both total and vacuolar fractions were insignificant between the parent and avt6Delta cells when cultured in nutrient-rich conditions. However, in nitrogen-starved conditions, the amount of glutamate and aspartate in the vacuolar fraction was notably increased in the avt6Delta cells. Avt6 is thus involved in vacuolar amino acid compartmentalization in S. cerevisiae cells, especially under conditions of nitrogen starvation.

SOS2 Promotes Salt Tolerance in Part by Interacting with the Vacuolar H+-ATPase and Upregulating Its Transport Activity▿

PubMed Central

Batelli, Giorgia; Verslues, Paul E.; Agius, Fernanda; Qiu, Quansheng; Fujii, Hiroaki; Pan, Songqin; Schumaker, Karen S.; Grillo, Stefania; Zhu, Jian-Kang

2007-01-01

The salt overly sensitive (SOS) pathway is critical for plant salt stress tolerance and has a key role in regulating ion transport under salt stress. To further investigate salt tolerance factors regulated by the SOS pathway, we expressed an N-terminal fusion of the improved tandem affinity purification tag to SOS2 (NTAP-SOS2) in sos2-2 mutant plants. Expression of NTAP-SOS2 rescued the salt tolerance defect of sos2-2 plants, indicating that the fusion protein was functional in vivo. Tandem affinity purification of NTAP-SOS2-containing protein complexes and subsequent liquid chromatography-tandem mass spectrometry analysis indicated that subunits A, B, C, E, and G of the peripheral cytoplasmic domain of the vacuolar H+-ATPase (V-ATPase) were present in a SOS2-containing protein complex. Parallel purification of samples from control and salt-stressed NTAP-SOS2/sos2-2 plants demonstrated that each of these V-ATPase subunits was more abundant in NTAP-SOS2 complexes isolated from salt-stressed plants, suggesting that the interaction may be enhanced by salt stress. Yeast two-hybrid analysis showed that SOS2 interacted directly with V-ATPase regulatory subunits B1 and B2. The importance of the SOS2 interaction with the V-ATPase was shown at the cellular level by reduced H+ transport activity of tonoplast vesicles isolated from sos2-2 cells relative to vesicles from wild-type cells. In addition, seedlings of the det3 mutant, which has reduced V-ATPase activity, were found to be severely salt sensitive. Our results suggest that regulation of V-ATPase activity is an additional key function of SOS2 in coordinating changes in ion transport during salt stress and in promoting salt tolerance. PMID:17875927

The Habc Domain of the SNARE Vam3 Interacts with the HOPS Tethering Complex to Facilitate Vacuole Fusion*

PubMed Central

Lürick, Anna; Kuhlee, Anne; Bröcker, Cornelia; Kümmel, Daniel; Raunser, Stefan; Ungermann, Christian

2015-01-01

Membrane fusion at vacuoles requires a consecutive action of the HOPS tethering complex, which is recruited by the Rab GTPase Ypt7, and vacuolar SNAREs to drive membrane fusion. It is assumed that the Sec1/Munc18-like Vps33 within the HOPS complex is largely responsible for SNARE chaperoning. Here, we present direct evidence for HOPS binding to SNAREs and the Habc domain of the Vam3 SNARE protein, which may explain its function during fusion. We show that HOPS interacts strongly with the Vam3 Habc domain, assembled Q-SNAREs, and the R-SNARE Ykt6, but not the Q-SNARE Vti1 or the Vam3 SNARE domain. Electron microscopy combined with Nanogold labeling reveals that the binding sites for vacuolar SNAREs and the Habc domain are located in the large head of the HOPS complex, where Vps16 and Vps33 have been identified before. Competition experiments suggest that HOPS bound to the Habc domain can still interact with assembled Q-SNAREs, whereas Q-SNARE binding prevents recognition of the Habc domain. In agreement, membranes carrying Vam3ΔHabc fuse poorly unless an excess of HOPS is provided. These data suggest that the Habc domain of Vam3 facilitates the assembly of the HOPS/SNARE machinery at fusion sites and thus supports efficient membrane fusion. PMID:25564619

Ypq3p-dependent histidine uptake by the vacuolar membrane vesicles of Saccharomyces cerevisiae.

PubMed

Manabe, Kunio; Kawano-Kawada, Miyuki; Ikeda, Koichi; Sekito, Takayuki; Kakinuma, Yoshimi

2016-06-01

The vacuolar membrane proteins Ypq1p, Ypq2p, and Ypq3p of Saccharomyces cerevisiae are known as the members of the PQ-loop protein family. We found that the ATP-dependent uptake activities of arginine and histidine by the vacuolar membrane vesicles were decreased by ypq2Δ and ypq3Δ mutations, respectively. YPQ1 and AVT1, which are involved in the vacuolar uptake of lysine/arginine and histidine, respectively, were deleted in addition to ypq2Δ and ypq3Δ. The vacuolar membrane vesicles isolated from the resulting quadruple deletion mutant ypq1Δypq2Δypq3Δavt1Δ completely lost the uptake activity of basic amino acids, and that of histidine, but not lysine and arginine, was evidently enhanced by overexpressing YPQ3 in the mutant. These results suggest that Ypq3p is specifically involved in the vacuolar uptake of histidine in S. cerevisiae. The cellular level of Ypq3p-HA(3) was enhanced by depletion of histidine from culture medium, suggesting that it is regulated by the substrate.

Multisensor fusion for 3-D defect characterization using wavelet basis function neural networks

NASA Astrophysics Data System (ADS)

Lim, Jaein; Udpa, Satish S.; Udpa, Lalita; Afzal, Muhammad

2001-04-01

The primary objective of multi-sensor data fusion, which offers both quantitative and qualitative benefits, has the ability to draw inferences that may not be feasible with data from a single sensor alone. In this paper, data from two sets of sensors are fused to estimate the defect profile from magnetic flux leakage (MFL) inspection data. The two sensors measure the axial and circumferential components of the MFL. Data is fused at the signal level. If the flux is oriented axially, the samples of the axial signal are measured along a direction parallel to the flaw, while the circumferential signal is measured in a direction that is perpendicular to the flaw. The two signals are combined as the real and imaginary components of a complex valued signal. Signals from an array of sensors are arranged in contiguous rows to obtain a complex valued image. A boundary extraction algorithm is used to extract the defect areas in the image. Signals from the defect regions are then processed to minimize noise and the effects of lift-off. Finally, a wavelet basis function (WBF) neural network is employed to map the complex valued image appropriately to obtain the geometrical profile of the defect. The feasibility of the approach was evaluated using the data obtained from the MFL inspection of natural gas transmission pipelines. Results show the effectiveness of the approach.

Characterization of Avt1p as a vacuolar proton/amino acid antiporter in Saccharomyces cerevisiae.

PubMed

Tone, Junichi; Yoshimura, Ayumi; Manabe, Kunio; Murao, Nami; Sekito, Takayuki; Kawano-Kawada, Miyuki; Kakinuma, Yoshimi

2015-01-01

Several genes for vacuolar amino acid transport were reported in Saccharomyces cerevisiae, but have not well been investigated. We characterized AVT1, a member of the AVT vacuolar transporter family, which is reported to be involved in lifespan of yeast. ATP-dependent uptake of isoleucine and histidine by the vacuolar vesicles of an AVT exporter mutant was lost by introducing avt1∆ mutation. Uptake activity was inhibited by the V-ATPase inhibitor: concanamycin A and a protonophore. Isoleucine uptake was inhibited by various neutral amino acids and histidine, but not by γ-aminobutyric acid, glutamate, and aspartate. V-ATPase-dependent acidification of the vesicles was declined by the addition of isoleucine or histidine, depending upon Avt1p. Taken together with the data of the amino acid contents of vacuolar fractions in cells, the results suggested that Avt1p is a proton/amino acid antiporter important for vacuolar compartmentalization of various amino acids.

Vacuolar morphology of Saccharomyces cerevisiae during the process of wine making and Japanese sake brewing.

PubMed

Izawa, Shingo; Ikeda, Kayo; Miki, Takeo; Wakai, Yoshinori; Inoue, Yoshiharu

2010-09-01

Although ethanol and osmotic stress affect the vacuolar morphology of Saccharomyces cerevisiae, little information is available about changes in vacuolar morphology during the processes of wine making and Japanese sake (rice wine) brewing. Here, we elucidated changes in the morphology of yeast vacuoles using Zrc1p-GFP, a vacuolar membrane protein, so as to better understand yeast physiology during the brewing process. Wine yeast cells (OC-2 and EC1118) contained highly fragmented vacuoles in the sake mash (moromi) as well as in the grape must. Although sake yeast cells (Kyokai no. 9 and no. 10) also contained highly fragmented vacuoles during the wine-making process, they showed quite a distinct vacuolar morphology during sake brewing. Since the environment surrounding sake yeast cells in the sake mash did not differ much from that surrounding wine yeast cells, the difference in vacuolar morphology during sake brewing between wine yeast and sake yeast was likely caused by innate characters.

Vacuolar processing enzyme in plant programmed cell death

PubMed Central

Hatsugai, Noriyuki; Yamada, Kenji; Goto-Yamada, Shino; Hara-Nishimura, Ikuko

2015-01-01

Vacuolar processing enzyme (VPE) is a cysteine proteinase originally identified as the proteinase responsible for the maturation and activation of vacuolar proteins in plants, and it is known to be an ortholog of animal asparaginyl endopeptidase (AEP/VPE/legumain). VPE has been shown to exhibit enzymatic properties similar to that of caspase 1, which is a cysteine protease that mediates the programmed cell death (PCD) pathway in animals. Although there is limited sequence identity between VPE and caspase 1, their predicted three-dimensional structures revealed that the essential amino-acid residues for these enzymes form similar pockets for the substrate peptide YVAD. In contrast to the cytosolic localization of caspases, VPE is localized in vacuoles. VPE provokes vacuolar rupture, initiating the proteolytic cascade leading to PCD in the plant immune response. It has become apparent that the VPE-dependent PCD pathway is involved not only in the immune response, but also in the responses to a variety of stress inducers and in the development of various tissues. This review summarizes the current knowledge on the contribution of VPE to plant PCD and its role in vacuole-mediated cell death, and it also compares VPE with the animal cell death executor caspase 1. PMID:25914711

Biolayer interferometry of lipid nanodisc-reconstituted yeast vacuolar H+ -ATPase.

PubMed

Sharma, Stuti; Wilkens, Stephan

2017-05-01

Vacuolar H + -ATPase (V-ATPase) is a large, multisubunit membrane protein complex responsible for the acidification of subcellular compartments and the extracellular space. V-ATPase activity is regulated by reversible disassembly, resulting in cytosolic V 1 -ATPase and membrane-integral V 0 proton channel sectors. Reversible disassembly is accompanied by transient interaction with cellular factors and assembly chaperones. Quantifying protein-protein interactions involving membrane proteins, however, is challenging. Here we present a novel method to determine kinetic constants of membrane protein-protein interactions using biolayer interferometry (BLI). Yeast vacuoles are solubilized, vacuolar proteins are reconstituted into lipid nanodiscs with native vacuolar lipids and biotinylated membrane scaffold protein (MSP) followed by affinity purification of nanodisc-reconstituted V-ATPase (V 1 V 0 ND). We show that V 1 V 0 ND can be immobilized on streptavidin-coated BLI sensors to quantitate binding of a pathogen derived inhibitor and to measure the kinetics of nucleotide dependent enzyme dissociation. © 2017 The Protein Society.

The V-ATPase subunit A is essential for salt tolerance through participating in vacuolar Na+ compartmentalization in Salicornia europaea.

PubMed

Lv, Sulian; Jiang, Ping; Tai, Fang; Wang, Duoliya; Feng, Juanjuan; Fan, Pengxiang; Bao, Hexigeduleng; Li, Yinxin

2017-12-01

The V-ATPase subunit A participates in vacuolar Na + compartmentalization in Salicornia europaea regulating V-ATPase and V-PPase activities. Na + sequestration into the vacuole is an efficient strategy in response to salinity in many halophytes. However, it is not yet fully understood how this process is achieved. Particularly, the role of vacuolar H + -ATPase (V-ATPase) in this process is controversial. Our previous proteomic investigation in the euhalophyte Salicornia europaea L. found a significant increase of the abundance of V-ATPase subunit A under salinity. Here, the gene encoding this subunit named SeVHA-A was characterized, and its role in salt tolerance was demonstrated by RNAi directed downregulation in suspension-cultured cells of S. europaea. The transcripts of genes encoding vacuolar H + -PPase (V-PPase) and vacuolar Na + /H + antiporter (SeNHX1) also decreased significantly in the RNAi cells. Knockdown of SeVHA-A resulted in a reduction in both V-ATPase and vacuolar H + -PPase (V-PPase) activities. Accordingly, the SeVHA-A-RNAi cells showed increased vacuolar pH and decreased cell viability under different NaCl concentrations. Further Na + staining showed the reduced vacuolar Na + sequestration in RNAi cells. Taken together, our results evidenced that SeVHA-A participates in vacuolar Na + sequestration regulating V-ATPase and V-PPase activities and thereby vacuolar pH in S. europaea. The possible mechanisms underlying the reduction of vacuolar V-PPase activity in SeVHA-A-RNAi cells were also discussed.

Avt5p is required for vacuolar uptake of amino acids in the fission yeast Schizosaccharomyces pombe.

PubMed

Chardwiriyapreecha, Soracom; Mukaiyama, Hiroyuki; Sekito, Takayuki; Iwaki, Tomoko; Takegawa, Kaoru; Kakinuma, Yoshimi

2010-06-03

We identified SPBC1685.07c of Schizosaccharomyces pombe as a novel vacuolar protein, Avt5p, with similarity to vacuolar amino acid transporters Avt5p from Saccharomyces cerevisiae. Avt5p localizes to the vacuolar membrane and upon disruption of avt5, uptake of histidine, glutamate, tyrosine, arginine, lysine or serine was impaired. During nitrogen starvation, the transient increase of vacuolar lysine transport observed for wild-type cells still occurred in the mutant cells, however, uptake of glutamate did not significantly increase in response to nitrogen starvation. Our results show that under diverse growth conditions Avt5p is involved in vacuolar transport of a selective set of amino acids. Copyright 2010 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.

Effect of vacuolar ATPase subunit H (VmaH) on cellular pH, asexual cycle, stress tolerance and virulence in Beauveria bassiana.

PubMed

Zhu, Jing; Zhu, Xiao-Guan; Ying, Sheng-Hua; Feng, Ming-Guang

2017-01-01

Vacuolar ATPase (V-ATPase) is a conserved multi-subunit protein complex that mediates intracellular acidification in fungi. Here we show functional diversity of V-ATPase subunit H (BbVmaH) in Beauveria bassiana, a filamentous fungal insect pathogen. Deletion of BbvmaH resulted in elevated vacuolar pH, increased Ca 2+ level in cytosol but not in vacuoles, accelerated culture acidification and reduced accumulation of extracellular ammonia. Aerial conidiation and submerged blastospore production were largely delayed and reduced in the deletion mutant, respectively, accompanied with a significant delay in conidial germination, alterations of conidia and blastospores in morphology, size and/or density, and severe growth defects in minimal media with different carbon and nitrogen sources. Despite null responses to osmotic, oxidative and cell wall perturbing stresses, the deletion mutant showed increased sensitivity to Ca 2+ , Zn 2+ and Cu 2+ during growth while its conidia were less tolerant to a wet-heat stress at 45°C and UV-B irradiation. Intracellular glycerol and mannitol contents also decreased significantly. Its virulence to Galleria mellonella larvae was significantly attenuated when conidia were topically applied for normal cuticle infection or injected into haemocoel for cuticle-bypassing infection. All phenotypic changes were restored by targeted gene complementation. Our results indicate that BbVmaH plays an important role in sustaining not only vacuolar acidification but also cytosolic calcium accumulation, ambient pH homeostasis, in vitro asexual cycle and virulence in B. bassiana. Copyright © 2016 Elsevier Inc. All rights reserved.

Rapid Nuclear Exclusion of Hcm1 in Aging Saccharomyces cerevisiae Leads to Vacuolar Alkalization and Replicative Senescence

PubMed Central

Ghavidel, Ata; Baxi, Kunal; Prusinkiewicz, Martin; Swan, Cynthia; Belak, Zach R.; Eskiw, Christopher H.; Carvalho, Carlos E.; Harkness, Troy A.

2018-01-01

The yeast, Saccharomyces cerevisiae, like other higher eukaryotes, undergo a finite number of cell divisions before exiting the cell cycle due to the effects of aging. Here, we show that yeast aging begins with the nuclear exclusion of Hcm1 in young cells, resulting in loss of acidic vacuoles. Autophagy is required for healthy aging in yeast, with proteins targeted for turnover by autophagy directed to the vacuole. Consistent with this, vacuolar acidity is necessary for vacuolar function and yeast longevity. Using yeast genetics and immunofluorescence microscopy, we confirm that vacuolar acidity plays a critical role in cell health and lifespan, and is potentially maintained by a series of Forkhead Box (Fox) transcription factors. An interconnected transcriptional network involving the Fox proteins (Fkh1, Fkh2 and Hcm1) are required for transcription of v-ATPase subunits and vacuolar acidity. As cells age, Hcm1 is rapidly excluded from the nucleus in young cells, blocking the expression of Hcm1 targets (Fkh1 and Fkh2), leading to loss of v-ATPase gene expression, reduced vacuolar acidification, increased α-syn-GFP vacuolar accumulation, and finally, diminished replicative lifespan (RLS). Loss of vacuolar acidity occurs about the same time as Hcm1 nuclear exclusion and is conserved; we have recently demonstrated that lysosomal alkalization similarly contributes to aging in C. elegans following a transition from progeny producing to post-reproductive life. Our data points to a molecular mechanism regulating vacuolar acidity that signals the end of RLS when acidification is lost. PMID:29519938

Vacuolar transporter Avt4 is involved in excretion of basic amino acids from the vacuoles of Saccharomyces cerevisiae.

PubMed

Sekito, Takayuki; Chardwiriyapreecha, Soracom; Sugimoto, Naoko; Ishimoto, Masaya; Kawano-Kawada, Miyuki; Kakinuma, Yoshimi

2014-01-01

Basic amino acids (lysine, histidine and arginine) accumulated in Saccharomyces cerevisiae vacuoles should be mobilized to cytosolic nitrogen metabolism under starvation. We found that the decrease of vacuolar basic amino acids in response to nitrogen starvation was impaired by the deletion of AVT4 gene encoding a vacuolar transporter. In addition, overexpression of AVT4 reduced the accumulation of basic amino acids in vacuoles under nutrient-rich condition. In contrast to AVT4, the deletion and overexpression of AVT3, which encodes the closest homologue of Avt4p, did not affect the contents of vacuolar basic amino acids. Consistent with these, arginine uptake into vacuolar membrane vesicles was decreased by Avt4p-, but not by Avt3p-overproduction, whereas various neutral amino acids were excreted from vacuolar membrane vesicles in a manner dependent on either Avt4p or Avt3p. These results suggest that Avt4p is a vacuolar amino acid exporter involving in the recycling of basic amino acids.

Biolayer interferometry of lipid nanodisc‐reconstituted yeast vacuolar H+‐ATPase

PubMed Central

Sharma, Stuti

2017-01-01

Abstract Vacuolar H+‐ATPase (V‐ATPase) is a large, multisubunit membrane protein complex responsible for the acidification of subcellular compartments and the extracellular space. V‐ATPase activity is regulated by reversible disassembly, resulting in cytosolic V 1‐ATPase and membrane‐integral V 0 proton channel sectors. Reversible disassembly is accompanied by transient interaction with cellular factors and assembly chaperones. Quantifying protein‐protein interactions involving membrane proteins, however, is challenging. Here we present a novel method to determine kinetic constants of membrane protein–protein interactions using biolayer interferometry (BLI). Yeast vacuoles are solubilized, vacuolar proteins are reconstituted into lipid nanodiscs with native vacuolar lipids and biotinylated membrane scaffold protein (MSP) followed by affinity purification of nanodisc‐reconstituted V‐ATPase (V 1 V 0ND). We show that V 1 V 0ND can be immobilized on streptavidin‐coated BLI sensors to quantitate binding of a pathogen derived inhibitor and to measure the kinetics of nucleotide dependent enzyme dissociation. PMID:28241399

Involvement of MoVMA11, a Putative Vacuolar ATPase c’ Subunit, in Vacuolar Acidification and Infection-Related Morphogenesis of Magnaporthe oryzae

PubMed Central

Chen, Guoqing; Liu, Xiaohong; Zhang, Lilin; Cao, Huijuan; Lu, Jianping; Lin, Fucheng

2013-01-01

Many functions of vacuole depend on the activity of vacuolar ATPase which is essential to maintain an acidic lumen and create the driving forces for massive fluxes of ions and metabolites through vacuolar membrane. In filamentous fungus Magnaporthe oryzae , subcellular colocalization and quinacrine staining suggested that the V1V0 domains of V-ATPase were fully assembled and the vacuoles were kept acidic during infection-related developments. Targeted gene disruption of MoVMA11 gene, encoding the putative c’ subunit of V-ATPase, impaired vacuolar acidification and mimicked the phenotypes of yeast V-ATPase mutants in the poor colony morphology, abolished asexual and sexual reproductions, selective carbon source utilization, and increased calcium and heavy metals sensitivities, however, not in the typical pH conditional lethality. Strikingly, aerial hyphae of the MoVMA11 null mutant intertwined with each other to form extremely thick filamentous structures. The results also implicated that MoVMA11 was involved in cell wall integrity and appressorium formation. Abundant non-melanized swollen structures and rare, small appressoria without penetration ability were produced at the hyphal tips of the ΔMovma11 mutant on onion epidermal cells. Finally, the MoVMA11 null mutant lost pathogenicity on both intact and wounded host leaves. Overall, our data indicated that MoVMA11, like other fungal VMA genes, is associated with numerous cellular functions and highlighted that V-ATPase is essential for infection-related morphogenesis and pathogenesis in M . oryzae . PMID:23826342

Disruption of the vacuolar calcium-ATPases in Arabidopsis results in the activation of a salicylic acid-dependent programmed cell death pathway.

PubMed

Boursiac, Yann; Lee, Sang Min; Romanowsky, Shawn; Blank, Robert; Sladek, Chris; Chung, Woo Sik; Harper, Jeffrey F

2010-11-01

Calcium (Ca(2+)) signals regulate many aspects of plant development, including a programmed cell death pathway that protects plants from pathogens (hypersensitive response). Cytosolic Ca(2+) signals result from a combined action of Ca(2+) influx through channels and Ca(2+) efflux through pumps and cotransporters. Plants utilize calmodulin-activated Ca(2+) pumps (autoinhibited Ca(2+)-ATPase [ACA]) at the plasma membrane, endoplasmic reticulum, and vacuole. Here, we show that a double knockout mutation of the vacuolar Ca(2+) pumps ACA4 and ACA11 in Arabidopsis (Arabidopsis thaliana) results in a high frequency of hypersensitive response-like lesions. The appearance of macrolesions could be suppressed by growing plants with increased levels (greater than 15 mm) of various anions, providing a method for conditional suppression. By removing plants from a conditional suppression, lesion initials were found to originate primarily in leaf mesophyll cells, as detected by aniline blue staining. Initiation and spread of lesions could also be suppressed by disrupting the production or accumulation of salicylic acid (SA), as shown by combining aca4/11 mutations with a sid 2 (for salicylic acid induction-deficient2) mutation or expression of the SA degradation enzyme NahG. This indicates that the loss of the vacuolar Ca(2+) pumps by itself does not cause a catastrophic defect in ion homeostasis but rather potentiates the activation of a SA-dependent programmed cell death pathway. Together, these results provide evidence linking the activity of the vacuolar Ca(2+) pumps to the control of a SA-dependent programmed cell death pathway in plants.

Vacuolar Chloride Fluxes Impact Ion Content and Distribution during Early Salinity Stress1

PubMed Central

Baetz, Ulrike; Tohge, Takayuki; Martinoia, Enrico; De Angeli, Alexis

2016-01-01

The ability to control the cytoplasmic environment is a prerequisite for plants to cope with changing environmental conditions. During salt stress, for instance, Na+ and Cl− are sequestered into the vacuole to help maintain cytosolic ion homeostasis and avoid cellular damage. It has been observed that vacuolar ion uptake is tied to fluxes across the plasma membrane. The coordination of both transport processes and relative contribution to plant adaptation, however, is still poorly understood. To investigate the link between vacuolar anion uptake and whole-plant ion distribution during salinity, we used mutants of the only vacuolar Cl− channel described to date: the Arabidopsis (Arabidopsis thaliana) ALMT9. After 24-h NaCl treatment, almt9 knock-out mutants had reduced shoot accumulation of both Cl− and Na+. In contrast, almt9 plants complemented with a mutant variant of ALMT9 that exhibits enhanced channel activity showed higher Cl− and Na+ accumulation. The altered shoot ion contents were not based on differences in transpiration, pointing to a vacuolar function in regulating xylem loading during salinity. In line with this finding, GUS staining demonstrated that ALMT9 is highly expressed in the vasculature of shoots and roots. RNA-seq analysis of almt9 mutants under salinity revealed specific expression profiles of transporters involved in long-distance ion translocation. Taken together, our study uncovers that the capacity of vacuolar Cl− loading in vascular cells plays a crucial role in controlling whole-plant ion movement rapidly after onset of salinity. PMID:27503602

The defects and microstructure in the fusion zone of multipass laser welded joints with Inconel 52M filler wire for nuclear power plants

NASA Astrophysics Data System (ADS)

Li, Gang; Lu, Xiaofeng; Zhu, Xiaolei; Huang, Jian; Liu, Luwei; Wu, Yixiong

2017-09-01

The defects and microstructure in the fusion zone of multipass laser welded joints with Inconel 52M filler wire are investigated for nuclear power plants. Experimental results indicate that the incomplete fusion forms as the deposited metals do not completely cover the groove during multipass laser welding. The dendritic morphologies are observed on the inner surface of the porosity in the fusion zone. Many small cellular are found in the zones near the fusion boundary. With solidification preceding, cellular gradually turn into columnar dendrites and symmetrical columnar dendrites are exhibited in the weld center of the fusion zone. The fine equiaxed grains form and columnar dendrites disappear in the remelted zone of two passes. The dendrite arm spacing in the fusion zone becomes widened with increasing welding heat input. Nb-rich carbides/carbonitrides are preferentially precipitated in the fusion zone of multipass laser welded joints. In respect to high cooling rate during multipass laser welding, element segregation could be insufficient to achieve the component of Laves phase.

Functional Expression and Characterization of Schizosaccharomyces pombe Avt3p as a Vacuolar Amino Acid Exporter in Saccharomyces cerevisiae.

PubMed

Chardwiriyapreecha, Soracom; Manabe, Kunio; Iwaki, Tomoko; Kawano-Kawada, Miyuki; Sekito, Takayuki; Lunprom, Siriporn; Akiyama, Koichi; Takegawa, Kaoru; Kakinuma, Yoshimi

2015-01-01

In Saccharomyces cerevisiae, Avt3p and Avt4p mediate the extrusion of several amino acids from the vacuolar lumen into the cytosol. SpAvt3p of Schizosaccharomyces pombe, a homologue of these vacuolar amino acid transporters, has been indicated to be involved in spore formation. In this study, we confirmed that GFP-SpAvt3p localized to the vacuolar membrane in S. pombe. The amounts of various amino acids increased significantly in the vacuolar pool of avt3Δ cells, but decreased in that of avt3+-overexpressing avt3Δ cells. These results suggest that SpAvt3p participates in the vacuolar compartmentalization of amino acids in S. pombe. To examine the export activity of SpAvt3p, we expressed the avt3+ gene in S. cerevisiae cells. We found that the heterologously overproduced GFP-SpAvt3p localized to the vacuolar membrane in S. cerevisiae. Using the vacuolar membrane vesicles isolated from avt3+-overexpressing S. cerevisiae cells, we detected the export activities of alanine and tyrosine in an ATP-dependent manner. These activities were inhibited by the addition of a V-ATPase inhibitor, concanamycin A, thereby suggesting that the activity of SpAvt3p is dependent on a proton electrochemical gradient generated by the action of V-ATPase. In addition, the amounts of various amino acids in the vacuolar pools of S. cerevisiae cells were decreased by the overproduction of SpAvt3p, which indicated that SpAvt3p was functional in S. cerevisiae cells. Thus, SpAvt3p is a vacuolar transporter that is involved in the export of amino acids from S. pombe vacuoles.

Regulation of Vacuolar pH in Citrus limon

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

Lincoln Taiz

The primary objective of this grant was to characterize the vacuolar V-ATPase of lemon fruits. Lemon fruit vacuoles have an internal pH of about 2.5. Since a typical plant vacuole has a luminal pH of around 5.5, the lemon fruit V-APTase must have special properties which allow it to acidify the lumen to such a low pH: (1) it might have a different structure; (2) it might have a different H{sup +}/ATP stoichiometry; and (3) it might be regulated differently. During the course of the investigations (which began in 1996) they characterized these aspects of the V-ATPases of both lemonmore » fruits and lime fruits. They examined lime fruits because of the availability of both acidic limes with a low vacuolar pH and sweet limes, which have a much higher vacuolar pH. The existence of two types of lime fruits allowed a comparison of the V-ATPases of the two varieties. In this report they are including two publications from 1996 and 1997 as background for the later publications. A review article with Heven Sze on V-ATPase nomenclature was also generated during the funding period. In addition to the studies on citrus fruit vacuoles, they also initiated studies in two new areas: polar auxin transport and the regulation of stomatal opening by UV-B irradiation. These studies were intended to serve as a basis of future separate grants, but the proposals they submitted on these topics were not funded.« less

Dictyostelium LvsB has a regulatory role in endosomal vesicle fusion

PubMed Central

Falkenstein, Kristin; De Lozanne, Arturo

2014-01-01

ABSTRACT Defects in human lysosomal-trafficking regulator (Lyst) are associated with the lysosomal disorder Chediak–Higashi syndrome. The absence of Lyst results in the formation of enlarged lysosome-related compartments, but the mechanism for how these compartments arise is not well established. Two opposing models have been proposed to explain Lyst function. The fission model describes Lyst as a positive regulator of fission from lysosomal compartments, whereas the fusion model identifies Lyst as a negative regulator of fusion between lysosomal vesicles. Here, we used assays that can distinguish between defects in vesicle fusion versus fission. We compared the phenotype of Dictyostelium discoideum cells defective in LvsB, the ortholog of Lyst, with that of two known fission defect mutants (μ3- and WASH-null mutants). We found that the temporal localization characteristics of the post-lysosomal marker vacuolin, as well as vesicular acidity and the fusion dynamics of LvsB-null cells are distinct from those of both μ3- and WASH-null fission defect mutants. These distinctions are predicted by the fusion defect model and implicate LvsB as a negative regulator of vesicle fusion. PMID:25086066

Identifying Novel Regulators of Vacuolar Trafficking by Combining Fluorescence Imaging-Based Forward Genetic Screening and In Vitro Pollen Germination.

PubMed

Feng, Qiang-Nan; Zhang, Yan

2017-01-01

Subcellular targeting of vacuolar proteins depends on cellular machinery regulating vesicular trafficking. Plant-specific vacuolar trafficking routes have been reported. However, regulators mediating these processes are obscure. By combining a fluorescence imaging-based forward genetic approach and in vitro pollen germination system, we show an efficient protocol of identifying regulators of plant-specific vacuolar trafficking routes.

The R2R3-MYB transcription factor MdMYB73 is involved in malate accumulation and vacuolar acidification in apple.

PubMed

Hu, Da-Gang; Li, Yuan-Yuan; Zhang, Quan-Yan; Li, Ming; Sun, Cui-Hui; Yu, Jian-Qiang; Hao, Yu-Jin

2017-08-01

Malate, the predominant organic acid in many fruits, is a crucial component of the organoleptic quality of fruit, including taste and flavor. The genetic and environmental mechanisms affecting malate metabolism in fruit cells have been studied extensively. However, the transcriptional regulation of malate-metabolizing enzymes and vacuolar transporters remains poorly understood. Our previous studies demonstrated that MdMYB1 modulates anthocyanin accumulation and vacuolar acidification by directly activating vacuolar transporters, including MdVHA-B1, MdVHA-E, MdVHP1 and MdtDT. Interestingly, we isolated and identified a MYB transcription factor, MdMYB73, a distant relative of MdMYB1 in this study. It was subsequently found that MdMYB73 protein bound directly to the promoters of MdALMT9 (aluminum-activated malate transporter 9), MdVHA-A (vacuolar ATPase subunit A) and MdVHP1 (vacuolar pyrophosphatase 1), transcriptionally activating their expression and thereby enhancing their activities. Analyses of transgenic apple calli demonstrated that MdMYB73 influenced malate accumulation and vacuolar pH. Furthermore, MdCIbHLH1 interacted with MdMYB73 and enhanced its activity upon downstream target genes. These findings help to elucidate how MdMYB73 directly modulates the vacuolar transport system to affect malate accumulation and vacuolar pH in apple. © 2017 The Authors The Plant Journal © 2017 John Wiley & Sons Ltd.

In vitro assay using engineered yeast vacuoles for neuronal SNARE-mediated membrane fusion

PubMed Central

Ko, Young-Joon; Lee, Miriam; Kang, KyeongJin; Song, Woo Keun; Jun, Youngsoo

2014-01-01

Intracellular membrane fusion requires not only SNARE proteins but also other regulatory proteins such as the Rab and Sec1/Munc18 (SM) family proteins. Although neuronal SNARE proteins alone can drive the fusion between synthetic liposomes, it remains unclear whether they are also sufficient to induce the fusion of biological membranes. Here, through the use of engineered yeast vacuoles bearing neuronal SNARE proteins, we show that neuronal SNAREs can induce membrane fusion between yeast vacuoles and that this fusion does not require the function of the Rab protein Ypt7p or the SM family protein Vps33p, both of which are essential for normal yeast vacuole fusion. Although excess vacuolar SNARE proteins were also shown to mediate Rab-bypass fusion, this fusion required homotypic fusion and vacuole protein sorting complex, which bears Vps33p and was accompanied by extensive membrane lysis. We also show that this neuronal SNARE-driven vacuole fusion can be stimulated by the neuronal SM protein Munc18 and blocked by botulinum neurotoxin serotype E, a well-known inhibitor of synaptic vesicle fusion. Taken together, our results suggest that neuronal SNARE proteins are sufficient to induce biological membrane fusion, and that this new assay can be used as a simple and complementary method for investigating synaptic vesicle fusion mechanisms. PMID:24821814

Expression analysis of Arabidopsis vacuolar sorting receptor 3 reveals a putative function in guard cells.

PubMed

Avila, Emily L; Brown, Michelle; Pan, Songqin; Desikan, Radhika; Neill, Steven J; Girke, Thomas; Surpin, Marci; Raikhel, Natasha V

2008-01-01

Vacuolar sorting receptors (VSRs) are responsible for the proper targeting of soluble cargo proteins to their destination compartments. The Arabidopsis genome encodes seven VSRs. In this work, the spatio-temporal expression of one of the members of this gene family, AtVSR3, was determined by RT-PCR and promoter::reporter gene fusions. AtVSR3 was expressed specifically in guard cells. Consequently, a reverse genetics approach was taken to determine the function of AtVSR3 by using RNA interference (RNAi) technology. Plants expressing little or no AtVSR3 transcript had a compressed life cycle, bolting approximately 1 week earlier and senescing up to 2 weeks earlier than the wild-type parent line. While the development and distribution of stomata in AtVSR3 RNAi plants appeared normal, stomatal function was altered. The guard cells of mutant plants did not close in response to abscisic acid treatment, and the mean leaf temperatures of the RNAi plants were on average 0.8 degrees C lower than both wild type and another vacuolar sorting receptor mutant, atvsr1-1. Furthermore, the loss of AtVSR3 protein caused the accumulation of nitric oxide and hydrogen peroxide, signalling molecules implicated in the regulation of stomatal opening and closing. Finally, proteomics and western blot analyses of cellular proteins isolated from wild-type and AtVSR3 RNAi leaves showed that phospholipase Dgamma, which may play a role in abscisic acid signalling, accumulated to higher levels in AtVSR3 RNAi guard cells. Thus, AtVSR3 may play an important role in responses to plant stress.

A vacuolar membrane protein Avt7p is involved in transport of amino acid and spore formation in Saccharomyces cerevisiae.

PubMed

Tone, Junichi; Yamanaka, Atsushi; Manabe, Kunio; Murao, Nami; Kawano-Kawada, Miyuki; Sekito, Takayuki; Kakinuma, Yoshimi

2015-01-01

Active transport systems for various amino acids operate in the vacuolar membrane of Saccharomyces cerevisiae. The gene families for vacuolar amino acid transporters were identified by reverse genetics experiments. In the AVT transporter family, Avt1p works for active uptake of amino acid into vacuole, and Avt3p, Avt4p, and Avt6p for active extrusion of amino acid from vacuole to cytosol. Here, we found green fluorescent protein-tagged Avt7p, an unidentified member of the AVT family, localized to the vacuolar membrane of S. cerevisiae. Disruption of the AVT7 gene enhanced both vacuolar contents of several amino acids and uptake activities of glutamine and proline by vacuolar membrane vesicles. Efficiency of spore formation was impaired by the disruption of the AVT7 gene, suggesting the physiological importance of Avt7p-dependent efflux of amino acid from vacuoles under nutrient-poor condition.

Spatiotemporal dynamics of membrane remodeling and fusion proteins during endocytic transport

PubMed Central

Arlt, Henning; Auffarth, Kathrin; Kurre, Rainer; Lisse, Dominik; Piehler, Jacob; Ungermann, Christian

2015-01-01

Organelles of the endolysosomal system undergo multiple fission and fusion events to combine sorting of selected proteins to the vacuole with endosomal recycling. This sorting requires a consecutive remodeling of the organelle surface in the course of endosomal maturation. Here we dissect the remodeling and fusion machinery on endosomes during the process of endocytosis. We traced selected GFP-tagged endosomal proteins relative to exogenously added fluorescently labeled α-factor on its way from the plasma membrane to the vacuole. Our data reveal that the machinery of endosomal fusion and ESCRT proteins has similar temporal localization on endosomes, whereas they precede the retromer cargo recognition complex. Neither deletion of retromer nor the fusion machinery with the vacuole affects this maturation process, although the kinetics seems to be delayed due to ESCRT deletion. Of importance, in strains lacking the active Rab7-like Ypt7 or the vacuolar SNARE fusion machinery, α-factor still proceeds to late endosomes with the same kinetics. This indicates that endosomal maturation is mainly controlled by the early endosomal fusion and remodeling machinery but not the downstream Rab Ypt7 or the SNARE machinery. Our data thus provide important further understanding of endosomal biogenesis in the context of cargo sorting. PMID:25657322

Calmodulin-stimulated Ca(2+)-ATPases in the vacuolar and plasma membranes in cauliflower.

PubMed

Askerlund, P

1997-07-01

The subcellular locations of Ca(2+)-ATPases in the membranes of cauliflower (Brassica oleracea L.) inflorescences were investigated. After continuous sucrose gradient centrifugation a 111-kD calmodulin (CaM)-stimulated and caM-binding Ca(2+)-ATPase (BCA1; P. Askerlund [1996] Plant Physiol 110: 913-922; S. Malmström, P. Askerlund, M.G. Plamgren [1997] FEBS Lett 400: 324-328) comigrated with vacuolar membrane markers, whereas a 116-kD caM-binding Ca(2+)-ATPase co-migrated with a marker for the plasma membrane. The 116 kD Ca(2+)-ATPase was enriched in plasma membranes obtained by aqueous two-phase partitioning, which is in agreement with a plasma membrane location of this Ca(2+)-ATPase. Countercurrent distribution of a low-density intracellular membrane fraction in an aqueous two-phase system resulted in the separation of the endoplasmic reticulum and vacuolar membranes. The 111-kD Ca(2+)-ATPase co-migrated with a vacuolar membrane marker after countercurrent distribution but not with markers for the endoplasmic reticulum. A vacuolar membrane location of the 111-kD Ca(2+)-AtPase was further supported by experiments with isolated vacuoles from cauliflower: (a) Immunoblotting with an antibody against the 111-kD Ca(2+)-ATPase showed that it was associated with the vacuoles, and (b) ATP-dependent Ca2+ uptake by the intact vacuoles was found to be CaM stimulated and partly protonophore insensitive.

LC3 and p62 as diagnostic markers of drug-induced autophagic vacuolar cardiomyopathy: a study of 3 cases.

PubMed

Daniels, Brianne H; McComb, Rodney D; Mobley, Bret C; Gultekin, Sakir Humayun; Lee, Han S; Margeta, Marta

2013-07-01

Autophagic vacuolar cardiomyopathy is an underrecognized, but potentially fatal, complication of treatment with chloroquine (CQ) and its derivative hydroxychloroquine (HCQ), which are used as therapy for malaria and common connective tissue disorders. Currently, the diagnosis of autophagic vacuolar cardiomyopathy is established through an endomyocardial biopsy and requires electron microscopy, which is not widely available and has a significant potential for sampling error. Recently, we have reported that immunohistochemistry for autophagic markers LC3 and p62 can replace electron microscopy in the diagnosis of HCQ-induced and colchicine-induced autophagic vacuolar skeletal myopathies. In the current study, we use 3 cases of CQ-induced or HCQ-induced cardiomyopathy and 1 HCQ-treated control case to show that the same two markers can be used to diagnose autophagic vacuolar cardiomyopathies by light microscopy. CQ-induced or HCQ-induced autophagic vacuolar cardiomyopathy is not universally fatal, but successful treatment requires early detection. By lowering the barriers to diagnosis, the application of these immunohistochemical markers will decrease the number of misdiagnosed patients, thus increasing the likelihood of favorable clinical outcomes.

Cloning, localization and expression analysis of vacuolar sugar transporters in the CAM plant Ananas comosus (pineapple).

PubMed

Antony, Edna; Taybi, Tahar; Courbot, Mikaël; Mugford, Sam T; Smith, J Andrew C; Borland, Anne M

2008-01-01

In photosynthetic tissues of the CAM plant pineapple (Ananas comosus), storage of soluble sugars in the central vacuole during the daytime and their remobilization at night is required to provide carbon skeletons for nocturnal CO(2) fixation. However, soluble sugars produced photosynthetically must also be exported to support growth processes in heterotrophic tissues. To begin to address how vacuolar sugar storage and assimilate partitioning are regulated in A. comosus, degenerate PCR and cDNA library screening were used to clone three candidate sugar transporters from the leaves of this species. Subcellular localization of the three transporters was investigated via expression of YFP-fusion proteins in tobacco epidermal cells and their co-localization with subcellular markers by confocal microscopy. Using this strategy, a putative hexose transporter (AcMST1) and a putative inositol transporter (AcINT1) were identified that both localized to the tonoplast, whereas a putative sucrose transporter (AcSUT1) was found to localize to prevacuolar compartments. A cDNA (AcMST2) with high similarity to a recently characterized tonoplast hexose transporter in Arabidopsis was also identified from an A. comosus fruit EST database. Analyses of transcript abundance indicated that AcMST1 was more highly expressed in fruits compared to leaves of A. comosus, whilst transcripts of AcINT1, AcSUT1, and AcMST2 were more abundant in leaves. Transcript abundance of AcINT1, the putative inositol transporter, showed day-night changes comparable to those of other CAM-related transcripts described in Mesembryanthemum crystallinum. The results are discussed in terms of the role of vacuolar sugar transporters in regulating carbon flow during the diel cycle in CAM plants.

Fe deficiency differentially affects the vacuolar proton pumps in cucumber and soybean roots

PubMed Central

Dell’Orto, Marta; Nisi, Patrizia De; Vigani, Gianpiero; Zocchi, Graziano

2013-01-01

Iron uptake in dicots depends on their ability to induce a set of responses in root cells including rhizosphere acidification through H+ extrusion and apoplastic Fe(III) reduction by Fe(III)-chelate reductase. These responses must be sustained by metabolic rearrangements aimed at providing the required NAD(P)H, ATP and H+. Previous results in Fe-deficient cucumber roots showed that high H+ extrusion is accompanied by increased phosphoenolpyruvate carboxylase (PEPC) activity, involved in the cytosol pH-stat; moreover 31P-NMR analysis revealed increased vacuolar pH and decreased vacuolar [inorganic phosphate (Pi)]. The opposite was found in soybean: low rhizosphere acidification, decreased PEPC activity, vacuole acidification, and increased vacuolar [Pi]. These findings, highlighting a different impact of the Fe deficiency responses on cytosolic pH in the two species, lead to hypothesize different roles for H+ and Pi movements across the tonoplast in pH homeostasis. The role of vacuole in cytosolic pH-stat involves the vacuolar H+-ATPase (V-ATPase) and vacuolar H+-pyrophosphatase (V-PPase) activities, which generating the ΔpH and ΔΨ, mediate the transport of solutes, among which Pi, across the tonoplast. Fluxes of Pi itself in its two ionic forms, H2PO4- predominating in the vacuole and HPO42- in the cytosol, may be involved in pH homeostasis owing to its pH-dependent protonation/deprotonation reactions. Tonoplast enriched fractions were obtained from cucumber and soybean roots grown with or without Fe. Both V-ATPase and V-PPase activities were analyzed and the enrichment and localization of the corresponding proteins in root tissues were determined by Western blot and immunolocalization. V-ATPase did not change its activity and expression level in response to Fe starvation in both species. V-PPase showed a different behavior: in cucumber roots its activity and abundance were decreased, while in Fe-deficient soybean roots they were increased. The distinct role of

Evolution of tonoplast P-ATPase transporters involved in vacuolar acidification.

PubMed

Li, Yanbang; Provenzano, Sofia; Bliek, Mattijs; Spelt, Cornelis; Appelhagen, Ingo; Machado de Faria, Laura; Verweij, Walter; Schubert, Andrea; Sagasser, Martin; Seidel, Thorsten; Weisshaar, Bernd; Koes, Ronald; Quattrocchio, Francesca

2016-08-01

Petunia mutants (Petunia hybrida) with blue flowers defined a novel vacuolar proton pump consisting of two interacting P-ATPases, PH1 and PH5, that hyper-acidify the vacuoles of petal cells. PH5 is similar to plasma membrane H(+) P3A -ATPase, whereas PH1 is the only known eukaryoticP3B -ATPase. As there were no indications that this tonoplast pump is widespread in plants, we investigated the distribution and evolution of PH1 and PH5. We combined database mining and phylogenetic and synteny analyses of PH1- and PH5-like proteins from all kingdoms with functional analyses (mutant complementation and intracellular localization) of homologs from diverse angiosperms. We identified functional PH1 and PH5 homologs in divergent angiosperms. PH5 homologs evolved from plasma membrane P3A -ATPases, acquiring an N-terminal tonoplast-sorting sequence and new cellular function before angiosperms appeared. PH1 is widespread among seed plants and related proteins are found in some groups of bacteria and fungi and in one moss, but is absent in most algae, suggesting that its evolution involved several cases of gene loss and possibly horizontal transfer events. The distribution of PH1 and PH5 in the plant kingdom suggests that vacuolar acidification by P-ATPases appeared in gymnosperms before flowers. This implies that, next to flower color determination, vacuolar hyper-acidification is required for yet unknown processes. © 2016 European Union. New Phytologist © 2016 New Phytologist Trust.

Proton Gradient-Driven Nickel Uptake by Vacuolar Membrane Vesicles of Saccharomyces cerevisiae

PubMed Central

Nishimura, Ken; Igarashi, Kazuei; Kakinuma, Yoshimi

1998-01-01

A vacuolar H+-ATPase-negative mutant of Saccharomyces cerevisiae was highly sensitive to nickel ion. Accumulation of nickel ion in the cells of this mutant of less than 60% of the value for the parent strain arrested growth, suggesting a role for this ATPase in sequestering nickel ion into vacuoles. An artificially imposed pH gradient (interior acid) induced transient nickel ion uptake by vacuolar membrane vesicles, which was inhibited by collapse of the pH difference but not of the membrane potential. Nickel ion transport into vacuoles in a pH gradient-dependent manner is thus important for its detoxification in yeast. PMID:9537401

Modeling of Powder Bed Manufacturing Defects

NASA Astrophysics Data System (ADS)

Mindt, H.-W.; Desmaison, O.; Megahed, M.; Peralta, A.; Neumann, J.

2018-01-01

Powder bed additive manufacturing offers unmatched capabilities. The deposition resolution achieved is extremely high enabling the production of innovative functional products and materials. Achieving the desired final quality is, however, hampered by many potential defects that have to be managed in due course of the manufacturing process. Defects observed in products manufactured via powder bed fusion have been studied experimentally. In this effort we have relied on experiments reported in the literature and—when experimental data were not sufficient—we have performed additional experiments providing an extended foundation for defect analysis. There is large interest in reducing the effort and cost of additive manufacturing process qualification and certification using integrated computational material engineering. A prerequisite is, however, that numerical methods can indeed capture defects. A multiscale multiphysics platform is developed and applied to predict and explain the origin of several defects that have been observed experimentally during laser-based powder bed fusion processes. The models utilized are briefly introduced. The ability of the models to capture the observed defects is verified. The root cause of the defects is explained by analyzing the numerical results thus confirming the ability of numerical methods to provide a foundation for rapid process qualification.

Appropriate vacuolar acidification in Saccharomyces cerevisiae is associated with efficient high sugar fermentation.

PubMed

Nguyen, Trung D; Walker, Michelle E; Gardner, Jennifer M; Jiranek, Vladimir

2018-04-01

Vacuolar acidification serves as a homeostatic mechanism to regulate intracellular pH, ion and chemical balance, as well as trafficking and recycling of proteins and nutrients, critical for normal cellular function. This study reports on the importance of vacuole acidification during wine-like fermentation. Ninety-three mutants (homozygous deletions in lab yeast strain, BY4743), which result in protracted fermentation when grown in a chemically defined grape juice with 200 g L -1 sugar (pH 3.5), were examined to determine whether fermentation protraction was in part due to a dysfunction in vacuolar acidification (VA) during the early stages of fermentation, and whether VA was responsive to the initial sugar concentration in the medium. Cells after 24 h growth were dual-labelled with propidium iodide and vacuolar specific probe 6-carboxyfluorescein diacetate (6-CFDA) and examined with a FACS analyser for viability and impaired VA, respectively. Twenty mutants showed a greater than two-fold increase in fluorescence intensity; the experimental indicator for vacuolar dysfunction; 10 of which have not been previously annotated to this process. With the exception of Δhog1, Δpbs2 and Δvph1 mutants, where dysfunction was directly related to osmolality; the remainder exhibited increased CF-fluorescence, independent of sugar concentration at 20 g L -1 or 200 g L -1 . These findings offer insight to the importance of VA to cell growth in high sugar media. Copyright © 2017 Elsevier Ltd. All rights reserved.

Cytosolic Nucleotides Block and Regulate the Arabidopsis Vacuolar Anion Channel AtALMT9*

PubMed Central

Zhang, Jingbo; Martinoia, Enrico; De Angeli, Alexis

2014-01-01

The aluminum-activated malate transporters (ALMTs) form a membrane protein family exhibiting different physiological roles in plants, varying from conferring tolerance to environmental Al3+ to the regulation of stomatal movement. The regulation of the anion channels of the ALMT family is largely unknown. Identifying intracellular modulators of the activity of anion channels is fundamental to understanding their physiological functions. In this study we investigated the role of cytosolic nucleotides in regulating the activity of the vacuolar anion channel AtALMT9. We found that cytosolic nucleotides modulate the transport activity of AtALMT9. This modulation was based on a direct block of the pore of the channel at negative membrane potentials (open channel block) by the nucleotide and not by a phosphorylation mechanism. The block by nucleotides of AtALMT9-mediated currents was voltage dependent. The blocking efficiency of intracellular nucleotides increased with the number of phosphate groups and ATP was the most effective cellular blocker. Interestingly, the ATP block induced a marked modification of the current-voltage characteristic of AtALMT9. In addition, increased concentrations of vacuolar anions were able to shift the ATP block threshold to a more negative membrane potential. The block of AtALMT9-mediated anion currents by ATP at negative membrane potentials acts as a gate of the channel and vacuolar anion tune this gating mechanism. Our results suggest that anion transport across the vacuolar membrane in plant cells is controlled by cytosolic nucleotides and the energetic status of the cell. PMID:25028514

Cytosolic nucleotides block and regulate the Arabidopsis vacuolar anion channel AtALMT9.

PubMed

Zhang, Jingbo; Martinoia, Enrico; De Angeli, Alexis

2014-09-12

The aluminum-activated malate transporters (ALMTs) form a membrane protein family exhibiting different physiological roles in plants, varying from conferring tolerance to environmental Al(3+) to the regulation of stomatal movement. The regulation of the anion channels of the ALMT family is largely unknown. Identifying intracellular modulators of the activity of anion channels is fundamental to understanding their physiological functions. In this study we investigated the role of cytosolic nucleotides in regulating the activity of the vacuolar anion channel AtALMT9. We found that cytosolic nucleotides modulate the transport activity of AtALMT9. This modulation was based on a direct block of the pore of the channel at negative membrane potentials (open channel block) by the nucleotide and not by a phosphorylation mechanism. The block by nucleotides of AtALMT9-mediated currents was voltage dependent. The blocking efficiency of intracellular nucleotides increased with the number of phosphate groups and ATP was the most effective cellular blocker. Interestingly, the ATP block induced a marked modification of the current-voltage characteristic of AtALMT9. In addition, increased concentrations of vacuolar anions were able to shift the ATP block threshold to a more negative membrane potential. The block of AtALMT9-mediated anion currents by ATP at negative membrane potentials acts as a gate of the channel and vacuolar anion tune this gating mechanism. Our results suggest that anion transport across the vacuolar membrane in plant cells is controlled by cytosolic nucleotides and the energetic status of the cell. © 2014 by The American Society for Biochemistry and Molecular Biology, Inc.

Functional expression of Schizosaccharomyces pombe Vba2p in the vacuolar membrane of Saccharomyces cerevisiae.

PubMed

Pongcharoen, Pongsanat; Kawano-Kawada, Miyuki; Iwaki, Tomoko; Sugimoto, Naoko; Sekito, Takayuki; Akiyama, Koichi; Takegawa, Kaoru; Kakinuma, Yoshimi

2013-01-01

A vacuolar membrane protein, Vba2p of Schizosaccharomyces pombe, is involved in basic amino acid uptake by intact cells. Here we found evidence that Vba2p mediated ATP-dependent lysine uptake by vacuolar membrane vesicles of Saccharomyces cerevisiae. Vba2p was also responsible for quinidine sensitivity, and the addition of lysine improved cell growth on quinidine-containing media. These findings should be useful for further characterization of Vba2p.

Subcellular localization and vacuolar targeting of sorbitol dehydrogenase in apple seed.

PubMed

Wang, Xiu-Ling; Hu, Zi-Ying; You, Chun-Xiang; Kong, Xiu-Zhen; Shi, Xiao-Pu

2013-09-01

Sorbitol is the primary photosynthate and translocated carbohydrate in fruit trees of the Rosaceae family. NAD(+)-dependent sorbitol dehydrogenase (NAD-SDH, EC 1.1.1.14), which mainly catalyzes the oxidation of sorbitol to fructose, plays a key role in regulating sink strength in apple. In this study, we found that apple NAD-SDH was ubiquitously distributed in epidermis, parenchyma, and vascular bundle in developing cotyledon. NAD-SDH was localized in the cytosol, the membranes of endoplasmic reticulum and vesicles, and the vacuolar lumen in the cotyledon at the middle stage of seed development. In contrast, NAD-SDH was mainly distributed in the protein storage vacuoles in cotyledon at the late stage of seed development. Sequence analysis revealed there is a putative signal peptide (SP), also being predicated to be a transmembrane domain, in the middle of proteins of apple NAD-SDH isoforms. To investigate whether the putative internal SP functions in the vacuolar targeting of NAD-SDH, we analyzed the localization of the SP-deletion mutants of MdSDH5 and MdSDH6 (two NAD-SDH isoforms in apple) by the transient expression system in Arabidopsis protoplasts. MdSDH5 and MdSDH6 were not localized in the vacuoles after their SPs were deleted, suggesting the internal SP functions in the vacuolar targeting of apple NAD-SDH. Copyright © 2013 Elsevier Ireland Ltd. All rights reserved.

Internalization and vacuolar targeting of the brassinosteroid hormone receptor BRI1 are regulated by ubiquitination.

PubMed

Martins, Sara; Dohmann, Esther M N; Cayrel, Anne; Johnson, Alexander; Fischer, Wolfgang; Pojer, Florence; Satiat-Jeunemaître, Béatrice; Jaillais, Yvon; Chory, Joanne; Geldner, Niko; Vert, Grégory

2015-01-21

Brassinosteroids are plant steroid hormones that control many aspects of plant growth and development, and are perceived at the cell surface by the plasma membrane-localized receptor kinase BRI1. Here we show that BRI1 is post-translationally modified by K63 polyubiquitin chains in vivo. Using both artificial ubiquitination of BRI1 and generation of an ubiquitination-defective BRI1 mutant form, we demonstrate that ubiquitination promotes BRI1 internalization from the cell surface and is essential for its recognition at the trans-Golgi network/early endosomes (TGN/EE) for vacuolar targeting. Finally, we demonstrate that the control of BRI1 protein dynamics by ubiquitination is an important control mechanism for brassinosteroid responses in plants. Altogether, our results identify ubiquitination and K63-linked polyubiquitin chain formation as a dual targeting signal for BRI1 internalization and sorting along the endocytic pathway, and highlight its role in hormonally controlled plant development.

Formation of vacuolar tannin deposits in the chlorophyllous organs of Tracheophyta: from shuttles to accretions.

PubMed

Brillouet, Jean-Marc; Romieu, Charles; Lartaud, Marc; Jublanc, Elodie; Torregrosa, Laurent; Cazevieille, Chantal

2014-11-01

Most Tracheophyta synthesize-condensed tannins (also called proanthocyanidins), polymers of catechins, which appear in the vacuole as uniformly stained deposits-termed tannin accretions-lining the inner face of the tonoplast. A large body of evidence argues that tannins are formed in recently described thylakoid-derived organelles, the tannosomes, which are packed in membrane-bound shuttles (Brillouet et al. 2013); it has been suggested that shuttles agglomerate into tannin accretions. The aim of the study was to describe the ontogenesis of tannin accretions in members of the Tracheophyta. For this purpose, fresh specimens of young tissues from diverse Tracheophyta were cut, gently lacerated in paraformaldehyde, and examined using light, epifluorescence, confocal, and transmission electron microscopy. Fresh samples were also incubated with gelatin-Oregon Green, a fluorescent marker of condensed tannins. Our observations showed that vacuolar accretions (1 → 40 μm), that constitute the typical form of tannin storage in tannin-producing Tracheophyta, are formed by agglomeration (not fusion) of shuttles containing various proportions of chlorophylls and tannins.

Arabidopsis adaptor protein 1G is critical for pollen development.

PubMed

Feng, Chong; Wang, Jia-Gang; Liu, Hai-Hong; Li, Sha; Zhang, Yan

2017-09-01

Pollen development is a pre-requisite for sexual reproduction of angiosperms, during which various cellular activities are involved. Pollen development accompanies dynamic remodeling of vacuoles through fission and fusion, disruption of which often compromises pollen viability. We previously reported that the Y subunit of adaptor protein 1 (AP1G) mediates synergid degeneration during pollen tube reception. Here, we demonstrate that AP1G is essential for pollen development. AP1G loss-of-function resulted in male gametophytic lethality due to defective pollen development. By ultrastructural analysis and fluorescence labeling, we demonstrate that AP1G loss-of-function compromised dynamic vacuolar remodeling during pollen development and impaired vacuolar acidification of pollen. Results presented here support a key role of vacuoles in gametophytic pollen development. © 2017 Institute of Botany, Chinese Academy of Sciences.

Job Sharing in the Endomembrane System: Vacuolar Acidification Requires the Combined Activity of V-ATPase and V-PPase.

PubMed

Kriegel, Anne; Andrés, Zaida; Medzihradszky, Anna; Krüger, Falco; Scholl, Stefan; Delang, Simon; Patir-Nebioglu, M Görkem; Gute, Gezahegn; Yang, Haibing; Murphy, Angus S; Peer, Wendy Ann; Pfeiffer, Anne; Krebs, Melanie; Lohmann, Jan U; Schumacher, Karin

2015-12-01

The presence of a large central vacuole is one of the hallmarks of a prototypical plant cell, and the multiple functions of this compartment require massive fluxes of molecules across its limiting membrane, the tonoplast. Transport is assumed to be energized by the membrane potential and the proton gradient established by the combined activity of two proton pumps, the vacuolar H(+)-pyrophosphatase (V-PPase) and the vacuolar H(+)-ATPase (V-ATPase). Exactly how labor is divided between these two enzymes has remained elusive. Here, we provide evidence using gain- and loss-of-function approaches that lack of the V-ATPase cannot be compensated for by increased V-PPase activity. Moreover, we show that increased V-ATPase activity during cold acclimation requires the presence of the V-PPase. Most importantly, we demonstrate that a mutant lacking both of these proton pumps is conditionally viable and retains significant vacuolar acidification, pointing to a so far undetected contribution of the trans-Golgi network/early endosome-localized V-ATPase to vacuolar pH. © 2015 American Society of Plant Biologists. All rights reserved.

Phosphatidylinositol 3-Kinase Promotes V-ATPase Activation and Vacuolar Acidification and Delays Methyl Jasmonate-Induced Leaf Senescence1

PubMed Central

Liu, Jian; Ji, Yingbin; Zhou, Jun; Xing, Da

2016-01-01

PI3K and its product PI3P are both involved in plant development and stress responses. In this study, the down-regulation of PI3K activity accelerated leaf senescence induced by methyl jasmonate (MeJA) and suppressed the activation of vacuolar H+-ATPase (V-ATPase). Yeast two-hybrid analyses indicated that PI3K bound to the V-ATPase B subunit (VHA-B). Analysis of bimolecular fluorescence complementation in tobacco guard cells showed that PI3K interacted with VHA-B2 in the tonoplasts. Through the use of pharmacological and genetic tools, we found that PI3K and V-ATPase promoted vacuolar acidification and stomatal closure during leaf senescence. Vacuolar acidification was suppressed by the PIKfyve inhibitor in 35S:AtVPS34-YFP Arabidopsis during MeJA-induced leaf senescence, but the decrease was lower than that in YFP-labeled Arabidopsis. These results suggest that PI3K promotes V-ATPase activation and consequently induces vacuolar acidification and stomatal closure, thereby delaying MeJA-induced leaf senescence. PMID:26739232

The vacuolar ATPase from Entamoeba histolytica: molecular cloning of the gene encoding for the B subunit and subcellular localization of the protein.

PubMed

Meléndez-Hernández, Mayra Gisela; Barrios, María Luisa Labra; Orozco, Esther; Luna-Arias, Juan Pedro

2008-12-23

Entamoeba histolytica is a professional phagocytic cell where the vacuolar ATPase plays a key role. This enzyme is a multisubunit complex that regulates pH in many subcellular compartments, even in those that are not measurably acidic. It participates in a wide variety of cellular processes such as endocytosis, intracellular transport and membrane fusion. The presence of a vacuolar type H+-ATPase in E. histolytica trophozoites has been inferred previously from inhibition assays of its activity, the isolation of the Ehvma1 and Ehvma3 genes, and by proteomic analysis of purified phagosomes. We report the isolation and characterization of the Ehvma2 gene, which encodes for the subunit B of the vacuolar ATPase. This polypeptide is a 55.3 kDa highly conserved protein with 34 to 80% identity to orthologous proteins from other species. Particularly, in silico studies showed that EhV-ATPase subunit B displays 78% identity and 90% similarity to its Dictyostelium ortholog. A 462 bp DNA fragment of the Ehvma2 gene was expressed in bacteria and recombinant polypeptide was used to raise mouse polyclonal antibodies. EhV-ATPase subunit B antibodies detected a 55 kDa band in whole cell extracts and in an enriched fraction of DNA-containing organelles named EhkOs. The V-ATPase subunit B was located by immunofluorescence and confocal microscopy in many vesicles, in phagosomes, plasma membrane and in EhkOs. We also identified the genes encoding for the majority of the V-ATPase subunits in the E. histolytica genome, and proposed a putative model for this proton pump. We have isolated the Ehvma2 gene which encodes for the V-ATPase subunit B from the E. histolytica clone A. This gene has a 154 bp intron and encodes for a highly conserved polypeptide. Specific antibodies localized EhV-ATPase subunit B in many vesicles, phagosomes, plasma membrane and in EhkOs. Most of the orthologous genes encoding for the EhV-ATPase subunits were found in the E. histolytica genome, indicating the

8-Dehydrosterols induce membrane traffic and autophagy defects through V-ATPase dysfunction in Saccharomyces cerevisae.

PubMed

Hernández, Agustín; Serrano-Bueno, Gloria; Perez-Castiñeira, José Román; Serrano, Aurelio

2015-11-01

8-Dehydrosterols are present in a wide range of biologically relevant situations, from human rare diseases to amine fungicide-treated fungi and crops. However, the molecular bases of their toxicity are still obscure. We show here that 8-dehydrosterols, but not other sterols, affect yeast vacuole acidification through V-ATPases. Moreover, erg2Δ cells display reductions in proton pumping rates consistent with ion-transport uncoupling in vitro. Concomitantly, subunit Vph1p shows conformational changes in the presence of 8-dehydrosterols. Expression of a plant vacuolar H(+)-pumping pyrophosphatase as an alternative H(+)-pump relieves Vma(-)-like phenotypes in erg2Δ-derived mutant cells. As a consequence of these acidification defects, endo- and exo-cytic traffic deficiencies that can be alleviated with a H(+)-pumping pyrophosphatase are also observed. Despite their effect on membrane traffic, 8-dehydrosterols do not induce endoplasmic reticulum stress or assembly defects on the V-ATPase. Autophagy is a V-ATPase dependent process and erg2Δ mutants accumulate autophagic bodies under nitrogen starvation similar to Vma(-) mutants. In contrast to classical Atg(-) mutants, this defect is not accompanied by impairment of traffic through the CVT pathway, processing of Pho8Δ60p, GFP-Atg8p localisation or difficulties to survive under nitrogen starvation conditions, but it is concomitant to reduced vacuolar protease activity. All in all, erg2Δ cells are autophagy mutants albeit some of their phenotypic features differ from classical Atg(-) defective cells. These results may pave the way to understand the aetiology of sterol-related diseases, the cytotoxic effect of amine fungicides, and may explain the tolerance to these compounds observed in plants. Copyright © 2015 Elsevier B.V. All rights reserved.

Vacuolar zinc transporter Zrc1 is required for detoxification of excess intracellular zinc in the human fungal pathogen Cryptococcus neoformans.

PubMed

Cho, Minsu; Hu, Guanggan; Caza, Mélissa; Horianopoulos, Linda C; Kronstad, James W; Jung, Won Hee

2018-01-01

Zinc is an important transition metal in all living organisms and is required for numerous biological processes. However, excess zinc can also be toxic to cells and cause cellular stress. In the model fungus Saccharomyces cerevisiae, a vacuolar zinc transporter, Zrc1, plays important roles in the storage and detoxification of excess intracellular zinc to protect the cell. In this study, we identified an ortholog of the S. cerevisiae ZRC1 gene in the human fungal pathogen Cryptococcus neoformans. Zrc1 was localized in the vacuolar membrane in C. neoformans, and a mutant lacking ZRC1 showed significant growth defects under high-zinc conditions. These results suggested a role for Zrc1 in zinc detoxification. However, contrary to our expectation, the expression of Zrc1 was induced in cells grown in zinc-limited conditions and decreased upon the addition of zinc. These expression patterns were similar to those of Zip1, the high-affinity zinc transporter in the plasma membrane of C. neoformans. Furthermore, we used the zrc1 mutant in a murine model of cryptococcosis to examine whether a mammalian host could inhibit the survival of C. neoformans using zinc toxicity. We found that the mutant showed no difference in virulence compared with the wildtype strain. This result suggests that Zrc1-mediated zinc detoxification is not required for the virulence of C. neoformans, and imply that zinc toxicity may not be an important aspect of the host immune response to the fungus.

The actin cytoskeleton inhibits pore expansion during PIV5 fusion protein-promoted cell-cell fusion

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

Wurth, Mark A.; Schowalter, Rachel M.; Smith, Everett Clinton

2010-08-15

Paramyxovirus fusion (F) proteins promote both virus-cell fusion, required for viral entry, and cell-cell fusion, resulting in syncytia formation. We used the F-actin stabilizing drug, jasplakinolide, and the G-actin sequestrant, latrunculin A, to examine the role of actin dynamics in cell-cell fusion mediated by the parainfluenza virus 5 (PIV5) F protein. Jasplakinolide treatment caused a dose-dependent increase in cell-cell fusion as measured by both syncytia and reporter gene assays, and latrunculin A treatment also resulted in fusion stimulation. Treatment with jasplakinolide or latrunculin A partially rescued a fusion pore opening defect caused by deletion of the PIV5 F protein cytoplasmicmore » tail, but these drugs had no effect on fusion inhibited at earlier stages by either temperature arrest or by a PIV5 heptad repeat peptide. These data suggest that the cortical actin cytoskeleton is an important regulator of fusion pore enlargement, an energetically costly stage of viral fusion protein-mediated membrane merger.« less

The actin cytoskeleton inhibits pore expansion during PIV5 fusion protein-promoted cell-cell fusion

PubMed Central

Wurth, Mark A.; Schowalter, Rachel M.; Smith, Everett Clinton; Moncman, Carole L.; Dutch, Rebecca Ellis; McCann, Richard O.

2010-01-01

Paramyxovirus fusion (F) proteins promote both virus-cell fusion, required for viral entry, and cell-cell fusion, resulting in syncytia formation. We used the F-actin stabilizing drug, jasplakinolide, and the G-actin sequestrant, latrunculin A, to examine the role of actin dynamics in cell-cell fusion mediated by the parainfluenza virus 5 (PIV5) F protein. Jasplakinolide treatment caused a dose-dependent increase in cell-cell fusion as measured by both syncytia and reporter gene assays, and latrunculin A treatment also resulted in fusion stimulation. Treatment with jasplakinolide or latrunculin A partially rescued a fusion pore opening defect caused by deletion of the PIV5 F protein cytoplasmic tail, but these drugs had no effect on fusion inhibited at earlier stages by either temperature arrest or by a PIV5 heptad repeat peptide. These data suggest that the cortical actin cytoskeleton is an important regulator of fusion pore enlargement, an energetically costly stage of viral fusion protein-mediated membrane merger. PMID:20537366

Topological defects in open string field theory

NASA Astrophysics Data System (ADS)

Kojita, Toshiko; Maccaferri, Carlo; Masuda, Toru; Schnabl, Martin

2018-04-01

We show how conformal field theory topological defects can relate solutions of open string field theory for different boundary conditions. To this end we generalize the results of Graham and Watts to include the action of defects on boundary condition changing fields. Special care is devoted to the general case when nontrivial multiplicities arise upon defect action. Surprisingly the fusion algebra of defects is realized on open string fields only up to a (star algebra) isomorphism.

AtALMT9 is a malate-activated vacuolar chloride channel required for stomatal opening in Arabidopsis

PubMed Central

De Angeli, Alexis; Zhang, Jingbo; Meyer, Stefan; Martinoia, Enrico

2013-01-01

Water deficit strongly affects crop productivity. Plants control water loss and CO2 uptake by regulating the aperture of the stomatal pores within the leaf epidermis. Stomata aperture is regulated by the two guard cells forming the pore and changing their size in response to ion uptake and release. While our knowledge about potassium and chloride fluxes across the plasma membrane of guard cells is advanced, little is known about fluxes across the vacuolar membrane. Here we present the molecular identification of the long-sought-after vacuolar chloride channel. AtALMT9 is a chloride channel activated by physiological concentrations of cytosolic malate. Single-channel measurements demonstrate that this activation is due to a malate-dependent increase in the channel open probability. Arabidopsis thaliana atalmt9 knockout mutants exhibited impaired stomatal opening and wilt more slowly than the wild type. Our findings show that AtALMT9 is a vacuolar chloride channel having a major role in controlling stomata aperture. PMID:23653216

ERG2 and ERG24 Are Required for Normal Vacuolar Physiology as Well as Candida albicans Pathogenicity in a Murine Model of Disseminated but Not Vaginal Candidiasis

PubMed Central

Luna-Tapia, Arturo; Peters, Brian M.; Eberle, Karen E.; Kerns, Morgan E.; Foster, Timothy P.; Marrero, Luis; Noverr, Mairi C.; Fidel, Paul L.

2015-01-01

Several important classes of antifungal agents, including the azoles, act by blocking ergosterol biosynthesis. It was recently reported that the azoles cause massive disruption of the fungal vacuole in the prevalent human pathogen Candida albicans. This is significant because normal vacuolar function is required to support C. albicans pathogenicity. This study examined the impact of the morpholine antifungals, which inhibit later steps of ergosterol biosynthesis, on C. albicans vacuolar integrity. It was found that overexpression of either the ERG2 or ERG24 gene, encoding C-8 sterol isomerase or C-14 sterol reductase, respectively, suppressed C. albicans sensitivity to the morpholines. In addition, both erg2Δ/Δ and erg24Δ/Δ mutants were hypersensitive to the morpholines. These data are consistent with the antifungal activity of the morpholines depending upon the simultaneous inhibition of both Erg2p and Erg24p. The vacuoles within both erg2Δ/Δ and erg24Δ/Δ C. albicans strains exhibited an aberrant morphology and accumulated large quantities of the weak base quinacrine, indicating enhanced vacuolar acidification compared with that of control strains. Both erg mutants exhibited significant defects in polarized hyphal growth and were avirulent in a mouse model of disseminated candidiasis. Surprisingly, in a mouse model of vaginal candidiasis, both mutants colonized mice at high levels and induced a pathogenic response similar to that with the controls. Thus, while targeting Erg2p or Erg24p alone could provide a potentially efficacious therapy for disseminated candidiasis, it may not be an effective strategy to treat vaginal infections. The potential value of drugs targeting these enzymes as adjunctive therapies is discussed. PMID:26231054

Using Laser Ultrasound to Detect Subsurface Defects in Metal Laser Powder Bed Fusion Components

NASA Astrophysics Data System (ADS)

Everton, Sarah; Dickens, Phill; Tuck, Chris; Dutton, Ben

2018-03-01

Laser powder bed fusion offers many advantages over conventional manufacturing methods, such as the integration of multiple parts that can result in significant weight-savings. The increased design freedom that layer-wise manufacture allows has also been seen to enhance component performance at little or no added cost. For such benefits to be realized, however, the material quality must first be assured. Laser ultrasonic testing is a noncontact inspection technique that has been proposed as suitable for in situ monitoring of metal additive manufacturing processes. This article explores the current capability of this technique to detect manufactured, subsurface defects in Ti-6Al-4V samples, ex situ. The results are compared with x-ray computed tomography reconstructions and focus variation microscopy. Although laser ultrasound has been used to identify material discontinuities, further work is required before this technique could be implemented in situ.

Cloning, 3D modeling and expression analysis of three vacuolar invertase genes from cassava (Manihot Esculenta Crantz).

PubMed

Yao, Yuan; Wu, Xiao-Hui; Geng, Meng-Ting; Li, Rui-Mei; Liu, Jiao; Hu, Xin-Wen; Guo, Jian-Chun

2014-05-15

Vacuolar invertase is one of the key enzymes in sucrose metabolism that irreversibly catalyzes the hydrolysis of sucrose to glucose and fructose in plants. In this research, three vacuolar invertase genes, named MeVINV1-3, and with 653, 660 and 639 amino acids, respectively, were cloned from cassava. The motifs of NDPNG (β-fructosidase motif), RDP and WECVD, which are conserved and essential for catalytic activity in the vacuolar invertase family, were found in MeVINV1 and MeVINV2. Meanwhile, in MeVINV3, instead of NDPNG we found the motif NGPDG, in which the three amino acids GPD are different from those in other vacuolar invertases (DPN) that might result in MeVINV3 being an inactivated protein. The N-terminal leader sequence of MeVINVs contains a signal anchor, which is associated with the sorting of vacuolar invertase to vacuole. The overall predicted 3D structure of the MeVINVs consists of a five bladed β-propeller module at N-terminus domain, and forms a β-sandwich module at the C-terminus domain. The active site of the protein is situated in the β-propeller module. MeVINVs are classified in two subfamilies, α and β groups, in which α group members of MeVINV1 and 2 are highly expressed in reproductive organs and tuber roots (considered as sink organs), while β group members of MeVINV3 are highly expressed in leaves (source organs). All MeVINVs are highly expressed in leaves, while only MeVINV1 and 2 are highly expressed in tubers at cassava tuber maturity stage. Thus, MeVINV1 and 2 play an important role in sucrose unloading and starch accumulation, as well in buffering the pools of sucrose, hexoses and sugar phosphates in leaves, specifically at later stages of plant development.

The Arabidopsis cax1 Mutant Exhibits Impaired Ion Homeostasis, Development, and Hormonal Responses and Reveals Interplay among Vacuolar Transporters

PubMed Central

Cheng, Ning-Hui; Pittman, Jon K.; Barkla, Bronwyn J.; Shigaki, Toshiro; Hirschi, Kendal D.

2003-01-01

The Arabidopsis Ca2+/H+ transporter CAX1 (Cation Exchanger1) may be an important regulator of intracellular Ca2+ levels. Here, we describe the preliminary localization of CAX1 to the tonoplast and the molecular and biochemical characterization of cax1 mutants. We show that these mutants exhibit a 50% reduction in tonoplast Ca2+/H+ antiport activity, a 40% reduction in tonoplast V-type H+-translocating ATPase activity, a 36% increase in tonoplast Ca2+-ATPase activity, and increased expression of the putative vacuolar Ca2+/H+ antiporters CAX3 and CAX4. Enhanced growth was displayed by the cax1 lines under Mn2+ and Mg2+ stress conditions. The mutants exhibited altered plant development, perturbed hormone sensitivities, and altered expression of an auxin-regulated promoter-reporter gene fusion. We propose that CAX1 regulates myriad plant processes and discuss the observed phenotypes with regard to the compensatory alterations in other transporters. PMID:12566577

The Arabidopsis cax1 mutant exhibits impaired ion homeostasis, development, and hormonal responses and reveals interplay among vacuolar transporters.

PubMed

Cheng, Ning-Hui; Pittman, Jon K; Barkla, Bronwyn J; Shigaki, Toshiro; Hirschi, Kendal D

2003-02-01

The Arabidopsis Ca(2+)/H(+) transporter CAX1 (Cation Exchanger1) may be an important regulator of intracellular Ca(2+) levels. Here, we describe the preliminary localization of CAX1 to the tonoplast and the molecular and biochemical characterization of cax1 mutants. We show that these mutants exhibit a 50% reduction in tonoplast Ca(2+)/H(+) antiport activity, a 40% reduction in tonoplast V-type H(+)-translocating ATPase activity, a 36% increase in tonoplast Ca(2+)-ATPase activity, and increased expression of the putative vacuolar Ca(2+)/H(+) antiporters CAX3 and CAX4. Enhanced growth was displayed by the cax1 lines under Mn(2+) and Mg(2+) stress conditions. The mutants exhibited altered plant development, perturbed hormone sensitivities, and altered expression of an auxin-regulated promoter-reporter gene fusion. We propose that CAX1 regulates myriad plant processes and discuss the observed phenotypes with regard to the compensatory alterations in other transporters.

Loss of ATP-dependent lysine uptake in the vacuolar membrane vesicles of Saccharomyces cerevisiae ypq1∆ mutant.

PubMed

Sekito, Takayuki; Nakamura, Kyosuke; Manabe, Kunio; Tone, Junichi; Sato, Yumika; Murao, Nami; Kawano-Kawada, Miyuki; Kakinuma, Yoshimi

2014-01-01

Saccharomyces cerevisiae Ypq1p is a vacuolar membrane protein of the PQ-loop protein family. We found that ATP-dependent uptake activities of amino acids by vacuolar membrane vesicles were impaired by ypq1∆ mutation. Loss of lysine uptake was most remarkable, and the uptake was recovered by overproduction of Ypq1p. Ypq1p is thus involved in transport of amino acids into vacuoles.

Modeling the vacuolar storage of malate shed lights on pre- and post-harvest fruit acidity.

PubMed

Etienne, Audrey; Génard, Michel; Lobit, Philippe; Bugaud, Christophe

2014-11-18

Malate is one of the most important organic acids in many fruits and its concentration plays a critical role in organoleptic properties. Several studies suggest that malate accumulation in fruit cells is controlled at the level of vacuolar storage. However, the regulation of vacuolar malate storage throughout fruit development, and the origins of the phenotypic variability of the malate concentration within fruit species remain to be clarified. In the present study, we adapted the mechanistic model of vacuolar storage proposed by Lobit et al. in order to study the accumulation of malate in pre and postharvest fruits. The main adaptation concerned the variation of the free energy of ATP hydrolysis during fruit development. Banana fruit was taken as a reference because it has the particularity of having separate growth and post-harvest ripening stages, during which malate concentration undergoes substantial changes. Moreover, the concentration of malate in banana pulp varies greatly among cultivars which make possible to use the model as a tool to analyze the genotypic variability. The model was calibrated and validated using data sets from three cultivars with contrasting malate accumulation, grown under different fruit loads and potassium supplies, and harvested at different stages. The model predicted the pre and post-harvest dynamics of malate concentration with fairly good accuracy for the three cultivars (mean RRMSE = 0.25-0.42). The sensitivity of the model to parameters and input variables was analyzed. According to the model, vacuolar composition, in particular potassium and organic acid concentrations, had an important effect on malate accumulation. The model suggested that rising temperatures depressed malate accumulation. The model also helped distinguish differences in malate concentration among the three cultivars and between the pre and post-harvest stages by highlighting the probable importance of proton pump activity and particularly of the free

Case Study: Organotypic human in vitro models of embryonic morphogenetic fusion

EPA Science Inventory

Morphogenetic fusion of tissues is a common event in embryonic development and disruption of fusion is associated with birth defects of the eye, heart, neural tube, phallus, palate, and other organ systems. Embryonic tissue fusion requires precise regulation of cell-cell and cell...

The Loss of Vacuolar Protein Sorting 11 (vps11) Causes Retinal Pathogenesis in a Vertebrate Model of Syndromic Albinism

PubMed Central

Thomas, Jennifer L.; Vihtelic, Thomas S.; denDekker, Aaron D.; Willer, Gregory; Luo, Xixia; Murphy, Taylor R.; Gregg, Ronald G.; Hyde, David R.

2011-01-01

Purpose. To establish the zebrafish platinum mutant as a model for studying vision defects caused by syndromic albinism diseases such as Chediak-Higashi syndrome, Griscelli syndrome, and Hermansky-Pudlak syndrome (HPS). Methods. Bulked segregant analysis and candidate gene sequencing revealed that the zebrafish platinum mutation is a single-nucleotide insertion in the vps11 (vacuolar protein sorting 11) gene. Expression of vps11 was determined by RT-PCR and in situ hybridization. Mutants were analyzed for pigmentation defects and retinal disease by histology, immunohistochemistry, and transmission electron microscopy. Results. Phenocopy and rescue experiments determined that a loss of Vps11 results in the platinum phenotype. Expression of vps11 appeared ubiquitous during zebrafish development, with stronger expression in the developing retina and retinal pigmented epithelium (RPE). Zebrafish platinum mutants exhibited reduced pigmentation in the body and RPE; however, melanophore development, migration, and dispersion occurred normally. RPE, photoreceptors, and inner retinal neurons formed normally in zebrafish platinum mutants. However, a gradual loss of RPE, an absence of mature melanosomes, and the subsequent degradation of RPE/photoreceptor interdigitation was observed. Conclusions. These data show that Vps11 is not necessary for normal retinal development or initiation of melanin biosynthesis, but is essential for melanosome maturation and healthy maintenance of the RPE and photoreceptors. PMID:21330665

The vacuolar channel VvALMT9 mediates malate and tartrate accumulation in berries of Vitis vinifera.

PubMed

De Angeli, Alexis; Baetz, Ulrike; Francisco, Rita; Zhang, Jingbo; Chaves, Maria Manuela; Regalado, Ana

2013-08-01

Vitis vinifera L. represents an economically important fruit species. Grape and wine flavour is made from a complex set of compounds. The acidity of berries is a major parameter in determining grape berry quality for wine making and fruit consumption. Despite the importance of malic and tartaric acid (TA) storage and transport for grape berry acidity, no vacuolar transporter for malate or tartrate has been identified so far. Some members of the aluminium-activated malate transporter (ALMT) anion channel family from Arabidopsis thaliana have been shown to be involved in mediating malate fluxes across the tonoplast. Therefore, we hypothesised that a homologue of these channels could have a similar role in V. vinifera grape berries. We identified homologues of the Arabidopsis vacuolar anion channel AtALMT9 through a TBLASTX search on the V. vinifera genome database. We cloned the closest homologue of AtALMT9 from grape berry cDNA and designated it VvALMT9. The expression profile revealed that VvALMT9 is constitutively expressed in berry mesocarp tissue and that its transcription level increases during fruit maturation. Moreover, we found that VvALMT9 is targeted to the vacuolar membrane. Using patch-clamp analysis, we could show that, besides malate, VvALMT9 mediates tartrate currents which are higher than in its Arabidopsis homologue. In summary, in the present study we provide evidence that VvALMT9 is a vacuolar malate channel expressed in grape berries. Interestingly, in V. vinifera, a tartrate-producing plant, the permeability of the channel is apparently adjusted to TA.

Engineering human cell spheroids to model embryonic tissue fusion in vitro.

EPA Science Inventory

Epithelial-mesenchymal interactions drive embryonic fusion events during development and upon perturbation can result in birth defects. Cleft palate and neural tube defects can result from genetic defects or environmental exposures during development, yet very little is known abo...

Creating Drought- and Salt-Tolerant Crops by Overexpressing a Vacuolar Pyrophosphatase Gene

USDA-ARS?s Scientific Manuscript database

Increased expression of an Arabidopsis vacuolar pyrophosphatase gene, AVP1, leads to increased drought and salt tolerance in transgenic plants, which has been demonstrated in laboratory and field conditions. The molecular mechanism of AVP1-mediated drought resistance is likely due to increased proto...

Transient anterior subcapsular vacuolar change of the crystalline lens in patients after posterior chamber phakic intraocular lens implantation.

PubMed

Chung, Jin Kwon; Shin, Jin Hee; Lee, Sung Jin

2013-10-25

We present two cases of transient vacuolar changes in the anterior subcapsular space of the crystalline lens in patients after posterior chamber phakic intraocular lens implantation. Implantable collamer lenses (ICL) were implanted in healthy myopic patients. Vacuolar changes developed just after the irrigating procedure through the narrow space between the ICL and the crystalline lens. Slit-lamp examinations and spectral domain optical coherence tomography showed bleb-like lesions in the anterior subcapsular space of one eye in each case, though the lesions gradually improved without visual deterioration. Consequently, the lesions turned into a few anterior subcapsular small faint opacities. Direct irrigation of the narrow space confined by the ICL and the crystalline lens is at risk for the development of vacuolar changes in the crystalline lens. The observed spontaneous reversal indicates that surgeons should not rush to surgical intervention but rather opt for close follow over several weeks.

Atg22p, a vacuolar membrane protein involved in the amino acid compartmentalization of Schizosaccharomyces pombe.

PubMed

Sugimoto, Naoko; Iwaki, Tomoko; Chardwiriyapreecha, Soracom; Shimazu, Masamitsu; Kawano, Miyuki; Sekito, Takayuki; Takegawa, Kaoru; Kakinuma, Yoshimi

2011-01-01

The fission yeast Schizosaccharomyces pombe has a homolog of the budding yeast Atg22p, which is involved in spore formation (Mukaiyama H. et al., Microbiology, 155, 3816-3826 (2009)). GFP-tagged Atg22p in the fission yeast was localized to the vacuolar membrane. Upon disruption of atg22, the amino acid levels of the cellular fraction as well as the vacuolar fraction decreased. The uptake of several amino acids, such as lysine, histidine, and arginine, was impaired in atg22Δ cells. S. pombe Atg22p plays an important role in the compartmentalization of amino acids.

Vba2p, a vacuolar membrane protein involved in basic amino acid transport in Schizosaccharomyces pombe.

PubMed

Sugimoto, Naoko; Iwaki, Tomoko; Chardwiriyapreecha, Soracom; Shimazu, Masamitsu; Sekito, Takayuki; Takegawa, Kaoru; Kakinuma, Yoshimi

2010-01-01

A recent study filling the gap in the genome sequence in the left arm of chromosome 2 of Schizosaccharomyces pombe revealed a homolog of budding yeast Vba2p, a vacuolar transporter of basic amino acids. GFP-tagged Vba2p in fission yeast was localized to the vacuolar membrane. Upon disruption of vba2, the uptake of several amino acids, including lysine, histidine, and arginine, was impaired. A transient increase in lysine uptake under nitrogen starvation was lowered by this mutation. These findings suggest that Vba2p is involved in basic amino acid transport in S. pombe under diverse conditions.

ERG2 and ERG24 Are Required for Normal Vacuolar Physiology as Well as Candida albicans Pathogenicity in a Murine Model of Disseminated but Not Vaginal Candidiasis.

PubMed

Luna-Tapia, Arturo; Peters, Brian M; Eberle, Karen E; Kerns, Morgan E; Foster, Timothy P; Marrero, Luis; Noverr, Mairi C; Fidel, Paul L; Palmer, Glen E

2015-10-01

Several important classes of antifungal agents, including the azoles, act by blocking ergosterol biosynthesis. It was recently reported that the azoles cause massive disruption of the fungal vacuole in the prevalent human pathogen Candida albicans. This is significant because normal vacuolar function is required to support C. albicans pathogenicity. This study examined the impact of the morpholine antifungals, which inhibit later steps of ergosterol biosynthesis, on C. albicans vacuolar integrity. It was found that overexpression of either the ERG2 or ERG24 gene, encoding C-8 sterol isomerase or C-14 sterol reductase, respectively, suppressed C. albicans sensitivity to the morpholines. In addition, both erg2Δ/Δ and erg24Δ/Δ mutants were hypersensitive to the morpholines. These data are consistent with the antifungal activity of the morpholines depending upon the simultaneous inhibition of both Erg2p and Erg24p. The vacuoles within both erg2Δ/Δ and erg24Δ/Δ C. albicans strains exhibited an aberrant morphology and accumulated large quantities of the weak base quinacrine, indicating enhanced vacuolar acidification compared with that of control strains. Both erg mutants exhibited significant defects in polarized hyphal growth and were avirulent in a mouse model of disseminated candidiasis. Surprisingly, in a mouse model of vaginal candidiasis, both mutants colonized mice at high levels and induced a pathogenic response similar to that with the controls. Thus, while targeting Erg2p or Erg24p alone could provide a potentially efficacious therapy for disseminated candidiasis, it may not be an effective strategy to treat vaginal infections. The potential value of drugs targeting these enzymes as adjunctive therapies is discussed. Copyright © 2015, American Society for Microbiology. All Rights Reserved.

To Be Cytosolic or Vacuolar: The Double Life of Listeria monocytogenes.

PubMed

Bierne, Hélène; Milohanic, Eliane; Kortebi, Mounia

2018-01-01

Intracellular bacterial pathogens are generally classified into two types: those that exploit host membrane trafficking to construct specific niches in vacuoles (i.e., "vacuolar pathogens"), and those that escape from vacuoles into the cytosol, where they proliferate and often spread to neighboring cells (i.e., "cytosolic pathogens"). However, the boundary between these distinct intracellular phenotypes is tenuous and may depend on the timing of infection and on the host cell type. Here, we discuss recent progress highlighting this phenotypic duality in Listeria monocytogenes , which has long been a model for cytosolic pathogens, but now emerges as a bacterium also capable of residing in vacuoles, in a slow/non-growing state. The ability of L. monocytogenes to enter a persistence stage in vacuoles might play a role during the asymptomatic incubation period of listeriosis and/or the carriage of this pathogen in asymptomatic hosts. Moreover, persistent vacuolar Listeria could be less susceptible to antibiotics and more difficult to detect by routine techniques of clinical biology. These hypotheses deserve to be explored in order to better manage the risks related to this food-borne pathogen.

Vacuolar myelinopathy in waterfowl from a North Carolina impoundment

USGS Publications Warehouse

Augspurger, T.; Fischer, John R.; Thomas, Nancy; Sileo, L.; Brannian, Roger E.; Miller, Kimberli J.; Rocke, Tonie E.

2003-01-01

Vacuolar myelinopathy was confirmed by light and electron microscopic examination of mallards (Anas platyrhynchos), ring-necked ducks (Aythya collaris), and buffleheads (Bucephala albeola) collected during an epizootic at Lake Surf in central North Carolina (USA) between November 1998 and February 1999. Clinical signs of affected birds were consistent with central nervous system impairment of motor function (incoordination, abnormal movement and posture, weakness, paralysis). This is the first report of this disease in wild waterfowl (Anseriformes).Aug

A vacuolar carboxypeptidase mutant of Arabidopsis thaliana is degraded by the ERAD pathway independently of its N-glycan

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

Yamamoto, Masaya; Kawanabe, Mitsuyoshi; Hayashi, Yoko

2010-03-12

Misfolded proteins produced in the endoplasmic reticulum (ER) are degraded by a mechanism, the ER-associated degradation (ERAD). Here we report establishment of the experimental system to analyze the ERAD in plant cells. Carboxypeptidase Y (CPY) is a vacuolar enzyme and its mutant CPY* is degraded by the ERAD in yeast. Since Arabidopsis thaliana has AtCPY, an ortholog of yeast CPY, we constructed and expressed fusion proteins consisting of AtCPY and GFP and of AtCPY*, which carries a mutation homologous to yeast CPY*, and GFP in A. thaliana cells. While AtCPY-GFP was efficiently transported to the vacuole, AtCPY*-GFP was retained inmore » the ER to be degraded in proteasome- and Cdc48-dependent manners. We also found that AtCPY*-GFP was degraded by the ERAD in yeast cells, but that its single N-glycan did not function as a degradation signal in yeast or plant cells. Therefore, AtCPY*-GFP can be used as a marker protein to analyze the ERAD pathway, likely for nonglycosylated substrates, in plant cells.« less

Multi site polyadenylation and transcriptional response to stress of a vacuolar type H+-ATPase subunit A gene in Arabidopsis thaliana

PubMed Central

Magnotta, Scot M; Gogarten, Johann Peter

2002-01-01

Background Vacuolar type H+-ATPases play a critical role in the maintenance of vacuolar homeostasis in plant cells. V-ATPases are also involved in plants' defense against environmental stress. This research examined the expression and regulation of the catalytic subunit of the vacuolar type H+-ATPase in Arabidopsis thaliana and the effect of environmental stress on multiple transcripts generated by this gene. Results Evidence suggests that subunit A of the vacuolar type H+-ATPase is encoded by a single gene in Arabidopsis thaliana. Genome blot analysis showed no indication of a second subunit A gene being present. The single gene identified was shown by whole RNA blot analysis to be transcribed in all organs of the plant. Subunit A was shown by sequencing the 3' end of multiple cDNA clones to exhibit multi site polyadenylation. Four different poly (A) tail attachment sites were revealed. Experiments were performed to determine the response of transcript levels for subunit A to environmental stress. A PCR based strategy was devised to amplify the four different transcripts from the subunit A gene. Conclusions Amplification of cDNA generated from seedlings exposed to cold, salt stress, and etiolation showed that transcript levels for subunit A of the vacuolar type H+-ATPase in Arabidopsis were responsive to stress conditions. Cold and salt stress resulted in a 2–4 fold increase in all four subunit A transcripts evaluated. Etiolation resulted in a slight increase in transcript levels. All four transcripts appeared to behave identically with respect to stress conditions tested with no significant differential regulation. PMID:11985780

Spatial correlation between chemical and topological defects in vitreous silica: UV-resonance Raman study

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

Saito, M., E-mail: makina.saito@elettra.eu; D’Amico, F.; Bencivenga, F.

2014-06-28

A spatial correlation between chemical and topological defects in the tetrahedron network in vitreous silica produced by a fusion process of natural quartz crystals was found by synchrotron-based UV resonance Raman experiments. Furthermore, a quantitative correlation between these defects was obtained by comparing visible Raman and UV absorption spectra. These results indicate that in vitreous silica produced by the fusion process the topological defects disturb the surrounding tetrahedral silica network and induce further disorder regions with sub nanometric sizes.

Glycolytic control of vacuolar-type ATPase activity: A mechanism to regulate influenza viral infection

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

Kohio, Hinissan P.; Adamson, Amy L., E-mail: aladamso@uncg.edu

As new influenza virus strains emerge, finding new mechanisms to control infection is imperative. In this study, we found that we could control influenza infection of mammalian cells by altering the level of glucose given to cells. Higher glucose concentrations induced a dose-specific increase in influenza infection. Linking influenza virus infection with glycolysis, we found that viral replication was significantly reduced after cells were treated with glycolytic inhibitors. Addition of extracellular ATP after glycolytic inhibition restored influenza infection. We also determined that higher levels of glucose promoted the assembly of the vacuolar-type ATPase within cells, and increased vacuolar-type ATPase proton-transportmore » activity. The increase of viral infection via high glucose levels could be reversed by inhibition of the proton pump, linking glucose metabolism, vacuolar-type ATPase activity, and influenza viral infection. Taken together, we propose that altering glucose metabolism may be a potential new approach to inhibit influenza viral infection. - Highlights: • Increased glucose levels increase Influenza A viral infection of MDCK cells. • Inhibition of the glycolytic enzyme hexokinase inhibited Influenza A viral infection. • Inhibition of hexokinase induced disassembly the V-ATPase. • Disassembly of the V-ATPase and Influenza A infection was bypassed with ATP. • The state of V-ATPase assembly correlated with Influenza A infection of cells.« less

Failure analysis of knee arthrodesis with the WichitaFusion Nail.

PubMed

Parcel, Ted W; Levering, Melissa; Polikandriotis, John A; Gustke, Kenneth A; Bernasek, Thomas L

2013-11-01

Arthrodesis is a salvage procedure for failed total knee arthroplasty with the intent to create a stable, pain-free limb on which to ambulate or transfer. For many patients, the alternative to arthrodesis may be an above-knee amputation. Available techniques for knee arthrodesis include compression plating, external fixators, and intramedullary fixation. The purpose of this study was to report the knee fusion rate of consecutive patients at 1 institution using an intramedullary fusion nail and to identify patient risk factors for fusion failure. Between November 1998 and November 2008, twenty-eight patients undergoing knee arthrodesis with an average follow-up of 18 months (range, 3-64 months) were retrospectively studied. Demographic information, presence of fusion, clinical function, pain level, and bone defect data were collected and analyzed. Eighty-two percent (23/28) of patients had radiographic evidence of successful fusion with an average time to fusion of 21 weeks (range, 10-58 weeks). When examining patient variables that could correlate with fusion rates, patients with an Anderson Orthopaedic Research Institute type 3 femoral or type 3 tibial defect had a statistically significant lower fusion rate. The intramedullary fusion nail is an effective device for knee arthrodesis that offers ease of insertion through the knee wound with the advantages of initial bone compression and rigid fixation. Although the use of intramedullary fusion nails leads to a high fusion rate, significant bone deficiency limits successful fusion. Copyright 2013, SLACK Incorporated.

Peroxisome Degradation by Microautophagy in Pichia pastoris: Identification of Specific Steps and Morphological Intermediates

PubMed Central

Sakai, Yasuyoshi; Koller, Antonius; Rangell, Linda K.; Keller, Gilbert A.; Subramani, Suresh

1998-01-01

We used the dye N-(3-triethylammoniumpropyl)-4-(p-diethylaminophenylhexatrienyl) pyridinium dibromide (FM4-64) and a fusion protein, consisting of the green fluorescent protein appended to the peroxisomal targeting signal, Ser-Lys-Leu (SKL), to label the vacuolar membrane and the peroxisomal matrix, respectively, in living Pichia pastoris cells and followed by fluorescence microscopy the morphological and kinetic intermediates in the vacuolar degradation of peroxisomes by microautophagy and macroautophagy. Structures corresponding to the intermediates were also identified by electron microscopy. The kinetics of appearance and disappearance of these intermediates is consistent with a precursor–product relationship between intermediates, which form the basis of a model for microautophagy. Inhibitors affecting different steps of microautophagy did not impair peroxisome delivery to the vacuole via macroautophagy, although inhibition of vacuolar proteases affected the final vacuolar degradation of green fluorescent protein (S65T mutant version [GFP])-SKL via both autophagic pathways. P. pastoris mutants defective in peroxisome microautophagy (pag mutants) were isolated and characterized for the presence or absence of the intermediates. These mutants, comprising 6 complementation groups, support the model for microautophagy. Our studies indicate that the microautophagic degradation of peroxisomes proceeds via specific intermediates, whose generation and/or processing is controlled by PAG gene products, and shed light on the poorly understood phenomenon of peroxisome homeostasis. PMID:9566964

Splenogonadal fusion with limb deficiency and micrognathia.

PubMed

Moore, P J; Hawkins, E P; Galliani, C A; Guerry-Force, M L

1997-11-01

Splenogonadal fusion (SGF) is a rare abnormality with two known types. In the continuous type, the spleen is connected to the gonad, and there are often limb defects, micrognathia, or other congenital malformations such as ventricular septal defect, anal atresia, microgastria, spina bifida, craniosynostosis, thoracopagus, diaphragmatic hernia, hypoplastic lung and abnormal lung fissures, polymicrogyria, deficient coccyx, and bifid spine C6-T3. The discontinuous type is usually not associated with congenital defects, and the gonad that fused with an accessory spleen has no connection with the native spleen. The etiology of SGF is not known. Conceivably, a teratogenic insult occurring between 5 weeks' and 8 weeks' gestation could interfere with the normal development of the spleen, gonads, and limb buds. We describe a case of splenogonadal fusion in a stillborn black boy with associated micrognathia and limb deformities. Also, we review the possible teratogenic etiologies and embryonic basis of SGF.

Aggregation of endosomal-vacuolar compartments in the Aovps24-deleted strain in the filamentous fungus Aspergillus oryzae

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

Tatsumi, Akinori; Shoji, Jun-ya; Kikuma, Takashi

2007-10-19

Previously, we found that deletion of Aovps24, an ortholog of Saccharomyces cerevisiae VPS24, that encodes an ESCRT (endosomal sorting complex required for transport)-III component required for late endosomal function results in fragmented and aggregated vacuoles. Although defective late endosomal function is likely responsible for this phenotype, critical lack of our knowledge on late endosomes in filamentous fungi prevented us from further characterization. In this study, we identified late endosomes of Aspergillus oryzae, by expressing a series of fusion proteins of fluorescent proteins with orthologs of late endosomal proteins. Using these fusion proteins as markers, we observed late endosomes in themore » wild type strain and the Aovps24 disruptant and demonstrated that late endosomes are aberrantly aggregated in the Aovps24 disruptant. Moreover, we revealed that the aggregated late endosomes have features of vacuoles as well. As deletion of another ESCRT-III component-encoding gene, Aovps2, resulted in similar phenotypes to that in the Aovps24 disruptant, phenotypes of the Aovps24 disruptant are probably due to defective late endosomal function.« less

ATP-binding cassette-like transporters are involved in the transport of lignin precursors across plasma and vacuolar membranes

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

Miao, Y.C.; Liu, C.

2010-12-28

Lignin is a complex biopolymer derived primarily from the condensation of three monomeric precursors, the monolignols. The synthesis of monolignols occurs in the cytoplasm. To reach the cell wall where they are oxidized and polymerized, they must be transported across the cell membrane. However, the molecular mechanisms underlying the transport process are unclear. There are conflicting views about whether the transport of these precursors occurs by passive diffusion or is an energized active process; further, we know little about what chemical forms are required. Using isolated plasma and vacuolar membrane vesicles prepared from Arabidopsis, together with applying different transporter inhibitorsmore » in the assays, we examined the uptake of monolignols and their derivatives by these native membrane vesicles. We demonstrate that the transport of lignin precursors across plasmalemma and their sequestration into vacuoles are ATP-dependent primary-transport processes, involving ATP-binding cassette-like transporters. Moreover, we show that both plasma and vacuolar membrane vesicles selectively transport different forms of lignin precursors. In the presence of ATP, the inverted plasma membrane vesicles preferentially take up monolignol aglycones, whereas the vacuolar vesicles are more specific for glucoconjugates, suggesting that the different ATP-binding cassette-like transporters recognize different chemical forms in conveying them to distinct sites, and that glucosylation of monolignols is necessary for their vacuolar storage but not required for direct transport into the cell wall in Arabidopsis.« less

Post-translational regulation of acid invertase activity by vacuolar invertase inhibitor affects resistance to cold-induced sweetening of potato tubers.

PubMed

McKenzie, Marian J; Chen, Ronan K Y; Harris, John C; Ashworth, Matthew J; Brummell, David A

2013-01-01

Cold-induced sweetening (CIS) is a serious post-harvest problem for potato tubers, which need to be stored cold to prevent sprouting and pathogenesis in order to maintain supply throughout the year. During storage at cold temperatures (below 10 °C), many cultivars accumulate free reducing sugars derived from a breakdown of starch to sucrose that is ultimately cleaved by acid invertase to produce glucose and fructose. When affected tubers are processed by frying or roasting, these reducing sugars react with free asparagine by the Maillard reaction, resulting in unacceptably dark-coloured and bitter-tasting product and generating the probable carcinogen acrylamide as a by-product. We have previously identified a vacuolar invertase inhibitor (INH2) whose expression correlates both with low acid invertase activity and with resistance to CIS. Here we show that, during cold storage, overexpression of the INH2 vacuolar invertase inhibitor gene in CIS-susceptible potato tubers reduced acid invertase activity, the accumulation of reducing sugars and the generation of acrylamide in subsequent fry tests. Conversely, suppression of vacuolar invertase inhibitor expression in a CIS-resistant line increased susceptibility to CIS. The results show that post-translational regulation of acid invertase by the vacuolar invertase inhibitor is an important component of resistance to CIS. © 2012 Blackwell Publishing Ltd.

Identification and functional expression of the Arabidopsis thaliana vacuolar glucose transporter 1 and its role in seed germination and flowering.

PubMed

Aluri, Sirisha; Büttner, Michael

2007-02-13

Sugar compartmentation into vacuoles of higher plants is a very important physiological process, providing extra space for transient and long-term sugar storage and contributing to the osmoregulation of cell turgor and shape. Despite the long-standing knowledge of this subcellular sugar partitioning, the proteins responsible for these transport steps have remained unknown. We have identified a gene family in Arabidopsis consisting of three members homologous to known sugar transporters. One member of this family, Arabidopsis thaliana vacuolar glucose transporter 1 (AtVGT1), was localized to the vacuolar membrane. Moreover, we provide evidence for transport activity of a tonoplast sugar transporter based on its functional expression in bakers' yeast and uptake studies in isolated yeast vacuoles. Analyses of Atvgt1 mutant lines indicate an important function of this vacuolar glucose transporter during developmental processes like seed germination and flowering.

49 CFR 229.75 - Wheels and tire defects.

Code of Federal Regulations, 2010 CFR

2010-10-01

... crack or break in the flange, tread, rim, plate, or hub. (l) A loose wheel or tire. (m) Fusion welding... 49 Transportation 4 2010-10-01 2010-10-01 false Wheels and tire defects. 229.75 Section 229.75....75 Wheels and tire defects. Wheels and tires may not have any of the following conditions: (a) A...

49 CFR 229.75 - Wheels and tire defects.

Code of Federal Regulations, 2012 CFR

2012-10-01

... crack or break in the flange, tread, rim, plate, or hub. (l) A loose wheel or tire. (m) Fusion welding... 49 Transportation 4 2012-10-01 2012-10-01 false Wheels and tire defects. 229.75 Section 229.75....75 Wheels and tire defects. Wheels and tires may not have any of the following conditions: (a) A...

49 CFR 229.75 - Wheels and tire defects.

Code of Federal Regulations, 2011 CFR

2011-10-01

... crack or break in the flange, tread, rim, plate, or hub. (l) A loose wheel or tire. (m) Fusion welding... 49 Transportation 4 2011-10-01 2011-10-01 false Wheels and tire defects. 229.75 Section 229.75....75 Wheels and tire defects. Wheels and tires may not have any of the following conditions: (a) A...

49 CFR 229.75 - Wheels and tire defects.

Code of Federal Regulations, 2014 CFR

2014-10-01

... crack or break in the flange, tread, rim, plate, or hub. (l) A loose wheel or tire. (m) Fusion welding... 49 Transportation 4 2014-10-01 2014-10-01 false Wheels and tire defects. 229.75 Section 229.75....75 Wheels and tire defects. Wheels and tires may not have any of the following conditions: (a) A...

49 CFR 229.75 - Wheels and tire defects.

Code of Federal Regulations, 2013 CFR

2013-10-01

... crack or break in the flange, tread, rim, plate, or hub. (l) A loose wheel or tire. (m) Fusion welding... 49 Transportation 4 2013-10-01 2013-10-01 false Wheels and tire defects. 229.75 Section 229.75....75 Wheels and tire defects. Wheels and tires may not have any of the following conditions: (a) A...

A Novel Arabidopsis Vacuolar Glucose Exporter Is Involved in Cellular Sugar Homeostasis and Affects the Composition of Seed Storage Compounds1[W][OA

PubMed Central

Poschet, Gernot; Hannich, Barbara; Raab, Sabine; Jungkunz, Isabel; Klemens, Patrick A.W.; Krueger, Stephan; Wic, Stefan; Neuhaus, H. Ekkehard; Büttner, Michael

2011-01-01

Subcellular sugar partitioning in plants is strongly regulated in response to developmental cues and changes in external conditions. Besides transitory starch, the vacuolar sugars represent a highly dynamic pool of instantly accessible metabolites that serve as energy source and osmoprotectant. Here, we present the molecular identification and functional characterization of the vacuolar glucose (Glc) exporter Arabidopsis (Arabidopsis thaliana) Early Responsive to Dehydration-Like6 (AtERDL6). We demonstrate tonoplast localization of AtERDL6 in plants. In Arabidopsis, AtERDL6 expression is induced in response to factors that activate vacuolar Glc pools, like darkness, heat stress, and wounding. On the other hand, AtERDL6 transcript levels drop during conditions that trigger Glc accumulation in the vacuole, like cold stress and external sugar supply. Accordingly, sugar analyses revealed that Aterdl6 mutants have elevated vacuolar Glc levels and that Glc flux across the tonoplast is impaired under stress conditions. Interestingly, overexpressor lines indicated a very similar function for the ERDL6 ortholog Integral Membrane Protein from sugar beet (Beta vulgaris). Aterdl6 mutant plants display increased sensitivity against external Glc, and mutant seeds exhibit a 10% increase in seed weight due to enhanced levels of seed sugars, proteins, and lipids. Our findings underline the importance of vacuolar Glc export during the regulation of cellular Glc homeostasis and the composition of seed reserves. PMID:21984725

Engineering human cell spheroids to model embryonic tissue fusion in vitro

PubMed Central

Wolf, Cynthia J.; Wood, Carmen; Ren, Hongzu; Grindstaff, Rachel; Padgett, William; Swank, Adam; MacMillan, Denise; Fisher, Anna; Winnik, Witold; Abbott, Barbara D.

2017-01-01

Epithelial-mesenchymal interactions drive embryonic fusion events during development, and perturbations of these interactions can result in birth defects. Cleft palate and neural tube defects can result from genetic defects or environmental exposures during development, yet very little is known about the effect of chemical exposures on fusion events during human development because of a lack of relevant and robust human in vitro assays of developmental fusion behavior. Given the etiology and prevalence of cleft palate and the relatively simple architecture and composition of the embryonic palate, we sought to develop a three-dimensional culture system that mimics the embryonic palate and could be used to study fusion behavior in vitro using human cells. We engineered size-controlled human Wharton’s Jelly stromal cell (HWJSC) spheroids and established that 7 days of culture in osteogenesis differentiation medium was sufficient to promote an osteogenic phenotype consistent with embryonic palatal mesenchyme. HWJSC spheroids supported the attachment of human epidermal keratinocyte progenitor cells (HPEKp) on the outer spheroid surface likely through deposition of collagens I and IV, fibronectin, and laminin by mesenchymal spheroids. HWJSC spheroids coated in HPEKp cells exhibited fusion behavior in culture, as indicated by the removal of epithelial cells from the seams between spheroids, that was dependent on epidermal growth factor signaling and fibroblast growth factor signaling in agreement with palate fusion literature. The method described here may broadly apply to the generation of three-dimensional epithelial-mesenchymal co-cultures to study developmental fusion events in a format that is amenable to predictive toxicology applications. PMID:28898253

Tributyltin sensitivity of vacuolar-type Na(+)-transporting ATPase from Enterococcus hirae.

PubMed

Chardwiriyapreecha, Soracom; Inoue, Tomohiro; Sugimoto, Naoko; Sekito, Takayuki; Yamato, Ichiro; Murata, Takeshi; Homma, Michio; Kakinuma, Yoshimi

2009-10-01

Tributyltin chloride (TBT), an environmental pollutant, is toxic to a variety of eukaryotic and prokaryotic organisms. Some members of F-ATP synthase (F-ATPase)/vacuolar type ATPase (V-ATPase) superfamily have been identified as the molecular target of this compound. TBT inhibited the activities of H(+)-transporting or Na(+)-transporting F-ATPase as well as H(+)-transporting V-ATPase originated from various organisms. However, the sensitivity to TBT of Na(+)-transporting V-ATPase has not been investigated. We examined the effect of TBT on Na(+)-transporting V-ATPase from an eubacterium Enterococus hirae. The ATP hydrolytic activity of E. hirae V-ATPase in purified form as well as in membrane-bound form was little inhibited by less than 10 microM TBT; IC50 for TBT inhibition of purified enzyme was estimated to be about 35 microM. Active sodium transport by E. hirae cells, indicating the in vivo activity of this V-ATPase, was not inhibited by 20 microM TBT. By contrast, IC50 of H(+)-transporting V-ATPase of the vacuolar membrane vesicles from Saccharomyces cerevisiae was about 0.2 microM. E. hirae V-ATPase is thus extremely less sensitive to TBT.

A defect in myoblast fusion underlies Carey-Fineman-Ziter syndrome

PubMed Central

Di Gioia, Silvio Alessandro; Connors, Samantha; Matsunami, Norisada; Cannavino, Jessica; Rose, Matthew F.; Gilette, Nicole M.; Artoni, Pietro; de Macena Sobreira, Nara Lygia; Chan, Wai-Man; Webb, Bryn D.; Robson, Caroline D.; Cheng, Long; Van Ryzin, Carol; Ramirez-Martinez, Andres; Mohassel, Payam; Leppert, Mark; Scholand, Mary Beth; Grunseich, Christopher; Ferreira, Carlos R.; Hartman, Tyler; Hayes, Ian M.; Morgan, Tim; Markie, David M.; Fagiolini, Michela; Swift, Amy; Chines, Peter S.; Speck-Martins, Carlos E.; Collins, Francis S.; Jabs, Ethylin Wang; Bönnemann, Carsten G.; Olson, Eric N.; Andrews, Caroline V.; Barry, Brenda J.; Hunter, David G.; Mackinnon, Sarah E.; Shaaban, Sherin; Erazo, Monica; Frempong, Tamiesha; Hao, Ke; Naidich, Thomas P.; Rucker, Janet C.; Zhang, Zhongyang; Biesecker, Barbara B.; Bonnycastle, Lori L.; Brewer, Carmen C.; Brooks, Brian P.; Butman, John A.; Chien, Wade W.; Farrell, Kathleen; FitzGibbon, Edmond J.; Gropman, Andrea L.; Hutchinson, Elizabeth B.; Jain, Minal S.; King, Kelly A.; Lehky, Tanya J.; Lee, Janice; Liberton, Denise K.; Narisu, Narisu; Paul, Scott M.; Sadeghi, Neda; Snow, Joseph; Solomon, Beth; Summers, Angela; Toro, Camilo; Thurm, Audrey; Zalewski, Christopher K.; Carey, John C.; Robertson, Stephen P.; Manoli, Irini; Engle, Elizabeth C.

2017-01-01

Multinucleate cellular syncytial formation is a hallmark of skeletal muscle differentiation. Myomaker, encoded by Mymk (Tmem8c), is a well-conserved plasma membrane protein required for myoblast fusion to form multinucleated myotubes in mouse, chick, and zebrafish. Here, we report that autosomal recessive mutations in MYMK (OMIM 615345) cause Carey-Fineman-Ziter syndrome in humans (CFZS; OMIM 254940) by reducing but not eliminating MYMK function. We characterize MYMK-CFZS as a congenital myopathy with marked facial weakness and additional clinical and pathologic features that distinguish it from other congenital neuromuscular syndromes. We show that a heterologous cell fusion assay in vitro and allelic complementation experiments in mymk knockdown and mymkinsT/insT zebrafish in vivo can differentiate between MYMK wild type, hypomorphic and null alleles. Collectively, these data establish that MYMK activity is necessary for normal muscle development and maintenance in humans, and expand the spectrum of congenital myopathies to include cell-cell fusion deficits. PMID:28681861

A defect in myoblast fusion underlies Carey-Fineman-Ziter syndrome.

PubMed

Di Gioia, Silvio Alessandro; Connors, Samantha; Matsunami, Norisada; Cannavino, Jessica; Rose, Matthew F; Gilette, Nicole M; Artoni, Pietro; de Macena Sobreira, Nara Lygia; Chan, Wai-Man; Webb, Bryn D; Robson, Caroline D; Cheng, Long; Van Ryzin, Carol; Ramirez-Martinez, Andres; Mohassel, Payam; Leppert, Mark; Scholand, Mary Beth; Grunseich, Christopher; Ferreira, Carlos R; Hartman, Tyler; Hayes, Ian M; Morgan, Tim; Markie, David M; Fagiolini, Michela; Swift, Amy; Chines, Peter S; Speck-Martins, Carlos E; Collins, Francis S; Jabs, Ethylin Wang; Bönnemann, Carsten G; Olson, Eric N; Carey, John C; Robertson, Stephen P; Manoli, Irini; Engle, Elizabeth C

2017-07-06

Multinucleate cellular syncytial formation is a hallmark of skeletal muscle differentiation. Myomaker, encoded by Mymk (Tmem8c), is a well-conserved plasma membrane protein required for myoblast fusion to form multinucleated myotubes in mouse, chick, and zebrafish. Here, we report that autosomal recessive mutations in MYMK (OMIM 615345) cause Carey-Fineman-Ziter syndrome in humans (CFZS; OMIM 254940) by reducing but not eliminating MYMK function. We characterize MYMK-CFZS as a congenital myopathy with marked facial weakness and additional clinical and pathologic features that distinguish it from other congenital neuromuscular syndromes. We show that a heterologous cell fusion assay in vitro and allelic complementation experiments in mymk knockdown and mymk insT/insT zebrafish in vivo can differentiate between MYMK wild type, hypomorphic and null alleles. Collectively, these data establish that MYMK activity is necessary for normal muscle development and maintenance in humans, and expand the spectrum of congenital myopathies to include cell-cell fusion deficits.

Simulation of the evolution of fused silica's surface defect during wet chemical etching

NASA Astrophysics Data System (ADS)

Liu, Taixiang; Yang, Ke; Li, Heyang; Yan, Lianghong; Yuan, Xiaodong; Yan, Hongwei

2017-08-01

Large high-power-laser facility is the basis for achieving inertial confinement fusion, one of whose missions is to make fusion energy usable in the near future. In the facility, fused silica optics plays an irreplaceable role to conduct extremely high-intensity laser to fusion capsule. But the surface defect of fused silica is a major obstacle limiting the output power of the large laser facility and likely resulting in the failure of ignition. To mitigate, or event to remove the surface defect, wet chemical etching has been developed as a practical way. However, how the surface defect evolves during wet chemical etching is still not clearly known so far. To address this problem, in this work, the three-dimensional model of surface defect is built and finite difference time domain (FDTD) method is developed to simulate the evolution of surface defect during etching. From the simulation, it is found that the surface defect will get smooth and result in the improvement of surface quality of fused silica after etching. Comparatively, surface defects (e.g. micro-crack, scratch, series of pinholes, etc.) of a typical fused silica at different etching time are experimentally measured. It can be seen that the simulation result agrees well with the result of experiment, indicating the FDTD method is valid for investigating the evolution of surface defect during etching. With the finding of FDTD simulation, one can optimize the treatment process of fused silica in practical etching or even to make the initial characterization of surface defect traceable.

The antiwrinkle effect of topical concentrated 2-dimethylaminoethanol involves a vacuolar cytopathology.

PubMed

Morissette, G; Germain, L; Marceau, F

2007-03-01

The 'cosmeceutical' agent 2-dimethylaminoethanol (DMAE) is a tertiary amine found in high concentration in numerous topical antiwrinkle preparations. We hypothesized that a 337 mmol L(-1) (3%) DMAE reservoir applied to the skin could reproduce the cytopathology induced by other amines by maintaining a millimolar drug concentration within a certain depth of the skin layers, and that vacuolar cell expansion could account for the very rapid effect on the apparent skin fullness. Morphological and functional assays were applied to cultured rabbit dermal fibroblasts treated with tertiary amines in vitro. A morphological verification of the vacuolization caused by topical DMAE was also attempted in vivo using the inner skin of the rabbit ear and in vitro using primary cultures of human cutaneous epithelial cells. Fibroblasts responded to DMAE (2.5-10 mmol L(-1)) by massive vacuolization (0.5-4 h; phase contrast observations). Triethanolamine, another chemical frequently used topically, was also active in this respect (10 mmol L(-1)). The vacuolar adenosine triphosphatase inhibitor bafilomycin A1 prevented DMAE- or triethanolamine-induced vacuolization; adding bafilomycin A1 or cell washout slowly reversed the established vacuolization induced by DMAE. Further effects of DMAE in cultured fibroblasts included a moderate cytotoxicity (10 mmol L(-1)) that was abated by bafilomycin A1 cotreatment, a concentration-dependent mitotic arrest (2.5 mmol L(-1)) and transient and mild effects on cell ploidy. The epidermis of the rabbit external ear was significantly thickened and exhibited clear perinuclear swelling indicative of vacuolization in response to 3% DMAE (1 h; paraffin tissue sections). Cultured human cutaneous epithelial cells responded to DMAE by vacuolization (inhibited by bafilomycin A1 cotreatment). The vacuolar cytopathology induced by concentrated organic amines may be the cellular basis of the antiwrinkle effect of DMAE.

A Grapevine TTG2-Like WRKY Transcription Factor Is Involved in Regulating Vacuolar Transport and Flavonoid Biosynthesis.

PubMed

Amato, Alessandra; Cavallini, Erika; Zenoni, Sara; Finezzo, Laura; Begheldo, Maura; Ruperti, Benedetto; Tornielli, Giovanni Battista

2016-01-01

A small set of TTG2-like homolog proteins from different species belonging to the WRKY family of transcription factors were shown to share a similar mechanism of action and to control partially conserved biochemical/developmental processes in their native species. In particular, by activating P-ATPases residing on the tonoplast, PH3 from Petunia hybrida promotes vacuolar acidification in petal epidermal cells whereas TTG2 from Arabidopsis thaliana enables the accumulation of proanthocyanidins in the seed coat. In this work we functionally characterized VvWRKY26 identified as the closest grapevine homolog of PhPH3 and AtTTG2 . When constitutively expressed in petunia ph3 mutant, VvWRKY26 can fulfill the PH3 function in the regulation of vacuolar pH and restores the wild type pigmentation phenotype. By a global correlation analysis of gene expression and by transient over-expression in Vitis vinifera , we showed transcriptomic relationships of VvWRKY26 with many genes related to vacuolar acidification and transport in grapevine. Moreover, our results indicate an involvement in flavonoid pathway possibly restricted to the control of proanthocyanidin biosynthesis that is consistent with its expression pattern in grape berry tissues. Overall, the results show that, in addition to regulative mechanisms and biological roles shared with TTG2-like orthologs, VvWRKY26 can play roles in fleshy fruit development that have not been previously reported in studies from dry fruit species. This study paves the way toward the comprehension of the regulatory network controlling vacuolar acidification and flavonoid accumulation mechanisms that contribute to the final berry quality traits in grapevine.

A Grapevine TTG2-Like WRKY Transcription Factor Is Involved in Regulating Vacuolar Transport and Flavonoid Biosynthesis

PubMed Central

Amato, Alessandra; Cavallini, Erika; Zenoni, Sara; Finezzo, Laura; Begheldo, Maura; Ruperti, Benedetto; Tornielli, Giovanni Battista

2017-01-01

A small set of TTG2-like homolog proteins from different species belonging to the WRKY family of transcription factors were shown to share a similar mechanism of action and to control partially conserved biochemical/developmental processes in their native species. In particular, by activating P-ATPases residing on the tonoplast, PH3 from Petunia hybrida promotes vacuolar acidification in petal epidermal cells whereas TTG2 from Arabidopsis thaliana enables the accumulation of proanthocyanidins in the seed coat. In this work we functionally characterized VvWRKY26 identified as the closest grapevine homolog of PhPH3 and AtTTG2. When constitutively expressed in petunia ph3 mutant, VvWRKY26 can fulfill the PH3 function in the regulation of vacuolar pH and restores the wild type pigmentation phenotype. By a global correlation analysis of gene expression and by transient over-expression in Vitis vinifera, we showed transcriptomic relationships of VvWRKY26 with many genes related to vacuolar acidification and transport in grapevine. Moreover, our results indicate an involvement in flavonoid pathway possibly restricted to the control of proanthocyanidin biosynthesis that is consistent with its expression pattern in grape berry tissues. Overall, the results show that, in addition to regulative mechanisms and biological roles shared with TTG2-like orthologs, VvWRKY26 can play roles in fleshy fruit development that have not been previously reported in studies from dry fruit species. This study paves the way toward the comprehension of the regulatory network controlling vacuolar acidification and flavonoid accumulation mechanisms that contribute to the final berry quality traits in grapevine. PMID:28105033

Repair of rabbit cartilage defect based on the fusion of rabbit bone marrow stromal cells and Nano-HA/PLLA composite material.

PubMed

Zhu, Weimin; Guo, Daiqi; Peng, Liangquan; Chen, Yun Fang; Cui, Jiaming; Xiong, Jianyi; Lu, Wei; Duan, Li; Chen, Kang; Zeng, Yanjun; Wang, Daping

2017-02-01

Objective To assess the effect of the fusion of rabbit bone marrow stromal cells (rBMSCs) and Nano-hydroxyapatite/poly (l-lactic acid) (Nano-HA/PLLA) in repairing the rabbit knee joint with full-thickness cartilage defect. Method The rBMSCs were isolated and cultured in vitro, and the third generation of rBMSCs was co-cultured with the Nano-HA/PLLA to construct the tissue-engineered cartilage (TEC). Eighteen New Zealand white rabbits were selected and randomly divided into three groups, namely, TEC group, Nano-HA/PLLA group, and control group. A cartilage defect model with the diameter of 4.5 mm and depth of 5 mm was constructed on the articular surface of medial malleolus of rabbit femur. General observation, histological observation, and Wakitani's histological scoring were conducted in the 12th and 24th week postoperatively. Results The results of TEC group indicated that new cartilage tissue was formed on the defect site and subchondral bone achieved physiological integration basically. Histological and immunohistochemical analyses indicated the generation of massive extracellular matrix. In contrast, limited regeneration and reconstruction of cartilage was achieved in the Nano-HA/PLLA group and control group, with a significant difference from the TEC group (p < 0.05). Moreover, the effect of cartilage repair was positively correlated with time. Conclusion The porous Nano-HA/PLLA combined with BMSCs promoted the repair of weight-bearing bone of adult rabbit's knee joint with cartilage defect.

Technique for writing of fiber Bragg gratings over or near preliminary formed macro-structure defects in silica optical fibers

NASA Astrophysics Data System (ADS)

Evtushenko, Alexander S.; Faskhutdinov, Lenar M.; Kafarova, Anastasia M.; Kazakov, Vadim S.; Kuznetzov, Artem A.; Minaeva, Alina Yu.; Sevruk, Nikita L.; Nureev, Ilnur I.; Vasilets, Alexander A.; Andreev, Vladimir A.; Morozov, Oleg G.; Burdin, Vladimir A.; Bourdine, Anton V.

2017-04-01

This work presents method for performing precision macro-structure defects "tapers" and "up-tapers" written in conventional silica telecommunication multimode optical fibers by commercially available field fusion splicer with modified software settings and following writing fiber Bragg gratings over or near them. We developed technique for macrodefect geometry parameters estimation via analysis of photo-image performed after defect writing and displayed on fusion splicer screen. Some research results of defect geometry dependence on fusion current and fusion time values re-set in splicer program are represented that provided ability to choose their "the best" combination. Also experimental statistical researches concerned with "taper" and "up-taper" diameter stability as well as their insertion loss values during their writing under fixed corrected splicer program parameters were performed. We developed technique for FBG writing over or near macro-structure defect. Some results of spectral response measurements produced for short-length samples of multimode optical fiber with fiber Bragg gratings written over and near macro-defects prepared by using proposed technique are presented.

V-ATPase-dependent luminal acidification is required for endocytic recycling of a yeast cell wall stress sensor, Wsc1p

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

Ueno, Kazuma; Saito, Mayu; Nagashima, Makiko

Highlights: •A targeted genome screen identified 5 gene groups affecting Wsc1p recycling. •V-ATPase-dependent luminal acidification is required for Wsc1p recycling. •Activity of V-ATPase might be required for cargo recognition by the retromer complex. -- Abstract: Wsc1p is a major cell wall sensor protein localized at the polarized cell surface. The localization of Wsc1p is maintained by endocytosis and recycling from endosomes back to the cell surface, but changes to the vacuole when cells are subjected to heat stress. Exploiting this unique property of Wsc1p, we screened for yeast single-gene deletion mutants exhibiting defects in Wsc1p trafficking. By expressing 3GFP-tagged Wsc1pmore » in mutants with deleted genes whose function is related to intracellular trafficking, we identified 5 gene groups affecting Wsc1p trafficking, impaired respectively in endocytic internalization, multivesicular body sorting, the GARP complex, endosomal maturation/vacuolar fusion, and V-ATPase. Interestingly, deletion of the VPH1 gene, encoding the V{sub o} subunit of vacuolar-type H{sup +}-ATPase (V-ATPase), led to mis-localization of Wsc1p from the plasma membrane to the vacuole. In addition, disruption of other V-ATPase subunits (vma mutants) also caused defects of Wsc1p trafficking and vacuolar acidification similar to those seen in the vph1Δ mutant. Moreover, we found that deletion of the VPS26 gene, encoding a subunit of the retromer complex, also caused a defect in Wsc1p recycling and mis-localization of Wsc1p to the vacuole. These findings clarified the previously unidentified Wsc1p recycling pathway and requirement of V-ATPase-dependent luminal acidification for Wsc1p recycling.« less

Geometric Defects in Quantum Hall States

NASA Astrophysics Data System (ADS)

Gromov, Andrey

I will describe a geometric analogue of Laughlin quasiholes in fractional quantum Hall (FQH) states. These ``quasiholes'' are generated by an insertion of quantized fluxes of curvature - which can be modeled by branch points of a certain Riemann surface - and, consequently, are related to genons. Unlike quasiholes, the genons are not excitations, but extrinsic defects. Fusion of genons describes the response of an FQH state to a process that changes (effective) topology of the physical space. These defects are abelian for IQH states and non-abelian for FQH states. I will explain how to calculate an electric charge, geometric spin and adiabatic mutual statistics of the these defects. Leo Kadanoff Fellowship.

Singlet Oxygen-Induced Membrane Disruption and Serpin-Protease Balance in Vacuolar-Driven Cell Death.

PubMed

Koh, Eugene; Carmieli, Raanan; Mor, Avishai; Fluhr, Robert

2016-07-01

Singlet oxygen plays a role in cellular stress either by providing direct toxicity or through signaling to initiate death programs. It was therefore of interest to examine cell death, as occurs in Arabidopsis, due to differentially localized singlet oxygen photosensitizers. The photosensitizers rose bengal (RB) and acridine orange (AO) were localized to the plasmalemma and vacuole, respectively. Their photoactivation led to cell death as measured by ion leakage. Cell death could be inhibited by the singlet oxygen scavenger histidine in treatments with AO but not with RB In the case of AO treatment, the vacuolar membrane was observed to disintegrate. Concomitantly, a complex was formed between a vacuolar cell-death protease, RESPONSIVE TO DESSICATION-21 and its cognate cytoplasmic protease inhibitor ATSERPIN1. In the case of RB treatment, the tonoplast remained intact and no complex was formed. Over-expression of AtSerpin1 repressed cell death, only under AO photodynamic treatment. Interestingly, acute water stress showed accumulation of singlet oxygen as determined by fluorescence of Singlet Oxygen Sensor Green, by electron paramagnetic resonance spectroscopy and the induction of singlet oxygen marker genes. Cell death by acute water stress was inhibited by the singlet oxygen scavenger histidine and was accompanied by vacuolar collapse and the appearance of serpin-protease complex. Over-expression of AtSerpin1 also attenuated cell death under this mode of cell stress. Thus, acute water stress damage shows parallels to vacuole-mediated cell death where the generation of singlet oxygen may play a role. © 2016 American Society of Plant Biologists. All Rights Reserved.

Expression of an arabidopsis vacuolar H+-pyrophosphatase gene (AVP1) in cotton improves drought- and salt tolerance and increases fibre yield in the field conditions

USDA-ARS?s Scientific Manuscript database

The Arabidopsis gene AVP1 encodes a vacuolar pyrophosphatase that functions as a proton pump on the vacuolar membrane. Overexpression of AVP1 in Arabidopsis, tomato and rice enhances plant performance under salt and drought stress conditions, because up-regulation of the type I H+-PPase from Arabid...

Expression of an Arabidopsis Vacuolar H+-pyrophosphatase Gene (AVP1) in Cotton Improves Drought- and Salt Tolerance and Increases Fibre Yield in the Field Conditions.

USDA-ARS?s Scientific Manuscript database

The Arabidopsis gene AVP1 encodes a vacuolar pyrophosphatase that functions as a proton pump on the vacuolar membrane. Overexpression of AVP1 in Arabidopsis, tomato and rice enhances plant performance under salt and drought stress conditions, because up-regulation of the type I H+PPase from Arabido...

Listeria monocytogenes switches from dissemination to persistence by adopting a vacuolar lifestyle in epithelial cells.

PubMed

Kortebi, Mounia; Milohanic, Eliane; Mitchell, Gabriel; Péchoux, Christine; Prevost, Marie-Christine; Cossart, Pascale; Bierne, Hélène

2017-11-01

Listeria monocytogenes causes listeriosis, a foodborne disease that poses serious risks to fetuses, newborns and immunocompromised adults. This intracellular bacterial pathogen proliferates in the host cytosol and exploits the host actin polymerization machinery to spread from cell-to-cell and disseminate in the host. Here, we report that during several days of infection in human hepatocytes or trophoblast cells, L. monocytogenes switches from this active motile lifestyle to a stage of persistence in vacuoles. Upon intercellular spread, bacteria gradually stopped producing the actin-nucleating protein ActA and became trapped in lysosome-like vacuoles termed Listeria-Containing Vacuoles (LisCVs). Subpopulations of bacteria resisted degradation in LisCVs and entered a slow/non-replicative state. During the subculture of host cells harboring LisCVs, bacteria showed a capacity to cycle between the vacuolar and the actin-based motility stages. When ActA was absent, such as in ΔactA mutants, vacuolar bacteria parasitized host cells in the so-called "viable but non-culturable" state (VBNC), preventing their detection by conventional colony counting methods. The exposure of infected cells to high doses of gentamicin did not trigger the formation of LisCVs, but selected for vacuolar and VBNC bacteria. Together, these results reveal the ability of L. monocytogenes to enter a persistent state in a subset of epithelial cells, which may favor the asymptomatic carriage of this pathogen, lengthen the incubation period of listeriosis, and promote bacterial survival during antibiotic therapy.

Listeria monocytogenes switches from dissemination to persistence by adopting a vacuolar lifestyle in epithelial cells

PubMed Central

Mitchell, Gabriel

2017-01-01

Listeria monocytogenes causes listeriosis, a foodborne disease that poses serious risks to fetuses, newborns and immunocompromised adults. This intracellular bacterial pathogen proliferates in the host cytosol and exploits the host actin polymerization machinery to spread from cell-to-cell and disseminate in the host. Here, we report that during several days of infection in human hepatocytes or trophoblast cells, L. monocytogenes switches from this active motile lifestyle to a stage of persistence in vacuoles. Upon intercellular spread, bacteria gradually stopped producing the actin-nucleating protein ActA and became trapped in lysosome-like vacuoles termed Listeria-Containing Vacuoles (LisCVs). Subpopulations of bacteria resisted degradation in LisCVs and entered a slow/non-replicative state. During the subculture of host cells harboring LisCVs, bacteria showed a capacity to cycle between the vacuolar and the actin-based motility stages. When ActA was absent, such as in ΔactA mutants, vacuolar bacteria parasitized host cells in the so-called “viable but non-culturable” state (VBNC), preventing their detection by conventional colony counting methods. The exposure of infected cells to high doses of gentamicin did not trigger the formation of LisCVs, but selected for vacuolar and VBNC bacteria. Together, these results reveal the ability of L. monocytogenes to enter a persistent state in a subset of epithelial cells, which may favor the asymptomatic carriage of this pathogen, lengthen the incubation period of listeriosis, and promote bacterial survival during antibiotic therapy. PMID:29190284

Mathematical modeling of the central carbohydrate metabolism in Arabidopsis reveals a substantial regulatory influence of vacuolar invertase on whole plant carbon metabolism.

PubMed

Nägele, Thomas; Henkel, Sebastian; Hörmiller, Imke; Sauter, Thomas; Sawodny, Oliver; Ederer, Michael; Heyer, Arnd G

2010-05-01

A mathematical model representing metabolite interconversions in the central carbohydrate metabolism of Arabidopsis (Arabidopsis thaliana) was developed to simulate the diurnal dynamics of primary carbon metabolism in a photosynthetically active plant leaf. The model groups enzymatic steps of central carbohydrate metabolism into blocks of interconverting reactions that link easily measurable quantities like CO(2) exchange and quasi-steady-state levels of soluble sugars and starch. When metabolite levels that fluctuate over diurnal cycles are used as a basic condition for simulation, turnover rates for the interconverting reactions can be calculated that approximate measured metabolite dynamics and yield kinetic parameters of interconverting reactions. We used experimental data for Arabidopsis wild-type plants, accession Columbia, and a mutant defective in vacuolar invertase, AtbetaFruct4, as input data. Reducing invertase activity to mutant levels in the wild-type model led to a correct prediction of increased sucrose levels. However, additional changes were needed to correctly simulate levels of hexoses and sugar phosphates, indicating that invertase knockout causes subsequent changes in other enzymatic parameters. Reduction of invertase activity caused a decline in photosynthesis and export of reduced carbon to associated metabolic pathways and sink organs (e.g. roots), which is in agreement with the reported contribution of vacuolar invertase to sink strength. According to model parameters, there is a role for invertase in leaves, where futile cycling of sucrose appears to have a buffering effect on the pools of sucrose, hexoses, and sugar phosphates. Our data demonstrate that modeling complex metabolic pathways is a useful tool to study the significance of single enzyme activities in complex, nonintuitive networks.

[Function of transport H+-ATPases in plant cell plasma and vacuolar membranes of maize under salt stress conditions and effect of adaptogenic preparations].

PubMed

Rybchenko, Zh I; Palladina, T O

2011-01-01

Participations of electrogenic H+-pumps of plasma and vacuolar membranes represented by E1-E2 and V-type H+-ATPases in plant cell adaptation to salt stress conditions has been studied by determination of their transport activities. Experiments were carried out on corn seedlings exposed during 1 or 10 days at 0.1 M NaCl. Preparations Methyure and Ivine were used by seed soaking at 10(-7) M. Plasma and vacuolar membrane fractions were isolated from corn seedling roots. In variants without NaCl a hydrolytical activity of plasma membrane H+-ATPase was increased with seedling age and its transport one was changed insignificantly, wherease the response of the weaker vacuolar H+-ATPase was opposite. NaCl exposition decreased hydrolytical activities of both H+-ATPases and increased their transport ones. These results demonstrated amplification of H+-pumps function especially represented by vacuolar H+-ATPase. Both preparations, Methyure mainly, caused a further increase of transport activity which was more expressed in NaCl variants. Obtained results showed the important role of these H+-pumps in plant adaptation under salt stress conditions realized by energetical maintenance of the secondary active Na+/H+ -antiporters which remove Na+ from cytoplasm.

Pax2 regulates a fadd-dependent molecular switch that drives tissue fusion during eye development.

PubMed

Viringipurampeer, Ishaq A; Ferreira, Todd; DeMaria, Shannon; Yoon, Jookyung J; Shan, Xianghong; Moosajee, Mariya; Gregory-Evans, Kevin; Ngai, John; Gregory-Evans, Cheryl Y

2012-05-15

Tissue fusion is an essential morphogenetic mechanism in development, playing a fundamental role in developing neural tube, palate and the optic fissure. Disruption of genes associated with the tissue fusion can lead to congenital malformations, such as spina bifida, cleft lip/palate and ocular coloboma. For instance, the Pax2 transcription factor is required for optic fissure closure, although the mechanism of Pax2 action leading to tissue fusion remains elusive. This lack of information defining how transcription factors drive tissue morphogenesis at the cellular level is hampering new treatments options. Through loss- and gain-of-function analysis, we now establish that pax2 in combination with vax2 directly regulate the fas-associated death domain (fadd) gene. In the presence of fadd, cell proliferation is restricted in the developing eye through a caspase-dependent pathway. However, the loss of fadd results in a proliferation defect and concomitant activation of the necroptosis pathway through RIP1/RIP3 activity, leading to an abnormal open fissure. Inhibition of RIP1 with the small molecule drug necrostatin-1 rescues the pax2 eye fusion defect, thereby overcoming the underlying genetic defect. Thus, fadd has an essential physiological function in protecting the developing optic fissure neuroepithelium from RIP3-dependent necroptosis. This study demonstrates the molecular hierarchies that regulate a cellular switch between proliferation and the apoptotic and necroptotic cell death pathways, which in combination drive tissue morphogenesis. Furthermore, our data suggest that future therapeutic strategies may be based on small molecule drugs that can bypass the gene defects causing common congenital tissue fusion defects.

Yarn-dyed fabric defect classification based on convolutional neural network

NASA Astrophysics Data System (ADS)

Jing, Junfeng; Dong, Amei; Li, Pengfei; Zhang, Kaibing

2017-09-01

Considering that manual inspection of the yarn-dyed fabric can be time consuming and inefficient, we propose a yarn-dyed fabric defect classification method by using a convolutional neural network (CNN) based on a modified AlexNet. CNN shows powerful ability in performing feature extraction and fusion by simulating the learning mechanism of human brain. The local response normalization layers in AlexNet are replaced by the batch normalization layers, which can enhance both the computational efficiency and classification accuracy. In the training process of the network, the characteristics of the defect are extracted step by step and the essential features of the image can be obtained from the fusion of the edge details with several convolution operations. Then the max-pooling layers, the dropout layers, and the fully connected layers are employed in the classification model to reduce the computation cost and extract more precise features of the defective fabric. Finally, the results of the defect classification are predicted by the softmax function. The experimental results show promising performance with an acceptable average classification rate and strong robustness on yarn-dyed fabric defect classification.

The formation of Anthocyanic Vacuolar Inclusions in Arabidopsis thaliana and implications for the sequestration of anthocyanin pigments.

PubMed

Pourcel, Lucille; Irani, Niloufer G; Lu, Yuhua; Riedl, Ken; Schwartz, Steve; Grotewold, Erich

2010-01-01

Anthocyanins are flavonoid pigments that accumulate in the large central vacuole of most plants. Inside the vacuole, anthocyanins can be found uniformly distributed or as part of sub-vacuolar pigment bodies, the Anthocyanic Vacuolar Inclusions (AVIs). Using Arabidopsis seedlings grown under anthocyanin-inductive conditions as a model to understand how AVIs are formed, we show here that the accumulation of AVIs strongly correlates with the formation of cyanidin 3-glucoside (C3G) and derivatives. Arabidopsis mutants that fail to glycosylate anthocyanidins at the 5-O position (5gt mutant) accumulate AVIs in almost every epidermal cell of the cotyledons, as compared to wild-type seedlings, where only a small fraction of the cells show AVIs. A similar phenomenon is observed when seedlings are treated with vanadate. Highlighting a role for autophagy in the formation of the AVIs, we show that various mutants that interfere with the autophagic process (atg mutants) display lower numbers of AVIs, in addition to a reduced accumulation of anthocyanins. Interestingly, vanadate increases the numbers of AVIs in the atg mutants, suggesting that several pathways might participate in AVI formation. Taken together, our results suggest novel mechanisms for the formation of sub-vacuolar compartments capable of accumulating anthocyanin pigments.

Molecular basis of abnormal red-green color vision: a family with three types of color vision defects.

PubMed Central

Drummond-Borg, M; Deeb, S; Motulsky, A G

1988-01-01

The molecular nature of three different types of X-linked color-vision defects, protanomaly, deuteranomaly, and protanopia, in a large 3-generation family was determined. In the protanomalous and protanopic males the normal red pigment gene was replaced by a 5' red-3' green fusion gene. The protanomalous male had more red pigment DNA in his fusion gene than did the more severely affected protanopic individual. The deuteranomalous individual had four green pigment genes and one 5' green-3' red fusion gene. These results extend those of Nathans et al., who proposed that most red-green color-vision defects arise as a result of unequal crossing-over between the red and green pigment genes. The various data suggest that differences in severity of color-vision defects associated with fusion genes are caused by differences in crossover sites between the red and green pigment genes. Currently used molecular methodology is not sufficiently sensitive to define these fusion points accurately, and the specific color-vision defect within the deutan or protan class cannot be predicted. The DNA patterns for color-vision genes of female heterozygotes have not previously been described. Patterns of heterozygotes may not be distinguishable from those of normals. However, a definite assignment of the various color pigment gene arrays could be carried out by family study. Two compound heterozygotes for color-vision defects who tested as normal by anomaloscopy were found to carry abnormal fusion genes. In addition, a normal red pigment gene was present on one chromosome and at least one normal green pigment gene was present on the other.(ABSTRACT TRUNCATED AT 250 WORDS) Images Figure 3 PMID:2847528

Alteration of complex sphingolipid composition and its physiological significance in yeast Saccharomyces cerevisiae lacking vacuolar ATPase.

PubMed

Tani, Motohiro; Toume, Moeko

2015-12-01

In the yeast Saccharomyces cerevisiae, complex sphingolipids have three types of polar head group and five types of ceramide; however, the physiological significance of the structural diversity is not fully understood. Here, we report that deletion of vacuolar H+-ATPase (V-ATPase) in yeast causes dramatic alteration of the complex sphingolipid composition, which includes decreases in hydroxylation at the C-4 position of long-chain bases and the C-2 position of fatty acids in the ceramide moiety, decreases in inositol phosphorylceramide (IPC) levels, and increases in mannosylinositol phosphorylceramide (MIPC) and mannosyldiinositol phosphorylceramide [M(IP)2C] levels. V-ATPase-deleted cells exhibited slow growth at pH 7.2, whereas the increase in MIPC levels was significantly enhanced when V-ATPase-deleted cells were incubated at pH 7.2. The protein expression levels of MIPC and M(IP)2C synthases were significantly increased in V-ATPase-deleted cells incubated at pH 7.2. Loss of MIPC synthesis or an increase in the hydroxylation level of the ceramide moiety of sphingolipids on overexpression of Scs7 and Sur2 sphingolipid hydroxylases enhanced the growth defect of V-ATPase-deleted cells at pH 7.2. On the contrary, the growth rate of V-ATPase-deleted cells was moderately increased on the deletion of SCS7 and SUR2. In addition, supersensitivities to Ca2+, Zn2+ and H2O2, which are typical phenotypes of V-ATPase-deleted cells, were enhanced by the loss of MIPC synthesis. These results indicate the possibility that alteration of the complex sphingolipid composition is an adaptation mechanism for a defect of V-ATPase.

Mutant Fusion Proteins with Enhanced Fusion Activity Promote Measles Virus Spread in Human Neuronal Cells and Brains of Suckling Hamsters

PubMed Central

Shirogane, Yuta; Suzuki, Satoshi O.; Ikegame, Satoshi; Koga, Ritsuko

2013-01-01

Subacute sclerosing panencephalitis (SSPE) is a fatal degenerative disease caused by persistent measles virus (MV) infection in the central nervous system (CNS). From the genetic study of MV isolates obtained from SSPE patients, it is thought that defects of the matrix (M) protein play a crucial role in MV pathogenicity in the CNS. In this study, we report several notable mutations in the extracellular domain of the MV fusion (F) protein, including those found in multiple SSPE strains. The F proteins with these mutations induced syncytium formation in cells lacking SLAM and nectin 4 (receptors used by wild-type MV), including human neuronal cell lines, when expressed together with the attachment protein hemagglutinin. Moreover, recombinant viruses with these mutations exhibited neurovirulence in suckling hamsters, unlike the parental wild-type MV, and the mortality correlated with their fusion activity. In contrast, the recombinant MV lacking the M protein did not induce syncytia in cells lacking SLAM and nectin 4, although it formed larger syncytia in cells with either of the receptors. Since human neuronal cells are mainly SLAM and nectin 4 negative, fusion-enhancing mutations in the extracellular domain of the F protein may greatly contribute to MV spread via cell-to-cell fusion in the CNS, regardless of defects of the M protein. PMID:23255801

Investigation of defect rate of lap laser welding of stainless steel railway vehicles car body

NASA Astrophysics Data System (ADS)

Wang, Hongxiao

2015-02-01

In order to resolve the disadvantages such as poor appearance quality, poor tightness, low efficiency of resistance spot welding of stainless steel rail vehicles, partial penetration lap laser welding process was investigated widely. But due to the limitation of processing technology, there will be local incomplete fusion in the lap laser welding seam. Defect rate is the ratio of the local incomplete fusion length to the weld seam length. The tensile shear strength under different defect rate and its effect on the car body static strength are not clear. It is necessary to find the biggest defect rate by numerical analysis of effects of different defect rates on the laser welding stainless steel rail vehicle body structure strength ,and tests of laser welding shear tensile strength.

The biosynthetic gene cluster for the cyanogenic glucoside dhurrin in Sorghum bicolor contains its co-expressed vacuolar MATE transporter

PubMed Central

Darbani, Behrooz; Motawia, Mohammed Saddik; Olsen, Carl Erik; Nour-Eldin, Hussam H.; Møller, Birger Lindberg; Rook, Fred

2016-01-01

Genomic gene clusters for the biosynthesis of chemical defence compounds are increasingly identified in plant genomes. We previously reported the independent evolution of biosynthetic gene clusters for cyanogenic glucoside biosynthesis in three plant lineages. Here we report that the gene cluster for the cyanogenic glucoside dhurrin in Sorghum bicolor additionally contains a gene, SbMATE2, encoding a transporter of the multidrug and toxic compound extrusion (MATE) family, which is co-expressed with the biosynthetic genes. The predicted localisation of SbMATE2 to the vacuolar membrane was demonstrated experimentally by transient expression of a SbMATE2-YFP fusion protein and confocal microscopy. Transport studies in Xenopus laevis oocytes demonstrate that SbMATE2 is able to transport dhurrin. In addition, SbMATE2 was able to transport non-endogenous cyanogenic glucosides, but not the anthocyanin cyanidin 3-O-glucoside or the glucosinolate indol-3-yl-methyl glucosinolate. The genomic co-localisation of a transporter gene with the biosynthetic genes producing the transported compound is discussed in relation to the role self-toxicity of chemical defence compounds may play in the formation of gene clusters. PMID:27841372

Method for vacuum fusion bonding

DOEpatents

Ackler, Harold D.; Swierkowski, Stefan P.; Tarte, Lisa A.; Hicks, Randall K.

2001-01-01

An improved vacuum fusion bonding structure and process for aligned bonding of large area glass plates, patterned with microchannels and access holes and slots, for elevated glass fusion temperatures. Vacuum pumpout of all components is through the bottom platform which yields an untouched, defect free top surface which greatly improves optical access through this smooth surface. Also, a completely non-adherent interlayer, such as graphite, with alignment and location features is located between the main steel platform and the glass plate pair, which makes large improvements in quality, yield, and ease of use, and enables aligned bonding of very large glass structures.

Fusion bonding and alignment fixture

DOEpatents

Ackler, Harold D.; Swierkowski, Stefan P.; Tarte, Lisa A.; Hicks, Randall K.

2000-01-01

An improved vacuum fusion bonding structure and process for aligned bonding of large area glass plates, patterned with microchannels and access holes and slots, for elevated glass fusion temperatures. Vacuum pumpout of all the components is through the bottom platform which yields an untouched, defect free top surface which greatly improves optical access through this smooth surface. Also, a completely non-adherent interlayer, such as graphite, with alignment and location features is located between the main steel platform and the glass plate pair, which makes large improvements in quality, yield, and ease of use, and enables aligned bonding of very large glass structures.

Schisandrin B protects PC12 cells by decreasing the expression of amyloid precursor protein and vacuolar protein sorting 35★

PubMed Central

Yan, Mingmin; Mao, Shanping; Dong, Huimin; Liu, Baohui; Zhang, Qian; Pan, Gaofeng; Fu, Zhiping

2012-01-01

PC12 cell injury was induced using 20 μM amyloid β-protein 25–35 to establish a model of Alzheimer's disease. The cells were then treated with 5, 10, and 25 μM Schisandrin B. Methylthiazolyldiphenyl-tetrazolium bromide assays and Hoechst 33342 staining results showed that with increasing Schisandrin B concentration, the survival rate of PC12 cells injured by amyloid β-protein 25–35 gradually increased and the rate of apoptosis gradually decreased. Reverse transcription-PCR, immunocytochemical staining and western blot results showed that with increasing Schisandrin B concentration, the mRNA and protein expression of vacuolar protein sorting 35 and amyloid precursor protein were gradually decreased. Vacuolar protein sorting 35 and amyloid precursor protein showed a consistent trend for change. These findings suggest that 5, 10, and 25 μM Schisandrin B antagonizes the cellular injury induced by amyloid β-protein 25–35 in a dose-dependent manner. This may be caused by decreasing the expression of vacuolar protein sorting 35 and amyloid precursor protein. PMID:25745458

Cell fusion in the liver, revisited

PubMed Central

Lizier, Michela; Castelli, Alessandra; Montagna, Cristina; Lucchini, Franco; Vezzoni, Paolo; Faggioli, Francesca

2018-01-01

There is wide agreement that cell fusion is a physiological process in cells in mammalian bone, muscle and placenta. In other organs, such as the cerebellum, cell fusion is controversial. The liver contains a considerable number of polyploid cells: They are commonly believed to originate by genome endoreplication, although the contribution of cell fusion to polyploidization has not been excluded. Here, we address the topic of cell fusion in the liver from a historical point of view. We discuss experimental evidence clearly supporting the hypothesis that cell fusion occurs in the liver, specifically when bone marrow cells were injected into mice and shown to rescue genetic hepatic degenerative defects. Those experiments-carried out in the latter half of the last century-were initially interpreted to show “transdifferentiation”, but are now believed to demonstrate fusion between donor macrophages and host hepatocytes, raising the possibility that physiologically polyploid cells, such as hepatocytes, could originate, at least partially, through homotypic cell fusion. In support of the homotypic cell fusion hypothesis, we present new data generated using a chimera-based model, a much simpler model than those previously used. Cell fusion as a road to polyploidization in the liver has not been extensively investigated, and its contribution to a variety of conditions, such as viral infections, carcinogenesis and aging, remains unclear. PMID:29527257

Nuclear congression and membrane fusion: two distinct events in the yeast karyogamy pathway

PubMed Central

1994-01-01

Karyogamy is the process where haploid nuclei fuse to form a diploid nucleus during yeast mating. We devised a novel genetic screen that identified five new karyogamy (KAR) genes and three new cell fusion (FUS) genes. The kar mutants fell into two classes that represent distinct events in the yeast karyogamy pathway. Class I mutations blocked congression of the nuclei due to cytoplasmic microtubule defects. In Class II mutants, nuclear congression proceeded and the membranes of apposed nuclei were closely aligned but unfused. In vitro, Class II mutant membranes were defective in a homotypic ER/nuclear membrane fusion assay. We propose that Class II mutants define components of a novel membrane fusion complex which functions during vegetative growth and is recruited for karyogamy. PMID:8051211

Different sets of ER-resident J-proteins regulate distinct polar nuclear-membrane fusion events in Arabidopsis thaliana.

PubMed

Maruyama, Daisuke; Yamamoto, Masaya; Endo, Toshiya; Nishikawa, Shuh-ichi

2014-11-01

Angiosperm female gametophytes contain a central cell with two polar nuclei. In many species, including Arabidopsis thaliana, the polar nuclei fuse during female gametogenesis. We previously showed that BiP, an Hsp70 in the endoplasmic reticulum (ER), was essential for membrane fusion during female gametogenesis. Hsp70 function requires partner proteins for full activity. J-domain containing proteins (J-proteins) are the major Hsp70 functional partners. A. thaliana ER contains three soluble J-proteins, AtERdj3A, AtERdj3B, and AtP58(IPK). Here, we analyzed mutants of these proteins and determined that double-mutant ovules lacking AtP58(IPK) and AtERdj3A or AtERdj3B were defective in polar nuclear fusion. Electron microscopy analysis identified that polar nuclei were in close contact, but no membrane fusion occurred in mutant ovules lacking AtP58(IPK) and AtERdj3A. The polar nuclear outer membrane appeared to be connected via the ER remaining at the inner unfused membrane in mutant ovules lacking AtP58(IPK) and AtERdj3B. These results indicate that ER-resident J-proteins, AtP58(IPK)/AtERdj3A and AtP58(IPK)/AtERdj3B, function at distinct steps of polar nuclear-membrane fusion. Similar to the bip1 bip2 double mutant female gametophytes, the aterdj3a atp58(ipk) double mutant female gametophytes defective in fusion of the outer polar nuclear membrane displayed aberrant endosperm proliferation after fertilization with wild-type pollen. However, endosperm proliferated normally after fertilization of the aterdj3b atp58(ipk) double mutant female gametophytes defective in fusion of the inner membrane. Our results indicate that the polar nuclear fusion defect itself does not cause an endosperm proliferation defect. © The Author 2014. Published by Oxford University Press on behalf of Japanese Society of Plant Physiologists. All rights reserved. For permissions, please email: journals.permissions@oup.com.

Vacuolar H+-ATPase Protects Saccharomyces cerevisiae Cells against Ethanol-Induced Oxidative and Cell Wall Stresses

PubMed Central

Charoenbhakdi, Sirikarn; Dokpikul, Thanittra; Burphan, Thanawat; Techo, Todsapol

2016-01-01

ABSTRACT During fermentation, increased ethanol concentration is a major stress for yeast cells. Vacuolar H+-ATPase (V-ATPase), which plays an important role in the maintenance of intracellular pH homeostasis through vacuolar acidification, has been shown to be required for tolerance to straight-chain alcohols, including ethanol. Since ethanol is known to increase membrane permeability to protons, which then promotes intracellular acidification, it is possible that the V-ATPase is required for recovery from alcohol-induced intracellular acidification. In this study, we show that the effects of straight-chain alcohols on membrane permeabilization and acidification of the cytosol and vacuole are strongly dependent on their lipophilicity. These findings suggest that the membrane-permeabilizing effect of straight-chain alcohols induces cytosolic and vacuolar acidification in a lipophilicity-dependent manner. Surprisingly, after ethanol challenge, the cytosolic pH in Δvma2 and Δvma3 mutants lacking V-ATPase activity was similar to that of the wild-type strain. It is therefore unlikely that the ethanol-sensitive phenotype of vma mutants resulted from severe cytosolic acidification. Interestingly, the vma mutants exposed to ethanol exhibited a delay in cell wall remodeling and a significant increase in intracellular reactive oxygen species (ROS). These findings suggest a role for V-ATPase in the regulation of the cell wall stress response and the prevention of endogenous oxidative stress in response to ethanol. IMPORTANCE The yeast Saccharomyces cerevisiae has been widely used in the alcoholic fermentation industry. Among the environmental stresses that yeast cells encounter during the process of alcoholic fermentation, ethanol is a major stress factor that inhibits yeast growth and viability, eventually leading to fermentation arrest. This study provides evidence for the molecular mechanisms of ethanol tolerance, which is a desirable characteristic for yeast strains

Vacuolar H+-ATPase Protects Saccharomyces cerevisiae Cells against Ethanol-Induced Oxidative and Cell Wall Stresses.

PubMed

Charoenbhakdi, Sirikarn; Dokpikul, Thanittra; Burphan, Thanawat; Techo, Todsapol; Auesukaree, Choowong

2016-05-15

During fermentation, increased ethanol concentration is a major stress for yeast cells. Vacuolar H(+)-ATPase (V-ATPase), which plays an important role in the maintenance of intracellular pH homeostasis through vacuolar acidification, has been shown to be required for tolerance to straight-chain alcohols, including ethanol. Since ethanol is known to increase membrane permeability to protons, which then promotes intracellular acidification, it is possible that the V-ATPase is required for recovery from alcohol-induced intracellular acidification. In this study, we show that the effects of straight-chain alcohols on membrane permeabilization and acidification of the cytosol and vacuole are strongly dependent on their lipophilicity. These findings suggest that the membrane-permeabilizing effect of straight-chain alcohols induces cytosolic and vacuolar acidification in a lipophilicity-dependent manner. Surprisingly, after ethanol challenge, the cytosolic pH in Δvma2 and Δvma3 mutants lacking V-ATPase activity was similar to that of the wild-type strain. It is therefore unlikely that the ethanol-sensitive phenotype of vma mutants resulted from severe cytosolic acidification. Interestingly, the vma mutants exposed to ethanol exhibited a delay in cell wall remodeling and a significant increase in intracellular reactive oxygen species (ROS). These findings suggest a role for V-ATPase in the regulation of the cell wall stress response and the prevention of endogenous oxidative stress in response to ethanol. The yeast Saccharomyces cerevisiae has been widely used in the alcoholic fermentation industry. Among the environmental stresses that yeast cells encounter during the process of alcoholic fermentation, ethanol is a major stress factor that inhibits yeast growth and viability, eventually leading to fermentation arrest. This study provides evidence for the molecular mechanisms of ethanol tolerance, which is a desirable characteristic for yeast strains used in alcoholic

Singlet Oxygen-Induced Membrane Disruption and Serpin-Protease Balance in Vacuolar-Driven Cell Death1[OPEN

PubMed Central

Carmieli, Raanan; Mor, Avishai; Fluhr, Robert

2016-01-01

Singlet oxygen plays a role in cellular stress either by providing direct toxicity or through signaling to initiate death programs. It was therefore of interest to examine cell death, as occurs in Arabidopsis, due to differentially localized singlet oxygen photosensitizers. The photosensitizers rose bengal (RB) and acridine orange (AO) were localized to the plasmalemma and vacuole, respectively. Their photoactivation led to cell death as measured by ion leakage. Cell death could be inhibited by the singlet oxygen scavenger histidine in treatments with AO but not with RB. In the case of AO treatment, the vacuolar membrane was observed to disintegrate. Concomitantly, a complex was formed between a vacuolar cell-death protease, RESPONSIVE TO DESSICATION-21 and its cognate cytoplasmic protease inhibitor ATSERPIN1. In the case of RB treatment, the tonoplast remained intact and no complex was formed. Over-expression of AtSerpin1 repressed cell death, only under AO photodynamic treatment. Interestingly, acute water stress showed accumulation of singlet oxygen as determined by fluorescence of Singlet Oxygen Sensor Green, by electron paramagnetic resonance spectroscopy and the induction of singlet oxygen marker genes. Cell death by acute water stress was inhibited by the singlet oxygen scavenger histidine and was accompanied by vacuolar collapse and the appearance of serpin-protease complex. Over-expression of AtSerpin1 also attenuated cell death under this mode of cell stress. Thus, acute water stress damage shows parallels to vacuole-mediated cell death where the generation of singlet oxygen may play a role. PMID:26884487

Fusion neutron irradiation of Ni-Si alloys at high temperature*1

NASA Astrophysics Data System (ADS)

Huang, J. S.; Guinan, M. W.; Hahn, P. A.

1988-07-01

Two Ni-4% Si alloys, with different cold work levels, have been irradiated with 14-MeV fusion neutrons at 623 K, and their Curie temperatures have been monitored during irradiation. The results are compared to those of an identical alloy irradiated by 2-MeV electrons. The results show that increasing dislocation density increases the Curie temperature change rate. At the same damage rate, the Curie temperature change rate for the alloy irradiated by 14-MeV fusion neutrons is only 6-7% of that for an identical alloy irradiated by 2-MeV electrons. It is well known that the migration of radiation induced defects contributes to segregation of silicon atoms at sinks in this alloy, causing the Curie temperature changes. The current results imply that the relative free defect production efficiency decreases from one for the electron irradiated sample to 6-7% for the fusion neutron irradiated sample.

Isolation, characterization, and structure analysis of a vacuolar processing enzyme gene (MhVPEγ) from Malus hupehensis (Pamp) Rehd.

PubMed

Ran, Kun; Yang, Hongqiang; Sun, Xiaoli; Li, Qiang; Jiang, Qianqian; Zhang, Weiwei; Shen, Wei

2014-05-01

Vacuolar processing enzymes (VPEs) have received considerable attention recently, as they exhibit caspase-1-like cleavage activity and regulate the process of PCD. However, knowledge about their detailed characteristics and structures is relatively limited. In this study, a gamma vacuolar processing enzyme gene, MhVPEγ, has been isolated from the leaves of Malus hupehensis (Ramp) Rehd. var pinyiensis Jiang. MhVPEγ coded-translated protein sequence comprised of 494 amino acids with a signal peptide and a transmembrane helix structure at N-terminal, peptidase_C13 domain, and vacuolar sorting signal at C-terminal. Consequently, genomic walking approach was performed for the isolation of its upstream sequence. Computational analysis demonstrated several motifs of the promoter exhibiting hypothetic MeJA, ABA, and light-induced characteristics, as well as some typical domains universally discovered in promoter, such as TATA-box and CAAT-box. MhVPEγ transcript level was enhanced during wounding treatment, and WUN-motif, as one of the cis-acting regulatory elements existing in the upstream sequence perhaps regulates its expression. In silico-constructed 3D models revealed that MhCPYL successively interacts with MhVPEγ like that of "Induced Fit-Lock and Key" model, providing molecular conformation evidence that CPY is a direct substrate of VPEγ. This study is the first stride to understand the molecular mechanism of VPEγ and CPYL interactions.

Genotype-phenotype relationships in human red/green color-vision defects: molecular and psychophysical studies.

PubMed Central

Deeb, S S; Lindsey, D T; Hibiya, Y; Sanocki, E; Winderickx, J; Teller, D Y; Motulsky, A G

1992-01-01

The relationship between the molecular structure of the X-linked red and green visual pigment genes and color-vision phenotype as ascertained by anomaloscopy was studied in 64 color-defective males. The great majority of red-green defects were associated with either the deletion of the green-pigment gene or the formation of 5' red-green hybrid genes or 5' green-red hybrid genes. A rapid PCR-based method allowed detection of hybrid genes, including those undetectable by Southern blot analysis, as well as more precise localization of the fusion points in hybrid genes. Protan color-vision defects appeared always associated with 5' red-green hybrid genes. Carriers of single red-green hybrid genes with fusion in introns 1-4 were protanopes. However, carriers of hybrid genes with red-green fusions in introns 2, 3, or 4 in the presence of additional normal green genes manifested as either protanopes or protanomalous trichromats, with the majority being protanomalous. Deutan defects were associated with green-pigment gene deletions, with 5' green-red hybrid genes, or, rarely, with 5' green-red-green hybrid genes. Complete green-pigment gene deletions or green-red fusions in intron 1 were usually associated with deuteranopia, although we unexpectedly found three carriers of a single red-pigment gene without any green-pigment genes to be deuteranomalous trichromats. All but one of the other deuteranomalous subjects had green-red hybrid genes with intron 1, 2, 3, or 4 fusions, as well as several normal green-pigment genes. The one exception had a grossly normal gene array, presumably with a more subtle mutation. Amino acid differences in exon 5 largely determine whether a hybrid gene will be more redlike or more greenlike in phenotype. Various discrepancies as to severity (dichromacy or trichromacy) remain unexplained but may arise because of variability of expression, postreceptoral variation, or both. When phenotypic color-vision defects exist, the kind of defect (protan or

Vacuolar processing enzyme: an executor of plant cell death.

PubMed

Hara-Nishimura, Ikuko; Hatsugai, Noriyuki; Nakaune, Satoru; Kuroyanagi, Miwa; Nishimura, Mikio

2005-08-01

Apoptotic cell death in animals is regulated by cysteine proteinases called caspases. Recently, vacuolar processing enzyme (VPE) was identified as a plant caspase. VPE deficiency prevents cell death during hypersensitive response and cell death of limited cell layers at the early stage of embryogenesis. Because plants do not have macrophages, dying cells must degrade their materials by themselves. VPE plays an essential role in the regulation of the lytic system of plants during the processes of defense and development. VPE is localized in the vacuoles, unlike animal caspases, which are localized in the cytosol. Thus, plants might have evolved a regulated cellular suicide strategy that, unlike animal apoptosis, is mediated by VPE and the vacuoles.

Vacuolar status and water relations in embryonic axes of recalcitrant Aesculus hippocastanum seeds during stratification and early germination.

PubMed

Obroucheva, Natalie V; Lityagina, Snezhana V; Novikova, Galina V; Sin'kevich, Irina A

2012-01-01

In tropical recalcitrant seeds, their rapid transition from shedding to germination at high hydration level is of physiological interest but difficult to study because of the time constraint. In recalcitrant horse chestnut seeds produced in central Russia, this transition is much longer and extends through dormancy and dormancy release. This extended time period permits studies of the water relations in embryonic axes during the long recalcitrant period in terms of vacuolar status and water transport. Horse chestnut (Aesculus hippocastanum) seeds sampled in Moscow were stratified in cold wet sand for 4 months. Vacuole presence and development in embryonic axes were examined by vital staining, light and electron microscopy. Aquaporins and vacuolar H(+)-ATPase were identified immunochemically. Water channel operation was tested by water inflow rate. Vacuolar acid invertase was estimated in terms of activity and electrophoretic properties. Throughout the long recalcitrant period after seed shedding, cells of embryonic axes maintained active vacuoles and a high water content. Preservation of enzyme machinery in vacuoles was evident from retention of invertase activity, substrate specificity, molecular mass and subunit composition. Plasmalemma and tonoplast aquaporins and the E subunit of vacuolar H(+)-ATPase were also present. In non-dormant seeds prior to growth initiation, vacuoles enlarged at first in hypocotyls, and then in radicles, with their biogenesis being similar. Vacuolation was accompanied by increasing invertase activity, leading to sugar accumulation and active osmotic functioning. After growth initiation, vacuole enlargement was favoured by enhanced water inflow through water channels formed by aquaporins. Maintenance of high water content and desiccation sensitivity, as well as preservation of active vacuoles in embryonic axes after shedding, can be considered a specific feature of recalcitrant seeds, overlooked when studying tropical recalcitrants due

Vacuolar status and water relations in embryonic axes of recalcitrant Aesculus hippocastanum seeds during stratification and early germination

PubMed Central

Obroucheva, Natalie V.; Lityagina, Snezhana V.; Novikova, Galina V.; Sin'kevich, Irina A.

2012-01-01

Backgrounds and aims In tropical recalcitrant seeds, their rapid transition from shedding to germination at high hydration level is of physiological interest but difficult to study because of the time constraint. In recalcitrant horse chestnut seeds produced in central Russia, this transition is much longer and extends through dormancy and dormancy release. This extended time period permits studies of the water relations in embryonic axes during the long recalcitrant period in terms of vacuolar status and water transport. Methodology Horse chestnut (Aesculus hippocastanum) seeds sampled in Moscow were stratified in cold wet sand for 4 months. Vacuole presence and development in embryonic axes were examined by vital staining, light and electron microscopy. Aquaporins and vacuolar H+-ATPase were identified immunochemically. Water channel operation was tested by water inflow rate. Vacuolar acid invertase was estimated in terms of activity and electrophoretic properties. Principal results Throughout the long recalcitrant period after seed shedding, cells of embryonic axes maintained active vacuoles and a high water content. Preservation of enzyme machinery in vacuoles was evident from retention of invertase activity, substrate specificity, molecular mass and subunit composition. Plasmalemma and tonoplast aquaporins and the E subunit of vacuolar H+-ATPase were also present. In non-dormant seeds prior to growth initiation, vacuoles enlarged at first in hypocotyls, and then in radicles, with their biogenesis being similar. Vacuolation was accompanied by increasing invertase activity, leading to sugar accumulation and active osmotic functioning. After growth initiation, vacuole enlargement was favoured by enhanced water inflow through water channels formed by aquaporins. Conclusions Maintenance of high water content and desiccation sensitivity, as well as preservation of active vacuoles in embryonic axes after shedding, can be considered a specific feature of recalcitrant

Possible roles of vacuolar H+-ATPase and mitochondrial function in tolerance to air-drying stress revealed by genome-wide screening of Saccharomyces cerevisiae deletion strains.

PubMed

Shima, Jun; Ando, Akira; Takagi, Hiroshi

2008-03-01

Yeasts used in bread making are exposed to air-drying stress during dried yeast production processes. To clarify the genes required for air-drying tolerance, we performed genome-wide screening using the complete deletion strain collection of diploid Saccharomyces cerevisiae. The screening identified 278 gene deletions responsible for air-drying sensitivity. These genes were classified based on their cellular function and on the localization of their gene products. The results showed that the genes required for air-drying tolerance were frequently involved in mitochondrial functions and in connection with vacuolar H(+)-ATPase, which plays a role in vacuolar acidification. To determine the role of vacuolar acidification in air-drying stress tolerance, we monitored intracellular pH. The results showed that intracellular acidification was induced during air-drying and that this acidification was amplified in a deletion mutant of the VMA2 gene encoding a component of vacuolar H(+)-ATPase, suggesting that vacuolar H(+)-ATPase helps maintain intracellular pH homeostasis, which is affected by air-drying stress. To determine the effects of air-drying stress on mitochondria, we analysed the mitochondrial membrane potential under air-drying stress conditions using MitoTracker. The results showed that mitochondria were extremely sensitive to air-drying stress, suggesting that a mitochondrial function is required for tolerance to air-drying stress. We also analysed the correlation between oxidative-stress sensitivity and air-drying-stress sensitivity. The results suggested that oxidative stress is a critical determinant of sensitivity to air-drying stress, although ROS-scavenging systems are not necessary for air-drying stress tolerance. (c) 2008 John Wiley & Sons, Ltd.

Environmental Factors Influencing Blooms of a Neurotoxic Stigonematalan Cyanobacterium Responsible for Avian Vacuolar Myelinopathy

DTIC Science & Technology

2013-01-01

aquatic plants and subsequent ecological consequences. The authors of this technical note have linked avian vacuolar myelinopathy (AVM), a disease...additional cyanobacteria sequences to determine designations for probe development, to advance understanding of the species’ phylogeny , and to lay...groundwork for its formal description. Phylogeny data confirm that the species is in section V, order Stigonematales. Phylogeny also infers that the

Validating Inertial Confinement Fusion (ICF) predictive capability using perturbed capsules

NASA Astrophysics Data System (ADS)

Schmitt, Mark; Magelssen, Glenn; Tregillis, Ian; Hsu, Scott; Bradley, Paul; Dodd, Evan; Cobble, James; Flippo, Kirk; Offerman, Dustin; Obrey, Kimberly; Wang, Yi-Ming; Watt, Robert; Wilke, Mark; Wysocki, Frederick; Batha, Steven

2009-11-01

Achieving ignition on NIF is a monumental step on the path toward utilizing fusion as a controlled energy source. Obtaining robust ignition requires accurate ICF models to predict the degradation of ignition caused by heterogeneities in capsule construction and irradiation. LANL has embarked on a project to induce controlled defects in capsules to validate our ability to predict their effects on fusion burn. These efforts include the validation of feature-driven hydrodynamics and mix in a convergent geometry. This capability is needed to determine the performance of capsules imploded under less-than-optimum conditions on future IFE facilities. LANL's recently initiated Defect Implosion Experiments (DIME) conducted at Rochester's Omega facility are providing input for these efforts. Recent simulation and experimental results will be shown.

Choline but not its derivative betaine blocks slow vacuolar channels in the halophyte Chenopodium quinoa: implications for salinity stress responses.

PubMed

Pottosin, Igor; Bonales-Alatorre, Edgar; Shabala, Sergey

2014-11-03

Activity of tonoplast slow vacuolar (SV, or TPC1) channels has to be under a tight control, to avoid undesirable leak of cations stored in the vacuole. This is particularly important for salt-grown plants, to ensure efficient vacuolar Na(+) sequestration. In this study we show that choline, a cationic precursor of glycine betaine, efficiently blocks SV channels in leaf and root vacuoles of the two chenopods, Chenopodium quinoa (halophyte) and Beta vulgaris (glycophyte). At the same time, betaine and proline, two major cytosolic organic osmolytes, have no significant effect on SV channel activity. Physiological implications of these findings are discussed. Copyright © 2014 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.

Human NKCC2 cation–Cl– co-transporter complements lack of Vhc1 transporter in yeast vacuolar membranes.

PubMed

Petrezselyova, Silvia; Dominguez, Angel; Herynkova, Pavla; Macias, Juan F; Sychrova, Hana

2013-10-01

Cation–chloride co-transporters serve to transport Cl– and alkali metal cations. Whereas a large family of these exists in higher eukaryotes, yeasts only possess one cation–chloride co-transporter, Vhc1, localized to the vacuolar membrane. In this study, the human cation–chloride co-transporter NKCC2 complemented the phenotype of VHC1 deletion in Saccharomyces cerevisiae and its activity controlled the growth of salt-sensitive yeast cells in the presence of high KCl, NaCl and LiCl. A S. cerevisiae mutant lacking plasma-membrane alkali–metal cation exporters Nha1 and Ena1-5 and the vacuolar cation–chloride co-transporter Vhc1 is highly sensitive to increased concentrations of alkali–metal cations, and it proved to be a suitable model for characterizing the substrate specificity and transport activity of human wild-type and mutated cation–chloride co-transporters. Copyright © 2013 John Wiley & Sons, Ltd.

Root bacterial endophytes confer drought resistance and enhance expression and activity of a vacuolar H+ -pumping pyrophosphatase in pepper plants.

PubMed

Vigani, Gianpiero; Rolli, Eleonora; Marasco, Ramona; Dell'Orto, Marta; Michoud, Grégoire; Soussi, Asma; Raddadi, Noura; Borin, Sara; Sorlini, Claudia; Zocchi, Graziano; Daffonchio, Daniele

2018-05-22

It has been previously shown that the transgenic overexpression of the plant root vacuolar proton pumps H + -ATPase (V-ATPase) and H + -PPase (V-PPase) confer tolerance to drought. Since plant-root endophytic bacteria can also promote drought tolerance, we hypothesize that such promotion can be associated to the enhancement of the host vacuolar proton pumps expression and activity. To test this hypothesis, we selected two endophytic bacteria endowed with an array of in vitro plant growth promoting traits. Their genome sequences confirmed the presence of traits previously shown to confer drought resistance to plants, such as the synthesis of nitric oxide and of organic volatile organic compounds. We used the two strains on pepper (Capsicuum annuum L.) because of its high sensitivity to drought. Under drought conditions, both strains stimulated a larger root system and enhanced the leaves' photosynthetic activity. By testing the expression and activity of the vacuolar proton pumps, H + -ATPase (V-ATPase) and H + -PPase (V-PPase), we found that bacterial colonization enhanced V-PPase only. We conclude that the enhanced expression and activity of V-PPase can be favoured by the colonization of drought-tolerance-inducing bacterial endophytes. This article is protected by copyright. All rights reserved. © 2018 Society for Applied Microbiology and John Wiley & Sons Ltd.

Case report of a 22-week fetus with 47,XXX karyotype and multiple lower mesodermal defects.

PubMed

Hoang, M P; Wilson, K S; Schneider, N R; Timmons, C F

1999-01-01

A 22-week stillborn fetus with 47,XXX karyotype had lower mesodermal defects consisting of irregular fusion of the sacral vertebrae, anal agenesis, multicystic dysplasia of a horseshoe kidney, a single umbilical artery, dysplastic ovaries, and uterine hypoplasia. This case provides additional evidence for an association between trisomy X and genitourinary defects including lower mesodermal defects sequence.

Reduced vacuolar β-1,3-glucan synthesis affects carbohydrate metabolism as well as plastid homeostasis and structure in Phaeodactylum tricornutum.

PubMed

Huang, Weichao; Haferkamp, Ilka; Lepetit, Bernard; Molchanova, Mariia; Hou, Shengwei; Jeblick, Wolfgang; Río Bártulos, Carolina; Kroth, Peter G

2018-05-01

The β-1,3-glucan chrysolaminarin is the main storage polysaccharide of diatoms. In contrast to plants and green algae, diatoms and most other algal groups do not accumulate storage polysaccharides in their plastids. The diatom Phaeodactylum tricornutum possesses only a single gene encoding a putative β-1,3-glucan synthase ( Pt BGS). Here, we characterize this enzyme by expressing GFP fusion proteins in P. tricornutum and by creating and investigating corresponding gene silencing mutants. We demonstrate that Pt BGS is a vacuolar protein located in the tonoplast. Metabolite analyses of two mutant strains with reduced amounts of Pt BGS reveal a reduction in their chrysolaminarin content and an increase of soluble sugars and lipids. This indicates that carbohydrates are shunted into alternative pathways when chrysolaminarin production is impaired. The mutant strains show reduced growth and lower photosynthetic capacities, while possessing higher photoprotective abilities than WT cells. Interestingly, a strong reduction in Pt BGS expression also results in aberrations of the usually very regular thylakoid membrane patterns, including increased thylakoid thickness, reduced numbers of thylakoids per plastid, and increased numbers of lamellae per thylakoid stack. Our data demonstrate the complex intertwinement of carbohydrate storage in the vacuoles with carbohydrate metabolism, photosynthetic homeostasis, and plastid morphology.

Void migration in fusion materials

NASA Astrophysics Data System (ADS)

Cottrell, G. A.

2002-04-01

Neutron irradiation in a fusion power plant will cause helium bubbles and voids to form in the armour and blanket structural materials. If sufficiently large densities of such defects accumulate on the grain boundaries of the materials, the strength and the lifetimes of the metals will be reduced by helium embrittlement and grain boundary failure. This Letter discusses void migration in metals, both by random Brownian motion and by biassed flow in temperature gradients. In the assumed five-year blanket replacement time of a fusion power plant, approximate calculations show that the metals most resilient to failure are tungsten and molybdenum, and marginally vanadium. Helium embrittlement and grain boundary failure is expected to be more severe in steel and beryllium.

[Fetal atrioventricular septal defect associated with Patau and Edwards syndromes, as well as trisomy 22].

PubMed

Cesko, I; Hajdú, J; Marton, T; Tóth-Pál, E; Papp, C; Papp, Z

1998-05-03

The atrioventricular septal defect is usually associated with trisomy 21 and it may be observed in the heterotaxia syndromes. Atrioventricular septal defect may be associated with 8p deletion. There are reported cases of familial atrioventricular septal defect. Atrioventicular septal defect is rarely associated with other chromosomal abnormalities. We are reporting three unusual cases of atrioventricular septal defect that were associated with trisomy 13, 18 and 22. This association may be due to effect of genetic loci on the 13, 18 and 22 chromosome which could play the role in the development and fusion of endocardial cushion and atrioventricular septal defect.

First principles study of intrinsic defects in hexagonal tungsten carbide

NASA Astrophysics Data System (ADS)

Kong, Xiang-Shan; You, Yu-Wei; Xia, J. H.; Liu, C. S.; Fang, Q. F.; Luo, G.-N.; Huang, Qun-Ying

2010-11-01

The characteristics of intrinsic defects are important for the understanding of self-diffusion processes, mechanical strength, brittleness, and plasticity of tungsten carbide, which are present in the divertor of fusion reactors. Here, we use first-principles calculations to investigate the stability of point defects and their complexes in tungsten carbide. Our results confirm that the defect formation energies of carbon are much lower than that of tungsten and reveal the carbon vacancy to be the dominant defect in tungsten carbide. The C sbnd C dimer configuration along the dense a direction is the most stable configuration of carbon interstitial defect. The results of carbon defect diffusion show that the carbon vacancy stay for a wide range of temperature because of extremely high diffusion barriers, while carbon interstitial migration is activated at lower temperatures for its considerably lower activation energy. Both of them prefer to diffusion in carbon basal plane.

Genotype-phenotype relationships in human red/green color-vision defects: Molecular and psychophysical studies

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

Deeb, S.S.; Motulsky, A.G.; Lindsey, D.T.

1992-10-01

The relationship between the molecular structure of the X-linked red and green visual pigment genes and color-vision phenotype as ascertained by anomaloscopy was studied in 64 color-defective males. The great majority of red-green defects were associated with either the deletion of the green-pigment gene or the formation of 5[prime] red-green hybrid genes or 5[prime] green-red hybrid genes. A rapid PCR-based method allowed detection of hybrid genes, including those undetectable by Southern blot analysis, as well as more precise localization of the fusion points in hybrid genes. Protan color-vision defects appeared always associated with 5[prime] red-green hybrid genes. Carriers of singlemore » red-green hybrid genes with fusion in introns 1-4 were protanopes. However, carriers of hybrid genes with red-green fusions in introns 2, 3, or 4 in the presence of additional normal green genes manifested as either protanopes or protanomalous trichromats, with the majority being protanomalous. Deutan defects were associated with green-pigment gene deletions, with 5[prime] green-red hybrid genes, or, rarely, with 5[prime] green-red-green hybrid genes. Complete green-pigment gene deletions or green-red fusions in intron 1 were usually associated with deuteranopia, although the authors unexpectedly found three carriers of a single red-pigment gene without any green-pigment genes to be deuteranomalous trichromats. All but one of the other deuteranomalous subjects had green-red hybrid genes with intron 1, 2, 3, or 4 fusions, as well as several normal green-pigment genes. The one exception had a grossly normal gene array, presumably with a more subtle mutation. Amino acid differences in exon 5 largely determine whether a hybrid gene will be more redlike or more greenlike in phenotype. Various discrepancies as to severity (dichromacy or trichromacy) remain unexplained but may arise because of variability of expression, postreceptoral variation, or both.« less

Sensor Fusion Techniques for Phased-Array Eddy Current and Phased-Array Ultrasound Data

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

Arrowood, Lloyd F.

Sensor (or Data) fusion is the process of integrating multiple data sources to produce more consistent, accurate and comprehensive information than is provided by a single data source. Sensor fusion may also be used to combine multiple signals from a single modality to improve the performance of a particular inspection technique. Industrial nondestructive testing may utilize multiple sensors to acquire inspection data depending upon the object under inspection and the anticipated types of defects that can be identified. Sensor fusion can be performed at various levels of signal abstraction with each having its strengths and weaknesses. A multimodal data fusionmore » strategy first proposed by Heideklang and Shokouhi that combines spatially scattered detection locations to improve detection performance of surface-breaking and near-surface cracks in ferromagnetic metals is shown using a surface inspection example and is then extended for volumetric inspections. Utilizing data acquired from an Olympus Omniscan MX2 from both phased array eddy current and ultrasound probes on test phantoms, single and multilevel fusion techniques are employed to integrate signals from the two modalities. Preliminary results demonstrate how confidence in defect identification and interpretation benefit from sensor fusion techniques. Lastly, techniques for integrating data into radiographic and volumetric imagery from computed tomography are described and results are presented.« less

A Dual Microscopy-Based Assay To Assess Listeria monocytogenes Cellular Entry and Vacuolar Escape.

PubMed

Quereda, Juan J; Pizarro-Cerdá, Javier; Balestrino, Damien; Bobard, Alexandre; Danckaert, Anne; Aulner, Nathalie; Shorte, Spencer; Enninga, Jost; Cossart, Pascale

2016-01-01

Listeria monocytogenes is a Gram-positive bacterium and a facultative intracellular pathogen that invades mammalian cells, disrupts its internalization vacuole, and proliferates in the host cell cytoplasm. Here, we describe a novel image-based microscopy assay that allows discrimination between cellular entry and vacuolar escape, enabling high-content screening to identify factors specifically involved in these two steps. We first generated L. monocytogenes and Listeria innocua strains expressing a β-lactamase covalently attached to the bacterial cell wall. These strains were then incubated with HeLa cells containing the Förster resonance energy transfer (FRET) probe CCF4 in their cytoplasm. The CCF4 probe was cleaved by the bacterial surface β-lactamase only in cells inoculated with L. monocytogenes but not those inoculated with L. innocua, thereby demonstrating bacterial access to the host cytoplasm. Subsequently, we performed differential immunofluorescence staining to distinguish extracellular versus total bacterial populations in samples that were also analyzed by the FRET-based assay. With this two-step analysis, bacterial entry can be distinguished from vacuolar rupture in a single experiment. Our novel approach represents a powerful tool for identifying factors that determine the intracellular niche of L. monocytogenes. Copyright © 2015, American Society for Microbiology. All Rights Reserved.

The "Arabidopsis cax3" mutants display altered salt tolerance, pH sensitivity and reduced plasma membrane H(+)-ATPase activity

USDA-ARS?s Scientific Manuscript database

Perturbing CAX1, an "Arabidopsis" vacuolar H(+)/Ca(2+) antiporter, and the related vacuolar transporter CAX3, has been previously shown to cause severe growth defects; however, the specific function of CAX3 has remained elusive. Here, we describe plant phenotypes that are shared among "cax1" and "ca...

Development of a 3D co-culture model using human stem cells for studying embryonic palatal fusion.

EPA Science Inventory

Morphogenetic tissue fusion is a critical and complex event in embryonic development and failure of this event leads to birth defects, such as cleft palate. Palatal fusion requires adhesion and subsequent dissolution of the medial epithelial layer of the mesenchymal palatal shelv...

Genome-Wide Analysis Reveals the Vacuolar pH-Stat of Saccharomyces cerevisiae

PubMed Central

Brett, Christopher L.; Kallay, Laura; Hua, Zhaolin; Green, Richard; Chyou, Anthony; Zhang, Yongqiang; Graham, Todd R.; Donowitz, Mark; Rao, Rajini

2011-01-01

Protons, the smallest and most ubiquitous of ions, are central to physiological processes. Transmembrane proton gradients drive ATP synthesis, metabolite transport, receptor recycling and vesicle trafficking, while compartmental pH controls enzyme function. Despite this fundamental importance, the mechanisms underlying pH homeostasis are not entirely accounted for in any organelle or organism. We undertook a genome-wide survey of vacuole pH (pHv) in 4,606 single-gene deletion mutants of Saccharomyces cerevisiae under control, acid and alkali stress conditions to reveal the vacuolar pH-stat. Median pHv (5.27±0.13) was resistant to acid stress (5.28±0.14) but shifted significantly in response to alkali stress (5.83±0.13). Of 107 mutants that displayed aberrant pHv under more than one external pH condition, functional categories of transporters, membrane biogenesis and trafficking machinery were significantly enriched. Phospholipid flippases, encoded by the family of P4-type ATPases, emerged as pH regulators, as did the yeast ortholog of Niemann Pick Type C protein, implicated in sterol trafficking. An independent genetic screen revealed that correction of pHv dysregulation in a neo1ts mutant restored viability whereas cholesterol accumulation in human NPC1−/− fibroblasts diminished upon treatment with a proton ionophore. Furthermore, while it is established that lumenal pH affects trafficking, this study revealed a reciprocal link with many mutants defective in anterograde pathways being hyperacidic and retrograde pathway mutants with alkaline vacuoles. In these and other examples, pH perturbations emerge as a hitherto unrecognized phenotype that may contribute to the cellular basis of disease and offer potential therapeutic intervention through pH modulation. PMID:21423800

Relationship of the Membrane ATPase from Halobacterium saccharovorum to Vacuolar ATPases

NASA Technical Reports Server (NTRS)

Stan-Lotter, Helga; Bowman, Emma J.; Hochstein, Lawrence I.

1991-01-01

Polyclonal antiserum against subunit A (67 kDa) of the vacuolar ATPase from Neurospora crassa reacted with subunit I (87 kDa) from a membrane ATPase of the extremely halophilic archaebacterium Halobacterium saccharovorum. The halobacterial ATPase was inhibited by nitrate and N-ethylmaleimide; the extent of the latter inhibition was diminished in the presence of adenosine di- or triphosphates. 4-Chloro-7-nitrobenzofurazan in- hibited the hatobacterial ATPase also in a nucleotide- protectable manner; the bulk of inhibitor was associated with subunit II (60 kDa). The data suggested that this halobacterial ATPase may have conserved structural features from both the vacuotar and the F-type ATPases.

Regulation of cell protrusions by small GTPases during fusion of the neural folds

PubMed Central

Rolo, Ana; Savery, Dawn; Escuin, Sarah; de Castro, Sandra C; Armer, Hannah EJ; Munro, Peter MG; Molè, Matteo A; Greene, Nicholas DE; Copp, Andrew J

2016-01-01

Epithelial fusion is a crucial process in embryonic development, and its failure underlies several clinically important birth defects. For example, failure of neural fold fusion during neurulation leads to open neural tube defects including spina bifida. Using mouse embryos, we show that cell protrusions emanating from the apposed neural fold tips, at the interface between the neuroepithelium and the surface ectoderm, are required for completion of neural tube closure. By genetically ablating the cytoskeletal regulators Rac1 or Cdc42 in the dorsal neuroepithelium, or in the surface ectoderm, we show that these protrusions originate from surface ectodermal cells and that Rac1 is necessary for the formation of membrane ruffles which typify late closure stages, whereas Cdc42 is required for the predominance of filopodia in early neurulation. This study provides evidence for the essential role and molecular regulation of membrane protrusions prior to fusion of a key organ primordium in mammalian development. DOI: http://dx.doi.org/10.7554/eLife.13273.001 PMID:27114066

Activities of Vacuolar Cysteine Proteases in Plant Senescence.

PubMed

Martínez, Dana E; Costa, Lorenza; Guiamét, Juan José

2018-01-01

Plant senescence is accompanied by a marked increase in proteolytic activities, and cysteine proteases (Cys-protease) represent the prevailing class among the responsible proteases. Cys-proteases predominantly locate to lytic compartments, i.e., to the central vacuole (CV) and to senescence-associated vacuoles (SAVs), the latter being specific to the photosynthetic cells of senescing leaves. Cellular fractionation of vacuolar compartments may facilitate Cys-proteases purification and their concentration for further analysis. Active Cys-proteases may be analyzed by different, albeit complementary approaches: (1) in vivo examination of proteolytic activity by fluorescence microscopy using specific substrates which become fluorescent upon cleavage by Cys-proteases, (2) protease labeling with specific probes that react irreversibly with the active enzymes, and (3) zymography, whereby protease activities are detected in polyacrylamide gels copolymerized with a substrate for proteases. Here we describe the three methods mentioned above for detection of active Cys-proteases and a cellular fractionation technique to isolate SAVs.

Distinct roles for key karyogamy proteins during yeast nuclear fusion.

PubMed

Melloy, Patricia; Shen, Shu; White, Erin; Rose, Mark D

2009-09-01

During yeast mating, cell fusion is followed by the congression and fusion of the two nuclei. Proteins required for nuclear fusion are found at the surface (Prm3p) and within the lumen (Kar2p, Kar5p, and Kar8p) of the nuclear envelope (NE). Electron tomography (ET) of zygotes revealed that mutations in these proteins block nuclear fusion with different morphologies, suggesting that they act in different steps of fusion. Specifically, prm3 zygotes were blocked before formation of membrane bridges, whereas kar2, kar5, and kar8 zygotes frequently contained them. Membrane bridges were significantly larger and occurred more frequently in kar2 and kar8, than in kar5 mutant zygotes. The kinetics of NE fusion in prm3, kar5, and kar8 mutants, measured by live-cell fluorescence microscopy, were well correlated with the size and frequency of bridges observed by ET. However the kar2 mutant was defective for transfer of NE lumenal GFP, but not diffusion within the lumen, suggesting that transfer was blocked at the NE fusion junction. These observations suggest that Prm3p acts before initiation of outer NE fusion, Kar5p may help dilation of the initial fusion pore, and Kar2p and Kar8p act after outer NE fusion, during inner NE fusion.

A Conserved Region in the F2 Subunit of Paramyxovirus Fusion Proteins Is Involved In Fusion Regulation▿

PubMed Central

Gardner, Amanda E.; Dutch, Rebecca E.

2007-01-01

Paramyxoviruses utilize both an attachment protein and a fusion (F) protein to drive virus-cell and cell-cell fusion. F exists functionally as a trimer of two disulfide-linked subunits: F1 and F2. Alignment and analysis of a set of paramyxovirus F protein sequences identified three conserved blocks (CB): one in the fusion peptide/heptad repeat A domain, known to play important roles in fusion promotion, one in the region between the heptad repeats of F1 (CBF1) (A. E. Gardner, K. L. Martin, and R. E. Dutch, Biochemistry 46:5094-5105, 2007), and one in the F2 subunit (CBF2). To analyze the functions of CBF2, alanine substitutions at conserved positions were created in both the simian virus 5 (SV5) and Hendra virus F proteins. A number of the CBF2 mutations resulted in folding and expression defects. However, the CBF2 mutants that were properly expressed and trafficked had altered fusion promotion activity. The Hendra virus CBF2 Y79A and P89A mutants showed significantly decreased levels of fusion, whereas the SV5 CBF2 I49A mutant exhibited greatly increased cell-cell fusion relative to that for wild-type F. Additional substitutions at SV5 F I49 suggest that both side chain volume and hydrophobicity at this position are important in the folding of the metastable, prefusion state and the subsequent triggering of membrane fusion. The recently published prefusogenic structure of parainfluenza virus 5/SV5 F (H. S. Yin et al., Nature 439:38-44, 2006) places CBF2 in direct contact with heptad repeat A. Our data therefore indicate that this conserved region plays a critical role in stabilizing the prefusion state, likely through interactions with heptad repeat A, and in triggering membrane fusion. PMID:17507474

Hereditary vacuolar internal anal sphincter myopathy causing proctalgia fugax and constipation: a new case contribution.

PubMed

de la Portilla, Fernando; Borrero, Juan José; Rafel, Enrique

2005-03-01

Hereditary anal sphincter myopathy is rare. We present a family with one affected member with proctalgia fugax, constipation and internal anal sphincter hypertrophy. Ultrastructural findings show vacuolization of smooth muscle cells without the characteristic polyglucosan inclusion. Further relief of symptoms was obtained using an oral calcium antagonist. Based on clinical presentation, endosonography and morphological findings, we consider our case is a histological variant of the vacuolar myopathy originally described.

In-depth glycoproteomic characterisation of grape berry vacuolar invertase using a combination of mass spectrometry-based approaches.

PubMed

Hovasse, Agnès; Alayi, Tchilabalo Dilezitoko; Van Dorsselaer, Alain; Marchal, Richard; Jégou, Sandrine; Schaeffer-Reiss, Christine

2016-06-01

Vacuolar invertase is a key enzyme of sugar metabolism in grape berries. A full characterisation of this highly N-glycosylated protein is required to help understand its biological and biochemical significance in grapes. We have developed a mass spectrometry (MS)-based glycoproteomic approach wherein deglycosylated peptides are analysed by LC-MS/MS, while intact glycopeptides are characterised using a dedicated MS method to determine the attachment sites and micro-heterogeneity. For grape invertase, in parallel with deglycosylated peptides analysis, different enzymatic digestions were performed and glycopeptide detection was improved by enrichment method, nanoLC-MS and oxonium glycan ions. This MS-based glycoproteomic approach demonstrates that vacuolar invertase is glycosylated at all twelve potential N-glycosylation sites. Glycosylation is heterogeneous, with twelve glycoforms identified at six of the sites. The identification of several types of N-glycans is a major result to correlate with the surface and foaming properties of wine, the solubility, allergenicity, and protease resistance of wine proteins. Copyright © 2016 Elsevier Ltd. All rights reserved.

Functional characterization of a vacuolar invertase from Solanum lycopersicum: post-translational regulation by N-glycosylation and a proteinaceous inhibitor.

PubMed

Tauzin, Alexandra S; Sulzenbacher, Gerlind; Lafond, Mickael; Desseaux, Véronique; Reca, Ida Barbara; Perrier, Josette; Bellincampi, Daniela; Fourquet, Patrick; Lévêque, Christian; Giardina, Thierry

2014-06-01

Plant vacuolar invertases, which belong to family 32 of glycoside hydrolases (GH32), are key enzymes in sugar metabolism. They hydrolyse sucrose into glucose and fructose. The cDNA encoding a vacuolar invertase from Solanum lycopersicum (TIV-1) was cloned and heterologously expressed in Pichia pastoris. The functional role of four N-glycosylation sites in TIV-1 has been investigated by site-directed mutagenesis. Single mutations to Asp of residues Asn52, Asn119 and Asn184, as well as the triple mutant (Asn52, Asn119 and Asn184), lead to enzymes with reduced specific invertase activity and thermostability. Expression of the N516D mutant, as well as of the quadruple mutant (N52D, N119D, N184D and N516D) could not be detected, indicating that these mutations dramatically affected the folding of the protein. Our data indicate that N-glycosylation is important for TIV-1 activity and that glycosylation of N516 is crucial for recombinant enzyme stability. Using a functional genomics approach a new vacuolar invertase inhibitor of S. lycopersicum (SolyVIF) has been identified. SolyVIF cDNA was cloned and heterologously expressed in Escherichia coli. Specific interactions between SolyVIF and TIV-1 were investigated by an enzymatic approach and surface plasmon resonance (SPR). Finally, qRT-PCR analysis of TIV-1 and SolyVIF transcript levels showed a specific tissue and developmental expression. TIV-1 was mainly expressed in flowers and both genes were expressed in senescent leaves. Copyright © 2013 Elsevier Masson SAS. All rights reserved.

Regulation of Vacuolar H+-ATPase (V-ATPase) Reassembly by Glycolysis Flow in 6-Phosphofructo-1-kinase (PFK-1)-deficient Yeast Cells*

PubMed Central

Chan, Chun-Yuan; Dominguez, Dennis; Parra, Karlett J.

2016-01-01

Yeast 6-phosphofructo-1-kinase (PFK-1) has two subunits, Pfk1p and Pfk2p. Deletion of Pfk2p alters glucose-dependent V-ATPase reassembly and vacuolar acidification (Chan, C. Y., and Parra, K. J. (2014) Yeast phosphofructokinase-1 subunit Pfk2p is necessary for pH homeostasis and glucose-dependent vacuolar ATPase reassembly. J. Biol. Chem. 289, 19448–19457). This study capitalized on the mechanisms suppressing vacuolar H+-ATPase (V-ATPase) in pfk2Δ to gain new knowledge of the mechanisms underlying glucose-dependent V-ATPase regulation. Because V-ATPase is fully assembled in pfk2Δ, and glycolysis partially suppressed at steady state, we manipulated glycolysis and assessed its direct involvement on V-ATPase function. At steady state, the ratio of proton transport to ATP hydrolysis increased 24% after increasing the glucose concentration from 2% to 4% to enhance the glycolysis flow in pfk2Δ. Tighter coupling restored vacuolar pH when glucose was abundant and glycolysis operated below capacity. After readdition of glucose to glucose-deprived cells, glucose-dependent V1Vo reassembly was proportional to the glycolysis flow. Readdition of 2% glucose to pfk2Δ cells, which restored 62% of ethanol concentration, led to equivalent 60% V1Vo reassembly levels. Steady-state level of assembly (100% reassembly) was reached at 4% glucose when glycolysis reached a threshold in pfk2Δ (≥40% the wild-type flow). At 4% glucose, the level of Pfk1p co-immunoprecipitated with V-ATPase decreased 58% in pfk2Δ, suggesting that Pfk1p binding to V-ATPase may be inhibitory in the mutant. We concluded that V-ATPase activity at steady state and V-ATPase reassembly after readdition of glucose to glucose-deprived cells are controlled by the glycolysis flow. We propose a new mechanism by which glucose regulates V-ATPase catalytic activity that occurs at steady state without changing V1Vo assembly. PMID:27226568

Clinical Utility of LC3 and p62 Immunohistochemistry in Diagnosis of Drug-Induced Autophagic Vacuolar Myopathies: A Case-Control Study

PubMed Central

Lee, Han S.; Daniels, Brianne H.; Salas, Eduardo; Bollen, Andrew W.; Debnath, Jayanta; Margeta, Marta

2012-01-01

Background Some patients treated with chloroquine, hydroxychloroquine, or colchicine develop autophagic vacuolar myopathy, the diagnosis of which currently requires electron microscopy. The goal of the current study was to develop an immunohistochemical diagnostic marker for this pathologic entity. Methodology Microtubule-associated protein light chain 3 (LC3) has emerged as a robust marker of autophagosomes. LC3 binds p62/SQSTM1, an adapter protein that is selectively degraded via autophagy. In this study, we evaluated the utility of immunohistochemical stains for LC3 and p62 as diagnostic markers of drug-induced autophagic vacuolar myopathy. The staining was performed on archival muscle biopsy material, with subject assignment to normal control, drug-treated control, and autophagic myopathy groups based on history of drug use and morphologic criteria. Principal Findings In all drug-treated subjects, but not in normal controls, LC3 and p62 showed punctate staining characteristic of autophagosome buildup. In the autophagic myopathy subjects, puncta were coarser and tended to coalesce into linear structures aligned with the longitudinal axis of the fiber, often in the vicinity of vacuoles. The percentage of LC3- and p62-positive fibers was significantly higher in the autophagic myopathy group compared to either the normal control (p<0.001) or the drug-treated control group (p<0.05). With the diagnostic threshold set between 8% and 15% positive fibers (depending on the desired level of sensitivity and specificity), immunohistochemical staining for either LC3 or p62 could be used to identify subjects with autophagic vacuolar myopathy within the drug-treated subject group (p≤0.001). Significance Immunohistochemistry for LC3 and p62 can facilitate tissue-based diagnosis of drug-induced autophagic vacuolar myopathies. By limiting the need for electron microscopy (a time consuming and costly technique with high specificity, but low sensitivity), clinical use of these

Clinical utility of LC3 and p62 immunohistochemistry in diagnosis of drug-induced autophagic vacuolar myopathies: a case-control study.

PubMed

Lee, Han S; Daniels, Brianne H; Salas, Eduardo; Bollen, Andrew W; Debnath, Jayanta; Margeta, Marta

2012-01-01

Some patients treated with chloroquine, hydroxychloroquine, or colchicine develop autophagic vacuolar myopathy, the diagnosis of which currently requires electron microscopy. The goal of the current study was to develop an immunohistochemical diagnostic marker for this pathologic entity. Microtubule-associated protein light chain 3 (LC3) has emerged as a robust marker of autophagosomes. LC3 binds p62/SQSTM1, an adapter protein that is selectively degraded via autophagy. In this study, we evaluated the utility of immunohistochemical stains for LC3 and p62 as diagnostic markers of drug-induced autophagic vacuolar myopathy. The staining was performed on archival muscle biopsy material, with subject assignment to normal control, drug-treated control, and autophagic myopathy groups based on history of drug use and morphologic criteria. In all drug-treated subjects, but not in normal controls, LC3 and p62 showed punctate staining characteristic of autophagosome buildup. In the autophagic myopathy subjects, puncta were coarser and tended to coalesce into linear structures aligned with the longitudinal axis of the fiber, often in the vicinity of vacuoles. The percentage of LC3- and p62-positive fibers was significantly higher in the autophagic myopathy group compared to either the normal control (p<0.001) or the drug-treated control group (p<0.05). With the diagnostic threshold set between 8% and 15% positive fibers (depending on the desired level of sensitivity and specificity), immunohistochemical staining for either LC3 or p62 could be used to identify subjects with autophagic vacuolar myopathy within the drug-treated subject group (p ≤ 0.001). Immunohistochemistry for LC3 and p62 can facilitate tissue-based diagnosis of drug-induced autophagic vacuolar myopathies. By limiting the need for electron microscopy (a time consuming and costly technique with high specificity, but low sensitivity), clinical use of these markers will improve the speed and accuracy of

RhVI1 is a membrane-anchored vacuolar invertase highly expressed in Rosa hybrida L. petals

PubMed Central

Farci, Domenica; Collu, Gabriella; Kirkpatrick, Joanna; Esposito, Francesca; Piano, Dario

2016-01-01

Invertases are a widespread group of enzymes that catalyse the conversion of sucrose into fructose and glucose. Plants invertases and their substrates are essential factors that play an active role in primary metabolism and in cellular differentiation and by these activities they sustain development and growth. Being naturally present in multiple isoforms, invertases are known to be highly differentiated and tissue specific in such a way that every isoform is characteristic of a specific part of the plant. In this work, we report the identification of the invertase RhVI1 that was found to be highly expressed in rose petals. A characterization of this protein revealed that RhVI1 is a glycosylated membrane-anchored protein associated with the cytosolic side of the vacuolar membrane which occurs in vivo in a monomeric form. Purification yields have shown that the levels of expression decreased during the passage of petals from buds to mature and pre-senescent flowers. Moreover, the activity assay indicates RhVI1 to be an acidic vacuolar invertase. The physiological implications of these findings are discussed, suggesting a possible role of this protein during anthesis. PMID:27083698

Abscisic acid induction of vacuolar H+-ATPase activity in mesembryanthemum crystallinum is developmentally regulated

PubMed

Barkla; Vera-Estrella; Maldonado-Gama; Pantoja

1999-07-01

Abscisic acid (ABA) has been implicated as a key component in water-deficit-induced responses, including those triggered by drought, NaCl, and low- temperature stress. In this study a role for ABA in mediating the NaCl-stress-induced increases in tonoplast H+-translocating ATPase (V-ATPase) and Na+/H+ antiport activity in Mesembryanthemum crystallinum, leading to vacuolar Na+ sequestration, were investigated. NaCl or ABA treatment of adult M. crystallinum plants induced V-ATPase H+ transport activity, and when applied in combination, an additive effect on V-ATPase stimulation was observed. In contrast, treatment of juvenile plants with ABA did not induce V-ATPase activity, whereas NaCl treatment resulted in a similar response to that observed in adult plants. Na+/H+ antiport activity was induced in both juvenile and adult plants by NaCl, but ABA had no effect at either developmental stage. Results indicate that ABA-induced changes in V-ATPase activity are dependent on the plant reaching its adult phase, whereas NaCl-induced increases in V-ATPase and Na+/H+ antiport activity are independent of plant age. This suggests that ABA-induced V-ATPase activity may be linked to the stress-induced, developmentally programmed switch from C3 metabolism to Crassulacean acid metabolism in adult plants, whereas, vacuolar Na+ sequestration, mediated by the V-ATPase and Na+/H+ antiport, is regulated through ABA-independent pathways.

Normal myoblast fusion requires myoferlin

PubMed Central

Doherty, Katherine R.; Cave, Andrew; Davis, Dawn Belt; Delmonte, Anthony J.; Posey, Avery; Earley, Judy U.; Hadhazy, Michele; McNally, Elizabeth M.

2014-01-01

Summary Muscle growth occurs during embryonic development and continues in adult life as regeneration. During embryonic muscle growth and regeneration in mature muscle, singly nucleated myoblasts fuse to each other to form myotubes. In muscle growth, singly nucleated myoblasts can also fuse to existing large, syncytial myofibers as a mechanism of increasing muscle mass without increasing myofiber number. Myoblast fusion requires the alignment and fusion of two apposed lipid bilayers. The repair of muscle plasma membrane disruptions also relies on the fusion of two apposed lipid bilayers. The protein dysferlin, the product of the Limb Girdle Muscular Dystrophy type 2 locus, has been shown to be necessary for efficient, calcium-sensitive, membrane resealing. We now show that the related protein myoferlin is highly expressed in myoblasts undergoing fusion, and is expressed at the site of myoblasts fusing to myotubes. Like dysferlin, we found that myoferlin binds phospholipids in a calcium-sensitive manner that requires the first C2A domain. We generated mice with a null allele of myoferlin. Myoferlin null myoblasts undergo initial fusion events, but they form large myotubes less efficiently in vitro, consistent with a defect in a later stage of myogenesis. In vivo, myoferlin null mice have smaller muscles than controls do, and myoferlin null muscle lacks large diameter myofibers. Additionally, myoferlin null muscle does not regenerate as well as wild-type muscle does, and instead displays a dystrophic phenotype. These data support a role for myoferlin in the maturation of myotubes and the formation of large myotubes that arise from the fusion of myoblasts to multinucleate myotubes. PMID:16280346

Renal Atp6ap2/(Pro)renin Receptor Is Required for Normal Vacuolar H+-ATPase Function but Not for the Renin-Angiotensin System.

PubMed

Trepiccione, Francesco; Gerber, Simon D; Grahammer, Florian; López-Cayuqueo, Karen I; Baudrie, Véronique; Păunescu, Teodor G; Capen, Diane E; Picard, Nicolas; Alexander, R Todd; Huber, Tobias B; Chambrey, Regine; Brown, Dennis; Houillier, Pascal; Eladari, Dominique; Simons, Matias

2016-11-01

ATPase H + -transporting lysosomal accessory protein 2 (Atp6ap2), also known as the (pro)renin receptor, is a type 1 transmembrane protein and an accessory subunit of the vacuolar H + -ATPase (V-ATPase) that may also function within the renin-angiotensin system. However, the contribution of Atp6ap2 to renin-angiotensin-dependent functions remains unconfirmed. Using mice with an inducible conditional deletion of Atp6ap2 in mouse renal epithelial cells, we found that decreased V-ATPase expression and activity in the intercalated cells of the collecting duct impaired acid-base regulation by the kidney. In addition, these mice suffered from marked polyuria resistant to desmopressin administration. Immunoblotting revealed downregulation of the medullary Na + -K + -2Cl - cotransporter NKCC2 in these mice compared with wild-type mice, an effect accompanied by a hypotonic medullary interstitium and impaired countercurrent multiplication. This phenotype correlated with strong autophagic defects in epithelial cells of medullary tubules. Notably, cells with high accumulation of the autophagosomal substrate p62 displayed the strongest reduction of NKCC2 expression. Finally, nephron-specific Atp6ap2 depletion did not affect angiotensin II production, angiotensin II-dependent BP regulation, or sodium handling in the kidney. Taken together, our results show that nephron-specific deletion of Atp6ap2 does not affect the renin-angiotensin system but causes a combination of renal concentration defects and distal renal tubular acidosis as a result of impaired V-ATPase activity. Copyright © 2016 by the American Society of Nephrology.

Vacuolar CAX1 and CAX3 influence auxin transport in guard cells via regulation of apoplastic pH

USDA-ARS?s Scientific Manuscript database

Cation exchangers CAX1 and CAX3 are vacuolar ion transporters involved in ion homeostasis in plants. Widely expressed in the plant, they mediate calcium transport from the cytosol to the vacuole lumen using the proton gradient across the tonoplast. Here, we report an unexpected role of CAX1 and CAX3...

Salinity Tolerance of Two Potato Cultivars (Solanum tuberosum) Correlates With Differences in Vacuolar Transport Activity

PubMed Central

Jaarsma, Rinse; de Boer, Albertus H.

2018-01-01

Potato is an important cultivated crop species and since it is moderately salt sensitive there is a need to develop more salt tolerant cultivars. A high activity of Na+ transport across the tonoplast in exchange for H+ is essential to reduce Na+ toxicity. The proton motive force (PMF) generated by the V-H+-ATPase and the V-H+-PPase energizes the Na+(K+)/H+ antiport. We compared the activity, gene expression, and protein levels of the vacuolar proton pumps and the Na+/H+ antiporters in two potato cultivars (Solanum tuberosum) contrasting in their salt tolerance (cv. Desiree; tolerant and Mozart; sensitive) grown at 0 and 60 mM NaCl. Tonoplast-enriched vesicles were used to study the pump activity and protein levels of the V-H+-ATPase and the V-H+-PPase and the activity of the Na+/H+ antiporter. Although salt stress reduced the V-H+-ATPase and the V-H+-PPase activity in both cultivars, the decline in H+ pump activity was more severe in the salt-sensitive cultivar Mozart. After salt treatment, protein amounts of the vacuolar H+ pumps decreased in Mozart but remained unchanged in the cultivar Desiree. Decreased protein amounts of the V-H+-PPase found in Mozart may explain the reduced V-H+-PPase activity found for Mozart after salt stress. Under non-stress conditions, protein amounts of V-H+-PPase were equal in both cultivars while the V-H+-PPase activity was already twice as high and remained higher after salt treatment in the cultivar Desiree as compared to Mozart. This cultivar-dependent V-H+-PPase activity may explain the higher salt tolerance of Desiree. Moreover, combined with reduced vacuolar H+ pump activity, Mozart showed a lower Na+/H+ exchange activity and the Km for Na+ is at least twofold lower in tonoplast vesicles from Desiree, what suggests that NHXs from Desiree have a higher affinity for Na+ as compared to Mozart. From these results, we conclude that the higher capacity in combination with the higher affinity for Na+ uptake can be an important factor

Salinity Tolerance of Two Potato Cultivars (Solanum tuberosum) Correlates With Differences in Vacuolar Transport Activity.

PubMed

Jaarsma, Rinse; de Boer, Albertus H

2018-01-01

Potato is an important cultivated crop species and since it is moderately salt sensitive there is a need to develop more salt tolerant cultivars. A high activity of Na + transport across the tonoplast in exchange for H + is essential to reduce Na + toxicity. The proton motive force (PMF) generated by the V-H + -ATPase and the V-H + -PPase energizes the Na + (K + )/H + antiport. We compared the activity, gene expression, and protein levels of the vacuolar proton pumps and the Na + /H + antiporters in two potato cultivars ( Solanum tuberosum ) contrasting in their salt tolerance (cv. Desiree; tolerant and Mozart; sensitive) grown at 0 and 60 mM NaCl. Tonoplast-enriched vesicles were used to study the pump activity and protein levels of the V-H + -ATPase and the V-H + -PPase and the activity of the Na + /H + antiporter. Although salt stress reduced the V-H + -ATPase and the V-H + -PPase activity in both cultivars, the decline in H + pump activity was more severe in the salt-sensitive cultivar Mozart. After salt treatment, protein amounts of the vacuolar H + pumps decreased in Mozart but remained unchanged in the cultivar Desiree. Decreased protein amounts of the V-H + -PPase found in Mozart may explain the reduced V-H + -PPase activity found for Mozart after salt stress. Under non-stress conditions, protein amounts of V-H + -PPase were equal in both cultivars while the V-H + -PPase activity was already twice as high and remained higher after salt treatment in the cultivar Desiree as compared to Mozart. This cultivar-dependent V-H + -PPase activity may explain the higher salt tolerance of Desiree. Moreover, combined with reduced vacuolar H + pump activity, Mozart showed a lower Na + /H + exchange activity and the K m for Na + is at least twofold lower in tonoplast vesicles from Desiree, what suggests that NHXs from Desiree have a higher affinity for Na + as compared to Mozart. From these results, we conclude that the higher capacity in combination with the higher

MFN1 deacetylation activates adaptive mitochondrial fusion and protects metabolically challenged mitochondria.

PubMed

Lee, Joo-Yong; Kapur, Meghan; Li, Ming; Choi, Moon-Chang; Choi, Sujin; Kim, Hak-June; Kim, Inhye; Lee, Eunji; Taylor, J Paul; Yao, Tso-Pang

2014-11-15

Fasting and glucose shortage activate a metabolic switch that shifts more energy production to mitochondria. This metabolic adaptation ensures energy supply, but also elevates the risk of mitochondrial oxidative damage. Here, we present evidence that metabolically challenged mitochondria undergo active fusion to suppress oxidative stress. In response to glucose starvation, mitofusin 1 (MFN1) becomes associated with the protein deacetylase HDAC6. This interaction leads to MFN1 deacetylation and activation, promoting mitochondrial fusion. Deficiency in HDAC6 or MFN1 prevents mitochondrial fusion induced by glucose deprivation. Unexpectedly, failure to undergo fusion does not acutely affect mitochondrial adaptive energy production; instead, it causes excessive production of mitochondrial reactive oxygen species and oxidative damage, a defect suppressed by an acetylation-resistant MFN1 mutant. In mice subjected to fasting, skeletal muscle mitochondria undergo dramatic fusion. Remarkably, fasting-induced mitochondrial fusion is abrogated in HDAC6-knockout mice, resulting in extensive mitochondrial degeneration. These findings show that adaptive mitochondrial fusion protects metabolically challenged mitochondria. © 2014. Published by The Company of Biologists Ltd.

Fusion in posttraumatic foot and ankle reconstruction.

PubMed

Thordarson, David B

2004-01-01

Despite appropriate acute treatment, many foot and ankle injuries result in posttraumatic arthritis. Arthrodesis remains the mainstay of treatment of end-stage arthritis of the foot and ankle. An understanding of the biomechanics of the foot and ankle, particularly which joints are most responsible for optimal function of the foot, can help guide reconstructive efforts. A careful history and physical examination, appropriate radiographs, and, when necessary, differential selective anesthetic blocks help limit fusion to only those joints that are causing pain. Compression fixation, when possible, remains the treatment of choice. When bone defects are present, however, neutralization fixation may be necessary to prevent a secondary deformity that could result from impaction into a bone defect.

When intracellular logistics fails--genetic defects in membrane trafficking.

PubMed

Olkkonen, Vesa M; Ikonen, Elina

2006-12-15

The number of human genetic disorders shown to be due to defects in membrane trafficking has greatly increased during the past five years. Defects have been identified in components involved in sorting of cargo into transport carriers, vesicle budding and scission, movement of vesicles along cytoskeletal tracks, as well as in vesicle tethering, docking and fusion at the target membrane. The nervous system is extremely sensitive to such disturbances of the membrane trafficking machinery, and the majority of these disorders display neurological defects--particularly diseases affecting the motility of transport carriers along cytoskeletal tracks. In several disorders, defects in a component that represents a fundamental part of the trafficking machinery fail to cause global transport defects but result in symptoms limited to specific cell types and transport events; this apparently reflects the redundancy of the transport apparatus. In groups of closely related diseases such as Hermansky-Pudlak and Griscelli syndromes, identification of the underlying gene defects has revealed groups of genes in which mutations lead to similar phenotypic consequences. New functionally linked trafficking components and regulatory mechanisms have thus been discovered. Studies of the gene defects in trafficking disorders therefore not only open avenues for new therapeutic approaches but also significantly contribute to our knowledge of the fundamental mechanisms of intracellular membrane transport.

Dissection of autophagy in tobacco BY-2 cells under sucrose starvation conditions using the vacuolar H+-ATPase inhibitor concanamycin A and the autophagy-related protein Atg8

PubMed Central

Yano, Kanako; Yanagisawa, Takahiro; Mukae, Kyosuke; Niwa, Yasuo; Inoue, Yuko; Moriyasu, Yuji

2015-01-01

Tobacco BY-2 cells undergo autophagy in sucrose-free culture medium, which is the process mostly responsible for intracellular protein degradation under these conditions. Autophagy was inhibited by the vacuolar H+-ATPase inhibitors concanamycin A and bafilomycin A1, which caused the accumulation of autophagic bodies in the central vacuoles. Such accumulation did not occur in the presence of the autophagy inhibitor 3-methyladenine, and concanamycin in turn inhibited the accumulation of autolysosomes in the presence of the cysteine protease inhibitor E-64c. Electron microscopy revealed not only that the autophagic bodies were accumulated in the central vacuole, but also that autophagosome-like structures were more frequently observed in the cytoplasm in treatments with concanamycin, suggesting that concanamycin affects the morphology of autophagosomes in addition to raising the pH of the central vacuole. Using BY-2 cells that constitutively express a fusion protein of autophagosome marker protein Atg8 and green fluorescent protein (GFP), we observed the appearance of autophagosomes by fluorescence microscopy, which is a reliable morphological marker of autophagy, and the processing of the fusion protein to GFP, which is a biochemical marker of autophagy. Together, these results suggest the involvement of vacuole type H+-ATPase in the maturation step of autophagosomes to autolysosomes in the autophagic process of BY-2 cells. The accumulation of autophagic bodies in the central vacuole by concanamycin is a marker of the occurrence of autophagy; however, it does not necessarily mean that the central vacuole is the site of cytoplasm degradation. PMID:26368310

Dissection of autophagy in tobacco BY-2 cells under sucrose starvation conditions using the vacuolar H(+)-ATPase inhibitor concanamycin A and the autophagy-related protein Atg8.

PubMed

Yano, Kanako; Yanagisawa, Takahiro; Mukae, Kyosuke; Niwa, Yasuo; Inoue, Yuko; Moriyasu, Yuji

2015-01-01

Tobacco BY-2 cells undergo autophagy in sucrose-free culture medium, which is the process mostly responsible for intracellular protein degradation under these conditions. Autophagy was inhibited by the vacuolar H(+)-ATPase inhibitors concanamycin A and bafilomycin A1, which caused the accumulation of autophagic bodies in the central vacuoles. Such accumulation did not occur in the presence of the autophagy inhibitor 3-methyladenine, and concanamycin in turn inhibited the accumulation of autolysosomes in the presence of the cysteine protease inhibitor E-64c. Electron microscopy revealed not only that the autophagic bodies were accumulated in the central vacuole, but also that autophagosome-like structures were more frequently observed in the cytoplasm in treatments with concanamycin, suggesting that concanamycin affects the morphology of autophagosomes in addition to raising the pH of the central vacuole. Using BY-2 cells that constitutively express a fusion protein of autophagosome marker protein Atg8 and green fluorescent protein (GFP), we observed the appearance of autophagosomes by fluorescence microscopy, which is a reliable morphological marker of autophagy, and the processing of the fusion protein to GFP, which is a biochemical marker of autophagy. Together, these results suggest the involvement of vacuole type H(+)-ATPase in the maturation step of autophagosomes to autolysosomes in the autophagic process of BY-2 cells. The accumulation of autophagic bodies in the central vacuole by concanamycin is a marker of the occurrence of autophagy; however, it does not necessarily mean that the central vacuole is the site of cytoplasm degradation.

Amyloplasts and Vacuolar Membrane Dynamics in the Living Graviperceptive Cell of the Arabidopsis Inflorescence StemW⃞

PubMed Central

Saito, Chieko; Morita, Miyo T.; Kato, Takehide; Tasaka, Masao

2005-01-01

We developed an adequate method for the in vivo analysis of organelle dynamics in the gravity-perceptive cell (endodermis) of the Arabidopsis thaliana inflorescence stem, revealing behavior of amyloplasts and vacuolar membranes in those cells. Amyloplasts in the endodermis showed saltatory movements even before gravistimulation by reorientation, and these movements were confirmed as microfilament dependent. From our quantitative analysis in the wild type, the gravity-oriented movement of amyloplasts mainly occurred during 0 to 3 min after gravistimulation by reorientation, supporting findings from our previous physiological study. Even after microfilament disruption, the gravity-oriented movement of amyloplasts remained. By contrast, in zig/sgr4 mutants, where a SNARE molecule functioning in vacuole biogenesis has been disrupted, the movement of amyloplasts in the endodermis is severely restricted both before and after gravistimulation by reorientation. Here, we describe vacuolar membrane behavior in these cells in the wild-type, actin filament–disrupted, and zig/sgr4 mutants and discuss its putatively important features for the perception of gravity. We also discuss the data on the two kinds of movements of amyloplasts that may play an important role in gravitropism: (1) the leading edge amyloplasts and (2) the en mass movement of amyloplasts. PMID:15689424

RhVI1 is a membrane-anchored vacuolar invertase highly expressed in Rosa hybrida L. petals.

PubMed

Farci, Domenica; Collu, Gabriella; Kirkpatrick, Joanna; Esposito, Francesca; Piano, Dario

2016-05-01

Invertases are a widespread group of enzymes that catalyse the conversion of sucrose into fructose and glucose. Plants invertases and their substrates are essential factors that play an active role in primary metabolism and in cellular differentiation and by these activities they sustain development and growth. Being naturally present in multiple isoforms, invertases are known to be highly differentiated and tissue specific in such a way that every isoform is characteristic of a specific part of the plant. In this work, we report the identification of the invertase RhVI1 that was found to be highly expressed in rose petals. A characterization of this protein revealed that RhVI1 is a glycosylated membrane-anchored protein associated with the cytosolic side of the vacuolar membrane which occurs in vivo in a monomeric form. Purification yields have shown that the levels of expression decreased during the passage of petals from buds to mature and pre-senescent flowers. Moreover, the activity assay indicates RhVI1 to be an acidic vacuolar invertase. The physiological implications of these findings are discussed, suggesting a possible role of this protein during anthesis. © The Author 2016. Published by Oxford University Press on behalf of the Society for Experimental Biology.

Co-overexpressing a plasma membrane and a vacuolar membrane sodium/proton antiporter significantly improves salt tolerance in transgenic Arabidopsis plants.

USDA-ARS?s Scientific Manuscript database

The Arabidopsis gene AtNHX1 encodes a vacuolar membrane bound sodium/proton (Sodium/Hydrogen) antiporter that transports sodium into the vacuole and exports hydrogen into the cytoplasm. The Arabidopsis gene SOS1 encodes a plasma membrane bound sodium/hydrogen antiporter that exports sodium to the ex...

Targeting vacuolar H+-ATPases as a new strategy against cancer.

PubMed

Fais, Stefano; De Milito, Angelo; You, Haiyan; Qin, Wenxin

2007-11-15

Growing evidence suggests a key role of tumor acidic microenvironment in cancer development, progression, and metastasis. As a consequence, the need for compounds that specifically target the mechanism(s) responsible for the low pH of tumors is increasing. Among the key regulators of the tumor acidic microenvironment, vacuolar H(+)-ATPases (V-ATPases) play an important role. These proteins cover a number of functions in a variety of normal as well as tumor cells, in which they pump ions across the membranes. We discuss here some recent results showing that a molecular inhibition of V-ATPases by small interfering RNA in vivo as well as a pharmacologic inhibition through proton pump inhibitors led to tumor cytotoxicity and marked inhibition of human tumor growth in xenograft models. These results propose V-ATPases as a key target for new strategies in cancer treatment.

Fusion silicide coatings for tantalum alloys.

NASA Technical Reports Server (NTRS)

Warnock, R. V.; Stetson, A. R.

1972-01-01

Calculation of the performance of fusion silicide coatings under simulated atmospheric reentry conditions to a maximum temperature of 1810 K (2800 F). Both recently developed and commercially available coatings are included. Data are presented on oxidation rate with and without intentional defecting, the influence of the coatings on the ductile-brittle bend transition temperature, and the mechanical properties. Coatings appear capable of affording protection for at least 100 simulated cycles to 2600 F and 63 cycles to 2800 F.

Nanoscale domain formation of phosphatidylinositol 4-phosphate in the plasma and vacuolar membranes of living yeast cells.

PubMed

Tomioku, Kan-Na; Shigekuni, Mikiko; Hayashi, Hiroki; Yoshida, Akane; Futagami, Taiki; Tamaki, Hisanori; Tanabe, Kenji; Fujita, Akikazu

2018-05-01

In budding yeast Saccharomyces cerevisiae, PtdIns(4)P serves as an essential signalling molecule in the Golgi complex, endosomal system, and plasma membrane, where it is involved in the control of multiple cellular functions via direct interactions with PtdIns(4)P-binding proteins. To analyse the distribution of PtdIns(4)P in yeast cells at a nanoscale level, we employed an electron microscopy technique that specifically labels PtdIns(4)P on the freeze-fracture replica of the yeast membrane. This method minimizes the possibility of artificial perturbation, because molecules in the membrane are physically immobilised in situ. We observed that PtdIns(4)P is localised on the cytoplasmic leaflet, but not the exoplasmic leaflet, of the plasma membrane, Golgi body, vacuole, and vesicular structure membranes. PtdIns(4)P labelling was not observed in the membrane of the endoplasmic reticulum, and in the outer and inner membranes of the nuclear envelope or mitochondria. PtdIns(4)P forms clusters of <100 nm in diameter in the plasma membrane and vacuolar membrane according to point pattern analysis of immunogold labelling. There are three kinds of compartments in the cytoplasmic leaflet of the plasma membrane. In the present study, we showed that PtdIns(4)P is specifically localised in the flat undifferentiated plasma membrane compartment. In the vacuolar membrane, PtdIns(4)P was concentrated in intramembrane particle (IMP)-deficient raft-like domains, which are tightly bound to lipid droplets, but not surrounding IMP-rich non-raft domains in geometrical IMP-distributed patterns in the stationary phase. This is the first report showing microdomain formations of PtdIns(4)P in the plasma membrane and vacuolar membrane of budding yeast cells at a nanoscale level, which will illuminate the functionality of PtdIns(4)P in each membrane. Copyright © 2018 Elsevier GmbH. All rights reserved.

A robust vision-based sensor fusion approach for real-time pose estimation.

PubMed

Assa, Akbar; Janabi-Sharifi, Farrokh

2014-02-01

Object pose estimation is of great importance to many applications, such as augmented reality, localization and mapping, motion capture, and visual servoing. Although many approaches based on a monocular camera have been proposed, only a few works have concentrated on applying multicamera sensor fusion techniques to pose estimation. Higher accuracy and enhanced robustness toward sensor defects or failures are some of the advantages of these schemes. This paper presents a new Kalman-based sensor fusion approach for pose estimation that offers higher accuracy and precision, and is robust to camera motion and image occlusion, compared to its predecessors. Extensive experiments are conducted to validate the superiority of this fusion method over currently employed vision-based pose estimation algorithms.

Vacuolar H(+)-Pyrophosphatase AVP1 is Involved in Amine Fungicide Tolerance in Arabidopsis thaliana and Provides Tridemorph Resistance in Yeast.

PubMed

Hernández, Agustín; Herrera-Palau, Rosana; Madroñal, Juan M; Albi, Tomás; López-Lluch, Guillermo; Perez-Castiñeira, José R; Navas, Plácido; Valverde, Federico; Serrano, Aurelio

2016-01-01

Amine fungicides are widely used as crop protectants. Their success is believed to be related to their ability to inhibit postlanosterol sterol biosynthesis in fungi, in particular sterol-Δ(8),Δ(7)-isomerases and sterol-Δ(14)-reductases, with a concomitant accumulation of toxic abnormal sterols. However, their actual cellular effects and mechanisms of death induction are still poorly understood. Paradoxically, plants exhibit a natural resistance to amine fungicides although they have similar enzymes in postcicloartenol sterol biosynthesis that are also susceptible to fungicide inhibition. A major difference in vacuolar ion homeostasis between plants and fungi is the presence of a dual set of primary proton pumps in the former (V-ATPase and H(+)-pyrophosphatase), but only the V-ATPase in the latter. Abnormal sterols affect the proton-pumping capacity of V-ATPases in fungi and this has been proposed as a major determinant in fungicide action. Using Saccharomyces cerevisiae as a model fungus, we provide evidence that amine fungicide treatment induced cell death by apoptosis. Cell death was concomitant with impaired H(+)-pumping capacity in vacuole vesicles and dependent on vacuolar proteases. Also, the heterologous expression of the Arabidopsis thaliana main H(+)-pyrophosphatase (AVP1) at the fungal vacuolar membrane reduced apoptosis levels in yeast and increased resistance to amine fungicides. Consistently, A. thaliana avp1 mutant seedlings showed increased susceptibility to this amine fungicide, particularly at the level of root development. This is in agreement with AVP1 being nearly the sole H(+)-pyrophosphatase gene expressed at the root elongation zones. All in all, the present data suggest that H(+)-pyrophosphatases are major determinants of plant tolerance to amine fungicides.

Fusion Rates of Different Anterior Grafts in Thoracolumbar Fractures.

PubMed

Antoni, Maxime; Charles, Yann Philippe; Walter, Axel; Schuller, Sébastien; Steib, Jean-Paul

2015-11-01

Retrospective CT analysis of anterior fusion in thoracolumbar trauma. The aim of this study was to compare fusion rates of different bone grafts and to analyze risk factors for pseudarthrosis. Interbody fusion is indicated in anterior column defects. Different grafts are used: autologous iliac crest, titanium mesh cages filled with cancellous bone, and autologous ribs. It is not clear which graft offers the most reliable fusion. Radiologic data of 116 patients (71 men, 45 women) operated for type A2, A3, B, or C fractures were analyzed. The average age was 44.6 years (range, 16-75 y) and follow-up was 2.7 years (range, 1-9 y). All patients were treated by posterior instrumentation followed by an anterior graft: 53 cases with iliac crest, 43 cases with mesh cages, and 20 with rib grafts. Fusion was evaluated on CT and classified into complete fusion, partial fusion, unipolar pseudarthrosis, and bipolar pseudarthrosis. Iliac crest fused in 66%, cages in 98%, and rib grafts in 90%. The fusion rate of cages filled with bone was significantly higher as the iliac graft fusion rate (P=0.002). The same was applied to rib grafts compared with iliac crest (P=0.041). Additional bone formation around the main graft, bridging both vertebral bodies, was observed in 31 of the 53 iliac crests grafts. Pseudarthrosis occurred more often in smokers (P=0.042). A relationship between fracture or instrumentation types, sex, age, BMI, and fusion could not be determined. Tricortical iliac crest grafts showed an unexpected high pseudarthrosis rate in thoracolumbar injuries. Their cortical bone is dense and their fusion surface is small. Rib grafts led to a better fusion when used in combination with the cancellous bone from the fractured vertebral body. Titanium mesh cages filled with cancellous bone led to the highest fusion rate and built a complete bony bridge between vertebral bodies. Smoking seemed to influence fusion. Case control study, Level III.

Vacuolar sequestration capacity and long-distance metal transport in plants

PubMed Central

Peng, Jia-Shi; Gong, Ji-Ming

2014-01-01

The vacuole is a pivotal organelle functioning in storage of metabolites, mineral nutrients, and toxicants in higher plants. Accumulating evidence indicates that in addition to its storage role, the vacuole contributes essentially to long-distance transport of metals, through the modulation of Vacuolar sequestration capacity (VSC) which is shown to be primarily controlled by cytosolic metal chelators and tonoplast-localized transporters, or the interaction between them. Plants adapt to their environments by dynamic regulation of VSC for specific metals and hence targeting metals to specific tissues. Study of VSC provides not only a new angle to understand the long-distance root-to-shoot transport of minerals in plants, but also an efficient way to biofortify essential mineral nutrients or to phytoremediate non-essential metal pollution. The current review will focus on the most recent proceedings on the interaction mechanisms between VSC regulation and long-distance metal transport. PMID:24550927

Vacuolar sequestration capacity and long-distance metal transport in plants.

PubMed

Peng, Jia-Shi; Gong, Ji-Ming

2014-01-01

The vacuole is a pivotal organelle functioning in storage of metabolites, mineral nutrients, and toxicants in higher plants. Accumulating evidence indicates that in addition to its storage role, the vacuole contributes essentially to long-distance transport of metals, through the modulation of Vacuolar sequestration capacity (VSC) which is shown to be primarily controlled by cytosolic metal chelators and tonoplast-localized transporters, or the interaction between them. Plants adapt to their environments by dynamic regulation of VSC for specific metals and hence targeting metals to specific tissues. Study of VSC provides not only a new angle to understand the long-distance root-to-shoot transport of minerals in plants, but also an efficient way to biofortify essential mineral nutrients or to phytoremediate non-essential metal pollution. The current review will focus on the most recent proceedings on the interaction mechanisms between VSC regulation and long-distance metal transport.

[Image fusion of gated-SPECT and CT angiography in coronary artery disease. Importance of anatomic-functional correlation].

PubMed

Nazarena Pizzi, M; Aguadé Bruix, S; Cuéllar Calabria, H; Aliaga, V; Candell Riera, J

2010-01-01

A 77-year old patient was admitted for acute coronary syndrome without ST elevation. His risk was stratified using the myocardial perfusion gated SPECT, mild inferior ischemia being observed. Thus, medical therapy was optimized and the patient was discharged. He continued with exertional dyspnea so a coronary CT angiography was performed. It revealed severe lesions in the proximal RCA. SPECT-CT fusion images correlated the myocardial perfusion defect with a posterior descending artery from the RCA, in a co-dominant coronary area. Subsequently, cardiac catheterism was indicated for his treatment. The current use of image fusion studies is limited to patients in whom it is difficult to attribute a perfusion defect to a specific coronary artery. In our patient, the fusion images helped to distinguish between the RCA and the circumflex artery as the culprit artery of ischemia. Copyright © 2010 Elsevier España, S.L. y SEMNIM. All rights reserved.

Abscisic Acid Induction of Vacuolar H+-ATPase Activity in Mesembryanthemum crystallinum Is Developmentally Regulated1

PubMed Central

Barkla, Bronwyn J.; Vera-Estrella, Rosario; Maldonado-Gama, Minerva; Pantoja, Omar

1999-01-01

Abscisic acid (ABA) has been implicated as a key component in water-deficit-induced responses, including those triggered by drought, NaCl, and low- temperature stress. In this study a role for ABA in mediating the NaCl-stress-induced increases in tonoplast H+-translocating ATPase (V-ATPase) and Na+/H+ antiport activity in Mesembryanthemum crystallinum, leading to vacuolar Na+ sequestration, were investigated. NaCl or ABA treatment of adult M. crystallinum plants induced V-ATPase H+ transport activity, and when applied in combination, an additive effect on V-ATPase stimulation was observed. In contrast, treatment of juvenile plants with ABA did not induce V-ATPase activity, whereas NaCl treatment resulted in a similar response to that observed in adult plants. Na+/H+ antiport activity was induced in both juvenile and adult plants by NaCl, but ABA had no effect at either developmental stage. Results indicate that ABA-induced changes in V-ATPase activity are dependent on the plant reaching its adult phase, whereas NaCl-induced increases in V-ATPase and Na+/H+ antiport activity are independent of plant age. This suggests that ABA-induced V-ATPase activity may be linked to the stress-induced, developmentally programmed switch from C3 metabolism to Crassulacean acid metabolism in adult plants, whereas, vacuolar Na+ sequestration, mediated by the V-ATPase and Na+/H+ antiport, is regulated through ABA-independent pathways. PMID:10398716

Vacuolar amino acid transporter Avt5p is responsible for lithium uptake in Schizosaccharomyces pombe.

PubMed

Iwaki, Tomoko; Sekito, Takayuki; Kakinuma, Yoshimi

2010-01-01

The fission yeast Schizosaccharomyces pombe was sensitive to salinity; cell growth was stopped by 0.5 M NaCl and by 10 mM LiCl. The avt5+ gene encodes a vacuolar transporter with a broad specificity for amino acids. We found that the avt5Delta mutant became highly tolerant of Li+ and Na+ in growth. Concanamycin A-sensitive Li+ uptake as well as cellular Li+ content was lower in the avt5 mutant, suggesting a role of Avt5p in cellular uptake of toxic Li+.

MTV1 and MTV4 encode plant-specific ENTH and ARF GAP proteins that mediate clathrin-dependent trafficking of vacuolar cargo from the trans-Golgi network.

PubMed

Sauer, Michael; Delgadillo, M Otilia; Zouhar, Jan; Reynolds, Gregory D; Pennington, Janice G; Jiang, Liwen; Liljegren, Sarah J; Stierhof, York-Dieter; De Jaeger, Geert; Otegui, Marisa S; Bednarek, Sebastian Y; Rojo, Enrique

2013-06-01

Many soluble proteins transit through the trans-Golgi network (TGN) and the prevacuolar compartment (PVC) en route to the vacuole, but our mechanistic understanding of this vectorial trafficking step in plants is limited. In particular, it is unknown whether clathrin-coated vesicles (CCVs) participate in this transport step. Through a screen for modified transport to the vacuole (mtv) mutants that secrete the vacuolar protein VAC2, we identified MTV1, which encodes an epsin N-terminal homology protein, and MTV4, which encodes the ADP ribosylation factor GTPase-activating protein nevershed/AGD5. MTV1 and NEV/AGD5 have overlapping expression patterns and interact genetically to transport vacuolar cargo and promote plant growth, but they have no apparent roles in protein secretion or endocytosis. MTV1 and NEV/AGD5 colocalize with clathrin at the TGN and are incorporated into CCVs. Importantly, mtv1 nev/agd5 double mutants show altered subcellular distribution of CCV cargo exported from the TGN. Moreover, MTV1 binds clathrin in vitro, and NEV/AGD5 associates in vivo with clathrin, directly linking these proteins to CCV formation. These results indicate that MTV1 and NEV/AGD5 are key effectors for CCV-mediated trafficking of vacuolar proteins from the TGN to the PVC in plants.

Nuclear inner membrane fusion facilitated by yeast Jem1p is required for spindle pole body fusion but not for the first mitotic nuclear division during yeast mating.

PubMed

Nishikawa, Shuh-ichi; Hirata, Aiko; Endo, Toshiya

2008-11-01

During mating of budding yeast, Saccharomyces cerevisiae, two haploid nuclei fuse to produce a diploid nucleus. The process of nuclear fusion requires two J proteins, Jem1p in the endoplasmic reticulum (ER) lumen and Sec63p, which forms a complex with Sec71p and Sec72p, in the ER membrane. Zygotes of mutants defective in the functions of Jem1p or Sec63p contain two haploid nuclei that were closely apposed but failed to fuse. Here we analyzed the ultrastructure of nuclei in jem1 Delta and sec71 Delta mutant zygotes using electron microscope with the freeze-substituted fixation method. Three-dimensional reconstitution of nuclear structures from electron microscope serial sections revealed that Jem1p facilitates nuclear inner-membrane fusion and spindle pole body (SPB) fusion while Sec71p facilitates nuclear outer-membrane fusion. Two haploid SPBs that failed to fuse could duplicate, and mitotic nuclear division of the unfused haploid nuclei started in jem1 Delta and sec71 Delta mutant zygotes. This observation suggests that nuclear inner-membrane fusion is required for SPB fusion, but not for SPB duplication in the first mitotic cell division.

A fluorescent reporter protein containing AtRMR1 domains is targeted to the storage and central vacuoles in Arabidopsis thaliana and tobacco leaf cells.

PubMed

Scabone, Camila María; Frigerio, Lorenzo; Petruccelli, Silvana

2011-10-01

To develop a new strategy to target recombinant proteins to the vacuolar storage system in transgenic plants, the ability of the transmembrane and cytosolic domains of Arabidopsis receptor homology-transmembrane-RING H2-1 (AtRMR1) was evaluated. A secreted version of RFP (secRFP) and a fusion of it to the transmembrane and cytosolic domains of AtRMR1 (RFP-TMCT) were produced and studied both in transient and stable expression assays. Transient expression in leaves of Nicotiana tabacum showed that secRFP is secreted to the apoplast while its fusion to TMCT of AtRMR1 is sufficient to prevent secretion of the reporter. In tobacco leaves, RFP-TMCT reporter showed an endoplasmic reticulum pattern in early expression stages while in late expression stages, it was found in the vacuolar lumen. For the first time, the role of TM and CT domains of AtRMR1 in stable expression in Arabidopsis thaliana is presented; the fusion of TMCT to secRFP is sufficient to sort RFP to the lumen of the central vacuoles in leaves and roots and to the lumen of PSV in cotyledons of mature embryos. In addition, biochemical studies performed in extract from transgenic plants showed that RFP-TMCT is an integral membrane protein. Full-length RFP-TMCT was also found in the vacuolar lumen, suggesting internalization into destination vacuole. Not colocalization of RFP-TMCT with tonoplast and plasma membrane markers were observed. This membrane vacuolar determinant sorting signal could be used for future application in molecular pharming as an alternative means to sort proteins of interest to vacuoles.

Delayed Induction of Human NTE (PNPLA6) Rescues Neurodegeneration and Mobility Defects of Drosophila swiss cheese (sws) Mutants.

PubMed

Sujkowski, Alyson; Rainier, Shirley; Fink, John K; Wessells, Robert J

2015-01-01

Human PNPLA6 gene encodes Neuropathy Target Esterase protein (NTE). PNPLA6 gene mutations cause hereditary spastic paraplegia (SPG39 HSP), Gordon-Holmes syndrome, Boucher-Neuhäuser syndromes, Laurence-Moon syndrome, and Oliver-McFarlane syndrome. Mutations in the Drosophila NTE homolog swiss cheese (sws) cause early-onset, progressive behavioral defects and neurodegeneration characterized by vacuole formation. We investigated sws5 flies and show for the first time that this allele causes progressive vacuolar formation in the brain and progressive deterioration of negative geotaxis speed and endurance. We demonstrate that inducible, neuron-specific expression of full-length human wildtype NTE reduces vacuole formation and substantially rescues mobility. Indeed, neuron-specific expression of wildtype human NTE is capable of rescuing mobility defects after 10 days of adult life at 29°C, when significant degeneration has already occurred, and significantly extends longevity of mutants at 25°C. These results raise the exciting possibility that late induction of NTE function may reduce or ameliorate neurodegeneration in humans even after symptoms begin. In addition, these results highlight the utility of negative geotaxis endurance as a new assay for longitudinal tracking of degenerative phenotypes in Drosophila.

Stability of concentration-related self-interstitial atoms in fusion material tungsten

NASA Astrophysics Data System (ADS)

Hong, Zhang; Shu-Long, Wen; Min, Pan; Zheng, Huang; Yong, Zhao; Xiang, Liu; Ji-Ming, Chen

2016-05-01

Based on the density functional theory, we calculated the structures of the two main possible self-interstitial atoms (SIAs) as well as the migration energy of tungsten (W) atoms. It was found that the difference of the <110> and <111> formation energies is 0.05-0.3 eV. Further analysis indicated that the stability of SIAs is closely related to the concentration of the defect. When the concentration of the point defect is high, <110> SIAs are more likely to exist, <111> SIAs are the opposite. In addition, the vacancy migration probability and self-recovery zones for these SIAs were researched by making a detailed comparison. The calculation provided a new viewpoint about the stability of point defects for self-interstitial configurations and would benefit the understanding of the control mechanism of defect behavior for this novel fusion material. Project supported by the Fundamental Research Funds for the Central Universities of Ministry of Education of China (Grant Nos. A0920502051411-5 and 2682014ZT30), the Program of International Science and Technology Cooperation, China (Grant No. 2013DFA51050), the National Magnetic Confinement Fusion Science Program, China (Grant Nos. 2011GB112001 and 2013GB110001), the National High Technology Research and Development Program of China (Grant No. 2014AA032701), the National Natural Science Foundation of China (Grant No. 11405138), the Southwestern Institute of Physics Funds, China, the Western Superconducting Technologies Company Limited, China, the Qingmiao Plan of Southwest Jiaotong University, China (Grant No. A0920502051517-6), and the China Postdoctoral Science Foundation (Grant No. 2014M560813).

The emerging structure of vacuolar ATPases.

PubMed

Drory, Omri; Nelson, Nathan

2006-10-01

Bioenergetics and physiology of primary pumps have been revitalized by new insights into the mechanism of energizing biomembranes. Structural information is becoming available, and the three-dimensional structure of F-ATPase is being resolved. The growing understanding of the fundamental mechanism of energy coupling may revolutionize our view of biological processes. The F- and V-ATPases (vacuolar-type ATPase) exhibit a common mechanical design in which nucleotide-binding on the catalytic sector, through a cycle of conformation changes, drives the transmembrane passage of protons by turning a membrane-embedded rotor. This motor can run in forward or reverse directions, hydrolyzing ATP as it pumps protons uphill or creating ATP as protons flow downhill. In contrast to F-ATPases, whose primary function in eukaryotic cells is to form ATP at the expense of the proton-motive force (pmf), V-ATPases function exclusively as an ATP-dependent proton pump. The pmf generated by V-ATPases in organelles and membranes of eukaryotic cells is utilized as a driving force for numerous secondary transport processes. V- and F-ATPases have similar structure and mechanism of action, and several of their subunits evolved from common ancestors. Electron microscopy studies of V-ATPase revealed its general structure at low resolution. Recently, several structures of V-ATPase subunits, solved by X-ray crystallography with atomic resolution, were published. This, together with electron microscopy low-resolution maps of the whole complex, and biochemistry cross-linking experiments, allows construction of a structural model for a part of the complex that may be used as a working hypothesis for future research.

Detecting wood surface defects with fusion algorithm of visual saliency and local threshold segmentation

NASA Astrophysics Data System (ADS)

Wang, Xuejuan; Wu, Shuhang; Liu, Yunpeng

2018-04-01

This paper presents a new method for wood defect detection. It can solve the over-segmentation problem existing in local threshold segmentation methods. This method effectively takes advantages of visual saliency and local threshold segmentation. Firstly, defect areas are coarsely located by using spectral residual method to calculate global visual saliency of them. Then, the threshold segmentation of maximum inter-class variance method is adopted for positioning and segmenting the wood surface defects precisely around the coarse located areas. Lastly, we use mathematical morphology to process the binary images after segmentation, which reduces the noise and small false objects. Experiments on test images of insect hole, dead knot and sound knot show that the method we proposed obtains ideal segmentation results and is superior to the existing segmentation methods based on edge detection, OSTU and threshold segmentation.

Polar-Direct-Drive Defect Implosions at OMEGA inPreparation for Experiments at NIF

NASA Astrophysics Data System (ADS)

Cobble, J. A.; Schmitt, M. J.; Murphy, T. J.; Tregillis, I. L.; Wysocki, F. J.; Obrey, K. D.; Magelssen, G. R.; Glebov, V.; Bradley, P. A.; Hsu, S. C.; Krasheninnikova, N. V.; Batha, S. H.

2011-10-01

The Defect-Implosion (DIME) campaign involves compressing perturbed spherical capsules with polar direct drive (PDD). For direct-drive implosions at NIF, PDD will be used. We have done simulations and experiments at OMEGA to test our modeling capability for equatorial-plane defects in fusion capsules and for PDD at NIF. Since PDD is anisotropic, we show the results of 0th hydrodynamics of implosions and perturbation-driven features near stagnation. Later presentations discuss defect-induced mix and neutronics, and laser pointing for NIF experiments. Prototype OMEGA shots used 865- μm diameter CH shells filled with 5 atm of D2. Machined channels 30- μm wide and up to 9- μm deep formed the defects. This work has been performed under the auspices of the US DOE, contract number DE-AC52-06NA25396.

Vacuolar Protein Sorting Genes in Parkinson's Disease: A Re-appraisal of Mutations Detection Rate and Neurobiology of Disease.

PubMed

Gambardella, Stefano; Biagioni, Francesca; Ferese, Rosangela; Busceti, Carla L; Frati, Alessandro; Novelli, Giuseppe; Ruggieri, Stefano; Fornai, Francesco

2016-01-01

Mammalian retromers play a critical role in protein trans-membrane sorting from endosome to the trans-Golgi network (TGN). Recently, retromer alterations have been related to the onset of Parkinson's Disease (PD) since the variant p.Asp620Asn in VPS35 (Vacuolar Protein Sorting 35) was identified as a cause of late onset PD. This variant causes a primary defect in endosomal trafficking and retromers formation. Other mutations in VPS genes have been reported in both sporadic and familial PD. These mutations are less defined. Understanding the specific prevalence of all VPS gene mutations is key to understand the relevance of retromers impairment in the onset of PD. A number of PD-related mutations despite affecting different biochemical systems (autophagy, mitophagy, proteasome, endosomes, protein folding), all converge in producing an impairment in cell clearance. This may explain how genetic predispositions to PD may derive from slightly deleterious VPS mutations when combined with environmental agents overwhelming the clearance of the cell. This manuscript reviews genetic data produced in the last 5 years to re-define the actual prevalence of VPS gene mutations in the onset of PD. The prevalence of p.Asp620Asn mutation in VPS35 is 0.286 of familial PD. This increases up to 0.548 when considering mutations affecting all VPS genes. This configures mutations in VPS genes as the second most frequent autosomal dominant PD genotype. This high prevalence, joined with increased awareness of the role played by retromers in the neurobiology of PD, suggests environmentally-induced VPS alterations as crucial in the genesis of PD.

FIRST-PRINCIPLES CALCULATIONS OF INTRINSIC DEFECTS AND Mg TRANSMUTANTS IN 3C-SiC

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

Hu, Shenyang Y.; Setyawan, Wahyu; Van Ginhoven, Renee M.

2013-09-25

Silicon carbide (SiC) possesses many desirable attributes for applications in high-temperature and neutron radiation environments. These attributes include excellent dimensional and thermodynamic stability, low activation, high strength, and high thermal conductivity. Therefore, SiC based materials draw broad attention as structural materials for the first wall (FW) and blanket in fusion power plants. Under the severe high-energy neutron environment of D-T fusion systems, SiC suffers significant transmutation resulting in both gaseous and metallic transmutants. Recent calculations by Sawan, et al. [2] predict that at a fast neutron dose of ~100 dpa, there will be about 0.5 at% Mg generated in SiCmore » through nuclear transmutation. Other transmutation products, including 0.15 at% Al, 0.2 at% Be and 2.2 at% He, also emerge. Formation and migration energies of point defects in 3C-SiC have been widely investigated using density functional theory (DFT). However, the properties of defects associated with transmutants are currently not well understood. Fundamental understanding of where the transmutation products go and how they affect microstructure evolution of SiC composites will help to predict property evolution and performance of SiC-based materials in fusion reactors.« less

Epizootiologic studies of avian vacuolar myelinopathy in waterbirds

USGS Publications Warehouse

Rocke, Tonie E.; Thomas, N.J.; Augspurger, T.; Miller, Kimberli J.

2002-01-01

Epizootic avian vacuolar myelinopathy (AVM) was first recognized as a neurologic disease in bald eagles (Haliaeetus leucocephalus) and American coots (Fulica americana) in Arkansas, USA in 1994 and 1996, respectively, but attempts to identify the etiology of the disease have been unsuccessful to date. Between 1998 and 2001, wing clipped sentinel birds (wild American coots and game farm mallards [Anas platyrhynchos]) were released at Lake Surf, North Carolina, a lake with recurrent outbreaks of AVM, in order to gain a better understanding of the epizootiology of the disease. As early as 5-7 days post-release, sentinel coots and mallards showed neurologic signs of disease and were confirmed with AVM upon histologic examination of their brains. Serial releases of sentinel mallards during the summer, fall, and winter of 2000-01 demonstrated that exposure to the causative agent at a threshold sufficient to manifest disease was seasonal and occurred over about a 2 mo period, during November and December. Our findings that disease onset can be very rapid (5-7 days) and that exposure to the causative agent of AVM is site-specific, seasonal (late fall to early winter), and occurs over a relatively short duration (several months) supports the hypothesis that the disease is caused by a chemical substance, most likely of natural origin.

Helium bubbles aggravated defects production in self-irradiated copper

NASA Astrophysics Data System (ADS)

Wu, FengChao; Zhu, YinBo; Wu, Qiang; Li, XinZhu; Wang, Pei; Wu, HengAn

2017-12-01

Under the environment of high radiation, materials used in fission and fusion reactors will internally accumulate numerous lattice defects and bubbles. With extensive studies focused on bubble resolution under irradiation, the mutually effects between helium bubbles and displacement cascades in irradiated materials remain unaddressed. Therefore, the defects production and microstructure evolution under self-irradiation events in vicinity of helium bubbles are investigated by preforming large scale molecular dynamics simulations in single-crystal copper. When subjected to displacement cascades, distinguished bubble resolution categories dependent on bubble size are observed. With the existence of bubbles, radiation damage is aggravated with the increasing bubble size, represented as the promotion of point defects and dislocations. The atomic mechanisms of heterogeneous dislocation structures are attributed to different helium-vacancy cluster modes, transforming from the resolved gas trapped with vacancies to the biased absorption of vacancies by the over-pressured bubble. In both cases, helium impedes the recombination of point defects, leading to the accelerated formation of interstitial loops. The results and insight obtained here might contribute to understand the underlying mechanism of transmutant solute on the long-term evolution of irradiated materials.

Activity of the C-terminal-dependent vacuolar sorting signal of horseradish peroxidase C1a is enhanced by its secondary structure.

PubMed

Matsui, Takeshi; Tabayashi, Ayako; Iwano, Megumi; Shinmyo, Atsuhiko; Kato, Ko; Nakayama, Hideki

2011-02-01

Plant class III peroxidase (PRX) catalyzes the oxidation and oxidative polymerization of a variety of phenolic compounds while reducing hydrogen peroxide. PRX proteins are classified into apoplast type and vacuole type based on the absence or the presence of C-terminal propeptides, which probably function as vacuolar sorting signals (VSSs). In this study, in order to improve our understanding of vacuole-type PRX, we analyzed regulatory mechanisms of vacuolar sorting of a model vacuole-type PRX, the C1a isozyme of horseradish (Armoracia rusticana) (HRP C1a). Using cultured transgenic tobacco cells and protoplasts derived from horseradish leaves, we characterized HRP C1a's VSS, which is a 15 amino acid C-terminal propeptide (C15). We found that the C-terminal hexapeptide of C15 (C6), which is well conserved among vacuole-type PRX proteins, forms the core of the C-terminal-dependent VSS. We also found that the function of C6 is enhanced by the remaining N-terminal part of C15 which probably folds into an amphiphilic α-helix.

Vacuolar SPX-MFS transporters are essential for phosphate adaptation in plants.

PubMed

Liu, Jinlong; Fu, Shaomin; Yang, Lei; Luan, Mingda; Zhao, Fugeng; Luan, Sheng; Lan, Wenzhi

2016-08-02

To survive in most soils in which inorganic phosphate (Pi) levels are limited and constantly changing, plants universally use the vacuoles as cellular Pi "sink" and "source" to maintain Pi homeostasis. However, the transporters that mediate Pi sequestration into the vacuoles remain unknown. Recently, we and other 2 groups independently identified the members of SPS-MSF family as the candidates for tonoplast Pi transporters in Arabidopsis thaliana and Oryza sativa. We and Liu et al. demonstrated that one of SPS-MSF member, VPT1 (Vacuolar Phosphate Transporter 1), also named as PHT5;1 (Phosphate Transporter 5;1), plays a predominant role in Pi sequestration of vacuoles in Arabidopsis. Here we show that vpt1 mutants and VPT1-GFP overexpressing lines displayed sensitive to Pi stress under the hydroponic system containing the medium with low iron, supporting that VPT1 is essential for Arabidopsis to adapt phosphate stress.

Bone bridge formation across the neuroforamen 14 years after instrumented fusion for isthmic spondylolisthesis—a case report

PubMed Central

Lim, Joel Louis; Tan, Kimberly-Anne

2017-01-01

This case report describes the first case of a bone bridge formation across the left L5/S1 neuroforamen after instrumented posterolateral fusion for L5/S1 isthmic spondylolisthesis. Our patient was a 70-year-old lady who had grade 2, L5/S1 isthmic spondylolisthesis and bilateral S1 nerve root compression. She suffered from mechanical low back pain and neurogenic claudication, with radicular pain over both S1 dermatomes. She underwent in-situ, instrumented, posterolateral fusion and was asymptomatic for more than 13 years before developing progressive onset of left radicular pain over the L5 dermatome. Imaging revealed a bisected left L5/S1 neuroforamen secondary to a bone bridge formation resulting in stenosis. The pars defect in this case may have had sufficient osteogenic and osteoinductive factors to heal following spinal stabilization. Although in-situ posterolateral fusion is an accepted surgical treatment for isthmic spondylolisthesis, surgeons should consider reduction of the spondylolisthesis and excision of the pars defects to avoid this possible long-term complication. PMID:28435923

Bone bridge formation across the neuroforamen 14 years after instrumented fusion for isthmic spondylolisthesis-a case report.

PubMed

Lim, Joel Louis; Tan, Kimberly-Anne; Hey, Hwee Weng Dennis

2017-03-01

This case report describes the first case of a bone bridge formation across the left L5/S1 neuroforamen after instrumented posterolateral fusion for L5/S1 isthmic spondylolisthesis. Our patient was a 70-year-old lady who had grade 2, L5/S1 isthmic spondylolisthesis and bilateral S1 nerve root compression. She suffered from mechanical low back pain and neurogenic claudication, with radicular pain over both S1 dermatomes. She underwent in-situ, instrumented, posterolateral fusion and was asymptomatic for more than 13 years before developing progressive onset of left radicular pain over the L5 dermatome. Imaging revealed a bisected left L5/S1 neuroforamen secondary to a bone bridge formation resulting in stenosis. The pars defect in this case may have had sufficient osteogenic and osteoinductive factors to heal following spinal stabilization. Although in-situ posterolateral fusion is an accepted surgical treatment for isthmic spondylolisthesis, surgeons should consider reduction of the spondylolisthesis and excision of the pars defects to avoid this possible long-term complication.

Defect detection of helical gears based on time-frequency analysis and using multi-layer fusion network

NASA Astrophysics Data System (ADS)

Ebrahimi Orimi, H.; Esmaeili, M.; Refahi Oskouei, A.; Mirhadizadehd, S. A.; Tse, P. W.

2017-10-01

Condition monitoring of rotary devices such as helical gears is an issue of great significance in industrial projects. This paper introduces a feature extraction method for gear fault diagnosis using wavelet packet due to its higher frequency resolution. During this investigation, the mother wavelet Daubechies 10 (Db-10) was applied to calculate the coefficient entropy of each frequency band of 5th level (32 frequency bands) as features. In this study, the peak value of the signal entropies was selected as applicable features in order to improve frequency band differentiation and reduce feature vectors' dimension. Feature extraction is followed by the fusion network where four different structured multi-layer perceptron networks are trained to classify the recorded signals (healthy/faulty). The robustness of fusion network outputs is greater compared to perceptron networks. The results provided by the fusion network indicate a classification of 98.88 and 97.95% for healthy and faulty classes, respectively.

Defect evolution in single crystalline tungsten following low temperature and low dose neutron irradiation

DOE PAGES

Hu, Xunxiang; Koyanagi, Takaaki; Fukuda, Makoto; ...

2016-01-01

The tungsten plasma-facing components of fusion reactors will experience an extreme environment including high temperature, intense particle fluxes of gas atoms, high-energy neutron irradiation, and significant cyclic stress loading. Irradiation-induced defect accumulation resulting in severe thermo-mechanical property degradation is expected. For this reason, and because of the lack of relevant fusion neutron sources, the fundamentals of tungsten radiation damage must be understood through coordinated mixed-spectrum fission reactor irradiation experiments and modeling. In this study, high-purity (110) single-crystal tungsten was examined by positron annihilation spectroscopy and transmission electron microscopy following low-temperature (~90 °C) and low-dose (0.006 and 0.03 dpa) mixed-spectrum neutronmore » irradiation and subsequent isochronal annealing at 400, 500, 650, 800, 1000, 1150, and 1300 °C. The results provide insights into microstructural and defect evolution, thus identifying the mechanisms of different annealing behavior. Following 1 h annealing, ex situ characterization of vacancy defects using positron lifetime spectroscopy and coincidence Doppler broadening was performed. The vacancy cluster size distributions indicated intense vacancy clustering at 400 °C with significant damage recovery around 1000 °C. Coincidence Doppler broadening measurements confirm the trend of the vacancy defect evolution, and the S–W plots indicate that only a single type of vacancy cluster is present. Furthermore, transmission electron microscopy observations at selected annealing conditions provide supplemental information on dislocation loop populations and visible void formation. This microstructural information is consistent with the measured irradiation-induced hardening at each annealing stage. This provides insight into tungsten hardening and embrittlement due to irradiation-induced matrix defects.« less

A method based on IHS cylindrical transform model for quality assessment of image fusion

NASA Astrophysics Data System (ADS)

Zhu, Xiaokun; Jia, Yonghong

2005-10-01

Image fusion technique has been widely applied to remote sensing image analysis and processing, and methods for quality assessment of image fusion in remote sensing have also become the research issues at home and abroad. Traditional assessment methods combine calculation of quantitative indexes and visual interpretation to compare fused images quantificationally and qualitatively. However, in the existing assessment methods, there are two defects: on one hand, most imdexes lack the theoretic support to compare different fusion methods. On the hand, there is not a uniform preference for most of the quantitative assessment indexes when they are applied to estimate the fusion effects. That is, the spatial resolution and spectral feature could not be analyzed synchronously by these indexes and there is not a general method to unify the spatial and spectral feature assessment. So in this paper, on the basis of the approximate general model of four traditional fusion methods, including Intensity Hue Saturation(IHS) triangle transform fusion, High Pass Filter(HPF) fusion, Principal Component Analysis(PCA) fusion, Wavelet Transform(WT) fusion, a correlation coefficient assessment method based on IHS cylindrical transform is proposed. By experiments, this method can not only get the evaluation results of spatial and spectral features on the basis of uniform preference, but also can acquire the comparison between fusion image sources and fused images, and acquire differences among fusion methods. Compared with the traditional assessment methods, the new methods is more intuitionistic, and in accord with subjective estimation.

Failure to transmit avian vacuolar myelinopathy to mallard ducks

USGS Publications Warehouse

Larsen, R.S.; Nutter, F.B.; Augspurger, T.; Rocke, T.E.; Thomas, N.J.; Stoskopf, M.K.

2003-01-01

Avian vacuolar myelinopathy (AVM) is a neurologic disease that has been diagnosed in free-ranging birds in the southeastern United States. Bald eagles (Haliaeetus leuocephalus), American coots (Fulica americana), and mallards (Anas platyrhynchos) have been affected. Previous investigations have not determined the etiology of this disease. In November and December 2002, we attempted to induce AVM in game-farmed mallards through four, 7-day exposure trials. Mallards were housed in six groups of eight, with two of these groups serving as controls. One group was housed with AVM-affected coots; one group was tube fed daily with water from the lake where affected coots were captured; one group was tube fed daily with aquatic vegetation (Hydrilla verticillata) from the same lake; and another group was tube fed daily with sediment from the lake. No ducks exhibited clinical neurologic abnormalities consistent with AVM and no evidence of AVM was present at histopathologic examination of brain tissue. Although limitations in sample size, quantity of individual doses, frequency of dose administration, duration of exposure, and timing of these trials restrict the interpretation of the findings, AVM was not readily transmitted by direct contact, water, hydrilla, or sediment in this investigation.

Involvement of Vacuolar Sequestration and Active Transport in Tolerance of Saccharomyces cerevisiae to Hop Iso-α-Acids▿ † ¶

PubMed Central

Hazelwood, Lucie A.; Walsh, Michael C.; Pronk, Jack T.; Daran, Jean-Marc

2010-01-01

The hop plant, Humulus lupulus L., has an exceptionally high content of secondary metabolites, the hop α-acids, which possess a range of beneficial properties, including antiseptic action. Studies performed on the mode of action of hop iso-α-acids have hitherto been restricted to lactic acid bacteria. The present study investigated molecular mechanisms of hop iso-α-acid resistance in the model eukaryote Saccharomyces cerevisiae. Growth inhibition occurred at concentrations of hop iso-α-acids that were an order of magnitude higher than those found with hop-tolerant prokaryotes. Chemostat-based transcriptome analysis and phenotype screening of the S. cerevisiae haploid gene deletion collection were used as complementary methods to screen for genes involved in hop iso-α-acid detoxification and tolerance. This screening and further analysis of deletion mutants confirmed that yeast tolerance to hop iso-α-acids involves three major processes, active proton pumping into the vacuole by the vacuolar-type ATPase to enable vacuolar sequestration of iso-α-acids and alteration of cell wall structure and, to a lesser extent, active export of iso-α-acids across the plasma membrane. Furthermore, iso-α-acids were shown to affect cellular metal homeostasis by acting as strong zinc and iron chelators. PMID:19915041

Calcium-Dependent Rubella Virus Fusion Occurs in Early Endosomes.

PubMed

Dubé, Mathieu; Etienne, Loïc; Fels, Maximilian; Kielian, Margaret

2016-07-15

The E1 membrane protein of rubella virus (RuV) is a class II membrane fusion protein structurally related to the fusion proteins of the alphaviruses, flaviviruses, and phleboviruses. Virus entry is mediated by a low pH-dependent fusion reaction through E1's insertion into the cell membrane and refolding to a stable homotrimer. Unlike the other described class II proteins, RuV E1 contains 2 fusion loops, which complex a metal ion between them by interactions with residues N88 and D136. Insertion of the E1 protein into the target membrane, fusion, and infection require calcium and are blocked by alanine substitution of N88 or D136. Here we addressed the requirements of E1 for calcium binding and the intracellular location of the calcium requirement during virus entry. Our results demonstrated that N88 and D136 are optimally configured to support RuV fusion and are strongly selected for during the virus life cycle. While E1 has some similarities with cellular proteins that bind calcium and anionic lipids, RuV binding to the membrane was independent of anionic lipids. Virus fusion occurred within early endosomes, and chelation of intracellular calcium showed that calcium within the early endosome was required for virus fusion and infection. Calcium triggered the reversible insertion of E1 into the target membrane at neutral pH, but E1 homotrimer formation and fusion required a low pH. Thus, RuV E1, unlike other known class II fusion proteins, has distinct triggers for membrane insertion and fusion protein refolding mediated, respectively, by endosomal calcium and low pH. Rubella virus causes a mild disease of childhood, but infection of pregnant women frequently results in miscarriage or severe birth defects. In spite of an effective vaccine, RuV disease remains a serious problem in many developing countries. RuV infection of host cells involves endocytic uptake and low pH-triggered membrane fusion and is unusual in its requirement for calcium binding by the membrane

Calcium-Dependent Rubella Virus Fusion Occurs in Early Endosomes

PubMed Central

Dubé, Mathieu; Etienne, Loïc; Fels, Maximilian

2016-01-01

ABSTRACT The E1 membrane protein of rubella virus (RuV) is a class II membrane fusion protein structurally related to the fusion proteins of the alphaviruses, flaviviruses, and phleboviruses. Virus entry is mediated by a low pH-dependent fusion reaction through E1's insertion into the cell membrane and refolding to a stable homotrimer. Unlike the other described class II proteins, RuV E1 contains 2 fusion loops, which complex a metal ion between them by interactions with residues N88 and D136. Insertion of the E1 protein into the target membrane, fusion, and infection require calcium and are blocked by alanine substitution of N88 or D136. Here we addressed the requirements of E1 for calcium binding and the intracellular location of the calcium requirement during virus entry. Our results demonstrated that N88 and D136 are optimally configured to support RuV fusion and are strongly selected for during the virus life cycle. While E1 has some similarities with cellular proteins that bind calcium and anionic lipids, RuV binding to the membrane was independent of anionic lipids. Virus fusion occurred within early endosomes, and chelation of intracellular calcium showed that calcium within the early endosome was required for virus fusion and infection. Calcium triggered the reversible insertion of E1 into the target membrane at neutral pH, but E1 homotrimer formation and fusion required a low pH. Thus, RuV E1, unlike other known class II fusion proteins, has distinct triggers for membrane insertion and fusion protein refolding mediated, respectively, by endosomal calcium and low pH. IMPORTANCE Rubella virus causes a mild disease of childhood, but infection of pregnant women frequently results in miscarriage or severe birth defects. In spite of an effective vaccine, RuV disease remains a serious problem in many developing countries. RuV infection of host cells involves endocytic uptake and low pH-triggered membrane fusion and is unusual in its requirement for calcium

Pollination in Nicotiana alata stimulates synthesis and transfer to the stigmatic surface of NaStEP, a vacuolar Kunitz proteinase inhibitor homologue

PubMed Central

Busot, Grethel Yanet; McClure, Bruce; Ibarra-Sánchez, Claudia Patricia; Jiménez-Durán, Karina; Vázquez-Santana, Sonia; Cruz-García, Felipe

2008-01-01

After landing on a wet stigma, pollen grains hydrate and germination generally occurs. However, there is no certainty of the pollen tube growth through the style to reach the ovary. The pistil is a gatekeeper that evolved in many species to recognize and reject the self-pollen, avoiding endogamy and encouraging cross-pollination. However, recognition is a complex process, and specific factors are needed. Here the isolation and characterization of a stigma-specific protein from N. alata, NaStEP (N. alata Stigma Expressed Protein), that is homologous to Kunitz-type proteinase inhibitors, are reported. Activity gel assays showed that NaStEP is not a functional serine proteinase inhibitor. Immunohistochemical and protein blot analyses revealed that NaStEP is detectable in stigmas of self-incompatible (SI) species N. alata, N. forgetiana, and N. bonariensis, but not in self-compatible (SC) species N. tabacum, N. plumbaginifolia, N. benthamiana, N. longiflora, and N. glauca. NaStEP contains the vacuolar targeting sequence NPIVL, and immunocytochemistry experiments showed vacuolar localization in unpollinated stigmas. After self-pollination or pollination with pollen from the SC species N. tabacum or N. plumbaginifolia, NaStEP was also found in the stigmatic exudate. The synthesis and presence in the stigmatic exudate of this protein was strongly induced in N. alata following incompatible pollination with N. tabacum pollen. The transfer of NaStEP to the stigmatic exudate was accompanied by perforation of the stigmatic cell wall, which appeared to release the vacuolar contents to the apoplastic space. The increase in NaStEP synthesis after pollination and its presence in the stigmatic exudates suggest that this protein may play a role in the early pollen–stigma interactions that regulate pollen tube growth in Nicotiana. PMID:18689443

Pollination in Nicotiana alata stimulates synthesis and transfer to the stigmatic surface of NaStEP, a vacuolar Kunitz proteinase inhibitor homologue.

PubMed

Busot, Grethel Yanet; McClure, Bruce; Ibarra-Sánchez, Claudia Patricia; Jiménez-Durán, Karina; Vázquez-Santana, Sonia; Cruz-García, Felipe

2008-01-01

After landing on a wet stigma, pollen grains hydrate and germination generally occurs. However, there is no certainty of the pollen tube growth through the style to reach the ovary. The pistil is a gatekeeper that evolved in many species to recognize and reject the self-pollen, avoiding endogamy and encouraging cross-pollination. However, recognition is a complex process, and specific factors are needed. Here the isolation and characterization of a stigma-specific protein from N. alata, NaStEP (N. alata Stigma Expressed Protein), that is homologous to Kunitz-type proteinase inhibitors, are reported. Activity gel assays showed that NaStEP is not a functional serine proteinase inhibitor. Immunohistochemical and protein blot analyses revealed that NaStEP is detectable in stigmas of self-incompatible (SI) species N. alata, N. forgetiana, and N. bonariensis, but not in self-compatible (SC) species N. tabacum, N. plumbaginifolia, N. benthamiana, N. longiflora, and N. glauca. NaStEP contains the vacuolar targeting sequence NPIVL, and immunocytochemistry experiments showed vacuolar localization in unpollinated stigmas. After self-pollination or pollination with pollen from the SC species N. tabacum or N. plumbaginifolia, NaStEP was also found in the stigmatic exudate. The synthesis and presence in the stigmatic exudate of this protein was strongly induced in N. alata following incompatible pollination with N. tabacum pollen. The transfer of NaStEP to the stigmatic exudate was accompanied by perforation of the stigmatic cell wall, which appeared to release the vacuolar contents to the apoplastic space. The increase in NaStEP synthesis after pollination and its presence in the stigmatic exudates suggest that this protein may play a role in the early pollen-stigma interactions that regulate pollen tube growth in Nicotiana.

Increased Activity of the Vacuolar Monosaccharide Transporter TMT1 Alters Cellular Sugar Partitioning, Sugar Signaling, and Seed Yield in Arabidopsis1[OA

PubMed Central

Wingenter, Karina; Schulz, Alexander; Wormit, Alexandra; Wic, Stefan; Trentmann, Oliver; Hoermiller, Imke I.; Heyer, Arnd G.; Marten, Irene; Hedrich, Rainer; Neuhaus, H. Ekkehard

2010-01-01

The extent to which vacuolar sugar transport activity affects molecular, cellular, and developmental processes in Arabidopsis (Arabidopsis thaliana) is unknown. Electrophysiological analysis revealed that overexpression of the tonoplast monosaccharide transporter TMT1 in a tmt1-2::tDNA mutant led to increased proton-coupled monosaccharide import into isolated mesophyll vacuoles in comparison with wild-type vacuoles. TMT1 overexpressor mutants grew faster than wild-type plants on soil and in high-glucose (Glc)-containing liquid medium. These effects were correlated with increased vacuolar monosaccharide compartmentation, as revealed by nonaqueous fractionation and by chlorophyllab-binding protein1 and nitrate reductase1 gene expression studies. Soil-grown TMT1 overexpressor plants respired less Glc than wild-type plants and only about half the amount of Glc respired by tmt1-2::tDNA mutants. In sum, these data show that TMT activity in wild-type plants limits vacuolar monosaccharide loading. Remarkably, TMT1 overexpressor mutants produced larger seeds and greater total seed yield, which was associated with increased lipid and protein content. These changes in seed properties were correlated with slightly decreased nocturnal CO2 release and increased sugar export rates from detached source leaves. The SUC2 gene, which codes for a sucrose transporter that may be critical for phloem loading in leaves, has been identified as Glc repressed. Thus, the observation that SUC2 mRNA increased slightly in TMT1 overexpressor leaves, characterized by lowered cytosolic Glc levels than wild-type leaves, provided further evidence of a stimulated source capacity. In summary, increased TMT activity in Arabidopsis induced modified subcellular sugar compartmentation, altered cellular sugar sensing, affected assimilate allocation, increased the biomass of Arabidopsis seeds, and accelerated early plant development. PMID:20709831

Non-selective cation channels in plasma and vacuolar membranes and their contribution to K+ transport.

PubMed

Pottosin, Igor; Dobrovinskaya, Oxana

2014-05-15

Both in vacuolar and plasma membranes, in addition to truly K(+)-selective channels there is a variety of non-selective channels, which conduct K(+) and other ions with little preference. Many non-selective channels in the plasma membrane are active at depolarized potentials, thus, contributing to K(+) efflux rather than to K(+) uptake. They may play important roles in xylem loading or contribute to a K(+) leak, induced by salt or oxidative stress. Here, three currents, expressed in root cells, are considered: voltage-insensitive cation current, non-selective outwardly rectifying current, and low-selective conductance, activated by reactive oxygen species. The latter two do not only poorly discriminate between different cations (like K(+)vs Na(+)), but also conduct anions. Such solute channels may mediate massive electroneutral transport of salts and might be involved in osmotic adjustment or volume decrease, associated with cell death. In the tonoplast two major currents are mediated by SV (slow) and FV (fast) vacuolar channels, respectively, which are virtually impermeable for anions. SV channels conduct mono- and divalent cations indiscriminately and are activated by high cytosolic Ca(2+) and depolarized voltages. FV channels are inhibited by micromolar cytosolic Ca(2+), Mg(2+), and polyamines, and conduct a variety of monovalent cations, including K(+). Strikingly, both SV and FV channels sense the K(+) content of vacuoles, which modulates their voltage dependence, and in case of SV, also alleviates channel's inhibition by luminal Ca(2+). Therefore, SV and FV channels may operate as K(+)-sensing valves, controlling K(+) distribution between the vacuole and the cytosol. Copyright © 2014 Elsevier GmbH. All rights reserved.

ER-associated SNAREs and Sey1p mediate nuclear fusion at two distinct steps during yeast mating.

PubMed

Rogers, Jason V; Arlow, Tim; Inkellis, Elizabeth R; Koo, Timothy S; Rose, Mark D

2013-12-01

During yeast mating, two haploid nuclei fuse membranes to form a single diploid nucleus. However, the known proteins required for nuclear fusion are unlikely to function as direct fusogens (i.e., they are unlikely to directly catalyze lipid bilayer fusion) based on their predicted structure and localization. Therefore we screened known fusogens from vesicle trafficking (soluble N-ethylmaleimide-sensitive factor attachment protein receptors [SNAREs]) and homotypic endoplasmic reticulum (ER) fusion (Sey1p) for additional roles in nuclear fusion. Here we demonstrate that the ER-localized SNAREs Sec20p, Ufe1p, Use1p, and Bos1p are required for efficient nuclear fusion. In contrast, Sey1p is required indirectly for nuclear fusion; sey1Δ zygotes accumulate ER at the zone of cell fusion, causing a block in nuclear congression. However, double mutants of Sey1p and Sec20p, Ufe1p, or Use1p, but not Bos1p, display extreme ER morphology defects, worse than either single mutant, suggesting that retrograde SNAREs fuse ER in the absence of Sey1p. Together these data demonstrate that SNAREs mediate nuclear fusion, ER fusion after cell fusion is necessary to complete nuclear congression, and there exists a SNARE-mediated, Sey1p-independent ER fusion pathway.

Toxoplasma gondii sequesters lysosomes from mammalian hosts in the vacuolar space.

PubMed

Coppens, Isabelle; Dunn, Joe Dan; Romano, Julia D; Pypaert, Marc; Zhang, Hui; Boothroyd, John C; Joiner, Keith A

2006-04-21

The intracellular compartment harboring Toxoplasma gondii satisfies the parasite's nutritional needs for rapid growth in mammalian cells. We demonstrate that the parasitophorous vacuole (PV) of T. gondii accumulates material coming from the host mammalian cell via the exploitation of the host endo-lysosomal system. The parasite actively recruits host microtubules, resulting in selective attraction of endo-lysosomes to the PV. Microtubule-based invaginations of the PV membrane serve as conduits for the delivery of host endo-lysosomes within the PV. These tubular conduits are decorated by a parasite coat, including the tubulogenic protein GRA7, which acts like a garrote that sequesters host endocytic organelles in the vacuolar space. These data define an unanticipated process allowing the parasite intimate and concentrated access to a diverse range of low molecular weight components produced by the endo-lysosomal system. More generally, they identify a unique mechanism for unidirectional transport and sequestration of host organelles.

Clathrin coat controls synaptic vesicle acidification by blocking vacuolar ATPase activity

PubMed Central

Farsi, Zohreh; Rammner, Burkhard; Woehler, Andrew; Lafer, Eileen M; Mim, Carsten; Jahn, Reinhard

2018-01-01

Newly-formed synaptic vesicles (SVs) are rapidly acidified by vacuolar adenosine triphosphatases (vATPases), generating a proton electrochemical gradient that drives neurotransmitter loading. Clathrin-mediated endocytosis is needed for the formation of new SVs, yet it is unclear when endocytosed vesicles acidify and refill at the synapse. Here, we isolated clathrin-coated vesicles (CCVs) from mouse brain to measure their acidification directly at the single vesicle level. We observed that the ATP-induced acidification of CCVs was strikingly reduced in comparison to SVs. Remarkably, when the coat was removed from CCVs, uncoated vesicles regained ATP-dependent acidification, demonstrating that CCVs contain the functional vATPase, yet its function is inhibited by the clathrin coat. Considering the known structures of the vATPase and clathrin coat, we propose a model in which the formation of the coat surrounds the vATPase and blocks its activity. Such inhibition is likely fundamental for the proper timing of SV refilling. PMID:29652249

A vacuolar iron-transporter homologue acts as a detoxifier in Plasmodium

PubMed Central

Slavic, Ksenija; Krishna, Sanjeev; Lahree, Aparajita; Bouyer, Guillaume; Hanson, Kirsten K.; Vera, Iset; Pittman, Jon K.; Staines, Henry M.; Mota, Maria M.

2016-01-01

Iron is an essential micronutrient but is also highly toxic. In yeast and plant cells, a key detoxifying mechanism involves iron sequestration into intracellular storage compartments, mediated by members of the vacuolar iron-transporter (VIT) family of proteins. Here we study the VIT homologue from the malaria parasites Plasmodium falciparum (PfVIT) and Plasmodium berghei (PbVIT). PfVIT-mediated iron transport in a yeast heterologous expression system is saturable (Km∼14.7 μM), and selective for Fe2+ over other divalent cations. PbVIT-deficient P. berghei lines (Pbvit−) show a reduction in parasite load in both liver and blood stages of infection in mice. Moreover, Pbvit− parasites have higher levels of labile iron in blood stages and are more sensitive to increased iron levels in liver stages, when compared with wild-type parasites. Our data are consistent with Plasmodium VITs playing a major role in iron detoxification and, thus, normal development of malaria parasites in their mammalian host. PMID:26786069

A vacuolar iron-transporter homologue acts as a detoxifier in Plasmodium.

PubMed

Slavic, Ksenija; Krishna, Sanjeev; Lahree, Aparajita; Bouyer, Guillaume; Hanson, Kirsten K; Vera, Iset; Pittman, Jon K; Staines, Henry M; Mota, Maria M

2016-01-20

Iron is an essential micronutrient but is also highly toxic. In yeast and plant cells, a key detoxifying mechanism involves iron sequestration into intracellular storage compartments, mediated by members of the vacuolar iron-transporter (VIT) family of proteins. Here we study the VIT homologue from the malaria parasites Plasmodium falciparum (PfVIT) and Plasmodium berghei (PbVIT). PfVIT-mediated iron transport in a yeast heterologous expression system is saturable (Km ∼ 14.7 μM), and selective for Fe(2+) over other divalent cations. PbVIT-deficient P. berghei lines (Pbvit(-)) show a reduction in parasite load in both liver and blood stages of infection in mice. Moreover, Pbvit(-) parasites have higher levels of labile iron in blood stages and are more sensitive to increased iron levels in liver stages, when compared with wild-type parasites. Our data are consistent with Plasmodium VITs playing a major role in iron detoxification and, thus, normal development of malaria parasites in their mammalian host.

Planarization of Isolated Defects on ICF Target Capsule Surfaces by Pulsed Laser Ablation

DOE PAGES

Alfonso, Noel; Carlson, Lane C.; Bunn, Thomas L.

2016-08-09

Demanding surface quality requirements for inertial confinement fusion (ICF) capsules motivated the development of a pulsed laser ablation method to reduce or eliminate undesirable surface defects. The pulsed laser ablation technique takes advantage of a full surface (4π) capsule manipulation system working in combination with an optical profiling (confocal) microscope. Based on the defect topography, the material removal rate, the laser pulse energy and its beam profile, a customized laser raster pattern is derived to remove the defect. The pattern is a table of coordinates and number of pulses that dictate how the defect will be vaporized until its heightmore » is level with the capsule surface. This paper explains how the raster patterns are optimized to minimize surface roughness and how surface roughness after laser ablation is simulated. The simulated surfaces are compared with actual ablated surfaces. Large defects are reduced to a size regime where a tumble finishing process produces very high quality surfaces devoid of high mode defects. The combined polishing processes of laser ablation and tumble finishing have become routine fabrication steps for National Ignition Facility capsule production.« less

Yarn-dyed fabric defect classification based on convolutional neural network

NASA Astrophysics Data System (ADS)

Jing, Junfeng; Dong, Amei; Li, Pengfei

2017-07-01

Considering that the manual inspection of the yarn-dyed fabric can be time consuming and less efficient, a convolutional neural network (CNN) solution based on the modified AlexNet structure for the classification of the yarn-dyed fabric defect is proposed. CNN has powerful ability of feature extraction and feature fusion which can simulate the learning mechanism of the human brain. In order to enhance computational efficiency and detection accuracy, the local response normalization (LRN) layers in AlexNet are replaced by the batch normalization (BN) layers. In the process of the network training, through several convolution operations, the characteristics of the image are extracted step by step, and the essential features of the image can be obtained from the edge features. And the max pooling layers, the dropout layers, the fully connected layers are also employed in the classification model to reduce the computation cost and acquire more precise features of fabric defect. Finally, the results of the defect classification are predicted by the softmax function. The experimental results show the capability of defect classification via the modified Alexnet model and indicate its robustness.

Multi-sensor information fusion method for vibration fault diagnosis of rolling bearing

NASA Astrophysics Data System (ADS)

Jiao, Jing; Yue, Jianhai; Pei, Di

2017-10-01

Bearing is a key element in high-speed electric multiple unit (EMU) and any defect of it can cause huge malfunctioning of EMU under high operation speed. This paper presents a new method for bearing fault diagnosis based on least square support vector machine (LS-SVM) in feature-level fusion and Dempster-Shafer (D-S) evidence theory in decision-level fusion which were used to solve the problems about low detection accuracy, difficulty in extracting sensitive characteristics and unstable diagnosis system of single-sensor in rolling bearing fault diagnosis. Wavelet de-nosing technique was used for removing the signal noises. LS-SVM was used to make pattern recognition of the bearing vibration signal, and then fusion process was made according to the D-S evidence theory, so as to realize recognition of bearing fault. The results indicated that the data fusion method improved the performance of the intelligent approach in rolling bearing fault detection significantly. Moreover, the results showed that this method can efficiently improve the accuracy of fault diagnosis.

Reovirus FAST Proteins Drive Pore Formation and Syncytiogenesis Using a Novel Helix-Loop-Helix Fusion-Inducing Lipid Packing Sensor

PubMed Central

Sarker, Muzaddid; de Antueno, Roberto; Langelaan, David N.; Parmar, Hiren B.; Shin, Kyungsoo; Rainey, Jan K.; Duncan, Roy

2015-01-01

Pore formation is the most energy-demanding step during virus-induced membrane fusion, where high curvature of the fusion pore rim increases the spacing between lipid headgroups, exposing the hydrophobic interior of the membrane to water. How protein fusogens breach this thermodynamic barrier to pore formation is unclear. We identified a novel fusion-inducing lipid packing sensor (FLiPS) in the cytosolic endodomain of the baboon reovirus p15 fusion-associated small transmembrane (FAST) protein that is essential for pore formation during cell-cell fusion and syncytiogenesis. NMR spectroscopy and mutational studies indicate the dependence of this FLiPS on a hydrophobic helix-loop-helix structure. Biochemical and biophysical assays reveal the p15 FLiPS preferentially partitions into membranes with high positive curvature, and this partitioning is impeded by bis-ANS, a small molecule that inserts into hydrophobic defects in membranes. Most notably, the p15 FLiPS can be functionally replaced by heterologous amphipathic lipid packing sensors (ALPS) but not by other membrane-interactive amphipathic helices. Furthermore, a previously unrecognized amphipathic helix in the cytosolic domain of the reptilian reovirus p14 FAST protein can functionally replace the p15 FLiPS, and is itself replaceable by a heterologous ALPS motif. Anchored near the cytoplasmic leaflet by the FAST protein transmembrane domain, the FLiPS is perfectly positioned to insert into hydrophobic defects that begin to appear in the highly curved rim of nascent fusion pores, thereby lowering the energy barrier to stable pore formation. PMID:26061049

Application of ECT inspection to the first wall of a fusion reactor with wavelet analysis

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

Chen, G.; Yoshida, Y.; Miya, K.

1994-12-31

The first wall of a fusion reactor will be subjected to intensive loads during fusion operations. Since these loads may cause defects in the first wall, nondestructive evaluation techniques of the first wall should be developed. In this paper, we try to apply eddy current testing (ECT) technique to the inspection of the first wall. A method based on current vector potential and wavelet analysis is proposed. Owing to the use of wavelet analysis, a new theory developed recently, the accuracy of the present method is shown to be better than a conventional one.

ATG14 promotes membrane tethering and fusion of autophagosomes to endolysosomes.

PubMed

Diao, Jiajie; Liu, Rong; Rong, Yueguang; Zhao, Minglei; Zhang, Jing; Lai, Ying; Zhou, Qiangjun; Wilz, Livia M; Li, Jianxu; Vivona, Sandro; Pfuetzner, Richard A; Brunger, Axel T; Zhong, Qing

2015-04-23

Autophagy, an important catabolic pathway implicated in a broad spectrum of human diseases, begins by forming double membrane autophagosomes that engulf cytosolic cargo and ends by fusing autophagosomes with lysosomes for degradation. Membrane fusion activity is required for early biogenesis of autophagosomes and late degradation in lysosomes. However, the key regulatory mechanisms of autophagic membrane tethering and fusion remain largely unknown. Here we report that ATG14 (also known as beclin-1-associated autophagy-related key regulator (Barkor) or ATG14L), an essential autophagy-specific regulator of the class III phosphatidylinositol 3-kinase complex, promotes membrane tethering of protein-free liposomes, and enhances hemifusion and full fusion of proteoliposomes reconstituted with the target (t)-SNAREs (soluble N-ethylmaleimide-sensitive factor attachment protein receptors) syntaxin 17 (STX17) and SNAP29, and the vesicle (v)-SNARE VAMP8 (vesicle-associated membrane protein 8). ATG14 binds to the SNARE core domain of STX17 through its coiled-coil domain, and stabilizes the STX17-SNAP29 binary t-SNARE complex on autophagosomes. The STX17 binding, membrane tethering and fusion-enhancing activities of ATG14 require its homo-oligomerization by cysteine repeats. In ATG14 homo-oligomerization-defective cells, autophagosomes still efficiently form but their fusion with endolysosomes is blocked. Recombinant ATG14 homo-oligomerization mutants also completely lose their ability to promote membrane tethering and to enhance SNARE-mediated fusion in vitro. Taken together, our data suggest an autophagy-specific membrane fusion mechanism in which oligomeric ATG14 directly binds to STX17-SNAP29 binary t-SNARE complex on autophagosomes and primes it for VAMP8 interaction to promote autophagosome-endolysosome fusion.

Interrelationships between mitochondrial fusion, energy metabolism and oxidative stress during development in Caenorhabditis elegans.

PubMed

Yasuda, Kayo; Hartman, Philip S; Ishii, Takamasa; Suda, Hitoshi; Akatsuka, Akira; Shoyama, Tetsuji; Miyazawa, Masaki; Ishii, Naoaki

2011-01-21

Mitochondria are known to be dynamic structures with the energetically and enzymatically mediated processes of fusion and fission responsible for maintaining a constant flux. Mitochondria also play a role of reactive oxygen species production as a byproduct of energy metabolism. In the current study, interrelationships between mitochondrial fusion, energy metabolism and oxidative stress on development were explored using a fzo-1 mutant defective in the fusion process and a mev-1 mutant overproducing superoxide from mitochondrial electron transport complex II of Caenorhabditis elegans. While growth and development of both single mutants was slightly delayed relative to the wild type, the fzo-1;mev-1 double mutant experienced considerable delay. Oxygen sensitivity during larval development, superoxide production and carbonyl protein accumulation of the fzo-1 mutant were similar to wild type. fzo-1 animals had significantly lower metabolism than did N2 and mev-1. These data indicate that mitochondrial fusion can profoundly affect energy metabolism and development. Copyright © 2010 Elsevier Inc. All rights reserved.

Excess cholesterol inhibits glucose-stimulated fusion pore dynamics in insulin exocytosis.

PubMed

Xu, Yingke; Toomre, Derek K; Bogan, Jonathan S; Hao, Mingming

2017-11-01

Type 2 diabetes is caused by defects in both insulin sensitivity and insulin secretion. Glucose triggers insulin secretion by causing exocytosis of insulin granules from pancreatic β-cells. High circulating cholesterol levels and a diminished capacity of serum to remove cholesterol from β-cells are observed in diabetic individuals. Both of these effects can lead to cholesterol accumulation in β-cells and contribute to β-cell dysfunction. However, the molecular mechanisms by which cholesterol accumulation impairs β-cell function remain largely unknown. Here, we used total internal reflection fluorescence microscopy to address, at the single-granule level, the role of cholesterol in regulating fusion pore dynamics during insulin exocytosis. We focused particularly on the effects of cholesterol overload, which is relevant to type 2 diabetes. We show that excess cholesterol reduced the number of glucose-stimulated fusion events, and modulated the proportion of full fusion and kiss-and-run fusion events. Analysis of single exocytic events revealed distinct fusion kinetics, with more clustered and compound exocytosis observed in cholesterol-overloaded β-cells. We provide evidence for the involvement of the GTPase dynamin, which is regulated in part by cholesterol-induced phosphatidylinositol 4,5-bisphosphate enrichment in the plasma membrane, in the switch between full fusion and kiss-and-run fusion. Characterization of insulin exocytosis offers insights into the role that elevated cholesterol may play in the development of type 2 diabetes. © 2017 The Authors. Journal of Cellular and Molecular Medicine published by John Wiley & Sons Ltd and Foundation for Cellular and Molecular Medicine.

Role of lipid phase separations and membrane hydration in phospholipid vesicle fusion.

PubMed

Hoekstra, D

1982-06-08

The relationship between lipid phase separation and fusion of small unilamellar phosphatidylserine-containing vesicles was investigated. The kinetics of phase separation were monitored by following the increase of self-quenching of the fluorescent phospholipid analogue N-(7-nitro-2,1,3-benzoxadiazol-4-yl)phosphatidylethanolamine, which occurs when the local concentration of the probe increases upon Ca2+-induced phase separation in phosphatidylserine (PS) bilayers [Hoekstra, D. (1982) Biochemistry 21, 1055-1061]. Fusion was determined by using the resonance energy transfer fusion assay [Struck, D. K., Hoekstra, D., & Pagano, R. E. (1981) Biochemistry 20, 4093-4099], which monitors the mixing of fluorescent lipid donor and acceptor molecules, resulting in an increase in energy transfer efficiency. The results show that in the presence of Ca2+, fusion proceeds much more rapidly (t 1/2 less than 5 s) than the process of phase separation (T 1/2 congruent to 1 min). Mg2+ also induced fusion, albeit at higher concentrations than Ca2+. Mg2+-induced phase separation were not detected, however. Subthreshold concentrations of Ca2+ (0.5 mM) or Mg2+ (2 mM) induced extensive fusion of PS-containing vesicles in poly(ethylene glycol) containing media. This effect did not appear to be a poly(ethylene glycol)-facilitated enhancement of cation binding to the bilayer, and consequently Ca2+-induced phase separation was not observed. The results suggest that macroscopic phase separation may facilitate but does not induced the fusion process and is therefore, not directly involved in the actual fusion mechanism. The fusion experiments performed in the presence of poly(ethylene glycol) suggest that the degree of bilayer dehydration and the creation of "point defects" in the bilayer without rigorous structural rearrangements in the membrane are dominant factors in the initial fusion events.

Fusion of the septic knee with external hybrid fixator.

PubMed

Kutscha-Lissberg, F; Hebler, U; Esenwein, S A; Muhr, G; Wick, M

2006-10-01

Arthrodesis is a well-recognized salvage procedure in patients with septic destruction of knee joints. It offers the opportunity for restoring weight bearing capability and significant pain relief as well as eradication of infection, but at the expense of joint motion. However, arthrodesis in order to joint sepsis may be difficult to achieve because of poor bone stock, persistent infection and soft tissue compromise. From 2000 to June 2004, in 70 consecutive patients arthrodesis of the knee was indicated. Nineteen patients were considered to have external hybrid fixator (EHF) and were therefore included to the prospective study protocol. Forty-three stabilisations of destructed knee joints were done by a modular titanium rod. In these cases bony fusion was very unlikely to be achieved because of devastating defects due to infected total knee arthroplasties. Eight patients were treated by compression nailing. In these situations EHF was not indicated. In three patients minor complication occurred. In two out of 17 patients fusion failed because of primary underestimated bony defects (11.8%). The use of EHF for arthrodeses after septic destruction of knee joints can be recommended according to our results. However, EHF will not be successful or applicable in each case. Therefore, physicians and institutions that offer this special method should have not only experiences with EHF but also with arthrodeses and alternative procedures.

Impaired Mitochondrial Dynamics Underlie Axonal Defects in Hereditary Spastic Paraplegias.

PubMed

Denton, Kyle; Mou, Yongchao; Xu, Chong-Chong; Shah, Dhruvi; Chang, Jaerak; Blackstone, Craig; Li, Xue-Jun

2018-05-02

Mechanisms by which long corticospinal axons degenerate in hereditary spastic paraplegia (HSP) are largely unknown. Here, we have generated induced pluripotent stem cells (iPSCs) from patients with two autosomal recessive forms of HSP, SPG15 and SPG48, which are caused by mutations in the ZFYVE26 and AP5Z1 genes encoding proteins in the same complex, the spastizin and AP5Z1 proteins, respectively. In patient iPSC-derived telencephalic glutamatergic and midbrain dopaminergic neurons, neurite number, length and branching are significantly reduced, recapitulating disease-specific phenotypes. We analyzed mitochondrial morphology and noted a significant reduction in both mitochondrial length and their densities within axons of these HSP neurons. Mitochondrial membrane potential was also decreased, confirming functional mitochondrial defects. Notably, mdivi-1, an inhibitor of the mitochondrial fission GTPase DRP1, rescues mitochondrial morphology defects and suppresses the impairment in neurite outgrowth and late-onset apoptosis in HSP neurons. Furthermore, knockdown of these HSP genes causes similar axonal defects, also mitigated by treatment with mdivi-1. Finally, neurite outgrowth defects in SPG15 and SPG48 cortical neurons can be rescued by knocking down DRP1 directly. Thus, abnormal mitochondrial morphology caused by an imbalance of mitochondrial fission and fusion underlies specific axonal defects and serves as a potential therapeutic target for SPG15 and SPG48.

ER-associated SNAREs and Sey1p mediate nuclear fusion at two distinct steps during yeast mating

PubMed Central

Rogers, Jason V.; Arlow, Tim; Inkellis, Elizabeth R.; Koo, Timothy S.; Rose, Mark D.

2013-01-01

During yeast mating, two haploid nuclei fuse membranes to form a single diploid nucleus. However, the known proteins required for nuclear fusion are unlikely to function as direct fusogens (i.e., they are unlikely to directly catalyze lipid bilayer fusion) based on their predicted structure and localization. Therefore we screened known fusogens from vesicle trafficking (soluble N-ethylmaleimide–sensitive factor attachment protein receptors [SNAREs]) and homotypic endoplasmic reticulum (ER) fusion (Sey1p) for additional roles in nuclear fusion. Here we demonstrate that the ER-localized SNAREs Sec20p, Ufe1p, Use1p, and Bos1p are required for efficient nuclear fusion. In contrast, Sey1p is required indirectly for nuclear fusion; sey1Δ zygotes accumulate ER at the zone of cell fusion, causing a block in nuclear congression. However, double mutants of Sey1p and Sec20p, Ufe1p, or Use1p, but not Bos1p, display extreme ER morphology defects, worse than either single mutant, suggesting that retrograde SNAREs fuse ER in the absence of Sey1p. Together these data demonstrate that SNAREs mediate nuclear fusion, ER fusion after cell fusion is necessary to complete nuclear congression, and there exists a SNARE-mediated, Sey1p-independent ER fusion pathway. PMID:24152736

Macrophage fusion is controlled by the cytoplasmic protein tyrosine phosphatase PTP-PEST/PTPN12.

PubMed

Rhee, Inmoo; Davidson, Dominique; Souza, Cleiton Martins; Vacher, Jean; Veillette, André

2013-06-01

Macrophages can undergo cell-cell fusion, leading to the formation of multinucleated giant cells and osteoclasts. This process is believed to promote the proteolytic activity of macrophages toward pathogens, foreign bodies, and extracellular matrices. Here, we examined the role of PTP-PEST (PTPN12), a cytoplasmic protein tyrosine phosphatase, in macrophage fusion. Using a macrophage-targeted PTP-PEST-deficient mouse, we determined that PTP-PEST was not needed for macrophage differentiation or cytokine production. However, it was necessary for interleukin-4-induced macrophage fusion into multinucleated giant cells in vitro. It was also needed for macrophage fusion following implantation of a foreign body in vivo. Moreover, in the RAW264.7 macrophage cell line, PTP-PEST was required for receptor activator of nuclear factor kappa-B ligand (RANKL)-triggered macrophage fusion into osteoclasts. PTP-PEST had no impact on expression of fusion mediators such as β-integrins, E-cadherin, and CD47, which enable macrophages to become fusion competent. However, it was needed for polarization of macrophages, migration induced by the chemokine CC chemokine ligand 2 (CCL2), and integrin-induced spreading, three key events in the fusion process. PTP-PEST deficiency resulted in specific hyperphosphorylation of the protein tyrosine kinase Pyk2 and the adaptor paxillin. Moreover, a fusion defect was induced upon treatment of normal macrophages with a Pyk2 inhibitor. Together, these data argue that macrophage fusion is critically dependent on PTP-PEST. This function is seemingly due to the ability of PTP-PEST to control phosphorylation of Pyk2 and paxillin, thereby regulating cell polarization, migration, and spreading.

Validity of the Worth 4 Dot Test in Patients with Red-Green Color Vision Defect.

PubMed

Bak, Eunoo; Yang, Hee Kyung; Hwang, Jeong-Min

2017-05-01

The Worth four dot test uses red and green glasses for binocular dissociation, and although it has been believed that patients with red-green color vision defects cannot accurately perform the Worth four dot test, this has not been validated. Therefore, the purpose of this study was to demonstrate the validity of the Worth four dot test in patients with congenital red-green color vision defects who have normal or abnormal binocular vision. A retrospective review of medical records was performed on 30 consecutive congenital red-green color vision defect patients who underwent the Worth four dot test. The type of color vision anomaly was determined by the Hardy Rand and Rittler (HRR) pseudoisochromatic plate test, Ishihara color test, anomaloscope, and/or the 100 hue test. All patients underwent a complete ophthalmologic examination. Binocular sensory status was evaluated with the Worth four dot test and Randot stereotest. The results were interpreted according to the presence of strabismus or amblyopia. Among the 30 patients, 24 had normal visual acuity without strabismus nor amblyopia and 6 patients had strabismus and/or amblyopia. The 24 patients without strabismus nor amblyopia all showed binocular fusional responses by seeing four dots of the Worth four dot test. Meanwhile, the six patients with strabismus or amblyopia showed various results of fusion, suppression, and diplopia. Congenital red-green color vision defect patients of different types and variable degree of binocularity could successfully perform the Worth four dot test. They showed reliable results that were in accordance with their estimated binocular sensory status.

Three-Dimensional Image Fusion of 18F-Fluorodeoxyglucose-Positron Emission Tomography/Computed Tomography and Contrast-Enhanced Computed Tomography for Computer-Assisted Planning of Maxillectomy of Recurrent Maxillary Squamous Cell Carcinoma and Defect Reconstruction.

PubMed

Yu, Yao; Zhang, Wen-Bo; Liu, Xiao-Jing; Guo, Chuan-Bin; Yu, Guang-Yan; Peng, Xin

2017-06-01

The purpose of this study was to describe new technology assisted by 3-dimensional (3D) image fusion of 18 F-fluorodeoxyglucose (FDG)-positron emission tomography (PET)/computed tomography (CT) and contrast-enhanced CT (CECT) for computer planning of a maxillectomy of recurrent maxillary squamous cell carcinoma and defect reconstruction. Treatment of recurrent maxillary squamous cell carcinoma usually includes tumor resection and free flap reconstruction. FDG-PET/CT provided images of regions of abnormal glucose uptake and thus showed metabolic tumor volume to guide tumor resection. CECT data were used to create 3D reconstructed images of vessels to show the vascular diameters and locations, so that the most suitable vein and artery could be selected during anastomosis of the free flap. The data from preoperative maxillofacial CECT scans and FDG-PET/CT imaging were imported into the navigation system (iPlan 3.0; Brainlab, Feldkirchen, Germany). Three-dimensional image fusion between FDG-PET/CT and CECT was accomplished using Brainlab software according to the position of the 2 skulls simulated in the CECT image and PET/CT image, respectively. After verification of the image fusion accuracy, the 3D reconstruction images of the metabolic tumor, vessels, and other critical structures could be visualized within the same coordinate system. These sagittal, coronal, axial, and 3D reconstruction images were used to determine the virtual osteotomy sites and reconstruction plan, which was provided to the surgeon and used for surgical navigation. The average shift of the 3D image fusion between FDG-PET/CT and CECT was less than 1 mm. This technique, by clearly showing the metabolic tumor volume and the most suitable vessels for anastomosis, facilitated resection and reconstruction of recurrent maxillary squamous cell carcinoma. We used 3D image fusion of FDG-PET/CT and CECT to successfully accomplish resection and reconstruction of recurrent maxillary squamous cell carcinoma

The mechanical consequence of failure of ossified union in attempted posterior spinal fusion. A canine model.

PubMed

Stonecipher, T K; Vanderby, R; Sciammarella, C A; Lei, S S; Fisk, J R

1983-01-01

The mechanical behavior of pseudarthrosis in posterior spinal fusion was investigated. A canine model was developed in which an incompletely ossified posterior fusion mass was consistently produced. The spines were excised, and the motion segments were mechanically tested using a specially developed loading apparatus. Tests were performed to evaluate stiffness of the segments to loading with compression, torsion, and anterioposterior and lateral bending shear stiffness. Changes in other modes of loading were less consistent. The motion characteristics of the pseudarthrosis could not be predicted from the extent of the osseous defect noted on roentgenograms. These findings correlate clinically with the progression of curvature seen with pseudarthrosis in scoliosis surgery and the unpredictable results of pseudarthrosis in posterior fusion performed in treatment of degenerative disc disease.

Inorganic polyphosphate in the yeast Saccharomyces cerevisiae with a mutation disturbing the function of vacuolar ATPase.

PubMed

Tomaschevsky, A A; Ryasanova, L P; Kulakovskaya, T V; Kulaev, I S

2010-08-01

A mutation in the vma2 gene disturbing V-ATPase function in the yeast Saccharomyces cerevisiae results in a five- and threefold decrease in inorganic polyphosphate content in the stationary and active phases of growth on glucose, respectively. The average polyphosphate chain length in the mutant cells is decreased. The mutation does not prevent polyphosphate utilization during cultivation in a phosphate-deficient medium and recovery of its level on reinoculation in complete medium after phosphate deficiency. The content of short chain acid-soluble polyphosphates is recovered first. It is supposed that these polyphosphates are less dependent on the electrochemical gradient on the vacuolar membrane.

Fusion 2.0: The Next Generation of Fusion in California: Aligning State and Regional Fusion Centers

DTIC Science & Technology

2010-03-01

bible ” for fusion center management, as evidenced by the theme of the 2009 National Fusion Center Conference; appropriately called “Achieving Baseline...NAVAL POSTGRADUATE SCHOOL MONTEREY, CALIFORNIA THESIS FUSION 2.0: THE NEXT GENERATION OF FUSION IN CALIFORNIA: ALIGNING STATE AND...Master’s Thesis 4. TITLE AND SUBTITLE Fusion 2.0: The Next Generation of Fusion in California: Aligning State and Regional Fusion

Complex cardiac defects after ethanol exposure during discrete cardiogenic events in zebrafish: Prevention with folic acid

PubMed Central

Sarmah, Swapnalee; Marrs, James A.

2014-01-01

BACKGROUND Fetal alcohol spectrum disorder (FASD) describes a range of birth defects including various congenital heart defects (CHDs). Mechanisms of FASD-associated CHDs are not understood. Whether alcohol interferes with a single critical event or with multiple events in heart formation is not known. RESULTS Our zebrafish embryo experiments showed that ethanol interrupts different cardiac regulatory networks and perturbed multiple steps of cardiogenesis (specification, myocardial migration, looping, chamber morphogenesis and endocardial cushion formation). Ethanol exposure during gastrulation until cardiac specification or during myocardial midline migration did not produce severe or persistent heart development defects. However, exposure comprising gastrulation until myocardial precursor midline fusion or during heart patterning stages produced aberrant heart looping and defective endocardial cushions. Continuous exposure during entire cardiogenesis produced complex cardiac defects leading to severely defective myocardium, endocardium, and endocardial cushions. Supplementation of retinoic acid with ethanol partially rescued early heart developmental defects, but the endocardial cushions did not form correctly. In contrast, supplementation of folic acid rescued normal heart development, including the endocardial cushions. CONCLUSIONS Our results indicate that ethanol exposure interrupted divergent cardiac morphogenesis events causing heart defects. Folic acid supplementation was effective in preventing a wide spectrum of ethanol-induced heart developmental defects. PMID:23832875

Designing Radiation Resistance in Materials for Fusion Energy

NASA Astrophysics Data System (ADS)

Zinkle, S. J.; Snead, L. L.

2014-07-01

Proposed fusion and advanced (Generation IV) fission energy systems require high-performance materials capable of satisfactory operation up to neutron damage levels approaching 200 atomic displacements per atom with large amounts of transmutant hydrogen and helium isotopes. After a brief overview of fusion reactor concepts and radiation effects phenomena in structural and functional (nonstructural) materials, three fundamental options for designing radiation resistance are outlined: Utilize matrix phases with inherent radiation tolerance, select materials in which vacancies are immobile at the design operating temperatures, or engineer materials with high sink densities for point defect recombination. Environmental and safety considerations impose several additional restrictions on potential materials systems, but reduced-activation ferritic/martensitic steels (including thermomechanically treated and oxide dispersion-strengthened options) and silicon carbide ceramic composites emerge as robust structural materials options. Materials modeling (including computational thermodynamics) and advanced manufacturing methods are poised to exert a major impact in the next ten years.

Structure of the vacuolar H+-ATPase rotary motor reveals new mechanistic insights.

PubMed

Rawson, Shaun; Phillips, Clair; Huss, Markus; Tiburcy, Felix; Wieczorek, Helmut; Trinick, John; Harrison, Michael A; Muench, Stephen P

2015-03-03

Vacuolar H(+)-ATPases are multisubunit complexes that operate with rotary mechanics and are essential for membrane proton transport throughout eukaryotes. Here we report a ∼ 1 nm resolution reconstruction of a V-ATPase in a different conformational state from that previously reported for a lower-resolution yeast model. The stator network of the V-ATPase (and by implication that of other rotary ATPases) does not change conformation in different catalytic states, and hence must be relatively rigid. We also demonstrate that a conserved bearing in the catalytic domain is electrostatic, contributing to the extraordinarily high efficiency of rotary ATPases. Analysis of the rotor axle/membrane pump interface suggests how rotary ATPases accommodate different c ring stoichiometries while maintaining high efficiency. The model provides evidence for a half channel in the proton pump, supporting theoretical models of ion translocation. Our refined model therefore provides new insights into the structure and mechanics of the V-ATPases. Copyright © 2015 The Authors. Published by Elsevier Ltd.. All rights reserved.

Study of modulation property to incident laser by surface micro-defects on KH2PO4 crystal

NASA Astrophysics Data System (ADS)

Chen, Ming-Jun; Cheng, Jian; Li, Ming-Quan; Xiao, Yong

2012-06-01

KH2PO4 crystal is a crucial optical component of inertial confinement fusion. Modulation of an incident laser by surface micro-defects will induce the growth of surface damage, which largely restricts the enhancement of the laser induced damage threshold. The modulation of an incident laser by using different kinds of surface defects are simulated by employing the three-dimensional finite-difference time-domain method. The results indicate that after the modulation of surface defects, the light intensity distribution inside the crystal is badly distorted, with the light intensity enhanced symmetrically. The relations between modulation properties and defect geometries (e.g., width, morphology, and depth of defects) are quite different for different defects. The modulation action is most obvious when the width of surface defects reaches 1.064 μm. For defects with smooth morphology, such as spherical pits, the degree of modulation is the smallest and the light intensity distribution seems relatively uniform. The degree of modulation increases rapidly with the increase of the depth of surface defects and becomes stable when the depth reaches a critical value. The critical depth is 1.064 μm for cuboid pits and radial cracks, while for ellipsoidal pits the value depends on both the width and the length of the defects.

The ESCRT regulator Did2 maintains the balance between long-distance endosomal transport and endocytic trafficking

PubMed Central

Haag, Carl

2017-01-01

In highly polarised cells, like fungal hyphae, early endosomes function in both endocytosis as well as long-distance transport of various cargo including mRNA and protein complexes. However, knowledge on the crosstalk between these seemingly different trafficking processes is scarce. Here, we demonstrate that the ESCRT regulator Did2 coordinates endosomal transport in fungal hyphae of Ustilago maydis. Loss of Did2 results in defective vacuolar targeting, less processive long-distance transport and abnormal shuttling of early endosomes. Importantly, the late endosomal protein Rab7 and vacuolar protease Prc1 exhibit increased shuttling on these aberrant endosomes suggesting defects in endosomal maturation and identity. Consistently, molecular motors fail to attach efficiently explaining the disturbed processive movement. Furthermore, the endosomal mRNP linker protein Upa1 is hardly present on endosomes resulting in defects in long-distance mRNA transport. In conclusion, the ESCRT regulator Did2 coordinates precise maturation of endosomes and thus provides the correct membrane identity for efficient endosomal long-distance transport. PMID:28422978

Detection of defects in laser powder deposition (LPD) components by pulsed laser transient thermography

NASA Astrophysics Data System (ADS)

Santospirito, S. P.; Słyk, Kamil; Luo, Bin; Łopatka, Rafał; Gilmour, Oliver; Rudlin, John

2013-05-01

Detection of defects in Laser Powder Deposition (LPD) produced components has been achieved by laser thermography. An automatic in-process NDT defect detection software system has been developed for the analysis of laser thermography to automatically detect, reliably measure and then sentence defects in individual beads of LPD components. A deposition path profile definition has been introduced so all laser powder deposition beads can be modeled, and the inspection system has been developed to automatically generate an optimized inspection plan in which sampling images follow the deposition track, and automatically control and communicate with robot-arms, the source laser and cameras to implement image acquisition. Algorithms were developed so that the defect sizes can be correctly evaluated and these have been confirmed using test samples. Individual inspection images can also be stitched together for a single bead, a layer of beads or multiple layers of beads so that defects can be mapped through the additive process. A mathematical model was built up to analyze and evaluate the movement of heat throughout the inspection bead. Inspection processes were developed and positional and temporal gradient algorithms have been used to measure the flaw sizes. Defect analysis is then performed to determine if the defect(s) can be further classified (crack, lack of fusion, porosity) and the sentencing engine then compares the most significant defect or group of defects against the acceptance criteria - independent of human decisions. Testing on manufactured defects from the EC funded INTRAPID project has successful detected and correctly sentenced all samples.

Macrophage Fusion Is Controlled by the Cytoplasmic Protein Tyrosine Phosphatase PTP-PEST/PTPN12

PubMed Central

Rhee, Inmoo; Davidson, Dominique; Souza, Cleiton Martins; Vacher, Jean

2013-01-01

Macrophages can undergo cell-cell fusion, leading to the formation of multinucleated giant cells and osteoclasts. This process is believed to promote the proteolytic activity of macrophages toward pathogens, foreign bodies, and extracellular matrices. Here, we examined the role of PTP-PEST (PTPN12), a cytoplasmic protein tyrosine phosphatase, in macrophage fusion. Using a macrophage-targeted PTP-PEST-deficient mouse, we determined that PTP-PEST was not needed for macrophage differentiation or cytokine production. However, it was necessary for interleukin-4-induced macrophage fusion into multinucleated giant cells in vitro. It was also needed for macrophage fusion following implantation of a foreign body in vivo. Moreover, in the RAW264.7 macrophage cell line, PTP-PEST was required for receptor activator of nuclear factor kappa-B ligand (RANKL)-triggered macrophage fusion into osteoclasts. PTP-PEST had no impact on expression of fusion mediators such as β-integrins, E-cadherin, and CD47, which enable macrophages to become fusion competent. However, it was needed for polarization of macrophages, migration induced by the chemokine CC chemokine ligand 2 (CCL2), and integrin-induced spreading, three key events in the fusion process. PTP-PEST deficiency resulted in specific hyperphosphorylation of the protein tyrosine kinase Pyk2 and the adaptor paxillin. Moreover, a fusion defect was induced upon treatment of normal macrophages with a Pyk2 inhibitor. Together, these data argue that macrophage fusion is critically dependent on PTP-PEST. This function is seemingly due to the ability of PTP-PEST to control phosphorylation of Pyk2 and paxillin, thereby regulating cell polarization, migration, and spreading. PMID:23589331

The effects of local insulin application to lumbar spinal fusions in a rat model.

PubMed

Koerner, John D; Yalamanchili, Praveen; Munoz, William; Uko, Linda; Chaudhary, Saad B; Lin, Sheldon S; Vives, Michael J

2013-01-01

The rates of pseudoarthrosis after a single-level spinal fusion have been reported up to 35%, and the agents that increase the rate of fusion have an important role in decreasing pseudoarthrosis after spinal fusion. Previous studies have analyzed the effects of local insulin application to an autograft in a rat segmental defect model. Defects treated with a time-released insulin implant had significantly more new bone formation and greater quality of bone compared with controls based on histology and histomorphometry. A time-released insulin implant may have similar effects when applied in a lumbar spinal fusion model. This study analyzes the effects of a local time-released insulin implant applied to the fusion bed in a rat posterolateral lumbar spinal fusion model. Our hypothesis was twofold: first, a time-released insulin implant applied to the autograft bed in a rat posterolateral lumbar fusion will increase the rate of successful fusion and second, will alter the local environment of the fusion site by increasing the levels of local growth factors. Animal model (Institutional Animal Care and Use Committee approved) using 40 adult male Sprague-Dawley rats. Forty skeletally mature Sprague-Dawley rats weighing approximately 500 g each underwent posterolateral intertransverse lumbar fusions with iliac crest autograft from L4 to L5 using a Wiltse-type approach. After exposure of the transverse processes and high-speed burr decortication, a Linplant (Linshin Canada, Inc., ON, Canada) consisting of 95% microrecrystalized palmitic acid and 5% bovine insulin (experimental group) or a sham implant consisting of only palmitic acid (control group) was implanted on the fusion bed with iliac crest autograft. As per the manufacturer, the Linplant has a release rate of 2 U/day for a minimum of 40 days. The transverse processes and autograft beds of 10 animals from the experimental and 10 from the control group were harvested at Day 4 and analyzed for growth factors. The

How to improve the irradiation conditions for the International Fusion Materials Irradiation Facility

NASA Astrophysics Data System (ADS)

Daum, Eric

2000-12-01

The accelerator-based intense D-Li neutron source International Fusion Materials Irradiation Facility (IFMIF) provides very suitable irradiation conditions for fusion materials development with the attractive option of accelerated irradiations. Investigations show that a neutron moderator made of tungsten and placed in the IFMIF test cell can further improve the irradiation conditions. The moderator softens the IFMIF neutron spectrum by enhancing the fraction of low energy neutrons. For displacement damage, the ratio of point defects to cascades is more DEMO relevant and for tritium production in Li-based breeding ceramic materials it leads to a preferred production via the 6Li(n,t) 4He channel as it occurs in a DEMO breeding blanket.

The prokaryote-to-eukaryote transition reflected in the evolution of the V/F/A-ATPase catalytic and proteolipid subunits

NASA Technical Reports Server (NTRS)

Hilario, E.; Gogarten, J. P.

1998-01-01

Changes in the primary and quarternary structure of vacuolar and archaeal type ATPases that accompany the prokaryote-to-eukaryote transition are analyzed. The gene encoding the vacuolar-type proteolipid of the V-ATPase from Giardia lamblia is reported. Giardia has a typical vacuolar ATPase as observed from the common motifs shared between its proteolipid subunit and other eukaryotic vacuolar ATPases, suggesting that the former enzyme works as a hydrolase in this primitive eukaryote. The phylogenetic analyses of the V-ATPase catalytic subunit and the front and back halves of the proteolipid subunit placed Giardia as the deepest branch within the eukaryotes. Our phylogenetic analysis indicated that at least two independent duplication and fusion events gave rise to the larger proteolipid type found in eukaryotes and in Methanococcus. The spatial distribution of the conserved residues among the vacuolar-type proteolipids suggest a zipper-type interaction among the transmembrane helices and surrounding subunits of the V-ATPase complex. Important residues involved in the function of the F-ATP synthase proteolipid have been replaced during evolution in the V-proteolipid, but in some cases retained in the archaeal A-ATPase. Their possible implication in the evolution of V/F/A-ATPases is discussed.

Chondroitin sulphate-mediated fusion of brain neural folds in rat embryos.

PubMed

Alonso, M I; Moro, J A; Martín, C; de la Mano, A; Carnicero, E; Martínez-Alvarez, C; Navarro, N; Cordero, J; Gato, A

2009-01-01

Previous studies have demonstrated that during neural fold fusion in different species, an apical extracellular material rich in glycoconjugates is involved. However, the composition and the biological role of this material remain undetermined. In this paper, we show that this extracellular matrix in rat increases notably prior to contact between the neural folds, suggesting the dynamic behaviour of the secretory process. Immunostaining has allowed us to demonstrate that this extracellular matrix contains chondroitin sulphate proteoglycan (CSPG), with a spatio-temporal distribution pattern, suggesting a direct relationship with the process of adhesion. The degree of CSPG involvement in cephalic neural fold fusion in rat embryos was determined by treatment with specific glycosidases.In vitro rat embryo culture and microinjection techniques were employed to carry out selective digestion, with chondroitinase AC, of the CSPG on the apical surface of the neural folds; this was done immediately prior to the bonding of the cephalic neural folds. In all the treated embryos, cephalic defects of neural fold fusion could be detected. These results show that CSPG plays an important role in the fusion of the cephalic neural folds in rat embryos, which implies that this proteoglycan could be involved in cellular recognition and adhesion. (c) 2008 S. Karger AG, Basel.

Tonoplast Na+/H+ Antiport Activity and Its Energization by the Vacuolar H+-ATPase in the Halophytic Plant Mesembryanthemum crystallinum L.

PubMed Central

Barkla, B. J.; Zingarelli, L.; Blumwald, E.; Smith, JAC.

1995-01-01

Tonoplast vesicles were isolated from leaf mesophyll tissue of the inducible Crassulacean acid metabolism plant Mesembryanthemum crystallinum to investigate the mechanism of vacuolar Na+ accumulation in this halophilic species. In 8-week-old plants exposed to 200 mM NaCl for 2 weeks, tonoplast H+-ATPase activity was approximately doubled compared with control plants of the same age, as determined by rates of both ATP hydrolysis and ATP-dependent H+ transport. Evidence was also obtained for the presence of an electroneutral Na+/H+ antiporter at the tonoplast that is constitutively expressed, since extravesicular Na+ was able to dissipate a pre-existing transmembrane pH gradient. Initial rates of H+ efflux showed saturation kinetics with respect to extravesicular Na+ concentration and were 2.1-fold higher from vesicles of salt-treated plants compared with the controls. Na+-dependent H+ efflux also showed a high selectivity for Na+ over K+, was insensitive to the transmembrane electrical potential difference, and was more than 50% inhibited by 200 [mu]M N-amidino-3,5-diamino-6-chloropyrazinecarboxamide hydrochloride. The close correlation between increased Na+/H+ antiport and H+-ATPase activities in response to salt treatment suggests that accumulation of the very high concentrations of vacuolar Na+ found in M. crystallinum is energized by the H+ electrochemical gradient across the tonoplast. PMID:12228611

Tonoplast Na+/H+ Antiport Activity and Its Energization by the Vacuolar H+-ATPase in the Halophytic Plant Mesembryanthemum crystallinum L.

PubMed

Barkla, B. J.; Zingarelli, L.; Blumwald, E.; Smith, JAC.

1995-10-01

Tonoplast vesicles were isolated from leaf mesophyll tissue of the inducible Crassulacean acid metabolism plant Mesembryanthemum crystallinum to investigate the mechanism of vacuolar Na+ accumulation in this halophilic species. In 8-week-old plants exposed to 200 mM NaCl for 2 weeks, tonoplast H+-ATPase activity was approximately doubled compared with control plants of the same age, as determined by rates of both ATP hydrolysis and ATP-dependent H+ transport. Evidence was also obtained for the presence of an electroneutral Na+/H+ antiporter at the tonoplast that is constitutively expressed, since extravesicular Na+ was able to dissipate a pre-existing transmembrane pH gradient. Initial rates of H+ efflux showed saturation kinetics with respect to extravesicular Na+ concentration and were 2.1-fold higher from vesicles of salt-treated plants compared with the controls. Na+-dependent H+ efflux also showed a high selectivity for Na+ over K+, was insensitive to the transmembrane electrical potential difference, and was more than 50% inhibited by 200 [mu]M N-amidino-3,5-diamino-6-chloropyrazinecarboxamide hydrochloride. The close correlation between increased Na+/H+ antiport and H+-ATPase activities in response to salt treatment suggests that accumulation of the very high concentrations of vacuolar Na+ found in M. crystallinum is energized by the H+ electrochemical gradient across the tonoplast.

Electron tomographic characterization of a vacuolar reticulum and of six vesicle types that occupy different cytoplasmic domains in the apex of tip-growing Chara rhizoids.

PubMed

Limbach, Christoph; Staehelin, L Andrew; Sievers, Andreas; Braun, Markus

2008-04-01

We provide a 3D ultrastructural analysis of the membrane systems involved in tip growth of rhizoids of the green alga Chara. Electron tomography of cells preserved by high-pressure freeze fixation has enabled us to distinguish six different types of vesicles in the apical cytoplasm where the tip growth machinery is accommodated. The vesicle types are: dark and light secretory vesicles, plasma membrane-associated clathrin-coated vesicles (PM-CCVs), Spitzenkoerper-associated clathrin-coated vesicles (Sp-CCVs) and coated vesicles (Sp-CVs), and microvesicles. Each of these vesicle types exhibits a distinct distribution pattern, which provides insights into their possible function for tip growth. The PM-CCVs are confined to the cytoplasm adjacent to the apical plasma membrane. Within this space they are arranged in clusters often surrounding tubular plasma membrane invaginations from which CCVs bud. This suggests that endocytosis and membrane recycling are locally confined to specialized apical endocytosis sites. In contrast, exocytosis of secretory vesicles occurs over the entire membrane area of the apical dome. The Sp-CCVs and the Sp-CVs are associated with the aggregate of endoplasmic reticulum membranes in the center of the growth-organizing Spitzenkoerper complex. Here, Sp-CCVs are seen to bud from undefined tubular membranes. The subapical region of rhizoids contains a vacuolar reticulum that extends along the longitudinal cell axis and consists of large, vesicle-like segments interconnected by thin tubular domains. The tubular domains are encompassed by thin filamentous structures resembling dynamin spirals which could drive peristaltic movements of the vacuolar reticulum similar to those observed in fungal hyphae. The vacuolar reticulum appears to serve as a lytic compartment into which multivesicular bodies deliver their internal vesicles for molecular recycling and degradation.

EDITORIAL: The Nuclear Fusion Award The Nuclear Fusion Award

NASA Astrophysics Data System (ADS)

Kikuchi, M.

2011-01-01

The Nuclear Fusion Award ceremony for 2009 and 2010 award winners was held during the 23rd IAEA Fusion Energy Conference in Daejeon. This time, both 2009 and 2010 award winners were celebrated by the IAEA and the participants of the 23rd IAEA Fusion Energy Conference. The Nuclear Fusion Award is a paper prize to acknowledge the best distinguished paper among the published papers in a particular volume of the Nuclear Fusion journal. Among the top-cited and highly-recommended papers chosen by the Editorial Board, excluding overview and review papers, and by analyzing self-citation and non-self-citation with an emphasis on non-self-citation, the Editorial Board confidentially selects ten distinguished papers as nominees for the Nuclear Fusion Award. Certificates are given to the leading authors of the Nuclear Fusion Award nominees. The final winner is selected among the ten nominees by the Nuclear Fusion Editorial Board voting confidentially. 2009 Nuclear Fusion Award nominees For the 2009 award, the papers published in the 2006 volume were assessed and the following papers were nominated, most of which are magnetic confinement experiments, theory and modeling, while one addresses inertial confinement. Sabbagh S.A. et al 2006 Resistive wall stabilized operation in rotating high beta NSTX plasmas Nucl. Fusion 46 635-44 La Haye R.J. et al 2006 Cross-machine benchmarking for ITER of neoclassical tearing mode stabilization by electron cyclotron current drive Nucl. Fusion 46 451-61 Honrubia J.J. et al 2006 Three-dimensional fast electron transport for ignition-scale inertial fusion capsules Nucl. Fusion 46 L25-8 Ido T. et al 2006 Observation of the interaction between the geodesic acoustic mode and ambient fluctuation in the JFT-2M tokamak Nucl. Fusion 46 512-20 Plyusnin V.V. et al 2006 Study of runaway electron generation during major disruptions in JET Nucl. Fusion 46 277-84 Pitts R.A. et al 2006 Far SOL ELM ion energies in JET Nucl. Fusion 46 82-98 Berk H.L. et al 2006

Fusion

NASA Astrophysics Data System (ADS)

Herman, Robin

1990-10-01

The book abounds with fascinating anecdotes about fusion's rocky path: the spurious claim by Argentine dictator Juan Peron in 1951 that his country had built a working fusion reactor, the rush by the United States to drop secrecy and publicize its fusion work as a propaganda offensive after the Russian success with Sputnik; the fortune Penthouse magazine publisher Bob Guccione sank into an unconventional fusion device, the skepticism that met an assertion by two University of Utah chemists in 1989 that they had created "cold fusion" in a bottle. Aimed at a general audience, the book describes the scientific basis of controlled fusion--the fusing of atomic nuclei, under conditions hotter than the sun, to release energy. Using personal recollections of scientists involved, it traces the history of this little-known international race that began during the Cold War in secret laboratories in the United States, Great Britain and the Soviet Union, and evolved into an astonishingly open collaboration between East and West.

New interatomic potentials of W, Re and W-Re alloy for radiation defects

NASA Astrophysics Data System (ADS)

Chen, Yangchun; Li, Yu-Hao; Gao, Ning; Zhou, Hong-Bo; Hu, Wangyu; Lu, Guang-Hong; Gao, Fei; Deng, Huiqiu

2018-04-01

Tungsten (W) and W-based alloys have been considered as promising candidates for plasma-facing materials (PFMs) in future fusion reactors. The formation of rhenium (Re)-rich clusters and intermetallic phases due to high energy neutron irradiation and transmutations significantly induces the hardening and embrittlement of W. In order to better understand these phenomena, in the present work, new interatomic potentials of W-W, Re-Re and W-Re, suitable for description of radiation defects in such alloys, have been developed. The fitted potentials not only reproduce the results of the formation energy, binding energy and migration energy of various radiation defects and the physical properties from the extended database obtained from DFT calculations, but also predict well the relative stability of different interstitial dislocation loops in W, as reported in experiments. These potentials are applicable for describing the evolution of defects in W and W-Re alloys, thus providing a possibility for the detailed understanding of the precipitation mechanism of Re in W under irradiation.

Dual-Tree Complex Wavelet Transform and Image Block Residual-Based Multi-Focus Image Fusion in Visual Sensor Networks

PubMed Central

Yang, Yong; Tong, Song; Huang, Shuying; Lin, Pan

2014-01-01

This paper presents a novel framework for the fusion of multi-focus images explicitly designed for visual sensor network (VSN) environments. Multi-scale based fusion methods can often obtain fused images with good visual effect. However, because of the defects of the fusion rules, it is almost impossible to completely avoid the loss of useful information in the thus obtained fused images. The proposed fusion scheme can be divided into two processes: initial fusion and final fusion. The initial fusion is based on a dual-tree complex wavelet transform (DTCWT). The Sum-Modified-Laplacian (SML)-based visual contrast and SML are employed to fuse the low- and high-frequency coefficients, respectively, and an initial composited image is obtained. In the final fusion process, the image block residuals technique and consistency verification are used to detect the focusing areas and then a decision map is obtained. The map is used to guide how to achieve the final fused image. The performance of the proposed method was extensively tested on a number of multi-focus images, including no-referenced images, referenced images, and images with different noise levels. The experimental results clearly indicate that the proposed method outperformed various state-of-the-art fusion methods, in terms of both subjective and objective evaluations, and is more suitable for VSNs. PMID:25587878

Dual-tree complex wavelet transform and image block residual-based multi-focus image fusion in visual sensor networks.

PubMed

Yang, Yong; Tong, Song; Huang, Shuying; Lin, Pan

2014-11-26

This paper presents a novel framework for the fusion of multi-focus images explicitly designed for visual sensor network (VSN) environments. Multi-scale based fusion methods can often obtain fused images with good visual effect. However, because of the defects of the fusion rules, it is almost impossible to completely avoid the loss of useful information in the thus obtained fused images. The proposed fusion scheme can be divided into two processes: initial fusion and final fusion. The initial fusion is based on a dual-tree complex wavelet transform (DTCWT). The Sum-Modified-Laplacian (SML)-based visual contrast and SML are employed to fuse the low- and high-frequency coefficients, respectively, and an initial composited image is obtained. In the final fusion process, the image block residuals technique and consistency verification are used to detect the focusing areas and then a decision map is obtained. The map is used to guide how to achieve the final fused image. The performance of the proposed method was extensively tested on a number of multi-focus images, including no-referenced images, referenced images, and images with different noise levels. The experimental results clearly indicate that the proposed method outperformed various state-of-the-art fusion methods, in terms of both subjective and objective evaluations, and is more suitable for VSNs.

Accumulation of specific sterol precursors targets a MAP kinase cascade mediating cell-cell recognition and fusion.

PubMed

Weichert, Martin; Lichius, Alexander; Priegnitz, Bert-Ewald; Brandt, Ulrike; Gottschalk, Johannes; Nawrath, Thorben; Groenhagen, Ulrike; Read, Nick D; Schulz, Stefan; Fleißner, André

2016-10-18

Sterols are vital components of eukaryotic cell membranes. Defects in sterol biosynthesis, which result in the accumulation of precursor molecules, are commonly associated with cellular disorders and disease. However, the effects of these sterol precursors on the metabolism, signaling, and behavior of cells are only poorly understood. In this study, we show that the accumulation of only ergosterol precursors with a conjugated double bond in their aliphatic side chain specifically disrupts cell-cell communication and fusion in the fungus Neurospora crassa Genetically identical germinating spores of this fungus undergo cell-cell fusion, thereby forming a highly interconnected supracellular network during colony initiation. Before fusion, the cells use an unusual signaling mechanism that involves the coordinated and alternating switching between signal sending and receiving states of the two fusion partners. Accumulation of only ergosterol precursors with a conjugated double bond in their aliphatic side chain disrupts this coordinated cell-cell communication and suppresses cell fusion. These specific sterol precursors target a single ERK-like mitogen-activated protein (MAP) kinase (MAK-1)-signaling cascade, whereas a second MAP kinase pathway (MAK-2), which is also involved in cell fusion, is unaffected. These observations indicate that a minor specific change in sterol structure can exert a strong detrimental effect on a key signaling pathway of the cell, resulting in the absence of cell fusion.

Accumulation of specific sterol precursors targets a MAP kinase cascade mediating cell–cell recognition and fusion

PubMed Central

Weichert, Martin; Lichius, Alexander; Priegnitz, Bert-Ewald; Brandt, Ulrike; Gottschalk, Johannes; Nawrath, Thorben; Groenhagen, Ulrike; Read, Nick D.; Schulz, Stefan; Fleißner, André

2016-01-01

Sterols are vital components of eukaryotic cell membranes. Defects in sterol biosynthesis, which result in the accumulation of precursor molecules, are commonly associated with cellular disorders and disease. However, the effects of these sterol precursors on the metabolism, signaling, and behavior of cells are only poorly understood. In this study, we show that the accumulation of only ergosterol precursors with a conjugated double bond in their aliphatic side chain specifically disrupts cell–cell communication and fusion in the fungus Neurospora crassa. Genetically identical germinating spores of this fungus undergo cell–cell fusion, thereby forming a highly interconnected supracellular network during colony initiation. Before fusion, the cells use an unusual signaling mechanism that involves the coordinated and alternating switching between signal sending and receiving states of the two fusion partners. Accumulation of only ergosterol precursors with a conjugated double bond in their aliphatic side chain disrupts this coordinated cell–cell communication and suppresses cell fusion. These specific sterol precursors target a single ERK-like mitogen-activated protein (MAP) kinase (MAK-1)-signaling cascade, whereas a second MAP kinase pathway (MAK-2), which is also involved in cell fusion, is unaffected. These observations indicate that a minor specific change in sterol structure can exert a strong detrimental effect on a key signaling pathway of the cell, resulting in the absence of cell fusion. PMID:27708165

Prediction of olive oil sensory descriptors using instrumental data fusion and partial least squares (PLS) regression.

PubMed

Borràs, Eva; Ferré, Joan; Boqué, Ricard; Mestres, Montserrat; Aceña, Laura; Calvo, Angels; Busto, Olga

2016-08-01

Headspace-Mass Spectrometry (HS-MS), Fourier Transform Mid-Infrared spectroscopy (FT-MIR) and UV-Visible spectrophotometry (UV-vis) instrumental responses have been combined to predict virgin olive oil sensory descriptors. 343 olive oil samples analyzed during four consecutive harvests (2010-2014) were used to build multivariate calibration models using partial least squares (PLS) regression. The reference values of the sensory attributes were provided by expert assessors from an official taste panel. The instrumental data were modeled individually and also using data fusion approaches. The use of fused data with both low- and mid-level of abstraction improved PLS predictions for all the olive oil descriptors. The best PLS models were obtained for two positive attributes (fruity and bitter) and two defective descriptors (fusty and musty), all of them using data fusion of MS and MIR spectral fingerprints. Although good predictions were not obtained for some sensory descriptors, the results are encouraging, specially considering that the legal categorization of virgin olive oils only requires the determination of fruity and defective descriptors. Copyright © 2016 Elsevier B.V. All rights reserved.

Vacuolar Localization of Endoproteinases EP(1) and EP(2) in Barley Mesophyll Cells.

PubMed

Thayer, S S; Huffaker, R C

1984-05-01

The localization of two previously characterized endoproteinases (EP(1) and EP(2)) that comprise more than 95% of the protease activity in primary Hordeum vulgare L. var Numar leaves was determined. Intact vacuoles released from washed mesophyll protoplasts by gentle osmotic shock and increase in pH, were purified by flotation through a four-step Ficoll gradient. These vacuoles contained endoproteinases that rapidly degraded purified barley ribulose-1,5-bisphosphate carboxylase (RuBPCase) substrate. Breakdown products and extent of digestion of RuBPCase were determined using 12% polyacrylamide-sodium dodecyl sulfate gels. Coomassie brilliant blue- or silver-stained gels were scanned, and the peaks were integrated to provide quantitative information. The characteristics of the vacuolar endoproteinases (e.g. sensitivity to various inhibitors and activators, and the molecular weights of the breakdown products, i.e. peptide maps) closely resembled those of purified EP(1) and partially purified EP(2). It is therefore concluded that EP(1) and EP(2) are localized in the vacuoles of mesophyll cells.

Molecular Basis of ADP Inhibition of Vacuolar (V)-type ATPase/Synthase*

PubMed Central

Kishikawa, Jun-ichi; Nakanishi, Atsuko; Furuike, Shou; Tamakoshi, Masatada; Yokoyama, Ken

2014-01-01

Reduction of ATP hydrolysis activity of vacuolar-type ATPase/synthase (V0V1) as a result of ADP inhibition occurs as part of the normal mechanism of V0V1 of Thermus thermophilus but not V0V1 of Enterococcus hirae or eukaryotes. To investigate the molecular basis for this difference, domain-swapped chimeric V1 consisting of both T. thermophilus and E. hirae enzymes were generated, and their function was analyzed. The data showed that the interaction between the nucleotide binding and C-terminal domains of the catalytic A subunit from E. hirae V1 is central to increasing binding affinity of the chimeric V1 for phosphate, resulting in reduction of the ADP inhibition. These findings together with a comparison of the crystal structures of T. thermophilus V1 with E. hirae V1 strongly suggest that the A subunit adopts a conformation in T. thermophilus V1 different from that in E. hirae V1. This key difference results in ADP inhibition of T. thermophilus V1 by abolishing the binding affinity for phosphate during ATP hydrolysis. PMID:24247239

Tks5-dependent formation of circumferential podosomes/invadopodia mediates cell-cell fusion.

PubMed

Oikawa, Tsukasa; Oyama, Masaaki; Kozuka-Hata, Hiroko; Uehara, Shunsuke; Udagawa, Nobuyuki; Saya, Hideyuki; Matsuo, Koichi

2012-05-14

Osteoclasts fuse to form multinucleated cells during osteoclastogenesis. This process is mediated by dynamic rearrangement of the plasma membrane and cytoskeleton, and it requires numerous factors, many of which have been identified. The underlying mechanism remains obscure, however. In this paper, we show that Tks5, a master regulator of invadopodia in cancer cells, is crucial for osteoclast fusion downstream of phosphoinositide 3-kinase and Src. Expression of Tks5 was induced during osteoclastogenesis, and prevention of this induction impaired both the formation of circumferential podosomes and osteoclast fusion without affecting cell differentiation. Tyrosine phosphorylation of Tks5 was attenuated in Src-/- osteoclasts, likely accounting for defects in podosome organization and multinucleation in these cells. Circumferential invadopodia formation in B16F0 melanoma cells was also accompanied by Tks5 phosphorylation. Co-culture of B16F0 cells with osteoclasts in an inflammatory milieu promoted the formation of melanoma-osteoclast hybrid cells. Our results thus reveal an unexpected link between circumferential podosome/invadopodium formation and cell-cell fusion in and beyond osteoclasts.

Tks5-dependent formation of circumferential podosomes/invadopodia mediates cell–cell fusion

PubMed Central

Oyama, Masaaki; Kozuka-Hata, Hiroko; Uehara, Shunsuke; Udagawa, Nobuyuki; Saya, Hideyuki; Matsuo, Koichi

2012-01-01

Osteoclasts fuse to form multinucleated cells during osteoclastogenesis. This process is mediated by dynamic rearrangement of the plasma membrane and cytoskeleton, and it requires numerous factors, many of which have been identified. The underlying mechanism remains obscure, however. In this paper, we show that Tks5, a master regulator of invadopodia in cancer cells, is crucial for osteoclast fusion downstream of phosphoinositide 3-kinase and Src. Expression of Tks5 was induced during osteoclastogenesis, and prevention of this induction impaired both the formation of circumferential podosomes and osteoclast fusion without affecting cell differentiation. Tyrosine phosphorylation of Tks5 was attenuated in Src−/− osteoclasts, likely accounting for defects in podosome organization and multinucleation in these cells. Circumferential invadopodia formation in B16F0 melanoma cells was also accompanied by Tks5 phosphorylation. Co-culture of B16F0 cells with osteoclasts in an inflammatory milieu promoted the formation of melanoma–osteoclast hybrid cells. Our results thus reveal an unexpected link between circumferential podosome/invadopodium formation and cell–cell fusion in and beyond osteoclasts. PMID:22584907

Proton pump inhibitors as anti vacuolar-ATPases drugs: a novel anticancer strategy.

PubMed

Spugnini, Enrico P; Citro, Gennaro; Fais, Stefano

2010-05-08

The vacuolar ATPases are ATP-dependent proton pumps whose functions include the acidification of intracellular compartments and the extrusion of protons through the cell cytoplasmic membrane. These pumps play a pivotal role in the regulation of cell pH in normal cells and, to a much greater extent, in tumor cells. In fact, the glucose metabolism in hypoxic conditions by the neoplasms leads to an intercellular pH drift towards acidity. The acid microenvironment is modulated through the over-expression of H+ transporters that are also involved in tumor progression, invasiveness, distant spread and chemoresistance. Several strategies to block/downmodulate the efficiency of these transporters are currently being investigated. Among them, proton pump inhibitors have shown to successfully block the H+ transporters in vitro and in vivo, leading to apoptotic death. Furthermore, their action seems to synergize with conventional chemotherapy protocols, leading to chemosensitization and reversal of chemoresistance. Aim of this article is to critically revise the current knowledge of this cellular machinery and to summarize the therapeutic strategies developed to counter this mechanism.

An Arf-GAP promotes endocytosis and hyphal growth of Ashbya gossypii.

PubMed

Oscarsson, Therese; Walther, Andrea; Lengeler, Klaus B; Wendland, Jürgen

2017-12-29

The ADP-ribosylation factor (ARF) family of GTPases are highly conserved from yeast to human and regulate vesicle budding. Sec7 domain containing proteins stimulate the guanine nucleotide exchange on Arf proteins, while ARF-GTPase activating proteins stimulate the hydrolysis of GTP. Since vesicle trafficking is important for hyphal growth, we studied the Ashbya gossypii homolog of Saccharomyces cerevisiae ARF3 along with its putative GEF and GTPase-activating protein (GAP) encoded by YEL1 and GTS1, respectively. Deletion of YEL1 had no discernible phenotype and deletion of ARF3 had only a minor defect in vacuolar fusion. In contrast, deletion of GTS1 severely impaired hyphal growth, and mutants showed defects in the maintenance of polarity and the localization of cortical actin patches. The uptake of the lipophilic dye FM4-64 was delayed in gts1 hyphae, indicating a defect in endocytosis. Gts1 has several protein domains, of which the Arf-GAP domain is required for complementation of the gts1 mutant phenotype. GFP-tagged GTS1 under control of its endogenous promoter localized to the plasma membrane but was enriched at hyphal tips and septal sites corresponding to a role in polarized vesicle trafficking. Our results indicate that this ARF-GTPase module plays an important role for filamentous hyphal growth. © FEMS 2017. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

Addressing Common Technical challenges in Inertial Confinement Fusion

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

Haynes, Donald A.

2016-09-22

The implosion phase for Inertial Confinement Fusion (ICF) occurs from initiation of the drive until just before stagnation. Evolution of the shell and fusion fuel during the implosion phase is affected by the initial conditions of the target, the drive history. Poor performing implosions are a result of the behavior that occurs during the implosion phase such as low mode asymmetries, mixing of the ablator into the fuel, and the hydrodynamic evolution of initial target features and defects such as the shell mounting hardware. The ultimate results of these effects can only be measured at stagnation. However, studying the implosionmore » phase can be effective for understanding and mitigating these effects and for of ultimately improving the performance of ICF implosions. As the ICF program moves towards the 2020 milestone to “determine the efficacy of ignition”, it will be important to understand the physics that occurs during the implosion phase. This will require both focused and integrated experiments. Focused experiments will provide the understanding and the evidence needed to support any determination concerning the efficacy of ignition.« less

Molecular cloning, functional expression and subcellular localization of two putative vacuolar voltage-gated chloride channels in rice (Oryza sativa L.).

PubMed

Nakamura, Atsuko; Fukuda, Atsunori; Sakai, Shingo; Tanaka, Yoshiyuki

2006-01-01

We isolated two cDNA clones (OsCLC-1 and OsCLC-2) homologous to tobacco CLC-Nt1, which encodes a voltage-gated chloride channel, from rice (Oryza sativa L. ssp. japonica, cv. Nipponbare). The deduced amino acid sequences were highly conserved (87.9% identity with each other). Southern blot analysis of the rice genomic DNA revealed that OsCLC-1 and OsCLC-2 were single-copy genes on chromosomes 4 and 2, respectively. OsCLC-1 was expressed in most tissues, whereas OsCLC-2 was expressed only in the roots, nodes, internodes and leaf sheaths. The level of expression of OsCLC-1, but not of OsCLC-2, was increased by treatment with NaCl. Both genes could partly substitute for GEF1, which encodes the sole chloride channel in yeast, by restoring growth under ionic stress. These results indicate that both genes are chloride channel genes. The proteins from both genes were immunochemically detected in the tonoplast fraction. Tagged synthetic green fluorescent protein which was fused to OsCLC-1 or OsCLC-2 localized in the vacuolar membranes. These results indicate that the proteins may play a role in the transport of chloride ions across the vacuolar membrane. We isolated loss-of-function mutants of both genes from a panel of rice mutants produced by the insertion of a retrotransposon, Tos17, in the exon region, and found inhibition of growth at all life stages.

Clinical features of avian vacuolar myelinopathy in American coots

USGS Publications Warehouse

Larsen, R.S.; Nutter, F.B.; Augspurger, T.; Rocke, T.E.; Tomlinson, L.; Thomas, N.J.; Stoskopf, M.K.

2002-01-01

Objectivea??To characterize clinical features of avian vacuolar myelinopathy (AVM) in American coots. Designa??Case-control study. Animalsa??26 AVM-affected American coots and 12 unaffected coots. Proceduresa??Complete physical, neurologic, hematologic, and plasma biochemical evaluations were performed. Affected coots received supportive care. All coots died or were euthanatized, and AVM status was confirmed via histopathologic findings. Resultsa??3 severely affected coots were euthanatized immediately after examination. Seventeen affected coots were found dead within 7 days of admission, but 5 affected coots survived > 21 days and had signs of clinical recovery. Abnormal physical examination findings appeared to be related to general debilitation. Ataxia (88%), decreased withdrawal reflexes (88%), proprioceptive deficits (81%), decreased vent responses (69%), beak or tongue weakness (42%), and head tremors (31%), as well as absent pupillary light responses (46%), anisocoria (15%), apparent blindness (4%), nystagmus (4%), and strabismus (4%) were detected. Few gross abnormalities were detected at necropsy, but histologically, all AVM-affected coots had severe vacuolation of white matter of the brain. None of the control coots had vacuolation. Conclusions and Clinical Relevancea??Although there was considerable variability in form and severity of clinical neurologic abnormalities, clinical signs common in AVM-affected birds were identified. Clinical recovery of some AVM-affected coots can occur when supportive care is administered. Until the etiology is identified, caution should be exercised when rehabilitating and releasing coots thought to be affected by AVM.

A strategy for targeting recombinant proteins to protein storage vacuoles by fusion to Brassica napus napin in napin-depleted seeds.

PubMed

Hegedus, Dwayne D; Baron, Marcus; Labbe, Natalie; Coutu, Cathy; Lydiate, Derek; Lui, Helen; Rozwadowski, Kevin

2014-03-01

Seeds are capable of accumulating high levels of seed storage proteins (SSP), as well as heterologous proteins under certain conditions. Arabidopsis thaliana was used to develop a strategy to deplete seeds of an endogenous SSP and then replenish them with the same protein fused to a heterologous protein. In several other studies, competition with endogenous SSP for space and metabolic resources was shown to affect the accumulation of recombinant proteins in seeds. We used RNAi to reduce the expression of the five napin genes and deplete the seeds of this SSP. Targeting a recombinant protein to a vacuole or structure within the seed where it can be protected from cytosolic proteases can also promote its accumulation. To achieve this, a synthetic Brassica napus napin gene (Bn napin) was designed that was both impervious to the A. thaliana napin (At napin) RNAi construct and permitted fusion to a heterologous protein, in this case green fluorescent protein (GFP). GFP was placed in several strategic locations within Bn napin with consideration to maintaining structure, processing sites and possible vacuolar targeting signals. In transgenic A. thaliana plants, GFP was strongly localized to the seed protein storage vacuole in all Bn napin fusion configurations tested, but not when expressed alone. This SSP depletion-replenishment strategy outlined here would be applicable to expression of recombinant proteins in industrial crops that generally have large repertoires of endogenous SSP genes. Crown Copyright © 2014. Published by Elsevier Inc. All rights reserved.

High Level Information Fusion (HLIF) with nested fusion loops

NASA Astrophysics Data System (ADS)

Woodley, Robert; Gosnell, Michael; Fischer, Amber

2013-05-01

Situation modeling and threat prediction require higher levels of data fusion in order to provide actionable information. Beyond the sensor data and sources the analyst has access to, the use of out-sourced and re-sourced data is becoming common. Through the years, some common frameworks have emerged for dealing with information fusion—perhaps the most ubiquitous being the JDL Data Fusion Group and their initial 4-level data fusion model. Since these initial developments, numerous models of information fusion have emerged, hoping to better capture the human-centric process of data analyses within a machine-centric framework. 21st Century Systems, Inc. has developed Fusion with Uncertainty Reasoning using Nested Assessment Characterizer Elements (FURNACE) to address challenges of high level information fusion and handle bias, ambiguity, and uncertainty (BAU) for Situation Modeling, Threat Modeling, and Threat Prediction. It combines JDL fusion levels with nested fusion loops and state-of-the-art data reasoning. Initial research has shown that FURNACE is able to reduce BAU and improve the fusion process by allowing high level information fusion (HLIF) to affect lower levels without the double counting of information or other biasing issues. The initial FURNACE project was focused on the underlying algorithms to produce a fusion system able to handle BAU and repurposed data in a cohesive manner. FURNACE supports analyst's efforts to develop situation models, threat models, and threat predictions to increase situational awareness of the battlespace. FURNACE will not only revolutionize the military intelligence realm, but also benefit the larger homeland defense, law enforcement, and business intelligence markets.

Enhanced salt stress tolerance of rice plants expressing a vacuolar H+-ATPase subunit c1 (SaVHAc1) gene from the halophyte grass Spartina alterniflora Löisel

USDA-ARS?s Scientific Manuscript database

The physiological role of a vacuolar ATPase subunit c1 (SaVHAc1) from a halophyte grass Spartina alterniflora was studied through its expression in rice. The SaVHAc1– expressing plants showed enhanced tolerance to salt stress than the wild-type plants, mainly through adjustments in early stage and p...

Reconstruction of juxta-articular huge defects of distal femur with vascularized fibular bone graft and Ilizarov's distraction osteogenesis.

PubMed

Lai, Davy; Chen, Chuan-Mu; Chiu, Fang-Yao; Chang, Ming-Chau; Chen, Tain-Hsiung

2007-01-01

We evaluate the effect of reconstructing huge defects (mean, 15.8 cm) of the distal femur with Ilizarov's distraction osteogenesis and free twin-barreled vascularized fibular bone graft (TVFG). We retrospectively reviewed a consecutive series of five patients who had cases of distal femoral fractures with huge defects and infection that were treated by the Ilizarov's distraction osteogenesis. After radical debridement, two of the five cases had free TVFG and monolocal distraction osteogenesis, and another two cases had multilocal distraction osteogenesis with knee fusion because of loss of the joint congruity. The other case with floating knee injury had bilocal distraction osteogenesis and a preserved knee joint. The mean defect of distal femur was 15.8 cm (range, 14-18 cm) in length. The mean length of distraction osteogenesis by Ilizarov's apparatus was 8.2 cm. The mean length of TVFG was 8 cm. The average duration from application of Ilizarov's apparatus to achievement of bony union was 10.2 months (range, 8-13 months). At the end of the follow-up, ranges of motion of three knees were 0 to 45 degrees, 0 to 60 degrees, and 0 to 90 degrees. Two cases had knee arthrodesis with bony fusion because of loss of the joint congruity. There were no leg length discrepancies in all five patients. In addition, three patients had pin tract infections and one case had a 10 degree varus deformity of the femur. Juxta-articular huge defect (>10 cm) of distal femur remains a challenge to orthopedic surgeons. Ilizarov's technique provides the capability to maintain stability, eradicate infection, restore leg length, and to perform adjuvant reconstructive procedure easily. In this study, we found that combining Ilizarov's distraction osteogenesis with TVFG results in improved patient outcome for patients with injuries such as supracondylar or intercondylar infected fractures or nonunion of distal femur with huge bone defect.

Critical Roles of Vacuolar Invertase in Floral Organ Development and Male and Female Fertilities Are Revealed through Characterization of GhVIN1-RNAi Cotton Plants.

PubMed

Wang, Lu; Ruan, Yong-Ling

2016-05-01

Seed number and quality are key traits determining plant fitness and crop yield and rely on combined competence in male and female fertilities. Sucrose metabolism is central to reproductive success. It remains elusive, though, how individual sucrose metabolic enzymes may regulate the complex reproductive processes. Here, by silencing vacuolar invertase (VIN) genes in cotton (Gossypium hirsutum) reproductive organs, we revealed diverse roles that VIN plays in multiple reproductive processes. A set of phenotypic and genetic studies showed significant reductions of viable seeds in GhVIN1-RNAi plants, attributed to pollination failure and impaired male and female fertilities. The former was largely owing to the spatial mismatch between style and stamen and delayed pollen release from the anthers, whereas male defects came from poor pollen viability. The transgenic stamen exhibited altered expression of the genes responsible for starch metabolism and auxin and jasmonic acid signaling. Further analyses identified the reduction of GhVIN expression in the seed coat as the major cause for the reduced female fertility, which appeared to disrupt the expression of some key genes involved in trehalose and auxin metabolism and signaling, leading to programmed cell death or growth repression in the filial tissues. Together, the data provide an unprecedented example of how VIN is required to synchronize style and stamen development and the formation of male and female fertilities for seed development in a crop species, cotton. © 2016 American Society of Plant Biologists. All Rights Reserved.

Potential Pitfalls and Solutions for Use of Fluorescent Fusion Proteins to Study the Lysosome

PubMed Central

Huang, Ling; Pike, Douglas; Sleat, David E.; Nanda, Vikas; Lobel, Peter

2014-01-01

Use of fusion protein tags to investigate lysosomal proteins can be complicated by the acidic, protease-rich environment of the lysosome. Potential artifacts include degradation or release of the tag and acid quenching of fluorescence. Tagging can also affect protein folding, glycosylation and/or trafficking. To specifically investigate the use of fluorescent tags to reveal lysosomal localization, we tested mCherry derivatives as C-terminal tags for Niemann-Pick disease type C protein 2 (NPC2), a luminal lysosomal protein. Full-length mCherry was released from the NPC2 chimera while deletion of the 11 N-terminal residues of mCherry generated a cleavage-resistant (cr) fluorescent variant. Insertion of proline linkers between NPC2 and crmCherry had little effect while Gly-Ser linkers promoted cleavage. The NPC2-crmCherry fusion was targeted to the lysosome and restored function in NPC2-deficient cells. Fusion of crmCherry to known and candidate lysosomal proteins revealed that the linkers had different effects on lysosomal localization. Direct fusion of crmCherry impaired mannose 6-phosphorylation and lysosomal targeting of the lysosomal protease tripeptidyl peptidase I (TPP1), while insertion of linkers corrected the defects. Molecular modeling suggested structural bases for the effects of different linkers on NPC2 and TPP1 fusion proteins. While mCherry fusion proteins can be useful tools for studying the lysosome and related organelles, our findings underscore the potential artifacts associated with such applications. PMID:24586430

Investigation of defect modes in a defective photonic crystal with a semiconductor metamaterial defect

NASA Astrophysics Data System (ADS)

Wu, Meng-Ru; Wu, Chien-Jang; Chang, Shoou-Jinn

2014-11-01

In this work, we theoretically investigate the properties of defect modes in a defective photonic crystal containing a semiconductor metamaterial defect. We consider the structure, (LH)N/DP/(LH)N, where N and P are respectively the stack numbers, L is SiO2, H is InP, and defect layer D is a semiconductor metamaterial composed of Al-doped ZnO (AZO) and ZnO. It is found that, within the photonic band gap, the number of defect modes (transmission peaks) will decrease as the defect thickness increases, in sharp contrast to the case of using usual dielectric defect. The peak height and position can be changed by the variation in the thickness of defect layer. In the angle-dependent defect mode, its position is shown to be blue-shifted as the angle of incidence increases for both TE and TM waves. The analysis of defect mode provides useful information for the design of tunable transmission filter in semiconductor optoelectronics.

Advanced phase change composite by thermally annealed defect-free graphene for thermal energy storage.

PubMed

Xin, Guoqing; Sun, Hongtao; Scott, Spencer Michael; Yao, Tiankai; Lu, Fengyuan; Shao, Dali; Hu, Tao; Wang, Gongkai; Ran, Guang; Lian, Jie

2014-09-10

Organic phase change materials (PCMs) have been utilized as latent heat energy storage and release media for effective thermal management. A major challenge exists for organic PCMs in which their low thermal conductivity leads to a slow transient temperature response and reduced heat transfer efficiency. In this work, 2D thermally annealed defect-free graphene sheets (GSs) can be obtained upon high temperature annealing in removing defects and oxygen functional groups. As a result of greatly reduced phonon scattering centers for thermal transport, the incorporation of ultralight weight and defect free graphene applied as nanoscale additives into a phase change composite (PCC) drastically improve thermal conductivity and meanwhile minimize the reduction of heat of fusion. A high thermal conductivity of the defect-free graphene-PCC can be achieved up to 3.55 W/(m K) at a 10 wt % graphene loading. This represents an enhancement of over 600% as compared to pristine graphene-PCC without annealing at a comparable loading, and a 16-fold enhancement than the pure PCM (1-octadecanol). The defect-free graphene-PCC displays rapid temperature response and superior heat transfer capability as compared to the pristine graphene-PCC or pure PCM, enabling transformational thermal energy storage and management.

Graphene's Viability for Fusion Applications

NASA Astrophysics Data System (ADS)

Navarro, Marcos; Hall, Karla; Rojas, Richard; Santarius, John; Kulcinski, Gerald

2015-11-01

Graphene is a source of interest for multiple applications due to its unusual electronic and physical properties. As a coating material, it has reduced oxidation of the main substrate, though no effort has been reported of testing it under fusion conditions. A number of experimental studies have established that defect-free graphene is an excellent barrier material for gases. We explore its viability to maintain a significant pressure difference under ion irradiation. Deuterium is used as a projectile on graphene coated silicon over a range of 10-50 keV energies and various fluences. The vacancy yield (amount of damage) and natural resonance for graphene are found at around 1350 cm-1 and 1550 cm-1, respectively. Damage of each sample is quantified via Raman spectroscopy (RS) using the ratio of the intensities at these wavenumbers. Graphene is also tested here as a coating for some fusion components. Though tungsten is a very promising divertor and first wall candidate, after intense irradiation, it is prone to developing fuzz or grass structures, leading to a diminished lifetime. Graphene grown on tungsten is tested under reactor conditions with 30 keV He ions at several fluences, and the sputtering of both materials is studied via RS and Scanning Electron Microscopy. This work was supported by the Graduate Engineering Research Scholars and the TEAM-Science program at the University of Wisconsin-Madison.

The membrane fusion enigma: SNAREs, Sec1/Munc18 proteins, and their accomplices--guilty as charged?

PubMed

Rizo, Josep; Südhof, Thomas C

2012-01-01

Neurotransmitter release is governed by proteins that have homo-logs in most types of intracellular membrane fusion, including the Sec1/Munc18 protein Munc18-1 and the SNARE proteins syntaxin-1, synaptobrevin/VAMP, and SNAP-25. The SNAREs initiate fusion by forming tight SNARE complexes that bring the vesicle and plasma membranes together. SNARE maintenance in a functional state depends on two chaperone systems (Hsc70/αCSP/SGT and synuclein); defects in these systems lead to neurodegeneration. Munc18-1 binds to an autoinhibitory closed conformation of syntaxin-1, gating formation of SNARE complexes, and also binds to SNARE complexes, which likely underlies the crucial function of Munc18-1 in membrane fusion by an as-yet unclear mechanism. Syntaxin-1 opening is mediated by Munc13s through their MUN domain, which is homologous to diverse tethering factors and may also have a general role in fusion. MUN domain activity is likely modulated in diverse presynaptic plasticity processes that depend on Ca(2+) and RIM proteins, among others.

The human immunodeficiency virus antigen Nef forms protein bodies in leaves of transgenic tobacco when fused to zeolin

PubMed Central

de Virgilio, Maddalena; Bellucci, Michele; Mainieri, Davide; Rossi, Marika; Benvenuto, Eugenio; Arcioni, Sergio; Vitale, Alessandro

2008-01-01

Protein bodies (PB) are stable polymers naturally formed by certain seed storage proteins within the endoplasmic reticulum (ER). The human immunodeficiency virus negative factor (Nef) protein, a potential antigen for the development of an anti-viral vaccine, is highly unstable when introduced into the plant secretory pathway, probably because of folding defects in the ER environment. The aim of this study was to promote the formation of Nef-containing PB in tobacco (Nicotiana tabacum) leaves by fusing the Nef sequence to the N-terminal domains of the maize storage protein γ-zein or to the chimeric protein zeolin (which efficiently forms PB and is composed of the vacuolar storage protein phaseolin fused to the N-terminal domains of γ-zein). Protein blots and pulse–chase indicate that fusions between Nef and the same γ-zein domains present in zeolin are degraded by ER quality control. Consistently, a mutated zeolin, in which wild-type phaseolin was substituted with a defective version known to be degraded by ER quality control, is unstable in plant cells. Fusion of Nef to the entire zeolin sequence instead allows the formation of PB detectable by electron microscopy and subcellular fractionation, leading to zeolin–Nef accumulation higher than 1% of total soluble protein, consistently reproduced in independent transgenic plants. It is concluded that zeolin, but not its γ-zein portion, has a positive dominant effect over ER quality control degradation. These results provide insights into the requirements for PB formation and avoidance of quality-control degradation, and indicate a strategy for enhancing foreign protein accumulation in plants. PMID:18540021

Non-canonical role of the SNARE protein Ykt6 in autophagosome-lysosome fusion

PubMed Central

Takáts, Szabolcs; Glatz, Gábor; Szenci, Győző; Boda, Attila; Horváth, Gábor V.; Hegedűs, Krisztina; Kovács, Attila L.

2018-01-01

The autophagosomal SNARE Syntaxin17 (Syx17) forms a complex with Snap29 and Vamp7/8 to promote autophagosome-lysosome fusion via multiple interactions with the tethering complex HOPS. Here we demonstrate that, unexpectedly, one more SNARE (Ykt6) is also required for autophagosome clearance in Drosophila. We find that loss of Ykt6 leads to large-scale accumulation of autophagosomes that are unable to fuse with lysosomes to form autolysosomes. Of note, loss of Syx5, the partner of Ykt6 in ER-Golgi trafficking does not prevent autolysosome formation, pointing to a more direct role of Ykt6 in fusion. Indeed, Ykt6 localizes to lysosomes and autolysosomes, and forms a SNARE complex with Syx17 and Snap29. Interestingly, Ykt6 can be outcompeted from this SNARE complex by Vamp7, and we demonstrate that overexpression of Vamp7 rescues the fusion defect of ykt6 loss of function cells. Finally, a point mutant form with an RQ amino acid change in the zero ionic layer of Ykt6 protein that is thought to be important for fusion-competent SNARE complex assembly retains normal autophagic activity and restores full viability in mutant animals, unlike palmitoylation or farnesylation site mutant Ykt6 forms. As Ykt6 and Vamp7 are both required for autophagosome-lysosome fusion and are mutually exclusive subunits in a Syx17-Snap29 complex, these data suggest that Vamp7 is directly involved in membrane fusion and Ykt6 acts as a non-conventional, regulatory SNARE in this process. PMID:29694367

Defect processes in Be12X (X = Ti, Mo, V, W)

NASA Astrophysics Data System (ADS)

Jackson, M. L.; Burr, P. A.; Grimes, R. W.

2017-08-01

The stability of intrinsic point defects in Be12X intermetallics (where X  =  Ti, V, Mo or W) are predicted using density functional theory simulations and discussed with respect to fusion energy applications. Schottky disorder is found to be the lowest energy complete disorder process, closely matched by Be Frenkel disorder in the cases of Be12V and Be12Ti. Antitisite and X Frenkel disorder are of significantly higher energy. Small clusters of point defects including Be divacancies, Be di-interstitials and accommodation of the X species on two Be sites were considered. Some di-interstitial, divacancy and X2Be combinations exhibit negative binding enthalpy (i.e. clustering is favourable), although this is orientationally dependent. None of the Be12X intermetallics are predicted to exhibit significant non-stoichiometry, ruling out non-stoichiometry as a mechanism for accommodating Be depletion due to neutron transmutation.

Fusion breeder

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

Moir, R.W.

1982-02-22

The fusion breeder is a fusion reactor designed with special blankets to maximize the transmutation by 14 MeV neutrons of uranium-238 to plutonium or thorium to uranium-233 for use as a fuel for fission reactors. Breeding fissile fuels has not been a goal of the US fusion energy program. This paper suggests it is time for a policy change to make the fusion breeder a goal of the US fusion program and the US nuclear energy program. The purpose of this paper is to suggest this policy change be made and tell why it should be made, and to outlinemore » specific research and development goals so that the fusion breeder will be developed in time to meet fissile fuel needs.« less

Fusion breeder

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

Moir, R.W.

1982-04-20

The fusion breeder is a fusion reactor designed with special blankets to maximize the transmutation by 14 MeV neutrons of uranium-238 to plutonium or thorium to uranium-233 for use as a fuel for fission reactors. Breeding fissile fuels has not been a goal of the US fusion energy program. This paper suggests it is time for a policy change to make the fusion breeder a goal of the US fusion program and the US nuclear energy program. The purpose of this paper is to suggest this policy change be made and tell why it should be made, and to outlinemore » specific research and development goals so that the fusion breeder will be developed in time to meet fissile fuel needs.« less

Viral membrane fusion

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

Harrison, Stephen C., E-mail: harrison@crystal.harvard.edu

2015-05-15

Membrane fusion is an essential step when enveloped viruses enter cells. Lipid bilayer fusion requires catalysis to overcome a high kinetic barrier; viral fusion proteins are the agents that fulfill this catalytic function. Despite a variety of molecular architectures, these proteins facilitate fusion by essentially the same generic mechanism. Stimulated by a signal associated with arrival at the cell to be infected (e.g., receptor or co-receptor binding, proton binding in an endosome), they undergo a series of conformational changes. A hydrophobic segment (a “fusion loop” or “fusion peptide”) engages the target-cell membrane and collapse of the bridging intermediate thus formedmore » draws the two membranes (virus and cell) together. We know of three structural classes for viral fusion proteins. Structures for both pre- and postfusion conformations of illustrate the beginning and end points of a process that can be probed by single-virion measurements of fusion kinetics. - Highlights: • Viral fusion proteins overcome the high energy barrier to lipid bilayer merger. • Different molecular structures but the same catalytic mechanism. • Review describes properties of three known fusion-protein structural classes. • Single-virion fusion experiments elucidate mechanism.« less

The Arabidopsis cax3 mutants display altered salt tolerance, pH sensitivity and reduced plasma membrane H+-ATPase activity.

PubMed

Zhao, Jian; Barkla, Bronwyn J; Marshall, Joy; Pittman, Jon K; Hirschi, Kendal D

2008-02-01

Perturbing CAX1, an Arabidopsis vacuolar H+/Ca2+ antiporter, and the related vacuolar transporter CAX3, has been previously shown to cause severe growth defects; however, the specific function of CAX3 has remained elusive. Here, we describe plant phenotypes that are shared among cax1 and cax3 including an increased sensitivity to both abscisic acid (ABA) and sugar during germination, and an increased tolerance to ethylene during early seedling development. We have also identified phenotypes unique to cax3, namely salt, lithium and low pH sensitivity. We used biochemical measurements to ascribe these cax3 sensitivities to a reduction in vacuolar H+/Ca2+ transport during salt stress and decreased plasma membrane H+-ATPase activity. These findings catalog an array of CAX phenotypes and assign a specific role for CAX3 in response to salt tolerance.

Role of the Simian Virus 5 Fusion Protein N-Terminal Coiled-Coil Domain in Folding and Promotion of Membrane Fusion

PubMed Central

West, Dava S.; Sheehan, Michael S.; Segeleon, Patrick K.; Dutch, Rebecca Ellis

2005-01-01

Formation of a six-helix bundle comprised of three C-terminal heptad repeat regions in antiparallel orientation in the grooves of an N-terminal coiled-coil is critical for promotion of membrane fusion by paramyxovirus fusion (F) proteins. We have examined the effect of mutations in four residues of the N-terminal heptad repeat in the simian virus 5 (SV5) F protein on protein folding, transport, and fusogenic activity. The residues chosen have previously been shown from study of isolated peptides to have differing effects on stability of the N-terminal coiled-coil and six-helix bundle (R. E. Dutch, G. P. Leser, and R. A. Lamb, Virology 254:147-159, 1999). The mutant V154M showed reduced proteolytic cleavage and surface expression, indicating a defect in intracellular transport, though this mutation had no effect when studied in isolated peptides. The mutation I137M, previously shown to lower thermostability of the six-helix bundle, resulted in an F protein which was properly processed and transported to the cell surface but which had reduced fusogenic activity. Finally, mutations at L140M and L161M, previously shown to disrupt α-helix formation of isolated N-1 peptides but not to affect six-helix bundle formation, resulted in F proteins that were properly processed. Interestingly, the L161M mutant showed increased syncytium formation and promoted fusion at lower temperatures than the wild-type F protein. These results indicate that interactions separate from formation of an N-terminal coiled-coil or six-helix bundle are important in the initial folding and transport of the SV5 F protein and that mutations that destabilize the N-terminal coiled-coil can result in stimulation of membrane fusion. PMID:15650180

Vacuolar transport of the glutathione conjugate of trans-cinnamic acid.

PubMed

Walczak, H A; Dean, J V

2000-02-01

Red beet (Beta vulgaris L.) tonoplast membrane vesicles and [14C]trans-cinnamic acid-glutatione were used to study the vacuolar transport of phynylpropanoid-glutathione conjugates which are formed in peroxidase-mediated reactions. It was determined that the uptake of [14C]trans-cinnamic acid-glutathione into the tonoplast membrane vesicles was MgATP dependent and was 10-fold faster than the uptake of non-conjugated [14C]trans-cinnamic acid. Uptake of the conjugate in the presence of MgATP was not dependent on a trans-tonoblast H+-electrochemical gradient, because uptake was not affected by the addition of NH4Cl (1 mM; 0% inhibition) and was only slightly affected by gramicidin-D (5 microM; 14% inhibition). Uptake of the conjugate was inhibited 92% by the addition of vanadate (1 mM) and 71% by the addition of the model substrate S-(2,4-dinitrophenyl) glutathione (500 microM). Uptake did not occur when a nonhydrolyzable analog of ATP was used in place of MgATP. The calculated Km and Vmax values for uptake were 142 microM amd 5.95 nmol mg(-1) min(-1), respectively. Based on these results, phenylpropanoid-glutation conjugates formed in peroxidase-mediated reactions appear to be transported into the vacuole by the glutathione S-conjugate pump(s) located in the tonoplast membrane.

Structures of Trypanosome Vacuolar Soluble Pyrophosphatases: Anti-Parasitic Drug Targets

PubMed Central

Yang, Yunyun; Ko, Tzu-Ping; Chen, Chun-Chi; Huang, Guozhong; Zheng, Yingying; Liu, Weidong; Wang, Iren; Ho, Meng-Ru; Danny Hsu, Shang-Te; O’Dowd, Bing; Huff, Hannah C.; Huang, Chun-Hsiang; Docampo, Roberto; Oldfield, Eric; Guo, Rey-Ting

2016-01-01

Trypanosomatid parasites are the causative agents of many neglected tropical diseases including the leishmaniases, Chagas disease, and human African trypanosomiasis. They exploit unusual vacuolar soluble pyrophosphatases (VSPs), absent in humans, for cell growth and virulence and as such, are drug targets. Here, we report the crystal structures of VSP1s from Trypanosoma cruzi and T. brucei, together with that of the T. cruzi protein bound to a bisphosphonate inhibitor. Both VSP1s form a hybrid structure containing an (N-terminal) EF-hand domain fused to a (C-terminal) pyrophosphatase domain. The two domains are connected via an extended loop of about 17 residues. Crystallographic analysis and size exclusion chromatography indicate that the VSP1s form tetramers containing head-to-tail dimers. Phosphate and diphosphate ligands bind in the PPase substrate-binding pocket and interact with several conserved residues, and a bisphosphonate inhibitor (BPH-1260) binds to the same site. Based on Cytoscape and other bioinformatics analyses it is apparent that similar folds will be found in most if not all trypanosomatid VSP1s, including those found in insects (Angomonas deanei, Strigomonas culicis), plant pathogens (Phytomonas spp.) and Leishmania spp. Overall, the results are of general interest since they open the way to structure-based drug design for many of the neglected tropical diseases. PMID:26907161

Regulation of vacuolar H{sup +}-ATPase in microglia by RANKL

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

Serrano, Eric M.; Ricofort, Ryan D.; Zuo, Jian

2009-11-06

Vacuolar H{sup +}-ATPases (V-ATPases) are large electrogenic proton pumps composed of numerous subunits that play vital housekeeping roles in the acidification of compartments of the endocytic pathway. Additionally, V-ATPases play specialized roles in certain cell types, a capacity that is linked to cell type selective expression of isoforms of some of the subunits. We detected low levels of the a3 isoform of the a-subunit in mouse brain extracts. Examination of various brain-derived cell types by immunoblotting showed a3 was expressed in the N9 microglia cell line and in primary microglia, but not in other cell types. The expression of a3more » in osteoclasts requires stimulation by Receptor Activator of Nuclear Factor {kappa}B-ligand (RANKL). We found that Receptor Activator of Nuclear Factor {kappa}B (RANK) was expressed by microglia. Stimulation of microglia with RANKL triggered increased expression of a3. V-ATPases in microglia were shown to bind microfilaments, and stimulation with RANKL increased the proportion of V-ATPase associated with the detergent-insoluble cytoskeletal fraction and with actin. In summary, microglia express the a3-subunit of V-ATPase. The expression of a3 and the interaction between V-ATPases and microfilaments was modulated by RANKL. These data suggest a novel molecular pathway for regulating microglia.« less

Drosophila Kette coordinates myoblast junction dissolution and the ratio of Scar-to-WASp during myoblast fusion

PubMed Central

Hamp, Julia; Löwer, Andreas; Dottermusch-Heidel, Christine; Beck, Lothar; Moussian, Bernard; Flötenmeyer, Matthias

2016-01-01

ABSTRACT The fusion of founder cells and fusion-competent myoblasts (FCMs) is crucial for muscle formation in Drosophila. Characteristic events of myoblast fusion include the recognition and adhesion of myoblasts, and the formation of branched F-actin by the Arp2/3 complex at the site of cell–cell contact. At the ultrastructural level, these events are reflected by the appearance of finger-like protrusions and electron-dense plaques that appear prior to fusion. Severe defects in myoblast fusion are caused by the loss of Kette (a homolog of Nap1 and Hem-2, also known as NCKAP1 and NCKAP1L, respectively), a member of the regulatory complex formed by Scar or WAVE proteins (represented by the single protein, Scar, in flies). kette mutants form finger-like protrusions, but the electron-dense plaques are extended. Here, we show that the electron-dense plaques in wild-type and kette mutant myoblasts resemble other electron-dense structures that are known to function as cellular junctions. Furthermore, analysis of double mutants and attempts to rescue the kette mutant phenotype with N-cadherin, wasp and genes of members of the regulatory Scar complex revealed that Kette has two functions during myoblast fusion. First, Kette controls the dissolution of electron-dense plaques. Second, Kette controls the ratio of the Arp2/3 activators Scar and WASp in FCMs. PMID:27521427

Paramyxovirus fusion: Real-time measurement of parainfluenza virus 5 virus-cell fusion

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

Connolly, Sarah A.; Lamb, Robert A.

2006-11-25

Although cell-cell fusion assays are useful surrogate methods for studying virus fusion, differences between cell-cell and virus-cell fusion exist. To examine paramyxovirus fusion in real time, we labeled viruses with fluorescent lipid probes and monitored virus-cell fusion by fluorimetry. Two parainfluenza virus 5 (PIV5) isolates (W3A and SER) and PIV5 containing mutations within the fusion protein (F) were studied. Fusion was specific and temperature-dependent. Compared to many low pH-dependent viruses, the kinetics of PIV5 fusion was slow, approaching completion within several minutes. As predicted from cell-cell fusion assays, virus containing an F protein with an extended cytoplasmic tail (rSV5 F551)more » had reduced fusion compared to wild-type virus (W3A). In contrast, virus-cell fusion for SER occurred at near wild-type levels, despite the fact that this isolate exhibits a severely reduced cell-cell fusion phenotype. These results support the notion that virus-cell and cell-cell fusion have significant differences.« less

The vacuole system is a significant intracellular pathway for longitudinal solute transport in basidiomycete fungi.

PubMed

Darrah, P R; Tlalka, M; Ashford, A; Watkinson, S C; Fricker, M D

2006-07-01

Mycelial fungi have a growth form which is unique among multicellular organisms. The data presented here suggest that they have developed a unique solution to internal solute translocation involving a complex, extended vacuole. In all filamentous fungi examined, this extended vacuole forms an interconnected network, dynamically linked by tubules, which has been hypothesized to act as an internal distribution system. We have tested this hypothesis directly by quantifying solute movement within the organelle by photobleaching a fluorescent vacuolar marker. Predictive simulation models were then used to determine the transport characteristics over extended length scales. This modeling showed that the vacuolar organelle forms a functionally important, bidirectional diffusive transport pathway over distances of millimeters to centimeters. Flux through the pathway is regulated by the dynamic tubular connections involving homotypic fusion and fission. There is also a strongly predicted interaction among vacuolar organization, predicted diffusion transport distances, and the architecture of the branching colony margin.

Macroautophagy and microautophagy in relation to vacuole formation in mesophyll cells of Dendrobium tepals.

PubMed

van Doorn, Wouter G; Kirasak, Kanjana; Ketsa, Saichol

2015-04-01

Prior to flower opening, mesophyll cells at the vascular bundles of Dendrobium tepals showed a large increase in vacuolar volume, partially at the expense of the cytoplasm. Electron micrographs indicated that this increase in vacuolar volume was mainly due to vacuole fusion. Macroautophagous structures typical of plant cells were observed. Only a small part of the decrease in cytoplasmic volume seemed due to macroautophagy. The vacuoles contained vesicles of various types, including multilamellar bodies. It was not clear if these vacuolar inclusions were due to macroautophagy or microautophagy. Only a single structure was observed of a protruding vacuole, indicating microautophagy. It is concluded that macroautophagy occurs in these cells but its role in vacuole formation seems small, while a possible role of microautophagy in vacuole formation might be hypothesized. Careful labeling of organelle membranes seems required to advance our insight in plant macro- and microautophagy and their roles in vacuole formation. Copyright © 2015 Elsevier GmbH. All rights reserved.

[Experimental study on repair of the defect of the pars interarticularis in rat with bone morphogenetic protein and fibrin glue].

PubMed

Nakamura, T

1992-07-01

The possibility of repairing the defect of the pars interarticularis (pars defect) with Bone Morphogenetic Protein (BMP) and fibrin glue was studied. The pars defect established in the 5th lumbar vertebra of Wistar rat was treated with surgical implantation of a composite consisting of BMP, fibrin glue and autologous cancellous bone. At 3, 6, 9 and 12 weeks after implantation, the osteoinductive activity in the pars defect was observed histologically and compared with that of other composite implants such as BMP with fibrin glue, autologous cancellous bone alone and autologous cancellous bone with fibrin glue. Although perfect bone fusion was not obtained with any of the composites employed, a significant increase in bone formation was seen in a composite of BMP, fibrin glue and autologous cancellous bone (p less than 0.01) as compared with that seen in the others. Consequently, implantation of BMP and fibrin glue combined with some biomaterials which support osteo-induction of BMP and stabilize the pars defect might be successfully applied to repair the pars defect.

GABARAPs regulate PI4P-dependent autophagosome:lysosome fusion.

PubMed

Wang, Hanzhi; Sun, Hui-Qiao; Zhu, Xiaohui; Zhang, Li; Albanesi, Joseph; Levine, Beth; Yin, Helen

2015-06-02

The Atg8 autophagy proteins are essential for autophagosome biogenesis and maturation. The γ-aminobutyric acid receptor-associated protein (GABARAP) Atg8 family is much less understood than the LC3 Atg8 family, and the relationship between the GABARAPs' previously identified roles as modulators of transmembrane protein trafficking and autophagy is not known. Here we report that GABARAPs recruit palmitoylated PI4KIIα, a lipid kinase that generates phosphatidylinositol 4-phosphate (PI4P) and binds GABARAPs, from the perinuclear Golgi region to autophagosomes to generate PI4P in situ. Depletion of either GABARAP or PI4KIIα, or overexpression of a dominant-negative kinase-dead PI4KIIα mutant, decreases autophagy flux by blocking autophagsome:lysosome fusion, resulting in the accumulation of abnormally large autophagosomes. The autophagosome defects are rescued by overexpressing PI4KIIα or by restoring intracellular PI4P through "PI4P shuttling." Importantly, PI4KIIα's role in autophagy is distinct from that of PI4KIIIβ and is independent of subsequent phosphatidylinositol 4,5 biphosphate (PIP2) generation. Thus, GABARAPs recruit PI4KIIα to autophagosomes, and PI4P generation on autophagosomes is critically important for fusion with lysosomes. Our results establish that PI4KIIα and PI4P are essential effectors of the GABARAP interactome's fusion machinery.

Gain-of-function mutations identify amino acids within transmembrane domains of the yeast vacuolar transporter Zrc1 that determine metal specificity

PubMed Central

Lin, Huilan; Burton, Damali; Li, Liangtao; Warner, David E.; Phillips, John D.; Ward, Diane McVEY; Kaplan, Jerry

2015-01-01

Cation diffusion facilitator transporters are found in all three Kingdoms of life and are involved in transporting transition metals out of the cytosol. The metals they transport include Zn2+, Co2+, Fe2+, Cd2+, Ni2+ and Mn2+; however, no single transporter transports all metals. Previously we showed that a single amino acid mutation in the yeast vacuolar zinc transporter Zrc1 changed its substrate specificity from Zn2+ to Fe2+ and Mn2+ [Lin, Kumanovics, Nelson, Warner, Ward and Kaplan (2008) J. Biol. Chem. 283, 33865–33873]. Mutant Zrc1 that gained iron transport activity could protect cells with a deletion in the vacuolar iron transporter (CCC1) from high iron toxicity. Utilizing suppression of high iron toxicity and PCR mutagenesis of ZRC1, we identified other amino acid substitutions within ZRC1 that changed its metal specificity. All Zrc1 mutants that transported Fe2+ could also transport Mn2+. Some Zrc1 mutants lost the ability to transport Zn2+, but others retained the ability to transport Zn2+. All of the amino acid substitutions that resulted in a gain in Fe2+ transport activity were found in transmembrane domains. In addition to alteration of residues adjacent to the putative metal-binding site in two transmembrane domains, alteration of residues distant from the binding site affected substrate specificity. These results suggest that substrate selection involves co-operativity between transmembrane domains. PMID:19538181

Expression, crystallization and phasing of vacuolar H(+)-ATPase subunit C (Vma5p) of Saccharomyces cerevisiae.

PubMed

Drory, Omri; Mor, Adi; Frolow, Felix; Nelson, Nathan

2004-10-01

The expression, crystallization and phasing of subunit C (Vma5p) of the yeast (Saccharomyces cerevisiae) vacuolar proton-translocating ATPase (V-ATPase) is described. The expressed protein consists of 412 residues: 392 from the reading frame of Vma5p and 20 N-terminal residues originating from the plasmid. Diffraction-quality crystals were obtained using the hanging-drop and sitting-drop vapour-diffusion methods assisted by streak-seeding, with PEG 3350 as precipitant. The crystals formed in hanging drops diffracted to 1.80 A and belong to space group P4(3)2(1)2(1), with unit-cell parameters a = b = 62.54, c = 327.37 A, alpha = beta = gamma = 90 degrees. The structure was solved using SIRAS with a Lu(O2C2H3)2 heavy-atom derivative.

Prm3p is a pheromone-induced peripheral nuclear envelope protein required for yeast nuclear fusion.

PubMed

Shen, Shu; Tobery, Cynthia E; Rose, Mark D

2009-05-01

Nuclear membrane fusion is the last step in the mating pathway of the yeast Saccharomyces cerevisiae. We adapted a bioinformatics approach to identify putative pheromone-induced membrane proteins potentially required for nuclear membrane fusion. One protein, Prm3p, was found to be required for nuclear membrane fusion; disruption of PRM3 caused a strong bilateral defect, in which nuclear congression was completed but fusion did not occur. Prm3p was localized to the nuclear envelope in pheromone-responding cells, with significant colocalization with the spindle pole body in zygotes. A previous report, using a truncated protein, claimed that Prm3p is localized to the inner nuclear envelope. Based on biochemistry, immunoelectron microscopy and live cell microscopy, we find that functional Prm3p is a peripheral membrane protein exposed on the cytoplasmic face of the outer nuclear envelope. In support of this, mutations in a putative nuclear localization sequence had no effect on full-length protein function or localization. In contrast, point mutations and deletions in the highly conserved hydrophobic carboxy-terminal domain disrupted both protein function and localization. Genetic analysis, colocalization, and biochemical experiments indicate that Prm3p interacts directly with Kar5p, suggesting that nuclear membrane fusion is mediated by a protein complex.

Construction, expression, and localization of a CycA::PhoA fusion protein in Rhodobacter sphaeroides and Escherichia coli.

PubMed Central

Varga, A R; Kaplan, S

1989-01-01

We demonstrated the utility of Escherichia coli alkaline phosphatase, encoded by phoA, as a reporter molecule for genetic fusions in Rhodobacter sphaeroides. A portion of the R. sphaeroides cycA gene was fused to phoA, yielding a fusion protein comprising the putative signal sequence and first 10 amino acids of the cytochrome c2 apoprotein joined to the sixth amino acid of alkaline phosphatase. The fusion protein was efficiently transported to the periplasm of R. sphaeroides as determined by enzyme activity, Western immunoblot analysis, and immunogold electron microscopy. We also documented the ability of an R. sphaeroides mutant, RS104, with gross defects in photosynthetic membrane morphology to efficiently recognize and translocate the fusion protein to the periplasmic compartment. The inclusion of 500 base pairs of R. sphaeroides DNA in cis to the cycA structural gene resulted in a 2.5-fold increase in alkaline phosphatase activity in photosynthetically grown cells compared with the activity in aerobically grown cells, demonstrating that the fusion protein is regulated in a manner similar to that of cytochrome c2 regulation. We also constructed two pUC19-based plasmids suitable for the construction of translational fusions to phoA. In these plasmids, translational fusions of phoA to the gene under consideration can be made in all three reading frames, thus facilitating construction and expression of fusion protein systems utilizing phoA. Images PMID:2553661

Fusion between perinuclear virions and the outer nuclear membrane requires the fusogenic activity of herpes simplex virus gB.

PubMed

Wright, Catherine C; Wisner, Todd W; Hannah, Brian P; Eisenberg, Roselyn J; Cohen, Gary H; Johnson, David C

2009-11-01

Herpesviruses cross nuclear membranes (NMs) in two steps, as follows: (i) capsids assemble and bud through the inner NM into the perinuclear space, producing enveloped virus particles, and (ii) the envelopes of these virus particles fuse with the outer NM. Two herpes simplex virus (HSV) glycoproteins, gB and gH (the latter, likely complexed as a heterodimer with gL), are necessary for the second step of this process. Mutants lacking both gB and gH accumulate in the perinuclear space or in herniations (membrane vesicles derived from the inner NM). Both gB and gH/gL are also known to act directly in fusing the virion envelope with host cell membranes during HSV entry into cells, i.e., both glycoproteins appear to function directly in different aspects of the membrane fusion process. We hypothesized that HSV gB and gH/gL also act directly in the membrane fusion that occurs during virus egress from the nucleus. Previous studies of the role of gB and gH/gL in nuclear egress involved HSV gB and gH null mutants that could potentially also possess gross defects in the virion envelope. Here, we produced recombinant HSV-expressing mutant forms of gB with single amino acid substitutions in the hydrophobic "fusion loops." These fusion loops are thought to play a direct role in membrane fusion by insertion into cellular membranes. HSV recombinants expressing gB with any one of four fusion loop mutations (W174R, W174Y, Y179K, and A261D) were unable to enter cells. Moreover, two of the mutants, W174Y and Y179K, displayed reduced abilities to mediate HSV cell-to-cell spread, and W174R and A261D exhibited no spread. All mutant viruses exhibited defects in nuclear egress, enveloped virions accumulated in herniations and in the perinuclear space, and fewer enveloped virions were detected on cell surfaces. These results support the hypothesis that gB functions directly to mediate the fusion between perinuclear virus particles and the outer NM.

A vacuolar H(+)-pyrophosphatase differential activation and energy coupling integrate the responses of weeds and crops to drought stress.

PubMed

Venancio, Josimara Barcelos; Catunda, Michelle Guedes; Ogliari, Juarez; Rima, Janaína Aparecida Hottz; Okorokova-Facanha, Anna Lvovna; Okorokov, Lev Alexandrovitich; Facanha, Arnoldo Rocha

2014-06-01

Cyperus rotundus L. is a C4 weed of large vegetative and reproductive vigor endowed with competitive advantages over most crop species mainly under adverse environmental conditions. Vacuole functions are critical for the mechanisms of drought resistance, and here the modulation of the primary system of vacuolar ion transport is investigated during a transient water stress imposed to this weed and to C4 crop species (Zea mays L.). The vacuolar H(+) pumps, the H(+)-ATPase and H(+)-PPiase, expression, activities and the energy coupling were spectrophotometrically investigated as key elements in the differential drought-resistance mechanisms developed by weeds and crops. In C. rotundus tonoplasts, ATP hydrolysis was more sensitive to drought than its coupled H(+) transport, which was in turn at least 3-folds faster than that mediated by the H(+)-PPiase. Its PPi hydrolysis was only slightly affected by severe water deficit, contrasting with the disruption induced in the PPi-dependent H(+)-gradient. This effect was antagonized by plant rehydration as the H(+)-PPiase activity was highly stimulated, reassuming a coupled PPi-driven H(+) pumping. Maize tonoplasts exhibited 2-4 times lower hydrolytic activities than that of C. rotundus, but were able to overactivate specifically PPi-dependent H(+) pumping in response to stress relief, resulting in an enhanced H(+)-pumps coupling efficiency. These results together with immunoanalysis revealed profiles consistent with pre- and post-translational changes occurring on the tonoplast H(+)-pumps, which differ between weeds and crops upon water deficit. The evidences highlight an unusual modulation of the H(+)-PPiase energy coupling as a key biochemical change related to environmental stresses adaptive capacity of plants. Copyright © 2014 Elsevier B.V. All rights reserved.

Ankle fusion for definitive management of non-reconstructable pilon fractures.

PubMed

Bozic, Vladimir; Thordarson, David B; Hertz, Jennifer

2008-09-01

Highly comminuted pilon fractures, especially with a compromised soft tissue envelope, present a challenging treatment scenario. This study presents our results for patients managed with ankle fusion rather than ORIF. Fourteen patients with ankle joint incongruence after non-reconstructable tibia pilon fractures were treated with primary tibiotalar arthrodesis using a fixed-angle cannulated blade plate. Delayed metaphyseal unions due to bone defects were treated concurrently. The subtalar joint was preserved in all cases. Metaphyseal healing and stable arthrodesis was obtained in each case. There was one case of blade plate breakage in a patient who still achieved successful arthrodesis without reoperation. Union was achieved at an average of 15 weeks. No secondary procedures were required to obtain union. All 14 patients were ambulatory at last followup. Average followup was 39 weeks. Primary ankle arthrodesis can be achieved using a cannulated blade plate to address a non-reconstructable articular surface and metaphyseal bone defects in complex tibia pilon fractures.

Promoter regions of potato vacuolar invertase gene in response to sugars and hormones.

PubMed

Ou, Yongbin; Song, Botao; Liu, Xun; Xie, Conghua; Li, Meng; Lin, Yuan; Zhang, Huiling; Liu, Jun

2013-08-01

Potato vacuolar acid invertase (StvacINV1) (β-fructofuranosidase; EC 3.2.1.26) has been confirmed to play an important role in cold-induced sweetening of potato tubers. However, the transcriptional regulation mechanisms of StvacINV1 are largely unknown. In this study, the 5'-flanking sequence of StvacINV1 was cloned and the cis-acting elements were predicted. Histochemical assay showed that the StvacINV1 promoter governed β-glucuronidase (GUS) expression in potato leaves, stems, roots and tubers. Quantitative analysis of GUS expression suggested that the activity of StvacINV1 promoter was suppressed by sucrose, glucose, fructose, and cold, while enhanced by indole-3-acetic acid (IAA), and gibberellic acid (GA3). Further deletion analysis clarified that the promoter regions from -118 to -551, -551 to -1021, and -1021 to -1521 were required for responding to sucrose/glucose, GA3, and IAA, respectively. These findings provide essential information regarding transcriptional regulation mechanisms of StvacINV1. Copyright © 2013 Elsevier Masson SAS. All rights reserved.

Amino Acid Availability Modulates Vacuolar H+-ATPase Assembly*

PubMed Central

Stransky, Laura A.; Forgac, Michael

2015-01-01

The vacuolar H+-ATPase (V-ATPase) is an ATP-dependent proton pump composed of a peripheral ATPase domain (V1) and a membrane-integral proton-translocating domain (V0) and is involved in many normal and disease processes. An important mechanism of regulating V-ATPase activity is reversible assembly of the V1 and V0 domains. Increased assembly in mammalian cells occurs under various conditions and has been shown to involve PI3K. The V-ATPase is necessary for amino acid-induced activation of mechanistic target of rapamycin complex 1 (mTORC1), which is important in controlling cell growth in response to nutrient availability and growth signals. The V-ATPase undergoes amino acid-dependent interactions with the Ragulator complex, which is involved in recruitment of mTORC1 to the lysosomal membrane during amino acid sensing. We hypothesized that changes in the V-ATPase/Ragulator interaction might involve amino acid-dependent changes in V-ATPase assembly. To test this, we measured V-ATPase assembly by cell fractionation in HEK293T cells treated with and without amino acids. V-ATPase assembly increases upon amino acid starvation, and this effect is reversed upon readdition of amino acids. Lysosomes from amino acid-starved cells possess greater V-ATPase-dependent proton transport, indicating that assembled pumps are catalytically active. Amino acid-dependent changes in both V-ATPase assembly and activity are independent of PI3K and mTORC1 activity, indicating the involvement of signaling pathways distinct from those implicated previously in controlling assembly. By contrast, lysosomal neutralization blocks the amino acid-dependent change in assembly and reactivation of mTORC1 after amino acid starvation. These results identify an important new stimulus for controlling V-ATPase assembly. PMID:26378229

Multilayer porous UHMWPE scaffolds for bone defects replacement.

PubMed

Maksimkin, A V; Senatov, F S; Anisimova, N Yu; Kiselevskiy, M V; Zalepugin, D Yu; Chernyshova, I V; Tilkunova, N A; Kaloshkin, S D

2017-04-01

Reconstruction of the structural integrity of the damaged bone tissue is an urgent problem. UHMWPE may be potentially used for the manufacture of porous implants simulating as closely as possible the porous cancellous bone tissue. But the extremely high molecular weight of the polymer does not allow using traditional methods of foaming. Porous and multilayer UHMWPE scaffolds with nonporous bulk layer and porous layer that mimics cancellous bone architecture were obtained by solid-state mixing, thermopressing and washing in subcritical water. Structural and mechanical properties of the samples were studied. Porous UHMWPE samples were also studied in vitro and in vivo. The pores of UHMWPE scaffold are open and interconnected. Volume porosity of the obtained samples was 79±2%; the pore size range was 80-700μm. Strong connection of the two layers in multilayer UHMWPE scaffolds was observed with decreased number of fusion defects. Functionality of implants based on multilayer UHMWPE scaffolds is provided by the fixation of scaffolds in the bone defect through ingrowths of the connective tissue into the pores, which ensures the maintenance of the animals' mobility. Copyright © 2016 Elsevier B.V. All rights reserved.

Extreme-value statistics reveal rare failure-critical defects in additive manufacturing

DOE PAGES

Boyce, Brad L.; Salzbrenner, Bradley C.; Rodelas, Jeffrey M.; ...

2017-04-21

Additive manufacturing enables the rapid, cost effective production of large populations of material test coupons such as tensile bars. By adopting streamlined test methods including ‘drop-in’ grips and non-contact extensometry, testing these large populations becomes more efficient. Unlike hardness tests, the tensile test provides a direct measure of yield strength, flow properties, and ductility, which can be directly incorporated into solid mechanics simulations. In the present work, over 1000 nominally identical tensile tests were used to explore the effect of process variability on the mechanical property distributions of a precipitation hardened stainless steel, 17-4PH, produced by a laser powder bedmore » fusion process, also known as direct metal laser sintering. With this large dataset, rare defects are revealed that affect only ~2% of the population, stemming from a single build lot of material. Lastly, the rare defects caused a substantial loss in ductility and were associated with an interconnected network of porosity.« less

Synaptotagmin-mediated bending of the target membrane is a critical step in Ca2+-regulated fusion

PubMed Central

Hui, Enfu; Johnson, Colin P.; Yao, Jun; Dunning, F. Mark; Chapman, Edwin R.

2009-01-01

Summary Decades ago it was proposed that exocytosis involves invagination of the target membrane, resulting in a highly localized site of contact between the bilayers destined to fuse. The vesicle protein synaptotagmin-I (syt) bends membranes in response to Ca2+, but whether this drives localized invagination of the target membrane to accelerate fusion has not been determined; previous studies relied on reconstituted vesicles that were already highly curved and used mutations in syt that were not selective for membrane-bending activity. Here, we directly address this question by utilizing vesicles with different degrees of curvature. A tubulation-defective syt mutant was able to promote fusion between highly curved SNARE-bearing liposomes, but exhibited a marked loss of activity when the membranes were relatively flat. Moreover, bending of flat membranes by adding an N-BAR domain rescued the function of the tubulation-deficient syt mutant. Hence, syt-mediated membrane bending is a critical step in membrane fusion. PMID:19703397

Myristoylation of the Arenavirus Envelope Glycoprotein Stable Signal Peptide Is Critical for Membrane Fusion but Dispensable for Virion Morphogenesis.

PubMed

York, Joanne; Nunberg, Jack H

2016-09-15

Arenaviruses are responsible for severe and often fatal hemorrhagic disease. In the absence of effective antiviral therapies and vaccines, these viruses pose serious threats to public health and biodefense. Arenaviruses enter the host cell by fusion of the viral and endosomal membranes, a process mediated by the virus envelope glycoprotein GPC. Unlike other class I viral fusion proteins, GPC retains its stable signal peptide (SSP) as an essential third subunit in the mature complex. SSP spans the membrane twice and is myristoylated at its cytoplasmic N terminus. Mutations that abolish SSP myristoylation have been shown to reduce pH-induced cell-cell fusion activity of ectopically expressed GPC to ∼20% of wild-type levels. In order to examine the role of SSP myristoylation in the context of the intact virus, we used reverse genetics to generate Junín viruses (Candid #1 isolate) in which the critical glycine-2 residue in SSP was either replaced by alanine (G2A) or deleted (ΔG2). These mutant viruses produced smaller foci of infection in Vero cells and showed an ∼5-fold reduction in specific infectivity, commensurate with the defect in cell-cell fusion. However, virus assembly and GPC incorporation into budded virions were unaffected. Our findings suggest that the myristate moiety is cryptically disposed in the prefusion GPC complex and may function late in the fusion process to promote merging of the viral and cellular membranes. Hemorrhagic fever arenaviruses pose significant threats to public health and biodefense. Arenavirus entry into the host cell is promoted by the virus envelope glycoprotein GPC. Unlike other viral envelope glycoproteins, GPC contains a myristoylated stable signal peptide (SSP) as an essential third subunit. Myristoylation has been shown to be important for the membrane fusion activity of recombinantly expressed GPC. Here, we use reverse genetics to study the role of SSP myristoylation in the context of the intact virion. We find that

iTRAQ Analysis Reveals Mechanisms of Growth Defects Due to Excess Zinc in Arabidopsis1[W][OA

PubMed Central

Fukao, Yoichiro; Ferjani, Ali; Tomioka, Rie; Nagasaki, Nahoko; Kurata, Rie; Nishimori, Yuka; Fujiwara, Masayuki; Maeshima, Masayoshi

2011-01-01

The micronutrient zinc is essential for all living organisms, but it is toxic at high concentrations. Here, to understand the effects of excess zinc on plant cells, we performed an iTRAQ (for isobaric tags for relative and absolute quantification)-based quantitative proteomics approach to analyze microsomal proteins from Arabidopsis (Arabidopsis thaliana) roots. Our approach was sensitive enough to identify 521 proteins, including several membrane proteins. Among them, IRT1, an iron and zinc transporter, and FRO2, a ferric-chelate reductase, increased greatly in response to excess zinc. The expression of these two genes has been previously reported to increase under iron-deficient conditions. Indeed, the concentration of iron was significantly decreased in roots and shoots under excess zinc. Also, seven subunits of the vacuolar H+-ATPase (V-ATPase), a proton pump on the tonoplast and endosome, were identified, and three of them decreased significantly in response to excess zinc. In addition, excess zinc in the wild type decreased V-ATPase activity and length of roots and cells to levels comparable to those of the untreated de-etiolated3-1 mutant, which bears a mutation in V-ATPase subunit C. Interestingly, excess zinc led to the formation of branched and abnormally shaped root hairs, a phenotype that correlates with decreased levels of proteins of several root hair-defective mutants. Our results point out mechanisms of growth defects caused by excess zinc in which cross talk between iron and zinc homeostasis and V-ATPase activity might play a central role. PMID:21325567

Critical Roles of Vacuolar Invertase in Floral Organ Development and Male and Female Fertilities Are Revealed through Characterization of GhVIN1-RNAi Cotton Plants1[OPEN

PubMed Central

2016-01-01

Seed number and quality are key traits determining plant fitness and crop yield and rely on combined competence in male and female fertilities. Sucrose metabolism is central to reproductive success. It remains elusive, though, how individual sucrose metabolic enzymes may regulate the complex reproductive processes. Here, by silencing vacuolar invertase (VIN) genes in cotton (Gossypium hirsutum) reproductive organs, we revealed diverse roles that VIN plays in multiple reproductive processes. A set of phenotypic and genetic studies showed significant reductions of viable seeds in GhVIN1-RNAi plants, attributed to pollination failure and impaired male and female fertilities. The former was largely owing to the spatial mismatch between style and stamen and delayed pollen release from the anthers, whereas male defects came from poor pollen viability. The transgenic stamen exhibited altered expression of the genes responsible for starch metabolism and auxin and jasmonic acid signaling. Further analyses identified the reduction of GhVIN expression in the seed coat as the major cause for the reduced female fertility, which appeared to disrupt the expression of some key genes involved in trehalose and auxin metabolism and signaling, leading to programmed cell death or growth repression in the filial tissues. Together, the data provide an unprecedented example of how VIN is required to synchronize style and stamen development and the formation of male and female fertilities for seed development in a crop species, cotton. PMID:26969720

Accelerated leukemogenesis by truncated CBFβ-SMMHC defective in high-affinity binding with RUNX1

PubMed Central

Kamikubo, Yasuhiko; Zhao, Ling; Wunderlich, Mark; Corpora, Takeshi; Hyde, R. Katherine; Paul, Thomas A.; Kundu, Mondira; Garrett, Lisa; Compton, Sheila; Huang, Gang; Wolff, Linda; Ito, Yoshiaki; Bushweller, John; Mulloy, James C.; Liu, P. Paul

2010-01-01

SUMMARY Dominant RUNX1 inhibition has been proposed as a common pathway for CBF-leukemia. CBFβ-SMMHC, a fusion protein in human acute myeloid leukemia (AML), dominantly inhibits RUNX1 largely through its RUNX1 high-affinity binding domain (HABD). However, the type I CBFβ-SMMHC fusion in AML patients lacks HABD. Here we report that the type I CBFβ-SMMHC protein binds RUNX1 inefficiently. Knock-in mice expressing CBFβ-SMMHC with a HABD deletion developed leukemia quickly, even though hematopoietic defects associated with Runx1-inhibition were partially rescued. A larger pool of leukemia initiating cells, increased MN1 expression, and retention of RUNX1 phosphorylation are potential mechanisms for accelerated leukemia development in these mice. Our data suggest that RUNX1 dominant inhibition may not be a critical step for leukemogenesis by CBFβ-SMMHC. PMID:20478528

Birth Defects

MedlinePlus

... both. Some birth defects like cleft lip or neural tube defects are structural problems that can be ... during pregnancy is a key factor in causing neural tube defects. For most birth defects, the cause ...

Score level fusion scheme based on adaptive local Gabor features for face-iris-fingerprint multimodal biometric

NASA Astrophysics Data System (ADS)

He, Fei; Liu, Yuanning; Zhu, Xiaodong; Huang, Chun; Han, Ye; Chen, Ying

2014-05-01

A multimodal biometric system has been considered a promising technique to overcome the defects of unimodal biometric systems. We have introduced a fusion scheme to gain a better understanding and fusion method for a face-iris-fingerprint multimodal biometric system. In our case, we use particle swarm optimization to train a set of adaptive Gabor filters in order to achieve the proper Gabor basic functions for each modality. For a closer analysis of texture information, two different local Gabor features for each modality are produced by the corresponding Gabor coefficients. Next, all matching scores of the two Gabor features for each modality are projected to a single-scalar score via a trained, supported, vector regression model for a final decision. A large-scale dataset is formed to validate the proposed scheme using the Facial Recognition Technology database-fafb and CASIA-V3-Interval together with FVC2004-DB2a datasets. The experimental results demonstrate that as well as achieving further powerful local Gabor features of multimodalities and obtaining better recognition performance by their fusion strategy, our architecture also outperforms some state-of-the-art individual methods and other fusion approaches for face-iris-fingerprint multimodal biometric systems.

Dissection of the Role of the Stable Signal Peptide of the Arenavirus Envelope Glycoprotein in Membrane Fusion

PubMed Central

Messina, Emily L.; York, Joanne

2012-01-01

The arenavirus envelope glycoprotein (GPC) retains a stable signal peptide (SSP) as an essential subunit in the mature complex. The 58-amino-acid residue SSP comprises two membrane-spanning hydrophobic regions separated by a short ectodomain loop that interacts with the G2 fusion subunit to promote pH-dependent membrane fusion. Small-molecule compounds that target this unique SSP-G2 interaction prevent arenavirus entry and infection. The interaction between SSP and G2 is sensitive to the phylogenetic distance between New World (Junín) and Old World (Lassa) arenaviruses. For example, heterotypic GPC complexes are unable to support virion entry. In this report, we demonstrate that the hybrid GPC complexes are properly assembled, proteolytically cleaved, and transported to the cell surface but are specifically defective in their membrane fusion activity. Chimeric SSP constructs reveal that this incompatibility is localized to the first transmembrane segment of SSP (TM1). Genetic changes in TM1 also affect sensitivity to small-molecule fusion inhibitors, generating resistance in some cases and inhibitor dependence in others. Our studies suggest that interactions of SSP TM1 with the transmembrane domain of G2 may be important for GPC-mediated membrane fusion and its inhibition. PMID:22438561

Kar5p is required for multiple functions in both inner and outer nuclear envelope fusion in Saccharomyces cerevisiae.

PubMed

Rogers, Jason V; Rose, Mark D

2014-12-02

During mating in the budding yeast Saccharomyces cerevisiae, two haploid nuclei fuse via two sequential membrane fusion steps. SNAREs (i.e., soluble N-ethylmaleimide-sensitive factor attachment protein receptors) and Prm3p mediate outer nuclear membrane fusion, but the inner membrane fusogen remains unknown. Kar5p is a highly conserved transmembrane protein that localizes adjacent to the spindle pole body (SPB), mediates nuclear envelope fusion, and recruits Prm3p adjacent to the SPB. To separate Kar5p's functions, we tested localization, Prm3p recruitment, and nuclear fusion efficiency in various kar5 mutants. All domains and the conserved cysteine residues were essential for nuclear fusion. Several kar5 mutant proteins localized properly but did not mediate Prm3p recruitment; other kar5 mutant proteins localized and recruited Prm3p but were nevertheless defective for nuclear fusion, demonstrating additional functions beyond Prm3p recruitment. We identified one Kar5p domain required for SPB localization, which is dependent on the half-bridge protein Mps3p. Electron microscopy revealed a kar5 mutant that arrests with expanded nuclear envelope bridges, suggesting that Kar5p is required after outer nuclear envelope fusion. Finally, a split-GFP assay demonstrated that Kar5p localizes to both the inner and outer nuclear envelope. These insights suggest a mechanism by which Kar5p mediates inner nuclear membrane fusion. Copyright © 2015 Rogers and Rose.

Kar5p Is Required for Multiple Functions in Both Inner and Outer Nuclear Envelope Fusion in Saccharomyces cerevisiae

PubMed Central

Rogers, Jason V.; Rose, Mark D.

2014-01-01

During mating in the budding yeast Saccharomyces cerevisiae, two haploid nuclei fuse via two sequential membrane fusion steps. SNAREs (i.e., soluble N-ethylmaleimide–sensitive factor attachment protein receptors) and Prm3p mediate outer nuclear membrane fusion, but the inner membrane fusogen remains unknown. Kar5p is a highly conserved transmembrane protein that localizes adjacent to the spindle pole body (SPB), mediates nuclear envelope fusion, and recruits Prm3p adjacent to the SPB. To separate Kar5p’s functions, we tested localization, Prm3p recruitment, and nuclear fusion efficiency in various kar5 mutants. All domains and the conserved cysteine residues were essential for nuclear fusion. Several kar5 mutant proteins localized properly but did not mediate Prm3p recruitment; other kar5 mutant proteins localized and recruited Prm3p but were nevertheless defective for nuclear fusion, demonstrating additional functions beyond Prm3p recruitment. We identified one Kar5p domain required for SPB localization, which is dependent on the half-bridge protein Mps3p. Electron microscopy revealed a kar5 mutant that arrests with expanded nuclear envelope bridges, suggesting that Kar5p is required after outer nuclear envelope fusion. Finally, a split-GFP assay demonstrated that Kar5p localizes to both the inner and outer nuclear envelope. These insights suggest a mechanism by which Kar5p mediates inner nuclear membrane fusion. PMID:25467943

Scanning electron microscope automatic defect classification of process induced defects

NASA Astrophysics Data System (ADS)

Wolfe, Scott; McGarvey, Steve

2017-03-01

With the integration of high speed Scanning Electron Microscope (SEM) based Automated Defect Redetection (ADR) in both high volume semiconductor manufacturing and Research and Development (R and D), the need for reliable SEM Automated Defect Classification (ADC) has grown tremendously in the past few years. In many high volume manufacturing facilities and R and D operations, defect inspection is performed on EBeam (EB), Bright Field (BF) or Dark Field (DF) defect inspection equipment. A comma separated value (CSV) file is created by both the patterned and non-patterned defect inspection tools. The defect inspection result file contains a list of the inspection anomalies detected during the inspection tools' examination of each structure, or the examination of an entire wafers surface for non-patterned applications. This file is imported into the Defect Review Scanning Electron Microscope (DRSEM). Following the defect inspection result file import, the DRSEM automatically moves the wafer to each defect coordinate and performs ADR. During ADR the DRSEM operates in a reference mode, capturing a SEM image at the exact position of the anomalies coordinates and capturing a SEM image of a reference location in the center of the wafer. A Defect reference image is created based on the Reference image minus the Defect image. The exact coordinates of the defect is calculated based on the calculated defect position and the anomalies stage coordinate calculated when the high magnification SEM defect image is captured. The captured SEM image is processed through either DRSEM ADC binning, exporting to a Yield Analysis System (YAS), or a combination of both. Process Engineers, Yield Analysis Engineers or Failure Analysis Engineers will manually review the captured images to insure that either the YAS defect binning is accurately classifying the defects or that the DRSEM defect binning is accurately classifying the defects. This paper is an exploration of the feasibility of the

Rho GTPase activity modulates paramyxovirus fusion protein-mediated cell-cell fusion

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

Schowalter, Rachel M.; Wurth, Mark A.; Aguilar, Hector C.

2006-07-05

The paramyxovirus fusion protein (F) promotes fusion of the viral envelope with the plasma membrane of target cells as well as cell-cell fusion. The plasma membrane is closely associated with the actin cytoskeleton, but the role of actin dynamics in paramyxovirus F-mediated membrane fusion is unclear. We examined cell-cell fusion promoted by two different paramyxovirus F proteins in three cell types in the presence of constitutively active Rho family GTPases, major cellular coordinators of actin dynamics. Reporter gene and syncytia assays demonstrated that expression of either Rac1{sup V12} or Cdc42{sup V12} could increase cell-cell fusion promoted by the Hendra ormore » SV5 glycoproteins, though the effect was dependent on the cell type expressing the viral glycoproteins. In contrast, RhoA{sup L63} decreased cell-cell fusion promoted by Hendra glycoproteins but had little affect on SV5 F-mediated fusion. Also, data suggested that GTPase activation in the viral glycoprotein-containing cell was primarily responsible for changes in fusion. Additionally, we found that activated Cdc42 promoted nuclear rearrangement in syncytia.« less

Exocytosis of vacuolar apical compartment (VAC): a cell-cell contact controlled mechanism for the establishment of the apical plasma membrane domain in epithelial cells

PubMed Central

1988-01-01

The vacuolar apical compartment (VAC) is an organelle found in Madin- Darby canine kidney (MDCK) cells with incomplete intercellular contacts by incubation in 5 microM Ca++ and in cells without contacts (single cells in subconfluent culture); characteristically, it displays apical biochemical markers and microvilli and excludes basolateral markers (Vega-Salas, D. E., P. J. I. Salas, and E. Rodriguez-Boulan. 1987. J. Cell Biol. 104:1249-1259). The apical surface of cells kept under these culture conditions is immature, with reduced numbers of microvilli and decreased levels of an apical biochemical marker (184 kD), which is, however, still highly polarized (Vega-Salas, D. E., P. J. I. Salas, D. Gundersen, and E. Rodriguez-Boulan. 1987. J. Cell Biol. 104:905-916). We describe here the morphological stages of VAC exocytosis which ultimately lead to the establishment of a differentiated apical domain. Addition of 1.8 mM Ca++ to monolayers developed in 5 microM Ca++ causes the rapid (20-40 min) fusion of VACs with the plasma membrane and their accessibility to external antibodies, as demonstrated by immunofluorescence, immunoperoxidase EM, and RIA with antibodies against the 184-kD apical plasma membrane marker. Exocytosis occurs towards areas of cell-cell contact in the developing lateral surface where they form intercellular pockets; fusion images are always observed immediately adjacent to the incomplete junctional bands detected by the ZO-1 antibody (Stevenson, B. R., J. D. Siliciano, M. S. Mooseker, and D. A. Goodenough. 1986. J. Cell Biol. 103:755-766). Blocks of newly incorporated VAC microvilli and 184-kD protein progressively move from intercellular ("primitive" lateral) spaces towards the microvilli-poor free cell surface. The definitive lateral domain is sealed behind these blocks by the growing tight junctional fence. These results demonstrate a fundamental role of cell-cell contact-mediated VAC exocytosis in the establishment of epithelial surface polarity

The Fusion Gain Analysis of the Inductively Driven Liner Compression Based Fusion

NASA Astrophysics Data System (ADS)

Shimazu, Akihisa; Slough, John

2016-10-01

An analytical analysis of the fusion gain expected in the inductively driven liner compression (IDLC) based fusion is conducted to identify the fusion gain scaling at various operating conditions. The fusion based on the IDLC is a magneto-inertial fusion concept, where a Field-Reversed Configuration (FRC) plasmoid is compressed via the inductively-driven metal liner to drive the FRC to fusion conditions. In the past, an approximate scaling law for the expected fusion gain for the IDLC based fusion was obtained under the key assumptions of (1) D-T fuel at 5-40 keV, (2) adiabatic scaling laws for the FRC dynamics, (3) FRC energy dominated by the pressure balance with the edge magnetic field at the peak compression, and (4) the liner dwell time being liner final diameter divided by the peak liner velocity. In this study, various assumptions made in the previous derivation is relaxed to study the change in the fusion gain scaling from the previous result of G ml1 / 2 El11 / 8 , where ml is the liner mass and El is the peak liner kinetic energy. The implication from the modified fusion gain scaling on the performance of the IDLC fusion reactor system is also explored.

Phagocytosis of antibody‐opsonized tumor cells leads to the formation of a discrete vacuolar compartment in macrophages

PubMed Central

Velmurugan, Ramraj; Ramakrishnan, Sreevidhya; Kim, Mingin

2018-01-01

Despite the rapidly expanding use of antibody‐based therapeutics to treat cancer, knowledge of the cellular processes following phagocytosis of antibody‐opsonized tumor cells is limited. Here we report the formation of a phagosome‐associated vacuole that is observed in macrophages as these degradative compartments mature following phagocytosis of HER2‐positive cancer cells in the presence of the HER2‐specific antibody, trastuzumab. We demonstrate that this vacuole is a distinct organelle that is closely apposed to the phagosome. Furthermore, the size of the phagosome‐associated vacuole is increased by inhibition of the mTOR pathway. Collectively, the identification of this vacuolar compartment has implications for understanding the subcellular trafficking processes leading to the destruction of phagocytosed, antibody‐opsonized cancer cells by macrophages. PMID:29437282

A systematic screen for morphological abnormalities during fission yeast sexual reproduction identifies a mechanism of actin aster formation for cell fusion

PubMed Central

Groux, Raphaël; Vincenzetti, Vincent

2017-01-01

In non-motile fungi, sexual reproduction relies on strong morphogenetic changes in response to pheromone signaling. We report here on a systematic screen for morphological abnormalities of the mating process in fission yeast Schizosaccharomyces pombe. We derived a homothallic (self-fertile) collection of viable deletions, which, upon visual screening, revealed a plethora of phenotypes affecting all stages of the mating process, including cell polarization, cell fusion and sporulation. Cell fusion relies on the formation of the fusion focus, an aster-like F-actin structure that is marked by strong local accumulation of the myosin V Myo52, which concentrates secretion at the fusion site. A secondary screen for fusion-defective mutants identified the myosin V Myo51-associated coiled-coil proteins Rng8 and Rng9 as critical for the coalescence of the fusion focus. Indeed, rng8Δ and rng9Δ mutant cells exhibit multiple stable dots at the cell-cell contact site, instead of the single focus observed in wildtype. Rng8 and Rng9 accumulate on the fusion focus, dependent on Myo51 and tropomyosin Cdc8. A tropomyosin mutant allele, which compromises Rng8/9 localization but not actin binding, similarly leads to multiple stable dots instead of a single focus. By contrast, myo51 deletion does not strongly affect fusion focus coalescence. We propose that focusing of the actin filaments in the fusion aster primarily relies on Rng8/9-dependent cross-linking of tropomyosin-actin filaments. PMID:28410370

Novel cooperative neural fusion algorithms for image restoration and image fusion.

PubMed

Xia, Youshen; Kamel, Mohamed S

2007-02-01

To deal with the problem of restoring degraded images with non-Gaussian noise, this paper proposes a novel cooperative neural fusion regularization (CNFR) algorithm for image restoration. Compared with conventional regularization algorithms for image restoration, the proposed CNFR algorithm can relax need of the optimal regularization parameter to be estimated. Furthermore, to enhance the quality of restored images, this paper presents a cooperative neural fusion (CNF) algorithm for image fusion. Compared with existing signal-level image fusion algorithms, the proposed CNF algorithm can greatly reduce the loss of contrast information under blind Gaussian noise environments. The performance analysis shows that the proposed two neural fusion algorithms can converge globally to the robust and optimal image estimate. Simulation results confirm that in different noise environments, the proposed two neural fusion algorithms can obtain a better image estimate than several well known image restoration and image fusion methods.

Inhibition of bone resorption in vitro by antisense RNA and DNA molecules targeted against carbonic anhydrase II or two subunits of vacuolar H(+)-ATPase.

PubMed Central

Laitala, T; Väänänen, H K

1994-01-01

The bone resorbing cells, osteoclasts, express high levels of carbonic anhydrase II (CA II) and vacuolar H(+)-ATPase (V-ATPase) during bone resorption. We have used antisense RNA and DNA molecules targeted against CA II, and against 16- and 60-kD subunits of vacuolar H(+)-ATPase (V-ATPase), to block the expression of these proteins in vitro. Osteoclastic bone resorption was studied in two in vitro culture systems: release of 45Calcium from prelabeled newborn mouse calvaria cultures, and resorption pit assays performed with rat osteoclasts cultured on bovine bone slices. Both antisense RNA and DNA against CA II and the V-ATPase were used to compare their specificities as regards inhibiting bone resorption in vitro. The antisense molecules inhibited the synthesis of these proteins by decreasing the amounts of mRNA in the cells in a highly specific manner. In osteoclast cultures treated with the 16-kD V-ATPase antisense RNA, acidification of an unknown population of intracellular vesicles was highly stimulated. The acidification of these vesicles was not sensitive to amiloride or bafilomycin A1. This suggests the existence of a back-up system for acidification of intracellular vesicles, when the expression of the V-ATPase is blocked. Our results further indicate that blocking the expression of CA II and V-ATPase with antisense RNA or DNA leads to decreased bone resorption. Images PMID:8200964

Magnetic-confinement fusion

NASA Astrophysics Data System (ADS)

Ongena, J.; Koch, R.; Wolf, R.; Zohm, H.

2016-05-01

Our modern society requires environmentally friendly solutions for energy production. Energy can be released not only from the fission of heavy nuclei but also from the fusion of light nuclei. Nuclear fusion is an important option for a clean and safe solution for our long-term energy needs. The extremely high temperatures required for the fusion reaction are routinely realized in several magnetic-fusion machines. Since the early 1990s, up to 16 MW of fusion power has been released in pulses of a few seconds, corresponding to a power multiplication close to break-even. Our understanding of the very complex behaviour of a magnetized plasma at temperatures between 150 and 200 million °C surrounded by cold walls has also advanced substantially. This steady progress has resulted in the construction of ITER, a fusion device with a planned fusion power output of 500 MW in pulses of 400 s. ITER should provide answers to remaining important questions on the integration of physics and technology, through a full-size demonstration of a tenfold power multiplication, and on nuclear safety aspects. Here we review the basic physics underlying magnetic fusion: past achievements, present efforts and the prospects for future production of electrical energy. We also discuss questions related to the safety, waste management and decommissioning of a future fusion power plant.

A major defect in mast cell effector functions in CRACM1-/- mice

PubMed Central

Vig, Monika; Dehaven, Wayne I; Bird, Gary S; Billingsley, James M; Wang, Huiyun; Rao, Patricia E; Hutchings, Amy B; Jouvin, Marie-Hélène; Putney, James W; Kinet, Jean-Pierre

2008-01-01

CRACM1 (Orai1) constitutes the pore subunit of CRAC channels that are crucial for many physiological processes 1-6. A point mutation in CRACM1 has been associated with SCID disease in humans 2. We have generated CRACM1 deficient mice using gene trap, where β-galactosidase (LacZ) activity identifies CRACM1 expression in tissues. We show here that the homozygous CRACM1 deficient mice are considerably smaller in size and are grossly defective in mast cell degranulation and cytokine secretion. FcεRI-mediated in vivo allergic reactions were also inhibited in CRACM1-/- mice. Other tissues expressing truncated CRACM1-LacZ fusion protein include skeletal muscles, kidney and regions in the brain and heart. Surprisingly, no CRACM1 expression was seen in the lymphoid regions of thymus. Accordingly, we found no defect in T cell development. Thus, our data reveal novel crucial roles for CRAC channels including a putative role in excitable cells. PMID:18059270

Data fusion for automated non-destructive inspection

PubMed Central

Brierley, N.; Tippetts, T.; Cawley, P.

2014-01-01

In industrial non-destructive evaluation (NDE), it is increasingly common for data acquisition to be automated, driving a recent substantial increase in the availability of data. The collected data need to be analysed, typically necessitating the painstaking manual labour of a skilled operator. Moreover, in automated NDE a region of an inspected component is typically interrogated several times, be it within a single data channel due to multiple probe passes, across several channels acquired simultaneously or over the course of repeated inspections. The systematic combination of these diverse readings is recognized to offer an opportunity to improve the reliability of the inspection, but is not achievable in a manual analysis. This paper describes a data-fusion-based software framework providing a partial automation capability, allowing component regions to be declared defect-free to a very high probability while readily identifying defect indications, thereby optimizing the use of the operator's time. The system is designed to applicable to a wide range of automated NDE scenarios, but the processing is exemplified using the industrial ultrasonic immersion inspection of aerospace turbine discs. Results obtained for industrial datasets demonstrate an orders-of-magnitude reduction in false-call rates, for a given probability of detection, achievable using the developed software system. PMID:25002828

Vacuolar biogenesis and aquaporin expression at early germination of broad bean seeds.

PubMed

Novikova, Galina V; Tournaire-Roux, Colette; Sinkevich, Irina A; Lityagina, Snejana V; Maurel, Christophe; Obroucheva, Natalie

2014-09-01

A key event in seed germination is water uptake-mediated growth initiation in embryonic axes. Vicia faba var. minor (broad bean) seeds were used for studying cell growth, vacuolar biogenesis, expression and function of tonoplast water channel proteins (aquaporins) in embryonic axes during seed imbibition, radicle emergence and growth. Hypocotyl and radicle basal cells showed vacuole restoration from protein storage vacuoles, whereas de novo vacuole formation from provacuoles was observed in cells newly produced by root meristem. cDNA fragments of seven novel aquaporin isoforms including five Tonoplast Intrinsic Proteins (TIP) from three sub-types were amplified by PCR. The expression was probed using q-RT-PCR and when possible with isoform-specific antibodies. Decreased expression of TIP3s was associated to the transformation of protein storage vacuoles to vacuoles, whereas enhanced expression of a TIP2 homologue was closely linked to the fast cell elongation. Water channel functioning checked by inhibitory test with mercuric chloride showed closed water channels prior to growth initiation and active water transport into elongating cells. The data point to a crucial role of tonoplast aquaporins during germination, especially during growth of embryonic axes, due to accelerated water uptake and vacuole enlargement resulting in rapid cell elongation. Copyright © 2014 Elsevier Masson SAS. All rights reserved.

Suppression of the vacuolar invertase gene delays senescent sweetening in chipping potatoes.

PubMed

Wiberley-Bradford, Amy E; Bethke, Paul C

2018-01-01

Potato chip processors require potato tubers that meet quality specifications for fried chip color, and color depends largely upon tuber sugar contents. At later times in storage, potatoes accumulate sucrose, glucose, and fructose. This developmental process, senescent sweetening, manifests as a blush of color near the center of the fried chip, becomes more severe with time, and limits the storage period. Vacuolar invertase (VInv) converts sucrose to glucose and fructose and is hypothesized to play a role in senescent sweetening. To test this hypothesis, senescent sweetening was quantified in multiple lines of potato with reduced VInv expression. Chip darkening from senescent sweetening was delayed by about 4 weeks for tubers with reduced VInv expression. A strong positive correlation between frequency of dark chips and tuber hexose content was observed. Tubers with reduced VInv expression had lower hexose to sucrose ratios than controls. VInv activity contributes to reducing sugar accumulation during senescent sweetening. Sucrose breakdown during frying may contribute to chip darkening. Suppressing VInv expression increases the storage period of the chipping potato crop, which is an important consideration, as potatoes with reduced VInv expression are entering commercial production in the USA. © 2017 Society of Chemical Industry. © 2017 Society of Chemical Industry.

FuzzyFusion: an application architecture for multisource information fusion

NASA Astrophysics Data System (ADS)

Fox, Kevin L.; Henning, Ronda R.

2009-04-01

The correlation of information from disparate sources has long been an issue in data fusion research. Traditional data fusion addresses the correlation of information from sources as diverse as single-purpose sensors to all-source multi-media information. Information system vulnerability information is similar in its diversity of sources and content, and in the desire to draw a meaningful conclusion, namely, the security posture of the system under inspection. FuzzyFusionTM, A data fusion model that is being applied to the computer network operations domain is presented. This model has been successfully prototyped in an applied research environment and represents a next generation assurance tool for system and network security.

The Pathogenesis of Atrial and Atrioventricular Septal Defects with Special Emphasis on the Role of the Dorsal Mesenchymal Protrusion

PubMed Central

Briggs, Laura E.; Kakarla, Jayant; Wessels, Andy

2012-01-01

Partitioning of the four-chambered heart requires the proper formation, interaction and fusion of several mesenchymal tissues derived from different precursor populations that together form the atrioventricular mesenchymal complex. This includes the major endocardial cushions and the mesenchymal cap of the septum primum, which are of endocardial origin, and the dorsal mesenchymal protrusion (DMP), which is derived from the Second Heart Field. Failure of these structures to develop and/or fully mature results in atrial septal defects (ASDs) and atrioventricular septal defects (AVSD). AVSDs are congenital malformations in which the atria are permitted to communicate due to defective septation between the inferior margin of the septum primum and the atrial surface of the common atrioventricular valve. The clinical presentation of AVSDs is variable and depends on both the size and/or type of defect; less severe defects may be asymptomatic while the most severe defect, if untreated, results in infantile heart failure. For many years, maldevelopment of the endocardial cushions was thought to be the sole etiology of AVSDs. More recent work, however, has demonstrated that perturbation of DMP development also results in AVSD. Here, we discuss in detail the formation of the DMP, its contribution to cardiac septation and describe the morphological features as well as potential etiologies of ASDs and AVSDs. PMID:22709652

Hendra virus fusion protein transmembrane domain contributes to pre-fusion protein stability

PubMed Central

Webb, Stacy; Nagy, Tamas; Moseley, Hunter; Fried, Michael; Dutch, Rebecca

2017-01-01

Enveloped viruses utilize fusion (F) proteins studding the surface of the virus to facilitate membrane fusion with a target cell membrane. Fusion of the viral envelope with a cellular membrane is required for release of viral genomic material, so the virus can ultimately reproduce and spread. To drive fusion, the F protein undergoes an irreversible conformational change, transitioning from a metastable pre-fusion conformation to a more thermodynamically stable post-fusion structure. Understanding the elements that control stability of the pre-fusion state and triggering to the post-fusion conformation is important for understanding F protein function. Mutations in F protein transmembrane (TM) domains implicated the TM domain in the fusion process, but the structural and molecular details in fusion remain unclear. Previously, analytical ultracentrifugation was utilized to demonstrate that isolated TM domains of Hendra virus F protein associate in a monomer-trimer equilibrium (Smith, E. C., Smith, S. E., Carter, J. R., Webb, S. R., Gibson, K. M., Hellman, L. M., Fried, M. G., and Dutch, R. E. (2013) J. Biol. Chem. 288, 35726–35735). To determine factors driving this association, 140 paramyxovirus F protein TM domain sequences were analyzed. A heptad repeat of β-branched residues was found, and analysis of the Hendra virus F TM domain revealed a heptad repeat leucine-isoleucine zipper motif (LIZ). Replacement of the LIZ with alanine resulted in dramatically reduced TM-TM association. Mutation of the LIZ in the whole protein resulted in decreased protein stability, including pre-fusion conformation stability. Together, our data suggest that the heptad repeat LIZ contributed to TM-TM association and is important for F protein function and pre-fusion stability. PMID:28213515

Characterization of fusion genes and the significantly expressed fusion isoforms in breast cancer by hybrid sequencing

PubMed Central

Weirather, Jason L.; Afshar, Pegah Tootoonchi; Clark, Tyson A.; Tseng, Elizabeth; Powers, Linda S.; Underwood, Jason G.; Zabner, Joseph; Korlach, Jonas; Wong, Wing Hung; Au, Kin Fai

2015-01-01

We developed an innovative hybrid sequencing approach, IDP-fusion, to detect fusion genes, determine fusion sites and identify and quantify fusion isoforms. IDP-fusion is the first method to study gene fusion events by integrating Third Generation Sequencing long reads and Second Generation Sequencing short reads. We applied IDP-fusion to PacBio data and Illumina data from the MCF-7 breast cancer cells. Compared with the existing tools, IDP-fusion detects fusion genes at higher precision and a very low false positive rate. The results show that IDP-fusion will be useful for unraveling the complexity of multiple fusion splices and fusion isoforms within tumorigenesis-relevant fusion genes. PMID:26040699

Dynamic trafficking of wheat γ-gliadin and of its structural domains in tobacco cells, studied with fluorescent protein fusions

PubMed Central

Francin-Allami, Mathilde; Saumonneau, Amélie; Lavenant, Laurence; Bouder, Axelle; Sparkes, Imogen; Hawes, Chris; Popineau, Yves

2011-01-01

Prolamins, the main storage proteins of wheat seeds, are synthesized and retained in the endoplasmic reticulum (ER) of the endosperm cells, where they accumulate in protein bodies (PBs) and are then exported to the storage vacuole. The mechanisms leading to these events are unresolved. To investigate this unconventional trafficking pathway, wheat γ-gliadin and its isolated repeated N-terminal and cysteine-rich C-terminal domains were fused to fluorescent proteins and expressed in tobacco leaf epidermal cells. The results indicated that γ-gliadin and both isolated domains were able to be retained and accumulated as protein body-like structures (PBLS) in the ER, suggesting that tandem repeats are not the only sequence involved in γ-gliadin ER retention and PBLS formation. The high actin-dependent mobility of γ-gliadin PBLS is also reported, and it is demonstrated that most of them do not co-localize with Golgi body or pre-vacuolar compartment markers. Both γ-gliadin domains are found in the same PBLS when co-expressed, which is most probably due to their ability to interact with each other, as indicated by the yeast two-hybrid and FRET-FLIM experiments. Moreover, when stably expressed in BY-2 cells, green fluorescent protein (GFP) fusions to γ-gliadin and its isolated domains were retained in the ER for several days before being exported to the vacuole in a Golgi-dependent manner, and degraded, leading to the release of the GFP ‘core’. Taken together, the results show that tobacco cells are a convenient model to study the atypical wheat prolamin trafficking with fluorescent protein fusions. PMID:21617248

Muon Catalyzed Fusion

NASA Technical Reports Server (NTRS)

Armour, Edward A.G.

2007-01-01

Muon catalyzed fusion is a process in which a negatively charged muon combines with two nuclei of isotopes of hydrogen, e.g, a proton and a deuteron or a deuteron and a triton, to form a muonic molecular ion in which the binding is so tight that nuclear fusion occurs. The muon is normally released after fusion has taken place and so can catalyze further fusions. As the muon has a mean lifetime of 2.2 microseconds, this is the maximum period over which a muon can participate in this process. This article gives an outline of the history of muon catalyzed fusion from 1947, when it was first realised that such a process might occur, to the present day. It includes a description of the contribution that Drachrnan has made to the theory of muon catalyzed fusion and the influence this has had on the author's research.

Type II integral membrane protein, TM of J paramyxovirus promotes cell-to-cell fusion.

PubMed

Li, Zhuo; Hung, Cher; Paterson, Reay G; Michel, Frank; Fuentes, Sandra; Place, Ryan; Lin, Yuan; Hogan, Robert J; Lamb, Robert A; He, Biao

2015-10-06

Paramyxoviruses include many important animal and human pathogens. Most paramyxoviruses have two integral membrane proteins: fusion protein (F) and attachment proteins hemagglutinin, hemagglutinin-neuraminidase, or glycoprotein (G), which are critical for viral entry into cells. J paramyxovirus (JPV) encodes four integral membrane proteins: F, G, SH, and transmembrane (TM). The function of TM is not known. In this work, we have generated a viable JPV lacking TM (JPV∆TM). JPV∆TM formed opaque plaques compared with JPV. Quantitative syncytia assays showed that JPV∆TM was defective in promoting cell-to-cell fusion (i.e., syncytia formation) compared with JPV. Furthermore, cells separately expressing F, G, TM, or F plus G did not form syncytia whereas cells expressing F plus TM formed some syncytia. However, syncytia formation was much greater with coexpression of F, G, and TM. Biochemical analysis indicates that F, G, and TM interact with each other. A small hydrophobic region in the TM ectodomain from amino acid residues 118 to 132, the hydrophobic loop (HL), was important for syncytial promotion, suggesting that the TM HL region plays a critical role in cell-to-cell fusion.

Temporary Percutaneous Instrumentation and Selective Anterior Fusion for Thoracolumbar Fractures.

PubMed

Charles, Yann Philippe; Walter, Axel; Schuller, Sébastien; Steib, Jean-Paul

2017-05-01

Prospective clinical trial in thoracolumbar trauma with 5-year follow-up. To analyze clinical and radiographic outcomes of minimal invasive surgery, and the rational of circumferential fracture treatment with regard to age, degenerative changes, bone mineral density, and global sagittal balance. Non-neurologic fractures with anterior column defect can be treated by posterior percutaneous instrumentation and selective anterior fusion. After consolidation, instrumentation can be removed at 1 year to provide mobility in non-fused segments. Fifty-one patients, 47 (18-75) years, were operated for A2, A3, or B-type fractures. Visual analog scale (VAS) for back pain and Oswestry Disability Index (ODI) were assessed. Radiographic measurements were: sagittal index, regional kyphosis, T4-T12 kyphosis, L1-S1 lordosis, pelvic incidence, pelvic tilt, sacral slope, and T9 tilt. Anterior fusion and facet joints were analyzed on computed tomography (CT) at 1 year. The ODI was 8.8 before accident, 35.4 at 3 months, 17.8 at 2 years, 14.4 at 5 years. The VAS was 2.0 at 3 months and 1.0 at 5 years. The sagittal index was 18.0° preoperatively and 1.0° at 3 months (P < 0.0001). A loss of reduction of 1.1° occurred after implant removal (P = 0.009). Global sagittal balance remained unchanged. Ten patients with osteopenia or osteoporosis had a worse ODI: 24.7 versus 11.9 (P = 0.016), and a greater loss of correction: 4.9° versus 1.3° (P = 0.007). Cages filled with cancellous bone from the fractured vertebra fused regularly. Spontaneous facet joint fusions were observed in two patients at the fracture level in B-type injuries. Percutaneous instrumentation and selective anterior fusion using autologous bone and mesh cages lead to high fusion rates, which provided good long-term clinical results in younger patients with thoracolumbar fractures. Sagittal alignment was maintained after instrumentation removal without damaging paravertebral muscles. Outcomes were worse in

Hendra virus fusion protein transmembrane domain contributes to pre-fusion protein stability.

PubMed

Webb, Stacy; Nagy, Tamas; Moseley, Hunter; Fried, Michael; Dutch, Rebecca

2017-04-07

Enveloped viruses utilize fusion (F) proteins studding the surface of the virus to facilitate membrane fusion with a target cell membrane. Fusion of the viral envelope with a cellular membrane is required for release of viral genomic material, so the virus can ultimately reproduce and spread. To drive fusion, the F protein undergoes an irreversible conformational change, transitioning from a metastable pre-fusion conformation to a more thermodynamically stable post-fusion structure. Understanding the elements that control stability of the pre-fusion state and triggering to the post-fusion conformation is important for understanding F protein function. Mutations in F protein transmembrane (TM) domains implicated the TM domain in the fusion process, but the structural and molecular details in fusion remain unclear. Previously, analytical ultracentrifugation was utilized to demonstrate that isolated TM domains of Hendra virus F protein associate in a monomer-trimer equilibrium (Smith, E. C., Smith, S. E., Carter, J. R., Webb, S. R., Gibson, K. M., Hellman, L. M., Fried, M. G., and Dutch, R. E. (2013) J. Biol. Chem. 288, 35726-35735). To determine factors driving this association, 140 paramyxovirus F protein TM domain sequences were analyzed. A heptad repeat of β-branched residues was found, and analysis of the Hendra virus F TM domain revealed a heptad repeat leucine-isoleucine zipper motif (LIZ). Replacement of the LIZ with alanine resulted in dramatically reduced TM-TM association. Mutation of the LIZ in the whole protein resulted in decreased protein stability, including pre-fusion conformation stability. Together, our data suggest that the heptad repeat LIZ contributed to TM-TM association and is important for F protein function and pre-fusion stability. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

FusionAnalyser: a new graphical, event-driven tool for fusion rearrangements discovery

PubMed Central

Piazza, Rocco; Pirola, Alessandra; Spinelli, Roberta; Valletta, Simona; Redaelli, Sara; Magistroni, Vera; Gambacorti-Passerini, Carlo

2012-01-01

Gene fusions are common driver events in leukaemias and solid tumours; here we present FusionAnalyser, a tool dedicated to the identification of driver fusion rearrangements in human cancer through the analysis of paired-end high-throughput transcriptome sequencing data. We initially tested FusionAnalyser by using a set of in silico randomly generated sequencing data from 20 known human translocations occurring in cancer and subsequently using transcriptome data from three chronic and three acute myeloid leukaemia samples. in all the cases our tool was invariably able to detect the presence of the correct driver fusion event(s) with high specificity. In one of the acute myeloid leukaemia samples, FusionAnalyser identified a novel, cryptic, in-frame ETS2–ERG fusion. A fully event-driven graphical interface and a flexible filtering system allow complex analyses to be run in the absence of any a priori programming or scripting knowledge. Therefore, we propose FusionAnalyser as an efficient and robust graphical tool for the identification of functional rearrangements in the context of high-throughput transcriptome sequencing data. PMID:22570408

FusionAnalyser: a new graphical, event-driven tool for fusion rearrangements discovery.

PubMed

Piazza, Rocco; Pirola, Alessandra; Spinelli, Roberta; Valletta, Simona; Redaelli, Sara; Magistroni, Vera; Gambacorti-Passerini, Carlo

2012-09-01

Gene fusions are common driver events in leukaemias and solid tumours; here we present FusionAnalyser, a tool dedicated to the identification of driver fusion rearrangements in human cancer through the analysis of paired-end high-throughput transcriptome sequencing data. We initially tested FusionAnalyser by using a set of in silico randomly generated sequencing data from 20 known human translocations occurring in cancer and subsequently using transcriptome data from three chronic and three acute myeloid leukaemia samples. in all the cases our tool was invariably able to detect the presence of the correct driver fusion event(s) with high specificity. In one of the acute myeloid leukaemia samples, FusionAnalyser identified a novel, cryptic, in-frame ETS2-ERG fusion. A fully event-driven graphical interface and a flexible filtering system allow complex analyses to be run in the absence of any a priori programming or scripting knowledge. Therefore, we propose FusionAnalyser as an efficient and robust graphical tool for the identification of functional rearrangements in the context of high-throughput transcriptome sequencing data.

Characterization of fusion genes and the significantly expressed fusion isoforms in breast cancer by hybrid sequencing.

PubMed

Weirather, Jason L; Afshar, Pegah Tootoonchi; Clark, Tyson A; Tseng, Elizabeth; Powers, Linda S; Underwood, Jason G; Zabner, Joseph; Korlach, Jonas; Wong, Wing Hung; Au, Kin Fai

2015-10-15

We developed an innovative hybrid sequencing approach, IDP-fusion, to detect fusion genes, determine fusion sites and identify and quantify fusion isoforms. IDP-fusion is the first method to study gene fusion events by integrating Third Generation Sequencing long reads and Second Generation Sequencing short reads. We applied IDP-fusion to PacBio data and Illumina data from the MCF-7 breast cancer cells. Compared with the existing tools, IDP-fusion detects fusion genes at higher precision and a very low false positive rate. The results show that IDP-fusion will be useful for unraveling the complexity of multiple fusion splices and fusion isoforms within tumorigenesis-relevant fusion genes. © The Author(s) 2015. Published by Oxford University Press on behalf of Nucleic Acids Research.

Atrx deficiency induces telomere dysfunction, endocrine defects, and reduced life span

PubMed Central

Watson, L. Ashley; Solomon, Lauren A.; Li, Jennifer Ruizhe; Jiang, Yan; Edwards, Matthew; Shin-ya, Kazuo; Beier, Frank; Bérubé, Nathalie G.

2013-01-01

Human ATRX mutations are associated with cognitive deficits, developmental abnormalities, and cancer. We show that the Atrx-null embryonic mouse brain accumulates replicative damage at telomeres and pericentromeric heterochromatin, which is exacerbated by loss of p53 and linked to ATM activation. ATRX-deficient neuroprogenitors exhibited higher incidence of telomere fusions and increased sensitivity to replication stress–inducing drugs. Treatment of Atrx-null neuroprogenitors with the G-quadruplex (G4) ligand telomestatin increased DNA damage, indicating that ATRX likely aids in the replication of telomeric G4-DNA structures. Unexpectedly, mutant mice displayed reduced growth, shortened life span, lordokyphosis, cataracts, heart enlargement, and hypoglycemia, as well as reduction of mineral bone density, trabecular bone content, and subcutaneous fat. We show that a subset of these defects can be attributed to loss of ATRX in the embryonic anterior pituitary that resulted in low circulating levels of thyroxine and IGF-1. Our findings suggest that loss of ATRX increases DNA damage locally in the forebrain and anterior pituitary and causes tissue attrition and other systemic defects similar to those seen in aging. PMID:23563309

Nuclear Fusion prize laudation Nuclear Fusion prize laudation

NASA Astrophysics Data System (ADS)

Burkart, W.

2011-01-01

Clean energy in abundance will be of critical importance to the pursuit of world peace and development. As part of the IAEA's activities to facilitate the dissemination of fusion related science and technology, the journal Nuclear Fusion is intended to contribute to the realization of such energy from fusion. In 2010, we celebrated the 50th anniversary of the IAEA journal. The excellence of research published in the journal is attested to by its high citation index. The IAEA recognizes excellence by means of an annual prize awarded to the authors of papers judged to have made the greatest impact. On the occasion of the 2010 IAEA Fusion Energy Conference in Daejeon, Republic of Korea at the welcome dinner hosted by the city of Daejeon, we celebrated the achievements of the 2009 and 2010 Nuclear Fusion prize winners. Steve Sabbagh, from the Department of Applied Physics and Applied Mathematics, Columbia University, New York is the winner of the 2009 award for his paper: 'Resistive wall stabilized operation in rotating high beta NSTX plasmas' [1]. This is a landmark paper which reports record parameters of beta in a large spherical torus plasma and presents a thorough investigation of the physics of resistive wall mode (RWM) instability. The paper makes a significant contribution to the critical topic of RWM stabilization. John Rice, from the Plasma Science and Fusion Center, MIT, Cambridge is the winner of the 2010 award for his paper: 'Inter-machine comparison of intrinsic toroidal rotation in tokamaks' [2]. The 2010 award is for a seminal paper that analyzes results across a range of machines in order to develop a universal scaling that can be used to predict intrinsic rotation. This paper has already triggered a wealth of experimental and theoretical work. I congratulate both authors and their colleagues on these exceptional papers. W. Burkart Deputy Director General Department of Nuclear Sciences and Applications International Atomic Energy Agency, Vienna

Measuring time of flight of fusion products in an inertial electrostatic confinement fusion device for spatial profiling of fusion reactions

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

Donovan, D. C.; Boris, D. R.; Kulcinski, G. L.

2013-03-15

A new diagnostic has been developed that uses the time of flight (TOF) of the products from a nuclear fusion reaction to determine the location where the fusion reaction occurred. The TOF diagnostic uses charged particle detectors on opposing sides of the inertial electrostatic confinement (IEC) device that are coupled to high resolution timing electronics to measure the spatial profile of fusion reactions occurring between the two charged particle detectors. This diagnostic was constructed and tested by the University of Wisconsin-Madison Inertial Electrostatic Confinement Fusion Group in the IEC device, HOMER, which accelerates deuterium ions to fusion relevant energies inmore » a high voltage ({approx}100 kV), spherically symmetric, electrostatic potential well [J. F. Santarius, G. L. Kulcinski, R. P. Ashley, D. R. Boris, B. B. Cipiti, S. K. Murali, G. R. Piefer, R. F. Radel, T. E. Radel, and A. L. Wehmeyer, Fusion Sci. Technol. 47, 1238 (2005)]. The TOF diagnostic detects the products of D(d,p)T reactions and determines where along a chord through the device the fusion event occurred. The diagnostic is also capable of using charged particle spectroscopy to determine the Doppler shift imparted to the fusion products by the center of mass energy of the fusion reactants. The TOF diagnostic is thus able to collect spatial profiles of the fusion reaction density along a chord through the device, coupled with the center of mass energy of the reactions occurring at each location. This provides levels of diagnostic detail never before achieved on an IEC device.« less

Novel Tonoplast Transporters Identified Using a Proteomic Approach with Vacuoles Isolated from Cauliflower Buds1[W][OA

PubMed Central

Schmidt, Ulrike G.; Endler, Anne; Schelbert, Silvia; Brunner, Arco; Schnell, Magali; Neuhaus, H. Ekkehard; Marty-Mazars, Daniéle; Marty, Francis; Baginsky, Sacha; Martinoia, Enrico

2007-01-01

Young meristematic plant cells contain a large number of small vacuoles, while the largest part of the vacuome in mature cells is composed by a large central vacuole, occupying 80% to 90% of the cell volume. Thus far, only a limited number of vacuolar membrane proteins have been identified and characterized. The proteomic approach is a powerful tool to identify new vacuolar membrane proteins. To analyze vacuoles from growing tissues we isolated vacuoles from cauliflower (Brassica oleracea) buds, which are constituted by a large amount of small cells but also contain cells in expansion as well as fully expanded cells. Here we show that using purified cauliflower vacuoles and different extraction procedures such as saline, NaOH, acetone, and chloroform/methanol and analyzing the data against the Arabidopsis (Arabidopsis thaliana) database 102 cauliflower integral proteins and 214 peripheral proteins could be identified. The vacuolar pyrophosphatase was the most prominent protein. From the 102 identified proteins 45 proteins were already described. Nine of these, corresponding to 46% of peptides detected, are known vacuolar proteins. We identified 57 proteins (55.9%) containing at least one membrane spanning domain with unknown subcellular localization. A comparison of the newly identified proteins with expression profiles from in silico data revealed that most of them are highly expressed in young, developing tissues. To verify whether the newly identified proteins were indeed localized in the vacuole we constructed and expressed green fluorescence protein fusion proteins for five putative vacuolar membrane proteins exhibiting three to 11 transmembrane domains. Four of them, a putative organic cation transporter, a nodulin N21 family protein, a membrane protein of unknown function, and a senescence related membrane protein were localized in the vacuolar membrane, while a white-brown ATP-binding cassette transporter homolog was shown to reside in the plasma membrane

Enzyme Replacement for Craniofacial Skeletal Defects and Craniosynostosis in Murine Hypophosphatasia

PubMed Central

Liu, Jin; Campbell, Cassie; Nam, Hwa Kyung; Caron, Alexandre; Yadav, Manisha C; Millán, José Luis; Hatch, Nan E.

2015-01-01

Hypophosphatasia (HPP) is an inborn-error-of-metabolism disorder characterized by deficient bone and tooth mineralization due to loss-of function mutations in the gene (Alpl) encoding tissue-nonspecific alkaline phosphatase (TNAP). Alpl−/− mice exhibit many characteristics seen in infantile HPP including long bone and tooth defects, vitamin B6 responsive seizures and craniosynostosis. Previous reports demonstrated that a mineral-targeted form of TNAP rescues long bone, verterbral and tooth mineralization defects in Alpl−/− mice. Here we report that enzyme replacement with mineral-targeted TNAP (asfotase-alfa) also prevents craniosynostosis (the premature fusion of cranial bones) and additional craniofacial skeletal abnormalities in Alpl−/− mice. Craniosynostosis, cranial bone volume and density, and craniofacial shape abnormalities were assessed by microsocopy, histology, digital caliper measurements and micro CT. We found that craniofacial shape defects, cranial bone mineralization and craniosynostosis were corrected in Alpl−/− mice injected daily subcutaneously starting at birth with recombinant enzyme. Analysis of Alpl−/− calvarial cells indicates that TNAP deficiency leads to aberrant osteoblastic gene expression and diminished proliferation. Some but not all of these cellular abnormalities were rescued by treatment with inorganic phosphate. These results confirm an essential role for TNAP in craniofacial skeletal development and demonstrate the efficacy of early postnatal mineral-targeted enzyme replacement for preventing craniofacial abnormalities including craniosynostosis in murine infantile HPP. PMID:25959417

Spinal Fusion

MedlinePlus

... concept of fusion is similar to that of welding in industry. Spinal fusion surgery, however, does not ... bone taken from the patient has a long history of use and results in predictable healing. Autograft ...

Thermodynamic and Kinetic Properties of Intrinsic Defects and Mg Transmutants in 3C-SiC Determined by Density Functional Theory

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

Hu, Shenyang Y.; Setyawan, Wahyu; Van Ginhoven, Renee M.

2014-02-20

Density functional theory (DFT) is used to calculate the thermodynamic and kinetic properties of transmutant Mg in 3C-SiC due to high-energy neutron irradiation associated with the fusion nuclear environment. The formation and binding energies of intrinsic defects, Mg-related defects, and clusters in 3C-SiC are systematically calculated. The minimum energy paths and activation energies during point defect migration and small cluster evolution are studied using a generalized solid-state elastic band (G-SSNEB) method with DFT energy calculations. Stable defect structures and possible defect migration mechanisms are identified. The evolution of binding energies during Mg2Si formation demonstrates that the formation of Mg2Si needsmore » to overcome a critical nucleus size and nucleation barrier. It is also found that a compressive stress field exists around the Mg2Si nucleus. These data are important inputs in meso- and macro-scale modeling and experiments to understand and predict the impact of Mg on phase stability, microstructure evolution, and performance of SiC and SiC-based materials during long-term neutron exposures.« less

Review of fusion synfuels

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

Fillo, J.A.

1980-01-01

Thermonuclear fusion offers an inexhaustible source of energy for the production of hydrogen from water. Depending on design, electric generation efficiencies of approx. 40 to 60% and hydrogen production efficiencies by high-temperature electrolysis of approx. 50 to 65% are projected for fusion reactors using high-temperatures blankets. Fusion/coal symbiotic systems appear economically promising for the first generation of commercial fusion synfuels plants. Coal production requirements and the environmental effects of large-scale coal usage would be greatly reduced by a fusion/coal system. In the long term, there could be a gradual transition to an inexhaustible energy system based solely on fusion.

Arthroscopic partial wrist fusion.

PubMed

Ho, Pak-Cheong

2008-12-01

The wide intraarticular exposure of the wrist joint under arthroscopic view provides an excellent ground for various forms of partial wrist fusion. Combining with percutaneous fixation technique, arthroscopic partial wrist fusion can potentially generate the best possible functional outcome by preserving the maximal motion pertained with each type of partial wrist fusion because the effect of extraarticular adhesion associated with open surgery can be minimized. From November 1997 to May 2008, the author had performed 12 cases of arthroscopic partial wrist fusion, including scaphotrapeziotrapezoid fusion in 3, scaphoidectomy and 4-corner fusion in 4, radioscapholunate fusion in 3, radiolunate fusion in 1, and lunotriquetral fusion in 1 case. Through the radiocarpal or midcarpal joint, the corresponding articular surfaces were denuded of cartilage using arthroscopic burr and curette. Carpal bones involved in the fusion process were then transfixed with K wires percutaneously after alignment corrected and confirmed under fluoroscopic control. Autogenous cancellous bone graft or bone substitute were inserted and impacted to the fusion site through cannula under direct arthroscopic view. Final fixation could be by multiple K wires or cannulated screw system. Early mobilization was encouraged. Surgical complications were minor, including pin tract infection, skin burn, and delay union in 1 case. Uneventful radiologic union was obtained in 9 cases, stable fibrous union in 2, and nonunion in 1. The average follow-up period was 70 months. Symptom was resolved or improved, and functional motion was gained in all cases. All surgical scars were almost invisible, and aesthetic outcome was excellent.

Magneto-Inertial Fusion

DOE PAGES

Wurden, G. A.; Hsu, S. C.; Intrator, T. P.; ...

2015-11-17

In this community white paper, we describe an approach to achieving fusion which employs a hybrid of elements from the traditional magnetic and inertial fusion concepts, called magneto-inertial fusion (MIF). Furthermore, the status of MIF research in North America at multiple institutions is summarized including recent progress, research opportunities, and future plans.

Immobile defects in ferroelastic walls: Wall nucleation at defect sites

NASA Astrophysics Data System (ADS)

He, X.; Salje, E. K. H.; Ding, X.; Sun, J.

2018-02-01

Randomly distributed, static defects are enriched in ferroelastic domain walls. The relative concentration of defects in walls, Nd, follows a power law distribution as a function of the total defect concentration C: N d ˜ C α with α = 0.4 . The enrichment Nd/C ranges from ˜50 times when C = 10 ppm to ˜3 times when C = 1000 ppm. The resulting enrichment is due to nucleation at defect sites as observed in large scale MD simulations. The dynamics of domain nucleation and switching is dependent on the defect concentration. Their energy distribution follows the power law with exponents during yield between ɛ ˜ 1.82 and 2.0 when the defect concentration increases. The power law exponent is ɛ ≈ 2.7 in the plastic regime, independent of the defect concentration.

Membrane fusion and exocytosis.

PubMed

Jahn, R; Südhof, T C

1999-01-01

Membrane fusion involves the merger of two phospholipid bilayers in an aqueous environment. In artificial lipid bilayers, fusion proceeds by means of defined transition states, including hourglass-shaped intermediates in which the proximal leaflets of the fusing membranes are merged whereas the distal leaflets are separate (fusion stalk), followed by the reversible opening of small aqueous fusion pores. Fusion of biological membranes requires the action of specific fusion proteins. Best understood are the viral fusion proteins that are responsible for merging the viral with the host cell membrane during infection. These proteins undergo spontaneous and dramatic conformational changes upon activation. In the case of the paradigmatic fusion proteins of the influenza virus and of the human immunodeficiency virus, an amphiphilic fusion peptide is inserted into the target membrane. The protein then reorients itself, thus forcing the fusing membranes together and inducing lipid mixing. Fusion of intracellular membranes in eukaryotic cells involves several protein families including SNAREs, Rab proteins, and Sec1/Munc-18 related proteins (SM-proteins). SNAREs form a novel superfamily of small and mostly membrane-anchored proteins that share a common motif of about 60 amino acids (SNARE motif). SNAREs reversibly assemble into tightly packed helical bundles, the core complexes. Assembly is thought to pull the fusing membranes closely together, thus inducing fusion. SM-proteins comprise a family of soluble proteins that bind to certain types of SNAREs and prevent the formation of core complexes. Rab proteins are GTPases that undergo highly regulated GTP-GDP cycles. In their GTP form, they interact with specific proteins, the effector proteins. Recent evidence suggests that Rab proteins function in the initial membrane contact connecting the fusing membranes but are not involved in the fusion reaction itself.

Review of the magnetic fusion program by the 1986 ERAB Fusion Panel

NASA Astrophysics Data System (ADS)

Davidson, Ronald C.

1987-09-01

The 1986 ERAB Fusion Panel finds that fusion energy continues to be an attractive energy source with great potential for the future, and that the magnetic fusion program continues to make substantial technical progress. In addition, fusion research advances plasma physics, a sophisticated and useful branch of applied science, as well as technologies important to industry and defense. These factors fully justify the substantial expenditures by the Department of Energy in fusion research and development (R&D). The Panel endorses the overall program direction, strategy, and plans, and recognizes the importance and timeliness of proceeding with a burning plasma experiment, such as the proposed Compact Ignition Tokamak (CIT) experiment.

A Toxoplasma gondii protein with homology to intracellular type Na{sup +}/H{sup +} exchangers is important for osmoregulation and invasion

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

Francia, Maria E.; Wicher, Sarah; Pace, Douglas A.

2011-06-10

The obligate intracellular parasite Toxoplasma gondii is exposed to a variety of physiological conditions while propagating in an infected organism. The mechanisms by which Toxoplasma overcomes these dramatic changes in its environment are not known. In yeast and plants, ion detoxification and osmotic regulation are controlled by vacuolar compartments. A novel compartment named the plant-like vacuole or vacuolar compartment (PLV/VAC) has recently been described in T.gondii, which could potentially protect extracellular tachyzoites against salt and other ionic stresses. Here, we report the molecular characterization of the vacuolar type Na{sup +}/H{sup +} exchanger in T. gondii, TgNHE3, and its co-localization withmore » the PLV/VAC proton-pyrophosphatase (TgVP1). We have created a TgNHE3 knockout strain, which is more sensitive to hyperosmotic shock and toxic levels of sodium, possesses a higher intracellular Ca{sup 2+} concentration [Ca{sup 2+}]{sub i}, and exhibits a reduced host invasion efficiency. The defect in invasion correlates with a measurable reduction in the secretion of the adhesin TgMIC2. Overall, our results suggest that the PLV/VAC has functions analogous to those of the vacuolar compartments of plants and yeasts, providing the parasite with a mechanism to resist ionic fluctuations and, potentially, regulate protein trafficking.« less

Regulatory assembly of the vacuolar proton pump VoV1-ATPase in yeast cells by FLIM-FRET

NASA Astrophysics Data System (ADS)

Ernst, Stefan; Batisse, Claire; Zarrabi, Nawid; Böttcher, Bettina; Börsch, Michael

2010-02-01

We investigate the reversible disassembly of VOV1-ATPase in life yeast cells by time resolved confocal FRET imaging. VOV1-ATPase in the vacuolar membrane pumps protons from the cytosol into the vacuole. VOV1-ATPase is a rotary biological nanomotor driven by ATP hydrolysis. The emerging proton gradient is used for secondary transport processes as well as for pH and Ca2+ homoeostasis in the cell. The activity of the VOV1-ATPase is regulated through assembly / disassembly processes. During starvation the two parts of VOV1-ATPase start to disassemble. This process is reversed after addition of glucose. The exact mechanisms are unknown. To follow the disassembly / reassembly in vivo we tagged two subunits C and E with different fluorescent proteins. Cellular distributions of C and E were monitored using a duty cycle-optimized alternating laser excitation scheme (DCO-ALEX) for time resolved confocal FRET-FLIM measurements.

Vacuolar and cytoskeletal dynamics during elicitor-induced programmed cell death in tobacco BY-2 cells.

PubMed

Higaki, Takumi; Kadota, Yasuhiro; Goh, Tatsuaki; Hayashi, Teruyuki; Kutsuna, Natsumaro; Sano, Toshio; Hasezawa, Seiichiro; Kuchitsu, Kazuyuki

2008-09-01

Responses of plant cells to environmental stresses often involve morphological changes, differentiation and redistribution of various organelles and cytoskeletal network. Tobacco BY-2 cells provide excellent model system for in vivo imaging of these intracellular events. Treatment of the cell cycle-synchronized BY-2 cells with a proteinaceous oomycete elicitor, cryptogein, induces highly synchronous programmed cell death (PCD) and provide a model system to characterize vacuolar and cytoskeletal dynamics during the PCD. Sequential observation revealed dynamic reorganization of the vacuole and actin microfilaments during the execution of the PCD. We further characterized the effects cryptogein on mitotic microtubule organization in cell cycle-synchronized cells. Cryptogein treatment at S phase inhibited formation of the preprophase band, a cortical microtubule band that predicts the cell division site. Cortical microtubules kept their random orientation till their disruption that gradually occurred during the execution of the PCD twelve hours after the cryptogein treatment. Possible molecular mechanisms and physiological roles of the dynamic behavior of the organelles and cytoskeletal network in the pathogenic signal-induced PCD are discussed.

A thermo-physical analysis of the proton pump vacuolar-ATPase: the constructal approach.

PubMed

Lucia, Umberto; Ponzetto, Antonio; Deisboeck, Thomas S

2014-10-24

Pumping protons across a membrane was a critical step at the origin of life on earth, and it is still performed in all living organisms, including in human cells. Proton pumping is paramount to keep normal cells alive, e.g. for lysosomal digestion and for preparing peptides for immune recognition, but it goes awry in cancer cells. They acidify their microenvironment hence membrane voltage is lowered, which in turn induces cell proliferation, a hallmark of cancer. Proton pumping is achieved by means of rotary motors, namely vacuolar ATPases (V-ATPase), which are present at many of the multiple cellular interfaces. Therefore, we undertook an examination of the thermodynamic properties of V-ATPases. The principal result is that the V-ATPase-mediated control of the cell membrane potential and the related and consequent environmental pH can potentially represent a valuable support strategy for anticancer therapies. A constructal theory approach is used as a new viewpoint to study how V-ATPase can be modulated for therapeutic purposes. In particular, V-ATPase can be regulated by using external fields, such as electromagnetic fields, and a theoretical approach has been introduced to quantify the appropriate field strength and frequency for this new adjuvant therapeutic strategy.

Ontological Issues in Higher Levels of Information Fusion: User Refinement of the Fusion Process

DTIC Science & Technology

2003-01-01

fusion question, the thing that is separates the Greek We explore the higher-level purpose offusion systems by philosophical questions and modem day...the The Greeks focused on both data fusion and the Fusion02 conference there are common fusion questions philosophical questions of an ontology - the...data World of Visible Things Belief (pistis) fusion - user refinement. The rest of the paper is as Appearances follows: Section 2 details the Greek

The Vacuolar-Type H+-ATPase in Ovine Rumen Epithelium is Regulated by Metabolic Signals

PubMed Central

Kuzinski, Judith; Zitnan, Rudolf; Warnke-Gurgel, Christina; Schweigel, Monika

2010-01-01

In this study, the effect of metabolic inhibition (MI) by glucose substitution with 2-deoxyglucose (2-DOG) and/or application of antimycin A on ovine rumen epithelial cells (REC) vacuolar-type H+-ATPase (vH+-ATPase) activity was investigated. Using fluorescent spectroscopy, basal pHi of REC was measured to be 7.3 ± 0.1 in HCO3−-free, glucose-containing NaCl medium. MI induced a strong pHi reduction (−0.44 ± 0.04 pH units) with a more pronounced effect of 2-DOG compared to antimycin A (−0.30 ± 0.03 versus −0.21 ± 0.03 pH units). Treatment with foliomycin, a specific vH+-ATPase inhibitor, decreased REC pHi by 0.21 ± 0.05 pH units. After MI induction, this effect was nearly abolished (−0.03 ± 0.02 pH units). In addition, membrane-associated localization of vH+-ATPase B subunit disappeared. Metabolic control of vH+-ATPase involving regulation of its assembly state by elements of the glycolytic pathway could provide a means to adapt REC ATP consumption according to energy availability. PMID:20069127

Distinct Requirements for HIV-Cell Fusion and HIV-mediated Cell-Cell Fusion*

PubMed Central

Kondo, Naoyuki; Marin, Mariana; Kim, Jeong Hwa; Desai, Tanay M.; Melikyan, Gregory B.

2015-01-01

Whether HIV-1 enters cells by fusing with the plasma membrane or with endosomes is a subject of active debate. The ability of HIV-1 to mediate fusion between adjacent cells, a process referred to as “fusion-from-without” (FFWO), shows that this virus can fuse with the plasma membrane. To compare FFWO occurring at the cell surface with HIV-cell fusion through a conventional entry route, we designed an experimental approach that enabled the measurements of both processes in the same sample. The following key differences were observed. First, a very small fraction of viruses fusing with target cells participated in FFWO. Second, whereas HIV-1 fusion with adherent cells was insensitive to actin inhibitors, post-CD4/coreceptor binding steps during FFWO were abrogated. A partial dependence of HIV-cell fusion on actin remodeling was observed in CD4+ T cells, but this effect appeared to be due to the actin dependence of virus uptake. Third, deletion of the cytoplasmic tail of HIV-1 gp41 dramatically enhanced the ability of the virus to promote FFWO, while having a modest effect on virus-cell fusion. Distinct efficiencies and actin dependences of FFWO versus HIV-cell fusion are consistent with the notion that, except for a minor fraction of particles that mediate fusion between the plasma membranes of adjacent cells, HIV-1 enters through an endocytic pathway. We surmise, however, that cell-cell contacts enabling HIV-1 fusion with the plasma membrane could be favored at the sites of high density of target cells, such as lymph nodes. PMID:25589785

Technologies for Army Knowledge Fusion

DTIC Science & Technology

2004-09-01

interpret it in context and understand the implications (Alberts et al., 2002). Note that the knowledge / information fusion issue arises immediately here...Army Knowledge Fusion Richard Scherl Department of Computer Science Monmouth University Dana L. Ulery Computational and Information Sciences...civilian and military sources. Knowledge fusion, also called information fusion and multisensor data fusion, names the body of techniques needed to

Identification and Characterization of LFD-2, a Predicted Fringe Protein Required for Membrane Integrity during Cell Fusion in Neurospora crassa

PubMed Central

Palma-Guerrero, Javier; Zhao, Jiuhai; Gonçalves, A. Pedro; Starr, Trevor L.

2015-01-01

The molecular mechanisms of membrane merger during somatic cell fusion in eukaryotic species are poorly understood. In the filamentous fungus Neurospora crassa, somatic cell fusion occurs between genetically identical germinated asexual spores (germlings) and between hyphae to form the interconnected network characteristic of a filamentous fungal colony. In N. crassa, two proteins have been identified to function at the step of membrane fusion during somatic cell fusion: PRM1 and LFD-1. The absence of either one of these two proteins results in an increase of germling pairs arrested during cell fusion with tightly appressed plasma membranes and an increase in the frequency of cell lysis of adhered germlings. The level of cell lysis in ΔPrm1 or Δlfd-1 germlings is dependent on the extracellular calcium concentration. An available transcriptional profile data set was used to identify genes encoding predicted transmembrane proteins that showed reduced expression levels in germlings cultured in the absence of extracellular calcium. From these analyses, we identified a mutant (lfd-2, for late fusion defect-2) that showed a calcium-dependent cell lysis phenotype. lfd-2 encodes a protein with a Fringe domain and showed endoplasmic reticulum and Golgi membrane localization. The deletion of an additional gene predicted to encode a low-affinity calcium transporter, fig1, also resulted in a strain that showed a calcium-dependent cell lysis phenotype. Genetic analyses showed that LFD-2 and FIG1 likely function in separate pathways to regulate aspects of membrane merger and repair during cell fusion. PMID:25595444

Retention of a Bean Phaseolin/Maize γ-Zein Fusion in the Endoplasmic Reticulum Depends on Disulfide Bond Formation[W

PubMed Central

Pompa, Andrea; Vitale, Alessandro

2006-01-01

Most seed storage proteins of the prolamin class accumulate in the endoplasmic reticulum (ER) as large insoluble polymers termed protein bodies (PBs), through mechanisms that are still poorly understood. We previously showed that a fusion between the Phaseolus vulgaris vacuolar storage protein phaseolin and the N-terminal half of the Zea mays prolamin γ-zein forms ER-located PBs. Zeolin has 6 Cys residues and, like γ-zein with 15 residues, is insoluble unless reduced. The contribution of disulfide bonds to zeolin destiny was determined by studying in vivo the effects of 2-mercaptoethanol (2-ME) and by zeolin mutagenesis. We show that in tobacco (Nicotiana tabacum) protoplasts, 2-ME enhances interactions of newly synthesized proteins with the ER chaperone BiP and inhibits the secretory traffic of soluble proteins with or without disulfide bonds. In spite of this general inhibition, 2-ME enhances the solubility of zeolin and relieves its retention in the ER, resulting in increased zeolin traffic. Consistently, mutated zeolin unable to form disulfide bonds is soluble and efficiently enters the secretory traffic without 2-ME treatment. We conclude that disulfide bonds that lead to insolubilization are a determinant for PB-mediated protein accumulation in the ER. PMID:17041149

Influence of incomplete fusion on complete fusion at energies above the Coulomb barrier

NASA Astrophysics Data System (ADS)

Shuaib, Mohd; Sharma, Vijay R.; Yadav, Abhishek; Sharma, Manoj Kumar; Singh, Pushpendra P.; Singh, Devendra P.; Kumar, R.; Singh, R. P.; Muralithar, S.; Singh, B. P.; Prasad, R.

2017-10-01

In the present work, excitation functions of several reaction residues in the system 19F+169Tm, populated via the complete and incomplete fusion processes, have been measured using off-line γ-ray spectroscopy. The analysis of excitation functions has been done within the framework of statistical model code pace4. The excitation functions of residues populated via xn and pxn channels are found to be in good agreement with those estimated by the theoretical model code, which confirms the production of these residues solely via complete fusion process. However, a significant enhancement has been observed in the cross-sections of residues involving α-emitting channels as compared to the theoretical predictions. The observed enhancement in the cross-sections has been attributed to the incomplete fusion processes. In order to have a better insight into the onset and strength of incomplete fusion, the incomplete fusion strength function has been deduced. At present, there is no theoretical model available which can satisfactorily explain the incomplete fusion reaction data at energies ≈4-6 MeV/nucleon. In the present work, the influence of incomplete fusion on complete fusion in the 19F+169Tm system has also been studied. The measured cross-section data may be important for the development of reactor technology as well. It has been found that the incomplete fusion strength function strongly depends on the α-Q value of the projectile, which is found to be in good agreement with the existing literature data. The analysis strongly supports the projectile-dependent mass-asymmetry systematics. In order to study the influence of Coulomb effect ({Z}{{P}}{Z}{{T}}) on incomplete fusion, the deduced strength function for the present work is compared with the nearby projectile-target combinations. The incomplete fusion strength function is found to increase linearly with {Z}{{P}}{Z}{{T}}, indicating a strong influence of Coulomb effect in the incomplete fusion reactions.

Cluster-impact fusion, or beam-contaminant fusion? (abstract)a),b)

NASA Astrophysics Data System (ADS)

Lo, Daniel H.; Petrasso, Richard D.; Wenzel, Kevin W.

1992-10-01

Beuhler, Friedlander, and Friedman (BFF) reported anomalously huge D-D fusion rates while bombarding deuterated targets with (D2O)N+ clusters (N˜25-1000) accelerated to ≊325 keV [R. J. Beuhler et al., Phys. Rev. Lett. 63, 1292 (1989); R. J. Beuhler et al., J. Phys. Chem. 94, 7665 (1990)] [i.e., ≊0.3 keV lab energy for D in (D2O)100+]. However, from our analysis of BFF's fusion product spectra, we conclude that their D lab energy was ˜50 keV. Therefore, no gross anomalies exist. Also, from our analysis of the BFF beam-ranging experiments through 500 μg/cm2 of Au, we conclude that light-ion-beam contaminants (e.g., D+ of order 100 keV) have not been ruled out, and are the probable cause of their fusion reactions. This work was supported by LLNL Subcontract B116798, Department of Energy (DOE) Grant No. DE-FG02-91ER54109, DOE Magnetic Fusion Energy Technology Fellowship Program (D. H. Lo), and DOE Fusion Energy Postdoctoral Research Program (Kevin W. Wenzel).

Ventral Dural Injury After Oblique Lumbar Interbody Fusion.

PubMed

Chang, JaeChil; Kim, Jin-Sung; Jo, Hyunjin

2017-02-01

Oblique lumbar interbody fusion (OLIF) through the oblique corridor between the aorta and anterior border of psoas muscle is favored among spinal surgeons who employ minimally invasive techniques. We report a case of ventral dural tear after OLIF that was associated with the inaccurate trajectory direction of endplate preparation. This is the first report to our knowledge of ventral dural tear associated with OLIF. A 72-year-old woman presented with right leg pain and numbness. X-rays showed degenerative spondylolisthesis and loss of disc height at L4-L5 and L5-S1 levels. Magnetic resonance imaging revealed right-sided paracentral disc herniation at the L3-L4 level and foraminal disc herniation at L4-L5. The initial surgical plan was OLIF of L3-L4 and L4-L5 after percutaneous screw fixation without laminectomy. With the patient in the lateral position, discectomy and endplate preparation were done successfully at the L3-L4 level, and the same procedure was done at the L4-L5 level for OLIF. A sharp Cobbs elevator for endplate preparation triggered a ventral dural defect at the L4-L5 level. We changed the patient's position to attempt dural repair. The ventral dural defect could not be repaired because it was too large. After the herniated rootlets were repositioned, TachoComb was patched over the defect site. Postoperatively, the patient has no definite neurologic deficits. When a surgeon performs OLIF, ventral dural injury should be avoided during the procedure of endplate preparation and contralateral annular release. Copyright © 2016 Elsevier Inc. All rights reserved.

Fusion Science Education Outreach

NASA Astrophysics Data System (ADS)

Danielson, C. A.; DIII-D Education Group

1996-11-01

This presentation will focus on education outreach activities at General Atomics that have been expanded to include the general population on science education with a focus on fusion energy. Outreach materials are distributed upon request both nationally and internationally. These materials include a notebook containing copies of DIII--D tour panels, fusion poster, new fusion energy video, new fusion energy brochure, and the electromagnetic spectrum curriculum. The 1996 Fusion Forum (held in the House Caucus Room) included a student/ teacher lunch with Energy Secretary Hazel O'Leary and a private visit to the Forum exhibits. The continuing partnership with Kearny High School includes lectures, job shadowing, internship, equipment donations and an award-winning electric car-racing program. Development of distribution by CD of the existing interactive fusion energy kiosk and a virtual reality tour of the DIII--D facility are underway. The DIII--D fusion education WWW site includes e-mail addresses to ``Ask the Wizard,'' and/or receive GA's outreach materials. Steve Rodecker, a local science teacher, aided by DIII--D fusion staff, won his second Tapestry Award; he also was named the ``1995 National Science Teacher of the Year'' and will be present to share his experiences with the DIII--D educational outreach program.

In Situ 3D Monitoring of Geometric Signatures in the Powder-Bed-Fusion Additive Manufacturing Process via Vision Sensing Methods

PubMed Central

Li, Zhongwei; Liu, Xingjian; Wen, Shifeng; He, Piyao; Zhong, Kai; Wei, Qingsong; Shi, Yusheng; Liu, Sheng

2018-01-01

Lack of monitoring of the in situ process signatures is one of the challenges that has been restricting the improvement of Powder-Bed-Fusion Additive Manufacturing (PBF AM). Among various process signatures, the monitoring of the geometric signatures is of high importance. This paper presents the use of vision sensing methods as a non-destructive in situ 3D measurement technique to monitor two main categories of geometric signatures: 3D surface topography and 3D contour data of the fusion area. To increase the efficiency and accuracy, an enhanced phase measuring profilometry (EPMP) is proposed to monitor the 3D surface topography of the powder bed and the fusion area reliably and rapidly. A slice model assisted contour detection method is developed to extract the contours of fusion area. The performance of the techniques is demonstrated with some selected measurements. Experimental results indicate that the proposed method can reveal irregularities caused by various defects and inspect the contour accuracy and surface quality. It holds the potential to be a powerful in situ 3D monitoring tool for manufacturing process optimization, close-loop control, and data visualization. PMID:29649171

Fusion Studies in Japan

NASA Astrophysics Data System (ADS)

Ogawa, Yuichi

2016-05-01

A new strategic energy plan decided by the Japanese Cabinet in 2014 strongly supports the steady promotion of nuclear fusion development activities, including the ITER project and the Broader Approach activities from the long-term viewpoint. Atomic Energy Commission (AEC) in Japan formulated the Third Phase Basic Program so as to promote an experimental fusion reactor project. In 2005 AEC has reviewed this Program, and discussed on selection and concentration among many projects of fusion reactor development. In addition to the promotion of ITER project, advanced tokamak research by JT-60SA, helical plasma experiment by LHD, FIREX project in laser fusion research and fusion engineering by IFMIF were highly prioritized. Although the basic concept is quite different between tokamak, helical and laser fusion researches, there exist a lot of common features such as plasma physics on 3-D magnetic geometry, high power heat load on plasma facing component and so on. Therefore, a synergetic scenario on fusion reactor development among various plasma confinement concepts would be important.

Fusion Rate and Clinical Outcomes in Two-Level Posterior Lumbar Interbody Fusion.

PubMed

Aono, Hiroyuki; Takenaka, Shota; Nagamoto, Yukitaka; Tobimatsu, Hidekazu; Yamashita, Tomoya; Furuya, Masayuki; Iwasaki, Motoki

2018-04-01

Posterior lumbar interbody fusion (PLIF) has become a general surgical method for degenerative lumbar diseases. Although many reports have focused on single-level PLIF, few have focused on 2-level PLIF, and no report has covered the fusion status of 2-level PLIF. The purpose of this study is to investigate clinical outcomes and fusion for 2-level PLIF by using a combination of dynamic radiographs and multiplanar-reconstruction computed tomography scans. This study consisted of 48 consecutive patients who underwent 2-level PLIF for degenerative lumbar diseases. We assessed surgery duration, estimated blood loss, complications, clinical outcomes as measured by the Japanese Orthopaedic Association score, lumbar sagittal alignment as measured on standing lateral radiographs, and fusion status as measured by dynamic radiographs and multiplanar-reconstruction computed tomography. Patients were examined at a follow-up point of 4.8 ± 2.2 years after surgery. Thirty-eight patients who did not undergo lumbosacral fusion comprised the lumbolumbar group, and 10 patients who underwent lumbosacral fusion comprised the lumbosacral group. The mean Japanese Orthopaedic Association score improved from 12.1 to 22.4 points by the final follow-up examination. Sagittal alignment also was improved. All patients had fusion in the cranial level. Seven patients had nonunion in the caudal level, and the lumbosacral group (40%) had a significantly poorer fusion rate than the lumbolumbar group (97%) did. Surgical outcomes of 2-level PLIF were satisfactory. The fusion rate at both levels was 85%. All nonunion was observed at the caudal level and concentrated at L5-S level in L4-5-S PLIF. Copyright © 2018 Elsevier Inc. All rights reserved.

Identification and classification of genes required for tolerance to freeze-thaw stress revealed by genome-wide screening of Saccharomyces cerevisiae deletion strains.

PubMed

Ando, Akira; Nakamura, Toshihide; Murata, Yoshinori; Takagi, Hiroshi; Shima, Jun

2007-03-01

Yeasts used in bread making are exposed to freeze-thaw stress during frozen-dough baking. To clarify the genes required for freeze-thaw tolerance, genome-wide screening was performed using the complete deletion strain collection of diploid Saccharomyces cerevisiae. The screening identified 58 gene deletions that conferred freeze-thaw sensitivity. These genes were then classified based on their cellular function and on the localization of their products. The results showed that the genes required for freeze-thaw tolerance were frequently involved in vacuole functions and cell wall biogenesis. The highest numbers of gene products were components of vacuolar H(+)-ATPase. Next, the cross-sensitivity of the freeze-thaw-sensitive mutants to oxidative stress and to cell wall stress was studied; both of these are environmental stresses closely related to freeze-thaw stress. The results showed that defects in the functions of vacuolar H(+)-ATPase conferred sensitivity to oxidative stress and to cell wall stress. In contrast, defects in gene products involved in cell wall assembly conferred sensitivity to cell wall stress but not to oxidative stress. Our results suggest the presence of at least two different mechanisms of freeze-thaw injury: oxidative stress generated during the freeze-thaw process, and defects in cell wall assembly.

A comparison of defect size and film quality obtained from Film digitized image and digital image radiographs

NASA Astrophysics Data System (ADS)

Kamlangkeng, Poramate; Asa, Prateepasen; Mai, Noipitak

2014-06-01

Digital radiographic testing is an acceptable premature nondestructive examination technique. Its performance and limitation comparing to the old technique are still not widely well known. In this paper conducted the study on the comparison of the accuracy of the defect size measurement and film quality obtained from film and digital radiograph techniques by testing in specimens and known size sample defect. Initially, one specimen was built with three types of internal defect; which are longitudinal cracking, lack of fusion, and porosity. For the known size sample defect, it was machined various geometrical size for comparing the accuracy of the measuring defect size to the real size in both film and digital images. To compare the image quality by considering at smallest detectable wire and the three defect images. In this research used Image Quality Indicator (IQI) of wire type 10/16 FE EN BS EN-462-1-1994. The radiographic films were produced by X-ray and gamma ray using Kodak AA400 size 3.5x8 inches, while the digital images were produced by Fuji image plate type ST-VI with 100 micrometers resolution. During the tests, a radiator GE model MF3 was implemented. The applied energy is varied from 120 to 220 kV and the current from 1.2 to 3.0 mA. The intensity of Iridium 192 gamma ray is in the range of 24-25 Curie. Under the mentioned conditions, the results showed that the deviation of the defect size measurement comparing to the real size obtained from the digital image radiographs is below than that of the film digitized, whereas the quality of film digitizer radiographs is higher in comparison.

Role of transmembrane segment 5 of the plant vacuolar H+-pyrophosphatase.

PubMed

Van, Ru C; Pan, Yih J; Hsu, Shen H; Huang, Yun T; Hsiao, Yi Y; Pan, Rong L

2005-08-15

Vacuolar H+-translocating inorganic pyrophosphatase (V-PPase; EC 3.6.1.1) is a homodimeric proton translocase consisting of a single type of polypeptide with a molecular mass of approximately 81 kDa. Topological analysis tentatively predicts that mung bean V-PPase contains 14 transmembrane domains. Alignment analysis of V-PPase demonstrated that the transmembrane domain 5 (TM5) of the enzyme is highly conserved in plants and located at the N-terminal side of the putative substrate-binding loop. The hydropathic analysis of V-PPase showed a relatively lower degree of hydrophobicity in the TM5 region as compared to other domains. Accordingly, it appears that TM5 is probably involved in the proton translocation of V-PPase. In this study, we used site-directed mutagenesis to examine the functional role of amino acid residues in TM5 of V-PPase. A series of mutants singly replaced by alanine residues along TM5 were constructed and over-expressed in Saccharomyces cerevisiae; they were then used to determine their enzymatic activities and proton translocations. Our results indicate that several mutants displayed minor variations in enzymatic properties, while others including those mutated at E225, a GYG motif (residues from 229 to 231), A238, and R242, showed a serious decline in enzymatic activity, proton translocation, and coupling efficiency of V-PPase. Moreover, the mutation at Y230 relieved several cation effects on the V-PPase. The GYG motif presumably plays a significant role in maintaining structure and function of V-PPase.

Control of mechanically activated polymersome fusion: Factors affecting fusion

DOE PAGES

Henderson, Ian M.; Paxton, Walter F.

2014-12-15

Previously we have studied the mechanically-activated fusion of extruded (200 nm) polymer vesicles into giant polymersomes using agitation in the presence of salt. In this study we have investigated several factors contributing to this phenomenon, including the effects of (i) polymer vesicle concentration, (ii) agitation speed and duration, and iii) variation of the salt and its concentration. It was found that increasing the concentration of the polymer dramatically increases the production of giant vesicles through the increased collisions of polymersomes. Our investigations also found that increasing the frequency of agitation increased the efficiency of fusion, though ultimately limited the sizemore » of vesicle which could be produced due to the high shear involved. Finally it was determined that salt-mediation of the fusion process was not limited to NaCl, but is instead a general effect facilitated by the presence of solvated ionic compounds, albeit with different salts initiating fusion at different concentration.« less

Image fusion

NASA Technical Reports Server (NTRS)

Pavel, M.

1993-01-01

The topics covered include the following: a system overview of the basic components of a system designed to improve the ability of a pilot to fly through low-visibility conditions such as fog; the role of visual sciences; fusion issues; sensor characterization; sources of information; image processing; and image fusion.

50 years of fusion research

NASA Astrophysics Data System (ADS)

Meade, Dale

2010-01-01

Fusion energy research began in the early 1950s as scientists worked to harness the awesome power of the atom for peaceful purposes. There was early optimism for a quick solution for fusion energy as there had been for fission. However, this was soon tempered by reality as the difficulty of producing and confining fusion fuel at temperatures of 100 million °C in the laboratory was appreciated. Fusion research has followed two main paths—inertial confinement fusion and magnetic confinement fusion. Over the past 50 years, there has been remarkable progress with both approaches, and now each has a solid technical foundation that has led to the construction of major facilities that are aimed at demonstrating fusion energy producing plasmas.

SEMATECH produces defect-free EUV mask blanks: defect yield and immediate challenges

NASA Astrophysics Data System (ADS)

Antohe, Alin O.; Balachandran, Dave; He, Long; Kearney, Patrick; Karumuri, Anil; Goodwin, Frank; Cummings, Kevin

2015-03-01

Availability of defect-free reflective mask has been one of the most critical challenges to extreme ultraviolet lithography (EUVL). To mitigate the risk, significant progress has been made on defect detection, pattern shifting, and defect repair. Clearly such mitigation strategies are based on the assumption that defect counts and sizes from incoming mask blanks must be below practical levels depending on mask specifics. The leading industry consensus for early mask product development is that there should be no defects greater than 80 nm in the quality area, 132 mm x 132 mm. In addition less than 10 defects smaller than 80 nm may be mitigable. SEMATECH has been focused on EUV mask blank defect reduction using Veeco Nexus TM IBD platform, the industry standard for mask blank production, and assessing if IBD technology can be evolved to a manufacturing solution. SEMATECH has recently announced a breakthrough reduction of defects in the mask blank deposition process resulting in the production of two defect-free EUV mask blanks at 54 nm inspection sensitivity (SiO2 equivalent). This paper will discuss the dramatic reduction of baseline EUV mask blank defects, review the current deposition process run and compare results with previous process runs. Likely causes of remaining defects will be discussed based on analyses as characterized by their compositions and whether defects are embedded in the multilayer stack or non-embedded.

Functional mechanism of lung mosaic CT attenuation: assessment with deep-inspiration breath-hold perfusion SPECT-CT fusion imaging and non-breath-hold Technegas SPECT.

PubMed

Suga, K; Yasuhiko, K; Iwanaga, H; Tokuda, O; Matsunaga, N

2009-01-01

The functional mechanism of lung mosaic computed tomography attenuation (MCA) in pulmonary vascular disease (PVD) and obstructive airway disease (OAD) has not yet been fully clarified. To clarify the mechanism of MCA in these diseases by assessing the relationship between regional lung function and CT attenuation change at MCA sites with the use of automated deep-inspiratory breath-hold (DIBrH) perfusion single-photon emission computed tomography (SPECT)-CT fusion images and non-breath-hold Technegas SPECT. Subjects were 42 PVD patients (31 pulmonary thromboembolism, four primary/two secondary pulmonary hypertension, and five Takayasu arteritis), 12 OAD patients (five acute asthma, four obliterative bronchiolitis, and three bronchiectasis), and 12 normal controls, all of whom had MCA on DIBrH CT. The relationship between regional lung function and CT attenuation change at the lung slices with MCA was assessed using DIBrH perfusion SPECT-CT fusion images and non-breath-hold Technegas SPECT. The severity of perfusion defects with or without MCA was quantified by regions-of-interest analysis. On DIBrH CT and perfusion SPECT, in contrast to no noticeable CT attenuation abnormality and fairly uniform perfusion in controls, 60 MCA and 274 perfusion defects in PVD patients, and 18 MCA and 61 defects in OAD patients were identified, with a total of 77 ventilation defects on Technegas SPECT in all patients. SPECT-CT correlation showed that, throughout the 78 MCA sites of all patients, lung perfusion was persistently decreased at low CT attenuation and preserved at intervening high CT attenuation, while lung ventilation was poorly correlated with CT attenuation change. The radioactivity ratios of reduced perfusion and the intervening preserved perfusion at the 78 perfusion defects with MCA were significantly lower than those at the remaining 257 defects without MCA (P<0.0001). Although further validation is required, our results indicate that heterogeneous pulmonary arterial

A new level of plasticity: Drosophila smooth-like testes muscles compensate failure of myoblast fusion

PubMed Central

Kuckwa, Jessica; Fritzen, Katharina; Buttgereit, Detlev; Rothenbusch-Fender, Silke; Renkawitz-Pohl, Renate

2016-01-01

The testis of Drosophila resembles an individual testis tubule of mammals. Both are surrounded by a sheath of smooth muscles, which in Drosophila are multinuclear and originate from a pool of myoblasts that are set aside in the embryo and accumulate on the genital disc later in development. These muscle stem cells start to differentiate early during metamorphosis and give rise to all muscles of the inner male reproductive system. Shortly before the genital disc and the developing testes connect, multinuclear nascent myotubes appear on the anterior tips of the seminal vesicles. Here, we show that adhesion molecules are distinctly localized on the seminal vesicles; founder cell (FC)-like myoblasts express Dumbfounded (Duf) and Roughest (Rst), and fusion-competent myoblast (FCM)-like cells mainly express Sticks and stones (Sns). The smooth but multinuclear myotubes of the testes arose by myoblast fusion. RNAi-mediated attenuation of Sns or both Duf and Rst severely reduced the number of nuclei in the testes muscles. Duf and Rst probably act independently in this context. Despite reduced fusion in all of these RNAi-treated animals, myotubes migrated onto the testes, testes were shaped and coiled, muscle filaments were arranged as in the wild type and spermatogenesis proceeded normally. Hence, the testes muscles compensate for fusion defects so that the myofibres encircling the adult testes are indistinguishable from those of the wild type and male fertility is guaranteed. PMID:26657767

Fusion for Space Propulsion

NASA Technical Reports Server (NTRS)

Thio, Y. C. Francis; Schafer, Charles (Technical Monitor)

2001-01-01

There is little doubt that humans will attempt to explore and develop the solar system in this century. A large amount of energy will be required for accomplishing this. The need for fusion propulsion is discussed. For a propulsion system, there are three important thermodynamical attributes: (1) The absolute amount of energy available, (2) the propellant exhaust velocity, and (3) the jet power per unit mass of the propulsion system (specific power). For human exploration and development of the solar system, propellant exhaust velocity in excess of 100 km/s and specific power in excess of 10 kW/kg are required. Chemical combustion can produce exhaust velocity up to about 5 km/s. Nuclear fission processes typically result in producing energy in the form of heat that needs to be manipulated at temperatures limited by materials to about 2,800 K. Using the energy to heat a hydrogen propellant increases the exhaust velocity by only a factor of about two. Alternatively the energy can be converted into electricity which is then used to accelerate particles to high exhaust velocity. The necessary power conversion and conditioning equipment, however, increases the mass of the propulsion system for the same jet power by more than two orders of magnitude over chemical system, thus greatly limits the thrust-to-weight ratio attainable. The principal advantage of the fission process is that its development is relatively mature and is available right now. If fusion can be developed, fusion appears to have the best of all worlds in terms of propulsion - it can provide the absolute amount, the propellant exhaust velocity, and the high specific jet power. An intermediate step towards pure fusion propulsion is a bimodal system in which a fission reactor is used to provide some of the energy to drive a fusion propulsion unit. The technical issues related to fusion for space propulsion are discussed. The technical priorities for developing and applying fusion for propulsion are

C-terminal tyrosine residues modulate the fusion activity of the Hendra virus fusion protein

PubMed Central

Popa, Andreea; Pager, Cara Teresia; Dutch, Rebecca Ellis

2011-01-01

The paramyxovirus family includes important human pathogens such as measles, mumps, respiratory syncytial virus and the recently emerged, highly pathogenic Hendra and Nipah viruses. The viral fusion (F) protein plays critical roles in infection, promoting both the viral-cell membrane fusion events needed for viral entry as well as cell-cell fusion events leading to syncytia formation. We describe the surprising finding that addition of the short epitope HA tag to the cytoplasmic tail (CT) of the Hendra virus F protein leads to a significant increase in cell-cell membrane fusion. This increase was not due to alterations in surface expression, cleavage state, or association with lipid microdomains. Addition of a Myc tag of similar length did not alter Hendra F fusion activity, indicating that the observed stimulation was not solely a result of lengthening the CT. Three tyrosine residues within the HA tag were critical for the increase in fusion, suggesting C-terminal tyrosines may modulate Hendra fusion activity. The effects of HA tag addition varied with other fusion proteins, as parainfluenza virus 5 F-HA showed decreased surface expression and no stimulation in fusion. These results indicate that additions to the C-terminal end of the F protein CT can modulate protein function in a sequence specific manner, reinforcing the need for careful analysis of epitope tagged glycoproteins. In addition, our results implicate C-terminal tyrosine residues in modulation of the membrane fusion reaction promoted by these viral glycoproteins. PMID:21175223

Opioids delay healing of spinal fusion: a rabbit posterolateral lumbar fusion model.

PubMed

Jain, Nikhil; Himed, Khaled; Toth, Jeffrey M; Briley, Karen C; Phillips, Frank M; Khan, Safdar N

2018-04-19

Opioid use is prevalent for management of pre- and post-operative pain in patients undergoing spinal fusion. There is evidence that opioids downregulate osteoblasts in-vitro, and one previous study found that morphine delays the maturation and remodeling of callus in a rat femur fracture model. However, the effect of opioids on healing of spinal fusion has not been investigated before. Isolating the effect of opioid exposure in humans would be limited by the numerous confounding factors that affect fusion healing. Therefore, we have used a well-established rabbit model to study the process of spinal fusion healing that closely mimics humans. To study the effect of systemic opioids on the process of healing of spinal fusion in a rabbit posterolateral spinal fusion model. Pre-clinical animal study. 24 adult New Zealand white rabbits were studied in two groups after approval from the Institutional Animal Care and Use Committee (IACUC). The opioid group (n=12) received four-weeks pre-operative and six-weeks post-operative transdermal fentanyl. Serum fentanyl levels were measured just before surgery and four-weeks post-operatively to ensure adequate levels. The control group (n=12) received only peri-operative pain control as necessary. All animals received a bilateral L5-L6 posterolateral spinal fusion using iliac crest autograft. Animals were euthanized at the six-week post-operative time point, and assessment of fusion was done by manual palpation, plain radiographs, micro-computed tomography (microCT), and histology. 12 animals in control group and 11 animals in the opioid group were available for analysis at the end of six weeks. The fusion scores on manual palpation, radiographs, and microCT were not statistically different. Three-dimensional microCT morphometry found that the fusion mass in the opioid group had a lower bone volume (p=0.09), lower trabecular number (p=0.02) and higher trabecular separation (p=0.02) as compared to control. Histological analysis

A network model for the specification of vulval precursor cells and cell fusion control in Caenorhabditis elegans

PubMed Central

Weinstein, Nathan; Mendoza, Luis

2013-01-01

The vulva of Caenorhabditis elegans has been long used as an experimental model of cell differentiation and organogenesis. While it is known that the signaling cascades of Wnt, Ras/MAPK, and NOTCH interact to form a molecular network, there is no consensus regarding its precise topology and dynamical properties. We inferred the molecular network, and developed a multivalued synchronous discrete dynamic model to study its behavior. The model reproduces the patterns of activation reported for the following types of cell: vulval precursor, first fate, second fate, second fate with reversed polarity, third fate, and fusion fate. We simulated the fusion of cells, the determination of the first, second, and third fates, as well as the transition from the second to the first fate. We also used the model to simulate all possible single loss- and gain-of-function mutants, as well as some relevant double and triple mutants. Importantly, we associated most of these simulated mutants to multivulva, vulvaless, egg-laying defective, or defective polarity phenotypes. The model shows that it is necessary for RAL-1 to activate NOTCH signaling, since the repression of LIN-45 by RAL-1 would not suffice for a proper second fate determination in an environment lacking DSL ligands. We also found that the model requires the complex formed by LAG-1, LIN-12, and SEL-8 to inhibit the transcription of eff-1 in second fate cells. Our model is the largest reconstruction to date of the molecular network controlling the specification of vulval precursor cells and cell fusion control in C. elegans. According to our model, the process of fate determination in the vulval precursor cells is reversible, at least until either the cells fuse with the ventral hypoderm or divide, and therefore the cell fates must be maintained by the presence of extracellular signals. PMID:23785384

Inorganic-organic shape memory polymers and foams for bone defect repairs

NASA Astrophysics Data System (ADS)

Zhang, Dawei

The ultimate goal of this research was to develop a "self-fitting" shape memory polymer (SMP) scaffold for the repair of craniomaxillofacial (CMF) bone defects. CMF defects may be caused by trauma, tumor removal or congenital abnormalities and represent a major class of bone defects. Their repair with autografts is limited by availability, donor site morbidity and complex surgical procedures. In addition, shaping and positioning of these rigid grafts into irregular defects is difficult. Herein, we have developed SMP scaffolds which soften at T > ˜56 °C, allowing them to conformally fit into a bone defect. Upon cooling to body temperature, the scaffold becomes rigid and mechanically locks in place. This research was comprised of four major studies. In the first study, photocrosslinkable acrylated (AcO) SMP macromers containing a poly(epsilon-caprolactone) (PCL) segment and polydimethylsiloxane (PDMS) segments were synthesized with the general formula: AcO-PCL40-block-PDMS m-block-PCL40-OAc. By varying the PDMS segment length (m), solid SMPs with highly tunable mechanical properties and excellent shape memory abilities were prepared. In the second study, porous SMP scaffolds were fabricated based on AcO-PCL 40-block-PDMS37-block-PCL 40-OAc via a revised solvent casting particulate leaching (SCPL) method. By tailoring scaffold parameters including salt fusion, macromer concentration and salt size, scaffold properties (e.g. pore features, compressive modulus and shape memory behavior) were tuned. In the third study, porous SMP scaffolds were produced from macromers with variable PDMS segment lengths (m = 0 -- 130) via an optimized SCPL method. The impact on pore features, thermal, mechanical, and shape memory properties as well as degradation rates were investigated. In the final study, a bioactive polydopamine coating was applied onto pore surfaces of the SMP scaffold prepared from PCL diacrylate. The thin coating did not affect intrinsic bulk properties of the

Systematic analysis of advanced fusion fuel in inertial fusion energy

NASA Astrophysics Data System (ADS)

Velarde, G.; Eliezer, S.; Henis, Z.; Piera, M.; Martinez-Val, J. M.

1997-04-01

Aneutronic fusion reactions can be considered as the cleanest way to exploit nuclear energy. However, these reactions present in general two main drawbacks.—very high temperatures are needed to reach relevant values of their cross sections—Moderate (and even low) energy yield per reaction. This value is still lower if measured in relation to the Z number of the reacting particles. It is already known that bremsstrahlung overruns the plasma reheating by fusion born charged-particles in most of the advanced fuels. This is for instance the case for proton-boron-11 fusion in a stoichiometric plasma and is also so in lithium isotopes fusion reactions. In this paper, the use of deuterium-tritium seeding is suggested to allow to reach higher burnup fractions of advanced fuels, starting at a lower ignition temperature. Of course, neutron production increases as DT contents does. Nevertheless, the ratio of neutron production to energy generation is much lower in DT-advanced fuel mixtures than in pure DT plasmas. One of the main findings of this work is that some natural resources (as D and Li-7) can be burned-up in a catalytic regime for tritium. In this case, neither external tritium breeding nor tritium storage are needed, because the tritium inventory after the fusion burst is the same as before it. The fusion reactor can thus operate on a pure recycling of a small tritium inventory.

A voltage-gated calcium channel regulates lysosomal fusion with endosomes and autophagosomes and is required for neuronal homeostasis.

PubMed

Tian, Xuejun; Gala, Upasana; Zhang, Yongping; Shang, Weina; Nagarkar Jaiswal, Sonal; di Ronza, Alberto; Jaiswal, Manish; Yamamoto, Shinya; Sandoval, Hector; Duraine, Lita; Sardiello, Marco; Sillitoe, Roy V; Venkatachalam, Kartik; Fan, Hengyu; Bellen, Hugo J; Tong, Chao

2015-03-01

Autophagy helps deliver sequestered intracellular cargo to lysosomes for proteolytic degradation and thereby maintains cellular homeostasis by preventing accumulation of toxic substances in cells. In a forward mosaic screen in Drosophila designed to identify genes required for neuronal function and maintenance, we identified multiple cacophony (cac) mutant alleles. They exhibit an age-dependent accumulation of autophagic vacuoles (AVs) in photoreceptor terminals and eventually a degeneration of the terminals and surrounding glia. cac encodes an α1 subunit of a Drosophila voltage-gated calcium channel (VGCC) that is required for synaptic vesicle fusion with the plasma membrane and neurotransmitter release. Here, we show that cac mutant photoreceptor terminals accumulate AV-lysosomal fusion intermediates, suggesting that Cac is necessary for the fusion of AVs with lysosomes, a poorly defined process. Loss of another subunit of the VGCC, α2δ or straightjacket (stj), causes phenotypes very similar to those caused by the loss of cac, indicating that the VGCC is required for AV-lysosomal fusion. The role of VGCC in AV-lysosomal fusion is evolutionarily conserved, as the loss of the mouse homologues, Cacna1a and Cacna2d2, also leads to autophagic defects in mice. Moreover, we find that CACNA1A is localized to the lysosomes and that loss of lysosomal Cacna1a in cerebellar cultured neurons leads to a failure of lysosomes to fuse with endosomes and autophagosomes. Finally, we show that the lysosomal CACNA1A but not the plasma-membrane resident CACNA1A is required for lysosomal fusion. In summary, we present a model in which the VGCC plays a role in autophagy by regulating the fusion of AVs with lysosomes through its calcium channel activity and hence functions in maintaining neuronal homeostasis.

FusionHub: A unified web platform for annotation and visualization of gene fusion events in human cancer.

PubMed

Panigrahi, Priyabrata; Jere, Abhay; Anamika, Krishanpal

2018-01-01

Gene fusion is a chromosomal rearrangement event which plays a significant role in cancer due to the oncogenic potential of the chimeric protein generated through fusions. At present many databases are available in public domain which provides detailed information about known gene fusion events and their functional role. Existing gene fusion detection tools, based on analysis of transcriptomics data usually report a large number of fusion genes as potential candidates, which could be either known or novel or false positives. Manual annotation of these putative genes is indeed time-consuming. We have developed a web platform FusionHub, which acts as integrated search engine interfacing various fusion gene databases and simplifies large scale annotation of fusion genes in a seamless way. In addition, FusionHub provides three ways of visualizing fusion events: circular view, domain architecture view and network view. Design of potential siRNA molecules through ensemble method is another utility integrated in FusionHub that could aid in siRNA-based targeted therapy. FusionHub is freely available at https://fusionhub.persistent.co.in.

Gene Fusion Markup Language: a prototype for exchanging gene fusion data.

PubMed

Kalyana-Sundaram, Shanker; Shanmugam, Achiraman; Chinnaiyan, Arul M

2012-10-16

An avalanche of next generation sequencing (NGS) studies has generated an unprecedented amount of genomic structural variation data. These studies have also identified many novel gene fusion candidates with more detailed resolution than previously achieved. However, in the excitement and necessity of publishing the observations from this recently developed cutting-edge technology, no community standardization approach has arisen to organize and represent the data with the essential attributes in an interchangeable manner. As transcriptome studies have been widely used for gene fusion discoveries, the current non-standard mode of data representation could potentially impede data accessibility, critical analyses, and further discoveries in the near future. Here we propose a prototype, Gene Fusion Markup Language (GFML) as an initiative to provide a standard format for organizing and representing the significant features of gene fusion data. GFML will offer the advantage of representing the data in a machine-readable format to enable data exchange, automated analysis interpretation, and independent verification. As this database-independent exchange initiative evolves it will further facilitate the formation of related databases, repositories, and analysis tools. The GFML prototype is made available at http://code.google.com/p/gfml-prototype/. The Gene Fusion Markup Language (GFML) presented here could facilitate the development of a standard format for organizing, integrating and representing the significant features of gene fusion data in an inter-operable and query-able fashion that will enable biologically intuitive access to gene fusion findings and expedite functional characterization. A similar model is envisaged for other NGS data analyses.

Gene Fusion Markup Language: a prototype for exchanging gene fusion data

PubMed Central

2012-01-01

Background An avalanche of next generation sequencing (NGS) studies has generated an unprecedented amount of genomic structural variation data. These studies have also identified many novel gene fusion candidates with more detailed resolution than previously achieved. However, in the excitement and necessity of publishing the observations from this recently developed cutting-edge technology, no community standardization approach has arisen to organize and represent the data with the essential attributes in an interchangeable manner. As transcriptome studies have been widely used for gene fusion discoveries, the current non-standard mode of data representation could potentially impede data accessibility, critical analyses, and further discoveries in the near future. Results Here we propose a prototype, Gene Fusion Markup Language (GFML) as an initiative to provide a standard format for organizing and representing the significant features of gene fusion data. GFML will offer the advantage of representing the data in a machine-readable format to enable data exchange, automated analysis interpretation, and independent verification. As this database-independent exchange initiative evolves it will further facilitate the formation of related databases, repositories, and analysis tools. The GFML prototype is made available at http://code.google.com/p/gfml-prototype/. Conclusion The Gene Fusion Markup Language (GFML) presented here could facilitate the development of a standard format for organizing, integrating and representing the significant features of gene fusion data in an inter-operable and query-able fashion that will enable biologically intuitive access to gene fusion findings and expedite functional characterization. A similar model is envisaged for other NGS data analyses. PMID:23072312

Herpes simplex virus glycoproteins gB and gH function in fusion between the virion envelope and the outer nuclear membrane.

PubMed

Farnsworth, Aaron; Wisner, Todd W; Webb, Michael; Roller, Richard; Cohen, Gary; Eisenberg, Roselyn; Johnson, David C

2007-06-12

Herpesviruses must traverse the nuclear envelope to gain access to the cytoplasm and, ultimately, to exit cells. It is believed that herpesvirus nucleocapsids enter the perinuclear space by budding through the inner nuclear membrane (NM). To reach the cytoplasm these enveloped particles must fuse with the outer NM and the unenveloped capsids then acquire a second envelope in the trans-Golgi network. Little is known about the process by which herpesviruses virions fuse with the outer NM. Here we show that a herpes simplex virus (HSV) mutant lacking both the two putative fusion glycoproteins gB and gH failed to cross the nuclear envelope. Enveloped virions accumulated in the perinuclear space or in membrane vesicles that bulged into the nucleoplasm (herniations). By contrast, mutants lacking just gB or gH showed only minor or no defects in nuclear egress. We concluded that either HSV gB or gH can promote fusion between the virion envelope and the outer NM. It is noteworthy that fusion associated with HSV entry requires the cooperative action of both gB and gH, suggesting that the two types of fusion (egress versus entry) are dissimilar processes.

Line tension at lipid phase boundaries as driving force for HIV fusion peptide-mediated fusion

NASA Astrophysics Data System (ADS)

Yang, Sung-Tae; Kiessling, Volker; Tamm, Lukas K.

2016-04-01

Lipids and proteins are organized in cellular membranes in clusters, often called `lipid rafts'. Although raft-constituent ordered lipid domains are thought to be energetically unfavourable for membrane fusion, rafts have long been implicated in many biological fusion processes. For the case of HIV gp41-mediated membrane fusion, this apparent contradiction can be resolved by recognizing that the interfaces between ordered and disordered lipid domains are the predominant sites of fusion. Here we show that line tension at lipid domain boundaries contributes significant energy to drive gp41-fusion peptide-mediated fusion. This energy, which depends on the hydrophobic mismatch between ordered and disordered lipid domains, may contribute tens of kBT to fusion, that is, it is comparable to the energy required to form a lipid stalk intermediate. Line-active compounds such as vitamin E lower line tension in inhomogeneous membranes, thereby inhibit membrane fusion, and thus may be useful natural viral entry inhibitors.

Plasma-wall interaction in laser inertial fusion reactors: novel proposals for radiation tests of first wall materials

NASA Astrophysics Data System (ADS)

Alvarez Ruiz, J.; Rivera, A.; Mima, K.; Garoz, D.; Gonzalez-Arrabal, R.; Gordillo, N.; Fuchs, J.; Tanaka, K.; Fernández, I.; Briones, F.; Perlado, J.

2012-12-01

Dry-wall laser inertial fusion (LIF) chambers will have to withstand strong bursts of fast charged particles which will deposit tens of kJ m-2 and implant more than 1018 particles m-2 in a few microseconds at a repetition rate of some Hz. Large chamber dimensions and resistant plasma-facing materials must be combined to guarantee the chamber performance as long as possible under the expected threats: heating, fatigue, cracking, formation of defects, retention of light species, swelling and erosion. Current and novel radiation resistant materials for the first wall need to be validated under realistic conditions. However, at present there is a lack of facilities which can reproduce such ion environments. This contribution proposes the use of ultra-intense lasers and high-intense pulsed ion beams (HIPIB) to recreate the plasma conditions in LIF reactors. By target normal sheath acceleration, ultra-intense lasers can generate very short and energetic ion pulses with a spectral distribution similar to that of the inertial fusion ion bursts, suitable to validate fusion materials and to investigate the barely known propagation of those bursts through background plasmas/gases present in the reactor chamber. HIPIB technologies, initially developed for inertial fusion driver systems, provide huge intensity pulses which meet the irradiation conditions expected in the first wall of LIF chambers and thus can be used for the validation of materials too.

Single-dose replication-defective VSV-based Nipah virus vaccines provide protection from lethal challenge in Syrian hamsters.

PubMed

Lo, Michael K; Bird, Brian H; Chattopadhyay, Anasuya; Drew, Clifton P; Martin, Brock E; Coleman, Joann D; Rose, John K; Nichol, Stuart T; Spiropoulou, Christina F

2014-01-01

Nipah virus (NiV) continues to cause outbreaks of fatal human encephalitis due to spillover from its bat reservoir. We determined that a single dose of replication-defective vesicular stomatitis virus (VSV)-based vaccine vectors expressing either the NiV fusion (F) or attachment (G) glycoproteins protected hamsters from over 1000 times LD50 NiV challenge. This highly effective single-dose protection coupled with an enhanced safety profile makes these candidates ideal for potential use in livestock and humans. Published by Elsevier B.V.

Neural Tube Defects

MedlinePlus

Neural tube defects are birth defects of the brain, spine, or spinal cord. They happen in the ... that she is pregnant. The two most common neural tube defects are spina bifida and anencephaly. In ...

Gradient-based multiresolution image fusion.

PubMed

Petrović, Valdimir S; Xydeas, Costas S

2004-02-01

A novel approach to multiresolution signal-level image fusion is presented for accurately transferring visual information from any number of input image signals, into a single fused image without loss of information or the introduction of distortion. The proposed system uses a "fuse-then-decompose" technique realized through a novel, fusion/decomposition system architecture. In particular, information fusion is performed on a multiresolution gradient map representation domain of image signal information. At each resolution, input images are represented as gradient maps and combined to produce new, fused gradient maps. Fused gradient map signals are processed, using gradient filters derived from high-pass quadrature mirror filters to yield a fused multiresolution pyramid representation. The fused output image is obtained by applying, on the fused pyramid, a reconstruction process that is analogous to that of conventional discrete wavelet transform. This new gradient fusion significantly reduces the amount of distortion artefacts and the loss of contrast information usually observed in fused images obtained from conventional multiresolution fusion schemes. This is because fusion in the gradient map domain significantly improves the reliability of the feature selection and information fusion processes. Fusion performance is evaluated through informal visual inspection and subjective psychometric preference tests, as well as objective fusion performance measurements. Results clearly demonstrate the superiority of this new approach when compared to conventional fusion systems.

A Cell-Cell Fusion Assay to Assess Arenavirus Envelope Glycoprotein Membrane-Fusion Activity.

PubMed

York, Joanne; Nunberg, Jack H

2018-01-01

For many viruses that enter their target cells through pH-dependent fusion of the viral and endosomal membranes, cell-cell fusion assays can provide an experimental platform for investigating the structure-function relationships that promote envelope glycoprotein membrane-fusion activity. Typically, these assays employ effector cells expressing the recombinant envelope glycoprotein on the cell surface and target cells engineered to quantitatively report fusion with the effector cell. In the protocol described here, Vero cells are transfected with a plasmid encoding the arenavirus envelope glycoprotein complex GPC and infected with the vTF7-3 vaccinia virus expressing the bacteriophage T7 RNA polymerase. These effector cells are mixed with target cells infected with the vCB21R-lacZ vaccinia virus encoding a β-galactosidase reporter under the control of the T7 promoter. Cell-cell fusion is induced upon exposure to low-pH medium (pH 5.0), and the resultant expression of the β-galactosidase reporter is quantitated using a chemiluminescent substrate. We have utilized this robust microplate cell-cell fusion assay extensively to study arenavirus entry and its inhibition by small-molecule fusion inhibitors.

ITER Fusion Energy

ScienceCinema

Holtkamp, Norbert

2018-01-09

ITER (in Latin “the way”) is designed to demonstrate the scientific and technological feasibility of fusion energy. Fusion is the process by which two light atomic nuclei combine to form a heavier over one and thus release energy. In the fusion process two isotopes of hydrogen – deuterium and tritium – fuse together to form a helium atom and a neutron. Thus fusion could provide large scale energy production without greenhouse effects; essentially limitless fuel would be available all over the world. The principal goals of ITER are to generate 500 megawatts of fusion power for periods of 300 to 500 seconds with a fusion power multiplication factor, Q, of at least 10. Q ? 10 (input power 50 MW / output power 500 MW). The ITER Organization was officially established in Cadarache, France, on 24 October 2007. The seven members engaged in the project – China, the European Union, India, Japan, Korea, Russia and the United States – represent more than half the world’s population. The costs for ITER are shared by the seven members. The cost for the construction will be approximately 5.5 billion Euros, a similar amount is foreseen for the twenty-year phase of operation and the subsequent decommissioning.

VCP-dependent muscle degeneration is linked to defects in a dynamic tubular lysosomal network in vivo

PubMed Central

Johnson, Alyssa E; Shu, Huidy; Hauswirth, Anna G; Tong, Amy; Davis, Graeme W

2015-01-01

Lysosomes are classically viewed as vesicular structures to which cargos are delivered for degradation. Here, we identify a network of dynamic, tubular lysosomes that extends throughout Drosophila muscle, in vivo. Live imaging reveals that autophagosomes merge with tubular lysosomes and that lysosomal membranes undergo extension, retraction, fusion and fission. The dynamics and integrity of this tubular lysosomal network requires VCP, an AAA-ATPase that, when mutated, causes degenerative diseases of muscle, bone and neurons. We show that human VCP rescues the defects caused by loss of Drosophila VCP and overexpression of disease relevant VCP transgenes dismantles tubular lysosomes, linking tubular lysosome dysfunction to human VCP-related diseases. Finally, disruption of tubular lysosomes correlates with impaired autophagosome-lysosome fusion, increased cytoplasmic poly-ubiquitin aggregates, lipofuscin material, damaged mitochondria and impaired muscle function. We propose that VCP sustains sarcoplasmic proteostasis, in part, by controlling the integrity of a dynamic tubular lysosomal network. DOI: http://dx.doi.org/10.7554/eLife.07366.001 PMID:26167652

Electron-impact excitation and recombination of molecular cations in edge fusion plasma: application to H2+and BeD+

NASA Astrophysics Data System (ADS)

Pop, Nicolina; Iacob, Felix; Mezei, Zsolt; Motapon, Ousmanou; Niyonzima, Sebastien; Schneider, Ioan

2017-10-01

Dissociative recombination, ro-vibrational excitation and dissociative excitation of molecular cations with electrons are major elementary process in the kinetics and in the energy balance of astrophysically-relevant ionized media (supernovae, interstellar molecular clouds, planetary ionospheres, early Universe), in edge fusion and in many other cold media of technological interest. For the fusion plasma edge, extensive cross sections and rate coefficients have been produced for reactions induced on HD+, H2+ and BeD+ using the Multichannel Quantum Defect Theory (MQDT). Our calculations resulted in good agreement with the CRYRING (Stockholm) and TSR (Heidelberg) magnetic storage ring results, and our approach is permanently improved in order to face the new generation of electrostatic storage rings, as CSR (Heidelberg) and DESIREE (Stockholm). Member of APS Reciprocal Society: European Physics Society.

Paramyxovirus F1 protein has two fusion peptides: implications for the mechanism of membrane fusion.

PubMed

Peisajovich, S G; Samuel, O; Shai, Y

2000-03-10

Viral fusion proteins contain a highly hydrophobic segment, named the fusion peptide, which is thought to be responsible for the merging of the cellular and viral membranes. Paramyxoviruses are believed to contain a single fusion peptide at the N terminus of the F1 protein. However, here we identified an additional internal segment in the Sendai virus F1 protein (amino acids 214-226) highly homologous to the fusion peptides of HIV-1 and RSV. A synthetic peptide, which includes this region, was found to induce membrane fusion of large unilamellar vesicles, at concentrations where the known N-terminal fusion peptide is not effective. A scrambled peptide as well as several peptides from other regions of the F1 protein, which strongly bind to membranes, are not fusogenic. The functional and structural characterization of this active segment suggest that the F1 protein has an additional internal fusion peptide that could participate in the actual fusion event. The presence of homologous regions in other members of the same family suggests that the concerted action of two fusion peptides, one N-terminal and the other internal, is a general feature of paramyxoviruses. Copyright 2000 Academic Press.

Distal Renal Tubular Acidosis and Calcium Nephrolithiasis

NASA Astrophysics Data System (ADS)

Moe, Orson W.; Fuster, Daniel G.; Xie, Xiao-Song

2008-09-01

Calcium stones are commonly encountered in patients with congenital distal renal tubular acidosis, a disease of renal acidification caused by mutations in either the vacuolar H+-ATPase (B1 or a4 subunit), anion exchanger-1, or carbonic anhydrase II. Based on the existing database, we present two hypotheses. First, heterozygotes with mutations in B1 subunit of H+-ATPase are not normal but may harbor biochemical abnormalities such as renal acidification defects, hypercalciuria, and hypocitraturia which can predispose them to kidney stone formation. Second, we propose at least two mechanisms by which mutant B1 subunit can impair H+-ATPase: defective pump assembly and defective pump activity.

Cell fusion and nuclear fusion in plants.

PubMed

Maruyama, Daisuke; Ohtsu, Mina; Higashiyama, Tetsuya

2016-12-01

Eukaryotic cells are surrounded by a plasma membrane and have a large nucleus containing the genomic DNA, which is enclosed by a nuclear envelope consisting of the outer and inner nuclear membranes. Although these membranes maintain the identity of cells, they sometimes fuse to each other, such as to produce a zygote during sexual reproduction or to give rise to other characteristically polyploid tissues. Recent studies have demonstrated that the mechanisms of plasma membrane or nuclear membrane fusion in plants are shared to some extent with those of yeasts and animals, despite the unique features of plant cells including thick cell walls and intercellular connections. Here, we summarize the key factors in the fusion of these membranes during plant reproduction, and also focus on "non-gametic cell fusion," which was thought to be rare in plant tissue, in which each cell is separated by a cell wall. Copyright © 2016 Elsevier Ltd. All rights reserved.

[Bone morphogenetic proteins (BMP): clinical application for reconstruction of bone defects].

PubMed

Sierra-García, Gerardo Daniel; Castro-Ríos, Rocío; Gónzalez-Horta, Azucena; Lara-Arias, Jorge; Chávez-Montes, Abelardo

2016-01-01

Since the introduction of bone morphogenetic proteins, their use has become an invaluable ally for the treatment of bone defects. These proteins are potent growth factors, related to angiogenic and osteogenic activity. The osteoinductive capacity of recombinant bone morphogenetic protein (rhBMP) in the formation of bone and cartilage has been confirmed in in vitro studies and evaluated in clinical trials. To obtain a therapeutic effect, administration is systemic, by injection over the physiological dose. Among the disadvantages, ectopic bone formation or high morbidity in cases of spinal fusion is observed. In this review, the roles of bone morphogenetic proteins in bone repair and clinical applications are analyzed. These findings represent advances in the study of bone regeneration and application of growth factors for more predictable results.

Single Ventricle Defects

MedlinePlus

... Your Risk • Symptoms & Diagnosis • Care & Treatment • Tools & Resources Web Booklets on Congenital Heart Defects These online publications ... to you or your child’s defect and concerns. Web Booklet: Adults With Congenital Heart Defects Web Booklet: ...

Investigations of image fusion

NASA Astrophysics Data System (ADS)

Zhang, Zhong

1999-12-01

The objective of image fusion is to combine information from multiple images of the same scene. The result of image fusion is a single image which is more suitable for the purpose of human visual perception or further image processing tasks. In this thesis, a region-based fusion algorithm using the wavelet transform is proposed. The identification of important features in each image, such as edges and regions of interest, are used to guide the fusion process. The idea of multiscale grouping is also introduced and a generic image fusion framework based on multiscale decomposition is studied. The framework includes all of the existing multiscale-decomposition- based fusion approaches we found in the literature which did not assume a statistical model for the source images. Comparisons indicate that our framework includes some new approaches which outperform the existing approaches for the cases we consider. Registration must precede our fusion algorithms. So we proposed a hybrid scheme which uses both feature-based and intensity-based methods. The idea of robust estimation of optical flow from time- varying images is employed with a coarse-to-fine multi- resolution approach and feature-based registration to overcome some of the limitations of the intensity-based schemes. Experiments show that this approach is robust and efficient. Assessing image fusion performance in a real application is a complicated issue. In this dissertation, a mixture probability density function model is used in conjunction with the Expectation- Maximization algorithm to model histograms of edge intensity. Some new techniques are proposed for estimating the quality of a noisy image of a natural scene. Such quality measures can be used to guide the fusion. Finally, we study fusion of images obtained from several copies of a new type of camera developed for video surveillance. Our techniques increase the capability and reliability of the surveillance system and provide an easy way to obtain 3-D

Quantitative Analysis of Lipid Droplet Fusion: Inefficient Steady State Fusion but Rapid Stimulation by Chemical Fusogens

PubMed Central

Murphy, Samantha; Martin, Sally; Parton, Robert G.

2010-01-01

Lipid droplets (LDs) are dynamic cytoplasmic organelles containing neutral lipids and bounded by a phospholipid monolayer. Previous studies have suggested that LDs can undergo constitutive homotypic fusion, a process linked to the inhibitory effects of fatty acids on glucose transporter trafficking. Using strict quantitative criteria for LD fusion together with refined light microscopic methods and real-time analysis, we now show that LDs in diverse cell types show low constitutive fusogenic activity under normal growth conditions. To investigate the possible modulation of LD fusion, we screened for agents that can trigger fusion. A number of pharmacological agents caused homotypic fusion of lipid droplets in a variety of cell types. This provided a novel cell system to study rapid regulated fusion between homotypic phospholipid monolayers. LD fusion involved an initial step in which the two adjacent membranes became continuous (<10 s), followed by the slower merging (100 s) of the neutral lipid cores to produce a single spherical LD. These fusion events were accompanied by changes to the LD surface organization. Measurements of LDs undergoing homotypic fusion showed that fused LDs maintained their initial volume, with a corresponding decrease in surface area suggesting rapid removal of membrane from the fused LD. This study provides estimates for the level of constitutive LD fusion in cells and questions the role of LD fusion in vivo. In addition, it highlights the extent of LD restructuring which occurs when homotypic LD fusion is triggered in a variety of cell types. PMID:21203462

SCFTIR1/AFB-auxin signalling regulates PIN vacuolar trafficking and auxin fluxes during root gravitropism

PubMed Central

Baster, Paweł; Robert, Stéphanie; Kleine-Vehn, Jürgen; Vanneste, Steffen; Kania, Urszula; Grunewald, Wim; De Rybel, Bert; Beeckman, Tom; Friml, Jiří

2013-01-01

The distribution of the phytohormone auxin regulates many aspects of plant development including growth response to gravity. Gravitropic root curvature involves coordinated and asymmetric cell elongation between the lower and upper side of the root, mediated by differential cellular auxin levels. The asymmetry in the auxin distribution is established and maintained by a spatio-temporal regulation of the PIN-FORMED (PIN) auxin transporter activity. We provide novel insights into the complex regulation of PIN abundance and activity during root gravitropism. We show that PIN2 turnover is differentially regulated on the upper and lower side of gravistimulated roots by distinct but partially overlapping auxin feedback mechanisms. In addition to regulating transcription and clathrin-mediated internalization, auxin also controls PIN abundance at the plasma membrane by promoting their vacuolar targeting and degradation. This effect of elevated auxin levels requires the activity of SKP-Cullin-F-boxTIR1/AFB (SCFTIR1/AFB)-dependent pathway. Importantly, also suboptimal auxin levels mediate PIN degradation utilizing the same signalling pathway. These feedback mechanisms are functionally important during gravitropic response and ensure fine-tuning of auxin fluxes for maintaining as well as terminating asymmetric growth. PMID:23211744

Enhanced image capture through fusion

NASA Technical Reports Server (NTRS)

Burt, Peter J.; Hanna, Keith; Kolczynski, Raymond J.

1993-01-01

Image fusion may be used to combine images from different sensors, such as IR and visible cameras, to obtain a single composite with extended information content. Fusion may also be used to combine multiple images from a given sensor to form a composite image in which information of interest is enhanced. We present a general method for performing image fusion and show that this method is effective for diverse fusion applications. We suggest that fusion may provide a powerful tool for enhanced image capture with broad utility in image processing and computer vision.

Positional effects of fusion partners on the yield and solubility of MBP fusion proteins

PubMed Central

Raran-Kurussi, Sreejith; Keefe, Karina; Waugh, David S.

2015-01-01

Escherichia coli maltose-binding protein (MBP) is exceptionally effective at promoting the solubility of its fusion partners. However, there are conflicting reports in the literature claiming that 1) MBP is an effective solubility enhancer only when it is joined to the N-terminus of an aggregation-prone passenger protein, and 2) MBP is equally effective when fused to either end of the passenger. Here, we endeavor to resolve this controversy by comparing the solubility of a diverse set of MBP fusion proteins that, unlike those analyzed in previous studies, are identical in every way except for the order of the two domains. The results indicate that fusion proteins with an N-terminal MBP provide an excellent solubility advantage along with more robust expression when compared to analogous fusions in which MBP is the C-terminal fusion partner. We find that only intrinsically soluble passenger proteins (i.e., those not requiring a solubility enhancer) are produced as soluble fusions when they precede MBP. We also report that even subtle differences in inter-domain linker sequences can influence the solubility of fusion proteins. PMID:25782741

Complementation of DNA repair defect in xeroderma pigmentosum cells of group C by the transfer of human chromosome 5

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

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

1993-01-01

Complementation of DNA excision repair defect in xeroderma pigmentosum cells of group C (XP-C) has been achieved by the transfer of human chromosome 5. Individual human chromosomes tagged with a selectable marker were transferred to XP-C cells by microcell fusion from mouse-human hybrid cell lines each bearing a single different human chromosome. Analysis of the chromosome transfer clones revealed that introduction of chromosome 5 into XP-C cells corrected the DNA repair defect as well as UV-sensitive phenotypes, while chromosomes 2, 6, 7, 9, 13, 15, 17, and 21 failed to complement. The introduced chromosome 5 in complemented UV[sup r] clonesmore » was distinguished from the parental XP-C chromosomes by polymorphism for dinucleotide (CA)[sub n] repeats at two loci, D5S117 and D5S209. In addition, an intact marked chromosome 5 was rescued into mouse cells from a complemented UV[sup r] clone by microcell fusion. Five subclones of a complemented clone that had lost the marked chromosome 5 exhibited UV-sensitive and repair-deficient phenotypes identical to parental XP-C cells. Concordant loss of the transferred chromosome and reappearance of XP-C phenotype further confirmed the presence of a DNA repair gene on human chromosome 5. 38 refs., 7 figs., 1 tab.« less

Fusion or confusion: knowledge or nonsense?

NASA Astrophysics Data System (ADS)

Rothman, Peter L.; Denton, Richard V.

1991-08-01

The terms 'data fusion,' 'sensor fusion,' multi-sensor integration,' and 'multi-source integration' have been used widely in the technical literature to refer to a variety of techniques, technologies, systems, and applications which employ and/or combine data derived from multiple information sources. Applications of data fusion range from real-time fusion of sensor information for the navigation of mobile robots to the off-line fusion of both human and technical strategic intelligence data. The Department of Defense Critical Technologies Plan lists data fusion in the highest priority group of critical technologies, but just what is data fusion? The DoD Critical Technologies Plan states that data fusion involves 'the acquisition, integration, filtering, correlation, and synthesis of useful data from diverse sources for the purposes of situation/environment assessment, planning, detecting, verifying, diagnosing problems, aiding tactical and strategic decisions, and improving system performance and utility.' More simply states, sensor fusion refers to the combination of data from multiple sources to provide enhanced information quality and availability over that which is available from any individual source alone. This paper presents a survey of the state-of-the- art in data fusion technologies, system components, and applications. A set of characteristics which can be utilized to classify data fusion systems is presented. Additionally, a unifying mathematical and conceptual framework within which to understand and organize fusion technologies is described. A discussion of often overlooked issues in the development of sensor fusion systems is also presented.

Dicentric breakage at telomere fusions

PubMed Central